Datasets:

sandy37

/

elife

like 0

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "epidemiology and global health" ]

[ [ "It is well-established that death rates vary throughout the year , and in temperate climates there tend to be more deaths in winter than in summer ( Campbell , 2017; Fowler et al . , 2015; Healy , 2003; McKee , 1989 ) .", "It has therefore been hypothesized that a warmer world may lower winter mortality in temperate climates ( Langford and Bentham , 1995; Martens , 1998 ) .", "In a large country like the USA , which possesses distinct climate regions , the seasonality of mortality may vary geographically , due to geographical variations in mortality , localized weather patterns , and regional differences in adaptation measures such as heating , air conditioning and healthcare ( Davis et al . , 2004; Braga et al . , 2001; Kalkstein , 2013; Medina-Ramón and Schwartz , 2007 ) .", "The presence and extent of seasonal variation in mortality may also itself change over time ( Bobb et al . , 2014; Carson et al . , 2006; Seretakis et al . , 1997; Sheridan et al . , 2009 ) .", "A thorough understanding of the long-term dynamics of seasonality of mortality , and its geographical and demographic patterns , is needed to identify at-risk groups , plan responses at the present time as well as under changing climate conditions .", "Although mortality seasonality is well-established , there is limited information on how seasonality , including the timing of minimum and maximum mortality , varies by local climate and how these features have changed over time , especially in relation to age group , sex and medical cause of death ( Rau , 2004; Rau et al . , 2018 ) .", "In this paper , we comprehensively characterize the spatial and temporal patterns of all-cause and cause-specific mortality seasonality in the USA by sex and age group , through the application of wavelet analytical techniques , to over three decades of national mortality data .", "Wavelets have been used to study the dynamics of weather phenomena ( Moy et al . , 2002 ) and infectious diseases ( Grenfell et al . , 2001 ) .", "We also used centre of gravity analysis and circular statistics methods to understand the timing of maximum and minimum mortality .", "In addition , we identify how the percentage difference between death rates in maximum and minimum mortality months has changed over time ." ], [ "The strengths of our study are its innovative methods of characterizing seasonality of mortality dynamically over space and time , by age group and cause of death; using wavelet and centre of gravity analyses; using ERA-Interim data output to compare the association between seasonality of death rates and regional temperature .", "A limitation of our study is that we did not investigate seasonality of mortality by socioeconomic characteristics which may help with understanding its determinants and planning responses ." ], [ "We used wavelet and centre of gravity analyses , which allowed systematically identifying and characterizing seasonality of total and cause-specific mortality in the USA , and examining how seasonality has changed over time .", "We identified distinct seasonal patterns in relation to age and sex , including higher all-cause summer mortality in young men ( Feinstein , 2002; Rau et al . , 2018 ) .", "Importantly , we also showed that all-cause and cause-specific mortality seasonality is largely similar in terms of both timing and magnitude across diverse climatic regions with substantially different summer and winter temperatures .", "Insights of this kind would not have been possible analysing data averaged over time or nationally , or fixed to pre-specified frequencies .", "Prior studies have noted seasonality of mortality for all-cause mortality and for specific causes of death in the USA ( Feinstein , 2002; Kalkstein , 2013; Rau , 2004; Rau et al . , 2018; Rosenwaike , 1966; Seretakis et al . , 1997 ) .", "Few of these studies have done consistent national and subnational analyses , and none has done so over time , for a comprehensive set of age groups and causes of death , and in relation to regional temperature differences .", "Our results on strong seasonality of cardiorespiratory diseases deaths and weak seasonality of cancer deaths , restricted to older ages , are broadly consistent with these studies ( Feinstein , 2002; Rau et al . , 2018; Rosenwaike , 1966; Seretakis et al . , 1997 ) , which had limited analysis on how seasonality changes over time and geography ( Feinstein , 2002; Rau et al . , 2018; Rosenwaike , 1966 ) .", "Similarly , our results on seasonality of injury deaths are supported by a few prior studies ( Feinstein , 2002; Rau et al . , 2018; Rosenwaike , 1966 ) , but our subnational analysis over three decades revealed variations in when injury deaths peaked and in how seasonal differences in these deaths have changed over time in relation to age group which had not been reported before .", "A study of 36 cities in the USA , aggregated across age groups and over time , also found that excess mortality was not associated with seasonal temperature range ( Kinney et al . , 2015 ) .", "In contrast , a European study found that the difference between winter and summer mortality was lower in colder Nordic countries than in warmer southern European nations ( Healy , 2003; McKee , 1989 ) ( the study’s measure of temperature was mean annual temperature which differed from the temperature difference between maximum and minimum mortality used in our analysis although the two measures are correlated ) .", "The absence of variation in the magnitude of mortality seasonality indicates that different regions in the USA are similarly adapted to temperature seasonality , whereas Nordic countries may have better environmental ( e . g . housing insulation and heating ) and health system measures to counter the effects of cold winters than those in southern Europe .", "If the observed absence of association between the magnitude of mortality seasonality and seasonal temperature difference across the climate regions also persists over time , the changes in temperature as a result of global climate change are unlikely to affect the winter-summer mortality difference .", "The cause-specific analysis showed that the substantial decline in seasonal mortality differences in adolescents and young adults was related to the diminishing seasonality of ( unintentional ) injuries , especially from road traffic crashes , which are more likely to occur in the summer months ( Liu et al . , 2005 ) and are more common in men .", "The weakening of seasonality in boys under five years of age was related to two phenomena: first , the seasonality of death from cardiorespiratory diseases declined , and second , the proportion of deaths from perinatal conditions , which exhibit limited seasonality ( Figure 9—figure supplement 2 and Figure 10—figure supplement 3 ) , increased ( MacDorman and Gregory , 2015 ) .", "In contrast to young and middle ages , mortality in older ages , where death rates are highest , maintained persistent seasonality over a period of three decades ( we note that although the percent seasonal difference in mortality has remained largely unchanged in these ages , the absolute difference in death rates between the peak and minimum months has declined because total mortality has a declining long-term trend ) .", "This finding demonstrates the need for environmental and health service interventions targeted towards this group irrespective of geography and local climate .", "Examples of such interventions include enhancing the availability of both environmental and medical protective factors , such as better insulation of homes , winter heating provision and flu vaccinations , for the vulnerable older population ( Katiyo et al . , 2017 ) .", "Social interventions , including regular visits to the isolated elderly during peak mortality periods to ensure that they are optimally prepared for adverse conditions , and responsive and high-quality emergency care , are also important to protect this vulnerable group ( Healy , 2003; Lerchl , 1998; Katiyo et al . , 2017 ) .", "Emergent new technologies , such as always-connected hands-free communications devices with the outside world , in-house cameras , and personal sensors also provide an opportunity to enhance care for the older , more vulnerable groups in the population , especially in winter when the elderly have fewer social interactions ( Morris , 2013 ) .", "Such interventions are important today , and will remain so as the population ages and climate change increases the within- and between-season weather variability ." ], [ "We used data on all 85 , 854 , 176 deaths in the USA from 1980 to 2016 from the National Center for Health Statistics ( NCHS ) .", "Age , sex , state of residence , month of death , and underlying cause of death were available for each record .", "The underlying cause of death was coded according to the international classification of diseases ( ICD ) system ( 9th revision of ICD from 1980 to 1998 and 10th revision of ICD thereafter ) .", "Yearly population counts were available from NCHS for 1990 to 2016 and from the US Census Bureau prior to 1990 ( Ingram et al . , 2003 ) .", "We calculated monthly population counts through linear interpolation , assigning each yearly count to July .", "We also subdivided the national data geographically into nine climate regions used by the National Oceanic and Atmospheric Administration ( Figure 18 and Table 2 ) ( Karl and Koss , 1984 ) .", "On average , the Southeast and South are the hottest climate regions with average annual temperatures of 18 . 4°C and 18°C respectively; the South also possesses the highest average maximum monthly temperature ( 27 . 9°C in July ) .", "The lowest variation in temperature throughout the year is that of the Southeast ( an average range of 17 . 5°C ) .", "The three coldest climate regions are West North Central , East North Central and the Northwest ( 7 . 6°C , 8 . 0°C , 8 . 2°C respectively ) .", "Mirroring the characteristics of the hottest climate regions , the largest variation in temperature throughout the year is that of the coldest region , West North Central ( an average range of 30 . 5°C ) , which also has the lowest average minimum monthly temperature ( −6 . 5°C in January ) .", "The other climate regions , Northeast , Southwest , and Central , possess similar average temperatures ( 10°C to 14°C ) and variation within the year of ( 23°C to 26°C ) , with the Northeast being the most populous region in the United States ( with 19 . 8% total population in 2016 ) .", "Data were divided by sex and age in the following 10 age groups: 0–4 , 5–14 , 15-24 , 25–34 , 35–44 , 45–54 , 55–64 , 65–74 , 75–84 , 85+ years .", "We calculated monthly death rates for each age and sex group , both nationally and for sub-national climate regions .", "Death rate calculations accounted for varying length of months , by multiplying each month’s death count by a factor that would make it equivalent to a 31 day month .", "For analysis of seasonality by cause of death , we mapped each ICD-9 and ICD-10 codes to four main disease categories ( Table 1 ) and to a number of subcategories which are presented in the Supplementary Note .", "Cardiorespiratory diseases and cancers accounted for 56 . 4% and 21 . 2% of all deaths in the USA , respectively , in 1980 , and 40 . 3% and 22 . 4% , respectively , in 2016 .", "Deaths from cardiorespiratory diseases have been associated with cold and warm temperatures ( Basu , 2009; Basu and Samet , 2002; Bennett et al . , 2014; Braga et al . , 2002; Gasparrini et al . , 2015 ) .", "Injuries , which accounted for 8% of all deaths in the USA in 1980 and 7 . 3% in 2016 , may have seasonality that is distinct from so-called natural causes .", "We did not further divide other causes because the number of deaths could become too small to allow stable estimates when divided by age group , sex and climate region .", "We obtained data on temperature from ERA-Interim , which combines predictions from a physical model with ground-based and satellite measurements ( Dee et al . , 2011 ) .", "We used gridded four-times-daily estimates at a resolution of 80 km to generate monthly population-weighted temperature by climate region throughout the analysis period .", "We used wavelet analysis to investigate seasonality for each age-sex group .", "Wavelet analysis uncovers the presence , and frequency , of repeated maxima and minima in each age-sex-specific death rate time series ( Hubbard , 1998; Torrence and Compo , 1998 ) .", "In brief , a Morlet wavelet , described in detail elsewhere ( Cazelles et al . , 2008 ) , is equivalent to using a moving window on the death rate time series and analysing periodicity in each window using a short-form Fourier transform , hence generating a dynamic spectral analysis , which allows measuring dynamic seasonal patterns , in which the periodicity of death rates may disappear , emerge , or change over time .", "In addition to coefficients that measure the frequency of periodicity , wavelet analysis estimates the probability of whether the data are different from the null situation of random fluctuations that can be represented with white ( an independent random process ) or red ( autoregressive of order one process ) noise .", "For each age-sex group , we calculated the p-values of the presence of 12 month seasonality for the comparison of wavelet power spectra of the entire study period ( 1980–2016 ) with 100 simulations against a white noise spectrum , which represents random fluctuations .", "We used the R package WaveletComp ( version 1 . 0 ) for the wavelet analysis .", "Before analysis , we de-trended death rates using a polynomial regression , and rescaled each death rate time series so as to range between 1 and −1 .", "To identify the months of maximum and minimum death rates , we calculated the centre of gravity and the negative centre of gravity of monthly death rates .", "Centre of gravity was calculated as a weighted average of months of deaths , with each month weighted by its death rate; negative centre of gravity was also calculated as a weighted average of months of deaths , but with each month was weighted by the difference between its death rate and the year’s maximum death rate .", "In taking the weighted average , we allowed December ( month 12 ) to neighbour January ( month 1 ) , representing each month by an angle subtended from 12 equally-spaced points around a unit circle .", "Using a technique called circular statistics , a mean ( θ- ) of the angles ( θ1 , θ2 , θ3… , θn , ) representing the deaths ( with n the total number of deaths in an age-sex group for a particular cause of death ) is found using the relation below:θ-=arg∑j=1nexp⁡ ( iθj ) , where arg denotes the complex number argument and θj denotes the month of death in angular form for a particular death j .", "The outcome of this calculation is then converted back into a month value ( Fisher , 1995 ) .", "Along with each circular mean , a 95% confidence interval ( CI ) was calculated by using 1000 bootstrap samples .", "The R package CircStats ( version 0 . 2 . 4 ) was used for this analysis .", "For each age-sex group and cause of death , and for each year , we calculated the percent difference in death rates between the maximum and minimum mortality months .", "We fitted a linear regression to the time series of seasonal differences from 1980 to 2016 , and used the fitted trend line to estimate how much the percentage difference in death rates between the maximum and minimum mortality months had changed from 1980 to 2016 .", "We weighted seasonal difference by the inverse of the square of its standard error , which was calculated using a Poisson model to take population size of each age-sex group through time into account .", "This method gives us a p-value for the change in seasonal difference per year , which we used to calculate the seasonal difference at the start ( 1980 ) and end ( 2016 ) of the period of study .", "Our method of analysing seasonal differences avoids assuming that any specific month or group of months represent highest and lowest number of deaths for a particular cause of death , which is the approach taken by the traditional measure of Excess Winter Deaths .", "It also allows the maximum and minimum mortality months to vary by age group , sex and cause of death ." ] ]

[ "In temperate climates , winter deaths exceed summer ones .", "However , there is limited information on the timing and the relative magnitudes of maximum and minimum mortality , by local climate , age group , sex and medical cause of death .", "We used geo-coded mortality data and wavelets to analyse the seasonality of mortality by age group and sex from 1980 to 2016 in the USA and its subnational climatic regions .", "Death rates in men and women ≥ 45 years peaked in December to February and were lowest in June to August , driven by cardiorespiratory diseases and injuries .", "In these ages , percent difference in death rates between peak and minimum months did not vary across climate regions , nor changed from 1980 to 2016 .", "Under five years , seasonality of all-cause mortality largely disappeared after the 1990s .", "In adolescents and young adults , especially in males , death rates peaked in June/July and were lowest in December/January , driven by injury deaths ." ]

[ "In the USA , more deaths happen in the winter than the summer .", "But when deaths occur varies greatly by sex , age , cause of death , and possibly region .", "Seasonal differences in death rates can change over time due to changes in factors that cause disease or affect treatment .", "Analyzing the seasonality of deaths can help scientists determine whether interventions to minimize deaths during a certain time of year are needed , or whether existing ones are effective .", "Scrutinizing seasonal patterns in death over time can also help scientists determine whether large-scale weather or climate changes are affecting the seasonality of death .", "Now , Parks et al . show that there are age and sex differences in which times of year most deaths occur .", "Parks et al . analyzed data on US deaths between 1980 and 2016 .", "While overall deaths in a year were highest in winter and lowest in summer , a greater number of young men died during summer – mainly due to injuries – than during winter .", "Seasonal differences in deaths among young children have largely disappeared and seasonal differences in the deaths of older children and young adults have become smaller .", "Deaths among women and men aged 45 or older peaked between December and February – largely caused by respiratory and heart diseases , or injuries .", "Deaths in this older age group were lowest during the summer months .", "Death patterns in older people changed little over time .", "No regional differences were found in seasonal death patterns , despite large climate variation across the USA .", "The analysis by Parks et al . suggests public health and medical interventions have been successful in reducing seasonal deaths among many groups .", "But more needs to be done to address seasonal differences in deaths among older adults .", "For example , by boosting flu vaccination rates , providing warnings about severe weather and better insulation for homes .", "Using technology like hands-free communication devices or home visits to help keep vulnerable elderly people connected during the winter months may also help ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "microbiology and infectious disease", "immunology and inflammation" ]

[ [ "Dysregulated complement activation is increasingly recognized as a significant pathological event in a variety of neurodegenerative and neuroinflammatory diseases of the central nervous system ( Tenner et al . , 2018 ) .", "These include conditions from Alzheimer’s disease and age-related macular degeneration ( AMD ) to amyotrophic lateral sclerosis and multiple sclerosis ( Hong et al . , 2016; Knickelbein et al . , 2015; Lee et al . , 2018; Loveless et al . , 2018 ) .", "Complement may orchestrate peripheral nervous system ( PNS ) disorders as well .", "For instance , nociceptive hypersensitivities , sensory neuropathies , and Guillain-Barré syndrome have been linked to aberrant complement activation ( Rosoklija et al . , 2000; Ramaglia et al . , 2008; Jang et al . , 2010; Fritzinger and Benjamin , 2016; Xu et al . , 2018; Susuki et al . , 2007 ) .", "However , the mechanistic role of complement is not well understood in neuroinflammatory pathologies impacting the PNS .", "Moreover , the pathophysiological trigger of sensory fiber retraction which contributes to sensation loss and in some instances chronic pain is unclear ( Stepp et al . , 2017 ) .", "Available treatments for peripheral neuropathies primarily focus on clinical symptoms without addressing the underlying pathomechanisms ( Colloca et al . , 2017 ) .", "Accordingly , elucidation of relevant pathomechanisms underlying sensory neuropathies may translate into efficacious mechanism-based therapeutics .", "The complement cascade is comprised of dozens of soluble and membrane-bound factors essential for host defense and efficient clearance of cellular debris .", "However , proper regulation of complement activation is necessary to balance immune responses and prevent collateral tissue damage ( Ricklin et al . , 2016 ) .", "The complement cascade pivots upon activation of complement component 3 ( C3 ) for its downstream proinflammatory effects .", "The pathogenic contributions of complement in systemic disease have been reviewed extensively in recent years ( Dobó et al . , 2018; Hajishengallis et al . , 2017; McGeer et al . , 2017; Ricklin et al . , 2016 ) .", "Nonetheless , emerging concepts centering on complement effector synthesis within inflamed tissue microenvironments have ushered in a ‘renaissance’ of novel complement-targeted drug development strategies ( Ricklin et al . , 2018; Tomlinson and Thurman , 2018 ) .", "Therefore , elucidating whether the complement cascade is a viable therapeutic target in inflammation-associated peripheral nerve impairment remains medically important .", "The eye has been labeled a ‘complement dysregulation hotspot’ due to complement’s contributions to many ophthalmic diseases , but complement activation is restricted in the healthy cornea ( Clark and Bishop , 2018 ) .", "As observed in AMD , subtle inflammatory reactions in the eye can mediate significant visual morbidity .", "Accordingly , ocular inflammation is tightly regulated by various anatomic , physiologic , and immunologic mechanisms in order to maintain visual acuity ( Amouzegar et al . , 2016; Streilein , 2003; Taylor et al . , 2018 ) .", "These homeostatic mechanisms , collectively dubbed ‘ocular immune privilege , ’ help preserve the ocular surface and enable the cornea to properly focus incoming light onto the retina .", "Furthermore , corneal nerves are increasingly recognized as central regulators of immune privilege at the ocular surface ( Guzmán et al . , 2018; Neelam et al . , 2018; Paunicka et al . , 2015 ) .", "Consequently , inflammatory events that damage corneal nerves can have insidious consequences in terms of ocular surface health and transparency ( Müller et al . , 2003; Shaheen et al . , 2014; Stepp et al . , 2017 ) .", "One such pathway that can rapidly initiate inflammation following activation is the complement cascade .", "Corneal nerves originate predominantly from the ophthalmic branch of the trigeminal ganglion ( TG ) , and these peripheral nerves provide the cornea with the highest density of sensory fibers in the human body .", "While the cornea and peripheral nerves constitutively synthesize complement proteins ( Bora et al . , 2008; de Jonge et al . , 2004 ) , the role of the complement cascade in corneal nerve damage has not been explored .", "Nonetheless , the complement pathway is poised for vigorous activation in the cornea in response to noxious or inflammatory stimuli ( Bora et al . , 2008 ) .", "Moreover , C3 activation has been reported in the cornea soon after infection with herpes simplex virus type 1 ( HSV-1 ) , which is a common cause of corneal sensory nerve damage in patients ( Royer et al . , 2017; Sacchetti and Lambiase , 2014 ) .", "Given the cornea’s high density of sensory nerves , it is particularly amenable for investigating the mechanistic role of complement in peripheral nerve damage .", "To this end , we evaluated corneal nerve integrity and mechano-sensory function using a murine model of ocular HSV-1 infection to test the hypothesis that local complement activation and T cell engagement coordinate corneal nerve damage .", "The pathobiology underlying non-penetrating corneal nerve damage is not well understood , although inflammation is generally recognized as an important feature ( Neelam et al . , 2018; Shaheen et al . , 2014; Cruzat et al . , 2015; Chucair-Elliott et al . , 2016; Chucair-Elliott et al . , 2017b; Chucair-Elliott et al . , 2017a ) .", "While HSV-1 is a clinically prominent cause of corneal nerve damage and sensation loss , a variety of pathogenic microbes impair the corneal nerve architecture upon ocular infection ( Cruzat et al . , 2015 ) .", "This observation adds to evidence that the neurotropism of HSV-1 is not directly responsible for nerve damage ( Chucair-Elliott et al . , 2016 ) .", "In addition to sensation loss evoked by HSV-1 , corneal nerve alterations in other contexts such as dry eye disease are associated with a broad array of neuropathic clinical symptoms including dryness , itch , and pain ( Andersen et al . , 2017 ) .", "To further qualify the possible role of complement in corneal nerve damage independent of infection , we evaluated corneal mechanosensory function in two noninfectious T cell-dependent ocular surface inflammatory diseases .", "For this purpose , we utilized established murine models of allergic eye disease ( AED ) and ocular graft-versus-host disease ( GVHD ) ( Herretes et al . , 2015; Lee et al . , 2015 ) .", "Our rationale for this is that complement has been implicated in the etiology of systemic GVHD and allergic inflammation ( Gour et al . , 2018; Kwan et al . , 2012; Ma et al . , 2014; Nguyen et al . , 2018; Zhang and Köhl , 2010 ) .", "However , a neuroinflammatory role of complement has not been described for either disease within the eye .", "The translational relevance of this study is underscored by in vivo confocal microscopy data from multiple clinical studies showing architectural changes in the corneal nerves of patients with herpetic keratitis , chronic ocular allergy , and ocular GVHD ( Hamrah et al . , 2010; Müller et al . , 2015; Moein et al . , 2018; Hu et al . , 2008; Le et al . , 2011; Leonardi et al . , 2012; Tepelus et al . , 2017; He et al . , 2017a ) .", "Elucidating the pathomechanisms underlying corneal nerve damage may enable development of more effective therapeutics to mitigate progression of such ocular surface inflammatory diseases .", "Each of the animal models utilized herein mimic clinically important , chronic ocular surface morbidities .", "Herpes simplex virus type 1 ( HSV-1 ) is a common cause of neurotropic keratitis and remains a leading cause of infectious corneal blindness ( Sacchetti and Lambiase , 2014; Farooq and Shukla , 2012 ) .", "The incidence of ocular allergy exceeds twenty percent of the population with varying degrees of neurogenic ocular surface discomfort that can severely diminish quality of life ( Craig et al . , 2017; Patel et al . , 2017; Saban et al . , 2013 ) .", "Finally , GVHD is the greatest cause of non-relapse morbidity following hematopoietic stem cell transplantation ( HSCT ) used to treat life-threatening malignancies and immunologic diseases ( MacDonald et al . , 2017 ) .", "A majority of patients with chronic presentations of GVHD suffer from ocular surface involvement ( Shikari et al . , 2013 ) ." ], [ "Adaptive immunity has been shown to prevent recovery of corneal sensory function following ocular HSV-1 infection ( Yun et al . , 2014 ) , but the initial pathophysiological triggers of denervation and sensation loss are not definitively characterized .", "Ocular HSV-1 infection provokes corneal denervation and sensation loss between days 5 to 8 post-infection ( p . i . ) in immunologically naive C57BL/6 mice ( Chucair-Elliott et al . , 2015 ) .", "This tempo appears to be synchronized with the host transition from innate to adaptive immunity following infection .", "Indeed , corneal nerve fiber retraction was evident upon T cell infiltration in the cornea ( Figure 1A ) .", "Accordingly , we hypothesized that local complement activation and T cell engagement coordinate corneal nerve damage during HSV-1 infection .", "To address our hypothesis , corneal sensation and pathogen burden were evaluated in C57BL/6 wildtype ( WT ) and complement C3-deficient ( C3-/- ) mice following ocular HSV-1 infection .", "Progressive loss of corneal mechano-sensitivity was evident in WT mice by days 5 to 8 p . i . , but corneal sensation was conserved in the C3-/- cohort ( Figure 1B ) .", "The same trend revealing preservation of corneal sensation in C3-/- animals was also observed with an increased HSV-1 challenge inoculum ( Figure 1—figure supplement 1 ) .", "Viral titers were measured at time points before and after the onset of corneal sensation loss to determine whether the divergence in sensation stemmed from a differential susceptibility to infection .", "On day 3 p . i . , the HSV-1 burden was similar in the tear film , corneas , and TG of WT and C3-/- animals ( Figure 1C–D ) .", "By day 7 p . i . , HSV-1 titers were elevated in the tear film and TG of C3-/- mice relative to WT ( Figure 1C , E ) .", "Corneal buttons from WT mice exhibited extensive denervation with CD3+ T cell infiltration at day 8 p . i . ( Figure 1F ) .", "However , T cell infiltration was observed without widespread denervation in C3-/- corneas ( Figure 1F ) .", "The number of cornea-infiltrating CD4+ or CD8+ T cells were similar among WT and C3-/- mice ( Figure 1G ) .", "Together , these data indicate that corneal sensation loss in herpetic keratitis involves a C3-dependent inflammatory process independent of viral burden .", "Although T cells successfully extravasate into the corneas of WT and C3-/- mice ( Figure 1F , G ) , C3-deficiency can have broad impacts on T cell priming , clonal expansion , and recruitment ( Clarke and Tenner , 2014; West et al . , 2018 ) .", "To investigate this possibility , corneas from HSV-1-infected WT and C3-/- mice were evaluated for chemokines associated with T cell recruitment , and the eye-draining mandibular lymph nodes ( MLN ) were harvested to evaluate T cell responses .", "Chemokines associated with T cell recruitment were elevated in corneas from both WT and C3-/- mice at day 5 p . i . —a time consistent with onset of sensation loss ( Figure 2A ) .", "Moreover , CD4+ and CD8+ T cell expansion was comparable within the eye-draining mandibular lymph nodes ( MLN ) from WT and C3-/- mice ( Figure 2B ) .", "Similarly , T cells harvested from WT and C3-/- mice at day 8 p . i . responded equally to in vitro stimulation in terms of IFNγ production ( Figure 2C ) .", "Collectively , these data show that C3-/- T cells exhibit normal expansion and recruitment to the cornea during ocular HSV-1 infection .", "In support of our hypothesis that C3 and T cells jointly coordinate corneal sensation loss , frank corneal sensation loss was not observed following ocular HSV-1 infection in C3-sufficient , alpha-beta T cell receptor-deficient mice ( TCRα-/- ) which congenitally lack classical CD4+ and CD8+ T cells ( Figure 2D ) .", "To investigate potential functional defects of C3-/- T cells in an in vivo context , CD3+ T cells were harvested from HSV-infected WT or C3-/- mice at day 8 p . i . and adoptively transferred into TCRα-/- mice .", "Six days following cell transfer , TCRα-/- recipients were infected with HSV-1 and corneal sensation monitored longitudinally .", "Adoptive transfer of CD3+ T cells from either WT or C3-/- donors evoked progressive corneal sensation loss in TCRα-/- recipients by day 8 p . i . ( Figure 2D ) .", "These findings further corroborate that C3-/- T cells remain functional in vivo .", "Analysis of corneal sensation was not feasible beyond 8 days p . i . , as the TCRα-/- mice succumbed to herpetic encephalitis .", "However , engraftment of donor cells was confirmed by analysis of T cells in the eye-draining MLN of recipient mice by flow cytometry at day 8 p . i . The baseline lymphocyte counts in MLN from TCRα-/- controls likely reflect expansion of non-classical T cell populations ( Viney et al . , 1994 ) .", "An increase in total cell number was observed for CD4+ but not CD8+ T cells upon adoptive transfer of purified CD3+ T cells into TCRα-/- recipient mice ( Figure 2—figure supplement 1A ) .", "Consistent with this observation , our data show that transfer of CD3+ T cells from HSV-infected mice have no appreciable impact on HSV-1 titers in the TG of TCRα-/- recipients by day 8 p . i . ( Figure 2—figure supplement 1B ) .", "Notably , bulk transfer of HSV-specific CD8+ T cells has been shown to reduce viral burden in the peripheral nervous system of C57BL/6 mice during acute HSV-1 infection ( Conrady et al . , 2009; Royer et al . , 2016 ) .", "Our collective findings reveal that C3 and T cells are individually necessary and interdependent in the pathogenesis of HSV-1-associated corneal sensation loss .", "Whether by direct or indirect mechanisms , these data support a paradigm in which C3 activation and T cell engagement coordinate corneal nerve damage in herpetic keratitis .", "Our data show that T cells contribute to corneal sensation loss during acute HSV-1 infection , yet whether this pathology is dependent upon CD4+ or CD8+ T cells remained unclear .", "To better discern the contributions of each subset , CD4+ and CD8+ T cells were harvested from infected WT mice , transferred into separate groups of TCRα-/- mice , and recipient mice subsequently infected with HSV-1 .", "Corneal sensation loss during acute HSV-1 infection was only observed upon reconstitution with CD4+ T cells ( Figure 3A ) .", "Donor cell engraftment was confirmed by flow cytometry , although a statistically significant increase in cell number within the eye-draining MLN was only observed upon transfer of CD4+ T cells ( Figure 3—figure supplement 1 ) .", "The requirement for antigen specificity was subsequently evaluated during acute HSV-1 infection by monitoring corneal sensation in WT and OT-II transgenic mice that generate ovalbumin ( OVA ) -specific CD4 T cells .", "Transgenic OT-II mice did not exhibit corneal sensation loss following infection despite evidence of bystander CD4+ T cell activation and recruitment into the cornea ( Figure 3B–D ) .", "Adoptive transfer of CD4+ T cells from HSV-infected WT and OT-II mice into TCRα-/- recipients corroborated these findings ( Figure 3—figure supplement 2 ) .", "Abrasion of the corneal epithelium , which is required to mediate infection in this model , induces local chemokine expression capable of recruiting T cells to the cornea even in the absence of viral infection ( Liu et al . , 2012 ) .", "Nonetheless , active corneal HSV-1 infection was necessary to provoke corneal sensation loss , as TCRα-/- mice reconstituted with CD4+ T cells harvested from HSV-infected WT mice did not elicit auto/allo-antigen-associated sensation loss within two weeks of corneal scratch injury ( i . e . mock infection ) ( Figure 3—figure supplement 3 ) .", "Collectively , our data show that complement C3 and antigen-specific CD4+ T cells are simultaneously necessary to drive corneal sensation loss during HSV-1 infection .", "These results strongly portend that a coordinated inflammatory axis exists involving C3 and antigen-specific CD4+ T cells , and that it is responsible for sensory neuropathy in herpetic keratitis .", "Complement-mediated tissue pathology can arise from complement activators synthesized by the liver ( systemic complement ) or in tissue microenvironments ( local complement ) .", "While local complement activation and regulation have been reported in the cornea in health and disease ( Bora et al . , 1993; Clark and Bishop , 2018; Mondino and Brady , 1981; Verhagen et al . , 1992 ) , the expression profile and cellular sources of various complement components have not been investigated in the context of ocular HSV-1 infection .", "To this end , a modest array of complement component transcripts was evaluated by semiquantitative real-time PCR on corneal buttons from healthy and HSV-infected mice at day 2 and 7 p . i . Upregulation of complement effectors and anaphylatoxin receptors were noted including C3 , C5 , C3ar1 , and C5ar1 ( Figure 4A , B ) .", "Constitutive expression of complement receptor 2 ( CD21 ) has been reported in the corneal epithelium ( Levine et al . , 1990 ) , but variances in its expression during HSV-1 infection were not statistically significant across the time points evaluated ( Figure 4B ) .", "Likewise , no differences in the local expression of various complement regulators were observed in HSV-infected corneas aside from the C1-inhibitor SerpinG1 ( Table 1 , Figure 4C ) .", "Collectively , this expression profile favors local complement activation , as effectors are upregulated without proportional enhancement of pathway regulatory components .", "As C3 is the central component of the complement activation pathway , the cellular sources of complement C3 were evaluated in corneas infected with HSV-1 .", "Monocytes and tissue macrophages are known sources of C3 ( Einstein et al . , 1977; Lubbers et al . , 2017; Morgan and Gasque , 1997; Verschoor et al . , 2001 ) , thus CSF1R+ cells from the peripheral blood of transgenic CSF1R-GFP mice ( MAFIA ) were utilized as the relative standard for C3 transcript expression .", "Expression of C3 was noted in CD45+ leukocytes isolated from infected corneas , yet detection of C3 expression was lost when CSF1R+ cells were removed from the total CD45+ pool .", "In addition , isolated EpCAM+ corneal epithelial cells expressed C3 at levels comparable to blood monocytes .", "However , the C3 expression level was greater in whole-cornea preparations than among individual cell fractions ( Figure 4D ) .", "Taken together , our data show that both tissue-resident non-hematopoietic cells and resident/infiltrating CSF1R+ leukocytes contribute to local C3 expression in the cornea during acute HSV-1 infection .", "Ocular HSV-1 infection amplifies local complement gene expression , yet whether local control of complement activation can be harnessed to prevent corneal nerve damage has not been explored .", "From a clinical perspective , modulating complement activation at the ocular surface may be a viable therapeutic option .", "As a proof of concept , daily ocular cobra venom factor ( CVF ) treatment was explored as a putative method to deplete C3 and preserve corneal sensation during acute HSV-1 infection .", "Vehicle ( PBS ) -treated mice exhibited corneal sensation loss following HSV-1 infection , but CVF treatment preserved corneal sensation ( Figure 5A ) .", "Protein levels of C3 remained near baseline in the cornea following CVF treatment during HSV-1 infection , yet vehicle-treated animals exhibited a 300–600% increase in C3 protein levels in the cornea at days 3 and 7 p . i . ( Figure 5B ) .", "Ocular CVF treatment did not significantly impact systemic serum C3 concentrations throughout the study ( Figure 5C ) .", "Ocular CVF treatment limited corneal edema during HSV-1 infection relative to the vehicle control ( Figure 5D ) .", "Consistent with this observation , CVF-treated animals had less leukocytic infiltrate ( CD45+ ) into the cornea at day 3 p . i . and , specifically , fewer infiltrating CSF1R+ cells ( Figure 5E , F ) .", "By day 7 p . i . , no difference in total CD45+ or CSF1R+ cells were observed in the cornea , yet there was a reduction in the total number of infiltrating CD4+ T cells ( Figure 5G ) .", "Despite the difference in CSF1R+ cell infiltration into the corneas at day 3 p . i . , HSV-1 titers were similar in the corneas and TG comparing vehicle- and CVF-treated animals ( Figure 5—figure supplement 1A ) .", "By day 7 p . i . CVF-treated animals had less virus in the corneas and TG than vehicle controls ( Figure 5—figure supplement 1B ) , suggesting that CVF may have unexpected antiviral effects .", "However , CVF treatment did not have any discernable impact on T cell expansion in the eye-draining MLN or the total number of circulating CXCR3+ CD4+ T cells in HSV-1 infected animals ( Figure 5—figure supplement 2 ) .", "Collectively , these findings imply that complement-targeted therapeutics could be tailored for ophthalmic use to limit tissue inflammation and preserve corneal sensation .", "While complement C3 and antigen-specific CD4+ T cells appear to coordinate corneal sensation loss in herpetic keratitis , it remained to be determined whether this phenomenon was specific to HSV-1 infection .", "To this end , corneal sensation was measured independent of infection in two T cell-dependent ocular surface inflammatory diseases .", "Murine models of allergic eye disease ( AED ) and ocular graft-versus-host disease ( GVHD ) were employed to further delineate the hypothesized role of T cells in coordinating corneal nerve damage ( Herretes et al . , 2015; Lee et al . , 2015 ) .", "First , AED was induced using an established systemic ovalbumin ( OVA ) -sensitization and ocular challenge model ( Figure 6A ) mimicking aspects of chronic keratoconjunctivitis ( Ahadome et al . , 2016; Lee et al . , 2015 ) .", "Clinical signs of ocular allergy developed as anticipated in OVA-challenged mice ( Figure 6B ) , yet corneal sensation loss was not observed ( Figure 6C ) .", "Consistent with development of AED , corneas from OVA-challenged mice exhibited CD3+ T cell infiltration by challenge day 7 ( Figure 6D ) .", "However , frank corneal nerve loss was not observed in AED ( Figure 6D ) .", "Morphometric analysis of confocal image stacks confirmed the preservation of corneal nerve density during AED ( Figure 6E ) , although CD3+ T cell numbers were elevated in sensitized animals relative to healthy controls ( Figure 6F ) .", "Flow cytometry was used to verify that the cornea-infiltrating T cells were predominately CD4+ in the AED model ( Figure 6G ) .", "Although clinical data show that corneal nerve remodeling can occur in patients with chronic ocular allergy ( Hu et al . , 2008; Le et al . , 2011 ) , allergy symptoms were not accompanied by frank loss of corneal mechano-sensation in the AED animal model .", "Corneal sensation was also evaluated in a T cell-dependent allogeneic model of chronic GVHD with systemic and ocular manifestations following hematopoietic stem cell ( HSC ) transplantation ( Perez et al . , 2016 ) .", "This minor histocompatibility antigen mismatch model was established by transferring HSC-rich bone marrow ( BM ) and purified T cells from C57BL/6 ( H2b ) donors into lethally irradiated sex-matched C3 . SW-H2b recipients .", "Recipient controls that receive BM without T cells recover and do not develop GVHD ( Figure 7A ) .", "Onset of GVHD was established by transfer of BM with CD4+ T cells only , CD8+ T cells only , or both CD4+ and CD8+ T cells .", "Progressive corneal sensation loss was observed following transfer of CD4+ T cells with and without addition of CD8+ T cells .", "Addition of CD8+ T cells alone evoked a transient corneal sensation deficit that recovered by day 26 post-transplant .", "However , corneal sensation loss was not observed in the BM only control group ( Figure 7B ) .", "External signs of ocular disease ( see Table 2 ) were also apparent in groups receiving CD4+ T cells by the study endpoint ( Figure 7C ) .", "The presence of tissue-infiltrating CD3+ T cells in corneas from animals with GVHD was verified by confocal microscopy at the study endpoint .", "Consistent with sensation loss , corneal nerve integrity was markedly reduced in groups receiving CD4+ T cells ( Figure 7D ) .", "Morphometric analysis of confocal image stacks confirmed the CD4-dependent decrease in corneal nerve density ( Figure 7E ) and concomitant increase in total CD3+ cells ( Figure 7F ) .", "Taken together , our data show for the first time that corneal sensation loss occurs early in ocular GVHD and that this pathology is instigated in part by allogeneic CD4+ T cells .", "The pharmacologic impact of local CVF treatment on ocular GVHD progression was consequently explored in order to identify whether the CD4+ T cell-associated corneal sensation loss observed in both herpetic keratitis and ocular GVHD shared a common C3-codependent pathomechanism .", "For these experiments , GVHD was established in C3 . SW-H2b recipients by transfer of C57BL/6-derived BM and CD3+ T cells .", "Recipients from the BM only control and GVHD cohorts received ophthalmic treatment with either PBS or CVF ( Figure 8A ) .", "Systemic disease progressed equally in both GVHD cohorts regardless of treatment ( Figure 8B ) .", "Although CVF administration was limited to the eye , systemic serum C3 concentrations were lower in the GVHD cohort treated with CVF compared to the PBS-treated GVHD groups at the study endpoint .", "Nonetheless , the inverted CD4:CD8 T cell ratio ( Herretes et al . , 2015 ) in the spleens and eye-draining mandibular lymph nodes of both groups of GVHD mice confirmed the presence of systemic disease ( Figure 8D ) .", "Despite development of systemic disease , local CVF treatment preserved corneal nerve integrity and mechano-sensory function in ocular GVHD ( Figure 8E , F ) .", "Morphometric analysis of confocal image stacks from the study endpoint corroborated the preservation of corneal nerve density in CVF-treated mice from the GVHD cohort ( Figure 8G ) , although CVF did not reduce the number of total CD3+ cells during GVHD relative to PBS ( Figure 8H ) .", "In addition , CVF treatment also limited GVHD-associated periocular disease in female but not in male mice ( Figure 8—figure supplement 1 ) .", "In summary , these preclinical models establish that CD4+ T cells and complement C3 coordinate corneal sensory nerve damage in both herpetic keratitis and ocular GHVD .", "Moreover , our data provide a proof of principle that complement-targeted therapeutics may limit the severity of immune-mediated sensory nerve damage at the ocular surface ." ], [ "Mechanistic advances in our understanding of the complement cascade’s physiologic regulation and pathologic contributions in disease have predictably spawned substantial investments in complement-targeted drug development over the past decade ( Harris et al . , 2018; Ricklin et al . , 2018; Tomlinson and Thurman , 2018 ) .", "The ocular surface is a unique milieu in which pharmacologic modulation of the complement pathway may limit the severity of inflammatory disease and improve clinical outcomes .", "This study provides proof of concept that such interventions may have important clinical impacts on ocular surface disease , and specifically neuropathic sensory disorders affecting the cornea .", "Complement-targeted drug development for ophthalmic use has almost exclusively focused on AMD with some interest in neuromyelitis optica ( NMO ) , Stargardt disease , and autoimmune uveitis ( Harris et al . , 2018; Pittock et al . , 2013 ) .", "By July 2019 , there were no complement-specific therapeutics indicated for ocular surface use registered on www . clinicaltrials . gov ( search strings: ‘complement’ / ‘C3’ / ‘C5’ / ‘eculizumab’ AND ‘cornea’ , ‘conjunctiva’ , ‘dry eye’ , ‘keratitis’ , OR ‘ocular surface’ ) .", "Nonetheless , the complement pathway is implicated in the pathophysiology of several ocular surface diseases ( Bora et al . , 2008 ) .", "The current investigation provides an important advancement in this arena by demonstrating that dysregulated complement activation can specifically contribute to sensory nerve damage in the cornea .", "While corneal nerves are increasingly implicated in maintenance of corneal immune privilege ( Paunicka et al . , 2015; Neelam et al . , 2018; Guzmán et al . , 2018 ) , this study is the first to identify that C3 is involved in the pathobiology of corneal sensory nerve damage .", "However , the individual fates of damaged corneal nerves were not explored herein .", "Lessons from peripheral nerve injury in other settings indicate that some damaged nerves undergo apoptosis , yet effectively promoting functional regeneration of surviving neurons remains a major clinical hurtle ( Doron-Mandel et al . , 2015; Menorca et al . , 2013; Scheib and Höke , 2013; Shacham-Silverberg et al . , 2018 ) .", "Likewise , promoting functional regeneration of damaged corneal nerves is important for restoration of immune privilege and ocular surface health .", "Our approach evaluated sensory nerve damage in murine models of T cell-dependent ocular surface disease including HSV-1 keratitis , OVA-induced AED , and ocular GHVD .", "The lack of a sensory phenotype in the AED model was unanticipated in light of evidence that patients with severe allergy exhibit changes in corneal nerve morphology ( Hu et al . , 2008; Le et al . , 2011; Leonardi et al . , 2012 ) .", "This contrasting model underscores the context-dependency of sensation loss during corneal inflammation .", "While we focused on C3 in HSV-1 keratitis and ocular GVHD due to the direct link to overt corneal sensation loss , complement may still have an important role in mediating neurogenic hypersensitivities in AED .", "In addition to loss of mechano-sensory function , corneal nerve pathologies can involve a broad array of neuropathic clinical symptoms including dryness , itch , and pain ( Andersen et al . , 2017 ) .", "Inflammatory mediators facilitate neuropathic hypersensitivities , but the therapeutic potential of complement inhibition is often overlooked ( Baral et al . , 2019; Fritzinger and Benjamin , 2016 ) .", "Moreover , the cornea produces many neurotrophic factors that influence sensory innervation in health and disease ( Sacchetti and Lambiase , 2017; Yu et al . , 2015 ) .", "Given the involvement of C3 activation in sensory nerve damage , future investigation into crosstalk between complement and other neurotropic factors in the cornea is warranted .", "Furthermore , AED is driven primarily by a combined Th2/Th17 CD4 T cell response that differs from the Th1 bias observed in HSV-1 keratitis and ocular GVHD ( Herretes et al . , 2015; Saban et al . , 2013; Tang and Hendricks , 1996 ) .", "This suggests that Th1 cytokines such as IFNγ , IL-2 , and lymphotoxin-alpha may be important in coordinating corneal sensation loss .", "Nonetheless , the unique inflammatory milieus and differential polarizations of cornea-infiltrating T cells across these disease models may also influence the downstream effects of complement activation in the tissue microenvironment ( Tomlinson and Thurman , 2018 ) .", "Future studies are needed to broaden the scope of these observations to other peripheral nerve diseases .", "For example , complement-mediated microvascular damage in diabetes correlates with neuropathy ( Rasmussen et al . , 2018; Rosoklija et al . , 2000 ) .", "While diabetic peripheral neuropathy can lead to neurotropic corneal ulcerations ( Gao et al . , 2016b ) , the role of complement in diabetic corneal disease is currently unknown .", "Complement enhances clearance of infected cells and nascent virions during HSV-1 infection ( Kostavasili et al . , 1997; Lubinski et al . , 2002; McNearney et al . , 1987 ) .", "Importantly , HSV-1 can also evade C3 activation through endogenous expression of glycoprotein C ( gC ) .", "By limiting C3b deposition on infected cells and nascent virions , gC can inhibit subsequent C5 binding and membrane attack complex ( MAC ) formation ( Kostavasili et al . , 1997; Lubinski et al . , 2002; McNearney et al . , 1987; Tegla et al . , 2011 ) .", "This could explain why herpetic keratitis is less severe in rabbits infected with a gC-deficient strain of HSV-1 during acute infection ( Drolet et al . , 2004 ) .", "However , this gC-mediated viral evasion strategy may also be cell type-dependent .", "Cell culture modeling indicates that neuronal cells maintain host-encoded complement regulator expression more efficiently and are more resistant to MAC deposition than epithelial cells upon HSV-1 infection ( Rautemaa et al . , 2002 ) .", "Accordingly , complement-mediated sensory nerve damage in HSV-1 keratitis may reflect a maladaptive outcome of host-defense .", "This is consistent with our data showing that immunologically naive C3-/- mice maintain corneal sensation and innervation , yet they exhibit concomitant enhancement of viral shedding in the tear film and increased viral burden in the TG by day 7 p . i . The absence of a concordant difference in HSV-1 titers in corneas from WT and C3-/- mice is likely explained by the potent antiviral effects of type-1 interferon ( Conrady et al . , 2011; Royer and Carr , 2016 ) .", "This suggests that the maintenance of corneal innervation in C3-/- mice enables enhanced shedding of nascent virions produced within the TG .", "Moreover , there is no difference in the amount of latent HSV-1 in the TG of naive WT and C3-/- mice 30 days after ocular HSV-1 challenge ( Royer et al . , 2019 ) .", "Taken together , this evidence indicates that although C3 is involved in progression of keratitis and sensory nerve fiber retraction during acute infection , it likely does not contribute to complete elimination of infected nerves .", "The studies reported herein involve immunologically naive mice , but the impacts of preexisting humoral immune responses on complement pathway activation and regulation in the cornea during HSV-1 infection remain incompletely understood .", "This point is of considerable importance clinically , as herpes-associated neurotropic keratitis typically develops as a result of recurrent corneal infections in patients ( Hamrah et al . , 2010 ) .", "Furthermore , we have recently reported that C3 is essential for optimal antibody-dependent viral clearance following ocular HSV-1 challenge in mice ( Royer et al . , 2019; Royer et al . , 2017 ) .", "While vaccinated animals did not exhibit corneal sensation loss following ocular HSV-1 infection in those studies , deposition of the terminal C3 cleavage product C3d was present in the corneal epithelium ( Royer et al . , 2017 ) .", "Host gene expression data herein corroborates that HSV-1 infection creates an imbalance in the local complement effector to regulator expression ratios that contribute to aberrant complement pathway activation in the cornea .", "This imbalance in ‘complement proteostasis’ may favor deposition of complement fragments and sublytic MAC on corneal sensory nerve fibers ( Tegla et al . , 2011; Triantafilou et al . , 2013 ) .", "Notwithstanding , the nerve-intrinsic molecular mechanisms responsible for trigeminal sensory fiber retraction , neuronal death , or axonal regeneration in the cornea are largely unknown ( Stepp et al . , 2017 ) .", "Future work is needed to identify the respective complement activation pathways involved in pathologic and protective immune responses to HSV-1 in the cornea .", "Identification of the complement pathway’s role in initiating corneal sensation loss following HSV-1 infection is an important advancement in the mechanistic understanding of this disease process , as it introduces an array of potential drug targets for local therapy .", "Previous observations note that complement activation in antigen-induced keratitis may be mediated by cellular immune mechanisms ( Verhagen et al . , 1992 ) .", "The identification of CSF1R+ macrophages/monocytes as a local source of C3 during infection ( Figure 4D ) is also critical , as these cells are recruited to the cornea in the early stages of HSV-1 infection and are associated with corneal nerve damage in HSV-1 keratitis ( Chucair-Elliott et al . , 2017b; Conrady et al . , 2013 ) .", "Moreover , CD4 T cells coordinate this pathology .", "Our data corroborate findings from the Hendricks’ lab showing that CD4 T cells are associated with corneal sensation loss in HSV-1 keratitis ( Yun et al . , 2014 ) , yet our adoptive transfer experiments establish that this process is not dependent upon endogenous C3 production by cornea-infiltrating donor T cells ( Figure 2D ) .", "Notably , the tempo of HSV-associated sensation loss was consistent among models once initiated ( Figure 1B , Figure 2D , Figure 3A ) .", "However , the initial onset was delayed by one day in the adoptive transfer model .", "This likely reflects the relative lymphopenic status of T cell-reconstituted TCRα-/- mice compared to WT mice ( compare Figure 2B and Figure 3—figure supplement 1 ) .", "Corneal sensation loss in HSV-1 infection and ocular GVHD shared a common mechanism dependent upon CD4+ T cells and complement C3 .", "The role of intracellular C3 in regulating human Th1 responses has received much attention in recent years ( Elvington et al . , 2017; Hansen et al . , 2019; Liszewski et al . , 2013 ) .", "Such studies involve CD3 and CD46 stimulation to activate human CD4 T cells in vitro .", "However , mice do not express CD46 .", "Instead , anaphylatoxin receptor signaling has been shown to modulate Th1 cytokine production in murine T cells ( Strainic et al . , 2008 ) .", "Our ex vivo re-stimulation data clearly show that there is no defect in IFNγ production by CD4 T cells from C3-/- mice following HSV-1 infection .", "Others have shown similar data based on T cell proliferation in response to re-stimulation with HSV-1 antigen ( Da Costa et al . , 1999 ) .", "In contrast , a reduction in IFNγ production by CD4 T cells from C3-/- mice was reported following in vitro expansion under Th1 polarizing conditions ( Liszewski et al . , 2013 ) .", "Nonetheless , it is possible that anaphylatoxin receptor signaling from locally produced C3a ( and potentially C5a generated downstream ) modulates T cell effector function upon extravasation into the inflamed cornea .", "The precise relationships between T cells and C3 in our mouse models of corneal neuropathic disease remain to be identified .", "Our working hypothesis involves an indirect mechanism whereby sublytic MAC deposition from local complement activation damages sensory nerves , which are then cleared by T cell-activated phagocytes or other cytolytic cells .", "These could include tissue-resident and infiltrating leukocytes ( macrophages , monocytes , dendritic cells , and NK cells ) as well as nerve-associated corneal epithelial cells ( Buela and Hendricks , 2015; Chucair-Elliott et al . , 2017b; Gao et al . , 2016a; Koyama and Hill , 2016; Liu et al . , 2017; Royer et al . , 2018; Seyed-Razavi et al . , 2014; Stepp et al . , 2017 ) .", "Emerging evidence indicates that the plasma membranes of corneal nerves and epithelial cells fuse thereby enabling cytosolic exchange ( Stepp et al . , 2017 ) .", "In light of this , it is plausible that complement-mediated damage to corneal epithelial cells has a direct impact on sensory nerves .", "Although corneal nerve damage is an established pathology in animal models of HSV-1 infection ( Chucair-Elliott et al . , 2015; He et al . , 2017b; Yun et al . , 2014 ) , this is the first report documenting loss of corneal mechano-sensory function in ocular GVHD .", "Notably , patients rarely exhibit neurological manifestations of GVHD affecting other tissues/organs ( Grauer et al . , 2010 ) .", "Corneal sensation loss may provide a clinical benchmark for initiation of targeted therapies to curb progression of ocular GVHD in patients .", "Data from our animal model of GVHD suggest that corneal sensation loss is an early warning sign of progressive ocular and systemic GVHD .", "Corneal sensation measurements are warranted in patients following HSCT to substantiate this finding among those who develop chronic GVHD .", "Published imaging studies confirm that corneal nerve remodeling occurs in patients with ocular GVHD ( He et al . , 2017a; Tepelus et al . , 2017 ) ; therefore , clinical studies to delineate the kinetics of disease onset are needed .", "Complement C3 is a known driver of systemic GVHD ( Kwan et al . , 2012; Ma et al . , 2014; Seignez et al . , 2017 ) , and our data show that aberrant complement pathway activation also contributes to the pathogenesis of ocular GVHD including corneal sensation loss .", "Furthermore , localized CVF treatment preserved corneal sensation in both sexes during ocular GVHD , but treatment only abrogated other facets of ocular surface disease in female mice .", "This dimorphic outcome may reflect insufficient maintenance of C3 depletion in the ocular surface microenvironment , as animals were only treated twice weekly .", "Furthermore , complement activity is reportedly limited by terminal pathway components in female mice compared to males ( Kotimaa et al . , 2016 ) .", "Collectively , our data suggest that sensory nerve involvement may be a unique facet and treatment target in ocular GVHD .", "While the complement pathway has many components , C3 is the heart of the pathway and its activation products mediate virtually all downstream pathway functions .", "Accordingly , we used CVF to target complement activation in models of HSV-1 keratitis and ocular GVHD as a proof of concept for local delivery of complement-targeted therapeutics for ocular surface disease .", "In contrast to the neurotoxic effects of complete cobra venom , purified CVF is a ‘nontoxic’ derivative .", "CVF forms a biochemically stable convertase to rapidly hydrolyze mammalian C3 and C5 .", "This ultimately results in complement inactivation via effector consumption ( Vogel and Fritzinger , 2010 ) .", "The only recorded side effect of purified CVF administration in rodents is transitory anaphylatoxin-mediated pulmonary inflammation resulting in acute respiratory distress ( Proctor et al . , 2006; Vogel and Fritzinger , 2010 ) .", "In our hands , respiratory distress was observed in some mice following the initial sub-conjunctival CVF injection irrespective of group assignment ( animals were euthanized ) .", "However , topical CVF administration did not provoke respiratory or corneal abnormalities in any animals .", "Previous findings also show that topical CVF administration has no obvious effects on corneal health or transparency ( Zaidi et al . , 2010 ) .", "Experimental CVF administration was once thought to directly stimulate T cells , but such effects were later attributed to mitogen contamination ( Rumjanek et al . , 1978; Cauvi et al . , 2012 ) .", "Because our disease models were T cell-dependent , we utilized highly-purified CVF from a commercial source for our studies .", "Moreover , we found no evidence to suggest that CVF impacted T cell responses outside of the eye .", "Local CVF treatment reduced corneal T cell infiltration following HSV-1 infection ( Figure 5G ) .", "However , this is likely due to reduced inflammation overall in C3-depleted corneas ( Figure 5D ) .", "Likewise , ocular CVF treatment in the GVHD model did not impact on the total numbers of T cells in the corneas at the experiment endpoint .", "However , we have not excluded the possibility that CVF treatment may delay the tempo of T cell influx .", "Nonetheless , CVF administration preserved corneal sensation in both disease models .", "By addressing the ‘heart’ of the complement pathway ( C3 ) , this study opens the door to future investigation to further elucidate the relevant pathomechanisms and corresponding therapeutic targets .", "The relevant complement activation pathways ( classical , alternative , lectin , terminal ) , cellular targets , and impacts of anaphylatoxin receptor signaling are topics of future investigation .", "Complement activation is a double-edged sword in the cornea , as activation can be either protective or harmful .", "Low-level complement turnover is observed in healthy corneas/tears , and complement may even contribute to the relative ‘paucibacterial’ state of the ocular surface microbiome ( Doan et al . , 2016; McDermott , 2013; Willcox et al . , 1997 ) .", "The mechanisms regulating each complement-mediated effect remain incompletely understood .", "Elucidation of how complement activation is triggered , the relevant molecular mechanisms of its downstream signaling , and contributions to pathology will promote an important advancement in drug development for ocular surface disease .", "Animal models are necessary for mechanistic studies when it comes to understanding complement pathway regulation , especially in dynamic tissue microenvironments; however , differences in complement regulation exist between mouse and man that will also have to be resolved to further assess the translational potential ( Jacobson and Weis , 2008 ) .", "This certainly applies to putative complement-targeted therapeutics for ocular surface treatment .", "Even among individual patients , small nucleotide polymorphisms evoke changes in complement activity that have major impacts on disease risks .", "Such genetic linkages , dubbed ‘complotype , ’ are important in differential susceptibility to AMD ( Harris et al . , 2012; Paun et al . , 2016 ) .", "Accordingly , complotype differences may help explain differential susceptibility patterns in a variety of ocular surface diseases .", "Our data herein have demonstrated successful prophylactic intervention in repressing complement-mediated sensory nerve pathology in two well characterized experimental T cell-dependent corneal disease models .", "Other avenues of future investigation are also needed to determine the efficacy of therapeutic intervention on established ocular surface diseases .", "Successful implementation of complement-targeted therapeutics for topical ophthalmic use may provide the benefit of controlling insidious ocular surface diseases without the risks associated with systemic therapies ." ], [ "Mouse strains were originally purchased from The Jackson Laboratory ( Bar Harbor , ME ) .", "These include: C57BL/6 , C3-/- ( stock #029661 ) , TCRα-/- ( stock # 002116 ) , OT-II ( stock #004194 ) , MAFIA/CSF1R-GFP ( stock #005070 ) , C3 . SW-H2b ( stock #000438 ) , and B6-GFP ( stock #003291 ) .", "Colonies were maintained in select-pathogen-free vivaria at the University of Oklahoma Health Sciences Center or Duke University Medical Center .", "Research was performed in accordance with protocols approved by respective institutional animal care and use committees .", "For all procedures relating to ocular HSV-1 infection , C57BL/6-background mice were anesthetized with ketamine and xylazine and euthanized by cardiac perfusion ( Royer et al . , 2018 ) .", "For infection , corneas were scratched to expose the epithelium and 1000 plaque forming units ( PFU ) of HSV-1 McKrae was applied to each eye .", "Viral stocks were propagated and infectious titers quantified by standard plaque assay on Vero cells ( American Type Culture Collection , Manassas , VA ) as previously reported ( Royer et al . , 2015 ) .", "For adoptive transfers , T cells were purified from splenocyte preparations using a BioRad S3e cell sorter ( Hercules , CA ) .", "Corneal complement C3 depletion was achieved by subconjunctival injection of CVF ( Cat . # 233552 , Millipore Sigma , Burlington , MA ) followed by topical maintenance dosing as described in respective figure legends .", "Allergic eye disease was initiated in B6 mice as previously described ( Ahadome et al . , 2016; Lee et al . , 2015; Schlereth et al . , 2012 ) .", "Briefly , hypersensitivity was elicited via intraperitoneal immunization with 10 μg ovalbumin ( OVA ) adjuvanted with 1 mg Alum and 300 ng pertussis toxin ( all from Sigma-Aldrich Corp . , St . Louis , MO ) .", "Ocular allergy was subsequently induced via daily eyedrop challenge containing 250 μg OVA ( Figure 6A ) .", "Clinical signs of AED were scored daily using established criteria ( Schlereth et al . , 2012 ) .", "Animals were euthanized by CO2 asphyxiation for tissue collection .", "Induction of GVHD was based on an established T-dependent , MHC-matched unrelated donor model ( Perez et al . , 2016 ) .", "Briefly , C3-SW . H2b mice were lethally irradiated ( 10 . 5 Gy , Cs-137 ) and reconstituted with 5 × 106 T cell-depleted bone marrow ( TCD-BM ) cells from allogeneic C57BL/6 ( H2b ) mice .", "T cell depletion was achieved using BD anti-mouse CD90 . 2 IMag particles ( Cat . # 551518 , San Jose , CA ) .", "Control groups received TCD-BM only , but GVHD onset required reconstitution with TCD-BM and T cells .", "T cells were isolated from B6 splenocytes using immunomagnetic microbeads ( Miltenyi Biotec , San Diego , CA ) targeting CD4 ( Cat . #130-049-201 ) , CD8 ( Cat . #130-116-478 ) , or CD90 . 2 ( Cat . #130-049-101 ) according to the manufacturer’s instructions .", "Cell purity was evaluated by flow cytometry ( ~80% ) and adjusted such that 1 . 3 × 106 CD4+ or CD8+ T cells or 2 . 3 × 106 CD3+ T cells were injected into each recipient with TCD-BM .", "Irradiated animals received water containing gentamicin ( 6 . 6 μg/ml ) for ten days following irradiation ( Henry Schein Animal Health , Dublin , OH ) .", "Supplical nutritional paste ( Henry Schein ) was also provided ad libitum to reduce weight loss .", "Systemic manifestations of GVHD were scored using established criteria ( Perez et al . , 2016 ) and ocular disease scored according to Table 2 .", "Corneal mechano-sensory function was measured on non-anesthetized mice with a Luneau Cochet-Bonnet esthesiometer ( Western Ophthalmics , Lynwood , WA ) in 0 . 5 cm increments on a scale of 0 to 6 cm as previously described ( Chucair-Elliott et al . , 2015 ) .", "Sensation scores reflect the longest filament length capable of eliciting replicate blink reflexes when applied to the central cornea .", "A Bioptigen spectral domain optical coherence tomography ( SD-OCT ) system ( Leica Microsystems , Buffalo Grove , IL ) was utilized to evaluate corneal inflammation and edema in vivo ( Downie et al . , 2014 ) .", "Digital photography of the external eye was captured with a Leica MZ16 FA stereomicroscope .", "Corneal nerves were imaged in flat-mounted corneal buttons as described ( Chucair-Elliott et al . , 2015 ) .", "Briefly , corneas were harvested with the limbus intact , fixed , permeabilized , and labeled with Abcam anti-mouse beta-III tubulin primary antibody ( Cat . # ab18207 , Cambridge , MA ) and a corresponding AlexaFluor647-conjugated secondary antibody ( Cat . # 711-605-152 , Jackson Immunoresearch , West Grove , PA ) .", "Corneas were also labeled with an eBioscience FITC-conjugated anti-mouse CD3ε antibody ( ThermoFisher , Waltham , MA ) .", "Slides were imaged using an Olympus Fluoview1200 laser scanning confocal ( Center Valley , PA ) or a Nikon AR1 HD resonant scanning confocal ( Melville , NY ) microscope with sequential channel scanning .", "Confocal z-stack images were evaluated using Imaris software ( Bitplane , Concord , MA ) to quantify corneal nerves densities and the total numbers of CD3+ cells .", "Briefly , images were displayed as maximum intensity projections .", "Nerve volume per field of view was measured with the ‘create surface tool’ with manual thresholding for voxel area coverage of βIII tubulin+ fibers ( default parameters; smooth surface area detail set to 0 . 5 μm; background subtraction set to 8 . 0 μm ) .", "Total CD3+ cells were quantified using the spots tool with default parameters , an estimated cell diameter of 8 μm , and classification using the quality filter method with manual thresholding .", "Morphometric nerve volume and cell count summary statistics were reported by the software per image ( 20x field of view ) .", "Nerve densities were normalized to the respective control group average .", "Secondary lymphoid organs were harvested and mechanically processed into single-cell suspensions ( Royer et al . , 2015 ) .", "Blood was collected from the facial vein and treated with erythrocyte lysis buffer prior to labeling .", "Corneas were digested in RPMI1640 media containing 0 . 26 Wünsch units of Roche LiberaseTL enzyme blend ( Sigma Aldrich ) at 37° C as described ( Royer et al . , 2017 ) .", "Cell suspensions were filtered and labeled with Fc block and fluorochrome-conjugated antibodies ( eBioscience ) .", "In vitro T cell functional assays were performed as previously described ( Royer et al . , 2015 ) .", "Briefly , cells were stimulated using 50 ng PMA and 800 ng ionomycin for three hours and monensin added after one hour ( BD Biosciences ) .", "Intracellular IFNγ expression was evaluated by flow cytometry using a BD cytofix/cytoperm staining kit .", "Samples were analyzed on a Miltenyi Biotec MacsQuant10 or a BD LSRFortessa flow cytometer with MacsQuantify or FacsDiva software , respectively .", "For downstream gene expression studies , cornea digests were fractionated with Miltenyi microbeads by sequentially targeting CD45 ( Cat . # 130-052-301 ) and EpCAM ( Cat . # 130-105-958 ) as described ( Royer et al . , 2018 ) .", "Alternatively , GFP-expressing cells were purified or depleted using a BioRad S3e cell sorter .", "Tissues were harvested and homogenized in phosphate-buffered saline ( PBS ) containing 1x Calbiochem protease inhibitor cocktail ( Millipore Sigma ) .", "Serum was obtained by collecting blood from the facial vein into BD microtainer serum separator tubes .", "Tissue homogenates and serum were clarified by centrifugation at 10 , 000×g .", "Chemokine concentrations were determined using a BioRad Luminex system and Millipore Milliplex MAP technology .", "Concentrations of C3 were measured by ELISA ( Abcam , Cat . # ab157711 ) .", "Gene expression studies were performed using RNA isolated from tissue and cells using the Trizol ( ThermoFisher ) method and converted to cDNA with iScript ( BioRad ) .", "Real-time PCR was performed using PrimePCR technology with commercially validated primer sequences ( Biorad ) and a Biorad CFX-Connect thermocycler as directed .", "Relative expression was calculated using the 2–ΔΔCt method with GAPDH ( glyceraldehyde 3-phosphate dehydrogenase ) as a reference gene .", "Final PCR products were resolved on a 2% agarose gel and imaged with an Azure Biosystems C-series gel documentation system ( Dublin , CA ) to confirm C3 expression in cell subsets .", "All data were evaluated using Prism six software ( GraphPad , San Diego , CA ) .", "Statistical tests and post hoc analyses utilized are listed in each figure legend .", "Significance thresholds are indicated as follows *=P < 0 . 05 , **=P < 0 . 01 , ***=P < 0 . 001 for all data .", "Supporting data are available online ." ] ]

[ "Whether complement dysregulation directly contributes to the pathogenesis of peripheral nervous system diseases , including sensory neuropathies , is unclear .", "We addressed this important question in a mouse model of ocular HSV-1 infection , where sensory nerve damage is a common clinical problem .", "Through genetic and pharmacologic targeting , we uncovered a central role for C3 in sensory nerve damage at the morphological and functional levels .", "Interestingly , CD4 T cells were central in facilitating this complement-mediated damage .", "This same C3/CD4 T cell axis triggered corneal sensory nerve damage in a mouse model of ocular graft-versus-host disease ( GVHD ) .", "However , this was not the case in a T-dependent allergic eye disease ( AED ) model , suggesting that this inflammatory neuroimmune pathology is specific to certain disease etiologies .", "Collectively , these findings uncover a central role for complement in CD4 T cell-dependent corneal nerve damage in multiple disease settings and indicate the possibility for complement-targeted therapeutics to mitigate sensory neuropathies ." ]

[ "Most people have likely experienced the discomfort of an eyelash falling onto the surface of their eye .", "Or that gritty sensation when dust blows into the eye and irritates the surface .", "These sensations are warnings from sensory nerves in the cornea , the transparent tissue that covers the iris and pupil .", "Corneal nerves help regulate blinking , and control production of the tear fluid that protects and lubricates the eye .", "But if the cornea suffers damage or infection , it can become inflamed .", "Long-lasting inflammation can damage the corneal nerves , leading to pain and vision loss .", "If scientists can identify how this happens , they may ultimately be able to prevent it .", "To this end , Royer et al . have used mice to study three causes of hard-to-treat corneal inflammation .", "The first is infection with herpes simplex virus ( HSV-1 ) , which also causes cold sores .", "The second is eye allergy , where the immune system overreacts to substances like pollen or pet dander .", "And the third is graft-versus-host disease ( GVHD ) , an immune disorder that can affect people who receive a bone marrow transplant .", "Royer et al . showed that HSV-1 infection and GVHD – but not allergies – made the mouse cornea less sensitive to touch .", "Consistent with this , microscopy revealed damage to corneal nerves in the mice with HSV-1 infection and those with GVHD .", "Further experiments showed that immune cells called CD4 T cells and a protein called complement C3 were contributing to this nerve damage .", "Treating the mice with an experimental drug derived from cobra venom protected the cornea from the harmful effects of inflammation .", "It did so by blocking activation of complement C3 at the eye surface .", "Identifying factors such as complement C3 that are responsible for corneal nerve damage is an important first step in helping patients with inflammatory eye diseases .", "Many drugs that target the complement pathway are currently under development .", "Some of these drugs could potentially be adapted for delivery as eye drops .", "But first , experiments must test whether complement also contributes to corneal nerve damage in humans .", "If it does , work can then begin on testing these drugs for safety and efficacy in patients ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "microbiology and infectious disease", "immunology and inflammation" ]

[ [ "HOIL-1 ( encoded by the RBCK1 gene ) , HOIP ( RNF31 ) and SHARPIN form the linear ubiquitin chain assembly complex ( LUBAC ) , which linearly ubiquitinates receptor signaling complex components such as NEMO to enhance NF-κB activation after engagement of immune receptors including TNF-R1 , IL-1R , CD40 , TLRs and NOD2 ( Tokunaga et al . , 2011; Tokunaga et al . , 2009; Ikeda et al . , 2011; Gerlach et al . , 2011; Haas et al . , 2009; Zak et al . , 2011; Hostager et al . , 2011; Boisson et al . , 2012; Damgaard et al . , 2012; Tian et al . , 2007 ) .", "Recently , HOIL-1/LUBAC was also shown to be important for activation of the NLRP3/ASC inflammasome in macrophages via linear ubiquitination of ASC ( Rodgers et al . , 2014 ) .", "These data suggest that HOIL-1 plays multiple roles in inflammation and infection .", "In mice , SHARPIN deficiency results in auto-inflammation involving multiple organs including the liver , esophagus , lung and , most noticeably , chronic proliferative dermatitis of the skin ( Seymour et al . , 2007 ) .", "The development and organization of secondary lymphoid organs and antibody isotype switching are also impaired in these mice ( HogenEsch et al . , 1999 ) .", "Loss of HOIP catalytic activity in B cells results in the impaired development of B1 B cells and antibody responses to antigen ( Sasaki et al . , 2013 ) .", "However , HOIL-1-deficient mice have not been analyzed extensively to date .", "Sixteen patients with bi-allelic mutations in the gene encoding HOIL-1 have been reported ( Boisson et al . , 2012; Nilsson et al . , 2013; Wang et al . , 2013 ) .", "Three patients exhibited cardiomyopathy , amylopectinosis , hyper-inflammation and mild immunodeficiency associated with an increased frequency of bacterial infections , whereas other patients presented with amylopectinosis and myopathy alone ( Figure 1—figure supplement 1 ) .", "The role of HOIL-1 in inflammation and immunity to infection in vivo is , therefore , uncertain .", "Although there are multiple possible explanations for the variable clinical presentations of the reported patients including hypomorphic expression of HOIL-1 or effects of mutations on protein function , another possibility was that environmental factors alter the clinical presentation of HOIL-1 deficiency .", "In this study we define the function of HOIL-1 in murine immunity to infection and explore the potential role of the virome in determining HOIL-1 deficiency-associated phenotypes .", "The bacterial microbiome and the virome regulate inflammation and immunity ( Virgin , 2014; Virgin et al . , 2009; Belkaid and Hand , 2014 ) .", "Within the virome , herpesviruses persistently infect most humans , and exert significant effects on innate immunity in mice during experimental chronic infection , including increasing resistance to tumors and a range of pathogens ( Barton et al . , 2007; White et al . , 2010; Yager et al . , 2009; Nguyen et al . , 2008; Haque et al . , 2004 ) .", "However , the potential effects of chronic infection on the phenotypic manifestations of immune deficiencies have not been considered .", "In this study , we show that chronic herpesvirus infection can alter the presentation of several genetic immunodeficiencies in mice .", "We first found that , in naïve mice , HOIL-1 is essential during infection with Listeria monocytogenes , Toxoplasma gondii and Citrobacter rodentium and for efficient induction of pro-inflammatory cytokines that are known to be essential for resistance to lethal infection by hematopoietic cells during Listeria infection .", "In contrast , HOIL-1 knock-out ( KO ) mice , with null mutations in the Rbck1 gene that encodes HOIL-1 , were resistant to infection with murine γ-herpesvirus 68 ( MHV68 ) and Mycobacterium tuberculosis .", "Although HOIL-1 KO mice raised in a barrier facility did not display signs of auto-inflammation , chronic infection with MHV68 or M . tuberculosis resulted in elevated inflammatory cytokines circulating in the serum , similar to that observed in some patients with mutations in RBCK1 ( HOIL1 ) .", "Interestingly , latent infection with MHV68 rescued HOIL-1 deficient mice from lethality during Listeria infection and induced high levels of the protective cytokine , interferon-gamma ( IFNγ ) .", "MHV68 latency also protected IL-6 , Caspase-1 and Caspase-1;Caspase-11 deficient mice from Listeria-induced lethality , indicating that the ability of latent infection to complement a genetic immunodeficiency is not restricted to mutation of Hoil-1 .", "These data indicate that chronic infections can modify the clinical presentations of genetic variations , thereby opening a new avenue for the analysis and interpretation of human genotype-phenotype association studies .", "We speculate that the protective effect of chronic herpesvirus infection is due to the stimulation of the function of the innate immune system in a manner that compensates for deficient early cytokine responses associated with multiple immunodeficiencies ." ], [ "HOIL-1 KO mice ( Tokunaga et al . , 2009 ) were born at Mendelian ratios and , in contrast to SHARPIN-deficient mice , failed to develop TNFα-driven inflammatory skin disease ( Ikeda et al . , 2011; Gerlach et al . , 2011; Tokunaga et al . , 2011; Tokunaga and Iwai , 2012 ) and exhibited normal histology of lymphoid organs , liver , lung , and kidney , and the presence of Peyer's patches along the small intestine ( not shown ) .", "Aged HOIL-1 KO mice exhibited deposits of material that stained with periodic acid-Schiff reagent and was resistant to digestion with diastase , similar to the amylopectin-like material observed in humans with HOIL-1 deficiency ( Figure 1—figure supplement 2 ) ( Boisson et al . , 2012 ) .", "Importantly , these barrier-raised mice showed minimal signs of baseline hyper-inflammation .", "In this regard , HOIL-1 KO mice exhibited normal numbers of lymphoid and myeloid cells in the spleen and thymus , normal complete blood counts ( Figure 1—figure supplement 3A , B , D ) , and no detectable increase of tumor necrosis factor alpha ( TNFα ) or interleukin 6 ( IL-6 ) in serum ( discussed below ) .", "However , in the peritoneum , HOIL-1 KO mice contained about twofold more B cells , T cells and resident macrophages without changes in other cell types ( Figure 1—figure supplement 3C ) .", "Expression of neighboring genes , Trib3 and Tbc1d20 , was unaffected by disruption of the Rbck1 ( Hoil1 ) gene ( Figure 1—figure supplement 4 ) .", "To determine the requirement for HOIL-1 during the immune response to infection in vivo , we challenged HOIL-1 KO mice with a number of different pathogens .", "Strikingly , HOIL-1 KO mice were highly susceptible to even low dose infection with the facultative gram-positive intracellular bacterium , Listeria monocytogenes ( Listeria ) , with 80% , 80% and 50% of mice succumbing to infection within 10 days of intraperitoneal ( i . p . ) inoculation with 105 , 104 and 103 CFU , respectively ( Figure 1A ) .", "Although bacterial burdens in the spleens and livers of control and HOIL-1 KO mice were similar 1 and 3 days post-infection with 105 CFU , bacterial CFUs were elevated in HOIL-1 KO mice by 6 days post-infection , indicating that HOIL-1 KO mice were unable to control and clear the bacteria ( Figure 1B ) .", "Further , these mutant mice developed large inflammatory lesions in the liver , elevated liver enzymes in the serum , and widespread tissue destruction in the spleen ( Figure 1—figure supplement 5 , not shown ) . 10 . 7554/eLife . 04494 . 006Figure 1 . HOIL-1 KO mice are highly susceptible to acute infection with Listeria monocytogenes , Toxoplasma gondii and Citrobacter rodentium .", "( A ) Survival of control ( blue circles ) and HOIL-1 KO ( red squares ) mice following i . p . inoculation with 105 ( left panel; control n = 35 , HOIL-1 KO n = 19 ) , 104 ( middle panel; control n = 15 , HOIL-1 KO n = 15 ) or 103 ( right panel; control n = 15 , HOIL-1 KO n = 15 ) CFU Listeria strain EGD .", "( B ) Listeria CFU in spleen and liver from control ( blue circles ) and HOIL-1 KO ( red squares ) mice infected with 105 CFU i . p . for 1 day ( left panel ) , 3 days ( middle panel ) or 6 days ( right panel ) .", "Each symbol represents an individual mouse and the mean log10 CFU is indicated .", "The dashed line indicates the limit of detection .", "( C ) Survival of control ( blue circles ) and HOIL-1 KO ( red squares ) mice following inoculation with 5000 ( left panel; control n = 17 , HOIL-1 KO n = 5 ) or 100 ( middle panel; control n = 10 , HOIL-1 KO n = 10 ) tachyzoites T . gondii strain Pru-luc .", "( D ) Log10 total flux ( luciferase activity; photons per second ) as a measure of parasite burden 8 days post-infection with 100 tachyzoites .", "Each symbol represents an individual mouse and the mean log10 is indicated .", "( E , F )", "Survival ( E ) and weight ( F ) of control ( blue circles ) and HOIL-1 KO ( red squares ) mice following oral gavage with 2 × 109 CFU C . rodentium .", "n = 20/group for survival and n = 10/group for weight .", "*p ≤ 0 . 05 , **p ≤ 0 . 01 , ***p ≤ 0 . 001 , ****p ≤ 0 . 0001 .", "Statistical analyses were performed using logrank Mantel–Cox test ( A , C and E ) , Mann–Whitney test ( B ) , or t-test ( D ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 04494 . 00610 . 7554/eLife . 04494 . 007Figure 1—figure supplement 1 . Comparison of RBCK1/HOIL1 alleles from RBCK1/HOIL1-mutant patients . Npl4 zinc finger ubiquitin binding domain , RING; really interesting new gene E3 ligase domain , IBR; Inbetween-RING domain .", "HOIL1L/RBCK1 isoform 2 ( reference sequences NM_031229 . 2/NP_112506 . 2 ) was used for annotation . DOI: http://dx . doi . org/10 . 7554/eLife . 04494 . 00710 . 7554/eLife . 04494 . 008Figure 1—figure supplement 2 . Myocardium from aged HOIL-1 KO mice contains amylopectin-like deposits . Representative PAS ( top ) , PAS plus diastase digestion ( middle ) and H&E-stained sections of myocardium from 18 month-old HOIL-1 KO ( right ) and control ( left ) mice .", "The scale bar ( inset ) represents 100 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 04494 . 00810 . 7554/eLife . 04494 . 009Figure 1—figure supplement 3 . Analysis of hematopoietic cell populations from naïve HOIL-1 KO mice .", "( A–C )", "Flow cytometric analysis of cell populations in the spleen ( A ) , thymus ( B ) and peritoneum ( C ) of HOIL-1 KO ( red squares ) and control ( blue circles ) mice .", "DP; CD4 , CD8 double-positive .", "( D ) Complete blood counts from HOIL-1 KO ( red squares ) and control ( blue circles ) mice .", "WBC , white blood cells , ×103/mm3 , RBC , red blood cells , ×106/mm3; HGB , hemoglobin , g/dl; HCT , hematocrit , %; MCV , mean corpuscular volume , µm3; MCH , mean corpuscular hemoglobin , pg; MCHC , mean corpuscular hemoglobin concentration , %; Seg Neu , segmented neutrophils , %; Lymphos , lymphocytes , % , Monos , monocytes , % .", "Eosinophils , basophils or band neutrophils were not detected .", "Each symbol represents an individual mouse and the mean is indicated . DOI: http://dx . doi . org/10 . 7554/eLife . 04494 . 00910 . 7554/eLife . 04494 . 010Figure 1—figure supplement 4 . Hoil1/Rbck1 and neighboring gene ( Trib3 and Tbc1d20 ) transcript expression in control and HOIL-1 KO bone marrow derived macrophages . Data represent the mean ± SEM from cells derived from four pairs of mice each analyzed in triplicate .", "*p ≤ 0 . 05 , **p ≤ 0 . 01 , ***p ≤ 0 . 001 , ****p ≤ 0 . 0001 .", "Statistical analyses were performed using t-test . DOI: http://dx . doi . org/10 . 7554/eLife . 04494 . 01010 . 7554/eLife . 04494 . 011Figure 1—figure supplement 5 . Pathology of HOIL-1 KO mice during Listeria infection . Alanine aminotransferase ( ATL; left panel ) and aspartate aminotransferase ( AST; right panel ) levels in serum from control ( blue circles ) and HOIL-1 KO ( red squares ) mice 6 days after infection with 105 CFU Listeria .", "Each symbol represents an individual mouse and the mean is indicated .", "*p ≤ 0 . 05 , **p ≤ 0 . 01; t-test with Welch's correction . DOI: http://dx . doi . org/10 . 7554/eLife . 04494 . 011 HOIL-1 KO mice were also highly susceptible to infection with a relatively avirulent type II strain of the intracellular apicomplexan parasite Toxoplasma gondii ( T . gondii ) ( Figure 1C ) .", "Despite infection with 5000 parasites resulting in lethality in only 20% of control mice , 100 parasites was sufficient to induce lethality in 100% of HOIL-1 KO mice .", "Quantification of parasite-encoded luciferase expression in vivo revealed that HOIL-1 KO mice failed to control T . gondii replication by 8 days post-infection ( Figure 1D ) .", "HOIL-1 KO mice also succumbed to infection with the enteric gram-negative pathogen Citrobacter rodentium , whereas control mice were highly resistant ( Figure 1E , F ) .", "These data indicated that loss of HOIL-1 expression confers profound immunodeficiency in barrier-raised mice .", "To define the role of HOIL-1 in immunity , we examined the response to Listeria in more detail .", "One patient with HOIL-1-associated immunodeficiency showed signs of recovery from hyper-inflammation after hematopoietic stem cell transplantation ( Boisson et al . , 2012 ) .", "In mice , reciprocal bone marrow transplantation revealed that expression of HOIL-1 in radiation-sensitive hematopoietic cells was critical for resistance to Listeria ( Figure 2A , Figure 2—figure supplement 1 ) .", "We noted that control mice that received control bone marrow were slightly more susceptible to infection than non-irradiated control mice ( compare with Figure 1A ) , suggesting that reconstitution does not fully restore the immune system of a lethally irradiated mouse to that of a non-irradiated animal .", "Despite this caveat , irradiated wild-type control mice that received HOIL-1 KO bone marrow and were challenged with 105 Listeria 8 weeks later succumbed to infection at the same rate as HOIL-1 KO mice that had received HOIL-1 KO bone marrow .", "HOIL-1 KO mice that received control bone marrow had an increased survival rate , but still succumbed more readily than control mice that received control bone marrow .", "These data indicate that , while HOIL-1 expression is essential in bone marrow-derived cells , HOIL-1 may also play a role in radiation resistant cells during Listeria infection . 10 . 7554/eLife . 04494 . 012Figure 2 . HOIL-1 is required in an innate immune cell compartment during Listeria infection .", "( A ) Survival of control and HOIL-1 KO reciprocal bone marrow chimeric mice following infection with 105 CFU Listeria .", "*p ≤ 0 . 0083; logrank Mantel–Cox test corrected for multiple comparisons .", "( B ) Survival of RAG1 KO HOIL-1 WT ( blue circles; n = 12 ) and RAG1 KO HOIL-1 KO ( red squares; n=11 ) mice following infection with 104 CFU Listeria .", "( C ) Listeria CFU in spleen and liver from RAG1 KO HOIL-1 WT ( blue circles ) and RAG1 KO HOIL-1 KO ( red squares ) mice infected with 104 CFU for 3 days .", "Each symbol represents an individual mouse and the mean log10 is indicated .", "For B and C , *p ≤ 0 . 05 , **p ≤ 0 . 01 , ***p ≤ 0 . 001 , ****p ≤ 0 . 0001; logrank Mantel–Cox test and Mann–Whitney test , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 04494 . 01210 . 7554/eLife . 04494 . 013Figure 2—figure supplement 1 . Confirmation of hematopoietic reconstitution of bone marrow chimeric mice . Percent Hoil1/Rbck1+/+ ( Hoil1/Rbck1 intron 7; top panel ) and percent Hoil1/Rbck1−/− ( neomycin-resistance cassette; bottom panel ) genomic DNA ( gDNA ) in peripheral blood from control and HOIL-1 KO reciprocal bone marrow chimeric mice determined by qPCR . DOI: http://dx . doi . org/10 . 7554/eLife . 04494 . 01310 . 7554/eLife . 04494 . 014Figure 2—figure supplement 2 . HOIL-1 KO mice are capable of generating an adaptive immune response to Listeria . Listeria titers in spleen and liver of naïve ( circles ) or pre-immunized ( 103 CFU for 28 days , squares ) control ( blue symbols ) and HOIL-1 KO ( red symbols ) mice challenged with 106 CFU Listeria for 3 days .", "*p ≤ 0 . 05 , **p ≤ 0 . 01 , ***p ≤ 0 . 001 , ****p ≤ 0 . 0001 .", "Statistical analyses were performed using two-way ANOVA with Holm-Sidak's multiple comparison test . DOI: http://dx . doi . org/10 . 7554/eLife . 04494 . 014 To determine whether HOIL-1 deficiency resulted in a defect in innate or adaptive immunity , we bred the HOIL-1 KO mice onto a RAG1-deficient background .", "T and B cell-deficient RAG1 HOIL-1 double KO mice succumbed to infection significantly faster than RAG1 KO mice ( Figure 2B ) , and exhibited elevated bacterial burden in the spleen and liver 3 days post-infection ( Figure 2C ) , indicating that HOIL-1 plays an essential role in innate immunity during Listeria infection .", "Indeed , HOIL-1-deficient mice succumbed to infection at the same rate regardless of the presence or absence of the adaptive immune system ( compare Figures 1A and 2B ) .", "Further , HOIL-1 KO mice immunized with a low dose of Listeria were capable of mounting a protective adaptive response to a high dose secondary challenge with Listeria 28 days later ( Figure 2—figure supplement 2 ) .", "We noted that 1000 CFU administered i . p . was a borderline dose required to successfully immunize control mice in our experiments , despite being sufficient to induce lethality in 50% of HOIL-1 KO mice ( Figure 1A ) .", "Together , these data do not rule out a role for HOIL-1 in adaptive immunity , but show that HOIL-1 plays a major role in hematopoietic cells to foster innate immunity to Listeria infection .", "Innate immunity to Listeria in mice depends on tissue-resident macrophages and CD8α+ dendritic cells responding to Listeria infection by secreting pro-inflammatory cytokines including TNFα , IL-12 , and IL-6 .", "These cytokines are each well recognized to be essential for survival after Listeria infection ( Unanue , 1997; Williams et al . , 2012 ) through their role in coordinating activation of NK cells , NKT cell and T cells to produce IFNγ required for the bactericidal activity of phagocytic cells .", "Therefore , to further define a role for HOIL-1 in the innate immune system , we determined whether HOIL-1 KO bone marrow-derived macrophages ( macrophages herein ) produced cytokines in response to Listeria infection with or without IFNγ treatment .", "Compared to control cells , HOIL-1 KO macrophages secreted only 50% , 20% and 10% of the expected levels of TNFα , IL-6 and IL-12p70 protein , respectively ( Figure 3A ) .", "Consistent with a role for HOIL-1 in the activation of the NF-κB transcription factor following TLR stimulation , Listeria-infected HOIL-1 KO macrophages expressed decreased levels of Tnf , Il6 and Il12b mRNA ( Figure 3B ) .", "The defects in cytokine transcript levels were of smaller magnitude than the decreases in secreted protein , particularly for Tnf , suggesting that HOIL-1 may also be involved in cytokine translation or secretion .", "However , HOIL-1 KO macrophages killed Listeria after activation with IFNγ as effectively as control cells , indicating the selectivity of HOIL-1 effects on macrophage function ( Figure 3—figure supplement 1 ) . 10 . 7554/eLife . 04494 . 015Figure 3 . HOIL-1 is important for induction of pro-inflammatory cytokines following Listeria infection .", "( A ) TNFα ( 6 hr ) , IL-6 ( 6 hr ) and IL-12p70 ( 24 hr ) protein in macrophage culture supernatants following infection with Listeria ( LM ) ± IFNγ co-treatment .", "( B ) Induction of Tnf , Il6 and Il12b transcripts in macrophages over 12 hr following infection with Listeria plus IFNγ .", "Data represent the mean ± SEM of macrophages derived from two mice per genotype analyzed in triplicate and are representative of at least three independent experiments .", "( C ) Induction of cytokine transcripts in peritoneal cells over 12 hr following infection of control ( blue circles ) and HOIL-1 KO ( red squares ) mice with 105 Listeria .", "Each symbol represents an individual mouse .", "*p ≤ 0 . 05 , **p ≤ 0 . 01 , ***p ≤ 0 . 001 , ****p ≤ 0 . 0001 .", "Statistical analyses were performed using t-test . DOI: http://dx . doi . org/10 . 7554/eLife . 04494 . 01510 . 7554/eLife . 04494 . 016Figure 3—figure supplement 1 . HOIL-1 is not required for listericidal activity of bone marrow-derived macrophages . Growth and killing of Listeria in untreated or IFNγ-pre-treated control and HOIL-1 KO macrophages at 0 and 6 hr post-infection .", "Data are from two independent experiments performed in duplicate . DOI: http://dx . doi . org/10 . 7554/eLife . 04494 . 01610 . 7554/eLife . 04494 . 017Figure 3—figure supplement 2 . Analysis of peritoneal cell populations following Listeria infection . Flow cytometric analysis of peritoneal cell populations in control ( blue circles; 0 hr n = 20 , 6 hr n = 10 , 12 hr n = 7 ) and HOIL-1 KO mice ( red squares; 0 hr n = 9 , 6 hr n = 12 , 12 hr n = 7 ) over 12 hr after infection with 105 CFU i . p . Data represent the mean ± SEM .", "*p ≤ 0 . 05 , **p ≤ 0 . 01 , ***p ≤ 0 . 001 , ****p ≤ 0 . 0001 .", "Statistical analyses were performed using t-test . DOI: http://dx . doi . org/10 . 7554/eLife . 04494 . 01710 . 7554/eLife . 04494 . 018Figure 3—figure supplement 3 . HOIL-1 is important for induction of pro-inflammatory cytokines by innate cells following Listeria infection in vivo . Induction of cytokine transcripts in peritoneal cells from uninfected ( 0 hr ) RAG1 KO HOIL-1 WT ( blue circles ) and RAG1 KO HOIL-1 KO ( red squares ) mice or 3 hr after infection with 104 Listeria .", "Each symbol represents an individual mouse . DOI: http://dx . doi . org/10 . 7554/eLife . 04494 . 018 We confirmed that induction of Tnf and Il6 mRNA was significantly impaired following Listeria infection in vivo by measuring cytokine transcripts in peritoneal cells from mice 3 to 12 hr after infection ( Figure 3C ) .", "This reduction in cytokine transcripts could not be explained by a decrease in macrophage numbers ( Figure 3—figure supplement 2 ) .", "Surprisingly , Il12b transcript levels were similar in HOIL-1 KO mice , with a significant difference being detected at only 6 hr after infection .", "Similar decreases in Tnf and Il6 mRNA were observed in mice on the Rag1−/− background at 3 hr post-infection , confirming that these differences are due to a defect in innate immunity in the absence of HOIL-1 ( Figure 3—figure supplement 3 ) .", "We also noted that fewer NK cells and neutrophils were present in the peritoneum 6 hr after Listeria infection , suggesting delayed recruitment or proliferation of these cell types ( Figure 3—figure supplement 2 ) .", "These decreases in cytokine production and delayed cell recruitment likely synergize with defects in IL-1β and TNFα signaling observed by others ( Tian et al . , 2007; Haas et al . , 2009; Tokunaga et al . , 2009 , 2011 ) to compromise antibacterial immunity , and may contribute to the impaired induction of Ifng mRNA observed by 12 hr ( Figure 3C ) .", "These data indicate that HOIL-1 plays a critical role in coordinating essential early cytokine responses after Listeria infection .", "The above data demonstrate that HOIL-1 KO mice have a severe immunodeficiency after certain types of infection .", "To assess the generality of this phenotype we infected HOIL-1 KO mice with murine γ-herpesvirus 68 ( MHV68 ) , a genetic relative of the common persistent human herpesviruses , Epstein–Barr virus and Kaposi's sarcoma-associated herpesvirus ( EBV , KSHV ) ( Barton et al . , 2011; Speck and Ganem , 2010 ) .", "HOIL-1 KO mice survived MHV68 infection for at least 3 months .", "MHV68 replication was unaffected by HOIL-1 deficiency in cultured macrophages , and was suppressed only slightly in vivo ( Figure 4—figure supplement 1 ) .", "Despite normal establishment of latency as determined by the number of cells carrying MHV68 genome 28 days after infection ( Figure 4—figure supplement 2 ) , the efficiency of MHV68 reactivation from latency in explanted peritoneal cells was significantly impaired ( approximately 50-fold , Figure 4A ) .", "Similarly , HOIL-1 KO mice failed to succumb to infection with M . tuberculosis over 70 days of infection , and in fact exhibited lower bacterial colony counts in the spleen while counts in the lung were no different than controls ( Figure 4B ) .", "Thus HOIL-1 KO mice are fully able to control , and may have an enhanced ability to control , specific aspects of acute and chronic MHV68 and M . tuberculosis infection , in striking contrast to the immunodeficiency apparent after infection with Listeria , Toxoplasma , and Citrobacter . 10 . 7554/eLife . 04494 . 003Figure 4 . Enhanced inflammatory response and control of MHV68 and M . tuberculosis by HOIL-1 KO mice .", "( A ) Limiting dilution assay of peritoneal cells from control ( blue circles ) and HOIL-1 KO ( red squares ) mice infected with MHV68 for 28 days onto mouse embryonic fibroblast monolayers to measure the frequency of cells capable of MHV68 reactivation .", "The dashed line indicates 63 . 2% , which was used to determine the frequency of cells reactivating virus by the Poisson distribution .", "Data represent the mean from three independent experiments each with cells combined from three mice/group .", "*p ≤ 0 . 05 .", "Statistical analyses were performed by calculating the number of control and HOIL-1 KO cells required for 63 . 2% of wells to contain complete cytopathic effect for each individual experiment by non-linear regression , then comparing these values by paired t-test .", "Preformed virus was not detected in disrupted samples ( not shown ) .", "( B ) M . tuberculosis titers in the lung and spleen of HOIL-1 KO ( red squares ) and control ( blue circles ) mice 70 days post-infection .", "*p ≤ 0 . 05 .", "Statistical analyses were performed using t-test .", "( C ) TNFα , IL-6 , IL-12/IL-23p40 and IFNγ protein detected in serum from naïve or latently-infected ( 28 days ) control ( blue circles ) and HOIL-1 KO ( red squares ) mice .", "Each symbol represents an individual mouse and the mean is indicated .", "*p ≤ 0 . 05 , t-test with Welch's correction ( IL-12/IL-23p40 ) or Mann Whitney test ( TNFα , IL-6 , IFNγ ) .", "( D ) TNFα , IL-6 , IL-12/IL-23p40 and IFNγ protein in serum from mice from ( B ) .", "Each symbol represents an individual mouse .", "Data are combined from two independent experiments .", "*p ≤ 0 . 05 , **p ≤ 0 . 01 .", "Statistical analyses were performed using t-test ( TNFα , IL-12p40 ) with Welch's correction ( IFNγ ) or Mann Whitney test ( IL-6 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 04494 . 00310 . 7554/eLife . 04494 . 004Figure 4—figure supplement 1 . Acute MHV68 replication in vitro and in vivo is minimally affected by HOIL-1-deficiency .", "( A ) MHV68 growth in HOIL-1 KO ( red symbols ) and control ( blue symbols ) bone marrow-derived macrophages with ( squares ) or without ( circles ) IFNγ pre- and post-treatment .", "Data represent the mean ± SEM from three independent experiments performed in triplicate .", "( B ) MHV68 titers in spleen during acute infection of HOIL-1 KO ( red squares ) and control ( blue circles ) mice .", "**p ≤ 0 . 01 , Mann Whitney test .", "The dashed line indicates the limit of detection . DOI: http://dx . doi . org/10 . 7554/eLife . 04494 . 00410 . 7554/eLife . 04494 . 005Figure 4—figure supplement 2 . Establishment of MHV68 latency is similar in control and HOIL-1 KO mice . Limiting dilution PCR to determine the frequency of peritoneal cells from latently infected ( 28 days ) HOIL-1 KO ( red squares ) and control ( blue circles ) mice containing MHV68 genomes .", "The dashed line indicates 63 . 2% , which was used to determine the frequency of cells containing viral genome by the Poisson distribution .", "Data represent the mean from three independent experiments each with cells combined from three mice/group .", "Statistical analyses were performed by calculating the number of control and HOIL-1 KO cells required for 63 . 2% of reactions to be positive for viral genome for each individual experiment by non-linear regression , then comparing these values by paired t-test . DOI: http://dx . doi . org/10 . 7554/eLife . 04494 . 005 The ability of HOIL-1 KO mice to effectively control chronic herpesvirus infection allowed us to test the hypothesis that persistent virus infection might alter two phenotypes , hyper-inflammation and immunodeficiency , in which HOIL-1 KO mice appear to differ from some reported patients with bi-allelic mutations in RBCK1 ( HOIL1 ) ( Boisson et al . , 2012 ) .", "Notably , patients with HOIL-1 deficiency and hyper-inflammation exhibited increased expression of IL-6 and TNFα in the serum and increased expression of mRNA for Il6 in blood cells ( Boisson et al . , 2012 ) .", "We therefore examined the serum of MHV68-infected HOIL-1 KO mice for cytokines essential for resistance to Listeria but deficient in Listeria-infected HOIL-1 KO mice .", "As previously observed ( Barton et al . , 2007 ) , latent infection of control mice with MHV68 was associated with an increase in circulating levels of TNFα , IL-6 , IL-12 and IFNγ compared with uninfected mice ( Figure 4C ) .", "In HOIL-1 KO mice , MHV68 latently resulted in small but significant increases in TNFα , IL-6 and IL-12p70 levels , and a more striking increase in IFNγ levels compared to latently infected controls .", "HOIL-1 KO mice chronically infected with M . tuberculosis also exhibited increased expression of both IL-6 and TNFα in serum at 70 days post-infection ( Figure 4D ) .", "Therefore , there is an overlap between cytokines expressed in hyper-inflammatory patients and in chronically-infected HOIL-1 KO mice .", "MHV68 latency has been shown previously to induce symbiotic protection against Listeria infection in wild-type mice ( Barton et al . , 2007 ) .", "Because expression of TNFα , IL-6 and IFNγ are essential for control of Listeria infection in mice ( Kopf et al . , 1994; Unanue , 1997; Williams et al . , 2012 ) , and were impaired in Listeria-infected HOIL-1 KO mice ( Figure 3C ) , but elevated in MHV68-infected HOIL-1 KO mice , we considered whether chronic MHV68 infection could complement the profound immunodeficiency observed in Listeria-infected barrier-raised HOIL-1 KO mice .", "As observed previously ( Barton et al . , 2007 ) , MHV68 latency , 1 month after infection , protected control mice from an otherwise lethal dose of Listeria ( Figure 5A ) .", "MHV68 latency also protected HOIL-1 KO mice from a dose of Listeria at least 1000-fold higher than the LD50 for MHV68-negative mice ( Figures 5A and 1A ) .", "Both control and HOIL-1 KO mice were still partially protected from Listeria challenge 6 months after MHV68 infection ( Figure 5—figure supplement 1 ) .", "A viral mutant capable of acute lytic infection but unable to efficiently establish latency ( ORF73 . stop ) ( Moorman et al . , 2003 ) was unable to efficiently protect HOIL-1 KO or control mice from Listeria infection , demonstrating that latent MHV68 is required to complement HOIL-1-associated immunodeficiency to Listeria ( Figure 5B ) . 10 . 7554/eLife . 04494 . 019Figure 5 . MHV68 latency rescues HOIL-1 KO , IL-6 , Caspase-1 and Caspase-1;Caspase-11-deficient mice from Listeria-induced lethality .", "( A ) Survival of control ( blue symbols; mock n = 9 , MHV68 n = 15 ) and HOIL-1 KO ( red symbols; mock n = 10 , MHV68 n = 20 ) mice challenged with 106 CFU Listeria 28 days following mock infection ( circles ) or infection with 106 PFU MHV68 ( squares ) .", "*p ≤ 0 . 0083; logrank Mantel–Cox test corrected for multiple comparisons .", "( B ) Survival of control ( blue symbols ) and HOIL-1 KO ( red symbols ) mice challenged with 106 CFU Listeria 28 days following intranasal mock infection ( circles ) or infection with 5 × 104 PFU wild-type ( squares ) or ORF73 . stop ( triangles ) MHV68 .", "Significantly different groups were: control mock infected and control MHV68wt infected , control mock infected and HOIL-1 KO MHV68wt infected , control mock infected and HOIL-1 KO MHV68orf73 . stop infected , HOIL-1 KO mock infected and control MHV68wt infected , HOIL-1 KO mock infected and HOIL-1 KO MHV68wt infected , control MHV68wt infected and control MHV68orf73 . stop infected , control MHV68wt infected and HOIL-1 KO MHV68orf73 . stop infected , HOIL-1 KO MHV68wt infected and control MHV68orf73 . stop infected , HOIL-1 KO MHV68wt infected and HOIL-1 KO MHV68orf73 . stop infected , control MHV68orf73 . stop infected and HOIL-1 KO MHV68orf73 . stop infected .", "*p ≤ 0 . 0033; logrank Mantel–Cox test corrected for multiple comparisons .", "( C ) Cytokine transcript levels in peritoneal cells from mock ( circles ) and MHV68-infected ( squares ) control ( blue symbols ) and HOIL-1 KO ( red symbols ) mice ( 28 days post-infection ) .", "( D ) Induction of cytokine transcripts in peritoneal cells from mock ( circles ) and MHV68-infected ( squares ) control ( blue symbols ) and HOIL-1 KO ( red symbols ) mice ( 28 days ) 3 hr after infection with 105 Listeria .", "Each symbol represents an individual mouse .", "For ( C ) and ( D ) , *p ≤ 0 . 05 , **p ≤ 0 . 01 , ***p ≤ 0 . 001 , ****p ≤ 0 . 0001 .", "Statistical analyses were performed using one-way ANOVA .", "( E ) Survival of control ( blue symbols ) and Il6−/− ( purple symbols ) mice challenged with 106 CFU Listeria 28 days following mock infection ( circles ) or infection with 106 PFU MHV68 ( squares ) .", "*p ≤ 0 . 0083; logrank Mantel–Cox test corrected for multiple comparisons .", "( F ) Survival of control ( blue symbols ) , Caspase-1;Caspase-11 ( orange symbols ) and Caspase-1 ( green symbols ) –deficient mice challenged with 106 CFU Listeria 28 days following mock infection ( circles ) or infection with 106 PFU MHV68 ( squares ) .", "*p ≤ 0 . 0033; logrank Mantel–Cox test corrected for multiple comparisons . DOI: http://dx . doi . org/10 . 7554/eLife . 04494 . 01910 . 7554/eLife . 04494 . 020Figure 5—figure supplement 1 . MHV68 latency-induced cross-protection is maintained for at least 6 months . Survival of control ( blue symbols ) and HOIL-1 KO ( red symbols ) mice challenged with 106 CFU Listeria 6 months after mock infection ( circles ) or infection with 106 PFU MHV68 ( squares ) .", "Statistical analyses were performed by logrank Mantel–Cox test corrected for multiple comparisons , with p ≤ 0 . 0083 considered significant .", "Control mock vs HOIL-1 KO mock , p = 0 . 1824; control mock vs control MHV68 infected , p = 0 . 0408; control mock vs HOIL-1 KO MHV68 infected , p = 0 . 0070; HOIL-1 KO mock vs control MHV68 infected , p = 0 . 0028; HOIL-1 KO mock vs HOIL-1 KO MHV68 infected , p = 0 . 0003; control MHV68 infected vs HOIL-1 KO MHV68 infected , p = 0 . 9303 . DOI: http://dx . doi . org/10 . 7554/eLife . 04494 . 02010 . 7554/eLife . 04494 . 021Figure 5—figure supplement 2 . MHV68 latency enhances the listericidal activity of peritoneal macrophages . Listeria CFU in control ( blue symbols ) and HOIL-1 KO ( red symbols ) ex vivo cultures of peritoneal macrophages from mock ( circles ) or MHV68-infected ( 32 days , squares ) mice at 0 and 6 hr post-infection .", "Data are combined from two independent experiments .", "Each symbol represents an individual mouse analyzed in duplicate .", "Cells from the same mouse were used for both 0 hr and 6 hr time points .", "Statistical analyses were performed by one-way ANOVA with Holm-Sidak's multiple comparison test for each time point . DOI: http://dx . doi . org/10 . 7554/eLife . 04494 . 02110 . 7554/eLife . 04494 . 022Figure 5—figure supplement 3 . Il6−/− mice have increased susceptibility to Listeria infection . Survival of control ( blue circles ) and Il6−/− ( purple squares ) mice following i . p . inoculation with 105 CFU Listeria .", "*p ≤ 0 . 0083; logrank Mantel–Cox test corrected for multiple comparisons . DOI: http://dx . doi . org/10 . 7554/eLife . 04494 . 022 To determine whether MHV68 latency rescued pro-inflammatory cytokine induction by HOIL-1 KO mice following Listeria infection , we quantitated cytokine transcripts in peritoneal cells from latently infected mice before ( Figure 5C ) and 3 hr after ( Figure 5D ) infection with Listeria .", "As predicted from the cytokine levels in the serum , MHV68 latency resulted in small but significant increases in Tnf , Il6 and Il12b transcripts prior to Listeria challenge ( Figure 5C ) .", "While MHV68 latency did not rescue the induction of Tnf or Il6 transcripts in HOIL-1 KO mice following infection with Listeria , Il12b transcript levels were increased approximately twofold , and were comparable to levels in control mice .", "More significantly , Ifng and Nos2 ( encoding iNOS ) transcripts were elevated approximately 200-fold and 1000-fold , respectively , in latently infected control and HOIL-1 KO mice ( Figure 5C ) , and further induced by 3 hr after infection with Listeria ( Figure 5D ) .", "Ifng transcript levels were significantly higher in latently infected HOIL-1 KO mice following infection with Listeria than in control mice .", "These data suggest that MHV68 latency by-passes the requirement for TNFα and IL-6 during early Listeria infection by enhancing the induction of IFNγ and downstream effector molecules important for controlling Listeria infection .", "To test whether peritoneal macrophages from latently infected HOIL-1 KO mice had an increased capacity to kill Listeria , we explanted peritoneal macrophages from mock or latently infected mice , infected them with Listeria , killed extracellular bacteria with gentamycin treatment , and compared the number of CFU at 6 hr to the number of CFU at the beginning of the experiment .", "As expected , cells from mock infected control mice exhibited mild listericidal activity and cells from latently infected control mice had an enhanced ability to kill Listeria ( Figure 5—figure supplement 2 , ( Barton et al . , 2007 ) ) .", "Macrophages from mock-infected HOIL-1 KO mice had a slightly impaired ability to control Listeria infection .", "However , MHV68 latency in HOIL-1 KO mice enhanced the ability of macrophages to kill Listeria , generating a capacity to kill similar to that observed with macrophages from control mice .", "Together , these data suggest that MHV68 latency induces an environment that enhances the ability of HOIL-1-deficient cells to kill and respond to Listeria .", "To determine whether the viral complementation of immunodeficiency was unique to HOIL-1 , we latently infected IL-6 , Caspase-1-deficient and Caspase-1;Caspase-11-double-deficient mice ( Kayagaki et al . , 2013 ) , which survive MHV68 infection but are all highly susceptible to Listeria infection ( Figure 5—figure supplement 3 ) ( Kopf et al . , 1994; Sarawar et al . , 1998; Edelson and Unanue , 2002; Tsuji et al . , 2004; Sauer et al . , 2011 ) .", "IL-6 , Caspase-1 and Caspase-1;Caspase-11-deficient mice were also protected from lethality following Listeria infection by chronic MHV68 infection ( Figure 5E , F ) .", "These data indicate that the capacity of chronic MHV68 to reverse a significant immunodeficiency is not restricted to mutations in Rbck1 ( Hoil1 ) , and suggest that chronic viral infections may alter phenotypes of many host allelic variants ." ], [ "We report that HOIL-1 is essential during infection with Listeria , T . gondii and C . rodentium , but not with MHV68 or M . tuberculosis in mice .", "Expression of HOIL-1 was critical in innate , hematopoietic-derived cells during Listeria infection in vivo .", "The requirement of HOIL-1 for the induction of protective inflammatory cytokines , TNFα , IL-6 and IL-12 , following infection of macrophages with Listeria in vitro is consistent with reports that LUBAC is required for efficient NF-κB activation following TLR engagement ( Zak et al . , 2011; Boisson et al . , 2012 ) , but not with a recent report that NF-κB activation following stimulation of TLR4 and TNF-R1 on macrophages by LPS and TNFα , respectively , is unaffected by HOIL-1 deficiency ( Rodgers et al . , 2014 ) .", "These apparently conflicting results suggest that HOIL-1 and LUBAC may not only have cell-type specific functions ( Boisson et al . , 2012; Rodgers et al . , 2014 ) , but also stimulus-specific roles that vary between different cell types .", "This may be further highlighted by the excessive inflammatory response and enhanced control of MHV68 and M . tuberculosis infection—two pathogens that also infect macrophages—by HOIL-1-deficient mice .", "It has been proposed that low levels of viral reactivation during MHV68 latency are responsible for the low level of constant immune activation and cytokine production ( Barton et al . , 2007 ) .", "It is paradoxical , then , that reactivation is almost undetectable in HOIL-1-deficient animals , and yet their inflammatory response is elevated .", "Since the negative regulator of NF-κB signaling , A20 , binds to linear ubiquitin chains ( Tokunaga et al . , 2012; Verhelst et al . , 2012 ) , HOIL-1/LUBAC may also be important for recruiting A20 to receptor signaling complexes to turn off signaling .", "Therefore , receptor signaling may be sustained in cells in HOIL-1 KO mice , resulting in the increase in TNFα , IL-6 and IL-12 , and ultimately IFNγ protein , observed in the serum of chronically infected mice .", "Decreased viral reactivation may be the result of this increased IFNγ , the inability of latently infected cells to response to a stimulus of reactivation or a combination thereof .", "It is unclear why HOIL-1 KO mice are extremely susceptible to some pathogens , yet control other infections remarkably well .", "This might be due to HOIL-1/LUBAC having differential roles in transducing signals from different immune sensors stimulated by different infections .", "Other possibilities include a differential requirement for the innate immune system to hold the acute infection in check while an adaptive response is being generated , the speed at which the pathogen replicates , the tissues that it damages , and whether pathology during acute infection is mostly immune- or pathogen-derived .", "Further studies will be required to address these possibilities .", "We further show that chronic infection with MHV68 rescued HOIL-1 , IL-6 , Caspase-1 and Caspase-1;Caspase-11-deficient mice from lethal Listeria infection , thereby masking the genetic immunodeficiency observed in MHV68-negative mice .", "As reported previously ( Barton et al . , 2007 ) , MHV68 latency was associated with increased basal levels of pro-inflammatory cytokines TNFα , IL-6 , IL-12 and IFNγ in the serum of wild-type animals , which were further increased in HOIL-1 KO mice .", "These cytokines appear to increase the activation status of the innate immune system such that their induction following Listeria infection is no longer as important as would be the case in a naïve mouse .", "Indeed , MHV68 infection did not enhance the transcription of Tnf , and only marginally enhanced the transcription of Il6 and Il12b even in control mice following Listeria infection , and did not rescue the defect in Tnf and Il6 induction observed in HOIL-1 KO mice .", "Conversely , transcription of Ifng and the effector molecule , iNOS ( encoded by Nos2 ) , was elevated in cells from latently infected mice and enhanced substantially by both HOIL-1 KO and control animals very rapidly following infection with Listeria .", "Macrophages require priming with IFNγ to induce IL-12 in response to Listeria infection .", "In a latently infected animal , IFNγ is already present and so IL-12 and additional IFNγ may be induced more rapidly .", "Furthermore , as reported previously for wild-type mice ( Barton et al . , 2007 ) , MHV68 latency enhanced the ability of peritoneal macrophages from control and HOIL-1 KO mice to kill Listeria .", "Together , these data suggest that the constant presence of low levels of IFNγ driven by latent virus infection results in an increase in the basal expression levels of downstream effector molecules and the priming of cells for the enhanced immediate killing of Listeria upon infection , as well as for a more rapid further induction of IFNγ and its effector molecules in response to the bacterial challenge .", "In this sense , chronic virus infection sets the level of innate immunity to subsequent infection .", "HOIL-1 KO mice bred in a high grade barrier facility failed to exhibit certain phenotypes of HOIL-1 deficient patients , specifically by not exhibiting baseline hyper-inflammation and by displaying a striking immunodeficiency out of proportion to that observed in some humans with bi-allelic mutations in RBCK1 ( HOIL1 ) .", "Most humans are infected life-long with multiple herpesviruses ( Virgin et al . , 2009; Virgin , 2014 ) , and many also carry other chronic or latent infections such as tuberculosis .", "Importantly , we observed complementation of immunodeficiency to Listeria by chronic herpesvirus infection in four different strains of immunodeficient mice , revealing virus infection as one possible environmental factor that might alter the genotype-phenotype relationship for patients with mutations in immune system genes .", "HOIL-1 KO mice chronically infected with either a herpesvirus or M . tuberculosis also exhibited increases in some of the same cytokines reported in the serum of HOIL-1 deficient humans .", "At least three of the HOIL-1 mutant patients were infected with at least one herpesvirus ( Boisson et al . , 2012 ) , and it is likely that other chronic infections were present .", "However , in the absence of data regarding the complete infection status of the HOIL-1 mutant patients , the relevance of the mouse studies to the human phenotypes is unclear .", "Nevertheless , perhaps the presence of the virome , and potentially variations in the virome or other chronic infections between people , confers significant phenotypic variation by complementing mutations in host genes responsible for innate immunity ( Virgin , 2014 ) .", "The genes involved in immunity and inflammation are the most rapidly evolving in the mammalian genome ( Lindblad-Toh et al . , 2011; Casanova et al . , 2013; Quintana-Murci and Clark , 2013 ) .", "Survival from infection requires a trade-off between alleles that promote or limit inflammation to balance immunity vs immunopathology .", "We speculate that the virome or other chronic infections hide or enhance the effects of genetic variations in immune responsiveness by complementing chromosomal variations in immune response genes .", "As the nature of the virome changes in persons growing up in different cultural and economic environments , it is possible that the immunophenotype of the host changes , and the beneficial or deleterious effects of existing genetic variation are unmasked by removal of complementation provided by chronic virus infection .", "It is also plausible that the striking auto-inflammation observed in humans with a variety of immune defects could be due to even well controlled herpesvirus infection alone or in combination with other chronic infections .", "Our data suggest that analysis of the metagenome , including the virome , may be of value in linking human phenotype and genotype ( Virgin , 2014; Virgin and Todd , 2011 ) .", "Recent rapid advances in sequencing and analysis of the metagenome will make integration of data from the virome into human genetic studies practical ( Virgin , 2014 ) ." ], [ "HOIL-1 KO mice , with null mutations in the Rbck1 gene that encodes HOIL-1 , have been described previously ( Tokunaga et al . , 2009 ) .", "C57BL/6J mice or HOIL-1 WT littermates were used as wild type controls .", "Rag1−/− mice were purchased from The Jackson Laboratory ( Bar Harbor , ME ) and bred to HOIL-1 KO mice .", "Il6−/− mice were purchased from The Jackson Laboratory .", "Caspase 1;Caspase 11-deficient mice with or without a Caspase 11 transgene were kindly provided by Vishva Dixit , Genentec , San Francisco USA .", "All mice were housed and bred at Washington University in Saint Louis in specific pathogen-free conditions in accordance with Federal and University guidelines and protocols were approved by the Animal Studies Committee of Washington University under protocol number 20140244 .", "Mice were inoculated between 8 and 11 weeks of age .", "L . monocytogenes wild type strain EGD was used for this study .", "Listeria glycerol stocks were stored at −80 °C , and thawed and diluted into PBS for intraperitoneal ( i . p . ) injection into mice .", "To determine tissue burden , spleens and livers were homogenized in 10 ml PBS containing 0 . 05% Triton X-100 and serial dilutions were plated on brain heart infusion agar plates .", "Listeria CFU were counted after overnight growth at 37°C .", "Small sections of spleen and liver were also fixed in 10% buffered formalin for histological analysis .", "The type II Prugniaud strain of T . gondii expressing a firefly luciferase ( PRU-Fluc-GFP , provided by J . Boothroyd , Stanford University , Palo Alto , CA ) ( Saeij et al . , 2005 ) was used in all in vivo T . gondii experiments .", "Tachyzoites were grown by 2-day serial passage in human foreskin fibroblasts .", "For infections , freshly egressed parasites were filtered , counted , and injected i . p . into mice .", "Mice were with inoculated orally with 2 × 109 CFU C . rodentium strain DBS100 ( ATCC , Manassas , VA ) from a fresh culture and monitored for morbidity and mortality .", "MHV68 WUMS ( ATCC VR1465 ) , MHV68 ORF73 . stop and γHV68 M3-Fluc were passaged and titered by plaque assay on NIH 3T12 cells .", "Virus stocks were stored at −80 °C , and thawed and diluted into PBS for i . p . or intranasal ( i . n . ) inoculation of mice .", "For experiments involving MHV68 ORF73 . stop , i . n . inoculation with 5 × 104 CFU was performed due to the low titer of the virus stock .", "To determine γHV68 titers in tissues , organs were placed in 1 ml of complete DMEM and frozen at −80°C .", "Samples were thawed prior to disruption with silica beads and virus titration by plaque assay .", "Before infection , exponentially replicating M . tuberculosis Erdman strain bacteria were washed in PBS + 0 . 05% Tween 80 , and sonicated to disperse clumps .", "Mice were exposed to 8 × 107 CFU of M . tuberculosis in an Inhalation Exposure System ( Glas-Col , Terre Haute , IN ) , which delivers ∼100 bacteria to the lung per animal .", "After 24 hr post infection , two mice per group were sacrificed , and lungs were harvested to determine infection efficiency , which was about 100 CFU/lung/mouse .", "Experimental mice were sacrificed 70 days after infection , and lungs and spleen were harvested for CFU , and serum was collected for cytokine analysis .", "Bacterial burdens were determined by plating serial dilutions of lung and spleen homogenates onto 7H10 agar plates and incubated at 37°C in 5% CO2 for 3 weeks prior to counting colonies .", "MHV68 reactivation from latency and preformed virus was assayed as described previously ( Weck et al . , 1996 ) .", "Briefly , peritoneal exudate cells were plated in twofold serial dilutions ( 24-wells per dilution ) onto permissive mouse embryonic fibroblast ( MEF ) monolayers and scored for cytopathic effect ( CPE ) 3 weeks later .", "Reactivation of lytic virus from a peritoneal cell leads to complete CPE of the MEF monolayer .", "To measure preformed infectious virus in the sample , parallel samples of cells were mechanically disrupted to kill the cells but keep any infectious virus intact .", "These samples were plated and scored as described above .", "Using the Poisson distribution , CPE in 63 . 2% of wells indicates that one reactivation event is likely to have occurred per well , and is used to determine the frequency of reactivating cells in the sample .", "To determine the frequency of cells harboring viral genome , peritoneal cells were assayed by nested PCR for viral genome as described previously ( Weck et al . , 1999 ) .", "The detection of PCR product in 63 . 2% of wells indicates that one genome was present per well .", "Primary bone marrow-derived macrophages were prepared as described previously ( Hwang et al . , 2012 ) .", "Briefly , bone marrow was extracted from mouse femurs and allowed to differentiate in DMEM containing 10% FBS , 10% CMG14-12 cell-conditioned media as a source of M-CSF ( Takeshita et al . , 2000 ) , 5% horse serum , 1 mM sodium pyruvate and 2 mM L-Glutamine for 7 days .", "For cytokine and transcript analyses following Listeria infection of macrophages , adherent cells were scraped and seeded in tissue culture-treated plates in the absence of M-CSF .", "After 3 days , macrophages were infected with 106/ml Listeria in the presence or absence of 100 U/ml IFNγ .", "2 hr post-infection , 50 U/ml Penicillin and 50 μg/ml streptomycin were added to kill the Listeria .", "Cell supernatants were harvested at indicated times and frozen at −80 °C prior to cytokine analysis .", "Cells were lyzed in TRI-Reagent for RNA extraction .", "For Listeria growth/killing assays , macrophages were seeded in non-tissue culture treated dishes in the absence of M-CSF .", "After 1 day , cells were treated with 300 µ/ml IFNγ or untreated , and 48 hr later scraped replated on sterile coverslips .", "After 3 hr , cells were infected with 105/ml Listeria from an overnight standing culture and centifuged to synchronize the infection .", "50 μg/ml gentamycin was added after 30 min to kill extracellular bacteria .", "At the indicated times , coverslips were washed with warm PBS , then lyzed in 10 ml cold water to release the bacteria .", "Serial dilutions were plated on brain heart infusion agar plates , and Listeria CFU were counted after overnight growth at 37°C .", "For MHV68 growth analysis , adherent cells were scraped and seeded in tissue culture-treated plates in the presence of M-CSF .", "After 2 days , macrophages were treated with 0 . 1 U/ml IFNγ or untreated , and 12 hr later infected with MHV68 at a multiplicity of infection ( MOI ) of 0 . 05 for 1 hr with occasional rocking at 37°C and 5% CO2 .", "Cells were washed once with medium and incubated in DMEM supplemented with 10% FBS and 2 mM L-glutamine ( with or without 0 . 1 U/ml IFNγ ) for the indicated period of time at 37°C and 5% CO2 , before being frozen at −80 °C .", "Virus titers were determined by plaque assay following two freeze–thaw cycles .", "Cells were flushed from the peritoneum of mice that had been mock infected or infected with MHV68 for 32 days with ice cold DMEM containing 10% FBS and 2 mM L-Glutamine .", "5 × 105 cells were plated on glass coverslips in 24 well plates in duplicate wells per timepoint and allowed to adhere overnight .", "Non-adherent cells were washed away with warm medium , and the remaining cells were infected with 105 CFU Listeria from a overnight standing culture by spinocculation at 600×g for 10 min at room temperature , and then incubated at 37 °C and 5% CO2 .", "50 μg/ml gentamycin was added after 30 min to kill extracellular bacteria .", "Coverslips were washed in warm PBS prior to hypotonic lysis of the cells in ice cold water to release the bacteria .", "Serial dilutions were plated on brain heart infusion agar plates , and Listeria CFU were counted after overnight growth at 37°C .", "Recipient mice were exposed to 1200 rad of whole body irradiation , and injected intravenously with 10 million whole bone marrow cells from donor mice .", "Mice were allowed to reconstitute for 8 to 10 weeks before Listeria challenge .", "Mice were bled at 7 weeks post-irradiation to determine percent chimerism .", "Genomic DNA was isolated from peripheral blood and analyzed by quantitative real-time PCR ( qRT-PCR ) for the presence of Rbck1/Hoil1 intron 7 ( in control cells; 5′-ATG CTG GAG TAG AGG CTG GA-3′ and 5′-TGA CTG CTG CTT GGA GAG TG-3′ ) , or the neomycin-resistance cassette ( in HOIL-1 KO cells; 5′-CAA GAT GGA TTG CAC GCA GG-3′ and 5′-GCA GCC GAT TGT CTG TTG TG-3′ ) .", "Rag2 was used as a normalization control ( 5′-GGG AGG ACA CTC ACT TGC CAG TA-3′ and 5′-AGT CAG GAG TCT CCA TCT CAC TGA-3′ ) .", "Imaging was performed as described previously ( Saeij et al . , 2005 ) .", "Briefly , mice were injected i . p . with 150 mg/kg D-Luciferin ( Biosynth AG , Switzerland ) and allowed to remain active for 5 min .", "Animals were subsequently anesthetized with 2% isoflurane for 5 min and then imaged with a Xenogen IVIS 200 machine ( Caliper Life Sciences , Hopkinton , MA ) .", "Data were analyzed using Living Image software ( Caliper Life Sciences ) .", "Peritoneal exudate cells were harvested by peritoneal lavage with 10 ml ice cold FACS buffer ( PBS supplemented with 2% FBS and 50 U/ml Penicillin and 50 µg/ml Streptomycin ) .", "Splenocytes were isolated by filtering through two 100 μm cell strainers into 10 ml ice cold FACS buffer .", "Residual red blood cells were lysed with Red Blood Cell lysis buffer ( Sigma , St Louis , MO ) , counted and stained for flow cytometry .", "Cells were incubated with FACS buffer plus 1% rat serum , 1% hamster serum and 1% Fc-block for 15 min .", "Surface staining was performed for 30 min at room temperature .", "Cells were then washed and fixed with 2% formaldehyde .", "Cells were analyzed on an LSRII or LSR Fortessa flow cytometer ( BD , Franklin Lakes , NJ ) and the data were analyzed using FlowJo software ( Tree Star , Inc , Ashland , OR ) .", "The following antibodies were used: anti-CD3e ( clone 145-2C11 , Biolegend , San Diego , CA ) , anti-CD4 ( clone RM4-5 , BD Pharmingen ) , anti-CD8 ( clone 53-6 . 7 , Biolegend ) , anti-IgM ( clone II/41 , BD Pharmingen ) , anti-CD19 ( clone 6D5 , Biolegend ) , anti-NK1 . 1 ( clone PK136 , BD Pharmingen ) , anti-NKp46 ( clone 29A1 . 4 , eBioscience , San Diego , CA ) , anti-Ly6C ( clone HK1 . 4 , Biolegend ) , anti-Ly6G ( clone 1A8 , Biolgend , anti-CD11b ( clone M1/70 , BD Pharmingen ) , anti-F4/80 ( clone BM8 , Biolegend ) .", "Cytokines in mouse serum and cell supernatants were quantitated using a custom Procarta Immunoassay Kit ( Affymetrix , Santa Clara , CA ) and analyzed on a Bio-Plex 200 System ( BioRad , Hercules , CA ) or by ELISA ( BD , Franklin Lakes , NJ ) , respectively , according to the manufacturers’ instructions .", "Spleen sections were homogenized , and macrophages were lysed in TRI-Reagent ( Sigma ) , and processed according to the manufacturer's instructions to isolate total RNA .", "RNA was isolated from peritoneal cells using RNeasy mini kit ( Qiagen , Netherlands ) .", "RNA samples were treated with Turbo DNA-free DNase ( Ambion , Austin , TX ) prior to first strand cDNA synthesis with ImProm-II ( Promega , Madison , WI ) and random hexamer primers .", "Quantitative PCR was performed on a StepOnePlus machine using Power SYBR Green master mix ( Applied Biosystems , Waltham , MA ) and primers specific for ribosomal protein S29 ( Rps29; 5′-AGC AGC TCT ACT GGA GTC ACC-3′ and 5′-AGG TCG CTT AGT CCA ACT TAA TG-3′ ) , Rbck1/Hoil-1 ( 5′-ATT CGG CGG AAT GGA GAC GG-3′ and 5′-CTG GTT GGT CCT GGG CTT CG-3′ ) , Trib3 ( 5′-CAC ACT GCC ACA AGC ACG GG-3′ and 5′-CAC GCA GGC ATC TTC CAG G-3′ ) , Tbc1d20 ( 5′-TGA GGG AGG GCT CCT GAC TG-3′ and 5′-AGC AGC ACT TGC TGG TAG TCC-3′ ) , Il12b ( 5′-GCA CGG CAG CAG AAT AAA TAT GAG-3′ and 5′-TTC AAA GGC TTC ATC TGC AAG TTC-3′ ) , Tnf ( 5′-GGG TGA TCG GTC CCC AAA GG-3′ and 5′- CTG AGT GTG AGG GTC TGG GC-3′ ) , Il6 ( 5′-GCC AGA GTC CTT CAG AGA GAT ACA-3′ and 5′-CTT GGT CCT TAG CCA CTC CTT C-3′ ) , Ifng ( 5′-ATG AAC GCT ACA CAC TGC ATC-3′ and 5′-CCA TCC TTT TGC CAG TTC CTC-3′ ) and iNos ( Nos2 ) ( 5′-GTT CTC AGC CCA ACA ATA CAA GA-3′ and 5′-GTG GAC GGG TCG ATG TCA C-3′ ) .", "Transcript levels were analyzed using the ΔΔCT method , with Rps29 as the reference gene .", "Tissues were fixed in 10% buffered formalin followed by 70% ethanol , paraffin embedded , sectioned and stained with Periodic acid-Schiff ( PAS ) .", "Alanine aminotransferase and aspartate aminotransferase were measured on a Liasys 330 ( AMS Diagnostics , Weston , FL ) , complete blood counts were measured on a Hemavet 1700 ( Drew Scientific , Waterbury , CT ) , and white blood cell differential counts were performed by Washington University Division of Comparative Medicine Animal Diagnostic Laboratory staff .", "Statistical significance was determined using GraphPad Prism software .", "The specific tests performed are noted in the figure legends ." ] ]

[ "Variation in the presentation of hereditary immunodeficiencies may be explained by genetic or environmental factors .", "Patients with mutations in HOIL1 ( RBCK1 ) present with amylopectinosis-associated myopathy with or without hyper-inflammation and immunodeficiency .", "We report that barrier-raised HOIL-1-deficient mice exhibit amylopectin-like deposits in the myocardium but show minimal signs of hyper-inflammation .", "However , they show immunodeficiency upon acute infection with Listeria monocytogenes , Toxoplasma gondii or Citrobacter rodentium .", "Increased susceptibility to Listeria was due to HOIL-1 function in hematopoietic cells and macrophages in production of protective cytokines .", "In contrast , HOIL-1-deficient mice showed enhanced control of chronic Mycobacterium tuberculosis or murine γ-herpesvirus 68 ( MHV68 ) , and these infections conferred a hyper-inflammatory phenotype .", "Surprisingly , chronic infection with MHV68 complemented the immunodeficiency of HOIL-1 , IL-6 , Caspase-1 and Caspase-1;Caspase-11-deficient mice following Listeria infection .", "Thus chronic herpesvirus infection generates signs of auto-inflammation and complements genetic immunodeficiency in mutant mice , highlighting the importance of accounting for the virome in genotype-phenotype studies ." ]

[ "The immune system protects an individual from invading bacteria , viruses and parasites , as well as malfunctioning or cancerous host cells .", "However , some people inherit genetic defects that cause part of the immune system to be missing or to not work properly .", "This is called a genetic immunodeficiency , and puts individuals at a higher risk of infection and disease .", "The symptoms of immunodeficiencies can vary substantially between individuals , even when they have defects in the same gene .", "For example , only some of the individuals who have defects in both of their copies of a gene called HOIL-1—which has been linked to several roles in the body's immune response—are reported to suffer from an altered susceptibility to bacterial infections and chronic ( persistent ) inflammation .", "Gaining a clear understanding of the possible factors that influence such variations in the symptoms of genetic immune deficiencies could help to speed up their diagnosis , as well as helping to develop more effective treatments .", "MacDuff et al . studied mice that had mutations in both copies of the mouse equivalent of the HOIL-1 gene .", "These mice , when raised in a clean barrier facility that reduces their exposure to viruses , were severely immunodeficient and died when infected by certain bacteria and parasites , including Listeria monocytogenes .", "However , they were able to tolerate infections with a herpesvirus or the bacterium that causes tuberculosis .", "The immunodeficiency to L . monocytogenes was linked to problems producing protective molecules called cytokines , which form a crucial part of the immune response .", "Unexpectedly , MacDuff et al . found that a chronic herpesvirus infection substantially protected these very immunodeficient animals from infection with Listeria monocytogenes , and the mice were able to efficiently produce protective cytokines .", "Mice with two other distinct genetic deficiencies that affect their immune system were also better able to survive otherwise lethal bacterial infections if they had a long-term herpesvirus infection .", "Macduff et al . suggest that the chronic herpesvirus infection stimulates the immune system , and so allows it to compensate for the lack of cytokine production associated with various immunodeficiencies , including those caused by mutations in the HOIL-1 gene .", "This suggests that the presence of viruses or other long-term infections may be responsible for some of the variability seen in the symptoms of different individuals with the same genetic immunodeficiency .", "This is an important concept since essentially all humans have life-long chronic infections from various herpesviruses , as well as other viruses that form the human virome ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "neuroscience" ]

[ [ "Flexible control of cognitive processes is fundamental to daily activities , including the execution of goal-directed tasks according to stimulus inputs and context dependencies .", "An important case of cognitive control arises when input stimuli elicit conflicting responses and subjects must select the task-relevant response despite competition from an often stronger but task-irrelevant response ( Miller , 2000; Miller and Cohen , 2001 ) .", "A canonical example of this type of conflict is the Stroop task: subjects are asked to name the font color of a word where the semantic meaning conflicts with the color signal ( e . g . the word 'red' shown in green versus red ) .", "Such incongruent inputs lead to longer reaction times , attributed to weaker signals ( color processing ) that must be emphasized over the automatic processing of word information ( Stroop , 1935 ) .", "The Stroop task is frequently used in cognitive neuroscience and clinical psychology and forms the foundation for theories of cognitive control .", "Neurophysiological , neuroimaging , and lesion studies have ascribed a critical role in cognitive control to networks within frontal cortex ( Miller , 2000; Miller and Cohen , 2001 ) , yet the neural circuit dynamics and mechanisms responsible for orchestrating control processes remain poorly understood .", "Lesion studies ( Cohen and Servan-Schreiber , 1992; Perrett , 1974 ) , human neuroimaging measurements ( Egner and Hirsch , 2005a; MacDonald , 2000 ) , and macaque single unit recordings ( Johnston et al . , 2007 ) implicate the dorsolateral prefrontal cortex ( dlPFC ) in providing top-down signals to bias processing in favor of the task-relevant stimuli ( Botvinick et al . , 2001; Miller and Cohen , 2001 ) .", "The medial frontal cortex ( mFC ) also participates in cognitive control , possibly in a conflict monitoring capacity ( Botvinick et al . , 2001; Ridderinkhof et al . , 2004; Rushworth et al . , 2004 ) .", "Recordings and lesions studies in the macaque anterior cingulate cortex ( ACC ) ( Ito et al . , 2003; Nakamura et al . , 2005 ) suggest that ACC neurons are principally involved in monitoring for errors and making between-trial adjustments ( Brown and Braver , 2005; Ito et al . , 2003; Johnston et al . , 2007; Rothé et al . , 2011 ) —an idea that has received support by a recent study in the human ACC ( Sheth et al . , 2012 ) .", "Recent work has also demonstrated that the supplementary motor area and the medial frontal cortex play an important role in monitoring for errors ( Bonini et al . , 2014 ) .", "An alternative and influential theoretical framework posits that the ACC monitors for potential conflicts and subsequently directs the dlPFC to engage control processes ( Botvinick et al . , 2001; Shenhav et al . , 2013 ) .", "Several human neuroimaging studies are consistent with this notion ( Botvinick et al . , 1999; Kerns , 2006; Kerns et al . , 2004; MacDonald , 2000 ) but the relative contributions of dlPFC , mFC , and ACC to cognitive control remain a matter of debate ( Aarts et al . , 2008; Cole et al . , 2009; Fellows and Farah , 2005; Mansouri et al . , 2007; Milham and Banich , 2005; Milham et al . , 2003; Rushworth et al . , 2004 ) .", "Previously , some neuroimaging studies have suggested that these frontal cortex regions can be differentiated based on the presence or absence of conflict signals ( MacDonald , 2000 ) .", "The challenge in dissociating the relative roles of these regions during Stroop-like tasks is that increased task difficulty recruits a host of executive functions ( attention , decision-making , uncertainty , cognitive control ) .", "These functions are associated with neural activity spanning tens to hundreds of milliseconds and the underlying dynamics are difficult to untangle with the low temporal resolution of existing neuroimaging techniques ( Shenhav et al . , 2013 ) .", "Human single neuron studies provide millisecond resolution but have focused on individual regions ( Sheth et al . , 2012 ) .", "We took advantage of the high spatiotemporal resolution of intracranial recordings in human epilepsy patients and the ability to record simultaneously from multiple regions to directly investigate the dynamics of conflict responses during cognitive control .", "We hypothesized that subregions of frontal cortex could be differentiated based on the temporal profile of their conflict responses .", "We recorded intracranial field potentials from 1397 electrodes in 15 subjects while they performed the Stroop task or a variation in which they were asked to read the word instead of focusing on its color .", "We observed conflict-selective activity throughout several regions in frontal cortex: ACC , mFC , dlPFC , and also orbitofrontal cortex ( OFC ) .", "Several analyses link these signals to cognitive control .", "Neural responses increased for incongruent compared to congruent trials , and these signals correlated with behavioral reaction time , depended on the task , and exhibited adaptation over trials .", "We compared pairs of simultaneously recorded electrodes to disassociate these different regions based on the timing of these conflict responses rather than their presence or absence .", "Conflict responses emerged first in the ACC and subsequently emerged in dlPFC and mFC and finally in OFC .", "These observations propose a plausible flow of signals underlying cognitive control ." ], [ "We focused on 469 electrodes located in areas within frontal lobe which have been previously implicated in executive function: medial frontal cortex ( mFC , n = 111 ) , orbitofrontal cortex ( OFC , n = 156 ) , dorsolateral prefrontal cortex ( dlPFC , n = 168 ) and the anterior cingulate cortex ( ACC , n = 34 ) .", "We applied a non-parametric analysis of variance ( ANOVA ) to measure whether and when the physiological responses differed between congruent and incongruent trials .", "An electrode was considered conflict-selective if the F-statistic was greater than a significance threshold computed by a permutation test with P = 0 . 001 for 50 consecutive milliseconds ( Materials and methods ) .", "The latency was defined as the first time of this threshold-crossing .", "Figure 2 shows an example electrode from the left Anterior Cingulate Cortex that responded differentially between congruent and incongruent trials during the Stroop task .", "These signals were better aligned to the speech onset than to the stimulus onset , as shown in the response-aligned view ( compare Figure 2A–C with Figure 2D–F ) .", "During the Stroop task , the response-aligned signals were significantly stronger for the incongruent ( brown ) trials compared to the congruent ( black ) trials ( Figure 2D , P < 10–5 , ANOVA ) , and were invariant to the particular word/color combinations ( Figure 2G ) .", "Incongruent trials could be discriminated from congruent trials at a latency of 669 ± 31 ms ( mean ± s . e . m . ) before the onset of the response ( Figure 2D ) .", "This conflict response was also specific to the Stroop task; there was a significant interaction between congruency and task ( F = 13 . 5 , P = 0 . 007 , ANOVA ) .", "The same stimuli did not elicit differential activity during the Reading task ( Figure 2F ) .", "We assessed the correlation between the neural signal strength and behavioral reaction times in single trials .", "The maximal gamma power during each incongruent trial ( using the average gamma power yielded similar results ) was positively correlated with the behavioral reaction times ( Figure 2H , ρ = 0 . 25 , P = 0 . 02 ) . 10 . 7554/eLife . 12352 . 006Figure 2 . Example electrode in left Anterior Cingulate Cortex .", "( A ) Average gamma power signals aligned to the stimulus onset from an electrode during the Stroop task , for congruent ( black ) or incongruent ( brown ) stimuli .", "For display purposes only , we z-scored the gamma power by subtracting the average and dividing by the standard deviation of power during the baseline period ( 500 ms prior to stimulus onset ) .", "Shaded areas indicate s . e . m . The total number of trials for each condition is indicated in the upper right .", "( B ) Single-trial data for congruent ( left ) and incongruent ( right ) trials .", "Each row is a trial , and the color indicates the z-scored gamma power ( color scale on upper right ) .", "Trials are sorted by behavioral response time ( black line ) .", "( C ) Same as ( A ) , but showing data from the Reading task .", "( D-F )", "Same as in A-C , but aligning the data to behavioral response time .", "Gamma power was better aligned to the behavioral response , and was stronger for incongruent compared to congruent trials .", "The dashed line indicates the response-aligned latency , defined as the first time point at which incongruent and congruent trials can be discriminated .", "( G ) Signals elicited by each of the 9 possible stimulus combinations .", "( H ) There was a correlation between the maximal z-scored gamma power and behavioral reaction times during incongruent trials ( Pearson correlation coefficient = 0 . 25 , P = 0 . 02 , permutation test ) .", "Each point in this plot represents a single trial . DOI: http://dx . doi . org/10 . 7554/eLife . 12352 . 00610 . 7554/eLife . 12352 . 007Figure 2—source data 1 . Conflict-selective electrode data . DOI: http://dx . doi . org/10 . 7554/eLife . 12352 . 00710 . 7554/eLife . 12352 . 008Figure 2—figure supplement 1 . Example conflict-selective electrode in the right dorsolateral Prefrontal Cortex . Here we show a different conflict selective electrode , located in the dlPFC ( format as in Figure 2 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 12352 . 00810 . 7554/eLife . 12352 . 009Figure 2—figure supplement 2 . Example conflict-selective electrode in the Orbitofrontal Cortex comparing responses in the Theta and Gamma Bands .", "( A-F )", "Responses in the theta power frequency band , z-scored .", "Same format as Figure 2—figure supplement", "1 . ( G-L ) Responses in the gamma power frequency band , z-scored .", "Same format as Figure 2—figure supplement 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 12352 . 009 Any differences between congruent and incongruent trials in the stimulus-aligned analyses can be confounded by the reaction time differences; therefore , we focus subsequent analyses on the response-aligned signals .", "More example electrodes are shown in Figure 2—figure supplement 1 ( dlPFC ) and Figure 2—figure supplement 2 ( OFC ) .", "Using the aforementioned criteria , we identified n = 51 conflict selective frontal cortex electrodes during the Stroop task , with contributions from 13 subjects ( Supplementary files 2 and 3 ) .", "These electrodes were distributed throughout different subregions within frontal cortex ( Figure 3A ) .", "To evaluate whether random variation in the signals could give rise to apparent conflict-selective electrodes , we randomly shuffled the congruent/incongruent trial labels 10 , 000 times and applied the same statistical criteria ( Materials and methods ) .", "Across our population , we found n = 4 . 4 ± 0 . 03 false positive electrodes ( mean ± s . e . m . , out of 469 electrodes ) , which corresponds to a false discovery rate ( FDR ) of q = 0 . 01 , which is significantly less than our observation of n = 51 electrodes .", "The number of conflict-selective electrodes within each subregion was significantly greater than expected by chance ( Figure 3B , P < 0 . 01 , all regions ) .", "We repeated the analyses during the Reading task .", "In contrast with the Stroop task , we only observed n = 3 conflict-selective frontal cortex electrodes during the Reading task ( out of 469 electrodes ) , a number that is within the false positive rate . 10 . 7554/eLife . 12352 . 010Figure 3 . Electrode locations .", "( A ) Location of conflict-selective electrodes ( black/gray ) shown on a reference brain , with each region colored ( Materials and methods ) .", "Electrodes from the right hemisphere were mapped to the left hemisphere for display purposes .", "For more detail , see Supplementary file", "2 . ( B ) Percent of total electrodes in each region that were selective for conflict .", "Chance levels were computed using a permutation test ( black line ) .", "The number of observed electrodes was significantly above chance for all regions ( P < 0 . 01 , permutation test , Materials and methods ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 12352 . 01010 . 7554/eLife . 12352 . 011Figure 3—source data 1 . Population gamma-power data . DOI: http://dx . doi . org/10 . 7554/eLife . 12352 . 011 To account for within-subject and across-subject variation , we used a multilevel model ( Aarts et al . , 2014 ) to conduct a group analysis of the physiological responses , with electrodes nested within subjects ( Materials and methods ) .", "Across the population , we observed a significant interaction between the factors congruency and task on the gamma power ( χ2=9 . 2 , P = 0 . 002 ) .", "Consistent with the single electrode examples , gamma power was greater for incongruent compared to congruent trials , but only during the Stroop task ( Figure 4A , Stroop: P < 10–3 , Reading: P = 0 . 56 ) .", "We computed the average response in each region ( Figure 4B ) .", "Each electrode’s response was normalized by dividing the power during incongruent trials by the power in congruent trials ( dividing the brown curve by the black curve in Figure 2 ) , computing the logarithm and finally pooling within each region .", "The pooled responses in the OFC are visually less compelling ( Figure 4B , bottom right subplot ) due to the heterogeneity in the latency of the individual electrodes but the responses in the OFC were as vigorous as the ones in other areas ( e . g . Figure 2—figure supplement 2 ) .", "Similar conclusions were reached when plotting the pooled responses aligned to stimulus onset ( Figure 4—figure supplement 3 ) . 10 . 7554/eLife . 12352 . 012Figure 4 . Gamma power in frontal cortex correlates with behavior .", "( A ) Distribution of gamma power log-ratio ( Incongruent/Congruent ) for the Stroop task ( blue ) and Reading task ( green ) .", "Bin size = 0 . 05 .", "Gamma power showed a significant interaction between Congruency and Task ( P = 0 . 002 , multilevel model , Materials and methods ) .", "Power was larger for incongruent versus congruent trials during the Stroop task ( P < 0 . 001 , n = 51 frontal cortex electrodes ) but not during the Reading task ( green , P = 0 . 56 ) .", "The statistical analyses directly compare the gamma power , we show the log-ratios here for display purposes only .", "( B ) Normalized gamma power log-ratio averaged across electrodes from each of the four different frontal cortex regions during the Stroop task .", "We divided the power during incongruent trials by the power during congruent trials , then computed the log and finally averaged across electrodes .", "Data are aligned to the behavioral response onset ( t=0 ) .", "( C ) Distribution of Pearson correlation coefficients between the maximal gamma power and behavioral reaction time during incongruent trials for n = 51 frontal cortex electrodes .", "These correlations were significantly positive ( P < 10–5 , sign-rank test ) .", "Bin size = 0 . 1 .", "( D ) For incongruent trials , there was a significant interaction between trial history and task ( P = 0 . 03 , multilevel model ) .", "Gamma power was larger for incongruent trials preceded by congruent trials ( cI ) compared to incongruent trials preceded by incongruent trials ( iI ) , particularly during the Stroop task ( blue , P = 0 . 001 ) , compared to the Reading task ( green , P = 0 . 72 ) .", "Data beyond the range of the x-axis are shown in the first or last bins .", "( E ) For congruent trials , there was no interaction between trial history and task ( P = 0 . 17 , multilevel model ) .", "Gamma power was similar in congruent trials preceded by incongruent trials ( iC ) compared to congruent trials preceded by congruent trials ( cC ) during the Stroop task ( blue , P = 0 . 16 ) and during the Reading task ( green , P = 0 . 19 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 12352 . 01210 . 7554/eLife . 12352 . 013Figure 4—source data 1 . Population gamma-power data . DOI: http://dx . doi . org/10 . 7554/eLife . 12352 . 01310 . 7554/eLife . 12352 . 014Figure 4—figure supplement 1 . Theta and Beta band population results .", "( A ) Distribution of theta power log-ratio ( Incongruent/Congruent ) for the Stroop task ( blue ) and Reading task ( green ) .", "Bin size = 0 . 05 .", "P values in black denote interaction statistics whereas P values in blue and green denote the statistics for the Stroop and Reading tasks respectively .", "As discussed in Figure 4 , the average log-ratios are presented here for display purposes only and the statistical tests are based on the raw power values .", "( B ) Distribution of the gamma power log-ratio between incongruent trials preceded by congruent trials ( cI ) compared to incongruent trials preceded by incongruent trials ( iI ) .", "( C ) Distribution of the gamma power log-ratio between congruent trials preceded by incongruent trials ( iC ) compared to congruent trials preceded by congruent trials ( cC ) .", "( D-F )", "Same as ( A-C ) , but for power in the beta band . DOI: http://dx . doi . org/10 . 7554/eLife . 12352 . 01410 . 7554/eLife . 12352 . 015Figure 4—figure supplement 2 . Cross-frequency coupling analyses . For the anterior cingulate cortex electrode in Figure 2: ( A ) Phase-amplitude distribution during the Stroop task for the example electrode shown in Figure 2 ( see Materials and methods for calculation of cross-frequency coupling ) .", "( B ) The observed Modulation Index ( MI , black arrow ) is significantly greater than the surrogate distribution generated by adding a lag between the phase and amplitude measurements , demonstrating that the amplitude of the gamma band is strongly coupled to the phase of the theta band .", "( C ) During the Stroop task , the difference in Modulation Index between congruent and incongruent trials ( black arrow ) was not significantly different from 0 ( P = 0 . 61 ) .", "The null distribution ( gray bars ) was generated by randomly permuting the congruent and incongruent labels .", "Across the population of electrodes: ( D ) The percent of total electrodes in each region ( Frontal cortex or non-frontal cortex ) that had significant phase-amplitude coupling .", "Shown on the right is the percentage of the n = 51 conflict selective electrodes that showed significant coupling .", "( E ) The MI of congruent compared to incongruent trials for all Frontal cortex electrodes ( gray dots ) and the subset that were conflict-selective in the gamma band ( blue dots ) .", "For both groups , there was no significant difference in the MI between congruent and incongruent trials ( Frontal Cortex , P = 0 . 45; Conflict-selective , P = 0 . 52; signed-rank test ) .", "For this comparison , the number of congruent and incongruent trials was equalized before computing the MI . DOI: http://dx . doi . org/10 . 7554/eLife . 12352 . 01510 . 7554/eLife . 12352 . 016Figure 4—figure supplement 3 . Stimulus-aligned population averages . Same as in Figure 4B , but data are aligned to the stimulus response onset ( t=0 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 12352 . 016 Several lines of evidence demonstrate a link between the neural signals described in the previous section and cognitive control: the neural signals correlated with reaction times , showed behavioral adaptation , and demonstrated error monitoring .", "As shown in previous studies , there was a wide distribution of behavioral reaction times ( Figure 1B ) .", "Consistent with the example electrode in Figure 2 , behavioral reaction times across the population correlated with the strength of the physiological signals , even after controlling for trial history ( Figure 4C , P < 10–5 , sign-rank test ) .", "The strength of these neural signals also revealed a neural correlate of the behavioral Gratton effect documented in Figure 1D: gamma power was greater in cI compared to iI trials ( Figure 4D ) .", "Using the aforementioned multilevel model , we found a significant interaction between trial history ( cI or iI ) and task ( χ2=4 . 4 , P = 0 . 03 ) .", "This Gratton effect was stronger in the Stroop task ( P < 0 . 001 ) than in the Reading task ( P = 0 . 72 ) .", "These differences were not observed for cC versus iC trials , where the interaction was not significant ( χ2=1 . 9 , P = 0 . 17 ) ( Figure 4E ) .", "This analysis was performed after removing stimulus repetition trials .", "The Gratton effect was present in all four frontal regions and there were no statistically significant differences in the strength of the effect across regions ( F = 0 . 25 , P = 0 . 86 , ANOVA ) .", "To control for reaction time effects on these comparisons , we ran an analysis of covariance ( ANCOVA ) to test for a main effect of trial history on the gamma power with the behavioral reaction time as a covariate ( Materials and methods ) .", "The neural Gratton effect during the Stroop task persisted under these controlled conditions ( P = 0 . 0002 , multilevel model ) .", "We also explicitly ruled out reaction time differences by subsampling to match the reaction time distribution between conditions , with similar results ( P = 0 . 01 , multilevel model ) .", "Together , these results suggest that the neural signals described here code for an internally perceived level of conflict that exhibits conflict adaptation and correlates with the across-trial variability in reaction times .", "The results presented above focus on the neural signals filtered within the gamma frequency band ( 70–120 Hz ) .", "We also examined the responses elicited in the broadband signals ( 1 to 100 Hz ) as well as in the theta , ( 4 to 8 Hz ) , beta , ( 9 to 30 Hz ) , and low gamma ( 30–70 Hz ) bands .", "No conflict selective responses were observed in the broadband signals or low gamma band .", "We found conflict-selective responses both in the theta and beta bands ( see example in Figure 2—figure supplement 2a–f ) .", "Across theta and beta frequency bands , we also observed a significant interaction between Congruency and Task ( theta: P < 10–5 , beta: P < 10–4 , multilevel model ) .", "Consistent with the results reported in the gamma frequency band , conflict responses in the theta and beta bands were more prominent during the Stroop task compared to the Reading task ( Figure 4—figure supplement 1 ) .", "In contrast to the results in the gamma band , power in the theta and beta bands decreased during incongruent trials .", "Furthermore , power in the theta and beta frequency bands was not correlated with reaction times ( theta: P = 0 . 43 , beta: P = 0 . 09 , sign-rank test ) .", "In addition to separately examining the responses in different frequency bands , an important aspect of encoding of cognitive information is the relationship between signals across frequencies .", "In particular , several studies have demonstrated that the amplitude of the gamma band is coupled to the phase of slower oscillations in the theta band ( Canolty et al . , 2006; Oehrn et al . , 2014; Tort et al . , 2008 ) .", "We therefore examined the degree of cross-frequency coupling between the signals in the gamma and theta bands ( Figure 4—figure supplement 2 ) .", "Consistent with previous studies , we found that 50% of the electrodes demonstrated significant theta-gamma coupling .", "However , the strength of this coupling was not different between congruent and incongruent trials across the population of conflict-selective electrodes ( P = 0 . 52 , sign-rank test ) .", "The conflict responses reported above are based on correct trials only .", "Yet , error monitoring has also been ascribed to frontal cortical circuits ( Bonini et al . , 2014; Shenhav et al . , 2013; Yeung et al . , 2004 ) .", "To investigate whether the same electrodes responding to conflict are also involved in successful error monitoring , we analyzed the neural signals during self-corrected trials .", "In these trials , subjects initially made an erroneous response and rapidly corrected themselves with the right answer .", "Given the high performance level of all subjects , the number of such trials is low .", "However , these trials are particularly interesting because we can be certain that there was successful error detection ( as opposed to error trials without any self-correction ) .", "An example self-corrected trial from the ACC electrode shown previously is illustrated in Figure 5A .", "The subject initially made an incorrect response ( green ) , which was rapidly followed with the correct response ( red ) .", "Increased gamma power was observed after onset of the erroneous response .", "In contrast , the following corrected behavioral response exhibited no such post-response signal .", "Additionally , these error-monitoring signals were not observed in correct incongruent trials ( Figure 2D ) , and were consistent across the n = 11 self-corrected trials for this subject ( Figure 5B , P = 0 . 001 , signed rank test ) .", "Another example electrode is shown in Figure 5C–D .", "There were only two subjects contributing n = 7 conflict-signaling electrodes that had a sufficient number of self-correction trials ( greater than five trials ) for this analysis .", "For each electrode , we compared the difference in neural signals during the one-second post-response window between the initial error and the following self-correction .", "Of those n = 7 electrodes , n = 5 electrodes showed evidence of error monitoring ( Figure 5E , P < 0 . 05 , sign-rank test ) .", "Although the number of electrodes and trials in this analysis is small , these results provide a direct correlate of error monitoring signals .", "Furthermore , these results highlight that the same electrodes that respond to conflict leading up to the behavioral response can also show post-response error monitoring . 10 . 7554/eLife . 12352 . 017Figure 5 . Responses during self-corrected error trials .", "( A ) An example self-correction trial from the ACC electrode in Figure 2 when the word Green colored in red was presented .", "The single trial gamma power is shown on top , with the speech waveform below .", "The dashed lines indicate the onset of the initially incorrect response ( 'green' ) and the following corrected response in bold ( 'no – red' ) .", "Note the increased gamma power after an error response .", "( B ) Average gamma power aligned to the onset of the initial error response ( blue ) and the onset of the corrected response ( black ) for n = 11 self-correction trials .", "Shaded areas indicate s . e . m . The post-response power was significantly greater after the error ( P = 0 . 001 , signed-rank test ) .", "( C-D )", "Same as ( A-B ) for another example electrode in the dorsolateral prefrontal cortex .", "The post-response power was significantly greater after the error response ( P = 0 . 002 , signed-rank test ) .", "( E ) Across the n = 7 electrodes with n = 10 or greater self-correction trials , the z-scored gamma power during the initial error response was larger than during the corrected response .", "Electrodes with significant differences ( P < 0 . 05 , signed-rank test ) are colored black .", "Letters mark the examples in ( A ) and ( C ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 12352 . 01710 . 7554/eLife . 12352 . 018Figure 5—source data 1 . Data for self-correcting trials . DOI: http://dx . doi . org/10 . 7554/eLife . 12352 . 018 We observed conflict-selective responses in the anterior cingulate cortex , medial frontal cortex , dorsolateral prefrontal cortex and orbitofrontal cortex .", "To examine the dynamics of cognitive control orchestrating the transformation of conflicting visual signals to motor outputs , we compared , across those four regional groups , the latencies relative to behavioral response onset at which the congruent and incongruent trials could be discriminated .", "Comparing latencies across regions is difficult , especially across subjects with varying reaction times .", "For a controlled and direct comparison , we restricted the analysis to compute the latency differences between pairs of simultaneously recorded electrodes .", "This within-subject pairwise analysis had increased power to examine the relative dynamics between frontal lobe areas ( Figure 6 ) .", "The relative latencies were significantly different across the regions ( P = 0 . 01 , permutation test , post-hoc testing was controlled for multiple comparisons using the Benjamin-Hochberg procedure , Materials and methods ) .", "Conflict responses in the ACC preceded those in all the other frontal lobe regions , followed 207 ± 40 ms later by dorsolateral prefrontal cortex and 388 ± 83 ms later by medial frontal cortex .", "Signals in orbitofrontal cortex emerged 319 ± 78 ms after dlPFC .", "This entire processing cascade took approximately 500 ms . For comparison , subjects’ behavioral reaction times to incongruent trials were 1105 ± 49 ms . The latency difference between ACC and dlPFC is based on 6 electrode pairs: one ACC electrode and six simultaneously recorded dlPFC electrodes .", "There was only one pair of simultaneous recordings between ACC and OFC and we do not report this value in Figure 6 .", "The other region comparisons have contributions from multiple electrodes in multiple subjects ( Supplementary file 3 ) .", "These results suggest a temporal hierarchy of cognitive control mechanisms culminating in speech onset . 10 . 7554/eLife . 12352 . 019Figure 6 . Latency Comparisons across regions . Latency differences between different regions computed from all pairs of simultaneously recorded electrodes .", "np denotes the number of electrode pairs .", "Because we only consider simultaneously recorded electrodes here , not all the electrodes modulated by conflict can be paired with any other electrode .", "Supplementary file 3 shows the number of electrodes modulated by conflict in each area and subject .", "There was only one electrode pair between ACC and OFC and therefore we do not show the latency difference between these two regions here .", "Significant latency differences ( P < 0 . 05 , permutation test , Materials and methods ) are shown in black , and non-significant differences in gray .", "ACC leads both mFC ( P = 0 . 001 ) and dlPFC ( P = 0 . 02 ) , with OFC following dlPFC ( P = 0 . 009 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 12352 . 01910 . 7554/eLife . 12352 . 020Figure 6—source data 1 . Data for region latency comparisons . DOI: http://dx . doi . org/10 . 7554/eLife . 12352 . 020" ], [ "We used intracranial field potentials to measure the dynamics of conflict responses across frontal cortex leading up to the behavioral response in the Stroop task .", "Previous physiological and functional neuroimaging studies have documented the involvement of multiple of these frontal cortex areas in the Stroop or similar tasks ( Botvinick et al . , 1999; Kolling et al . , 2012; MacDonald , 2000; Niendam et al . , 2012; Oehrn et al . , 2014; Sheth et al . , 2012 ) .", "The intracranial field potential recordings reported here show conflict-selective signals in ACC ( e . g . Figure 2 ) , dlPFC ( e . g . Figure 2—figure supplement 1 ) , mFC ( e . g . Figure 4B ) and OFC ( e . g . Figure 2—figure supplement 2 ) .", "The mFC and dlPFC have been previously implicated in cognitive control , and these structures are extensively connected to the rest of frontal cortex areas ( Ridderinkhof et al . , 2004 ) .", "The role of the OFC in cognitive control during Stroop-like tasks has not been reported previously , possibly because of technical challenges in neuroimaging near this area ( Weiskopf et al . , 2006 ) .", "We presented several lines of evidence that demonstrate that these conflict-selective physiological signals are relevant for behavior during the Stroop task .", "Longer behavioral reaction times were correlated with greater gamma power on a trial-by-trial basis during the Stroop task but not during the Reading task , even after accounting for trial history and for differences between congruent and incongruent stimuli ( Figure 2H , 4C ) .", "The same identical stimuli can elicit a range of behavioral reaction times and this internal degree of conflict can be captured , at least partly , by the strength of gamma power in frontal cortex in each trial .", "The neural correlates of behavioral adaptation ( Gratton effect [Gratton et al . , 1992] ) were observed in the ACC , consistent with prior studies based on human single neuron recordings ( Sheth et al . , 2012 ) , neuroimaging ( Botvinick et al . , 1999; Kerns , 2006 ) and also in accordance with the behavioral effects of ACC resection ( Sheth et al . , 2012 ) .", "Conflict responses throughout the other frontal cortex regions also demonstrated the neural Gratton effect , suggesting a more distributed network involved in across-trial adaptation than previously hypothesized .", "The physiological responses in these areas were stronger in cI trials ( incongruent trials that were preceded by congruent trials ) than iI trials ( Figure 4D ) .", "While the increased activity in cI trials compared to iI trials is consistent with neuroimaging studies ( Botvinick et al . , 1999 ) , single neuron recordings in a different Stroop-like task report the opposite relationship ( iI > cI ) ( Sheth et al . , 2012 ) .", "These differences point to potentially interesting distinctions between the activity of individual neurons and coarser population measures that warrant further investigation .", "Another discrepancy between neuroimaging studies and single unit recordings is the presence of conflict responses and error signals .", "Single unit recording in macaque ACC typically find error monitoring signals but not conflict-selective responses ( Cole et al . , 2009; Emeric et al . , 2010; Ito et al . , 2003; Taylor et al . , 2006 ) , see however ( Ebitz and Platt , 2015 ) , whereas human neuroimaging studies report both types of signals in ACC .", "There has been significant debate concerning whether action monitoring and conflict detection represent distinct processes ( Carter et al . , 1998; Carter et al . , 2000; Nee et al . , 2011; Swick and Turken , 2002 ) .", "Because both processes may co-occur on the same trials , high temporal resolution is required to disassociate the two computations .", "A recent human intracranial study has found error signals in supplementary motor area and medial frontal cortex ( Bonini et al . , 2014 ) , and a human single unit study reported conflict signals in ACC ( Sheth et al . , 2012 ) .", "The current work demonstrates the coexistence of both error signals and conflict signals .", "The analysis of the few self-correction trials in our data suggests that the same areas responsible for pre-behavioral conflict signals can also produce post-behavioral response error-monitoring signals ( Figure 5 ) .", "In addition , the relative timing of the conflict and error signals surrounding the neural responses confirms computational predictions based on a connectionist architecture to explain the mechanisms of conflict ( Yeung et al . , 2004 ) and scalp EEG studies ( Hughes and Yeung , 2011 ) .", "These results are consistent with computational models suggesting that these signals may represent a general error-likelihood prediction , of which conflict and error detection are special cases ( Brown and Braver , 2005 ) .", "It has been suggested that ACC and supplementary eye field neurons in macaque monkeys respond to specific stimulus and/or behavioral combinations but are not directly modulated by conflict ( Cole et al . , 2009; Nakamura et al . , 2005 ) .", "At the level of the intracranial field potentials reported here , the modulation of conflict trials observed in the four frontal cortex regions could not be ascribed to specific stimulus or behavioral responses ( e . g . Figure 2G ) and were also task dependent ( compare Figure 2A versus 2C ) .", "In these patients , we did not have access to single neuron responses and we therefore cannot rule out the possibility that individual neurons show distinct patterns of responses that are averaged out at the field potential level .", "Besides the high gamma band , we also observed conflict responses in the beta and theta bands , but not the low gamma band ( e . g . Figure 4—figure supplement 1 ) .", "Previous work has suggested differential roles for distinct oscillatory components of the local field potential ( Cavanagh and Frank , 2014; Kahana et al . , 2001; Ullsperger et al . , 2014; von Stein and Sarnthein , 2000 ) .", "There were clear differences in the type of information conveyed by distinct frequencies components .", "Lack of significant correlations with reaction time in the theta and beta bands suggests that the gamma band better captures the behavior .", "Additionally , conflict responses were characterized by increased power in the gamma band , but decreased power in the theta and beta bands ( Figure 4—figure supplement 1 ) .", "Previous scalp EEG recordings ( Cavanagh and Frank , 2014; Ullsperger et al . , 2014; van Driel et al . , 2015 ) have demonstrated that conflict and/or error trials elicit increased theta power , suggesting potentially interesting differences in how theta is captured across spatial scales .", "We also observed a decrease in beta power , which is consistent with previous studies that correlate frontal cortex activation with desynchronization in the beta band and increased synchronization in the gamma bands ( Crone et al . , 1998a; Crone et al . , 1998b ) .", "Differences across tasks , recording methods , and targeted regions should be interpreted with caution .", "The roles of different oscillatory components in neocortex are not clearly understood .", "One possibility is that lower frequency bands reflect the summed dendritic input of the nearby neural population ( Logothetis et al . , 2001; Mitzdorf , 1987 ) and can act as channels for communication ( Cavanagh and Frank , 2014 ) , whereas higher frequency bands represent the population spiking rate ( Buzsaki et al . , 2012; Ray and Maunsell , 2011 ) .", "Along these lines , we speculate that the theta desynchronization we observe could reflect a reduction of inputs , leading to inhibition of the prepotent but erroneous response .", "While we observed conflict responses throughout frontal cortex , the spatiotemporal resolution of our intracranial recordings allowed us to separate regions by the latency at which conflict-selective responses emerge with respect to speech onset .", "By comparing pairs of simultaneously recorded electrodes , we found that conflict responses in the ACC lead the dlPFC by ~200 ms . Medial frontal cortex is anatomically close and extensively connected to the ACC , and the two regions are often grouped together ( Cavanagh et al . , 2009; Ridderinkhof et al . , 2004 ) .", "Yet , conflict responses in the mFC trail the ACC by hundreds of milliseconds , suggesting an important distinction between the two regions ( Rushworth et al . , 2004 ) .", "The relative latency measurements place the OFC at the bottom of this cascade .", "The hierarchical cascade of processes described here is consistent with predictions from mechanistic models of cognitive control ( e . g . see Figure 2 in Shenhav et al , Neuron 2013 ) .", "In particular , stimulus related signals are evident along the ventral visual stream early on and feed onto frontal cortex , where we find that ACC activity precedes activity in other frontal regions , followed by dlPFC , and finally mFC , and OFC .", "Since the local field potential pools over many neurons , latency measures can be influenced by a variety of factors , such as the proportion of neurons selective for conflict and their laminar organization .", "Yet , at least in the ACC , the temporal profile of conflict responses we observed is similar to responses from human single unit recordings ( Sheth et al . , 2012 ) .", "The relatively long delays between regions are also particularly intriguing .", "There are monosynaptic connections that link these four regions within frontal cortex and yet , it takes 100–200 ms to detect the relative activation between these areas ( Figure 6 ) .", "Daily decisions require integration of different goals , contexts , input signals , and the consequences of the resulting actions .", "The current study provides initial steps to elucidate not only which brain areas participate in cognitive control on a trial-by-trial basis but also their relative interactions and differential roles .", "The relative latency measurements and correlations between neural activity and reaction time provide a framework to constrain theories of cognitive control , and propose a plausible flow of conflict responses through frontal cortex ." ], [ "Subjects were 15 patients ( 10 male , Ages 10–50 , Supplementary file", "1 ) with pharmacologically intractable epilepsy treated at Children’s Hospital Boston ( CHB ) , Johns Hopkins Medical Institution ( JHMI ) , Brigham and Women’s Hospital ( BWH ) , or Taipei Veterans General Hospital ( TVGH ) .", "These subjects were implanted with intracranial electrodes in frontal cortex for clinical purposes .", "Five other subjects participated in this task but they were excluded from the analyses because they did not have any electrodes in frontal cortex .", "All studies were approved by each hospital’s institutional review boards and were carried out with the subjects’ informed consent .", "Subjects were implanted with 2 mm diameter intracranial subdural electrodes ( Ad-Tech , Racine , WI , USA ) that were arranged into grids or strips with 1 cm separation .", "Electrode locations were determined by clinical considerations .", "There were 1397 electrodes ( 15 subjects ) .", "Sampling rates ranged from 256 Hz to 1000 Hz depending on the equipment at each institution: CHB ( XLTEK , Oakville , ON , Canada ) , BWH ( Bio-Logic , Knoxville , TN , USA ) , JHMI ( Nihon Kohden , Tokyo , Japan ) , and TVGH ( Natus , San Carlos , CA ) .", "All the data were collected during periods without any seizure events or immediately following any seizures .", "A schematic of the task is shown in Figure 1 .", "After 500 ms of fixation , subjects were presented with a word stimulus for 2 s .", "The stimulus presentation was 3 s in two subjects .", "Stimuli were one of three words ( Red , Blue , Green ) presented in the subjects’ primary language ( CHB , BWH , JHMI: English; TVGH: Mandarin ) either in red , blue , or green font color .", "Stimuli subtended approximately 5 degrees of visual angle and were centered on the screen .", "Trials were either congruent ( C ) , where the font color matched the word , or incongruent ( I ) , where the font color conflicted with the word .", "The order of congruent and incongruent trials was randomized .", "Approximately 40% of the trials were incongruent trials .", "Within congruent trials and within incongruent trials all color-word combinations were counter balanced and randomly interleaved .", "Subjects were asked to either name the color ( Stroop task ) or read the word ( Reading task ) within the time limit imposed by the stimulus presentation time .", "Each block contained 18 trials , and the two tasks were completed in separate blocks .", "Most subjects completed 18 blocks of the Stroop task and 9 blocks of the Reading task ( Supplementary file 1 ) .", "Audio was recorded using a microphone at 8192 Hz sampling rate .", "No correct/incorrect feedback was provided .", "Electrodes were localized by co-registering the preoperative magnetic resonance imaging ( MRI ) with the postoperative computer tomography ( CT ) ( Destrieux et al . , 2010; Liu et al . , 2009 ) .", "In 4 subjects without a postoperative CT , electrodes were localized using intraoperative photographs and preoperative MRI .", "For each subject , the brain surface was reconstructed from the MRI and then assigned to one of 75 regions by Freesurfer .", "Depth electrodes were assigned to either a subcortical structure or to gyri/sulci .", "We focused on those electrodes in four frontal cortex regions ( ACC: anterior and middle-anterior cingulate gyrus , mFC: superior frontal gyrus , dlPFC: middle frontal gyrus , and OFC: orbitofrontal gyrus ) .", "To determine the behavioral reaction time for each trial , the short-time energy was computed from the audio recordings .", "For an audio signal x ( t ) , the short-time energy E ( t ) is defined as: E ( t ) =∑m=0m=T[x ( m ) w ( t−m ) ]2 , where T is the length of the recording and w ( t ) is a 300-point Hamming window ( ~40 ms ) .", "Speech onset was defined as the first time when the energy crossed a threshold set as 1 standard deviation above the baseline .", "Only trials where the subject gave a single verbal response and the speech onset could be identified were considered correct trials .", "Unless otherwise noted , analyses in this manuscript used correct trials only .", "Electrodes with significant spectral noise were excluded from analysis ( n = 25 out of 1397 total electrodes ) .", "For each electrode , a notch filter was applied at 60 Hz , and the common average reference computed from all channels was subtracted .", "Power in the theta ( 4–8 Hz ) , beta ( 9–30 Hz ) , and high-gamma band ( 70–120 Hz ) was extracted using a moving window multi-taper Fourier transform ( Chronux toolbox [Mitra and Bokil , 2008] ) with a time-bandwidth product of five and seven tapers .", "The window size was 200 ms with 10 ms increments .", "In several figures , the gamma power was z-scored for display purposes ( see figure legends ) .", "To determine whether and when an electrode responded selectively to conflict , we used a sliding F-statistic procedure ( Liu et al . , 2009 ) .", "Electrodes with differential responses between congruent and incongruent trials were selected by computing the F-statistic , for each time bin , comparing the neural responses between congruent and incongruent trials .", "Electrodes were denoted as ‘conflict selective’ if ( 1 ) the F-statistic exceeded a significance threshold for 50 consecutive milliseconds , and ( 2 ) the average neural response exceeded one standard deviation above the baseline period at least once during the trial .", "A null distribution generated by randomly permuting the labels was used to set the significance threshold with P = 0 . 001 .", "The latency at which congruent and incongruent stimuli could be discriminated was defined as the first time of this threshold crossing .", "For the response-aligned view , only electrodes where the latency preceded the response were included in subsequent analysis .", "This selection process was independently performed for each electrode in both stimulus-aligned and response-aligned analyses , and separately for the Stroop and Reading task .", "We used a permutation test with 10 , 000 shuffles to obtain a false discovery rate for our selection process .", "The congruent/incongruent trial labels were randomized 10 , 000 times and we measured the average number of electrodes across our population that passed the selection procedure .", "For the selected electrodes obtained with the procedure described in the previous section , we performed a number of within-electrode analyses .", "We measured single-trial correlations with behavioral reaction times , assessed the significance of interactions and simple/main effects , and controlled for confounds in measuring the neural Gratton effect .", "To account for both within-subject and across-subject variance , statistical testing of the electrophysiological data was conducted with multilevel models ( Aarts et al . , 2014; Goldstein , 2011 ) ( also known as random effect models ) .", "Random factors included electrodes nested within subjects .", "Significance of interactions and/or main effects was assessed with a likelihood ratio test against a null model excluding that particular term .", "For comparison of latency across regions , we restricted our analyses to simultaneous measurements made within each subject .", "We computed the latency difference for each pair of simultaneously recorded electrodes from different regions .", "The F-statistic of this latency difference across the groups was compared against a null distribution generated by shuffling , within each subject , the region labels ( n = 10 , 000 shuffles ) .", "Post hoc testing used the Benjamin-Hochberg procedure to control for multiple comparisons .", "To measure cross-frequency coupling between the theta and gamma frequency bands , we used the Modulation Index ( MI ) defined previously ( Tort et al . , 2008 ) .", "Activity in the theta ( 4–8 Hz ) and high gamma ( 70–120 Hz ) bands was obtained with a zero-phase least-squares finite impulse response ( FIR ) filter .", "Instantaneous phase and amplitude was extracted with the Hilbert Transform .", "For the Stroop and Reading Task separately , the MI was computed as the Kullback-Leiber distance between the phase-amplitude histogram and a uniform distribution .", "For comparison between tasks , the number of trials was equalized .", "This MI was compared against a surrogate distribution generated by randomly lagging the time series across 1000 repetitions .", "Similar results were obtained with the measure defined in Canolty et al . ( Canolty et al . , 2006 ) .", "Results were also similar when a surrogate distribution was created by randomly pairing low-frequency phase with high-frequency power from different trials .", "To compare the strength of cross-frequency coupling between congruent and incongruent conditions , we computed the difference in MI between the two conditions while equalizing the trial count .", "This difference was compared against a null distribution generated by randomly shuffling the congruent and incongruent labels ." ] ]

[ "Rapid and flexible interpretation of conflicting sensory inputs in the context of current goals is a critical component of cognitive control that is orchestrated by frontal cortex .", "The relative roles of distinct subregions within frontal cortex are poorly understood .", "To examine the dynamics underlying cognitive control across frontal regions , we took advantage of the spatiotemporal resolution of intracranial recordings in epilepsy patients while subjects resolved color-word conflict .", "We observed differential activity preceding the behavioral responses to conflict trials throughout frontal cortex; this activity was correlated with behavioral reaction times .", "These signals emerged first in anterior cingulate cortex ( ACC ) before dorsolateral prefrontal cortex ( dlPFC ) , followed by medial frontal cortex ( mFC ) and then by orbitofrontal cortex ( OFC ) .", "These results disassociate the frontal subregions based on their dynamics , and suggest a temporal hierarchy for cognitive control in human cortex ." ]

[ "The brain adapts to control our behavior in different ways depending on the specific situation , which is particularly useful when deciding how to interpret conflicting sets of information .", "The 'Stroop task' is a classic demonstration of this process .", "In this task , individuals are shown words where the color and the meaning of the text conflict – for example , the word 'green' is written in blue .", "When asked what the color of the text is , individuals must suppress the instinct to read the word .", "This causes them to make more mistakes and take longer to decide on an answer than when they perform the same task using words that have no conflict ( for example , when “red” is written in red ) .", "Previous work has suggested that several regions within part of the brain called the frontal cortex play a role in this cognitive control process .", "However , the relative contributions of each of these regions , and the order in which they are activated , remain unclear .", "This is in part due to the fact that accurately measuring the electrical activity of the frontal cortex requires implanting electrodes into the brain .", "Tang et al . took advantage of a rare opportunity to record this activity from a group of patients who had electrodes implanted in their frontal cortex to treat epilepsy .", "The electrical signals recorded by these electrodes as the subjects performed the Stroop task revealed that four regions in the frontal cortex altered their activity during trials where the color and the meaning of a word conflicted .", "These responses corresponded with the subject’s reaction time , changed depending on the exact nature of the task , and even reflected the subjects’ errors .", "These responses arose at different times in different regions , allowing Tang et al . to suggest how signals flow through the frontal cortex during cognitive control .", "In the future it will be important to further understand how the regions of the frontal cortex identified by Tang et al . interact with each other and to establish their roles in cognitive control .", "These observations could then be used to produce a theoretical framework that describes how the brain adapts behavior to different circumstances ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "structural biology and molecular biophysics" ]

[ [ "Myosin 5a moves in a hand-over-hand fashion with individual heads moving by 74 nm and the center of mass by 37 nm for each step ( Vale , 2003; Sellers and Veigel , 2006; Hammer and Sellers , 2012 ) .", "A finely tuned kinetic sequence of ATP binding , hydrolysis , phosphate release and eventually ADP release by the enzymatic heads enables processive motion towards the ‘+’ end of actin filaments ( De La Cruz et al . , 1999; De La Cruz and Ostap , 2004; Rosenfeld and Sweeney , 2004; Sakamoto et al . , 2008; Forgacs et al . , 2008 ) .", "One of the most remarkable aspects of myosin 5a is the efficient conversion of chemical energy by the molecular-sized enzymatic heads into a translation of 74 nm along a 7 nm diameter actin filament in the presence of Brownian motion and a crowded cellular environment .", "Crystal structures of myosin 5a exist either with or without nucleotide , but only in the detached state ( Coureux et al . , 2003 , 2004 ) .", "Although these structures served as a model for the structural changes potentially induced by binding to actin , ( Volkmann et al . , 2005 ) the internally coupled rearrangement of the subdomains leading to ADP and phosphate release or dissociation from actin upon ATP binding could only be studied in silico ( Cecchini et al . , 2008 , 2010; Sweeney and Houdusse , 2010; Preller and Holmes , 2013 ) .", "The experimental challenge in revealing such structural dynamics has been largely of spatiotemporal origin , since they are likely on the Ångstrom scale and may be very transient .", "Similarly , there has been considerable interest in directly revealing the motion of the individual heads as myosin steps along actin in an attempt to unravel the origins of efficient motion on the nanoscale .", "Single molecule studies employing optical trapping ( Mehta et al . , 1999; Rief et al . , 2000 ) and fluorescence imaging ( Forkey et al . , 2003; Yildiz et al . , 2003; Snyder et al . , 2004; Warshaw et al . , 2005 ) have either reported periods of increased flexibility , ( Veigel et al . , 2002; Dunn and Spudich , 2007; Beausang et al . , 2013 ) or partitioning of the step into sub-events ( Veigel et al . , 2002; Uemura et al . , 2004; Cappello et al . , 2007; Sellers and Veigel , 2010 ) .", "All resulting models involve a forward aiming power stroke followed by a Brownian search mechanism ( Veigel et al . , 2002; Okada et al . , 2007; Shiroguchi and Kinosita , 2007; Karagiannis et al . , 2014 ) .", "The power stroke of the attached head contributes only partially to the step by moving the pivot point that facilitates Brownian rotation of the unbound head .", "The bias towards the next binding site is believed to be provided by the recovery stroke and its stability , but how unidirectional stepping is achieved in the presence of such a large and mostly random motion remains unclear ( Shiroguchi et al . , 2011 ) .", "The first passage time of the translocating head is expected to be on the order of 100 µs , ( Veigel et al . , 2002; Hinczewski et al . , 2013 ) although the head spends tens of ms in the unbound state given the maximum rates of ATP hydrolysis and binding to actin ( Veigel et al . , 2002; Dunn and Spudich , 2007; Beausang et al . , 2013 ) .", "Any technique aiming to visualize the structural dynamics of the motor domain or the motion of the unattached head would thus have to achieve simultaneous millisecond temporal and nanometer spatial precision .", "Optical trapping operates in this spatiotemporal regime , but it is not possible to monitor the motion of the unattached head without significant perturbation .", "Smaller labels affixed to the myosin motor domain or to calmodulin molecules on the lever arm , as available in fluorescence imaging , do not suffer from this limitation , but cannot provide sufficient localization precision on the millisecond time scale .", "We therefore designed an assay based on interferometric scattering microscopy ( iSCAT ) , ( Kukura et al . , 2009; Ortega-Arroyo and Kukura , 2012 ) a technique that has recently been shown to enable simultaneous high-speed and high-precision imaging of 20 nm diameter or smaller nanoscale scattering labels ( Andrecka et al . , 2013; Ortega Arroyo et al . , 2014 ) ." ], [ "We attached a 20 nm gold label functioning as an efficient light scatterer to the N-terminus of the myosin 5 head and tracked its motion as the motor travels along actin filaments ( Figure 1A , inset ) .", "From the centre of mass of the signal produced by the label , we determined the position of the head as a function of time revealing discrete 74 nm steps at 1000 frames/s imaging speed with ∼4 nm positional precision ( Figure 1A , Figure 1—figure supplement 1A , Video 1 ) .", "We verified that the addition of the 20 nm label did not interfere with the mechano–chemical cycle of myosin 5a by characterizing the speed of movement at different ATP concentrations .", "Our results compare well with those from a series of other single molecule studies where the molecule was labeled on the lever arm , the tail , or attached to a surface ( Figure 1—figure supplement 1B ) . 10 . 7554/eLife . 05413 . 003Figure 1 . High-speed nanometric tracking of myosin 5 with interferometric scattering ( iSCAT ) microscopy .", "( A ) Distance traveled as a function of time for a single myosin 5 molecule biotinylated at the N-terminus and labeled with a 20 nm streptavidin-functionalized gold particle .", "The lateral localisation precision , σ , defined as the standard deviation of the positional fluctuations of the label while bound to actin is given above each of the actin-attached periods .", "Inset: schematic of gold-labeled myosin 5 stepping along actin .", "( B ) Corresponding 2D-trajectory with the arrow indicating the direction of movement .", "( C–E )", "Close-up of the transient states indicated in A and B . ATP concentration: 10 μM .", "Scale bar: 50 nm .", "Imaging speed: 1000 frames/s ( corresponding video: Video 1 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 05413 . 00310 . 7554/eLife . 05413 . 004Figure 1—figure supplement 1 . Activity of myosin 5 labeled with 20 nm gold nanoparticles at the N-terminus .", "( A ) Sample trajectories at [ATP] = 10 µM and at 1000 frames/s .", "Arrow indicates direction of motion .", "Scale bar: 50 nm .", "( B ) Comparison of myosin 5 activity recorded in this work with previous single molecule studies .", "Black circles: this study .", "Symbols are defined as: purple rectangle , ( Mehta et al . , 1999 ) grey squares , ( Forkey et al . , 2003 ) dark blue square , ( Warshaw et al . , 2005 ) orange triangle , ( Komori et al . , 2007 ) pink circle , ( Ohmachi et al . , 2012 ) red open squares , ( Ortega Arroyo et al . , 2014 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 05413 . 00410 . 7554/eLife . 05413 . 005Figure 1—figure supplement 2 . Detection of the transient state with a molecular sized fluorescent label .", "( A ) Representative distance time series of a myosin 5 molecule labelled with an atto-647N/streptavidin conjugate at the N-terminus and tracked using single molecule total internal fluorescence microscopy .", "( B ) Corresponding 2D trajectory .", "( C ) Additional traces exhibiting the transient state marked by arrows .", "ATP concentration: 10 μM .", "Scale bar: 50 nm .", "Imaging speed: 100 frames/s . DOI: http://dx . doi . org/10 . 7554/eLife . 05413 . 00510 . 7554/eLife . 05413 . 006Figure 1—figure supplement 3 . Rare transient unbinding events for the leading head of myosin 5 . ( A ) Representative time trace segment of an event in which the leading head detaches from the actin filament and exhibits the intermediate state behaviour reported in the main text .", "( B ) Corresponding 2D—trajectory of the segment highlighted in A . ATP concentration: 1 μM .", "Scale bar: 50 nm .", "Imaging speed: 1000 frames/s . DOI: http://dx . doi . org/10 . 7554/eLife . 05413 . 00610 . 7554/eLife . 05413 . 007Video 1 . This file contains the iSCAT video from which the trajectory shown in Figure 1 of the main manuscript was obtained . The left panel presents the raw video format after referencing with the flat field image .", "The flat field accounts for illumination inhomogeneities and was generated as described in the Materials and Methods section .", "The right panel shows the static background subtracted video format .", "The background includes the average of 100 frames before the particle binds and removes the constant features which are part of the sample ( a . g . actin , glass roughness ) by simple subtraction ( Ortega Arroyo et al . , 2014 ) .", "The video was taken at 1000 frames/s and at a magnification of 31 . 8 nm/pixel .", "The corresponding field of view shown is 1 . 30 × 1 . 30 μm2 and corresponds to a total recording time of 1 . 3 s .", "The video playback speed is 1000 frames/s . DOI: http://dx . doi . org/10 . 7554/eLife . 05413 . 007 In addition to the expected 74 nm steps , we observed periods of increased positional fluctuations between detachment and reattachment of the labeled head , previously interpreted as a signature of Brownian search ( Dunn and Spudich , 2007 ) .", "Our effective lateral localization precision of 4 nm at 1000 frames/s , however , enabled us to visualize the motion of the unbound head precisely and revealed a transient state with a center of mass just over half way between the two binding sites and offset by 40 nm perpendicular to the actin filament ( Figure 1B ) .", "From this position , the myosin head repeatedly moved back and forth between the next actin binding site and the transient state position ( Figure 1C , D ) .", "The same behavior sometimes occurred just before myosin detached from the filament at the end of a trajectory ( Figure 1E ) , although detachment of myosin with the labeled head bound was equally likely .", "On the rare occasions that the labelled leading head of myosin detached and reattached , it occupied a similar position in space to a translocating head ( Figure 1—figure supplement 3 ) , suggesting that the transient state represents a true potential minimum of the one head bound state of myosin .", "In addition to providing nanometer precise information on the lateral position of the head through the center of mass of the signal , the iSCAT signal magnitude is very sensitive to the label-to-surface distance allowing for overall nanometric localization in three-dimensions ( Krishnan et al . , 2010 ) .", "This property arises from the interferometric nature of the technique , which scales the iSCAT signal as sinΦ , where Φ is the phase difference between scattered and reflected light fields .", "As the label moves perpendicular to the sample plane , the optical path length and with it the phase difference between the two fields changes by the following relation Φ = 4πηz/λ ( Figure 2A , inset ) .", "A complete signal inversion occurs for a displacement of z = λ/4η = 84 nm , where λ is the illumination wavelength ( 445 nm ) and η the refractive index of the medium ( 1 . 33 ) .", "Thus , variations in the axial distance of the motor domain with respect to the filament bound to the sample surface lead to changes in the scattering contrast .", "We often observed these changes in contrast between the actin bound and unbound states ( Figure 2A ) and obtained an average change in iSCAT signal during the step ( Figure 2B ) .", "The drop of 3 . 5% in iSCAT contrast ( ∼40% of the total signal ) suggests that the myosin head lifts on average by 24 ± 10 nm from its actin bound position .", "Although the precision of the measurement was in principle higher , we could not accurately determine the additional phase contributions to the interferometric signal on an individual label basis , which are required for a robust calibration . 10 . 7554/eLife . 05413 . 008Figure 2 . Three-dimensional interferometric tracking of the myosin head .", "( A ) Distance trace ( upper panel ) with the simultaneously recorded iSCAT contrast ( lower panel ) .", "The red subset of the traces corresponds to the unbound head state .", "Inset: schematic illustrating how the difference in optical path difference for the bound ( zbound ) and unbound ( zunbound ) state leads to changes in iSCAT contrast caused by the interference of the reflected ( Ereflected ) and scattered ( Escattered ) electric fields .", "( B ) Normalized histogram of the average iSCAT contrast while the head is in the transient state ( red , N = 329 ) or bound to actin ( black , N = 303 ) .", "ATP concentration: 10 μM .", "Imaging speed: 1000 frames/s . DOI: http://dx . doi . org/10 . 7554/eLife . 05413 . 00810 . 7554/eLife . 05413 . 009Figure 2—figure supplement 1 . Dual colour iSCAT imaging of myosin 5 . ( A ) 2D-trajectory of myosin 5 stepping along actin recorded simultaneously with 445 nm ( blue trace ) and 635 nm ( red trace ) illumination .", "Although the latter channel exhibits higher noise due to a lower scattering signal , the transient state is evident in every step even though much of the data are lost in the trace recorded at 445 nm due to the major drop in scattering intensity .", "( B ) Schematic of myosin stepping along actin with different azimuthal orientations relative to the actin filament .", "( C ) Corresponding iSCAT contrast as a function of time .", "Although the contrast is large in the blue channel , it drops much more significantly during population of the transient state .", "( D–F )", "Zoom of the contrast behaviour during the transient state from ( C ) .", "ATP concentration: 10 μM .", "Scale bar: 50 nm .", "Imaging speed: 1000 frames/s . DOI: http://dx . doi . org/10 . 7554/eLife . 05413 . 009 We occasionally encountered tracks where the transient state could not be localized because the iSCAT contrast dropped to zero .", "We interpret these cases as corresponding to molecules oriented parallel to the glass surface based on simultaneous iSCAT measurements using blue ( 445 nm ) and red ( 635 nm ) illumination .", "The particle remained visible in the red channel while it disappeared in the blue .", "At the same time , the off-axis component of the transient state was much smaller compared to those trajectories when the transient state remained visible in both channels , which is most consistent with a parallel bound molecule lifting the detached head up by ∼40 nm ( Figure 2—figure supplement 1B ) .", "To investigate whether subsequent steps occur on the same or opposite sides of the actin filament , commonly referred to as symmetric and asymmetric hand-over-hand stepping , ( Hua et al . , 2002 ) we labeled the two heads of a single molecule differently , one with a quantum dot and the other with a 20 nm gold particle ( Figure 3 , top ) .", "Simultaneous detection of quantum dot fluorescence and iSCAT signal was only possible with a longer wavelength scattering beam which is only weakly absorbed by the quantum dot , thereby avoiding excessive blinking and bleaching ( 660 vs 445 nm ) .", "In addition , the imaging speed was lowered to 500 frames/s to ensure <10 nm localization precision in the fluorescence channel while imaging over several seconds .", "We tracked both the scattering from the gold particle of one head ( upper traces in Figure", "3 ) and the fluorescence of the quantum dot-labeled head ( lower traces in Figure 3 ) .", "In all recorded trajectories of doubly-labeled molecules that exhibited clear transient states in both the fluorescence and scattering channels , the transient states for both heads appeared on the same side of the actin filament ( Figure 3—figure supplement 1 ) . 10 . 7554/eLife . 05413 . 010Figure 3 . Simultaneous scattering and fluorescence tracking of a single myosin 5a . One head was labeled with a 20 nm streptavidin-functionalized gold particle ( red ) and the other with a fluorescent quantum dot ( blue ) .", "Reported values correspond to the standard deviation σ in the position of the bound state ( nm ) and the shaded regions encompass an area of 3σ .", "The traces are colored according to the label colors in the inset .", "ATP concentration: 10 μM .", "Scale bar: 100 nm .", "Imaging speed: 500 frames/s . DOI: http://dx . doi . org/10 . 7554/eLife . 05413 . 01010 . 7554/eLife . 05413 . 011Figure 3—figure supplement 1 . Additional traces of simultaneous scattering and fluorescence tracking of a single myosin 5a . One head was labelled with a 20 nm streptavidin-functionalized gold particle ( red ) and the other with a fluorescent quantum dot ( blue ) .", "Reported values correspond to the standard deviation σ in the position of the bound state ( nm ) and the shaded regions encompass an area of 3σ .", "The traces are colored according to the label colors in the inset .", "ATP concentration: 10 μM .", "Scale bar: 100 nm .", "Imaging speed: 100 frames/s . DOI: http://dx . doi . org/10 . 7554/eLife . 05413 . 011 Close inspection of the time traces occasionally revealed what appeared to be small backwards movements of the attached head , such as the transition starting at 480 ms in the trace of Figure 1A .", "Since our localisation precision at 1000 frames/s is limited by the intrinsic motion of the label about its attachment point to the motor domain and diffusion of background scatterers , we repeated the tracking assay at 100 frames/s .", "At this imaging speed , we could frequently observe a transition between two distinct states during each 74 nm step in both an x–y projection ( Figure 4A ) and the time trace ( Figure 4B ) .", "As shown in the lateral trajectory , the transition between these states ( AB transition ) involves a small , <10 nm , off-axis backward motion of the gold particle attached to the bound head .", "As a result , the overall step size appears larger than the expected 74 nm step ( Figure 4—figure supplement 1 ) . 10 . 7554/eLife . 05413 . 012Figure 4 . Two states of the motor domain during myosin movement .", "( A ) Sample 2D trajectories for a 20 nm gold particle immobilised on the surface and attached to the N-terminus of myosin 5a .", "The arrow indicates the direction of myosin movement .", "The trace shown is a fragment of the trajectory obtained from Video 2 .", "( B ) The same trajectory depicted as distance travelled vs time .", "Two distinct states ( A and B ) of the bound head are evident ( black arrows ) .", "Standard deviations ( σ ) are given to compare both fixed ( blue ) and myosin bound particles ( black ) .", "( C ) Corresponding iSCAT contrast time trace for the trajectories in ( B ) .", "A brief reduction in iSCAT contrast coincides with 74 nm steps taken by the labeled head ( red vertical lines ) .", "The behaviour of a non-specifically surface-bound 20 nm gold particle that is completely immobilized is shown for comparison ( blue traces in upper portion of the graphs in panels B and C ) .", "Repeated localization of the particle throughout suggests a nominal sub-nm lateral localization precision and a constant scattering contrast .", "ATP concentration: 10 μM .", "Scale bar: 20 nm .", "Imaging speed: 100 frames/s . DOI: http://dx . doi . org/10 . 7554/eLife . 05413 . 01210 . 7554/eLife . 05413 . 013Figure 4—figure supplement 1 . ( A ) Step size histograms for post to pre-power stroke and post to post power stroke states . Data was collected at 100 Hz and 10 μM ATP , using 20 nm diameter gold nanoparticles .", "Number of steps analysed: 554 .", "( B ) The positional fluctuations of the A and B states presented as histograms of lateral localization precision , σ , show no measurable difference in mechanical stability of the two states . DOI: http://dx . doi . org/10 . 7554/eLife . 05413 . 01310 . 7554/eLife . 05413 . 014Video 2 . This file contains a full video from which the trajectory shown in Figure 4 of the main manuscript was obtained . The video was taken at 100 frames/s and at a magnification of 31 . 8 nm/pixel .", "The corresponding field of view shown is 2 × 2 μm2 and corresponds to a total recording time of 5 s .", "The video playback speed is 100 frames/s . DOI: http://dx . doi . org/10 . 7554/eLife . 05413 . 014 The positional fluctuations ( σ = 0 . 91 nm SD ) of a surface-attached label recorded in the same field of view as the trajectory suggest that we achieved sub-nm lateral localization precision of 20 nm gold at 100 frames/s ( Figure 4B , blue ) .", "The localization noise increased for a gold bead attached to an actin-bound myosin ( σ = 1 . 6 ± 0 . 3 nm in Figure 4B ) , but remained small enough such that transitions on the order of 5 nm were clearly visible ( Figure 4A–B ) .", "The slightly larger positional fluctuations for gold bound to the actomyosin complex compared to immobilized gold were likely caused by a combination of a flexible protein-label connection and a limited ability in to completely immobilize actin .", "We observed a clear drop in the iSCAT contrast when the labeled trailing head detached and transitioned to the leading position ( Figure 4C ) .", "We also measured a much smaller change in iSCAT contrast during the AB transition , likely due to a three-dimensional reorientation of the label .", "We then performed simultaneous tracking of the head and the tail of myosin 5a , which showed that the small backwards transition corresponds to the power stroke of the bound motor domain .", "To achieve this , we labeled the myosin head with a 20 nm gold particle as previously and the tail with a GFP booster ( Ries et al . , 2012 ) .", "We reduced our frame rate to 20 frames/s in order to increase the fluorescence signal from the tail , since single molecule fluorescence tracking of GFP with <5 nm precision at 100 frames/s is challenging .", "The resulting traces showed that both the large-scale translocation of the head and the subsequent smaller backwards transition coincide with translation of the tail ( Figure 5A ) .", "This implies that the small-scale transition accompanies the power stroke of the attached labeled head ( AB transition ) and is representative of a pre- to post-power stroke transition ( Figure 5B ) . 10 . 7554/eLife . 05413 . 015Figure 5 . Simultaneous iSCAT and fluorescence tracking of myosin 5a .", "( A ) Tracking of the scattering signal from the gold nanoparticle attached to the N-terminus ( black ) and fluorescence signal from the GFP moiety located at the C-terminus of the same myosin 5a molecule ( green ) .", "Movement of the tail correlates with the labeled head taking its step ( 74 nm displacement ) and with the AB transition ( power stroke of the labeled head ) .", "Green arrows represent the tail movement , which corresponds to the step of either the labelled or unlabeled head .", "When the unlabeled head takes its step it coincides with the AB transition within the labeled head ( red arrows ) .", "Static localization precisions determined as in Figure 2: 1 . 6 nm ( iSCAT ) , 8 nm ( GFP ) .", "( B ) Labelling scheme and schematic of the stepping mechanism and the corresponding observables .", "ATP concentration: 1 μM .", "Imaging speed for both channels: 20 frames/s . DOI: http://dx . doi . org/10 . 7554/eLife . 05413 . 015 To determine the structural origin of the AB transition , we repeated the tracking experiments with differently sized labels .", "We found that the average size of the AB transition increased from 7 . 4 ± 3 . 2 nm to 9 . 5 ± 3 . 1 nm and 11 . 5 ± 2 . 5 nm for 20 , 30 and 40 nm diameter labels , respectively ( Figure 6A ) .", "This suggests that the AB transition reports on a conformational change of the bound head , which is a rotation of the N-terminal domain , rather than a translation of the head itself along actin .", "The consequences of a rotational movement of the label are presented schematically in Figure 6B .", "The red circles indicate the positions of two labels in the pre-power stroke A state ( with radii r1 and r2 , where r2 = 2 × r1 ) .", "The grey circles correspond to the post-power stroke B state .", "Red and grey dots indicate their respective centers .", "For a larger label ( r2 ) , the same movement of the N-terminus leads to an overall similar motion but with a larger displacement of the center of mass ( d2 ) than for a small label ( d1 ) .", "Using this model and our data , we could extract an angle of rotation , α , as well as a distance , x , that defines the origin of rotation , since sin ( α/2 ) = ½ d1/ ( r1 + x ) and d2/d1 = ( r2 + x ) / ( r1 + x ) .", "Using the hydrodynamic radii for the labels obtained by dynamic light scattering ( 20 , 26 and 32 nm ) , we obtained α = 20° for the angle and x = 1 . 7 nm . 10 . 7554/eLife . 05413 . 016Figure 6 . Conformational change within the N-terminal domain during the power stroke .", "( A ) Histograms for the distances between A and B states for 20 , 30 and 40 nm gold nanoparticle labels located at the N-terminus .", "Total number of steps recorded: 124 , 103 and 116 , respectively .", "( B ) Expected movement for two differently sized labels attached to myosin 5a during a conformational change in the head domain associated with the power stroke .", "The red circle represents the position of the label in the A state , and the grey circle corresponds to the B state with dots indicating their respective centres of mass .", "The labels r1 and r2 correspond to the radii of both labels where r2 = 2 × r1 , and d1 and d2 correspond to the AB distance after rotation by an angle α around an origin located within the head domain at a distance x from the nanoparticle surface .", "The myosin 5a head domain pre-power stroke conformation is shown in orange ( PDB: 1W7J ) .", "The lever arm is pointing out ( shown in dark blue ) and the blue arrow indicates its movement during the power stroke . DOI: http://dx . doi . org/10 . 7554/eLife . 05413 . 016 Trajectories recorded at 100 frames/s frequently showed both the position of the transient state and the direction of the AB transition , revealing that they were invariably in line ( Figure 7A , B ) .", "During the recording of 351 traces that exhibited clear signatures of the transient state at 100 frames/s , we found a preference ( 66% vs 33% ) of right over left-handed walking .", "Within individual traces , we only observed the transient state on the same side of actin .", "In some cases , however , we could observe individual myosin molecules switch from one actin filament to another .", "As previously , the transient state always appeared on one side while on one actin filament , but either switched to the other ( Figure 7C ) or remained on the same side ( Figure 7D ) of the actin track when the molecule moved from one filament to another .", "For 102 switching events , we found that the transient state remained on the same side of the filament 60% of the time , while changing sides in 40% of the events . 10 . 7554/eLife . 05413 . 017Figure 7 . Directionality and kinetics of the AB transition and the transient state .", "( A and B )", "Simultaneous observation of the AB transition and the transient state for right and left handed walking molecules .", "( C and D )", "Position of the transient state for the same molecule before and after switching actin tracks .", "ATP concentration: 10 μM .", "Scale bar: 100 nm .", "Imaging speed: 100 frames/s . DOI: http://dx . doi . org/10 . 7554/eLife . 05413 . 017 Given that the transient state could always be clearly identified either to the right or to the left side of actin , we could align all recorded steps from the 1000 frames/s tracking data by flipping them about the actin filament when necessary to obtain a 2D spatial probability distribution maps of the location of both the unbound and bound myosin head compared to the actin binding sites during a single step ( Figure 8 ) .", "The resulting contour plot contained two maxima .", "For the transient state one was located ∼5 nm away from the final actin binding site and the other 40 nm off-axis from the actin filament .", "For the A and B states the bound head orientations were in line with the position of the transient state , with the lateral projection connecting the A , B and transient states approximately in a straight line ( see also Figure 7 ) . 10 . 7554/eLife . 05413 . 018Figure 8 . Probability density contour maps of the myosin step . Upper panel represents the transient state of the unbound head .", "Contour map of a two-dimensional histogram with a 10 × 10 nm2 bin width obtained from the 1000 frames/s data ( N = 486 ) .", "Lower panel shows the AB transition within the bound head , a two-dimensional histogram with a 1 × 1 nm2 bin width generated using the 100 frames/s data ( N = 129 ) .", "All contributing steps were aligned and those to the right of the filament when viewed in the direction of motion were mirrored .", "The arrow represents direction of movement ( from left to right ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 05413 . 01810 . 7554/eLife . 05413 . 019Figure 8—figure supplement 1 . Spatiotemporal dynamics of the transient state as a function of ATP concentration .", "( A ) Contour maps of the transient state at three different ATP concentrations .", "Obtaining traces at high ATP concentration was challenging due to the small available field of view and rapid detachment of myosin from actin caused by the faster stepping rate .", "( B ) Dwell time histograms at three different ATP concentrations ( 1 μM , N = 116; 10 μM , N = 223; 1 mM , N = 90 ) .", "Fitting the dwell time distribution to a single exponential yields an average lifetime of 17 . 5 ± 0 . 6 ms , independent of ATP concentration . DOI: http://dx . doi . org/10 . 7554/eLife . 05413 . 01910 . 7554/eLife . 05413 . 020Figure 8—figure supplement 2 . Dwell time distributions for pre and post-power stroke states at different ATP concentrations . In all cases , data and fits for the pre-power stroke state are shown in red; data and fits for the post-power stroke state are shown in blue .", "At saturating ( 1 mM ) ATP concentration ( A ) both dwell time distributions exhibited single exponential behaviour in line with ADP release being the rate limiting step .", "The constants for the A state dwell times are given by kA and that for the B state by kB .", "At lower ATP concentrations , sequential ADP release and ATP binding result in bi-exponential behaviour .", "At 10 μM ATP ( B ) , both kinetic constants , k1 , k2 , are almost identical therefore the dwell time distribution is fit to two sequential process with the same rate constant ( termed kA for the dwell time of the A state and kB for that of the B state ) .", "( C ) At 1 μM ATP the process is limited by ATP binding .", "Data taken at 100 frames/s , we increased the imaging speed to 400 Hz at 1 mM ATP concentration .", "Total number of steps recorded: 110 , 245 and 104 , for 1 μM , 10 μM and 1 mM , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 05413 . 02010 . 7554/eLife . 05413 . 021Figure 8—figure supplement 3 . Geometrical considerations assuming myosin molecules moving parallel to the glass surface . If the head searched the full spherical space , the average position would be simply about half way between the leading head and the next binding site .", "For a parallel orientation this would be 56 nm forward and 15 nm off axis and for a perpendicular orientation 56 nm forward but ‘in line’ with the bound head positions .", "These numbers arise from simple geometric considerations but also agree with much more sophisticated simulations based on a free rotational diffusion model ( Hinczewski et al . , 2013 ) .", "We , however , observed a transient state half way between the two binding sites , 40 nm off axis ( marked in orange ) , which rules out this geometry . DOI: http://dx . doi . org/10 . 7554/eLife . 05413 . 02110 . 7554/eLife . 05413 . 022Figure 8—figure supplement 4 . Probability density contour map of the transient state . Contour map of a two-dimensional histogram with a 18 × 18 nm2 bin width was obtained using all , left- and right- handed walking traces , without flipping ( left panel ) .", "The resulting contour plot shows good agreement with previous results ( Dunn and Spudich , 2007 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 05413 . 022 Our ability to clearly distinguish between bound and unbound states during processive motion , allowed us to extract the dwell times of the transient state .", "We found that it followed a single exponential distribution with a lifetime of 17 . 5 ± 0 . 6 ms . We found no ATP concentration dependence on both the spatial and temporal distributions of the transient state within our experimental error ( Figure 8—figure supplement 1 ) .", "The corresponding dwell time distributions for the A and B states followed the behavior expected for processes limited by ATP binding and ADP release ( Figure 8—figure supplement 2 ) .", "In all cases , the dwell times of the A and the B states were identical suggesting that the 20 nm gold particle had no effect on the stepping kinetics ." ], [ "Label-sizes of a few tens of nm are traditionally avoided in single particle tracking experiments to prevent the label from perturbing the dynamics of interest .", "By monitoring the lifetimes of the A and B states , which effectively provides the dwell times of the labeled and unlabeled heads , respectively , we demonstrate that the 20 nm gold particle attached to the N-terminus of myosin has no measureable effect on the kinetics of stepping .", "If detrimental effects can be excluded experimentally , as in this case , our results show that the larger size of the label can actually be advantageous in revealing structural dynamics that would otherwise remain invisible .", "Here , the label probes the structural change occurring in the bound head during the power stroke and amplifies it into a nanometer scale motion .", "Previous studies using gold labels were not able to observe this transition likely due to a combination of a different labeling strategy ( 40 nm or 60 nm gold nanoparticle attached to a calmodulin on the lever arm ) and much lower spatial precision ( >10 nm ) ( Dunn and Spudich , 2007 ) .", "Several EM studies ( Walker et al . , 2000; Burgess et al . , 2002; Oke et al . , 2010 ) failed to detect any obvious conformational differences in the position of the SH3 ( N-terminal domain ) of lead and trail heads , whereas we now demonstrate a movement of this domain associated with the power stroke taken after the trail head dissociates .", "This change is consistent with results of molecular dynamic simulations of both myosin 5a and myosin 2 based on crystal structures suggesting that the N-terminus undergoes a rotation during the power stroke transition ( Coureux et al . , 2003; Cecchini et al . , 2008; Preller and Holmes , 2013 ) .", "Although the original myosin 5 tracking performed with a fluorescently-labeled calmodulin bound to the lever arm at 1 . 5 nm precision revealed 74 nm steps , ( Yildiz et al . , 2003 ) a later study with rigidly attached dye labels on the calmodulin bound to the lever arm suggested that they in fact represent 64–10 nm steps ( Syed et al . , 2006 ) .", "During the acquisition of 4728 steps in 635 trajectories , we never observed a large followed by a small forward step .", "In our work , we labeled the motor domain directly , while in the previous studies , calmodulin bound to the lever arm was used as an attachment point .", "Since the lever arm moves significantly during the power stroke , attachment of the dye at this location may suggest a sub-step along actin , even though the motor domain itself does not move .", "Thus the term ‘sub step’ is inappropriate for this pattern of movement .", "In addition , the orientation of single dipoles can have a significant impact on the position obtained from standard Gaussian fitting and exacerbate even very small translocations ( Enderlein et al . , 2006 ) .", "A recent AFM study ( Kodera et al . , 2010 ) reported that detachment and reattachment of the leading head , a so-called ‘foot stomp’ contributes to the mechanochemical cycle of myosin 5 .", "Such behavior was also observed in the single molecule tracking study discussed above , ( Syed et al . , 2006 ) although the frequency of occurrence was not discussed in that work .", "The sensitivity of our tracking methodology to detachment from actin in three dimensions , however , shows that myosin remains firmly bound to actin with both heads during the mechanochemical cycle irrespective of whether the head is leading or trailing ( Figure 4—figure supplement 1 ) , except when the head takes a 74 nm step .", "Only on very rare occasions did we observe detachment of the leading head .", "Specifically , only ~3% of steps taken at 1 µM ATP , ~0 . 6% of steps taken at 10 µM ATP , none at saturating ATP exhibited this behavior .", "The unbinding event was not necessarily followed by reattachment at a different site suggesting that these events can be explained simply by the binding equilibria between myosin and actin , rather than being an active component of the stepping mechanism .", "One likely explanation for the increased occurrence of lead head detachment and reattachment in recent AFM studies ( Kodera et al . , 2010; Ando et al . , 2013 ) is the much lower ATP concentration used or the non-negligible interaction of the AFM tip with the protein .", "The combination of temporal and spatial precision achieved in this work directly reveals the motion of the unbound head of myosin during translocation .", "From the two head bound state of myosin 5a , the trailing head detaches from actin upon binding of ATP ( Figure 9A ) .", "The leading head then undergoes a power stroke that leans the protein forward , a motion that exhibits a strong torsional component ( Komori et al . , 2007; Ohmachi et al . , 2012 ) .", "After the power stroke , the unbound head arrives at a minimum in the potential energy from where it approaches the next actin binding site in what appears to be a one dimensional biased search .", "The transient state lifetime agrees with the duration of increased flexibility reported in optical trapping experiments probing the attachment stiffness at high time-resolution ( Veigel et al . , 2002 ) and single molecule tracking of the motor domain ( Dunn and Spudich , 2007; Beausang et al . , 2013 ) .", "The measured lifetime also correlates well with previously reported ensemble rate constant for the weak-to-strong binding transition ( 47 s−1 ) ( Rosenfeld and Sweeney , 2004 ) .", "The presence of an additional high probability density very close to the final binding site further favors such an interpretation .", "This density very likely corresponds to the weakly bound state from which the head frequently moves back to the side position . 10 . 7554/eLife . 05413 . 023Figure 9 . Mechanism of the myosin step .", "( A ) ATP binding to the trailing head ( yellow ) and its detachment releases strain stored in the molecule .", "( B ) The bound head ( black with a gold nanoparticle attached ) performs its power stroke which is accompanied by the AB transition ( inset ) .", "The labeled head becomes a new trailing head ( C ) which detaches after ATP binding .", "( D ) It moves forward in a partially twisting motion and occupies an off axis position ( transient state ) .", "From this position , the head binds the desired binding site while ATP hydrolyses .", "( E ) The step completes as the head binds actin and is repeated by the other head in the same direction dictated by the initial torsional strain .", "( F ) Schematic representation of both heads' movement ( lateral projection ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 05413 . 023 An important implication of our data is that the position of the transient state , displaced 40 nm along and perpendicular to the filament is inconsistent with a purely rotational diffusion search .", "In that case we would require most molecules to be bound parallel to the surface and then expect the average position of the detached head to be displaced ∼56 nm along and not more than 20 nm perpendicular to the filament ( Figure 8—figure supplement 3 ) .", "We emphasize however , that our data does not contradict previous dark field results , which lead to the rotational diffusion model ( Dunn and Spudich , 2007 ) .", "We can closely reproduce such results by adding 17 nm of localization noise to our traces .", "At such localization precision , left- and right- handed walking is no longer distinguishable leading to a probability density map that is no longer one-sided ( Figure 8—figure supplement 4 ) .", "Why the unattached head occupies a positon 40 nm away from the actin filament is a priori puzzling .", "There is neither an intrinsic reason for the head to pause there , nor is there a binding site present at this position .", "Our experimental data provides no evidence that the transient state is induced or measurably affected by unwanted interactions caused by the particle or the surface as suggested by the similarity of our transient state lifetime to previous studies with alternative labeling strategies , ( Forkey et al . , 2003; Snyder et al . , 2004; Warshaw et al . , 2005 ) the presence of the transient state for much smaller labels ( Figure 1—figure supplement 2 ) , the change in contrast during the step ( Figure 2 ) , the lack of non-specific binding of our label to the surface or actin and any localizations during the one head bound state that coincide with the underlying actin track .", "This location and the resulting movement , however , become more intuitive when considering that the intrinsic angle between the lever arms is similar to that found when the two heads are bound to actin , that is , ∼37 nm away from each other .", "Comparison of electron micrographs of myosin 5 bound to actin ( Walker et al . , 2000 ) and non-specifically bound to a surface in the absence of actin ( Takagi et al . , 2014 ) show clearly peaked angle distributions around 105 and 115° , respectively , suggesting a built-in preference for such a spatial arrangement of the two heads , rather than a truly flexible linkage , another indication that a completely free swivel at the neck-linker is unlikely .", "In this way , keeping the angle between the lever arms roughly intact during stepping and twisting partially about the leading head causes the translocating motor domain to preferentially reach the desired binding site .", "As a result a full three dimensional search mechanism is minimized to one defined by a one dimensional arc .", "The position of the transient state together with what appears to be constrained diffusion suggests that myosin side steps along actin in a combined twisting and leaning motion , much more like a drawing compass rather than a freely jointed swivel or human-like gait .", "In hindsight , such a mechanism has numerous advantages over a search based on full rotational diffusion .", "It generates a built-in bias for finding the correct binding site and ensures that the head needs to travel and search only along one dimension to find the desired binding site defined by the torsion about the leading head and neck linker rather than a lower probability three-dimensional Brownian search .", "Even if the motor domain fails to bind strongly on first passage , it swings back and forth between the transient state and the desired actin site until tight binding can occur .", "This suggests that the structure of myosin 5a facilitates and controls the motion of the unbound head to achieve such high specificity in finding the desired binding site .", "Our results do not imply that the head is effectively stationary at the transient state position while in the one head bound state .", "Some degree of diffusional search is inherent to the system and cannot be captured even at our low exposure time of 0 . 56 ms , which is comparable to the positional autocorrelation time we measured ( 0 . 3 ms ) , and predicted theoretically ( Craig and Linke , 2009 ) .", "The location of the transient state , the fact that in several thousand steps we never observed a single step with transient localizations on both sides of the actin filament , and a predominantly perpendicular orientation of myosin relative to the surface caused by our surface preparation , however , suggest that diffusion is constrained .", "On the contrary , we believe that this constraint is a key to myosin 5's efficiency in terms of processive motion in contrast to a completely free , spherical search space .", "The fact that the AB transition and the relative location of the rear-binding site and the transient state are in line has two further important implications .", "It suggests that the AB transition cannot solely be a consequence of a steric interaction between the label and the lever arm .", "In that case , we should only observe one type of AB transition , irrespective of whether right or left-handed walking occurs .", "Furthermore , it shows a connection between the directionality of the N-terminus and lever arm movement .", "The observed N-terminus domain movement could thus be a signature of the release of this strain .", "This conformational change , critical for rearrangement of the nucleotide binding pocket and/or closure of the internal cleft , would then allow for finalizing the actomyosin energy transduction .", "In other words , the energy from the ATPase reaction , stored in the actomyosin complex can only be fully used after the trailing head detaches and the system can relax .", "Our data shows that the AB transition is associated with the power stroke but it is unlikely that the 20° rotation of the N-terminus is directly responsible for the side step per se .", "The fact that the transient state is either to the left or to the right of the filament implies that it cannot be a pure consequence of the attached lever arm swing .", "We therefore propose that the position of the transient state and the direction of the AB transition reflect the strain release upon rear head detachment .", "The initial strain is built up within the head domain and transmitted up to the lever arm during the first binding when one of the heads twists in order to bind to the same actin filament ( Liu et al . , 2006 ) .", "The simultaneous tracking of the movement of both heads using the combination of gold scattering and fluorescence demonstrates that myosin walks in a symmetric manner where each head movement is accompanied by an 180° swing on the same side of the actin filament , unlike a human gait .", "This type of movement would imply that the cargo also rotates with each step or that a swivel exists to relieve the strain that would otherwise develop between the myosin and the cargo .", "Such a rotation has been measured by the orientation of quantum rods attached to the myosin 5a tail , although , in this work the source of the rotational movement was thought to be thermal ( Ohmachi et al . , 2012 ) .", "Our observation that the orientation of the transient state relative to actin may change for the same molecule by binding to another actin filament shows that the position of the transient state relative to actin is not an intrinsic property of individual myosin molecules .", "Otherwise , we would expect each molecule to preserve the orientation of the transient state even when changing from one filament to another .", "Instead , the decision is made upon binding to actin , with both cases following the right and left hand preference previously mentioned .", "With respect to the source of the symmetry breaking , one possible explanation involves the steric interaction with the surface .", "Depending on the initial binding angle the steric interactions with the surface would determine the direction of rotation .", "However , if the sidedness was only a consequence of an interaction with the surface , then we would at least expect the molecules that are bound perpendicular to the glass surface and thus have the least interaction with it , to show no preference .", "Since we never observed transient states on both sides of the filament during individual runs , the reported mechanism is likely to be an intrinsic feature of the myosin 5a stepping .", "Based on our observation that the transient state for both heads occurs on the same side of the actin filament we propose that the twisting that takes place during the initial binding event of both heads to actin determines the directionality .", "This initial twist , possibly driven by thermal fluctuations , establishes the direction in which the initial torsional strain is built up and is then repeatedly stored and released as the protein moves along actin .", "Once the directionality of the transient state has been established , both heads follow the same constrained route resulting in an efficient , unidirectional motion ." ], [ "Rabbit skeletal muscle actin was prepared as described ( Spudich and Watt , 1971 ) and stored in liquid nitrogen until used .", "A 20 μM actin stock solution was prepared in polymerisation buffer ( 10 mM imidazole , 50 mM KCl , 1 mM MgCl2 , 1 mM EGTA [pH 7 . 3] containing 1 . 7 mM DTT , 3 mM ATP ) .", "Actin was diluted in motility buffer ( MB; 20 mM MOPS pH 7 . 3 , 5 mM MgCl2 , 0 . 1 mM EGTA ) 20–50 times .", "Mouse myosin 5a HMM with a C-terminal GFP was expressed in the presence of calmodulin and purified as described ( Wang , 2000 ) .", "In addition , the N-terminus was modified by the addition of a nucleotide sequence encoding an AviTag peptide ( GLNDIFEAQKIEWHE ) for site-specific biotinylation with BirA ligase ( Avidity , Aurora , Colorado ) .", "After biotinylation , the sample was aliquoted , flash frozen in 20 μl drops and stored at −80°C .", "Before labeling , the myosin sample was diluted in MB containing 40 mM KCl , 5 mM DTT , 0 . 1 mg/ml BSA and 5 μM calmodulin .", "Gold nanoparticles of 20 nm , 30 nm and 40 nm in diameter conjugated with streptavidin were purchased from BBI ( UK ) and directly mixed with biotinylated myosin 5a sample in a 4:1 , gold to myosin ratio , consistent with one or zero myosin molecules per gold particle .", "The mixture was incubated on ice for at least 15 min ( sample volume 50 μl , final concentration of myosin 300 pM ) .", "The same procedure was used for double gold/Qdot labeling .", "The quantum dots ( emission maximum 565 ) conjugated with streptavidin were purchased from Invitrogen ( Lifetechnologies , UK ) .", "For fluorescence only imaging , myosin was incubated for 10 min with Atto-647 streptavidin ( Atto-TEC , Germany ) .", "The following oxygen scavenger system was used to increase the fluorescent dye stability: 0 . 2 mg/ml glucose oxidase , 0 . 4% wt/vol glucose , 0 . 04 mg/ml catalase , all purchased from Sigma-Aldrich ( UK ) .", "The flow cell was prepared as described ( Dunn and Spudich , 2011 ) .", "It was first rinsed with 1 mg/ml solution of poly ( ethylene glycol ) -poly-l-lysine ( PEG-PLL ) branch copolymer ( Surface Solutions SuSoS , Switzerland ) in PBS and incubated for 30 min .", "Next , it was washed twice with MB before adding the actin solution .", "After 5 min of incubation , the chamber was washed with MB and the surface was blocked by adding 1 mg/ml BSA in MB buffer .", "Finally , the chamber was inspected and myosin-gold conjugate solution containing ATP was added .", "All assays were performed at room temperature .", "Interferometric scattering microscopy was performed as detailed previously ( Ortega-Arroyo and Kukura , 2012 ) .", "For two-colour imaging a second diode laser ( 635 nm ) was overlapped with the 445 nm beam path with a dichroic mirror .", "In the detection arm , the images were separated by an identical optic before being imaged onto two separate CMOS cameras ( MV-D1024-160-CL-8 , Photonfocus , Switzerland ) at 333× magnification ( 31 . 8 nm/pixel ) .", "The incident power was adjusted to 17 . 9 kW/cm2 at the sample to achieve near-saturation of the CMOS camera , which ensured shot noise limited detection .", "Fluorescence only imaging and tracking was achieved with a home-built TIRF microscope using a 635 nm diode laser .", "The incident power was set to 5 kW/cm2 and the fluorescence imaged onto an Andor iXon3 860 EM-CCD camera at 72 . 1 nm/pixel with a 9 . 2 μm × 9 . 2 μm field of view using dielectric filters ( Thorlabs , Germany ) to separate illumination and emission .", "Sample-specific images with shot noise limited sensitivity were obtained by dividing the raw image by a reference flat field image .", "The flat field reference image containing non-sample specific illumination inhomogeneities , fixed pattern noise and any constant background caused by residual reflections was produced by first recording 2000 images while moving the sample stage with a piezo-driven XY translation stage .", "The resulting image stack was temporally averaged to 100 frames and each pixel of the flat field reference image was computed as the temporal median value of the frame sequence .", "Removal of sample specific constant background and thereby increase in the image signal to noise ratio was achieved by obtaining the static background from the sample by averaging at least 10 frames that lacked a signal from the processive myosin 5a molecule labeled with a gold nanoparticle of interest ( Ortega Arroyo et al . , 2014 ) .", "Nanometric tracking of labeled myosin 5a was achieved by non-linear least square fitting of the point spread function to a two-dimensional Gaussian .", "Spatiotemporal characterization of the transient state was achieved by alignment of several steps and separation of the states .", "We segmented each trajectory into step pairs defined as the set containing the data before and after each 74 nm step .", "These steps were detected manually by using a time interval slider along the 2D trajectory and checking for changes in positional fluctuations .", "The orientation of the transient state relative to actin was determined and later used to align all steps .", "To determine the spatiotemporal dynamics of the side step only step pair traces that showed transient states lasting longer than 10 ms were used .", "First , we fixed the center of mass of the beginning of the step to coordinate position ( 0 , 0 ) .", "Then , we applied a rotation matrix to the entire step pair trace so that both bound states lie along the horizontal axis .", "Finally , all aligned step pair traces were overlapped to produce the contour plots shown in Figure 8 and Figure 8—figure supplement 1 ." ] ]

[ "Myosin 5a is a dual-headed molecular motor that transports cargo along actin filaments .", "By following the motion of individual heads with interferometric scattering microscopy at nm spatial and ms temporal precision we found that the detached head occupies a loosely fixed position to one side of actin from which it rebinds in a controlled manner while executing a step .", "Improving the spatial precision to the sub-nm regime provided evidence for an ångstrom-level structural transition in the motor domain associated with the power stroke .", "Simultaneous tracking of both heads revealed that consecutive steps follow identical paths to the same side of actin in a compass-like spinning motion demonstrating a symmetrical walking pattern .", "These results visualize many of the critical unknown aspects of the stepping mechanism of myosin 5 including head–head coordination , the origin of lever-arm motion and the spatiotemporal dynamics of the translocating head during individual steps ." ]

[ "Cells use motor proteins that to move organelles and other cargos from one place to another .", "The myosins are a family of motor proteins that pull cargo along filaments made of another protein called actin .", "The ‘head’ end of myosin attaches to the actin filament and the ‘tail’ end binds to the cargo .", "The head and tail are connected by a flexible linker that allows the protein to change shape .", "The tails of two myosin molecules bind together to form a two-headed motor that can move along an actin filament by taking 74 nm steps .", "At the start of each step , both heads are attached to the actin with one in front of the other .", "The leading head remains attached while the rear head detaches from actin and moves in front of the leading head reattaching to actin .", "These movements are repeated many times to allow the motor to move along the filament , much like a tightrope walker walking along a wire .", "However , it is not known how the motor can move so efficiently along the actin while managing to avoid falling off its track .", "Here , Andrecka et al . analyzed the movement of the myosin 5 motor in real time using a method called ‘interferometric scattering microscopy’ .", "The experiments show that when a head detaches from the actin , it is temporarily held out to one side of the actin filament .", "From here , this detached head sways back and forth , until it takes a step forward and binds firmly to the next position on the filament .", "Both heads follow identical paths along the actin filament , and so the movement resembles the way that a drawing or dividing compass can be used to measure distances on a map .", "Andrecka et al . 's findings shed new light on how myosin motors move along actin .", "This may become a blueprint for efficient nano-mechanical motion and could therefore be important for designing artificial machines that operate on the nanoscale ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "microbiology and infectious disease" ]

[ [ "Combination antiretroviral therapy ( ART ) suppresses HIV replication but does not eliminate the latent reservoir , which persists for decades in CD4+ T cells and forms a major barrier to HIV cure ( Chun et al . , 1997; Finzi et al . , 1997; Wong et al . , 1997 ) .", "Our understanding of the phenotypic features of latent cells in vivo is limited , in large part because the lack of a universal surface biomarker makes it impossible to directly identify these cells .", "As a result , fundamental questions remain , such as whether latent cells distribute equally among all CD4+ T cell subsets , and to what extent the reservoir is similar between individuals and between blood and tissues .", "In the absence of direct ways to phenotype latent cells , characterizing the composition of the HIV reservoir has largely entailed sorting CD4+ T cells based on predefined sets of cell-surface proteins , followed by quantitating HIV through a variety of methods .", "A vast majority of infected cells in virally suppressed individuals harbor defective HIV genomes ( Hiener et al . , 2017; Ho et al . , 2013; Imamichi et al . , 2016; Lee et al . , 2017 ) .", "Older quantitation methods based on measuring total HIV DNA/RNA levels , regardless of provirus integrity , are being replaced with methods quantitating levels of replication-competent or genome-intact provirus .", "These methods afford a more accurate picture of the cells leading to viral rebound during treatment interruption , which are the most relevant targets for HIV cure .", "The ‘gold standard’ in virus quantitation has been the quantitative viral outgrowth assay ( qVOA; Finzi et al . , 1997 ) .", "qVOA has not identified significant differences in the amount of replication-competent HIV in the most commonly studied subsets of memory CD4+ T cells – central memory ( Tcm ) , effector memory ( Tem ) , and transitional memory ( Ttm ) ( Buzon et al . , 2014; Kwon et al . , 2020 ) .", "Such observations have led many researchers to consider the replication-competent reservoir as equally residing in all cell subsets , which if true makes targeting it more difficult .", "The analysis of proviral sequences , on the other hand , has revealed differences in the frequencies of genome-intact provirus in different sorted subsets .", "For instance , sequencing of near full-length HIV provirus has identified Th1 and Tem , particularly those that are HLADR+ , as subsets preferentially harboring intact viral genomes ( Hiener et al . , 2017; Horsburgh et al . , 2020; Lee et al . , 2017 ) .", "Viral sequencing has also revealed many intact proviruses within clonally expanded populations , particularly within the Tem and Th1 subsets , and those expressing Ox40 or CD161 ( De Scheerder et al . , 2019; Hiener et al . , 2017; Kuo et al . , 2018; Lee et al . , 2017; Li et al . , 2019 ) .", "These and many other studies ( Cohn et al . , 2020; Liu et al . , 2020 ) have solidified the view that clonal expansion plays a major role in maintaining the long-lived HIV reservoir .", "Although analyses of sorted populations have provided meaningful insights into HIV persistence , they have been limited to comparing cell populations that differ in one or two markers .", "Thus , they do not provide a comprehensive view of the phenotypic features of latent cells .", "Several methods have recently enabled the direct phenotyping of reservoir cells at single-cell resolution ( Baxter et al . , 2016; Cohn et al . , 2018; Grau-Expósito et al . , 2017; Pardons et al . , 2019 ) , providing deeper analyses of the reservoir .", "These methods entail stimulating cells from ART-suppressed patients with a mitogen to induce the expression of HIV RNA and/or proteins , which serve as markers for identifying or isolating cells using FACS .", "However , one important limitation with these methods is that they require ex vivo stimulation before phenotyping as viral proteins are not typically expressed at detectable levels in unstimulated latent cells ( Baxter et al . , 2016; Cohn et al . , 2018; Pardons et al . , 2019 ) .", "This stimulation will induce phenotypic changes associated with T cell activation , and expression of HIV accessory genes from reactivation will result in host cell remodeling ( e . g . downregulation of cell-surface CD4 ) , which will further alter the phenotypes of the cells ( Cavrois et al . , 2017; Ma et al . , 2020 ) .", "Therefore , although current methods enable phenotyping of reactivated cells , there is at present no methodology to directly assess the phenotypes of individual latent cells in their original dormant state .", "To overcome this limitation , we applied the principles behind Predicted Precursor as determined by SLIDE ( PP-SLIDE ) , an analytic method we recently established that combines high-dimensional single-cell phenotyping and computational approaches .", "We have used PP-SLIDE to predict the state of CD4+ T cells before phenotypic changes induced by productive HIV infection ( Cavrois et al . , 2017; Ma et al . , 2020 ) .", "Here , we applied it to determine the features of in vivo latent CD4+ T cells before their reactivation by ex vivo stimulation , and validated these findings with patient specimens .", "Through PP-SLIDE analysis , we were able to address questions fundamental to our understanding of HIV latency and to isolate highly enriched populations of replication-competent latent cells for mechanistic studies of reservoir maintenance ." ], [ "PP-SLIDE was recently validated as an approach to trace HIV-remodeled cells to their original pre-infection states ( Cavrois et al . , 2017; Ma et al . , 2020 ) .", "To determine if it can trace reactivated cells to their original latent state , we tested it first in J-Lat cells , an in vitro model of HIV latency .", "J-Lat cells are Jurkat cells harboring a single integrated HIV provirus that can be reactivated upon stimulation .", "Multiple lines of J-Lats have been cloned ( Chan et al . , 2013; Jordan et al . , 2003 ) that have diverged slightly over successive passages .", "We tested how well PP-SLIDE traces reactivated cells back to their original clone when given the choice of two parental clones ( Figure 1A ) .", "Cells of clone 6 . 3 were reactivated by stimulation with PMA/ionomycin and phenotyped using a CyTOF T cell panel ( Ma et al . , 2020 ) , along with unstimulated cells of clone 6 . 3 and another clone 5A8 .", "Using PP-SLIDE ( see Materials and methods ) , the high-dimensional CyTOF-generated information from reactivated 6 . 3 cells ( identified by expression of HIV Gag ) was matched against an atlas of concatenated unstimulated 5A8 and 6 . 3 cells to identify the most similar unstimulated cell for each reactivated 6 . 3 cell .", "These unstimulated cells , identified using a k-nearest neighbor ( kNN ) approach , are collectively termed ‘kNN latent cells’ .", "The error rate , defined as the % of reactivated 6 . 3 cells that matched to unstimulated 5A8 cells , was 0 . 8% ( Figure 1B ) .", "These results suggest that although reactivation greatly changes the phenotype of a latent cell , some of the original ‘identity’ of the cell is still retained and that PP-SLIDE can be used to capture this information and thereby identify precursors of reactivated latent cells .", "Next , we applied PP-SLIDE to characterize latent cells from HIV-infected individuals , using a CyTOF panel ( Supplementary file 1 ) that is directed at markers of the major subsets and differentiation states of CD4+ T cells and includes receptors and intracellular proteins associated with latency ( Banga et al . , 2016; Buzon et al . , 2014; Chomont et al . , 2009; Descours et al . , 2017; Fromentin et al . , 2016; Hogan et al . , 2018; Iglesias-Ussel et al . , 2013; Khoury et al . , 2016; Li et al . , 2019; Serra-Peinado et al . , 2019; Sun et al . , 2015 ) .", "Since reactivation events are rare and reducing background was key , we conjugated different anti-Gag antibodies to three different metal lanthanides and only considered cells displaying a Gag signal in all three channels as true events .", "The full panel was validated in vitro ( Figure 2—figure supplement 1 ) .", "We collected via leukapheresis CD4+ T cells from the blood of four ART-suppressed HIV-infected individuals ( Supplementary file 2 ) , purified memory CD4+ T cells by negative selection , and separated the cells into two pools ( Figure 2A ) .", "One pool was used to generate the atlas of unstimulated cells .", "This pool was phenotyped within 2 hours of cell isolation , and never went through any cryopreservation or ex vivo culture .", "The other pool was stimulated with PMA/ionomycin under fully suppressive ART ( Figure 2—figure supplement 2A ) for 40 hr , a time point that precedes mitogen-induced cellular proliferation ( Figure 2—figure supplement 2B ) .", "The CyTOF profiles of reactivated cells were matched against those of the unstimulated atlas cells .", "Assuming each reactivated cell retained some of its original pre-stimulation identity and that a phenotypically similar cell was present in the atlas of unstimulated cells , we used PP-SLIDE to identify , for each reactivated cell , the nearest-neighbor within the atlas ( Figure 2A ) .", "These identified cells , the ‘kNN latent cells’ , harbor the predicted phenotypes of latent cells before their reactivation from latency .", "We identified between 20 and 96 reactivated cells per specimen , corresponding to a mean frequency of 11 . 3 per million memory ( CD45RA-CD45RO+ ) CD4+ T cells ( range 5 . 6–17 . 6 , Figure 2B , Figure 2—figure supplement 3 ) .", "When visualized against the atlas of unstimulated cells by t-distributed stochastic neighbor embedding ( tSNE; van der Maaten , 2009 ) , Gag+ reactivated cells occupied a unique region , confirming their distinct phenotype relative to unstimulated cells ( Figure 2C , left ) .", "kNN latent cells mapped to several distinct regions of the atlas ( Figure 2C , right ) , suggesting a non-random distribution among memory CD4+ T cells .", "To verify this , we implemented FlowSOM ( Van Gassen et al . , 2015 ) to subdivide the atlases from each of the four donors into 20 clusters , and visualized the distribution of kNN latent cells on the tSNE maps or on pie graphs ( Figure 2C , center , and 2D ) .", "We found large clusters lacking kNN latent cells as well as small clusters harboring kNN latent cells , consistent with a non-random distribution of latent cells , which was confirmed using a chi-squared test ( Figure 2D ) .", "For a detailed description of the extent of enrichment of kNN latent cells in clusters and validation of non-random distribution of the reservoir , see Materials and methods .", "Next , we examined the composition of the kNN latent cells .", "We focused first on the Tcm , Tem , and Ttm subsets , as they have been the most commonly studied memory CD4+ T cells in the context of HIV latency ( Chomont et al . , 2009; Josefsson et al . , 2013; Kwon et al . , 2020; Pardons et al . , 2019; Soriano-Sarabia et al . , 2014; von Stockenstrom et al . , 2015 ) .", "In all four donors , the contribution of Tem to the reservoir was greater than its contribution to total memory CD4+ T cells ( Figure 3—figure supplement 1 ) .", "We did not identify any kNN latent cells with the Ttm phenotype , likely because this subset is relatively rare in blood and not efficiently captured in our samples , each of which harbored fewer than 100 kNN latent cells .", "When the three FlowSOM clusters of atlas cells with the highest frequencies of kNN latent cells were examined for the relative contributions of the Tcm , Tem , and Ttm subsets , we found that they were all comprised of multiple subsets ( Figure 3—figure supplement 2 ) .", "Collectively , these data demonstrate that although Tem are over-represented among kNN latent cells , the clusters most enriched for kNN latent cells – as classified by unbiased clustering based on ~40 markers – are a mixture of Tcm/Tem/Ttm subsets .", "These findings could suggest that the reservoir is quite different between individuals .", "Alternatively , they could mean that latency markers shared between individuals are not those used to define Tcm/Tem/Ttm cells .", "Consistent with the latter , when datasets from the four donors were combined and re-run within the same tSNE , the kNN latent cells from the four donors resided within a similar region of the plot ( Figure 3A ) .", "These results argue that kNN latent cells between different donors are in fact similar .", "Consistent with this conclusion , relative to total memory CD4+ T cells , kNN latent cells consistently expressed higher levels of select markers of immune checkpoint ( PD1 , CTLA4 ) , activation ( CD69 , CD25 , HLADR ) , and T cell differentiation states ( Tbet , CRTH2 , CCR6; Figure 3B ) .", "We conclude that in vivo latent cells are not randomly distributed but instead share specific features between individuals .", "Even though latent cells are similar between individuals , the inter-donor variability in latent cells could still be greater than the intra-donor variability .", "To test this idea , we obtained leukapheresis specimens from donor PID5003 at two time points spaced 2 months apart and compared in an unbiased manner the kNN latent cells from those specimens to kNN latent cells from donor PID3010 ( Figure 3—figure supplement 3A , B ) .", "For each kNN latent cell in the PID3010 sample , we calculated the Euclidean distances ( based on CyTOF parameters ) to every kNN latent cell in the PID5003 sample .", "Similar calculations were conducted between kNN latent cells from the two time points sampled in PID5003 ( Figure 3—figure supplement 3A ) .", "The median distances were lower between specimens from the same donor than from different donors ( Figure 3—figure supplement 3C ) , demonstrating that despite the similarity of kNN latent cells between donors , the kNN latent cells differ more between donors than within a donor .", "Next , we took advantage of our ability to obtain high-resolution maps of the phenotypic features of latent cells to compare their reactivation by different ex vivo stimulations .", "Longitudinal blood specimens were obtained from the same suppressed individual and stimulated with the broad-spectrum mitogens PMA/ionomycin or anti-CD3/CD28 .", "Euclidean distance calculations ( Figure 3—figure supplement 3A ) revealed that PMA/ionomycin-reactivatable kNN latent cells from different collection time points were more similar to each other than to anti-CD3/CD28-reactivatable kNN latent cells from the same time point ( Figure 3—figure supplement 3D , E ) .", "Next , we compared the latent cells reactivatable by PMA/ionomycin to those reactivatable by a combination LRA consisting of an HDAC inhibitor together with an ingenol ( Romdepsin/PEP005 ) using three longitudinal blood specimens each spaced 2 months apart .", "tSNE visualization of the kNN latent cells revealed a region of the plot that harbored kNN latent cells from the PMA/ionomycin samples but not from the Romidepsin/PEP005 samples ( Figure 4A ) .", "Euclidean distance calculations ( Figure 3—figure supplement 3A ) were again used to verify that kNN latent cells reactivatable by the same stimulation at different time points were more similar than those reactivatable by different stimulations within the same time point ( Figure 4B ) .", "These data suggest that Romdepsin/PEP005 reactivates only a subset of latent cells reactivatable by broad-spectrum mitogens such as PMA/ionomycin , although these findings should be confirmed with longitudinal specimens from additional donors .", "Since most of the HIV reservoir persists within tissues ( Estes et al . , 2017 ) , we next applied the PP-SLIDE method of latent cell characterization to tissue specimens .", "One of the four suppressed participants who had donated a leukapheresis specimen , PID3010 , also agreed to the collection of two sets of lymph node specimens via fine needle aspirates ( FNAs ) spaced 6 months apart .", "Upon isolation , each FNA was immediately mock-treated or stimulated for 40 hr with PMA/ionomycin and then subjected to PP-SLIDE .", "Unlike with blood specimens , memory CD4+ T cells were not purified ahead of time since FNAs yielded limited numbers of cells .", "However , we pre-gated on memory CD4+ T cells to compare with purified blood cells .", "The atlases from the three specimens ( one blood , two FNAs ) showed that blood and FNA cells were phenotypically distinct ( Figure 4C ) .", "The kNN latent cells from the FNAs , similar to those from blood , were not randomly distributed and remained similar across the longitudinal specimens as demonstrated by their occupying similar regions of tSNE space ( Figure 4C ) .", "Several antigens distinguished kNN latent cells in the blood versus tissues .", "For instance , CD27 , an antigen expressed on Tcm cells , and CD69 , a marker of activation in blood but a T resident memory ( Trm ) marker in tissues ( Cantero-Pérez et al . , 2019 ) , were more highly expressed on FNA kNN latent cells ( Figure 4D , Figure 4—figure supplement 1A ) .", "PD1 and CXCR5 , markers of Tfh that are preferentially expressed in latent cells particularly in tissues ( Banga et al . , 2016 ) , were also expressed at especially high levels on kNN latent cells from FNAs ( Figure 4D , Figure 4—figure supplement 1A ) .", "The co-stimulatory molecule ICOS was expressed at high levels on kNN latent cells exclusively in FNAs , while the exhaustion marker TIGIT was more variably expressed ( Figure 4D , Figure 4—figure supplement 1A ) .", "These results reveal marked differences between the phenotypic features of kNN latent cells in blood versus lymph nodes , with the notable exception of PD1 , which was high in both compartments .", "We also characterized the reservoir in the gut , the primary site of viral persistence during suppressive therapy ( Estes et al . , 2017 ) .", "As longitudinal gut specimens were not available , we conducted a cross-sectional analysis comparing gut specimens from four donors to the blood specimens previously analyzed by PP-SLIDE .", "While differences in the reservoir between the blood and gut were observed , some interesting similarities also emerged .", "kNN latent cells from the gut were mostly CD69hiCD27lo , while those from blood exhibited a much more variable expression pattern of these two antigens ( Figure 4—figure supplement 1B ) .", "By contrast , kNN latent cells from both the gut and blood expressed high levels of PD1 but relatively low levels of CXCR5 ( Figure 4—figure supplement 1B ) .", "Another similarity was that CTLA4 and CCR6 , previously reported to be preferentially expressed on latent cells from tissues ( Gosselin et al . , 2017; McGary et al . , 2017 ) , were both highly expressed in kNN latent cells from the gut and blood .", "Taken together with the FNA results , these analyses identify CD69 and PD1 as antigens expressed at high levels in kNN latent cells from both gut and lymph nodes , suggesting shared features of the reservoir between different tissue compartments .", "To compare the global similarities between kNN latent cells from the three compartments ( blood , lymph nodes , gut ) , we visualized the atlas and kNN latent cells of all donors combined using tSNE ( Figure 4E ) .", "Interestingly , the FNA atlas cells appeared equidistant to the blood and gut atlas cells , an observation confirmed by Euclidean distance calculations .", "By contrast , kNN latent cells from FNA and gut tended to cluster more closely together , away from those from blood , which we also confirmed by Euclidean distance calculations ( Figure 4E ) .", "Together with the above analyses suggesting that kNN latent cells from both tissue compartments express high levels of CD69 and PD1 , these results suggest that phenotypic features of the reservoir are shared between different tissue sites .", "That said , a notable difference was that kNN latent cells from FNAs included both naïve and memory cells , while those from the gut only included memory cells , potentially due to the under-representation of naïve cells within the gut compartment ( Figure 4—figure supplement 1C ) .", "To determine whether surface markers differentially expressed on kNN latent cells can enrich for the replication-competent reservoir , we custom-designed sorting strategies for each of the four donor blood specimens .", "Because PD1 is arguably the most validated surface marker of latent cells ( Banga et al . , 2016; Chomont et al . , 2009; Fromentin et al . , 2016; Pardons et al . , 2019 ) , we compared the additional enrichment afforded by our sorting strategy to that afforded by PD1 selection alone .", "Our panels took into account the expression levels of markers in kNN latent cells , the numbers of cryopreserved cells available from the participant , and the overall abundance of the populations in each sequential gate .", "The latter two aspects were important considerations as an infrequent population of cells may be highly enriched for kNN latent cells but too rare to sort in sufficient numbers for viral outgrowth and other downstream assays .", "We arrived at panels of 8–10 markers individualized for each participant .", "For each donor specimen , we isolated a PD1+ population that we submitted to further selection with the relevant marker panel ( enriched ) and two PD1+ populations that we did not subject to further selection ( disenriched ) .", "The disenriched populations harbored markedly lower frequencies of kNN latent cells than the enriched one ( Figure 5 ) .", "These populations were sorted ( Figure 6—figure supplement 1 ) and subjected to multiple assays .", "We first conducted a viral outgrowth assay .", "Because sorting based on our 8–10 marker panels yielded few cells , we modified the qVOA accordingly .", "Sorted cells were stimulated in bulk for 2 days with anti-CD3/CD28 , and plated into individual wells at various dilutions .", "The HIV-permissive MOLT-CCR5 cell line was added to propagate viral replication , and after 2 weeks supernatants were quantitated for viral outgrowth by p24 ELISA .", "Replication was scored as the proportion of wells that had outgrowth and as the concentration of p24 in each well .", "Both scoring methods revealed strikingly more outgrowth in the enriched relative to both disenriched populations in all four donors ( Figure 6A ) .", "In fact , most disenriched populations did not result in any outgrowth even at the highest concentration tested ( 200 , 000 cells/well ) .", "The enriched populations also harbored more replication-competent HIV than total memory CD4+ T cells ( Figure 6—figure supplement 2 ) .", "Because recent studies suggest that the in vivo reservoir is transcriptionally active ( Yukl et al . , 2018 ) , we assessed HIV transcription initiation , elongation , and completion in the sorted populations .", "In the absence of stimulation , HIV transcriptional activity was observed in the sorted cells , with more transcription initiation in the enriched than disenriched ones in 7/8 instances ( Figure 6B ) .", "These results suggest that populations highly enriched for the replication-competent reservoir initiate HIV transcription in the absence of stimulation .", "We then assessed to what extent our 8–10 marker panels enrich for latent cells harboring intact genomes .", "We sequenced by FLIPS ( Hiener et al . , 2017 ) proviruses from the enriched and from one of the disenriched populations , for each of three donors .", "Consistent with the PP-SLIDE and outgrowth results , enriched cells harbored higher frequencies of intact p24 sequences than disenriched ones ( Figure 6C ) .", "To assess the extent of clonal expansion , a major driver of HIV persistence ( Cohn et al . , 2020; Liu et al . , 2020 ) , we determined the proportions of expanded identical sequences ( EIS ) .", "EIS were prominent in both populations , but the proportions of EIS with intact p24 sequences were consistently higher in the enriched ones ( Figure 6—figure supplement 3 ) .", "A phylogenetic analysis of the sequences revealed that in two of the donors ( PID1695 and PID5003 ) , EIS from the enriched and disenriched populations formed distinct lineages , while in donor PID3010 multiple EIS were shared between the enriched and disenriched populations ( Figure 6—figure supplement 4 ) .", "As the enriched population from PID5003 harbored a particularly high frequency of intact p24 sequences , we analyzed this donor in more detail .", "We categorized all the sequences analyzed from this individual into those that were full-length intact ( i . e . no defects along the entire proviral genome ) , and those harboring large internal deletions , hypermutations , premature stop codons , and inversions .", "Remarkably , 65 . 2% of the enriched population was fully intact , compared to only 0 . 9% for the disenriched population ( Figure 6D ) .", "This corresponded to an overall frequency of 214 full-length intact sequences per million in the enriched population , as compared to only two per million in the disenriched population .", "The vast majority ( 95 . 5% ) of the proviruses in the disenriched population harbored large internal deletions .", "EIS were prominent among both populations ( Figure 6E ) .", "Among the EIS in the enriched population , 66 . 7% were fully intact , compared to 0% in the disenriched population .", "Fully-intact EIS in the enriched population comprised two groups , suggesting two major expansions of replication-competent reservoir cells in this population of cells highly enriched for replication-competent HIV ( Figure 6E ) .", "Collectively , these results show that surface markers identified by PP-SLIDE can be used to enrich for replication-competent latent cells and that these cells are enriched for the transcriptionally active reservoir and exhibit clear evidence of clonal expansion of fully-intact proviruses .", "The sort panels implemented above were tailored for each donor specimen .", "Given that some features of kNN latent cells are shared between individuals ( Figure 3 ) , we set out to design a universal panel to enrich for latent cells in a donor-independent fashion .", "We designed a sorting strategy incorporating eight surface antigens that enrich for kNN latent cells from all four blood specimens analyzed ( Figure 6—figure supplement 5 ) .", "As TIGIT was found to be expressed at high levels on kNN latent cells in some but not all donors ( Figure 6—figure supplement 5 ) , we designed a strategy to isolate two enriched populations – one TIGIThigh and one TIGITmed/low – to test the hypothesis that TIGIT is indeed not a marker of latent cells shared between donors .", "These populations were compared to a PD1+ population disenriched for kNN latent cells ( Figure 6—figure supplement 5 ) .", "We applied this ‘universal’ panel on four donor blood specimens ( Supplementary file 2 ) that had not undergone PP-SLIDE analysis ( Figure 6—figure supplement 6 ) .", "Viral outgrowth assays revealed robust outgrowth in the enriched but not disenriched populations ( Figure 6F ) .", "Furthermore , the TIGIThigh population harbored more viruses in some donors and the TIGITmed/low one in others , validating the notion that TIGIT is not a marker of latent cells shared between donors .", "These results suggest that shared features between kNN latent cells can be exploited to enrich for the replication-competent latent reservoir across donors ." ], [ "Although the latent reservoir has long been recognized as a major barrier to HIV cure , the phenotype of these latently infected cells in vivo has remained somewhat obscure .", "A common viewpoint , based on the limited number of studies that have conducted viral outgrowth assays on sorted cell populations , is that latent HIV persists in all types of CD4+ T cells with no preference for one subset over another .", "If so , targeting latently infected cells as a curative strategy would be all the more difficult .", "Taking advantage of recent developments in high-parameter single-cell phenotyping and our PP-SLIDE analysis pipeline , we now show that latently infected cells are not randomly distributed among CD4+ T cells , and provide an in-depth view of the phenotypic features of the in vivo blood and tissue reservoir .", "Multiple observations make us confident that PP-SLIDE identifies the physiologically relevant reservoir .", "First , our in vitro assay with J-Lat cells showed the method could predict the correct precursor cell 99 . 2% of the time .", "Second , cells sorted based on PP-SLIDE were enriched for genome-intact and replication-competent reservoir cells in the expected populations .", "Of note , our PP-SLIDE characterization had charted the translation-competent reservoir , defined as latent cells able to produce viral proteins upon stimulation ( Baxter et al . , 2018 ) , which may include some replication-incompetent HIV provirus ( Baxter et al . , 2016; Imamichi et al . , 2020 ) .", "Nevertheless , our viral outgrowth results closely aligned with the enrichment of kNN latent cells , confirming that PP-SLIDE identifies markers of the replication-competent HIV reservoir most relevant for a cure .", "Third , using our ‘universal panel , ’ we validated the preferential presence of the replication-competent reservoir in expected populations in four untested donors , and PP-SLIDE’s prediction that the latent reservoir is preferentially maintained in TIGIT-expressing cells in some but not all donors .", "The sorting strategies used in our validation experiments were designed to enable comparison of different populations of memory CD4+ T cells expressing high levels of PD1 , a marker preferentially expressed on reservoir cells ( Banga et al . , 2016; Chomont et al . , 2009; Fromentin et al . , 2016; Pardons et al . , 2019 ) and which we found expressed at high levels on kNN latent cells .", "This was implemented for two reasons: First , to demonstrate that we can enrich for latent cells beyond the selection afforded by PD1 alone .", "Indeed both our viral outgrowth and proviral sequencing results confirmed PP-SLIDE’s prediction that some but not all PD1-expressing memory CD4+ T cells harbor high frequencies of latent HIV .", "Second , to enable mechanistic comparisons of highly similar populations of sorted cells harboring vastly different levels of replication-competent HIV .", "We found that in 7/8 cases , the enriched populations initiated more HIV transcription than their disenriched counterparts , suggesting that the transcriptionally active reservoir associates most closely with the replication-competent reservoir and therefore should be a target of HIV curative strategies .", "We also found that relative to disenriched populations , the enriched ones harbored higher proportions of EIS with intact p24 , consistent with the importance of clonal expansion in driving the maintenance of the replication-competent HIV reservoir ( Bui et al . , 2017; Hosmane et al . , 2017; Lorenzi et al . , 2016 ) , although it should be noted that cells with different integration sites can harbor the same proviral sequence ( Patro et al . , 2019 ) .", "Of particular interest was our in-depth comparison of two PD1-expressing memory CD4+ T cell populations from PID5003 .", "A striking 65 . 2% of proviruses in the enriched population from this donor were fully intact , versus 0 . 9% in the disenriched , despite both being PD1 expressors .", "To put these numbers in perspective , on average only 2–3% of sequences from infected individuals treated during chronic infection are intact ( Bruner et al . , 2016; Hiener et al . , 2017 ) .", "The overall frequency of intact provirus in the enriched PID5003 population was also extremely high: at 214 per million cells , it was more than an order of magnitude higher than what is observed in Tem ( range = 1–26 , median , 13 per million ) , the subset with the highest frequencies of intact provirus as assessed by FLIPS ( Hiener et al . , 2017 ) .", "This high frequency is not due to an unusually large overall reservoir in this donor , as the phenotypically-similar ( PD1+ ) disenriched population sequenced from this donor exhibited a frequency of only two per million .", "Our observation that this enriched population , but not the disenriched one , exhibited robust viral outgrowth , further suggests that the intact proviruses in this population are replication-competent .", "Interestingly , the intact sequences in this enriched population consisted of two large clusters of EIS , suggesting two major clonal expansions in this well-defined population of memory CD4+ cells sorted based on eight surface markers .", "As these two clusters of viral sequences likely integrated at different genomic sites , these observations support a host rather than virus-driven expansion of these latent cells .", "That we could find such vastly different levels of intact and replication-competent reservoir cells in our enriched versus disenriched populations goes against the notion that the reservoir is ‘everywhere’ and randomly distributed in all subsets .", "But how do our observations fit with prior reports that the frequencies of replication-competent provirus , as determined from outgrowth assays , were similar between sorted subsets ( Buzon et al . , 2014; Kwon et al . , 2020 ) ?", "We believe that the previously sorted subsets were not ones that differ markedly in reservoir frequencies .", "Most prior subset analyses had focused on the Tem and Tcm subsets , and when focusing on just these subsets our PP-SLIDE results indeed support the notion that both Tem and Tcm were prominent among kNN latent cells .", "However , our finding that the proportions of Tem were greater among the kNN latent cells than among atlas cells suggests that latency is somewhat biased toward the Tem subset , a finding in line with the higher prevalence of intact provirus in Tem over Tcm ( Hiener et al . , 2017 ) .", "Further supporting a non-random distribution of the reservoir is our identification of markers consistently expressed at higher levels on kNN latent cells relative to memory CD4+ T cells .", "Such markers include ones previously implicated in latency ( PD1 , CTLA4 , Ox40 ) ( Banga et al . , 2016; Fromentin et al . , 2016; Kuo et al . , 2018; McGary et al . , 2017 ) , some activation markers ( CD25 , CD69 ) , and markers of Th1 , Th2 , and Th17 cells ( Tbet , CRTh2 , and CCR6 , respectively ) suggesting preferential persistence of HIV in more differentiated cells .", "The reservoir may persist in these cells due to antigen- or homeostatically-driven expansion of these cells which can maintain the reservoir through repeated cycles of clonal expansion .", "Our analysis of longitudinal specimens by PP-SLIDE also provided insights into the stability of the reservoir and its capacity for reactivation .", "By comparing kNN latent cells from different donors versus longitudinal samples , we found intra-individual variability to be less than inter-individual variability .", "This observation implies that the phenotypic properties of the reservoir are stable over time , a feature that may facilitate its targeting .", "In fact , longitudinal stability also held for the reservoir’s susceptibility to the LRAs we tested .", "Our observation that different stimulatory signals reactivate different latent cells further suggests that ‘shock and kill’ strategies may require multiple LRAs , each preferentially targeting different cell subsets , as recently suggested ( Baxter et al . , 2016; Grau-Expósito et al . , 2019; Pardons et al . , 2019 ) .", "Although our study focused on the blood reservoir for logistical and technical reasons , we also conducted PP-SLIDE analyses on tissue specimens .", "Perhaps not surprising given the known phenotypic differences between T cells from blood versus tissues ( Wong et al . , 2015 ) , we found that blood and tissue kNN latent cells differ quite dramatically .", "High-dimensional distance calculations revealed that kNN latent cells were more similar between lymph node and gut than between the tissues and blood , suggesting phenotypic commonalities in the latent reservoir found in tissues .", "Indeed , kNN latent cells from both lymph node and gut expressed high levels of the Trm marker CD69 , suggesting that Trm in these tissues , like those from the cervix ( Cantero-Pérez et al . , 2019 ) , preferentially harbor the reservoir .", "Together with our observation that kNN latent cells from all sites examined expressed high levels of PD1 , these results suggest PD1-expressing Trm as possible targets to eliminate the tissue reservoir .", "Given recent findings that blood , lymph nodes , and gut are the major sources of HIV dissemination in ART-suppressed individuals ( Chaillon et al . , 2020 ) , targeting latent cells in the compartments analyzed in this study may be sufficient to make a meaningful reduction in the reservoir .", "Our study has some limitations , in particular , a relatively small sample size of only eight blood and five tissue donors , although some donors were characterized longitudinally .", "Nonetheless , our findings do not support the existence of a single surface biomarker that can be used to target the entire HIV reservoir at once .", "However , PP-SLIDE enables in-depth charting of the latent reservoir residing in the blood and , likely , any tissue we can access .", "For therapeutic applications , surface antigens identified by PP-SLIDE as preferentially co-expressed on latent cells can be targeted with a series of bi- or multi-specific antibodies ( Klein et al . , 2016 ) .", "More importantly , our ability to isolate highly enriched populations of replication-competent reservoir cells enables in-depth functional analyses of the reservoir and potentiates future implementation of discovery-based approaches such as scRNAseq and PCR-activated cell sorting ( PACS; Bradley et al . , 2018; Clark and Abate , 2017 ) .", "These approaches hold great promise for identifying novel markers for HIV eradication but require a high frequency of latent cells that was not achievable previously .", "We envision that implementing PP-SLIDE on cells phenotyped by scRNAseq together with Antibody-seq approaches ( Peterson et al . , 2017; Stoeckius et al . , 2017 ) will enable even higher-resolution mapping , with the added advantage that whole-transcriptome approaches do not require pre-defining markers of interest , and allow for the discovery of unanticipated—and perhaps unique—markers of latent cells ." ], [ "Multiple leukapheresis samples were obtained from eight HIV-1 subtype-B individuals on long-term ART who initiated therapy during chronic ( >1 year ) infection and were stably suppressed ( HIV RNA <40 copies/mL; Supplementary file 2 ) .", "Leukapheresis specimens from four of these eight individuals were subjected to full analyses using CyTOF/PP-SLIDE .", "Lymph node specimens were obtained from one of these four participants under local anesthesia using ultrasound-guided fine needle aspiration .", "All fine needle aspirate ( FNA ) procedures were performed without any adverse effects , and the subject reported minimal to no discomfort both during and after the procedure and was able to perform their usual activities after the procedure .", "Gut specimens were obtained by sigmoid biopsies from four ART-suppressed HIV-infected participants ( Supplementary file 2 ) .", "All participants provided informed consent before participation .", "The study was approved by the University of California , San Francisco ( IRB # 10–01330 ) and the University of North Carolina ( IRB # 12–1660 ) .", "Memory CD4+ T cells from the blood of two HIV-seronegative donors were purified as described above , and then resuspended at 2 × 107 cells/mL in PBS containing 0 . 1% FBS .", "The cells were then loaded for 8 min with 1 . 5 μM of the proliferation dye CFSE ( ThermoFisher ) .", "Labeling was then stopped by the addition of an equal volume of pre-warmed , 100% FBS , and the labeled cells were incubated at 37°C for an additional 10 min .", "The sample was then washed three times in RP10 .", "Memory CD4+ T cells not exposed to CFSE were treated identically in parallel .", "The time = 0 specimen was immediately stained as described below .", "The remaining cells were cultured with or without activation with 16 nM PMA , 1 μM ionomycin , and 100 IU/mL IL2 ( all three reagents from Thermofisher ) .", "At the indicated time points , cells were stained with APC/Cyanine7 anti-human CD3 ( Clone SK7 , Biolegend ) , PE/Cy7 anti-human CD4 ( Clone A161A1 , Biolegend ) , Alexa Fluor 700 anti-human CD8 ( Clone SK1 , Biolegend ) , and Zombie Red ( Biolegend ) as a Live/Dead discriminator .", "Stained cells were fixed and analyzed by FACS on an LSRII ( BD Biosciences ) .", "Flowjo ( BD Biosciences ) was used for analysis .", "Live , singlet CD3+CD4+CD8- cells were assessed for proliferation by monitoring the loss of CFSE signal .", "Results shown are from one of two donors which gave similar results .", "293 T cells were seeded in 6-well plates ( Falcon ) at a concentration of 3 × 105 cells/well , and transfected the next day using FuGENE ( Promega ) with 0 . 5 μg/well of the F4 . GFP or F4 . HSA proviral constructs previously described ( Cavrois et al . , 2017; Ma et al . , 2020; Neidleman et al . , 2017 ) .", "Supernatants from the cultures were harvested after 2 days and p24Gag concentrations were measured with a Lenti-X p24 Rapid Titer kit ( Clontech ) .", "To demonstrate that the cocktail of ART used in this study was fully suppressive , PBMC-derived CD4+ T cells from HIV-seronegative donors were activated for 2 days with RP10 containing 10 μg PHA ( PeproTech ) and 100 IU/mL IL2 ( Thermofisher ) , washed with RP10 , and then cultured in 20 IU/mL IL2 for one additional day .", "The cells were then either left untreated , or pretreated for 1 hr with an ART cocktail consisting of 5 μM AZT , 5 μM Ritonavir , 8 nM Efavirenz , 10 μM Lamivudine , 50 nM Raltegravir , and 0 . 5 μg/mL T-20 ( all from NIH AIDS reagent program ) .", "Cells were then cultured for 4 days at a concentration of 2 × 106 cells/mL with RP10 alone or in the presence of 250 ng/mL p24Gag F4 . GFP in RP10 containing 20 IU/mL IL2 .", "The cells were then stained for 30 min at 4°C at a concentration of 106 cells/well in FACS buffer , with anti-CD3 APC/Cyanine7 , anti-CD4 PE/Cy7 , anti-CD8 Alexa Fluor 700 , and Zombie Aqua as a Live/Dead discriminator ( all from Biolegend ) .", "The cells were then washed three times with FACS buffer and fixed overnight in 2% PFA ( Electron Microscopy Sciences ) .", "The cells were analyzed on an LSRII ( BD Biosciences ) and Flowjo software was used to gate on live , singlet CD3+CD8- cells and infection rates in these cells were monitored by GFP expression .", "For atlas generation , we used freshly isolated and purified memory CD4+ T cells from blood , or freshly isolated total cells from tissues ( as described above ) .", "A portion of these cells was immediately treated with cisplatin ( Sigma-Aldrich ) as a Live/Dead marker and fixed .", "Briefly , 6 × 106 cells were resuspended at room temperature in 2 mL PBS ( Rockland ) with 2 mM EDTA ( Corning ) .", "Next , 2 mL of PBS containing 2 mM EDTA and 25 μM cisplatin ( Sigma-Aldrich ) were added to the cells .", "The cells were quickly mixed and incubated at room temperature for 60 s , after which 10 mL of CyFACS ( metal contaminant-free PBS [Rockland] supplemented with 0 . 1% FBS and 0 . 1% sodium azide [Sigma-Aldrich] ) was added to quench the reaction .", "The cells were then centrifuged and resuspended in 2% PFA in CyFACS and incubated for 10 min at room temperature .", "The cells were then washed twice in CyFACS , after which they were resuspended in 100 μL of CyFACS containing 10% DMSO .", "These fixed cells were stored at −80°C until analysis by CyTOF .", "The rest of the freshly isolated cells were stimulated to allow for latent cell reactivation .", "These cells were diluted to 2 × 106 cells/mL and stimulated with 16 nM PMA ( Sigma-Aldrich ) and 1 μM ionomycin ( Sigma-Aldrich ) for 40 hr .", "This stimulation time was chosen as it is similar to times previously implemented ( Cohn et al . , 2018 ) and shorter stimulations under these conditions did not lead to reactivation .", "Where indicated , cells were instead stimulated with Dynabeads Human T-Activator CD3/CD38 beads ( Gibco ) , or 12 nM PEP005 ( Ingenol 3-angelate , Sigma-Aldrich ) with 40 nM romidepsin ( Sigma-Aldrich ) .", "For gut specimens , to minimize cellular toxicity , cells were instead stimulated for 16 hr with 160 nM PMA and 1 μM ionomycin .", "All stimulations were conducted in the presence of 100 IU/mL IL2 ( Thermofisher ) .", "To prevent spreading infection , a cocktail of ART consisting of 5 μM AZT , 5 μM Ritonavir , 8 nM Efavirenz , 10 μM Lamivudine , 50 nM Raltegravir , and 0 . 5 μg/mL T-20 was added .", "To limit the death of reactivated cells , 10 μM of the pan-caspase inhibitor Z-VAD-FMK ( R and D Systems Inc ) was added as previously described ( Cohn et al . , 2018; Grau-Expósito et al . , 2019 ) .", "Staining of cells for analysis by CyTOF was conducted similar to recently described methods ( Cavrois et al . , 2017; Ma et al . , 2020; Trapecar et al . , 2017 ) .", "Briefly , cisplatin-treated cells were thawed and washed in Nunc 96 DeepWell polystyrene plates ( Thermo Fisher ) with CyFACS buffer at a concentration of 6 × 106 cells/800 μL per well .", "Cells were then pelleted and blocked with mouse ( Thermo Fisher ) , rat ( Thermo Fisher ) , and human AB ( Sigma-Aldrich ) sera for 15 min at 4°C .", "The samples were then washed twice in CyFACS , pelleted , and stained in a 100 μL cocktail of surface antibodies ( Supplementary file 1 ) for 45 min at 4°C .", "The samples were then washed 3× with CyFACS and fixed overnight at 4°C in 100 μL of freshly prepared 2% PFA in PBS ( Rockland ) .", "Samples were then washed twice with Intracellular Fixation and Permeabilization Buffer ( eBioscience ) and incubated in this buffer for 45 min at 4°C .", "Next , samples were washed twice in Permeabilization Buffer ( eBioscience ) .", "The samples were then blocked for 15 min at 4°C in 100 μL of mouse and rat sera diluted in Permeabilization Buffer , washed 1× with Permeabilization buffer , and incubated for 45 min at 4°C in a 100 μL cocktail of intracellular antibodies ( Supplementary file 1 ) diluted in Permeabilization Buffer .", "The cells were then washed with CyFACS and stained for 20 min at room temperature with 250 nM of Cell-ID Intercalator-IR ( Fluidigm ) .", "Finally , the cells were washed 2× with CyFACS buffer , once with MaxPar cell staining buffer ( Fluidigm ) , and once with Cell acquisition solution ( CAS , Fluidigm ) , and then resuspended in EQ Four Element Calibration Beads ( Fluidigm ) diluted to 1× in CAS .", "The sample concentration was adjusted to acquire at a rate of 200–350 events/sec using a wide-bore ( WB ) injector on a CyTOF2 instrument ( Fluidigm ) at the UCSF Parnassus flow core facility .", "For tissue specimens where multiple specimens were combined to minimize cell loss , the Cell-ID 20-Plex Pd Barcoding Kit ( Fluidigm ) was used according to the manufacturer’s instructions to barcode the individual cisplatin-treated samples before mixing .", "Briefly , the frozen and fixed samples were thawed and washed once with CyFACS and 2× with Maxpar Barcode Perm Buffer ( Fluidigm ) , and then resuspended in 800 μL of Barcode Perm Buffer .", "A total of 10 μL of each barcode was diluted in 100 μL of Barcode Permeabilization Buffer and added to each sample .", "The samples were barcoded for 30 min at room temperature , washed once with MaxPar cell staining buffer , and once with CyFACS .", "The samples were then combined subjected to the CyTOF staining protocol as described above .", "Memory CD4+ T cells purified from cryopreserved PBMCs as described above were sorted using the following antibodies .", "Antibody clone numbers are indicated in the table further below: Dead cells were excluded using the Zombie Aqua fixable viability kit ( BioLegend ) , while doublets were excluded based on FSC-A/FSC-H .", "Cells were sorted on an Aria FACS sorter ( Becton Dickinson ) .", "The purity of the subsets was confirmed by analysis on the Aria immediately post-sort .", "Antigen targetCloneVendor ( catalog number ) Live/Dead ( Zombie Aqua ) BioLegend ( #423102 ) PE-CF594 Mouse Anti-Human CD27M-T271BD ( #562297 ) BUV737 Mouse Anti-Human CD38HB7BD ( #564686 ) BUV395 Mouse Anti-Human CD45ROUCHL1BD ( #564291 ) FITC Mouse Anti-Human CD57NK-1BD ( #555619 ) BV650 Mouse Anti-Human CD62LDREG-56BD ( #563808 ) APC Mouse Anti-Human CD69FN50BioLegend ( #310910 ) BV421 Mouse Anti-Human CD69FN50BD ( #562884 ) APC Mouse Anti-Human CD103Ber-ACT8BD ( #563883 ) APC-R700 Rat Anti-Human CXCR5 ( CD185 ) RF8B2BD ( #565191 ) PE-Cy7 Mouse Anti-Human CD134 ( OX40 ) ACT35BD ( #563663 ) PE Mouse anti-Human CD279 ( PD-1 ) EH12 . 1BD ( #560795 ) BUV737 Mouse Anti-Human CD28CD28 . 2BD ( #612815 ) BB515 Mouse Anti-Human CD49d9F10BD ( #564593 ) PE/Dazzle 594 Mouse Anti-Human CD197 ( CCR7 ) G043H7BioLegend ( #353236 ) Brilliant Violet 421 Rat Anti-Human CD195 ( CCR5 ) J418F1BioLegend ( #359118 ) PE-Cy7 Mouse Anti-Human CD57TB01eBioscience ( 25-0577-42 ) BUV395 Mouse Anti-Human CD252A3BD ( #564034 ) APC-H7 Mouse Anti-Human CD7M-T701BD ( #564020 ) BV605 Mouse Anti-Human CD127HIL-7R-M21BD ( #562662 ) APC Mouse Anti-Human TIGITA15153GBioLegend ( #372706 ) PE-Cy5 Mouse Anti-Human CD28CD28 . 2BioLegend ( #302910 ) APC-Cy7 Mouse Anti-Human CD69FN50BD ( #560737 ) BV421 Mouse Anti-Human CD2TS1/8BioLegend ( #309218 ) PE-Cy7 Mouse Anti-Human CD57TBO1Invitrogen ( #25-0577-42 ) Cells sorted from blood were cultured at a density of 106 cells/mL in RPMI in the presence of 60 IU/mL recombinant human IL-2 ( Thermofisher ) , and activated with Dynabeads Human T-Activator CD3/CD28 ( Gibco ) at a ratio of 1 bead/cell .", "Of note , all populations of cells were sorted before stimulation to account for any effects of the sorting process on viral outgrowth .", "After 48 hr of culture , cells were diluted to a concentration of 105 cells/mL in RPMI supplemented with 60 IU/mL recombinant human IL-2 , and then plated at 2 × 105 cells per well ( dilution A ) , 4 × 104 cells per well ( dilution B ) , 8 × 103 cells per well ( dilution C ) , and 1 . 6 × 103 cells per well ( dilution D ) .", "Molt4/CCR5 cells ( Laird et al . , 2013 ) were then added at 105 cells per well .", "Cultures were replenished at days 3 , 6 , 12 , and 15 by replacing half of the media with RPMI containing 5 IU/mL IL-2 without disturbing the cellular layer .", "On day 9 , half the cultures , inclusive of both cells and media , were discarded , and the remaining cells were replenished with the same volume of RPMI containing 105 Molt4/CCR5 cells .", "At the end of the culture period ( days 15–18 ) , supernatants were quantitated for levels of p24 antigen by ELISA ( Takara ) .", "A viral outgrowth assay was conducted for one of the gut specimens ( PID1223 ) to demonstrate the presence of replication-competent virus in the tissue .", "To this end , a Digital ELISA Viral Outgrowth ( DEVO ) assay was implemented .", "Pooled biopsy cells were plated in a limiting dilution format at five replicates at 470 , 000 cells per well , 12 replicates at 100 , 000 cells per well , and 12 replicates at 25 , 000 cells per well .", "Cells were stimulated for 24 hr with 3 µg/mL phytohemagglutinin ( PHA ) ( Thermofisher ) , 100 IU/mL IL-2 , and irradiated PBMCs from an HIV-seronegative donor .", "The cells were washed and Molt4/CCR5 target cells were added to amplify outgrowth of HIV .", "The cultures were replenished with fresh media every 3–4 days , and another round of Molt4/CCR5 cells added on day 9 post-stimulation .", "Supernatants were harvested on days 9 , 13 , 16 , 20 , 26 , and 29 post-stimulation and assessed for HIV p24 using the SIMOA HD-1 Analyzer ( Quanterix , Billerica , MA ) .", "Only wells exhibiting sustained and increasing p24 expression over time were scored as positive .", "The frequency of infection reported as Infectious Units Per Million ( IUPM ) of rectal-sigmoid cells was calculated using the SLDAssay R software package ( Trumble et al . , 2017 ) .", "The IUPM from this analysis was 0 . 265 , with 95% confidence intervals between 0 . 0216 and 1 . 400 .", "Of note , this IUPM corresponds to that of total gut cells .", "Pellets of sorted cells were lysed in 1 mL TRI reagent with 2 . 5 μL polyacryl carrier ( Molecular Research Center ) .", "Total cellular RNA was subsequently extracted per the TRI Reagent protocol , while total cellular DNA was extracted using the ‘back extraction buffer’ ( 4M guanidine thiocyanate , 50 mM sodium citrate , 1M Tris ) as described ( Yukl et al . , 2018 ) .", "A common reverse transcriptase ( RT ) reaction was used to generate cDNA for all droplet digital PCR ( ddPCR ) assays except TAR , where a separate two-step RT was performed as described ( Yukl et al . , 2018 ) .", "Briefly , each 50 µL RT reaction contained cellular RNA , 5 µL of 10× Superscript III buffer ( Invitrogen ) , 5 µL of 50 mM MgCl2 , 2 . 5 µL of 50 ng / µL random hexamers ( Invitrogen ) , 2 . 5 µL of 50 µM dT15 , 2 . 5 µL of 10 mM dNTPs , 1 . 25 µL of 40 U / µL RNaseOUT ( Invitrogen ) , and 2 . 5 µL of 200 U / µL Superscript III RT ( Invitrogen ) .", "PBMCs from uninfected donors , and water that was subjected to nucleic extraction by TRI Reagent , served as negative controls for each transcript .", "The thermocycling conditions were as follows: 25°C for 10 min , 50°C for 50 min , and an inactivation step at 85°C for 5 min .", "For the ddPCR reactions , cDNA from each sample was assayed in duplicate wells for TAR , Long LTR , Pol , and PolyA regions using validated assays ( Kaiser et al . , 2017; Telwatte et al . , 2018; Yukl et al . , 2018 ) .", "Total cell equivalents in the DNA extracted from the same samples were determined by measuring the absolute copy numbers of a nonduplicated cellular gene , Telomere Reverse Transcriptase ( TERT ) , similar to previously described methods ( Telwatte et al . , 2018; Yukl et al . , 2018 ) .", "To account for the effects of deletions or hypermutations on the HIV RNA measurements , all HIV RNAs were further normalized to HIV DNA measured by the same assay used for the RNA .", "Each 20 µL ddPCR reaction contained 5 µL cDNA or DNA , 10 µL of ddPCR Supermix for Probes ( no dUTP ) ( Bio-Rad , Hercules ) , 900 nM of primers , and 250 nM of probe .", "Following production of droplet emulsions using the QX100 Droplet Generator ( Bio-Rad ) , samples were amplified under the following thermocycling conditions using a 7900 Thermal Cycler ( Life Technologies ) : 10 min at 95°C , 45 cycles of 30 s at 95°C and 59°C for 60 s , and a final droplet cure step of 10 min at 98°C .", "Droplets were quantified using the QX100 Droplet Reader ( Bio-Rad ) and analyzed using the QuantaSoft software ( version 1 . 6 . 6 , Bio-Rad ) in the ‘Rare Event Detection’ quantification mode .", "The FLIPS assay was performed as previously reported ( Hiener et al . , 2017 ) .", "Briefly , HIV-1 proviruses within sorted CD4+ T cell subsets were amplified and sequenced at limiting dilution to near full-length ( 9 kb; 92% of the genome ) .", "A median of 140 individual proviruses ( range 18–254 ) was sequenced per sorted population of cells from three of the four participant blood specimens analyzed by PP-SLIDE .", "The next-generation sequencing was conducted using the Illumina MiSeq platform with individual proviruses de novo assembled using a specifically designed workflow in CLC Genomics .", "Proviruses were characterized as defective if they contained INDELs , stop codons , or APOBEC3G hypermutations , or intact ( full-length ) if they lacked such defects .", "Expanded identical sequences ( EIS ) were identified using ElimDupes from the Los Alamos database ( https://www . hiv . lanl . gov/content/sequence/elimdupesv2/elimdupes . html ) .", "All sequences that were 100% identical were considered part of an EIS cluster .", "Maximum likelihood phylogenetic trees using the generalized time-reversible model were estimated for each participant using Geneius .", "Branch support was inferred using 1000 bootstrap replicates .", "Annotated tree images were constructed using the iTOL software Version 5 . 5 . 1 ( Letunic and Bork , 2019 ) .", "The CyTOF data were exported as FCS files , and samples were de-barcoded according to manufacturer’s instructions ( Fluidigm ) .", "For the J-Lat experiments , equal numbers of the unstimulated live , singlet intact 6 . 3 and 5A8 were concatenated together as a source of the ‘atlas cells’ .", "Reactivated 6 . 3 cells were identified by gating on the Gag-expressing cells in the stimulated 6 . 3 sample .", "For patient samples , events corresponding to live , singlet intact CD3+CD19-CD8- T cells from unstimulated samples were exported as a source of the atlas cells .", "Events corresponding to reactivated cells were identified by gating upon live , singlet intact CD3+CD19-CD8- T cells expressing Gag on all three Gag channels , and exported as the population of reactivated cells .", "Where indicated , naïve cells ( CD45RA+CD45RO- ) were gated out before export .", "Data export was conducted using FlowJo ( BD Biosciences ) and Cytobank software .", "tSNE analyses were performed using the Cytobank software with default settings .", "All cellular markers not used in the upstream gating strategy were included in generating the tSNE plots .", "Non-cellular markers ( e . g . live/dead stain ) and the HIV gag proteins were excluded for the generation of tSNE plots .", "Dot plots were generated using both Cytobank and FlowJo .", "PP-SLIDE analysis to identify kNN latent cells followed our previously described method ( Cavrois et al . , 2017; Ma et al . , 2020 ) .", "Using a custom script in R ( source code made available as part of manuscript ) , each reactivated ( Gag+ ) cell from the stimulated sample was mapped against every cell in the unstimulated atlas generated immediately after sample procurement , to identify the kNN latent cells for each patient sample .", "The steps implemented for PP-SLIDE are summarized below: To assess the intra-individual and inter-individual variability in kNN latent cells and to compare kNN latent cells reactivated following treatment with PMA/ionomycin , anti-CD3/CD28 , and Romidepsin/PEP005 , empirical cumulative distributions were calculated .", "The distributions of median distances in 32-dimensional space ( corresponding to measured protein levels of antigens not used in the upstream gating strategy ) corresponded to those between cells of the indicated pairs of samples .", "The median ( Euclidean ) distance over all cells in the first sample was computed for each cell in the second sample using their corresponding 32-dimensional protein level vectors .", "Then , similarly , the median ( Euclidean ) distance over all cells in the second sample was computed for each cell in the first sample using their corresponding 32-dimensional protein level vectors .", "The union of these two sets of median distances was used to determine the distribution of median distances between the sample pair .", "These resulting datasets were visualized in terms of a cumulative distribution function using the stat_ecdf function of the ggplot2 package in R . This section describes in detail how FlowSOM was implemented to cluster the atlas and kNN latent cells , and how this information was used to demonstrate non-random distribution of kNN latent cells among memory CD4+ T cells .", "For simplicity , we have illustrated the process with one of the four leukapheresis donors ( PID1695 ) , but similar methods were implemented for the other three .", "The FlowSOM analysis for all four donor specimens revealed a non-random distribution of kNN latent cells among memory CD4+ T cells .", "TermDescriptionNlatent ( Cluster DX ) Number of kNN latent cells in Cluster DXPlatent ( Cluster DX ) Percent of kNN latent cells belonging to Cluster DXPlatent_null ( Cluster X ) Percent of kNN latent cells belonging to Cluster X under null hypothesis of stochastic distributionNlatent_null ( Cluster X ) Number of kNN latent cells in Cluster X under null hypothesis of stochastic distributionNday0 ( Cluster DX ) Number of atlas cells in Cluster X that were runNatlas ( Cluster DX ) Number of atlas cells in Cluster X that would have been run to capture 20 reactivated cellsFlatent ( Cluster DX ) Frequency of kNN latent cells per million cells of Cluster DX" ] ]

[ "The latent reservoir is a major barrier to HIV cure .", "As latently infected cells cannot be phenotyped directly , the features of the in vivo reservoir have remained elusive .", "Here , we describe a method that leverages high-dimensional phenotyping using CyTOF to trace latently infected cells reactivated ex vivo to their original pre-activation states .", "Our results suggest that , contrary to common assumptions , the reservoir is not randomly distributed among cell subsets , and is remarkably conserved between individuals .", "However , reservoir composition differs between tissues and blood , as do cells successfully reactivated by different latency reversing agents .", "By selecting 8–10 of our 39 original CyTOF markers , we were able to isolate highly purified populations of unstimulated in vivo latent cells .", "These purified populations were highly enriched for replication-competent and intact provirus , transcribed HIV , and displayed clonal expansion .", "The ability to isolate unstimulated latent cells from infected individuals enables previously impossible studies on HIV persistence ." ]

[ "There is no cure for the human immunodeficiency virus infection ( HIV ) , but anti-retroviral drugs allow infected people to keep the virus at bay and lead a normal life .", "These drugs suppress the growth of HIV , but they do not eliminate the virus .", "If the treatment is interrupted , the virus bounces back within weeks in most individuals .", "HIV can start growing again because it hides within particular immune cells , called T cells .", "These infected cells stay in the infected person’s body for their whole life in a dormant or “latent” state , and represent the main barrier to an HIV cure .", "If these cells could be eliminated or prevented from producing more virus without daily treatment , then HIV could be cured .", "The fact that HIV hides inside T cells has been known for a long time , but it has remained unclear exactly what kinds of T cells the virus prefers .", "One challenge to characterizing latently-infected cells is that there is no single protein made by them that is not also made by uninfected T cells .", "The latently-infected T cells are also very rare: HIV mainly attaches to a protein called CD4 , but only one in about a million T cells with CD4 contain the virus .", "To figure out which CD4-carrying T cells in a patient sample are latently infected , the cells are extracted from the patient’s body and ‘reactivated’ so the virus will start growing again .", "Unfortunately , the mixture of drugs used to reactivate the T cells changes the cells and the proteins they are producing , which obscures the features the latently-infected T cells had before reactivation .", "Neidleman , Luo et al . developed a new approach to trace the infected , reactivated T cells back to their state before reactivation .", "Using computational methods and a laboratory technique called mass cytometry , the levels of approximately 40 different proteins were measured in millions of T cells from people living with HIV .", "These experiments provided an ‘atlas’ of overall T cell features onto which each reactivated cell could be mapped .", "The population of latently-infected T cells exhibited common features among all the participants .", "Selecting a few of the most abundant proteins on the surface of the latently-infected cells allowed these cells to be physically separated from all other immune cells .", "In the future , this relatively pure population of infected T cells could be used to study how HIV can persist for many decades .", "The ‘map’ of these cells’ features will provide a valuable resource for HIV researchers and might enable the discovery of new drugs to eliminate the latent T cells ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "ecology", "computational and systems biology" ]

[ [ "Animals produce conspicuous intraspecific communication signals whose intensity is maximised through sexual selection .", "Several insect species , including tree crickets , use sound to advertise for mates .", "The louder the call , the farther it travels and the more detectable and attractive it is to listening females ( Farris et al . , 1997; Forrest , 1991; Mhatre and Balakrishnan , 2007 ) .", "Several strategies exist to enhance the radiation of acoustic energy ( Bennet-Clark , 1999 ) , but the principal strategy insects use is to exploit the mechanical resonance either of adapted body parts , such as wings , or tymbal organs ( Mhatre et al . , 2012; Sueur et al . , 2006 ) , or of their immediate environment as mole crickets do with their calling burrow ( Daws et al . , 1996 ) .", "Tree crickets use a unique and distinct strategy to enhance the reach of their call; they make and use their own acoustic baffles ( Forrest , 1991; Prozesky-Schulze et al . , 1975; Forrest , 1982 ) .", "Tree crickets , like other crickets , produce sound by rubbing raised forewings together and setting them into resonant vibration ( Mhatre et al . , 2012 ) .", "The sound that radiates from either side of the vibrating wing , however , is of opposite phase; positive pressure or compression in the direction of the wing’s motion and rarefaction or negative pressure on the other side ( see Video 1 ) .", "The wing’s sound production capability is thus made inefficient by ‘acoustic short-circuiting’ , whereby the two opposing sound waves emanating from the wing destructively interfere at its edges , thus reducing the total radiated sound pressure ( Forrest , 1991; Prozesky-Schulze et al . , 1975; Forrest , 1982 ) ( Video 1 ) .", "This short-circuiting is particularly potent in tree crickets because their wings are small ( ~9 mm in length ) compared to the wavelength of their call ( ~110 mm at 3 . 1 kHz ) .", "One way to circumvent the limitation is to separate the front and back faces of the sound source ( the resonating wings ) thus precluding deleterious interaction between the two pressure waves .", "A baffle provides exactly such acoustic isolation ( Video 1 ) .", "In effect , male tree crickets cut a hole in a leaf to make a baffle .", "They then place their head and forelegs through the hole and call with their wings parallel to the leaf surface and centred within the hole ( Figure 1A , Video 2 ) .", "The leaf surface area provides acoustic isolation between the two sides , increasing call loudness ( Forrest , 1991; Prozesky-Schulze et al . , 1975 ) .", "Given that the only function of the baffle is to prevent acoustic short circuiting , and not to resonate with the call , a tree cricket could use any leaf , or flat surface for this purpose .", "Here , we show that not all leaves confer equal benefits to sound radiation .", "Baffle design has a profound effect on radiation efficiency , and tree crickets can select the best leaf to make a near optimal baffle .", "We show that we can quantitatively predict the relationship between calling effort ( measured as wing vibration velocity ) , baffle design , and the resulting sound using biomechanical and acoustical measurements , and finite element analysis ( FEA ) .", "Using FEA models , we calculate and test the efficiency of tree crickets calling from different natural baffle designs , accounting for the complex geometries of cricket wings , their self-made baffles , and the leaves crickets chose to call from ." ], [ "To characterize calling effort , wing vibrations during calling were measured using scanning laser Doppler vibrometry .", "Call was recorded simultaneously at a distance of 200 mm ( Figure 1A , Figure 1—figure supplement 1 ) .", "Measurements were made from freely behaving animals who sang when offered pre-made paper baffles ( Figure 1B , C , Materials and methods ) .", "The sound pressure produced by a vibrating structure is dependent on its space and time averaged velocity .", "It also depends on the size of the radiator and the wavelength of the sound being produced and is captured by a constant called the radiator resistance ( Hambric and Fahnline , 2007 ) .", "In tree crickets , the average velocity over the entire wing surface for a fixed period of one second was 0 . 13 ± 0 . 06 m·s−1 , and it produced an average sound pressure level ( SPL ) of 22 . 8 ± 11 . 0 mPa ( 60 . 4 ± 4 . 1 dB ) .", "Wing velocity was thus related to sound pressure level by a constant of 0 . 18 ± 0 . 03 Pa/m/s ( all measures are given as mean ±SD , n = 5 ) .", "To better understand the acoustic benefit offered by baffles , FEA models were developed of a cricket calling from a baffle identical to that used in the vibrometry experiment ( Figure 1—figure supplement 2 ) .", "Vibration measurements served as inputs for FEA models and were used to compute the radiated SPL .", "Models predicted a bi-lobed acoustic field , consistent with previous acoustic measurements ( Figure 2 ) ( Forrest , 1991; Prozesky-Schulze et al . , 1975; Forrest , 1982 ) .", "A male calling in free space with average wing velocity was predicted to produce an SPL of 14 . 6 mPa ( 57 . 2 dB at 200 mm ) ( Figure 2A ) , commensurate with field measurements ( Deb and Balakrishnan , 2014 ) .", "The virtual oval baffle increased SPL at the same distance to 24 . 9 mPa ( 61 . 9 dB ) ( Figure 2B ) , consistent with the actual sound pressures recorded in the experiments ( 22 . 8 ± 11 . 0 mPa or 60 . 4 ± 4 . 1 dB , n = 5 ) .", "Real baffles , however , vary given that crickets encounter leaves of different sizes and produce dissimilar holes .", "Hence , baffles are also expected to vary in their capacity to enhance call amplitude .", "Larger leaves and cricket-size baffle holes are thought to make the best baffles ( Prozesky-Schulze et al . , 1975 ) .", "Using FEA , we produced a quantitative comparison between different baffle designs using a metric we defined , sound radiation efficiency ( SRE ) .", "Males are expected to maximize the reach of their mate attraction calls per unit calling effort .", "Therefore , we defined SRE as the average absolute sound pressure around the calling cricket over a fixed volume normalised to wing vibration amplitude ( expressed in mPa/m∙s−1 , Materials and methods ) .", "SRE was calculated only for the range of leaf and hole sizes that encompasses natural variation ( Figure 3A , Figure 3—figure supplement 1 ) .", "This could also be considered an index of male conspicuousness in terms of active acoustic space ( Deb and Balakrishnan , 2014; Mhatre and Balakrishnan , 2006 ) , and all else being equal , the volume of each male’s active space is covariant with SRE .", "We found that SRE increased with leaf size but became asymptotic for leaves longer than 100 mm , i . e . longer than most natural leaves ( Figure 3A , B ) .", "Thus , males choosing larger leaves would always make better baffles under natural conditions ( Figure 3A ) .", "FEA also predicted that a baffle hole of length ~10 mm , the same as the model wing length ( and the size of a typical male cricket wing ) , was optimal ( Figure 3A ) .", "The maximum SRE of 386 . 3 mPa/m∙s−1 was achieved by a baffle made with a leaf 110 . 2 mm long , with a centrally located hole of length 9 . 7 mm .", "Having constructed a geometrically-resolved map of SRE for feasible baffles , we investigated whether male tree crickets optimized the acoustics of their baffles .", "SRE with a baffle of any size is higher than unbaffled calling ( Figure 3A , B ) , and the expectation is that males should always baffle given an opportunity .", "In natural populations observed over three field seasons , however , the probability of finding a baffling male was as low as 26% ( 27 of 105 males observed calling ) and while baffling males tended to be on larger leaves , there was wide variation in leaf size ( Figure 3—figure supplement 1A ) .", "Thus baffled-calling is not a stereotyped and obligate calling behaviour; tree cricket males must decide whether to call with or without a baffle .", "In the wild , large leaves that can make acoustically optimal baffles are extremely rare and search costs are likely to be high ( Figure 3B ) .", "However , if we eliminate search costs , the propensity to make a baffle may increase with leaf size , a hypothesis that we tested under controlled laboratory conditions .", "Males were offered leaves of four size classes , one leaf at a time , over four nights .", "Each night , they could choose either to make a baffle or to call from the edge of the leaf .", "In this situation , in the absence of search costs , calling males were only balancing the effort expended in making a baffle against the increased SRE .", "Baffling probability varied depending on the leaf size ( Cochran’s Q: 63 . 9 , df = 3 , p<0 . 001 , Figure 3B ) and was significantly different between extra-large and large leaf sizes ( Mc-Nemars χ2 = 4 . 9 , df = 1 , p=0 . 02 ) , large and medium leaf sizes ( Mc-Nemars χ2 = 15 . 05 , df = 1 , p<0 . 001 ) and medium and small leaf sizes ( Mc-Nemars χ2 = 5 . 14 , df = 1 , p=0 . 02 ) .", "The larger the leaves , the more likely the crickets were to make a baffle ( Figure 3B ) .", "However , the probability of making a baffle approached 50% only with leaves between 61 and 69 mm long , where SRE increased by 54% .", "Even with the largest leaves , baffling probability did not exceed 67% , and 33% of the males missed the opportunity to more than double their SRE and thus their active acoustic space ( Figure 3B ) .", "Next , we tested whether males preferred larger leaves or whether they could also select the best leaf when given a choice .", "Tree crickets could indeed select the leaves that yielded higher SREs .", "19 males were presented with a choice between a small ( 61 . 2 ± 4 . 8 mm ) and a large leaf ( 115 . 2 ± 11 . 7 mm ) of their host plant ( Hyptis suaveolens ) .", "Males always selected the leaves yielding the higher SRE .", "All males that made a baffle ( 15 of 19 ) used the larger leaf .", "The holes they made were also acoustically optimal in size ( 10 . 0 ± 1 . 9 mm , n = 15 ) .", "Thus , given the opportunity males would indeed optimize the SRE of their baffles in terms of both selecting the appropriate materials , i . e . the largest leaf and then modifying it appropriately i . e . cutting a hole of acoustically optimal size ( Figure 3A ) .", "Males , however , did not make holes at the exact centre of leaves ( Figure 1E ) .", "We used our FEA to further explore whether eccentric hole positions came at a cost to SRE .", "An SRE map was constructed by varying hole position across an average leaf in the large size class .", "This map revealed that SRE was highest at the leaf centre and decreased for eccentric hole positions ( Figure 3C , Figure 2—figure supplement 1 ) .", "Thus , eccentric hole positions come at a cost to SRE .", "A central-hole baffle on the smaller leaf would have achieved an SRE of 226 . 3 ± 20 . 9 mPa/m∙s−1 .", "On the large leaf , it would achieve an SRE of 375 . 1 ± 11 . 1 mPa/m∙s−1 .", "The radiation efficiency of the real baffles made by the crickets was 332 . 2 ± 30 . 3 mPa/m∙s−1 i . e . 88 . 6 ± 7 . 6% of the maximum achievable SRE with a skew towards higher efficiencies ( Figure 3D ) ( mean ± SD , range = 72 . 8% to 97 . 7% , n = 15 all measures ) .", "Although theoretically optimal , a central hole position is impractical as it cuts through the midrib .", "This compromises the leaf’s structural rigidity and its main water transport system , causing it to wilt , thus reducing the lifetime of the baffle .", "Additionally , cutting through the rigid midrib may be mechanically difficult .", "Indeed , closer observation of baffle holes ( Figure 3—figure supplement 1B ) showed that males never cut through major leaf veins .", "Surprisingly , most males made only one hole and thus made a baffle in a single attempt , without any progressive trial and error optimization of baffle design .", "There was , however , one exception , one male made two holes ( Figure 4A ) .", "The first hole was far from the centre and achieved 72 . 1% of the maximum possible SRE .", "The male then made another hole closer to the centre which on its own would have achieved 96 . 9% of the maximum SRE .", "Why did the other males make no effort to boost their call amplitude further ?", "For instance , mole crickets gradually restructure their burrows to bring burrow resonance closer to call frequency ( Daws et al . , 1996; Bennet-Clark , 1987 ) .", "Baffles however , are unique and cannot be ‘edited’ unlike cavities; once a hole has been cut in a leaf , it cannot be erased or repositioned .", "Only a new hole can be made .", "Here we show that cutting a new hole entails the risk of making a sub-optimal baffle by reducing current SRE through increased acoustic short-circuiting .", "We studied the effect of a second hole on SRE by simulating a scenario where an eccentric hole was abandoned and a second , optimally centred hole was constructed .", "The map depicts at each primary hole position , the percent change in SRE if the male abandoned that position and made a centrally positioned secondary hole ( Figure 4B ) .", "A second baffle hole would accrue a large increase in SRE only if the primary hole was near a leaf margin and produced very low SRE to begin with ( Figure 4B ) .", "The only two-holed baffle observed was from a male that made an unusually eccentric primary baffle hole ( highlighted in Figure 4B ) .", "In this example , making a more central hole improved SRE by 37 . 5% , despite increased acoustic short-circuiting .", "Interestingly , our simulations predict that the more central the primary hole , the less beneficial a secondary baffle hole becomes and in the central zone of the leaf , a second hole becomes deleterious to SRE ( Figure 4B ) .", "This is because the second hole would fuse with the primary hole effectively enlarging it to a suboptimal size ( Figure 3A ) .", "Our observations show that the first holes made by males are already close to the centre; if they made a second central hole , six males would have decreased SRE by 6 . 73 ± 2 . 77% and the other nine males stood to increase SRE but only by 19 . 17 ± 11 . 15% ( Figure 4B ) .", "This finding supports the notion that crickets position their baffle holes with geometric optimality , but also avoid jeopardising this optimality .", "This may be why large , and sought-after , leaves only ever have one baffle hole .", "Near-optimal SREs are achieved by most males at the first baffling attempt ( Figure 3D ) and most attempts at improvement can be predicted as being futile ( Figure 4B ) ." ], [ "The question naturally arises as to how tree crickets make acoustically optimal baffles in a single attempt ?", "In the tree cricket genus Oecanthus , baffle making is common ( Forrest , 1991; Prozesky-Schulze et al . , 1975; Forrest , 1982 ) and expressed by many individuals within the species , suggesting that it is an inherited trait .", "Thus , the optimization procedure may also be ‘hard-wired’ , rather than learned and may have developed via sexual selection .", "We surmise that tree crickets accomplish acoustic optimization using hard-wired heuristics or ‘rules of thumb’ ( Hutchinson and Gigerenzer , 2005; Cross and Jackson , 2005 ) , which require them to make a series of choices .", "If a male decides to make a baffle , three simple rules can produce an optimal baffle in a single attempt:", "( i ) find the largest available leaf ,", "( ii ) place the hole as close to the centre of the leaf as possible and", "( iii ) cut a hole that can just accommodate wings ( Figure 3 , Figure 3—figure supplement 1B ) .", "These rules encode sufficient information to capture the shape of SRE landscape within baffle design space into a simple , yet high-accuracy heuristic that always produces the baffle with the highest possible SRE .", "The accuracy of this heuristic is higher than observed in the progressive optimization procedure observed in mole crickets burrows which always perform just sub-optimally ( Daws et al . , 1996 ) .", "At 3 kHz , the burrow resonance is always higher than call frequency at 2 . 5 kHz and the actual burrow dimensions do not fit optimal dimensions for sound radiation at either frequency ( Daws et al . , 1996 ) .", "The explanation for this sub-optimal performance lies in Weber’s law , whereby sensory systems cannot distinguish between small differences in signal amplitude or frequency at high signal amplitudes due to receptor saturation and habituation ( Imaizumi and Pollack , 2001; Imaizumi and Pollack , 1999; Givois and Pollack , 2000 ) .", "This neurophysiological limitation is likely to be a common driver of suboptimal performance in any method that uses feedback for optimization ( Nachev et al . , 2017 ) .", "It is possible that the tree crickets learn the SRE landscape , however , we believe that this is unlikely since the opportunity for learning is small due to life-history and time constraints .", "Tree crickets call only as adults , and for only a few hours each day .", "Finding and testing each leaf size with every baffle position to find the optimal solution , especially with large leaf sizes being so rare , would require a formidably large number of learning trials and considerable memory .", "Another possibility is that they can abstract these general rules from a few trials .", "While we believe that inherited heuristic optimization is a more parsimonious explanation , however , repeating these experiments with naïve males would provide a more definitive answer .", "Heuristics are , in essence , rational search methods ( McFarland , 1991 ) .", "When an object or behaviour has different efficiencies depending on a set of parameters , heuristics guide the search for the optimal position in the landscape described by these efficiencies ( McFarland , 1991 ) .", "Therefore , using a heuristic demonstrates the implicit or explicit knowledge that a behaviour can be accomplished in multiple ways and that each way differs in performance .", "Therefore heuristics , whether invertebrate , or vertebrate including humans , inherited or learned , imply not only an expectation of performance variability , but also choice and behavioural flexibility .", "One context in which this may be important is tool use; the conventional view is that invertebrates generally inherit tool use behaviour ( Hunt et al . , 2013 ) and hence have highly stereotyped tool use , whereas vertebrates inherit a propensity to ‘innovate’ tool behaviour and hence have flexible tool use ( Shettleworth , 2009 , 2010 ) .", "Inflexible or stereotyped tool use implies that optimization would not be possible for any invertebrate tool-users .", "It is suggested that baffles may be tools or borderline tools ( Bentley-Condit and Smith , 2010; Pierce , 1986; Crain et al . , 2013 ) , therefore baffle optimization suggests that inherited mechanisms such as heuristics may enable flexibility and even optimization in invertebrate tool use .", "Flexible tool use has indeed already been observed in invertebrates , first in cephalopods ( Finn et al . , 2009 ) and more recently , from bees ( Loukola et al . , 2017; Alem et al . , 2016 ) .", "Bees have a large capacity for learning , which enables significant behavioural flexibility , and it has now been shown that bees can be taught to use tools .", "They can even improve upon the tool use demonstrated to them ( Loukola et al . , 2017; Alem et al . , 2016 ) .", "Indeed , we argue that the behavioural flexibility that enables bees to improve upon demonstrated tool use emerges from a heuristic strategy .", "Bumble bees were trained to roll a ball into a specific position to gain a reward .", "The bees spontaneously used the ball nearest to the target even when they had been trained on a different ball ( Loukola et al . , 2017 ) , an excellent example of the well-known greedy heuristic ( Cormen et al . , 2009 ) in which the nearest solution is the most preferred .", "Indeed , tree cricket behaviour shows that even inherited heuristic mechanisms may enable flexibility and even optimization , prompting a re-examination of invertebrate tool use .", "In summary , tree cricket males that call from the edge of leaves tend to have low efficiencies ( 94–171 mPa/m∙s−1 depending on leaf size ) and those that make baffles can achieve much higher efficiencies ( 91–386 mPa/m∙s−1 over same size range ) .", "Thus , by finding the largest leaf and making a near optimal baffle , a male tree cricket can increase his active acoustic area by as much as four times ( 12 dB ) , greatly increasing his chances of attracting a mate .", "The increase in call amplitude that baffling confers can work in two ways .", "Baffling can either add to the existing variability , broadening the distribution of male call amplitudes or it can function as a compensatory mechanism: males unable to call loudly may make a baffle instead .", "Behavioural experiments show that despite the advantage baffles confer , only a few animals choose to make a baffle under natural conditions .", "Non-host plants with larger leaves are available to the tree crickets , yet are rarely used .", "The rarity of baffling suggests that manufacturing a baffle may be a costly activity ( Figure 3—figure supplement 1A ) .", "In contrast , all mole cricket males stereotypically make acoustic burrow chambers that resonate and boost call amplitude ( Hill et al . , 2006; Hill , 1999 ) .", "The low incidence of baffling and high variability in baffle design observed in natural baffling behaviour ( Figure 3—figure supplement 1A ) are quite distinct .", "Baffling may be used to garner even higher mate-attraction benefits on males whose high quality or condition already allows them to make larger energetic investments .", "Thus , baffle making may represent a unique system in which ‘good’ males use an acoustically optimized object to further enhance their mate-attraction signal .", "Another enticing possibility is that baffle-making may have evolved as an alternative strategy allowing smaller males , or those in poorer condition , to sound bigger or louder than they actually are ." ], [ "Vibration velocities from the forewings of freely calling tree cricket ( Oecanthus henryi ) males calling from baffles were measured using a micro-scanning laser Doppler vibrometer ( Polytec , PSV-400 , Waldbronn , Germany ) with a Polytec PSV-I-400 scanning head fitted with a close-up attachment , and digitized using the Polytec Scanning Vibrometer software ( version 8 . 8 , Polytec Gmbh , Waldbronn , Germany ) through a data acquisition board ( National Instruments , PCI-6110 , Austin , Texas , USA ) .", "Acoustic measurements were made simultaneously using two calibrated 1/8th inch precision pressure microphones ( Brüel and Kjær , 4138 , [frequency range 6 Hz to 140 kHz] , Naerum , Denmark ) and preamplifier ( Brüel and Kjær , 2633 , Naerum , Denmark ) .", "All data were collected at a sampling frequency of 128 kHz for 1 . 024 s .", "The microphones have a flat response in the measured frequency range .", "All experiments were carried out on a vibration isolation table ( Technical Manufacturing Corp , 784-443-12R , Peabody , Massachusetts , USA ) .", "The experimental set-up ( Figure 1E , D ) allowed the calling cricket to be oriented such that the upraised wings were normal to the path of the laser beam .", "The two microphones were placed coaxially ≈ 20 mm below the path of the laser beam , at a distance of ±200 mm from the center of the turn-table , hence from the position of the calling male .", "The laser was aligned using a video feed and it could then be remotely positioned by the vibrometer software across a grid of points placed over the wings of the calling insect .", "An average of 245 measurement points across the wing surface were used per animal leading to an average scan duration of just over 4 min .", "Paper baffles ( made using white 80 g/m2 paper ) were used in the study .", "These premade baffles were oval in shape and had a length of 68 . 6 ± 3 . 6 mm and width of 40 . 8 ± 2 . 2 mm on average ( n = 5 ) and the holes were 17 . 4 ± 1 . 9 mm in length and 11 . 4 ± 1 . 9 mm in width ( n = 5 ) .", "During calling , the peak baffle surface displacements were considerably lower ( 1/600th ) than the wings leading us to conclude that the baffles did not act as significant secondary acoustic radiators .", "The sound pressure radiating from a vibrating body depends on the space-time average of the body’s vibration velocity ( Hambric and Fahnline , 2007 ) .", "The peak value , which we also report in the main paper , is taken from the point of maximum deflection of the wing , as shown in Figure 1A .", "The rest of the wing does not move with this magnitude .", "The resulting sound is a consequence of the whole wings movement not just of this one point .", "If we used the peak value alone , the magnitude of wing vibration would be over-estimated .", "Taking an average in space resolves this issue .", "Since pressure is conventionally measured after integration over a fixed time period for amplitude-modulated signals , to be comparable , we also performed a time average of the vibration velocity .", "Hence we calculated the space-time average of the velocity in the z-direction ( Figure 1—figure supplement 1 ) from all the measurement points placed over the entire surface of the wing during calling .", "Finite element analysis ( FEA ) models of a cricket calling in free space or on different baffles were created to investigate pressures produced by vibrating cricket wings .", "FEA modeling was carried out using a commercial package ( COMSOL 4 . 3 , Burlington , Massachusetts , USA ) .", "The acoustics module was used implementing harmonic analysis with the Helmholtz equation as the governing equation .", "Oecanthus henryi males are observed to call and manufacture baffles from Hyptis suaveolens leaves in the wild .", "To better understand their interactions with the leaves , we performed three independent studies ." ] ]

[ "Object manufacture in insects is typically inherited , and believed to be highly stereotyped .", "Optimization , the ability to select the functionally best material and modify it appropriately for a specific function , implies flexibility and is usually thought to be incompatible with inherited behaviour .", "Here , we show that tree-crickets optimize acoustic baffles , objects that are used to increase the effective loudness of mate-attraction calls .", "We quantified the acoustic efficiency of all baffles within the naturally feasible design space using finite-element modelling and found that design affects efficiency significantly .", "We tested the baffle-making behaviour of tree crickets in a series of experimental contexts .", "We found that given the opportunity , tree crickets optimised baffle acoustics; they selected the best sized object and modified it appropriately to make a near optimal baffle .", "Surprisingly , optimization could be achieved in a single attempt , and is likely to be achieved through an inherited yet highly accurate behavioural heuristic ." ]

[ "Male tree crickets produce sounds at a specific pitch to attract females .", "The louder the call , the further the sound travels and the more females he can attract .", "But making loud sounds is difficult for small animals like insects .", "To produce sounds , tree crickets rub their wings together and set them into vibration .", "As the wings vibrate , their motion creates changes in the surrounding air pressure , which is perceived as sound .", "As the wings move forwards , they compress the air in front of them and thin the air behind them , working much like the membrane of a loudspeaker .", "However , when the compressed and thinned air meet at the edges of the wings , the sound cancels out .", "This problem is known as acoustic short-circuiting , and the smaller the wings , the larger this effect and the less efficient the broadcast of sound becomes .", "Tree crickets overcome acoustic short-circuiting by making baffles , for which they cut a hole near the centre of a leaf .", "The cricket then sings from inside this hole with its wings flat against the leaf surface , so that the sound has to travel to the leaf edge before short-circuiting .", "Not all baffles work equally well though , and scientists are interested to know whether tree crickets know how to make the best possible baffle to attract more females .", "To find out what makes an ideal baffle , Mhatre et al . first measured the wing vibrations and sounds of real tree crickets , and used them to simulate a cricket singing from different baffles .", "From these tests , three simple rules emerged that led to the best baffle: use the largest available leaf , make a hole the size of the wings , and place it at the centre of the leaf .", "Mhatre et al . then discovered that the crickets did not make a baffle every time – only when the leaves were large enough .", "This suggests that rather than being solely ‘robotic’ in their behaviour and the use of objects , insects can behave flexibly .", "When faced with a choice between two leaves , the crickets followed the same three decision rules that the scientists had discovered , and achieved near optimal baffles .", "Insects are thought to only be able to gradually improve an object or behaviour , but rarely to optimize it .", "However , the discovery that tree crickets can make optimal acoustic baffles in a single attempt means that we are only beginning to unravel the underappreciated abilities of insects .", "An enticing next step will be to see whether the creation of baffles could be considered as tool-making ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "neuroscience" ]

[ [ "IBMPFD is an autosomal dominant disease that afflicts multiple body systems ( Kimonis et al . , 2008a ) .", "90% of IBMPFD patients display skeletal muscle weakness ( myopathy ) , the primary and earliest symptom of IBMPFD ( Weihl et al . , 2009 ) .", "With progression of the disease , 50% of patients will develop Paget’s disease of bone , affecting the skull , spine , hips and long bones of all four limbs .", "One-third of the patients will also develop frontotemporal dementia ( Weihl et al . , 2009; Kimonis et al . , 2008b ) .", "Single missense mutations of p97/cdc48/Valosin-containing protein ( VCP ) cause fully penetrant IBMPFD ( Watts et al . , 2004 ) .", "VCP mutations are also associated with 1–2% of cases of amyotrophic lateral sclerosis ( ALS ) , familial hereditary spastic paraplegia ( HSP ) and Charcot-Marie-Tooth 2 ( CMT2 ) disease ( Abramzon et al . , 2012; de Bot et al . , 2012; Gonzalez et al . , 2014 ) .", "VCP encodes a highly conserved and abundant AAA+ ATPase which participates in multiple cellular processes ( Meyer et al . , 2012 ) .", "Human p97/VCP and its Drosophila homologue have 85% identity and 93% similarity in protein sequence .", "VCP has three major domains: the regulatory N domain , and the D1 and D2 ATPase domains .", "VCP hexameric rings utilize the energy from ATP hydrolysis to promote protein and RNA homeostasis , often by directly or indirectly modifying the fate of ubiquitin-labeled proteins ( Meyer et al . , 2012 ) .", "VCP functions in multiple contexts that include protein quality control in the endoplasmic reticulum ( Ye et al . , 2001; Shih and Hsueh , 2016 ) , chromatin modification ( Puumalainen et al . , 2014; Dobrynin et al . , 2011; Vaz et al . , 2013 ) , endolysosomal sorting ( Ritz et al . , 2011 ) , membrane fusion ( Zhang et al . , 2014 ) , autophagosome/lyosome function ( Ju et al . , 2009; Johnson et al . , 2015 ) , ER protein translocation ( DeLaBarre et al . , 2006; Weihl et al . , 2006 ) , formation of stress granules ( Buchan et al . , 2013 ) and ciliogenesis ( Raman et al . , 2015 ) .", "VCP interacts with a number of co-factors to regulate these processes ( Meyer and Weihl , 2014; Meyer et al . , 2012 ) , making it challenging to identify the molecular basis of phenotypes associated with disease mutations .", "Disease-causing , single missense mutations of VCP are mainly located in the N-terminal half of the protein , either in the N domain or the D1 domain .", "Among them , the R155H mutation is the most frequently identified in IBMPFD patients , while the A232E mutation is associated with the most severe clinical manifestation ( Kimonis et al . , 2008a; Ritson et al . , 2010 ) .", "In vitro assays show that disease mutants have enhanced ATPase activity ( Weihl et al . , 2006; Zhang et al . , 2015; Niwa et al . , 2012; Manno et al . , 2010; Tang and Xia , 2013 ) .", "However , because VCP assembles as a hexamer , it is controversial whether disease mutants with increased ATPase activity cause disease through a dominant-active ( Chang et al . , 2011 ) or dominant-negative mechanism ( Ritz et al . , 2011; Ju et al . , 2009; Kim et al . , 2013; Bartolome et al . , 2013; Kimura et al . , 2013 ) .", "VCP disease mutants predominantly affect organs that have a high level of energy expenditure , such as brain and muscle .", "Mitochondria provide the bulk of the ATP to these tissues through oxidative phosphorylation , and mitochondrial functional defects , including mitochondrial uncoupling and decreased ATP production , are observed in IBMPFD patient fibroblasts ( Bartolome et al . , 2013; Nalbandian et al . , 2015a ) .", "Abnormal mitochondria are also observed in transgenic VCP disease mutant R155H mice as well as VCP R155H knock-in mice ( Custer et al . , 2010; Nalbandian et al . , 2012 ) .", "These observations suggest that mitochondrial dysfunction is important for the pathogenesis of IBMPFD , but the mechanism by which VCP mutation alters mitochondrial function is not clear .", "Mitochondrial morphology is controlled by dynamic cycles of fusion , controlled by Mitofusin ( Mfn ) , and fission , regulated by DRP1 ( Chan , 2012 ) .", "Recent studies have uncovered the roles of mitochondria fusion and fission defects in the pathogenesis of multiple neurodegenerative disorders ( Davies et al . , 2007; Chen et al . , 2003; Wakabayashi et al . , 2009 ) , particularly Parkinson's disease , the second most common neurodegenerative disorder ( Guo , 2012; Pickrell and Youle , 2015; Deng et al . , 2008; Yang et al . , 2008; Poole et al . , 2008 , 2010; Park et al . , 2009 ) .", "In mammals , homologous proteins Mitofusin 1 and 2 ( Mfn1 and Mfn2 ) mediate mitochondrial outer membrane fusion , with loss of function of Mfn 1 and 2 resulting in fragmented mitochondria and multiple defects in mitochondrial function ( Chen et al . , 2003b ) .", "In Hela cells , VCP promotes Mfn 1 degradation ( Xu et al . , 2011 ) .", "VCP also mediates Mfn 1 and 2 degradation when mitophagy is stimulated in mammalian cells , and overexpression of VCP in Drosophila leads to downregulation of a tagged Mfn-transgene ( Kim et al . , 2013; Kimura et al . , 2013 ) .", "These observations led us to investigate the mitochondrial basis and molecular mechanisms for VCP disease mutants function using both Drosophila and IBMPFD patient cell models .", "As IBMPFD show the highest penetrance in muscle , with 90% of patients manifesting phenotypes in this tissue , we generated Drosophila models of IBMPFD in muscle , which recapitulate disease pathologies .", "We provide evidence in both Drosophila and human patient cells that VCP disease mutants have an enhanced ability to promote Mfn degradation , loss of which is associated with defects in mitochondrial fusion and physiology .", "Consistent with the hypothesis that VCP disease phenotypes are due to increased activity on substrates , we find that VCP ATPase activity inhibitors such as NMS-873 and ML240 can significantly rescue mitochondrial defects , disrupted muscle integrity and muscle cell death in vivo in Drosophila , and mitochondrial fusion and respiration defects in IBMPFD patient fibroblasts ." ], [ "Since VCP disease mutants have dramatic muscle phenotypes in humans and mouse models , and these are associated with defects in mitochondrial structure and function , we first examined the consequences of manipulating VCP levels .", "The Drosophila adult indirect flight muscle ( IFM ) is a non-essential , post-mitotic , and energy-intense tissue containing a high density of mitochondria .", "It also shows strong and consistent phenotypes in response to altered expression of genes required for mitochondrial fusion , fission and quality control ( Clark et al . , 2006; Deng et al . , 2008; Yang et al . , 2008; Poole et al . , 2008; Park et al . , 2009; Poole et al . , 2010; Yun et al . , 2014 ) .", "Using the UAS-Gal4 system ( Brand and Perrimon , 1993 ) , we expressed VCP under the control of an IFM promoter derived from the flightin gene we previously generated ( Yun et al . , 2014 ) .", "While expression of VCP under the control of the pan-muscle Gal4 drivers 24B-Gal4 or Mef2-Gal4 resulted in 100% adult lethality , expression of wildtype VCP under IFM control , which provides a pulse of expression in late pupal stages and early adulthood , gave rise to viable flies with intact and healthy muscle following adult eclosion .", "We utilized mitochondrially targeted GFP ( mitoGFP ) as a mitochondrial marker , as well as transmission electron microscopy ( EM ) at the ultrastructual levels for enhanced resolution particularly for cristae morphology .", "In 2-day-old flies , expression of VCP results in muscle with small mitochondria with intact cristae ( Figure 1—figure supplement 1A–B’ ) .", "Similar phenotypes are also observed in muscle from 6-day-old VCP-expressing flies ( Figure 1A–B’’ ) .", "Conversely , expression of UAS-VCP RNAi under IFM control ( VCP RNAi flies ) results in mitochondria with an elongated phenotype ( Figure 1C–C’’ , Figure 1—figure supplement 1C and C’ ) .", "Two independent VCP RNAi lines were utilized and both showed significant knockdown ( Figure 1—figure supplement 2 ) . 10 . 7554/eLife . 17834 . 003Figure 1 . Endogenous VCP regulates mitochondrial fusion via negative regulation of Mfn protein levels .", "( A–C )", "MitoGFP localization to mitochondria serves as a marker for healthy mitochondria and their morphology in the indirect flight muscle .", "VCP overexpression ( VCP OE , B ) results in small mitochondria compared to wildtype ( WT ) flies ( A ) ; VCP RNAi expression results in elongated mitochondria ( C ) .", "Scale Bar: 5 µm .", "( A’–C’’ )", "Electronic microscopic ( EM ) images show that VCP OE ( B’–B’’ ) results in smaller mitochondria as compared with WT ( A’–A’’ ) .", "VCP RNAi generates elongated mitochondria with intact cristae ( outlined with white dashed lines in C’ and C’’ ) .", "( A’–C’ )", "Lower magnification; ( A’’–C’’ ) Higher magnification of mitochondria outlined with black solid lines in A’–C’ .", "Scale bar: 1 µm .", "( D ) : Schematic diagram of nurse cell mosaic analysis in Drosophila female germline .", "A stage 10A egg chamber has three types of cells .", "The monolayer follicle cells coat the surface of the egg chamber; 15 nurse cells provide proteins and RNAs to the oocyte during oogenesis .", "Heat shock induction of mitotic recombination during larvae stages results in the creation of a mosaic pattern in the adult nurse cells .", "Ubi-mRFP . NLS ( Red ) is used as a clone marker .", "Wildtype ( WT ) cells are labeled with two copies of RFP , +/+; heterozygous mutant cells have one copy of RFP and one copy of vcp loss-of-function mutant , vcpK15502/+; homozygous mutant cells are RFP negative and have two copies of vcp loss-of-function mutant , vcpK15502/vcpK15502 .", "( E ) : A stage 10A egg chamber with a mosaic pattern of nurse cells .", "Red signal is Ubi-mRFP . NLS .", "Scale bar: 20 µm .", "( E’ )", "Anti-ATP5A antibody staining is used to visualize mitochondrial morphology in nurse cells .", "Scale bar: 20 µm .", "( F–I )", "Higher magnification view of mitochondrial morphology in E’ ( outlined in white solid lines ) .", "Mitochondria appear as discrete and punctate structures in wildtype ( G ) and heterozygous vcp mutant cells ( F ) , but becomes elongated and clumped in homozygous vcp loss-of-function mutant cells ( H and I ) .", "Scale bar: 5 µm .", "( J ) A stage 10B egg chamber with a mosaic pattern of nurse cells .", "Red signal is Ubi-mRFP . NLS; Green signal is anti-GFP staining of pCasper-Mfn-eGFP .", "Scale bar: 20 µm .", "( J’–J’’ )", "Higher magnification of the egg chamber in J ( outlined in white solid lines ) .", "Wildtype cells are RFP positive and homozygous vcp loss-of-function mutant cells are RFP negative ( J’ ) .", "pCasper-Mfn-eGFP levels significantly increase in homozygous vcp loss-of-function mutant cells ( J’’ ) .", "Scale bar: 10 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 17834 . 00310 . 7554/eLife . 17834 . 004Figure 1—figure supplement 1 . VCP overexpression leads to smaller mitochondria , while VCP RNAi leads to elongated mitochondria in 2-day-old indirect flight muscles .", "( A–C’ )", "EM shows that compared to WT ( IFM-Gal4 control , A and A’ ) , VCP WT has smaller mitochondria with intact cristae ( B and B’ ) .", "VCP RNAi results in mitochondrial elongation ( C and C’ ) in 2-day-old flies .", "Scale bar: 1 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 17834 . 00410 . 7554/eLife . 17834 . 005Figure 1—figure supplement 2 . IFM-Gal4 driven UAS-VCP RNAi results in a significant decrease in VCP levels . Expression of VCP RNAi line 1 and RNAi line 2 result in a decrease in VCP levels to 45 . 06 ± 11 . 17% ( p=0 . 014 ) and 42 . 29 ± 1 . 12% ( p=0 . 037 ) of those present in WT .", "Independent t test is used for statistical analysis , N = 3 .", "Normalized VCP levels are shown as mean±½SD . DOI: http://dx . doi . org/10 . 7554/eLife . 17834 . 00510 . 7554/eLife . 17834 . 006Figure 1—figure supplement 3 . pCasper-Mfn-eGFP colocalizes with the mitochondria marker ATP5A , and is silenced by expression of dsRNA targeting endogenous Mfn .", "( A ) One nurse cell of a stage 10B egg chamber in transgenetic flies carrying the genomic rescue of Mfn ( pCasper-Mfn-eGFP ) .", "The pCasper-Mfn-eGFP construct expresses Mfn-eGFP under the control of the endogenous Mfn promoter .", "GFP signal colocalizes with anti-ATP5A staining , indicating mitochondrial localization .", "Scale bar: 20 µm .", "( B ) S2 cells were transfected with pCasper-Mfn-eGFP and blotted and probed with anti-GFP antibody .", "Mfn dsRNA treatment results in loss of Mfn-eGFP expression . DOI: http://dx . doi . org/10 . 7554/eLife . 17834 . 006 To explore the generality of VCP’s ability to regulate mitochondrial morphology we also examined mitochondrial morphology phenotypes of a vcp loss-of-function mutation .", "vcp null mutants in Drosophila are embryonic lethal ( Ruden et al . , 2000 ) .", "The indirect flight muscle is a cellular syncytium and therefore cannot be used for mosaic analysis .", "As an alternative , we focused on the Drosophila female germline , in which a vcp loss-of-function mutant can be monitored in individual nurse cells , which are relatively large and offer great resolution for examining mitochondrial morphology .", "vcpK15502 is a strong loss-of-function allele that has a P element insertion at the 5’-UTR of Drosophila VCP gene ( Chang et al . , 2011; Ruden et al . , 2000 ) and has been used to generate vcp loss-of-function clones in imaginal discs ( Zhang et al . , 2013 ) .", "The Drosophila ovary consists of numerous developing egg chambers .", "Each egg chamber develops from stage 1 to stage 14 and has three types of cells: follicle cells , nurse cells and the oocyte ( Frydman and Spradling , 2001 ) .", "The germline complement of each egg chamber ( 15 nurse cells and the oocyte ) derives from four sequential divisions of a single daughter of a stem cell .", "We utilized the FLP/FRT system ( Theodosiou and Xu , 1998 ) to create mitotic recombinants in which nurse cells were either wildtype ( +/+ ) , heterozygous ( vcpK15502/+ ) or homozygous ( vcpK15502/vcpK15502 ) for the vcp loss-of-function mutation ( Figure 1D ) .", "In this system wildtype and heterozygous vcp loss-of-function mutant cells carry 2 or 1 copies of ubi-mRFP . NLS ( RFP ) respectively , whereas the homozygous vcp mutant cells are RFP negative ( Figure 1E ) .", "Anti-ATP5A antibody is used to visualize mitochondrial morphology in nurse cells ( Figure 1E’ ) .", "Mitochondria in wildtype and heterozygous vcp mutant cells have a punctate morphology ( Figure 1F and G ) .", "In contrast , in homozygous vcp loss-of-function mutants mitochondria are more tubular and clumped ( Figure 1H and I ) .", "Together , these results suggest that endogenous VCP negatively regulates mitochondrial fusion or positively regulates fission .", "Next , we investigated how vcp loss-of-function leads to elongated mitochondria .", "First , we examined the expression levels of Mfn in the female germline , using mosaic analysis , as above .", "We used a tagged genomic rescue transgene ( pCasper-Mfn-eGFP , a transgene construct with a GFP tagged Mfn under the control of its endogenous promoter , a kind gift from Dr . CK Yao ) to monitor the endogenous Mfn level as the existing anti-Mfn antibody lacked sufficient sensitivity .", "As expected , the genomic rescue Mfn-eGFP signal colocalizes with the mitochondria marker ATP5A in the nurse cells ( Figure 1—figure supplement 3A ) .", "This signal is eliminated following Mfn RNAi , indicating that genomic rescue Mfn-eGFP is a reliable marker for endogenous Mfn ( Figure 1—figure supplement 3B ) .", "In each egg chamber , vcp loss-of-function cells are adjacent to wildtype cells , providing an ideal opportunity to unambigously compare the levels of Mfn .", "As shown in Figure 1J–J’’ , nurse cells homozygous for a vcp loss-of-function mutant have greatly increased Mfn levels compared to wildtype and heterozygous vcp loss-of-function cells .", "The findings that endogenous VCP regulates Mfn in vivo are unexpected and important , as VCP is a highly abundant cytosolic protein with many targets and VCP does not show appreciable localization to mitochondria in unstressed cells .", "Second , we examined whether alteration of vcp can regulate Mfn levels in tissue lysate .", "Overexpression of wildtype VCP ( VCP OE ) in the flight muscle resulted in a decrease in endogenous Mfn levels ( Figure 2A ) .", "VCP overexpression also caused a decrease in the levels of Mfn when Mfn was overexpressed ( Mfn OE ) using the IFM promoter , suggesting that VCP regulates Mfn post transcriptionally ( Figure 2A ) .", "Conversely , VCP RNAi resulted in an increase in endogenous Mfn levels in a wildtype background , and an increase in total Mfn levels in the presence of Mfn OE ( Figure 2B ) .", "In contrast , VCP overexpression does not alter the levels of pro-fission protein DRP1 ( Figure 2C ) .", "In addition , VCP’s regulation of Mfn is specific and not part of a general mitophagy response since the levels of other mitochondrial proteins , such as the outer membrane protein Porin , the inner membrane protein NDUSF3 , and the matrix protein MnSOD , are not altered ( Figure 2D ) .", "To explore the possibility that Mfn is a direct target for VCP , we asked if Mfn could physically interact with VCP in immunoprecipitation assays from S2 cells .", "Indeed , full length VCP strongly interacts with Mfn ( Figure 2E ) .", "This is consistent with previous findings ( Kim et al . , 2013 ) .", "VCP has been shown to interact with substrate through the D1 ATPase domain , with the presence of the N domain being essential for the interaction ( Meyer et al . , 2012 ) .", "As shown in Figure 2E , this is also the case with the VCP-Mfn interaction .", "When the N domain of VCP is deleted , the strength of VCP-Mfn interaction is decreased , and it is undetectable when N and D1 are both deleted .", "In contrast , removal of the D2 domain has no effect on interaction between VCP and Mfn .", "Interestingly , Mfn levels in the input blot are decreased under conditions when VCP and Mfn interact , but not under conditions in which interactions are not observed , consistent with the hypothesis that direct interaction between VCP and Mfn is required for Mfn degradation .", "Together , these observations suggest that Mfn is a specific , direct target of VCP . 10 . 7554/eLife . 17834 . 007Figure 2 . Mfn is a specific target of VCP .", "( A ) In fly thoraxes , VCP OE in wildtype ( IFM-Gal4 control ) and Mfn OE background results in a significant decrease in Mfn levels .", "Hsp60 was used as a mitochondria control; Actin was used as a loading control .", "( B ) VCP RNAi leads to enhanced Mfn levels ( 1 . 72 ± 0 . 37 compared to wildtype , set as 1; p=0 . 021 , independent t-test , N = 4 ) .", "VCP RNAi results in accumulation of Mfn in the Mfn OE background ( 16 . 15 ± 0 . 85 as compared to Mfn OE alone , 9 . 02 ± 1 . 63 , wildtype set as 1 , p=0 . 0101 , independent t test , N = 3 ) .", "*p<0 . 05 .", "Mfn levels are normalized with Hsp60 and are displayed as mean±½SD .", "( C ) VCP OE also does not result in changes in the levels of the pro-fission protein DRP1 level ( p=0 . 088 , independent t test , N = 3 ) .", "DRP1 was detected using an HA tagged genomic rescue transgene expressed under the control of the endogenous DRP1 promoter ( pCasper-DRP1-HA ) .", "n . s . : no statistical difference .", "( D ) Markers for various mitochondrial compartments , including Porin ( outer membrane ) , MnSOD ( matrix ) , and NDUSF3 ( inner membrane ) were not altered by VCP overexpression .", "( E ) Schematic diagram of full length and truncated VCP forms used in protein interaction assays in S2 cells .", "Protein interactions are assayed between full length and truncated VCP forms and Mfn .", "N and D1 domains are essential domains for the VCP-Mfn interaction . DOI: http://dx . doi . org/10 . 7554/eLife . 17834 . 007 Mutations in PINK1 and Parkin lead to autosomal recessive forms of Parkinson’s disease ( PD ) ( Kitada et al . , 1998; Valente et al . , 2004 ) .", "We and others have shown that PINK1 and Parkin function in the same pathway , with PINK1 positively regulating Parkin to control mitochondrial integrity and quality control in Drosophila ( Clark et al . , 2006; Yang et al . , 2006; Park et al . , 2006 ) .", "Importantly , levels of Mfn are significantly increased in PINK1 and parkin null mutants and downregulation of Mfn protein levels suppresses phenotypes in both mutants ( Deng et al . , 2008; Yang et al . , 2008; Park et al . , 2009; Yu et al . , 2011 ) .", "The results presented above suggest that expression of VCP , by decreasing the level of Mfn , could also suppress PINK1/parkin phenotypes .", "Kim et al . have argued that VCP expression suppresses PINK1 , but not parkin loss-of-function phenotypes .", "This , along with their observation that VCP levels at mitochondria increased following expression of Parkin in the presence of CCCP , suggested a model in which VCP action on mitochondria requires recruitment by Parkin ( Kim et al . , 2013 ) .", "Because the EDTP-Gal4 driver used in these earlier experiments does not express at significant levels in flight muscle ( Seroude et al . , 2002 ) ( Figure 3J , see GFP signals ) , we reexamined this issue .", "We expressed VCP in the flight muscle of PINK1 and parkin mutants .", "As previously shown ( Yang et al . , 2006; Clark et al . , 2006; Park et al . , 2006 ) , PINK1 and parkin null mutants show mitochondrial defects and tissue disintegration with vacuolation in muscle ( Figure 3A–B’’ and D–D’’ ) .", "At the ultrastructural level , PINK1 and parkin mutants display swollen mitochondria with a broken cristae ( Figure 3A’’’ , B’’’ and D’’’ ) .", "VCP OE completely suppressed tissue damage and mitochondrial defects ( Figure 3C–C’’’ and E–E’’’ ) .", "The thorax indentation phenotype in PINK1/parkin mutants is also completely suppressed by VCP overexpression under the control of the IFM-Gal4 driver , but not the EDTP-Gal4 driver ( Figure 3—figure supplement 1 ) .", "The suppression observed by Kim , et al . is probably due to non-disjunction of the PINK1 null flies .", "As predicted for a mechanism of VCP action that occurs through regulation of Mfn , VCP OE significantly reduced the accumulation of Mfn normally observed in both mutants ( Figure 3F ) . 10 . 7554/eLife . 17834 . 008Figure 3 . VCP overexpression suppresses mitochondrial defects in PINK1 null , parkin null and parkin mul1 double null mutants .", "( A–E’ )", "Compared to wildtype ( A and A’ ) , parkin and PINK1 mutants lose MitoGFP signal and accumulate large aggregates .", "VCP OE ( IFM-Gal4>UAS-VCP ) significantly rescues the MitoGFP phenotype in both mutants .", "Filamentous actin is stained with Rhodamine Phalloidin ( Red ) .", "( A–E )", "Lower magnification .", "Scale bar: 20 µm .", "( A’–E’ )", "Higher magnification .", "Scale bar: 5 µm .", "( A’’–E’’ )", "Toluidine Blue shows that vacuole formation in the muscle tissue in parkin and PINK1 mutants is robustly suppressed by VCP OE .", "Thoraxes are assayed 2 days after eclosion .", "Scale bar: 30 µm .", "( A’’’–E’’’ )", "At the ultrastructural level , wildtype ( WT , IFM-Gal4 control ) mitochondria are well aligned with compact cristae ( A’’’ ) .", "parkin and PINK1 mutants display swollen mitochondria with broken cristae ( B’’’ and D’’’ ) .", "VCP OE ( IFM-Gal4>UAS-VCP ) completely rescued the mitochondrial defects in both mutants ( C’’’ and E’’’ ) .", "Thoraxes are sectioned at 2 days after eclosion .", "Scale bar: 1 µm .", "( F ) VCP OE caused a decrease in Mfn accumulation in parkin and PINK1 mutants .", "Mfn protein levels in the parkin mutant increased to 2 . 12 ± 0 . 69 as compared with wildtype ( set as 1 ) .", "VCP OE caused a decrease in Mfn levels in the parkin mutant to 1 . 26 ± 0 . 34 as compared with parkin ( p=0 . 005 , independent t test , N = 3 . **p<0 . 01 ) .", "In PINK1 mutants , Mfn levels increased to 2 . 69 ± 0 . 11 as compared with wildtype , while VCP OE in PINK1 mutant caused Mfn levels to decrease ( 1 . 77 ± 0 . 29 as compared with PINK1 , p=0 . 023 , independent t test , N = 3 , *p<0 . 05 ) .", "Normalized Mfn levels are shown as mean±½SD .", "( G–I’ )", "Compared to wildtype , the parkin mul1 mutant lacks the most MitoGFP signal and large aggregates are present ( G–H’ ) .", "VCP OE ( IFM-Gal4>UAS-VCP ) rescues the MitoGFP phenotype in parkin mul1 mutants ( I and I’ ) .", "Myofibrils are stained with Rhodamine Phalloidin ( Red ) .", "( G–I )", "Lower Magnification , Scale bar: 20 µm; ( G’–I’ ) Higher Magnification , Scale bar: 5 µm .", "( J ) Expression pattern of EDTP-Gal4 and IFM-Gal4 in 3-day-old flies .", "EDTP-Gal4 driven UAS-MitoGFP signal is barely detected in the thorax , where the indirect flight muscle is located , suggesting that EDTP-Gal4 does not have sufficient flight muscle specific expression .", "MitoGFP is present at high levels in the thoraxes of IFM-Gal4 driven flies .", "Thorax structure is outlined with dashed white lines in white light and indicated with a white arrowhead in GFP field .", "( K ) Schematic diagram showing that PINK/Parkin in parallel with Mul1 negatively regulates Mfn protein levels; VCP negatively regulates Mfn protein levels independent of these modifiers . DOI: http://dx . doi . org/10 . 7554/eLife . 17834 . 00810 . 7554/eLife . 17834 . 009Figure 3—figure supplement 1 . IFM-Gal4 driven UAS-VCP expression but not EDTP-Gal4 driven UAS-VCP rescues thorax defects in PINK1 and parkin mutants in Drosophila . Thorax indentation ( white arrows ) is observed in both PINK1 and parkin mutants , indicative of muscle tissue damage .", "EDTP-Gal4 driven UAS-VCP does not rescue this defect in PINK1 and parkin mutants .", "IFM-Gal4 driven UAS-VCP completely rescues the thorax indentation in both mutants . DOI: http://dx . doi . org/10 . 7554/eLife . 17834 . 009 To further substantiate the finding that overexpression of VCP suppresses parkin null mutants , we also examined VCP’s ability to suppress mitochondrial phenotypes associated with loss of both parkin and mul1 .", "MUL1 ( MULAN/MAPL ) is a RING-containing E3 ligase that acts as a negative regulator of Mfn in both Drosophila and mammals ( Yun et al . , 2014 ) .", "When overexpressed , it suppresses PINK1 and parkin null mutant phenotypes by degrading Mfn , while loss of mul1 leads to increased levels of Mfn .", "Thus , parkin mul1 double null mutants have strikingly more severe mitochondrial phenotypes than those observed in flies lacking parkin alone ( Yun et al . , 2014 ) .", "Remarkably , expression of VCP resulted in a dramatic rescue of mitochondrial morphology due to lack of both parkin and mul1 ( Figure 3G–I’ ) .", "Together , these results suggest that VCP-dependent suppression of PINK1 null , parkin null and parkin mul1 double phenotypes occurs through downregulaton of Mfn levels ( Figure 3K ) .", "Together , our observations argue that VCP regulates Mfn levels under non-stress conditions .", "They also show that VCP-dependent regulation of Mfn does not require PINK1 or Parkin .", "However , these results do not exclude the possibility that Parkin activation further promotes VCP-dependent Mfn degradation under some situations .", "To explore the basis of myopathy induced by VCP disease mutants we sought to create a model of IBMPFD in Drosophila muscle , as 90% of the IBMPFD patients show muscle phenotypes .", "Since VCP disease mutations are autosomal dominant , we characterized the consequences of disease mutant overexpression .", "The pathologies observed in IBMPFD patient muscle , and in muscle from VCP disease mutant transgenic and mouse knock-in models , include muscle cell death , changes in mitochondrial morphology , damaged tissue integrity , TAR DNA-binding protein 43 ( TDP43 ) mislocalization to the cytosol , and formation of autophagic marker p62 and ubiquitin aggregates , signs of multisystem proteinopathy ( Ju et al . , 2009; Ritson et al . , 2010; Nalbandian et al . , 2012; Ritz et al . , 2011; Nalbandian et al . , 2015b; Weihl , 2011; Ahmed et al . , 2016 ) .", "Previous studies of VCP disease mutants in Drosophila muscle showed effects on tissue integrity and mitochondria cristae structure ( Chang et al . , 2011; Kim et al . , 2013 ) , but not other pathologies .", "We thus investigated the pathologies due to mutant VCP expression .", "We focused on expression of VCP R152H and A229E ( corresponding to Human VCP R155H and A232E , hereafter referred to as VCP RH and VCP AE ) , as they are the most frequent and the most severe mutations identified , respectively ( Figure 4A ) .", "Anti-VCP antibody blotting shows that VCP WT , VCP RH and VCP AE are expressed at comparable level in the fly's thorax ( Figure 4B ) .", "In wildtype flies , flies with IFM-Gal4 insertion , and flies with VCP WT expression , the muscle structure was healthy and intact 6 days after eclosion .", "No cell death was observed and mitochondria were densely packed and contained high levels of MitoGFP ( Figure 4C , D and D’ ) .", "Muscles also displayed a well-aligned myofibril structure ( Figure 4E and E’ ) .", "Together these observations indicate that increased expression of VCP is not overtly toxic .", "In contrast , muscle from flies expressing VCP RH or VCP AE under IFM-Gal4 control ( thereafter called VCP RH or VCP AE flies ) showed extensive cell death ( 97 ± 3 . 6% and 95 . 7 ± 5 . 1% muscle are TUNEL-positive ) , defective MitoGFP signals ( Figure 4D’’ and D’’’ ) , and severely disrupted muscle integrity ( Figure 4E’’ and E’’’ ) .", "In addition , while in wildtype and VCP WT flies TDP43 was found in IFM nuclear and sarcoplasmic compartments , IFMs from VCP RH and AE flies showed a decrease in the intensity of the nuclear signal , and an increase in the intensity of staining associated with puncta in the sarcoplasmic area ( Figure 4C , F–F’’’ ) .", "Muscle from VCP RH and AE flies also contained large aggregates of anti-p62 and anti-ubiquitin staining .", "These were not observed in wildtype or VCP WT flies ( Figure 4G–H’’’ ) .", "Importantly , the pathologies observed in flight muscle at day 6 post eclosion were not present at day 2 ( Figure 4—figure supplement 1 ) , suggesting the degenerative nature of the pathology , as with the human disease and mouse models ( Kimonis et al . , 2008a; Custer et al . , 2010; Nalbandian et al . , 2012 ) .", "Together , these observations indicate that IFM-specific expression of VCP RH and AE recapitulates a broad spectrum of IBMPFD disease pathologies and forms the strong basis for further investigation of disease mechanisms and treatment studies . 10 . 7554/eLife . 17834 . 010Figure 4 . Expression of VCP disease mutants and mfn RNAi knocking down lead to pathology in adult muscle tissue .", "( A ) Diagram of Human p97/VCP protein domains and two disease mutants , VCP R155H and A232E .", "Their corresponding Drosophila homologues are VCP R152H and A229E , hereafter referred to as VCP RH and AE .", "( B ) Expression levels of UAS-VCP WT , RH and AE disease mutants expressed under IFM-Gal4 are comparable .", "( C ) Diagram of Drosophila indirect flight muscle structure .", "Mitochondria ( Green ) and nuclei ( Orange ) are densely packed in between myofibrils ( which contain large amounts of actin , Black ) .", "( D–D''' )", "MitoGFP ( Green ) and TUNEL staining ( Red ) .", "6-day-old WT ( IFM-Gal4 control ) flies and VCP WT flies have healthy muscles ( no TUNEL staining ) and are MitoGFP positive ( D and D' ) .", "6-day-old VCP RH and AE expressing flies show high levels of TUNEL staining .", "97 ± 3 . 6% and 95 . 7 ± 5 . 1% of RH ( p=0 . 03 . RH v . s . WT , independent t test ) and AE ( p=0 . 015 , AE v . s . WT , independent t test ) and are MitoGFP negative ( D'' and D''' ) .", "Scale bar: 10 µm .", "( E–E''' )", "Toluidine Blue staining of muscle in WT , VCP WT , RH and AE flies .", "Myofibrils are well aligned with densely packed mitochondria in WT and VCP WT flies 6 days after eclosion ( E and E' ) .", "In VCP RH and AE flies fiber structure is disrupted , and mitochondria are misaligned and lightly stained , with empty spaces in between ( E'' and E''' ) .", "Scale bar: 40 µm .", "( F–F''' )", "Anti-TDP43 antibody staining shows nuclear ( white arrowhead ) and sarcoplasmic localization of TDP-43 in WT and VCP WT adult fly muscle ( F and F' ) .", "In VCP RH and AE flies the nuclear signal disappears and the signal is increased in muscle sarcoplasm ( F'' and F''' ) .", "Scale bar: 5 µm .", "( G–G''' )", "Anti-Ref ( 2 ) P/p62 antibody staining .", "The signal is weak and uniform in WT and VCP WT flies ( G and G' ) , and punctate in VCP RH and AE flies ( G'' and G''' ) .", "Scale bar: 5 µm .", "( H–H''' )", "Anti-P4D1 ubiquitin antibody staining .", "Signal is weak and uniform in WT and VCP WT flies ( H' and H' ) , and punctate in VCP RH and AE flies ( H'' and H''' ) .", "Scale bar: 5 µm .", "( I–M )", "Effects of mfn RNAi in 8-day-old adult muscle tissue .", "( I–M )", "Wildtype muscle visualized with TUNEL and mitoGFP ( I ) , anti-TDP43 ( J ) , Toluidine blue ( K ) , anti-p62 ( L ) , and anti-ubiquitin ( M ) .", "( I'–M' ) mfn RNAi muscle visualized with the same probes as above .", "Scale bar: 30 µm in K-K’ , 5 µm in the rest . DOI: http://dx . doi . org/10 . 7554/eLife . 17834 . 01010 . 7554/eLife . 17834 . 011Figure 4—figure supplement 1 . Muscle isolated from flies expressing VCP disease mutants does not show gross defects at 2 days post eclosion .", "( A–D )", "Toluidine Blue staining of overall muscle morphology shows that VCP RH and AE expression does not result in tissue damage .", "Scale bar: 40 µm .", "( E–H )", "Cell death , as visualized using TUNEL/MitoGFP assay , is absent ( No Red Signal ) and MitoGFP localization ( Green ) is normal in VCP RH and AE .", "Scale bar: 10 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 17834 . 011 Next , we investigated the cellular basis of mitochondrial defects in our IBMPFD flies using electron microscopy .", "6 days after eclosion , flies expressing VCP WT have smaller mitochondria as compared with control flies , but with intact cristae ( Figure 5A–B’ ) .", "In VCP RH and AE muscle , however , mitochondria are smaller in size , and more fragmented than in VCP WT flies .", "In addition , they also have severe ultrastructural defects under EM studies with a broken cristae ( Figure 5C–D’ ) .", "This phenotype is similar to that of flies with a muscle-specific Mfn knock down ( Figure 5E and E’ ) ( Deng et al . , 2008 ) .", "Muscle from 2-day-old VCP RH and VCP AE flies also show similar phenotypes to Mfn knock down ( Figure 5—figure supplement 1 ) . 10 . 7554/eLife . 17834 . 012Figure 5 . VCP disease mutants are hyperactive in downregulating Mfn protein levels and inhibiting mitochondrial fusion .", "( A–B’ )", "Electronic microscopic images of muscle of different genotypes .", "Compared to WT ( IFM-Gal4 control , A and A’ ) , mitochondria are smaller with intact cristea in VCP WT ( B and B’ ) 6 days after eclosion .", "Scale bar: 1 µm .", "( C–E’ )", "Expression of VCP RH and AE leads to distorted fiber structure , and small mitochondria with broken or empty cristae .", "mfn RNAi flies have similar morphological defects in mitochondria ( E and E’ ) .", "Scale bar: 1 µm .", "( F ) Expression of VCP RH and AE lead to a further reduction in Mfn as compared to VCP WT .", "Mfn levels in VCP WT are reduced to 0 . 73 ± 0 . 21 as compared with Gal4 control , which is set as 1 ( p=0 . 037 , independent t test , N = 3 ) .", "Mfn levels in VCP RH and AE are reduced to 0 . 52 ± 0 . 14 ( p=0 . 037 , independent t test , N = 3 ) and 0 . 32 ± 0 . 16 ( p=0 . 003 , independent t test , N = 3 ) as compared with Gal4 control .", "Mfn levels are normalized with those of Hsp60 , a mitochondria marker .", "Expression of VCP ATPase defective mutant E2Q ( p=0 . 097 , independent t test , N = 3 ) does not significantly change the Mfn level in fly thoraxes as compared with Gal4 control .", "n . s . : no statistical significance , p>0 . 05 .", "Normalized Mfn levels are shown as mean±½SD .", "( G–L )", "MitoGFP localization assay shows expression of VCP WT ( H ) , RH ( I ) and AE ( J ) potently rescues the mitochondrial defects in parkin mutant ( G ) .", "Expression of the ATPase defective mutant E2Q ( K ) or VCP RNAi ( L ) does not .", "Scale bar: 20 µm .", "( M–R )", "MitoGFP assay shows expression of VCP WT ( N ) , RH ( O ) and AE ( P ) potently rescues the mitochondrial defects in parkin mul1 double mutants ( M ) .", "Expression of VCP E2Q ( Q ) and VCP RNAi ( R ) do not .", "Scale bar: 20 µm .", "( S ) Western blot shows that increased Mfn levels normally present in a parkin null mutant are significantly decreased in VCP WT , RH and AE flies , but not in VCP E2Q flies .", "Mfn levels are further decreased in a parkin mutant expressing VCP RH ( p=0 . 0228 , independent t test , N = 3 ) or AE ( p=0 . 00565 , independent t test , N = 3 ) , as compared to VCP WT .", "Samples are from 2-day-old fly thoraces .", "Normalized Mfn levels are shown as mean±½SD .", "*p<0 . 05; **p<0 . 01 . DOI: http://dx . doi . org/10 . 7554/eLife . 17834 . 01210 . 7554/eLife . 17834 . 013Figure 5—figure supplement 1 . Expression of VCP disease mutants leads to small mitochondria with abnormal cristae , phenotypes similar to mfn RNAi knocking down .", "( A–B’ ) 2 days after eclosion , EM shows that compared to WT ( IFM-Gal4 control , A and A’ ) , VCP WT expression results in smaller mitochondrial with intact cristae ( B and B’ ) .", "( C–E’ )", "VCP RH and AE also have smaller and fragmented mitochondria , but the cristae are abnormal ( C–D’ ) , similar to the phenotype of mfn RNAi flies ( E and E’ ) .", "Scale bar: 1 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 17834 . 01310 . 7554/eLife . 17834 . 014Figure 5—figure supplement 2 . Mfn expression can suppress mitochondrial defects observed in VCP RH and AE flies .", "( A ) 2 copies of genomic rescue Mfn-HA ( pCasper-Mfn-HA ) lead to a heterogeneous mitochondrial phenotype in which some mitochondria are elongated .", "A’ shows a typical elongated mitochondrion .", "( B–E’ )", "In 2-day-old flies , expression of 2 copies pCasper-Mfn-HA ( C , C’ and E , E’ ) results in suppression of mitochondrial defects ( fragmented mitochondria with abnormal cristae ) in VCP RH ( B and B’ ) and AE ( D and D’ ) flies .", "Scale bar: 1 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 17834 . 01410 . 7554/eLife . 17834 . 015Figure 5—figure supplement 3 . Mfn expression does not significantly rescue the pathology in VCP RH and AE fly muscles . 2 copies of the pCasper-Mfn-HA/eGFP transgene in VCP RH and AE flies at 6 days of age does not significantly alter muscle cell death assayed by TUNEL/MitoGFP ( A , A’ and F , F’ , Scale bar: 10 µm ) , overall tissue disintegration ( B , B’ and G , G’ , Scale bar: 40 µm ) , TDP43 mislocalization ( C , C’ and H , H’ Scale bar: 5 µm ) , or the frequency of p62 ( D , D' and I , I' , Scale bar: 5 µm ) and ubiquitin positive aggregates ( E , E’ and J-J’ , Scale bar: 5 µm ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 17834 . 015 The strong mitochondrial phenotypes observed in VCP RH or VCP AE predicts that expression of VCP RH or VCP AE may result in a greater decrease in Mfn levels as compared with the expression of VCP WT .", "This is indeed what we observed ( Figure 5F ) .", "This raises the possibility that VCP RH and AE are hyperactive alleles .", "To further substantiate these findings , we compared the Mfn levels in flies expressing VCP E2Q , a well-established ATPase defective mutant containing two mutations ( fly or human residues by number E305Q and E578Q ) that abolish the ATPase activity of the D1 and D2 domains .", "Expression of E2Q did not significantly change the Mfn levels ( Figure 5F ) .", "As a control , the expression of any of the above forms of VCP did not alter expression levels of mitochondrial matrix protein Hsp60 ( Figure 5F ) .", "These results suggest that VCP disease mutants are hyperactive with respect to promoting downregulation of Mfn levels .", "Based on these observations , we hypothesized that VCP disease mutants should also rescue mitochondrial phenotypes in parkin and parkin mul1 double mutants .", "As shown in Figure 5G–J , expression of VCP RH and AE mutants , but not VCP E2Q or VCP RNAi , robustly restored mitochondrial structure , as indicated by MitoGFP fluorescence , in the parkin null mutant ( Figure 5K and L ) .", "Moreover , corresponding to the mitochondrial phenotypes , the accumulation of Mfn in parkin mutants was significantly reduced in the presence of VCP WT , RH and AE , with VCP RH and AE causing greater decreases in Mfn levels than VCP WT ( Figure 5S ) .", "In addition , VCP RH and AE expression also robustly rescued the mitochondrial defects in the parkin mul1 double null mutants , but an expression of VCP E2Q or VCP RNAi did not ( Figure 5M–R ) .", "Taken together , these results indicate that VCP RH and AE disease mutants do not behave as ATPase defective or loss-of-function mutants , and instead are hyperactive with respect to regulation of Mfn .", "A prediction from our finding that Mfn downregulation is important for IBMPFD pathology is that downregulation of Mfn should result in phenotypes similar to those observed with an expression of VCP disease mutants .", "Indeed , mfn muscle-specific knockdown flies have fragmented mitochondria ( Figure 4I–I’ ) , TDP43 mislocalization ( Figure 4J–J’ ) , vacuole formation ( Figure 4K–K’ ) , p62 and ubiquitin accumulation ( Figure 4L–L’ and M–M’ ) , as with the VCP disease mutants .", "Next we asked if mfn overexpression could rescue the phenotypes due to VCP RH and AE .", "Since mfn overexpression results in significant mitochondrial phenotypes , tissue disintegration and cell death on its own ( Yun et al . , 2014 ) , we are unable to address this question completely due to a technical limitation .", "We were , however , able to bring about a mild increase in Mfn levels through addition of 2 copies of an mfn genomic rescue transgene in the background of VCP RH and AE expressing flies .", "Interestingly , this resulted in a suppression of the mitochondrial fragmentation and cristae phenotypes in 2-day-old VCP RH and AE flies ( Figure 5—figure supplement 2 ) but not the pathology observed in VCP RH and AE fly muscles at 6 days ( Figure 5—figure supplement 3 ) .", "Together , our data suggest that downregulation of Mfn is an important contributor to IBMPFD muscle pathology .", "Next , we extended our studies in IBMPFD patient fibroblasts .", "We focused our studies on VCPR155H/+ cells ( thereafter called RH cells ) , as fibroblasts harboring the VCPA232E/+ mutation are not available .", "First , we characterized mitochondrial respiration in control and immortalized patient fibroblasts .", "As shown in Figure 6A , the basal level Oxygen Consumption Rate ( OCR ) in the VCP RH patient cells is slightly decreased compared to the healthy control , suggesting that baseline mitochondrial respiratory chain function is compromised in patient cells .", "Maximum OCR , measured in the presence of the uncoupler FCCP , is significantly decreased in VCP RH IBMPFD patient cells .", "The spare OCR , which is calculated by subtracting the basal OCR from the maximum OCR , provides a measure of reserve mitochondrial capacity for ATP generation .", "The spare OCR in IBMPFD patient cells is significantly decreased as compared with controls .", "Others have recently reported similar observations ( Nalbandian et al . , 2015a ) . 10 . 7554/eLife . 17834 . 016Figure 6 . IBMPFD patient cells carrying the VCPR155H/+ mutation have decreased Mfn 1 and Mfn 2 levels and reduced mitochondrial fusion .", "( A ) Oxygen consumption rates ( OCR ) in healthy control and IBMPFD patient fibroblasts .", "Inhibitory drugs were added at the time points indicated .", "Basal levels of OCR ( p=0 . 026 , independent t test , N = 8 ) and maximum ( CCCP-stimulated ) OCR ( p=3 . 74E-06 , independent t test , N = 8 ) are both decreased in the IBMPFD patient fibroblasts .", "The spare OCR is also significantly decreased ( p=0 . 000024 , independent t test , N = 8 ) .", "( B ) Mfn 1 and 2 levels are consistently decreased in three independent lysates from the IBMPFD patient fibroblasts when compared to the healthy control .", "Mfn 1 levels in patient fibroblasts are reduced to 0 . 66 ± 0 . 14 of the controls ( p=0 . 001 , independent t test , N = 5 ) ; Mfn 2 levels are reduced to 0 . 60 ± 0 . 14 of the controls ( p=0 . 008 , independent t test , N = 5 ) .", "**p<0 . 01 .", "Normalized Mfn 1 and 2 levels are shown as mean±½SD .", "( C–D’ )", "Mitochondrial fusion assay utilizing photoactivatable mitoGFP ( PA-GFP ) in healthy control and IBMPFD patient fibroblasts .", "After laser photoactivation of a region ( Red hollow square in C and D ) , cells were tracked for 30 min . ( E ) PA-GFP signal intensity ( throughout the whole cell ) is recorded every 3 minutes , and the rate of decrease of average GFP signal is a measure of mitochondrial fusion .", "Fusion rates are significantly decreased in the IBMPFD patient fibroblasts when compared to the healthy control ( C’ , D’ and E , independent t test , *p<0 . 05 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 17834 . 016 Disruption of mitochondrial fusion can result in defects in respiration similar to those observed in patient cells ( Chen et al . , 2003b ) .", "Mfn 1 and 2 levels in VCP RH cells were reduced to 0 . 66 ± 0 . 14 and 0 . 60 ± 0 . 14 respectively , as compared with age and gender matched healthy control cells ( set as 1 , Figure 6B ) .", "To explore the functional significance of this decrease , we carried out mitochondrial fusion assays using fibroblasts from healthy controls and patients .", "Both cell populations were transfected with a plasmid encoding a mitochondrial matrix targeted , photoactivated GFP ( PA-GFP ) .", "Following photoactivation of GFP within a region of the cell , the GFP fluorescence signal spreads and the intensity decreases as these mitochondria fuse with non-activated neighboring mitochondria .", "Thus , in this assay an increased rate of fluorescence loss corresponds to increased mitochondrial fusion activity ( Mishra et al . , 2014a ) .", "In control cells , PA-GFP gradually decreases after photoactivation; in the VCP RH IBMPFD patient cells , the GFP signal decrease is significantly delayed ( Figure 6C–D’ , E ) .", "These data suggest that the VCP RH mutation results in a decrease in Mfn 1 and 2 levels that lead to impaired mitochondrial fusion .", "In addition , the VCP mutant impairs both basal and maximal mitochondrial respiratory function .", "It has been controversial in the field whether VCP disease mutants with increased ATPase activity behave as dominant-active or dominant-negative mutants .", "Some studies show that VCP RH and AE , with enhanced ATPase activity ( Weihl et al . , 2006; Zhang et al . , 2015; Niwa et al . , 2012; Manno et al . , 2010; Tang and Xia , 2013 ) , result in hyperactivity in animal disease models ( Chang et al . , 2011 ) .", "However , other cell-based studies suggest they behave in a dominant negative fashion ( Ritz et al . , 2011; Ju et al . , 2009; Kim et al . , 2013; Bartolome et al . , 2013; Kimura et al . , 2013 ) .", "Understanding how VCP disease mutants behave is critical for understanding the disease process and finding possible therapies .", "We hypothesize that if an abnormally enhanced ATPase activity causes pathology , disease severity should ameliorate if ATPase activity is decreased through the use of VCP ATPase inhibitors .", "To test this hypothesis , we characterized the effects of VCP inhibitors in vivo .", "Multiple VCP ATPase activity inhibitors have recently been described ( Chapman et al . , 2015 ) .", "NMS-873 , 3-[3-Cyclopentylsulfanyl-5- ( 4'-methanesulfonyl-2-methyl-biphenyl-4-yloxymethyl ) -[1 , 2 , 4] triazol-4-yl]-pyridine , an allosteric inhibitor of VCP ATPase activity , is a highly specific and robust VCP ATPase inhibitor ( Magnaghi et al . , 2013 ) .", "ML240 , 2- ( 2-Amino-1H-benzimidazol-1-yl ) −8-methoxy-N- ( phenylmethyl ) −4-quinazolinamine is another potent inhibitor , which competitively blocks ATP binding to VCP ( Chou et al . , 2013 ) .", "These drugs and their derivatives were developed for treatment of cancers ( Magnaghi et al . , 2013; Chou et al . , 2013; Deshaies , 2014; Zhou et al . , 2015 ) .", "The ability of these compounds to rescue defects in IBMPFD models has not been reported .", "We first examined the effects of VCP inhibitors delivered to Drosophila through feeding .", "Various concentrations of drug or DMSO were included in food during the first to third larval instars , and muscle mitochondrial morphology was characterized at 2 and 6 days after eclosion .", "NMS-873 or ML240 feeding resulted in dramatic mitochondrial elongation in wildtype animals as visualized with MitoGFP using light microscopy ( Figure 7A–A’’ ) and at the ultrastructural level using EM ( Figure 7B–B’’’ ) .", "These phenotypes are similar to those observed following VCP RNAi ( Figure 1C–C’’ ) .", "In another respect , drug-treated animals developed normally ( data not shown ) .", "The smaller mitochondria phenotype associated with overexpression of VCP WT was also significantly suppressed following feeding with 30 µM NMS-873 or 30 µM ML240 ( Figure 7C–C’’’ ) .", "Feeding with NMS-873 and ML240 also reversed the phenotypic rescue of mitochondrial defects caused by expression of VCP WT , RH and AE in PINK1 mutants ( Figure 7—figure supplement 1 ) .", "Supporting these results , feeding with NMS-873 also resulted in a dose-dependent increase in the levels of Mfn ( Figure 7D ) .", "Together , these observations suggest that NMS-873 and ML240 inhibit endogenous VCP in vivo , resulting in a phenocopy of loss of VCP function . 10 . 7554/eLife . 17834 . 017Figure 7 . VCP inhibitors promote mitochondrial elongation .", "( A–A’’ )", "MitoGFP assay for mitochondrial morphology .", "Compared to DMSO-fed flies , 10 µM NMS-873 or ML240 feeding results in more fused mitochondria in 2-day-old flies .", "Scale bar: 5 µm .", "( B–B’’ and C–C’’ )", "In 6-day-old flies fed with 30 µM NMS-873 ( B’ ) or 30 µM ML240 ( B’’ ) , elongated mitochondria ( outlined in dashed white lines ) are observed in muscle .", "Mitochondria are small in VCP WT flies ( C ) as compared with WT ( IFM-Gal4 control , B ) .", "This phenotype is reversed by 30 µM NMS-873 ( C’ ) or 30 µM ML240 ( C’’ ) feeding and shifted towards a pro-fusion direction , as the mitochondria are also elongated as compared with WT ( B ) .", "( B’’’ and C’’’ )", "Statistical analysis of mitochondrial size in EM cross sections .", "In wildtype flies , 30 µM NMS-873 or ML240 feeding results in a mitochondrial size increase to 6 . 46 ± 0 . 44 µm2 ( p=0 . 007 , independent t test , N = 45 ) or 5 . 75 ± 0 . 48 µm2 ( p=0 . 032 , independent t test , N = 39 ) as compared to the DMSO group ( 3 . 45 ± 0 . 27 µm2 , N = 47 ) .", "VCP WT expression results in small mitochondria ( 1 . 78 ± 0 . 05 µm2 , N = 68 ) as compared with WT ( B ) .", "Feeding of 30 µM NMS-873 ( 5 . 41 ± 0 . 40 µm2 , p=0 . 000 , independent t test , N = 44 ) or ML240 ( 4 . 90 ± 0 . 42 µm2 , p=0 . 000 , independent t test , N = 45 ) reverses these effects .", "Mitochondria size is shown as mean±½SEM .", "( D ) pCasper-Mfn-eGFP levels accumulate in a dose dependent manner when treated with NMS-873 at 0 . 5 µM and 1 µM for 14 hr . DOI: http://dx . doi . org/10 . 7554/eLife . 17834 . 01710 . 7554/eLife . 17834 . 018Figure 7—figure supplement 1 . VCP inhibitor treatment blocks VCP WT and disease mutant rescue of mitochondrial defects in a PINK1 mutant .", "( A–B’ )", "MitoGFP assay shows that NMS-873 and ML240 feeding blocks the ability of VCP WT to rescue PINK1 mutant mitochondrial phenotypes in a dose-dependent manner .", "2 . 5 µM NMS-873 ( A ) and 10 µM ML240 ( B ) partially inhibit the rescue effect .", "The rescue effect is significantly blocked with 10 µM NMS-873 ( A’ ) and 20 µM ML240 ( B’ ) .", "Scale bar: 20 µm .", "( C–E’’ ) 10 µM NMS-873 blocks VCP WT ( C’ ) , RH ( D’ ) and AE ( E’ ) rescue of PINK1 mutant mitochondrial phenotypes ( C–E ) as does feeding with 15 µM ML240 in rescue flies of VCP WT ( C’’ ) , RH ( D’’ ) and AE ( E’’ ) in PINK1 mutant .", "Scale bar: 5 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 17834 . 018 We then fed NMS-873 and ML240 to VCP RH and AE disease mutant flies and analyzed mitochondrial phenotypes , muscle viability and integrity in 6-day-old flies .", "Flies were fed with 30 µM NMS-873 or 30 µM ML240 from first instar larvae until 6 days after eclosion .", "In control and VCP WT expressing flies , NMS-873 and ML240 feeding resulted in normal development to adulthood ( data not shown ) and did not cause muscle death or tissue damage; all muscles were TUNEL negative and had an intact tissue structure ( Figure 8—figure supplement 1 ) .", "Strikingly , NMS-873 and ML240 feeding largely prevented muscle cell death caused by expression of VCP RH or AE ( Figure 8A–B’’ ) .", "It also significantly rescued disrupted muscle integrity ( Figure 8C–D’’ ) .", "Rescue of mitochondrial size , cristae structure , and myofibril organization was also observed at the ultrastructural level ( Figure 8E–F’’ ) .", "It is interesting to note that in the VCP disease mutants feeding groups ( Figure 8E’ , E’’ , F’ and F’’ ) , mitochondria remained somewhat smaller as compared to wildtype in Figure 7B .", "Elongated mitochondria , such as those observed in the wildtype and VCP WT feeding groups ( Figure 7B’ , B’’ , C’ and C’’ ) , were never observed in the VCP RH and VCP AE groups even though all were fed the same concentration of inhibitors .", "This partial suppression further suggests that these mutants are hyperactive .", "Finally , we note that VCP inhibitor feeding also resulted in significant rescue of a number of other phenotypes in VCP RH and AE expressing flies , including those associated with p62 , ubiquitin and TDP43 ( Figure 8G–L’’ ) .", "Together , these observations suggest that VCP disease mutants are hyperactive in multiple aspects , and that disease phenotypes can be suppressed by inhibition of VCP ATPase activity . 10 . 7554/eLife . 17834 . 019Figure 8 . VCP inhibitors block mitochondrial defects , muscle tissue damage and muscle cell death in VCP disease mutant flies .", "( A–B’’ )", "Expression of VCP RH and AE leads to muscle cell death by 6 days after eclosion , as visualized through loss of MitoGFP and TUNEL positive signal in nuclei .", "In the DMSO feeding groups 3 . 4 ± 0 . 5% of VCP RH and 4 . 8 ± 0 . 6% of VCP AE flies are TUNEL negative .", "30 µM NMS-873 or ML240 feeding since first instar larvae till 6 days after eclosion significantly blocks the muscle cell death , with 64 . 3 ± 17 . 0% ( p=0 . 017 ) and 85 . 1 ± 19 . 0% ( p=0 . 000 ) of VCP RH , and 80 . 9 ± 10 . 1% ( p=0 . 002 ) and 95 . 6 ± 3 . 1% ( p=0 . 000 ) of VCP AE muscles TUNEL negative .", "Data are shown as mean±½SD .", "Independent t test is used .", "Three independent rounds are performed , 15–20 flies are used each round .", "*p<0 . 05; **p<0 . 01 .", "Scale bar: 10 µm .", "( C–D’’ )", "Toluidine Blue staining shows that 30 µM NMS-873 or ML240 feeding significantly rescue the disrupted muscle structure in VCP RH and AE flies .", "Scale bar: 40 µm .", "( E–F’’ )", "EM shows that 30 µM NMS-873 or ML240 feeding significantly rescue the broken myofibril structure and small mitochondria with broken cristae phenotypes in VCP RH and AE flies .", "Scale bar: 1 µm .", "Note that the mitochondria are still smaller than those from WT flies ( Figure 6B ) .", "( G–L’’ )", "In 6-day-old flies , 30 µM NMS-873 ( G’ , H’ , J’ and K’ ) or ML240 ( G’’ , H’’ , J’’ and K’’ ) treatments result in a significant decrease in Ref ( 2 ) P/p62 ( G and J ) and ubiquitin ( H and K ) aggregates in VCP RH and AE expressing flies; TDP43 mislocalization ( I and L ) is partially rescued as the large sarcoplasmic signal decreases , but no nuclear signal is observed ( I’ , I’’ , L’ and L’’ ) .", "Scale bar: 5 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 17834 . 01910 . 7554/eLife . 17834 . 020Figure 8—figure supplement 1 . VCP inhibitor feeding does not affect muscle viability and tissue integrity in 6-day-old flies .", "( A–B’’ )", "TUNEL/MitoGFP assay shows that feeding of 30 µM NMS-873 or ML240 does not cause muscle cell death in WT ( IFM-Gal4 control , A–A’’ ) and VCP WT flies ( B–B’’ ) .", "Scale bar: 10 µm .", "( C–D’’ )", "Toluidine Blue assay shows that feeding of 30 µM NMS-873 or ML240 does not disrupt muscle integrity in WT ( IFM-Gal4 control , C–C’’ ) and VCP WT flies ( D–D’’ ) .", "Scale bar: 40 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 17834 . 020 The above data demonstrated a decrease in Mfn 1 and 2 levels , mitochondrial fusion defects and impaired mitochondrial respiratory function in IBMPFD patient cells .", "Next , we explored the effects of these drugs on these changes in IBMPFD patient fibroblasts .", "In patient cells , a high concentration of ML240 ( 10 µM , with IC50 of 110 nM ) or NMS-873 ( 10 µM , with IC50 of 30 nM ) led to cell death , and NMS-873 directly inhibits mitochondrial respiration ( data not shown ) .", "However , when lower concentrations of ML240 are used , robust rescue effects are observed ( Figure 9 ) .", "As shown in Figure 9A , immortalized IBMPFD patient fibroblasts have decreased Mfn 1 levels , and these are increased by treatment with 250 nM ML240 for 6 hr .", "Overall mitochondrial morphology of IBMPFD fibroblasts does not change in response to inhibitor treatment ( Figure 9—figure supplement 1 ) .", "However , in a more sensitive assay in which rates of fusion are measured directly , mitochondrial fusion defects in IBMPFD patient fibroblasts were significantly rescued after 6 hr of treatment with 250 nM ML240 ( Figure 9B ) .", "Finally , 50 nM and 100 nM ML240 treatment for 4 days also significantly enhanced the basal and maximal oxygen consumption rate ( OCR ) in patient fibroblasts ( Figure 9C ) .", "Similarly , 1 nM and 10 nM NMS-873 treatment for 6 days significantly enhanced the maximal oxygen consumption rate ( OCR ) in patient fibroblasts as well ( Figure 9D ) .", "The fact that two inhibitors with different mechanisms generate similar results in terms of rescuing IBMFPD pathologies reduces the likelihood that these are off-targeting effects .", "Together , these data strongly support the effects of VCP inhibitors as potential therapeutic tools for IBMPFD disease . 10 . 7554/eLife . 17834 . 021Figure 9 . VCP inhibitor treatment significantly suppresses mitochondrial respiratory chain and fusion defects in VCPR155H/+ IBMPFD patient fibroblasts .", "( A ) After treatment of 250 nM ML240 for 6 hr , Mfn 1 level of VCPR155H/+ IBMPFD patient fibroblasts is elevated from 0 . 77 ± 0 . 02 to 1 . 05 ± 0 . 11 ( p=0 . 037 , independent t test , N = 3 ) , as compared with healthy controls , for which values are set as 1 .", "( B ) After treatment with 250 nM ML240 for 6 hr , the mitochondria fusion assay shows that the decreased mitochondrial fusion observed in IBMPFD patient’s fibroblasts is significantly reversed ( independent t test , *p<0 . 05 ) .", "( C ) 50 nM and 100 nM ML240 treatment for 4 days significantly increases basal ( p=0 . 0001 , DMSO v . s . 50 nM ML240; p=0 . 0027 , DMSO v . s . 100 nM ML240 , Welch's unpaired t test , N = 8 ) and maximal oxygen consumption rate ( p=0 . 0415 , DMSO v . s . 50 nM ML240; p=0 . 0028 , DMSO v . s . 100 nM ML240 , Welch’s unpaired t test , N = 8 ) in the IBMPFD patient fibroblasts harboring VCPR155H/+ mutation .", "( D ) 1 nM and 10 nM NMS-873 treatment for 6 days significantly increases maximal oxygen consumption rate ( p=0 . 0021 , DMSO v . s . 1 nM NMS-873; p=0 . 0395 , DMSO v . s . 10 nM NMS-873 , N = 8 ) , but not basal oxygen consumption rate ( p=0 . 0994 , DMSO v . s . 1 nM NMS-873; p=0 . 1804 , DMSO v . s . 10 nM NMS-873 , N = 8 ) .", "Welch’s unpaired t test is used .", "*p<0 . 05 , **p<0 . 01 , n . s , no statistical significance . DOI: http://dx . doi . org/10 . 7554/eLife . 17834 . 02110 . 7554/eLife . 17834 . 022Figure 9—figure supplement 1 . ML240 and NMS-873 treatments do not significantly change the mitochondrial morphology in IBMPFD patients . In healthy control fibroblasts mitochondria display an elongated and interconnected phenotype ( A–A’ ) .", "IBMPFD patient’s fibroblasts harboring VCPR155H/+ mutation do not show a significant mitochondrial morphological difference ( B–B’ ) .", "Treatment of these cells with 100nM ML240 treatment for 4 days ( C-C’ ) or 10 nM NMS-873 treatment for 6 days ( D–D’ ) also does not result in a change in mitochondrial morphology ( though see Figure 9 for evidence that rates of fusion are altered ) .", "Scale bar: ( A–D ) 20 µm; Scale bar: ( A’–D’ ) : 5 µm .", "Anti-Tom20 antibody is used for mitochondial morphology assay . DOI: http://dx . doi . org/10 . 7554/eLife . 17834 . 022" ], [ "Missense mutations of VCP lead to the autosomal dominant disease IBMPFD .", "However , it is controversial whether these mutations cause disease through a dominant active or dominant negative manner .", "Moreover , the molecular mechanisms by which VCP mutations alter mitochondrial function are unclear .", "Here , we have investigated the endogenous function of VCP , cellular effects of expression of VCP disease mutants , and the ability of VCP inhibitors to suppress VCP disease pathology in Drosophila and patient fibroblasts .", "In summary , we have made the following findings: Endogenous VCP regulates mitochondrial fusion ( Figure 10A ) .", "Loss of VCP function leads to increased Mfn levels and increased mitochondrial size in multiple tissues .", "Conversely , VCP overexpression results in reduced Mfn levels and decreased mitochondrial size .", "VCP physically interacts with Mfn via domains that are also used to bind its substrates in other contexts .", "Thus , it is likely that Mfn is an endogenous VCP substrate .", "Here we provide the first in vivo evidence in muscle , the major disease tissue in IBMPFD , that Mfn is a specific endogenous target of VCP .", "Regulation of endogenous Mfn by VCP is unexpected , given that VCP is an abundant protein implicated in multiple cellular processes , but has not been shown to localize to mitochondria that are not depolarized by CCCP .", "We show that VCP-dependent removal of Mfn does not require Parkin or Mul1 , E3 ligases known to ubiquitinate Mfn .", "How VCP brings about Parkin and Mul1-independent removal of Mfn requires further investigation . 10 . 7554/eLife . 17834 . 023Figure 10 . Proposed mechanisms of VCP disease mutants mediated mitochondrial defects and potential therapeutic role of VCP inhibitors .", "( A ) Under physiological conditions , VCP , independent of PINK1/parkin and Mul1 , negatively regulates Mfn protein levels , which are critical for the proper balance of mitochondrial fusion and fission .", "( B ) In the IBMPFD disease , the enhanced ATPase activity of a VCP disease mutant protein results in excessive loss of Mfn and mitochondrial fusion , leading to mitochondrial fusion defects .", "Besides the mitochondrial defects , VCP disease mutants also generate pathology including adult muscle tissue damage and muscle cell death .", "( C ) VCP ATPase inhibitors robustly relieve the pathology caused by VCP disease mutants associated with hyperactive VCP activity while leaving normal VCP-dependent functions intact . DOI: http://dx . doi . org/10 . 7554/eLife . 17834 . 023 IBMPFD is 100% penetrant , and myopathy is the most prevalent ( affecting 90% of patients ) and primary symptom of IBMPFD patients .", "We generated an IBMPFD disease model in adult Drosophila muscle , and used this system to monitor tissue pathology , and investigate the molecular mechanisms by which VCP disease mutants function .", "We show that compared to VCP WT , expression of VCP disease mutants results in tissue damage , mitochondrial fusion defects and decreased levels of Mfn ( Figure 10B ) .", "These phenotypes are opposite to those associated with decreased levels of VCP , or expression of an ATPase defective mutant version of VCP .", "Expression of VCP disease mutants , but not a VCP ATPase defective mutant or VCP RNAi , also suppresses the mitochondrial defects mediated by accumulation of Mfn in PINK1 null , parkin null , and parkin mul1 double null mutants .", "Taken together , these results suggest that VCP disease mutants do not function as loss-of-function or dominant negative mutants , but rather as dominant activated mutants in regulating Mfn levels and bringing about mitochondrial fusion defects .", "This conclusion is further supported by our findings that mitochondrial fusion and Mfn levels are also decreased in VCP disease mutant fibroblasts .", "Our finding that VCP overexpression suppresses a parkin null mutant in addition to a PINK1 null stands in contrast to what was reported in Kim et al . , who proposed that VCP-dependent degradation of Mfn required recruitment by Parkin .", "In addition , we also find that VCP overexpression suppresses phenotypes due to double mutants of parkin and mul1 , which acts in a parallel pathway to degrade Mfn ( Yun et al . , 2014 ) .", "Together , these observations show that VCP-dependent regulation of Mfn does not require PINK1 or parkin .", "The VCP-dependent degradation of Mfn we observe is also specific and unlikely to be mediated through a more general process such as mitophagy , as levels of several other mitochondrial proteins are not altered by VCP overexpression .", "However , these results do not exclude models in which Parkin activation further promotes VCP-dependent Mfn degradation , which may or may not involve mitophagy , under some conditions .", "Regarding the mechanisms of VCP disease mutant action , in vitro biochemistry studies suggest that they have increased ATPase activities , thus favoring a dominant active model ( Weihl et al . , 2006; Zhang et al . , 2015; Niwa et al . , 2012; Manno et al . , 2010; Tang and Xia , 2013 ) .", "In addition , in vivo data from Drosophila shows that eye phenotypes caused by VCP disease mutant expression can be alleviated through loss of one copy of wildtype VCP .", "This also is consistent with models in which VCP mutants have increased activity ( Chang et al . , 2011 ) .", "In contrast , in cell culture studies Bartolome , et al showed that VCP disease mutant expression led to mitochondria respiratory chain defects , as did VCP RNAi ( Bartolome et al . , 2013 ) ; Ritz , et al showed that VCP disease mutant expression impaired endocytosis to the same extent as that of VCP ATPase defective mutants ( Ritz et al . , 2011 ) .", "While the above studies can be interpreted as showing that VCP disease mutants function as loss-of-function or dominant negative mutants , these results do not rule out the possibility that disease mutants are hyperactive alleles .", "For example , an alternative interpretation of the impairment of respiratory chain function observed following VCP disease mutant expression or following VCP RNAi is that either an increase or a decrease in the levels of VCP activity leads to mitochondrial defects , which is what we observed in this study .", "Currently there is no treatment that can halt the progression of IBMPFD .", "Our findings that VCP disease mutants are hyperactive provide important therapeutic implications .", "Indeed , we find VCP inhibitors potently rescue multiple VCP disease phenotypes in flies and patient cells ( Figure 10C ) .", "Importantly , suppression includes phenotypes beyond mitochondria ( Figure 10C ) , involving TDP43 , p62 and ubiquitin , suggesting that these inhibitors are likely to be effective in therapeutic settings .", "Of course , in order for VCP inhibitors to be useful as therapeutics , it will be necessary to inhibit mutant forms of VCP to an extent sufficient to suppress disease without bringing about a deleterious decrease in the activity of wildtype VCP .", "Our observation that Drosophila fed inhibitors are suppressed with respect to VCP disease phenotypes , but otherwise develop normally , suggests that this therapeutic goal may be possible .", "Finally , VCP disease mutants are associated with sporadic amyotrophic lateral sclerosis ( Abramzon et al . , 2012 ) , and hereditary spastic paraplegia ( de Bot et al . , 2012 ) and Charcot-Marie-Tooth disease ( Gonzalez et al . , 2014 ) .", "Our findings suggest possible therapeutic values of VCP inhibitors for these diseases ." ], [ "Full length VCP ( 1-802aa ) , and truncated VCP cDNAs ∆N-VCP ( 186–802 aa ) , ∆N + D1-VCP ( 456–802 aa ) and ∆D2-VCP ( 1–477 aa ) were amplified from VCP cDNA derived from pUASt-VCP WT , a gift from Dr . Yun Nung Jan ( Rumpf et al . , 2011 ) .", "All cDNAs were subcloned into pUASt vectors as translational fusions fused with the 6 Myc tag using the Gateway cloning system ( Invitrogen ) .", "To generate pUASt-Mfn-3Flag , Mfn cDNA was obtained from an EST clone ( Drosophila Genome Research Center , RE04414 ) , and subcloned into pUASt with the 3Flag tag .", "To generate VCP RNAi 2 , microRNA precursors targeting the coding region of VCP transcripts were cloned into the pUASt vector .", "The MicroRNA-based silencing technology has been described previously ( Chen et al . , 2006; Ganguly et al . , 2008 ) .", "All constructs generated above were verified with sequencing .", "UASt-VCP WT , VCP RH , AE and E2Q lines were gifts from Dr . Tzu Kang Sang ( Chang et al . , 2011 ) .", "IFM-Gal4 , UAS-Mfn RNAi , pCasper-DRP1-HA , PINK15 , parkin25 , dpk21 , parkin25mulA6 were described previously ( Clark et al . , 2006; Deng et al . , 2008; Yun et al . , 2014 ) .", "VCP RNAi line1 flies were obtained from the Vienna Drosophila RNAi Center ( VDRC 24354 ) .", "VCP RNAi 2 was generated in the lab ( see above ) .", "Efficiency of RNAi knocking down was verified by Western blot .", "24B-Gal4 , Mef2-Gal4 and EDTP-Gal4 were obtained from the Bloomington Stock Center at Indiana University .", "The pCasper-Mfn-HA flies ( Sandoval et al . , 2014 ) were a kind gift from Dr . Hugo Bellen at Baylor College of Medicine , Texas .", "The pCasper-Mfn-eGFP construct was a kind gift from Dr . C . K . Yao at Academia Sinica , Taipei .", "Flies carrying pCasper-Mfn-eGFP and pUASt-VCP RNAi two constructs were created through injection in a w1118 genetic background ( pCasper-Mfn-eGFP , X chromosome; VCP RNAi line 2 , second chromosome , Rainbow Transgenic Flies , Inc . ) .", "vcpK15502 , hsFLP22 , neoFRT42D , and Ubi-mRFP . NLS , neoFRT42D were obtained from Bloomington stock center .", "Drosophila strains were maintained in a 25°C humidified incubator or at room temperature .", "vcpK15502 was recombined with neoFRT42D .", "Female flies with the genotype: hsFLP22 / +; vcpK15502 , neoFRT42D / Ubi-mRFP . NLS , neoFRT42D or pCasper-Mfn-eGFP / hsFLP22; vcpK15502 , neoFRT42D / Ubi-mRFP . NLS , neoFRT42D were heat-shocked at 37°C for 2 hr every day from second instar larvae until pupae formation .", "After eclosion , flies were fed with dry yeast paste for 24 hr to stimulate oogenesis .", "The egg chambers then were dissected and fixed in 3 . 7% formaldehyde/Schneider’s Buffer for immunoflurescence assays .", "Fly thoraxes of the relevant genotypes were cut and fixed in 4% paraformaldehyde/Schneider’s Buffer at desired time points .", "Indirect flight muscles were then dissected out and separated .", "Muscles were further blocked with blocking buffer [50 mM Tris-Cl ( pH 7 . 4 ) , 0 . 1% Triton X-100 , 188 mM NaCl] and assayed using the In Situ Cell Death Detection Kit ( Roche ) .", "15–20 fly thoraxes are dissected for each round .", "For each experiment , at least three rounds were performed .", "Fly thoraxes of the relevant genotypes were fixed in 1% paraformaldehyde/1% glutaraldehyde/0 . 1 M Phosphate Buffer , post-fixed in 1% osmium tetroxide/ddH20 , dehydrated in gradient ethanol and embedded in Epon 812 .", "After polymerization , sections were obtained using either glass knives or diamond knife ( Diatome ) .", "1 . 0–1 . 5 µm sections were stained with Toluidine blue .", "80–90 nm sections were stained with uranyl acetate and lead citrate and examined by transmission electron microscope ( UCLA Brain Research Institute Electron Microscopy Facility ) .", "At least 3 thoraxes of each genotype were examined .", "Electron microscopy images ( 10 , 000X magnification ) were analyzed in Image J Software ( National Institute of Health ) .", "After setting the scale , each mitochondrion on the image was selected and its area calculated .", "An independent t test was used to test for statistical significance .", "At least three images were analyzed for each thorax and at least 3 thoraxes of each genotype were examined .", "For assays in indirect flight muscles , muscles of the corresponding age and genotypes were fixed in 4% paraformaldehyde/Schneider’s Buffer , permeabilized in 0 . 3% Triton-X100/PBS .", "Rhodamine Phalloidin ( Invitrogen , 1:500 ) was used for myofibrile staining .", "Muscle pieces were incubated with anti-TDP43 rabbit monoclonal antibody ( ProteinTech , 1:100 ) , anti-Ref ( 2 ) P/p62 rabbit polyclonal antibody ( Abcam , 1:100 ) , anti-P4D1 monoclonal antibody ( ENZO , 1:100 ) at 4°C overnight .", "Washes were followed by Goat anti-rabbit/mouse Alexa Fluor 546 secondary antibody ( Invitrogen 1:200 ) at room temperature for 2 hr .", "For assays of egg chambers , egg chambers were fixed in 3 . 7% formaldehyde/Schneider’s Buffer for 30 min and permeabilized with 0 . 4% Triton-X100/PBS for 4 hr .", "Egg chambers were incubated with anti-GFP rabbit polyclonal antibody ( Invitorgen 1:100 ) , anti-ATP5A mouse monoclonal antibody ( Abcam , 1:100 ) at 4°C overnight and followed by goat anti-rabbit/mouse Alexa Fluor 488/546 secondary antibodies ( Invitrogen 1:200 ) at 4°C overnight .", "Fibroblasts were fixed in 10% formalin for 10 min , 0 . 2% Triton-X100/PBS permeabilization for 15 min , 5% FBS/PBS blocking for 1 hr , anti-Tom20 mouse monoclonal antibody ( BD , 1:200 ) at 4°C overnight and followed by goat anti-mouse Alexa Fluor 488 secondary antibodies ( Invitrogen 1:200 ) at 4°C overnight .", "Images were taken using a LSM5 confocal microscope ( Zeiss ) .", "S2 cells were cultured in Schneider’s Buffer , 10% FBS , 1% Penicillin and Streptomycin , 50 µM Tetracyclin at 25°C .", "Qiagen Effectene and was used as a transfection reagent according to the producer’s instruction .", "Mfn DsRNA were designed according to a protocol from www . flyrrnai . org and synthesized using the Mega T7 kit from Ambion .", "Cells were harvested 96 hr after dsRNA treatment .", "Mfn dsDNA-F: TGA GCA AAT ACC CCC AAA AG; Mfn dsDNA-R: GAT CTG GAG CGG TGA TTT GT .", "Human primary fibroblasts from IBMPFD ( GM21752 ) and age-matched control ( GM00024 ) were obtained from the Coriell Institute for Medical Research ( https://catalog . coriell . org ) and grown in Dulbecco’s Modified Eagle Medium ( DMEM , Gibco ) or Minimum Essential Media ( MEM , Gibco ) supplemented with 10% fetal bovine serum ( FBS ) , 2 mM L-glutamine , and penicillin/streptomycin at 37°C and 5% CO2 .", "Primary fibroblasts were immortalized by infection with retrovirus expressing hTERT ( Addgene plasmid #1771 ) .", "Infected cells were selected in puromycin ( 1 . 5 ug/mL ) for 1 week and maintained in 1 ug/ml puromycin .", "S2 cells , fly thoraxes or fibroblasts were lysed in RIPA Buffer with Protease Inhibitors ( Roche ) and 200 mM PMSF ( Sigma ) .", "Protein lysates were centrifuged at 16 , 000g for 15 mins , and the supernatants boiled with 6XSDS sample Buffer ( Bioland ) at 95°C for 5 mins .", "Proteins were separated in SDS-PAGE Gels .", "Gels were transferred to PVDF membrane ( Millipore ) and incubated with primary antibody at 4°C overnight .", "Following several washes , blots were then incubated with secondary antibodies for 2 hr at room temperature and then washed extensively .", "Primary antibodies used include: Anti-HA mouse monoclonal antibody ( Millipore , 1:1000 ) , Anti-Myc mouse monoclonal antibody ( Millipore , 1:3000 ) , Anti-Flag rabbit polyclonal antibody ( Genscript , 1:2000 ) , Anti-GFP rabbit polyclonal antibody ( Invitrogen , 1:3000 ) , Anti-Actin rabbit polyclonal antibody ( Sigma , 1:2000 ) , Anti-Tubulin mouse monoclonal antibody ( Sigma , 1:4000 ) , Anti-Porin mouse monoclonal antibody ( Abcam , 1:2000 ) , Anti-VCP rabbit monoclonal antibody ( Cell Signalling Technology , 1:2000 ) , Anti-Human Mfn 1 and 2 mouse monoclonal antibody ( Abcam , 1:2000 ) , Anti-MnSOD rabbit polyclonal antibody ( Abcam , 1:2000 ) and Anti-NDUSF3 mouse monoclonal antibody ( Abcam , 1:2000 ) .", "Anti-Drosophila Mfn Rabbit polyclonal antibody ( 1:3000 ) was a gift from Dr . Alexander Whitworth ( Ziviani et al . , 2010 ) .", "Donkey anti-mouse and anti-rabbit HRP conjugated secondary antibodies ( GE Healthcare , 1:10000 or 20000 ) were used .", "48 hr after transfection , S2 cells were harvested and lysed in the RIPA Buffer with protease inhibitors .", "Protein lysates were incubated with Dynabeads G and primary antibody ( Anti-Myc mouse monoclonal antibody , Millipore 1:300 ) at 4°C overnight and washed in 0 . 1%Tween/PBS four times and eluted in 2XSDS sample buffer ( BioRad ) and denatured at 95°C .", "The samples are assayed by western blot .", "Powdered forms of NMS-873 ( Selleckchem ) and ML240 ( Sigma-Aldrich ) were dissolved in DMSO as stocks .", "Stock solution and DMSO as the vehicle control were diluted in ethanol/ddH2O and mixed with Drosophila food .", "Food dye was added to ensure thorough mix .", "Parents of desired genotypes were put in DMSO or inhibitors containing food for 3 days and removed .", "Thus , all growth following egg laying occurred in the presence of inhibitors .", "Immediately after eclosion , the progeny were transferred to newly prepared food vials containing the same concentration of DMSO or inhibitors .", "The progeny was assayed at the time points stated in the text .", "For human fibroblast treatment , stock solution of ML240 , NMS-873 and DMSO vehicle control were diluted to the desired concentration and added to the culture media .", "Media were changed each day if the treatment was longer than 24 hr .", "The mitochondrial fusion assay was performed using photoactivatable mitoGFP ( PA-GFP ) localized to the mitochondrial matrix as previously described ( Mishra et al . , 2014b; Karbowski et al . , 2004 ) .", "Briefly , immortalized fibroblasts were infected with retroviruses expressing matrix-treated DsRed and matrix-targeted PA-GFP to create stable cell lines .", "Cells were plated on glass coverslips ( LabTek ) and imaged live at 37°C on an LSM 710 confocal microscope ( Carl Zeiss , Inc . ) .", "PA-GFP is activated in a region of interest ( 5 µm x 5 µm ) by illumination with a 405 nm laser .", "The activated signal is collected in z-stacks every 3 min over the next 30 min .", "Fusion events result in the dilution of the activated signal , and the average pixel intensity ( for the entire cell ) over time is a measurement of fusion rates ( Karbowski et al . , 2004 ) .", "P-values are calculated using a Student’s t-test on the slopes of intensity versus time for individual measurements ( >20 per genotype ) .", "Oxygen consumption rates ( OCR ) were measured in immortalized fibroblasts using a Seahorse Biosciences Extracellular Flux Analyzer ( Model XF96 ) .", "Briefly , 10 , 0000 cells/well were plated the day before measurement into a 96 well culture plate .", "On the day of measurement , the media was exchanged for non-bicarbonate-containing DMEM ( Sigma-Aldrich Cat . #D5030 ) media using the Seahorse PrepStation , and allowed to equilibrate for 1 hr at 37°C in room air prior to initiating measurements .", "Oxygen levels were measured over 5 min periods .", "Oxygen consumption rate was measured under basal and stress conditions as indicated .", "Oligomycin inhibits complex V and blocks ATP production dependent on respiration .", "CCCP uncouples oxidative phosphorylation and ATP production .", "It maximizes respiration without ATP synthesis .", "Antimycin A inhibits Complex III and thus blocks proton pumping and membrane potential formation .", "Drugs were injected automatically as indicated in the figure legends ( Oligomycin 5 µM , CCCP 10 µM , Antimycin A 1 µM ) .", "For drug pre-treatment studies ( Figure 9C–D ) , 5000 cells/well were plated into 96 well culture plates and pre-treated with ML240 for 4 days and NMS-873 for 6 days prior to OCR measurement .", "OCR values were normalized from total cellular content via sulforhodamine B measurements .", "The overall study design was a series of controlled laboratory experiments in Drosophila and human fibroblasts , as described in detail in the Figure legends sections .", "In all experiments , animals were randomly assigned to various experimental groups .", "The experiments were replicated at least three times ( N was noted in each experiment ) and the final analysis was presented .", "For in vivo experiments , 10–20 flies per group were used for each experiment .", "For the human fibroblasts experiments , details are described in the above methods sections .", "The data were analyzed using Statistical Package for the Social Sciences ( SPSS ) 16 . 0 .", "The P values were assessed using a two-tailed unpaired Student’s t test with P values considered significant as follows: *p<0 . 05 and **p<0 . 01 .", "For seahorse assay in human fibroblasts , Welch’s unpaired t test was used ." ] ]

[ "Missense mutations of valosin-containing protein ( VCP ) cause an autosomal dominant disease known as inclusion body myopathy , Paget disease with frontotemporal dementia ( IBMPFD ) and other neurodegenerative disorders .", "The pathological mechanism of IBMPFD is not clear and there is no treatment .", "We show that endogenous VCP negatively regulates Mitofusin , which is required for outer mitochondrial membrane fusion .", "Because 90% of IBMPFD patients have myopathy , we generated an in vivo IBMPFD model in adult Drosophila muscle , which recapitulates disease pathologies .", "We show that common VCP disease mutants act as hyperactive alleles with respect to regulation of Mitofusin .", "Importantly , VCP inhibitors suppress mitochondrial defects , muscle tissue damage and cell death associated with IBMPFD models in Drosophila .", "These inhibitors also suppress mitochondrial fusion and respiratory defects in IBMPFD patient fibroblasts .", "These results suggest that VCP disease mutants cause IBMPFD through a gain-of-function mechanism , and that VCP inhibitors have therapeutic value ." ]

[ "A disease called “inclusion body myopathy , Paget disease and frontotemporal dementia ( IBMPFD ) ” is an inherited disorder that can affect the muscles , brain and bones .", "People affected by the disease find that their muscles become progressively weaker , and may go on to develop a bone disorder and a form of dementia .", "The disease is caused by mutations in a gene that codes for Valosin-Containing Protein ( VCP ) – a protein that carries out many different roles in cells .", "Mutated forms of VCP predominantly affect tissues and organs that need a lot of energy , such as the muscles and the brain .", "Within cells , structures called mitochondria generate energy .", "A number of studies suggest that the mitochondria in cells taken from individuals with IBMPFD do not generate energy properly .", "However , it is not known how mutant VCP disrupts mitochondria or how this leads to disease .", "Fruit flies and humans have similar versions of the gene that produces VCP .", "Studying flies that have mutations that affect the gene can therefore help researchers to understand how these mutations might affect humans .", "Zhang et al . have now engineered fruit flies whose muscle cells made a mutant form of VCP .", "These flies showed many of the symptoms of IBMPFD ( such as the death of muscle cells ) .", "In addition , the mitochondria in the muscle cells were smaller and more fragmented than normal .", "This led Zhang et al . to look at a protein called Mitofusin that controls how mitochondria fuse .", "VCP normally degrades Mitofusin; the mutant form of VCP caused Mitofusin to degrade excessively .", "This resulted in mitochondria experiencing reduced levels of fusion , leading to cell malfunction and death .", "In further experiments , Zhang et al . treated the disease-modeled flies and cells from human patients with IBMPFD with inhibitor drugs that prevent the activity of VCP .", "This treatment reversed the defects that affected the mitochondria and prevented the death of muscle cells .", "This opens up the possibility of using VCP inhibitors – which are already being investigated in clinical trials as a treatment for cancer – as drugs to treat IBMPFD ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "structural biology and molecular biophysics", "microbiology and infectious disease" ]

[ [ "In Homo sapiens the skin of the underarm ( axilla ) provides a unique niche for bacteria .", "Through the secretions of various glands , that open onto the skin or into hair follicles , this environment is nutrient-rich and hosts a unique microbial community ( Taylor et al . , 2003 ) .", "Esterified fatty acids and other lipids are secreted by the sebaceous gland , while the eccrine glands produce a dilute salt solution with lactate and other organic solutes being secreted ( Bovell , 2015 ) .", "A third exocrine gland , the apocrine , which is more specialized and found only in the axilla , areolae , genitalia and external auditory meatus ( ear canal ) ( Collins , 1989 ) , starts secreting an odourless lipid-rich viscous secretion with the onset of puberty ( Figure 1A ) .", "The lipid rich secretion is not involved in thermoregulation like the eccrine gland and in mammals has a likely role in scent generation ( Collins , 1989 ) .", "In humans , the link between the axillary apocrine gland , underarm bacteria and body odour ( BO ) , was first recognised over 50 years ago ( Shelley et al . , 1953 ) and early culture-based studies on this relationship found the axillary microbiota to be dominated by Staphylococcus , Corynebacterium and Propionibacterium species , with the Corynebacteria in particular correlating with malodour intensity ( Taylor et al . , 2003; Leyden et al . , 1981 ) .", "More recent culture-independent meta-taxonomic studies have confirmed that the axilla is dominated by these genera , although the presence of additional taxa not previously indicated by culture-based methods is also evident , notably Gram-positive anaerobic cocci ( GPAC ) belonging to the Anaerococcus and Peptoniphilus genera ( Costello et al . , 2009; Egert et al . , 2011; Troccaz et al . , 2015 ) .", "Various molecules produced in the axilla have been linked to malodour , including 16-androstene steroids ( Gower et al . , 1994 ) and short-chain ( C2-C5 ) volatile fatty acids ( VFAs ) produced by axillary bacteria via several metabolic routes ( James et al . , 2004 ) .", "However , 16-androstenes are now believed to be only minor contributors to axillary malodour ( James et al . , 2013 ) , and it is accepted that the VFAs most heavily involved in BO are a series of structurally-unusual medium-chain ( C6-C10 ) acids originating from N-acylglutamine precursors present in apocrine sweat ( Natsch et al . , 2003; Natsch et al . , 2006 ) .", "A third class of molecule , the thioalcohols ( also called sulfanylalkanols ) , have recently been identified as important components of human body odour and a number of these have been detected within the volatiles released from the underarm ( Troccaz et al . , 2004; Natsch et al . , 2004; Hasegawa et al . , 2004 ) .", "They are particularly pungent and can be detected in pg . l−1 quantities , an order of magnitude lower than many other volatile chemicals emanating from the skin ( Natsch et al . , 2004 ) .", "The most abundant and pungent of these thioalcohols is 3-methyl-3-sulfanylhexan-1-ol ( 3M3SH ) ( Figure 1B ) , which is a primary causal molecule of axillary malodour and subsequent BO ( Troccaz et al . , 2004; Hasegawa et al . , 2004 ) .", "Thioalcohols are known to be released as a peptide-conjugated precursor from the apocrine gland , which secretes directly into the hair follicle ( Figure 1A ) .", "The biosynthetic origin of the thioalcohol precursors is currently poorly understood , but they are thought to be secreted into the axilla via the ABC transporter ABCC11 as odourless glutathione conjugates ( Baumann et al . , 2014a ) .", "The glutathione conjugated form ( SG-3M3SH ) has never been detected in axillary sweat; however , the L-cysteinylglycine dipeptide conjugated form , S-[1- ( 2-hydroxyethyl ) −1-methylbutyl]-L-cysteinylglycine ( S-Cys-Gly-3M3SH ) ( Figure 1B ) , is readily detectable ( Starkenmann et al . , 2005 ) and recently a γ-glutamyl transferase ( GGT1 ) has been demonstrated to localise to the apocrine sweat gland and convert SG-3M3SH to S-Cys-Gly-3M3SH ( Baumann et al . , 2014a ) ( Figure 1C ) .", "We and others have demonstrated that S-Cys-Gly-3M3SH can be taken up and metabolised by a highly limited range of staphylococcal species present in the axilla , namely Staphylococcus hominis , Staphylococcus haemolyticus and Staphylocooccus lugdunensis , to liberate 3M3SH , that subsequently exits the cell through an undefined mechanism ( Troccaz et al . , 2004; Starkenmann et al . , 2005; Bawdon et al . , 2015 ) .", "A strong correlation between S . hominis and body odour production has recently been established using culture-independent taxonomic studies of the axillary microbiota ( Troccaz et al . , 2015 ) , confirming the biological significance of this low-abundance species within the axilla in BO production in humans .", "However , the mechanism by which the S-Cys-Gly-3M3SH precursor molecule is taken up into S . hominis and related thioalcohol producers is currently unknown , hampering efforts to identify novel mechanisms to disrupt this process and control odour production in humans .", "Here we identify the biochemical and structural basis for the recognition and specific transport of the malodour precursor peptide , S-Cys-Gly-3M3SH , into S . hominis .", "We first show that S-Cys-Gly-3M3SH is actively taken up by S . hominis through a specific secondary active transporter , STAH0001_1446 ( SH1446 ) , a member of the proton coupled oligopeptide transporter ( POT ) family ( Hagting et al . , 1994; Paulsen and Skurray , 1994 ) .", "Through a combination of structural , biochemical and cell based assays we reveal how this peptide transporter is able to recognise a thioalcohol-conjugated peptide and further , show that transport is coupled to the inwardly directed proton electrochemical gradient .", "Our results suggest that SH1446 is a clear target for inhibitor design to prevent malodour production , thus providing further molecular insights into the role of the skin microbiota in human scent generation ." ], [ "To elucidate how thiol-conjugated dipeptides are transported by bacteria , we first examined the model organism Escherichia coli , which is able to recognise and metabolise the dipeptide version of the non-physiological malodour precursor S-benzyl-L-cysteine , namely , S-benzyl-L-cysteinylglycine ( James et al . , 2013 ) .", "Resting cells of E . coli were indeed able to remove S-benzyl-L-cysteinylglycine from solution and break it down to release the thiol active product , benzyl mercaptan ( Figure 2—figure supplement 1A , B ) .", "Following transport into the cell , the dipeptide must be cleaved to release the benzyl mercaptan , most likely through a cysteine-S-conjugate β-lyase-type activity , for which a number have been described in E . coli , including MetC and MalY ( Dwivedi et al . , 1982; Zdych et al . , 1995; Awano et al . , 2003 ) .", "Interestingly , TnaA which is a tryptophanase known to have L-cysteine desulfhydrase activity ( Awano et al . , 2005 ) is also capable of releasing benzyl mercaptan .", "Individual disruption of metC and malY did not decrease the amount of benzyl mercaptan produced by E . coli , while disruption of tnaA led to an almost complete loss of biotransformation ( Figure 2—figure supplement 1C ) .", "This suggests that E . coli takes up the S-benzyl-L-cysteinylglycine , whereupon a host dipeptidase cleaves the glycine residue prior to cleavage of the S-benzyl-cysteine by TnaA to release the benzyl mercaptan .", "To discover the genetic basis of the transport and metabolism of these thiol-conjugated dipeptides , we exploited the knock out ( KO ) library of single gene disruptions in E . coli K-12 ( Baba et al . , 2006 ) .", "We hypothesised that the dipeptide moiety might direct the molecule through a peptide transporter .", "In E . coli the main peptide systems are relatively well characterised , the two main transport systems belong to the ATP binding cassette ( ABC ) family and the proton dependent oligopeptide transporter ( POT ) family ( Figure 2B ) .", "The E . coli Opp and Dpp ABC transporters have been extensively studied in E . coli and the closely related bacterium Salmonella typhimurium ( Smith et al . , 1999; Klepsch et al . , 2011; Abouhamad et al . , 1991; Goodell and Higgins , 1987; Tame et al . , 1995 ) and an E . coli BW25113 strain with non-functional Opp and Dpp system ( ΔDB1 ) , was used to assess the role of these transporters in the production of benzyl mercaptan .", "Surprisingly , benzyl mercaptan production was similar to the wild-type strain , suggesting that neither ABC transporter was important for Cys-Gly-conjugate transport ( Figure 2A ) .", "Having ruled out transport via the ABC peptide transporters , we then broadened our search to include additional peptide transporters that have been described in E . coli .", "These were the four members of the proton dependent oligopeptide transporter , or POT family , encoded by dtpA-D , that have poorly defined physiological roles during growth on peptides ( Harder et al . , 2008; Prabhala et al . , 2014; Casagrande et al . , 2009 ) , the CstA protein , orthologues of which have been demonstrated to be peptide transporters ( Garai et al . , 2016 ) and the Sap system , which is implicated in uptake of cationic antimicrobial peptides ( Groisman et al . , 1992 ) .", "Most of these strains had similar levels of benzyl mercaptan production to the wild-type , with the exception of dtpB which had an overall 50% drop in thiol production ( Figure 2A ) .", "These data suggest that transporters belonging to the POT family are most likely responsible for Cys-Gly-conjugate transport , revealing a new biological role for bacterial POT transporters in the uptake of Cys-Gly- ( S ) conjugates .", "Having established that E . coli has the ability to transport and cleave a Cys-Gly- ( S ) conjugated substrate , we reasoned that overexpression of a POT transporter might confer the ability to recognise the physiological malodour precursor S-Cys-Gly-3M3SH .", "The four dtp genes were cloned into a pBAD vector commonly used for overexpression of membrane transporters ( Mulligan et al . , 2009; Mulligan et al . , 2012 ) and transformed into the wild-type E . coli background .", "The ability of the resulting strains to convert S-Cys-Gly-3M3SH to 3M3SH was subsequently measured ( Bawdon et al . , 2015 ) .", "Consistent with the reduction in benzyl mercaptan production reported in Figure 2A , the dtpB gene alone was able to confer a malodour production ( BO+ ) phenotype to E . coli , which was not seen with any of the other dtp genes ( Figure 2C ) .", "The same pattern was observed with S-benzyl-Cys-Gly as a substrate ( Figure 2—figure supplement 2B ) , suggesting that DtpB has a unique ability to transport these unusual conjugated peptides .", "The molecular basis for S-Cys-Gly-3M3H transport into malodour producing staphylococci is unknown , and the genetic intractability of these poorly studied coagulase negative Staphylococcal species makes it difficult to study using genetic approaches .", "We therefore used an alternative approach exploiting our discovery that expression of a transporter in trans could produce a BO+ phenotype to E . coli .", "Given our discovery that E . coli POT family peptide transporters can transport S-Cys-Gly-3M3SH , we examined the genome of S . hominis SK1119 , as S . hominis are the most abundant malodour producing Staphylococcal species in the underarm microbiota .", "Unlike E . coli , S . hominis contains only a single POT family member DtpT ( SH1446 ) , orthologues of which were seen in all sequenced staphylococci ( Figure 2—figure supplement 3A ) .", "The POT peptide systems remain largely uncharacterised in staphyoloccci , although in the well-studied pathogen S . aureus , the single POT transporter encoded by dtpT is the sole route for dipeptide uptake in this species ( Hiron et al . , 2007 ) .", "We also identified a second system , encoded by STAHO0001_0415 ( SH0415 ) , which is annotated as an MFS transporter and only present in S . hominis and S . haemolyticus , two known thioalcohol producers , and which is genetically linked to genes involved in peptide and amino acid metabolism ( Figure 2—figure supplement 3B ) .", "Both transporters were cloned into pBADcLIC2005 and transformed into E . coli for functional analysis .", "When levels of 3M3SH production were measured in these strains , uptake was only enhanced by the presence of SH1446 , with SH0415 having little effect on 3M3SH activity ( Figure 2D ) .", "In contrast , with S-benzyl-cysteinlyglycine as a substrate , there was no increase in production of benzyl mercaptan with SH1446 but a significant increase was observed with SH0415 ( Figure 2—figure supplement 2B ) .", "Together these data strongly support the conclusion that the S-Cys-Gly-3M3SH transporter in S . hominis is the POT transporter STAH0001_1446 ( SH1446 ) .", "To further demonstrate the importance of SH1446 in the physiological transport of S-Cys-Gly-3M3SH we used a biochemical approach to examine whether peptides could inhibit 3M3SH production by competing with S-Cys-Gly-3M3SH uptake .", "Firstly , we used our E . coli system where 3M3SH production is seen when SH1446 is expressed in trans and we observed clear inhibition of 3M3SH production with both di- and tri-Ala peptides ( Figure 2—figure supplement", "4 ) but not with L-Ala alone nor tetra-Ala .", "The purified TnaA enzyme that cleaves Cys-3M3SH was not inhibited by any of these peptides ( Figure 2—figure supplement", "5 ) , supporting the hypothesis that the whole cell inhibition is due to inhibition of peptide transport .", "Similarly , we measured the ability of the same peptides to inhibit 3M3SH production directly in S . hominis ( Figure 2—figure supplement 4 ) and observed a comparable profile .", "It should be noted that a significant affect was observed for 25 mM tetra-Ala in S . hominis wild-type , however this affect is due to the acidic pH that was required to dissolve the peptide which appears to inhibit metabolism in S . hominis but not E . coli .", "POT transporters belong to the Major Facilitator Superfamily ( MFS ) and use an inwardly directed proton electrochemical gradient to drive the concentrative uptake of di- and tri-peptides across the cell membrane for metabolic assimilation ( Daniel and Kottra , 2004 ) .", "To establish SH1446 as the protein responsible for S-Cys-Gly-3M3SH recognition and transport , the gene was cloned and overexpressed in E . coli for functional and structural studies .", "The purified protein , hereafter referred to as PepTSh ( Peptide Transporter Staphylococcus hominis ) was purified and reconstituted into liposomes .", "Transport was studied using a previously employed pyranine based reporter assay , which measures transport via changes to the internal pH of the liposomes ( Parker et al . , 2014 ) ( Figure 3A ) .", "Acidification of the lumen of the liposome was observed in the presence of a membrane potential ( ΔΨ , negative inside ) , and addition of external di- or tri-peptide substrate , demonstrating that PepTSh is a proton coupled peptide transporter .", "Importantly we also observed acidification in the presence of the S-Cys-Gly-3M3SH precursor peptide , confirming our earlier identification of PepTSh as the transporter for this molecule in S . hominis .", "To understand the recognition of S-Cys-Gly-3M3SH compared to natural peptides , we determined IC50 values using competition with 3H-di-alanine .", "The IC50 value for tri-alanine , di-alanine and Cys-Gly were 23 . 7 ± 3 . 4 , 72 . 2 ± 9 . 7 and 115 ± 22 . 2 μM , respectively ( Figure 3B ) , indicating that PepTSh is able to recognise tri-Ala more favourably than di-Ala .", "However , for S-Cys-Gly-3M3SH the IC50 value was 362 ± 21 . 8 μM , three-fold higher than the Cys-Gly peptide .", "Our data therefore show that while PepTSh can transport the modified peptide , the addition of the thioalcohol group on the cysteine side chain has substantially impaired the recognition and transport versus natural peptides .", "To gain further insight into the mechanism of substrate binding we successfully obtained a high-resolution co-crystal structure of PepTSh at 2 . 5 Å resolution , bound to S-Cys-Gly-3M3SH using the in meso crystallisation method ( Figure 4A , Table 1 and Figure 4—figure supplement 1 ) .", "PepTSh adopts the canonical MFS fold with helices H1-H6 forming the N-terminal bundle and H7-H12 the C-terminal bundle .", "PepTSh was crystallised in an inward open state , observed previously for other members of the POT family ( Newstead , 2015 ) , wherein the extracellular gate , consisting of helices H1-2 and H7-8 from the N- and C-terminal bundles respectively , is closed and the intracellular gate , consisting of helices H4-5 and H10-11 is open .", "Similar to other bacterial POT family members , PepTSh contains two additional helices inserted between the N- and C-terminal bundles , termed HA and HB ( Figure 4B and Figure 4—figure supplement 2 ) .", "Unlike previous POT structures however , PepTSh also contains a well-structured β-hairpin motif between helices H7 and H8 on the extracellular side of the membrane , extending out in line with the lipid head groups .", "Interestingly we observed a number of monoolein lipid molecules that cluster around both the β- hairpin and the cavity created between helices HA and HB with the transporter domain .", "The role of helices HA and HB in prokaryotic POT family transporters still remains unclear .", "However , the observation of lipid molecules bound between these helices and the transport domain suggests a possible role in stabilising these transporters in the membrane , which may be an adaption specific to prokaryotic members of the POT family ( Newstead et al . , 2011 ) .", "Following extensive screening and co-crystallisation we observed clear mFo-DFc difference electron density for the S-Cys-Gly-3M3SH ligand in the peptide binding site ( Figure 4C and Figure 4—figure supplement 3 ) .", "The Cys-Gly part of the precursor is positioned such that the carboxy termini of the peptide faces towards the intracellular side of the binding site .", "The terminal carboxyl group makes two hydrogen bond interactions , one with Tyr41 on H1 and a second interaction to a water molecule ( W107 ) , which links the carboxyl group with Asn347 on H8 .", "The carbonyl group of the CG peptide also makes a hydrogen bond to this water molecule , creating a well-coordinated interaction network between the peptide and Asn347 ( Figure 4D ) .", "The terminal amino group makes two further polar interactions , with a water molecule that sits next to Tyr41 and also to the carbonyl group of Gln344 , which sits further up on H8 .", "In contrast , the thioalcohol moiety points towards the extracellular gate , with the sulphur atom sitting close to Gln310 on H7 , whereas the alcohol group extends towards helix H8 making interactions with backbone carbonyl and amide groups of Phe343 and Asn347 , respectively .", "The acyl group extends into a large pocket nestled between helices H7 and H10 ( Figure 4E ) .", "This long pocket ( ~10 Å ) extends from the central peptide binding site into the C-terminal bundle of the transporter and is both hydrophobic and polar in character .", "Previously , we showed that a related POT family transporter , PepTSt , is able to bind peptides in different orientations in the binding site , with a di-peptide adopting a horizontal position and a tri-peptide a vertical one ( Lyons et al . , 2014 ) and suggested this flexibility may be responsible for the accomodation of larger peptide ligands ( Newstead , 2017 ) .", "Here we see that the S-Cys-Gly-3M3SH peptide adopts a more vertical orientation , similar to the tri-alanine position in PepTSt .", "However , there are significant differences in the interactions .", "A structural overlay of the S-Cys-Gly-3M3SH peptide shows that it sits much further up in the binding site compared to the natural peptides captured in PepTSt ( Figure 4—figure supplement 4A & B ) , most likely as a result of the requirement to accommodate the thioalcohol group in the extended pocket .", "This has a knock-on effect with respect to the position of the amino and carboxy termini .", "In PepTSt , the amino terminal group of the di-peptide directly interacts with helix H8 and H10 , whereas the tri-peptide makes a much weaker interaction through a carbonyl group .", "In contrast , in PepTSh this interaction is achieved through a water mediated hydrogen bond ( Figure 4D ) .", "The role of water in mediating interactions between peptides and their binding sites has been previously observed in the ABC transporter binding domains ( Tame et al . , 1995 ) , albeit for side chain independent binding .", "A similar mechanism may be employed by PepTSh to facilitate transport of modified peptides by replacing direct interactions between the peptide and the transporter when these are sterically restricted , as we observe with S-Cys-Gly-3M3SH .", "The interaction of the amino termini of peptides with both the mammalian and bacterial POT family transporters has been identified as facilitating high affinity binding ( Weitz et al . , 2007; Meredith et al . , 2000 ) .", "The reduced IC50 value for the modified peptide in PepTSh ( Figure 3B ) , may therefore be a combination of steric restraints resulting in the accommodation of the extended side chain group , and the subsequent knock on effects on the position of the interactions between the amino and carboxy groups with conserved side chains in the binding site .", "S-Cys-Gly-3M3SH is the first human derived peptide with a modified side chain to be captured in a POT family transporter , and contains biochemical features similar to physiological peptides ( Cys-Gly ) , but also incorporates the non-peptide thioalcohol group ( 3M3SH ) .", "We therefore wished to explore whether S-Cys-Gly-3M3SH is recognised using a similar mechanism to physiological peptides .", "Several mutations in the binding site that have previously been shown to play an important role in peptide recognition within the bacterial POT family were therefore generated ( Newstead , 2017 ) .", "Tyrosine 41 forms part of a conserved E33xxERFxYY41 motif on TM1 ( Solcan et al . , 2012 ) , which plays a role in proton coupling and influences peptide uptake in several POT family members ( Doki et al . , 2013; Ernst et al . , 2009; Guettou et al . , 2014; Lyons et al . , 2014 ) .", "In PepTSh Tyr41 hydrogen bonds with the carboxyl terminus of the terminal glycine amino acid in S-Cys-Gly-3M3SH ( Figure 4C , D ) .", "Mutating Tyr41 to phenylalanine or alanine severely reduced transport of di-alanine compared to WT protein ( Figure 5A ) .", "A similar negative effect on transport was observed in the related POT family transporter PepTSt , from Streptococcus thermophilus ( Solcan et al . , 2012 ) , suggesting a common role for the conserved tyrosine in peptide recognition within the POT family .", "We then used the same variants in our whole cell assay for 3M3SH production .", "Interestingly , while both Tyr41Phe and Tyr41Ala variants had reduced activity , at about 50% of the wild-type protein ( Figure 5B ) , the phenotype was not as severe as that observed in the liposome based assay .", "We interpret this result to indicate that recognition of S-Cys-Gly-3M3SH is less dependent on this residue than di-Ala .", "We next wanted to test the role of other residues we observed sitting close to the S-Cys-Gly-3M3SH ligand .", "In particular , we were interested in the contribution made by the Asn347 and Glu418 , which coordinate a water molecule ( W107 ) linking both the carboxyl and carbonyl groups of the ligand to the binding site ( Figure 4B ) .", "The equivalent residues in homologous POT family transporters have been implicated in both peptide recognition ( Lyons et al . , 2014 ) and gating ( Solcan et al . , 2012 ) , and in the case of Glu418 may facilitate the coupling between peptide recognition , proton movement and transport ( Parker et al . , 2017 ) .", "Although a Glu418Ala variant had no activity in our assays , consistent with its proposed role in coordinating the intracellular gate ( Fowler et al . , 2015 ) , we observed reduced uptake of di-Ala in the Asn347Ala variant ( Figure 5A ) .", "Interestingly , this reduction was far more severe in the in vivo assay ( Figure 5B ) , indicating that this interaction is more important for transport of the S-Cys-Gly-3M3SH peptide than di-Ala .", "This may reflect the requirement of Asn347 to coordinate the water molecule linking the peptide to the intracellular gate side chain Glu418 , which in the case of the di-peptide may interact directly as in PepTSt , lessening the requirement for the Asn347 interaction .", "The co-crystal structure also identified the acyl chain portion of the thioalcohol group was accommodated within an extended pocket , formed near the extracellular side of the binding site ( Figure 4E ) .", "To test the importance of this pocket on S-Cys-Gly-3M3SH transport , we made a Tyr411Trp variant , which would block the cavity .", "Uptake was noticeably reduced , but still measurable at ~19% compared to WT ( Figure 5A ) .", "The same mutation resulted in a similar large decrease in the biotransformation of S-Cys-Gly-3M3SH , while a Tyr411Ala mutation had wild-type biotransformation rates ( Figure 5B ) .", "It is clear that the hydrophobic pocket in PepTSh plays an important role in facilitating the recognition of the thioalcohol group in the binding pocket and , as discussed below , is a potential site for inhibitor design .", "Taken together the current data lend further support to our model of an adaptable binding site within the POT family that can accommodate chemically diverse peptides and their derivatives through the use of both side chain and water mediated interactions with conserved residues and different specificity pockets to accommodate the different chemical side chains ." ], [ "The production of malodour by humans is an evolutionary ancient olfactory process that likely was involved in mate selection and evolutionary processes that relied on chemical signalling ( Stoddart , 1990 ) .", "Evidence for the microbial origins of chemical signalling molecules is also seen in other animals such as foxes , deer , cats and beetles ( Wyatt , 2003 ) , but the details of this process in humans is less well understood .", "The thioalcohol 3M3SH , a major component of body odour , is produced in the human axilla as a glutathione-conjugated precursor and is processed to a unique dipeptide structure ( Figure 1 ) .", "The discovery that axilla-isolated strains of S . hominis , S . lugdunensis and S . heamolyticus are able to produce 3M3SH prompted the search for the molecular basis for this process ( Bawdon et al . , 2015 ) .", "While S-Cys-Gly-3M3SH is a highly modified and uniquely human dipeptide , we reasoned that this moiety might dictate the uptake mechanism into malodour producing bacteria and here we identify a peptide transporter , PepTSh ( SH1446 ) , from the POT family of secondary active transporters as being responsible for this process .", "Each of these strains have a POT family transporter that is closely related to PepTSh ( Figure 4—figure supplement 2 ) , and we would predict they will also be able to uptake S-Cys-Gly-3M3SH .", "The genetic intractability of S . hominis dictated that we use a biochemical approach to demonstrate that SH1446 is likely the sole route of uptake of S-Cys-Gly-3M3SH .", "First , this staphylococci has a similar repertoire of peptide transporters in S . aureus , in that it has a single POT transporter and one Opp ABC transporter , and previous work in S . aureus has demonstrated that the POT transporter , DtpT , is essential for growth on dipeptides and some tripeptides , while the ABC transporter ( Opp3 ) has a function in transporting longer oligopeptides ( Hiron et al . , 2007 ) .", "The use of L-Ala containing peptides as inhibitors in this study demonstrates an inhibitory effect on 3M3SH production that is consistent with the properties of a POT family transporter .", "Several attempts were made to genetically disrupt SH1446 in S . hominis , using tools developed for S . aureus and S . epidermidis , but these were unsuccessful , most likely due to the presence of multiple species-specific restriction modification ( RM ) systems which render this organism , as with many other coagulase negative ( CoNS ) species , genetically intractable .", "The specific role for POT transporters in the transport of these unusually modified dipeptides is supported from the inability of the E . coli ABC transporters for oligopeptides and dipeptides , Opp and Dpp , to recognise and transport S-Cys-Gly-3M3SH .", "Given that S-Cys-Gly-3M3SH is effectively a dipeptide with a highly extended R-group on the first amino acid , it is noteworthy that only the promiscuous nature of the POT binding site is able to recognise this ligand .", "For OppA and DppA , the substrate binding proteins that initially recognise peptides transported by the ABC systems , the binding pocket is well defined and makes general main chain-main chain interactions to ensure that the protein can bind peptides in a sequence independent manner ( Tame et al . , 1995; Sleigh et al . , 1997; Sleigh et al . , 1999 ) .", "However , the pocket has evolved to accommodate regular amino acid side chains and the unusually large structure of the thioalcohol attachment would likely be sterically incompatible with recognition by either OppA or DppA .", "The data presented here further support the idea that POT transporters have an unusually promiscuous binding site that may play an evolutionary advantageous role in accepting modified peptides as substrates .", "Recently , the human POT family transporters , PepT1 and PepT2 , have gained additional attention due to their role in the absorption and retention of several important classes of drug molecule ( Smith et al . , 2013 ) , including peptide modified pro-drugs .", "Similar to S-Cys-Gly-3M3SH , these pro-drugs are drug molecules conjugated to a peptide or amino acid ( Brandsch , 2013 ) .", "Pro-drugs are able to compete with natural peptides for transport via PepT1 and PepT2 , and are therefore actively transported into the body ( Ganapathy et al . , 1998; Gupta et al . , 2013; Terada and Inui , 2012 ) .", "The promiscuous nature of the POT family binding site has been shown to derive , in part , from their ability to recognise peptides in different conformations and through the availability of different specificity pockets , which are able to accommodate chemically different classes of peptides ( Newstead , 2017 ) .", "Our results here provide the first evidence that specificity pockets can also accommodate exotic chemical extensions .", "The observation that water plays a role in facilitating the interaction between the S-Cys-Gly-3M3SH peptide and PepTSh may also indicate a broader role for water in facilitating the recognition of xenobiotic peptides .", "In particular , the observation that water may bridge interactions between the binding site and either the amino or carboxy group of the ligand would greatly increase the number of conformations a peptide could adopt in the transporter and still trigger proton coupled transport .", "Finally , we can ask the questions , why do these bacteria transport S-Cys-Gly-3M3SH and can this process be inhibited to provide effective malodour control ?", "The presumed biochemical pathway for breakdown of the S-Cys-Gly-3M3SH is outlined in Figure 6 .", "The firststep is the likely removal of the glycine by a dipeptidase and then the lyase reaction that cleaves the carbon-sulphur bond to release pyruvate and ammonia in addition to 3M3SH .", "The route of export of 3M3SH is unknown , but currently assumed to be diffusion across the bacterial inner membrane .", "The bacterium will gain a nutritional benefit from the catabolism of S-Cys-Gly-3M3SH via the release of both nitrogen and carbon as glycine , pyruvate and ammonia ( Figure 6 ) , which may have been the evolutionary trigger for the adaptation to catabolise this chemical .", "Asian individuals with single nucleotide polymorphisms ( SNPs ) in ABCC11 , the transporters involved in the secretion of S-Cys-Gly-3M3SH ( Figure 1 ) have been studied ( Nakano et al . , 2009; Baumann et al . , 2014b ) .", "Recently , a microbiological investigation of individuals with wild-type alleles of ABCC11 compared to heterozygous and double mutant genotypes revealed that the proportion of Staphylococcus sp .", "is higher in the underarms of humans that produce the precursor ( Harker et al . , 2014 ) .", "While these studies are limited in that they do not provide species level identification , they are at least consistent with the idea that the production of the precursor positively correlates with the presence of Staphylococcus sp .", "in the underarm .", "The discovery of the transporter and molecular understanding of the mode of S-Cys-Gly-3M3SH binding now suggests a specific route to develop inhibitors of malodour production by directly inhibiting the transporter SH1446 ( Grice , 2014 ) .", "Importantly , such intervention would not impact on the overall axillar microbiome , but instead specifically target the reduction in body odour production ." ], [ "Escherichia coli K-12 was used as the model organism throughout this study .", "Escherichia coli K-12 single gene deletions were obtained from the in-house E . coli Keio collection at University of York .", "Bacterial strains and plasmids used in this study are listed in Supplementary file 1 and", "2 . All strains were cultured in LB ( 1% tryptone , 0 . 5% yeast extract , 1% NaCl ) unless otherwise stated , Antibiotics were used at the following concentrations Ampicillin 100 μg/mL and Kanamycin 50 μg/mL; L-arabinose concentrations are indicated .", "PCR , plasmid extractions , and transformations were carried out as per standard protocols .", "For routine cloning , genomic DNA was extracted from BW25113 and high fidelity KOD polymerase was used ( Novagen ) .", "Correct clones were confirmed by DNA sequencing .", "Specific details of plasmid constructions are detailed below .", "Multiple gene knockouts were created in single keio deletion strains .", "To create the double KO strain DB1 ( ΔoppC ΔdppC ) , the KanR cassette was removed from a target gene using pCP20 ( encoding Flp recombinase ) at 30°C , loss of antibiotic cassette was confirmed by PCR and sequencing .", "The second gene to be disrupted was PCR amplified with 100 bp homology including the FRT flanked KanR cassette from the single gene deletion strain in the keio collection .", "Targeted gene disruptions were created using λ Red recombination as previously described ( Datsenko and Wanner , 2000 ) .", "Overexpression of the E . coli POT transporters were cloned into pBADcLIC2005 vector under the control of arabinose .", "Full-length genes were amplified from E . coli BW25113 genomic DNA using high fidelity KOD polymerase ( Merck ) using the corresponding primers listed in Supplementary file", "3 . In silico analysis of the available Staphylococcus hominis SK1119 genome identified STAH0001_1446 ( SH1446 ) as a POT family member which orthologues present in all sequenced staphylococci and a MFS transporter STAH0001_0415 ( SH0415 ) present in only S . hominis and S . haemolyticus .", "SH0415 and SH1446 genes were amplified from S . hominis genomic DNA using high fidelity KOD polymerase ( Merck ) using primers listed in Supplementary file", "3 . All PCR products were purified using Wizard SV Gel and PCR Clean-Up System ( Promega ) , cloned into pBADcLIC2005 vector using standard ligation-independent cloning ( LIC ) techniques .", "All fragments were confirmed by PCR and DNA sequencing ( GATC Biotech ) .", "The wildtype STAHO0001_1446 ( SH1446 ) cloned onto the expression plasmid pBADcLIC2005 was used to individually generate the Y411W , Y411A , Y41A , Y41F , N167A and N347A mutants .", "To introduce the mutation , high fidelity inverse PCR was performed using divergent primers with one per pair being mutagenic ( Supplementary file 3 ) .", "The resultant products were then circularised by performing a blunt end ligation and transformed into E . coli BW25113 .", "The mutant sequences were verified by DNA sequencing ( GATC Biotech ) .", "S-Benzyl-L-cysteinylglycine and ( S ) - ( 1- ( 2-hydroxyethyl ) −1-methylbutyl ) -L-cysteinylglycine ( S-Cys-Gly-3M3SH ) ( Peakdale Molecular ) were prepared at a stock concentration of 10 mM in M9 salts ( 6 g Na2HPO4 g/L , 3 g KH2PO4 g/L , 0 . 5 g NaCl g/L ) and stored at 4°C .", "E . coli were grown overnight in LB , cells were harvested by centrifugation at 3 , 500 rpm 10 min and resuspended in sterile M9 salts .", "Where appropriate , E . coli strains harbouring inducible genes on the pBADcLIC2005 plasmid were pre-induced during overnight growth with 0 . 0001% L-arabinose .", "To a sterile Eppendorf the following was added; 2 mM substrate ( S-Benzyl-L-cysteinylglycine or S-Cys-Gly-3M3SH ) , cells to an OD650nm of 5 and M9 salts up to 200 μL .", "Cells only ( no added substrate ) and substrate only ( no added cells ) reactions were also included in every individual experiment .", "The reactions were incubated at 37°C for up to 48 hr with samples taken at appropriate time points .", "Liberated thiols were quantified using either MTS/PMS or DTNB labelling as detailed below .", "Liberated benzyl mercaptan was labelled as follows , 500 μl from each reaction was taken at the appropriate time point and centrifuged at 13 000 r . p . m . for 2 min .", "To chemically label benzyl-mercaptan , 50 µl of labelling reagent , consisting of 4 . 09 mM 3- ( 4 , 5-dimethylthiazol-2-yl ) −5- ( 3-carboxymethoxyphenyl ) −2- ( 4-sulfophenyl ) −2H-tetrazolium ( MTS ) ( Promega ) and 0 . 2 mM phenazine methosulfate ( PMS ) ( Sigma ) ( Goodwin et al . , 1995 ) , dissolved in dH2O , was aliquoted into either a 96 well microplate or 0 . 5 ml Eppendorf tubes .", "For reactions using a 96 well microplate , 200 µl reaction supernatant was added to the pre- aliquoted 50 µl MTS/PMS .", "The plate was sealed with a semi-permeable film lid , incubated at room temperature for 50 min and the A492nm recorded on a Biotek PowerWave XS microplate spectrophotometer .", "The data was captured by KC junior and exported to Microsoft Excel .", "For reactions in Eppendorf tubes , 20 µl - 50 µl reaction supernatant was added to 50 µl MTS/PMS .", "The reaction was incubated at room temperature for 50 min . 20 µl was subsequently removed and diluted in 980 µl dH2O in a 1 . 5 ml disposable plastic cuvette .", "The A492nm was recorded .", "To quantify free 3M3SH , a 75 µl volume from each reaction ( isolated as described above ) was taken at the appropriate time point and centrifuged at 13 , 000 rpm for 2 min . 950 µl labelling solution , consisting of: 800 µl dH2O , 100 µl UltraPure Tris ( pH 8 . 0 ) and 50 µl DTNB ( Ellman , 1958 ) stock solution ( 50 mM sodium acetate , 2 mM DTNB , dissolved in dH2O ) was added to a 1 . 5 ml disposable cuvette .", "50 µl reaction supernatant was added to the cuvette , mixed by pipetting and incubated at room temperature for 5 min .", "The A412nm was recorded on a Jenway 6305 spectrophotometer .", "( Van Horn , 2003 ) .", "Where appropriate , E . coli strains harbouring inducible genes on the pBADcLIC2005 plasmid were pre-induced during overnight growth with 0 . 0001% L-arabinose .", "In all experiments cells only ( no added substrate ) and substrate only ( no added cells ) were also included .", "The gene encoding S . hominis SH1446 was cloned into a C-terminal octa histidine GFP fusion vector ( pWaldo-GFPe ) ( Drew et al . , 2006 ) and subsequently transformed into Escherichia coli C43 ( DE3 ) cells ( Miroux and Walker , 1996 ) .", "Mutations were introduced using site directed mutagenesis followed by DpnI digestion .", "An overnight starter culture was used to inoculate 4 L of Terrific Broth ( TB ) .", "Cells were left to grow at 37°C and aerated by shaking at 250 rpm .", "Overexpression of the fusion protein was induced when the culture reached an OD600 of ~0 . 6 , by adding isopropyl-β-D-thiogalactopyranoside ( IPTG ) to a final concentration of 0 . 4 mM .", "Following induction , the temperature was reduced to 25°C .", "Cells were harvested 16 hr later , and resuspended in phosphate buffered saline ( PBS ) containing deoxyribonuclease I from bovine pancreas , and stored at −80°C . Unless stated , the following steps were carried out at 4°C .", "Cells were thawed and disrupted at 32 kpsi ( Constant Systems , UK ) .", "Cellular debris was removed by centrifugation at 30 , 000 x g for 30 min , and membranes were isolated by ultracentrifugation at 230 , 000 x g for 120 min .", "Membranes were resuspended in PBS using a dounce homogeniser , flash frozen , and stored at −80°C .", "Thawed membranes were diluted with 1x PBS ( 10 mL per gram of membrane ) , 20 mM imidazole ( pH 8 . 0 ) , 150 mM NaCl , and 1% n-Dodecyl-β-D-maltoside ( DDM ) and left to solubilise for 60 min , stirring .", "Non-solubilised membranes were removed by ultracentrifugation at 230 , 000 g for 60 min .", "Nickel-NTA resin ( ThermoFisher Scientific ) was added to the sample , using a ratio of 1 mL resin per gram of membrane , and left to bind , stirring , for approximately 90 min .", "The resin was then packed into a glass econo-column ( BioRad , USA ) under gravity .", "The resin was washed with 20 column volumes ( CV ) of wash buffer ( 1x PBS , 20 mM imidazole ( pH 8 . 0 ) , 150 mM NaCl and 0 . 05% DDM ) .", "The resin was further washed with 10CV of wash buffer containing 30 mM imidazole .", "The bound GFP-SH1446 fusion protein was eluted with wash buffer containing 250 mM imidazole .", "The eluent was combined with an equivalent concentration of hexa histidine tagged TEV protease and dialysed overnight against 20 mM Tris-HCl ( pH 7 . 5 ) , 150 mM NaCl , and 0 . 03% DDM .", "The cleaved protein was filtered using a 0 . 22 μm filter ( Millipore , USA ) and passed through a Ni-NTA 5 mL HisTrap column ( GE Healthcare ) to remove uncleaved GFP-SH1446 protein and TEV protease .", "The flow through was collected and concentrated to 300 μl using a 50 kDa MWCO concentrator ( Vivapsin 20 , Sartorius AG ) and applied to a size exclusion column ( Superdex 200 10/30 , GE Healthcare ) pre-equilibrated with 20 mM Tris-HCl ( pH 7 . 5 ) , 150 mM NaCl , and 0 . 03% DDM .", "Fractions containing purified SH1446 protein were pooled and concentrated to a final concentration of 15–20 mg . ml−1 .", "For expression and purification of TnaA , overnight cultures were grown in LB at 37°C , 200 rpm .", "1 L of LB was inoculated 1:50 with the starter culture and grown to an OD600nm ~0 . 6–0 . 8 , target protein expression was induced with 0 . 001% arabinose .", "Following induction the culture was further incubated at 37°C for ~8 hr .", "Cells were harvested by centrifugation at 4 , 500 rpm for 15 min and resuspended in 35 mL resuspension buffer ( 50 mM Kpi , 20% Glycerol , 200 mM NaCl and 10 mL Imidazole , pH 7 . 8 ) and stored at −80°C .", "All subsequent steps were carried out at 4°C , for protein purification cells were thawed and lysed using sonication with 3 s pulsed and 7 s pause for 3mins .", "Lysates were clarified by centrifugation 25 , 000 rpm ~30 min .", "The clarified supernatant was loaded onto a 5 mL HisTrap column ( GE Healthcare ) and affinity purified on an AKTA Start ( GE Healthcare ) using a standard protocol as per manufacturer’s instructions .", "Crystals of SH1446 were obtained in-meso phase .", "Briefly , concentrated protein was mixed with 9 . 9 monoacylglycerol ( Monoolein , Sigma-Aldrich ) at a protein:lipid ratio of 2:3 , in a mechanical syringe mixer at 20°C ( Ai and Caffrey , 2000 ) .", "50 nL of mesophase and 800 nL of precipitant solution was dispensed on to a 96-well glass plate using an in meso robot ( Gryphon , Art Robbins Instruments ) .", "SH1446 S-Cys-Gly-3M3SH complex was crystallised in precipitant solution containing 26–27% ( v/v ) PEG 200 , 220 mM ( NH4 ) 2HPO4 , 110 mM sodium citrate ( pH 5 . 0 ) and 5 mM S-Cys-Gly-3M3SH ( Peakdale Molecular ) and left overnight at 4°C , prior to crystallisation .", "Crystals appeared after 2–3 days , and grew to its maximum size after 10 days .", "Crystallisation wells were accessed using a tungsten-carbide glasscutter , and crystals were directly mounted onto 50–100 μm microloops ( MiTeGen ) and flash frozen at 100 K . X-ray diffraction data were collected at ID23-2 at the European Synchrotron Radiation Facility on a Pilatus3 2M detector with an exposure setting of 0 . 05 s with a 0 . 2° oscillation range over 360° Diffraction data was indexed and integrated in XDS ( Kabsch , 2010 ) and merged in Aimless ( Evans , 2011 ) .", "The ligand bound complex was solved by molecular replacement in PHASER ( McCoy et al . , 2007 ) using a modified search model of the bacterial peptide transporter from Streptococcus thermophilus , PepTSt ( Lyons et al . , 2014 ) , PDB: 4D2B .", "Iterative rounds of manual model building in COOT ( Emsley et al . , 2010 ) followed by refinement in autoBUSTER ( Bricogne G . 2017 ) followed by refinement in Phenix ( Adams et al . , 2010 ) , resulted in a final model , which was validated using the Molprobity server ( Chen et al . , 2010 ) .", "Additional difference density was observed in the binding site and matched the stereo chemical properties and size of S-CG3M3SH .", "Ligand restraints were generated using the Grade Web Server ( http://grade . globalphasing . org ) .", "Figures were prepared using PyMOL ( Schrödinger , LLC ) .", "Purified SH1446 was detergent exchanged into n-Decyl-β-D-maltopyranoside ( DM , Anatrace ) using a size exclusion column ( Superdex 200 10/30 , GE Healthcare ) pre-equilibrated with 20 mM Tris-HCl ( pH 7 . 5 ) , 150 mM NaCl , and 0 . 3% DM .", "Lipids ( POPE:POPG in a 3:1 ratio ) were mixed with the eluted protein at a ratio of 60:1 .", "The protein:lipid mixture was rapidly diluted with 50 mM KPO4 ( pH 7 . 0 ) buffer to below the CMC value of DM .", "Proteoliposomes were harvested by ultracentrifugation ( 234 , 000 xg ) for 120 min .", "The pellet was resuspended in 50 mM KPO4 and dialysed overnight against 3L of 50 mM KPO4 ( pH 7 . 0 ) .", "Proteoliposomes were recovered and subjected to three rounds of freeze thawing before storage at −80°C .", "Proteoliposomes were thawed and harvested by ultracentrifugation at 150 , 000 g .", "The resulting pellet was carefully resuspended in INSIDE buffer ( 120 mM KCl , 2 mM MgSO4 , 5 mM HEPES pH 6 . 8 ) and 1 mM pyranine ( trisodium 8-hydroxypyrene-1 , 3 , 6-trisulfonate ) .", "Proteoliposomes underwent eleven freeze-thaw cycles in liquid nitrogen before the sample was extruded through a 0 . 4 μm polycarbonate membrane and harvested by ultracentrifugation as before .", "Excess pyranine dye was removed with a Microspin G-25 column ( GE Healthcare ) pre-equilibrated with INSIDE buffer without pyranine .", "For the assay , proteoliposomes were diluted into OUTSIDE buffer ( 120 mM NaCl , 5 mM HEPES ( pH 6 . 8 ) , 2 mM MgSO4 ) .", "A Cary Eclipse fluorescence spectrophotometer ( Agilent Technologies ) equipped with a cuvette and magnetic flea was used to carry out the transport assay .", "Dual fluorescence excitation was set to 460/415 nm with emission at 510 nm .", "Transport was initiated with 1 μM valinomycin , the resulting data was exported and analysed using Prism ( GraphPad Software ) .", "Transport data was normalised to one to facilitate comparison with data collected from multiple conditions .", "Proteoliposomes were harvested as before , the resulting pellet was resuspended in INSIDE buffer , subjected to three freeze-thaw cycles in liquid nitrogen , extruded through a 0 . 4 μm polycarbonate membrane and harvested by ultracentrifugation once more .", "For the assay , proteoliposomes were diluted in external buffer ( 110 mM NaCl , 10 mM NaPO4 and 2 mM MgSO4 ) supplemented with 50 μM di-alanine peptide containing trace amounts of tritiated 3H-di-alanine ( specific activity 30 Ci/mmol ) and the competing substrate at increasing concentrations as indicated .", "Competition assays were performed at 30°C , samples were taken at specified time points and transport was stopped by addition of 2 mL H2O .", "Proteoliposomes were immediately filtered onto a 0 . 22 μm cellulose filter ( Merck Millipore ) using a vacuum manifold and subsequently washed twice more with 2 mL H2O .", "The amount of peptide transported into the liposomes was calculated based on the specific activity for each peptide as detailed by the manufacturer and counting efficiency for the radioisotope in Ultima Gold ( PerkinElmer ) counted in a Wallac scintillation counter ( 3H , 45% counting efficiency ) .", "Transport assays were performed a minimum of three times to generate an overall mean and S . E . M . Atomic coordinates for the atomic models have been deposited in the Protein Data Bank under accession numbers 6EXS ." ] ]

[ "Mammals produce volatile odours that convey different types of societal information .", "In Homo sapiens , this is now recognised as body odour , a key chemical component of which is the sulphurous thioalcohol , 3-methyl-3-sulfanylhexan-1-ol ( 3M3SH ) .", "Volatile 3M3SH is produced in the underarm as a result of specific microbial activity , which act on the odourless dipeptide-containing malodour precursor molecule , S-Cys-Gly-3M3SH , secreted in the axilla ( underarm ) during colonisation .", "The mechanism by which these bacteria recognise S-Cys-Gly-3M3SH and produce body odour is still poorly understood .", "Here we report the structural and biochemical basis of bacterial transport of S-Cys-Gly-3M3SH by Staphylococcus hominis , which is converted to the sulphurous thioalcohol component 3M3SH in the bacterial cytoplasm , before being released into the environment .", "Knowledge of the molecular basis of precursor transport , essential for body odour formation , provides a novel opportunity to design specific inhibitors of malodour production in humans ." ]

[ "Human body odour contains a number of chemicals , but the most pungent and recognisable are thioalcohols .", "These molecules are created through a series of chemical reactions that start with an odourless precursor , a compound produced in glands located in our armpits .", "Then , a type of bacteria called Staphylococcus hominis takes in these molecules and transforms them into smelly thioalcohols .", "The precise details of how the bacteria do this are not clear .", "Now , Minhas , Bawdon et al . show how S . hominis uses a transport protein in its membrane to bring the odourless precursor inside .", "In the experiments , tools such as X-ray crystallography captured snapshots of this transporter as it was moving the compound into the bacteria .", "This helped to understand how the bacteria recognize these precursors , as well as the exact structure of these molecules and of their transporters .", "The experiments also reveal that bacteria which do not create odour can also ingest the precursors through this same process .", "This suggests that the odour production is a unique process that happens once these molecules are inside S . hominis .", "The findings imply that humans and their body odour-producing bacteria likely evolved together .", "In other mammals , the bacterial production of bodily smells is linked to the release of pheromones , which are chemicals involved in communication and in selecting sexual partners .", "It is not clear whether this is also true for humans .", "Ultimately , learning more about how S . hominis converts precursor molecules into thioalcohols could lead to new ways of nipping body odours in the bud ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "cell biology", "immunology and inflammation" ]

[ [ "The mononuclear phagocytic system ( MPS ) consists of a family of cells—including monocytes , macrophages , and dendritic cells ( DC ) —that are derived from common committed bone marrow progenitors and perform related functions ( Hume , 2006; Hashimoto et al . , 2011 ) .", "The lung is a mucosal surface of the body , exposed constantly to inhaled particles including pathogens , as well as other potential toxins ( Nelson et al . , 2012 ) .", "Lung mononuclear phagocytes have been shown to adapt specifically to the lung environment , and contribute to lung homeostasis , scavenging , and immune surveillance ( Kopf et al . , 2014; The lungs at the frontlines of immunity , 2014 ) .", "Monocytes were originally considered to be circulating precursors of macrophages , participating in renewal of tissue-resident macrophages in steady state and recruited in large numbers in response to inflammatory stimuli ( Auffray et al . , 2009; Geissmann et al . , 2010 ) .", "Fate mapping approaches and parabiosis experiments have been used to argue that tissue-resident macrophages such as alveolar macrophages ( AM ) may be seeded from the yolk sac or foetal liver during embryonic development and can be maintained in the absence of monocyte recruitment ( Guilliams et al . , 2013; Hashimoto et al . , 2013; Yona et al . , 2013 ) .", "However , others have suggested the models used in these studies may disturb the availability of the key growth factor , CSF1 , and do not necessarily reflect the steady state ( Jenkins and Hume , 2014 ) .", "Whatever their normal contribution to the resident macrophage pool , monocytes derived from the bone marrow ( Chow et al . , 2011 ) and spleen ( Swirski et al . , 2009 ) can clearly replenish tissue macrophages after their cytotoxic depletion .", "Beyond this precursor role , monocytes carry out specific effector functions during infection ( Serbina et al . , 2008 ) and may be involved in steady-state tissue surveillance by capturing and transporting antigen from tissue to lymphoid organs ( Jakubzick et al . , 2013 ) .", "Macrophages isolated from different organs have distinct expression profiles which can be distinguished further if the cells are separated according to their surface markers ( Gautier et al . , 2013 ) .", "Unfortunately , no available surface marker is well correlated with any other marker , at either the protein or mRNA level ( Hume , 2008 , 2012; Hume et al . , 2010 ) , so the number of macrophage subsets definable by flow cytometry is essentially infinite ( Becher et al . , 2014 ) .", "Peripheral blood monocytes , on the other hand , can be subdivided into two broad functional classes .", "In the mouse , one subset called classical monocytes , expresses high levels of both Ly6C and the chemokine receptor CCR2 but a low level of the fractalkine ( CX3CL1 ) receptor CX3CR1 , while the second , the so-called non-classical monocytes , lacks Ly6C but expresses a high level of CX3CR1 ( Geissmann et al . , 2003 ) .", "In contrast to what we know about tissue macrophages , very little information is available on how monocytes behave after entering tissues .", "Myeloid-restricted fluorescent reporter genes based upon various lineage-restricted genes have been used in live imaging and functional genomics ( Hume , 2011 ) .", "A Csf1r-EGFP reporter gene serves as a definitive marker of MPS cells ( Sasmono et al . , 2003 ) , while a Cx3cr1-EGFP reporter labels the non-classical monocytes as well as subsets of tissue macrophages , including microglia , as well as a subset of natural killer ( NK ) lymphocytes ( Jung et al . , 2000 ) .", "The Itgax-YFP transgenic mouse was considered to provide a marker for classical ‘dendritic cells’ ( Lindquist et al . , 2004 ) and has been used to image so-called interstitial DC in the lung ( Looney et al . , 2011; Thornton et al . , 2012 ) .", "However , this reporter is rather uniformly expressed in tissue macrophages associated with mucosa and in the lung provides a generic MPS marker ( Hume , 2008 , 2012 ) .", "The deletion of a conserved distal element of the Csf1r promoter in the Csf1r-ECFPtg/+ mouse ( MacBlue ) ablates expression of a reporter gene in trophoblasts , osteoclasts , granulocytes , and many tissue macrophages ( Ovchinnikov et al . , 2008 ) .", "This deleted promoter was used to construct an amplified binary transgene in which Csf1r promoter elements direct the expression of gal4-VP16 , which in turn activates expression of a UAS-ECFP transgene .", "All blood monocytes in these MacBlue mice are strongly ECFP+ , whereas most tissue macrophages do not express the reporter protein ( Sauter et al . , 2014 ) .", "In the current study , we combined the myeloid-specific fluorescent reporters from MacBlue mice with either Cx3cr1gfp/+ or Itgax-YFP transgenic mice to support in situ imaging of lung monocyte cell trafficking and compare their phagocytic activity with that of resident mononuclear phagocytes ." ], [ "The MacBlue binary transgene ( Csf1r-gal4VP16/UAS-ECFP ) provides a unique marker of blood monocytes ( Jenkins and Hume , 2014 ) .", "To confirm this restricted expression in the lung , we generated MacBlue×Cx3cr1gfp/+ double transgenic mice .", "Two-photon laser scanning microscopy 3D-reconstruction of fresh explanted lung and histological section of cryo-preserved lungs from these mice identified distinct subsets with distinct morphologies and distributions within the organ ( Figure 1 ) .", "Stellar EGFP+ECFPneg cells were seen in the collagen membrane surrounding the lung pleura ( Figure 1A , B , green squares ) , deeper in the lung parenchyma with small round shapes or stellar shapes ( Figure 1B , green squares ) , and along the basal membranes of bronchial airways ( Figure 1C , green squares ) .", "The luminal side of airways and alveoli contained large round ECFP+ cells , likely AM ( Figure 1A–D , pink squares ) .", "Smaller amoeboid-like ECFP+ cells were located in the interstitial space of the lung parenchyma ( Figure 1A–D , purple squares ) .", "In overview , the pattern was consistent with previous evidence that the MacBlue ECFP transgene was expressed only in AM and monocyte-like cells , whereas most interstitial CX3CR1-EGFP expressing cells lacked expression . 10 . 7554/eLife . 07847 . 003Figure 1 . MacBlue×Cx3cr1gfp/+ transgenic mouse discriminates lung mononuclear phagocyte subsets with specific tissue localization .", "( A ) Front and side views of two-photon laser scanning microscopy ( TPLSM ) 3D reconstruction from pleura to alveolar space of explanted lung from a MacBlue×Cx3cr1gfp/+ mouse .", "( B ) MacBlue×Cx3cr1gfp/+ mouse lung cryo-section showing lung pleura and parenchyma .", "( C ) MacBlue×Cx3cr1gfp/+ mouse lung cryo-section showing longitudinal view of bronchial airway .", "( D ) MacBlue×Cx3cr1gfp/+ mouse lung cryo-section showing interstitial space near bronchial airways and alveoli .", "Satellite images represent higher magnification of the corresponding coloured squares for each image .", "Images are representatives of more than three mice . DOI: http://dx . doi . org/10 . 7554/eLife . 07847 . 003 To establish the relationship between the cells that could be imaged in situ and their cellular phenotypes in MacBlue×Cx3cr1gfp/+ mice , we applied a panel of phenotypic markers including CD11b , CD115 , Ly6C , Ly6G , F4/80 , CD64 , CD11c , IAb , CD62L , NK1 . 1 , and SiglecF to discriminate four different subsets of the lung based on their EGFP/ECFP signature in the double transgenic line ( Figure 2A ) and compared them to blood populations ( Figure 2B and Table 1 ) .", "The lungs contained two ECFPbright populations , either EGFPbright or EGFPdim ( Figure 2A , purple gate ) resembling those observed in the blood ( Figure 2B ) .", "The CX3CR1-EGFPlow population was Ly6ChighCD11b+Ly6GnegF4/80intNK1 . 1negCD64+ ( blue gate ) in both the blood and the lungs ( Table 1 ) , consistent with identity as ‘classical’ Ly6Chigh monocytes ( Ly6Chigh Mo ) .", "The CX3CR1-EGFPhigh population was Ly6ClowCD11b+Ly6GnegF4/80intNK1 . 1negCD64+CD11c+ phenotype ( red gate ) , consistent with the phenotype of the Ly6Clow monocyte subset ( Ly6Clow Mo ) ( Guilliams et al . , 2014 ) .", "For both subsets , the expression of CD115 and CD62L was down modulated in the lung cells compared to their circulating counterparts .", "Downregulation of surface CSF1R ( CD115 ) could reflect the down-modulation of the surface receptor both by its ligand and by the many inflammatory stimuli present in the lung ( Sester et al . , 1999 ) .", "As expected , the lung also contained an ECFPhighEGFPneg signature ( pink gate ) .", "These cells were larger than monocytes and CD11b+Ly6CnegLy6GnegF4/80highNK1 . 1negCD11chighCD64highSiglecFhigh cells , consistent with their identity as AM . 10 . 7554/eLife . 07847 . 004Figure 2 . Differential fluorescent reporter expression discriminates mononuclear phagocyte subsets . ECFP and EGFP expression in ( A ) the lungs and ( B ) the blood of MacBlue×Cx3cr1gfp/+ mice .", "Colour-coded gating identifies the main subsets according to EGFP/ECFP signature .", "Percentages ± SD of total cells according to colour code are indicated ( n = 6 from two independent experiments ) .", "Dot plots showing spectral overlap of EGFP and ECFP fluorescence are depicted using separated MacBlue and Cx3cr1gfp/+ transgenic mice .", "Overlay of histogram plots of indicated markers shows the phenotype of the respective colour-coded gated cell populations .", "Grey histograms present the FMO ( full minus one ) signal gated on total monocytes .", "For lungs , lower grey histograms present the FMO signal gated on alveolar macrophages ( AM ) ( pink gate ) .", "For the blood , the cyan subset expressing a low level of ECFP represents blood neutrophils .", "( C ) Dot plots show the intensity and the frequency of ECFP expression on Ly6G+ gated cells in the lungs .", "Means of percentage ± SD are indicated ( n = 6 from two independent experiments ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 07847 . 00410 . 7554/eLife . 07847 . 005Table 1 . Comparative phenotype of mononuclear phagocyte ( MP ) subsets in the blood and the lungDOI: http://dx . doi . org/10 . 7554/eLife . 07847 . 005TissueLungBloodLungBloodLungBloodLungCD11b++++++++++/+++/++CD115−++−++−/−+/−−Ly6C+++++/−+/−+/++/+−Ly6G−−−−−−−F4/80++++++/−+/−++CD64++++++/−+/−++CD11c−−+++/−+/−++IAb+/−+/−+/−+/−+/−−/−−CD62L+++−−−/++/+−NK1 . 1−−−−−/++−/++−SiglecF−−−−−/−−/−++ConclusionLy6Chigh MoLy6Clow MoCx3cr1+ MP/NKAMThe relative expression of the markers for each subset in the blood and the lungs are compared and specific subsets defined according to the phenotype .", "++ : high expression; + : positive expression; − : below FMO ( full minus one ) signal .", "AM , alveolar macrophages; NK , natural killer .", "The EGFPbright cells that lacked detectable ECFP ( green gate ) were a heterogeneous population .", "A subset of these cells in the lung labelled with NK1 . 1 , but many were CD64+F4/80+ ( Figure 2A and Table 1 ) interstitial and pleural macrophages , as observed on histological reconstruction ( Figure 1A ) .", "In the blood , the majority of ECFPnegEGFPbright cells labelled with NK1 . 1 ( Figure 2B ) , but the population also included immature myeloid cells as previously reported ( Sauter et al . , 2014 ) .", "Note that the ECFP transgene is expressed at low but detectable levels on granulocytes in the blood ( cyan gate ) ; these cells were positive for Ly6G ( Figure 2B , C ) .", "In summary , the binary expression of the two transgenic reporters in MacBlue×Cx3cr1gfp/+ mice permits the identification of monocytes in tissues , and their distinction from other mononuclear phagocyte subsets as well as from NK cells .", "In order to confirm the monocyte origin of the ECFP+EGFPlow/high -Ly6Chigh and -Ly6Clow monocytes , we generated parabionts of the double transgenic MacBlue×Cx3cr1gfp/+ with C57Bl6 mice and analysed the reconstitution after 1 month of parabiosis .", "In the lung , donor-derived ECFP cells displayed Ly6Chigh and Ly6Clow monocyte phenotypes ( Figure 3A ) , were small in size , with ameboid-like morphologies , and were all located in the interalveolar space ( Figure 3B ) .", "By contrast , neither AM nor interstitial macrophages were derived from the donor as there was no expression of the transgenes in these compartments ( Figure 3B ) .", "The only cells expressing EGFP but not ECFP , were NK cells derived from the donor ( Figure 3A ) . 10 . 7554/eLife . 07847 . 006Figure 3 . Interstitial ECFP+ cells are monocyte-derived .", "( A ) Dot plot shows the ECFP/EGFP chimerism in the blood and the lungs of C57Bl6 host parabiont with MacBlue×Cx3cr1gfp/+ mouse .", "Histograms represent the expression of monocyte markers on the CX3CR1low ( blue gate ) and CX3CR1high cells ( red gate ) .", "( B ) Pictures compare different magnifications of lung cryo-section from MacBlue×Cx3cr1gfp/+ mouse ( left ) with C57Bl6 host parabiont with MacBlue×Cx3cr1gfp/+ mouse ( right ) .", "Up to six parabionts were prepared independently . DOI: http://dx . doi . org/10 . 7554/eLife . 07847 . 006 To evaluate the function of chemotactic signals in the lung , we compared the frequency of the different subsets defined above in Ccr2−/− , Cx3cr1−/− , and Ccr2−/−Cx3cr1−/− ( dKO ) mice in the blood and the lung ( Figure 4 ) .", "Consistent with published studies ( Kim et al . , 2011; Yona et al . , 2013 ) , the frequency of circulating Ly6Chigh monocytes was similar in wild-type ( WT ) and Cx3cr1−/− mice .", "By contrast , the absence of CX3CR1 did impact on the yield of Ly6Chigh cells in the lung .", "As expected , this subset was strongly reduced in Ccr2−/− mice and dKO mice in both the blood and the lungs ( Figure 4B , C ) .", "Ly6Clow monocyte frequency was similar in Ccr2−/− mice but significantly reduced in Cx3cr1−/− and dKO mice in both the blood and the lungs compared to WT mice ( Figure 4B , C ) .", "AM frequencies were similar in all strains ( Figure 4C ) consistent with their reported monocyte-independent homeostasis ( Guilliams et al . , 2013 ) .", "Together , these results showed that CCR2 and CX3CR1 control the steady-state trafficking and survival of both Ly6Chigh and Ly6Clow monocyte-derived cells in the lungs . 10 . 7554/eLife . 07847 . 007Figure 4 . CCR2 and CX3CR1 control the accumulation of lung mononuclear phagocytes .", "( A ) Gating strategy defines ( I ) Ly6Chigh monocytes , ( II ) Ly6Clow monocytes , ( III ) alveolar macrophages ( AM ) , and ( IV ) CX3CR1+ lung macrophages gated on CD45+CD11b+NK1 . 1negLy6Gneg cells .", "Bars represent quantification as a percentage of CD45+ cells of the defined cell subsets from ( B ) the blood and ( C ) the lungs in Cx3cr1gfp/+Ccr2rfp/+ ( WT ) , Cx3cr1gfp/gfpCcr2rfp/+ ( Cx3cr1−/− ) , Cx3cr1gfp/+Ccr2rfp/- ( Ccr2−/− ) , and Cx3cr1gfp/gfpCcr2rfp/rfp ( dKO ) mice .", "Bars represent means ± SEM ( n = 10–13 mice per group from four independent experiments ) .", "ANOVA with Bonferroni adjustment was used .", "Mo , monocytes; MP , mononuclear phagocytes; WT , wild-type . DOI: http://dx . doi . org/10 . 7554/eLife . 07847 . 007 Having demonstrated the utility of the MacBlue marker to define monocyte-derived cells , we performed live imaging on explanted lungs to characterize the behaviour of these cells ( Figure 5A and Video 1 ) .", "Small interstitial ECFP+ cells ( area 61 ± 17 μm² ) with an amoeboid-like shape displayed a bimodal behaviour: they were either highly motile , going backwards and forwards between the alveoli , or relatively sessile with protrusions extending towards the lumen of the alveoli ( Figure 5B–C and Videos 1 and 2 ) .", "Based upon these distinct behaviours , we defined ‘patrolling’ cells with high velocity ( 10 ± 4 . 6 μm/min ) and a low arrest coefficient ( 14 ± 13% ) and interstitial ECFP+ cells with lower velocity ( 4 ± 2 . 2 μm/min ) and a higher arrest coefficient ( 64 ± 21% ) ( Figure 5C ) .", "Intravenous injection of rhodamine dextran prior to mouse sacrifice permitted , during a short time frame before vascular leakage , to determine that fast moving patrolling ECFP+ cells were located inside large vessels , whereas slow motile ECFP+ cells appeared to be extravascular but in close contact with the vasculature ( Video 3 ) .", "Aside from monocytes , large round ECFPbright AM ( area 121 ± 23 μm² ) were detected in the alveolar lumina , leisurely surveying the surface of the airways with a velocity of 2 ± 1 . 5 μm/min and a high arrest coefficient ( 76 ± 19% ) ( Video 4 ) .", "Interstitial EGFP+ cells also migrated slowly , with comparable velocity and arrest coefficient to AM ( Figure 5C ) .", "To determine their motility coefficient ( MC ) , we plotted the mean square displacement as a function of the square root of time .", "The MC of patrolling monocytes was 30-fold higher than the MC of interstitial ECFP+ cells ( 39 vs 1 . 3 μm²/min ) and a further fourfold higher than the MC of AM ( 0 . 3 μm²/min ) ( Figure 5D ) .", "This higher MC in interstitial ECFP+ monocytes was attributable to the protrusive activity and slow displacement suggesting scanning of the interalveolar space ( Video 2 ) .", "Time lapse imaging of explanted non-transgenic lungs from parabiont mice showed similar scanning behaviour ( Videos 5 and 6 ) , supporting the monocyte origin of these cells .", "In overview , while AM survey the luminal side of the alveoli , interstitial monocyte-derived cells survey the lung tissue through either active patrolling in the vasculature or protrusive activity toward the alveolar space . 10 . 7554/eLife . 07847 . 008Figure 5 . Lung mononuclear phagocytes constitutively survey the entire space of the alveolar areas through distinct migratory patterns .", "( A ) Time series two-photon laser scanning microscopy ( TPLSM ) pictures with overlaid tracks of cell motility of the alveolar area from explanted lung .", "Pink , blue , red , and green squares surround alveolar macrophages , patrolling monocytes , interstitial ECFP+ cells , and interstitial EGFP+ cells , respectively .", "( B ) Time series TPLSM pictures show representative protrusive activity ( upper panel ) and patrolling ( lower panel ) behaviour by interstitial ECFP+ cells .", "( C ) Quantification of the mean velocity and arrest coefficient of cell subsets .", "Bars indicate the medians .", "Data are pooled from six independent mice .", "Kruskal–Wallis tests followed by Dunn’s multiple comparison tests were performed .", "( D ) Mean displacement ± SEM as a function of the square root of time for alveolar macrophages ( AM ) ( pink ) , scanning interstitial ECFP+ cells ( red ) , EGFP+ cells ( green ) ( left scale ) , and patrolling interstitial ECFP+ cells ( blue ) ( right scale ) .", "Coloured lines represent the linear regression of the curves .", "r2 and motility coefficients ( MC = x²/4t ) are indicated . DOI: http://dx . doi . org/10 . 7554/eLife . 07847 . 00810 . 7554/eLife . 07847 . 009Video 1 . Mononuclear phagocyte immune surveillance of alveolar space . Live 3D imaging of mononuclear phagocyte behaviour in the alveolar space of the lung of a MacBlue×Cx3cr1gfp/+ mouse .", "The ECFP signal is in cyan , the EGFP signal in green , and the SHG signal ( blue ) indicates interstitial tissue and defines alveoli .", "Representative behaviour of alveolar macrophages ( AM ) ( pink squares ) , patrolling ECFP+ cells ( blue squares ) , interstitial ECFP+ cells ( red squares ) , and interstitial EGFP+ cells ( green square ) are indicated . DOI: http://dx . doi . org/10 . 7554/eLife . 07847 . 00910 . 7554/eLife . 07847 . 010Video 2 . Migratory behaviour of lung interstitial ECFP+ cells . Live 3D imaging shows interstitial monocyte-derived cells scanning with protrusive activity or patrolling in the steady-state alveolar space of the lung of a MacBlue×Cx3cr1gfp/+ mouse .", "The ECFP signal is in cyan , the EGFP signal in green , and the SHG signal ( blue ) indicates interstitial tissue , while white line drawings define the limits of alveoli . DOI: http://dx . doi . org/10 . 7554/eLife . 07847 . 01010 . 7554/eLife . 07847 . 011Video 3 . Migratory behaviour of ECFP+ cells in lung vasculature . Live 3D imaging shows ECFP+ cells patrolling in the lumen of lung vessels ( blue arrow ) and interstitial ECFP+ cells in the vicinity of the vasculature .", "MacBlue×Cx3cr1gfp/+ mice were injected with 2 MkDa rhodamine dextran 1 min before sacrifice .", "Lungs were imaged rapidly before leakage of the dye into the alveoli .", "The ECFP signal is in cyan , the EGFP signal in green , lung capillaries and vessels are in red , and the SHG signal is in blue . DOI: http://dx . doi . org/10 . 7554/eLife . 07847 . 01110 . 7554/eLife . 07847 . 012Video 4 . Alveolar macrophage surveillance of the alveolar lumen . Live high-resolution 3D imaging of alveolar macrophages ( indicated by pink arrows ) in the alveoli of a MacBlue×Cx3cr1gfp/+ mouse .", "The ECFP signal is in cyan , the EGFP signal in green , and the SHG signal in blue . DOI: http://dx . doi . org/10 . 7554/eLife . 07847 . 01210 . 7554/eLife . 07847 . 013Video 5 . Monocyte patrolling behaviour in host parabiont . Live 3D imaging shows interstitial monocyte patrolling behaviour ( blue square ) in the steady-state alveolar space of the lung of a C57Bl6 host parabiont with MacBlue×Cx3cr1gfp/+ mouse 1 month after parabiosis . DOI: http://dx . doi . org/10 . 7554/eLife . 07847 . 01310 . 7554/eLife . 07847 . 014Video 6 . Monocyte cell scanning behaviour in host parabiont . Live 3D imaging shows interstitial monocyte protrusive activity ( red squares ) in the steady-state alveolar space of the lung of a C57Bl6 host parabiont with MacBlue×Cx3cr1gfp/+ mouse 1 month after parabiosis . DOI: http://dx . doi . org/10 . 7554/eLife . 07847 . 014 In order to determine the molecular mechanism involved in the steady-state lung surveillance , we analysed the behaviour of lung mononuclear phagocytes in MacBlue×Cx3cr1gfp/gfp mice ( Figure 6A and Video 7 ) .", "The absence of functional CX3CR1 did not change the arrest coefficient ( Figure 6B ) but did slightly reduce the MC of patrolling cells ( Figure 6C ) .", "As might be anticipated given their lack of CX3CR1 expression , AM behaviour was also comparable in WT and Cx3cr1−/− mice ( Figure 6B–C ) .", "On the other hand , deletion of the CX3CR1 clearly altered the behaviour of the interstitial ECFP+ cells , which showed a greatly increased arrest coefficient and reduced MC ( Figure 6B–C ) .", "The scanning behaviour was also strongly affected .", "In the absence of CX3CR1 , the remaining interstitial ECFP+ cells present had greatly reduced protrusive activity , as indicated by increased global ‘sphericity’ ( Figure 6D , E ) and reduced sphericity variation ( Figure 6F ) . 10 . 7554/eLife . 07847 . 015Figure 6 . Interalveolar space scanning by monocyte-derived cells is CX3CR1 dependent .", "( A ) Time series two-photon laser scanning microscopy ( TPLSM ) pictures show interstitial ECFP+ cell activity in explanted lungs of MacBlue×Cx3cr1gfp/gfp mice .", "( B ) Comparative box and whiskers analysis of the arrest coefficient of indicated cell subsets between MacBlue×Cx3cr1gfp/+ ( full boxes ) and MacBlue×Cx3cr1gfp/gfp ( empty boxes ) .", "Mann–Whitney tests were performed .", "Data are pooled from four to six mice from different experiments .", "( C ) Comparison of the mean displacement ± SEM as a function of the square root of time for patrolling interstitial ECFP+ cells ( blue ) , scanning interstitial ECFP+ cells ( red ) , and alveolar macrophages ( AM ) ( pink ) between MacBlue×Cx3cr1gfp/+ ( full circles ) and MacBlue×Cx3cr1gfp/gfp ( empty circles ) .", "Coloured dashed lines represent the linear regression of the curves .", "r2 and motility coefficients ( MC = x²/4t ) are indicated for the full versus empty circles , respectively .", "( D ) Time series volume rendering image of interstitial ECFP+ cell showing shape modifications .", "( E ) Graph representing the mean sphericity of individual cells determined by measuring sphericity at each time point .", "Red bars represent the mean .", "( F ) Graph representing the coefficient of sphericity variation for each cell tracked during 10 consecutive planes ( 5 min ) .", "Data are pooled from different movies from at least three different mice in each group from independent days .", "Student’s t test was used . DOI: http://dx . doi . org/10 . 7554/eLife . 07847 . 01510 . 7554/eLife . 07847 . 016Video 7 . Migratory behaviour of lung interstitial monocyte-derived cells in a MacBlue×Cx3cr1gfp/gfp mouse . Live 3D imaging shows reduced interstitial monocyte-derived cell protrusive activity ( red square ) in the steady-state alveolar space of the lung of a MacBlue×Cx3cr1gfp/gfp mouse .", "The ECFP signal is in cyan , the EGFP signal is in green , and the SHG signal ( blue ) indicates interstitial tissue and defines alveoli . DOI: http://dx . doi . org/10 . 7554/eLife . 07847 . 016 Lung DC defined by CD11c expression have been previously shown to participate in antigen uptake ( Thornton et al . , 2012 ) .", "CD11c is expressed on all tissue macrophages in the lung , but is absent from monocytes .", "To compare the roles of these cells in particle clearance , we combined MacBlue with the Itgax-YFP transgene ( MacBlue×Itgax-YFP ) and again compared the fluorescent signatures of different mononuclear phagocyte populations ( Figure 7A ) .", "According to previous phenotyping , Ly6Chigh monocytes were CD11b+Ly6G−SiglecF-IAblowCD64lowLy6Chigh , Ly6Clow monocytes were CD11b+Ly6G−SiglecF-IAblowCD64lowLy6Clow , interstitial macrophages were CD11b+Ly6G−SiglecF-IAbhighCD64+ , and AM were CD11blowCD11chighSiglecFhighCD64+ .", "Based upon published markers ( Guilliams et al . , 2014 ) , two sets of classical myeloid DC have been defined: CD11b+ DC that were CD11b+CD11c+Ly6G−SiglecF-IAbhighCD64−CD24+ and CD11b− DC that were CD11b−CD11c+Ly6G−SiglecF-IAbhighCD64−CD24+CD103+ ( Figure 7A ) .", "We determined the mean fluorescence intensity ( MFI ) of EGFP , ECFP , and YFP reporters from the MacBlue×Cx3cr1gfp/+ and MacBlue×Itgax-YFP in these subsets ( Figure 7B ) .", "EGFP and ECFP fluorescent reporter expressions were mainly restricted to lung macrophage and monocyte subsets as previously described ( Figure 2 ) .", "Although CD11c mRNA and surface protein are expressed widely among tissue macrophages in the lung , YFP expression was mainly restricted to the defined classical DC subsets with a brighter expression for CD11b− DC ( Figure 7C ) .", "Interestingly , AM only express the MacBlue transgene and despite expression of CD11c protein on their surface , YFP was detectable in only a small subset ( Figure 7B–C ) .", "Thus the combination of these different transgenes permits the distinction of lung mononuclear phagocytes and lung DC .", "AM represented the most abundant mononuclear phagocyte subset of the lung ( Figure 7D ) .", "Lung monocytes represented the second main mononuclear phagocyte subset and were even more numerous than interstitial macrophages , CD11b+ DC and CD11b−CD103+ DC ( Figure 7D ) .", "Histological analysis of a lung section of MacBlue×Itgax-YFP showed that YFPbright cells were mainly located along bronchial airways and poorly distributed in the alveolar space in contrast to ECFP+ monocytes and AM ( Figure 7E ) . 10 . 7554/eLife . 07847 . 017Figure 7 . Interstitial monocyte-derived cells localized in the alveolar space whereas lung dendritic cells preferentially localized near large airways .", "( A ) Dot plots show the gating strategy to define the different lung mononuclear phagocytes and dendritic cells from either MacBlue×Cx3cr1gfp/+ or MacBlue×Itgax-YFP mice .", "( I ) Ly6Clow monocytes ( red ) ; ( II ) Ly6Chigh monocytes ( blue ) ; ( III ) interstitial macrophages ( light green ) ; ( IV ) alveolar macrophages ( AM ) ( pink ) ; ( V ) CD11b+ DC ( yellow ) ; ( VI ) CD11b− DC ( purple ) ; and ( VII ) neutrophils ( grey ) .", "For CD11b− DC , AM , CD11b+ DC , and interstitial macrophages ( Inter . Mac ) , FMO ( full minus one ) signals gated on the specific subset were overlaid in black ( for the x-axis ) and grey ( for the y axis ) .", "( B ) Histogram plots represent the EGFP , ECFP , and YFP fluorescent reporter expression by each defined subset .", "YFP and EGFP were measured on individual mice .", "( C ) Mean fluorescent intensity of EGFP ( green ) , ECFP ( blue ) , and YFP ( yellow ) .", "Bars are mean ± SEM ( n = 3 MacBlue×Cx3cr1gfp/+ and MacBlue×Itgax-YFP mice ) .", "The experiment has been repeated at least three times .", "( D ) Absolute number of indicated subset per mg of lung ( pooled data of n = 11 mice from at least three independent preparations ) .", "( E ) Wide field image of MacBlue×Itgax-YFP mouse lung cryo-section showing ECFP+ and YFP+ cell distributions in alveolar space and near bronchial airways .", "Satellite images represent higher magnification of the corresponding white squares .", "Images are representative of three different mice .", "Mo , monocytes . DOI: http://dx . doi . org/10 . 7554/eLife . 07847 . 017 Particle size and the physical properties of inhaled particles are critical in the uptake by lung phagocytes and trafficking to regional lymph nodes ( Jakubzick et al . , 2008; Blank et al . , 2013 ) .", "We hypothesized that regional segregation of monocytes and classical DC could be also important in antigen uptake .", "We inoculated mice with fluorescent beads either by intravenous or airway routes and compared the proportion of phagocytic cells in different lung subsets 4 hr after bead inoculation ( Figure 8A ) .", "After i . v . inoculation , only monocytic cells appeared to have taken up particles ( Figure 8B–C ) .", "After airway inhalation , AM were the main phagocytic subset ( Figure 8B–C ) , but particles were also detected among interstitial cells .", "The numbers of phagocytic Ly6Clow and Ly6Chigh monocytes were significantly higher compared to the number of phagocytic DC after airway inhalation , showing that lung monocytes are the only subsets that effectively capture both blood-derived and airway-derived fluorescent particles ( Figure 8C ) . 10 . 7554/eLife . 07847 . 018Figure 8 . Lung monocyte-derived cells survey both airways and vascular routes whereas dendritic cells ( DC ) survey only airways .", "( A ) Representative dot plots showing fluorescent bead uptake by lung mononuclear phagocytes and dendritic cells 4 hr after airway inoculation .", "Respective control subsets without beads are overlaid in black .", "( B ) Graph represents the frequency of phagocytic cells as a percentage of the respective subset after intravenous injection ( empty bars ) or airway inhalation ( full bars ) .", "( C ) Graph represents the number of phagocytic cell subset per mg of collected tissue .", "Bars are mean ± SEM ( n = 6–7 mice in each group from two to three different experiments ) .", "Student’s t tests were performed to compare the phagocytic activity of all subsets to the referent population of Ly6Clow monocytes .", "Mo , monocytes . DOI: http://dx . doi . org/10 . 7554/eLife . 07847 . 018 In order to further discriminate lung monocytes from circulating monocytes , we performed blood/tissue partitioning using in vivo CD45 labelling as reported by others ( Anderson et al . , 2014; Girgis et al . , 2014 ) .", "This approach was reported to be technically challenging due to the vascular permeability of the lung ( Jakubzick et al . , 2013 ) .", "Lung mononuclear phagocyte subsets and neutrophils were labelled in different proportions ( Figure 9A ) .", "AM were not labelled , confirming that the antibody did not reach the airways .", "Overall , 27% of interstitial macrophages , 12% of CD11b+ DC , and only 1% of CD11b− DC were labelled , suggesting a distinct location of these subsets related to the vasculature ( Figure 9A ) .", "Up to 90% of Ly6Chigh and Ly6Clow lung monocytes were labelled by the anti-CD45 antibody in vivo; however , the MFI was significantly lower compared to that of blood monocytes ( Figure 9B ) .", "We excluded that this effect was due to collagen treatment , as similar results were obtained in non-digested lungs ( Figure 9B ) .", "Furthermore , the phenotype of both Ly6Chigh and Ly6Clow monocytes was slightly distinct from their circulating counterparts .", "Indeed lung monocytes displayed reduced expression of Ly6C , CD62L , and CD115 as previously observed , in addition to a higher level of CD11b , whereas only Ly6Clow monocytes displayed higher CD11c expression ( Figure 9C ) .", "The same differences were observed in monocytes isolated from lung without enzymatic digestion ( data not shown ) .", "These results suggest that monocytes can be either extravascular but in close vicinity to the vessels or still intravascular and trapped in capillaries with reduced access to the bloodstream and submitted to the lung environment .", "MacBlue×Cx3cr1gfp/+ mice were inoculated intravenously 5 min before sacrifice with a mixture of 10 µm and 0 . 2 µm red fluorescent beads to differentiate large vessel areas identified by the presence of both 10 µm and 0 . 2 µm beads , and lung capillary areas identified by only 0 . 2 µm beads ( Figure 9D ) .", "In large vessels , ECFP+ cells were mainly round shaped , whereas in the vicinity of capillaries , interstitial ECFP+ cells displayed an elongated or amoeboid-like shape ( Figure 9D ) .", "Confocal analysis of the microvascular area using CD31 staining showed that interstitial ECFP+ cells locate at the interface between the capillaries and the alveoli either intra- or extravascularly ( Figure 9E and Video 8 ) .", "To confirm that this positioning allows the sampling of particles inoculated by the airways route , we performed in vivo CD45 staining of monocytes that had captured beads 4 hr after intranasal inoculation ( Figure 9F ) .", "CD45 in vivo labelling was exactly the same for both Ly6Chigh and Ly6Clow monocytes that had captured the beads compared to non-phagocytic monocytes ( Figure 9F ) .", "This result confirmed that lung monocytes are strategically positioned at the interface between the bloodstream and airways to survey both compartments . 10 . 7554/eLife . 07847 . 019Figure 9 . Lung monocyte-derived cells are located at the interface between blood and airways .", "( A ) Representative overlayed dot plots of in vivo CD45 staining gated on blood and lung mononuclear phagocytes and neutrophils ( coloured ) .", "CD45 staining control from mice not injected with anti-CD45 are shown ( black ) .", "Percentage of CD45+ labelled cells according to control are indicated .", "Mac , macrophages .", "( B ) Bars represent CD45 mean fluorescence intensity ( MFI ) after in vivo staining in Ly6Chigh ( blue ) and Ly6Clow ( red ) monocytes from the blood ( full bars ) and the lungs ( empty bars ) with or without enzymatic digestion .", "( C ) Bars represent MFI of the indicated markers gated on Ly6Chigh ( blue ) and Ly6Clow ( red ) monocytes from the blood ( full bars ) and the lungs ( empty bars ) .", "Bars represent mean ± SEM ( n = 10 mice from two different experiments ) .", "ANOVA with Bonferroni adjustment was used .", "( D ) MacBlue×Cx3cr1gfp/+ mouse lung cryo-section showing ECFP+ cell localisation in distinct vascular areas , according to bead distribution after intravenous injection of a mixture of 10 µm and 0 . 2 µm red fluorescent beads .", "Satellite images represent higher magnification of large vessels containing 10 µm and 0 . 2 µm beads , indicated by white arrows ( left ) , and capillaries containing only 0 . 2 µm beads , indicated by white arrows ( right ) .", "( E ) Confocal volume rendering reconstitution image of CD31 ( red ) ( left ) or isotype staining ( right ) showing ECFP+ monocytes ( blue ) in the vicinity of lung capillaries .", "Volume rendering reconstruction has been determined according to the isotype staining .", "( F ) Dot plots represent in vivo CD45 staining of Ly6Chigh ( blue ) and Ly6Clow ( red ) monocytes 4 hr after intranasal inoculation of fluorescent beads .", "CD45 staining control from mice not injected with anti-CD45 is shown ( black ) .", "Bars represent the CD45 MFI gated on phagocytic ( beads+ ) and non-phagocytic ( beads− ) monocytes .", "Bars represent mean ± SEM ( n = 4 mice from two different experiments ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 07847 . 01910 . 7554/eLife . 07847 . 020Video 8 . Confocal 3D reconstruction of lung ECFP+ cells in the lung vasculature . High resolution 3D reconstruction shows lung ECFP+ cell localisation at the interface between lung capillaries and airways .", "Lung vasculature has been stained with anti-CD31 ( right ) or isotype control ( left ) in a lung cryo-section of a MacBlue×Cx3cr1gfp/+ mouse .", "Acquisition parameters were identical for both stainings .", "Volume rendering parameters were determined based on CD31 isotype staining .", "Capillaries are in red and ECFP+ cells are in blue . DOI: http://dx . doi . org/10 . 7554/eLife . 07847 . 020" ], [ "Jakubzick et al . ( 2013 ) recently reported that monocytes enter and survey non-lymphoid organs ( such as the lungs ) at steady state .", "How this property differs from the already known functions of AM and DC was still unknown .", "Fluorescent myeloid-specific reporter transgenic mice have been widely used to study the behaviour of myeloid cells in vivo , although no transgenic reporter is restricted to a particular myeloid lineage ( Hume , 2011 ) .", "LysM reporters can be used to track either neutrophils or monocytes ( Faust et al . , 2000 ) .", "A wide diversity of cell subsets are labelled in the Cx3cr1gfp/+ system , including NK cells , which can be excluded only with a specific γc-deficient background ( Auffray et al . , 2007 ) .", "Similarly , the MacGreen ( Csf1r-EGFP ) transgene enables labelling of a wide range of mononuclear phagocytes as well as granulocytes ( Sasmono et al . , 2003 ) .", "The MacBlue binary transgene , based upon a deleted Csf1r promoter , produced a transgenic line in which the reporter gene is extinguished in the majority of mature tissue macrophages and greatly reduced in granulocytes ( Ovchinnikov et al . , 2008; Sauter et al . , 2014 ) .", "Despite the fact that ECFP expression is not directly related to the level of Csf1r mRNA , it provides a unique marker of cells that have recently derived from monocytes .", "In the current study , we intercrossed Cx3cr1gfp/+ and MacBlue mice to obtain an additional dimension of resolution and specificity .", "Monocytes were easily distinguishable from other subsets due to their common expression of ECFP .", "The differential expression of EGFP enabled a further distinction between Ly6Chigh and Ly6Clow monocytes .", "Although our imaging approach did not allow us to distinguish between the two monocyte subsets , the shape , size , tissue localization , and behaviour of the ECFP+ cells provided a unique opportunity to track lung tissue mononuclear phagocytes and to compare their migratory patterns .", "The dual reporter also avoids potential artefacts created by the γc-deficient background excluding NK cells which we have shown to be highly represented in the CX3CR1-GFP+ fraction within the lungs .", "The combination of CCR2 and CX3CR1 deficient mice and parabionts permitted a more precise characterization of the nature and origin of the different subsets and confirmed that interstitial ECFP+ were monocyte-derived according to the proposed unified nomenclature ( Guilliams et al . , 2014 ) .", "Krummel’s group previously performed functional imaging of lung DC using the Itgax-YFP reporter ( Thornton et al . , 2012 ) .", "The combination of this reporter with the MacBlue mouse confirmed that interstitial monocyte-derived cells were distinct from lung DC .", "Histological analysis showed that monocyte-derived cells and DC share the surveillance activity through distinct regional distribution , the former located in the alveolar space at the interface with the bloodstream and the latter near larger bronchial airways .", "In vivo CD45 labelling added a new dimension of regional segregation between AM , interstitial macrophages , CD11b+ DC , CD11b− DC , and lung monocytes .", "This specific regional segregation at steady state is likely the result of the rapid circulation of monocytes that arrest constitutively in the capillaries of the alveolar space with relatively short life span ( Jakubzick et al . , 2013 ) , whereas DC are slow motile cells ( Thornton et al . , 2012 ) .", "Of course regional segregation is likely less marked in the allergic asthma model for which important DC recruitment was observed in the vicinity of the alveoli , likely originating from monocytes ( Jakubzick et al . , 2008 ) , where they exert longer transepithelial dendrites formation toward airways ( Thornton et al . , 2012 ) .", "Our data demonstrate that the interstitial space of the lung is intensively surveyed by monocytes actively migrating or extending processes .", "The strategic positioning of lung monocytes at the interface allows for efficient capture of both blood and airway-derived particles .", "Four hours after intranasal inoculation , fluorescent particles remained undetectable in the mediastinal lymph nodes , as previously observed ( Jakubzick et al . , 2008; data not shown ) , however after 1 d , DC seem to prevail over monocytes in the transport of the inhaled particles .", "This suggests that the majority of monocytes may scavenge particles to clean up the tissue and filter the blood , whereas the majority of DC aim to transport antigen to the draining lymph node .", "Due to the vascular permeability of the lung , it is challenging to address the blood/tissue partitioning of the subsets ( Jakubzick et al . , 2013 ) .", "We combined in vivo CD45 labelling and confocal imaging to determine the precise location of lung monocytes ( Anderson et al . , 2014; Girgis et al . , 2014 ) .", "We determined that lung permeability does not allow it to be definitely concluded that lung monocytes share their time either intra- or extravascularly or even both .", "However , we showed that lung monocytes have a slightly differentiated phenotype compared to the circulating monocytes with a downregulation of CD115 and CD62L and an upregulation of CD11b and CD11c .", "Secondly , the proportions of Ly6Chigh and Ly6Clow monocytes were different in the lungs compared to the blood .", "Finally , monocyte localization in the vicinity of the bloodstream allows them to capture beads inoculated via the intranasal route .", "Several studies used in situ imaging of the lung ( Kreisel et al . , 2010; Looney et al . , 2011 ) , but it is likely that working on lung explants may somehow affect the dynamics of the studied cells .", "For instance , fast circulating monocytes in the blood flow could not be observed .", "Nevertheless , we still observed high velocity patrolling cells that were present within large vessels , suggesting that this migration is independent of the blood flow .", "This approach provides at least the advantage of better stability without breathing constraints and avoids any accumulation of inflammatory monocytes due to inflammation .", "Other monocyte behaviour might be observed using in vivo imaging .", "By contrast to the CX3CR1-dependence of endothelium patrolling ( Auffray et al . , 2007 ) as well as transepithelial dendrite formation in the gut ( Kim et al . , 2011 ) , the patrolling activity of interstitial monocytes in the lung was unaffected by the absence of this receptor , whereas their scanning activity was severely impaired .", "Kim et al . showed that CX3CL1 was expressed by the epithelial cells of bronchioles and in the alveolar space using the CX3CL1-RFP reporters ( Kim et al . , 2011 ) , arguing in favour of the role of this axis in protrusive activity of the CX3CR1+ monocyte-derived cells .", "Because CX3CR1 is an important survival pathway , it is unclear whether these modifications are related to defects in chemotactic signal or cell survival .", "We did not observe defects in phagocytosis in CX3CR1-deficient mice ( unpublished data ) , suggesting that the recently recruited monocytes are functional , arguing in favour of a role of CX3CR1 in their survival ( Kim et al . , 2011 ) .", "Overall , our study provided important fundamental insights into the behaviour of tissue monocytes during the process of immune surveillance in comparison to resident macrophages and DC in a key biological tissue .", "We concluded that tissue monocytes represent a major first line phagocytic compartment in comparison to DC .", "Monocyte-derived cells developed an organized distribution at the interface between blood and airways with a specific pattern of movements in the lungs to enable them to rapidly detect danger , trigger inflammation , capture antigen , and undergo subsequent immune response .", "Understanding these activities is a key step towards improving the treatment of a wide range of inflammatory diseases and also vaccination strategies targeting the route of antigen uptake ." ], [ "Cx3cr1-GFP-Kin ( Cx3cr1gfp/+ ) or Itgax-YFP transgenic mice ( CD11c-YFP ) ( Lindquist et al . , 2004 ) and Csf1r-Gal4VP16/UAS-ECFP ( MacBlue ) ( Ovchinnikov et al . , 2008 ) were intercrossed to generate Cx3cr1gfp/+×Csf1r-Gal4VP16/UAS-ECFP mice herein called MacBlue×Cx3cr1gfp/+ or MacBlue×Cx3cr1gfp/gfp , and MacBlue×Itgax-YFP , respectively .", "These new strains were bred in the Nouvelle Animalerie Commune animal facility at Pitié-Salpêtrière .", "Cx3cr1gfp/+-Ccr2rfp/+ mice were kindly provided by Israel Charo ( Gladstone Institute , San Francisco , CA , USA ) ( Saederup et al . , 2010 ) to generate Cx3cr1gfp/gfp-Ccr2rfp/+ , Cx3cr1gfp/+-Ccr2rfp/- , and Cx3cr1gfp/gfp-Ccr2rfp/rfp mouse strains .", "All mice were used between 8 and 12 weeks of age .", "C57Bl6 female host parabionts were generated with MacBlue×Cx3cr1gfp/+females and analysed after 1 month of parabiosis .", "Blood leukocyte chimerism was evaluated 2 weeks after surgery , and showed a T cell chimerism of 50 ± 10% .", "In these settings , monocyte chimerism from B6 to MacBlue×Cx3cr1gfp/+ was around 37 ± 14% and MacBlue×Cx3cr1gfp/+ to B6 was reduced to 14 ± 13% .", "All experiment protocols were approved by the French animal experimentation and ethics committee and validated by Service Protection et Santé Animales , Environnement ( no . A-75-2065 ) .", "Sample sizes were chosen to ensure the reproducibility of the experiments and according to the 3Rs of animal ethics regulation .", "A total of 1011 FluoroSpheres carboxylate–modified microspheres ( 200 nm ) ( Invitrogen , Eugene , OR , USA ) were inoculated either by intravenous or airway routes .", "Phagocytosis by lung subsets was analysed 4 hr after inoculation by flow cytometry .", "Bead inhalation was performed by loading a 10 µl drop of NaCl 0 . 9% bead solution in each nostril of mice anaesthetized by an intraperitoneal injection of a mixture of ketamine/xylazine ( 100 and 10 mg/kg body weight , respectively ) .", "Mice for which bead inhalation failed have been excluded from the analysis .", "For in vivo CD45 labelling , mice were injected intravenously with 1 µg of anti-CD45 ( clone 30-F11 ) .", "Mice were sacrificed 2 min after blood was drawn .", "Lungs were harvested and bathed in a large volume of PBS to dilute free antibody .", "Flow cytometry was performed with the flow cytometer FACScanto or Fortessa ( BD , Franklin Lakes , NJ , USA ) and DIVA Flow Cytometry acquisition software , and was analysed with FlowJo software ( Tree Star , Ashland , OR , USA ) .", "After blood was drawn via retro-orbital puncture with heparin , the mouse lung vasculature was gently flushed with an intracardiac injection of PBS until complete blood clearance .", "For in vivo CD45 staining experiments , the lung vasculature was not flushed .", "Lungs were then harvested and digested in RPMI medium ( Gibco , Invitrogen , Cergy Pontoise , France ) with 1 mg/ml collagenase IV ( Sigma , St Quentin Fallavier , France ) for 30 min at 37°C and/or directly ( without collagenase incubation ) dissociated through a 70 μm-pore cell strainer ( Becton Dickinson , Rungis , France ) to obtain the cell suspension .", "Similarly , for some experiments blood was or was not incubated with 1 mg/ml collagenase IV .", "For antibody staining , 50 μL of cell suspension was incubated with 1 μg/mL purified anti-CD16/32 ( 2 . 4G2 ) ( BD Biosciences , San Jose , CA , USA ) for 10 min at 4°C and for an additional 20 min with the appropriate dilution of specific antibodies .", "The panel of antibodies used was: anti-CD11b ( clone M1/70 ) , anti-Ly6C ( clone AL-21 ) , anti-Ly6G ( clone 1A8 ) , anti-NK1 . 1 ( clone PK136 ) , anti-CD45 ( clone 30-F11 ) , anti-CD11c ( clone HL3 ) , anti-I-Ab ( clone AF6-120-1 ) , anti-CD 62L ( clone MEL-14 ) , anti-SiglecF ( clone E50-2440 ) , anti-CD24 ( cloneM1/69 ) , anti-CD103 clone ( M290 ) , rat IgG2b isotype control ( BD Biosciences ) , F4/80 ( clone BM8 ) , CD115 ( clone AFS98 ) , rat IgG2a isotype control ( clone eBr2a; eBioscience , San Diego , CA , USA ) , and anti-CD64 ( clone X54-5/7 . 1 . 1 ) ( BioLegend , San Diego , CA , USA ) .", "After incubation , cell suspensions were washed once in 0 . 5% BSA/2 mM EDTA in PBS and analysed directly by flow cytometry .", "For blood samples , erythrocytes were lysed with a lysis buffer containing 0 . 15 M NH4Cl , 0 . 01 mM KHCO3 and 0 . 1 mM EDTA and resuspended in 0 . 5% BSA/2 mM EDTA in PBS .", "FMO staining controls ( full minus one ) have been performed for all sets of experiment and are indicated in dot plots or histogram plots when necessary .", "Specific FMO gating for alveolar macrophages was required due to their bright autofluorescence .", "Absolute numbers were calculated by adding to each vial a fixed number ( 10 , 000 ) of non-fluorescent 10-µm Polybead Carboxylate Microspheres ( Polysciences , Niles , IL , USA ) according to the formula: no .", "cells = ( no . acquired cells × 10 , 000 ) / ( no . acquired beads ) .", "Briefly , organs were harvested and fixed in 10% formalin for 4 hr then incubated in 30% sucrose-PBS overnight at 4°C before being embedded in Tissue–Tek OCT compound ( Sakura Finetek , Alphen aan den Rijn , Netherlands ) and frozen at −80°C .", "Sectioning was completed on a HM550 Cryostat ( Thermo Fisher Scientific , Waltham , MA , USA ) at −20°C and 5 μm sections were collected on Superfrost Plus Slides ( Thermo Fisher Scientific ) and stored at −20°C until use .", "Vascular staining using beads was performed on histological sections of lung from MacBlue×Cx3cr1gfp/+ mice injected 5 min before sacrifice with a mixture of 10 µm Polybead Carboxylate Microspheres and 0 . 2 µm FluoroSpheres carboxylate .", "Lung sections were rehydrated with PBS for 10 min , counterstained and mounted with Vectashield Mounting Medium with DAPI ( 4 , 6-diamidino-2-phenylindole; Vector Laboratories ) .", "Imaging used a Zeiss Axio Microscope ( Carl Zeiss , Oberkochen , Germany ) .", "CD31 vascular staining was performed on lung cryo-sections from MacBlue×Cx3cr1gfp/+ mice .", "A first blocking step was performed with 3% BSA/PBS solution for 30 min .", "Slides were then incubated for 1 hr at 37°C with the primary antibody rat anti-mouse CD31 ( 4 µg/ml ) ( clone MEC 13 . 3; Becton Dickinson , San Jose , CA , USA ) or the isotype control ( 4 µg/ml ) ( clone eBR2a; eBioscience ) .", "Slides were next incubated with Avidin/Biotin Blocking Kit ( SP-2001; Vector Laboratories , Burlingame , CA , USA ) and then stained with a biotinylated donkey anti-rat IgG at 3 µg/ml for 30 min at room temperature .", "After three ( 5 min ) washes in PBS , slides were incubated with Cy3-conjugated streptavidin at 2 . 6 µg/ml for 30 min at room temperature ( Jackson ImmunoResearch Laboratories , West Grove , PA , USA ) .", "Slides were counterstained and mounted with Vectashield Mounting Medium with DAPI and analysed by using a Zeiss LSM 710 NLO confocal microscope coupled with 458 nm , 488 nm , and 543 nm lasers to detect ECFP , EGFP , and Cy3 simultaneously on three photomultipliers .", "Acquisition settings were identical for both isotype and CD31 staining .", "Volume rendering was performed using Imaris software ( Bitplane , Zurich , Switzerland ) and parameters were set according to CD31 isotype staining .", "Freshly explanted lungs were immobilized in an imaging chamber perfused with oxygenated ( 95% O2 plus 5% CO2 ) RPMI medium containing 10% FCS .", "The local temperature was monitored and maintained at 37°C .", "For some experiments , 2 MkDa rhodamine dextran was injected intravenously 1 min before euthanasia and lung vasculature was ligatured to reduce leakage of the dye .", "The two-photon laser scanning microscopy ( TPLSM ) set-up used consisted of a Zeiss LSM 710 NLO multiphoton microscope ( Carl Zeiss ) coupled to a Ti:Sapphire crystal laser ( Coherent Chameleon Ultra , Santa Clara , CA , USA ) , which provides 140 fs pulses of NIR light , selectively tunable between 680 and 1080 nm , and an acousto-optic modulator to control laser power .", "The system included three external non-descanned detectors that enabled the simultaneous recording of three fluorescent channels with a combination of two dichroic mirrors ( 565 nm and 690 nm ) , 565/610 and 500/550 bandpass filters , and a 485 lowpass filter .", "The excitation wavelength was 870 nm .", "Cell motility was measured every 30 s by five consecutive 3 μm z spacing stacks ( total thickness of 12 μm ) with a plan apochromat × 20 ( NA = 1 ) water immersion objective .", "Fluorescent cells were monitored over time with three-dimensional automatic tracking and manual correction with Imaris software ( Bitplane ) .", "The different cell subsets were defined according to the fluorescent signature , the size , the shape , the localization , and the behaviour .", "Typically AM are ECFPbright , large , round , and sessile cells located in the lumen of alveoli .", "Patrolling monocytes are ECFP+ and small with an amoeboid-like shape displaying strong displacements in the interalveolar space .", "Interstitial monocyte-derived cells are ECFP+ sessile cells displaying protrusive activity in the interalveolar space .", "Interstitial EGFP+ are either small and round ( likely NK ) in the lung alveolar space , or dendritic-shaped in the pleura and along airways .", "Cells that could not be tracked for more than 2 min were not considered .", "The arrest coefficient was defined as the proportion of time each cell’s instantaneous velocity ( calculated for every 30 s interval ) was less than 2 μm/min .", "The MC was determined on a 2D-based analysis by z projection of the 3D stacks , using the formula ( x²/4 t ) ( where x represents the slope of the mean displacement as a function of the square root of time ) ( Cahalan and Parker , 2008 ) ( see statistical section ) .", "Coefficient of sphericity variation was determined by calculating the coefficient of variation with Graphpad Prism ( Graphpad , San Diego , CA , USA ) of the sphericity determined for each cell on 10 consecutive planes ( 5 min ) .", "Velocity and sphericity were determined using Imaris ( Bitplane ) .", "The acquisition and analysis protocols for all experimental conditions to be compared were identical .", "All statistical analyses were performed with Graphpad Prism .", "For intravital analysis of cell behaviour , each sample value was first tested for Gaussian distribution by the D'Agostino and Pearson omnibus normality test .", "Accordingly , multigroup comparison tests were performed by one-way ANOVA for parametric distribution followed by Bonferroni multiple comparison test or Kruskal–Wallis test for non-parametric distributions , followed by Dunn’s multiple comparison test .", "For simple comparison analysis , Student’s t test was performed to compare parametric distribution and Mann–Whitney rank sum tests were performed to compare non-parametric distribution .", "For MC measurement , the slope of the mean displacement as a function of the square root of time ( Cahalan and Parker , 2008 ) was calculated by linear regression statistical analysis .", "Linearity was considered significant for r² >0 . 9 .", "Symbols used: * , p<0 . 05; ** , p<0 . 01; *** , p<0 . 001 ." ] ]

[ "Monocytes are phagocytic effector cells in the blood and precursors of resident and inflammatory tissue macrophages .", "The aim of the current study was to analyse and compare their contribution to innate immune surveillance of the lung in the steady state with macrophage and dendritic cells ( DC ) .", "ECFP and EGFP transgenic reporters based upon Csf1r and Cx3cr1 distinguish monocytes from resident mononuclear phagocytes .", "We used these transgenes to study the migratory properties of monocytes and macrophages by functional imaging on explanted lungs .", "Migratory monocytes were found to be either patrolling within large vessels of the lung or locating at the interface between lung capillaries and alveoli .", "This spatial organisation gives to monocytes the property to capture fluorescent particles derived from both vascular and airway routes .", "We conclude that monocytes participate in steady-state surveillance of the lung , in a way that is complementary to resident macrophages and DC , without differentiating into macrophages ." ]

[ "White blood cells form part of the immune system , which protects the body against infectious diseases and other harmful agents .", "Some of these cells , including ‘mononuclear phagocytes’ , can reside within different tissues of the body , such as the lungs .", "Other less specialized cells , called monocytes , circulate in the bloodstream .", "It had long been thought that once these monocytes had taken up residence in a tissue , they could only develop into tissue-resident phagocytes .", "Several researchers , however , recently reported that monocytes can also reside within tissues without becoming more specialized .", "Nevertheless , it remained unclear what these cells did when they were in these tissues .", "Rodero , Poupel , Loyher et al . investigated the activities of tissue-resident monocytes found in the lungs of mice .", "First , mice were genetically engineered to produce fluorescent markers that meant that their monocytes could be easily distinguished from the mononuclear phagocytes in their lungs when viewed under a microscope .", "Rodero , Poupel , Loyher et al . then showed that the monocytes and the other mononuclear phagocytes localized to different regions of the lung .", "Further experiments showed that these two groups of cells also moved around the lungs in different ways .", "The tissue-resident monocytes surveyed both the blood vessels and airways , while the other tissue-resident mononuclear phagocytes only surveyed the airways .", "These findings show that lung-resident monocytes perform a different role to those found in the bloodstream .", "The findings also open the way to improving our understanding of what tissue-resident monocytes do in other organs , and in healthy or diseased animals ." ]

[ "Introduction", "Results", "Discussion", "Material and methods" ]

[ "biochemistry and chemical biology", "genetics and genomics" ]

[ [ "Many steps throughout the transcription cycle of RNA polymerase II ( Pol II ) can be regulated , and modulation of any step has the potential to alter the timing and output of mRNA production .", "After initiation of Pol II , transcription regulation is mediated mostly by the dynamics of Pol II elongation .", "For example , +20 to 100 nts downstream of the transcription start site ( TSS ) , Pol II can be slowed down and paused by negative elongation factor ( NELF ) , DRB-sensitivity inducing factor ( DSIF ) and core promoter components ( Adelman and Lis , 2012; Kwak et al . , 2013 ) .", "The escape of Pol II from the paused state into productive elongation can be rate-limiting , and is dependent on the positive elongation factor P-TEFb , which consists of the Cdk9 kinase and CyclinT1 ( Marshall et al . , 1996; Lis et al . , 2000; Ni et al . , 2008 ) .", "P-TEFb is recruited directly or indirectly to the paused Pol II complex by transcription activators , where it phosphorylates the C-Terminal domain ( CTD ) , as well as DSIF and NELF , transforming DSIF into a positive elongation factor and evicting NELF ( Peterlin and Price , 2006 ) .", "P-TEFb appears to be both necessary and sufficient for paused Pol II escape into productive elongation; blocking P-TEFb kinase activity with the drug flavopiridol ( FP ) ( Chao and Price , 2001 ) in Drosophila causes an increase of promoter proximal Pol II at the majority of genes ( Henriques et al . , 2013 ) .", "The accumulation of paused Pol II has been proposed to be the result of rapid rounds of termination and re-initiation , creating a highly dynamic Pol II peak at the promoter proximal region ( Brannan et al . , 2012; Davidson et al . , 2012 ) .", "However , paused Pol II in Drosophila seems remarkably stable as shown by extensive kinetic and in vivo analysis at the Drosophila Hsp70 locus ( Buckley et al . , 2014 ) and by estimation of decay rates of over a dozen Drosophila genes by blocking TFIIH helicase activity , and thereby initiation , with the drug triptolide ( Trp ) ( Henriques et al . , 2013 ) .", "Thus , Drosophila Pol II transcription can be regulated by the promoter proximal , stable pausing and by transcription factor-controlled entry of paused Pol II into productive elongation in Drosophila .", "In mammals , promoter proximal pausing also seems to be a P-TEFb dependent and rate-limiting step during early elongation for many genes ( Core et al . , 2008; Rahl et al . , 2010 ) .", "Expressed genes without a peak of paused Pol II in one cell type , may acquire pausing in another ( Min et al . , 2011 ) , indicating that genes have the potential of becoming regulated by promoter proximal pausing even when a promoter proximal Pol II peak is absent .", "However , it is unclear if all genes undergo this P-TEFb dependent step in mammals , and how stable the paused Pol II is .", "Downstream of the promoter proximal region , the rate of Pol II elongation has been proposed to influence ( co- ) transcriptional processes such as splicing ( Howe et al . , 2003; de la Mata et al . , 2003; Shukla and Oberdoerffer , 2012 ) , 3′ end processing ( Nag et al . , 2007 ) , termination ( Hazelbaker et al . , 2012 ) , and overall levels of mRNA ( Danko et al . , 2013 ) .", "Genic features that could potentially influence elongation rates are often , but not exclusively , associated with exons .", "Examples are histone modifications around exons and within the gene body ( Kolasinska-Zwierz et al . , 2009; Schor et al . , 2009; Schwartz et al . , 2009; Kim et al . , 2011; Saint-André et al . , 2011; Schor et al . , 2013 ) , nucleosome occupancy ( Schwartz et al . , 2009; Tilgner et al . , 2009 ) , GC content of both intronic and exonic DNA ( Schwartz et al . , 2009; Amit et al . , 2012 ) , and DNA methylation ( Maunakea et al . , 2013 ) .", "Many exonic chromatin features have been implicated in regulation of splicing as well ( Kolasinska-Zwierz et al . , 2009; Schor et al . , 2009; Shukla et al . , 2011 ) .", "Moreover , Pol II has been shown to accumulate preferentially at spliced exons ( Brodsky et al . , 2005; Alexander et al . , 2010; Chodavarapu et al . , 2010; Kwak et al . , 2013 ) , leading to the hypothesis that reduction of Pol II elongation rate facilitates splice site recognition and spliceosome assembly , and thus , splicing of the associated intron ( Shukla and Oberdoerffer , 2012 ) .", "Indeed , transcription with a slow mutant of Pol II promotes alternative splicing in human and yeast cells ( Howe et al . , 2003; de la Mata et al . , 2003; de la Mata et al . , 2010 ) .", "The relationship of elongation rates , exons and other features of transcription units is still in dispute .", "Despite observations of Pol II accumulation at exons , three studies that directly measured elongation rates at multiple genes could not clearly demonstrate a correlation between exons and elongation rate ( Singh and Padgett , 2009; Brody et al . , 2011; Danko et al . , 2013 ) .", "Furthermore , although in vitro studies clearly show the effect of nucleosomes and histone modifications on elongation rate ( Orphanides et al . , 1998; Hodges et al . , 2009; Bintu et al . , 2012 ) , the in vivo consequences of chromatin on elongation rate are less understood , primarily because of the inability to measure elongation rates at many genes simultaneously .", "Previous studies have measured elongation rates ranging from 1 to 4 kb/min at individual genes in various organisms ( Ardehali and Lis , 2009 ) .", "Recently , elongation rates for over 160 genes were measured simultaneously by following the induction wave of Pol II after estradiol or TNF-alpha treatment ( Danko et al . , 2013 ) .", "Interestingly , this study showed a broad range of elongation rates between and within cell types , suggesting that the control of elongation rate may be used to regulate transcription and co-transcriptional processes .", "Thus far , elongation rates have only been measured in rapidly inducible genes , which limits the analytical power to reveal the associations to various features of transcription .", "Therefore , it is critical to expand the number of elongation rates measured simultaneously in vivo , to allow systematic analysis of the correlation between exons , chromatin and elongation rate .", "In this study , we use Trp and FP , two highly specific drugs to block initiation or pause escape , in combination with the sensitive GRO-seq assay ( Core et al . , 2008 ) to examine the drug-induced kinetic changes in Pol II distribution over promoter proximal regions and in the gene body .", "While FP blocks escape of paused Pol II , elongating Pol II can still clear the gene , and both changes can easily be followed by the sensitive and transcription orientation specific GRO-seq assay .", "We definitively show that P-TEFb is required for paused Pol II to escape into a productive elongation , providing a platform for transcription regulatory input on the early elongation rate , even for genes not previously known to be paused , confirming and extending earlier results using ChIP-seq of Pol II ( Rahl et al . , 2010 ) .", "Similarly , use of Trp to block Pol II initiation and entry to the pause , allows kinetic analysis of paused Pol II stability on nearly 3200 genes , showing that paused Pol II has a relatively long half-life and excluding rapid termination mechanisms as a major factor of Pol II regulation at the promoter .", "Furthermore , inhibition of Pol II gene body entry causes a ‘wave’ of elongating Pol II that , when assayed as a function of time after FP or Trp addition , allows measurement of elongation rates at over 1000 genes simultaneously , and over different regions within genes .", "We show that Pol II elongation rates increase within the gene body , suggesting a gradual maturation of the elongation complex as it progresses through the gene .", "In addition , we analyzed elongation rate variation as a function of a large number of genic and chromatin characteristics .", "Elongation rates correlate negatively with exon density and CpG content and methylation , and positively with active transcription mark H3K79me2 .", "Overall , we can explain ∼30% of the gene-by-gene variation in elongation rates .", "Our study of the dynamics of Pol II shows that elongation rate is highly dynamic at all genes , both at the promoter proximal region and within the gene body ." ], [ "When a gene is activated , P-TEFb kinase is recruited to promoters and phosphorylates the paused Pol II·NELF·DISF complex , allowing paused Pol II to more rapidly escape into productive elongation .", "To identify all genes dependent on P-TEFb , we inhibited P-TEFb kinase activity with the drug FP .", "For comparison , we also inhibited pre-initiation complex formation with Trp .", "We isolated replicates of nuclei from untreated mESCs and cells treated for 2 , 5 , 12 . 5 , 25 and 50 min with 300 nM FP , as well as nuclei treated for 12 . 5 , 25 , and 50 min with 500 nM Trp ( Figure 1A ) and performed GRO-seq with these nuclei ( Figure 1—source data 1 ) .", "To minimize off-target and secondary effects , we first determined the minimum concentrations of FP and Trp required to clear Pol II from the ActB gene body using ChIP-qPCR with an antibody to total Pol II ( not shown ) , and then used these concentrations , which were at the lower spectrum compared to previous studies ( Chao and Price , 2001; Rahl et al . , 2010; Titov et al . , 2011 ) , in our genome-wide analyses .", "Furthermore , we ensured that drug treated mESCs were morphologically indistinguishable from untreated cells .", "Biological replicates correlated extremely well ( Figure 1—source data 2 and 3 ) and were combined for further analysis .", "Because inhibition of P-TEFb and initiation were anticipated to have large genome-wide effects on Pol II transcription , we normalized treated and control libraries to in vitro transcribed Arabidopsis thaliana RNAs added during the run-on . 10 . 7554/eLife . 02407 . 003Figure 1 . Timed inhibition of pause escape ( P-TEFb ) or initiation ( TFIIH ) has similar effects on the gene body Pol II distribution , but opposite effect at the promoter-proximal region .", "( A ) Experimental set-up , 300 nM flavopiridol ( FP ) and 500 nM of triptolide ( Trp ) were used to block pause escape or transcription initiation in mES cells .", "Nuclei were isolated for GRO-seq at timepoints after treatment as specified .", "( B ) Screenshot of genes Pkp4 and Ppp2r5e with or without Trp or FP treatment for 12 . 5 , 25 or 50 min , with sense reads in red and antisense reads in blue .", "( C ) Composite profile of GRO-seq read density of all genes >12 . 5 kb ( top panel ) or >150 kb ( bottom panel ) after treatment with Trp for various durations of time .", "The middle panel is a zoom-in of the top panel .", "The bottom panel shows the region downstream of the TSS for genes longer than 150 kb .", "( D ) As ( C ) , but after timed treatment with FP . DOI: http://dx . doi . org/10 . 7554/eLife . 02407 . 00310 . 7554/eLife . 02407 . 006Figure 1—source data 1 . Sequencing and alignment of GRO-seq replicates in the Trp and FP time courses . The top tables depict the total reads sequenced , and the alignment to the spike-in controls , ribosomal , reference genomes for each replicate .", "One of the 25 min Trp replicates was of poor quality due to RNA degradation during the library preparation and wasn’t included in further analysis . DOI: http://dx . doi . org/10 . 7554/eLife . 02407 . 00610 . 7554/eLife . 02407 . 007Figure 1—source data 2 . Pearson correlation of GRO-seq replicates in the Trp and FP time courses . Pearson correlations between the replicates for each time course after drug treatment in either the gene body or promoter regions . DOI: http://dx . doi . org/10 . 7554/eLife . 02407 . 00710 . 7554/eLife . 02407 . 008Figure 1—source data 3 . Spearman correlation of GRO-seq replicates in the Trp and FP time courses . Spearman correlations between the replicates for each time course after drug treatment in either the gene body or promoter regions . DOI: http://dx . doi . org/10 . 7554/eLife . 02407 . 00810 . 7554/eLife . 02407 . 004Figure 1—figure supplement 1 . Inhibition of the P-TEFb kinase by Flavopiridol .", "( A ) Western blots of chromatin bound or unbound protein fractions of mES cells after 50 min of Trp , FP or DMSO ( no Trp ) treatment or no treatment ( no FP ) , with antibodies against Ser2- , and Ser5-phosphorylated CTD , or N-terminal Pol II .", "( B ) Analysis of triplicate of western blots as shown in A . After FP or Trp treatment the bound fraction of total Pol II was reduced to ∼50 and 30% , respectively , while the free fraction was increased after treatment with FP , but not with Trp .", "( C ) Fraction of Pol II bound to chromatin that is phosphorylated at Ser2 or Ser5 of the CTD after 50 min of Trp or FP treatment .", "FP treatment causes an overall ∼50% reduction of phosphorylation of bound Pol II at both Ser2 and Ser5 . DOI: http://dx . doi . org/10 . 7554/eLife . 02407 . 00410 . 7554/eLife . 02407 . 005Figure 1—figure supplement 2 . Stability of Pol II elongation after the inhibition of initiation or pause escape .", "( A ) Composite profile of Pol II GRO-seq density from 230 to 240 kb downstream of the TSS in genes larger than 250 kb after treatment with Trp for 0 , 12 . 5 , 25 or 50 min shows that levels downstream of the inhibition wave are unaffected .", "( B ) As in ( A ) , but for FP time course . DOI: http://dx . doi . org/10 . 7554/eLife . 02407 . 005 To assess the cellular effects of the drugs on Pol II and the phosphorylation of its CTD , we fractionated insoluble chromatin from the soluble cytoplasmic/nucleoplasmic fractions of control and drug treated mESCs and performed western blots with antibodies against the N-terminus of Rpb1 , and the Serine5 or Serine2 phosphorylated CTD ( Figure 1—figure supplement 1A ) .", "Chromatin bound Pol II is reduced after treatment with either FP or Trp ( Figure 1—figure supplement 1B ) .", "However , phosphorylation of the CTD of chromatin bound Pol II was reduced only after FP treatment , but not Trp ( Figure 1—figure supplement 1C ) .", "Overall , these results indicate that FP and Trp exert the intended effects on the phosphorylation of the Pol II CTD .", "Two long genes , Pkp4 and Ppp2r5e ( Figure 1B ) , and composite profiles of all genes ( Figure 1C , D ) illustrate the effects of P-TEFb inhibition by FP ( pause escape ) and TFIIH inhibition by Trp ( initiation ) on Pol II distribution .", "Both drugs work rapidly , as gene body density near the TSS approaches background levels within 12 . 5 min .", "The rapid drug action and immediate measurements thereafter minimize the possibility of major secondary effects within the short timeframe of the experiment ( Figure 1C , D ) .", "Furthermore , as the FP and Trp dependent block of Pol II entry into the gene persists , elongating Pol II forms ‘inhibition waves’ that are very similar between treatments ( Figure 1C , D , lower panels ) .", "Downstream of these inhibition waves , Pol II density levels remain equal throughout the time course , indicating that elongating Pol II in the gene body is not affected ( Figure 1—figure supplement 2A , B ) .", "Conversely , the two drugs have opposite effects at the promoter proximal region .", "The composite paused Pol II peak increases after FP treatment ( Figure 1B , D ) , but disappears after Trp treatment ( Figure 1B , C ) .", "These results confirm that Trp blocks transcription initiation and causes the time dependent clearing of Pol II from the promoter and gene body , while FP’s prominent effect is to block escape from the pause , causing a time dependent gene body clearance but enhanced promoter proximal Pol II pausing .", "Recently , Henriques et al . ( 2013 ) observed in Drosophila that the majority of genes are susceptible to P-TEFb inhibition .", "To extend this result to mammals , we quantified changes in Pol II distribution near the TSS after FP treatment and compared it to the effects of Trp treatment .", "We selected the top 75% actively transcribed genes that are over 3 . 5 kb in length and well resolved ( n = 6380 , Figure 2—figure supplement 1A ) , and generated heat-maps of the Pol II density ± 2 kb around the TSSs of each gene before and after 50 min of drug treatment on the sense or antisense strand ( Figure 2 , Figure 2—figure supplement 1B ) .", "The antisense strand shows the presence of upstream divergent transcription , a well-documented feature of mammalian promoters ( Core et al . , 2008; Seila et al . , 2009; Flynn et al . , 2011 ) .", "While Trp causes a reduction in the promoter and downstream regions of the annotated gene ( sense strand ) , and the upstream divergent region ( antisense strand ) ( Figure 2A , Figure 2—figure supplement 1B ) , FP inhibition causes the opposite effect on the promoter regions ( Figure 2B , Figure 2—figure supplement 1B ) .", "Pol II density in the gene body is decreased after FP treatment , but Pol II increases in promoter proximal and divergent regions at the majority of genes ( Figure 2B , Figure 2—figure supplement 1B; Figure 2—source data 1 ) .", "These results indicate that FP generally allows newly recruited Pol II to enter into early elongation and pausing , but blocks the entry into productive elongation .", "While a smaller fraction ( ∼20% ) of genes displayed an unexpected decrease in paused and divergent Pol II peaks ( Figure 2—source data 1; Figure 2B ) , these genes generally display lower levels of pausing and gene body transcription ( Figure 2B ) .", "This suggests that FP can reduce initiation or increase termination of weakly paused and less active genes , either directly or indirectly .", "Generally , the change in Pol II promoter density after FP inhibition correlates positively with the preexisting level of pausing and productive transcription of a gene ( Figure 2C ) , suggesting that efficient initiation is a prerequisite for an FP-induced increase in paused Pol II . 10 . 7554/eLife . 02407 . 009Figure 2 . Pol II distribution and change at the TSS region of active genes after pause escape ( P-TEFb ) or initiation ( TFIIH ) inhibition .", "( A ) Left panel shows a density plot of the log10 of reads of the no Trp dataset , in 10 bp windows ±2 kb around TSSs of active genes .", "The two right panels show the log10 difference in 10 bp windows between 50 min Trp treatment and no treatment for the sense strand ( middle ) and antisense strand ( right panel ) with decrease in blue and increase in red .", "Genes are ordered by the maximum decrease after Trp treatment at the promoter-proximal region .", "The density scales and color keys for each panel are depicted the bottom .", "( B ) As in ( A ) , log10 reads in 10 bp windows around the TSS of the no FP control dataset ( left panel ) , and the log10 difference in reads between 50 min FP treatment and no treatment for the sense ( middle panel ) and antisense strand ( right panel ) .", "Genes are ordered by maximum increase after FP treatment at the promoter-proximal region .", "( C ) The average fold change of promoter proximal Pol II in a 250 bp region in the sense direction after 50 min FP treatment in quartiles of pausing , as measured by the pausing index ( y-axis ) , and activity , as measured by GRO-seq gene body density ( x-axis ) .", "( D ) Paused Pol II decay rates are calculated by non-linear regression with an exponential decay model over the read counts in the promoter-proximal region of each replicate time point in the Trp time course for each individual gene .", "Four examples of decay rate measurement with the standard deviation and the regression are shown , with the reads per time point per replicate shown as black dots , and the blue line as the regression of the decay .", "( E ) The half-life’s of Pol II in promoter proximal regions of 3181 genes that have a high confidence decay rate with standard deviations ( <0 . 5 times the decay rate ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 02407 . 00910 . 7554/eLife . 02407 . 011Figure 2—source data 1 . Change in promoter proximal and gene body read density , and pausing index after treatment with FP or Trp in time . The gene body change was measured in the region from 1 to 3 . 5 kb downstream of the TSS for each timepoint , and was called significantly increased when the read density ratio was greater then 1 with a Fisher Exact p-value <0 . 05 , or significantly decreased when the read density ratio was smaller then 1 with a Fisher Exact p-value <0 . 05 .", "The same for the pause peak increase and decrease , but using a 250 bp region with maximal read density in a window of ±500 bp around the annotated TSS .", "The divergent peak was found by using a 250 bp region with maximal read density in a window of −1000 to +500 bp around the annotated TSS on the antisense strand , and change was defined similarly as the gene body change .", "Pausing indexes ( PI ) are the ratio between gene body and promoter proximal Pol II density , and the change in PI has therefore no Fisher Exact p-value associated with it .", "PI’s are increased or decreased when the ratio between untreated and treated PI’s is greater or smaller then 1 , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 02407 . 01110 . 7554/eLife . 02407 . 010Figure 2—figure supplement 1 . Differential dynamics of paused Pol II upon the inhibitions of initiation or the pause escape .", "( A ) Scheme of selection process of active , long and well-resolved genes .", "( B ) Log10 of GRO-seq read count in 10 bp bins ± 2 kb around the TSSs of active genes ( n = 6380 ) before and after 50 min treatment with Trp ( left panel ) , or before and after 50 min of FP treatment ( right panel ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 02407 . 010 Quantification of the decrease in read density directly downstream of the TSS shows that 96% of genes are significantly decreased after Trp treatment , while 95% of genes are decreased after FP treatment ( Figure 2—source data 1 ) .", "This demonstrates that P-TEFb-dependent Pol II entry into productive elongation is as universal a step in gene transcription as TFIIH helicase-dependent initiation .", "After blocking Pol II initiation with Trp , the level of promoter proximal Pol II will decrease in time with a rate comprising both escape and termination .", "This decay rate indicates the stability of paused Pol II , from which we can infer whether pausing is regulated by continuous cycling of termination and re-initiation , characterized by a high decay rate and a minimal half-life of paused Pol II , or whether paused Pol II is relatively stable , and is released into productive elongation by a positive signal like recruitment and phosphorylation by P-TEFb .", "We calculated decay rates of Pol II after Trp treatment using a first order exponential decay model , and found high confidence decay rates for 3181 genes ( standard deviation <0 . 5 times the decay rate ) ( Figure 2D , E ) .", "The mean half-life of Pol II is 6 . 9 min , in agreement with the decay rates measured in Drosophila ( Henriques et al . , 2013; Buckley et al . , 2014 ) .", "Thus , promoter proximal Pol II stability is comparable between species and paused Pol II is relatively stable .", "Pol II that is already transcribing when FP is added generates a clearly distinguishable wave as time progresses .", "We tracked the rate of this wave’s progress in an unbiased manner with a hidden Markov model ( HMM , Figure 3—figure supplement 1A ) at more than 1000 long and actively transcribed genes ( Figure 3A ) .", "The HMM was applied to each biological replicate , and only genes with reproducible results were used .", "We also tracked the wave following Trp addition as an independent strategy for blocking Pol II entry into genes ( Figure 3—figure supplement 1B , C ) .", "The Trp inhibition wave was less clearly defined than the FP wave , likely due to the fact that Pol II initiated and paused before Trp treatment will gradually escape after TFIIH inhibition , resulting in a less defined block of Pol II entry into the gene body ( Figure 3A , Figure 3—figure supplement 1B , see Med13l ) .", "The middle of the inhibition wave identified by the HMM was defined as the transition point between the affected and unaffected region of the gene body . 10 . 7554/eLife . 02407 . 012Figure 3 . Genome-wide determination of Pol II elongation rates after FP and Trp treatment .", "( A ) Three representative genes used for measurement of transition points with the HMM .", "In green , the affected region after FP treatment as established by the HMM , the transition point being the endpoint of this region .", "In black , repeat regions where reads couldn’t be aligned uniquely .", "( B ) Average of HMM derived transition points of the FP timecourse ( red ) or Trp timecourse ( blue ) plotted against the time of drug treatment .", "Error bars are standard deviation from the mean .", "The numbers of the genes with high confidence HMM measurements are next to each data point .", "( C ) Elongation rates derived from the FP time course .", "Elongation rate is the distance traveled in the time spanning 5–12 . 5 min ( top; n = 141 ) , 12 . 5–25 min ( middle; n = 938 ) and 25–50 min ( bottom; n = 245 ) .", "All elongation rates can be found in the Figure 3—source data 1 .", "( D ) RNA-seq intron gradient relative to the 3′ splicing site ( 3′SS ) .", "Introns longer than 10 kb ( n = 17 , 828 ) in the refseq gene list are grouped by their sizes , and the average RNA-seq read count per 100 bp bins are plotted by the distance from the 3′SS for each group .", "Read density in the windows is normalized to the level of reads at the 3′ SS , to compensate for expression differences between genes .", "The average and the standard deviation of the slope are shown .", "( E ) The RNA-seq gradients of the mid elongation rate genes ( 12 . 5–25 min ) in the introns ( 1650 introns longer than 10 kb in 938 genes ) grouped by the quartiles of the elongation rate ( n = 413 , 415 , 411 , 411 respectively for the slowest , slower , faster , and the fastest ) .", "Note that the slower genes have greater negative slope than the faster genes .", "( F ) The RNA-seq intron gradients of the refseq introns upstream of 25 kb from TSS ( n = 380 ) and introns downstream of 50 kb ( n = 854 ) on the same gene for a smaller region . DOI: http://dx . doi . org/10 . 7554/eLife . 02407 . 01210 . 7554/eLife . 02407 . 013Figure 3—source data 1 . Full list of elongation rates derived after FP treatments between times spanning 5–12 . 5 min , 12 . 5–25 min , and 25–50 min . DOI: http://dx . doi . org/10 . 7554/eLife . 02407 . 01310 . 7554/eLife . 02407 . 014Figure 3—figure supplement 1 . Determination of elongation rates using the Hidden Markov Model of inhibition time-points .", "( A ) Scheme of the Hidden Markov Model ( HMM ) , in which each hidden state follows a binomial distribution B of 20 observed states n of the read ratio .", "We assumed two hidden states as ‘inhibition affected’ and ‘inhibition unaffected’ , with two emission probabilities that leads to low or high levels of Pol II densities , respectively .", "The transition probabilities between the ‘affected’ and ‘unaffected’ states is unidirectional .", "( B ) Three representative genes used for measurement of transition points with the HMM .", "In green , the affected region after Trp treatment as established by the HMM , the transition point being the endpoint of this region .", "( C ) Elongation rates derived from the Trp time course .", "Elongation rate is the distance traveled in kb divided by the time spanning 12 . 5 to 25 min ( top; n = 466 ) or 25 to 50 min ( bottom; n = 239 ) .", "( D ) Average of HMM derived transition points of all the transition points in the FP time course ( red ) or of genes ( n = 91 ) that had three consecutive transition points ( blue ) plotted against the time of drug treatment .", "Error bars are standard deviation from the mean .", "( E ) Average of HMM derived transition points of the FP time course ( red ) or Trp time course ( blue ) plotted against the time of drug treatment of genes we derived transition points for in both the Trp and FP time course .", "Error bars are standard deviation from the mean . DOI: http://dx . doi . org/10 . 7554/eLife . 02407 . 01410 . 7554/eLife . 02407 . 015Figure 3—figure supplement 2 . Determination of elongation rates using the intron RNA-seq gradients .", "( A ) PolyA ( − ) RNA-seq pattern in genes with long introns display a ‘saw-tooth’ pattern as shown by Ameur et al . ( 2011 ) and represented here for a typical gene ( AUTS2 ) .", "( B ) Intronic reads of poly ( A ) - RNA-seq form a gradient as a consequence of co-transcriptional splicing and elongation rates .", "Slow Pol II ( top ) will take longer than fast Pol II ( bottom ) to reach the nearest exon , allowing for a larger build-up of intronic reads and a steeper gradient before the intron is spliced out and degraded ( top ) than will fast Pol II ( bottom ) ( C ) The GRO-seq density after 25 min FP treatment of genes with a transition point from the TSS to 60 kb is shown on the left .", "Genes are ordered by distance traveled after 25 min FP treatment and grouped in quartiles .", "On the right , intron reads of the genes in the rate quartiles are plotted with read density in bins within introns >10 kb as a function of the distance to the 3′ SS .", "Read density in the windows is normalized to the level of reads at the 3′ SS , to compensate for expression differences between genes .", "The slope of a linear regression model of the data points is depicted with standard deviation in the right hand corner . DOI: http://dx . doi . org/10 . 7554/eLife . 02407 . 01510 . 7554/eLife . 02407 . 016Figure 3—figure supplement 3 . Acceleration of Pol II and its accompanying CTD phosphorylation .", "( A ) Elongation rates of genes from which more than one elongation rate could be determined .", "p-values are calculated using Wilcox test .", "( B ) Acceleration constant calculated in genes that have at least two elongation rates , see ( A ) .", "Early acceleration is from early elongation rates to mid elongation rates , and late acceleration is from mid to late elongation rates .", "Elongation rates were corrected for exon density in the regions where elongation rates were measured .", "( C ) As in ( B ) , but for genes that have three elongation rates , and both acceleration constants are calculated within the same gene ( n = 6 ) .", "( D ) Mid elongation rates ( 12 . 5–25 min ) , but not late elongation rates ( 25–50 min ) , correlate negatively with pausing .", "Boxplots of mid elongation rates ( left ) or late elongation rates ( right ) in quartiles of pausing index .", "( E ) Composite profile of total Pol II , Serine 2 and Serine 5 phosphorylated Pol II ChIP-seq in mid elongation rate genes shows that total and Serine 5 phosphorylated Pol II accumulate near the TSS , while Serine 2 phosphorylated Pol II gradually increases in the gene body .", "( F ) Same as ( E ) , but normalized to total Pol II levels , showing that Serine 2 phosphorylation increase is independent from total Pol II levels .", "The most dramatic increase in Serine 2 phosphorylation is over the first 10 kb and is the interval corresponding to the largest increase in Pol II acceleration . DOI: http://dx . doi . org/10 . 7554/eLife . 02407 . 016 On average , Pol II travels about 100 kb during 50 min of FP treatment .", "This corresponds to an elongation rate of about 2 kb/min , which is comparable to Pol II rates from previous studies ( Ardehali and Lis , 2009; Danko et al . , 2013 ) .", "The average Pol II inhibition wave travels at identical rates after either Trp or FP treatment , indicating that inhibition of P-TEFb or off-target effects of FP are unlikely to have significant effects on elongation rates downstream of the promoter proximal region ( Figure 3B ) .", "Although the HMM did not always derive transition points for each timepoint of drug treatment within genes , the average transition points of genes with detectable inhibition waves at three time-points behaved identically to the average of the genes where the HMM derived only one or two transition points ( Figure 3—figure supplement 1D ) .", "Similarly , for genes where both FP and Trp transition points were reliably measured , the average FP and Trp transition points aligned well ( Figure 3—figure supplement 1E ) .", "Both observations indicate that the measurements of transition points are unaffected by the groups of genes for which we measured the transition points after either treatment , or the method of inhibition .", "Next , we calculated elongation rates for genes that had two or more consecutive transition points .", "Since we do not include 0 min as a baseline , and start measuring transition points after 5 min when significant changes in Pol II distribution are observed , the lag time of the inhibitor action does not affect the elongation rate estimations .", "We directly measured 141 early ( 5–12 . 5 min ) , 938 mid ( 12 . 5–25 min ) , and 245 late ( 25-50 min ) elongation rates , with average elongation rates of 0 . 5 , 1 . 8 and 2 . 4 kb/min respectively ( Figure 3—source data 1 ) .", "The variation within each group of elongation rates was considerable , with standard deviations of 0 . 43 kb/min , 0 . 69 kb/min and 0 . 79 kb/min for the early , mid and late rates , respectively .", "Thus , the direct measurement of elongation rates after inhibition of Pol II gene entry by FP or Trp leads to two main observations .", "First , elongation rates vary considerably between genes , and second , the elongation complex seems to accelerate as it transcribes through the gene .", "We proceeded to confirm both observations independently .", "To verify our directly measured elongation rates , we compared the mid elongation rates group ( n = 938 ) , to an independent measurement of relative elongation rates ( Ameur et al . , 2011 ) , using intronic reads of ribosomal RNA depleted total RNA-seq data from Sigova et al . ( 2013 ) in mESCs .", "This method is based on the observation that the majority of intronic RNA is spliced and degraded co-transcriptionally ( Djebali et al . , 2012; Tilgner et al . , 2012 ) , producing a saw-tooth pattern corresponding to RNA-seq reads in introns across genes ( Figure 3—figure supplement 2A; Ameur et al . , 2011 ) .", "The gradient of RNA density from the 5′ to 3′ splice sites ( SS ) of the intron , normalized for gene expression by dividing intronic reads with the average read counts near the 3′ end of the introns , reflects the time Pol II spends during elongation along the intron .", "Slow Pol II will take longer to transcribe an intron , accumulating more RNA-seq reads prior to splicing and degradation , and resulting in a steep intronic RNA-seq read gradient .", "Fast Pol II will spend less time in an intron , reaching the 3′ SS and subsequent splicing and degradation of intronic RNA faster , resulting in a flatter intronic read pattern and a lower slope ( Figure 3—figure supplement 2B ) .", "Because the saw tooth pattern was more clearly distinguishable in larger introns and because intronic reads are much less abundant than exonic reads in typical RNA-seq datasets , we grouped introns and used pooled intronic read counts on all introns longer than 10 kb .", "Similar to what was shown in an individual gene example ( Figure 3—figure supplement 2A ) , we found a negative slope of the read densities as a function of the distance from the 3′ SS ( Figure 3D ) .", "GRO-seq density after 25 min FP treatment in the mid elongation rate genes ordered from slow to fast shows clear alignment of the Pol II inhibition wave ( Figure 3—figure supplement 2C , left ) .", "To verify the observed gene-to-gene variation in elongation rates of these genes , we split the 938 mid elongation rates genes into quartiles .", "The slope of the intronic density plots of the quartiles decreases as the directly measured elongation rates become faster ( Figure 3E , Figure 3—figure supplement 2C , right ) , independently confirming the gene-to-gene variation in elongation rates .", "Next , we explored the acceleration of Pol II within the gene body in independent ways: ( 1 ) Directly measuring elongation rates in different regions within genes , ( 2 ) Analyzing relative elongation rates within different regions of the gene body with intronic RNA-seq read gradients .", "First , our direct measurement of early , mid and late elongation rates after either FP or Trp treatment suggests that Pol II accelerates as it transcribes through the gene body ( Figure 3C , Figure 3—figure supplement 1C ) , which is also evident from the non-linear increase of all the HMM-derived transition points plotted against time ( Figure 3B , Figure 3—figure supplement 1D , E ) .", "Moreover , elongation rates derived from multiple regions within the same gene were significantly greater further downstream in the gene body ( Figure 3—figure supplement 3A ) , showing that this acceleration occurs within most genes and is not a consequence of elongation rate variation between genes .", "Also , Danko et al . ( 2013 ) recently showed a similar acceleration of elongation rates within the gene body for estrogen or TNF-alpha induced genes .", "Second , we examined the read density in introns , as described above , in the region from the TSS to 25 kb , and in introns 50 kb downstream of the TSS within the same genes .", "The slope of intron read density as a function of distance to the 3′ SS is higher in the upstream region compared to the downstream region ( Figure 3F ) , indicative of slower elongation rates upstream in genes , and acceleration as Pol II travels within the gene body .", "The acceleration is not constant , but decreases as transcription proceeds ( Figure 3—figure supplement 3B , C ) , suggesting that the Pol II elongation complex ‘matures’ and reaches its maximum speed while transcribing a gene .", "The maturation could be caused by the stochastic accumulation of elongation factors to the Pol II complex , or by gradual stochastic loss of pausing factors .", "Indeed , pausing negatively correlates with mid elongation rates , while late elongation rates are not affected ( Figure 3—figure supplement 3D ) , suggesting that pausing delays the maturation of Pol II .", "Furthermore , ChIP-seq composite profiles of total Pol II and of Ser5 or Ser2 phosphorylated CTD ( Rahl et al . , 2010 ) show that Pol II Ser2 phosphorylation , which is a product of P-TEFb activity and other kinases ( Bartkowiak et al . , 2010; Devaiah et al . , 2012 ) and presumably coincides with the loss of NELF and DSIF pausing activity , increases gradually within the gene body within the same region as the acceleration of the elongation complex ( Figure 3—figure supplement 3E , F ) .", "This suggests that Pol II is modified gradually while it transcribes , leading to maturation and increased elongation rates .", "The GRO-seq density on the gene body shows the amount of productively elongating Pol II and reflects the transcriptional activity of genes .", "However , the average GRO-seq density shows a decrease as the Pol II travels into the body of the gene ( Figure 1C , D , top panels ) .", "The decreasing pattern of gene body Pol II density in itself could be explained by the gradual termination of Pol II ( Figure 4A , top; Figure 4—figure supplement 1B ) , but alternatively , an accelerating Pol II could on its own produce a decreasing gene body Pol II density ( Figure 4A , bottom; Figure 4—figure supplement 1D ) .", "Thirdly , a mixed model can be envisioned where a decrease in termination and acceleration go hand in hand as Pol II progresses in the gene body ( Figure 4—figure supplement 1C; Mason and Struhl , 2005 ) .", "The directly measured elongation rates ( Figure 3C ) can be used to assess the degree to which termination and acceleration models contribute to the observed Pol II density decrease .", "If there is no loss of Pol II through termination , then Pol II density should be inversely proportional to the elongation rate .", "Therefore , comparing the inverse of the measured elongation rates ( v−1 ) with the Pol II density ( D ) can be used to determine which model of Pol II elongation is more probable ( Figure 4A ) .", "On average , the inverse of the elongation rate ( v−1 ) does not fall below the density plot ( D ) in the gene body region ( Figure 4B ) where the rates are measured ( starting from the average 5 min transition point 2 . 3 kb downstream of TSS ) .", "This indicates that the bulk of the change in Pol II density stems from the acceleration of stably elongating Pol II molecules . 10 . 7554/eLife . 02407 . 017Figure 4 . Elongation rate explains transcriptional stability and the steady-state output .", "( A ) Models of Pol II elongation ( left ) , and the expected steady-state GRO-seq density ( D ) and the inverse elongation rates ( v−1 ) for each model ( right ) .", "The termination model proposes that the decrease in D is a combination of Pol II termination and increasing elongation rate , while the acceleration proposes that the decrease in D is primarily a consequence of increasing elongation rate .", "( B ) The steady-state GRO-seq density ( D ) and inverse elongation rates ( v−1 ) from average transition points of the FP measurements .", "( C ) Stages of transcription determining the mRNA level following the productive elongation stage .", "( D ) Correlation plot between GRO-seq gene body density as a measure of nascent transcription and RNA-seq as a measure of mRNA steady state level .", "( E ) Non-linear correlation between nascent transcription level and mRNA level in highly transcribed genes .", "To determined the monomial degree of the correlation , a LOESS fit was used for the scatterplot in D , and the slopes of the LOESS fit in the higher 50 percentile and the lower 50 percentile were derived .", "( F ) Elongation rate correlates with GRO-seq density .", "Correlation plot was determined from the z-scores of the elongation rates and the gene body GRO-seq densities .", "( G ) Correlation of mRNA steady state level and GRO-seq of the 938 mid elongation rate genes ( 12 . 5–25 min ) , and the monomial degree of the correlation derived from the slope .", "( H ) Correlation plot of the mRNA production rate ( GRO-seq density multiplied by the elongation rate ) and mRNA steady state level of the same genes , and the monomial degree of the correlation . DOI: http://dx . doi . org/10 . 7554/eLife . 02407 . 01710 . 7554/eLife . 02407 . 018Figure 4—figure supplement 1 . Monte-Carlo modeling of the acceleration and the termination hypotheses .", "( A ) Simulation time-course of change in Pol II distribution after a block of Pol II entry into the gene body assuming an intrinsic acceleration model ( top ) and a termination model ( bottom ) .", "See supplementary methods for the details of the simulation .", "( B ) Pol II density D and inverse elongation rate v−1 plot of a simulation assuming a termination model as explained in the top panel of Figure 4A .", "( C ) Pol II density D and inverse elongation rate v−1 plot of a simulation assuming a mixed rates model .", "In this model , slower and faster populations of Pol II co-exists , and slower Pol II terminates more frequently leaving the faster Pol II more abundant in more downstream regions .", "( D ) Pol II density D and inverse elongation rate v−1 plot of a simulation assuming a intrinsic acceleration model as explained in the bottom panel of Figure 4A .", "( E ) Scatterplot of the slope of v and the slope of D−1 for multiple acceleration models and termination models .", "See supplementary methods for the details of the simulation . DOI: http://dx . doi . org/10 . 7554/eLife . 02407 . 018 We further assessed the contribution of termination and acceleration by simulating these three models of Pol II dynamics and density profiles with different levels of termination .", "The measured elongation rates fit better to the model when the termination was insignificant compared to the acceleration ( Figure 4—figure supplement 1 ) .", "Therefore , Pol II termination does not appear to have a global contribution to the amount of Pol II in the regions more downstream of 2–3 kb from the TSS .", "Elongation rates can influence the total mRNA output .", "The steady state mRNA level is determined by the balance of nascent RNA production rate , which is represented by GRO-seq density times the elongation rate , as well as RNA processing and mRNA degradation ( Figure 4C ) .", "Thus , deviation from a log–log slope of 1 in a correlation plot between steady state mRNA levels as measured by RNA-seq and GRO-seq density ( Figure 4D ) could potentially be explained by differences in elongation rates .", "Interestingly , genes that are relatively highly expressed show a slope of 1 . 8 indicating that mRNA level is not a simple function of GRO-seq density for these genes ( Figure 4E ) .", "We observed a modest correlation between elongation rate and GRO-seq ( Figure 4F ) , indicating that highly active genes have faster elongation rates and produce more mRNA , in agreement with a previous analysis of a smaller set of inducible genes ( Danko et al . , 2013 ) .", "To further assess the influence of elongation rates on steady state mRNA level , we examined the correlation between mRNA-seq and GRO-seq in the large group of mid elongation rate genes ( n = 938 ) .", "These highly expressed genes display a log–log slope of 2 . 2 ( Figure 4G ) .", "In contrast , when plotting the production rates , that is the mid elongation rates multiplied by the corresponding GRO-seq densities , vs mRNA level , the slope is reduced to 1 . 6 ( Figure 4H ) , indicating that elongation rate can partially explain the divergence between Pol II density and the mRNA level at highly transcribed genes .", "The remaining positive correlation might be explained either by increased mRNA stability or RNA processing efficiency in highly transcribed genes .", "The large number of measured elongation rates allows us to identify major factors that influence variation in elongation rates .", "We used genes for which we measured the large number of mid elongation rates ( 12 . 5–25 min , n = 938 ) to increase statistical power , and to rule out major effects of intrinsic acceleration in the comparison between elongation rates .", "Existing genome-wide data of transcription factors , chromatin modifiers , and chromatin modifications were used to detect determinants of elongation rate ( see for full list of features Figure 5—source data 1 ) ." ], [ "Inhibition of P-TEFb , like inhibition of initiation , affects >95% of actively transcribed genes , showing that escape from pause is an integral step in the transcription cycle .", "However , not all active genes show signs of promoter proximal accumulation of paused Pol II in mES cells ( Min et al . , 2011 , this study ) .", "Clearly , this accumulation of Pol II depends on both the rate with which pause complex is formed ( the transcription initiation rate ) and its rate of escape to elongation .", "Some transcription factors stimulate the formation of paused Pol II , others recruit P-TEFb directly or indirectly to facilitate the escape of paused Pol II , while others do both ( Blau et al . , 1996; Rahl et al . , 2010; Danko et al . , 2013; Li and Gilmour , 2013 ) .", "The balance of these factors at promoters , together with pausing factors that stabilize Pol II ( Yamaguchi et al . , 1999; Lee et al . , 2008; Kwak et al . , 2013 ) determine the relative accumulation paused Pol II .", "As we ( Figure 2 ) and others ( Henriques et al . , 2013; Buckley et al . , 2014 ) have shown , pausing of Pol II is remarkably stable at many genes , indicating that termination does not have a great effect on promoter proximal Pol II levels .", "This stably-paused Pol II may help maintain accessibility of the promoter to regulatory factors that tune transcriptional output by simply changing the escape rate of paused Pol II into the body of the gene .", "Indeed , heat shock has recently been shown both optically and biochemically to increase the escape of paused Pol II into the gene body at the Drosophila Hsp70 locus without decreasing termination of paused Pol II , arguing against a role for termination in the regulation of the accumulation and escape of paused Pol II .", "If Pol II undergoes a pausing step in the transcription cycle at all genes , then blocking P-TEFb kinase activity should lead to an increase of paused Pol II .", "Indeed , this is what we observe for the majority of genes ( Figure 2 ) .", "However , a small fraction of poorly expressed and weakly paused genes show a reduction in paused Pol II after FP treatment .", "We speculate that transcription initiation , Pol II pausing , and escape into productive elongation are not necessarily uncoupled processes for all genes .", "Instead , disruption of pausing dynamics by FP may feed back on transcription initiation and decrease the formation of paused Pol II .", "Other observations support a cross-talk of pausing and initiation .", "Depletion of Nelf can decrease expression of a subset of genes , indicative of a dependency of initiation on pausing , in this case through the maintenance of nucleosome free promoters by paused Pol II ( Gilchrist et al . , 2008 ) .", "Furthermore , precise mapping of Pol II reveals that core promoter elements appear to contribute to stronger and more promoter proximal pausing in Drosophila ( Kwak et al . , 2013; Venters and Pugh , 2013 ) .", "Overall , the specific inhibitors used in this manuscript inhibit either initiation or pause escape seemingly with few secondary effects , as the Pol II inhibition profile within gene bodies is remarkably similar after either drug treatment .", "Nonetheless , perturbations of one process could possibly have effects on the other , at least for some genes .", "Because P-TEFb inhibition affects all active genes , we could determine over 1300 individual elongation rates of Pol II , often at multiple regions within the same gene .", "Danko et al . ( 2013 ) has used GRO-seq following rapid induction of transcription to examine the rate of the Pol II induction wave front .", "Our elongation rates derived after inhibition of P-TEFb were slightly lower in comparison to the elongation rates derived after induction which may point to a model in which lagging Pol II pushes leading Pol II thereby increasing overall elongation rates ( Saeki and Svejstrup , 2009 ) .", "Indeed , elongation rates in highly expressed genes , where elongating Pol II is more tightly packed , are faster compared to rates in poorly expressed genes in both studies ( Figure 4; Danko et al . , 2013 ) .", "Moreover , we have shown that chromatin composition , specifically H3K79me2 and CpG content , of genes influences elongation rates .", "Interestingly , mouse ESCs have high levels of DNA methylation ( Stadler et al . , 2011 ) .", "DNA methylation and CG content negatively influence elongation rate , and could partially explain the difference between mESC and differentiated cell line derived elongation rates ( Danko et al . , 2013 ) .", "Finally , repression of H3K79me2 has recently been observed to increase reprogramming efficiency of fibroblasts to iPSCs , by reducing expression of lineage specific genes in the first stages of reprogramming ( Onder et al . , 2012 ) .", "We have shown that elongation rates can influence steady state mRNA levels ( Figure 4 ) , and that H3K79me2 positively influences elongation rate ( Figure 6 ) .", "We therefore argue that reprogramming efficiency by reducing H3K79me2 levels could be a result of elongation rate modulation .", "Finally , we emphasize that the variation of elongation rates revealed in this study have enormous potential in timing of mRNA production .", "Mammalian genes of over 200 kb and twofold differences in elongation rates between genes are not uncommon , leading to variation in response time of mRNA production of an hour or more .", "This timing can be very relevant in development and the stress response ( Thummel , 1992; Swinburne and Silver , 2008 ) , and could not only be dependent on intron length , but also be regulated by elongation rate modulation .", "The hypothesis that Pol II elongation rate regulates splicing is longstanding ( reviewed in Shukla and Oberdoerffer 2012 ) .", "Here , we show directly that exon density is the greatest predictor of elongation rates ( Figure 6 ) , strongly suggesting that Pol II slows down at each exon .", "Exonic features such as CpG methylation , H3K36me3 and H3K4me1 could work synergistically to establish a transient slow down at exons ( Figure 6 ) .", "Although we demonstrate that Pol II slows down at exons , presumably to facilitate splicing , we could not determine whether a slowly transcribing Pol II increases inclusion of exons .", "Although rates of elongation have been shown to influence splicing outcomes ( Howe et al . , 2003; de la Mata et al . , 2003 ) , the effects on specific genes , we suggest , will be governed by competing processes such as rates of splicing complex assembly , RNA secondary and tertiary structure formation , and regulatory factor binding .", "While our data cannot on its own assess the outcomes on alternative splicing of such competing and cooperating processes , our observed slow down of transcription at exons supports the general view that Pol II elongation rates are coupled to splicing at all exons ." ], [ "Cell culturing of the V6 . 5 mES cell line was done as in Monkhorst et al . ( 2008 ) , and drug treatment was performed on pre-plated mES cells to remove irradiated MEF-feeder cells , grown for one passage on 15 cm2 plates up to ∼70% confluence before isolation of nuclei .", "Drugs treatment was done by replacing ES medium with pre-heated ES medium containing 300 nM FP , 500 nM Trp or 0 . 0125% DMSO as no Trp control .", "Nuclei isolation was done according to Min et al . ( 2011 ) .", "Nuclear run-on and nascent RNA library preparation was performed as in Core et al . ( 2008 ) .", "In brief , after rinsing the 15 cm2 plates with drug-treated cells with ice-cold PBS , pH 7 . 4 , cells were scraped off in 15 ml cell lysis buffer ( 10 mM Tris-Cl , pH 7 . 5 , 300 mM Sucrose , 3 mM CaCl2 , 2 mM MgAc2 , 0 . 5% NP-40 , 5 mM DTT , 1 mM PMSF , protease inhibitors ) , and spun down at 4°C .", "Cells were dounced 50x in 5 ml fresh cell lysis buffer on ice and spun down , after which supernatant was discarded and nuclei were taken up in ∼250 μl of glycerol storage buffer ( 50 mM Tris-Cl , pH 8 . 3 , 40% glycerol , 0 . 1 mM EDTA , 5 mM MgAc2 , 5 mM DTT , 1 mM PMSF , protease inhibitors ) and snap frozen .", "For each nuclear run-on ( NRO ) , 107 nuclei were mixed with an equal volume of reaction buffer ( 10 mM Tris-Cl pH 8 . 0 , 5 mM MgCl2 , 1 mM DTT , 300 mM KCL , 20 units of SUPERaseIn , 1% sarkosyl , 500uM ATP , GTP , and Br-UTP , 2 µM CTP and 0 . 33 µM α-32P-CTP [3000 Ci/mmole] ) .", "The NRO was performed at 30°C for 5 min , and a population of ∼100 different in vitro transcribed Arabidopsis thaliana spike-in RNAs with and without Br-UTP was added to the nascent RNA .", "The RNA was fragmented to ∼150 nts with 0 . 2N NaOH and BrU-RNA was isolated three consecutive times with BrdU-antibody beads ( sc-32323; Santa Cruz Technologies , Dallas , TX ) , with enzymatic TAP and PNK treatments to remove the cap and 3′-phosphate and to add a 5′-phosphate , as well as Illumina adaptor ligations between the BrU-RNA isolation steps .", "The three consecutive isolation steps lead to an approximate 500 . 000x enrichment of BrU-RNA over background RNA .", "BrU-RNA was reverse transcribed , amplified , barcoded and Illumina sequenced .", "Each dataset was done in replicate .", "All the GRO-seq libraries were sequenced in 50 nt runs on the Illumina HiSeq and split by barcode .", "Reads were trimmed to 32-mers and Illumina adaptors were removed with the cutadapt tool ( https://code . google . com/p/cutadapt/ ) and aligned uniquely with two mismatches with bowtie to the mm9 reference genome .", "Replicates were highly correlated and were pooled for further analysis ( Figure 1—figure supplement 1 ) , with exception of the extensively degraded 25 min Trp-treated #1 replicate .", "Normalization between datasets was done with uniquely aligned spike-in RNA reads .", "Sequence datasets can be found under GEO admission number GSE48895 .", "The mm9 RefSeq genelist was used as reference genelist for all analysis .", "Unmappable regions of the genome were identified and excluded by aligning the genome to itself in 30-mers and reads aligned to these regions were not used in analysis .", "To establish which genes were active above background with a Fisher exact p-value of <0 . 05 , we mapped reads from control datasets to gene-poor regions , took 10 × the average read density of these two datasets as a safe threshold for background ( 5 × 10−4 reads/bp ) .", "The genes for analysis of FP and Trp sensitivity are the top 75% active genes larger than 3 . 5 kb present in both the FP as the Trp control dataset , without genes that have an annotated TSS within 1000 bp on the opposite strand ( bidirectional genes ) and genes that have an annotated polyA site upstream of its TSS within 10 kb ( tandem genes ) ( Figure 2—figure supplement 1A , n = 6380 ) .", "Pause and divergent peak locations were found by searching for maximum sense or antisense strand read density in 10 bp windows from ±500 bp or −1000 to +500 around the annotated TSS of the 6380 selected genes , respectively .", "The peak was defined as a 250 bp region centered on the maximum 10 bp window .", "The pausing index is the ratio of the pause peak density and the annotated gene body region density ( +1 kb from the TSS to polyA site ) .", "To calculate the fold change after FP or Trp treatment for each timepoint , we added a pseudo-count to the divergent , pause or gene body region close to the TSS ( +1 kb to +3 . 5 kb only ) , calculated the density in the mappable region of each and took the ratio of the read densities .", "The change was significant if the Fisher Exact p-value was <0 . 05 .", "Decay rates were calculated by selecting seven points randomly of the seven Trp datasets for 1000 times and doing non-linear regression using an exponential decay equation ( Rt = RWT × e− ( λt ) , with pause peak read density R , decay rate λ , and Trp treatment time t ) with each of the seven points .", "The mean of the regressions is the decay rate and the standard deviation of the decay rate is the standard deviation of the 1000 individual regressions .", "Composite profiles of all genes >12 . 5 kb or >150 kb were made in R with read density taken in 50 bp windows .", "Density plots of the 6380 selected genes around the TSS were made by taking the log10 of counted reads plus a pseudo-count in 10 bp windows around the annotated TSS for the sense and antisense strands in the control and 50 min treated datasets .", "The change in each 10 bp window was calculated by subtracting the no FP/Trp from the 50 min FP/Trp log10 read count .", "Genes were ordered by maximum pause peak read density decrease or increase after 50 min Trp or FP treatment .", "The density in windows was plotting using the R packages gplots and RColorBrewer .", "First , we selected genes longer than a sufficient cut-off for each time-point ( 30 kb for 5 min and 12 . 5 min , 60 kb for 25 min , and 150 kb for 50 min ) , that have corresponding transcription units at the annotated TSS , but do not contain intragenic transcription units defined by the genome-wide transcription unit calling algorithm ( described below in this section ) .", "Also , genes that have premature termination before the annotated 3′ ends and/or the 60 kb/150 kb mark are removed using a regional transcription unit calling algorithm ( described below in this section ) .", "After filtering , the number of selected genes are n = 4461 , 2769 and 571 for genes longer than 30 kb , 60 kb , and 150 kb , respectively .", "The transition points from the drug affected ( inhibited ) region to the drug unaffected ( uninhibited ) region of the gene body were determined by a regional transition point calling algorithm ( described below in this section ) for each replicate of FP or Trp GRO-seq time courses .", "The following is the description of the custom made HMM algorithms in c++ .", "We analyzed relative elongation rates with methods described in Ameur et al . ( 2011 ) , using intronic reads of total poly-A ( − ) RNA-seq ( Sigova et al . 2013 ) .", "Intronic reads are sparse , so we pooled multiple genes to assess the relative elongation rate .", "First , introns longer than 10 kb are selected .", "Introns containing annotated alternative exons were split in a 5′ and 3′ intron to exclude the interference from exonic RNA-seq reads .", "Second , selected introns are subgrouped by length in 10 kb bins; that is 10–20 kb , 20–30 kb , 30–40 kb groups , etc .", "We used up to 50 kb for the estimation of elongation rates ( up to 80 kb are plotted ) .", "Third , introns are aligned at the 3′ splicing sites ( 3′SS ) , and intronic reads are counted in 100 bp windows in each intron length subgroup .", "To normalize for differences in the transcription level in each group , the read counts in windows are normalized to the read density 1 kb upstream of the 3′ SS to the 3’SS in each group ( Figure 3—figure supplement 2B ) .", "Finally , we plotted read counts per window for each intronic position in all the subgroups ( Figure 3D ) .", "The slope of the RNA-seq gradient was obtained using the linear regression in the R statistics package .", "Apart from the termination and acceleration models described in Figure 4A , we also considered a mixed model , in which elongating Pol II could consist of different populations; fast Pol II , transcribing at maximum speed throughout the entire gene body , and slow , non-processive Pol II .", "If the slow population terminates prematurely leaving the fast Pol IIs , then the overall apparent elongation rate would increase as transcription proceeds in the gene body .", "To differentiate between the termination , acceleration and mixed model , we used a Monte Carlo simulation describing the dynamics of Pol II movement through the gene body .", "In short , we simulated the time course of the inhibition wave of Pol II ( Figure 4—figure supplement 1A ) and used the regional transition point calling algorithm ( Figure 3—figure supplement 1A ) to define the transition points of inhibition waves and elongation rates .", "The relationship between the simulated Pol II density and the inverse of average elongation rates of 1000 simulated experiments shows a clear difference between the models ( Figure 4—figure supplement 1B , C , D ) .", "When we plotted the slope of simulated elongation rates vs the slope of inverse density in various simulated acceleration and termination models along with actual observations , the observations appear to fit better with the distribution of the acceleration models than the termination models ( Figure 4—figure supplement 1E ) .", "In detail , the dynamics of elongating Pol II in gene body is simulated using a newly designed modeling program to describe Pol II transcription through a gene .", "First , we modeled a Pol II transcription complex entering the gene body region with an entry rate ( r ) as a function of time .", "For the steady state assumption , r is a constant over time ( t ) , while for the simulation of the inhibition wave r is an exponential decay function of t .", "Each Pol II molecule was generated with the rate r , and has a randomly assigned activity parameter ( A ) , from 0 to 100 as a percentile .", "This activity parameter A is an intrinsic value that determines the relative elongation and the termination rates for each Pol II molecule .", "The termination constant ( kt ) and the elongation rate ( v ) are the functions dependent on the activity ( A ) as well as the position ( x ) within the gene body of the Pol II molecule .", "For instance , in a simple acceleration model where all Pol II molecules accelerate uniformly , kt = 0 for all A and x , while v is an increasing function of x but a constant function for A . In a termination model , intrinsically active Pol II molecules elongate faster while less active ones elongate at a slower rate and terminate more frequently .", "In this case , kt is a decreasing function of A , and v is an increasing function of A regardless of x .", "When running a simulation , the entry , termination , and progression events are assessed after each time increment of Δt .", "For each event , a pseudorandom number between 0 and 1 is generated and compared to the probability of initiation as described by 1-exp ( -rΔt ) , termination as described by 1-exp ( -ktΔt ) , and processive elongation as described by 1-exp ( -vΔt ) respectively .", "If the number is less than the probability of any of the processes , the status of the polymerase changes accordingly .", "For the approximation of the progression event , the polymerase can move k bases following the Poisson distribution if the pseudorandom number is in the range ( F ( k; λ ) , F ( k+1; λ ) ) , where F is the cumulative Poisson distribution function and λ = vΔt .", "If there is a collision event between two polymerases , the leading polymerase terminates .", "The distribution of simulated Pol II in N = 1000 DNA templates are equilibrated for 10 , 000s .", "The average Pol II distribution at this point is recorded as D . Upon the simulation of the decay of entry , average Pol II distribution is recorded every 100 s over 100 , 000 bp region .", "The average distribution at each time point is analyzed using the HMM , and the transition points are estimated .", "From the time course of transition points , the apparent elongation rates ( va ) are calculated as a function of the position ( x ) .", "The slope plot ( Figure 4—figure supplement 1E ) is generated by calculating ΔD−1/Δx and Δva/Δx using linear interpolation between x = 5 , 15 , 25 , 35 , 45 , 55 kb or t = 5 , 12 . 5 , 25 , and 50 min .", "We tested the following parameter spaces for the simulation .", "Only one examples of the results for each simple termination , mixed , and stable acceleration models are shown in Figure 4—figure supplement 1B–D .", "However , all the described models were used to generate the scatterplot in Figure 4—figure supplement 1E .", "We calculated the intrinsic acceleration constant a of Pol II with vend = vstart × a Δt for corrected elongation rates between 5 to 12 . 5 min and 12 . 5 to 25 min FP treatment , and for 12 . 5 to 25 min and 25 to 50 min FP treatment .", "Elongation rates were corrected for exon density within the transition region where the rate was measured by using the linear regression formulas in Figure 5B and Figure 5—figure supplement 1A , B .", "For the linear regressions the elongation rates over transition regions that had an exon density >0 were taken into account to assess the additive time delay per exon .", "ChIPseq datasets listed in Figure 5—source data 1 were downloaded and aligned to the mm9 genome when necessary .", "Read density was derived in the mappable promoter region or the average 12 . 5–25 min or 25—50 min transition regions .", "We correlated read density of ChIPseq factors in the promoter or transition regions with elongation rates .", "For factors that correlated with elongation rates , we made additional density profiles from −2 to 30 kb from the TSS in 25 bp windows in the 12 . 5–25 min mid elongation rate genes , ordered by mid elongation rate from slow to fast .", "Also , we looked at the elongation rates in quartiles of correlating factors in the promoter or transition regions .", "To apply the linear modeling methods , we converted individual genomic features to fit the Gaussian distributions using rank ordered z-statistics ( R statistics package ) .", "Briefly , the percentile rank of a gene’s specific genomic feature is converted to a z-score through the inverse cumulative Gaussian distribution function .", "This conversion enables the comparison of different non-linear features , yet also allows us to use the linear modeling tools such as least square methods and principal component analysis .", "Genes with tied rank orders are randomly assigned to have different ranks .", "The major features taken into account have a p-value<0 . 01 from the simple linear regression ( function lm ( ) in R ) .", "The z-score distribution between the elongation rate and a major feature is plotted in a [−4 , 4] × [−4 , 4] range ( Figure 6A ) .", "The principal component analysis ( function princomp ( ) in R ) between the two features was used to identify the eigenvector that shows the direction of correlation , and a red arrow was depicted to represent the direction as well as the magnitude of the correlation coefficient .", "For the iterative linear modeling , we took the residual elongation rates after fitting to the linear modeling using a 1° feature ( Figure 6—figure supplement 1A , first row ) , and the residuals are again converted using the rank order z-statistics .", "The converted residuals are fitted to a 2° feature linear modeling ( Figure 6—figure supplement 1A , second row ) .", "The correlation coefficient of the iterative linear modeling was compared to the correlation coefficient of a single linear modeling using the 2° feature alone , and the percent reduction of the R2 by the 1° feature was color scaled in a combinatorial manner to produce the heatmap in Figure 6B .", "The predicted values from the linear models using one or multiple features are plotted against the actual z-scores of the measured elongation rates to show the convergence of the prediction to the observation ( Figure 6C ) .", "Chromatin bound and nucleoplasmic/cytoplasmic free proteins were extracted after treatment of pre-plated mES cells for 50 min with 300 nM FP , 500 nM Trp or 0 . 0125% DMSO as no Trp control .", "Cells were rinsed twice in ice-cold PBS , pH 7 . 4 , scraped off the plates , spun and resuspended in nuclei lysis buffer ( 20 mM Tris-Cl pH 7 . 5 , 3 mM EDTA , 10% glycerol , 150 mM KAc , 1 . 5 mM MgCl2 , 1 mM DTT , 0 . 1% NP-40 , and phosphatase and protease inhibitors ) , dounced 60 times on ice and centrifuged at 13 , 000 rpm for 5 min at 4°C .", "Supernatant was snap frozen as the unbound fraction , while the remaining pellet was resuspended in nuclei lysis buffer and sonicated to break up the chromatin and solubilize the pellet , and snap frozen .", "Western blot analysis was done in triplicate with antibodies against the Ser5 ( 3E8; EMD Millipore , Billerica , MA ) or Ser2 ( 3E10; EMD Millipore , Billerica , MA ) phosphorylated CTD , or N-terminal Pol II ( N-20; Santa Cruz Technologies , Dallas , TX ) ." ] ]

[ "Production of mRNA depends critically on the rate of RNA polymerase II ( Pol II ) elongation .", "To dissect Pol II dynamics in mouse ES cells , we inhibited Pol II transcription at either initiation or promoter-proximal pause escape with Triptolide or Flavopiridol , and tracked Pol II kinetically using GRO-seq .", "Both inhibitors block transcription of more than 95% of genes , showing that pause escape , like initiation , is a ubiquitous and crucial step within the transcription cycle .", "Moreover , paused Pol II is relatively stable , as evidenced from half-life measurements at ∼3200 genes .", "Finally , tracking the progression of Pol II after drug treatment establishes Pol II elongation rates at over 1000 genes .", "Notably , Pol II accelerates dramatically while transcribing through genes , but slows at exons .", "Furthermore , intergenic variance in elongation rates is substantial , and is influenced by a positive effect of H3K79me2 and negative effects of exon density and CG content within genes ." ]

[ "Many different factors determine how quickly the DNA in genes is transcribed to produce molecules of messenger RNA .", "The start of the transcription process features two milestones: first , an enzyme called RNA Polymerase II starts the process; shortly afterwards , however , the process pauses and only starts again when other proteins are recruited .", "This provides two levels of control over the production of messenger RNA and , it also allows the transcription process to be interrupted in order to study the rate of transcription .", "Here , Jonkers , Kwak and Lis used two drugs to block either the start of transcription or the release from the paused state in mouse cells .", "Both drugs prevented new transcription and disrupted about 95% of the total number of genes .", "However , RNA Polymerase II that was already copying DNA could continue to copy , and did so at an average rate of 2000 bases per minute .", "Transcription rates were , however , shown to vary between different genes—highly active genes are transcribed faster .", "Transcription rates also varied within individual genes , with the enzyme accelerating as it moves along the gene .", "This suggests that the transcription machinery , including other proteins that improve the enzyme’s efficiency , are recruited or modified after transcription has already started , and that these proteins help the enzyme to reach its maximum transcription speed .", "Other factors also affected the transcription rate: the genetic code is written in four letters—A , C , G and T—and genes that contained more Cs and Gs were transcribed slower than those with lots of As and Ts .", "Genes also contain regions called exons that code for proteins , and regions called introns that do not: Jonkers , Kwak and Lis found that genes with lots of exons were transcribed slower .", "Furthermore , DNA is wrapped around proteins into a compacted structure , and genes that had certain chemical markings added to these proteins were transcribed faster .", "The work of Jonkers , Kwak and Lis is the first in-depth look at how transcription is affected by gene structure , and leads the way to uncovering how transcription rates throughout genes are regulated to influence production of messenger RNA ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "neuroscience" ]

[ [ "Bilaterian animals have two major photoreceptor cell-types , the rhabdomeric- and the ciliary-type photoreceptor cells ( rPRC and cPRC , respectively ) ( Arendt , 2003; Arendt et al . , 2004; Eakin , 1979; Erclik et al . , 2009 ) .", "These cells have distinct morphologies and express different classes of opsins ( light-sensitive proteins ) .", "Rhabdomeric PRCs have apical microvillar specializations ( rhabdom ) that store the opsin photopigments .", "The visual photoreceptor cells in most protostome eyes , including the compound eyes of arthropods , the pigment-cup eyes of annelids and the camera or stalk eyes of mollusks , are rhabdomeric and express rhadomeric ( r- ) opsins ( Arendt et al . , 2002; Cowman et al . , 1986; Katagiri et al . , 2001; Katagiri et al . , 1995; Ovchinnikov et al . , 1988; Pollock and Benzer , 1988; Randel et al . , 2013 ) .", "Rhabdomeric PRCs also exist in the pigmented eyespots of both protostomes ( e . g . annelids , flatworms ) and some non-vertebrate deuterostomes ( hemichordates , cephalochordates ) ( Arendt and Wittbrodt , 2001; Braun et al . , 2015; Nakao , 1964 ) .", "In contrast , the visual eyes of vertebrates have cPRCs ( rods and cones ) where the ciliary ( c- ) opsin photopigment is stored in specialized ciliary membrane compartments ( Jan and Revel , 1974; Nir et al . , 1984 ) .", "Ciliary PRCs also occur in some invertebrates , where they can be part of pigmented eyespots ( e . g . some mollusks and flatworms ) , in simple eyes ( e . g . some nemerteans ) , or as brain photoreceptors not associated with pigment cells ( e . g . some annelids ) ( Arendt et al . , 2004; Barber et al . , 1967; Döhren and Bartolomaeus , 2018; Randel and Jékely , 2016 ) .", "The class of opsin expressed in a PRC generally correlates with the cell’s morphological type , with cPRCs usually expressing c-opsins and rPRCs expressing r-opsins ( Arendt , 2003; Arendt et al . , 2004; Randel et al . , 2013; Vopalensky et al . , 2012 ) .", "However some exceptions to this rule are known , such as the occasional coexpression of melanopsin ( an r-opsin ) with c-opsins or r-opsins with Go- or xenopsins ( Davies et al . , 2011; Gühmann et al . , 2015; Vöcking et al . , 2017 ) .", "Given their broad phylogenetic distribution and shared opsin expression , both photoreceptor cell types likely coexisted in the last common ancestor of bilaterians .", "The two cell types still coexist in several marine animals , including cephalochordates , some mollusks , flatworms , and annelids ( Arendt et al . , 2004; McReynolds and Gorman , 1970; Randel and Jékely , 2016; Vopalensky et al . , 2012 ) and form parts of pigmented or non-pigmented light-sensitive structures .", "Understanding how the two photoreceptor cell types integrate at the functional and circuit levels in these animals will help to clarify the history of eyes and photoreceptor cells .", "Here we study the planktonic larva of Platynereis dumerilii , a marine annelid that has both photoreceptor cell types .", "In Platynereis , non-pigmented brain cPRCs with ramified cilia express a ciliary type opsin ( c-opsin1 ) ( Arendt et al . , 2004 ) and coexist with r-opsin-expressing rPRCs that are part of the pigmented visual eyes ( adult eyes ) and eyespots ( Arendt et al . , 2002; Jékely et al . , 2008; Randel et al . , 2014 , 2013 ) .", "The pigmented larval eyespots and the adult eyes mediate early- and late-stage larval phototaxis , respectively ( Gühmann et al . , 2015; Jékely et al . , 2008; Randel et al . , 2014 ) .", "The mechanism and neuronal circuitry of both early- and late-stage larval phototaxis is well understood .", "Trochophore larvae ( approximately 1–2 . 5 days post fertilization ) have a pair of pigmented eyespots with a rPRC that directly innervates the adjacent ciliary band .", "This rPRC is cholinergic and expresses r-opsin3 ( Jékely et al . , 2008; Randel et al . , 2013 ) .", "When the rPRC is activated during helical swimming , the ciliary beating changes on the illuminated side , so that the larva reorients its trajectory towards the light source .", "Nectochaete larvae ( approximately 3–5 days post fertilization ) develop two pairs of adult eyes with several rPRCs coexpressing r-opsin1 , r-opsin3 and Go-opsin ( Gühmann et al . , 2015; Randel et al . , 2013 ) .", "These eyes mediate visual phototaxis by comparing the intensity of light on the two sides of the body .", "The adult eyes and their downstream neuronal circuitry regulate the contraction of the longitudinal muscles of the trunk to steer the larva towards or away from a light source ( Randel et al . , 2014 ) .", "The function of cPRCs in Platynereis is much less clear .", "These and the surrounding cells ( the ‘cPRC region’ ) have been proposed to produce melatonin and to entrain the circadian clock ( Arendt et al . , 2004; Tosches et al . , 2014 ) .", "C-opsin1 was recently shown to absorb UV light .", "This suggests that the c-opsin1-expressing cPRCs mediate circadian entrainment by ambient UV light ( Tsukamoto et al . , 2017 ) .", "However , the precise function of the cPRCs in Platynereis larvae and how they interact with rPRCs is still unknown ." ], [ "To identify synaptic connections between the cPRC and rPRC circuits , we used a serial-section transmission electron microscopy ( ssTEM ) dataset spanning the entire body of a 72 hr post fertilization ( hpf ) Platynereis larva ( Randel et al . , 2015 ) .", "Previously , we reported the synaptic connectome of the rPRCs from the visual eyes and eyespots , ( Randel et al . , 2015 , 2014 ) and the direct postsynaptic circuit of the four cPRCs with ramified cilia ( Williams et al . , 2017a ) .", "Briefly , the glutamatergic rPRCs of the adult eyes connect through three layers of interneurons to cholinergic motoneurons in the ventral head ( vMN ) that innervate longitudinal trunk muscles .", "The main premotor interneurons in this circuit are the Schnörkel interneurons ( INsn ) ( Figure 1C , G ) ( Randel et al . , 2015; Randel et al . , 2014 ) .", "The rPRCs of the eyespot synapse directly on the ciliary band and the vMNs ( Figure 1G ) ( Jékely et al . , 2008; Randel et al . , 2015 , 2013 ) .", "The four cholinergic cPRCs send axons to the neurosecretory plexus in the anterior nervous system and synapse on four peptidergic/cholinergic RGW interneurons ( labelled INRGW , and named after the expression of the RGWamide neuropeptide [Shahidi et al . , 2015; Williams et al . , 2017] ) and four NOS interneurons ( labelled INNOS , and named after the expression of the nitric oxide synthase ( NOS ) gene; unpublished ) .", "The cPRCs also synapse on a group of flask-shaped sensory-neurosecretory neurons that are part of the neurosecretory anterior nervous system .", "These cells express diverse neuropeptides but have no postsynaptic partners ( Williams et al . , 2017 ) .", "The RGW cells synapse on two serotonergic cells ( Ser-h1 ) that , together with their postsynaptic partner , the cholinergic MC neuron , are part of the ciliomotor circuitry of the larva ( Verasztó et al . , 2017 ) .", "To analyze the possible synaptic integration of the rPRC and cPRC circuits , we searched for all synaptic connections between neurons of these circuits ( Figure 1 ) in a synapse-level skeleton reconstruction of all cells in the larval head ( 2359 cells of which approximately 1230 are neurons; unpublished data ) .", "We did a systematic network search in Catmaid ( Schneider-Mizell et al . , 2016 ) for all possible synaptic paths ( two hops ) between the rPRC or the cPRC circuit .", "We identified three sites of contact .", "First , the RGW interneurons synapse on the Schnörkel premotor interneurons ( INsn ) ( Figure 1E–G and Video 1 ) .", "Second , we identified six interneurons ( INpreMN ) that are postsynaptic to the RGW interneurons and presynaptic to the ventral motoneurons ( vMNs ) of the visual circuit ( Figure 1E–G ) .", "Third , two putative mechanosensory neurons bearing a sensory cilium and a collar of microvilli and located in the median head ( MS1 and MS2 ) ( Bezares-Calderon et al . , 2018 ) are postsynaptic to the RGW cells and presynaptic to the vMNs and INpro interneurons of the visual circuit ( Figure 1E–G and Video 1 ) .", "Our graph search did not reveal any neurons that were directly postsynaptic to the rPRC circuit ( from rPRC to vMN ) and presynaptic to any neuron of the cPRC circuit .", "Thus , the cPRC circuit feeds hierarchically into the visual rPRC circuit ( Figure 1G ) .", "This suggests that the cPRCs could influence phototaxis , a behavior mediated by the rhabdomeric eyes and eyespots ( Gühmann et al . , 2015; Jékely et al . , 2008; Randel et al . , 2014 ) .", "What is the role of cPRCs in larval behavior and how do they influence phototaxis ?", "First , to estimate the light sensitivity of the cPRCs , we reconstructed the morphology with ssTEM and measured the total sensory membrane surface-area of a cPRC ( Figure 2A–D ) .", "Each cPRC has 12–15 basal bodies each with a root and an extensively branched sensory cilium .", "The branches are supported by single microtubule doublets ( Figure 2—figure supplement 3 ) .", "Based on the average diameter and total length of all branches in one cPRC we estimated a membrane area of 276 μm2 , which is approximately 10 times smaller than the total disk membrane surface area of rat rods ( Mayhew and Astle , 1997 ) .", "This suggests that Platynereis cPRCs are sensitive enough to mediate acute light sensation .", "Next , we expressed Platynereis c-opsin1 in COS1 cells , reconstituted it with 11-cis-retinal and purified it .", "The reconstituted pigment absorbed in the UV range with a λ-max of 384 nm in the dark spectrum and a λ-max of 370 nm in the dark-light difference spectrum ( Figure 2E ) , in agreement with a recent report ( Tsukamoto et al . , 2017 ) .", "To investigate how the cPRCs respond to light , we did calcium imaging with larvae ubiquitously expressing the calcium sensor GCaMP6s ( Chen et al . , 2013; Verasztó et al . , 2017 ) .", "When imaged with a low-intensity 488 nm laser , the cPRCs had a high resting calcium level and the GCaMP6s signal highlighted their sensory cilia .", "We could thus identify the four cPRCs without stimulation ( Figure 2F ) .", "When the cPRC cilia were locally stimulated ( Figure 2—figure supplement 1 ) for 5 min with 405 nm light ( 140–250 times more photons on the sensory cilia than by the imaging laser ) , the calcium level dropped transiently at the cPRC somata and then increased strongly .", "This indicates transient cPRC hyperpolarization followed by depolarization .", "However , when the cPRCs were stimulated with a 488 nm laser of equal photon flux , they showed prolonged hyperpolarization only , without depolarization ( Figure 2G ) .", "When the 405 nm stimulation was switched off during the hyperpolarization phase ( after 20 s ) , the cPRCs still depolarized afterwards ( Figure 2H ) .", "405 nm stimulation of the cPRCs also changed the activity of other neurons in the larval brain .", "Four neurons followed the activity pattern of the cPRCs ( Figure 2J; Figure 2—figure supplement 2 ) .", "These cells correspond by position to the four RGW interneurons , which together with the four NOS interneurons are direct postsynaptic targets of the cPRCs ( Figure 1G ) ( Williams et al . , 2017a ) .", "Additionally , two flask-shaped sensory neurons ( SNearly ) in the middle of the anterior nervous system depolarized upon stimulus onset .", "Two further sensory cells ( SNlate ) flanking the SNearly cells depolarized later , in parallel with the rising phase of cPRC activity ( Figure 2I ) .", "These four SN cells correspond by position to four sensory-neurosecretory neurons that are postsynaptic to the cPRCs in the anterior nervous system ( SNMIP1l , SNASTC1r and two SNNS20 cells ) ( Figure 1D ) ( Williams et al . , 2017a ) .", "We next compared the responses of the RGW interneurons and the four SN neurons to 405 and 488 nm stimulation of the cPRCs .", "The RGW interneurons and the SNearly sensory neurons only responded to 405 nm stimulation ( with opposite sign ) but not to 488 nm stimulation .", "The SNlate neurons in some larvae also responded to 488 nm stimulation but these responses were weaker ( Figure 2—figure supplement 2 ) .", "Thus , the cPRCs and their postsynaptic neurons respond differentially to violet and blue stimulation , with only violet light inducing cPRC depolarization and consistent changes in the activity of postsynaptic neurons .", "To characterize how Platynereis larvae react to UV-violet light , we assayed larval swimming behavior in a vertical column setup .", "Since Platynereis larvae show strong directional phototaxis to a broad spectrum of light ( between 380–540 nm ) ( Gühmann et al . , 2015; Jékely et al . , 2008 ) , we illuminated the setup equally from two opposite sides with non-directional UV light so that the larvae could not respond with directional phototaxis ( Figure 3A ) .", "When the larvae were stimulated with non-directional UV light , they started to swim downward .", "To characterize the wavelength dependence of this behavior , we assayed larvae in a vertical cuvette and stimulated them with monochromatic light of different wavelengths from two sides .", "The larvae swam down to UV-violet light ( between 340–420 nm ) but not to longer wavelengths ( >420 nm , Figure 3B ) .", "This downward swimming UV-avoidance behavior to non-directional UV-violet light has not been previously reported in Platynereis .", "The observations that UV-avoidance can be triggered by non-directional light and has an action spectrum that closely matches the absorption spectrum of c-opsin1 ( Figure 2E ) suggest that the response is mediated by the non-pigmented cPRCs .", "If the cPRCs indeed mediate UV-avoidance , then the developmental onset of this response should correlate with the morphological differentiation of cPRCs .", "To test this , we assayed UV-avoidance ( 395 nm light from the side ) as well as phototaxis ( 480 nm light from the top ) at different larval stages ( Figure 3C ) and correlated the behaviors to photoreceptor differentiation .", "Phototaxis , but not UV-avoidance was already present at 27 hpf , at a stage when the larval eyespots are already functional ( Jékely et al . , 2008 ) .", "UV avoidance appeared at 36 hpf , approximately coinciding with the morphological differentiation of cPRCs ( after 32 hpf , as judged by the appearance of the ramified cilia ) ( Figure 3C ) , but long before the differentiation of the adult eyes ( at 72 hpf ) ( Fischer et al . , 2010; Jékely et al . , 2008; Randel et al . , 2014 ) .", "Thus , Platynereis larvae show UV-violet-light avoidance that is independent of phototaxis , can be induced by non-directional stimulus light , and is likely mediated by the UV-violet-responding non-pigmented cPRCs .", "To study how UV avoidance interacts with rhabdomeric-eye-mediated phototaxis , we stimulated the larvae in the vertical column with directional monochromatic light from above .", "When we used 380 nm stimulus light , both early- ( 41 hpf ) and late-stage ( 3 and 4 . 5 dpf ) larvae first swam upward towards the light ( for approximately one minute ) , and then swam downward ( Figure 3D , Video 2 ) .", "This upward phase was not observed when the larvae were illuminated uniformly from the side ( data not shown ) .", "These results indicate that the upward-swimming phase is phototaxis , which is then overwritten by the UV-avoidance response .", "To test the wavelength-dependence of this effect , we measured larval responses to different wavelengths of directional light coming from the top of the vertical column .", "We plotted responses 1 . 5–3 . 5 min after stimulus onset to focus on the phase when UV-avoidance has potentially overwritten phototaxis ( Figure 3D ) .", "In response to illumination with 340–400 nm light , larvae swam downward after prolonged stimulation .", "In response to illumination with 440–540 nm light ( early-stage ) or 440–600 nm light ( late-stage ) , larvae swam upward ( Figure 3E ) .", "420 nm light did not trigger the vertical displacement of the larvae .", "The swimming direction could be switched several times by changing the wavelength of the light from 380 nm to 520 nm ( Figure 3F ) , demonstrating that this behavioral switch does not habituate even after sustained exposure to light .", "These results indicate that directional UV-violet light first triggers upward-swimming phototaxis ( the pigmented eyes are sensitive in the UV-violet range [Jékely et al . , 2008; Gühmann et al . , 2015] ) , which is then overridden by the downward-swimming UV-avoidance response likely mediated by the cPRCs .", "Directional blue/cyan light only triggers phototaxis .", "Importantly , the switching in behavioral response cannot be explained as a wavelength-dependent alternation between positive and negative phototaxis , since early-stage larvae are exclusively positively phototactic ( Jékely et al . , 2008 ) ( until 72 hpf [Randel et al . , 2014] ) , yet already show the behavioral switch in swimming direction ( Figure 3E , F ) .", "Next we asked if the antagonistic phototaxis and UV-avoidance behaviors could form a potential mechanism to measure depth by the larvae .", "Since blue and UV-violet light attenuate differently in seawater , the ratio of blue to UV-violet light increases with depth ( field data from the Mediterranean , where our Platynereis strain comes from , were shown before [Gühmann et al . , 2015] ) .", "Strong UV-violet light at the ocean’s surface is expected to cause larvae to swim downward as an avoidance response , and relatively strong blue light in deeper waters is expected to trigger phototactic upward swimming .", "Such depth-dependent behavioral switching could form a ratio-chromatic depth-gauge ( Nilsson , 2009 ) .", "To test this , we exposed larvae to mixed wavelength light containing different photon ratios of UV ( 380 nm ) and blue ( 480 nm ) light coming from the top of the vertical column .", "At high 380/480 ratios , larvae swam downward , while at low ratios larvae swam upward .", "At a 40% 380/480 ratio , larvae remained at a constant average depth ( Figure 3G ) , despite being exposed to a directional light stimulus .", "Mixing the same intensity UV light as before with 660 nm red light ( a wavelength to which larvae do not respond phototactically at the intensity used ( Figure 3E ) ) did not induce upward swimming at any 380/660 ratio ( Figure 3G ) .", "This experiment shows that a reduction in UV intensity alone does not cause a switch in swimming direction .", "These results suggest that UV avoidance and phototaxis act antagonistically and cancel each other out under certain wavelength ratios , resulting in no net vertical swimming .", "This is consistent with the presence of a ratio-chromatic depth gauge in Platynereis larvae .", "To test whether c-opsin1 mediates the UV-violet response in Platynereis larvae , we used a c-opsin1 Platynereis knockout line generated by TALEN-mediated genome editing .", "The TALENs targeted the third exon of c-opsin1 and induced an 8 bp deletion ( Figure 4A ) .", "In homozygous c-opsin1Δ8/Δ8 larvae , the cPRCs had low resting calcium level and neither hyperpolarized nor depolarized upon 405 nm stimulation ( Figure 4B , C ) .", "Thus , c-opsin1 in the Platynereis cPRCs is required for an elevated resting calcium level in the dark state and for hyperpolarization and subsequent depolarization upon 405 nm exposure .", "The high resting calcium level in the cPRCs indicates a dark current , a characteristic of vertebrate rods and cones ( Hagins et al . , 1970 ) .", "Next , we tested behavioral responses to light in c-opsin1Δ8 mutant larvae .", "Similar to wild type larvae , homozygous c-opsin1Δ8/Δ8 larvae swim upward ( positive phototaxis ) in response to 480 nm light .", "However , c-opsin1Δ8/Δ8 larvae have a defective UV-avoidance response .", "Whereas wild type larvae swim downward after an initial upward-swimming phase , c-opsin1Δ8/Δ8 larvae continue to swim upward , showing sustained positive phototaxis in response to UV light ( Figure 4D ) .", "The magnitude of upward swimming diminishes , with close to 0 vertical displacement between 1 . 5–2 min after UV onset .", "This indicates that an additional , c-opsin1-independent UV-avoidance mechanism may exist in Platynereis larvae ( cf . the last bin under 480 and 395 nm stimulation ) .", "These results show that c-opsin1 is a critical mediator of UV-avoidance .", "The loss of c-opsin1 is expected to disrupt the depth gauge since in c-opsin1 mutants both cyan and UV light induce sustained positive phototactic behavior which is likely mediated by the pigmented eyes with a broad spectral sensitivity ( 380–540 nm [Jékely et al . , 2008] ) ." ], [ "Our results are consistent with the presence of a ratio-chromatic depth-gauge in the planktonic larvae of Platynereis dumerilii .", "The depth gauge is formed by the antagonistic interaction of two distinct behaviors , mediated by two distinct types of photoreceptor systems with different spectral sensitivities .", "The rPRCs mediate phototaxis to a broad range of wavelengths between UV and green light , while the cPRCs mediate UV-violet avoidance .", "Under UV-violet light , after approximately 30 s the UV-violet avoidance response overrides an initial phototactic response .", "At a set UV/blue ratio , the two opposing behaviors cancel each other out .", "Due to the differential attenuation of UV and blue light in seawater , this mechanism could allow the larvae to stay at a certain depth in the ocean .", "While the function of the rPRCs in the larval and adult eyes has previously been described ( Jékely et al . , 2008; Randel et al . , 2014 ) , here we defined , both physiologically and behaviorally , the function of the cPRCs and unraveled how they interact with the rPRC system .", "The cPRCs express a UV-absorbing opsin , have a high resting calcium level and react by hyperpolarization and subsequent depolarization to violet light ( 405 nm ) .", "The cPRCs mediate an UV-violet avoidance response characterized by downward swimming .", "The action spectrum of this behavior matches the absorption spectrum of c-opsin1 and the response is severely impaired in c-opsin1 knockouts , demonstrating a critical role for c-opsin1 in mediating it .", "The depolarizing response in the cPRCs shows a similar delayed onset as UV-violet avoidance , suggesting that the behavior is due to cPRC depolarization .", "Intriguingly , the depolarization also occurs if the stimulus light is switched off during the hyperpolarizing phase .", "This implies that cPRC depolarization is not due to direct opsin activation but to a sustained cell-autonomous or non-autonomous signal .", "The activation of cPRCs also induces complex differential responses in postsynaptic neurons .", "The RGW cells follow the activity of cPRCs under violet but not blue illumination , whereas two groups of sensory neurons respond to violet light only ( SNearly ) or to both violet and , to a lesser extent , blue light ( SNlate ) .", "This implies that different postsynaptic mechanisms operate in the different cPRC targets .", "The high calcium level in the cPRCs suggests the possibility of the tonic release of a neurotransmitter ( probably acetylcholine ) in the dark , similar to the tonic release of glutamate by vertebrate photoreceptors ( Heidelberger et al . , 2005 ) .", "Blue illumination ( 488 nm ) also induces cPRC hyperpolarization , but no subsequent depolarization .", "This response may be mediated by another opsin expressed in the cPRCs .", "One candidate is c-opsin2 with a maximal absorption at 490 nm ( Ayers et al . , 2018 ) .", "One surprising observation is that cPRC hyperpolarization induced by violet or blue light has different downstream consequences .", "This may be due to the engagement of different signaling cascades by the different opsins .", "How cPRC activation and the postsynaptic responses lead to downward swimming during UV-avoidance is unclear .", "The cPRC circuit connects to the Ser-h1 serotonergic ciliomotor neurons ( Verasztó et al . , 2017 ) , suggesting that the modulation of ciliary activity may contribute to UV avoidance .", "In addition , we have genetic evidence implicating neuroendocrine volume transmission in UV avoidance .", "The cPRCs and their direct postsynaptic targets are part of the neurosecretory brain center of the larva ( Williams et al . , 2017a ) , and knocking out one neuroendocrine signaling component leads to strong defects in UV avoidance ( unpublished results ) .", "It also remains to be elucidated how the UV response overrides phototaxis at the neuronal level .", "We found that the cPRC circuit connects to the circuit of the rhabdomeric eyes via the cholinergic RGW interneurons and their distinct downstream synaptic pathways .", "The RGW neurons provide inputs to the phototactic circuit at the level of the INsn , INpro and the vMN cells .", "The strongest and most direct connection between the cPRC and rPRC circuits is through synapses from the RGW cells to the INsn neurons .", "Cholinergic input from the RGW cells to these premotor interneurons may antagonize phototaxis .", "Other sensory inputs may also tune phototaxis via the RGW-INsn pathway since neuropeptides derived from sensory-neurosecretory cells can influence RGW neuron activity ( Williams et al . , 2017 ) .", "The RGW neurons also connect to the putatively mechanosensory MS cells which strongly synapse on the vMNs .", "UV light may thus tune mechanosensation that may in turn modulate phototaxis .", "One outstanding question in eye evolution is why did the ciliary and rhabdomeric PRC types originally evolve ( Nilsson , 2013; 2009 ) ?", "Our findings suggest that the two types may have evolved antagonistic functions early in evolution .", "Invertebrates then elaborated on the rhabdomeric , and vertebrates on the ciliary type ( Arendt et al . , 2004 ) .", "According to one hypothesis , as the brain cPRCs were recruited for vision in the vertebrate lineage , the rPRCs evolved into the retinal ganglion cells ( Arendt , 2008; Arendt , 2003 ) .", "This scenario is supported by the expression of melanopsin , an r-opsin ortholog , in the intrinsically photosensitive retinal ganglion cells ( Hattar et al . , 2003; 2002; Koyanagi et al . , 2005; Lucas et al . , 2001; Panda et al . , 2003 ) .", "Although we need more comparative data to test this model , we hypothesize that the cell-type mosaic of the vertebrate retina may have originated from the hierarchical integration of distinct cPRC and rPRC circuits mediating antagonistic behaviors , as observed in Platynereis larvae ." ], [ "Larvae of Platynereis dumerilii were cultured at 18°C in a 16 hr light 8 hr dark cycle until experiments .", "Larvae were raised to sexual maturity according to established breeding procedures ( Fischer and Dorresteijn , 2004; Hauenschild and Fischer , 1969 ) .", "We used serial-sectioning transmission electron microscopy to analyze cPRC sensory morphology ( Randel et al . , 2015 ) .", "Each cPRC has 12–15 basal bodies , each basal body gives rise to one cilium that branches close to its base to 3–9 branches .", "Each branch is supported by at least one microtubule doublet .", "The branches have an average diameter of 130 nm ( st . dev . 19 nm ) and a total length of 677 μm ( measured in one cPRC ) .", "This represents a total membrane surface area of approximately 276 μm2 .", "Recombinant c-opsin1 was purified and analyzed following ( Yokoyama , 2000 ) .", "Full-length Platynereis c-opsin1 was amplified with primers that introduced EcoRI , Kozak and SalI sequences , and cloned into the expression plasmid pMT5 .", "C-opsin1 was expressed in COS1 cells and incubated with 11-cis-retinal ( a gift from Dr . Rosalie K . Crouch at Storm Eye Institute ) to regenerate the photopigment .", "The pigment was purified with immobilized 1D4 ( The Culture Center , Minneapolis , MN ) in buffer W1 ( 50 mM N- ( 2-hydroxyethyl ) piperazine-N’−2-ethanesulfonic acid ( HEPES ) ( pH 6 . 6 ) , 140 mM NaCl , 3 mM MgCl2 , 20% ( w/v ) glycerol and 0 . 1% dodecyl maltoside ) .", "The UV/VIS spectrum of the pigment was recorded at 20°C with a Hitachi U-3000 dual beam spectrophotometer .", "The pigment was bleached for 3 min with a 60 W standard light bulb equipped with a Kodak Wratten #3 filter at a distance of 20 cm .", "COS1 cells ( ATCC CRL1650 ) , established from the kidney cells of the African green monkey ( Cercopithecus aethiops ) , were authenticated by the American Type Culture Collection ( Manassas , VA ) with the COI assay .", "The mycoplasma contamination test was negative .", "We used a variety of light sources for photostimulation , depending on the experimental setup .", "In the calcium imaging experiments , we used the laser lines available in our Olympus FV1200 confocal microscope ( 405 and 488 nm; Showa Optronics , Tokyo ) .", "The lasers were operating in continuous mode .", "The power of the lasers was measured with a microscope slide power sensor ( S170C; Thorlabs , Newton , USA ) .", "The typical power for stimulation was 5 . 59 µwatts for the 405 nm laser and 4 . 62 µwatts for the 488 nm laser .", "These values correspond to 1 . 1e + 13 photons/sec .", "For behavioral assays , we used either UV LEDs ( 395 nm peak wavelength ) or a monochromator ( Polychrome II , Till Photonics ) for which we quantified photon irradiances across the spectrum ( 3−4 × 1018 photons/sec/m2 ) ( Gühmann et al . , 2015 ) .", "We refer to color according to these wavelength ranges: UV <400 nm , violet 400–450 nm , blue 450–490 nm ( 450–460 royal blue ) , cyan 490–520 nm .", "For calcium imaging , 36–52 hpf larvae were used .", "Experiments were conducted at room temperature in filtered natural seawater .", "Larvae were immobilized between a slide and a coverslip spaced with adhesive tape .", "GCaMP6s mRNA ( 1 mg/μl ) was injected into zygotes as described previously ( Randel et al . , 2014 ) .", "Larvae were imaged on an Olympus FV1200 microscope ( with a UPLSAPO 60X water-immersion objective , NA 1 . 2 ) with a frame rate of 1 . 25/sec and an image size of 254 × 254 pixels .", "The larvae were stimulated in a region of interest ( a circle with 18–24 pixel diameter ) with continuous 405 nm or 488 nm lasers controlled by the SIM scanner of the Olympus FV12000 confocal microscope while scanning .", "The imaging laser had a similar intensity than the stimulus laser but covered an area that was 140–250 times larger than the stimulus ROI .", "The calcium-imaging movies were analyzed with Fiji ( Schindelin et al . , 2012 ) ( RRID:SCR_002285 ) and a custom Python script , as described previously ( Gühmann et al . , 2015 ) , with the following modifications .", "The movies were motion-corrected in Fiji with moco ( Dubbs et al . , 2016 ) and Descriptor-based registration ( https://github . com/fiji/Descriptor_based_registration ) .", "The data are shown as ΔF/F0 .", "For the calculation of the normalized ΔF/F0 with a time-dependent baseline , F0 was set as the average of an area of the brain with no calcium activity to normalize for the additional lasers ( 405 nm and 488 nm ) and potential artefacts from the microscope's detector .", "Spatial correlation analyses of neuron activities were done in Fiji and Python as previously described ( Verasztó et al . , 2017 ) .", "The ROI was manually defined and was correlated with every pixel of the time series .", "Finally , a single image was created with the Pearson correlation coefficients and a [−1 , 1] heatmap plot with two colors .", "Scripts are available at ( Gühmann and Verasztó , 2018; copy archived at https://github . com/elifesciences-publications/Veraszto_et_al_2018 ) .", "Photoresponses of larvae of different ages were assayed in a vertical Plexiglas column ( 31 mm x 10 mm x 160 mm water height ) .", "The column was illuminated from above with light from a monochromator ( Polychrome II , Till Photonics ) .", "The monochromator was controlled by AxioVision 4 . 8 . 2 . 0 ( Carl Zeiss MicroImaging GmbH ) via analog voltage or by a custom written Java program via the serial port .", "The light passed a collimator lens ( LAG-65 . 0–53 . 0 C with MgF2 Coating , CVI Melles Griot ) .", "The column was illuminated from both sides with light-emitting diodes ( LEDs ) .", "The LEDs on each side were grouped into two strips .", "One strip contained UV ( 395 nm ) LEDs ( SMB1W-395 , Roithner Lasertechnik ) and the other infrared ( 810 nm ) LEDs ( SMB1W-810NR-I , Roithner Lasertechnik ) .", "The UV LEDs were run at 4 V to stimulate the larvae in the column from the side .", "The infrared LEDs were run at 8 V ( overvoltage ) to illuminate the larvae for the camera ( DMK 22BUC03 , The Imaging Source ) , which recorded videos at 15 frames per second and was controlled by IC Capture ( The Imaging Source ) .", "27-hour-old , 36-hour-old , 2-day-old , and 3-day-old Platynereis dumerilii larvae were stimulated in the column with UV ( 395 nm ) light from the LEDs on the side .", "Afterwards , the larvae were stimulated with monochromatic blue ( 480 nm ) light coming from above to assay for phototaxis .", "Each stimulus lasted 4 min .", "The LEDs were controlled manually and the monochromator ( Polychrome II , Till Photonics ) was controlled via AxioVision .", "To compare the behavior of wildtype and c-opsin1-knockout larvae in the vertical column , we tested individual batches of larvae .", "We distributed the larvae in the vertical column by mixing and dark adapted them for 5 min .", "The larvae were recorded for 1 min in the dark followed by exposure to collimated blue ( 480 nm ) light from the top of the column for 2 min , then 2 min darkness , and finally collimated UV ( 395 nm ) light from above for 2 min .", "Stimulus light was provided by a monochromator ( Polychrome II , Till Photonics ) .", "Early and late-stage Platynereis dumerilii larvae were assayed in the vertical columns .", "The larvae were stimulated six times alternatively with UV ( 380 nm ) and green ( 520 nm ) light .", "Each stimulus lasted 4 . 5 min .", "The last 3 min within each stimulus were analyzed for vertical displacement of the larvae .", "The light was provided by a monochromator ( Polychrome II , Till Photonics ) , which was controlled by AxioVision .", "2-day-old and 3-day-old Platynereis dumerilii larvae were assayed in vertical columns .", "The larvae were stimulated with monochromatic light from above between 340 nm and 480 nm in 20 nm steps .", "Between the stimuli , additional 520 nm stimuli were introduced to avoid the accumulation of the larvae at the bottom after UV treatment .", "The larvae were also stimulated with monochromatic light from above between 400 nm and 680 nm in 20 nm steps .", "Between these stimuli , additional 400 nm stimuli were introduced to avoid the accumulation of the larvae at the top due to phototaxis .", "Each stimulus lasted 3 . 5 min .", "The last 2 min of each stimulus were analyzed for vertical displacement of the larvae .", "The light was provided by a monochromator ( Polychrome II , Till Photonics ) , which was controlled by AxioVision .", "3-day-old Platynereis dumerilii larvae were stimulated with UV-blue ( 380 nm , 480 nm ) or UV-red ( 380 nm , 660 nm ) mixed light from above .", "The larvae were mixed to be uniformly distributed in the column and dark adapted for 5 min .", "In each step , 10% UV-light was replaced by blue or red light .", "Each step was followed by a blue ( 480 nm ) light stimulus to avoid the accumulation of the larvae at the bottom after UV treatment .", "Different ratios were created by rapidly switching between wavelengths within 500 ms periods ( e . g . , for a 10% UV 90% blue ratio we provided UV-light for 50 ms followed by blue light for 450 ms ) .", "Each stimulus condition lasted 4 min .", "The monochromator was controlled by a custom Java program via the serial port .", "2-day-old Platynereis dumerilii larvae were assayed in a vertical cuvette of 10 mm x 10 mm x 42 mm ( L x W x H ) .", "The larvae were kept at 18°C and had been exposed to daylight before the experiment .", "The larvae were illuminated with a monochromator ( Polychrome II , Till Photonics ) from one side of the cuvette .", "A mirror ( PFSQ20-03-F01 , Thorlabs ) placed at the opposite side reflected the light to provide near-uniform bilateral illumination .", "The light passed a diffuser ( Ø1’ 20° Circle Pattern Diffuser , ED1-C20; Thorlabs ) and a collimating lens ( LAG-65 . 0–53 . 0 C with MgF2 Coating , CVI Melles Griot ) before it hit the cuvette .", "The cuvette was illuminated with infrared ( 850 nm ) light-emitting diodes ( LEDs ) ( L2 × 2-I3CA , Roithner Lasertechnik ) from the side .", "The LEDs were run at 6 . 0 V . The larvae were stimulated with light from 340 nm to 560 nm in 20 nm steps .", "Each step lasted 1 min .", "The steps were separated by 1 min darkness , so that the larvae could redistribute after each stimulus .", "The larvae were recorded at 16 frames per second with a DMK 23GP031 camera ( The Imaging Source ) controlled by IC Capture .", "The camera was equipped with a macro objective ( Macro Zoomatar 1:4/50–125 mm , Zoomar Muenchen ) .", "It was mounted with a close-up lens ( +2 52 mm , Dörr close-up lens set 368052 ) .", "The larvae were tracked and their vertical displacement was analyzed during the last 45 s of each stimulus period .", "Scripts are available at ( Gühmann and Verasztó , 2018 ) .", "The genomic region of c-opsin1 was amplified to screen for putative size-polymorphic alleles or single nucleotide polymorphisms ( SNPs ) from different Platynereis strains ( PIN and VIO strains ) with the following screening primers: cops1_F1/R1 , cops1_F2/R2 , cops1_F3/R3 , cops1_F4/R4 and cops1_F5/R5 ( For a detailed protocol for SNP screening see: ( Bannister et al . , 2014 ) .", "c-opsin1 TALEN pairs were designed in several non-polymorphic exon regions with the TALE-NT prediction tool ( TAL Effector Nucleotide Targeter 2 . 0; https://tale-nt . cac . cornell . edu/ ) ( Doyle et al . , 2012 ) .", "The in silico predictions were done with customized design conditions - 15 left/right Repeat Variable Diresidue ( RVD ) length , 15–25 bp spacer length , G substitute by NN RVD and presence of a restriction enzyme site within the spacer region .", "The predicted c-opsin1 TALENs were constructed in vitro with the Golden Gate assembly protocol ( Golden Gate TAL Effector Kit 2 . 0 , Addgene #1000000024 ) ( Cermak et al . , 2011 ) .", "The final TALEN repeats were cloned to heterodimeric FokI expression plasmids pCS2TAL3-DD for left TALEN array and pCS2TAL3-RR for right TALEN array ( Dahlem et al . , 2012 ) .", "All cloned TALEN plasmids were sequence-verified with the TAL_F1 and TAL_R2 primers .", "c-opsin1 TALEN mRNA for each array was generated by linearizing the corresponding plasmid ( NotI ) and transcribing it in vitro with the mMESSAGE mMACHINE Sp6 kit ( Thermofisher ) .", "200 ng/μl/TALEN mRNA was microinjected into Platynereis zygotes ( Backfisch et al . , 2013 ) and screened for TALEN-induced mutations ( Bannister et al . , 2014 ) with the PCR primers cops1_TAL_R1/cops1_TAL_L2 followed by restriction digest by the BanI enzyme for the TAL_pair3 and MluC1 for the TAL_pair4 .", "The injected embryos were raised to maintain knockout cultures .", "For genotyping of the c-opsin1 locus , genomic DNA was isolated from single larvae , groups of 6–20 larvae , or from the tails of adult worms .", "The DNA was amplified by PCR with the cops1_del8m_F/cops1_com_R or the cops1_ del8_2F/cops1_ del8_2R primers with the dilution protocol of the Phusion Human Specimen Direct PCR Kit ( Thermo Scientific ) .", "The PCR product was sequenced directly with a nested sequencing primer cops1_ nest_seq .", "A mixture of wild-type and deletion alleles in a sample gave double peaks in the sequencing chromatograms , with the relative height of the double peaks reflecting the relative allele ratio in the sample ." ] ]

[ "Ciliary and rhabdomeric photoreceptor cells represent two main lines of photoreceptor-cell evolution in animals .", "The two cell types coexist in some animals , however how these cells functionally integrate is unknown .", "We used connectomics to map synaptic paths between ciliary and rhabdomeric photoreceptors in the planktonic larva of the annelid Platynereis and found that ciliary photoreceptors are presynaptic to the rhabdomeric circuit .", "The behaviors mediated by the ciliary and rhabdomeric cells also interact hierarchically .", "The ciliary photoreceptors are UV-sensitive and mediate downward swimming in non-directional UV light , a behavior absent in ciliary-opsin knockout larvae .", "UV avoidance overrides positive phototaxis mediated by the rhabdomeric eyes such that vertical swimming direction is determined by the ratio of blue/UV light .", "Since this ratio increases with depth , Platynereis larvae may use it as a depth gauge during vertical migration .", "Our results revealed a functional integration of ciliary and rhabdomeric photoreceptor cells in a zooplankton larva ." ]

[ "The animal kingdom contains many different types of eyes , but all share certain features in common .", "All detect light using specialized cells called photoreceptors , of which there are two main kinds: ciliary and rhabdomeric .", "Crustaceans and their relatives , including insects , have rhabdomeric photoreceptors; while animals with backbones , including humans , have ciliary photoreceptors .", "There are also several groups of animals , mostly sea-dwellers , that inherited both types of photoreceptors from their ancestors that lived millions of years ago .", "These include the marine ragworm , Platynereis dumerilii .", "The larvae of Platynereis are free-swimming plankton .", "Each has a transparent brain and six small , pigmented eyes .", "The eyes contain rhabdomeric photoreceptors .", "These enable the larvae to detect and swim towards light sources .", "Yet the larval brain also contains ciliary photoreceptors , the role of which was unknown .", "Verasztó , Gühmann et al . now show that ultraviolet light activates ciliary photoreceptors , whereas cyan , or blue-green , light inhibits them .", "Shining ultraviolet light onto Platynereis larvae makes the larvae swim downwards .", "By contrast , cyan light makes the larvae swim upwards .", "In the ocean , ultraviolet light is most intense near the surface , while cyan light reaches greater depths .", "Ciliary photoreceptors thus help Platynereis to avoid harmful ultraviolet radiation near the surface .", "Though if the larvae swim too deep , cyan light inhibits the ciliary photoreceptors and activates the rhabdomeric pigmented eyes .", "This makes the larvae swim upwards again .", "Using high-powered microscopy , Verasztó , Gühmann et al . confirm that neural circuits containing ciliary photoreceptors exchange messages with circuits containing rhabdomeric photoreceptors .", "This suggests that the two work together to form a depth gauge .", "By enabling the larvae to swim at a preferred depth , the depth gauge influences where the worms end up as adults .", "Its discovery should also stimulate new ideas about the evolution of eyes and photoreceptors ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "medicine", "microbiology and infectious disease" ]

[ [ "Renewed interest in exploring avenues for curing Human Immunodeficiency Virus Type 1 ( HIV-1 ) infection ( Henrich et al . , 2013; Persaud et al . , 2013; Saez-Cirion et al . , 2013; Denton et al . , 2014; Tebas et al . , 2014 ) has resulted in the investigation of interventions to eradicate cells in which HIV-1 persists despite antiretroviral therapy ( ART ) .", "Once HIV-1 has infected a cell and integrated its genome into the cellular DNA , that cell may revert to a resting state , only producing replication competent virions when activated at a later date .", "These cells have been labeled the ‘HIV reservoir’ .", "There is , however , a lack of clarity relating to the cell types that might harbour the ‘reservoir’ , as well as the tissues in which these cells might be located .", "For clarity , we will use the term ‘reservoir’ to describe the population of HIV-1-infected cells that persist during ART and which are the source of rebound viraemia on stopping therapy .", "Current understanding is that the majority of cells comprising this reservoir are CD4 T memory cells of a resting phenotype ( Avettand-Fenoel et al . , 2009; Liszewski et al . , 2009; Eriksson et al . , 2013 ) .", "Many assays have been developed to quantify the HIV-1 reservoir , ranging from simple quantitative PCR ( qPCR ) estimation of cell associated HIV-1 DNA to labour-intensive viral outgrowth assays ( VOA ) ( Siliciano and Siliciano , 2005; Avettand-Fenoel et al . , 2009; Liszewski et al . , 2009; Eriksson et al . , 2013 ) .", "Whereas measurement of plasma viraemia ( ‘viral load’ ) and CD4 T cell count are documented surrogate markers of HIV clinical progression , the clinical relevance and utility of measuring the reservoir—regardless of assay–remains less clear .", "As cell-associated HIV-1 DNA precedes plasma viraemia in the viral life cycle , it is tantalizing to speculate whether measuring HIV-1 DNA ( as a surrogate for reservoir size ) might have significant clinical relevance .", "It is well documented that HIV-1 DNA persists in patients on antiretroviral therapy ( ART ) even when the plasma viral load is undetectable using the most sensitive assays ( Palmer et al . , 2008; Saez-Cirion et al . , 2013 ) .", "Much of this detectable HIV-1 DNA has been found to be mutated and replication-incompetent calling into question its biological relevance ( Ho et al . , 2013 ) .", "However , as a simple surrogate measure of the reservoir it may still have a role to play .", "As new interventions to cure HIV-1 infection are developed and taken into clinical trials , a means to measure their efficacy is needed .", "Stopping ART to await the return of viraemia would be the ‘gold standard’ approach , but has been associated with risk in certain ( Strategies for Management of Antiretroviral Therapy ( SMART ) Study Group et al . , 2006 ) , although not all studies ( SPARTAC Trial Investigators et al . , 2013 ) .", "Ideally , the clinician would have access to an algorithm of biomarker assays to help identify those patients who might ( or , alternatively , should not ) be candidates for a safe treatment interruption ( TI ) .", "The best way to assess the patient successfully managed on ART is unclear but , with the viral load rendered undetectable , it is plausible that HIV-1 DNA might be an alternative biomarker for disease progression .", "For example , compared with individuals with uncontrolled viraemia , HIV-1 DNA levels are much lower in cohorts such as VISCONTI in which apparently persistent aviraemia has been reported following TI ( Saez-Cirion et al . , 2013 ) , and in the case of the Mississippi baby extremely low DNA levels were associated with a prolonged period of virological remission .", "However , this contrasts with cases in which undetectable DNA on ART was associated with prompt rebound viraemia on stopping ( Chun et al . , 2010; Henrich et al . , 2014 ) .", "We therefore wished to gain a broader picture of the utility of measuring HIV-1 DNA levels by studying participants in a large , randomized trial of primary HIV-1 infection .", "We measured both Total and Integrated HIV DNA levels in peripheral blood CD4 T cells in participants in the Short Pulse Antiretroviral Treatment at HIV-1 Seroconversion ( SPARTAC ) trial ( SPARTAC Trial Investigators et al . , 2013 ) –the largest randomized clinical trial of short-course ART in primary HIV-1 infection ( PHI ) .", "Studying individuals recruited at PHI , randomized to no treatment or ART , and who subsequently underwent treatment interruption , allowed us to ask two questions .", "Was HIV-1 DNA independently predictive of clinical progression , and did HIV-1 DNA predict the time taken for viraemic rebound on stopping therapy , advocating its role in future treatment interruption protocols ?" ], [ "154 participants across all the SPARTAC trial arms were studied based on infection with subtype B HIV-1 and sample availability .", "All 154 patients were sampled at the pre-therapy baseline at trial enrolment .", "The demographics of the 154 participants are shown in Table 1 .", "Participants who were randomised to receive no therapy or 48 weeks of ART and for whom samples were available ( n = 51 and n = 47 , respectively; Supplementary file 1 ) were studied in separate analyses described below .", "Assays of both Total and Integrated HIV-1 DNA were conducted at pre-therapy ‘baseline’ ( trial week 0 ) and then at weeks 12 , 48 , 52 , 60 and 108 , where samples permitted .", "As detailed in Supplementary file 2 , not all patients were assayed at all time-points , dependent on the analyses being conducted and sample availability . 10 . 7554/eLife . 03821 . 003Table 1 . Patient demographicsDOI: http://dx . doi . org/10 . 7554/eLife . 03821 . 003Total participants available for analysis*Number154Patients with a Total HIV-1 DNA test154 ( 100% ) Patients with an Integrated HIV-1 test111 ( 72% ) Log10 baseline Total HIV-1 DNA copies/ml3 . 88 ( 3 . 42–4 . 24 ) Log10 baseline Integrated HIV-1 DNA copies/ml3 . 6 ( 3 . 26–3 . 79 ) Time since seroconversion ( days ) 73 . 82 ( 49 . 2–95 . 8 ) Log10 Baseline Viral Load copies/ml4 . 62 ( 3 . 95–5 . 25 ) Baseline CD4 Cell count ( cells/μl ) 558 ( 428–680 . 9 ) Country of recruitment Australia21 ( 13 . 6% ) Italy18 ( 12% ) Brazil13 ( 8 . 4% ) UK102 ( 66 . 2% ) Viral Subtype ( % ) B ( 100% ) Sex Female4 ( 3% ) Male150 ( 97% ) Data shown are values ( % of non-missing values ) for categorical data or medians and interquartile ranges in brackets for continuous variables .", "*At the week 0 ‘baseline’ timepoint’ .", "A subset of these patients ( Supplementary files 1 and 2 ) was used for analyses at later time-points .", "Traditionally , plasma viral load ( VL ) ( Mellors et al . , 1996 ) and CD4 cell count ( Frater et al . , 2014 ) are the only validated surrogate markers of progression used in the HIV-1 clinic .", "We therefore measured these biomarkers as well as HIV-1 DNA in 154 SPARTAC participants at enrolment to the trial and prior to any ART being given .", "The median ( interquartile range ) values of Total and Integrated HIV-1 DNA values in PHI ( Figure 1—figure supplement 1 ) were 7707 ( 2477–18187 ) and 3830 ( 1563–6325 ) copies of HIV-1 DNA per million CD4 T cells , respectively .", "Total and Integrated HIV-1 DNA levels were closely associated ( p < 0 . 0001; r2 = 0 . 72; Pearson correlation ) ( Figure 1—figure supplement 2 ) in these pre-therapy samples .", "Total and Integrated HIV-1 DNA were significantly associated with plasma viral load ( both p < 0 . 001; r2 = 0 . 48 and 0 . 64 , respectively; linear regression ) ( Figure 1A ) , and inversely with CD4 T cell count ( both p < 0 . 001; r2 = 0 . 20 and 0 . 27 , respectively; linear regression ) ( Figure 1B ) .", "Interestingly , the estimated time since seroconversion at recruitment did not correlate with HIV-1 DNA ( both Integrated and Total ) ( Figure 1—figure supplement 3 ) . 10 . 7554/eLife . 03821 . 004Figure 1 . HIV-1 DNA correlates with baseline plasma viral load and CD4 T cell count . Pre-therapy ‘baseline’ Total HIV-1 DNA ( black points and line ) ( n = 154 ) and Integrated HIV-1 DNA ( n = 111 ) ( red points and line ) correlated with log10 plasma HIV-1 RNA ( A ) and CD4 cell count ( B ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 03821 . 00410 . 7554/eLife . 03821 . 005Figure 1—figure supplement 1 . Distribution of log10 total and integrated HIV-1-DNA levels in untreated patients at baseline . Kernel density plot to show distribution of Total ( blue ) and Integrated ( red ) HIV-1-DNA at baseline . DOI: http://dx . doi . org/10 . 7554/eLife . 03821 . 00510 . 7554/eLife . 03821 . 006Figure 1—figure supplement 2 . Pearson correlation for total and integrated HIV-1 DNA levels in untreated patients at baseline . DOI: http://dx . doi . org/10 . 7554/eLife . 03821 . 00610 . 7554/eLife . 03821 . 007Figure 1—figure supplement 3 . Relationship between estimated time since seroconversion and HIV-1 DNA levels . Linear regression of HIV-1 DNA levels ( Total in black; Integrated in Red ) vs the estimated time since seroconversion ( weeks ) ( total n = 154 , integrated n = 109 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 03821 . 007 For this analysis , disease progression was defined according to the primary end-point of the SPARTAC trial , that is , a composite end-point of either a CD4 T cell count of 350 cells/µl or the commencement of long-term ART ( for any clinical determined decision ) ( SPARTAC Trial Investigators et al . , 2013 ) .", "We carried out Kaplan–Meier survival analyses with patients randomised to receive no ART , and stratified according to median HIV-1 DNA level at time of recruitment ( n = 51 for Total HIV-1 DNA , and n = 38 for Integrated [due to limited sample availability] ) ( patient demographics detailed in Supplementary file 1 ) .", "There was a significant delay in clinical progression in those with lower Total and Integrated HIV-1 DNA at baseline ( p = 0 . 0016 and 0 . 0022 , respectively; log-rank test ) ( Figure 2 ) .", "The median time from randomization to primary endpoint stratified by low and high Total HIV-1 DNA levels was 187 . 0 ( IQR 127 . 0–222 . 0 ) and 77 . 9 ( IQR 35 . 0–172 . 8 ) weeks , respectively , and for low and high Integrated levels was 187 . 7 ( IQR 132 . 7–214 . 9 ) and 52 . 0 ( IQR 32 . 4–161 . 3 ) weeks , respectively . 10 . 7554/eLife . 03821 . 008Figure 2 . HIV-1 DNA predicts clinical progression in absence of ART . Kaplan–Meier survival analyses for ( A ) Total ( n = 51 ) and ( B ) Integrated ( n = 38 ) HIV-1 DNA and clinical progression , based on time from randomization to the SPARTAC trial primary endpoint of a CD4 T cell count of 350 cells/μl or starting long-term ART .", "HIV-1 DNA data was divided into two ‘high’ and ‘low’ at the median level , which was 4 . 02 and 3 . 61 copies HIV-1 DNA per million CD4 T cells for Total and Integrated , respectively .", "Significance was determined by log rank test . DOI: http://dx . doi . org/10 . 7554/eLife . 03821 . 008 Univariable Cox analyses showed Total HIV-1 DNA ( HR 4 . 16 per log10 increase [CI 2 . 10–8 . 26]; p < 0 . 0001 ) , Integrated HIV-1 DNA ( HR 5 . 41 per log10 increase ( CI 1 . 65–18 . 04 ) ; p = 0 . 006 ) and plasma viral load ( HR 1 . 74 per log10 increase ( CI 1 . 13–2 . 68 ) p = 0 . 011 ) predicted the trial primary endpoint ( Table 2 ) .", "Multivariable analyses were carried out with the baseline covariates , Total HIV-1 DNA , viral load , and CD4 T cell count .", "Here , Total HIV-1 DNA ( HR = 3 . 57 ( 1 . 58–8 . 08 ) ; p = 0 . 002 ) and CD4 count ( HR = 0 . 67 ( 0 . 53–0 . 84 ) ; p < 0 . 001 ) , but not plasma viral load ( HR = 1 . 25 ( 0 . 80–1 . 95 ) ; p = 0 . 33 ) predicted time to primary endpoint ( Table 2 ) .", "In a similar multivariable analysis , Integrated DNA did not associate significantly with the trial endpoint . 10 . 7554/eLife . 03821 . 009Table 2 . Cox regression models for variables associated with clinical progression in untreated individuals followed up from PHIDOI: http://dx . doi . org/10 . 7554/eLife . 03821 . 009Univariable unadjustedMultivariable adjustedCovariateHR ( 95% CI ) p ValueHR ( 95% CI ) p ValueTotal DNA ( log10 DNA copies ) 4 . 16 ( 2 . 1–8 . 26 ) <0 . 0013 . 57 ( 1 . 58–8 . 08 ) 0 . 002Viral load ( log10 RNA copies ) 1 . 74 ( 1 . 14–2 . 67 ) 0 . 0111 . 25 ( 0 . 80–1 . 95 ) 0 . 33CD4+ T cell count/100 cells0 . 66 ( 0 . 53–0 . 82 ) <0 . 0010 . 67 ( 0 . 53–0 . 84 ) <0 . 001Univariable and multivariable cox regression models were used to determine predictors of clinical progression in untreated individuals followed up from Primary HIV-1 Infection .", "Progression was determined according to reaching the SPARTAC trial primary endpoint ( Chun et al . , 2010 ) .", "Co-variables analysed were baseline ( i . e . first pre-therapy trial sample ) Total HIV-1 DNA , baseline plasma viral load and baseline CD4+ T cell count .", "One third of the participants recruited to SPARTAC were randomised according to the trial protocol to receive 48 weeks of ART before undertaking a treatment interruption ( SPARTAC Trial Investigators et al . , 2013 ) .", "This allowed us not only to study the impact of ART on HIV-1 DNA levels in this cohort ( which has been reported in different cohorts [Siliciano et al . , 2003; Chun et al . , 2007] ) , but also to characterise what happens on stopping therapy after treatment initiated during PHI .", "Prior to starting ART , Total and Integrated HIV-1 DNA levels were significantly different ( p < 0 . 0001; Students t test ) ( Figure 3 ) , most likely explained by the presence of unintegrated circular and linear DNA forms .", "As expected , HIV-1 DNA levels after 48 weeks of ART were significantly lower than those measured at baseline ( p < 0 . 0001 for all comparisons; Students t test ) by 0 . 63 log copies/million CD4 cells for Total , and 0 . 59 log copies/million CD4 cells for Integrated ( Figure 3 ) .", "After 48 weeks of ART , Total DNA levels remained significantly greater than Integrated levels in patients despite undetectable viraemia ( 0 . 027; paired t test ) ( Figure 3 ) .", "This is consistent with other reports of residual unintegrated HIV-1 DNA up to a year after ART initiation ( Agosto et al . , 2011 ) . 10 . 7554/eLife . 03821 . 010Figure 3 . Analysis of impact on HIV-1 DNA of antiretroviral therapy . Total and Integrated HIV-1 DNA levels and plasma viral load ( HIV-1 RNA ) were measured at Week 0 ‘baseline’ ( in participants from all three trial arms prior to any therapy ) and also in those receiving 48 weeks of ART ( weeks 48 , 52 , 60 and 108 after baseline ) .", "DNA levels ( log10 copies/million CD4 T cells ) and viral load ( log10 copies/ml plasma ) were measured at all time-points , but not all participants were sampled at all time-points dependent on sample availability .", "Significance was determined by unpaired Students t Tests or paired t test ( marker with * ) when samples at the two time-points being compared were matched . DOI: http://dx . doi . org/10 . 7554/eLife . 03821 . 01010 . 7554/eLife . 03821 . 011Figure 3—figure supplement 1 . Impact of stopping and re-starting ART on HIV-1 DNA . Total HIV-1 DNA levels were calculated in 15 patients on ART before TI ( pre-TI ) These patients subsequently started long-term ART ( ltx ) .", "DNA was sampled at least 6 months post ltx start date .", "Significance was established using a paired students t test . DOI: http://dx . doi . org/10 . 7554/eLife . 03821 . 011 Having ascertained that in untreated individuals HIV-1 DNA was a predictor of progression , we now asked whether the lower HIV-1 DNA levels following ART would predict progression if therapy was stopped .", "This has potentially greater utility , as the majority of individuals on successful ART will have undetectable plasma viraemia using standard assays .", "We measured DNA levels in participants who received a median of 48 ( IQR 47 . 7–48 . 7 ) weeks of ART with successfully suppressed viraemia ( VL < 50 copies/ml plasma ) , immediately prior to treatment interruption .", "The demographics of the subset of individuals ( n = 47 ) studied in this analysis are detailed in Supplementary file 1 .", "Kaplan–Meier survival analyses were undertaken in which participants were again divided into two groups ( low and high ) based on median HIV-1 DNA levels at TI .", "Both low Total and Integrated HIV-1 DNA levels associated with a longer time to trial endpoint ( p = 0 . 039 and 0 . 031 , respectively; log-rank test ) ( Figure 4 ) .", "The median time from TI to primary endpoint stratified by low and high Total HIV-1 DNA levels was 159 . 2 ( IQR 111 . 9–200 . 6 ) and 117 . 8 ( IQR 67 . 8–173 . 8 ) weeks , respectively , and by low and high Integrated levels was 166 ( IQR 124 . 9–200 . 6 ) and 101 . 1 ( IQR 65 . 5–156 . 8 ) weeks , respectively . 10 . 7554/eLife . 03821 . 012Figure 4 . HIV-1 DNA on ART predicts clinical progression following treatment interruption . Kaplan–Meier survival analyses for ( A ) Total ( n = 47 ) and ( B ) Integrated ( n = 47 ) HIV-1 DNA and clinical progression , based on time to the SPARTAC trial primary endpoint of a CD4 T cell count of 350 cells/μl or starting back on long-term ART .", "HIV-1 DNA data was divided into ‘high’ and ‘low’ at the median .", "Significance was determined by log rank test .", "Participants had received a median of 48 weeks of ART and then undertook a treatment interruption .", "DNA levels were measured at week 48 , at the point of stopping ART .", "Time from TI to primary endpoint is plotted on the x-axis . DOI: http://dx . doi . org/10 . 7554/eLife . 03821 . 012 In univariable Cox regression analyses , Total and Integrated HIV-1 DNA both predicted clinical progression from TI , determined by time to reaching the trial primary endpoint ( Total HR 3 . 52 [1 . 32–9 . 37]; p = 0 . 012; Integrated HR 3 . 01 ( 1 . 13–7 . 95 ) ; p = 0 . 027 ) .", "Multivariable cox regression models were constructed with HIV-1 DNA and CD4 cell count at TI .", "Viral load was not included as it was undetectable at TI .", "Both Integrated ( HR 2 . 81 CI ( 1 . 05–7 . 55 ) p = 0 . 04 ) and Total ( HR 3 . 42 CI ( 1 . 29–9 . 05 ) p = 0 . 013 ) HIV-1 DNA retained significance , and in both cases CD4 T cell count at TI was not a significant predictor ( HR 1 . 04 CI ( 0 . 83–1 . 11 ) p = 0 . 58 and HR 0 . 94 CI 0 . 825–1 . 08 p = 0 . 4 ) .", "At TI , HIV-1 DNA was the only predictor of the primary end point .", "One of the concerns around the viral rebound following a TI is the risk of ‘re-seeding’ the reservoir in individuals who might have extremely low HIV-1 DNA levels , and who might be candidates for ‘post-treatment control’ of viraemia ( Hocqueloux et al . , 2010 ) .", "We therefore measured HIV-1 DNA in those participants who had received 48 weeks of ART at the point of TI and then again 4 , 12 and 60 weeks post TI , where samples were available .", "Total and Integrated HIV-1 DNA levels were not significantly greater than at the time of ART cessation for up to 12 weeks post TI , although had significantly increased 60 weeks after TI ( p < 0 . 0001 for Total and Integrated DNA; Students t test ) , returning approximately to the Week 0 pre-therapy levels ( Figure 3 ) .", "The increase in Total and Integrated HIV-1 DNA 4 weeks after TI was not significant ( p = 0 . 30 ) , in contrast to the rebound in plasma viraemia ( p < 0 . 001 ) , which may be re-assuring for those implementing a TI strategy in which ART would be re-introduced when plasma VL became detectable .", "Of note , in an analysis of those individuals who subsequently restarted ART after the TI–and for whom we had samples ( n = 15 ) –there was no significant difference between the HIV-1 reservoir size pre-TI and at least 6 months after re-starting ART ( p = 0 . 58; paired students t test; Figure 3—figure supplement 1 ) , suggesting that any increase in HIV-1 DNA on stopping ART may be reversible if therapy is re-commenced .", "However , larger studies will be needed to confirm these data .", "Although almost all participants in SPARTAC experienced VL rebound on stopping ART , we have previously shown that of those who received >12 weeks of therapy , 14% still had undetectable viraemia 12 months later ( Stohr et al . , 2013 ) .", "We therefore wished to establish—albeit in this different , although overlapping , sub-group of SPARTAC participants—whether HIV-1 DNA predicted the return of plasma viraemia post-TI .", "As our previous findings included participants in centres using both 50 and 400 copies/ml as the lower limit of detection for plasma viral load assays , we studied both cut-offs for the HIV-1 DNA analyses .", "No patients were censored before viral rebound was detected and all were aviraemic ( <50 copies/ml plasma ) at the point of stopping ART .", "Levels of Total ( but not Integrated ) HIV-1 DNA at TI predicted time to viral rebound to 400 copies/ml by univariable Cox regression analysis ( HR 2 . 43 ( 1 . 23–4 . 79 ) p = 0 . 010 ) .", "CD4 T cell count at TI was not predictive ( HR 0 . 92 ( 0 . 78–1 . 08 ) p = 0 . 32 ) ( Supplementary file 3 ) .", "In a multivariable Cox regression model including Total HIV-1 DNA and CD4 count , both sampled at the point of TI , only Total HIV-1 DNA significantly predicted time to viral rebound to 400 copies ( HR 2 . 68 [1 . 31–5 . 48] p = 0 . 0069 ) ( Supplementary file 3 ) .", "When using values from pre-therapy baseline rather than at the time of TI in the model , neither plasma viral load nor CD4 T cell count predicted time to viral rebound ( >400 copies/ml ) from TI ( HR 1 . 38 [0 . 96–1 . 99] p = 0 . 080 ) and ( HR 1 . 03 [0 . 92-1 . 12] p = 0 . 60 ) , respectively .", "Kaplan–Meier survival analyses showed similar results , with a low Total HIV-1 DNA ( based on stratification around the median level ) associated with a slower time to a viral rebound of 400 copies/ml ( p = 0 . 0038; log-rank test ) but not to 50 copies per ml ( p = 0 . 18 ) ( Figure 5 ) . 10 . 7554/eLife . 03821 . 013Figure 5 . HIV-1 DNA at ART interruption predicts time to viral rebound . Survival analyses of time to viral rebound ( weeks ) in participants undertaking TI after 48 weeks of ART .", "HIV-1 DNA levels are presented divided at the median level into high ( red ) and low ( black ) .", "Rebound to 400 HIV-1 RNA copies ( n = 46 ) is presented for Total ( A ) and Integrated ( B ) HIV-1 DNA .", "Rebound to 50 HIV-1 RNA copies ( n = 45 ) is presented for Total ( C ) and Integrated ( D ) HIV-1 DNA .", "Significance was determined by log rank test . DOI: http://dx . doi . org/10 . 7554/eLife . 03821 . 013 It was unclear why Total HIV-1 DNA should predict rebound to 400 copies but not to 50 .", "In an attempt to explain this we studied those individuals with data available at both cut-offs .", "In this small post-hoc analysis ( n = 45 ) , we found that rebound varied according to the HIV-1 DNA level at the time of TI .", "Patients with high Total HIV-1 DNA levels were more likely to have a first detectable VL greater than 400 copies/ml , whereas those with lower HIV-1 DNA levels were more likely initially to rebound below 400 but above 50 copies/ml ( p = 0 . 0074; Fisher's exact test ) ( Supplementary file 4 ) .", "In summary , we find evidence that HIV-1 DNA is a significant predictor of the duration of viral remission and magnitude of the initial rebound following TI .", "This , if confirmed in larger studies , would have implications for those designing protocols for ART-reintroduction following viral rebound in TI studies ." ], [ "Since first described nearly two decades ago a persistent reservoir of HIV-1-infected cells remains the main reason that HIV-1 infection cannot be cured ( Chun et al . , 1997; Finzi et al . , 1997; Wong et al . , 1997 ) .", "The simplest measure of the reservoir is a qPCR assay that detects all intracellular HIV-1 DNA regardless of whether it is integrated into host chromosomes or is in unintegrated linear or circular forms .", "A modification of this assay incorporates an initial step to prime host Alu repeats in order to quantify only viral DNA that has been integrated into host DNA .", "These assays are open to criticism as the vast majority of intracellular HIV-1 DNA is thought to be replication incompetent , and qPCR is not able to discriminate between replication competent and incompetent viral DNA genomes .", "This has led to the development of alternative approaches such as viral outgrowth assays ( which are considered the gold standard , but are expensive and time-consuming , even with recent improvements to their protocols [Laird et al . , 2013] ) and assays to measure intracellular HIV-1 RNA , which may more accurately reflect an infected cell's ability to produce new virions , especially under conditions where viral transcription is stimulated ( Bullen et al . , 2014 ) .", "Despite the debate over the biological relevance of measuring HIV-1 DNA—and bearing in mind that none of these assays have been standardised for clinical use–a number of reports have attributed clinical meaning to HIV-1 DNA assays .", "Over a decade ago Tierney and colleagues suggested that proviral DNA in PBMCs from 111 participants receiving limited nucleoside analogue therapy was an independent predictor of clinical progression , although it is unclear how suppressive the ART regimes were in this study ( Tierney et al . , 2003 ) .", "Havlir et al studied 100 individuals with chronic HIV-1 infection and viral suppression on ART and showed that HIV-1 DNA independently predicted residual viraemia on ART ( Havlir et al . , 2005 ) .", "However there has not been a comprehensive analysis of both HIV-1 Total and Integrated HIV-1 DNA in individuals randomised to treatment or no treatment soon after seroconversion .", "We applied both Total and Integrated DNA measures to a unique cohort of individuals with evidence of PHI randomised to immediate interrupted ART or no therapy with longitudinal follow-up for a median of 4 . 5 years .", "As participants were randomised to different short course ART therapies prior to TI , we were able to determine how well HIV-1 DNA correlated with accepted surrogate markers of progression such as VL and CD4 count , and also whether HIV-1 DNA was an independent predictor of disease progression within the SPARTAC trial in both treated and untreated participants .", "Our first finding that HIV-1 DNA associated closely with both plasma VL and CD4 cell counts ( Figure", "1 ) was not surprising as this is reported elsewhere ( Chun et al . , 2010; Parisi et al . , 2012 ) .", "Our findings that both baseline and pre-TI HIV-1 DNA strongly predicted the trial primary endpoint ( Figure 4 and Table", "2 ) are supported by data from other smaller , discrete observational studies , in which low HIV-1 DNA levels associated with a longer time to clinical progression ( Goujard et al . , 2006; Minga et al . , 2008; Piketty et al . , 2010 ) , a lower viral set point and reduced chance of virological failure on ART re-initiation ( Yerly et al . , 2004 ) at PHI .", "We are aware of one other report associating HIV-1 DNA with time to viral rebound on stopping ART ( Yerly et al . , 2004 ) .", "In this study Yerly and colleagues studied chronically-infected individuals with sequential treatment interruptions and reported that DNA was a predictor of the peak of viraemia following therapy cessation and failure to reach undetectable viraemia on re-starting ART–they do not report on the actual duration of viral suppression after TI .", "In a smaller study at PHI , Lafeuillade et al also associated HIV-1 DNA with time to rebound , however this study is complicated by other interventions such as IL-2 and hydroxyurea in addition to ART ( Lafeuillade et al . , 2003 ) .", "We measured plasma viral load in the pre-therapy ‘baseline’ sample closest to the estimated time of infection .", "One possible criticism—and explanation for why plasma VL was less predictive in this study—is that other studies have associated progression with the ‘set-point’ viral load , the value at which the VL stabilizes following the dynamic PHI stage .", "However , in our untreated participants we found that the ‘baseline’ and ‘set-point’ VL values were highly correlated , although the former was higher , as would be expected ( data not shown ) .", "From a clinical perspective , it is worth noting that if individuals with PHI are commenced on ART immediately , then their ‘set-point’ VL will not be known , potentially placing greater impact on the less dynamic HIV-1 DNA measure .", "After TI , we observed a period of at least 12 weeks where no significant increases in the HIV-1 reservoir level were detected by both assays ( Figure 3 ) .", "However , we found little evidence of longer term post-treatment control ( Persaud et al . , 2013; Saez-Cirion et al . , 2013 ) , as levels of HIV-1 DNA 1-year after therapy interruption were not significantly different to that seen at pre-therapy baseline .", "Nevertheless , the potential for there to be a short window period during which plasma viraemia has rebounded but HIV-1 DNA levels have not risen significantly is encouraging , if future closely-monitored TI studies are to be undertaken .", "Concerns around ‘re-seeding’ the reservoir are very real , and it is important that any possible harm associated with a TI is limited .", "It is therefore also re-assuring that in our admittedly small sub-study , re-initiation of ART subsequently restored HIV-1 DNA to pre-TI levels .", "Finally , a low ‘Total’ HIV-1 reservoir at TI resulted in a longer time to a viral rebound to 400 copies/ml ( Figure 5 ) .", "In univariable and multivariable Cox regression models Total HIV-1 DNA at TI predicted time to rebound to 400 copies/ml , whereas CD4 T cell count did not ( Supplementary file 3 ) .", "Of interest , the baseline VL and CD4 prior to therapy were also not predictive of time to rebound .", "In contrast to other studies exploring TI , we have a larger and randomly allocated patient group who have received similar durations of ART at PHI and hence can be directly compared .", "Although viral rebound was observed in all individuals after TI ultimately , this is the first report of a randomised cohort that has shown that time to viral rebound and primary study end point could be predicted by HIV-1 DNA measurement at TI .", "Our findings of an association with HIV-1 DNA and time to viral rebound raise a number of other questions .", "Why was Total DNA predictive of rebound but not Integrated ?", "Why was Total DNA predictive for rebound to 400 copies/ml but not 50 copies/ml ?", "Much larger studies will be needed to answer most of these questions , however our sub-analysis of rebounding patients suggested that a high Total DNA at TI was more indicative of a higher VL rebound ( i . e . >400 copies ) , whereas a low DNA level was not associated with a lower rebound .", "These data might indicate that a Total DNA level at TI is better at predicting the patients who will be quick to rebound rather than those who will maintain suppression .", "A question for larger studies to answer will be to define what the viral load cut-off should be for considering rebound , rather than just assuming the assay with the lowest limit of detection is best .", "Data from at least one other study ( Riabaudo et al . , 2009 ) indicate that a level greater than 50 copies/ml may be more relevant .", "The difference between the Integrated and Total DNA is also interesting .", "Integrated DNA should be the most biologically relevant marker , based on the assumption that unintegrated HIV-1 DNA forms are thought not to contribute to rebound viraemia .", "However , the assay for Total HIV-1 DNA is much simpler and with tighter coefficients of variation , possibly due to the lack of a pre-amplification PCR stage .", "Another important factor impacting our data is that the median estimated time from seroconversion was 73 . 8 days , and so most of our patients would be starting therapy at Fiebig stage IV or later .", "It is possible that earlier identification of PHI and initiation of ART would have a greater impact on the reservoir and post-treatment control , and it is important that large studies are undertaken to determine this .", "In light of observational cohorts such as VISCONTI ( Saez-Cirion et al . , 2013 ) where treatment cessation revealed individuals who remain aviraemic post TI , there is increasing interest in undertaking closely monitored treatment interruption studies in which ART would be re-started based on a detectable plasma VL .", "These do not , however , have an encouraging history with previous studies set in the context of therapeutic vaccination or CD4 T cell restoration , resulting in rapid viraemic rebound and even harm ( Strategies for Management of Antiretroviral Therapy ( SMART ) Study Group et al . , 2006; Angel et al . , 2011; Garcia et al . , 2013 ) .", "Additionally , the recent report of viral rebound in the case of the Mississippi baby , means that greater understanding of mechanisms behind post-treatment control is needed .", "The potential , therefore , to develop an algorithm to combine various biomarkers to help predict individuals suitable for such studies is appealing .", "These data are evidence that such an algorithm may be possible , and that a marker as simple as HIV-1 DNA could be an important component ." ], [ "The design of the SPARTAC trial is reported elsewhere ( SPARTAC Trial Investigators et al . , 2013 ) .", "In brief , SPARTAC was an international open Randomised Controlled Trial enrolling adults with PHI within 6 months of a last negative , equivocal or incident HIV-1 test .", "All participants gave written informed consent .", "Research ethics committees in each country approved the trial .", "Time of seroconversion was estimated as the midpoint of last negative/equivocal and first positive tests , or date of incident test .", "Participants were randomised to receive ART for 48 weeks ( ART-48 ) , 12 weeks ( ART-12 ) or no therapy ( standard of care , SOC ) .", "The primary endpoint was a composite of two events: if participants either reached a CD4 count of <350 cells/mm3 ( >3 months after randomization and confirmed within 4 weeks ) or initiated long-term ART .", "This provided an immunological surrogate of clinical progression , but also allowed inclusion of those participants who commenced ART at CD4 cell counts greater than 350 cells/mm3 .", "Time to virological failure of participants randomized to ART-48 ( two analyses using both 50 and 400 HIV-1 RNA copies/ml as the cut-off [two consecutive readings] ) was a secondary endpoint .", "Participants for this sub-study of SPARTAC were those infected with subtype B HIV-1 and for whom adequate samples were available .", "For those in the analysis of progression and viral rebound at TI , we only selected participants who had viral load suppression ( <50 copies/ml; Chiron bDNA ) at point of stopping ART ( Table 1 and Supplementary file 1 ) .", "CD4 T cells isolated from peripheral blood mononuclear cells ( PBMC ) were sampled for HIV-1 DNA in all participants at baseline , regardless of trial arm .", "Participants randomised to the ART-48 arm were sampled at week 48 at the point of stopping ART and at a further 4 , 12 and 60 weeks post ART interruption ( 52 , 60 and 108 weeks post-ART initiation ) .", "Participants who were viraemic using the Chiron bDNA , ( Bayer , Leverkusen , Germany ) ( LLD 50 copies/ml ) at the point of TI were excluded .", "CD4 T cells were enriched from frozen PBMC samples by negative selection ( Dynabeads , Invitrogen , Carlsbad , CA ) to a purity of >97% .", "CD4 T cell DNA was extracted ( Qiagen , Venlo , Netherlands ) and used as input DNA for PCR .", "Cell copy number and total HIV-1 DNA levels were quantified both in triplicate using previously published assays ( Duncan et al . , 2013; Jones et al . , 2014 ) .", "Integrated HIV-1 was measured using an assay based on that previously published ( Liszewski et al . , 2009 ) but with some minor modifications .", "40 repeated integration measurements per patient sample were performed along with five PCR reactions to which no Alu primer was added , to serve as a background control for determination of sample positivity .", "The first round master mix contained 1 . 5 U platinum taq per 50 µl reaction .", "The second round qPCR reaction was the same as the Total reaction described above , but with 10 µl of first round product being the input DNA .", "To quantify patient samples , one standard curve was generated by plotting the average cycle threshold ( Ct ) values for all integration signals at each Integration Standard ( IS ) dilution ( 70–0 . 2 copies of IS standard per well , diluted in 2 µg/ml PBMC DNA ) , so long as at least one integration signal was significantly different ( two standard deviations ) to the average gag-only background signal .", "The IS was a kind gift from Una O’Doherty .", "Ln ( Copy number ) was plotted vs Ln ( average Ct ) and each point on the standard curve was repeated in duplicate .", "The standard curve fitted extremely well to a line of best fit ( r2 = 0 . 987 ) , which was then used to calculate copy numbers in patient samples .", "Each patient sample replicate was quantified individually using the standard curve to generate error .", "Plate to plate variation was assessed using quadruplicate replicates of 8e5 cells , which have one copy of HIV-1 per cell , diluted to 100 copies per well as a first round PCR DNA input .", "The average coefficient of variance was 8 . 31% .", "HIV-1 DNA ( Total and Integrated ) distributed normally following log10 transformation .", "The association between Total and Integrated HIV-1 DNA levels was tested using Pearson correlations .", "Linear regression was used to examine the association between continuous clinical baseline covariates and HIV-1 DNA .", "Tests between grouped variables and DNA levels were tested with Mann–Whitney , Kruskal–Wallis and t tests where appropriate .", "For the association between baseline DNA and the SPARTAC primary endpoint , Kaplan–Meier plots and univariable Cox models were constructed , and subsequently adjusted for baseline covariates .", "Where participants received ART , the time to primary endpoint was calculated from the time of TI .", "Association with time to rebound was also assessed using Kaplan–Meier plots and Cox models .", "All statistics were calculated using R version 3 . 1 . 0 .", "Plots were drawn using Prism version 5 . 0 ." ] ]

[ "In HIV-1 infection , a population of latently infected cells facilitates viral persistence despite antiretroviral therapy ( ART ) .", "With the aim of identifying individuals in whom ART might induce a period of viraemic control on stopping therapy , we hypothesised that quantification of the pool of latently infected cells in primary HIV-1 infection ( PHI ) would predict clinical progression and viral replication following ART .", "We measured HIV-1 DNA in a highly characterised randomised population of individuals with PHI .", "We explored associations between HIV-1 DNA and immunological and virological markers of clinical progression , including viral rebound in those interrupting therapy .", "In multivariable analyses , HIV-1 DNA was more predictive of disease progression than plasma viral load and , at treatment interruption , predicted time to plasma virus rebound .", "HIV-1 DNA may help identify individuals who could safely interrupt ART in future HIV-1 eradication trials .", "Clinical trial registration: ISRCTN76742797 and EudraCT2004-000446-20 DOI: http://dx . doi . org/10 . 7554/eLife . 03821 . 001" ]

[ "HIV is a virus that can hide in , and hijack , the cells of the immune system and force them to make new copies of the virus .", "This eventually destroys the infected cells and weakens the ability of a person with HIV to fight off infections and disease .", "If diagnosed early and treated , most people with HIV now live long and healthy lives and do not develop AIDS—the last stage of HIV infection when previously harmless , opportunistic infections can become life-threatening .", "However , there are still numerous hurdles and challenges that must be overcome before a cure for HIV/AIDS can be developed .", "Treatment with drugs called antiretrovirals can reduce the amount of the HIV virus circulating in an infected person's bloodstream to undetectable levels .", "However , when HIV infects a cell , the virus inserts a copy of its genetic material into the cell's DNA—and , for most patients , antiretroviral treatment does not tackle these ‘hidden viruses’ .", "As such , and in spite of their side-effects , antiretroviral drugs have to be taken for life in case the hidden viruses re-emerge .", "As research into a cure for HIV/AIDS gathers momentum , patients who might be candidates for new experimental treatments will need to be identified .", "Although it is not recommended as part of standard clinical care , the only way to test if a patient's viral levels would remain suppressed without the drugs would be to temporarily stop the treatment under the close supervision of a physician .", "As such , a new method is needed to identify if there are patients who might benefit from stopping antiretroviral therapy , and more importantly , those who might not .", "Williams , Hurst et al . have now tested whether measuring the levels of HIV DNA directly might help to predict if , and when , the virus might re-emerge ( or rebound ) .", "In a group of HIV patients participating in a clinical trial , those with higher levels of HIV DNA at the point that the treatment was stopped were found to experience faster viral rebound than those with lower levels of HIV DNA .", "This method could therefore identify those patients who are at the greatest risk of HIV viral rebound , and are therefore unlikely to benefit if their treatment is interrupted .", "Williams , Hurst et al . also found that measuring the levels of HIV DNA could help to predict how the disease would progress in treated and untreated patients .", "Furthermore , these predictions were more accurate than those based on measuring the amount of the virus circulating in a patient's body .", "The next challenge is to identify other methods to distinguish patients who may remain ‘virus-free’ for a period without treatment , from those who would not .", "With this achieved , it might be possible to identify the mechanisms that determine why the virus comes back and so develop new treatments to stop this happening .", "This would make developing a cure for HIV/AIDS a much more tangible prospect ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "physics of living systems", "neuroscience" ]

[ [ "One of the great promises of personalized medicine is the delivery of a maximally efficacious and minimally harmful dose of appropriate medication to every patient ( Fitzgerald et al . , 2006 ) .", "Traditionally , dosing decisions are based on the relationship between drug concentration and the magnitude of effect observed in a population expressed as the sigmoidal dose-response curve ( Goodman , 1996 ) .", "An implicit assumption of this approach is that population averages adequately reflect the processes operating within each individual patient .", "This is not always true .", "For many drug classes such as antiepileptics , anesthetics , and antiarrhythmics , the response is binary at the level of an individual—the desired effect is either present or not ( Löscher , 2011; Sunderam et al . , 2001; Jürgens et al . , 2003; Sonner , 2002 ) .", "The population-based dose-response curve , in contrast , is a smooth graded function of drug concentration .", "Therefore , in order to deliver on the promise of optimal drug dosing at the individual level , the relationship between individual binary responses and the population-based graded estimates of drug potency have to be more rigorously defined ( Koch-Weser , 1975 ) .", "Many examples of graded population-level responses co-existing with discrete , binary individual responses are seen in biophysics .", "A priori , one might have expected that the current flow through a single ion channel molecule would simply be a scaled version of the current recorded in a cell containing many such ion channels .", "Yet , this is not the case ( Sakmann and Neher , 1984 ) .", "While at the whole cell level , current varies smoothly as a function of voltage and time ( Hodgkin and Huxley , 1952 ) , each ion channel molecule stochastically switches between conductive and nonconductive states ( Fukushima , 1982; Hoshi et al . , 1990; Aldrich et al . , 1983 ) .", "Ligand-gated ion channels also stochastically fluctuate between discrete conductive and non-conductive states at a constant concentration of a ligand ( Brickley et al . , 1999; Swanson et al . , 1996; Papke et al . , 1989 ) .", "Stochastic transitions between discrete states extend to higher levels of organization .", "For example , synaptic transmission is mediated by stochastic vesicular fusion events ( del Castillo and Katz , 1954; Kuffler and Yoshikami , 1975; Rizzoli and Betz , 2005 ) .", "At the cellular level , stochastic fluctuations in gene expression and protein abundance are observed even in a clonal population of cells ( Elowitz et al . , 2002; McAdams and Arkin , 1997; Newman et al . , 2006; Raser and O'Shea , 2004 ) .", "For instance , stochastic forces influence differentiation patterns across identical cells and give rise to drastically different proteomic responses of clonal cancer cells to drugs ( Cohen et al . , 2008; Losick and Desplan , 2008 ) .", "Thus , stochastic fluctuations among discrete states even in a constant environment are the rule at the microscopic level in many , if not all , biological processes .", "Detailed quantitative models of stochastic switching between different states of an individual molecule ( Hille , 2001; Colquhoun and Hawkes , 1995 ) are required to forge the relationship between discrete microscopic events and graded responses in the population .", "Macroscopic constructs such as pharmacologic efficacy depend upon drug-induced changes in discrete states of single molecules as well as stochastic switches among them .", "Yet , drug efficacy does not uniquely specify responses of individual molecules ( Colquhoun , 1998 ) .", "Thus , while discrete responses at the individual level can be linked to the smooth population-level responses using techniques from statistical mechanics , individual responses cannot be readily inferred from population-level analyses .", "Here , we asked whether stochastic fluctuations among discrete states , similar to those postulated to act at the level of individual molecules , organelles , and cells influence the responses of intact multicellular organisms exposed to a constant drug concentration .", "For this purpose , we chose to focus on anesthetics .", "Anesthesia is thought to consist of four basic components: amnesia , immobility , analgesia , and unconsciousness .", "Responses to stimuli are typically used to quantify the various components of anesthesia .", "At lower anesthetic doses that produce light sedation , a patient can respond to salient verbal commands and less noticeable auditory commands ( Wong et al . , 2014 ) , albeit with longer latency .", "When the state of general anesthesia is attained , the patient is unresponsive even to a painful surgical stimulus ( Miller , 2014 ) .", "Clinical assessment of anesthetic depth ultimately collapses to binary outcomes: amnestic or not , immobile or not , and conscious or not .", "We consequently studied binary responses to a simple stimulus at a fixed anesthetic concentration across individuals to determine whether they are anesthetized or not .", "These all-or-none responses can be assessed by determining whether a human patient reacts to a simple verbal command ( Sanders et al . , 2017; Russell , 2013; Flaishon et al . , 1997 ) .", "The concentration of anesthetic at which 50% of patient lose their ability to respond to verbal commands is known at MAC-awake ( Franks , 2008 ) .", "In rodent literature , the righting reflex ( RR ) is typically used to establish whether a mouse is awake or not ( Figure 1A ) ( Wasilczuk et al . , 2018; Franks , 2006 ) .", "MAC-awake and the EC50 for loss of RR are closely correlated across mechanistically distinct anesthetics ( Franks , 2008 ) .", "Discrete responses to simple stimuli can be used in other organisms such as larval zebrafish to assess anesthetic potency ( Yang et al . , 2018 ) .", "In contrast to discrete measures used in individual subjects , anesthetic potency at a population level is expressed as a smooth sigmoid function ( Miller , 2014 ) .", "Traditionally , the dichotomy between the graded population-level response on the one hand , and the binary responses of individuals on the other , has been interpreted as inter-subject variability ( Sonner et al . , 2000 ) .", "For instance , at the population level half maximal effective concentration ( EC50 ) , it is assumed that half of all the subjects will be able to consistently respond to the stimulus while the other half will consistently fail to do so ( Figure 1B ) .", "Yet , this is not the only possibility .", "It is possible that at EC50 , each individual subject will respond to 50% of stimuli ( Figure 1C ) .", "Finally , it is possible that the probability of response is influenced by the state of the subject at the moment when the stimulus is applied ( Figure 1D ) .", "To distinguish among these possibilities , we exposed both mice and zebrafish to fixed concentrations of two mechanistically distinct anesthetics and repeatedly tested their responsiveness .", "The results unequivocally demonstrate that at a fixed drug concentration , each individual mouse or zebrafish stochastically switches between being responsive and unresponsive .", "The probability of response depends upon the subject’s state .", "Therefore , stochastic state switching is apparently present at all organizational levels , from receptor to cell to whole animal .", "Unlike identical ion channel molecules , however , individual animals both in highly genetically inbred and across outbred populations exhibited dramatic variability in parameters that describe the stochastic switching between the responsive and unresponsive states .", "The inter-individual variability in parameters of the stochastic model fit to each individual was highly structured in both mice and in zebrafish .", "One manifestation of this structure is that while the overall sensitivity to anesthetics varied among individuals , the amount of noise that drives state switches was the same across individuals exposed to the same anesthetic concentration .", "As a result of the inter-subject variability and stochastic intra-subject fluctuations in responsiveness , the population-level concentration response cannot be used to reliably determine the probability that any given individual will ( or will not ) be anesthetized .", "Detailed quantification of stochastic forces that shape the within-subject fluctuations in responsiveness and between-subject variability lays the foundation for the construction of more informative stochastic models that can reconcile binary responses of individual organisms with population-based measures of drug potency .", "These models can in turn be used to deliver on the promise of personalized medicine to deliver the appropriate dose of medication tailored to each individual patient ." ], [ "The presence of the righting reflex ( RR ) during continuous isoflurane administration was used as the measure of responsiveness in mice .", "After 2 hr of equilibration at 0 . 6% isoflurane , the probability of an intact RR was 44 ± 6% ( mean ± SD across trials ) .", "This did not correspond to 44% of mice being consistently responsive and the rest being consistently unresponsive , as is commonly assumed .", "Instead , in every mouse , the outcome of the RR test fluctuated over time at a fixed anesthetic concentration ( Figure 2A ) while the population response probability at 0 . 6% isoflurane remained stable over time ( Figure 2B ) .", "Individual fluctuations in RR at concentrations deviating from EC50 were less frequent , but nevertheless were reliably observed at concentrations below 0 . 9% ( Figure 2—figure supplement 1 ) .", "In an analogous experiment using a mechanistically distinct anesthetic , propofol , we determined the responsiveness in larval zebrafish using the startle reflex ( SR ) triggered by mechanical stimulation .", "Individual larval zebrafish demonstrated fluctuating responses to identical tap stimuli ( Figure 2C ) .", "Zebrafish exposed to no propofol had a significantly higher response probability to the tap stimulus ( Figure 2—figure supplement 2A , U = 55 , nE3 = n3µM=360 trials , p<0 . 0001 ) .", "Drug concentration remained constant in the propofol exposures ( Figure 2—figure supplement 2B ) .", "The response probability across the population remained constant ( 45 ± 5% mean ± SD across trials ) for three hours after an initial hour of equilibration in 3 μM propofol ( Figure 2D ) .", "Effective concentration , defined as the concentration of the drug required to produce an effect of a given intensity , is a universally used population-based measure of drug potency ( Goodman , 1996 ) .", "We sought to apply this measure to each individual .", "At the individual level , effective concentration is equivalent to the average across-trial probability of observing a response to a stimulus at a given drug concentration .", "Therefore , we compared the experimental results in Figure 2A and C to simulations of a Bernoulli process ( Materials and methods ) constructed such that the probability of positive RR ( or SR ) was identical on each trial and the same as that observed experimentally for a population ( Figure 3A ) .", "In mice for instance , this simulation was constructed such that the probability of responding to a stimulus is 44% on every trial .", "As expected , both simulations ( Figure 3A ) and the experimental results ( Figure 3B ) have a similar overall response probability ( 45 ± 3% and 44 ± 6% mean ± SD for simulations and mouse experiments , respectively ) .", "The median response probability in simulations was not statistically different from experimental observations in both mice ( Figure 3C , U = 197 . 5 , nsim = nexp = 20 , p=0 . 95 ) and in zebrafish ( Figure 3D , U = 2276 , nsim = nexp = 72 , p=0 . 34 ) .", "Note , however , that the experimentally observed switches between positive and negative RR occur less frequently than in the simulation ( Figure 3E , U = 48 . 5 , nsim = nexp = 20 , p<0 . 0001 ) .", "This was also true of zebrafish ( Figure 3F , U = 300 . 5 , nsim = nexp = 72 p<0 . 0001 ) .", "To further quantify this resistance to state transitions , we compared the probability of becoming unresponsive after responding to a stimulus on a previous trial , P ( U|R ) ( Figure 3B , red arrow ) , to the probability of failing to respond on two consecutive trials , P ( U|U ) ( Figure 3B , purple arrow ) .", "In both mice ( Figure 3G , U = 103 , nP ( U|R ) = nP ( U|U ) =20 , p<0 . 001 ) and zebrafish ( Figure 3H , U = 501 , nP ( U|R ) = nP ( U|U ) =72 , p<0 . 0001 ) , the probability of being unresponsive on the next trial was significantly higher if the animal was found to be unresponsive on the preceding trial .", "Hence , fluctuations in responsiveness under constant anesthetic concentration are inconsistent with a Bernoulli process .", "Therefore , while effective concentration is a useful measure of population-based drug potency , it cannot be adequately applied to an individual .", "Specifically , effective concentration cannot account for the apparent resistance to state transitions .", "We now turn to the relationship between trial-to-trial fluctuations and population level variability .", "One common assumption is that observing a single mouse over long period of time is equivalent to observing a snapshot of the population of mice .", "In other words , an experiment on each individual mouse can be thought of as a different realization of the same process .", "In an apparent departure from this assumption , we observed high inter-individual variability in responsiveness in mice and in zebrafish .", "For instance , at 0 . 6% isoflurane some mice were able to right themselves on fewer than 20% of trials , while other mice within the same highly inbred population , exposed to the same anesthetic concentration , during the same experiment , were able to right themselves on ~70% of trials ( Figure 3C ) .", "Experimentally observed inter-individual variability was significantly higher than in simulations constrained to have the same number of trials ( Figure 3C , F ( 1 , 38 ) =27 . 8 , p<0 . 0001 for mice , Figure 3D , F ( 1 , 142 ) =52 . 5 , p<0 . 0001 for zebrafish , Brown-Forsythe test ) .", "This implies that the observed inter-subject variability in responsiveness is unlikely to be solely due to finite sample size .", "Rather , anesthetic sensitivity can differ significantly between individuals .", "We then sought to determine how inter-individual variability is reflected in the parameters of a model of trial-to-trial fluctuations in responsiveness fit to each animal individually ( Materials and methods ) .", "The dwell times in responsive and unresponsive states for both mice ( Figure 4—figure supplement 1A ) and zebrafish ( Figure 4—figure supplement 1B ) were approximately exponential .", "No significant autocorrelations in fluctuations were observed ( Figure 4—figure supplement 1C , D ) .", "Thus , switching between states of responsiveness and unresponsiveness in each individual can be well approximated by a two-state transition probability matrix ( Materials and methods ) .", "Because the sum of transition probabilities in each row of this matrix is exactly one , the two-state transition probability matrix is completely specified by knowing the two transition probabilities along the diagonal , P ( U|U ) and P ( R|R ) .", "The plane spanned by these two diagonal transition probabilities , therefore , is the parameter space for models of stochastic fluctuations in responsiveness in mice and in zebrafish ( Figure 4A ) .", "Movement along the x-axis to the right , results in decrease in the overall probability of response ( e . g . from I to II and from III to IV , Figure 4A ) .", "Conversely , movement up along the y-axis results in the increase in the overall probability of response ( e . g . from III to I and from IV to II , Figure 4A ) .", "Moving along the dotted lines within the parameter space does not affect the overall probability of being responsive – that is systems II and III have the same overall probability of response .", "The key difference between II and III is the number of transitions between the responsive and the unresponsive state .", "In contrast , movement from I to IV does not affect the sum of P ( U|U ) and P ( R|R ) .", "As a result , while the overall probability of responsiveness in I and IV is different , the amount of noise that drives state transitions between the responsive and the unresponsive states is the same .", "To realize why this is the case , we can visualize the two-by-two transition probability matrix as an energy landscape with two wells which correspond to the responsive and the unresponsive states ( Proekt and Hudson , 2018 ) .", "Conservation of the sum of P ( U|U ) and P ( R|R ) reflects the fact that the sum of the depths of the wells is conserved .", "To characterize inter-individual variability , we estimated transition probability matrices for each animal individually .", "This yields a single estimate of P ( U|U ) and P ( R|R ) for each individual .", "The simplest model of inter-individual variability is that the transition probability matrix , M , for each individual is a random sample taken independently from the distribution of P ( U|U ) and P ( R|R ) .", "This null hypothesis corresponds to a cloud of points in Figure 4B .", "Yet , in stark departure from this prediction , the observed joint distribution of transition probabilities in individual mice lies on a diagonal ( Figure 4C ) .", "Therefore , P ( U|U ) and P ( R|R ) are strongly negatively correlated among individuals ( R2 = −0 . 85 , p<0 . 0001 , Pearson’s R ) .", "The same relationship was observed in larval zebrafish ( Figure 4E , R2 = −0 . 77 , p<0 . 0001 , Pearson’s R ) .", "A similar strong negative correlation is observed when comparing decay rates of the responsive and unresponsive states estimated from individual dwell time distributions fit to single exponential decay functions ( dwell times from Figure 4—figure supplement 1A , B , R2mouse = −0 . 64 , p=0 . 002 , Pearson’s R , R2zebrafish = −0 . 71 , p<0 . 0001 , Pearson’s R ) .", "Note that P ( U|U ) and P ( R|R ) quantify the propensity of the system to stay in its previously observed state .", "The fact that their sum is constant across individuals implies that the amount of noise that drives state transitions between responsive and unresponsive states was consistent in all individuals exposed to a fixed anesthetic concentration .", "Yet , sensitivity to anesthetics measured as the overall probability of responding to a stimulus varied broadly in the same population of individuals .", "Altogether these results indicate that , while transitions between states of responsiveness are noise-driven and therefore unpredictable , the amount of noise is tightly controlled in all individuals .", "Too little noise would result in individuals being trapped in a single state , whereas too much noise would overpower the intrinsic dynamics of the brain , leading to a noise dominated process characterized by rapid state switching .", "Sigmoid dose-response curves are one-to-one functions—knowing drug concentration is sufficient to estimate probability of a response in a population .", "Critically , the converse is also true; knowing the probability of a response in a population is sufficient to determine the concentration of the drug to which this population is exposed .", "While this one-to-one relationship may hold for a population , we sought to determine whether the large inter-individual variability ( Figures 4–5 ) complicates this one-to-one relationship at the level of an individual .", "We compared individual transition probability matrices estimated at 0 . 6% and 0 . 3% isoflurane .", "Note that here , mice exposed to 0 . 6% isoflurane were distinct from mice exposed to 0 . 3% isoflurane .", "Individual matrices estimated for 0 . 3% isoflurane , much like those estimated at 0 . 6% isoflurane , exhibited strong negative correlations ( R2 = −0 . 86 , p<0 . 0001 , Pearson’s R ) between the diagonal elements of the transition probability matrices ( Figure 6A ) .", "At the level of the population , there were statistically significant differences between righting probability observed at the two anesthetic concentrations ( Figure 6B , U = 54 , n0 . 3% iso = 18 , n0 . 6% iso = 20 , p<0 . 001 ) .", "Note , however , that there is significant overlap ( 57% ) between the two distributions .", "As a consequence of this overlap , it is not possible to reliably determine whether an individual was exposed to 0 . 6% or 0 . 3% isoflurane over a large range of individual anesthetic sensitivities ( Figure 6C ) .", "The same phenomenon was observed for individual zebrafish exposed to medium alone or 3 μM propofol ( Figure 6—figure supplement 1 ) .", "This observation is in stark contrast to the population-based measures of anesthetic potency .", "Hill slopes of the population-based dose-response curves for anesthetics are some of the steepest among clinically useful drugs ( Friedman et al . , 2010; Joiner et al . , 2013 ) .", "Thus , small increases in drug concentration are expected to result in a dramatic change in the probability that an individual will be anesthetized .", "Yet , as demonstrated here , there is considerable overlap in sensitivity to anesthetics .", "This illustrates the fundamental inconsistency between population-based and individual-based measures of drug potency .", "Because results in Figure 6 were obtained in two separate cohorts of mice each exposed to a single anesthetic concentration , we sought to determine whether it is possible to reliably infer drug concentration from behavioral responses for the same individual exposed to two drug concentrations .", "To address this , we exposed a separate cohort of 20 mice on eight occasions , four times each to 0 . 4% and 0 . 7% isoflurane .", "Strong negative correlations between P ( U|U ) and P ( R|R ) existed at both 0 . 4% isoflurane ( R2 = −0 . 69 , p<0 . 0001 , Pearson’s R ) and 0 . 7% isoflurane ( R2 = −0 . 79 , p<0 . 0001 , Pearson’s R ) .", "Population-level response probability at 0 . 4% and 0 . 7% isoflurane differed significantly ( Figure 7A , U = 7 , n0 . 4% iso = n0 . 7% iso = 20 , p<0 . 0001 ) .", "Overlap of response probabilities at the population level between different isoflurane concentrations did exist ( 18% ) , but was approximately three times smaller than that observed across separate mouse cohorts exposed to 0 . 3% and 0 . 6% isoflurane ( Figure 6A ) .", "Overlap at the individual level varied widely , from close to zero to nearly complete ( Figure 7B , C , Figure 7—figure supplement 1 ) .", "Thus , observing the same individual exposed to different drug concentrations , improves the reliability of distinguishing between drug concentrations on the basis of righting probability .", "Comparing different , albeit highly genetically similar individuals , exposed to different isoflurane concentrations increases the response variability and therefore decreases the reliability of classification .", "In Figures 4 and 6 , we observe a strong negative correlation between the two conditional probabilities that express the tendency of staying in the previously observed state ( P ( U|U ) and P ( R|R ) ) .", "To investigate the origins of this observation , we constructed a simple mathematical model that can explain this striking correlation .", "The Markov process defined by a two-state transition probability matrix can be thought of as a discrete approximation of a continuous system that fluctuates stochastically between two stable states .", "To illustrate how such a multistable system can be embodied in the brain , we consider a simple network consisting of two neuronal populations ( α and β Figure 8A ) .", "The two neuronal populations inhibit each other and excite themselves .", "Moreno-Bote et al . ( 2007 ) demonstrate that activity of such networks can be parameterized as a function of difference in activity of the two neuronal populations ( Figure 8B ) .", "Because of self-excitation and mutual inhibition , the network exhibits two stable activity patterns: one where activity of α dominates , and the other , where activity of β dominates .", "We operationally define these stable network patterns as awake and anesthetized respectively .", "The likelihood of every activity pattern of the network is expressed by an ‘energy function’ ( Figure 8B ) .", "The more likely activity patterns are associated with lower energy and the less likely activity patterns are associated with higher energy .", "In the absence of noise such networks stay in one of the two stable states indefinitely .", "When noise is added to the system , it switches between the two stable network configurations stochastically .", "While the amount of noise changes the frequency of switching between the two states ( Figure 8C ) , it does not have a dramatic effect on the shape of the overall distribution of states .", "In both cases the distribution of states of the system is bimodal akin to what we observe in the startle reflex data in the zebrafish ( Figure 2—figure supplement 2C ) .", "To model the effects of anesthetics on different individuals , we assume that anesthetics activate sleep-active β neurons and inhibit wake-active α neurons .", "To account for the strong negative correlation between P ( U|U ) and P ( R|R ) , we assume that the degree of anesthetic-induced excitation and inhibition is correlated .", "Thus , individual differences in anesthetic sensitivity can be modeled by modulating the anesthetic effect on the network .", "In an attempt to model the results in Figures 4 and 6 , we simulate the behavior of such networks with various amounts of noise .", "We then approximate the data of the kind seen in Figure 8C obtained for different anesthetic sensitivities and noise levels using a Markov model akin to that deployed for analysis of righting and startle reflex throughout this work .", "To accomplish this , the continuous fluctuations in the state of the network are binarized using a threshold ( Figure 8C ) into the ‘responsive’ and the ‘unresponsive’ states .", "The transition probability matrix is then estimated from these binary time-series .", "The results of this analysis are shown in Figure 8D .", "When the noise amounts are small , the system tends to stay in its previously observed state .", "Thus , for low-noise simulations , the points are found around the periphery of the plane spanned by P ( U|U ) and P ( R|R ) .", "When the noise is exceedingly high , the dynamics of the system become independent of the shape of the energy landscape and tends toward the point ( 0 . 5 , 0 . 5 ) in the plot in Figure 8C .", "This would be expected if the system behaved like a Bernoulli process at EC50 .", "In order to reproduce the results in Figures 4 and 6 , the amount of noise needs to be tuned and maintained at an approximately the same level across individuals .", "In the simulations , this corresponds to Gaussian noise with mean 0 and standard deviation ( σ ) =0 . 4 .", "These simulations support our conjecture that while individuals are different in terms of anesthetic sensitivity , the amount of noise that drives fluctuations between the responsive and the unresponsive states is conserved .", "Note , that in the units of trial number , the dwell time distributions were similar in fish and in mice .", "However , in mice the trials were spaced 3 minutes apart whereas in fish the inter-trial interval was 30 seconds .", "Thus , in units of seconds , mice dwelled signficantly longer in the awake and the unresponsive state than zebrafish .", "In order to account for this difference in the characteristic dwell times , the time scale of fluctuations shown in Figure 8C would have to be scaled by a specie-specific diffusion constant ( Methods ) ." ], [ "Here , we demonstrate that even when an animal is exposed to a fixed drug concentration , its response fluctuates stochastically .", "The probability of response depends on the state of the animal at the moment of stimulation .", "Specifically , the fluctuations exhibit inertia – the animal is more likely to be stuck in its current state than to transition between states of responsiveness .", "In contrast to most drugs that are administered either intravenously or orally , the route of administration of many anesthetics is inhalational .", "This allows us to assure that the drug concentration in the animal is at equilibrium with the ambient concentration in a closed chamber ( Friedman et al . , 2010 ) .", "We utilize this significant experimental advantage to focus on the dynamics of responses within each individual at a constant drug concentration .", "However , stochastic fluctuations are not unique to a volatile anesthetic .", "An intravenous anesthetic , propofol , administered to zebrafish equilibrated with a fixed concentration bath , was also associated with dynamic fluctuations in responsiveness .", "The stochastic processes that govern the fluctuations observed under two mechanistically distinct anesthetics were remarkably similar between mice and zebrafish .", "It is curious to note that while the dwell times in the responsive and the unresponsive state in mice and in zebrafish were similarly correlated , the absolute duration of these states varied signficantly between mice and zebrafish .", "The duration of responsive and unresponsive states was approximately similar to the duration of sleep episodes in these two species ( Zhang et al . , 2007; Yokogawa et al . , 2007 ) .", "It is possible that this relationship between duration of sleep episodes and the duration of the episodes of unresponsiveness arises because similar neurobiological mechanisms are responsible for both transitions between sleep and wakefulness and fluctuations between the responsive and the unresponsive states observed under anesthesia .", "Our observations of stochastic switching between responsive and unresponsive states are consistent with previous findings showing that at a fixed anesthetic concentration , spectral characteristics of electrical activity within thalamocortical networks switch stochastically between several discrete activity patterns ( Hudson et al . , 2014; Clement et al . , 2008 ) .", "Similar observations have been made using electroencephalography ( EEG ) of patients under anesthesia ( Chander et al . , 2014; Li et al . , 2019; Vlisides et al . , 2019 ) .", "Our findings in this study suggest that such stochastic fluctuations have a behavioral counterpart expressed as fluctuating ability to respond to a stimulus .", "It has been suggested previously that anesthetics stabilize neuronal dynamics in humans ( Alonso et al . , 2014; Tagliazucchi et al . , 2016 ) and in non-human primates ( Solovey et al . , 2015; Alonso et al . , 2019 ) .", "Stabilization of neuronal dynamics may contribute to the behavioral inertia observed in fluctuations in responsiveness of both mice and zebrafish .", "This resistance to state transitions may contribute ( Proekt and Hudson , 2018 ) to anesthetic hysteresis ( Friedman et al . , 2010; Joiner et al . , 2013; Kuizenga et al . , 2018; Warnaby et al . , 2017 ) – a left-shift in a dose-response curve for emergence relative to induction of anesthesia that has been observed across taxa from Drosophila to humans .", "Stochastic responses at a fixed drug concentration help explain several otherwise puzzling phenomena in clinical anesthesiology .", "On the one hand , Hill slopes of dose-response curves for anesthetics are reported between 10 and 40 ( Miller , 2014 ) ; some of the steepest of the clinically used drugs .", "This is traditionally interpreted as a sign of low inter-subject variability in responsiveness .", "Nonetheless , approximately ten percent of patients transiently wake up during surgery as measured by their ability to respond to a verbal command ( Sanders et al . , 2017; Russell , 2013; Gaskell et al . , 2017 ) .", "Luckily , incidence of awareness accompanied by post-operative recall is much less frequent ( Avidan et al . , 2008 ) .", "However , even episodes of awareness associated with recall and high risk of developing post-traumatic stress disorder ( Osterman et al . , 2001 ) are not reliably detected by the existing EEG-based monitors of anesthetic state ( Avidan et al . , 2011 ) .", "While anesthetics are thought to impart dose-dependent effects on neuronal activity ( Stanski et al . , 1984; Katoh et al . , 1998 ) , only weak correlations are observed between EEG characteristics and anesthetic concentration ( Whitlock et al . , 2011 ) .", "Indeed , both inter-individual differences ( Whitlock et al . , 2011 ) and fluctuations of EEG measures of depth of anesthesia at a constant drug concentration ( Bloom et al . , 2005 ) obscure the relationship between drug concentration and the observed state of the EEG .", "It is likely that the traditional population-based approach of estimating the relationship between the concentration of the drug and the response dramatically under-represents the within and inter-subject variability .", "Indeed , under most circumstances , each animal is tested only once at each drug concentration .", "The repeated testing of each animal under a fixed drug concentration is more akin to the clinical scenario where the patient is exposed to many surgical stimuli that can elicit stochastic fluctuations in the level of consciousness .", "The Hill slope value observed in our study was approximately an order of magnitude lower than the previously published data ( Friedman et al . , 2010; Joiner et al . , 2013 ) .", "This relative decrease in Hill slope reflects high inter-individual variability and stochastic fluctuations in each individual .", "There are several significant differences between our experimental paradigm and those explored in the previous work .", "Previous work , guided by purely pharmacokinetic considerations , used shorter isoflurane exposures .", "In contrast , here we measured anesthetic responsiveness after a two-hour equilibration time .", "Under most circumstances , Markov processes lead to a single equilibrium distribution of states ( Roman , 1989 ) .", "The time it takes to reach this equilibrium , however , is dictated by , among other things , the degree of resistance to state transitions .", "Thus , one reason for the discrepancy between our Hill slope estimate and that published previously is that our analysis assures that the population of animals is at a behavioral steady state .", "In order to reliably anesthetize an individual after a short exposure , higher concentrations of anesthetics are necessary .", "Indeed , our EC50 estimate ( 0 . 54–0 . 55% isoflurane ) is significantly lower than that published in the previous work ( 0 . 9% for induction and 0 . 83% for emergence ) ( Friedman et al . , 2010 ) .", "Another fundamental difference between our approach and that used in the previous work is that we tested mice repeatedly .", "In contrast , in the previous work , mice are typically tested only once at each concentration .", "Repeated testing revealed trial-to-trial fluctuations .", "These fluctuations increase the apparent variability in responsiveness , thereby decreasing the apparent Hill slope of the population .", "The detailed mechanism of these stochastic fluctuations is not known as anesthetics exert effects on many molecular targets distributed broadly throughout the brain and spinal cord .", "A number of lines of evidence converge on the fact that anesthetics at least in part hijack the sleep-wake circuitry by exciting sleep-promoting and inhibiting wake-promoting neurons ( Zhang et al . , 2015; Moore et al . , 2012; Vazey and Aston-Jones , 2014; Nelson et al . , 2002 ) .", "Switching between sleep and wake is thought to be mediated by the reciprocal inhibition between these neuronal populations .", "Networks consisting of reciprocally inhibitory neuronal populations tend to exhibit self-reinforcing behavior .", "Once sleep-active neurons activate beyond a certain threshold , they shut down the wake-active neurons thereby decreasing their inhibitory effects and further strengthen mutual excitation amongst the sleep-active neurons ( Saper et al . , 2005 ) .", "The converse happens during wakefulness .", "In the absence of perturbations , the network consisting of such mutually inhibitory self-reinforcing neuronal populations will remain in the same state indefinitely .", "Once sufficient noise is added , however , the system will stochastically switch between consolidated states of sleep and wakefulness .", "Theoretical investigations of bistable neuronal networks suggest that activity within such mutually inhibitory neuronal populations can be well approximated by a diffusion on an energy landscape with two potential wells; one for each stable activity pattern ( Moreno-Bote et al . , 2007 ) .", "Anesthetics can therefore be thought of as stabilizing ( deepening ) the wells associated with unresponsiveness and de-stabilizing the wells associated with wakefulness .", "The degree to which these states are stabilized by clinically relevant doses of anesthetics is apparently insufficient to consistently keep all animals in the anesthetized state .", "Hence , stochastic fluctuations may be responsible for episodes of awareness that occur during surgeries .", "The importance of noise in systems near a bifurcation between two stable behaviors is not limited to circuits that control sleep and wakefulness ( Chialvo , 2010; Destexhe and Contreras , 2006 ) .", "Neuronal architectures similar to those involved in sleep and wakefulness are thought to play a role in diverse processes such as sensory perception ( Moreno-Bote et al . , 2007 ) , decision making ( Wong and Wang , 2006 ) , seizure generation ( Suffczynski et al . , 2004; Fröhlich et al . , 2010 ) , and working memory ( Camperi and Wang , 1998 ) to name a few .", "Mathematically similar phenomena govern transitions between normal sinus rhythm and arrhythmias ( Kim et al . , 2009 ) .", "Thus , it is likely that stochastic fluctuations between distinct responses observed under constant drug concentration are not unique to anesthetics .", "To determine whether a bistable system with noise can explain the striking correlations between P ( U|U ) and P ( R|R ) observed herein , we simulated such a neural-network-inspired system with parametrically varied amount of noise .", "The results of this simulation show that in order to obtain such correlations , the amount of noise must be conserved among individuals .", "Another issue addressed by the modeling approach is the binary nature of behavioral responses in each individual .", "In zebrafish , this binarization was motivated by the observation of a bimodal distribution of distances travelled by each zebrafish after the tap stimulus .", "This bimodality naturally suggests that the responses fall into two distinct classes .", "Yet , similar measurements are much more difficult to perform for the righting reflex .", "Thus , it is not clear from behavioral observations of RR alone , whether the underlying processes that give rise to a response on a RR trial are continuous or discrete .", "Furthermore , even if the underlying processes are discrete it is not a priori clear that only two states are present .", "We observed that dwell times in both the responsive and the unresponsive state are well-approximated by a single exponential distribution .", "Thus , we only find empirical evidence for two states .", "Longer recordings may be required in future work to determine whether other states are needed to completely describe the data .", "The two state Markov model used to analyze behavioral responses , can be seen as a natural discretization of the continuous bistable system exemplified by our modeling approach .", "For most reasonable choices of threshold , the continuous system and its discrete approximation will yield similar results .", "In some ways , our observations of stochastic switching between different states at a fixed drug concentration are similar to those well known for single receptor molecules ( Hoshi et al . , 1990; Papke et al . , 1989; Hille , 2001; Colquhoun and Hawkes , 1995; Tank et al . , 1982 ) .", "There is an important distinction , however , between molecular scale state transitions and those observed at the behavioral level in animals .", "At the molecular scale , differences between receptors are largely immaterial—the same stochastic model can be used to describe state transitions in all receptors of a particular kind .", "Thus , observing a single receptor molecule over time is sufficient to determine the response of a population of such receptors .", "Within intact multicellular organisms , in contrast , we show that the transition probabilities were significantly different amongst highly genetically similar ( Uchimura et al . , 2015 ) and genetically outbred individuals exposed to the same anesthetic concentration .", "Observation of one individual on many trials does not equate to observing a population of individuals .", "Indeed , at population level EC50 , some animals were three times as likely to be responsive as other individuals from the same population .", "Furthermore , these individual differences were consistent across time in each individual .", "The inter-individual variability in the transition probabilities that govern switching between responsive and unresponsive states was constrained such that the sum of the diagonal elements was a constant .", "This constraint is evolutionarily conserved across vertebrates from zebrafish to mice .", "Note that the sum of the diagonal elements in a square matrix is known as the trace and is equal to the sum of its eigenvalues .", "The largest eigenvalue of a transition probability matrix for a reversible Markov process is 1 ( Levin and Peres , 2017 ) .", "The fact that the trace is approximately the same in all individuals under similar experimental conditions implies that the spectral gap of the 2 × 2 transition probability matrix defined as , λ1−λ2 , where λn is the nth eigenvalue is also a constant conserved among individuals .", "The spectral gap of the matrix sets its mixing time , or the time it takes for a system starting out in a random distribution of states to approach its equilibrium distribution .", "In this particular case , the equilibrium distribution is the overall probability that a given animal will be able to respond to a stimulus .", "Boltzmann equation asserts that diffusive systems of the kind used in our model come to a single equilibrium distribution that depends just on the energy function .", "The time it takes to reach this equilibrium distribution of states , however , depends on the amount of noise .", "The more noise is added to the system , the quicker it reaches the equilibrium distribution .", "Thus , there is a fundamental relationship between the conservation of the spectral gap across individuals and the tightly controlled noise in a continuous system characterized by a two well potential .", "In a clear departure from the predictions made by the population-level dose-response curve , our findings indicate that the equilibrium probability of responding to a stimulus at a given drug concentration is different for distinct individuals .", "The time it takes to reach this behavioral equilibrium , however , is conserved amongst animals .", "This strongly implies that the noise which drives state transitions between responsive and unresponsive states is tightly biologically controlled .", "A similar observation has been made for stochastic influences on gene expression ( Elowitz et al . , 2002 ) .", "On the one hand , our results may be seen as disappointing for the clinical practice of anesthesiology .", "Because of the strong influence of stochastic forces , it does not appear possible to keep a subject reliably in an anesthetized state without exposing them to the potentially dangerous high concentrations of anesthetics associated with subsequently impaired cognition ( Whitlock et al . , 2014; Chan et al . , 2013; Fritz et al . , 2016 ) .", "Yet , the fact that variability in transition probabilities is constrained offers a possible novel avenue for improvement by developing therapies specifically aimed at state stabilization .", "Selective stabilization of the unconscious state could provide a solution to minimize the risk of spontaneously shifting into a brain state where awareness is possible , without requiring drug concentrations prone to adverse effects .", "How might this be achieved ?", "One possibility is to target the mechanisms that are known to affect the stability of the sleep-wake circuitry .", "For instance , interference with orexinergic signaling destabilizes both sleep and wake states thereby increasing the frequency of spontaneous transitions between sleep and wakefulness ( Mochizuki et al . , 2004 ) .", "Interestingly , interference with orexin signaling also affects responses to anesthetics ( Kelz et al . , 2008; Shirasaka et al . , 2011; Tose et al . , 2009; Dong et al . , 2009 ) .", "Detailed investigation of the synergy between anesthetics and modification of noise inherent in neuronal networks that control sleep and wakefulness may help develop novel therapies that will allow clinicians better control over the state of each individual patient ." ], [ "Studies were approved by the Institutional Animal Care and Use Committee at the University of Pennsylvania and were conducted in accordance with National Institutes of Health guidelines .", "For larval zebrafish ( Danio rerio ) experiments , Tübingen long fin wild-type zebrafish were mated ( Zebrafish International Resource Center , OR ) , and the embryos raised for 5 days in constant darkness .", "At 5 days post-fertilization ( dpf ) , the larvae were used for the experiments ( n = 120 ) .", "For mouse experiments , inbred male wild-type C57Bl/6 mice ( Jackson Laboratories , ME ) aged 16–24 weeks ( n = 60 ) were used in righting reflex behavioral assays .", "One group of 20 mice was exposed to 0 . 6% and 0 . 9% isoflurane .", "A second set of 20 mice were exposed to 0 . 3% isoflurane .", "A third set of 20 mice were exposed to 0 . 4% and 0 . 7% isoflurane .", "All mice were acclimatized to sealed , temperature-controlled , 200 mL cylindrical chambers with 100% oxygen flowing at 200 mL/minute , as previously described ( Sun et al . , 2006 ) .", "This flow rate ensures isoflurane chamber equilibration within 5 min ( Figure 2—figure supplement 3 ) .", "Mice were exposed to 0 . 90% , 0 . 7% , 0 . 60% , 0 . 4% or 0 . 30% isoflurane in 100% oxygen for 4 hr , beginning at ZT12-ZT14 , corresponding to the period of maximal activity and wakefulness .", "Chamber isoflurane concentrations in all assays were confirmed using a Riken FI-21 refractometer ( AM Bickford , NY . ) .", "As tolerance to repeated isoflurane exposures does not occur ( Smith et al . , 1979 ) , mice were exposed to each isoflurane concentration a total of four times over the course of 3 weeks .", "The presence or absence of the righting reflex ( RR ) was checked every 3 min , starting after 2 hr of isoflurane exposure to assure pharmacokinetic equilibration .", "RR was assessed as described previously ( Sun et al . , 2006 ) .", "A mouse was considered to have an intact righting reflex if it was able to restore its upright posture twice in a row after being turned on its back by rotating the anesthetic chamber without interrupting anesthetic delivery .", "Otherwise , the righting reflex was considered to be absent .", "Forty RR assessments were performed per animal per exposure .", "In total , 160 RR assessments were performed on each mouse at each anesthetic concentration .", "At 5 days post fertilization ( dpf ) , individual larvae were placed into a 96-well glass plate ( JG Finneran , 500 μL volume wells ) .", "Seventy-two zebrafish were exposed to 3 μM propofol in E3 medium , while 48 zebrafish were exposed to E3 alone ( Kaufman et al . , 2009 ) .", "Larvae were then placed into the DanioVision ( Noldus , Leesburg VA ) behavioral system .", "Startle reflex experiments were performed at a maximal intensity tap every 30 s for 4 hr .", "This choice of inter-stimulus interval was based upon the lack of habituation to acoustic and vibrational stimuli in larval zebrafish when these stimuli are delivered every 15 s ( Burgess and Granato , 2007; Woods et al . , 2014 ) .", "The histogram of the total distance travelled in the first second after each stimulus was bimodal suggesting an all-or-none response ( Figure 2—figure supplement 2C ) .", "A threshold of 0 . 4 mm ( approximately 1/10 of a 5 dpf zebrafish’s body length ) , was chosen on the basis of this histogram to distinguish between responsive and unresponsive startle reflex assessments ( Kimmel et al . , 1995 ) .", "The outcomes of the behavioral response assays , righting reflex ( RR ) and startle reflex ( SR ) , are binary .", "The output is a series of zeros and ones ( one for intact RR or SR and zero otherwise ) .", "The simplest model that describes this time series is a stochastic Markov process .", "Markov models relate the state of the system at time t to the state of the system observed at the previous time step t − 1 , as Xt = MX t−1 , where the 2x2 transition probability matrix , M , constructed as shown below is the fundamental quantity of interest . M=[a1−a1−bb] In the above matrix , a denotes the probability that the mouse determined to be responsive on trial one will stay responsive on the next trial .", "b denotes the probability that an animal found to be unresponsive on trial one will stay unresponsive on the next behavioral assessment .", "Note that because the sum of probabilities in a row of M must be 1 , probabilities of transitions between the awake and the anesthetized state are completely determined by finding a and b in a system with just two states .", "The simplest possibility is that the probability of being in a particular state at time t is the same for all trials ( i . e . independent of the state of the system ) ( Roman , 1989 ) .", "This corresponds to a scenario where , b = 1 − a .", "This particular type of process is called a Bernoulli process .", "Conversely , the future outcome of a behavioral trial may depend on the outcome of the previous trial .", "One manifestation of this history-dependence is inertia; the system tends to persist in a particular state .", "Inertia increases as the diagonal elements ( a and b ) of the transition probability matrix approach one .", "a and b were empirically estimated from the binary sequence of behavioral outcomes .", "For instance , to calculate a , we computed the fraction of trials where the animal responded to the stimulus on two consecutive trials .", "Formally , this corresponds to P ( Rt|Rt−1 ) or P ( R|R ) .", "By definition , a Bernoulli process is a stochastic process in which the probability of a given outcome ( positive RR or SR ) is the same for every trial .", "This is equivalent to adapting the concept of effective concentration to an individual .", "For a given effective concentration , ec , computed as the overall probability of positive RR or SR , the Bernoulli process can be expressed as the following transition probability matrix . Mb=[ec1−ecec1−ec] Mb is completely specified by a single experimentally determined parameter , ec .", "For the purposes of simulations , we experimentally determined drug potency as the average response probability across all RR or SR trials and simulated the Bernoulli process given by Mb .", "Mbwas used to simulate 80 experiments consisting of 40 trials each to mimic experimental conditions in mice ( 72 experiments each consisting of 360 trials were simulated for the zebrafish experiments ) .", "Initial states were randomized in each simulated experiment .", "To verify that the simulated Bernoulli process and experimental observations give rise to similar drug potency , we compared the simulated and observed ec .", "To test the hypothesis that behavioral observations exhibit inertia or resistance to state transitions , we compared the observed probability of state transitions , P ( U|R ) +P ( R|U ) P ( R|R ) +P ( U|U ) +P ( U|R ) +P ( R|U ) , in the observed and the simulated time series .", "As an additional test of adequacy for describing experimentally observed time series by a Bernoulli process , we compared the tendency of the system to stay in the unresponsive state , P ( U|U ) , to probability of becoming unresponsive after being responsive on the previous trial , P ( U|R ) .", "The transition probability matrix for a two-state Markov process has two free parameters .", "A transition probability matrix estimated for each specific individual can therefore be represented by a point on a plane spanned by the diagonal elements of the matrix .", "Principal component analysis ( PCA ) was used to capture the maximal inter-individual variance between transition probability matrices .", "To determine whether the transition probability matrix is a consistent trait of each individual animal , we determined the correlation in position of each individual along the first principal component ( PC ) across time .", "Statistical significance of this correlation was assessed using a permutation test ( 10 , 000 permutations ) .", "To quantify how reliably the drug concentration can be inferred from the observed behavioral responses of each individual , we applied Bayes’ theorem:P ( Drug|Response ) =P ( Response|Drug ) P ( Drug ) P ( Response ) This analysis was performed to determine how reliably one can distinguish between exposures to 0 . 6% and 0 . 3% isoflurane in mice or 0 vs . 3 μM propofol in zebrafish .", "Since mice exposed to 0 . 6% and 0 . 3% isoflurane were from two separate populations , we wanted to further investigate whether it is possible to infer drug concentration from behavioral responses within an individual exposed to two drug concentrations .", "For this purpose , we exposed a separate cohort of twenty mice on eight occasions , four times each to 0 . 4% and 0 . 7% isoflurane .", "Here , the collective trials at each concentration were pooled together for each individual ( 160 trials ) , and a bootstrap resampling technique with replacement was used to generate a response probability distribution for each concentration for each individual mouse .", "1000 bootstraps consisting of 20 randomly chosen trials were taken , where the average response probability of each bootstrap was recorded .", "Probability distribution functions were then fit to each mouse’s bootstrapped response probabilities for both isoflurane concentrations .", "Overlap between the two concentrations was computed for each mouse by finding the union of the two distributions .", "At the population level , response probability distributions were computed based on average response probabilities for each animal , and overlap was computed in a similar fashion .", "The population response probabilities observed in Figure 2B and Figure 2—figure supplement 1 suggest a shallower dose-response as compared to previously published population-based dose-response curves ( Friedman et al . , 2010; Joiner et al . , 2013; Sun et al . , 2006 ) .", "In order to compute the aggregate dose-response , we fit sigmoid curves to jackknifed subsamples across all tested isoflurane concentrations .", "Data from one animal at each concentration was removed for each subsample , and the remaining subset of data was fitted to a sigmoidal curve:1−R=11+ ( EC50/Iso ) Hwhere R is the righting probability , Iso is the isoflurane concentration and the two paramters EC50 and H are the half-maximal effect concentration and the Hill slope respectively .", "Fits were calculated such that the sum of the squared error was minimized between the subsample values and sigmoid .", "We constrained the fit such that the sigmoid value was 1 at 0 . 0% isoflurane , and 0 at 0 . 9% isoflurane .", "Mean and 95% confidence intervals were calculated for the EC50 and the Hill slope .", "The modeling approach here is essentially identical to that in Proekt and Hudson ( 2018 ) and Moreno-Bote et al . ( 2007 ) .", "Activity patterns of the bistable neuronal network consisting of two neurons can be parametrized as a function of the difference between activity of α neurons and β neurons x = Activityα-Acivityβ .", "To express the fact that mutual inhibition and self-excitation give rise to the winner-take-all behavior , Moreno-Bote et al . ( 2007 ) used the following energy function that we adapt here:E ( x ) =x2 ( X22−2 ) This function has two energy minima x= ( −1 , 1 ) corresponding to β-dominated ( ‘anesthetized’ ) and α-dominated ( ‘awake’ ) activity states , respectively .", "To add the effects of anesthetics we modified this function as follows ( Proekt and Hudson , 2018 ) E ( x , i ) =x2 ( X22−2 ) +i ( x−1 ) 2+ ( 1−i ) ( x+1 ) 2where i is the anesthetic concentration i ∈ [0 , 1] in arbitrary units .", "Note that in this equation , the effect of anesthetic on stabilizing the anesthetized state is proportional to that destabilizing the awake state .", "This was done to reflect the results shown in Figures 4 and 6 which show strong negative correlation between P ( U|U ) and P ( R|R ) in both mice and zebrafish .", "Individual differences in righting probability were simulated by changing i .", "This results in deepening the energy well in the vicinity of x=-1 and making the well in the vicinity of x = 1 proportionally shallower .", "Time evolution of the state of the network , x , was simulated as diffusion on an energy function using the standard approach:dxdt=−D∂E ( x , i ) ∂x+ϵwhere D is the diffusion constant ( assumed to be one in all simulations for simplicity ) .", "The first term in the equation reflects the energy gradient .", "This assures that the system tends towards energy minima .", "The second term , ε , is zero mean Gaussian noise .", "To modulate the amount of noise in the system , the standard deviation , σ , of noise was altered parametrically .", "Simulations of this diffusion equation were performed for 1 , 000 , 000 time steps .", "While x varies continuously , the distribution of x is bimodal with peaks near the energy troughs x= ( −1 , 1 ) .", "To compare simulations to our experimental results on RR and SR modeled using a two-state Markov process , we binarized x such that x > 0 was classified as ‘anesthetized’ and x ≤ 0 was classified as ‘awake’ .", "To further mimic the fact that RR and SR are performed intermittently , the binarized time series from the simulation was decimated 100 fold .", "This binary time series was modeled by a two-state transition probability matrix using the same methods as for the analysis of the RR and SR .", "The results of these simulations for different anesthetic concentrations and σ’s is shown in Figure 8D .", "Analyses were performed using custom code written in Matlab using the Statistics and Machine Learning toolboxes ( Mathworks , MA ) .", "Steady state population response probabilities were confirmed through Pearson correlation coefficient analysis between the trial index and the response probability averaged across animals .", "R2 values approaching zero indicate that the population is near a steady state .", "Statistical comparisons of medians were performed using the nonparametric Mann-Whitney U test .", "The Brown-Forsythe test for equal variances was used to compare variability in simulated and experimental response probabilities .", "p<0 . 05 were considered statistically significant for all comparisons ." ] ]

[ "Traditionally , drug dosing is based on a concentration-response relationship estimated in a population .", "Yet , in specific individuals , decisions based on the population-level effects frequently result in over or under-dosing .", "Here , we interrogate the relationship between population-based and individual-based responses to anesthetics in mice and zebrafish .", "The anesthetic state was assessed by quantifying responses to simple stimuli .", "Individual responses dynamically fluctuated at a fixed drug concentration .", "These fluctuations exhibited resistance to state transitions .", "Drug sensitivity varied dramatically across individuals in both species .", "The amount of noise driving transitions between states , in contrast , was highly conserved in vertebrates separated by 400 million years of evolution .", "Individual differences in anesthetic sensitivity and stochastic fluctuations in responsiveness complicate the ability to appropriately dose anesthetics to each individual .", "Identifying the biological substrate of noise , however , may spur novel therapies , assure consistent drug responses , and encourage the shift from population-based to personalized medicine ." ]

[ "Every year , millions of patients undergo general anesthesia for complex or life-saving surgeries .", "In the vast majority of cases , the drugs work as intended .", "But a minority of patients take longer than expected to regain consciousness after anesthetic , and a few wake up during the surgery itself .", "It is unclear what causes these unintended events .", "When choosing an anesthetic dose for each patient , physicians rely on data from large clinical studies .", "These studies expose many patients to different doses of an anesthetic drug .", "At higher doses , fewer and fewer patients remain conscious .", "This enables physicians to identify the dose at which an average person will lose consciousness .", "But this approach ignores the difference between the response of an individual and that of the population as a whole .", "At the population level , the likelihood of a patient being awake decreases smoothly as the concentration of anesthetic increases .", "But within that population , each individual patient can only ever show a binary response: awake or not awake .", "To compare anesthetic effects on individuals versus populations , McKinstry-Wu , Wasilczuk et al . exposed mice to a commonly used anesthetic called isoflurane .", "During prolonged exposure to a constant dose of the drug , each mouse was sometimes unconscious and sometimes awake .", "These fluctuations in responsiveness seemed to occur at random .", "Exposing zebrafish to propofol , an anesthetic that works via a different mechanism , had a similar effect .", "Notably , the responses of both species to anesthesia showed a phenomenon known as inertia .", "If an individual was unresponsive at one point in time , they were likely to still be unresponsive when assessed again after three minutes .", "The amount of inertia was similar in mice and zebrafish .", "This suggests that the mechanism responsible for inertia has remained unchanged over more than 400 million years of evolution .", "The results reveal similarities between how individuals respond to anesthetics and how individual anesthetic molecules act on cells .", "When a molecule binds to its receptor protein on a cell , the receptor fluctuates spontaneously between active and inactive states .", "Studying how individuals respond to drugs could thus provide clues to how the drugs themselves work .", "Future studies should explore the biological basis of fluctuations in anesthetic responses .", "Understanding how these arise will help us tailor anesthetics to individual patients ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "neuroscience" ]

[ [ "Visual contrast is usually referred to as the difference in brightness between an object and the background ( Hofstetter et al . , 2000 ) .", "Classical vision research experiments systematically investigated how visual contrast affects objects motion perception ( Thompson , 1982; Stone and Thompson , 1992; Blakemore and Snowden , 1999; Anstis , 2003 ) .", "These studies have shown that the perceived speed of two-dimensional moving objects—for example , plaid patterns on a computer screen—is underestimated when visual contrast is reduced .", "More recent studies based on driving scenarios have suggested that the underestimation of visual speed at low contrast applies also to perceived self-motion in three-dimensional environments ( Snowden et al . , 1998; Horswill and Plooy , 2008; Owens et al . , 2010 ) .", "This finding was proposed—and is still considered—as a possible explanation for excessive driving speed in fog .", "In the abovementioned studies , and more generally in all studies having assessed the effect of visual contrast on motion perception , contrast was reduced uniformly for all objects of the visual scene , irrespective of their distance from the observer .", "While uniform contrast reduction is a valid model to assess the perception of two-dimensional patterns moving on a computer screen , it is a poor model to investigate how an atmospheric phenomenon like fog affects motion perception in three-dimensional environments .", "Specifically , fog alters visual contrast because tiny water droplets suspended in the air are interposed between the observer and the surrounding objects .", "The quantity of droplets increases along the line-of-sight as distance from the observer increases .", "As a consequence , the contrast of the visual scene decreases with distance , and visibility is better for close than for distant objects .", "Therefore , the distance-dependent attenuation of contrast experienced in fog is obviously very different from the uniform reduction traditionally adopted in vision research .", "Uniform contrast reduction resembles more closely what one could experience when looking at the environment through a dirty glass or a foggy windshield .", "To date , the effects of distance-dependent contrast reduction on motion perception are unknown .", "In other words , we still do not know how fog affects perceived self-motion .", "Here , we tested the perceptual and behavioural effects of distance-dependent contrast reduction—that is , fog—on speed perception .", "We compared these effects with those of the distance-independent—that is , uniform—contrast reduction that has been used in previous studies to simulate fog ( Snowden et al . , 1998; Horswill and Plooy , 2008; Owens et al . , 2010 ) .", "To perform these experiments , we used a state-of-the-art virtual reality setup allowing us to realistically simulate fog in an ecological driving scenario ( Loomis et al . , 1999 ) ." ], [ "In the first experiment , we used a standard psychophysical procedure to test how contrast affects perceived visual speed .", "Twelve experienced drivers were presented with pairs of driving scenes and instructed to estimate which scene moved faster ( Figure 1A ) .", "One of the scenes ( reference ) had clear visibility and moved at one of three target speeds ( 40 , 60 , or 90 km/hr ) .", "The other scene ( test ) had either clear or reduced visibility , and its speed was adjusted for each trial using a Bayesian adaptive method ( Kontsevich and Tyler , 1999 ) .", "This method allowed us to determine the point of subjective equality ( PSE ) as well as the just-noticeable difference ( JND ) .", "The PSE corresponded to the speed at which the two scenes were perceived as moving equally fast .", "Therefore , PSEs higher than the actual speed of the reference scene indicated speed underestimation , whereas PSEs lower than the speed of the reference scene indicated speed overestimation .", "The JND corresponded to the smallest detectable difference between two different speeds .", "High JNDs indicated low discrimination sensitivity , whereas low JNDs indicated high discrimination sensitivity . 10 . 7554/eLife . 00031 . 003Figure 1 . Experimental design and time course of trials .", "( A ) Experiments 1 and 3: for each trial , the first scene was presented for 700 ms , which included a 100-ms fade-in phase at the beginning and a 100-ms fade-out phase at the end .", "The second scene was presented 300 ms after the end of the first scene and had the same temporal structure as the first one .", "Participants had to fixate a central cross for the whole duration of the trial .", "The order of presentation of the reference and test scene was randomized .", "( B ) Experiments 2 and 4: three driving sessions ( i . e . , one per target speed ) were performed in random order for experiment 2 , and one session for experiment 4 .", "Before each test session , the drivers performed a training phase in which a numerical feedback indicated the driving speed when it did not match the target speed ( white digits at the bottom of the screen , left panel ) .", "In each training phase , the drivers had to drive a total of 5 min at target speed .", "In addition , at the beginning of each test trial , the scene was shown for 7 s moving at target speed with clear visibility ( memory refresher ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 00031 . 003 The contrast of the scene was reduced either in a distance-dependent manner , as would happen in natural fog , or in a distance-independent manner , as has been done in previous experiments .", "The difference between these two types of contrast reduction is represented in Figure 2B and C . Moderate and severe levels of reduction were implemented for each type of contrast alteration .", "Importantly , for each level of reduction , the overall visual contrast was the same for distance-dependent and distance-independent alteration ( Root mean square [RMS] contrast = 0 . 31 for moderate visibility reduction and 0 . 19 for severe visibility reduction , vs 0 . 46 for clear visibility ) .", "In total , the experiment consisted of five visibility conditions: clear ( no contrast reduction ) , moderate and severe fog ( distance-dependent contrast reduction ) , moderate and severe uniform reduction ( distance-independent contrast reduction ) . 10 . 7554/eLife . 00031 . 004Figure 2 . Visibility conditions .", "( A ) Clear weather conditions ( clear visibility ) : contrast is unaltered and the visibility is optimal in all directions ( brown line ) .", "( B ) Distance-independent contrast reduction ( uniform contrast ) : visibility drops equally for all objects of the visual scene , irrespective of their distance from the observer ( green lines ) .", "( C ) Distance-dependent contrast reduction ( fog ) : visibility is good for close objects , and worsens as distance from the observer increases ( red lines ) .", "( D ) Reversed distance-dependent contrast reduction ( anti-fog ) : visibility is poor for close objects , and improves as distance from the observer increases ( blue lines ) .", "( E ) Pictures of the actual setup: side view ( left ) and driver's view ( right ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 00031 . 004 Reducing the contrast of the visual scene altered speed perception [F ( 4 , 44 ) = 52 . 086 , p<0 . 001 , ηG2 = 0 . 61] .", "However , as shown in Figure 3A , perceived speed was affected differently by the two types of contrast reduction .", "Specifically , when contrast reduction depended on distance , participants matched lower speeds ( PSE mean = 54 . 7 and 41 . 7 km/hr for moderate and severe fog , respectively ) to the perceived speed under clear visibility ( mean = 65 km/hr ) .", "This indicates that natural fog led to an overestimation of speed ( equivalent to 76 . 4 and 94 . 3 km/hr for moderate and severe fog , respectively; Figure 3A ) .", "Conversely , when visibility was reduced in a distance-independent manner , higher speeds ( PSE mean = 95 . 5 and 95 . 2 km/hr for moderate and severe uniform reduction , respectively ) were matched to the perceived speed under clear visibility .", "Therefore , speed was underestimated with uniform contrast reduction ( perceived speed equivalent to 41 . 4 and 41 . 5 km/hr for moderate and severe reduction , respectively ) .", "Each condition differed from each of the others ( p<0 . 05 ) , except for uniform-moderate and uniform-severe reduction that did not differ significantly from one another .", "The same pattern of results was observed for all three target speeds .", "These results show that the two types of contrast reduction gave rise to opposite perceptual effects . 10 . 7554/eLife . 00031 . 005Figure 3 . Opposite effects of distance-dependent and distance-independent contrast reduction .", "Experiments 1 and 2 . ( A ) Mean perceived driving speed across subjects as a function of visibility: for each subject , PSE values were averaged across the three target speeds ( i . e . , 40 , 60 , and 90 km/hr ) , then perceived speed was calculated using the following equation: Speedperceived = PSEclear + PSEclear × ln ( PSEclear/PSEreduced visibility ) .", "As compared to clear visibility ( brown dashed line ) , speed was overestimated with distance-dependent visibility reduction ( red bars ) and underestimated with distance-independent visibility reduction ( green bars ) .", "( B ) Mean produced driving speed across subjects as a function of visibility: for each subject , measured speed values were averaged across the three target speeds .", "As compared to their driving speed with clear visibility ( brown dashed line ) , drivers drove slower with distance-dependent visibility reduction ( red bars ) and faster with distance-independent visibility reduction ( green bars ) .", "In both ( A ) and ( B ) , the error bars represent the standard error of the mean .", "PSE: point of subjective equality . DOI: http://dx . doi . org/10 . 7554/eLife . 00031 . 005 Reducing visibility also affected speed discrimination sensitivity [F ( 4 , 44 ) = 29 . 58 , p<0 . 001 , ηG2 = 0 . 37] , which was significantly lower ( higher JNDs ) in the two conditions in which visibility was reduced in a distance-independent manner ( mean = 0 . 58 km/hr for both moderate and severe uniform reduction ) , as compared to the other three conditions that did not differ from one another ( mean = 0 . 41 , 0 . 40 , and 0 . 42 km/hr for clear , moderate , and severe fog , respectively ) .", "This indicates that the participants had more difficulties estimating the driving speed when visibility reduction was independent of distance .", "This reduction of speed discrimination sensitivity might result from the fact that distance-independent contrast alteration is seldom encountered in real life .", "Specifically , although entering a car with a foggy windshield is common , especially due to cold weather , drivers usually demist their windshield before starting to drive .", "Unfortunately , drivers' options to reduce environmental fog are much more restricted .", "In the second experiment , we tested whether the perceptual changes measured in the first experiment affect actual driving behaviour .", "Ten experienced drivers who did not participate in the first experiment were instructed to drive at target speeds with either clear or reduced visibility .", "The visibility conditions and target speeds were the same as in experiment 1 .", "In a preliminary training phase , drivers learned to reach and maintain each target speed with clear visibility ( Figure 1B , left pane ) .", "In the test phase , drivers freely controlled their driving speed with the gas pedal , and pressed a button when thinking they were driving at target speed ( Figure 1B , right pane ) .", "To refresh drivers' memory , the scene was shortly presented moving at the target speed with clear visibility at the beginning of each trial .", "Driving speed was affected by the contrast of the visual scene [F ( 4 , 36 ) = 43 . 18 , p<0 . 001 , ηG2 = 0 . 44] .", "However , as for perceived speed , the direction of the effect depended on the type of contrast reduction .", "As compared to clear visibility ( mean = 85 . 1 km/hr ) , the participants drove significantly slower with distance-dependent contrast reduction ( mean = 77 . 3 and 70 . 9 km/hr for moderate and severe fog , respectively ) , but faster with distance-independent reduction ( mean = 93 . 6 and 101 . 3 km/hr for moderate and severe uniform reduction , respectively; see Figure 3B ) .", "The five visibility conditions differed significantly from one another ( p<0 . 05 ) , and the same pattern was observed for all three target speeds .", "These behavioural results are consistent with the perceptual results of the first experiment and can be interpreted as follows: distance-dependent contrast reduction induces an overestimation of visual speed that prompts drivers to drive slower , whereas distance-independent contrast reduction evokes an underestimation of visual speed , prompting drivers to drive faster .", "The results of the first two experiments highlight that the distance-dependent contrast reduction experienced in fog evokes perceptual and behavioural effects that are radically opposite to those resulting from the distance-independent contrast reduction traditionally adopted in vision research .", "Such a radical difference is striking because in our experiments , the two types of contrast reduction resulted in the same global attenuation of visual contrast .", "This suggests that the global level of contrast is not the only factor affecting perceived speed .", "The spatial distribution of contrast over the visual scene also seems to play a critical role .", "Yet , what are the perceptual mechanisms underlying speed overestimation in fog ?", "In fog , visibility—that is , contrast—is reduced with distance .", "Therefore , when the driver looks straight ahead , the regions of better visibility in the proximity of the vehicle are typically viewed in peripheral vision , whereas the distant regions where visibility is impaired fall in the centre of the driver's visual field ( Snowden and Freeman , 2004 ) .", "In this situation , fog acts as a mask obscuring the central region of the visual field .", "Recently , we have shown that speed perception strongly depends on the visible portion of the visual field ( Pretto et al . , 2009 ) .", "In particular , we found that speed is overestimated when the central area is occluded , and underestimated when it is the peripheral area that is masked .", "A similar phenomenon could explain why speed is overestimated in fog , that is , when contrast is reduced in a distance-dependent manner .", "More specifically , speed overestimation in fog could result from the relative contrast between the central and peripheral areas of the visual field .", "To test this hypothesis , we created an anti-fog , that is , a distance-dependent contrast reduction characterized by an increase of visibility with distance ( see Figure 2D ) .", "With anti-fog , visibility was better for distant than for close objects , that is , visibility was good for the portion of road situated at a distance ahead , and poor for the direct surroundings of the vehicle .", "In other words , normal fog and anti-fog resulted in opposite spatial distributions of contrast over the visual scene , although the global contrast reduction was the same for both fog types .", "We hypothesized that if the relative contrast between central and peripheral areas of the visual field underlies the speed overestimation observed with fog , then speed should be underestimated with the anti-fog .", "In the third experiment , we used the same psychophysical procedure as in the first experiment to compare the effect of natural fog and anti-fog on perceived speed .", "Ten experienced drivers who had not participated in the first two experiments were presented with pairs of driving scenes and instructed to estimate which scene moved faster ( see methods of experiment 1 ) .", "Three visibility conditions were used: clear , fog , and anti-fog .", "Only one target speed was used ( 60 km/hr ) as the first two experiments revealed the same pattern of results for all three target speeds .", "As shown in Figure 4A , perceived speed depended on visibility [F ( 2 , 18 ) = 65 . 64 , p<0 . 001 , ηG2 = 0 . 81] .", "With fog , lower speeds ( mean PSE = 47 . 7 km/hr ) were matched to the perceived speed with clear visibility ( mean PSE = 60 . 1 km/hr ) , whereas higher speeds were matched to it with anti-fog ( mean PSE = 121 . 6 km/hr ) .", "Therefore , as compared to clear visibility , the scenes were perceived as moving faster with fog ( equivalent to 74 . 3 km/hr , Figure 4A ) and slower with anti-fog ( equivalent to 19 km/hr ) .", "All three visibility conditions differed significantly from one another ( p<0 . 05 ) .", "Visibility also affected speed discrimination sensitivity [F ( 2 , 18 ) = 82 . 85 , p<0 . 001 , ηG2 = 0 . 79] .", "JND was twice as high in the anti-fog condition ( mean = 0 . 543 km/hr ) than in the other two conditions that did not differ from one another ( mean = 0 . 248 and 0 . 225 km/hr for clear and fog , respectively ) .", "This indicates that estimating driving speed was more difficult in the anti-fog condition , which likely results from the completely artificial nature of this type of contrast reduction .", "Indeed , anti-fog is a type of contrast alteration that never occurs in real life .", "In that respect , it is interesting to mention that reduced speed discrimination performance was observed with both unusual types of contrast reduction , namely anti-fog here and uniform contrast reduction in the first experiment . 10 . 7554/eLife . 00031 . 006Figure 4 . Opposite effects of fog and anti-fog .", "Experiments 3 and 4 . ( A ) Mean perceived driving speed across subjects as a function of visibility: Perceived speed was calculated from the measured PSEs using the following equation: Speedperceived = PSEclear + PSEclear × ln ( PSEclear/PSEreduced visibility ) .", "As compared to clear visibility ( brown dashed line ) , speed was overestimated when visibility was better for close than for distant objects , that is , in fog ( red bars ) , and underestimated when visibility was better for distant than for close objects , that is , anti-fog ( blue bars ) .", "( B ) Mean produced driving speed across subjects as a function of visibility: As compared to their driving speed with clear visibility ( brown dashed line ) , drivers drove slower when visibility was better for close than for distant objects , that is , in fog ( red bars ) , and faster when visibility was better for distant than for close objects , that is , anti-fog ( blue bars ) .", "In both ( A ) and ( B ) , the error bars represent the standard error of the mean .", "PSE: point of subjective equality . DOI: http://dx . doi . org/10 . 7554/eLife . 00031 . 006 The same 10 participants took part in a fourth experiment where they were instructed to drive at target speeds with either clear or reduced visibility ( same driving procedure as experiment 2 ) .", "This experiment aimed to compare the effects of fog and anti-fog on produced driving speed .", "The visibility and speed conditions were identical to those used in experiment 3 .", "The order of experiments 3 and 4 was counterbalanced between participants .", "Driving speed was affected by visibility [F ( 2 , 18 ) = 39 . 99 , p<0 . 001 , ηG2 = 0 . 71] .", "Specifically , as compared to clear visibility ( mean = 67 . 9 km/hr ) , participants drove slower with fog ( mean = 53 . 1 km/hr ) and faster with anti-fog ( mean = 104 . 4 km/hr , see Figure 4B ) .", "All three conditions differed from one another ( p<0 . 05 ) .", "Visibility also affected the variability of driving speed [F ( 2 , 18 ) = 9 . 56 , p<0 . 01 , ηG2 = 0 . 33] , which was twice as large in the anti-fog condition ( mean = 17 . 6 km/hr ) as in the other two conditions that did not differ from one another ( mean = 9 and 7 . 5 km/hr for clear visibility and fog , respectively ) ." ], [ "Previous studies suggested that the speed of visual motion in depth is underestimated when the global level of contrast is reduced ( Snowden et al . , 1998; Horswill and Plooy , 2008; Owens et al . , 2010 ) .", "These studies were directly inspired by classical vision research experiments that assessed the effect of contrast on the perceived speed of two-dimensional objects on a monitor ( Thompson , 1982; Stone and Thompson , 1992; Blakemore and Snowden , 1999; Anstis , 2003 ) .", "In the present study , we reproduced this perceptual bias , showing that visual speed is indeed underestimated when contrast is reduced in a distance-independent manner .", "However , we show that this is only part of the whole picture .", "In particular , we demonstrate here for the first time that an identical global loss of visibility can evoke opposite percepts , depending on the nature of the underlying visual contrast reduction .", "Therefore , contrarily to what has been consistently reported in previous studies , a global contrast reduction can also lead to an overestimation of visual speed .", "This is notably the case when contrast is not reduced uniformly for all objects of the visual scene but varies according to their distance from the viewer .", "For instance , in fog , contrast reduction is more important for distant than for close objects .", "This generates a distance-dependent visibility gradient between the peripheral and central area of the visual field .", "Our results show that in this situation , perceived speed is not determined by the global level of contrast per se , but rather by the spatial distribution of contrast over the visual scene .", "More specifically , perceived speed is determined by the relative contrast between the central and peripheral areas of the visual field .", "When visibility is better in the peripheral than in the central visual field , as is the case in fog , speed is overestimated .", "Inverting the direction of the contrast gradient with anti-fog and thereby obscuring more the peripheral than the central region of the visual field , inverts the perceptual bias such that speed is now underestimated .", "This highlights the critical role of the visibility gradient in perceived speed , explaining why speed is unexpectedly overestimated in fog despite a global reduction of visibility .", "Importantly , our results also evidence the direct relationship between perceived and produced speed .", "Specifically , speed overestimation systematically prompted drivers to drive slower , whereas speed underestimation led to faster driving paces .", "This demonstrates that driving speed is strongly affected by perceived visual speed .", "In ‘real-life’ driving , the roadsides usually include various objects and landmarks as trees , buildings , traffic signs , or pedestrians .", "Such objects next to the road can have a cognitive influence on driving speed because they constitute potential obstacles that increase the risk of collision ( e . g . , tree or building if one drives out of the road and cars or pedestrians suddenly crossing the road ) .", "However , we were interested in the perceptual and not in the cognitive effects of contrast reduction on driving speed .", "Therefore , the roadsides of our driving scenario consisted of a grass texture clear of any object and landmark .", "This prevented cognitive factors as those mentioned above to bias our results , but importantly , it also prevented subjects of using these landmarks to estimate moving speed .", "Indeed , adding landmarks or other ‘higher-level’ cues would have given the subjects the possibility to use some cognitive strategies to estimate speed .", "For instance , subjects could have used relative size information of landmarks to infer distances and counted passing them to assess speed .", "Undoubtedly , this would have biased our results .", "Yet , one could argue that at a ‘pure’ perceptual level , objects next to the road can also contribute to increase peripheral visual flow , thereby altering perceived speed .", "This would be mostly true with a driving environment that provides only little visual flow information ( e . g . , very smooth road pavement and roadsides ) .", "However , the scene we used consisted of a rough asphalt pavement and roughly textured grass roadsides ( see Figure 2 ) .", "These rough textures provided participants with rich visual flow information when driving , as attested by our results .", "Specifically , in the ‘clear visibility’ condition , participants estimated visual speed with great accuracy .", "In addition , the variability of speed estimates was small ( i . e . , low JNDs ) in all conditions , and even more with clear visibility .", "Those elements indicate that our driving scenario provided robust visual flow information allowing for reliable speed estimates .", "Contrast-dependent modulation of neural activity has been reported at early stages of visual processing , as the retina ( Shapley and Victor , 1978 ) , the lateral geniculate nucleus ( Solomon et al . , 2002 ) , and the primary visual cortex ( Levitt and Lund , 1997; Polat et al . , 1998; Sceniak et al . , 1999 ) .", "It has also been reported in the middle temporal area ( MT or V5 ) , an area playing an important role in motion processing ( Pack et al . , 2005; Bartels et al . , 2008 ) and speed detection ( Maunsell and Van Essen , 1983b ) , notably via feedforward projections to the medial superior temporal area ( Maunsell and van Essen , 1983a; Ungerleider and Desimone , 1986 ) .", "Yet , linking these neurophysiological findings with the effect of contrast on perceived speed in humans is not straightforward .", "For instance , the observation that MT neurons tuned to high speeds are strongly activated by slow stimuli at low contrast ( Pack et al . , 2005 ) would predict speed overestimation at low contrast .", "This is the opposite of what has been usually observed with uniform contrast reduction , including in the current study .", "As stated by Pack et al . ( 2005 ) , this inconsistency might be resolved by assuming a bias towards slow speeds when the total MT population activity is low .", "Such a bias towards slow speed is precisely what is proposed by Bayesian models of speed perception ( Weiss et al . , 2002; Stocker and Simoncelli , 2006 ) .", "Specifically , these models rely on the assumption that speed ‘measurements’ are intrinsically noisy , and that based on our everyday experience , slower motions are more likely to occur than faster ones—resulting in an elevated prior for slow speeds .", "Whereas Bayesian models of speed perception can accurately predict speed underestimation at low contrast ( Stocker and Simoncelli , 2006 ) , speed overestimation observed at higher speeds ( Thompson et al . , 2006 ) and at low luminance ( Hammett et al . , 2007 ) are more difficult to account for .", "Alternative models have proposed that speed is encoded as ‘the ratio of the responses of physiologically plausible temporal filters’ ( Hammett et al . , 2000; Hammett et al . , 2005 ) .", "These ‘ratio models’ account for both under- and overestimation of speed at low contrast ( Thompson et al . , 2006 ) .", "However , none of the abovementioned models addressed situations in which the amount of contrast reduction differed for different areas of the moving visual scene , as is the case when driving in fog .", "In that respect , these models do not explain the differences in speed perception observed here with uniform and distance-dependent contrast reduction .", "They do not account for opposite biases evoked with the same global contrast reduction at the same actual speed .", "Poor visibility conditions affect millions of drivers around the world .", "Thousands of them die each year in a car accident .", "Excessive speed constitutes a major causal factor for these car accidents .", "We show here for the first time how fog biases speed perception , and we reveal the perceptual mechanisms underlying this bias , providing important insights into the human visual system .", "In particular , we show that contrarily to what was previously believed , speed is overestimated in fog because visibility is poorer in the central than in the peripheral area of the visual field .", "We also show that the behavioural consequence of this speed overestimation is a natural tendency to drive at a slower pace .", "Therefore , drivers should probably listen to their visual system when it prompts them to decelerate ." ], [ "Thirty-two experienced drivers ( 23 males and 9 females; aged 21–35 years , mean = 25 . 3 years ) participated voluntarily in the study ( 12 in experiment 1 , 10 in experiment 2 , and 10 in experiments 3 and 4 ) .", "All had normal or corrected-to-normal vision .", "They were paid , naive as to the purpose of the research , and gave their informed consent before taking part in the experiment .", "The study was performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki , in line with Max Planck Society policy and in compliance with all relevant German legislation .", "The participants had the option to withdraw from the study at any time without penalty and without having to give a reason .", "All experiments were performed in an immersive virtual environment .", "For all experiments , the participants were seated in a simplified vehicle mock-up equipped with steering wheel and pedals .", "The steering wheel haptic feedback was disabled so that no speed information could be inferred from the wheel feedback .", "Also , the steering wheel rotation amplitude was linearly mapped to the vehicle turning speed and applied on the centre of mass of the vehicle .", "This way , a small rotation of the steering wheel resulted in a slow rotation of the vehicle around its vertical axis , whereas a large steering wheel rotation led to a fast turning , independently of the actual longitudinal speed of the vehicle .", "The mock-up was located at the centre of a large semi-spherical screen equipped with a multi-projection system .", "The panoramic screen surrounded the observer to provide an image that embraces almost the entire human visual field .", "More specifically , a cylindrical screen with a curved extension onto the floor provided a projection surface of 230° ( horizontally ) × 125° ( vertically ) .", "The resulting surface was entirely covered by four LCD projectors , with a resolution of 1400 × 1050 pixels each .", "Overlapping regions were blended by openWARP technology ( Eyevis , Reutlingen , Germany ) .", "The geometry of the scene was adjusted for an eye height of 0 . 8 m at a distance of 3 m from the vertical screen .", "The virtual environment was created using 3DVIA Virtools 4 . 1 ( Dassault Systemes , Vélizy-Villacoublay , France ) behavioural engine , which was running distributed on a 5-PCs cluster , one for each of the projectors and a supervisor .", "The visual stimulus consisted of a virtual environment where a textured plane reproducing a straight single-lane road was scrolled at different speeds .", "The central surface of the plane was a rough asphalt pavement , whereas the sides were covered with grass-like textures .", "The sky consisted of a homogenous grey texture with the same colour of the fog and the plane used for the uniform contrast reduction ( RGB = [128 , 128 , 128] ) .", "Visual contrast was reduced by blending the fog colour ( RGB = [128 , 128 , 128] ) into the virtual scene , according to the alpha blending model Cr = Co α + Cf ( 1 − α ) , where , for each pixel of the projected image , Cr is the resulting colour , Co is the original colour , Cf is the colour of the fog , and α is the blending factor .", "For the distance-dependent contrast reduction , the blending factor was determined by e−f·d , where f is the density of the fog and d is the distance from the observer to the depicted object .", "The colour of each pixel was converted into the corresponding brightness ( the arithmetic mean of the red , green , and blue colour coordinates in the RGB colour space ) , and the scene luminance distribution was computed based on the empirically determined function between luminance and brightness .", "The function was determined in two phases:", "( i ) a plane model with uniform colour ( brightness ) was displayed on the screen facing the observer , and luminance was measured on its surface with a Minolta LS-100 photometer ( Konica Minolta , Tokyo , Japan ) for several brightness levels;", "( ii ) the readings of the photometer were plotted against the corresponding brightness and fitted ( R2 = 0 . 999 ) by a quadratic function that was then used to compute the luminance distribution of the scene for each visibility condition .", "The contrast of the scene was then computed as the RMS of the luminance ( in cd/m2 ) of the pixels in the virtual scene ( similarly to what Snowden et al . , 1998 did in their work ) .", "We set the fog density value to 0 . 1 for the medium visibility condition and 0 . 3 for the poor condition , in a range from 0 ( full visibility ) to 1 ( no visibility ) .", "These values correspond to a meteorological visibility range ( MVR ) of 30 and 10 m , respectively .", "The MVR indicates the distance at which a white object appears with a 5% contrast ( Kovalev and Eichinger , 2004 ) .", "The distance-independent ( uniform ) contrast reduction was implemented by inserting a transparent virtual plane in front of the scene .", "The plane brightness was composited with the brightness of the background image , according to the standard alpha blending model described previously .", "The opacity of the plane was adjusted to 0 . 28 and 0 . 52 in order to match the contrast of the moderate and severe fog conditions , respectively .", "The global attenuation of visibility was identical for both types of contrast reduction ( RMS = 0 . 46 for clear visibility , 0 . 31 for moderate contrast reduction , and 0 . 19 for severe contrast reduction ) .", "Anti-fog ( experiments 3 and", "4 ) was obtained using a vertex shader technique ( Engel , 2005 ) .", "The blending factor was set to 1 − e−af·d , where af is the density of anti-fog , which was adjusted to match the overall scene contrast of the fog condition ( RMS = 0 . 24 ) .", "For the two-interval forced-choice ( 2IFC ) procedure used in experiments 1 and 3 , 80 trials per condition were performed , for a total of 1200 randomly interleaved trials in experiment 1 ( two sessions of six blocks each , total duration of 2 . 5 hr ) and 240 in experiment 3 ( two blocks , total duration of 30 min ) .", "For experiments 2 and 4 , each subject performed five trials per condition , and mean driving speed was computed for each subject and condition .", "Experiment 2 consisted of 25 trials , performed in five consecutive blocks , and lasted 2 hr in total .", "Experiment 4 lasted 1 . 5 hr , consisting of three blocks of five trials each .", "For all experiments , the order of presentation of the trials was fully randomized and different for all subjects .", "During the 5-min breaks between two consecutive blocks and the 15-min break between two sessions , the lights of the experimental room were switched on and subjects could walk and relax .", "Mean PSE and JND values in experiment 1 and mean driving speed values in experiment 2 were analysed using a 5 × 3 ( contrast reduction [clear visibility , moderate fog , severe fog , moderate uniform reduction , and severe uniform reduction] , target speed [40 , 60 , and 90 km/hr] ) repeated-measures ( within subjects design ) analysis of variance ( ANOVA ) .", "Mean PSE and JND values in experiment 3 and mean driving speed values in experiment 4 were analysed using a 3 ( contrast reduction [clear visibility , fog , and anti-fog] ) repeated-measures ANOVA .", "The reported values are Huynh–Feldt corrected , and post hoc tests using the Holm adjustment method for multiple comparisons ( p<0 . 05 ) were performed when necessary ." ] ]

[ "Visual speed is believed to be underestimated at low contrast , which has been proposed as an explanation of excessive driving speed in fog .", "Combining psychophysics measurements and driving simulation , we confirm that speed is underestimated when contrast is reduced uniformly for all objects of the visual scene independently of their distance from the viewer .", "However , we show that when contrast is reduced more for distant objects , as is the case in real fog , visual speed is actually overestimated , prompting drivers to decelerate .", "Using an artificial anti-fog—that is , fog characterized by better visibility for distant than for close objects , we demonstrate for the first time that perceived speed depends on the spatial distribution of contrast over the visual scene rather than the global level of contrast per se .", "Our results cast new light on how reduced visibility conditions affect perceived speed , providing important insight into the human visual system ." ]

[ "The ways people respond to conditions of reduced visibility is a central topic in vision research .", "Notably , it has been shown that people tend to underestimate speeds when visibility is reduced equally at all distances , as for example , when driving with a fogged up windshield .", "But what happens when the visibility decreases as you look further into the distance , as happens when driving in fog ?", "Fortunately , as new research reveals , people tend to overestimate their speed when driving in fog-like conditions , and show a natural tendency to drive at a slower pace .", "Pretto et al . performed a series of experiments involving experienced drivers and high-quality virtual reality simulations .", "In one experiment , drivers were presented with two driving scenes and asked to guess which scene was moving faster .", "In the reference scene , the car was driving at a fixed speed through a landscape under conditions of clear visibility; in the test scene , it was moving through the same landscape , again at a fixed speed , but with the visibility reduced in different ways .", "The experiments showed that drivers overestimated speeds in fog-like conditions , and they underestimated speeds when the reduction in visibility did not depend on distance .", "Further experiments confirmed that these perceptions had an influence on driving behaviour: drivers recorded an average speed of 85 . 1 km/hr when the visibility was good , and this dropped to 70 . 9 km/hr in severe fog .", "However , when visibility was reduced equally at all distances , as happens with a fogged up windshield , the average driving speed increased to 101 . 3 km/hr .", "Based on previous work , Pretto et al . developed the theory that the perception of speed is influenced by the relative speeds of the visible regions in the scene .", "When looking directly into the fog , visibility is strongly reduced in the distant regions , where the relative motion is slow , and is preserved in the near regions , where the motion is fast .", "This visibility gradient would lead to speed overestimation .", "To test this theory , the experiments were repeated with new drivers under three different conditions: good visibility , fog , and an artificial situation called ‘anti-fog’ in which visibility is poor in the near regions and improves as the driver looks further into the distance .", "As predicted , the estimated speed was lower in anti-fog than in clear visibility and fog .", "Conversely , the driving speed was 104 . 4 km/hr in anti-fog compared with 67 . 9 km/hr in good visibility and 51 . 3 km/hr in fog .", "Overall , the results show that the perception of speed is influenced by spatial variations in visibility , and they strongly suggest that this is due to the relative speed contrast between the visible and covert areas within the scene ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "stem cells and regenerative medicine", "neuroscience" ]

[ [ "An improved understanding of brain regeneration holds the potential to stimulate new therapies geared toward the treatment of stroke , neurological disease , and brain injury .", "Brain regeneration , as a process , must occur through several interrelated steps: wound signaling and healing; production of new cells by proliferation , dedifferentiation or transdifferentiation; specification of cell lineages of the proper type ( s ) and ratio ( s ) ; organization of new cells in three-dimensional space; differentiation of precursors; reestablishment of connections required for functional restoration; and , finally , cessation of regeneration to avoid overgrowth or damage to nearby healthy tissue .", "Of these steps , production of new CNS cells has been a major focus .", "The advent of mammalian stem cell-based approaches and advances in cell reprogramming enabled the creation of new , often functional , neurons in vitro ( Wichterle et al . , 2002; Zhang et al . , 2001; Yan et al . , 2005; Han et al . , 2012; Vierbuchen et al . , 2010; Elkabetz and Studer , 2008; Piña-Crespo et al . , 2012; Wernig et al . , 2004 ) .", "Additionally , the discovery of endogenous stem cell pools in the mammalian brain ( Altman and Das , 1965; Eriksson et al . , 1998 ) raises the possibility of enhancing the brain’s ability to create new neurons from within ( for a review of this progress , see Gage and Temple , 2013 ) .", "Despite these advances , functional repair in the human CNS remains an extremely ambitious goal and requires the development of new therapies aimed not only at generation of new cells , but also placing and integrating these cells appropriately within a broader regenerative context .", "To understand how the cell biological processes of regeneration are regulated and coordinated , we look to organisms that successfully regenerate in nature .", "While some species , like zebrafish and axolotls have a greater regenerative capacity than do mammals , species with the ability to regenerate an entire brain de novo are rare in the animal kingdom and in the realm of established model organisms ( Bely and Nyberg , 2010; Tanaka and Ferretti , 2009; Egger et al . , 2007 ) .", "Of the small number of animals that regenerate their brains after injury , planarians have emerged as a tractable model system in which to study this remarkable process .", "Scientists have appreciated the extreme capacity for regeneration found in planarians for over two hundred years ( Elliott and Sánchez Alvarado , 2013 ) .", "A nascent brain structure appears soon after amputation ( Cebrià et al . , 2002b ) and reproducible brain wiring is indicated by full functional recovery , with resumed movement and feeding after one week ( Inoue et al . , 2004 ) .", "Anatomical studies have revealed details of the structure and molecular complexity of the planarian central nervous system ( CNS ) , which consists of bi-lobed cephalic ganglia ( the brain ) and two ventral nerve cords ( Figure 1A; Agata et al . , 1998 ) .", "The planarian brain is composed of a diverse array of neural subtypes that produce conserved neurotransmitters and dozens of neuropeptides ( Nishimura et al . , 2007a , 2007b; 2008a; 2010; 2008b; Collins et al . , 2010 ) .", "These cell types are regionally organized , with subtypes of cells appearing medially or in lateral structures called brain branches that project toward the margin of the head ( MacRae , 1967; Cebrià et al . , 2002a ) .", "Planarians also regenerate missing brain tissue regardless of the amputation plane or the severity of the injury to the CNS ( Reddien and Sánchez Alvarado , 2004 ) . 10 . 7554/eLife . 17002 . 003Figure 1 . A transcriptional view of planarian head regeneration .", "( A ) Diagrams depicting the overall organization of the planarian CNS ( left ) and the principal cell types known to play roles in brain regeneration ( right ) .", "( B ) A schematic illustrating the samples used for RNA-Seq analysis .", "The CNS is shown in green and anterior polarity signals are represented in blue .", "Whole animals and animals killed immediately after postpharyngeal amputation were used for controls .", "( C ) A Venn diagram depicts the 933 contigs upregulated during head regeneration compared to cut controls ( ≥2x upregulated , p≤0 . 01 ) .", "Most identified genes were upregulated to this threshold at only one time point .", "( D ) Pie charts showing the proportion of genes upregulated at each time point ( vs . cut control ) that are also upregulated when compared to uninjured ( whole ) animals ( ≥2x upregulated , p≤0 . 01 ) .", "The fractions are 44% ( 12 hr , 36 hr ) and 48% ( 72 hr ) .", "( E ) Workflow depicting our characterization of upregulated genes .", "( F ) Pie chart showing the percentage of upregulated genes with neural expression patterns .", "362 genes gave clear expression patterns .", "Of these , 47% had expression in the central nervous system—132 genes with expression in the brain and 38 with expression in a subset of CNS cells .", "( G ) Examples of expression patterns ( by in situ hybridization , ISH ) are shown for genes expressed in the CNS alone , in the CNS and other tissues ( arrow indicates the parenchyma for xp-C and arrowhead indicates the gut for hyp-c30620 ) , and in neural subsets .", "Scale bar: 500 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 17002 . 00310 . 7554/eLife . 17002 . 004Figure 1—figure supplement 1 . Time courses of anterior regeneration .", "( A ) Diagram of the postpharyngeal amputation used for the time courses in this figure , as well as the transcriptomic experiments .", "The CNS is diagrammed in green .", "( B ) Time courses using immunofluorescence and ISH to illustrate the timing of events in head regeneration .", "Anterior markers appear early ( e . g . , nou darake , ndk ) .", "Neural gene expression initiates at around 36 hr ( e . g . , prohormone convertase 2 , PC-2 ) and becomes stronger at 2 d post-amputation .", "By 3 d after amputation , a brain primordium is visible by DAPI staining and neural markers are all reexpressed .", "Synapsin staining indicating further maturation of the neurons continues to 5 d after amputation . The innervated pharynx is also visible 5 d after amputation .", "( Scale bars: 500 µm ( B , bottom rows ) , 100 µm ( B , top row ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 17002 . 00410 . 7554/eLife . 17002 . 005Figure 1—figure supplement 2 . Expression patterns of genes upregulated during head regeneration .", "( A ) The eight largest clusters of upregulated genes .", "For each gene in the cluster , the mean expression across the five conditions is set to zero and relative expression is calculated for each experiment .", "The expression profile across all genes in the cluster is averaged to form a representation typical of the group .", "Each bar represents the average gene expression relative to the mean for that time point , with one unit representing one standard deviation from the mean across time points .", "( B ) 31/362 upregulated genes ( 8 . 5% ) show head enrichment .", "( C ) Examples of genes enriched in the anterior of the animal are shown .", "( D ) 63/362 upregulated genes ( 17 . 4% ) are expressed during homeostasis in the parenchyma of the planarian body .", "( E ) Examples of genes expressed in the parenchyma are shown .", "( F ) Other expression patterns seen in our analysis are the pharynx ( 19 genes ) , intestine ( 46 ) , protonephridia ( 7 ) , epithelium ( 13 ) and eye ( 17 ) .", "( G ) Examples of genes with the aforementioned gene expression patterns are shown .", "Scale bars: 500 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 17002 . 005 In the past two decades , the introduction of molecular techniques and availability of genomic and transcriptomic data ( Newmark et al . , 2003 , Umesono et al . , 1997; Sánchez Alvarado and Newmark , 1999; Robb et al . , 2008 ) have enabled great progress in understanding the molecular underpinnings of planarian regeneration .", "The regenerative ability of planarians depends on a pool of stem cells called neoblasts , at least some of which are pluripotent ( Wagner et al . , 2011 ) .", "This stem cell pool is heterogeneous ( van Wolfswinkel et al . , 2014; Moritz et al . , 2012; Hayashi et al . , 2010 ) and stem cells respond to injury by migrating , proliferating , and producing daughter cells that differentiate to replace all cell types of the body ( Saló and Baguñà , 1985; Baguñà et al . , 1989; Baguñà , 1976 ) .", "Additionally , planarians restore their axial polarity after injury; polarity markers reappear early during the regenerative process and are responsible for resetting the body plan ( reviewed in Adell et al . , 2010 ) .", "Specifically , anterior cues ( including Notum and secreted Frizzled-related proteins ) inhibit posterior Wnt ligands to enable head and brain regeneration ( Petersen and Reddien , 2011 ) .", "Planarian brain regeneration requires stem cell activity and proper specification of axial polarity , but beyond these generalities , the mechanisms underlying specific regeneration of the brain remain mysterious .", "Several regulators of planarian brain regeneration were uncovered through candidate-gene approaches , including axon guidance cues like Slit/Robo and Netrin/DCC as well as transcriptional regulators of certain neural subtypes , including Lhx1/5–1 , Coe , and others ( Cebrià and Newmark , 2005 , 2007; Cebrià et al . , 2007; Currie and Pearson , 2013; Cowles et al . , 2013 ) .", "Despite the identification of these molecules , we know little about the mechanisms that coordinate cell biological events required for regenerative success .", "We do not yet understand how neoblasts are directed toward particular neural fates or how cells of different neural subtypes become distributed to ensure proper ratios of diverse cell types .", "Though polarity cues certainly play a key role , we still lack an understanding of how cells within the organism interpret such cues .", "Furthermore , the signaling molecules that promote regeneration generally and instruct replacement of specific cell types are largely unknown .", "While muscle cells produce polarity cues during homeostasis and in response to injury ( Wurtzel et al . , 2015; Witchley et al . , 2013 ) , it is not yet clear whether they also produce molecules that promote regeneration , or whether other cells carry out or assist in this key signaling role .", "Finally , once nascent neural tissue is formed , we do not yet understand how it is patterned , reconnected , and restored to function—especially in the context of pre-existing tissue .", "To begin answering these important questions , we examined gene expression during the early stages of head regeneration and employed a functional screen to identify key factors in an unbiased manner .", "We identified several genes required broadly for brain regeneration , including a novel transcriptional regulator of neural progenitors and several putative signaling molecules , some of which are produced by one or more uncharacterized , differentiated cell types in the planarian parenchyma .", "Furthermore , we identified factors required to promote connectivity and function in specific brain regions .", "Finally , by investigating genes downregulated during regeneration , we identified a marker that led us to discover a population of cells that we determined to be glia , based on their gene expression and morphology .", "Taken together , our work uncovered players in diverse aspects of planarian neural regeneration and neurobiology ." ], [ "To devise an unbiased list of genes that could promote brain regeneration , we first identified transcripts upregulated during planarian head regrowth .", "We chose an approach in which we amputated planarians postpharyngeally ( Figure 1—figure supplement 1A ) , reasoning that this amputation would allow us to identify global gene-expression changes , as well as changes that occur in the regenerating region itself .", "To identify key time points , we performed several time courses to examine morphological restoration and resumed expression of known brain markers following this particular injury .", "We examined regenerating animals at 12 hr or 24 hr intervals by immunofluorescence and in situ hybridization ( ISH , Figure 1—figure supplement 1B ) .", "We confirmed reestablishment of anterior polarity by 12 hr , initial neural gene expression at 36 hr , primordial brain formation after 3 days ( 72 hr ) , and further maturation ( with synapsin staining and broad neural gene expression ) at 5 days post-amputation ( Figure 1—figure supplement 1B ) .", "Based on these time courses , we chose 12 hr , 36 hr , and 72 hr as key time points that span the early stages of brain regeneration ( Figure 1B ) .", "To identify transcripts upregulated during head regeneration , we performed RNA sequencing on regenerating planarians at three time points ( 12 , 36 , and 72 hr ) as well as whole ( non-regenerating ) planarians and planarians immediately after amputation ( ‘cut’ ) as controls ( Supplementary file 1 ) .", "Over 900 transcripts were upregulated ( ≥2x , p≤0 . 01 ) at one or more time points in regenerating animals compared to cut ( 0 hr ) controls that were initially identical to the regenerating animals ( Figure 1C and Supplementary file 1 ) .", "Furthermore , ~45% of transcripts upregulated vs . cut controls at each time point were also upregulated vs . whole animals ( Figure 1D ) , indicating that differentially expressed genes do not simply reflect a return to homeostatic gene expression .", "Our data were also subjected to weighted gene correlation network analysis ( Supplementary file 2 ) so that clusters of similarly regulated genes could be identified .", "Trends for the most abundant clusters of upregulated genes are shown ( Figure 1—figure supplement 2A ) ; these modules contain known regulators of neural biology and anterior polarity including coe and lhx1/5–1 ( module 6 ) , foxD ( module 7 ) , and notum ( module 8 ) ( Petersen and Reddien , 2008; Gurley et al . , 2008; Petersen and Reddien , 2011; Koinuma et al . , 2003; Cowles et al . , 2013; Currie and Pearson , 2013 ) .", "We next sought to examine upregulated genes in more detail .", "Of the genes upregulated compared to cut controls , we eliminated transcripts that were very low abundance , parts of repetitive sequences , or housekeeping genes and cloned 428/933 transcripts for further analyses ( Figure 1E ) .", "We examined the gene expression patterns of these genes by ISH and were able to establish expression patterns for ~85% of them ( 362/428 ) .", "We hypothesized that our dataset would be enriched in genes expressed in the CNS and , indeed , found that 47% of genes with clear expression patterns showed enrichment in the CNS ( 170/362 , Figure 1F–G ) .", "Of the 170 genes with CNS expression , 132 were expressed broadly and 38 showed enrichment in subsets of CNS cells ( Figure 1F–G ) .", "Additionally , genes expressed in the CNS were often expressed elsewhere , for example in the parenchyma or in the intestine ( Figure 1G ) .", "Of upregulated genes with detectable expression patterns , we also found that 9% showed enriched expression in the head ( Figure 1—figure supplement 2B–C ) and 17% were expressed in the parenchyma , some in a pattern similar to neoblast genes ( Figure 1—figure supplement 2D–E ) .", "Additional genes were expressed in tissue-specific patterns that included the pharynx , intestine , protonephridia , epithelium , and eyespots ( Figure 1—figure supplement 2F–G ) .", "Some non-CNS expression patterns could still reflect neural tissue in the pharynx , body wall , or eyes , but we have not investigated neural regeneration outside the CNS at this point .", "However , the variety of expression patterns reflects the diverse physiological changes that occur concurrently during head regeneration ( Supplementary file 3A ) .", "To determine whether the upregulated genes promote brain regeneration , we performed RNA interference ( RNAi ) experiments to knock down 326 of the upregulated transcripts ( Figure 2A ) .", "These genes included all those enriched in the CNS , head , or parenchyma , as well as a subset of genes with other expression patterns or for which no pattern was detected .", "After RNAi we examined brain regeneration by performing ISH to detect choline acetyltransferase ( ChAT ) mRNA , which is expressed in an abundant cell type in the CNS ( Nishimura et al . , 2010 ) .", "We validated genes that caused a small brain upon regeneration by repeating RNAi and ISH , this time quantifying brain area after regeneration ( Figure 2B , Supplementary file 3B ) .", "Of the genes screened , 9 . 2% ( 30/326 ) caused a significant reduction in brain area after RNAi ( Figure 2C ) .", "A wide range of severity was observed for genes associated with this phenotype , with relative brain areas ranging from 16–80% of controls .", "RNAi targeting two additional genes caused nervous system regeneration phenotypes without a small brain ( Supplementary file 3B ) : sp6-9 ( RNAi ) caused defects in eye regeneration ( Lapan and Reddien , 2011 ) and arrowhead ( RNAi ) caused defects at the midline of the brain which will be described below .", "If RNAi animals showed gross phenotypes like lysis or curling prior to amputation or regeneration , they were killed and fixed when a phenotype was first observed ( Supplementary file 3C , Figure 2—figure supplement 2 ) . 10 . 7554/eLife . 17002 . 006Figure 2 . A screen for genes required for regeneration of the planarian brain .", "( A ) A diagram depicting the RNAi protocol used for our functional screen .", "For each of the 326 genes analyzed in our study , dsRNA was synthesized and fed to animals three times over 10–11 days .", "Five days after the final dsRNA feeding , animals were cut prepharyngeally and allowed to regenerate for 6 days .", "The animals were then killed and fixed for ISH to visualize the CNS .", "Animals were also monitored for behavioral defects prior to the termination of the experiment .", "When animals manifested phenotypes earlier than the conclusion of the experiment ( e . g . , lysis or curling ) , they were fixed when such a phenotype was first observed .", "( B ) Example of RNAi animals used for brain area quantification .", "Control ( RNAi ) and soxB2-2 ( RNAi ) animals were subjected to ISH with the choline acetyltransferase ( ChAT ) probe .", "After imaging , the body area ( red line ) and brain area ( yellow line ) were calculated for each sample .", "( C ) Brain area/body area ratios were averaged across ~8 worms per sample and were normalized to control ( RNAi ) animals .", "All 30 genes for which RNAi caused a significant reduction in brain area are shown here , with error bars representing SEM .", "Genes for which RNAi also caused reduction in an anterior marker are indicated with blue bars .", "Red bars indicate genes for which RNAi also affected stem cell maintenance .", "Bars with grey diagonal lines indicate genes for which RNAi also caused small blastemas after amputation .", "Scale bar: 500 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 17002 . 00610 . 7554/eLife . 17002 . 007Figure 2—figure supplement 1 . Further analysis of genes with functions in CNS regeneration .", "( A ) The expression patterns of the genes identified as having roles in CNS regeneration are shown in the main text ( Figures 3 , 4 ) or in this panel .", "Expression patterns of uninjured worms are shown in the top panels , while expression of each gene in a 2 d regenerating worm ( cut postpharyngeally ) is shown below .", "( B ) Reestablishment of anterior polarity , using sFRP-1 as a marker , after gene knockdown with the indicated dsRNA .", "Knockdown of several genes caused reduction of sFRP-1 expression which ranged from minor to severe .", "( C ) The stem cell pool was examined using smedwi-1 after RNAi targeting control or target genes .", "These three genes showed some decrease in smedwi-1 staining .", "Scale bars: 500 µm ( A–C ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 17002 . 00710 . 7554/eLife . 17002 . 008Figure 2—figure supplement 2 . Upregulated genes play roles in tissue maintenance and stem cell biology .", "( A ) Knockdowns of the indicated genes show phenotypes including head regression , curling , and lysis .", "Live animals are shown after dsRNA feedings prior to amputation or after regeneration as indicated .", "( B–C ) smedwi-1 expression was used to determine the effect of each RNAi knockdown on the stem cell pool .", "Animals were prepared for ISH either after regeneration ( B ) or in uninjured animals ( C ) depending on the timing of phenotype presentation .", "Many genes identified in this study perturb smedwi-1 expression and likely disrupt stem cell maintenance and/or function .", "( D ) Gene expression patterns of the genes with roles in homeostasis are shown .", "Scale bars: 500 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 17002 . 008 Genes associated with CNS-regeneration phenotypes were expressed in a variety of patterns , including neural , parenchymal , and ubiquitous expression ( Figures 3–4 , Figure 2—figure supplement 1A ) .", "Most were upregulated in the anterior-most tissue in regenerating tail fragments ( Figures 3–4 , Figure 2—figure supplement 1A ) , though patterns ranged from small subsets of cells ( e . g . , AADC , Figure 2—figure supplement 1A ) to broad upregulation throughout the remaining tissue ( e . g . , tRNA synthase , Figure 2—figure supplement 1A ) .", "A prior study examined the expression of genes in stem cells and their progeny ( Labbé et al . , 2012 ) , and a number of genes identified in our functional screen are also expressed in these cell populations ( Supplementary file 3B ) .", "Genes with roles in regeneration belonged to several expression modules in our clustering analysis ( Supplementary file 2 and Supplementary file 3B ) with module 7 appearing most frequently ( 7/30 , Figure 1—figure supplement 2A ) .", "This module includes 600 genes that are modestly upregulated at 12 hr and more dramatically so at 36 and 72 hr post-amputation ( Supplementary file 2 ) .", "The spatiotemporal diversity of gene expression patterns suggests that these genes likely affect regeneration through multiple mechanisms . 10 . 7554/eLife . 17002 . 009Figure 3 . Genes important for brain regeneration exert their effects through multiple mechanisms .", "( A ) Expression of soxB2-2 by ISH during homeostasis .", "soxB2-2 cells are scarce , but mostly localized near the brain .", "( B ) soxB2-2 expression was also determined by ISH in animals cut postpharyngeally and allowed to regenerate for two days .", "( C ) RNA was prepared from whole control ( RNAi ) and soxB2-2 ( RNAi ) animals at the end of a treatment course depicted in Figure 2A .", "qPCR was used to examine the expression of 134 genes after RNAi .", "Selected qPCR results are shown here .", "The bar graph is color coded to indicate the expression of the gene targeted by RNAi ( black/grey ) , anterior polarity genes ( blue ) , genes important for neurotransmitter biogenesis ( red/pink ) , and genes with suspected roles in neural differentiation or neural progenitors ( dark/light green ) .", "soxB2-2 was knocked down to 44% of control ( RNAi ) levels after RNAi .", "Significant decreases in hlh-1 , otxA , and POU2/3-2 were also seen after soxB2-2 ( RNAi ) .", "Similarly , expression patterns and downstream effects were determined for runt ( D–F ) , hesl-3 ( G–I ) , mblk ( J–L ) , and CRELD ( M–O ) .", "runt also affected neural progenitor genes , while hesl-3 primarily affected polarity genes .", "No downregulated genes were identified after mblk ( RNAi ) or CRELD ( RNAi ) .", "Targets were knocked down to 31% ( runt ) , 34% ( hesl-3 ) , 20% ( mblk ) and 6% ( CRELD ) of control ( RNAi ) levels .", "Asterisks mark genes with a significant difference in expression ( p≤0 . 05 ) after RNAi .", "Scale bars: 500 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 17002 . 00910 . 7554/eLife . 17002 . 010Figure 3—figure supplement 1 . Upregulated genes play roles in diverse processes to promote regeneration .", "( A ) hlh-1 , POU2/3–2 , TH , and otxA were detected in subsets of cells in the planarian CNS by ISH .", "( B ) hlh-1 FISH ( green ) revealed cells near the neuropil in control ( RNAi ) worms but not soxB2-2 ( RNAi ) worms .", "Background speckles are present due to background because of the low abundance of the hlh-1 transcript .", "DAPI is shown in blue .", "( C ) Photoreceptor neurons were visualized after control ( RNAi ) and hesl-3 ( RNAi ) treatment using VC-1 antibody ( green ) .", "Eyespots were fused medially after hesl-3 ( RNAi ) .", "DAPI is shown in blue .", "( D ) By in situ hybridization ( ISH ) , foxD expression at the anterior-most point of the planarian body is reestablished after control ( RNAi ) but is diminished or absent after hesl-3 ( RNAi ) .", "( E ) Diagrams depict translations of the two isoforms of CRELD supported by transcriptomics ( Brandl et al . , 2016 ) .", "TM marks a predicted transmembrane domain .", "Epidermal growth factor-like domains ( EGF ) and a domain of unknown function ( DUF ) are also marked .", "Scale bars: 100 µm ( A , C ) , 10 µm ( B ) , 500 µm ( D ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 17002 . 01010 . 7554/eLife . 17002 . 011Figure 4 . Secreted molecules with roles in planarian brain regeneration .", "( A ) Expression patterns of LDLRR-1 , LDLRR-2 , LDLRR-3 , and F-spondin were determined by ISH for uninjured worms ( top ) and tail fragments two days after amputation ( bottom ) .", "( B ) The domain architectures of LDLRR-1 and LDLRR-3 .", "( C ) Coexpression of the indicated genes by fluorescent in situ hybridization ( FISH ) .", "All LDLRR-3+ cells express LDLRR-1 ( 16/16 ) , but only about half of LDLRR-1+cells express LDLRR-3 ( 16/31 ) .", "LDLRR-3+ cells do not express smedwi-1 ( 0/34 ) , although these cells are often found in proximity to one another .", "LDLRR-1 , LDLRR-3 , and F-spondin are not expressed in mhc+ muscle cells ( 0/8 , 0/23 , and 0/6 respectively ) .", "LDLRR FISH experiments were performed on whole worms and F-spondin FISH was performed on 2d regenerating animals .", "( D ) The domain architecture of F-spondin .", "Scale bars: 500 µm ( A ) , 20 µm ( C ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 17002 . 01110 . 7554/eLife . 17002 . 012Figure 4—figure supplement 1 . Parenchymal genes are irradiation-insensitive and do not influence wound response .", "( A ) Planarians were subjected to ISH with smedwi-1 , F-spondin , and LDLRR-3 probes after mock irradiation as well as 1 , 3 , and 7 days post-irradiation .", "While smedwi-1 transcript is lost quickly , F-Spondin and LDLRR-3 transcripts are still present at 7 dpi , when most animals were lysing and dying .", "( B ) Diagram of amputation schema for wound-response genes .", "Animals were fed dsRNA three times before amputation as in Figure 2A .", "( C ) Wound-response genes follistatin , jun , inhibin , and runt were examined by ISH after RNAi and amputation .", "All genes were upregulated despite knockdown of the genes indicated , suggesting that LDLRR and F-spondin genes do not inhibit the general wound response .", "Scale bars: 500 µm ( A , C ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 17002 . 012 As noted above , successful brain regeneration depends on several interrelated processes , not all of which are specific to the CNS .", "Thus , to distinguish between target genes whose knockdown specifically affected CNS regeneration and those which had indirect effects through other mechanisms , we repeated RNAi experiments and examined effects of each of the 30 genes on anterior polarity , stem cell maintenance , and regeneration of tail tissue .", "First , we examined the anterior marker sFRP-1 ( Petersen and Reddien , 2008; Gurley et al . , 2008 ) after RNAi of each gene .", "RNAi knockdown of 5 genes ( argininosuccinate synthase , autophagy-related 13 , smarcb1 , fzd-5/8–2 , and hesl-3 ) reduced sFRP-1 expression during regeneration , with reductions ranging from minor to severe ( Figure 2C-blue bars , Figure 2—figure supplement 1B ) .", "Thus , for these five genes , the small brain RNAi phenotype could result from insufficient reestablishment of anterior identity .", "We also investigated whether the stem cell pool was affected after RNAi knockdown of these 30 genes using the neoblast marker smedwi-1 ( Reddien et al . , 2005 ) .", "We determined that knockdown of three genes caused a reduction in smedwi-1 signal: snRNPC and rbp-1-like , which have not been described , and the previously identified gene soxP-1 ( Figure 2C-red bars , Figure 2—figure supplement 1C; Wagner et al . , 2012; Wenemoser et al . , 2012 ) .", "We concluded that RNAi of these three genes led to brain regeneration phenotypes secondary to stem cell defects .", "Finally , to confirm that brain regeneration phenotypes did not merely result from a general impairment in blastema formation ( i . e . general defects in neoblast proliferation or migration ) , we repeated each RNAi experiment and amputated animals anterior and posterior to the pharynx and subsequently measured blastema size after six days .", "RNAi of five genes ( tRNA synthase , GTPbp , argininosuccinate synthase , wdr24 , and smarcb1 ) caused smaller blastemas after regeneration and only GTPbp ( RNAi ) had an anterior-specific reduced blastema size ( Supplementary file 3B ) .", "Thus , these five genes likely play a role in regeneration that is not specific to the CNS ( Figure 2C-gray diagonal lines ) .", "RNAi of the remaining 19 genes caused no overt polarity or stem cell defects by ISH and no evidence of generally reduced regenerative activity ( Figure 2C-black bars ) .", "We thus prioritized these genes for further study , as these represent the most promising candidates for factors specifically influencing brain regeneration .", "Though our current work is focused on regeneration of the planarian CNS , the functional screen we performed resulted in the identification of 26 additional genes for which RNAi caused defects during homeostasis and/or regeneration ( Supplementary file 3C , Figure 2—figure supplement 2 ) .", "RNAi targeting 12 genes caused head regression and/or curling , while knockdown of 7 genes caused general lysis ( Supplementary file 3C , Figure 2—figure supplement 2A ) .", "We used ISH with the smedwi-1 probe and detected loss or reduction of neoblasts after knockdown of 13 of these genes ( Supplementary file 3C , Figure 2—figure supplement 2B–C ) ; in many of these cases ( e . g . , zfp-4 ) , the causative gene had not been implicated in planarian neoblast biology .", "As expected , most genes identified in this portion of the screen showed ubiquitous or parenchymal expression patterns ( Figure 2—figure supplement 2D ) with enrichment in stem cells and/or stem cell progeny ( Labbé et al . , 2012; Supplementary file 3C ) .", "Other knockdowns ( e . g . , exo70 , hnRNPK , and smarcc2 ) caused tissue homeostasis phenotypes despite the presence of stem cells ( Figure 2—figure supplement 2C ) , suggesting potential roles for these genes in proper stem cell function , tissue maintenance , or differentiation .", "One of these genes ( hnRNPK ) is required for pharynx regeneration ( Adler et al . , 2014 ) and here we show that it is important for head maintenance and regeneration .", "Therefore , hnRNPK could have a broad role in the maintenance or renewal of differentiated cell types .", "RNAi of one gene , jouberin , also caused bloating reminiscent of protonephridial defects ( Figure 2—figure supplement 2A ) .", "All other genes had no detectable phenotypes in our assays ( Supplementary file 3D ) .", "We investigated genes important for brain regeneration to determine whether we could assign them to known pathways or processes .", "To determine which downstream cells or pathways are affected , we repeated RNAi experiments for five genes ( soxB2-2 , runt , hesl-3 , mblk , and CRELD ) and used quantitative RT-PCR ( qPCR ) to examine the expression of ~134 genes after the 6-day regeneration period ( Supplementary file 3E–F ) .", "The genes chosen for qPCR analysis represent neural markers ( including neuropeptide genes and genes important for neurotransmitter production ) , markers of neural progenitors ( genes expressed in differentiating neoblasts and also associated with neural expression or differentiation ) , and polarity markers .", "We also included neoblast transcripts and genes expressed in progenitors of other tissues ( protonephridia , intestine , etc . ) to rule out general roles in stem cell maintenance or differentiation ( Supplementary file 3E–F ) .", "One of the biggest outstanding questions in understanding planarian regeneration is: what molecular cues regulate the process ?", "Among the 30 genes identified in our functional analysis as being required for proper brain regeneration , several genes ( including CRELD ) encoded putative signaling molecules .", "Of these genes , a few are expressed in differentiated cells ( Supplementary file 3B; Labbé et al . , 2012 ) .", "One such group of genes is the LDLRR ( low density lipoprotein receptor-related ) family .", "LDLRR-1 and LDLRR-2 were previously identified in an analysis of wound-response genes ( Wenemoser et al . , 2012 ) .", "LDLRR-1 is expressed in the parenchyma , while LDLRR-2 is expressed in a subset of intestinal cells and in epithelial cells , particularly in the head ( Figure 4A ) .", "Both show redistribution of expression after injury , with punctate expression in the blastema during regeneration ( Figure 4A ) .", "We also identified a gene with similarity to LDLRR-1 in our functional screen , which we have named LDLRR-3; this gene is also expressed in a pattern resembling that of LDLRR-1 ( Figure 4A ) .", "LDLRR-1 was so named for an LDLR ( Low Density Lipoprotein Receptor ) domain ( Wenemoser et al . , 2012 ) ; however , the current gene model for LDLRR-1 includes a signal peptide sequence but no transmembrane domain , suggesting that LDLRR-1 is likely to be a secreted molecule or to remain attached to the cell surface by other means ( Figure 4B ) .", "We were unable to detect known domains within the existing LDLRR-2 sequence , but LDLRR-3 has a domain architecture similar to LDLRR-1 and also lacks a transmembrane domain ( Figure 4B ) .", "Furthermore , LDLRR-1 and LDLRR-3 share closest similarity with proteins of the Heparan Sulfate Proteoglycan ( HSPG ) family ( Supplementary file 3G ) .", "HSPGs play diverse roles in regulation of the extracellular matrix and signaling ( for review , see Lin , 2004 ) .", "Thus , the planarian family of LDLRR molecules could serve to modulate signaling in a way that promotes regeneration .", "We asked whether LDLRR-3 is expressed in cell types already known to play key roles during regeneration .", "LDLRR-3 is not irradiation sensitive ( Figure 4—figure supplement 1A; Labbé et al . , 2012 ) and we confirmed that LDLRR-3+ cells in the parenchyma are distinct from ( but sometimes adjacent to ) smedwi-1+ stem cells ( Figure 4C ) .", "Because muscle cells secrete position-control factors that set up the body axis of planarians ( Witchley et al . , 2013 ) , we tested whether LDLRR-3 could be produced by muscle cells .", "We found that LDLRR-3+cells do not express the muscle marker mhc ( myosin heavy chain; Figure 4C; Kobayashi et al . , 1998 ) ; thus , LDLRR-3 is expressed in differentiated , non-muscle cells to promote regeneration .", "Furthermore , while LDLRR-1 and LDLRR-3 are expressed in similar patterns ( Figure 4A ) , their expression is only partly overlapping ( Figure 4C ) ; all LDLRR-3+ cells express LDLRR-1 , but only half of LDLRR-1+ cells express LDLRR-3 .", "As expected based on this finding , LDLRR-1 is also expressed in differentiated , mhc- cells ( Figure 4C; Labbé et al . , 2012 ) .", "Because LDLRR-1 was identified as a wound response gene ( Wenemoser et al . , 2012 ) , we examined other wound response genes ( follistatin , jun , inhibin , and runt ) in LDLRR-1 ( RNAi ) and LDLRR-3 ( RNAi ) animals .", "We were not able to observe any diminished wound response after these perturbations ( Figure 4—figure supplement 1B–C ) .", "In addition to the LDLRR family , a gene encoding planarian F-spondin was identified as important for CNS regeneration in our functional screen .", "F-spondin is also expressed in parenchymal , differentiated cells that are not mhc+ ( Figure 4A , C , Figure 4—figure supplement 1A ) .", "Furthermore , the predicted planarian F-spondin protein possesses a signal peptide but lacks a transmembrane domain ( Figure 4D ) , suggesting that it is secreted , like its homologs in other species ( Feinstein and Klar , 2004 ) .", "We thus conclude that one or more parenchymal cell types produce cues ( including planarian LDLRR and F-spondin proteins ) that promote regeneration of the CNS and/or regeneration more generally .", "In addition to exploring genes that are required for brain regeneration , we were interested in examining genes that function in more specific aspects of neural architecture .", "We hypothesized that regionally expressed genes might affect local connectivity or function in the regenerating planarian CNS .", "One subset of genes upregulated during regeneration was expressed medially in the planarian brain ( Figure 5A ) .", "RNAi targeting these genes did not cause small brain phenotypes ( data not shown ) .", "However , we did detect a specific phenotype after targeting the planarian homolog of arrowhead , a gene that encodes a LIM-homeodomain transcription factor ( Curtiss and Heilig , 1995 , 1997 ) .", "Planarian arrowhead is expressed in neurons , at least some of which are ChAT+ ( Figure 5A–B ) .", "Despite normal brain size , arrowhead ( RNAi ) animals regenerated with a gap between the lobes of the cephalic ganglia ( Figure 5C , arrows ) .", "The medial gap was more clearly visualized with an anti-synapsin antibody that stains the planarian neuropil , a region rich in axons , dendrites , and synapses ( Figure 5D–E ) .", "The anterior commissure is the largest commissure in the planarian CNS and connects the two lobes of the cephalic ganglia ( Figure 1A ) .", "Many axons cross the midline at the anterior commissure , including photoreceptor axons ( Agata et al . , 1998 ) .", "We detected photoreceptor neurons using an anti-arrestin antibody ( VC-1; Agata et al . , 1998 ) and determined that photoreceptor axons appeared frayed and/or failed to cross the midline in a majority of arrowhead ( RNAi ) animals ( Figure 5F–G ) .", "Failure of photoreceptor axons to cross at the anterior commissure was never detected after control ( RNAi ) ( Figure 5F–G ) .", "One factor previously shown to regulate axon guidance in planarians is slit ( Cebrià et al . , 2007 ) .", "While some arrowhead+ cells are slit+ , the overall slit expression pattern was unaffected after arrowhead ( RNAi ) ( Figure 5—figure supplement 1A , B ) .", "Taken together , our results suggest that arrowhead regulates cells or factors that drive reconnection of the planarian brain lobes and organization of newly regenerated axons at the anterior commissure . 10 . 7554/eLife . 17002 . 015Figure 5 . arrowhead is required for regeneration of medial structures in the planarian CNS .", "( A ) Five upregulated genes were shown to be expressed medially in the CNS .", "These expression patterns were not overlapping ( data not shown ) .", "( B ) arrowhead is expressed in neurons , many of which are ChAT+ ( 12/27 ) .", "( C ) arrowhead ( RNAi ) did not cause a small brain phenotype , but ISH with ChAT revealed a gap between the cephalic ganglia after regeneration .", "Arrows denote the location between cephalic ganglia in both samples .", "( D ) The gap at the midline in arrowhead ( RNAi ) regenerates was visualized with an anti-synapsin antibody that stains the neuropil of the CNS .", "( E ) 86 . 8% of arrowhead ( RNAi ) animals showed a gap at the midline , while this gap was not visible in any control ( RNAi ) animals ( N = 38 and 40 , respectively ) .", "( F ) Photoreceptor neurons were visualized with the anti-arrestin ( VC-1 ) antibody after regeneration of control ( RNAi ) and arrowhead ( RNAi ) animals .", "( G ) Photoreceptor neurons were disorganized ( 19% ) and often failed to cross the midline ( 38% ) in arrowhead ( RNAi ) animals ( N = 21 ) .", "Scale bars: 500 µm ( A , C ) , 20 µm ( B ) , 100 µm ( D , F ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 17002 . 01510 . 7554/eLife . 17002 . 016Figure 5—figure supplement 1 . arrowhead and slit at the midline of the planarian brain .", "( A ) arrowhead and slit are coexpressed in some cells near the anterior midline of the cephalic ganglia ( arrow ) .", "( B ) slit expression is not visibly altered in arrowhead ( RNAi ) animals vs . controls .", "Scale bars: 10 µm ( A ) , 500 µm ( B ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 17002 . 016 As described previously , brain regeneration involves not only the production of properly patterned tissue , but also a restoration of function .", "Planarian brain branches have been ascribed functions in mechanosensing and/or chemosensing ( Oviedo et al . , 2003 , Umesono et al . , 1997 , Nakazawa et al . , 2003 ) and the auricles at the edge of the head of other planarian species have been shown to be important for chemosensory behavior ( Koehler , 1932 , Farnesi and Tei , 1980 ) .", "The expression of CNG1 , which encodes a cyclic nucleotide-gated ( CNG ) channel , as well as other homologs of olfactory signaling proteins in the brain branches ( Figure 6A ) , supported the hypothesis that these structures mediate functions specific to chemosensing .", "We observed a particularly interesting expression pattern for CNG1 , which was expressed in regions at the very edge of the head ( Figure 6A–B ) , a location expected for cells serving in a sensory capacity .", "CNG channels play important signaling roles in the retina and olfactory neurons of other organisms , being activated by cyclic nucleotides ( cyclic AMP or cyclic GMP ) downstream of sensory signaling ( Pifferi et al . , 2006; Figure 6—figure supplement 1C , D ) . 10 . 7554/eLife . 17002 . 017Figure 6 . FLI-1 is expressed in the brain branches and functions in reacquisition of chemosensory behavior during regeneration .", "( A ) Fourteen upregulated genes were expressed in the brain branches of the planarian head .", "Expression patterns ranged from single rows of cells to broad expression in these structures .", "( B ) CNG1 is expressed in large , sub-epithelial regions of the planarian head margin .", "( C ) Knockdown of FLI-1 prior to regeneration caused a reduction of feeding in a maze-based experiment ( shown in Figure 6—figure supplement 1E–H ) .", "Only 40% of FLI-1 ( RNAi ) animals ate in this assay , compared to 90% of control ( RNAi ) animals ( 9–10 animals each condition for each replicate , 3 replicates ) .", "Error bars depict SEM; results were significant with p=6 . 8 × 10−5 .", "( D ) FLI-1 is expressed in lateral structures of the planarian brain ( here marked with ChAT ) .", "( E ) Some FLI-1+ cells express ChAT and GluR1 ( 7/42 and 12/44 respectively ) .", "Scale bars: 500 µm ( A ) , 20 µm ( B , E ) , 100 µm ( D ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 17002 . 01710 . 7554/eLife . 17002 . 018Figure 6—figure supplement 1 . Assaying chemosensory function in the brain branches .", "( A ) Upper panel: brain branch markers like hyp-c47858 are expressed lateral to the main cephalic ganglia structures ( here marked with PC-2 ) .", "Lower panel: brain branch structures are labeled with GluR1 and newly identified brain branch genes are expressed in these structures .", "( B ) Some hyp-c47858+ cells express GluR1 ( 12/44 ) .", "( C ) CNG channels function downstream of cyclic AMP or cyclic GMP signaling ( reviewed in Pifferi et al . , 2006 ) .", "( D ) Conserved components of olfactory signaling are also expressed in cells of the brain branches .", "( E–G )", "Diagrams and photo of the maze used for feeding assay that relies on chemosensory behavior .", "Dimensions are shown in ( E ) and the placement of food and animals is shown in ( G ) .", "( H ) The time course used for behavioral RNAi experiments .", "( I ) control ( RNAi ) and FLI-1 ( RNAi ) animals were placed in the interior-most 1 . 5 cm of a 6 cm petri dish and were allowed 15 min to move about the petri dish .", "At the conclusion of the time period , the number of worms outside the inner-most circle were counted; 59 . 2% of control ( RNAi ) animals ( 16/27 ) were outside the circle at this point and 56% of FLI-1 ( RNAi ) animals were outside the circle in this assay , indicating similar movement after FLI-1 ( RNAi ) ( J ) .", "The difference between samples was not significant .", "Scale bars: 500 µm ( D ) , 40 µm ( A , B ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 17002 . 018 Because of the parallels between brain branches and chemosensory organs of other species , we sought to explore the roles of brain branch-expressed genes following regeneration .", "The 14 brain branch genes ( Figure 6A ) were expressed in a broad variety of patterns , ranging from single rows of cells ( hyp-c47858 and cdc45-like ) to broad expression throughout the brain branches ( DL1 and FLI-1 ) .", "We detected these transcripts in cells lateral to the neuropil of the cephalic ganglia , in or near cells expressing the classical brain branch marker GluR1 ( Cebrià et al . , 2002a; Figure 6—figure supplement 1A–B and data not shown ) .", "To understand the function of these genes , we first examined whether the 14 brain branch genes affected regeneration of overall brain branch structure .", "As mentioned previously , mblk ( RNAi ) affects brain size during regeneration , but the other genes caused no such defect .", "Brain branches , detected by GluR1 ISH , were also present after knockdown of these 14 genes ( data not shown ) .", "To test whether brain branch-expressed genes promote chemosensory behavior , we repeated all RNAi treatments , this time assaying regenerated worms for their ability to find food in a simple maze ( Figure 6—figure supplement 1E–H ) .", "The maze was designed so that planarians had to detect chemicals from their food ( liver paste ) and travel ~45 cm toward the food in order to eat .", "FLI-1 ( RNAi ) animals consistently showed significantly reduced completion of the maze-based feeding assay , compared to control ( RNAi ) animals ( Figure 6C ) .", "We confirmed the ability of FLI-1 ( RNAi ) animals to move and to eat food ( without traveling a maze ) to rule out general defects in movement ( Figure 6—figure supplement 1I–J , data not shown ) .", "FLI-1 encodes an ETS-family transcription factor that is expressed in numerous cells of the planarian brain branches ( Figure 6A , D ) .", "A minority of FLI-1+ cells express ChAT or GluR1 ( Figure 6E , arrowheads ) , suggesting that the FLI-1+ population likely represents a heterogeneous population of neural cells .", "Taken together , our findings:", "i ) support a role for the planarian brain branches in chemosensory behavior and", "ii ) suggest that FLI-1 is important for regeneration of some aspect of CNS structure or connectivity that restores chemosensory function during regeneration .", "Furthermore , our analysis of these genes reveals a previously underappreciated complexity to the brain branch structures , which must be reestablished during regeneration .", "In addition to exploring the genes that are upregulated during regeneration as potential drivers of the process , we also investigated genes downregulated ( ≥2x , p≤0 . 01 ) after injury as potential inhibitors of regeneration .", "As proof of principle , we found that the planarian homolog of activin , an inhibitor of regeneration ( Roberts-Galbraith and Newmark , 2013 , Gavino et al . , 2013 ) , is downregulated during regeneration ( Supplementary file 1 ) .", "We identified 534 downregulated transcripts , with the number of downregulated genes increasing between 12 hr and 72 hr post-amputation ( Figure 7A ) .", "We were interested in the types of cells that express these downregulated genes , so we cloned 70 genes and determined the expression patterns of 58 by ISH ( Supplementary file 3H ) .", "The most frequent expression patterns for these genes were in the peripharyngeal secretory cells and the intestine ( 39% and 24% , respectively , Figure 7—figure supplement 1A–C ) , suggesting that cell loss or a change in the physiology of these organ systems occurs during the early stages of planarian head regeneration .", "We also detected a number of genes expressed in posterior regions ( Figure 7—figure supplement 1D ) , an expected pattern as posterior genes likely need to be downregulated after amputation to permit rescaling of the body axis .", "Ultimately , we determined that 8/58 genes were expressed in the CNS ( 14% , Figure 7B–C ) .", "This proportion was about one third that of CNS expression patterns in the upregulated gene dataset . 10 . 7554/eLife . 17002 . 019Figure 7 . Expression profiling of genes downregulated during head regeneration .", "( A ) Venn diagram showing the 534 genes significantly downregulated compared to cut controls ( ≥2x downregulated , p≤0 . 01 ) .", "The number of downregulated genes increased over time .", "( B ) Pie chart depicting the fraction of downregulated genes examined that are expressed in the CNS ( 8 of 58 with clear expression patterns ) .", "( C ) ISH showing expression of several downregulated genes in the planarian CNS .", "IF-1 is expressed in a pattern closely resembling the neuropil , while other markers ( OBP9 , elav-4 , and hyp-22479 ) are typical of neural expression .", "Scale bars: 500 µm ( C ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 17002 . 01910 . 7554/eLife . 17002 . 020Figure 7—figure supplement 1 . Expression patterns of genes downregulated during head regeneration .", "( A ) The predominant gene expression patterns of downregulated genes were in the peripharyngeal secretory region ( 20/58 , 34 . 4% ) and gut ( 16/58 , 27 . 6% ) .", "Secretory expression patterns are shown in ( B ) and intestinal expression patterns are shown in ( C ) .", "Some genes were also expressed in posterior patterns ( D ) .", "Scale bars: 500 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 17002 . 020 Of the downregulated genes expressed in the planarian CNS , one expression pattern was distinct .", "Expression of an intermediate filament gene ( IF-1 ) was detected in the CNS in a pattern similar to antibody markers of the neuropil and different than neural gene expression , which typically marks cell bodies around the CNS ( Figure 7C ) .", "The expression pattern of IF-1 led us to hypothesize that it is expressed in a non-neural subset of cells in the CNS .", "To test this possibility , we examined whether IF-1 was coexpressed with markers of abundant neural types , including PC-2 and ChAT .", "Indeed , IF-1 was expressed in cells present in the neuropil that did not express neural markers ( Figure 8A , Figure 8—figure supplement 1A , data not shown ) . 10 . 7554/eLife . 17002 . 021Figure 8 . Intermediate Filament ( IF-1 ) gene expression marks planarian glial cells .", "( A ) FISH was used to show that IF-1 is not coexpressed with prohormone convertase-2 ( PC-2 , 0/25 IF-1+ cells were PC-2+ ) .", "( B ) slc1a-5 and glutamine synthetase 1 ( GS-1 ) , genes required for glutamate uptake and conversion to glutamine are also expressed in an IF-1-like neuropil pattern .", "( C ) IF-1 is co-expressed with slc1a-5 and GS-1 ( 46/62 IF-1+ cells were slc1a-5+ and 56/56 IF-1+ cells were GS-1+ ) .", "IF-1+cells also express the solute carrier genes slc1a-3 ( 33/33 ) , slc2a-1 ( 76/76 ) , slc6a-2 ( 31/35 ) , slc6a-8 ( 48/48 ) and slc7a-8 ( 63/63 ) .", "Overlap was near perfect for some genes ( all slc7a-8+ cells express IF-1 ) , but other genes are more widely expressed ( only 48/347 slc6a-8+ cells express IF-1 ) .", "( D ) Two genes from module 2 ( a downregulated gene cluster ) are also expressed in a neuropil-like pattern .", "jagged-like is expressed in puncta and a novel gene , estrella , is expressed strongly in the neuropil and in cells around the planarian body .", "( E ) IF-1+ cells express jagged-like and estrella ( 48/48 and 61/61 , respectively ) .", "( F–G )", "FISH with the estrella probe marks cells with extensive cytoplasmic projections in the neuropil , in the periphery of the animal body , near the brain branches , and in the eye , suggesting that estrella+ cells surround many components of the planarian nervous system .", "( H ) An electron micrograph of a putative planarian glial cell in the neuropil of the ventral nerve cord .", "The cell membrane ( determined by images acquired at higher magnification ) is marked in magenta and the nuclear envelope is shown in cyan .", "The cytoplasm is more electron dense than that in surrounding axons .", "The nuclear morphology is elongated , with heterochromatin at the nuclear periphery; these features are not present in neurons visualized by EM .", "( I ) A partial reconstruction of the cell in ( H ) through ten serial sections .", "The cell membrane and nuclear envelope are colored as above .", "Scale bars: 500 µm ( B , D ) , 20 µm ( A , C , E , G ) , 100 µm ( F ) , 5 µm ( H–I ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 17002 . 02110 . 7554/eLife . 17002 . 022Figure 8—figure supplement 1 . Characterization of planarian glial cells .", "( A ) A whole-animal double FISH showing the expression of IF-1 in PC-2- cells throughout the neuropil of the animal .", "( B ) slc1a-5 is also expressed in cells that do not express PC-2 or ChAT ( 0/8 for each gene ) .", "( C ) ISH showing solute carrier genes ( slc ) expressed in glial cells .", "( D ) Module 2 from our clustering analysis contains IF-1 and many other downregulated genes .", "High gene expression is shown in red while downregulated gene expression is shown in green .", "( E ) The average expression levels of genes in module 2 across samples is shown .", "This graph is configured as in Figure 1—figure supplement 2A .", "Scale bars: 100 µm ( A ) , 20 µm ( B ) , 500 µm ( C ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 17002 . 022 The nervous systems of many metazoans contain non-neural support cells that are broadly called glia .", "The existence of glial cells in planarians has been a matter of debate: electron microscopy analyses have been used to argue both for and against the presence of such cells ( Morita and Best , 1966 , Lentz , 1967 , Baguñà and Ballester , 1978 , Golubev , 1988 ) .", "The identification of IF-1+ glia is significant , especially given", "( i ) the downregulation of IF-1 as an injury response and", "( ii ) the tendency of glial cells in other organisms to affect neural regeneration ( positively or adversely depending on the situation – for review , see Pekny et al . , 2014 , and for more recent results see Anderson et al . ( 2016 ) ) .", "IF-1 itself shares some similarity with intermediate filament genes expressed in neurons ( vertebrate neurofilament and C . elegans intermediate filament , ifa-1 ) and glia ( vertebrate glial fibrillary acid protein ) ( Supplementary file 3G ) .", "Thus , to determine whether the IF-1+ cells of the planarian nervous system share similarities with glial cells in other organisms , we sought to identify genes coexpressed with IF-1 .", "One frequent function of glial cells is to take up excess neurotransmitters or other chemicals that can be toxic to nearby neurons .", "For example , astrocytes take up glutamate and convert it to glutamine ( Schousboe et al . , 1977 ) .", "Glutamate uptake occurs through excitatory amino acid transporters and conversion of glutamate to glutamine is catalyzed by glutamine synthetase ( Rothstein et al . , 1994 , Norenberg and Martinez-Hernandez , 1979 ) .", "We therefore investigated the expression patterns of planarian slc1a-5 ( solute carrier 1a-5 / EAAT ) and GS-1 ( glutamine synthetase-1 ) and found that they were also expressed in a pattern similar to IF-1 ( Figure 8B ) .", "Furthermore , we detected IF-1 coexpression with both slc1a-5 and GS-1 ( Figure 8C ) .", "We also confirmed non-neural expression of slc1a-5 using both PC-2 and ChAT as markers of neurons ( Figure 8—figure supplement 1B ) .", "Recently , a comprehensive characterization of solute carrier proteins in S . mediterranea was completed ( Vu et al . , 2015 ) .", "We assayed the expression patterns of several additional solute carrier genes and determined that IF-1+ cells express slc1a-3 ( an additional glutamate/neutral amino acid transporter ) , slc2a-1 ( a facilitative glutamate transporter ) , slc6a-2 and slc6a-8 ( sodium and chloride-dependent sodium:neurotransmitter transporters ) , and slc7a-8 ( a cationic amino acid transporter ) ( Figure 8C , Figure 8—figure supplement 1C ) .", "Importantly , many of the genes coexpressed with IF-1 are not differentially expressed ( slc1a-5 and slc6a-2 ) or are even slightly upregulated during regeneration ( slc2a-1 , GS-1 , and slc7a-8 ) .", "Taken together , these results indicate that IF-1+ glial cells in planarians are similar to glial cells ( and in particular astrocytes ) in other organisms , in that they express a variety of transporters of the solute carrier family ( Cahoy et al . , 2008 ) .", "Furthermore , because most markers coexpressed with IF-1 are not reduced after amputation , we conclude that glial cells are not lost after injury , but instead alter their gene expression .", "We also sought an unbiased way of identifying genes coexpressed with IF-1 , particularly additional genes downregulated during head regeneration .", "Reasoning that genes that behave like IF-1 during regeneration might meet both criteria , we turned to our clustering analysis , and focused on module 2 , a cluster of 1520 genes ( including IF-1 ) that are downregulated by 12 hr post-amputation and even more significantly downregulated at 36 hr and 72 hr ( Figure 8—figure supplement 1D–E ) .", "As a pilot study , we cloned 48 genes from this module and examined their expression patterns .", "The majority of these genes showed expression patterns irrelevant to our glial studies .", "However , two transcripts were expressed in IF-1+cells ( Figure 8D–E ) .", "jagged-like encodes a protein with weak similarity to Jagged that contains a signal peptide , two EGF domains , and a transmembrane domain .", "We also identified a gene that encodes a signal peptide-containing protein; we named this novel gene estrella ( 'star' in Catalan ) based on its expression pattern and the origin of our S . mediterranea strain in Barcelona .", "The expression pattern of estrella highlights the extensive processes of glial cells and their wide distribution in the neuropil , the peripheral nervous system , near the brain branches , and near photoreceptors ( Figure 8F–G; Cowles et al . , 2014 ) .", "Together , our results indicate that IF-1+ glial cells respond to injury by downregulating a variety of genes ( including genes that encode potential signaling molecules ) over the course of several days .", "Finally , we used electron microscopy to confirm the existence of glial cells in the planarian neuropil .", "Indeed , we detected cells in the neuropil that had numerous fine cellular processes that intermingled with axons ( Figure 8H–I , Video 3–4 ) .", "Planarian glial cells were also distinct from nearby neurons in that they had electron-dense cytoplasm and elongated nuclei with abundant peripheral heterochromatin ( Figure 8H ) .", "Throughout the neuropil , where neuronal processes are a dominant feature , electron-dense projections were also common ( Video 5 ) , suggesting extensive projection of glial cells throughout the neuropil of the planarian nervous system .", "Though we have not yet shown that the cells identified by electron microscopy are IF-1+ , the morphology seen by electron microscopy is consistent with the cell shapes revealed by estrella gene expression ( Figure 8F–G ) .", "We therefore conclude that both our gene expression data and electron microscopic analysis support the existence of glia in the planarian CNS . 10 . 7554/eLife . 17002 . 023Video 3 . Electron micrographs of the glial cell shown in Figure 8H through serial sections . DOI: http://dx . doi . org/10 . 7554/eLife . 17002 . 02310 . 7554/eLife . 17002 . 024Video 4 . Rotation of the reconstruction shown in Figure 8I . DOI: http://dx . doi . org/10 . 7554/eLife . 17002 . 02410 . 7554/eLife . 17002 . 025Video 5 . Close-up from serial sections showing electron-dense projections of glial cells among axons in the neuropil . DOI: http://dx . doi . org/10 . 7554/eLife . 17002 . 025" ], [ "To better understand regeneration , it will be important to identify the signals that initiate and direct the process .", "Some signals , from small molecules to proteins , have been identified in other regenerative model organisms ( e . g . , Wnt3 in Hydra , H2O2 and cysteinyl leukotriene in zebrafish , a MARCKS-like protein in axolotl , and newt anterior gradient protein; Chera et al . , 2009 , Kyritsis et al . , 2012 , Niethammer et al . , 2009 , Kumar et al . , 2007 , Sugiura et al . , 2016 ) .", "Surprisingly , the signals that promote regeneration in planarians remain mysterious .", "Notwithstanding a host of signaling molecules known to establish polarity in the regenerating worm , only a few signaling molecules or their receptors have been determined to regulate regeneration , including opposing Follistatin and Activin signals and EGF- and FGF-receptors that regulate various aspects of homeostasis and regeneration ( Roberts-Galbraith and Newmark , 2013 , Gavino et al . , 2013 , Fraguas et al . , 2011 , Ogawa et al . , 2002 , Wagner et al . , 2012 , Lei et al . , 2016 ) .", "Importantly , though planarian neoblasts have been hypothesized to integrate a wide variety of signals to choose between daughter cell fates ( Reddien , 2013 ) , whether neoblasts express the diversity of receptors required to integrate numerous signals remains unknown .", "Neoblasts do express FGF-receptors that are important for tissue maintenance ( Ogawa et al . , 2002 , Wagner et al . , 2012 ) , but no ligands for these receptors have been identified through homology searches .", "Thus , further exploring the signals required for regeneration , as well as the cellular origin ( s ) and receiver ( s ) of each signal will greatly enhance our understanding of how diverse cellular events ( i . e . , proliferation , migration , organization , differentiation ) are triggered and coordinated during the regenerative response .", "One goal of our screen was to identify putative signals that influence regeneration , generally , or for the CNS in particular .", "To that end , we identified several putative signaling molecules encoded by CRELD , F-spondin , and LDLRR family genes that each encode a secreted or transmembrane protein that could function in a signaling capacity .", "CRELD genes are conserved among metazoans but are not very well characterized .", "Two CRELD homologs exist in mammals .", "CRELD1 serves an essential function during mouse heart development and encodes a protein reported to have two transmembrane domains and a cytoplasmic EGF-like domain ( Mass et al . , 2014 ) .", "Conversely , CRELD2 encodes a secreted protein ( Oh-hashi et al . , 2011 ) .", "In mice , CRELD1 is expressed in the CNS in addition to its expression in the heart , and the Drosophila CRELD homolog ( CG11377 ) is expressed across multiple tissues , including the nervous system ( Mass et al . , 2014; Brown et al . , 2014 ) .", "Planarian CRELD is expressed in diverse cell types ( Wurtzel et al . , 2015 ) , including stem cell progeny ( Labbé et al . , 2012 ) , and is enriched in the brain and head .", "Smed-CRELD has two predicted isoforms ( Figure 3—figure supplement 1E; Brandl et al . , 2016 ) that encode a transmembrane protein and a secreted protein , each with extracellular EGF-like domains .", "Further studies will be required to determine how CRELD exerts its function in the regenerating head and to what extent this function might be conserved .", "Other signaling molecules identified in our study include homologs of matrix-associated proteins .", "F-spondin proteins are conserved among metazoans , localize to ECM-rich areas , and play roles in cell adhesion , including promoting axon extension from the neural floor plate ( Klar et al . , 1992; Umemiya et al . , 1997; Burstyn-Cohen et al . , 1998; Burstyn-Cohen et al . , 1999; Woo et al . , 2008 ) .", "Despite their names , planarian LDLRR proteins are most closely related to basement membrane-specific Heparan Sulfate Proteoglycans ( HSPGs ) in other metazoans .", "In other organisms , HSPGs are also ECM components and modulate signaling of many ligands , including Wnt , Hedgehog , and FGF ( Tsuda et al . , 1999; Reichsman et al . , 1996; The et al . , 1999; Kiefer et al . , 1990 ) .", "F-spondin and the LDLRR proteins are predicted to be secreted molecules and could promote regeneration by serving as signals themselves , by binding to and changing the activity of other signaling molecules , and/or by contributing to an unknown regenerative function of the planarian ECM .", "Importantly , F-spondin , LDLRR-1 , and -3 are each expressed in differentiated , non-muscle cells in the parenchyma , suggesting that these uncharacterized cells perform some signaling function important for proper regeneration .", "Cells expressing LDLRR-3 and F-Spondin clustered with 'gut' cells in a recent transcriptomic analysis of single cells ( Wurtzel et al . , 2015 ) , but the overall expression patterns of these genes are not reflective of the intestine .", "Thus , further work will be required to identify and characterize these cells .", "Planarian stem cells are present throughout the parenchyma and are surrounded by other parenchymal cell types , one of which has been termed 'fixed parenchyma cells' in the electron microscopy literature ( Pedersen , 1961 ) .", "We have yet to determine what kind of cell-cell interactions or local signaling events in the parenchyma influence the activity of planarian stem cells , either in homeostasis or in regeneration .", "The parenchymal signaling cells and molecules revealed by our screen might be a valuable entry point in determining whether or how neighboring cells promote the maintenance , cycling , differentiation , or migration of planarian neoblasts .", "Another area of investigation concerns whether the CNS influences regeneration by playing a signaling role , as it does in other organisms ( Kumar and Brockes , 2012 ) .", "A recent single-cell analysis suggested that neurons do not respond to injury with gene expression changes ( Wurtzel et al . , 2015 ) .", "However , these studies were performed on worm fragments that were missing their cephalic ganglia , and only addressed gene expression changes in the first 12 hr after amputation .", "We identified one gene upregulated later in regeneration , arrowhead , that is required at the midline of the cephalic ganglia for axon organization across the anterior commissure .", "This non-cell-autonomous role likely involves signaling from arrowhead+ neurons at this location to organize the projections of other cells , including the photoreceptor neurons .", "Our preliminary finding that some arrowhead+ cells express slit offers one clue about how this might occur , but a deeper investigation of Arrowhead targets will distinguish between this and other possibilities .", "Thus , at a minimum , some neurons of the CNS likely influence the organization of one another during regeneration .", "Further work will be required to identify the molecules that govern this influence and to study other contributions of the CNS to the signaling environment required for a proper regenerative response .", "One of the crucial events required for regeneration of nervous tissue is coordinated neurogenesis .", "In the planarian field , a major question is how neoblasts are directed to specific cell fates , including neurons , and how the appropriate number and mixture of cells are reestablished .", "In planarians , some work has been done to identify a handful of factors that regulate commitment to certain neural subtypes , including coe , lhx1/5–1 , and others ( Currie and Pearson , 2013; Cowles et al . , 2013; Scimone et al . , 2014 ) .", "Additionally , a new category of specialized neoblast has recently been proposed to contribute to neural fates ( Molinaro and Pearson , 2016 ) .", "However , we lack a fundamental understanding of the basic neurogenesis program ( s ) in planarians .", "It is not yet clear whether neoblasts commit to specific neural fates in one step or through a series of binary choices .", "Additionally , we do not know how or whether these fate choices are reoriented in response to injury .", "In our screen , we expected to identify factors that could play roles in neural cell fate decisions .", "We identified a transcription factor , soxB2-2 , required for full expression of several neural progenitor genes .", "Importantly , soxB2-2 ( RNAi ) animals display behavioral phenotypes ( decreased sensitivity to vibrations ) and regenerate brains smaller than those resulting from many other perturbations .", "These observations suggest that soxB2-2 likely regulates gene expression even more broadly than we have so far observed .", "The homolog of soxB2-2 is expressed during embryonic development of Schmidtea polychroa ( Monjo and Romero , 2015 ) .", "Related Sox family members sox14 and sox21 play roles in the neurobiology of diverse animal species ( Hargrave et al . , 2000 , Kirilly et al . , 2009 , Sandberg et al . , 2005 , Richards and Rentzsch , 2015 ) .", "In particular , sox21 is upregulated during spinal cord regeneration in Xenopus ( Lee-Liu et al . , 2014 ) .", "It will be important to determine whether roles for soxB2-2 in neurogenesis and regeneration are indeed conserved .", "We also expanded the known roles for planarian runt in brain regeneration .", "runt is expressed in response to different injuries in planarians ( Wenemoser et al . , 2012 , Sandmann et al . , 2011 ) and modulates factors , including sp6-9 and ovo , that are essential for eye development ( Figure 3F; Sandmann et al . , 2011 , Wenemoser et al . , 2012 , Lapan and Reddien , 2011 , Lapan and Reddien , 2012 ) .", "In addition to its role in the eye , runt is important for proper expression of ap2 and klf ( Figure 3F; Wenemoser et al . , 2012 ) ; ap2 drives respecification of medial TrpA+ neurons , and klf is important for regeneration of cintillo+ cells in the brain branches ( Wenemoser et al . , 2012 , Scimone et al . , 2014 ) .", "Thus , runt influences neural progenitors to contribute to multiple neural types across different regions of the brain .", "Neither soxB2-2 nor runt appears to be required globally for neurogenesis , but each controls a distinct array of fates and is required for optimal brain regeneration , suggesting that these two genes’ products promote distinct neurogenesis pathways .", "Both soxB2-2 and runt are upregulated during regeneration , implying that they are activated in neoblasts or early progenitors to skew the balance of specialized progeny and promote neural fates needed for rebuilding the brain .", "Future studies could use soxB2-2 and runt as starting points for defining neurogenesis pathways; by exploring both upstream regulators and targets that control downstream progenitor and neural fates , these middle-out studies could define differentiation trajectories from neoblast to neuron .", "Dozens of neural cell types in the planarian CNS can be defined by the expression patterns of neuropeptide and neurotransmitter biosynthesis genes ( Nishimura et al . , 2007a , 2007b , 2008a , 2010 , 2008b , Collins et al . , 2010 ) .", "Most of these neural subtypes arise during regeneration through mechanisms that have not yet been uncovered .", "Exceptions include the specification of serotonergic neurons by lhx1/5–1 and pitx ( Currie and Pearson , 2013 , Marz et al . , 2013 ) and the specification of dopamine ß-hydroxylase+ neurons by pax3/7 ( Scimone et al . , 2014 ) .", "In our study , RNAi knockdown of runt failed to significantly influence any of the specific markers of differentiated neurons that we tested in this analysis ( e . g . , ChAT to mark cholinergic neurons ) .", "Although knockdown of soxB2-2 decreased several markers , only tyrosine hydroxylase ( TH ) dropped past the threshold of statistical significance ( Figure 3C ) .", "That RNAi would affect presumptive neural progenitors without a detectable effect on a neural marker could simply be due to a lack of tools required to delineate the affected neural cell types , or due to the sensitivity of the assay .", "However , other transcription factors identified in this screen as having no brain-size phenotype ( e . g . , glass ) could play more subtle roles in specification or function of neural subtypes .", "FLI-1 expression in neoblasts could shift them to certain fates important for brain-branch architecture or wiring .", "In contrast , transcription factors like arrowhead and mblk are each expressed predominantly in differentiated cell types ( Labbé et al . , 2012 ) , and likely function later in differentiation or in mature neurons to affect maintenance , connectivity , or function .", "Along these lines , a homolog of mblk in C . elegans is important for neurite pruning ( Kage et al . , 2005 ) .", "Surprisingly , though we anticipated that hesl-3 would affect gene expression in neural progenitors ( Cowles et al . , 2013 ) , we found that the clearest impact of hesl-3 ( RNAi ) is on polarity genes like sFRP and foxD .", "hesl-3 expression is highest in neoblasts ( Cowles et al . , 2013 , Labbé et al . , 2012 ) , though expression in the CNS is also evident ( Figure 3G; Cowles et al . , 2013 ) .", "However , no expression at the anterior tip is visible .", "One straightforward model for the function of hesl-3 would be that it is required for specification of muscle cells that eventually contribute to the anterior pole .", "Another possibility is that hesl-3+ cells contribute to regeneration of the brain and that the regenerating brain is important for resetting or maintaining anterior polarity .", "Neural tissue is critical for regeneration in a variety of other model organisms ( Kumar and Brockes , 2012 ) , but we have not been able to determine whether it is so in planarians .", "Future work will be required to identify targets of hesl-3 and to determine whether its roles in head patterning and brain regeneration occur through an influence on neurogenesis , polarity , or both .", "Glia exist across animal phyla and play diverse , crucial roles in supporting the differentiation , survival , and function of neurons .", "We investigated genes downregulated during planarian regeneration and discovered a marker ( Intermediate Filament-1 ) that is expressed in non-neural cells that have several hallmarks of glial identity .", "First , IF-1+ cells express a range of solute carrier family genes , including genes that are important for reuptake of glutamate; this function is carried out by astrocytes in the mammalian nervous system ( Schousboe et al . , 1977 , Norenberg and Martinez-Hernandez , 1979 ) .", "Second , IF-1+ cells are present in the neuropil and have extensive cytoplasmic projections , positioning them ideally to support axonal or synaptic activity of neurons in the CNS .", "Third , IF-1+ cells respond to injury by changing their gene expression ( downregulating IF-1 , jagged-like , and estrella ) , much like astrocytes in mammals ( for reviews , Sofroniew , 2009 , Pekny and Nilsson , 2005 ) .", "The discovery of glia in the planarian CNS raises many interesting possibilities for future study .", "Glia are an abundant , diverse , and essential population of cells in the mammalian nervous system , so functional regeneration of human neural tissue would require regeneration of glia alongside regeneration of neurons themselves .", "Thus , it will be informative to understand how glia repopulate the regenerating planarian CNS and in particular how glial and neural regeneration are coordinated .", "Additionally , activation of astrocytes in the mammalian nervous system protects tissue after injury but also contributes to glial scarring that can promote or prohibit axonal regrowth or functional recovery ( Pekny et al . , 2014 , Anderson et al . , 2016 ) .", "Though it is not yet clear whether the planarian glial injury-response program is important for driving or permitting regeneration , contrasting the planarian glial response with that of glia in non-regenerating organisms might shed light on how glial activation can be harnessed for purely regeneration-promoting functions .", "For example , planarian IF-1 is downregulated after injury , while mammalian GFAP is upregulated during astrocyte activation ( Condorelli et al . , 1990 ) .", "Finally , RNAi of the glial genes identified in this study has so far failed to reveal phenotypes ( data not shown ) .", "This is not surprising , because the downregulation of IF-1 and other glial genes during regeneration suggests that gain-of-function treatments might be more likely to perturb regeneration .", "More nuanced assays and identification of genes required for glial specification or maintenance will be required to assess whether and how planarian glia support neuronal biology and behavior in planarians .", "Planarians regenerate their central nervous system de novo , prodigiously replacing neurons and reconnecting neural networks to restore function in only a week .", "As such , they provide a simple , in vivo model for understanding neural regeneration .", "In this study , we used a transcriptomic approach to guide an unbiased functional screen .", "Through RNAi studies , we identified several factors that influence reestablishment of cell types , reconnection of a major commissure , and restoration of chemosensory behavior .", "Genes identified in our study also mark new cell types , including planarian glia , putative chemosensory cells , and previously unknown parenchymal cells that promote regeneration .", "In sum , we have advanced our understanding of diverse aspects of planarian neurobiology and neural regeneration; furthermore , we have laid the foundation for a wide range of studies geared toward understanding success and failure in neural regeneration ." ], [ "Planarians from an asexual , clonal line ( Schmidtea mediterranea , CIW4; Sánchez Alvarado et al . , 2002 ) were maintained at 21ºC in ultrapure water with 0 . 5 g/L Instant Ocean salts ( Spectrum Brands , Blacksburg , VA ) .", "Regeneration experiments were completed in the presence of 50 µg/mL gentamicin ( Gemini Bio-Products , West Sacramento , CA ) .", "Animals were stored in unsealed Ziploc containers or 60 mm petri dishes .", "For regeneration experiments , animals were amputated using sterile scalpels .", "Animals were between 2–5 mm for all experiments unless otherwise noted and were starved for at least one week prior to use .", "For stem cell ablation , animals were irradiated with 60 gray at 150 kV , 5 mA on the top shelf of a Gammacell-220 with a cobalt-60 source ( Nordion , Ottawa , ON , Canada ) .", "Large worms ( ~1 cm ) were used for transcriptomic studies .", "Animals were amputated post-pharyngeally and were allowed to regenerate for 12 , 36 , or 72 hr .", "Whole animals and animals killed immediately after amputation were used for two separate controls .", "Either 10 whole animals or 30 tail pieces were used per condition , in triplicate .", "Planarians were killed in TRIzol reagent ( Thermo Fisher Scientific , Waltham , MA ) and samples were frozen on dry ice and stored at −80ºC until RNA purification was completed as per the manufacturer’s protocol .", "After purification , RNA was DNase-treated ( Promega , Madison , WI ) and column purified ( Zymo Research , Irvine , CA ) .", "RNA samples were submitted to the W . M . Keck Center for Comparative and Functional Genomics ( UIUC ) for quality analysis using a bioanalyzer ( Agilent , La Jolla , CA ) and subsequent library construction and sequencing ( Illumina , San Diego , CA ) .", "Sequencing depth was 13 . 5–40 . 1 million reads ( 100 nt ) per sample , for a total of 325 . 5 million reads across all conditions .", "All Illumina sequencing was deposited in the Sequence Reads Archive as study PRJNA319973 .", "Initially , reads were mapped via RNA-Seq ( Marioni et al . , 2008 ) in the CLC genomics workbench ( QIAgen , Hilden , Germany ) to a previously generated transcriptome ( Rouhana et al . , 2012; available online at www . ideals . illinois . edu/handle/2142/28689 and within the PlanMine database ( Brandl et al . , 2016 ) , planmine . mpi-cbg . de ) .", "RPKM ( reads per kilobase of transcript per million mapped reads ) values were calculated and compared using the CLC suite , with p values calculated for each pair of samples using two-sided , unpaired T-tests with Bonferroni correction ( Supplementary file 1 ) .", "From this analysis , contigs ≥two-fold upregulated or downregulated ( p≤0 . 01 ) were pursued for initial functional analysis .", "In parallel , raw reads for each contig were input into R 3 . 1 . 1 ( R Core Team , 2013 ) for data pre-processing and statistical analysis using packages from Bioconductor ( Gentleman et al . , 2004 ) as below .", "After trimmed mean of M-values normalization ( Robinson and Oshlack , 2010 ) and filtering of low abundance transcripts ( <0 . 5 counts per million were discarded ) , the remaining 31 , 862 ( of 55 , 949 ) contigs were analyzed using edgeR v 3 . 6 . 8 ( Robinson et al . , 2010 ) .", "A one-way ANOVA was calculated across the five groups for each contig using a quasi-likelihood negative binomial generalized log-linear model ( Lund et al . , 2012 ) with robust estimation of the sample variances .", "Multiple hypothesis test correction was completed using the False Discovery Rate ( FDR ) method ( Benjamini and Hochberg , 1995 ) .", "To examine global gene expression patterns across the dataset , we selected 9 , 850 contigs with one-way ANOVA FDR p-value ≤0 . 1 for weighted gene correlation network analysis ( WGCNA ) ( Zhang and Horvath , 2005 , Langfelder and Horvath , 2008 ) .", "We performed WGCNA ( v 1 . 41–1 ) using the default values of the blockwiseModules ( ) function except for: soft thresholding power beta = 2- , TOMType = “signed , ” a minimum module size of 20 , and merging similar modules at 0 . 15; this approach yielded 40 modules , each with genes that share similar expression dynamics across the time course .", "An eigengene value was computed for each sample from the first principal component score of the expression values of the contigs in the module .", "For each gene of interest , a 300–1000 bp fragment was cloned from cDNA into pJC53 . 2 , a vector designed to allow TA-cloning and subsequent production of riboprobes or dsRNA ( Collins et al . , 2010 ) , using standard molecular biological methods .", "Riboprobes and dsRNA were synthesized as previously described ( Collins et al . , 2010 , Rouhana et al . , 2013 ) .", "For all RNAi experiments , dsRNA matching GFP or bacterial ccdB genes was used for negative controls .", "Riboprobes were synthesized with digoxigenin-12-UTP ( Roche , Basel , Switzerland ) or dinitrophenol-11-UTP ( Perkin Elmer , Waltham , MA ) .", "Quantitative PCR experiments were performed as previously described ( Miller and Newmark , 2012 ) .", "Briefly , RNA was purified as described above and cDNA was synthesized using an iScript kit ( Bio-Rad , Hercules , CA ) .", "qPCR was performed using GoTaq Mastermix ( Promega ) on a StepOnePlus real-time PCR machine and software ( Applied Biosystems , Foster City , CA ) .", "ß-tubulin was used as a normalization control and qPCR primers are listed in Supplementary file 3E .", "Initial qPCR experiments were performed 2–4 times per gene knockdown; validation qPCR experiments ( Figure 3 ) were performed in biological triplicate and technical triplicate , except soxB2-2 ( RNAi ) and control , for which we had 6 biological replicates and technical triplicates .", "p values were calculated by t test in Prism ( GraphPad , La Jolla , CA ) .", "Sequence analysis was performed to detect protein domains ( pFam 29 . 0; Finn et al . , 2016 ) , transmembrane domains ( TMHMM 2 . 0; Krogh et al . , 2001 ) , and signal peptides ( SignalP 4 . 1; Petersen et al . , 2011 ) .", "Sequences for genes identified in this analysis have been deposited at Genbank .", "Colorimetric in situ hybridization ( ISH ) experiments were done as per Pearson et al . ( 2009 ) , but with the following changes described in King and Newmark ( 2013 ) : the reduction step was eliminated; animals were bleached in a formamide-containing solution for 2 . 5–3 hr under light; and proteinase K was used at a concentration of 5 µg/mL ( for regenerating worms ) or 10 µg/mL ( whole animals ) .", "For colorimetric ISH , Digoxigenin-containing probes were used in combination with an anti-DIG-AP antibody ( 1:2000 , Roche ) .", "Fluorescent in situ hybridization ( FISH ) was performed as above , but with the following changes: digoxigenin- and/or dinitrophenol-containing probes were used; riboprobes were detected by anti-digoxigenin-POD ( 1:2000 , Roche ) or anti-dinitrophenol-HRP ( 1:300 , Perkin Elmer ) ; the blocking solution was TNTx ( 100 mM Tris pH 7 . 5 , 150 mM NaCl , 0 . 3% Triton X-100 ) with 5% horse serum and 0 . 5% Roche western blocking reagent ( Roche ) ; and TNTx was also used for post-antibody washes .", "For FISH , tyramide conjugates were synthesized and signal amplification reactions were performed as described ( King and Newmark , 2013 ) .", "Immunofluorescence experiments were performed as described ( Roberts-Galbraith and Newmark , 2013 ) .", "The following primary antibodies were used: anti-phospho-histone H3 ( 1:5000 , S10; Cell Signaling Technology , Danvers , MA ) , anti-synapsin ( 1:100 , 3C11 concentrate; Developmental Studies Hybridoma Bank , Iowa City , IA ) , and anti-arrestin ( 1:10000 , VC1; kindly provided by Kiyokazu Agata ) .", "Secondary antibodies ( Molecular Probes , Eugene , OR ) were used at a dilution of 1:800 ( goat anti-mouse Alexa Fluor 488 ) or 1:2500 ( goat anti-rabbit Alexa Fluor 568 ) .", "Animals ( 10–12 ) were placed in a 60 mm petri dish in salts and were fed 3 µg dsRNA in 30 µL of a liver puree:salts mixture ( 3:1 , tinted with green food coloring ) .", "RNAi experiments are diagrammed in Figure 2A and Figure 6—figure supplement 1H .", "Briefly , animals were fed three times over the course of 10–11 days , amputated prepharyngeally 5 days after the last feeding , and allowed to regenerate for 6 hr ( Figure 4—figure supplement 1B–C ) , 6 days ( for all other in situ or immunofluorescence experiments ) , or 10 days ( behavioral assays ) .", "For all experiments , animals were observed for obvious behavioral or physiological defects before they were fixed or otherwise assayed .", "To assay blastema size , animals were fixed after regeneration for imaging .", "Mazes were constructed at the Life Sciences Machine Shop ( University of Illinois ) out of clear and black acrylic sheets according to the measurements shown ( Figure 6—figure supplement 1E–F ) .", "Prior to a behavioral experiment , we added 70 mL salts to each maze and pipetted 60 µL of a liver puree:salts mixture ( 3:1 , tinted green ) into the far side of the maze as shown ( Figure 6—figure supplement 1G ) .", "The mazes were incubated at room temperature for fifteen minutes .", "RNAi-treated , regenerated animals ( 10–12 per replicate ) were placed in the maze in the position shown ( Figure 6—figure supplement 1G ) .", "Mazes were covered to allow complete darkness and animals were permitted 1 hr to complete the maze and eat .", "Animals that had eaten were scored based on the presence of green in their intestines .", "Experiments were performed in biological triplicate and differences between the samples were evaluated using a Chi-squared test .", "In a separate experiment , control and FLI-1 ( RNAi ) animals were added to the center of 60 mm petri dishes on a sheet with concentric circles ( the smallest of which was 1 . 5 cm in diameter ) .", "After 15 min , animals were scored for their ability to move beyond the starting circle .", "A Chi-squared test was also used to evaluate the difference between these samples .", "We used a Leica M205A stereomicroscope to image animals live or after colorimetric ISH .", "Images were captured using LAS 3 . 6 . 0 software ( Leica , Wetzlar , Germany ) .", "We quantified brain area and blastema size in ImageJ ( Schneider et al . , 2012 ) .", "Areas were averaged across a condition and were compared to control using a one-way ANOVA .", "Fluorescence images ( immunofluorescence and FISH ) were captured using a Zeiss LSM 710 confocal microscope and either a 20X ( Plan-Apochromat 206/0 . 8 ) or a 63X objective ( Plan-Apochromat 636/1 . 4 ) .", "Zen software ( Carl Zeiss , Oberkochen , Germany ) was used for these experiments .", "We fixed and processed planarians for transmission electron microscopy as described in Brubacher et al . , 2014 .", "Serial 70 nm sections were cut on a Leica Ultracut S ultramicrotome , and collected on formvar- and carbon-coated copper slot grids ( Ted Pella , Redding , CA ) and stained with lead citrate for 2 min at room temperature ( Venable and Coggeshall , 1965 ) .", "We imaged the trunk ventral nerve cord using a Hitachi H-7000 STEM at 75 kV , equipped with an AMT 1600 M CCD camera ( Hitachi , Tokyo , Japan ) .", "IMOD software version 4 . 3 . 7 ( Kremer et al . , 1996 ) was used to stack and align images , and to segment and mesh digital models .", "Image processing and animations were conducted with Adobe Photoshop CS5 software ( Adobe Systems , San Jose , CA ) ." ] ]

[ "Planarians regenerate all body parts after injury , including the central nervous system ( CNS ) .", "We capitalized on this distinctive trait and completed a gene expression-guided functional screen to identify factors that regulate diverse aspects of neural regeneration in Schmidtea mediterranea .", "Our screen revealed molecules that influence neural cell fates , support the formation of a major connective hub , and promote reestablishment of chemosensory behavior .", "We also identified genes that encode signaling molecules with roles in head regeneration , including some that are produced in a previously uncharacterized parenchymal population of cells .", "Finally , we explored genes downregulated during planarian regeneration and characterized , for the first time , glial cells in the planarian CNS that respond to injury by repressing several transcripts .", "Collectively , our studies revealed diverse molecules and cell types that underlie an animal’s ability to regenerate its brain ." ]

[ "Animals differ in the extent to which they can regenerate missing body parts after injury .", "Humans regenerate poorly after many injuries , especially when the brain becomes damaged after stroke , disease or trauma .", "On the other hand , planarians – small worms that live in fresh water – regenerate exceptionally well .", "A whole planarian can regenerate from small pieces of tissue .", "The ability of planarians to regenerate their nervous system relies on stem cells called neoblasts , which can migrate through the body and divide to replace lost cells .", "However , the specific mechanisms responsible for regenerating nervous tissue are largely unknown .", "Roberts-Galbraith et al . carried out a screen to identify genes that tell planarians whether to regenerate a new brain , what cells to make and how to arrange them .", "The study revealed over thirty genes that allow planarians to regenerate their brains after their heads have been amputated .", "These genes play several different roles in the animal .", "Some of the genes help neoblasts to make decisions about what kinds of cells they should become .", "One gene is needed to make an important connection in the planarian brain after injury .", "Another helps to restore the ability of the planarian to sense its food .", "The experiments also show that some key genes are switched on in a new cell type that might produce signals to support regeneration .", "Lastly , Roberts-Galbraith et al . found that the planarian nervous system contains cells called glia .", "Previous studies have shown that many of the cells in the human brain are glia and that these cells help nerve cells to survive and work properly .", "The discovery of glia in planarians means that it will be possible to use these worms to study how glia support brain regeneration and how glia themselves are replaced after injury .", "In the long term , this work might lead to discoveries that shed light on how tissue regeneration could be improved in humans ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "biochemistry and chemical biology", "neuroscience" ]

[ [ "Adenosine 5’ triphosphate ( ATP ) is the essential energy currency of intracellular metabolism of all living systems , but also plays important roles as an extracellular signaling molecule in a wide variety of physiological and pathological processes , including neurotransmission ( Burnstock , 2007 ) , neuron–glia signaling ( Fields and Burnstock , 2006; Khakh and North , 2012 ) , tissue circulation ( Cheng et al . , 2018; González-Alonso et al . , 2002; MacVicar and Newman , 2015 ) , regulation of the immune system ( Idzko et al . , 2014; Junger , 2011 ) , and cancer development ( Di Virgilio et al . , 2018; Stagg and Smyth , 2010 ) .", "In order to elucidate the mechanisms of these processes , an understanding of the spatiotemporal dynamics of ATP is essential .", "To date , a variety of techniques for measurement of ATP have been developed: bioluminescence assay methods using firefly luciferase ( Holton , 1959; Pellegatti et al . , 2005; Wang et al . , 2000 ) , electrochemical methods including voltammetry ( Singhal and Kuhr , 1997 ) , enzyme-coupled electrode techniques ( Kueng et al . , 2004; Llaudet et al . , 2005 ) , cell-based electrophysiological techniques ( Brown and Dale , 2002; Pangrsic et al . , 2007 ) , and whole cell-based fluorescence assay methods using Ca2+ indicator ( Anselmi et al . , 2008; Huang et al . , 2007 ) .", "Some of them have been applied to measure extracellular ATP concentrations in vivo ( Gourine et al . , 2005; Melani et al . , 2005; Pellegatti et al . , 2008 ) .", "For example , bioluminescence assay is able to quantify the extracellular ATP levels in microdialysis samples ( Melani et al . , 2005 ) , and enzyme-coated electrodes inserted in tissues allow continuous monitoring of extracellular ATP levels in the brain ( Gourine et al . , 2005 ) .", "However , these methods do not provide spatial information .", "Imaging of the bioluminescence generated by luciferin-luciferase reaction might be a solution for spatially resolved measurement of ATP ( Rajendran et al . , 2016 ) , and indeed , engineered cells displaying luciferase on the plasma membrane were used to visualize extracellular ATP in vivo by inoculating the cells into living mice ( Pellegatti et al . , 2008 ) .", "However , both spatial resolution and temporal resolution in luciferase-based imaging are inherently compromised by the low photon flux of bioluminescence emission ( Rajendran et al . , 2016 ) .", "On the other hand , imaging techniques using fluorescent sensors can provide excellent spatiotemporal resolution ( Giepmans et al . , 2006 ) , and a variety of fluorescent ATP sensors based on fluorescent proteins have been developed ( Arai et al . , 2018; Berg et al . , 2009; Imamura et al . , 2009; Lobas et al . , 2019; Tantama et al . , 2013; Yaginuma et al . , 2015 ) .", "These genetically encoded ATP sensors can visualize intracellular ATP dynamics , and have facilitated our understanding of ATP signaling inside cells ( Depaoli et al . , 2018; Zala et al . , 2013 ) .", "In contrast , attempts to adapt the existing sensors for extracellular use have achieved limited success , and in vivo imaging of extracellular ATP is still difficult ( Conley et al . , 2017; Lobas et al . , 2019 ) .", "One problem is that the affinities of these extracellular ATP sensors are too low to monitor extracellular ATP , the concentration of which likely ranges from hundreds of nanomolar to micromolar levels ( Yegutkin , 2008 ) .", "Moreover , these extracellular ATP sensors are designed to be synthesized inside the cells and transported to the plasma membrane to face the extracellular space ( Conley et al . , 2017; Lobas et al . , 2019 ) .", "Imperfect membrane transport as well as potential posttranslational modifications such as glycosylation during this process can compromise the performance of the sensors ( Morciano et al . , 2017 ) .", "Another problem is the strong pH dependency of these genetically encoded ATP sensors , due to the innate pH sensitivity of the fluorescent proteins used ( Chudakov et al . , 2010; Tsien , 1998 ) .", "This pH dependence is especially problematic in in vivo ATP imaging , because changes in ATP concentration in tissues are often accompanied by pH fluctuations ( Berg et al . , 2009; Di Virgilio et al . , 2018; Gourine et al . , 2010 ) .", "Another strategy to develop fluorescent sensors is a hybrid-type probe design in which a small-molecule fluorescent dye is conjugated to a ligand-binding protein ( Takikawa et al . , 2014; Wang et al . , 2009 ) .", "We previously developed a hybrid-type glutamate sensor , in which the AMPA receptor GluR2 subunit is employed as a glutamate-binding protein ( Namiki et al . , 2007 ) , and visualized extrasynaptic glutamate dynamics in vivo ( Okubo et al . , 2010 ) .", "In this study , to develop a hybrid-type ATP sensor , we adopted bacterial FoF1-ATP synthase ε subunit as an ATP-binding protein , because fluorescent-dye conjugation has been used to study conformational changes of the protein upon ATP binding ( Iino et al . , 2005; Kato et al . , 2007 ) .", "We screened fluorescent dyes and their labeling sites within the ATP-binding protein , and obtained a highly sensitive ATP sensor with a large fluorescence response , high selectivity for ATP , and insensitivity to pH fluctuation , suitable for detection of extracellular ATP .", "The sensor can be anchored on the plasma membrane to detect extracellular ATP .", "To illustrate potential applications of the sensor , we performed extracellular ATP imaging in the brain of living mice , and observed the propagation of a wave-like extracellular ATP increase through the cerebral cortex in response to neuronal excitation evoked by electrical stimulation .", "The results confirm that the ATP sensor is suitable for in vivo imaging of the dynamics of extracellular ATP signaling ." ], [ "To visualize extracellular ATP , we designed a hybrid-type fluorescent ATP sensor composed of an ATP-binding protein and a small-molecule fluorescent dye .", "We adopted the ε subunit of FoF1-ATP synthase of thermophilic Bacillus PS3 , which has high affinity for ATP ( Kato-Yamada and Yoshida , 2003 ) , as the ATP-binding protein , and introduced a single cysteine substitution in its amino-acid sequence to enable the conjugation of a small-molecule fluorescent dye bearing a cysteine-reactive maleimide group ( Figure 1A ) .", "The performance of a hybrid-type sensor is determined by the location of the fluorophore within the ligand-binding protein ( Namiki et al . , 2007; Takikawa et al . , 2014 ) .", "To obtain a high-sensitivity ATP sensor , we used our previously developed high-throughput screening system , HyFInD ( Takikawa et al . , 2014 ) , in which a large variety of fluorescent conjugates can be generated by varying both the fluorophore conjugation sites and the fluorophores , and then evaluated for fluorescence response to a ligand .", "We generated 134 cysteine mutants covering every position in the amino-acid sequence except for the first position ( the initiating methionine ) .", "We used four small-molecule fluorescent dyes , Oregon Green ( OG ) , Alexa Fluor 488 ( Alexa488 ) , Cy3 and SiR650 , resulting in the generation of 536 fluorescent conjugates in total .", "For the initial screening we employed a high concentration ( 2 mM ) of ATP to select sensors having a large dynamic range of fluorescence response .", "For each of the four fluorophores , we found a moderate number of fluorescent conjugates showing detectable fluorescence changes upon application of ATP , but with a large variation in the response ( Figure 1B ) .", "Notably , even if the fluorophore labeling site was the same , the fluorescence response varied depending on the type of fluorophore .", "We then selected eight fluorescent conjugates as candidate sensors showing a large fluorescence response in the initial screening , and tested their fluorescence response to a low ATP concentration ( 200 nM ) ( Figure 1—figure supplement 1A ) .", "Variation in the fluorescence response was again observed , as in the case of the higher concentration of ATP .", "The conjugate exhibiting the largest fluorescence response at 200 nM ATP was the Cy3-labeled cysteine substitution mutant at glutamine 105 ( Q105C-Cy3 ) .", "We hereafter designate Q105C-Cy3 as ATPOS ( ATP Optical Sensor ) .", "We next examined the characteristics of ATPOS in more detail .", "In the absence of ATP , ATPOS displayed a fluorescence excitation peak at 556 nm and an emission peak at 566 nm ( Figure 1C; Table 1 ) .", "At 1 mM ATP , the fluorescence intensity of ATPOS increased without any appreciable change in the peak wavelengths ( excitation peak at 556 nm , emission peak at 564 nm ) , while the absorption spectrum showed almost no change upon the addition of ATP ( Figure 1—figure supplement 1B ) .", "On the other hand , the fluorescence quantum yield increased from 0 . 29 to 0 . 62 ( Table 1 ) .", "The results indicate that the change in fluorescence intensity is due to the change in the quantum yield .", "To estimate the sensitivity of ATPOS for ATP , we examined the dependence of the fluorescence change of ATPOS on the concentration of ATP ( Figure 1D ) .", "Fluorescence changes were observed at submicromolar concentrations , and fitting the Hill equation to the dose-response data yielded an apparent dissociation constant of ~150 nM and a Hill coefficient of 1 . 08 .", "The results suggest that ATPOS has sufficiently high affinity for ATP to be applicable for the visualization of extracellular ATP .", "The Hill coefficient value of ~1 suggests that ATPOS binds to ATP with 1:1 stoichiometry .", "We also compared the affinity for ATP with those for ATP metabolites and other nucleotides to examine the selectivity of ATPOS .", "The affinity for ATP was about one hundred-fold higher than that for ADP , and ATPOS was almost insensitive to other metabolites of ATP , such as AMP and adenosine , and other nucleotides such as GTP , CTP and UTP ( Figure 1D ) .", "To study the kinetics of ATPOS , we performed stopped-flow measurements ( Figure 1—figure supplement 1C , D and E ) .", "The observed rate constants showed a dose-dependent increase ranging from 5 . 0 s−1 at 0 . 5 μM ATP to 8 . 9 s−1 at 2 . 0 μM ATP .", "We also measured fluorescence decay of ATPOS upon a decrease in ATP concentration , and determined the decay rate constant to be 2 . 0 s−1 .", "These results suggest that the association and dissociation kinetics of ATPOS is fast enough to monitor ATP dynamics on a time scale of seconds .", "The pH dependence of ATP sensors is a critical determinant of the accuracy of ATP quantification , because pH fluctuations often occur at the same time as changes in ATP concentration ( Berg et al . , 2009; Di Virgilio et al . , 2018; Gourine et al . , 2010 ) .", "Therefore , we assessed the pH sensitivity of ATPOS and observed no fluorescence response to changes in pH from 6 . 0 to 8 . 5 in the presence or absence of ATP ( Figure 1E ) .", "For in vivo ATP imaging with ATPOS , we introduced two additional features .", "Firstly , in order to anchor ATPOS to the outer surface of cell membranes , enabling it to report extracellular ATP in brain tissue , we employed BoNT/C-Hc , a nontoxic subunit of Clostridium botulinum neurotoxin , which binds to the plasma membrane of neurons ( Tsukamoto et al . , 2005 ) .", "Secondly , we introduced a capability for dual-color ratiometric imaging , which should mitigate the effect of spatial inhomogeneity of sensor concentration , as well as movement artifacts .", "For this purpose , we employed Alexa488 as a fluorophore for a reference color channel .", "Accordingly , we assembled a molecular complex of ATPOS with BoNT/C-Hc and Alexa488 labeled-streptavidin ( Figure 2A ) .", "The ATPOS complex exhibited a large fluorescence change in the red ( Cy3 ) channel and high sensitivity for ATP , comparable to those of ATPOS itself , whereas it showed no fluorescence change in the green ( Alexa488 ) channel ( Figure 2—figure supplement 1 ) .", "Therefore , the ratio of red to green fluorescence provides a measure of ATP concentration .", "We next tested the performance of the ATPOS complex anchored to cellular membranes using primary cultures of rat hippocampal neurons .", "After the ATPOS complex was applied , followed by wash-out , both red and green fluorescence was observed along the neurons ( Figure 2B ) , indicating successful anchoring of the ATPOS complex on the neuronal cell surface .", "The fluorescence intensity in the red channel increased upon ATP addition , but that in the green channel did not; consequently the fluorescence ratio was increased .", "These results indicate that the ATPOS complex anchored on the neuronal surface can work as a ratiometric sensor of extracellular ATP .", "We then applied the ATPOS complex for extracellular ATP imaging in the cerebral cortex of living mice .", "The ATPOS complex was pressure-injected into layer 2/3 of the cerebral cortex with a fine glass pipette through a craniotomy ( Figure 3A ) .", "The area labeled by ATPOS , identified by its fluorescence , gradually spread across the cortex to occupy a region 2 mm in diameter ( Figure 3—figure supplement 1A ) .", "The fluorescence of ATPOS was stably observed after the end of the injection .", "To examine the distribution of ATPOS in the cerebral cortex with cellular resolution , we performed two-photon imaging of the target cortical area , and found that the blood vessels and cell bodies remained unstained ( Figure 3—figure supplement 1B ) .", "These results indicate that ATPOS is immobilized in the extracellular space of the cortex .", "To confirm that ATPOS introduced into the cerebral cortex in this way works as a sensor of extracellular ATP , we next injected ATP into the cortex with a glass pipette , and visualized the ratiometric fluorescence response of ATPOS by means of wide-field fluorescence microscopy .", "Injection of ATP elicited an increase of the fluorescence ratio to a peak amplitude of 6 . 5 ± 0 . 6% ( n = 18 from five mice ) ( Figure 3B and C; Figure 3—videos 1 and 2 ) .", "To check the selectivity of ATPOS’s response , we applied apyrase , which sequentially hydrolyzes ATP to ADP and ADP to AMP , to the cortex , and observed whether it reduced the fluorescence response of ATPOS to the ATP injection .", "In the presence of apyrase , the peak amplitude of the ratio was reduced to 2 . 4 ± 0 . 5% ( n = 14 from three mice ) , while a sham injection containing bovine serum albumin had almost no effect ( Figure 3D and E ) .", "These results support the view that ATPOS applied in the mouse cortex does indeed report the extracellular ATP concentration .", "We then attempted to visualize extracellular ATP endogenously released during neuronal excitation in living mice .", "Previous reports indicate that a propagating wave of neuronal depolarization evoked by electrical stimulation or KCl application to the brain ( Pietrobon and Moskowitz , 2014 ) causes elevation of the extracellular ATP concentration ( Heinrich et al . , 2012; Schock et al . , 2007 ) .", "Accordingly , we delivered electrical stimulation to the cortex with a metal microelectrode to induce a wave of neuronal depolarization , and observed the fluorescence response of ATPOS during the neuronal excitation under a wide-field microscope ( Figure 4A ) .", "The ratiometric images show a wave-like propagation starting around the electrode ( Figure 4B and C; Figure 4—video 1 ) .", "A precipitous rise of the fluorescence intensity is seen at the wave front , and the increase shifts in a radial direction away from the stimulation site ( Figure 4D ) .", "The increase of the ratio , which showed a peak amplitude of ~30% , lasted about 2 min .", "To verify that this wave-like propagation represented the dynamics of extracellular ATP , we compared the fluorescence responses of ATPOS to the stimulation before and after the application of apyrase .", "In the presence of apyrase , although a wave-like propagation could still be discerned ( Figure 4E and F; Figure 4—video 2 ) , its peak amplitude was substantially reduced to 15 . 8 ± 2 . 5% ( n = 7 from five mice ) while that of the controls was 31 . 4 ± 1 . 4% ( n = 11 from five mice ) ( Figure 4G and H ) .", "These results suggest that the wave-like propagation of fluorescence changes of ATPOS reflects the dynamics of extracellular ATP signaling during neuronal excitation .", "In addition , to visualize the dynamics of extracellular ATP during the neuronal excitation at a single cross section , we performed two-photon imaging of ATPOS upon electrical stimulation in layer 2/3 , where ATPOS was injected ( Figure 4—figure supplement 1 ) .", "The wave-like propagation of fluorescence changes of ATPOS was also observed at a single cross section in layer 2/3 .", "These results are consistent with those obtained by wide-field microscopy , showing the wave-like propagation of extracellular ATP during neuronal excitation ." ], [ "In the present study , we developed a fluorescent ATP sensor that enables real-time imaging of extracellular ATP dynamics in vivo , and we showed that this sensor , ATPOS , can visualize wave-like extracellular ATP signaling in the cerebral cortex of living mice in response to electrical stimulation .", "ATPOS has at least four advantages for in vivo visualization of extracellular ATP .", "First , ATPOS has a very high affinity for ATP; its apparent dissociation constant is ~150 nM , while those of previously reported fluorescent ATP sensors , such as ATeam , QUEEN and iATPSnFR , are in the range of several micromolar to several millimolar ( Arai et al . , 2018; Conley et al . , 2017; Imamura et al . , 2009; Lobas et al . , 2019; Yaginuma et al . , 2015 ) .", "The low affinity of the conventional sensors makes them unsuitable for in vivo extracellular ATP imaging .", "Second , ATPOS displays high brightness; its fluorescence quantum yield ( QY ) in the presence of ATP was around 0 . 62 , while that of Cy3 itself , which is employed in ATPOS , is only around 0 . 04 ( Table 1 ) .", "The QY of ATPOS is equivalent to that of Cy3B ( QY = 0 . 67 ) , a rigidified analog of Cy3 , which was designed to prevent nonradiative energy loss due to rotational and translational vibration modes ( Cooper et al . , 2004 ) .", "Therefore the high brightness of ATPOS may result from restriction of the intramolecular vibration modes of Cy3 within ATPOS .", "Third , ATPOS shows high selectivity for ATP over ATP metabolites and other nucleotides .", "Therefore , compared with conventional fluorescent sensors targeting adenine compounds ( Tokunaga et al . , 2012 ) or nucleoside polyphosphates ( Kurishita et al . , 2012 ) , ATPOS should be better suited to study the specific roles of extracellular ATP , because derivatives of ATP also work as extracellular signaling molecules ( Dunwiddie and Masino , 2001 ) .", "This characteristic of ATPOS is likely attributable to the ligand selectivity of the ε subunit of FoF1-ATP synthase ( Kato-Yamada and Yoshida , 2003 ) , because existing ATP sensors with high selectivity , including ATeam , QUEEN and iATPSnFR , also adopt the ε subunit as an ATP-binding protein ( Arai et al . , 2018; Conley et al . , 2017; Imamura et al . , 2009; Lobas et al . , 2019; Yaginuma et al . , 2015 ) .", "Lastly , ATPOS is insensitive to pH changes .", "Existing fluorescent protein-based ATP sensors all show pH-dependent changes of fluorescence intensity ( Arai et al . , 2018; Berg et al . , 2009; Imamura et al . , 2009; Lobas et al . , 2019; Tantama et al . , 2013; Yaginuma et al . , 2015 ) .", "The pH insensitivity of ATPOS can be ascribed to its hybrid-type design , in which a pH-insensitive small-molecule fluorescent dye is employed instead of a fluorescent protein .", "This feature of ATPOS enables precise quantitative analysis of ATP dynamics regardless of pH fluctuations .", "Although pH in the brain is homeostatically regulated , pH fluctuations are known to occur concomitantly with neuronal activity ( Chesler and Kaila , 1992; Magnotta et al . , 2012 ) .", "Furthermore , the brain pH can change drastically in pathological conditions such as ischemia ( Nedergaard et al . , 1991 ) .", "Thus , its pH insensitivity greatly extends the applicability of ATPOS to various biological conditions .", "Despite these great advantages over genetically encoded sensors , there are drawbacks in the use of ATPOS .", "One is that the injection procedure required for delivery of ATPOS might cause tissue damage .", "Although this possibility should be taken into account , we suppose that the tissue damage is likely to be limited , given that the glass micropipette used for the injection procedure is similar in shape to glass electrodes conventionally used in electrophysiological experiments .", "Another drawback is that targeting ATPOS to specific cell types is not readily feasible .", "We anticipate that combining technology for cell type-specific display of tag proteins might enable the specific targeting of ATPOS ( Hinner and Johnsson , 2010 ) .", "The screening of fluorophore labeling sites enabled us to select ATP sensors exhibiting a large fluorescence response to ATP .", "The maximal fluorescence response of ATPOS to ATP is about 2-fold , which is sufficient for the detection of ATP dynamics in vivo .", "Some ATP sensors found through the screening exhibited a large fluorescence change , but showed low sensitivity .", "For example , the Alexa488-labeled sensor in which serine-64 was replaced by cysteine ( S64C-Alexa488 ) , whose EC50 was tens of micromolar , showed about 200% maximal fluorescence response to ATP .", "These low-affinity ATP sensors obtained in the screening may be useful for ATP imaging under circumstances where the ATP concentration is elevated to such high levels .", "We also obtained different-colored ATP sensors displaying green or near-infrared fluorescence .", "This rich color variation should enable multiplexed imaging of ATP together with Ca2+ and other signaling molecules , and should also be compatible with the manipulation of cellular activities by means of optogenetics .", "ATPOS revealed the dynamic response of extracellular ATP to electrical stimulation ( 10 mA for 1 s ) in the cerebral cortex of living mice .", "Such electrical stimulation has been used to evoke a propagating wave of neuronal depolarization ( Reid et al . , 1987 ) , known as cortical spreading depression ( CSD ) , which is involved in the pathogenesis of migraine and stroke ( Lauritzen et al . , 2011 ) .", "We found that the elevation of extracellular ATP around the stimulation site propagates through the cortex in a concentric fashion .", "Although the elevation of extracellular ATP concentration during CSD is already known ( Heinrich et al . , 2012; Schock et al . , 2007 ) , its spatial dynamics have not been reported .", "We found that the ATP wave propagated at the speed of ~2 mm/min , which is comparable to the reported rate of neuronal propagation of CSD , 2 to 5 mm/min ( Khennouf et al . , 2016; Takagaki et al . , 2014; van den Maagdenberg et al . , 2004 ) , suggesting a role of the ATP-release mechanism in CSD .", "The precipitous rise of ATP at the wave front suggests that the ATP wave is likely to be formed by active propagation of ATP release , rather than passive diffusion .", "In addition , the ATP wave was observed in the entire field of view , suggesting that the elevation of ATP concentration occurs across a broad area of the brain .", "Given that ATP works as a signaling molecule for both neurons and glial cells ( Burnstock , 2007; Fields and Burnstock , 2006; Khakh and North , 2012 ) , the ATP wave during CSD may play an important role in the cellular processes involved in the pathogenesis of migraine and stroke ( Charles and Baca , 2013; Pedata et al . , 2016 ) .", "We anticipate that ATPOS will be a useful tool not only for studies of these pathophysiological processes , but also for investigating the feasibility of novel clinical treatments targeting ATP signaling ." ], [ "All procedures used in animal experiments were in accordance with the guidelines established by the Animal Welfare Committee of the University of Tokyo .", "Hippocampal neuronal cultures were prepared from Sprague-Dawley rats ( SLC; embryonic , 21 days old ) .", "The embryos were decapitated , and the hippocampi were dissected out and treated with trypsin ( Life Technologies ) and DNase I ( Sigma ) .", "The dissociated neurons were incubated on glial cell monolayers plated on coverslips coated with laminin ( Life Technologies ) and poly-L-lysine ( Nacalai tesque ) in Neurobasal A medium ( Life Technologies ) supplemented with B-27 supplement ( Life Technologies ) , 0 . 5 mM Glutamax ( Life Technologies ) , 1 mM sodium pyruvate ( Nacalai tesque ) , and 1% penicillin/streptomycin ( Nacalai tesque ) at 37°C with 5% CO2 .", "HyFInD screening followed the protocols described in our previous work with a few modifications ( Takikawa et al . , 2014 ) .", "A synthetic gene encoding the ε subunit of FoF1-ATP synthase of thermophilic Bacillus PS3 was obtained from Genescript , and inserted into the NdeI/XhoI site of a modified pET23a vector ( Novagen ) with an 8 × His tag .", "Cysteine mutants of the ATP-binding protein were comprehensively generated by inverse PCR for site-directed mutagenesis .", "The pair of primers used in the inverse PCR was designed in a back-to-back orientation , and the original codon at the 5’-end of the forward primer was replaced with a cysteine codon .", "After 5’-kination using T4 polynucleotide kinase ( Toyobo ) , the PCR products were recircularized with a DNA ligation kit ver . 2 . 1 ( Toyobo ) .", "For bacterial production of the recombinant proteins , Escherichia coli BL21 ( DE3 ) cells ( Stratagene ) were transformed with the circularized PCR products .", "The transformed cells were grown overnight in a semisolid medium containing 2% tryptone , 0 . 5% yeast extract , 8 . 6 mM NaCl , 2 . 5 mM KCl , 1 mM NaOH , 20 mM MgSO4 , 100 μg/ml carbenicillin and 0 . 8% agarose ( SeaPrep ) in a 96-deep-well plate at 30°C .", "Fifty microliters of the melted medium was added to 2 × YT medium ( 1 . 6% tryptone , 1% yeast extract , and 86 mM NaCl ) containing 100 μg/ml carbenicillin in a 96-deep-well plate .", "The bacteria were incubated at 37°C until the OD600 reached 0 . 4–0 . 8 .", "Further incubation at 25°C for 12 hr was performed in the presence of 0 . 4 mM isopropyl β-D-thiogalactoside ( IPTG ) ( Nacalai tesque ) .", "The cells were harvested by centrifugation and lysed in phosphate-buffered saline ( PBS; 137 mM NaCl , 2 . 7 mM KCl , 1 . 5 mM KH2PO4 , and 8 . 1 mM Na2HPO4; pH 7 . 4 ) with lysis mix buffer ( 10 mM MgCl2 , 10 μg/ml lysozyme , 0 . 027% BriJ 58 , and 3 . 3 U/ml DNase I ) .", "For purification of the recombinant proteins , the cleared lysate was incubated for 45 min in a 96-well filter plate containing TALON metal affinity resin ( Clontech ) , followed by washing with PBS containing 10 mM imidazole ( Nacalai tesque ) .", "For fluorophore labeling , the recombinant proteins absorbed on the resin were incubated at 25°C for 45 min with Oregon Green ( OG ) maleimide ( Life Technologies ) , Alexa Fluor 488 ( Alexa488 ) maleimide ( Life Technologies ) , Cy3 maleimide ( GE Healthcare ) , or SiR650-PEG3-maleimide , which were dissolved in PBS at a concentration of 4 μM .", "SiR650-PEG3-maleimide was prepared by conjugating a maleimide group to SiR-carboxy ( Lukinavičius et al . , 2013 ) via a PEG3 linker .", "The resin was washed with PBS containing 10 mM imidazole , and the fluorescent conjugates were eluted with PBS containing 150 mM imidazole and 0 . 1% bovine serum albumin .", "For the performance test of the fluorescent conjugates , the fluorescence intensity in each well of a 96-well plate was automatically scanned with a custom-built fluorescence plate reader system .", "OG- or Alexa488-labeled conjugates were excited at 460–480 nm and emission was measured at 495–540 nm .", "Cy3-labeled conjugates were excited at 535–555 nm and emission was measured at 570–625 nm .", "SiR650-labeled conjugates were excited at 608–648 nm and emission was measured at 672–712 nm .", "In the first screening , fluorescence changes upon addition of ATP ( Sigma ) at a final concentration of 2 mM were measured .", "In the second screening , fluorescence changes upon addition of ATP at a final concentration of 200 nM were measured .", "Escherichia coli BL21 ( DE3 ) cells were transformed with plasmids encoding the ATP-binding protein with Q105C mutation ( ATPBP-Q105C ) .", "The transformed cells were incubated in 2 × YT media containing 100 μg/ml ampicillin at 37°C until the OD600 reached 0 . 5–0 . 8 .", "Further incubation at 25°C for 16 hr was performed in the presence of 0 . 4 mM IPTG , followed by centrifugation at 4°C for 15 min at 2 , 070 g .", "The harvested cells were lysed by means of a freeze-thaw procedure in PBS containing 1 mg/ml lysozyme and then incubated at 4°C for 15 min in PBS containing 0 . 1% Triton X100 , 10 mM MgCl2 , and 3 U/ml DNaseI ( Takara ) .", "After centrifugation of the cell lysate at 4°C for 30 min at 15 , 000 g , the collected supernatant was purified by using HiTrap TALON crude ( BD Biosciences ) .", "The purified ATPBP-Q105C proteins ( 10 μM ) were incubated with Cy3 maleimide ( 50 μM ) in PBS at 25°C for 45 min .", "The labeled proteins were separated from unreacted dyes on a PD-10 desalting column ( GE Healthcare ) .", "If further purification was necessary , anion-exchange chromatography was carried out on COSMOGEL IEX Type Q ( Nacalai tesque ) by applying a linear salt gradient .", "The chromatography was performed with an AKTA purifier ( GE Healthcare ) according to the instructions of the manufacturer .", "To create the ATPOS complex , which enables the anchoring of ATPOS on neuronal surfaces , ATPOS ( 4 μM ) was biotinylated with NHS-PEG4-biotin ( Thermo Scientific ) at a concentration of 10 μM in PBS at 25°C for 1 hr , followed by separation from unreacted NHS-PEG4-biotin on a NAP-5 desalting column ( GE Healthcare ) .", "Biotinylated BoNT/C-Hc was prepared as described previously ( Takikawa et al . , 2014 ) .", "Alexa Fluor 488 streptavidin ( degree of labeling 5 , Thermo Scientific ) , the biotinylated BoNT/C-Hc , and the biotinylated ATPOS were mixed in a buffer ( 10 mM HEPES , 150 mM NaCl , and 2 . 5 mM KCl; pH 7 . 4 ) at final concentrations of 2 μM , 2 μM , and 6 μM , respectively .", "Absorption spectra were obtained in HEPES-buffered saline ( HBS; 50 mM HEPES , 125 mM NaCl , 4 mM KCl , 2 mM CaCl2 , and 1 mM MgCl2 ) containing 0 . 1% bovine serum albumin at pH 7 . 4 with a spectrometer ( V-550 , JASCO ) at 25–26°C .", "Fluorescence spectroscopic measurements were performed in HBS containing 0 . 1% bovine serum albumin at pH 7 . 4 with a spectrofluorometer ( FP-6500 , JASCO ) .", "The excitation wavelengths of Cy3 and Alexa488 were 550 nm and 490 nm , respectively , and the emission wavelengths were 570 nm and 519 nm , respectively .", "Fluorescence intensities were measured three times at each concentration of ATP , ADP ( Sigma ) , AMP ( Sigma ) , adenosine ( Sigma ) , CTP ( Apollo Scientific ) , GTP ( Nacalai tesque ) , or UTP ( Combi-Blocks ) at 25–26°C .", "Mean values of the triplicate measurements were calculated for each sample .", "The apparent dissociation constant and Hill coefficient were determined by fitting to the Hill equation .", "For the assessment of pH dependence , HEPES ( pH 7 . 0–8 . 5 ) or MES ( pH 6 . 0–6 . 5 ) was used as a buffer compound .", "The fluorescence quantum yield ( QY ) was determined by using Rhodamine B in basic ethanol ( QY = 0 . 65 ) as a standard ( Kubin and Fletcher , 1982 ) .", "Measurements for the kinetic analysis were performed with a stopped-flow apparatus ( SFM 2000 , BioLogic ) , whose flow path was pretreated with a solution ( 155 mM NaCl , 3 mM Na2HPO4 , and 1 mM KH2PO4; pH 7 . 4 ) containing 1% gelatin ( Sigma ) or with Bullet Blocking One ( Nacalai tesque ) for blocking , followed by extensive washing with Milli-Q water .", "For measuring association kinetics , equal volumes of HBS with 0 . 1% bovine serum albumin containing either ATPOS or ATP were mixed at ~20 nM final concentration of ATPOS .", "For measuring dissociation kinetics , HBS containing 5 nM ATPOS and 200 nM ATP was diluted with an equal volume of HBS .", "Cy3 was excited at 546 nm and the emission was collected through a 570–625 nm band-pass filter ( Olympus ) .", "Fluorescence intensity was measured five times at each concentration of ATP at 25–26°C .", "Averaged traces of fluorescence intensity , F , were fitted with the following equation:F=c0+c1t+c2e−kt ( cn:constant ) where k is the observed rate constant .", "Cultured neurons were incubated with ATPOS complex at a final ATPOS concentration of 600 nM in physiological salt solution ( 50 mM HEPES , 125 mM NaCl , 2 . 5 mM KCl , 2 mM CaCl2 , 1 mM MgCl2 , and 25 mM glucose; pH 7 . 4 ) containing 0 . 1% bovine serum albumin at room temperature .", "After 10 min incubation , the cells were washed four times with physiological salt solution containing 0 . 1% bovine serum albumin .", "Fluorescence images were acquired with an inverted microscope ( IX-71 , Olympus ) equipped with an EM-CCD camera ( iXon EM+ , Andor ) and a 75 W xenon light source ( U-LH75XEAPO , Olympus ) , using a × 40/0 . 95 NA dry objective lens ( UPlanSApo , Olympus ) .", "Cy3 was excited at 520–550 nm and the emission was measured at wavelengths longer than 580 nm with a filter set ( Olympus ) .", "Alexa488 was excited at 460–480 nm and the emission was measured at 495–540 nm with a filter set ( Olympus ) .", "The extracellular solution was physiological salt solution containing 0 . 1% bovine serum albumin and 100 μM ARL 67156 ( Sigma ) at pH 7 . 4 .", "Male C57BL/6 mice ( SLC; postnatal 1–2 months old ) were anesthetized with an intraperitoneal injection of a mixture of medetomidine ( Nippon Zenyaku; 0 . 75 mg/kg ) , midazolam ( Sandoz; 4 mg/kg ) , and butorphanol ( Meiji Seika Pharma; 5 mg/kg ) .", "The depth of anesthesia was assessed by tail pinch .", "The body temperature was maintained at 37°C with a heating pad ( BWT-100A , Bio Research Center ) throughout the experiment .", "After the scalp was removed , a custom-made metal frame was attached to the exposed skull with dental acrylic ( Fuji LUTE BC , GC ) for head fixation .", "A 4-mm-diameter craniotomy centered 3 mm posterior to the bregma and 3 mm lateral to the midline was performed with a dental drill .", "The dura was left intact during the procedure .", "The ATPOS complex solution was pressure-injected into the cerebral cortex for 20–30 min through a glass micropipette whose tip was inserted to a depth of 300 μm from the pia .", "The surface of the cortex was covered with artificial cerebrospinal fluid ( aCSF; 125 mM NaCl , 4 . 5 mM KCl , 1 . 25 mM NaH2PO4 , 26 mM NaHCO3 , 2 mM CaCl2 , 1 mM MgCl2 , and 20 mM glucose; pH 7 . 4 ) .", "Fluorescence images were captured with a wide-field microscope ( M165FC , Leica ) equipped with a 150 W xenon lamp with a high-speed scanning polychromatic light source ( C7773 , Hamamatsu Photonics ) and an EM-CCD camera ( Evolve 512 , Roper Scientific ) , using a × 1 . 0 objective lens ( Plan APO , Leica ) .", "Cy3 and Alexa488 were excited at 555 nm and 490 nm , respectively , and the emissions of Cy3 and Alexa488 were sequentially collected at 2 . 5 Hz ( 200 ms duration for each emission ) through a filter set ( Leica ) at 592–682 nm and at 505–540 nm , respectively .", "Two-photon ATP imaging was performed using a two-photon laser scanning microscope ( Leica TCS SP8 MPO , Leica ) equipped with an optical parametric oscillator ( OPO ) laser system and a HyD detector ( Leica ) , using a × 25/0 . 95 NA water-immersion objective lens ( HCX IR APO , Leica ) .", "Cy3 was excited at 1 , 045 nm and the emission was collected through a filter set ( Semrock ) at 565–605 nm .", "Test compounds or reagents dissolved in aCSF were pressure-injected at 1 psi into the cerebral cortex with a glass micropipette ( 10 μm inner tip diameter ) inserted at the depth of 300 μm from the surface .", "For ATP application , 10 mM ATP was pressure-injected for 10 s .", "In some experiments , 100 U/ml apyrase ( Nacalai tesque ) or bovine serum albumin at a concentration equivalent to that of apyrase was pressure-injected for 15 min .", "For electrical stimulation , a monopolar tungsten microelectrode ( 1–3 MΩ , FHC ) was placed below the dura , and a ground electrode was attached to the custom-made metal frame for head fixation .", "A train of 100 μs pulses at 200 Hz lasting for 1 s with an intensity of 10 mA was delivered using a stimulus isolator ( ISO-Flex , AMPI ) .", "Image data were analyzed with ImageJ software ( NIH; ver . 1 . 50e ) .", "After background fluorescence was subtracted from images , fluorescence response ( R/R0 or F/F0 ) was calculated relative to the baseline before ATP application or electrical stimulation .", "All data are presented as mean ± SEM .", "Wilcoxon’s rank-sum tests are used to determine statistical significance ." ] ]

[ "Adenosine 5’ triphosphate ( ATP ) is a ubiquitous extracellular signaling messenger .", "Here , we describe a method for in-vivo imaging of extracellular ATP with high spatiotemporal resolution .", "We prepared a comprehensive set of cysteine-substitution mutants of ATP-binding protein , Bacillus FoF1-ATP synthase ε subunit , labeled with small-molecule fluorophores at the introduced cysteine residue .", "Screening revealed that the Cy3-labeled glutamine-105 mutant ( Q105C-Cy3; designated ATPOS ) shows a large fluorescence change in the presence of ATP , with submicromolar affinity , pH-independence , and high selectivity for ATP over ATP metabolites and other nucleotides .", "To enable in-vivo validation , we introduced BoNT/C-Hc for binding to neuronal plasma membrane and Alexa Fluor 488 for ratiometric measurement .", "The resulting ATPOS complex binds to neurons in cerebral cortex of living mice , and clearly visualized a concentrically propagating wave of extracellular ATP release in response to electrical stimulation .", "ATPOS should be useful to probe the extracellular ATP dynamics of diverse biological processes in vivo ." ]

[ "Biologists often refer to a small molecule called adenosine triphosphate – or ATP for short – as ‘the currency of life’ .", "This molecule carries energy all through the body , and most cells and proteins require ATP to perform their various roles .", "Nerve cells ( also known as neurons ) in the brain release ATP when activated , and use this molecule to send signals to other active neurons or other cells in the brain .", "But ATP can also signal danger in the brain .", "A molecule derived from ATP is involved in transmitting the pain signals of migraines and severe headaches; and ATP levels can become imbalanced after strokes , when parts of the brain are deprived of blood .", "Despite its importance , ATP remains difficult to visualize in the body , and monitoring the molecule in the active brain in real time is challenging .", "To address this issue , Kitajima et al . designed an optical sensor that could monitor ATP in the healthy brain , and was sensitive enough to detect when and where it was released .", "First , Kitajima et al . made several potential sensors by attaching various fluorescent tags to different locations on a protein that binds ATP .", "Next each sensor was tested to determine whether it could bind ATP tightly and get bright upon binding .", "This is important because previous sensors could not detect ATP release in the brains of living animals .", "To illustrate the new sensors’ potential , Kitajima et al . used the sensor to image ATP in the brains of live mice .", "A ‘wave’ of ATP was seen spreading through the brain after neurons were stimulated with a small electric pulse , mimicking a sudden migraine or stroke .", "The results confirm that this new sensor is suitable for imaging how ATP signals in the brain , and it may help resolve the underlying mechanisms of migraines and strokes .", "This sensor could also be used to understand other cellular process which rely on ATP to carry out their role ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "immunology and inflammation" ]

[ [ "The regulation of protein expression is essential for development , differentiation , maintenance , division and death of cells as well as homeostasis of tissues ( Buszczak et al . , 2014 ) .", "Regulation of protein biogenesis is controlled on transcriptional , translational and post-translational level ( Day and Tuite , 1998 ) .", "Critical players in this complex process of protein biogenesis are RNA binding proteins ( RPBs ) that control many important aspects such as RNA trafficking , stability and translation rate ( Gerstberger et al . , 2014 ) .", "The RBPs are also involved in immunological recognition ( Kafasla et al . , 2014 ) .", "IMP3 ( also known as IGF2BP3 ) is a member of the family of the insulin-like growth factor 2 mRNA-binding proteins .", "It binds mRNAs via 6 RNA-binding domains consisting of 2 RNA recognition motif domains ( RRM ) and 4 K Homology domains ( KH ) ( Müeller-Pillasch et al . , 1997 ) .", "Besides the critical role of IMP3 in fetal development ( Mueller-Pillasch et al . , 1999 ) and fertility ( Li et al . , 2014; Hammer et al . , 2005 ) , its importance as an oncogene in several kinds of cancer was determined in the last few years: Expression of IMP3 could be observed in colon carcinoma ( Li et al . , 2009; Lochhead et al . , 2012; Kumara et al . , 2015 ) , adenocarcinomas ( Bellezza et al . , 2009; Lu et al . , 2009; Gao et al . , 2014 ) , urothelial carcinomas ( Sitnikova et al . , 2008; Xylinas et al . , 2014 ) , lymphomas ( King et al . , 2009; Tang et al . , 2013; Hartmann et al . , 2012 ) , renal cell carcinomas ( Hoffmann et al . , 2008; Jiang et al . , 2006; 2008 ) , and many more ( Hammer et al . , 2005; Chen et al . , 2013; Walter et al . , 2009; Köbel et al . , 2009; Zhou et al . , 2014 ) .", "Additionally , high expression of IMP3 often correlates with poor survival prognosis for patients ( Hoffmann et al . , 2008; Jiang et al . , 2006; Köbel et al . , 2009; Yuan et al . , 2009; Lin et al . , 2013 ) .", "So far , the role of IMP3 as regulator of cell proliferation , migration and invadopodia formation was mainly studied due to its ability to bind and stabilize mRNAs coding for insulin like growth factor 2 ( IGF2 ) or CD44 ( Liao et al . , 2005; Vikesaa et al . , 2006 ) .", "Practically nothing is known on its immune evasion properties .", "NK cells are important for the immune surveillance of transformed cells .", "They belong to the innate immune system ( although they possess some adaptive features as well [Sun et al . , 2011; Min-Oo et al . , 2013] ) and are able to kill transformed cells , virus-infected cells and to interact with bacteria and fungi ( Wu and Lanier , 2003; Lodoen and Lanier , 2006; Lanier , 2008; Seidel et al . , 2012; Schmidt et al . , 2013; Koch et al . , 2013 ) .", "The killing by NK cells is mediated by several NK -activating receptors , among them is NKG2D ( Seidel et al . , 2012; Koch et al . , 2013; Bauer et al . , 1999 ) .", "The ligands of NKG2D are MHC class I polypeptide-related sequences A and B ( MICA , MICB ) and the family of unique length 16 ( UL16 ) binding proteins 1 – 6 ( ULBP 1–6 ) , collectively known as stress-induced ligands ( Elias and Mandelboim , 2012 ) .", "The stress-induced ligands are differentially expressed on the cell surface following stresses like viral infection , heat-shock or genotoxic stress ( Raulet et al . , 2013 ) .", "Accordingly , these ligands are important for immune surveillance and both cancer cells and viruses often suppress stress ligand surface expression ( Elias and Mandelboim , 2012; Salih et al . , 2002; Fuertes et al . , 2008; Stern-Ginossar and Mandelboim , 2009 ) .", "Although mRNAs for some of these stress ligands are found almost in every cell , these ligands are barely expressed on the surface of healthy cells ( Stern-Ginossar and Mandelboim , 2009 ) .", "This suggests that the expression of the NKG2D ligands is also controlled at mRNA level , and indeed , we have previously shown that 10 different cellular miRNAs negatively regulate the expression of MICA and MICB proteins ( Stern-Ginossar et al . , 2007; 2009; Nachmani et al . , 2009 ) .", "We further demonstrated that several RBPs are involved in the regulation of MICB expression ( Nachmani et al . , 2014 ) .", "In this study , we show a new mechanism of IMP3 that facilitates immune evasion of cancerous cells by downregulation of the NKG2D ligand ULBP2 in a direct manner and MICB in an indirect manner .", "Thereby , we give new insights into the complex biological processes that are regulated by this powerful oncogene .", "Notably , we also discovered the first cellular mechanism acting on ULBP2 ." ], [ "Our group has previously demonstrated that the stress-induced ligand MICB is regulated by numerous RBPs that directly affect its stability and expression ( Nachmani et al . , 2014 ) .", "In order to identify additional regulators of other NKG2D ligands , particularly of the ULBP family , we performed RNA binding protein affinity purification ( RNA-AP ) with subsequent analysis by mass spectrometry ( schematically described in Figure 1A ) . 10 . 7554/eLife . 13426 . 003Figure 1 . RNA affinity purification using the 3 ‘UTR of ULBP2 . ( A ) Schematic representation of the workflow in RNA affinity purification ( RNA-AP ) . ( B ) Cytoplasmic extracts of RKO cells were incubated with RNA coding for the 3‘UTR of ULBP2 or control UTRs . The enriched RNA-binding proteins were run on 10% polyacrylamide gel and stained with Coomassie BB G-250 . A specific band ( indicated with an arrow , 64 kDa ) was excised and analyzed by mass spectrometry . For facilitated visualization , a detail of the Coomassie gel is shown in false color and contrast was increased . ( C ) Top-listed results from mass spectrometry analysis of the excised gel part . IMP3 ( here called IGF2BP3 ) is the hit with the highest coverage and amount of specific and total peptides in the analyzed gel band . DOI: http://dx . doi . org/10 . 7554/eLife . 13426 . 003 For that purpose , we cloned the 3’UTR sequences of ULBP1 , 2 and 3 as well as control UTRs and generated in vitro RNAs in presence of biotin-UTP ( Figure 1A ) . About 10 percent of the incorporated UTPs were biotinylated . Next , we generated cytoplasmic extracts of RKO cells ( since they express numerous NKG2D ligands , see figures below ) and incubated the RNAs with the extracts ( Figure 1A ) . We purified the biotinylated RNAs with the specifically bound RBPs using streptavidin-coated sepharose beads and separated the enriched proteins by running the eluates on SDS PAGE gels using reducing conditions . We performed Coomassie staining and excised protein bands that selectively appeared in the lane of ULBP2 ( Figure 1B , a band at 64 kDa , indicated by an arrow ) . The band that can be seen in the ULBP2-3’UTR at about 120 kDa was not specific . Mass spectrometry analysis of an excised gel slice revealed IMP3 ( Figure 1C , named Insulin-like growth factor mRNA-binding protein 3 ) as the protein with the highest coverage ( percentage of protein sequence covered by identified peptides , named 'ΣCoverage' in Figure 1C ) , the most unique peptides ( number of peptides unique to a specific protein , named Σ# Unique peptides in Figure 1C ) and total peptides ( number of peptides that can be attributed to a particular protein , named Σ# Peptides in Figure 1C ) , suggesting that IMP3 precipitates with the 3’UTR of ULBP2 . In order to verify that IMP3 indeed regulates the expression of ULBP2 , we transduced RKO cells with a plasmid containing a small hairpin RNA ( shRNA ) that selectively targets IMP3 ( RKO shIMP3 ) . As control , we transduced RKO cells with a scrambled shRNA ( RKO scrambled shRNA ) . We confirmed the knockdown using Western Blot ( WB ) with two different antibodies targeting IMP3 and used GAPDH as reference ( Figure 2A , shown is the WB with one of these anti-IMP3 antibodies ) . Quantification of the WB revealed a remaining protein expression of less than 10 percent ( Figure 2B ) . Additionally , we analyzed mRNA levels of IMP3 using qRT-PCR and observed a residual amount of about 13% mRNA of IMP3 compared to scrambled shRNA ( Figure 2C ) . Next , we analyzed the mRNA levels of the ULBP ligands in RKO shIMP3 compared to scrambled shRNA and observed a significant increase of ULBP2 mRNA levels following knockdown of IMP3 but no changes in mRNA levels of the other ULBP family members 1 and 3 ( Figure 2D ) . To assess the significance of this knockdown , we performed flow cytometry analysis of the NKG2D ligands ULBP1 , 2 and 3 . We observed an elevation in levels of ULBP2 upon IMP3 knockdown of more than 30% . The surface expression of ULBP3 was unchanged , while little or no expression of ULBP1 was observed on RKO cells prior and after the IMP3 knockdown ( Figure 2E ) . We quantified changes in ULBP2 levels in Figure 2F . In order to exclude a possible off-target effect of the shRNA that might cause the observed effects on the stress-ligand expression , we performed a rescue experiment of IMP3 expression in RKO ( Figure 2—figure supplement 1 ) . Expectedly , stress-ligand levels decreased back after the transduction of IMP3 in shIMP3 cells . 10 . 7554/eLife . 13426 . 004Figure 2 . Knockdown of IMP3 in RKO cells and surface staining of NKG2D ligands . ( A ) Western Blot analysis of IMP3 ( 64 kDa ) in RKO cells transduced with a scrambled shRNA or with a shRNA against IMP3 ( shIMP3 ) . GAPDH ( 36 kDa ) was used as a reference . Sections were cropped . ( B ) Quantification of IMP3 levels of Western Blots assays of two independent experiments performed with two different antibodies recognizing IMP3 , relative to GAPDH expression *p=0 . 02 in student’s t-test . ( C ) qRT-PCR analysis of mRNA levels of IMP3 levels in RKO cells transduced with a scrambled shRNA or with shIMP3 , normalized to GAPDH . *p<0 . 0001 in student’s t-test . ( D ) qRT-PCR analysis of mRNA levels for the NKG2D ligands in RKO transduced with shIMP3 compared to RKO transduced with scrambled shRNA . Combined data out of 4 replicates , statistics are calculated by comparing transcript levels of the same mRNA in RKO shIMP3 and RKO scrambled shRNA . *p ( ULBP2 ) <0 . 001 in one-sample t-test . ( E ) Surface expression of NKG2D ligands ULBP1 , 2 and 3 on RKO cells analyzed by FACS . Expression is shown on RKO cells transduced with shIMP3 ( red histogram ) and on cells transduced with a scrambled shRNA ( black histogram ) . The grey filled histogram is the background staining determined for an isotype mouse IgG antibody on shIMP3 RKO . Figure shows one representative experiment out of 3 performed . ( F ) Quantification of ULBP2 surface expression on transduced with a scrambled or a IMP3 targeting shRNA . *p ( ULBP2 ) =0 . 015DOI: http://dx . doi . org/10 . 7554/eLife . 13426 . 00410 . 7554/eLife . 13426 . 005Figure 2—figure supplement 1 . Rescue IMP3 in RKO shIMP3 cells reverses increase in ULBP2 expression . RKO transduced with a shRNA targeting IMP3 were selected using puromycin and subsequently transduced with a plasmid coding for IMP3 . FACS analysis of stress-ligand expression of ULBP2 is shown; cells that expressed the IMP3 targeting hairpin RNA were transduced either with an empty vector as control ( black histogram ) or with a IMP3 expression vector ( red histogram ) . The grey filled histogram is the background staining determined for an isotype mouse IgG antibody . Cells were gated according to their appearance in forward and side scatter and for the expression of GFP as reporter for the IMP3-overexpression . Figure shows each one representative plot out of 3 experiments performed . DOI: http://dx . doi . org/10 . 7554/eLife . 13426 . 005 To further validate the findings seen for the knockdown of IMP3 we transduced RKO cells with an IMP3 overexpression vector . We verified the overexpression using WB with GAPDH as reference ( Figure 3A ) . The WB quantification revealed a 9-fold overexpression of this RBP ( Figure 3B ) . The RNA levels of IMP3 were also significantly increased after transduction ( Figure 3C ) . Compared to the knockdown of IMP3 , RNA levels and FACS analysis of the overexpression revealed inverse effects on the stress-ligand expression ( Figure 3D and E ) . RNA levels in overexpression cells were about 20% reduced compared to the control cells for ULBP2 , but not for ULBP3 . In FACS analysis , the surface expression of ULBP2 decreased by about 10% . The surface expression of ULBP1 and ULBP3 was unchanged as seen for the knockdown . 10 . 7554/eLife . 13426 . 006Figure 3 . Surface staining of stress-induced ligands in IMP3 overexpressing RKO cells . ( A ) Western Blot analysis of IMP3 in parental RKO cells and RKO cells overexpressing IMP3 ( 64 kDa ) . GAPDH ( 36 kDa ) was used as a reference , sections were cropped . ( B ) Quantification of IMP3 levels of Western Blots assays of two independent experiments performed relative to GAPDH expression . *p<0 . 03 in student's t-test . ( C ) IMP3 RNA levels in the RKO-IMP3 compared to RKO analyzed by qRT-PCR . ( D ) RNA levels of ULBP2 and ULBP3 in RKO-IMP3 compared to RKO-dsRed analyzed by qRT-PCR . *p ( ULBP2 ) =0 . 044 , *p ( ULBP3 ) =0 . 550 in student's t-test . ( E ) Surface expression of NKG2D ligands ULBP1 , 2 and 3 on RKO cells analyzed by FACS . Expression is shown on RKO cells overexpressing IMP3 ( red histogram ) and on parental cells ( black histogram ) . The grey filled histogram is the background staining determined for an isotype mouse IgG antibody on parental RKO . Figure shows one representative experiment out of 3 performed . ( F ) Quantification of ULBP2 surface expression on RKO or RKO-IMP3 , *p ( ULBP2 ) =0 . 034 . DOI: http://dx . doi . org/10 . 7554/eLife . 13426 . 006 To see if the effect of IMP3 on the stress-induced ligands ULBP2 is specific for the colon carcinoma RKO cell line , we performed a knockdown of IMP3 in the colorectal carcinoma HCT116 and in the embryonic kidney-derived cell line 293T and verified the knockdowns using WB . In HCT116 cells , the shRNA targeting IMP3 caused a loss of about 85%; in 293T cells , we could virtually not detect any remaining IMP3 protein ( Figure 4A , quantified in Figure 4B ) . 10 . 7554/eLife . 13426 . 007Figure 4 . ULBP2 after IMP3 knockdown and overexpression in HCT116 and 293T cells . ( A ) Western Blot analysis of IMP3 knockdown in HCT116 and 293T cells compared to cells transduced with a scrambled shRNA . For HCT116 cells , Vinculin was used as reference ( 130kDa ) ; for 293T cells , GAPDH was used ( 36kDa ) . Sections were cropped . ( B ) Quantification of IMP3 WB in HCT116 and 293T , a single experiment was performed . ( C ) FACS analysis of ULBP2 on HCT116 and 293T cell lines with or without IMP3 knockdown . ( D ) Quantification of FACS analysis on HCT116 and 293T cell lines with a knockdown for IMP3 shown if figure C . Statistical analysis for data of three replicates was performed using student’s t-test . *p ( ULBP2 , HCT116 ) =0 . 0014 , *P ( ULBP2 , 293T ) =8 . 97 E-4 . ( E ) Cell surface staining for ULBP2 in IMP3-overexpressing cell lines HCT116 and 293T . Cells were gated according to their appearance in forward and side scatter and to their GFP levels in FACS that correlate with IMP3 expression . ( F ) Quantification of surface expression of ULBP2 in IMP3 overexpressing HCT116 and 293T cells shown in figure E . Statistical analysis for data of three replicates was performed using student’s t-test . *p ( ULBP2 , HCT116 ) =9 . 46 E-9 , *p ( ULBP2 , 293T ) =0 . 0002 . DOI: http://dx . doi . org/10 . 7554/eLife . 13426 . 00710 . 7554/eLife . 13426 . 008Figure 4—figure supplement 1 . Rescue IMP3 in 293T shIMP3 cells reverses ULBP2 increase . 293T transduced with a shRNA targeting IMP3 were selected using puromycin and subsequently transduced with IMP3 using a lentiviral vector . FACS analysis of stress-ligand expression of ULBP2 is shown; staining of shIMP3 cells transduced with an empty vector as black histogram and cells with rescued IMP3 expression as red histogram . The grey filled histogram is the background staining determined for an isotype mouse IgG antibody . Cells were gated according to their appearance in forward and side scatter and for expression of GFP as reporter for the IMP3-overexpression . Figure shows each one representative plot out of three experiments performed . DOI: http://dx . doi . org/10 . 7554/eLife . 13426 . 008 Notably , we observed similar effects in all IMP3 knockdown cell lines on stress-ligand expression ( Figure 4C ) . In HCT116 colorectal carcinoma cell lines , the knockdown resulted in a significant increase of ULBP2 of more than 30%; similarly , we observed an increase of about 15% for ULBP2 in 293T upon IMP3 loss this cell line ( Figure 4D ) . In order to exclude off-target effects of the shRNA in this cell line as well , we performed a rescue experiment of IMP3 expression in 293T as well ( Figure 4—figure supplement 1 ) . As we observed for RKO , ULBP2 levels in 293T cells decreased back after the transduction of IMP3 in shIMP3 cells . To confirm our findings , we performed the overexpression of IMP3 also in HCT116 and 293T cells . In both cell lines , we saw an even stronger decrease of ULBP2 in comparison to RKO after overexpression of IMP3 ( Figure 4D ) : 25% for HCT116 and 35% loss for 293T cells . The results are quantified in Figure 4F . We next investigated the mechanism by which the RBP IMP3 affects ULBP2 expression . To this end , we incubated RKO shIMP3 and RKO scrambled shRNA for 16 hr with the transcription inhibitor D-Actinomycin or with DMSO as diluent control . D-Actinomycin blocks the elongation process of the RNA polymerase II thereby suppressing the generation of new mRNAs . Consequently , a comparison of the RNA levels in the diluent control and the D-Actinomycin treated cells shows the rate of mRNA decay of a specific gene of interest tested in qRT-PCR . After 16 hr , we isolated total RNA and performed subsequently qRT-PCR analysis on the corresponding cDNAs . We defined the amount of mRNAs in the DMSO-treated control as 1 and calculated the remaining mRNA levels after D-Actinomycin treatment for RKO shIMP3 and RKO scrambled shRNA . Since no new transcripts can be synthesized due to D-Actinomycin treatment , all observed differences are due to alterations in the RNA decay rate . Interestingly , we observed significantly increased amounts of transcript coding for ULBP2 in absence of IMP3 ( shIMP3 ) , but – expectedly – no difference in the levels of ULBP3 ( Figure 5 ) . For ULBP2 , about 45% of the initial RNA levels were still present after 16 hr of treatment in the IMP3-knockdown cell lines , but only about 30% in the RKO cell line with a scrambled shRNA ( Figure 5 ) . Therefore , we assumed a direct IMP3 binding to ULBP2 mRNA that decreases RNA half-life . 10 . 7554/eLife . 13426 . 009Figure 5 . Stability determinations of mRNA transcripts of ULBP2 and 3 . RKO transfected with a scrambled shRNA ( control ) or with shIMP3 were treated with D Actinomycin or with DMSO as control . 16 hr later , mRNAs were isolated and cDNA was prepared . The various mRNA transcripts were analyzed using qRT-PCR . Transcript levels were compared by normalization to GAPDH and by setting transcript levels determined for DMSO treatment as 1 . Figure shows merged data of three replicates . *p<0 . 01 for ULBP2 in student’s t-test; for ULBP3 , no significant differences were observed ( NS ) . DOI: http://dx . doi . org/10 . 7554/eLife . 13426 . 009 To further demonstrate that IMP3 directly interacts with the 3’UTRs of ULBP2 , we fused the 3’UTRs of ULBP2 and two control UTRs - ULBP3 and CD247 3’UTRs ( we used the 3’UTR of CD247 ( CD3ζ ) that seemed unlikely to be affected by IMP3 ) to luciferase genes in the vector pGL3 ( schematically described in Figure 6A ) . The constructs were transiently expressed in RKO shIMP3 cells or RKO scrambled shRNA cells and luciferase activity was determined . Following the knockdown of IMP3 , we observed a significant increase if the luciferase gene was fused to the ULBP2-3’UTR only ( Figure 6B ) . The luciferase activity of all other constructs was not affected . Taking the results of the stability determination in consideration ( Figure 5 ) together with the luciferase assay , we concluded that IMP3 decreases directly the mRNA stability of ULBP2 . 10 . 7554/eLife . 13426 . 010Figure 6 . Post-transcriptional effects of IMP3 on ULBP2 mRNA assessed by luciferase assay . ( A ) Schematic representation of the luciferase constructs used for this assay with the 3’UTRs of the ULBP family members including their lengths in nucleotides ( nt ) . ( B ) The 3‘UTRs of ULBP 1 , 2 , 3 and CD247 ( as control ) were fused to the luciferase gene as shown in A and expressed in RKO cells transduced either with scrambled shRNA or with shIMP3 . 28 hr after transfection of the luciferase vectors , luciferase activity was measured . The results were normalized to empty vector control and statistics were performed based on data acquired for the control UTR ( CD247 ) . Figure shows merged data of three independent replicates *p=0 . 023 in student’s t-test . ( C ) Schematic representation of the truncation mutants of the ULBP2 3’UTR . ( D ) Luciferase assay with the 3’UTR of ULBP2 and shortened variants presented in ( C ) , expressed in RKO cells transduced either with scrambled shRNA or with shIMP3 . Shown is merged data of three independent replicates . The luciferase activity of the fragment ranging from 1–100 bp was significantly lower than the activity of the fragment 1–200 bp ( *p=0 . 002 ) , 1 300 bp ( *p=0 . 006 ) , 1–400 bp ( *p=0 . 041 ) and the full 3'UTR ( *p=0 . 010 ) ( E ) Schematic representation of the ULBP2 3’UTR sequences areas that were mutated . The putative binding motif CATT is shown in loose characters ( positions 161 – 164 ) . The introduced mutations in the 3’ UTRs ( CAGG ) are shown in red letters .", "( F ) The wild type ( WT ) or mutated 3’UTRs ( as shown in A ) of ULBP2 was transiently expressed in RKO cells transduced with scrambled shRNA or with shRNA IMP3 .", "Luciferase activity was assayed 28 hr after transfection .", "Shown is merged data of three independent replicates .", "*p<0 . 001 in student’s t-test . DOI: http://dx . doi . org/10 . 7554/eLife . 13426 . 010 Next , we wanted to identify the binding site of IMP3 within the 3’UTRs of ULBP2 .", "To narrow down the exact binding site , we decided to perform a continuous shortening of the ULBP2-3’UTR .", "Out of the full length 3’UTR of 535 base pairs , we generated four short variants covering the range from 1–100 , 1–200 , 1–300 , 1–400 base pairs and compared it to the constructs with the complete 3’UTR ( schematically shown in Figure 6C ) .", "We observed that the construct ranging from 1 to 100 base pairs yielded an equal ratio of luciferase activity in RKO shIMP3 and RKO shRNA scrambled ( Figure 6D ) .", "All other constructs , including the construct ranging from 1 to 200bp , had significantly higher luciferase activities in the IMP3 knockdown cells compared to the shRNA scrambled ( Figure 6D ) .", "Thus , we concluded that the binding site for IMP3 must be located between 100 and 200bp of the 3’UTR of ULBP2 .", "In 2010 , Hafner et al . used PAR-CLIP technology to identify putative binding sites of RNA binding proteins and proposed the binding motif ‘CAUU’ for IMP3 equivalent to CATT on DNA level ( Hafner et al . , 2010 ) .", "This motif exists twice in the 3’UTR ULBP2 , at the positions 161–164 and 292–295 of the 3’UTR .", "Since we determined that the IMP3 binding site in the 3’UTR of ULBP2 is located between 100 and 200 base pairs ( Figure 6D ) , we replaced by PCR the TT nucleotides of the CATT motif found at position 164/165 with GG yielding in CAGG ( schematically shown in Figure 6E ) .", "Consequently , the ULBP2-3’UTR mutation abrogated the effect of IMP3-dependent luciferase activity ( Figure 6F ) completely .", "Therefore , we concluded from this assay that there is only a single binding site for IMP3 in the 3’UTR of ULBP2 .", "Next , we tested the functional relevance of ULBP2 targeting IMP3 .", "To this end , we co-incubated primary activated bulk NK cells that express the activating receptor NKG2D with RKO , HCT116 and 293T cells expressing shIMP3 or a scrambled shRNA and performed NK cytotoxicity assays .", "We observed a significantly higher lysis of shIMP3-expressing RKO cells ( Figure 7A ) , HCT116 cells ( Figure 7B ) and 293T cells ( Figure 7C ) consistent with the increased surface expression levels of ULBP2 on RKO and HCT116 ( Figure 2E and Figure 4B ) and ULBP2 only on 293T ( Figure 4B ) .", "By using a blocking antibody for NKG2D , we demonstrated that the differences observed are due to NKG2D recognition since when NKG2D was blocked killing of the cells was almost identical .", "The observed drastic decrease in NK cell activation was remarkable taking the moderate shift of ULBP2 following knockdown into account .", "For that reason , effect of IMP3 on the remaining NKG2D ligands MICA and MICB ( MHC class I polypeptide-related sequence A and B ) was investigated as well . 10 . 7554/eLife . 13426 . 011Figure 7 . Knockdown of IMP3 enhances NK cell-mediated killing of cancer cells in a NKG2D dependent manner .", "( A-C )", "Primary human NK cells were incubated with an isotype antibody ( left columns , αIsotype ) or with anti-hNKG2D monoclonal antibody ( right column , αNKG2D ) for one hour on ice before target cells – either transduced with a control shRNA or shIMP3 – were added .", "35S released into the supernatant upon target cell lysis by NK cells , was assessed 3 hr later ( A ) 35S release by RKO cells co-cultured with NK cells in the ratio 1:25 .", "*p=0 . 023 in student’s t-test .", "( B ) 35S release by HCT116 cells co-cultured with NK cells in the ratio 1:10 .", "*p=0 . 001 in student’s t-test .", "( C ) 35S release by 293T cells co-cultured with NK cells in the ratio 1:10 .", "*P=0 . 013 in student’s t-test .", "All experiments were performed at least twice and one representative replicate is shown . DOI: http://dx . doi . org/10 . 7554/eLife . 13426 . 011 To assess if IMP3 affects the expression of MICA and MICB , we stained RKO and 293T cells with IMP3 knockdown or a transduced scrambled control for expression of these NKG2D ligands .", "We found RKO to be negative for MICA but highly positive for MICB .", "In contrast , 293T cells express MICA but lack MICB expression ( Figure 8A ) .", "Interestingly , we observed an increase of about 50% for MICB following IMP3 knockdown in RKO ( quantified in Figure 8B ) , but no effect on MICA .", "We also validated these results by performing the rescue experiments of IMP3 in these cell lines .", "In agreement with the KD experiments MICB expression was reduced after the restoration of IMP3 expression in RKO cells and the no effect was seen regarding MICA ( Figure 8—figure supplement 1 ) .", "To further confirm that IMP3 affects MICB expression , we overexpressed this RBP in the parental RKO cell line .", "A dramatic reduction of MICB expression was observed ( Figure 8C ) and only about 20% of the original MICB expression remained ( Figure 8D ) .", "Consistent with our observations for the surface expression of MICB , we could also detect an elevation MICB , but not MICA , RNA levels in RKO cells following IMP3 knockdown ( Figure 8E ) .", "Surprisingly , we could neither detect a IMP3-dependent change in stability of the MICB mRNA using D-Actinomycin treatment ( Figure 8F ) nor IMP3-dependent effects on transcript stability , processing or translation efficacy that would be observed in a luciferase experiment ( Figure 8G ) .", "Consequently , we conclude that the IMP3 uses different mechanisms to affect mRNA and protein levels of ULBP2 and MICB . 10 . 7554/eLife . 13426 . 012Figure 8 . IMP3 regulates MICB in a functionally distinct mechanism .", "( A ) FACS analysis of MICA and MICB surface expression on RKO and 293T cells .", "Expression is shown on cells transduced with shIMP3 ( red histogram ) and on cells transduced with a scrambled shRNA ( black histogram ) .", "The grey-filled histogram is the background staining determined for an isotype mouse IgG antibody .", "Figure shows one representative experiment out of three performed .", "( B ) Quantification of MICB surface expression of RKO cells transduced either with a scrambled or an IMP3 targeting shRNA .", "*P ( MICB ) =0 . 029 in student’s t-test ( C ) FACS analysis of MICB surface protein levels in IMP3 overexpressing RKO cells ( red histograms ) or controls ( black histogram ) , Shown is one representative experiment of at least three performed ones .", "( D ) Quantification of MICB surface expression of IMP3 overexpressing RKO cells or control cells *P ( MICB ) =1 . 24 E-6 in student’s t-test .", "( E ) RNA levels of MICA and MICB in RKO transduced with an IMP3 targeting shRNA relative to the scrambled shRNA control and normalized to GAPDH .", "*P ( MICB ) =0 . 0005 .", "( F ) RKO transfected with a scrambled shRNA ( control ) or with an IMP3 targeting shRNA were treated with D-Actinomycin or with DMSO as control .", "After 16 hr , mRNAs were isolated and cDNA was prepared .", "The various mRNA transcripts were analyzed using qRT-PCR .", "Transcript levels were compared by normalization to GAPDH and by setting transcript levels determined for DMSO treatment as 1 .", "Figure shows merged data of three replicates .", "( G ) The 3‘UTRs of MICA and MICB and CD247 ( as control ) were fused to the luciferase gene and expressed in RKO cells transduced either with scrambled shRNA or with shIMP3 .", "28 hr after transfection of the luciferase vectors , luciferase activity was measured .", "Results were normalized to empty vector control and statistics were performed based on data acquired for the control UTR ( CD247 ) .", "No significant changes were obtained ( NS ) .", "Figure shows merged data of three independent replicates . DOI: http://dx . doi . org/10 . 7554/eLife . 13426 . 01210 . 7554/eLife . 13426 . 013Figure 8—figure supplement 1 . Rescue IMP3 in RKO and 293T shIMP3 cells reverses MICB downregulation but does not affect MICA expression . RKO and 293T cells transduced with a shRNA targeting IMP3 were selected using puromycin and subsequently transduced with a plasmid coding for IMP3 .", "FACS analysis of stress-ligand expression MICB and ULBP2 for RKO cells or MICA and ULBP2 in 293T cells is shown .", "As shown before , MICA is not expressed on RKO cells and 293T cells are negative for MICB .", "RKO and 293T shIMP3 cells were transduced with either with an empty vector ( black histogram ) or rescued with an IMP3 expression plasmid ( red histogram ) .", "The grey filled histogram is the background staining determined for an isotype mouse IgG antibody .", "Figure shows each one representative plot out of three experiments performed . DOI: http://dx . doi . org/10 . 7554/eLife . 13426 . 013" ], [ "The understanding of the diverse mechanistic details of oncogenes in all stages of carcinogenesis: tumor initiation , tumor promotion , malignant conversion and tumor progression ( Multistage Carcinogenesis , 2015 ) , and the complex interplay of oncogenes with tumor suppressing genes is one of most challenging but also most important objectives in cancer research .", "Immunotherapy is at the forefront of current cancer research and treatment , with numerous and diverse approaches aimed at harnessing the immune system to fight cancer ( Rosenberg et al . , 2004; Rosenberg et al . , 2008; Weiner et al . , 2010; Mellman et al . , 2011; Pardoll , 2012 ) .", "Accordingly , understanding the regulation of stress-induced ligands has immense importance as they pose potential therapeutic targets on tumor cells .", "The stress-induced ligands that are bound by the activating receptor NKG2D seem to play a critical role in immune surveillance and immune escape .", "Tumors and viruses alike developed numerous mechanisms to avoid surface expression ( Seidel et al . , 2012; Salih et al . , 2002; Fuertes et al . , 2008; Nachmani et al . , 2009; Fernandez-Messina et al . , 2010; Nachmani et al . , 2010; Bauman and Mandelboim , 2011; Bauman et al . , 2011 ) and even to effectively suppress NK cells activity by the release of soluble or exosomal NKG2D ligands ( Fernandez-Messina et al . , 2010; Kloess et al . , 2010 ) .", "A complete knowledge of the complex mechanisms that tightly regulate the expression of surface markers in health or completely deregulate the expression in disease is essential to decide about the application of current therapeutic strategies and for the development of new therapeutics which could increase surface expression of these proteins .", "In this study , we present a new mechanism of immune escape that is mediated by the well-established oncogene IMP3 .", "IMP3 is absent in almost all healthy , adult tissues .", "However , expression of IMP3 could be observed in several kinds of cancer like in colon carcinoma ( Li et al . , 2009; Lochhead et al . , 2012; Kumara et al . , 2015 ) , adenocarcinomas ( Bellezza et al . , 2009; Lu et al . , 2009; Gao et al . , 2014 ) , urothelial carcinomas ( Sitnikova et al . , 2008; Xylinas et al . , 2014 ) , lymphomas ( King et al . , 2009; Tang et al . , 2013; Hartmann et al . , 2012 ) or renal cell carcinomas ( Hoffmann et al . , 2008; Jiang et al . , 2006; Jiang et al . , 2008 ) .", "We demonstrate in several human cell lines of different origins that the knockdown of IMP3 up-regulates surface expression of the NKG2D ligand ULBP2 .", "Consequently , we observed inverse effects on this stress-ligand following the overexpression of IMP3 .", "We demonstrate a direct interaction of IMP3 with ULBP2 mRNA .", "IMP3 was precipitated with biotinylated ULBP2 3’ UTR .", "We observed an increased half-life of ULBP2 mRNA transcripts and , consequently , elevated luciferase activity in absence of IMP3 .", "We also showed that only a single binding site for IMP3 in the 3’UTR of ULBP2 at the position 161–164 exists since its mutation abolished differences in IMP3-dependent luciferase activity .", "We tested the effect of IMP3 on ULBP2 in several cell lines using knockdown and overexpression of this oncogene .", "The effect on ULBP2 expression in RKO , HCT116 and 293T cells were in a comparable magnitude .", "The impact of IMP3 was capable of altering ULBP2 expression up to 35% in the cell lines tested .", "However , we also observed an effect of IMP3 on another stress-induced ligand , MICB .", "Higher expression levels of IMP3 correlated with a reduction of MICB mRNA and surface expression .", "In contrast to ULBP2 , we did not observe differences in half-life or a change in the luciferase activity in presence or absence of IMP3 for MICB .", "Also , the surface expression changes between knockdown and overexpression for ULBP2 and MICB varied tremendously: In the knockdown , the up-regulation of ULBP2 and MICB was more or less similar , but the IMP3 overexpression decreased only moderately ULBP2 expression , but drastically affected MICB expression .", "Thus , we concluded that the IMP3-dependant alterations in MICB expression are probably indirect .", "Most likely , IMP3 acts on a so far unidentified transcription factor or regulator that subsequently leads to alterations in MICB gene transcription .", "The identification and validation of this putative mediating factor requires further investigation which is beyond the scope of this manuscript .", "Finally , we assessed the functional significance in an in vitro system by co-culturing NK cells with RKO cells , HCT116 cells or 293T cells with or without IMP3 knockdown .", "For all knockdown cell lines , we observed higher killing compared to the cells with natural levels of IMP3 .", "The physiological significance of IMP3 in healthy , adult tissues is still not completely understood .", "Obviously , the widely held belief that IMP3 is a strictly oncofetal gene could be disproved by reports that showed expression both in testis and placenta of healthy individuals ( Li et al . , 2014; Hammer et al . , 2005 ) .", "The recent discoveries about the role in placental trophoblasts might also shed a light on the array of target genes that are known to be affected by IMP3 , for instant CD44 expression that is up-regulated by IMP3 is important in migration of trophoblasts within the placenta .", "Accordingly , the destabilization of ULBP2 mRNA and regulation of MICB expression described in this study might also protect trophoblasts in the placenta from NK cells , which are the most abundant population of lymphocytes present in the decidua during early pregnancy ( Varla-Leftherioti , 2005; Sánchez-Rodríguez et al . , 2011 ) .", "This assumption requires further experimental support; nevertheless , it would explain the protection of cancerous cells by IMP3 expression that we spotlighted .", "Although we could investigate a new effect of IMP3 , its role in carcinogenesis is still incompletely understood as well .", "Our knowledge about its multiple targets that are discovered up to now raise the hypothesis that the complexity of action of an oncogenic RNA-binding protein might exceed the complexity of classical oncogenes like cytosolic or receptor tyrosine kinases .", "Further research is required to elucidate additional target RNAs that this RNA-binding protein can regulate and to ultimately develop an inhibitor for IMP3 that prevents its impacts on tumor cells .", "For cancer therapy , IMP3 might be an excellent drug target due to its restricted occurrence in healthy tissues and its widespread occurrence in different kinds of cancer and especially in cases of unfavorable prognosis .", "In accordance with our results , an inhibition of IMP3 would - next to other potential benefits - lead to an elevated stress ligand expression and thereby enlighten recent approaches of NKG2D ligand-based cancer treatment , for instance , immunotherapy by Natural Killer cell infusion ( Locatelli et al . , 2013; Spear et al . , 2013 ) ." ], [ "All cells were cultivated in 37°C , >95% humidity and 5% CO2 in Dulbecco’s Modified Eagle Medium ( DMEM , Sigma , Israel ) supplemented with 10% heat-inactivated fetal calf serum ( Sigma-Aldrich , Israel ) and addition of non-essential amino acids , L-glutamine , sodium pyruvate and penicillin/streptomycin according to manufacturer’s instruction ( all Biological Industries , Israel ) .", "RKO is a human colon carcinoma cell line ( ATCC CRL-2577 ) , HCT116 is a human colorectal carcinoma cell line ( ATCC CLL-247 ) , 293T is a human cell line established from embryonic kidney ( ATCC CRL-3216 ) .", "All cells were directly received from the ATCC , Virginia , USA , therefore no further authentication was considered .", "Prior to transduction , cells were tested negative for mycoplasma contamination .", "All knockdown and overexpression cell lines showed good viability and proliferation .", "The interactions between RNA and RNA-binding proteins were analyzed by RNA affinity purification as previously described ( Hämmerle et al . , 2013 ) .", "In short , the 3’UTRs of ULBP2 ( both sense and antisense ) was cloned into pBluescriptII vector .", "Additionally , a UTR of a control gene with similar length and GC-content ( GBP2 ) was used .", "In vitro RNA transcription was performed using the MEGAscript T7 transcription kit ( Life Technologies , CA , USA ) after linearization of the plasmids with PspOMI restriction enzyme ( Thermo Scientific ( Fermentas ) , MA , USA ) .", "Around 10 percent of totally incorporated UTPs were Biotin-16-UTPs ( GE Healthcare , UK ) .", "The biotinylated RNAs were coupled with streptavidin-sepharose beads ( GE Healthcare , UK ) and incubated with cytoplasmic extracts prepared from 80% confluent RKO cells for at least 12 hr .", "After purification and elution of proteins that bound specifically to the RNAs , a SDS gel analysis was performed and specific bands detected with Coomassie Brilliant Blue G-250 ( Sigma Aldrich , Israel ) .", "Specific bands were excised and analyzed by mass spectrometry using either a LTQ Orbitrap or a Q Exative LC-MS/MS ( Thermo Scientific , Israel ) .", "Analysis was performed by the Smoler Proteomics Center , Haifa , Israel .", "Knockdown of IMP3 was executed with MISSION shRNA clones ( Sigma Aldrich , Israel ) for IMP3 in the vector pLKO ( MISSION clone: NM_006547 . 2-2284s21c1 with following sequence: CCGGTGTTGTAGTCTCACAGTATAACTCGAGTTATACTGTGAGACTACAACATTTTTG ) .", "IMP3 mRNA is targeted within the 3’UTR ( 3’ untranslated region ) .", "Lentiviruses were generated in 293T cells and used for transduction of RKO , 293T and HCT116 that express IMP3 as detected by Western Blot analysis .", "Next to the transduction of a shRNA specifically targeting IMP3 ( shIMP3 ) , a control vector containing a scrambled shRNA was transduced ( hairpin sequence: CCTAAGGTTAAGTCGCCCTCGCTCGAGCGAGGGCGACTTAACCTTAGG ) .", "Transduced cells were selected using 2 . 5 µg/mL puromycin in DMEM for HCT116 and 293T cells and 3 µg/mL for RKO cells .", "The vector containing the coding sequence for IMP3 was a kind donation of Prof . Joel K Yisraeli ( Hebrew University , Jerusalem , Israel ) .", "The insert was transferred into a lentiviral vector and stably transfected into RKO cells .", "An empty vector served as control .", "The efficacy of the transduction could be determined using the GFP reporter on the vector .", "Using limiting dilution , a single RKO clone expressing high GFP was selected and used in all assays described .", "The overexpression was confirmed using Western Blot .", "In order to confirm the effects observed in RKO cells , IMP3 was over-expressed as well in HCT116 and 293T cell line without selection of GFP expressing cells .", "To rescue IMP3 expression in RKO and 293T , cells that stably express the shRNA targeting IMP3 were transduced with an empty vector as control or the vector containing the coding DNA sequence ( CDS ) of IMP3 .", "Since the shRNA against IMP3 targets the 3’UTR of the mRNA , the overexpression using the CDS only can’t be targeted and downregulated .", "Lysates of the various RKO , HCT116 and 293T cells were prepared and SDS gel electrophoresis was executed .", "Proteins were transferred onto a nitrocellulose membrane with the tank blot procedure and specific protein bands were detected using antibodies detecting IMP3 ( sc-47893 , Santa Cruz , TX , USA ( 1:200 in 5% BSA in PBS ) and 07–104 , Millipore , MA , USA [1:2000 in 5% BSA in PBS] ) or GAPDH ( Santa Cruz , TX , USA [1:1000 in 5% BSA in PBS] ) or Vinculin ( Abcam , UK , [1:1000 in 5% BSA in PBS] ) as loading control .", "Chemiluminesce caused by detection antibody-linked horse-reddish peroxidase ( HRP , Jackson ImmunoResearch , PA , USA ) was detected .", "For Fluorescent Activated Cell Sorting ( FACS ) staining of NKG2D ligands on RKO cells , 3 x 105 were seeded 18 hr prior to analysis in 6-well plates .", "On the day of analysis , cells were diluted in FACS buffer ( PBS , 1% BSA , 0 . 05% NaN3 ) and about 1 x 105 cells were stained with 0 . 25 µg of the antibody of interest for 1 hr .", "The cells were always analyzed in a confluence of about 60–80% .", "Anti-hULBP1 , anti-hULBP2 , anti-hULBP3 , anti-MICA and anti-MICB antibodies were obtained from R&DSystems ( MN , USA ) as well as the mouse isotype IgG antibody .", "For detection , a goat-anti-mouse IgG antibody coupled to Alexa Fluor 488 or Alexa Fluor 647 ( Jackson ImmunoResearch , PA , USA ) was incubated in a dilution of 1:250 for 1 hr .", "Analysis was performed with a FACSCalibur flow cytometer ( BD Biosciences , CA , USA ) .", "Cells were gated according to their appearance in forward and side scatter ( FSC/SSC ) ; to analyze the effects of the overexpression of IMP3 on stress-ligand expression , only GFP-positive cells were gated ( both for the overexpression and the rescue of IMP3 after knockdown in RKO and 293T ) .", "RNAs for the detection of mRNA levels were prepared using the QuickRNA Kit ( Zymo Research , CA , USA ) .", "For the generation of cDNA , M-MLV reverse transcriptase ( Invitrogen ) was used in the presence of anchored Oligo dT primers ( Thermo Scientific ( Fermentas ) , MA , USA ) .", "Both procedures were performed according to manufacturers’ protocols .", "For quantitative Real Time-PCR , freshly prepared cDNAs were used for SYBR Green-based detection in a QuantStudio 12k Flex Real-time PCR cycler ( Life Technologies , CA , USA ) with primers targeting GAPDH , HPRT , ULBP1 , ULBP2 , ULBP3 , IMP3 , MICA and MICB .", "GAPDH forward: GAGTCAACGGATTTGGTCGTGAPDH reverse: GATCTCGCTCCTGGAAGATG HPRT forward: TGACACTGGCAAAACAATGCA HPRT reverse: GGTCCTTTTCACCAGCAAGCT ULBP1 forward: GCGTTCCTTCTGTGCCTC ULBP1 reverse: GGCCTTGAACTTCACACCAC ULBP2 forward: CCCTGGGGAAGAAACTAAATGTC ULBP2 reverse: ACTGAACTGCCAAGATCCACTGC ULBP3 forward: AGATGCCTGGGGAAAACAACTG ULBP3 reverse: GTATCCATCGGCTTCACACTCAC IMP3 forward: AGACACCTGATGAGAATGACC IMP3 reverse: GTTTCCTGAGCCTTTACTTCC MICA forward: ATCTTCCCTTTTGCACCTCC MICA reverse: AACCCTGACTGCACAGATCC MICB forward: CTGCTGTTTCTGGCCGTC MICB reverse: ACAGATCCATCCTGGGACAG The 3’UTRs of MICA , MICB , ULBP2 , ULBP3 and CD247 were cloned downstream to a Firefly reporter cassette in the vector pGL3 ( Promega , WI , USA ) as described ( Stern-Ginossar et al . , 2007 ) .", "The vector contains a SV40 promoter replacing the natural gene promoter and a SV40 polyadenylation site .", "1 . 25 x 105 RKO cells transfected with scrambled or IMP3-specific shRNA were seeded into 24-well-plates .", "The next day , the cells were transfected using TransIT-LT1 reagent ( MIRUS Bio , WI , USA ) with 250 ng of Firefly-Luciferase vector containing the 3’UTRs , and 50 ng of Renilla Luciferase vector ( pRL-CMV ) as reference .", "28 hr post transfection , cells were lysed and luciferase activity measured using Dual-Luciferase Reporter Assay System ( Promega , WI , USA ) .", "For the assessment of differences of luciferase activity of the shortened 3’UTRs of ULBP2 , following UTRs were cloned into pGL3: 1–100 bp ( basepairs ) , 1–200 bp , 1–300 bp , 1–400 bp and full length UTR ( 540 bp ) .", "1 x 105 RKO cells were seeded and the following day transfected with constructs coding for these shortened UTRs .", "Cells were harvested and analyzed 28 hr post transfection .", "For the binding site analysis of IMP3 in ULBP2-3’UTR , the putative-binding motif CATT at position 162 – 165 was mutated into CAGG to prevent binding of IMP3 to the RNA .", "1 x 105 RKO cells were transfected with the unchanged , wild type UTR of ULBP2 or the CAGG-mutated UTR and harvested 28 hr post transfection .", "The empty vector pGL3 was used as control and for normalization .", "For the readout , a LB 940 Mithras device ( Berthold Technologies , Germany ) was used .", "For technical reasons , the device needed to be exchanged between the performance of Figure 6B and Figure 6D/Figure 6F .", "For the transcription inhibition with D-Actinomycin , RKO cells transfected with a scrambled or IMP3 specific shRNA were seeded in a density of 2 . 5 x 105 in 6-well-plates .", "The next day , medium was exchanged to fresh DMEM containing either 5 µg/mL D-Actinomycin ( Sigma Aldrich , Israel ) or equal volume DMSO as diluent control .", "D-Actinomycin effectively inhibits the elongation process of the RNA polymerase II , thereby suppressing the synthesis of new mRNA transcripts ( Sobell , 1985 ) .", "After 16 hr incubation , medium was removed , and cells were harvested following the manufacturers’ protocol for RNA extraction ( Zymo Research , CA , USA ) .", "cDNA was prepared using M-MLV reverse transcriptase ( Invitrogen , CA , USA ) according to manufacturer’s protocol .", "qRT-PCR was performed as mentioned in the corresponding section .", "For analysis , we defined the level of a specific mRNA in the DMSO-treated control cells ( diluent control ) in both the scrambled shRNA- and shIMP3-transduced cells as 1 .", "By calculating the ratio of mRNA level after 16 hr D-Actinomycin treatment and 16 hr DMSO treatment , we assessed the rate of mRNA decay in dependence of IMP3 .", "NK cells were purified from whole blood of healthy donors via MACS separation ( Miltenyi Biotec , Germany ) .", "Separated NK cells were activated by co-cultivation with 50 000 irradiated PBMCs of two autologous donors , 5 000 irradiated 8866 B cell lymphoma cells and 0 . 2 μg/mL phytohemagglutinin ( PHA , DYN diagnostics , Israel ) .", "NK cells were further cultivated for three weeks in IL-2 containing medium .", "For the 35S release assay , 2 x 105 of the transduced RKO , HCT116 and 293T cells were labeled with 1 µCi/mL EasyTag L-[35S]-Methionine ( Perkin Elmer , Israel ) in methionine-free RPMI for about 12 hr .", "After removing excess labeling solution , NK cells were co-incubated with the labeled RKO cells in the ratio 25:1 , with labeled HCT116 or 293T cells in the ratio 10:1 .", "Before the assay was set up , NK cells were blocked for 1 hr on ice with 1 µg IgG1 isotype control or a blocking NKG2D antibody .", "After about 3 hr co-cultivation of cells , plates were centrifuged to pellet cells and 50 µL of the supernatants were transferred to Optiplate-96-plates ( Perkin Elmer ) .", "150 µL of Microscint-40 solution ( Perkin Elmer ) per well were added .", "The readout was performed using a MicroBeta2 device ( Perkin Elmer ) .", "The percentage of specifically lysed cells in a certain well was calculated according to following formula: [%] specific lysis = ( ( count – 'spontaneous' ) / ( 'total' – 'spontaneous' ) ) *100% .", "Spontaneous release was assessed by culturing labeled RKO cells without NK cells; total release was assessed by adding 150 µl 1M NaOH to RKO cells to lyse all cells .", "Unless stated otherwise , student’s t-tests were applied for statistical analysis .", "P-values are stated in the corresponding figure legends .", "The statement 'replicate' defines a biological replicate meaning that cells between experiments were separately seeded and grown and antibodies were diluted discretely .", "The number of technical replicates within a biological replicate is one for FACS analyses and four for qRT-PCR analyses , luciferase experiments and 35S release assay ." ] ]

[ "Expression of the stress-induced ligands MICA , MICB and ULBP 1–6 are up-regulated as a cellular response to DNA damage , excessive proliferation or viral infection; thereby , they enable recognition and annihilation by immune cells that express the powerful activating receptor NKG2D .", "This receptor is present not exclusively , but primarily on NK cells .", "Knowledge about the regulatory mechanisms controlling ULBP expression is still vague .", "In this study , we report a direct interaction of the oncogenic RNA binding protein ( RBP ) IMP3 with ULBP2 mRNA , leading to ULBP2 transcript destabilization and reduced ULBP2 surface expression in several human cell lines .", "We also discovered that IMP3 indirectly targets MICB with a mechanism functionally distinct from that of ULBP2 .", "Importantly , IMP3-mediated regulation of stress-ligands leads to impaired NK cell recognition of transformed cells .", "Our findings shed new light on the regulation of NKG2D ligands and on the mechanism of action of a powerful oncogenic RBP , IMP3 ." ]

[ "Tumor cells differ from healthy cells in many aspects .", "Importantly , tumor cells have the ability to divide and grow much faster than normal cells .", "To protect ourselves from full-grown cancers , our bodies have developed a surveillance system: when a tumor cell starts to divide without restraint , “stress-induced” proteins start to appear on its surface .", "These proteins help the immune system recognize abnormal or damaged cells , allowing the immune cells to eliminate the defective cells .", "Despite this system of protection , a tumor cell sometimes manages to avoid having stress-induced proteins placed on its surface , allowing it to remain undetected by the immune system .", "By studying several different types of human cancer cells , Schmiedel et al . found that a protein called IMP3 is present in cancer cells but not in healthy cells .", "Further investigation revealed that IMP3 prevents the production of some stress-induced proteins and stops them moving to the cell surface .", "Schmiedel et al . also show that the presence of the IMP3 protein in cancer cells causes nearby immune cells to become much less active .", "This suggests that developing drugs that block the activity of IMP3 could help the immune system to fight back and destroy cancer cells ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "ecology", "computational and systems biology" ]

[ [ "Two sub-populations comprise our model: the nomadic organisms , and the colonial ones .", "In a similar vein to habitat-patch models , organisms that exist in multiple sub-populations can be modelled as follows: ( 1 ) d⁢nid⁢t=gi⁢ ( ni ) +∑jsi⁢j⁢nj-∑jsj⁢i⁢niwhere ni is the size of sub-population i , gi is the function describing the growth rate of ni in isolation , and si⁢j is the rate of switching to sub-population i from sub-population j .", "Population sizes are assumed to be large enough that Equation 1 adequately approximates the underlying stochasticity .", "Let n1 be the nomadic population size .", "In the absence of behavioral switching , the nomadic growth rate is given by ( 2 ) g1⁢ ( n1 ) =-r1⁢n1where r1 is the nomadic growth constant .", "Nomadism is modelled as a losing strategy by setting −r1 < 0 , such that n1 decays with time .", "In the context of Parrondo’s paradox , nomadism corresponds to the ‘agitating’ strategy , or Game A . Importantly , competition among nomads , as well as between nomads and colonists , is taken to be insignificant , due to the independence of a nomadic lifestyle .", "Colonial population dynamics will be modelled by the well-known logistic equation , with carrying capacity K , but with two important modifications .", "Firstly , the Allee effect is taken into account .", "This serves two roles: it captures the cooperative effects that occur among colonial organisms , and it ensures that the growth rate is negative when the population falls below a critical capacity A . Let n2 be the colonial population size .", "In the absence of behavioral switching , the colonial growth rate is given by ( 3 ) g2 ( n2 ) =r2n2 ( n2min ( A , K ) −1 ) ( 1−n2K ) where r2 is the colonial growth constant .", "Setting r2>0 , we have a positive growth rate when A<n2<K , and a negative growth rate otherwise .", "The min ( A , K ) term ensures that when K<A , g2 is always zero or negative , as would be expected .", "Secondly , the carrying capacity K changes at a rate dependent upon the colonial population size , n2 , accounting for the destruction of environmental resources over the long run .", "The rate of change of K with respect to t is given by ( 4 ) d⁢Kd⁢t=α-β⁢n2where α>0 is the default growth rate of K , and β>0 is the per-organism rate of habitat destruction .", "An alternative interpretation of this equation is that K , the short-term carrying capacity , is dependent on some essential nutrient in the environment , and that this nutrient is slowly depleted over time at a rate proportional to β⁢n2 .", "Let n*=αβ , the critical population level at which no habitat destruction occurs .", "d⁢Kd⁢t is zero when n2=n* , positive when n2<n∗ , and negative when n2>n∗ .", "n∗ can thus also be interpreted as the long-term carrying capacity .", "Clearly , if the long-term carrying capacity n*<A , the only stable point of the system becomes n2=0 .", "Under this condition , colonialism is a losing strategy as well .", "Note that g2 increases as K increases , and that K increases more quickly as n2 decreases .", "In the context of Parrondo’s paradox , colonialism can thus serve as a ‘ratcheting’ strategy , or Game B , because the rate of growth is implicitly dependent upon the colonial population in the past .", "Another way of understanding the ‘ratcheting’ behavior is through the lens of positive reactivity ( Williams and Hastings , 2011; Hastings , 2001 , Hastings , 2004 ) .", "In the short-term , n2=A is a positively reactive equilibrium , because small upwards perturbations of n2 away from A will result in rapid growth towards K before a slow decrease back down towards A . Organisms are able to detect the amount of environmental resources available to them , and by proxy , the carrying capacity of the population .", "Thus , they can undergo behavioral changes in response to the current carrying capacity .", "Here , we model organisms that switch to nomadic behavior from colonial behavior when the carrying capacity is low ( K < L1 ) , and switch to colonial behavior from nomadic behavior when the carrying capacity is high ( K > L2 ) , where L1≤L2 are the switching levels .", "Let rs > 0 be the switching constant .", "Using the notation from Equation 1 , switching rates can then be expressed as follows: ( 5 ) s12={rsif K < L10otherwises21={rsif K > L20otherwise A variety of mechanisms might trigger this switching behavior in biological systems .", "For example , since the nomadic organisms are highly mobile , they could frequently re-enter their original colonial habitat after leaving it , and thus be able to detect whether resource levels are high enough for recolonization .", "It should also be noted that the decision to switch need not always be ‘rational’ ( i . e . result in a higher growth rate ) for each individual .", "Switching behavior could be genetically programmed , such that ‘involuntary’ individual sacrifice ends up promoting the long-term survival of the species .", "Without loss of generality , we scale all parameters such that α=β=1 .", "Equation 4 thus becomes: ( 6 ) d⁢Kd⁢t=1-n2 Hence , n*=αβ=1 .", "All other population sizes and capacities can then be understood as ratios with respect to this critical population size .", "Additionally , since β=1 , r1 , r2 and rs can be understood as ratios to the rate of habitat destruction .", "For example , if r2≫1 , this means that colonial growth occurs much faster than habitat destruction .", "Time-scale separation between the population growth dynamics and the habitat change dynamics can thus be achieved by setting r1 , r2≫1 .", "Similarly , the separation between the behavioral switching dynamics and the population growth dynamics can be achieved by setting rs≫r1 , r2 ." ], [ "As described earlier , both nomadic and colonial behaviors can be modelled as losing strategies given the appropriate parameters .", "Simulations across a range of parameters elucidated the conditions which resulted in extinction for both strategies .", "Figure 1a shows an example when both strategies are losing , resulting in extinction , while Figure 1b shows an example where only the colonial sub-population survives .", "It is clear from Equation 2 that the growth rate of the nomadic population n1 is always negative , because of the restriction that r1", "> 0 . Hence , nomadism is always a losing strategy .", "However , the conditions under which colonial behavior is a losing strategy are more complicated .", "Complex dynamics occur when the critical capacity A is just below 1 that can result in either survival or extinction .", "Nonetheless , it can be shown that when A > 1 , extinction occurs ( as in Figure 1a ) , and that survival is only possible when A is significantly less than 1 ( as in Figure 1b ) .", "That is: ( 7 ) A>1 ( colonial extinction guaranteed ) ( 8 ) A<1 ( colonial survival possible ) The intuition behind this is straightforward .", "Suppose that initially , A < n2 < K , so that the growth rate is positive .", "When A < 1 , the colonial population n2 increases until it reaches the carrying capacity K , following which they converge in tandem until stabilizing at the critical population size , n2=K=1 .", "However , when A > 1 , n2=K=1 is no longer a stable equilibrium , since dn2/dt < 0 when n2 < A , resulting in the eventual extinction of the population .", "For a formal proof , refer to Theorem A . 3 .", "We now restrict our analysis to the case where A > 1 . Under this condition , both nomadism ( Game A ) and colonialism ( Game B ) are losing strategies when played individually .", "Paradoxically , it is possible to combine these two strategies through behavioral switching such that population survival is ensured , thereby producing an overall strategy that wins .", "Simulation results over a range of parameters have predicted this paradoxical behavior , and also elucidated the conditions under which it occurs .", "Figure 2a is a typical example where the population becomes extinct , even though it undergoes behavioral switching , while Figure 2b is a typical example where behavioral switching ensures population survival .", "Conceptually , this paradoxical survival is possible because the colonial strategy , or Game B , is history-dependent .", "In particular , the colonial growth rate d⁢n2/d⁢t is dependent upon the carrying capacity K , which in turn is dependent upon previous levels of n2 .", "Behavioral switching to a nomadic strategy decreases the colonial population size , allowing the resources in the colonial environment , represented by K , to recover .", "Switching back to a colonial strategy then allows the population to take advantage of the newly generated resources .", "Because switching occurs periodically , as can be seen in Figure 2b , it should be noted that the organisms need not even detect the amount of resources present in the environment to implement this strategy .", "A biological clock would be sufficient to trigger switching behavior .", "The exact process by which survival is ensured can be understood by analysing the simulation results in detail .", "In the nomadic phase , the colonial population n2 is close to zero , the nomadic population n1 undergoes slow exponential decay , and the carrying capacity K undergoes slow linear growth .", "K increases until it reaches L2 , which triggers the switch to colonial behavior .", "The population thus enters the colonial phase .", "If the colonial population n2 exceeds the critical capacity A at this point , then n2 will grow until it slightly exceeds the carrying capacity K . Subsequently , n2 decreases in the tandem with K until K drops to L1 , triggering the switch back to the nomadic phase .", "However , if n2 < A when the colonial phase begins , the colonial population goes extinct , as can be seen in Figure 2a .", "Hence , a basic condition for survival is that n2≥A at the start of each colonial phase .", "This implies that , by the end of the nomadic phase , n1 needs to be greater by a certain amount than A as well .", "Otherwise , there will be insufficient nomads to form a colony which can overcome the Allee effect .", "Under the reasonable assumption that the rate of behavioral switching is much faster than either colonial or nomadic growth ( rs≫r1 , r2 ) , it can be shown more precisely that at the end of the nomadic phase , n1 needs to be greater than a critical level B , which is related to A by the equation: ( 9 ) A=B− ( 1−B ) W0 ( B1−BexpB1−B ) A full derivation is provided in the Appendix ( Theorem A . 4 ) .", "Here , W0⁢ ( x ) is the principal branch of the Lambert W function .", "Qualitatively speaking , B is a function of A on the interval ( 1 , ∞ ) that increases in an exponential-like manner , and that approaches 1 when A does as well .", "Thus , B≥A , as expected .", "The greater the difference between the switching levels , the longer the nomadic phase will last , because it takes more time for K to increase to the requisite value for switching , L2 .", "And the longer the nomadic phase lasts , the more n1 will decay .", "If , at the end of the nomadic phase , the value that n1 decays to happens to be less than B , then the population will fail to survive .", "It follows that there should be some constraint on the difference between the switching levels L1 and L2 .", "Under the same assumption that rs≫r1 , r2 , such a constraint can be derived: ( 10 ) L2 < L1+1r1ln⁡L1+W0 ( −L1e−L1 ) B Survival is ensured given the following additional condition: ( 11 ) There exists t∗≥t0:n2 ( t∗ ) =K ( t∗ ) ≥L1where t0 marks the start of an arbitrary colonial phase , and t* marks the time of intersection between n2 and K during that phase .", "In other words , n2 has to grow sufficiently quickly during the colonial phase such that it exceeds both K and L1 before switching begins .", "This can be seen occurring in Figure 2b .", "In accordance with intuition , numerical simulations predict that this occurs when the colonial growth constant is sufficiently large ( r2≫r1 ) , as can be seen in the Figures .", "( The Figures also show that r1 close to 1 , but this is not strictly necessary . )", "Collectively , Equations 10–11 are sufficient conditions for population survival .", "Mathematical details are provided in the Appendix ( Theorems A . 5 and A . 6 ) .", "Note that Equation 10 contains an implicit lower bound on L1 .", "Since L2≥L1 by stipulation , we must have ln⁡[L1+W0 ( −L1e−L1 ) ] > ln⁡ B for survival .", "The following bound is thus obtained: ( 12 ) L1 > BeBeB−1 On the other hand , under the assumptions made , there is no upper bound for L1 , and hence no absolute upper bound for L2 either .", "This suggests that given a sufficiently well-designed switching rule , K can grow larger over time while ensuring population survival .", "Such a rule is investigated in the following section .", "Suppose that , in addition to being able to detect the colonial carrying capacity , nomads and colonists are able to detect or estimate their current population size .", "This might happen by proxy , by communication , or by built-in estimation of the time required for growth or decay to a certain population level .", "The following switching rule then becomes possible: ( 13 ) When n2=K , dn2/dt > 0 , set L1=K , L2=∞When n1=B , dn1/dt < 0 , set L2=K That is , L1 is set to the carrying capacity K whenever n2 rises to K , resulting immediately in a switch to nomadic behavior , and that L2 is in turn set to K whenever n1 falls to B , resulting in an immediate switch to colonial behavior .", "This switching rule is optimal according to several criteria .", "Firstly , by switching to nomadic behavior just as n2 reaches K , it ensures that d⁢n2/d⁢t≥0 for the entirety of the colonial phase .", "As such , it avoids the later portion of the colonial phase where K and n2 decrease in tandem , and maximizes the ending value n2 .", "Consequently , it also maximizes the value of n1 at the start of each nomadic phase .", "Furthermore , by switching to colonial behavior right when n1 decays to B , the rule maximizes the duration of the nomadic phase while ensuring survival .", "This in turn means that the growth of K is maximized , since the longer the nomadic phase , the longer that K is allowed to grow .", "In fact , this switching rule is a paradigmatic example of how Parrondo’s paradox can be achieved .", "It plays Game A , the nomadic strategy , for as long as possible , in order to maximize K and hence the returns from Game B . And then it switches to Game B , the colonial strategy , only for as long as the returns are positive ( dn2/dt > 0 ) , thereby using it as a kind of ratchet .", "Suppose that K grows more during each nomadic phase than it falls during each colonial phase .", "Then the switching rule is not just optimal , but it also enables long-term growth .", "Simulation results predict that this can indeed occur .", "Figure 3a shows long-term growth of K from t=0 to t=10 , while Figure 3b shows that with the same initial conditions , this continues until t=300 with no signs of abating .", "Together with K , the per-phase maximal values of n1 and n2 increase as well .", "In the cases shown , long-term growth is achieved because K indeed grows more during each nomadic phase than it falls during the subsequent colonial phase .", "As can be seen from Figure 3a , this is , in turn , because the nomadic phase lasts much longer than the colonial phase , such that the amount of environmental destruction due to colonialism is limited .", "Simulation results predict that this generally occurs as long as the colonial growth rate is sufficiently large ( r2≫r1 ) .", "An interesting phenomenon that can be observed from Figure 3b is how the nomadic population size n1 , which peaks at the start of each nomadic phase , eventually exceeds the carrying capacity K , and then continues doing so by increasing amounts at each peak .", "This is , in fact , a natural consequence of the population model .", "When n2 grows large , the assumption that switching is much faster than colonial growth starts to break down .", "This occurs even though rs≫r2 , due to the increasing contribution of the ( n2A-1 ) factor in Equation", "3 . The result is that when a large colonial population begins switching to nomadism , a significant number of colonial offspring are simultaneously being produced .", "These offspring also end up switching to a nomadic strategy , resulting in more nomadic organisms than there were colonial organisms before .", "A particularly pronounced example of this is shown in Figure", "4 . However , this same phenomenon also introduces a limiting behavior to the pattern of long-term growth .", "As Figure 5 shows , when the same simulation as in Figure 3a and b is continued to t=1000 , peak levels of n1 , n2 and K eventually plateau around t=650 .", "This occurs because sufficiently high levels of n2 cause a qualitative change in the dynamics of behavioral switching .", "Normally , switching to nomadic behavior starts when K falls below L1 , and ends when K rises above it again .", "K rises towards the end of the switch , when n2 levels fall below the critical level of n*=1 .", "But when n2 is sufficiently large , the faster production of colonial offspring drags out the duration of switching , as seen in Figure", "4 . The higher levels of n2 , combined with the longer switching duration , causes an overall drop in K by the end of the switching period .", "Because the increase in K during the subsequent nomadic phase is unable to overcome this drop , K stops increasing in the long-run .", "Nonetheless , it is clear that significant long-term gains can be achieved via the optimal switching rule .", "Under the conditions of fast colonial growth and even faster switching ( rs≫r2≫r1≃1 , as in Figures 3a–5 ) , these gains are several orders of magnitude larger than the initial population levels , a huge departure from the long-term extinction that occurs in purely colonial or nomadic populations .", "Limiting behavior eventually emerges , but this is to be expected in any realistic biological system .", "Our proposed model is convenient for the functional understanding of growth and survival , and can be easily modified for a variety of applications .", "Additional constraints can be imposed under which survival and long-term growth are still observed .", "For example , in many biological systems , the dynamics of habitat change might occur on a slower timescale than both colonial and nomadic growth ( i . e . r1 , r2≫1 ) .", "Figure 6 shows the simulation results when this timescale separation exists ( r1=10 , r2=100 for", "( a ) , r1=100 , r2=1000 for", "( b ) ) .", "It can clearly be seen that survival is still possible under such conditions .", "Another practical constraint that can be imposed is limiting the growth of the carrying capacity to some maximal value Kmax , capturing the fact that the resources in any one habitat do not grow infinitely large .", "This can be achieved by modifying Equation 6 as follows: ( 14 ) dKdt= ( 1−n2 ) ( 1−KKmax ) Figure 6 already takes this constraint into account , showing that survival through periodic alternation is achievable under both bounded carrying capacity and slow habitat change , as long as the maximum carrying capacity is sufficiently high ( Kmax=20 ) .", "As Figure 7 shows , even long-term growth is possible , under both fast habitat change ( Figure 7a ) and slow habitat change ( Figure 7b ) .", "In both cases , the carrying capacity K converges towards a maximum value as it approaches Kmax ." ], [ "The results presented in this study demonstrate the theoretical possibility of Parrondo’s paradox in an ecological context .", "Many evolutionary strategies correspond to the strategies that we have termed here as ‘nomadism’ and ‘colonialism’ .", "In particular , any growth model that is devoid of competitive or collaborative effects is readily captured by Equation 2 ( nomadism ) , while any logistic growth model which includes both the Allee effect and habitat destruction can be described using Equations 3 and 4 ( colonialism ) .", "Many organisms also exhibit behavioral change or phenotypic switching in response to changing environmental conditions .", "By incorporating this into our model , we have demonstrated that nomadic-colonial alternation can ensure the survival of a species , even when nomadism or colonialism alone would lead to extinction .", "Furthermore , it has been demonstrated that an optimal switching rule can lead to long-term population growth .", "The switching rules which lead to survival and long-term growth are analogous to the periodic alternation between games that produces a winning expectation in Parrondo’s paradox .", "If one views the carrying capacity K as the capital of the population , then it is clear that Equation 5 is a capital-dependent switching rule .", "By setting the appropriate amounts of capital at which switching should occur , survival and growth can be achieved .", "Survival is achieved by ensuring that Game A , or nomadism , is never played beyond the point where extinction is inevitable , that is , the point where n1 falls below the critical level B . Long-term growth is additionally achieved by ensuring that Game B , or colonialism , is only played in the region where gains are positive , that is , when A < n2 < K such that dn2/dt > 0 .", "The history-dependent dynamics of Game B are thus optimally exploited .", "Several limitations of the present study should be noted .", "Firstly , the study only focuses on cases where nomadism and colonialism are individually losing strategies , despite the abundance of similar strategies that do not lose in the real world .", "This is because assuming individually losing strategies in fact leads to a stronger result – if losing variants of nomadism and colonialism can be combined into a winning strategy , it follows that non-losing variants can be combined in a similar way too ( see Theorem A . 7 in the Appendix ) .", "Secondly , the population model does not encompass all variants of qualitatively similar behavior .", "For example , many other equations can be used to model the Allee effect ( Boukal and Berec , 2002 ) .", "Nonetheless , our proposed model is general enough that it can be adapted for use with other equations and be expected to produce similar results .", "Even the presence of the Allee effect is not strictly necessary , since the colonial population might die off at low levels because of stochastic fluctuations , rather than because of the effect .", "Theorem A . 7 in the Appendix also demonstrates that paradoxical behavior can occur even without the Allee effect causing long-term death of the colonial population .", "Thirdly , though it is trivially the case that pure nomadism and pure colonialism cannot out-compete a behaviorally-switching population , a more complex analysis of the evolutionary stability of behavioral switching is beyond the scope of this paper .", "Finally , spatial dynamics are not accounted for in this study .", "Exploring such dynamics is a goal for future work ." ], [ "Our comprehensive model captures both capital and history-dependent dynamics within a realistic ecological setting , thereby exhibiting Parrondo's paradox without the need for exogenous environmental influences .", "The possibility of an ecological Parrondo’s paradox has wide-ranging applications across the fields of ecology and population biology .", "Not only could it provide evolutionary insight into strategies analogous to nomadism , colonialism , and behavioral diversification , it potentially also explains why environmentally destructive species , such as Homo sapiens , can thrive and grow despite limited environmental resources .", "By providing a theoretical model under which such paradoxes occur , our approach may enable new insights into the evolution of cooperative colonies , as well as the conditions required for sustainable population growth ." ] ]

[ "Organisms often exhibit behavioral or phenotypic diversity to improve population fitness in the face of environmental variability .", "When each behavior or phenotype is individually maladaptive , alternating between these losing strategies can counter-intuitively result in population persistence–an outcome similar to the Parrondo’s paradox .", "Instead of the capital or history dependence that characterize traditional Parrondo games , most ecological models which exhibit such paradoxical behavior depend on the presence of exogenous environmental variation .", "Here we present a population model that exhibits Parrondo’s paradox through capital and history-dependent dynamics .", "Two sub-populations comprise our model: nomads , who live independently without competition or cooperation , and colonists , who engage in competition , cooperation , and long-term habitat destruction .", "Nomads and colonists may alternate behaviors in response to changes in the colonial habitat .", "Even when nomadism and colonialism individually lead to extinction , switching between these strategies at the appropriate moments can paradoxically enable both population persistence and long-term growth ." ]

[ "Many organisms , from slime molds to jellyfish , alternate between life as free-moving “nomadic” individuals and communal life in a more stationary colony .", "So what evolutionary reasons lie behind such stark behavioral diversity in a single species ?", "What benefits are obtained by switching from one behavior to another ?", "Tan and Cheong have now developed a mathematical model that suggests an intriguing possibility: under conditions that would cause the extinction of both nomadic individuals and colonies , switching between these life strategies can enable populations to survive and grow – a counter-intuitive phenomenon called Parrondo’s paradox .", "Parrondo’s paradox says that it is possible to follow two losing strategies in a specific order such that success is ultimately achieved .", "For example , slot machines are designed to ensure that players lose in the long run .", "What the paradox says is that two slot machines can be configured in such a way that playing either slot machine will lead to financial disaster , but switching between them will leave the player richer in the long run .", "Most studies of similar phenomena suggest that switching between two ‘losing’ lifestyle strategies can only improve the chances of survival if the environment keeps changing in unpredictable ways .", "However , Tan and Cheong’s model shows that this unpredictability is an unnecessary condition – paradoxes also occur when organisms form colonies that predictably destroy their habitat .", "The basic mechanism for survival is elegant .", "The organism periodically exploits its habitat as part of a colony , then switches to a nomadic lifestyle to allow the environment to regenerate .", "Through mathematical analysis and simulations , Tan and Cheong confirm that this strategy is viable as long as two conditions hold: that colonies grow sufficiently quickly when environmental resources are abundant; and that colonists switch to a nomadic lifestyle before allowing the resource levels to dip dangerously low .", "The results produced by Tan and Cheong’s model help to explain how behavior-switching organisms can survive and thrive , even in harsh conditions .", "Further work needs to be done to adapt this general model to specific organisms and to investigate the possible evolutionary origins of behavior-switching lifestyles ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "neuroscience" ]

[ [ "Learning to navigate within the spatial organization of different habitats is essential for animals’ survival ( Geva-Sagiv et al . , 2015 ) .", "Electric fish , for example , occupied a lucrative ecological niche by evolving the ability to navigate and localize food sources in complete darkness using short-range electrosensation ( Jun et al . , 2016 ) .", "The spatial acuity that they exhibit , along with their reliance on learned landmark positions , strongly suggest that they memorize the relative arrangement of the landmarks and the environmental borders .", "The information animals use to generate such allocentric knowledge include sensory experiences collected during object encounters ( Jun et al . , 2016; Petreanu et al . , 2012; Save et al . , 1998 ) and motor actions ( heading changes and distance traveled ) executed between such encounters; utilization of these motor variables in spatial learning and navigation is termed path integration ( Collett and Graham , 2004; Etienne and Jeffery , 2004 ) .", "This acquired information , however , is always egocentric in nature .", "Fittingly , the primary brain regions dedicated to sensory and motor processing , such as the optic tectum ( OT ) of all vertebrates and many cortical regions in mammals are topographically organized along an egocentric coordinate system ( Knudsen , 1982; Sparks and Nelson , 1987; Stein , 1992 ) .", "Unravelling the neural operations underlying the transformation of egocentric sensory and motor information streams into an allocentric representation of the environment has been a central theme in studies of spatial learning and navigation .", "Recent studies in the mouse ( Peyrache et al . , 2017 ) have suggested that the key computations include vestibular input that defines the animal’s head direction ( head direction cells , egocentric ) and external sensory input that signals the presence of stable environmental features ( i . e . , landmarks ) .", "Linking the head directions that orient the animal to different environmental features are then hypothesized to generate an allocentric representation of those features .", "The neural circuits that have been hypothesized to implement these computations are , however , exceedingly complicated and include thalamic ( head direction ) and cortical ( external sensory ) input to the hippocampal formation .", "The proposed wiring diagrams are highly speculative and very far from providing a well-defined mechanistic model of how spatial maps are created .", "Equivalent computations appear to be carried out in Drosophila ( Seelig and Jayaraman , 2015 ) .", "Visual orientation to landmarks and body direction via path integration are combined in the ellipsoid body with dynamics suggestive of a ring attractor .", "While these studies in the simpler nervous system of the fly are now closer to providing a mechanistic explanation of how egocentric and external ( visual ) inputs are combined , it is not clear if the fly has a full representation of the allocentric relations of different environmental features .", "It is also not at all clear that the dynamics of the ellipsoid body can be mapped onto the cortical and hippocampal circuitry of mammals .", "Teleost fish offer an attractive model for studying this question , as their related brain circuitry is relatively tractable: lesion studies point to the dorsolateral pallium ( DL ) as the key telencephalic region required for allocentric spatial learning ( Broglio et al . , 2010; Durán et al . , 2010; Rodríguez et al . , 2002 ) , similarly to the medial cortex in reptiles and the hippocampus in mammals ( see Discussion ) .", "DL has strong excitatory recurrent connectivity ( Elliott et al . , 2017; Giassi et al . , 2012b ) .", "Importantly , DL receives sensory and motor information related to electrosensory and visual object motion from OT ( Bastian , 1982 ) via a single structure – the diencephalic preglomerular complex ( PG , Giassi et al . , 2012b , Figure 1A ) .", "The tectal recipient portion of PG projects solely to DL ( Giassi et al . , 2012a ) in agreement with the lesion studies .", "Importantly , PG receives very little feedback from areas associated with DL ( Giassi et al . , 2012b ) and therefore functions as an exclusive feed-forward bottleneck between OT and the pallium .", "DL in turn projects to the central pallium ( DC , Giassi et al . , 2012b ) ; DL also has reciprocal connections with the dorsal pallium ( DD ) and DD itself has strong recurrent connectivity .", "DC is the only route by which DL can control motor activity and it does so solely via its projections to the OT ( Giassi et al . , 2012b ) .", "We hypothesize that egocentric object-related information ( OT ) conveyed by PG to DL , is converted to a learned allocentric spatial map by the recurrent circuitry of DL , DD and DC; DC , in turn , then controls the fish’s spatial behavior via its projections to OT ." ], [ "We first examined the spatial representation in PG cells by measuring their receptive fields ( RFs; measured in 27 cells ) .", "The OT electrosensory cells driving PG have spatially-restricted , topographically organized RFs ( Bastian , 1982 ) , and thus provide labeled-line information on the egocentric position of objects .", "In PG , by contrast , only 11% of the cells ( 3/27 ) were topographic with a spatially restricted RF ( Figure 2A ) ; the majority of PG cells ( 89% , 24/27 ) responded across most or all of the fish’s body ( Figure 2B and Figure 2—figure supplement 1 ) .", "Therefore , PG activity does not convey a topographic ‘labeled-line’ code of object position .", "We also checked whether object location is encoded by the firing-rate of PG neurons ( i . e . , a rate code ) .", "The firing-rate was significantly correlated with object position only in one non-topographic cell ( 4% , 1/24 neurons; p < 0 . 05 , random-permutations test , Figure 2C ) .", "Similarly , mutual information between object position and firing-rate was significant only in two non-topographic cells ( 8% , 2/24 neurons; p < 0 . 05 , random-permutations test , Figure 2D ) .", "Therefore , almost all PG cells have whole-body RFs and lack topographic spatial information– the hallmark of all electrosensory regions from the sensory periphery up to OT .", "We attempted to systematically sample throughout the full extent of PG .", "However , we cannot absolutely determine whether the subset of topographic cells represent a small distinct sub-nucleus or are sparsely distributed throughout the PG complex .", "Next , we checked what information PG neurons convey about object motion .", "OT cells , which drive PG , respond to an object moved parallel to the fish ( longitudinal motion ) while the object traverses their RFs ( Bastian , 1982 ) .", "Only a minority of recorded PG units ( 26% , 7 out of 27 tested with longitudinal motion ) responded in this manner ( Figure 3A ) .", "Rather , the majority of PG units ( 78% , 20/27 ) exhibited a strikingly different behavior , emitting a brief burst response confined to the onset ( and sometimes to the offset ) of object motion , but not during motion itself ( Figure 3B ) .", "This is further demonstrated when motion in each direction was broken into four segments separated by wait periods ( Figure 3C ) , evoking responses at the onset ( yellow arrowheads ) and offset ( red arrows ) of each segment across the entire body .", "Interestingly , we have encountered several lateral-line responsive PG units that , unlike most of their electrosensory counterparts , did respond persistently throughout ( and even after ) object motion ( Figure 3—figure supplement 1 ) .", "We next applied transverse motion , which mimics an object looming/receding ( incoming/outgoing ) into/from the electrosensory receptive field ( tested in 40 cells ) .", "Three types of responses to such motion were identified: proximity detection , encounter detection , and motion-change detection; 50% ( 20/40 ) displayed more than one type of response .", "Proximity detectors ( 75% , 30/40 ) responded when an object was encountered very close to the skin ( < 2 cm , Figure 3D ) ; encounter detectors ( 32% , 13/40 ) responded when an object either entered to or departed from their electroreceptive range ( ~4 cm , Figure 3E ) ; lastly , motion-change detectors ( 57 . 5% , 32/40 ) displayed a response similar to that observed in longitudinal motion , firing at the onset/offset of motion ( i . e . , when the object accelerated/decelerated , Figure 3F ) ; remarkably , this type of response was relatively distance-invariant , yielding comparable responses both very close to ( 0 . 5 cm ) and very far from ( 5 . 5 cm ) the skin despite the drastic effects of distance on both the magnitude and spread of the object’s electrical image ( Figure 2—figure supplement 1 , see Chen et al . , 2005 ) .", "This discrete , nearly stereotypic response stands in contrast to the finely-tuned rate code of object distance previously reported in the primary electrosensory cells in ELL ( Clarke et al . , 2014; Clarke and Maler , 2017 ) .", "Taken together , the variety of PG response-types mentioned above may constitute a distributed representation of object proximity .", "Unlike the loss of egocentric topographic mapping discussed in the previous section , this representation does provide DL with coarse-grained , non-directional egocentric information .", "The possible role ( s ) of this information was not further considered in our analyses .", "We conclude that PG electrosensory cells predominantly respond to novelty: onset/offset of object motion within their receptive field or the introduction/removal of objects into/from this receptive field; we use the term object encounters to designate all such events .", "Most PG cells responded both to conductive and non-conductive objects ( Figure 4 ) and would therefore not discriminate between different object types , for example , plants versus rocks .", "We can , therefore , infer that during active exploration of the environment , PG reports to the dorsal pallium whenever the fish encounters or leaves a prey or a landmark ( e . g . , root mass or rock ) or alters its swimming trajectory near a landmark .", "While topographic egocentric information was scarce in PG , temporal information was prevalent .", "Many of the PG cells exhibited pronounced adaptation to repeated motion ( 45% , 15/33 neurons tested with repeated-motion protocol; Figure 5—figure supplement 1 ) .", "We delivered a sequence of object encounters ( motions in and out of the RF , Figure 5A ) ; time-intervals between sequential encounters were drawn randomly and independently ( in the range of 1–30 s ) .", "The response intensity ( number of spikes within an individually determined time window ) of these adapting cells to each encounter was strongly correlated with the time-interval immediately prior to that encounter ( Figure 5B ) .", "By contrast , there was no significant correlation with intervals prior to the last one ( inset of Figure 5B and Figure 5C ) .", "Thus , a large subset of PG cells encoded the duration of the time-interval preceding the last encounter – but did not convey information about the intervals prior to that .", "We term this history-independent adaptation .", "Interestingly , similar behavior was also found in visually-responsive cells ( ventral PG , Figure 5—figure supplement 2 ) .", "This result contrasts with many studies in the mammalian cortex , where adaptation is incremental and responses depend on multiple preceding intervals ( Lampl and Katz , 2017; Ulanovsky et al . , 2004 ) .", "Moreover , a spatial ‘oddball’ experiment ( Methods ) , which we conducted on 14 neurons ( 6 of which were adapting ) revealed that this adaptation is also spatially non-specific – that is , encounters at one body location adapt the response to encounters at all other locations ( Figure 3D–F ) .", "Therefore , this thalamic adaptation mechanism enables neurons to encode the time between the two most recent successive stimuli , without contamination by prior encounter history and irrespective of the objects’ egocentric position .", "Can the sequence of time intervals between encounters be accurately decoded from the activity of the adapting PG cells ?", "To answer this question , we first constructed a simple model to describe the cells’ response dynamics .", "This model assumes each cell’s responsiveness is governed by a resource variable x which is depleted following each encounter and recovers exponentially between encounters ( Tsodyks and Markram , 1997 ) .", "A parameter β indicates the fraction of the resource remaining after depletion , while a time-constant τ sets the speed of recovery .", "The resource variable x is converted into a firing rate variable λ using static rectification with a gain parameter a and baseline activity parameter c .", "Finally , each encounter generates a spike count according to a Poisson distribution with the current value of the rate parameter λ ( Figure 6A ) .", "The parameter β determines the extent to which past intervals in the sequence affect the current state ( Figure 6B ) .", "For instance , β=0 signifies complete exhaustion of the resource at each and every encounter .", "For such a cell , therefore , the firing rate increases monotonically as a function of the last time interval ( with the dependence taking the form of a saturating exponential function ) regardless of past intervals .", "In other words , β=0 indicates a completely history-independent cell , as defined above .", "As β approaches unity the memory of past intervals becomes more dominant , for example , a succession of short intervals will yield progressively weaker responses .", "All 15 adapting cells had statistically significant correlations between the responses and the fitted rate parameter {Tn} ( p < 0 . 05 , random permutations , see Figure 6—figure supplement 1A , B ) .", "Consistent with the results in the previous section , most adapting cells had an extremely low value of β ( see parameter distributions in Figure 6—figure supplement 1 ) ; 33% of the cells ( 5/15 ) were best fitted with β = 0 ( see example in Figure 5B ) , the median β was 0 . 12 and only 13% ( 2/15 ) had β>0 . 5 .", "Hence , most cells were indeed predominantly affected only by the very last time interval .", "The fitted recovery time-scales were in the range 2 . 6-25 . 3s ( see Figure 6—figure supplement 1C ) .", "Note , however , that this largely reflects the range explored with our stimulation protocol ( 1-30s intervals ) ; it is quite probable that PG contains faster cells ( i . e . , with τ<1s ) that appear to be ‘non-adapting’ under this protocol , as well as slower cells ( i . e . , with τ>30s ) that quickly cease responding and were therefore not recorded from .", "Using this model , we simulated the response of a population of adapting PG cells to random interval sequences .", "A Maximum-Likelihood Estimator ( MLE , see Materials and methods ) was used to decode the most recent interval from the population response to each encounter .", "To demonstrate this approach , we used a homogeneous population of identical memoryless neurons ( β=0 ) .", "At intervals shorter than the cells’ adaptation time-constant τ the MLE was approximately unbiased ( Figure 6C ) and saturated the Cramér-Rao lower error bound ( CRLB , Figure 6D ) .", "The error increased with the decoded time interval T and baseline spontaneous activity c and decreased with population size N and response gain a ( Figure 6E-G ) .", "Finally , we checked the effect of non-zero adaptation memory ( β>0 ) while still using the same memoryless MLE decoder ( constructed by fitting a memoryless model to the simulated responses ) .", "The estimation error and bias were nearly unchanged up to β=0 . 2 ( Figure 6H ) .", "We conclude that the response of the majority of PG adapting cells to an encounter can be used to estimate the time interval elapsed prior to that encounter , using a simple , memoryless decoder .", "It is important to note that the population model mentioned above assumes that the responses of individual cells are statistically independent given the interval sequence .", "This assumption may not hold in certain scenarios , such as the existence of a latent state variable ( e . g . arousal ) modulating the overall population responsiveness .", "Such correlations across the population , if not taken into account in the decoding algorithm , would degrade the estimation accuracy .", "One possible solution to this could be based on the population of non-adapting cells ( 55% , 18/33 ) which , by definition , are not significantly affected by the temporal pattern of stimulation but might be affected by such global modulations .", "Thus , this population may convey sufficient information to enable estimation of such confounding variables and correct the temporal estimation accordingly .", "Can PG activity be used to explain features of spatial behavior in freely-swimming electric fish ?", "To check this possibility , we must first measure the time-intervals being memorized by fish conducting a spatial learning task , as well as the error in the fish’s estimation of these intervals .", "To this end we used previously published data from spatial learning experiments conducted in our lab ( Jun et al . , 2016 ) .", "In these experiments fish were trained to find food in a specific location in complete darkness .", "This was performed either with or without the aid of landmarks .", "Since only the short-range electrosense was available to the fish , they had to use path-integration from the encountered objects to the food location .", "In other words , the fish had to memorize the intervals/distances between these encounters in order to improve its behavioral performance and find the food more efficiently .", "Therefore , we measured the time-interval and the distance traveled by the trained fish from the last encounter with an object ( tank wall or landmark ) to the food in each trial , either with ( Figure 7A ) or without landmarks ( Figure 7B ) .", "As one might expect , these intervals were longer in the absence of landmarks ( Figure 7C ) .", "Next , in order to estimate the fish’s temporal accuracy , we randomly performed ‘probe’ trials in which no food was present in the arena .", "In these trials the fish vigorously searched for the missing food around its estimated position .", "We measured the distance between the center of the searched area and the designated food location to obtain the fish’s spatial error ( Figure 7D , with landmarks; Figure 7E , without landmarks ) .", "This error increased in the absence of landmarks as well ( Figure 7F ) .", "Dividing this error by the median velocity in each trial yielded the temporal error , that is the error in the fish’s estimation of the time elapsed between object encounters and the estimated food location ( Figure 7F , inset ) .", "Thus , this behavioral analysis yielded the relation between the memorized time-intervals and the estimation error of this variable in two different scenarios .", "PG is the only source of sensory information for the dorsolateral pallium ( DL ) , the likely site of spatial memory ( Rodriguez et al . , 2002 ) .", "Therefore , we hypothesized that the history-independent adapting PG cells provide DL with one necessary component for path integration: the temporal sequence of object encounters ( the two other components are heading-direction and linear velocity ) .", "How many adapting PG cells are required to achieve the behavioral temporal acuity observed as described above ?", "We constructed a heterogeneous population model using the empirically obtained parameters ( β was set to 0 for all neurons to simplify the simulations ) .", "This model was then stimulated at random time-intervals , which were then decoded from the population response using MLE .", "This estimation was approximately unbiased across the entire range tested ( Figure 8A ) .", "We compared the simulated estimation error , as well as the analytically computed CRLB for the heterogeneous population , to the behavioral temporal results obtained with and without landmarks ( Figure 8B ) .", "We also performed the comparison in spatial terms by using the fishes’ average velocity to convert the model’s results into units of distance ( Figure 8C ) .", "The simulated PG population yielded temporal and spatial estimation errors comparable to those displayed in behavior ( boxplots in Figure 8B , C ) , both with and without landmarks , using only ~500 adapting cells .", "The lateral subdivision of PG ( PGl ) , in which most motion-responsive cells were found , contains about 60 , 000 cells ( Trinh et al . , 2016 ) .", "Based on our data , we can estimate that over 9 , 000 of these are memoryless adapting cells ( a conservative estimate using only strictly history-independent cells with β=0 , 5/33 or 15% ) .", "Thus , we hypothesize that the number of PGl cells is sufficient to attain the observed behavioral precision , even when additional encoding errors ( e . g . in heading and velocity estimation ) are taken into account .", "Taken together , our simulations suggest that time-interval encoding in PG can consistently account for the observed behavioral precision of spatial learning .", "It should be noted that the validity of this computational analysis relies on our ( admittedly simple ) model correctly capturing the neuronal response dynamics .", "Finer details , such as the inter-dependency between the model’s various parameters , were not taken into account in this study .", "However , it seems reasonable to suggest that the plentitude of PGl cells provides a large margin of error to accommodate such extensions ." ], [ "Our results suggest the following space-to-time transformation scheme: PG derives a sequence of discrete novelty events ( encounters ) from OT activity .", "The remarkable history-independent adaptation process provides an accessible , accurate and unbiased representation of the time intervals between encounters .", "We have found visually-responsive PG cells displaying similar adaptation features ( Figure 5—figure supplement 2 ) , suggesting that this mechanism is implemented across multiple sensory modalities .", "The elimination of egocentric topographic information in PG – both in the response itself and in its adaptation – ensures that the encoding of time is invariant to the specific body part encountering the object .", "This temporal information is then transmitted to DL , which can use it to integrate the fish’s swim velocity to obtain distance-traveled , a key allocentric variable .", "For fish , the necessary velocity information may be provided by the lateral-line system ( Chagnaud et al . , 2007; Oteiza et al . , 2017 ) ; several lateral-line responsive PG units were encountered in our recordings ( Figure 3—figure supplement 1 ) .", "Finally , DL can combine the distance information with instantaneous heading-direction ( vestibular system , Straka and Baker , 2013; Yoder and Taube , 2014 ) to yield the animal’s allocentric spatial position ( Etienne and Jeffery , 2004 ) .", "A recent study in zebrafish suggests that DL neurons can indeed process temporal information on the long time-scales discussed here ( Cheng et al . , 2014 ) .", "Our computational analysis demonstrates that the PG temporal information is sufficient to account for the spatial acuity displayed in behavioral studies of gymnotiform fish utilizing electrosensory information alone ( Figure 8 ) .", "This space-to-time mechanism may shed light on the primitive basis of egocentric-to-allocentric transformations .", "Short-range sensing , used by ancestor species living in low-visibility environments , necessitated the perception of space through temporal sequences of object encounters .", "With the evolution of long-range sensory systems such as diurnal vision ( MacIver et al . , 2017 ) , simultaneous apprehension of the spatial relations of environmental features became possible .", "The neural mechanisms implementing sequential ( space-to-time ) spatial inference and simultaneous spatial inference presumably both exist in mammals , for example , we can acquire a map of relative object locations by looking at a scene from afar , or by walking and sequentially encountering landmarks with our eyes closed .", "Whether the sequential or the simultaneous spatial-inference is more dominant may depend on the species ( e . g . , nocturnal or diurnal ) and on context ( e . g . , open field or underground burrows ) .", "However , it is not clear whether sequentially versus simultaneously-acquired spatial knowledge is processed in a common circuit .", "Indeed , clinical case studies on the regaining of eyesight in blind humans indicate that sequential and simultaneous spatial perceptions are fundamentally different , and may therefore involve two distinct computations and neuronal pathways ( Sacks , 1995 ) .", "The population of thalamic neurons that we discovered may provide an essential component underlying one of these two major computations – the encoding of sequential temporal information – and we hypothesize that such neurons underlie sequential spatial learning in all vertebrates .", "There is substantial evidence indicating that the pathway studied here indeed has parallels in other vertebrates , and specifically in mammals .", "PG’s homology to posterior thalamic nuclei is supported by previously published anatomical and developmental findings ( Giassi et al . , 2012a; Ishikawa et al . , 2007; Mueller , 2012 ) , as well as by physiological ( Figure 1B , C ) and molecular ( Figure 1—figure supplement 1 ) results presented here .", "The thalamic pulvinar nucleus is particularly similar to PG in that it receives direct tectal input ( Berman and Wurtz , 2011 ) .", "Its involvement in visual attention and saliency in primates ( Robinson and Petersen , 1992 ) corresponds to PG’s involvement in novelty detection ( Figure 2 ) .", "Moreover , pulvinar lesions are associated with saccadic abnormalities and deficits in the perception of complex visual scenes ( Arend et al . , 2008 ) , suggesting a link to the sequential mode of spatial learning .", "Komura et al . ( 2001 ) demonstrated that posterior thalamic regions ( including the rodent equivalent of the pulvinar ) can implement interval timing computations over long time-scales ( >>1 s ) ; however , the mechanistic basis for these computations has not been identified ( Simen et al . , 2011 ) and potential contributions to path integration have not been explicated .", "A recent paper ( Paton and Buonomano , 2018 ) reviewed models of temporal encoding based on recurrent neural networks .", "Further studies will be required to determine whether the novel adaptation encoding mechanism in PG engages the downstream recurrent networks of DD and DL to produce refined estimates of the time interval between salient sensory and/or motor events .", "Finally , a recent study in rodents demonstrated spatially non-specific adaptation in VPM ( Jubran et al . , 2016 ) , a posterior thalamic nucleus responding to vibrissal object encounters ( Yu et al . , 2006 ) .", "Taken together , we hypothesize that thalamic space-to-time mechanisms akin to those presented here play an important role in mammalian sequential spatial learning , especially in nocturnal animals relying on sparse sensory cues ( Save et al . , 1998 ) .", "The telencephalic target of PG , DL , resembles the mammalian hippocampus not only in function , as revealed in lesion studies ( Rodríguez et al . , 2002 ) , but also in development , gross circuitry and gene expression ( Elliott et al . , 2017 ) .", "The role of the hippocampus in spatial learning and navigation is well established , and hippocampal neural correlates of allocentric spatial variables have been exquisitely described ( Barry and Burgess , 2014; Buzsáki and Moser , 2013 ) .", "There is also evidence for the importance of time coding in the mammalian hippocampus: ‘Time cells’ responsive to elapsed time have been reported and , in some cases , these cells also respond at specific spatial loci ( Eichenbaum , 2014 ) .", "Furthermore , a recent study on the representation of goals in the hippocampus found cells encoding the length/duration of the traveled path ( Sarel et al . , 2017 ) .", "The mechanism we have found may therefore contribute to creating temporal coding in the hippocampus , not just in the context of egocentric-to-allocentric transformations but rather whenever expectations associated with specific time intervals need to be generated .", "It should be noted , however , that unlike DL’s direct thalamic input via the PG bottleneck , the hippocampus receives sensory and motor information primarily via the cortex .", "Furthermore , multiple bi-directional pathways connect the mammalian sensory and motor cortical regions with the hippocampal network .", "Pinpointing the exact loci where egocentric-to-allocentric transformations may take place in the mammalian brain is therefore extremely challenging .", "We propose that this transformation is initiated in the mammalian thalamus where history-independent adaptation also encodes time between encounter events .", "Finally , we propose that this thalamic output contributes to the generation of an allocentric spatial representation in the mammalian hippocampus .", "In this contribution , we propose a hypothesis about how gymnotiform fish , and perhaps vertebrates in general , generate their representation of position relative to the environment .", "Future experiments could test the predictions entailed by this hypothesis .", "Behaviorally , our model implies that the fish's sense of position is critically dependent on its last encounter with an object .", "Further behavioral studies of spatial learning could elucidate this relationship , for example , by manipulating the objects' arrangement relative to the navigation target .", "Combining these studies with chronic recordings of PG and its pallial targets in freely navigating fish will permit testing of our proposed space-to-time neural transformation scheme ." ], [ "All procedures were approved by the University of Ottawa Animal Care and follow guidelines established by the Society for Neuroscience .", "Apteronotus leptorhynchus fish ( imported from natural habitats in South America ) were kept at 28°C in community tanks .", "Fish were deeply anesthetized with 0 . 2% 3-aminobenzoic ethyl ester ( MS-222; Sigma-Aldrich , St . Louis , MO; RRID: SCR_008988 ) in water just before surgery or tissue preparation .", "Surgery was performed to expose the rostral cerebellum , lateral tectum and caudal pallium .", "Immediately following surgery , fish were immobilized with an injection of the paralytic pancuronium bromide ( 0 . 2% weight/volume ) , which has no effect on the neurogenic discharge of the electric organ that produces the fish’s electric field .", "The animal was then transferred into a large tank of water ( 27°C; electrical conductivity between 100–150 μS cm−1 ) and a custom holder was used to stabilize the head during recordings .", "All fish were monitored for signs of stress and allowed to acclimatize before commencing stimulation protocols .", "Custom made stereotrodes or tritrodes were made of 25 μm diameter Ni-Cr wire ( California Fine Wires ) .", "Each electrode was manually glued to a pulled filamented glass pipette ( P-1000 Micropipette Puller , Sutter Instrument , Novato , CA ) ; the glass pipette provided mechanical rigidity that allowed advancing the tetrode to the deep-lying PG .", "Prior to recording , tetrode tips were gold-plated ( NanoZ 1 . 4 , Multi Channel Systems , Reutlingen , Germany ) to obtain 200–300 kOhm impedance at 1 kHz .", "The electrode was positioned above the brain according to stereotaxic brain atlas coordinates ( 150–300 µm caudal to T26 and 800–1000 µm lateral to midline [Maler et al . , 1991] ) , and lowered using a micropositioner ( Model 2662 , David Kopf Instruments , Tujunga , CA ) while delivering visual and electrosensory stimuli .", "Tectal responses to such stimuli ( Bastian , 1982 ) were usually detected twice , around 1200 µm and around 1900 µm ventral to the top of cerebellum ( as expected from the curved shape of OT ) .", "The electrode usually then transversed nucleus Electrosensorius , producing weak multi-unit responses to electrocommunication stimuli around 2300–2500 µm ( Heiligenberg et al . , 1991 ) .", "PG units were usually encountered between 2800 µm and 3400 µm ventral to the top of the cerebellum , and were easily identified due to their characteristic rapid spike bursts ( Figure 1B , C ) .", "Differential extracellular voltage was obtained by using one stereotrode/tritrode channel as reference .", "This enabled near-complete cancellation of the electric organ discharge ( EOD ) interference .", "We report on the responses of 84 PG neurons responsive to object motion; several motion protocols were used and the sample size for each protocol is mentioned in context .", "We also found PG cells responding to electrocommunication signals , mostly within the medial subdivision of PG ( PGm ) .", "As expected from the sparse retinal input to OT ( Sas and Maler , 1986 ) , we recorded only a small number ( n = 19 ) of PG cells responsive to visual input ( Figure 5—figure supplement 2 ) , mostly in more ventral portions of PG ( Figure 1—figure supplement 4 ) .", "In addition , we identified a small number of cells responsive to passive electrosensory ( Grewe et al . , 2017 ) ( ampullary receptors , n = 27 ) , acoustic ( n = 7 ) and lateral line ( n = 7 , Figure 3—figure supplement 1 ) stimulation – but did not attempt to further characterize their coding properties .", "Cell responses were initially manually tested with brass and plastic spheres .", "Cells responding to both were also tested with an electrically neutral gel ball made of 15% agarose in tank water to exclude lateral-line responses ( Heiligenberg , 1973 ) ( Figure 3—figure supplement 1 ) .", "A plastic or brass sphere ( 1 . 21 cm diameter ) was connected to an electromechanical positioner ( Vix 250IM drive and PROmech LP28 linear positioner , Parker Hannifin , Cleveland , OH ) , which was pre-programmed for the appropriate motion sequence and initiated by outputs from our data acquisition software ( Spike2 , Cambridge Electronic Designs , Cambridge , UK ) .", "Typically , a trapezoidal velocity profile was used with 150 cm/s2 acceleration and 5 cm/s peak velocity , and total distance in either direction of 8 cm ( longitudinal ) or 5 cm ( transverse ) ; due to technical limitations , each cell was recorded using only one direction of motion ( longitudinal or transverse ) .", "For protocols involving motion in two axes ( receptive field sampling , Oddball ) , the object was attached to a second electromechanical positioner ( L12-100-50-12-P linear actuator , Firgelli Technologies , Ferndale , WA ) , which was mounted perpendicularly to the first one .", "In order to measure the receptive field ( RF ) size , we used one motor to repeatedly perform transverse object motion ( towards and away from the fish ) while a second motor was used to randomly change the longitudinal position between repetitions .", "This protocol was performed on a total of 27 cells .", "To check the spatial specificity of the adaptation process ( i . e . if it is affecting only the body location experiencing the encounters or a whole-body effect ) , we performed a spatial oddball experiment: First , a series of N ‘standard’ encounters ( in and out transverse object motion , N = 5–9 ) were given in rapid succession at one location ( e . g . the head ) ; the ( N + 1 ) th encounter was given at a different location ( e . g . the trunk ) while maintaining the same time-interval between encounters ( 3–5 s , the second motor was used to quickly switch positions ) .", "Each such series was followed by a long recovery period ( ≥ 30 s ) and then repeated in the opposite direction .", "This was performed on a total of 14 cells , out of which six were found to be adapting ( in either direction ) .", "In the initial experiments the location of PG was verified by preparing histological sections and locating the electrode track marks ( Figure 1—figure supplement 4 ) .", "After recordings were complete , the fish was deeply anesthetized using tricaine methanesulfonate ( MS-222 0 . 2 g/L; Sigma-Aldrich , St-Louis , MO ) and transcardially perfused with 4% paraformaldehyde , 0 . 1% glutaraldehyde , and 0 . 2% picric acid in 0 . 1 M PBS pH 7 . 4 .", "Brains were removed and incubated overnight in a solution of 4% paraformaldehyde , 0 . 2% picric acid , and 15% sucrose in 0 . 1 M PBS pH 7 . 4 at 4°C .", "Cryostat sections were cut at 25 μm in the transverse plane and mounted on Superfrost Plus glass slides ( Fisher Scientific , Pittsburgh , PA ) .", "All sections were counterstained with green fluorescent Nissl reagent 1:300 ( Molecular Probes , Eugene , OR; NeuroTrace 500/525 green-fluorescent Nissl Stain #N21480 , RRID: SCR_013318 ) in PBS for 20 min at room temperature .", "Three adult male fish were anesthetized .", "Ice-cold ACSF was dripped on the head while the skull was removed and brains quickly removed and submerged in ice-cold ACSF .", "PG and DL brain regions were superficially located ( Maler et al . , 1991 ) , identified , dissected out and stored at −20°C .", "Tissues were weighted and total RNA was purified using Trizol ( Sigma-Aldrich ) according to the manufacturer’s recommendations .", "Contaminating genomic DNA was digested with DNAse1 , total RNA was then precipitated overnight at −20°C , resuspended in nuclease-free water and quantified by spectroscopy .", "300 ng of total RNA were used for first strand cDNA synthesis using the Maxima H Minus First Strand cDNA Synthesis Kit ( K1681; ThermoFisher ) .", "PCR were made with the Taq polymerase ( EP0402; ThermoFisher ) according to manufacturer’ recommendations for 35 cycles using the following primer pairs: G amplicon ( 301 bp ) , direct: 5’-CGACACCTTCCGCAAAATCG-3’ , reverse: 5’-AGCACAGACAGACCTCCGc-3’; H amplicon ( 338 bp ) , direct: 5’-GGGACGATTTCAGGGACAGG-3’ , reverse: 5’-CACTCGCAGCAGACGGAA-3’; I amplicon ( 355 bp ) , direct: 5’-TGGGATGAGATTGGAG TGAAAC-3’ , reverse: 5’-AGCGGACCAGCTTAATGACC-3’ .", "Amplicons were then migrated on a 1% agarose Et-Br gel and photographed with a BIO-RAD Gel Doc System .", "In all paired comparisons , the bootstrap method ( 5000 random redistributions without replacement ) was employed to estimate statistical significance .", "Random permutations were used to evaluate significance of correlations ( 5000 random permutations without replacement ) .", "Sample sizes were determined by statistical requirements , aiming at confidence levels > 95% .", "No statistical methods were used to pre-determine sample sizes but our sample sizes are similar to those generally employed in the field .", "No randomization or blinding was used is this study .", "The random interval protocol ( Figure 5A ) produced for each cell an interval ( input ) vector {Tn} and a spike count response ( output ) vector {Rn} ( count computed for each unit in a time-window determined by its response type , see Figure 3 ) .", "For some of the cells , a slow decline in response due to experimental instability was corrected by dividing the response time course by a least-squared fitted slow ( > 10 min ) exponential decay .", "The model ( Figure 6 ) assumes each neuron has a latent state variable x with dynamics following each encounter given byxn=1−e−Tnτ ( 1−βxn−1 ) where Tn is the nth time interval in the sequence , β is the memory coefficient ( 0≤β≤1 ) and τ the recovery time-constant .", "Note that x is always in the range [0 , 1] .", "Equation ( 1 ) is in fact the between-event , discrete-time solution of the standard resource-based depression model of Tsodyks and Markram ( Tsodyks and Markram , 1997 ) , where β=1-UEI; we chose this formalization so that the extent of history-dependence in the adaptation dynamics will be emphasized ( i . e . , β=0 signifies complete history independence and history dependence increases as β→1 ) .", "The neuron’s firing parameter λn at the nth encounter is λn = axn+c+ , where a > 0 , c∈R and ∙+ denotes non-negative rectification .", "Fitting of a and c was performed using linear least squares , while β and τ were found by exhaustive search on a 2-D grid .", "Fitting was deemed successful if the parameter values generated by the model were correlated with the actual responses with P < 0 . 05 ( random permutations , Figure 6—figure supplement 1 ) .", "This was true for 15/33 cells .", "Note that when β=0 ( ‘history-independent’ adaptation ) , we get: ( 2 ) λn=[a ( 1−e−Tnτ ) +c]+ , which corresponds to the solid blue line in Figure 5B .", "Finally , {τn} were used as the parameters of Poisson random variables to generate stochastic spike-counts {Rn} , so that the number of spikes emitted at each encounter was distributed according to: ( 3 ) p ( Rn=k ) =λnk e−λnk ! , k=0 , 1 , 2 , 3… Now we assume a population of N history-independent ( β=0 ) neurons , with aj , cj , and τj are the individual parameters of the jth neuron and Rnj is its response to the nth encounter .", "We also assume that Rnjj=1N are independent Poisson random variables ( r . v . ’s ) , with each r . v . distributed according to equation ( 3 ) .", "The likelihood of this population response is thereforeLTn|Rnj=PRnj|Tn=∏j=1Naj1-e-Tnτj+cj+Rnje-aj1-e-Tnτj+cj+Rnj !", ", and the log-likelihood islTn|Rnj=∑j=1NRnjlog⁡aj1-e-Tnτj+cj+-aj1-e-Tnτj+cj+-log⁡Rnj !", ".", "The Maximum-Likelihood Estimator of the last time interval is therefore obtained by finding the time interval Tn that maximizes this likelihood:TnMLE=argmaxTn>0 ( ∑j=1NRnjlog ( [aj ( 1−e−Tnτj ) +cj]+ ) −[aj ( 1−e−Tnτj ) +cj]+ ) .", "This maximum was found numerically for each generated time interval .", "To explore the coding properties of the model we used a population of N identical cells ( τj=τ; aj=a; cj=c ) .", "Assuming for a moment a ( 1−e−Tnτ ) +c>0 , the MLE for the population is the solution of: ( 4 ) dl ( Tn ) dTn=∑j=1Na ( −1τe−TnMLEτ ) ( 1−Rnja ( 1−e−TnMLEτ ) +c ) =0N-∑j=1NRnja1-e-TnMLEτ+c=0a+c-1N∑j=1NRnj=ae-TnMLEτTnMLE=-τlog⁡a+c-1N∑j=1NRnja=τlog⁡aa+c-1N∑j=1NRnj One can easily show that this solution indeed maintains a ( 1−e−TnMLEτ ) +c>0 .", "Note that when 1N∑j=1NRnj> ( a+c ) then TnMLE has no real solution ( i . e . , likelihood function has no finite maximum ) .", "In these cases , the estimator output is ignored .", "Since for f ( X ) =log⁡ ( 11−x ) , E ( f ( X ) ) ≠f ( E ( X ) the MLE is biased .", "However , if we assume T≪τ , then we can approximate:TnMLE≅τ1N∑j=1NRnj-cafor which:ERnjTnMLE≅τλn-ca= τaxn+c-ca=τ1-e-Tnτ≅Tn To compute the Fisher information for any time T>0 , recall the identity: I ( T ) =−E{Rj}{d2l ( {Rj} , T ) dT2}=−E{Rj}{ddT ( ∑j−ajτje−Tτj ( 1−Rjaj ( 1−e−Tτj ) +cj ) ) } , where the sum is restricted to those neurons with positive activity on that value of T , that is , {j|j∈[1:N]∧aj ( 1−e−Tτj ) +cj>0} .", "Therefore:I ( T ) =−E{Rj}{∑jajτj2e−Tτj ( 1−Rjaj ( 1−e−Tτj ) +cj−ajRj ( aj ( 1−e−Tτj ) +cj ) 2e−Tτj ) }=-∑jajτj2e-Tτj1-ERjaj1-e-Tτj+cj-ajERjaj1-e-Tτj+cj2e-Tτj Since Rj are Poisson r . v . with rate λj=aj1-e-Tτj+cj:IT=∑jaj2τj2aj1-e-Tτj+cje-2Tτj For a homogeneous population ( Figure 6C–G ) , this becomes:IT=a2Nτ2a1-e-Tτ+c+e-2Tτ Therefore , the CRLB ( assuming zero bias ) becomes:VarTmle≥∑jaj2τj2aj1-e-Tτj+cje-2Tτj-1 The set of parameter values obtained for all 15 fitted adapting cells ( a , c and τ ) were randomly drawn from to generate a bootstrap population of N cells ( N=100 , 500 and 2000 ) .", "Gaussian noise with standard deviation of 25% was added to the parameters to obtain smoother distributions .", "The memory variable β was set to 0 for all cells to simplify simulations .", "We also assumed that spiking across the population was statistically independent with Poisson statistics given the last interval , that is pRni , Rnj|Tn=pRni , |TnpRnj , |Tn where Rni is the response of neuron i to time interval n .", "Random intervals were drawn in the range 1–30 s , and for each interval a vector of responses ( spike counts ) across the population Rnjj=1N was generated , where N is the population size .", "Published spatial learning data ( Jun et al . , 2016 ) were used; methods used for the generation of these data are explained in detail in that study .", "Briefly , South American weakly electric fish ( Gymnotus sp . ) were trained to find food ( a mealworm restrained to a suction cup ) in complete darkness , at a specific location within a 150 cm diameter custom made circular arena .", "In experiments with landmarks , four acrylic objects ( two square prisms , 5 . 6 and 9 . 0 cm/side and two cylinders , 7 . 6 cm and 10 . 2 cm diameter ) were placed in fixed locations within the arena .", "In each daily session , each fish was given four trials to find the food .", "After a 12-session training stage , animal performance stabilized , and four test sessions were performed in which food was omitted in one randomly assigned ‘probe’ trial .", "Only data from these four last sessions were analyzed here .", "A total of four fish were used with landmarks and eight fish were used without landmarks .", "Fish behavior was video recorded and tracked as previously described ( Jun et al . , 2014 ) .", "The cruising time/distance from the last encounter ( with either a landmark or the tank wall ) to the location of the food is the epoch/trajectory the fish had to memorize in order to perform path integration .", "This was measured in each of the ‘food’ trials ( Figure 7A , B ) ; the segment from the last encounter ( 3 cm from a landmark or 6 cm from the arena wall ) to the detection of food ( 3 cm from food location ) was found and the trajectory’s total length and duration were computed .", "The spatial ‘decoding’ errors were obtained by measuring where the fish searched for the missing food in the probe trials ( Figure 7D , E ) ; the normalized histogram of the visiting frequency across space ( heat map ) was fitted with a two-dimensional Gaussian function:Px , y=A∙exp-121-θ2x-μx2σx2+y-μy2σy2-2θx-μxy-μyσxσywhere μx and μy are mean parameters; σx and σy are variance parameters; A is a gain parameter and θ is the cross correlation parameter .", "The distance between the Gaussian center ( μx , μy ) ( white ‘x’ mark ) and the food location ( white ‘+’ mark ) is the spatial error; dividing this error by the median velocity in the trial produces the temporal error ." ] ]

[ "Learning the spatial organization of the environment is essential for most animals’ survival .", "This requires the animal to derive allocentric spatial information from egocentric sensory and motor experience .", "The neural mechanisms underlying this transformation are mostly unknown .", "We addressed this problem in electric fish , which can precisely navigate in complete darkness and whose brain circuitry is relatively simple .", "We conducted the first neural recordings in the preglomerular complex , the thalamic region exclusively connecting the optic tectum with the spatial learning circuits in the dorsolateral pallium .", "While tectal topographic information was mostly eliminated in preglomerular neurons , the time-intervals between object encounters were precisely encoded .", "We show that this reliable temporal information , combined with a speed signal , can permit accurate estimation of the distance between encounters , a necessary component of path-integration that enables computing allocentric spatial relations .", "Our results suggest that similar mechanisms are involved in sequential spatial learning in all vertebrates ." ]

[ "Finding their way around is an essential part of survival for many animals and helps them to locate food , mates and shelter .", "Animals have evolved the ability to form a 'map' or representation of their surroundings .", "For example , the electric fish Apteronotus leptorhynchus , is able to precisely learn the location of food and navigate there .", "It can do this in complete darkness by generating a weak electric field .", "As it swims , every object it encounters generates an ‘electric image’ that is detected on the skin and processed in the brain .", "However , all the cues the fish comes across are from its own point of view – the information about its environment is processed with respect to its location .", "And yet , the map that it generates needs to be independent of the fish’s position – it has to work regardless of where the animal is .", "The way animals translate ‘self-centered’ experiences to form a general representation of their surroundings is not yet fully understood .", "Now , Wallach et al . studied how internal brain maps are generated in A . leptorhynchus .", "Information about the fish's environment passes through a structure in the brain called the preglomerular complex .", "Measuring the activity of this region revealed that the preglomerular complex does not process much self-centered information .", "Instead , whenever the fish passed any object – regardless of where it was in relation to the fish – the event triggered a brief burst of preglomerular activity .", "The intensity of the activity depended on how recently the fish had encountered another object .", "This information , combined with the dynamics of the fish's movement , could be what allows the fish to convert a sequence of encounters into a general spatial map .", "These findings could help to inform research on learning and navigation .", "Further research could also reveal whether other species , including humans , generate their mental maps in a similar way .", "This may be relevant for people suffering from diseases such as Alzheimer’s , in which a sense of orientation has become impaired ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "neuroscience" ]

[ [ "Perceptual representations of the environment are critical to an animal's survival and are believed to occur through coactivated neuronal groups known as cell assemblies .", "Human neuronal firing ( Ekstrom et al . , 2007; Kraskov et al . , 2007; Chan et al . , 2011; Rey et al . , 2014 ) and increases in high-frequency activity ( HFA ) in the gamma range ( above 30 Hz; Jacobs and Kahana , 2009; Jacobs et al . , 2012; van Gerven et al . , 2013 ) carry information about perceptual and mnemonic representations .", "Several recent studies have shown that these two signals are positively correlated ( Ray et al . , 2008; Manning et al . , 2009; Whittingstall and Logothetis , 2009; Miller et al . , 2014; Rey et al . , 2014; Burke et al . , 2015 ) and are each modulated by the phase of low frequency oscillations ( LFO ) ( O'Keefe and Recce , 1993; Bragin et al . , 1995; Skaggs et al . , 1996; Canolty et al . , 2006; Jacobs et al . , 2007; Tort et al . , 2009; Axmacher et al . , 2010; Rutishauser et al . , 2010; McGinn and Valiante , 2014 ) .", "This modulation is detectable as phase-amplitude coupling ( PAC ) of gamma amplitude to LFO phase ( Buzsaki , 2010; Miller et al . , 2014; Aru et al . , 2015 ) .", "Together , these findings have motivated models positing that LFO phase may organize cell assemblies ( Kayser et al . , 2012; Lisman and Jensen , 2013; Jensen et al . , 2014; Watrous et al . , 2015 ) , a form of phase coding ( O'Keefe and Recce , 1993 ) .", "Supporting this view , LFO phase can be used to decode behaviorally relevant information ( Belitski et al . , 2008 , 2010; Fell et al . , 2008; Schyns et al . , 2011; Lopour et al . , 2013; Ng et al . , 2013 ) and phase coded neural activity has been demonstrated in rodents ( O'Keefe and Recce , 1993; Skaggs et al . , 1996 ) and monkeys ( Kayser et al . , 2009; Siegel et al . , 2009 ) .", "Although the PAC observed in humans ( Canolty et al . , 2006; Axmacher et al . , 2010 ) has been thought to reflect phase-coding , this assumption has yet to be validated because prior studies have not investigated the relation between PAC and decoding from LFO phases .", "We have recently proposed that the frequency-specific phase of LFO coordinates neural firing to support neural representations ( Watrous and Ekstrom , 2014; Watrous et al . , 2015 ) .", "Here , we tested this prediction , a form of the phase-coding hypothesis in humans , by examining the relation between PAC and neural representations for categories .", "We analyzed intracranial recordings from 167 electrodes in six patients with pharmaco-resistant epilepsy as they viewed pictures of houses , tools , scenes , and faces .", "First , we identified PAC on individual electrodes by using a recently developed metric which allows for the characterization of PAC across individual HFA events .", "On electrodes exhibiting PAC , we then assessed the distinctiveness of each category's phase-coded representation during periods with and without pronounced HFA .", "Our results suggest that during periods with pronounced HFA , categorical representations can be recovered based on the phase of low-frequency oscillations , supporting the idea of phase-coded neural representations in humans ." ], [ "Testing the phase-coding hypothesis , we asked if high frequency activity occurred during category-specific phases of the modulatory LFO .", "Figure 3A shows two traces from an example electrode which are color-coded by the instantaneous phase at Fmax .", "HFA windows ( boxes color coded by 1 Hz phase ) occurred during different modulatory phases depending on stimulus category .", "On this electrode , phases extracted during HFA windows were clustered for each category to different phases , resulting in category-specific phase-clustering ( Figure 3B ) .", "Similar findings were observed in other patients ( Figure 3C ) , and appeared distinct from representations using power or phase ( Figure 3—figure supplements 1 , 2 ) . 10 . 7554/eLife . 07886 . 009Figure 3 . HFA occurs at category-specific low-frequency phases .", "( A ) Two example trials from patient #6 demonstrating that HFA windows occur at different phases for different categories .", "The signal is color-coded by the phase of 1 Hz oscillation only during the stimulus period .", "Times prior to stimulus period are shown in order to visualize the 1 Hz modulatory signal .", "HFA windows are indicated by the boxes , color-coded by the 1 Hz phase at which they occur .", "( B ) Summary circular histograms and resultant vectors for this electrode .", "Categorical phase-clustering to different phases was prominent at Fmax , allowing for the decoding of categorical information based on the phase at which HFA events occur .", "DSs are plotted for each category in the lower panel .", "( C ) Another example , from a different patient ( #4 ) , showing phase-clustered HFA windows for different categories ( upper ) along with DSs ( lower ) .", "( D ) Proportion of electrodes in each patient showing category specific phase-clustered HFA .", "( E ) Average absolute phase difference across categories and electrodes for increasingly distinct phase representations ( PRs ) .", "( F ) Circular distribution of phases for each level of DS , pooled over electrodes and categories .", "Phase coded representations were equally likely to occur at each phase . DOI: http://dx . doi . org/10 . 7554/eLife . 07886 . 00910 . 7554/eLife . 07886 . 010Figure 3—figure supplement 1 . Decoding categorical information using delta power , phase , or HFA power on example electrode shown in Figure 3A–B .", "( Left )", "Time-resolved and trial-averaged values for delta power ( upper ) , phase ( middle ) , or HFA ( 50–200 Hz ) power ( lower ) .", "Shaded areas show standard error of the mean .", "Time-resolved difference scores for each neural measure are plotted below each panel and are conceptually similar to DS values reported in Figure 3 and Figure 1—figure supplement 1 , with the following exceptions .", "Power values were compared between conditions using a two-sample t-test and significant differences between conditions were assessed for all neural measures using trial-label permutation testing ( n = 1000 shuffles , p < 0 . 05 ) .", "( Right )", "HFA windows , color coded by 1 Hz phase , for each category .", "Note that although some categorical information can be recovered when considering delta power , phase , or HFA power , these effects do not correspond to HFA time windows and more categorical information exists in the phase at which HFA windows occur ( i . e . , PAC , Figure 3B lower ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 07886 . 01010 . 7554/eLife . 07886 . 011Figure 3—figure supplement 2 . Decoding categorical information using delta power , phase , or HFA power on example electrode shown in Figure 3C . See caption for Figure 3—figure supplement 1 for description of figure layout . DOI: http://dx . doi . org/10 . 7554/eLife . 07886 . 01110 . 7554/eLife . 07886 . 012Figure 3—figure supplement 3 . Category-specific phase locking analysis . Proportion of 167 electrodes showing significant phase-clustering ( Rayleigh test , p < 0 . 000001 ) exclusively for houses , tools , scenes , or faces as a function of time and frequency . DOI: http://dx . doi . org/10 . 7554/eLife . 07886 . 012 These findings imply that representations might occur by the category-specific phase at which HFA events occur .", "In order to further quantify this effect , we developed a simple metric , the difference score ( ‘DS’ ) , which allowed us to identify the distinctiveness of each category's phase distribution during HFA windows .", "We applied this metric to the subset of 63 PAC+ electrodes showing significant phase-clustered HFA for each category .", "This was necessary in order to exclude spurious phase differences between categories occurring in the absence of phase clustering .", "Across all patients , 78% ( 49/63 ) of PAC+ electrodes showed a unique phase-clustering profile for one category compared with each other category ( e . g . , Figure 1B; DS = 3 for at least one category , p < 10−9 , Watson Williams test , Bonferroni corrected across comparisons ) .", "This pattern was consistent both within and across patients , with at least 15% of electrodes in each patient showing these effects ( Figure 3D ) .", "We next calculated the average phase difference between categories , expecting this measure to increase with increasing DS .", "Indeed , categories with larger DSs exhibited larger phase differences with other categories such that maximally distinct representations were 35° phase offset from all other categories ( Figure 3E ) .", "As described above , PAC was most likely to occur at the oscillatory trough ( Figure 2I and Figure 2—figure supplement 1 ) .", "Nonetheless , on individual electrodes or for individual categories , HFA could occur at different phases .", "In fact , across electrodes , phase-coding was equally likely to occur at all phases and for all categories; phase-coded categories were not clustered at particular phases at any level of DS ( Rayleigh test , all p > 0 . 19; Figure 3F ) and phase-coding was equally likely for each category ( χ2 ( 3 ) = 1 . 6 , p = 0 . 64 ) .", "Thus , a large proportion of PAC+ electrodes also show category-specific phase clustering of HFA events to different phases ( Video 1 ) , suggesting that PAC is related to phase-coding ( Figure 1B , middle ) . 10 . 7554/eLife . 07886 . 013Video 1 . Significant electrodes rendered onto a glass brain . Each point represents an electrode , and each color represents different effects .", "Black electrodes ( n = 95 ) did not show significant phase-amplitude coupling ( PAC ) .", "Green electrodes ( n = 9 ) only showed significant PAC .", "Yellow electrodes ( n = 14 ) showed significant PAC and phase-clustering of HFA for all 4 categories .", "Red electrodes ( n = 49 ) showed significant PAC , phase-clustering for all 4 categories , and phase-coding of high-frequency activity ( i . e . , difference score of 3 for at least one category ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 07886 . 013 To link these findings more directly to neural coding , we used pattern classification to determine if the phase at which HFA events occur is sufficient to recover categorical information ( see ‘Materials and methods’ ) .", "As expected from the analysis using DS , 42 ( 25% of all ) electrodes showed significant decoding accuracy ( using LFO phases during HFA windows as features ) compared to category label shuffled surrogates and this proportion was significantly higher than would be expected by chance ( p < 10−10 , binomial test , chance level: 8 . 3 electrodes , Cohen's d = 0 . 6 ) .", "Next , we assessed whether phase-coding of categorical information indeed depended on HFA , as would be expected if PAC supports phase-coding .", "We compared decoding accuracy during HFA events to decoding accuracy during randomly selected surrogate events .", "19 ( 11% ) electrodes showed significantly higher decoding accuracy during HFA events as compared to random event surrogates , and this proportion was significantly higher than would be expected by chance ( p < 0 . 0003 , binomial test , chance = 8 . 3 electrodes , Cohen's d = 0 . 22 ) .", "Moreover , 17 ( 10% ) electrodes showed significant enhancements of decoding accuracy during HFA events relative to both label and event shuffled surrogates , with at least two electrodes in each patient showing this pattern .", "This proportion of electrodes far exceeded that expected by chance ( p < 10−10 , binomial test , chance = 0 . 41 electrodes , Cohen's d = 0 . 46 ) .", "These findings complement the above results using DS and indicate that the phase at which HFA events occur carries sufficient information to decode image category , suggesting such information may be a relevant component of the neural code .", "We performed several control analyses to rule out alternative explanations .", "First , if slow oscillatory phase relates to category-specific representations , we expect phase-locking across trials to different categories .", "We observed significant phase locking on many electrodes to specific categories ( Figure 3—figure supplement 3 , Rayleigh test , p < 0 . 000001 ) , similar to previous studies which have identified phase-locked activity ( e . g . , Fell et al . , 2008 ) .", "Second , we excluded the possibility that our PAC+ or phase-clustering inclusion criteria biased our findings by computing a composite measure of phase representation ( PR ) on each electrode ( see ‘Supplement results’ ) .", "This analysis again revealed that phase coding is largest on PAC+ electrodes and is enhanced during HFA windows .", "Third , for comparison with prior PAC methods , we recomputed PAC using the modulation index ( MI , Tort et al . , 2009 ) in different low-frequency bands , again finding PAC that was most prevalent in the delta band ( Figure 2—figure supplement 3 ) .", "Lastly , several models predict that neural processes forming representations will show frequency-specificity ( Siegel et al . , 2012; Watrous and Ekstrom , 2014; Womelsdorf et al . , 2014 ) .", "We therefore recalculated phase-clustering and DS at the minimum modulatory frequency ( FMIN; see ‘Materials and methods’ and Figure 4—figure supplement 1 for individual subject values ) using the same criteria detailed above .", "As one would expect , on PAC+ electrodes , phase clustering was larger at FMAX compared to at FMIN , both on individual electrodes ( Figure 4A , B ) and at the group level ( Figure 4C; paired t-test on resultant vector lengths , t ( 287 ) = 8 , p < 10−10 , Cohen's d = 0 . 32 ) .", "Moreover , only 20% ( 15/72 ) of PAC+ electrodes showed significant phase-clustering at FMIN for all 4 categories and only 1 electrode showed category-selective phase-clustering of HFA events .", "Given that the phase of slower frequencies varies less over time and that we primarily identified Fmax at slow frequencies , this result might be biased towards finding enhanced phase clustering at Fmax .", "We therefore recalculated phase clustering across the full range of frequencies ( 1–12 Hz , 0 . 1 Hz steps ) .", "Again , we found enhanced phase-clustering around 0 . 5 and 1 Hz ( Figure 4—figure supplement 2 ) , but not at adjacent frequencies as would be expected from this alternative account .", "Taken together , these results support the conclusion that HFA at distinct phases and frequencies reflect representations for different categories . 10 . 7554/eLife . 07886 . 014Figure 4 . HFA clusters to specific phases and frequencies for different categories .", "( A ) Example electrode showing phase clustering at the maximum modulatory signal ( Fmax; frequency with maximum power in the FFT , see Figure 2E ) but not at the minimum modulatory signal ( panel B; Fmin; frequency with minimum power in the FFT ) .", "HFA events are marked in color as the phase of the oscillation at the respective frequencies .", "( C ) At the group level , phase clustering was more prominent at the maximum frequency ( Fmax; maroon ) compared to the minimum frequency ( Fmin; black ) across categories and PAC+ electrodes . DOI: http://dx . doi . org/10 . 7554/eLife . 07886 . 01410 . 7554/eLife . 07886 . 015Figure 4—figure supplement 1 . Fmax and Fmin values by subject . Average Fmax and Fmin values for PAC+ electrodes in individual subjects .", "Black dots show values for individual electrodes and have been slightly jittered vertically in order to show all points; true values range from 0 . 5–12 Hz in 0 . 5 Hz increments . DOI: http://dx . doi . org/10 . 7554/eLife . 07886 . 01510 . 7554/eLife . 07886 . 016Figure 4—figure supplement 2 . Phase-locking analysis across all frequencies from 0 . 1–12 Hz . Phase-locked activity as a function of frequency and electrode .", "Values are expressed as percentage of maximal phase clustering .", "We calculated the resultant vector length for each category , summed these values across categories , and divided by the maximum value of 4 .", "If our analysis was biased towards observing effects at slow frequencies , we would expect a smooth gradient of large phase clustering values at low frequencies , trailing off to smaller values at higher frequencies .", "Instead , there are clear peaks in phase clustering around 0 . 5 and 1 Hz and not at adjacent frequencies , particularly at frequencies lower than 0 . 5 Hz . DOI: http://dx . doi . org/10 . 7554/eLife . 07886 . 016" ], [ "We tested the hypothesis that PAC reflects a phase-coding mechanism , measuring both PAC and categorical PR in intracranial recordings from six patients who viewed pictures from different categories .", "Our analyses show that on a large subset of electrodes showing PAC , the frequency-specific phase at which HFA occurs varies with categorical information .", "Therefore , to the extent that HFA reflects increases in local neuronal activity ( Crone et al . , 1998; Miller et al . , 2014 ) , our results suggest that neural representations for categories might occur by the phase at which neurons fire .", "These findings thus provide a novel link between PAC and phase-coded neural representations in humans .", "Critically , although PAC and phase-coded representations share some attributes , such as phase-clustering of activity , they are not necessarily identical processes .", "High frequency activity could occur at particular phases of LFOs , as reflected by PAC , but these phases may not vary with stimulus category ( Figure 1B , left ) .", "In other words , there could be PAC without phase coding .", "This is in fact the null hypothesis we have tested and would manifest as PAC with DSs of zero .", "On the other hand , categorical information may be represented by specific low-frequency phases independent of HFA , leading to DS without phase-clustering across HFA events ( PAC; Figure 1B , right ) .", "We did not find a complete overlap between PAC+ and phase-coding electrodes , indicating that each can occur in isolation , but instead found a compromise between these extremes ( Figure 1B , middle ) .", "These results suggest that PAC in many cases reflects phase coding because of the significant overlap between the two phenomena ( Video 1 ) .", "Phase-coding , in the form of phase-modulated neuronal firing , has been identified in rodents , monkeys , and humans ( O'Keefe and Recce , 1993; Skaggs et al . , 1996; Jacobs et al . , 2007; Kayser et al . , 2009; Rutishauser et al . , 2010 ) .", "Although the mechanisms which guide such a neuronal phase preference remain poorly understood , previous studies have found enhanced PAC during learning and memory tasks ( Tort et al . , 2008; Axmacher et al . , 2010; Kendrick et al . , 2011; Friese et al . , 2013; Lega et al . , 2014 ) .", "Our findings provide a potentially unifying account of these observations , suggesting that PAC may be promoting the formation of phase-coded neural assemblies ( Canolty and Knight , 2010; Watrous and Ekstrom , 2014; Watrous et al . , 2015 ) .", "Follow-up studies will need to test this account of PAC as it relates to other putative roles for PAC ( Canolty and Knight , 2010; Voytek et al . , 2015 ) .", "While epilepsy is marked by increased synchronized neuronal activity which could potentially manifest as HFA or PAC , we believe several factors weigh against this interpretation .", "First , we only analyzed electrodes overlying putatively healthy tissue , typically from the hemisphere contralateral to the epileptic focus , as assessed by our clinical team .", "Electrodes showing epileptic spiking were systematically removed from our analysis and all analyzed trials were visually inspected for artifacts related to epilepsy .", "Next , the PAC metric allows for assessment of the modulatory signal .", "Visual inspection of these signals did not reveal a similarity to epileptic spikes ( Figure 2—figure supplement 1 ) .", "Finally , it seems unlikely that epileptic activity at different phases would systematically differ by category .", "Similar reasoning excludes saccade-related artifacts ( Yuval-Greenberg et al . , 2008; Kovatch et al . , 2011 ) as a parsimonious account of our results .", "We therefore conclude that similar findings would translate into healthy human populations .", "Another caveat is that our results provide evidence for categorical phase-coding based on a restricted image set .", "This was necessary in the present study to maximize the chances of identifying category-selective responses while still ensuring that these responses were generalizable across a few exemplars .", "Follow-up studies should test the generalizability of these findings using more exemplars within a category and using other categories .", "PAC has typically been investigated using pre-defined low and high-frequency filters which may optimize statistical power for detecting PAC but do not adequately deal with the time-resolved nature of cognition ( Aru et al . , 2015 ) .", "Here , we leveraged a recent method which can identify PAC and subsequently test mechanistically interesting questions related to the modulation of HFA , such as its temporal profile and its dependence on phase , frequency , and behavioral requirements .", "Notably , this method may conservatively estimate PAC because it is based on transient increases in HFA , which do not necessarily occur in all cases of PAC .", "Our findings demonstrate that PAC and large HFA events can be identified and subsequently linked to categorically distinct representations .", "These results thus extend previous research which has decoded neural representations using either low or high frequency activity ( Jacobs and Kahana , 2009; Schyns et al . , 2011; van Gerven et al . , 2013 ) and may provide new avenues for decoding the human representational system .", "Intriguingly , phase-coding of categorical information extended beyond brain areas associated with higher-order vision .", "Thus , our findings of category-specificity do not appear to exclusively relate to perception but may also involve other more complex , and idiosyncratic , associations to these stimuli .", "Our findings are nonetheless in line with prior work ( Majima et al . , 2014; Yaffe et al . , 2014; Zhang et al . , 2015 ) which has found spatially-distributed content-specific representations .", "We identified frequency-specific PRs in humans , consistent with a growing body of evidence implicating the relevance of frequency-specific oscillatory activity to human cognition ( Daitch et al . , 2013; Watrous et al . , 2013; Fontolan et al . , 2014; Freudenburg et al . , 2014 ) .", "These findings are therefore consistent with models implicating frequency-specific oscillations as central to higher-order cognition ( Siegel et al . , 2012; Watrous and Ekstrom , 2014; Watrous et al . , 2015 ) .", "It has recently been shown that the frequency of LFOs contributes to several neuronal properties such that relatively slower LFOs lead to decreased firing threshold and increased spike timing variability ( Cohen , 2014 ) .", "It is not immediately clear how this relates to our finding that PAC predominantly occurs with modulating frequencies in the delta band , particularly around 1 Hz .", "It is possible that our findings reflect the activation of assemblies during ‘up’ states which show a similar frequency profile ( Destexhe et al . , 2007 ) or that the applied method of identifying peaks in the spectrum biased our findings to find PAC at lower frequencies .", "A third possibility , more likely in our view based on our results indicating multiple modulating frequencies per electrode ( Figure 2G ) , is that the timing of our task ( 1 image per second with a jittered inter-stimulus interval ) partially entrained slow oscillations forming an oscillatory hierarchy ( Lakatos et al . , 2005 ) .", "Similarly , our results showing PAC at a variety of phases and frequencies ( Maris et al . , 2011; van der Meij et al . , 2012 ) , particularly near 32 Hz , might reflect a form of ‘nested coupling’ ( Kopell et al . , 2010 ) distinct from ‘broadband’ high gamma , which has been suggested to reflect population spiking ( Manning et al . , 2009; Miller et al . , 2014 ) .", "Future research may clarify this issue by comparing single neuron activity and HFA modulation during different perceptual tasks and by investigating their relation to hierarchical cross-frequency coupling .", "To summarize , by identifying electrodes exhibiting both PAC and phase-coded neural representations for categories , our results employing direct brain recordings explicitly link phase-coupled neural activity to phase coding in humans ." ], [ "Six right handed patients with pharmacoresistant epilepsy ( mean age 31 . 8 years; 3 female ) participated in the study .", "All patients were stereotactically implanted for diagnostic purposes .", "Medial temporal depth electrodes ( AD-Tech , Racine , WI , USA ) with 10 cylindrical platinum-iridium contacts ( diameter: 1 . 3 mm ) were implanted in 1 patient , and 5 patients were implanted with subdural grid and strip electrodes with stainless-steel contacts ( diameter: 4 mm ) at temporal , frontal , and parietal sites ( Video 2 ) .", "Recordings were performed using a Stellate recording system ( Stellate GmbH , Munich , Germany ) at the Department of Epileptology , University of Bonn , Germany .", "The study was conducted according to the latest version of the Declaration of Helsinki and approved by the ethical committee of the medical faculty at the University of Bonn ( approval identifier 280/08 ) .", "All patients provided written informed consent to participate in the study and for the results to be published in a pseudonymized manner . 10 . 7554/eLife . 07886 . 017Video 2 . Electrode locations for each patient , rendered onto a glass brain of the average MNI template . Each point represents an electrode , and each color represents a different patient .", "Electrodes were primarily located in the temporal lobe . DOI: http://dx . doi . org/10 . 7554/eLife . 07886 . 017 Patients performed an object–location association task , though here we focus on neural representations for categories independent of memory encoding per se .", "Patients viewed greyscale images taken from four different categories ( houses , tools , scenes , and faces ) and each category had four unique stimuli , resulting in a stimulus set of 16 unique images .", "Example images from each category are shown in Figure 1A .", "Each image was presented 30 times in pseudo random order ( total of 480 trials ) and was followed by a white square in a fixed location .", "Patients were instructed to form object–location associations and to rate if they liked or disliked each image , thus ensuring that they were attending to each image presentation .", "Images were presented on a laptop placed in front of the patient .", "Each image was presented for 1 s , followed by the white square presented for 1 s , and finally a jittered inter-stimulus interval ranging from 1800–2200 ms . A fixation cross was presented between images .", "Intracranial EEG recordings ( sampled at 1000 Hz ) were referenced to linked mastoids and band-pass filtered ( 0 . 01 Hz [6 dB/octave] to 300 Hz [12 dB/octave] ) .", "Recordings from the hemisphere contralateral to the epileptogenic focus were analyzed .", "To boost our electrode sampling , an additional 32 electrodes from an ipsilateral left lateral temporal grid were included from patient 5 based on the physicians' report , which indicated a left hippocampal focus and no evidence of neocortical lesion based on an magnetic resonance imaging ( MRI ) .", "Signals from this grid were carefully visually inspected for artifacts and did not show increased artifacts associated with epilepsy .", "Qualitatively similar results were observed when excluding these electrodes from the analysis , with the proportions of electrodes showing any reported effect changing by no more than 3% .", "Electrode locations were determined by post-implantation MRI such that electrodes were mapped by co-registering pre- and post-implantation MRIs , normalizing the pre-implantation MRI and applying the normalization matrix to the post-implantation MRI .", "The anatomical locations of contacts were then identified by comparison with standardized anatomical atlases and using custom software ( published at http://pylocator . thorstenkranz . de/ ) .", "In total , 167 implanted electrode contacts were analyzed across all patients ( Video 2 ) .", "Raw EEG signals were extracted from 750 ms before to 1500 ms after image onset .", "EEG trials were visually inspected for artifacts ( e . g . , epileptiform spikes ) , and trials with artifacts were excluded from further analysis ( 15% of all trials on average ) .", "Trial epochs were then concatenated for subsequent analysis described below .", "We analyzed an average of 103 trials per category and subject and there were no differences in total number of trials analyzed across categories ( F ( 3 , 20 ) = 0 . 38 , p = 0 . 76 ) .", "PAC was detected using the methods described by Dvorak and Fenton ( 2014 ) .", "This method is conceptually similar to an event-locked analysis around periods of enhanced HFA .", "All analyses were conducted using standard routines in EEGLab ( Delorme and Makeig , 2004 ) and Matlab based on previously published algorithms ( Rizzuto et al . , 2006; Berens , 2009; Tort et al . , 2009; Dvorak and Fenton , 2014 ) .", "In brief , the power and phase of the signal on each electrode was computed in the low frequency ( Fphase , 0 . 5–12 Hz , 0 . 5 Hz steps ) and gamma ( Famp , center frequencies at 32–120 Hz , 4 Hz steps ) bands using Morlet wavelet convolution with 7 cycles .", "At each center HFA frequency , the time course of power values was z-scored and time periods exceeding the 95th percentile of these values were identified ( we refer to these as ‘HFA windows’ , red shaded area in Figure 2C ) .", "The time point of the largest power value within each window was identified and taken as the time-locking ‘HFA event’ for OTC analyses ( arrow , Figure 2C ) .", "Two-second segments ( 1 s before to 1 s after each HFA event ) of the raw signal were extracted around these timestamps and raw signal segments were summed at each time point across segments , resulting in the modulatory signal at each center HFA frequency .", "The strength of modulation was determined based on the peak to trough height of the modulatory signal .", "Surrogate modulatory signals ( n = 100 ) were constructed at each modulated frequency based on choosing an equal number of pseudo-HFA events at random timestamps and repeating the above procedure .", "Surrogate modulation strengths were extracted and used to z-score the observed modulation strength .", "PAC+ electrodes were identified as electrodes ( 1 ) with a modulation strength z-score >4 . 35 for at least one gamma frequency and ( 2 ) with a clear peak in the power spectrum of the raw signal at Fphase ( Aru et al . , 2015 ) .", "This z-score threshold was calculated by identifying the z value equivalent to a Bonferroni corrected ( across 23 gamma frequencies and 167 electrodes ) alpha threshold of p < 0 . 05 and corresponded to p < 0 . 00005 .", "Peaks were identified by normalizing the power spectrum of both the raw and modulatory signal to their respective maxima and ensuring that both normalized signals were maximal ( i . e . ,", "1 ) at the same frequency .", "We identified the peak HFA modulation frequency as the frequency with the largest z-score and extracted the modulatory signal ( see Figure 2B ) .", "The phase and frequency content of the modulatory signal was determined using a Hilbert transform and fast Fourier transform , respectively .", "The modulatory signal was mean-centered prior to FFT in order to remove DC components .", "The maximum ( ‘FMAX’ ) and minimum ( ‘FMIN’ ) of this FFT output indicate the strongest and weakest slow-modulating frequencies in the 0 . 5–12 Hz band , respectively .", "Phase comparisons were conducted using a Watson Williams test following Rizzuto et al ( 2006 ) and using code taken from these eegtoolbox available at ( http://memory . psych . upenn . edu/Software ) .", "Statistical testing was performed between the phases extracted during HFA windows for all pairs of conditions ( 4 categories; 6 total category pairs ) .", "DSs were computed for each category as the total number of significant differences ( p < 0 . 001 ) between the phase distribution for one category and the remaining categories and thus ranged from 0 ( no difference to any other category ) to 3 ( significant difference to all other categories ) .", "Phase clustering scores were defined as the resultant vector length for each category's phase distribution .", "We used a pattern classification approach for comparison with our DS metric , classifying image category based on the phase at which HFA events occur .", "To this end , we trained support vector machines using a linear kernel and fivefold cross-validation .", "Phase values at Fmax were extracted at moments in time when HFA events occurred during image presentation and were used as input features for the classifier .", "Similar to previous approaches ( Lopour et al . , 2013; Majima et al . , 2014 ) , the sine and cosine of the phase values were used as input features for phase .", "Classifiers were run separately on each electrode and the classifier output was a prediction of the category label for each HFA event .", "Classification accuracy was defined as the average proportion of correctly classified HFA events across folds .", "Chance classification performance varies across electrodes because we classified the category label associated with each HFA event and the number of HFA events per category varied across electrodes .", "We thus opted to report significance based on permutation testing which accounts for the varying chance level across electrodes and assessed the significance of classification using two separate analyses which both utilized permutation testing .", "First , we randomized the category labels associated with HFA events and assessed classification accuracy .", "Second , we used random time points ( also corresponding to random phases ) as surrogate HFA events and assessed classification accuracy .", "Each type of permutation test was performed 50 times , resulting in a distribution of pseudo classification accuracy values for each test .", "Observed classification accuracies at or above the 95th percentile of each of these distributions were deemed significant .", "Thus , we fixed the type 1 error rate for each test at 5% and , assuming independence between tests , we would therefore expect 0 . 0025 × 167 = 0 . 42 electrodes to show significance by chance for both permutation tests ." ] ]

[ "Prior studies have shown that high-frequency activity ( HFA ) is modulated by the phase of low-frequency activity .", "This phenomenon of phase-amplitude coupling ( PAC ) is often interpreted as reflecting phase coding of neural representations , although evidence for this link is still lacking in humans .", "Here , we show that PAC indeed supports phase-dependent stimulus representations for categories .", "Six patients with medication-resistant epilepsy viewed images of faces , tools , houses , and scenes during simultaneous acquisition of intracranial recordings .", "Analyzing 167 electrodes , we observed PAC at 43% of electrodes .", "Further inspection of PAC revealed that category specific HFA modulations occurred at different phases and frequencies of the underlying low-frequency rhythm , permitting decoding of categorical information using the phase at which HFA events occurred .", "These results provide evidence for categorical phase-coded neural representations and are the first to show that PAC coincides with phase-dependent coding in the human brain ." ]

[ "Electrocorticography , or ECoG , is a technique that is used to record the electrical activity of the brain via electrodes placed inside the skull .", "This electrical activity repeatedly rises and falls , and can therefore be represented as a series of waves .", "All waves have three basic properties: amplitude , frequency and phase .", "Amplitude describes the height of a wave's peaks ( and the depth of its troughs ) , and frequency defines how many waves are produced per second .", "The phase of a wave changes from 0° to 360° between two consecutive peaks of that wave and then repeats , similar to the phases of the moon .", "Previous studies have shown that brain activity at different frequencies can interact .", "For instance , neural firing ( when nerve impulses are sent from one neuron to the next ) is related to ‘high frequency activity’; and the amplitude of high frequency activity can be altered by the phase of other , lower frequency brain activity .", "It has been suggested that this phenomenon , called ‘phase-amplitude coupling’ , might be one way that the brain uses to represent information .", "This ‘phase coding’ hypothesis has been demonstrated in rodents but is largely untested in humans .", "Now , Watrous et al . have explored this hypothesis in epilepsy patients who had ECoG electrodes implanted in their brains for a diagnostic procedure before surgery .", "These electrodes were used to record brain activity while the patients viewed images from four different categories ( houses , scenes , tools and faces ) .", "Watrous et al . found that phase-amplitude coupling occurred in over 40% of the recordings of brain activity .", "The analysis also revealed that the phase of the lower frequency activity at which the high frequency activity occurred was different for each of the four image categories .", "This provides support for the phase-coding hypothesis in humans .", "Furthermore , it suggests that not only how much neural firing occurs but also when ( or specifically at what phase ) it occurs is important for how the brain represents information .", "Future studies could now build on this analysis to see if phase-amplitude coupling also supports phase coding and neural representations in other thought processes , such as memory and navigation ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "short report", "structural biology and molecular biophysics", "microbiology and infectious disease" ]

[ [ "Plasmodium falciparum is the causative agent of the most severe form of malaria with over 700 , 000 deaths each year , mostly in sub-Saharan Africa .", "The asexual blood cycle of this parasite begins with the invasion of human erythrocytes by the merozoite form of P . falciparum in a complex multistep process involving a cascade of protein–protein interactions between the parasite and host cell ( reviewed in Cowman and Crabb , 2006 ) .", "This process requires members of the reticulocyte binding-like homologues ( PfRh or PfRBP ) and erythrocyte binding-like ( EBL ) ligand families .", "PfRh5 is a member of the PfRh family and binds specifically to the receptor basigin on the human erythrocyte surface ( Crosnier et al . , 2011 ) .", "This protein plays an essential role in merozoite invasion ( Baum et al . , 2009 ) and host tropism of P . falciparum ( Wanaguru et al . , 2013 ) .", "Polymorphisms in PfRh5 can convert a non-virulent Plasmodium falciparum parasite into a virulent form upon infection of Aotus monkeys , supporting the view that this ligand is a determinant of virulence and host specificity ( Hayton et al . , 2008 ) .", "PfRh5 has distinct characteristics suggesting that it plays a different role to other members of the family .", "In particular , PfRh5 is a much smaller protein ( ∼60 kDa compared to the average of ∼300 kDa for the family ) and lacks a transmembrane region .", "It forms a complex with the cysteine-rich protein PfRipr during merozoite invasion; the complex is peripherally associated with parasite membranes and is released at the apical end of the merozoite during invasion of the human erythrocyte ( Chen et al . , 2011 ) .", "Antibodies to PfRh5 can block merozoite invasion , suggesting that it is a potential vaccine candidate ( Douglas et al . , 2011; Williams et al . , 2012; Patel et al . , 2013; Reddy et al . , 2014 ) .", "This is supported by clinical data showing that antibodies to PfRh5 are associated with protection against malaria , indicating that PfRh5 may be a component of acquired protective immunity ( Chiu et al . , 2014; Tran et al . , 2014 ) .", "To provide a molecular basis for understanding the function of PfRh5 , we have determined the crystal structure of PfRh5 using diffraction data to 2 . 18 Å resolution , the first three-dimensional structure in PfRh protein family .", "We show that it exhibits a novel fold ." ], [ "Initially , the full-length 60-kDa protein was expressed in insect cells .", "Although the recombinant protein was capable of binding red blood cells , it was unstable , with the N-terminal region proteolytically degraded to yield a 48-kDa fragment that had a higher erythrocyte binding affinity than that of the full-length protein ( Figure 1—figure supplement 1 ) .", "We determined the N-terminal amino acid sequence by mass spectrometry and re-expressed this region in insect cells to produce a highly stable module of PfRh5 that we denote PfRh5-C ( Figure 1A ) .", "PfRh5-C likely reflects the 45-kDa processed form present in P . falciparum and released into culture supernatant during merozoite invasion ( Baum et al . , 2009 ) . 10 . 7554/eLife . 04187 . 003Figure 1 . Production of functional recombinant PfRh5 . ( A ) Purified recombinant PfRh5 was analysed by SDS-PAGE analyses and by erythrocyte binding assays .", "( B ) Formation of the PfRh5–basigin complex was monitored by size-exclusion chromatography .", "The chromatographic profiles are shown for PfRh5 ( panel 1 ) , basigin ( panel 2 ) , and the PfRh5-basigin complex ( panel 3 ) .", "The fractions eluted from the column in panel 3 were analysed by SDS-PAGE .", "( C ) The binding affinity of the recombinant PfRh5 to human basigin was measured by SPR on Biacore 3000 with the basigin coupled to a sensor chip .", "( D ) In vitro growth inhibition assays were performed to assess the abilities of the polyclonal antibodies to the recombinant PfRh5 in blocking P . falciparum parasite invasion into erythrocytes . DOI: http://dx . doi . org/10 . 7554/eLife . 04187 . 00310 . 7554/eLife . 04187 . 004Figure 1—figure supplement 1 . Production of full-length PfRh5 . ( A ) SDS-PAGE analyses of the FLAG-tagged recombinant full-length PfRh5 eluted from an anti-FLAG affinity column .", "( B ) Size-exclusion chromatography analyses of the anti-FLAG bead affinity purified PfRh5 .", "The fractions eluted from the affinity column were pooled , concentrated , and stored at 4°C for 2 days before the analyses .", "( C ) Red blood cell binding assay with PfRh5 purified by anti-FLAG bead affinity chromatography .", "Both full-length protein and the breakdown fragments bound red blood cells with a 48-kDa species having the highest affinity . DOI: http://dx . doi . org/10 . 7554/eLife . 04187 . 00410 . 7554/eLife . 04187 . 005Figure 1—figure supplement 2 . PfRh5 and human basigin form a 1:1 complex . PfRh5-C and basigin were cross-linked with EDC in the presence of NHS .", "The mixture was then analysed on a SDS-PAGE gel and the band of the crosslinked complex was excised for trypsin/chymotrypsin digestion followed by mass spectrometric analysis . DOI: http://dx . doi . org/10 . 7554/eLife . 04187 . 005 To show that PfRh5-C is functional , we demonstrated that it could bind red blood cells and the receptor human basigin , which is also produced in insect cells ( Figure 1A ) .", "PfRh5-C formed a stable complex with basigin as evidenced by size-exclusion chromatographic analysis .", "In these experiments , PfRh5-C was incubated with excess basigin and the stable PfRh5-basign complex eluted ahead of free PfRh5 and basigin ( Figure 1B ) .", "The stoichiometry of the complex was shown to be 1:1 by chemical cross-linking ( Figure 1—figure supplement 2 ) .", "The binding affinity of the PfRh5-C–basigin interaction was determined by surface plasmon resonance ( SPR ) to be KD = 43 . 4 nM ( Figure 1C ) .", "This KD value is higher than that previously reported ( Crosnier et al . , 2011 ) .", "We note that while the PfRh5 sample used for the SPR measurement was prepared in monomeric form by gel-filtration chromatography , it is possible that a dynamic equilibrium with oligomeric forms within the sample has contributed to the higher affinity measurement .", "Antibodies to the recombinant PfRh5-C block growth of 3D7 and W2mef strains of P . falciparum at levels comparable to previous studies ( Douglas et al . , 2011; Bustamante et al . , 2013; Douglas et al . , 2014; Reddy et al . , 2014 ) ( Figure 1D ) .", "Taken together , these data imply that PfRh5-C is functionally competent .", "PfRh5-C was crystallized and its structure determined through single-wavelength anomalous diffraction ( SAD ) phasing using iodine-derivatized crystals with subsequent refinement against native diffraction data to a resolution of 2 . 18 Å ( Figure 2—source data 1 ) .", "The shape of PfRh5-C approximates an elliptical disk ( Figure 2 ) , the core consisting of nine mostly anti-parallel α-helices that encase a small β-hairpin located near the N-terminus ( Figure 2 and Figure 2—figure supplement 1 ) .", "We denote these nine helices α1 , α2a , α2b , α3a , α3b , α4 , α5 , α6 , and α7 , where the numeric order indicates progression from N- to C-terminus along the polypeptide and the a and b suffixes indicate that α2a and α2b as well as helices α3a and α3b arise from breaks in the canonical ( i , i+4 ) hydrogen bonding pattern of longer ‘parent helices’ α2 and α3 , respectively . 10 . 7554/eLife . 04187 . 006Figure 2 . The crystal structure of PfRh5 . ( A ) Ribbon representation of the PfRh5 structure .", "Color scheme is rainbow ( N-terminus: blue; C-terminus: red ) except for the β-hairpin that is colored magenta for clarity .", "The cysteine residues that form disulfide bridges ( Cys345–Cys351 and Cys224–Cys317 ) are shown as spheres .", "Helices α4 , α5 , α6 , and α7 assemble as a triplet-helical coiled-coil domain running the length of the long axis of the molecule , helices α1 , α2a , and α3b assemble to form a short triplet-helical bundle and helices α2b and α3a assemble to form a short two-helix coiled coil .", "( B ) Ribbon representation of the PfRh5 structure viewed after a 180o rotation relative to that in ( A ) .", "The residues between Ser257 and Asp294 are disordered .", "( C ) Ribbon representation of the PfRh5 structure viewed from the side with the C-terminus on the left .", "The disulfide bridges are indicated with arrows .", "( D ) Ribbon representation of the PfRh5 structure viewed after a 180o-rotation relative to that in ( C ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 04187 . 00610 . 7554/eLife . 04187 . 007Figure 2—source data 1 . Data collection and refinement statistics of Rh5 and Rh5_KI . DOI: http://dx . doi . org/10 . 7554/eLife . 04187 . 00710 . 7554/eLife . 04187 . 008Figure 2—figure supplement 1 . The secondary structure of PfRh5 . Density for the N-terminal residues Asp127–Leu145 , loop residues Glu258–Asn293 , and C-terminal residues Met512–Gln526 was not observed .", "The helices and β-strands are colored red and green respectively .", "The vertical line at residue 119 and 319 indicates the position of a helical break . DOI: http://dx . doi . org/10 . 7554/eLife . 04187 . 00810 . 7554/eLife . 04187 . 009Figure 2—figure supplement 2 . Two free cysteine residues ( Cys203 on α2a and Cys329 on α3b ) within the crystal structure of PfRh5 . The side chain of Cys329 is completely buried inside the three-dimensional fold of PfRh5 and the side chain of Cys203 is only partly exposed , consistent with the fact that no intermolecular disulfide bond was observed for the native and recombinant PfRh5 . DOI: http://dx . doi . org/10 . 7554/eLife . 04187 . 00910 . 7554/eLife . 04187 . 010Figure 2—figure supplement 3 . Superimposition of the PfRh5 structure with N-terminal coiled-coil domain of SipB . The PfRh5 ( green ) coiled-coil helix bundle formed by helices α5 , α6 , and α7 has a very similar fold to the N-terminal coiled-coil domain of SipB ( magenta ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 04187 . 01010 . 7554/eLife . 04187 . 011Figure 2—figure supplement 4 . A unique pocket on the surface of the PfRh5 molecule .", "( A ) Location of the pocket on the surface formed by the β-hairpin , the triple-helical bundle , and the triple-helical coiled coil .", "( B ) Close-up view of the pocket; key residues lining the pocket are labeled . DOI: http://dx . doi . org/10 . 7554/eLife . 04187 . 011 Helices α4 , α5 , α6 , and α7 assemble as a triplet-helical coiled-coil domain running the length of the long axis of the molecule .", "On the opposite side to the α4/α5/α6/α7 coiled-coil domain , helices α1 , α2a , and α3b assemble to form a short three-helix bundle and helices α2b and α3a assemble to form a short two-helix coiled-coil domain , these domains being approximately half the length of the α4/α5/α6/α7 coiled-coil domain .", "The ‘absence’ of a third helix to the α2b/α3a coiled-coil is necessary to accommodate the small β-hairpin formed by residues 161–175 in the overall tertiary structure .", "Within each of the three-helical domains , the central pairwise interactions between the constituent helices are overwhelmingly hydrophobic in nature .", "In contrast , the interactions between the helical domains and the β-hairpin and between the α4/α5/α6/α7 coiled-coil domain and the α1/α2a/α3b bundle domain are of mixed hydrophilicity .", "Inspection shows these interfaces to be relatively loosely packed .", "There are two disulfide bonds within the structure .", "The first disulphide bond is Cys345–Cys351 , located at one apex of the helical bundle .", "Cys345 lies at the C-terminal end of helix α3b and Cys351 at the N-terminal end of helix α4 .", "The second disulphide bond is Cys224–Cys317; Cys224 lies close to the N-terminus of helix α2b and Cys317 close to the C-terminus of helix α3a , that is , in proximity to ‘kink points’ of the parent helices α2 and α3 .", "A large loop ( residues 240-297 ) , located at the opposite apex of molecule to the Cys345–Cys351 disulphide bond , interconnects helices α2b and α3a ( Figure 2A ) .", "Electron density for residues 258–293 is absent ( Figure 2—figure supplement 1 ) , indicating partial disorder of this loop within the crystal structure .", "Two free cysteine residues ( Cys203 in α3 and Cys329 in α4 ) occur within the structure; the side chain of the Cys329 residue is completely buried , whereas that of the Cys203 is only partly exposed ( Figure 2—figure supplement 2 ) .", "Burial of these sulfhydryls is consistent with the monomeric nature of the native protein isolated from parasites and of the recombinant protein described here .", "A search using DALI ( Holm and Rosenstrom , 2010 ) indicates that the PfRh5 fold is novel .", "The only element of PfRh5-C found to have a structural homologue within the Protein Data Bank ( PDB ) is the coiled-coil domain formed by helices α5 , α6 , and α7 ( Figure 2 ) : this element can be superimposed with a root mean square deviation of all backbone atoms of 3 . 4 Å on the N-terminal coiled-coil domain ( residues 82–226 ) of the SipB protein of the bacterial type III secretion system ( TTSS ) of Salmonella enterica ( PDB entry 3TUL , chain A ) ( Barta et al . , 2012 ) ( Figure 2—figure supplement 3 ) .", "Analyses of molecular surface using DoGSiteScorer ( Volkamer et al . , 2012 ) detected a number of pockets of dimensions suitable for targeting with small molecules on the surface of the PfRh5 structure .", "These pockets arise from the relatively loose packing of the four constituent domains of the PfRh5 .", "One of these ( Figure 2—figure supplement 4A ) is lined by residues of mixed hydrophilicity and has a surface area of ∼430 Å2 , of which ∼60% is lipophilic ( Figure 2—figure supplement 4B ) .", "If this pocket is in proximity to the basigin binding site , small molecules targeting it may have the potential to interfere the interaction between the two molecules , either through disrupting the relatively loose packing of the constituent domains of PfRh5 or through steric interference .", "Alternatively , as PfRh5 functions in complex with PfRipr and at least one further parasite protein ( Chen et al . , 2011 ) , if this pocket is involved in binding these partner/s , a small molecule targeting this pocket may likewise interfere with the complex formation and therefore ultimately with its function .", "To explore the structure–function relationship of PfRh5 , we examined the relevance of the disulphide bonds and cysteine residues for the function of PfRh5 by reducing and alkylating PfRh5-C followed by measuring the binding affinity of the modified protein to basigin using surface plasmon resonance ( Figure 3A ) .", "The reduced and alkylated PfRh5-C has an affinity for basigin with KD = 127 nM .", "A threefold reduction in affinity as compared to untreated PfRh5-C ( KD = 43 . 4 nM ) was consistent with the two disulphide-bonds being important for the stability of the overall fold of the protein rather than being directly involved in basigin binding . 10 . 7554/eLife . 04187 . 012Figure 3 . Binding of mutant PfRh5 to basigin .", "( A ) The binding affinity of the reduced and alkylated PfRh5 to basigin measured by SPR .", "( B ) The binding affinity of the PfRh5 mutant , in which the disordered loop has been deleted , to basigin measured by SPR . DOI: http://dx . doi . org/10 . 7554/eLife . 04187 . 012 We also investigated whether the disordered region of 35 amino acids ( Glu258–Asn293 ) ( Figure 2—figure supplement 1 ) is involved in the binding of PfRh5 to human basigin .", "A mutant form of PfRh5-C lacking amino acids Asp261–Asn289 ( Figure 2—figure supplement 1 ) was produced in insect cells; its binding affinity for basigin was determined by SPR to be 25 . 9 nM ( Figure 3B ) , that is , comparable to that of the non-mutated PfRh5 .", "These data suggest that the disordered region ( Glu258–Asn293 ) is not involved in receptor binding ." ], [ "Invasion of P . falciparum merozoite into human erythrocytes involves several ligand–receptor interactions including the PfRh family of proteins , which are important in binding to and identifying the appropriate host cell for invasion ( reviewed in Cowman and Crabb , 2006 ) .", "Whilst PfRh5 is a member of the PfRh family , it appears to have distinct functions to other family members and it plays an essential role for invasion ( Baum et al . , 2009; Crosnier et al . , 2011 ) .", "To provide a structural basis for understanding the function of this protein , we determined its three-dimensional structure , the first crystal structure in the PfRh protein family .", "PfRh5 exhibits a novel fold comprising three-helical domains surrounding a small β-hairpin .", "PfRh5 appears to function as a multi-protein complex .", "We have previously identified one partner , PfRipr , for the complex ( Chen et al . , 2011 ) .", "The PfRh5–PfRipr complex is essential for merozoite invasion as the genes encoding both proteins cannot be disrupted and antibodies to both inhibit this process ( Baum et al . , 2009; Chen et al . , 2011 ) .", "Consequently , both proteins are potential vaccine candidates and worth consideration for a combination vaccine because specific antibodies to both molecules would inhibit the same functional process and likely to be at least additive and potentially synergistic .", "The structural similarity of the larger PfRh5 coiled-coil domain with the N-terminal coiled-coil domain of SipB may potentially provide an indication of the function of the complex .", "SipB forms part of the Salmonella type III secretion system ( TTSS ) that is responsible for transport of bacterial effector proteins across the host cell membrane .", "Currently , there is no direct evidence that the PfRh5–PfRipr complex is involved in transport of proteins or molecules across the erythrocyte membrane during invasion .", "Nevertheless , it is interesting that P . falciparum does inject proteins , including members of the RON complex , into the erythrocyte during merozoite invasion and these proteins are required for formation of the tight junction that bring the parasite membrane and host membrane together in a tight interaction through binding of RON2 to apical membrane antigen-1 ( AMA-1 ) ( Narum et al . , 2008; Richard et al . , 2010; Riglar et al . , 2011 ) .", "By analogy to the bacterial type III secretion system , the PfRh5–PfRipr complex may play a role in transfer of components such as the RON complex to the host cell during merozoite invasion .", "In summary , this work has elucidated the structure of PfRh5 and may provide a model for the remainder of the PfRh family members .", "During the review process of this manuscript , a study was published reporting the structure of the Rh5–basigin complex ( Wright et al . , 2014 ) .", "In contrast , our work describes the structure of Rh5 not bound to the receptor basigin .", "Comparison of the Rh5 structure in the bound and unbound state shows that there are no changes in the core structure on binding of receptor .", "A pocket identified on the surface of PfRh5 may provide an opportunity for development of a new anti-malaria drug .", "Structural similarity with SipB has provided the tantalizing possibility that PfRh5–PfRipr complex may play a similar role to the TTSS system of bacteria that secrete effector proteins into the host cell .", "However , further evidence will be required to support such speculation ." ], [ "A synthetic gene encoding Plasmodium falciparum ( 3D7 ) full-length mature PfRh5 ( residues 24–526 ) , PfRh5-C ( residues 127–526 ) , or its mutant was inserted into insect/mammalian cell expression vector pgpHFT ( Xu et al . , 2010 ) using Kpn I and Xho I sites to produce pgpHFT-PfRh5 .", "The pgpHFT-PfRh5 was then co-transfected with FlashBAC ( Oxford Expression Technologies ) into Sf21 insect cells as per supplier's manual .", "The seed virus was amplified to obtain high-titer viral stocks , which were then used to infect Hi5 cells grown in express Five SFM medium ( Life Technologies Pty Ltd , Australia ) supplemented with 1 mM glutamine .", "The supernatant containing the secreted recombinant protein was harvested , centrifuged , and passed over anti-FLAG M2 agarose ( Sigma-Aldrich , Australia ) column .", "After extensive washing , bound proteins were eluted from the column with the FLAG peptide at a concentration of 100 μg/ml , concentrated and further purified by size-exclusion chromatography with a Superdex 200 column ( GL 10/300 , GE Healthcare , Australia ) in 50 mM Tris , 100 mM NaCl , pH 8 . 5 .", "For crystallization of PfRh5 , the tandem 6xHis and FLAG tags were removed by digestion with a TEV protease and the pure protein was recovered by Ni-resin and/or size-exclusion chromatography purification .", "Human basigin isoform 2 ( BSG-S ) ( Crosnier et al . , 2011 ) was also expressed in insect cells and purified as described for PfRh5 .", "Crystallization trials were performed in sitting drop within a 96-well format at 8°C .", "Crystals were obtained from drops containing 8–12% PEG3350 or PEG4000 and 0 . 2 M DL-malate-imidazole , pH 6 . 5-7 . 5 .", "X-ray diffraction data were collected on beamline MX2 at the Australian Synchrotron .", "For the phase determination , derivative crystals were prepared by quick-soaking native crystals in potassium iodide ( KI ) solutions prepared in cryoprotection solution for 1–5 min and data collected at the K-edge of 1 . 55 Å .", "Diffraction data were processed and scaled with XDS ( Kabsch , 2010 ) .", "Diffraction data were included to a maximum resolution of 2 . 18 Å based on significance of the CC1/2 criterion at the p = 0 . 001 level of significance ( Karplus and Diederichs , 2012 ) .", "Three iodine binding sites were found using SOLVE ( Terwilliger and Berendzen , 1999 ) ; phases were then improved using RESOLVE ( Terwilliger , 2000 ) .", "The initial model generated by the RESOLVE autobuild utility comprised only a partial set of the nine helices .", "Model building and refinement then continued with PHENIX ( Adams et al . , 2010 ) , with automated building and morphing routines leading to a model comprising approximately 50% of the PfRh5 sequence .", "Further rounds of refinement and manual rebuilding were undertaken using REFMAC5 ( Vagin et al . , 2004 ) and COOT ( Emsley et al . , 2010 ) .", "The final refinement was undertaken with AutoBUSTER ( v2 . 10 . 0 ) ( Bricogne et al . , 2011 ) .", "Data processing and refinement statistics are in Figure 2—source data 1 .", "Rabbits were immunised three times with 200 μg PfRh5 in Freund's adjuvant .", "IgG was purified from serum , concentrated and dialysed against RPMI-Hepes for growth inhibition assays .", "One cycle growth inhibition assay was performed as described ( Healer et al . , 2013 ) .", "Serial dilutions of IgG in RPMI-HEPES , starting at 10 mg/ml were added to P . falciparum-infected RBC ( 3D7 and W2mef ) at a parasitaemia of 0 . 5% .", "Parasitaemia was counted after 48 hr and specific growth inhibition calculated relative to parasites grown in non-immune IgG .", "Erythrocyte binding assays were performed as described previously ( Triglia et al . , 2001 ) .", "To prepare and analyse the PfRh5/basigin complex , two anti-flag affinity beads purified proteins were mixed , incubated at 4°C overnight and loaded to a Superdex 200 size-exclusion chromatography column in 50 mM Tris , 150 mM NaCl , pH 8 . 5 or 25 mM HEPES , 150 mM NaCl , pH 7 . 2 .", "The complex eluted from the column was collected for analysis .", "Affinity ( KD ) measurements were performed at room temperature on a Biacore 3000 Biosensor with HBS ( 10 mM HEPES pH 7 . 2 , 150 mM NaCl , 3 . 4 mM EDTA , 0 . 005% Tween 20 ) as the running buffer ( Chen et al . , 2011 ) .", "The human basigin was immobilized onto a CM5 sensorchip using amine-coupling ( EDC/NHS ) chemistry .", "PfRh5 or its mutants ( purified by anti-FLAG M2 agarose beads followed by gel-filtration chromatography ) were injected at 20 µl/min into the sensorchip containing a channel immobilized with basigin .", "A blank channel was used as control .", "After each injection , the chip was regenerated with 2 M NaCl supplemented with 3 mM NaOH in the running buffer , followed by two washes with the running buffer .", "Affinity ( KD ) was derived from sensorgrams , following subtraction of baseline responses , using BIA evaluation software ( version 4 . 1: Biacore Life Sciences , GE Healthcare , Australia ) .", "To an aliquot of 50 µl at 1 mg/ml PfRh5 was added DTT to a final concentration of 2 mM and incubated at room temperature for 2 hr .", "Iodoacetamide was then added to a final concentration of 10 mM , incubated at room temperature for 2 hr and then left at 4°C overnight .", "For SPR experiment , the excess DTT and iodoacetamide were removed by a desalting column equilibrated and eluted with HEPES buffer , pH 7 . 4 containing 150 mM NaCl .", "For cross-linking of PfRh5 to Basigin , to approximately 50 µg of purified PfRh5/Basigin complex in 25 mM MES , 150 mM NaCl , pH 7 . 4 was added 1-ethyl-3- ( dimethylaminopropyl ) carbodiimide ( EDC ) and N-hydroxysuccinimide ( NHS ) to a final concentration of 2 mM .", "The reaction was allowed to occur at room temperature for 30 min before quenching with 100 mM Tris , pH 8 . 0 .", "The cross-linked sample was then analysed on a SDS-PAGE gel ." ] ]

[ "Plasmodium falciparum causes the most severe form of malaria in humans and is responsible for over 700 , 000 deaths annually .", "It is an obligate intracellular parasite and invades erythrocytes where it grows in a relatively protected niche .", "Invasion of erythrocytes is essential for parasite survival and this involves interplay of multiple protein–protein interactions .", "One of the most important interactions is binding of parasite invasion ligand families EBLs and PfRhs to host receptors on the surface of erythrocytes .", "PfRh5 is the only essential invasion ligand within the PfRh family and is an important vaccine candidate .", "PfRh5 binds the host receptor basigin .", "In this study , we have determined the crystal structure of PfRh5 using diffraction data to 2 . 18 Å resolution .", "PfRh5 exhibits a novel fold , comprising nine mostly anti-parallel α-helices encasing an N-terminal β-hairpin , with the overall shape being an elliptical disk .", "This is the first three-dimensional structure determined for the PfRh family of proteins ." ]

[ "Malaria is a disease caused by a single-celled parasite called Plasmodium , which is transmitted between humans by mosquitoes .", "It is estimated that 3 . 4 billion people worldwide live in regions where they are at risk of malaria , and malaria infections cause hundreds of thousands of deaths each year .", "When a mosquito carrying Plasmodium parasites in its salivary glands bites a human , the parasite is injected into the person's bloodstream with the mosquito's saliva .", "The parasite then travels through the bloodstream to the liver , where it infects liver cells and multiplies without causing any symptoms for up to 4 weeks .", "After this period , the parasites break out of each infected liver cell , re-enter the bloodstream , and begin infecting red blood cells .", "When another mosquito bites the infected individual to feed on their blood , the parasite moves into the mosquito with the red blood cells and the cycle of infection continues .", "While prevention and control measures have dramatically reduced the incidence of malaria in some countries , many people in African countries—and especially young children—die from malaria each year .", "Finding ways to reduce the spread of Plasmodium parasites , and in particular Plasmodium falciparum ( which is responsible for the deadliest type of malaria ) , is critical for the global effort to control and eliminate this disease .", "As such , many researchers are trying to gain a better understanding of how the parasite both invades host cells and evades the immune system .", "In this study , Chen et al . reveal the high-resolution structure of PfRh5 , the protein from Plasmodium falciparum that forms a complex with other proteins to allow the parasite to bind to , and invade , red blood cells .", "This is one of the first three-dimensional structures that have been uncovered for this family of proteins—and reveals that the PfRh5 protein is shaped like an elliptical disk .", "Solving the structure of PfRh5 is the first step in understanding the role of this protein , and the other protein components , involved in invading red blood cells .", "These proteins are molecules that could potentially be used to vaccinate people against malaria , and understanding these proteins' functions will help efforts to design vaccines to prevent malarial disease ." ]

[ "Introduction", "Materials and methods", "Results", "Discussion" ]

[ "ecology", "epidemiology and global health" ]

[ [ "While it is well known that predation reduces vector populations through consumptive effects , non-consumptive effects of predators can also greatly impact prey demographics ( Preisser et al . , 2005 ) .", "Mosquitoes are vectors of a variety of debilitating and deadly diseases , including malaria , lymphatic filariasis , and arboviruses , such as chikungunya , Zika , and dengue ( Weaver and Reisen , 2010; WORLD HEALTH ORGANIZATION , 2020 ) .", "Consequently , there is motivation from a public health perspective to better understand the different drivers of variation in mosquito traits that can ultimately impact vector population growth and disease transmission .", "In addition , recent work has suggested that incorporation of vector trait variation into disease models can improve the reliability of their predictions ( Cator et al . , 2020 ) .", "In this study , systematic review and meta-analysis methods are used to synthesize a clearer understanding of the consumptive and non-consumptive effects of predators on mosquito traits , including survival , oviposition , development , and size .", "Mosquito insecticide resistance is recognized as a growing problem ( Hancock et al . , 2018; Hemingway and Ranson , 2000; Liu , 2015 ) leading some to suggest that control efforts should rely more heavily on ‘non-insecticide based strategies’ ( Benelli et al . , 2016 ) .", "The consumptive effects of predators on mosquitoes have previously been harnessed for biocontrol purposes .", "Past biocontrol efforts have used predators such as cyclopoid copepods ( Kay et al . , 2002; Marten , 1990; Russell et al . , 1996; Veronesi et al . , 2015 ) and mosquitofish ( Pyke , 2008 , Seale , 1917 ) to target the mosquito’s aquatic larval stage .", "The strength of the consumptive effects of these predators on mosquitoes can be influenced by multiple factors , including predator-prey size ratio and temperature .", "Predator-prey body size ratios tend to be higher in freshwater habitats than other types of habitats ( Brose et al . , 2006 ) , and attack rate tends to increase with temperature ( Kalinoski and DeLong , 2016; Dam and Peterson , 1988 ) , although other studies suggest a unimodal response to temperature ( Uiterwaal and Delong , 2020; Englund et al . , 2011 ) .", "Predators can also have non-consumptive effects on prey ( Peacor and Werner , 2001 ) , and these effects are thought to be more pronounced in aquatic ecosystems than in terrestrial ecosystems ( Preisser et al . , 2005 ) .", "Non-consumptive effects of predators are the result of the prey initiating anti-predator behavioral and/or physiological trait changes that can aid in predator avoidance ( Hermann and Landis , 2017; Lima and Dill , 1990 ) .", "Such plasticity in certain prey traits may also result in energetic costs ( Lima , 1998 ) .", "Predator detection is key for these trait changes to occur and can be mediated by chemical , tactile , and visual cues ( Hermann and Thaler , 2014 ) .", "In mosquitoes , exposure to predators is known to affect a variety of traits including behavior , size , development , and survival ( Arav and Blaustein , 2006; Bond et al . , 2005; Roberts , 2012; Roux et al . , 2015 , Zuharah et al . , 2013 ) .", "Experimental observations of predator effects on mosquito size and development are inconsistent and results sometimes vary by mosquito sex .", "For example , exposure to predation was found to increase the size of Culex pipiens mosquitoes ( Alcalay et al . , 2018 ) but decrease the size of Culiseta longiareolata ( Stav et al . , 2005 ) .", "In addition , female Aedes triseriatus exhibited shorter development times when exposed to predation at high nutrient availability ( Ower and Juliano , 2019 ) , but male C . longiareolata had longer development times in the presence of predators ( Stav et al . , 2005 ) .", "In some cases , a shared evolutionary history between predator and prey organisms can strengthen the non-consumptive effects of predators on mosquitoes ( Buchanan et al . , 2017; Sih , 1986 ) .", "This investigation assesses the consumptive and non-consumptive effects of predators on mosquito traits and describes how these effects could impact disease transmission .", "The roles of vector genus , predator family , mosquito larval instar ( an indicator of prey size ) , and temperature are also examined as potential moderators of predator effects .", "Non-consumptive effects of predators are expected to cause a smaller reduction in mosquito survival than consumptive effects because , in practice , measures of consumptive effects always include both consumptive and non-consumptive effects .", "Based on previous findings , larger predators are more likely to consumptively reduce mosquito survival ( Kumar et al . , 2008 ) .", "In addition , Aedes mosquito larvae may be more vulnerable to consumption than other genera because of the high degree of motility observed in this genus ( Dieng et al . , 2003; Marten and Reid , 2007; Soumare and Cilek , 2011 ) .", "The oviposition response to predation is expected to be weakest among Aedes species that oviposit above the water line , due in part to their delayed-hatching eggs ( Vonesh and Blaustein , 2010 ) .", "Predation is predicted to reduce mosquito size and lengthen development time , consistent with the reduced growth response observed in other insect systems ( Hermann and Landis , 2017 ) .", "Certain non-consumptive effects of predation , particularly oviposition site selection and decreased vector size , are likely to play important roles in the dynamics of mosquito-borne disease ." ], [ "A systematic search was conducted for studies on predation of mosquitoes that were published between 1970 and July 1 , 2019 using both PubMed and Web of Science search engines , according to the PRISMA protocol ( Moher et al . , 2009 ) .", "Mosquito vectors of the Anopheles and Aedes genera were specifically highlighted in our search terms because these genera contain the vector species that transmit malaria , yellow fever , and dengue – the three most deadly mosquito-borne diseases worldwide ( Hill et al . , 2005 ) .", "Searches included 18 combinations of three vector predation terms ( mosquito predat* , Anopheles predat* , Aedes predat* ) and six trait terms ( survival , mortality , development , fecundity , dispers* , host preference ) .", "Abstracts from the 1136 studies were each screened by two different co-authors , using the ‘metagear’ package in R ( Lajeunesse , 2016 , R Development Core Team , 2020 ) .", "If either screener thought the study had information relevant to predation of mosquitoes , or both screeners thought the abstract was ambiguous , the study was read in full .", "This resulted in 306 studies that were fully reviewed to determine if any predation data could be extracted ( Figure 1 ) .", "Data were extracted from studies that collected data on non-consumptive and/or consumptive effects of predators on mosquitoes .", "Studies were required to have a mean , error measurement , and at least two replicates for both control and predator treatments .", "The control treatment was required to have all the same conditions as the predator treatment , such as prey density and type of water , without the predators .", "Studies that were not published in English and studies that did not differentiate between predators of multiple families were excluded .", "Studies were also excluded if oviposition by free-flying female mosquitoes could have interfered with observing the consumptive effects of predators on vector survival .", "The final database comprised data extracted from 60 studies ( Supplementary file 1 ) .", "The data included observations from laboratory experiments , as well as semi-field experiments , in which mesocosms of different treatments were observed in outdoor settings .", "Variables related to the publication , the vector , the predator , and the effect size ( Table 1 ) were extracted from each study .", "Data from tables and text were recorded as they were published , and data from figures were extracted using WebPlotDigitizer ( Rohatgi , 2020 ) .", "Error measurements that were not originally presented as standard deviations were converted to standard deviations prior to the effect size calculation .", "A PRISMA plot of literature inclusion and exclusion is provided in Figure 1 .", "Observations where insecticide was used were excluded because insecticides are known to interfere with consumptive and non-consumptive effects of predators ( Delnat et al . , 2019; Janssens and Stoks , 2012 ) .", "In addition , observations from experiments with mosquito prey of two or more species were excluded because it was not possible to account for effects from apparent competition or prey-switching .", "Observations of vector fecundity , vector competence , behavioral traits other than oviposition , as well as observations where the vector trait was marked as ‘other’ were not analyzed because each of these traits were only recorded from three or fewer studies .", "Due to protandry , the earlier emergence of males to maximize their reproductive success , mosquitoes respond to sex-specific selective forces that influence their development time and body size ( Kleckner et al . , 1995 ) .", "Under low resource conditions , female mosquitoes are likely to maximize body mass by extending their development time , whereas males tend to minimize their development time at the expense of lower body mass ( Kleckner et al . , 1995 ) .", "Observations of mosquito development time and body size in our database that were not sex-specific were excluded so that these vector traits could be analyzed while controlling for sex .", "In addition , among the observations of development time and body size , some predator means did not necessarily represent an evenly weighted average of the replicates .", "For example , if a total of 20 mosquitoes from three different predator replicates survived to adulthood , the mean development time and size of those 20 individuals may have been reported .", "To represent an evenly weighted average of the replicates , it is necessary to first calculate summary statistics among multiple individuals that emerge from the same replicate , and then report the average of the replicate-specific means .", "Observations that might have been influenced by uneven representation of replicates were excluded to prevent pseudo-replication from altering later meta-analyses .", "For consumptive observations where life stage-specific survival was reported after more than 10 days of predator exposure , only data on survival marked by adult emergence were included for analysis .", "Effects observed among immature vector stages after such a long period of predator exposure were not analyzed because they could have resulted from a combination of non-consumptive effects on development , and consumptive effects on survival .", "Development time observations that were reported as the inverse of development time ( units of days–1 ) were excluded because although their means could be converted to units of days , their standard deviations could not be converted to match units of days .", "In cases where multiple body sections of the same mosquitoes were measured to produce multiple size observations , only the wing measurement was included in the analysis to prevent pseudo-replication .", "Observations in which both the control and the predator treatments had standard deviations of zero were excluded because the meta-analysis methods did not support non-positive sampling variances .", "One study that was included in our database reported egg survival data as the hatch rate of field collected Culex pervigilans rafts ( Zuharah et al . , 2013 ) .", "However , mosquitoes have been shown to lay eggs independent of mating ( O’Meara , 1979 ) , and hatch rates of zero have previously been observed in rafts laid by Culex females that were held separately from males ( Su and Mulla , 1997 ) .", "Thus , hatch rates of zero were excluded from further analysis because these values may represent unfertilized egg rafts , rather than a strong impact of predators on survival .", "Twenty of the 187 consumptive survival observations had a predation mean of zero , and each of these zeros resulted from experiments that began with a specified number of live larvae .", "Consumptive survival zeros were each replaced with 0 . 5% of the starting number of mosquito prey to avoid undefined effect sizes .", "In addition , there was one zero out of the 36 oviposition predation means; this value had units of ‘number of egg rafts laid’ and was replaced with 0 . 5 rafts .", "Similar methods for replacing zero values in the treatment mean with small non-zero values have previously been employed ( Thapa et al . , 2018 ) .", "The final analysis dataset included seven subsets: consumptive effects on survival , non-consumptive effects on survival , oviposition , development ( female and male ) , and size ( female and male ) .", "The data included 187 observations from 34 studies of consumptive survival , 24 observations from seven studies of non-consumptive survival , 36 observations from 12 studies of oviposition , 14 observations from seven studies of female development , 14 observations from seven studies of male development , 27 observations from 10 studies of female size , and 18 observations from nine studies of male size ( Figure 1 ) .", "These observations covered seven different classes of predator families ( Figure 2 ) ." ], [ "Each data subset ( Figure 1 ) had an I2 statistic of greater than 75% , indicating high heterogeneity ( Higgins et al . , 2003 ) .", "Random effects model results showed that predators consumptively decreased mosquito survival with an effect size of –1 . 23 ( 95% CI −1 . 43 , –1 . 03 ) , p-value < 0 . 0001 , and non-consumptively reduced survival with a smaller effect size of –0 . 11 ( 95% CI −0 . 17 , –0 . 04 ) , p-value = 0 . 0016 .", "In addition , predators non-consumptively reduced oviposition behavior with an effect size of –0 . 87 ( 95% CI −1 . 31 , –0 . 42 ) , p-value = 0 . 0001 , and mosquito body size was non-consumptively reduced by predators in both males and females; the female effect size was –0 . 13 ( 95% CI −0 . 19 , –0 . 06 ) , p-value = 0 . 0002 , and the male effect size was –0 . 03 ( 95% CI −0 . 06 , –0 . 01 ) , p-value = 0 . 0184 .", "There was not a significant non-consumptive effect of predators on either male or female development time; the female effect size was –0 . 01 ( 95% CI –0 . 09 , 0 . 07 ) , p-value = 0 . 7901 , and the male effect size was –0 . 04 ( 95% CI –0 . 12 , 0 . 04 ) , p-value = 0 . 3273 .", "The Egger’s regression test results showed that the non-consumptive survival subset , both development time subsets ( male and female ) , and the female size subset exhibited funnel plot asymmetry indicative of publication bias .", "The ‘trim and fill’ procedure identified missing studies in the non-consumptive survival subset and the female size subset , but the procedure did not identify any missing studies in either of the development time subsets .", "Three studies were estimated to be missing from the non-consumptive survival data , and accounting for imputed values from missing studies resulted in a shift in the predation effect size from –0 . 11 ( 95% CI −0 . 17 , –0 . 04 ) , p-value = 0 . 0016 , to -0 . 13 ( 95% CI −0 . 20 , –0 . 07 ) , p-value < 0 . 0001 .", "Two studies were estimated to be missing from the female size data , and accounting for imputed values from these missing studies shifted the predation effect size from –0 . 13 ( 95% CI −0 . 19 , –0 . 06 ) , p-value = 0 . 0002 , to -0 . 10 ( 95% CI −0 . 17 , –0 . 03 ) , p-value = 0 . 0083 .", "Shifts in effect size estimates due to the trim and fill procedure were minor and did not cause any of the observed effects of predators to change direction or become insignificant .", "The consumptive survival and oviposition data subsets met the criteria of high heterogeneity , observations from at least 10 studies , and no evidence of publication bias .", "Therefore , these data subsets were tested for moderators using multilevel mixed effects models .", "Predator families that decreased mosquito survival included Cyprinidae: –3 . 44 ( 95% CI −5 . 79 , –1 . 09 ) , p-value = 0 . 0042; Poeciliidae: –1 . 42 ( 95% CI −2 . 67 , –0 . 16 ) , p-value = 0 . 0270; Ambystomatidae: –5 . 18 ( 95% CI −7 . 94 , –2 . 42 ) , p-value = 0 . 0002; Aeshnidae: –2 . 93 ( 95% CI −4 . 80 , –1 . 07 ) , p-value = 0 . 0020; and Notonectidae: –2 . 14 ( 95% CI −3 . 07 , –1 . 21 ) , p-value < 0 . 0001 ( Figure 3a ) .", "Vector genera that experienced significant decreases in survival due to consumptive effects of predators included Aedes: –1 . 23 ( 95% CI −1 . 81 , –0 . 65 ) , p-value < 0 . 0001; Anopheles: –1 . 34 ( 95% CI −2 . 01 , –0 . 66 ) , p-value = 0 . 0001; and Culex: –1 . 41 ( 95% CI −1 . 96 , –0 . 86 ) , p-value < 0 . 0001 ( Figure 3b ) .", "Among all 187 consumptive survival observations from 34 studies , the best model fit , according to AICc value , was achieved when an interaction between predator family and vector genus was included in the model ( Table 2 ) .", "However , among the 163 larval stage consumptive survival observations from 30 studies , adding an interactive term between larval instar ( an indicator of prey size ) and predator family had a greater improvement on model fit than adding an interactive term between vector genus and predator family ( Figure 3c , Table 3 ) .", "Temperature did not affect the heterogeneity of consumptive survival data , either as a linear moderator: –0 . 01 ( 95% CI –0 . 10 , 0 . 07 ) , p-value = 0 . 7559 , or a quadratic moderator: 0 . 00 ( 95% CI 0 . 00 , 0 . 00 ) , p-value = 0 . 8184 .", "The best oviposition model fit , according to AICc value , was achieved when vector genus was added as a moderator ( Table 4 ) .", "The mean oviposition effect size was not significantly different than zero for Aedes: 0 . 32 ( 95% CI –2 . 14 , 2 . 79 ) , p-value = 0 . 7970 , or Culiseta: –0 . 61 ( 95% CI –1 . 83 , 0 . 62 ) , p-value = 0 . 3329 , but for Culex mosquitoes , oviposition was significantly decreased by predator presence: –1 . 69 ( 95% CI −2 . 82 , –0 . 56 ) , p-value = 0 . 0033 ( Figure 4 ) ." ], [ "Several larger predators reduced mosquito survival , including freshwater fish ( Cyprinidae and Poeciliidae ) , salamander larvae ( Ambystomatidae ) , dragonfly larvae ( Aeshnidae ) , and backswimmers ( Notonectidae ) ( Figure 3a ) .", "This finding is consistent with a previous analysis which showed a positive linear relationship between predator body mass and ingestion rate across taxa ( Peters , 1983 ) .", "In addition , more effect size heterogeneity in the consumptive survival data was explained by an interaction between predator family and larval instar than was explained by an interaction between predator family and vector genus ( Table 3 ) .", "This result suggests that the relative sizes of predator and prey groups could play a more important role in determining consumptive mosquito survival than variations in predator responses to different behaviors of prey genera , which are likely to be shaped by the degree of shared evolutionary history between trophic levels ( Buchanan et al . , 2017 ) .", "Larval instar is an indicator of mosquito size , and previous modeling work has provided evidence of prey size selection by predators to maximize energetic gain ( Mittelbach , 1981 ) .", "While smaller cyclopoid copepods are more effective against early instar mosquito larvae ( Dieng et al . , 2002 ) , larger predators including tadpoles , giant water bugs , dragonfly larvae , fish , and backswimmers are more effective against late instar larvae ( Kweka et al . , 2011 ) .", "Exposure to predation cues significantly lowered mosquito survival , and this non-consumptive effect has also been observed in dragonfly larvae prey ( Leucorrhinia intacta ) that were exposed to caged predators ( McCauley et al . , 2011 ) .", "The reduction in mosquito survival from non-consumptive effects of predators was significantly smaller than the reduction that was observed from consumptive effects .", "This is partially due to the practical constraints of most experimental designs , which cause consumptive and non-consumptive effects of predators on survival to be grouped together and reported as consumptive effects .", "The greater impact of combined consumptive and non-consumptive effects , in comparison to only non-consumptive effects , has previously been observed in pea aphids ( Acyrthosiphon pisum ) ( Nelson et al . , 2004 ) .", "While predators did not significantly impact mosquito development time through non-consumptive effects in either sex , mosquito body size was decreased by the non-consumptive effects of predators in both sexes .", "Smaller body size is associated with lower reproductive success in mosquitoes because smaller females lay fewer eggs ( Blackmore and Lord , 2000; Lyimo and Takken , 1993; Oliver and Howard , 2011; Styer et al . , 2007; Tsunoda et al . , 2010 ) , and smaller males produce less sperm ( Hatala et al . , 2018; Ponlawat and Harrington , 2007 ) .", "These effects suggest that predation could non-consumptively reduce mosquito population growth .", "The smaller size of mosquitoes exposed to predators could also limit disease transmission .", "Vector lifespan contributes disproportionately to disease transmission because older vectors are more likely to have been exposed to pathogens , more likely to already be infectious after having survived the extrinsic incubation period , and more likely to survive long enough to bite subsequent hosts ( Cator et al . , 2020 ) .", "It is well-established that smaller mosquito body size is associated with shorter mosquito lifespan ( Araújo et al . , 2012; Hawley , 1985 , Reisen et al . , 1984; Reiskind and Lounibos , 2009; Xue et al . , 2010 ) .", "Therefore , non-consumptive effects of predators may limit the transmission of mosquito-borne diseases .", "Predator presence also non-consumptively reduced oviposition behavior in adult female mosquitoes .", "Meta-regression results showed that Culex females significantly avoid oviposition sites that contain predators or predator cues , but Aedes and Culiseta females do not avoid these sites , despite a slight non-significant trend toward predator avoidance in Culiseta ( Figure 4 ) .", "Both Culex and Culiseta mosquitoes have an ‘all-or-none’ oviposition strategy ( Johnson and Fonseca , 2014 ) , in which they lay hundreds of rapidly hatching eggs in rafts on the water’s surface ( Day , 2016 ) .", "Such an oviposition strategy is conducive to evolving predator avoidance behaviors , and a previous meta-analysis showed significant predator avoidance in both Culex and Culiseta during oviposition ( Vonesh and Blaustein , 2010 ) .", "Conversely , it is likely that an oviposition response to predation is not particularly advantageous for Aedes because the delayed hatching of their eggs ( Day , 2016 ) can prevent the level of predation risk at the time of oviposition from matching the level of predation risk present in the eventual larval environment ( Vonesh and Blaustein , 2010 ) .", "The predator avoidance response in Aedes species that lay their eggs above the water’s edge in containers has previously been described as ‘non-existent’ ( Vonesh and Blaustein , 2010 ) .", "Both Aedes species included in this study’s oviposition data subset , Ae .", "albopictus and Ae .", "aegypti , meet the criterion of ovipositing above water in containers ( Juliano , 2009 ) .", "Predator avoidance during oviposition has previously been found to increase the mosquito population size at equilibrium ( Spencer et al . , 2002 ) .", "However , this study’s results and those of a previous meta-analysis ( Vonesh and Blaustein , 2010 ) suggest that models of oviposition site selection , such as those using parameters from Notonectidae predators and Culiseta prey ( Kershenbaum et al . , 2012 ) , are not generalizable to Aedes vectors .", "Predator avoidance during oviposition by Culex mosquitoes ( Figure 4 ) may be of particular importance to West Nile virus ( WNV ) disease dynamics .", "Previous work has shown that Cx .", "pipiens , Cx .", "restuans , and Cx .", "tarsalis all avoid predator habitats ( Vonesh and Blaustein , 2010 ) , and that Cx .", "pipiens is the primary bridge vector of WNV responsible for spill-over transmission from avian reservoir hosts to humans ( Fonseca et al . , 2004; Hamer et al . , 2008a , Kramer et al . , 2008; Andreadis , 2012 ) .", "Cx .", "pipiens mosquitoes can live in permanent aquatic environments , such as ground pools ( Amini et al . , 2020; Barr , 1967; Dida et al . , 2018; Sulesco et al . , 2015 ) , ponds ( Lühken et al . , 2015 ) , stream edges ( Amini et al . , 2020 ) , and lake edges ( Vinogradova , 2000 ) that are more common in rural areas , but Cx .", "pipiens are also found in urban and suburban residential areas , where they typically breed in artificial containers ( Sulesco et al . , 2015 ) , including tires ( Lühken et al . , 2015; Nikookar et al . , 2017; Verna , 2015 ) , rainwater tanks ( Townroe and Callaghan , 2014 ) , and catch basins ( Gardner et al . , 2012 ) .", "Small artificial containers , such as discarded tires , are generally unlikely to harbor larger predators , including freshwater fish ( Cyprinidae and Poeciliidae ) , salamander larvae ( Ambystomatidae ) , dragonfly larvae ( Aeshnidae ) , and backswimmers ( Notonectidae ) , because temporary aquatic environments cannot support the relatively long development times of these organisms .", "The mean dispersal distance of adult Culex mosquitoes is greater than one kilometer ( Ciota et al . , 2012; Hamer et al . , 2014 ) , and female Cx .", "pipiens have exhibited longer dispersal distances after developing in the presence of a fish predator ( Alcalay et al . , 2018 ) .", "Therefore , predator avoidance during oviposition may cause Cx .", "pipiens populations to disperse from permanent aquatic environments in more rural areas to artificial container environments in urbanized areas , where the risk of human WNV infection is higher ( Brown et al . , 2008 ) .", "Predator cue levels may be altered by climate conditions , and these changes in cue levels can impact WNV transmission to humans .", "Drought has previously been associated with human WNV cases ( Johnson and Sukhdeo , 2013; Marcantonio et al . , 2015; Roehr , 2012; Shaman et al . , 2005; Epstein and Defilippo , 2001; Paull et al . , 2017 ) , but the association has thus far lacked a clear underlying mechanism .", "Under drought conditions , the density of aquatic organisms increases and predation pressures can intensify due to compressed space and high encounter rates ( Amundrud et al . , 2019 ) .", "A previous study of a stream ecosystem found that impacts of fish predation are more severe during the dry season ( Dudgeon , 1993 ) .", "In addition , reductions in water volume can facilitate consumption of mosquito larvae by crane fly larvae ( Tipulidae ) , whereas mosquito consumption by tipulids was not observed at a higher water level ( Amundrud et al . , 2019 ) .", "Laboratory and semi-field studies have shown that mosquitoes respond to a gradient of predator cues ( Roux et al . , 2014; Silberbush and Blaustein , 2011 ) .", "The frequency of larval anti-predator behavior is correlated with the concentration of predator cues ( Roux et al . , 2014 ) , and adult female mosquitoes prefer oviposition sites with lower predator densities ( Silberbush and Blaustein , 2011 ) .", "Therefore , as predator cue levels increase due to drought , permanent aquatic habitats are likely to transition from suitable oviposition sites for one generation of female mosquitoes , to unsuitable oviposition sites for the next generation .", "When suitable oviposition sites are absent , females retain their eggs until sites become available ( Bentley and Day , 1989 ) .", "Cx .", "pipiens females can retain their eggs for up to five weeks , allowing them enough time to find container sites with low predation risk , often located in residential areas ( Johnson and Fonseca , 2014 ) .", "The movement of gravid female Cx .", "pipiens to residential areas increases the risk of WNV spill-over to humans because these vectors are likely to have already blood-fed at least once ( Clements , 1992 ) , suggesting that they have a higher risk of WNV infection , relative to non-gravid mosquitoes .", "This is consistent with studies that have reported associations between drought and WNV-infected mosquitoes in urban and residential areas ( Johnson and Sukhdeo , 2013; Paull et al . , 2017 ) .", "In addition , vertical transmission of WNV from gravid females to their progeny may occur during oviposition ( Rosen , 1988 ) , when the virus is transmitted by an accessory gland fluid that attaches eggs to one another ( Nelms et al . , 2013 ) .", "Because the rate of vertical transmission in Cx .", "pipiens increases with the number of days following WNV infection ( Anderson et al . , 2008 ) , extended searches for oviposition sites due to drought could increase the frequency of vertical transmission .", "However , the impact of vertical transmission on WNV epidemics is thought to be minimal because when transmission to an egg raft did occur , only 4 . 7% of the progeny were found to be infected as adults ( Anderson et al . , 2008 ) , and only about half of those infected adults are estimated to be female .", "In summary , the movement of Cx .", "pipiens females toward more residential areas , combined with potential limited WNV amplification from increased vertical transmission , suggests that the vector trait of predator avoidance during oviposition can serve as a plausible explanation for associations between drought and human WNV cases .", "Another theory for the association between drought and human WNV cases is based on the hypothesis that increased contact between mosquito vectors and passerine reservoir hosts occurs during drought conditions ( Paull et al . , 2017; Shaman et al . , 2005 ) .", "The proposed aggregation of bird and mosquito populations during drought was originally thought to occur in humid , densely vegetated hammocks – a type of habitat that is specific to southern Florida ( Shaman et al . , 2005 ) , but WNV incidence is more consistently clustered in other regions of the US , particularly the Northern Great Plains ( CENTERS FOR DISEASE CONTROL AND PREVENTION , 2021; Sugumaran et al . , 2009 ) .", "Northern cardinals ( Cardinalis cardinalis ) , American robins ( Turdus migratorius ) , and house sparrows ( Passer domesticus ) were among the bird species that most frequently tested seropositive for WNV antibodies in 2005 and 2006 in Chicago , where high numbers of human cases were reported ( Hamer et al . , 2008b ) , and these passerine species are more abundant in residential areas , regardless of precipitation patterns ( Anderson , 2006b; Beddall , 1963; Lepczyk et al . , 2008 ) .", "Apart from drought , landowners’ participation in supplemental bird feeding , providing bird houses , gardening , and maintaining vegetation can strongly influence passerine abundance in residential areas ( Lepczyk et al . , 2004 ) .", "Furthermore , as terrestrial foragers that can obtain hydration from their diet of insects , fruits , and other plant material ( Anderson , 2006a; Brzek et al . , 2009; Malmborg and Willson , 1988; Renne et al . , 2000 ) , passerine reservoir hosts of WNV are less likely to move in response to drought than the mosquito vectors of WNV , which have obligate aquatic life stages .", "While hatch-year birds are more vulnerable to mosquito biting , and thus contribute to the amplification of WNV ( Hamer et al . , 2008b ) , it is illogical to expect an increased abundance of hatch-year birds during drought conditions .", "However , some have argued that in cases where drought decreases the abundance of juvenile birds , the ratio of mosquitoes to birds increases , and this could lead to higher WNV prevalence in the mosquito population ( Paull et al . , 2017 ) .", "Although reductions in both hatching success ( George et al . , 1992 ) and survival of recently fledged birds ( Yackel Adams et al . , 2006 ) have been observed during drought conditions , the impact of drought on avian abundance varies widely by species ( Verner and Purcell , 1999 ) .", "In particular , synanthropic species , such as those likely to harbor WNV , are less negatively affected by drought ( Albright et al . , 2009 ) .", "Additionally , the droughts that impact avian abundance often occur over much longer periods of time than the seasonal droughts that predict WNV transmission to humans .", "For example , avian abundance has been modeled based on precipitation metrics spanning 32 weeks , and house wren ( Troglodytes aedon ) abundance has been predicted by precipitation averages spanning four years ( Verner and Purcell , 1999 ) .", "Finally , birds with higher levels of stress hormones are more likely to be fed on by mosquitoes , and certain factors associated with residential areas , such as road noise , light pollution , and pesticide exposure , can cause avian stress ( Gervasi et al . , 2016 ) .", "Therefore , elevated avian stress hormones in these habitats may contribute to WNV prevalence in the mosquito population , independent of drought conditions .", "Although the aquatic phase of the mosquito life cycle is often overlooked in mathematical models of mosquito-borne pathogen transmission ( Reiner et al . , 2013 ) , vector survival at immature stages plays an important role in determining mosquito population abundance , which is an essential factor for predicting disease transmission ( Beck-Johnson et al . , 2013 ) .", "The results of this study show that mosquito survival decreases among the Aedes , Anopheles , and Culex genera due to consumptive effects of predators ( Figure 3b ) , and that there is also a reduction in mosquito survival due to non-consumptive effects .", "Other studies have demonstrated that aquatic predators dramatically impact mosquito survival and abundance .", "For example , a biocontrol intervention relying on the application of copepod predators eliminated Aedes albopictus from three communes in Nam Dinh , Vietnam , where dengue transmission was previously detected , and reduced vector abundance by 86–98% in three other communes ( Kay et al . , 2002 ) .", "Conversely , the annual abundance of Culex and Anopheles mosquitoes was observed to increase 15-fold in semi-permanent wetlands in the year following a drought , likely because the drought eliminated aquatic predators from wetlands that dried completely , and mosquitoes were able to re-colonize newly formed aquatic habitats more quickly than their most effective predators ( Chase and Knight , 2003 ) .", "While relationships between temperature and different vector traits , such as fecundity and lifespan , have been incorporated into models of temperature effects on mosquito population density ( El Moustaid and Johnson , 2019 ) , models of predator effects on vector borne disease transmission have focused primarily on the impacts of predation on vector survival .", "Previous models have shown that predators of vector species can decrease or eliminate pathogen infection in host populations as vector fecundity increases ( Moore et al . , 2010 ) .", "The findings of this meta-analysis suggest that predators also decrease vector fecundity through non-consumptive effects on vector body size .", "In addition , the entomological inoculation rate ( EIR ) is likely to be reduced by effects of predators on mosquito fecundity and lifespan , as well as effects of predators on mosquito survival .", "The EIR has been defined as the product of three variables: ( m ) the number of mosquitoes per host , ( a ) the daily rate of mosquito biting , and ( s ) the proportion of mosquitoes that are infectious ( Beck-Johnson et al . , 2013 ) .", "Based on this study’s findings , predators are likely to decrease the number of mosquitoes per host by reducing mosquito survival through both consumptive and non-consumptive effects , and by reducing mosquito fecundity through non-consumptive effects on body size .", "In addition , predators are likely to decrease the proportion of mosquitoes that are infectious by shortening the vector lifespan through non-consumptive effects on body size .", "The relationship between mosquito body size and biting rate is unclear , with some studies showing higher biting rates among larger mosquitoes ( Araújo et al . , 2012; Gunathilaka et al . , 2019 ) , and others reporting higher biting rates among smaller mosquitoes ( Farjana and Tuno , 2013; Leisnham et al . , 2008 ) .", "The links between factors that influence the EIR and observed effects of predators on mosquito prey demonstrate the necessity of including both consumptive and non-consumptive effects of predators in models of mosquito-borne disease .", "This meta-analysis on mosquito predation demonstrates that predators not only play an important role in directly reducing mosquito populations , but also have non-consumptive effects on surviving mosquitoes that may ultimately reduce further population growth and decrease disease transmission .", "While families of larger sized predators were effective in reducing mosquito survival , other factors , such as impacts on native species , as well as the economic cost of mass-rearing and field applications ( Kumar and Hwang , 2006; Pyke , 2008 ) , should be carefully considered before selecting a predator as a suitable biocontrol agent .", "Predictive disease models are likely to be more reliable when the non-consumptive effects of predation are incorporated .", "Although exposure of mosquito larvae to predators is commonplace in outdoor field settings , it remains rare in most laboratory-based assessments of vector traits .", "Therefore , mosquitoes observed in nature are likely to have smaller body sizes than those observed under optimal laboratory conditions .", "It is important for disease modelers to recognize these impacts of predation on vector traits as they can reduce mosquito population growth and limit disease transmission due to shorter vector lifespans .", "Within the WNV disease system , consideration of the oviposition behavioral response to predation cues by Culex vectors can improve current understanding of the association between drought and human cases .", "This study provides general estimates of the effects of predators on selected mosquito traits for use in predictive disease models .", "Modeling efforts that aim to optimize the application of biocontrol predators should also consider incorporating predator effects on vector survival , fecundity , and lifespan .", "These additions to predictive models of various biocontrol interventions are likely to help public health officials choose the most cost-effective strategies for limiting disease transmission .", "In the 60-study database that was compiled , only one study was designed to directly measure the effect of larval-stage predation on vector competence ( Roux et al . , 2015 ) .", "Therefore , future efforts to assess the impact of predators on mosquito-borne disease transmission should prioritize experimental studies in which infected mosquito larvae are observed throughout an initial period of aquatic exposure to predators , followed by a period of blood-feeding in the adult stage .", "Two studies from the compiled database examined the compatibility of predators with Bacillus thuringiensis var .", "israelensis ( Bti ) , a commonly used bacterial biocontrol agent ( Chansang et al . , 2004; Op de Beeck et al . , 2016 ) .", "Previous studies have supported the simultaneous application of cyclopoid copepod predators and Bti ( Marten et al . , 1993; Tietze et al . , 1994 ) , but additional analyses are needed on the use of Bti with other families of mosquito predators .", "Populations of other insect pests , such as the southern green stink bug ( Nezara viridula ) , are known to be regulated by both predators and parasites ( Ehler , 2002 ) .", "The literature search conducted for this meta-analysis returned studies on water mite parasites ( Rajendran and Prasad , 1994 ) and nematode parasitoids ( de Valdez , 2006 ) of mosquitoes , and ascogregarine parasites have previously been evaluated as biocontrol agents against Aedes mosquitoes ( Tseng , 2007 ) .", "A more thorough review of the impacts of parasites and parasitoids on vector traits , such as survival , fecundity , and lifespan , is needed before incorporating these potential biocontrol agents into integrated vector control plans .", "Three studies in the 60-study database included experiments where two mosquito prey species were made available to the predator species ( Grill and Juliano , 1996; Griswold and Lounibos , 2005 , Micieli et al . , 2002 ) .", "In these cases , the effect size measurement for each mosquito species could be influenced by interspecific competition , or a preference of the predator species for a certain prey species .", "Hetero-specific prey observations were excluded from this meta-analysis , but future analyses centered on the concepts of interspecific competition or predator preferences might further evaluate these data .", "In addition , this meta-analysis investigated consumptive and non-consumptive effects of predators separately .", "More research is needed to determine how models should combine these different types of predator effects to accurately reflect predation interactions as they occur in natural environments ." ] ]

[ "Predator-prey interactions influence prey traits through both consumptive and non-consumptive effects , and variation in these traits can shape vector-borne disease dynamics .", "Meta-analysis methods were employed to generate predation effect sizes by different categories of predators and mosquito prey .", "This analysis showed that multiple families of aquatic predators are effective in consumptively reducing mosquito survival , and that the survival of Aedes , Anopheles , and Culex mosquitoes is negatively impacted by consumptive effects of predators .", "Mosquito larval size was found to play a more important role in explaining the heterogeneity of consumptive effects from predators than mosquito genus .", "Mosquito survival and body size were reduced by non-consumptive effects of predators , but development time was not significantly impacted .", "In addition , Culex vectors demonstrated predator avoidance behavior during oviposition .", "The results of this meta-analysis suggest that predators limit disease transmission by reducing both vector survival and vector size , and that associations between drought and human West Nile virus cases could be driven by the vector behavior of predator avoidance during oviposition .", "These findings are likely to be useful to infectious disease modelers who rely on vector traits as predictors of transmission ." ]

[ "Mosquitoes are often referred to as the deadliest animals on earth because some species spread malaria , West Nile virus or other dangerous diseases when they bite humans and other animals .", "Adult mosquitoes fly to streams , ponds and other freshwater environments to lay their eggs .", "When the eggs hatch , the young mosquitoes live in the water until they are ready to grow wings and transform into adults .", "In the water , the young mosquitoes are particularly vulnerable to being eaten by dragonfly larvae , fish and other predators .", "When adult females are choosing where to lay their eggs , they can use their sense of smell to detect these predators and attempt to avoid them .", "Along with eating the mosquitoes , the predators may also reduce mosquito populations in other ways .", "For example , predators can disrupt feeding among young mosquitoes , which may affect the time that it takes for them to grow into adults or the size of their bodies once they reach the adult stage .", "Although the impacts of different predators have been tested separately in multiple settings , the overall effects of predators on the ability of mosquitoes to spread diseases to humans remain unclear .", "To address this question , Russell , Herzog et al . used an approach called meta-analysis on data from previous studies .", "The analysis found that along with increasing the death rates of mosquitoes , the presence of predators also leads to a reduction in the body size of those mosquitoes that survive , causing them to have shorter lifespans and fewer offspring .", "Russell , Herzog et al . found that one type of mosquito known as Culex – which carries West Nile virus – avoided laying its eggs near predators .", "During droughts , increased predation in streams , ponds and other aquatic environments may lead adult female Culex mosquitoes to lay their eggs closer to residential areas with fewer predators .", "Russell , Herzog et al . propose that this may be one reason why outbreaks of West Nile virus in humans are more likely to occur during droughts .", "In the future , these findings may help researchers to predict outbreaks of West Nile virus , malaria and other diseases carried by mosquitoes more accurately .", "Furthermore , the work of Russell , Herzog et al . provides examples of mosquito predators that could be used as biocontrol agents to decrease numbers of mosquitoes in certain regions ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "evolutionary biology", "biochemistry and chemical biology" ]

[ [ "Minimalist pathogens like RNA viruses survive dynamic host environments by virtue of their extreme adaptability .", "This adaptability is driven by a high rate of genetic variation , mediated by error-prone genome replication ( Sanjuán et al . , 2010 ) .", "Most missense mutations have deleterious consequences for protein function , often owing to either thermodynamic ( reduced stability of the native state or enhanced stability of unfolded/misfolded states ) or kinetic ( slow folding or enhanced misfolding/aggregation ) effects on folding ( DePristo et al . , 2005 ) .", "In the context of viruses , these phenomena may underpin the observation that the distribution of mutational fitness effects can be largely accounted for by considering protein folding biophysics ( Wylie and Shakhnovich , 2011; Chéron et al . , 2016; Tokuriki and Tawfik , 2009a ) .", "Indeed , stable proteins tend to be more evolvable , as any given missense mutation is less likely to severely disrupt protein folding or structure ( Bloom et al . , 2006; Gong et al . , 2013 ) .", "In cells , protein folding challenges are addressed by proteostasis networks composed of chaperones and quality control factors that work in concert to shepherd nascent proteins to folded , functional conformations ( Balch et al . , 2008; Hartl et al . , 2011; Powers and Balch , 2013 ) .", "Important work focused primarily on the Hsp90 chaperone has suggested a critical role for chaperones in modulating the evolution of their endogenous clients , ( Cowen and Lindquist , 2005; Queitsch et al . , 2002; Lachowiec et al . , 2015; Sangster et al . , 2007 , 2008a , 2008b; Rohner et al . , 2013; Whitesell et al . , 2014; Geiler-Samerotte et al . , 2016; Rutherford and Lindquist , 1998 ) in part by buffering deleterious effects of non-synonymous mutations .", "The consequences of Hsp90 activity for protein evolution may be due to Hsp90 directly engaging an evolving client protein ( termed a primary effect ) .", "Alternatively , the effects of Hsp90 may be secondary , mediated indirectly by Hsp90 influencing the folding of other endogenous clients that themselves engage relevant evolving proteins .", "For instance , Hsp90-dependent azole resistance in Candida albicans is mediated by secondary effects of Hsp90 on calcineurin , an Hsp90 client that regulates responses to environmental stimuli ( Cowen and Lindquist , 2005 ) .", "Efforts to look beyond Hsp90 to understand how other components of the metazoan proteostasis machinery modulate evolution ( e . g . , Hsp40/70 chaperones or protein misfolding stress responses like the heat shock response ) have been slowed by the paucity of chemical biology tools to perturb the activities of these systems .", "However , Tawfik and coworkers have shown that the GroEL/ES chaperonin system can govern the fitness of certain client protein variants in bacteria ( Tokuriki and Tawfik , 2009b ) , and computational modeling suggests that other chaperones may also have roles in evolution ( Bogumil and Dagan , 2012; Cetinbaş and Shakhnovich , 2013 ) .", "Chaperones and other proteostasis mechanisms are theoretically well-positioned to address the biophysical challenges created by high mutation rates in viruses .", "Intriguingly , most RNA viruses lack autonomous chaperones or other co-factors to assist their proteins with folding .", "Instead , viral proteins engage host chaperones , ( Melville et al . , 1999; Momose et al . , 2002; Naito et al . , 2007; York et al . , 2014; Watanabe et al . , 2010 ) and host chaperone inhibitors have been shown to limit the viability of certain RNA viruses ( Geller et al . , 2007; Heaton et al . , 2016; Taguwa et al . , 2015; Chase et al . , 2008; Geller et al . , 2012 ) .", "However , the possibility that host chaperones can shape the evolution of viral pathogens has not been investigated .", "In summary , it is clear that: ( 1 ) high genetic variability is essential to support RNA virus adaptability; ( 2 ) missense mutations important for viral adaptation are often biophysically deleterious , constraining the accessible mutational landscape; and ( 3 ) many viruses engage host chaperones to fold their proteins .", "An important but still untested hypothesis is that host proteostasis modulates RNA virus evolutionary trajectories .", "Experimentally testing this hypothesis requires methods to regulate the host cell’s proteostasis network without significantly perturbing cell health or the ability of an RNA virus to propagate .", "Here , we achieve this goal in the context of long-term influenza propagation by using small molecules to either modulate the heat shock response in a stress-independent manner ( Shoulders et al . , 2013; Moore et al . , 2016 ) or to inhibit Hsp90 at sub-lethal concentrations ( Ying et al . , 2012 ) .", "We find that the resulting perturbations to host proteostasis mechanisms significantly impact both the extent and the nature of selection pressure on the influenza genome .", "We conclude that host proteostasis is a critical , under-appreciated player in influenza evolution , with significant implications for our ability to predict and prevent the evolution of influenza and other RNA viral pathogens ." ], [ "Eukaryotic cells dynamically match proteostasis network capacity to demand via compartment-specific stress responses .", "Thus , one biologically relevant strategy to create an altered proteostasis environment is to induce such responses .", "We focused on the heat shock response , ( Åkerfelt et al . , 2010 ) because numerous influenza proteins must fold and/or function in the cytosol and nucleus ( Watanabe et al . , 2010 ) .", "Typical methods to induce heat shock factor 1 ( HSF1 ) , the master regulator of the heat shock response and thus of cytosolic and nuclear chaperone and quality control protein levels , involve treatment with toxins or acute heat stress .", "These methods are not useful for our studies because they engender massive protein misfolding stress in host cells that rapidly become apoptotic , preventing influenza propagation .", "An alternative strategy is to over-express a constitutively active form of HSF1 lacking amino acids 186–202 ( termed cHSF1 ) , ( Voellmy , 2005 ) but high levels of cHSF1 over-expression outside the physiologically relevant regime are typically toxic ( Ryno et al . , 2014 ) .", "Instead , we took a chemical genetic approach , fusing cHSF1 to a destabilized variant of E . coli dihydrofolate reductase ( DHFR ) ( Shoulders et al . , 2013; Iwamoto et al . , 2010 ) .", "The DHFR . cHSF1 fusion is targeted for rapid proteasomal degradation and is therefore non-functional ( Figure 1A ) , unless cells are treated with the DHFR-stabilizing pharmacologic chaperone trimethoprim ( TMP ) .", "We created a stable , clonal Madin Darby canine kidney ( MDCK ) cell line expressing DHFR . cHSF1 .", "In these cells , termed MDCKHSF1 cells , we can dosably induce cHSF1 transcriptional activity by TMP treatment in a stress-independent manner within the biologically relevant regime ( Figure 1B ) , avoiding cytotoxicity that would be induced by stressors or cHSF1 overexpression and yet still providing robust access to cells expressing enhanced levels of HSF1 targets ( Figure 1—figure supplement 1A–C ) .", "To control for any possible unintended consequences of TMP treatment or expression of a DHFR-fusion protein in our evolution experiments , we also created a control MDCK cell line expressing DHFR . YFP ( MDCKYFP ) .", "This MDCKYFP cell line does not display TMP-dependent upregulation of HSF1-dependent chaperones , indicating that TMP induces HSF1 activity in our MDCKHSF1 cells specifically by stabilizing DHFR . cHSF1 ( Figure 1—figure supplement 1B ) .", "A second approach to create an altered folding environment is to inhibit individual chaperones .", "Here , we employed the Hsp90 inhibitor STA-9090 , a small molecule capable of targeting multiple Hsp90 isoforms ( Ying et al . , 2012 ) .", "Notably , Hsp90 inhibition using high concentrations of STA-9090 can cause an undesirable compensatory heat shock response ( Moore et al . , 2016 ) , resulting in activation of HSF1 and upregulation of Hsp70 , Hsp40 , and other HSF1 targets .", "Such compensatory HSF1 activation would convolute interpretation of our results .", "Thus , we treated with the highest possible STA-9090 concentration that does not induce a compensatory heat shock response in MDCK cells .", "We selected the concentration used via a functional assay for all experiments , examining Hsp70 protein levels by immunoblotting to ensure the absence of any heat shock response signature ( Figure 1C ) .", "To confirm that we are still engaging Hsp90 at the low STA-9090 concentration used in our serial passaging experiments , we performed a cellular thermal shift assay ( Martinez Molina et al . , 2013 ) and observed a small but highly reproducible increase in Hsp90 thermal stability upon STA-9090 treatment ( Figure 1—figure supplement 1D ) .", "Because Hsp90 inhibition can be employed in our MDCKHSF1 cell line , these methods access three distinctive host proteostasis environments in a single cell line dependent only on small molecule treatment .", "The use of just a single cell line for experiments ( MDCKHSF1 ) and for controls ( MDCKYFP ) minimizes any possible cell line-dependent bias in our evolution experiments .", "Importantly , monitoring chaperone levels in the context of influenza A/Wuhan/1995 ( H3N2 ) infection shows that , under our infection conditions , influenza itself neither induces heat shock protein transcripts nor interferes with our method to activate HSF1 .", "Thus , we have full user control of the host proteostasis environment during a progressing infection ( Figure 1—figure supplement 2 ) .", "To further characterize these perturbed host environments , we performed RNA-Seq for each treatment in the MDCKHSF1 and MDCKYFP cell lines .", "We observed a total of only 118 transcripts whose expression is altered ≥2 fold with a p-value <10–5 in any one or more of the treatments employed , indicating that we are remodeling only a small portion of the transcriptome ( comprehensive quality control and RNA-Seq results are provided in Figure 2—source data 1–2 ) .", "A heat map for these 118 genes highlights that two distinctive cellular environments are indeed created by HSF1 activation and Hsp90 inhibition in MDCKHSF1 cells ( Figure 2A ) .", "Moreover , only two transcripts meet these cutoffs upon TMP treatment in MDCKYFP cells , confirming that the remodeled proteostasis environment upon TMP treatment of MDCKHSF1 cells is specifically due to HSF1 activation .", "The volcano plots in Figure 2B show the distribution of differentially expressed genes for HSF1 activation and Hsp90 inhibition in MDCKHSF1 cells , relative to the basal environment , with selected transcripts labeled ( for a list of all transcripts meeting these thresholds see Figure 2—source data 3 ) .", "As expected , stress-independent HSF1 activation upregulates numerous classic heat shock response genes ( Ryno et al . , 2014 ) , including HSP70 , BAG3 , HSP90AA1 , DNAJB1 , and FKBP4 ( Figure 2B ) .", "This remodeling of the cellular proteostasis network is limited to nuclear and cytosolic proteostasis mechanisms , as ER proteostasis network components are not induced by TMP treatment of the MDCKHSF1 cells ( Figure 2C ) .", "Moreover , transcript-level proteostasis network remodeling is not observed upon STA-9090 treatment , with the exception of a 2 . 2-fold induction of HSPB1 , as expected given the low concentration of inhibitor employed .", "We observe modest upregulation of several transcripts involved in transcription factor regulation and DNA damage upon HSF1 activation , such as CAMK1 , RELB , and PARP3 , consistent with the intimate role of HSF1 in cytoprotection ( Morimoto and Santoro , 1998 ) .", "STA-9090 slightly upregulates several interferon response-related transcripts , including OAS2 and MX2 ( 2 . 9 and 2 . 2 fold-change , respectively ) , a phenomenon that has been previously reported upon treatment with other Hsp90 inhibitors ( Yang et al . , 2006; Donzé et al . , 2001; Shang and Tomasi , 2006 ) .", "In summary , our RNA-Seq data are fully consistent with the chemically-controlled creation of three unique host cell proteostasis environments in MDCKHSF1 cells .", "We next serially passaged influenza A/Wuhan/1995 in our three distinctive , small molecule-controlled host environments to test the hypothesis that host proteostasis impacts influenza evolution .", "Prior to each infection , we split a population of MDCKHSF1 cells and treated with TMP to activate HSF1 , STA-9090 to inhibit Hsp90 , or vehicle ( Figure 3A ) .", "We infected at a low multiplicity of infection ( MOI ) , ranging from 0 . 001 to 0 . 04 infectious virions/cell , to minimize non-viable variants hitchhiking with functional variants owing to co-infection of a single cell ( Figure 3—figure supplement 1A ) .", "We performed 23 serial passages in biological triplicate in each proteostasis environment .", "We also performed identical passaging experiments ± TMP in our MDCKYFP cells to control for any possible off-target effects of TMP treatment or expression of a DHFR-fusion protein in our evolution experiments ( Figure 3B ) .", "Following each passage , clarified viral supernatant was harvested for hemagglutination titering and subsequent infection to initiate the next passage ( Figure 3C , Figure 3—figure supplement 1B ) .", "Importantly , infectious titering at intermittent passages confirmed that the MOI did not vary systematically between the three distinctive proteostasis environments ( Figure 3—figure supplement 1A ) .", "Thus , differences observed in evolutionary trajectories cannot be attributed to either systematically altered rates of viral growth in the three different host proteostasis environments studied here or to gross differences in cell health .", "Next , we sought to evaluate whether our serial passaging strategy provides a valid platform for modeling influenza evolution .", "In the absence of a strong exogenous selection pressure , such as an antiviral drug or antibody , we would predict that the influenza genome experiences purifying selection , meaning that most amino acid substitutions result in reduced fitness relative to the wild-type consensus sequence .", "We assessed the extent of purifying selection by determining the ratio of non-synonymous to synonymous substitutions at each passage , normalized to the ratio of non-synonymous sites to synonymous sites in the influenza genome ( 3 . 5: 1 ) , defined as Dn/Ds .", "As expected , non-synonymous mutations are selected against in all folding environments , as indicated by a Dn/Ds < 1 throughout our serial passaging ( Figure 3D ) ( Breen et al . , 2012 ) .", "To comparatively evaluate the selection pressure placed on influenza by our three distinctive host proteostasis environments , we constructed variant frequency distributions ( site frequency spectra; SFS ) for each viral population ( Nielsen , 2005 ) .", "In the SFS ( Figure 4A–B ) , we separate mutations present above our sequencing error threshold ( 1 . 5% ) in a given viral population into either non-synonymous or synonymous groups .", "The bars in the SFS charts represent the portion of variants in a viral population that fall within a given frequency bin , averaged across biological triplicates .", "As expected , the resulting SFS for non-synonymous mutations ( Figure 4A ) show that early passages consist entirely of low frequency variants .", "As the passaging experiment progresses , the high mutation rate of influenza maintains a substantial population of low frequency variants .", "However , the proportion of low frequency variants in the viral population decreases as selected mutations increase in frequency and become fixed .", "This phenomenon is highlighted in later passages as variants begin to occupy high frequency bins and a bimodal , U-shaped distribution emerges .", "The time resolution afforded by our serial passaging and sequencing strategy provides the opportunity to assess the strength of selection pressure on the influenza genome .", "Non-synonymous mutations fix latest in the Hsp90-inhibited environment and earliest in the HSF1-activated environment ( Figure 4A; outlined in green ) .", "For instance , in the Hsp90-inhibited environment , no mutations have exceeded 60% frequency by passage 9 or 80% frequency by passage 11 .", "In contrast , in the HSF1-activated environment , mutations exceed 80% frequency as early as passage 9 and become fixed ( >90% ) by passage 11 .", "The basal environment lies between these two extremes , with mutations exceeding only 70% frequency by passage 9 and with no mutations yet being fixed at passage 11 .", "As mutations in each environment increase in frequency , all the distributions achieve similar U-shaped , bimodal distributions by passage 23 .", "For the basal and HSF1-activated environments , the distributions become U-shaped by passage 11 , whereas for the Hsp90-inhibited environment , this distribution emerges only after passage 19 .", "To assess the significance of these differences in the shape of the SFS , we applied the Mann-Whitney test , which is a statistical test for comparing two distributions ( Mann and Whitney , 1947 ) .", "Indeed , by passage 11 , the first passage at which we observe fixed mutations in any environment , the shape of the non-synonymous SFS for influenza evolved in the Hsp90-inhibited environment is significantly different from that of influenza evolved in the basal and HSF1-activated environments ( Figure 4A ) .", "This reduced rate of adaptation in Hsp90-inhibited versus basal and HSF1-activated environments is also evident from plotting individual mutation trajectories ( Figure 4—figure supplement 1 ) , revealing specific mutations that increase in frequency more gradually when Hsp90 is inhibited .", "In contrast , the overall shapes of the basal and HSF1-activated SFS are not significantly different .", "As an internal control , we also examined the SFS and mutation trajectories for synonymous variants ( Figure 4B ) .", "We find that synonymous mutations for each environment have similar SFS within each passage , and that the Mann-Whitney test cannot distinguish between the synonymous SFS in different proteostasis environments at any stage of the serial passaging experiment ( Figure 4B ) .", "Moreover , unlike non-synonymous mutations ( see below and also see Figure 4—source data 1 ) , specific synonymous mutations do not fix reproducibly in one particular environment ( Figure 4—source data 2 ) .", "This observation is consistent with our expectation that , although the influenza genome does undergo selection at the RNA level ( Air et al . , 1990 ) , the host proteostasis-based perturbations of HSF1 activation and Hsp90 inhibition should primarily affect influenza evolution by modulating protein-level folding/function .", "The similar SFS for synonymous mutations also confirm that differences between folding environments for non-synonymous mutations cannot be attributed to altered viral growth rates .", "Taken together , these observations draw an intriguing picture of the impact of host proteostasis on viral evolution .", "On the one hand , there is little effect on strongly deleterious mutations , as illustrated by the similar Dn/Ds ratios across environments ( Figure 3D ) .", "On the other hand , the rate of adaptation by accumulation of advantageous mutations is significantly reduced in the Hsp90-inhibited environment , as compared to the basal and HSF1-activated environments .", "This signature of altered selection pressure in distinctive host proteostasis environments highlights the importance of this under-appreciated factor impacting viral evolution .", "The data in Figure 4 show that the host proteostasis environment is a determinant of the nature of selection pressure placed on the influenza genome .", "Next , we examined how the mutational landscapes of individual influenza proteins are influenced by these same environments .", "We anticipated perturbations of HSF1- and Hsp90-mediated proteostasis mechanisms might affect the mutational landscape of polymerase subunits , as their interactions with cytosolic chaperones are well-established ( Momose et al . , 2002; Naito et al . , 2007; Chase et al . , 2008 ) .", "Moreover , our sequencing coverage of the polymerase subunits PA and PB1 was sufficient to rigorously analyze the distribution of mutations in those two genes .", "We aligned all non-synonymous variants present in passage 23 to the secondary structure , the amino acid relative surface accessibility , and the amino acid conservation score ( or site entropy; Figure 5A–B ) .", "Using this analysis , we identified regions of PA and PB1 ( outlined in Figure 5A–B ) with a high density of non-synonymous , but not synonymous , mutations with frequencies above our sequencing error threshold ( 1 . 5% ) .", "We termed these regions mutational hotspots .", "Most mutational hotspots appear in all host environments , indicating sites under strong positive selection in this experimental setting and/or suggesting inherent mutational tolerance of those protein regions that is independent of host proteostasis .", "For example , in polymerase PA , residues 590–640 comprise a mutational hotspot present in every host environment ( Figure 5A ) .", "The region is surface-exposed prior to polymerase complex assembly ( Figure 5A , relative surface accessibility; RSA ) and the mutations largely occur at sites known to be hypervariable ( Figure 5A , site entropy; SE ) ( Pei and Grishin , 2001; Bao et al . , 2008 ) .", "In contrast , certain other mutational hotspots are specific to or absent from one or more host proteostasis environments .", "For example , we observe eight mutations in the N-terminal 100 residues of PB1 in the HSF1-activated environment , two mutations in the baseline environment , and only one mutation in the Hsp90-inhibited environment ( Figure 5B ) .", "The identification of mutational hotspots in the N-terminal domain of PB1 and residues 590–640 of PA is interesting , as their interaction is required for assembly of the mature polymerase complex ( Figure 5C ) ( Reich et al . , 2014; Perez and Donis , 2001; Obayashi et al . , 2008 ) .", "In particular , residues 9–13 of PB1 span the loop portion of a helix-loop-helix motif ( Figure 5B , secondary structure; SS ) that critically defines the interface ( Figure 5C , purple ) with PA ( Figure 5C , red ) ( Reich et al . , 2014; Perez and Donis , 2001; Obayashi et al . , 2008 ) .", "Our observation that amino acid substitutions in PA at the interface are tolerated in all host environments while substitutions in PB1 at the interface are tolerated only when HSF1 is activated ( Figure 5A–B ) prompted us to further investigate the consequences of these amino acid substitutions .", "First , we selected mutations that alter amino acid residues known to make direct contacts at the PA–PB1 interface ( Liu and Yao , 2010 ) and that exceed 1 . 5% frequency ( our sequencing error threshold ) for at least two passages ( Figure 5D ) .", "To estimate their effects on PA:PB1 complex stability , we performed molecular dynamics simulations between wild-type and mutant forms of PAC ( specifically , a C-terminal PA domain covering residues 257–716 ) and PB1N ( an N-terminal PB1 domain spanning the first 15 residues ) ( Liu and Yao , 2010 ) .", "We observed that the PA variants , which occur in both the HSF1-activated and Hsp90-inhibited environments , have slightly less favorable binding energy with wild-type PB1N ( Figure 5E ) .", "The PB1 variants , which occur exclusively in the HSF1-activated environment , are also destabilizing , with the K11E substitution very strongly destabilizing the complex ( Figure 5E ) .", "These data suggest that , whereas our perturbations of host proteostasis may be necessary to accommodate strongly destabilizing mutations in PB1 , they may not be important for moderately destabilizing variants in PA .", "We note that co-occurrence of certain PA variants ( e . g . , E629K in PA ) can compensate for the strongly destabilizing effects of observed PB1 substitutions ( e . g . , the combination of E629K in PA and K11E in PB1 results in a stable complex; Figure 5—source data 1 ) .", "However , the mutation frequencies are not strongly correlated in our data and likely occur in distinctive influenza genomes .", "Cumulatively , these observations indicate that host proteostasis impacts the fitness of destabilizing amino acid substitutions in influenza proteins in a protein-specific manner .", "Next , we analyzed the mutational trajectories of variants present at high frequency in our founder virus to identify those with significantly altered fitness between proteostasis environments .", "Such variants had the opportunity to be selected for or against in all environments and thus are less subject to the stochasticity inherent in a serial passaging-based evolution experiment .", "As we would expect , some non-synonymous variants ( e . g . , HA N262K , NS1 F150S , and PB2 F14S ) follow similar trajectories regardless of host proteostasis ( Figure 4—source data 1 ) .", "Much more interesting are the HA and PA variants that exhibit divergent trajectories upon serial passaging in distinct folding environments ( Figure 6A–B and Figure 4—source data 1 ) .", "Notably , we do not observe any synonymous variants with consistently divergent trajectories between environments ( Figure 4—source data", "2 ) indicating that the divergent PA and HA trajectories are in fact due to selection at the protein level .", "In HA , three variants ( N144T , V242I , and M246I ) present at 20% frequency in our founder virus become fixed by passage 19 in all replicates of the HSF1-activated and basal environments , but in only one replicate of the Hsp90-inhibited environment ( Figure 6A ) .", "Moreover , these variants completely fall out of two replicates of the Hsp90-inhibited environment by passage 9 .", "All three mutations share identical mutational trajectories and V242I and M246I occur together in sequencing reads .", "Hence , the trajectory of only one variant , N144T , is shown in Figure 6A .", "The three mutations occur in the globular head domain of HA that binds cellular sialic acid and has inherently high mutational tolerance ( Thyagarajan and Bloom , 2014 ) .", "These mutations may increase the affinity of HA for MDCK sialic acid receptors , but may be less fit when Hsp90 is inhibited .", "HA folding occurs in the endoplasmic reticulum , which contains an Hsp90 isoform ( Grp94 ) that is also inhibited by STA-9090 ( Marzec et al . , 2012 ) .", "In the absence of active Grp94 , these HA variants may have compromised folding and/or intracellular trafficking , resulting in diminished fitness relative to wild-type HA regardless of any functional advantage they may confer in other host proteostasis environments .", "In PA , we observed that the H452Q variant is present at 20% frequency in the founder virus and becomes fixed in two replicates of the Hsp90-inhibited environment ( Figure 6B ) .", "This variant does not become fixed in the basal environment , and is selected against in the HSF1-activated environment .", "Interestingly , although the H452Q mutation is far from the PA endonuclease site , ( Reich et al . , 2014 ) it is known to occur preferentially in reassorted viruses compared to pure strains , which is indicative of a fitness cost ( Zeldovich et al . , 2015 ) .", "To unequivocally determine the fitness of the H452Q variant relative to wild-type PA in HSF1-activated and Hsp90-inhibited host cells , we performed reverse genetics to enable head-to-head competition of the variants , thereby quantitatively establishing their relative fitness in our three distinct host proteostasis environments .", "We prepared wild-type and H452Q PA-containing influenza populations that were otherwise genetically identical .", "Next , we co-infected host cells displaying HSF1-activated , basal , or Hsp90-inhibited folding environments with equivalent amounts of each virus at low MOI and sequenced the resulting populations after the competition to quantify variant fitness relative to wild-type .", "We found that Hsp90 inhibition does indeed significantly enhance the fitness of the H452Q variant relative to wild-type PA , while HSF1 activation significantly reduces it ( Figure 6C ) .", "One likely possibility is that Hsp90 delays subunit assembly or directs destabilized PA variants to degradation , as H452Q has a predicted ΔΔG of +1 . 10 kcal/mol relative to wild-type PA ( Yin et al . , 2007 ) .", "In either scenario , this observation that the fitness of an otherwise deleterious amino acid substitution in a non-autonomous Hsp90 client can be enhanced by chaperone inhibition but reduced by HSF1 activation is provocative ." ], [ "Considerable evidence suggests that autonomous chaperone networks can critically influence the evolution of their endogenous client protein partners ( Cowen and Lindquist , 2005; Queitsch et al . , 2002; Lachowiec et al . , 2015; Sangster et al . , 2007 , 2008a , 2008b; Rohner et al . , 2013; Whitesell et al . , 2014; Geiler-Samerotte et al . , 2016; Rutherford and Lindquist , 1998; Tokuriki and Tawfik , 2009b ) .", "However , prior to this work , the possibility that host chaperones significantly modulate pathogen evolution had not been rigorously investigated .", "Moreover , in eukaryotic systems , research has focused largely on Hsp90 inhibition .", "Here , we not only define a new role for host proteostasis in influenza evolution , but we also show that two unique proteostasis perturbations , HSF1 activation and Hsp90 inhibition , have distinctive consequences for client protein evolution .", "These consequences are revealed at the levels of the whole genome , individual genes , and specific mutations .", "At the whole genome-level , our data indicate that non-synonymous mutations are fixed more slowly when Hsp90 is inhibited and more quickly when HSF1 is activated .", "Moreover , the overall shape of the Hsp90-inhibited passage 11 SFS is significantly different from that observed in the other two environments .", "Changes in the rate of adaptation could be caused by buffering of mildly deleterious mutations or by potentiation of advantageous variants ( Cowen and Lindquist , 2005; Queitsch et al . , 2002; Lachowiec et al . , 2015; Sangster et al . , 2007 , 2008a , 2008b; Rohner et al . , 2013; Whitesell et al . , 2014; Geiler-Samerotte et al . , 2016; Rutherford and Lindquist , 1998; Cetinbaş and Shakhnovich , 2013 ) .", "In the case of buffering , the rate of fixation of advantageous variants ( driver mutations ) would be decreased if the effects of mildly deleterious variants linked with the driver ( passenger mutations ) are rendered more damaging by Hsp90 inhibition ( McFarland et al . , 2013 , 2014 ) .", "In the case of potentiation , Hsp90 activity could necessitate emergence of the observed high frequency variants .", "Alternatively , Hsp90 activity may reduce weakly deleterious effects of driver mutations on their carrier proteins ( e . g . , disrupted folding caused by an otherwise beneficial mutation ) , thereby increasing the selective advantage provided by the drivers .", "Though we cannot yet fully resolve which of these factors , or combinations thereof , are at play here , overall , we observe that host proteostasis indeed modulates the pace of influenza evolution .", "At the level of individual genes , we find that mutational tolerance at the interface of the PA:PB1 influenza polymerase complex , a region essential for polymerase assembly and activity , is impacted by host proteostasis .", "Interestingly , moderately destabilizing variants in PA are tolerated in all three host proteostasis environments studied .", "In contrast , amino acid substitutions in the N-terminus of PB1 are observed only when HSF1 is activated .", "These PB1 variants strongly destabilize the PB1:PA complex and thus may be accessible only in host cells with high chaperone levels .", "Indeed , polymerase assembly is known to be mediated by cytosolic host chaperones ( Naito et al . , 2007 ) .", "These observations indicate that the evolution of the influenza polymerase complex may be modulated by host proteostasis in significant ways .", "At the level of individual mutations , our data demonstrate that host proteostasis impacts the fitness of specific mutations in at least two viral proteins .", "Intriguingly , the directionality of this effect seems to be specific to a given variant or protein .", "For example , the apparently destabilizing H452Q PA variant is significantly more fit when Hsp90 is inhibited , but significantly less fit when HSF1 is activated .", "These results highlight that the impact of proteostasis perturbation on evolution is currently difficult to predict a priori and demands further study , as one might by default assume that chaperone inhibition would enhance the fitness costs of such a mutation .", "We also identified variants in HA that behave in the opposite manner , displaying apparently enhanced fitness when HSF1 is activated .", "These divergent effects of host proteostasis on the fitness of individual variants illustrate the complexity of the interactions between viral proteins and host proteostasis mechanisms .", "Thus , we observe that host proteostasis modulates the nature of selection on the influenza genome , the mutational tolerance of specific influenza proteins , and the trajectories of particular variants .", "These results are especially compelling because consequences of altered host proteostasis for influenza evolution are emerging in the course of a relatively short-timescale evolution experiment , and without exerting additional selection pressure such as treatment with antiviral drugs .", "As in previous studies on the role of Hsp90 in the evolution of their endogenous protein clients , our observations may derive from direct interactions between host proteostasis components and influenza proteins , or from indirect consequences of perturbing proteostasis .", "Untangling these possibilities will require detailed biophysical and mechanistic studies .", "Regardless of its precise origin , this role for host proteostasis in modulating both the pace and the directionality of influenza evolution is provocative .", "Our observations raise a number of intriguing questions for future work .", "How do host proteostasis mechanisms beyond the heat shock response modulate influenza evolution , and what specific chaperones beyond Hsp90 are involved ?", "Can we quantitatively evaluate the magnitude and ultimately predict the directionality of these effects ?", "Are these effects direct consequences of host chaperones engaging influenza clients ?", "If not , what are the mediators ?", "How does host proteostasis impact the evolution of rapidly evolving viruses beyond just influenza ?", "Do physiological states involving altered or perturbed proteostasis ( e . g . , fever or host-switching ) impact viral evolution ?", "Defining the molecular details of this interplay between host proteostasis and viral evolution will be essential to fully elucidate the factors potentiating and constraining viral evolution .", "Moreover , such studies may eventually enable design of improved antiviral therapeutics and antibodies that are refractory to the evolution of resistance ." ], [ "MDCK cells were generously provided by Prof . Jianzhu Chen ( MIT ) , and were originally purchased from American Type Culture Collection ( Manassas , VA ) .", "The identity of these cells was authenticated by STR profiling .", "MDCK cells were cultured at 37°C in a 5% CO2 atmosphere in DMEM ( CellGro ) supplemented with 10% fetal bovine serum ( CellGro ) and 1% penicillin/streptomycin/glutamine ( CellGro ) .", "Cells were transduced with lentiviruses encoding either the DHFR . HSF1 ( Δ186–202 ) or DHFR . YFP gene .", "Heterostable cells expressing the construct of interest were then selected using 4 μg/mL puromycin .", "Single colonies were generated by diluting cells to ~40 cells per 96-well plate , expanding the resulting colonies , and functionally testing by qPCR or fluorescence microscopy in the presence or absence of trimethoprim ( TMP; 10 μM ) .", "All cell lines were periodically tested for mycoplasma using the MycoSensor PCR Assay Kit from Agilent ( 302109 ) .", "All experiments were performed with influenza A/Wuhan/1995 ( H3N2 ) , which was generously provided by Prof . Jianzhu Chen ( MIT ) .", "STA-9090 was purchased from MedChem Express , sodium arsenite 0 . 1 N standardized solution was purchased from Alfa Aesar , TPCK-trypsin was purchased from Sigma Aldrich , TMP was purchased from Alpha Aesar .", "Mouse monoclonal anti-β-actin was obtained from Sigma ( A1978 ) .", "Rabbit polyclonal anti-HSP70/72 and rabbit polyclonal anti-Hsp40 antibodies were obtained from Enzo Life Sciences ( ADI-SPA-811-D and ADI-SPA-400D , respectively ) .", "The rabbit monoclonal anti-HSP90 antibody was obtained from Cell Signaling Technologies ( C45G5 ) .", "MDCKHSF1 cells were seeded at 200 , 000 cells/well in a 6-well plate and treated with 0 . 01% DMSO , 10 μM TMP , or 10 and 25 nM STA-9090 for 48 hr prior to harvesting cell lysates .", "100 μg of protein lysate was separated on a 12% SDS-PAGE polyacrylamide gel , followed by transfer to a nitrocellulose membrane .", "Hsp70 , Hsp40 , Hsp90 , and actin protein levels were determined using the antibodies described above .", "Membranes were incubated with 680 or 800 nm fluorophore-labeled secondary antibodies ( LI-COR Biosciences , Lincoln , NE ) prior to detection using a LI-COR Biosciences Odyssey Imager .", "Band intensity quantification was performed using Image J . MDCKHSF1 cells were seeded at 100 , 000 cells/well in a 12-well plate and treated with 0 . 01% DMSO , 10 μM TMP , or 10 nM STA-9090 for 24 hr .", "MDCKYFP cells were treated with 0 . 01% DMSO or 10 μM TMP for 24 hr , or 100 μM arsenite for 2 hr as a positive control for heat shock response activation .", "To monitor chaperone levels during influenza infection , MDCKHSF1 cells were infected with influenza A/Wuhan/1995 at an MOI of 1 for 8 hr to properly mimic the actual environment of a cell infected with a single influenza virion as in our serial passaging experiment .", "Cellular RNA was harvested using the Omega RNA Extraction kit with Homogenizer Columns .", "1 μg RNA was used to prepare cDNA using random primers ( total reaction volume = 20 μL; Applied Biosystems High-Capacity Reverse Transcription kit ) .", "The reverse transcription reaction was diluted to 80 μL with water , and 2 μL of each sample was used for qPCR with 2 × Sybr Green ( Roche ) and primers for canis RPLP2 ( housekeeping gene ) , Hsp70 , Hsp40 , Hsp90 , and influenza Matrix ( Supplementary file 1 ) .", "Hsp transcript levels were normalized to RPLP2 .", "For qPCR of influenza-infected cells , a standard curve was prepared with a pDZ plasmid backbone containing the Influenza PR8 M segment to determine influenza Matrix copy number , which was used as a positive control for productive infection .", "For the MDCKHSF1 cell line characterization ( Figure 2 ) , MDCKHSF1 cells were seeded at 100 , 000 cells/well in a 12-well plate and treated with 0 . 01% DMSO , 10 μM TMP , or 10 nM STA-9090 for 24 hr .", "MDCKYFP cells were treated with 0 . 01% DMSO or 10 μM TMP .", "Each treatment was done in biological triplicate .", "Cellular RNA was harvested using the Qiagen RNeasy Plus Mini Kit with QIAshredder homogenization columns .", "RNA-Seq libraries were prepared using the Illumina NeoPrep system and were sequenced on an Illumina HiSeq SE40 .", "Quality control: Reads were aligned against canFam3 ( Sept . 2011 ) using bwa mem v . 0 . 7 . 12-r1039 [RRID:SCR_010910] with flags –t 16 –f .", "Mapping rates , fraction of multiply-mapping reads , number of unique 20-mers at the 5’ end of the reads , insert size distributions and fraction of ribosomal RNAs were calculated using dedicated perl scripts and bedtools v . 2 . 25 . 0 [RRID:SCR_006646] ( Quinlan and Hall , 2010; Huang et al . , 2009 ) .", "In addition , each resulting bam file was randomly down-sampled to a million reads , which were aligned against canFam3 and read density across genomic features were estimated for RNA-Seq-specific quality control metrics ( Figure 2—source data 1 ) .", "RNA-Seq mapping and quantitation: Reads were aligned against canFam3/ENSEMBL 86 ( Aken et al . , 2017 ) annotation using STAR v . 2 . 5 . 3a with the following flags -runThreadN 8 --runMode alignReads --outFilterType BySJout --outFilterMultimapNmax 20 --alignSJoverhangMin 8 --alignSJDBoverhangMin 1 --outFilterMismatchNmax 999 --alignIntronMin 10 --alignIntronMax 1000000 --alignMatesGapMax 1000000 --outSAMtype BAM SortedByCoordinate --quantMode TranscriptomeSAM with --genomeDir pointing to a 75nt-junction canFam3 STAR suffix array ( Dobin et al . , 2013 ) .", "Gene expression was quantitated using RSEM v . 1 . 3 . 0 [RRID:SCR_013027] with the following flags for all libraries: rsem-calculate-expression --calc-pme --alignments -p 8 --forward-prob 0 against an annotation matching the STAR SA reference ( Li and Dewey , 2011 ) .", "Posterior mean estimates ( pme ) of counts and estimated RPKM were retrieved .", "Differential expression analysis: Treatments were compared against DMSO for MDCKHSF1 and MDCKYFP cell lines independently .", "Briefly , differential expression analysis was performed in the R statistical environment ( R v . 3 . 2 . 3 ) using Bioconductor’s DESeq 2 package on the protein-coding genes only [RRID:SCR_000154] ( Love et al . , 2014 ) .", "Dataset parameters were estimated using the estimateSizeFactors ( ) , and estimateDispersions ( ) functions; read counts across conditions were modeled based on a negative binomial distribution and a Wald test was used to test for differential expression ( nbinomWaldtest ( ) , all packaged into the DESeq ( ) function ) , using the treatment type as a contrast .", "Fold-changes , p-values and Benjamin-Hochberg-adjusted p-values ( BH ) were reported for each protein-coding gene ( Figure 2—source data 2 ) .", "Transcripts changing ≥2 fold with a p-value<10–5 are included in Figure 2—source data 3 .", "For the annotation of these transcripts , the reference gene annotation was canFam3/ENSEMBL 86 [RRID:SCR_002344] .", "Canine genes lacking an official gene symbol were manually annotated by individual inspection of the orthology tracks in the UCSC genome browser and reviewing the orthology and paralogy evidence in the ENSEMBL database ( release 89 ) .", "Presumed genes/gene families were assigned based on the depth of gene model conservation across species and orthologs and paralogs assigned by ENSEMBL .", "If transcripts not identified in the reference annotation displayed very strong homology across multiple species , transcripts were annotated with a single asterisk ‘*' or termed ‘gene , by homology’ in Figure 2B and Figure 2—source data 3 , respectively .", "Alternatively , if transcripts not identified in the reference annotation fell within paralog gene families with partial homology they were annotated with a double asterisk ‘**' or termed ‘gene-like’ in Figure 2B and Figure 2—source data 3 , respectively .", "For each biological replicate , MDCKHSF1 cells were seeded at 3 , 000 , 000 cells/plate in 15 cm plates and treated with 0 . 01% DMSO or 10 nM STA-9090 for 4 hr .", "After drug treatment , cells were harvested by trypsinizing , washed twice with PBS , and resuspended in PBS ( with DMSO or STA-9090 ) at a concentration of 20 , 000 , 000 cells/mL .", "This cell suspension was heated in a thermocycler at a temperature gradient ( 100 μL per temperature per treatment condition ) for 3 min , followed by 3 min at room temperature ( Martinez Molina et al . , 2013 ) .", "Samples were then lysed in a modified RIPA buffer without SDS .", "Lysate was then separated by centrifugation and run ( in technical triplicate ) on an SDS-PAGE gel in reducing loading buffer .", "Protein bands were transferred to a nitrocellulose membrane , which was probed with an Hsp90 primary antibody ( C45G5 ) .", "Membranes were incubated with 800 nm fluorophore-labeled secondary antibodies ( LI-COR Biosciences ) prior to detection using a LI-COR Biosciences Odyssey Imager .", "Band intensity quantification was performed using Image J , and the signal was normalized to the band intensity at 37°C .", "Technical replicates were averaged within each biological replicate; biological triplicates were then averaged , with the SEM propagated .", "Serial passaging experiments were performed on a 12-well scale in biological triplicate , at 100 , 000 cells/well and an MOI of 0 . 002 virions/cell , as estimated by hemagglutination titering .", "Cells were pre-treated with TMP or DMSO for 24 hr or with STA-9090 for 90 min prior to influenza infection to establish altered proteostasis environments .", "All infections were performed in DMEM supplemented with penicillin-streptomycin , glutamine , 1 μg/mL TPCK-trypsin , and the relevant small molecules for modulating proteostasis capacity .", "Infections were allowed to proceed for 48 hr , after which the viral supernatant was harvested , cleared of cellular debris by centrifugation , and titered using a hemagglutination assay ( Eisfeld et al . , 2014 ) .", "Viral supernatant was diluted 2-fold across round-bottom 96-well plates with PBS and incubated with human red blood cells ( 3 . 92 × 107 RBC/mL; Innovative Research ) for 30–120 min .", "Wells displaying agglutination were marked influenza-positive , and the titer was determined based on the lowest dilution that was still influenza-positive .", "Influenza was serially passaged in both the MDCKHSF1 and MDCKYFP cell lines in biological triplicate for each treatment condition ( 0 . 01% DMSO , 10 μM TMP , and 10 nM STA-9090 ) for 23 passages .", "Titering was performed in technical duplicate for each biological replicate , with minimal variation observed between technical replicates .", "We employed a TCID50 assay based on that described by Thyagarajan and Bloom ( Thyagarajan and Bloom , 2014 ) .", "Briefly , eight 10-fold dilutions of each virus were prepared in quadruplicate in 96-well plates .", "5 , 000 MDCKHSF1 cells were then added to each well and incubated at 37°C for 72 hr , after which the wells were scored for the presence of cytopathic effect .", "The dilutions of virus displaying cytopathic effect in the MDCKHSF1 cells were then used to calculate the TCID50/μL using https://github . com/jbloomlab/reedmuenchcalculator as described by Thyagarajan and Bloom , ( Thyagarajan and Bloom , 2014 ) where virions/μL = 0 . 69*TCID50/μL .", "The H452Q PA variant was introduced by site directed mutagenesis on a wild-type PA pHW2000 reverse genetics plasmid for the influenza A/Wuhan/1995 H3N2 strain ( generous gift from Prof . Hui-Ling Yen at Hong Kong University ) ( Cheung et al . , 2014 ) .", "The corresponding mutant and wild-type viruses were made by co-transfection on a co-culture of MDCK and HEK 293T cells , previously described by Hoffman et al ( Hoffmann et al . , 2000 ) .", "Viruses were titered using a TCID50 assay ( Thyagarajan and Bloom , 2014 ) to perform competition experiments starting with approximately the same amount of wild-type and mutant virus .", "Competitions were performed under conditions identical to that of the serial passaging experiments , in biological triplicate .", "RNA from the P0 and P1 viral supernatant was harvested and prepared for sequencing , as described below .", "RNA was extracted from 140 μL influenza supernatant from passages P0 , P2 , P9 , P11 , P19 and P23 using the Qiagen RNA Mini Kit and eluted in 40 μL molecular biology grade H2O .", "dsDNA was made from 2 . 5 μL template RNA using universal influenza primers as previously described , ( Zhou et al . , 2009 ) except that the small segments were amplified separately from the polymerase segments and pooled following PCR .", "The amplicons were separated on a 0 . 8% agarose analytical gel to verify the presence of each influenza genomic segment ( 8 total ) .", "1 ng of each sample was prepared using the Illumina NexteraXT Sample Preparation kit , omitting the bead normalization step .", "The concentration of dsDNA in each sample was quantified by Qubit; samples were pooled in sets of 24 and sequenced on an Illumina MiSeq 300v2 cartridge to obtain 2 × 150 base pair paired-end reads .", "RNA from the reverse genetics competition experiments was sequenced using the same protocol , except primers that specifically amplified ~900 bp of the PA gene spanning the mutation site were used for PCR ( Supplementary file 1 ) .", "Sequencing reads were aligned against the influenza A/Wuhan/1995 complete CDS sequence , or the influenza A/Wuhan/1995 PA sequence ( for reverse genetics sequencing ) , using bwa mem 0 . 7 . 10-r789 [RRID:SCR_010910] .", "Allele pileups were generated using samtools v . 0 . 1 . 19 mpileup [RRID:SCR_002105] with flags -d 10000000 --excl-flags 2052 , and allele counts/frequencies were extracted ( Li , 2011; Li et al . , 2009 ) .", "Only positions with greater than 600-fold coverage in all replicates of each sample were included in the analysis .", "Variant alleles present at greater than 1 . 5% frequency are included in the site frequency spectra ( Figure 4 ) , protein alignment ( Figure 5 ) , and mutational trajectories ( Figure 6 ) analyses .", "This frequency threshold is the lowest mutation frequency at which a mutation can be reliably detected in a sample that is sequenced in technical duplicate , for our specific sequencing method and instrument .", "A 600-fold coverage threshold requires that we observe such a mutation a minimum of nine times .", "All trajectories for mutations that increase in frequency during passaging are included in Figure 4—figure supplement 1 .", "Increasing trajectories are those best fitting an increasing exponential model aebx , where a > 0 and b > 0 . 1 .", "Best fit was determined by comparing least squares regression value .", "Selected mutations with divergent trajectories between environments are plotted in Figure 6 .", "Site frequency spectra were generated by binning all mutations meeting our coverage ( 600-fold ) and frequency ( >1 . 5% ) thresholds into 10% frequency bins and averaging the portion of mutations within a given frequency bin across biological triplicates .", "To quantify differences in the passage 11 site frequency spectra , the Mann-Whitney test was performed using Graph Pad Prism software ( details in statistics section below ) .", "Alignment analyses were performed by aligning mutations meeting our coverage and frequency thresholds to the corresponding secondary structure and relative surface accessibility using DSSP , ( Kabsch and Sander , 1983; Joosten et al . , 2011; Bloom , 2014 ) as well as to the site entropy , which was computed using all full-length protein sequences from the Influenza Virus Resource for all Influenza A PA and PB1 sequences except bat influenza sequences ( Pei and Grishin , 2001; Bao et al . , 2008; Katoh and Standley , 2013 ) .", "Observed mutations were also mapped onto the corresponding protein crystal structure ( PA-PB1 PBDID 4WSB ) ( Reich et al . , 2014 ) .", "Mutational hotspots were manually determined as regions with a high density of exclusively non-synonymous mutations .", "Sequencing reads from reverse genetics competition experiments were aligned to the PA reference sequence to determine %-frequency of mutant and wild-type alleles .", "The ratio of mutant to wild-type PA was calculated for each replicate of each proteostasis environment; the resulting ratio was normalized to the average ratio of mutant to wild-type PA in the basal proteostasis environment .", "Ratios were then averaged for each set of biological replicates and plotted as a bar chart with SEM .", "An unpaired t-test was used to assess statistical significance between host environments .", "Mutant protein stability predictions were made using Eris , ( Yin et al . , 2007 ) employing the fixed backbone setting .", "All experiments were performed in at least biological triplicate , which we define as replicates that are independent for the entirety of the experiment ( i . e . , from plating the cells , to treating the cells , to acquiring the data ) .", "To quantify differences in the passage 11 site frequency spectra ( Figure 4 ) , the Mann-Whitney test was performed using Graph Pad Prism software ( nonparametric , one-tailed ) .", "This test was performed on the passage 11 site frequency spectra because this is the first passage at which we observe fixed mutations in any environment .", "A one-tailed test was used as we expect mutation frequency spectra to shift in a one-directional manner as mutations become fixed and the distribution becomes U-shaped .", "All mutation frequencies observed in the two distributions ( where each distribution represents three biological triplicate data sets ) to be compared were ranked .", "Each set of ranks was then compared to determine if the distributions are significantly different .", "The Mann-Whitney ( Mann and Whitney , 1947 ) U values were: 2109 ( HSF1 ( N = 73; median = 4 . 92 ) vs . Hsp90-inhibited ( N = 71; median = 3 . 472 ) non-synonymous ) , 661 ( HSF1 ( N = 41; median = 4 . 01 ) vs . Hsp90-inhibited ( N = 34; median = 3 . 212 ) synonymous ) , 2340 ( basal ( N = 79; median = 4 . 787 ) vs . Hsp90-inhibited ( N = 71; median = 3 . 472 ) non-synonymous ) , 776 ( basal ( N = 48; median = 3 . 934 ) vs . Hsp90-inhibited ( N = 34; median = 3 . 212 ) synonymous ) , 2819 ( HSF1 ( N = 73; median = 4 . 92 ) vs . basal ( N = 79; median = 4 . 787 ) non-synonymous ) , 970 ( HSF1 ( N = 41; median = 4 . 01 ) vs . basal ( N = 48; median = 3 . 934 ) synonymous ) .", "To assess statistical significance for the reverse genetics competition experiment ( Figure 6C ) , an unpaired t-test was performed between each set of conditions , each with three biological replicate data sets ( basal vs . HSF1: p-value =0 . 0019; t = 4 . 192; df = 10; basal vs . Hsp90-inhibited: p-value =0 . 0091; t = 3 . 224; df = 10; HSF1 vs . Hsp90-inhibited: p-value <0 . 0001; t = 12 . 45; df = 10 ) .", "To assess statistical significance for the CETSA ( Figure 1—figure supplement 1D ) , the vehicle and STA-9090 conditions were compared across biological triplicates ( technical triplicates for each biological triplicate ) by an F-test ( p-value <0 . 0001 , F ( DFn , DFd ) =16 . 48 ( 1 , 194 ) ) .", "The wild type PAC–PB1N complex was taken from the crystal structure with PDBID 2ZNL ( Obayashi et al . , 2008 ) .", "PAC is the C-terminal PA domain ( residues 257–716 ) , and PB1N is the N-terminal domain ( residues 1–15 ) of PB1 .", "In 2ZNL there are some missing residues ( residues 349–353; 372–397; 550–557 ) , but they were distant from the PAC–PB1N binding site .", "As all of these missing residues were far from the binding site they were not added back in .", "Furthermore , as described below , only residues 398–716 of PAC were allowed to move and the rest of PAC was position-restrained throughout the simulations .", "For each segment of PAC , the termini were far away from the interaction site and thus were left uncapped .", "The N-terminus of PB1N was also left uncapped; however , the C-terminus of the short 15-residue PB1N was capped with an N-methyl group to remove the artificial negative charge .", "All mutations of the wild-type complex were made using the Pymol molecular modeling package ( DeLano , 2002 ) .", "All MD simulations were performed using the GROMACS 4 . 6 . 7 suite ( Hess et al . , 2008 ) .", "All simulations were performed using the AMBER99 force field ( Hornak et al . , 2006 ) and TIP3P ( Jorgensen et al . , 1983 ) water .", "Each initial complex structure ( wild-type and mutant subunits ) was first immersed in a cubic box containing pre-equilibrated water molecules .", "The dimensions of the water box were 102 Å ×109 Å × 79 Å .", "Counter-ions were added as necessary to neutralize the system .", "The solvated system was then energy-minimized using the steepest descent algorithm for a maximum of 5000 iterations with a force constant of 1000 kJ mol–1 nm–2 applied to all non-hydrogen atoms .", "Next , a further energy minimization was performed without any constraints where the steepest descent method switched to conjugate gradient every 500 steps for a maximum of 2500 total steps .", "The system then underwent a two-stage equilibration process .", "The first stage of equilibration was a 100 ps NVT ( isochoric–isothermal ) simulation and consisted of a gentle annealing of the system from 0 to 300 K over the first 50 ps .", "Throughout the first stage of equilibration , a position restraint was applied to all non-hydrogen atoms with a force constant of 1000 kJ mol–1 nm–2 .", "The temperature was maintained at 300 K using the V-rescale thermostat ( Bussi et al . , 2007 ) with a coupling time constant of 1 . 0 ps .", "The complex and solvent molecules were coupled to separate thermostats to avoid the ‘hot solvent-cold solute’ problem ( Cheng and Merz , 1996; Lingenheil et al . , 2008 ) .", "The second stage of equilibration was a 500 ps NPT ( isobaric–isothermal ) simulation where position restraints were applied to all non-hydrogen atoms of residues 257–348 and 354–371 of PAC , as these residues are far from the PAC–PB1N interaction site .", "The pressure was regulated using the Berendsen barostat ( Berendsen et al . , 1984 ) with a reference pressure of 1 bar and a pressure coupling constant of 2 . 0 ps .", "The leapfrog algorithm ( Hockney , 1970 ) with a time step of 2 fs was used for dynamics evolution .", "All bonds involving hydrogen were constrained using the LINCS algorithm ( Hess et al . , 1997 ) .", "All neighbor searching , electrostatic interactions and van der Waals interactions were truncated at 1 . 4 nm .", "Long-range Coulomb interactions were treated using the particle mesh Ewald ( PME ) summation ( Essmann et al . , 1995 ) with a Fourier spacing of 0 . 12 nm and a PME order of 4 .", "A long-range dispersion correction for energy and pressure was applied to account for the 1 . 4 nm cut-off of Lennard-Jones interactions ( Allen and Tildesley , 1987 ) .", "All production runs followed the same scheme as the second equilibration stage and were run for 20 ns total .", "Binding free energy calculations were performed on the last 10 ns of each production simulation .", "The last 10 ns of each simulation were stripped of water molecules and counter ions .", "From these , snapshots were extracted every 10 ps giving 1000 snapshots for the last 10 ns .", "The binding free energy was calculated as described by Liu and Yao ( Liu and Yao , 2010 ) .", "Here , the binding free energy was calculated as Htotal , GB = Egas + Gsol , GB , and thus did not contain any contribution from solute configuration entropy .", "Since our focus here is on the relative order of binding affinities and the complexes have similar binding poses , the solute configuration entropy is thus omitted ( Rastelli et al . , 2010; Hou et al . , 2011a , 2011b; Wang et al . , 2001 ) .", "The GBSA implicit solvent model ( Still et al . , 1990 ) was used with a dielectric constant of 80 .", "The OBC ( II ) ( Onufriev et al . , 2004 ) algorithm was used to calculate the Born radii with a frequency of 1 and a cutoff of 1 . 4 nm .", "A surface tension of 3 . 01248 kJ mol–1 nm–2 was set using the Ace-approximation .", "Averages and standard error of mean were calculated based on 200 snapshot increments ." ] ]

[ "Predicting and constraining RNA virus evolution require understanding the molecular factors that define the mutational landscape accessible to these pathogens .", "RNA viruses typically have high mutation rates , resulting in frequent production of protein variants with compromised biophysical properties .", "Their evolution is necessarily constrained by the consequent challenge to protein folding and function .", "We hypothesized that host proteostasis mechanisms may be significant determinants of the fitness of viral protein variants , serving as a critical force shaping viral evolution .", "Here , we test that hypothesis by propagating influenza in host cells displaying chemically-controlled , divergent proteostasis environments .", "We find that both the nature of selection on the influenza genome and the accessibility of specific mutational trajectories are significantly impacted by host proteostasis .", "These findings provide new insights into features of host–pathogen interactions that shape viral evolution , and into the potential design of host proteostasis-targeted antiviral therapeutics that are refractory to resistance ." ]

[ "Influenza viruses , commonly called flu , can evade our immune system and develop resistance to treatments by changing frequently .", "Specifically , mutations in their genome cause influenza proteins to change in ways that can help the virus evade our defences .", "However , these mutations come at a cost and can prevent the viral proteins from forming functional and stable three-dimensional shapes – a process known as protein folding – thereby hampering the virus’ ability to replicate .", "In human cells , proteins called chaperones can help our other proteins fold properly .", "Influenza viruses do not have their own chaperones and , instead , hijack those of their host .", "Host chaperones are therefore crucial to the virus’ ability to replicate .", "However , until now , it was not known if host chaperones can influence how these viruses evolve .", "Here , Phillips et al . used mammalian cells to study how host chaperones affect an evolving influenza population .", "First , cells were engineered to either have normal chaperone levels , elevated chaperone levels , or inactive chaperones .", "Next , the H3N2 influenza strain was grown in these different conditions for nearly 200 generations and sequenced to determine how the virus evolved in each distinctive host chaperone environment .", "Phillips et al . discovered that host chaperones affect the rate at which mutations accumulate in the influenza population , and also the types of mutations in the influenza genome .", "For instance , when a chaperone called Hsp90 was inactivated , mutations became prevalent in the viral population more slowly than in cells with normal or elevated chaperone levels .", "Moreover , some specific mutations fared better in cells with high chaperone levels , whilst others worked better in cells with inactivated chaperones .", "These results suggest that influenza evolution is affected by host chaperone levels in complex and important ways .", "Moreover , whether chaperones will promote or hinder the effects of any single mutation is difficult to predict ahead of time .", "This discovery is significant , as the chaperones available to influenza can vary in different tissues , organisms and infectious conditions , and may therefore influence the virus' ability to change and evolve in a context-specific manner .", "The findings are likely to extend to other viruses such as HIV and Ebola , which also hijack host chaperones for the same purpose .", "More work is now needed to systematically quantify these effects so that we can better predict how specific chaperones will affect the ability of viruses to adapt , especially in pathologically relevant conditions like fever or viral host-switching .", "In the future , such insights could help shape the design of treatments to which viruses do not evolve resistance ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "short report", "neuroscience" ]

[ [ "Schwann cells , the myelinating cells of the peripheral nervous system ( PNS ) , contain peroxisomes in distant cytoplasmic compartments , including myelin channels and cytosolic loop regions close to nodes of Ranvier ( Kassmann et al . , 2011 ) .", "Peroxisomes degrade fatty acids derived from myelin lipids and generate precursors of myelin plasmalogens ( Waterham et al . , 2016 ) .", "Lysosomal compartments are also present in these nodal regions ( Gatzinsky et al . , 1997 ) , and growing evidence from in vitro studies suggests interactions between both types of organelles ( Baarine et al . , 2012; Chu et al . , 2015; Thai et al . , 2001 ) .", "Peroxisomal dysfunction is caused by mutations of genes encoding peroxisomal proteins or peroxisomal biogenesis factors ( Waterham et al . , 2016 ) .", "In humans , loss-of-function mutations of ABCD1 are responsible for the disease X-linked Adrenoleukodystrophy ( X-ALD ) .", "This peroxisomal ATP-binding cassette ( ABC- ) transporter mediates the import of very long-chain fatty acids ( VLCFA ) into the organelle .", "In consequence , ABCD1-deficient peroxisomes are not capable of importing and degrading VLCFA that are specific substrates of peroxisomal β-oxidation ( Kemp et al . , 2012 ) .", "A more severe impairment of peroxisomes is caused by lack of the Hsd17b4 gene ( also called multifunctional protein 2; Mfp2 gene ) that encodes a central enzyme of peroxisomal β-oxidation .", "In MFP2-deficient cells , the β-oxidation of virtually all peroxisome-specific substrates , including VLCFA , is inhibited ( Verheijden et al . , 2013 ) .", "A complete disruption of the organelle is observed in the absence of peroxisome biogenesis factor peroxin 5 ( PEX5 ) .", "This cycling receptor recognizes proteins with a peroxisomal targeting sequence type 1 ( PTS1 ) and is involved in their transfer into peroxisomes .", "PEX5-dependent protein import applies to the majority of peroxisomal enzymes .", "Thus , PEX5-deletion disrupts peroxisomal function substantially ( Waterham et al . , 2016 ) .", "Schwann cell lipid metabolism is rate-limiting for myelination and is important for maintenance of axonal integrity ( Saher et al . , 2011; Viader et al . , 2013 ) , which requires in addition to membrane wrapping the assembly of nodal , paranodal , and juxtaparanodal membrane proteins ( Rasband and Peles , 2015 ) .", "The juxtaparanodal domain of myelinated axons harbors voltage-gated shaker-type potassium channels , Kv1 . 1 ( KCNA1 ) and Kv1 . 2 ( KCNA2; Chiu and Ritchie , 1980; Robbins and Tempel , 2012 ) , which also align the inner mesaxon as a thin band ( Arroyo et al . , 1999 ) .", "Associated with connexin-29 hemichannels ( Rash et al . , 2016 ) , their clustering and anchoring at juxtaparanodes requires the neuronal membrane proteins CASPR2 and TAG-1 , the latter expressed by glia and neurons ( Poliak et al . , 1999b; Traka et al . , 2003 ) .", "Kv1 channels have been proposed to play a role in regulating fiber excitability ( Baker et al . , 2011; Glasscock et al . , 2012 ) , but the exact in vivo function of these fast-opening/slowly inactivating channels remains unknown ( Arancibia-Carcamo and Attwell , 2014 ) ." ], [ "Cnp-Cre::Pex5flox/flox mice , termed cKO or 'mutants' in the following , lack peroxisomal protein import in Schwann cells ( Figure 1a; Figure 1—figure supplement 1a ) .", "The PNS of these mice is well myelinated and unlike the CNS ( Kassmann et al . , 2007 ) without immune-mediated injury , in agreement with pilot observations ( Kassmann et al . , 2011 ) .", "Upon closer inspection , we determined about 50% genomic recombination , corresponding to the fraction of Schwann cell ( SC ) nuclei in sciatic nerves ( Figure 1—figure supplement 1b ) .", "Teased fiber preparations , stained for PMP70 , revealed peroxisomes as puncta .", "In mutant nerves , these were import-deficient 'ghosts' , as evidenced by cytoplasmic catalase , normally a luminal peroxisomal marker ( Figure 1b ) . 10 . 7554/eLife . 23332 . 003Figure 1 . Schwann cell-specific PEX5-deficiency causes peroxisome dysfunction and peripheral neuropathy in the absence of axonal loss or dysmyelination .", "( A ) Scheme of normal ( left ) and impaired ( right ) PEX5-dependent peroxisomal protein import .", "PTS1 , peroxisomal targeting signal type 1; PEX5 , peroxisomal biogenesis factor peroxin 5 .", "( B ) Catalase ( red ) is present in peroxisomes ( PMP70; green ) of controls , but is localized in the cytoplasm of mutant fibers .", "PMP70 , peroxisomal membrane protein 70; DAPI-stained SC nuclei are depicted in blue; Scale bar , 10 µm .", "( C , D )", "Lipid mass spectrometry of nerve lysates from controls and mutants aged 9 months indicates peroxisomal dysfunction .", "Peroxisomal products ( PEO- ) and its corresponding plasmalogens ( PEP- ) are reduced .", "Specific substrates of peroxisomal β-oxidation , VLCFA , are accumulated in mutant nerves .", "Statistics: means ± s . d . ; n = 6; Student’s T-test; ***p<0 . 001 .", "( E–G )", "Functional impairment of mutant compared to control sciatic nerves is assessed by electrophysiology at the age of 2 months ( n = 4 ) .", "To evoke significant responses of all measured nerves , larger stimulus intensity is required for mutant ( 0 . 155mA ) as compared to control ( 0 . 135mA ) nerves .", "Peak amplitudes plotted against increasing stimulus intensity indicates earlier response saturation of mutant nerves .", "CAP , compound action potentials; au , arbitrary units; NCV , nerve conduction velocities ( each curve representing single-nerve mean responses across intensities ) .", "( H ) g-ratio analysis by electron microscopy of sciatic nerves as measure of myelin thickness ( n = 3; ≥100 randomly chosen axons per nerve at age 2 months ) .", "( I ) Axonal analysis by methylene blue-stained semithin cross-sections of sciatic nerves of control and mutant mice at the age of 2 months ( n = 3 ) .", "Scale bar , 10 µm .", "( J ) Immunostained teased sciatic nerve fibers of mutants and controls at 2 and 9 months show normal paranodal localization of CASPR ( red ) and anchoring protein neurofascin ( NF155; green ) .", "( K , L )", "Intact transverse bands ( arrows ) at paranodal loops ( PNLs ) between axon ( Ax ) and myelin ( M ) by electron microscopy in a 9-month mutant nerve , even when PNLs harbor enlarged vesicles ( top inset , arrowheads ) .", "Mutant nerves display normal adherens junctions ( AJ ) between loops ( bottom inset , asterisk ) .", "Scale bars , 500 nm; scale bars in insets , 250 nm . DOI: http://dx . doi . org/10 . 7554/eLife . 23332 . 00310 . 7554/eLife . 23332 . 004Figure 1—figure supplement 1 . Successful PEX5 ablation causes peroxisomal dysfunction .", "( A ) PCR on genomic tail DNA using primers specifically generating a 300 bp amplicon after Cre-mediated excision of Pex5-floxed exons 11–14 .", "( B ) qRT-PCR reveals a 50% ( corresponding to the fraction of SC nuclei ) reduction of Pex5 mRNA level in mutant nerves ( n = 6 ) .", "( C ) Quantitative mass spectrometry of sciatic nerve lysates .", "Plasmalogens were abundant in a control nerve ( top , green arrows at peaks ) .", "The same plasmalogens were hardly detectable in a mutant nerve lysate ( bottom , red arrows at peaks ) .", "( D ) Analysis of VLCFA by gas chromatography–mass spectrometry in control and mutant mice .", "Statistics: means ± s . e . m . ; n = 6; *p<0 . 05; **p<0 . 01; ***p<0 . 001 , Student’s T-test ( B , D ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 23332 . 00410 . 7554/eLife . 23332 . 005Figure 1—figure supplement 2 . Electrophysiology of mouse sciatic nerves .", "( A ) Photographs of electrophysiology setup with a pair of suction electrodes .", "( B ) Pex5 mutant nerves display smaller and delayed responses when elicited at stimulus intensities of 0 . 17mA ( left ) or 0 . 22mA ( right ) .", "( C ) In vivo electrophysiology of controls and PEX5 cKO shows a greater decline of the compound muscle action potential ( CMAP ) amplitudes measured at more proximal versus distal stimulation indicating conduction blocks .", "The ratios of the amplitudes elicited by proximal vs . distal stimulation are depicted .", "Statistics: n = 5 , means ± s . d . **p<0 . 01 , Student’s T-test . DOI: http://dx . doi . org/10 . 7554/eLife . 23332 . 00510 . 7554/eLife . 23332 . 006Figure 1—figure supplement 3 . Myelin analysis of mouse sciatic nerves .", "( A ) Chromogenic immune-staining for myelin protein zero ( MPZ/P0 ) on paraffin-embedded sciatic nerve sections at 2 months .", "Scale bar , 50 µm .", "( B ) Abundance of major PNS myelin proteins P0 and PMP22 , as well as fatty-acid-binding protein P2 , and DM20 were not significantly altered by quantification of Western blot analysis of sciatic nerve lysates obtained at the age of 2 months ( n = 4; each normalized to control levels; 1 . 0 ) .", "Only the covalently lipid-binding protein PLP showed significant reduction in mutant as compared to control nerves .", "Alpha-Tubulin served as loading control .", "Statistics: mean ± s . e . m . *p<0 . 05; **p<0 . 01; ***p<0 . 001; n . s . , not significant , Student’s T-test .", "( C ) Electron micrographs of sciatic nerve cross-section showing myelin sheaths of animals aged 2 months .", "Scale bars , 10 nm .", "( D ) Internodal lengths measured using teased fibers of 2-month-old PEX5 mutant and control animals .", "Statistics: n = 3 , mean ± s . d . *p<0 . 05 , Student’s T-test .", "( E ) Chromogenic immune-staining for APP on longitudinal paraffin-embedded sciatic nerve sections .", "Scale bar , 50 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 23332 . 00610 . 7554/eLife . 23332 . 007Figure 1—figure supplement 4 . Young Pex5 mutant nerves lack signs of inflammation .", "( A , D )", "Chromogenic immunostaining for MAC-3 ( A ) and CD3 ( D ) on longitudinal paraffin-embedded sciatic nerve sections of mice aged 2 and 9 months .", "( B , C )", "Mast cells ( arrow ) visualized and quantified using entire semithin ( 500 nm ) methylene-blue-stained cross-sections of mouse sciatic nerves at 2 months of age ( n = 4 ) .", "Statistics: means ± s . e . m . *p<0 . 05; **p<0 . 01; ***p<0 . 001; Student’s T-test . DOI: http://dx . doi . org/10 . 7554/eLife . 23332 . 007 Peroxisomal dysfunction in myelinating SC was confirmed by lipid mass spectrometry ( Figure 1c , Figure 1—figure supplement 1c ) , showing reduced plasmalogens ( PEP- ) and its precursor alkylated phosphatidyl-ethanolamines ( PEO-; Wanders , 2014 ) .", "Also VLCFA were increased , indicating the accumulation of peroxisomal β-oxidation substrates ( Figure 1d; Figure 1—figure supplement 1d ) .", "We determined nerve conduction velocity ( NCV ) by electrophysiology of isolated sciatic nerves ( to avoid possible contributions of altered muscle endplates ) at the age of 2 months ( Figure 1e–g; Figure 1—figure supplement 2a ) .", "For all stimulus intensities tested , responses of mutant nerves were different from controls ( Figure 1—figure supplement 2b ) .", "Compound action potentials ( CAPs ) and NCV were diminished in mutants ( mean: 28 ± 4 . 7 m/s ) compared to controls ( 41 . 5 ± 3 . 6 m/s; Figure 1e ) .", "Thresholds to evoke a signal were only slightly elevated ( 155µA versus 135µA ) , but the maximal response was 50% of control ( Figure 1f , g ) .", "Also , in vivo recordings revealed significantly reduced compound muscle action potentials ( CMAPs ) in mutant mice ( Kassmann et al . , 2011 ) .", "This was more enhanced when stimulating proximally than distally , which clinically defines conduction blocks ( Figure 1—figure supplement 2c ) .", "We suspected that reduced nerve conduction would be explained by demyelination .", "Surprisingly , by immunohistochemistry and Western blot analysis structural myelin proteins , including PMP22 , MPZ/P0 , and P2 , were not significantly altered ( Figure 1—figure supplement 3a , b ) .", "Only PLP , a minor PNS myelin protein , showed significant reduction .", "Also by electron microscopy ( EM ) , myelin thickness , periodicity , and compaction were indistinguishable ( Figure 1h , Figure 1—figure supplement 3c ) .", "Next , we determined internodal length in teased fiber preparations , which was shorter in mutant ( 623 nm ) than in control fibers ( 691 nm; Figure 1—figure supplement 3d ) , but unlikely sufficiently reduced to cause a slower conduction by itself ( Wu et al . , 2012 ) .", "Importantly , while the reduced CAP suggested significant axon loss at 2 months , the morphometric analysis of entire nerve cross-sections revealed normal axonal numbers and calibers ( Figure 1i ) .", "Thus , reduced CAPs were most likely caused by functional conduction blocks rather than physical axon loss .", "Signs of axonal transport defects ( APP accumulations ) were not observed ( Figure 1—figure supplement 3e ) , and also , indirect signs of neurodegeneration ( neuroinflammation ) were not significantly enhanced in nerves at 2 months ( Figure 1—figure supplement 4a–d ) .", "Disruptions of axo-glial junctions might cause leak currents and thus conduction failure ( Arancibia-Carcamo and Attwell , 2014 ) .", "Yet , both neurofascin-155 ( NF155 ) and axonal CASPR , that is adhesion proteins that mediate axo-glial contacts ( Desmazieres et al . , 2014; Normand and Rasband , 2015 ) , were normally localized ( Figure 1j ) .", "By EM , we never found the detachment of paranodal loops ( Figure 1k , l ) , even where abnormally enlarged by accumulated vesicles ( Kassmann et al . , 2011 ) .", "Next , we examined the distribution of ion channels ( Devaux et al . , 2004; Rasband and Peles , 2015 ) in sciatic nerve teased fibers from 2- and 9-month-old mutants and controls .", "Nodal Nav1 . 6 and Kv7 . 2 , as well as their anchoring proteins , were normally flanked by CASPR-positive paranodes ( Figure 2—figure supplement 1a–c ) .", "Surprisingly , the juxtaparanodal potassium channel protein Kv1 . 1 displayed an abnormal localization , frequently displaced into the adjacent internodal region , and to a minor extent into paranodes .", "Kv1 . 1 clusters were in the majority of cases still maintained at juxtaparanodes , suggesting that internodal mislocalization is secondary ( Figure 2—figure supplement 1d , e; Figure 2a ) .", "Indeed , the pathology of internodal Kv1 channels progressively increased with age frequently presenting more than one extra cluster along one internode in aged mutants , while shifts into the nodal direction were not progressive ( Figure 2a , b; Figure 2—figure supplement 1e ) .", "We confirmed the observation of displaced juxtaparanodal Kv1 channels using Kv1 . 2 antibody ( data not shown ) .", "At 9 months , we determined a twofold overall increase of Kv1 . 1 in nerve lysates ( Figure 2c , d ) .", "However , as most Kv1 . 1 is expressed by unmyelinated fibers , the increase of internodal Kv1 . 1 in myelinated axons may be significantly higher .", "Neuronal CASPR2 , the cis-anchor for Kv1 channels in the axonal membrane , and TAG-1 on SC are essential for channel clustering at juxtaparanodes ( Hivert et al . , 2016; Poliak et al . , 1999a; Savvaki et al . , 2010 ) .", "Both proteins colocalized with Kv1 . 1 at regular and ectopic internodal positions ( Figure 2e–g ) .", "TAG-1 expression was slightly but not significantly elevated ( Figure 2h ) .", "In contrast , staining for myelin-associated glycoprotein ( MAG ) did not reveal a connection between Schmidt-Lanterman incisures and the ectopic Kv1 . 1/TAG-1/CASPR2-positive clusters ( data not shown ) .", "Although , we cannot exclude that enhanced expression of TAG-1 contributes to the formation of ectopic protein clusters , the observations suggest that in mutant nerves entire membrane patches of the juxtaparanodal compartment accumulate , break-off , and drift into the internodal region . 10 . 7554/eLife . 23332 . 008Figure 2 . Abnormal distribution of axonal ion channels Kv1 . 1 , its anchoring proteins , and associated lipids in conditional Pex5 mutant nerves .", "( A , B )", "Immune-stained sciatic teased fibers of 2- and 9-month-old mice reveal normally localized nodal sodium channels ( Nav1 . 6; blue ) flanked by paranodal CASPR ( red; scale bar , 10 µm ) .", "Only mutant nerves show extra Kv1 . 1+ patches within internodes .", "This is progressive with age shown by the corresponding quantification at different ages , starting at P19 , revealing a shift of Kv1 . 1 primarily to internodes ( IN ) , and ( not significantly ) to paranodes ( PN ) .", "n = 3 for P19 and 1 month; n = 5 for 2 months; n = 7 for>9 months .", "Statistics: means ± s . e . m , two-way ANOVA followed by Student’s T-test .", "*p<0 . 05; **p<0 . 01; ***p<0 . 001; n . s . , not significant .", "( C , D )", "Western blot analysis of sciatic nerve lysates of 9-month-old mice shows increased Kv1 . 1 protein abundance in mutant nerve lysates .", "β-III Tubulin served as loading control for normalization ( n = 4 ) .", "( E–G )", "Kv1-anchoring proteins ( green ) CASPR2 and TAG-1 showed in addition to normal juxtaparanodal localization an ectopic internodal ( arrow heads ) and occasionally a partial overlap ( asterisk ) with paranodal CASPR ( red ) in mutant sciatic teased fibers .", "Both proteins colocalized at original ( white arrows ) and ectopic ( arrow heads ) positions ( bottom panels ) .", "Quantification of TAG-1/Kv1 . 1-positive ectopic clusters is depicted , ( G ) .", "Blue arrows indicate nodes of Ranvier .", "Scale bars , 10 µm .", "( H ) qRT-PCR analysis ( n = 5 ) for TAG-1 showing a tendency of increased mRNA expression in mutant compared to control nerves at 2 months .", "( I ) Mass spectrometry of sciatic nerve lysates ( n = 3 ) obtained at the age of 9 months shows percentage of GD1 species per genotype containing 41 , 43 , or 44 C-atoms ( ‘:’ separates number of double-bonds and ‘;’ separates number of hydroxyl-groups ) .", "Only 0 . 74% of GD1 in control lysates contain 41 carbons , species with more carbons are absent , while highly enriched in mutant nerve lysates .", "( J ) Antibody-staining for gangliosides of nerves from mice aged 9 months shows GD1a+ vesicles abnormally distended into mutant internodes ( green , top ) .", "GM1 is localized at paranodes of control fibers , but extends into mu internodes .", "Dotted lines indicate borders of myelinated fibers .", "Blue arrows indicate nodes of Ranvier .", "Scale bars , 10 µm .", "Statistics: means ± s . d . ; *p<0 . 05; **p<0 . 01; ***p<0 . 001; n . s . , not significant , Student’s T-test . DOI: http://dx . doi . org/10 . 7554/eLife . 23332 . 00810 . 7554/eLife . 23332 . 009Figure 2—figure supplement 1 . Normal distribution of nodal proteins and associated anchors in Pex5 mutant nerves . Immune-stained sciatic teased fibers of 2- and 9-month-old mice .", "( A ) Nodal sodium ( Nav1 . 6; left ) and potassium ( Kv7 . 2; right ) channels are normally localized and flanked by paranodal CASPR ( red , scale bars , 5 µm ) .", "( B , C )", "Ion channel anchoring proteins βIV-spectrin ( green ) ( B ) and ankyrinG ( green ) ( C ) are normally localized to nodes of Ranvier and flanked by paranodal CASPR ( red ) .", "Scale bars , 5 µm .", "( D ) Staining for 4 . 1G ( green ) , which unlike 4 . 1B is abundantly present at paranodes , shows normal localization ( arrowheads ) .", "Potassium channel Kv1 . 1 clusters ( red ) are located at juxtaparanodes but in mutants additionally localize along internodes .", "( E ) Triple-staining of Nav1 . 6 ( blue ) , paranodal CASPR ( red ) , and juxtaparanodal potassium channels Kv1 . 1 ( green ) shows preservation of Kv1 . 1 at juxtaparanodes and at mesaxonal lines ( arrows ) of mutant fibers .", "In addition , ectopic internodal Kv1 . 1 patches are depicted .", "Scale bar , 10 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 23332 . 00910 . 7554/eLife . 23332 . 010Figure 2—figure supplement 2 . Abnormal distribution of GM1 ganglioside in the perinodal region of mutant nerves by CTB-staining . GM1-labeling by CTB ( red ) of sciatic teased fibers from mice aged 9 months shows a defined paranodal staining in control ( left ) but is more distended in mutant fibers ( right ) .", "Area in rectangles ( top ) is magnified in bottom panels .", "Axons are stained for TUJ1 ( green ) .", "CTB , fluorescently tagged subunit B of cholera toxin . DOI: http://dx . doi . org/10 . 7554/eLife . 23332 . 010 Gangliosides are glycosphingolipids important for stabilizing membrane-spanning proteins and axon-glia interactions ( Susuki et al . , 2007 ) .", "We analyzed sciatic nerve lysates from 9-month-old mutants by lipid mass spectrometry and noted that many more gangliosides contained VLCFA ( a 14-fold enrichment; Figure 2h ) .", "To determine their subcellular distribution , we stained teased fibers with a fluorescently tagged cholera-toxin subunit B ( CTB ) , which binds GM1 gangliosides .", "Also by immunostaining , GM1 was restricted to paranodes in wild-type nerves but widely dispersed into the internodal myelin of mutant nerves ( Figure 2—figure supplement 2; Figure 2i ) .", "Likewise , ganglioside GD1a could be immunostained as enlarged puncta ( >5 µm ) in internodal myelin of mutant nerves ( Figure 3a ) .", "These GD1a-containing vesicles that colocalized with LAMP1 were a frequent finding in mutant internodes , but rarely observed in controls ( Figure 3a , left ) . 10 . 7554/eLife . 23332 . 011Figure 3 . Hallmarks of lysosomal storage disorders in Pex5 mutant nerves .", "( A ) Double-staining of sciatic teased fibers displayed giant GD1a+ ( green ) vesicles that colocalized with LAMP1 ( red ) in internodes only of mutant animals ( rectangular inset magnified in right panels ) .", "Dotted lines indicate borders of myelinated fibers; scale bar in inset , 5 µm .", "( B ) Electron microscopy of a teased and sectioned mutant sciatic nerve depicting paranodal vesicle accumulations .", "Scale bar , 500 nm .", "( C ) Immuno-electron microscopy of sciatic nerves from mice aged 9 months identifies enlarged LAMP1+ vesicles ( arrows ) within paranodal loops ( PNLs ) .", "M , myelin; Ax , axon .", "Scale bar , 500 nm .", "( D , E )", "Immunolabeling of teased sciatic nerves at the age of 2 months shows enlarged LAMP1+ ( left , green ) or LIMP-2+ ( right , green ) compartments close to CASPR+ paranodes ( red ) of mutant nerves .", "Scale bars , 10 µm .", "( F–H )", "Analysis of sciatic nerve lysates by Western blotting ( n = 4; β-III Tubulin , served as loading control for normalization ) and qRT-PCR ( n = 6 ) for LAMP1 and LIMP-2 showing increased protein , but decreased mRNA levels in mutants .", "( I ) Immunostaining of control sciatic fibers shows close association of peroxisomal PMP70 ( green ) and LAMP1 on lysosomal membranes ( red ) in the paranodal region , suggesting physical interactions .", "A wide-field flourescent image after deconvolution ( 250 nm z-stack width ) is shown .", "Scale bar , 10 µm .", "Dotted lines indicate borders of the myelinated fiber .", "( J ) Lysosomal enzyme activities of nerve lysates was enhanced in mutants compared to controls as assessed by assays using substrates of α-Mannosidase ( left ) and β-Hexosaminidase ( right; n = 4 ) .", "Statistics: means ± s . e . m . *p<0 . 05; **p<0 . 01; ***p<0 . 001; n . s . , not significant , Student’s T-test ( G , H , J ) .", "Blue arrows indicate nodes of Ranvier ( D , E , I ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 23332 . 01110 . 7554/eLife . 23332 . 012Figure 3—source data 1 . Lysosomal accumulation within paranodal loops . DOI: http://dx . doi . org/10 . 7554/eLife . 23332 . 01210 . 7554/eLife . 23332 . 013Figure 3—figure supplement 1 . Characterization of paranodal vesicle inclusions in Pex5 mutant nerves .", "( A–C )", "Immunofluorescence showing normal abundance of early/late endosomal and autophagosomal vesicles ( green ) in the vicinity of nodes of Ranvier ( indicated by paranodal CASPR or blue arrow ) of teased fibers obtained from 9-month-old animals .", "RAB7 , Ras-related protein 7 ( late endosomes; top ) ; EEA1 , early endosome-associated protein 1 ( middle ) ; ATG5 , autophagy-related protein 5 ( autophagosomes; bottom ) .", "Scale bars , 10 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 23332 . 013 Paranodal loops in conditional mutants showed vesicular inclusions of variable size and electron-density ( Figure 3b ) .", "By immune-labeling of ultra-thin cryosections and teased fibers , the majority of these were LAMP1-positive ( Figure 3c , d; Figure 3—source data 1 ) .", "Even more striking , lysosomal LIMP-2 marked small puncta in controls ( <500 nm ) , but giant compartments within mutant paranodes ( Figure 3e , right ) .", "Strongly increased abundance of LAMP1 and LIMP-2 was confirmed by Western blotting ( Figure 3f , g ) .", "Decreased mRNA levels ( Figure 3h ) suggest that the enrichment of LAMP1 and LIMP-2 marks organelle accumulation rather than enhanced lysosomal biogenesis .", "Lysosomal markers are found in autophagosomes and early/late endosomes ( Saftig and Klumperman , 2009 ) .", "We thus phenotyped accumulated vesicles with antibodies specific for EEA1 , RAB7 , and ATG5 .", "Neither marker was increased at mutant paranodes ( Figure 3—figure supplement 1a–c ) .", "Therefore , the majority of accumulating vesicles within mutant paranodes are likely bona fide lysosomes , serving the degradation of myelin-associated proteins/lipids , including gangliosides ( Luzio et al . , 2007; Saftig and Klumperman , 2009 ) .", "To determine the relationship between paranodal lysosomes ( Gatzinsky et al . , 1997 ) and peroxisomes , we immunostained for PMP70 and LAMP1 , revealing a close spatial association ( Figure 3i ) as reported for cultured cells ( Chu et al . , 2015 ) .", "We determined higher activities of lysosomal α-mannosidase and β-hexosaminidase in mutant nerve lysates ( Figure 3j ) , and note that increased compartment size and enzymatic activities are known phenomena of lysosomal disorders ( Sardiello and Ballabio , 2009 ) , yet associated with organelle dysfunction .", "Theoretically , complete peroxisomal failure in Cnp-Cre::Pex5flox/flox mice could perturb many aspects of SC metabolism in the observed neuropathy .", "However , we found that Cnp-Cre::Hsd17b4flox/flox mice ( referred to as MFP2 conditional knockout mice in the following ) , which specifically lack peroxisomal β-oxidation ( Verheijden et al . , 2013 ) , share the key features of this novel phenotype , including ectopic internodal Kv1 . 1 clusters ( Figure 4—figure supplement 1a–e ) , confirming the important role for glial lipid metabolism in maintaining axonal membrane composition , and function .", "To investigate whether this new pathomechanism , identified in two mouse models with a glia-specific mutation , is also relevant for a human genetic disease , we analyzed ABCD1-deficient mice , a model for X-ALD/AMN ( Forss-Petter et al . , 1997 ) .", "Sciatic nerve axons and myelin were morphologically intact , even at 22 months of age ( Figure 4a–c ) .", "However , ABCD1-mutants exhibited a similar mislocalization of Kv1 . 1 positive channels as observed in both conditional ( Pex5 and Mfp2 ) mutants .", "The quantification showed that mislocalization of internodal Kv1 . 1 was temporally progressive .", "In accordance with our model of membrane patches drifting into internodes , significant alterations were observed only at 22 months of age , but not yet at 2 months ( Figure 4d–e ) .", "This pathology was accompanied by lysosomal accumulates in myelinated fibers ( Figure 4f–h ) . 10 . 7554/eLife . 23332 . 014Figure 4 . Normal nerve morphology , but ectopic ion channels and lysosomal accumulates in 22 months aged Abcd1 null mutants .", "( A–C )", "Analysis of nerve morphology using entire methylene blue-stained semithin cross-sections ( A , C ) and g-ratios as measure of myelin thickness by electron microscopy of sciatic nerves ( ≥200 randomly chosen axons per nerve ) obtained from control and Abcd1 knockout mice ( n = 4 ) .", "Scale bar , 10 µm .", "( D , E )", "Immune-stained teased sciatic nerve fibers obtained from mutants and controls with corresponding quantifications show progressively abnormal Kv1 . 1 localization ( green ) , while Nav1 . 6 ( blue ) and CASPR ( red ) are normally localized .", "Scale bars , 10 µm .", "( n = 4 , 2 mo; n = 6 , 22 mo ) .", "Statistics: means ± s . e . m , Student’s T-test *p<0 . 05; **p<0 . 01; ***p<0 . 001; n . s . , not significant .", "( F–H )", "Analysis of lysosomes reveals LAMP1+ and LIMP-2+ accumulations ( green ) by immune-stained sciatic teased fibers ( n = 3 ) and a mild increase of lysosomal β-Hexosaminidase activity measured using sciatic nerve lysates ( n = 4 ) .", "Blue arrows indicate nodes of Ranvier .", "Scale bars , 10 µm .", "Statistics: means ± s . e . m , two-way ANOVA followed by Bonferroni ( B ) or Student’s T-test ( H ) .", "*p<0 . 05; **p<0 . 01; ***p<0 . 001; n . s . , not significant . DOI: http://dx . doi . org/10 . 7554/eLife . 23332 . 01410 . 7554/eLife . 23332 . 015Figure 4—figure supplement 1 . Ectopic , internodal Kv1-channel clusters in nerves of conditional MFP2 knockout mice .", "( A ) Fluorescent image of aged ( 19 months ) Cnp-Cre::Mfp2flox/flox ( MFP2 cKO ) sciatic teased nerve fibers show normal distribution of nodal Nav1 . 6 ( blue ) , paranodal CASPR ( red ) , and juxtaparanodal Kv1 . 1 ( green ) , as in controls .", "Mutant fibers exhibit in addition ectopic internodal Kv1 . 1 clusters ( arrow head ) .", "Scale bar , 10 µm .", "( B ) Quantification of ectopic Kv1 . 1 channel distribution at paranodes ( PN ) or internodes ( IN ) .", "Statistics: means ± s . e . m . n = 3; *p<0 . 05; **p<0 . 01; ***p<0 . 001 , Student’s T-test .", "( C ) Enzyme assay measuring lysosomal β-Hexosaminidase in nerve lysates from animals aged 16 months showing increased activity in mutant nerve lysates .", "( D , E )", "Functional impairment of MFP2 mutant compared to control sciatic nerves assessed by ex vivo electrophysiology at age 16 months ( n = 6 ) .", "Mfp2 mutant nerves display smaller responses ( red curves ) as compared to controls ( black curves ) when elicited at stimulus intensities of 0 . 15mA ( left ) or 0 . 155mA ( right ) .", "Peak amplitudes plotted against increasing stimulus intensity indicate reduced compound action potentials of MFP2 mutant nerves ( red dots ) compared to controls ( black dots ) at all intensities measured .", "Statistics: Two-way ANOVA , effect of genotype , F ( 1200 ) =27 . 37; p=0 . DOI: http://dx . doi . org/10 . 7554/eLife . 23332 . 01510 . 7554/eLife . 23332 . 016Figure 4—figure supplement 2 . Hypothetical model for lipid turnover of the juxtaparanodal anchoring complex of Kv1-channels . Myelin TAG-1 is stabilized by gangliosides ( GS , light green ) within juxtaparanodes ( JXP ) .", "For normal turnover ( left ) of JXP domains , SC require lysosomes ( purple ) and peroxisomes ( dark green ) that degrade GS ( indicated by curved pink arrow ) .", "When GS degradation is perturbed ( right ) , indicated by impaired peroxisomes ( red ) , GS accumulate within enlarged lysosomes ( purple ) , storage vesicles ( gray ) , and likely within JXP .", "Excess GS within JXP stabilize the protein complex containing TAG-1 ( yellow ) , CASPR2 ( red ) and Kv1 . 1 ( blue ) proteins , which thereby escape normal turnover , leading to domains breaking-off and drifting into internodes .", "Extra clusters possibly allow more K+ ( blue dots ) efflux ( curved gray arrow ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 23332 . 016" ], [ "Our data suggest an important pathomechanism of peroxisomal dysfunction in peripheral nerves is the secondary impairment of lysosomes .", "For example , gangliosides , which are frequently esterified with VLCFA and 2-hydroxy fatty acids ( Chrast et al . , 2011; Sandhir et al . , 2000 ) , must be degraded in lysosomes .", "However , subsequent β-oxidation of VLCFA and 2-hydroxy fatty acids requires peroxisomes ( Foulon et al . , 2005; Wanders , 2014 ) .", "Thus , our mouse model provides first in vivo evidence for functional interactions between lysosomes and peroxisomes , so far only discussed for cultured cells ( Chu et al . , 2015 ) .", "One consequence of lysosomal impairment is the perturbation of normal ganglioside turnover .", "Gangliosides are in close association with Kv1-anchoring proteins ( Loberto et al . , 2003 ) and provide stability to the juxtaparanodal clusters ( Susuki et al . , 2007 ) , which resemble giant membrane 'lipid rafts' .", "As mechanism for ectopic localization of axonal Kv1 channels , we suggest the following working model ( Figure 4—figure supplement 2 ) : Lack of ganglioside breakdown causes their accumulation in the glial juxtaparanodal membrane .", "Thereby , clusters of Kv1 and associated anchoring proteins ( TAG-1 and CASPR2 ) on the axonal side gain abnormal stability , break-off , diffuse into the internodal region , and escape from regular turnover .", "It is likely that also glial TAG-1 remains anchored to the axonal Kv1-/CASPR2-/TAG-1 clusters , but this cannot be distinguished by TAG-1 immunofluorescence .", "Kv1-channels are fast-opening , voltage-gated channels that are activated by mild voltage-shifts and thought to regulate fiber excitability ( Glasscock et al . , 2012 ) .", "The integrity of Kv1 channels is also impaired in CASPR2-/- mice , but perturbation of nerve function is not a feature ( Poliak et al . , 2003 ) .", "Unlike PEX5 mutant nerves , CASPR2-/- nerves do not exhibit elevated levels of Kv1 . 1 channel protein .", "Also , the ectopic Kv1 . 1+ clusters are situated directly adjacent to juxtaparanodes in CASPR2-deficient fibers , not along the internodes as observed in PEX5 mutants .", "Considering the severe progressive decline of nerve function in PEX5 mutant mice , which becomes clearly more affected with age ( data not shown ) , it is unlikely that a rather non-progressive pathology , that is the shift of Kv1 into paranodes , is the underlying cause of conduction slowing .", "In contrast , the progressive shift of Kv1 toward internodes correlates well with disease progression .", "Theoretically , Kv1-mediated currents in the internode could hyperpolarize the axonal membrane and dampen the conductivity of spiking axons .", "Such ‘feedback regulation’ might prevent hyperexcitability , which is a feature of mice lacking Kv1 . 1 function ( Zhou et al . , 1998 ) .", "On the other hand , a ‘gain-of-function’ effect by opening additional ectopic internodal Kv1 channels , may contribute to a disturbed equilibrium of axonal ions causing slowed NCV and conduction blocks .", "Direct proof of this model would require patch clamping single axons underneath myelin , which is impossible at present .", "Peripheral neuropathy has been associated with numerous metabolic diseases , including AMN .", "In this clinically milder variant of ABCD1 loss-of-function mutations , the reduced NCV was assumed to reflect demyelination , which is a hallmark in the brain of severely affected patients .", "Similarly , conditional PEX5 knockout mice display a cerebral inflammatory demyelination , while such pathology is absent in sciatic nerves .", "This remarkable discrepancy between the peripheral and central nervous system pathology is currently unexplained and presumably related to differently responding cell populations ( Kassmann , 2014 ) .", "Also in ABCD1-deficient nerves neither demyelination nor axon degeneration , but ectopic axonal Kv1 channels was a progressive pathological feature as early as at 2 months of age , preceding the disease onset by more than 1 year ( Pujol et al . , 2002 ) .", "Our present findings therefore expand the emerging role of myelinating glial cells in maintaining axonal membrane composition and thereby influencing axonal function , independent of myelin itself ." ], [ "Cnp1Cre/+ and Pex5flox/flox mice with C57/Bl6 genetic background were genotyped as described ( Kassmann et al . , 2007 ) .", "To check for recombination , genomic DNA was isolated from tail biopsies using the nexttec kit according to the manufacturer’s instructions .", "PCR was performed with sense ( 5’- CCAACGTCTGCCCATTCCTCCACCTG-3’ ) and antisense primers ( 5’- TTTGAGGATGGGAAGCAGTGCT −3’ ) generating a 330 bp amplicon after Cre-mediated excision of the floxed Pex5 gene in conditional knockout mice .", "Hsd17b4flox/flox ( Mfp2flox/flox ) mice and Abcd1 knockout mice with C57/Bl6 genetic background were genotyped by PCR as described ( Verheijden et al . , 2013; Forss-Petter et al . , 1997 ) .", "Animals were maintained in individually ventilated cages under SPF conditions .", "For visualization of gangliosides sciatic nerves of Cnp-Cre::Pex5flox/flox mice and controls were dissected and pre-teased in artificial cerebrospinal fluid ( ACSF; 126 mM NaCl , 3 mM KCl , 1 , 25 mM NaH2PO4 , 26 mM NaHCO3 , 2 mM MgSO4 , 10 mM Glucose , 2 mM CaCla2 ) .", "Fiber bundles were incubated for 1 . 5 hr at room temperature with antibodies specific for GM1 or GD1a ( kindly provided by Hugh Willison , Glasgow ) .", "Samples were washed in ACSF , incubated with isotype-specific fluorescent secondary antibodies ( Alexa-488 IGg3 and IG2b goat anti-mouse; Thermo Fisher Scientific , Massachusetts , USA ) , again washed in ACSF , fixed in 4% paraformaldehyde ( PFA ) for 5 min , then washed in PBS containing 0 . 1 M glycine , teased on glass slides , air-dried , and stored at −20°C .", "For other antibody-labeling , sciatic nerves were dissected , teased and stored at −20°C as described previously ( Kassmann et al . , 2011 ) .", "For staining protein antigens , frozen teased fibers were fixed in 4% paraformaldehyde ( PFA ) for 5 min , permeabilized in methanol at −20°C for 3–5 min , PBS-washed , and blocked for 1 hr in PBS containing 10% horse serum and 0 . 05% Triton X-100 .", "Primary antibodies diluted in blocking solution were applied overnight at 4°C , specimens were washed in PBS , and incubated for 1 hr with fluorescent secondary antibodies Alexa-488 , Alexa-555 ( Thermo Fisher Scientific , 1:2000 ) , and DyLight 633 ( 1:1000 , YO Proteins , Sweden ) .", "Cell nuclei were visualized with 4′ , 6′- diamidino-2-phenylindole ( DAPI; 1:10 . 000 , Thermo Fisher Scientific ) .", "Fibers were mounted in AquaPolymount ( Polysciences , Pennsylvania , USA ) .", "Fluorescent images were acquired with an inverted Zeiss Axio Observer . Z1 equipped with an Axiocam MRm ( Zeiss , Germany ) .", "Mouse sciatic nerves were fixed overnight in 4% PFA , embedded in paraffin , and longitudinally sectioned ( 5 µm ) .", "For labeling , Dako LSAB2 system ( Dako , Denmark ) was used according to the manufacturer’s directions .", "All samples were analyzed by light microscopy ( Zeiss Axiophot ) .", "Mice were anesthetized with avertin ( 100 μl/10 g bodyweight ) and perfused intracardially with 15 ml HBSS ( Lonza , Switzerland ) , followed by 30 ml of fixative ( 0 . 2% glutaraldehyde , 4% paraformaldehyde in PBS ) .", "Sciatic nerves were dissected and post-fixed for 2–4 hr in 4% followed by overnight incubation in 1% PFA .", "Epon embedding of sciatic nerves was performed as described previously ( Kassmann et al . , 2007 ) .", "Semithin cross-sections ( 500 nm ) of sciatic nerves were incubated with methylene blue/Azur II and axons of entire cross-sections of sciatic nerves were analyzed by light microscopy ( Zeiss Axiophot ) .", "Quantification of axon number was performed using Fiji software .", "Preparation of ultrathin cryosections ( 50 nm ) was performed according to the Tokuyasu technique , and immunolabeling was carried out as previously described ( Werner et al . , 2007 ) .", "Antibody binding was visualized with protein A-gold ( 1:60 , 10 nm ) .", "Ultrathin sections ( 50 nm ) were contrasted with 1% uranyl acetate and lead citrate .", "Sections were examined with a LEO EM 912AB electron microscope ( Zeiss ) , and pictures were taken with an on-axis 2048 X 2048 CCD camera ( Proscan ) .", "Myelin thickness was evaluated using Fiji software by g-ratio analysis ( dividing the axonal by the fiber diameter ) derived from at least 100 randomly chosen fibers per nerve .", "Sciatic nerves were transferred to 200 µl ( per nerve ) ice-cold lysis buffer containing 1% Triton X-100 , 50 mM Tris-HCl pH 7 . 5 , 150 mM NaCl , 1% NP-40 , and protease inhibitors ( Complete , Roche , Switzerland ) .", "Samples were homogenized using Precellys 24 ( VWR International , Dealware , USA ) , centrifuged for 10 min at 1000 g to remove cellular nuclei , and protein concentration of the supernatant was determined using Lowry assay ( Bio-rad Laboratories , California , USA ) .", "Blotting membranes were incubated overnight at 4°C with primary antibodies .", "Western Lightning + ECL Kit ( Perkin Elmer , Massachusetts , USA ) was used to label protein bands , which were detected using Intas ChemoCam Image system .", "At least four biological replicates were analyzed .", "Quantification was performed in Fiji .", "4 . 1G ( 1:100; kindly provided by E . Peles ) AnkG ( 1:100; kindly provided by M . Rasband ) , APP ( 1:1000; MAB348 , Merck Millipore , Massachusetts , USA ) , ATG5 ( 1:100; pab50264 Covalab , France ) , CASPR ( mk , m; 1:1000; clone K65/35 NeuroMab , California , USA ) , CASPR2 ( 1:500; kindly provided by E . Peles ) , Catalase ( 1:200; C0979 Sigma-Aldrich , Missouri , USA ) , CD3 ( 1:150; MCA1477 Serotec , MorphoSys , Germany ) , EEA1 ( 1:300; ab2900 Abcam , Cambridge , United Kingdom ) , Kv1 . 1 ( 1:50; sc11184 Santa Cruz Biotechnology , Texas , USA ) , Kv1 . 1 ( 1:50; clone 20/78 NeuroMab ) , Kv7 . 2 ( 1:2000; PA1-929 ThermoSicentific ) , LAMP1 ( WB , 1:400; IF , 1:200; IEM , 1:200; 553792 BD Bioscience , New Jersey , USA ) , LIMP-2 ( WB , 1:250; IF , 1:2000; kindly provided by J . Blanz ) , MAC-3 ( 1:400; 553322 BD Bioscience ) , Nav1 . 6 ( 1:500; ASC-009 Alomone labs , Israel ) , NF155 ( 1:1000; kindly provided by P . Brophy ) , P0 ( 1:1000 [Archelos et al . , 1993] ) ; P2 ( 1:500; sc-49304 Santa Cruz ) , PLP ( 1:5000 [Jung et al . , 1996] ) , PMP22 ( 1:1000; SAB4502217 Sigma ) , PMP70 ( 1:600; ab3421 Abcam ) , RAB7 ( 1:300; R4779 Sigma ) , TAG-1 ( 1:500; kindly provided by E . Peles ) , ßiv spectrin ( 1:400; kindly provided by M . Rasband ) , α-Tubulin ( 1:1000; T 5168 Sigma ) , III β-Tubulin ( 1:1000; Covance , New Jersey , USA ) .", "Sciatic nerves were homogenized using Precellys 24 ( twice for 20 s at 5000 rpm ) in 150 µl ice-cold lysis buffer containing 10 mM Tris-HCl pH 7 . 4 , 150 mM NaCl , 5 mM EDTA , 0 . 5% Triton X-100 , 1 x PEFA , and Protease inhibitors ( Complete , Roche ) .", "Nerve lysates were centrifuged for 10 min at 15 , 000 g to remove cellular debris and nuclei .", "The supernatant was incubated with 10 mM of the substrate ( i . e . p-nitrophenyl-a-D-mannopyranosid or p-Nitrophenyl-N-acetyl-ß-D-glucosaminide; Sigma ) dissolved in 0 . 2 M citrate buffer , and the reaction was stopped by addition of 500 µl 0 . 4 M glycine NaOH ( pH 10 . 4; adjusted with 1 M NaOH ) to stop enzyme activity .", "Samples were centrifuged for 10 min at 15 , 000 g , and the supernatants of two technical replicates were measured at 405 nm with an Eon microplate spectrophotometer ( BioTek , Vermont , USA ) .", "Total RNA was extracted from sciatic nerve lysates as described previously ( Fünfschilling et al . , 2012 ) .", "Reactions were performed with four technical replicates of nerves obtained from six different animals per group .", "PCR primers were specific for β-actin ( forward 5′-CTTCCTCCCTGGAGAAGAGC and reverse 5′-ATGCCACAGGATTCCATACC ) , Lamp1 ( forward 5’-CCTACGAGACTGCGAATGGT and reverse 5’-CCACAAGAACTGCCATTTTTC ) , Limp-2 ( forward 5’-TGGAGATCCTAACGTTGACTTG and reverse 5’-GGCCAGATCCACGACAGT ) , and Pex5 ( forward 5’-CACATCCGCTTCCTATGACA and reverse 5’-AAAAGGCTGAGGGTGGTCA ) .", "To quantify PE ( diacyl ) , PE O- ( alkanyl-acyl ) , and PE P- ( alkenyl-acyl ) species , acidic ( PE and PE O- ) or neutral ( PE P- ) lipid extractions were performed as described ( Bligh and Dyer , 1959 ) .", "Typically , 1–5 µl of a 1:10 dilution of total lysates were measured .", "As lipid standards , 50 pmol of PE standards and 40–60 pmol of a PE P- standards were used .", "Standard syntheses were performed as described ( Özbalci et al . , 2013; Paltauf and Hermetter , 1994 ) .", "After solvent evaporation under a gentle nitrogen flow at 37°C , lipids were re-suspended in 50 µl 10 mM ammonium acetate in methanol .", "PE/PE O- and PE P- species were analyzed in neutral loss or precursor ion mode selecting for class-specific fragments on a QTRAP5500 ( Sciex , Massachusetts , USA ) equipped with a NanoMate devise ( Advion , New York , USA ) , employing MS settings as described ( Özbalci et al . , 2013 ) .", "Data evaluation was done using LipidView ( AB Sciex ) and an in-house-developed software ( ShinyLipids ) .", "Species annotated as PE O- mainly contain plasmenylethanoamines but also minor amounts of PE P- species and PE species with odd-numbered fatty acids .", "Extraction of gangliosides was performed as described ( Sampaio et al . , 2011 ) , with the exception that the first neutral extraction was performed using chloroform:methanol as a 17:1 ( v/v ) solution , followed by a chloroform:methanol 2:1 ( v/v ) extraction .", "For quantification of gangliosides , GD1a and GD1b 50 pmol N-CD3-Stearoyl GM3 and N-CD3-Stearoyl-GM1 ( Matreya , Pennsylvania , USA ) were used as internal ganglioside standards .", "Following evaporation , samples were re-suspended in 100 µl methanol .", "Gangliosides were subjected to UHPLC-MS analysis , using a CSH C18 column ( 1 × 150 mm , 1 . 7 µm particles , Waters ) coupled to a QExactive high-resolution Orbitrap mass spectrometer ( Thermo ) equipped with an ESI source .", "For GD1a/b quantification , 30 µl aliquots of the aqueous and the chloroform:methanol ( 2:1 ) phase were transferred to Eppendorf cups , evaporated and resuspended in 50 µl buffer containing 60% of mobile phase A ( acetonitrile:water; 60:40 ( v/v ) with 10 mM ammonium formate and 0 . 1% formic acid ) and 40% mobile phase B ( isopropanol:acetonitrile , 90:10 ( v/v ) with 10 mM ammonium formate and 0 . 1% formic acid ) .", "Of each sample , 10 µl was subjected to UPLC separation ( Dionex , California , USA ) , using a multistep gradient .", "Full MS scans were acquired for 30 min in negative ion mode ( 600–1800 m/z ) with automatic gain control target set to 1 × 106 ions .", "The maximum injection time was set to 200 ms and a FHWM resolution of 140 , 000 ( at m/z 200 ) .", "In addition to Full MS scans , all ion fragmentation scans in negative ion mode were performed at a resolution of 70 , 000 , scanning a mass range of 120–600 m/z with a normalized collision energy set to 30 eV .", "Data evaluation of Full-MS scans ( profile spectra ) was performed using MassMap ( MassMap , Germany ) .", "Therefore , data files obtained were converted to mzXML-files using the software Proteowizard .", "mzXML-Files were then converted to mmp-Files using the LC-MS data evaluation software MassMap .", "VLCFA determination was performed using 10 µl of a 1:10 dilution of total membrane fractions .", "500 pmol of C25 fatty acid ( Dr . Ehrenstorfer GmbH , Germany ) and C23 fatty acid ( Matreya ) was used as internal standards .", "Prior to extraction , glass tubes where washed with chloroform containing 1% acetic acid .", "The samples were resuspended in 80 µl of toluene .", "1 ml acetonitrile-hydrochloric acid ( conc . ) ( 4:1 ) was added and incubated for 2 hr at 90°C .", "After cooling to room temperature , 2 ml hexane were added , the samples were vortexed , incubated for 5 min and centrifuged for 2 min at 2000 rpm .", "The upper phase was transferred to a new glass tube .", "After evaporation of the organic phase under a gentle stream of nitrogen , lipid extracts were resuspended in 100 µl chloroform:methanol:water ( 50:45:5 , v/v/v ) with 0 . 1% ammonium hydroxide solution .", "Of each sample , 5 µl was transferred to a 96-well plate containing 10 µl 0 . 01% piperidine in methanol and 5 µl MeOH .", "Samples were subjected to mass spectrometric analysis on a QSTAR Elite ( Sciex ) instrument equipped with a NanoMate ( Advion ) .", "TOF scans were performed over a mass range of 100–500 m/z in negative ion mode .", "Data sets were processed and evaluated using LipidView ( Sciex ) .", "Following cervical dislocation mouse sciatic nerves were rapidly transferred into a perfusion chamber filled with gassed ACSF at 37°C containing in mM: 126 NaCl , 3 KCl , 26 NaHCO3 , 1 . 25 NaH2PO4 , 25 glucose , 2 . 5 MgSO4 and 2 CaCl2; a pH of 7 . 4 was ensured by continuous gassing with carbogen ( 95% O2/5% CO2 ) .", "Nerves’ lengths were measured and were then allowed to adapt for approximately 30 min before electrophysiological recordings began .", "Suction electrodes backfilled with ACSF were used for stimulation and recording .", "Electrical stimulation was performed at different intensities ranging between 0 . 13 mA and 0 . 3 mA , and increased manually in step-width of 0 . 05 mA .", "Inter-stimulus intervals were 3 s and each of the stimulus intensities was used 10 times .", "CAPs were continuously measured .", "Recorded signals were acquired at 100 kHz , amplified x100 by Ext-2F ( NPI Electronic , Germany ) , and a further 50-fold by SR560 ( Stanford Research Systems , California , USA ) , and filtered .", "Recordings were controlled by patchmaster software ( HEKA , Germany ) with an EPC9 amplifier interface ( HEKA ) .", "Stimulus intensities were altered using Stimulus Isolator A385 ( World Precision Instruments ) .", "Analysis of electrophysiology was performed using Matlab .", "Each nerve was analyzed separately .", "Responses triggered but the same stimulus repetitions were averaged together .", "Nerve conduction velocity was calculated for each of the stimulus intensities as the mean across trials of the ratio between the length of the nerve and the time between peaks of stimulus-artifact ( first small positive peak ) and response ( third positive peak ) .", "Mice aged 9 months were anesthetized with ketaminhydrochloride ( 100 mg kg−1 ) /xylazin hydrochloride ( 8 mg kg−1 ) .", "Steel needle electrodes were placed subcutaneously , one pair at sciatic notch ( proximal stimulation ) , a second pair at the tibial nerve above the ankle ( distal stimulation ) .", "Supramaximal square wave pulses lasting 100 ms were delivered using a Toennies Neuroscreen ( Jaeger , Germany ) .", "Compound muscle action potential ( CMAP ) was recorded from the intrinsic foot muscles .", "Digital images were processed and quantified with ZEN 2012 software ( Zeiss ) and/or Fiji .", "All numerical values are shown as the mean ± s . e . m . ; n = 3–6 , unless stated otherwise .", "Statistical significance was determined using GraphPad Prism5 by two-way ANOVA or the two-tailed Student's t test for unpaired samples assuming unequal variance and p values below 0 . 05 were marked as significant: *p<0 . 05; **p<0 . 01; ***p<0 . 001 ) .", "Normal distribution was assumed , but not formally tested .", "Mice were kept in the mouse keeping facility of the Max-Planck-Institute of Experimental Medicine at 12 hr light/dark cycle .", "All experiments were executed according to the German Animal Protection Law and approved by the government agency of the State of Lower Saxony , Germany ." ] ]

[ "Impairment of peripheral nerve function is frequent in neurometabolic diseases , but mechanistically not well understood .", "Here , we report a novel disease mechanism and the finding that glial lipid metabolism is critical for axon function , independent of myelin itself .", "Surprisingly , nerves of Schwann cell-specific Pex5 mutant mice were unaltered regarding axon numbers , axonal calibers , and myelin sheath thickness by electron microscopy .", "In search for a molecular mechanism , we revealed enhanced abundance and internodal expression of axonal membrane proteins normally restricted to juxtaparanodal lipid-rafts .", "Gangliosides were altered and enriched within an expanded lysosomal compartment of paranodal loops .", "We revealed the same pathological features in a mouse model of human Adrenomyeloneuropathy , preceding disease-onset by one year .", "Thus , peroxisomal dysfunction causes secondary failure of local lysosomes , thereby impairing the turnover of gangliosides in myelin .", "This reveals a new aspect of axon-glia interactions , with Schwann cell lipid metabolism regulating the anchorage of juxtaparanodal Kv1-channels ." ]

[ "Nerve cells transmit messages along their length in the form of electrical signals .", "Much like an electrical wire , the nerve fiber or axon is coated by a multiple-layered insulation , called the myelin sheath .", "However , unlike electrical insulation , the myelin sheath is regularly interrupted to expose short regions of the underlying nerve .", "These exposed regions and the adjacent regions underneath the myelin contain ion channels that help to propagate electrical signals along the axon .", "Peroxisomes are compartments in animal cells that process fats .", "Genetic mutations that prevent peroxisomes from working properly can lead to diseases where the nerves cannot transmit signals correctly .", "This is thought to be because the nerves lose their myelin sheath , which largely consists of fatty molecules .", "The nerves outside of the brain and spinal cord are known as peripheral nerves .", "Kleinecke et al . have now analyzed peripheral nerves from mice that had one of three different genetic mutations , preventing their peroxisomes from working correctly .", "Even in cases where the mutation severely impaired nerve signaling , the peripheral nerves retained their myelin sheath .", "The peroxisome mutations did affect a particular type of potassium ion channel and the anchor proteins that hold these channels in place .", "The role of these potassium ion channels is not fully known , but normally they are only found close to regions of the axon that are not coated by myelin .", "However , the peroxisome mutations meant that the channels and their protein anchors were now also located along the myelinated segments of the nerve’s axons .", "This redistribution of the potassium ion channels likely contributes to the peripheral nerves being unable to signal properly .", "In addition , Kleinecke et al . found that disrupting the peroxisomes also affected another cell compartment , called the lysosome , in the nerve cells that insulate axons with myelin sheaths .", "Lysosomes help to break down unwanted fat molecules .", "Mutant mice had more lysosomes than normal , but these lysosomes did not work efficiently .", "This caused the nerve cells to store more of certain types of molecules , including molecules called glycolipids that stabilize protein anchors , which hold the potassium channels in place .", "A likely result is that protein anchors that would normally be degraded are not , leading to the potassium channels appearing inappropriately throughout the nerve .", "Future work is now needed to investigate whether peroxisomal diseases cause similar changes in the brain .", "The results presented by Kleinecke et al . also suggest that targeting the lysosomes or the potassium channels could present new ways to treat disorders of the peroxisomes ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "stem cells and regenerative medicine" ]

[ [ "Adult stem cells reside in most if not all tissues .", "They are endowed with the ability to self-renew long-term and differentiate into specialized cell lineages in order to maintain , renew and repair tissues .", "In turn , stem cells are heavily influenced by the tissue microenvironment in which they reside .", "Together , the tight interaction and cooperativity between stem cells and their niche allow stem cells to perform their critical task of maintaining tissue homeostasis and function throughout the lifetime of the organism .", "To date , most studies on the niche have focused on the various factors it secretes to control stem cell behavior during normal homeostasis .", "These factors include both positive and negative signals emanating from a diverse array of niche cells , including stromal cells , blood vessels , nerves and stem cell progeny ( Asada et al . , 2017; Hsu et al . , 2014a; Yu and Scadden , 2016 ) .", "Far less is known about how stem cells cope with perturbations in niche integrity and orchestrate responses to restore niche homeostasis .", "For epithelial tissue stem cells , this is particularly important , as they must respond quickly to and repair a breach in their epithelial barrier that excludes harmful microbes and retains essential fluids .", "In the current study , we tackle this problem by taking advantage of the relatively simple architecture of the hair follicle ( HF ) bulge , the niche in which stem cells of the HF reside ( Cotsarelis et al . , 1990; Hsu et al . , 2014a ) .", "The main part of the bulge is bilayered .", "It consists of a single concentric layer of stem cells that is sandwiched between a basement membrane on the outer side and a layer of terminally differentiated epithelial barrier ‘niche’ cells on the inner side .", "These ‘inner bulge’ niche cells are exposed to the external environment through an open channel that enables the hair to protrude from the skin surface ( Figure 1A ) .", "The cells derive from the stem cells , but they do not do so directly within the bulge .", "Rather , the barrier cells are generated late in the terminal differentiation pathway of stem cell progeny that produce the new hair ( Hsu et al . , 2011 ) .", "Indeed , besides forming this epithelial barrier , the bulge stem cells also fuel the episodic bouts of hair follicle ( HF ) regeneration ( ‘anagen’ ) needed to replenish the protective hair coat of the animal .", "In between episodic cycles of hair growth , the bulge exists in a resting state ( ‘telogen’ ) , anchoring the hair made in the previous hair cycle ( Figure 1A ) .", "At the bulge base ( ‘hair germ’ ) , quiescent stem cells interact with a specialized underlying mesenchymal structure , called the dermal papilla ( DP ) .", "This cross-talk is necessary to yield a threshold of activating factors ( BMP inhibitors , WNTs ) that launch a new hair cycle ( Hsu et al . , 2011 ) .", "Stem cells tether to their inner bulge niche through cadherins , the core transmembrane components of adherens junctions ( AJs ) , which coordinate intercellular adhesion , junctional integrity and proliferation in epithelial tissues ( Stepniak et al . , 2009; Takeichi , 2014 ) .", "AJs also stabilize tight junctions , which prevent passage of bacteria and other entities across an epithelial sheet ( Rübsam et al . , 2017; Tunggal et al . , 2005 ) .", "While bulge stem cells express both E- and P-cadherin , we discovered that the inner bulge niche expresses only E-cadherin .", "Here , we report that selective loss of E-cadherin within the inner bulge niche triggers a barrier breach that results in microbial invasion .", "We show that breached niche cells elevate an adhesive and cytoskeletal transcriptome .", "By contrast , the resident stem cells , which are normally quiescent , begin to proliferate in a fashion that we show is independent of microbial influence .", "Strikingly , they also activate a transcriptome that , when compared to proliferating anagen-phase stem cells in a normal niche , is similar in cell cycle genes , but differs in a cohort of immune-signaling genes .", "Moreover , although the cell cycle transcriptome lowers their threshold for quiescence , pushing them into proliferation requires this additional facet of the immune-signaling transcriptome .", "Indeed , the myriad of chemo-attractants , cytokines and growth factors induced by these stem cells when they experience a damaged niche is required to recruit immune cells into this otherwise purportedly ‘immune privileged’ site ( Meyer et al . , 2008; Paus and Bertolini , 2013 ) .", "The outcome of this atypical two-pronged path to stem cell proliferation , namely enhanced immune signaling and cell cycling , is a repurposing of new daughter stem cells from their normal task of tissue regeneration and towards patching the breached barrier .", "Our findings have strong relevance to a breadth of human skin conditions where progenitor cell proliferation is coupled to barrier breach .", "Our data reveal that stem cells have the potential to sense defects in the integrity of their niche and respond by activating two branches of a transcriptional program , which together cope with the compromised tissue function surrounding them .", "Additionally and importantly , we show that the niche is not merely a source of secreted factors that control stem cell activity and tissue growth .", "Rather , it is a key structural entity whose integrity is closely monitored by its vigilant stem cell residents ." ], [ "We became interested in the possibility that cadherins may function critically in HF stem cell niche integrity when we analyzed bulge cadherin expression during the extended , synchronized telogen phase of the second hair cycle in mice ( Figure 1A ) .", "E-cadherin localized to most epithelial cell-cell boundaries , displaying the strongest immuno-labeling at the interface between niche cells and stem cells .", "Conversely , P-cadherin specifically concentrated at stem cell intercellular junctions and was absent from niche cells .", "This was consistent with the ~20X higher Cdh3 transcript level in the stem cells versus niche cells .", "Indeed , as judged by enzyme-linked immunosorbent assays ( ELISAs ) on protein lysates of bulge stem cells [purified by fluorescence-activated cell sorting ( FACS ) of skin cell suspensions] , P-cadherin levels were even higher than E-cadherin ( Figure 1—figure supplement 1A and B ) .", "As expected from our expression data and the functional redundancy of these cadherins ( Tinkle et al . , 2008 ) , P-cadherin ( Cdh3 ) null mice ( Radice et al . , 1997 ) displayed a normal hair coat and hair cycle ( Figure 1—figure supplement 1C ) .", "E-cadherin was maintained at all cell-cell junctions within the P-cadherin depleted bulge , and hair germ cells became proliferative on cue during early anagen ( Figure 1—figure supplement 1D ) .", "To explore the functional consequences of E-cadherin loss of function ( loss of function ) in the bulge , we conditionally ablated its gene ( Cdh1 ) in Cdh1fl/fl; Rosa26lox-STOP-lox-YFP reporter mice by using a tamoxifen ( TAM ) -inducible CreER knocked into the endogenous locus of Sox9 .", "By inducing Cdh1 ablation near the beginning of 2nd telogen ( postnatal day P50 ) , E-cadherin was efficiently depleted throughout the bulge when analyzed 3 weeks later ( Figure 1B ) .", "Cdh1-null HF stem cells continued to display AJs along stem cell-stem cell borders , reflective of P-cadherin compensation ( Figure 1C and Figure 1—figure supplement 2A and B ) .", "In fact , by immunoblot analyses and quantifications of proteins from FACS-purified α6+CD34+ HF stem cells , P-cadherin levels in E-cadherin deficient HF stem cells were even higher than normal ( Figure 1D and Figure 1—figure supplement 2C ) , a feature confirmed by knocking down Cdh1 transcripts through shRNA ( Figure 1—figure supplement 2D ) .", "By contrast , the Cdh1-null inner bulge layer lacked P-cadherin and in turn AJs on all cell-cell borders ( Figure 1C ) .", "Since AJ formation requires intercellular cadherin-cadherin binding , this also left the stem cell-niche interface devoid of AJs .", "This was best exemplified by the paucity of p120-catenin at this interface ( arrows in Figure 1C ) .", "Notably , p120-catenin binds directly to classical cadherins and is required for AJ stabilization .", "Immunoblot analyses confirmed the overall reduction in these other AJ proteins , despite upregulation of P-cadherin in stem cells within the Cdh1-depleted bulge ( Figure 1D ) .", "AJs form an intercellular network of F-actin across an epithelial sheet of keratinocytes ( Vaezi et al . , 2002; Vasioukhin et al . , 2000 ) .", "In wild-type HFs , F-actin organization was particularly robust within the inner bulge , suggesting a special role for the cytoskeletal-intercellular networks within this layer of niche cells .", "However , in the absence of E-cadherin , the actin cytoskeletal organization was markedly perturbed throughout the inner bulge , including the stem cell-niche interface ( Figure 1E ) .", "Probing deeper , we next examined tight junctions .", "As shown in Figure 1F and Figure 1—figure supplement 2E , the expression , localization , continuity and stability of tight junction proteins were largely intact in the CD34+ stem cells , but disrupted in the niche cells of the bulge .", "Moreover , when we tested for tight junction integrity by exposing HFs to Lucifer yellow , the dye penetrated the inner bulge layer deficient for E-cadherin ( Figure 1G ) .", "Although our findings underscored the importance of E-cadherin in maintaining AJs , tight junctions and the integrity of the niche , niche cells were not lost from the bulge: desmosomes were still present in seemingly normal morphology , numbers and localization , and intercellular membranes at the niche-stem cell interface were still sealed ( Figure 1—figure supplement 3A and B ) .", "The inner bulge layer also remain adhered to the club hair via ‘half-desmosomal’ structures as previously described for WT bulge ( Hsu et al . , 2011 ) ( Figure 1—figure supplement 3C ) .", "As in the normal hair cycle , telogen-phase stem cells in Cdh1-heterozygous HFs were quiescent until anagen onset , when their proliferation initiated first within the hair germ ( AnaI ) , and several days later within the bulge ( AnaII-III; Figure 2A , first three panels ) ( Hsu et al . , 2014b ) .", "Upon homozygous Cdh1 ablation , however , telogen-phase bulge stem cell residents began proliferating ( Figure 2A , fourth panel; quantifications at right ) .", "In striking contrast to the normal hair cycle , this was neither preceded nor accompanied by hair germ proliferation .", "Normally , whether in homeostasis or in response to a wound , for example hair plucking , HF stem cell proliferation results in progeny that exit their niche and launch a new round of hair cycling ( Hsu et al . , 2011; Morris et al . , 2004; Tumbar et al . , 2004 ) .", "By contrast , the daughters of proliferative events in the telogen Cdh1-null bulge remained within the niche ( Figure 2A ) .", "Moreover , these HFs actually spent longer than normal in telogen prior to entering a new round of hair growth ( Figure 2B ) .", "These results indicated that the normal telogen phase cues were still intact , which we later document further .", "Instead of being utilized to launch a new hair cycle , this telogen-phase stem cell proliferation produced additional bulge layers , readily evident by 5 weeks post-Cdh1 ablation ( Figure 2C ) .", "Daughters retained stem cell identity as judged by bulge stem cell markers CD34 and LHX2 , and by factors such as TCF4 and SOX9 , which are expressed but not exclusively by HF stem cells ( Figure 2D and Figure 2—figure supplement 1A ) .", "Quantifications revealed a significant expansion of stem cell numbers within the Cdh1-null bulge ( Figure 2D ) .", "Despite the increased proliferation in telogen , Cdh1-null HF stem cells were still sensitive to the activating cues of the hair cycle , and returned to a non-dividing state as inhibitory levels rose in subsequent anagen and telogen ( Figure 2—figure supplement 1B ) .", "This further suggested that the normal cues that govern hair cycling were still in place .", "We next addressed whether the bulge stem cells proliferated because of their own Cdh1 loss or that of the niche .", "Since changes in AJs can impact proliferation ( Stepniak et al . , 2009 ) , we first considered whether the elevated P-cadherin seen in the Cdh1-null stem cells might be involved .", "However , when we engineered mice whose skin epithelium was selectively transduced with doxycycline ( Doxy ) -inducible Cdh3 , we saw no significant effect on stem cell proliferation upon induction of P-cadherin over-expression ( Figure 2—figure supplement 1C ) .", "Next , we used Krt15-CrePGR mice ( Morris et al . , 2004 ) to selectively ablate Cdh1 in bulge stem cells during the extended 2nd telogen ( Figure 2E and Figure 2—figure supplement 1D ) .", "Strikingly , the ectopic stem cell proliferation and expansion of bulge layers that we had observed upon Cdh1 ablation in both stem cells and inner bulge , did not occur upon stem cell-specific ablation ( Figure 2F ) .", "These data pointed to perturbations within the niche as the source of the stimulus for neighboring stem cells to proliferate .", "Since the inner bulge niche is derived from stem cell progeny that terminally differentiate towards the end of the hair cycle ( Hsu et al . , 2011 ) , we could test our hypothesis by monitoring Krt15CrePGR-activated Cdh1 cKO mice through subsequent hair cycles , when E-cadherin was also lost in the niche ( Figure 2E ) .", "Indeed , bulge stem cells proliferated and expanded concomitant with E-cadherin loss within the inner bulge ( Figure 2G ) .", "Finally , by titrating down the TAM dosage administered to Sox9CreER; Cdh1fl/fl; Rosa26lox-STOP-lox-YFP mice , we restricted Cre activity to the inner bulge layer ( Hsu et al . , 2011 ) ( Figure 2—figure supplement 1E ) .", "Notably , selective Cdh1 ablation in the inner bulge niche was sufficient to elicit an architectural shift to a triple layered bulge , even though the stem cells were now WT ( Figure 2H ) .", "Together , these findings suggest that bulge stem cell proliferation may rely at least in part upon the direct interface between the stem cells and their defective inner bulge niche .", "The inability of the Cdh1 null inner bulge to retain dye ( Figure 1G ) suggested that its integrity is needed to provide a protective barrier to the external environment .", "Upon testing this possibility , we found that in contrast to controls , Cdh1 cKO HFs were infiltrated with microbes , as shown both by Gram-positive ( deep violet ) and Gram-negative ( pink ) bacterial stains and by fluorescence in situ hybridization ( FISH ) for a pan-bacterial 16S rRNA ( Figure 3A and B ) .", "These findings highlighted the importance of a healthy inner bulge niche in guarding its stem cells and the skin against microbial infiltration .", "A priori , the stem cells might be responding to these microbes that infiltrate the breached barrier , rather than the damaged niche per se .", "To test for the impact of these infiltrating bacteria , we treated mice with antibiotics to deplete bacteria from the skin .", "As judged by in situ hybridization for 16S ribosomal RNA , microbes were efficiently thwarted ( Figure 3B ) .", "Despite efficient microbial clearing , the Cdh1 cKO bulge still remained proliferative and hyper-thickened ( Figure 3C ) .", "Altogether , while our findings do not discount a role for bacterial signals , they rule them out as the instigating factor in causing stem cell proliferation when the niche barrier was breached .", "Upon barrier breach , a swarm of immune cells , positive for the pan-leukocyte marker CD45 , selectively surrounded the bulge ( Figure 4A ) .", "Although CD45+ numbers per unit area of skin varied , when we quantified the number of CD45+ cells within a 25 μm radius of the bulge , we consistently observed a marked and statistically significant increase in immune cells surrounding the Cdh1 null bulge as compared to the WT control bulge ( Figure 4C , Cdh1-null bulge [Sox9CreER] vs control ) .", "Given the microbial invasion , an immune response was expected .", "Yet surprisingly , we found that immune cells had already begun to accumulate by D23 post Cdh1 ablation , before the increase in 16S rRNA and shift to a triple layered bulge architecture which were robust only by D35 ( Figures 2C and 4B ) .", "Moreover , when we depleted bacteria with antibiotics , immune cells were still recruited to the breached niche ( Figure 4C and Figure 4—figure supplement 1A ) .", "By contrast , when we used Krt15CrePGR-activated mice to selectively ablate Cdh1 from 2nd telogen HF stem cells , immune cells were not recruited until subsequent hair cycles , when the inner bulge layer became Cdh1-null ( Figure 4C and Figure 4—figure supplement 1B and C ) .", "A robust immune response also encircled the bulge when we ablated Cdh1 selectively in the inner bulge and not in stem cells ( Figure 4C and Figure 4—figure supplement 1D ) .", "While many immune cells , including dendritic cells ( DCs ) , macrophages , γδ dendritic epidermal T cells ( DETCs ) , γδ TCR+ dermal T cells , and αβTCR+ dermal T cells , are known to be residents of the skin ( Naik et al . , 2015; Scharschmidt et al . , 2017; Scharschmidt et al . , 2015 ) , this immune response arising from the breached niche went well beyond the normal surveillance status of the immune system .", "Moreover , the composition of the CD45+ immune cells that infiltrated the breached bulge showed clear differences from the normal resident immune cell patterns .", "Most notable was the increase in immunostaining for CD3+ T cells and CD11b+ myeloid lineage cells , including MHC-class II+ dendritic cells and F4/80+ macrophages ( Figure 4D and E and Figure 4—figure supplement 1E ) .", "To further characterize , distinguish and quantify these cell types , we used these and additional markers to analyze immune cells by flow cytometry ( Figure 4D–4F and Figure 4—figure supplement 1F and G ) .", "We observed a significant increase in the MHCII+ CD11c+ cells ( Figure 4D ) which are defined as dendritic cells since these cells are also negative/low for CD64 , Mertk or Ly6C ( Figure 4—figure supplement 1F ) .", "Additionally , although CD64+ Mertk+ macrophage numbers were more variable , and hence not statistically significant , they did follow a trend of being higher than normal , and preferentially associating with the breached niche ( Figure 4D and Figure 4—figure supplement 1E ) .", "Focusing on T cells that were concentrated near breached bulge niches , γδ T cell and DETC numbers were comparable between WT and Cdh1 cKO skins , although DETC numbers were quite variable even among control samples ( Figure 4E ) .", "By contrast , αβ T cells showed marked and statistically significant increases in Cdh1 cKO skins ( Figure 4E ) .", "Many of these T cells were positive for FOXP3 , a marker for regulatory T cells ( Tregs ) , and further quantification revealed statistically significant increases in both absolute numbers and percentages of Tregs relative to the total αβ T cell population ( Figure 4F and Figure 4—figure supplement 1G ) .", "Moreover , Treg numbers surrounding the bulge were markedly higher than the normal skin resident population ( Figure 4F ) .", "Our data thus far indicated that the ability to elicit an immune response was independent of microbial presence , but dependent upon the barrier breach within the niche .", "A priori , this could mean that Cdh1-null inner bulge cells produce the chemokines that recruit the immune cells .", "Alternatively , upon detecting an AJ-deficient interface with the breached niche , stem cells , whether Cdh1-null or wild-type , might respond by producing and transmitting stress signals to the immune system .", "To distinguish between these possibilities , we FACS-purified and transcriptionally profiled inner bulge niche cells ( Figure 5—figure supplement 1A ) and stem cells ( Figure 1—figure supplement 2C ) from telogen-phase Cdh1 cKO and control littermate bulges .", "To tease out changes specific to stem cells residing in the breached niche , we also performed RNA-sequencing ( RNA-seq ) on WT bulge stem cells in anagen II/III , that is the only time when they naturally proliferate ( Figure 5—figure supplement 1B ) .", "Our data were consistent across independent replicates , enabling us to generate molecular signatures of transcripts that were differentially expressed ( p-value<0 . 05 , absolute fold-change [FC]≥1 . 5 ) in either niche cells ( 1070↑ , 1058↓ ) or stem cells ( 1194↑ , 793↓ ) from Cdh1-null versus control bulges ( Figure 5—figure supplement 1C ) .", "Scrutinizing the specific genes within each of the top four KEGG pathway categories of the Cdh1-null inner bulge profile , we found that transcripts encoding cell adhesion , proteoglycans and cytoskeletal-associated proteins were featured highly , consistent with a feedback attempt by these junction-compromised cells to restore the barrier , while metabolic pathways were downregulated ( Figure 5A and Figure 5—figure supplement 1D ) .", "By contrast , cell cycle genes and immune signaling and response genes dominated the most highly changed pathways in the stem cells from Cdh1-null bulge niches ( Figure 5B–5D , upper Venn diagram and Figure 5—figure supplement 1E and F ) .", "Moreover , in contrast to these proliferative stem cells within the telogen Cdh1-null niche , neither proliferative WT stem cells within an Ana II/III WT bulge nor quiescent WT stem cells within a telogen WT bulge showed expression of these immune pathway genes ( Figure 5—figure supplement 1G ) .", "Principal Component Analyses further highlighted these marked distinctions between proliferative bulge stem cells devoted to normal tissue growth and those repurposed here to add cell layers to the damaged niche ( Figure 5E ) .", "Our analyses also revealed salient differences in the transcriptional responses of niche versus stem cells within the damaged bulge ( Figure 5D , bottom Venn diagram ) .", "This was true even for categories with some apparent overlap , such as cytokine:cytokine receptor interaction transcripts ( Figure 5A and B ) .", "Indeed , closer inspection of individual genes within this and related immune cell categories of the niche transcriptome revealed that levels of these niche transcripts were often considerably lower and less significant than in their stem cell neighbors ( Figure 5F ) .", "By contrast , of the cytokine and chemokine genes that were highly changed in the stem cell transcriptome ( Figure 5C ) , most were substantially more highly expressed by these stem cells than their Cdh1-null niche neighbors , even when the stem cells surrounding a Cdh1-null inner bulge niche were wild-type ( Figure 5G ) .", "Ccl1 and Ccl2 were the top two most highly upregulated genes in this stem cell cohort ( 184X , 38X; Figure 5C ) , and like most of this transcriptome , were barely expressed by the neighboring breached niche .", "Notably , CCL1 and CCL2 are established chemokines for both DCs and macrophages ( Cantor and Haskins , 2007; Gombert et al . , 2005; Li and Tai , 2013; Moser et al . , 2004 ) .", "Importantly , not only were Ccl1 , Ccl2 and many other features of these signatures robustly expressed by the HF stem cells experiencing a breached niche , but this response still occurred even when microbial infiltration was repressed ( Figure 5H ) .", "This suggested that the stem cells recruited immune cells in direct response to the damaged niche rather than the bacteria that infiltrated .", "Since CCL2 is an established chemokine for the CCR2 receptor , expressed by both DCs and macrophages , we focused first on the potential interactions between stem cells and these immune cells .", "To mimic the damaged niche microenvironment in vitro , we used Cdh1-null HF stem cells , which retained elevated Ccl2 in culture ( Figure 5—figure supplement 1H ) .", "We then tested their ability against WT HF stem cells to recruit bone marrow-derived DCs and macrophages in transwell migration assays .", "As shown in Figure 5I , a significant increase occurred in chemotaxis of bone marrow-derived DCs .", "Moreover , this difference was abolished when we used DCs that lacked CCR2 .", "On the other hand , while the migration of macrophages in response to Cdh1-null HF stem cells followed a similar trend , the difference was highly variable and thus not statistically significant .", "Strikingly , these in vitro patterns of DC and macrophage migration were consistent with our in vivo immune cell profiling ( Figure 4D ) , and revealed that Cdh1-null but not WT stem cells can directly attract DCs , and to a lesser extent macrophages , through a CCL2-CCR2-dependent mechanism .", "Conversely , CCL1 stimulates CCR8 receptors , which are on the surface of DCs and macrophages .", "To test the significance of HF stem cell CCL1 , we repeated the cell migration assays as above , this time using a CCR8 blocking antibody to disrupt a potential CCL1-CCR8 interaction .", "Interestingly , while the reduction in chemotactic response of DCs and macrophages was not as robust as blocking the CCL2-CCR2 axis ( Figure 5J ) , treatment of CCR2-null DCs or macrophages with CCR8 blocking antibody to simultaneously block both CCL1 and CCL2-mediated chemotaxis further reduced their migration to statistically significant levels ( Figure 5I ) .", "These findings were important as they indicated that HF stem cells experiencing a barrier breach in their niche can activate chemokines and directly recruit the immune cells that we see elevated upon a barrier breach .", "Why DC recruitment in vivo appeared to be more consistent than macrophage recruitment is not clear .", "However , it is notable that DCs and macrophages respond to similar chemokines during recruitment , and depending upon the particular cytokine milieu , differentiation of inflammatory monocytes can be skewed to DCs or macrophages ( Geissmann et al . , 2010 ) .", "We next addressed whether immune cells are required to trigger the stem cell proliferation that generated a thickened bulge .", "We waited until D18 post-Tam treatment , when Cdh1 was ablated , but immune cells had not yet been recruited to the bulge ( Figure 4B ) .", "We then administered the immunosuppressant dexamethasone ( Dex ) daily for ~2 . 5 weeks , and analyzed mice .", "At this time , untreated counterparts were swarmed by CD45+ immune cells and the bulge was hyperthickened ( Figure 6A ) .", "While not eliminating the resident skin immune cells , Dex effectively blocked the immune infiltration that otherwise surrounded the breached niche barrier in Cdh1 cKO HFs ( Figure 6A ) .", "Significantly , Dex-treated Cdh1 cKO HFs also displayed normal bulge architecture ( Figure 6B ) .", "Accordingly , both the telogen-phase proliferation displayed by stem cells in their deficient niche , and the concomitant increase in stem cell numbers , were now abolished .", "This was further reflected by the enhanced BMP-signaling effector phosphorylated-SMAD1 ( Figure 6B ) , which in normal stem cells only diminishes in Anagen II/III when the natural activating cues override BMP quiescence signals from the inner bulge niche ( Hsu et al . , 2014b ) .", "That said , even in the absence of Dex , there were still signs that the normal telogen signals were intact in the Cdh1-null bulge .", "Thus , inner bulge-derived BMP6 , a prerequisite for these pSMAD1 signaling dynamics , was still expressed by the Cdh1-null inner bulge cells , and correspondingly , the HF stem cells experiencing the breached niche still expressed BMP target genes , including Nfatc1 and Foxc1 ( Figure 6—figure supplement 1A ) .", "Since ablating only one immune component can have a profound effect on the remaining immune cell repertoire , it was necessary to use total immune cell suppression , rather than genetic ablation of specific immune cell populations .", "Although Dex impacts full-anagen phase HFs by inducing their early entry into the destructive phase ( Kwack et al . , 2017 ) , our study focused on telogen , where no adverse effects of Dex had been reported .", "However , it was important to assess whether it was suppression of the immune response or Dex itself that was impacting stem cell proliferation .", "As shown in the growth curves in Figure 6B , Dex alone did not inhibit HF stem cell proliferation directly .", "Moreover , bacteria were still present in immunosuppressed skin , arguing against their involvement in the proliferation dynamics observed here ( Figure 6—figure supplement 1B ) .", "This finding also added to the evidence that the microbial infiltration arose from the loss of junctional integrity within the Cdh1-null niche , and not from the associated architectural disturbances caused by stem cell proliferation in the Cdh1-null niche .", "Given these collective results , we focused on immune cell infiltration as the root of stem cell proliferation and expanded bulge structure .", "Thus far , our immune analyses indicated that DCs and Tregs were elevated prominently and specifically around the breached barrier ( Figure 4 ) .", "Closer inspection revealed that DCs and Tregs were closely associated with each other when surrounding the breached niche , and Dex treatment markedly blocked their infiltration into the Cdh1-null bulge niche ( Figure 6C ) .", "Moreover , the immunosuppressive effects of Dex appeared to be specific for the immune infiltration to the bulge , as immune cells elsewhere in the skin remained during the Dex treatment ( Figure 6C ) .", "In this regard , the Dex-treated Cdh1-cKO pattern of CD3+ and MHCII+ immune cells resembled that of untreated control skin ( Figure 6—figure supplement 1C ) .", "Since Dex blocked immune infiltration into the breached niche , and since immune infiltration was essential for the specific proliferation of bulge stem cells that we observed , we turned to in vitro studies to address whether DCs or Tregs might directly affect HF stem cell proliferation .", "For this purpose , we purified and activated DCs as before ( Figure 5I and J ) , and employed established protocols ( see Materials and methods ) to stimulate freshly isolated splenic naïve CD4+ T cells with CD3/CD28 antibodies ( conventional T cells; Tconv ) .", "For Treg induction , we also added TGFβ ( Fantini et al . , 2007 ) .", "By compartmentalizing the immune cells , we then assessed the effects of their secreted factors on the proliferation of HF stem cells .", "As shown in Figure 6D , while DCs showed no appreciable effect on HF stem cells , Tregs appeared to be particularly robust in soliciting a proliferative response .", "In wild-type skin , resident Tregs , which are positive for NOTCH ligand JAG1 , have been proposed to promote HF stem cell proliferation through direct engagement and activation of Notch target genes ( Ali et al . , 2017 ) .", "If so , we might have expected to see an elevation of Notch target genes in the stem cells of the Cdh1 null bulge , given the marked elevation of Tregs within close proximity .", "That said , the most striking canonical NOTCH target gene showing sensitivity to dexamethasone was Hey1 , and in this case , transcripts were up , rather than down when the Treg swarm was diminished ( Figure 6—figure supplement 2A ) .", "We also examined the status of a cohort of HF stem cell genes reported to bind activated NOTCH in a ChIP-seq analysis of a muscle cell line ( Castel et al . , 2013 ) ( Figure 6—figure supplement 2B ) .", "Indeed , most of these and established NOTCH target genes were very lowly expressed in normal telogen phase HF stem cells , consistent with the well-documented role for NOTCH signaling in HF differentiation , rather than bulge stem cell proliferation ( Demehri and Kopan , 2009 ) .", "Importantly , NOTCH target genes were not elevated appreciably in HF stem cells when the niche barrier was altered and swarmed with Tregs .", "Rather , we surmise that Tregs’ proliferative effects that we observed on HF stem cells here are more likely to be rooted in amphiregulin and/or keratinocyte growth factor , known to be robustly expressed by Tregs in an inflammatory setting ( Arpaia et al . , 2015; Dial et al . , 2017 ) .", "Similarly , in some contexts , the tension sensor YAP , is retained by α-catenin in the cytoplasm and becomes nuclear upon a loss of adherens junctions ( Li et al . , 2016; Neto et al . , 2018; Schlegelmilch et al . , 2011 ) , leading to a marked increase in target genes of the YAP/TEAD transcription factor complex ( Liu and Wang , 2015; Zanconato et al . , 2015; Zhang et al . , 2011 ) .", "Since the telogen-phase HF stem cells in a Cdh1-mutant niche were proliferative , and YAP is known to influence proliferation , the elevation in Ccne1 and other YAP target genes governing proliferation seemed at first glance to suggest altered YAP activity .", "However , Dex treatment largely normalized these cell cycle-related differences , and most other YAP target genes showed no major changes in expression irrespective of immune suppression ( Figure 6—figure supplement 2C ) .", "In the absence of expression changes attributable to Cdh1 status , and given the multiple alternative and possibly confounding means by which YAP is known to be regulated ( Deng et al . , 2018; Totaro et al . , 2018 ) , we did not pursue this further .", "Finally , to identify the portion of the stem cell transcriptome signature that is independent of immune cell infiltration , we performed RNA-seq on stem cells isolated from Dex-treated Cdh1-null and control bulges of telogen-phase HFs and compared their transcriptomes to their non-treated counterparts .", "A total of 509 genes were found to be significantly upregulated in stem cells from Dex-treated Cdh1-null versus control niches .", "Of these , 392 genes ( 77% ) were also upregulated in their non-treated counterparts ( Figure 7A ) .", "Despite stem cells being un-proliferative under Dex treatment , the top KEGG pathway enriched among these 392 overlapping genes was still cell cycle ( Figure 7B ) .", "Although the expression of these cell cycle transcripts was dampened by immunosuppressive conditions ( Figure 7C , purple vs . red bars ) , they were still significantly upregulated , independent of both Dex treatment ( Figure 7C , purple vs . green bars ) and Cdh1 loss ( Figure 7C , orange vs . blue bars ) .", "A similar trend was observed for many of the immune response genes , whose expression persisted in HF stem cells in a breached niche even under immunosuppressive conditions ( Figure 7D ) .", "Taken together , our findings suggested that the necessary threshold level of cell cycle transcripts required to push telogen-phase stem cells into proliferation was contributed in part by the Cdh1-null niche and in part by the ability of these stem cells to recruit immune cells .", "Seeking why telogen-phase proliferation of stem cells was dependent upon not only immune cells but also niche status , we were drawn once again to the reduction in adherens junction proteins that stem cells experienced when they resided in the Cdh1-null bulge niche ( Figure 1C and D ) .", "An inverse correlation between adherens junctions and cell proliferation is well-established ( Stepniak et al . , 2009 ) , and our findings here pointed to a potential link at the transcriptional level .", "Of additional note , many of the immune response transcripts that remained elevated in the HF stem cells of Dex-treated cKO skin were putative NFkB target genes , including Tnfa , Ccl2 , Ccl1 , Csf1 , Cxcl1 and Ccl20 ( Figure 7E ) .", "Previously , we showed that when p120-catenin ( Ctnnd1 ) is conditionally ablated in skin epithelia , IkB kinase ( IKK ) activity is elevated , resulting in the phosphorylation of IkBα and the release of the transcription factor NFkB into the nucleus ( Perez-Moreno et al . , 2006 ) .", "Intriguingly , by immunofluorescence , NFkB showed nuclear localization in telogen-phase Cdh1-null HF stem cells ( Figure 7F ) , which also had reduced p120 levels ( Figure 1C and D ) .", "Although dexamethasone has been reported to dampen NFkB signaling by elevating IkBα transcription ( Auphan et al . , 1995; Scheinman et al . , 1995 ) , it only had a partial effect in Cdh1-null HF stem cells under conditions where IkBα phosphorylation was enhanced ( Figure 7E ) .", "While further details are beyond the scope of the present study , our results suggest a mechanism whereby when the barrier is breached , reductions in adherens junctions impacts the activity of transcription factors such as NFkB , that can directly alter the transcriptional landscape of the HF stem cells in a way that promotes immune cell recruitment , and reduces , but does not overcome , the threshold for proliferation ( Figure 8 and Figure 6—figure supplement 2D ) ." ], [ "The most important function of the skin epithelium is to provide the barrier that excludes harmful microbes and retains body fluids .", "When the barrier is breached , it must be repaired expeditiously to restore fitness to the animal .", "HFs extend deep into the skin , leaving an entry site for pathogens .", "The bulge niche of HF stem cells is strategically positioned at the base of this orifice that opens to the external environment .", "In our current study , we show that in addition to its role as a source of inhibitory signals for its stem cells ( Hsu et al . , 2011 ) , the inner bulge provides the barrier that protects stem cells from microbial invasion .", "In exploring how stem cells cope when their barrier is breached , we learned that by co-expressing P- and E-cadherin , the stem cells themselves are protected from the many situations where E-cadherin levels are naturally downregulated , which includes stem cell activation at the start of the hair cycle ( Lay et al . , 2016 ) .", "Our new data substantiates this further , as when E-cadherin is lost from HF stem cells , they markedly upregulate P-cadherin .", "Through their immediate proximity to the inner bulge layer , the HF stem cells are poised to sense a barrier breach .", "Remarkably , our studies revealed that neither bacteria nor immune cells were required for this sensing to occur .", "Although the precise details await further investigation , the roots are likely to reside in the inability of stem cells to form adherens junctions with their Cdh1-null niche neighbors , which lack a cadherin backup system .", "As our data revealed , intercellular cadherin-cadherin junctions are featured prominently at the stem cell-niche interface of healthy HF bulges .", "Such junctions are known to act as key signaling centers and integrators of proliferation , polarity and the actin cytoskeleton across an epithelial tissue ( Padmanabhan et al . , 2015; Stepniak et al . , 2009 ) , and our current findings add to the mounting evidence that these junctions also serve as key regulators of inflammatory sensors ( Perez-Moreno et al . , 2006 ) .", "As we learned , once stem cells face a breach in their niche barrier , they trigger a distress transcriptional program that we show occurs even in the face of antibiotics or immune suppressive drugs and is strikingly different from the response elicited in the neighboring niche cells .", "Our findings are interesting in light of prior studies showing that under a number of different conditions , keratinocytes can express cytokines and chemokines that harbor potential to communicate with the immune system in normal homeostasis and in injury ( Adachi et al . , 2015; Ali et al . , 2017; Chen et al . , 2015; Pasparakis et al . , 2002; Zhang et al . , 2004 ) .", "Of additional relevance to our study are previous observations that hair plucking enhances hair cycling , an effect which has been postulated on the one hand to be triggered by the loss of the BMP-expressing inner bulge niche cells ( Hsu et al . , 2011 ) but on the other hand to an elevation of Ccl2 and inflammatory macrophages in the skin ( Chen et al . , 2015 ) .", "While intriguing , none of these prior studies have revealed whether stem cells are specifically capable of altering their transcriptional program in response to perturbations in their microenvironment and if they can , whether this endows them with the ability to adjust their behavior by directly recruiting immune cells .", "Notably , Ccl2 was also at the top of our changed genes , along with related Ccl1 , underscoring their sensitivity to skin perturbations , and tracing their expression to stem cells .", "However , in our system , when the entire immune repertoire was analyzed and quantified relative to bulge proximity , DCs were among the most consistently changed early immune respondents .", "Like M1 macrophages , DCs have surface receptors that respond to CCL1/2 ( Gombert et al . , 2005; Moser et al . , 2004 ) .", "Moreover , by in vitro co-culture and genetic targeting of the corresponding DC-CCL receptors , we showed that DCs use this crosstalk with HF stem cells to specifically congregate around the bulge .", "In this case , however , recruitment of DCs and ( more variably ) macrophages did not trigger a new hair cycle , pointing to a major difference between disrupting the barrier function of the BMP6-expressing inner niche layer and removing this layer altogether as in hair plucking which does initiate a new hair cycle .", "During normal epithelial homeostasis , resident DCs can dialogue with Tregs ( Leventhal et al . , 2016; Seneschal et al . , 2012 ) , and thus may also be capable on their own to shape the adaptive immune response that swarmed the damaged niche .", "Although our co-localization data and accumulation of Tregs with DCs around the bulge are supportive of this mechanism , our transcriptomics revealed that independently of immune cells , stem cells within a damaged niche are also well-equipped with a complex arsenal of cytokines and chemokines to communicate with both the adaptive and innate immune systems .", "Such chemokines include CCL1 ( Hoelzinger et al . , 2010; Scharschmidt et al . , 2017 ) and CCL20 , which are both potent stimuli for Tregs .", "Recently , it was shown that in normal homeostasis , Tregs come into close proximity of the bulge towards the telogen to anagen transition , where in addition to BMP-inhibitors , WNTs and other activating cues , they help to fuel hair regeneration ( Ali et al . , 2017 ) .", "Although Treg ablation studies suggested a role for Tregs in hair cycling , it has remained unknown whether such effects are direct or indirect .", "Our findings here showed that proliferative effects of Tregs on HF stem cells can be direct .", "It is curious that in homeostasis ( Ali et al . , 2017 ) and hair plucking ( Chen et al . , 2015 ) , stem cells are stimulated to proliferate and launch new hair growth , while in the face of a barrier breach , stem cells direct their efforts towards containing the damage .", "Based upon the evidence at hand , this difference likely resides in the plethora of growth factors , cytokines , reactive oxygen species , and lipid mediators that are generated by immune cells under different conditions ( Demehri et al . , 2008; Yoon et al . , 2016 ) .", "Although the precise nature of this complex communication between the stem cells and immune cells is beyond the scope of the current study , we have shown that it is considerably more robust and diverse than that which functions in normal hair cycling or transient injury response .", "That said , the collective evidence points to an important role for stem cells in sensing and responding to their local environment .", "Their ability to subsequently recruit distinct immune cell repertoires enables stem cells to receive new sets of instructions that deviate from their normal homeostatic state , thereby channeling their efforts in new directions .", "This intricate feedback mechanism enables the recruited immune cells to repurpose stem cells from their normal job of making hair to instead participate in curbing the breached barrier .", "This diversion of HF stem cells away from their normal regenerative function had two important outcomes .", "First , it added a new layer of AJ-competent cells to line/reinforce the damaged bulge .", "Second , because the activated stem cells transcribed an array of immune signaling genes , their amplification within the niche bolstered the local production of distress signals .", "Altogether , this captivating communication network appeared to be aimed at patching the breached barrier and limiting tissue damage that would otherwise be caused by chronic inflammation .", "Indeed , the ensuing immune cell-associated ‘hyperthickening’ phenotype was locally restricted to the site of the barrier breach ( Figure 8 and Figure 6—figure supplement 2D ) .", "Since our breached HF barrier model was a genetic one , it was not surprising that the efforts of the HF stem cells to patch the barrier were futile , as evidenced by the bacterial infiltration seen in the Cdh1-null HFs and their surrounding tissue ( Figure 3 ) .", "Indeed , there are a number of genetic disorders involving breached skin barriers in which stem cells hyper-proliferate and yet are unable to repair the barrier .", "In this regard , it is intriguing to consider epidermolytic hyperkeratosis patients and mice , which harbor suprabasal epidermal keratin one and/or keratin 10 mutations .", "In this case , the healthy epidermal stem cells respond to the barrier breach , but they maintain a single layer of proliferative progenitors , while dramatically expanding the ( defective ) layers of terminally differentiating cells ( Fuchs et al . , 1992 ) .", "By contrast , the proliferative Cdh1 null HF stem cells cannot directly generate the terminally differentiated barrier cells ( Hsu et al . , 2011 ) , and instead generate multiple layers of stem cells .", "While neither of these responses are successful , they likely reflect a general , evolutionarily conserved mechanism intrinsic within epithelial stem cells to respond to and repair barrier defects wherever they emerge .", "Studies in Drosophila testis and ovary have long established a role for E-cadherin in adhering germline stem cells to their niche , a process critical to balancing stem cell self-renewal and differentiation ( Song et al . , 2002; Yamashita et al . , 2003 ) .", "Although the roles of AJs in mammalian stem cell niches have not been studied extensively , the increased HF stem cell proliferation could reflect a direct response to the reduced AJ levels that appeared to emanate from the perturbed inner bulge:stem cell interface .", "Thus it was surprising that immune suppression kept the ectopic proliferation of telogen HF stem cells largely in check .", "These findings expose an additional layer of complexity in the proliferative control of HF stem cells not seen in the invertebrate stem cell systems studied to date .", "Previous reports have described immune , hyperproliferative responses of mammalian epithelial tissues following genetic perturbations in various AJ components ( Perez-Moreno et al . , 2006; Vasioukhin et al . , 2001 ) .", "However , the responses documented occur directly at the level of genetic alterations and are thus largely cell-autonomous .", "The specific roles for α-catenin and p120-catenin in signaling also appear to be independent of barrier function .", "Notably , as we have shown here , the stem cells displaying a hyperproliferative response can themselves be wild-type and depend upon their border with damaged niche neighbors to trigger the subsequent cascade of events we identified .", "Despite the critical role that the immune response plays to induce stem cell proliferation , the telogen-phase stem cells within the damaged niche still maintained in part a transcriptome reflective of cell proliferation even when the immune response was repressed .", "This aspect of their transcriptome paralleled that of their natural proliferative state in the hair cycle .", "However , in early anagen , the levels of hair cycle stimulatory factors outweigh the quiescence factors emanating from the bulge niche .", "By contrast , within a damaged telogen-phase niche , the quiescence factors still remained high , and in the absence of anagen-stimulating factors , the reduction in AJs and elevation in cell cycle transcriptome were not sufficient on their own to activate these stem cells .", "In this way , the proliferative trigger for repurposing the stem cells became contingent upon the unleashing of immune distress signals upon the failure of their niche neighbors to provide a proper barrier .", "Our studies exposed a fascinating interplay between the terminally differentiated cells that provide the niche barrier , and its neighboring stem cells that fuel regenerative processes in hair cycling and wound repair .", "Through their ability to respond to their damaged niche , stem cells are able to alter their program of gene expression in a way that both primes the stem cells for proliferation and enables them to recruit immune cells .", "In turn , the immune cells bestow new keratinocyte growth factors and cytokines on the local niche microenvironment .", "The strong presence of Tregs may also enable stem cell niches to limit tissue damage that might otherwise be caused by massive inflammation .", "Our findings suggest that through their elevated cell cycle transcriptome , the primed stem cells within the damaged niche have a reduced threshold for proliferation and therefore selectively respond to recruited immune cells , even though normal homeostatic quiescent controls are still sufficient to keep other nearby cells ( e . g . the hair germ ) in check .", "This selective self-renewal behavior by stem cells sustains the immune response and also reinforces the damaged barrier with layers of cells whose intercellular junctions are intact .", "Our findings explain a number of pathological conditions involving perturbations in terminally differentiating cells that are manifested in healthy stem/progenitor cells ." ], [ "Cdh3null , Cdh1Flox , Rosa26lox-STOP-lox-YFP , Sox9-CreER , Krt15-CrePGR and Krt14-rtTA were described previously ( Boussadia et al . , 2002; Mao et al . , 1999; Morris et al . , 2004; Nguyen et al . , 2006; Radice et al . , 1997; Soeda et al . , 2010 ) .", "Sox9-CreER was activated by tamoxifen ( Sigma ) administered either by intraperitoneal ( i . p . ) injection ( 75 μg/g body weight in corn oil ) once a day for 3 days or by single topical application ( 1 . 5 mg in ethanol ) on hair coat of mice .", "Krt15-CrePGR was activated by mifepristone ( RU486 , TCI America ) administered by i . p . injection ( 75 μg/g body weight in sesame oil ) and topically ( 12 mg in ethanol ) on shaved dorsal back of mice once a day for 7 days .", "Antibiotics treatment was performed by feeding mice with chow containing 0 . 025% trimethoprim and 0 . 1242% sulfamethoxazole; feeding with antibiotics water containing metronidazole ( 500 mg/L , Sigma ) , sulfamethoxazole ( 400 mg/L , Sigma ) , trimethoprim ( 100 mg/L , Sigma ) and cephalexin ( 500 mg/L , Sigma ) with Splenda sweetener to improve the taste of the water; and i . p . injection of vancomycin ( 0 . 75 mg , Sigma ) and metronidazole ( 0 . 75 mg , Sigma ) daily .", "Sox9CreER mice were treated with antibiotics starting one week post-tamoxifen until time of harvest .", "Immunosuppression was achieved by daily i . p . injections of dexamethasone at 1 mg/kg body weight .", "rtTA was activated by feeding mice with doxycycline ( 2 mg/kg ) chow .", "To track hair cycles ( Müller-Röver et al . , 2001 ) , full-length telogen hairs were trimmed with electric clippers to reveal dorsal skin .", "HF entry into anagen was determined by darkening of skin and reappearance of hair .", "Completion of anagen and re-entry into telogen were determined by appearance of full-length hairs and loss of pigmentation in skin .", "EdU ( 5-ethynyl-2’-deoxyuridine; Thermo Fisher , 25 μg/g body weight ) was injected i . p . into mice twice a day before obtaining skin biopsies or lethal administration of CO2 .", "All mice were maintained in a facility approved by The Association for Assessment and Accreditation of Laboratory Animal Care ( AAALAC ) , and procedures were performed with protocols approved by Rockefeller University’s institutional animal care and use committee ( IACUC ) members .", "Cdh1-shRNA was obtained from the Broad Institute’s Mission TRC-1/2 mouse library and cloned into LV-pLKO-PGK-H2B-RFP vector ( Moffat et al . , 2006 ) .", "Cdh1-shRNA sequence is CCGAGAGAGTTACCCTACATA .", "For myc-tagged PCAD overexpression , Cdh3 coding DNA sequence ( CDS ) containing myc tag sequence inserted after its pro-peptide sequence was synthesized and cloned into LV-TRE vector .", "To tag PCAD with myc at the C-terminal end , Cdh3 CDS was amplified with myc inserted before its STOP codon , then cloned into LV-TRE vector .", "LV production , concentration and ultrasound-guided transduction of mouse embryos in utero were performed as described previously ( Beronja et al . , 2010 ) .", "To prepare sagittal skin sections for immunofluorescence microscopy , backskins were either freshly embedded and frozen in OCT , or pre-fixed in 4% paraformaldehyde ( PFA ) in PBS for 2 hr at 4°C then treated with 30% sucrose in PBS overnight at 4°C prior to embedding and freezing in OCT .", "Backskins were cryosectioned ( 12 μm ) and sections from freshly embedded tissues were fixed for 10 min in 4% PFA .", "To prepare whole-mounts for immunofluorescence microscopy , subcutaneous fat was scraped from backskins , which were then incubated ( dermis side down ) on 2 . 5 U/ml dispase +20 mM EDTA for 2 hr at 37°C .", "Epidermis and HFs were separated from dermis , fixed in 4% PFA for 30 min at room temperature ( RT ) and permeabilized for 30 min in PBS + 0 . 5% Triton ( PBST ) .", "Sections and whole-mounts were blocked for 1–2 hr at RT in 2% fish gelatin , 5% donkey serum , 1% BSA and 0 . 2% Triton in PBS .", "Primary antibody incubation was performed overnight at 4°C .", "Incubation with secondary antibodies conjugated to rhodamine red-X , Alexa 488 , 546 or 647 was performed for 1–2 hr at RT .", "Mouse antibodies were incubated with M . O . M . block according to manufacturer’s directions ( Vector Laboratories ) .", "Images were acquired with Zeiss Axio Observer Z1 equipped with ApoTome . 2 through a 20x air objective or Zeiss LSM780 laser-scanning confocal microscope through a 40x water objective .", "To prepare sagittal skin sections for hematoxylin and eosin staining and immunohistochemistry , backskin was fixed in 4% PFA overnight at 4°C .", "Subsequent dehydration , paraffin embedding , sectioning and staining were performed by Histowiz Inc .", "Stained slides were scanned at 40x magnification using Aperio AT2 and visualized using Aperio Image Scope software .", "Modified Gram staining method has been described by ( Becerra et al . , 2016 ) .", "Briefly , paraffin sections were applied , in the following order , with crystal violet , Gram iodine , Gram decolorizer and safranin ( Remel ) .", "To dehydrate , sections were immersed in 95% ethanol , 100% ethanol , then treated with alcoholic saffron ( American Master Tech Scientific Inc . ) before further dehydration with 100% ethanol and citrus clearing solvent .", "Images were acquired with an Axioplan two upright microscope equipped with a Spot Insight QE color digital camera through 40x N . A . 1 . 3 oil and 63x N . A . 1 . 4 oil objectives .", "The following antibodies and dilutions were used for immunofluorescence: ECAD ( rabbit , 1:1000 , Cell Signaling , AB_2291471 ) , PCAD ( goat , 1:400 , Sigma , AB_355581 ) , CD34 ( rat , 1:100 , eBioscience , AB_466426 ) , K6 ( guinea pig , 1:2000 , Fuchs lab ) , TCF4 ( rabbit , 1:250 , Cell Signaling , AB_2199816 ) , SOX9 ( rabbit , 1:1000 , Fuchs lab ) , LHX2 ( rabbit , 1:1000 , Fuchs lab ) , p120-catenin ( mouse , 1:1000 , Zymed , AB_87178 ) , β-catenin ( mouse , 1:1000 , BD , AB_397555 ) , α-catenin ( rabbit , 1:1000 , Sigma , AB_476830 ) , DSG3 ( mouse , 1:500 , MBL , AB_591238 ) , plakoglobin ( mouse , 1:500 , BD , AB_397649 ) , DP1 and 2 ( mouse , 1:500 , Millipore , AB_93346 ) , myc-tag ( rabbit , 1:300 , Cell Signaling , AB_490778 ) , CLDN1 ( rabbit , 1:500 , Abcam , AB_301644 ) , ZO1 ( rabbit , 1:250 , Zymed , AB_2533938 ) , CD45 ( rat , 1:100 , Biolegend , AB_312969 ) , MHC II ( rat , 1:100 , Biolegend , AB_493727 ) , F4/80 ( rat , 1:100 , Biolegend , AB_893504 ) , CD3 ( rat , 1:100 , Biolegend , AB_1877072 ) , FOXP3 ( rat , 1:25 , eBioscience , AB_467576 ) , GFP ( chicken , 1:2000 , Abcam , AB_300798 ) , pSMAD1 ( rabbit , 1:100 , Cell Signaling , AB_331671 ) and phospho-p65 ( rabbit , 1:50 , Cell Signaling , AB_330559 ) .", "Phalloidin ( 1:100 , Thermo Fisher ) was used to detect F-actin .", "Nuclei were stained with 4’6’-diamidino-2-phenylindole ( DAPI ) .", "EdU click-iT reaction was performed according to manufacturer’s directions ( Thermo Fisher ) .", "β-catenin antibody ( rabbit , 1:50 , Cell Signaling , AB_11127855 ) was used for immunohistochemistry .", "To prepare single cell suspensions from telogen backskin , subcutaneous fat was scraped off with a scalpel and backskin was placed ( dermis side down ) on 0 . 25% trypsin ( Gibco ) for 35–45 min at 37°C .", "If inner bulge cells were to be isolated , backskin was placed on trypsin overnight at 4°C , then 1 hr at 37°C .", "To prepare single cell suspensions from anagen backskin , backskin was placed ( dermis side down ) on collagenase ( Sigma ) for 45 min at 37°C , dermal side was scraped off with a scalpel , and remaining epidermal side was transferred to trypsin for 20 min at 37°C .", "For both telogen and anagen backskins , trypsinized HF and epidermal cells were then scraped off gently and filtered with strainers ( 70 μm , followed by 40 μm ) .", "Dissociated cells were incubated with antibodies for 20 min at 4°C .", "The following antibodies were used: CD34-eFluor660 ( 1:100 , eBioscience , AB_10596826 ) , α6-PE ( 1:100 , BD Bioscience , AB_396079 ) , α6-PerCP-Cy5 . 5 ( 1:250 , Biolegend , AB_2249260 ) , β1-PE-Cy7 ( 1:400 , eBioscience , AB_1234962 ) , Sca1-PerCP-Cy5 . 5 ( 1:1000 , eBioscience , AB_914372 ) , Sca1-APC-Cy7 ( 1:1000 , Biolegend , AB_10645327 ) , CD200-PE ( 1:300 , eBioscience , AB_1907362 ) , CD45-biotin ( 1:200 , eBioscience , AB_466446 ) , CD31-biotin ( 1:200 , eBioscience , AB_466423 ) , CD117-biotin ( 1:200 , eBioscience , AB_466569 ) , CD140a-biotin ( 1:200 , eBioscience , AB_466606 ) and Streptavidin-PE-Cy7 ( 1:500 , eBioscience , AB_1011648 ) .", "DAPI was used to exclude dead cells .", "Cell purification was performed on FACS Aria sorters equipped with Diva software ( BD Bioscience ) .", "To analyze immune cells , backskin was minced in RPMI1640 media with L-glutamine ( Gibco ) , 1 mM sodium pyruvate ( Lonza ) , 10 mM acid-free HEPES ( Gibco ) , 100 U/ml penicillin and streptomycin ( Gibco ) .", "Liberase TL ( Roche ) was added to the media ( 25 g/ml ) and backskin was digested for 120 min at 37°C .", "Digestion was stopped by addition of 20 ml of 0 . 5 M EDTA ( Life Technologies ) and 1 ml of 10% DNase ( Sigma ) solution .", "Dissociated cells were filtered with 70 μm strainer and stained with the following antibodies: Ly6c-FITC ( 1:100 , Biolegend , AB_1186134 ) , Ly6g-PE ( 1:200 , Biolegend , AB_1186104 ) , CD11c-PE-Cy7 ( 1:150 , Biolegend , AB_493569 ) , CD11b-PacBlue ( 1:300 , Biolegend , AB_755985 ) , MHCII-AF700 ( 1:300 , Biolegend , AB_493727 ) , CD45-AF750 ( 1:100 , Biolegend , AB_2572115 ) , CD64-PerCP-Cy5 ( 1:200 , Biolegend , AB_2561962 ) , TCRβ-PerCP-Cy5 . 5 ( 1:200 , Biolegend , AB_1575176 ) , γδTCR-APC ( 1:400 , Biolegend , AB_1731813 ) and MerTK-PE ( 1:100 , Biolegend , AB_2617035 ) .", "Dead cells were excluded using LIVE/DEAD Fixable Blue Dead Cell Stain Kit ( Molecular Probes ) for UV excitation .", "FACS analyses were performed using LSRII FACS Analyzers and results were analyzed using FlowJo software .", "FACS-purified cell protein lysates were prepared using RIPA buffer containing protease inhibitor ( Roche ) and split equally for ECAD-ELISA and PCAD-ELISA .", "ECAD-ELISA was performed as per manufacturer’s protocol ( R and D Systems ) .", "PCAD-ELISA was performed as per ECAD-ELISA protocol , but instead using PCAD monoclonal antibody ( rat , Life Technologies , AB_2533006 ) as capture antibody , PCAD-biotin polyclonal antibody ( goat , R and D Systems , AB_442232 ) as detection antibody , and recombinant mouse PCAD Fc chimera protein ( R and D Systems ) to calibrate standard curve for quantification .", "Gel electrophoresis of FACS-purified cell protein lysates was run on 4–12% NuPAGE Bis-Tris gradient gel ( Thermo Fisher ) and transferred to nitrocellulose membrane ( Amersham ) .", "Membrane was blocked in TBS containing 2% bovine serum albumin ( BSA ) and 0 . 1% Tween-20 for 1 hr at RT , incubated with primary antibodies overnight at 4°C and with secondary antibodies conjugated to horseradish peroxidase ( HRP ) for 1 hr at RT .", "HRP activity was detected using enhanced chemiluminescence ( ECL ) substrate ( Amersham ) .", "Quantification of bands was performed on ImageJ software .", "The following antibodies were used: ECAD ( rabbit , 1:1000 , Cell Signaling , AB_2291471 ) , PCAD ( goat , 1:1000 , R and D Systems , AB_355581 ) , p120 ( mouse , 1:1000 , Zymed , AB_87165 ) , β-catenin ( mouse , 1:1000 , BD Biosciences , AB_11127855 ) , α-catenin ( rabbit , 1:4000 , Sigma , AB_476881 ) , claudin 1 ( rabbit , 1:500 , Abcam , AB_301644 ) , ZO1 ( rabbit , 1:500 , Life Technologies , AB_2533938 ) and GAPDH ( mouse , 1:4000 , Abcam . AB_2107448 ) .", "To prepare samples for 16S-FISH , backskins , including those of germ-free mice as negative control , were fixed in 4% PFA overnight at 4°C , then washed with PBS before being permeabilized in 0 . 3% PBST overnight at 4°C .", "Backskins were additionally fixed and permeabilized through a methanol series: 100% for 4 × 5 min , 100% for 1 × 30 min , 75% for 1 × 5 min , 50% for 1 × 5 min , and 30% for 1 × 5 min , followed by 3 × 5 min of 0 . 3% PBST washes .", "To probe for bacterial 16S , FISH with hybridization chain reactions ( HCR ) was employed ( Choi et al . , 2010 ) .", "Samples were pre-hybridized with probe hybridization buffer for 30 min at 45°C before incubating with 1 pmol of each probe , mixed together , overnight at 45°C .", "The sequences of the probes ( probeBase ) were: EB338: GCUGCCUCCCGUAGGAGU Univ1390: GACGGGCGGUGUGUACAA Scrambled ( non-EUB , negative control ) : ACUCCUACGGGAGGCAGC Following hybridization , samples were washed with probe wash buffer for 2 × 5 min and 2 × 30 min at 45°C , then washed with 5X SSCT ( containing 0 . 1% Tween-20 ) for 3 × 5 min at RT .", "Samples were pre-amplified with amplification buffer for 30 min at RT before incubating with 30 pmol of fluorescently labeled hairpins overnight at RT .", "Samples were washed with 5X SSCT for 2 × 5 min , 2 × 30 min and 1 × 5 min at RT before fixation in 4% PFA for 5–15 min at RT and incubation with DAPI ( 2 μg/ml ) overnight at 4°C .", "Samples were then washed with 0 . 3% PBST before being cleared for imaging .", "Probe hybridization buffer , probe wash buffer and probes were purchased from Molecular Technologies .", "For tissue clearing , backskins were transferred through increasing concentrations of ethanol diluted in distilled water and adjusted to pH9 . 0: 30% for 20 min , 50% for 20 min , and 70% for 20 min .", "Backskins were then incubated in 100% ethanol for 1 hr before transferring into ethyl cinnamate ( Sigma ) for clearing .", "Images were acquired with an inverted spinning-disk confocal system driven by iQ Live Cell Imaging software ( Andor ) using a 20x N . A . 0 . 75 air objective and 561 nm , 642 nm and UV laser lines , and analyzed using Imaris software .", "For spot analysis and quantification , sphere sizes were set to 2 . 5–3 , and the estimated x-y diameter for spots was 0 . 6–1 . 5 μm .", "WT bulge RNA-seq data is from Lay et al . ( 2016 ) .", "For all others , total RNA was extracted from FACS-purified cells by directly sorting cells into TrizolLS ( Sigma ) and processing with Direct-Zol RNA mini-prep kit ( Zymo Research ) .", "RNA quality was determined using an Agilent 2100 Bioanalyzer and all samples sequenced had RNA integrity numbers", "> 8 . mRNA library preparation using Illumina TrueSeq mRNA sample preparation kit and single-end sequencing on Illumina HiSeq 4000 were performed at Weill Cornell Medical College Genomic Core Facility ( New York ) .", "Alignment of reads was done using STAR version 2 . 5 . 2a ( Spliced Transcripts Alignment to a Reference ) ( Dobin et al . , 2013 ) and transcripts were annotated using Gencode release M10 of the mouse genome .", "Differential gene expression analysis was performed with DESeq2 package version 1 . 18 . 0 ( Love et al . , 2014 ) using the gene count output from STAR read aligner .", "Gene ontology analysis was performed using DAVID ( Huang et al . , 2009a; Huang et al . , 2009b ) .", "To perform qRT-PCR , equal amounts of RNA were reverse transcribed using Superscript III with oligo-dT primer ( Invitrogen ) or Superscript VILO ( Invitrogen ) cDNA Synthesis kits .", "cDNAs were mixed with indicated primers and SYBR green PCR Master Mix ( Sigma ) , and qRT-PCR was performed on an Applied Biosystems 7900HT Fast Real-Time PCR system .", "cDNAs were normalized to equal amounts using primers against Ppib .", "The following primer sequences were used ( 5’ → 3’ ) : Ccl1 forward: AGTGTTACAGAAAGATGGGCTC , Ccl1 reverse: GAGGACTGAGGGAAACTGC Ccl2 forward: GTCCCTGTCATGCTTCTGG , Ccl2 reverse: GTGATCCTCTTGTAGCTCTCC Ppib forward: GTGAGCGCTTCCCAGATGAGA , Ppib reverse: TGCCGGAGTCGACAATGATG Bmp6 forward: GGGGCTCCGGTTCTTCAGA , Bmp6 reverse: GGACGTACTCGGGATTCATAAGGT , Fgf18 forward: CTGTGCTTCCAGGTTCAGGT , Fgf18 reverse: TGCTTCCGACTCACATCATC , Skin biopsies were treated with dispase with EDTA , and epidermis was separated from dermis , in the same way as preparing tissue for whole-mount immunofluorescence .", "HFs in the epidermal fraction were then submerged in 1 mM Lucifer yellow ( Thermo Fisher ) in PBS for 3 hr at 37°C before fixation , mounting and imaging .", "HF stem cells were FACS purified and co-cultured with feeder fibroblasts in 300 μM Ca2+ as described previously ( Nowak and Fuchs , 2009 ) .", "For growth curve assays , equal numbers of Cdh1 cKO and control HF stem cells were cultured in triplicates in the absence or presence of 10 nM dexamethasone , and cell numbers were counted every other day from Days 5 to", "9 . For transwell migration assay , bone marrow-derived dendritic cells ( BMDCs ) or bone marrow-derived macrophages ( BMDM ) were obtained from femur and tibiae bone marrow of 8-week-old C57BL/6 mice and cultured in BMDC/macrophage medium ( RPMI 1640 with L-glutamine , 10% heat-inactivated FBS , 10 mM HEPES , and 100 U/ml penicillin/streptomycin ) at a density of 1 × 106 cells/ml with addition of 20 ng/ml recombinant murine GM-CSF ( R and D Systems ) and 10 ng/mL recombinant murine IL-4 ( R and D Systems ) for BMDC and 10 ng/ml recombinant murine M-CSF ( R and D Systems ) for BMDM derivation .", "The maturation of BMDCs or BMDM was induced by 100 ng/ml of LPS 24 hr prior to co-culture with HF stem cell medium .", "5 × 105 mature WT or CCR2-nullBMDCs or BMDM were seeded onto upper chambers of 5 μm pore size in transwell plates with or without isotype control antibody or CCR8 blocking antibody ( Biolegend , AB_2566246 ) .", "WT or Cdh1-null HF stem cells were cultured in lower chambers and incubated for 3 . 5 hr at 37°C .", "BMDCs or BMDM that migrated into the bottom chamber were harvested and counted by flow cytometry , using CD45-APC ( 1:500 , eBioscience , AB_469392 ) and DAPI to exclude dead cells .", "For co-culture proliferation assays , BMDCs were isolated in the same way as described above for transwell migration assay .", "Naive CD4+ T cells were isolated from the spleen of the 8-week-old C57BL/6 mice by Mojo CD4 T cell Negative Selection Kit ( Biolegend ) , followed by in vitro activation on plates coated with 10 μg/ml anti-CD3 antibody ( Thermo Fisher Scientific , AB_468847 ) and 2 μg/ml anti-CD28 antibody ( Thermo Fisher Scientific , AB_468921 ) , with or without 5 ng/ml TGF-β1 ( R and D Systems ) , and incubation for 5 days to obtain regulatory T cells ( Treg ) and conventional T cells ( Tconv ) respectively .", "Tregs were then enriched by FACS using CD45-APC/Cy7 ( 1:200 , Biolegend , AB_312981 ) , CD4-PE/Cy7 ( 1:200 , Biolegend , AB_312707 ) , CD25-APC ( 1:200 , Biolegend , AB_2280288 ) , CD127-PE ( 1:200 , Biolegend , AB_493509 ) , and DAPI to exclude dead cells .", "Both Tconv and FACS-purified Tregs were cultured in T cell medium ( RPMI-1640 with L-glutamine , 10% heat-inactivated FBS , 1 mM HEPES , 1 mM sodium pyruvate , 100 U/ml penicillin/streptomycin , and 55 μM 2-mercaptoethanol ) .", "To measure the effects of BMDCs , Tconv and Tregs on HF stem cell proliferation , 5000 Cdh1 cKO HF stem cells were seeded onto feeder fibroblast layers in lower chambers of transwell plates .", "2 days later , HF stem cells were co-cultured with 2 × 105 LPS-treated BMDCs seeded in BMDC media , or 1 × 104 Tconv or FACS-isolated Tregs seeded in T cell media in upper chambers of 0 . 8 μm pore size for 4–5 days before their numbers were counted .", "For EM , backskins were fixed in 2% glutaraldehyde , 4% PFA , and 2 mM CaCl2 in 0 . 05 M sodium cacodylate buffer , pH7 . 2 , at RT for >1 hr , post-fixed in 1% osmium tetroxide , and processed for Epon embedding .", "Ultrathin sections ( 60–65 nm ) were counterstained with uranyl acetate and lead citrate .", "EM images were taken with a transmission electron microscope ( Tecnai G2-12; FEI ) equipped with a digital camera ( Ultrascan , Gatan Inc . ) .", "Data were analyzed and statistics were performed using unpaired two-tailed Student’s t-test ( when comparing two groups ) , one-way ANOVA with Tukey’s post-hoc test ( when comparing >2 groups ) or two-way ANOVA with Sidak test ( for quantifications of EdU +HF stem cell numbers ) in GraphPad Prism versions 6 and 7 , with 0 . 05 level of confidence being accepted as a significant difference .", "The accession number for the RNA-seq data reported in this paper is NCBI GEO: GSE106767 ." ] ]

[ "Adult stem cells are responsible for life-long tissue maintenance .", "They reside in and interact with specialized tissue microenvironments ( niches ) .", "Using murine hair follicle as a model , we show that when junctional perturbations in the niche disrupt barrier function , adjacent stem cells dramatically change their transcriptome independent of bacterial invasion and become capable of directly signaling to and recruiting immune cells .", "Additionally , these stem cells elevate cell cycle transcripts which reduce their quiescence threshold , enabling them to selectively proliferate within this microenvironment of immune distress cues .", "However , rather than mobilizing to fuel new tissue regeneration , these ectopically proliferative stem cells remain within their niche to contain the breach .", "Together , our findings expose a potential communication relay system that operates from the niche to the stem cells to the immune system and back .", "The repurposing of proliferation by these stem cells patch the breached barrier , stoke the immune response and restore niche integrity ." ]

[ "Most , if not all , tissues of an adult animal contain stem cells .", "These stem cells regenerate and repair damaged tissues and organs for the entire lifetime of an animal , contributing to a healthy life .", "They divide to make daughter cells that become either new stem cells or specialized cells of that organ .", "Adult stem cells exist in specific areas within tissues known as niches , where they interact with surrounding cells and molecules that inform their behavior .", "For example , cells and molecules within these niches can signal stem cells to remain in a ‘dormant’ state , but upon injury , they can mobilize stem cells to form new tissue and repair the wound .", "So far , it has been unclear how stem cells sense damage and stress and direct their efforts away from their normal duties towards repair .", "Here , Lay et al . studied the stem cells in the mouse skin that are responsible to regenerate hair .", "Every hair follicle contains a niche ( the ‘bulge’ ) , where these stem cells live and share their environment with cells that anchor the hair .", "The niche tethers to the stem cells through specific adhesion molecules that also help the niche to form a tight seal to prevent bacteria from entering .", "Lay et al . removed one of the adhesion molecules called E-cadherin , which caused a breach in the niche’s barrier .", "The stem cells sensed their damaged niche , prepared to multiply , and sent out stress signals to the immune system .", "The immune cells then arrived at the niche and sent signals back to the stem cells , prodding them to multiply and patch the barrier , while at the same time , keeping the inflammation in check .", "This remarkable ability of the stem cells to recruit immune cells and initiate a dialogue with them enabled the stem cells to divert their attention from regenerating hair and instead directing it towards the site of the tissue damage .", "Other stem cells , such as those in the lung or gut , may have similar mechanisms to detect and respond to physical damage .", "It will be interesting to investigate the underlying mechanism of how immune cells are involved in balancing stem cell regenerative capacity and response to physical damage .", "A better knowledge of these processes could help to regenerate tissues or even entire organs ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "developmental biology" ]

[ [ "In many animals , development from a single cell to a complex multicellular organism requires the graded distribution and activity of diffusible proteins , which are known as morphogens .", "How morphogen gradients are formed is not fully understood .", "Studies in many organisms have suggested three major mechanisms to establish morphogen gradients:", "1 ) diffusion ,", "2 ) transcytosis and", "3 ) via cytonemes ( Green , 2002; Müller et al . , 2013; Rogers and Schier , 2011; Wartlick et al . , 2009 ) .", "For example , the gradient of fibroblast growth factors ( FGFs ) is established by diffusion and is regulated by extracellular heparan sulfate proteoglycans ( HSPGs ) ( Dowd et al . , 1999; Duchesne et al . , 2012; Makarenkova et al . , 2009; Miura et al . , 2009; Nowak et al . , 2011; Yu et al . , 2009 ) , whereas the gradient of Drosophila Decapentaplegic ( Dpp ) is established not only by diffusion , but also via transcytosis ( Dierick and Bejsovec , 1998; Kruse et al . , 2004 ) and cytonemes ( Hsiung et al . , 2005; Ramirez-Weber and Kornberg , 1999; Roy et al . , 2011 ) .", "Nodal proteins , which belong to the TGF-β family of signaling proteins , play critical roles in vertebrate development ( Arnold and Robertson , 2009; Wakefield and Hill , 2013 ) .", "They serve as mesendoderm inducers in vertebrates , and are involved in many aspects of embryonic axis formation during development ( Kumari et al . , 2013; Sampath and Robertson , 2016 ) .", "Nodal proteins are translated as precursors and function as dimers ( Massagué , 1990 ) .", "The Nodal precursors are cleaved by extracellular convertases , and convertase processing was found to be essential for Nodal activation in zebrafish and mouse embryonic tissues ( Beck et al . , 2002; Le Good et al . , 2005 ) .", "A recent report found that FurinA convertase activity regulates long range signaling by the zebrafish left-right patterning Nodal , Southpaw ( Spaw ) , but not other Nodal factors ( Tessadori et al . , 2015 ) .", "Upon activation , Nodal proteins form complexes with type II and type I Activin receptors ( Acvr1b; Acvr2a/b ) , which are serine/threonine kinases ( Reissmann et al . , 2001; Yan et al . , 2002; Yeo and Whitman , 2001 ) and activate the Nodal pathway ( Jia et al . , 2008; Kumar , 2000; Massagué et al . , 2005; Whitman , 1998 ) .", "Nodal target genes include nodal itself and lefty , which encodes a feedback inhibitor of Nodal signaling ( Branford and Yost , 2002; Meno et al . , 1999 ) .", "In zebrafish , of the three nodal homologs , cyclops ( cyc ) , squint ( sqt ) and southpaw ( spaw ) , sqt and cyc are expressed in an overlapping pattern in the gastrula margin where presumptive mesoderm and endoderm cells are located ( Erter et al . , 1998; Feldman et al . , 1998; Gritsman et al . , 2000; Long et al . , 2003; Rebagliati et al . , 1998a; 1998b; Sampath et al . , 1998; van Boxtel et al . , 2015 ) .", "However , Sqt and Cyc elicit differential responses in target cells: Sqt acts at long-range whereas Cyc only affects cells immediately adjacent to the source of the signal ( Chen and Schier , 2001; Jing et al . , 2006; Müller et al . , 2012; Tian et al . , 2008 ) .", "So far , there is no evidence for a requirement for transcytosis and cytonemes in distributing the Nodal factors and the Nodal morphogen gradient has been proposed to be established by simple diffusion ( Williams et al . , 2004 ) .", "The diffusion coefficient of a molecule is a measure of its ability to move freely across a defined region .", "The free diffusion coefficient of the zebrafish Nodals has been suggested to be faster than their effective diffusion coefficient ( Müller et al . , 2012; 2013 ) , resulting in fast diffusion over short distances but slow diffusion over longer distances presumably by morphogen trapping at high affinity binding sites .", "These observations led to the hypothesis that Nodal diffusion is hindered either by cell surface interactions or by molecules in the extracellular matrix ( Müller et al . , 2013 ) .", "How Nodal diffusion is hindered , and to what extent it shapes the Nodal gradient is unclear .", "In contrast to the differential diffusion model , a recent study suggested that a temporal signal activation window created by microRNA-430 ( miRNA-430 ) delays translation of the Nodal antagonist Lefty to determine the dimensions of Nodal signaling in the gastrula ( van Boxtel et al . , 2015 ) .", "Repression by miRNA-430 likely plays a key role in regulation of Nodal signaling .", "However , miRNA-430 is not exclusive to lefty1 but also targets nodal/sqt ( Choi et al . , 2007 ) .", "Moreover , reporter protein expression and ribosome-profiling data from zebrafish embryos indicate that Nodal/Sqt and Lefty1 are translated in a similar temporal window in the early gastrula ( Choi et al . , 2007; Bazzini et al . , 2012; Chew et al . , 2013 ) .", "As such , it is unclear how the proposed temporal activation window might be converted into a spatial Nodal gradient .", "Some studies have suggested that in addition to diffusion , the gradient of a morphogen is related to the rate of ligand clearance or stability ( Callejo et al . , 2006; Chamberlain et al . , 2008; Gregor et al . , 2007; Kicheva et al . , 2007; Wartlick et al . , 2009 ) , and a role for stability and clearance of Nodals in vivo has been proposed ( Jing et al . , 2006; Le Good et al . , 2005; Tian and Meng , 2006 ) .", "Previously , we reported an atypical lysosome-targeting region located in the pro-domain of Cyc , which targets this Nodal protein for destruction , and regulates target gene induction ( Tian et al . , 2008 ) .", "How the lysosome-targeting region regulates Nodal clearance and how it influences the Nodal morphogen gradient was not known .", "In this study , we have examined the diffusion coefficient of Nodals in live zebrafish embryos by fluorescence correlation spectroscopy ( FCS ) .", "FCS is a widely used single molecule sensitive technique that can quantitatively measure diffusion and concentrations in vivo by determining how fast particles diffuse through a fixed observation volume ( Shi et al . , 2009c; Yu et al . , 2009 ) .", "We estimated the affinity of Nodals to the type II receptor Acvr2b on the cell surface and to Lefty inhibitors in the extracellular space by single wavelength fluorescence cross-correlation spectroscopy ( SW-FCCS ) .", "SW-FCCS uses a single laser to excite proteins labeled with spectrally different fluorophores within the observation volume ( e . g . , the confocal volume ) ( Shi et al . , 2009a ) .", "By analyzing the correlated movement of various labeled proteins ( ligands/receptor/inhibitor ) , we determined the fraction of the proteins that were free or bound , and calculated the dissociation constants in live zebrafish embryos .", "We also investigated the contribution of ligand stability in forming the Nodal gradient .", "By analyzing diffusion and binding in vivo and from computational simulations , we show that diffusivity alone is insufficient to generate the Nodal morphogen gradient .", "Our findings show that in order to generate and maintain a robust Nodal morphogen gradient , ligand clearance by degradation is balanced against the binding and release of Nodal ligands with the receptor and inhibitors ." ], [ "To visualize Nodal ligands in vivo , we fused the enhanced green fluorescent protein ( EGFP ) with Sqt , Cyc , SqtCyc2 and CycΔ2 .", "The CycΔ2mutant , which lacks a lysosomal targeting region in the Cyc pro-domain , shows significantly increased stability and signaling range over wild type Cyc protein ( Tian et al . , 2008 and Figure 1 ) .", "SqtCyc2 chimeric protein harbors the atypical lysosome-targeting region from Cyc , and shows reduced stability and signaling range in comparison to Sqt ( Tian et al . , 2008 ) .", "We tested the activity of the fusion proteins by comparing nodal target gene induction by the various fusion proteins to that of their untagged counterparts , and found similar activity ( Figure 1A–D and Figure 1—figure supplement 1 ) . 10 . 7554/eLife . 13879 . 003Figure 1 . Activity range and diffusion of Nodal-GFP fusion proteins .", "( A ) Constructs used for profiling fluorescent Nodal fusion proteins in embryos .", "S , signal peptide; Pro , pro-domain; Mat , mature-domain; sec-EGFP , secreted EGFP .", "Red arrow indicates convertase cleavage sites .", "( B ) Injection procedure .", "( C ) Confocal image of an injected embryo at 30% epiboly stage .", "White crosses mark the extracellular spots where the FCS measurements were taken .", "( D ) Representative images of RNA in situ hybridization showing the activity range of Sqt , Cyc and mutant Nodals .", "Source cells are marked in brown and blue staining indicates expression of the Nodal target ntl .", "Scale bars , 50 μm .", "( E ) Representative auto-correlation functions ( dots ) and fittings ( line ) of Sqt-EGFP ( green ) and sec-EGFP ( black ) .", "( F ) Table showing diffusion coefficients of the Nodal and Lefty fusion proteins as measured by FCS . DOI: http://dx . doi . org/10 . 7554/eLife . 13879 . 00310 . 7554/eLife . 13879 . 004Figure 1—source data 1 . Individual FCS measurements and diffusion coefficient values for EGFP-tagged Nodals and Leftys compared to control secreted EGFP . DOI: http://dx . doi . org/10 . 7554/eLife . 13879 . 00410 . 7554/eLife . 13879 . 005Figure 1—figure supplement 1 . ( Related to Main Figure 1 ) .", "Tagged Nodals show similar activity compared to their untagged counterparts .", "( A ) Induction of ntl in embryos overexpressing Nodal or Nodal fusions .", "Five picogram aliquots of RNA was injected into one-cell stage wild-type embryos and ntl transcript expression was examined at 50% epiboly .", "Animal pole views of embryos showing endogenous ntl expression ( I ) , and mild ( II ) or massive ( III and IV ) expansion of the ntl expression domain .", "Embryos were assessed and counted accordingly .", "Percentages for each class are shown in the histogram .", "( C ) Induction of gsc in embryos overexpressing Nodal or Nodal fusions .", "Animal pole views of embryos showing endogenous gsc expression ( I ) , mild expansion ( II ) or massive expansion ( III and IV ) of gsc expression domains .", "( C ) Five picogram aliquots of RNA encoding Nodal or Nodal fusions were injected into one-cell at the 128-cell stage with a lineage tracer ( Biotin-Dextran , brown color staining ) .", "Range of signaling was examined by detecting ntl transcription ( blue/purple color staining ) .", "Scale bars represent 100 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 13879 . 005 The diffusivity of extracellular signaling molecules can determine their distribution and activity range .", "To examine the diffusivity of the Nodal ligands , we determined the diffusion coefficients of Sqt- , Cyc- , Sqt Cyc2- and CycΔ2-EGFP fusion proteins in vivo using FCS .", "EGFP-tagged Nodal fusion proteins were expressed from a localized source and FCS measurements were acquired in the extracellular space at various distances from the source cells ( Figure 1 and Figure 1—source data 1 ) .", "All the Nodal-GFP fusions , including Sqt , Cyc , SqtCyc2 and CycΔ2 , as well as Lefty1 and Lefty2 fusions show very similar diffusion coefficients ( Figure 1F ) .", "These results suggest that the free diffusivity alone is unlikely to differentiate the range and activity of the Nodal proteins .", "Cell surface receptors of extracellular signaling molecules can bind to the diffusible ligands , and thereby reduce their distribution range .", "In this scenario , the mobility of ligands that bind with higher affinity to the receptors will be more effectively retarded .", "To quantitate the binding affinity of Nodal ligands to the receptors , we determined the apparent dissociation constant ( Kd ) of Sqt and Cyc in vivo ( Foo et al . , 2012b ) with the predominant Nodal receptor Acvr2b , using FCCS .", "To uncouple binding from signaling events within the cytoplasm , we fused the extracellular and trans-membrane domains of Acvr2b lacking the intracellular kinase domain , to a red fluorescent protein , mCherry ( Figure 2A–C ) .", "Sqt-EGFP , Cyc-EGFP or control secreted eGFP ( sec-EGFP ) fusion proteins were expressed from a localized source in embryos that uniformly expressed Acvr2b-mCherry , and correlation curves were obtained to infer the Kd .", "Surprisingly , the Kd of Sqt-Acvr2b is 65 ± 7 nM and the Kd of Cyc-Acvr2b is 124 ± 12 nM ( Figure 2D–I ) .", "This result suggests Sqt binds with Acvr2b with an approximately twofold higher affinity compared to Cyc . 10 . 7554/eLife . 13879 . 006Figure 2 . Sqt has higher affinity to the Acvr2b receptors than Cyc .", "( A ) Sqt-/Cyc-/sec-EGFP and Acvr2b-mCherry constructs .", "S , signal peptide; Pro , pro-domain; Mat , mature-domain; ECM , extracellular and transmembrane domain .", "Red arrows indicate convertase cleavage sites .", "Sqt signal peptides and pro-domain were used in sec-EGFP constructs .", "( B ) Injection procedure .", "( C ) Representative image of an injected embryo at 30% epiboly stage showing the expression patterns of the fusion proteins .", "Scale bar represents 50 μm .", "( D , E , F )", "Representative auto-correlation ( ACF ) and cross-correlation functions ( CCF ) and fits .", "( G , H , I )", "Individual Ln ( Kd ) frequency histogram and Gaussian fitting ( red curve ) .", "Inset , concentration plot and linear regression ( red line ) .", "X axis , concentration of bound protein ( Cgr ( x10-9 M ) ) ; Y axis , products of concentrations of free proteins ( Cg x Cr ( x10-16 M ) ) .", "n = number of data points ( number of embryos ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 13879 . 006 The Nodal antagonist Lefty prevents Nodal proteins from binding to their receptors and has the potential to influence the distribution of Nodal ligands .", "To test if binding to the inhibitor affects Nodal ligand distribution , we determined the affinity of Sqt and Cyc to Lefty2 in vivo by co-expressing Lefty-mCherry with Sqt-EGFP or Cyc-EGFP from a localized source and measuring the Kdin the extracellular space of embryonic blastula cells at various distances from the source ( Figure 3A–C ) .", "The Kd of Sqt-Lefty2 is 29 ± 1 . 2 nM and Cyc-Lefty2 Kd is 50 ± 3 nM ( Figure 3D–I ) , indicating an approximately twofold higher affinity of Sqt-Lefty2 binding in comparison to Cyc-Lefty2 binding .", "The differential affinity of the Nodals for Lefty could fine-tune their activity range by removing freely diffusing Nodals from the signaling pool . 10 . 7554/eLife . 13879 . 007Figure 3 . FCCS measurements reveal that Lefty has higher affinity to Sqt compared to Cyc .", "( A ) Constructs used for injection .", "S , signal peptide; Pro , pro-domain; Mat , mature-domain .", "Red arrows indicate the convertase cleavage sites .", "( B ) Injection procedure .", "( C ) Confocal image of an injected embryo at 30% epiboly showing the expression patterns of the fusion proteins .", "Scale bar represents 50 μm .", "( D , E , F )", "Representative auto- and cross-correlation functions ( ACF; CCF ) and fittings .", "( G , H , I )", "Individual Ln ( Kd ) frequency histogram and Gaussian fits ( red curve ) .", "Inset , concentration plot and linear regression ( red line ) .", "X axis , concentration of bound protein ( Cgr ( x10-9 M ) ) ; Y axis , products of concentrations of free proteins ( Cg x Cr ( x10-17 M ) ) .", "n = number of data points ( i . e . , number of embryos ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 13879 . 007 To visualize Nodal gradients in zebrafish embryos , we expressed Sqt , Cyc , SqtCyc2 and CycΔ2-EGFP fusion proteins from a localized source ( Figure 4A–D ) .", "Consistent with findings by Müller et al . , Sqt-EGFP was found to reach the edges of the blastula with no more than 50–60% loss in intensity , whereas the intensity of Cyc-EGFP fusion protein falls steeply from the source ( Figure 4B and Figure 4—source data 1 ) .", "Interestingly , the gradient of the deletion mutant , CycΔ2-EGFP , which has a longer signaling range than Cyc-EGFP , was significantly shallower than that of Cyc , and the gradient of the SqtCyc2-EGFP chimera ( which has reduced signaling range compared to Sqt ) was steeper than that of Sqt-EGFP ( Figure 4B and Figure 4—source data 1 ) . 10 . 7554/eLife . 13879 . 008Figure 4 . The distribution of Nodal proteins correlates with clearance .", "( A ) Upper , representative image and region of interest ( red rectangle ) for measuring distribution; lower , inset showing magnified region of interest .", "( B ) Normalized distribution profiles and fitting .", "Error bars indicate standard error of mean ( s . e . m ) .", "( C ) Representative western blots of Nodal proteins harvested from HEK293T cell culture medium at different time points after removal of the source .", "The Nodal proteins were immuno-precipitated with anti-FLAG antibody and detected by western blot with the same antibody .", "Schematics on the left show the position of the FLAG epitope tags in each construct .", "( D ) The profile of Nodal protein levels over time after source removal .", "The data points were fitted with an exponential decay model .", "Error bars indicate s . e . m . DOI: http://dx . doi . org/10 . 7554/eLife . 13879 . 00810 . 7554/eLife . 13879 . 009Figure 4—source data 1 . Gradient data for tagged wild type and mutant Nodals . DOI: http://dx . doi . org/10 . 7554/eLife . 13879 . 00910 . 7554/eLife . 13879 . 010Figure 4—figure supplement 1 . ( Related to Main Figure 4 ) .", "Representative immunoblots showing protein degradation over time .", "Left , representative immunoblots .", "Equal amounts of sec-GFP-3xFLAG supernatant was added in all of the samples as input controls .", "Right , quantitation and fitting of protein expression levels .", "Each experiment was repeated three times , and the band intensity of the Nodal or Lefty proteins was normalized to GFP ( input control ) and to time 0 hr .", "The plots were then fitted with exponential decay ( equation shown on the upper right corner ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 13879 . 010 To determine the relative stability of Sqt , Cyc , CycΔ2 and SqtCyc2 , we expressed FLAG-tagged versions of the proteins in HEK293T cells and examined the amount of secreted protein in the supernatant after various periods ( Figure 4C , D and S2 ) .", "By fitting the normalized band intensity acquired from immune-blots with an exponential decay model , we inferred the degradation rates ( 0 . 166 × 10−4 /s for Cyc-FLAG , 0 . 093 × 10−4 /s for CycΔ2-FLAG 0 . 090 × 10−4 /s for SqtCyc2-FLAG and 0 . 003 × 10−4 /s for Sqt-FLAG ) .", "The decay rate of these proteins shows a trend consistent with their gradient profile and their signaling range ( Figure 4A–D , Figure 4—source data 1 , Figure 1 and S2 ) .", "These results indicate a strong correlation between Nodal ligand stability and the gradient .", "To test the validity of our measurements , we performed simulations to model the Nodal gradient .", "Modeling of the Nodal morphogen gradient requires a range of different parameters , of which some have been measured in vivo and are available , and we have in this study determined binding affinities , and inferred concentrations ( Table 1 ) .", "We found the dissociation constants , KD , of Sqt and Cyc to their major cell surface receptor Acvr2 , to be ~60 and 120 nM , respectively .", "In addition , we determined the diffusion coefficients of the Nodals to be ~60 μm2/s .", "From the amplitude of our FCS measurements , we estimated the concentration of the Nodal factors to be on the order of 102 nM .", "We determined that the degradation rate for Cyc is higher than that for Sqt , confirming previous work ( Tian et al . , 2008; Jing et al . , 2006 ) .", "Nonetheless , because our degradation rate values were estimated from cell culture , in our simulations we use the values of 0 . 0001/s and 0 . 0005/s for Sqt and Cyc , respectively , documented in or estimated from previous reports ( Jing et al . , 2006; Müller et al . , 2012 ) .", "In the simulations we produced particles with 0 . 07/s to 0 . 7/s in the simulation volume ( corresponding to a production rate of 0 . 3–3 pM/s ) to obtain sufficient number of particles for statistical analysis .", "The number of particles at equilibrium is given by the ratio of production over degradation rate , which was 700–7000 for a degradation rate of 0 . 0001/s and 140–1400 for 0 . 0005/s .", "Importantly , the production rate itself does not change the gradient shape and only the gradient amplitude is altered .", "Therefore , the gradient shape is determined by the degradation rate and diffusion . 10 . 7554/eLife . 13879 . 011Table 1 . Simulation parametersDOI: http://dx . doi . org/10 . 7554/eLife . 13879 . 011ParameterSqtCycReferenceKd60 nM120 nMThis workDegradation rate0 . 0001/s0 . 0005/sEstimated from Jing et al . , 2006; Müller et al . , 2012Ligand concentration~100 nM~100 nMEstimated from this workReceptor concentration40 μM40 μMEstimated from this workDfree [μm2/s]6060This workDtortuosity [μm2/s]3030This work and Müller et al . , 2013Deff [μm2/s]0 . 0450 . 09This work First , we determined how the fact that particles have to transverse longer paths around obstacles ( e . g . , cells ) during diffusion , renders diffusion apparently slower , and influences the effective diffusion coefficient ( Figure 5A , B and Videos 1 , 2 ) .", "This is referred to as tortuosity by Müller et al . ( 2013 ) and reduces diffusion maximally by a factor 2 ( Müller et al . , 2013 ) .", "In agreement with this , we determined that for cells with 10 μm diameter and cell membrane-to cell membrane distance of 2 μm , we obtain a reduction of diffusion by a factor 1 . 84 ( Figure 5D ) . 10 . 7554/eLife . 13879 . 012Figure 5 . Simulations of morphogen diffusion .", "( A ) Free diffusion with a diffusion coefficient D = 60 μm2/s .", "( Dashed circles indicate positions of cells in later simulations but not taken account of in this case ) .", "( B ) Diffusion in the presence of cells .", "( C ) Diffusion in the presence of cells and binding with an average number of free particles of 0 . 003 , i . e . 99 . 7% of all particles are bound on average .", "Simulations were done in a 3D space as described in the text and the diffusion coefficient was D = 60 μm2/s .", "( D ) Comparison of the spread of the particles as a function of the distance from the source ( the left border in panels A–C ) .", "The concentration curves were fit with a bell curve that describes the diffusion of particles from the source .", "For free diffusion ( A ) we recover a diffusion coefficient of D = 63 . 4 μm2/s close to the input value , and in the presence of cells ( B ) this reduces to an effective diffusion coefficient of Deff = 33 . 8 μm2/s , demonstrating the effect of tortuosity .", "( E ) Simulations of diffusion in the presence of cells , and with different amounts of binding .", "The simulated diffusion coefficient was D = 60 μm2/s .", "The concentration curves were fit with Equation 8 .", "The recovered effective diffusion coefficients for a fraction of free particles of 0 . 1 , 0 . 003 , and 0 . 0015 were Deff = 2 . 99 μm2/s , Deff = 0 . 09 μm2/s , and Deff = 0 . 042 μm2/s , respectively , demonstrating the effect of binding on the effective diffusion coefficient .", "( F ) Gradient formation using the effective diffusion coefficients determined from graph E and degradation rates of 0 . 0001/s and 0 . 0005/s , respectively .", "The blue curve represents Cyc , the red curve Sqt .", "Although Sqt has higher binding affinity and consequently a lower free mobile fraction , its lower degradation rate ensures that Sqt has a less steep gradient .", "The data was fit with an exponential function yielding gradients of 19 μm for Cyc and and 30 μm for Sqt , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 13879 . 01210 . 7554/eLife . 13879 . 013Video 1 . Simulation of particles moving and freely diffusing from the source . Dashed circles indicate positions of cells in later simulations ( included here for illustration only , but have no influence on the simulation ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 13879 . 01310 . 7554/eLife . 13879 . 014Video 2 . Particle movement is hindered by cells ( black circles ) around which they move . DOI: http://dx . doi . org/10 . 7554/eLife . 13879 . 014 Secondly , we determined how binding affects the effective diffusion coefficient ( Figure 5C , E and Video 3 ) .", "For quantitative analysis , we simulated particles whose diffusion coefficient was recued from 60 to 30 μm2/s due to tortuosity and assumed that a fraction of the particles is bound to binding sites that are homogeneously distributed .", "The effective diffusion coefficient is reduced more for higher affinities , i . e . when more particles are bound on average .", "For instance , when 90 or 99% of particles are bound , morphogen diffusion is reduced by a factor 10 or 100 , respectively .", "The actual amount of bound ligand depends on the total concentration of ligand ( Lt ) , receptor ( Rt ) and the KD: ( 1 ) fbound=Kd+Lt+Rt2Lt− ( Kd+Lt+Rt ) 24Lt2−RtLt Thirdly , for the simulations we assume that Sqt and Cyc share the receptors , and a ligand concentration of 100 nM is used for both Nodal ligands .", "The receptor concentration is on the order of 10 μM or more ( see next paragraph ) , and is much higher than that of the ligands .", "As such , the exact ligand amount does not change the outcome significantly for ligand concentration changes within a factor of ~5 .", "The differences in the gradient length are therefore , a result of diffusion , differential binding of Sqt and Cyc , as well as different degradation rates . 10 . 7554/eLife . 13879 . 015Video 3 . Particle movement is hindered further by binding . DOI: http://dx . doi . org/10 . 7554/eLife . 13879 . 015 Next , we determined the effective diffusion coefficient that results in a Sqt gradient length consistent with our measured values of about 30 μm .", "The gradient length is described by a model previously used for Fgf8 diffusion in zebrafish embryos ( Müller et al . , 2013; Yu et al . , 2009 ) : ( 2 ) λ=D/kR/Kd+1 where λ is the gradient length , D is the free diffusion coefficient , k is the clearance rate which is assumed constant , R is the concentration of the receptor , and Kd is the equilibrium dissociation constant , respectively ( Table 1 ) .", "Using a gradient length of about 30 μm and the other values as given in Table 1 , we estimate the effective diffusion coefficient to be on the order of 0 . 045 μm2/s .", "With a Kd of 60 nM for Sqt and an Lt of 100 nM , this requires a bound fraction fbound of 99 . 85% and a receptor concentration of ~40 μM .", "The estimation of the receptor number of 40 μM is based on the value of Deff required to establish the gradient of appropriate dimensions for Sqt .", "At this time it is not clear whether this concentration comprises only membrane receptors or whether additional binding sites ( e . g . , in interstitial spaces between cells ) contribute to it as shown for Fgf8 ( Yu et al . , 2009 ) .", "Any corrections in binding affinities for the receptor or different affinities for additional binding sites would alter the required concentration .", "Importantly , this number is dependent on the clearance/degradation rates that we have based upon previous reports , and which could change with more precise in vivo measurements .", "Based on the above , we assume that 40 μM is the upper limit for the receptor concentration .", "At this receptor concentration , Cyc , with a Kd of 120 nM will have a bound fraction of 99 . 7% and an effective diffusion coefficient of 0 . 09 μm2/s .", "Finally , we used these effective diffusion coefficients and the degradation rates of 0 . 0001/s and 0 . 0005/s for Sqt and Cyc , respectively , to simulate gradient formation ( see simulation parameters in Table 1 ) .", "Using these parameters , Sqt produced a gradient of ~30 μm length and Cyc gradient was ~19 μm ( Figure 5F ) .", "These values are consistent with actual in vivo measurements ( Figure 4B and Figure 4—source data 1 ) , and support our hypothesis ." ], [ "In our study , we found that the diffusion coefficients of free Nodal and Lefty proteins measured by FCS are very similar ( ~60 µm2/s ) , consistent with their similar apparent molecular weight and with previous reports ( Müller et al . , 2013 ) .", "However , their mobility is too great to account for the sharp gradients observed in developing embryos .", "The diffusion coefficient values determined by FCS are ~3 to 85 times higher than the effective diffusion coefficients reported using FRAP ( 18 . 9 ± 3 . 0 µm2/s for Lefty2 , 3 . 2 ± 0 . 5 µm2/s for Sqt and 0 . 7 ± 0 . 2 µm2/s for Cyc ) ( Müller et al . , 2013 ) .", "FCS and FRAP produce different readouts because they measure diffusion in different contexts , time windows and scales .", "FCS determines diffusion within a small volume ( <1 µm3 ) and on a short timescale ( <1 s ) , whereas FRAP measures net diffusion over a large area ( >1 , 000 µm3 ) over a longer time period ( >>10 s ) .", "FCS has been very useful to determine local diffusion , to infer the concentration of molecules within a defined confocal volume , and to determine the affinity of molecular interactions within the defined confocal volume .", "FRAP has been very useful for examining large-scale movement of molecules in tissues .", "The diffusion coefficients determined by the two techniques are known to vary dramatically .", "For example , in Drosophila imaginal discs , the measured diffusion coefficient of Dpp-GFP is 10 ± 1 µm2/s from FCS measurements ( Zhou et al . , 2012 ) , and 0 . 1 ± 0 . 05 µm2/s by FRAP ( Kicheva et al . , 2007 ) .", "This difference is thought to arise from other factors ( e . g . , by degradation of the molecules during diffusion ) .", "Taken together , these findings strongly suggest there must be other molecules and mechanisms in the embryo that refine and shape the Nodal morphogen gradient .", "The diffusional movement of morphogens can also be altered by transient binding to other molecules such as receptors or to components of the extracellular matrix ( Baeg et al . , 2004; Belenkaya et al . , 2004; Han et al . , 2004; Lander et al . , 2002; Wang et al . , 2008; Yu et al . , 2009 ) , so that one possible mechanism to shape the gradient is transient binding of Nodal proteins to immobilized diffusion regulators , as found for the fibroblast growth factor Fgf8 ( Yu et al . , 2009 ) .", "To explain the long range distribution of Sqt compared to Cyc , it was also proposed that Sqt might have a lower binding affinity to its receptors , ( Müller et al . , 2012 ) .", "However , we find that Sqt in fact binds in vivo to Acvr2b with a higher affinity than the short-range Nodal , Cyc .", "Sqt also binds to Lefty with higher affinity , raising the possibility that Lefty-binding might alter Sqt activity .", "Another potential mechanism for gradient formation is rapid clearance of molecules during diffusion , as observed for Dpp in Drosophila ( Kicheva et al . , 2007 ) .", "Our measurements and calculations strongly support this mechanism .", "Previous studies have suggested that the Nodal gradient might be influenced by its stability: Le Good et al . showed that increasing the stability of mouse Nodal protein increases its range of activity ( Le Good et al . , 2005 ) ; Tian et al . found a lysozyme targeting signal in Cyc that accelerates its degradation and reduces the signaling activity of chimeric SqtCyc2 protein ( Tian et al . , 2008 ) ; Jing et al . determined the half-life of Sqt and Cyc to be ~8 hr ( ~480 min ) and ~2 hr ( ~120 min ) , respectively , which somewhat correlates with the difference in target induction by the two proteins ( Jing et al . , 2006 ) .", "However , the difference in clearance rates of Sqt , Cyc , Lefty1 and Lefty2 determined by photo-conversion assays is not pronounced enough to explain their very different decay lengths ( Müller et al . , 2012 ) .", "Interestingly , Müller et al . found that their fluorescent Cyc fusion protein was expressed at very low levels in the extracellular matrix , but exhibited an unusual punctate distribution close to the plasma membrane and in the cytosol , whereas their Sqt fusion showed a strong , uniform and mainly intracellular distribution .", "The punctate distribution of Cyc suggests that Cyc might undergo a much faster and/or sustained endocytosis process compared to Sqt .", "This supports our finding that cells selectively destroy Nodal ligands by recognizing the lysosome-targeting signal , since the ligands have to be internalized .", "Simulations of the Nodal gradient show that Sqt generates a gradient of 30 μm and Cyc 19 . 1 μm , consistent with our measurements of 29 . 5 ± 5 μm for Sqt and 19 . 7 ± 2 μm for Cyc ( from Figure 4B ) , as well as the estimated signaling range of these proteins ( Chen and Schier 2001; Tian et al . , 2008 ) .", "In the simulations , 80% and 95% of steady state levels for Cyc is achieved at 0 . 7 and 1 . 25 hr , respectively , which is consistent with the timing of mesoderm induction in the gastrula .", "By the same predictions , Sqt reaches the 80% and 95% levels at ~4 hr and 7 hr , respectively , which is longer than expected .", "However , these simulations have not taken into consideration cell divisions or binding to other factors that could influence the gradient .", "Despite some differences in absolute values , the overall agreement between our experimental results , theory , and simulation supports our conclusion that the Nodal gradient is dependent upon diffusion , binding , and degradation of the morphogen .", "An important point to note from Equation ( 2 ) is that the clearance rate k and the receptor concentration R are inversely related to each other .", "Thus , a higher clearance rate would predict a lower receptor concentration and vice versa .", "It will be interesting to determine these values with higher accuracy in live zebrafish embryos .", "In addition , some aspects of the system have not been taken into account in our simulations .", "In particular , we found that Lefty binds the Nodals with high affinity .", "This may not influence gradient formation as the Sqt/Lefty and Cyc/Lefty complexes likely diffuse very similarly to Sqt and Cyc given that their size difference is within a factor of two .", "However , Lefty will influence Sqt and Cyc signaling when in complex , even if this is not directly evident in the gradient of fluorescent molecules .", "We also have not considered how the Nodal co-receptor ( Yan et al . , 1999 ) influences gradient formation .", "It will be interesting to determine how Oep/Cripto co-receptors and Lefty shape the active signaling gradient .", "The extracellular matrix ( ECM ) has been shown to play a key role in regulating diffusion of FGFs , presumably via interactions with heparan sulphate proteoglycans ( HSPGs ) ( Makarenkova et al . , 2009; Yu et al . , 2009 ) .", "It is not known if the ECM or HSPGs play a role in modulating the Nodal morphogen gradient although sulfated proteoglycans have been proposed to provide directional cues for left-asymmetric Nodal in Xenopus ( Marjoram and Wright . , 2011 ) .", "In conclusion , we find that in addition to hindered diffusion via binding to the receptors and inhibitors , the differential stability of Nodal ligands play key roles in shaping the Nodal gradient and activity range .", "Our experimental findings together with theoretical and computational simulations show that diffusion , extracellular interactions i . e . , Nodal-receptor binding , Nodal-Lefty inhibitor binding , and selective ligand destruction collectively shape and refine the Nodal morphogen gradient ." ], [ "All the constructs were PCR amplified and cloned into pCS2+ vector with Kozak sequence gccacc immediate 5’ of the start codon .", "For Cyc and CycΔ2 fusions , EGFP or 3xFLAG ( DYKDHDGDYKDHD-IDYKDDDDK ) tag was inserted 4 amino acids after the cleavage site ( RRGRR ) .", "For Sqt and SqtCyc2 fusions , EGFP or 3xFLAG tag was inserted 1 amino acid after the cleavage site ( RRHRR ) .", "For Lefty1 and Lefty2 fusions , EGFP or 3xFLAG tag was fused to the C-terminus of the protein as previously described ( Müller et al . , 2012 ) .", "For Acvr2b fusion , mCherry was fused to the C-terminus of Acvr2b ( 1–188 aa ) .", "For generating the sec-EGFP construct , the EGFP tag was fused to the C-terminus of 4 amino acids after the cleavage site ( RRGRR ) of Sqt .", "For sec-EGFP-3xFLAG construct , 3xFLAG tag was fused to the C-terminus of Sec-EGFP ( Yu et al . , 2009 ) .", "Wild-type ( AB ) fish were maintained at 28 . 5°C and embryos were obtained from natural matings according to standard procedures and in accordance with institutional animal care regulations .", "The plasmids were linearized with NotI restriction endonuclease ( NEB ) and transcribed using the mMessage mMachine SP6 Kit ( Ambion ) to produce capped RNA .", "Synthetic RNA was purified with P-30 Bio-Spin columns ( Bio-Rad , Hercules , CA ) followed by phenol/chloroform extraction and ethanol precipitation , and RNA concentration was quantified by Nanodrop ( Thermo Fisher Scientific , Waltham , MA ) and estimation of agarose gel electrophoresis bands .", "To test overall inductivity of the various Nodal fusions , 5 ng of the RNA was mixed with 0 . 25% phenol red ( Sigma , Aldrich , St Louis , MO ) and injected into the yolk of 1-cell stage AB wild type embryos .", "To test the signaling range of the Nodal fusions , single cells of de-chorionated 128-cell stage embryos were injected with 2 . 5 pg RNA and 0 . 25% 10kDa biotin-Dextran ( Thermo Fisher Scientific ) .", "Un-injected embryos from the same batch were used as controls and as reference for staging the embryos .", "Embryos were fixed at the 50% epiboly stage in 4% paraformaldehyde ( PFA ) in PBS at 4°C for at least 24 hr .", "The embryos were subjected to in situ hybridization to detect gsc and ntl expression levels as previously described ( Le Good et al . , 2005; Müller et al . , 2012; Tian et al . , 2008 ) .", "To generate clones of cells expressing Nodal or Lefty , Sqt , Cyc , Lefty1 or Lefty2 -EGFP RNA ( 2 . 5 pg ) was injected into single cells of 32–128-cell stage embryos .", "To determine the dissociation constant of Nodal and Acvr2b , 50 pg RNA encoding Acvr2b-mCherry was injected into 1-cell stage embryos prior to clone generation .", "The embryos were mounted on glass-bottom dishes ( World Precision Instruments ) in 0 . 75% low melting temperature agarose ( in 30% Danieau’s solution ) at the 30% epiboly stage for confocal imaging and FCS/FCCS measurements .", "HEK293T cells were transfected with plasmid DNA encoding 3xFLAG tagged proteins using FuGene HD ( Promega , Madison , WI ) transfection reagent .", "The medium with transfection reagent was removed and replaced with fresh Opti-MEM medium ( Life Technologies , Carlsbad , CA ) 24 hr after addition of transfection reagent .", "Cell culture supernatants were collected and flash frozen in liquid nitrogen 24 hr after the removal of the transfection reagent .", "Small aliquots of frozen supernatants were immunoprecipitated with anti-FLAG M2 antibody ( Sigma ) and protein G dynabeads ( Life Technologies ) , and eluted with 3xFLAG peptide ( Sigma ) .", "The samples were immunobloted with the same antibody and the signals were detected with a Syngene PXi gel imaging system .", "The band intensity was quantified using ImageJ .", "To determine the clearance rate of the proteins , the remaining supernatant was diluted to the same concentration as supernatants from non-transfected cells , mixed with sec-EGFP-3xFLAG supernatant for input control , added to dishes with non-transfected cells and collected at different time points .", "Proteinase inhibitor cocktail ( Roche , Switzerland ) was added immediately after the supernatants were collected and flash frozen in liquid nitrogen .", "The supernatants were enriched and detected as described above .", "The intensity of individual protein bands was normalized against EGFP to correct for differences in sample volume and immunoprecipitation , and normalized to time 0 for relative changes .", "A custom-built single wavelength fluorescence cross-correlation spectroscopy ( SW-FCCS ) system was used for the FCS and FCCS measurements as described ( Shi et al . , 2009a; 2009b ) .", "We obtained the correlation curve of the various fusion proteins by focusing the detection volume on the cell membrane at various distances from the source ( Figure 2A–C ) .", "The correlation curves were analyzed and fitted with a bimolecular binding model to calculate the Kd .", "We co-expressed Lefty-mCherry with Sqt-EGFP or Cyc-EGFP from a localized source and measured the Kd in the extracellular space of blastula cells at various distances from the source .", "The experimental raw auto-correlation data was fitted with defined correlation function models .", "In FCS , a one-component 3D diffusion model with triplet state was used for free diffusing molecules: ( 3 ) G3D , 1C , 1trip ( τ ) =1N[1+ ( Ftrip1−Ftrip ) e−τ/τtrip] ( 1+ττd ) −1[1+ ( ω0z0 ) 2ττd]−1/2+G∞ , where N is the number of particles in the confocal volume; Ftrip is the fraction of the particles that have entered the triplet state; τtrip is the triplet state relaxation time; τd is the average time required for one particle to diffuse through the confocal volume , ω0 and z0 are the radial and axial distances where the excitation intensity reaches 1/e2 of its value from the center of the confocal volume; and G∞ is the convergence value of the ACF for long times .", "In FCCS , a one-component 2D diffusion model and a two-component 3D model were used for the membrane anchored receptors and Nodal ligands , respectively: ( 4 ) G2D , 1C , 1trip ( τ ) =1N[1+ ( Ftrip1−Ftrip ) e−τ/τtrip] ( 1+ττd ) −1+G∞ ( 5 ) G3D , 2C , 1trip ( τ ) =1N[1+ ( Ftrip1−Ftrip ) e−τ/τtrip]{∑i⁡Fi ( 1+ττdi ) −1[1+ ( ω0z0 ) 2ττdi]−1/2}+G∞ where τdi and Fi are the diffusion time and the amplitude of the ith component .", "The cross-correlation data was fitted by a one-component 2D model: ( 6 ) G3D , 1C , 1trip ( τ ) =1N ( 1+ττd ) −1+G∞ Data was fit with the Levenberg-Marquardt algorithm using the described models in Igor Pro 6 . 0 ( WaveMetrics ) ( Wohland et al . , 2001 ) .", "The procedure of calibration and quantification of diffusion coefficient and dissociation constants were as previously described ( Foo et al . , 2012a; Shi et al . , 2009a ) .", "EGFP fusion proteins were excited with a 488 nm laser beam and the emitted fluorescence was collected through a 10X objective lens ( Olympus , UPLSAPO NA = 0 . 40 ) and a long-pass 505 emission filter with a 2 . 5X digital zoom .", "Images were acquired in planes ~15 μm below the enveloping layer of the embryos at 512 × 512 pixels with a corresponding size of 1 . 4 μm2/pixel .", "Acquired images were analyzed using the ImageJ package .", "A rectangular region of interest ( ROI ) with a fixed height of 50 . 4 μm ( 36 pixels ) adjacent to the source was drawn .", "The width of the ROI differed depending on the size of the embryo .", "Windows of 7 × 50 . 4 μm2 ( 5 × 36 pixels ) were binned and the average intensity of each binned window was calculated .", "Background auto-fluorescence was estimated from images of un-injected embryos and subtracted from all measurements .", "The data was normalized to the value closest to the source boundary , plotted on the intensity-distance coordinate with ImageJ .", "The data was pooled and fitted , or individual data sets were fitted and the gradient length was calculated as the mean of all fits .", "Both procedures yielded similar results .", "Fits were performed with an exponential decay: ( 7 ) C ( x ) =A∗exp[−xλ]+C where A is the amplitude of the gradient , λ is the gradient decay length and C is a possible offset .", "Simulations were performed with Mathematica 10 . 0 ( Wolfram , Champaign , IL ) .", "Initial simulations to determine effective diffusion coefficients in the presence of cells as obstacles ( tortuosity ) and morphogen binding were conducted in 3D .", "For this purpose , we simulated a 3D slab of 2 μm height ( z-axis ) , 44 μm width ( y-axis ) , and 86 . 7 μm length ( x-axis ) , for 5 s ( Figure 5A , B ) .", "We used a diffusion coefficient of D = 60 μm2/s , and created 1000 particles at the left border of the simulation volume .", "The particles were allowed to perform a random walk for 5 s with a time resolution of 5 ms per step .", "At the left and right borders ( along the x-axis ) , particles were reflected .", "At the other four borders we used periodic boundary conditions .", "Based upon actual measurements from early gastrula embryos , we assumed that the space is packed with cells of ~10 μm diameter , and an intercellular space ( cell membrane-to-cell membrane distance ) of ~2 μm .", "As the height of the simulation volume was only 2 μm , we used cylinders to represent the cells within this space .", "Under these circumstances , ligand diffusion was reduced by a factor of 1 . 84 .", "This value is consistent with the findings of Müller et al . who reported tortuosity to reduce diffusion maximally by a factor of 2 ( Müller et al . , 2013 ) .", "Therefore , for further modeling we assumed the effective diffusion coefficient of the Nodals to be ~30 μm2/s .", "In the case of binding we used the values in Table 1 and Equation 1 to determine the average number of free particles at each step .", "All concentration profiles were normalized and fitted by the following equation to determine the effective diffusion coefficient: ( 8 ) C ( x , t ) =exp[−x24Defft] Here C ( x , t ) is the concentration profile , x is the coordinate along which the particle diffusion is observed , t is the time at which the profile is measured ( i . e . , 5", "s ) , and Deff is the effective diffusion coefficient .", "Final simulations , including continuing particle production and degradation , used the values given in Table 1 and were run in 1D with an extent of 200 μm , assuming a reduced diffusion coefficient of 30 μm2/s due to tortuosity , and an average number of particles bound as determined by Equation 1 . .", "To ensure that the gradients reached equilibrium , the simulation time was 16 hr 40 min .", "The normalized concentration gradients C ( x ) were fitted with a simple exponential function to determine the gradient length λ .", "( 9 ) C ( x ) =exp[−xλ]" ] ]

[ "The correct distribution and activity of secreted signaling proteins called morphogens is required for many developmental processes .", "Nodal morphogens play critical roles in embryonic axis formation in many organisms .", "Models proposed to generate the Nodal gradient include diffusivity , ligand processing , and a temporal activation window .", "But how the Nodal morphogen gradient forms in vivo remains unclear .", "Here , we have measured in vivo for the first time , the binding affinity of Nodal ligands to their major cell surface receptor , Acvr2b , and to the Nodal inhibitor , Lefty , by fluorescence cross-correlation spectroscopy .", "We examined the diffusion coefficient of Nodal ligands and Lefty inhibitors in live zebrafish embryos by fluorescence correlation spectroscopy .", "We also investigated the contribution of ligand degradation to the Nodal gradient .", "We show that ligand clearance via degradation shapes the Nodal gradient and correlates with its signaling range .", "By computational simulations of gradient formation , we demonstrate that diffusivity , extra-cellular interactions , and selective ligand destruction collectively shape the Nodal morphogen gradient ." ]

[ "Animals develop from a single fertilized egg cell into multicellular organisms .", "This process requires chemical signals called “morphogens” that instruct the cells how to behave during development .", "The morphogens move across cells and tissues to form gradients of the signal .", "Cells then respond in different ways depending on how much of the signal they receive .", "This , in turn , depends on several factors: first , how quickly or slowly the signal moves; second , how well the morphogen binds to responding cells and other molecules in its path; and third , how much signal is lost or destroyed during the movement .", "Many researchers study morphogen gradients in the transparent zebrafish , since it grows quickly and it is easy to see developmental changes .", "However , until now it was not fully clear how the well-known morphogen called Nodal moves in live zebrafish as they develop .", "Wang , Wang et al . have now investigated how well Nodal signals bind to the surface of cells that receive the signal and to a molecule called “Lefty” , which is present in the same path and interferes with Nodal signals .", "Advanced techniques called fluorescence correlation and cross-correlation spectroscopy were used to measure Nodal signals at the level of single molecules in growing zebrafish .", "The experiments gave insights into how far Nodal signals move and remain active .", "The results showed that , in addition to Nodal diffusing and binding to receiving cells , one of the most important factors determining how far and quickly Nodal moves is its inactivation and destruction .", "Lastly , Wang , Wang et al . built computational models to test their observations from live zebrafish .", "The current work was based on forcing zebrafish to produce molecules including Nodal at locations within the fish that normally do not make them .", "Therefore future experiments will aim to examine these molecules and their interactions when they are produced at their normal locations in the animal over time ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "evolutionary biology", "cell biology" ]

[ [ "Clathrin-coated vesicles ( CCVs ) are key players in eukaryotic intracellular membrane traffic ( Brodsky , 2012 ) .", "Their characteristic lattice-like coat is self-assembled from cytoplasmic clathrin proteins , captures membrane-embedded protein cargo and deforms the membrane into a vesicle .", "This process enables CCVs to mediate protein transport to and from the plasma membrane and between organelles .", "The triskelion-shaped clathrin molecule is formed from three identical clathrin heavy chain ( CHC ) subunits .", "Humans have two genes ( CLTC and CLTCL1 ) that respectively encode CHC17 and CHC22 clathrins ( Wakeham et al . , 2005 ) .", "CHC17 clathrin , which has three bound clathrin light chain ( CLC ) subunits , is expressed uniformly in all tissues and forms CCVs that control receptor-mediated endocytosis , as well as lysosome biogenesis and maturation of regulated secretory granules .", "These pathways are conventionally associated with clathrin function and are mediated by clathrin in all eukaryotic cells ( Brodsky , 2012 ) .", "In humans CHC22 clathrin is most highly expressed in muscle and adipose tissue and forms separate CCVs that are not involved in endocytosis ( Dannhauser et al . , 2017 ) .", "In these tissues , CHC22 CCVs regulate targeting of the glucose transporter 4 ( GLUT4 ) to an intracellular compartment where it is sequestered until released to the cell surface in response to insulin ( Vassilopoulos et al . , 2009 ) .", "This insulin-responsive GLUT4 pathway is the dominant mechanism in humans for clearing blood glucose into muscle and fat tissues after a meal ( Shepherd and Kahn , 1999 ) .", "In addition to its distinct tissue expression pattern and biological function , CHC22 does not bind the CLC subunits that associate with CHC17 clathrin , even though the CHC protein sequences are 85% identical ( Dannhauser et al . , 2017; Liu et al . , 2001 ) .", "This remarkable biochemical and functional divergence evolved since the gene duplication event that gave rise to the two different clathrins during the emergence of chordates ( Wakeham et al . , 2005 ) .", "Notably , however , the CLTCL1 gene encoding CHC22 evolved into a pseudogene in the Mus genus , although mice maintain an insulin-responsive GLUT4 pathway for clearing blood glucose .", "This observation suggests that , despite the importance of the CLTCL1 gene product , backup pathways have evolved to compensate for loss of the CHC22 protein .", "To understand the evolution of the specialized function of CHC22 , and the potential selective processes involved , we here explore the phylogenetic history of the CLTCL1 gene in vertebrates and its population genetics in humans , non-human primates and bears .", "Ecological shifts create selective forces that filter variation in cellular genes .", "These include changes in nutritional conditions ( Babbitt et al . , 2011 ) , as well as encounters with pathogens ( Fumagalli et al . , 2011 ) ; both documented as selective forces that affect membrane traffic genes ( Elde and Malik , 2009; Liu et al . , 2014 ) .", "Recent studies of the evolution of genes involved in membrane traffic have focused on an overview of all eukaryotes with the goals of establishing the origins of membrane-traffic regulating proteins in the last common eukaryotic ancestor and defining the species distribution of various families of traffic-regulating proteins ( Rout and Field , 2017; Dacks and Robinson , 2017 ) .", "These studies have identified common features of proteins that regulate membrane traffic ( Rout and Field , 2017 ) and revealed that extensive gene duplication has allowed lineage-specific diversification of coat proteins and other membrane traffic regulators , such as the Rab GTPases ( Diekmann et al . , 2011; Guerrier et al . , 2017 ) .", "Our earlier study of available annotated genomes in 2005 suggested that the gene duplication giving rise to the two CHC-encoding genes occurred as a result of one of the whole genome duplications contributing to vertebrate evolution ( Wakeham et al . , 2005 ) .", "Here we focus on the more recent evolutionary history of these genes , as well as analyze the increased number of fully annotated vertebrate genomes .", "We establish that the loss of CLTCL1 in the Mus genus is not unique in vertebrates , identifying at least one additional independent gene loss event in the clade of Cetartiodactyla affecting pigs , cows , sheep , porpoise , and possibly additional related species .", "Nonetheless , there is strong evidence for CHC22 sequence conservation amongst those species that retain CLTCL1 ( Wakeham et al . , 2005 ) .", "This evolutionarily recent gene loss in some lineages and retention of the functional form in others suggested that CLTCL1 may still be under purifying selection , so we examined CLTCL1 variation between individuals within vertebrate populations .", "Comparing populations , we found CLTCL1 to be considerably more polymorphic than CLTC , which encodes the clathrin found in all eukaryotes , with evidence for strong ancient purifying selection for CHC17 clathrin function and relaxed purifying selection on CHC22 function .", "Additionally , we identified two common allotypes of human CHC22 , which have different functional properties .", "The derived allele arose in ancient humans and is more frequent in farming populations when compared to hunter-gatherers .", "We previously observed that CHC22 accumulates at sites of GLUT4 retention in the muscle of insulin-resistant patients with type two diabetes ( Vassilopoulos et al . , 2009 ) in addition to its active role in membrane traffic of GLUT4 .", "Thus , CHC22 variation has potential to differentially affect membrane traffic pathways involved in insulin resistance , as well as alter normal glucose metabolism within human and other vertebrate populations .", "The analyses reported here lead us to propose that variation in the CHC22 clathrin coat may be a response to changing nutritional pressures both between and within vertebrate species ." ], [ "Identification of CHC-encoding genes in 62 vertebrate and non-vertebrate species ( ( Figure 1—figure supplement 1 , Figure 1—figure supplement 2 ) indicates a dynamic history of gene duplications and losses ( Figure 1 ) .", "The CLTCL1 gene was detected only in jawed vertebrates ( bony vertebrates and cartilaginous fish ) , while the two jawless vertebrate genomes available – lamprey ( Petromyzon marinus ) and hagfish ( Eptatretus burgeri ) – have only one CHC-encoding gene .", "This distribution refines the timing of the CHC-encoding gene duplication to the period after the Agnatha split off the vertebrate lineage , estimated at 493 . 8MYA ( 95% HPD: 459 . 3 , 533 . 8 ) , and before the evolution of jawed vertebrates 450 . 8MYA ( 95% HPD: 432 . 1 , 468 . 1 ) ( Hedges et al . , 2015; dos Reis et al . , 2015 ) .", "Of the ten species of bony fish that split off the spotted gar ( Lepisosteus oculatus ) lineage ( Amores et al . , 2011 ) , whose genomes are generally tetraploid , all had two versions of CLTC and at least one CLTCL1 gene , except for cave fish ( Astyanax mexicanus ) apparently lacking CLTCL1 .", "Eight additional species of vertebrates with high genome coverage and reliable annotation had the CLTC gene but no identifiable CLTCL1 gene .", "CLTCL1 genes are present in the Caviomorpha and Sciuridae rodent suborders , and lost in the Muroidea suborder from the entire Mus genus ( Wakeham et al . , 2005 ) and from rat ( Rattus norvegicus ) .", "The Cetartiodactyla clade also appears to have lost CLTCL1 , as CLTCL1 is absent from the four representative genomes in our dataset ( pig ( Sus scrofa ) , sheep ( Ovis aries ) , cow ( Bos taurus ) , Yangtze finless porpoise ( Neophocaena asiaeorientalis ) ) .", "This suggests a loss event before the Cetartiodactyla lineage split , independent of the loss event preceding split of the Muroidea lineage .", "The absence of CLTCL1 in rat clarifies why CHC22 could not be biochemically identified in rat and indicates that antibodies against CHC22 that react with rat cells must cross-react with other proteins ( Towler et al . , 2004 ) .", "CLTCL1 was also not detected in the genomes of the little brown bat ( Myotis lucifugus ) and the duck-billed platypus ( Ornithorhynchus anatinus ) .", "Assuming the genome annotations for the species analyzed are reliable , these data indicate that there have been at least five independent losses of CLTCL1 that are clade- or species-specific .", "The intermittent loss of CLTCL1 and the retention of CLTC raises the question of whether their patterns of evolution are typical for genes with related functions that duplicated in the same time frame as CLTC/CLTCL1 .", "CLTC and CLTCL1 are located on paralogous regions of human chromosomes 17 and 22 , respectively .", "For these two genes , the evolutionary rates ( rate of non-synonymous substitutions to rate of synonymous substitutions; dN/dS ) across vertebrates at each position were determined and plotted along the length of the protein sequences ( Figure 2A–B ) .", "Several adjacent paralogs have been maintained in these chromosomal regions , some of which are involved in membrane traffic , including the gene pair of MTMR4 and MTMR3 , encoding myotubularin lipid phosphatases .", "Also , CLC subunits of CHC17 clathrin are encoded by paralogous genes on different chromosomes ( CLTA and CLTB ) that arose from a local gene duplication , mapped to the same time frame as the CHC-encoding duplication ( Wakeham et al . , 2005 ) .", "Comparison of the distribution of dN/dS ratios for the three pairs revealed stronger purifying selection on the CLTC/CLTCL1 genes than on MTMR4/MTMR3 and CLTA/CLTB ( Figure 2C–E ) , suggesting the CHC-encoding clade is more evolutionarily constrained .", "This observation is consistent with our previous identification of conserved signature residues in CLTCL1 using DIVERGE analysis ( Wakeham et al . , 2005 ) and indicates conserved functions for both the CLTC and CLTCL1 gene products .", "Furthermore , there is a striking difference in the distribution and average of evolutionary rates , as measured by dN/dS , between CLTC and CLTCL1 ( Kolmogorov-Smirnov test p-value<2 . 2e-16 ) , with CLTC being significantly more constrained by purifying selection than CLTCL1 .", "In contrast , there is minimal difference in the distribution and average of evolutionary rates between the two paralog pairs MTMR4/MTMR3 and CLTA/CLTB ( Kolmogorov-Smirnov test yields p-values 0 . 003643 and 0 . 9959 , respectively ) .", "To follow up the indication that CLTC and CLTCL1 are subject to different degrees of purifying selection , we investigated their variation in human populations .", "We analyzed 2504 genomes from the 1000 Genomes Project database , phase 3 ( 1000 Auton et al . , 2015 ) and identified alleles resulting from non-synonymous substitutions for CLTC and CLTCL1 .", "This dataset included individuals from each of five human meta-populations: European ( EUR , 503 ) , East Asian ( EAS , 504 ) , Admixed American ( AMR , 347 ) , South Asian ( SAS , 489 ) and African ( AFR , 661 ) .", "Individual populations with their abbreviations are listed in Supplementary file 1a .", "The reference sequences for chimpanzee ( Pan troglodytes ) and pseudo-references for two archaic humans , Altai Neanderthal and Denisovan , were also included to relate allelic variation to the ancestral state .", "A median-joining network for all the inferred CLTC human alleles showed a very common allele ( sample frequency 0 . 997 ) with only five low-frequency variants generating a total of six alleles ( Figure 3A ) .", "Each allele encodes a variant of a CHC ( allotype ) , which includes one or more single nucleotide polymorphisms ( SNPs ) .", "In contrast to CLTC , we identified 46 non-synonymous SNPs in CLTCL1 , present in 52 distinct haplotypes ( referred to here as alleles , following the definition given above , Supplementary file 2b-c ) .", "A median-joining network for the most common CLTCL1 alleles showed that they are widely distributed within the human meta-populations ( Figure 3B ) .", "Each meta-population tends to have private , less frequent alleles .", "Nevertheless , all the meta-populations comprised two main allelic clades , together constituting a sample frequency of 77% .", "These two main alleles differ by a single methionine to valine substitution at position 1316 ( M1316V ) in the protein sequence ( SNP ID rs1061325 with genomic location chr22:19184095 on hg19 assembly ) .", "The valine at position 1316 is predicted to have a functional effect on the protein since it was categorized as ‘probably damaging’ with a probability of 0 . 975 by PolyPhen ( Adzhubei et al . , 2010 ) and as ‘damaging’ by SIFT ( Kumar et al . , 2009 ) .", "Sequences in chimpanzee and archaic humans have the M1316 allotype , suggesting that M1316 is likely to represent the ancestral state .", "To further investigate this , we inspected raw sequencing data from both Altai Neanderthal and Denisovan ( Supplementary file 1d ) .", "We inferred the most likely genotype to be homozygous for the M1316 amino acid ( minimum sequencing depths equal to 40 and 28 , respectively ) .", "We then extracted sequencing data for an additional 13 archaic and ancient humans ( Supplementary file 1d ) .", "We found that the V1316 amino acid is present in Pleistocene hunter-gatherers and Neolithic farmers but not in other Neanderthals or Holocene hunter-gatherers in this limited data set .", "The equivalent residue in human CHC17 ( encoded by CLTC ) is also methionine , suggesting that methionine at this position likely pre-dated the initial duplication generating CLTCL1 .", "For the non-human species analyzed ( Figure 1 ) , all CHC-encoding genes present would produce clathrins with M1316 , further indicating its ancient and conserved role in CHC structure or function .", "To quantify the levels of nucleotide and allelic diversity for non-synonymous sites within human populations , several summary statistics of diversity were calculated .", "For populations within each meta-population , we separately calculated Watterson’s and Nei’s estimators of genetic diversity ( TW and PI , respectively ) , Tajima’s D ( TD ) , Fu and Li’s D* ( FLDs ) and F* ( FLFs ) , the sum of squared allele frequencies including the most common allele ( H1 ) and excluding it ( H2 ) , and the normalized ratio between H2 and H1 ( H2H1 ) ( Supplementary file 2a ) .", "To assess whether observed summary statistics are expected or not under neutral evolution in each population , we calculated the empirical null distribution from a set of 500 control genes with the same coding length as CLTCL1 ( Supplementary file 2b ) .", "High or low percentile rank values for CLTCL1 in the empirical distribution indicate that the summary statistic for CLTCL1 is unlikely to occur by mutation and neutral genetic drift alone .", "Summary statistics and populations were then clustered according to their empirical ranks and plotted on a heat map ( Figure 4 ) .", "All populations tend to display high genetic diversity for CLTCL1 , as summarized by PI and TW , and an unusually high frequency for the second most common allele , as summarized by H2 and H2H1 .", "Such configuration is likely to occur under balancing selection ( Charlesworth , 2006 ) or because of a soft sweep ( Messer and Petrov , 2013 ) .", "That CLTCL1 was low ranking in all populations for H1 , a statistic representing the frequency of the most common allele , also supported diversifying selection rather than hard sweeps .", "On the other hand , all populations display negative TD values with many populations exhibiting negative FLDs and FLFs values .", "These values are consistent with low diversity within common alleles and an excess of low-frequency variants .", "Finally , we calculated a measure of genetic differentiation ( fixation index FST ) between pairs of canonical reference populations , namely Yoruba from Nigeria ( YRI ) , North Americans with European ancestry ( CEU ) , and Han Chinese from Beijing ( CHB ) .", "We did not find any evidence that FST values for CLTCL1 ( YRI-CEU 0 . 15 , YRI-CHB 0 . 077 , CEU-CHB 0 . 065 ) are outliers in the empirical distribution of control genes .", "Such inconsistent patterns could be partly explained by the fact that we considered only non-synonymous changes , and the limited number of SNPs considered per gene may create a larger variance in the empirical distributions , especially for allele-based statistics .", "We therefore further examined the high frequency of the second most common allele by investigating whole genomic variation , including silent SNPs .", "We observed a local increase of H2 statistics in CLTCL1 for European populations , which already shows a large value based on non-synonymous changes ( Figure 4—figure supplement 1 ) .", "This analysis also indicates that any selection signatures are restricted to a local genomic region encompassing CLTCL1 .", "Another reason for the summary statistics not being strong outliers in the empirical distribution is the high recombination rate ( sex-average rate of 2 . 5 cM/Mb ) inferred for the genomic region encompassing CLTCL1 ( Kong et al . , 2002 ) .", "We therefore performed coalescent simulations under neutrality of a putative 100kbp genomic region surrounding the SNP encoding the M1316V variation , taking into account the local recombination rate and a previously proposed demographic model for Africans ( YRI ) , Europeans ( CEU ) and East Asians ( CHB ) ( Gutenkunst et al . , 2009 ) with a mutation rate of 1 . 5 × 10−8 per base pair per generation .", "The observed values for TW and PI were significantly greater than expected under neutral evolution for all populations ( p-values<0 . 001 ) , while TD was greater than expected for CHB only , although with a marginally non-significant statistical support ( p-value 0 . 056 ) .", "All these results are suggestive of a genetic diversity higher than expected under neutrality for a region encompassing M1316V , although possible complex evolutionary scenarios may limit the power of summary statistics to detect such selective events .", "One plausible explanation of the high genetic diversity and frequency of the two major alleles of CLTCL1 that occur in all modern human populations ( Figure 3B , Supplementary file 1b ) is balancing selection ( Charlesworth , 2006 ) .", "Such a distribution of allele frequency was confirmed using a different data set of more than 50 sampled human populations ( Figure 4—figure supplement 2 ) .", "In several populations , we also observed an apparent excess of heterozygosity at SNP rs1061325 ( Supplementary file 2c ) , compatible with heterozygote advantage ( overdominance ) for the two encoded allotypes differing at residue 1316 .", "Specifically , all European populations show a ratio of observed versus expected ( assuming Hardy-Weinberg equilibrium ) heterozygosity greater than 1 , with the highest value of 1 . 24 ( chi-squared test nominal p-value 0 . 047 ) for Iberic Spanish ( IBS ) ( Figure 4—figure supplement 3 ) .", "Selective pressures that might be acting on CLTCL1 , irrespective of population distribution , could be changes in human diet , a number of which have been inferred over the last 2 . 6 million years ( Hardy et al . , 2015 ) .", "Perhaps the best known of these dietary transitions are the introduction of cooking ~450 KYA , the development of farming ~12 , 500 YA , and more recently industrialized food processing , which gradually and then dramatically increased carbohydrate availability and consumption by humans .", "As CHC22 clathrin , the gene product of CLTCL1 , is required for formation of the intracellular pathway critical for an insulin response , its genetic history could potentially be influenced by these changes .", "To address the hypothesis that nutritional habits conferred selective pressure on CLTCL1 , we compared the frequency of SNP rs1061325 ( M1316V ) in farming versus hunter-gatherer population samples from ancient and modern humans .", "Although the appearance of SNP rs1061325 predates the advent of farming ( Supplementary file 1d ) , the observed frequencies of this allele , which encodes the CHC22-V1316 allotype , are consistent with a tendency for it to increase once farming became common practice for a population ( Figure 5 ) , although the small sample size for modern humans limits the power to reach statistical significance .", "The highest difference in allele frequency was observed between early farmers and hunter-gatherers from West Eurasia .", "However , as these two populations are highly diverged , it remains possible that this significant difference in allele frequency is due to genetic drift shaped by population history , rather than natural selection .", "To test this model , using the same dataset , we extracted 2500 control SNPs with a global minor allele frequency similar to rs1061325 ( M1316V ) ( up to an error of 5% ) and minimum global sequencing depth of 100X .", "We obtained statistical significance ( p-value 0 . 036 ) when testing the difference in derived allele frequency in farmers compared to hunter-gatherers ( +26 . 58% ) , while we found no statistical support ( p-value 0 . 080 ) when testing the absolute difference in allele frequency between these two populations .", "We analyzed allelic variation for CLTC and CLTCL1 in the genomes of 79 individuals representing six species of great ape , two species each for chimpanzees , gorillas and orangutans ( Pan troglodytes , Pan paniscus , Gorilla beringei , Gorilla gorilla , Pongo abellii , Pongo pygmaeus ) .", "After data filtering and haplotype phasing , we found no non-synonymous SNPs for CLTC and 64 putative non-synonymous SNPs for CLTCL1 ( Supplementary file 3a ) .", "In three species of great apes analyzed , one of the non-synonymous changes in CLTCL1 leads to a premature stop-codon at amino position 41 , with an overall frequency of 36% .", "However , sequences containing the stop-codon exhibited only a marginal increase of nucleotide diversity ( +4 . 7% as measured by Watterson’s index; Watterson , 1975 ) compared to the full-length sequences , suggesting that these are relatively new variants .", "Notably , for all the non-human primates analyzed , CLTCL1 variants do not encode the V1316 allotype , which appears private to humans .", "However , in all three types of great ape we found a common but different substitution , threonine ( T1316 ) , at the same amino acid position .", "To further investigate variation in non-human primates , we increased the sample size per species by analyzing CLTC and CLTCL1 variation in 70 chimpanzee and bonobo genomes , including four subspecies of chimpanzee .", "While no variation was observed for CLTC ( Figure 3C ) , a median-joining network for the inferred 8 CLTCL1 alleles ( Supplementary file 3b ) showed a major allele common to different species and subspecies with less frequent alleles primarily restricted to individual ones ( Figure 3D ) .", "In this chimpanzee data set , we observed considerable diversity , with a potential tendency towards multiple variants .", "However , amino acid 1316 was not covered in this data set , possibly due to poor data mapping quality associated with the high nucleotide diversity observed .", "In another data set of 20 individuals ( Teixeira et al . , 2015 ) , we found a frequency of 10% for the T1316 allotype in chimpanzees but not in bonobos .", "We further investigated CLTCL1 variation in polar bears ( Ursus maritimus ) and their closest related species , brown bears ( Ursus arctos ) .", "These two species , which diverged 479–343 KYA ( Liu et al . , 2014 ) , have very different diets ( Liu et al . , 2014; Bojarska and Selva , 2012 ) and are phylogenetically closer to each other than chimpanzees and humans .", "Polar bears subsist on a high fat , low carbohydrate diet , whereas brown bears consume a more varied diet of carbohydrate , protein and fat .", "Analysis of 21 bear genomes ( seven polar bears and 14 brown bears ) ( Benazzo et al . , 2017 ) , revealed three positions ( 1267 , 1389 , and 1522 ) which are fixed in polar bears but are either polymorphic or have a different residue in brown bears ( Supplementary file 3c ) .", "Genetic differentiation between polar and brown bears , as measured by FST , is markedly higher for CLTCL1 ( 0 . 56 ) than for CLTC ( 0 . 26 ) ( Liu et al . , 2014 ) .", "Furthermore , a phylogenetic tree of both bear species in our sample exhibits more diversification for CLTCL1 compared to CLTC ( Figure 3—figure supplement 1 ) .", "This sample of bear populations may support the emergence of multiple CLTCL1 variants within a species and a potential role for diet-related selection .", "One expectation for selection of the human-specific CLTCL1 allele encoding the CHC22-V1316 allotype is that this amino acid change might confer a functional change in the clathrin lattice .", "This was predicted by the PolyPhen and SIFT analyses , highlighting the change as potentially structure-altering .", "As many humans are heterozygous for the M1316 and V1316 allotypes ( 44% based on all individuals from 1000 Genomes project ) , there may potentially be special properties for mixed lattices formed from the two protein allotypes .", "To address the possibility that the M1316V polymorphism affects protein function , we used MODELLER ( Benjamin and Sali , 2014 ) to produce a homology model of the two CHC22 allotypes based on the crystal structure of CHC17 clathrin ( PDB 1B89 ) ( Ybe et al . , 1999 ) , taking advantage of the 85% protein sequence identity between human CHC17 and CHC22 ( Figure 6 ) .", "Modeling using UCSF Chimera ( Pettersen et al . , 2004 ) showed that residue 1316 is found at a key interface between triskelion legs in assembled clathrin ( Figure 6A and top of panel B ) .", "If M1316 is substituted by V1316 , the smaller side chain creates a void that would be energetically unfavorable ( Figure 6 , bottom of panel B ) , such that the triskelion leg might twist slightly to close the void .", "In the clathrin lattice , the legs have a torque that rotates the assembly interface along the protein sequence ( Wilbur et al . , 2005 ) , so a further twist could slightly adjust the interface , altering assembly interactions .", "Changes in the assembly interface could affect integrity of the lattice and potentially influence kinetics of assembly and disassembly .", "Mixed lattices of the two CHC22 allotypes would therefore have different properties from CHC22 coats formed in homozygotes for the two major CLTCL1 alleles .", "CHC22 is needed for the traffic of GLUT4 to its intracellular storage compartment , where GLUT4 awaits release to the plasma membrane in response to insulin .", "However , CHC22 also accumulates at the GLUT4 storage compartment ( GSC ) when it expands due to impaired GLUT4 release in cases of insulin-resistant type two diabetes ( T2D ) ( Vassilopoulos et al . , 2009 ) .", "Thus , genetic variation of CHC22 could alter rates of retention and release of GLUT4 in both healthy and disease states .", "To test whether the evolutionary change from M1316 to V1316 in CHC22 clathrin alters its properties , three aspects of CHC22 biochemistry and function were compared for the two allotypes .", "HeLa cells were transfected with constructs encoding each CHC22 variant or CHC17 , tagged with green fluorescent protein ( GFP ) .", "Atypically for their epithelial cell origin but not for transformed cells , HeLa cells express CHC22 clathrin ( they are homozygous for the M1316 allotype ) ( Adey et al . , 2013; Landry et al . , 2013 ) .", "We observed that the transfected fluorescently tagged CHC22 allotypes were both concentrated in the perinuclear region of the cell , similar to endogenous CHC22-M1316 detected by antibody , and did not overlap with endogenous CHC17 ( Figure 7A ) .", "Conversely , transfected GFP-CHC17 did not overlap with endogenous CHC22 , so expression of the transfected CHCs reflected their natural distribution ( Dannhauser et al . , 2017 ) .", "Using these constructs , the dynamics of membrane association for the two allotypes of CHC22 and for CHC17 was assessed by Fluorescence Recovery After Photobleaching ( FRAP ) .", "To assess clathrin turnover , as an indicator of clathrin coat stability , cells expressing fluorescent proteins were photobleached in the perinuclear area ( Figure 7B ) and their rate of fluorescence recovery was measured .", "Recovery of CHC17 fluorescence was the fastest , consistent with its more soluble properties compared to CHC22 ( Dannhauser et al . , 2017 ) .", "CHC22-M1316 showed the slowest recovery and CHC22-V1316 was intermediate ( Figure 7C–E ) , suggesting that it is more exchangeable in the CHC22 coat than the M1316 allotype .", "The impact of CHC22 variation on GLUT4 retention was then assessed .", "Because HeLa cells express CHC22 , they can form a GSC , when transfected to express GLUT4 .", "These cells sequester GLUT4 intracellularly , and then release it to the plasma membrane in response to insulin , behaving like muscle and adipocytes , though with more modest insulin response ( Camus et al . , 2018; Trefely et al . , 2015; Haga et al . , 2011 ) .", "To detect GLUT4 release to the cell surface , we used a construct expressing GLUT4 tagged with mCherry and a hemagglutinin ( HA ) epitope embedded in an exofacial loop of the transporter ( HA-GLUT4-mCherry ) .", "Appearance of surface GLUT4 in response to insulin was detected by fluorescence-activated cell sorting ( FACS ) using an antibody to the HA epitope ( Figure 8A ) .", "Transfection of HeLa cells with siRNA depleting CHC22 ablates this insulin-responsive pathway ( Camus et al . , 2018 ) ( Figure 8A ) .", "We then assessed if siRNA inhibition of insulin-responsive GLUT4 release can be rescued by expression of CHC22-M1316-GFP or CHC22-V1316-GFP .", "These constructs , the same as characterized in Figure 7A , are siRNA-resistant , as well as being GFP-tagged .", "We observed that , when endogenous CHC22 was depleted , CHC22-M1316 was able to restore the insulin response but CHC22-V1316 was not , when the rescue constructs were expressed at the same levels in cells ( measured by intensity of GFP fluorescence ) ( Figure 8A ) .", "CHC17 expression also did not rescue insulin-induced GLUT4 expression , as shown previously ( Vassilopoulos et al . , 2009 ) .", "However , CHC22-V1316 is functional for trapping GLUT4 intracellularly because CHC22-transgenic mice that express CHC22-V1316 in muscle , using the natural human promoter , show excessive GLUT4 sequestration in muscle compared to wild-type mice without CHC22 , leading to higher blood glucose in the transgenic animals ( Vassilopoulos et al . , 2009 ) .", "To analyze GLUT4 sequestration in another way , cells depleted for CHC22 and then transfected with mCherry-GLUT4 plus either CHC22 allotype or CHC17 were each divided into three populations expressing equivalently low , medium and high levels of the transfected CHC-GFP .", "Then , the total GLUT4 content of the cells was measured by mCherry fluorescence .", "We observed higher levels of GLUT4 in CHC22-depleted cells expressing CHC22-M1316-GFP , compared to cells expressing either CHC22-V1316-GFP or CHC17-GFP at both medium and high levels of CHC expression ( Figure 8B ) .", "This suggests that GLUT4 is sequestered more effectively from degradative membrane traffic pathways when trafficked by CHC22-M1316 than by CHC22-V1316 , indicating that the M1316 variant is more efficient at targeting GLUT4 to the GSC .", "As indicated by their weak insulin response compared to muscle or fat cells , HeLa cells are only just able to form a functional GSC from which GLUT4 can be released .", "For these cells , the less effective CHC22-V1316 is inadequate to restore GSC formation when their endogenous CHC22-M1316 is depleted .", "Use of this HeLa model was necessitated by the lack of natural models for the CHC22-dependent GLUT4 pathway in myoblasts and adipocytes , as well as a lack of antibodies that detect surface GLUT4 .", "Nonetheless , these experiments demonstrate a functional difference between CHC22-M1316 and CHC22-V1316 and suggest that CHC22-V1316 is less efficient at GLUT4 sequestration ." ], [ "We studied the phylogenetics and population genetics of CHC22 clathrin to understand the functional variation of this protein in relation to its evolutionary history .", "CHC22 clathrin is a key player in post-prandial blood glucose clearance in humans through its role in intracellular packaging of the GLUT4 glucose transporter in muscle and fat , the tissues in which CHC22 and GLUT4 are expressed ( Vassilopoulos et al . , 2009 ) .", "The CHC22 pathway positions GLUT4 for cell surface release in response to insulin and consequent uptake of glucose into these tissues ( Bryant et al . , 2002 ) .", "The CLTCL1 gene encoding CHC22 resulted from gene duplication that we have now dated to 494–451 MYA , early in vertebrate evolution when jawed vertebrates emerged .", "We had previously shown that CLTCL1 is a pseudogene in mice ( Wakeham et al . , 2005 ) .", "Expanding analysis to 56 jawed vertebrate genomes ( >5X coverage ) we could not detect CLTCL1 in nine of them .", "Six of these absences can be ascribed to two independent gene loss events in branches of the Rodentia and the Cetartidactylae .", "The three others may represent additional gene losses or incomplete genome annotation .", "All vertebrate and non-vertebrate eukaryotes considered here have retained the parent CLTC gene encoding CHC17 clathrin , which mediates endocytosis and other housekeeping membrane traffic pathways .", "The analysis described here establishes that CLTC is under strong purifying selection .", "Notable is our evidence for purifying selection on CLTCL1 in the species in which it has been retained , supporting its functional importance in those species .", "Compared to CLTC , extensive allelic diversity was observed for CLTCL1 in all species for which populations were analyzed , including humans , chimpanzees and bears .", "Variant alleles were species-specific in most cases .", "In all human populations , two allelic variants of CLTCL1 are present in high frequency , differing only at one nucleotide , resulting in CHC22 protein with either methionine or valine at position 1316 .", "The V1316 allotype appears specific to humans , but some non-human primates have a different variation at the position 1316 .", "Analysis of ancient humans dated the appearance of the V1316 variant to 500–50 KYA and indicated that M1316 , which is fixed in CHC17 clathrin , is the ancestral state .", "Analyses of human population genetic data provided support for the maintenance of high genetic diversity and two allotypes of CHC22 .", "We hypothesize that selective pressure on CHC22 clathrin comes from its role in nutrient metabolism .", "Consistent with this hypothesis , we observed functional differences between the two CHC22 allotypes in their capacity to control GLUT4 membrane traffic , as predicted by structural modeling and differences in cellular dynamics of the two allotypes .", "Retention of CLTC in all vertebrate species is consistent with the encoded CHC17 mediating cellular housekeeping clathrin functions shared by all eukaryotes .", "On the other hand , CHC22 , encoded by the paralogous gene CLTCL1 , operates in the specialized insulin-responsive GLUT4 pathway to make the pathway more efficient in those species that retained CLTCL1 .", "Data presented here ( Figure 8 ) and our recent mapping of a novel intracellular location for CHC22 function ( Camus et al . , 2018 ) indicate that , in human cells , CHC22 clathrin promotes transport from the secretory pathway to the insulin-responsive GSC .", "This CHC22 pathway complements the endocytic pathway for GLUT4 targeting to the GSC , so species without CHC22 can rely primarily on endocytosis for GLUT4 trafficking to the GSC , while species with CHC22 use both pathways .", "Thus , we hypothesize that species with functional CHC22 clathrin are more efficient at intracellular GLUT4 sequestration , resulting in lower surface GLUT4 in the absence of insulin , and tighter regulation of GLUT4 release in response to insulin .", "The trade-off is that these species have an inherent increased tendency to insulin resistance as their GLUT4 is sequestered more effectively .", "The two main vertebrate branches that have lost CHC22 comprise the Muridae ( mice and rats ) who are incessant herbivores and the Cetartiodactyla ( sheep , cattle , porpoise and pigs ) which include the ruminants ( sheep and cattle ) whose muscle uptake of glucose is critical for muscle function , but is not a main pathway for glucose clearance ( Hocquette et al . , 1995 ) .", "These two groups of species require greater availability of GLUT4 on their cell surfaces , so that more efficient GLUT4 sequestration by CHC22 would not be favorable to their nutritional needs .", "The fact that CHC22 alters the balance of membrane traffic to the GSC means that species losing CLTCL1 could evolve compensatory pathways more compatible with their diets .", "Thus , transgenic mice expressing CHC22 over-sequester GLUT4 in their muscle and develop hyperglycemia with aging ( Vassilopoulos et al . , 2009 ) .", "The cave fish , which appears to lack CLTCL1 , has independently evolved mutations in the insulin receptor , creating natural insulin resistance , such that the presence of CHC22 on top of this mechanism might be detrimental ( Riddle et al . , 2018 ) .", "The loss of CLTCL1 from cave fish is consistent with the insulin responsive GLUT4 pathway being a target for natural selection driven by diet , which might also explain CLTCL1 variation or loss for additional vertebrate species during vertebrate evolution .", "The allelic variation reported here for CLTCL1 in human and bear populations further supports the hypothesis that CLTCL1 has undergone continued selection during vertebrate evolution in relation to diet .", "While purifying selection appears to be operating on CLTCL1 in those species that retain it , CLTCL1 is far more variable than CLTC in these species .", "In humans , we find two major and functionally distinct alleles at remarkably similar frequencies in all populations studied .", "Statistical analysis comparing early farmer and hunter-gatherer populations shows an apparent increase of the V1316 variant , suggesting a correlation with regular consumption of digestible carbohydrate .", "Notably , the SNP distinguishing these alleles is human-specific and likely arose 550–50 KYA ( i . e . post-Neanderthal split , pre-Neolithic ) .", "Other dramatic increases in digestible carbohydrate utilization have been inferred for humans in this timeframe; in particular the advent of cooking ( which gelatinizes crystalized starch , making it much easier to digest ) , salivary amylase gene copy number increase ( allowing increased starch digestion capacity ) and accelerated brain size increase ( which would increase demands for blood glucose ) ( Hardy et al . , 2015 ) .", "While the co-evolution of these cultural and genetic traits was originally proposed to have occurred some 800 KYA , recent studies indicate a time frame of 450–300 KYA years for cooking ( Shahack-Gross et al . , 2014 ) , increased oral amylase activity ( Inchley et al . , 2016 ) and accelerated brain size increase ( Dunbar , 2019 ) .", "The fact that the two major human CLTCL1 alleles are functionally distinct is consistent with diversifying selection operating on CLTCL1 , with a balancing selection possibly caused by heterozygote advantage .", "While population genetic signatures for balancing or overdominant selection were not entirely robust , some summary statistics were suggestive of an increased diversity that was unlikely to have occurred under neutrality .", "Other statistics , such as the ones based on allele frequencies , would not be expected to gain significance within the timeframe of the human-specific diversifying selection we detect .", "The allelic diversity of CLTCL1 in other primate species could have the potential effect of diluting its function .", "Whilst chimpanzees are omnivores and gorillas herbivores , both rely for nutrition on extensive foraging for carbohydrate .", "Also notable is that polar bears , who have a very low carbohydrate diet compared to their brown bear relatives , have distinct CHC22 variants with unknown functionality , again consistent with CLTCL1 undergoing selection driven by nutritional ecology .", "Clathrins are self-assembling proteins and function as a latticed network in the protein coat that they form on transport vesicles .", "Our structural modeling predicts that the single amino acid difference between the two main human CHC22 allotypes could influence the strength of molecular interactions in the CHC22 clathrin lattice , as position 1316 occurs at a lattice assembly interface ( Figure 6 ) .", "When expressed in cells , both CHC22 variants gave the same overall intracellular distribution , but CHC22-V1316 shows faster turnover from membranes than CHC22-M1316 ( Figure 7 ) and is less effective at GLUT4 sequestration ( Figure 8B ) .", "These properties are consistent with the methionine to valine change attenuating GLUT4 retention .", "This interpretation is further supported by a GLUT4 translocation assay , which indicates that the V1316 variant is less effective in forming the insulin-responsive GSC than the ancestral M1316 form of CHC22 ( Figure 8A ) .", "Thus , mixed lattices occurring in heterozygous individuals , potentially reflect balancing selection and overdominance , might reduce GLUT4 sequestration compared to M1316 homozygotes .", "This would have the effect of improving glucose clearance .", "It can be argued that human consumption of digestible carbohydrate on a regular basis ( Hardy et al . , 2015 ) , requiring increased glucose clearance , might be a selective force driving this genetic adaptation .", "This view is consistent with the increased frequency of the V1316 variant in early farmers .", "It is also possible that some forms of polar bear CHC22 are super-active at GLUT4 sequestration , providing a route to maintain high blood glucose , as occurs through other mutations in the cave fish ( Riddle et al . , 2018 ) .", "Regulators of fundamental membrane traffic pathways have diversified through gene duplication in many species over the timespan of eukaryotic evolution .", "Retention and loss can , in some cases , be correlated with special requirements resulting from species differentiation , such as the extensive elaboration of genes in the secretory pathway of Tetrahymena ( Dacks and Robinson , 2017; Bright et al . , 2010 ) .", "The evolutionary history of CLTCL1 , following vertebrate-specific gene duplication , suggests that differentiation of nutritional habits has shaped selection for the presence and absence of CLTCL1 in some vertebrate species , and its diversification in humans and potentially other species .", "Though its highest expression is in muscle and adipose tissue , transient expression of CHC22 during human brain development has also been documented ( Nahorski et al . , 2015 ) .", "This was noted in a study of a very rare null mutant of CLTCL1 that caused loss of pain sensing in homozygotes and no symptoms for heterozygotes ( Nahorski et al . , 2015 ) .", "Attenuated CHC22 function of the V1316 variant might lead to a spectrum of pain sensing in humans but this is unlikely to be a strong selective force affecting reproductive success , whereas glucose homeostasis , as suggested by our analysis , is more likely .", "By exerting efficient control of blood glucose levels , the presence of CHC22 clathrin was likely beneficial in providing the nutrition required to develop the large human brain , as well as affecting reproduction by influencing glucose availability during pregnancy ( Hardy et al . , 2015 ) .", "However , over the last 12 , 500 years in association with farming , or perhaps over the last 450 , 000 years in association with cooking , salivary amylase activity and starch digestion ( Hardy et al . , 2015; Shahack-Gross et al . , 2014; Inchley et al . , 2016 ) , readily available carbohydrate has increased our need to clear glucose from the blood , such that selection continues to act on CLTCL1 in humans .", "Our cell biology studies have also demonstrated that CHC22 increases GLUT4 retention .", "While we would not expect the major CLTCL1 polymorphism to directly influence the development of T2D , CHC22 accumulates on the expanded GSC that forms in cases of insulin-resistant T2D ( Vassilopoulos et al . , 2009 ) , so its variation could potentially exacerbate insulin resistance to different degrees .", "The genetic diversity that we report here may reflect evolution towards reversing a human tendency to insulin resistance and have relevance to coping with increased carbohydrate in modern diets ." ], [ "Vertebrate genomes as well as genomes of Drosophila melanogaster , Caenorhabditis elegans , Ciona intestinalis and Ciona savignyi were downloaded from Ensembl ( Yates et al . , 2016 ) , all accessed on 23/04/2016 except for pig ( 14/12/2017 ) , marmoset and hagfish ( both 09/08/2018 ) , excluding vertebrate species sequenced below five-fold genome coverage , that is with less than five reads per site on average .", "In addition , we downloaded the genomes of the elephant shark ( Venkatesh et al . , 2014 ) , whale shark ( Read et al . , 2017 ) , marmot ( The Alpine Marmot Genome , BioProject PRJEB8272 on NCBI ) and porpoise ( Yuan et al . , 2018 ) .", "All potential orthologs for the human isoforms of CLTC/CLTCL1 , MTMR4/MTMR3 , and CLTA/CLTB , in the above genomes were retrieved via BLAST ( Boratyn et al . , 2013 ) .", "An e-value threshold of 0 . 001 with additional constraints applied by default in InParanoid version 7 ( Ostlund et al . , 2010 ) were used ( at least 50% of the sequences are covered by the alignment , and at least 25% of the residues are aligned ) .", "The polar bear ( Ursus maritimus ) ( Liu et al . , 2014 ) , brown bear ( Ursus arctos ) ( Benazzo et al . , 2017 ) and black bear ( Ursus americanus ) CHC17 and CHC22 protein sequences were manually added .", "For CLTCL1 only the elephant and horse sequences ( XP_023397213 . 1 and XP_023502410 . 1 respectively ) were manually added .", "The sequences corresponding to the longest transcripts were aligned with MAFFT ( Katoh and Standley , 2013 ) and phylogenetic trees generated with PhyML ( Guindon et al . , 2010 ) .", "The last two steps were repeated after manually removing outlier sequences lying on long branches ( CLTC/CLTCL1: ENSTNIP00000007811 . 1 , ENSTGUP00000014952 . 1 , XP_023397213 . 1 , XP_023502410 . 1; CLTA/CLTB: ENSPSIP00000012669 . 1 ) and , in the case of genomes not retrieved from Ensembl ( therefore lacking the gene-to-transcript mapping ) , sequences most likely corresponding to alternative transcripts ( XP_015350877 . 1 , XP_007899998 . 1 , XP_007899997 . 1 , XP_007904368 . 1 , XP_007904367 . 1 , XP_020375861 . 1 , XP_020375865 . 1 , XP_020375862 . 1 , XP_020392037 . 1 , XP_020375864 . 1 , XP_020375859 . 1 ) .", "Trees were manually reconciled based on the Ensembl species tree extended by elephant shark , whale shark , brown bear , black bear , porpoise and marmot with TreeGraph ( Stöver and Müller , 2010 ) .", "Branch lengths were estimated based on the multiple sequence alignment ( MSA ) with PhyML fixing the manually reconciled topology , with options ‘-u’ and ‘--constraint_file’ .", "With this approach no support values for splits are calculated .", "The resulting trees were used as input to generate a new phylogeny-aware MSAs with PRANK ( Löytynoja and Goldman , 2005 ) .", "Branch lengths of the reconciled topologies were then re-estimated based on the MSA generated by PRANK .", "To compute evolutionary rates , the sequences and subtrees corresponding to CLTC and CLTCL1 clades after duplication ( i . e . excluding non-vertebrates ) were extracted and sequences from species without either CLTC or CLTCL1 were removed .", "The same procedure was performed for MTMR4/MTMR3 and CLTA/CLTB .", "A phylogeny-aware MSA was computed with PRANK on the remaining sequences , and the amino acid alignment was converted to a codon alignment with PAL2NAL ( Suyama et al . , 2006 ) .", "Finally , dN/dS ratios ( i . e . the ratio of the rate of nonsynonymous substitutions to the rate of synonymous substitutions ) were inferred based on the codon alignments with PAML ( Yang , 2007 ) for the six proteins independently using the site model M7 .", "Model M7 fits a Beta-distribution to the site rates by estimating the two Beta parameters shape and scale .", "Rates are estimated per site over the entire phylogeny , and therefore represent time averages .", "Phylogenetic trees of consensus amino acid sequences for bear samples only were computed using PhyML 3 . 1 ( Guindon et al . , 2010 ) with default values as implemented in Phylogeny . fr ( Dereeper et al . , 2008 ) .", "Phased genotypes were obtained by querying Variant Call Format ( VCF ) files ( Danecek et al . , 2011 ) from the 1000 Genomes Project database Phase 3 ( 1000 Auton et al . , 2015 ) for all available 2504 samples .", "Only high-quality variants were retained using vcflib ( https://github . com/vcflib/vcflib ) with options ‘VT = SNP and QUAL > 1 and AC >1 and DP >5000’ .", "Missing genotypes were assigned to homozygotes for the reference alleles .", "Finally , only sites with a recorded annotated function of being missense , nonsense , stop-loss or frame-shift for tested genes according to the UCSC Table Browser were retained ( Speir et al . , 2016 ) ( tables snp150 and snp150CodingDbSnp ) .", "For each retained position , the reference sequence for chimpanzee from the UCSC Table Browser ( Speir et al . , 2016 ) ( table snp150OrthoPt5Pa2Rm8 ) was initially used to infer the putative ancestral state .", "For ambiguous or multiallelic states in the chimpanzee sequence , the human reference base was used as an initial proxy for the ancestral state .", "The predicted functional impact of amino acid replacements was obtained by using Polyphen ( Adzhubei et al . , 2010 ) and SIFT ( Kumar et al . , 2009 ) .", "Additional frequency information for a single mutation of interest in more than 50 human populations was retrieved from the HGDP CEPH Panel ( Cann et al . , 2002 ) from http://hgdp . uchicago . edu/cgi-bin/gbrowse/HGDP/ .", "Genotype data for farmer and hunter-gatherer individuals were collected from the Simons Genome Diversity Project Dataset ( Mallick et al . , 2016 ) .", "Populations were merged based on their assigned geographical region with the following classification for hunter-gatherers: Africa ( Biaka , Ju|‘hoan North , Khomani San , Mbuti ) , Central Asia and Siberia ( Aluet , Chukchi , Eskimo Chaplin , Eskimo Naukan , Eskimo Sireniki , Even , Itelman , Tlingit , Tubalar , Ulchi , Atayal ) , East and South Asia ( Atayal , Kusunda ) .", "Farmer and hunter-gatherer allele frequencies were compared following a previously described approach ( Raineri et al . , 2014 ) .", "Briefly , we analytically computed the probability that the V allele is more frequent in farmers than in hunter gatherers while fully accounting for the uncertainty in the individual frequency estimates .", "V allele frequencies were inferred from allele counts of M and V in a Bayesian framework with a conjugate Beta uniform prior .", "We recorded maximum a posteriori estimates with 95% highest posterior density credible intervals computed with the Smisc R library , version 0 . 3 . 9 .", "We collected further published ancient DNA data from Western Eurasia and classified into three genetic grouping: hunter-gatherer ( HG ) , early farmer ( EF ) and steppe , using supervised ADMIXTURE ( Alexander et al . , 2009 ) as previously described ( Mathieson and Mathieson , 2018 ) .", "These are genetic groups and not directly based on differences in material culture or subsistence , but importantly in the case of HG and EF , these genetic classifications correspond closely to hunter-gatherer and agricultural subsistence strategies ( Haak et al . , 2015; Skoglund et al . , 2014; Skoglund et al . , 2012 ) .", "We then restricted analysis to samples dated between 10 , 000 and 5 , 000 years before present that were classified as either HG or EF , leading to a dataset of 119 HG and 316 EF of which 85 and 188 respectively had coverage at rs1061325 .", "Frequencies for South-East Asians and ancient Eurasians were down-sampled to ensure numerical stability .", "The HeLa genomic data were accessed through the NIH database of Genotypes and Phenotypes ( dbGaP at http://www . ncbi . nlm . nih . gov/sites/entrez ? db=gap ) through dbGaP accession number phs000640 .", "High-coverage VCF files for 79 individuals from six species of great apes were retrieved ( Prado-Martinez et al . , 2013 ) .", "Data was filtered using vcflib on the combined data set with the options ‘QUAL > 32 and DP >50 and DP <7000 and FS <27 and MQ > 25 and AC >1’ , similarly to the original manuscript describing this data set ( Prado-Martinez et al . , 2013 ) .", "To retrieve nonsynonymous changes , only variants where the translated proteins for each allele differ were retained .", "We finally phased the data and assigned individual haplotypes using shapeit v2 . r837 with the options ‘-burn 50 -prune 20 -main 100 -window 0 . 5 -effective-size 20000’ .", "Additional 110 genomes of chimpanzees and bonobos were analyzed ( Teixeira et al . , 2015; de Manuel et al . , 2016 ) .", "Data filtering , functional annotation and haplotype phasing were performed as described above .", "Full genome VCF files for two high-coverage archaic humans , namely one Altai Neanderthal ( Prüfer et al . , 2014 ) and one Denisova were retrieved ( Meyer et al . , 2012 ) .", "Low-quality sites were filtered out using vcflib with the options ‘QUAL > 1 and DP >10’ .", "A pseudo-reference sequence for each archaic human was constructed by replacing the heterozygous sites with the previously inferred human ancestral state .", "Sequencing data information for additional ancient human samples were obtained from previously published high-quality whole genome sequences ( Skoglund et al . , 2014; Broushaki et al . , 2016; Hofmanová et al . , 2016; Lazaridis et al . , 2014; Olalde et al . , 2014; Raghavan et al . , 2014; Seguin-Orlando et al . , 2014; Fu et al . , 2014 ) .", "Genotype likelihoods were calculated using the standard GATK model ( McKenna et al . , 2010 ) .", "Median-joining network plots were generated in R using pegas package ( Paradis , 2010 ) .", "Several summary statistics were calculated on the inferred alleles to describe their levels of nucleotide diversity .", "Specifically , for each population separately , Watterson’s estimator of population mutation parameter ( TW ) ( Watterson , 1975 ) , Nei’s genetic diversity index ( PI ) ( Nei , 1973 ) , Tajima’s D ( TD ) ( Tajima , 1989 ) , Fu and Li’s D* ( FLDs ) and F* ( FLFs ) ( Fu and Li , 1993 ) , the sum of squared allele frequencies including the most common allele ( H1 ) and excluding it ( H2 ) and their normalized ratio ( H2H1 ) ( Garud and Rosenberg , 2015; Garud et al . , 2015 ) were calculated .", "We also computed genetic differentiation ( FST ) ( Reynolds et al . , 1983 ) between pairs of canonical reference populations , namely Yoruban ( YRI ) , Europeans ( CEU ) , and Han Chinese ( CHB ) .", "To assess whether the observed summary statistics are expected under neutral evolution , genes with a coding length approximately equal ( ±5% ) to the one observed for the tested gene , CLTCL1 , were selected .", "For this analysis , the longest isoform for each gene , and its annotation was considered according to refGene table from the UCSC Genome Browser .", "We discarded genes on chromosome six and on sex chromosomes , as well as CLTA , CLTB and CLTC .", "This set was further reduced to the first 500 genes with the closest genomic length to CLTCL1 .", "As summary statistics can be calculated only in case of genetic variability , genes showing no non-synonymous SNPs within each population were discarded .", "For each summary statistic , the empirical percentile rank for the value observed in CLTCL1 compared to the whole distribution of control genes was calculated .", "Low or high values are suggestive of CLTCL1 being an outlier in the empirical distribution .", "For plotting purposes , summary statistics and populations were clustered according to a dendrogram inferred from their respective distances based on the calculated matrix of empirical percentile ranks .", "That is , populations clustering together exhibit similar patterns of percentile ranks , and thus of summary statistics .", "The underlying dendrograms are not reported .", "The heatmap plot was generated using the function heatmap . 2 in R with the package gplots .", "Cells with an empirical percentile rank lower than 0 . 10 or greater than 0 . 90 were filled with the exact rank value .", "We also obtained a null distribution of summary statistics by performing coalescent simulations using msms ( Ewing and Hermisson , 2010 ) under a previously derived demographic model for human populations ( Gutenkunst et al . , 2009 ) .", "MODELLER v9 . 13 ( Benjamin and Sali , 2014 ) was used to model the structure of the proximal leg segment of CHC22 , using the crystal structure of bovine CHC17 ( PDB 1B89 ) ( Ybe et al . , 1999 ) as a template .", "The model of the M1316V mutant was derived in a similar way using a mutated sequence .", "Structure visualization and analysis of residue interactions at the mutation site M1316 were performed using UCSF Chimera ( Pettersen et al . , 2004 ) .", "The wild type and mutant homology models were positioned in the cryo-electron microscopy map of the bovine clathrin lattice ( EMD: 5119 ) ( Fotin et al . , 2004 ) by structural superposition on the atomic model originally fitted in the map ( PDB 1XI4 ) ." ] ]

[ "CHC22 clathrin plays a key role in intracellular membrane traffic of the insulin-responsive glucose transporter GLUT4 in humans .", "We performed population genetic and phylogenetic analyses of the CHC22-encoding CLTCL1 gene , revealing independent gene loss in at least two vertebrate lineages , after arising from gene duplication .", "All vertebrates retained the paralogous CLTC gene encoding CHC17 clathrin , which mediates endocytosis .", "For vertebrates retaining CLTCL1 , strong evidence for purifying selection supports CHC22 functionality .", "All human populations maintained two high frequency CLTCL1 allelic variants , encoding either methionine or valine at position 1316 .", "Functional studies indicated that CHC22-V1316 , which is more frequent in farming populations than in hunter-gatherers , has different cellular dynamics than M1316-CHC22 and is less effective at controlling GLUT4 membrane traffic , altering its insulin-regulated response .", "These analyses suggest that ancestral human dietary change influenced selection of allotypes that affect CHC22’s role in metabolism and have potential to differentially influence the human insulin response ." ]

[ "When we eat carbohydrates , they are digested into sugars that circulate in the blood to provide energy for the brain and other parts of the body .", "But too much blood sugar can be poisonous .", "The body regulates blood sugar balance using the hormone insulin , which triggers the removal of sugar from the blood into muscle and fat cells .", "This removal process involves a pore in membranes at the surface of muscle and fat tissue , called a glucose transporter , through which the sugar molecules can pass .", "During fasting , the glucose transporter remains inside muscle and fat .", "But after a meal , insulin acts to release the transporter from its storage area to the surface of the tissue .", "How efficiently this process happens reflects how efficiently sugar can be removed from the blood .", "When this pathway breaks down , it can lead to diabetes .", "In humans , a protein called CHC22 is needed to deliver the glucose transporter to its storage area .", "In mice , CHC22 is absent .", "The question arises: do different animals' eating habits influence CHC22's role in controlling blood sugar ?", "The evolutionary history of CHC22 in a number of different animals could reveal what is special about glucose transport after a meal in humans , and how it might fail in diabetes .", "By analyzing the genomes of several different species , Fumagalli et al . found that the gene encoding CHC22 first evolved around the time animals began developing a backbone and complex nervous systems .", "Afterwards , it was lost by some animals – including mice , sheep and pigs .", "Fumagalli et al . also discovered that CHC22 varies between individual people .", "A new form of CHC22 , which first appeared in ancient humans , is less effective at holding the glucose transporter inside muscle and fat – leading to a tendency to reduce blood sugar levels .", "This new form became more common in humans over a period witnessing the introduction of cooking , and later farming; both of these technologies are associated with increased sugar in the diet .", "But not everyone has this new variant of the gene – both the old and newer variants are present in people today .", "The history of CHC22 suggests that it was useful for early humans to hold the glucose transporter inside muscle and fat , keeping blood sugar levels high , which contributed to the development of a large brain .", "But as humans became exposed to higher dietary levels of sugar the newer form of CHC22 allowed blood sugar to be lowered more readily .", "People with different forms of CHC22 are likely to differ in their ability to control blood sugar after a meal .", "In some cases , this could lead to heightened blood sugar levels , which in turn can lead to diabetes ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "chromosomes and gene expression", "tools and resources" ]

[ [ "A major goal in human genetics research is to understand genetic contributions to complex diseases , specifically the molecular mechanisms by which common DNA variants impact disease etiology .", "Most genome-wide association studies ( GWAS ) implicate non-coding variants that are far from genes , complicating interpretation of their mode of action and correct identification of the target gene ( Maurano et al . , 2012 ) .", "Mounting evidence suggests that disease variants disrupt the function of cis-acting regulatory elements , such as enhancers , which in turn affects expression of the specific gene or genes that are functional targets of these elements ( Wright et al . , 2010; Musunuru et al . , 2010; Cowper-Sal lari et al . , 2012; Smemo et al . , 2014; Claussnitzer et al . , 2015 ) .", "However , because cis-acting regulatory elements can be located kilobases ( kb ) away from their target gene ( s ) , identifying the true functional targets of regulatory elements remains challenging ( Smemo et al . , 2014 ) .", "Chromosome conformation capture techniques such as Hi-C ( Lieberman-Aiden et al . , 2009 ) enable the genome-wide mapping of long-range chromatin contacts and therefore represent a promising strategy to identify distal gene targets of disease-associated genetic variants .", "Recently , Hi-C maps have been generated in numerous human cell types including embryonic stem cells and early embryonic lineages ( Dixon et al . , 2012 , 2015 ) , immune cells ( Rao et al . , 2014 ) , fibroblasts ( Jin et al . , 2013 ) and other primary tissue types ( Schmitt et al . , 2016 ) .", "However , despite the increasing abundance of Hi-C maps , most datasets are of limited resolution ( >40 kb ) and do not precisely identify the genomic regions in contact with gene promoters .", "More recently , promoter capture Hi-C ( PCHi-C ) was developed which greatly increases the power to detect interactions involving promoter sequences ( Schoenfelder et al . , 2015; Mifsud et al . , 2015 ) .", "PCHi-C in different cell types identified thousands of enhancer-promoter contacts and revealed extensive differences in promoter architecture between cell types and throughout differentiation ( Schoenfelder et al . , 2015; Mifsud et al . , 2015; Javierre et al . , 2016; Freire-Pritchett et al . , 2017; Rubin et al . , 2017; Siersbæk et al . , 2017 ) .", "These studies collectively demonstrated that genome architecture reflects cell identity , suggesting that disease-relevant cell types are critical for successful interrogation of the gene regulatory mechanisms of disease loci .", "In support of this notion , several recent studies utilized high-resolution promoter interaction maps to identify tissue-specific target genes of GWAS associations .", "Javierre et al . generated promoter capture Hi-C data in 17 primary human blood cell types and identified 2604 potentially causal genes for immune- and blood-related disorders , including many genes with unannotated roles in those diseases ( Javierre et al . , 2016 ) .", "Similarly , Mumbach et al . interrogated GWAS SNPs associated with autoimmune diseases using HiChIP where they identified ~10 , 000 promoter-enhancer interactions that linked several hundred SNPs to target genes , most of which were not the nearest gene ( Mumbach et al . , 2017 ) .", "Importantly , both studies reported cell-type specificity of SNP-target gene interactions .", "Cardiovascular diseases , including cardiac arrhythmia , heart failure , and myocardial infarction , continue to be the leading cause of death world-wide .", "Over 50 GWAS have been conducted for these specific cardiovascular phenotypes alone , with more than 500 loci implicated in cardiovascular disease risk ( NHGRI GWAS catalog , https://www . ebi . ac . uk/gwas/ ) , most of which map to non-coding genomic regions .", "To begin to dissect the molecular mechanisms by which genetic variants contribute to CVD risk , a comprehensive gene regulatory map of human cardiac cells is required .", "Here , we present high-resolution promoter interaction maps of human iPSCs and iPSC-derived cardiomyocytes ( CMs ) .", "Using PCHi-C , we identified hundreds of thousands of promoter interactions in each cell type .", "We demonstrate the physiological relevance of these datasets by functionally interrogating the relationship between gene expression and long-range promoter interactions , and demonstrate the utility of long-range chromatin interaction data to resolve the functional targets of disease-associated loci ." ], [ "We used iPSC-derived CMs ( Burridge et al . , 2014 ) as a model to study cardiovascular gene regulation and disease genetics .", "The CMs generated in this study were 86–94% pure based on cardiac Troponin T protein expression and exhibited spontaneous , uniform beating ( Figure 1—figure supplement 1A , Video 1 ) .", "To demonstrate that iPSCs and CMs recapitulate transcriptional and epigenetic profiles of matched primary cells , we conducted RNA-seq and ChIP-seq for the active enhancer mark H3K27ac in both cell types and compared these data with similar cell types from the Epigenome Roadmap Project ( Kundaje et al . , 2015 ) .", "RNA-seq profiles of iPSCs clustered tightly with H1 embryonic stem cells , whereas CMs clustered with both left ventricle ( LV ) and fetal heart ( FH ) profiles ( Figure 1—figure supplement 1B ) .", "Furthermore , we observed that matched cell types exhibited three-fold greater overlap in the number of promoter-distal H3K27ac ChIP-seq peaks than non-matched cell types ( Figure 1—figure supplement 1C , D ) , indicating that both iPSCs and CMs recapitulate tissue-specific epigenetic states of human stem cells and primary cardiomyocytes , respectively .", "To further validate our system , we analyzed differentially expressed genes between iPSCs and CMs .", "Among the top 10% of over-expressed genes in CMs were genes directly related to cardiac function including essential cardiac transcription factors ( GATA4 , MEIS1 , TBX5 , and TBX20 ) and differentiation products ( TNNT2 , MYH7B , MYL7 , ACTN2 , NPPA , HCN4 , and RYR2 ) ( fold-change >1 . 5 , Padj <0 . 05 , Figure 1—figure supplement 2A–C ) .", "Gene Ontology ( GO ) enrichment analysis for genes over-expressed in CMs relative to iPSCs further confirmed the cardiac-specific phenotypes of these cells with top terms relating to the development of the cardiac conduction system and cardiac muscle cell contraction ( Figure 1—figure supplement 2D ) .", "To comprehensively map long-range regulatory elements in iPSCs and CMs , we performed in-situ Hi-C ( Rao et al . , 2014 ) in triplicate iPSC-CM differentiations; importantly , we used the four-cutter restriction enzyme MboI which generates ligation fragments with an average size of 422 bp , enabling enhancer-level resolution of promoter contacts .", "We enriched iPSC and CM in situ Hi-C libraries for promoter interactions through hybridization with a set of 77 , 476 biotinylated RNA probes ( ‘baits’ ) targeting 22 , 600 human RefSeq protein-coding promoters ( see Materials and methods ) and sequenced each library to an average depth of ~413 million ( M ) paired-end reads .", "After removing duplicates and read-pairs that did not map to a bait , we obtained an average of 31M and 41M read-pairs per replicate for iPSC and CM , respectively .", "We used CHiCAGO ( Cairns et al . , 2016 ) , a computational pipeline which accounts for bias from the sequence capture , to identify significant interactions and further filtered for those significant in at least two out of three replicates ( see Materials and methods ) .", "Finally , we exclusively focused on interactions that were separated by a distance of at least 10 kb .", "This criterion addresses the high frequency of close-proximity ligation evets in Hi-C data , which are difficult to distinguish as random Brownian contacts or functional chromatin interactions ( Cairns et al . , 2016 ) .", "In total , we identified 350 , 062 promoter interactions in iPSCs and 401 , 098 in CMs .", "A large proportion ( ~55% ) of interactions were shared between the two cell types , indicating that even at high resolution many long-range interactions are stable across cell types ( Figure 1A ) .", "Approximately 20% of all interactions were between two promoters , demonstrating the high connectivity between genes and supporting the recently suggested role of promoters acting as regulatory inputs for distal genes ( Dao et al . , 2017; Diao et al . , 2017 ) ( Figure 1B ) .", "Most interactions were promoter-distal , with a median of ~170 kb between the promoter and the distal-interacting region ( Figure 1C ) .", "To compare the PCHi-C maps with known features of genome organization , we sequenced our pre-capture Hi-C libraries to an average depth of 665M reads per cell type and identified topologically associating domains ( TADs ) with TopDom ( see Materials and methods ) .", "TADs are organizational units of chromosomes defined by <1 megabase ( Mb ) genomic blocks that exhibit high self-interacting frequencies with a very low interaction frequency across TAD boundaries ( Dixon et al . , 2012; Nora et al . , 2012 ) .", "Notably , this organization is thought to constrain the activity of cis-regulatory elements to target genes within the same TAD , as disruption of TAD boundaries has been shown to lead to aberrant activation of genes in neighboring TADs ( Nora et al . , 2012; Lupiáñez et al . , 2015; Franke et al . , 2016; Symmons et al . , 2016; Tsujimura et al . , 2015 ) .", "We found that the majority of PCHi-C interactions occurred within TADs ( 73 and 77% in iPSCs and CMs , respectively; Figure 1D and Figure 1—figure supplement 3A ) .", "TAD-crossing interactions ( ‘inter-TAD’ ) contained proportionally more promoter-promoter interactions than intra-TAD interactions , and were more likely to overlap promoter-distal CTCF sites; however , they were similarly enriched for looping to distal H3K27ac sites , a mark of active chromatin ( Figure 1—figure supplement 3B–D ) .", "Inter-TAD interactions had slightly lower CHiCAGO scores , reflecting a lower number of reads supporting these interactions , and spanned greater genomic distances than intra-TAD interactions ( Figure 1—figure supplement 3E , F ) .", "Additionally , promoters with inter-TAD interactions were preferentially located close to TAD boundaries ( Figure 1—figure supplement 3G ) and had higher expression levels compared to promoters with intra-TAD interactions , particularly in CMs ( Figure 1—figure supplement 3H ) .", "These observations are consistent with previous studies which demonstrated that highly expressed genes , specifically housekeeping genes , are enriched at TAD boundaries ( Dixon et al . , 2012 ) .", "To illustrate the utility of high-resolution PCHi-C interaction maps , we highlight the GATA4 locus in Figure 1D and E . GATA4 is a master regulator of heart development ( Watt et al . , 2004; Pikkarainen et al . , 2004 ) and the GATA4 gene is located in a TAD structure that is relatively stable between iPSCs and CMs ( Figure 1D ) .", "However , PCHi-C identified increased interaction frequencies between the GATA4 promoter and several H3K27ac-marked regions , including four in vivo validated heart enhancers from the Vista enhancer browser ( Visel et al . , 2007 ) , specifically in CMs and coincident with strong up-regulation of GATA4 ( Figure 1—figure supplement 2C ) .", "Although TAD-based analyses help define a gene’s cis-regulatory landscape , high-resolution promoter interaction data provides the resolution necessary to precisely map enhancer-promoter interactions in the context of cellular differentiation .", "To validate the CM interaction map as a resource for cardiovascular disease genetics we next extensively characterized several important aspects of genetic architecture in CMs .", "We compared CMs with iPSCs in each analysis as a measure of cell-type specificity .", "These analyses serve as benchmarks that build on established features of genome organization and aid interpretations of the roles that long range interactions play in gene regulation .", "Distal enhancers activate target genes through DNA looping , a mechanism that enables distally bound transcription factors to contact the transcription machinery of target promoters ( Pennacchio et al . , 2013; Miele and Dekker , 2008; Deng et al . , 2012 ) .", "To assess whether this feature of gene regulation was reflected in the iPSC and CM interactions , we conducted motif analysis using HOMER ( Heinz et al . , 2010 ) on the set of promoter-distal interacting sequences in each cell type .", "We initially focused on interactions for genes differentially expressed between iPSCs and CMs ( fold-change >1 . 5 , Padj <0 . 05 ) .", "We identified CTCF as the most enriched motif in each case ( Figure 2A , B ) , consistent with the known role of this factor in mediating long-range genomic interactions ( Phillips and Corces , 2009; Phillips-Cremins et al . , 2013; Nora et al . , 2017 ) .", "Among the other top motifs , we identified the pluripotency factor motifs OCT4-SOX2-TCF-NANOG ( OSN ) and SOX2 as preferentially enriched in distal sequences looping to genes over-expressed in iPSCs ( Figure 2A , C ) , whereas top motifs in distal sequences looping to genes over-expressed in CMs included TBX20 , ESRRB and MEIS1 ( Figure 2B , C ) .", "TBX20 and MEIS1 transcription factors are important regulators of heart development and function ( Cai et al . , 2005; Sakabe et al . , 2012; Mahmoud et al . , 2013 ) and ESRRB was previously identified as a potential binding partner of TBX20 in adult mouse cardiomyocytes ( Shen et al . , 2011 ) .", "We also observed that distal interactions unique to either iPSCs or CMs were similarly enriched for tissue-specific transcription factor motifs ( Figure 2D ) .", "In line with a recent report that AP-1 contributes to dynamic loop formation during macrophage development ( Phanstiel et al . , 2017 ) , both iPSC- and CM-specific interactions were enriched for AP-1 motifs ( Figure 2D ) , suggesting that AP-1 transcription factors may represent a previously unrecognized genome organizing complex .", "Functionally active cis-regulatory elements are characterized by the presence of specific histone modifications; active enhancers are generally associated with H3K4me1 and H3K27ac ( Creyghton et al . , 2010; Heintzman et al . , 2009 ) , whereas inactive ( e . g . poised or silenced ) elements are often associated with H3K27me3 ( Rada-Iglesias et al . , 2011; Erceg et al . , 2017 ) .", "In support of the gene-regulatory function of long-range interactions , we found that the promoter-distal MboI fragments involved in significant promoter interactions were enriched for these three histone modifications in both iPSCs and CMs ( Figure 3A–C ) .", "When promoters were grouped by expression level , we observed that this enrichment increased with increasing expression for H3K27ac and H3K4me1 , and decreased with increasing expression for H3K27me3 , consistent with an additive nature of enhancer-promoter interactions ( Schoenfelder et al . , 2015; Javierre et al . , 2016 ) , and validating that PCHi-C enriches for likely functional long-range chromatin contacts .", "A strong correlation ( Pearson correlation coefficient r > 0 . 7 ) between the degree of histone modifications and gene expression was first reported nearly 10 years ago ( Karlić et al . , 2010 ) ; however , that analysis only considered histone modifications within 2 kb of promoters .", "To understand whether this relationship extends beyond promoter-proximal regions , we correlated the number of histone ChIP-seq peaks within 300 kb of promoters with the promoter’s expression level ( Figure 3—figure supplement 1A , B ) .", "H3K27ac and H3K4me1 both positively correlated with expression level ( Spearman’s ρ = 0 . 22 and 0 . 16 , respectively in iPSC and ρ = 0 . 23 and 0 . 24 , respectively in CMs , p<2 . 2−16 ) ; in contrast , H3K27me3 negatively correlated with expression level in CMs ( Spearman’s ρ = −0 . 20 , p<2 . 2−16 ) ; however , this relationship was not present in iPSCs ( Spearman’s ρ = 0 . 02 , p=0 . 06 ) .", "Although moderate , these correlations could partially explain why higher expressed genes show stronger enrichment for promoter interactions overlapping histone peaks when using a genome-wide background model ( see Materials and methods ) , and lends support to the notion that active genes are located in generally active genomic environments ( Stevens et al . , 2017; Gilbert et al . , 2004 ) .", "We next investigated the relationship between cell-type-specific interactions and enrichment for tissue-specific CTCF , H3K27ac , and H3K27me3 marks , hypothesizing that interactions unique to iPSCs or CMs would be most enriched for tissue-specific chromatin features .", "Indeed , we observed that cell-type-specific interactions preferentially involved H3K27ac peaks from the matched cell type , and were either not enriched ( iPSC ) or depleted ( CM ) for H3K27ac marks that were specific to the non-matched cell type ( Figure 3E , middle panel ) .", "However , the strongest enrichment was for cell-type-specific interactions to overlap chromatin features that were present in both cell types ( Figure 3E ) .", "Additionally , interactions that were shared between iPSCs and CMs were most enriched for shared chromatin features .", "These results suggest that all interactions , whether shared or unique to one cell type , preferentially contact regulatory regions that are active in both cell types , whereas cell-type-specific interactions are not likely to occur in regions specifically marked in the non-matched cell type .", "An example of a gene that encompasses these observations is the atrial natriuretic peptide gene NPPA ( Figure 3F ) which is specifically expressed in cells of the heart atrium and is upregulated in CMs ( Figure 1—figure supplement 2C ) .", "NPPA makes numerous cell-type-specific interactions to a distal region that is only marked with active chromatin ( H3K27ac and H3K4me1 ) in CMs; furthermore , functional characterization showed that this region corresponds to an in vivo enhancer recapitulating NPPA’s endogenous expression in the developing heart ( Visel et al . , 2007 ) .", "Taken together , these results illuminate the complex relationship between long-range promoter interactions and gene regulation and provide evidence that promoter architecture reflects cell-type-specific gene expression .", "As a final benchmark of our datasets , we analyzed large-scale differences in genome organization between iPSCs and CMs .", "The first Hi-C studies revealed that the genome is organized in two major compartments , A and B , that correspond to open and closed regions of chromosomes , respectively ( Lieberman-Aiden et al . , 2009; Rao et al . , 2014 ) .", "Although most compartments are stable across different cell types , some compartments switch states in a cell-type-specific manner which may reflect important gene regulatory changes ( Dixon et al . , 2015 ) .", "To assess whether capture Hi-C data , which is more cost-effective for capturing promoter-centered interactions , is able to identify A/B compartments , we compared our capture Hi-C data with pre-capture , genome-wide Hi-C libraries .", "A/B compartments identified using HOMER ( Heinz et al . , 2010 ) were remarkably similar in the whole-genome and PCHi-C datasets ( 97% correspondence , Figure 4A , top panel , and Figure 4—figure supplements 1 and 2 ) , demonstrating that PCHi-C data contains sufficient information to identify broadly active and inactive regions of the genome .", "As an example , we highlight a 10 Mb region on chromosome 4 containing the CAMK2D gene locus ( Figure 4A ) .", "Compartments were relatively stable across this region in iPSCs and CMs; however , the CAMK2D gene itself was located in a dynamic compartment that switched from inactive in iPSCs to active in CMs .", "Correspondingly , this gene was highly upregulated during differentiation to CMs ( Figure 4A , inset ) .", "We observed this effect on a global level , as genes located in A compartments were expressed at significantly higher levels than genes located in the B compartments in both iPSCs and CMs ( Figure 4B ) .", "Additionally , genes that switched A/B compartments between cell types were correspondingly up- or down-regulated ( Figure 4C ) .", "GO analysis of the 1008 genes that switched from B to A compartments during iPSC-CM differentiation revealed enrichment for terms such as ‘cardiovascular system development’ and ‘heart contraction’ ( Figure 4D , Supplementary file 5 ) .", "Importantly , these genes were identified based solely on their location in a dynamic genomic compartment and not from gene expression data .", "GO analysis for genes that switched from A to B compartments during iPSC-CM differentiation related to non-cardiac processes , such as skin development , epithelial cell differentiation and sex determination ( Figure 4—figure supplement 3 , Supplementary file 5 and 6 ) .", "These data show that PCHi-C accurately captured tissue-specific interactions and indicate that compartmentalization of genes in spatially regulated regions of the nucleus may be one mechanism to ensure tissue-specific gene expression ( Dixon et al . , 2015 ) .", "In summary , our analyses demonstrated that CM promoter interactions recapitulate key features of cardiac gene regulation and function , validating the CM map as an important tool to investigate CVD genetics .", "A particularly relevant application of high-resolution promoter interaction maps is to guide post-GWAS studies by identifying the target genes of disease-associated variants .", "We employed this approach to link GWAS SNPs for several major cardiovascular diseases to their target gene ( s ) using the CM interaction map .", "We compiled 524 lead SNPs from the NHGRI database ( https://www . ebi . ac . uk/gwas/ ) for three important classes of CVDs: cardiac arrhythmias , heart failure , and myocardial infarction ( Table 1 , Supplementary files 7 and 8 ) .", "Because of linkage disequilibrium ( LD ) patterns , the true causal SNP could be any SNP in high LD with the lead variant .", "Therefore , we expanded this set of SNPs to include all variants in high LD ( r2 >0 . 9 , within 50 kb of lead SNP ) , increasing the number of putatively causal variants to 10 , 475 ( hereafter called LD SNPs ) .", "We found that 1999 ( 19% ) of the LD SNPs were located in promoter-distal MboI fragments that interacted with the promoters of 347 genes in CMs ( Supplementary file 8 ) , hereafter referred to as target genes .", "The majority ( 89% ) of LD SNP-target gene pairs were located within the same TAD , with a median distance of 185 kb between each SNP-target gene pair ( Figure 5A ) .", "Importantly , 90 . 4% of SNP-target gene interactions skipped at least one gene promoter and 42% of SNPs interacted with at least two different promoters ( Figure 5B ) .", "To confirm that the CM PCHi-C interactions linked SNPs to CVD-relevant target genes , we performed GO analysis and found that target genes were highly and specifically enriched for biological processes related to cardiac function , such as membrane repolarization and cardiac conduction ( Figure 5C , left panel and Supplementary file 5 and 6 ) .", "As a control , we used iPSC interactions to link the same SNPs to target genes and observed a completely different set of unrelated biological processes for these genes ( Figure 5C , right panel ) .", "To further characterize the biological relevance of target genes , we mined mouse knock-out data from the Mouse Genome Informatics ( MGI ) database ( Blake et al . , 2017 ) , which revealed that a statistically significant number of target genes resulted in a cardiovascular phenotype when knocked-out in the mouse ( 78 genes ( 22 . 4% ) , p=1 × 10−5 , Figure 5D ) .", "Finally , we examined expression quantitative trait loci ( eQTL ) data from human left ventricle ( LV ) tissue obtained as part of the Genotype-Tissue Expresion ( GTEx ) Project ( Carithers et al . , 2015 ) and found that of the 1999 LD SNPs in interactions , 410 ( 20 . 5% ) corresponded to LV eQTLs; in comparison , only 12 . 2% of the full set of LD SNPs corresponded to LV eQTLs ( p<0 . 00001 , Figure 5E ) .", "We next assessed whether eQTLs loop to their associated gene .", "For this analysis , we considered the full set of LV eQTLs , as the 410 LD SNP eQTLs represent too small of a proportion of the full set ( <0 . 1% of all LV eQTLs ) to fully ascertain significance .", "On a genome-wide level , LV eQTLs in promoter-distal interactions were significantly more likely to loop to their associated gene than expected by chance ( p<0 . 00001 , Figure 5F , left panel ) .", "Importantly , this significance decreased when LV eQTLs were analyzed with iPSC promoter interactions ( p=0 . 035 , Figure 5F , right panel ) .", "Taken together , these results indicate that CM promoter interactions identify a subset of disease-relevant SNPs most likely to be functional and support the use of the CM map to assign distal CVD-associated SNPs to putative target genes .", "Based on an enrichment of target genes with known cardiac function , we next assessed whether expression level is an informative metric to further prioritize functional follow-up studies .", "We examined the expression level of the 347 target genes and found that they were moderately over-expressed in CMs compared to iPSCs ( median log2 fold change = 1 . 08 , mean log2 fold change = 1 . 44 , mean TPM values were 40 . 6 in iPSCs and 60 . 1 in CMs , p=0 . 12 , Figure 6A and B ) .", "Although not significant , this result reflects the enrichment of known cardiac-related genes that interact with CVD loci .", "However , because a subset of target genes was over-expressed in iPSCs relative to CMs ( Figure 6C ) , we predicted that gene expression level alone may be an insufficient metric to gauge the relevance of target genes to CVD biology .", "Indeed , we found that 21 of the 78 target genes ( 27% ) that cause cardiovascular phenotypes when knocked-out in mice were overexpressed in iPSCs compared to CMs ( Supplementary file 8 ) .", "This result indicates that putatively causal genes may not appear as obvious candidates based solely on gene expression data .", "To illustrate this point , we highlight two genes: TBX5 , a gene directly linked to cardiac arrhythmia ( Figure 6D ) ( Smemo et al . , 2012; Arnolds et al . , 2012 ) , and LITAF , a gene that , until recently , had no obvious role in cardiac biology ( Moshal et al . , 2017 ) ( Figure 6E ) .", "Both genes formed long-range interactions to LD SNPs identified in arrhythmia GWAS , making both genes candidate functional targets of the GWAS associations .", "TBX5 , which is over-expressed in CMs ( Figure 6C ) , is the most likely target gene of the LD SNPs nearby based on the interaction data but also because of its known role in directing proper development of the cardiac conduction system .", "LITAF , on the other hand , was over-expressed in iPSCs compared to CMs ( Figure 6C ) and was not known to contribute to cardiac function until a recent study identified this gene as a regulator of cardiac excitation in zebrafish hearts ( Moshal et al . , 2017 ) .", "Because the three disease classes that we analyzed represent diverse pathologies , we predicted that the target genes identified for each class individually may relate to different biological processes .", "Specifically , we considered that cardiac arrhythmias – which directly result from defects in cardiomyocytes specialized for electrical conduction – may uncover the most cardiac-relevant target genes compared to heart failure and myocardial infarction , two CVDs that also involve non-cardiac systems .", "When broken down into the respective disease classes , we confirmed that the majority of the GO enrichment for cardiac terms was driven by the cardiac arrhythmia SNPs ( Figure 7A ) , with terms directly related to the cardiac conduction system .", "Myocardial infarction ( Figure 7B ) and heart failure ( Figure 7C ) analyses uncovered a set of genes that were slightly enriched for regulation of growth and morphogenesis , respectively .", "Despite these seemingly non-specific processes , each set of target genes contained important disease-relevant candidates .", "For example , one of the strongest associations for myocardial infarction lies in-between the CELSR2 and PSRC1 genes on chromosome 1p13 , but a careful screen of genes whose expression was affected by the risk allele implicated the more distal SORT1 gene ( Musunuru et al . , 2010 ) .", "SORT1 encodes a sorting receptor that is expressed in many tissues and has been shown to act in the liver to regulate cholesterol levels ( Petersen et al . , 1997; Musunuru et al . , 2010 ) .", "Despite functioning in the liver , we identified multiple promoter interactions between SORT1 and the myocardial infarction GWAS locus in CMs ( Figure 7D ) , directly implicating SORT1 as the target gene and lending further support to experimental validation of this locus as a SORT1 enhancer ( Musunuru et al . , 2010 ) .", "Additionally , the ACTA2 gene is located 220 kb away from the heart failure GWAS locus proximal to the CH25H and LIPA genes on chromosome 10q21 ( Smith et al . , 2010 ) ( Figure 7E ) .", "ACTA2 encodes the smooth muscle cell-specific actin protein and mutations in this gene have been shown to cause coronary artery disease , among other vascular diseases ( Guo et al . , 2009 ) .", "Despite its location at a considerable distance from the GWAS association , chromatin interactions provide an important level of evidence that ACTA2 is a putative causal gene in the development of heart failure .", "Therefore , the CM interaction map is not only useful to interrogate diseases directly related to cardiomyocytes , as in the case of cardiac arrhythmias , but also aids interpretation of target genes that may act in non-cardiac tissues ." ], [ "Gene regulation by distant regulatory elements involves the bridging of linearly separated DNA sequences , for example between a promoter and its distal enhancers , through chromatin looping mechanisms ( Spitz and Furlong , 2012 ) .", "In support of this model , we report an enrichment of tissue-defining transcription factor motifs in the distally interacting sequences of differentially expressed promoters both for CMs and iPSCs , providing an important level of evidence to validate the functional relevance of iPSC and CM interactions .", "One explanation for this enrichment is that our interaction maps are high resolution .", "We generated Hi-C libraries with the 4 bp cutter MboI , which generates fragments with an average size of 422 bp; this increased specificity of the captured region likely leads to better resolution of the underlying enhancer sequence and , consequently , increased power to detect short transcription factor binding motifs .", "The majority of capture Hi-C studies to date have reported that gene expression level correlates with enrichment for various histone marks .", "We observed the same trend in our data , with highly expressed genes exhibiting strong enrichment for looping to distal H3K4me1 and H3K27ac-marked regions , and lowly expressed genes exhibiting strong enrichment for looping to H3K27me3-marked regions .", "These data are consistent with a model in which the number of long-range interactions to enhancers or repressors additively contributes to gene expression level ( Schoenfelder et al . , 2015; Javierre et al . , 2016 ) .", "The forces that drive increased association between promoters and distal cis-regulatory elements are not completely understood and have been topics of investigation in the genome organization and chromatin biology fields for several years ( Dekker and Mirny , 2016; Calo and Wysocka , 2013 ) .", "One possibility is that this increasing enrichment is driven by genomic compartmentalization of active and inactive chromatin .", "We showed that a gene’s expression level correlates with the number of histone ChIP-seq peaks within a large window ( 300 kb ) surrounding each promoter .", "Thus , highly expressed genes are more likely to contact active chromatin regions compared to lowly expressed genes , corresponding to the observed increasing enrichment of contacts and expression we and others have reported .", "This local increase in active or repressive chromatin may be one driving force underlying the expression level-dependent increase in association between promoters and cis-regulatory elements , akin to a phase separation-mediated model of enhancer-promoter interactions ( Hnisz et al . , 2017 ) .", "We demonstrated several ways in which promoter interaction data can be used to better understand disease genetics , specifically addressing the major requirement for a high-resolution map of the gene-regulatory network in human cardiomyocytes .", "Although iPSC-derived CMs are known to be relatively immature and do not fully reflect the diverse structural and functional aspects of adult cardiac cells ( Gherghiceanu et al . , 2011; Karakikes et al . , 2015 ) , the difficulty in obtaining pure sub-populations of primary cardiomyocytes with high integrity necessitates the use of an in vitro system .", "We showed that the CMs used in this study were highly pure and recapitulate known gene regulatory properties of primary cardiomyocytes .", "Because of this purity , we were able to integrate CVD-associated SNPs with CM promoter interactions with high confidence , assigning nearly 20% of the variants in high LD with these associations to 347 target genes .", "Supporting the physiological relevance of CMs to the cardiac conduction system , we found that target genes were most relevant for GWAS loci associated with cardiac arrhythmias , in line with previous findings in immune cells that many target gene interactions were unique to relevant immune cell subtypes ( Javierre et al . , 2016; Mumbach et al . , 2017 ) .", "Our data also revealed that even for diseases whose etiology involves cell types other than cardiomyocytes , such as myocardial infarction and heart failure , we identified interactions involving loci associated with these diseases that recapitulate the enhancer-promoter interactions in non-cardiac cell types .", "As an example , we showed that a validated myocardial infarction locus interacts with the distal SORT1 promoter in CMs even though this locus has been extensively characterized in the context of cholesterol metabolism in hepatocytes .", "Therefore , the promoter interactions we observe linking the disease locus to SORT1 may represent tissue-invariant genome architecture , likely reflecting that genome organization in general is relatively stable ( Dixon et al . , 2015; Jin et al . , 2013; Ghavi-Helm et al . , 2014 ) .", "While we advocate the use of the CM map for investigating gene regulatory mechanisms of diseases related to cardiomyocyte biology , we also emphasize that , where identified , any interaction between a promoter and a putative disease-associated genomic region serves as an important level of evidence to prioritize that gene for future follow-up studies .", "The PCHi-C technique holds great promise to identify with high resolution and throughput all gene regulatory elements in any tissue or developmental stage of interest .", "However , due to technical and biological limitations , there are important caveats to PCHi-C that should be considered when interpreting the iPSC or CM interaction data .", "The most important caveat is that there are likely to be many false negatives , or ‘missing’ interactions .", "Although the capture step greatly enriches for promoter-containing ligation fragments in a Hi-C library , the total landscape of promoter contacts in a population of cells is still under-sampled , even with a sequencing depth of ~400M reads per replicate conducted for this study .", "This is due to several factors , including the hybridization efficiency of each bait , ability to design sufficient baits per promoter , and the transient nature of many regulatory interactions .", "This latter issue is confounded by the distance-dependent effect on ligation frequency: as the distance between two fragments increases , the read-depth required to robustly identify that interaction also increases .", "The feasibility of deeper sequencing and modifications to computational pipelines will continue to improve the coverage and resolution of Hi-C data .", "Additionally , because the CHiCAGO program does not incorporate TAD boundaries into its background model , it may slightly underestimate the expected number of reads corresponding to intra-TAD interactions which could lead to potential false positives .", "However , we note that there is a strong correspondence between TADs called on pre-capture Hi-C data and PCHi-C interactions identified with CHiCAGO ( Figure 1—figure supplement 3A ) ; this suggests that accounting for TAD boundaries may only marginally improve our ability to identify significant interactions .", "A final consideration is the interpretation of interactions involving inactive genes .", "Although most regulatory elements are thought of as activating , it is possible that long-range interactions may also contribute to gene silencing; this is supported by the observation that silent genes are enriched for long-range interactions to H3K27me3 marked regions ( Figure 3D ) .", "Alternatively , silent genes may contact regulatory elements that are not active in the analyzed cell type or developmental stage; these may represent ‘pre-formed’ loops between genes and their regulatory elements as characterized in Ghavi-Helm et al . ( 2014 ) .", "Despite these limitations , the data sets we provide here represent a highly enriched set of ~350 , 000 and~400 , 000 promoter interactions in iPSC and CMs , respectively; although there are likely missing interactions , the interactions that we did identify should be considered as very high confidence , as they were independently identified in at least two biological replicates and show strong signal of enrichment for known features of genome architecture and gene regulation .", "In conclusion , the promoter interaction maps we generated in this study represent important resources for any investigation into the gene regulatory mechanisms underlying cardiovascular disease traits .", "The list of candidate regulatory variants and their target genes may serve as an entry point for several hypotheses related to CVD GWAS , and can be readily tested in experimental settings .", "To provide both the iPSC and CM maps as an accessible resource , we have hosted the full set of data presented in this study as a public track hub at the WashU EpiGenome Browser ( Zhou et al . , 2015 ) , accessible at the following link: http://epigenomegateway . wustl . edu/browser/ ?", "genome=hg19&publichub=Lindsey .", "Additionally , we provide the significant PCHi-C interaction files used in all analyses in the Supplementary Material ( Supplementary files 1 and 2 ) ; these can be applied to future multi-omics analyses of gene regulation and disease genetics ." ], [ "We used the Yoruban iPSC line 19101 , kindly provided by the laboratory of Yoav Gilad .", "This iPSC line was reprogrammed from lymphoblastoid cells as part of a previous study , where it was shown to differentiate into all three germ layers , displayed a normal karyotype , and expressed markers characteristic of pluripotency ( Banovich et al . , 2018 ) .", "iPSCs were grown in Essential 8 ( E8 ) Medium ( Thermo Fisher #A1517001 ) supplemented with 1X Penicillin-Streptomycin ( Pen/Strep , Gibco ) on Matrigel-coated tissue culture dishes ( Corning #354277 ) .", "Cells were passaged when they were ~80% confluent using enzyme-free dissociation solution ( 30 mM NaCl , 0 . 5 mM EDTA , 1X PBS minus Magnesium and Calcium ) and maintained in E8 Medium with 10 μM Y-27632 dihydrochloride ( Abcam #ab120129 ) for 24 hr .", "Medium was replaced daily .", "iPSC cultures routinely tested negative for mycoplasma contamination using the Universal Mycoplasma Detection Kit ( ATCC #30–1012K ) .", "Cardiomyocyte differentiations were based on the protocol of Burridge et al . ( 2014 ) with modifications described in Banovich et al . ( Banovich et al . , 2018 ) .", "iPSCs were expanded in 60 mm dishes in E8 media until they reached 60–70% confluency at which time the differentiation was started ( day 0 ) .", "On day 0 , E8 media was replaced with 10 mL of basic heart media/12 μM GSK-3 inhibitor CHIR-99021 trihydrochloride ( Tocris #4953 ) /Matrigel overlay [basic heart media: RPMI 1640 minus L-glutamine ( HyClone #SH30096 . 01 ) with 1X GlutaMax ( Life Technologies #11879020 ) supplemented with 1X B27 minus insulin ( Thermo Fisher #A1895601 ) and 1X Pen/Strep; Matrigel overlay was accomplished by dissolving Matrigel in 50 mL basic heart media at a concentration of 0 . 5X according to the lot-specific dilution factor] .", "After 24 hr ( day 1 ) , the GSK-3 inhibitor was removed by replacing media with 10 mL basic heart media .", "On day 3 , media was replaced with 10 mL basic heart media supplemented with 2 μM Wnt-C59 ( Tocris #5148 ) .", "On day 5 ( 48 hours later ) , media was replaced with 10 mL basic heart media .", "On day 7 , cells were washed once with 1X PBS and then 15 mL basic heart media was added .", "Media was replaced every other day in this way until day 15 at which time cardiomyocytes were selected for by replacing basic heart media with 10 mL lactate media ( RPMI 1640 minus D-glucose , plus L-glutamine ( Life Technologies #11879020 ) , supplemented with 0 . 5 mg/mL recombinant human albumin ( Sigma 70024-90-7 ) , 5 mM sodium DL-lactate ( Sigma 72-17-3 ) , 213 μg/mL L-ascorbic acid 2-phosphate ( Sigma 70024-90-7 ) and 1X Pen/Strep ) .", "Lactate media was replaced every other day until day 20 at which point cardiomyocytes were harvested .", "Cells from successful differentiations exhibited spontaneous beating around days 7–10 .", "Cardiomyocytes were harvested by washing once with 1X PBS followed by incubation in 4 mL TrypLE ( Life Technologies 12604–021 ) at 37°C for 5 min .", "After incubation , 4 mL lactate media was added to the TrypLE and a 1 mL pipet was used to dislodge cells .", "Cells were strained once with a 100 μM strainer and then once with a 40 μM strainer .", "Cells were pelleted at 500xg and then resuspended in PBS and counted .", "For each batch of differentiation , 5 million cells were taken for promoter-capture Hi-C and 1 million cells were taken for RNA-seq .", "To assess purity , 2 million cells were taken for flow cytometry analysis using an antibody for cardiac Troponin T ( BD Biosciences 564767 ) .", "All cells used in downstream experiments were at least 86% Troponin T positive ( Figure 1—figure supplement 1A ) .", "We carried out three independent differentiations of the same iPSC line and generated promoter-capture Hi-C and RNA-seq libraries in iPSCs and CMs from each triplicate .", "We used HiCUP v0 . 5 . 9 ( Wingett et al . , 2015 ) to align and filter Hi-C reads ( total and filtered read counts are presented in Supplementary file 9 . 2 ) .", "Unique reads were given to CHiCAGO version 1 . 2 . 0 ( Cairns et al . , 2016 ) and significant interactions were called with default parameters .", "In this study , we focused exclusively on cis-interactions as the evidence that trans-chromosomal interactions contribute to gene expression regulation is limited .", "CHiCAGO reports interactions for each captured restriction fragment; to summarize interactions by gene , we considered the interval spanning all captured fragments ( i . e . the set of probes spanning each TSS ) as the promoter region ( ‘merged TSS’ ) .", "This means the promoter regions created have variable lengths .", "In cases where multiple genes were annotated to the same promoter region , we report the interaction for each gene individually .", "This annotation allowed us to perform gene-level analyses , for example based on expression level .", "We removed this redundancy as necessary , for example in motif enrichment analyses of the promoter-interacting fragments .", "Using the ‘merged TSS’ interaction files , we filtered interactions to retain those that mapped within 1 kb of each other in at least two replicates .", "Specifically , we extended each promoter-interacting fragment by 1 kb on each end and then used BEDTools ( Quinlan and Hall , 2010 ) pairToPair functionality to identify interactions where both ends matched across replicates .", "To identify cell type-specific interactions , we required that the interaction ( with the 1 kb extension ) was not present in any of the three replicates of the other cell type .", "The number of read-pairs per promoter and the corresponding number of significant interactions identified is presented in Supplementary file 9 . 3 .", "The TAD analyses , motif enrichment , ChIP-seq peak enrichment , and eQTL analyses ( related to Figures 1 , 2 , 3 and 5 ) were conducted with fragment-level interactions ( no 1 kb extension ) .", "The GWAS SNP analyses were conducted with 1kb-extended interactions , as we aimed to be as inclusive as possible when linking CVD SNPs to target genes .", "PCHi-C interactions , TADs , RNA-seq , publicly available ChIP-seq , and GWAS SNPs are hosted by the WashU EpiGenome Browser ( Zhou et al . , 2015 ) as a public track hub .", "This can be accessed by going to http://epigenomegateway . wustl . edu/browser/ .", "The public hub ( ‘A promoter interaction map for cardiovascular disease genetics’ ) can be found under the Human Hg19 browser .", "To generate the by-gene read counts displayed in the genome-browser figures , all read-pairs mapping to captured MboI fragments for a given promoter were summed across replicates .", "Specifically , we summed reads for each MboI fragment where the read was part of a paired-read that mapped to a bait for the given gene .", "The arcs that are displayed underneath the 4C-style plot represent significant interactions that were identified in at least two replicates as detailed above in ‘Interaction calling’ .", "To identify TADs , we pooled reads across replicates for each cell type using the pre-capture Hi-C data ( 600M reads for iPSC and 733M reads for CM ) and used HiCUP v0 . 5 . 9 ( Wingett et al . , 2015 ) to align and filter Hi-C reads .", "HOMER v4 . 8 . 3 ( Heinz et al . , 2010 ) was used to generate normalized interaction matrices at a resolution of 40 kb and then TopDom v0 . 0 . 2 ( Shin et al . , 2016 ) was used with a window size w = 10 to identify topological domains , boundaries and gaps .", "We only considered domains for the analyses in this paper .", "We considered a promoter capture Hi-C interaction to be ‘intra-TAD’ if the entire span of the interaction was fully contained in a single domain .", "‘Inter-TAD’ interactions are defined as interactions where each end maps to a different domain .", "The program runHiCpca . pl from the HOMER ( Heinz et al . , 2010 ) v4 . 8 . 3 package was used to call A/B compartments with -res 50000 for both whole-genome and capture Hi-C data .", "Total RNA was extracted from flash-frozen pellets of 1 million cells using TRI Reagent ( Sigma #T9424 ) and a homogenizer followed by RNA isolation and clean-up using the Direct-zol RNA Kit ( Zymo Research #11–331 ) .", "RNA-seq libraries were generated with the Illumina TruSeq V2 kit ( Illumina , RS-122–2001 ) and 1 μg of RNA , following manufacturer’s instructions .", "Libraries were made from RNA isolated from three independent iPSC-CM differentiations ( triplicates of iPSC and of cardiomyocytes ) .", "Libraries were sequenced on an Illumina HiSeq 4000 .", "Gene counts were quantified with Salmon 0 . 7 . 2 ( Patro et al . , 2017 ) and imported with tximport 1 . 2 . 0 ( Soneson et al . , 2015 ) into DESeq2 1 . 12 . 4 ( Love et al . , 2014 ) to call differentially expressed genes .", "A minimum 1 . 5-fold-difference between CMs and iPSC triplicates and a minimum adjusted p-value of 0 . 05 were required to select differentially expressed genes for downstream analyses .", "TPMs ( transcripts per million ) were also estimated by Salmon .", "Because the samples clearly clustered according to their known tissues of origin ( Figure 1—figure supplement 2A ) , no correction for batch effects was performed .", "We performed ChIP-seq on 2 . 5 million cells each for iPSCs and CMs using H3K27ac antibodies ( Wako #306–34849 ) .", "Briefly , cells were crosslinked with 1% formaldehyde for 10 min at room temperature , quenched with 0 . 2M glycine for 5 min , pelleted and snap-frozen in liquid nitrogen .", "Cells were lysed in Lysis Buffer 1 ( 50 mM HEPES-KOH , pH 7 . 5 , 140 mM NaCl , 1 mM EDTA , 10% glycerol , 0 . 5% NP-40 , 0 . 25% Triton X-100 ) .", "Crosslinked chromatin was sheared to an average size of 300 bp using a Bioruptor with 30\" on/30\" off at high setting and then incubated overnight at 4°C with 1 μg antibody .", "Dynabeads M-280 Sheep Anti-Mouse IgG ( ThermoFisher #11201D ) were used to pull down chromatin and ChIP DNA was eluted and prepared for sequencing using the NEBNext Ultra II DNA Library prep kit ( NEB #E7645S ) .", "ChIP-seq reads were aligned with Bowtie 2–2 . 2 . 3 ( Langmead and Salzberg , 2012 ) and peaks were called with HOMER ( Heinz et al . , 2010 ) v4 . 8 . 3 on unique reads with mapping quality >10 using the –region and –style histone parameters .", "Significant peaks were overlapped with H3K27ac peaks from Epigenome Roadmap samples which demonstrated high concordance between matched tissue types ( Figure 1—figure supplement 2C , D ) .", "Because we performed a low level of sequencing , we did not identify as many peaks as the Roadmap samples .", "Therefore , we used Roadmap ChIP-seq data in all of our analyses .", "The human Gene Ontology ( GO ) associations of GO terms ( Ashburner et al . , 2000 ) to genes and the GO database were downloaded on January 22 , 2016 from http://geneontology . org/gene-associations .", "GO terms were associated with RefSeq genes via gene symbols .", "Using the GO annotation graph , all parent terms were assigned to the terms annotated to a gene .", "A hypergeometric test was used to calculate the statistical significance of the difference of the number of genes associated with a given GO term in a particular gene set and the universe of all RefSeq genes ( p<0 . 05 ) .", "p-Values were corrected with the R package p . adjust function using the ‘fdr’ method .", "For two of the GWAS disease groups ( heart failure and myocardial infarction ) , the list of genes looping to LD SNPs included many histone genes .", "This is because there is a tag SNP located in the middle of a histone gene cluster ( containing >30 histone genes located close together ) in each case .", "After expanding the tag SNP to all SNPs in LD , many of the histone genes in that cluster looped to the LD SNPs , resulting in a high representation of these genes in the final gene list .", "The resulting Gene Ontology enrichment analysis gave terms relating to nucleosome and chromatin organization because of this over-representation .", "We therefore chose to remove these genes from the final gene lists of heart failure and myocardial infarction target genes .", "The program findMotifsGenome . pl from the HOMER ( Heinz et al . , 2010 ) v4 . 8 . 3 package was used with –size given parameter to identify overrepresented motifs in the distal ( non-promoter ) interacting sequences of promoter interactions .", "As stated above , this analysis was performed on fragment-level promoter-interacting sequences .", "We obtained publicly available ChIP-seq data in the form of processed peak calls for H3K27ac , H3K4me1 and H3K27me3 from the Roadmap Epigenomics Project ( Kundaje et al . , 2015 ) , and for CTCF from ENCODE ( ENCODE Project Consortium , 2012 ) ( Supplementary file 10 ) .", "We only considered peaks that mapped outside of the captured region of promoters to ensure our results were not driven by the strong peak signal over most promoters .", "As a proxy for iPSCs , we used data from the H1 embryonic stem cell line and for CMs we used data from Left Ventricle tissue .", "We grouped genes into five expression categories based on the average TPM values: group 1 ( 0 TPM ) , group 2 ( TPM 0–3 ) , group 3 ( TPM 3–25 ) , group 4 ( TPM 25–150 ) and group 5 ( TPM >150 ) and for each group of genes , we calculated the enrichment for promoter interactions to overlap a given feature .", "To calculate enrichment of interactions overlapping an epigenetic feature , we compared the observed proportion of MboI fragments in significant interactions overlapping a feature to the proportion of random MboI fragments overlapping the feature .", "Specifically , we randomly selected MboI fragments from a set that excluded fragments mapping within captured regions ( promoters ) or within unmappable genomic regions ( gaps ) .", "The number of randomly selected fragments matched the number of interacting fragments considered for the analysis .", "We performed 100 iterations of overlapping random fragments with a feature and report the average fold-enrichment .", "We refer to this method of enrichment as a ‘genome-wide’ background model because for each gene expression group , the observed proportion of fragments containing a peak is compared to randomly selected fragments from the whole genome .", "To calculate the correlation between expression and histone ChIP-seq peak density , we calculated the Spearman’s rank correlation between the expression value for each gene ( the average TPM value ) and the number of peaks mapping within 300 kb of each gene TSS .", "We only considered genes with at least one significant interaction in the respective cell type to allow for generalizations to the enrichment analysis presented in Figure 3 .", "We compiled genome-wide significant SNPs associated with GWAS for cardiac arrhythmia , heart failure , and myocardial infarction from the NHGRI-EBI database ( http://www . ebi . ac . uk/gwas/ ) ; see Supplementary file 7 for list of terms used to identify specific GWAS .", "We expanded each set of SNPs to all SNPs in high LD ( r2 >0 . 9 ) using phase 3 data of the 1000 genomes project ( Nikpay et al . , 2015 ) ( Supplementary file 3 ) .", "For each lead SNP from the GWAS we analyzed , we selected a 100 kb interval centered on the SNP ( SNP ± 50 kb ) .", "For each 100 kb interval , Tabix ( Li , 2011 ) was used to retrieve genotypes .", "We then used PLINK ( Purcell et al . , 2007 ) v1 . 90p on phase three data from the 1000 genomes project ( Nikpay et al . , 2015 ) ( ftp . 1000genomes . ebi . ac . uk/vol1/ftp/release/20130502 , v5a ) to select SNPs in LD ( r2 >0 . 9 ) with the tag SNP and a minimum allele frequency of 0 . 01 .", "We only included the populations primarily studied in the GWASs: CEU ( central European ) , ASW ( African American ) and JPT ( Japanese ) .", "We assigned all SNPs in promoter-distal interactions to their interacting gene ( s ) ( ‘target genes’ ) using cardiomyocyte promoter capture Hi-C data .", "We did not require the SNP to map to regions associated with open chromatin or enhancer marks as these types of data are highly cell-type specific and we did not wish to exclude SNPs in regions that may be active in non-assayed cell types .", "We note that one major GWAS for dilated cardiomyopathy was not included in the NHGRI-EBI database ( Meder et al . , 2014 ) , likely because there is an error obtaining the online methods of the paper .", "After careful inspection of the study , we concluded that the GWAS met the NHGRI-EBI criteria and included the associations from that study in our analysis .", "A complete list of all studies used in this analysis can be found in Supplementary file 8 .", "To calculate enrichment of target genes to cause cardiovascular phenotypes when deleted in mice ( Mouse Genome Informatics database ) , we randomly selected 347 genes from the list of starting genes ( i . e . genes with at least one promoter-distal interaction in CMs , meaning it could be a target gene ) , and calculated the proportion that caused a cardiovascular phenotype in mice .", "We performed this randomized selection for 1000 iterations to generate the randomized ( expected ) values .", "Random genes were not required to be expressed , as the set of target genes contains genes that are not expressed .", "p-Value was calculated with a Z test .", "For eQTLs used in comparisons with GWAS variants and Hi-C interactions , we used the set of GTEx v7 eQTLs identified as significant in the left ventricle of the heart ( Carithers et al . , 2015 ) .", "eQTLs were called significant if q < 0 . 05 after false discovery rate correction ( Storey and Tibshirani , 2003 ) .", "We only considered promoter-distal eQTLs that were at least 10 kb from their associated gene to allow for that eQTL to map to an interaction with it’s associated gene .", "To calculate enrichment for eQTLs to loop to their associated gene , we used a background model whereby each promoter’s set of interactions were re-mapped to a different promoter , keeping the distance and strand orientation consistent .", "We performed this re-mapping of all promoter interactions 1000 times and calculated the proportion of all eQTLs that mapped to interactions for their eQTL-associated gene in each permutation .", "We either used the CM interactions or the iPSC interactions with the same set of left ventricle eQTLs to compare cell-type specificity of the promoter interaction data .", "Raw and processed sequencing data are provided at ArrayExpress through accession numbers E-MTAB-6014 ( Hi-C ) and E-MTAB-6013 ( RNA-seq ) ." ] ]

[ "Over 500 genetic loci have been associated with risk of cardiovascular diseases ( CVDs ) ; however , most loci are located in gene-distal non-coding regions and their target genes are not known .", "Here , we generated high-resolution promoter capture Hi-C ( PCHi-C ) maps in human induced pluripotent stem cells ( iPSCs ) and iPSC-derived cardiomyocytes ( CMs ) to provide a resource for identifying and prioritizing the functional targets of CVD associations .", "We validate these maps by demonstrating that promoters preferentially contact distal sequences enriched for tissue-specific transcription factor motifs and are enriched for chromatin marks that correlate with dynamic changes in gene expression .", "Using the CM PCHi-C map , we linked 1999 CVD-associated SNPs to 347 target genes .", "Remarkably , more than 90% of SNP-target gene interactions did not involve the nearest gene , while 40% of SNPs interacted with at least two genes , demonstrating the importance of considering long-range chromatin interactions when interpreting functional targets of disease loci ." ]

[ "Our genomes contain around 20 , 000 different genes that code for instructions to create proteins and other important molecules .", "When changes , or mutations , occur within these genes , malfunctioning proteins that are damaging to the cell may be produced .", "Researchers of human genetics have tried to spot the genetic mutations that are associated with illnesses , for example heart diseases .", "However , they found that most of these mutations are actually located outside of genes , in the ‘non-coding’ areas that make up the majority of our genome .", "These mutations do not modify proteins directly , which makes it challenging to understand how they may be related to heart conditions .", "One possibility is that the genetic changes affect regions called enhancers , which control where , when and how much a gene is turned on by physically interacting with it .", "Mutations in enhancers could lead to a gene producing too much or too little of a protein , which might create problems in the cell .", "Yet , it is difficult to match an enhancer with the gene or genes it controls .", "One reason is that a non-coding region can influence a gene placed far away on the DNA strand .", "Indeed , the long DNA molecule precisely folds in on itself to fit inside its compartment in the cell , which can bring together distant sequences .", "Montefiori et al . take over 500 non-coding areas , which can carry mutations associated with heart diseases , and use a technique called Hi-C to try to identify which genes these regions may control .", "The tool can model the 3D organization of the genome , and it was further modified to capture only the regions of the genome that contain genes , and the DNA sequences that interact with them , in human heart cells .", "This helped to create a 3D map of 347 genes which come in contact with the non-coding areas that carry mutations associated with heart diseases .", "In fact , deleting those genes often causes heart disorders in mice .", "In addition , Montefiori et al . reveal that 90% of the non-coding regions examined were influencing genes that were far away .", "This shows that , despite a common assumption , enhancers often do not regulate the coding sequences they are nearest to on the DNA strand .", "Pinpointing the genes regulated by the non-coding regions involved in cardiovascular diseases could lead to new ways of treating or preventing these conditions .", "The 3D map created by Montefiori et al . may also help to visualize how the genetic information is organized in heart cells .", "This will contribute to the current effort to understand the role of the 3D structure of the genome , especially in different cell types ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "ecology" ]

[ [ "Mimicry complexes ( i . e . a composite of several Müllerian and Batesian mimics ) have long fascinated biologists for the theoretical and empirical challenges they pose .", "Classic Müllerian mimicry was thought to only include equally defended species ( Müller , 1878 ) , while Batesian mimicry ( Bates , 1862 ) describes undefended species resembling defended models .", "In reality , this simplistic dichotomy ( sensu Speed , 1999 ) represents the end-points of a mimetic spectrum that contains species that vary in their mimetic fidelity and/or their degree of defensiveness .", "A topic of considerable debate ( Sherratt , 2008 ) is the putative influence that moderately defended mimics have on the stability of mimicry rings ( i . e . a composite of several similar Müllerian mimics ) .", "Discrepancy in protection between two mimetic species may dilute the protection of a more defended species ( Speed , 1999 ) .", "Great tits ( Parus major , Paridae , Passeriformes ) for instance , attacked artificial models more often when less defended mimics were frequently encountered and food was restricted ( Rowland et al . , 2010 ) .", "In direct contrast , associative learning mechanisms predict a mutualistic relationship between moderately and highly defended members of the ring ( Rowland et al . , 2007 ) : wild caught great tits equally avoided four species of true bugs with varying levels of defence but which shared similar warning colours ( Hodová Svádová et al . , 2013 ) .", "Müllerian mimicry rings typically consist of a few closely related species ( Symula et al . , 2001; Williams , 2007; Alexandrou et al . , 2011 ) , with the exceptions of the species-rich mutillid wasp ( Wilson et al . , 2012 , 2015 ) , Heliconius butterfly ( Mallet and Gilbert , 1995 ) and bumblebee ( Williams , 2007 ) complexes .", "These complexes , despite being speciose , are taxonomically limited because their members often belong to only a single family or genus ( Lepidoptera or Hymenoptera ) .", "The mutillid wasp complex is suspected of including species from other orders but this has not been formally presented ( Wilson et al . , 2012 , 2015 ) .", "A mathematical model suggests that mimicry complexes should be common in multispecies communities with distantly related taxa ( Beatty et al . , 2004 ) .", "The general lack of data on multi-order mimicry complexes may represent an observer bias .", "For example , ants have been largely overlooked as Müllerian mimics , yet they are diverse , typically well defended through a range of defensive measures including a sting , noxious chemicals , spines , a thickened cuticle , mandibles and communal attack ( Hölldobler and Wilson , 1990 ) .", "There is also a rich diversity of invertebrate taxa that mimic ants .", "Species within Müllerian rings thus far described use the same defensive traits .", "Heliconius butterflies , for instance , rely on their distastefulness while mutillid wasps have a venomous sting .", "The degree of distastefulness or level of venom toxicity within a mimicry ring is expected to vary among members .", "Experimental investigations , that started a hundred years ago ( e . g . McAtee , 1912 , 1932 ) , have tended to focus on a single visually-oriented predatory guild and yet most environments contain a wide range of potential predators of mimics .", "For example , species that mimic ants may encounter several ant-averse and ant-eating predators ( Pekár et al . , 2011 ) .", "In some microhabitats , such as leaf litter , the ant-eating predators ( which often use chemical cues to recognise prey ) and ant-adverse nonvisually-orienting predators are indeed more common than visually-orienting ones , thus exerting strong selection pressure on mimics to signal their unpalatability in sensory modalities other than visual .", "Thus one of the major outstanding questions regarding the contribution of mimics to the function of mimetic rings is evaluating the success of Müllerian and/or Batesian mimicry by analysing the selection pressure exerted by all predators in a given environment ( Blumstein , 2006 ) .", "Here , we describe a novel diverse and species rich mimicry complex consisting of golden ant , wasp , true bug , treehopper , and spider mimics and we investigate hypotheses concerning their unpalatability and resulting efficacy against predation .", "Mimetic complexes are often composed of rings – groups of species that draw advantages from similar aposematic signals ( Williams , 2007; Wilson et al . , 2015 ) .", "Thus , our first step was the identification of separate rings to understand how the mimicry complex evolved and is reinforced .", "We therefore tested whether the rings have a sympatric distribution , which would indicate a mutualistic relationship between mimics and whether mimics within a ring share common ancestry .", "Our second step was to quantify the relationship between the proposed aposematic signal ( golden/black colouration ) and the level of noxiousness or distastefulness of the mimics that display the signal .", "Aposematic warning signals and distastefulness are expected to evolve together within a species ( e . g . Ruxton et al . , 2004 ) and thus , the strength of the distasteful stimulus should coincide with the conspicuousness of the warning signal ( Speed and Ruxton , 2007 ) .", "In this mimicry complex , we investigated whether there is a positive relationship between signal intensity and unpalatability across species as has recently been found among lady-bird beetles ( María Arenas et al . , 2015 ) .", "This would mean that truly Müllerian mimics are expected to honestly signal their distastefulness but truly Batesian mimics display a conspicuous warning signal without the noxious stimulus .", "We tested these assumptions by correlating the size of the warning signal ( golden/back colouration ) with a measure of distastefulness derived from the sum of defences found in the mimics .", "Our critical third step was to extend the scope of previous studies of mimicry complexes by experimentally testing the efficacy of the aposematic signal , and indeed the validity of the Müllerian mimicry claim , through staged predator-prey trials .", "Based on the broad diversity of defences used by mimics in this complex , we predict that predators should learn quickly to avoid mimics ( Beatty et al . , 2004 ) , and that mimics should be protected from a range of predators .", "Finally , we examined how the aposematic signal and the level of defensiveness interplay with a community of predators that rely on multiple sensory modalities .", "Mimicry complexes are typically characterised by a distinct colour pattern and hence experiments are often biased towards visual-oriented predators and usually vertebrates .", "In reality , the members of a complex are exposed to predators that use a variety of sensory modalities and foraging modes to detect and subdue their prey .", "We combined staged predatory trials with a dietary analysis of wild populations of a community of predators to test the hypothesis that more unpalatable species are attacked less frequently by a community of predators ." ], [ "We uncovered a cross-order mimicry complex , consisting of at least 140 arthropod species including 126 ant species , seven spider species ( and several forms ) , three species of true bugs , one mutillid wasp and one treehopper ( Table 1 ) .", "Members of the complex are all ant-like in appearance and characterised by a dorsal patch of golden coloration combined with blackish colouration ( Figure 1 , Video 1 ) , which we recognise as an aposematic signal ( RGB contrast: mean = 241 . 4 , SD = 58 . 1 , N = 55 ) .", "Related non-mimetic species are typically uniformly black or brown . 10 . 7554/eLife . 22089 . 003Table 1 . List of mimic species belonging to the golden complex arranged according to the order , family ( subfamily ) , and genus ( subgenus ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 22089 . 003OrderFamily ( Subfamily ) Genus ( Subgenus ) SpeciesHymenopteraFormicidae ( Formicinae ) Camponotus aeneopilosus Mayr; aurocinctus ( Smith ) ; bigenus Santschi; ephippium ( Smith ) ; fergusoni McArthur; nigroaeneus ( Smith ) ; oxleyi Forel; piliventris ( Smith ) ; setosus Shattuck and McArthur; suffusus ( Smith ) ; tasmani Forel; thadeus Shattuck; wiederkehri Forel Polyrhachis ( Chariomyrma ) appendiculata Emery; arcuata ( Le Guillou ) ; aurea Mayr; bedoti Forel; constricta Emery; cydista Kohout; cyrus Forel; guerini Roger; heinlethii Forel; lata Emery; obtusa Emery; pallescens Mayr; schoopae Forel; senilis Forel; vermiculosa Mayr Polyrhachis ( Hagiomyrma ) ammon ( Fabricius ) ; ammonoeides Roger; anderseni Kohout; angusta Forel; archeri Kohout; aurora Kohout; brisbanensis Kohout; brutella Kohout; burwelli Kohout; callima Kohout; capeyorkensis Kohout; conciliata Kohout; cracenta Kohout; crawleyi Forel; darlingtoni Kohout; denticulata Karavaiev; diversa Kohout; dougcooki Kohout; electra Kohout; elengatula Kohout; feehani Kohout; hoffmani Kohout; melanura Kohout; nourlangie Kohout; penelope Forel; pilbara Kohout; placida Kohout; seducta Kohout; semiaurata Mayr; stricta Kohout; tanami Kohout; tenebra Kohout; thusnelda Forel; trapezoidea Mayr; tubifera Forel; unicaria Kohout; vernoni Kohout; weiri Kohout Polyrhachis ( Hedomyrma ) argentosa Forel; barretti Clark; cleopatra Forel; consimilis Smith; cupreata Emery; daemeli Mayr; erato Forel; euterpe Forel; hermione Emery; mjobergi Forel; ornata Mayr; rufifemur Forel; terpsichore Forel; thais Forel Formicidae ( Formicinae ) Polyrhachis ( Myrma ) andromache Roger; foreli Kohout; inusitata Kohout Polyrhachis ( Myrmhopla ) dispar Kohout; dives Smith; reclinata Emery; sexspinosa ( Latreille ) Polyrhachis ( Polyrhachis ) bellicosa Smith Formicidae ( Dolichoderinae ) Dolichoderus angusticornis Clark; clarki Wheeler; dentatus Forel; doriae Emery; extensispinus Forel; inferus Shattuck and Marsden; niger Crawley; rufotibialis Clark; scabridus Roger; scrobiculatus ( Mayr ) ; turneri Forel Iridomyrmex anderseni Shattuck; azureus Viehmeyer; coeruleus Heterick and Shattuck; roseatus Heterick and Shattuck; ypsilon Forel Formicidae ( Myrmeciinae ) Myrmecia athertonensis Forel; auriventris Mayr; borealis Ogata and Taylor; chrysogaster ( Clark ) ; cydista ( Clark ) ; eungellensis Ogata and Taylor; fabricii Ogata and Taylor; flavicoma Roger; fulviculis Forel; fulvipes Roger; gilberti Forel; harderi Forel; luteiforceps Wheeler; mandibularis Smith; michaelseni Forel; petiolata Emery; piliventris Smith; rugosa Wheeler; tepperi Emery; tridentata Ogata and Taylor FormicidaeDiacamma schoedli Shattuck and Barnett ( Ponerinae ) Pachycondyla sublaevis ( Emery ) MutillidaeEphutomorpha aurata ( Fabricius ) HemipteraEurymelidaeEurymela rubrolimbata Kirkaldy RhyparochromidaeDaerlac apicalis ( Distant ) ; cephalotes ( Dallas ) ; nigricans DistantAraneaeSalticidaeMyrmarachne erythrocephala forma erato ( L . Koch ) ; erythrocephala forma ornata ( L . Koch ) ; erythrocephala forma daemeli ( L . Koch ) ; luctuosa forma aeneopilosa ( L . Koch ) ; luctuosa forma aurea ( Ceccarelli ) ; macleayana forma foreli ( Bradley ) Ligonipes illustris Karsch Ohilimia scutellata ( Kritscher ) CorinnidaeNyssus luteofinis Raven GnaphosidaeEilica sp . 10 . 7554/eLife . 22089 . 004Figure 1 . Projection of the mimicry complex on a pruned phylogenetic tree . The tips of the tree represent species and full circles are coloured according to putative ring classification .", "Representatives of each ring are shown .", "Four types of defence are projected on the branches . DOI: http://dx . doi . org/10 . 7554/eLife . 22089 . 00410 . 7554/eLife . 22089 . 005Video 1 . Overview of selected mimetic species belonging to the golden complex . DOI: http://dx . doi . org/10 . 7554/eLife . 22089 . 005 The golden sheen is achieved by diverse modes .", "Among the hymenopteran representatives ( ants and wasps ) , the golden sheen is generated only by pubescence of varying density ( Figure 2 ) .", "Consequently , this visual effect entirely disappears when they are submerged in ethanol .", "In contrast , true bugs and spiders use a combination of hairs and yellow pigmentation ( in the exocuticle of the abdomen/gaster ) , while the treehopper appears to use only pigment as the golden shine is lost after death . 10 . 7554/eLife . 22089 . 006Figure 2 . Modes of production of the golden shine .", "( A ) Polyrhachis ammon , gaster , SEM .", "( B ) P . ammon , detail of hairs .", "( C ) Dolichoderus clarki , gaster , SEM .", "( D ) D . clarki , detail of hairs .", "( E ) Myrmecia tepperi , gaster , SEM .", "( F ) M . tepperi , detail of hairs .", "( G ) Camponotus aeneopilosus , gaster , SEM .", "( H ) C . aeneopilosus , detail of hairs .", "( I ) Ephutomorpha aurata , gaster , SEM .", "( J ) E . aurata , detail of hairs .", "( K ) Daerlac nigricans , gaster , SEM .", "( L ) D . nigricans , pigment .", "( M ) Myrmarachne luctuosa , abdomen , SEM .", "( N ) M . luctuosa , pigment on abdomen .", "( O ) Eurymela rubrolimbata , pigment on gaster .", "SEM ( A–K , M ) and light photography ( L , N , O ) of the dorsal side of mimics . DOI: http://dx . doi . org/10 . 7554/eLife . 22089 . 006 The cluster analysis suggested the existence of ten putative rings within the complex , according to variation in the dorsal colour pattern but the differences were rather small ( Figure 3A ) .", "These rings were distributed across the Australian continent with a considerable overlap .", "For instance , rings in the east coast of Australia overlapped where species richness was the greatest ( Figure 3B ) .", "However , the degree of sympatry in a ring was not related to colour pattern ( estimated from 24 measures of the colour pattern , body size and area of golden patch ) similarity ( Mantel test , r = −0 . 03 , p=0 . 79 ) .", "We did not find a strong effect of phylogeny on the classification into rings ( GLS , Pagel’s λ = 0 . 22 , Figure 1 ) .", "All this indicates that the splitting into ten putative rings was artificial and there is only one complex . 10 . 7554/eLife . 22089 . 007Figure 3 . Mimetic rings and their distribution .", "( A ) NMDS ordination of species classified into ten putative rings ( stress = 0 . 16 ) .", "The rings were distinguished by k-means clustering using ssi criterion ( in which the maximum value indicates the correct number of clusters ) , which is shown on the right of the plot .", "( B ) Map of distribution of ten putative rings in Australia . DOI: http://dx . doi . org/10 . 7554/eLife . 22089 . 007 We determined the unpalatability of 100 species belonging to the golden mimicry complex .", "Because several complementary forms of defence are likely , we estimated total unpalatability for each mimic based on the sum of morphological , chemical , and behavioural defensive traits .", "Our analyses uncovered a spectrum of mimics from non-defended ( Batesian ) to highly defended ( Müllerian ) species .", "For instance , the spider and treehopper species had the lowest unpalatability score ( i . e . sum of normalised measurements on defensive traits ) and were therefore classified as Batesian mimics .", "Daerlac bugs , mutillid wasps and all ant species were unpalatable , but we found considerable variation in the degree of unpalatability ranging from quasi-Müllerian to unambiguously Müllerian , mimics .", "The index of unpalatability had an overall normal distribution ( Shapiro-Wilk normality test , W = 0 . 98 , p=0 . 21 ) with the highest frequency consisting of moderately-palatable species .", "Unpalatability varied in each ring but the variation was not significantly different among the ten rings suggested previously ( Bartlett test , K26 = 11 , p=0 . 09 , Figure 4A ) supporting further the existence of only one complex .", "We discovered that the absolute area of the golden colouration increased with unpalatability ( controlling for the effect of phylogeny and body size: GLS , F1 , 91 = 21 . 1 , p<0 . 0001 , Figure 4B ) supporting the hypothesis that the golden colour is an aposematic signal . 10 . 7554/eLife . 22089 . 008Figure 4 . Effect of unpalatability .", "( A ) Histogram of unpalatability of the mimetic complex with horizontal boxplots for each ring .", "( B ) Relationship between the absolute area of dorsal golden colouration of mimics and their unpalatability .", "( C ) Relationship between unpalatability and post-attack response of skinks to the six prey species from being eaten ( 0 ) , to cleaning its mouth ( 0 . 5 ) and spitting out the prey item ( 1 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 22089 . 008 We quantified predation of mimics by the 12 most abundant predatory species that co-occurred with 13 sympatric mimetic species belonging to the complex ( Table 2 ) .", "We analysed gut content or faeces of these predators ( 553 spiders , 50 skinks , and 48 birds ) , representing three different guilds , using Next Generation Sequencing , which allows identification of prey to species .", "We found that individuals of all predatory species were positive for primers targeting the DNA of ants , spiders , and mutillid wasps .", "The frequency of captured mimics was not significantly different from their availability in the environment ( X212 = 1 . 5 , p=0 . 99 ) because mimics were very rare in the field .", "Only three mimetic species ( Camponotus aeneopilosus , Myrmarachne erythrocephala , Myrmarachne luctuosa ) were detected in the faeces/gut of predators .", "Visually-oriented and non-visually oriented euryphagous ( ant-adverse ) predators captured mimics at less than 4% frequency , whereas specialized ant-eating predators captured mostly Batesian mimics ( Table 2 ) .", "The overall attack rate estimated for all predators was marginally significantly negatively related to the unpalatability of mimics when it included two Batesian mimics ( GAM , F3 . 8 , 4 . 6 = 3 . 8 , p=0 . 045 , Figure 5A ) . 10 . 7554/eLife . 22089 . 009Table 2 . The percentage of predator individuals found with DNA ( Next Generation Sequencing ) of mimics in their gut/faeces .", "The primer specific for ants amplified on average 9 . 3% individuals of predators , the primer specific for Myrmarachne spiders amplified 3 . 9% individuals of predators , and the primer specific for mutillids amplified 9 . 2% individuals of predators .", "The predators ( 553 spiders , 50 skinks , 48 birds ) were collected on the Macquarie University campus .", "They are grouped according to their guild .", "N gives the number of individual predators screened .", "The mimics are arranged from the most to the least palatable ( left to right ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 22089 . 009Predatory guild Species ( Family ) P .", "ornata P . vermiculosa P . aurea P . ammon P . erato M . piliventris M . tepperi C . aeneopilosus E . aurata D . cephalotes D . nigricans M . erythrocephala M . luctuosa NVisually-oriented euryphagous Eulamprus quoyii ( Scincidae ) 000000000000050Manorina melanocephala ( Meliphagidae ) 00000000 . 020000 . 04048Sandalodes superbus ( Salticidae ) 000000000000037Holoplatys planissima ( Salticidae ) 000000000000012Ocrisiona sp .", "( Salticidae ) 000000000003 . 83 . 826Specialised ant-eating Servaea incana ( Salticidae ) 0000000000001 . 8114Euryopis umbilicata ( Theridiidae ) 000000000009 . 98 . 6101Euryopis sp .", "( Theridiidae ) 000000000000012Hemicloea sp .", "1 ( Gnaphosidae ) 00000005 . 00000020Hemicloea sp .", "2 ( Gnaphosidae ) 00000002 . 40002 . 42 . 442Non-visually oriented euryphagous Lampona murina ( Lamponidae ) 000000000002 . 24 . 346Clubionia robusta ( Clubionidae ) 0000000000001 . 758Clubiona sp .", "( Clubionidae ) 000000000002 . 42 . 48510 . 7554/eLife . 22089 . 010Figure 5 . Relationship between unpalatability and predation pressure .", "( A ) Relationship between unpalatability and the total predation pressure ( K-value ) on 13 mimics by three guilds of predators ( visually-oriented euryphagous , specialized ant-eating , non-visually-oriented ant-adverse ) .", "Estimated non-parametric regression model ( GAM ) is displayed .", "See Table 2 for unpalatability of mimics .", "( B ) Relationship between unpalatability of five mimics and total predation pressure ( K-value ) from three representatives of predators ( see Table 3 for unpalatability of mimics ) .", "A high K-value indicates high predation while a low K-value indicates low predation .", "Solid symbols = Batesian mimics , hollow symbols = Müllerian mimics . DOI: http://dx . doi . org/10 . 7554/eLife . 22089 . 010 We exposed five species of mimics ( ants , spiders and bugs ) of varying unpalatability ( Table 3 ) and one non-mimic ( spider ) to one representative of each of three guilds of naïve predator species ( euryphagous skink , ant-adverse spider , ant specialist spider ) .", "Eastern water skinks ( N = 26 ) are visually-oriented generalist predators and captured ( i . e . ate ) non-mimetic spiders at significantly higher frequency than members of the mimetic complex such as ants , spiders and bugs ( GEE-b , X25 = 125 . 8 , p<0 . 0001 , Figure 6 ) .", "In accordance with our classification of unpalatability , we found a strong positive relationship between prey unpalatability and the post-attack response of skinks ( GLM , F1 , 4 = 19 . 4 , p=0 . 012 , Figure 4C ) .", "Mimic or non-mimic spiders were immediately eaten , but skinks showed an adverse response to mimic ants and bugs .", "These prey were clearly distasteful because the skinks immediately cleaned their mouths and frequently spat the prey out . 10 . 7554/eLife . 22089 . 011Table 3 . A list of traits used to assess the unpalatability of five mimics .", "Values are means ( ±SE ) estimated from 10 measurements .", "The species are arranged from the most to the least palatable . DOI: http://dx . doi . org/10 . 7554/eLife . 22089 . 011SpeciesFrequency of bitingSpray chemicalsNumber of spinesTotal spines length [mm]Cuticle thickness [mm]Total body size [mm]Mandible size [mm]Gland size [mm2]P .", "ammon 0 . 1143 . 54 ( 0 . 08 ) 0 . 04 ( 0 . 002 ) 9 . 04 ( 0 . 08 ) 1 . 00 ( 0 . 02 ) 2 . 84 ( 0 . 16 ) P . vermiculosa 0162 . 96 ( 0 . 08 ) 0 . 03 ( 0 . 002 ) 5 . 98 ( 0 . 05 ) 0 . 71 ( 0 . 03 ) 1 . 62 ( 0 . 06 ) C . aeneopilosus 0 . 31000 . 02 ( 0 . 001 ) 8 . 04 ( 0 . 17 ) 0 . 84 ( 0 . 02 ) 2 . 43 ( 0 . 09 ) D . nigricans 00000 . 02 ( 0 . 0002 ) 7 . 40 ( 0 . 14 ) 00 . 57 ( 0 . 03 ) M . luctuosa 00000 . 02 ( 0 . 001 ) 7 . 04 ( 0 . 28 ) 0010 . 7554/eLife . 22089 . 012Figure 6 . Capture of mimics by three predators . Comparison of frequency of attacks on five mimics and one non-mimic by skinks ( euryphagous visually-oriented predator ) ; Lampona spiders ( ant-adverse non-visually oriented predators ) , and Servaea spiders ( specialised ant-eating predators ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 22089 . 012 We found similar patterns in the non-visually oriented ant-adverse Lampona spiders ( N = 25 ) , which captured non-mimic spiders significantly more frequently than all mimics ( Cochran test , X25 = 104 , p<0 . 0001 , Figure 6 ) , showing no differentiation between the mimics .", "By contrast , the visually oriented ant-eating specialist Servaea spiders ( N = 27 ) readily attacked and captured mimics as well as non-mimics .", "Although these spiders readily attacked the mimics they did appear to differentiate between their levels of defence ( attack: GEE-b , X24 = 15763 , p<0 . 0001 , Figure 6 ) .", "The most unpalatable ant was attacked at a significantly lower frequency than other mimics ( contrasts , p<0 . 008 ) and moderately unpalatable ant species were captured at a significantly lower frequency than bug and spider mimics ( contrasts , p<0 . 006 ) .", "In summary , euryphagous and ant-adverse predators were less likely to capture a mimic than a non-mimic while the ant-specialist attacked the ants and ant-mimics alike but the likelihood of attack varied according to the mimic’s actual unpalatability .", "There was no relationship between levels of unpalatability and the sum of predation pressure ( K-values ) exerted by the three predatory guilds ( LM , F1 , 3 = 1 . 83 , p=0 . 27 , Figure 5B ) ." ], [ "Ants have not been considered in Müllerian mimetic rings , which is perhaps surprising given their diversity and the number of known ant-mimics .", "The lack of ant fauna in mimetic rings possibly reflects an overall lack of conspicuous defensive coloration in ants .", "However , our study supports a species rich mimicry complex of golden coloured ant models and mimics , adding to the small number of documented large-scale complexes .", "Mimetic complexes ( among several families of beetles ) were first recognised by Nicholson ( 1927 ) for Australian insect fauna .", "Such complexes are presumably far more widespread than appreciated , but have only been subject to more intensive study relatively recently .", "Many more large-scale mimetic complexes among ants and other animals ( particularly arthropods ) are likely to be described in future .", "The species richness of this golden complex exceeds those of fish or millipedes ( Marek and Bond , 2009; Alexandrou et al . , 2011 ) but is currently smaller than in mutillid wasp ( Wilson et al . , 2012 , 2015 ) , heliconid butterfly ( Mallet and Gilbert , 1995 ) and bumblebee ( Williams , 2007 ) complexes .", "The key members in the golden complex are ants but the complex is taxonomically broad and includes species with distant phylogenetic origins .", "Beside the species mentioned , it may include a few other groups , such as bees , flies , and cerambycid beetles , in which some species also have a golden colouration .", "This is in contrast to complexes of similar scale that only include members from a single order .", "The distribution of the majority of the ten putative mimetic rings in the complex overlapped with at least one other ring .", "Although the cluster analysis distinguished rings , the distances between the rings were not large suggesting that the entire complex is just a single ring .", "Our findings are not unlike those from other complexes that include rings of different colour pattern , which overlap in geographic distribution ( Williams , 2007; Wilson et al . , 2012 , 2015 ) .", "An overlap of golden rings or actually their non-existence potentially suggests that the variable golden colour patterns produce similar aposematic signals .", "Large mimetic complexes include taxa of close and distant phylogenetic origins ( e . g . , Linsley et al . , 1961; Rodriguez et al . , 2014 ) in which the colour pattern is not only a product of common ancestry but also of convergence .", "A shared ancestry is apparent for two species of Myrmarachne spiders ( Pekár et al . , 2017 ) , three species of Daerlac bugs ( Casis and Symonds , 2012 ) , and several species of Polyrhachis ants ( Robson et al . , 2015 ) .", "The majority though , including Camponotus , Diacamma , Pachycondyla , and Iridomyrmex ants , other spider species , Eurymela treehopers , and Ephutomorpha wasps , have converged on the same phenotype as evidenced by their isolation within lineages of non-mimetic colour patterns .", "The diversity and geographic pattern of the golden phenotype is likely a result of co-divergence as found in other complexes ( e . g . , Rodriguez et al . , 2014 ) .", "Species in Müllerian rings described to date , e . g . heliconid butterflies , use a single defensive trait to teach predators that they are distasteful ( e . g . , Van Zandt Brower , 1958 ) .", "The lesser-defended individuals in this context are less distasteful .", "Species in our golden complex use multiple defensive traits including a variety of structural and chemical defences .", "To understand how these traits interplay with the aposematic signal we created an index that summed all traits known to serve a defensive function .", "We acknowledge this approach is somewhat simplistic as various traits are differentially effective against various predators .", "For example , a thick cuticle is effective against arthropod predators but ineffective against vertebrates ( Evans and Sanson , 2005 ) .", "On the other hand , spines are effective against vertebrates but ineffective in the defence against arthropod predators ( Mikolajewski et al . , 2006 ) .", "Different species of closely related predators may also use a variety of adaptations to deal with the defences of prey .", "For example , some spiders are able to catch ants even with a thick cuticle while others are not ( Pekár and Toft , 2015 ) .", "A true index of unpalatability requires an estimation of the total effectiveness of a combination of defensive structures , such as spines and thick cuticle and the toxicity of chemical compounds for each predator-prey combination .", "Estimates including spines and cuticle thickness may be relatively straight forward but information on the toxicity of chemical compounds is largely not available ( see http://toxnet . nlm . nih . gov ) .", "Although our approach is simplistic , the positive relationship between the index of unpalatability and the response of skinks strongly suggests that the measure is a good proxy for unpalatability .", "Some of the defensive traits were strongly correlated with body size .", "Body size alone is an anatomical constraint that may prevent predation , thus it is difficult to separate the effect of pure body size from the relative size of some defensive traits ( e . g . , spines ) .", "A strong correlation between body size and the size of defensive traits is natural , because larger spines , for example , require a larger body to support them .", "It is , however , the absolute size of defences not their relative size that is ecologically important ( e . g . , a skink will swallow larger spines with greater difficulty than smaller ones ) .", "Whatever their single effect is , all these traits together , add to an overall unpalatability of mimics .", "Our study describes a new complex with a golden/black colour combination .", "This combination is somewhat similar to the classic aposematic yellow/black phenotype ( Ruxton et al . , 2004; Williams , 2007 ) .", "Accordingly , our findings support the idea the coverage of golden colour acts as an honest signal of aposematism .", "All the predators in staged trials exhibited an aversion to the prey with the greatest coverage of golden colour .", "Furthermore , golden individuals were rarely found in the diet of natural populations of predators .", "Honest signalling is further supported by the positive correlation between the extent of the golden colour and unpalatability , controlling for body size in the analyses via phylogenetic correlation or as a covariate .", "The theory of honest signalling , however , predicts such a relationship within rather than across species .", "However , recent evidence shows that this relationship can also exist across closely related aposematic species ( Cortesi and Cheney , 2010; María Arenas et al . , 2015 ) .", "It remains to be tested what speciation process and selection forces have driven this pattern .", "Given the fact , that the complex is composed of many unrelated taxonomic groups that occur in different micro-habitats , the processes are likely to vary .", "It is necessary to investigate whether such a relationship between species holds for other known mimetic complexes .", "Unlike the classic yellow/black combination , members of the golden complex are defined by a metallic golden sheen .", "This sheen reflects brightly under direct sunlight making the animals very conspicuous , at least to a human observer .", "Just how important this sheen is in deterring a predator over and above just a matt colour has yet to be determined for this system .", "In other systems though , reflective iridescence has a significant additive effect ( e . g . Fabricant et al . , 2014 ) so it is likely that the sheen enhances the detectability of the signal .", "A possible additional benefit , or even an alternative explanation , is that the sheen is important for reflecting harmful radiation and dissipating heat ( Shi et al . , 2015 ) .", "Irrespective of the explanation the importance of this colour variant , further experimental investigation is warranted .", "The aversion to prey with golden colour was apparent irrespective of the predator’s visual sensitivity .", "Visually- and non-visually orienting predators equally avoided the golden prey suggesting that golden mimics advertise their unpalatability via multiple sensory modalities .", "The non-visual predator still proportionally avoided the mimics despite not being able to detect the visual signal .", "This is an area of promising future investigation .", "Some non-visual predators potentially recognize mimics via the patterns of vibrations generated by their movements ( Pekár et al . , 2011 ) .", "Predators typically use multisensory modalities to identify prey ( Bro-Jørgensen , 2010 ) such as movement , shape and defensive chemicals they emit .", "Our measure of unpalatability suggested that the highest proportion of individuals was moderately defended and defensiveness is distributed from highly defended to weakly defended .", "This is in agreement with previous claims that mimicry rings are dominated by moderately defended species ( Sherratt , 2008 ) .", "With a predominance of moderately defended prey , there is an expectation that their presence will erode the effectiveness of the model .", "Our findings , based on a limited number of mimics , multiple predatory guilds when taking into account , did not support this notion .", "Instead , all mimics investigated in our predator-prey trials experienced significantly lower mortality than the non-mimetic species , and this was irrespective of the coverage of gold .", "We found the skinks were more likely to attack the lesser-defended individuals but when the suite of predators was taken into account there was little variation in the overall predation pressure .", "This is potentially because some predators may not use the visual signal as a reference and are cuing in on other signals .", "Diet analyses of several potential predators also showed predators of all guilds , including non-visually oriented predators , captured mimics with very low frequency .", "The predators that we used are known to feed on non-mimetic prey too ( Jackson and Harding , 1982; Daniels , 1987; Platnick , 2000; Higgins et al . , 2001; McGinley et al . , 2015 ) .", "Indeed , they fed on prey related to the mimics as evidenced by the positive results of designed primers targeting the mimics .", "We did not measure the availability of alternative prey so we do not have an absolute indication of relative encounter rates .", "However , birds learn to avoid both Müllerian models and mimics irrespective of the availability of alternative prey ( Lindström et al . , 2004 ) .", "In addition , there are multiple species of ant and ant models distributed across the sampling habitat occupying both terrestrial and arboreal habitats .", "The majority of the species in the complex are also ants and ants are typically super abundant due to their sociality .", "So it seems likely the predators would see these animals on a regular basis .", "Using a multi-disciplinary approach here , we faced several constraints .", "For example , measurements of mimic colour were based on photographs as we were unable to collect all mimics .", "To correct for inherent differences in illumination used during photographing mimics , the obtained RGB values were standardized but not linearised , and thus should be interpreted with caution .", "Similarly , the results of experiments with skinks must be interpreted with caution due to constrained randomisation used ( Müllerian mimics first ) .", "The response of skinks could be affected by the constrained prey order ( due to some kind of habituation ) .", "Yet , other predators used showed similar response to mimics as skinks , though the order of offered prey was fully randomised .", "Here we present the first account of the golden mimetic complex , which we believe is an exciting model for future research .", "Our work to date is mainly descriptive , and it requires more explicit experimental approaches to generate robust conclusions about the evolution of this golden aposematic signal .", "For example , the relationship between the level of unpalatability and the quality of the golden signals needs rigorous testing with a greater number of mimics .", "This would also help in deciphering the role of less-defended mimics in the protection of the entire complex .", "Similarly , exploring the function of non-visual signals in deterring predators will elucidate how this mimicry ring works .", "In conclusion , we present the first account of the golden mimicry complex .", "It consists of species that show considerable variation in their level and mode of defence , ranging from undefended Batesian mimics to highly defended Müllerian mimics , with most members of the complex somewhere in between .", "Our data suggest that all members of the golden mimicry complex receive quite similar net protection from a variety of predators presumably because they have a variety of defensive traits .", "We predict that mimetic complexes that incorporate a range of different forms of defence are not only more common than previously believed , but also more likely result in mutualism among the unequally defended members of the complex ." ], [ "Arthropod species forming the complex were selected based on the presence of a golden colour pattern on the dorsal side of their body and body size falling within the range 3–15 mm .", "All Australian ants , and known ant-like mimics , were examined for a dorsal golden appearance .", "The specimens we examined were either field collected by the authors ( Sydney , Blue Mountains , NSW outback , NSW eastern coast , Queensland eastern coast ) or from museum collections ( Australian Museum , Sydney ) .", "All other specimens we viewed were images from AntWiki ( www . antwiki . org ) , Atlas of Living Australia ( www . ala . org . au ) , or taxonomic papers ( Ogata and Taylor , 1991; Shattuck and Barnett , 2006; McArthur , 2007; Kohout , 2010a , 2010b , 2012 , 2013a , 2013b; Heterick and Shattuck , 2011; Casis and Symonds , 2012; Shattuck and Marsden , 2013 ) .", "The dorsal colour pattern was estimated from digital images of specimens ( Figure 7 ) as performed in other studies on mimetic complexes ( Wilson et al . , 2012 , 2015; Rodriguez et al . , 2014 ) .", "Field collected specimens ( of 41 species ) were killed by ethylacetate and arranged in their natural body position .", "Photographs were taken with a Canon Legria HF G10 against a white background and lit from above by two full spectrum light bulbs ( Repti Glo 2 . 0 UVB , Eko Terra ) .", "Photographs of the remaining species ( 59 species ) were obtained mostly from the personal collection of R . Kohout , followed by AntWiki , and Atlas of Living Australia .", "Prior to taking measurements all photographs were calibrated for the white and/or black colour within ImageJ to correct for different lighting conditions .", "The average value of each RGB colour component was estimated on the following body parts: dorsal side of head , appendages ( femur II ) , pronotum , mesonotum , metanotum , petiole , first segment of the gaster , and the remaining segments of the gaster/abdomen in ants and wasps using ImageJ .", "Likewise , for hemipterans and spiders the average colour value of each RGB colour component of corresponding body parts ( to that of ants ) were examined including the pedipalps/chelicera , frontal third of prosoma , second third and last third of the prosoma , legs ( femur III ) , the first half of the abdomen and the second half of the abdomen .", "In each species , we also measured the length of each body part ( listed above ) and an absolute area of the golden colouration on the dorsal side using ImageJ . 10 . 7554/eLife . 22089 . 013Figure 7 . Selected list of 100 species representing the ten putative mimetic rings . Species are arranged within each ring from the most to the least unpalatable ( left to right ) .", "Some pictures of ants are displayed with the permission of AntWiki and R . Kohout . DOI: http://dx . doi . org/10 . 7554/eLife . 22089 . 013 To assess colour pattern similarity among mimics and to find how many rings there are , we subjected 24 measures of the colour pattern , body size and area of golden patch of 100 mimics ( Pekár et al . , 2016 ) to the Non-Metric Multidimensional Scaling based on Bray-Curtis distance matrix .", "Because various body parts have different area and are thus differently conspicuous to predators , we used weighting of the measures by their relative size .", "Then we estimated the number of putative rings via k-means clustering using the vegan R package ( Oksanen et al . , 2013 ) .", "This method used the simple structure index ( ssi ) as a partition criterion ( Legendre and Legendre , 1998 ) as AIC is not available .", "Red Green Blue ( RBG ) contrast was estimated using 55 species of mimics .", "It was computed as the sum of differences in red , green , and blue values representing the golden and black colouration found on the body of mimics .", "The phylogenetic relationship at the genus level among the golden mimics was reconstructed from a combination of published phylogenies ( Moreau et al . , 2006; Regier et al . , 2010; Robson et al . , 2015; Pekár et al . , 2017 ) .", "If species level phylogeny was not available , we used polytomic splitting at the species level .", "To test for phylogenetic inertia of classification to putative rings we used generalised least squares ( GLS ) with Pagel’s phylogenetic correlation structure ( Paradis , 2006 ) , in which low value of λ indicates low phylogenetic signal .", "The mode by which the animals generated the golden colour ( pigments or structural colours ) was determined via microscopy .", "One specimen of each species from each genus ( Ephutomorpha aurata , Camponotus aeneopilosus , Daerlac nigricans , Dolichoderus doriae , Polyrhachis ammon , P . vermiculosa , Ligonipes illustris , Myrmarachne luctuosa , Myrmecia tepperi , Eurymela rubrolimbata ) was further examined via SEM .", "The golden gaster/abdomen was dried at a room temperature for 10 days , then mounted on a stub and coated with gold prior to viewing under a SEM Jeol JSM 648OLA .", "The presence of pigments on the gaster/abdomen was examined in ethanol-preserved specimens under Olympus stereomicroscope SZX9 and photographed with a digital camera mounted on the stereomicroscope .", "The distribution of mimics across Australia was estimated using occurrence records from collections made by the authors , taxonomic papers ( Ogata and Taylor , 1991; Shattuck and Barnett , 2006; McArthur , 2007; Kohout , 2010a , 2010b , 2012 , 2013a , 2013b; Heterick and Shattuck , 2011; Casis and Symonds , 2012; Shattuck and Marsden , 2013 ) , museum material deposited in the Australian Museum and Queensland Museum and records stored in the database of the Atlas of Living Australia .", "To test for an association between colour similarity and distribution overlap of species in each mimicry-ring in Australia we used a grid cell of 400 × 400 km .", "Within each grid occurrence of a species was classified as 1 ( if present ) or 0 ( if not recorded ) ( Pekár et al . , 2016 ) .", "Then a distance matrix was created from the data using the Bray-Curtis method ( Legendre and Legendre , 1998 ) .", "Using the colour pattern data , we created a matrix and generated a distance matrix between species using Euclidean distance .", "To test a hypothesis that putative rings overlap in their geographic distribution , we studied the association between the geographical occurrence of mimics and colour similarity matrices by means of a Mantel test based on the Pearson correlation coefficient .", "We estimated unpalatability in 100 mimics ( workers and adults ) from the complex using the following seven traits ( xi ) : length of the sting , number of spines , total length of spines , size of mandibles , cuticle thickness , size of poison/pygidial gland , and performance of a communal attack ( Pekár et al . , 2016 ) .", "Size , number of spines and size of mandibles were measured in one individual from each species .", "Cuticle thickness and size of the poison/pygidial gland could only be measured from freshly collected specimens .", "So we collected the mimics ( workers in the case of ants ) from a variety of places across Australia ( Cairns , Townsville , Brisbane , Sydney , Blue Mountains , Broken Hill , Hay , Macquarie Port , Tweed Heads , Sunshine Coast , Hervey Bay , Agnes Water ) .", "In total , 75 specimens belonging to 41 species , eight genera , and four subfamilies were collected alive .", "Individuals were killed by freezing at −20°C .", "Cuticle thickness was measured by slicing the thorax ( mesosoma ) perpendicularly using a blade and then measuring it under a stereomicroscope to the nearest 0 . 01 mm .", "To measure the size of the gland , the gland was dissected from the gaster ( thorax in bugs ) using fine forceps , placed on a glass slide in a drop of water , and covered with a coverslip .", "The gland was approximately oval; thus the two perpendicular lengths of the gland were measured using an ocular ruler .", "We estimated gland area assuming a regular ellipsoid shape .", "We measured the sting length after pulling it from the abdomen using forceps .", "In each of these individuals we also measured the gaster/abdomen length , and thorax length .", "The ability to perform a communal attack ( i . e . attack by several individuals ) was taken from the literature and was assumed to be consistent throughout each genus .", "To estimate these traits for the remaining ant species that we failed to collect we used an interpolation based on proxy measures using 55 specimens: the size of both the poison/pygidial gland and sting length were regressed ( using a linear model ) from the size of the gaster , and cuticle thickness was regressed from the size of the thorax .", "These regression models ( Figure 8 ) were then used to predict values for species we were unable to sample .", "The gland content varies among species of ants from different subfamilies: Formicinae ants possess formic acid ( O'Rourke , 1950; Stumper , 1952 ) , Dolichoderinae ants possess terpens , such as iridodial and dolichodial ( Cavill , 1960; Cavill and Hinterberger , 1960 ) , and Myrmeciinae ants possess indole ( Jackson et al . , 1990 ) .", "The metathoracic scent gland in true bugs produces pungent pyrazines and alkenals ( Millar , 2005 ) .", "As the relative potency of these chemicals is unknown , we considered all these chemicals to be similarly noxious . 10 . 7554/eLife . 22089 . 014Figure 8 . Relationship between cuticle thickness and thorax length .", "( A ) poison/pygidial gland area and gaster length ( B ) sting length and gaster length ( C ) with estimated linear regression models . DOI: http://dx . doi . org/10 . 7554/eLife . 22089 . 014 Various defensive traits measured here are not similarly effective against predators .", "Yet we assumed that their effect is similar because different predators adapted to deal with different defences of prey .", "We further assumed that the effect of these traits on predators is additive .", "So to quantify unpalatability ( uj ) of the jth mimic we summed the ith trait values , which were scaled by their maximal value due to difference in range: uj=∑j=1nxijmax ( xi ) .", "We also computed a simple sum of defensive traits and found that the two measures are highly correlated ( Pearson correlation , r = 0 . 72 , p<0 . 0001 ) .", "As the index of unpalatability includes more details we used it in all analyses .", "In order to test that the complex is dominated by moderately-defended species we used a Shapiro-Wilk test to test for a shape of a normal distribution of the unpalatability .", "To test whether the rings are composed of a similar range of unpalatable species , we used Bartlett test of variance homogeneity .", "To test a hypothesis that unpalatability is related to the extent of gold colouration we used GLS to model the relationship between index of unpalatability and the absolute golden area ( which is more ecologically relevant than relative area ) .", "The linear predictor included a quadratic term as the response was an area .", "A GLS model with Brownian motion model correlation structure was used to correct for the effect of phylogeny ( Pekár and Brabec , 2012 ) .", "To account for the fact that for some species , some defensive traits could not be measured ( and were interpolated ) we used weights that corresponded to the proportion of observed characteristics .", "Both variables were affected strongly by the body size so this measure needed to be included in the analysis .", "The body size had strong phylogenetic inertia ( GLS , Pagel’s λ = 1 . 08 ) , thus by controlling for phylogeny the body size effect was included too , therefore use of the body size as a covariate was not necessary .", "We investigated the diet of several predators on the campus of Macquarie University , North Ryde , Sydney , Australia .", "The campus in addition to buildings , contains a patch of native forest , several rocky streams , and open green space interspersed with trees where the predators could have been collected and observed which would be very difficult or even impossible in the pristine forest where the selection have taken place .", "The predators included noisy miners , lizards ( skinks ) , and a number of arthropods that occur on the trunks and the immediate vicinity of Eucalyptus trees , where the mimics occur ( see Table 2 ) .", "We sampled arthropods from under the bark of more than 50 trees and selected those that were predatory .", "Arthropods were immediately placed into 99% pure ethanol and then in a freezer at −20°C .", "Co-existing predators were selected among the most abundant species to represent three guilds according to their trophic strategy and prey detection modality: ( 1 ) visually-oriented euryphagous predators , ( 2 ) non-visually oriented ant-adverse predators , and ( 3 ) specialized ant-eating predators .", "We used the information of their gut content and data from the literature ( Jackson and Harding , 1982; Platnick , 2000; Higgins et al . , 2001; McGinley et al . , 2015 ) to identify those that feed on prey related to the mimics .", "If more than 10% of individuals of a specific predator species were positive for primers designed for ants , the species was classified as ant-eating .", "Otherwise , the species was classified as euryphagous .", "We collected 48 faecal samples from Eulamprus quoyii ( Duméril et Bibron ) ( Eastern Water Skink , Scincidae , Squamata ) and 48 samples from Manorina melanocephala Latham ( Noisy Miner , Meliphagidae , Passeriformes ) from across the Macquarie University campus during 2014 and 2015 .", "These two predators were selected because they feed on arthropods , were most abundant among congeners and foraged on tree trunks .", "Eulamprus quoyii were collected from both rocky streams ( human-altered ) and forest-edge by noosing .", "Lizards typically defecated immediately upon capture and in some cases we gently massaged the abdomen to expel a faecal pellet .", "Manorina melanocephala faeces were collected at temporary feeding stations to avoid handling the birds .", "We lured birds to a large canvas ( 3 × 5 m ) by placing small amounts of sweet cake near a water bowl and perches made from branches .", "The stations were monitored continuously .", "We sampled across a wide area in order to target specific family groups and the sampling was short enough to ensure that only droppings produced from previous meals were collected .", "We stored both the lizard and bird faecal samples individually in Eppendorf tubes in 80% ethanol in a −20°C freezer .", "This work was approved by the Animal Ethics Committee of Macquarie University ( ARA 2014/031 ) .", "The gut of predators progressively degrades DNA through digestion ( Sint et al . , 2011 ) .", "To account for the shortened DNA fragments we designed specific primers , which amplified fragments no longer than 300 bp ( Deagle et al . , 2006 ) .", "This length is known to be a good compromise that increases the likelihood of amplification while still being long and variable enough to distinguish different prey taxa ( Herbert et al . , 2003; Zeale et al . , 2011 ) .", "A fragment of the COI gene was PCR amplified using LCO1490 ( 5´-GGTCAACAAATCATAAAGATATTGG-3´ ) and HCO2198 ( 5´-TAAACTTCAGGGTGACCAAAAAATCA-3´ ) primers ( Folmer et al . , 1994 ) from representatives of potential prey found across the Macquarie University campus ( Hemiptera: Daerlac apicalis , D . cephalotes , D . nigricans; Formicidae: Myrmecia piliventris , M . tepperi , Camponotus aeneopilosus , Polyrhachis ammon , P . aurea , P . ornata , P . vermiculosa; Araneae: Myrmarachne luctuosa , M . erythrocephala ) and predators ( Araneae: Clubiona robusta L . Koch , Clubiona sp .", "; Euryopis umbilicata L . Koch , Euryopis sp .", "; Hemicloea sp .", "1 , Hemicloea sp .", "2; Holoplatys planissima ( L . Koch ) ; Lampona murina L . Koch; Ocrisiona sp .", "; Sandalodes superbus ( Karsch ) ; Servaea incana ( Karsch ) ) .", "Each PCR ( 20 μL total volume ) consisted of 5 μL of DNA , 1 μL of each primer ( 10 μM ) , 0 . 4 μL of 10 mM dNTP´s , 2 . 2 μL of 25 mM MgCl2 , 2 μL of 10x PCR buffer , 1 μL of bovine albumin serum ( BSA ) , 0 . 3 μL of Taq Polymerase ( 5 u/μL ) , and 7 . 1 μL of DNA-free water .", "PCR conditions were as follows: initial denaturation at 94°C for 3 . 5 min; 38 cycles of 94°C for 1 min , 44°C for 1 min ( annealing temperature ) , 72°C for 1 . 5 min; a final extension at 72°C for 7 min .", "PCR products were detected by electrophoresis in 2% GoldView-stained agarose gels .", "Amplified products were purified using the QIAquick PCR Purification Kit ( Quiagen ) and sequenced in both directions with BigDye Terminator v3 . 1 Sequence Kit ( Applied Biosystems ) .", "Sequencing was carried out on an ABI Prism 3130 Genetic Analyzer ( Applied Biosystems ) .", "Sequences were assembled in Sequencher 4 . 8 ( Gene Codes Corporation , Ann Arbor , MI ) and aligned using ClustalW ( Thompson et al . , 1994 ) implemented in MEGA 5 . 1 ( Tamura et al . , 2011 ) .", "Based on sequence similarities/differences , group-specific primers , which would amplify DNA of mimics in the predator guts , were designed using Amplicon . b08 ( Jarman , 2004 ) .", "Four primer pairs were tested with all prey and predator specimens used in this study to ensure that the primers amplify DNA of the studied prey groups only: primers MyrmF and MyrmR amplified Myrmarachne spiders , DaerF and DaerR amplified Daerlac true bugs , FormF and FormR amplified Camponotus , Polyrhachis and Myrmecia ants , and BothmF and BothmR primers amplified Bothriomutilla mutillid wasps ( for primer sequences see Table 4 ) and to reject cross-amplifications with the predators .", "The sequences of cytochrome c oxidase obtained from the studied predators and the mimics are deposited in GenBank ( Accession numbers KX980351--KX980391 ) . 10 . 7554/eLife . 22089 . 015Table 4 . List of primer pairs used to identify mimics in the gut or faeces of potential predators . DOI: http://dx . doi . org/10 . 7554/eLife . 22089 . 015PrimerSequenceFragment length ( bp ) Amplified generaDaerF5´- GAT CAA ATT TAT AAT AC −3´204Daerlac DaerR5´- TTC TGA TTA ATA AGG −3´FormF5´- GAT CAA ACY TTT AAY TC −3´220Camponotus , Myrmecia , Polyrhachis FormR5´- CCW GAT CCT TCA TTA ATA AA −3´MyrmF5´- ATT AGC TTC TAT TAT TG −3´142Myrmarachne MyrmR5´- TCT ATA GAA ATW CCT TCA G −3´BothmF5´- TCC TCA TGT TCA GGA ATA ATT AA −3´215Bothriomutilla BothmR5´- ATT AAG AGC ATA ATG GAT ATT GGG −3´ DNA was extracted from 553 spider abdomens using DNeasy Blood and Tissue Kit ( Qiagen ) following the manufacturer’s animal tissue protocol with a change in final step when only 50 μL of elution buffer was used .", "DNA from fecal samples ( 50 samples of skinks and 48 samples of noisy miner ) was extracted using the DNeasy mericon Food Kit ( Qiagen ) following manufacturer’s Small Fragment protocol .", "PCRs were performed using Multiplex PCR kit ( Qiagen ) under the following conditions: initial denaturation at 95°C for 15 min; 42 cycles of 94°C for 30 s , annealing temperature for 90 s ( 47 . 5°C when using Bothm primers , 43 and 45°C with the other primers ) , 72°C for 90 s; and a final extension at 72°C for 10 min .", "The reaction mixture total volume of 20 μL consisted of 10 . 6 μL of Multiplex PCR Master Mix , 1 . 8 μL of Q-Solution , 2 . 1 μL of RNase-free water , 0 . 5 μL of 10 μM forward and 0 . 5 μL of reverse primers , and 4 . 5 μL of DNA .", "PCR with Bothm primers was done separately whereas three remaining primer pairs were used in one multiplex reaction .", "PCR products were detected by electrophoresis in 2% GoodView-stained agarose gels .", "PCR was repeated for each sample and primer combination at least three times to exclude falsely negative results .", "In positive samples , PCR was repeated once again using primers modified with MID identifiers ( i . e . multiplex identifiers , 10bp-long barcoding sequences ) .", "Each sample was thus marked with a unique combination of barcodes .", "PCR products were purified using a QIAquick PCR Purification Kit ( Qiagen ) according to the manufacturer’s protocols .", "The concentration of each PCR product was measured using NanoDrop 8000 UV-Vis Spectrophotometer ( Thermo Scientific , Waltman , MA ) and assessed comparing to 50 bp ladder ( Fermentas ) .", "Five μL of 50 μg/μL PCR products was pooled into the same sterile vial and sent for sequencing .", "Sequencing library was prepared using Ion Plus Fragment Library Kit and Ion PI Template OT2 200 Kit v3 ( Thermo Fisher Scientific ) .", "Sequencing on the Ion Proton System was provided by the SEQme company ( Dobříš , Czech Republic ) .", "Sequencing data were processed using the Galaxy platform ( https://usegalaxy . org/ ) , BioEdit 7 . 2 . 5 ( Hall , 1999 ) and MEGA 5 . 10 .", "Reads were split according to their MIDs resulting in files corresponding to individual predators .", "Then , forward and reverse primers were removed and sequences were filtered according to their length ( <120 or 180 bp ) to remove dimers or too short reads .", "The sequences were collapsed and rare haplotypes ( containing <2 identical sequences ) were removed .", "Furthermore , sequences with stop codons and indels causing frameshifts were also removed .", "Remaining haplotypes were clustered into molecular operational taxonomic units ( MOTU ) using jMOTU 4 . 1 ( Jones et al . , 2011 ) with a 4 bp cut-off ( corresponding to 3% sequence divergence ) .", "Each MOTU was compared to the GenBank database using megablast and BOLD database and to the sequences of mimics obtained within this study .", "NGS produced 11 068 904 informative reads from all predators of which 614 , 273 reads belonged to the mimics .", "Availability of mimics to the predators was estimated by visual search on the trunks and below bark of 20 gum trees randomly selected on the campus .", "On each tree , abundance of mimics was surveyed by one person up to the height of 2 m from the ground for a period of 30 min on a sunny day .", "All mimics were collected , brought to the laboratory and identified to species .", "To test a hypothesis that the frequencies of captured mimics by all predators and those available at the microhabitat of predators are similar we used Chi-square test .", "In order to estimate the attack rate exerted by the whole community of predators as precisely as possible , we had to take into account two important variables that affect the predation: ( 1 ) the relative frequency ( fij ) of positively screened ith predator individual for each jth mimic; ( 2 ) the capture probability of araneophagous spider predator ( Lampona ) , where body size is a strong predictor of the likelihood of predation .", "The capture probability was estimated for each spider predator and mimic ( Pekár et al . , 2016 ) .", "This latter adjustment was necessary in order to take into account the fact that spiders could have captured juvenile Myrmarachne or Daerlac individuals , which are not golden mimics and could not be distinguished from adult specimens by molecular methods .", "The functional model for the capture probability was obtained from data on eight spider species representing different guilds ( Nentwig and Wissel , 1986 ) .", "The logistic function was 11+exp ( −1 . 99+2 . 11ratioij ) , where ratioij is the mimic/predator body size ratio .", "The ratio was estimated by measuring the body size of ten collected specimens of each predator and mimic .", "Then the overall pressure by the community of predators was estimated as a sum of all attack rates by means of the killing value , Kj ( Varley and Gradwell , 1960 ) : Kj=∑j=1n−log ( 1−fij ) .", "To test whether natural mortality caused by predation is related to unpalatability of mimics we used nonparametric regression as the response variable had non-standard distribution , namely generalized additive model ( GAM ) from the mgcv package ( Wood , 2011 ) .", "We regressed log-transformed K-values ( in order to stay within positive bounds ) against unpalatability index .", "For predation trials we selected five species of mimics found on the Macquarie University campus .", "The five species represented a gradient of unpalatability ( Table", "3 ) within the mimetic complex .", "We included examples of the most unpalatable to the least palatable mimics: the ants Polyrhachis ammon , Polyrhachis vermiculosa , and Camponotus aeneopilosus , the true bug Daerlac nigricans , and the spider Myrmarachne luctuosa .", "Based on their unpalatability , the ants and bugs were predicted to be Müllerian mimics , while the spider was predicted to be a Batesian mimic .", "Unpalatability was re-estimated for these five species using ten individuals from each species .", "All attributes as measured previously were re-measured with the addition of a behavioural measure .", "The behavioural measure was the frequency of biting in the field following agitation with forceps .", "The addition of an extra measure adjusted the previous estimated unpalatability ( uj ) but the relative relationship of unpalatability between species remained the same .", "We used Eulamprus quoyii as our visually-oriented euryphagous vertebrate predator .", "At least 14 days prior to the experiment adult skinks were placed singly in a white container ( 70 × 50 × 40 cm ) at constant temperature 26°C and L:D = 12:12 regime .", "They were fed regularly with three crickets ( Acheta domestica ) three times a week .", "The skinks were then starved five days prior to the predation trials .", "One day prior to the trial a layer of butter was applied to the sides of the white container to prevent arthropods from climbing the walls and escaping .", "At the start of a trial a lizard was left to settle down for a period of 5 min .", "A prey item was released into the container and the behaviour of the skink during the 5 min trial was recorded with a fixed video camera positioned above the container .", "Five minutes was deemed sufficient , as previous feeding trials with crickets have shown this species of lizard will attempt to capture prey within 1 min .", "If the potential prey survived it was removed , and the skink was left for 2 min to settle down .", "Another prey item was then released and so on until all five mimic prey types were used .", "As a non-mimic control , we used juveniles of the spider , Badumna insignis L . Koch , which was of a similar size to the other species but was not a mimic as it lacked golden colouration .", "At the end a cricket was released as a positive control .", "Results of trials were used in the analysis only if the cricket was captured .", "The prey was offered to skinks using a constrained randomisation .", "There was randomisation within Müllerian mimics ( ants ) followed by randomisation within less defended mimics ( Daerlac , Myrmarachne ) and non-mimic prey ( Badumna ) because the predator must learn unpalatability by encountering the unpalatable models first .", "The learned aversion was necessary because skinks were collected in the wild a number of months prior to the trials and fed with crickets since then .", "We used 26 skinks in total .", "Ants , spiders and bugs were collected a few days prior to the trial .", "From the video footage we recorded whether prey was consumed or spat out .", "The spider Lampona murina L . Koch was used as a nonvisually-oriented ant-adverse predator that will not capture ants but other spiders .", "Twenty-five late-instar juvenile Lampona spiders were collected from the bark of gum ( Eucalyptus sp . ) trees on the Macquarie University campus and kept individually in tubes ( 1 cm diameter , 10 cm long ) .", "Spiders were placed in a chamber set to 23°C and L:D = 16:8 regime .", "Spiders were fed with a clubionid spider once a week and provided with a moistened piece of gauze .", "Lampona was not fed seven days prior to a trial .", "During a trial , an individual from the five mimetic prey species and one non-mimic spider ( Clubiona sp . ) was released into the Petri dish in a random order ( diameter 6 cm ) .", "The prey was offered to Lampona in a random order .", "Induction of learned aversion was not necessary because the spiders were collected from the field only a week prior to the experiment .", "If the prey was not captured within 30 min we replaced it with a new one until one prey was captured .", "If the prey was captured , another prey was offered seven days later .", "In each trial we recorded capture frequency of Lampona .", "Finally , we used the spider Servaea incana ( Keyserling ) as a specialised ant-eating predator .", "Twenty-seven adult individuals of Servaea spiders were collected from gum trees on the Macquarie University campus and placed singly in a Petri dish ( 8 cm diameter ) and kept at 23°C and L:D = 16:8 .", "Spiders were fed with small crickets once a week and provided with a moistened piece of gauze .", "We did not feed Servaea for five days prior to a trial .", "One individual of a mimic prey species was released into the dish .", "The prey was offered to Servaea in a random order .", "Induction of learned aversion was not necessary because spiders were collected from the field only a week prior to the experiment .", "If the prey was not captured within 5 min we replaced it with a new one .", "If the prey was captured another prey was offered five days later .", "In each trial we recorded whether the prey was captured as well as the behaviour of Servaea and prey .", "To compare the frequency of capture among used prey types for each predator , relative frequencies of prey capture from Eulamprus quoyi or Servaea experiments were subjected to generalised estimating equations ( GEE ) from the geepack package with a binomial error structure due to repeated use of the same skink individuals with different prey .", "The exchangeable association structure was used ( Varley and Gradwell , 1960 ) .", "To test whether the post-attack reaction of the skinks was related to prey unpalatability we used generalized linear model with binomial error structure ( GLM ) of scores of the post-attack reaction of the skinks and the unpalatability index .", "The post-attack reaction was ranked as follows: 0 if the mimic was eaten , one if it was eaten but the skink cleaned its mouth , and two if the skink spat out the mimic .", "The ranks were then scaled to 0–1 range .", "To compare the relative capture frequencies of Lampona on the prey species a Cochran Q test was used .", "To test whether overall mortality of prey is related to its unpalatability we used a general linear model ( LM ) of the sum of all probabilities of capture ( pij ) by the three predators and unpalatability index .", "The sum of capture rate was expressed as a killing value , Kj using the formula: Kj=∑j=1n−log ( 1−pij ) .", "All statistical analyses were performed in R ( R Core Team , 2013 ) .", "For each model , diagnostic plots were used to assess model adequacy , such as variance homogeneity and distribution of residuals ." ] ]

[ "Mimicry complexes typically consist of multiple species that deter predators using similar anti-predatory signals .", "Mimics in these complexes are assumed to vary in their level of defence from highly defended through to moderately defended , or not defended at all .", "Here , we report a new multi-order mimicry complex that includes at least 140 different putative mimics from four arthropod orders including ants , wasps , bugs , tree hoppers and spiders .", "All members of this mimicry complex are characterised by a conspicuous golden body and an ant Gestalt , but vary substantially in their defensive traits .", "However , they were similarly effective at deterring predators - even mildly defended mimics were rarely eaten by a community of invertebrate and vertebrate predators both in the wild and during staged trials .", "We propose that despite the predominance of less defended mimics the three predatory guilds avoid the mimics because of the additive influence of the various defensive traits ." ]

[ "Many animals use bright colours to warn a potential predator that they can defend themselves .", "Wasps , for instance , are armed with a harmful sting and advertise this fact via their distinctive yellow and black stripes .", "Predators often learn to heed such warnings and avoid these unpalatable animals in future .", "As a result , animals that mimic another animal’s warning signals can reap the benefit of being left alone by predators even if they are otherwise undefended .", "Textbooks on evolution are typically full of different examples of mimicry .", "However , the specifics of these examples are often poorly understood .", "Ninety years ago a famous Australian entomologist , Alexander Nicholson , suggested the existence of large groups of mimics in the Australian wildlife .", "More of these so-called “mimetic complexes” have recently been recognized among several species of insect , but not previously in ants .", "Now , Pekár et al . have looked at all known ants and ant-like mimics in Australia and discovered over 140 species that use gold and black colours as a warning signal .", "Most of the species were ants , but the collection of mimics also includes wasps , spiders , true bugs and insects called treehoppers .", "Some of the mimics were less palatable than others , and they possessed a range of defences , including spines and foul-tasting chemicals .", "Pekár et al . then looked in the guts of 12 species of predators in the wild , and found that very few of them ate the mimics .", "When mimics were offered to three different predators ( specifically a lizard and two species of spider ) , most avoided the mimics regardless of whether they were palatable or unpalatable .", "Instead , the predators preferred to eat a spider that was not a member of the group of mimics because it lacked the gold colouration .", "Further studies are now needed to continue to document the details of this and other mimetic complexes .", "For example , this includes revealing how the different defences protect the members of the complex from predators do not use vision to recognize their prey and so cannot see the warning colouration .", "All this is needed to understand evolutionary processes that have fascinated biologists for decades , and explain how such large mimetic complexes evolved and persisted in spite of the influence of the community of predators ." ]

[ "Results", "Discussion" ]

[ "computational and systems biology", "neuroscience" ]

[ [ "The spontaneous dynamics produced by the network is constrained to the low dimensional manifold codified in the connectivity matrix and spanned by the parameter x→ .", "The short-range interactions and the uniform inhibition enforced by the firing threshold h0 produce a localized 'bump' of activity in the manifold .", "The presence of an asymmetry in the connection strengths prevents the system from remaining in a stationary equilibrium .", "Instead , it generates a steady flow of activity in the direction of the asymmetry .", "This flow is illustrated in Figure 2", "( a ) ,", "( b ) and", "( c ) , obtained with numerical simulation of a network encoding a one- , two- , or three-dimensional manifold , respectively .", "In the simulation , each neuron is assigned to a preferential firing location xi→ in the manifold to encode , and the plots show the activity of the network organized according to this disposition .", "The activity of the population clusters in a bump around a certain position at each time point ( t1 , t2 , and t3 ) , and the bump shifts by effect of the asymmetric component of the interactions .", "In this way , the neural population collectively encodes an evolving coordinate on the manifold spanned by x→ .", "The coherence of the representation is not affected by the presence of the asymmetric term: the movement of the activity bump happens without dissipation .", "The speed of movement of the bump is modulated by the value of the parameter γ and by the sparsity level of the representation f , that is the fraction of neurons active at each given time during the dynamics .", "The dependence of the speed on these two parameters is illustrated in 2", "( d ) .", "Stronger asymmetry ( high γ ) produces a faster shift .", "Interestingly , the sparsity value f acts as a modulator of the influence of γ: sparser representations move more slowly than dense ones .", "While γ describes a feature of the synaptic interactions , determined during the learning phase and relatively fixed at the short timescales of retrieval , f can be instantaneously modulated during retrieval dynamics .", "A change in the gain or the excitability of the population can be used to produce dynamic retrieval at different speeds .", "Thus , the model predicts an interaction between the sparsity and the speed of the reactivation of a continuous memory sequence , with increased activity leading to faster replay .", "It is worth noting , however , that the direction of the dynamics is fixed with J: the model is able to retrieve either forward or backward sequences , but not alternate between them .", "The dependence of the retrieval speed s on γ and f is well described by the approximate functional form ( 6 ) s⁢ ( γ , f ) =A⁢γ⁢fb⁢γ+c⁢f+d⁢γ⁢f+e⁢γ⁢f2 This dependence is shown in 2", "( b ) , where the dots are the values obtained with numerical simulations and the full curves the fitted relationship .", "The full understanding of the nature of this functional form remains an open challenge for future analysis .", "As we will see in the next section , the analytical solution of the model yields the same form to describe the dependence of speed on γ and f , but a closed-form solution is still lacking .", "In the model presented , the asymmetry in the interactions is enforced uniformly along a single direction also for two- and three-dimensional manifolds , representing the case in which neural dynamics follows a forced trajectory along one dimension , but is free to move without energy costs along the others .", "However , the same mechanism can be used to produce one-dimensional trajectories embedded in low-dimensional manifolds , with the introduction of a positional dependence in the direction of the asymmetry ( Blum and Abbott , 1996 ) .", "In this case , an interesting problem is posed by the intersection of two trajectories embedded in the same manifold: is the network , during the retrieval of one trajectory , able to cross these intersections , or do they hinder dynamical retrieval ?", "The investigation of the full phenomenology of position-dependent asymmetric kernels with intersecting trajectories is beyond the scope of the present work , but we present in Figure 2 ( e ) a numerical study of a minimal version of this problem , with two orthogonal trajectories ( Figure 2 ( e ) , black arrows ) embedded in a 2D manifold and memorized simultaneously in the network .", "Notice that in this case the two trajectories are parallel to the main axis of the square environment , but they do not need to be: any pair of orthogonal trajectories will behave in the same manner .", "When the network is cued to retrieve the horizontal trajectory , the behavior at the intersection depends on the strength γ and scale ξ of the asymmetric component .", "At low γ , the dynamics spontaneously switch trajectory at the intersection ( Figure 2 ( e ) , blue curve ) , while for γ sufficiently large the retrieval of the horizontal trajectory is successful ( Figure 2 ( e ) , orange curve ) .", "The value γ* required for a successful crossing depends on the spatial scale ξ: larger ξ allow for crossing with lower values of γ , as shown in the top-right inset of Figure 2 ( e ) , in which γ*⁢ ( ξ ) is plotted .", "Intuitively , the ability of the network to retrieve crossing trajectories depends on the shape of the activity bump , which needs to be sufficiently elongated in the direction of retrieval for the successful crossing of the intersection .", "The blue and orange insets of Figure 2 ( e ) show the difference in shape of the bump in the case of a trajectory switch ( blue ) and successful crossing ( orange ) .", "The simplicity of continuous attractor models often allows to extract important computational principles from their analytical solution ( Wu et al . , 2008; Fung et al . , 2010 ) .", "In our case , the dynamic behavior of the system and its features can be fully described analytically with a generalization of the framework developed by Battaglia and Treves , 1998 .", "For this purpose , it is easier to formulate the problem in the continuum , and describe the population activity {Vi} by its profile V⁢ ( x→ ) on the attractive manifold parametrized by the coordinate x→ , and the dynamical evolution as a discrete step map , equivalent to Equation 1 . ( 7 ) V⁢ ( x→ , t+1 ) =g⁢[h⁢ ( x→ , t ) ]+ ( 8 ) h⁢ ( x→ , t ) =∫𝑑x→′⁢K⁢ ( x→-x→′ ) ⁢V⁢ ( x→′ , t ) -h0 The requirement of a rigid shift of population activity is then imposed by setting the activity at time t+1 to be equal at the activity at time t , but translated by an amount Δ⁢x→=τ⁢s⁢n→ , proportional to the speed s of the shift and in the direction n→ of the asymmetry in the connections .", "The timescale τ sets the time unit in which the duration of the evolution is measured and does not have an impact on the behavior of the system .", "The activity profile V⁢ ( x→ ) is then found as the self-consistent solution to the integral equation ( 9 ) V⁢ ( x→+Δ⁢x→ ) =g⁢[∫𝑑x→′⁢K⁢ ( x→-x→′ ) ⁢V⁢ ( x→′ ) -h0]+ Equation 9 is valid in general .", "We will focus here , for the explicit derivation ( reported in appendix C ) , on the case of a one dimensional manifold with an exponential interaction kernel ( 10 ) K⁢ ( x-x′ ) =e-|x-x′|+γ⁢sign⁢ ( x-x′ ) ⁢e-|x-x′| In this case , the activity bump will take the form: ( 11 ) V⁢ ( x ) ={C⁢ek1⁢x⁢cos⁡ ( k2⁢x ) +g⁢θ1-2⁢gif -R≤x≤R0if -R>x⁢ or ⁢x>R The parameters k1=k1⁢ ( γ , s ) and k2=k2⁢ ( γ , s ) determine the properties of the solution and they depend on the values of γ and speed s=Δ⁢x/τ .", "k2 is related to the bump width by the relation ( 12 ) R=π2⁢k2where R is the point at which V⁢ ( x ) =0 .", "k1 is related to the asymmetry of the bump: in the limit case γ=0 , s=0 ( Figure 3", "( a ) , first column ) k1=0 , and we recover the cosine solution of the symmetric kernel case studied in Battaglia and Treves , 1998 .", "Larger k1 values result in more and more asymmetric shapes ( Figure 3", "( a ) , second and third columns ) .", "From this analytical solution , we can determine the dependence of the speed s on the asymmetry strength γ and on the sparsity f=2⁢R/L ( note that in the continuum case the fraction of active neurons is given by the ratio between the bump size 2⁢R and the manifold size L ) .", "The sparsity f is modulated by the value of the gain g , as shown in Figure 3", "( b ) : a larger gain in the transfer function corresponds to a sparser activity .", "The exact relation s⁢ ( γ , f ) can be obtained from the numerical solution of a transcendental equation ( see appendix C ) , and can be approximated with a functional shape analogous to the one used for the simulated network: ( 13 ) s⁢ ( γ , f ) =A⁢γ⁢fb⁢γ+c⁢f+d⁢γ⁢f+e⁢γ⁢f2 The full transcendental solution and the fitted curves are reported in Figure 3", "( c ) .", "The analytical solution of the model shows that the network performs dynamical retrieval for all values of the asymmetry strength γ , and that this parameter influences the retrieval speed and the shape of the activity bump .", "To further investigate the robustness of dynamical retrieval to parameter changes , we investigate with numerical simulations the behavior of the model with respect to another important parameter: the scale ξ of the anti-symmetric component .", "We run several dynamics of a network with interaction kernel given by ( 14 ) K⁢", "( d ) =e-d+γ⁢sign⁢", "( d ) ⁢e-d/ξvarying the parameters γ and ξ and measuring the retrieval speed , the peak value of the activity bump and its skeweness .", "The joint effects of γ and ξ are shown in Figure 4 .", "In the whole range of parameters analyzed , spanning four orders of magnitude for both parameters , the network was able to produce dynamic retrieval .", "γ and ξ affect the speed of the shift , the peak values of the activity distribution and the skewness of the activity bump , without hindering network functionality .", "Moreover , all these feature vary gradually and mildly with the parameters values , producing dynamical behavior qualitatively similar in the full parameter range .", "This analysis shows that dynamical retrieval does not require any fine tuning of network parameters , but relies on the assumption of an exponential shape for the interaction kernel .", "How robust is the behavior of the network to the details of the kernel shape ?", "We addressed this question by simulating the network dynamics with alternative kernel choices .", "We kept fixed the symmetric component , and explored three different anti-simmetric shapes: a gaussian-derivative shape ( Figure 5a ) , a sinusoidal shape ( Figure 5b ) and a double step function ( Figure 5c ) .", "Each of these simulations produced the same retrieval dynamics ( a stable bump shifting at constant speed ) , the only effect of the kernel shape being on the details of the shape of the activity bump ( Figure 5 , bottom row ) .", "This shows that the dynamic retrieval mechanism , much like standard continuous attractors , is robust with respect to the precise shape of the interactions .", "Importantly , no particular relationship is required between the shape of the symmetric and the anti-symmetric components of the kernel .", "We described in detail the behavior of a neural network with asymmetric connectivity in the case of a single manifold encoded in the synaptic connectivity .", "For the network to behave as an autoassociative memory , however , it needs to be able to store and dynamically retrieve multiple manifolds .", "This is possible if we construct the interaction matrix Ji⁢j as the sum of the contributions from p different , independently encoded manifolds: ( 15 ) Ji⁢j=1N⁢∑μ=1pK⁢ ( x→iμ-x→jμ ) Here , each xiμ represent the preferred firing location of neuron i in the manifold μ , and K is the same interaction kernel as in Equation 4 , containing a symmetric and anti-symmetric component .", "The resulting dynamics show multiple continuous attractors , corresponding to the stored manifolds .", "Given an initial configuration , the networks rapidly converges to the nearest ( i . e . most correlated ) attractor , forming a coherent bump that then moves along the manifold as a consequence of the asymmetric component of the connectivity .", "The same dynamics , if projected on the other unretrieved manifolds , appear as random noise .", "This is illustrated in Figure 6 obtained with numerical simulations of a network encoding three different manifolds ( of dimension one in", "( a ) , dimension two in", "( b ) ) , and dynamically retrieving the first one .", "How does this shifting activity bump relate to the activity of single cells ?", "To clarify this aspect we simulated an electrophysiological recording from the dynamical retrieval of Figure 6", "( a ) .", "We selected a random subset of 15 of the 1000 cells of the network , and generated spike trains using a poisson point process with an instantaneous firing rate proportional to the activity level of each cell yielded by the retrieval dynamics at each time step , plus a small noise .", "With this procedure we obtain the spike trains of each cell , that we can visualize with a rasterplot ( Figure 6c ) .", "We can sort the cells according to their firing field position on each of the three manifolds ( if these were , e . g . , linear tracks , this would correspond to sorting according to place field positions in each of the tracks ) .", "When ordered as in the retrieved manifold , the recorded cells show a structured pattern that is considered the hallmark of sequential activity in experimental studies ( Figure 6c , first column ) .", "If the same spikes are ordered according to the unretrieved manifolds , the pattern is lost ( Figure 6c , second and third column ) , indicating that the population activity is retrieving the dynamical structure of the first manifold specifically .", "Multiple dynamic manifolds can be memorized and retrieved by the network , with different speeds .", "Figure 7", "( b ) shows the result of the numerical simulation of a network with five different one-dimensional manifold stored in its connectivity matrix , each encoded with a different value of γ ( see appendix D ) .", "These manifold are dynamically retrieved by the network at different speeds , depending on the corresponding γ .", "This allows the model to simultaneously store memories without the constraint of a fixed dynamical timescale , an important feature for the description of biological circuits that need to be able to operate at different temporal scales .", "Different memories stored in the same neural population can interact with each other , building more retrieval schemes in which , for example , the retrieval of a memory cues the retrieval of another one .", "To investigate this possibility , we have incorporated in the model a mechanism for interaction between memories , in which the endpoint of a dynamical , one-dimensional manifold elicits the activation of the start point of a different one ( see Appendix E ) .", "This results in the sequential retrieval of multiple memories , one after the other , as illustrated in Figure 7", "( d ) .", "The top row shows the evolution in time of the overlaps mμ: ( 16 ) mμ⁢ ( t ) =1N2⁢∑i , jKS⁢ ( xiμ-xjμ ) ⁢Vi⁢ ( t ) ⁢Vj⁢ ( t ) These order parameters quantify the coherence of the population activity V⁢ ( t ) with each of the manifolds .", "Localized activity in manifold μ results in a large mμ , while a low mμ corresponds to an incoherent scattering of the activity .", "The network retrieves the manifolds in sequence , one at a time , following the instructed transitions encoded in its connectivity .", "The all-or-nothing behavior of the coherence parameters segments the continuous dynamics of the network into a sequence of discrete states .", "The bottom row shows the evolution of the retrieved position , given in each manifold by the center of mass: ( 17 ) <x>μ⁢ ( t ) =1N⁢∑ixiμ⁢Vi⁢ ( t ) The dynamic runs across the retrieved manifold , from its beginning to its end , then jumps to next one and repeats the process .", "Note that the position in each of the un-retrieved manifold fluctuates around L/2 , as a consequence of the incoherence of the activity .", "Within each of the retrieved manifolds , the dynamic retains its continuous nature in the representation of the evolving position .", "This sequential dynamic goes beyond the simple cued retrieval of independent memories that is the focus of most autoassociative memory models , and provides an example of a hybrid computational system , encoding both continuous and discrete features .", "The interaction mechanism introduced here provides the opportunity to investigate the effect of more complex interactions than the simple memory chain presented here .", "We present here this first example as a proof of principle of the possibility of storing interacting dynamical memories , and will proceed to the investigation of more complex structures ( e . g . interaction networks , probabilistic interactions , etc . ) in future studies .", "The number of maps that can be stored and retrieved by an attractor network of this kind is typically proportional to the number of inputs per neuron C ( Treves and Rolls , 1991 ) .", "The memory load α=p/C crucially determines the behavior of the system: when α is increased above a certain threshold value αc , the network is not able to retrieve any of the stored memories , falling instead into a disordered state .", "Therefore it is the magnitude of αc , that is the storage capacity of the system , that determines how effectively it can operate as a memory .", "To estimate the storage capacity of dynamic continuous attractors , and to investigate how it is impacted by the presence of asymmetric connections , we proceed along two complementary paths .", "In the case a fully connected network , where the analytical tools developed for equilibrium systems are not applicable , we take advantage of the fact that numerical simulations can be effective for the estimation of the capacity , since the number of connections per neuron C ( the relevant parameters in the definition of the storage capacity αc=p/C ) coincides with the number of neurons , minus one .", "For a highly diluted system , on the other hand , the number of neurons is much larger than C , making the simulation of the system very difficult in practice .", "We then resort to an analytical formulation based on a signal-to-noise analysis ( Battaglia and Treves , 1998 ) , that exploits the vanishing correlations between inputs of different neurons in a highly diluted network , and does not require symmetry in the connectivity ( Derrida et al . , 1987 ) .", "The quantification of the effect of loops in the dense connectivity regime , developed in Shiino and Fukai , 1992 and Roudi and Treves , 2004 for the case of static , discrete attractors , is beyond the scope of the present work and remains an interesting open direction .", "In both the fully connected and the highly diluted case we study the dependence of the capacity on two important parameters: the map sparsity , that is the ratio between the width of the connectivity kernel ( fixed to one without loss of generality ) and the size L of the stored manifolds , and the asymmetry strength γ .", "Note that the map sparsity 1/L is different from the activity sparsity f: the former is a feature of the stored memories , that we will treat as a control parameter in the following analysis; the latter is a feature of the network dynamics , and its value will be fixed by an optimization procedure in the calculation of the maximal capacity .", "The signal-to-noise approach we follow , illustrated in details in Battaglia and Treves , 1998 , involves writing the local field hi as the sum of two contributions: a signal term , due to the retrieved – ‘condensed’ – map , and a noise term consisting of the sum of the contributions of all the other , ‘uncondensed’ maps .", "In the diluted regime ( C/N→0 ) , these contributions are independent and can be summarized by a Gaussian term ρ⁢z , where z is a random variable with zero mean and unit variance .", "In the continuous limit , assuming without loss of generality that map μ=1 is retrieved , we can write: ( 18 ) h⁢ ( x1 ) =g⁢∫L𝑑x1⁣′⁢K⁢ ( x1-x1⁣′ ) ⁢V⁢ ( x1⁣′ ) +ρ⁢z The noise will have variance: ( 19 ) ρ2=α⁢y⁢L2⁢⟨⟨K2⁢ ( x-x′ ) ⟩⟩where L is the size of the map , ⟨⟨K2⁢ ( x-x′ ) ⟩⟩ is the spatial variance of the kernel and ( 20 ) y=1N⁢∑iVi2is the average square activity .", "We can write the fixed point equation for the average activity profile m1⁢ ( x ) , incorporating the dynamic shift with an argument similar to the one made for the single map case: ( 21 ) m1⁢ ( x+Δ⁢x ) =g⁢∫+D⁢z⁢ ( h⁢ ( x ) -h0 ) where D⁢z= ( e-z2/2/2⁢π ) ⁢d⁢z and ∫+f⁢ ( x ) ⁢𝑑x=∫f⁢ ( x ) ⁢θ⁢ ( x ) ⁢𝑑x .", "The average square activity y , entering the noise term , reads ( 22 ) y=g2L⁢∫𝑑x⁢∫+D⁢z⁢ ( h⁢ ( x ) -h0 ) 2 Introducing the rescaled variables ( 23 ) w=-h0ρ ( 24 ) v⁢ ( x ) =m1⁢ ( x ) ρ And the functions ( 25 ) 𝒩⁢ ( x ) =x⁢Φ⁢ ( x ) +σ⁢ ( x ) ( 26 ) ℳ⁢ ( x ) = ( 1+x2 ) ⁢Φ⁢ ( x ) +x⁢σ⁢ ( x ) where Φ⁢ ( x ) and σ⁢ ( x ) are the Gaussian cumulative and the Gaussian probability mass function respectively , we can rewrite the fixed-point equation as ( 27 ) v⁢ ( x+Δ⁢x ) =g⁢𝒩⁢ ( ∫𝑑x′⁢K⁢ ( x-x′ ) ⁢v⁢ ( x′ ) +w ) ( 28 ) y=ρ2⁢g2⁢∫d⁢xL⁢ℳ⁢ ( ∫𝑑x′⁢K⁢ ( x-x′ ) ⁢v⁢ ( x′ ) +w ) Substituting Equation 28 in the expression for the noise variance 19 we obtain ( 29 ) 1α=g2⁢L⁢⟨⟨K2⟩⟩⁢∫𝑑x⁢ℳ⁢ ( ∫𝑑x′⁢K⁢ ( x-x′ ) ⁢v⁢ ( x′ ) +w ) If we are able to solve Equation 27 for the rescaled activity profile v⁢ ( x ) , we can use Equation 29 to calculate α .", "We can then maximize α with respect to g and w: this yields the maximal value αc for which retrieval solutions can be found .", "These equations are valid in general and have to be solved numerically .", "Here we present the results for the case of one-dimensional manifolds and interactions given by the exponential kernel of Equation 36 .", "In this case , we have ( 30 ) ⟨⟨K2⁢ ( x-x′ ) ⟩⟩= ( 1+γ2 ) ⁢⟨⟨KS2⁢ ( x-x′ ) ⟩⟩ .", "where KS⁢ ( x-x′ ) =e-|x-x′| is the symmetric component of the kernel .", "A simple approximation , illustrated in appendix F along with the detailed solution procedure , allows to decouple the dependence of αc on γ and L , with the former given by the spatial variance given by Equation 30 and the latter by the solution of Equations 27 and 29 in the γ=0 case .", "We therefore have: ( 31 ) αc⁢ ( L , γ ) ∼αc⁢ ( L , 0 ) / ( 1+γ2 ) The storage capacity is plotted in Figure 8", "( a ) as a function of γ and L . For sparse maps and small values of the asymmetry , the capacity scales as ( 32 ) αc∼-1ln⁡ ( 1/L ) ⁢ ( 1+γ2 ) The scaling with 1/L is the same found by Battaglia and Treves , 1998 in the analysis of the symmetric case , as expected: for γ=0 the two models are equivalent .", "The presence of asymmetry decreases the capacity , but does not have a catastrophic effect: the decrease is continuous and scales with a power of γ .", "There is therefore a wide range of values of asymmetry and map sparsity in which a large number of dynamic manifolds can be stored and retrieved .", "To estimate the storage capacity for a fully connected network , we proceed with numerical simulations .", "For a network of fixed size N , and for given γ , L and number of maps p , we run a number of simulations NS , letting the network evolve from a random initial configuration .", "We consider a simulation to have performed a successful retrieval if the global overlap ( 33 ) mμ=1N2⁢∑i≠jVi⁢Vj⁢KS⁢ ( xiμ-xjμ ) that quantifies the coherence of the activity with map μ , is large for one map μ* ( at least 95% of the overlap value obtained in the case of a single map ) and low in all others maps μ≠μ* .", "We then define the retrieval probability as pr=NR/NS , where NR is the number of observed retrievals .", "We repeat the process varying the storage load , that is the number of stored manifolds p .", "As p is increased , the system reaches a transition point , at which the retrieval probability rapidly goes to zero .", "This transition is illustrated , for various values of γ , in Figure 8", "( b ) .", "The number of maps pc at which the probability reaches zero defines the storage capacity αc⁢ ( γ , L ) =pc⁢ ( γ , L ) /N .", "Repeating this procedure for a range of values of γ and L , we obtain the plots shown in Figure 8", "( c ) , for networks encoding one dimensional and two dimensional dynamical memories .", "The first thing that can be noticed is that the network can store a large number of maps in the fully connected case as well , for a wide range of γ and L . A network with size in the order of ten thousand neurons could store from tens up to hundreds of dynamical memories .", "The capacity for one dimensional attractors is higher than the one for their two dimensional counterparts .", "This is in line with what was found for symmetric networks ( Battaglia and Treves , 1998 ) .", "Finally , we see that the peak of the capacity is found not only for intermediate values of map sparsity – again in line with what is known from the symmetric case – but also for intermediate values of the coefficient γ .", "This shows that moderate values of asymmetry can be beneficial for the storage of multiple continuous attractors , a non-trivial phenomenon that may be crucial for the memory capacity of biological networks .", "In particular this suggests that the natural tendency of the neural activity to show a rich spontaneous dynamics not only does not hinder the possibility for multiple memories to coexist in the same population , but can be a crucial ingredient for the correct functioning of memory mechanisms ." ], [ "The results presented show how a continuous attractor neural network with memory-dependent asymmetric components in the connectivity can function as a dynamic memory .", "Our model is simple enough to be treated analytically , robustly produces dynamic retrieval for a large range of the relevant parameters and shows a storage capacity that is comparable to – and in some cases higher than – the capacity for static continuous attractors .", "The analytical solution of the single manifold case shows that the interaction between the strength of the asymmetry and the velocity of the shift can be modulated by global features of the network activity such as its sparsity .", "This makes the network able to retrieve at different velocities in different regimes , without necessarily requiring short term synaptic modifications .", "The dependence of the retrieval speed from the sparsity of the activity yields a testable prediction in the context of hippocampal replay: faster reactivations are to be expected in association with an increase in the excitability of the population .", "The insensitivity of the general features of the dynamics to the fine details of the shape of the interactions suggests that this mechanism could robustly emerge from learning or self organization processes in the presence of noise .", "The quantitative analysis of the learning process needed to effectively memorize low-dimensional dynamic manifolds is an interesting open direction , which goes beyond the scope of this work .", "However , the asymmetric Hebbian plasticity rule used here provides a simple and biologically realistic starting point .", "Our analysis shows that the simple introduction of an aysmmetric Hebbian plasticity rule is sufficient to describe a dynamic memory able to store and retrieve multiple manifolds with different speeds , and it can incorporate interactions between them , producing the chained retrieval of a sequence of continuous memories .", "The central result of the paper is the quantification of the storage capacity for dynamic continuous attractors , that we find to be large in magnitude , only mildly impacted by the asymmetry in diluted networks , and even higher than the capacity for static attractors in fully connected networks with moderate degrees of asymmetry .", "The storage capacity of out of equilibrium continuous attractors has been calculated , in a different scenario , by Zhong et al . , 2020 .", "The authors considered the case of an external signal driving the activity bump along the attractor , in a network of binary neurons , and proceeded to calculate the storage capacity with several assumptions that allowed to model the interference of multiple maps as thermal noise .", "Interestingly , their main result is broadly compatible with what we show here: in the highly diluted regime the velocity of the external signal has a mild – detrimental – effect on the capacity .", "This hints that out of equilibrium effects could show some form of universality across different network models and implementations of the shift mechanism .", "Moreover , a high capacity for dynamical sequences has shown to be achievable also in the case of discrete items ( Gillett et al . , 2020 ) .", "Together these results suggest that the introduction of a temporal structure is compatible with the functioning of autoassociative memory in recurrent networks , and they open the way to the use of attractor models for the quantitative analysis of complex memory phenomena , such as hippocampal replay and memory schemata .", "The model we propose suggests that the tendency of the activity to move in the neural population is a natural feature of networks with asymmetric connectivity , when the asymmetry is organized along a direction in a low dimensional manifold , and that static memories could be the exception rather than the rule .", "Indeed , Mehta et al . , 2000 have shown that place fields can become more asymmetric in the course of spatial learning , demonstrating that the idea that symmetry emerges from an averaging of trajectory-dependent effects ( Sharp , 1991 ) does not always hold true .", "The structural role of the asymmetry has important implications for the analysis methods used to describe the activity of large populations of neurons , which often rely on the assumption of symmetry in the interactions ( e . g . in the analysis of pairwise correlations ) or equilibrium of the neural activity ( e . g . the standard inverse Ising inference ) .", "In most of the two- and three-dimensional cases analysed here , the asymmetry is constant along a single direction in each attractor .", "This can describe the situation in which the temporal evolution of the memory is structured along a certain dimension , and free to diffuse , without energy costs , in the remaining ones .", "The description of several one-dimensional trajectories embedded in a two dimensional or three dimensional space requires a position-dependent asymmetric component .", "A systematic analysis of this situation is left for future analysis .", "However , the simple case of two intersecting trajectories embedded in a 2D map , analysed here , provides a proof of concept that several intersecting trajectories can be correctly retrieved , provided that the activity bump is sufficiently elongated in the direction of the trajectory .", "A progressive elongation of the place fields in the running direction has been observed in rats running on a linear track ( Mehta et al . , 1997 ) , and our analysis predicts that an analogous effect would be observed also in open-field environments , when restricting the analysis to trajectories in the same running direction .", "Another challenge is posed by the evidence that replayed sequences can be organized both forward and backward in time ( Foster and Wilson , 2006 ) .", "The model in its current formulation can produce the retrieval of a given sequence either forward or backward , but cannot alternate between the two .", "This suggests that , if replay relies on asymmetric connections , the hippocampus would have to use different representations for the forward and the backward component .", "The fact that a change in reward uniquely modulates backward replay ( Ambrose et al . , 2016 ) provides some evidence in this direction , but this question remains open to experimental investigation .", "The dynamical retrieval of the model generalizes , in the framework of attractor networks , the idea of cognitive maps , incorporating a temporal organization in the low-dimensional manifold encoding the structure of the memory .", "This feature is reminiscent of the idea of memory schemata – constructs that can guide and constrain our mental activity when we reminisce about the past , imagine future or fictional scenarios or let our minds free to wander ( Ciaramelli and Treves , 2019 ) .", "The use of the present model to describe memory schemata will require further steps , such as an account of the interaction between hippocampus and neocortex , and the expansion of the mechanism describing the transition between different dynamical memories .", "Nevertheless , the idea of dynamic retrieval of a continuous manifold and the integration of the model presented here with effective models of cortical memory networks ( Boboeva et al . , 2018 ) open promising perspectives .", "Finally , the full analytical description of a densely connected asymmetric attractor network is a challenge that remains open , and can yield valuable insights on the workings of the neural circuits underlying memory ." ] ]

[ "Episodic memory has a dynamic nature: when we recall past episodes , we retrieve not only their content , but also their temporal structure .", "The phenomenon of replay , in the hippocampus of mammals , offers a remarkable example of this temporal dynamics .", "However , most quantitative models of memory treat memories as static configurations , neglecting the temporal unfolding of the retrieval process .", "Here , we introduce a continuous attractor network model with a memory-dependent asymmetric component in the synaptic connectivity , which spontaneously breaks the equilibrium of the memory configurations and produces dynamic retrieval .", "The detailed analysis of the model with analytical calculations and numerical simulations shows that it can robustly retrieve multiple dynamical memories , and that this feature is largely independent of the details of its implementation .", "By calculating the storage capacity , we show that the dynamic component does not impair memory capacity , and can even enhance it in certain regimes ." ]

[ "When we recall a past experience , accessing what is known as an ‘episodic memory’ , it usually does not appear as a still image or a snapshot of what occurred .", "Instead , our memories tend to be dynamic: we remember how a sequence of events unfolded , and when we do this , we often re-experience at least part of that same sequence .", "If the memory includes physical movement , the sequence combines space and time to remember a trajectory .", "For example , a mouse might remember how it went down a hole and found cheese there .", "However , mathematical models of how past experiences are stored in our brains and retrieved when we remember them have so far focused on snapshot memories .", "‘Attractor network models’ are one type of mathematical model that neuroscientists use to represent how neurons communicate with each other to store memories .", "These models can provide insights into how circuits of neurons , for example those in the hippocampus ( a part of the brain crucial for memory ) , may have evolved to remember the past , but so far they have only focused on how single moments , rather than sequences of events , are represented by populations of neurons .", "Spalla et al . found a way to extend these models , so they could analyse how networks of neurons can store and retrieve dynamic memories .", "These memories are represented in the brain as ‘continuous attractors’ , which can be thought of as arrows that attract mental trajectories first to the arrow itself , and once on the arrow , to the arrowhead .", "Each recalled event elicits the next one on the arrow , as the mental trajectory advances towards the arrowhead .", "Spalla et al . determined that memory networks in the hippocampus of mammals can store large numbers of these ‘arrows’ , up to the same amount of ‘snapshot’ memories predicted to be stored with similar models .", "Spalla et al . ’s results may allow researchers to better understand memory storage and recall , since they allow for the modelling of complex and realistic aspects of episodic memories .", "This could provide insights into processes such as why our minds wander , as well as having implications for the study of how neurons physically interact with each other to transmit information ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "developmental biology", "physics of living systems" ]

[ [ "During morphogenesis , tissues with complex morphologies are formed from the collective interplay of many cells .", "This process involves spatial patterns of signaling activity , and previous work has discovered mechanisms for generating tissue-scale patterns of activity in signaling pathways such as Hedgehog , TGFβ , and Wnt ( Green and Sharpe , 2015 ) .", "In addition , patterns of cellular morphology arise during morphogenesis .", "Such patterns can be important for ensuring the function of the resulting tissue .", "For example , the compound eye of Drosophila consists of hundreds of ommatidia organized in a precise hexagonal array that is required to fully sample the visual field ( Kumar , 2012 ) .", "Patterns of cellular morphology that arise during morphogenesis can also guide the morphogenetic processes itself .", "For example , spatial patterns of cell morphology emerge during growth of the Drosophila larval imaginal discs , which are precursors of adult tissues ( Aegerter-Wilmsen et al . , 2010; Condic et al . , 1991; Legoff et al . , 2013; Mao et al . , 2013 ) .", "These patterns have been proposed to be involved in the eversion process , during which these flattened epithelial sacs turn themselves inside out when the animal transitions from larva to pupa ( Condic et al . , 1991 ) .", "While extensive work has studied the emergence of biochemical signaling patterns , how patterns of cellular morphology arise during tissue development is poorly understood .", "Here , we investigate tissue-scale patterning of cell morphology in the Drosophila larval wing imaginal disc , which has a geometry that is ideal for studying spatial patterns of epithelial cell morphology .", "We focus specifically on the cell shape patterns in the central ‘pouch’ region , which is the precursor of the adult wing blade .", "To a good approximation , this region is planar and ellipsoidal .", "Cells near the center have smaller cell areas and are more isotropic in shape , whereas cells near the periphery have larger cell areas and are elongated tangentially ( Aegerter-Wilmsen et al . , 2010; Legoff et al . , 2013; Mao et al . , 2013 ) .", "Cell shape has been correlated with mechanical stress: tangentially oriented bonds of elongated cells in the periphery are under higher tension than radially oriented bonds ( Legoff et al . , 2013 ) .", "It has been previously proposed that this pattern of cell morphology in the wing pouch could stem from differential proliferation: if the center grows faster than the rest , the resulting area pressure could stretch peripheral cells tangentially ( Mao et al . , 2013 ) .", "Indeed , there is evidence to suggest that cells divide slightly faster closer to the center during very early stages ( before 80hr after egg laying , AEL ) .", "It was suggested that this early growth differential is sufficient to account for the persistence of the cell morphology pattern through the remaining ∼40hr of development .", "However , it has since been shown that cell rearrangements occur ( Dye et al . , 2017; Heller et al . , 2016 ) , which could relax stress patterns once growth has become uniform .", "Furthermore , stress patterns may even relax during homogeneous growth in the absence of cell rearrangements ( Ranft et al . , 2010 ) .", "Thus , it remains unclear how cell morphology patterns generated early by differential growth could be maintained through later stages , and alternative mechanisms for the establishment of these patterns must be considered .", "Here , we measure the spatial patterns of cell morphology , cell divisions , and cell rearrangements during the middle of the third larval instar ( starting at 96hr AEL ) .", "We quantify the pattern of tangential cell elongation and show that it becomes stronger over time , even though growth is spatially uniform and cell rearrangements are frequent .", "Strikingly , this change in tangential cell elongation is coupled to a radially biased pattern of cell neighbor exchanges .", "Using a physical model of tissue dynamics , we show that active patterning of radial cell neighbor exchanges can account for the observed morphology patterns in the absence of differential growth .", "Lastly , using a combination of experiment and theory , we provide evidence that this active patterning could be self-organized by mechanosensitive feedback ." ], [ "Cell morphology patterns in the wing disc have been previously analyzed using static images ( Aegerter-Wilmsen et al . , 2010; Legoff et al . , 2013; Mao et al . , 2013 ) .", "However , relating cell morphology patterns to patterns of growth , cell divisions , and cell rearrangements requires dynamic data .", "We therefore performed long-term timelapse imaging of growing explanted wing discs using our previously described methods ( Dye et al . , 2017 ) , starting at 96hr AEL and continuing for ∼13hr of imaging .", "We used Ecadherin-GFP as an apical junction marker ( Figure 1A ) .", "To quantify cell morphology , we averaged apical cell area and cell elongation locally in space , using data from five wing discs , and in time using ∼2hr intervals .", "Over the course of the 13hr time-lapse , the qualitative features of the morphological patterns do not change .", "Therefore , in Figure 1B–C , we present the spatial patterns calculated for the middle timepoint .", "Cell area is represented as a color code .", "Cell elongation is characterized by a tensor Q , which defines an axis and a strength of elongation and is represented by bars in Figure 1C ( see Materials and methods , Figure 1—figure supplement 1B; Etournay et al . , 2015; Merkel et al . , 2017 ) .", "To quantify the radial symmetry of this pattern , we first determined the center of symmetry ( Figure 1—figure supplement 1 and Materials and methods ) .", "We then introduce a polar coordinate system at the center with the radial coordinate r and present the radial projection Qrr of the cell elongation tensor as a color code in Figure 1C ( see also Figure 1—figure supplement 1 ) .", "This figure highlights the pattern of tangential cell elongation , with cells elongating on average perpendicular to the radial axis ( blue in Figure 1C ) .", "It also reveals that this pattern is interrupted around the Dorsal-Ventral ( DV ) boundary , where cells are elongated parallel to this boundary ( red in Figure 1C ) .", "We quantify this region separately ( Figure 1—figure supplement 2 ) and exclude it from our analysis of the circular patterns ( Figure 1D–F ) .", "The Anterior-Posterior ( AP ) boundary also affects cell morphology ( Landsberg et al . , 2009 ) , but the effect is weaker and more variable at this stage; thus , we do not quantify it separately .", "The spatial maps of cell morphology reveal that both cell area and cell elongation magnitude are largest at the periphery and decrease toward the center ( Figure 1B–C ) .", "We quantified this radial gradient in cell area and observe that cell area ranges from ∼3−7μm2 when moving toward the periphery ( Figure 1E ) .", "We also observe a radial gradient in cell elongation starting from Qrr≈0 at r=10 μm and extending to about Qrr≈-0 . 1 at r=40 μm ( Figure 1F ) .", "The negative value corresponds to tangential elongation ( Figure 1C ) .", "When evaluated over the timelapse , we find that these radial gradients grow slightly more pronounced over time ( Figure 1—figure supplement 3C–D ) .", "As previously proposed , differential growth can generate such patterns of cell elongation ( Mao et al . , 2013 ) .", "However , indirect metrics of tissue growth in vivo do not indicate that differential growth still occurs at this later stage ( Mao et al . , 2013 ) .", "We directly measured the spatial pattern of growth during timelapse .", "Cell division rate has been previously used as an indicator of growth; however , tissue growth actually results from a combination of cell division , cell area changes , and cell extrusions ( Figure 1G ) .", "Thus , we evaluated the spatial pattern ( Figure 1H , I , K , L ) and radial profiles ( Figure 1J , M ) of total tissue growth and its cellular contributions .", "These data show that tissue area growth , as well as cell division rate , are to a good approximation independent of the distance to the center .", "In summary , we quantified cell morphology patterns in mid-third instar wing explants during live imaging .", "We confirm previous static observations of the pattern and further identify a region around the DV boundary with a morphological pattern that differs from the rest of the wing pouch .", "We quantify the radial gradients in cell area and cell elongation existing outside of the DV boundary region and show that they strengthen in time in the absence of differential growth , raising the question of what mechanism underlies the persistence of these cell morphology patterns during mid to late stages of wing growth .", "To directly relate the observed cell morphology patterns with cell rearrangements , we next analyzed the spatial patterns in cellular contributions to tissue shear ( Figure 2A ) .", "Tissue shear can be decomposed into contributions from cell divisions , cell elongation changes , T1 transitions , and so-called correlation effects ( Etournay et al . , 2015; Merkel et al . , 2017 ) .", "Here , correlation effects result mainly from correlated fluctuations in cell elongation and cell rotation ( see Appendix 3 and Merkel et al . , 2017 ) .", "We find that the spatial patterns of tissue growth and its cellular contributions exhibit overall anisotropies perpendicular to the DV boundary , as reported previously ( Dye et al . , 2017 ) .", "In addition , the patterns of cell elongation changes and T1 transitions can be described as a superposition of a uniformly oriented pattern and an approximately radial or tangential pattern ( Figure 2C–D ) .", "To determine the magnitude of the radial or tangential patterns in all quantities , we quantified their average radial projections as cumulative plots over time ( Figure 2B ) .", "The radial component of tissue shear is small , and cell divisions do not contribute to radial tissue shear .", "In contrast , we observe a pronounced buildup of a tangential pattern of cell elongation accompanied by a radial pattern of T1 transitions and of correlation effects .", "As shown above ( Figure 1J ) , tissue area growth does not have a radial gradient and thus does not contribute to the increase in tangential cell elongation that we observe at this time ( Figure 2A–B , Figure 1—figure supplement 3C–D ) .", "Furthermore , we observe numerous T1 transitions ( on average 1 . 0 cell-1 hr-1 ) , and their radially biased orientation increases rather than relaxes tangential cell elongation ( Figure 2E–F ) .", "Thus , we are not observing the relaxation of a pattern of cell elongation caused by early differential growth .", "Rather , our data support a model whereby a radially patterned morphogenetic cue actively biases the direction of T1 transitions and consequently the complementary pattern of cell shape changes .", "We next apply a biophysical model to determine whether radially patterned T1 transitions could account for the observed cell morphology patterns in the wing disc .", "This model takes into account the interplay of T1 transitions , cell shape changes , and tissue shear in a continuum description ( Etournay et al . , 2015; Popović et al . , 2017 ) .", "Active anisotropic force-generating processes that bias cell rearrangements in the tissue , such as polarization of the actomyosin cytoskeleton , are captured by a nematic cell polarity q , defined by a magnitude and an orientation axis .", "We propose that such a patterning cue leads to the radially oriented pattern of T1 transitions we observe in the wing disc .", "In our model , we consider the spatial patterns of tissue shear rate v~ , the patterns of cell shape Q , and the patterns of cell rearrangements R , which have nematic symmetry and are represented by traceless symmetric tensors that describe the magnitude and orientation .", "Tissue shear is defined as a velocity gradient tensor that results from a combination of cell shape changes and cell rearrangements ( Etournay et al . , 2015; Merkel et al . , 2017 ) : ( 1 ) v~=DQDt+R Here , DQ/Dt is a co-rotational time derivative of Q , and the shear due to cell rearrangements R includes contributions from T1 transitions , cell divisions and extrusions , and also correlation effects .", "Tissue material properties are described by constitutive equations for the tissue shear stress σ~ and the shear due to cell rearrangements R ( Figure 3A–B ) : ( 2 ) σ~=2KQ+ζq ( 3 ) R= 1τQ + λ q Here , the shear stress tensor σ~ is the sum of the elastic and active stress .", "The elastic stress is associated with cell elongation Q and characterized by the shear elastic modulus K . The active stress associated with the cell polarity cue q ( Figure 3A ) is described by the coefficient ζ .", "The shear rate due to cell rearrangements R given by Equation 3 is driven in part by shear stress , and therefore depends on the cell elongation Q , and in part by active processes that are oriented by the nematic cell polarity q ( Figure 3B ) .", "τ is a relaxation time for cell rearrangements over which elastic stresses are relaxed , and λ is the rate of cell rearrangements driven by cell polarity .", "In this coarse-grained picture , subcellular processes are captured by effective coefficients .", "For simplicity , we do not include dissipative processes on cellular scales , such as cytoskeletal viscosity or cell-cell friction .", "Such dissipative processes are relevant on short timescales and are small in comparison with elastic stresses on tissue relevant timescales .", "To discuss the wing disc , we consider a radially symmetric geometry and average the oriented quantities after projection onto the radial axis .", "Radial tissue shear is small compared to that associated with cell shape changes and T1 transitions during our observed time window ( Figure 2B ) .", "We therefore consider a steady state with v~rr=0 , DQrr/Dt =0 , and Rrr=0 .", "In this case , cell elongation becomes: ( 4 ) Qrr=-τλ qrr Thus , we find that the steady state cell elongation pattern is a result of cell rearrangements that are oriented by the cell polarity cue q ( Figure 3C–D ) .", "Note that our data show that the wing disc is not exactly at steady state: cells slowly change their shape and rearrange radially ( Figure 2A–B ) .", "However , as we show in Appendix 1 part 1-2 , Equation 4 holds to a good approximation .", "Can the radial pattern of T1 transitions defined by q also explain the observed radial profile of cell area ( Figure 1B , E ) ?", "To answer this question , we then considered force balances in the tissue .", "We consider tissue area pressure: ( 5 ) P=−K¯ ln ( aa0 ) where P is the difference in pressure from a reference value , K¯ is tissue area compressibility , a is the average cell area , and a0 is a reference cell area .", "As pressure increases , cell area decreases .", "To calculate the cell area profile , we again approximate the wing pouch as a radially symmetric disc .", "In the radially symmetric geometry , force balance can be expressed as: ( 6 ) ∂rP=∂rσ~rr+2rσ~rr A radial profile of pressure determined from this equation implies a radial pattern of cell area via Equation 5 ( see Figure 3C–E and Appendix 1 part 1-2 ) .", "To test this implication , we first quantify the radial profile of cell elongation Qrr to estimate the profile of qrr using Equation 4 ( Figure 3F ) .", "We represent the cell elongation data in a functional form using an empirical power law that is fit to the data .", "Then , using this functional form in Equations 2 , 5 , and 6 , we solve for the cell area profile ( Figure 3G and Appendix 1 part 1-2 ) .", "Finally , we show that this function can account for the observed pattern of cell area ( see Figure 3G ) .", "From this analysis , we conclude that the cell morphology patterns observed in the wing disc could be generated by radially biased cell rearrangements .", "Next , we test whether the stress profile predicted by the model ( Equation 2 ) exists in the tissue , and we measure key mechanical parameters of the model .", "Later , we address the potential molecular origin of the cell polarity cue orienting the cell rearrangements .", "Our model predicts a stress pattern in the wing disc that results from active processes that are radially oriented by a cell polarity cue .", "To compare this prediction to experiment , we infer tissue stress using laser ablation .", "Tissue stress has been estimated previously by laser ablation techniques that are based on determining the initial retraction velocity ( Bonnet et al . , 2012; Etournay et al . , 2015; Farhadifar et al . , 2007; Legoff et al . , 2013; Mao et al . , 2013; Shivakumar and Lenne , 2016 ) .", "However , to compare theory and experiment , ideally one should measure quantities that are well-captured by the model .", "Therefore , instead of using initial retraction velocity , we perform circular cuts and analyze the final , relaxed position of the inner and outer elliptical contours of tissue formed by the cut ( Figure 4A , Appendix 2 ) .", "From the size and the anisotropy of the cut , we can infer anisotropic and isotropic tissues stress , normalized by the respective elastic constants , as well as the ratio of elastic constants ( see Appendix 2 ) .", "Furthermore , we can also infer the existence of polarity-driven stress .", "We name this method ESCA ( Elliptical Shape after Circular Ablation ) .", "We perform local measurements of tissue mechanics by cutting the tissue in the smallest possible circle that would still allow us to measure the shape of the inner piece left by the cut ( radius = 7μm , encircling ∼5−15 cells , Figure 4A ) .", "To relate measurements of tissue stress to cell elongation , we calculate the average cell elongation in the ablated region before it is cut ( Figure 4A ) .", "In Figure 4B , we present the measured cell elongation and shear stress tensors at the position of ablation .", "We find that local average cell elongation correlates well with the principal direction of shear stress .", "Also , we observe that the cells in the band around the DV boundary , which are exposed to high Wg and Notch signaling , have different mechanical properties than elsewhere in the tissue .", "Near the DV boundary , cells elongate less than outside this region for comparable amounts of stress ( Figure 4B and Figure 4—figure supplement 1B , E ) .", "The ratio of elastic constants in this region is also smaller: near the DV boundary 2K/K¯=2 . 3±0 . 3 , whereas outside this region , 2K/K¯=3 . 4±0 . 4 ( see also Figure 4—figure supplement 1C , F ) .", "We focus hereafter on the radial patterns of elongation and stress outside of the DV boundary region .", "The relationship between cell elongation and stress normalized by the elastic modulus has a slope 1 in the absence of polarity-driven stress ( see Equation 2 ) .", "We observe a much smaller slope for this relationship in our data ( Figure 4C ) , indicating that polarity-driven stress is significant .", "We now use these data to estimate the parameters of our mechanical model .", "We write the shear stress defined in Equation 2 in terms of cell elongation and cell rearrangements , eliminating the orientational cue qrr using Equation 3 .", "For the radial components , we have: ( 7 ) σ~rr=2K*Qrr+2K-K*τRrrwhere K* = 1-ζ/ ( 2Kτλ ) K is an effective shear elastic coefficient .", "The difference between K* and K depends on the parameters ζ and λ associated with the nematic cell polarity .", "We fit Equation 7 to the data and find K*/K=0 . 05±0 . 02 and ( 1-K*/K ) τRrr=0 . 011±0 . 002 ( Figure 4C , see Appendix 1 part 3 ) .", "Combined with data from Figure 1 , we find an estimate for the tissue relaxation time τ=2±2 hr , which is roughly consistent with that found during pupal morphogenesis ( Etournay et al . , 2015 ) .", "From our data , we can also infer the radial profile of tissue area pressure , revealing that pressure increases toward the center ( Figure 4D and Appendix 1 part 3 ) .", "This finding is consistent with the observed cell area profile , with smaller cell areas toward the center ( Figure 1B , E ) .", "In sum , we find a stress profile in the wing disc that is consistent with the observed measurements of both cell elongation and area .", "Further , we use these data to measure certain parameters of our biophysical model , including the tissue relaxation timescale and the effective shear elastic coefficient .", "In our model , the radial orientation cue is required to generate the observed patterns of cell morphology , cell rearrangement , and tissue stress .", "Candidates for such an orientational cue are the planar cell polarity pathways ( PCP ) , which are groups of interacting proteins that polarize within the plane of the epithelium .", "There are two well-characterized PCP pathways: Fat and Core ( Butler and Wallingford , 2017; Eaton , 2003 ) .", "In the wing , these systems form tissue-scale polarity patterns during growth ( Brittle et al . , 2012; Merkel et al . , 2014; Sagner et al . , 2012 ) and are required to position the hairs and cuticle ridges on the adult wing ( Adler et al . , 1998; Doyle et al . , 2008; Eaton , 2003; Gubb and García-Bellido , 1982; Hogan et al . , 2011 ) .", "To determine whether either of these pathways could function as the orientational cue described in our model , we analyzed cell elongation patterns after their removal .", "We perturbed the Fat pathway using nub-Gal4 to drive the expression of RNAi constructs targeting both fat ( ft ) and dachs ( d ) in the pouch region throughout the third larval instar .", "This perturbation results in almost complete loss of Dachsous from the apical membrane , and any residual signal is no longer polarized , confirming the loss of PCP ( Figure 5—figure supplement 1A–B ) .", "Furthermore , we observe a suppression of tissue growth upon ft+d double RNAi knockdown ( visible at the end of larval development in Figure 5A and in the resulting adult wings in Figure 5—figure supplement 1C ) , consistent with previous work on the loss of both Dachs and Fat ( Cho and Irvine , 2004 ) .", "Nonetheless , the pattern of tangential cell elongation persists to the end of larval development ( Figure 5A–C ) .", "Using scaled coordinates , we find that the radial profiles of cell elongation in ft+d RNAi and control wings are similar ( Figure 5D ) .", "We perturbed the Core PCP pathway using a previously characterized null mutation in prickle ( pk30 ) , which causes defects in adult wing hair orientation ( Gubb et al . , 1999 ) .", "We found that the cell elongation pattern in the pk mutant is similar to the wild-type control ( Figure 5E–I ) .", "In the pk mutant , the region of tangential cell elongation extends even further into the center than in control wings .", "We conclude that the tangential cell elongation pattern persists in the absence of either PCP pathway .", "This result excludes these pathways as orienting cues for the cell elongation patterns .", "We have shown that perturbing PCP pathways does not affect the radial patterns of morphology , raising the question of how orientational cues might arise .", "In previous sections , we have considered the orientational cue to be provided by a cell polarity system that is independently patterned .", "However , cell polarity in general would be affected by stresses in the tissue .", "Indeed , there are many examples of cells polarizing in response to mechanical stress ( Duda et al . , 2019; Hirashima and Adachi , 2019; Ladoux et al . , 2016; Ohashi et al . , 2017 ) .", "Here , we show that introducing mechanosensitive feedback to the model of tissue mechanics can give rise to spontaneous emergence of the cell polarity cue ( Figure 6 ) .", "Mechanosensitivity is incorporated into our model of tissue mechanics through a dynamic equation for the orientational cue q that becomes stress-dependent: ( 8 ) dqdt=−1τqq−μσ~−α|q|2q+D∇2q Here , τq is a relaxation timescale for q , μ is a mechanosensitive feedback strength , the coefficient α>0 ensures stability , and D is a coupling strength locally aligning orientational cues .", "Now , Equation 8 provides a mechanosensitive feedback to Equations 1 , 2 and 3 .", "These combined equations show a novel behavior .", "Specifically , the orientational cue can emerge spontaneously by self-organization ( Figure 6A–B ) .", "Beyond a critical value μc of the mechanosensitive feedback strength μ , an isotropic tissue with q=0 is no longer stable , and a state with an orientational cue q≠0 emerges instead ( Figure 6B and Appendix 1 part 4 ) .", "The magnitude of this spontaneous polarization is |q|=q0 , where q02= ( τqμ ( 2Kτλ-ζ ) -1 ) / ( ατq ) , where a positive coefficient α is needed to stabilize the polarized state .", "By this mechanism , the anisotropic cue introduced earlier in our model can be locally generated by mechanosensitive self-organization and does not require the existence of pre-patterned polarity cues .", "To generate a large-scale pattern from locally generated anisotropic cues , they need to be aligned in neighboring regions .", "This local alignment is captured in Equation 8 by the orientation coupling term with strength D , which is similar to alignment terms found in anisotropic physical systems , such as liquid crystals ( Gennes and Prost , 1993; Jülicher et al . , 2018; Marchetti et al . , 2013 ) .", "To discuss cell morphology profiles in the wing disc , we consider a simplified tissue model with radial symmetry , where the rate of radial cell rearrangement Rrr is given ( as estimated in Appendix 1 part 2 ) and the cell shape pattern and tissue stress pattern are calculated .", "Using a fit of cell elongation to the experimental data , we find a set of parameter values that accounts for the observed cell elongation patterns in the wing disc ( Table 1 , Appendix 1 , Figure 6C ) .", "From this cell elongation pattern also follows the cell area pattern ( as described above , Figure 3 ) .", "We conclude that our mechanosensitive model can account for the radial pattern of cell morphology in the wing disc .", "Due to the relatively large number of parameters used to fit a single experimental curve , there are large uncertainties when estimating parameter values .", "Nonetheless , these uncertainties do not affect the qualitative prediction that a reduction in mechanosensitivity μ would lead to less polarization and thereby reduced cell elongation ( see Appendix 1 part 4 ) .", "We next test this prediction of our model experimentally .", "In order to test our prediction that the reduction of mechanosensitivity will reduce the magnitude of cell elongation , we used RNAi to reduce the levels of Myosin VI ( MyoVI ) , a molecular motor implicated in mechanosignaling .", "MyoVI , encoded by jaguar ( jar ) in Drosophila , is an upstream component of a Rho-dependent signaling pathway that reorganizes the actin-myosin cytoskeleton in response to mechanical stress ( Acharya et al . , 2018 ) .", "Experiments in wing discs also indicate that mechanosensation involves Rho polarization and signaling ( Duda et al . , 2019 ) .", "We performed RNAi targeting MyoVI in the wing pouch using nub-Gal4 and evaluated cell morphology at the end of larval development ( ∼120hr AEL ) .", "We observe a clear reduction in the magnitude of tangential cell elongation as compared to wild type at this stage ( Figure 7A–D , Figure 7—figure supplement 1 ) .", "In addition , our model predicts that such reduction of cell elongation would result in an increase of cell area in the wing ( Equations 1-6 ) .", "The observed pattern of increased cell area upon reducing MyoVI levels with RNAi is consistent with this prediction ( Figure 7E–F ) .", "Therefore , the qualitative predictions of our model upon reducing the mechanosensitive feedback strength μ are confirmed by the experimental downregulation of the mechanosensitive motor MyoVI ." ], [ "Our work shows that the spatial pattern of T1 transitions is an integral part of the emergence of tissue organization during wing development .", "In contrast to situations such as germband extension , where T1 transitions exhibit clearly discernible patterns , the patterns of T1 transitions in the wing disc have been elusive .", "Many T1 transitions occur in the tissue in seemingly random orientations .", "However , on average , they exhibit a spatial pattern .", "We revealed these patterns by quantifying the nematics of T1 transitions and cell shape changes using the previously-described triangle method ( Merkel et al . , 2017 ) and then quantified them with radial averaging ( Figure 2A–B ) .", "In this way , we revealed that a radial pattern of T1 transitions is linked to a tangential pattern of cell elongation .", "Given this radial pattern of T1 cell rearrangements , the observed cell morphology pattern follows from a continuum tissue model based on a radially oriented nematic cell polarity field ( Figure 3 ) .", "The polarity-oriented radial T1s create a cell shape pattern with corresponding patterns of tissue stress and tissue area pressure .", "The 2D area pressure is higher in the center and is lower toward the periphery .", "Note that this pressure profile does not rely on differential proliferation , as was previously proposed ( Mao et al . , 2013 ) but instead relies on a radial pattern of T1 transitions .", "We test our model using a novel circular laser ablation method .", "This method allows us to determine specific combinations of tissue parameters .", "In particular , we estimate the ratio of elastic constants 2K/K¯=3 . 4±0 . 4 and K*/K=0 . 05±0 . 02 , as well as the cell shape relaxation timescale τ=2±2hr .", "This analysis raised the question of which nematic cell polarity cues guide the cell rearrangement and cell elongation patterns .", "PCP pathways are required for the proper orientation of T1 transitions in other contexts ( Bosveld et al . , 2012 ) .", "However , we found that neither of the two known PCP pathways in the wing are required for the observed tangential cell elongation patterns ( Figure 5 ) .", "We instead show that an orientation cue can arise through self-organization via mechanosensitivity and identify MyoVI as a key molecular player .", "Cell polarity cues can emerge via mechanosensitive feedback by transforming mechanical cues into chemical anisotropies .", "Nematic cell polarity can then orient active stresses and thereby amplify the mechanical stimulus ( Figure 6 ) .", "We introduce this mechanosensitive feedback in our continuum theory , which quantitatively describes the emergence of patterns of cell shape and cell rearrangements .", "The strength of this mechanosensitive feedback is described by a parameter μ .", "If μ exceeds a critical value μc , an orientation cue and elongated cell shapes spontaneously emerge ( Figure 6B ) .", "This model can account for the observed patterns of cell area and cell elongation in the wing disc and predicts that the reduction of mechanosensitivity will result in reduced cell elongation .", "To test this prediction , we perturbed a RhoA-dependent mechanotransduction pathway by lowering levels of an upstream component , MyoVI , using RNAi ( Figure 7 ) .", "We find a clear phenotype of reduced cell elongation and increased cell areas in the center region , as predicted by our model .", "In the spatially resolved model , which includes the term coupling polarity orientation of neighboring cells ( Figure 6C ) , a polarity pattern can be induced even below μc by imposing polarity at the tissue boundaries .", "Thus , the residual pattern of cell elongation that we observe after removal of MyoVI in the wing pouch could be due to polarity existing outside of this perturbed region .", "In addition , the residual pattern could also indicate an incomplete knockdown from RNAi or the presence of other mechanosensitive elements in the tissue .", "Nevertheless , given the clear phenotype that is fully consistent with our model , we propose that the mechanosensitive feedback mechanism is a significant determinant of the cell shape patterns in the wing pouch .", "Our data , together with the fact that MyoVI is involved in Rho-dependent activation of actin-myosin cytoskeleton ( Acharya et al . , 2018 ) , suggest that MyoVI is a molecular component of the mechanosensitive feedback we describe in our self-organized model .", "However , the molecular nature of the cue that defines the nematic cell polarity is unknown .", "This cue may organize the structure or dynamics of the actin-myosin cytoskeleton or the actin-myosin system itself could define nematic cell polarity .", "Indeed , it has been shown that Myosin II ( MyoII ) localizes to long cell boundaries in the wing ( Legoff et al . , 2013 ) , corresponding to a nematic polarity aligned with the nematic cell polarity q .", "Also , wing disc stretching experiments have shown that MyoII can polarize in response to exogenous stress in a Rho-dependent manner ( Duda et al . , 2019 ) .", "Furthermore , it has been suggested that MyoII polarity arises as a consequence of cell stretching and functions as a negative mechanical feedback ( Legoff et al . , 2013 ) , consistent with the role of q in our model .", "Precisely how the actin-myosin cytoskeleton is affected by MyoVI in this system and how these cytoskeletal elements together guide cell rearrangements in response to anisotropic tissue stresses and cell shape changes remain open questions for future research .", "Lastly , our laser ablation analysis shows that the region around the DV boundary has a different ratio of elastic constants than the rest of the tissue , which could affect the self-organized pattern formation we describe .", "Therefore , it will be interesting to study how Wingless/Notch signaling , which defines the DV boundary , may influence the mechanical properties that lead to mechanosensitive self-organization of polarity and morphology .", "In addition , we observe a richer pattern emerging very late in development ( see Figure 7—figure supplement 1 , Figure 5 ) , including a region anterior to the AP boundary that is radially elongated .", "Future research will expand upon the model presented here to explore the dynamics of these patterns .", "In summary , we used the Drosophila wing disc to identify a mechanism by which tissue morphology can arise from the self-organization of a mechanical feedback coupling cell polarity to active cell rearrangements .", "This mechanism is general and could be employed in other tissues and organisms to generate patterns of cell shape and cell area .", "Thus , we hope our work inspires new avenues of research that integrate theory and experiment to understand biological self-organization ." ], [ "This study did not generate any new unique reagents .", "All requests for further information and reagents may be directed to the lead author , natalie_anne . dye@tu-dresden . de .", "All experiments were performed with Drosophila melanogaster , using lines that are publicly available and previously published .", "Our Drosophila lines were fed with a standard media containing cornmeal , molasses agar and yeast extract and grown under a 12hr light/dark cycle .", "All experiments were performed at 25∘C .", "Both males and females were analyzed , and the sex of the animals was not recorded , as we have no reason to believe there is any sexual dimorphism in the studied phenomenon .", "To synchronize development , we collected eggs deposited within a defined time window on apple juice agar plates .", "To do so , we transferred the flies from standard food vials to cages covered by apple juice agar plates containing a dollop of yeast paste for food .", "After at least 2hr , the plates were replaced , and the timing of collection started .", "Eggs laid within a ∼2hr time window were collected by cutting out a piece of the agar and transferring it to a standard food vial .", "We limited the number of eggs per vial to <15 to avoid crowding .", "The middle of the time window for egg collection was considered to be 0hr AEL .", "Experiments from timelapse imaging and laser ablation ( Figures 1 , 2 , and 4 ) were captured after explanting at 96hr AEL , whereas those involving RNAi ( Figures 5 and 7 ) were explanted at 110−120hr AEL to allow the maximal amount of time for the RNAi phenotype to emerge .", "The specific genotypes used for each experiment are indicated in the Key Resources Table and in the figures ." ] ]

[ "Tissue organization is often characterized by specific patterns of cell morphology .", "How such patterns emerge in developing tissues is a fundamental open question .", "Here , we investigate the emergence of tissue-scale patterns of cell shape and mechanical tissue stress in the Drosophila wing imaginal disc during larval development .", "Using quantitative analysis of the cellular dynamics , we reveal a pattern of radially oriented cell rearrangements that is coupled to the buildup of tangential cell elongation .", "Developing a laser ablation method , we map tissue stresses and extract key parameters of tissue mechanics .", "We present a continuum theory showing that this pattern of cell morphology and tissue stress can arise via self-organization of a mechanical feedback that couples cell polarity to active cell rearrangements .", "The predictions of this model are supported by knockdown of MyoVI , a component of mechanosensitive feedback .", "Our work reveals a mechanism for the emergence of cellular patterns in morphogenesis ." ]

[ "During development , carefully choreographed cell movements ensure the creation of a healthy organism .", "To determine their identity and place across a tissue , cells can read gradients of far-reaching signaling molecules called morphogens; in addition , physical forces can play a part in helping cells acquire the right size and shape .", "Indeed , cells are tightly attached to their neighbors through connections linked to internal components .", "Structures or proteins inside the cells can pull on these junctions to generate forces that change the physical features of a cell .", "However , it is poorly understood how these forces create patterns of cell size and shape across a tissue .", "Here , Dye , Popovic et al . combined experiments with physical models to examine how cells acquire these physical characteristics across the developing wing of fruit fly larvae .", "This revealed that cells pushing and pulling on one another create forces that trigger internal biochemical reorganization – for instance , force-generating structures become asymmetrical .", "In turn , the cells exert additional forces on their neighbors , setting up a positive feedback loop which results in cells adopting the right size and shape across the organ .", "As such , cells in the fly wing can spontaneously self-organize through the interplay of mechanical and biochemical signals , without the need for pre-existing morphogen gradients .", "A refined understanding of how physical forces shape cells and organs would help to grasp how defects can emerge during development .", "This knowledge would also allow scientists to better grow tissues and organs in the laboratory , both for theoretical research and regenerative medicine ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "microbiology and infectious disease", "immunology and inflammation" ]

[ [ "Vpr is a highly conserved HIV accessory protein that is necessary for optimal replication in macrophages ( Balliet et al . , 1994 ) but its mechanism of action is poorly understood .", "Studies using human lymphoid tissue ( HLT ) , which are rich in both T cells and macrophages , have found that loss of Vpr decreases virus production ( Rucker et al . , 2004 ) but only when the virus strain used is capable of efficiently infecting macrophages ( Eckstein et al . , 2001 ) .", "These studies provide evidence that Vpr enhances infection of macrophages and increases viral burden in tissues where macrophages reside .", "Because Vpr is packaged into the virion ( Cohen et al . , 1990 ) and localizes to the nucleus ( Lu et al . , 1993 ) , it may enhance early viral replication events .", "However , in mononuclear phagocytes vpr-null virus in which Vpr protein is provided by trans-complementation in the producer cells replicates poorly compared to wild-type virus ( Connor et al . , 1995 ) , indicating that Vpr’s role in the HIV replication cycle continues into late stages .", "Previous work by our group demonstrated that Vpr counteracts an unidentified macrophage-specific restriction factor that targets Env and Env-containing virions for lysosomal degradation ( Mashiba et al . , 2014; Collins et al . , 2015 ) .", "This restriction could be conferred to permissive 293T cells by fusing them with MDM to create 293T-MDM heterokaryons .", "A follow up study demonstrated that by increasing steady state levels of Env , Vpr increases formation of virological synapses between infected MDM and autologous uninfected T cells , enhancing HIV infection of T cells ( Collins et al . , 2015 ) .", "This enhances spread to T cells and dramatically increases levels of Gag p24 in the culture supernatant .", "This finding helps explain the paradoxical observations that Vpr is required for maximal infection of T cells in vivo ( Hoch et al . , 1995 ) but numerous studies have shown Vpr only marginally impacts infection of pure T cell cultures in vitro ( e . g . Mashiba et al . , 2014 ) .", "Our goal in the current study was to identify and characterize the myeloid restriction factor targeting Env that is counteracted by Vpr .", "We reasoned that macrophage-specific Env-binding proteins , including the carbohydrate binding protein mannose receptor ( MR ) , were candidates .", "MR is expressed on several types of macrophages in vivo ( Liang et al . , 1996; Linehan et al . , 1999 ) and is known to mediate innate immunity against various pathogens ( Macedo-Ramos et al . , 2014; Subramanian et al . , 2019 ) .", "MR recognizes mannose-rich structures including high-mannose glycans , which are incorporated in many proteins during synthesis .", "In eukaryotic cells most high-mannose glycans are cleaved by α-mannosidases and replaced with complex-type glycans as they transit through the secretory pathway .", "By contrast , in prokaryotic cells , high-mannose residues remain intact , making them a useful target of pattern recognition receptors including MR . Some viral proteins , including HIV-1 Env , evade mannose trimming ( Coss et al . , 2016 ) and retain enough high-mannose to bind MR ( Trujillo et al . , 2007; Lai et al . , 2009 ) .", "There is evidence that HIV-1 proteins Nef and Tat decrease expression of MR based on studies performed in monocyte derived macrophages ( MDM ) and the monocytic U937 cell line , respectively ( Caldwell et al . , 2000; Vigerust et al . , 2005 ) .", "Nef dysregulates MR trafficking using an SDXXLΦ motif in MR’s cytoplasmic tail ( Vigerust et al . , 2005 ) , which is similar to the sequence in CD4’s tail that Nef uses to remove it from the cell surface ( Bresnahan et al . , 1998; Greenberg et al . , 1998; Cluet et al . , 2005 ) .", "Whether MR or its modulation by viral proteins alters the course of viral replication has not been established .", "Here we confirm that Nef reduces MR expression in primary human MDM , although in our system , the effect of Nef alone was relatively small .", "In contrast , we report that co-expression of Vpr and Nef dramatically reduced MR expression .", "In the absence of both Vpr and Nef , MR levels normalized indicating that Tat did not play a significant , independent role in MR downmodulation .", "Deleting mannose residues on Env or silencing MR alleviated mannose-dependent interactions between MR and Env and reduced the requirement for Vpr .", "Although the post-infection interactions between MR and Env reduced Env levels and inhibited viral release , we provide evidence that these same interactions were beneficial for initial infection of MDM .", "Together these results reveal that mannose residues on Env and the accessory proteins Nef and Vpr are needed for HIV to utilize and then disable an important component of the myeloid innate response against pathogens intended to thwart infection ." ], [ "Because we had previously determined that Vpr functions in macrophages to counteract a macrophage specific restriction factor that targets Env , we reasoned that Env-binding proteins selectively expressed by macrophages were potential candidate restriction factors .", "To determine whether any factors fitting this description were targeted by Vpr , we cultured macrophages under conditions that achieve a saturating infection by both wild-type and Vpr-null mutant viruses ( Figure 1A and B ) .", "We found that mannose receptor ( MR ) , which is highly expressed on macrophages and has been previously shown to bind Env ( Trujillo et al . , 2007; Fanibunda et al . , 2008; Lai et al . , 2009 ) , was significantly decreased by wild-type HIV 89 . 6 but not by 89 . 6 vpr-null ( Figure 1C and D , p<0 . 01 ) .", "In contrast , we observed no significant effect of Vpr on the expression of GAPDH .", "We also observed that stimulator of interferon genes ( STING ) was unaffected by Vpr ( Figure 1—figure supplement 1 ) .", "Relative expression of known restriction factors GBP5 and IFITM3 varied in infected MDM from multiple donors ( Figure 1—figure supplement 1 ) , but unlike MR they were not consistently reduced in the wild-type condition , indicating they are not targeted by Vpr .", "To confirm the effect of Vpr on Env during HIV infection of primary human macrophages in which MR was downmodulated , we performed quantitative western blot analysis .", "As shown in Figure 1E and F , we confirmed that amounts of Vpr sufficient for MR downmodulation were also sufficient for stabilizing expression of Env ( gp160 , gp120 , gp41 ) .", "Compiled data from nine donors clearly demonstrated results that were similar to our prior publication ( Mashiba et al . , 2014 ) ; under conditions of matched infection in which there was no significant difference in HIV Gag pr55 levels between wild-type and vpr-null infections , all three forms of Env were significantly more abundant in the wild-type infection ( gp160: 4-fold , p<0 . 002; gp120: 6-fold , p<0 . 002; gp41: 3-fold , p<0 . 001 ) .", "Because an earlier report indicated that Nef decreases surface expression of MR ( Vigerust et al . , 2005 ) , we asked whether Nef was playing a role in MR downmodulation in our systems .", "Because HIVs lacking Vpr and Nef spread too inefficiently in MDM to observe effects on host proteins by western blot analysis , we utilized a replication defective HIV with a GFP marker ( NL4-3 ∆GPE-GFP , Figure 2A ) to allow measurement of MR expression via flow cytometry following single-round transduction .", "This construct has the additional advantage that it eliminates potentially confounding effects of differences between wild-type and mutant HIV viral spread .", "We generated truncation mutations in nef and vpr and confirmed that these mutations only affected expression of the altered gene product in transfected 293T ( Figure 2B ) .", "For these experiments , primary MDM were harvested earlier than the experiments described in Figure 1 ( five days versus ten days ) because the viruses could not replicate and the GFP marker allowed identification of transduced cells ( Figure 2C ) .", "Under these conditions , we found that MR expression was dramatically reduced in a subset of GFP+ cells when both Vpr and Nef were expressed ( Figure 2C–E ) .", "Both Nef and Vpr contributed to MR downmodulation; loss of function mutation in either Vpr or Nef reduced the severity of MR downmodulation similarly , and there was no statistical difference between MR levels in macrophages expressing either Vpr or Nef alone ( Figure 2E ) .", "In addition , complete elimination of downmodulation required mutation of both Vpr and Nef ( Figure 2C–E ) .", "These results indicate that both Vpr and Nef are required for maximal MR downmodulation in HIV-infected macrophages and that neither alone is sufficient .", "Vpr was previously demonstrated to interact with a cellular co-factor called DCAF1 , a component of the cellular DCAF1-DDB1-CUL4 E3 ubiquitin ligase complex .", "( Hrecka et al . , 2007; Le Rouzic et al . , 2007; McCall et al . , 2008; Lahouassa et al . , 2016; Wu et al . , 2016; Zhou et al . , 2016 ) .", "The interaction between Vpr and DCAF1 can be disrupted through a Vpr mutation ( Q65R ) that inhibits many Vpr-dependent functions , including reversal of Env degradation in macrophages ( Mashiba et al . , 2014 ) .", "To determine whether this mutant is defective at MR downmodulation , we generated the mutation in the NL4-3 ∆GPE-GFP parent ( Figure 2A ) , confirmed expression in transfected 293T cells ( Figure 2F ) and tested the effect of the mutation on MR levels in macrophages .", "As expected , we found that in transduced MDM the vpr-Q65R mutant behaves similarly to vpr-null ( Figure 2E ) .", "These results indicate interactions between Vpr and DCAF1 are required to mediate Vpr’s effects on MR . The differences in MR downmodulation we observed using this system were not due to variations in multiplicity of infection of the different viral constructs as MDM transduced with the mutant viral constructs had roughly similar transduction rates as the parental construct ( Figure 2G ) but demonstrated less MR downmodulation ( Figure 2E ) .", "To determine whether the relatively modest effect of Nef alone on MR levels was due to using HIV to deliver Nef as compared to an adenoviral vector delivery system used in a prior publication ( Vigerust et al . , 2005 ) , we repeated the experiment using an adenoviral vector expressing Nef .", "These experiments confirmed that levels of Nef sufficient to downmodulate the HIV receptor , CD4 , on nearly all MDM in the culture achieved only modest effects on MR in a subset of cells ( Figure 2H ) similar to what was observed using the HIV reporter construct ( Figure 2E ) .", "Thus , Nef and Vpr have modest but significant effects on MR when expressed individually , however the combined effects of both proteins can achieve nearly complete downmodulation at least in a subset of infected cells .", "While the effect of Nef has been previously reported and found to be due to disruption of MR intracellular trafficking ( Vigerust et al . , 2005 ) , the effect of Vpr on MR is a novel observation .", "Vpr is known to target cellular proteins involved in DNA repair pathways for proteasomal degradation via interactions with Vpr binding protein [DCAF1 , ( McCall et al . , 2008 ) ] .", "Using this mechanism , Vpr degrades the uracil deglycosylases UNG2 and SMUG1 in 293T cells following co-transfection ( Schröfelbauer et al . , 2005; Schröfelbauer et al . , 2007 ) .", "To determine whether Vpr directly targets MR using a similar strategy , we co-transfected NL4-3 ∆GPE-GFP or a vpr-null derivative with expression vectors encoding an UNG2-FLAG fusion protein or MR ( Liu et al . , 2004 ) in 293T cells .", "We then analyzed expression of MR or UNG2 by flow cytometry and western blot ( Figure 2—figure supplement 1 ) .", "We found that Vpr in 293T cells virtually eliminated UNG2 expression when measured by flow cytometry and noticeably reduced UNG2 by western blot .", "However , Vpr had no effect on expression of MR measured by either method .", "Thus , we concluded that Vpr does not degrade MR by the direct , proteasomal mechanism it uses to degrade UNG2 .", "Because MR expression in this system is controlled by a heterologous CMV promoter; the lack of effect by Vpr suggested its action may depend on MR’s native promoter .", "In addition to targeting proteins for degradation , Vpr also functions to inhibit transcription of genes such as IFNA1 ( Laguette et al . , 2014; Mashiba et al . , 2014 ) .", "Therefore , we hypothesized that Vpr may reduce MR expression via inhibition of transcription .", "To examine this , we assessed transcriptional activity in primary human MDM transduced with the wild-type or Vpr-null reporter virus ( Figure 3A ) using cells isolated based on GFP expression ( Figure 3B ) .", "We found that the MR gene ( MRC1 ) was consistently reduced in cells transduced by vpr-competent virus compared to cells transduced by vpr-null virus ( Figure 3C and D , p=0 . 001 ) .", "In contrast , any effects of Vpr on the housekeeping genes ACTB ( β-actin ) and POL2A ( RNA polymerase 2A ) were significantly smaller ( Figure 3D , p<0 . 01 ) .", "Similar results were obtained when each gene was normalized to ACTB instead of GAPDH ( Figure 3—figure supplement 1A–B ) .", "The magnitude of the effect on MRC1 is consistent with prior reports of HIV-1 inhibiting MRC1 transcription− though this was not previously linked to Vpr ( Koziel et al . , 1998; Sukegawa et al . , 2018 ) .", "Relative MRC1 expression in untransduced MDM was heterogeneous , varying over a ten-fold range .", "When compiled across donors , MRC1 levels in mock-transduced samples were not significantly different than transduced ( Figure 3—figure supplement 1C–F ) .", "To determine whether the striking downmodulation of MR we observed with expression of both Nef and Vpr affected viral spread in MR+ macrophages , we generated additional mutations in HIV-1 89 . 6 to create a nef-null mutant and a vpr-nef-null double mutant .", "As expected , in transfected 293T cells these mutations did not alter Env protein levels ( Figure 4A ) or release of virions as assessed by measuring Gag p24 in the supernatant by ELISA ( Figure 4B ) .", "However , in primary human MDM infected with these HIVs , the mutants demonstrated defects in viral spread , with the double mutant having the greatest defect ( Figure 4C and D ) .", "The defect in spread was caused in part by diminished virion release , which we previously showed occurred in the absence of Vpr ( Mashiba et al . , 2014 ) ; MDM infected with the HIV mutants released less Gag p24 even after adjusting for the frequency of infected cells ( Figure 4D , right panel ) .", "To determine whether combined effects of Nef and Vpr on MR expression affected Env restriction , we assessed Env levels in primary human MDM infected with each construct .", "Because the frequency of infected cells as assessed by intracellular Gag staining ( Figure 4C ) and Gag pr55 western blot ( Figure 4E ) was lower in the mutants than in the wild-type infection , lysate from the wild-type sample was serially diluted to facilitate comparisons .", "Remarkably , we found that the vpr-nef-null double mutant , which retains near normal MR levels , exhibited the greatest defect in Env expression ( Figure 4E , compare lanes with similar Gag as indicated ) .", "In sum , Vpr and Nef-mediated downmodulation of MR correlated inversely with Env levels , consistent with MR being the previously described but unidentified HIV restriction factor that targets Env for lysosomal degradation in macrophages and is counteracted by Vpr ( Mashiba et al . , 2014 ) .", "Combined effects of Nef on MR and other Env binding proteins including CD4 ( Aiken et al . , 1994 ) and chemokine receptors ( Michel et al . , 2006 ) may also play a role in stabilization of Env .", "A particularly dense mannose-containing structure on Env , known as the mannose patch , may mediate interactions between Env and MR . This structure is present on all HIV Env proteins that require Vpr for stability in macrophages [89 . 6 , NL4-3 and AD8 ( Mashiba et al . , 2014; Collins et al . , 2015 ) . Interestingly , a macrophage tropic strain YU-2 , which was isolated from the CNS of an AIDS patient ( Li et al . , 1991 ) , lacks a mannose patch . This structure is the target of several broadly neutralizing antibodies including 2G12 , to which YU-2 is highly resistant ( Trkola et al . , 1996 ) . If Vpr targets MR to counteract detrimental interactions between MR and mannose residues on Env , we hypothesized that HIV Envs lacking a mannose patch would have a reduced requirement for Vpr . To test this hypothesis , we first examined the extent to which virion release and Env expression were influenced by Vpr in primary human MDM infected with YU-2 or 89 . 6 HIVs . Consistent with our hypothesis , we observed no significant difference in Gag p24 release between wild-type and vpr-null YU-2 infection of MDM ( Figure 5A ) . Moreover , the vpr-null mutant of YU2 displayed only a minor defect in Env expression compared to Vpr null versions of 89 . 6 and NL4-3 ( Figure 5B ) . Because there are a number of other genetic differences between YU-2 and the other HIVs , we constructed a chimeric virus , which restricted the differences to the env open reading frame . As shown in Figure 5C , a fragment of the YU-2 genome containing most of env but none of vpr ( Figure 5C , shaded portion ) was cloned into NL4-3 and NL4-3 vpr-null . As expected , these genetic alterations did not affect Env protein levels or virion release in transfected 293T cells ( Figure 5D and E ) . To confirm that the chimeric Env was still functional , we examined infectivity in T cells prior to performing our analyses in primary human MDM . Conveniently , sequence variation within the gp120 region allows YU-2 Env to only utilize the co-receptor CCR5 for entry , whereas NL4-3 can only utilize CXCR4 . Thus , we expected the NL4 3envYU2 chimera would switch from being CXCR4- to CCR5-tropic . To test this , we utilized a T cell line expressing both chemokine receptors ( MOLT4-R5 ) and selectively blocked entry via CXCR4 and CCR5 entry inhibitors [AMD3100 and maraviroc , respectively ( Figure 5F ) ] .", "As expected , entry into MOLT4-R5 cells by NL4-3 was blocked by AMD3100 but not maraviroc , indicating CXCR4-tropism .", "The chimeric NL4-3 envYU2 and wild-type YU-2 demonstrated the inverse pattern , indicating CCR5-tropism .", "These results demonstrated that we had made the expected changes in the chimeric Env without disrupting its capacity to infect cells .", "To determine whether swapping a limited portion of YU-2 env into NL4-3 alleviated the requirement for Vpr , we examined Env expression and virion release in primary human MDM infected with these viruses .", "Because the parental NL4-3 virus required pseudotyping with a macrophage-tropic Env for entry and was unable to spread in MDM , all infections were treated with entry inhibitors AMD3100 and maraviroc starting at 48 hr after inoculation and maintained throughout the culture period to block subsequent rounds of infection .", "Consistent with our hypothesis that YU-2 Env lacked determinants necessary for the restriction that was alleviated by Vpr , we observed that wild-type NL4-3 Env but not chimeric NL4-3 envYU2 required Vpr for maximal expression ( Figure 5G ) .", "Moreover , MDM infected with the chimeric HIV had a reduced requirement for Vpr for maximal virion release ( Figure 5H and Figure 5—figure supplement 1 ) .", "This experiment provides strong evidence that the requirement for Vpr can be alleviated by genetic changes within the env open reading frame .", "These results are consistent with a model in which YU-2 env confers resistance to the effects of MR due to the absence of the mannose-rich structure on the YU-2 Env glycoprotein .", "To more directly assess the role of mannose in restricting expression of Env in HIV-1 infected primary human MDM , we engineered a version of 89 . 6 Env in which two N-linked glycosylation sites , N230 and N339 ( HIV HxB2 numbering ) were deleted by substituting non-glycosylated amino acids found at analogous positions in YU-2 Env ( Figure 6A ) .", "The glycosylation sites N230 and N339 were selected because they contain high-mannose glycan structures ( Leonard et al . , 1990 ) that are absent in YU-2 Env .", "Loss of N230 limits neutralization by glycan specific antibodies ( Huang et al . , 2014 ) .", "Loss of N339 decreases the amount of oligomannose ( Man9GlcNAc2 ) present on gp120 by over 25% , presumably by opening up the mannose patch to processing by α-mannosidases ( Pritchard et al . , 2015 ) .", "These substitutions ( N230D and N339E ) in 89 . 6 did not alter virion production ( Figure 6B ) or Env protein expression ( Figure 6C ) in transfected 293T cells .", "To confirm that mutation of N230 and N339 disrupted the mannose patch on Env , we assayed the ability of 2G12 , which recognizes epitopes in the mannose patch ( Sanders et al . , 2002; Scanlan et al . , 2002 ) to neutralize wild-type and mutant Env .", "As shown in Figure 6D , wild-type but not mannose deficient N230D N339E Env was neutralized by 2G12 .", "In addition , we found that these substitutions did not disrupt infection of a T cell line that does not express MR ( Figure 6E ) .", "However , somewhat unexpectedly , we found that HIV containing the N230D N339E Env substitutions was approximately 40% less infectious to primary human macrophages expressing MR than the wild-type parental virus ( Figure 6E , p=0 . 002 ) .", "This macrophage-specific difference in infectivity suggested that mannose on Env may facilitate initial infection through interactions with MR , which is highly expressed on differentiated macrophages .", "To examine this possibility further , we asked whether soluble mannan , which competitively inhibits MR interactions with mannose containing glycans ( Shibata et al . , 1997 ) , was inhibitory to HIV infection of macrophages .", "As a negative control , we tested 89 . 6 ∆env pseudotyped with vesicular stomatitis virus G-protein Env ( VSV-G ) which has only two N-linked glycosylation sites , both of which contain complex-type rather than high-mannose glycans ( Reading et al . , 1978 ) .", "Therefore VSV-G should not bind MR or be inhibited by mannan .", "As expected , we found that infection of a T cell line lacking MR was not sensitive to mannan ( Figure 6F , left panel ) .", "However , infection of MDM by wild-type HIV-1 was inhibited up to 16-fold by mannan ( Figure 6F , right panel ) .", "This was specific to HIV Env because mannan did not inhibit infection by HIV lacking env and pseudotyped with heterologous VSV-G Env .", "Interestingly , mannan also inhibited baseline macrophage infection by mannose-deficient Env ( 89 . 6 Env N230D N339E ) , indicating that N230D N339E substitutions did not completely abrogate glycans on Env that are beneficial to initial infection .", "In sum , our results demonstrate that interactions with mannose binding receptors are advantageous for initial HIV infection of macrophages and that the glycans remaining on Env N230D N339E retain some ability to bind glycan receptors on macrophages that facilitate infection .", "While interactions between high-mannose residues on Env and MR were advantageous for viral entry , we hypothesized that they interfered with intracellular Env trafficking and were deleterious to egress of Env-containing virions in the absence of Vpr and/or Nef .", "To test this , we examined virion release and Env expression by HIVs encoding the mannose-deficient Env N230D N339E in the presence or absence of Vpr .", "In a spreading infection of MDM , we found that virus expressing mannose-deficient Env had a reduced requirement for Vpr for maximal virus release compared with the parental wild-type virus ( Figure 6G , p<0 . 001 ) .", "In addition , in single-round infections of MDM , the mannose-deficient Env had a reduced requirement for both Nef and Vpr ( Figure 6H and Figure 6—figure supplement 1 , p<0 . 001 ) .", "Single round infection assays cultured for ten days were used to assess the vpr-nef double mutant because depletion of mannose on Env did not rescue spread under conditions that were most comparable to our ten day spreading infections .", "The defect in spread is likely due to pleiotropic effects of Nef that disrupt interference by the HIV receptors , CD4 , CXCR4 and CCR5 ( Lama et al . , 1999; Michel et al . , 2005; Venzke et al . , 2006 ) combined with the reduced infectivity of the mannose deficient Env .", "Finally , we asked whether the mannose-deficient Env had increased stability in primary human MDM lacking Vpr and/or Nef by western blot analysis .", "We found that the Env mutant ( N230D . N339E ) was more stable in the absence of Vpr ( Figure 6I , right side , black bars ) and Nef ( Figure 6I , right side , gray bars ) once differences in infection frequency were accounted for by matching pr55 expression in the dilution series .", "These data provide strong support for a model in which MR restricts Env expression via direct interaction with high-mannose residues on Env and this restriction is counteracted by Vpr and Nef .", "To directly test the hypothesis that MR is a restriction factor in MDM that is counteracted by Vpr , we examined the effect of MR silencing on Env expression in HIV-infected MDM lacking Vpr .", "Consistent with our hypothesis , we observed that silencing MR stabilized Env relative to Gag pr55 ( Figure 7A ) .", "These results support the conclusion that the Env restriction observed in the absence of Vpr is dependent on expression of MR . Previous work in our laboratory demonstrated that restriction of Env in primary human MDM disrupted formation of virological synapses and cell-to-cell spread of HIV from infected MDM to T cells ( Collins et al . , 2015 ) .", "Expression of Vpr alleviated these effects , dramatically increasing viral transmission – especially under conditions of low initial inoculum of free virus .", "To expand on these findings , we measured Vpr-dependent HIV-1 spread from primary human MDM to autologous T cells , as diagrammed in Figure 7—figure supplement 1A .", "Co-cultured cells were stained for CD3 to distinguish T cells and CD14 to distinguish MDM as shown in Figure 7—figure supplement 1B , accounting for differences in autofluorescent background in the two cell types by using isotype controls ( Figure 7—figure supplement 1C ) We confirmed our prior finding that Vpr enhances HIV-1 89 . 6 spread from MDM to T cells ( Figure 7—figure supplement 1D ) and extended this finding to the transmitted/founder ( T/F ) clone REJO ( Figure 7—figure supplement 1E ) .", "Consistent with our previous findings , we observed that a higher frequency of T cells became infected following co-culture with infected MDM as compared to incubation with high titer cell free virus [47-fold ( 89 . 6 , p=0 . 0002 ) and 38-fold ( REJO , p=0 . 048 ) ] .", "To determine whether Vpr stimulated spread from macrophages to T cells by counteracting MR restriction , we measured spread to T cells from macrophages in which MR had been silenced as diagrammed in Figure 7B .", "Using the gating strategy shown in Figure 7—figure supplement 1B , infected MDM and infected T cells were identified by intracellular Gag stain ( Figure 7C ) .", "We found that silencing MR reduced the difference between wild type and Vpr-null infected macrophage spread to T cells from 7-fold ( p=0 . 003 ) to 2-fold ( p=0 . 02 ) ( Figure 7D ) .", "These results provide strong evidence that MR is the previously described but unidentified restriction factor in macrophages that reduces HIV spread from macrophages to T lymphocytes in the absence of Vpr ." ], [ "We previously reported that Env and Env-containing virions are degraded in macrophage lysosomes in the absence of Vpr , impairing virion release , virological synapse formation , and spread of HIV to T cells ( Mashiba et al . , 2014; Collins et al . , 2015 ) .", "Moreover , this requirement for Vpr was conferred to heterokaryons comprised of macrophages and permissive cells , suggesting the existence of a previously unidentified host restriction factor that is counteracted by Vpr in macrophages ( Mashiba et al . , 2014 ) .", "Results presented here clearly define mannose receptor ( MR ) as the HIV restriction factor counteracted by Vpr in macrophages to enhance viral dissemination .", "We provide strong evidence that Env mannosylation is required for restriction of Env and virion release in macrophages in the absence of Vpr , and that MR silencing relieves a requirement for Vpr to overcome this restriction .", "Moreover , we confirm and extend a prior report that Nef also acts to downmodulate MR from the macrophage cell surface ( Vigerust et al . , 2005 ) and demonstrate that Vpr and Nef cooperate to counteract MR in an additive fashion through independent mechanisms .", "Other investigators have reported that HIV inhibits MRC1 transcription in macrophages and that MR inhibits virion egress upon exogenous expression in 293T cells ( Sukegawa et al . , 2018 ) .", "In contrast to results we report here , the prior study observed effects on virions that were Env-independent and did not examine effects of Vpr on MR . In primary macrophages , Vpr-sensitive virion restriction only occurs when virions contain Env ( Mashiba et al . , 2014 ) and genetic changes in the env open reading frame – especially those that alter N-linked glycosylation sites – critically affect the requirement for Vpr .", "The effect of MR on Env and Env-containing virion release reported here helps explain previous observations that primate lentivirus infection reduces MR activity in humans ( Koziel et al . , 1993; Koziel et al . , 1998 ) and monkeys ( Holder et al . , 2014 ) .", "By confirming and extending our prior finding that Vpr-mediated stabilization of Env promotes macrophage to T cell spread ( Collins et al . , 2015 ) we also provide an explanation for how Vpr increases infection of human lymphoid tissue ex vivo ( Eckstein et al . , 2001; Rucker et al . , 2004 ) , which contain macrophages and T cells in a highly physiological , three-dimensional environment .", "As Nef had already been shown to reduce MR surface expression ( Vigerust et al . , 2005 ) , the observation that HIV encodes a second protein , Vpr , to reduce MR expression was unanticipated , but not unprecedented; other host proteins are known to be affected by more than one lentiviral accessory protein .", "The HIV receptor , CD4 , is simultaneously targeted by Vpu , Nef and Env in HIV-1 ( Chen et al . , 1996 ) and tetherin is alternately targeted by Vpu , Nef , or Env in different strains of primate lentiviruses ( Harris et al . , 2012 ) .", "Nef has also been shown to downmodulate the viral co-receptors CXCR4 ( Venzke et al . , 2006 ) and CCR5 ( Michel et al . , 2005 ) , which may also interfere with Env expression and viral egress in infected cells .", "Nef’s activity against CXCR4 , CCR5 , and MR presumably has the same ultimate purpose as its activity against CD4 , namely to stabilize Env , enhance virion release and prevent superinfection of the producer cell ( Lama et al . , 1999; Ross et al . , 1999 ) .", "The impact of these deleterious interactions is clearly demonstrated by the profound loss of Env we observed in HIV-infected macrophages lacking both Vpr and Nef .", "The need for both Vpr and Nef to counteract MR may be explained by the high level of MR expression , estimated at 100 , 000 copies per macrophage ( Stahl et al . , 1980 ) .", "The potent combined effect likely derives from synergistic targeting of MR at two different stages of MR synthesis .", "Nef was shown to alter MR trafficking ( Vigerust et al . , 2005 ) and we show Vpr inhibits MR transcription .", "In addition , our results suggest that maximal MR downmodulation is time-dependent in macrophages , which have the capacity to survive while infected for weeks; western blot analysis of whole cell lysates from saturated , ten-day infected cultures achieved a more striking reduction than was observed by flow cytometric analysis of five day cultures of macrophages infected with non-spreading viruses expressing GFP .", "This time dependency is potentially explainable in part by the very long half-life of MR [33 hr ( Lennartz et al . , 1989 ) ] combined with the large amount of MR expressed per cell discussed above .", "In sharp contrast to the effect we observed in MDM , Vpr did not affect MR protein levels when MR was expressed via a heterologous promoter in the 293T cell line , which is derived from human embryonic kidney cells and is not a natural target of HIV .", "The cell type selectivity in these experiments is likely due to differences in the promoters driving MR expression , however , we cannot rule out the existence of other macrophage specific pathways required to recreate the effect of Vpr on MR . Further work will be needed to examine these questions and determine other mechanistic details .", "Our findings also implicate the Vpr binding protein VprBP/DCAF1 ( McCall et al . , 2008 ) , a component of the cellular DCAF1-DDB1-CUL4 E3 ubiquitin ligase complex , in downmodulation of MR by Vpr .", "This complex is required for most of the known functions of Vpr , including: disruption of the cell cycle , disruption of cellular DNA repair pathways in dividing cells ( Belzile et al . , 2007; Hrecka et al . , 2007; Le Rouzic et al . , 2007; Wen et al . , 2007; Lahouassa et al . , 2016; Wu et al . , 2016; Zhou et al . , 2016 ) and transcriptional inhibition of type I interferons in response to infection in macrophage cultures ( Laguette et al . , 2014; Mashiba et al . , 2014 ) .", "Additional research is now needed to determine how interactions between Vpr and DCAF1 mediate these pleiotropic effects .", "Deleterious interactions between MR and Env that are alleviated by Vpr and Nef , likely occur along the secretory pathway and continue at the cell surface .", "This is based on previously published work showing that Env-containing virions are retained at the cell surface and targeted to lysosomes in macrophages lacking Vpr ( Collins et al . , 2015 ) .", "Our prior studies also provided evidence that unprocessed Env gp160 is affected and targeted to lysosomal compartments albeit to a lesser degree ( Mashiba et al . , 2014 ) .", "Because Env processing occurs via furin-mediated cleavage in the trans-Golgi network ( TGN ) , the effect on unprocessed Env provides evidence that in addition to acting at the surface , MR likely also interacts with Env along the secretory pathway prior to its arrival and processing in the TGN .", "MR’s interaction with Env appears to be mediated by the unusually high density of N linked glycosylation sites on Env that retain high-mannose glycans , which is a known pathogen-associated molecular pattern ( Stahl and Ezekowitz , 1998; McGreal et al . , 2006 ) .", "Here , we show that selective deletion of mannose residues alleviated the requirement for Vpr .", "Deletion of individual glycosylation sites is known to lead to changes in the processing of neighboring glycans and deletions at certain sites lead to larger than expected losses of oligomannose ( Balzarini , 2007 ) presumably because their removal allows greater access to mannosidases and facilitates trimming of surrounding glycans .", "Selective pressure to maintain mannose residues on Env may be due to the enhanced attachment they mediate .", "Indeed , we provide strong evidence that Env’s interaction with MR boosts initial infection of MDM .", "This finding is supported by a prior report that MR enhances HIV-1 binding to macrophages and transmission of the bound virus to co-cultured T cells ( Nguyen and Hildreth , 2003 ) .", "Our study adds to these findings by providing evidence that interactions with mannose binding receptors also enhance direct infection of macrophages .", "Moreover , the capacity of Vpr and Nef to mitigate the effect of detrimental intracellular interactions during viral egress limits the negative impact of retaining high-mannose on Env .", "In addition , the dense glycan packing , which is privileged from antibody recognition through immune tolerance , is believed to play a role in evasion of the antibody response ( Stewart-Jones et al . , 2016 ) .", "Because MR has both positive and negative effects on infection , the interpretation of some experiments examining spreading infection in the setting of MR silencing or mutations in Env that reduced mannose content were complex to interpret .", "Some donors had increased infection resulting from MR silencing whereas others had a small decrease at the ten-day time point ( data not shown ) .", "By using viral systems that allowed us to focus independently on viral entry and exit , we nevertheless clearly discerned that MR can serve as a positive factor for entry and a negative factor for egress .", "Thus far , all viral Envs we have tested ( NL4-3 , AD8 and 89 . 6 ) require Vpr for stable expression in macrophages except YU-2 .", "We show here that genetically altering the mannose patch on 89 . 6 so that it mirrored changes in the YU-2 mannose patch altered the behavior of 89 . 6 to resemble that of YU-2 with respect to Vpr phenotypes .", "This is strong evidence supporting our model that Vpr alleviates deleterious interactions caused by the Env mannose patch .", "Interestingly , YU-2 was cloned from the central nervous system and 89 . 6 was directly cloned from peripheral blood .", "Because the blood-brain barrier limits exposure to antibodies , CNS isolates may have a diminished requirement for high mannose residues , which protect from antibody responses .", "Here we also confirm and extend our prior observation ( Collins et al . , 2015 ) that co-culturing T cells with infected MDM boosted HIV infection compared to direct infection of T cells with cell-free virus .", "Similar to clone 89 . 6 , T cell infection by the transmitter/founder virus REJO was enhanced by co-culture with MDM , and spread from MDM to T cells was enhanced by Vpr .", "In the context of natural person-to-person transmission , accelerated spread to T cells may be critical to establishing a persistent infection before innate and adaptive immune responses are activated .", "The strong selective pressure to retain Vpr despite its limited effect on T cell-only cultures indicates there is more to learn about the role of Vpr , macrophages and T/F viruses in HIV transmission and pathogenesis .", "Collectively , these studies suggest that novel therapeutic approaches to inhibit the activity of Vpr and Nef in macrophages would potentially represent a new class of antiretroviral drug that could be an important part of a treatment or prophylactic cocktail ." ], [ "The following molecular clones were obtained via the AIDS Reagent Program: p89 . 6 [cat# 3552 from Dr . Ronald G . Collman ) , pNL4-3 ( cat# 114 from Dr . Malcolm Martin ) , pREJO . c/2864 ( cat# 11746 from Dr . John Kappes and Dr . Christina Ochsenbauer ) and pYU2 ( cat# 1350 from Dr . Beatrice Hahn and Dr . George Shaw ) . Vpr-null versions of 89 . 6 , NL4-3 , and YU2 were created by cutting the AflII site within vpr and filling in with Klenow fragment . The vpr-null version of REJO was created by doing the same at the AvrII site . A nef-null version of 89 . 6 was created by deleting nef from its start codon to the XhoI site . To do this , a PCR amplicon was generated from the XhoI site in env to env’s stop codon . The 3’ reverse primer added a XhoI site after the stop codon . The 89 . 6 genome and the amplicon were digested with XhoI and ligated together . ( 5’ primer CACCATTATCGTTTCAGACCCT and 3’ primer TCTCGAGTTTAAACTTATAGCAAAGCCCTTTCCA ) . The NL4-3 envYU2 chimera consists of the pNL4-3 plasmid in which the fragment from the KpnI site in env to the BamHI site in env has been replaced with the equivalent fragment of pYU-2 . Because the KpnI site is not unique within the plasmid , the fragment from the SalI site to BamHI site ( which are unique ) was cloned into pUC19 , the change was made in env , and the fragment from SalI to BamHI was inserted back into pNL4-3 . To generate p89 . 6 N230D N339E a synthetic DNA sequence ( ThermoFisher , Waltham , Massachusetts ) was purchased commercially . The synthetic gene contained the following nucleotide mutations , counting from the start of 89 . 6 env: 694 A > G , 701 C > A , 1018 A > G , 1020 T > A . This sequence was substituted into p89 . 6 using the KpnI and BsaBI sites within env . pSIV3+ , pSPAX2 , pAPM-1221 and pMD2 . G were obtained from Dr . Jeremy Luban ( Pertel et al . , 2011 ) . pSIV3+ vpr-null was generated using a synthesized DNA sequence ( ThermoFisher ) containing a fragment of the SIV genome in which the Vpr start codon was converted to a stop codon ( TAG ) . This was substituted into pSIV3+ using the sites BstBI and SapI . pYU2 env was obtained from Dr . Joseph Sodroski ( Sullivan et al . , 1995 ) . Creation of pNL4-3 ∆GPE-GFP was described previously ( Zhang et al . , 2004; McNamara et al . , 2012 ) . Notably , the transcript containing the gfp gene retains the first 42 amino acids of env , including the signal peptide , which creates a fully fluorescent Env-GFP fusion protein . The vpr-Q65R mutant of NL4-3 ∆GPE-GFP was created using the Q5 site-directed mutagenesis kit from New England Biolabs ( Ipswich , MA ) . The forward primer was AGAATTCTGCGACAACTGCTG and the reverse primer TATTATGGCTTCCACTCC . After synthesis by PCR , the entire provirus was confirmed by sequencing . pCDNA . 3 . hMR was obtained from Dr . Johnny J . He ( Liu et al . , 2004 ) . pPROA-3FLAG-UNG2-EYFP was obtained from Dr . Marit Otterlei ( Akbari et al . , 2010 ) and 3x FLAG tagged UNG2 was amplified using the 5’ primer CTAGCTCGAGACCATGGACTACAAAGACCATGAC , which added an XhoI site , and the 3’ primer GTTAACTCACAGCTCCTTCCAGTCAATGGGCTT , which added an HpaI site . The amplicon was cloned into the XhoI and HpaI sites of pMSCV IRES-GFP ( Van Parijs et al . , 1999 ) to generate pMSCV 3xFLAG UNG2 IRES-GFP . Leukocytes isolated from anonymous donors by apheresis were obtained from the New York Blood Center Component Laboratory . The use of human blood from anonymous , de-identified donors was classified as non-human subject research in accordance with federal regulations and thus not subjected to formal IRB review . Peripheral blood mononuclear cells ( PBMCs ) were purified by Ficoll density gradient . CD14+ monocytes were positively selected using a CD14 sorting kit ( cat# 17858 , StemCell Technologies , Vancouver , Canada ) following the manufacturer’s instructions . Monocyte-derived macrophages ( MDM ) were obtained by culturing monocytes in R10 [RPMI-1640 with 10% certified endotoxin-low fetal bovine serum ( Invitrogen , ThermoFisher ) , penicillin ( 100 Units/mL ) , streptomycin ( 100 μg/mL ) , L-glutamine ( 292 μg/mL ) , carrier-free M-CSF ( 50 ng/mL , R and D Systems , Minneapolis , Minnesota ) and GM-CSF ( 50 ng/mL , R and D Systems ) ] for seven days .", "Monocytes were plated at 5 × 105 cells/well in a 24 well dish , except for those to be transduced with lentivirus and puromycin selected , which were plated at 1 × 106 cells/well .", "CD4+ T lymphocytes were prepared from donor PBMCs as follows: anti-CD8 Dynabeads ( cat# 11147D , ThermoFisher ) were used to deplete CD8+ T lymphocytes and the remaining cells , which were mainly CD4+ lymphocytes , were maintained in R10 until the time of stimulation .", "Lymphocytes were stimulated with 5 μg/mL phytohemagglutinin ( PHA-L , Calbiochem , Millipore Sigma , Burlington , Massachusetts ) overnight before addition of 50 IU/mL recombinant human IL-2 ( R and D Systems ) .", "The 293T cell line was obtained from ATCC and independently authenticated by STR profiling .", "It was maintained in DMEM medium ( Gibco ) supplemented with 100 U/mL penicillin , 100 μg/mL streptomycin , 2 mM glutamine ( Pen-Strep-Glutamine , Invitrogen ) , 10% fetal bovine serum ( Invitrogen ) , and 0 . 022% plasmocin ( Invivogen ) .", "The MOLT-R5 cell line was obtained from the NIH AIDS Reagent Repository , which confirmed the lot is mycoplasma negative .", "It was maintained in RPMI-1640 medium ( Gibco ) supplemented with 100 U/mL penicillin , 100 μg/mL streptomycin , 2 mM glutamine ( Pen-Strep-Glutamine , Invitrogen ) , 10% fetal bovine serum ( Invitrogen ) , and 0 . 022% plasmocin ( Invivogen ) .", "Sequences within MRC1 suitable for shRNA-based targeting were identified using the program available at http://katahdin . mssm . edu/siRNA/RNAi . cgi ?", "type=shRNA maintained by the laboratory of Dr . Ravi Sachidanandam .", "The sequence chosen , 5’-AGTAACTTGACTGATAATCAAT-3’ was synthesized as part of larger DNA oligonucleotides with the sequences TCGAGAAGGTATATTGCTGTTGACAGTGAGCGAGTAACTTGACTGATAATCAATTAGTGAAGCCACAGATGTAATTGATTATCAGTCAAGTTACTTGCCTACTGCCTCGG ( forward ) and AATTCCGAGGCAGTAGGCAAGTAACTTGACTGATAATCAATTACATCTGTGGCTTCACTAATTGATTATCAGTCAAGTTACTCGCTCACTGTCAACAGCAATATACCTTC ( reverse ) .", "These oligos were annealed , which created overhangs identical to those produced by digestion with the enzymes EcoRI and XhoI .", "This double stranded DNA oligomer was inserted into the EcoRI and XhoI sites of pAPM-1221 to generate pAPM-MRC1-C .", "Short hairpin RNA-mediated silencing was performed as previously described ( Pertel et al . , 2011; Mashiba and Collins , 2013; Collins et al . , 2015 ) .", "Briefly , we spinoculated freshly isolated primary monocytes with VSV-G-pseudotyped SIV3+ vpr-null at 2500 rpm for 2 hr with 4 μg/mL polybrene to allow Vpx-dependent degradation of SAMHD1 .", "Cells were then incubated overnight in R10 with M-CSF ( 50 ng/mL ) and GM-CSF ( 50 ng/mL ) plus VSV-G-pseudotyped lentivirus containing an shRNA cassette targeting luciferase ( pAPM-1221 or ‘shNC’ ) or MR ( pAPM-MRC1-C or ‘shMR’ ) .", "The following day , media was removed and replaced with fresh R10 with M-CSF ( 50 ng/mL ) and GM-CSF ( 50 ng/mL ) .", "Three days later 10 μg/mL puromycin was added and cells were cultured for three additional days prior to HIV-1 infection .", "shRNA target sequences used: Luciferase: 5'-TACAAACGCTCTCATCGACAAG-3' , MRC1: 5’-ATTGATTATCAGTCAAGTTACT-3’ .", "Virus stocks were obtained by transfecting 293T cells ( ATCC , Manassas , Virginia ) with viral DNA and polyethylenimine ( PEI ) .", "Cells were plated at 2 . 5 × 106 cells per 10 cm dish and incubated overnight .", "The following day 12 µg of total DNA was combined with 48 µg of PEI , mixed by vortexing , and added to each plate of cells .", "For NL4-3 ∆GPE-GFP , cells were transfected with 4 µg viral genome , 4 µg pCMV-HIV , and 4 µg pHCMV-V ( VSV-G expression plasmid ) .", "For SIV3+ vpr-null the cells were transfected with 10 . 5 µg of viral genome and 1 . 5 µg pHCMV-V .", "For shLentivirus ( shNC or shMR ) cells were transfected with 6 µg pAPM-1221 or pAPM-MRC1-C , 4 . 5 µg pSPAX2 , and 1 . 5 µg pMD2 . G .", "Viral supernatant was collected 48 hr post-transfection and centrifuged at 1500 rpm ( 500 x g ) 5 min to remove cellular debris .", "SIV3+ vpr-null was pelleted by centrifugation at 14 , 000 rpm ( 23 , 700 x g ) for 4 hr at 4°C and resuspended at 10x concentration .", "Virus stocks were aliquoted and stored at −80°C .", "Co-transfections of HIV and MR or UNG2 were performed in 293T cells .", "Cells were plated at 1 . 6 × 105 per well in a 12-well dish .", "The following day 10 ng of pcDNA . 3 . hMR or 10 ng of pMSCV 3xFLAG UNG2 IRES-GFP , 250 ng of NL4-3 ∆GPE-GFP , and 740 ng pUC19 plasmid was combined with 4 µg PEI , mixed by vortexing , and added to each well .", "48 hr later , cells were lifted using enzyme free cell dissociation buffer ( ThermoFisher , cat# 13150016 ) and analyzed by flow cytometry or lysed in 500 µL blue loading buffer ( cat# 7722 , Cell Signaling Technology , Danvers , Massachusetts ) and analyzed by western blot .", "Prior to infection , 500 µL of medium was removed from each well and this ‘conditioned’ medium was saved to be replaced after the infection .", "MDM were infected by equal inocula of HIV as measured by Gag p24 mass in 500 µL of R10 for 6 hr at 37°C .", "After 6 hr , infection medium was removed and replaced with a 1:2 mixture of conditioned medium and fresh R10 .", "Where indicated , HIV spread was blocked by AMD3100 ( 10 µg/mL , AIDS Reagent Program cat# 8128 ) and/or maraviroc ( 20 µM , AIDS Reagent Program cat# 11580 ) added 48 hr post-infection and replenished with each media change every three days .", "MDM were centrifuged at 2500 rpm ( 1049 x g ) for 2 hr at 25°C with equal volume of NL4-3 ∆GPE-GFP or an isogenic mutant in 500 uL total medium .", "Following infection , medium was removed and replaced with a 1:2 mixture of conditioned medium and fresh R10 .", "Adenovirus was prepared by the University of Michigan Vector Core , and the transduction of MDM was performed as previously described ( Leonard et al . , 2011 ) at an MOI of 1000 based on 293T cell infection estimations and the concentration of particles as assessed by OD280 .", "Activated T cells were infected by two methods as indicated .", "For direct infection , 5 × 105 cells were plated per well with 50 µg HIV p24 in 500 µL R10 +50IU/mL of IL-2 and incubated at 37°C for 48 hr .", "For co-culture with autologous , infected MDM medium was removed from MDM wells and 5 × 105 T cells were added in 1mL R10 + 50IU/mL of IL-2 .", "All T cell infections were collected 48 hr post infection .", "Intracellular staining of cells using antibodies directed against HIV Gag p24 , MR and FLAG-UNG2 was performed by permeabilizing paraformaldehyde-fixed cells with 0 . 1% Triton-X in PBS for 5 min , followed by incubation with antibody for 20 min at room temperature .", "For Gag and MR , PE-conjugated primary antibodies were used .", "For FLAG-UNG2 , cells were stained with a PE-conjugated goat anti-mouse IgG1 secondary antibody for 20 min at room temperature .", "Surface staining for CD4 , CD3 and CD14 was performed before fixation as described previously ( Collins et al . , 2015 ) .", "Flow cytometric data was acquired using a FACSCanto instrument with FACSDiva collection software ( BD , Franklin Lakes , New Jersey ) or a FACScan ( Cytek , BD ) with FlowJo software ( TreeStar , Ashland , Oregon ) and analyzed using FlowJo software .", "Live NL4-3 ∆GPE-GFP transduced cells were sorted using a FACSAria III ( BD ) or MoFlo Astrios ( Beckman Coulter ) and gating on GFP+ cells .", "MDM sorted as described above in ‘Flow cytometry’ were collected into tubes containing RLT buffer ( Qiagen , Hilden , Germany ) and RNA was isolated using RNeasy Kit ( Qiagen ) with on-column DNase I digestion .", "RNA was reverse transcribed using qScript cDNA SuperMix ( Cat #95048 , Quantabio , Beverly , Massachusetts ) .", "Quantitative PCR was performed using TaqMan Gene Expression MasterMix ( ThermoFisher , cat# 4369016 ) on an Applied Biosystems 7300 Real-Time PCR System using TaqMan Gene Expression primers with FAM-MGB probe .", "The primer/probe sets for ACTB ( Hs99999903 ) , MRC1 ( Hs00267207 ) , POL2A ( Hs02786624 ) , and GAPDH ( Hs00172187 ) were purchased from ThermoFisher .", "Reactions were quantified using ABI Sequence Detection software compared to serial dilutions of cDNA from mock-treated cells .", "Measured values for all genes were normalized to measured values of GAPDH or ACTB as indicated .", "MDM cultures were lysed in Blue Loading Buffer ( cat# 7722 , Cell Signaling Technology ) , sonicated with a Misonix sonicator ( Qsonica , LLC . , Newtown , Connecticut ) , boiled for 5 min at 95°C and clarified by centrifugation at 8000 RPM ( 7 , 000 x g ) for 3 min .", "Lysates were analyzed by SDS-PAGE immunoblot .", "The proteins MR , GAPDH and pr55 were visualized using AlexFluor-647 conjugated secondary antibodies on a Typhoon FLA 9500 scanner ( GE , Boston , Massachusetts ) and quantified using ImageQL ( GE ) .", "The proteins gp160 , gp120 , gp41 , Nef , Vpr , GFP , Env-GFP , STING , GBP5 , and IFITM3 were visualized using HRP-conjugated secondary antibodies on film .", "Immunoblot films were scanned and the mean intensity of each band , minus the background , was calculated using the histogram function of Photoshop CC ( Adobe , San Jose , California ) .", "Supernatant containing viral particles was lysed in Triton X lysis buffer ( 0 . 05% Tween 20 , 0 . 5% Triton X-100 , 0 . 5% casein in PBS ) .", "Gag p24 antibody ( clone 183-H12-5C , AIDS Reagent Program cat# 1519 from Dr . Bruce Cheseboro and Dr . Hardy Chen ) was bound to Nunc MaxiSorp plates ( ThermoFisher cat# 12-565-135 ) at 4°C overnight .", "Lysed samples were captured for 2 hr and then incubated with biotinylated antibody to Gag p24 ( clone 31-90-25 , ATCC cat# HB-9725 ) for 1 hr .", "Clone 31-90-25 was biotinylated with the EZ-Link Micro Sulfo-NHS-Biotinylation Kit ( ThermoFisher cat# PI-21925 ) .", "Clones 31-90-25 and 182-H12-5C were purified using Protein G columns ( GE Healthcare , cat# 45-000-054 ) following the manufacturer’s instructions .", "Samples were detected using streptavidin-HRP ( Fitzgerald , Acton , Massachusetts ) and 3 , 3′ , 5 , 5′-tetramethylbenzidine substrate ( Sigma cat# T8665-IL ) .", "CAp24 concentrations were measured by comparison to recombinant CAp24 standards ( cat# 00177 V , ViroGen , Watertown , Massachusetts ) .", "Antibodies to CAp24 ( clone KC57-PE cat# 6604667 and KC57-FITC cat# 6604665 , Beckman Coulter , Brea , California ) , CD3 ( clone OKT3-Pacific Blue , cat# 317313 , BioLegend , San Diego , California ) , CD14 ( clone HCD14-APC , cat# 325608 , BioLegend ) , CD4 ( clone OKT4 , cat#17-0048-42 , Invitrogen , ThermoScientific ) , FLAG ( clone M2 , cat#F3165 , Sigma ) , and MR ( clone 19 . 2-PE , cat# 555954 , BD ) were used for flow cytometry .", "Antibodies to the following proteins were used for immunoblot analysis: MR ( cat# ab64693 , Abcam , Cambridge , Massachusetts ) , GAPDH ( clone 3C2 , cat# H00002597-M01 , Abnova , Taipei , Taiwan ) , Gag pr55 ( HIV-Ig AIDS Reagent Program cat# 3957 ) , Env gp160/120 ( AIDS Reagent Program cat# 288 from Dr . Michael Phelan ) , 89 . 6 and YU-2 Env gp41 ( clone z13e1 , AIDS Reagent Program cat# 11557 from Dr . Michael Zwick ) , NL4-3 Env gp41 ( clone CHESSIE-8 , AIDS Reagent Program cat# 526 from Dr . George Lewis ) , Vpr ( AIDS Reagent Program cat# 3951 from Dr . Jeffrey Kopp ) , GFP ( cat# ab13970 , Abcam ) , Nef ( AIDS Reagent Program cat# 2949 from Dr . Ronald Swanstrom ) , FLAG ( clone M2 , cat# F3165 , Sigma ) , STING ( D2P2F , cat# 13647 , Cell Signaling Technology ) , GBP5 ( sc-160353 , which was a generous gift from Dr . Frank Kirchhoff ) , and IFITM3 ( cat# 11714–1-AP , Proteintech , Rosemont , IL ) .", "Neutralizing antibody 2G12 ( AIDS Reagent Program cat# 1476 from Dr . Hermann Katinger ) was used at a 1 μg/mL at the time of infection .", "Antibody clone CHESSIE-8 was purified using Protein G columns ( GE Healthcare , cat# 45-000-054 ) following the manufacturer’s instructions ." ] ]

[ "HIV-1 Vpr is necessary for maximal HIV infection and spread in macrophages .", "Evolutionary conservation of Vpr suggests an important yet poorly understood role for macrophages in HIV pathogenesis .", "Vpr counteracts a previously unknown macrophage-specific restriction factor that targets and reduces the expression of HIV Env .", "Here , we report that the macrophage mannose receptor ( MR ) , is a restriction factor targeting Env in primary human monocyte-derived macrophages .", "Vpr acts synergistically with HIV Nef to target distinct stages of the MR biosynthetic pathway and dramatically reduce MR expression .", "Silencing MR or deleting mannose residues on Env rescues Env expression in HIV-1-infected macrophages lacking Vpr .", "However , we also show that disrupting interactions between Env and MR reduces initial infection of macrophages by cell-free virus .", "Together these results reveal a Vpr-Nef-Env axis that hijacks a host mannose-MR response system to facilitate infection while evading MR’s normal role , which is to trap and destroy mannose-expressing pathogens ." ]

[ "Human cells have defense mechanisms against viral infection known as restriction factors .", "These are proteins that break down parts of a virus including its DNA or proteins .", "To evade these defenses , viruses in turn make proteins that block or break down restriction factors .", "This battle between human and viral proteins determines which types of cells are infected and how quickly a virus can multiply and spread to new cells .", "HIV produces a protein called Vpr that counteracts a restriction factor found in immune cells called macrophages .", "However , the identity of the restriction factor targeted by Vpr is a mystery .", "When Vpr is missing , this unknown restriction factor breaks down a virus protein called Env .", "Env is a glycoprotein , which is a protein with sugars attached .", "When Env levels are low , HIV cannot spread to other cells and multiply .", "Identifying the restriction factor that breaks down Env may lead to new ways of treating and preventing HIV infections .", "Now , Lubow et al . reveal that the unknown restriction factor in macrophages is a protein called the mannose receptor .", "This protein binds and destroys proteins containing mannose , a type of sugar found on bacteria and some viruses .", "The experiments revealed that the mannose receptor grabs mannose on the HIV protein Env .", "This causes Env to be broken down and stops HIV from spreading .", "Lubow et al . also find that Vpr works with another protein produced by HIV called Nef to reduce the number of mannose receptors on macrophages .", "The two proteins do this by targeting different steps in the assembly of mannose receptors , allowing the virus to multiply and spread more efficiently .", "The experiments suggest that drugs that simultaneously block Vpr and Nef might prevent or suppress HIV infections .", "More studies are needed to develop and test potential HIV-treatments targeting Vpr and Nef ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "cell biology", "neuroscience" ]

[ [ "Neurogenesis is the process that leads to a regionalized brain from an embryonic neuroepithelium .", "In the mouse developing brain , most neurons are generated between the 10th day of embryonal life ( E10 ) and birth ( Martynoga et al . , 2012 ) , even though adult neurogenesis persists in restricted areas near the lateral ventricles , the ventricular ( VZ ) and subventricular ( SVZ ) zones ( Bjornsson et al . , 2015 ) , and in the subgranular zone of the dentate gyrus of the hippocampus ( Urbán and Guillemot , 2014 ) .", "Neurogenesis is fueled by specialized stem cells that are collectively named Neural Stem/Progenitor Cells ( NPCs ) ( Johansson et al . , 2010; Taverna et al . , 2014 ) .", "As embryonic neurogenesis starts , neuroepithelial cells at first , and radial glia cells ( RGCs ) later , divide symmetrically in the VZ , to generate cells retaining self-renewal capacity , or asymetrically to give rise to intermediate progenitor cells ( IPCs ) or differentiated cells ( Paridaen and Huttner , 2014; Taverna et al . , 2014 ) .", "IPCs divide away from the VZ , one or more times , before they generate post-mitotic neurons , which localize in the basal compartment of the developing cortex ( cortical layer ) .", "These divisions of neural precursors need to be tightly modulated , as proper regulation of neurogenesis is of paramount importance to achieve the right number of neurons and the correct expansion and stratification of the cortex during development .", "In particular , fine-tuned control of NPC fate is required to balance self-renewing divisions with asymmetric divisions ( Taverna et al . , 2014; Fernández et al . , 2016 ) .", "Conversely , dysregulation of neurogenesis can result in developmental disorders linked to neurological pathologies ( Sun and Hevner , 2014 ) .", "Thus , understanding the molecular mechanisms that control neurogenesis may pave the path to novel approaches to these diseases .", "Multiple mechanisms , including regulation of metabolic routes ( Shyh-Chang et al . , 2013 ) and of gene expression ( Paridaen and Huttner , 2014 ) , cooperate to create an interconnected network that balances NPC self-renewal and differentiation in the correct time and space during cortical expansion .", "Mounting evidence documents that modulation of RNA metabolism , and particularly of alternative splicing , plays a key role in neurogenesis and during formation of neuronal circuits , as highlighted by the global changes in the splicing signature that accompany the transition from NPCs to neurons ( Zheng and Black , 2013; Raj and Blencowe , 2015 ) .", "Splicing is operated by the spliceosome , a large ribonucleoprotein machinery that mediates sequentially ordered reactions to excise introns and ligate exons ( Wahl et al . , 2009 ) .", "Through regulated assortment of multiple exons and introns during pre-mRNA processing , alternative splicing allows the production of many splice variants from most genes , thus greatly amplifying the coding potential and plasticity of the genome ( Fu and Ares , 2014 ) .", "Notably , brain is among the tissues displaying the largest extent of alternative splicing , which likely contributes to the complexity of neural circuits ( Raj and Blencowe , 2015 ) .", "In support of this notion , several splicing factors have been shown to play key roles during neurogenesis and/or specific brain functions ( Raj and Blencowe , 2015 ) .", "For instance , a temporal switch in the expression of two homologous polypyrimidine-tract-binding proteins , PTBP1 and PTBP2 , governs splicing of a large set of neural-specific exons in genes involved in neuronal functions ( Boutz et al . , 2007 ) .", "Knockout of the Ptbp2 gene caused premature neurogenesis and depletion of the NPC pool ( Licatalosi et al . , 2012 ) , proving the crucial role played by this splicing factor in the developing brain .", "Similarly , the neural-specific serine-arginine ( SR ) -related protein of 100 kDa ( nSR100 ) regulates a network of exons in genes involved in neuronal functions and knockout of this gene in mice leads to widespread neurodevelopmental defects ( Calarco et al . , 2009; Quesnel-Vallières et al . , 2015 ) .", "Another splicing factor involved in neuronal functions is Sam68 , encoded by the Khdrbs1 gene , which is highly expressed in brain and testis ( Richard et al . , 2005; Paronetto et al . , 2009 ) , and it was shown to be involved in the pathogenesis of fragile X tremor/ataxia syndrome ( Sellier et al . , 2010 ) and spinal muscular atrophy ( Pedrotti et al . , 2010; Pagliarini et al . , 2015 ) .", "Furthermore , Sam68 modulates splicing of the neurexin one gene ( Nrxn1 ) in response to neuronal activity ( Iijima et al . , 2011 ) and its ablation caused altered synaptic plasticity and motor coordination defects ( Lukong and Richard , 2008; Iijima et al . , 2011 ) .", "In this study , we have investigated the role of Sam68 during the development of the central nervous system .", "Sam68 is strongly expressed during cortical expansion ( E10 . 5–15 . 5 ) , whereas its levels decline after birth .", "We found that Sam68 regulates the switch between self-renewal and differentiation of mouse NPCs both in vivo and in vitro .", "Splicing-sensitive microarrays identified a subset of genes and exons whose expression is dependent on Sam68 in NPCs .", "In particular , Sam68 prevents usage of a cryptic polyadenylation signal ( PAS ) in intron 7 of the Aldehyde Dehydrogenase 1A3 ( Aldh1a3 ) gene , thus promoting the expression of a functional enzyme .", "We also found that ALDH1A3 enhances anaerobic glycolytic metabolism in NPCs and that enforcing its activity rescued the self-renewal defect of Sam68 knockout ( Khdrbs1-/- ) NPCs .", "Conversely , Aldh1a3 knockdown in wild-type cells mimicked the phenotype of Sam68 knockout NPCs , by reducing glycolytic activity and promoting neuronal differentiation .", "Thus , our work unveils a key role of Sam68 in neurogenesis through regulation of Aldh1a3 pre-mRNA processing , which results in the modulation of glycolytic metabolism and NPC fate during cortical development ." ], [ "Sam68 is a KH-domain RNA-binding protein involved in several steps of RNA metabolism ( Bielli et al . , 2011; Frisone et al . , 2015 ) .", "Developmental analysis of the mouse cortex showed that Sam68 mRNA and protein levels peak between E13 . 5 and E15 . 5 , whereas its expression slowly declines thereafter and is minimal from 9 days post-partum ( 9dpp ) until adulthood ( Figure 1A , B ) .", "The peak of Sam68 expression corresponds to stages of intense neurogenesis in the developing cortex ( Paridaen and Huttner , 2014; Taverna et al . , 2014 ) and parallels that of the NPC marker SOX2 , which is also high between E10 . 5 and E15 . 5 and sharply decreases in post-natal stages ( Figure 1B ) .", "Furthermore , Sam68 is strongly expressed in neurogenic periventricular regions of E13 . 5 brain , like SOX2 ( Figure 1C ) .", "Sam68 and SOX2 co-localized in most cells of the VZ and SVZ of E13 . 5 cortex ( Figure 1D ) , and their expression was even more restricted to these cortical zones at 1dpp ( Figure 1E ) .", "These results suggested that Sam68 expression is high in NPCs and declines upon differentiation .", "To test this hypothesis , NPCs were isolated from E13 . 5 cortex and cultured in vitro under proliferating or differentiating conditions ( Bertram et al . , 2012 ) .", "Sam68 , like SOX2 , was abundant in proliferating NPC ( 0d ) and steadily decreased when cells were induced to differentiate ( 1d-6d in Figure 1F , G ) .", "Conversely , expression of the neuronal marker TUBB3 ( βIII-tubulin ) was barely detectable in proliferating NPCs and augmented upon differentiation ( Figure 1F , G ) .", "Thus , Sam68 is highly expressed in embryonic NPCs . 10 . 7554/eLife . 20750 . 003Figure 1 . Sam68 is highly expressed in NPCs and decreases during differentiation .", "( A ) qPCR analysis of Khdrbs1 mRNA levels in the cortex of embryonic ( E10 . 5-E15 . 5 ) and post-natal ( P0-25dpp ) mouse brain .", "Khdrbs1 relative expression was evaluated by △CT method using L34 expression for normalization .", "( B ) Western blot analysis of Sam68 and SOX2 expression in lysates from embryonic ( E10 . 5-E15 . 5 ) and post-natal ( P0-25dpp ) mouse cortices .", "GAPDH was used as loading control .", "( C and D )", "Immunofluorescence analyses of Sam68 and SOX2 expression in E13 . 5 mouse brain .", "( C ) Horizontal sections of whole brain; white arrows point to periventricular zones where both proteins are highly expressed .", "Scale bar = 250 µm .", "( D ) High-magnification confocal images confirm Sam68 and SOX2 colocalization in most cells of the VZ and SVZ .", "Scale bar = 25 µm .", "( E ) High magnification of confocal images of 1 dpp mouse VZ-SVZ , show the colocalization of Sam68 and SOX2 in NPCs ( white arrows ) .", "Scale bar = 25 µM .", "( F ) Analysis of Sam68 , SOX2 and TUBB3 mRNA ( left panels ) and protein levels ( right panels ) in NPCs cultured under proliferating condition ( 0d ) or during 1–6 days of differentiation ( 1-6d ) .", "( G ) qPCR analysis of Khdrbs1 , Sox2 and Tubb3 mRNA levels in NPCs under proliferation conditions ( 0d ) and 1–6 days of differentiation ( 1 , 3 , 6d ) .", "Relative expression was evaluated using △△CT method and 0d as reference point .", "L34 expression was used for the initial △CT normalization .", "NPCs , Neural progenitor cells . DOI: http://dx . doi . org/10 . 7554/eLife . 20750 . 003 To investigate whether Sam68 plays a role during neurogenesis , we first assessed whether its ablation affected the rate of NPC proliferation in the embryonic cortex .", "To this end , proliferating cells were labeled by injecting BrdU in pregnant females at 13 . 5 days post-coitum ( dpc ) 2 hr before embryo ( E13 . 5 ) collection .", "Proliferating cells ( BrdU-positive cells ) were significantly decreased in the cortex of Khdrbs1-/- embryos ( Figure 2—figure supplement 1A ) .", "Likewise , SOX2+-NPCs , as well as the number of NPCs undergoing DNA synthesis ( SOX2+/BrdU+ cells ) , were also markedly affected ( Figure 2—figure supplement 1A ) , indicating that fewer NPCs are present and proliferate in the E13 . 5 Khdrbs1-/- cortex .", "At this developmental stage , two main types of proliferating NPCs have been described in the mouse cortex ( Paridaen and Huttner , 2014; Taverna et al . , 2014 ) : RGCs and IPCs .", "RGCs express the transcription factor PAX6 and divide in the VZ and SVZ ( apical layers ) .", "IPCs express the transcription factor TBR2 , are derived from RGCs and divide one or more times away from the VZ before they generate post-mitotic neurons , which localize in the basal compartment of the cortex ( cortical layer ) and express the transcription factor TBR1 ( Englund et al . , 2005 ) .", "Thus , these three markers are expressed in sequentially ordered manner from the apical ( PAX6 ) to the basal ( TBR1 ) layer of the E13 . 5 neuroepithelium ( Figure 2A ) .", "Strikingly , total ( PAX6+ cells ) and proliferating RGCs ( PAX6+/BrdU+ cells ) and IPCs ( TBR2+ and TBR2+/BrdU+ cells ) were markedly reduced in the knockout cortex ( Figure 2B , C ) , whereas differentiated neurons ( TBR1+ cells ) were almost doubled ( Figure 2D ) .", "No TBR1-positive cell was labeled by BrdU in both wild-type and knockout embryos ( Figure 2—figure supplement 1B ) , confirming that these cells are post-mitotic neurons ( Englund et al . , 2005 ) . 10 . 7554/eLife . 20750 . 004Figure 2 . Ablation of Khdrbs1 perturbs neurogenesis in the embryonic cortex .", "( A ) Schematic representation of the stratification of the VZ/SVZ , intermediate zone ( IZ ) and cortical plate ( CP ) in the E13 . 5 embryonic cortex .", "Molecular markers , expressed by the respective cell types are represented on the right side of the scheme .", "( B–D )", "Immunofluorescence analyses on sections of Khdrbs1+/+and Khdrbs1-/- brain from E13 . 5 embryos treated for 2 hr with BrdU to label proliferating cells .", "Sections were stained with anti-PAX6 ( B ) , anti-TBR2 ( C ) or anti-TBR1 ( D ) .", "All sections were co-stained with anti-BrdU antibody .", "Bar graphs on the right side show the number of BrdU/PAX6 ( B ) , BrdU/TBR2 ( C ) and TBR1 ( D ) positive cells counted in 100 μm2 .", "N = 3; ***p<0 . 001 .", "Scale bar = 100 µm .", "SVZ , Subventricular Zone; VZ , Ventricular Zone; IZ , Intermediate Zone; CP , Cortical Plate; RGCs , Radial Glia Cells; IPCs , Intermediate Progenitor Cells . DOI: http://dx . doi . org/10 . 7554/eLife . 20750 . 00410 . 7554/eLife . 20750 . 005Figure 2—figure supplement 1 . Ablation of Khdrbs1 perturbs neurogenesis in the embryonic cortex .", "( A ) Representative images of immunofluorescence analysis of BrdU ( left panel ) and SOX2 ( center panel ) performed in E13 . 5 cortex of embryos treated with BrdU for 2 hr .", "Bar graphs represent ( mean±SD ) measurement of the number of BrdU+ cells ( Graph on the left ) , SOX2+ ( Graph on the center ) and BrdU+/SOX2+ cells ( Graph on the right ) .", "N = 3; *** p<0 . 001 .", "Scale bar = 100 µm .", "( B ) Representative immunofluorescence analysis of BrdU incorporation and TBR1 expression in E13 . 5 cortex of embryos treated for 2 hr with BrdU . DOI: http://dx . doi . org/10 . 7554/eLife . 20750 . 005 The concomitant decrease in proliferating cells and increase in post-mitotic neurons observed in the knockout embryos suggested that , in the absence of Sam68 , NPCs are prone to exit the cell cycle and differentiate .", "To directly test this hypothesis , we injected pregnant females at 12 . 5dpc with BrdU and collected the embryos 48 hr later ( E14 . 5 ) ( Figure 3A ) .", "Co-staining with the Ki67 proliferation marker revealed that the fraction of NPCs that exited the cell cycle ( Ki67-/BrdU+/ BrdU+ cells ) in the E12 . 5-E14 . 5 time-frame was significantly increased in Khdrbs1-/- embryos ( Figure 3B ) .", "Furthermore , in wild-type embryos , ~30% of post-mitotic neurons were also labeled with BrdU ( Figure 3C ) , indicating that only a subset of NPCs underwent terminal differentiation within 48 hr .", "By contrast , this process was strongly enhanced in knockout embryos , with more than 80% of TBR1-positive cells that were labeled with BrdU ( Figure 3C ) .", "These results suggest that Sam68 is required to promote NPC self-renewal and to delay their differentiation into post-mitotic neurons in the mouse embryonic cortex . 10 . 7554/eLife . 20750 . 006Figure 3 . Differentiation of NPCs in post-mitotic neurons is accelerated in the Khdrbs1-/- mouse cortex .", "( A ) Diagram of the experimental design to investigate the fate on NPCs during neurogenesis .", "Pregnant females were treated with BrdU at E12 . 5 , and embryos were collected after 2 days for immunofluorescence analyses .", "( B and C )", "Confocal images of immunofluorescence analysis of Ki67 ( B ) and TBR1 ( C ) in Khdrbs1+/+ and Khdrbs1-/- E14 . 5 cortex .", "All sections were co-stained with anti-BrdU antibody .", "Bar graphs on the right side show the number of BrdU+/Ki67- cells ( B ) and of BrdU+/TBR1+ cells ( C ) counted in 100 μm2 .", "N = 3; ***p<0 . 001; scale bar = 100 µm .", "( D ) Representative images of embryonic cortex of Khdrbs1+/+ andKhdrbs1-/- E17 . 5 embryos treated with BrdU 2 hr before collection .", "Sections were processed for immunofluorescence analysis of BrdU and SOX2 and co-stained with Hoechst to detect nuclei .", "( E ) Bar graphs represent ( mean±SD ) measurement of cortex length from ventricular to pial surface ( left ) , number of BrdU+ cells ( middle ) and SOX2+ cells ( right ) .", "N = 3; *** p<0 . 001; ** p<0 . 01 .", "Scale bar = 100 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 20750 . 00610 . 7554/eLife . 20750 . 007Figure 3—figure supplement 1 . Ablation of Khdrbs1 does not affect overall embryo development . Weight analysis of Khdrbs1+/+ , Khdrbs1+/- and Khdrbs1-/- mouse embryos at E13 . 5 , 15 . 5 and E17 . 5 .", "At least 5 embryos for each genotype were weighted .", "Statistical analysis was performed using the ANOVA one-way test and Tuckey post-test . DOI: http://dx . doi . org/10 . 7554/eLife . 20750 . 007 To determine if premature exit from the cell cycle and differentiation of NPCs has an effect on cortical expansion , we examined embryos at E17 . 5 , when neurogenesis is almost completed ( Englund et al . , 2005 ) .", "A 2-hr pulse showed that BrdU-positive proliferating cells were strongly decreased in Khdrbs1-/- cortex also at this stage , which was reflected in lower number of SOX2-positive NPCs ( Figure 3D , E ) .", "Furthermore , although they were indistinguishable in size from the wild type and heterozygote littermates ( Figure 3—figure supplement 1 ) , cortical expansion , measured as length from ventricular to pial surface , resulted significantly reduced in E17 . 5 Khdrbs1-/- embryos ( Figure 3D , E ) .", "These observations strongly suggest that ablation of Sam68 depletes the NPC pool and limits cortical expansion during neurogenesis .", "To elucidate the function of Sam68 in NPCs , we isolated them from wild type and knockout E13 . 5 cortices ( Bertram et al . , 2012 ) .", "NPCs grown under stemness conditions form characteristic neurospheres that rapidly grow in volume .", "We observed that knockout neurospheres grew at slower rate than wild type ones ( Figure 4—figure supplement 1A ) , even though no differences were detected in the extent of cell death ( Figure 4—figure supplement 1B ) , and appeared irregular in shape with a tendency to form protrusions , due to cells that attached to the substrate and exited the sphere ( Figure 4A ) .", "Furthermore , when seeded at single cell level , knockout NPCs produced cytoplasmic extensions and attached to the plate , while wild-type cells remained in suspension ( Figure 4—figure supplement 1C ) .", "Collectively , these features suggested that Khdrbs1-/- NPCs display a tendency to lose stemness and differentiate .", "To directly test this possibility , we performed clonogenic assays at different passages from NPC isolation .", "The clonogenic potential of wild-type NPCs slowly declined from ~45% to~25% after 7 passages in culture .", "However , loss of clonogenicity was markedly accelerated in knockout NPCs , reaching ~10% by passage 7 ( Figure 4B ) . 10 . 7554/eLife . 20750 . 008Figure 4 . Sam68-/-NPCs lose stemness and are prone to differentiate in culture .", "( A ) Bright field images of Khdrbs1+/+ and Khdrbs1-/- NPCs cultured in proliferating condition for 3 days in culture .", "The insets show higher magnification of a selected neurosphere .", "Black arrowheads point to Khdrbs1-/- NPCs exiting the sphere and attaching to the surface .", "( B ) Clonogenic assay of Khdrbs1+/+ and Khdrbs1-/- NPCs .", "Clonogenicity was expressed as the percentage of neurospheres obtained from the seeded NPCs at each indicated passage .", "Data represent mean±SD of 3 independent experiments; * p<0 . 05 .", "( C and D )", "Immunofluorescence analysis of the stemness markers PAX6 ( C ) and Nestin ( D ) in Khdrbs1+/+ andKhdrbs1-/- NPCs cultured in differentiating condition for 1 day .", "Arrowheads point to high-PAX6+ cells ( C ) and high-Nestin+ cells ( D ) .", "Bar graphs represent ( mean±SD ) measurement of number of high-PAX6+ cells ( C ) and high-Nestin+ cells ( D ) .", "N = 3; * p<0 . 05; scale bars 50 µm .", "( E and F )", "Immunofluorescence analysis of expression of the neuronal marker TUBB3 ( E ) and of the oligodendrocyte marker MBP ( F ) in Khdrbs1+/+ and Khdrbs1-/- NPCs , differentiated for 3 days .", "Bar graphs represent ( mean±SD ) measurement of number of TUBB3+ cells ( E ) and MBP+ cells ( F ) .", "Analysis results were reported on the graphs on the right of the images .", "N = 3; * p<0 . 05; scale bars 50 µm .", "NPCs , Neural progenitor cells . DOI: http://dx . doi . org/10 . 7554/eLife . 20750 . 00810 . 7554/eLife . 20750 . 009Figure 4—source data 1 . List of the genes upregulated and downregulated inSam68 knockout NPCs . Yellow=upregulated genes; green=downregulated genes .", "NPCs , Neural progenitor cells . DOI: http://dx . doi . org/10 . 7554/eLife . 20750 . 00910 . 7554/eLife . 20750 . 010Figure 4—source data 2 . List of exons regulated by SAM68 in mouse NPCs . Green: genes downregulated at expression level; Yellow: genes up-regulated at expression level; White: genes unchanged at expression level .", "NPCs , Neural progenitor cells . DOI: http://dx . doi . org/10 . 7554/eLife . 20750 . 01010 . 7554/eLife . 20750 . 011Figure 4—figure supplement 1 . Khdrbs1-/- NPCs lose stemness and are prone to differentiate in culture .", "( A ) Bar graph reporting the analysis of the diameter of neurospheres formed by Khdrbs1+/+ and Khdrbs1-/- NPCs in 1–3 days of culture .", "N = 3; *p<0 . 05 .", "( B ) Cell death analysis of Khdrbs1+/+ and Khdrbs1-/- NPCs assessed during the indicated days of proliferation .", "( C ) Confocal images of immunofluorescence analysis of actin cytoskeleton stained with phalloidin in Khdrbs1+/+ and Khdrbs1-/- NPCs seeded as single cell on polyornithine/laminin-coated dishes .", "The bar graphs on the right show the average perimeter , area and number of cells with a perimeter > 150 µm , respectively .", "At least 10 cells/experiment were measured .", "N = 5; * p<0 . 05 .", "Scale bar = 10 µm .", "NPCs , Neural progenitor cells . DOI: http://dx . doi . org/10 . 7554/eLife . 20750 . 01110 . 7554/eLife . 20750 . 012Figure 4—figure supplement 2 . Khdrbs1-/- NPCs-derived neurons display more complex morphology .", "( A ) Images representing a typical neuron obtained through the differentiation of Khdrbs1-/- NPCs ( upper image ) and the representation of the scheme used to quantify the complexity of its structure for the evaluation of branching level ( lower image ) .", "( B ) Analysis of the complexity and of the differentiation stage of Khdrbs1+/+ and Khdrbs1-/- .", "Bar graphs represent the total length of neurites per cell , the average length of each branch per cell , the average number of neurites and the average branching level , as reported in ( A ) of neurons obtained after 3 days of differentiation .", "At least 10 cells/experiment were measured .", "N = 5; * p<0 . 05 . DOI: http://dx . doi . org/10 . 7554/eLife . 20750 . 01210 . 7554/eLife . 20750 . 013Figure 4—figure supplement 3 . High-throughput analyses of the transcriptomic of Khdrbs1-/-NPCs .", "( A ) Analysis of the gene and exon expression in Khdrbs1-/- vs Khdrbs1+/+ NPCs reporting upregulated and downregulated events .", "( B ) Overlap between the genes regulated at expression and splicing level in Khdrbs1-/- vs Khdrbs1+/+ NPCs .", "( C ) Pie chart representing the percentage of each splicing pattern among the regulated events identified by the exon array experiment .", "( D ) Cluster Gene Ontology ( GO ) terms enrichment analysis performed using DAVID bioinformatics resources .", "( E ) Validation of exon array experiment performed by qPCR on representative genes .", "Validation rate was 80% .", "*p<0 . 05 , **p<0 . 01 , ***p<0 . 001 .", "NPCs , Neural progenitor cells . DOI: http://dx . doi . org/10 . 7554/eLife . 20750 . 013 Next , we performed differentiation assays by switching NPCs to serum-containing medium for 1–3 days ( Bertram et al . , 2012; Compagnucci et al . , 2013 ) .", "Khdrbs1-/- NPCs differentiated with higher efficiency than wild-type cells , as indicated by the fewer cells that retained high expression of the stemness markers PAX6 and Nestin after 1 day ( Figure 4C , D ) .", "When differentiation was evaluated after 3 days , Khdrbs1-/- NPCs gave rise to significantly more neurons and oligodendrocytes than the corresponding wild-type NPCs ( Figure 4E , F ) .", "Furthermore , neurons generated by knockout NPCs exhibited higher complexity and more advanced differentiation stage , as documented by their increased branching level ( Figure 4E , Figure 4—figure supplement 2 ) .", "These findings indicate that Khdrbs1-/- NPCs have reduced self-renewal proficiency and enhanced tendency to differentiate both in vivo and in vitro .", "We hypothesized that the reduced self-renewal capacity of Khdrbs1-/- NPCs was due to dysregulation of gene expression .", "Splicing-sensitive exon arrays identified 254 annotated genes regulated at the expression levels and 117 exons in 87 genes regulated at the splicing level in knockout NPCs ( Figure 4—figure supplement 3A ) .", "Sam68 generally acted as a repressor of expression and the majority of targets were upregulated in knockout NPCs ( Figure 4—figure supplement 3A ) .", "By contrast , Sam68 functioned as splicing enhancer for target exons , with little overlap between genes regulated at expression and splicing level ( Figure 4—figure supplement 3A , B ) .", "Exon cassette was the predominant splicing event regulated by Sam68 in NPCs , followed by alternative last exon selection ( Figure 4—figure supplement 3C ) .", "Notably , gene ontology analysis highlighted significant enrichment of functional categories related to neuron and oligodendrocyte properties ( cluster 1 in Figure 4—figure supplement 3D ) , thus supporting the pro-differentiation effect of Sam68 depletion in NPCs .", "For instance , genes encoding neuronal proteins like FXYD , a transmembrane modulator of the Na , K-ATPase enzyme linked to RETT syndrome ( Deng et al . , 2007 ) , and DDC , the DOPA decarboxilase that catalyzes dopamine production ( Bertoldi , 2014 ) , were upregulated ( Supplementary file 1 ) .", "Similarly , the oligodendrocyte-specific myelin-associated glycoprotein ( MAG ) and myelin basic protein ( MBP ) were strongly induced in Khdrbs1-/- NPCs ( Figure 4—figure supplement 3E; Figure 4-source data file 1 ) .", "These results confirmed at the molecular level the pro-differentiation phenotype exhibited by Khdrbs1-/- NPCs .", "Sam68 is mainly known for its direct role in the regulation of pre-mRNA processing ( Frisone et al . , 2015 ) .", "Thus , we further investigated the functional relevance of splicing-regulated genes to identify targets that could cause the phenotype of knockout NPCs .", "Among the 87 genes identified in the array ( Figure 4—figure supplement 3B; Figure 4-source data file 2 ) , we focused on Aldh1a3 for its potential relevance in NPCs .", "Indeed , ALDH1A3 was shown to support self-renewal and clonogenic potential of cancer stem cells ( Duan et al . , 2016 ) , including glioma stem cells ( Mao et al . , 2013 ) , which can differentiate into neurons and glial cells like NPCs ( Lathia et al . , 2015 ) .", "The 5' region of the Aldh1a3 transcript ( exons 1–7 ) was upregulated in Khdrbs1-/- NPCs , whereas the 3' region ( exons 8–13 ) was downregulated ( Figure 5A , B , Figure 5—figure supplement 1A ) .", "Closer inspection of the array results highlighted the potential upregulation of a cryptic alternative last exon ( ALE ) residing in the proximal part of intron 7 , suggesting the existence of an alternative PAS in this intron and premature termination of the transcript ( Figure 5A ) .", "To verify this hypothesis , we performed 3'-RACE experiments using an oligo-dT anchor as reverse primer and forward primers located in the 5' region of intron 7 ( primer", "1 ) and at the exon-intron junction ( primer", "2 ) ( Figure 5C ) .", "Both primers amplified a distinct band from Khdrbs1-/- NPCs RNA , whereas only faint bands of various sizes were detected in wild type cells ( Figure 5D ) .", "Sequencing of the 3'-RACE products amplified in knockout NPCs revealed usage of an alternative PAS in intron 7 that is followed by two potential cleavage sites ( Figure 5E ) .", "To confirm this result , and to rule out accumulation of intron 7-containing pre-mRNA , we performed RT-PCR analyses using a forward primer in exon 6 and reverse primers located either upstream ( primers 1 and", "2 ) or downstream ( primer", "3 ) of the alternative PAS and cleavage sites ( Figure 5—figure supplement 1B ) .", "A spliced transcript containing intron 7 sequences could be amplified only with the primers situated upstream of the alternative PAS ( Figure 5—figure supplement 1B ) .", "Furthermore , incubation of Khdrbs1-/- NPCs with 5 , 6-dichloro-1-β-D-ribofuranosylbenzimidazole ( DRB ) to block RNA transcription led to rapid decay of transcripts containing exon 3-intron 3 and intron 8-exon 8 sequences , indicative of pre-mRNA amplification , whereas the amplicon corresponding to exon 7-intron 7 sequences was stable up to 6 hr of incubation ( Figure 5—figure supplement 1C ) .", "These results confirm the presence of an alternative mRNA originating from premature termination of the Aldh1a3 transcript in knockout NPCs . 10 . 7554/eLife . 20750 . 014Figure 5 . Sam68 regulates the alternative 3'-end processing of ALDH1A3 pre-mRNA .", "( A ) Diagram of ALDH1A3 gene structure .", "Red and green squares represent exons , respectively , upregulated and downregulated in the microarray analysis .", "Proximal polyadenylation site ( pPAS ) and distal ( dPAS ) polyadenylation site are also represented .", "Black arrows indicate the regions were primers for RT-PCR validation were designed .", "( B ) Conventional RT-PCR analysis for the validation of the canonical and alternative ALDH1A3 transcripts expressed in Khdrbs1+/+ and Khdrbs1-/- NPCs .", "( C ) Schematic representation of 3'-RACE PCR experimental design .", "Arrows indicate the position of the P1 and P2 forward primers used for the RACE experiment .", "( D ) 3'-RACE PCR analysis of ALDH1A3 alternative transcripts expressed in Khdrbs1+/+ and Khdrbs1-/- NPCs .", "Asterisks mark the bands that were sequenced to identify the pPAS .", "( E ) Sequence obtained through DNA sequencing of bands amplified by 3'-RACE PCR .", "Blue and green letters represent exon and intron sequences , respectively .", "Exon ( blue ) , intron ( green ) , alternative PAS ( highlighted in yellow ) and the two putative cleavage sites ( red ) are indicated .", "( F ) CLIP assay of Sam68 binding to the ALDH1A3 pre-mRNA .", "E13 . 5 mouse cortices were UV-crosslinked and immunoprecipitated with control IgGs or anti-Sam68 IgGs .", "The upper panel shows a schematic representation of ALDH1A3 gene structure and primers ( black arrows ) used in the assay .", "The bar graph shows qPCR signals amplified from the CLIP assays expressed as percentage of amplification in the input RNA . DOI: http://dx . doi . org/10 . 7554/eLife . 20750 . 01410 . 7554/eLife . 20750 . 015Figure 5—figure supplement 1 . Sam68 regulates the alternative 3'-end processing of ALDH1A3 pre-mRNA .", "( A ) Diagramatic scheme of the Aldh1a3 gene reporting the up- ( red ) and down-regulated ( green ) exons identified by the microarray experiment .", "The bar graphs represent the results of qPCR analyses reported to validate the alternative 3-end processing event suggested by the microarray experiment in Khdrbs1-/- NPCs .", "N = 3; *p<0 . 05 .", "( B ) RT-PCR to validate the alternative polyadenylation of ALDH1A3 and to rule out accumulation of the unspliced pre-mRNA .", "Reactions were performed using a forward primer in exon 6 ( E6 ) and reverse primers annealing upstream ( I7a and I7b ) or downstream ( I7c ) of the alternative PAS and RNA extracted from Khdrbs1+/+ and Khdrbs1-/- NPCs .", "( C ) RT-PCR of Khdrbs1-/- NPCs treated for 2 or 6 hr with 75 µM DRB to block transcription .", "Amplification of ~150 bp bands was performed using primers spanning the exon-intron regions indicated in the diagram . DOI: http://dx . doi . org/10 . 7554/eLife . 20750 . 015 The canonical PAS sequences ( AAUAAA and AUUAAA ) correspond to optimal binding sites for Sam68 ( Ray et al . , 2013; Feracci et al . , 2016 ) .", "We hypothesized that Sam68 can bind the cryptic PAS in intron 7 in wild-type NPCs , thus preventing its recognition and blocking premature termination of the pre-mRNA .", "In line with this possibility , UV crosslink immunoprecipitation ( CLIP ) experiments showed strong recruitment of Sam68 in proximity of the cryptic PAS in intron 7 of the Aldh1a3 pre-mRNA , whereas binding in other regions was much weaker ( Figure 5F ) .", "These observations strongly suggest that Sam68 binding is required to mask the cryptic PAS and avoid premature termination of the Aldh1a3 transcript in mouse NPCs .", "Alternative PAS usage should lead to lower expression of the full-length ALDH1A3 protein and potential expression of a truncated ALDH1A3 protein .", "Western blot analysis confirmed the reduction of the full-length ALDH1A3 protein ( Figure 6A ) .", "However , we did not detect a band corresponding to the truncated protein ( ALDH1A3△ ) originating from the ALE in intron 7 ( Figure 6—figure supplement 1A ) .", "Blocking the proteasome by incubation with MG132 did not lead to ALDH1A3△ accumulation , whereas interference with autophagy using chloroquine caused a mild accumulation of a protein product slightly smaller than the recombinant ALDH1A3△ ( Figure 6—figure supplement 1A ) .", "Thus , ALDH1A3△ appears to be either very unstable or not expressed in NPCs .", "Nevertheless , to verify if such truncated protein could be functional , we evaluated its enzymatic activity .", "Full-length ALDH1A3 overexpressed in HEK293 exhibited enzymatic activity , as measured by reduction of NADPH ( Lindahl et al . , 1982 ) , whereas the ALDH1A3△ protein was completely inactive ( Figure 6—figure supplement 1B ) .", "Since ALDH1A3 is a multimeric protein and homodimerization domains are found also in the N-terminus of the protein , which is present in ALDH1A3△ , we checked if ALDH1A3△ could contribute to ALDH activity in combination with the full-length protein .", "ALDH1A3△ efficiently co-immunoprecipitated with ALDH1A3 .", "However , its co-expression with the full-length protein reduced ALDH activity in a dose-dependent manner ( Figure 6—figure supplement 1C , D ) .", "These observations suggest that , even if expressed at low levels , ALDH1A3△ could not contribute to ALDH activity in NPCs , but it could eventually exert a dominant-negative effect . 10 . 7554/eLife . 20750 . 016Figure 6 . Sam68 promotes ALDH1A3 expression and activity .", "( A ) Western blot analysis of the expression of ALDH1A3 and Sam68 in lysates from Khdrbs1+/+ and khdrbs1-/- NPCs .", "Vinculin was used as loading control .", "The bar graph represent densitometric analyses of ALDH1A3 expression ( mean±SD of 3 independent experiments; * p<0 . 05 ) .", "( B ) Representative flow cytometric analysis of ALDH activity performed on Khdrbs1+/+ and khdrbs1-/-NPCs ( left and center panel ) by ALDEFLUOR assay .", "Gate was determined using NPCs treated with DEAB as negative control .", "SSC: side scatter value to measure cell complexity .", "Results ( mean±SD; * p<0 . 05 ) of 4 independent experiments are reported in the bar graph on the right .", "( C ) Horizontal sections of E13 . 5 Khdrbs1+/+ and khdrbs1-/- cortex immunostained with ALDH1A3 and SOX2 antibodies .", "Scale bar 100 µm .", "Graph on the right represents counts of ALDH1A3+/SOX2+-positive cells .", "N = 3; *** p<0 . 001 .", "( D ) RT-PCR analysis of the expression of full length Aldh1a3 , ALDH1A3△ , and Khdrbs1 in Khdrbs1+/+ NPCs cultured under proliferating ( 0d ) and differentiating conditions ( 1-6d ) .", "( E ) Correlation analysis between the expression levels of Khdrbs1 and full length Aldh1a3 determined by qPCR analysis of RNA extracted from the indicated brain regions .", "Pearson’s correlation test was used .", "Trend line is represented in red and the inset shows the magnification of the respective portion of the graph .", "( F ) qPCR analysis of the expression of full length Aldh1a3 ( Ex 9–10 ) and ALDH1A3△ ( Ex7– In7 ) in Khdrbs1+/+ and khdrbs1-/-cerebellum and olfactory bulbs .", "N = 3; *p<0 . 05 .", "( E–F )", "Khdrbs1 and Aldh1a3 relative expression was evaluated by △CT method using L34 expression for normalization . DOI: http://dx . doi . org/10 . 7554/eLife . 20750 . 01610 . 7554/eLife . 20750 . 017Figure 6—figure supplement 1 . Sam68 promotes ALDH1A3 expression and activity .", "( A ) Western blot analysis of ALDH1A3△ expression in Khdrbs1-/- NPCs treated with 10 µM proteasome inhibitor MG132 and 2 µM autophagy inhibitor Chloroquine .", "HEK293 cells transfected with pCI-ALDH1A3△ were used as positive control for ALDH1A3△ expression .", "P53 ( MG132 ) and LC3 ( chloroquine ) accumulation was used as control of the inhibitors activity; Vinculin was used as loading control .", "( B ) Enzymatic assay ( bar graph on the left ) and western blot analysis ( right panel ) to analyze the activity and the expression of Flag-tagged FL ALDH1A3 and ALDH1A3△ recombinant proteins transfected in Hek293 cells .", "( C ) Co-immunoprecipitation assays performed in HEK293 cell co-transfected with GFP-ALDH1A3 full length plasmid or GFP plasmid as control , Flag-ALDH1A3 full length plasmid and raising concentration ( 2 . 5 and 5 µg ) of Flag-ALDH1A3 △ plasmid .", "After transfection cell lysates were immunoprecipitated with GFP antibody and the proteins in the complexes were revealed with either anti-GFP or anti-FLAG antibodies .", "( D ) Enzymatic assay of the activity of full length ( FL ) ALDH1A3 and ALDH1A3△ recombinant proteins transfected either alone or in combination in HEK293 cells .", "( mean±SD of 3 independent experiments; * p<0 . 05; ** p<0 . 01 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 20750 . 017 In line with the reduced expression of full length ALDH1A3 , ALDH activity was markedly decreased in Khdrbs-/- NPCs ( Figure 6B ) .", "Furthermore , ALDH1A3 staining , which is restricted to the SOX2+ cells in VZ and SVZ of the developing cortex , was also significantly reduced in E13 . 5 knockout embryos with respect to wild-type littermates ( Figure 6C ) .", "These results indicate that Sam68 promotes ALDH1A3 protein expression in NPCs .", "The localization of ALDH1A3 suggests that its expression is restricted to RGCs .", "Moreover , we found that the full-length Aldh1a3 transcript rapidly declined upon differentiation of wild-type NPCs in culture ( Figure 6D ) .", "Importantly , this decline was accompanied by reduced Sam68 expression and increased expression of ALDH1A3△ ( Figure 6D ) , indicating that alternative 3'-end processing of the Aldh1a3 transcript is a physiological process of NPC differentiation controlled by fluctuations in Sam68 expression .", "In agreement with a direct modulation of Aldh1a3 expression by Sam68 , we also observed a highly significant correlation between expression of these genes in post-natal brain , with highest levels of both transcripts in olfactory bulb and cerebellum and lowest expression in the hypothalamus ( Figure 6E ) .", "Furthermore , expression of full-length Aldh1a3 transcript was reduced in olfactory bulb and cerebellum of Khdrbs1-/- mice , while the ALE-containing shorter transcript was induced ( Figure 6F ) .", "These results indicate that Sam68 is required to promote the expression and activity of ALDH1A3 through modulation of its pre-mRNA 3'-end processing in both embryonic and post-natal mouse brain .", "ALDH1A3 promotes self-renewal and clonogenic potential of glioma stem cells ( Mao et al . , 2013 ) , suggesting that its reduced expression may account for the phenotype of Khdrbs1-/- NPCs .", "To test this hypothesis , we transfected Khdrbs1-/- NPCs with constructs expressing either GFP-ALDH1A3 or GFP alone as control .", "Notably , expression of GFP-ALDH1A3 almost completely rescued the growth and clonogenic defects of knockout NPCs ( Figure 7A–C ) .", "In line with its negative effect of ALDH activity , expression of ALDH1A3△ in wild-type NPCs blocked proliferation and interfered with formation of neurospheres , even though the cells remained alive ( Figure 7—figure supplement 1 ) .", "Moreover , knockdown of endogenous ALDH1A3 in wild-type NPCs promoted neuronal differentiation to an extent similar to that observed in Khdrbs1-/- NPCs ( Figure 7D , E ) .", "Collectively , these results support the notion that Sam68 promotes NPC proliferation and represses their differentiation by maintaining high levels of full-length ALDH1A3 . 10 . 7554/eLife . 20750 . 018Figure 7 . ALDH1A3-dependent glycolytic metabolism rescues the self-renewal potential of Khdrbs1-/- NPCs .", "( A ) Representative images of Khdrbs1+/+ NPCs transfected with GFP vector and Khdrbs1-/- NPCs transfected with GFP and GFP-ALDH1A3 vectors , cultured for 3 days in proliferating condition .", "Scale bar 50 µm .", "( B ) Bar graph reporting the analysis of the diameter of neurospheres formed by transfected NPCs in 1–3 days of culture .", "N = 3; *p<0 . 05; **p<0 . 01 .", "( C ) Clonogenic assay of Khdrbs1+/+ and Khdrbs1-/- NPCs transfected as indicated .", "N = 3; *p<0 . 05 .", "( D ) RT-PCR analysis of Aldh1a3 expression in Khdrbs1+/+and Khdrbs1-/- NPCs transfected with si-CTRL or si-ALDH1A3 siRNAs .", "( E ) Differentiation assay of Khdrbs1+/+ and Khdrbs1-/- NPCs transfected with the same siRNAs used in ( D ) .", "Images are representative of the third day of culture in differentiation condition .", "Scale bar 50 µm .", "Bar graph represent ( mean±SD ) measurement of number of TUBB3+ cells .", "N = 3; * p<0 . 05 ( F ) Glycolytic activity measured by lactate accumulation in the medium of Khdrbs1+/+ and Khdrbs1-/- NPCs transfected as indicated .", "( G ) Bar graph reporting the analysis of the diameter of neurospheres formed by Khdrbs1+/+ and Khdrbs1-/- NPCs cultured in proliferating condition for 3 days in the presence or absence of the ALDH inhibitor DEAB ( 50 µM ) .", "N = 3; *p<0 . 05 .", "( H ) Clonogenic assay of Khdrbs1+/+ and Khdrbs1-/- NPCs cultured in proliferating condition in the presence or absence of 50 µM DEAB .", "N = 3; *p<0 . 05 . DOI: http://dx . doi . org/10 . 7554/eLife . 20750 . 01810 . 7554/eLife . 20750 . 019Figure 7—figure supplement 1 . ALDH1A3-dependent glycolytic metabolism rescues the self-renewal potential of Khdrbs1-/- NPCs .", "( A ) Representative images of Khdrbs1-/- NPCs transfected with GFP vector and Khdrbs1+/+ NPCs transfected with GFP and GFP-ALDH1A3△ vectors , cultured for 3 days in proliferating condition .", "Scale bar 50 µm .", "( B ) Bar graph reporting the analysis of the diameter of neurospheres formed by transfected NPCs in 1–3 days of culture .", "N = 3; *p<0 . 05; **p<0 . 01; ***p<0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 20750 . 01910 . 7554/eLife . 20750 . 020Figure 7—figure supplement 2 . ALDH1A3-dependent glycolytic metabolism rescues the self-renewal potential of Khdrbs1-/- NPCs .", "( A ) Evaluation of the relative lactate production of Khdrbs1+/+ and Khdrbs1-/- NPCs cultured under proliferating condition .", "( B ) Relative lactate production in Khdrbs1+/+ or Khdrbs1-/- NPCs treated or not with the indicated concentration of the ALDH inhibitor DEAB .", "( C ) Analysis of the relative lactate production in Khdrbs1+/+ in proliferation condition ( 0d ) and during differentiation process ( 1-6d ) .", "N = 3 , *p<0 . 05 , **p<0 . 01 , ***p<0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 20750 . 020 Stem cells often rely on anaerobic glycolysis for their proliferation and self-renewal ( Ito and Suda , 2014 ) and high glycolytic activity is required to prevent precocious NPC differentiation in vivo ( Lange et al . , 2016 ) .", "Since ALDH1A3 is involved in this metabolic pathway ( Mao et al . , 2013 ) , we investigated whether Sam68 expression affects anaerobic glycolysis in NPCs .", "Lower accumulation of the end-product lactate in the culture medium indicated that glycolytic activity was significantly reduced in Khdrbs1-/- NPCs compared to wild-type cells ( Figure 7—figure supplement 2A ) .", "A similar reduction in lactate production could be obtained by inhibiting ALDH activity using N , N-diethylaminobenzaldehyde ( DEAB ) ( Figure 7—figure supplement 2B ) , a commonly used inhibitor of ALDH enzymes ( Morgan et al . , 2015 ) .", "Furthermore , knockdown of Aldh1a3 expression in wild type NPCs was sufficient to reduce anaerobic glycolysis to the levels of knockout cells , whereas overexpression of GFP-ALDH1A3 fully rescued lactate production in Khdrbs1-/- NPCs ( Figure 7F ) .", "Notably , reduced glycolytic activity accompanied differentiation of NPCs in vitro ( Figure 7—figure supplement 2C ) , concomitant with the decline in Sam68 and Aldh1a3 expression ( Figure 6D ) .", "These findings indicate that Sam68 maintains high levels of anaerobic glycolysis through modulation of ALDH1A3 protein expression .", "Next , to determine whether inhibition of ALDH activity and glycolytic metabolism could recapitulate the phenotype of Khdrbs1-/- NPCs , we treated wild-type cells with 50 µM DEAB , a dose that lowered anaerobic glycolysis to the levels observed in knockout NPCs ( Figure 7—figure supplement 2B ) .", "Strikingly , pharmacologic inhibition of ALDH activity in wild-type NPCs was sufficient to recapitulate the neurosphere growth ( Figure 7G ) and clonogenic defect ( Figure 7H ) of Khdrbs1-/- NPCs .", "These results strongly indicate that Sam68-dependent modulation of ALDH1A3 expression is required to maintain self-renewal capacity of NPCs through enhanced glycolytic metabolism ( Figure 8 ) . 10 . 7554/eLife . 20750 . 021Figure 8 . Sam68 modulates neurogenesis through regulating ALDH1A3 expression . Schematic model of the function of Sam68 in the modulation of NPCs stemness .", "High Sam68 expression in NPCs of the neocortex insures high expression of ALDH1A3 protein and anaerobic glycolysis , thus promoting NPC self-renewal and cortical expansion .", "Upon differentiation , Sam68 expression declines , causing premature termination of Aldh1a3 transcription , reduced ALDH activity and glycolytic metabolism , thus enhancing NPC differentiation into post-mitotic neurons . DOI: http://dx . doi . org/10 . 7554/eLife . 20750 . 021" ], [ "The multifunctional RNA-binding protein Sam68 has been involved in several biological processes ( Bielli et al . , 2011; Frisone et al . , 2015 ) .", "Analysis of the mouse knockout model revealed that Sam68 is physiologically required for adipogenesis ( Richard et al . , 2005; Huot et al . , 2012 ) , male and female gametogenesis ( Paronetto et al . , 2009; Bianchi et al . , 2010; Paronetto et al . , 2011 ) and for regulation of synapse morphology and function in neurons ( Iijima et al . , 2011; Klein et al . , 2013 , 2015 ) .", "In most cases , its role was shown to require direct binding to select mRNAs and regulation of their splicing or translation .", "Notably , Khdrbs1-/- mice also displayed high perinatal lethality ( Richard et al . , 2005 ) , but the biological processes and the genes regulated by Sam68 during embryonic development are completely unknown .", "Our studies have now unveiled an unpredicted function of Sam68 as key regulator of the balance between stem and differentiated cells in the developing cortex .", "Ablation of this function in vivo caused anticipated NPC differentiation into post-mitotic neurons and limited the expansion of the neocortex .", "These findings highlight a novel Sam68-dependent pathway required for stem cell fate during brain development .", "Sam68 is strongly expressed in NPCs during stages of intense neurogenesis and cortical expansion ( E13 . 5–E15 . 5 ) .", "These processes rely on the dual ability of RGCs to divide symmetrically and amplify their number , or asymmetrically to produce IPCs and post-mitotic neurons ( Paridaen and Huttner , 2014; Taverna et al . , 2014 ) .", "Knockout of Sam68 function significantly decreased proliferating NPCs ( both PAX6-positive RGCs and TBR2-positive IPCs ) at E13 . 5 , while leading to a sharp increase in TBR1-positive post-mitotic neurons .", "Birth-dating experiments indicated that Khdrbs1-/- NPCs were prone to exit the cell cycle and differentiate into neurons .", "The consequence of this premature NPC loss was a reduced expansion of the cortex at E17 . 5 , indicating that Sam68 is strictly required for the correct development of the mouse brain .", "Of note , the same loss of NPC stemness features could be recapitulated in vitro .", "First , Sam68 was strongly expressed in self-renewing E13 . 5 NPCs and its expression declined upon their differentiation .", "Second , Khdrbs1-/- NPCs were deficient in proliferation and clonogenicity while displaying enhanced tendency to differentiate .", "These observations indicate that Sam68 is required to maintain the proper balance between NPC self-renewal and differentiation and suggest that this protein supports stem-like properties in embryonic NPCs .", "Analysis of splicing-sensitive microarrays identified a relatively small subset of genes that are modulated by Sam68 in NPCs .", "These genes were selectively enriched in functional categories typical of neurons and glial cells and the majority of them were upregulated in Khdrbs1-/- NPCs , in line with the role of Sam68 in the promotion of stem-like features .", "Among these targets , we focused on ALDH1A3 , a metabolic enzyme that was identified as marker of cancer stem cells .", "Since ALDH1A3 supports the clonogenic and tumorigenic potential of cancer stem cells ( Duan et al . , 2016; Mao et al . , 2013 ) , we hypothesized that it might also promote stemness in NPCs and that Sam68 exerted its effects through modulation of ALDH1A3 expression .", "Indeed , Khdrbs1-/- NPCs displayed lower ALDH enzymatic activity and ALDH1A3 protein expression .", "Furthermore , by performing knockdown and gain-of-function studies , we demonstrated that regulation of ALDH1A3 expression accounts for many of the phenotypes of Khdrbs1-/- NPCs .", "Thus , our results link the physiological defect of knockout embryos to regulation of a specific target that promotes NPC self-renewal .", "Although it is generally accepted that pre-mRNA processing is a key regulatory step in brain development ( Raj and Blencowe , 2015 ) , few studies so far have directly linked these processes .", "Knockout of Ptbp2 was shown to alter the dynamics of embryonic neurogenesis and splicing of several genes , but the specific target gene ( s ) of PTBP2 that are responsible for defective neurogenesis were not identified ( Licatalosi et al . , 2012 ) .", "Likewise , the Pnky long noncoding RNA is required to expand the IPC population and it functions by interacting with PTBP1 and by regulating the expression and splicing of several neuronal genes ( Ramos et al . , 2015 ) , but the specific targets involved in its neurogenic function are unknown .", "Our study extends beyond these correlations and establishes a direct link between Sam68 and ALDH1A3 functions in the maintenance of NPC self-renewal capacity during neurogenesis .", "ALE selection is a prominent and unexpected pattern of regulation by Sam68 in NPCs .", "Mechanistically , we found that Sam68 directly binds to Aldh1a3 pre-mRNA in proximity of a cryptic PAS located in the 5' portion of intron 7 .", "Ablation of Sam68 unmasked this intronic PAS , causing alternative polyadenylation and premature termination of the Aldh1a3 transcript .", "Notably , the same regulation of Aldh1a3 alternative 3'-end processing occurred also in wild-type NPCs undergoing differentiation , concomitantly with the decline in expression of endogenous Sam68 and with acquisition of neuronal features .", "Thus , our results indicate that high levels of Sam68 are required to maintain Aldh1a3 expression and activity by suppressing premature termination of transcription of its pre-mRNA ( Figure 8 ) .", "This function of Sam68 resembles that of the U1 snRNP , which prevents global recognition of a large number of cryptic intronic PASs , thus insuring proper transcription termination of pre-mRNAs in the last exon ( Kaida et al . , 2010; Berg et al . , 2012 ) .", "However , Sam68 appears to play a much more restricted role than U1 snRNP , by controlling 3'-end processing of only a limited subset of transcripts .", "It will be interesting to investigate whether or not Sam68 functionally interacts with U1snRNP in this regulation and why its expression affects the alternative 3'-end processing of only few transcripts .", "Nevertheless , the studies shown here unveil a previously unappreciated role of Sam68 in alternative polyadenylation with physiological relevance for NPC differentiation .", "ALDH1A3 was shown to support clonogenicity and tumorigenicity of glioma stem cells by enhancing the glycolytic pathway ( Mao et al . , 2013 ) .", "Notably , very recent studies demonstrated that anaerobic glycolysis is required for NPC self-renewal in vivo ( Lange et al . , 2016 ) .", "We observed that lactate production through the glycolytic pathway was strongly reduced in Khdrbs1-/- NPCs .", "This phenotype could be recapitulated by Aldh1a3 knockdown or ALDH pharmacological inhibition in wild-type NPCs , or by promoting NPC differentiation in culture .", "Furthermore , the clonogenic potential of both wild-type and knockout NPCs was tightly linked to the extent of glycolysis , as modulation of this metabolic pathway by up- or down-regulating ALDH1A3 activity paralleled their self-renewal potential .", "By linking ALDH1A3 activity to anaerobic glycolysis in NPCs , we identify a novel metabolic route required for maintenance of stemness in neural precursors that is strictly under the control of Sam68 .", "Our work suggests that during neurogenesis , high levels of Sam68 expression are required for the correct processing of Aldh1a3 pre-mRNA , thus insuring high ALDH1A3 activity and fueling of the glycolytic pathway .", "Conversely , a decline in Sam68 expression leads to reduced expression and activity of ALDH1A3 and inhibits the glycolytic pathway , priming NPCs to enter the differentiation program ( Figure 8 ) .", "Importantly , the impaired balance between self-renewal and differentiation in Khdrbs1-/- mice results in depletion of the NPC pool and reduced expansion of the neocortex .", "Since altered neurogenesis and cortical development are associated to severe neuronal disorders like epilepsy , schizophrenia and autism ( Sun and Hevner , 2014; Fernández et al . , 2016 ) , our findings suggest that fine-tuned regulation of Sam68 expression represents a safeguard mechanism during brain development .", "In conclusion , our study identifies Sam68 as a novel regulator of neurogenesis and highlights an unprecedented link between Sam68 , ALDH1A3 and the glycolytic pathway that supports maintenance of the NPC pool in the embryonic cortex ." ], [ "Animal experiments were performed according to protocol number 809_2015PR , following the Institutional guidelines of the Fondazione Santa Lucia and the approval of the Ethical Committee .", "E12 . 5 , E13 . 5 and E17 . 5 pregnant mice were treated with 90 mg/kg BrdU ( Sigma-Aldrich , St . Louis , MO ) .", "After 2 days ( E12 . 5 mice ) or 2 hr ( E13 . 5 and E17 . 5 mice ) , mice were sacrified and whole embryo ( E12 . 5 and E13 . 5 ) or embryonic brains ( E17 . 5 ) were collected and fixed in formaldehyde 4%/PBS ( v/v ) for 8 hr , transferred for 24 hr to a Sucrose/PBS 30% ( w/v ) solution and then frozen to −80°C .", "Brains were embedded in Tissue-Tek OCT before collecting the cryosections ( 40–45 µm thick ) in PBS 0 . 01% sodium azide ( w/v ) .", "Sections were treated with 0 . 1 N HCl for 20 min at 37° C and then in 1 M sodium borate for 20 min at room temperature ( RT ) .", "Floating sections were incubated overnight at 4°C with the following antibodies: goat anti-SOX2 ( 1:300; Santa Cruz , Santa Cruz , CA , RRID:AB_2286684 ) , rabbit anti-TBR1 ( 1:300; Abcam , Cambridge , MA , RRID:AB_2200219 ) or anti-TBR2 ( 1: 300; Abcam , RRID:AB_778267 ) , rat anti-BrdU ( 1:400; AbDSerotech , Hercules , CA , RRID:AB_323427 ) , rabbit anti-PAX6 ( 1:100; Covance , Princeton , NJ , RRID:AB_2313780 ) , rabbit anti-Ki67 ( 1:200; LabVision , Waltham , MA , RRID:AB_2335745 ) , rabbit anti-Sam68 ( 1:1000; Santa Cruz , RRID:AB_631869 ) and rabbit anti-ALDH1A3 ( 1:100; Sigma-Aldrich , RRID:AB_10607146 ) .", "Secondary antibodies ( Jackson ImmunoResearch , West Grove , PA ) were used as follows: Cy3-conjugated donkey anti-rat , 1:300; Cy2-conjugated donkey anti-rabbit , 1:300; Alexa647-conjugated donkey anti-goat , 1:300 .", "Images were collected by laser-scanning confocal microscopy using a TCS SP5 microscope ( LEICA microsystem , Switzerland ) and elaborated with Photoshop ( Adobe , San Jose , CA ) .", "NPCs were isolated from Khdrbs1+/+ and Khdrbs1-/- C57/BL6 ( Charles River Laboratories , RRID:MGI:3696370 ) E13 . 5 ( Bard et al . , 1998 ) mouse embryos as previously reported ( Compagnucci et al . , 2013 ) .", "Briefly , after olfactory bulbs , ganglionic eminences and meninges removal , embryonic cortices were enzymatically digested with Papain 30 U/ml ( Sigma-Aldrich ) , 0 . 24 mg/ml L-Cysteine ( Sigma-Aldrich ) , 40 mg/ml DNaseI ( Sigma-Aldrich ) dissolved in MEM ( Sigma-Aldrich ) for 10 min at 37°C to obtain cell suspensions .", "After centrifugation , cells were seeded in neurosphere medium consisting of DMEM:F12 ( 1:1 ) ( Sigma-Aldrich ) containing 0 . 2 mg/ml L-glutamine ( Sigma-Aldrich ) , B27 ( 1 ml/50 ml , Gibco ) , penicillin ( 100 U/ml ) , streptomycin ( 100 mg/ml ) ( all from Lonza , Switzerland ) , and supplemented with 20 ng/ml epidermal growth factor ( EGF ) ( Peprotech , United Kingdom ) and 20 ng/ml basic fibroblast growth factor ( bFGF ) ( Peprotech ) , and passaged every 4–5 days .", "For clonal analysis , 5000 NPCs were plated in 35-mm wells for each experimental point .", "After 5 days of culture in proliferating conditions , neurosphere number was evaluated and NPC clonogenity was calculated as ratio between plated cells and neurospheres formed , expressed as percentage .", "For differentiation assays , 20 , 000 NPCs/well were plated in 4-well dishes , pre-coated with poly-ornithine ( Sigma-Aldrich ) in H2O and with laminin-1 ( Sigma-Aldrich ) in PBS for 1 hr each at 37°C , in neurosphere medium containing 1% v/v fetal bovine serum ( FBS ) ( Gibco , United Kingdom ) and incubated in a humidified atmosphere with 6% CO2 at 37uC for 1 to 6 days .", "For immunofluorescence analysis , NPCs cultured in differentiating condition were fixed with 4% ( v/v ) formaldehyde ( Sigma-Aldrich ) and permeabilized with 0 . 1% Triton X-100 in PBS , supplemented with 1% BSA .", "Primary antibodies ( 1 hr at RT ) : mouse anti-Nestin ( 1:1000; Millipore , Billerica , MA , RRID:AB_94911 ) , rabbit anti-PAX6 ( 1:500; Covance , RRID:AB_2313780 ) , rabbit anti-GFP ( 1:300; Abcam , RRID:AB_303395 ) , mouse anti-GFP ( 1:300; Santa Cruz , RRID:AB_627695 ) , mouse anti-TUBB3 ( 1:300; Sigma-Aldrich , RRID:AB_1841228 ) , chicken anti-MBP ( 1:50; Millipore , RRID:AB_2140366 ) .", "Specimens were then incubated with Cy3- ( 1:500 ) and FITC-conjugated ( 1:250 ) secondary antibodies ( Jackson ImmunoResearch ) for 1 hr at RT and with Hoechst ( Invitrogen , United Kingdom ) for 15 min and preserved using Prolong Gold mounting solution ( Invitrogen ) .", "10 randomly taken fields for each sample were taken using a DMI6000B inverted microscope ( LEICA Geosystems ) equipped with a Pan-Neofluar 20× /0 . 75 objective lens .", "Data are represented as percentage of positive cells/total cells ( evaluated by the number of total nuclei ) .", "For transfection , NPCs were electroporated using AMAXA nucleoflector device II and AMAXA mouse NSC nucleoflector kit ( Lonza ) , following manufacturer instructions .", "Briefly , 5×106 NSCs were centrifuged and resuspended in 100 µl of P3 primary solution with 5 µg of GFP or ALDH1A3-GFP vectors or 300 nM of si-ctrl or a mix of 3 siALDH1A3 siRNAs .", "Electroporated NPCs were resuspended in 500 µl of pre-warmed medium , centrifuged at 1300 rpm for 5 min at RT and resuspended in NPC proliferation medium .", "siRNAs sequence is reported in Supplementary file 1 .", "Total RNA was extracted from NPCs or brain tissues using Trizol reagent ( Invitrogen ) following manufacturer instructions .", "1 µg of RNA was retrotranscribed using M-MLV reverse transcriptase ( Invitrogen ) and used in RT-PCR experiments using GoTaq Flexi DNA Polymerase ( Promega Corporation , Madison , WI ) or in quantitative RT-PCR ( qPCR ) experiments , using SIBR green PCR master mix ( Applied Biosystem ) following manufacturers instructions .", "All primers used in RT-PCR and qPCR experiments are reported in Supplementary file 1 .", "L34 gene expression was for normalization in qPCR experiments .", "For 3' RACE PCR , 2 µg of total RNA isolated from Sam68+/+ and Sam68-/- NPCs was used for retrotranscription with 0 . 5 µg of oligo dT18XbaI-KpnI-BamHI primer , comprising an oligo-dT tail followed by an anchor sequence ( see Supplementary file 1 ) .", "PolyA enriched/anchor tagged cDNA was subsequently used in RT-PCR experiments using gene-specific forward primers in presence of anchor reverse primer ( see Supplementary file 1 ) .", "PCR products were resolved in agarose gel and bands of interest were purified and sequenced .", "NPCs or brain tissues were lysed in 50 mM Tris–HCl , pH 7 . 4; 100 mM NaCl; 1 mM MgCl2; 0 . 1m MCaCl2; 1% NP-40; 0 . 5% sodium deoxycholate; 0 . 1% SDS , protease inhibitor cocktail , plus phosphatase and protease inhibitors ( Sigma-Aldrich ) .", "After protein separation by SDS–PAGE and transfer to polyvinyl difluoride ( PVDF ) membranes ( Amersham , United Kingdom ) , the following antibodies were incubated in 5% BSA in TBS-0 . 1% Tween buffer: rabbit anti-Sam68 ( 1:1000 , Santa Cruz , RRID:AB_631869 ) , rabbit anti-SOX2 ( 1:1000 , Millipore , RRID:AB_2286686 ) , rabbit anti-ALDH1A3 ( 1:1000 , Abgent , San Diego , CA , RRID:AB_2224040 ) , mouse anti-GAPDH ( 1:1000 , Santa Cruz , RRID:AB_627679 ) , mouse anti-Vinculin ( 1:1000 , Sigma-Aldrich , RRID:AB_10603627 ) .", "Signals were detected by enhanced chemiluminescence ( ECL ) ( Biorad ) .", "Total RNA was extracted and retrotranscribed as reported above and cDNA was used to clone ALDH1A3 FL and △ isoforms in pEGFP-C3 , p3xFlag-CMV or pCI vectors .", "Inserts were amplified using primers containing HindIII ( forward ) or PstI ( reverse ) recognition sites in pEGFP-C3 , p3xFlag-CMV vectors cloning and EcorI ( forward ) or NotI ( reverse ) recognition sites in pCI vector cloning and High fidelity Phusion DNA Polymerase ( Thermo Scientific , Waltham , MA ) , following manufacturer instructions .", "All products were verified by sequencing .", "Sequence of primers used is reported in Supplementary file 1 .", "ALDH activity using the ALDEFLUOR assay kit ( Stem cells , Canada ) .", "1×106 NPCs were resuspended in Aldefluor assay buffer containing BAAA ( 1 µmol/l ) for 60 min and maintained on ice .", "For each sample , the same number of cells was treated with 50 mmol/l of diethylaminobenzaldehyde ( DEAB ) ALDH inhibitor , to set the negative control gate .", "Flow cytometric analyses were conducted by exciting samples at 488 nm and detecting emission light using a standard fluorescein isothiocyanate ( FITC ) channel .", "To evaluate glycolytic metabolism , a glycolysis cell-based assay kit ( Sigma-Aldrich ) was used .", "1×104 NPCs were seeded in a 96-well plate with 200 µL of neurosphere medium and cultured overnight .", "25 μl of supernatant from cultured cell plates and 25 μl of assay buffer were mixed together and then 50 μL of reaction solution were added .", "6 samples were used to set up the standard curve by reading absorbance of each well at 490 nm was read and L-lactate concentrations of each sample were calculated using the corrected absorbance of each sample and interpolating it on the standard curve .", "CLIP assays were performed as previously described ( Wang et al . , 2009 ) , with some modification .", "E13 . 5 cortices were dissected , freed from meninges and dissociated mechanically .", "After irradiation with UV light on ice ( 400 mJ/cm2 ) , samples were incubated with lysis buffer ( 50 mM Tris–HCl , pH 7 . 4; 100 mM NaCl; 1 mM MgCl2; 0 . 1mMCaCl2; 1% NP-40; 0 . 5% sodium deoxycholate; 0 . 1% SDS , protease inhibitor cocktail , RNase inhibitor ) , briefly sonicated and treated with DNase-RNase free for 3 min at 37°C .", "After centrifugation at 15 , 000xg for 3 min at 4°C , 500 µg of extract was treated with Proteinase K ( PK ) for 30 min at 37°C and RNA was purified by standard procedure ( input ) or diluted to 1 ml with lysis buffer for immunoprecipitation with anti-Sam68 ( Santa Cruz , RRID:AB_631869 ) or control rabbit IgGs ( Sigma-Aldrich ) as negative control , in presence of protein-G magnetic dynabeads ( Life Technologies , United Kingdom ) and 10 μl RNase I ( 1:1000 , Ambion , Waltham , MA ) , for 2 hr at 4°C under rotation .", "After stringent washes in high salt , dynabeads were resuspended in PK buffer .", "10% of each sample was kept as control of immunoprecipitation and the rest was treated with 50 μg PK for 1 hr at 55°C .", "RNA was then isolated and used for qPCR analysis .", "Total RNA was isolated from independent NPCs obtained from 3 wild type and 3 knockout E13 . 5 mouse embryos .", "RNA was hybridized to GeneChip Mouse Exon 1 . 0 ST Arrays ( Affymetrix , Santa Clara , CA ) at Genosplice , Paris .", "Bioinformatics analyses for gene expression and splicing index was performed at Genosplice using the EASANA software and FAST-DB as reference database .", "All data are expressed as mean ± SD .", "Student’s t-test and ANOVA were performed using Graphpad Prism software ( RRID:SCR_002798 ) ." ] ]

[ "The balance between self-renewal and differentiation of neural progenitor cells ( NPCs ) dictates neurogenesis and proper brain development .", "We found that the RNA- binding protein Sam68 ( Khdrbs1 ) is strongly expressed in neurogenic areas of the neocortex and supports the self-renewing potential of mouse NPCs .", "Knockout of Khdrbs1 constricted the pool of proliferating NPCs by accelerating their cell cycle exit and differentiation into post-mitotic neurons .", "Sam68 function was linked to regulation of Aldh1a3 pre-mRNA 3'-end processing .", "Binding of Sam68 to an intronic polyadenylation site prevents its recognition and premature transcript termination , favoring expression of a functional enzyme .", "The lower ALDH1A3 expression and activity in Khdrbs1-/- NPCs results in reduced glycolysis and clonogenicity , thus depleting the embryonic NPC pool and limiting cortical expansion .", "Our study identifies Sam68 as a key regulator of NPC self-renewal and establishes a novel link between modulation of ALDH1A3 expression and maintenance of high glycolytic metabolism in the developing cortex ." ]

[ "Neurons develop from cells called neural progenitors .", "These cells can either divide to produce more progenitor cells or develop into specific types of neurons .", "These two activities – known as self-renewal and differentiation – must be balanced to produce the right number of specialized neurons , without depleting the pool of progenitor cells .", "The self-renewal and differentiation of progenitor cells is balanced by essentially regulating which genes are active , or expressed , within the cells .", "In the first step of gene expression , the genetic instructions are copied to form a molecule of pre-messenger RNA ( or pre-mRNA for short ) .", "Each pre-mRNA molecule is then processed to produce a final product that can be translated into protein .", "Importantly , two copies of the same pre-mRNA may sometimes be processed in different ways , which allows multiple proteins to be produced from a single gene .", "RNA-binding proteins control pre-mRNA processing .", "The expression of one such protein , called Sam68 , oscillates during the development of the nervous system , such that its expression peaks when there is intense production of new neurons and then declines .", "However , it was not known whether Sam68 actually helps neurons to develop .", "La Rosa et al . have now analysed the role of Sam68 in the developing brain of mice .", "The experiments confirmed that Sam68 is highly expressed in neural progenitor cells and showed that its levels dictate the cell’s fate: high expression encourages a cell to self-renew , while low expression triggers it to develop into a specialized neuron .", "Further investigation revealed that Sam68 works by promoting the expression of a metabolic enzyme called Aldehyde Dehydrogenase 1A3 or ALDH1A3 .", "This enzyme promotes the release of energy from molecules of glucose via a process known as anaerobic glycolysis .", "La Rosa et al . found that cells that lack Sam68 make a truncated version of the pre-mRNA encoding ALDH1A3 .", "This truncated pre-mRNA encodes a shortened version of the enzyme that is inactive .", "Further experiments confirmed that Sam68 normally prevents this from happening by binding to the pre-mRNA and processing it to produce the full-length , working version of the ALDH1A3 enzyme .", "Also , La Rosa et al . found that progenitor cells need working ALDH1A3 to keep them dividing , and to stop them from developing into specialized neurons too soon .", "Finally , because the processing of pre-RNA plays a major role in brain development , problems with this process often lead to intellectual disabilities and neurodegenerative diseases , such as autism spectrum disorder and amyotrophic lateral sclerosis .", "The next step following on from these new findings will be to investigate whether defects in Sam68 contribute to such conditions and , if so , to look for ways to counteract these defects ." ]

[ "Introduction", "Results", "Discussion", "Materials and method" ]

[ "short report", "neuroscience" ]

[ [ "We investigated whether the presence of an inaccessible goal in an unvisited portion of an environment was sufficient to elicit pre-activation ( ‘preplay’ ) of hippocampal place cell sequences that will subsequently represent runs through the unvisited environment .", "To this end , we recorded from ensembles of place cells ( O'Keefe and Dostrovsky , 1971 ) ( 4 rats , 37–66 place cells each , 212 cells in total ) while rats ran along a T-shaped track ( Figure 1—figure supplement 1 , Table 1 ) with visible yet inaccessible arms ( Figure 1A ) —RUN1 .", "One arm ( counter balanced between animals ) was subsequently cued with food while the animal remained on the track—GOAL-CUE .", "During a rest period before RUN1 ( REST1 ) and after GOAL-CUE ( REST2 ) , spiking events—periods of 300 ms or less , where at least 15% of cells were active ( Foster and Wilson , 2006; Diba and Buzsaki , 2007 ) —were analysed .", "These spiking events were associated with significantly higher power in the ripple spectrum ( 80–250 Hz ) than other comparable periods ( Figure 1—figure supplement 2 ) .", "To investigate whether paths on the cued and uncued arms were preplayed we assessed the match between the order in which cells fired during spiking events and during future runs on the arms ( RUN2 , Figure 1—figure supplement 3 ) .", "Specifically , we computed the rank-order correlations between spiking events and sequences of place cells active on the arms , referred to as templates ( Lee and Wilson , 2002; Foster and Wilson , 2006; Diba and Buzsaki , 2007; Dragoi and Tonegawa , 2011 ) ( Figure 1—figure supplement 4 ) .", "Preplay events were identified as those with either a significant positive or negative correlation—a two-tailed test , each tail tested at the 97 . 5% level .", "These preplay events were found to exhibit higher power in the ripple spectrum than non-significant spiking events ( Figure 1—figure supplement 2 ) .", "To establish significance at the population level , the proportion of preplay events measured was compared to a null distribution generated by calculating correlations between place cell templates and shuffled sequences from events ( see Figure 1B–C ) . 10 . 7554/eLife . 06063 . 003Table 1 . Experimental parametersDOI: http://dx . doi . org/10 . 7554/eLife . 06063 . 003R1838R505R584R504All rats ( mean ) Cue bias ( dwell time ) RUN10 . 330 . 32−0 . 05−0 . 200 . 10 GOAL-CUE0 . 460 . 200 . 310 . 320 . 33Cue bias ( looking time ) RUN1−0 . 09−0 . 020 . 01−0 . 36−0 . 11 GOAL-CUE0 . 060 . 310 . 090 . 050 . 13RUN2 arm bias RUN21 . 00 . 841 . 01 . 00 . 96Session duration ( min ) SLEEP16088757474 RUN1131013911 GOAL-CUE1017121113 SLEEP26067716065 RUN23419353130Template length ( number of cells ) Up cued arm2036534338 Down cued arm1535453332 Up uncued arm1926454033 Down uncued arm153241433310 . 7554/eLife . 06063 . 004Figure 1 . Preferential preplay of a behaviourally relevant , unvisited environment .", "( A ) Experimental protocol .", "( i ) Prior to running on the track , the animals rested for at least an hour ( REST1 ) .", "( ii ) Following REST1 , animals ran 20 laps on the stem ( RUN1 ) .", "Access to the arms was blocked by a barrier at the end of the stem which the animals could see through but not pass .", "( iii ) Following RUN1 , the experimenter baited one arm so to provoke the animals' interest in that arm ( GOAL-CUE ) .", "( iv ) Following goal-cueing , the animal rested for at least another hour ( REST2 ) .", "( v ) Following REST2 , the barrier was removed and the animals traversed the extent of the track , in alternate L-shaped laps ( RUN2 ) .", "( B ) Left: an example template for a run to the cued arm .", "x-axis shows location on the track and y-axis cell IDs .", "Right: Example raster plots of preplay events—the title shows the correlation between the preplay event and the template sequence .", "C same as B but for the uncued template . DOI: http://dx . doi . org/10 . 7554/eLife . 06063 . 00410 . 7554/eLife . 06063 . 005Figure 1—figure supplement 1 . Schematic of experimental apparatus black rectangles on track represent texture cues , dotted line transparent barrier , circle rest enclosure ( 18 cm wide ) , wiggly border demarcates the white curtains surrounding the environment , with distal landmarks fixed to the curtain ( ‘X’ and ‘0’ ) .", "Note , the rest enclosure was only present during rest periods . DOI: http://dx . doi . org/10 . 7554/eLife . 06063 . 00510 . 7554/eLife . 06063 . 006Figure 1—figure supplement 2 . Ripple power is elevated during preplay events power in the ripple spectrum ( 80–250 Hz ) is higher during preplay events ( red ) than during both non-significant spiking events ( black , p < 0 . 001 ) and non-event periods ( dashed line , p < 0 . 001 ) .", "Vertical lines represent start and end of spiking events . DOI: http://dx . doi . org/10 . 7554/eLife . 06063 . 00610 . 7554/eLife . 06063 . 007Figure 1—figure supplement 3 . Place cell templates place cell sequences for each template . Cells are ordered according to the location of their place field .", "x-axis shows location on arm ( cm ) —left side closer to the stem , and y-axis cell IDs .", "UCA = Up Cued Arm , DCA = Down Cued Arm , UUA = Up Uncued Arm , DUA = Down Uncued Arm . DOI: http://dx . doi . org/10 . 7554/eLife . 06063 . 00710 . 7554/eLife . 06063 . 008Figure 1—figure supplement 4 . Spiking events for cued arm in REST2 centre: bootstrapped cumulative distribution of ( absolute ) correlations between spiking events and the cued template in REST2 ( red = data , black = shuffle ) .", "Lighter areas of the curve show 1 standard deviation of the mean .", "Raster plots: spiking events of varying correlations with the future cued arm template .", "The title of each quadrant shows the correlation range for raster plots in that quadrant .", "Labels above the top row in each quadrant show which animal the spiking events belong to .", "Red rasters = first spike emitted from a cell in an event , grey rasters = consecutive spikes emitted from a cell . DOI: http://dx . doi . org/10 . 7554/eLife . 06063 . 008" ], [ "During GOAL-CUE , all four animals displayed more interest in the cued arm than the uncued arm ( as indexed by the difference in time spent on the cued side of the stem vs the uncued side , divided by the total time spent on either side , mean bias = 0 . 33 ) .", "In contrast , prior to goal-cueing , two animals spent more time on the uncued side of the stem ( mean bias = 0 . 10 , see Table 1 for results for individual animals ) .", "Moreover , during GOAL-CUE all animals also spent more time looking towards the cued arm than the uncued arm ( mean bias = 0 . 13 ) , again this bias was not observed prior to goal-cueing when only one animal spent more time looking towards the cued arm ( mean bias = −0 . 11 , see Table 1 for results of individual animals ) .", "Furthermore , during RUN2 when the barrier was first removed and the food cue was no longer present , all four animals initially turned towards the cued arm and spent more time on the cued rather than the uncued arm ( mean bias = 0 . 96 , see Table 1 for results for individual animals ) .", "Consistent with the behavioural bias , in REST2 we found significant preplay of the yet unvisited cued arm ( 7 . 37% preplay events , p < 0 . 001 , binomial test vs chance , Figure 2A , D ) .", "Conversely , the uncued arm was not significantly preplayed ( 4 . 41% preplay events p = 0 . 33 , vs cued arm: p < 0 . 001 , Figure 2B , D ) .", "Similarly significant effects were found when animals were analysed individually ( see Figure 2D , Table 2 ) , although the results for one animal were based on a relatively small sample ( number of cued preplay events = 9 , R1838 ) , it still showed significant preplay of the cued arm .", "Moreover , the results were corroborated by a distribution-based analysis; namely , comparing the area under the curve ( AUC ) of bootstrapped cumulative distributions of absolute correlations for each arm to that of their shuffle distribution ( Figure 2A , B , cued: p < 0 . 001 , uncued: p = 0 . 22 , cued vs uncued: p = 0 . 0044 , Figure 2—figure supplement 1 ) .", "Preplay events of the cued arm were equally likely to represent paths to and from the cued arm ( 7 . 34% vs 7 . 40% p = 0 . 49 ) and to run towards ( ‘forward’ ) and away ( ‘reverse’ ) from the ends of the arms ( 6 . 93% vs 7 . 80% , p = 0 . 18 , Figure 3A ) .", "Moreover , the amount of preplay exhibited by each animal appeared to be predicted by the interest they displayed for the cued arm during GOAL-CUE ( Figure 3—figure supplement 3 ) .", "Importantly , preferential preplay of the cued arm could not be explained by differences in the number of cells with fields on the arms ( Figure 3—figure supplement 2A–B ) , spike-sorting quality ( cells with neighbouring place fields were as well separated in cluster-space as those with distant fields , p = 0 . 45 , 2-sample Kolmogorov–Smirnov test ) , place field stability on the two arms ( cued arm stability r = 0 . 54 vs uncued arm stability r = 0 . 49 , p = 0 . 15 ) or the location of place fields on the cued arm ( Figure 3B , p = 0 . 22 two-sample Kolmogorov–Smirnov test ) .", "In sum , we found during rest after goal-cueing , significant and preferential preplay of an unvisited and motivationally relevant portion of the environment . 10 . 7554/eLife . 06063 . 009Figure 2 . Preplay is a function of goal-cueing .", "( A ) Bootstrapped cumulative distribution of ( absolute ) correlations between spiking events and the cued template in REST2 ( red = data , black = shuffle ) .", "Lighter areas of the curve show 1 standard deviation of the mean .", "Inset: difference between the data and shuffle distributions .", "If there are more high correlations in the data compared to the shuffle then the data distribution will deviate below the shuffle distribution .", "( B–C ) same as A but for the uncued template in REST2 and the cued template in REST1 , respectively .", "( D ) Proportion of spiking events categorised as preplay events in REST2 for the cued and uncued arms .", "Bars show mean for all animals , and the black lines show the result for each animal .", "The grey dashed line shows the proportion of preplay events expected by chance .", "( E ) Same as D but comparing proportion of preplay events for the cued template in REST1 and REST2 . DOI: http://dx . doi . org/10 . 7554/eLife . 06063 . 00910 . 7554/eLife . 06063 . 010Figure 2—figure supplement 1 . Preplay of cued arm in REST2—distribution-based analysis A mean difference between the bootstrapped cumulative distributions of absolute correlations for the data and the bootstrapped shuffle for the cued and uncued arms in REST2 and the cued arm in REST1 . If a place cell template is preplayed then the AUC of the data distribution should be smaller than that of the shuffled distribution ( i . e . , negative difference score ) .", "To assess significance we computed 95% confidence intervals for the difference scores ( obtained by subtracting the AUC of the bootstrapped data from the AUC of the bootstrapped shuffle ) .", "If the confidence interval did not contain 0 we deemed the data distribution to be significantly different from the shuffle distribution .", "The distribution for the cued arm in REST2 was found to be significantly different from the shuffle ( p < 0 . 001 ) , yet that of the uncued arm in REST2 and the cued arm in REST1 were not ( uncued REST2: p = 0 . 22 , cued REST1: p = 0 . 14 ) .", "Black lines on bars show one standard deviation , *** = p < 0 . 001 .", "( B ) Frequency distribution of absolute correlation coefficients between spiking events and future cued arm templates .", "Red bars represent data and the grey bars the shuffle distribution .", "p-value above histogram is based on a two-sample Kolmogorov–Smirnov test comparing the data distribution to the shuffle distribution . DOI: http://dx . doi . org/10 . 7554/eLife . 06063 . 01010 . 7554/eLife . 06063 . 011Figure 2—figure supplement 2 . Preplay of stem during REST1 bootstrapped cumulative distribution of absolute correlations for spiking events of the stem , recorded during REST1 . Green line shows the data and the black line the bootstrapped distribution obtained after shuffling events 100 times .", "Light green regions show 1 standard deviation of the distribution .", "Inset: difference between the two distributions .", "If the stem was preplayed during REST1 then one would expect the data distribution to deviate below the shuffle distribution ( p = 0 . 053 , area under the curve ( AUC ) test ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 06063 . 01110 . 7554/eLife . 06063 . 012Table 2 . REST period resultsDOI: http://dx . doi . org/10 . 7554/eLife . 06063 . 012AnimalArm# spiking events# preplay events% preplay% chancep-valueREST2 R1838Cued44920 . 456 . 866 . 56 × 10−4Uncued26311 . 546 . 770 . 10 R505Cued631386 . 024 . 570 . 037Uncued860293 . 373 . 820 . 72 R584Cued437327 . 324 . 736 . 20 × 10−3Uncued398225 . 534 . 740 . 19 R504Cued516417 . 955 . 150 . 0027Uncued373195 . 094 . 400 . 21 All ratsCued16281207 . 374 . 864 . 12 × 10−6Uncued1657734 . 414 . 220 . 33REST1 R1838Cued6334 . 767 . 050 . 66Uncued3525 . 717 . 370 . 48 R505Cued664395 . 874 . 340 . 025Uncued1215383 . 133 . 450 . 70 R584Cued269145 . 204 . 670 . 28Uncued247218 . 504 . 720 . 0035 R504Cued31161 . 934 . 440 . 99Uncued173116 . 364 . 210 . 063 All ratsCued1307624 . 744 . 560 . 34Uncued1670724 . 313 . 800 . 12Summary results from REST1 and REST2 for the cued and uncued arms for individual animals .", "# Spiking events = total number of spiking events recorded .", "# preplay events = number of significant spiking events .", "% preplay = Proportion of the spiking events that qualified as preplay events ( i . e . , that were significant ) , expressed as a percentage .", "% chance = proportion of spiking events from the shuffled data that qualified as preplay events , expressed as a percentage .", "p-value = probability , derived from a binomial test , of obtaining the observed number of preplay events for each template given the chance level calculated from the shuffled data . 10 . 7554/eLife . 06063 . 013Figure 3 . Spatial and temporal dynamics of preplay .", "( A ) The proportion of preplay events when negative ( ‘reverse’ ) and positive ( ‘forward’ ) spiking event correlations are analysed separately .", "Bars show means for all data and black lines the results for each animal .", "( B ) Frequency of preplay events vs location on the cued ( red ) and uncued ( blue ) arms normalised by the density of place field centres—100% indicates the expected number of preplay events under an even distribution across each arm .", "No bias towards particular sections of the arms was evident ( cued p = 0 . 22 , uncued p = 0 . 15 , based on a two-sample Kolmogorov–Smirnov test ) .", "Lighter areas show standard error of the mean ( SEM ) and the black line the expected distribution .", "( C ) Bootstrapped cumulative distribution of ( absolute ) correlations between spiking events and the cued template during GOAL-CUE ( red = data , black = shuffle ) .", "Lighter areas of the curve show 1 standard deviation of the mean .", "Inset: difference between the data and shuffle distributions .", "( D ) Ratio of activity levels between cued and uncued arm cells ( cued/uncued ) during events for the first and second half of each experimental period .", "Red line shows mean ratio , derived from bootstrapped data , obtained for each period , and the shaded areas 1sd of the bootstrapped data .", "The black horizontal line indicates equal rates for the two arms .", "* = significantly different from 1 based on 95% confidence intervals . DOI: http://dx . doi . org/10 . 7554/eLife . 06063 . 01310 . 7554/eLife . 06063 . 014Figure 3—figure supplement 1 . Preplay of cued arm during GOAL-CUE—distribution-based analysis A mean difference between the cumulative distributions of absolute correlations for the bootstrapped data and shuffle for the cued and uncued arms during GOAL-CUE . If a place cell template is preplayed then AUC of the data distribution should be smaller than that of the shuffled distribution ( i . e . , negative difference score ) .", "To assess significance we computed 95% confidence intervals for the difference scores .", "If the confidence interval did not contain 0 we deemed the data distribution to be significantly different from the shuffle distribution .", "The distribution for the cued arm was found to be significantly different from the shuffle ( p = 0 . 02 ) , yet that of the uncued arm was not ( uncued p = 0 . 24 ) .", "Black lines on bars show one standard deviation , * < 0 . 05 .", "( B ) Frequency distribution of absolute correlation coefficients between spiking events and future cued arm templates .", "Red bars represent data and the grey bars the shuffle distribution .", "p-value above histogram is based on a two-sample Kolmogorov–Smirnov test comparing the data distribution to the shuffle distribution . DOI: http://dx . doi . org/10 . 7554/eLife . 06063 . 01410 . 7554/eLife . 06063 . 015Figure 3—figure supplement 2 . Preferential preplay of the cued arm is not confounded by the number and activity of cells on the cued arm .", "( A ) Proportion of preplay events for the cued and uncued arm in REST2 following down-sampling to equate the number of cells on the two arms .", "Bars show means , black lines the results for individual animals and the dashed grey lines the proportion of preplay events expected by chance .", "( B ) same as a but comparing the proportion of preplay events for the cued arm in REST1 and REST2 .", "C–D same as A–B but following down-sampling to equate firing rates during rest . DOI: http://dx . doi . org/10 . 7554/eLife . 06063 . 01510 . 7554/eLife . 06063 . 016Figure 3—figure supplement 3 . Animals' interest in cued arm during GOAL-CUE is associated with subsequent preplay for each animal . The proportion of preplay events for the cued arm recorded during REST2 is shown plotted against that animal's interest in the cued arm assessed by body position during GOAL-CUE .", "−0 indicates animal showed equal interest in the cued and uncued arm , values above 0 indicate more interest in the cued arm .", "Animals exhibiting a greater interest in the cued arm subsequently appear to experience a greater proportion of preplay for that same arm ( Spearman's rank order correlation r = 1 , p = 0 . 0833 , note correlation is based on only 4 data points ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 06063 . 01610 . 7554/eLife . 06063 . 017Figure 3—figure supplement 4 . Tetrode tracts example histology showing the location of tetrode recording from area CA1 in rat R504 . Staining: cresyl violet . DOI: http://dx . doi . org/10 . 7554/eLife . 06063 . 017 Does goal-cueing trigger preplay ?", "If so , there should be a greater number of significant pre-play events in REST2 compared to REST1 which was recorded before animals had visited or seen any part of the environment .", "Preplay of the cued arm was higher in REST2 than REST1 ( 7 . 37% vs 4 . 74% , p < 0 . 001 , Figure 2C , E ) , an effect that was seen for all animals ( Table 2 ) .", "Indeed , the cued arm was not significantly preplayed during REST1 ( 4 . 74% , p = 0 . 34 ) .", "Again , the result was corroborated using an AUC analysis ( Figure 2C , Figure 2—figure supplement 1 ) .", "Thus , we find preplay only occurs during rest periods recorded after goal-cueing .", "However , it is possible that the frequency of preplay might decrease as a function of the temporal gap between rest and behaviour .", "As such our failure to detect preplay in REST1 might be due to the greater delay between REST1 and RUN2 than between REST2 and RUN2 .", "To address this we analysed preplay of the stem ( i . e . , RUN1 ) during REST1 .", "We did not find preplay of the stem ( 4 . 12% preplay events , p = 0 . 44 , AUC analysis p = 0 . 053 , Figure 2—figure supplement 2 , Table 3 , RUN1 REST1 vs cued REST2: p < 0 . 001 ) .", "Consequently , these results imply that the preplay of the unvisited , yet visible , environment we observed in REST2 was driven by behavioural cueing of that environment . 10 . 7554/eLife . 06063 . 018Table 3 . REST1 stem resultsDOI: http://dx . doi . org/10 . 7554/eLife . 06063 . 018Animal# spiking events# preplay events% preplay% chancep-valueR18386834 . 416 . 010 . 59R505980353 . 574 . 030 . 74R584329175 . 174 . 530 . 24R504396184 . 553 . 500 . 11All rats1773734 . 124 . 080 . 44Summary results from REST1 analysing preplay of the stem .", "# Spiking events = total number of spiking events recorded .", "# Preplay events = number of significant spiking events .", "% preplay = Proportion of the spiking events that qualified as preplay events ( i . e . , that were significant ) , expressed as a percentage .", "% chance = proportion of spiking events from the shuffled data that qualified as preplay events , expressed as a percentage .", "p-value = probability , derived from a binomial test , of obtaining the observed number of preplay events for each template given the chance level calculated from the shuffled data .", "At what point does preferential preplay of the cued arm emerge ?", "Plausibly preplay might be initiated immediately when the cued arm is baited ( start of GOAL-CUE ) and simply persist into the subsequent REST2 period , alternatively the bias may only arise during rest .", "Due to the short duration of the goal-cueing period ( ∼10 min ) a relatively small number of spiking events were recorded for the two arms during this period ( 172 and 170 for the cued and uncued arm respectively ) .", "However , based on a bootstrapped comparison of the AUC for absolute correlations from the cued and uncued arm vs shuffled distributions , we found that the cued but not the uncued arm was preplayed ( p = 0 . 02 , p = 0 . 24 respectively , Figure 3C , Figure 3—figure supplement 1 ) .", "A direct comparison of the proportion of preplay events for the cued vs uncued arm was marginally not significant ( 6 . 4% vs 4 . 12% , p = 0 . 052 , see Table 4 for results for individual animals ) .", "Finally , to validate the results from this smaller dataset we carried out a further , more inclusive , analysis .", "Specifically , we tracked the temporal evolution of the bias in preplay by comparing the activity of cells from the cued and uncued arms at different points during the experiment .", "For every spiking event we computed the mean rate for cells that would subsequently have fields on the cued arm compared to those with fields on the uncued arm .", "During REST1 and RUN1 the future cued and uncued arm cells did not differ in activity , this was true for both the first and second half of these periods ( mean cued/uncued rate ratio: REST1 early ratio = 0 . 96 , p = 0 . 88 , REST1 late ratio = 1 . 04 p = 0 . 09 , RUN1 early ratio = 1 . 09 p = 0 . 32 , RUN1 late ratio = 1 . 18 p = 0 . 22 , Figure 3D ) .", "However , during GOAL-CUE cued arm cells were significantly more active than uncued arm cells , an effect that was most pronounced during the first half ( 5 min ) of the cueing period ( GOAL-CUE early ratio = 1 . 78 , p = 0 . 01 , late = 1 . 46 , p < 0 . 01 ) .", "Finally , the difference between the two groups persisted through the subsequent rest period , albeit attenuating with time ( REST2 early ratio = 1 . 30 p < 0 . 001 , REST2 late ratio = 1 . 10 p < 0 . 01 , Figure 3D ) .", "Importantly , control analyses showed that the bias in sequential preplay is not a mere product of differing activity levels of the cells for the two arms ( Figure 3—figure supplement 2C–D ) .", "Together , these findings indicate that biased pre-activation of future experiences is instantiated at the point when an environment becomes motivationally-relevant . 10 . 7554/eLife . 06063 . 019Table 4 . GOAL-CUE resultsDOI: http://dx . doi . org/10 . 7554/eLife . 06063 . 019AnimalArm# spiking events# preplay events% preplay% chancep-valueR1838Cued50070 . 30Uncued50070 . 30R505Cued4536 . 674 . 020 . 11Uncued4812 . 083 . 570 . 52R584Cued11187 . 214 . 810 . 087Uncued11254 . 464 . 820 . 46R504Cued11004 . 360 . 39Uncued7114 . 295 . 000 . 044All ratsCued172116 . 404 . 640 . 11Uncued17074 . 124 . 490 . 50Summary results from GOAL-CUE analysis # Spiking events = total number of spiking events recorded .", "# Preplay events = number of significant spiking events .", "% preplay = Proportion of the spiking events that qualified as preplay events ( i . e . , that were significant ) , expressed as a percentage .", "% chance = proportion of spiking events from the shuffled data that qualified as preplay events , expressed as a percentage .", "p-value = probability , derived from a binomial test , of obtaining the observed number of preplay events for each template given the chance level calculated from the shuffled data .", "Finally , to corroborate the results obtained from the rank-order analysis of spike sequences , we applied a Bayesian spatial reconstruction algorithm ( Davidson et al . , 2009; Bendor and Wilson , 2012 ) to the data from the two rest sessions .", "In contrast to the rank-order method , which utilised only the first spike emitted by each cell , the Bayesian decoding approach used all spikes emitted during an event .", "The Bayesian decoding approach uses the spiking activity of all simultaneously recorded place cells to calculate the posterior probability of an animal being at any position in the environment , based on a Poisson spiking framework .", "( See Materials and methods; R1838 was excluded from this analysis due to low cell yield ) .", "The Bayesian decoder performed equally well on the cued and uncued arms ( Figure 4A , B , median error for both arms = 10 . 0 cm ) .", "Next , we applied the Bayesian decoding to spiking events during the rest sessions .", "First , we calculated the posterior probabilities for 5 ms non-overlapping bins , which generated a posterior probability matrix for each event ( Figure 4C , D ) .", "A spiking event that reflects a constant speed run through the environment will show an increased posterior along a line in the decoded posterior matrix .", "Therefore , for each spiking event we fit a line that accounted for the maximum variance and calculated its goodness of fit ( Figure 4C , D ) .", "To assess if that event represented a significant preplay event: we generated 1000 posterior probability matrices by shuffling the identities of cells included in the event , fit lines on all matrices , and calculated their goodness of fits .", "Events whose goodness of fits exceeded the 95th percentile of the shuffled distributions were labelled as preplay events .", "Again , during REST2 , we found preplay of the cued arm ( 7 . 64% of events , p < 0 . 001 , binomial test ) but not of the uncued arm ( 4 . 78% of events , p = 0 . 55 , vs cued = p < 0 . 001 , Figure 4E ) .", "Moreover , neither the cued nor the uncued arm were significantly preplayed in REST1 ( cued = 5 . 04% , p = 0 . 43 , uncued = 4 . 69% , p = 0 . 55 , Figure 4F ) .", "Thus , this alternative , more powerful , analysis replicates the exact same pattern of results as demonstrated by our main analysis; indicating that goal-triggered pre-activation of future place cell sequences is a robust and reliable phenomenon . 10 . 7554/eLife . 06063 . 020Figure 4 . Bayesian reconstruction confirms preferential cued arm preplay .", "( A–B )", "Confusion matrices based on RUN2 data showing decoding accuracy for the cued ( A ) and uncued arms ( B ) .", "Columns show the mean posterior probability distribution across the arm ordered by the true position of the rat ( 2 cm bins , data for R504 ) .", "Decoding errors appear as power away from the identity diagonal—here both arms yield accurate estimates of the animal's position at all points on the track .", "( C–D )", "Representative preplay events for the cued ( C ) and uncued ( D ) arms showing , ( left ) the straight line trajectory that best fits the decoded event ( x-axis indicates time bin ( 5 ms ) within the event , y-axis position on arm , title indicates probability of obtaining a better trajectory by chance ) and , ( right ) the null distribution of trajectory fits obtained by shuffling the cell identities for each event ( x-axis , quality of trajectory fit , y-axis , proportion of shuffled events ) .", "( E ) Proportion of spiking events categorised as preplay events for the cued and uncued arms in REST2 .", "Bars show mean for all data with SEM .", "( F ) Same as E but comparing preplay in REST1 and REST2 for the cued arm . DOI: http://dx . doi . org/10 . 7554/eLife . 06063 . 020" ], [ "Preplay of a visible , unvisited environment that has motivational relevance to an animal accords with various reports from neuropsychological and imaging studies indicating a crucial role for the hippocampus in processing future goals ( Spiers and Barry , 2015 ) , imagination , and future thinking ( Hassabis et al . , 2007; Schacter et al . , 2012 ) .", "Moreover , these findings agree with previous studies showing replay can be modulated by reward ( Cheng and Frank , 2008; Singer and Frank , 2009; Pfeiffer and Foster , 2013 ) but extend this modulatory influence to preplay of future , goal-oriented routes .", "In our data , sequences of place cells leading to and away from the goal were preplayed with similar frequency and occurred in both forward and reverse directions ( i . e . , positive and negative correlations ) .", "Replay of sequences leading towards goal locations has previously been linked with the planning of future trajectories ( Pfeiffer and Foster , 2013 ) .", "Plausibly the elevated proportion of preplay events we observed leading to the cued goal might also reflect route planning—especially given the animals' subsequent preference for the cued arm .", "However , preplay of sequences leading from the cued goal towards the stem are less easily explained .", "On one hand it is possible that these sequences simply reflect planning for a return trip to the familiarity of the stem after retrieval of the food .", "However , reverse replay has often been characterised as a solution to the temporal credit assignment problem ( Foster and Wilson , 2006; Foster and Knierim , 2012 ) —providing a learning mechanism to evaluate a sequence of actions that led to a successful outcome .", "In the context of preplay , when the cued arm has not been physically visited , an intriguing possibility is that paths towards the goal might be simulated using forwards preplay then evaluated and learnt from during reverse preplay , in a manner similar to that proposed to exist for periods of exploration and post-exploration rest ( Foster and Wilson , 2006; Diekelmann and Born , 2010; Buhry et al . , 2011 ) .", "Recently several studies have described ‘de novo preplay’—preplay of environments that have yet to be experienced ( Dragoi and Tonegawa , 2011 , 2013 , 2013 ) —which is believed to reflect the activity of preconfigured cell ensembles ( McNaughton et al . , 1996 ) that subsequently become bound to sensory cues in the environment .", "We did not observe de novo preplay in the current experiment ( no preplay of stem during REST1 ) .", "Although the origin of this discrepancy is unclear , we note that several other studies also do not find de novo preplay for a completely novel environment ( Wilson and McNaughton , 1994; Lee and Wilson , 2002 ) .", "However , it seems likely that other environmental factors present here but absent in the do novo preplay studies ( Dragoi and Tonegawa , 2011 , 2013 , 2014 ) , such as the barrier , and visible but inaccessible arms , might have ameliorated this phenomenon .", "Indeed , the AUC analysis comparing spiking events recorded during REST1 against the stem template ( RUN1 , Figure 2—figure supplement 2 ) was narrowly not significant ( p = 0 . 053 ) , suggesting that perhaps with more data or a simpler environment we might have found de novo preplay for the stem .", "In a similar vein one might also have expected to observe a background rate of de novo preplay for the uncued arm during REST2 .", "Again it seems possible that the complexity of the environment worked against such an effect in that the motivationally relevant cued arm was preplayed at the expense of the uncued arm .", "What remains to be understood is if this bias reflects an active process , simulating trajectories associated with the goal , or if the relevance of the cued arm simply allows it to more effectively capture preconfigured cell assemblies .", "In sum , our data indicates that preplay of an unvisited environment can be initiated in response to viewing that environment if it is relevant to future goals—features that were absent in previous studies investigating the formation of hippocampal representations for novel spaces ( Wilson and McNaughton , 1994; Lee and Wilson , 2002; Rowland et al . , 2011 ) .", "Furthermore , although our data does not provide direct support for de novo preplay it also does not argue against it , and is consistent with the hypothesis that the hippocampus supports the construction of novel spatial experiences as suggested by other authors ( for example see Dragoi and Tonegawa ( 2014 ) ) .", "These results are consistent with the notion that place cells form preconfigured assemblies or ‘charts’ ( McNaughton et al . , 1996 ) —an active but unassigned chart being associated with the cued arm during GOAL-CUE , but do not preclude the possibility that place cell firing is driven in a feed-forward manner ( e . g . , Hartley et al . , 2000 ) .", "We conclude that these findings support the hypothesised role of preplay for preparation for future experiences ( Diba and Buzsaki , 2007; Erdem and Hasselmo , 2012; Foster and Knierim , 2012; Pfeiffer and Foster , 2013 ) but extend it to simulating future experiences in environments yet to be actively explored .", "It remains to be seen if the ability of the hippocampus to construct future spatial sequences is related to the generation of more abstract temporal associations ( Kraus et al . , 2013; Eichenbaum and Cohen , 2014 ) ." ], [ "Four male lister-hooded rats were used in this study .", "All procedures were approved by the UK Home Office , subject to the restrictions and provisions contained in the Animals ( Scientific Procedures ) Act of 1986 .", "Two rats ( 330–333 g at implantation ) received two microdrives , each carrying four tetrodes of twisted 25 µm HM-L coated platinum iridium wire ( 90% and 10% , respectively; California Fine Wire ) , targeted to the left and right CA1 .", "One animal ( 350 g ) received a single microdrive , carrying eight tetrodes , targeting right CA1 .", "Finally , one animal ( 425 g ) received one microdrive carrying four tetrodes of 12- µm wire targeted to the mEC and one carrying four tetrodes of 17- µm wire targeted to contralateral CA1 .", "Only single units from the CA1 implant were included in this study .", "The 12 and 17- µm wires were platinum-plated to reduced impedance to 200–300 kΩ at 1 kHz .", "Rats were maintained at 90% of free-feeding weight , and were housed individually on a 12-hr light/dark cycle .", "After surgery , rats were food deprived with ad libitum access to water .", "Screening was performed post-surgically after a 1-week recovery period .", "An Axona recording system ( Axona Ltd . , UK ) was used to acquire the single-units , and positional data ( for details of the recording system see Barry et al . [Barry et al . , 2007] ) .", "The position and head direction of the rats were captured using an overhead video camera to record the position using a light-emitting diode ( LED ) on the animals' head-stages ( 50 Hz ) .", "Tetrodes were gradually advanced over days until place cells were found .", "We used a T-shaped track , raised 43 cm off the ground .", "The track consisted of 10 cm wide runways covered in black rubber , the stem was 222 cm long and the arms 200 cm from tip to tip .", "Animals were not habituated to running on linear tracks prior to the experiment .", "However , all animals had been trained to forage for rice in a square enclosure located in the same room but outside the curtained area .", "Initially , to prevent animals from accessing the arms , a barrier was placed on the stem 22 cm from the junction with the arms .", "The barrier consisted of a grill of vertical wooden bars spaced so the animals could easily see through but could not scale it .", "The apparatus was located in a curtained environment , and two distal landmarks were mounted on the curtain on either side of the maze for orientation .", "To encourage even distribution of place fields along the track , textured local cues were placed along the stem and arms .", "These five different cues were sheets of: black rubber , grey cardboard , black polypropylene , plaster tape , and sandpaper each approximately 20 cm long and 10 cm wide .", "For rest periods , the animal was placed in a cylindrically shaped enclosure ( 18 cm diameter × 61 cm high ) with a towel placed in the bottom .", "Prior to the experiment the animal was familiarised with the rest enclosure for a minimum of 120 min .", "The rest enclosure was located beside the track during rest periods , but was not present during the other periods .", "Prior to running on the track , animals were placed in the rest enclosure for at least 60 min—REST1 ( if the animals rested for less than 40 min during a session then the session was extended in order to obtain at least 40 min worth of recording ) .", "During this period the animals' quiescence was assessed based on speed estimates , derived by measuring the movement of the LED attached to the animals' headstages .", "Following REST1 , RUN1 was initiated by placing the rats at the end of the stem facing away from the track .", "During RUN1 animals were encouraged to run up and down the stem by the experimenter rewarding alternate ends of the stem with rice grains .", "During this period animals were prevented from running onto the arms by the barrier .", "Once animals had completed 20 laps , the experimenter stopped baiting the stem and instead baited the end of one of the arms ( GOAL-CUE ) , which was inaccessible yet visible to the animal ( cue located ca . 1 m from the animal ) .", "The cued arm was counterbalanced between animals .", "The animals had good visual access to the inaccessible arms as they could see between the wooden dowels of the barrier .", "The experimenter stood by the end of the cued arm to elicit further interest .", "Animals were allowed to remain on the track for at least a further 10 min ( GOAL-CUE ) .", "Following RUN1 , the animals were placed back into the rest enclosure for at least 60 min—REST2 .", "Following REST2 , animals were put back on the end of the stem facing away from the track .", "The barrier at the end of the stem was removed and the animals were allowed to traverse both the stem and arms in alternate L-shaped laps for each arm—RUN2 .", "On the first lap during RUN2 , animals were allowed to choose which arm they turned to when reaching the end of the stem .", "On subsequent laps animals were forced to alternate—a barrier ( the same as the one used for RUN1 ) was placed at the junction of the T-maze to occlude each arm in turn , and was moved between trials by the experimenter .", "The animals completed 20 L-shaped laps for each arm .", "For each rest period , times where at least 15% of cells from a given template fired within 300 ms and were bound by at least 50 ms of silence were selected as ‘spiking events’ ( for R1838 , which had a lower cell yield than the other rats , a minimum of 4 cells were required to be active ) .", "If a single cell fired more than one spike within this period , the first spike was counted and other spikes disregarded .", "The extent to which templates were represented in the spiking events was assessed using template-matching ( Lee and Wilson , 2002; Foster and Wilson , 2006; Diba and Buzsaki , 2007 ) .", "Specifically , the temporal sequence of cells in spiking events and the order of their corresponding peaks in the template was compared .", "If future template sequences are preplayed in rest the order in which cells fire during spiking events would be expected to resemble the order in which they fire on the track .", "In other words , one would expect the spike sequence in events and in future template sequence to be more correlated than predicted by chance .", "To determine if each template was being significantly preplayed we compared the proportion of spiking events that individually correlated with that template with the proportion expected by chance ( as predicted by a shuffling procedure ) .", "Specifically , the Spearman's rank-order correlations between each spiking event and the current template was computed and the number yielding significant correlations ( based on a two-tailed test , each tail tested at the 97 . 5% level ) was recorded; these were labelled preplay events .", "Both positive as well as negative significant correlations were categorised as preplay events , as previous studies have reported that replay can reactivate a place cell sequence in the forward or reverse order ( Lee and Wilson , 2002; Diba and Buzsaki , 2007 ) .", "Next , the sequence of spikes in each event was randomly permuted ( shuffled ) 100 times by randomly re-assigning cell IDs such that spikes from the same cell were never separated .", "For each permutation the Spearman's rank-order correlation and matching p-value were calculated , again the proportion of significant correlations was recorded; this value represented the proportion of preplay events expected by chance .", "Finally , the proportion of shuffled events that had a significant correlation with the future template sequence was compared to the number obtained for the unshuffled data using a one sample binomial test .", "To assess whether the cued arm was preplayed more than the uncued arm we directly compared the total number of events and number of preplay events , again using a binomial test .", "A similar approach was used for comparing number of preplay events in REST1 and REST2 .", "Subsequently , we corroborated these results using an alternative- distribution-based , analysis .", "Namely , we assessed whether the AUC of the cumulative distribution of absolute correlations for each arm in each of the two rest sessions differed from their shuffle distribution .", "Specifically , for each arm and each period , we bootstrapped the population of absolute correlations obtained between templates and spiking events 10 , 000 times .", "On each iterations we computed the difference between the AUC of the cumulative distribution of the bootstrapped data vs the AUC of the bootstrapped shuffled data correlations , which had been obtained previously .", "95% confidence intervals ( 2 . 5% and 97 . 5% percentiles used , i . e . , a two-tailed test ) were determined from these difference scores and were used to assess whether the two distributions differed significantly .", "Specifically , if the interval did not contain 0 then we concluded the two distributions were significantly different .", "To analyse preplay during GOAL-CUE we performed the same analysis for spiking events emitted while the animal was on the stem .", "Specifically , we only considered events recorded when an animal's velocity was below 10 cm/s and the animal was located within 20 cm ( 10% of track length ) of the barrier .", "As an additional control for differences in the number of cells contributing to each template we repeated the analyses described above after first down-sampling each template to equate the number of cells ( i . e . , to match the length of the shortest template ) .", "Specifically , if the shortest template consisted of 20 cells while another template consisted of 30 cells , we randomly removed 10 cells from the longer template .", "Moreover , we repeated this down-sampling process a 100 times , each time removing a random set of 10 cells .", "We then ran the preplay analysis as before—estimating the number of preplay events for each down-sampled cued and uncued template and comparing them to each other and chance levels .", "A similar procedure was used to equate activity rates for cued and uncued templates during the two rest periods .", "Namely , the distribution of events , containing different numbers of cells , were equated for the different templates .", "For example , if one template had 50 events with 5 cells active and another only had 40 events , we randomly removed 10 events from the former template .", "This process was then repeated for events of different lengths .", "As before the entire down sampling procedure was repeated 100 times .", "As well as controlling for quiescent rate differences this analysis also controls for the number of events for each template .", "To analyse the temporal evolution of preplay across the entire experiment we used an alternative approach to the template-matching described above .", "This was done due to the short duration of the RUN1 and GOAL-CUE periods ( ∼10 min ) , which limited the power of the template-matching approach .", "As an alternative , we computed the mean rate of future cued and uncued arm cells during spiking events for each half of all experimental periods ( i . e . , REST1 , RUN1 , GOAL-CUE and REST2 ) .", "If a cell was active on both arms we excluded it , unless the rate on one arm was at least twice as high as the rate on the other arm .", "To assess statistical significance of rate differences we bootstrapped the event rates for the cued and uncued arm cells 10 , 000 times .", "We then computed the mean ratio between the cued and uncued arm rates ( cued/uncued ) for each bootstrap , and estimated the 95% confidence interval .", "If the interval did not contain 1 then we deemed the rate difference significant .", "To corroborate results obtained from the rank-order correlation between spikes and field position we also applied a Bayesian reconstruction algorithm to spike sequences recorded during events .", "Following Zhang et al . , 1998 , and Davidson et al . , 2009 , using non-overlapping temporal windows , we computed the probability distribution across position based on ratemaps generated from RUN2 ( Davidson et al . , 2009; Zhang et al . , 1998 ) .", "Specifically during a time window ( T ) the spikes generated by N place cells was K = ( k1 , … , ki , … , kN ) , where ki was the number of spikes fired by the ith cell .", "The probability of observing K in time T given position ( x ) was taken as: ( 1 ) P ( K|x ) =∏Poisson ( ki , Tαi ( x ) ) =∏i=1N ( T×αi ( x ) ) kiki !", "×e−Tαi ( x ) , where x indexes the 1 cm spatial bins defined on the arms of the apparatus and αi ( x ) is the firing rate of the ith place cell at position x , derived from RUN2 ratemaps .", "To compute the probability of the animals' position given the observed spikes we applied Bayes' rule , assuming a flat prior for position ( P ( x ) ) , to give: ( 2 ) P ( x|K ) =R ( ∏i=1Nαi ( x ) ki ) exp ( −T∑i=1Nαi ( x ) ) , where R is a normalising constant depending on T and the number of spikes emitted .", "Note we do not use the historic postion of the animals' to contstrain P ( x|K ) thus the probability estimate in each T is independent of its neighbours .", "To validate the effectiveness of the reconstruction algorithm it was used to decode the animals' actual position during RUN2 .", "For each T ( 200 ms ) location was decoded by taking the peak of the P ( x|K ) distribution .", "Decoded estimates of location were compared with true location to obtain a measure of the absolute decoding error .", "To decode offline activity , posterior probability matrices were produced for events with ≥7 active cells using 5 ms non-overlapping time windows ( T ) .", "To decode the probability matrices we used a method similar to Davidson et al . ( 2009 ) , fitting a straight line—equivalent to a constant speed trajectory—to each matrix .", "Lines were defined with a gradient ( V ) and intercept ( c ) , equivalent to the velocity and starting location of the trajectory .", "The goodness of fit of a given line ( R ( V , c ) ) was defined as the proportion of the probability distribution that lay within 20 cm of it .", "Specifically where P is the probability matrix obtained from ( 2 ) : ( 3 ) R ( V , c ) =1n∑t=0n−1P ( |x ( t ) − ( V . t . T+c ) |≤d ) , where t index the time bins of width T and d is set to 20 cm .", "R ( V , c ) was maximised using an exhaustive search to test all combinations of V between −50 ms−1 and 50 ms−1 in 0 . 2 ms−1 increments ( excluding slow trajectories with speeds > −2 ms−1 and < 2 ms−1 ) and c between −15 m and 16 m in 0 . 01 m increments .", "To assess the presence of preplay in each event we compared the goodness of fit of the best straight line trajectory with the best fits made to 1000 posterior probability matrices generated by shuffling the identities of cells included in the event .", "Events whose best fit line exceeded the 95th percentile of its own shuffled distribution we labelled as preplay events .", "To eliminate the possibility that poor cluster isolation might contribute to the observed preplay ( such as mistakenly dividing a complex spike into two clusters ) , we analysed the cluster separation between every pair of place cells recorded from the same tetrode .", "Thus , for each spike the first three principle components of the waveform recorded on each of the four channels of the tetrode were calculated .", "Thus , 12 features ( 4 channels × 3 principle components ) defined each spike .", "Based on these features the squared Mahalanobis distance between that spike and the centre of another cluster was calculated , using the covariance matrix for all spikes belonging to the cluster .", "This process was then repeated for all spikes in a given cluster and the mean Mahalanobis' distance calculated .", "Clusters of spikes originating from a single cell but which have mistakenly been assigned to two or more cells will be identified by low a Mahalanobis distance and proximate place fields .", "We divided cell pairs into two groups—those with short Mahalanobis distances ( less than the median ) and those with long distances ( greater than or equal to the median ) .", "The distribution of place field separation for the two groups was then compared using a 2-sample Kolmogorov–Smirnov test .", "To eliminate the possibility that preplay of the cued arm during REST2 merely reflects familiarity with the proximal portions of the cued arm during GOAL-CUE we assessed whether off-line spikes were more likely to be associated with the first half of the arm ( i . e . , closest to the stem ) vs the second half ( excluding the first 22 cm that are common to both arms ) .", "We computed the proportion of spikes representing proximal locations and assessed whether the proportion differed significantly ( using a binomial test ) from what one would expect based on the place field distribution of the cued arm .", "We also repeated the same analysis for the uncued arm .", "Finally , to assess whether the distribution of preplayed locations ( normalised by place field distribution ) for the two arms differed significantly from a uniform distribution we used a two-sample Kolmogorov–Smirnov test .", "Finally , to assess place field stability on the cued and uncued arm we split the RUN2 session in half ( based on odd and even samples ) and generated ratemaps for each half .", "We then calculated the correlation between the two ratemaps ( bin-wise Pearson product–moment correlation coefficients were calculated after first removing unvisited bins and any bin pairs that had mutual 0 Hz firing rates ) and compared correlations obtained for the cued and uncued arms using a two-sample t-test .", "LFP from CA1 was recorded at 4 . 8 kHz throughout the experiment .", "To analyse sharp-wave ripples ( O'Keefe and Nadel , 1978; Buzsaki et al . , 1992 ) the LFP was band-pass filtered between 80 and 250 Hz .", "An analytic signal was constructed using the Hilbert transform , taking the form: ( 4 ) sa ( tk ) =s ( tk ) +iH[s ( tk ) ] , where H specifies the Hilbert transform , s ( tk ) is the filtered LFP signal , tk = kΔ , where k = 1 , . . . , K indexes the time-step and Δ is the inverse of the sampling rate .", "An instantaneous measure of power was found by taking the squared complex modulus of the signal at each time point .", "This measure was then down sampled to 50 Hz to match the position sampling rate , and finally was smoothed with a boxcar filter of width 0 . 1 s .", "Two-tailed independent samples t-tests were used to compare power in the ripple-band during significant ( i . e . , preplay events ) and non-significant spiking events and to compare power during significant spiking events and periods outside of spiking events .", "Rats were anaesthetised ( 4% isoflurane and 4 l/min O2 ) , injected intra-peritoneal with an overdose of Euthatal ( sodium pentobarbital ) after which they were transcardially perfused with saline followed by a 4% paraformaldehyde solution ( PFA ) .", "Brains were carefully removed and stored in PFA which was exchanged for a 4% PFA solution in PBS ( phosphate buffered saline ) with 20% sucrose 2–3 days prior to sectioning .", "Subsequently , 40–50 μm frozen coronal sections were cut using a cryostat , mounted on gelatine-coated glass slides and stained with cresyl violet .", "Images of the sections were acquired using an Olympus microscope , Xli digital camera ( XL Imaging Ltd . ) .", "Sections in which clear tracks from tetrode bundles could be seen were used to determine the location of cells recorded .", "Animals' interest in the cued arm during goal-cueing was assessed as follows .", "The portion of the stem within 20 cm of the barrier was notionally divided in two long-ways to yield a cued and uncued side .", "Time spent on the two sides was determined based on the animals' tracked positions .", "Finally , to express interest in the cued arm as a single behavioural measure , the difference between the time spent on the cued vs the uncued side was divided by the total time spent on that section of the track .", "Thus , a value above 0 indicates that an animal spent more time on cued side than the uncued side .", "As an additional measure of the animals' interest in the cued arm during the goal-cueing period we assessed the amount of time animals spent looking toward the cued arm ( while the animals' velocity was at least 5 cm/s , head direction was inferred by tracking a single LED which requires the animal to be moving in order to get accurate head direction estimates ) .", "Again , preference for the cued arm was expressed as a single behavioural measure by subtracting the amount of time spent with the head oriented towards the cued arm from the time spent with the head oriented towards the uncued arm and dividing by the total time spent with the head oriented towards either arm .", "Finally , to assess the animals' preference for the cued arm during the first lap of RUN2 , the difference between the time spent on the cued arm vs the uncued arm during the first 3 s following the animals' entry onto the arms was divided by the total time spent on either arm ." ] ]

[ "Dominant theories of hippocampal function propose that place cell representations are formed during an animal's first encounter with a novel environment and are subsequently replayed during off-line states to support consolidation and future behaviour .", "Here we report that viewing the delivery of food to an unvisited portion of an environment leads to off-line pre-activation of place cells sequences corresponding to that space .", "Such ‘preplay’ was not observed for an unrewarded but otherwise similar portion of the environment .", "These results suggest that a hippocampal representation of a visible , yet unexplored environment can be formed if the environment is of motivational relevance to the animal .", "We hypothesise such goal-biased preplay may support preparation for future experiences in novel environments ." ]

[ "As an animal explores an area , part of the brain called the hippocampus creates a mental map of the space .", "When the animal is in one location , a few neurons called ‘place cells’ will fire .", "If the animal moves to a new spot , other place cells fire instead .", "Each time the animal returns to that spot , the same place cells will fire .", "Thus , as the animal moves , a place-specific pattern of firing emerges that scientists can view by recording the cells' activity and which can be used to reconstruct the animal's position .", "After exploring a space , the hippocampus may replay the new place-specific pattern of activity during sleep .", "By doing so , the brain consolidates the memory of the space for return visits .", "Recent evidence now suggests that these mental rehearsals—or internal simulations of the space—may begin even before a new space has been explored .", "Now , Ólafsdóttir , Barry et al . report that whether an animal's brain simulates a first visit to a new space depends on whether the animal anticipates a reward .", "In the experiments , rats were allowed to run up to the junction in a T-shaped track .", "The animals could see into each of the arms , but not enter them .", "Food was then placed in one of the inaccessible arms .", "Ólafsdóttir , Barry et al . recorded the firing of place cells in the brain of the animals when they were on the track and during a rest period afterwards .", "The rats were then allowed onto the inaccessible arms , and again their brain activity was recorded .", "In the rest period after the rats first viewed the inaccessible arms , the place cell pattern that would later form the mental map of a journey to and from the food-containing arm was pre-activated .", "However , the place cell pattern that would become the mental map of the other inaccessible arm was not activated before the rat explored that area .", "Therefore , Ólafsdóttir , Barry et al . suggest that the perception of reward influences which place cell pattern is simulated during rest .", "An implication of these findings is that the brain preferentially simulates past or future experiences that are deemed to be functionally significant , such as those associated with reward .", "A future challenge will be to determine whether this goal-related simulation of unvisited spaces predicts and is needed for behaviour such as successful navigation to a goal ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "biochemistry and chemical biology", "structural biology and molecular biophysics" ]

[ [ "Replicative helicases and DNA polymerases ( DNAPs ) are not efficient at unwinding the duplex DNA when they are working independently .", "The unwinding rates are slower than their translocation rates on single-stranded ( ss ) DNA and slower than the rates of DNA replication ( Kim et al . , 1996; Delagoutte and von Hippel , 2001; Galletto et al . , 2004; Stano et al . , 2005; Lionnet et al . , 2007; Donmez and Patel , 2008 ) .", "Moreover , the unwinding rates of isolated helicases decrease steeply with increasing GC percentage in the duplex DNA , therefore , assisting forces that destabilize the junction base pairs stimulate the helicase ( Galletto et al . , 2004; Johnson et al . , 2007; Lionnet et al . , 2007; Donmez and Patel , 2008 ) .", "However , in the presence of an actively synthesizing replicative DNAP , the unwinding rates of the helicase become fast and GC independent ( Kim et al . , 1996; Delagoutte and von Hippel , 2001; Stano et al . , 2005; Manosas et al . , 2012b; Pandey and Patel , 2014 ) .", "Similarly , replicative DNAPs on their own have limited strand displacement synthesis activity , restricted to 4–6 base pairs in T7 DNAP ( Stano et al . , 2005; Yuan and McHenry , 2009; Pandey and Patel , 2014 ) .", "The isolated DNAPs often stall and move backward to excise the nascent DNA using their proofreading exonuclease activity when faced with downstream duplex DNA ( Manosas et al . , 2012a ) .", "The presence of helicase or single-stranded DNA-binding protein ( SSB ) inhibits processive excision of the nascent DNA and allows DNAP to catalyze fast and processive strand displacement synthesis ( Manosas et al . , 2012b; Pandey and Patel , 2014 ) .", "Originally identified in prokaryotic systems ( T7 , T4 bacteriophage , Escherichia coli ) , this functional coupling between the helicase and DNAP is found also in eukaryotic replication systems ( Kang et al . , 2012 ) .", "Several models have attempted to explain the functional coupling between replicative helicases and DNAPs .", "The underlying theme of the helicase only unwinding models is that the helicase unwinds the duplex DNA creating ssDNA template for the DNAP and the DNAP traps the displaced ssDNA through DNA synthesis ( Delagoutte and von Hippel , 2001; Stano et al . , 2005 ) .", "Recent studies of bacteriophage T7 and T4 DNAPs suggest alternative models indicating that DNAP aids the helicase by destabilizing the first few base pairs of the double-stranded ( ds ) DNA ( Manosas et al . , 2012a , 2012b ) .", "Exonuclease mapping showed that T7 DNAP is located with the T7 helicase in close proximity to the fork junction and in a position to influence the junction base pairs ( Pandey and Patel , 2014 ) .", "To understand functional coupling between helicase and DNAP , we need to understand the basic mechanism of DNA unwinding by each enzyme .", "Although the mechanism of replicative DNAPs is well characterized on ssDNA template ( Patel et al . , 1991; Doublie et al . , 1998; Delagoutte , 2012 ) , there is little known about the mechanism of DNA unwinding-synthesis on duplex DNA template .", "Similarly , there are no structures of replisomes with the exception of a small angle X-Ray scattering structure of the T7 helicase-T7 DNAP bound to ssDNA and primer template DNA , respectively ( Kulczyk et al . , 2012 ) .", "However , the low-resolution structure in the absence of a replication fork DNA does not provide the location of the DNAP and helicase at the replication fork junction to understand which enzyme is involved in melting the base pair at the fork junction .", "In addition to structural questions such as the positioning of the helicase and DNAP at the replication fork , many aspects of the mechanism of functional coupling remain unexplored .", "For example , T7 and E . coli DNAPs are capable of strand displacement synthesis in the presence of SSB with rates comparable to their replication rates ( Yuan and McHenry , 2009; Pandey and Patel , 2014 ) .", "Hence , the specific role of the helicase in stimulating the synthesis activity of DNAP remains unclear .", "As combined enzymes , T7 helicase and T7 DNAP exhibit highly coordinated catalysis , whereby helicase hydrolyzes one dNTP for every dNMP incorporated by the DNAP ( Pandey and Patel , 2014 ) .", "This implies that the two enzymes coordinate their steps of nucleotide binding ( 2′-deoxythymidine 5′-triphosphate ( dTTP ) binding to T7 helicase and dNTP binding to T7 DNAP ) and catalysis ( dTTP hydrolysis and dNMP incorporation ) , but there is no model that explains how these steps are coupled between the two enzymes during active leading strand synthesis .", "The studies in this paper use a combination of 2-aminopurine ( 2-AP ) fluorescence and transient-state kinetics to investigate the unwinding mechanisms of T7 DNAP and T7 helicase as isolated enzymes and as combined enzymes .", "The kinetics indicates that DNAP and helicase use different mechanisms to unwind DNA , but the mechanisms when coupled generate an efficient replisome .", "In the replisome , T7 DNAP stimulates the helicase by increasing the unwinding kcat , and T7 helicase stimulates the DNAP by decreasing the dNTPs Km .", "The 2-AP studies probe DNA melting with single base pair resolution and show that the isolated enzymes are not as efficient at melting the fork junction as compared to the combined enzymes .", "However , T7 DNAP with its ability to melt two base pairs ahead of the primer-end positions T7 helicase two nucleotides ahead with efficient and synergistic melting of the junction base pair .", "Overall , these studies provide both kinetic and structural basis to understand how helicase and polymerase mutually stimulate each other's activities during leading strand synthesis ." ], [ "To measure the DNA-unwinding kinetics in real time , we labeled the 3′-end of the lagging strand with fluorescein and the 5′-end of the leading strand with a black hole quencher ( BHQ1 ) ( Figure 1A ) .", "The fluorescence intensity of fluorescein-labeled lagging strand is quenched by BHQ1 when the DNA is duplexed , but when the lagging strand is unwound by T7 DNAP + E . coli SSB the fluorescence intensity increases ( Figure 1B ) .", "We find that T7 DNAP on its own does not unwind the 40 bp replication fork at fast rates , which is consistent with our previously reported gel-based studies showing T7 DNAP stalling after 4–6 base pairs synthesis ( Stano et al . , 2005; Pandey and Patel , 2014 ) and this can be overcome by adding SSB ( Pandey and Patel , 2014 ) .", "Indeed , T7 DNAP fully unwinds the fork DNA with E . coli SSB in the reactions .", "Note that we can replace E . coli SSB with T7 gp2 . 5 ( Figure 1—figure supplement 1A ) ( Myers and Romano , 1988; Nakai and Richardson , 1988; Pandey and Patel , 2014 ) .", "However , we have used E . coli SSB because of its higher affinity for ssDNA ( 0 . 1–10 nM ) ( Molineux et al . , 1975 ) , requiring lower amounts of SSB for the same degree of stimulation as with T7 gp2 . 5 ( 200 nM SSB vs 3 μM T7 gp2 . 5 ) ( Figure 1—figure supplement 1B ) .", "The E . coli SSB does not unwind DNA on its own ( Figure 1—figure supplement 1C ) .", "In these experiments , T7 DNAP and E . coli SSB were preassembled on the fork DNA , and DNA unwinding was initiated with dNTPs and Mg ( II ) in a stopped-flow instrument at 18°C .", "By preassembling the DNAP-SSB-DNA complex , we bypass the slow enzyme binding steps and synchronize the unwinding reactions .", "Therefore , the unwinding kinetics shows a presteady state time lag prior to fluorescence increase ( Figure 1B ) .", "The lag time represents the time of unwinding , because it gets shorter when the duplex DNA to be unwound is 25 bp rather than 40 bp ( Figure 1—figure supplement 1D ) .", "Therefore , the kinetics were fit to the n-step model ( Levin et al . , 2009 ) to extract the base pair unwinding rates ( Appendix—Section 1 and Figure 1—figure supplement 2A–C ) .", "These average unwinding rates include time spent in unwinding the 40 bp fork DNA and time spent in any paused states .", "The unwinding rates of T7 DNAP ( with SSB ) increase in a hyperbolic manner with increasing dNTPs concentrations with each of the GC forks ( Figure 1C ) .", "At low-dNTPs concentration , the unwinding rates are GC-sensitive , but the rates reach a similarly high value at saturating dNTPs concentrations .", "Thus , the unwinding kcat remains nearly constant and ranges from 190 to 140 bp/s as GC percentage increases ( Figure 1D ) .", "However , the dNTPs Km increases steeply from 40 μM to 270 μM as the GC percentage increases ( Figure 1E ) .", "This indicates that DNA unwinding-synthesis by T7 DNAP ( with SSB ) is rate-limited by base pair separation at low-dNTPs concentrations , but not at high dNTPs .", "For comparison , when the downstream DNA is single-stranded and does not need to be melted , the dNTPs Km is 10–20 μM and the rate of synthesis is ∼200 nt/s ( Patel et al . , 1991; Stano et al . , 2005 ) .", "Thus , the dNTPs Km on duplex DNA template is 2–10 times higher than on ssDNA template .", "The minimal mechanism of DNA synthesis contains three steps ( Figure 1F ) .", "The first step is capture of the templating base ( base-capture ) in the insertion site of the DNAP through translocation ( Poln ⇔ Poln . base ) , the second step is dNTP binding and base pairing with the templating-base ( Poln . base ⇔ Poln . base . dNTP ) , and the third step is chemistry where dNMP is added , PPi is released , and the primer-end is elongated by one nucleotide ( Poln . base . dNTP ⇔ Poln + 1 ) .", "The rate vs dNTPs dependencies of all GC forks fit well to this ordered three-step mechanism ( Appendix—Section 2 and Figure 1—figure supplement 3 ) with variable equilibrium constant for the base-capture step ( K1 ) , which we find decreases steadily with increasing GC percentage ( Figure 1G ) .", "This means that the downstream DNA stability mainly affects the base-capture step , and DNAP is less efficient at capturing the templating-base when the stability of the duplex DNA is higher .", "However , the nearly GC-independent DNA unwinding kcat indicates that dNTP binding stabilizes the base-captured state and drives DNA unwinding , but higher concentrations of dNTPs are required with higher GC percentage in the downstream DNA .", "This mechanism of DNAP resembles the mechanism proposed for DNA-dependent RNA polymerases ( Thomen et al . , 2005 ) .", "The unwinding rates of T7 helicase were measured using the same replication fork substrates , except fluorescein was moved to the end of the leading strand ( to avoid fluorescence changes from helicase binding to the end of the lagging strand ) and a GGG quencher was introduced opposite to the fluorescein in the lagging strand to ensure that the signal is quenched when the DNA is duplexed ( Figure 2A ) .", "The fork DNA was incubated with T7 helicase in the presence of dTTP without Mg ( II ) to avoid assembly lags , and the unwinding reactions were initiated with Mg ( II ) and dT90 trap DNA .", "The unwinding kinetics show a presteady state time lag followed by fluorescence increase due to strand separation ( Figure 2B ) .", "The kinetics were fit to the n-step model , and the average base pair unwinding rates were determined at various dTTP concentrations with all the GC forks ( Figure 2C ) . 10 . 7554/eLife . 06562 . 007Figure 2 . Kinetic mechanism of DNA unwinding by T7 helicase .", "( A ) Replication fork DNA for the measurement of the unwinding kinetics of T7 helicase .", "( B ) Representative kinetic trace of DNA unwinding by T7 helicase ( dots ) fit to the n-step model ( solid line ) .", "( C ) The unwinding rates against dTTP concentrations fit to Equation 4 ( solid line ) to obtain the unwinding kcat and dTTP Km values .", "( D , E )", "The kcat and dTTP Km plotted as a function of GC percentages .", "( F ) Schematic of the helicase model with random order of base-capture and dTTP binding .", "( G , H )", "Plots of equilibrium constants for the base-capture steps in the dTTP free ( K1 ) and dTTP bound ( K2 ) state as a function of GC percentages .", "Kd , dTTP was fixed at 90 μM , which corresponds to the Km , dTTP for the helicase when translocating on ssDNA ( Figure 2—figure supplement 2 and Appendix—Section 4 ) .", "kcat was fixed at 130 nt/s corresponding to the ssDNA translocation rate of the helicase ( Kim et al . , 2002 ) .", "Details of the fittings are provided in Appendix—Section 3 . DOI: http://dx . doi . org/10 . 7554/eLife . 06562 . 00710 . 7554/eLife . 06562 . 008Figure 2—figure supplement 1 . Fitting kinetics of unwinding by T7 helicase . The dTTP dependency data for unwinding by the helicase on the 20–65% GC DNA were fit to the model A ( A ) model B ( B and C ) and model C ( D ) .", "The data do not fit well to model A . Although it fits reasonably well to model B for the 20% and 35% GC DNA , the 50% and 65% GC DNA data do not fit well to the model .", "( C ) The best fits were obtained with model C . ( D ) The fitting was performed using SigmaPlot .", "The derivation of equations used in the fitting is shown in the Appendix—Section 3 . The Km , dTTP was fixed at 90 μM in the fitting based on the Km , dTTP for the helicase when translocating on ssDNA substrate ( Appendix—Section 4 ) .", "The kcat was fixed at 130 nt/s corresponding to the ssDNA translocation rate of the helicase . DOI: http://dx . doi . org/10 . 7554/eLife . 06562 . 00810 . 7554/eLife . 06562 . 009Figure 2—figure supplement 2 . Pi release kinetics . Burst Rate of Pi release plotted as a function of dTTP to obtain the Km , dTTP in the presence of dT90 ssDNA .", "The error represents fitting error . DOI: http://dx . doi . org/10 . 7554/eLife . 06562 . 009 Unlike T7 DNAP , the unwinding kcat of the helicase decreases from 65 bp/s to 15 bp/s with increasing GC percentage ( Figure 2D ) , but the dTTP Km remains relatively unchanged and decreases only two-fold from 300 μM to 160 μM ( Figure 2E ) .", "Thus , T7 helicase and T7 DNAP respond differently to increasing GC content , which indicates that their DNA-unwinding mechanisms are fundamentally different .", "T7 helicase moves on DNA through sequential nucleotide hydrolysis and translocation mechanism , where each subunit of the ring takes turn in binding the incoming nucleotide ( Liao et al . , 2005; Crampton et al . , 2006; Enemark and Joshua-Tor , 2006; Thomsen and Berger , 2009; Patel et al . , 2011 ) .", "Therefore at any given time , only the leading subunit of T7 helicase binds an incoming dTTP and reels in the nucleotide base from the fork junction ( Sun et al . , 2011 ) .", "There are three minimal steps during each base pair unwinding event .", "One , the leading helicase subunit binds to the nucleotide base from the fork junction ( base-capture ) ; two , the leading subunit binds to a molecule of dTTP; and three , dTTP hydrolysis and product release ( 2′-deoxythymidine 5′-diphosphate ( dTDP ) and Pi ) occur at distinct subunits around the hexameric ring ( Figure 2F ) .", "The order of events at the leading subunit is not known .", "In other words , it is not known whether base-capture by the leading subunit occurs in the dTTP-bound or dTTP-free state .", "Therefore , we considered all three models and our kinetic modeling shows that the rate vs dTTP data do not fit to models where dTTP binding and base-capture steps occur in a particular order ( Appendix—Section 3 and Figure 2—figure supplement 1A–C ) .", "Instead , the data fit well to the random order mechanism ( Figure 2F and Figure 2—figure supplement 1D ) , wherein base-capture can occur both in the dTTP-free and dTTP-bound states of the leading subunit .", "We show that the dTTP-bound state is slightly better at capturing the DNA-base than the dTTP-free state ( Figure 2H ) , but the equilibrium constants of the base-capture steps in the dTTP-free ( K1 ) and dTTP-bound ( K2 ) states both decrease with increasing GC percentage ( Figure 2G–H ) , which indicates that the rate of unwinding by the helicase is limited by base pair separation even at high concentrations of dTTP .", "Our results demonstrate that the DNA-unwinding activity of T7 helicase and T7 DNAP is rate-limited by inefficient base-capture at the fork junction .", "However , the two enzymes respond differently to increasing GC percentage , and this is because of their different kinetic mechanisms .", "T7 DNAP follows an ordered mechanism , wherein dNTP binding follows the base-capture step .", "Therefore , the kinetic outcome of increasing GC content is analogous to a pure competitive mechanism where inhibitor ( GC content ) increases the dNTPs Km without affecting the unwinding kcat .", "T7 helicase does not follow an ordered mechanism .", "Consequently , the kinetic outcome of increasing GC content is analogous to a mixed inhibition mechanism where inhibitor ( GC content ) decreases the unwinding kcat and mildly affects the dTTP Km .", "Therefore , T7 DNAP is able to achieve fast rates of unwinding at high dNTPs , but this is not the case with the helicase , whose unwinding kcat remains suboptimal even at high-dTTP concentrations .", "We used the same replication fork substrates used in the DNAP experiments to investigate the unwinding mechanism of the combined T7 helicase and T7 DNAP enzymes ( Figure 3A ) .", "In these experiments , helicase and DNAP were preassembled on the fork DNA using dTTP without Mg ( II ) , and unwinding was initiated with Mg ( II ) , dVTPs ( 3 dNTPs except dTTP ) , and dT90 trap DNA .", "The kinetic traces show an initial lag followed by an increase in fluorescence , but then a slight dip at the end of the reaction ( Figure 3A ) .", "The dip is more prominent with high-GC content forks than with the low-GC content forks ( Figure 3—figure supplement 1A ) .", "The dip was observed with the isolated helicase and with helicase-DNAP functioning together ( Figure 3—figure supplement 1B ) , but not with isolated DNAP .", "Therefore , we believe that the dip comes from interactions of the helicase with the fluorescein at the end of the lagging strand .", "We fit the lag and the increase in fluorescence to the n-step model to obtain the average base pair unwinding rates .", "These unwinding rates correlate well with the rates from the gel-based primer-extension assays ( Pandey and Patel , 2014 ) . 10 . 7554/eLife . 06562 . 010Figure 3 . The kinetics of DNA unwinding by the combined helicase and DNAP enzymes .", "( A ) The replication fork DNA substrate and representative kinetic trace of DNA unwinding by the combined T7 DNAP and T7 helicase enzymes .", "( B ) The base pair unwinding rates of the combined enzymes at 5 µM dVTPs plotted against dTTP concentrations and fit to Equation 4 ( solid lines ) to obtain the maximal rate of unwinding ( kcat ) and Km for dTTP .", "( C , D )", "The unwinding kcat and dTTP Km as a function of GC percentages .", "( E ) The unwinding rates of the isolated helicase ( red circles ) and helicase-DNAP ( green circles ) were measured using the 50% GC fork at constant 5 μM dVTP and increasing dTTP concentrations .", "( F ) The unwinding rates of the isolated helicase ( red circle ) and helicase-DNAP ( green circle ) were measured using the 50% GC fork at 50 μM dVTP concentrations and increasing dTTP concentrations .", "( G ) The unwinding rates of T7 DNAP with E . coli SSB ( red circle ) or with T7 helicase ( green circle ) were measured using the 50% GC fork at 500 μM dTTP and increasing dNTPs concentrations . DOI: http://dx . doi . org/10 . 7554/eLife . 06562 . 01010 . 7554/eLife . 06562 . 011Figure 3—figure supplement 1 . Unwinding-synthesis trace for helicase-DNAP .", "( A ) Representative trace showing time course of unwinding-synthesis by the helicase–polymerase on the 20% GC and 50% GC DNA substrate at 100 μM dTTP and 5 μM dVTP .", "The dip in signal is more prominent with the 50% GC DNA substrate compared to the 20% GC DNA substrate .", "This suggests that the first and second phase could represent the coupled and uncoupled activities of the helicase-DNAP , respectively .", "( B ) Representative trace showing time course of unwinding by the helicase at 500 μM dTTP on the 20% GC DNA .", "The presence of the dip in the helicase only reaction suggests that interaction of helicase with the fluorescein leads to a dip in the fluorescence signal once the strands are unwound .", "Together , these data indicate that the dip in the fluorescence signal could be due to", "( i ) helicase interaction with the fluorescein and", "( ii ) coupled and uncoupled activities of the helicase-DNAP . DOI: http://dx . doi . org/10 . 7554/eLife . 06562 . 011 The unwinding rates of the combined enzymes measured at increasing dTTP and constant dVTPs concentration show little dependency on the GC percentage ( Figure 3B ) .", "The unwinding kcat is ∼90 bp/s when GC percentage is low , and kcat decreases minimally to 70 bp/s when GC percentage is high .", "Similarly , the dTTP Km changes little from 120 to 90 μM as GC percentage increases ( Figure 3C–D ) .", "This indicates that the DNA-unwinding rates of the combined enzymes are not rate-limited by base pair separation at any dNTPs concentrations .", "Additionally , these results also show that the two enzymes mutually stimulate the activities of unwinding and synthesis .", "How does T7 DNAP stimulate the helicase ?", "The dTTP Km of the isolated helicase on the 50% GC fork is ∼160 μM , which decreases to ∼80 μM in the presence of T7 DNAP ( Figure 3E ) , which is close to the dTTP Km on ssDNA ( Figure 2—figure supplement 2 ) .", "Similarly , the unwinding kcat of the isolated helicase is ∼20 bp/s and increases substantially to ∼150 bp/s in the presence of T7 DNAP ( Figure 3F ) , which is close to the translocation rate of the helicase on ssDNA ( ∼130 nt/s ) ( Kim et al . , 2002 ) .", "Thus , T7 DNAP stimulates T7 helicase by increasing the DNA-unwinding kcat and by slightly decreasing the dTTP Km .", "The kinetic parameters of T7 helicase coupled to T7 DNAP resemble those of helicase translocating on ssDNA rather than unwinding duplex DNA .", "How does the helicase stimulate the DNAP ?", "The unwinding kcat ( with SSB ) is 160 bp/s and decreases minimally to 140 bp/s in the presence of T7 helicase , whereas the dNTPs Km decreases by 24-fold from ∼120 μM ( with SSB ) to 5 µM in the presence of T7 helicase ( Figure 3G ) .", "The 5 μM dNTPs Km is slightly lower than the DNAP's dNTP Km on ssDNA template ( ∼10–20 μM ) ( Patel et al . , 1991; Stano et al . , 2005 ) .", "Thus , T7 helicase stimulates T7 DNAP by promoting dNTP binding , and in the presence of helicase , T7 DNAP behaves like a motor translocating on ssDNA template .", "In summary , by measuring the unwinding kinetics of the individual enzymes and comparing it to the combined enzymes , we determine how T7 helicase and T7 DNAP mutually stimulate each other's activity .", "The T7 DNAP stimulates the T7 helicase by increasing the unwinding kcat , whereas the helicase stimulates the DNAP by decreasing the dNTPs Km .", "When functioning independently , the lower kcat of the helicase and higher dNTPs Km of DNAP are due to inefficient capture of the nucleotide base from the fork junction .", "This implies that the two enzymes help each other by increasing the efficiency of the base-capture step .", "What happens when one enzyme is faster than the other enzyme during leading strand synthesis ?", "Do the enzymes become functionally uncoupled ?", "Does the faster enzyme outrun the slower enzyme ?", "Because T7 helicase mainly uses dTTP as its substrate and the DNAP uses all dNTPs , we can change the concentrations of dTTP and dVTPs to control the speeds of helicase and DNAP , respectively .", "When the helicase rate is decreased by lowering dTTP , DNA unwinding slows down ( Figure 4A ) .", "At 500 μM dTTP , the kcat of the combined enzymes is fast ( 150 bp/s ) , but at 50 μM dTTP , the kcat is 60 bp/s .", "This implies that when helicase is slower than DNAP , the DNAP will not outrun the helicase .", "Interestingly , the dVTPs Km of the combined enzymes remains unchanged at ∼5 μM , both at low- and high-dTTP concentrations .", "This means that when helicase is slower than DNAP , the combined enzymes remain functionally coupled . 10 . 7554/eLife . 06562 . 012Figure 4 . Functionally coupled and uncoupled helicase-DNAP .", "( A ) The unwinding rates of the combined helicase-DNAP were measured at 50 µM dTTP ( red circle ) or 500 µM dTTP ( green circle ) at increasing dVTPs concentration on the 50% GC fork .", "The bar chart shows the unwinding kcat and dVTPs Km of the combined enzymes at low- ( grey bars ) and high-dTTP ( black bars ) concentrations .", "The cartoon shows that the enzymes remain functionally coupled when helicase is the slow motor .", "( B ) The unwinding rates of the combined enzymes ( green circles ) at zero dVTPs concentration are compared to the rates of helicase alone ( red circles ) at increasing dTTP concentrations on the 50% GC fork .", "The bar charts compare the unwinding kcat and dTTP Km of helicase-DNAP ( black ) and isolated helicase ( gray ) .", "Error bars represent fitting errors .", "The cartoon show that stalling DNAP leads to functional uncoupling between helicase-DNAP with or without physical uncoupling .", "( C ) The DNA unwinding rates of the isolated helicase and helicase-DNAP were measured at 1 mM dTTP and 0 . 5 µM dVTPs on the 20% GC fork .", "The bar chart shows the unwinding rate of the isolated helicase ( light gray ) , helicase-DNAP complex ( dark gray ) , and the predicted rate of DNA synthesis by helicase-DNAP assuming coupled synthesis ( black ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 06562 . 012 When DNAP cannot move forward due to lack of dNTPs , the associated helicase unwinds the replication fork with unstimulated rates ( Figure 4B ) .", "This means that the stalled DNAP does not pull the helicase back or prevent it from unwinding the DNA .", "With stalled DNAP , the unwinding rate of the 50% GC fork is 20 bp/s , which is similar to the rate of helicase functioning independently ( 18 bp/s ) .", "Similarly , with the stalled DNAP , the dTTP Km of the helicase is ~130 μM , which is similar to the dTTP Km in the absence of DNAP ( ~160 μM ) ( Figure 4B ) .", "Thus , stalling the DNAP functionally uncouples the helicase .", "When dVTPs concentrations are low ( 0 . 5 μM ) , the unwinding rate with the combined enzymes is similar to that of the isolated helicase ( Figure 4C ) .", "This indicates that when T7 DNAP is slow , the helicase is capable of moving faster and can outrun the DNAP .", "The functional data presented above indicate that the two enzymes help each other by increasing the base-capture efficiency .", "To understand the structural basis for the mutual stimulation of helicase and DNAP , we used 2-AP as a probe to monitor base pair melting .", "When 2-AP is base-paired ( Figure 5A ) , it has a low-fluorescence intensity , but when 2-AP:T base pair is melted and the 2-AP base is unstacked , the fluorescence increases ( Ward et al . , 1969 ) .", "Such changes in 2-AP fluorescence successfully monitor base unstacking and base pair separation in a variety of enzyme studies , including replication enzymes ( Reha-Krantz , 2009; Jose et al . , 2012 ) .", "However , there are no studies using this method to investigate base pair melting of downstream duplex DNA by replicative DNAPs or the helicase-DNAP complex .", "By systematically labeling the replication fork DNA with a single 2-AP probe at different positions near the fork junction ( Figure 5B ) , we are able to determine the base pair melting footprint of the individual and combined enzymes and deduce the precise positions of the helicase and DNAP at the replication fork . 10 . 7554/eLife . 06562 . 013Figure 5 . Base pair melting by isolated and combined T7 DNAP and T7 helicase using 2-aminopurine fluorescence changes .", "( A ) Structure of the 2-aminopurine ( 2-AP ) :T base pair .", "( B ) Structure of the replication fork substrate for 2-AP studies .", "The primer-end is N and subsequent base pairs are N + 1 , N + 2 , etc .", "The substrates contained a single 2-AP in the leading or the lagging strand .", "The primer-end is next to the junction base pair as shown or separated by gaps of one to three template strand nucleotides ( not shown ) .", "( C ) Crystal structure of T7 DNAP bound to primer-template DNA substrate ( PDB: 2AJQ ) .", "The N + 1 base ( blue ) is bound in the insertion site , and the N + 2 and N + 3 bases are bound in the template-binding channel .", "The figure was made using PyMOL ( Schrodinger , 2010 ) .", "( D ) Fluorescence intensities of 2-AP modified primer-template substrate without ( blue ) and with T7 DNAP ( green ) .", "The 2-AP probe is shown in red at the indicated positions .", "( E–H )", "Fluorescence intensities of 2-AP modified replication fork substrates with and without T7 DNAP and T7 helicase .", "The cartoons show the structure of fork DNA before and after binding of combined T7 DNAP and T7 helicase enzymes .", "Errors shown are standard deviations from average of 2–5 experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 06562 . 01310 . 7554/eLife . 06562 . 014Figure 5—figure supplement 1 . Determining optimal conditions for T7 DNAP and extent of influence of T7 DNAP on template bases in replication fork substrate .", "( A ) Effect of increasing ratio of the T7 DNAP processivity factor thioredoxin on the 2-AP fluorescence signal .", "A final ratio of 1:2 . 5 was used in all the experiments .", "( B ) Effect of DNAP concentration on fluorescence emission of 2-AP in the +1 lag 1 gap substrate .", "Final enzyme concentration of 200 nM was used in all experiments .", "( C ) Sample fluorescence intensity trace showing the effect of T7 DNAP binding to No gap replication fork DNA .", "The 2-AP intensity at 370 nm was used in all experiments .", "( D ) Fluorescence intensities of replication fork DNAs with 2-AP at various indicated positions in the leading strand with and without T7 DNAP . DOI: http://dx . doi . org/10 . 7554/eLife . 06562 . 01410 . 7554/eLife . 06562 . 015Figure 5—figure supplement 2 . Effect of T7 DNAP on 2-AP at the junction at N + 2 and N + 3 . ( A and B ) Fluorescence intensities of fork DNA with 2-AP part of the junction base pair at A . N + 2 and B . N + 3 positions in the lagging strand with and without T7 DNAP .", "The cartoons show the structure of fork DNA before and after binding of T7 DNAP . DOI: http://dx . doi . org/10 . 7554/eLife . 06562 . 01510 . 7554/eLife . 06562 . 016Figure 5—figure supplement 3 . Base pair melting by combined SSB—DNAP using 2-AP fluorescence change . Fluorescence intensities of 2-AP modified replication fork substrates with and without T7 DNAP and E . coli SSB .", "The cartoons show the structure of fork DNA before and after binding of combined T7 DNAP and E . coli SSB enzymes .", "Each experiment had 100 nM DNA , 200 nM DNAP , and 200 nM SSB ( tetramer ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 06562 . 01610 . 7554/eLife . 06562 . 017Figure 5—figure supplement 4 . Determining optimal conditions for T7 helicase .", "( A ) Effect of helicase concentration on fluorescence emission of 2-AP in the +1 lag 1 gap substrate .", "Final enzyme concentration of 200 nM was used in all experiments .", "( B ) Fold change on helicase binding to short 8 nt ssDNA substrates with probe at positions 2 , 3 , 4 , 5 , 6 , and 7 from the 5′ end .", "Error bars represent instrument fluctuations .", "( C ) Sample fluorescence intensity trace showing the effect of helicase binding to No gap replication fork DNA .", "The 2-AP intensity at 370 nm was used in all experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 06562 . 01710 . 7554/eLife . 06562 . 018Figure 5—figure supplement 5 . ( A–C ) Fluorescence intensities of fork DNA with 2-AP in the lagging strand at the fork junction and increasing distance between primer end and fork junction with ( red bars ) and without T7 helicase ( blue bars ) .", "The cartoons show the structure of fork DNA before and after binding of T7 helicase .", "The numbers above the bar refer to fold change of 2-AP intensity on protein binding to the DNA .", "( D ) Fluorescence intensities of 2-AP modified fork substrates with and without T7 Helicase and E . coli SSB .", "The cartoons show the structure of fork DNA before and after binding of combined T7 Helicase and E . coli SSB enzymes .", "Fork substrates with two single-stranded DNA overhangs were used in the experiments with SSB and helicase to provide a binding site for both enzymes on opposite strands . DOI: http://dx . doi . org/10 . 7554/eLife . 06562 . 018 The crystal structure of T7 DNAP ( Doublie et al . , 1998 ) shows interactions with two template-bases ( N + 1 and N + 2 ) immediately downstream of the primer-end at positon N and ∼90o bend between N + 1 and N + 2 template-bases ( Figure 5C ) .", "Thus , introducing a 2-AP at N + 1 results in a significant increase in fluorescence consistent with unstacking of the N + 1 base from bending of the template DNA ( Figure 5D ) .", "However , there is no increase in fluorescence of 2-AP at N + 2 and a decrease is observed at N + 3 ( Figure 5D ) , which is consistent with interactions of these template DNA-bases with the amino acids in the template-binding pocket of T7 DNAP ( Doublie et al . , 1998 ) .", "The change in 2-AP fluorescence at positions N + 4 and N + 5 is minimal upon DNAP binding , which indicates that T7 DNAP may not interact with these downstream positions .", "Thus , T7 DNAP influences only three template-bases immediately downstream from the primer-end .", "Experiments with 2-AP at N + 1 to N + 3 positions on the leading strand show that T7 DNAP makes similar interactions with the template-bases in replication fork substrates ( Figure 5—figure supplement 1D ) .", "To determine if T7 DNAP unwinds the duplex DNA downstream of the primer-end , we introduced 2-AP at various positions in the lagging strand in the replication fork substrates .", "The fluorescence intensity increases upon adding T7 DNAP when 2-AP is at the N + 1 , N + 2 , or N + 3 base pair , but not at N + 4 ( Figure 5E–H—Green bars ) .", "Similarly , when 2-AP is at N + 2 and is part of the fork junction , addition of T7 DNAP increases the fluorescence ( Figure 5—figure supplement 2A ) .", "However , when 2-AP is at N + 3 and is part of the fork junction , no increase in fluorescence is observed ( Figure 5—figure supplement 2B ) , which indicates that T7 DNAP does not melt the junction base pair three nucleotides downstream from the primer-end .", "The increase in 2-AP intensity at N + 3 in the internal position ( Figure 5G ) is due to N + 2 base unstacking and not from unstacking of the N + 3 base .", "Taken together , these results indicate that T7 DNAP binds to three template-bases downstream of primer-end and melts two base pairs .", "When 2-AP experiments with T7 DNAP were carried out with E . coli SSB , the fluorescence intensity changes were much larger , although SSB by itself did not increase the 2-AP fluorescence significantly ( Figure 5—figure supplement 3 ) .", "These results indicate that T7 DNAP on its own only partially melts the junction base pairs .", "Furthermore , these base pairs appear to be in dynamic equilibrium ( closed ↔ open ) , because SSB can shift the equilibrium towards the open state by simply binding to ssDNA .", "This provides direct proof that T7 DNAP can melt the junction base pair , but DNAP is not efficient at preventing base pair reannealing; thus , SSB stimulates the activity of DNAP by trapping the unwound bases .", "The 2-AP experiments with T7 helicase were carried out in the presence of dTMPPCP , the non-hydrolyzable dTTP analog , which does not support translocation or processive DNA unwinding , but is needed for hexamer formation and DNA binding ( Hingorani and Patel , 1993 ) .", "T7 helicase does not change the fluorescence intensity of 2-AP in ssDNA ( Figure 5—figure supplement 4B ) .", "However , T7 helicase increases the fluorescence of 2-AP at the fork junction , but not the second base pair from the junction , irrespective of the gap size between the primer end and fork junction ( Figure 5E–H—Red bars ) .", "Thus , unlike T7 DNAP that melts two base pairs upon binding to the replication fork substrate , T7 helicase melts only the junction base pair .", "However , T7 helicase melts the junction base pair even when the primer-end is separated from the fork junction by more than one nucleotide ( Figure 5—figure supplement 5A–C compare to Figure 5—figure supplement 2 ) .", "These results indicate that T7 helicase follows the fork junction and is not influenced by the position of the primer-end .", "Interestingly , SSB has no effect on the helicase catalyzed melting of the fork junction ( Figure 5—figure supplement 5D ) .", "This is consistent with the observation that SSB does not stimulate the unwinding rates of the helicase ( Donmez and Patel , 2008 ) .", "To investigate the effect of T7 DNAP and T7 helicase together on junction base pair melting , we measured 2-AP fluorescence after sequential addition of helicase and DNAP to the fork DNA in that order and the reverse order ( Figure 5E–H—light blue bars ) .", "The combined enzymes show a much larger increase in fluorescence intensity with 2-AP at N + 1 , N + 2 , or N + 3 base pairs , but not at N + 4 . As shown earlier with the DNAP , the increase in N + 3 is due to N + 2 base unstacking; therefore , the results indicate that the combined enzymes melt two base pairs downstream from the primer-end , just like T7 DNAP , but more efficiently .", "The 2-AP fluorescence intensity at steady state measures the equilibrium distribution of melted and annealed states of the junction base-pair .", "The small increase in fluorescence intensity with the isolated helicase and DNAP suggests that each enzyme shifts the equilibrium only moderately to the base-pair melted state .", "On the other hand , the striking increase in fluorescence intensity with the combined enzymes indicates that together the two enzymes shift the equilibrium strongly toward the base-pair melted state .", "Interestingly , the combined effect of helicase and DNAP on base pairs melting is greater than the sum , which indicates synergism in DNA melting .", "This synergism depends on the number of nucleotides between the primer-end and fork junction .", "Synergistic melting of the base pair is observed only when there is no gap or one nucleotide gap between the primer-end ( DNAP-binding site ) and fork junction ( helicase-binding site ) ( Figure 5E–G ) .", "Synergistic melting is not observed when there are two nucleotides between the primer-end and fork junction ( Figure 5H ) .", "The results also demonstrate that a replication fork with two ssDNA template-bases between the primer-end and fork junction can stably accommodate both enzymes of the T7 replisome .", "Therefore , this study defines the specific positions of helicase and DNAP at the replication fork junction with single-base resolution to create a structural model of the replisome ( Figure 6 ) that forms the basis for understanding how the helicase and DNAP mutually stimulate each other's activities as discussed below . 10 . 7554/eLife . 06562 . 019Figure 6 . Proposed model of DNA unwinding-synthesis by T7 replisome . The top cartoon of T7 replisome results after melting of the N + 2 base pair by helicase and DNAP .", "There are two unwound nucleotides ( N + 1 and N + 2 ) between the primer-end and fork junction at N + 3 . The N + 1 base of the leading strand is bound in the insertion site and serves as the templating nucleotide for the incoming dNTP , and the N + 2 is unstacked and bound in the template-binding channel of the DNAP .", "The complementary N + 1 and N + 2 nucleotides ( as well as N and N − 1 ) on the lagging strand are bound to individual subunits of the helicase hexamer , as shown .", "Stable binding to N + 2 base by the helicase triggers dTTP hydrolysis and products release at different subunits of the ring .", "The helicase subunit at the leading edge has weak interactions with the partially unwound N + 3 junction base ( dotted line ) , which gets stabilized after the next round of catalysis .", "When N is elongated by one nucleotide , the N + 2 moves into the insertion site after PPi release , and the helicase and DNAP cooperatively melt the N + 3 junction base pair , as shown in the bottom cartoon .", "This model explains the one-nucleotide step size where the combined enzymes translocate by one nucleotide for every dTTP hydrolyzed and nucleotide incorporated ( Pandey and Patel , 2014 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 06562 . 019" ], [ "When DNAP is in the post-translocated state , the N + 1 templating base ( blue ) is positioned in the polymerase active site ready to base pair with the incoming dNTP ( Figure 6 , upper cartoon ) .", "After the chemical step , the DNAP translocates downstream by one nucleotide to position the next templating-base N + 2 ( red ) in the active site ( Figure 6 , lower cartoon ) .", "This forward translocation step is coupled to unwinding of the N + 3 junction base pair ( green ) .", "Based on our results , we propose that the DNAP by itself is not efficient at preventing junction base pair reannealing , and this unfavorable equilibrium constant for DNA melting destabilizes the post-translocated state of DNAP and competes with incoming dNTP binding .", "When helicase is present at the fork junction , it helps both unwind and trap the junction bases .", "Thus , the associated helicase stimulates dNTP binding by stabilizing the post-translocated state of T7 DNAP , and the outcome is decrease in dNTPs Km .", "The helicase by itself is not efficient at unwinding the fork junction .", "However , the associated DNAP by providing an unwound base to the helicase at the fork junction facilitates the base-capture step and drives the reactions of dTTP binding-hydrolysis-product release around the helicase ring , and the outcome is an increase in the unwinding kcat .", "The combined binding energy of the two enzymes bound to opposite strands is sufficient to keep the unwound bases from reannealing , explaining the fast and GC-independent unwinding rates of helicase-DNAP .", "Interestingly , cooperative and enhanced efficiency of base pair melting is observed only when the helicase and DNAP are within one nucleotide distance from each other .", "In most cases , helicase is coupled physically to the DNAP , either directly as in the case of T7 replication system or indirectly through accessory proteins ( Kim et al . , 1996; Hamdan et al . , 2007; Gambus et al . , 2009; Sengupta et al . , 2013 ) .", "Some of these interactions aid in the assembly of the replisome ( Zhang et al . , 2011 ) and perhaps in proper positioning of the helicase with the DNAP in the replisome , but the consequences of breaking physical interactions on synergistic melting need to be investigated .", "One can imagine situations where flexible positioning is needed when one or the other enzyme pauses or stalls during leading strand synthesis .", "Our investigation of such situations reveals that when DNAP stalls or is the slower motor , the helicase becomes functionally uncoupled and outruns the DNAP by unwinding the replication fork at the unstimulated rates .", "Similar behaviors were observed in other replisome studies as well ( Byun et al . , 2005; McInerney and O'Donnell , 2007 ) .", "Whether the functionally uncoupled helicase remains physically coupled to the DNAP remains unknown .", "Interestingly , when the helicase slows down , the two enzymes remain functionally coupled as evident from the low dNTPs Km and that the DNAP does not outrun the helicase .", "In this case , the combined enzymes unwind the DNA with the stimulated rate of the helicase .", "Although SSB stimulates base pair melting by T7 DNAP , our studies find that the unwinding rates of T7 DNAP with SSB remain GC-sensitive at low-dNTPs concentrations .", "We propose that this is because SSB cannot trap the junction bases coordinately with DNA synthesis in the manner that T7 helicase does during leading strand synthesis .", "Similarly , it has been shown previously that SSB does not increase the unwinding rates of T7 helicase , which remain GC sensitive at all concentrations of dTTP ( Donmez and Patel , 2008 ) .", "These observations indicate that simply trapping the displaced strand by DNA binding is not sufficient , but coordination between the steps of junction base pair unwinding/trapping and synthesis is needed for rate acceleration .", "The replicative helicase is a central player in coordinating leading and lagging strand synthesis ( Pandey et al . , 2009 ) .", "The interdependency between helicase and DNAP assures that the DNA is not unwound in an uncoupled manner leading to disruption in the coordinated synthesis of the two strands .", "The mechanism of DNAP is conserved in all organisms where DNAPs elongate the primer in the 3′–5′ direction .", "On the other hand , replicative helicases of the prokaryotes and phages unwind DNA in the 5′–3′ direction , whereas those of eukaryotes unwind DNA in the opposite 3′–5′ direction .", "Our studies suggest that the leading edges of the T7 helicase and T7 DNAP are close together at the fork junction and this conformation is important for functional coupling of unwinding and synthesis reactions and preventing DNA reannealing .", "This model of the replication fork is likely to be generally applicable to replisomes of prokaryotes as most show functional coupling between helicase and DNAP ( Patel et al . , 2011 ) .", "In contrast to prokaryotic replisomes , the replicative helicase of eukaryotes and archaea binds to the same strand as the DNAP ( O'Donnell et al . , 2013 ) .", "In this case , both helicase and DNAP cannot be close to the fork junction , and there must be other mechanisms to functionally couple the two activities and prevent junction base pair reannealing .", "It is possible that although the MCM2-7 helicase encircles the leading strand , other subunits in the CMG ( Cdc45-MCM2-7-GINS ) complex may interact with the lagging strand and this could be a mechanism for preventing DNA reannealing at the fork junction ( Costa et al . , 2014 ) ." ], [ "Oligodeoxynucleotides labeled with fluorescein ( FAM ) on the 3′-end and BHQ1 on the 5′-end were purchased from Biosearch Technologies and RP-HPLC purified ( Novato , CA ) .", "Oligodeoxynucleotides labeled with fluorescein on the 5′-end , 2-AP labeled and unlabeled oligodeoxynucleotides were purchased from Integrated DNA Technologies ( Coralville , IA ) .", "These DNAs were gel-purified and extracted from the gel by electroelution ( Whatman Schleicher & Schuell ) .", "Replication fork substrates were created by heating the appropriate DNA strands to 95°C for 5 min and slowly cooling to room temperature .", "The DNA sequences are provided in Supplementary file 1 .", "T7 helicase ( gp4A′ ) , T7 gp5 exo- , and E . coli SSB were purified as described ( Lohman et al . , 1986; Patel et al . , 1991 , 1992; Kim et al . , 1992 ) .", "Thioredoxin was purchased from Sigma ( St . Louis , MO ) .", "The unwinding assays were carried out at 18°C in a stopped-flow instrument ( Kintek Corp , Austin , TX ) with excitation at 480 nm and fluorescence emission using a long pass 515 nm cut-off filter .", "Reaction buffer A for the helicase and helicase-DNAP experiments contained 50 mM Tris acetate , pH 7 . 6 , 50 mM potassium glutamate , 1 . 5 mM Ethylenediaminetetraacetic acid ( EDTA ) , 5 mM Dithiothreitol ( DTT ) , 10 mM total Mg ( II ) .", "For the helicase assays , a mixture of fork DNA , T7 helicase , dTTP , and EDTA from syringe A was mixed with Mg ( II ) , dVTPs ( dCTP , dGTP , and dATP ) , and dT90 trap from syringe B to initiate the reaction .", "The reactions with the combined enzymes were carried out similarly except syringe A contained T7 DNAP .", "For the T7 DNAP-unwinding assays , a mixture of fork DNA , T7 DNAP , and E . coli SSB ( pre-incubated at 18°C for 10 min ) from syringe A was mixed with Mg ( II ) and dNTPs from syringe B to initiate the reaction .", "Reaction buffer B for the DNAP reactions contained 50 mM Tris Cl , pH 7 . 6 , 40 mM NaCl , 1 . 5 mM EDTA , 5 mM DTT , 8 . 1–8 . 5 mM free Mg ( II ) ( MgCl2 ) .", "Reaction buffer B was used in the T7 DNAP-unwinding assays as SSB was observed to precipitate in buffer A . The final concentrations of enzymes and DNA were 10 nM fork DNA , 20 nM T7 helicase hexamer , 20 nM T7 DNAP , 200 nM E . coli SSB , and 2 μM dT90 trap DNA .", "The equilibrium fluorescence experiments were carried out on FluoroMax 4 ( Horiba Join Yvon Inc ) .", "The sample was excited at 315 nm ( 2 mm slit width ) , and emission was measured at 370 nm ( 6 mm slit width ) .", "The buffer contained 50 mM Tris Cl pH 7 . 6 , 40 mM NaCl , 10 mM MgCl2 , 5 mM DTT .", "The observed fluorescence was corrected for buffer and protein bound to unlabeled replication fork substrate .", "The proteins absorb minimally at the emission wavelength , and hence , the inner filter effect was negligible .", "The experiments were carried out with 100 nM DNA , 200 nM T7 helicase ( Figure 5—figure supplements 1B , 2A ) , 200 nM T7 DNAP/thioredoxin ( 2 . 5 times excess thioredoxin ) ( Figure 5—figure supplement 1A ) , and 10 μM dTMP-PCP at 25°C .", "A sample trace showing the effect of DNAP and helicase binding to a 2-AP DNA substrate is shown in Figure 5—figure supplements 1C , 2C .", "The DNA unwinding kinetics were fit to the n-step model ( Ali and Lohman , 1997 ) using gfit and model [unwinding . m] in MATLAB with Optimization toolbox ( The MathWorks , Inc . , Natick , MA ) ( Levin et al . , 2009 ) .", "Unwinding is modeled as a multistep process with equal step-size ( s ) and rate constant ( ki ) that are estimated from fittings as described previously ( Pandey et al . , 2010 ) .", "More information about the fitting is provided in the Appendix—Methods section .", "The average unwinding rates were plotted against dNTP concentration and fit to the hyperbolic equation to obtain kcat and Km values .", "( 1 ) unwinding rate= kcat∗[dNTP]Km+[dNTP] ." ] ]

[ "Leading strand DNA synthesis requires functional coupling between replicative helicase and DNA polymerase ( DNAP ) enzymes , but the structural and mechanistic basis of coupling is poorly understood .", "This study defines the precise positions of T7 helicase and T7 DNAP at the replication fork junction with single-base resolution to create a structural model that explains the mutual stimulation of activities .", "Our 2-aminopurine studies show that helicase and polymerase both participate in DNA melting , but each enzyme melts the junction base pair partially .", "When combined , the junction base pair is melted cooperatively provided the helicase is located one nucleotide ahead of the primer-end .", "The synergistic shift in equilibrium of junction base pair melting by combined enzymes explains the cooperativity , wherein helicase stimulates the polymerase by promoting dNTP binding ( decreasing dNTP Km ) , polymerase stimulates the helicase by increasing the unwinding rate-constant ( kcat ) , consequently the combined enzymes unwind DNA with kinetic parameters resembling enzymes translocating on single-stranded DNA ." ]

[ "DNA replication is the process whereby a molecule of DNA is copied to form two identical molecules .", "First , an enzyme called a DNA helicase separates the two strands of the DNA double helix .", "This forms a structure called a replication fork that has two exposed single strands .", "Other enzymes called DNA polymerases then use each strand as a template to build a new matching DNA strand .", "DNA polymerases build the new DNA strands by joining together smaller molecules called nucleotides .", "One of the new DNA strands—called the ‘leading strand’—is built continuously , while the other—the ‘lagging strand’—is made as a series of short fragments that are later joined together .", "Building the leading strand requires the helicase and DNA polymerase to work closely together .", "However , it was not clear how these two enzymes coordinate their activity .", "Now , Nandakumar et al . have studied the helicase and DNA polymerase from a virus that infects bacteria and have pinpointed the exact positions of the enzymes at a replication fork .", "The experiments revealed that both the polymerase and helicase contribute to the separating of the DNA strands , and that this process is most efficient when the helicase is only a single nucleotide ahead of the polymerase .", "Further experiments showed that the helicase stimulates the polymerase by helping it to bind to nucleotides , and that the polymerase stimulates the helicase by helping it to separate the DNA strands at a faster rate .", "The next challenge is to investigate the molecular setup that allows the helicase and polymerase to increase each other's activities ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "neuroscience" ]

[ [ "Infertility is a common clinical problem affecting 15% of couples; ovulatory disorders account for 25% of this total ( Macaluso et al . , 2010 ) .", "The hypothalamic-pituitary-gonadal axis controls reproduction and malfunction of this axis can cause ovulatory dysfunction and/or other disturbances of the reproductive cycle ( Helm et al . , 2009; Plant and Zeleznik , 2015 ) .", "Gonadotropin-releasing hormone ( GnRH ) neurons form the final common pathway for central neural regulation of reproduction .", "GnRH stimulates the pituitary to secrete follicle-stimulating hormone and luteinizing hormone ( LH ) , which regulate gonadal steroid and gamete production .", "Estradiol , via estrogen receptor alpha ( ERα ) , plays crucial roles in both homeostatic negative feedback and positive feedback action on GnRH/LH release in females ( Döcke and Dörner , 1965; Moenter et al . , 1990; Lubahn et al . , 1993; Krege et al . , 1998; Wintermantel et al . , 2006; Christian et al . , 2008; Glanowska et al . , 2012; Cheong et al . , 2015 ) .", "Low estradiol levels suppress pulsatile GnRH/LH release , whereas sustained elevations in estradiol during the late follicular phase of the cycle cause a switch of estradiol feedback action from negative to positive , inducing prolonged GnRH/LH surges , which ultimately triggers ovulation ( Christian and Moenter , 2010 ) .", "As GnRH neurons typically do not express detectable ERα ( Hrabovszky et al . , 2001 ) , estradiol feedback is likely transmitted to GnRH neurons by ERα-expressing afferents .", "Kisspeptin neurons in the arcuate and anteroventral periventricular ( AVPV ) regions are estradiolsensitive GnRH afferents that are postulated to mediate estradiol negative and positive feedback , respectively ( Oakley et al . , 2009; Lehman et al . , 2010 ) .", "Kisspeptin potently stimulates GnRH neurons and Kiss1 mRNA is differentially regulated in these nuclei by estradiol ( Han et al . , 2005; Messager et al . , 2005; Smith et al . , 2005; Pielecka-Fortuna et al . , 2008; Lehman et al . , 2010; Kumar et al . , 2015; Yip et al . , 2015 ) .", "ERα in kisspeptin cells is critical for estradiol negative and positive feedback , as kisspeptin-specific ERα knockout ( KERKO ) mice exhibit higher frequency LH pulses and fail to exhibit estradiol-induced LH surges ( Mayer et al . , 2010; Dubois et al . , 2015; Greenwald-Yarnell et al . , 2016; Wang et al . , 2018 ) .", "Although informative , the KERKO model has several caveats that limit interpretation .", "First , ERα is deleted as soon as Kiss1 is expressed , before birth in arcuate kisspeptin neurons ( also called KNDy neurons for coexpression of kisspeptin , neurokinin B and dynorphin ) and before puberty in AVPV kisspeptin neurons ( Semaan et al . , 2010; Kumar et al . , 2014 ) .", "This may cause developmental changes in these cells and/or their networks .", "Second , ERα is deleted from all kisspeptin cells , thus making it impossible to assess independently the role of AVPV and arcuate kisspeptin neurons .", "Combining CRISPR-Cas9 with targeted viral vector injection allows deletion of ERα in a nucleus-specific and temporally-controlled manner to address the above caveats ( Swiech et al . , 2015 ) .", "We designed Cre-dependent AAV vectors that carry single guide RNAs ( sgRNAs ) that target Esr1 ( encoding ERα ) or lacZ and delivered these vectors to the AVPV or arcuate of adult female mice that express Cas9 in kisspeptin cells .", "We then compared the reproductive phenotypes as well as kisspeptin neuronal physiology in AAV-Esr1 vs AAV-lacZ targeted mice and KERKO mice ." ], [ "We first used extracellular recordings to monitor the spontaneous firing rate of YFP-identified AVPV kisspeptin neurons in coronal brain slices from ovary-intact control and KERKO mice .", "As the persistent cornified vaginal cytology of KERKO mice is similar to that observed during estrus ( Greenwald-Yarnell et al . , 2016 ) , we used mice in the estrous stage of the reproductive cycle as controls .", "The firing frequency of AVPV kisspeptin neurons was lower in ovary-intact KERKO mice compared to controls ( Figure 1a , b , two-way ANOVA/Holm-Sidak , p=0 . 0001 ) .", "To test if the firing rate of AVPV kisspeptin neurons in KERKO mice responds to circulating estradiol , we repeated this study in ovariectomized ( OVX ) mice and OVX mice with an estradiol implant producing constant physiologic levels ( OVX + E ) ( Christian et al . , 2005 ) .", "OVX reduced and estradiol treatment increased firing rate in cells from control , but not KERKO , mice ( Figure 1a , b two-way ANOVA/Holm-Sidak , control intact vs OVX p=0 . 009 , intact vs OVX + E , p=0 . 02 , OVX vs OVX + E , p<0 . 0001 ) .", "As a result of this difference , the firing frequency is higher in cells from OVX + E control than OVX + E KERKO mice ( p<0 . 0001 ) .", "Statistical test parameters for all figures are in Tables 1 and", "2 . We next recorded the whole-cell firing signatures of neurons in these six groups in response to current injection .", "AVPV kisspeptin neurons in control mice mice exhibit a greater number of depolarization-induced bursts ( DIB ) and rebound bursts when estradiol is elevated , confirming previous observations ( Wang et al . , 2016 ) ( Figure 1c , d , e , Chi-square , DIB , p=0 . 02; rebound , p=0 . 02; Fisher’s exact post hoc test , DIB , OVX vs OVX +E , p=0 . 008 , rebound OVX vs OVX +E p=0 . 03 , for other paired comparisons , p>0 . 2 ) .", "In KERKO mice , these two types of bursts were rare ( <25% of cells ) in all steroid conditions tested and were not regulated by estradiol ( Figure 1c , d , e , Chi-square , DIB , p=0 . 4 , rebound , p=0 . 3 ) .", "We also compared the action potential output of these cells in response to current injection ( 0–50 pA , 10 pA increments , 500 ms ) .", "Cells from ovary-intact KERKO mice generated fewer action potentials compared to controls .", "Action potential generation as a function of current injection was similar in cells from OVX control and OVX KERKO mice but was increased by estradiol only in control mice ( Figure 1f , two-way repeated-measures ANOVA/Holm-Sidak , intact , 20 pA , p=0 . 03 , 30 pA , p=0 . 06; OVX +E , 20 pA to 50 pA , p≤0 . 04 ) .", "Reduced action potential firing of AVPV kisspeptin neurons from KERKO mice may be attributable at least in part to decreased input resistance compared to controls ( Figure 1—figure supplement 1 , two-way ANOVA/Holm-Sidak control vs KERKO , intact , p=0 . 006 , OVX , p=0 . 7 , OVX +E p=0 . 02 ) .", "As both depolarization-induced bursts and rebound bursts are sensitive to NiCl ( 100 µM ) ( Lee et al . , 1999 ) at levels that fairly specifically block T-type calcium channels , we measured T-type ( IT ) current density and voltage dependence .", "IT current density was decreased in AVPV kisspeptin cells from gonad-intact KERKO mice compared to controls ( Figure 2a , b , two-way repeated-measures ANOVA/Holm-Sidak , −50 mV , p=0 . 003; −40 mV , p=0 . 002; −30 mV , p=0 . 003 ) .", "The voltage dependence of activation was not different between groups , but the voltage dependence of inactivation was depolarized in cells from KERKO mice ( Figure 2c , control vs KERKO , two-tailed unpaired Student’s t-test , V1/2 activation −52 . 2 ± 1 . 6 vs −48 . 6 ± 1 . 4 mV , p>0 . 1; slope 5 . 5 ± 0 . 6 vs 5 . 5 ± 0 . 7 , p>0 . 1; V1/2 inactivation −74 . 8 ± 4 . 1 vs −61 . 9 ± 3 . 1 mV , p=0 . 03; slope −3 . 1 ± 0 . 5 vs −4 . 2 ± 0 . 3 , p=0 . 1 ) .", "A caveat of studying the role of ERα in AVPV kisspeptin neurons using KERKO mice is that the deletion of ERα ( encoded by Esr1 ) using cre recombinase under the control of the kisspeptin promoter is neither time- nor location-specific .", "We utilized the CRIPSR-Cas9 approach to achieve temporal and spatial control of Esr1 gene knockdown .", "Two sgRNAs were designed that target exon1 of Esr1 based on software prediction ( Ran et al . , 2013 ) ; sites predicted by FengZhang’s guide design software ( http://crispr . mit . edu ) as possible off-target regions for binding of these guides are listed in Table", "3 . The efficiency of each guide was tested in vitro in C2C12 mouse myoblast cells ( Milanesi et al . , 2008 ) .", "The sgRNAs that target Esr1 and a sgRNA that targets lacZ as a control were subcloned into the lentiCRISPRv2 plasmid ( Sanjana et al . , 2014 ) , from which Cas9 and the sgRNA are expressed after transfection of C2C12 cells .", "Puromycin was used to select construct-expressing cells .", "After a ~ 4-week selection period , DNA was harvested and the Esr1 region sequenced .", "Cells expressing either of the sgRNAs targeting Esr1 , but not lacZ , exhibited a peak-on-peak sequencing pattern , indicating disruption of the gene ( Figure 3a ) .", "As these in vitro experiments suggested these sgRNAs were able to mutate Esr1 , we designed Cre-dependent AAV vectors to express each sgRNA and mCherry ( to indicate infected cells ) under control of the U6 promoter ( Figure 3b ) .", "The AAV vector was bilaterally stereotaxically injected into the AVPV region of adult female mice that express Cas9 and GFP under control of the kisspeptin promoter ( Kiss1-Cre; Cas9 loxp-stop-Gfp , Figure 3—figure supplement 1 ) ; these groups are referred to as AVPV-AAV-Esr1 or AVPV-AAV-lacZ .", "Only one guide was injected per animal to allow comparison of phenotypes when different areas of Esr1 were targeted .", "The ERα knockdown efficiency of the two sgRNAs target Esr1 was comparable .", "The infection rate for AVPV-AAV-Esr1 was 81 ± 4% ( Figure 3d , Esr1-guide 1 [g1] 82 ± 2% , n = 3; Esr1-guide 2 [g2] 81 ± 8% , n = 3 ) and only 28 ± 1% of AVPV kisspeptin cells expressed ERα post infection ( Figure 3d , n = 3 , Esr1-guide1 27 ± 0 . 4%; n = 3 , Esr1-guide2 28 ± 2% ) .", "In mice that received AVPV-AAV-lacZ ( n = 3 ) , the infection rate was comparable at 82 ± 2% , but there was no disruption of ERα; 72 ± 2% of AVPV kisspeptin neurons expressed ERα , which is similar to control mice ( Kumar et al . , 2015 ) .", "Of note , the ERα antibody used recognizes the C-terminus , suggesting a lack of rare splice variants that were generated at low levels in initial ERKO mice ( Couse et al . , 1995 ) .", "We monitored the reproductive cycles of the mice injected with AAV-sgRNAs in the AVPV 12 days before and for up to eight weeks following surgery .", "Neither AVPV-AAV-Esr1 guide ( tested independently ) nor the AVPV-AAV-lacZ disrupted reproductive cyclicity ( Figure 3e ) , even in mice with a high rate of bilateral infection ( ~80% ) .", "These mice entered proestrus at the same frequency in the last four weeks compared to the first four weeks ( two weeks pre-surgery plus the first two weeks post-surgery , two-way repeated-measures ANOVA/Holm-Sidak , before vs after; g1 , n = 3 , 1 . 3 ± 0 . 1 vs 1 . 6 ± 0 . 1; g2 , n = 4 , 1 . 2 ± 0 . 1 1 . 4 ± 0 . 1; lacZ n = 4 , 1 . 3 ± 0 . 2 vs 1 . 3±0 . 1 , p>0 . 1 for each paired comparison ) .", "To test for the occurrence of estradiol-positive feedback , we monitored both proestrous ( preovulatory ) and estradiol-induced LH surges in these mice .", "Surge data were similar for guide 1 and guide 2 and data from both guides were combined for group comparisons .", "Both proestrous and estradiol-induced LH surges were blunted after ERα knockdown ( Figure 3f , g , two-way repeated-measures ANOVA/Holm-Sidak; f , lacZ , 3pm vs 5pm , p=0 . 04; lacZ vs Esr1 , 4pm , p=0 . 006 , 5pm , p<0 . 0001 , h , lacZ AM vs PM , p<0 . 0001 ) .", "There were fewer corpra lutea ( CL ) in mice with Esr1 guides targeted to the AVPV ( p<0 . 05 , guides 1 and 2 combined n = 6 , 5 . 2 ± 2 . 1 CL/mouse , vs lacZ guide n = 5 , 10 . 8 ± 0 . 4 CL/mouse , two-tailed paired Student’s t test with Welch’s correction , t = 2 . 598 , df = 5 . 305 ) .", "Of note , variation was high in the Esr1 mice , with two looking similar to controls , two having fewer CL and two not having any CL .", "This suggests ovulation is disrupted in a substantial subpopulation of these mice but can proceed with the blunted LH surge in some animals .", "To test if knockdown of ERα in adult AVPV kisspeptin neurons alters their intrinsic excitability , we recorded firing signatures of infected and uninfected cells in brain slices from AAV injected OVX + E mice .", "We again observed no difference between AVPV-AAV-Esr1 g1 vs g2 and combined these data .", "Some cells were loaded with neurobiotin during recording for identification and ERα protein detected post hoc with immunofluorescence ( Figure 4a , c , and IF post hoc portions of Figure 4e–j ) .", "Cells not infected with AVPV-AAV-Esr1 and cells infected with either AVPV-AAV-Esr1 guide but in which ERα protein was detected exhibited similar firing signatures in terms of DIB and rebound bursts ( Figure 4b , e , f ) .", "In contrast , cells infected by AVPV-AAV-Esr1 that had undetectable ERα protein had reduced burst firing compared to AVPV-AAV-lacZ or uninfected groups ( Figure 4e , f , Chi-square , DIB , p=0 . 008 , rebound bursts , p=0 . 0008; Fisher’s exact post hoc test , DIB , Esr1 vs lacZ , or vs uninfected , p≤0 . 03; rebound , Esr1 vs lacZ , or vs uninfected p≤0 . 04; for other paired comparisons , p>0 . 5 ) .", "The firing signature of AAV-Esr1-infected cells with successful deletion of ERα was comparable to cells from KERKO mice ( Chi-square , p>0 . 9 for both DIB and rebound bursts ) .", "Cells that lost detectable ERα after AVPV-AAV-Esr1 infection also produced fewer action potentials with current injection than cells infected with AAV-lacZ ( Figure 4d left and center , two-way repeated-measures ANOVA/Holm-Sidak , Esr1 vs lacZ , 20 pA , p=0 . 08; 30 to 50 pA p≤0 . 02 ) .", "This difference is not attributable to passive properties ( Figure 1—figure supplement 1a , b ) .", "The relationship between current injection and number of action potentials fired ( input-output curve ) in cells from KERKO and in AVPV-AAV-Esr1 knockdown mice was only different at the highest level of current injected , with AVPV-AAV-Esr1-infected cells being less excitable ( Figure 4d , right , two-way repeated-measures ANOVA/Holm-Sidak , 50 pA , p=0 . 01 ) , despite no change in input resistance ( Figure 1—figure supplement 1a , b KERKO vs AAV , p=0 . 08 ) .", "Action potential properties from AVPV-AAV-Esr1 knockdown cells with AVPV-AAV-lacZ control also differed .", "Specifically , loss of ERα led to decreased action potential rate of rise , a trend toward prolonged full-width half-maximum ( FWHM ) , and hyperpolarized afterhyperpolarization potential ( AHP ) ( Figure 4–j , two-tailed unpaired Student’s t-test , h , p=0 . 02 , j , p=0 . 0002; i , Mann-Whitney U-test , p=0 . 06 ) .", "In parallel , we performed whole-cell patch-clamp recording with single-cell PCR post hoc identification of Esr1 mRNA on a separate set of cells ( AVPV-AAV-Esr1 , 10 cells from four mice; AVPV-AAV-lacZ , 9 cells from three mice; primers are in Table 4 ) .", "A similar decrease in burst firing and action potential input-output curve was observed in Esr1 mRNA negative cells as was observed in cells verified to have undetectable ERα protein by immunofluorescence ( Figure 4e , f , Chi-square , DIB , p=0 . 04 , rebound p=0 . 03; Fisher’s exact post hoc test , DIB , Esr1 vs lacZ p=0 . 03 . Esr1 vs uninfected , p=0 . 07; rebound , Esr1 vs lacZ p=0 . 02; for other paired comparisons , p>0 . 2 ) .", "Absence of Esr1 mRNA expression was again associated with decreased number of action potentials in response to current injection ( Figure 4d middle , Esr1 vs lacZ , p<0 . 002 for 20 to 50 pA steps ) .", "Absence of Esr1 mRNA , similar to loss of ERα protein , led to decreased action potential rate of rise , prolonged FWHM , and AHP ( Figure 4g–j , two-tailed unpaired Student’s t-test , h , p=0 . 02; i , p=0 . 006; j , p=0 . 02 ) .", "Single-cell PCR analysis also indicates that a lower percent of AVPV-AAV-Esr1 knockdown cells express Kiss1 and a trend to increase in Esr2 mRNA ( AVPV-AAV-Esr1 , 23 cells from four mice; AVPV-AAV-lacZ , 16 cells from three mice; Figure 4—figure supplement 1 ) .", "Interestingly , expression of the mRNA for progesterone receptor ( Pgr ) did not differ between groups ( Figure 4—figure supplement 1 ) , suggesting the estradiol-dependence of this gene may be paracrine regulated in the brain as in other tissues ( Hilton et al . , 2015 ) .", "We also examined gene expression for several ion channels , but none showed any changes or patterns of expression among groups ( Figure 4—figure supplement 1 ) .", "To examine the role of estradiol feedback on arcuate kisspeptin neurons , we delivered the AAV-sgRNAs bilaterally to the arcuate region to knockdown ERα in these cells ( Figure 5—figure supplement 1 ) ; these groups are referred to as Arc-AAV-Esr1 or Arc-AAV-lacZ .", "The infection rate for Arc-AAV-Esr1 was 92 ± 3% ( Figure 5—figure supplement 1 , n = 3 Esr1-guide1 96 ± 2%; n = 3 Esr1-guide2 86 ± 2% ) and only 34 ± 3% of KNDy neurons expressed ERα post infection ( Figure 5a n = 3 Esr1-g1 , 38 ± 0 . 3%; n = 3 Esr1-g2 , 30 ± 5% ) .", "In mice that received Arc-AAV-lacZ , the infection rate was comparable ( Figure 5a n = 3 , 94 ± 3% ) , and 92 ± 1% of KNDy neurons expressed ERα , similar to control mice ( Kumar et al . , 2015 ) .", "Reproductive cycles were monitored for 12 days before and for up to eight weeks following surgery .", "In contrast to mice with Arc-AAV-Esr1 targeted to the AVPV region , mice with the same virus targeted to the arcuate began exhibiting disrupted cyclicity three to four weeks post surgery ( Figure 5b ) .", "These mice entered proestrus less frequently after surgery than before ( two weeks pre-surgery plus the first two weeks post-surgery , Figure 5d , two-way repeated-measures ANOVA/Holm-Sidak , g1 , p=0 . 002 , g2 , p=0 . 03 ) .", "There was no difference in LH pulse frequency measured on the day of estrus or mean levels between Arc-AAV-Esr1 vs Arc-AAV-lacZ injected mice on estrus ( Figure 5c , e , f ) .", "Notably , LH response to kisspeptin and to GnRH was reduced in Arc-AAV-Esr1 mice ( Figure 5g , two-way repeated measures ANOVA/Holm-Sidak , lacZ , control vs kisspeptin or GnRH , p<0 . 001; Esr1 vs lacZ for kisspeptin and GnRH both , p≤0 . 002 ) .", "Arcuate kisspeptin neurons are postulated to form an interconnected network that is steroid sensitive and utilizes glutamatergic transmission at least in part for intranetwork communication ( Qiu et al . , 2016 ) .", "We thus hypothesized that loss of ERα specifically from arcuate kisspeptin neurons would increase their spontaneous firing rate and increase glutamatergic transmission to these cells , similar to what is observed in these cells in KERKO mice ( Wang et al . , 2018 ) .", "As Arc-AAV-Esr1 knockdown mice spend the most time in estrus , similar to KERKO mice , we used estrus as the reproductive stage to examine the short-term ( ~10 min ) firing frequency of these neurons and AMPA-mediated excitatory glutamatergic postsynaptic currents ( EPSCs ) .", "The firing frequency of Arc-AAV-Esr1 infected cells was not different from Arc-AAV-lacZ infected cells ( Figure 6a , b , Mann-Whitney U-test , p=0 . 14 ) even though there tends to be more cells firing at >1 Hz in the Arc-AAV-Esr1 group compared to the Arc-AAV-lacZ group ( Figure 6c , Fisher’s exact test , Esr1 vs lacZ , p=0 . 07 ) .", "In contrast , the frequency and amplitude of glutamatergic EPSCs in arcuate kisspeptin cells in Arc-AAV-Esr1 infected mice was greater than in the Arc-AAV-lacZ group ( Figure 6d , e , f , two-tailed unpaired Student’s t-test , frequency p=0 . 0007 , amplitude p=0 . 014 ) ." ], [ "This study examined the roles of two hypothalamic kisspeptin neuronal populations in mediating estradiol feedback from cellular , molecular and whole-body physiology perspectives .", "We utilized both conventional kisspeptin-specific ERα knockout mice ( KERKO ) and CRISPR-Cas9-based viral vector-mediated knockdown of Esr1 .", "The latter approach allows both temporal control and nucleus-specific manipulations to distinguish the role of ERα within each population in negative and positive feedback regulation of LH release and neurobiological properties ( Figure 7 ) .", "AVPV kisspeptin neurons are postulated to convey estradiol positive feedback signals to generate the GnRH surge .", "Consistent with this postulate , these neurons are more excitable during positive feedback and also receive increased glutamatergic transmission ( Piet et al . , 2013; Zhang et al . , 2013; Wang et al . , 2016; Wang et al . , 2018 ) .", "AVPV kisspeptin cells in both KERKO and AVPV-AAV-Esr1 models are less excitable compared to controls , firing fewer bursts and single action potentials in response to the same current injection .", "Results from the AVPV-AAV-Esr1 model support and extend data from KERKO mice and provide evidence towards accepting the hypothesis that the role of ERα in shifting excitability is activational , independent of its role in the development of these cells ( Mayer et al . , 2010 ) .", "Kisspeptin expression in AVPV cells is estradiol activated and fewer cells expressing Kiss1 mRNA are detected in this region in KERKO mice ( Greenwald-Yarnell et al . , 2016 ) .", "In AVPV-AAV-Esr1 mice with adult knockdown , we also observed fewer cells express Kiss1 mRNA compared to AVPV-AAV-lacZ mice , further supporting an activational role for estradiol in the adult physiology of these cells .", "The inability to sense estradiol through ERα in AVPV kisspeptin neurons may reduce production and release of kisspeptin and ultimately impair the downstream GnRH/LH surge .", "This could explain the blunted LH surges in AVPV-AAV-Esr1 injected mice .", "Esr1 knockdown in the AVPV , however , did not alter reproductive cyclicity monitored by changes in vaginal cytology .", "Vaginal cytology reflects circulating steroids , in particular estradiol .", "Of note , only some of these mice had evidence of typical ovulation monitored by the number of corpra lutea .", "It is possible that sufficient estradiol is produced during these cycles to induce vaginal cytology , but not to trigger an LH surge .", "It is important to point out , however , that estradiol-induced LH surges , in which an established dose of estradiol was provided to the mouse , were also blunted in AVPV-AAV-Esr1 mice .", "This latter observation suggests that inappropriate response of the neuroendocrine system to estradiol is , at least in part , responsible for the blunting of the LH surge .", "With regard to the continuation of estrous cycles in the AVPV-AAV-Esr1 mice , typical function of the remaining ERα-positive AVPV kisspeptin neurons may be sufficient to drive maintain cyclicity .", "Alternatively , cyclicity and the associated changes in sex steroids may be controlled by other cells that express ERα .", "Of interest , stress or neuronal androgen receptor KO can similarly disrupt the LH surge without a change in estrous cyclicity ( Wagenmaker and Moenter , 2017; Walters et al . , 2018 ) .", "In support of a non-AVPV kisspeptin neuronal population being a primary driver of estrous cyclicity , several reproductive phenotypes of KERKO and AVPV-targeted ERα knockdown mice are different .", "KERKO mice tend to exhibit prolonged vaginal cornification and enlarged uteri , neither of which were observed in mice in which AAV-Esr1 infection was targeted to the AVPV ( Figure 1—figure supplement 1c ) .", "In contrast , prolonged estrus and enlarged uteri were observed in mice in which Arc-AAV-Esr1 infection was targeted to arcuate kisspeptin neurons .", "These latter neurons have been postulated to play a role in generating episodic GnRH output .", "Changes in episodic GnRH frequency drive gonadotropins and thus follicle development and steroidogenesis , including the estradiol rise , which triggers positive feedback and changes in vaginal cytology .", "Long-term firing output of arcuate kisspeptin neurons in brain slices is episodic and steroid modulated ( Vanacker et al . , 2017 ) , and activation of these cells in vivo generates a pulse of LH release ( Clarkson et al . , 2017 ) .", "Further evidence comes from Tac2-specific ERα KO mice , in which ERα is primarily deleted from the arcuate , not the AVPV , kisspeptin population .", "These mice also exhibit prolonged vaginal cornification ( Greenwald-Yarnell et al . , 2016 ) .", "We thus hypothesize that ERα in arcuate kisspeptin neurons contributes to maintaining pulsatile LH release and mediates central estradiol negative feedback .", "Consistent with this postulate , partial ( ~65% ) adult knockdown of ERα in these cells altered the reproductive cycle .", "As KERKO mice exhibit increased LH-pulse frequency , we were initially surprised we did not observe differences in pulse frequency or mean LH levels in mice receiving Arc-AAV-Esr1 .", "This may be attributable to single housing conditions in the present experiment , which may make mice prone to stress despite more than four weeks of handling before sampling .", "It is also possible that the pulse frequency during diestrus differs between Arc-AAV-Esr1 and Arc-AAV-lacZ mice .", "Despite this lack of statistical difference in LH-pulse frequency , ERα knockdown mice had a markedly reduced response to IP injection of both kisspeptin and GnRH , similar to KERKO mice ( Wang et al . , 2018 ) .", "This suggests loss of ERα function in arcuate kisspeptin neurons may disrupt GnRH neuronal response to kisspeptin and/or the pituitary response to GnRH .", "This could arise from a disruption of negative feedback leading to overstimulation and thus desensitization of the hypothalamo-pituitary-gonadal axis or blunting of the response to administered neuropeptides .", "Dissection of the electrophysiological properties of arcuate kisspeptin neurons revealed that glutamatergic transmission to these neurons was elevated when ERα is knocked down .", "This indicates the connectivity of these cells remains plastic even after puberty .", "The observation that targeted reduction of ERα in arcuate kisspeptin neurons increases glutamatergic transmission further suggests interconnections among these cells provide many of their glutamatergic inputs .", "Given this , it is intriguing that the short-term firing rate of these cells was not increased , although there was a strong trend toward a greater percent of higher frequency cells .", "The lack of change in mean firing rate may reflect the partial deletion of ERα in this population , with lower firing rate being preserved in cells with ERα , and elevated EPSC frequency arising at least in part from the high firing cells .", "It is also possible that long-term firing patterns of these Arc-AAV-Esr1 infected arcuate kisspeptin neurons , which may be associated with episodic neuroendocrine activity , are disrupted .", "These data support the idea that glutamatergic inputs to arcuate kisspeptin neurons play an important role on maintaining normal reproductive function .", "Although the CRISPR-Cas9-based knockdown approach allows spatial and temporal control , it too has caveats .", "For example , sgRNAs may have off-target actions on other regions of the genome beyond the sites predicted by the design software ( Anderson et al . , 2018 ) .", "To address this , we independently tested two sgRNAs that target Esr1 to address the possible off-target effects among groups .", "We did not observe any differences between Esr1 guide1 and guide2 groups .", "This suggests the phenotypes observed are primarily attributable to the deletion of ERα .", "Because of the nature of the nonhomologous end joining repair machinery activated after CRISPR-Cas9-initiated cuts , Esr1 gene editing in each cell varies .", "It is difficult to assess each individual neuron to test if mutations at other genes are potentially involved in changes of biophysical properties .", "Despite these variables , in the present study the systemic and cellular phenotypes in Esr1 guide1 vs guide2 infected mice were quite consistent .", "In conclusion , utilizing CRISPR-Cas9 AAV , we were able to successfully knockdown ERα in specific populations of kisspeptin neurons in adult female mice .", "Knockdown in each population recapitulated part of the KERKO model and furthers our understanding the role ERα in that population in regulating estradiol feedback ." ], [ "The University of Michigan Institutional Animal Care and Use Committee approved all procedures .", "Adult female mice ( 60–150 days ) were used .", "Mice were provided with water and Harlan 2916 chow ( VetOne ) ad libitum and were held on a 14L:10D light cycle ( lights on 0400 Eastern Standard Time ) .", "To delete ERα specifically from all kisspeptin cells ( Wang et al . , 2018 ) , mice with the Cre recombinase gene knocked-in after the Kiss1 promoter ( Kiss1-ires-Cre mice , Cravo et al . , 2011 ) were crossed with mice with a floxed Esr1 gene , which encodes ERα ( ERα floxed mice ) ( Greenwald-Yarnell et al . , 2016 ) .", "The expression of Cre recombinase mediates deletion of ERα in kisspeptin cells ( KERKO mice ) .", "To visualize kisspeptin neurons for recording , mice heterozygous for both Kiss-Cre and floxed ERα were crossed with Cre-inducible YFP mice .", "Crossing mice heterozygous for all three alleles yielded litters that contained some mice that were homozygous for floxed ERα and at least heterozygous for both Kiss1-Cre and YFP; these were used as KERKO mice .", "Littermates of KERKO mice with wild-type Esr1 , Kiss1-Cre YFP ( heterozygous or homozygous for either Cre or YFP ) were used as controls; no differences were observed among these controls and they were combined .", "To generate kisspeptin-specific S . pyogenes Cas9 ( Cas9 ) -expressing mice , mice with the Cre recombinase gene knocked-in after the Kiss1 promoter ( Kiss1-Cre mice ) were crossed with mice that have Cre recombinase-dependent expression of CRISPR-associated protein 9 ( Cas9 ) endonuclease , a 3X-FLAG epitope tag and eGFP directed by a CAG promoter .", "KERKO mice have disrupted estrous cycles with persistently cornified vaginal cytology typical of estrus; we thus used females in estrus as controls .", "Estrous cycle stage was determined by vaginal lavage .", "To examine the role of circulating estradiol , mice were ovariectomized ( OVX ) under isoflurane anesthesia ( Abbott ) and were either simultaneously implanted with a Silastic ( Dow-Corning ) capsule containing 0 . 625 µg of estradiol suspended in sesame oil ( OVX +E ) or not treated further ( OVX ) ( Christian et al . , 2005 ) .", "Bupivacaine ( 0 . 25% , APP Pharmaceuticals ) was provided local to incisions as an analgesic .", "These mice were studied 2-3 days after surgery .", "Mice for electrophysiology were sacrificed at the time of estradiol positive feedback in the late afternoon ( Christian et al . , 2005 ) .", "For free-floating immunochemistry staining , mice were perfused at 1700 EST 2-3d post OVX +E surgery at the expected peak of the estradiol-induced LH surge .", "For Cas9 target selection and generating single guide RNAs ( sgRNA ) , 20-nt target sequences were selected to precede a 5’NGG protospacer-adjacent motif ( PAM ) sequence .", "To minimize off-targeting effects and maximize sgRNA activity , two CRISPR design tools were used to evaluate sgRNAs ( Ran et al . , 2013; Doench et al . , 2014 ) targeting the first coding exon of mouse Esr1 .", "The two best candidates were selected based on lowest predicted off-target effects and highest activity .", "The target sequence for guide 1 is 5’-CACTGTGTTCAACTACCCCG-3’ ( referred to as g1 ) and the target sequence for guide 2 is 3’-CTCGGGGTAGTTGAACACAG-5’ ( referred to as g2 ) .", "Because g1 and g2 were similarly effective in Esr1 knockdown and effects on cycles , mice were combined for physiology studies .", "Control sgRNA sequence was designed to target lacZ gene from Escherichia coli ( target sequence: 5’-TGCGCAGCCTGAATGGCGAA −3’ ) .", "Mycoplasma-free C2C12 mouse myoblast cells ( generous gift of Dr . Daniel Michele , University of Michigan ) were grown in DMEM containing 10% FBS ( Thermo Fisher ) at 37°C in 5% CO2 .", "Each individual sgRNA was introduced to BsmBI site of the lentiCRISPRv2 construct .", "Cells were co-transfected with one of the lentiCRISPRv2 plasmids containing sgRNAs and a standard GFP plasmid construct ( Ramakrishnan et al . , 2016 ) using Lipofectamine 3000 ( Invitrogen ) according to the manufacturer’s instructions .", "Cells were selected for ~4 weeks with medium containing 1 μg/mL puromycin .", "Selected cells were harvested , DNA isolated using the Qiagen DNA Extraction Kit , and sequenced with primers for Esr1 .", "To construct the AAV plasmid , a mCherry-U6 promoter-sgRNA scaffold segment was synthesized by Integrated DNA Technologies ( IDT ) .", "After PCR amplification , the ligation product containing mCherry-U6 promoter-sgRNA scaffold was cloned in reverse orientation into a hSyn ( human Synapsin", "1 ) promoter driven Cre-inducible AAV vector backbone ( Flak et al . , 2017 ) .", "The individual sgRNAs ( with an extra G added to the 5'-end of each sgRNA to increase guide efficiency [Doench et al . , 2014] ) were then inserted into a designed SapI site between U6 promoter and sgRNA scaffold component .", "All three AAV viral vectors were prepared in AAV8 serotype at University of North Carolina Vector Core .", "Kiss1Cre/Cas9-GFP female animals ( >2 mo ) were checked for estrous cycles for >10 days before surgery; only mice with regular 4–5 day cycles were used .", "Mice were anesthetized with 1 . 5–2% isoflurane .", "AVPV injections were targeted to 0 . 55 mm posterior to Bregma , ±0 . 2 mm lateral to midline , and 4 . 7 and 4 . 8 mm ventral to dura .", "Arcuate injections were targeted to 1 . 5–1 . 7 mm posterior to Bregma , ±0 . 2 mm lateral to midline , and 5 . 9 mm ventral to dura .", "100 nl virus injected bilaterally at the target coordinates at ~5 nl/min .", "The pipette was left in place for 5 min after injection to allow viral diffusion into the brain .", "Carprofen ( Zoetis , Inc . , 5 mg/kg , sc ) was given before and 24 hr after surgery to alleviate postsurgical pain .", "Estrous cycle monitoring continued after surgery for up to 8 weeks .", "Stereotaxic hits were defined as ≥70% infection rate in both hemispheres; only bilateral hits were included for in vivo evaluation of reproductive parameters .", "Mice were anesthetized with isoflurane and then transcardially perfused with PBS ( 15–20 mL ) then 10% neutral-buffered formalin for 10 min ( ~50 mL ) .", "Brains were placed into the same fixative overnight , followed by 30% sucrose for ≥24 hr for cryoprotection .", "Sections ( 30 μm , four series ) were cut on a cryostat ( Leica CM3050S ) and stored at −20°C in antifreeze solution ( 25% ethylene glycol , 25% glycerol in PBS ) .", "Sections were washed with PBS , treated with 0 . 1% hydrogen peroxide , and then placed in blocking solution ( PBS containing 0 . 1% TritonX-100 , 4% normal goat serum , Jackson Immunoresearch ) for 1 hr at room temperature , then incubated with rabbit anti-ERα ( #06–935 , Millipore , 1:10 , 000; this antibody recognizes the C-terminus of ERα . ) in blocking solution 48 hr at 4°C .", "Sections were washed then incubated with biotinylated anti-rabbit antibody ( Jackson Immunoresearch , 1:500 ) followed by ABC amplification ( Vector Laboratories , 1:500 ) and nickel-enhanced diaminobenzidine ( Thermo Scientific ) reaction ( 4 . 5 min ) .", "Sections were washed with PBS and incubated overnight with chicken anti-GFP ( ab13970 , Abcam , 1:2000 ) and rat anti-mCherry ( M11217 , Invitrogen , 1:5000 ) in blocking solution .", "The next day , sections were washed and incubated with Alexa 594-conjugated anti-rat and Alexa 488-conjugated anti-chicken antibodies for 1 hr at room temperature ( Molecular Probes , 1:500 ) .", "Sections were mounted and coverslipped ( VWR International 48393 251 ) .", "Images were collected on a Zeiss AXIO Imager M2 microscope , and the number of immunoreactive GFP only , GFP/mCherry , and GFP/mCherry/ERα cells were counted in the injected region .", "The other kisspeptin region in the hypothalamus was examined and no infection of kisspeptin cell bodies was observed .", "All solutions were bubbled with 95%O2 and 5%CO2 for ≥15 min before exposure to tissue and throughout experiments .", "Brains were rapidly removed 1 . 5–2 hr before lights off and placed in ice-cold sucrose saline solution containing ( in mM ) : 250 sucrose , 3 . 5 KCl , 25 NaHCO3 , 10 D-glucose , 1 . 25 Na2HPO4 , 1 . 2 MgSO4 , and 3 . 8 MgCl2 .", "Coronal slides ( 300 μm ) were made with a Leica VT1200S .", "Slices were incubated in a 1:1 mixture of sucrose-saline and artificial cerebrospinal fluid ( ACSF ) containing ( in mM ) : 135 NaCl , 3 . 5 KCl , 26 NaHCO3 , 10 D-glucose , 1 . 25 Na2HPO4 , 1 . 2 MgSO4 , 2 . 5 CaCl2 for 30 min at room temperature .", "Slices were then transferred to 100% ACSF at room temperature for ≥30 min before recording .", "Slices were used within 6 hr of preparation .", "Slices were transferred to a recording chamber and perfused with oxygenated ACSF ( 3 mL/min ) and heated by an in-line heater ( Warner Instruments ) to 30 ± 1°C .", "GnRH-GFP neurons were identified by brief illumination at 470 nm using an upright fluorescence microscope Olympus BX51W1 .", "Recording pipettes were pulled from borosilicate glass ( type 7052 , 1 . 65 mm outer diameter and 1 . 12 mm inner diameter; World Precision Instruments , Inc ) using a P-97 puller ( Sutter Instruments ) to obtain pipettes with a resistance of 2–3 . 5 MΩ .", "Recordings were performed with an EPC-10 dual-patch clamp amplifier and Patchmaster acquisition software ( HEKA Elektronik ) .", "Recorded cells were mapped to a brain atlas ( Paxinos and Franklin , 2001 ) to determine if cell location was related to response to treatment .", "No such correlation was observed in this study .", "Extracellular recordings were used to characterize firing rate as they maintain internal milieu and have minimal impact neuronal firing rate ( Nunemaker et al . , 2003; Alcami et al . , 2012 ) .", "Recordings were made with receptors for ionotropic GABAA and glutamate synaptic transmission antagonized ( 100 µM picrotoxin , 20 µM D-APV [D- ( − ) −2-amino-5-phosphonopentenoic acid] , 10 µM CNQX [6-cyano-7-nitroquinoxaline] ) .", "Pipettes were filled with HEPES-buffered solution containing ( in mM ) : 150 NaCl , 10 HEPES , 10 D-glucose , 2 . 5 CaCl2 , 1 . 3 MgCl2 , and 3 . 5 KCl ( pH = 7 . 4 , 310 mOsm ) , and low-resistance ( 22 ± 3 MΩ ) seals formed between the pipette and neuron after first exposing the pipette to the slice tissue in the absence of positive pressure .", "Recordings were made in voltage-clamp mode ( 0 mV pipette holding potential ) and signals acquired at 20 kHz and filtered at 10 kHz .", "Resistance of the loose seal was checked frequently during the first 3 min of recordings to ensure a stable baseline , and also before and after a subsequent 10 min recording period; data were not used if seal resistance changed >30% or was >25 MΩ .", "The first 5 min of this 10-min recording were consistently stable among cells and were thus used for analysis .", "For whole-cell patch-clamp recordings , three different pipette solutions were used depending on the goal .", "Most recordings were done with a physiologic pipette solution containing ( in mM ) : 135 K gluconate , 10 KCl , 10 HEPES , 5 EGTA , 0 . 1 CaCl2 , 4 MgATP and 0 . 4 NaGTP , pH 7 . 2 with NaOH , 302 ± 3 mOsm .", "A similar solution containing 10 mM neurobiotin was adjusted to similar osmolarity .", "A solution in which cesium gluconate replaced potassium gluconate was used to reduce potassium currents and allow better isolation of calcium currents .", "Membrane potentials reported were corrected online for liquid junction potential of −15 . 7 mV , same among all solutions ( Barry , 1994 ) .", "After achieving a minimum 1 . 6 GΩ seal and the whole-cell configuration , membrane potential was held at −70 mV between protocols during voltage-clamp recordings .", "Series resistance ( Rs ) , input resistance ( Rin ) , holding current ( Ihold ) and membrane capacitance ( Cm ) were frequently measured using a 5 mV hyperpolarizing step from −70 mV ( mean of 16 repeats ) .", "Only recordings with Rin >500 MΩ , Ihold−40 to 10 pA and RS <20 MΩ , and stable Cm were accepted .", "Rs was further evaluated for stability and any voltage-clamp recordings with ∆Rs >15% were excluded; current-clamp recordings with ∆Rs >20% were excluded .", "There was no difference in Ihold , Cm , or Rs among any comparisons .", "For current-clamp recordings , depolarizing and hyperpolarizing current injections ( −50 to +50 pA , 500 ms , 10 pA increments ) were applied from an initial membrane potential of −71 ± 2 mV , near the resting membrane potential of these cells ( DeFazio et al . , 2014 ) .", "For voltage-clamp recordings of excitatory postsynaptic currents ( EPSCs ) , membrane potential was held at −68 mV , the reversal potential for GABAA-receptor mediated currents , and ACSF contained picrotoxin ( 100 μM ) , and D-APV ( 20 μM ) .", "For voltage-clamp recordings of IT , ACSF containing antagonists of ionotropic GABAA and glutamate receptors was supplemented with TTX ( 2 µM ) and the Cs-based pipette solution was used .", "Two voltage protocols were used to isolate IT as reported ( Wang et al . , 2016 ) .", "First , total calcium current activation was examined .", "Inactivation was removed by hyperpolarizing the membrane potential to −110 mV for 350 ms ( not shown in figures ) .", "Next , a 250 ms prepulse of −110 mV was given .", "Then membrane potential was varied in 10 mV increments for 250 ms from −110 to −30 mV .", "Finally , test pulse of −40 mV for 250 ms was given .", "From examination of the current during the test pulse , it was evident that no sustained ( high-voltage activated , HVA ) calcium current was activated at potentials more hyperpolarized than −40 mV .", "To remove HVA contamination from the step to −30 mV , a second protocol was used in which removal of inactivation ( −110 mV , 350 ms ) was followed by a 250 ms prepulse at −40 mV , then a step for 250 ms at −30 mV and finally a test pulse of −40 mV for 250 ms . IT was isolated by subtracting the trace following the −40 mV prepulse from those obtained after the −110 mV prepulse for the depolarized variable step to −30 mV; raw traces from the initial voltage protocol were used without subtraction for variable steps from −110 mV to −40 mV because of the lack of observed activation of HVA at these potentials .", "Activation of IT was assessed from the resulting family of traces by peak current during the variable step phase .", "Inactivation of IT was assessed from the peak current during the final −40 mV test pulse .", "The pipette solution containing neurobiotin was used for recordings cells from AAV-injected mice .", "An outside-out patch was formed after recording to reseal the membrane and the location of cells was marked on a brain atlas ( Paxinos and Franklin , 2001 ) .", "The brain slices were fixed overnight in 10% formalin at 4°C and changed to PBS .", "Slices were photo-bleached with a UV illuminator for ~72 hr and checked to ensure no visible fluorescent signal was observed .", "Slices were then placed in blocking solution for 1 hr , then incubated with rabbit anti-ERα for 48 hr at 4°C .", "Slices were washed and then incubated with Alexa 594-conjugated anti-rabbit and Alexa 350-conjugated neutravidin for 2 hr at room temperature ( Molecular Probes , 1:500 ) .", "Slices were mounted , coverslipped and imaged as above .", "Cells with neurobiotin-labeling were examined for ERα-immunoreactivity .", "Single-cell PCR Cells for single cell PCR were collected as previously described ( Ruka et al . , 2013 ) .", "Patch pipettes ( 2–3 MΩ ) were filled with 5–8 μL of an RNase free solution containing ( in mM ) : 135 K-gluconate , 10 KCl , 10 HEPES , 5 EGTA , 4 . 0 Mg-ATP , 0 . 4 Na-GTP , and 1 . 0 CaCl2 ( pH 7 . 3 , 305 mOsm ) .", "Additionally , just before use 1 U/µL Protector RNase Inhibitor ( Roche , Indianapolis , IN ) was added to the pipette solution .", "Single-cell RNA was harvested from the target cells in whole-cell configuration after recording membrane response in current-clamp; cytoplasm was aspirated into the pipette and expelled into a 0 . 2 mL tube containing reverse transcriptase buffer ( Superscript Vilo cDNA Synthesis Kit , Invitrogen/ThermoFisher ) , volume was adjusted to 20 µL with molecular grade water .", "Cell contents were reverse transcribed following manufacturer’s instructions .", "False harvests , in which the pipette was lowered into the slice preparation but no aspiration of cell contents occurred , were used to estimate background contamination .", "These were performed on each recording day .", "Additionally , a standard curve of mouse hypothalamic RNA ( 1 , 0 . 1 , 0 . 01 , 0 . 001 ng/μL final concentration ) and a water blank ( negative control ) were reverse transcribed .", "An equivalent volume of water or patch solution was reverse transcribed as a negative control .", "Single-cell cDNA , controls , and the standard curve were preamplified for 15 cycles using TaqMan PreAmp Master Mix ( Invitrogen/ThermoFisher ) as previously described ( Glanowska et al . , 2014 ) .", "Quantitative PCR was performed using 5 μL of diluted preamplified DNA ( 1:10 ) per reaction , in duplicate , for 50 cycles ( TaqMan Gene Expression Master Mix; Invitrogen ) .", "Single-cell cDNA was assayed for: Kiss1 , TH , Esr1 , Esr2 , Pgr , Cacna1g , Cacna1h , Cacna1i , Hcn1 Hcn2 Hcn3 Hcn4; Syn1 was used as housekeeping gene; only Syn1-positive cells were analyzed .", "Single cells were considered positive for a transcript if their threshold was a minimum of three cycles earlier ( eight fold greater ) than the false harvests and the reverse transcribed and preamplified water blank sample .", "TaqMan PrimeTime qPCR assays for mRNAs ( Table 4 ) were purchased from IDT .", "Ovary-intact Kiss1Cre-Cas9 adult female mice with AAV-lacZ and AAV-Esr1 targeted to the arcuate nucleus were singly-housed were handled daily ≥4 wks before sampling .", "Vaginal cytology was determined for ≥10 days before sampling .", "As the majority of AAV-Esr1 arcuate targeted mice ( 6 of 9 ) exhibit prolonged cornification typical of estrus , all mice ( Esr1 and lacZ ) were sampled during estrus .", "Repetitive tail-tip blood collecting was performed as described ( Steyn et al . , 2013 ) .", "After the excision of the very tip of the tail , blood ( 6 µL ) was collected every 6 min for 2 hr from 1pm to 3pm .", "At the end of this frequent sampling period , mice received a single intraperitoneal injection of kisspeptin ( 65 µg/kg ) ( Hanchate et al . , 2012 ) .", "Blood was collected just before and 15 min after kisspeptin injection .", "GnRH ( 150 µg/kg ) ( Glanowska et al . , 2014 ) was injected 40–45 min after kisspeptin , with blood collected immediately before and 15 min after GnRH injection .", "Ovary-intact Kiss1Cre-Cas9 adult female mice with AAV-lacZ and AAV-Esr1 targeted to the AVPV were singly-housed .", "Tail blood was collected as above on proestrus at 3 , 4 and 5pm EST ( lights are off at 5pm EST in the mouse room ) .", "One to two weeks later , these same mice were then subjected to OVX + E surgery and tail blood ( 6 µL ) was collected 2–3 days post-surgery at 9am and 5pm EST .", "Whole blood was immediately diluted in 54 μL of 0 . 1M PBS with 0 . 05% Tween 20% and 0 . 2% BSA , mixed and kept on ice .", "Samples were stored at −20°C for a subsequent ultrasensitive LH assay ( Steyn et al . , 2013 ) .", "Intraassay CV was 2 . 2%; interassay CVs were 7 . 3% ( low QC , 0 . 13 ng/mL ) , 5 . 0% ( medium QC , 0 . 8 ng/mL ) and 6 . 5% ( high QC , 2 . 3 ng/mL ) .", "Functional sensitivity was 0 . 016 ng/mL .", "Ovaries were fixed for 24 hr in 10% neutral-buffered formalin , then stored in 70% ethanol until paraffin embedding , sectioning ( 5 µm ) and H and E staining .", "Every fifth section was examined and corpra lutea counted .", "Data were analyzed offline using custom software written in IgorPro 6 . 31 ( Wavemetrics ) .", "For targeted extracellular recordings , mean firing rate in Hz was determined over 5 min of stable recording .", "In experiments examining IT , the peak current amplitude at each step potential ( V ) was first converted to conductance using the calculated reversal potential of Ca2+ ( ECa ) and G = I/ ( ECa - V ) , because driving force was linear over the range of voltages examined .", "The voltage dependencies of activation and steady-state inactivation were described with a single Boltzmann distribution: G ( V ) =Gmax/ ( 1- exp [ ( V1/2 - Vt ) /k] ) , where Gmax is the maximal conductance , V1/2 is the half-maximal voltage , and k is the slope .", "Current density of IT at each tested membrane potential was determined by dividing peak current by membrane capacitance .", "LH pulses were detected by a version of Cluster ( Veldhuis and Johnson , 1986 ) transferred to IgorPro using cluster sizes of two points for both peak and nadir and t-scores of two for detection of increases and decreases .", "Data were analyzed using Prism 7 ( GraphPad Software ) and reported as mean ± SEM .", "The number of cells per group is indicated by n and the number of mice by N in Table 5 .", "For two-by-two designs , data were normally distributed and analyzed by two-way ANOVA or two-way repeated-measures ( RM ) with Holm-Sidak post hoc .", "For two group comparisons , normally-distributed data were analyzed by two-tailed unpaired Student’s t-test; non-normal data were analyzed by Mann-Whitney U test .", "For categorical data , for more than three categories , Chi-square test of independence was used with Fisher’s exact test as post hoc analysis .", "For two categories , Fisher’s exact test was used .", "For each electrophysiological parameter comparison , no more than three cells per mouse was used in control and KERKO mice; no more than four cells per mouse was used for AAV-infected mice .", "No less than five mice were tested per parameter .", "The variance of the data was no smaller within an animal than among animals .", "For IF staining , LH surge and LH pulse measurements , and reproductive cyclicity , at least three mice were tested per AAV vector ." ] ]

[ "The brain regulates fertility through gonadotropin-releasing hormone ( GnRH ) neurons .", "Estradiol induces negative feedback on pulsatile GnRH/luteinizing hormone ( LH ) release and positive feedback generating preovulatory GnRH/LH surges .", "Negative and positive feedbacks are postulated to be mediated by kisspeptin neurons in arcuate and anteroventral periventricular ( AVPV ) nuclei , respectively .", "Kisspeptin-specific ERα knockout mice exhibit disrupted LH pulses and surges .", "This knockout approach is neither location-specific nor temporally controlled .", "We utilized CRISPR-Cas9 to disrupt ERα in adulthood .", "Mice with ERα disruption in AVPV kisspeptin neurons have typical reproductive cycles but blunted LH surges , associated with decreased excitability of these neurons .", "Mice with ERα knocked down in arcuate kisspeptin neurons showed disrupted cyclicity , associated with increased glutamatergic transmission to these neurons .", "These observations suggest that activational effects of estradiol regulate surge generation and maintain cyclicity through AVPV and arcuate kisspeptin neurons , respectively , independent from its role in the development of hypothalamic kisspeptin neurons or puberty onset ." ]

[ "Female reproduction relies on a complex balance of hormones that drive the reproductive cycle ( menstrual cycle in humans ) and influence fertility .", "A hormone called GnRH , which stands for gonadotropin-releasing hormone , plays a major role in regulating this balance .", "GnRH is transmitted from the brain and stimulates the release of other hormones from a nearby gland called the pituitary gland , which , in turn , activates the reproductive organs to produce steroid hormones , such as estrogen .", "Steroids do many things in the body , including regulating the release of GnRH and pituitary hormones through a process called feedback .", "In the case of negative feedback , steroids maintain the release of GnRH and pituitary hormone within a normal range .", "Once per reproductive cycle , estrogen will instead positively feed back into the system and activate GnRH , causing pituitary hormone levels to spike , and initiate the release of one or more eggs from the ovary by a process known as ovulation .", "The neurons that make GnRH do not directly respond to estrogen , but instead receive input from different upstream neurons that contain estrogen receptors .", "However , it is poorly understood how fertility is regulated by these neurons .", "To investigate the effects of estrogen on these upstream neurons , Wang et al . genetically removed the estrogen receptors from two separate populations of neurons in mice .", "Estrogen was found to affect each of these populations differently , inhibiting one and activating the other .", "Wang et al . showed that these two populations likely have different roles in reproduction: the population inhibited by estrogen regulates negative feedback and generates reproductive cycles , whilst the population activated by estrogen regulates positive feedback and stimulates ovulation .", "This knowledge furthers our understanding of how the brain regulates fertility , and the genetic approach used to remove the estrogen receptor could be applied to the study of other hormones that act on the brain ." ]

[ "Introduction", "Results", "Discussion" ]

[ "structural biology and molecular biophysics" ]

[ [ "An elaborate network of chaperones is present in organisms across all domains of life to oversee the health of the cellular proteome ( Balchin et al . , 2016 ) .", "The Hsp70 family of molecular chaperones occupies a central node in this network , steering proteins synthesized on the ribosome to their native conformations , as well as cooperating with the machinery of protein disaggregation , refolding and proteolysis to control the fate of improperly folded and aggregated cellular proteins ( Mayer and Bukau , 2005; Mayer , 2013; Mogk et al . , 2015; Rosenzweig et al . , 2013 ) .", "Hsp70 interacts with client substrates via an ATP-dependent chaperone cycle that is tightly regulated by Hsp40 co-chaperones and nucleotide exchange factors ( NEFs ) ( Balchin et al . , 2016; Mayer and Bukau , 2005; Mayer , 2013 ) .", "Substrates are brought to the cycle by either Hsp40 or ATP-bound Hsp70 and typically undergo multiple rounds of binding and release before either folding to the native state or being transferred to downstream chaperone systems such as GroEL/ES and ClpB ( bacteria ) or Hsp90 and Hsp104 ( eukaryotes ) .", "Hsp70 is a 70 kDa protein that consists of a 45 kDa N-terminal ATPase domain ( NBD ) and a 25 kDa C-terminal substrate binding domain ( SBD ) ( Figure 1A ) ( Mayer and Bukau , 2005 ) .", "Much of our understanding of client recognition by Hsp70 derives from studies of peptide-based substrates .", "Crystal ( Zhu et al . , 1996; Zahn et al . , 2013 ) and NMR structures ( Stevens et al . , 2003 ) of peptide-bound SBDs establish that the substrate binds in an extended conformation , with a 4–5 amino acid core of the substrate forming backbone hydrogen bonds as well as hydrophobic contacts with chaperone residues Val 436 , Ile 401 and Ile 438 that line the SBD binding pocket ( Figure 1B ) .", "The hydrophobic nature of the central binding pocket governs the preference of Hsp70 for substrate sequences enriched in large aliphatic hydrophobic sidechains such as Ile , Leu and Val ( Rüdiger et al . , 1997 ) .", "Studies on full length protein substrates have revealed that these are globally unfolded in the Hsp70-bound state ( Palleros et al . , 1994; Kurt et al . , 2006; Chen et al . , 2006; Kurt and Cavagnero , 2008; Sharma et al . , 2010; Kellner et al . , 2014; Lee et al . , 2015; Sekhar et al . , 2015 ) and lack long-range interactions ( Sekhar et al . , 2016 ) , though they can form local residual native ( Sekhar et al . , 2015 ) and non-native secondary structure ( Kurt and Cavagnero , 2008 ) in regions far from the Hsp70 binding site .", "The ability of Hsp70 to recognize commonly occurring sequences in typical proteins with a frequency of about 1 in 40 residues makes it a promiscuous interactor ( Rosenzweig et al . , 2017 ) , generating a highly heterogeneous Hsp70-substrate ensemble that ensures the efficient search of conformational space for an optimal folding pathway ( Lee et al . , 2015; Rosenzweig et al . , 2017; Sekhar et al . , 2017 ) .", "The structural constraints of the Hsp70 binding pocket ( Figure 1C ) are such that only an unfolded stretch of polypeptide can fit into the binding groove if the helical lid is closed ( Zhu et al . , 1996 ) , though more compact protein conformations can be accommodated if the lid is partially open ( Schlecht et al . , 2011 ) .", "Hsp70 function involves altering the conformation of client proteins ( Mayer , 2013; Rodriguez et al . , 2008; Goloubinoff and De Los Rios , 2007; Clerico et al . , 2015 ) during folding ( Sharma et al . , 2010; Szabo et al . , 1994 ) and disaggregation ( Mogk et al . , 2015 ) , as well as dismantling of oligomeric assemblies such as clathrin coats ( Böcking et al . , 2011; Sousa et al . , 2016 ) that surround vesicles involved in intra-cellular trafficking .", "However , the molecular mechanism by which Hsp70 causes these diverse conformational changes remains poorly understood .", "The structures of the Hsp70 chaperone in its ATP ( Kityk et al . , 2012; Qi et al . , 2013 ) ( Figure 1D ) and ADP states ( Bertelsen et al . , 2009 ) ( Figure 1A ) are substantially different and the conformational switch that occurs in Hsp70 upon ATP hydrolysis has been hypothesized to perform conformational work on the bound substrate and alter its structure ( Mayer , 2013; Clerico et al . , 2015 ) .", "Contrary to this hypothesis , NMR studies by our group have shown that the dominant native ( i . e . , unbound ) and bound conformations of a substrate can be significantly different despite the fact that the structure of the substrate in the ADP-bound , ATP-bound and nucleotide-free forms of Hsp70 are identical , showing that ATP hydrolysis does not have to be coupled to a substrate conformational change in the Hsp70 chaperone system ( Sekhar et al . , 2015 ) .", "Two limiting-case mechanisms have been proposed for the binding of Hsp70 to its target substrates , namely conformational selection ( CS ) and induced fit ( IF ) , also referred to in some reports as the holdase and the unfoldase models , respectively ( Mayer , 2013; Sharma et al . , 2010; Rodriguez et al . , 2008; Goloubinoff and De Los Rios , 2007; Clerico et al . , 2015; Böcking et al . , 2011; Slepenkov and Witt , 2002 ) .", "In some cases conformational transitions in the substrate may occur in the absence of Hsp70 binding but still form an important component of the binding process .", "For example , the binding of Hsp70 to the clathrin triskelion that leads to downstream uncoating of a clathrin-coated vesicle has been postulated to occur via local conformational fluctuations in individual clathrin molecules that transiently expose the Hsp70 binding site ( Böcking et al . , 2011 ) .", "The recognition of σ32 by E . coli Hsp70 ( DnaK ) has also been hypothesized to occur through transient excursions of the σ32 molecule from a compact conformation where the binding site is sequestered in a helix to a more accessible extended structure ( Rodriguez et al . , 2008 ) .", "In contrast , the IF-like unfoldase model is believed to operate in accelerating the folding of large protein substrates such as luciferase by transforming misfolded kinetically trapped states into unfolded but folding-competent conformations ( Sharma et al . , 2010 ) .", "In general , distinguishing between CS and IF mechanisms ( Changeux and Edelstein , 2011 ) requires a consideration of both structure and kinetics .", "For example , in order to establish that a particular binding interaction occurs via CS , it is necessary both to demonstrate that a very similar conformation to that in the bound state is sampled in the absence of the binder and to show that the formation of the complex occurs primarily via a kinetic pathway whereby the conformation of the ligand that resembles the bound form is selected for binding .", "While there is compelling evidence in a number of cases for the pre-sampling of a bound conformation in the unbound state ( Boehr et al . , 2009; Tzeng and Kalodimos , 2009; Ye et al . , 2016; Lange et al . , 2008; Anthis et al . , 2011; Venditti and Clore , 2012 ) , very few reports have unequivocally established that such a conformation is on-pathway to the bound state ( Birdsall et al . , 1980; Daniels et al . , 2014; Daniels et al . , 2015; Chakrabarti et al . , 2016 ) .", "Indeed , pre-sampling of a bound conformation does not automatically imply that the bound state forms from such a conformation .", "Several recent studies have sought to make this clarification by formulating the CS and IF mechanisms in terms of the flux of molecules flowing through the two pathways to the bound state ( Hammes et al . , 2009; Weikl and Paul , 2014 ) .", "Herein , in an effort to further characterize how Hsp70 chaperones bind to cognate substrates , we address which of the competing pathways , CS or IF , better explains the binding interaction between Hsp70 and its targets .", "We consider a pair of folding competent substrates with very different secondary structures ( α-helix and β-sheet ) and both E . coli and human ( Hsc70 ) chaperones and probe the binding reaction using magnetization exchange ( Farrow et al . , 1994; Palmer et al . , 2001 ) and chemical exchange saturation transfer ( CEST ) NMR methodology ( Vallurupalli et al . , 2017; Anthis and Clore , 2015 ) , which simultaneously provide structural information about each of the participating components as well as the kinetics of the recognition event .", "The NMR experiments exploit selective isotope labeling ( Kerfah et al . , 2015 ) and methyl transverse relaxation optimized spectroscopy ( methyl TROSY ) ( Tugarinov et al . , 2003; Ollerenshaw et al . , 2003 ) approaches so that large protein assemblies such as the Hsp70-substrate complex can be probed quantitatively at atomic resolution .", "We have carried out equilibrium measurements and analyzed the resulting kinetic parameters in terms of fluxes along the competing CS and IF pathways , where formation of the unfolded bound conformer is predominantly the result of chaperone binding to either unfolded or native substrate states , respectively .", "Our results establish that the dominant binding process is via the CS mechanism for both substrates considered , and for both DnaK and human Hsc70 , suggesting that the CS mode of Hsp70 recognition may be conserved .", "Our data highlight the importance of molecular dynamics in Hsp70 chaperone-substrate binding and emphasize the potential of NMR spectroscopy in providing unambiguous mechanistic insights into key biological processes that involve molecular interactions ." ], [ "In what follows we consider a solution comprising client protein substrate and Hsp70 ( referred to as K ) at equilibrium .", "The client protein is not characterized by a single structure , but rather by an ensemble of conformers , ranging from native ( N ) to unfolded ( U ) ; in the examples that follow both N and U states are approximately equally populated , and are in principle , both available to form a substrate-Hsp70 bound complex .", "A number of different biophysical experiments , performed on a variety of Hsp70 complexes , has established that the substrate is unfolded in the bound state , UK ( Palleros et al . , 1994; Chen et al . , 2006; Sharma et al . , 2010; Kellner et al . , 2014; Lee et al . , 2015; Sekhar et al . , 2015 ) .", "Our goal is to obtain mechanistic insights into the binding reaction by considering the flux from either conformers U or N to UK so as to evaluate what the dominant pathway for the Hsp70 - substrate interaction might be and hence establish whether binding is best described in terms of a CS or IF mechanism .", "As described in the Introduction , in the IF ( unfoldase ) model the dominant process involves Hsp70 binding to N to form a preliminary complex ( NK ) , where the bound substrate has an N-like structure , that is subsequently unfolded to form the dominant bound conformation ( UK ) .", "The reaction can be denoted as N→NK→UK , although in principle more complex schemes can also be included so long as they do not involve the U→UK step .", "In contrast , in the CS ( holdase ) model , as we consider it here , Hsp70 selects a substrate conformer that structurally resembles the bound state ( UK ) from an equilibrium mixture , with larger fluxes for reactions that proceed via this path ( U→UK ) than for those involving N→NK→UK .", "It is important to emphasize that a number of pathways fit the description of the CS mechanism , including both N→U→UK and the simpler U→UK .", "Indeed , in cases where the N , U conformational interconversion is limiting kinetically the flux through the U→UK pathway will be larger than the more complex CS scheme that begins with N . Since the distinction between the CS and IF models involves a consideration of both structure and kinetics , our strategy here is to use magnetization exchange ( zz-exchange ) ( Farrow et al . , 1994; Palmer et al . , 2001 ) or CEST NMR ( Vallurupalli et al . , 2017 ) experiments that both resolve different conformers through chemical shifts and facilitate extraction of rates of interconversion .", "Figure 2A shows a schematic of a zz-exchange spectrum for a two-site exchange reaction , A⇌kBAkABB .", "In this experiment the chemical shifts of NMR signals are measured both before and after a delay during which chemical exchange occurs .", "Because the experiment is constructed essentially as a 13C-1H HMQC , whereby 13C and 1H chemical shifts are recorded before and after the exchange period , respectively , a dataset is generated in which for each methyl group a pair of peaks ( diagonal-peaks ) is obtained at the resonance positions for the 1H and 13C methyl spins that are associated with the two interconverting states .", "An additional pair of peaks ( cross-peaks ) is also present that link the diagonal peaks , derived from the transfer of longitudinal magnetization from A to B or B to A during the exchange period .", "Quantification of the buildup and decay of the cross- and diagonal-peaks , respectively , as a function of the duration of the exchange period enables the extraction of rates of interconversion and equilibrium populations of states .", "Figure 2B illustrates a hypothetical CEST profile , again focusing on a two-state exchange process .", "In this experiment the intensity of an observed peak ( say from a proton of residue j of state A ) is monitored as a function of the position of a weak radio frequency ( rf ) 1H field that perturbs magnetization resonating at or close to the frequency of application of the weak field .", "The field , applied for a duration TEX , is scanned across all possible frequencies ( one frequency per experiment ) , eventually perturbing the corresponding proton magnetization from residue j of state B and this perturbation is subsequently transferred to the observed peak ( A ) due to chemical exchange .", "Thus , a plot of the intensity ratio of the A state peak ( I/Io , where I and Io are the intensities of peak A when the field is ( TEX ≠ 0 ) and is not ( TEX = 0 ) applied , respectively ) as a function of frequency shows a major dip at the resonance position of the target proton in state A and a minor dip at the position of the resonance frequency of the proton in state B . Note that there is an analogy between the CEST profiles from A and B and a zz-exchange dataset , with the major dips in CEST corresponding to the diagonal peaks in zz-exchange , connected via the minor dips in the two profiles , similar to the zz-exchange crosspeaks .", "The resulting curves from zz-exchange or CEST profiles can be fit to the Bloch-McConnell equations ( McConnell , 1958 ) that include the effects of chemical exchange to extract parameters of the interconversion process .", "There are a number of important experimental constraints which must be taken into account in the choice of Hsp70 substrates for study via these two approaches .", "For example , if the N , U interconversion is significantly faster than the binding step the distinction between N and U becomes ‘blurred’ and it becomes difficult to distinguish binding starting from N or from U . This is illustrated in the context of a set of reactions , N⇌kUNkNUUU+K⇌koffkonUKUKwhere N and U are DnaK-free conformations of the substrate and K refers to DnaK .", "Focusing on CEST , Figure 2C shows that the N ( red ) and U ( green ) profiles become indistinguishable for fast interconversion ( kex , NU >> kex , UK , where kex , NU = kNU + kUN , kex , UK=konUK[K]+koff; kex , NU << ΔωNU where ΔωNU is the chemical shift difference between corresponding peaks in states N and U ) , while if the interconversion is slow ( kex , NU << kex , UK ) then the mechanism of binding can clearly be discerned from a comparison of the CEST profiles of N and U , since only the latter shows a minor dip at the chemical shift of the reporter nucleus in the bound state in this case ( Figure 2D ) .", "Similarly , assuming kex , NU << kex , UK in a zz-exchange experiment for the same CS-based mechanism as above ( Figure 2E ) , cross-peaks are observed only between U and UK , while for an induced-fit mode of binding cross-peaks connect only N and UK ( Figure 2F ) .", "In addition , rates of interconversion must be on the order of ~1 s−1 or faster for zz-exchange ( Farrow et al . , 1994; Palmer et al . , 2001 ) if all of the exchange parameters are to be extracted robustly , or ~20 s−1 for the CEST experiment ( Vallurupalli et al . , 2017 ) in the general case where profiles from only one of the interconverting species can be measured .", "Notably , in cases where peaks from N , U and UK are visible in spectra this restriction is relaxed for the analysis of CEST profiles as we show below .", "Finally , it is worth emphasizing that , in the above discussion , we have assumed that exchange rates are slow compared to chemical shift differences between spins in interconverting states , N , U and UK , so that separate correlations are observed for each state in NMR datasets .", "The N and U states defined above may themselves be composed of sub-ensembles of interconverting conformers , for example {N1 , N2 , . . . } and {U1 , U2 , . . . } .", "In constructing the CS and IF models in terms of N and U states it is assumed that the interconversion between sub-states Ni or between Ui occurs much faster than the N-U exchange or than ligand binding , with a rate that is fast on the NMR chemical shift timescale .", "In this manner only single distinct NMR peaks are observed for spins in N or U states rather than separate peaks for each element of the sub-ensembles ( Ni ) or ( Ui ) so that N or U can thus be treated as single ‘averaged’ conformers .", "It naturally follows that the affinities and rates obtained from NMR experiments will be averages over the members of each sub-ensemble .", "It is also the case that additional intermediates such as alternate unfolded states ( U* ) can be explicitly included in any model of exchange only if their presence is detected either by distinct peaks in NMR spectra or by minor dips in CEST profiles .", "As a first substrate we focused on the slow-folding marginally stable L90A mutant of the 17th domain of chicken-brain α-spectrin ( referred to as R17* ) ( Scott et al . , 2006 ) .", "R17* is a three-helix bundle protein domain ( Figure 3A ) with kinetics and thermodynamics of folding that suggest that it may be an ideal candidate for distinguishing between different Hsp70 binding mechanisms using the NMR approaches described above ( Scott et al . , 2006 ) .", "R17* has a strong predicted ( Rüdiger et al . , 1997 ) Hsp70 binding site and the sidechains ( 41IQGLL45 ) forming this binding site are >40% solvent-exposed on average ( Figure 3B ) , potentially facilitating binding of Hsp70 to the native protein along an IF pathway .", "On the other hand the low stability of the folded domain suggests a large population of the U state in solution ( see below ) that might favour the CS route .", "E . coli DnaK is the Hsp70 ortholog that has been most extensively characterized at structural ( Kityk et al . , 2012; Qi et al . , 2013; Bertelsen et al . , 2009; Pellecchia et al . , 2000; Harrison et al . , 1997 ) , kinetic ( Sharma et al . , 2010; Slepenkov and Witt , 2002; Gisler et al . , 1998; Grimshaw et al . , 2001; Grimshaw et al . , 2003; Han and Christen , 2003; Pierpaoli et al . , 1998; Schmid et al . , 1994; Chesnokova and Witt , 2005; Slepenkov and Witt , 1998; Jordan and McMacken , 1995; Russell et al . , 1998 ) and mechanistic levels ( Sharma et al . , 2010; Szabo et al . , 1994; Landry et al . , 1992; Schröder et al . , 1993; Schönfeld et al . , 1995; Greene et al . , 1998; Montgomery et al . , 1999; Swain and Gierasch , 2006; Swain et al . , 2007; Smock et al . , 2010; Zhuravleva and Gierasch , 2015; Zhuravleva et al . , 2012; Moro et al . , 2003; Moro et al . , 2004; Taneva et al . , 2010; Mayer et al . , 2000; Laufen et al . , 1999 ) , and it was therefore selected for initial experiments .", "The ATP state of DnaK is an important entry point for substrates to the Hsp70 chaperone cycle ( Pierpaoli et al . , 1998 ) and was chosen as the chaperone nucleotide state in this study .", "In the ATP state DnaK substrate complexes have millisecond lifetimes and have been previously studied with CEST methodology ( Sekhar et al . , 2015 ) .", "In all experiments involving DnaK/ATP , the allosterically active T199A mutant of DnaK ( McCarty and Walker , 1991 ) that is deficient in ATP hydrolysis was used to minimize ATP turnover during NMR data collection .", "Figure 3C shows the Met methyl region of a 13C-1H HMQC spectrum of 250 μM U-2H , Ileδ1-13CH3 , Leu/Val-13CH3/12CD3 , Met-13CH3 ( ILVM-13CH3 ) labeled R17* , 25°C .", "There are two Met residues in R17* and five resonances in the spectral region .", "Three of the peaks were assigned to Met 87 and two to Met 26 based on the 13C-1H HMQC spectrum of M26L R17* ( Figure 3—figure supplement 1A ) .", "In addition , we carried out a temperature titration of R17* to gradually unfold the protein so as to ascertain whether the extra peaks arise from its unfolded state .", "Figure 3—figure supplement 1B shows that only two of the resonances remain in a 13C-1H HMQC spectrum recorded at 45°C and these are therefore assigned to the unfolded state of R17 .", "The other three resonances derive from the native conformer , with the weaker resonance from Met 87 ( M87NB ) possibly originating from an R17* dimer that is detected on the size exclusion column during purification .", "Upon addition of 500 μM U-2H DnaK/ATP a new peak appears in the Met region of the 13C-1H HMQC spectrum that can be unambiguously assigned to the bound state of R17* ( Figure 3D ) .", "The binding of R17* to DnaK can also be established from the 13C-1H HMQC spectrum of a sample prepared as 400 μM ILVM-13CH3 R17* and 800 μM I-13CH3 DnaK/ADP in which a number of new peaks are observed for the key Ile 401 and Ile 438 residues present at the central binding pocket of DnaK that are sensitive reporters of the binding event ( Rosenzweig et al . , 2017 ) ( Figure 3—figure supplement 2 , middle ) .", "The presence of several Ile 401 and Ile 438 resonances at distinct chemical shifts in the bound state demonstrates the existence of multiple bound conformations of the R17*-DnaK complex , interconverting with each other and with free DnaK in slow exchange on the NMR chemical shift timescale .", "Such conformational heterogeneity in DnaK-substrate interactions , observed previously in studies focused on the three-helix bundle client hTRF1 ( Rosenzweig et al . , 2017 ) , arises from the binding of DnaK to a number of sites on R17* that position different aliphatic hydrophobic amino acids of the client at the central groove of the DnaK binding site ( referred to as the 0 position ) .", "Indeed , the DnaK binding site prediction algorithm LIMBO ( Van Durme et al . , 2009 ) identifies at least two regions on R17* with a high propensity to interact with DnaK , 42QGLLKKH48 and 70EDLIKKN76 ( Figure 3B ) , each of which has two hydrophobic residues ( L44 and L45 in region 1 and L72 and I73 in region", "2 ) that could occupy the central cavity .", "Notably , substitution of the Leu pair at positions 44 and 45 with Ile leads to the appearance of new correlations in the Ile region of a 13C-1H HMQC spectrum recorded on a 200 μM ILVM-13CH3 L44I/L45I R17* sample to which 400 μM 2H DnaK/ADP has been added , confirming that the region containing residues 42–48 is a DnaK binding site .", "We have not been able to confirm DnaK binding to the second predicted site , as substitution of Leu 72 with Ile did not yield additional correlations in the Ile region of a spectrum recorded on a suitably prepared R17* , DnaK/ADP sample .", "In order to determine whether the new resonance in the HMQC spectrum of the R17*-DnaK complex belongs to a Met residue at the 0 position of the DnaK binding site we measured intermolecular NOEs between ILVM-13CH3 R17* and I-13CH3 DnaK/ADP ( Figure 3—figure supplement 2 ) .", "Here the ADP state of DnaK is used as it has higher affinity for substrates and the complex has lower koff rates ( Mayer and Bukau , 2005 ) .", "Previous X-ray and NMR structures of a variety of different complexes have established that Ile 401 and Ile 438 in the DnaK binding pocket are within 5 Å of the methyl moiety of the substrate residue located at position 0 ( Zhu et al . , 1996; Zahn et al . , 2013; Stevens et al . , 2003 ) and NOEs have been used to link Ile 401 , Ile 438 cross peaks with those from substrate residues that belong to the same conformation ( Rosenzweig et al . , 2017 ) .", "For R17* the bound Met methyl does not show NOE cross-peaks to Ile 401 and Ile 438 of DnaK , establishing that this Met residue is not located at the 0 position ( Figure 3—figure supplement 2 , left ) .", "However , NOE cross-peaks can be observed connecting pairs of Ile 401 and Ile 438 residues ( labeled as 'a' and 'b' ) with their substrate counterparts whose methyls are localized to the Leu region of the HMQC spectrum , establishing that at least two distinct Leu residues of R17* can be present at the DnaK 0 position in the R17*-DnaK ensemble ( Figure 3—figure supplement 2 , right ) .", "A number of biophysical studies using different spectroscopies such as NMR , fluorescence and circular dichroism have shown that DnaK-bound substrates are globally unfolded ( Palleros et al . , 1994; Chen et al . , 2006; Sharma et al . , 2010; Kellner et al . , 2014; Lee et al . , 2015; Sekhar et al . , 2015 ) .", "In order to determine the conformation of R17* in its DnaK-bound state we have compared the intensities of cross-peaks in 13C-1H HMQC spectra derived from native and unfolded states of R17* as probes .", "The intensities of native state Met and Ile resonances decrease by a factor of 1 . 6 ± 0 . 1 upon DnaK binding , consistent with solution equilibria shifting from N , and also with different 1H and 13C chemical shifts of R17* in the bound state from those in N . In contrast , intensities of Ile and Met correlations derived from the unfolded state increase by 2 . 2 ± 0 . 6 fold .", "The simplest interpretation of the data is that the Met and Ile residues of R17* in bound conformations have the same chemical shifts as U , with the degeneracy in shifts leading to an increase , rather than a decrease in unfolded peak intensities .", "This provides strong evidence that R17* is globally unfolded in the DnaK-bound state .", "The distinct 13C and 1H chemical shifts from the Met peak labeled 'Bound' in Figure 3D ( assigned as Met 26 , see below ) , that are not unfolded-like , reflect a conformation of R17*-DnaK whereby the Met is proximal to the DnaK binding site and hence shifted from the random coil region of the spectrum .", "Taken together , our data shows that DnaK-bound R17* is globally unfolded and resembles the U conformation of R17* that exists in equilibrium with N in the absence of DnaK .", "Having established that the bound form of R17* is structurally very similar to U we next turned to studying the binding reaction using zz-exchange magnetization transfer .", "This experiment is particularly powerful in the case of R17* because both the 1H and 13C chemical shifts of the one Met resonance that reports on the bound state are distinct from those derived from N and U , and the Met region of the 13C-1H HMQC spectrum is well resolved , facilitating the detection and quantification of cross-peaks between states .", "Figure 4A shows a 2D plane from the zz-exchange dataset acquired with a mixing time of 300 ms . A pair of cross-peaks can be observed , labeled 1 and 2 , connecting the unfolded state of Met 26 with the bound state and assigning the bound resonance to the methyl group of Met", "26 . Notably , there are no cross-peaks between N and the bound state , unequivocally demonstrating that DnaK interacts predominantly with the unfolded state of R17* .", "In order to obtain the binding and release rate constants for the reaction , U⇌kBUkUBUK , we measured diagonal ( unfolded and bound ) and cross-peak intensities for Met 26 as a function of mixing time values that ranged from 25 to 800 ms and fit the resulting profiles globally to extract kUB and kBU values of 0 . 8 ± 0 . 1 s−1 and 2 . 7 ± 0 . 2 s−1 , respectively ( Figure 4B ) .", "One dimensional reduced χ2 surfaces show that both rate constants can be obtained reliably from the exchange dataset ( Figure 4C ) , with errors in the pseudo-first order rate constant kUB and the first order constant kBU estimated by a bootstrapping procedure ( Efron and Tibshirani , 1986 ) , described in Materials and methods ( Figure 4D ) .", "On the basis of the relative fractional populations of N and U ( pN/pU ) , as estimated from the first plane of the pseudo-3D zz-exchange dataset with TMIX = 25 ms ( 1 . 8:1 ) along with pU/pB ( =kBU/kUB ) values from the time-dependent zz-exchange data ( 3 . 4:1 ) pN , pU and pB ( bound fraction ) are calculated to be 58 ± 2% , 33 ± 1% and 9 ± 2% , respectively .", "From the total DnaK concentration of 500 μM in the sample the bimolecular rate constant for the binding reaction is calculated to be konUB=kUB/[DnaK]=1600 ± 250 M−1s−1 .", "Note that this value is an underestimate because there are several bound conformations in solution ( see discussion above ) and we only focus here on the process for which the cross-peak of Met 26 of the bound state is resolved .", "Other complexes for which the Met 26 cross-peaks of the bound state are degenerate with the unfolded state are invisible to this analysis .", "Because there are no cross-peaks between states N and B only upper-estimates of kNB and kBN rates can be obtained by assuming that the N-B cross-peaks are at the noise level .", "Computations were performed , following the description outlined previously ( Huang et al . , 2016 ) , where relative intensities of the N and B diagonal-peaks and the N-B cross-peaks are calculated using experimentally measured values of relaxation times for longitudinal order , as a function of kNB and kBN .", "Upper bounds for kUN and kNU were calculated in a similar manner as for kNB , kBN since exchange-based cross-peaks reporting on the U , N interconversion are absent as well .", "The values for all of the rate constants ( including upper bounds in some cases ) are illustrated in Figure 4E that shows the R17* binding mechanism .", "The flux along the CS pathway is theoretically defined as the sum of fluxes for all pathways leading to the bound state via U that do not involve the NK→UK transition .", "Similarly , the net flux through the IF pathway is the sum of the flux values for all paths that do not involve the U→UK step .", "In practice , however , the U↔N transition is very slow for R17* with an upper bound of 0 . 01 s−1 for kex , UN so that the dominant flux contribution along the CS pathway derives from the U→UK transition while an upper bound for the IF pathway ( N→NK→UK ) can be estimated from the absence of an N , UK cross-peak .", "The relative partitioning of the flux along the CS ( FCS ) and IF ( FIF ) paths , Θ , is thus defined asΘ=FCSFIF=konUB[DnaK][U]konNB[DnaK][N]=kUBkNBpUpN From kUB = 0 . 8 s−1 and kNB <0 . 004 s−1 , as well as pN/pU = 1 . 8 , Θ >113 .", "Flux measurements thus confirm that the mechanism of R17* binding to DnaK is overwhelmingly biased in favour of CS ( Figure 4E ) .", "A histogram of the quantified flux values for the U→UK ( lower bound ) , N→U→UK and N→NK→UK pathways ( only upper bounds ) is presented in Figure 4F .", "Having established that the binding of the α-helical bundle domain R17* to DnaK proceeds through a CS mechanism we next addressed the dependence of binding on the secondary structure of the client protein by choosing the incomplete 5-stranded β-barrel SH3 domain of the Drosophila melanogaster Enhancer of sevenless 2B protein ( drkN SH3 , Figure 5A , B ) as a client .", "DrkN SH3 is a slow-folding protein with kex , UN ~ 1 s−1 , 20°C , and is marginally stable , populating a significant fraction of U at equilibrium ( pN/pU ~ 2 , pH 6 . 0 , 20°C ) ( Farrow et al . , 1994; Tollinger et al . , 2001 ) .", "Previous studies have established that the DnaK bound state of drkN SH3 is structurally similar to U ( Lee et al . , 2015 ) .", "Figure 5 shows the methyl spectrum of 250 μM IM-13CH3 drkN SH3 without ( C ) , and with ( D ) 500 μM U-2H DnaK/ATP .", "The binding of SH3 to DnaK can be clearly visualized through the appearance of at least four new peaks , labeled 1–4 in Figure 5D .", "Two of these , 1 and 2 , are particularly intense and separated in the 1H dimension from other resonances derived from the N and U states of drkN SH3 .", "The 13C-1H HMQC spectrum of a sample of 500 μM IM-13CH3 DnaK/ADP and 250 μM ILVM-13CH3 drkN SH3 is shown in Figure 5—figure supplement 1 ( middle ) .", "There are at least three new resonances for each of the DnaK binding site residues Ile 401 and Ile 438 when drkN SH3 is present , confirming that the SH3 domain binds to the canonical binding pocket on DnaK and highlighting the conformational heterogeneity in the SH3-DnaK ensemble , as has been seen in all other folding competent clients that we have studied to date .", "The identity of the residues of drkN SH3 at position 0 of the DnaK binding pocket have been obtained by recording a 3D 13C-edited NOESY dataset on the same sample ( Figure 5—figure supplement 1 ) .", "Notably , there are NOEs connecting the two Ile peaks 1 and 2 of drkN SH3 to Ile 438 of the chaperone , indicating that peaks 1 and 2 arise from Ile at the 0 position of the DnaK binding site .", "In contrast , NOEs from Leu residues in drkN SH3 to Ile 401 and Ile 438 are not observed .", "The absence of Leu residues at the binding site may possibly account for the low affinity of drkN SH3 for DnaK , even in the high affinity binding state where DnaK is ADP loaded .", "Cavagnero and coworkers report a KD of 243 μM for drkN SH3 and DnaK/ADP ( Lee et al . , 2015 ) that is at least two orders of magnitude weaker than literature values for peptides or proteins where Leu residues can occupy position 0 ( Pierpaoli et al . , 1998 ) .", "While the 1H chemical shifts of the bound Ile peaks 1 and 2 from drkN SH3 are unique , their 13C shifts are nearly degenerate with those from Ile 27 and Ile 4 of the N state , respectively , ( Figure 5D ) which complicates the quantification of potential N-B cross-peaks in a zz-exchange dataset .", "Thus , we have measured 1H CEST spectra to obtain the kinetics of drkN SH3 binding to DnaK/ATP using the pulse sequence shown in Figure 6—figure supplement 1A .", "Our choice of exploiting the 1H nucleus in CEST studies is motivated by the fact that the largest chemical shift differences between folded , unfolded and bound state peaks in methyl-based 13C-1H HMQC datasets of drkN SH3 are in the 1H dimension .", "However , 1H CEST can be prone to artifacts arising from NOE effects that lead to spurious minor dips that do not report on conformational exchange ( Sekhar et al . , 2016; Bouvignies and Kay , 2012 ) .", "In order to minimize these NOE-dips we have used highly deuterated I-13CH3 labeled drkN SH3 for acquiring CEST data and fortunately , there are only two pairs of Ile residues that are within 5 Å , considerably reducing the number of possible NOE dips .", "Finally , the mixing time during which exchange is quantified was set to 150 ms , a reasonably small value that further minimizes the sizes of these spurious dips .", "In principle it is possible to eliminate NOE effects completely using a spin-state selective 1H CEST scheme that has been proposed recently ( Yuwen et al . , 2017 ) .", "However , this comes at a considerable cost in sensitivity ( more than a factor of two ) and since signal-to-noise is limiting in applications involving very slowly exchanging conformers ( see below ) or when short mixing times are selected we have chosen not to use this approach here .", "In order to interpret CEST profiles it was necessary to first assign peaks 1 and 2 to specific Ile residues in drkN SH3 .", "This was achieved using a zz-exchange experiment that links peaks 1 and 2 to the corresponding U state correlations , that were already assigned by traditional NMR methods .", "In this way we could show that peaks 1 and 2 are derived from Ile 53 and Ile 27 , respectively ( Figure 6—figure supplement 2 ) .", "Since we know from NOE data that both peaks 1 and 2 are present at the 0 position , it follows that peaks 1 and 2 report on two distinct DnaK-bound conformations of drkN SH3 , labeled as B1 and B2 respectively .", "In the B1 conformer Ile 53 is at the 0 position while in B2 the 0 position is occupied by Ile", "27 . Figure 6 shows 1H CEST profiles from Ile 27 and Ile 53 methyl groups of folded ( A , D ) , unfolded ( B , E ) and DnaK-bound ( C , F ) drkN SH3 , acquired on a sample with 250 μM IM-13CH3 drkN SH3 and 500 μM U-2H-DnaK/ATP .", "Recall that in CEST spectra measured by recording the intensity of a peak from state j as a function of irradiation frequency the major dip derives from that state , while minor dips link state j with other states in the exchanging network .", "The native state CEST profiles of both Ile 27 and Ile 53 show only one minor dip each , at the resonances frequencies of the corresponding 1Hδ1 shifts in their respective unfolded states .", "These minor dips result from the U-N conformational exchange .", "However , the corresponding CEST traces from both residues in U show minor dips to N as well as to the bound state , unambiguously demonstrating that the major flux for the binding reaction involves a pathway whereby DnaK selects the unfolded state of drkN SH3 .", "This conclusion is reinforced by CEST profiles of the bound state peaks where only one strong minor dip is observed in each trace that links the bound state with U . In contrast to DnaK/ATP , the CEST profiles of drkN SH3 bound to DnaK/ADP do not show any signature of binding/release ( Figure 6—figure supplement 1B ) , though the presence of the bound states B1 and B2 can clearly be discerned from 13C-1H HMQC spectra recorded on the same sample .", "This is a consequence of the slow rates of substrate release from DnaK/ADP ( ~0 . 001 s−1 , 25°C ) ( Mayer and Bukau , 2005; Pierpaoli et al . , 1998 ) , which places the exchange process outside the window of detection via CEST methods ( Vallurupalli et al . , 2017 ) .", "The presence of CEST minor dips with DnaK/ATP and not with DnaK/ADP also confirms that DnaK is predominantly in the ATP-bound form in the sample used for acquiring CEST data in Figure", "6 . Analysis of the 1H CEST profiles for the drkN SH3 system is challenging because of the measurable exchange between U and N , and U and UK , and by the presence of two bound states .", "Accordingly , we fit the CEST profiles from the Ile 27 and Ile 53 N and U peaks , as well as the profiles of I53B1 and I27B2 peaks globally to the four-state model shown in Figure", "7 . The intermediate state along the IF pathway , corresponding to NK , is not included in the model because there is no evidence from 13C-1H HMQC spectra of SH3/DnaK samples or from the CEST profiles justifying the explicit inclusion of such a state; instead , the three-state N-NK-UK pathway was approximated as N-UK .", "The rate constants and populations that have been obtained from our modeling analysis are shown in Figure", "7 . The reliability of the kinetics parameters was evaluated by generating 1D reduced χ2 surfaces as a function of each of the eight global variables ( 5 rates , 3 populations; Figure 7—figure supplement 1 ) .", "These show pronounced minima for all five rate constants kex , UB1 , kex , NB1 , kex , UB2 , kex , NB2 and kex , UN . However , the minima are very shallow and broad for the populations , indicating that only the rate constants and not the populations can be obtained reliably from the CEST data .", "The flux ratio Θ for the U→UK and N→UK pathways based on the simultaneous fit of CEST profiles from Ile 27 and Ile 53 is given byΘ=konUB[DnaK][U]konNB[DnaK][N]=kex , UB ( pBpU+pB ) pUkex , NB ( pBpN+pB ) pN=kex , UBkex , NBΞwhere kex , UB and kex , NB for both the B1 and B2 arms of the binding scheme of Figure 7 can be obtained reliably .", "In order to estimate Ξ , we fit the CEST data using a bootstrap procedure ( Efron and Tibshirani , 1986 ) described in Materials and methods; the resulting distribution of Ξ values is narrow and centred around ~1 , with 98% of the values falling within the range of 0 . 84–1 . 27 for state B1 ( Ile 53 at the 0 position ) and 0 . 49–1 . 57 for B2 ( Ile 27 at the 0 position ) ( Figure 7—figure supplement 2 ) .", "The average and standard deviation of the Ξ distribution are 1 . 0 ± 0 . 1 and 1 . 0 ± 0 . 2 for the B1 and B2 arms respectively .", "This implies that Θ is well approximated by the ratio kex , UB/kex , NB .", "Rate constants and their errors , as reported in Figure 7 , are listed as the means and standard deviations of the respective distributions obtained from a bootstrapping analysis .", "Using these values , Θ1 and Θ2 for the formation of bound states B1 and B2 corresponding to Ile 53 and Ile 27 of drkN SH3 at position 0 of DnaK are found to be 4 and 21 respectively .", "Figure 7B and C show histograms of flux values for U→UK , N→U→UK and N→UK pathways .", "The flux through N→NK→UK could not be estimated because NK correlations are not observed , but an upper bound is given by the flow through N→UK .", "Thus our data strongly support the notion that the dominant flux is through the U→UK pathway whereby DnaK directly selects the U state , and therefore the binding mechanism can be described according to the CS model .", "The Hsp70 family of chaperones are integral to the quality control machinery in organisms ranging from bacteria to humans and the Hsp70 client binding site sequence and architecture have been highly conserved through evolution .", "We thus wondered if the mechanism of client substrate recognition by Hsp70 is also conserved and whether a CS mode of binding might also apply to other Hsp70 chaperones .", "To this end we choose the constitutively expressed human Hsc70 for study and focused on the drkN SH3 substrate .", "Upon addition of 500 μM U-2H , Hsc70/ATP to a solution of 170 μM IM-13CH3 drkN SH3 , no new peaks were observed ( Figure 8A , B ) , suggesting that the affinity of drkN SH3 for Hsc70/ATP is lower than that for DnaK/ATP ( Figure 5 ) .", "A reduced affinity would lower the population of UK and in the present case render it invisible to study by traditional NMR methods .", "For applications of this sort CEST is particularly powerful because the signals from sparsely populated protein conformations can be amplified by detecting them through NMR visible states ( Vallurupalli et al . , 2017 ) .", "Figure 8C , D shows 1H CEST profiles derived from Ile 27 of N and U drkN SH3 .", "Similar to our observations with DnaK , the native state CEST profile ( red ) shows a minor dip at the frequency of Ile 27 Hδ1 in the unfolded state , while the unfolded state profile ( green ) shows a pair of dips , with one at the position of the native state for Ile 27 Hδ1 and a second , initially unassigned .", "There are several lines of evidence that strongly indicate that the unassigned minor dip arises from Hsc70-bound drkN SH3 .", "First the chemical shift of the dip , 0 . 35 ppm , is close to 0 . 2 ppm that was measured for the DnaK bound states B1 and B2 ( Figure 5 ) .", "Second , when CEST profiles are recorded of drkN SH3 bound to DnaK/ADP , where the exchange kinetics are much slower than for the ATP loaded chaperone form and too slow to observe exchange derived dips ( see above ) , a minor dip in the region between 0 . 2–0 . 4 ppm is not observed in the unfolded state CEST profile ( Figure 6—figure supplement 1B , middle ) .", "Finally , a 13C-1H HMQC spectrum recorded after leaving the sample for approximately 2 days at room temperature , sufficient time for Hsc70/ATP to convert to Hsc70/ADP , shows a new peak at the 1H chemical shift of the minor dip , 0 . 35 ppm .", "This new peak belongs to Hsc70/ADP-bound drkN SH3 and is now visible because Hsc70/ADP has a higher affinity for substrate than Hsc70/ATP and the population of the bound state is higher .", "Notably , in the N state CEST profile a small broad peak is observed at the position of Ile 27 Hδ1 in the bound state ( ~0 . 35 ppm ) .", "This dip is not the result of exchange between N and UK but can be attributed , instead , to an NOE between proximal residues Ile 27 and Ile 48 in the native drkN SH3 state and the fact that the bound state Hδ1 chemical shift for Ile 27 is degenerate with the 1H Ile δ1 shift of residue 48 in the native state .", "Confirmation that this is an NOE dip and not due to exchange is obtained by measuring CEST profiles of drkN SH3 bound to DnaK/ADP where an NOE dip is observed as well ( Figure 8E ) but , as discussed above , where the rates of binding/release are too slow for the development of minor dips resulting from exchange and by the observation of a distinct NOE crosspeak between Ile 27 and Ile 48 in a 3D 13C-13C-1H NOESY spectrum of ILVM-13CH3 drkN SH3 .", "Taken together our results thus confirm that the major flux to UK proceeds through U so that human Hsc70 also employs a CS mode of interaction with the drkN SH3 client protein .", "As a final note , we have chosen not to quantify fluxes in this case because a bound state profile could not be obtained ( peaks from the bound state are not observed in spectra ) and because of the potential for contamination from NOE dips ." ], [ "The Hsp70 chaperone plays a ubiquitous role in maintaining cellular proteostasis by recognizing and modulating the structure of its client proteins in an ATP-dependent manner to effect a variety of downstream functions that include protein folding , translocation and oligomer disassembly ( Balchin et al . , 2016 ) .", "Structures of Hsp70 bound to a number of different peptide substrates have appeared ( Zhu et al . , 1996; Zahn et al . , 2013; Stevens et al . , 2003; Clerico et al . , 2015 ) and both biochemical and biophysical studies of Hsp70 with folding competent protein domains and intact proteins have been reported ( Kellner et al . , 2014; Lee et al . , 2015; Sekhar et al . , 2015; Sekhar et al . , 2016; Rodriguez et al . , 2008; Sekhar et al . , 2012a; Sekhar et al . , 2012b ) .", "However , the coupling between chaperone binding and substrate conformational changes remains poorly understood .", "Understanding the mechanisms by which flexible molecules bind their targets has been the focus of considerable attention in the literature ( Boehr et al . , 2009; Rauh et al . , 2004; Zhang et al . , 2007; Mittag et al . , 2010 ) .", "In general , such molecules exist in an array of different interconverting conformations in solution at equilibrium , yet binding to a protein generally selects one of the states over the others .", "In principle , NMR spectroscopy is a valuable tool for obtaining mechanistic information about the binding process if the kinetics happen to be in a regime that is amenable to the methodology .", "In the case of zz-exchange this requirement puts exchange rates on the order of 1 s−1 or larger , although in some cases significantly slower timescales have been studied using methionine methyl probes with very slow longitudinal relaxation rates ( Religa et al . , 2010 ) .", "The CEST experiment , in general , is most sensitive to slightly faster processes with rates of at least 20–30 s−1 , but in the present application accurate estimates for rates of ~1 s−1 could be obtained because profiles derived from spins in all of the interconverting states could be measured .", "An elegant illustration of the utility of NMR in this regard was provided close to forty years ago in studies by Birdsall and coworkers using a one-dimensional analogue of the CEST approach described here ( Birdsall et al . , 1980 ) .", "The authors were able to demonstrate that the enzyme dihydrofolate reductase ( DHFR ) selects one conformation of folinic acid , a product of the reaction with tetrahydrofolate that is catalyzed by DHFR , over a second , under conditions where both conformations exist in roughly equal proportions in solution in the absence of enzyme .", "Here we have extended this work by using state of the art NMR methods that include relaxation optimized techniques for recording spectra of methyl group probes ( methyl TROSY ) in high molecular weight complexes ( Rosenzweig and Kay , 2014 ) and two-dimensional spin relaxation experiments such as CEST ( Vallurupalli et al . , 2017 ) and magnetization exchange ( Farrow et al . , 1994; Palmer et al . , 2001 ) to probe the binding reaction of Hsp70 with cognate substrates .", "It is increasingly recognized that protein molecules , much like simpler small organic compounds such as folinic acid , exist in solution as an equilibrium mixture between multiple conformations , and that these diverse conformers may in some cases have different functional properties , including different propensities for binding to ligands .", "The targets of Hsp70 include an array of different protein substrates and elucidating what , if any , common structural features are recognized by the chaperone during the initial stages of binding to these targets would represent an important contribution towards understanding the overall binding mechanism .", "We have studied the binding reaction of Hsp70 from E . coli ( DnaK ) to a pair of substrates with very different secondary structures , including the three-helical bundle domain R17* from chicken-brain α-spectrin and the all β-sheet SH3 domain from the Drosophila adaptor protein Drk .", "Both of these protein domains exist in solution as an equilibrium between N and U states , with peaks from each conformer observed in 13C-1H correlation datasets so that probes are available that can report on preferred chaperone binding to either of these two predominant solution conformations .", "Relaxation based experiments establish that in both cases the dominant flux to the bound state , in which the substrate is unfolded in complex with DnaK ( Lee et al . , 2015 ) , proceeds through the single step binding reaction U →UK as opposed to any scheme in which binding to N is the first step in an NK to UK transition .", "In order to obtain insight into whether the DnaK binding mechanism is conserved among different Hsp70 orthologs we have carried out further studies using the constitutively expressed human Hsc70 protein and the drkN SH3 substrate .", "Notably , peaks from the bound state were not observed in spectra , reflecting the low affinity of the complex .", "However , an excited state dip was observed in the 1H CEST profile of Ile 27 Hδ1 in U that could be assigned to the ‘invisible’ bound state , while a corresponding peak from chemical exchange was not observed for N . This establishes that the flux to the Hsc70 bound state , like that for DnaK , also proceeds via a U →UK mechanism .", "It is worth noting that all of the flux measurements described here were done at equilibrium , with the stability of samples monitored by recording a series of 13C-1H correlation maps at different time points .", "It is , of course , possible to envision non-equilibrium measurements where the relative importance of different fluxes change over time as populations of states vary during the course of reactions , but this was not the case for the equilibrium studies conducted here .", "Our data thus point to the conformational selection model as a good descriptor of Hsp70-ligand binding , at least for the substrates and chaperones that are considered in the present study .", "It is important to emphasize that the analysis of flux through IF and CS pathways discussed here is somewhat different than what is typically described in the literature where a comparison of fluxes through N→U→UK ( CS ) and N→NK→UK ( IF ) pathways exclusively is made , without considering binding proceeding directly from other equilibrium states ( U in this case ) ( Hammes et al . , 2009; Weikl and Paul , 2014; Vogt and Di Cera , 2012 ) .", "Here we have considered proteins as equilibrium ensembles , with binding reactions proceeding from any member of the ensemble , rather than focusing exclusively on the N conformer as the initial state .", "An analysis that includes the possibility of fluxes from any of the existing equilibrium states of a protein in solution is required for distinguishing between different mechanisms of binding .", "In the case here U is thermally accessible from N and is significantly populated at equilibrium , with the N→U→UK reaction contributing only a small amount to the net flux by which UK is formed relative to the U→UK pathway for the substrates and Hsp70 chaperones considered .", "The preference of Hsp70 for the unfolded state of the substrates demonstrates a 'holdase' recognition mechanism whereby Hsp70 captures intrinsic fluctuations in its client protein .", "Our results argue against a simple N→NK→UK unfoldase scheme as a dominant binding mechanism , where the native state of the substrate is recognized by Hsp70 and induced to unfold upon binding .", "However , it must be emphasized that we have focused on elementary binding steps here and our results are not inconsistent with broader unfoldase mechanisms involving recognition of partially unfolded or misfolded regions of substrates that eventually result in unfolded Hsp70-bound clients via a multistep process .", "For example , such a mechanism may be operative in the conversion of a misfolded state of luciferase to a globally unfolded , folding-competent ensemble ( Sharma et al . , 2010 ) where the elementary binding step is a holdase-like CS reaction , U →UK , involving trapping by Hsp70 of local conformational fluctuations occurring in a small region of the protein .", "A CS-based mechanism for Hsp70-substrate interactions is consistent with expectations based on Hsp70 function .", "First , such a binding mode ensures that Hsp70 will not randomly unfold properly folded and functional cellular proteins .", "Second , a number of cognate Hsp70 substrates such as nascent polypeptides synthesized on the ribosome ( Teter et al . , 1999 ) and proteins translocated across membranes ( Pilon and Schekman , 1999 ) are unfolded and would therefore be poised for binding to Hsp70 .", "Third , the CS binding mode ensures that Hsp70 will select only misfolded intermediates lacking stable secondary and tertiary structure for refolding rather than stably folded native conformations with exposed hydrophobic sidechains so that refolding occurs in a unidirectional manner from the misfolded to the native state .", "Finally , Hsp70 recognition sites on proteins such as σ32 that bind in their native state are in long solvent-exposed loop regions ( Rodriguez et al . , 2008 ) where both sidechain and backbone motifs of the binding site residues are available for interaction .", "It should also be noted that there are likely to be small differences in structure between the Hsp70-bound ( UK ) and the globally unfolded state ( U ) of substrates .", "Indeed , conformational sampling in the bound state is expected to be different from the globally unfolded state because each Hsp70 molecule divides the substrate into two distinct polypeptide segments at the binding site .", "We have shown earlier that tertiary interactions that are transiently present in the U state of the unbound substrate are disrupted across the Hsp70 binding site ( Sekhar et al . , 2016 ) , so that the two segments could have different overall structural propensities than would be the case for the full-length protein in its unfolded state .", "We cannot determine through our measurements whether in this case these small changes have occurred as a result of binding ( IF ) or prior to it ( CS ) .", "Instead , our conclusions of CS being the prevalent mechanism , therefore , pertain to binding events involving interactions of the chaperone with states that have larger conformational differences such as N and U . The multiple Hsp70-bound conformations of R17* and drkN SH3 noted in this study reinforce our previous observations with the three-helix bundle substrate hTRF1 that Hsp70 binds promiscuously to its substrates , recognizing different sites containing aliphatic hydrophobic residues along the polypeptide sequence ( Rosenzweig et al . , 2017 ) .", "Our results show that each binding site of the substrate can be recognized by Hsp70 when it becomes exposed to the solvent in the thermally accessible unfolded state , highlighting the prominent role played by conformational dynamics in this crucial protein-protein interaction ." ] ]

[ "Molecular recognition is integral to biological function and frequently involves preferred binding of a molecule to one of several exchanging ligand conformations in solution .", "In such a process the bound structure can be selected from the ensemble of interconverting ligands a priori ( conformational selection , CS ) or may form once the ligand is bound ( induced fit , IF ) .", "Here we focus on the ubiquitous and conserved Hsp70 chaperone which oversees the integrity of the cellular proteome through its ATP-dependent interaction with client proteins .", "We directly quantify the flux along CS and IF pathways using solution NMR spectroscopy that exploits a methyl TROSY effect and selective isotope-labeling methodologies .", "Our measurements establish that both bacterial and human Hsp70 chaperones interact with clients by selecting the unfolded state from a pre-existing array of interconverting structures , suggesting a conserved mode of client recognition among Hsp70s and highlighting the importance of molecular dynamics in this recognition event ." ]

[ "Proteins are the workhorses of a cell and are involved in almost all biological processes .", "Newly made proteins need to ‘fold’ into precise three-dimensional shapes in order to carry out their roles .", "However , proteins sometimes fold incorrectly or unfold .", "These protein forms are not able to work effectively and in some cases may even cause diseases .", "Chaperone proteins help other proteins to fold correctly and are found in living organisms ranging in complexity from bacteria to humans .", "There are many different types of chaperones that play different roles inside cells .", "One , called Hsp70 , binds to proteins that are incorrectly folded to help them to mature into their correct structures .", "However , it was not clear whether Hsp70 can also associate with the mature , correctly folded form of the proteins .", "A technique called Nuclear Magnetic Resonance ( NMR ) spectroscopy can distinguish between mature , unfolded and chaperone-bound forms of the same protein .", "Sekhar et al . therefore used NMR to investigate which forms of a protein Hsp70 binds to .", "This revealed that both the bacterial and human versions of the Hsp70 chaperone interact only with unfolded proteins .", "The results presented by Sekhar et al . also explain why Hsp70 does not disrupt the routine workings of the cell: because it does not bind to mature forms of proteins .", "These observations extend our understanding of how chaperones assist in folding proteins , and fit into a broader research theme exploring how proteins recognize one another .", "It will now be interesting to see whether the same mechanism holds for more complex forms of proteins , such as aggregates , or larger protein structures with regions of both folded and unfolded elements ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "structural biology and molecular biophysics" ]

[ [ "An essential aspect of adaptive immunity is the ability of T cells to discriminate between structurally similar agonist and non-stimulatory self peptides bound to major histocompatibility complex ( pMHC ) molecules presented on the surface of antigen-presenting cells ( APCs ) ( Weiss and Littman , 1994 ) .", "Fewer than 10 agonist pMHC molecules can trigger T cells ( Irvine et al . , 2002; Manz et al . , 2011 ) , and the signaling response occurs on a timescale of seconds ( Huse et al . , 2007 ) .", "It has been recognized for decades that subtle , peptide-specific differences in binding parameters , especially kinetic rates for the pMHC and T cell receptor ( TCR ) reaction , seem to be the basis of antigen recognition ( Matsui et al . , 1994; Tian et al . , 2007 ) .", "Experimental correlations between molecular pMHC:TCR binding kinetics , typically determined from solution assays of soluble extracellular domains and bulk measurements of T cell activity , have provided the foundation for much of this understanding .", "From a physical point of view , the extreme sensitivity , selectivity , and apparent immunity to stochastic noise exhibited by TCR antigen recognition pose challenges to classical notions of ligand-receptor signaling ( van der Merwe and Dushek , 2011; Dustin and Groves , 2012 ) .", "For example , the maximum affinity a TCR can have for self pMHC ligands ( non-triggering ) is sharply defined and close to the affinity threshold for foreign pMHC ( triggering ) .", "Consequently , every TCR is likely to bind a subset of the ever-present self pMHC molecules with affinities differing only slightly from genuine foreign agonist pMHC .", "If TCR triggering and signaling were simply proportional to TCR occupancy by ligand , then the abundance of self pMHC could easily overwhelm genuine agonist signals , rendering this as an ineffective discriminatory mechanism .", "Alternatively , each engagement of pMHC with TCR ( serial triggering; Valitutti et al . , 1995 ) , perhaps with some minimum engagement time ( kinetic proof reading; McKeithan , 1995; Rabinowitz et al . , 1996 ) , might define the threshold for TCR triggering .", "Other proposed mechanisms elaborate further , suggesting ( pseudo ) heterodimers ( Irvine et al . , 2002; Krogsgaard et al . , 2005 ) of self and agonist pMHC molecules or pMHC-independent forms of trans activation ( Cooper and Qian , 2008 ) of multiple TCR by a single agonist pMHC may be at work .", "Proving or disproving any of these various possibilities based on current data is confounded by the vast difference between ensemble biochemical measurements and cell population behavior .", "Substantial ambiguity with respect to the actual molecular mechanisms responsible for antigen triggering of T cells remains .", "A physically accurate understanding of this remarkable process will require simultaneous observations of the molecular binding kinetics , stoichiometry , and movement of individual signaling complexes in living T cells .", "Here , we characterize the molecular interactions between pMHC and TCR , at the single molecule level , while simultaneously monitoring the local membrane recruitment of cytosolic Zeta-chain-associated protein kinase 70 ( ZAP70 ) in live primary T cells .", "Every pMHC can be individually resolved and tracked for up to minutes before photobleaching by using a multi-timescale single molecule fluorescence imaging approach .", "Key to this strategy is the variable control of excitation light dose and exposure time to achieve hardware discrimination of molecular species with different mobilities .", "The pMHC molecules fall into two unambiguously distinguished classes: one undergoing fast random motion and the second moving slowly along linear trajectories .", "These slow moving pMHC are the bound pMHC:TCR complexes; they are only observed with agonist peptide and they spatially correlate with both TCR and recruitment of ZAP70 .", "The linear trajectories of the pMHC:TCR complexes match the well-characterized cytoskeleton-driven movement of TCR during the formation of the immunological synapse ( Campi et al . , 2005; Yokosuka et al . , 2005; DeMond et al . , 2008; Yu et al . , 2012 ) .", "Thus a lone agonist pMHC bound to TCR leads to stable engagement of the resulting complex with the cytoskeleton .", "Single molecule intensity calibration of the number of ZAP70 recruited to the vicinity of each agonist pMHC indicates that TCR are triggered in a 1:1 stoichiometry with pMHC .", "Associations of pMHC with TCR exhibited molecular binding dwell times with mean durations of 53 . 8 ± 12 . 2 s and 5 . 2 ± 0 . 2 s for AND and 5c . c7 TCRs , respectively .", "Individual dwell times are roughly exponentially distributed and are in general agreement with bulk solution measurements of pMHC:TCR kinetic off-rates for both TCRs ( Corse et al . , 2010; Huppa et al . , 2010; Newell et al . , 2011 ) .", "However , dwell times measured from tracking experiments specifically correspond to spatial entrapment of pMHC with a TCR , or cluster of TCRs ( Schamel and Alarcon , 2013 ) , on the T cell surface .", "They do not necessarily correspond to individual molecular binding events with a single TCR .", "Indeed , recent studies ( e . g . , by FRET ) have suggested that pMHC:TCR kinetic off-rates may be accelerated in living cells relative to in vitro measurements , possibly as a result of actively applied forces from the cytoskeleton ( Huang et al . , 2010; Huppa et al . , 2010; Zhu et al . , 2013 ) .", "We explore the possibility that individual dwell times observed by tracking experiments could be composed of rapid unbinding and serial rebinding of pMHC to multiple TCR within a cluster .", "Results from a stochastic reaction-diffusion analysis , covering a wide range of parameter space , indicate that serial rebinding alone is unlikely to sustain entrapment .", "If pMHC thoroughly disengages from TCR , it will most likely escape .", "Structural flexibility within the pMHC:TCR complex ( Adams et al . , 2011; Hawse et al . , 2012; Reboul et al . , 2012 ) could give rise to apparently fast kinetics ( e . g . , in FRET measurements ) without actual unbinding of pMHC from TCR .", "The tracking observations reported here directly reveal that the functional interaction between agonist pMHC and TCR is long-lived in living cells and that triggered TCR remain localized with the same pMHC ." ], [ "We probe agonist pMHC:TCR complex dynamics in hybrid live cell—supported membrane junctions ( Grakoui et al . , 1999 ) ( Figure 1A ) .", "The supported membrane is functionalized with MHC ( IEk ) and intercellular adhesion molecule-1 fusion with a blue fluorescent protein ( ICAM1-TagBFP ) , both linked to the membrane via C-terminal poly-His tag binding to Ni-chelating lipids ( Nye and Groves , 2008 ) .", "The MHC is loaded with peptide ( moth cytochrome c [MCC] agonist or null ) , which is covalently coupled in a 1:1 stoichiometry ( verified by HPLC ) to the photostable fluorophores Atto647N or Atto488 using maleimide-thiol chemistry .", "Upon contact between the T cell and the supported membrane , Leukocyte function-associated antigen 1 ( LFA1 ) -ICAM1 binding leads to rapid cell spreading and formation of an essentially planar interface between the T cell and supported membrane , within which pMHC:TCR interactions occur . 10 . 7554/eLife . 00778 . 003Figure 1 . Agonist pMHC binding to TCR in T cells is revealed by changes in mobility .", "( A ) Schematic of the hybrid live cell–SLB system .", "Binding of agonist pMHC to TCR ( PDB , 3QIU ) leads to phosphorylation of ITAMs , on the intracellular domain of the TCR , which is followed by recruitment of the kinase ZAP70 ( PDB , 2OQI , 2OZO ) .", "We directly observe both pMHC:TCR binding and ZAP70:ITAM recruitment using single molecule fluorescence microscopy ( bilayer adapted from Domanski et al . , 2010 ) .", "( B ) At short exposure times ( 17 . 5 ms , left ) all agonist pMHC molecules are readily resolved .", "Imaging with a long exposure time ( 500 ms , right ) allows for unambiguous discrimination between the slow , TCR-bound fraction of agonist pMHC and the fast diffusing fraction .", "This also allows for long ( 1–10 s interval ) time-lapse sequences ( Video 1 ) .", "Automated detection of single molecule features ( red circles ) is discussed in methods .", "( C ) Representative intensity trace showing a single agonist pMHC molecule , identified by step photobleaching , bound continuously for ∼60 s .", "( D ) Step size histogram of single agonist pMHC molecules in a SLB is bimodal under T cells ( red ) and unimodal before addition of T cells ( blue ) .", "pMHC molecules in ( B ) – ( D ) were labeled with Atto647N on the MCC peptide . DOI: http://dx . doi . org/10 . 7554/eLife . 00778 . 003 At fast exposure times ( 17 . 5 ms ) and high excitation powers ( 0 . 2 W/cm2 ) , all pMHC molecules are readily resolved ( Figure 1B , left panel ) .", "The pMHC move as individual molecules , as identified by single-step photobleaching , and can be continuously tracked for up to hundreds of frames ( Figure 1C ) .", "In regions without a cell , pMHC exclusively exhibit random diffusive motion .", "The step-size distribution from these trajectories corresponds to a single lateral diffusion coefficient of 0 . 44 ( SEM = 0 . 002 ) µm2/s , which is typical for supported membranes ( Lin et al . , 2010 ) .", "Within the T cell junction , the step-size distribution becomes bimodal ( Figure 1D ) .", "In addition to a fast component from freely diffusing pMHC , a distinct slow-moving component also appears .", "At long exposure times ( 500 ms ) and low excitation powers ( 0 . 02 W/cm2 ) , the fast moving pMHC fraction in Figure 1D is averaged over several pixels to form a relatively homogenous background .", "The slow moving molecules remain highly localized and can be unambiguously tracked for longer than a minute , using 1–10 s time-lapses ( Figure 1B , right panel; Video 1 ) .", "The slow moving pMHC molecules colocalize with TCR ( Figure", "2 ) and move in linear trajectories toward the geometric center of the live cell-supported membrane junction; these are the pMHC:TCR complexes .", "When MHC is loaded with a mixture of agonist and null peptides , with different fluorescent labels , only the agonist peptides are observed in the slow-moving complexes ( Figure 3 ) .", "The result is identical when the fluorophores are reversed , excluding the possibility that fluorophore effects could be responsible for binding . 10 . 7554/eLife . 00778 . 004Video 1 . A 3 s time-lapse video ( 189 frames ) of agonist pMHC interacting with live 5c . c7 T cells ( labeled with MCC-Atto488 ) .", "A long ( 500 ms ) exposure time allows for unambiguous discrimination between TCR-bound and unbound agonist pMHC in the bilayer .", "The hardware filtering approach utilized here facilitates particle detection in a relatively dense field of fluorophores , allowing for linking particles between frames even when long ( 1–10s ) time lapses are introduced .", "This technique also has the advantage of using only one probe , compared to other techniques for detecting ligand binding , such as smFRET , which require two different color probes to detect one molecular binding event .", "These advantages allow for simultaneous two color single molecule tracking and kinetics measurements . DOI: http://dx . doi . org/10 . 7554/eLife . 00778 . 00410 . 7554/eLife . 00778 . 005Figure 2 . TCR and MCC agonist pMHC colocalization .", "( A ) and ( B ) TCR and agonist pMHC colocalize in bulk in the central supramolecular assembly cluster ( cSMAC ) and ( C ) at the single molecule level .", "Although TCR clusters were not readily observed in these experiments ( even using a 4 s camera integration time ) , previous reports indicate that TCR does cluster at the agonist density ( ∼0 . 2 molecules/µm2 ) used here ( Varma et al . , 2006 ) .", "However , these reports used GPI-MHC , which is problematic because this GPI-linked protein is associated with clustering in supported membranes ( Manz et al . , 2011; Dustin and Groves , 2012 ) .", "The Ni2+-chelating lipids used in supported membranes for the experiments reported here have been shown to increase the likelihood that attached proteins are monodispersed ( Manz et al . , 2011; Xu et al . , 2011 ) , and this is confirmed by direct single molecule observation in our experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 00778 . 00510 . 7554/eLife . 00778 . 006Figure 3 . Agonist binding is specific and independent of fluorophore . RICM images map the footprint of T cell adhesion to the SLB ( mediated through LFA1:ICAM1 binding ) .", "T cells engage SLBs conjugated with mixtures of independently labeled MCC agonist and null peptide MHC .", "Only the MCC agonist pMHC is observed in the slow moving fraction , irrespective of which fluorescent label ( Atto488 or Atto647N ) it carries . DOI: http://dx . doi . org/10 . 7554/eLife . 00778 . 006 The agonist pMHC densities used in these experiments range from 0 . 2 to 100 molecules/µm2 .", "The lowest densities are near thresholds for triggering Ca2+ flux ( Manz et al . , 2011 ) and below levels where stable TCR microclusters are readily visible ( Figure 2 ) , whereas the higher densities are well above the densities at which microclusters are observed ( Campi et al . , 2005; Yokosuka et al . , 2005; DeMond et al . , 2008; Yu et al . , 2012 ) .", "Nonetheless , the observed single molecule pMHC motion at all densities is reminiscent of the well-characterized actomyosin-driven TCR microcluster radial transport in cells that are activated ( Campi et al . , 2005; Yokosuka et al . , 2005; Kaizuka et al . , 2007; DeMond et al . , 2008; Yu et al . , 2012 ) .", "Two-color single molecule tracking is used to quantitatively monitor membrane recruitment of cytosolic ZAP70-EGFP ( incorporated by retroviral transfection ) to the locations of the pMHC:TCR complexes .", "Using a dual-view system in which chromatic aberrations have been mapped ( Figure 4—figure supplement 1 ) , spatial colocalization between the two channels to less than 105 nm is achieved .", "Immediately after cell landing , ZAP70 localizes to and moves together with the pMHC:TCR complexes ( Figure 4A; Videos 2 and 3 ) .", "For each frame in a tracking sequence , fluorescence intensity in the ZAP70-EGFP channel is integrated over a 315 nm square region centered on each pMHC:TCR complex ( Figure 4B ) .", "The resulting intensity traces reveal discrete changes in average intensity , which we attribute to the binding of one ZAP70-EGFP to the phosphorylated ITAM domains on the cytoplasmic side of TCR engaged with pMHC ( Iwashima et al . , 1994 ) .", "Representative traces of colocalized ZAP70-EGFP intensity along with the corresponding intensity trace from the pMHC are illustrated in Figure 4C .", "Stochastic transitions are identified using a change-point algorithm ( Ensign and Pande , 2009 ) and results from this analysis are superimposed on the raw intensity traces .", "Observed molecular binding dwell times of individual ZAP70-EGFP , as resolved by the stochastic transitions , range from 12 to 107 s .", "The majority of single molecule ZAP70-EGFP traces exhibit intensity fluctuations consistent with background ( Figure 4—figure supplement 2A ) , which would not be expected if cytosolic ZAP70-EGFP exchanged during exposure time .", "These dwell times are slightly longer than estimates of ∼10 s obtained from bulk fluorescence recovery after photobleaching ( FRAP ) experiments ( Bunnell et al . , 2002 ) .", "However , those experiments also identified a slower-exchanging fraction of ZAP70 that was not included in the average . 10 . 7554/eLife . 00778 . 007Figure 4 . ZAP70 recruitment , stoichiometry , and movement are consistent with 1:1 agonist pMHC:TCR stoichiometry .", "( A ) A spatial map of MCC-Atto647N single molecule ( red ) and ZAP70-EGFP ( blue ) puncta .", "Raw data are included as Videos 2 and 3 .", "Data were recorded at 1 frame/s , such that each adjacent blue or red dot was recorded 1 s apart .", "Both single MCC agonist pMHC molecules and ZAP70-EGFP puncta follow linear trajectories towards the geometric center of the 2D cell–supported bilayer interface .", "( B ) Single ZAP70-EGFP molecules recruited to single agonist pMHC molecules ( labeled with MCC-Atto647N ) are recorded using sub-pixel color registration ( Figure 4—figure supplement 1 ) .", "( C ) Representative single molecule ZAP70-EGFP ( blue ) and MCC-Atto647N agonist pMHC ( red ) localized fluorescence intensity traces .", "Change-points are detected using a Bayesian change point algorithm ( in black; see methods ) .", "Step decreases in MCC intensity ( red ) are most likely agonist pMHC:TCR unbinding events , since τoff≪τbl .", "Step increases in ZAP70 intensity ( blue ) are attributed to ZAP70:ITAM binding .", "( D ) ZAP70-EGFP puncta brightness histogram is symmetric and centered at 136 . 0 counts ± 0 . 04 SEM and corresponds to an average of 2 . 9 ± 0 . 04 SEM EGFP molecules ( using six single molecule ZAP70-EGFP traces from the same cell for intensity calibration ) .", "Bright ZAP70-EGFP speckles are detected from the raw data using an automated algorithm ( blue circles; see methods ) .", "Scale bar 5 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 00778 . 00710 . 7554/eLife . 00778 . 008Figure 4—figure supplement 1 . Dual View color registration .", "( A ) Tetraspec beads adhered to piranha-etched coverglass are used to spatially register the EGFP and Atto647N channels in our split camera apparatus .", "Ten consecutive images are recorded and the coordinate map is generated in post processing ( see ‘Materials and methods’ ) .", "( B ) and ( C ) Registration to less than one pixel ( 105 nm ) is achieved across the majority of the 26 × 52 µm imaging area .", "T cells are typically 10 µm in diameter , and are imaged at the center of the field of view . DOI: http://dx . doi . org/10 . 7554/eLife . 00778 . 00810 . 7554/eLife . 00778 . 009Figure 4—figure supplement 2 . Single molecule ZAP70-EGFP .", "( A ) The majority of single molecule agonist pMHC traces result in ZAP70-EGFP traces with intensity fluctuations consistent with the background .", "Step increases and decreases in ZAP70-EGFP intensity are rare , but single ZAP70-EGFP events are long-lived ( 12–107 s ) .", "Taken together , these observations indicate that rapid ZAP70-EGFP unbinding and rebinding in between observations is unlikely .", "If rapid unbinding/rebinding were to occur , then either single molecule ZAP70-EGFP traces would more frequent and shorter , or ZAP70-EGFP traces without single step intensity increases or decreases would be noisier .", "( B ) A small minority of agonist pMHC traces exhibit two-step photobleaching , indicating pMHC dimerization .", "However , the ZAP70-EGFP output for these traces is equivalent to the ZAP70-EGFP output for single agonist pMHC single molecule traces . DOI: http://dx . doi . org/10 . 7554/eLife . 00778 . 009 ZAP70-EGFP features brighter than single molecules are also observed and we calibrate their stoichiometries using single molecule ZAP70-EGFP intensities from the same cell .", "For example , the average ZAP70-EGFP feature intensity per frame ( 136 . 0 counts ± 0 . 04 SEM , integrated over a 315 nm square area for each feature ) for the cell shown in Figure 4D corresponds to 2 . 9 ± 0 . 04 SEM ZAP70-EGFP per feature , given that the single molecule intensity is 47 ± 2 counts ( SEM; n = 6 ) in that cell .", "Each feature therefore contains on average ∼6 ZAP70 molecules , since ZAP70-EGFP was expressed in a roughly 1:1 ratio with endogenous ZAP70 ( selected by FACS and confirmed by western blotting ) in these experiments .", "Each TCR complex has 10 ITAM domains that , when phosphorylated , can bind one ZAP70 each ( Weiss and Littman , 1994 ) .", "Thus observation of ∼6 ZAP70 per agonist pMHC suggests only a single TCR is triggered .", "Brighter ZAP70-EGFP features tend to be located towards the geometric center of the cell at later time points ( >5–10 min after T cell landing ) .", "These can be tracked for tens to hundreds of seconds , indicating that recruited ZAP70-EGFP remain stably associated with the pMHC:TCR complex while traveling along the same linear trajectories ( Figure 4A; Videos 2 and 3 ) .", "The observation of a range of ZAP70-EGFP stoichiometries ( from 1 to ∼10 ZAP70-EGFP per pMHC:TCR complex ) implies that some time-dependent accumulation of ZAP70 is likely to occur , although we have not definitively observed ZAP70-EGFP accumulation over time within individual intensity traces .", "Taken together , these observations demonstrate that engagement of TCR with an individual agonist pMHC molecule leads to stable association with the actin cytoskeleton , one-to-one TCR triggering ( ITAM phosphorylation ) , and subsequent ZAP70 recruitment .", "Moreover , since every pMHC:TCR:ZAP70 complex is individually resolved in these experiments , we demonstrate that a single pMHC:TCR complex can lead to TCR triggering without molecular-scale association with other MHC molecules . 10 . 7554/eLife . 00778 . 010Video 2 . Simultaneous observation of ZAP70-EGFP recruitment and pMHC:TCR binding immediately after a living AND T cell lands on the SLB . ZAP70-EGFP membrane recruitment ( Video", "2 ) and pMHC:TCR binding ( Video", "3 ) occur almost immediately after landing .", "Radial transport of pMHC:TCR:ZAP70 complexes commences immediately after landing .", "Data were recorded at 1 frame per second with a 500 ms integration time .", "These data were analyzed to create the spatial map of pMHC and ZAP70 positions displayed in Figure 4A . DOI: http://dx . doi . org/10 . 7554/eLife . 00778 . 01010 . 7554/eLife . 00778 . 011Video 3 . Simultaneous observation of ZAP70-EGFP recruitment and pMHC:TCR binding immediately after a living AND T cell lands on the SLB . This video shows binding of pMHC:TCR , and is from the same cell as the ZAP70-EGFP data in Video 2 .", "Agonist pMHC is labeled as MCC-Atto647N .", "These data were analyzed to create the spatial map of pMHC and ZAP70 positions displayed in Figure 4A . DOI: http://dx . doi . org/10 . 7554/eLife . 00778 . 011 Since the slow-moving pMHC can be clearly resolved from the fast moving component , the lifetime of molecules in this bound state is directly observable .", "The minimum detectable lifetime is limited by the fastest frame rate ( 17 . 5 ms per frame ) and the maximum measureable lifetime is limited by photobleaching .", "For these single molecule tracking experiments , the temporal dynamic range spans from ∼50 ms to ∼5 min .", "Unbinding and photobleaching are indistinguishable in fluorescence methods such as this .", "For molecular binding , characterized by a constant kinetic off-rate , the distribution of observed dwell times , τobs , is described byf ( τobs ) = ( 〈τbl〉−1+〈τoff〉−1 ) e−τobs ( 〈τbl〉−1+〈τoff〉−1 ) , where 〈τbl〉−1 is the photobleaching rate ( kb ) , 〈τoff〉−1 is the unbinding rate ( koff ) , and ( 〈τbl〉−1+〈τoff〉−1 ) −1=〈τobs〉 is the observed mean dwell time in this experiment .", "The observed dwell time distributions are roughly exponential , as is expected for molecular binding .", "Thus by measuring both kbl and 〈τobs〉 it is possible to determine 〈τoff〉 as long as 〈τobs〉≤〈τbl〉 .", "We determine 〈τoff〉 to be 53 . 8 ± 12 . 2 s for AND and 5 . 2 ± 0 . 2 s for 5c . c7 TCRs for Atto488-labeled peptide agonist with 〈τbl〉 of 300 and 30 s respectively ( Figure 5A , B ) .", "While fluorescent labels can affect binding kinetics , we measure similar values of 〈τoff〉 with both Atto647N and Atto488 labeled peptides ( see , e . g . , Figures 1C , 3 , and 4A , C ) .", "〈τoff〉 is also relatively unchanged at high agonist pMHC density ( 100 molecules/µm2 ) , which is far above minimal levels required for T cell activation and observation of stable TCR microclusters ( Manz et al . , 2011 ) ( Figure 5C ) .", "We observe that cytoskeleton disruption by the actin-binding molecule , Latrunculin A , moderately increases 〈τoff〉 with the AND TCR and had no significant effect on 5c . c7 kinetics ( Figure 5C ) .", "Similarly , the dwell time distribution was only modestly affected by anti-CD4 ( data not shown ) ; however , the total number of TCR:pMHC complexes per cell was smaller in the anti-CD4 experiments , suggesting that the antibody interfered with pMHC:TCR binding . 10 . 7554/eLife . 00778 . 012Figure 5 . The distribution of live cell single molecule agonist pMHC:TCR molecular binding dwell times is observed directly . Measured dwell time distributions for both the 5c . c7 ( A ) and AND ( B ) TCRs are roughly exponential and match reported solution measurements .", "Bleaching times , kbl−1 , ( grey circles ) are measured using agonist pMHC SLB standards without cells and with the same fluorescent label ( Atto488 ) and are significantly longer than observed dwell times , τobs , for both TCRs .", "( C ) Measured values for 5c . c7 and AND CD4+ T cells under varying conditions .", "Values in columns five and six represent ∼300–600 MCC agonist pMHC molecules per experimental condition from a population of 7–20 cells .", "Data are representative of at least 5 independent experiments performed on T cell blasts isolated from different mice for both the 5c . c7 and AND TCRs .", "Uncertainty in the average across different mice , shown in columns five and six , is calculated as the standard error of the mean of the molecular averages from different mice .", "In some cases ( e . g . for cytoskeleton disruption experiments with Latrunculin A ) one experiment ( representative of 7–10 cells and 100 s of single molecule measurements ) may be performed , but these data are always compared to a control sample recorded on the same day with T cell blasts from the same mouse .", "In these cases uncertainty is reported as the standard error of the mean of the molecular dwell time distribution .", "SPR measurements for 5c . c7 1 ( Huppa et al . , 2010 ) and AND-related 226 TCRs 2 ( Newell et al . , 2011 ) , along with single molecule FRET measurements for 5c . c7 1 ( Huppa et al . , 2010 ) , are shown for comparison . DOI: http://dx . doi . org/10 . 7554/eLife . 00778 . 012 Tracking observations reveal the time intervals over which individual agonist pMHC molecules remain physically trapped within the immediate vicinity of the same TCR .", "Thus , although our measured dwell times in live cells are in general agreement with bulk solution measurements of pMHC:TCR kinetic off-rates for both TCRs ( Figure 5C ) ( Corse et al . , 2010; Newell et al . , 2011 ) , this may not result from the same reasons in each case .", "Unbinding of pMHC from TCR followed by rapid rebinding to the same TCR or another TCR within the same signaling cluster could conceivably lead to entrapment of pMHC for timescales longer than the lifetime of the molecular interaction .", "This has been hypothesized as a potential mode by which a small number of agonist pMHC could trigger a larger number of TCR ( Govern et al . , 2010; Huppa et al . , 2010 ) .", "Furthermore , recently reported single molecule measurements of pMHC:TCR binding kinetics in live cells , by force probe and by FRET , have suggested accelerated kinetic-off rates ( Huang et al . , 2010; Huppa et al . , 2010 ) .", "We investigate this further below .", "We quantitatively assess the possibility of serial rebinding of agonist pMHC to multiple TCR within a TCR cluster using a stochastic reaction-diffusion simulation over a large range of 〈τoff〉 and TCR cluster size .", "The total time to escape for an individual molecule , which is the parameter directly measured in pMHC tracking experiments , is given by:τesc=∑i=0nτoffi+∑i=1nτoni+τexit In this representation , τoffi and τoni are the individual dwell times in the bound and unbound configurations , n is the number of rebinding events , and τexit is the duration of the final unbound period prior to ultimate escape .", "For the stochastic simulation , τoffi and τoni are treated as random variables with exponential distributions defined by the in situ measured values of koff and kon for pMHC:TCR binding , respectively .", "If the pMHC diffuses out of the TCR cluster prior to rebinding , it has escaped .", "Otherwise , the pMHC rebinds and the cycle repeats .", "Using the fastest kon ( 0 . 17 µm2s−1molecule−1 ) observed in similar hybrid live cell-SLB systems ( Huppa et al . , 2010 ) and the measured diffusion coefficient of pMHC in our supported membranes , we find that τesc≈τoff for TCR clusters of the sizes observed experimentally ( Varma et al . , 2006 ) ( ≤100 TCR molecules ) ( Figure 6 ) .", "Only for unrealistically large TCR clusters ( ∼1000 TCR molecules ) could rebinding within the same cluster lead to appreciable entrapment ( τesc>τexit ) ( Figure 6—figure supplement 1 ) . 10 . 7554/eLife . 00778 . 013Figure 6 . Stochastic reaction-diffusion simulation of time before MCC agonist pMHC escape from TCR clusters , τesc ( in color; log scale ) , as a function of τoff and TCR cluster size . For small TCR clusters ( 1–100 TCR molecules ) τoff ≈ τesc , indicating no serial rebinding .", "Only for unrealistically large TCR clusters ( 1000–10 , 000 molecules ) does τesc become appreciably longer than τoff . DOI: http://dx . doi . org/10 . 7554/eLife . 00778 . 01310 . 7554/eLife . 00778 . 014Figure 6—figure supplement 1 . Stochastic reaction-diffusion simulation of time before MCC agonist pMHC escape from TCR clusters . Simulations of τescτoff as a function of TCR cluster size , τoff , and kon .", "The ratio τescτoff , which is an indicator of agonist pMHC entrapment , is a function of TCR cluster size and kon , but not koff .", "kon = 0 . 51 µm2s−1molecule−1 corresponds to the fastest average kon that allows for rebinding to the same TCR .", "Only for unrealistically large TCR cluster sizes ( approaching 1000 TCRs per cluster ) and fast kon is τescτoff≫1 , meaning that entrapment due to millisecond-scale unbinding and rebinding is unlikely to result in the minute-scale dwell times observed in our experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 00778 . 014 These simulations indicate that the observed values of τoff are unlikely to be the result of rapid serial rebinding of one pMHC with many TCR within a TCR cluster .", "We note that although TCR clusters are not readily visible at the low antigen densities in these experiments , low level TCR clustering has been reported in the resting state by other methods ( Schamel and Alarcon , 2013 ) .", "Even with a very fast kon , if pMHC completely disengages from TCR for long enough to diffuse to an adjacent TCR , then the probability of complete escape from the TCR cluster is high .", "Only in the extreme limit , where kon is so fast that pMHC unbinding is predominantly followed by rebinding to the same TCR , is the escape time appreciably longer than the individual molecular dwell times .", "The distinction between rapidly rebinding the same TCR and a single engagement is largely semantic .", "However , it may reveal something about the mechanical stability of pMHC:TCR interactions , and could account for the apparently accelerated koff observed in other types of experiments ." ], [ "All evidence reported here suggests that individual agonist pMHC remain bound to the same TCR for at least several seconds for the 5c . c7 TCR and for approximately one minute for the AND TCR in live cells .", "Subsequent recruitment of ZAP70 indicates TCR are triggered and movement of the complexes along linear trajectories confirms stable association with the actin cytoskeleton .", "All of this occurs as a result of a lone agonist pMHC binding TCR , without involvement of other MHC .", "The measured average pMHC:TCR:ZAP70 stoichiometry indicates TCR triggering is most likely 1:1 with agonist pMHC .", "Supernumerary triggering of multiple TCR by a single pMHC is not observed on the timescales of minutes investigated in these experiments .", "The original serial triggering model ( Valitutti et al . , 1995 ) drew its conclusions from the extent of TCR down regulation measured 5 hr after exposure of T cells to antigen-pulsed APCs .", "Additionally , the lack of molecular-scale cooperativity in TCR triggering by agonist pMHC indicates that observed cooperativity at the level of cellular calcium response , at similar ligand densities and timescales ( Manz et al . , 2011 ) , is most likely due to intracellular feedback mechanisms ( Altan-Bonnet and Germain , 2005; Chan et al . , 2004; Das et al . , 2009; Stefanová et al . , 2003 ) .", "The timescales of the pMHC:TCR interactions we observe in live cells are consistent with SPR measurements of koff for the 5c . c7 TCR and 226 TCR , which is nearly identical to AND ( Figure 5C and Figure 7 ) ( Corse et al . , 2010; Newell et al . , 2011 ) .", "They are also consistent with reports of a ∼2 s kinetic threshold for thymic selection determined in vivo ( Williams et al . , 1999; Palmer and Naeher , 2009 ) .", "Stochastic reaction-diffusion analysis of the measured kinetic and mobility parameters indicates that rapid serial rebinding of agonist pMHC to multiple TCRs in a signaling cluster is unlikely to be a universal mechanism for ligand discrimination and rapid signal amplification in T cells . 10 . 7554/eLife . 00778 . 015Figure 7 . Interaction of the 226 TCR with the MCC peptide .", "( A ) CDR3α ( yellow ) and CDR3β ( orange ) loops form all the specific interactions with the peptide ( cyan ) .", "Hydrogen bonds between the CDR3 loops and the MCC residues are shown by dashed lines ( PDB , 3QIU ) ( B ) Comparison of CDR3 loops between the 226 and AND TCRs reveal identical sequences and suggest similar binding kinetics .", "226 and AND also share Vα , Jα , Vβ , and Jβ gene segments that encode the residues specific for interaction with IEk ( Newell et al . , 2011 ) .", "The residues involved in hydrogen bonds between 226 and MCC are shown in blue . DOI: http://dx . doi . org/10 . 7554/eLife . 00778 . 015 The intercellular geometry as well as active processes within the T cell have long been suspected to influence pMHC:TCR interactions ( Shaw and Dustin , 1997; Qi et al . , 2001; Burroughs and Wulfing , 2002; Zhu et al . , 2013 ) .", "Direct in situ measurements of individual pMHC:TCR binding kinetics , such as we report here , are extremely limited ( Huang et al . , 2010; Huppa et al . , 2010; Axmann et al . , 2012 ) but informative comparisons can be made .", "Notably , a recent intramolecular FRET study of the 5c . c7 TCR binding MCC pMHC reports short ( 〈τoff〉∼150 ms ) in situ 2D dwell times , nearly 35 times faster than the 〈τoff〉 = 5 . 2 ± 0 . 2 s we measured by tracking .", "This same study measures 〈τoff〉 25 times longer under conditions of cytoskeleton disruption , essentially in agreement with tracking observations .", "This result has been interpreted to mean that the actin cytoskeleton actively destabilizes agonist pMHC:TCR complexes .", "In contrast , we observe very small effects of actin disruption on agonist pMHC dwell times for AND and essentially no effect with 5c . c7 TCR ( Figure 5C ) .", "It is conceivable that the agonist pMHC:TCR complex does not remain bound in the same structural configuration for the duration of engagement .", "If mechanical coupling to actin significantly reduces the apparent 〈τoff〉 in a single molecule FRET measurement , but not in a single molecule tracking experiment , this raises the possibility that mechanical forces can induce conformational alterations in agonist pMHC:TCR without complete disengagement of the complex .", "Recent structural studies of pMHC:TCR indicate the possibility of such flexibility ( Adams et al . , 2011; Hawse et al . , 2012; Reboul et al . , 2012 ) .", "While putative conformational changes would not affect single-molecule tracking measurements , they could produce a FRET signature ( Ha et al . , 1999; Majumdar et al . , 2007 ) .", "Under such a scenario , apparently fast 〈τoff〉 observed by FRET imaging ( Huppa et al . , 2010 ) may not correspond to actual molecular unbinding and escape of agonist pMHC from TCR .", "In the aggregate , the data reported here indicate that spatially discrete pMHC:TCR:ZAP70 complexes form according to molecular mass action laws with relatively predictable chemical kinetics and stoichiometry in living cells .", "The observed pMHC:TCR molecular binding kinetics mirror solution measurements and we observe no evidence for molecular scale cooperativity in the triggering of TCR by agonist pMHC ( at low agonist density ) .", "Thus any amplification or other form of signal processing must occur downstream of TCR triggering ." ], [ "A plasmid containing enhanced green fluorescent protein fused to CD3 zeta-chain-associated protein of 70 kDa ( Zap70-EGFP ) was a gift of Takashi Saito , RIKEN Research Center for Allergy and Immunology , Yokohama , Japan ( Yokosuka et al . , 2005 ) .", "The Zap70-EGFP gene was amplified by PCR and subcloned into a murine stem cell virus parent vector ( pMSCV ) .", "Bi-hexahistidine-tagged major histocompatibility complex ( MHC ) class II I-Ek protein was produced and purified as previously described ( Nye and Groves , 2008 ) .", "A decahistidine-tagged ICAM1-TagBFP fusion protein was generated by PCR amplifying the TagBFP sequence ( Evrogen Inc . , Moscow , Russia ) and subcloning it into a pN1-ICAM1 vector .", "The entire Icam1-TagBFP gene was then further subcloned into the pFastBac1 vector ( Invitrogen Inc . , Carlsbad , CA ) , which was used to generate baculovirus for infection of High Five cells ( Invitrogen Inc . ) .", "The ICAM1-TagBFP was subsequently purified on a Ni-NTA-agarose affinity column , eluted with an imidazole gradient , dialyzed , and stored in Tris buffer containing 10% glycerol .", "AND CD4+ T cells ( Kaye et al . , 1989 ) and 5 c . c7 CD4+ T cells were harvested and cultured essentially as previously described ( Smith et al . , 2011; Smoligovets et al . , 2012 ) .", "T cells were transduced with Zap70-EGFP and sorted using fluorescence-activated cell sorting ( FACS ) according to viability and EGFP expression .", "The population of transduced cells that was used expressed EGFP at no more than 50% of the highest EGFP level in the overall EGFP-positive population .", "Using the basic sequence of moth cytochrome c ( amino acids 88–103 ) and previously described variants ( Huppa et al . , 2010 ) , the following peptides were synthesized by David King at the HHMI Mass Spectrometry Laboratory at UC Berkeley and/or commercially ( Elim Biopharmaceuticals , Hayward , CA ) : MCC ( ANERADLIAYLKQATK ) , MCC ( C ) ( ANERADLIAYLKQATKGGSC ) , MCC-null ( C ) ( ANERAELIAYLTQAAKGGSC ) .", "For fluorophore labeling , cysteine-containing peptides were dissolved in a small amount of phosphate buffer and mixed in a 1:2 molar ratio with Atto 647N resuspended in a small amount of 1-propanol or lyophilized Atto 488 ( Atto-Tec GmbH , Siegen , Germany ) and labeled using maleimide-thiol chemistry .", "The peptides were then incubated at room temperature for at least 1 hr and purified on a C18 reverse phase column ( Grace–Vydac , Deerfield , IL ) and H2O:acetonitrile gradient using ÄKTA explorer 100 FPLC system ( Amersham Pharmacia Biotech , Piscataway , NJ ) .", "Peptide identity was confirmed after purification using mass spectrometry .", "TIRF experiments were performed on an inverted microscope ( Nikon Eclipse Ti; Technical Instruments , Burlingame , CA ) using a custom-built laser launch with 488 nm ( Sapphire HP; Coherent Inc . , Santa Clara , CA ) and 640 nm ( Cube; Coherent Inc . ) diode lasers , as described previously ( Smith et al . , 2011 ) .", "Laser powers measured at the sample were 0 . 8 mW ( 640 nm ) and 0 . 5 mW ( 488 nm ) for 500 ms exposures and 4 . 4 mW ( 640 nm ) for 17 . 5 ms exposures .", "A dichroic beamsplitter ( z488/647rpc; Chroma Technology Corp . , Bellows Falls , VT ) reflected the laser light through the objective lens ( Nikon 1 . 49 NA TIRF; Technical Instruments , Burlingame , CA ) and fluorescence images were recorded using an EM-CCD ( iXon 597DU; Andor Inc . , South Windsor , CT ) after passing through a laser-blocking filter ( Z488/647 M; Chroma Technology Corp . , Bellows Falls , VT ) .", "Bandpass filters ( FF03 525/50; Semrock Inc . , Rochester , NY and ET 700/75 , Chroma Technology Corp . , Bellows Falls , VT ) were placed in a DualView 2 Simultaneous Imaging System ( Photometrics , Tuscon , AZ ) .", "Colors were registered before every two-color experiment by imaging 100 nm Tetraspec beads ( Invitrogen Inc . ) deposited on a coverslip patterned with a Cr grid with ∼80 nm width and 3–4 μm pitch Cr lines , since the Tetraspec beads preferentially bind the regular Cr pattern .", "Exposure times and time-lapse periods for most experiments were set using image collection software ( MetaMorph 7 . 5; Molecular Devices Inc . , Downingtown , PA ) , which drives an external shutter ( Uniblitz LS6; Vincent Associates , Rochester , NY ) .", "Exposure time and Fast Kinetics Mode for short ( 17 . 5 ms ) integration time experiments were set using Andor Solis ( Andor Inc . , South Windsor , CT ) .", "Exposure times were measured directly from the Fire output of the EM-CCD using an oscilloscope ( TDS 210; Tektronix , Inc . , Beaverton , OR ) .", "Single unilamellar vesicles ( SUVs ) were formed by tip sonication of a solution composed of 98 mol % 1 , 2-dioleoyl-sn-glycero-3-phosphocholine ( DOPC ) and 2 mol % 1 , 2-dioleoyl-sn-glycero-3-[ ( N- ( 5-amino-1-carboxypentyl ) iminodiacetic acid ) succinyl] ( nickel salt ) ( Ni2+-NTA-DOGS ) ( Avanti Polar Lipids , Alabaster , AL ) in Mill-Q water ( EMD Millipore , Billerica , MA ) .", "Tip sonication was preferred to vesicle extrusion due to the introduction of significant levels of fluorescent impurities into the SUVs during extrusion .", "Prior to experiments , #2 40 mm diameter round coverslips were ultrasonicated for 30 min in 50:50 isopropyl alcohol:water , rinsed thoroughly in Milli-Q water ( EMD Millipore , Billerica , MA ) , etched for 5 min in piranha solution ( 3:1 sulfuric acid:hydrogen peroxide ) , and again rinsed thoroughly in Milli-Q water .", "The coverslips were used in the assembly of FCS2 Closed Chamber Systems ( flow cells; Bioptechs , Butler , PA ) , which were pre-filled with Tris-buffered saline ( TBS; 19 . 98 mM Tris , 136 mM NaCl , pH 7 . 4; Mediatech Inc . , Herndon , VA ) .", "SUVs were then flowed into the chambers , and bilayers were allowed to form for at least 30 min .", "The bilayers were rinsed once with TBS , incubated for 5 min with 100 mM NiCl2 in TBS , rinsed with TBS , and then rinsed with a T cell imaging buffer composed of 1 mM CaCl2 , 2 mM MgCl2 , 20 mM HEPES , 137 mM NaCl , 5 mM KCl , 0 . 7 mM Na2HPO4 , 6 mM d-glucose , and 1% wt/vol bovine serum albumin .", "48 hr prior to experiments , MHC was loaded with peptide at 37°C in a buffer composed of 1% wt/vol bovine serum albumin in phosphate-buffered saline and brought to pH 4 . 5 with citric acid .", "Unbound peptide was separated from peptide loaded MHC ( pMHC ) using 10k spin concentrators ( Amicon Ultra , Cork , Ireland ) and then pMHC was diluted in imaging buffer .", "ICAM1-TagBFP and pMHC were further diluted with imaging buffer , introduced into the flow cells , and incubated for 35 min followed by a rinse with imaging buffer .", "T cells resuspended in imaging buffer and added to the flow cells 35 min after the final rinse and imaged immediately for 30–60 min .", "To visualize TCR , T cells were incubated in a solution of 1 μl Alexa 647 ( Invitrogen Inc . ) -labeled H57 anti-TCR Fab and 100 μl imaging buffer for 20 min at 4°C prior to the regular imaging buffer resuspension .", "All other incubations during this protocol were performed at room temperature , and imaging experiments were performed at 37°C .", "Single molecule diffraction-limited spots were detected in raw . tif image stacks of agonist pMHC labeled with MCC-Atto488 and MCC-647N molecules by filtering for both size and intensity and linked into tracks using published particle detection and tracking algorithms ( Crocker and Grier , 1996 ) adapted for MATLAB ( The Mathworks; Natick , MA ) by Daniel Blair and Eric Dufresne ( http://physics . georgetown . edu/matlab/; accessed 16 August 2012 ) .", "Size and intensity thresholds were first determined by eye using a test data set and then applied uniformly to all data collected with the same exposure time and incident laser intensities .", "Single molecules were identified by step photobleaching detected in an automated way using a Bayesian change point detection algorithm ( Ensign and Pande , 2009 ) .", "The brightness of ZAP70-EGFP features varies from a single molecule to several molecules , and different brightness features are detected using slightly different methods , despite the fact that the features are physically similar .", "Bright ZAP70-EGFP features ( as shown in Figure 4A , D ) were detected using the same algorithm as is used for single molecule pMHC .", "The lower signal-to-noise single molecule ZAP70-EGFP intensity traces like those in Figure 4C were obtained by summing the intensity of the ZAP70-EGFP channel using the agonist pMHC ( labeled with Atto647N ) molecule position as a mask , as is explained in the main text .", "The lifetime of the bright ZAP70-EGFP speckles is difficult to accurately assess due to the fluctuating background and varying speckle intensity ( which biases measurement towards brighter , longer-lived fluorescent features ) , but speckle lifetimes appear to be longer than the single molecule ZAP70-EGFP lifetimes .", "Single molecule ZAP70-EGFP molecules are uncorrected for photobleaching of both ZAP70-EGFP and agonist pMHC and therefore the range of binding times reported ( 12–107", "s ) only serves as a lower bound for the molecular ZAP70 dwell time .", "The agonist pMHC step size distribution at 17 . 5 ms resolution in Figure 1D is populated using similar particle detection and tracking methods to the 500 ms resolution analysis .", ", Agonist pMHC:TCR binding kinetics cannot be uniquely inferred from the step size distribution , since the step size distribution is a time-independent analysis .", "For instance , the step size distribution measured over a certain time period with 2kon and 2koff would appear identical to a scenario with kon and koff .", "Lifetime distributions are roughly exponential and of the form f ( τobs ) =〈τobs〉−1e−τobs〈τobs〉 , where τobs is the observed dwell time in our experiments .", "The individual kinetic transitions were derived assuming the following model:pMHCfree⇌koffkonpMHC:TCR→kblpMHCbleachedwhere pMHCfree is the fast-mobility state , pMHC:TCR is the slow-mobility state ( or the TCR stably bound state ) , pMHCbleached is the bleached slow-mobility state , koff and kon are the rates of transitions between the bound and the free pMHC , and kbl is the rate of transition from bound pMHC:TCR to photobleached pMHC .", "We assume that transititions between states follow a Markov memory-less process and derive a probability density function , f ( τobs ) , for the single molecule dwell time distribution: f ( τobs ) = ( 〈τbl〉−1+〈τoff〉−1 ) e−τobs ( 〈τbl〉−1+〈τoff〉−1 ) , where ( 〈τbl〉−1+〈τoff〉−1 ) −1=〈τobs〉 is the observed mean dwell time in our experiments .", "Agonist pMHC labeled with Atto488 and Atto647N SLB bleaching curves were background subtracted and then fit to an exponential decay function of the form f", "( t ) =kble−kblt .", "Fitting was done using MATLAB .", "Simulations were performed using MATLAB .", "Our simulation models a TCR cluster as a square lattice upon which agonist pMHC molecules bind discrete TCR lattice sites for duration τoffi , τoffi where is treated as a random variable drawn from an exponential distribution with mean equal to 〈τoff〉 .", "〈τoff〉 is varied over several orders of magnitude and is chosen to match measured values from the literature .", "pMHC are initially placed at a randomly lattice position drawn from a uniform distribution .", "After each time period ( determined by τoffi ) , the agonist pMHC molecule steps to a new lattice site or stays at the same lattice site ( the lattice spacing is set to 10 nm to roughly follow the size of the TCR complex [Newell et al . , 2011; Yin et al . , 2012] ) until the agonist pMHC is no longer on the TCR cluster , such that τesck=∑i=0nτoffi+∑i=1nτoni+τexit , where n indicates the number of steps an individual agonist pMHC molecule takes before exiting the TCR cluster , τon is the time period between unbinding and binding events , and τexit is the time between the last unbinding event and the ultimate exit from the TCR cluster .", "Step size is treated as a combination of two independent random variables , ( Δx , Δy , ) drawn from Gaussian distributions with mean 0 and standard deviation 2DSLBτon .", "The step size is then a random variable Δr=Δx2+Δy2 and the angle of displacement is drawn from a uniform distribution .", "The interval between binding events , τon , is treated as a random variable drawn from an exponential distribution with mean kon ρTCR where the density of TCR , ρTCR , is taken to be 10 , 000 molecules/µm2 ( as in the central supramolecular activation cluster ) .", "In this way 〈τoff〉=1k∑kτesck , where k is the number of iterations ( 100 in the case of Figure 6 and Figure 6—figure supplement 1 ) , is calculated for every combination of 〈τoff〉 , TCR cluster size , and kon .", "Note that since τoff≫τon , τescτoff≈〈n〉 , where 〈n〉 is equivalent to the TCR cluster size .", "This relationship between τescτoff and TCR cluster size can be seen in Figure 6—figure supplement 1 .", "It is possible that agonist pMHC binding interactions with CD4 could slow the mobility of an individual agonist pMHC within a TCR cluster relative to DSLB when the agonist pMHC are unbound from TCR .", "This could hypothetically lead to entrapment and long single molecule tracks ( like those reported here ) in the absence of direct , sustained agonist pMHC-TCR interactions .", "While such a mechanism is conceivable , there is no direct evidence for such a tethering mechanism in the literature ." ] ]

[ "T cells discriminate between self and foreign antigenic peptides , displayed on antigen presenting cell surfaces , via the TCR .", "While the molecular interactions between TCR and its ligands are well characterized in vitro , quantitative measurements of these interactions in living cells are required to accurately resolve the physical mechanisms of TCR signaling .", "We report direct single molecule measurements of TCR triggering by agonist pMHC in hybrid junctions between live primary T cells and supported lipid membranes .", "Every pMHC:TCR complex over the entire cell is tracked while simultaneously monitoring the local membrane recruitment of ZAP70 , as a readout of TCR triggering .", "Mean dwell times for pMHC:TCR molecular binding of 5 and 54 s were measured for two different pMHC:TCR systems .", "Single molecule measurements of the pMHC:TCR:ZAP70 complex indicate that TCR triggering is stoichiometric with agonist pMHC in a 1:1 ratio .", "Thus any signal amplification must occur downstream of TCR triggering ." ]

[ "The immune system identifies and combats foreign objects , including pathogens , in the body .", "T cells are key components of the immune system , and each has a unique variant of a signalling complex known as the T cell receptor on its surface .", "T cells scan the surfaces of other cells in search of antigens , which are peptides ( fragments of proteins ) that derive from foreign pathogens such as viruses .", "Successful recognition of a foreign peptide leads to an immune response that , in most cases , ultimately rids the body of the pathogen .", "Most importantly , however , the immune system must be able to discriminate between peptides that are produced naturally in the body ( ‘self’ peptides ) and foreign or ‘non-self’ peptides .", "This is challenging because self peptides may have similar structures to non-self peptides and are often much more abundant .", "Many models have been proposed to explain how T cells are able to detect just a few molecules of foreign peptide .", "According to some hypotheses the T cell receptors get together in clusters to function cooperatively; alternatively , it has been suggested that rapid binding of a foreign peptide to multiple T cell receptors sequentially can build up a strong signal .", "However , none of these phenomena have been directly observed .", "O'Donoghue et al . now image the interactions between T cell receptors and peptides bound to molecules called major histocompatibility complexes ( MHCs ) , and show that T cell activation can occur when a single foreign peptide binds to a single receptor .", "These interactions are long-lived and ultimately result in the recruitment of ZAP70 , which has an important role in the activation of T cells , to the complex formed by the T cell , the peptide and the MHC molecule .", "Therefore , any amplification of the activating signal transmitted by non-self peptides occurs following receptor binding , in contrast to previous models ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "physics of living systems", "microbiology and infectious disease" ]

[ [ "Many prokaryotes move directionally in response to a chemical or physical stimulus .", "However , it is generally assumed that bacteria are too small for direct sensing of a", "concentration gradient across the cell: instead they probe changes in stimulus", "concentration over time , as in the classic paradigm of flagella-mediated chemotaxis", "in Escherichia coli ( reviewed by Wadhams and Armitage , 2004 ) .", "When moving through a spatial concentration", "gradient of an attractant , E . coli cells experience temporal", "concentration changes , which they sense by employing a biochemical memory that", "directs a “biased random walk” .", "Swimming along a straight path ( run )", "alternates with random changes of direction ( tumble ) .", "Tumbles become less frequent", "when cells sense a temporal increase in attractant concentration , introducing a bias", "to movement up a concentration gradient ( Berg and", "Brown , 1972 ) .", "For phototrophic prokaryotes , light is the main source of energy but also potentially", "harmful , depending on intensity and wavelength .", "Unsurprisingly , many phototrophs can", "alter their movement in response to the light environment ( reviewed in Häder , 1987 ) .", "Bacterial phototaxis was", "first noted in 1883 ( Engelmann , 1883 ) and", "has been characterized in free-swimming phototrophs including purple bacteria and", "Halobacterium spp .", "( Hildebrand and Dencher , 1975; Alam and", "Oesterhelt , 1984 ) .", "Cyanobacteria , which are oxygenic phototrophs , do not", "swim with flagella .", "Instead , various species exhibit “twitching” or", "“gliding” motility over moist surfaces ( Pringsheim , 1968 ) .", "This movement can be directed towards a", "light source , thus constituting true phototaxis ( Choi et al . , 1999; Bhaya , 2004;", "Yoshihara and Ikeuchi , 2004 ) .", "The model unicellular cyanobacterium Synechocystis sp .", "PCC 6803", "( hereafter Synechocystis ) has spherical cells about 3 µm in", "diameter and moves using Type IV pili ( T4P ) ( Bhaya", "et al . , 2000; Yoshihara et al . ,", "2001 ) .", "The location of the T4P extension motor PilB1 implies that pili", "are extended at the leading edge of the cell , and therefore that movement is", "generated by pilus retraction ( Schuergers et al . ,", "2015; Wilde and Mullineaux ,", "2015 ) , as has been shown in other bacteria ( Merz et al . , 2000 ) .", "It has recently been established that the motility", "of a filamentous cyanobacterium is also T4P-dependent , suggesting that this form of", "motility is widespread in cyanobacteria ( Khayatan", "et al . , 2015 ) .", "One likely exception is marine Synechococcus", "spp .", ", which swims and exhibits chemotaxis without obvious surface", "appendages ( Willey and Waterbury , 1989;", "Ehlers and Oster , 2012 ) apart from", "short spicules found in one of the motile Synechococcus strains", "( Samuel et al . , 2001 ) .", "Synechocystis T4P-dependent phototaxis can be observed", "microscopically at the single cell level and macroscopically through the", "migration of cell colonies .", "Genetic studies have identified a number of", "photoreceptors that influence phototactic behavior under different light regimes", "( Bhaya , 2004 ) .", "While", "Synechocystis harbors signal transduction systems for pilus", "biogenesis that are homologous to the chemotaxis system in E . coli ,", "it lacks the CheR methyltransferase and the CheB methylesterase that are required in", "most chemotactic bacteria to sense temporal changes in attractant concentration", "( Wuichet and Zhulin , 2010 ) .", "This", "suggests a different mode of directional control .", "Previous studies of Synechocystis single cell phototaxis", "( Choi et al . , 1999; Chau et al . , 2015 ) have not addressed the", "question of how an individual cell might be able to perceive the direction of", "illumination .", "Here , we establish that individual Synechocystis", "cells can directly and accurately perceive the position of a unidirectional light", "source , and control their motility so as to move towards it .", "We then show that", "Synechocystis cells act as microlenses , and that the light", "intensity gradient across the cell due to this lensing effect is far greater than", "the effects of shading due to light absorption or reflection .", "Finally , we use", "highly-localized laser excitation to show that specific excitation of one side of", "the cell triggers movement away from the light , indicating that positive phototaxis", "results from movement away from an image of the light source focused on the opposite", "side of the cell .", "Essentially , the cell acts as a microscopic eyeball ." ], [ "Individual Synechocystis cells moving in two dimensions on", "an agarose surface in response to different light regimes were tracked", "microscopically to determine whether single cells are capable of direct", "perception of the position of a light source .", "First , we tested the response of", "cells to a light intensity gradient projected onto the surface from the", "microscope condenser ( Figure 1a ) , using a", "gradient of white light from 0–20 µmol photons", "m–2 s–1 , an intensity range that is", "relevant for positive phototaxis ( Choi et al . ,", "1999; Bhaya , 2004; Chau et al . , 2015 ) .", "However , the cells", "moved randomly without any significant directional bias ( Figure 1b; Video", "1 ) .", "If Synechocystis phototaxis were based on a", "biochemical memory like E . coli chemotaxis , cells would", "perceive temporal changes in light intensity as they move across the surface", "through the light gradient and would then accumulate in regions of optimal light", "intensity .", "This did not occur ( Figure 1b ,", "Video 1 ) .", "By contrast , when cells", "were illuminated by a unidirectional light source ( RGB illumination at 10", "µmol photons m–2 s–1 ) at an angle", "oblique to the surface ( Figure 1a ) , the", "majority of motile cells switched direction within about 1 min , and then moved", "directly towards the light source ( Figure", "1c , d , f; Video 1 ) .", "Under", "illumination from two equal-intensity orthogonal light sources , the majority of", "cells moved towards a point midway between the two light sources ( Figure 1e; Video 1 ) .", "These behaviors are not consistent with a", "run-and-tumble mechanism or any kind of biased random walk .", "In accord with", "previous studies ( Choi et al . , 1999;", "Chau et al . , 2015 ) , we conclude that", "individual cells can directly and accurately perceive the position of a light", "source and control their motility accordingly . 10 . 7554/eLife . 12620 . 003Figure", "1 . Movement of individual", "Synechocystis cells under different light", "regimes . Displacement over a 5 min time-frame was measured", "1 min after the onset of illumination .", "The mean resultant", "length from a Rayleigh test ( r ) and the number of", "tracked cells ( n ) are shown .", "See also Video 1 .", "All data were", "obtained with the same cells from a single continuous experiment .", "( a ) Schematic diagram to illustrate the optical", "set-up for a light gradient projected onto the agar surface ( as in", "b ) and oblique directional illumination ( as in c–e ) .", "( b ) Cells moving in a white light gradient from", "0–20 µmol photons m–2", "s–1 over a 165 µm interval from the top", "to the bottom of the plot , showing no significant directional bias .", "( c ) Illumination from an oblique RGB LED light", "source from the right , at intensity 10 µmol photons", "m–2 s–1 .", "( d )", "Illumination from a similar light source placed orthogonally to that", "in", "( b ) .", "( e ) Simultaneous illumination from", "both oblique light sources .", "( f ) Correlation of cell", "movement with light direction , as a function of time after applying", "the light .", "The shading indicates the time window plotted in", "b–e .", "The y-axis shows the mean resultant", "length from a Rayleigh test ( r ) where 0 indicates", "random displacements and 1 indicates maximal clustering in the", "direction of illumination .", "LED , light emitting", "diode . DOI:", "http://dx . doi . org/10 . 7554/eLife . 12620 . 00310 . 7554/eLife . 12620 . 004Video", "1 . Motility of Synechocystis", "cells under different illumination regimes . The video", "gives a schematic overview of the experimental set-up , followed by", "movement of cells in a projected light gradient , and with oblique", "illumination from two orthogonal directions , and then from both", "directions simultaneously .", "In each case , the raw video data is", "followed by the same movie clip with the tracks of cells", "superimposed .", "Time in minutes is indicated . DOI:", "http://dx . doi . org/10 . 7554/eLife . 12620 . 004 Synechocystis cells contain a dense lamellar system of thylakoid", "membranes packed with photosynthetic complexes , and we initially postulated that", "light direction sensing depends on shading by the thylakoids , with the motility", "apparatus activated by brighter light at the illuminated side of the cell and", "deactivated at the shaded side of the cell .", "To assess the plausibility of this", "idea , we estimated the transmission spectrum of a single motile cell , scaling", "the absorption spectrum for a cell suspension according to the mean cellular", "pigment content .", "To ensure that pigment content was appropriate for phototactic", "cells , we used cells taken from moving colonies on motility plates ( Figure 2 ) .", "The transmission spectrum shows", "that even at peak absorption wavelengths , a single cell can absorb only about", "20% of the photons that pass through it ( Figure", "2 ) .", "Although local pigment concentrations within the cell are quite", "high ( Figure 2 ) , the very short optical", "path length means that light absorption by the cell is low .", "Note", "that our estimate assumes a homogeneous distribution of pigments within the", "thylakoid region of the cell: in reality , pigment clustering will tend to", "decrease the cell absorbance due to enhanced self-shading of pigments ( Duysens , 1956 ) .", "Thus , 20% represents a", "maximum estimate of the proportion of photons that can be absorbed , and the", "light intensity gradient across the cell due to shading must be almost", "negligible .", "The direct measurement of single cell absorption spectra is", "technically challenging , and we are aware of only one such measurement in the", "literature for a cyanobacterium .", "Sugiura and", "Itoh ( 2012 ) show that the peak absorbance for a single cell of", "Nostoc sp .", "is about 0 . 04 .", "This corresponds to a peak", "absorption of about 10% of the photons that pass through the cell , which is even", "lower than our estimate for Synechocystis . 10 . 7554/eLife . 12620 . 005Figure", "2 . Estimated transmission spectrum of a", "single motile Synechocystis", "cell . Estimate obtained by scaling and converting the", "absorption spectrum for a suspension of cells from a moving", "Synechocystis colony , as detailed in Materials", "and methods .", "Pigments ( including chlorophyll: Chl and", "phycocyanin-coupled phycocyanobilin: PCB ) are assumed to be evenly", "distributed within the thylakoid region as shown in the diagram , and", "the estimated transmission is for a narrow beam of light passing", "straight through the center of the cell , with an optical", "path length through the thylakoid region of 1 μm .", "The", "spectrum represents a minimum estimate for transmission through the", "cell , since the estimate assumes a homogeneous distribution of", "pigments within the thylakoid region .", "In reality , inhomogeneous", "distribution of membranes and pigments will tend to decrease", "absorption due to enhanced self-shading ( Duysens , 1956 ) .", "DOI:", "http://dx . doi . org/10 . 7554/eLife . 12620 . 005 Most light microscopy uses illumination orthogonal to the surface on which the", "sample rests , with the exception of dark-field microscopy , which uses oblique", "illumination from all sides .", "While investigating Synechocystis", "phototaxis , we observed cells instead with oblique illumination from one side", "only .", "Observations with this unusual illumination suggest a solution to the", "problem of directional light perception in Synechocystis .", "These", "images reveal that each cell acts as a microscopic spherical lens , focusing an", "intense light spot close to the opposite side of the cell from", "the light source and the direction of movement ( Figure 3a ) .", "Images from two orthogonal light sources ( as employed in", "one of the motility assays in Figure 1e", "and Video 1 ) are focused at different", "points on the cell periphery ( Figure 3a ) ,", "indicating that the cell can focus an image of its surroundings at the plasma", "membrane . 10 . 7554/eLife . 12620 . 006Figure", "3 . Micro-optic effects in", "cyanobacteria .", "( a )", "Synechocystis cells viewed with oblique", "illumination from the different directions shown , with enlarged", "image on the right .", "Scale-bars: 5 µm .", "( b )", "Images of periplasmic GFP fluorescence in Synechocystis", "torA-gfp ( Spence et al . , 2003 ) .", "Left: two-channel confocal", "micrograph ( GFP fluorescence in green; chlorophyll fluorescence", "in red ) with GFP epifluorescence images of a single cell", "illuminated from the right , above and left ( arrows indicate", "illumination direction; see Figure 3—figure supplement 1 for the optical", "set-up ) .", "( c ) GFP fluorescence profile around the", "cell circumference , extracted from the epifluorescence image in", "( b ) with illumination from the right .", "The", "profile was taken in an anti-clockwise direction starting at the", "point nearest the light source .", "( d ) Schematic", "illustration of the measurement by photolithography of", "near-field optical effects of a Synechocystis", "cell .", "( e ) Height profile reconstructed from an AFM", "image of a photolithograph from the experiment illustrated in", "( d ) .", "( f ) Finite difference time", "domain model of the light path ( wavelength 365 nm )", "through a Synechocystis cell ( illumination", "direction indicated by the arrow ) .", "The color scale", "indicates relative light intensity obtained by time-averaging", "the amplitude of the Poynting vector for the electromagnetic", "field .", "The wave patterns represent a snapshot of the", "oscillating electromagnetic field propagating through the model", "cell .", "GFP , green fluorescent", "protein . DOI:", "http://dx . doi . org/10 . 7554/eLife . 12620 . 00610 . 7554/eLife . 12620 . 007Figure 3—figure", "supplement 1 . Optical set-up for", "oblique-angle excitation fluorescence microscopy to visualize", "periplasmic GFP fluorescence ( Figure 3c ) .", "Fluorescence excitation was", "achieved by near-total internal reflection fluorescence ( TIRF )", "( “highly inclined”/“oblique” )", "illumination of the samples , with fiber-coupled 488 nm laser", "light injected into the epifluorescence port of the microscope", "via the dichroic mirror .", "The fiber , collimator , quarter-wave", "plate and total internal reflection ( TIR ) -lens were mounted on a", "translation stage and positioned such that the focused beam was", "centered at the BFP of the Olympus 100x objective lens .", "By", "adjusting the translation stage , the position of the beam could", "be translated across the BFP to adjust the inclination angle of", "the beam through the sample .", "Images were captured using a GFP", "filter set .", "BFP , back focal plane; GFP , green", "fluorescent protein . DOI:", "http://dx . doi . org/10 . 7554/eLife . 12620 . 007 The images in Figure 3a do not give a", "quantitative picture of the lensing effect since the focused light spot is", "observed very indirectly via light reflected from the agar surface .", "Therefore ,", "to quantify the lensing effect , we first employed a", "Synechocystis mutant that accumulates green fluorescent", "protein ( GFP ) evenly distributed in the periplasm ( Spence et al . , 2003 ) .", "Thus , GFP can be used as local", "reporter of light intensity at the cell periphery ( Figure 3b ) since GFP fluorescence will be proportional to", "the local excitation light intensity .", "GFP fluorescence images with oblique laser", "excitation show a spot of light sharply focused at the opposite edge of the", "cell .", "Profiles of GFP fluorescence around the cell perimeter ( Figure 3c ) indicate that the mean ratio of", "the light intensity at the center of this focused spot to the intensity at the", "front of the cell facing the light source was 4 . 1 ± 1 . 5 ( mean and", "standard deviation , n = 13 cells from one representative", "experiment ) and the observed mean full width at half maximum ( FWHM ) of the", "focused spot was 609 ± 30 nm ( mean and standard deviation ,", "n = 11 cells ) .", "Since the measured FWHM for the point-spread", "function of the microscope was 270 nm , the true FWHM for the focused spot of 488", "nm light at the cell periphery can be estimated to be about 550 nm .", "This", "experiment shows that for 488 nm light , lensing effects leading to concentration", "of light at the opposite side of the cell from the light source are", "overwhelmingly greater than shading effects , which would lead to higher light", "intensity on the illuminated side of the cell .", "Shading effects at other", "wavelengths in the range from 400 to 750 nm could be only", "marginally greater than at 488 nm ( Figure", "2 ) .", "The spatial resolution of the fluorescence measurement in Figure 3b is limited by the optical point-spread function", "of the microscope .", "Therefore , for higher-resolution measurement of near-field", "light perturbation by Synechocystis cells , we used a", "photolithographic method that gives a very high resolution image of the light", "pattern adjacent to the cell , although it does not provide such a direct and", "quantitative measurement of relative light intensity as the fluorescence imaging", "in Figure 3b .", "Synechocystis cells were adsorbed onto the surface of a", "photopolymer and collimated UV light ( 365 nm ) was projected vertically down onto", "them ( Figure 3d ) .", "The UV light induces", "cross-linking of the photopolymer .", "After development , the surface relief of the", "photopolymer gives a high-resolution replica of the light field around", "and beneath each cell , which we examined by atomic force microscopy", "( Figure 3E ) .", "Synechocystis cells produced distinctive near-field optical", "scattering patterns on the polymer surface with a remarkably sharp and intense", "peak beneath the center of each cell .", "We measured an FWHM of 281 ± 33 nm", "( mean and standard deviation , n = 6 from profiles at", "different angles across the representative image shown ) ( Figure 3e ) .", "This experiment indicates that near-UV light", "is focused to a spot with diameter less than the wavelength .", "Figure 3e also confirms that near-UV light", "is concentrated at the side of the cell opposite to the light source in a", "similar manner to light in the visible range .", "As with visible light ( Figure 2b , c ) , the lensing effect", "predominates over any shading effects with near-UV light ( Figure 3e ) .", "To probe the physical basis for the lensing effect , we modeled light perturbation", "by the cell with finite difference time domain ( FDTD ) simulations , a method that", "uses the electromagnetic Maxwell equations without any geometrical", "simplifications ( Yee , 1966 ) .", "This", "electromagnetic description traces all observed effects back to the interference", "of incoming waves with waves scattered by their encounter with the object , which", "in this case was a simplified model of the cell as a microsphere with uniform", "refractive index .", "The method predicts a combination of effects that , on larger", "scales , are described as interference , refraction and internal reflection .", "We", "found that the observed near-field light pattern ( Figure 3e ) could be accurately reproduced by an FDTD", "simulation approximating the cell as a dielectric sphere with a diameter of 3", "µm and a refractive index of 1 . 4 ( Figure", "3f ) .", "Microspheres with similar dimensions to", "Synechocystis have been experimentally shown to produce", "similar sharply focused light beams at the edge of the object opposite to", "the light source: these are termed “photonic nanojets” ( Ferrand et al . , 2008; Heifetz et al . , 2009 ) .", "The micro-optic effects shown in Figure 3", "produce intensity differences across Synechocystis cells that", "are opposite in orientation and at least 20 times greater than those predicted", "from shading due to light absorption by the photosynthetic pigments ( compare", "Figure 3c with Figure 2 ) .", "Figure", "3a , b , c , e provide direct experimental confirmation that light", "intensity is highest at the edge of the cell furthest from the light source .", "This suggests that the basis for directional light perception by", "Synechocystis should depend on the lensing properties of", "the cells , with positive phototaxis based on the cell moving away from the light", "spot focused at its periphery .", "We tested this idea by using a", "highly focused laser light spot ( Lowe", "et al . , 2015 ) as an alternative way to selectively illuminate one", "edge of the cell .", "Synechocystis cells moving directionally on", "an agarose surface towards a red ( 625 nm ) light emitting diode ( LED ) light", "source were visualized by fluorescence from the photosynthetic pigments excited", "by the LED light .", "Focused spots of light at the rear periphery of the cell were", "again observed under this illumination regime ( Figure 4a , Video 2 ) ,", "showing that the focused light spots extend into the thylakoid membrane region .", "The moving cells were allowed to encounter a spot of 640 nm laser light focused", "on the agarose surface ( Figure 4; Video 2 ) .", "The intensity gradient at the", "edge of the laser spot was steep enough to ensure specific exposure of one side", "of the cell to the light ( Figure 4a ) .", "Whenever one edge of a cell encountered the edge of the laser spot , the cell", "changed direction to move away from the laser illumination ( Figure 4b; Video", "2 ) .", "Cells did not cross the center of the intense laser spot , but", "instead changed direction when the laser light intensity at the front edge of", "the cell exceeded the intensity of the light spot focused by the cell at its", "rear periphery by a factor of about 2–10 , as assessed from the brightness", "of fluorescence from the photosynthetic pigments ( Figure 4c ) .", "In accord with our hypothesis , this shows that", "Synechocystis phototaxis is essentially a photophobic", "response to selective excitation of one side of the cell .", "The data in Figure 4 and Video 2 indicate that this photophobic response is", "increasingly strong with stronger localized excitation: thus , when the cells", "encounter laser light that is stronger than the focused light spot at the rear", "edge of the cell , they change direction to move away from the laser light . 10 . 7554/eLife . 12620 . 008Video", "2 . Effects of a highly-focused laser spot on", "directional motility in", "Synechocystis . Cells are imaged by", "fluorescence from the photosynthetic pigments , and are moving", "towards an oblique LED light at the bottom of the frame: note the", "focused light spot at the rear edge of each cell .", "The superimposed", "red spot indicates the position of the laser , and time in min is", "shown at the top left .", "LED , light emitting", "diode . DOI:", "http://dx . doi . org/10 . 7554/eLife . 12620 . 00810 . 7554/eLife . 12620 . 009Figure", "4 . Direction switching triggered by", "specific excitation of one edge of the", "cell . Synechocystis cells were", "moving in response to an oblique 625 nm light source from the", "direction indicated by the red arrows and allowed to encounter a", "spot of 640 nm laser light focused on the agarose surface .", "See", "Video 2 for the", "full data and Figure", "4—figure supplement 1 for the optical set-up", "and the intensity profile of the laser spot .", "( a )", "False color image of fluorescence from the photosynthetic", "pigments , with the laser spot indicated by the red circle .", "The", "broken white lines indicate the approximate cell boundaries and", "the white arrows highlight examples of the focused images of the", "light source at the rear edge of the cell .", "Cells approaching the", "laser spot show strong selective excitation of the leading edge", "of the cell .", "( b ) Direction switching triggered by", "contact with the edge of the laser spot .", "The arrowed lines", "indicate net displacements of representative cells over", "time windows of 132 s before and after closest approach", "to the laser spot .", "The mean orientation of the tracks ( ±", "standard deviation , n = 29 ) is shown .", "( c ) Light intensity required to reverse the", "path of Synechocystis cells .", "Tracks of the", "mid-points of individual cells are shown over a 30 min", "time window , with a color scale indicating the", "ratio of laser intensity to the intensity of the phototactic LED", "light focused on the cell .", "Purple color indicates tracks of", "cells in which autofluorescence induced by the laser exceeds", "autofluorescence induced by the light focused on the cell for", "phototaxis by at least two-fold .", "LED , light emitting", "diodeDOI:", "http://dx . doi . org/10 . 7554/eLife . 12620 . 00910 . 7554/eLife . 12620 . 010Figure 4—figure", "supplement 1 . Optical set-up used to measure", "the response of Synechocystis cells to a", "highly focused laser light spot ( Figure 4; Video", "2 ) .", "( a ) The", "time lapse microscope was constructed on the base of an", "Olympus IX81 microscope , using a 40x× objective lens", "( Air , NA 0 . 75 ) .", "Two air-cooled electron-multiplying", "charge-coupled device ( EMCCD ) cameras were coupled to the camera", "port of the microscope via a magnifying relay .", "The cameras were", "simultaneously triggered using a TTL", "( transistor-transistor logic ) pulse from an", "external digital-to-analog converter , with an exposure time of", "50 ms and a frequency of 0 . 25 Hz .", "A dichroic mirror in the", "Fourier plane of the camera relay split the emission into red", "( fluorescence ) and green ( transmission bright-field imaging )", "channels .", "Each channel had a separate band pass filter .", "The focused laser spot was generated by expanding a collimated", "beam from a fiber-coupled continuous wave ( CW ) diode", "laser , injected via the epifluorescence port of the microscope", "and directed to the objective lens via a dichroic filter .", "The", "beam was expanded to overfill the back aperture of the objective", "lens to achieve a diffraction-limited spot at the focal plane .", "A", "combination of neutral density filters and a half-wave", "plate and polarizing beam splitter were used to adjust the power", "of the laser to approximately 0 . 01 μW .", "A 1:1 lens pair", "with one lens mounted on a Z-translation stage was used to", "adjust the axial position of the focus at the sample plane .", "A", "quarter-wave plate was used to circularly polarize the beam", "before it was injected into the microscope .", "The laser intensity", "profile is shown in", "( b ) .", "Bright-field", "trans-illumination was performed by fiber-coupling a 530 nm LED", "into a multimode fiber and imaging the magnified end of the", "fiber at the sample plane using a condenser lens .", "The LED was", "TTL triggered from via the camera acquisition to reduce light", "exposure to the cells , resulting in a synchronized 50 ms pulse", "during acquisition .", "A third LED ( 625 nm , 3 mW ) was mounted close", "to the sample at an oblique angle to provide directional", "illumination for the motility assay .", "LED , light emitting", "diode . DOI:", "http://dx . doi . org/10 . 7554/eLife . 12620 . 010" ], [ "Here we have shown that Synechocystis cells act as very", "effective spherical microlenses that focus a sharp image of a light source at", "the opposite edge of the cell .", "This implies that positive phototaxis ( i . e .", "movement towards a light source ) is actually triggered as a negative response to", "the focused spot of light at rear periphery of the cell .", "We directly tested this", "idea by exposing cells to a spot of red laser light that was", "sharply focused enough to selectively excite one edge of the cell ( Figure 4 , Video 2 ) .", "As predicted by our hypothesis , cells moved", "away from localized laser excitation that was only slightly brighter than the", "focused image of the light source ( Figure", "4 , Video 2 ) .", "This result", "is the opposite of what would be expected from a “shading” model", "for directional light perception , which would predict that light in this", "intensity range should attract the cells .", "Synechocystis motility depends on the extension , adhesion and", "retraction of T4P ( Bhaya , 2004 ) , which", "is powered by the motor ATPases PilB and PilT ( Merz et al . , 2000 ) .", "We previously examined the localization of one", "of these motor ATPases in Synechocystis with GFP-tagging ( Schuergers et al . , 2015 ) .", "We were unable", "to generate a strain with functional GFP-tagged PilT , but a strain expressing", "PilB1-GFP in a ∆pilB1 null background was motile and", "capable of phototaxis , albeit with lower efficiency than the wild type ( Schuergers et al . , 2015 ) .", "Imaging of", "PilB1-GFP in Synechocystis shows that it is localized in a", "crescent-like zone of the plasma membrane at one side of the cell , and that", "these patches can dynamically relocate to other areas of the membrane ( Schuergers et al . , 2015 ) .", "For cells", "without directional illumination , random relocation of the PilB1-GFP patch", "frequently occurs within a 5 min window .", "The direction of motility in", "Synechocystis strongly correlates with the position of the", "PilB1 patch , indicating that direction is determined by the localization of", "PilB1 , probably in concert with other T4P components such as PilT ( Schuergers et al . , 2015 ) .", "In", "Myxococcus xanthus , both PilB and PilT show dynamic", "relocalization between the two cell poles , on comparable timescales ( Bulyha et al . , 2009 ) .", "It is clear that multiple photoreceptors are involved in", "Synechocystis phototaxis , with light sensors that absorb at", "different wavelengths and trigger either positive or negative phototaxis ( Ng et al . , 2004 ) .", "In addition , motility", "is modulated by the second messengers cyclic diguanylate monophosphate", "( c-di-GMP ) ( Savakis et al . , 2012 ) and", "cyclic adenosine monophosphate ( cAMP ) ( Terauchi and Ohmori , 1999; Bhaya et al . , 2006 ) .", "However , the best-characterized", "candidate for a directional photoreceptor for positive phototaxis is PixJ1 ( also", "known as TaxD1 ) .", "Mutants in which the pixJ1 gene is inactivated", "show only negative phototaxis ( Yoshihara et", "al . , 2000; Bhaya et al . ,", "2001 ) .", "PixJ1 is a cyanobacteriochrome with two transmembrane domains , a", "chromophore-binding domain and a domain with similarity to the methyl-accepting", "chemotaxis proteins of enterobacteria at the C-terminus ( Bhaya et al . , 2001; Yoshihara and Ikeuchi , 2004 ) .", "Proteomic studies with cell", "fractionation indicate that PixJ1 is located in the plasma membrane ( Pisareva et al . , 2007 ) and this is an", "important consideration for our model .", "Downstream signal transduction from PixJ1 likely involves the products of", "neighboring genes in the tax1 locus , since mutants lacking", "these genes ( apart from pixI ) also show only negative", "phototaxis ( Bhaya et al . , 2001; Yoshihara et al . , 2000 ) .", "The respective", "gene products include homologs of the E . coli chemotaxis signal", "transducers CheW , CheA and CheY ( Bhaya et al . ,", "2001; Yoshihara and Ikeuchi ,", "2004 ) .", "A likely signal transduction pathway would proceed through", "light activation of PixJ1 ( sll0041 ) that regulates autophosphorylation of the", "CheA homolog sll0043 ( PixL ) via the CheW homolog sll0040 ( PixI ) .", "PixL could", "transfer the phosphate group to the CheY-like response regulators sll0038 ( PixG )", "and/or sll0039 ( PixH ) .", "By analogy with the E . coli chemotaxis", "system ( reviewed by Sourjik and Wingreen ,", "2012 ) , these response regulators might interact directly with the", "Synechocystis motility apparatus .", "Combining our current results with the previous study on PilB1 localization and", "motility in Synechocystis ( Schuergers et al . , 2015 ) and the likely scheme for signal", "transduction discussed above leads to a simple model for control of positive", "phototaxis in Synechocystis , which is illustrated in Figure 5 .", "The bright focused image of the", "light source is perceived by PixJ1 in the plasma membrane , resulting in local", "changes in the phosphorylation status of the response regulators PixG and/or", "PixH , which leads to local inactivation of the T4P motility apparatus and", "dispersal of the motor proteins ( PilB1 and likely also PilT ) .", "The motility", "apparatus therefore assembles at the side of the cell facing the light ,", "resulting in movement towards the source ( Figure", "5 ) .", "The 1 min timescale for direction switching in", "Synechocystis ( Figure", "1f ) is consistent with the rapid kinetics of relocalization of PilB1", "patches ( Schuergers et al . , 2015 ) .", "Responses to multiple light sources ( as in Figure 1e and Figure 4 ) can", "be explained if it is assumed that the dispersal signal is graded with light", "intensity .", "Thus when a cell is exposed to a stronger light source at one edge", "( as with cells encountering the focused laser spot in Figure 4 and Video", "2 ) , the motility apparatus is always most strongly inactivated in the", "region of strongest local illumination . 10 . 7554/eLife . 12620 . 011Figure", "5 . Model for control of positive phototaxis", "in Synechocystis . Directional", "illumination of the cell produces a sharply focused and", "intense spot of light ( resembling a photonic nanojet ) at the cell", "periphery on the opposite side from the light source .", "The focused", "spot is perceived by photoreceptors in the cytoplasmic membrane ( for", "example PixJ1 ) triggering signal transduction via CheY-like response", "regulators that locally inactivates the T4P motility apparatus ,", "dispersing T4P components including the extension motor PilB1 .", "Consequently , patches of the motor proteins can only form on the", "side of the cell facing the light source .", "Pili are extended and", "retracted at this side of the cell , which therefore moves towards", "the light .", "T4P , Type IV pili . DOI:", "http://dx . doi . org/10 . 7554/eLife . 12620 . 011 The model in Figure 5 implies that ,", "essentially the Synechocystis cell functions as a microscopic", "eyeball , with the spherical cell body as the lens and the cytoplasmic membrane", "as the retina .", "From the observed dimensions of the spot of 488 nm light focused by", "Synechocystis cells ( Figure", "3c ) we can estimate that Synechocystis", "“vision” has an angular resolution ( FWHM ) of about 21° ,", "essentially limited by light wavelength and the area of the plasma membrane ,", "which is tiny in comparison to an animal retina .", "However , this resolution is", "sufficient to incorporate quite complex spatial information into a 360°", "image of the cell’s surroundings , and our data in Figure 1e indicate that the cell can integrate information", "from distinct and spatially separated light sources .", "The directional motile", "responses of the cells ( Figure 1c , d ) show", "a distribution of displacement angles with FWHM ~30° .", "This is less", "accurate than the initial imaging of the light source , which suggests an", "unsurprising degree of spreading and noise during the signal transduction that", "comes between initial light perception and the response of the motility", "apparatus .", "Our model ( Figure 5 ) implies that the", "maximum efficiency of directional light sensing will be achieved in a", "spherical cell .", "Among the unicellular cyanobacteria , a number of species of the", "genera Synechocystis and Gloeocapsa are", "described as motile and phototactic , and these species do indeed have spherical", "or near-spherical cells ( Rippka et al . ,", "1979 ) .", "However , the rod-shaped unicellular thermophilic", "cyanobacterium Thermosynechococcus elongatus is also motile and", "appears capable of direct perception of light direction ( Kondou et al . , 2001 ) .", "The", "Thermosynechococcus homolog of", "Synechocystis PixJ1 ( TePixJ ) is clustered at the cell poles", "( Kondou et al . , 2002 ) .", "For our", "model to be applied to Thermosynechococcus , the cell would need", "a method to concentrate light at the pole furthest away from the light source .", "Micro-optic effects in rod-shaped cells such as those of", "Thermosynechococcus need further investigation .", "Cyanobacteria are not the only unicellular organisms capable of directional light", "perception .", "Rhodospirillum centenum cells form phototactic", "swarm colonies , with an unknown mechanism of light direction sensing ( Ragatz et al . , 1995 ) .", "Phototaxis in the", "larger cells of some unicellular eukaryotic green algae is mediated by clusters", "of photoreceptors known as eyespots , coupled with complex shading and reflecting", "devices ( Kreimer , 2009 ) .", "Lensing", "effects are implicated in phototactic orientation and phototropic responses in", "some other eukaryotes ( Shropshire ,", "1962; Häder and Burkart ,", "1983 ) .", "However , focused imaging at the cell periphery in the tiny", "spherical cells of Synechocystis represents a particularly", "elegant and compact solution to the problem of directional light-perception ,", "that is probably more ancient than the eukaryotic systems .", "Spherical", "cyanobacteria may have been the first organisms to see their world .", "The", "micro-optic effects that we observe in cyanobacterial cells are relevant not", "only to phototaxis but also to photosynthesis , since light distribution within", "the cell is so strongly affected .", "Future work will also need to explore the", "biological implications of optical lensing in non-photosynthetic bacteria , since", "such micro-optic effects are likely to be widespread in bacteria with cells of", "the appropriate size and shape ." ], [ "We used a motile sub-strain of the original Synechocystis sp .", "PCC 6803 wild type from the Pasteur Culture Collection , acquired from the lab of", "S . Shestakov ( Moscow ) in 1993 .", "This sub-strain ( PCC-M ) was recently", "characterized by complete genome sequencing ( Trautmann et al . , 2012 ) .", "Synechocystis cells were", "grown for at least 24 h on motility medium ( 0 . 3% ( w/v ) agarose in BG11 medium", "( Stanier et al . , 1971 ) supplemented", "with 0 . 2% glucose and 10 mM", "Tris ( hydroxymethyl ) methyl-2-aminoethanesulfonic acid ( TES ) ( pH", "8 . 0 ) buffer ) in dark boxes with a one sided opening leading to a directional", "illumination of 5–10 µmol photons m–2", "s–1 ( Phillips MASTER TL-D Super 80 18W/840 , Philips GmbH", "Market DACH , Germany ) .", "Cells from the moving front of a colony on a motility plate were resuspended in", "fresh BG11 medium and 3 µl aliquots were directly spotted on top of 5 ml", "motility medium in 35 mm glass-bottom plates .", "When liquid droplets were no", "longer visible ( 5–10 min ) a cover-slip was carefully placed on top of the", "cells and the surrounding surfaces of the plates were covered with a silicone", "ring to minimize surface oscillations and evaporation .", "Time-lapse videos were", "captured at room temperature ( ca . 22°C ) using an upright Nikon Eclipse", "Ni-U microscope ( Nikon Instruments , Germany ) fitted with a 40× objective", "( numerical aperture 0 . 75 ) .", "Gradient illumination was with white light from the", "microscope condenser lamp .", "RGB LEDs ( 470/525/625 nm at equal intensity )", "( World Trading Net GmbH , Germany ) for directional illumination were mounted in", "boreholes of a black plastic cylinder surrounding the motility plates .", "Light", "intensities were measured with a LiCOR light-meter with a planar quantum sensor", "( LI-COR Biosciences GmbH , Germany ) with a detection window from 400–700", "nm .", "Single cell movement was captured at 1 frame per 3 s .", "Unless otherwise", "stated , the exposure time was 200 ms to reduce the background light needed for", "visualizing the cells .", "We developed the BacteriaMobilityQuant software ( https://web . fe . up . pt/~dee11017/software/BacterialMobilityQuant . zip )", "implemented in MATLAB for tracking single cells in time lapse", "videos .", "It is based on a detection-association tracking approach that relies on", "the Laplacian of Gaussian filter ( LoG ) for the task of bacteria detection ( Esteves et al . , 2013a; Esteves et al . , 2013b ) .", "The LoG filter is", "based on the image scale-space representation to enhance the blob-like structure", "as introduced by Lindeberg ( Lindeberg ,", "1994 ) .", "The scale of the LoG filter is set to the expected range of", "the bacterial radius .", "The bacteria detection is performed by finding local", "maxima of LoG response in the input image ( number of patches for local maxima", "detection controlled manually by a parameter ) .", "The detected maxima enable the", "estimation of bacteria location .", "We performed bacteria tracking based on the", "spatial distance between detections in consecutive frames ( Esteves et al . , 2013b ) .", "Data analysis of the raw tracks", "was done using R software ( R CoreTeam ,", "2013 ) implementing the CIRCULAR package ( Agostinelli and Lund , 2013 ) .", "To eliminate artifacts due to", "wrong mapping of dividing cells or cells in densely populated areas , only cells", "that could be tracked for at least 25 consecutive frames with an average", "velocity below 0 . 4 µm s–1 and a maximum displacement of", "less than 8 µm between two frames ( 3 s ) were considered .", "Cells with an", "average velocity lower than 0 . 05 µm s–1 were regarded", "as immotile and discarded .", "Displacement and orientation were calculated for all", "cells that could be continuously tracked for 60 frames , during 5 min after the", "onset of the directional light .", "All the data shown in Figure 1 are from a single continuous experiment to ensure", "the reliability of quantitative comparisons .", "The experiment is representative of", ">10 such experiments carried out under comparable conditions .", "Synechocystis cells were scraped from a moving colony on a", "motility plate and resuspended in fresh BG11 medium .", "An absorption spectrum for", "the culture was recorded in an Aminco DW2000 spectrophotometer ( Olis Inc ,", "Bogart , GA ) , which is equipped with a wide detection window to minimize", "distortion due to light scattering .", "After subtraction of the optical density due", "to light scattering at 750 nm , the absorption spectrum was deconvoluted into", "chlorophyll and phycobilin components and chlorophyll and phycocyanobilin", "concentrations were estimated according to the formulae of Myers et al . ( 1980 ) .", "Cell density in the suspension was", "estimated by counting in a hemocytometer , leading to estimates of 2 . 94 ×", "107 chlorophyll molecules and 3 . 70 × 107", "phycocyanin-coupled phycocyanobilin molecules per cell .", "These numbers are", "slightly higher than previously estimated ( Mann et al . , 2000 ) ; however , this study used different", "growth conditions .", "Effective pigment concentrations in the thylakoid membranes", "were then estimated by approximating the multiple thylakoid lamellae around the", "periphery of the cytoplasm as a hollow sphere with inner diameter 1 µm", "and outer diameter 2 µm , corresponding to the dimensions estimated from", "fluorescence micrographs .", "Pigments were assumed to be evenly distributed within", "the hollow sphere .", "A narrow light beam passing straight through the", "center of the cell would then have a total path-length of 1 µm through an", "effective chlorophyll concentration of 6 . 7 mM and an effective phycocyanobilin", "concentration of 8 . 4 mM .", "This would give a peak single cell absorbance of", "0 . 106 at 620 nm from the published extinction coefficients ( Myers et al . , 1980 ) .", "To estimate the full", "single cell absorption spectrum , the absorption spectrum for the culture", "was scaled appropriately .", "The scaled spectrum was converted into the", "transmission spectrum shown in Figure 2 .", "Note that the calculation considers only light passing straight through the", "center of the cell and ignores any effects due to refraction and interference .", "In reality , the photosynthetic pigments will not be evenly distributed in the", "thylakoid region as we had to assume for the calculation , but rather clustered", "into membrane layers , reaction centers and light-harvesting complexes .", "Furthermore , the membranes themselves are usually not as regular and symmetrical", "as assumed in our model .", "A homogeneous distribution of pigments will maximize", "the absorption , since inhomogeneous clustering is liable to decrease absorption", "due to enhanced mutual shading of pigments ( Duysens , 1956 ) .", "Therefore , the spectrum shown represents a minimum", "estimate of light transmission through a single cell .", "The Synechocystis torA-gfp mutant , expressing GFP fused to the", "TorA leader sequence for export to the periplasm , was previously described", "( Spence et al . , 2003 ) .", "Cells were", "grown in liquid culture to OD750nm ~1 . 0 in BG11 medium", "supplemented with 50 µg ml–1 spectinomycin .", "100", "µl of culture was added to a chamber in an ibiTreat-coated 8-well", "µ-slide from ibidi and cells were left for 10 min to settle at the", "bottom .", "Suspended cells and excess medium were removed from the chamber , leaving", "adhered cells and a thin film of liquid , with some areas of the chamber bottom", "appearing dry .", "The samples were imaged immediately .", "Microscopy was performed", "using a modified version of the laser-spot time-lapse microscope ( see Figure 3—figure supplement 1 ) .", "Fluorescence excitation was achieved by near-TIRF ( “highly", "inclined”/”oblique” ) illumination of the samples .", "Briefly ,", "a fiber-coupled continuous wave ( CW ) diode laser ( Toptica iChrome", "HP , 488 nm , Toptica , Germany ) was injected into the epifluorescence port of the", "microscope via the dichroic mirror .", "The fiber , collimator , quarter-wave plate", "and TIR-lens ( Thorlabs AC254-200-A-ML , Thorlabs , UK ) were mounted on a", "translation stage and positioned such that the focused beam was centered at the", "back focal plane ( BFP ) of the Olympus 100× objective lens ( UPON TIRF 1 . 49", "NA oil ) ( Olympus , Japan ) .", "By adjusting the translation stage , the position of", "the beam could be translated across the BFP to adjust the inclination angle of", "the beam through the sample .", "Images were captured using the green channel using", "a GFP filter set ( Semrock FF01-520/35-25 , Semrock , Rochester , NY ) and an", "exposure time of 100 ms . Average images were generated by frame averaging over", "1–2 s .", "Measurements were taken from a single experimental run ,", "representative of images recorded for three separate cultures of", "Synechocystis torA-gfp .", "Fluorescent profiles for spot", "intensity and FWHM measurements were obtained from the intensity profile of", "hand-fitted spline curves to the circumference of the cell image starting at the", "edge opposite to the focus spot ( proximal to illumination source ) and tracing in", "an anti-clockwise direction back to the origin .", "The line width was 3 pixels and", "the analysis was done in ImageJ .", "Permanent EPON epoxy-based photosensitive resin ( SU-8 3000 series , MicroChem ,", "Westborough , MA ) with outstandingly low absorption in the near-UV range ( LaBianca and Gelorme , 1995 ) was applied", "on pre-cleaned silicon wafers by spin coating .", "SU-8 3005 was coated at a final", "rotation speed of 4000 rpm for 30 s , after which the photosensitive film was", "subjected to a soft bake at 95°C for 5 hr .", "Droplets ( 4 μl ) of an", "exponentially growing Synechocystis culture in BG11 medium were", "dispensed on top of the 5 µm thick photoresist .", "The BG11 medium", "evaporated under standard clean-room conditions after 30 min , leaving the", "Synechocystis cells on top of the wafer .", "Flood exposure of", "the photopolymer was performed at a center wavelength of 365 nm ( MA6 exposure", "system , Karl Suess , SÜSS MicroTec AG , Germany ) .", "The exposure dose of 85", "mJ cm–2 was found by a lithographic series , in order to", "achieve sharp rendering of scattering patterns on the SU-8 film .", "However , the", "SU-8 layer was not exposed entirely and rendered a height-dependent cross-linked", "structure similar to grayscale lithography ( Gal , 1994 ) .", "Height profiles of the scattering pattern were obtained", "by atomic force microscopy ( tapping-mode , Dimension Icon , Bruker Nano Surfaces", "Division , Santa Barbara , CA ) .", "Completely exposed SU-8 patterns had a median", "roughness of Ra = 3 . 2 nm .", "From the scattering patterns , a mean of six", "axially symmetric profiles was computed in order to average out the influence of", "surface roughness .", "The optical field distribution was computed by the FDTD method .", "The algorithm", "solves the time-dependent Maxwell curl equations by using discrete time steps", "and leads to a time-resolved evolution of the spatial electromagnetic field", "distribution ( Yee , 1966 ) .", "We used a", "freeware FDTD implementation by Schmidt ( Schmidt , 2013 ) .", "The material interface geometry , including", "assignment of refractive index values to material regions , as well as the", "incoming light source properties were required to initiate the simulation", "process .", "The subsequent FDTD solution process was controlled by specifying a", "space and time grid ( Δx = Δy = 10 nm , Δt =", "10–8 s ) .", "Wild-type motile Synechocystis cells were freshly plated in a", "line on motility plates and grown overnight at 30°C with directional", "illumination ( ~10 µmol photons m–2", "s–1 ) in a standing incubator .", "Fingers of cells projecting", "0 . 3–0 . 5 cm were seen after ~16 hrs .", "The leading edges of the", "fingers were collected and resuspended in 20–50 µl of BG11 medium", "and spotted onto freshly made motility plates and left to adsorb .", "Blocks of the", "motility agarose containing the spots were excised from the plate and inverted", "onto 35 mm glass-bottomed tissue-culture dishes for imaging in a custom-built", "inverted microscope .", "The time lapse microscope ( see Figure 4—figure supplement 1 ) was constructed on", "the base of an Olympus IX81 microscope , using an Olympus 40× ( UPLANFLUOR", "0 . 75 NA Air ) objective lens .", "Two air-cooled EMCCD cameras ( Andor iXon Ultra", "DU-897U-CS0-#BV , Andor , UK ) were coupled to the camera port of the microscope", "via a magnifying relay .", "The cameras were simultaneously triggered using a TTL", "pulse from an external digital-to-analog ( D/A ) converter ( Data", "Translation DT9834 , Data Translation , Germany ) , with an exposure time of 50 ms", "and a frequency of 0 . 25 Hz .", "A dichroic mirror ( Semrock FF560-FDi01-25x36 ) in the", "Fourier plane of the camera relay split the emission into red ( fluorescence ) and", "green ( transmission bright-field imaging ) channels .", "Each channel had a separate", "band pass filter ( Semrock BLP01-647R-25​ and Semrock", "FF01-520/35-25 ) .", "The focused laser spot was generated by expanding a collimated", "beam from a fiber-coupled CW diode laser ( Toptica iChrome HP , 640 nm ) , injected", "via the epifluorescence port of the microscope and directed to the objective", "lens via a dichroic filter ( Semrock Di01-R405/488/561/635-25x36 ) .", "The beam was", "expanded to overfill the back aperture of the objective lens to achieve a", "diffraction-limited spot at the focal plane .", "A combination of neutral density", "filters and a half-wave plate and polarizing beam splitter were used to adjust", "the power of the laser to approximately 0 . 01 μW as measured using a power", "meter .", "A 1:1 lens pair ( Thorlabs AC254-100-A-ML ) with one lens mounted on a", "Z-translation stage was used to adjust the axial position of the focus at the", "sample plane .", "A quarter-wave plate was used to circularly polarize the beam", "before it was injected into the microscope .", "Bright-field trans-illumination was", "performed by fiber-coupling a 530 nm LED ( Thorlabs M530F1 ) into a multimode", "fiber and imaging the magnified end of the fiber at the sample plane using a", "condenser lens .", "The LED was TTL triggered via the camera acquisition to reduce", "light exposure to the cells , resulting in a synchronized 50 ms pulse during", "acquisition .", "A third LED ( 625 nm , 3 mW ) was mounted close to the sample at an", "oblique angle to provide directional illumination for the motility assay .", "Light", "intensities were measured with a silicon photodiode-based power meter ( Thorlabs", "S120C and PM100D ) .", "Acquisition was performed over 30 min .", "All the data shown in", "Figure 4 are from a single continuous", "experiment , representative of 5 such experiments carried out under comparable", "conditions ." ] ]

[ "Bacterial phototaxis was first recognized over a century ago , but the method by", "which such small cells can sense the direction of illumination has remained", "puzzling .", "The unicellular cyanobacterium Synechocystis sp .", "PCC", "6803 moves with Type IV pili and measures light intensity and color with a range", "of photoreceptors .", "Here , we show that individual Synechocystis", "cells do not respond to a spatiotemporal gradient in light intensity , but rather", "they directly and accurately sense the position of a light source .", "We show that", "directional light sensing is possible because Synechocystis", "cells act as spherical microlenses , allowing the cell to see a light source and", "move towards it .", "A high-resolution image of the light source is focused on the", "edge of the cell opposite to the source , triggering movement away from the", "focused spot .", "Spherical cyanobacteria are probably the world’s smallest", "and oldest example of a camera eye .", "DOI:", "http://dx . doi . org/10 . 7554/eLife . 12620 . 001" ]

[ "Cyanobacteria are blue-green bacteria that are abundant in the environment .", "Cyanobacteria in the oceans are among the world’s most important oxygen", "producers and carbon dioxide consumers .", "Synechocystis is a", "spherical single-celled cyanobacterium that measures about three thousandths of", "a millimetre across .", "Because Synechocystis needs sunlight to", "produce energy , it is important for it to find places where the light is neither", "too weak nor too strong .", "Unlike some bacteria , Synechocystis", "can’t swim , but it can crawl across surfaces .", "It uses this ability to", "move to places where the light conditions are better .", "It was already known that Synechocystis cells move towards a", "light source that is shone at them from one side , which implies that the", "cyanobacteria can “see” where the light is .", "But how can such a", "tiny cell accurately detect where light is coming from ?", "Schuergers et al . tracked how Synechocystis moved in response to", "different light conditions , and found that the secret of “vision”", "in these cyanobacteria is that the cells act as tiny spherical lenses .", "When a", "light is shone at the cell , an image of the light source is focused at the", "opposite edge of the cell .", "Light-detecting molecules called photoreceptors", "respond to the focused image of the light source , and this provides the", "information needed to steer the cell towards the light .", "Although the details are", "different , and although a Synechocystis cell is in terms of", "volume about 500 billion times smaller than a human eyeball , vision in", "Synechocystis actually works by principles similar to", "vision in humans .", "Schuergers et al . ’s findings open plenty of further questions , as other", "types of bacteria may also act as tiny lenses .", "More also remains to be learnt", "about how the cyanobacteria process visual information .", "DOI:", "http://dx . doi . org/10 . 7554/eLife . 12620 . 002" ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "cell biology" ]

[ [ "Mitochondrial fission and fusion create a dynamic network that is necessary for mitochondrial distribution and homeostasis .", "During mitosis , fission is necessary to distribute mitochondria between daughter cells .", "In neurons , fission and transport maintains mitochondrial distribution throughout the length of these highly polarized cells .", "Mitochondrial fission is also a fundamental step during mitophagy , a quality control mechanism that removes damaged mitochondrial segments .", "Dysregulated mitochondrial dynamics are linked to multiple neurological disorders , such as Alzheimer’s , Huntington’s , Parkinson’s , hereditary ataxia , and Charcot-Marie Tooth disease ( Chen and Chan , 2009; Nunnari and Suomalainen , 2012; Girard et al . , 2012; Niemann et al . , 2005 ) .", "The dynamin-like GTPase Drp1 is a central component of the fission machinery , with Drp1 oligomerization and constriction providing a driving force for the process .", "Drp1 is a cytosolic protein that translocates to the outer mitochondrial membrane ( OMM ) ( Smirnova et al . , 2001 ) .", "In solution , Drp1 exists in a number of oligomeric states , including dimers , tetramers , and higher-order oligomers ( Fröhlich et al . , 2013; Macdonald et al . , 2014 ) .", "Membrane-bound oligomeric Drp1 induces tubulation of associated membrane , and constricts the membrane in the presence of GTP ( Francy et al . , 2015; Mears et al . , 2011 ) .", "Drp1 can bind to the OMM through several factors , including the mitochondrial-specific lipid cardiolipin ( Macdonald et al . , 2014; Stepanyants et al . , 2015; Bustillo-Zabalbeitia et al . , 2014; Ugarte-Uribe et al . , 2014 ) ; and several ‘receptors’ on the OMM , such as Mff , MiD49 , and MiD51 ( Richter et al . , 2015 ) .", "What mechanisms target specific sites on mitochondria for fission ?", "Recent findings have shed light on this question .", "The endoplasmic reticulum ( ER ) forms close contacts with mitochondria at the eventual fission site ( Friedman et al . , 2011 ) .", "Actin polymerization at the ER-mitochondrial interface may be a key component , controlled by the ER-bound formin INF2 ( Korobova et al . , 2013 ) and the mitochondrially-bound Spire1C protein ( Manor et al . , 2015 ) .", "Myosin II activity also plays a role in this pathway ( DuBoff et al . , 2012; Korobova et al . , 2014 ) .", "Inhibition of actin polymerization , myosin II activity , or INF2 causes decreased levels of mitochondrially-bound Drp1 oligomers , suggesting that actin and myosin are acting at the level of Drp1 recruitment .", "Two additional actin binding proteins , cortactin and cofilin , have also been linked to mitochondrial fission ( Li et al . , 2015 ) .", "Given the number of factors that can mediate Drp1 recruitment to the OMM , as well as the necessity for Drp1 oligomerization at the fission site , a fundamental question concerns the relationship between Drp1 recruitment , oligomerization , and fission .", "We envision two possible models for the process , termed de novo assembly and targeted equilibrium .", "In de novo assembly , cytosolic Drp1 is recruited specifically to fission sites , where it oligomerizes and mediates fission in a concerted series of steps in response to fission signals ( which include OMM receptors , cardiolipin and actin ) .", "In targeted equilibrium , Drp1 maintains a constant equilibrium between cytosolic and mitochondrial pools , as well as between dimer and oligomer .", "These equilibria are independent of fission .", "Fission signals shift these equilibria toward stable oligomerization at targeted fission sites on the OMM .", "While few studies address the order of events during Drp1 recruitment , de novo assembly is often tacitly assumed ( Otera et al . , 2013; Elgass et al . , 2015; Labbé et al . , 2014 ) .", "Here , we show evidence supporting targeted equilibrium .", "We observe that mitochondrially-bound Drp1 oligomers mature into stable oligomers through a series of merging events .", "Some of these Drp1 oligomers are motile—capable of translocating on the mitochondrial surface before engaging in fission .", "Notably , not all mitochondrially-bound Drp1 oligomers engage in fission .", "Using ionomycin treatment to induce mitochondrial fission , we show that actin polymerization precedes Drp1 oligomerization , and that actin enriches with Drp1 at fission sites .", "Inhibition of actin polymerization , INF2 or myosin II decreases ionomycin-induced Drp1 accumulation and mitochondrial fission .", "Biochemically , actin filaments bind Drp1 directly and enhance its GTPase activity , suggesting that actin filaments can organize Drp1 into a productive oligomer .", "These results suggest that actin promotes fission by facilitating productive oligomerization of Drp1 on the OMM ." ], [ "To monitor Drp1 dynamics in live cells , we developed a U2OS cell line stably expressing GFP-Drp1 with partial shRNA suppression of endogenous Drp1 ( gDrp-U2OS ) .", "By quantitative western blotting , gDrp-U2OS cells maintain 1 . 64-fold total Drp1 levels compared to control U2OS cells , with 56% endogenous Drp1 and 44% GFP-Drp1 ( Figure 1—figure supplement 1A , B ) .", "Mitochondrial morphology appears similar between control U2OS and gDrp-U2OS cells ( not shown ) .", "We also developed a live-cell assay to measure mitochondrial fission rate , in which peripheral cellular regions are imaged over 10 min for dynamics of a fluorescent mitochondrial marker .", "Fission events are scored based on stable separation of the marker , and similar fission rates are obtained with either a matrix marker or an OMM marker ( Figure 1—figure supplement 1C ) .", "Using this assay , the fission rates of control U2OS and gDrp1-U2OS cells are statistically similar ( 1 . 26 ± 1 . 01 versus 1 . 51 ± 0 . 69 fission events per mm mitochondrion per min , p = 0 . 49 ) ( Figure 1—figure supplement 1D ) .", "Additionally , a second clone displaying undetectable endogenous Drp1 levels and GFP-Drp1 levels ~3 . 5-fold higher than control Drp1 levels displays similar mitochondrial fission frequency to control cells ( Figure 1 , Figure 1—figure supplement 1E–G ) , suggesting that GFP-Drp1 functionally compensates for endogenous Drp1 in these cells .", "We used the gDrp1-U2OS line for subsequent studies , due to its overall Drp1 levels being closer to those of control cells .", "By live-cell confocal microscopy , much of the GFP-Drp1 in gDrp-U2OS cells appears diffuse in the cytoplasm ( Figure 1—figure supplement 2A ) .", "However , adjusting the fluorescence threshold to eliminate this diffuse signal reveals abundant Drp1 ‘puncta’ of varying intensity ( Figure 1A , Figure 1—figure supplement 2A , B ) , likely representing Drp1 oligomers .", "We arbitrarily defined two categories of puncta: ‘total’ puncta , including all Drp1 signal over the diffuse background; and ‘high-threshold’ puncta , representing the brightest 30% of Drp1 puncta .", "These designations do not represent two distinct pools of intensity , as the distribution of punctum intensity is continuous ( Figure 1—figure supplement 2B ) .", "Here , we use these categories to distinguish between smaller puncta and those approaching maximal size . 10 . 7554/eLife . 11553 . 003Figure 1 . Mitochondrial association of Drp1 puncta .", "( A ) Time-lapse image of region of gDrp1-U2OS cell transiently expressing mito-BFP and processed to remove background GFP ( described in Figure 1—figure supplement 2A ) .", "Arrows denote variety of Drp1 puncta: stable ( white arrow ) ; transient ( blue arrow ) ; punctum at a fission site ( yellow arrow ) .", "White arrowheads indicate unbound Drp1 puncta .", "Time in sec .", "Scale bar , 2 μm ( Video 1 ) .", "( B ) Pie charts of mitochondria-associated versus non-associated Drp1 puncta: left panel , total puncta; right panel , high threshold .", "Each percentage is calculated by averaging percentages of mitochondrial Drp1 puncta over 201 frames from a 10 min imaging time .", "Quantification is based on 11 ROIs from 4 cells .", "( C ) Histograms of lifetime distribution for mitochondrial Drp1 puncta , determined over 1200 s total recorded time .", "Left panel , total puncta; right panel , high threshold . DOI: http://dx . doi . org/10 . 7554/eLife . 11553 . 00310 . 7554/eLife . 11553 . 004Figure 1—figure supplement 1 . Characterization of stable GFP-Drp1 cell line ( gDrp1-U2OS cells ) .", "( A ) Western blot of control U2OS cells or gDrp1-U2OS cells showing expression level of GFP-Drp1 and endogenous Drp1 with varying amount of extract loaded ( 0 . 5 , 0 . 75 and 1 . 0x from left to right ) .", "( B ) Quantification of endogenous Drp1 and GFP-Drp1 in control U2OS and gDrp1-U2OS cells from western blots ( normalized to tubulin level ) .", "Error bars , S . D . ( C ) Comparison of mitochondrial fission rates in control U2OS cells transfected with a mitochondrial matrix marker ( mito-dsRed ) versus an OMM marker ( Tom20-GFP or Tom20-mApple ) .", "Mean ± standard deviation for each condition are ( in fission events mm mitochondrial length-1 min-1 ) : mito-dsRed , 2 . 56 ± 1 . 36 ( 14 ROIs/7 cells ) ; and Tom20 , 2 . 91 ± 1 . 73 ( 24/9 ) .", "N . S . , p = 0 . 52 , unpaired Student t-test .", "( D ) Comparison of fission rate in control U2OS and gDrp1-U2OS cells transiently transfected with mCherry-mito7 plasmid ( fission events mm mitochondrial length-1 min-1 ) .", "13 ROIs/7 cells for gDrp1-U2OS cells , and 12 ROIs/6 cells control U2OS cells .", "N . S . = not significant .", "p = 0 . 49 , unpaired Student t-test .", "( E-G )", "Analysis of a second GFP-Drp1 stable cell line ( called clone 8 ) .", "( E ) Western blots with varying amount of extract loaded ( 0 . 25 , 0 . 5 and 1 . 0x from left to right ) for both endogenous Drp1 and GFP-Drp1 level in WT U2OS versus stable clone #8 .", "( F ) Quantification of Drp1 and GFP-Drp1 levels in WT U2OS and clone #8 .", "Drp1 level was normalized to tubulin level when comparing samples , and combined Drp1/GFP-Drp1 level in clone #8 was normalized to the Drp1 level of WT cells .", "( G ) Live-cell analysis of mitochondrial fission rates in WT U2OS and in clone #8 in the absence and presence of ionomycin ( 4 μM ) .", "Quantification based on at least nine ROIs and 5 cells for all conditions .", "Imaging period was at least 21 min for all conditions .", "***p<0 . 001 , unpaired Student t-test .", "( H ) Quantification of number of total Drp1 puncta or high threshold Drp1 puncta ( y axis ) versus length of mitochondria ( x axis ) .", "Quantification was based on 26 ROIs . DOI: http://dx . doi . org/10 . 7554/eLife . 11553 . 00410 . 7554/eLife . 11553 . 005Figure 1—figure supplement 2 . Image processing for GFP-Drp1 intensity in gDrp1-U2OS cells .", "( A ) Example of image processing for analysis of Drp1 dynamics .", "Images were threshold-adjusted by subtracting background Drp1 in the cytosol using a rolling ball algorithm ( radius = 2 pixels or 0 . 304 mm ) , followed by smoothing ( ImageJ ) .", "( B ) Example of categorizing Drp1 puncta based on mean intensity over time .", "Bar graphs are histograms of mean Drp1 intensity over 90 s ( 3 s acquisition intervals ) , with the cytosolic “background” ( bar at extreme left , indicated by asterisk ) as the most abundant .", "Top histogram shows “total puncta” to include all puncta over background ( mean intensity >10 ) , and bottom histogram shows “high threshold” to examine only the brighter puncta ( mean intensity >100 ) .", "Micrographs show a merged image of mitochondria and mitochondrially bound puncta ( left ) , puncta included in the total puncta category ( circled in middle image ) , and puncta included in the high threshold category ( circled in right image ) .", "Scale bar , 10 μm in ( A ) , 5 μm in ( B ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 11553 . 005 We employed a particle-tracking algorithm to analyze punctum position and lifetime relative to a mitochondrial matrix marker ( Figure 1C ) .", "Approximately 49% of total puncta and 70% of high-threshold puncta co-localize with mitochondria at individual frames in the viewing period ( Figure 1B ) .", "For most puncta , mitochondrial association is transient ( Video 1 ) , with mean lifetimes of 45 . 0 ± 104 . 8 s and 64 . 4 ± 113 . 1 s for total and high-threshold puncta , respectively ( 3582 and 1601 puncta analyzed ) .", "The largest population of puncta remains bound < 12 s in both cases ( Figure 1C ) , although the high-threshold puncta are significantly more stably associated with mitochondria ( p<0 . 001 for lifetime comparison ) .", "Puncta density on mitochondria does not vary systematically with mitochondrial length , with approximately 0 . 41 total puncta and 0 . 17 high-threshold puncta per μm mitochondrial length ( Figure 1—figure supplement 1H ) . 10 . 7554/eLife . 11553 . 006Video 1 . Left: confocal time-lapse of Drp1 dynamics in gDrp1-U2OS cell transiently expressing mito-BFP; Right: mitochondrially-associated Drp1 puncta pseudo-colored in blue . Time lapse was taken in single z-plane every 3 s .", "Time min:sec .", "Bar , 2 μm ( Figure 1A ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 11553 . 006 How do Drp1 puncta assemble on mitochondria ?", "We observed three distinct dynamic behaviors associated with mitochondrially-bound Drp1 puncta: punctum merging , punctum motility , and mitochondrial fission .", "Mitochondrially-bound puncta frequently merge together , with successive merging events resulting in punctum growth ( Figure 2A , B , Figure 2—figure supplement 1A , Video 2 , 3 ) .", "Merging is a reversible process , and can be followed by splitting of the merged punctum ( Figure 2—figure supplement 1B , Video 4 ) .", "The combination of these results suggests that a population of Drp1 is in rapid equilibrium between the cytosol and the OMM , and that one mechanism for assembly of larger Drp1 oligomers is through merging of mitochondrially-bound oligomers .", "We refer to this process as ‘maturation’ .", "Imaging at higher resolution by Airyscan microscopy suggests that maturing puncta are loosely associated with the mitochondrion , and progressively encircle the mitochondrion as they mature ( Figure 2C , Video 5 ) . 10 . 7554/eLife . 11553 . 007Figure 2 . Maturation of mitochondrially-bound Drp1 puncta .", "( A ) Example of Drp1 maturation events ( arrowheads ) , followed by mitochondrial fission ( 63 s ) .", "Time in sec .", "Scale bar , 1 μm ( Video 2 ) .", "( B ) Quantification of mitochondrial Drp1 merging rate , defined as number of merging events per min per number of Drp1 puncta .", "5317 puncta from 11 ROIs in four cells based on Trakmate with parameters described before .", "Line indicates mean ( 0 . 045 ± 0 . 029 ) ( C ) Super-resolution Airyscan live-cell images showing Drp1 maturation ( yellow arrowheads ) ( Video 5 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 11553 . 00710 . 7554/eLife . 11553 . 008Figure 2—figure supplement 1 . Drp1 maturation events .", "( A ) Multiple Drp1 merging events , followed by fission at a looped junction ( arrow at 36 s ) ( Video 3 ) .", "( B ) Drp1 merging event , followed by splitting of the punctum .", "Three dim Drp1 puncta merge to form a bright punctum at 3 s ( blue arrow ) .", "Subsequently , the punctum splits between 6 and 12 s and the resulting two puncta move away from each other ( Video 4 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 11553 . 00810 . 7554/eLife . 11553 . 009Video 2 . Confocal time-lapse of Drp1 maturation in gDrp1-U2OS cell transiently expressing mCherry-mito7 ( red ) .", "Time lapse was taken in single z-plane every 3 s .", "Time min:sec .", "Bar , 1 μm ( Figure 2A ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 11553 . 00910 . 7554/eLife . 11553 . 010Video 3 . confocal time-lapse of multiple Drp1 merging events , followed by fission at a looped junction in gDrp1-U2OS cell transiently expressing mCherry-mito7 ( red ) .", "Time lapse was taken in single z-plane every 3 s .", "Time min:sec .", "Bar , 1 μm ( Figure 2—figure supplement 1A ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 11553 . 01010 . 7554/eLife . 11553 . 011Video 4 . Confocal live cell image of Drp1 merging event , followed by splitting of the punctum in gDrp1-U2OS cell transiently expressing mCherry-mito7 ( red ) .", "Time lapse was taken in single z-plane every 3 s .", "Time min:sec .", "Bar , 1 μm ( Figure 2—figure supplement 1B ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 11553 . 01110 . 7554/eLife . 11553 . 012Video 5 . Airyscan time-lapse of Drp1 maturation in gDrp1-U2OS cell ( GFP in green ) transiently expressing mCherry-mito-7 ( red ) .", "Time lapse was taken in single z-plane in dorsal region of cells every 7 s .", "Time min:sec .", "Bar , 1 μm ( Figure 2C ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 11553 . 012 Low-intensity puncta are motile along the OMM , allowing them to interact during merging events ( Figure 2A , B , Figure 2—figure supplement 1A , B ) .", "In addition , a sub-set of high-threshold Drp1 puncta translocates directionally along mitochondria for short distances at a mean velocity of 46 . 9 ± 12 . 8 nm/sec ( Figure 3A , B ) .", "Assessing the fraction of motile puncta is challenging due to the underlying motility of the mitochondrion itself , thus our analysis is limited to situations where mitochondria are visibly stationary during the viewing period .", "Motile high-threshold puncta are capable of stopping abruptly and engaging in fission ( Figure 3A , Video 6 ) . 10 . 7554/eLife . 11553 . 013Figure 3 . Drp1 puncta motility and relationship to mitochondrial fission .", "( A ) Example of motile Drp1 punctum ( yellow arrow ) engaging in fission at 60 s , and a stationary punctum ( white arrowhead ) .", "Lower panel maps tracks of these puncta .", "Time in sec .", "Scale bar , 1 μm ( Video 6 ) .", "( B ) Drp1 punctum translocation velocity on mitochondrion ( 12 motile puncta from 12 ROIs from 11 cells ) .", "( C ) Quantification of percentage of Drp1 puncta engaging in fission over the 10 min viewing period for total puncta and for high threshold puncta .", "11 ROIs from 4 cells , ***p<0 . 001 , unpaired Student t-test .", "( D ) Lag time between Drp1 punctum appearance and fission for productive Drp1 puncta .", "49 fission events from 11 cells .", "( E ) Relationship between lag time and mitochondrial length for 20 mitochondria .", "Line fit data: y = 83 . 7 + 0 . 22x , RMSD = 0 . 096 . DOI: http://dx . doi . org/10 . 7554/eLife . 11553 . 01310 . 7554/eLife . 11553 . 014Figure 3—figure supplement 1 . Correlation between mitochondrial length and fission , and Drp1 punctum persistence on mitochondrial ends after mitochondrial fission .", "( A ) Analysis of mitochondrial lengths for mitochondria not undergoing fission ( “Non-dividing” , left ) , and mitochondria undergoing fission ( “Dividing mitochondria , middle ) over a 10 min imaging period . For dividing mitochondria , mitochondrial length was measured at the frame prior to dividing; for non-dividing mitochondria , length was measured at first frame imaged . Histogram of all mitochondria shown in right panel . ( B ) Two examples of Drp1 remaining on both new mitochondrial ends after fission events ( white arrows ) . ( C ) Example of Drp1 remaining on only one new mitochondrial end after fission ( blue arrow indicating mitochondrial end without Drp1 punctum ) . ( D ) Frequency of Drp1 left on ends after fission . Quantification was based on 20 fission events , in which Drp1 puncta were readily resolved after fission . Bar , 1 μm . Time in sec . DOI: http://dx . doi . org/10 . 7554/eLife . 11553 . 01410 . 7554/eLife . 11553 . 015Video 6 . Confocal time-lapse of Drp1 movement along mitochondrion in gDrp1-U2OS cell transiently expressing mito-BFP ( red ) . Mitochondrially-bound Drp1 puncta were followed by Trackmate . Time lapse was taken in single z-plane every 1 . 5 s . Time min:sec . Bar , 1 μm ( Figure 3A ) . DOI: http://dx . doi . org/10 . 7554/eLife . 11553 . 015 We next assessed the correlation between Drp1 puncta and mitochondrial fission , and found that all observed fission events are associated with high-threshold Drp1 puncta ( 49 events , examples in Figure 1A , Figure 2A , Figure 2—figure supplement 1A , Figure 3A ) . However , only a low percentage of Drp1 puncta ( 2 . 5% of total puncta and 6 . 3% of high threshold puncta ) engages in observable fission events during the 10 min imaging period . Even for productive puncta , the lag between punctum establishment and mitochondrial fission varies significantly , with a mean of 76 . 0 ± 40 . 3 s ( Figure 3D ) that appears to be independent of mitochondrial length ( Figure 3E ) . These results suggest that establishment of an apparently stable mitochondrially-bound Drp1 oligomer is not sufficient , and that additional steps are required to drive fission . We made two additional observations concerning mitochondrial fission . First , mitochondria undergoing fission tend to be longer than the mean ( Figure 3—figure supplement 1A ) . Second , Drp1 remains at newly created mitochondrial ends for a significant time after fission . This residual Drp1 can be asymmetrically distributed , with one end inheriting most or all residual Drp1 ( Figure 3—figure supplement 1B–D ) . We examined Drp1 puncta at higher spatial resolution by live-cell 3D-structured illumination microscopy ( 3D-SIM ) , using Tom20-mCherry as the mitochondrial marker in order to label the OMM . We analyzed diameters of both mitochondria and associated Drp1 puncta for ‘productive’ events ( leading to fission ) and non-productive events ( sites of stable Drp1 puncta that did not undergo fission during observation ) . The mean mitochondrial diameter in the absence of Drp1 is 322 ± 64 nm ( Figure 4B ) . Non-productive high-threshold Drp1 puncta still cause significant constriction ( Figure 4—figure supplement 1A ) , with mean mitochondrial diameter of 215 ± 50 nm ( Figure 4B ) and Drp1 diameter of 270 ± 38 nm ( Figure 4C ) at these sites . This constriction is maintained over time ( Figure 4D ) . Productive Drp1 puncta undergo significant contraction immediately prior to fission , with mean Drp1 diameter of 200 ± 31 nm in the frame prior to fission ( Figure 4C , D , Figure 4—figure supplement 1B , Video 7 , 8 ) . The mitochondrial diameter in this frame is 134 ± 25 nm , which is at the limit of resolution for 3D-SIM . Our results are qualitatively similar to those obtained from fixed-cell PALM studies ( Rosenbloom et al . , 2014 ) , while adding the dynamic component of contraction prior to fission . 10 . 7554/eLife . 11553 . 016Figure 4 . Drp1 and mitochondrial diameter by live-cell 3D-SIM . ( A ) 3D-SIM image of region of a live gDrp1-U2OS cell transiently expressing Tom20-mCherry . Yellow arrowhead , Drp1 punctum engaged in fission . Green arrowhead , non-productive punctum . White arrowhead , unbound punctum . Time in sec . Scale bar , 1 μm . ( B ) Quantification of mitochondrial diameters from regions devoid of Drp1 ( “bulk mitochondria” , left ) or at sites of non-productive stationary puncta ( right ) .", "***p<0 . 001 unpaired Student t-test .", "( C ) Quantification of Drp1 diameters for productive Drp1 puncta ( 4 . 5 s before fission , 11 events ) versus non-productive puncta ( 82 events ) .", "***p<0 . 001 unpaired Student t-test .", "( D ) Diameter variation for seven productive Drp1 puncta over 30 s prior to fission ( at 0 s , left ) and for nine non-productive puncta over a similar time ( right ) .", "( E ) Time-lapse of fission event from ( A ) showing Drp1 punctum constriction .", "Bottom panel of Drp1 alone is further enlarged ( Video 7 ) .", "Time in sec .", "Scale bar , 1 μm ( top ) ; 0 . 5 μm ( bottom ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 11553 . 01610 . 7554/eLife . 11553 . 017Figure 4—figure supplement 1 . 3D-SIM imaging of Drp1 and mitochondria .", "( A ) Two close-up examples of non-productive stationary puncta , showing constriction of OMM ( left panel ) in the absence of fission .", "( B ) Additional example of mitochondrial fission event from 3D-SIM ( similar to Figure 4E ) shows morphological changes of Drp1 structures .", "The region in dashed rectangle is enlarged in the bottom panel .", "GFP-Drp1 ( green ) , mCherry-Tom20 ( red ) ( Video 8 ) .", "Time in sec .", "Scale bars , 0 . 5 μm in ( A ) and 1 μm ( top and middle panels ) and 0 . 5 μm ( bottom panel ) in ( B ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 11553 . 01710 . 7554/eLife . 11553 . 018Video 7 . 3D-SIM time-lapse of Drp1-mediated mitochondrial fission in gDrp1-U2OS cell transiently expressing Tom20-mCherry ( red ) .", "Time lapse was taken every 4 . 5 s .", "Time min:sec .", "Bar , 1 μm ( Figure 4A , E ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 11553 . 01810 . 7554/eLife . 11553 . 019Video 8 . 3D-SIM live cell image of Drp1-mediated mitochondrial fission in gDrp1-U2OS cell transiently expressing Tom20-mCherry ( red ) .", "Time lapse was taken every 4 . 5 s .", "Time min:sec .", "Bar , 1 μm ( Figure 4—figure supplement 1B ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 11553 . 019 We also observed motile puncta by 3D-SIM .", "These puncta appear to distort as they translocate , periodically separating into strands ( Figure 5A , Figure 5—figure supplement 1A , Video 9 , 10 ) .", "Dynamic shape changes are not characteristic of stable puncta , which can be most easily appreciated when viewing a punctum transitioning from motile to stationary ( Figure 5B ) .", "Motile puncta appear to encircle the mitochondrion to a significant degree , although the spatial resolution achieved here is not sufficient to answer this question definitively .", "Punctum velocity in 3D-SIM is similar to that measured by confocal microscopy ( 48 . 5 ± 17 . 1 nm/sec , Figure 5—figure supplement 1B ) . 10 . 7554/eLife . 11553 . 020Figure 5 . Drp1 motility by live-cell 3D-SIM .", "( A ) Motile Drp1 punctum on a stationary mitochondrion ( yellow arrow ) and stationary Drp1 punctum on a motile mitochondrion ( white arrow ) .", "White arrowhead indicates stationary Drp1 punctum on stationary mitochondrion .", "Lower panel is zoom of Drp1 alone , indicating changing Drp1 morphology during movement ( Video 9 ) .", "( B ) Punctum transitioning from motile to stationary , accompanied by change in morphology .", "Time in sec .", "Scale bar , 1 μm in A ( top ) ; 0 . 5 μm in A ( bottom ) ; 1 μm in B ( top ) ; 0 . 3 μm in B ( bottom ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 11553 . 02010 . 7554/eLife . 11553 . 021Figure 5—figure supplement 1 . ( A ) Additional example of motile Drp1 punctum from 3D-SIM ( similar to Figure 5A ) .", "Yellow arrow indicates the motile Drp1 punctum moving along the mitochondrion .", "White arrowhead indicates stationary Drp1 punctum .", "Time in sec .", "Scale bar , 1 μm ( Video 10 ) .", "( B ) Velocity quantification of mitochondrially-associated motile Drp1 puncta from 3D-SIM .", "Four ROIs from 2 cells are used in this quantification via Trackmate V2 . 7 . 3 . DOI: http://dx . doi . org/10 . 7554/eLife . 11553 . 02110 . 7554/eLife . 11553 . 022Video 9 . 3D-SIM time-lapse of Drp1 movement along mitochondrion in stable gDrp1-U2OS cell transiently expressing Tom20-mCherry ( red ) .", "Time lapse was taken every 4 . 5 s .", "Time min:sec .", "Bar , 1 μm ( Figure 5A ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 11553 . 02210 . 7554/eLife . 11553 . 023Video 10 . 3D-SIM live cell image of Drp1 movement along mitochondrion in gDrp1-U2OS cell transiently expressing Tom20-mCherry ( red ) .", "Time lapse was taken every 4 . 5 s .", "Time min:sec .", "Bar , 1 μm ( Figure 5—figure supplement 1A ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 11553 . 023 What signal might stimulate Drp1 punctum maturation ?", "We previously showed that mitochondrial length increased upon three cellular treatments: inhibition of actin polymerization by Latrunculin A ( LatA ) , siRNA suppression of the formin protein INF2 , or inhibition of myosin II by chemical inhibitors or siRNA ( Korobova et al . , 2013; Korobova et al . , 2014 ) .", "The increase in mitochondrial length suggests a fission defect , but could also reflect changes in mitochondrial fusion .", "Here , we establish that the effect is on mitochondrial fission , using our live-cell fission assay .", "Suppression of either INF2 or myosin IIA causes an ~50% decrease in fission rate , whereas Drp1 suppression or expression of the dominant-negative Drp1 K38A mutant cause near-complete inhibition ( Figure 6—figure supplement 1A , B ) .", "In addition , suppression of either INF2 or myosin IIA reduces mitochondrially-associated Drp1 puncta , with a moderate reduction of total Drp1 puncta ( 2 . 7-fold for both INF2 and myosin IIA ) and a larger reduction in high threshold puncta ( 6 . 7-fold and 8 . 3-fold , respectively , Figure 6—figure supplement 1C ) .", "LatA treatment also causes a reduction in mitochondrially-associated Drp1 puncta , although the degree of high threshold reduction is not as great as for INF2 or myosin IIA suppression ( Figure 6—figure supplement 1D ) .", "These results support the previous results suggesting a role for actin polymerization in mitochondrial Drp1 assembly .", "We next examined the relationship between actin filaments and Drp1 during mitochondrial fission by live-cell microscopy in gDrp-U2OS cells .", "One challenge is the abundance of actin-based structures in U2OS cells .", "To mitigate this issue , we imaged cells at a focal plane significantly above the ventral surface , where there are fewer actin-based structures such as stress fibers .", "In this region , we observed frequent examples of actin filaments accumulating prior to fission ( Figure 6—figure supplement 2A , Video 11 ) , but the overall actin filament abundance in the region remained a challenge for determining whether its presence might contribute to fission .", "To assess the significance of the actin accumulation , we calculated the mean percentage of mitochondrial area covered by actin ( Figure 6—figure supplement 2B ) , and compared this number to the percentage of fission events at which actin was present immediately prior to fission .", "We found that a significantly higher percentage of fission events ( 56% ) were associated with actin filaments than would be expected by random correlation ( 28% , n = 59 fission events , Figure 6— figure supplement 2C ) . 10 . 7554/eLife . 11553 . 024Video 11 . Confocal live cell image of mitochondrial fission in an un-stimulated U2OS cell transiently expressing mApple-F-tractin and mito-BFP . Time lapse was taken in single z-plane in dorsal region of cells to avoid massive actin based structures every 3 s .", "Time min:sec .", "Bar , 1 μm ( Figure 6—figure supplement 1A ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 11553 . 024 While this correlation is suggestive , the low frequency of fission events is challenging for temporal correlation of actin and Drp1 accumulation prior to fission .", "To examine this relationship in more detail , we used ionomycin , a calcium ionophore , to increase mitochondrial fission rate ( Tan et al . , 2011; Sanmartin et al . , 2014 ) .", "A recent publication showed that 3T3 cells respond to increased cytosolic calcium with an acute burst of actin polymerization throughout the cytosol , mediated by INF2 ( Shao et al . , 2015 ) .", "We find a similar ionomycin-induced actin burst in gDrp-U2OS cells ( 16 . 9 ± 8 . 7% signal increase , t1/2 28 . 1 ± 19 . 8 s ) followed by a rapid decline ( Figure 6A ) .", "Ionomycin treatment also increases mitochondrial fission rate 3 . 9-fold ( 2 . 61 ± 1 . 01 vs 10 . 23 ± 5 . 41 events mm-1 min-1 ) ( Figure 6B ) .", "Similar fission rate increases occur when using mitochondrial matrix markers tagged with either dsRed or BFP ( Figure 6—figure supplement 3 ) . 10 . 7554/eLife . 11553 . 025Figure 6 . Ionomycin treatment induces actin polymerization , Drp1 maturation and mitochondrial fission . gDrp1-U2OS cells transiently transfected with mApple-F-tractin and mito-blue plasmids .", "( A ) Time course of changes in Drp1 oligomer and actin filaments ( judged by changes in GFP and mApple signal over cytosolic background , fluorescence normalized to time 0 ) .", "GFP-Drp1 quantified over whole cell .", "Actin filaments were quantified from two or three ROIs per cell ( approximately 3 × 3 μm each ) , in which no stress fibers or cell edges were included .", "DMSO ( N = 10 cells ) or ionomycin ( 4 μM , N = 12 cells ) added at time 0 ( arrow ) .", "* denotes total Drp1 puncta or polymerized actin fluorescence ( as indicated for individual curves ) .", "( B ) Mitochondrial fission rate ( fission events per mm mitochondrial length per min ) upon ionomycin treatment in the absence or presence of LatA ( 2 μM ) or siRNA for INF2 or myosin IIA .", "***p<0 . 001 .", "( C ) Quantification of mitochondrially bound Drp1 puncta ( total puncta ( left ) and high threshold ( right ) ) in response to ionomycin treatment in control ( sc siRNA ) and INF2 suppressed ( INF2 siRNA ) cells .", "Nine ROIs from six control cells and six INF2 suppressed cells .", "Ionomycin was added at 0 s to all samples ( black arrow ) .", "Data first quantified as mitochondrially-bound puncta per mm mitochondrial length , then normalized such that 0 s value = 1 .", "Error bars , standard deviation ( S . D . ) .", "( D ) Airyscan microscopy of a fission site ( white arrow ) showing actin filament enrichment ( red arrows ) and Drp1 maturation ( yellow arrowheads ) upon ionomycin treatment ( 1 μM at t = 0 ) .", "Larger area shown in Figure 6—figure supplement 2 ( Video 12 ) .", "Time in sec .", "Bar , 1 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 11553 . 02510 . 7554/eLife . 11553 . 026Figure 6—figure supplement 1 . Suppression of INF2 or myosin IIA decreases fission rate in un-stimulated conditions .", "( A ) Western blots for INF2 ( left ) or myosinIIA ( right ) using lysates from siRNA-treated cells ( tubulin as loading control ) .", "( B ) Quantification of mitochondrial fission rates in U2OS cells transiently transfected with mito-dsRed in un-stimulated conditions .", "Fission rate defined as fission events per mm mitochondrial length per min in a defined ROI .", "Mean rate ± standard deviation for each condition are: control siRNA , 3 . 97 ± 1 . 65 ( 28 ROIs/4 independent experiments ) ; INF2-CAAX siRNA , 2 . 42 ± 1 . 99 ( 27/4 ) ; myosin IIA siRNA , 1 . 55 ± 1 . 52 ( 24/4 ) ; Drp1 siRNA , 0 . 43 ± 1 . 41 ( 7/2 ) ; and Drp1-K38A over-expression , 0 . 081 ± 0 . 20 ( 6/2 ) .", "**p<0 . 01 , ***p<0 . 001 , unpaired Student t-test .", "( C , D )", "Inhibition of actin , INF2 or myosin IIA reduces density of mitochondrially associated Drp1 in unstimulated conditions .", "( C ) Knock down of INF2 or MyosinII-A reduces both total Drp1 puncta ( left panel ) and high threshold Drp1 puncta ( right panel ) .", "Each value is calculated by averaging density of mitochondrial Drp1 puncta over 201 frames with 3 s time intervals over 10 min imaging time .", "Quantification is based on 11 ROIs from 4 scRNA treated cells , 9 ROIs from 3 INF2 siRNA treated cells; and 13 ROIs from 5 MyosinIIA siRNA treated cells from 2 independent experiments .", "( D ) Inhibition of actin polymerization by LatA reduces both total Drp1 puncta ( left panel ) and high threshold Drp1 puncta ( right panel ) .", "Quantification was based on 12 ROIs from five DMSO pre-treated cells; 12 ROIs from six 2 μM LatA pre-treated cell from 2 independent experiments .", "***p< 0 . 001 , unparied Student t-test . DOI: http://dx . doi . org/10 . 7554/eLife . 11553 . 02610 . 7554/eLife . 11553 . 027Figure 6—figure supplement 2 . Actin filament enrichment with Drp1 puncta at fission sites in un-stimulated conditions .", "( A ) Example of actin filaments enriching with Drp1 punctum prior to fission in gDrp1-U2OS cell transiently transfected with mApple-Ftractin and mito-BFP .", "Upper panel , mitochondrial signal only; middle panel , Drp1 and actin filaments; bottom panel , all three markers .", "Red arrows indicate fission sites in the upper panel .", "Yellow arrows indicate Drp1 punctum that overlays with actin filaments , and white arrows indicate Drp1 punctum that does not overlay with actin ( Video 12 ) .", "( B ) Two examples of image processing for determining the percentage of mitochondrial surface covered by actin filaments .", "Upper panels are raw images of mitochondria and actin filaments from the frame immediately before mitochondrial fission ( 3 s ) .", "Bottom panels show processed images with% of actin/mitochondrial overlap given .", "White arrows indicate fission sites .", "( C ) Table comparing the number of fission events at actin-mitochondrial contact sites compared to the expected number if there is no relationship between the presence of actin and mitochondrial fission ( predicted from the percent of mitochondrial coverage by actin filaments for 59 examples ) .", "**p=0 . 0026 , Fisher’s exact test .", "Time in sec .", "Scale bar , 1 μm in ( A and B ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 11553 . 02710 . 7554/eLife . 11553 . 028Figure 6—figure supplement 3 . Comparison of mitochondrial fission rates in mitoRed labeled versus mitoBFP labeled cells . Quantification was based on 11 ROIs from 7 DMSO treated , mitoRed labeled cells over 25 . 4 min; 9 ROIs from 7 Ionomycin treated , mitoRed labeled cells over 24 . 3 min; 11 ROIs from 10 DMSO treated , mitoBFP labeled cell over 24 . 2 min; 11 ROIs from 8 Ionomycin treated , mitoBFP labeled cell over 21 . 5 min .", "***p<0 . 001 , unpaired Student t-test . DOI: http://dx . doi . org/10 . 7554/eLife . 11553 . 02810 . 7554/eLife . 11553 . 029Figure 6—figure supplement 4 . Larger field of super resolution Airyscan images , including the region shown in Figure 6D . Actin filaments accumulate at three mitochondrial constriction sites ( red arrows ) prior to Drp1 maturation ( yellow arrowheads ) .", "Two of the constriction sites do not undergo fission in this time period ( Video 12 ) .", "Time in sec .", "Scale bar , 1 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 11553 . 029 We next asked whether the quantity of Drp1 oligomers also increased following ionomycin treatment , initially monitoring total Drp1 puncta .", "Indeed , ionomycin treatment causes Drp1 oligomer signal increases from 10-20% , depending on the experiment , with a consistent t1/2 of 99 . 9 ± 19 . 0 s ( Figure 6A , also see Figure 7A , C and D ) .", "In contrast to the transient actin increase , the Drp1 oligomer signal remains elevated for >10 min .", "These measurements are rapid to conduct , allowing analysis of every time point in the sequence , but do not provide information on the change in mitochondrially-bound Drp1 puncta .", "For this reason , we also quantified the change in the number of mitochondrially-associated Drp1 puncta upon ionomycin treatment on a more limited set of time points , and found ~40% increase for low- or high-threshold puncta ( Figure 6C ) . 10 . 7554/eLife . 11553 . 030Figure 7 . Inhibition of actin , INF2 or myosin IIA reduces ionomycin-induced Drp1 maturation .", "( A , B ) gDrp1-U2OS cells were transiently transfected with mitochondrial matrix marker , mito-BFP , and actin filament marker , mApple-F-tractin .", "Cells were treated with 0 , 0 . 1 , 0 . 2 , 1 and 2 μM LatA ( or DMSO ) for 15 min before imaging .", "At 60 s after starting imaging , cells were treated with 4 μM ionomycin ( in the presence of the appropriate concentration of LatA ) and imaged for 9 min .", "GFP-Drp1 signals over cytosolic background ( background subtract , ImageJ ) were measured per whole cell; actin filament signals were quantified from two or three ROIs per cell ( approximately 3 × 3 μm ) , in which no stress fibers or cell edges were included .", "Error bars , standard deviation for A and S . E . M . for B . ( C ) Time course of ionomycin-induced changes in Drp1 oligomer and actin filaments after INF2 siRNA treatment ( INF2 siRNA ) .", "11 control cells ( sc siRNA ) , six INF2 siRNA .", "Error bars , S . E . M . ( D ) Time course of ionomycin-induced changes in Drp1 oligomer and actin filaments after myosin IIA siRNA treatment .", "N = 11 control cells ( sc siRNA ) , 12 myosin IIA siRNA .", "* denotes total Drp1 puncta or polymerized actin ( as indicated for individual curves ) .", "Error bars , S . E . M for C and D . DOI: http://dx . doi . org/10 . 7554/eLife . 11553 . 030 In addition , we visually monitored the dynamics of actin and Drp1 at individual ionomycin-induced fission sites by Airyscan microscopy .", "Prior to ionomycin treatment , a number of low intensity Drp1 puncta dynamically associate with mitochondria , and a meshwork of actin exists throughout the cytoplasm ( Figure 6D , Figure 6—figure supplement 4 , Video 12 ) .", "This actin meshwork enriches at certain positions on mitochondria , and Drp1 puncta tend to be more abundant at these sites but are dynamic .", "Upon ionomycin treatment , the actin signal intensifies throughout the cytosol , particularly at sites of mitochondrial crossover .", "Drp1 puncta mature at these actin-enriched sites , and some of these sites undergo fission while others undergo constriction but no fission during the viewing period ( Figure 6D , Figure 6—figure supplement 4 , Video 12 ) . 10 . 7554/eLife . 11553 . 031Video 12 . Airyscan time-lapse of mitochondrial fission in response to Ionomycin treatment in gDrp1-U2OS cell ( GFP in blue ) transiently expressing mApple-F-tractin ( green ) and mito-BFP ( red ) .", "Time lapse was taken in single z-plane in dorsal region of cells every 18 s .", "Time min:sec .", "Bar , 1 μm ( Figure 6D ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 11553 . 031 We tested the association between actin and Drp1 further by inhibiting actin polymerization , followed by ionomycin stimulation .", "Pre-treatment for 15 min with LatA abolishes both Drp1 accumulation ( Figure 7A , B ) and the increase in mitochondrial fission ( Figure 6B ) .", "Suppression of INF2 by siRNA treatment potently inhibits the actin increase , but causes a smaller decrease in whole-cell Drp1 oligomer accumulation than that achieved by LatA ( Figure 7C ) .", "However , the effect of INF2 suppression on mitochondrially-associated high threshold Drp1 puncta is dramatic , with almost complete elimination of the ionomycin-induced increase ( Figure 6C Right ) .", "INF2 suppression also causes a decrease in ionomycin-induced mitochondrial fission ( Figure 6B ) .", "Interestingly , INF2 suppression has a negligible effect on Drp1 puncta of lower intensity ( Figure 6C Left ) .", "These results suggest that INF2-mediated actin polymerization plays a role in Drp1 maturation on mitochondria .", "We next tested the role of myosin II in this process .", "Suppression of myosin IIA does not alter the actin response to ionomycin ( signal intensity increase 20 . 0 ± 6 . 4% with t1/2 of 22 . 8 ± 12 . 7 s ) , but strongly reduces Drp1 oligomerization ( Figure 7D ) and mitochondrial fission ( Figure 6B ) .", "These experiments suggest that both INF2-induced actin polymerization and myosin II activity are important pre-requisites for productive Drp1 oligomer assembly .", "The mechanism by which actin might stimulate Drp1 accumulation is unclear .", "One possibility is that Drp1 binds directly to actin filaments at the fission site , similar to the direct binding between dynamin and actin ( Gu et al . , 2010; Palmer et al . , 2015; Mooren et al . , 2009 ) .", "Interestingly , a previous study demonstrates that actin accumulates at clathrin-mediated endocytosis sites prior to dynamin 2 arrival ( Grassart et al . , 2014 ) .", "We tested the possibility of a direct interaction using purified proteins .", "Through co-sedimentation assay , Drp1 binds actin with an apparent Kd of 0 . 8 ± 0 . 4 μM ( Figure 8A , Figure 8— figure supplement 1A ) .", "Interestingly , less than 50% of the Drp1 binds actin filaments even at saturating actin concentrations ( Figure 8A ) .", "This situation is not caused by a significant pool of ‘inactive’ Drp1 in our purified preparation , since >90% of the Drp1 pellets when incubated with a non-hydrolyzable GTP analogue ( Figure 8—figure supplement 1B ) .", "We postulate that partial binding is due to differential affinities of Drp1 oligomeric states for actin filaments .", "In these assays , Drp1 does not change the critical concentration of actin , since actin polymerizes to a similar extent in the absence or presence of Drp1 ( Figure 8—figure supplement 1C ) . 10 . 7554/eLife . 11553 . 032Figure 8 . Drp1 binds to actin filaments .", "( A ) Co-sedimentation assay in which Drp1 ( 1 . 3 μM ) is incubated with indicated concentration of pre-polymerized actin ( concentrations indicate total actin ) for 1 hr , then centrifuged at >100 , 000 ×g to sediment actin filaments .", "Pellets analyzed by SDS-PAGE ( Figure 8—figure supplement 1A ) .", "Each data point is the mean from 10 independent experiments .", "Error bars , standard deviation .", "( B ) Single time point images from TIRF microscopy assay of actin filaments ( 20% TAMRA-labeled ) mixed with saturating concentration of GFP-Drp1 .", "Scale bar , 2 μm .", "( C ) Negative stain electron microscopy of 2 μM actin filaments in the absence or presence of 1 μM Drp1 .", "Mean filament widths: 8 . 9 + 0 . 2 nm ( n = 44 filaments ) for actin alone; and 27 . 2 + 1 . 3 nm ( n = 49 ) for actin/Drp1 .", "Scale bar , 50 nm .", "( D ) TIRF microscopy time-lapse montage showing GFP-Drp1 dynamics on an actin filament .", "Time indicates seconds after GFP-Drp1 addition .", "Scale bar , 2 μm ( Video 13 ) .", "( E ) TIRF microscopy time-lapse montage showing GFP-Drp1 can bundle actin filaments ( 20% TAMRA-labeled ) .", "Time indicated in seconds .", "Scale bar , 2 μm ( Video 14 ) .", "( F ) TIRF microscopy time-lapse montage showing multiple bundling events by GFP-Drp1 ( denoted by red , orange and blue arrows ) .", "Actin filaments not shown .", "Note Drp1-coated filament denoted by blue arrow , which binds by its end to a second filament for ~50 s , before releasing , flipping , binding by its opposite end , then bundling into the second filament .", "Time in sec .", "Scale bar , 2 μm ( Video 15 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 11553 . 03210 . 7554/eLife . 11553 . 033Figure 8—figure supplement 1 . Binding of Drp1 to actin filaments by co-sedimentation assay .", "( A ) Example of an SDS-PAGE gel from a high-speed co-sedimentation assay , where 1 . 3 μM Drp1 was incubated with the indicated concentration of actin filaments for 1 hr at 23°C .", "Samples were ultra-centrifuged for 20min at 4°C .", "The amount of Drp1 in the pellet with actin filaments was analyzed using Colloidal blue staining SDS-PAGE and ImageJ Software .", "The standards indicate known amounts of Drp1 and actin , which were used to generate a standard curve to calculate the amount of Drp1 in the pellet .", "The concentrations of actin and Drp1 indicated in the ‘pellet’ lanes indicate the total amounts present in the assay before centrifugation .", "The Drp1 band is just below 75 kDa marker , and the actin band is between 37 . 5 and 50 kDa markers .", "( B ) High-speed sedimentation assay where 1 . 3 μM Drp1 was incubated with 500 μM GMPPCP for 1 hr at 23°C .", "Samples were ultra-centrifuged for 20min at 4°C .", "The pellet and supernatant samples were analyzed using Coomassie staining SDS-PAGE .", "The standards indicate known amounts of Drp1 .", "All samples were run on the same gel and stained identically ( intervening lanes were cut out for clarity ) .", "( C ) Pyrene-actin polymerization assay of 2 μM actin alone ( 10% pyrene label ) or with 1 μM Drp1 .", "Actin was monomeric at time 0 ( time of addition of Drp1 and polymerization buffer ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 11553 . 033 By total internal reflection ( TIRF ) microscopy , GFP-tagged Drp1 binds evenly throughout the actin filament ( Figure 8B ) , with no apparent preference for specific filament regions ( Figure 8C , Video 13 ) .", "By negative-stain electron microscopy , Drp1-bound filaments have mean widths of 27 . 2 ± 1 . 3 nm , compared to 8 . 9 ± 0 . 2 nm for actin alone ( Figure 8D ) .", "It is unclear whether this increased width is due to Drp1 binding a single filament or to Drp1-mediated filament bundling .", "The width of a Drp1 dimer is 22 . 9 nm ( Fröhlich et al . , 2013 ) , similar to our measured width .", "However , Drp1 can induce filament bundling , as evidenced by TIRF microscopy ( Figure 8E , Video 14 ) .", "Indeed , time-lapse imaging of the bundling process suggests that filaments have a preferred bundled orientation , and can be end-bound to the sides of Drp1-bound filaments prior to bundling ( Figure 8F , Video 15 ) . 10 . 7554/eLife . 11553 . 034Video 13 . TIRF microscopy time-lapse GFP-Drp1 ( 270 nM ) was added to TAMRA-actin filaments ( 1 μM , 20% TAMRA initially ) .", "Actin filaments were polymerized for 10 min prior to GFP-Drp1 addition .", "Dual-color simultaneous images were collected every 1 s .", "Scale bar , 2 μm .", "307 frames played at 75 ms frame rate ( 13 . 3-fold accelerated ) ( Figure 8C ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 11553 . 03410 . 7554/eLife . 11553 . 035Video 14 . TIRF microscopy time-lapse showing filament bundling . GFP-Drp1 ( 270 nM ) was added to TAMRA-actin filaments ( 1 μM , 20% TAMRA initially ) .", "Actin filaments were polymerized for 10 min prior to GFP-Drp1 addition .", "Dual-color simultaneous images were collected every 1 s .", "Scale bar , 2 μm .", "13 frames played at 250 ms frame rate ( fourfold accelerated ) ( Figure 8E ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 11553 . 03510 . 7554/eLife . 11553 . 036Video 15 . TIRF microscopy time-lapse showing filament bundling . GFP-Drp1 ( 2 μM ) was added to TAMRA-actin filaments ( 1 μM , 20% TAMRA initially ) .", "Actin filaments were polymerized for 10 min prior to GFP-Drp1 addition .", "Images were collected every 2 s .", "Scale bar , 2 μm .", "46 framed played at 100 ms frame rate ( 20-fold accelerated ) .", "Red , yellow and blue arrows indicate bundling events ( Figure 8F ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 11553 . 036 Drp1 binding to anionic lipids such as cardiolipin increases its GTPase activity ( Macdonald et al . , 2014 ) , presumably by placing its GTPase domains in close proximity as is found for other dynamin family proteins ( Bui and Shaw , 2013 ) .", "We tested whether actin filaments could also increase Drp1’s GTPase activity and found a ~3 . 5-fold increase ( Figure 9A ) .", "An additional question is whether actin filaments can synergize with Drp1 receptors on the OMM .", "We tested this possibility using the cytosolic portion of Mff .", "Mff alone causes only a slight increase in Drp1 GTPase activity at the concentrations tested .", "However , the combination of Mff and actin filaments causes a substantial increase in Drp1 activity , far beyond the additive effects of either Mff or actin alone ( Figure 9B ) .", "These results show that Drp1 binds actin filaments in a manner that stimulates its catalytic activity , and that actin filaments can synergize with Mff to increase productive Drp1 oligomerization . 10 . 7554/eLife . 11553 . 037Figure 9 . Actin fialments stimulate Drp1 GTP hydrolysis synergistically with Mff .", "( A ) GTPase assays containing 1 μM Drp1 in the presence or absence of 0 . 5 or 1 μM actin ( pre-polymerized for 1 hr ) for 5 min before GTP addition ( 250 μM ) .", "N = 6 experiments .", "( B ) GTPase assays containing 1 μM Drp1 in the presence or absence of 0 . 5 μM actin ( pre-polymerized for 1 hr ) and the indicated concentration of Mff ( cytosolic region ) for 5 min before GTP addition ( 250 μM ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 11553 . 037" ], [ "Overall , our results support several important and novel features of Drp1 oligomerization .", "Rather than being an ‘all or none’ process in which Drp1 oligomerization at the fission site is induced de novo by fission signals , we postulate that both Drp1 oligomerization and mitochondrial association are in constant equilibrium even in the absence of fission signals .", "Fission signals serve to ‘target’ this equilibrium to fission sites .", "Actin filaments are one such fission signal .", "We illustrate these features as a three-stage model for fission-productive Drp1: recruitment , maturation , and conversion ( Figure 10 ) .", "In recruitment , ‘units’ of Drp1 are in rapid equilibrium between cytosolic and mitochondrially-bound pools .", "These Drp1 units diffuse on the OMM , and merge to form larger units .", "Maturation is the progressive association of mitochondrially-bound Drp1 units , leading to assembly of an oligomer that fully encircles the mitochondrion .", "The size of the mature oligomer is unclear , but might be similar to the 26-40 subunits found for dynamin2 ( Grassart et al . , 2014; Cocucci et al . , 2014 ) .", "Mature oligomers are capable of movement along the OMM .", "Conversion represents a distinct process in which a site containing a stable Drp1 oligomer is rendered fission-competent . 10 . 7554/eLife . 11553 . 038Figure 10 . Model for assembly of fission-productive Drp1 on mitochondria . Step 1: recruitment .", "Drp1 units are in equilibrium between cytosol and OMM , possibly binding to OMM receptors such as Mff , MiD49 , MiD51 , or Fis1 , or to cardiolipin .", "We suggest that several distinct oligomeric species may interact with the OMM .", "Step 2: maturation .", "Mitochondrially-bound Drp1 oligomers grow through incorporation of other mitochondrially-bound oligomers , progressively encircling the OMM in the process .", "The larger Drp1 oligomers constrict the OMM but are not yet competent to drive fission .", "Step 3: conversion .", "A stable Drp1 oligomer becomes productive for mitochondrial fission ( change from green to blue ) , with increased Drp1 ring constriction driving membrane ingression .", "Actin filaments and myosin II clearly stimulate the maturation process , with possible effects on recruitment as well . DOI: http://dx . doi . org/10 . 7554/eLife . 11553 . 038 This model raises many new testable questions .", "First , what is the complete list of “fission signals” ?", "There is evidence that OMM proteins such as Mff , MiD49/51 , and Fis1 , as well as cardiolipin exposure on the OMM , can serve as fission signals .", "We postulate that actin filaments in proximity to the mitochondrion are another such signal .", "Other potential factors that might influence fission could include mitochondrial length and mitochondrial branches .", "We find that fission events occur disproportionately on longer mitochondria , but we do not find an increase in Drp1 puncta density on longer mitochondria , suggesting that maturation is not favored on long mitochondria .", "It should be realized , however , that most mitochondria exist in a branched network in these cells .", "The relationship between mitochondrial branching and mitochondrial fission is a fascinating question still to be addressed .", "Second , do the various fission signals act in series or in parallel ?", "In other words , do the signals work together within the same fission pathway , or act in alternate pathways ?", "Both options could be true , depending on the fission signal .", "For example , we postulate that actin filaments and cardiolipin might represent alternate maturation factors , since both are polyvalent anions .", "In contrast , actin filaments could work in concert with Mff , either in “coincidence detection” by Drp1 ( Hatch et al . , 2014 ) or by actin filaments serving as a reservoir for delivery of Drp1 oligomers to Mff .", "Recent work has shown that Mff binds preferentially to oligomerized Drp1 ( Liu and Chan , 2015 ) , which is consistent with this possibility .", "We show here that actin filaments and Mff synergize in stimulating Drp1’s GTPase activity .", "A final possibility is that specific fission factors mediate distinct steps in the process .", "For example , a recent study on the membrane-altering ability of the Drp1-cardiolipin interaction might suggest that cardiolipin triggers a conversion step ( Stepanyants et al . , 2015 ) .", "Third , what is the size of the Drp1 ‘unit’ that is recruited to mitochondria ?", "We cannot detect the smallest oligomers ( dimers , tetramers ) in this study for several reasons , including: our threshold procedure forcibly removes these from analysis , and only ~ half of the Drp1 in these cells is GFP-labeled .", "The available data suggest that the recruited unit may be mixed oligomers of GFP-labeled and endogenous Drp1 .", "Recent biochemical studies have shown that Drp1 exists in equilibrium between oligomeric states in solution , with some evidence that dimers are the relevant membrane-recruited unit in the case of cardiolipin binding ( Fröhlich et al . , 2013; Macdonald et al . , 2014 ) .", "On the other hand , in the present study we observe clear instances of Drp1 puncta apparently translocating from cytosol to mitochondrion .", "We state ‘apparently’ because it is possible that these oligomers transfer from another membrane or a cytoskeletal structure .", "Based on these findings , we postulate that a range of oligomeric species can be recruited to mitochondria , but that their on- and off-rates might vary considerably .", "Fourth , what steps are stimulated by actin ?", "Our data show that inhibiting actin polymerization inhibits the accumulation of Drp1 oligomers , suggestive of roles in either recruitment or maturation .", "Direct Drp1 binding to actin filaments near mitochondria might enhance initial mitochondrial binding , or might accelerate oligomerization steps of cytosolic Drp1 prior to mitochondrial interaction , both of which would aid recruitment .", "Alternately , actin binding to mitochondrially-bound Drp1 might enhance oligomerization on the mitochondrial surface , which would represent maturation .", "The two possibilities are not mutually exclusive .", "A related question is: what is myosin II’s role in the process ?", "One possibility is that myosin II activity might lead to Drp1-independent ‘pre-constriction’ of the OMM , as we have proposed previously ( Korobova et al . , 2014; Hatch et al . , 2014 ) .", "It is also possible that myosin II organizes the INF2-assembled actin filaments in a manner optimal for Drp1 recruitment and/or maturation .", "Fifth , what are the roles of other actin binding proteins that have been identified as contributing to mitochondrial fission , such as cortactin , cofilin , and Spire 1C ( Manor et al . , 2015; Li et al . , 2015 ) ?", "There is evidence that mitochondrially-bound Spire 1C and ER-bound INF2 work through the same pathway , suggesting that Spire 1C might serve as a nucleation factor and INF2 as an elongation factor and/or a severing protein .", "Spire proteins cooperate with other formin proteins in a similar manner ( Vizcarra et al . , 2011; Quinlan , 2013 ) .", "Roles for cortactin and cofilin in the same pathway as Spire 1C/INF2 are possible , but an alternative is that they contribute to a distinct actin-dependent pathway .", "The fact that steady-state fission rate is only partially reduced by INF2 or myosin IIA suppression ( Figure 6—figure supplement 1 ) suggests some degree of functional redundancy .", "Sixth , does the conversion step represent a change in Drp1 structure/activity , or changes in other fission components ?", "One possibility is recruitment of additional necessary molecules .", "For example , recent publications show that endophilin co-operates with actin and dynamin in clathrin-independent endocytosis ( Boucrot et al . , 2015; Renard et al . , 2015 ) .", "Alternately , exposure of cardiolipin on the OMM could trigger a conversion step , as mentioned above ( Macdonald et al . , 2014 ) .", "Seventh , how does ionomycin stimulate mitochondrial fission ?", "We postulate that , in addition to the known effect of calcium on Drp1 phosphorylation state ( Cribbs and Strack , 2007 ) , increased cytosolic calcium activates INF2 , as demonstrated in recent work ( Shao et al . , 2015 ) ( R . Wedlich-Söldner , personal communication ) .", "These effects are likely to be similar to those induced by the Listeria protein LLO ( Stavru et al . , 2013 ) , which is also a calcium ionophore .", "Eighth , what is the role of the ER in mitochondrial fission ?", "The tight association of ER with mitochondria at fission sites ( Friedman et al . , 2011 ) could contribute in two ways .", "First , ER supplies the INF2 isoform responsible for actin dynamics at the fission site ( Korobova et al . , 2013; Manor et al . , 2015 ) .", "Second , ER might contribute to the calcium dynamics necessary for ionomycin-mediated fission , considering the complex relationship between extracellular calcium entry , calcium release from ER , and ER-mitochondrial calcium communication ( Horne and Meyer , 1997; Csordás et al . , 1999 ) .", "Finally , what is the mechanism of Drp1 motility on mitochondria ?", "Our 3D-SIM time-lapse movies suggest that motile Drp1 oligomers are rings that encircle the mitochondrion , and that these rings change significantly during the motility process , in a manner similar to ‘walking’ along the mitochondrial surface .", "Another possible mechanism could be ‘treadmilling’ of Drp1 units , adding to one side of the Drp1 oligomer while dissociating from the other side , akin to cytoskeletal polymers .", "Furthermore , what is the purpose of this motility ?", "One possibility is something we call the ‘night watchman’ hypothesis , in which motility allows mature Drp1 oligomers to ‘patrol’ the mitochondrion in search of fission signals ." ], [ "Mito-DsRed and mito-BFP constructs were previously described ( Korobova et al . , 2014 ) , and consist of amino acids 1–22 of S . cerevisiae COX4 N-terminal to the respective fusion protein .", "mCherry-mito-7 was purchased from Addgene ( #55102 ) , and consists of the mitochondrial targeting sequence was from subunit VIII of human cytochrome C oxidase N-terminal to mCherry .", "Tom20-mCherry was a gift from Andrew G . York ( NIH , Bethesda , MD ) and described in York et al . ( 2013 ) .", "Drp1-containing plasmids ( GFP ( A206K ) -Drp1 and GFP ( A206K ) -Drp1 K38A ) were described in Strack et al . ( 2013 ) .", "These plasmids co-expresse H1 promoter–driven shRNA for endogenous Drp1 as well as GFP-tagged , RNAi-resistant rat Drp1 .", "mApple-F-tractin plasmid was a gift from Clare Waterman and Ana Pasapera ( NIH , Bethesda , MD ) , and described in ( Johnson and Schell , 2009 ) .", "Human Mff isoform 8 cDNA ( Gandre-Babbe and van der Bliek , 2008 ) was amplified through RT-PCR from Hela cell RNA .", "Mff protein containing amino acids 1-197 ( NP_001263994 ) and Ala-2 Cys substitution , for protein labeling , was cloned into the pET21a ( Novagen ) vector using the NdeI and XhoI sites .", "Oligonucleotides for human total INF2 siRNA were synthesized by IDT Oligo against target sequence 5’- GGAUCAACCUGGAGAUCAUCCGC-3’ ( siRNA#1 ) , and 5’- GCAGUACCGCUUCAGCAUUGUCA-3’ ( siRNA#2 ) .", "Oligonucleotides for INF2 CAAX isoform were 5’-ACAAAGAAACTGTGTGTGA-3’ ( siRNA#1 ) , and 5’- CCCTGATTCTGATGATAAT-3’ ( siRNA#2 ) .", "Oligonucleotides for human Drp1siRNA were synthesized by IDT Oligo against target sequence 5’-GCCAGCUAGAUAUUAACAACAAGAA-3’ ( siRNA#1 ) and 5’- GGAACGCAGAGCAGCGGAAAGAGCT-3’ ( siRNA#2 ) .", "Oligonucleotides for human myosin IIA siRNA were synthesized by IDT Oligo against target sequence 5’- GCCACGCCCAGAAGAACGAGAAUGC-3’ ( siRNA#1 ) and 5’- GCAAGCUGCCGAUAAGUAUCUCUAT-3’ ( siRNA#2 ) .", "As a control , Silencer Negative Control 5’-CGUUAAUCGCGUAUAAUACGCGUAT-3’ ( Ambion ) was used .", "Human osteosarcoma U2OS ( obtained directly from American Type Culture Collection ( HTB-96 ) in 2014 ) were grown in DMEM ( Invitrogen , Carlsbad , CA , USA ) supplemented with 10% calf serum ( Atlanta Biologicals ) .", "The U2OS line was tested at regular intervals for mycoplasma contamination using LookOut Mycoplasma PCR detection kit ( Sigma-Aldrich ) .", "U2OS cells are not on the list of mis-identified or cross-contaminated cell lines compiled by the International Cell Line Authentication Committee ( ICLAC ) , and we have not had them verified by a third party .", "The stable GFP-Drp1 U2OS cell line ( gDrp1-U2OS ) .", "The gDrp1-U2OS cell line was made by transient transfection of the GFP ( A206K ) -Drp1 plasmid into U2OS cells , followed by selection in G418 .", "Selected cells were then flow-sorted for GFP signal as single cells onto 96-well plates , and individual clones were analyzed for GFP-Drp1 expression and endogenous Drp1 suppression .", "Cell lines we used for a maximum of 20 passages .", "For transfection of the U2OS or gDrp1-U2OS lines , cells were seeded at 4 × 105 cells per well of a 6-well dish ~16 hr prior to transfection .", "Plasmid transfections were performed in OPTI-MEM media ( Invitrogen ) with 2 μL Lipofectamine 2000 ( Invitrogen ) per well for 6 hr , followed by trypsinization and re-plating onto concanavalin A ( ConA , Sigma/Aldrich , Cat . No . C5275 ) - coated coverslips ( 25 mm , Electron microscopy Sciences , Cat . No . #72225-01 ) , at ~3 . 5 × 105 cells per well .", "Cells were imaged in live cell media ( Cat . No . 21063-029 , Life technologies ) , ~16–24 hr after transfection .", "For all experiments , the following amounts of DNA were transfected per well ( individually or combined for co-transfection ) : 400 ng for mito-BFP; 300 ng for GFP-Drp1 and GFP-Drp1 K38A constructs; 850 ng for Tom20-mCherry; 900 ng for mCherry-mito7; 500 ng for mApple-F-Tractin .", "For siRNA transfections , cells were plated on 6 well plates with 30–40% density , and 2 μl RNAimax ( Invitrogen ) and 63 pg of siRNA were used per well .", "Cells were analyzed 72–84 hr post-transfection for suppression .", "For Ionomycin treatment , gDrp1-U2OS cells were transfected with mitoBFP and mApple-F-tractin , as described above the day before imaging .", "Cells were mounted on the microscope for imaging , then treated with 4 μM Ionomycin ( from a 4 mM stock in DMSO , Sigma/Aldrich , Cat . No . I6034 ) at 20 frames ( 1 or 2 min , depends on time interval used ) during imaging .", "Medium was pre-equilibrated for temp and CO2 content before use .", "DMSO was used as the negative control .", "For Latrunculin A ( LatA ) pre-incubation followed by ionomycin treatment , cells were transfected with mitoBFP and mApple-F-tractin the day before treatment .", "Cells were incubated with live cell medium containing LatA ( added from a 0 . 2 mM DMSO stock ) for 15 min before imaging , and ionomycin was added at frame 20 ( 1 or 2 min depends on time intervals ) with DMSO used as the negative control .", "Cells were grown on 25 mm coverslips coated with ConA ( coverslips treated for ~2 hr with 100 μg/mL ConA in water at room temperature ) .", "Coverslips were mounted into flow chambers , then onto a Wave FX spinning disk confocal microscope ( Quorum Technologies , Inc . , Guelph , Canada , on a Nikon Eclipse Ti microscope ) , equipped with Hamamatsu ImageM EM CCD cameras and Bionomic Controller ( 20/20 Technology , Inc ) temperature-controlled stage set to 37°C .", "After equilibrating to temperature for 10 min , cells were imaged with the 60x 1 . 4 NA Plan Apo objective ( Nikon ) using the 403 nm and 450/50 filter for BFP , 491 nm laser and 525/20 filter for GFP , and the 561 nm laser and 593/40 filter for mApple or mCherry .", "Super-resolution 3D-SIM images were acquired on a DeltaVision OMX V4 ( GE Healthcare ) equipped with a 60x/1 . 42 NA PlanApo oil immersion objective ( Olympus ) , 405 , 488 , 568 and 642 nm solid state lasers ( 100 mW ) and sCMOS cameras ( pco . edge ) .", "Image stacks of 1 µm with 0 . 125 µm thick z-sections and 15 images per optical slice ( 3 angles and 5 phases ) with were acquired using immersion oil with a refractive index 1 . 524 .", "Images were reconstructed using Wiener filter settings of 0 . 005 and optical transfer functions ( OTFs ) measured specifically for each channel with SoftWoRx 6 . 1 . 3 ( GE Healthcare ) to obtain super-resolution images with a twofold increase in resolution both axially and laterally .", "Images from different color channels were registered using parameters generated from a gold grid registration slide ( GE Healthcare ) and SoftWoRx 6 . 1 . 3 ( GE Healthcare ) .", "Super resolution images were acquired on LSM 880 equipped with 63x/1 . 4 NA plan Apochromat oil objective , using the Airyscan detector ( Carl Zeiss Microscopy , Thornwood , NY ) .", "The Airyscan uses a 32-channel array of GaAsP detectors configured as 0 . 2 Airy Units per channel to collect the data that is subsequently processed using the Zen2 software .", "The processing involves an online reassignment of the pixel information followed by linear deconvolution .", "The end result is a 1 . 7-fold improvement in resolution in X , Y and Z and at least fourfold improvement in signal to noise ratio .", "gDrp1-U2OS cells transiently transfected with mitochondrial markers were imaged live by spin disc confocal fluorescence microscopy every 3 s for 10 min in a single focal plane .", "Regions of interest with readily resolvable mitochondria and Drp1 were processed as described in Figure 1—figure supplement 2A .", "We thresholded mitochondrially associated Drp1 puncta by using a ImageJ plugin , Colocalization , with the following parameters: Ratio 50% ( 0–100% ) ; Threshold channel 1: 30 ( 0–255 ) ; Threshold channel 2: 30 ( 0–255 ) ; Display value: 255 ( 0–255 ) .", "Mitochondrially associated Drp1 puncta were further analyzed by Trackmate V2 . 7 . 3 ( as described in Figure 1—figure supplement 2B , C ) to separate into total puncta and high threshold categories .", "Parameters used in Trackmate are: estimated blob diameter of 0 . 7 microns for confocal and 0 . 5 microns for SIM; LoG detector settings: tracker – LAP tracker , frame to frame linking of 1 micron , track segment gap-closing of 1 micron max distance and 1 frame max frame gap , track segment splitting of 1 micron , and track segment merging of 1 micron .", "The number of Drp1 puncta in each category were automatically counted frame-by-frame by ImageJ macro , Find Stack Maxima .", "Suitable ROI’s were selected for analysis based on whether individual mitochondria were resolvable and did not leave the focal plane .", "Files of these ROIs were assembled , then coded and scrambled by one investigator , and analyzed for fission by a second investigator in a blinded manner as to the treatment condition .", "The second investigator scanned the ROIs frame-by-frame manually for fission events , and determined mitochondrial length within the ROI using the ImageJ macro , Mitochondrial Morphology ( described in Dagda et al . ( 2009 ) ) .", "The results were then given back to the first investigator for de-coding .", "For measuring lengths of individual mitochondria , ROIs were selected that enabled imaging of the entire mitochondrial length where possible .", "Due to the fact that the majority of mitochondrial mass is in the form of a branched mitochondrial ‘network’ in these cells , and that one end of the network is often in the peri-nuclear region which is difficult to resolve , it was frequently difficult to find both ends of the network , in which case the resolvable length was reported .", "Polyclonal antibodies against human INF2 N-terminus ( amino acids 1-424 ) or FH1-FH2- C ( amino acids 469-1249 , CAAX ) were raised in rabbits by Covance ( Denver , PA ) , and affinity purified using DID construct ( amino acids 1-269 ) or FH1-FH2 ( amino acids 469-940 ) coupled to Sulfolink ( Thermo/Pierce ) .", "Anti-Tubulin ( DM1-α , Sigma/Aldrich ) was used at 1:10 , 000 dilution .", "Drp1 was detected using a rabbit monoclonal antibody ( Cell Signaling ) at 1:500 dilution .", "MyosinII-A was detected using a rabbit polyclonal antibody ( Cell Signaling ) at 1:500 dilution .", "For Western Blotting , cells were grown on 6 well plate , trypsinized , washed with PBS and resuspended 50 μL PBS .", "50 μL was mixed with 34 μL of 10% SDS and 1 μL of 1 M DTT , boiled 5 min , cooled to 23°C , then 17 μl of 300 mM of freshly made NEM in water was added .", "Just before SDS-PAGE , the protein sample was mixed 1:1 with 2xDB ( 250 mM Tris-HCl pH 6 . 8 , 2 mM EDTA , 20% glycerol , 0 . 8% SDS , 0 . 02% bromophenol blue , 1000 mM NaCl , 4 M urea ) .", "Proteins were separated by 7 . 5% SDS-PAGE and transferred to a PVDF membrane ( polyvinylidine difluoride membrane , Millipore ) .", "The membrane was blocked with TBS-T ( 20 mM Tris-HCl , pH 7 . 6 , 136 mM NaCl , and 0 . 1% Tween-20 ) containing 3% BSA ( Research Organics ) for 1 hr , then incubated with the primary antibody solution at 4°C overnight .", "After washing with TBS-T , the membrane was incubated with horseradish peroxidase ( HRP ) -conjugated secondary antibody ( Bio-Rad ) for 1 hr at room temperature .", "Signals were detected by Chemiluminescence ( Pierce ) .", "We expressed and purified Human Drp1 000 isoform ( Strack et al . , 2013 ) from S . cerevisiae and E . coli .", "Yeast-purified Drp1 was used for TIRF microscopy and negative staining electron microscopy .", "E . coli-purified Drp1 was used in high-speed co-sedimentation experiments and GTPase assays .", "Actin filaments were assembled from monomers ( 20 μM ) for 1 hr at 23°C by addition of a 10x stock of polymerization buffer ( 500 mM NaCl , 10 mM MgCl2 , 10 mM EGTA , 100 mM imidazole pH 7 . 0 ) to a 1x final concentration .", "To maintain ionic strength across all samples , an actin blank was prepared in parallel using G-buffer in place of actin monomers , and used to dilute actin filaments as needed for each sample .", "Drp1 was diluted to 10 μM in 150 mM NaCl , 1 mM MgCl2 , 1 mM EGTA , 10 mM Imidazole , then centrifuged at 100 , 000 rpm for 20 min at 4°C in a TLA-120 rotor ( Beckman ) .", "The supernatant was stored on ice , and its protein concentration determined by Bradford assay ( Bio-Rad 500-0006 ) .", "Drp1 ( 1 . 3 μM ) was incubated with varying amounts of actin filaments ( 0 . 25–10 μM ) for 1 hr at 23°C in a 200 μl volume .", "The final ionic strength was adjusted to 75 mM using NaCl .", "Following incubation , samples were centrifuged at 80 , 000 rpm for 20 min at 4°C in a TLA-100 . 1 rotor ( Beckman ) .", "The supernatant was carefully removed , and 100 μl was mixed with SDS-PAGE sample buffer .", "Pellets were washed briefly and gently with 100 μl of 50 mM NaCl , 1 mM MgCl2 , 1 mM EGTA , 10 mM Imidazole pH 7 . 4 , then resuspended in 100 μl SDS-PAGE sample buffer and resolved by SDS-PAGE .", "Gels were stained with Colloidal blue staining SDS-PAGE ( Invitrogen LC6025 ) , and band intensity was analyzed using ImageJ software .", "TAMRA-labeled actin ( 1 μM , 20% TAMRA labeled ) was diluted in TIRF buffer ( 50 mM KCl , 1 mM MgCl2 , 1 mM EGTA , 10 mM Hepes pH 7 . 4 , 100 mM DTT , 0 . 2 mM ATP , 15 mM Glucose , 0 . 5% Methyl Cellulose , 0 . 01 mg/ml catalase ( Sigma C3515 ) , 0 . 05 mg/ml glucose oxidase ( Sigma G6125 ) , 0 . 1% BSA ) was polymerized for 10 min in glass flow chambers ( see below ) , at which point indicated concentrations of GFP-Drp1 ( diluted in TIRF buffer ) was added .", "The filaments were visualized using an Olympus IX-83 inverted microscope equipped with a 4-channel CellTIRF attachment , appropriate lasers and driven by Metamorph for Olympus software .", "Simultaneous dual-color images were acquired every 1 s with TIRF objective ( 60x , 1 . 49 N . A . ) and two Andor Zyla scMOS cameras with an Andor TuCam adapter .", "Glass flow chambers were assembled using VWR microcover glasses ( 22 × 22 mm and 18 × 18 mm No 1 . 5 ) with double-stick scotch tape to hold 10 μl volume .", "Prior to chamber assembly , the cover glasses were washed with acetone ( 50 min ) , ethanol ( 10 min ) , water ( 1 min ) , and incubated in a solution containing a 1:2 ratio of 30% H2O2: concentrated H2SO4 for one hour .", "Cover glasses were then rinsed with water , 0 . 1 M KOH , and water again .", "Inert gas was used to completely dry cover glasses before silanization .", "Glasses were silanized overnight in a solution of 0 . 0025% dichlorodimethyl silane ( Sigma 85126 ) in chloroform , then washed with methanol and dried with inert gas .", "Glasses were stored in a clean sealed container .", "Immediately before starting an assay , chambers were incubated with 1% Pluronic F127 ( Sigma P2443 ) in BRB80 buffer ( 80 mM Pipes/KOH , pH 6 . 9 , 1 mM EGTA , 1 mM MgCl2 ) for 1 min , and then equilibrated with TIRF buffer .", "Drp1 was diluted in 50 mM KCl , 1 mM MgCl2 , 1 mM EGTA , 10 mM Hepes pH 7 . 4 .", "To remove potential aggregates or small Drp1 nuclei , Drp1 was centrifuged at 100 , 000 rpm for 20 min at 4°C in a TLA-120 rotor ( Beckman ) , and the supernatant was stored on ice for use .", "4 μM actin was polymerized for 7 min in G-buffer plus 50 mM KCl , 1 mM MgCl2 , 1 mM EGTA , 10 mM Hepes pH 7 . 4 .", "Drp1 was added to polymerized actin for a final concentration of 1 . 3 μM Drp1 and 2 μM actin .", "Samples ( 30 μl ) were absorbed onto EM grids ( Electron Microscopy Sciences , CF300-Cu ) for 4 min , then blotted gently with filter paper .", "Grids were subsequently stained with 1% uranyl acetate solution for 1 min , and again blotted gently with filter paper .", "The prepared grids were imaged on a JEOL JEM 1010 transmission electron microscope operated at 100 keV acceleration .", "Images were collected using an XR-41B AMT digital camera and capture engine software ( AMTV 540; Advanced Microscopy Techniques ) .", "Filament widths were quantified using ImageJ .", "To remove ATP , actin monomers in G-buffer were incubated with Bio-Rad AG1-X2 100–200 mesh anion exchange resin ( Dowex ) ( Bio-Rad , 1401241 ) rotating at 4°C for 5 min , followed by low-speed centrifugation to remove resin .", "Actin filaments ( 20 μM ) were polymerized in 50 mM KCl , 1 mM MgCl2 , 1 mM EGTA , 10 mM Hepes pH 7 . 4 for 1 hr at 23°C .", "Drp1 was diluted in 150 mM KCl , 1 mM MgCl2 , 1 mM EGTA , 10 mM Hepes pH 7 . 4 , then centrifuged to remove aggregates as described above .", "Drp1 ( 1 . 3 μM ) was mixed with actin filaments ( 1 μM ) , and the final ionic strength was adjusted to the equivalent of 75 mM KCl using 4 M KCl stock .", "Samples were incubated at 37°C for 5 min .", "At this point , GTP was added to a final concentration of 250 μM to start reactions at 37°C .", "Reactions were quenched at various time points by mixing 20 μL of sample with 5 μL of 125 mM EDTA in a clear flat-bottomed 96-well plate ( Greiner ) .", "Six time points were acquired for each condition .", "Inorganic phosphate was determined by addition of 150 μl of malachite green solution ( 1 mM Malachite green ( Sigma Aldrich , 2290105–100 g ) , 10 mM ammonium molybdate ( [Sigma Aldrich , A7302–100 g] in 1N HCl ) to 25 μl quenched reactions .", "Absorbance at 650 nm was measured with a 96-well fluorescence plate reader ( TECAN Infinite M1000 , Mannedorf , Switzerland ) .", "GTP hydrolysis rates were determined by plotting phosphate concentration as a function of time .", "Described in detail in Gurel et al . ( 2014 ) .", "Briefly , unlabeled and pyrene-labeled actin were mixed to a concentration of 6 . 67 μM ( 5% pyrene label ) in G-buffer , then diluted to 6 μM by adding 0 . 1 volumes of 10 mM EGTA/1 mM MgCl2 for 2 min .", "Polymerization assays were initiated by diluting actin to 2 μM using 1 . 5x polymerization buffer containing the indicated concentrations of Drp1 .", "Polymerization was monitored in a TECAN Infinite M1000 fluorescence plate reader at 365 nm excitation and 410 nm emission at 23°C .", "Time between reagent mixing and fluorescence recording was < 30 s ." ] ]

[ "While the dynamin GTPase Drp1 plays a critical role during mitochondrial fission , mechanisms controlling its recruitment to fission sites are unclear .", "A current assumption is that cytosolic Drp1 is recruited directly to fission sites immediately prior to fission .", "Using live-cell microscopy , we find evidence for a different model , progressive maturation of Drp1 oligomers on mitochondria through incorporation of smaller mitochondrially-bound Drp1 units .", "Maturation of a stable Drp1 oligomer does not forcibly lead to fission .", "Drp1 oligomers also translocate directionally along mitochondria .", "Ionomycin , a calcium ionophore , causes rapid mitochondrial accumulation of actin filaments followed by Drp1 accumulation at the fission site , and increases fission rate .", "Inhibiting actin polymerization , myosin IIA , or the formin INF2 reduces both un-stimulated and ionomycin-induced Drp1 accumulation and mitochondrial fission .", "Actin filaments bind purified Drp1 and increase GTPase activity in a manner that is synergistic with the mitochondrial protein Mff , suggesting a role for direct Drp1/actin interaction .", "We propose that Drp1 is in dynamic equilibrium on mitochondria in a fission-independent manner , and that fission factors such as actin filaments target productive oligomerization to fission sites ." ]

[ "Inside cells , structures called mitochondria supply the energy needed to carry out the processes that sustain life .", "Mitochondria constantly divide ( a process known as fission ) or fuse together , which helps to keep them in good working condition and well distributed around the cell .", "Several neurological disorders , including Parkinson’s disease and Alzheimer’s , are associated with problems that affect mitochondrial fission .", "Many different molecules work together to help mitochondria divide , including a protein called Drp1 .", "A number of Drp1 molecules can associate with each other to form an “oligomer” in the shape of a ring around a mitochondrion .", "The ring then constricts to split the mitochondrion in two .", "It is often assumed that Drp1 molecules are recruited to the mitochondria immediately before fission and then form the oligomer ring .", "However , by using microscopy to track the movement of fluorescently labeled Drp1 molecules in human cells , Ji , Hatch et al . now suggest that Drp1 is continuously binding to and releasing from mitochondria , regardless of the need for fission .", "The experiments showed that when bound to surface of the mitochondrion , Drp1 switches between assembling and disassembling the oligomer ring .", "This process of Drp1 assembly and oligomerization on mitochondria is called maturation .", "Specific signals for fission can push Drp1 toward maturation , which then leads to fission .", "Ji , Hatch et al . found that one such signal is the assembly of filaments of a protein called actin .", "Preventing actin filaments from forming reduced the amount of Drp1 that accumulated at mitochondria , and resulted in the mitochondria dividing less frequently .", "Further biochemical experiments also revealed that actin interacts directly with Drp1 and stimulates Drp1 activity , helping the ring to organize and assist mitochondrial fission .", "The formation of actin filaments is not the only mechanism that can recruit Drp1 to mitochondria .", "Future work should investigate whether other mechanisms work with actin to recruit Drp1 .", "As with actin filaments , other signals might be predicted to influence the balance of maturation and disassembly of Drp1 oligomers ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "developmental biology" ]

[ [ "The morphological diversity of body wall muscles is necessary for precision , strength and coordination of body movements specific to each animal species .", "The development of the complex architecture of the body wall musculature of the Drosophila larva – 30 different muscles in each hemi-segment ( Bate , 1993 ) – is a classical model to decrypt transcription regulatory networks controlling muscle morphological diversity .", "Each muscle is a single multinucleated fiber built by fusion of a Founder Cell ( FC ) with fusion competent myoblasts ( FCMs ) .", "Muscle identity - orientation , shape , size , attachment sites - reflects the expression by each FC of a specific combination of identity Transcription Factors ( iTFs ) .", "Establishment of the FC iTF code starts with activation of specific muscle iTFs , in response to positional information from the ectoderm which defines equivalence groups of myoblasts within each segment , called promuscular clusters ( PMCs ) ( Carmena et al . , 1995; Baylies et al . , 1998 ) .", "The second step is the selection of progenitor cells ( PCs ) from each PMC , via interplay between Ras signaling and Notch ( N ) /Delta-mediated lateral inhibition , the unselected myoblasts becoming FCMs ( Carmena et al . , 2002 ) .", "The third step is the asymmetric division of each PC into two FCs or , in some cases , one FC and one adult muscle precursor cell ( AMP ) or pericardial cell .", "Asymmetric division leads to maintaining expression of some iTFs in one FC and their repression by N signaling in the sibling cell , thereby contributing to muscle lineage diversity ( Ruiz-Gomez et al . , 1997; Carmena et al . , 1998 ) .", "This henceforth classical , three-step model of muscle identity specification relies heavily on positional information conferring each muscle its identity ( Tixier et al . , 2010 ) .", "Interestingly , pioneering studies showed that specification of two nearby Even-skipped ( Eve ) expressing PCs was sequential ( Buff et al . , 1998; Halfon et al . , 2000 ) , but the link between PC birth time and muscle identity remained to be explored .", "We have previously shown that four PCs , at the origin of one dorsal muscle ( DA2 ) and one AMP , and the 6 dorso-lateral ( DL ) muscles , DA3 , DO3 , DO4 , DO5 , DT1 , and LL1 , are serially selected from a PMC expressing Collier ( Col/Kn , Early B-Cell Factor ( EBF ) in vertebrates ( Daburon et al . , 2008 ) .", "More precisely , the DA2/AMP , DA3/DO5 and LL1/DO4 PCs are sequentially selected at roughly identical positions in thoracic and abdominal segments , while the DO3/DT1 PC is selected at a slightly posterior position and only in abdominal segments ( Boukhatmi et al . , 2012; Enriquez et al . , 2012; Figure 1A ) .", "Beyond the PC step , col transcription is only maintained in the DA3 muscle ( Crozatier and Vincent , 1999 ) , while other iTFs , the C2H2 zinc finger protein Krüppel ( Kr ) , the homeodomain protein S59 ( vertebrate NKx1 . 1 ) and the Lim-homeodomain protein Tailup ( Tup/Islet1 ) are expressed in the LL1 , DT1 and DA2 lineages , respectively ( Dohrmann et al . , 1990; Ruiz Gomez and Bate , 1997; Boukhatmi et al . , 2012 ) .", "Serial emergence of DL PCs , followed by lineage-specific expression of different iTFs , raised the question of how PC selection timing and muscle identity were linked .", "The discovery that Tup expression led to col repression in the DA2/AMP PC , thereby distinguishing between DA2 and DA3 identities , provided a first insight into this question .", "We indeed found that the time lag between DA2/AMP and DA3/DO5 PC selection coincides with the period of dorsal regression of Tinman ( Tin; vertebrate Nkx2 . 5 ) expression ( Johnson et al . , 2011 ) , such that only the first-born DA2 PC inherits Tin levels above the threshold required for activation of tup and imposing a DA2 fate .", "Yet , our understanding of how conjunction of developmental time and position translates into muscle-specific iTF codes , remained fragmentary . 10 . 7554/eLife . 14979 . 003Figure 1 . Genetic identification of muscle identity genes .", "( A ) Diagrammatic representation of the sequential emergence of four PCs ( large cells ) from the Col expressing PMC , followed by PC into FC divisions ( embryonic stages ( st ) 10–12 . 5 ) and the corresponding muscle pattern at stage 15 .", "The name of each PC , FC and muscle is indicated .", "Col expression is in red , color intensity indicating expression level .", "( B ) Gene density along chromosome 2L and 2R , schematized by black bars .", "Position and size of each of 36 regions identified in our screen are indicated by red or blue bars .", "( C ) Pie chart showing repartition of the DA3 phenotypes into two classes of generic ( blue ) , and identity ( red ) defects .", "( D–I )", "Col immunostaining of late stage 15 embryos; ( D ) wt and ( E–I ) , representative examples ( deficiency name indicated ) of each phenotypic class .", "The asterisk in ( D , E , H ) labels a dorsal class IV md neuron expressing Col . In this , and following figures , lateral views of embryos are shown , anterior to the left .", "( J , K )", "Pie charts associating individual genes with generic myogenic ( K ) or identity ( L ) mutant phenotypes .", "See also Figure 1—source data 1 for phenotypes . DOI: http://dx . doi . org/10 . 7554/eLife . 14979 . 00310 . 7554/eLife . 14979 . 004Figure 1—source data 1 . 36 chromosomal deficiencies showing DA3 muscle phenotypes . Numbering indicates the position of the deleted region along the chromosome , schematized in Figure 1B .", "The phenotypes observed at embryonic stage 16 were classified as either identity or generic muscle defects , and ranked in different types ( Figure 1C ) .", "Identified genes responsible for the deficiency phenotype are indicated together with their known or predicted biochemical function . DOI: http://dx . doi . org/10 . 7554/eLife . 14979 . 00410 . 7554/eLife . 14979 . 005Figure 1—figure supplement 1 . EGF-R signaling is required for a normal pattern of DL muscles .", "( A , B ) wt DA3 Col expression at stage 16 ( A ) , is lost in star ( SIIN ) mutant embryos ( B ) .", "Insets show Col expression in the DA3/DO5 ( white arrowhead ) and DT1/DO3 ( black arrowhead ) PCs ( stage 11 ) .", "Col expression is only detected in the posterior-most DT1/DO3 PC in SIIN mutants ( B ) .", "( C–D )", "MHC staining of wt ( C ) and SIIN ( D ) embryos; the DA2 is present ( arrowhead in D compare to C ) in SIIN embryos , while the only DL muscle forming in absence of EGF-R signaling is DT1 ( arrow in D , compare to C ) .", "Of note , muscles issued from dorsal PCs , including the alary muscles , form . DOI: http://dx . doi . org/10 . 7554/eLife . 14979 . 005 Here , we identified several new muscle TFs , starting from a systematic deficiency screen of the second chromosome , i . e . , roughly 40% of the Drosophila genome .", "We describe the roles of No Ocelli ( Noc ) , a NET family zinc finger protein ( Cheah et al . , 1994 ) , Sine oculis ( So ) , a member of the Six family of homeodomain proteins ( Cheyette et al . , 1994; Serikaku and O'Tousa , 1994; Kenyon et al . , 2005 ) , and the co-factor ETS domain lacking Edl ( Baker et al . , 2001; Yamada et al . , 2003; Qiao et al . , 2006 ) in DL muscle development , and in more detail , roles of Eyes-absent ( Eya ) , a partner of Six proteins ( Pignoni et al . , 1997 ) , and Anterior open ( Aop ) , an Ets-domain transcription repressor ( Rebay and Rubin , 1995; Xu et al . , 2000 ) .", "Analysis of the aop , edl , eya , noc and so muscle mutant phenotypes and time windows of transcription , combined with col transcription in the different mutant contexts , revealed a cascade of regulations including coherent and incoherent feed-forward loops , which link PC selection time to muscle identity .", "aop and edl control the temporal sequence of DL PC selections , eya is required in PCs for maintaining iTF transcription , while so and one eya-specific isoform are deployed at the FC step .", "Finally , noc regulates expression of other iTFs , at the PC or FC step , depending upon the muscle lineage .", "Integration of these new data with pre-existing knowledge provides a comprehensive , dynamic view of the transcriptional control of muscle identity in Drosophila , and an extended framework for studies of interactions between general myogenic factors such as Nautilus ( Nau ) /MyoD and Eya , and iTFs in the diversification of muscle lineages during animal evolution ." ], [ "In order to identify new muscle identity genes , we screened a collection of 389 overlapping deficiencies , each deleting between 10 and 15 genes , and together covering about 80% of the Drosophila second chromosome ( Chanut-Delalande et al . , 2014 ) .", "Homozygous deficiency embryos were first examined for DA3 Col expression at the end of the fusion phase , embryonic stage 15 .", "Nuclear Col localization allowed appraisal both of DA3 formation and shape , the number and spatial distribution of DA3 nuclei , and the presence of ectopic Col-expressing muscles .", "General embryonic defects could be identified by the loss , or gross disturbance of Col expression elsewhere , in the central and peripheral nervous systems , and/or lymph gland ( Dubois and Vincent , 2001 ) , and the corresponding chromosomal deficiencies were not considered here .", "36 were retained ( Figure 1B and Figure 1—source data 1 ) .", "The observed DA3 phenotypes were divided into two broad classes ( Figure 1C ) : Class 1: Decreased number or abnormal repartition of nuclei ( Figure 1E–F compare to Figure 1D ) ; Class 2: Abnormal DA3 orientation and/or either loss of Col expression or ectopic Col expression in additional muscles ( Figure 1G–I compare to Figure 1D ) .", "Three deletions showing both DA3 abnormal orientation and low nuclei number were considered as class 2 ( regions 3 , 4 and 30 , Figure 1—source data 1 ) .", "Class 1 phenotypes have previously been observed in myoblast fusion or nuclei migration mutants which affect roughly equally all muscles ( Folker et al . , 2014; Rushton et al . , 1995 ) and were considered here as 'generic myogenesis' defects ( Figure 1C and Figure 1—source data 1 ) .", "Class 2 phenotypes were reminiscent of either iTF or Notch ( N ) mutants ( Ruiz-Gomez et al . , 1997; Crozatier and Vincent , 1999; Tixier et al . , 2010 ) and considered as 'muscle identity' defects ( Figure 1C and Figure 1—source data 1 ) .", "To identify the gene ( s ) whose loss caused a DA3 phenotype in mapped deletions , we tested the most promising candidates for which loss of function mutants were available .", "Genes for which mutants over the deficiency reproduced the deficiency phenotype were selected for further analysis .", "From a total of 36 different chromosomal regions , we identified 9 genes out of 10 regions linked to generic defects and 15 genes in 14 regions linked to identity defects ( Figure 1J , K and Figure 1—source data 1 ) .", "The relevant gene ( s ) in 12 other regions remain to be identified .", "Seven of the nine genes in the generic class encode cytoskeletal or membrane-associated proteins with an already well-known role in either myoblast fusion or nuclei repartition in muscle syncitia , validating our screen ( Figure 1J; Kim et al . , 2015 ) .", "The eigth gene is sin3A , a chromatin binding protein present in transcription repressor complexes , also required for a normal pattern of myoblast fusions ( Dobi et al . , 2014 ) .", "The 9th gene is basigin ( bsg ) , a predicted igG family plasma membrane protein , interacting with integrin ( Curtin et al . , 2005 ) , whose role in muscle development has not been assessed .", "Among the 15 genes associated with identity phenotypes ( Figure 1K ) , six encoded components of either the N or Robo/Slit signaling pathways , two pathways previously implicated at different steps of DA3 muscle formation ( Crozatier and Vincent , 1999; Ordan et al . , 2015 ) and were therefore not further studied .", "Four other encoded components of the EGF-R signaling pathway: spitz ( spi ) , Star ( S ) , aop ( also called yan; Flybase FBgn 000097 ) , and edl ( also called mae; Flybase FBgn0023214 ) , while a deficiency ( Df ( 2R ) BSC259 ) removing both mesodermal FGFs , Thisbe and Pyramus , ( Stathopoulos et al . , 2004 ) did not show a DA3 phenotype .", "A complete lack of DA3 , DO5 , DO4 and LL1 muscles in mutants for either spi ( spiIIA ) , the EGF-R signal , or Star ( SIIN ) , a chaperone protein required for Spi processing ( Heberlein and Rubin , 1991 ) , confirmed the central role of Epidermal Growth Factor-Receptor ( EGF-R ) signaling in specification of these DL muscles ( Figure 1—figure supplement 1 ) .", "Seven identity genes encoded transcriptional regulators , and potentially , new muscle iTFs: aop , edl , eya , noc , so , spalt major ( salm ) and tup ( Figures 1K and 2A–F ) .", "Previous studies showed that the Ets-domain transcription activator Pointed ( Pnt ) , and transcription repressor Aop/Yan ( Xu et al . , 2000; Rebay and Rubin , 1995 ) , promoted and inhibited the formation of Eve-expressing dorsal PCs , respectively , downstream of EGF-R signaling ( Halfon et al . , 2000; Carmena et al . , 2002 ) .", "The mesodermal function of edl remained , however , unknown .", "Comparing the aop and edl phenotypes thus provided an opportunity to further characterize outputs of EGF-R signaling in muscle identity specification .", "Whereas we previously reported tup function in dorsal muscle identity , neither function of noc , salm nor so in muscle development was previously characterized .", "Drosophila Six4 and So are orthologous to Six proteins which interact with Eya in mouse myogenic progenitors ( Heanue et al . , 1999; Relaix et al . , 2013 ) .", "eya was proposed to interact with Six4 in Drosophila somatic muscle development , both genes showing similar expression patterns ( Clark et al . , 2006; Liu et al . , 2009 ) .", "Our identification of so mutants in our screen and the difference between the eya and so phenotypes ( Figure 1—source data 1 ) called for a detailed comparison of eya and so expression and function in muscle PCs . 10 . 7554/eLife . 14979 . 006Figure 2 . Specific muscle patterning defects in aop , edl , eya , noc and so mutant embryos .", "( A–F )", "Late stage 15 embryos stained for Col , to visualize the DA3 muscle .", "( A ) wt , ( B–F ) embryos homozygous mutant for aop , edl , eya , noc and so null alleles with their names indicated .", "Inserts in ( A , B , D , E ) show Col expression in PCs , stage 11 .", "( G–L ) stage 16 embryos stained for Col ( red ) and β3-tubulin ( green ) to visualize all body wall muscles; arrowheads point to LT1 and LT2 , asterisks indicate DT1; DA2 is surrounded by a line in G , I , and LL1 by a dotted line in G . ( G ) wt embryo .", "( H ) aop1 ( I ) edll19; Col is expressed in DA2 and DA3 .", "( J ) eyaCII/Df ( 2L ) BSC354; DA3 Col expression is lost; inset , LL1>DA3 transformation .", "( K ) noc35ba2: Col expression is lost .", "( L ) so3; Col expression in DO5 ( arrow ) .", "( M–R )", "Stage 16 embryos stained for β3-tubulin ( blue ) , βPS integrin ( green ) , to visualize tendon cell-muscle connections and moeGFP ( red ) expressed under control of a DA3-specific col CRM ( colLCRM ) , abbreviated col-gfp .", "Only βPS integrin and moeGFP are shown , except insets .", "( M ) wt; DA3 ventral and dorsal attachment along the anterior and posterior segmental borders , respectively , are indicated by asterisks; inset , DT1 ( arrowhead ) .", "( N ) aop; DA3 with both DA3 and DA2-like ( arrowhead ) anterior attachments; DA3>DA2 transformation , inset .", "( O ) edl: moeGFP expression in DA2 and DA3 ( arrow and inset ) .", "The arrow indicates partial DA2>DA3 transformation; inset , bifid anterior DA3 attachment .", "( P ) eya: moeGFP is lost in most segments or indicates partial or complete ( inset ) DA3>DA2 transformation .", "( Q ) noc: DA3>DT1 transformation , resulting in DT1 ( arrowhead in inset ) duplication .", "( R ) so: moeGFP expression in DO5; DO5>DA3 transformation ( arrowhead ) in some segments .", "( S–X )", "Schematic diagram of the most frequent DA2 and DL muscle phenotypes in aop , edl , eya , noc and so mutants; Col expression is in red; see Figure 2—source data 1 for statistics .", "Bars: 30 μmDOI: http://dx . doi . org/10 . 7554/eLife . 14979 . 00610 . 7554/eLife . 14979 . 007Figure 2—source data 1 . Quantification of muscle phenotypes observed in aop , edl , eya , noc and so mutant stage 15 embryos . DA3>DA2: complete or partial DA3 into DA2 muscle orientation; DA2>DA3: complete or partial DA2 into DA3 muscle orientation; ColexpDO5: DO5 muscle expressing Col . Loss of Col: loss of DA3 muscle Col expression .", "DA3>DT1 transformations have been quantified in colLCRM-moeGFP embryos . DOI: http://dx . doi . org/10 . 7554/eLife . 14979 . 00710 . 7554/eLife . 14979 . 008Figure 2—figure supplement 1 . salm1 is required for proper skeletal attachment and morphology of the DA3 muscle .", "( A , C , C’ ) wt , and ( B , D , D’ ) salm1mutant stage 16 embryos .", "( A , B )", "Staining for Col ( red ) and β3-tubulin ( green ) .", "( B ) the white arrow and arrowhead point to a loose DA3 anterior attachment and a vertical DA3 fiber with no posterior attachment , respectively; the inset shows dorsal muscles without defined posterior attachment sites .", "( C , D )", "Staining for MoeGFP expressed under control of a DA3-specific CRM ( colLCRM ) ( green ) and βPS integrin ( red ) .", "No βPS integrin accumulation is detected at the position of DL muscle attachment sites ( white asterisks ) in salm1 mutant embryos ( C’ , D’ ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 14979 . 00810 . 7554/eLife . 14979 . 009Figure 2—figure supplement 2 . Snapshots for Videos 1–6 . Snapshots of Videos 1–6 , showing β3-tubulin staining of muscles ( green ) and Col expression ( red ) in stage 16 embryos .", "Five segments are shown in ( A–D ) , two in ( E–G ) .", "( A , E–E” ) wt embryos , Video", "1 . ( B ) aop1 mutant , Video", "2 . ( C ) edll19 , Video", "3 . ( D ) noc35ba2 , Video 5 .", "( F ) eyaCII/Df ( 2L ) BSC354 , Video 4 .", "( G ) so3 , Video 6 .", "The positions of DA3 , and DA2 and DA3 muscles are indicated in A , F , G , and C , respectively .", "DA2 is circles in A and LL1 in E , F .", "E , E” , F , F” , and G , G” are single channel in black and white , E’F’ , G’ green chanel , E” , F” , G” red channel .", "DO5>DA3 and LL1>DA3 transformations are indicated in F , F” and G , G” , and F , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 14979 . 009 To better assess the muscle phenotypes associated with each mutant , we examined the pattern of DL muscles in late stage 15 embryos immunostained for β3Tubulin , using Col staining to visualize DA3 ( Figure 2G–L , Video 1 ) .", "In addition , we introduced the DA3-specific colLCRM-moeGFP reporter gene ( Enriquez et al . , 2012 ) , to precisely visualize the DA3 contours in these mutant backgrounds .", "Stability of the MoeGFP fusion protein also allowed following 'DA3' muscles in noc and eya mutants which lack Col expression ( Figure 2M–R ) .", "In order to verify that muscle phenotypes were not associated with defective tendon cell differentiation , we stained mutant embryos for βPS integrin which accumulates at muscle-tendon junctions ( Leptin et al . , 1989 ) .", "Based on this analysis , we eliminated salm ( salm1 ) mutants which exhibited a phenotype reminiscent of defective tendon cells ( Schnorrer and Dickson , 2004; Staudt et al . , 2005; Figure 2—figure supplement 1 ) .", "Unlike salm , no βPS integrin accumulation defects were detected in null mutants for aop ( aop1 ) , edl ( edlL19 ) , noc ( noc35ba2 ) , eya ( eyaCII-IID ) , and so ( so3 ) mutants ( Figure 2M–R ) , confirming muscle identity defects . 10 . 7554/eLife . 14979 . 010Video 1 . 3-D view of the muscle pattern in stage 16 wt embryos , Figure 2G . DOI: http://dx . doi . org/10 . 7554/eLife . 14979 . 010 In aop mutants , the DA3 muscle ( s ) was misshapen in 2/3 of segments ( n = 123 ) , with cases of DA3 to DA2 transformation ( DA3>DA2; Figure 2B , Figure 2— figure supplement 2 , Video 2 and Figure 2—source data 1 ) .", "The DT1 and LL1 muscles were also malformed in 40% segments ( in 48/123 and 47/123 segments , respectively ) and lateral and ventral muscles were severely disorganized .", "The DA2 muscle was unaffected ( Figure 2H—and Figure 2—source data 1 ) .", "colLCRM-moeGFP expression confirmed an abnormal shape of the DA3 muscle suggestive of partial DA3>DA2 transformation ( Figure 2N , T ) .", "In edl mutants , a second Col-expressing muscle was observed in some segments , sometimes associated with morphological change suggestive of DA2>DA3 transformation ( Figure 2C , I , Figure 2— figure supplement 2 , Video 3 and Figure 2—source data 1 ) .", "The DT1 muscle was either absent or too de-structured to be assigned specific identities ( in 61/124 segments; Figure 2I ) .", "colLCRM-moeGFP expression confirmed a DA2>DA3 transformation in 47% of segments ( Figure 2O ) , but also revealed a number of reciprocal at least partial DA3>DA2 transformations ( 16/124 segments ) ( Figure 2O inset—and Figure 2—source data 1 ) .", "In eya mutants , DA3 Col expression was lost , a loss already observed at the PC stage ( Figure 2D , J ) .", "Consistent with loss of Col expression early during muscle specification , the DL muscle pattern was severely disorganized in most of segments .", "The LL1 was absent or oriented like DA3 ( in 129/140 segments; Figure 2J , Figure 2— figure supplement 2 , Video 4 and Figure 2—source data 1 ) a phenotype already observed in col mutant embryos ( Enriquez et al . , 2012 ) .", "The LT muscles were also often missing and some ventral muscles were absent or malformed , as previously reported ( Figure 2J; Figure 2—source data 1 ) ( Liu et al . , 2009 ) .", "Conversely , dorsal muscles and DT1 appeared normal ( Figure 2J ) .", "colLCRM-moeGFP expression revealed a , sometimes complete or partial , DA3>DA2 transformation ( Figure 2P , V ) .", "noc mutant embryos also lacked DA3 Col expression at stage 15 ( Figure 2E , K and Figure 2 , Figure 2— figure supplement 2 , Video 5 and Figure 2—source data 1 ) .", "Contrary to eya mutants , however , Col expression was detected at the PC stage ( insets in Figure 2E ) , indicating a role of noc in maintenance of Col expression in the DA3 lineage .", "colLCRM-moeGFP expression further revealed that the DA3 could orient like a DT1 in most segments , indicating a DA3>DT1 identity shift resulting in DT1 duplication ( Figure 2Q and and Figure 2—source data 1 ) .", "LT muscles were also affected ( Figure 2K ) .", "In so mutants , Col ectopic expression was specifically observed in DO5 ( Figure 2F , L , Figure 2—figure supplement 2 , Video 6 , and Figure 2—source data 1 ) .", "colLCRM-moeGFP expression both confirmed col ectopic expression in the DO5 muscle and its DA3-like orientation in a fraction of segments ( Figure 2R , arrowhead , indicating a partial DO5>DA3 identity shift in 79/116 segments ( Figure 2F , L and Figure 2—source data 1 ) . 10 . 7554/eLife . 14979 . 011Video 2 . 3-D view of the muscle pattern in stage 16 aop1 embryos , Figure 2H . DOI: http://dx . doi . org/10 . 7554/eLife . 14979 . 01110 . 7554/eLife . 14979 . 012Video 3 . 3-D view of the muscle pattern in stage 16 edlL19 embryos , Figure 2I . DOI: http://dx . doi . org/10 . 7554/eLife . 14979 . 01210 . 7554/eLife . 14979 . 013Video 4 . 3-D view of the muscle pattern in stage 16 eyaCII/Df ( 2L ) BSC354 embryos , Figure 2J . DOI: http://dx . doi . org/10 . 7554/eLife . 14979 . 01310 . 7554/eLife . 14979 . 014Video 5 . 3-D view of the muscle pattern in stage 16 noc35ba2 embryos , Figure 2KDOI: http://dx . doi . org/10 . 7554/eLife . 14979 . 01410 . 7554/eLife . 14979 . 015Video 6 . 3-D view of the muscle pattern in stage 16 so3 embryos , Figure 2LDOI: http://dx . doi . org/10 . 7554/eLife . 14979 . 015 In summary , we found that aop , edl , eya , noc , and so mutants display distinctive patterns of DL muscle defects and DA3 transformations , associated with modifications of Col expression ( schematized in Figure 2S–X ) , indicating that each gene acts in different subsets of DL muscles , and/or at different steps of muscle identity specification .", "Understanding the specific muscle transformations observed in aop , edl , eya , noc , and so mutants required determining their expression patterns at the PMC , PC and FC stages .", "In order to access dynamic aspects of this expression , we used FISH with intronic probes which detect nascent transcripts and allow precisely determining temporal windows of transcription .", "To follow PC delamination events , we used Nau , the Drosophila ortholog of vertebrate myogenic regulatory factors ( MRFs ) , a marker of PCs and FCs ( Michelson et al . , 1990; Nose et al . , 1998 ) .", "High-resolution 3-D analyses allow us to unambiguously identify the DA2/AMP and DL PCs and the derived DA2 , DA3 and DO5 FCs and AMP ( Enriquez et al . , 2012; Figure 1A and Figure 3— figure supplement 1 , Video 7 ) .", "In early stage 10 embryos , the first selected , DA2/AMP PC is recognizable as a large apical cell , expressing high Nau levels ( Figure 3B , B’ ) .", "At stage 11 , after the DA2/AMP PC has divided , the DA3/DO5 PC is observed , adjacent to the DA2 FC ( Figure 3C ) .", "3-D analyses also revealed previously undescribed , low level Nau expression in two or three cells surrounding each PC being selected ( Figure 3B , F; Figure 3—figure supplement 2 ) .", "This Nau expression pattern was reminiscent of subgroups of PMCs cells displaying higher level dpMAPK ( di-phospho mitogen-associated protein kinase ) , diagnostic of EGFR activity ( Carmena et al . , 1998 , 2002 ) and postulated to be cells primed to become PCs .", "Double staining confirmed that the Nau and dpMAPK patterns overlap , revealing that low level Nau expression corresponds to cells transitioning from PMC to PC , before reaching high level in selected PCs ( Figure 3—figure supplement 2 ) .", "Using Nau staining allowed us to follow these cells and PCs in subsequent FISH experiments . 10 . 7554/eLife . 14979 . 016Video 7 . Multiple FCs originate from the Col-expressing PMC . DOI: http://dx . doi . org/10 . 7554/eLife . 14979 . 01610 . 7554/eLife . 14979 . 017Figure 3 . aop and edl differential expression and roles during PC selection .", "( A ) Schematic representation of the positions of DL PCs and FCs , relative to the A/P , D/V and proximal/distal axes in stage 10 , 11 and 12 wt embryos ( Video 7 ) ; the blue trapeziums indicate planes of section shown in panels ( B–K ) and ( R–U ) ; Col expression is in red .", "( B–I )", "ISH to aop ( B–E ) and edl ( F–I ) primary transcripts ( red dots ) , in wt embryos stained for Col ( blue ) and Nau ( green ) , at stages indicated above; early stage is abbreviated ste; two different planes of the same embryo are shown in B and B’ , F and F’ .", "aop transcription in the Col PMC ( B ) , and the AMP ( D ) .", "( F–I ) edl transcription in all PCs , the AMP and the DA3 FC .", "( B , F )", "Nau accumulation in two to three Col PMC cells , below the emerging PC .", "( J , O ) 3D reconstruction of the Col PMC during DA2/AMP and DA3/DO5 PC selection; Col staining , red , Nau , green .", "The embryonic stage indicated in each panel .", "( J , K ) wt; ( J ) apical DA2/AMP PC ( dotted white circle ) ; ( K ) apical DA3/DO5 PC ( dotted circle ) , DA2 FC and AMP ( asterisks ) .", "( L , M ) aop embryos; ( L ) premature DA3/DO5 PC selection; additional Nau-expressing PMC cells ( arrow ) , become PCs , ( M ) .", "( N , O ) edl embryos; ( N ) no PC is selected; a group of 3 to 4 Nau-expressing cells is embedded in the Col PMC; ( O ) two PCs are simultaneously selected .", "( P–U )", "ISH to col primary transcripts ( red ) , Nau staining ( green ) ; ( P–R ) wt; sequential col transcription in the PMC and DA2/AMP PC ( P ) , DA3/DO5 PC ( Q ) , and DA3 FC ( R ) .", "( S ) aop mutant: simultaneous col transcription in two apical PCs; increased number of low level Nau-expressing cells ( green dotted circle ) .", "( T , U ) edl mutant; ectopic col transcription in the DA2/AMP PC ( T ) and DA2 FC ( U ) .", "( V ) Measurement of the diameter of Col ( red ) and Nau ( green ) expressing domains in early stage 10 wt and aop embryos .", "The Col expression domain is identical ( P value = 0 , 1410; ns ) and Nau domain expanded in aop compared to wt ( P value<0 , 0001; **** ) , schematized on top of the statistics .", "Bars: 5 μmDOI: http://dx . doi . org/10 . 7554/eLife . 14979 . 01710 . 7554/eLife . 14979 . 018Figure 3—figure supplement 1 . Snapshot for Video 7 . Stage 12 embryo .", "Col ( green ) is expressed in a large promuscular cluster and the FCs at the origin of the DA3 , DO5 , LL1 , DO3 , DT1 and DO4 muscles , indicated on the screenshot .", "Col expression has been lost from the DA2 FC and the AMP at this stage .", "All FCs and the AMP express Nau ( red ) .", "The Antero-Posterior , Dorso-Ventral and Distal-Proximal axes are indicated on the video by red , green and blue arrows , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 14979 . 01810 . 7554/eLife . 14979 . 019Figure 3—figure supplement 2 . Transient Nau expression in subsets of PMC cells .", "( A ) Schematic representation of the positions of the DA2/AMP PC , relative to the A/P , D/V and proximal/distal axis in stage early 10 ( st e10 ) and late 10 ( st l10 ) ; the trapezium indicates planes of sections shown in panels ( B–C ) .", "Nau expression is in blue .", "( B–C ) wt embryos at st e10 ( B ) and st l10 ( C ) stained for Nau ( blue ) and dpERK ( green ) .", "B and B' are two planes of section across a small equivalence group of cells both expressing Nau and accumulating dpERK .", "The selected PC ( B' ) is positioned apical to the equivalence group ( B ) .", "( C ) At a slightly later stage , only the PC maintains Nau and dpERK ( arrow ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 14979 . 01910 . 7554/eLife . 14979 . 020Figure 3—figure supplement 3 . Extended analysis of the aop muscle mutant phenotype .", "( A , B ) stage 16 embryos stained for Col ( red ) and F-actin ( green ) .", "( A ) wt , ( B ) aop1mutant embryo displaying several DA3 ( horizontal arrow ) or LL1 ( vertical arrow ) muscles . DOI: http://dx . doi . org/10 . 7554/eLife . 14979 . 020 FISH experiments indicated aop transcription in Col PMC cells ( Figure 3B ) , but not PCs ( Figure 3B’–E ) , indicating its repression during the PC selection process , consistent with a role in promoting FCM fate ( Carmena et al . , 2002 ) and being a target of the FCM TF Lameduck ( Ciglar et al . , 2014 ) .", "Conversely , edl transcription was not detected in PMC cells , but in PCs and the dorsal AMP and DA3 FC ( Figure 3F–I ) .", "Thus aop and edl show sequential , complementary transcription patterns .", "In stage 10 aop mutant embryos , col transcription was detected in PMC cells as in wt ( Figure 3P , S and V ) .", "However , a significantly increased number of cells expressing low Nau level revealed that aop down-regulation of EGF-R signaling was required to restrict Nau expression to PMC cells primed to become PCs ( Figure 3P , S , V ) .", "Furthermore , 3-D reconstructions confirmed the presence of two apical cells expressing high Nau Level ( 21/27 PMCs with 2 , and 6/27 with 1 apical PC ) , when only the DA2/AMP PC was observed in wt ( 30/35 PMC with 1 apical PC and 5/35 with no apical PC; Figure 3J , L ) .", "Thus , concomitant , early selection of two PCs in aop embryos prefigures the DA3>DA2 transformation ( Figure 2T ) .", "At stage 11 , Nau remained expressed at high level in several cells , showing that supplementary PMC cells are primed to become PCs ( Figure 3M , compare to ) .", "This corroborates the observation of several aligned DA3-like fibers revealed by phalloidin staining of stage 16 aop mutant embryos ( Figure 3—figure supplement 3 ) .", "Conversely , in edl mutant embryos , no high Nau-expressing apical cell was observed at stage 10 ( 30/37 segments ) , when the DA2/AMP PC is selected in wt embryos .", "Rather , three to four small low Nau-positive cells remained embedded in the Col PMC ( Figure 3N compare with Figure 3J ) ( Figure 3O compare to Figure 3K ) both of which transcribed col ( Figure 3T compare with Figure 3Q ) .", "Accordingly , col transcription was maintained in two cells at late stage 11 , at positions corresponding to DA2 and DA3 FCs in wt embryos ( Figure 3U compare with Figure 3R ) .", "In summary , we found that sequential PC selection is inversely compromised in aop and edl mutants .", "In absence of aop , the DA3/DO5 , and supernumerary PCs are selected early , and in absence of edl , the DA2/AMP is selected too late .", "We have previously shown that the time lag between DA2/AMP and DA3/DO5 PC selection coincided with a period of dorsal regression of Tin expression , such that only the first selected , DA2/AMP PC inherited Tin ( Boukhatmi et al . , 2012; Figure 4C–D' ) .", "Tin staining of aop mutant embryos showed that the two apical cells observed at stage 10 inherit Tin ( Figure 4E , E' ) , confirming advanced selection of the DA3/DO5 PC .", "Conversely , in edl mutants , none of the apical cells inherited Tin , confirming a delayed selection of the DA2/AMP PC ( Figure 4G , H' ) , consistent with both transcribing col ( Figure 3T , U ) .", "To verify that the observed shifts in PC selection timing lead to shifts of PC identity , we analyzed tup transcription .", "As previously shown , only the DA2/AMP PC inherits Tin levels above the threshold required for tup activation ( Figure 4I ) , leading in turn to col repression and initiation of tup auto-regulation ( Boukhatmi et al . , 2012 ) .", "We found that , in aop mutants , early selected PCs transcribed tup ( Figure 4J ) , while in edl mutants , late selected PCs did not ( Figure 4K ) , mirroring col transcription ( Figure 3T , U ) .", "Together , Tin , tup and col expression data , and the DA3>DA2 and DA2>DA3 muscle transformations predominantly observed in aop and edl embryos , respectively , show that timely PC selection is essential for each PC to inherit different Tin levels and either initiate tup ( DA2/AMP ) or col ( DA3/DO5 ) feed-forward positive loops .", "Positive auto-regulation , a hallmark of bistable systems ( Graham et al . , 2010; Park et al . , 2012 ) , of either tup or col distinguishes between DA2 and DA3 identities ( Boukhatmi et al . , 2012; Figure 4L ) . 10 . 7554/eLife . 14979 . 021Figure 4 . aop and edl control the temporal sequence of PC selection .", "( A , B )", "Schematic representation of the DA2/AMP PC and FCs and DA3/DO5 PC , at stages 10 ( A ) and 11 ( B ) ; the blue trapeziums indicate the planes of section shown below .", "( C–H’ )", "Tin ( green ) and Col ( red ) embryo staining .", "( C , C’ )", "Tin expression in the DA2/AMP PC and underlying PMC cells .", "( D , D’ )", "Tin expression has regressed dorsally; the DA3/DO5 PC and underlying PMC cells are Tin negative .", "( E–F’ ) aop mutants; ( E , E’ ) , stage 10 , two Col and Tin-expressing PCs are selected; ( F , F’ ) stage 11 , Col positive , Tin-negative cells are selected .", "( G–H’ ) edl mutants; ( G , G’ ) , No PC is selected .", "( H , H’ )", "Two PCs are selected after dorsal regression of Tin expression .", "( I–K )", "FISH to tup primary transcripts in wt ( I ) aop ( J ) and edl ( K ) stage 10 ( I , J ) and 11 ( K ) embryos , stained for Col ( blue ) and Nau ( green ) .", "In wt embryos ( I ) tup expression is only detected in the first selected Col positive PC ( DA2/AMP , 100% n = 27 ) .", "In aop mutants ( J ) , tup transcription is sometimes detected ( 17% n = 34 ) in a second Col positive PC ( arrow ) .", "In edl mutants ( K ) , tup transcription is frequently lost in Col positive PCs ( 82% n = 29 ) .", "( L ) Summary scheme of the aop and edl phenotypes; wt , late stage ( stl ) 10; only the first-born , DA2/AMP PC inherits Tin , and activates tup , preventing Col autoregulation ( left ) which occurs in the second born , DA3/DO5 PC , in absence of Tin and Tup , stage 11 ( Boukhatmi et al . , 2012 ) .", "In edl and aop mutants , the temporal sequence of PC selection is compromised; it occurs too early and too late in aop and edl mutants , respectively , leading to confusions of DA2 and DA3 fates .", "Bars: 10 μmDOI: http://dx . doi . org/10 . 7554/eLife . 14979 . 021 Drosophila eya gives rise to 3 different mRNA and protein isoforms ( eya-RA , eya-RB , eya-RC ) corresponding to the alternate use of different Transcription Start Sites ( TSS ) ( Flybase FBgn0000320; Figure 5—figure supplement 1 ) .", "FISH performed with a probe complementary to a common coding region , eyae , detected eya expression in the Col PMC , DA2/AMP , DA3/DO5 and LL1/DO4 PCs and DO5 lineage ( Figure 5B–E , O and Figure 5— figure supplement 2 ) .", "However , an intronic probe specific for transcripts initiated from the 5’-most TSS ( eya-RBi; Figure 5—figure supplement 1 ) revealed that eya-RB transcription was restricted to the AMP , DO5 FC/muscle and the LL1/DO4 PC ( Figure 5F–I , P and Figure 5— figure supplement 2 ) , implying that , conversely , eya-RA/RC is specifically expressed in the PMC , the DA2/AMP and the DA3/DO5 PCs .", "This indicated a temporal shift in TSS , leading to sequential production of different Eya protein isoforms .", "In eya mutants , col transcription was detected in PMC cells like in wt ( not shown ) , but prematurely lost in the DA3/DO5 PC , and undetectable in the DA3 FC ( Figure 5R , S ) , showing that eya-RA/RC is required for sustained col transcription during PC specification .", "Reciprocally , both eya-RA/RC transcripts in the DA3/DO5 PC ( de Taffin et al . , 2015 ) and eya-RB transcripts in the DO5 FC ( Figure 5T , U ) were lost in col mutants , revealing that Eya and Col positively regulate each other transcription .", "Whether , both eya-RA/RC , and eya-RB are under direct control of Col binding to a dedicated cis-regulatory module ( CRM ) ( de Taffin et al . , 2015; Figure 5—figure supplement 1 ) , or eya-RB control is indirect , and requires prior expression of eya-RA/RC at the PC stage ( Figure 5X ) , remains unknown . 10 . 7554/eLife . 14979 . 022Figure 5 . Sequential eya and so transcription and control of col transcription in distinct muscle lineages .", "( A ) Schematic representation of the positions of DL PCs and FCs in stage 10 , 11 and 12 wt embryos , reproduced from Figure 3A; the blue trapeziums indicate the planes of section shown below , panels ( B–M ) .", "( B–M and O–Q )", "ISH to eya and so transcripts ( red ) in wt embryos stained for Col ( blue ) and Nau ( green ) .", "( B–E ) , eya expression in the DA2/AMP ( B ) , DA3/DO5 ( C ) AMP and DO5 FC ( D ) LL1/DO3 PC ( E ) .", "( N ) Schematic representation of the DL muscle pattern , DA3 in red and DO5 in grey .", "( O ) DO5 eya expression .", "( F–I ) eya-RB transcription in the AMP DO5 FC ( H ) and LL1/DO4 PC ( I ) .", "( P ) DO5 eya-RB transcription .", "( J–M , Q ) so transcription in the DA2/AMP , DA3/DO5 and LL1/DO4 PCs , DO5 FC and muscle .", "( R , S )", "Loss of col transcription in the DA3/DO5 PC in eya mutants .", "( T–U )", "Loss of eya-RB transcription in col mutants ( U ) .", "( V , W ) col ectopic transcription in the DO5 FC in so mutants .", "Embryos in R-W are co-stained for Nau ( green ) .", "( X ) Summary diagram of eya and so expression and function in DL muscle lineages .", "Bars: 5 μmDOI: http://dx . doi . org/10 . 7554/eLife . 14979 . 02210 . 7554/eLife . 14979 . 023Figure 5—figure supplement 1 . Schematic representation of the eya genomic region and transcripts . Schematic representation of the eya genomic region chromosomal positions Chr2L:6 , 525 , 000–6 , 550 , 000 .", "The different eya transcripts ( RA , RB , RC ) initiated from different Transcription Start Sites are indicated .", "Red boxes indicate the position of the exonic ( eyae ) and intronic ( eya-RBi ) probes used for FISH experiments .", "Green boxes show RNA-PolII binding at the indicated embryonic stage ( Zinzen et al . , 2009 ) .", "Two previously characterized mesodermal CRMs are indicated by blue ( Liu et al . , 2009 ) and purple boxes , respectively .", "eya_Col CRM activity depends upon in vivo Col binding ( de Taffin et al . , 2015 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 14979 . 02310 . 7554/eLife . 14979 . 024Figure 5—figure supplement 2 . Sequential eya and so transcription .", "( A ) Schematic representation of the DL PCs , FCs and muscles as in Figure 5 .", "( B–Q )", "ISH to eya and so transcripts ( red ) in wt embryos stained for Col ( blue ) and Nau ( green ) .", "To complement Figure 5 , the red channel is show separately in black and white . DOI: http://dx . doi . org/10 . 7554/eLife . 14979 . 02410 . 7554/eLife . 14979 . 025Figure 5—figure supplement 3 . Loss of DA3 Col expression in Six4 mutant embryos .", "( A ) Stage 15 wt , and ( B ) homozygous mutant embryos for a null allele of Six4 , Six4289 .", "Col expression is detected in 100% of segments in wt embryos ( n = 117 ) , and lost in 84% of segments in Six4 mutants ( n = 249 ) .", "In most of the remaining muscles , residual Col expression indicates a DA3>DA2 transformation ( arrow in B ) .", "Col expression is already lost at the PC stage in Six4 mutants ( insets in A , B ) , similar to the loss observed in eya mutants ( See Figure 2D ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 14979 . 025 Eya protein phosphatases interact with Six family TFs , Six4 , Optix ( Op ) and So in Drosophila ( see introduction ) .", "We found that so was transcribed in the DA2/AMP , DA3/DO5 and LL1/DO4 PCs and subsequently maintained only in the DO5 FC and muscle ( Figure 5J–M , Q and Figure 5— figure supplement 2 ) .", "col transcription in DL PCs was normal in so mutants , showing that So is not required for Eya-RA/RC regulation of col transcription ( not shown ) .", "On the contrary , col was ectopically transcribed in the DO5 FC ( Figure 5V , W ) , showing that so activity contributes to repress col transcription in this lineage .", "so transcription in the DO5 FC and contribution to distinguishing between the DA3 and DO5 identities suggests that so could act downstream of N in this process ( Crozatier and Vincent , 1999 ) .", "The difference between the eya and so DA3 mutant phenotypes ( Figure 2D , F , Figure 2—figure supplement 2 , and Videos 4 and 6 ) suggested that eya was partnering with Six4 to positively regulate col . To verify this assertion , we analyzed DA3 Col expression in Six4 mutants .", "The loss of Col expression ( Figure 5—figure supplement 3 ) , very similar to that observed in eya mutants ( Figure 2D ) , supports the conclusion that Eya partners with Six4 to positively , and with So to negatively regulate col . Thus , Six4 and So play distinct roles during muscle specification ( Figure 5X ) .", "noc is required for DA3 Col expression and DA3 formation ( Figure 2 ) .", "noc and its paralog elbow ( elb ) , act as transcription repressors in morphogenesis of appendages , tracheal branches and specification of monochromatic receptors in the retina ( Dorfman et al . , 2002; Nakamura et al . , 2004; Wernet et al . , 2014 ) but a function in myogenesis was not reported .", "A deficiency removing elb ( Df ( 2R ) exel6035 ) had no DL muscle phenotype , while the phenotypes of noc35ba2 mutants and a deficiency removing both elb and noc were identical ( Figure 1—source data 1 and Figure 2E ) .", "Thus , only noc is required for DL muscle specification .", "FISH experiments revealed noc transcription in Col PMC cells which express low Nau level , followed by the DA2/AMP , the DA3/DO5 and the LL1/DO4 , but not the DT1/DO3 PC ( Figure 6B–E ) .", "In noc mutants , col transcription was detected in the PMC and DA3/DO5 PC ( not shown ) but completely lost from the DA3 FC ( Figure 6F , G ) , correlating with the loss of Col DA3 expression after the PC stage ( Figure 2E , K ) .", "The DA3>DT1 transformation observed at stage 15 in noc mutants ( Figure 2Q ) was therefore intriguing , since a DA3>DA2 identity shift was observed in other mutants where DA3 Col expression was lost , namely col , eya and Six4 ( Figure 2D and Figure 5—figure supplement 3 ) .", "Since DT1 identity requires S59 expression in the DT1 FC ( Knirr et al . , 1999 ) , we analyzed S59 expression in noc mutant embryos and found that it was ectopically expressed in the DA3 FC ( Figure 6H , I ) , consistent with DA3>DT1 transformation .", "This finding suggested that loss of DA3 Col expression in noc embryos was secondary to gain of S59 expression .", "To test this possibility , we expressed S59 in all myoblasts , using the Twist-Gal4 pan-mesodermal driver .", "While the muscle pattern was severely disorganized , loss of Col expression in Twi>S59 embryos demonstrated S59 ability to repress col mesodermal expression ( Figure 6—figure supplement 1A , B ) .", "We next analyzed col transcription in S59 mutants and found that it was ectopically transcribed in one posterior DL FC , likely DT1 ( Figure 6—figure supplement 1C , D ) .", "On the one hand , these data confirmed that S59 represses col transcription in the DT1 lineage , via an incoherent feed-forward loop initiated by Col activation of S59 in the DT1/DO3 PC ( Enriquez et al . , 2012 ) .", "On the other hand , noc and S59 loss-of-function and S59 gain-of-function data revealed a double negative regulatory cascade where noc repression of S59 maintains col transcription in the DA3 lineage and DA3 identity ( Figure 6J ) .", "Noc expression in the DA3/DO5 PC and not the DT1/DO3 PC ( Figure 6E ) thus distinguishes between DA3 and DT1 identities ( Figure 7 ) . 10 . 7554/eLife . 14979 . 026Figure 6 . noc transcription and control of col and S59 transcription in DL muscle lineages .", "( A ) Relative positions of DL PCs and FCs between stages 10 and 12 , reproduced from Figure 3A; the blue trapeziums indicate the planes of section shown below , panels ( B–E ) .", "( B–E ) , Embryos co-stained for Nau ( green ) and Col ( blue ) ; ( B ) noc transcription ( red ) in a small subset of Col PMC cells expressing low Nau level ( B ) , the DA2/AMP ( B’ ) , DA3/DO5 and DA3 and DO5 FCs ( C , D ) and LL1/DO4 but not the DT1/DO3 PC ( E ) .", "( F–I )", "Embryos stained for Nau ( green ) and ( H , I ) Col ( blue ) .", "( F , G ) loss of col transcription in the DA3 FC ( red dots ) in noc mutants .", "( H ) wt S59 transcription in DT1 ( red dots , arrow ) and ( I ) ectopic transcription in the DA3 FC ( arrowhead ) in noc mutant embryos .", "( J ) Summary diagram of noc expression and function in DL muscle lineages .", "Bars: 5 μmDOI: http://dx . doi . org/10 . 7554/eLife . 14979 . 02610 . 7554/eLife . 14979 . 027Figure 6—figure supplement 1 . S59 represses col transcription .", "( A , B ) stage 16 embryos stained for Col ( red ) and β3-tubulin ( green ) .", "( A ) wt , ( B ) embryo expressing S59 under control of the twist-gal4 mesodermal driver .", "The muscle pattern is strongly disorganized , and Col muscle expression is lost .", "( C , D ) stage 13 embryos stained for col primary transcripts ( red ) , Col ( green ) and Nau ( blue ) .", "( C ) wt; the arrow indicates the posterior group of cells giving rise to the DT1 and DO3 FCs .", "At this stage , col expression is rarely detected in these cells ( 6% of segments , n = 52 ) .", "( D ) S59 mutant embryo .", "col expression is maintained in at least one posterior cell , likely the DT1 FC ( 81% of segments , n = 65 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 14979 . 02710 . 7554/eLife . 14979 . 028Figure 6—figure supplement 2 . eya and noc are required for Kr expression in the LL1 FC .", "( A–F )", "Stage 12 embryos stained for Col ( green ) and Kr ( red ) .", "( A ) wt , co-expression of Col and Kr in the LL1 FC ( horizontal arrow ) .", "Kr expression is detected in aop1 ( B ) , edlL19 ( C ) and so3 ( F ) , and lost in eyaCII/Df ( 2L ) BSC354 ( D ) and noc35ba2 ( E ) mutant embryos .", "Of note , Kr expression is lost in DL , but not dorsal FCs ( asterisks ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 14979 . 02810 . 7554/eLife . 14979 . 029Figure 7 . Intertwined transcriptional control of DL muscle identity: A progressive resolution of the possible . Diagrammatic representation of transcription regulatory interactions and loops operating in DL muscle identity specification .", "One abdominal segment is considered .", "3 steps are indicated on top and color-shaded .", "Left , PC selection; the A/P axis is on the abscissa and the developmental time on the ordinate .", "aop and edl positively and negative regulate Nau expression ( blue circle ) in a subset of PMC cells , favoring and inhibiting selection of PCs from PMC cells , respectively .", "Center , PC specification: the dorsal DA2/AMP and dorso-lateral DA3/DO5 , LL1/DO4 and DT1/DO3 PCs are represented .", "Right , FC specification; only the DA3 and DO5 lineages are detailed .", "Color coding of expression of the different iTFs , and the names of their vertebrate orthologs are indicated .", "Genes and either positive ( arrow ) , or negative ( crossed line ) regulatory steps identified in this study are drawn in black; previously reported interactions are in grey . DOI: http://dx . doi . org/10 . 7554/eLife . 14979 . 029 Analysis of the DL muscle phenotypes showed that formation of the LL1 muscle is also affected in noc , aop and eya mutants ( Figure 2K , W and Figure 2—source data 1 ) .", "Kr was previously shown to be expressed in the LL1 PC and required for LL1 development ( Ruiz-Gomez et al . , 1997 ) .", "We found that Kr expression in the LL1/DO4 ( but not DA1 ) PC required both noc and eya activity , but neither aop nor col ( Figure 6—figure supplement 2 ) ( Enriquez et al . , 2012 ) .", "These data further underline the intricate wiring and combinatorial nature of transcriptional regulations specifying muscle identities ( Figure 7 ) ." ], [ "Noc and Elb are two transcription repressors belonging to the NET family of C2H2 zinc finger proteins ( Dorfman et al . , 2002; Nakamura et al . , 2004 ) .", "Until now , identification of a Tin-dependent mesodermal noc enhancer was the only suggestion of a possible function in muscle development ( Jin et al . , 2013 ) .", "We show here that noc is required for distinguishing between DA3 and DT1 muscle identities .", "Primary transcript analyses revealed that noc maintenance of col transcription in the DA3 lineage involved a double Noc —|S59 —|col negative loop .", "Col activation of S59 expression ( Enriquez et al . , 2012 ) initiates a negative , 'incoherent' feed-forward loop in the DT1/DO3 PC , resulting in Col repression and DT1 identity .", "Noc breaks this loop in the DA3 lineage ( Figure 7 ) .", "noc is transcribed in myoblasts expressing low Nau level whose number is controlled by EGF-R signaling , and selected PCs ( Figure 6 and Figure 3—figure supplement 2 ) .", "However , it is not transcribed in the DT1/DO3 PC which is specified under control of abdominal Hox proteins ( Enriquez et al . , 2010 ) .", "Thus noc regulation integrates multimodal signaling and positional inputs .", "Functions of vertebrate NET proteins Nolz-1/Nolz2 and ZNF503/nlz1 have , so far , been addressed in the central nervous system .", "Yet , zebrafish Znf503 and mouse nolz1 are expressed in branchial arches ( zfin ID: ZDB-Gene-031113-5 , genebank accession number NM-198840; Chang et al . , 2013 ) .", "Whether this expression corresponds to migrating myoblasts which contribute to facial muscle development and express orthologs of other iTFs studied here ( Nathan et al . , 2008; Sambasivan et al . , 2009; Tolkin and Christiaen , 2012; Razy-Krajka et al . , 2014; Diogo et al . , 2015 ) , remains to be explored .", "Our previous finding that several PCs are sequentially selected from the Col PMC and each express a specific iTF code brought to light the importance of time in muscle identity specification ( Boukhatmi et al . , 2012 ) .", "We report here that aop and edl , are required for the robustness of sequential selection of the DA2/AMP and DA3/DO5 PCs .", "A consequence of lack of Aop function is the premature , concomitant selection of several Nau-expressing PCs , when sequential in wt embryos .", "These data indicate that Aop acts to restrict EGF-R MAPK activity to prospective PCs , as previously suggested ( Carmena et al . , 2002 ) .", "Inversely , edl expression in selected cells and delayed PC delamination in edl mutant embryos indicate that Edl ensures timely progression of primed cells to a stable PC fate .", "Thus , proper regulation of EGF-R signaling effectors is both required for the proper number and temporal sequence of PC selections .", "During eye development , the differentiation of all ommatidial cell types is triggered in a stereotypical sequence by reiterated EGFR use ( Freeman , 1996 ) .", "Likewise , EGF-R signalling regulates pulses of cell delamination from the ectoderm ( Brodu et al . , 2004 ) .", "Whether pulses of EGF-R signalling control serial PC selection remains to be determined .", "Parallels between serial PC selection and successive waves of neuroblast ( NB ) selection ( Doe , 1992; Berger et al . , 2001 ) are striking .", "However , in NB lineages , the focus of investigation has shifted to the sequential expression of Temporal Transcription Factors ( TTFs ) in each NB progeny , which specifies the temporal identity of neurons ( Isshiki et al . , 2001 ) , and how birth time controls NB identity has not been determined .", "Our observation of an increased number of Nau-positive cells in aop mutants revealed that Nau/MyoD is expressed in myoblasts primed to a PC fate .", "Nau role in generic myogenesis versus identity aspects has been debated ( Balagopalan et al . , 2001; Wei et al . , 2007 ) .", "We have previously proposed that Nau iTF functions could reflect lineage-dependent cooperation with other iTFs ( Enriquez et al . , 2012 ) .", "Another b-HLH protein , Lethal of Scute ( L ( 1 ) sc ) , has been proposed to act as a promuscular gene ( Carmena et al . , 1995 ) , based on its pattern of expression and regulation in PMCs and PCs , and by analogy to the role of proneural b-HLH proteins in NB selection .", "Yet , only minor muscle patterning defects were observed in l ( 1 ) sc mutants ( Carmena et al . , 1995 ) .", "The detection of Nau expression in PMC cells subject to high EGF-R signaling raises the possibility that Nau could play earlier functions than previously thought in the PC specification process .", "The time lag between DA2/AMP and DA3/DO5 PC emergence coincides with dorsal regression of Tin expression , due to an auto-regulatory circuit in which Tin progressively limits its own transcription ( Johnson et al . , 2011 ) .", "We previously showed that only the DA2/AMP PC inherited Tin ( Boukhatmi et al . , 2012 ) .", "Considering Tin levels as a translation of developmental time , our new results show that EGF-R control of serial PC selection converts this translation into sharp transcriptional decisions and ultimately distinct muscle identities ( Figures 4 and 7 ) .", "Tup and Col direct up-regulation of their own transcription after the PC stage ( Enriquez et al . , 2010; Boukhatmi et al . , 2012 , 2014 ) can explain why small differences in initial expression levels are transformed into stable muscle fates .", "Our data thus provide a new paradigm for how birth timing controls cell identity during development ( Figure 4L ) .", "Drosophila Six proteins , Six4 , So and Op correspond to vertebrate Six1/2 , Six4/5 and Six3/6 , respectively ( Kenyon et al . , 2005 ) .", "Six1/2/4/5 proteins have been reported to interact with eya in controlling the myogenic progenitor cell population in mouse , while no role was found for Six3/6 in this process ( Relaix et al . , 2013 ) .", "Likewise , we did not observe DL muscle pattern defects in embryos lacking op ( Df ( 2R ) Exel6055 ) .", "Drosophila Six4 was previously proposed to interact with Eya in regulating somatic muscle development , both genes showing similar expression pattern ( Clark et al . , 2006; Liu et al . , 2009 ) .", "Tin in vivo binding to the so locus ( Liu et al . , 2009; Jin et al . , 2013 ) raised the possibility that so could also be involved in muscle development .", "Interestingly , temporal ChIP profiles indicated Tin binding to six4 earlier than to so ( Jin et al . , 2013 ) , suggesting sequential regulation .", "Our data show that one eya isoform , eya-RB , is transcribed later than the other isoforms ( eya-RA/RC , Figure 5—figure supplement 1 ) , owing to a switch in TSS , correlating with the profile of in vivo RNA polymerase II binding ( Bonn et al . , 2012 ) .", "Together , these expression data and our finding that eya and Six4 regulate positively , and so negatively , col transcription , suggest that Eya could switch from activator to repressor , by changing partner , from Six4 to So .", "We thus hypothesize that sequential partnering of different Eya isoforms and Six proteins could contribute the diversity of iTF codes and muscle morphologies ( Figure 7 ) .", "Various contributions of mouse Six1/2/4 to the Pax3/MyoD transcription regulatory network controlling early myogenesis in different embryonic territories have recently been reported ( Relaix et al . , 2013 ) .", "It would be interesting to determine whether different eya isoforms are also involved in different Six partnerships and control different facets of muscle development in vertebrates .", "Characterisation of aop , edl , eya , noc and so functions and transcription dynamics revealed that distinguishing between DA3 , versus DA2 , DT1 or DO5 identities involves specific sequences of transcriptional regulations integrating temporal and positional cues ( Figure 7 ) .", "The functions of these , and previously characterized iTFs , underline the intricacy of positive and negative regulatory loops acting at successive steps in different muscle lineages ( Figure 7 ) .", "Besides clear transformations suggestive of complete identity switch , a significant fraction of muscles show incomplete transformations in iTF mutant embryos .", "This supports the idea that , rather than lineage-specific 'master iTFs' , stereotypy of Drosophila muscle patterns relies upon combinatorial inputs of multiple iTFs during PC and FC specification .", "The finding that TFs combinatorically specify muscle identity ( Enriquez et al . , 2012; de Joussineau et al . , 2012; Boukhatmi et al . , 2014 , this report ) indicates that activation of their target genes is context-dependent and involves multiple cis-regulatory elements .", "Multiple levels of cross-regulation ( Figure 7 ) could provide robustness to the final muscle pattern .", "While a function of Nolz proteins in the mesoderm remains to be investigated , Nkx2 . 5 , Eya , Six1 , Islet1 , Col/Ebf and MyoD , are core components of transcriptional regulatory networks controlling the development of pharyngeal/facial muscles originating from the cardio-pharyngeal territory in chordates .", "Our data raise the possibility that these conserved mesodermal TFs could combinatorically control muscle regional diversity in vertebrates , attested by human muscular dystrophies ( Emery , 2002 ) .", "but whose molecular basis remains poorly understood .", "It is reasonable to speculate that these TFs have been co-opted in different wirings during evolution to generate the muscle lineage diversity found in the animal kingdom ." ], [ "Deficiency lines from the Bloomington Drosophila Stock Center were balanced over a CyO–wg–LacZ chromosome to genotype embryos ( Chanut-Delalande et al . , 2014 ) .", "Screening for embryonic phenotypes was as in Chanut-Delalande et al . ( 2014 ) , except that embryos were stained with a monoclonal mouse antibody against the Col protein ( Dubois et al . , 2007 ) .", "Genetic complementation assays were used to identify genes in chromosomal deficiencies whose loss led to DA3 phenotype .", "Homozygous aop1 , edlL19 , eyaCII/IID , so3 , noc35ba2 , salm1 , and col1 homozygous mutants showed muscle phenotypes identical to trans-heterozygous mutants over deficiency .", "They were thus considered as null alleles and consistently used for phenotypic analyses .", "For eya analysis , we used eyaCII/IID/Df ( 2l ) BSC354 trans-heterozygous embryos as eyaCII/IID homozygous embryos present a strong myoblast fusion defect ( not shown ) , not observed in transheterozygous and probably due to a secondary mutations on the eyaCII/IID chromosome .", "Col::moeGFP expression under control of a late mesodermal col CRM ( colLCRM; previously named 2 . 6_0 . 9c; Dubois et al . , 2007 ) was used to visualize the DA3 muscle contours in mutant embryos .", "The col PMC cells and PCs were visualized in col mutant embryos by LacZ expression under control of the early mesodermal col CRM , colECRM ( previously CRM276; Enriquez et al . , 2010 ) .", "For all deficiency screening , sample size was estimated empirically ( >100 stage 14–16 embryos , in duplicates ) .", "For aop , edl , eya , so , and noc , mutant analyses , sample sizes are indicated in the text and the legend of Figure 2—source data 1 .", "Antibody staining and in situ hybridization with intronic probes were as described previously ( Dubois et al . , 2007 ) .", "Primary antibodies were: mouse βPS integrin , anti-Col ( Dubois et al . , 2007 ) , anti-GFP ( Torrey Pines Biolabs ) , anti-β-galactosidase ( Promega , Madison , Wisconsin ) , rabbit anti-Tin ( Manfred Frasch , Erlangen , Germany ) , anti-Nau ( Bruce Paterson , Bethesda , USA ) , anti-Kr ( Ralf Pflanz , Goettingen , Germany ) , anti-β3-tubulin ( Renate Renkawitz-Pohl , Marburg , Germany ) .", "Secondary antibodies were: Alexa Fluor 488- and 555-conjugated antibodies ( Molecular Probes ) , and biotinylated goat anti-mouse ( Vector Laboratories ) .", "Digoxygenin-labelled antisense RNA probes were transcribed in vitro from PCR-amplified DNA sequences , using T7 polymerase ( Roche Digoxigenin labelling Kit ) .", "For aop , edl and eya-RB , 3 non overlapping 600 nucleotide ( nt ) probes were pooled together; for noc , a single probe spanning the entire 302 bp intron; for so , a 2771nt probe hydrolyzed to ~600nt fragments ( Cox et al . , 1984 ) .", "The primer pairs used to amplify the different intron fragments are listed below , with the T7 promoter indicated by small characters .", "aop1: CTCATTGTATGCACGGTACG aop1T7: ccgaattctaatacgactcactatagggATAGCTGCGGCAGAAGCAGG aop2: GCAACAGCAACACTCCAATC aop2T7: ccgaattctaatacgactcactatagggAGACGGTGCGGGCAGAAATTGGG aop3: AAGAGAAAGAGCACGGCAAG aop3T7: ccgaattctaatacgactcactatagggAGATCGGCGACGTTCTCCGAGAC edl1: GGGAGGTGGAAATGACAAAC edl1T7: ccgaattctaatacgactcactatagggCATCGTCTGCCTGACGTCTG edl2: CCAAATATCGCCGATAAGCC edl2T7: ccgaattctaatacgactcactatagggAGACTGCGCACAGGATGCACACC edl3: GAAGATCGACCAGACTTAGG edl3T7: ccgaattctaatacgactcactatagggAGAAGCGGCGTCGAGATTCCCAG eyaRB1: GTTCCTCTAGCTCCGAAATG eyaRB1T7: ccgaattctaatacgactcactatagggTTACGCCGGAGTTGTGAGGG eyaRB2: GACAGCATCGGAGACAACAC eyaRB2T7: ccgaattctaatacgactcactatagggCCCGGCCACAAACGAGAAAC eyaRB3: AGCCCAGTCAAATGCGAAAC eyaRB3T7: ccgaattctaatacgactcactatagggATGCGTGTCCGTGTCGCTAC noc1: CGACGGTTAGTATTGACTAAG noc1T7: ccgaattctaatacgactcactatagggGGCGTCCATCTGTTATGAATAAAATG so1: TCCACGTTTCCAAGTTGGCTACTC so1T7: ccgaattctaatacgactcactatagggAATGCGGCATGTTCGATGCTCGATAATCGG Confocal sections were acquired on Leica SP5 or SPE microscopes at 40× magnification , 1024/1024 pixel resolution .", "Images were assembled using ImageJ and Photoshop softwares .", "3-D reconstructions of the topology of DL PCs and FCs were made from optimized section , 'using volocity ( PerkinElmer ) or Imaris ( Bitplane ) Softwares' .", "Images presented are representative of observations of at least 10 embryos per genotype at a given stage and between five and six segments per embryo .", "To compare the size of the Col and Nau expression domains in wt and aop mutants , optimized stacks of double-stained embryos in the same orientation were flattened , and the largest diameter of each domain measured .", "Statistical analysis was with GraphPad Prism5 using unpaired t-test ." ] ]

[ "Each Drosophila muscle is seeded by one Founder Cell issued from terminal division of a Progenitor Cell ( PC ) .", "Muscle identity reflects the expression by each PC of a specific combination of identity Transcription Factors ( iTFs ) .", "Sequential emergence of several PCs at the same position raised the question of how developmental time controlled muscle identity .", "Here , we identified roles of Anterior Open and ETS domain lacking in controlling PC birth time and Eyes absent , No Ocelli , and Sine oculis in specifying PC identity .", "The windows of transcription of these and other TFs in wild type and mutant embryos , revealed a cascade of regulation integrating time and space , feed-forward loops and use of alternative transcription start sites .", "These data provide a dynamic view of the transcriptional control of muscle identity in Drosophila and an extended framework for studying interactions between general myogenic factors and iTFs in evolutionary diversification of muscle shapes ." ]

[ "Animals have many different muscles of various shapes and sizes that are suited to specific tasks and behaviors .", "The fruit fly known as Drosophila has a fairly simple musculature , which makes it an ideal model animal to investigate how different muscles form .", "In fruit fly embryos , cells called progenitor cells divide to produce the cells that will go on to form the different muscles .", "Proteins called identity Transcription Factors are present in progenitor cells .", "Different combinations of identity Transcription Factors can switch certain genes on or off to control the muscle shapes in specific areas of an embryo .", "However , progenitor cells born in the same area but at different times display different patterns of identity Transcription Factors; this suggests that timing also influences the orientation , shape and size of a developing muscle , also known as muscle identity .", "Dubois et al . used a genetic screen to look for identity Transcription Factors and the roles these proteins play in muscle formation in fruit flies .", "Tracking the activity of these proteins revealed a precise timeline for specifying muscle identity .", "This timeline involves cascades of different identity Transcription Factors accumulating in the cells , which act to make sure that distinct muscle shapes are made .", "In flies with specific mutations , the timing of these events is disrupted , which results in muscles forming with different shapes to those seen in normal flies .", "The findings of Dubois et al . suggest that the timing of when particular progenitor cells form , as well as their location in the embryo , contribute to determine the shapes of muscles .", "The next step following on from this work is to use video-microscopy to track identity Transcription Factors when the final muscle shapes emerge .", "Further experiments will investigate how identity Transcription Factors work together with proteins that are directly involved in muscle development ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "chromosomes and gene expression" ]

[ [ "Age-related hearing loss ( ARHL ) or presbycusis is one of the most prevalent chronic medical conditions associated with aging .", "Indeed , more than 30% of people aged over 65 years suffer ARHL ( Gates and Mills , 2005; Huang and Tang , 2010; Van Eyken et al . , 2007 ) .", "Clinically , ARHL is defined as a progressive bilateral sensorineural impairment of hearing in high sound frequencies mainly caused by a mixture of 3 pathological changes: loss of the hair cells of the organ of Corti ( sensory ) , atrophy of the stria vascularis ( metabolic ) and degeneration of spiral ganglion neurons ( SGN ) , as well as the central auditory pathway ( neural ) ( Gates and Mills , 2005; Schuknecht , 1955; Yamasoba et al . , 2013 ) .", "ARHL has a complex multifactorial etiology with both genetic and environmental factors contributing ( Cruickshanks et al . , 2010; Christensen et al . , 2001 ) .", "Although most people lose hearing acuity with age , it has been demonstrated that genetic heritability affects the susceptibility , onset and severity of ARHL ( Wingfield et al . , 2007; Cruickshanks et al . , 2001; Gates et al . , 1999; Karlsson et al . , 1997; Cruickshanks et al . , 1998 ) .", "Unfortunately , the complexity of the pathology coupled with highly variable nature of the environmental factors , which cause cumulative effects , increases the difficulty in identifying the genetic contributors underlying ARHL .", "Most of the findings from genome-wide association studies ( GWAS ) performed into adult hearing function could neither be replicated between populations , nor the functional validation of those candidates be confirmed ( Dawes and Payton , 2016 ) .", "Mouse models , including inbred strains , have been essential for the identification of several defined loci that contribute to ARHL ( Bowl and Dawson , 2015 ) .", "SLC7A8/SLC3A2 is a Na+‐independent transporter of neutral amino acids that corresponds to system L also known as LAT2 ( L-type Amino acid Transporter-2 ) ( Pineda et al . , 1999; Rossier et al . , 1999; Oxender and Christensen , 1963 ) .", "SLC7A8 is the catalytic subunit of the heterodimer and mediates obligatory exchange with 1:1 stoichiometry of all neutral amino acids , including the small ones ( e . g . alanine , glycine , cysteine and serine ) , which are poor substrates for SLC7A5 ( 18 ) , another exchanger with system L activity .", "Functional data indicate that the role of SLC7A8 is to equilibrate the relative concentrations of different amino acids across the plasma membrane instead of mediating their net uptake ( Pineda et al . , 1999; Meier et al . , 2002; Verrey , 2003 ) .", "The SLC7A8/SLC3A2 heterodimer is primarily expressed in renal proximal tubule , small intestine , blood-brain barrier and placenta , where it is thought to have a role in the flux of amino acids across cell barriers ( Rossier et al . , 1999; Bauch et al . , 2003; Kanai and Endou , 2001; del Amo et al . , 2008 ) .", "So far , SLC7A8 research has been focused mainly on amino acid renal reabsorption .", "However , in vitro studies demonstrated that SLC7A8 could have a role in cystine efflux in epithelial cells and the in vivo deletion of Slc7a8 in a mouse model showed a moderate neutral aminoaciduria ( Braun et al . , 2011 ) , suggesting compensation by other neutral amino acid transporters .", "Therefore , in order to better understand the physiology of SLC7A8 , we generated null Slc7a8 knockout mice ( Slc7a8−/− ) ( Font-LLitjós , 2009 ) and ( Figure 1—figure supplement 1A ) .", "Here , we describe the detection of a hypoacusic phenotype in the Slc7a8−/− mouse model and demonstrate that novel loss-of-function SLC7A8 mutations constitute a primary cause in the development of ARHL in a cohort of elderly people from two isolated villages in Italy ." ], [ "SLC7A8 is highly expressed in the kidney , intestine and brain , and neither full-length nor truncated SLC7A8 protein were detected in membrane samples of Slc7a8−/− mice ( Figure 1A ) .", "The Allen Brain Atlas ( Allen Institute for Brain Science , 2004 ) localizes mouse brain SLC7A8 to the cortical subplate , cerebellum , thalamus and olfactory bulb .", "Our results showed that SLC7A8 protein was localized to the plasma membrane of neuronal axons in different brain regions such as , the choroid plexus , subfornical organ , cerebral cortex and hypothalamus by immunohistochemistry ( Figure 1—figure supplement 2A ) .", "This specific localization in the brain pointed to the possibility that the absence of the transporter could potentially lead to neurological disorders .", "Behavioral screening showed that absence of SLC7A8 in mice does not affect either learning or memory ( Figure 1—figure supplement 3 ) .", "In contrast , a significant reduction in latency was observed in the rotarod acceleration test indicating impairment in motor coordination in Slc7a8−/− mice ( Figure 1—figure supplement 3G ) .", "Reaffirming poorer motor coordination performance in the Slc7a8−/− mice , an increased exposure to shock on the treadmill was also observed ( Figure 1—figure supplement 3B ) .", "Interestingly , a marked impairment was observed in the pre-pulse inhibition of acoustic startle response , which assesses the response to a high intensity acoustic stimulus ( pulse ) and its inhibition by a weaker pre-pulse .", "The response to a 120 dB single-pulse was significantly reduced in Slc7a8−/− mice ( Figure 1B ) .", "The higher threshold required for responding to the acoustic stimulus in the PPI tests in Slc7a8−/− animals could potentially be indicative of a hearing impairment or to a defect in the stress response signaling .", "Response to stress is modulated by the hypothalamic-pituitary-adrenal axis via the release of corticosterone from the adrenal cortex ( Smith and Vale , 2006 ) .", "As SLC7A8 is expressed in the murine pituitary gland ( Figure 1—figure supplement 2A and S3H ) , plasma corticosterone levels under stressing conditions were analyzed .", "No differences were observed in corticosterone levels at either basal conditions , nor under restraint stress in the Slc7a8−/− group , indicating a normal stress response in the absence of SLC7A8 ( Figure 1—figure supplement 3I ) .", "Thus , a hearing impairment in Slc7a8−/− animals was considered the most probable cause of the differences observed in the acoustic startle response test ( Figure 1B ) .", "The impact of the ablation of SLC7A8 on the auditory system was tested initially on mice with a mixed C57BL6/J‐129Sv genetic background .", "Auditory brainstem response ( ABR ) recording , which evaluates the functional integrity of the auditory system , was performed in Slc7a8−/− mice .", "Reinforcing our hypothesis , adult 4- to 6-month-old Slc7a8−/− mice showed significantly higher ( p≤0 . 01 ) ABR thresholds in response to click stimulus , compared with age matched Slc7a8+/− and wild type mice , which maintain normal hearing thresholds ( Figure 1C–E ) .", "The hearing loss observed in Slc7a8−/− mice affected the highest frequencies tested ( 20 , 28 and 40 kHz ) ( Figure 1F ) .", "The analysis of latencies and amplitudes of the ABR waves in response to click stimuli , showed increased latency and decreased amplitude of wave I , but similar II-IV interpeak latency , in the Slc7a8−/− mice when compared with the other genotypes , pointing to a hypoacusis of peripheral origin without affectation of the central auditory pathway ( Figure 1—figure supplement 4A to D ) .", "Mice were grouped according to genotype , age and ABR threshold level and descriptive statistics calculated , showing that the penetrance of the hearing phenotype in the Slc7a8−/− mice is incomplete ( Figure 1D and E ) .", "Therefore , mice were classified according to their hearing loss ( HL ) phenotype , defining normal hearing when ABR thresholds for all frequencies were <45 dB SPL , mild phenotype when at least two thresholds were between 45 and 60 dB SPL and severe hypoacusis when at least two thresholds were >60 dB SPL .", "At 4–6 months of age , Slc7a8−/− mice showed either severe ( 37 . 5% ) or mild ( 25% ) hearing loss , whilst mice from the other genotypic groups did not show hearing loss ( Figure 1E ) .", "Next we studied 7–13 month-old mice , 50% of Slc7a8−/− mice presented severe hypoacusis and the hearing loss spread to lower frequencies with age .", "Slc7a8−/− mice with hearing loss showed statistically significant differences in ABR parameters when compared to the other genotypes ( Figure 1F ) .", "Moreover , 43% of Slc7a8+/− mice developed mild hearing loss at 7–13 months , whereas the age-matched wild-type mice maintained intact hearing indicating a predisposition toward hearing loss in aged Slc7a8+/−mice ( Figure 1E ) .", "The onset and severity of ARHL is attributed to both environmental and genetic factors ( Cruickshanks et al . , 2010 ) .", "As the environmental factors were well controlled in all the experiments , thus the phenotypic variability could be attributed as the consequence of individual genetic differences .", "Indeed , it has been described that several strains of inbred mice present a predisposition to suffer ARHL dependent on multiple genetic factors ( Kane et al . , 2012; Murillo-Cuesta et al . , 2010 ) .", "Here , the hearing loss phenotype was confirmed in a second mouse strain , the inbread C57BL6/J genetic background ( Figure 1—figure supplement 5 ) .", "Additionally , longitudinal study of Slc7a8−/− mice into the inbred C57BL6/J genetic background showed higher penetrance than the mixed background throughout the ages studied ( Figure 2—figure supplement 2 ) .", "The presence of SLC7A8 has previously been reported in the mouse cochlea ( Yang et al . , 2011; Uetsuka et al . , 2015; Sharlin et al . , 2011 ) , and specifically localized to the stria vascularis by liquid chromatography tandem mass spectrophotometry and by Western blotting ( Uetsuka et al . , 2015 ) .", "Here , SLC7A8 was detected in wild-type mouse cochlea by immunofluorescence supporting its localization to the spiral ligament and spiral limbus from the basal to the apical regions of the cochlea ( Figure 2A and B ) .", "SLC7A8 immunolabeling was not observed in the stria vascularis .", "We observed an intense expression of SLC7A8 in the spiral ligament surrounding the stria indicating that the SLC7A8 epitope ( Figure 1—figure supplement 1B ) is either hidden or absent in the stria vascularis .", "Quantification of SLC7A8 expression in the cochlea showed half a dose of the transporter in the Slc7a8+/−than in wild-type mice , and its ablation in Slc7a8−/− mice ( Figure 2C ) .", "A closer study of SLC7A8 immunofluorescence showed that the transporter is also expressed in the spiral ganglia neurons area ( SGN ) ( Figure 1—figure supplement 2B ) .", "The early HL onset and the progressive ARHL phenotype observed in Slc7a8−/− and Slc7a8+/− mice respectively , prompted us to compare the expression of SLC7A8 in wild-type cochlea at different ages ( Figure 1D ) .", "Immunofluorescence quantification of SLC7A8 intensity at 2- and 12 months of age showed expression in the young mice and increased presence of the transporter in the older mice ( Figure 2D ) .", "In the same line , Slc7a8 mRNA quantification from cochlea extracts showed a progressive increased expression throughout mouse life ( Figure 2—figure supplement 1A ) .", "The cytoarchitecture of the inner ear was studied by hematoxylin/eosin staining ( Figure 3 ) , immunofluorescence ( Figure 4 and Figure 4—figure supplement 1 ) and mRNA detection of several cochlear markers ( Figure 3D and Figure 2—figure supplement 1 ) .", "Most of the structures of the cochlear duct , including spiral ligament , spiral limbus , tectorial and basilar membranes showed a normal gross cytoarchitecture in the Slc7a8−/− mice .", "In contrast , in the basal turns of the cochlea we observed that 3 out of 6 Slc7a8−/− mice evaluated showed complete loss of hair cells and flat epithelia , while only one Slc7a8−/− mouse showed intact epithelia in the organ of Corti ( Figure 3A ) .", "Likewise , loss of cells in the spiral ganglia , especially in the basal regions of the cochlea , was observed ( Figure 3A ) .", "Slc7a8−/− mice at 4 to 7 months of age presented ~50% of cell loss in the spiral ganglion compared with wild type mice ( Figure 3B ) .", "Decreased number of cells in the ganglia significantly correlates with ABR threshold and HL phenotype ( Figure 3—figure supplement 1and B ) .", "Concomitantly with the loss of hair cells and spiral ganglion ( SG ) nuclei in Slc7a8−/− mice , the messenger levels of cell type specific biomarkers , such as the potassium voltage-gated channels Kcnq2 , Kcnq3 and Kcnq5 , and the transporter Slc26a5 , which are expressed in the organ of Corti and SG were down-regulated respectively ( Figure 3D and Figure 2—figure supplement 2B ) .", "Less densely packed cells in the spiral ligament were observed in Slc7a8−/− than in wild-type mice ( Figure 3A ) .", "Reinforcing this observation , the expression of Kir4 . 1 , a potassium channel highly expressed in stria vascularis cells ( Ando and Takeuchi , 1999 ) , was also dramatically reduced by 50% in Slc7a8−/− ( Figure 4B and Figure 4—figure supplement 1A ) .", "Likewise , decreased expression of Kir4 . 1 marker correlates with HL phenotype ( Figure 3—figure supplement 1C ) .", "Phalloidin labeling of actin fibers in the basal cells of the stria vascularis was also decreased 50% in the base of the cochlea ( Figure 4C and Figure 4—figure supplement 1 ) .", "SLC7A8 is abundantly expressed in fibrocytes of the spiral ligament and limbus ( Figure 2 ) , accordingly the number of fibrocytes in the spiral ligament decreased by 2/3 and 1/3 in the null and Slc7a8+/− mice , respectively ( Figure 3C ) .", "Moreover , mice with severe HL phenotype showed 30% less number of fibrocytes in the spiral ligament ( Figure 3—figure supplement 1D ) .", "The expression of the transcription factor Tbx18 , essential for fibrocytes development and differentiation , was 50% less in Slc7a8−/− than in wild-type mouse cochleae ( Figure 3D ) .", "In contrast , the expression of s100 , fibrocyte types I and II marker , did not show significant differences ( Figure 4D and Figure 4—figure supplement 1C ) .", "Once we associated mouse SLC7A8 transporter with deafness and identified it as a potential ARHL gene , screening for mutations in human populations was initiated .", "Whole genome sequencing ( WGS ) and audiogram test data obtained from 147 individuals from isolated villages in Italy were included in the study .", "The inclusion criteria were people 50 years old or older with an audiogram test done at high frequencies ( Pure-tone audiometric PTA-H , 4 and 8 kHz ) .", "Individuals with pure-tone average for high frequencies ( PTA-H ) greater than or equal to 40 decibels hearing level ( dB HL ) were considered ARHL cases , whilst people with PTA-H less than 25 dB were considered as controls .", "A total of 66 cases suffering ARHL and 81 controls were selected .", "The gene-targeted studies conducted in this isolated cohort succeeded in detecting seven heterozygous missense variants ( Table 1 ) .", "Four of the variants: p . Val460Glu ( V460E ) , p . Thr402Met ( T402M ) , p . Val302Ile ( V302I ) and p . Arg418His ( R418C ) belong to ARHL cases ( see Audiogram in Figure 5—figure supplement 1A ) and other three: p . Arg8Pro ( R8P ) , p . Ala94Thr ( A94T ) and p . Arg185Gln ( R185L ) to the control group ( see Audiogram in Audiogram in Figure 5—figure supplement 1B ) .", "All the mutations found in SLC7A8 cases and controls from isolated villages of Friuli Venezia Giulia exhibited different frequencies in comparison to public data bases , such as ExAC among others ( see Table 1 ) .", "According to ExAC database’s constrain metrics ( Lek et al . , 2016 ) , the gene shows evidence of tolerance of both loss of function ( pLi = 0 ) and missense variation ( missense Z score = −0 . 14 ) .", "A structural model of human SLC7A8 protein built using the homologous protein AdiC ( Kowalczyk et al . , 2011 ) in the outward-facing conformation ( Rosell et al . , 2014 ) ( Figure 5—figure supplement 1C and D ) was used to localize all the mutations identified here .", "Interestingly , three of the four mutations found in ARHL patients were located in very striking places:", "( i ) V302 is a conserved amino acid located in the extracellular loop four which corresponds to the external lid that closes the substrate binding site when the transporter is open to the cytosol ,", "( ii ) T402 is located in transmembrane ( TM ) domain 10 facing to the substrate binding site , and", "( iii ) V460 is located at the very end of TM domain 12 , with potential interaction with the plasma membrane .", "In contrast , R418 is in the intracellular loop 5 , between TM domain 10 and TM domain 11 and with no functional role described in transporters with the LeuT-fold ( Krishnamurthy and Gouaux , 2012 ) .", "Thus , three of these mutations were promising candidates to affect the transporter function due to their crucial location .", "In vitro functional characterization of variants present in patients with ARHL and controls was performed by measuring amino acid uptake in HeLa cells co-transfected with the heavy subunit CD98hc and Strep tagged-SLC7A8 wild type and variants ( Figure 5 ) .", "Co-expression of the light ( SLC7A8 ) and the heavy ( CD98hc ) subunits in the same cell increases the plasma membrane localization of the transporter ( Rosell et al . , 2014 ) .", "All tested variants showed expression levels comparable to those of wild type , except for V460E that showed only 20% expression of wild -ype protein ( Figure 5—source data 1 ) , being the only variant that did not reach the plasma membrane as indicated by the lack of co-localization with wheat germ agglutinin staining ( Figure 5A ) .", "Amino acid transport induced by SLC7A8 was analyzed for wild type and the identified variants ( Figure 5B ) .", "All variants present in controls ( R8P , R186L and A94T ) conserved more than 80% of alanine transport compared with wild-type protein .", "Three variants found in patients with ARHL showed diminished alanine transport activity: T402M and V460E presented little residual transport activity ( 14 . 6 ± 2 . 6% and 3 . 6 ± 0 . 3% of wild-type activity , respectively ) and R418C showed 50 . 7 ± 5 . 4% of wild-type alanine transport .", "Surprisingly , V302I presented similar alanine transport levels to wild type SLC7A8 .", "Location of residue V302 within EL4 ( within the external substrate lid ( Figure 5—figure supplement 1D ) led us to additionally measure a larger size SLC7A8 substrate , whose transport could potentially be more compromised than that of a small substrate ( e . g . alanine ) .", "Interestingly , V302I transport activity of tyrosine was found to be only 40 . 0 ± 1 . 6% of wild-type SLC7A8 .", "Because the V302I mutation showed a substrate-dependent impact , tyrosine transport in the other variants was also tested ( Figure 5B ) .", "Other SLC7A8 variants found in patients with ARHL and controls showed similar decreased transport activity for alanine and tyrosine .", "Thus , the SLC7A8-induced tyrosine transport was clearly defective in the four variants found in patients with ARHL , whereas it was barely affected ( >85% of wild-type transport activity ) in the variants found in controls ." ], [ "The present work provides evidence that the amino acid transporter SLC7A8/SLC3A2 has a direct role in age-related hearing-loss ( ARHL ) .", "The ablation of SLC7A8 in a mouse model causes deafness with ARHL characteristics , defective audition at high-frequencies with early onset in homozygotes and progressive worsening in heterozygotes with age .", "Identification of rare variants in SLC7A8 gene together with amino acid transport loss-of-function in ARHL patients supports the concept that this gene has a role in the auditory system in association with other genetic and/or environmental factors .", "This study highlights amino acid transporters as new targets to study in largely uncharacterized hearing disorders .", "The description of SLC7A8 as a novel gene involved in a complex trait such as ARHL demonstrates the importance of amino acid homeostasis in preserving auditory function and suggests that genetic screening should be extended to consider other amino acid transporters as potential new genes involved in cochlear dysfunction .", "Our results may enable the identification of individuals susceptible to developing ARHL , allowing for early treatment or prevention of the disease ." ], [ "Animal experimentation complied with the ARRIVE guidelines and was conducted in accordance with Spanish ( RD 53/2013 ) and European ( Directive 2010/63/EU ) legislations .", "All protocols used in this study were reviewed and approved by the Institutional Animal Care and Use Committee at IDIBELL in a facility accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International ( AAELAC accredited facility , B900010 ) .", "Mice procedures were done according with scientific , humane , and ethical principles .", "The studied mouse model did not show phenotype differences comparing male and female .", "Thus , to ensure that our research represents both genders , the studies describes in this work were performed using both sexes equitably .", "The number of biological and experimental replicates is detailed in the legend of each figure .", "Generation of the null Slc7a8 ( Slc7a8−/− ) was done by gene disruption .", "A coding region that includes exon 1 of the Slc7a8 gene was replaced for a neomycin resistance cassette by homologous recombination using a pBlueScript vector with two homologous arms ( right: 6 . 1 kb and left: 2 . 3 kb ) and two resistances ( neomycin and thymidine kinase ) in 5’ region of the gene ( Figure 1—figure supplement 1A ) .", "ES cells transfection and microinjection experiments were done by GenOway ( Lyon-France ) .", "Chimera mouse was outcrossed with a wild‐type C57BL6/J mouse to obtain first generation ( F1 ) of Slc7a8 heterozygous ( Slc7a8+/− ) in a mixed C57BL6/J‐129Sv background .", "Intercross of F1 resulted in the analyzed F2 generation , which contemplates the three genotypes: wild type , Slc7a8+/− and Slc7a8−/− knockout mice .", "The pure inbred genetic background was generated backcrossing Slc7a8−/− F1 mice in the mixed C57BL6/J‐129Sv strain for 10 generations with pure C57BL6/J wild-type mice alternating male and females to avoid a genetic drift in the X and Y chromosomes .", "Mice genotype was confirmed by triplex-PCR using DNA from the tail .", "Primers used were forward: 5’GGAGCGATCTGCGGAGTGA3’; reverse: 5’ACAGAGTGCGCTCCTACCCT3’ and reverse KO-specific: 5’CGGTGGGCTCTATGGGTCTA3’ , and Standard DNA polymerase ( Biotools Ref:10 . 002 ) .", "The PCR products are 458 bp ( wild type allele ) and 180 bp ( Slc7a8−/− allele ) fragment .", "Protein analysis was done by western blotting using total membrane samples .", "Frozen tissues ( 50–100 mg ) were homogenized in 5 mL of membrane buffer ( 25 mM HEPES – 4 mM EDTA – 250 mM sucrose – and protease inhibitors ) and centrifuged at 10 , 000 rpm for 10 min at 4°C .", "Supernatant was centrifuged at 200 , 000xg for 1 hr at 4°C .", "The pellet was resuspended in 150 μL of membrane buffer using a 25G syringe .", "Pierce BCA Protein Assay Kit ( Thermo Scientific Ref:23225 ) was used for protein quantification .", "Polyclonal rabbit antibody against mouse SLC7A8 protein was generated using an antigen against the C‐terminal region ( peptide sequence: PIFKPTPVKDPDSEEQP ) ( Figure 1—figure supplement 1B ) .", "Serum extracts from inoculated rabbits were purified with protein G and used as primary antibody .", "Detection was by chemiluminescent reaction using ECL ( GE Healthcare Ref:RPN2232 ) and autoradiography ( Amersham Hyperfilm Ref:28906839 ) .", "For specific SLC7A8 light subunit detection , samples were run in the presence of 100 mM of dithiothreitol ( SigmaAldrich Ref:D9779 ) .", "Rotarod ( Panlab Ref:LE8500 ) .", "The experimental design consisted of two training trials ( TR ) at the minimum speed ( 4 rpm ) followed of two different tasks:", "( a ) motor coordination and balance were assessed by measuring the latency to fall off the rod in consecutive trials with increasing fixed rotational speeds ( FRS 4 , 10 , 14 , 19 , 24 , and 34 rpm ) .", "The animals were allowed to stay on the rod for a maximum period of 1 min per trial and a resting period of 5 min was left between trials .", "( b ) In the accelerating rod test , the rotation speed was increased from 4 to 40 rpm during two sessions of 1 min .", "For each trial , the elapsed time until the mouse fell off the rod was recorded .", "Treadmill ( Panlab Ref:LE8710MTS ) : During two training trials ( TR ) , the inclination of the treadmill was increased from 0° to 20° from the horizontal plane at different speeds ( 5 , 10 , 20 , 30 , 40 and 50 cm/s ) .", "Whenever an animal fell off the belt , foot shocks were applied for a maximal duration of 1 s .", "After the shock , mice were retrieved and placed back .", "Morris water maze ( MWM ) : Mice were tested over 4 days ( four trials/session , 10 min inter-trial intervals ) .", "The Morris Water Maze test consists of a circular tank ( 150 cm diameter , 100 cm high ) filled with opaque water ( with non-toxic white paint ) and maintained at 21 ± 2°C .", "A removable circular platform ( 8 cm diameter ) was located in a fixed position ( NE quadrant ) inside the pool .", "The pool was surrounded by white curtains , with cues affixed .", "The test was performed under low non-aversive lighting conditions ( 50 lux ) .", "An overhead camera connected to video-tracking software ( SMART , Panlab SL . , Spain ) will be used to monitor the animal’s behavior .", "Latency to reach the platform , total distance travelled , speed and time in zones will be recorded for posterior data analysis .", "The maze was surrounded by white curtains with black patterns affixed , to provide an arrangement of spatial cues .", "A pre-training session was performed in which the platform was visible in the center ( day 1 ) , followed by five acquisition sessions during which the platform was submerged 2 cm below the water ( days 2–6 ) .", "In each trial , mice were introduced in the pool from one of the random starting locations .", "Mice failing to find the platform within 60 s .", "were placed on it for 10 s .", "At the end of every trial the mice were dried for 15 min in a heater .", "Escape latencies , length of the swimming paths and swimming speed for each animal and trial were monitored and computed by a tracking system connected to a video camera placed above the pool .", "Pre-pulse inhibition of acoustic startle response ( PPI ) ( Panlab Ref:LE116 ) : Training was 5 min of habituation time to the apparatus with a background noise level of 70 dB and then exposed to six blocks of 7 trial types in pseudo-random order with 15 s .", "inter-trial intervals .", "The trials: 1 s of a 120 dB , 8000 kHz sound preceded 100 ms . by a 40 ms pre-pulse ( PP ) sound of 74 , 78 , 82 , 86 or 90 dB .", "The startle response was recorded for 65 ms , measuring every 1 ms . from the onset of the startle stimulus .", "Restrain stressor ( LabResearch Ref:G05 ) : Mice were habituated for 3 days prior the experiment collecting 10–15 μL of blood from tail .", "All sets were carried in the same room at the same time to minimize environmental variations and corticosterone fluctuations as a result of circadian rhythms .", "Mice were placed for 15 min in the conditioning unit and 75 μL of tail’s blood was collected .", "For recovery mice were placed into a clean cage for 90 min .", "Blood corticosterone were determined by Corticosterone EIA kit ( Enzo Ref:ADI900097 ) .", "Hearing was evaluated by recording the auditory brainstem responses ( ABR ) with a System 3 TDT Evoked Potential Workstation ( Tucker Davis Technologies TDT , Alachua , FL , USA ) as previously described ( Cediel et al . , 2006; Riquelme et al . , 2010 ) .", "Briefly , mice were anesthetized with intraperitoneal injection of ketamine ( 100 mg/kg ) and xylazine ( 10 mg/kg ) , and placed inside a sound chamber .", "Broadband click ( 0 . 1 ms ) and tone bursts ( 5 ms ) at 8 , 16 , 20 , 28 and 40 kHz were delivered with an open field speaker ( MF1 , TDT ) at an intensity range from 90 to 10 dB sound pressure level ( SPL ) in 5–10 dB SPL steps .", "The electrical responses were amplified and averaged and the ANABR recordings analyzed with BioSig software ( TDT ) to determine hearing thresholds in response to each stimulus , peak and interpeak latencies and peak amplitudes .", "Animals were kept thermostatized and monitored during both anesthesia and the following recovery period .", "Mice were perfused through vascular system with 4% PFA and inner ear and brain samples were collected .", "The cochlea was dissected , post-fixed and decalcified in 0 . 3 M EDTA pH 6 . 5 ( Sigma-Aldrich Ref:E1644 ) for seven days .", "Decalcified cochleae were embedded in OCT or paraffin as reported ( Murillo-Cuesta et al . , 2012 ) .", "Deparaffinized cochlear sections were stained with hematoxylin and eosin for general cytoarchitecture evaluation .", "Immunohistochemistry: Floating brain tissue sections were incubated with 3% H2O2 in 10% methanol in PBS for 10 min .", "Blocking buffer with: 0 . 2% gelatine , 0 . 2% Triton x-100% and 10% FBS for 30 min .", "Primary antibody: anti-SLC7A8 1/500 in blocking buffer ON at 4°C with agitation .", "Secondary antibody: 1/200 biotinylated anti rabbit in blocking buffer for 1 hr at RT .", "Third antibody: 1/100 of A + B conjugate ( Vectastain , Ref:ABC kit ) in blocking buffer for 1 hr at RT .", "Develop staining: 0 . 03%DAB in PBS for 5 min .", "Reaction: incubate 0 . 03%DAB +1/10 . 000 H2O2 for 2–7 min with agitation .", "Reaction was stopped by rinsing with PBS .", "Sections were dried and dehydrated before mounting .", "Detection was using a bright-light microscope .", "Immunofluorescence: OCT tissue sections were permeabilized by incubating for 10 min with 0 . 1% Triton X-100 and incubated as reported ( Sanchez-Calderon et al . , 2010; de Iriarte Rodríguez et al . , 2015b ) with the following primary antibodies: anti-SLC7A8 ( 1/200 ) , -s100 ( 1/1000 , Sigma-Aldrich Ref:S2532 ) , -Kir4 . 1 ( 1/200 , Merck Millipore Ref:AB5818 ) , -IBA1 ( 1/100 , Abcam Ref:ab5076 ) , or with Phalloidin ( 1/100 , Thermo Fisher Scientific Ref:A22287 ) , ON at 4°C .", "Sections were then incubated with secondary antibodies: ( 1:300 , Thermo Fisher Scientific Ref:A-11034 Goat anti-Rabbit Alexa Fluor 488 , Ref:A-11030 Goat anti-Mouse Alexa Fluor 546 , Ref:A-21206 Donkey anti-Rabbit Alexa Fluor 488 , Ref:A-11056 Donkey anti-Goat Alexa Fluor 546 ) for 2 hr at RT .", "Detection by confocal microscopy ( Leica , Ref:LSM 780 Zeiss ) .", "Four sections of apex , middle and basal turns of the cochlea were quantified using the same settings , including argon laser voltage , for the quantification .", "Using Fiji software , the sum of the intensity of all stacks ( 2 . 6 μm in the z axis along the 10 μm section ) from the spiral ligaments + stria vascularis area was extracted .", "Data were analyzed with Prism 7 statistic software package ( Graph Pad Software , Inc . ) .", "Statistical significance was determined by Student’s t test for unpaired samples .", "The number of biological and experimental replicates are detailed in the legend of each figure .", "RNA was isolated using RNeasy ( Qiagen ) from 1 to 2 cochleae; its integrity and concentration were assessed using an Agilent Bioanalyzer 2100 ( Agilent Technologies ) .", "At least , three mice per condition were used .", "cDNA was then generated by reverse transcription ( High Capacity cDNA Reverse Transcription Kit; Applied Biosystems ) and gene expression analyzed in triplicate by qPCR using TaqMan Gene Expression Assay kits ( Applied Biosystems ) .", "The following probes were used: potassium voltage-gated channel subfamily Q member 2 ( Kcnq2 ) Mm00440080_m1; potassium voltage-gated channel subfamily Q member 3 ( Kcnq3 ) Mm00548884_m1; potassium voltage-gated channel subfamily Q member 5 ( Kcnq5 ) Mm01226041_m1; prestin ( Slc26a5 ) Mm00446145_m1; T-box transcription factor TBX18 ( Tbx18 ) Mm00470177_m1; interleukin 1 beta ( Il1b ) Mm00434228m1; interleukin 6 ( Il6 ) Mm00446190m1; solute carrier family 7 ( cationic amino acid transporter , y + system ) , member 8 ( Slc7a8 ) Mm01318971m1 .", "PCR was performed on an Applied Biosystems 7900HT Real-Time PCR System using Hprt1 or RPLP0 as the endogenous housekeeping gene .", "Relative quantification values were calculated using the 2-ΔΔCt method .", "All procedures have been already reported ( de Iriarte Rodríguez et al . , 2015a ) .", "A total of 147 Subjects were recruited in North-Eastern Italy isolated villages ( FVG Genetic Park ) ( Esko et al . , 2013 ) and from one isolated village from Southern Italy ( Carlantino ) .", "Subjects underwent a clinical evaluation to exclude any syndromic form of hearing loss or other systemic illnesses linked with sensorineural hearing loss .", "Audiometric tests using standard audiometers were carried out for each subject .", "Measurements have been obtained after any acoustically obstructing wax was removed .", "Thresholds for six different frequencies ( 0 . 25 , 0 . 5 , 1 , 2 , 4 , 8 kHz ) were measured and then a pure-tone average for high frequencies ( P-TAH ) was computed by taking the average of 4 and 8 kHz .", "To avoid non-genetic variations in the hearing phenotype ( e . g . monolateral hearing loss ) , the best hearing ear was considered for each individual .", "Cases were defined as people older than 50 years old having PTAH ≥40 , while controls were subjects more than 50 years old with PTAH ≤25 .", "All studies were approved by the Institutional Review Board of IRCCS Burlo Garofolo , Trieste , Italy and consent forms for clinical and genetic studies have been signed by each participant .", "All research was conducted according to the ethical standards as defined by the Helsinki Declaration .", "Blood samples were collected and used to extract DNA using standard protocols .", "Low coverage whole genome sequence was generated using Illumina technology ( Genome Analyzer and HiSeq 2000 ) at the Welcome Trust Sanger Institute and Beijing Genomics Institute .", "Data coverage was ranging from 4 to 10X .", "A multi-sample genotype calling was performed and standard quality filters were applied .", "The detailed pipeline has already been described elsewhere ( Timpson et al . , 2014 ) .", "Variants belonging to SLC7A8 gene were extracted using bcftools [http://samtools . github . io/bcftools/] and annotated with ANNOVAR ( Wang et al . , 2010 ) .", "Only the exonic variants were further considered .", "Finally , variants of interest were confirmed by direct Sanger sequencing on a 3500 Dx Genetic Analyzer ( Life Technologies , CA ) , using ABI PRISM 3 . 1 Big Dye terminator chemistry ( Life Technologies ) per manufacturer’s instructions .", "Mutation frequencies were compared with public databases such as Esp6500siv2 ( NHLBI Exome Sequencing Project ) , 1000 g ( 1000 Genomes Project ) , Campion ( The Allele Frequency Net Database ) and ExAC ( The Exome Aggregation Consortium ) .", "For SLC7A8 we collected several statistics including the probability of loss of function intolerance ( pLI ) , where the closer pLI value is to 1 , the more LoF intolerant the gene could be considered .", "We also collected the missense Z score , a positive score indicates intolerance to missense variation whereas a negative Z score indicates that the gene had more missense variants than expected .", "The QuikChange site-directed mutagenesis kit ( Stratagene ) was used to introduce point mutations in SLC7A8 sequence , according to the manufacturer's protocol .", "The pcDNA3 . 1-StrepTag fused SLC7A8 construct was used as template ( Costa et al . , 2013 ) .", "Amino acid substitutions were introduced into SLC7A8 sequence using a compatible reverse primer and forward primers ( Figure 5—source data 1 ) .", "All primers annealed to the coding sequence , and the position of the mutated codon was underlined .", "All constructs were verified by DNA sequencing and then used for transient transfection .", "HeLa cells ( Sigma Aldrich , Ref: 93021013 ) were maintained at 37°C/5% CO2 in Dulbecco's modified Eagle's medium ( Life Technologies ) supplemented with 10% ( v/v ) fetal bovine serum , 50 units/ml penicillin , 50 μg/ml streptomycin , and 2 mM l-glutamine .", "HeLa cells were transiently transfected with plasmid constructions mentioned above with the use of Lipofectamine 2000 ( Invitrogen ) following the manufacturer's protocol .", "Amino acid transport and fluorescence microscopy analyses were carried out 48 hr after transfection .", "To analyze the effect of the mutations on SLC7A8 protein expression and plasma membrane localization , fluorescence microscopy of Strep-tagged wild type and mutant transporters was performed on a semiconfluent monolayer of transfected HeLa cells cultured on glass coverslips .", "Glass coverslip-grown cells were incubated with 1 mg/ml wheat germ agglutinin ( WGA ) labeled with Texas-Red ( Thermo Fisher Scientific ) at 37°C for 10 min , rinsed three times with phosphate-buffered saline-Ca2+-Mg2+ and fixed for 15 min in 4% paraformaldehyde .", "Fixed cells were blocked in blocking buffer ( 10% FBS and 0 . 1% saponin in PBS ) for 1 hr and then incubated for 1 hr with primary antibody ( anti-Strep Tag GT517 , 1/100; Abcam ) .", "Secondary goat-anti-mouse-FITC antibody ( Life Technologies ) was incubated for 2 hr protected from light and rinsed three times with phosphate-buffered saline .", "Nuclear staining was performed by incubating 1 µg/ml Hoechst ( Thermo Fisher Scientific ) for 10 min , rinsed three times with phosphate-buffered saline and then mounted with aqua-poly/mount coverslipping medium ( Polysciences Inc . ) .", "Images were taken using a Nikon E1000 upright epifluorescence microscope .", "All images were captured during 200 ms except for those corresponding to V460E that were overexposed to 2 s to reveal the subcellular localization of this very low expressing variant .", "To quantify SLC7A8 wild type and mutated transporters expression levels in cells , a single in-focus plane was acquired .", "Using ImageJ ( v1 . 48 , NIH ) , an outline was drawn around each cell and area and mean fluorescence measured , along with several adjacent background readings .", "The total corrected cellular fluorescence ( TCCF ) = integrated density – ( area of selected cell × mean fluorescence of background readings ) , was calculated .", "Amino acid uptake was measured by exposing replicate cultures at room temperature to L- [3H]-labeled alanine or [3H]-tyrosine ( 1 μCi/ml; Perkin Elmer ) in sodium-free transport buffer ( 137 mM choline chloride , 5 mM KCl , 2 mM CaCl2 , 1 mM MgSO4 , and 10 mM HEPES , pH 7 . 4 ) .", "Initial rates of transport were determined using an incubation period of 1 min and 50 µM of cold alanine or tyrosine .", "Assays were terminated by washing with an excess volume of chilled transport buffer .", "Cells were lysed using 0 . 1% SDS and 100 mM NaOH and radioactivity measured in a scintillation counter .", "Uptake values were corrected by their total corrected cellular fluorescence ( TCCF ) for all transporters except for V460E mutant , which does not reach the plasmatic membrane .", "Behavior and ABR experiments using mice were not performed blind to genotype and treatment conditions , but as data acquisition was automated this will not affect data processing and analysis .", "The sample size was chosen according to the standard sample sizes used in the field and without applying any statistical method .", "The general criteria of exclusion were pre-established: ( 1 ) samples with a value that differed by more/less than two standard deviations from the mean value were excluded from the study .", "The statistical tests used in each experiment were appropriate to the type of groups , data and samples .", "Unpaired Student t-test was used for experiments with only two independent groups .", "Repeated measures two-way ANOVA was applied when we had to compare two independent groups ( genotype as the between subjects factor ) where repeated measurements of the dependent variable were obtained ( Rotarod and PPI ) .", "Data were analyzed with IBM SPSS 23 . 0 statistic software package ( Chicago , IL ) .", "Statistical significance was determined by one-way analysis of variance ( ANOVA ) and Levene's F test to assess the equality of variances .", "When significant differences were obtained , post hoc comparisons were performed using Bonferroni or Tamhane tests to compare the three genotypes .", "Normal distribution of data and homogeneity of variances was assessed using Shapiro-Wilk and Levene tests , respectively .", "In most of the datasets these two assumptions were achieved .", "However , when not achieved and because we use comparable sample sizes and ANOVA is robust to normality violations , our results are still valid .", "Sphericity assumption was assessed using Mauchly’s test and when not achieved Greenhouse correction was taken .", "Posthoc tests were performed using Bonferroni correction for individual comparisons .", "Bonferroni p<0 . 05 was assumed as critical value for significance throughout the study .", "Statistical analyses were performed using SPSS package ." ] ]

[ "Age-related hearing loss ( ARHL ) is the most common sensory deficit in the elderly .", "The disease has a multifactorial etiology with both environmental and genetic factors involved being largely unknown .", "SLC7A8/SLC3A2 heterodimer is a neutral amino acid exchanger .", "Here , we demonstrated that SLC7A8 is expressed in the mouse inner ear and that its ablation resulted in ARHL , due to the damage of different cochlear structures .", "These findings make SLC7A8 transporter a strong candidate for ARHL in humans .", "Thus , a screening of a cohort of ARHL patients and controls was carried out revealing several variants in SLC7A8 , whose role was further investigated by in vitro functional studies .", "Significant decreases in SLC7A8 transport activity was detected for patient’s variants ( p . Val302Ile , p . Arg418His , p . Thr402Met and p . Val460Glu ) further supporting a causative role for SLC7A8 in ARHL .", "Moreover , our preliminary data suggest that a relevant proportion of ARHL cases could be explained by SLC7A8 mutations ." ]

[ "Age-related hearing loss affects about one in three individuals between the ages of 65 and 74 .", "The first symptom is difficulty hearing high-pitched sounds like children’s voices .", "The disease starts gradually and worsens over time .", "Changes in the ear , the nerve that connects it to the brain , or the brain itself can cause hearing loss .", "Sometimes all three play a role .", "Genetics , exposure to noise , disease , and aging may all contribute .", "The condition is so complex it is difficult for scientists to pinpoint a primary suspect or develop treatments .", "Now , Guarch , Font-Llitjós et al . show that errors in a protein called SLC7A8 cause age-related hearing loss in mice and humans .", "The SLC7A8 protein acts like a door that allows amino acids – the building blocks of proteins – to enter or leave a cell .", "This door is blocked in mice lacking SLC7A8 and damage occurs in the part of their inner ear responsible for hearing .", "As a result , the animals lose their hearing .", "Next , Guarch , Font-Llitjós et al . scanned the genomes of 147 people from isolated villages in Italy for mutations in the gene for SLC7A8 .", "The people also underwent hearing tests .", "Mutations in the gene for SLC7A8 that partially block the door and prevent the flow of amino acids were found in people with hearing loss .", "Some mutations in SLC7A8 that allow the door to stay open where found in people who could hear .", "The experiments suggest that certain mutations in the gene for SLC7A8 are likely an inherited cause of age-related hearing loss .", "It is possible that other proteins that control the flow of amino acids into or out of cells also may play a role in hearing .", "More studies are needed to see if it is possible to fix errors in the SLC7A8 protein to delay or restore the hearing loss ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "biochemistry and chemical biology", "structural biology and molecular biophysics" ]

[ [ "S-adenosyl methionine ( SAM ) plays many important roles in biology .", "It is an essential methyl donor for methyltransferases that act on nucleic acids , proteins , lipids , and small molecules , but is also involved in many other reactions , for example , as a substrate used in biosynthesis of polyamines and quorum sensing molecules ( reviewed in Loenen , 2006 ) .", "It is also involved in epigenetic changes in many organisms ( Janke et al . , 2015; Su et al . , 2016 ) and in bacterial restriction-modification systems where methylation of DNA is used to distinguish foreign DNA from host DNA ( Wilson and Murray , 1991 ) .", "In early studies of nucleic acid methylation , it was observed that while infection of Escherichia coli with phage T1 , T2 , and T4 induced higher levels of DNA methylation , infection with phage T3 reduced the degree of methylation of not only DNA , but also tRNA and rRNA ( Gold and Hurwitz , 1964 ) .", "The reduction of methylation by T3 infection was associated with an immediate and dramatic lowering of the level of SAM in the E . coli cell extract .", "The degradation products were , using paper chromatography and chemical tests , identified as 5’-methyl-thioadenosine ( MTA ) and L-homoserine ( Gold and Hurwitz , 1964; Figure 1 , top ) , which led to the claimed discovery of a potent T3-encoded SAM hydrolase enzyme ( Hausmann , 1967 ) .", "Subsequent work showed that the T3 SAMase was produced early in infection ( Gefter et al . , 1966 ) , encoded in the early transcribed portion of the phage genome ( Herrlich and Schweiger , 1970 ) and important for the counter defense of the bacteriophage against the type I restriction modification ( RM ) system of the host bacterium .", "In this type of RM system , SAM is essential for both methylation of host DNA and restriction of target sequences in the foreign DNA .", "Two potential mechanisms for how the RM system could be impaired were presented .", "First , the lowered SAM levels prevented methylation of the host genome and the SAM-dependent restriction of the phage genome ( Krueger et al . , 1975 ) .", "Second , inhibition was observed to be independent of SAM degradation and possibly linked to an interaction with the restriction enzyme ( Spoerel et al . , 1979; Studier and Movva , 1976 ) .", "Recently , three additional SAM degrading enzymes were identified in a screen for bacteriophage DNA that could rescue an auxotrophic E . coli mutant where the isoleucine biosynthetic ilvA gene was deleted .", "Investigations using proteomics and RNA-seq showed that the phage-encoded polypeptides induced up-regulation of the biosynthetic pathway for methionine by degradation of SAM ( Jerlström Hultqvist et al . , 2018 ) , which together with the repressor protein MetJ acts as a co-repressor of the met regulon ( Weissbach and Brot , 1991 ) .", "Isoleucine biosynthesis was rescued through a promiscuous activity of one of the up-regulated enzymes , MetB ( Figure 1—figure supplement 1 ) .", "One of the newly identified SAM degrading enzymes , Svi3-3 , was cloned , expressed , and purified .", "In vitro activity assays demonstrated that Svi3-3 catalyzed conversion of SAM to MTA .", "We herein present the first structure of a phage-encoded SAMase and explore the reaction mechanism using both computational and experimental biochemistry .", "Strikingly , the results unambiguously show that the phage-encoded SAMases are not hydrolases , as believed since the 1960s , but lyases ( Figure 1 ) ." ], [ "The Svi3-3 enzyme was originally expressed from a library of fragmented environmental phage DNA .", "For this reason , the exact size of the original open reading frame was unknown , but an N-terminally hexahistidine-tagged 162 amino acid construct including some vector-derived sequence was shown to have SAMase activity ( Jerlström Hultqvist et al . , 2018 ) .", "The phage-derived sequence was subcloned to allow proteolytic removal of the hexahistidine tag , and based on predictions of a disordered N terminus , truncated variants were made .", "Full-length and N-terminally truncated variants of Svi3-3 were expressed with an N-terminal hexahistidine tag and purified on a Ni-column for structural studies .", "When expressed from the T7-promoter expression plasmid , the proteins were highly toxic and the tightly regulated , arabinose-inducible BL21-AI cells had to be used to allow cell growth until induction .", "An N-terminally truncated 146 amino acid construct of Svi3-3 , where the hexahistidine tag had been removed ( Svi3-3_d19 , Supplementary file 1-table 1 ) , formed crystals in the presence of SAM or the analogue S-adenosyl homocysteine ( SAH ) .", "We here number its sequence starting at the N terminus of the TEV-cleaved protein , corresponding to an offset of −16 in relation to previous work ( Jerlström Hultqvist et al . , 2018 ) .", "The crystals diffracted to 1 . 45 Å resolution in space group F4132 and the structure was solved using ab initio methods with Arcimboldo ( Rodríguez et al . , 2009; Figure 2A , F , Table 1 ) .", "There is one monomer in the asymmetric unit , forming a trimer around the threefold crystallographic axis .", "Apart from the disordered C-terminal 16 residues , the full structure could be built .", "Svi3-3 forms a trimer of ferredoxin-like fold alpha–beta sandwiches , where each subunit has two helices on the outside and a five-stranded anti-parallel beta sheet at the center ( Figure 2A and B , Figure 2—video 1 ) .", "The subunits pack in a triangular manner , forming continuous beta sheets with their neighbors and are each in a ‘velcro-like’ topology where residues 2–6 at the N terminus of one subunit form beta-sheet interactions with residues 120–124 in β8 of another subunit ( Figure 2A ) .", "The long β5 forms beta-sheet interactions with both of the other monomers through interactions between the backbones around residues 51 and 55 .", "In addition , the end of the β5–β6 hairpin packs against α1 of another monomer .", "The trimer interfaces are each stabilized by a single salt bridge , numerous hydrogen bonds , and by hydrophobic interactions at the center .", "The substrate analogue SAH binds at the interface between two subunits ( Figure 2C and D ) , enclosed by the β5–β6 hairpin at the edge of the sheet .", "The adenosine base is recognized by interactions with the backbone carbonyl and amide of Ile77 and the side chain of Ser50 .", "The ribose forms interactions with Ser50 from one subunit and Glu69 and Ser71 from the other subunit .", "The homoserine moiety forms interactions with backbone groups and the side chains of Glu69 and Gln104 .", "In the structure from co-crystallization with SAM , catalysis has occurred and the product MTA is observed in identical position as the corresponding part of SAH , with the methyl group pointing away from Glu69 ( Figure 2E and F ) , while a proline molecule from the cryo buffer is bound in the second half of the active site , mimicking the second product .", "The protein structures are virtually identical in the presence of SAH and MTA ( root mean square deviation [RMSD] of 0 . 16 Å over 129 Cα atoms ) .", "To test if the structure contains all the elements needed for activity in vivo , and because the start and end of the native phage protein sequence remain unknown , constructs truncated to only contain the ordered parts were tested for their ability to rescue the ilvA knockout mutant .", "Indeed , the N-terminally truncated construct additionally lacking the disordered C terminus provided similar rescue to full-length Svi3-3 , indicating that neither tail is needed for SAMase activity ( data not shown ) .", "Based on this result , all further experiments were performed with the 146 amino acid Svi3-3_d19 construct ( Supplementary file 1-table 1 ) .", "Crystallization in the absence of ligands led to a lower-resolution apo structure .", "The structure of Svi3-3 in apo state is overall very similar to the complex structures ( RMSD of 0 . 84 Å over 124 Cα atoms ) , but there is a shift of the N terminus , a conformational change of residues 7–9 to form a more extensive interaction with β9 and the preceding loop of the neighboring subunit , leading to a slight loosening of the trimer ( Figure 3 ) .", "In addition , residues 64 and 65 in the β5–β6 hairpin are disordered .", "Small angle X-ray scattering ( SAXS ) data confirmed that Svi3-3 has a similar trimeric structure in apo state as in the presence of SAM ( Figure 3—figure supplement 1 ) , indicating that product release and substrate binding do not require trimer dissociation .", "A search for structures with similar fold and connectivity as Svi3-3 using PDBeFold ( Krissinel and Henrick , 2004 ) showed that bacterial PII signaling proteins and cation tolerance proteins of CutA type ( belonging to a PII-like family ) in the GlnB-like superfamily have similar trimeric architectures of ferredoxin-like folds .", "The closest structural neighbors superpose with RMSDs above 2 Å over no more than 86 Cα atoms .", "PII regulatory proteins bind ATP/ADP and 2-oxoglutarate at the trimer interface , and conformational changes of loops in response to ligand binding and modification modulates binding to other proteins ( Forchhammer and Lüddecke , 2016 ) .", "Intriguingly , the position of ATP is similar to where SAH binds to Svi3-3 , although the binding site appears non-conserved .", "The reaction mechanism of the presumed SAM hydrolases ( Figure", "1 ) is previously unexplored .", "Two possibilities would either be that the enzyme provides a catalytic base that by deprotonation activates a water molecule for nucleophilic attack on the γ carbon of SAM , or that a catalytic residue from the enzyme acts as nucleophile and attacks the γ carbon , forming a covalent intermediate with the substrate that subsequently gets hydrolyzed by reaction with a water molecule .", "In both scenarios , a catalytic amino acid in proximity to the site of hydrolysis would be needed .", "Inspection of the active site shows that Glu69 is relatively close to the site of cleavage ( Figure 2C ) , making this residue the most likely candidate .", "Another possible residue that could act as a base or a nucleophile is Tyr58 , which in such case would need to donate a proton to the nearby Glu105 ( Figure 2C ) .", "To test these possibilities , Svi3-3 mutants Y58F , E69Q , E69A , and E105Q were constructed , expressed , and purified for in vitro activity assays .", "The Y58F mutant eluted mainly as monomer , indicating a de-stabilized trimer , while all other mutants migrated as trimers .", "The activity of wild-type ( WT ) and mutant Svi3-3 variants in conversion of SAM to MTA was determined at 1 mM substrate concentration .", "The WT truncated Svi3-3 construct showed an average turnover of 9 . 5 s−1 at 25°C , nearly doubled compared to the full length , His-tagged construct ( Jerlström Hultqvist et al . , 2018 ) , but with significant deviation between batches .", "The mutant enzymes all showed reduced activity compared to WT ( Figure 4A , Figure 4—figure supplement 1 ) .", "For the Y58F and E105Q mutants , activity was fivefold and threefold reduced , whereas for the E69Q and E69A mutants , activity was nearly abolished ( 4500- and 1500-fold reduction ) .", "Thus , Glu69 is in vitro critical for substrate binding , catalysis , or both .", "To characterize the binding of substrate and product to Svi3-3 , thermal unfolding experiments were performed using differential scanning fluorimetry ( DSF ) ( Niesen et al . , 2007 ) .", "In the presence of the substrate SAM , its analogue SAH , or the reaction product MTA , the melting temperature of the WT enzyme increased by up to 30°C , demonstrating a major stabilizing effect on the structure ( Figure 4B ) .", "Under the assay conditions , the substrate is most likely turned over , and as a result , the sample with SAM is stabilized by binding of MTA .", "A large stabilization was also seen in thermal denaturation assays followed by circular dichroism in the presence of MTA ( data not shown ) .", "The DSF assay clearly demonstrates that the E69Q mutant is not impaired in binding of SAM or MTA , but that binding of SAH is abolished ( Figure 4B ) .", "However , despite extensive trials , a structure of Svi3-3 E69Q with SAM could not be obtained .", "A crystal structure of Svi3-3 E69Q with MTA shows no major conformational changes of the enzyme , but a minor shift of the mutated side chain close to the reaction site ( data not shown ) .", "A structure-guided multiple sequence alignment of Svi3-3 with T3 SAMase and two other polypeptides annotated with SAMase activity ( Orf1 and Svi3-7 [Jerlström Hultqvist et al . , 2018] ) but with barely detectable sequence similarity suggests that a similar core fold can be formed by all aligned enzymes ( Figure 5 ) .", "Only three amino acids are strictly conserved , and only a handful conservatively substituted .", "Based on the SAH structure , all the conserved residues are important for binding of SAM .", "Gly56 packs against the ribose , not allowing space for a side chain , Glu69 and Gln104 as described above form hydrogen bonds to the ligand , and Tyr58 packs against the γ carbon and forms a hydrogen bond to Glu105 , enclosing the ligand .", "The Svi3-3 structure allows interpretation of the effect of loss-of-function mutations identified in the previous complementation studies ( Jerlström Hultqvist et al . , 2018 ) .", "One group of disabling mutations would act to sterically disturb the interaction between the monomers and/or change the character of the interface ( R14C , V33D , V52D , E110K , and G115V ) .", "Mutation of the conserved glycine ( G56D ) would clash with the ribose in MTA and SAH and thereby disturb substrate binding .", "The remaining mutations ( A13V , A93G , and G95D ) are likely to affect folding or stability of the structure .", "To validate the structure-guided sequence alignment , Glu68 in T3 SAMase , predicted by the sequence alignment ( Figure", "5 ) to be the equivalent of Glu69 in Svi3-3 , was mutagenized and tested for in vitro activity .", "The E68Q mutant of T3 SAMase retained 20–30% activity compared to WT .", "In addition , mutant variants carried on an inducible plasmid were tested for their ability to rescue the ΔilvA auxotrophic mutant ( Figure 1—figure supplement 1; Jerlström Hultqvist et al . , 2018 ) under uninduced and induced conditions .", "Because of its toxicity at higher expression level , WT T3 SAMase only shows rescue under uninduced conditions ( Jerlström Hultqvist et al . , 2018 ) .", "Both T3 SAMase and Orf1 contain two consecutive acidic residues ( E67 and E68 in T3 SAMase , E50 and D51 in Orf1 , Figure 5 ) , both of which were conservatively mutated .", "No rescue was observed either for the Svi3-3 E69Q , Svi3-3 E69A , Orf1 E50Q , or the T3 SAMase E68Q mutants , suggesting a lost or reduced activity of these variants ( Table", "2 ) and validating the sequence alignment .", "Mutations of the neighboring acidic residues ( E67Q in T3 SAMase and D51N in Orf1 ) still allowed rescue under the same conditions as the corresponding WT .", "Given that we observed measurable activity for the T3 SAMase E68Q mutant in vitro , we decided to insert different variants of the T3 SAMase on the chromosome , to allow titration of the expression levels .", "To this end , the different mutant gene variants were placed downstream of the pBAD promoter in a ΔilvA knockout mutant .", "Different concentrations of L-arabinose were used to induce the expression of these variants to determine if the variants could show in vivo activity at higher expression levels .", "For the E68Q mutant , growth was only observed at the highest concentration of arabinose ( 0 . 1% ) , where low enzymatic activity can be compensated by increased enzyme levels ( Supplementary file 1-table 2 ) .", "To gain further insights into substrate binding to Svi3-3 , molecular dynamics ( MD ) simulations were performed .", "An initial complex of Svi3-3 with SAM was generated from docking calculations , which indicated a slight binding preference for SAM over SAH .", "A total of 100 ns of MD simulation was run for both apo Svi3-3 and the SAM complex ( Figure 6 ) .", "The simulation of the complex revealed that the SAM conformation in the active site is very stable , particularly the methionine part ( Figure 6—figure supplement 1 ) .", "The time evolution of the backbone RMSD with respect to the initial structure is shown in Figure 6A and reaches stability in about 30 ns .", "In agreement with the experimental structure with SAH , the carboxylate group is strongly stabilized by the backbone amide hydrogens of Glu105 and Ser106 , whereas the amino group is primarily stabilized by the side-chains of Glu69 and Gln104 ( Figure 6—figure supplement 2A ) .", "Moreover , the positively charged methionine S atom is consistently stabilized through a π-cation interaction with the Tyr58 side-chain ( Figure 6—figure supplement 1 , Figure 6—figure supplement 2A ) .", "The adenosine part of SAM is primarily stabilized by H-bond interactions with the side-chain of Ser50 and the backbone of Val57 and Ile77 ( Figure 6—figure supplement 1 ) , as in the crystal structures with SAH and MTA ( Figure 2C and E ) .", "Comparison of the average root mean square fluctuation ( RMSF ) of the protein backbone shows that Svi3-3 is more flexible throughout the 100 ns MD simulation in apo state ( RMSF for trimer 0 . 91 Å2 ) than with SAM in the active site ( RMSF for trimer 0 . 86 Å2 ) ( Figure 6B ) .", "Interestingly , the residues surrounding the active site cavity , and in particular the methionine-stabilizing residues 104–106 , become significantly more flexible in the apo simulations ( Figure 6B ) .", "Thus , 100 ns of MD simulation demonstrated that apo Svi3-3 shows the highest flexibility in the regions surrounding the active site , and that these regions become significantly more rigid upon substrate binding .", "The mechanism of enzymatic SAMase activity is previously unexplored .", "Thus , the observed active site conformations from the MD simulations were used to build a 194-atom cluster model ( Figure 6—figure supplement 2B ) to investigate the SAMase reaction mechanism with density functional theory ( DFT ) .", "The simulations revealed one potential , but not optimally positioned , water molecule for hydrolysis that was stabilized by H-bonds between the backbone of Ser106 and Gln104 , but there were no obvious residues within reach to activate this water by acting as a base .", "Glu69 was initially suspected to either work as a catalytic base , activating a water molecule for hydrolysis , or as a nucleophile , attacking the γ-carbon of SAM .", "However , no water molecule was observed with a suitable position with respect to Glu69 in the experimental structures or after the MD simulation , indicating that the role as a catalytic base is unlikely .", "Moreover , the observed SAM configuration relative to Glu69 was not optimal for attack on the γ-carbon of SAM .", "DFT geometry optimizations furthermore failed to locate any stationary point ( transition state ) for the attack , suggesting that the role as a nucleophile is also unlikely .", "Thus , it seems that the main role of Glu69 is to bind and orient the substrate in the active site by accepting H-bonds primarily from the amino group of SAM , but also from the 3’-hydroxyl group of the ribose ring ( Figure 6—figure supplement 2 ) .", "In fact , the only residue in the active site oriented properly for a potential hydrolase reaction mechanism is Tyr58 .", "However , DFT calculations could exclude the possibility of Tyr58 acting as a nucleophile in such a mechanism .", "Further DFT geometry optimization , however , revealed a completely different mechanism , a unimolecular reaction resulting in the formation of homoserine lactone ( Figure 7A ) .", "The DFT optimized stationary points shown in Figure 7A indicate that Tyr58 is deprotonated and bridged by a water molecule to the protonated Glu105 .", "Deprotonation of Tyr58 enhances its cation-π interaction with the S atom of SAM and the electrostatic preorganization weakens the bond to the ribose ring .", "Together with the amino group interaction with Glu69 this makes the configuration of SAM in the active site of Svi3-3 susceptible to intramolecular carboxylate oxygen attack on the γ-carbon .", "The activation energy for the formation of homoserine lactone in the DFT cluster model was calculated to be 16 . 8 kcal/mol with an exothermic reaction energy of −5 . 7 kcal/mol ( Figure 7B ) .", "These values were rather insensitive to the choice of dielectric constant , as a change from ε = 4 to ε = 80 yields an energy barrier of 16 . 1 kcal/mol and a reaction energy of −6 . 3 kcal/mol .", "However , since our active site cluster model by necessity is limited in size , it seems possible that the predicted net proton transfer from Tyr58 to Glu105 could be caused by the DFT optimization disfavoring ion pairs with charge separation over a larger distance ( Glu-…TyrOH…S+ ) , and instead moving the proton from Tyr58 to Glu105 ( the initial configuration had the proton on Tyr58 ) .", "In order to examine this issue , the reaction energetics were recalculated with the proton constrained to remain on Tyr58 , maintaining the standard protonation states of the tyrosine and Glu105 .", "This , in fact , yielded very similar energetics with a barrier of 15 . 5 kcal/mol and a reaction energy of −6 . 1 kcal/mol ( ε = 4 ) , indicating that the proton relay is not a necessary feature of the reaction mechanism .", "In the product state ( Figure 7A ) , Glu69 shares a proton with the amino group of SAM , and a water molecule makes strong hydrogen bonds with Tyr58 and Glu105 , irrespective of protonation state at the start of the reaction .", "This water appeared to be a good candidate for nucleophilic attack on the carbonyl carbon of homoserine lactone , thereby forming a tetrahedral intermediate which could break down to homoserine .", "The calculated barrier for this step is 17 . 5 kcal/mol , which is not unrealistically high , but the reaction energy for the tetrahedral intermediate is 25 . 2 kcal/mol relative to the homoserine lactone ( Figure 7B ) .", "Thus , the DFT calculations indicate that Svi3-3 forms homoserine lactone , but that further breakdown to homoserine does not occur in the active site .", "Most importantly , the DFT calculations clearly predict that Svi3-3 is not a SAM hydrolase , but rather a SAM lyase , catalyzing the unimolecular transformation of SAM to homoserine lactone , whereafter this product is released from the active site .", "One prediction from the suggested reaction mechanism is that the carboxyl group of SAM is essential for the degradation of SAM by Svi3-3 .", "To test this hypothesis , decarboxylated SAM ( dcSAM ) was produced enzymatically from SAM using SAM decarboxylase ( Cohn et al . , 1983 ) and used as a substrate in the MTA formation assay .", "In support of the proposed mechanism , we observed no Svi3-3-catalyzed production of MTA from dcSAM ( Figure 8 ) .", "However , in a DSF binding assay , dcSAM does not induce a thermal stabilization of Svi3-3 ( Figure 8—figure supplement 1 ) .", "Comparison with the strong stabilization by SAM/MTA and SAH ( Figure 4B ) suggests that the carboxyl group of SAM is required for binding to Svi3-3 .", "Our activity assay ( Jerlström Hultqvist et al . , 2018 ) is based on detection of MTA that is also observed in the structure from co-crystallization of Svi3-3 with SAM .", "Thus , MTA is indeed formed in the reaction .", "In order to test the predictions from the DFT calculations , we used thin-layer chromatography ( TLC ) , commonly used for separation of amino acids , to determine whether homoserine was formed .", "The substrate SAM and the products could be separated by TLC , the adenosyl-containing compounds were visualized under UV light , and the amines were stained with ninhydrin after pre-treatment with β-mercapto ethanol ( BME ) .", "Interestingly , we observed a reaction product with mobility and color upon staining distinct from homoserine , proving that Svi3-3 is not a SAM hydrolase .", "The same assay was performed with T3 SAMase and Orf1 and comparison with reference samples shows that for all three enzymes , a reaction product is observed that on TLC migrates and stains similar to homoserine lactone ( Figure 9A ) , supporting the hypothesis that Svi3-3 , T3 SAMase and Orf1 are indeed SAM lyases .", "To confirm the identity of the reaction product , the enzymatic reaction products for Svi3-3 were examined with 1H nuclear magnetic resonance ( NMR ) and the spectrum compared to reference spectra for SAM , homoserine , and homoserine lactone ( Figure 9B , Figure 9—figure supplement 1 , Figure 9—figure supplement 2 ) .", "The results confirm that homoserine lactone is formed on the same time scale as MTA .", "Upon incubation of homoserine lactone in phosphate buffer at pH 7 . 4 , the lactone is transformed to homoserine through spontaneous hydrolysis ( Figure 9—figure supplement 3 ) ." ], [ "Svi3-3 represents the first structure of a phage-encoded SAM degrading enzyme .", "There is no previously established reaction mechanism for these enzymes , and to our knowledge there are only two previous examples of enzymes that can cleave a trialkyl sulfonium substrate .", "A distinct type of SAM lyase , 1-aminocyclopropane-1-carboxylate synthase ( ACC synthase , EC 4 . 4 . 1 . 14 ) exists in higher plants and some fungi .", "The products of this enzyme are MTA and 1-aminocyclopropane-1-carboxylate that is used in the biosynthetic pathway for ethylene .", "The reaction is PLP-dependent and the enzyme is structurally and mechanistically unrelated to the phage encoded SAM lyases ( Capitani et al . , 1999 ) .", "Instead , Svi3-3 shows structural similarity to PII and PII-like proteins , many of which bind nucleotides or nucleotide-derived metabolites in the inter-subunit clefts .", "Despite very low levels of sequence identity within those families , they have been suggested to have arisen by divergent evolution from a common ancestor ( Forchhammer and Lüddecke , 2016 ) .", "Based on this similarity , we tested binding of ATP , ADP , and AMP to Svi3-3 by DSF , but detected no interaction .", "Still , these two families of enzymes may have a distant evolutionary relationship .", "Svi3-3 forms a trimer in solution both in the absence and in the presence of substrate ( Figure 3—figure supplement 1 ) .", "Thermal shift binding experiments showed a major stabilization of Svi3-3 upon binding of MTA or SAH ( Figure 4B ) .", "Comparisons of the apo and ligand-bound structures ( Figure 3 ) as well as MD simulations ( Figure 6B ) indicate that stabilization is caused by tightening of the trimer around the ligand .", "Results presented here show that E69 in Svi3-3 plays a critical role in the enzyme , and the failure in getting crystals of an E69Q mutant with SAM suggests that binding of SAM may be associated with conformational changes that are incompatible with crystal packing or with the crystallization conditions .", "The observation that the E69Q mutant binds SAM but does not bind SAH indicates that although SAH only lacks one methyl group compared to SAM , the complex structure of WT Svi3-3 with SAH may not fully mimic the substrate-bound state .", "Since MTA and SAH show a perfect overlap between the two structures ( Figure 2D ) , any such difference in binding mode between SAM and SAH is likely to involve the methionine end of the substrate , where MD indicates that Tyr58 forms a cation-π interaction with the positively charged sulfur .", "The reason why the E69Q mutant does not bind to SAH may be that two important interactions are lacking; Q69 cannot accept two hydrogen bonds ( Figure 2C ) and there is no positive charge on the sulfur that can participate in the cation-π interaction with Tyr58 ( Figure 6—figure supplement 2 ) .", "Prompted by DFT calculations producing high energy barriers for a hydrolysis reaction within the active site of Svi3-3 ( Figure 7B ) , the TLC assays and NMR show unambiguously that Svi3-3 is a SAM lyase forming homoserine lactone and MTA ( Figure 9 ) .", "The lactone is spontaneously converted to homoserine in solution ( Wu et al . , 1983; Figure 9—figure supplement 3 , Figure 10 ) .", "Previous attempts to set up a coupled SAM hydrolase activity assay for Svi3-3 using homoserine dehydrogenase or homoserine kinase failed to show homoserine production with the same rate as MTA production ( data not shown ) .", "The observed rates were 1000-fold lower , and we can now explain that this is due to the slow and un-catalyzed formation of homoserine from the homoserine lactone that is enzymatically formed .", "Computational work by others suggests that the non-enzymatic degradation of SAM to homoserine lactone and MTA is slowed down by the favorable interactions of the carboxylate group with water ( Lankau et al . , 2017 ) .", "Thus , Svi3-3 increases the reactivity of the carboxylate group by excluding water from the corresponding part of the active site , while stabilizing a reactive conformation of the substrate .", "For this unimolecular reaction mechanism ( Figure 10A ) , only very few strictly conserved residues are required , as illustrated in the multiple-sequence alignment ( Figure 5 ) .", "Both hydrogen bond acceptors of Glu69 seem critical for stabilization of the reactive state ( Figure 10 ) , and Svi3-3 E69Q has nearly abolished activity .", "However , the effect of the corresponding mutation in T3 SAMase is not quite as dramatic .", "Since the level of sequence identity between the two enzymes is low , there will be many differences in the active site , and additional interactions may contribute to stabilizing the reactive state for the same mechanism in T3 SAMase .", "The only previously partly characterized phage SAMase comes from phage T3 ( Hausmann , 1967 ) .", "It was identified based on its anti-restriction activity , but the mechanism was never fully clarified .", "Anti-restriction activity of the closely related T7 phage is based on the OCR protein that forms a structure that mimics B-form DNA and blocks DNA binding of EcoKI and other type I RM systems ( Walkinshaw et al . , 2002 ) .", "The first structure from the SAM lyase enzyme family that we present here clearly proves that they have no structural similarity to the OCR protein .", "Instead , our data shows that also the T3 SAMase is a lyase and not a hydrolase , but future studies are needed to elucidate whether these enzymes also have additional mechanisms of anti-restriction activity ( Spoerel et al . , 1979 ) .", "Around the same time as the SAM-degrading enzyme from bacteriophage T3 was discovered , the same enzymatic activity was also found in extract from bacteria ( Shapiro and Mather , 1958 ) and yeast ( Mudd , 1959a; Mudd , 1959b ) .", "In both of these systems , the reaction products were described as MTA and γ-aminobutyro-lactone ( homoserine lactone ) , and the conversion to homoserine was considered to be spontaneous .", "In contrast , the phage enzyme from T3 was early described as a SAM hydrolase , and referred to as such until this day .", "In the early literature , homoserine lactone was identified as an intermediate ( Gold et al . , 1964 ) but , perhaps due to the available methods at the time , it was not realized that homoserine was formed on a different time-scale from MTA , indicating a spontaneous and not enzyme-catalyzed reaction .", "For this reason , it is not until now that we can correct the functional annotation to SAM lyase .", "The SAM lyases show very low sequence conservation and large variations in size ( Figure 5 ) , and future studies will elucidate the relationship between structure and activity in this family of enzymes , their prevalence , and their exact biological roles in different organisms ." ], [ "For expression of an N-terminally truncated construct of Svi3-3 , the svi3-3 gene was PCR amplified using Pfu DNA polymerase with primers Svi3-3_d19f and Svi3-3_r1 and cloned into the pEXP5-NT/TOPO vector ( Invitrogen ) according to the manufacturer's protocol .", "Transformants were selected on LA plates supplemented with 100 μg/ml ampicillin .", "The correctness of plasmid pEXP5-Svi3-3_d19 was confirmed by sequencing ( Eurofins ) .", "The resulting plasmid encoded amino acid 20–162 of the original His-tagged Svi3-3 polypeptide ( corresponding to residues 5–147 from the phage-encoded sequence ) fused to an N-terminal hexahistidine tag followed by a TEV cleavage site .", "The sequence is numbered starting at the N terminus of the TEV-cleaved protein sequence , corresponding to an offset of −16 in relation to previous work ( Jerlström Hultqvist et al . , 2018 ) .", "Svi3-3-d19 mutants Y58F , E69Q , E69A , and E105Q were generated by site-directed mutagenesis of pEXP5-Svi3-3_d19 using the QuickChange II protocol ( Stratagene ) using the primers listed in Supplementary file 1-table 3 .", "Mutations in the plasmids pEXP5-Svi3-3_d19_Y58F , pEXP5-Svi3-3_d19_E69Q , pEXP5-Svi3-3_d19_E69A , and pEXP5-Svi3-3_d19_E105Q were confirmed by DNA sequencing .", "Expression plasmids were transformed into BL21-AI cells and plated on LA plates containing 50 μg/ml ampicillin and 0 . 1% glucose .", "For protein expression , 5 ml overnight culture in LB containing 50 μg/ml ampicillin and 0 . 1% glucose was used to inoculate 800 ml LB medium with the same composition and incubated at 37°C with shaking .", "When OD600 reached 0 . 9 , expression was induced with 0 . 2% L-arabinose and the culture was further incubated at 37°C for 4 hr before harvest by centrifugation .", "The cell pellet was resuspended in buffer A ( 50 mM Tris-HCl pH 7 . 5 , 300 mM NaCl , 20 mM imidazole , 5 mM BME ) including cOmplete EDTA-free protease inhibitor ( Roche ) and subjected to lysis by sonication .", "After centrifugation at 30 , 000 × g for 30 min , the supernatant lysate was clarified by filtration through a 0 . 45 μm syringe filter , loaded to a gravity column containing pre-equilibrated Ni Sepharose 6 Fast Flow ( GE Healthcare ) and incubated under slow rotation for 10 min at 4°C .", "The column was washed extensively with buffer A supplemented with 20 mM imidazole , and the His-tagged protein was eluted with buffer A containing 500 mM imidazole .", "Protein-containing fractions were loaded onto a HiLoad 16/60 Superdex 75 pg column equilibrated with buffer B ( 25 mM Tris-HCl , 150 mM NaCl , pH 8 . 0 ) .", "WT Svi3-3_d19 , E69Q , E69A , and E105Q mutants eluted as trimers , while the Y58F mutant eluted mainly as monomer .", "Peak fractions were pooled and concentrated to 2 mg/ml .", "To cleave off the His-tag , the protein was incubated at 4°C overnight with a 1:10 molar ratio of TEVSH protease ( van den Berg et al . , 2006 ) .", "The cleavage reaction was passed through Ni-Sepharose before being loaded onto a HiLoad 16/60 Superdex 75 pg column equilibrated in buffer B . Peak fractions were concentrated to 10 mg/ml for further use .", "T3 SAMase was produced by in vitro transcription–translation as previously described ( Jerlström Hultqvist et al . , 2018 ) .", "Crystallization was done using the sitting-drop vapor diffusion method at room temperature ( 293 K ) .", "Crystals grew in 2–10 days in drops containing 1 μl Svi3-3_d19 ( 10 mg/ml , with or without 5 mM SAH/SAM ) and 1 μl of reservoir solution containing 0 . 4–0 . 6 M ammonium phosphate .", "Crystals were cryo-protected in reservoir solution supplemented with 1 . 5 M proline and vitrified in liquid nitrogen for data collection .", "All data were collected at ESRF beamline ID23-1 at 100 K and processed with XDS ( Kabsch , 2010 ) .", "The Svi3-3_d19 structure with SAM was solved with ab initio methods using Arcimboldo_lite ( Rodríguez et al . , 2009 ) run on the National Supercomputer Center ( NSC ) in Linköping and a 15 amino acid helix as search model ( McCoy et al . , 2007 ) .", "The structure was manually rebuilt in Coot ( Emsley et al . , 2010 ) and refined using phenix . refine ( Afonine et al . , 2012 ) .", "Statistics for data collection and refinement are summarized in Table 1 .", "All structure figures were prepared using PyMol ( Schrödinger LLC , 2020 ) .", "SEC-SAXS data for Svi3-3_d19 samples were collected at the Diamond Light Source on beamline B21 .", "In-line SEC-SAXS was performed using an Agilent 1200 HPLC system connected Shodex KW403 column .", "Data were recorded on a Pilatus 2M detector with a fixed camera length of 4 . 014 m and 12 . 4 keV energy allowing the collection of the angular range q between 0 . 0038 and 0 . 42 Å−1 .", "His6-tagged Svi3-3_d19 samples at 10–13 mg/ml concentration with and without 5 mM SAM were loaded onto the size exclusion chromatography ( SEC ) column previously equilibrated in 25 mM Tris-HCl pH 8 . 0 , 150 mM NaCl .", "Initial buffer subtraction and data processing was performed using ScÅtter ( Förster et al . , 2010 ) .", "Further data analysis was performed with Primus ( Konarev et al . , 2003 ) and SAXSMoW ( Piiadov et al . , 2019 ) .", "The activity of WT and mutant versions of the N-terminally truncated Svi3-3 construct was determined according to the previously published discontinuous assay ( Jerlström Hultqvist et al . , 2018 ) , by separation of SAM and MTA using cation exchange chromatography .", "All experiments were conducted at 25°C and the enzyme concentration and duration of the experiment were adjusted to the level of activity , ranging from 50 to 500 nM enzyme and 10 min to 1 week incubation .", "The experiments were done in biological duplicates ( two separately purified batches of each protein ) and technical duplicates ( two independently pipetted and measured enzymatic reactions ) .", "The protocol was adopted from Niesen et al . , 2007 .", "Each 25 µl reaction consisted of 20 µM WT or mutant Svi3-3 in 25 mM HEPES ( 4–2-hydroxyethyl- 1-piperazineethanesulfonic acid ) , 150 mM NaCl , 0 . 2 µl 50× SYPRO orange dye and 0–2500 µM of SAM or SAH or 0–600 µM dcSAM mix .", "Reactions were done in technical triplicates ( for dcSAM technical duplicates ) in a BioRad CFx connect real-time system and subjected to a temperature gradient from 15°C to 95°C with an increment of 0 . 2°C per 30 s .", "Structure-guided sequence alignment was performed with PROMALS3D ( Pei et al . , 2008 ) and manually edited .", "The sequence alignment figure was prepared using ESPript ( Gouet et al . , 2003 ) .", "The different gene variants encoding the WT and mutant T3 SAMase were inserted on the chromosome of an ΔilvA auxotrophic mutant of E . coli K-12 MG1655 by λ-red recombineering as previously described ( Datsenko and Wanner , 2000; Koskiniemi et al . , 2011 ) .", "Each of the variants was used to replace the araBAD operon , so that the expression for these was under the control of the pBAD , the native promoter for the araBAD operon .", "Briefly , the first step involved replacing the araBAD operon in an ΔilvA E . coli K-12 MG1655 strain by cat-sacB-yfp cassette .", "The cassette was PCR amplified using the primers araBAD_cat_sacB_F and araBAD_cat_sacB_R ( Supplementary file 1-table 3 ) .", "Native T3 SAMase and variants with mutations at E67Q and E68Q were PCR amplified using specific primers that contained homologies surrounding the araBAD operon at the 5’ end followed by sequences that allowed amplification of the T3 SAMase variants from the respective plasmids ( ara_t3samF , ara_t3samR ) .", "PCR products were purified , DNA was transformed into the strain containing the cat-sacB-yfp cassette at the araBAD location , and transformants were selected on sucrose plates .", "Variants were confirmed by Sanger sequencing ( using test_primer_F , test_primer_R , Supplementary file 1-table 3 ) .", "Two different approaches were used to determine if the different variants of Svi3-3 , Orf1 , and T3 SAMase could complement the ΔilvA auxotrophic mutant .", "In cases where the variant was present on a plasmid , the plasmid was transformed into the ΔilvA auxotrophic mutant and was selected on LA-ampicillin ( 50 μg/ml ) or LA-chloramphenicol ( 15 μg/ml ) plates .", "Svi-3–3 and Orf1 were both cloned on a high copy number plasmid pCA24N –gfp , while T3 SAM hydrolase was cloned on an intermediate copy number plasmid pRD2 .", "In each case the genes were placed under the IPTG-inducible promoter PLlacO .", "The transformants were then re-streaked , and the re-streaked colonies were tested for growth on M9-Glucose ( 0 . 2% ) minimal media plates , with or without IPTG .", "On each test plate , the ΔilvA auxotrophic mutant containing the empty vector was used as a negative control .", "The same approach was used to test functionality of the T3 SAMase variants that were present on the chromosome , with induction being obtained using different concentrations of L-arabinose ( 0% , 0 . 01% , 0 . 05% , and 0 . 1% ) .", "E69A and E69Q variants were constructed for full-length Svi3-3 and Svi3-3_d19 to test for their ability to rescue the ΔilvA mutant ( Jerlström Hultqvist et al . , 2018 ) .", "Synthetic genes ( Eurofins ) containing the desired mutations and Kpn1 and Xba1 cleavage sites were cleaved-out of the original vector and ligated into a modified version of the pCA24N plasmid , purified on a spin-column and transformed directly into the ΔilvA mutant .", "Plasmids were extracted from isolated colonies and sequenced to confirm the correct sequence .", "The respective clones were then checked for their ability to grow on minimal glucose plates .", "All in vivo complementation experiments were done in biological duplicates .", "MD simulations of the Svi3-3 trimer with and without SAM in the active sites were performed with Desmond ( Bowers et al . , 2006; Schrödinger , 2018 ) using the OPLS3 force field ( Harder et al . , 2016; Jorgensen et al . , 1996; Jorgensen and Tirado-Rives , 1988; Shivakumar et al . , 2010 ) .", "The Svi3-3 crystal structure in complex with SAH was used as starting conformation for the simulations where missing hydrogens were automatically added using the protein preparation wizard tool in Maestro ( Sastry et al . , 2013 ) .", "The native substrate SAM was docked to the active site using Glide ( Friesner et al . , 2006; Friesner et al . , 2004; Halgren et al . , 2004; Schrödinger , 2018 ) .", "The docking grid was generated with the OPLS3 force field centered on the SAH inhibitor bound to the active site of the Svi3-3 crystal structure .", "Due to an error in our library file , the force field parameters of the base were taken from tubercidin instead of adenine , but the charge distributions of these two bases are almost identical .", "The Maestro system builder ( Schrödinger , n . d . ) was used to solvate the Svi3-3 trimer with TIP3P ( Jorgensen et al . , 1983 ) water molecules in an orthorhombic box with buffer distances of 10 Å to the boundary on all sides .", "The system was neutralized by addition of Na+ ions and the final simulation box consisted of 60 , 972 atoms .", "A total of 100 ns MD simulation at 298 K was run in the NPT ensemble using the reference system propagator algorithm ( RESPA ) time stepping scheme ( Tuckerman et al . , 1991 ) with time steps of 2 fs for bonded terms , 2 fs for van der Waals and short-range electrostatic interactions , and 6 fs for long-range electrostatic interactions .", "Short-range Coulomb interactions were treated with a cutoff radius of 9 Å .", "Long-range interactions were treated with the smooth Particle Mesh Ewald method ( Darden et al . , 1993 ) with a tolerance of 10–9 .", "The NPT ensemble was calculated with the Nose-Hoover chain thermostat method ( Hoover , 1985; Nosé , 1984 ) , using a relaxation time of 1 ps , and the Martyna-Tobias-Klein barostat method ( Martyna et al . , 1994 ) , using isotropic coupling with a relaxation time of 2 ps .", "The catalytic mechanism of Svi3-3 was investigated with DFT calculations .", "A cluster model ( Figure 6—figure supplement 2B ) was generated from a snapshot of the equilibrated Svi3-3 X-ray structure taken from the MD simulation described above ( Figure 6—figure supplement 2A ) .", "This snapshot is a typical representative displaying the key interactions shown in Figure 6—figure supplement 1 .", "The active site model was composed of the backbone atoms of Val17 , Gly18 , Leu19 , Asn20 , and Val21 chopped at the N and C termini , Tyr58 and Glu69 chopped at the CA position , and Gln104 , Glu105 , and Ser106 ( with backbone ) chopped at the N and C-termini .", "A smaller substrate mimicking SAM was used for the DFT calculations .", "Here , adenosine was deleted from SAM , resulting in 2-ammonio-4- ( ( R ) -ethyl ( methyl ) sulfonio ) butanoate , or S-ethylmethionine ( SEM ) .", "In addition , a total of six water molecules from the MD snapshot were included in the cluster model , which includes the first two solvation shells of the reaction center .", "To account for the steric effect of the surrounding parts of the protein , atoms in the chopped positions were kept fixed to their original positions ( Figure 6—figure supplement 2 ) .", "The final model after addition of methyl groups to the chopped protein positions consisted of 194 atoms ( 716 electrons ) .", "In general , cluster models comprising ~200 atoms have been found to be sufficiently reliable for mechanistic investigations , provided they include all key protein residues involved in the reaction , and have also been shown to be relatively insensitive to dielectric effects ( Siegbahn and Himo , 2009 ) .", "However , as noted above , ion-pair configurations with charge separation over a large distance may be disfavored , and we thus also recalculated the reaction energetics with the system constrained to the standard protonation state of Tyr58 and Glu105 , which was also the initial configuration for the optimization .", "All DFT calculations were performed using the Gaussian 09 ( Frisch et al . , 2009 ) package .", "Geometry optimizations and frequency calculations were computed with the B3LYP functional ( Becke , 1993 ) and the 6-31G ( d , p ) basis set .", "Dispersion effects were included in all calculations using Grimme’s B3LYP-D3 method ( Grimme et al . , 2011; Grimme et al . , 2010 ) .", "Intrinsic reaction coordinate calculations were performed in both directions from the transition state to verify that the correct minima are connected .", "Solvent effects were obtained by single-point calculations on the optimized stationary points with the solvent model based on density ( SMD ) ( Marenich et al . , 2009 ) .", "Electronic energies were calculated from single-point calculations on the optimized geometries ( RS , TS , and PS ) at the b3lyp/6-311G+ ( 2d , 2p ) level of theory .", "The final reported energies are the electronic energies with the large basis set corrected for zero-point energies ( ZPE ) and solvent effects in kcal/mol .", "E . coli K-12 MG1655 pCA24N:speD from the ASKA library ( Kitagawa et al . , 2005 ) encoding the hexahistidine tagged SAM decarboxylase ( SDC ) enzyme were inoculated in LB containing 34 µg/ml chloramphenicol .", "Five milliliters of saturated culture was used to inoculate 800 ml LB medium with the same composition and incubated at 37°C until OD600 reached 0 . 6 .", "Expression was induced with 0 . 5 mM IPTG and the culture was incubated overnight at 20°C .", "The cells were harvested by centrifugation at 4°C .", "The pellet was washed in 25 mM Tris pH 8 , 150 mM NaCl , and pelleted again at 8°C .", "The cells were resuspended in buffer A ( 50 mM Tris-HCl pH 8 , 300 mM NaCl , 10 mM MgSO4 , 5 mM BME ) including cOmplete EDTA-free protease inhibitor ( Roche ) and subjected to lysis by sonication .", "After centrifugation at 30 , 000 × g for 30 min , the supernatant was clarified by filtration through a 0 . 45-µm-syringe filter , loaded in a gravity column containing 2 ml of pre-equilibrated Ni-sepharose ( GE Healthcare ) , and incubated under slow rotation for 30 min at 8°C .", "The column was washed extensively with buffer A supplemented with 20 mM imidazole , and the His-tagged protein was eluted with buffer A containing 500 mM imidazole .", "Protein-containing fractions were loaded onto a HiLoad 16/60 Superdex 200 column previously equilibrated with buffer B ( 25 mM Tris-HCl pH 8 , 150 mM NaCl , 10 mM MgSO4 , 5 mM BME ) .", "Peak fractions were pooled and concentrated to 5 mg/ml .", "0 . 5 or 2 mM SAM was incubated with 20 µM SDC for 2 hr at 37°C in reaction buffer ( 20 mM HEPES pH 7 , 50 mM KCl , 10 mM MgSO4 ) .", "After the incubation , 40 µl of the reaction was quenched with the same volume of quenching buffer ( 50 mM citrate pH 2 . 6 ) .", "The rest of the reaction was filtered through a 3 kDa cutoff concentrator to remove the enzyme .", "The flow-through was collected and the concentration dcSAM + SAM + MTA was determined by the absorbance at 260 nm to 0 . 32 mM and 1 . 1 mM .", "The fraction of dcSAM was determined using ion exchange chromatography to 77% in the lower-concentration reaction and 72% in the higher-concentration reaction .", "0 . 32 mM dcSAM mix ( 77% dcSAM ) was incubated with 0 . 1 µM Svi3-3 in reaction buffer at 37°C .", "As a control , dcSAM without Svi3-3 was incubated for the same time .", "SAM ( 0 . 32 mM ) was incubated with 0 . 1 µM Svi3-3 and as a control only SAM ( 0 . 32 mM ) was incubated for the same period of time .", "Samples of 40 µl were quenched with the same volume of quenching buffer after 0 . 5 and 23 hr .", "Samples were analyzed with the same cation exchange method as for the standard activity assay ( Jerlström Hultqvist et al . , 2018 ) but using a linear buffer gradient over seven column volumes .", "For TLC experiments , reactions containing 2 mM SAM and 0 . 20 μM Svi3-3 , 0 . 20 μM Orf1 , or 0 . 25 μM T3 SAMase in 50 mM NaPi pH 7 . 4 were incubated for 20 min at 25°C .", "MTA product formation of >70% was verified using ion exchange chromatography .", "5 × 2 μl reaction was loaded on a TLC Silica gel 60 F254 plate and developed using a mobile phase of 55% n-butanol , 30% H2O , 15% acetic acid .", "The plate was treated with BME followed by ninhydrin staining ( Basak et al . , 2005 ) .", "Briefly , the dried plate was sprayed with 1% BME in acetone , heated with a hair dryer , sprayed with 0 . 25% ninhydrin in acetone , and heated again until spots were clearly visible .", "To analyze the reaction products by NMR spectroscopy , a 700 μl reaction mixture was prepared containing 4 mM SAM and 500 nM Svi3-3 d19 in 100 mM Na phosphate buffer pH 7 . 4% and 90% D2O .", "The pH of the sample was adjusted to 7 . 4 after addition of SAM with 5M NaOH .", "The reaction was incubated for 20 min at 25°C and shock-frozen in liquid N2 and stored at −80°C until measurement .", "Turnover of >90% of substrate was verified by ion exchange chromatography .", "1H NMR spectra were recorded at 600 . 18 MHz on a Bruker Avance Neo spectrometer equipped with a TCI ( CRPHe TR-1H and 19F/13C/15N 5mm-EZ ) cryogenic probe for samples in aqueous sodium phosphate buffer ( NaPi , 100 mM , pH 7 . 4 , solvent D2O ) at 25°C .", "Typically 64–128 scans were accumulated with a relaxation delay of 0 . 7 s and an acquisition time of 2 . 75 s , using the zg30 pulse sequence .", "Spectra were obtained by zero filling the recorded 32 k data points to 128 k , followed by multiplication with an exponential weighting function and Fourier transformation .", "The formation of homoserine lactone in the enzymatic reaction was confirmed by comparison with the 1H NMR spectrum of an authentic sample ( from Sigma ) dissolved in the same buffer , and by comparison with literature data ( Helms et al . , 1988 ) .", "Homoserine lactone spectra were also recorded for various concentrations ( 70 mM , 17 . 5 mM , 4 . 4 mM , 1 . 1 mM , and 0 . 3 mM ) and for various times after sample preparation ( Figure 9—figure supplement 3 ) .", "The spectra indicated a gradual hydrolysis of the lactone to homoserine that was identified by comparison with literature 1H NMR data ( Jamieson et al . , 2009 ) and an authentic homoserine sample ( Figure 9—figure supplement 2 ) ." ] ]

[ "The first S-adenosyl methionine ( SAM ) degrading enzyme ( SAMase ) was discovered in bacteriophage T3 , as a counter-defense against the bacterial restriction-modification system , and annotated as a SAM hydrolase forming 5’-methyl-thioadenosine ( MTA ) and L-homoserine .", "From environmental phages , we recently discovered three SAMases with barely detectable sequence similarity to T3 SAMase and without homology to proteins of known structure .", "Here , we present the very first phage SAMase structures , in complex with a substrate analogue and the product MTA .", "The structure shows a trimer of alpha–beta sandwiches similar to the GlnB-like superfamily , with active sites formed at the trimer interfaces .", "Quantum-mechanical calculations , thin-layer chromatography , and nuclear magnetic resonance spectroscopy demonstrate that this family of enzymes are not hydrolases but lyases forming MTA and L-homoserine lactone in a unimolecular reaction mechanism .", "Sequence analysis and in vitro and in vivo mutagenesis support that T3 SAMase belongs to the same structural family and utilizes the same reaction mechanism ." ]

[ "Bacteria can be infected by viruses known as bacteriophages .", "These viruses inject their genetic material into bacterial cells and use the bacteria’s own machinery to build the proteins they need to survive and infect other cells .", "To protect themselves , bacteria produce a molecule called S-adenosyl methionine , or SAM for short , which deposits marks on the bacteria’s DNA .", "These marks help the bacteria distinguish their own genetic material from the genetic material of foreign invaders: any DNA not bearing the mark from SAM will be immediately broken down by the bacterial cell .", "This system helps to block many types of bacteriophage infections , but not all .", "Some bacteriophages carry genes that code for enzymes called SAMases , which can break down SAM , switching off the bacteria’s defenses .", "The most well-known SAMase was first discovered in the 1960s in a bacteriophage called T3 .", "Chemical studies of this SAMase suggested that it works as a 'hydrolase' , meaning that it uses water to break SAM apart .", "New SAMases have since been discovered in bacteriophages from environmental water samples , which , despite being able to degrade SAM , are genetically dissimilar to one another and the SAMase in T3 .", "This brings into question whether these enzymes all use the same mechanism to break SAM down .", "To gain a better understanding of how these SAMases work , Guo , Söderholm , Kanchugal , Isaksen et al . solved the crystal structure of one of the newly discovered enzymes called Svi3-3 .", "This revealed three copies of the Svi3-3 enzyme join together to form a unit that SAM binds to at the border between two of the enzymes .", "Computer simulations of this structure suggested that Svi3-3 holds SAM in a position where it cannot interact with water , and that once in the grip of the SAMase , SAM instead reacts with itself and splits into two .", "Experiments confirmed these predictions for Svi3-3 and the other tested SAMases .", "Furthermore , the SAMase from bacteriophage T3 was also found to degrade SAM using the same mechanism .", "This shows that this group of SAMases are not hydrolases as originally thought , but in fact ‘lyases’: enzymes that break molecules apart without using water .", "These findings form a starting point for further investigations into how SAM lyases help bacteriophages evade detection .", "SAM has various different functions in other living organisms , and these lyases could be used to modulate the levels of SAM in future studies investigating its role ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "evolutionary biology", "microbiology and infectious disease" ]

[ [ "Mammalian genomes combat the persistent threat of viruses by encoding a battery of cell-intrinsic antiviral proteins , termed restriction factors , that recognize and inhibit viral replication within host cells .", "The potency of restriction factors places selective pressure on viruses to evade recognition in order to complete replication ( Duggal and Emerman , 2012 ) .", "In turn , viral escape spurs adaptation of restriction factors , by selecting for variants that re-establish viral recognition and thereby restriction ( McCarthy et al . , 2015 ) .", "Mutual antagonism between viruses and their hosts thus drives cycles of recurrent adaptation , in prey-predator-like genetic arms races ( Van Valen , 1973 ) .", "These arms races result in the rapid evolution of restriction factors , which accumulate amino acid mutations at their virus-binding interfaces at a higher than expected rate ( Daugherty and Malik , 2012 ) .", "Numerous restriction factors , including TRIM5α ( Sawyer et al . , 2005 ) , APOBEC3G ( Sawyer et al . , 2004 ) , and MxA ( Mitchell et al . , 2012 ) , evolve rapidly as a result of arms races with target viruses .", "The resulting divergence between restriction factor orthologs can result in cross-species barriers to viral infection ( Compton and Emerman , 2013; Kirmaier et al . , 2010 ) .", "Such barriers led to the initial identification of TRIM5α , during a screen for proteins that prevented HIV-1 ( human immunodeficiency virus ) from efficiently replicating in rhesus macaque cells ( Stremlau et al . , 2004 ) .", "Rhesus TRIM5α could potently restrict HIV-1 , whereas the virus almost completely escapes TRIM5α-mediated inhibition in its human host .", "Subsequent studies revealed that restriction of SIVs ( simian immunodeficiency viruses ) also varies across TRIM5α orthologs and that SIVs likely drove the rapid evolution of TRIM5α in Old World monkeys ( McCarthy et al . , 2015; Wu et al . , 2013 ) .", "TRIM5α disrupts retroviral replication early in infection by binding to the capsid core of retroviruses entering the cell ( Li et al . , 2016; Maillard et al . , 2007; Owens et al . , 2003 ) .", "Its binding causes the premature uncoating of the viral core ( Stremlau et al . , 2006 ) , preventing delivery of the viral genome to the nucleus for integration .", "TRIM5α binds to the capsid via the unstructured v1 loop within its B30 . 2 domain ( Biris et al . , 2012; Sebastian and Luban , 2005 ) .", "Experiments swapping the v1 loop between TRIM5α orthologs indicated that it is critical for recognition of capsid from many retroviruses ( Ohkura et al . , 2006; Perron et al . , 2006; Sawyer et al . , 2005 ) .", "In Old World monkeys and hominoids , rapid evolution of TRIM5α is concentrated within this v1 loop ( Sawyer et al . , 2005; Figure 1A ) .", "Single amino acid mutations at these rapidly evolving sites can cause dramatic gains of restriction against HIV-1 and other retroviruses ( Li et al . , 2006; Maillard et al . , 2007; Yap et al . , 2005 ) .", "However , it remains unclear whether such adaptive mutations are rare among all single mutational steps that might be randomly sampled during TRIM5α’s natural evolution .", "The functional consequence of all single mutations from a given protein sequence can be visualized as an evolutionary landscape ( Smith , 1970 ) , in which mutations are either beneficial ( fitness peak ) , detrimental ( fitness valley ) , or neutral .", "The topology of this evolutionary landscape , in terms of numbers of peaks and valleys and their connections , represents the adaptive potential of restriction factors in their evolutionary arms race with viruses .", "Previous studies that have empirically mapped evolutionary landscapes of conserved enzymes and transcription factors revealed that ligand-binding residues are highly intolerant to substitutions ( Fowler et al . , 2010; Guo et al . , 2004; McLaughlin et al . , 2012; Suckow et al . , 1996 ) .", "Moreover , mutations that allowed proteins to gain novel ligand specificity , even for closely related ligands , were rare among all possible substitutions ( McLaughlin et al . , 2012; Starr et al . , 2017; Stiffler et al . , 2015 ) .", "In contrast , TRIM5α and other restriction factors can dramatically change antiviral potency via single mutations at viral interaction interfaces ( Daugherty and Malik , 2012; Mitchell et al . , 2012 ) .", "However , since the frequency of such gain-of-function mutations is unknown , it is unclear whether virus-binding surfaces in rapidly evolving antiviral factors are subject to the same evolutionary constraints as previously mapped for other proteins .", "Here , we investigated the adaptive landscape of antiviral specificity conferred by the rapidly evolving , capsid-binding v1 loop of TRIM5α .", "To our surprise , we found that , rather than the evolutionary landscape of TRIM5α being narrowly constrained among all possible amino acid substitutions , the majority of random mutations in the v1 loop resulted in gains of antiviral restriction .", "We found that the primary v1 loop determinant for TRIM5α’s restriction of HIV-1 and other lentiviruses is its net electrostatic charge .", "Furthermore , both rhesus and human TRIM5α proteins are highly resilient to mutation , in that they withstand more than half of all possible single amino acid mutations in the v1 loop without compromising their antiviral restriction abilities .", "This unexpectedly permissive landscape allows TRIM5α to sample a wide variety of mutations to maximize its chances of success in arms races with retroviruses ." ], [ "Despite their rapid evolution , primate TRIM5α orthologs have sampled relatively limited amino acid diversity at rapidly evolving positions within the capsid-binding v1 loop ( Figure 1A ) .", "For example , although single amino acid changes at residue 332 are responsible for dramatic differences in antiviral restriction ( Li et al . , 2006 ) , this residue repeatedly toggles between just three amino acids .", "The limited diversity is not due to evolutionary inaccessibility , since most amino acids that can be sampled with single nucleotide changes are not observed among primate TRIM5α orthologs ( Figure 1B ) .", "There are two alternative explanations for this restricted diversity .", "First , it might suggest that adaptive gain-of-function mutations in TRIM5α are rare , with TRIM5α’s evolutionary landscape mainly consisting of fitness valleys with only a few mutational avenues to reach fitness peaks ( Figure 1C ) .", "Conversely , the limited diversity might be a consequence of epistatic interactions with other sites that constrain amino acid sampling , or even simple chance .", "Under this scenario , TRIM5α’s evolutionary landscape may consist of numerous , wide peaks that tolerate substantial mutational variation ( Figure 1D ) .", "We sought to differentiate between these possibilities by experimentally defining the evolutionary landscape of antiviral restriction over all possible single mutational steps in the v1 loop of both human and rhesus TRIM5α .", "We took a deep mutational scanning ( DMS ) approach ( Fowler et al . , 2010 ) to measure the effect on antiviral restriction of all v1 loop single mutations in a pooled assay .", "We first generated a library of all single amino acid variants ( including stop codons ) within the rapidly evolving portion of the v1 loop ( amino acids 330 to 340 , Figure 1A ) , with a library diversity of 231 amino acid ( 352 nucleotide ) variants ( Figure 2A ) .", "The resulting TRIM5α variants were stably expressed via transduction into CRFK ( cat renal fibroblast ) cells , which naturally lack TRIM5α ( McEwan et al . , 2009 ) .", "We transduced CRFK cells at a low dose to limit the integration of multiple variants into individual cells , thus generating a pool of cells each expressing a single TRIM5α point mutant .", "Libraries were represented with at least 500-fold coverage through all experimental steps to avoid bottlenecking library diversity .", "Human TRIM5α only weakly restricts HIV-1 ( Jimenez-Guardeño et al . , 2019; OhAinle et al . , 2018 ) .", "However , single amino acid mutations in the v1 loop can substantially increase restriction activity ( Li et al . , 2006; Pham et al . , 2010; Pham et al . , 2013 ) .", "To comprehensively assess how many single mutation variants of human TRIM5α had increased activity against HIV-1 , we first performed a gain-of-function screen .", "We challenged the library of human TRIM5α variant-expressing cells with HIV-1 bearing a GFP reporter , at a dose infecting 98% of cells ( Figure 2B ) .", "Because GFP expression becomes detectable only after integration of the HIV-1 proviral genome , cells expressing TRIM5α variants that restrict HIV-1 infection remain GFP-negative .", "However , ~2% of cells that were uninfected by chance would also be GFP-negative .", "Therefore , to enrich for cells expressing bona fide restrictive TRIM5α variants , we sorted the GFP-negative cells from the first round of infection and subjected them to a second round of HIV-1-GFP infection and sorting .", "Following this second round of selection , we deep sequenced the TRIM5α variants in the GFP-negative cell population .", "We normalized the count of each variant to its representation in the pre-selection cell population to determine its enrichment score , which should reflect the relative antiviral function of each TRIM5α variant .", "Enrichment scores were highly correlated between two independent biological replicates ( Figure 2C , Spearman r = 0 . 97 ) .", "Furthermore , mutants bearing premature stop codons , which should be non-functional and depleted from the restrictor pool , were all among the most depleted variants ( Figure 2D , red ) .", "Despite the weak ( ~2 fold ) restriction of HIV-1 by wildtype ( WT ) human TRIM5α , variants containing synonymous nucleotide changes ( no amino acid changes compared to WT , blue ) had significantly higher enrichment scores than those containing stop codons ( p<0 . 0001 , student’s unpaired t-test with Welch’s correction ) , confirming that the assay worked as expected .", "To investigate whether enrichment scores were truly representative of increased antiviral function , and to validate some of the novel amino acid changes that appeared to result in increased restriction , we made 16 targeted missense mutants from across the enrichment spectrum and challenged them individually with HIV-1-GFP .", "We determined their fold-restriction by determining the relative viral dose required to infect 10% of cells ( ID10 ) expressing a TRIM5α variant compared to an empty vector control; a larger viral dose is required to overcome TRIM5α-mediated restriction ( Figure 2E ) .", "We confirmed that several previously described gain-of-function variants ( Li et al . , 2006; Pham et al . , 2010; Pham et al . , 2013 ) had increased antiviral activity and were highly enriched ( Figure 2F: G330E , R332P , R332E , R335E ) .", "Moreover , we identified novel amino acid mutations that significantly increased antiviral activity , such as R335A and G333D , whereas moderately enriched variants ( e . g . G333Y , Q337N ) had correspondingly modest gains in HIV-1 restriction .", "Indeed , enrichment scores and fold-restriction were highly correlated across all mutants tested ( Spearman r = 0 . 90 ) .", "Thus , enrichment scores accurately reflect antiviral activity , validating our approach to simultaneously identify all single mutants with increased HIV-1 restriction .", "Therefore , in subsequent analyses , we use enrichment scores as a proxy for the antiviral restriction activity of TRIM5α mutants .", "Based on the limited amino acid diversity among primate TRIM5α v1 loops ( Figure 1A–B ) , we expected that our DMS assay would reveal only a few beneficial mutations that improve human TRIM5α restriction of HIV-1 .", "Contrary to this expectation , we found that more than half of all missense variants ( 115 , 57% ) had enrichment scores that fell more than two standard deviations above WT TRIM5α ( Figure 2D ) .", "Even if we limited our analysis to amino acid variants that are evolutionarily accessible via single-nucleotide changes from the WT TRIM5α sequence , this ratio did not change substantially ( 32 , 54% ) .", "These enrichment scores represent dramatic gains in HIV-1 restriction , with the most potent variants ( R332P and R335A ) improving HIV-1 restriction ~15 fold relative to WT ( 33- and 39-fold restriction , respectively ) .", "Our findings indicate that the fitness landscape of the v1 loop is not narrowly constrained , but rather is remarkably permissive ( Figure 1D ) , in that most single amino acid changes not seen in natural sequences enhance the ability of human TRIM5α to restrict HIV-1 .", "Thus , TRIM5α has the capacity to readily evolve antiviral potency against HIV-1 via single mutations .", "We analyzed whether a common biochemical mechanism could explain the unexpectedly high fraction of restrictive TRIM5α variants .", "We found that increased expression levels could explain some of the improvement in HIV-1 restriction , although several mutations ( G333D , G333Y ) improved restriction without increasing expression ( Figure 3—figure supplement 1A–B ) .", "In contrast , most gains in HIV-1 restriction could be completely accounted for by a reduction in the electrostatic charge of the v1 loop ( Figure 3A ) , regardless of expression level .", "Indeed , reducing the electrostatic charge always improved HIV-1 restriction , but did not always increase TRIM5α expression level ( Figure 3—figure supplement 1C–D ) .", "Among all variants tested , mutation of the positively-charged residues 332 or 335 from the WT arginine ( R ) to any amino acid except lysine ( K ) significantly improved HIV-1 restriction ( Figure 3A–B ) , consistent with previous reports on R332 variants ( Li et al . , 2006 ) .", "Mutation of uncharged sites to K or R decreased TRIM5α restriction of HIV-1 , whereas introducing a negatively charged aspartic acid ( D ) or glutamic acid ( E ) significantly increased HIV-1 restriction ( Figure 3C ) .", "Since our DMS assay tests only one mutation at a time , all mutations to D or E occur in the context of at least one proximal positively charged residue .", "Therefore , we infer that the position-independent benefit of introducing D or E derives from offsetting pre-existing positive charge in the v1 loop that is detrimental to HIV-1 restriction .", "Indeed , reducing the net charge of the v1 loop explains all the highest enrichment scores ( Figure 3D ) .", "Thus , we conclude that positive charge in the v1 loop is the dominant impediment to HIV-1 restriction by human TRIM5α .", "Removal of positive charge , however , could not explain all of the improved HIV-1 restriction we observed .", "For example , despite its strict conservation in primate TRIM5α ( Figure 1A ) , a glycine ( G ) at residue 333 compromises HIV-1 restriction .", "Mutation of G333 to most other amino acids significantly improves TRIM5α activity ( Figure 3B ) .", "We confirmed this finding for several individual variants ( Figure 2F: G333Y , G333D ) .", "We found a similar pattern for residue F339 , which is disfavored for HIV-1 restriction , albeit not to the same extent as G333 .", "Contrary to our initial expectations , there is only a weak association between rapidly evolving residues and residues whose mutation can significantly improve HIV-1 restriction: missense mutations in three of six rapidly evolving sites , versus one of five conserved sites , significantly improve HIV-1 restriction ( Figure 3B ) .", "This result suggests that conserved positions in the vicinity of rapidly evolving sites possess unexpected potential to improve antiviral potency .", "We also tested whether beneficial mutations might have additive effects on HIV-1 restriction by human TRIM5α .", "We combined several gain-of-function variants with the R332P mutation , previously described to potently restrict HIV-1 ( Yap et al . , 2005 ) .", "However , we found that no double mutants tested increased HIV-1 restriction beyond that of R332P alone ( Figure 3E ) .", "Instead , combination of one gain-of-function variant ( R335E ) with R332P resulted in loss of protein expression and HIV-1 restriction .", "Previous reports also found that most beneficial mutations are either non-additive or interfering , identifying only one combination ( R332G with R335G ) that was partially additive ( Li et al . , 2006; Pham et al . , 2010; Pham et al . , 2013 ) .", "Given that R332P is one of the strongest gain-of-function variants we identified , it remains possible that other , more modest gain-of-function variants might additively improve HIV-1 restriction .", "Nevertheless , these results suggest that single gain-of-function mutations , such as R332P , can confer most or all the increased HIV-1 restriction potential onto human TRIM5α .", "Thus , remarkably , human TRIM5α appears to be located only one mutational step away from fitness peaks in its evolutionary landscape of potential adaptation against HIV-1 .", "Finally , we investigated whether gain-of-function mutations for HIV-1 restriction also conferred protection against other lentiviruses .", "We focused on lentiviruses whose restriction is v1 loop-dependent: either the entire v1 loop ( Figure 4—figure supplement 1 ) or the R332P mutation from rhesus TRIM5α ( Stremlau et al . , 2005 ) could confer human TRIM5α with substantial antiviral function .", "In each case , WT human TRIM5α only weakly restricts these lentiviruses ( Figure 4A ) .", "However , the charge-altering mutations R332P and R335A increased restriction of all lentiviruses we tested , including HIV-2 , SIVcpz ( SIV infecting chimpanzees ) , and SIVmac ( SIV infecting rhesus macaques ) .", "Introduction of negative charge ( R332E , R335E , G330E , G333D , and G337D ) also selectively improved restriction of HIV-1 , SIVcpz , and HIV-2 but not SIVmac .", "Thus , positive charge at positions 332 and 335 appears to be generally detrimental for lentiviral restriction .", "Furthermore , although TRIM5α fitness landscapes are lentivirus-specific , many of the mutations we tested increased restriction against other lentiviruses .", "These data suggest that the evolutionary landscape for lentiviral restriction by TRIM5α is likely to be generally permissive , as it is for HIV-1 .", "Our data show that novel antiviral potency is readily attainable by single amino acid changes in human TRIM5α ( Figures 2D and 4A ) .", "However , these gains might be just as easily lost through further mutation , since rapidly evolving antiviral proteins like TRIM5α continually adapt in their arms race with viruses .", "Therefore , in order to test whether newly acquired antiviral potency is fragile or resistant to mutation , we investigated the mutational resilience of the R332P variant of human TRIM5α , which inhibits HIV-1 ~15 fold more than WT ( Figure 2F ) .", "To do so , we generated a v1 DMS library of human TRIM5α with R332P fixed in all variants .", "We challenged this pooled cell library with HIV-1-GFP , at a viral titer which human TRIM5α-R332P restricts to ~1% infection .", "In this case , we sorted and deep sequenced GFP-positive cells , so that enrichment ( relative to initial representation ) now reflects the degree to which each TRIM5α-R332P variant lost its antiviral function against HIV-1 ( Figure 4B ) .", "As expected , we observed strong enrichment of stop codons in the non-restrictor pool and good correlation between biological replicates ( Figure 4C ) .", "Addition of positive charge by mutations to K or R at most positions in the v1 loop reduced HIV-1 restriction ( Figure 4—figure supplement 2A–B ) .", "This preference against positive charge mirrors that of WT human TRIM5α , for which removal of positive charge increased HIV-1 restriction ( Figure 4—figure supplement 2C ) .", "However , we found no other consistent biochemical constraints for HIV-1 restriction by TRIM5α-R332P ( Figure 4—figure supplement 2A–B ) .", "Indeed , we found that the majority of missense variants ( 65% ) did not weaken HIV-1 restriction by the R332P variant of human TRIM5α ( Figure 4C ) .", "Thus , WT human TRIM5α is only one mutational step away from a fitness peak ( Figure 3 ) that , once achieved , also exhibits a surprising degree of resilience to mutation .", "This implies that gains of restriction by TRIM5α are not likely to be compromised by its continued adaptation .", "To determine if HIV-1 restriction is also resilient to mutation in a naturally occurring TRIM5α variant , we next assessed the likelihood that random mutations disrupt viral restriction by WT rhesus macaque TRIM5α , which strongly restricts HIV-1 in a manner that strictly requires the v1 loop ( Sawyer et al . , 2005 ) .", "Like with human TRIM5α , we constructed a library of cells each expressing a single rhesus TRIM5α variant , with each variant containing a single mutation in the v1 loop ( note that the v1 loop is slightly longer in macaques compared to humans , Figure 1A ) .", "We then challenged this pool of cells with HIV-1-GFP and sorted GFP-positive cells for subsequent deep sequencing .", "Rhesus TRIM5α variants containing premature stop codons were strongly enriched in the non-restrictor pool , whereas WT variants were significantly depleted ( Figure 5A ) .", "In contrast , half of all missense mutations ( 125 , 51% ) fell within two standard deviations of WT .", "Even missense mutations accessible by single-nucleotide changes reflected this pattern ( 40 , 55% ) .", "By re-testing individual variants , we confirmed that enrichment scores negatively correlate with antiviral potency ( Figure 5B ) .", "We tested seven variants enriched for loss-of-restriction ( more than two standard deviations above WT ) and found that six lost HIV-1 restriction .", "The seventh variant ( L337N ) was only slightly outside the two standard-deviation cut-off for enrichment and correspondingly only slightly worse than WT in terms of HIV-1 inhibition .", "Thus , enrichment in the non-restrictor pool represents bona fide loss of restriction .", "All the rhesus TRIM5α variants we report here represent novel loss-of-function mutations .", "Their loss of HIV-1 restriction cannot be explained by loss of expression or protein stability ( Figure 5C ) .", "For example , the F340D , P341I , and P341G variants were all expressed at WT levels but lost HIV-1 restriction .", "Moreover , the T344E variant retained restriction despite reduced expression levels .", "We also re-tested ten rhesus TRIM5α variants not significantly enriched for loss-of-restriction ( Figure 5B ) .", "Two variants ( P334M , G335I ) enriched one standard deviation above WT correspondingly retained only partial HIV-1 restriction relative to WT rhesus TRIM5α .", "The eight remaining variants retained HIV-1 inhibition , consistent with their lack of enrichment relative to WT .", "Based on this validation , we conclude that roughly half of all v1 loop single point mutations do not significantly reduce HIV-1 restriction by rhesus TRIM5α .", "Thus , a natural rhesus TRIM5α antiviral variant , much like the human TRIM5α-R332P variant , displays considerable mutational resilience .", "We expected that conserved residues should be less tolerant of changes than rapidly evolving sites .", "However , we found that mutations in only three of seven conserved sites , versus two of six rapidly evolving sites , led to significant loss of function ( Figure 5D ) .", "Collectively , these results indicate that rhesus TRIM5α restriction of HIV-1 is highly robust to changes within the critical v1 loop at both rapidly evolving and conserved sites .", "The biochemical preferences for HIV-1 restriction are similar but not identical between rhesus and human TRIM5α .", "In both cases , the introduction of positive charge , particularly R , weakened HIV-1 inhibition ( Figure 5D , compare to Figure 3C ) .", "In contrast , the introduction of bulky hydrophobic residues , including leucine ( L ) , phenylalanine ( F ) , and tryptophan ( W ) , significantly impaired HIV-1 restriction by rhesus TRIM5α but did not affect the potency of human TRIM5α .", "These data suggest that both universal as well as lineage-specific requirements for the v1 loop shape TRIM5α restriction of HIV-1 .", "Our findings with TRIM5α restriction of HIV-1 suggest that single mutations can readily achieve gain-of-function .", "In contrast , loss-of-function mutations are not so abundant as to make adaptation unlikely .", "Thus , the evolutionary landscape of TRIM5α appears to resemble 'rolling hills' ( Figure 1D ) rather than rare , sharp peaks ( Figure 1C ) .", "Our DMS analyses of human TRIM5α revealed unexpected ease of gaining antiviral potency against HIV-1 and potentially other lentiviruses .", "However , gains in potency against one virus might be offset by a concomitant loss of function against other viruses , as previously seen for the antiviral protein MxA ( Colón-Thillet et al . , 2019 ) .", "Such functional tradeoffs might partially explain the evolutionary constraints acting on primate TRIM5α sequences .", "To explore this possibility , we investigated the mutational resilience of N-tropic murine leukemia virus ( N-MLV ) restriction by TRIM5α .", "N-MLV is strongly inhibited by both rhesus and human TRIM5α , and this activity is at least partly dependent on the v1 loop ( Ohkura et al . , 2006; Perron et al . , 2006 ) .", "We infected cells expressing either the rhesus ( Figure 6A ) or WT human TRIM5α ( Figure 6B ) v1 DMS libraries with GFP-marked N-MLV , sorted GFP-positive cells , and sequenced the non-restrictor variants .", "For both selections , stop codon variants were significantly more enriched than WT variants in the non-restrictor pool .", "Similar to HIV-1 restriction , we found that most missense mutations ( 143 , 58% ) in rhesus TRIM5α were tolerated for N-MLV restriction ( Figure 6A ) .", "However , some missense mutations dramatically reduced N-MLV restriction , affirming that the v1 loop is indeed critical for inhibition of N-MLV ( Figure 6—figure supplement 1A–C ) .", "In particular , hydrophobic and especially aromatic residues at most positions in the v1 loop significantly decreased N-MLV restriction .", "This preference against aromatic residues is similar between HIV-1 and N-MLV restriction .", "However , N-MLV restriction is insensitive to the introduction of positively charged residues , which disrupt HIV-1 inhibition ( Figure 6—figure supplement 1D ) .", "These results indicate that the evolutionary landscape for rhesus TRIM5α against N-MLV is distinct from that of HIV-1 .", "Nevertheless , the overall degree of mutational resilience against both viruses is remarkably similar: less than half of all missense mutations disrupt restriction of either virus .", "Human TRIM5α restriction of N-MLV was even more resilient to mutation than rhesus TRIM5α .", "Almost all variants ( 187 , 92% ) had no effect on N-MLV restriction ( Figure 6B , Figure 6—figure supplement 2 ) .", "Indeed , our selection for non-restrictive variants only strongly enriched for stop codons .", "This extreme mutational resilience may reflect the massive potency ( >250 fold restriction , data not shown ) of human TRIM5α against N-MLV , and/or a decreased reliance on the v1 loop for N-MLV recognition by human TRIM5α ( Perron et al . , 2006 ) .", "We validated several human TRIM5α mutants as retaining nearly WT levels of N-MLV restriction ( Figure 6—figure supplement 2C ) .", "Thus , both rhesus and human TRIM5α inhibition of N-MLV is highly resistant to mutations , allowing mutational flexibility without loss of pre-existing antiviral restriction .", "These results , in conjunction with the substitution tolerance of HIV-1 restriction by human R332P and WT rhesus TRIM5α , indicate that mutational resilience is a general property of TRIM5α’s rapidly evolving v1 loop ." ], [ "Antiviral restriction factors are locked in high-stakes tit-for-tat evolutionary arms races with target viruses .", "However , viruses would appear to have the upper hand in these battles because of their higher mutation rates , shorter generation times , and larger population sizes .", "Although host genomes have the advantage of encoding a diverse , polygenic immune response , evolutionary constraints acting on innate immune genes could curtail their adaptive potential .", "Here , using deep-mutational scanning approaches combined with viral infection assays , we investigated the evolutionary landscape of adaptation of the most rapidly evolving segment , the disordered v1 loop , of the retroviral restriction factor TRIM5α .", "We focused on this loop because of its critical role in adapting to changing viral repertoires .", "We found two attributes of this evolutionary landscape that favor host immune evolution .", "First , human TRIM5α readily gains significant HIV-1 restriction: roughly half of all single missense mutations allow human TRIM5α to better restrict HIV-1 ( Figures 2–3 ) .", "Based on our results , we infer that positive charge is the dominant impediment to HIV-1 inhibition in human TRIM5α ( Figure 3D ) .", "Removal of this positive charge improved human TRIM5α restriction not only of HIV-1 but also of multiple lentiviruses ( Figure 4A ) .", "Recent findings revealed that cyclophilin A ( CypA ) protects the HIV-1 capsid from TRIM5α recognition ( Kim et al . , 2019; Selyutina et al . , 2020; Veillette et al . , 2013 ) .", "Although structural studies currently lack sufficient resolution to observe the molecular details of the TRIM5α–capsid interaction , we speculate that positive charge in the v1 loop impairs an interaction between TRIM5α and the capsid’s CypA-binding site via electrostatic repulsion .", "Alternatively , the detrimental effect of positive charge might largely be accounted for by lowering TRIM5α expression level ( Figure 3—figure supplement 1C–D ) .", "Increasing human TRIM5α expression has been shown to improve HIV-1 restriction ( Richardson et al . , 2014 ) , although we identified at least one mutation ( V340H ) that improved expression without increasing activity against HIV-1 and at least one mutation ( G333D ) that reduced positive charge and improved HIV-1 restriction without increasing expression level ( Figure 3—figure supplement 1B ) .", "Surprisingly , our comprehensive DMS analyses also revealed that both rapidly evolving and conserved residues can contribute to antiviral adaptation ( Figure 3B ) .", "For instance , many variants at position 333 of human TRIM5α led to increased restriction of HIV-1 and other lentiviruses ( Figures 3A and 4A ) .", "Thus , it is unclear why simian primates have retained a glycine at this position ( 333 in human , 335 in macaques ) .", "One possibility is that changes in this residue might be generally deleterious for TRIM5α function , yet subsequent analyses revealed little or no impairment of TRIM5α antiviral functions ( Figure 5D , Figure 6—figure supplement 1A , Figure 6—figure supplement 2A ) .", "More broadly , we identified many v1 loop mutations that improved human TRIM5α restriction of HIV-1 , and even other lentiviruses , without impairing N-MLV antiviral function ( Figure 6—figure supplement 2D ) .", "This permissivity stands in stark contrast to the natural evolution of TRIM5α , which has sampled relatively limited amino acid diversity ( Figure 1B ) .", "The discrepancy might be explained by recurrent selection by a viral lineage distinct from the viruses we tested here ( HIV-1 and N-MLV ) .", "For example , since the critical R332 was fixed at the common ancestor of humans , chimps , and bonobos ~7 million years ago , it is possible that R332 improved restriction of a paleovirus that has since gone extinct .", "Alternatively , TRIM5α’s constrained natural evolution could reflect a cellular function independent of viral capsid recognition .", "For example , TRIM5α has been shown to induce some innate immune signaling even in the absence of infection ( Lascano et al . , 2016; Pertel et al . , 2011; Tareen and Emerman , 2011 ) .", "Many gain-of-antiviral-function mutations increased TRIM5α expression levels ( Figure 3—figure supplement 1 ) , and this increased expression might increase aberrant signaling by TRIM5α , driving chronic immune activation that can be costly for the host ( Ashley et al . , 2012; Okin and Medzhitov , 2012 ) .", "Our analyses uncovered a second unexpected , advantageous aspect of TRIM5α’s evolutionary landscape: its antiviral restriction displays remarkable mutational resilience across multiple orthologs and against two divergent retroviruses .", "51–92% of all possible missense variants retain antiviral activity ( Figure 6—figure supplement 3 ) .", "This resilience is manifest even when potent antiviral activity is newly acquired via a single mutation , as with the R332P variant of human TRIM5α against HIV-1 .", "Therefore , we conclude that the fitness landscape of TRIM5α's rapidly evolving v1 loop resembles 'rolling hills' ( Figure 1D ) , in which valleys are infrequent and only one evolutionary step removed from mutationally tolerant plateaus .", "TRIM5α's permissive landscape contrasts with the relative inflexibility of ligand-binding domains in the core of evolutionarily conserved proteins ( Guo et al . , 2004; McLaughlin et al . , 2012; Suckow et al . , 1996 ) .", "However , these studies found increased mutational tolerance in peripheral , disordered loops not involved in critical functions like ligand recognition , where mutations are less likely to disrupt protein structure .", "The use of flexible loops for viral ligand binding by TRIM5α , as well as MxA ( Mitchell et al . , 2012 ) , thus grants rapidly evolving restriction factors mutational flexibility without significant risk of disrupting core protein structure .", "Nevertheless , the degree of mutational plasticity in TRIM5α's v1 loop is remarkable given that this loop is essential for ligand binding , suggesting that even functional constraints do not narrow its evolutionary landscape .", "Intriguingly , TRIM5α's reliance on the v1 loop for specificity mirrors that of antibodies' dependence on complementarity-defining loops for antigen recognition .", "Indeed , a high degree of mutational tolerance within complementarity-defining loops allows somatic hypermutation to significantly increase antibody-antigen affinity ( Daugherty et al . , 2000; NISC Comparative Sequencing Program et al . , 2017 ) .", "Overall , our analyses reveal not only many paths for TRIM5α to gain antiviral function but also an unexpectedly low probability of losing antiviral function via single mutations .", "Such landscapes should be highly advantageous to host genomes in evolutionary arms races with viruses .", "Mutational tolerance allows the accumulation of neutral variants that do not compromise antiviral function among antiviral genes in a population .", "Many of these novel variants may carry the capacity to restrict additional viruses , whether these result from cross-species transmissions or mutations that allow species-matched viruses to evade recognition by the dominant antiviral allele .", "Indeed , mutational tolerance has been shown to facilitate the evolution of de novo functions through the accumulation of neutral mutations ( Draghi et al . , 2010; Hayden et al . , 2011 ) .", "Although rare in human populations ( Clarke et al . , 2017 ) , extensive polymorphism within the v1 loop of TRIM5α in Old World monkeys results in diverse antiviral repertoires that have been maintained by balancing selection ( Newman et al . , 2006 ) .", "Thus , TRIM5α appears to evolve with low-cost , high-gain fitness landscapes that favor its success in co-evolutionary battles with rapidly evolving retroviruses ." ], [ "All virus-like particles ( VLPs ) were generated using three plasmids to ensure a single round of infectivity: a pseudotyping plasmid for transient expression of the VSV-G envelope protein ( pMD2 . G , Addgene plasmid #12259 , gift from Didier Trono ) , a plasmid for transient expression of the viral gag/pol , and a transfer vector encoding a green fluorescent protein ( GFP ) integration reporter between the corresponding viral LTRs .", "HIV-1 VLPs were made with the transfer vector pHIV-ZsGreen ( Welm et al . , 2008 ) ; HIV-2 and SIV VLPs used the pALPS-eGFP transfer vector ( McCauley et al . , 2018 ) ; and N-MLV was made with pQCXIP-eGFP , encoding GFP between the EcoRI and ClaI sites of pQCXIP ( P . S . Mitchell , unpublished ) .", "HIV-1 VLPs were made with p8 . 9NdSB bGH BlpI BstEII , encoding the NL4 . 3 HIV-1 gag/pol ( Berthoux et al . , 2004 ) .", "HIV-2 VLPs used a chimeric gag/pol , in which the HIV-1 CA sequence ( residues 1–202 ) was replaced by HIV-2ROD ( p8 . 9NdSB bGH BlpI BstEII HIV-2 CA ) ( Pizzato et al . , 2015 ) .", "For SIV VLPs , pCRV1-based gag/pol chimeric vectors replaced the HIV-1 CA-NTD ( residues 1–146 ) with the corresponding residues of either SIVmac239 , a virus passaged in rhesus macaques , here SIVmac ( pHIV-MAC , containing an A77V mutation ) ; or SIVcpzGab2 , a natural isolate from chimpanzee , here SIVcpz ( pHIV-Gb2 ) ( Kratovac et al . , 2008 ) .", "N-MLV VLPs were generated using pCIG3N , encoding the N-MLV gag/pol ( Bock et al . , 2000 ) .", "For stable expression of TRIM5α constructs from pQCXIP , the MLV gag/pol was transiently expressed from JK3; the pseudotyping envelope protein was transiently expressed from the L-VSV-G plasmid and driven by expression of Tat from the CMV-Tat plasmid .", "C-terminally HA-tagged human , human with the rhesus macaque v1 loop , and rhesus macaque TRIM5α ( Sawyer et al . , 2005 ) were amplified and cloned into pQCXIP ( Takara Bio , Kusatsu , Shiga , Japan ) , just upstream of the IRES–puromycin resistance cassette , between the EcoRI and NotI restriction sites .", "Targeted TRIM5α mutations were generated by Quikchange PCR using primers containing the desired point mutation flanked by 17–25 nucleotides of homology on each side of the mutation .", "Primestar polymerase ( Takara Bio ) was used to minimize errors during full-plasmid amplification , followed by DpnI digestion of unmodified parent DNA .", "All plasmids were cloned into high-efficiency chemically competent DH5α ( NEB , Ipswich , MA ) .", "Plasmids were purified using PureYield miniprep kits ( Promega , Madison , WI ) , and coding sequences were verified by complete sequencing .", "See Table 1 for all primers used in cloning and sequencing .", "Deep mutational scanning libraries were generated using degenerate primers to amplify TRIM5α-HA in pQCXIP using high-fidelity Q5 polymerase ( NEB ) .", "Degenerate primers contained a single NNS codon ( N = A/T/C/G , S = C/G ) , which encodes all 20 amino acids with only one stop codon among 32 possibilities .", "For each of the 11 or 13 codons in the v1 loop of human or rhesus TRIM5α , respectively , the two halves of TRIM5α ( on either side of randomized codon ) were amplified separately with shared flanking primers and unique internal primers for each codon ( Table 1 ) .", "For the human TRIM5α library in which R332P was fixed , internal primers matched the R332P variant of TRIM5α and codon 332 was not randomized .", "Internal primers encoded NNS at the designated codon flanked by 17–25 nucleotides of homology on each side; the forward and reverse internal primers shared 17–25 nucleotides of homology with each other to promote hybridization between the N- and C-terminal PCR fragments .", "The codon-matched N- and C-terminal fragments were combined and amplified into a single fragment using the same flanking primers as in the first amplification .", "PCR products were gel purified and cloned via Gibson assembly ( NEB ) into pQCXIP-TRIM5α-HA of the matching species , which had been digested with NotI and BamHI and gel purified .", "Gibson assembly products were transformed into high-efficiency chemically competent DH5α ( NEB ) with 30 min of heat shock recovery .", "Serial dilutions were plated to count the number of unique colonies , and transformations were repeated until at least 100x library coverage was achieved ( human: 32 × 11×100=3 . 52×104 colonies; rhesus: 32 × 13×100=4 . 16×104 colonies ) .", "To ensure library quality , 40 random colonies were sequenced from each library .", "Clones were verified to have insert by analytical restriction digest , and the coding sequence was fully sequenced to ensure that ( 1 ) each clone had only one mutation , ( 2 ) there were no mutations outside the v1 loop , and ( 3 ) the number of sites mutated once , twice , etc . among these 40 clones approximated a Poisson distribution .", "When libraries met these criteria , colonies were scraped from all transformation plates and plasmids were directly purified , without further growth to avoid amplification bias , using NucleoBond Xtra midiprep kits ( Takara Bio ) .", "HEK-293T/17 ( RRID:CVCL_1926 ) and CRFK ( RRID:CVCL_2426 ) cells were purchased from ATCC ( Manassas , VA ) and not further authenticated; cells were confirmed to be mycoplasma free by MycoProbe kit ( R and D Systems , Minneapolis , MN ) .", "Cells were grown on tissue-culture treated plates in high-glucose and L-glutamine containing DMEM ( Thermo Fisher , Waltham , MA ) supplemented with 1x penicillin/streptomycin ( Thermo Fisher ) and 10% fetal bovine serum ( Thermo Fisher ) .", "Cells were grown at 37°C , 5% CO2 in humidified incubators and passaged by digestion with 0 . 05% trypsin-EDTA ( Thermo Fisher ) .", "Cell counting was performed using a TC20 automated cell counter ( BioRad , Hercules , CA ) .", "HEK-293T/17 were seeded at 5 × 105 cells/well in 6-well plates the day prior to transfection .", "Transfections were performed with Trans-IT 293T transfection reagent ( Mirus Bio , Madison , WI ) according to manufacturer’s instructions , using 3 µL reagent per µg DNA .", "All transfected DNA was purified using PureYield mini or NuceloBond midi kits to minimize LPS contamination and quantified by NanoDrop ( Thermo Fisher ) A260 .", "To produce HIV-1 , each well was transfected with 1 µg of p8 . 9NdSB , 667 ng of pHIV-ZsGreen , and 333 ng of pMD2 . G .", "N-MLV transfections contained 1 µg of pQCXIP-eGFP , 667 ng of pCIG3N , and 333 ng of pMD2 . G .", "HIV-2 , SIVcpz , and SIVmac transfections contained 1 µg of pALPS-eGFP , 333 ng of pMD2 . G , and 667 ng of either p8 . 9NdSB HIV-2 CA , pHIV-Gb2 , or pHIV-MAC , respectively .", "TRIM5α-transducing virus was produced using 1 µg of the appropriate TRIM5α construct , 600 ng of JK3 , 300 ng of L-VSV-G , and 100 ng of CMV-Tat .", "After 24 hr , media was replaced with 1 mL of fresh media .", "Virus was harvested at 48 hr post-transfection .", "To harvest , cells were pelleted from virus-containing media at 500 x g , and supernatant was removed , aliquoted , and snap frozen in liquid nitrogen .", "To increase titers for HIV-2 and N-MLV , and in some cases HIV-1 , virus was concentrated prior to freezing .", "To concentrate , virus was pelleted through a 20% sucrose cushion at 23 , 000 rpm ( ~70 , 000 x g ) for 1 hr at 4°C .", "Pellets were air dried for 5 min , and then resuspended in fresh media for 24 hr with periodic gentle vortexing .", "All viruses were titered under conditions most closely mimicking their large-scale use .", "CRFK cells were seeded at 1 × 105 cells/mL the day prior to transduction .", "Freshly thawed viruses were serially diluted and replaced cellular media at ½ x volume .", "No transducing reagent was used for GFP-marked retroviral VLPs; TRIM5α-transducing VLPs were supplemented with 10 µg/mL polybrene .", "Plates were centrifuged at 1100 x g for 30 min and then incubated at 37°C .", "The following day , virus was removed and cells were fed fresh media , which contained 6 µg/mL puromycin for TRIM5α-transducing VLPs only .", "For GFP-marked retroviral VLPs , transduction efficiency was monitored by flow cytometry 72 hr after transduction .", "For TRIM5α-transducing VLPs , cell survival was monitored daily by estimating cell confluence , until untransduced cells were completely dead ( no surface-adhered cells ) .", "Media was replaced with fresh puromycin-containing media every 2–3 days , and cells were passaged into larger well format as needed .", "Multiplicity of infection ( MOI ) for serial dilutions was estimated by Poisson distribution; for example , ~63% of cells are expected to be transduced at least once and thus survive selection with an MOI of 1 .", "To stably transduce TRIM5α variants , we chose an MOI of ~0 . 33 ( 25–30% survival during titering ) to minimize multiple transductions per cell ( <5% probability ) .", "CRFK cells were seeded in six-well plates at 2 × 105 cells/well the day prior to transduction; for deep mutational scanning libraries , sufficient cells were seeded to generate at least 500x independent transductions for each nucleotide variant ( 32 codons x 13 sites x 500 ÷ 25% survival = 8 . 3×105 cells ) .", "Cells were transduced at the appropriate MOI with 10 µg/mL polybrene and spinoculation ( 1100 x g , 30 min ) , then underwent 6 µg/mL puromycin selection starting at 24 hr post-transduction and continuing until untransduced controls were completely dead ( usually ~7 days ) .", "Upon completion of selection , surviving cells were pooled and maintained in 2 µg/mL puromycin .", "Passages always maintained at least 5 × 105 cells ( 1000x library coverage ) to avoid bottlenecking library diversity .", "CRFK cells expressing a TRIM5α deep mutational scanning library were seeded in 12-well plates at 1 × 105 cells/well the day prior to viral infection .", "Sufficient wells were seeded for at least 1000x library coverage among target cells to be sorted 4 days later ( assuming at least two doublings in that time , with sorting frequency typically ~5% of cells as estimated beforehand by viral titering against DMS library-expressing cells ) .", "Thus , each biological replicate began with at least 2 . 4 × 106 cells ( 32 codons x 13 sites x 1000 ÷ 5% ÷ 4 ) seeded from the same CRFK library .", "Libraries were infected with HIV-1-GFP or N-MLV-GFP the following day .", "For loss-of-restriction experiments ( Figures 4–6 ) , we chose viral doses that were restricted by WT TRIM5α to <1% , as determined during preliminary titering experiments .", "For gain-of-HIV-1-restriction by human TRIM5α , we chose a viral dose in which WT TRIM5α was infected to ~98% , in order to minimize uninfected GFP-negative cells .", "Infection efficiency was monitored by parallel infection of controls ( empty vector , WT TRIM5α , uninfected negative control ) .", "Cells were infected by spinoculation ( 1100 x g , 30 min ) and media was replaced 24 hr post-infection .", "Infected cells were incubated an additional 48 hr to increase GFP expression levels .", "Cells were harvested by trypsinization , pelleted , and vigorously resuspended as well as filtered ( 0 . 7 µm ) to minimize aggregation .", "Cells were FACS sorted , with stringent gating on size , single cells , and presence or absence of GFP ( for loss- or gain-of-restriction , respectively ) .", "At least 4 × 105 cells ( 1000x library coverage ) were sorted for each biological replicate .", "For gain-of-HIV-1-restriction by human TRIM5α , sorted GFP-negative cells were pelleted and re-seeded at 1 × 105 cells/well for a second round of infection , at the same dose , the following day , in order to enrich true restrictors and deplete cells uninfected by chance .", "Infection , harvest , and FACS sorting were all performed identically , except that apparent HIV-1 restriction by pooled variants was improved in the second round of enrichment ( ~50% GFP-negative compared to ~10% in the first round of infection ) .", "Sorted cells were pelleted , resuspended in PBS , and genomic DNA was harvested using DNeasy Blood and Tissue kits ( Qiagen , Hilden , Germany ) .", "Input samples were harvested from infected but unsorted cells for each replicate .", "Illumina libraries were constructed from genomic DNA by 2-step PCR amplification using Q5 polymerase .", "The first PCR amplified the v1 loop of TRIM5α and added adapters; the second set of PCR primers annealed to these adapters and added a unique 8 bp i7 Nextera barcode as well as P5 and P7 adapters for flow cell binding ( see Table 1 ) .", "Genomic DNA from each sample ( two input replicates , two sorted replicates for each experiment ) was amplified in three separate PCR tubes , with 500 ng of genomic DNA per tube , to offset random PCR jackpotting .", "This sampled a total of 1 . 5 µg of DNA , which represents ~500 x library coverage , assuming 6 . 6 pg gDNA/cell and a single TRIM5α integration/cell .", "After 15 cycles of amplification , samples were digested for 15 min at 37°C with 5 µL of ExoI ( NEB ) to remove first round primers .", "PCR products were then pooled from triplicate tubes , purified by QIAquick PCR purification kit ( Qiagen ) , and the entire elution was divided between three separate PCR tubes for 18 cycles of second round amplification .", "Barcoded PCR products ( 234 bp ) were pooled from triplicate tubes and purified by double-sided size selection using Ampure beads ( Beckman Coulter , Pasadena , CA ) .", "In brief , large DNA was removed by incubation with 0 . 8x bead volume and magnetization; PCR products were bound from the supernatant with 1 . 5x bead volume , washed with 80% ethanol , and eluted in water .", "PCR product purity was confirmed by gel electrophoresis .", "Samples were then pooled at equimolar ratios and Illumina sequenced ( MiSeq-v2 ) with single-end reads .", "Estimated read depth ( equally distributed between libraries ) always exceeded 1 million counts per library to ensure >1000 x coverage , on average , per nucleotide sequence .", "One read was generated using the i7 index primer for the 8 bp barcode , and a second read used a custom sequencing primer , which annealed immediately adjacent to the v1 loop ( 33 bp read for human , 39 bp for rhesus TRIM5α ) .", "PhiX was included at 15% in sequencing runs to increase per-bp-diversity , since the majority ( 10/11 for human or 12/13 for rhesus ) of reads should not randomize any given codon .", "Reads counts for each unique nucleotide sequence from all four samples in an experiment were compiled into a single tsv file .", "Sequences that differed from WT by more than one codon , or sequences in which codons did not end in C or G , were filtered from the dataset; these largely had only a few reads per sample and represented sequencing errors .", "Reads counts were normalized to total counts per million ( cpm ) within each barcoded sample .", "Sequences with low read counts ( <50 cpm ) were excluded as they were found to introduce noise ( poor correlation between replicates and across codons ) .", "Enrichment was calculated as the ratio of sorted to input cpm .", "The average and standard deviation of enrichment was calculated across both replicates of all synonymous codons to determine statistics at the amino acid level , except for WT variants , where we show each synonymous variant separately ( averaged across replicates ) to better visualize WT variance .", "Amino acid enrichment values were plotted in waterfall plots ( descending order of enrichment ) , scatter plots ( comparing replicates ) , and double-gradient heat maps ( comparing amino acid variants at each position , with baseline value [white] set to the average for WT enrichment ) using GraphPad Prism .", "R scripts for data analysis , including all filtering , normalization , and calculations , as well as raw sequence reads have been uploaded to Github: https://github . com/jtenthor/T5DMS_data_analysis ( copy archived at https://github . com/jtenthor/T5DMS_data_analysis; Tenthorey , 2020 ) .", "Viral inhibition by TRIM5α constructs was always compared to CRFK cells transduced with empty vector .", "CRFK lines were seeded in 96-well plates at 1 × 104 cells/well the day prior to transduction .", "Media was removed and replaced with serial 3-fold dilutions of the appropriate GFP-marked retrovirus .", "Serial dilutions were started at titers that yielded ~95% infection in untransduced CRFK .", "Plates were centrifuged at 1100 x g for 30 min and then incubated at 37°C .", "The following day , virus was removed and cells were fed fresh media .", "Cells were harvested by trypsinization 72 hr after transduction and analyzed by flow cytometry for GFP fluorescence .", "Cells were gated on size ( FSC vs . SSC ) , single cells ( FSC height vs . area ) , and GFP-positive as compared to negative control ( FITC vs . PE empty channel ) .", "Fold inhibition was calculated by comparing ID10 , the amount of virus required to infect 10% of cells , between TRIM5α and empty vector .", "Infection ( % GFP-positive ) was plotted against viral dose , both on logarithmic scale , as in Figure 2E .", "Infection points < 0 . 5% or > 50% GFP-positive were excluded due to increased noise or curve saturation , respectively , yielding a simple linear relationship between viral dose and % GFP-positive; infections without at least three data points in this range were excluded from further analysis .", "A linear regression ( against log-transformed data ) was then used to calculate the viral dose corresponding to 10% infection ( back-calculated to linear scale ) , and the dose for TRIM5α was divided by that for empty vector .", "This method was used to calculate fold inhibition for all viruses except human TRIM5α against N-MLV , as we could not consistently achieve infection greater than 1% for WT human TRIM5α; we therefore report raw infection data .", "All fold inhibitions were calculated from at least three independent experiments , which were performed either in biological singlicate or duplicate .", "CRFK cells stably expressing TRIM5α-HA variants were harvested by trypsinization , washed in PBS , and counted; 106 cells were lysed for 15 min on ice in 100 µL pre-chilled lysis buffer ( 50 mM Tris , pH 8 , 150 mM NaCl , 1% Triton-X100 , 1x cOmplete EDTA-free protease inhibitor cocktail [Roche , Basel , Switzerland] ) .", "Lysates were pelleted at 20 , 000 x g for 15 min at 4°C .", "Supernatants were quantified by Bradford protein assay ( BioRad ) and normalized to load equal protein across all samples ( usually 10–25 µg per lane ) .", "Samples were boiled for 5 min in Laemmli Sample Buffer ( BioRad ) supplemented with 5% β-mercaptoethanol and loaded onto Mini-PROTEAN TGX stain-free gels ( BioRad ) .", "Gels were run in Tris/Glycine/SDS buffer ( BioRad ) for 50 min at 150 V , then transferred semi-dry for 7 min at 1 . 3 mV using Trans-Blot Turbo 0 . 2 µm nitrocellulose transfer packs and the Trans-Blot Turbo transfer system ( BioRad ) .", "Blots were blocked with Odyssey blocking buffer ( LI-COR , Lincoln , NE ) , then probed with mouse anti-HA at 1:1000 ( RRID:AB_2565335 , Biolegend , San Diego , CA ) and rabbit anti-β-actin at 1:5000 ( RRID:AB_2305186 , Abcam , Cambridge , UK ) .", "All antibodies were diluted in TBST with 5% bovine serum albumin ( Sigma Aldrich , St . Louis , MO ) .", "Blots were washed in TBST and probed with IRDye 680RD donkey anti-mouse ( RRID:AB_10953628 , LI-COR ) and IRDye 800CW donkey anti-rabbit ( RRID:AB_621848 , LI-COR ) , both diluted 1:10 , 000 .", "Blots were washed and scanned at 680 and 800 nm .", "HA intensities were quantified using ImageJ and normalized to actin , then compared to WT TRIM5α to determine relative expression levels .", "A tBLASTn search of human TRIM5α ( NP_149023 . 2 ) against primate genomes returned 29 unique simian primate orthologs of TRIM5α .", "We excluded New World monkey sequences as they share a 9-amino acid deletion in the v1 loop .", "Open reading frames of the following sequences were translation aligned using MUSCLE: human ( Homo sapiens , NM_033034 . 2 ) , chimpanzee ( Pan troglodytes , NM_001012650 . 1 ) , bonobo ( Pan paniscus , XM_003819046 . 3 ) , gorilla ( Gorilla gorilla , NM_001279549 . 1 ) , Sumatran orangutan ( Pongo abelii , NM_001131070 . 1 ) , Bornean orangutan ( Pongo pygmaeus , AY923179 . 2 ) , white-handed gibbon ( Hylobates lar , AY923180 . 1 ) , white-cheeked gibbon ( Nomascus leucogenys , NM_001280113 . 1 ) , crab-eating macaque ( Macaca fascicularis , NM_001283295 . 1 ) , rhesus macaque ( Macaca mulatta , NM_001032910 . 1 ) , olive baboon ( Papio anubis , NM_001112632 . 1 ) , collared mangabey ( Cercocebus torquatus , KP743974 . 1 ) , sooty mangabey ( Cercocebus atys , NM_001305964 . 1 ) , drill ( Mandrillus leucophaeus , XM_011971974 . 1 ) , Wolf’s guenon ( Cercopithecus wolfi , KP743973 . 1 ) , red guenon ( Erythrocebus patas , AY740619 . 1 ) , grivet ( Cercopithecus aethiops , AY669399 . 1 ) , tantalus monkey ( Chlorocebus tantalus , AY740613 . 1 ) , African green monkey ( Chlorocebus sabaeus , XM_008019877 . 1 ) , vervet monkey ( Chlorocebus pygerythrus , AY740612 . 1 ) , golden snub-nosed monkey ( Rhinopithecus roxellana , XM_010364548 . 1 ) , and Angola colobus ( Colobus angolensis , XM_011963593 . 1 ) .", "A PHYML tree was built using the HKY85 substitution model with 100 bootstraps and rooted on human TRIM6 ( NM_001003818 . 3 ) .", "The unrooted tree was used for site-specific PAML analysis ( Yang , 1997 ) using both F3 × 4 and F61 codon models to ensure robust results .", "We performed maximum likelihood ( ML ) tests comparing model 7 ( neutral selection beta distribution ) to model 8 ( beta distribution with positive selection allowed ) .", "In each case , the model allowing positive selection gave the best fit to the data ( p<0 . 0001 , chi-squared test on 2x ∆ML with two df ) .", "Model eight also identified rapidly evolving sites with a Bayes Empirical Bayes posterior probability >0 . 95 .", "We report residues that meet this threshold for rapid evolution under both the F3 × 4 and F61 codon models .", "Evolutionarily accessible amino acids were defined as one nucleotide substitution away from the wild-type sequence .", "For Figure 1B , we determined all amino acids that were accessible from any of the 22 aligned sequences , then determined the fraction of these amino acids that were represented in our alignment .", "Evolutionary landscapes for Figure 1C–D were generated from hypothetical data with 3D surface plots using the Plotly R package .", "All statistics were performed using GraphPad Prism .", "Correlations between replicates or across experiments were computed using non-log-transformed data , and using non-parametric Spearman correlations where data did not pass normality tests .", "Student’s unpaired t-tests were used to compare differences in mean values for normally distributed data from two groups; where variances were determined to be significantly different , Welch’s correction was applied .", "For comparisons of more than two groups , one-way ANOVA tests were used with Holm-Sidak’s multiple comparison correction; where variances were determined to be unequal , the Geisser-Greenhouse correction was applied .", "Exact p-values are reported in source data files ." ] ]

[ "Host antiviral proteins engage in evolutionary arms races with viruses , in which both sides rapidly evolve at interaction interfaces to gain or evade immune defense .", "For example , primate TRIM5α uses its rapidly evolving ‘v1’ loop to bind retroviral capsids , and single mutations in this loop can dramatically improve retroviral restriction .", "However , it is unknown whether such gains of viral restriction are rare , or if they incur loss of pre-existing function against other viruses .", "Using deep mutational scanning , we comprehensively measured how single mutations in the TRIM5α v1 loop affect restriction of divergent retroviruses .", "Unexpectedly , we found that the majority of mutations increase weak antiviral function .", "Moreover , most random mutations do not disrupt potent viral restriction , even when it is newly acquired via a single adaptive substitution .", "Our results indicate that TRIM5α’s adaptive landscape is remarkably broad and mutationally resilient , maximizing its chances of success in evolutionary arms races with retroviruses ." ]

[ "The evolutionary battle between viruses and the immune system is essentially a high-stakes arms race .", "The immune system makes antiviral proteins , called restriction factors , which can stop the virus from replicating .", "In response , viruses evolve to evade the effects of restriction factors .", "To counter this , restriction factors evolve too , and the cycle continues .", "The challenge for the immune system is that mammals do not evolve as fast as viruses .", "How then , in the face of this disadvantage , can the immune system hope to keep pace with viral evolution ?", "One human antiviral protein that seems to have struggled to keep up is TRIM5α .", "In rhesus macaques , it is very effective at stopping the replication of HIV-1 and related viruses .", "But in humans , it is not effective at all .", "But why ?", "Protein evolution happens due to small genetic mutations , but not every mutation makes a protein better .", "If a protein is resilient , it can tolerate lots of neutral or negative mutations without breaking , until it mutates in a way that makes it better .", "But , if a protein is fragile , even small changes can render it completely unable to do its job .", "It is possible that restriction factors , like TRIM5α , are evolutionarily 'fragile' , and therefore easy to break .", "But it is difficult to test whether this is the case , because existing mutations have already passed the test of natural selection .", "This means that either the mutation is somehow useful for the protein , or that it is not harmful enough to be removed .", "Tenthorey et al . devised a way to introduce all possible changes to the part of TRIM5α that binds to viruses .", "This revealed that TRIM5α is not fragile; most random mutations increased , rather than decreased , the protein’s ability to prevent viral infection .", "In fact , it appears it would only take a single mutation to make TRIM5α better at blocking HIV-1 in humans , and there are many possible single mutations that would work .", "Thus , it would appear that human TRIM5α can easily gain the ability to block HIV-1 .", "The next step was to find out whether these gains in antiviral activity are just as easily lost .", "To do this , Tenthorey et al . performed the same tests on TRIM5α from rhesus macaques and an HIV-blocking mutant version of human TRIM5α .", "This showed that the majority of random mutations do not break TRIM5α’s virus-blocking ability .", "Thus , TRIM5α can readily gain antiviral activity and , once gained , does not lose it easily during subsequent mutation .", "Antiviral proteins like TRIM5α engage in uneven evolutionary battles with fast-evolving viruses .", "But , although they are resilient and able to evolve , they are not always able to find the right mutations on their own .", "Experiments like these suggest that it might be possible to give them a helping hand .", "Identifying mutations that help human TRIM5α to strongly block HIV-1 could pave the way for future gene therapy .", "This step would demand significant advances in gene therapy efficacy and safety , but it could offer a new way to block virus infection in the future ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "biochemistry and chemical biology", "cell biology" ]

[ [ "Mitochondria cannot be created de novo and pre-existing mitochondria are used as templates for mitochondrial biogenesis .", "This genesis requires the ~1500 different mitochondrial proteins to be imported via dynamic translocation machines to one of four subcompartments of the organelle – outer and inner membrane , intermembrane space and matrix ( Chacinska et al . , 2009; Stojanovski et al . , 2012; Dolezal et al . , 2006; Harbauer et al . , 2014; Neupert and Herrmann , 2007; Baker et al . , 2014 ) .", "The Translocase of the Outer Membrane ( TOM ) complex is described as the general entry gate to mitochondria and provides a passageway through which precursors can cross the outer membrane .", "The mitochondrial inner membrane contains two translocase machines that are responsible for the import of a large fraction of the mitochondrial proteome; the Translocase of the Inner Membrane 23 ( TIM23 ) complex and the Translocase of the Inner Membrane 22 ( TIM22 ) complex .", "The TIM23 complex typically transports proteins that possess a matrix-targeting N-terminal presequence ( Chacinska et al . , 2009; Neupert and Herrmann , 2007; Wagner et al . , 2009; Mokranjac and Neupert , 2010 ) , while the TIM22 complex mediates the inner membrane insertion of multi-transmembrane spanning proteins that contain internal targeting elements ( Chacinska et al . , 2009; Neupert and Herrmann , 2007; Rehling et al . , 2004; Koehler , 2004 ) .", "Substrates of the TIM22 complex include the mitochondrial carrier family , such as the ADP/ATP carrier ( AAC ) and the phosphate carrier ( PiC ) , and multispanning inner membrane proteins like , Tim17 and Tim23 ( subunits of the TIM23 complex ) and Tim22 itself ( pore forming unit of the TIM22 complex ) ( Chacinska et al . , 2009; Stojanovski et al . , 2012; Koehler , 2004; Sirrenberg et al . , 1996; Káldi et al . , 1998 ) .", "In yeast cells , TIM22 is a 300-kDa complex , consisting of four membrane integral subunits , Tim22 , Tim54 , Tim18 and Sdh3 , and a peripheral chaperone complex consisting of the small TIM proteins , Tim9-Tim10-Tim12 ( Adam et al . , 1999; Gebert et al . , 2011; Jarosch et al . , 1997 , 1996; Kerscher et al . , 1997 , 2000; Koehler et al . , 2000 , 1998; Kovermann et al . , 2002 ) .", "The small TIM proteins are a family of intermembrane space chaperones that facilitate the passage of hydrophobic membrane proteins through this aqueous environment .", "Tim9 and Tim10 form a soluble hexameric complex , but a fraction also interacts with the TIM22 complex via assembly with Tim12 ( Adam et al . , 1999; Gebert et al . , 2008; Baud et al . , 2007 ) .", "Like yeast , the human TIM22 complex consists of the channel-forming hTim22 protein , along with subunits of the small TIM family , hTim9 , hTim10a , and hTim10b ( Mühlenbein et al . , 2004 ) , with hTim10b being the functional homologue of yeast Tim12 ( Koehler et al . , 1998; Baud et al . , 2007; Mühlenbein et al . , 2004; Gentle et al . , 2007 ) .", "However , homologues of yeast Tim54 or Tim18 are absent in human cells and there is no evidence to indicate that the Sdh3 homologue , SDHC , interacts with the human TIM22 translocase .", "Thus , the true architecture of the human TIM22 complex remains an open question .", "Given the many elaborate functions of mitochondria in human cells , including , cell death , metabolism , tumorigenicity and neurodegenerative disorders , we reasoned the composition of the TIM22 complex in human cells is likely different to yeast .", "This led us to investigate the subunit composition of the human TIM22 complex .", "Here we report on the identification of C19orf52 as a novel subunit of the human TIM22 complex , and accordingly rename the protein as Tim29 .", "Tim29 functions in the assembly of hTim22 and consequently the stability of the TIM22 complex .", "Furthermore , we propose that Tim29 links the TIM22 complex to the general entry gate of mitochondria , the TOM complex .", "Our findings highlight the importance of analysing mitochondrial protein import across phylogenetic boundaries , since it can reveal novel facilitators of this essential cellular process , in addition to novel mechanisms ." ], [ "We sought to investigate the composition of the human TIM22 complex using affinity enrichment approaches .", "Exogenous expression of hTim22 in cells proved challenging , with the protein displaying a propensity to aggregate ( data not shown ) .", "However , this was not the case for hTim10b and we therefore generated a stable tetracycline-inducible HEK293T cell line expressing hTim10b3XFLAG .", "The FLAG-tagged protein was exclusively localised to mitochondria when assessed by fluorescence microscopy ( Figure 1A , upper panel ) .", "Blue Native ( BN ) -PAGE of isolated mitochondria revealed that hTim10b3XFLAG formed the same assemblies as the untagged hTim10b protein ( assessed by in vitro import of hTim10b into isolated mitochondria ) , including the hTim9-hTim10a-hTim10b hexamer and a higher molecular weight 450-kDa TIM22 complex ( Figure 1B and C ) . 10 . 7554/eLife . 17463 . 003Figure 1 . The previously uncharacterised protein C19orf52 immunoprecipitates with hTim10b .", "( A ) Representative fluorescence images of tetracycline-induced HEK293T cells that contained the Control empty vector ( pCDNA5-FRT/TO ) or hTim10b3XFLAG .", "Cells were fixed prior to incubation with anti-FLAG ( left panel , green; to stain hTim10b3XFLAG ) and anti-NDUFAF2 ( middle panel , red; to visualise the mitochondria ) .", "Hoechst stain ( far right panel , blue ) was used to stain the nucleus .", "Primary antibodies were counterstained with Alexa Fluor 568 and 488 secondary antibodies prior to microscopy .", "Scale bar: 10 µm .", "( B ) Mitochondria isolated from control cells or cells expressing hTim10b3XFLAG were solubilised in digitonin-containing buffer before being analysed by BN-PAGE and immunoblotting using anti-FLAG antibodies .", "( C ) [35S]-labelled hTim10b precursor was imported into mitochondria isolated from wild type HeLa cells for the indicated times in the presence or absence of a membrane potential ( Δψ ) .", "Following import the mitochondria were re-isolated , lysed in digitonin-containing buffer and subjected to blue native electrophoresis and autoradiography .", "Asterisk ( * ) indicates non-specific band .", "( D ) Mitochondria isolated from control and hTim10b expressing cells were solubilised in digitonin-containing buffer .", "Mitochondrial lysates were subjected to immunoprecipitation with anti-FLAG resin .", "Collected fractions were analysed by SDS-PAGE and western blotting using anti-FLAG and anti-SDHA antibodies .", "T , Total; P , Pellet; S , Supernatant; UB , Unbound; WI and WII , Wash I and II and E , Elution .", "5% of the T , P , S , UB , WI and WII fractions and 100% of the E fraction were loaded for SDS-PAGE analysis .", "( E ) Volcano plot showing proteins enriched in hTim10b pull-down versus the empty vector control .", "All proteins were plotted and each circle represents one protein/gene .", "The X-axis shows the Log2 fold change of Tim10b interacting partners and Y-axis shows the −log10 of the p-values . DOI: http://dx . doi . org/10 . 7554/eLife . 17463 . 00310 . 7554/eLife . 17463 . 004Figure 1—source data 1 . Data from Figure 1E . Rows are ordered by t-test difference between empty vector and hTim10b3xFLAG triplicates . DOI: http://dx . doi . org/10 . 7554/eLife . 17463 . 004 We affinity-purified hTim10b3XFLAG and associated proteins using anti-FLAG resin ( Figure 1D ) and a stable cell line containing an empty vector was used as a negative control ( since we often observed leaky expression of hTim10b in un-induced cells ) .", "A single protein of the expected molecular weight was detected in the eluate fraction by western blotting with FLAG-specific antibodies ( Figure 1D , lane 7 , upper panel ) .", "Eluate fractions were then analysed by label-free quantitative mass spectrometry to reveal the interacting partners of hTim10b .", "Among proteins that co-purified specifically with hTim10b3XFLAG were known interacting partners hTim10a , hTim9 and hTim22 ( Figure 1E and Figure 1—source data 1 ) , which suggested the purification of functional TIM22 complexes .", "The uncharacterised protein , C19orf52 , also displayed significant enrichment in the hTim10b3XFLAG sample ( Figure 1E and Figure 1—source data 1 ) .", "Sequence similarity searches for C19orf52 did not reveal homologs in lower eukaryotes .", "However , C19orf52 is conserved in metazoan species ( Figure 2—figure supplement 1 ) .", "The previously uncharacterised protein is predicted to possess an N-terminal presequence at amino acids 1–16 , based on the MitoProt II bioinformatics program ( Claros , 1995; Claros and Vincens , 1996 ) and a single transmembrane domain spanning amino acids 61–79 ( Figure 2A ) .", "We first performed a mitochondrial in vitro import assay to assess import of the protein into mitochondria .", "[35S]-C19orf52 was synthesised in rabbit reticulocyte lysate and imported into mitochondria isolated from HeLa cells .", "C19orf52 could be imported into a protease-protected location and in a time-dependent manner , but we did not observe proteolytic processing of the protein ( Figure 2B , upper panel ) , as observed for the presequence-containing , pre-ornithine transcarbamylase ( pOTC ) ( Figure 2B , lower panel ) .", "However , the import of C19orf52 was inhibited under conditions where the membrane potential ( Δψ ) had been dissipated ( Figure 2B , upper panel , lane 8 ) in a similar manner to pOTC ( Figure 2B , lower panel , lane 8 ) , suggesting import via a Δψ-dependent import pathway .", "To confirm the mitochondrial localisation of C19orf52 and define the boundaries of the protein’s targeting information , we expressed the following constructs in HeLa cells as C-terminal FLAG-tagged fusion proteins:", "( i ) full length C19orf52 ( C19orf52WT ) ;", "( ii ) C19orf52 lacking the 16 amino acids that make up the predicted presequence ( C19orf52Δ16 ) ;", "( iii ) the N-terminal 89 amino acids of C19orf52 , which includes the predicted presequence and transmembrane domain ( aa 61–79 ) ( C19orf52aa1-89 ) ;", "( iv ) the N-terminal 89 amino acids of C19orf52 , but lacking the predicted presequence ( C19orf52aa17-89 ) and", "( v ) a N-terminal truncation of C19orf52 containing amino acids 80–260 ( C19orf52aa80-260 ) .", "C19orf52 showed exclusive localisation to mitochondria ( Figure 2C , upper panel ) , confirming the protein is indeed mitochondrial .", "C19orf52Δ16 and C19orf52aa17-89 , which both lack the N-terminal 16 amino acids were both targeted to mitochondria , suggesting that residues 1–16 are dispensable for targeting to the organelle ( Figure 2C ) .", "Expression of C19orf52aa1-89 also displayed exclusive mitochondrial localisation , while C19orf52aa80-260 could not be targeted to mitochondria and was localised to the cytosol ( Figure 2C ) . 10 . 7554/eLife . 17463 . 005Figure 2 . C19orf52 is a mitochondrial inner membrane protein .", "( A ) Schematic representation of the predicted domain structure for C19orf52 from Homo sapiens .", "( B ) In vitro import of [35S]-labelled C19orf52 and pre-ornithine transcarbamylase , pOTC into mitochondria isolated from HeLa cells for the indicated times in the presence or absence of membrane potential ( Δψ ) .", "Following import mitochondria were re-isolated and either left untreated ( lanes 1–4 ) or treated with Proteinase K ( lanes 5–8 ) .", "Samples were analysed using SDS-PAGE and autoradiography .", "p , precursor and m , mature .", "( C ) The indicated C19orf52 variants ( C19orf52WT , C19orf52Δ16 , C19orf52aa1-89 , C19orf52aa17-89 and C19orf52aa80-260 ) were transiently transfected and expressed as C-terminal 3XFLAG fusions in HeLa cells .", "Cells were immunostained with anti-FLAG and anti-NDUFAF2 ( mitochondria marker ) antibodies for visualisation using fluorescence microscopy .", "Scale bar: 10 µm .", "( D ) Mitochondria were isolated from stable tetracycline-inducible HEK293T cells expressing C19orf523XFLAG .", "Intact mitochondria ( lanes 1 and 2 ) , mitoplasts ( generated by hypotonic swelling of the outer membrane , lanes 3 and 4 ) and solubilised mitochondria ( Triton X-100 , lanes 5 and 6 ) were incubated with or without Proteinase K ( 50 µg/ml ) and analysed by SDS-PAGE and western blotting using the indicated antibodies .", "( E ) Mitochondria isolated from C19orf523XFLAG expressing cells were subjected to alkaline extraction using 100 mM Na2CO3 ( pH 11 and 12 ) .", "The membrane ( P ) and soluble ( S ) fractions were separated by ultra-centrifugation prior to SDS-PAGE and immunoblotting analysis using the indicated antibodies . DOI: http://dx . doi . org/10 . 7554/eLife . 17463 . 00510 . 7554/eLife . 17463 . 006Figure 2—figure supplement 1 . C19orf52 is conserved in metazoa . Alignment of C19orf52 homologous proteins from the indicated metazoa species using ClustalW2 .", "Dark grey indicates 100% identity , light grey either 80% or 60% identity ( conserved in 4 or 3 species , respectively ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 17463 . 00610 . 7554/eLife . 17463 . 007Figure 2—figure supplement 2 . C19orf52 lacking the N-terminal 16 amino acids is not sorted to the inner membrane . Mitochondria were isolated from cells transiently transfected with DNA encoding ( A ) C19orf52aa1-89 ( B ) C19orf52△16 and ( C ) C19orf52aa17-89 as 3XFLAG fusions .", "Mitochondria ( lanes 1 and 2 ) , mitoplasts ( lanes 3 and 4 ) and solubilised mitochondrial membranes ( lanes 5 and 6 ) were left untreated ( lanes 1 , 3 and 5 ) or treated with Proteinase K ( lanes 2 , 4 and 6 ) .", "Intact mitochondria were also treated with sodium carbonate to separate mitochondrial proteins into membrane integrated ( pellet , P ) or peripheral ( supernatant , S ) proteins .", "Samples were analysed by SDS-PAGE and western-blotting . DOI: http://dx . doi . org/10 . 7554/eLife . 17463 . 007 Next we addressed the sub-mitochondrial localisation of C19orf52 .", "We generated a stable tetracycline-inducible HEK293T cell line expressing C19orf523XFLAG .", "Hypo-osmotic swelling and carbonate extraction treatments were performed on the isolated mitochondria .", "C19orf523XFLAG was present in isolated mitochondria and only became accessible to external protease when the outer membrane was disrupted ( Figure 2D , compare lanes 2 and 4 ) , indicating the C-terminal FLAG tag is exposed to the intermembrane space .", "Outer membrane proteins Bak and Mfn2 , Opa1 that is exposed to the intermembrane-space and matrix-located NDUFAF2 served as controls .", "C19orf523XFLAG was resistant to carbonate treatment , like the membrane integrated Bak and Mfn2 , indicating that it is likely an integral membrane protein ( Figure 2E ) .", "In a similar manner , subfractionation and carbonate extraction of mitochondria isolated from cells expressing C19orf521-89 revealed that the truncated protein is also an integral membrane protein that exposes it’s C-terminal FLAG tag to the intermembrane space ( Figure 2—figure supplement 2A ) .", "Taken together this data indicates that C19orf52 is an integral inner membrane protein with a C-terminal domain exposed to the intermembrane space .", "Surprisingly , upon subfractionation and carbonate extraction of mitochondria isolated from cells expressing C19orf52△16 and C19orf5217-89 ( Figure 2—figure supplement 2C and D ) we noticed that these proteins failed to sort properly to the inner membrane , but appeared trapped in the outer membrane , most likely in the TOM complex ( indicated by accessibility to protease and presence in both the pellet and supernatant fractions upon carbonate treatment ) .", "Thus , although the first 16 amino acids originally appeared to be dispensable for mitochondrial targeting , closer examination revealed that these residues are indeed crucial for subsequent sorting of C19orf52 to the inner membrane .", "Next we investigated the association of C19orf52 with the human TIM22 complex .", "Mitochondria from hTim10b3XFLAG and C19orf523XFLAG expressing cells were solubilised in digitonin-containing buffer prior to analysis by BN-PAGE and immunodecoration ( Figure 3A ) .", "C19orf52 specific antibodies revealed the presence of a high molecular weight complex of approximately 450-kDa , reminiscent of the TIM22 complex in both control ( Figure 3A , lane 1 ) and hTim10b3XFLAG mitochondria ( Figure 3A , lane 2 ) .", "We observed a slight delay in the migration of the C19orf52-containing complex in mitochondria isolated from hTim10b3XFLAG expressing cells ( Figure 3A , lane 2 and 4 ) .", "This suggested that C19orf52 is located within a hTim10b3XFLAG-containing complex and the presence of the FLAG-tag is causing the observed delay in mobility .", "To interrogate this observation further , we performed antibody-shift analysis of the human TIM22 complex .", "In this case , [35S]-hTim22 was imported into mitochondria isolated from control , hTim10b3XFLAG and C19orf523XFLAG expressing cells where it assembled into the mature TIM22 complex ( Figure 3B , lane 1 , 4 and 7 ) .", "Following import , mitochondria were isolated , solubilised in digitonin-containing buffer and incubated with anti-FLAG or anti-SDHA antibodies prior to BN-PAGE .", "As can be seen ( Figure 3B , lanes 2 , and 8 ) , the FLAG antibodies shifted [35S]-hTim22 ( hTIM22 complex ) in both hTim10b3XFLAG and C19orf523XFLAG containing mitochondria , suggesting the presence of all three proteins within the same complex .", "In an alternative approach C19orf523XFLAG was shown to specifically immunoprecipitate hTim22 ( Figure 3C ) , while C19orf52 could be co-immunoprecipitated with hTim10b3XFLAG ( Figure 3D ) .", "As a final source of confirmation immunoprecipitation of C19orf523XFLAG and subsequent quantitative mass spectrometry-based proteomics analysis revealed the presence of all known TIM22 complex subunits , including hTim22 , hTim10b , hTim10a and hTim9 ( Figure 3—source data 1 ) .", "These findings confirm that C19orf52 is a bona fide subunit of the human TIM22 complex , and represents a novel metazoan specific subunit of the translocation machine .", "Given that the molecular weight of the protein is 29 kDa and the nomenclature of the mitochondrial protein import machinery ( Pfanner et al . , 1996 ) , we henceforth rename this protein as Tim29 and its gene as TIMM29 . 10 . 7554/eLife . 17463 . 008Figure 3 . C19orf52 is a subunit of the human TIM22 complex .", "( A ) Mitochondria isolated from control or hTim10b3XFLAG-expressing cells were solubilised in 1% digitonin-containing buffer and analysed by BN-PAGE and western blotting with the indicated antibodies .", "( B ) [35S]-labelled hTim22 was imported into mitochondria isolated from control , C19orf523XFLAG or hTim10b3XFLAG expressing cells .", "Following import at 37°C mitochondria were reisolated , solubilised in digitonin-containing buffer and incubated with either anti-FLAG or anti-SDHA antibodies .", "Samples were separated by BN-PAGE and analysed by autoradiography .", "# indicates antibody-shifted protein complex .", "( C ) Control and C19orf523XFLAG mitochondria were solubilised in digitonin-containing buffer and subjected to immunoprecipitation using anti-FLAG resin .", "Total ( 5% ) and Elution ( 100% ) fractions were analysed using SDS-PAGE and immunoblotting .", "( D ) Digitonin-solubilised mitochondrial lysates from control and hTim10b3XFLAG expressing cells were subjected to immunoprecipitation with anti-FLAG resin .", "Fractions were analysed by SDS-PAGE and western blotting using the indicated antibodies .", "T , Total; P , Pellet; S , Supernatant; UB , Unbound; W , Wash and E , Elution . DOI: http://dx . doi . org/10 . 7554/eLife . 17463 . 00810 . 7554/eLife . 17463 . 009Figure 3—source data 1 . C19orf52 immunoprecipitates TIM22 complex subunits . Data from C19orf523XFLAG immunoprecipitations looking at the presence of TIM22 complex subunits .", "The fold increase ( C19orf52/EV ) is shown in the last column . DOI: http://dx . doi . org/10 . 7554/eLife . 17463 . 009 To investigate the molecular function of Tim29 at the TIM22 complex we undertook RNA interference studies in HEK293FS cells .", "Knockdown ( KD ) of Tim29 was assessed relative to cells transfected with a non-targeting control and we also performed KD of hTim22 , which allowed us to monitor the implications of shutting down the carrier pathway ( Figure 4—figure supplement 1A and B ) .", "To address the global impact of Tim29 and hTim22 KD on cell health and viability we monitored the impact of Tim29 and hTim22 KD on mitochondrial respiration , cell proliferation and cell viability .", "We measured mitochondrial oxygen consumption rates in intact cells by high-resolution respirometry to determine if the KD of Tim29 and hTim22 affects mitochondrial metabolism .", "Knockdown of Tim29 significantly reduced basal mitochondrial oxygen consumption to 65% ( p<0 . 05 ) compared to cells transfected with a control scrambled siRNA ( Figure 4—figure supplement 1C ) .", "This reduction in basal oxygen consumption was similar to that observed in hTim22 KD cells ( 61% residual respiration , p<0 . 05 ) .", "Following addition of FCCP , the maximal mitochondrial oxygen consumption rate was also significantly lower in Tim29 KD cells compared to control cells ( 88% residual respiration , p<0 . 05 ) and in hTim22 knockdown cells compared to the control ( 63% , p<0 . 05 ) .", "KD of both Tim29 and hTim22 did not have any significant impact on cell proliferation ( Figure 4—figure supplement 1D ) , or cell viability ( Figure 4—figure supplement 1E ) .", "Next we assessed the impact of KD of Tim29 on TIM22 complex substrates at the steady state protein level .", "As can be seen , Tim29 ( Figure 4A ) and hTim22 ( Figure 4B ) were depleted from mitochondria while the control protein Mfn2 remained unaffected ( Figure 4A and B , bottom panels ) .", "Specific depletion of TIM22 complex substrates , including hTim23 , ANT3 and the glutamate carrier was observed in both Tim29 and hTim22 depleted mitochondria ( quantifications shown in Figure 4A and B , lower panels ) .", "The depletion of Tim29 also led to a significant reduction in the levels of hTim22 ( Figure 4A ) , while the lack of hTim22 had no obvious effect on the levels of Tim29 , indicating Tim29 is imported in a TIM22-independent manner ( Figure 4B ) .", "BN-PAGE analysis also revealed that depletion of Tim29 caused a reduction in the assembly of ANT3 and hTIM23 complexes ( Figure 4C ) , with the latter causing a reduction in the steady state level of TIM23 complex substrates ( COXIV , NDUFV2 and NDUFV1 ) ( Figure 4—figure supplement 2A ) and import of [35S]-NDUFV1 and [35S]-NDUFV2 ( Figure 4—figure supplement 2B and C ) . 10 . 7554/eLife . 17463 . 010Figure 4 . Knockdown of Tim29 reduces the steady state protein levels of TIM22 substrates .", "( A ) HEK293FS cells were transfected with scrambled ( control ) or Tim29 siRNA targets for 96 hr .", "Following knock-down mitochondria were isolated from cells and analysed using SDS-PAGE and western blotting analysis with the indicated antibodies .", "Protein levels were quantified and normalised against the loading control , Mfn2 .", "The amount of each protein in control cells was set to 100% .", "Data are expressed as mean ± SD , n = 3 .", "( B ) Mitochondria were isolated from control cells ( scrambled siRNA ) or cells transfected with hTim22 siRNA target for 96 hr .", "Mitochondrial proteins were subjected to SDS-PAGE and immunoblotting using the indicated antibodies and quantified as described above ( C & D ) Mitochondria were isolated from control or Tim29 knock down ( KD ) cells and solubilised in 1% digitonin-containing buffer .", "Protein complexes were analysed by BN-PAGE and western blotting . DOI: http://dx . doi . org/10 . 7554/eLife . 17463 . 01010 . 7554/eLife . 17463 . 011Figure 4—figure supplement 1 . Cellular consequences of Tim29 depletion .", "( A ) HEK293FS cells were transfected with scrambled ( control ) or Tim29 siRNA targets for 72 hr .", "Following knock-down mitochondria were isolated from cells and analysed using SDS-PAGE and western blotting analysis with the indicated antibodies .", "( B ) HEK293FS cells were transfected with scrambled ( control ) or hTim22 siRNA targets for 72 hr .", "Following knock-down mitochondria were isolated from cells and analysed using SDS-PAGE and western blotting analysis with the indicated antibodies .", "( C ) Oxygen consumption was measured in HEK293FS cells following knockdown of Tim29 or hTim22 ( as described in A and B ) .", "Cells transfected with a scrambled siRNA were used as the control .", "Knockdown of either Tim29 or hTim22 significantly reduced both basal and maximal mitochondrial oxygen consumption rates .", "Data expressed as mean ± S . D . , n = 3 .", "( D ) Proliferation of control , Tim29 and hTIm22 KD cells were determined by BrdU incorporation for 24 hr .", "The level of BrdU incorporation was measured at 450–550 nm ( background ) .", "Data reported as mean ± SD ( n = 3 ) .", "( E ) Cells transfected with scrambled ( control ) or Tim29 siRNA targets were treated with propidium iodide ( PI ) 72 hr post transfection and analysed using flow cytometry for% of cell death .", "Data are shown as mean ± SD ( n = 3 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 17463 . 01110 . 7554/eLife . 17463 . 012Figure 4—figure supplement 2 . TIM23 complex substrates are reduced in cells depleted of Tim29 . ( A ) Mitochondria were isolated from control cells ( scrambled siRNA ) or cells transfected with Tim29 siRNA target for 72 hr .", "Mitochondrial proteins were subjected to SDS-PAGE and immunoblotting using the indicated antibodies .", "The levels of the TIM23 complex substrates , COXIV , NDUFV2 and NDUFV1 was quantified relative to protein levels in control mitochondria and normalised against the loading control Mfn2 .", "Data are shown as mean ± SD ( n = 3 ) .", "( B and C ) [35S]-labelled NDUFV1 and NDUFV3 were imported into mitochondria isolated from control and Tim29 knockdown cells for the indicated times .", "Re-isolated mitochondria were treated with protease and separated by SDS-PAGE and analysed by autoradiography .", "For quantification of imported NDUFV1 and NDUFV3 , the longest incubation time ( 45 min ) was set to 100% .", "Data are shown as mean ± SD ( n = 2 , NDUFV1; n = 3 NDUFV3 ) .", "p , precursor and m , mature . DOI: http://dx . doi . org/10 . 7554/eLife . 17463 . 012 The decrease in Tim29 protein levels also revealed a reduction in the levels of the endogenous TIM22 complex on BN-PAGE ( Figure 4D ) .", "This observation and the decrease of endogenous TIM22 complex substrates in Tim29 KD cells , prompted us to address if this decrease was due to:", "( i ) an active role of Tim29 in the import of substrates into the inner membrane , or", "( ii ) a role of Tim29 in the biogenesis of the TIM22 complex itself .", "To address this , we investigated the in vitro import and assembly of two well-established substrates of the TIM22 pathway , ANT1 and hTim23 .", "While the assembly of ANT1 was reduced in the absence of hTim22 at 60 min ( Figure 5A; right panel ) , assembly of the protein proceeded like WT in the absence of Tim29 ( Figure 5A; left panel ) .", "In contrast , the assembly of hTim23 was mildly reduced in mitochondria depleted of either Tim29 or hTim22 ( Figure 5B ) .", "We next addressed the assembly of the TIM22 complex itself using the radiolabelled hTim22 .", "In mitochondria isolated from control cells , [35S]-hTim22 efficiently assembled into the 450-kDa TIM22 complex ( Figure 6A and B , lanes 1–4 ) , while there was a clear assembly defect into the TIM22 complex in mitochondria isolated from Tim29 KD cells ( Figure 6A , lanes 5–8 ) .", "The amount of assembled TIM22 complex in Tim29 KD mitochondria was lower than that in the control mitochondria by 53% , after a 60 min-import .", "To support this observation , we examined the difference in hTim22 protein assembly in control and Tim29 KD mitochondria using a two-tailed Student’s t-test .", "We obtained a t[2] = 6 . 02 ( p<0 . 05 ) , demonstrating a significant reduction in the assembly of hTim22 into the mature complex in Tim29 KD mitochondria .", "On the contrary , we consistently detected a moderate increase ( ~20% ) in assembled TIM22 complex in hTim22 KD mitochondria after a 60 min-import compared to control mitochondria ( t[2] = −5 . 07 , p<0 . 05 ) . 10 . 7554/eLife . 17463 . 013Figure 5 . Knockdown of Tim29 influences the assembly of the TIM23 complex , but not ANT1 . ( A ) [35S]-labelled ANT1 was imported into mitochondria isolated from control or Tim29 ( left panel ) or hTim22 ( right panel ) knock-down ( KD ) cells for the indicated times in the presence or absence of membrane potential ( Δψ ) .", "Re-isolated mitochondria were treated with 50 µg/ml of Proteinase K prior to solubilisation in digitonin-containing buffer .", "Mitochondrial lysates were analysed by BN-PAGE and autoradiography .", "Assembled ANT1 was quantified from three independent experiments .", "The amount of assembled ANT1 in control mitochondria at 60 min was set to 100% .", "Data are shown as mean ± SD ( n = 3 ) .", "( B ) Import of [35S]-hTim23 was performed and quantified as described above . DOI: http://dx . doi . org/10 . 7554/eLife . 17463 . 01310 . 7554/eLife . 17463 . 014Figure 6 . Tim29 is required for the assembly of hTim22 into the mature TIM22 complex .", "( A ) [35S]-hTim22 was imported into mitochondria isolated from control ( scrambled siRNA ) or Tim29 knock-down ( KD ) cells for the indicated times in the presence or absence of membrane potential ( Δψ ) .", "Re-isolated mitochondria were treated with 50 µg/ml of Proteinase K prior to solubilisation in digitonin-containing buffer .", "Mitochondrial lysates were analysed by BN-PAGE and autoradiography .", "Assembled hTim22 was quantified from three independent experiments .", "The amount of assembled hTim22 in control mitochondria at 60 min was set to 100% .", "Data are shown as mean ± SD ( n = 3 ) .", "( B ) [35S]-hTim22 was imported into mitochondria isolated from control ( scrambled siRNA ) or hTim22 knock-down cells and treated as described in ( A ) .", "The amount of assembled hTim22 in control mitochondria at 60 min was set to 100% .", "Data are represented as mean ± SD ( n = 3 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 17463 . 014 In the absence of Tim29 we did observe a minor decrease in the import kinetics of [35S]-hTim22 by SDS-PAGE ( Figure 7 ) , however , following treatment of isolated mitochondria with sodium carbonate it was apparent that [35S]-hTim22 protein efficiently integrated into the inner membrane in mitochondria isolated from both Tim29 and hTim22 KD cells ( Figure 7C and D ) .", "Based on these observations , we conclude that Tim29 is required for the assembly of the hTim22 protein into the TIM22 complex . 10 . 7554/eLife . 17463 . 015Figure 7 . hTim22 is integrated into the inner membrane in the absence of Tim29 . ( A and B ) [35S]-hTim22 was imported into mitochondria isolated from control ( scrambled siRNA ) , and Tim29 ( A ) or hTim22 ( B ) knock-down cells for the indicated times in the presence or absence of membrane potential ( Δψ ) and analysed by SDS-PAGE .", "The import efficiency of [35S]-hTim22 into control or Tim29/hTim22 knock-down mitochondria was quantified ( mean ± SD , n = 3 ) and the yield at the longest import time into the control mitochondria was set to 100% .", "( C and D ) [35S]-hTim22 was imported into mitochondria isolated from control ( scrambled siRNA ) , and Tim29 ( C ) or hTim22 ( D ) knock-down cells for 45 min .", "Mitochondria were isolated and treated with sodium carbonate and separated into a membrane integrated ( pellet , P ) and peripheral membrane protein ( supernatant , S ) .", "WB , indicates western blot . DOI: http://dx . doi . org/10 . 7554/eLife . 17463 . 015 Coupling of the TOM and TIM23 complexes in yeast mitochondria is believed to enhance the import efficiency of presequence-containing precursors ( Chacinska et al . , 2005 , 2003; Waegemann et al . , 2015; Mokranjac et al . , 2009; Popov-Čeleketić et al . , 2008 ) .", "More recently , coupling between the TOM and SAM complexes of the outer membrane in yeast mitochondria has been shown to be important for the biogenesis of β-barrel proteins ( Wenz et al . , 2015; Qiu et al . , 2013 ) .", "Interestingly , no interaction has ever been reported between the TOM and TIM22 translocases in yeast mitochondria .", "Rather , the carrier pathway is described as a series of five stages , with stage 3 encompassing the transfer of substrates between the TOM and TIM22 complexes by the soluble intermembrane space Tim9-Tim10 complex ( Chacinska et al . , 2009; Rehling et al . , 2004; Ryan et al . , 1999 ) .", "Given that Tim29 exposes a domain into the intermembrane space , we reasoned it could have a role in linking the TIM22 complex to the TOM complex in mammalian mitochondria .", "Mitochondria were isolated from control and Tim293XFLAG expressing cells , solubilised in digitonin and incubated with anti-FLAG resin for immunoprecipitation of the FLAG-tagged proteins and associated partners .", "As expected , we observed co-elution of hTim9 with Tim293XFLAG .", "Interestingly , we observed a fraction of both hTom40 and hTom22 co-eluting with Tim293XFLAG , suggesting a co-operation between Tim29 and the TOM complex ( Figure 8A ) .", "To confirm this interaction , we imported radiolabelled versions of hTom40 , hTom22 and hTim9 ( positive control ) into mitochondria isolated from control and Tim293XFLAG expressing cells and following import subjected the isolated mitochondria to immunoprecipitation with anti-FLAG resin ( Figure 8B ) .", "Indeed a fraction of both hTom40 and hTom22 were observed in the Tim29 elution fraction ( Figure 8B , middle and lower panels ) , like that of the control protein hTim9 ( Figure 8B , upper panel ) . 10 . 7554/eLife . 17463 . 016Figure 8 . Tim29 couples the TIM22 complex to the TOM complex .", "( A ) Mitochondria were isolated from control and Tim293XFLAG expressing cells and were solubilised in 0 . 5% digitonin-containing buffer prior to immunoprecipitation with anti-FLAG resin .", "Total ( 10% ) and Elution ( 100% ) fractions were collected and analysed using SDS-PAGE and immunoblotting using the indicated antibodies .", "( B ) [35S]-hTim9 , [35S]-hTom40 and [35S]-hTom22 were imported into mitochondria isolated from control cells or cells expressing Tim293XFLAG for 60 min .", "Following import samples were solubilised in 0 . 5% digitonin-containing buffer prior to immunoprecipitation with anti-FLAG resin .", "Total ( T; 5% ) , Unbound ( UB; 5% ) and Elution ( E; 100% ) fractions were separated on SDS-PAGE followed by autoradiography and western blotting using Mfn2 antibody .", "( C ) Control and 3xFLAGhTom22-harbouring mitochondria were lysed in 0 . 5% digitonin-containing buffer .", "Mitochondrial extracts were subjected to immunoprecipitation and were separated on SDS-PAGE prior to immunoblotting analysis .", "Total , 2% and Elution , 100% .", "DOI: http://dx . doi . org/10 . 7554/eLife . 17463 . 01610 . 7554/eLife . 17463 . 017Figure 8—figure supplement 1 . 3XFLAGhTom22 localises to mitochondria and assembles within the TOM complex .", "( A ) HeLa cells were transiently transfected with DNA encoding 3XFLAGhTom22 and analysed by immunofluorescence using anti-FLAG antibodies ( green ) and anti-NDUFAF2 ( mitochondrial marker- red ) .", "Cells were visualisation using fluorescence microscopy .", "Scale bar: 10 µm .", "( B ) Mitochondria were isolated from cells transiently transfected with DNA encoding 3XFLAGhTom22 .", "Mitochondria ( lanes 1 and 2 ) and mitoplasts ( lanes 3 and 4 ) were left untreated ( lanes 1 and 3 ) or treated with Proteinase K ( lanes 2 and 4 ) .", "Intact mitochondria were also treated with sodium carbonate to separate mitochondrial proteins into membrane integrated ( pellet , P ) or peripheral ( supernatant , S ) proteins .", "Samples were analysed by SDS-PAGE and western blotting .", "( C ) Mitochondria isolated from 3XFLAGhTom22 expressing cells were solubilised in digitonin-containing buffer and analysed by BN-PAGE and immunoblotting with the indicated antibodies . DOI: http://dx . doi . org/10 . 7554/eLife . 17463 . 017 If the interaction we observed between TIM22 and TOM was authentic , it should be possible to perform the reverse experiment , i . e . copurification of the TIM22 complex with a tagged TOM complex .", "We generated an N-terminally-tagged variant of TOM22 , which localised to mitochondria and assembles into the TOM complex ( Figure 8—figure supplement 1 ) .", "Upon solubilisation of the 3XFLAGhTom22 mitochondria and immunoprecipitation we observed co-purification of hTom40 and hTom70 ( Figure 8C ) as expected .", "We also observed a fraction of hTim9 , Tim29 and hTim22 co-purifying with the TOM complex , while numerous controls of the outer membrane ( hFis1 ) , intermembrane space ( cytochrome c ) , inner membrane ( OPA1 , hTim23 , Glutamate carrier , COXIV ) and matrix ( NDUFV1 , NDUFV2 ) did not co-elute with hTom22 ( Figure 8C ) .", "Given that the TIM22 complex was not purified in great amounts , the data suggest the interaction between TOM and TIM22 complexes is labile in the absence of precursor protein , or under the conditions employed for the immunoprecipitation .", "To explore the relationship between TOM and TIM22 further , we performed chemical cross-linking with dithiobis ( succinimidylpropionate ) ( DSP ) , an amino-group-specific homobifunctional crosslinker that is cleaved by reducing agents .", "Mitochondria isolated from control and Tim293XFLAG expressing cells were incubated with DSP , solubilised in SDS to dissociate non-covalent interactions and then incubated with anti-FLAG resin for purification of Tim293XFLAG-containing species .", "Eluate fractions were treated under reducing conditions in order to liberate cross-linked species .", "Interestingly , we found hTom40 cross-linked to Tim29 ( Figure 9A and B ) , but not hTom22 ( Figure 9B , third panel from top ) .", "We also observed crosslinks between Tim29 and hTim9 , but not hTim22 ( most likely due to the lack of primary amines in the predicted Tim29 transmembrane domain , which are essential for the amine-reactive crosslinker DSP ) ( Figure 9A ) .", "We next performed cross-linking using mitochondria isolated from cells expressing hTim10b3XFLAG and although we observed cross-links of hTim10b with both hTim9 and Tim29 we did not observe any crosslinks with hTom40 ( Figure 9C ) .", "Taken together , these results suggest that Tim29 acts as a bridge between the TIM22 and TOM complexes . 10 . 7554/eLife . 17463 . 018Figure 9 . Tim29 interacts with hTom40 . ( A and B ) Mitochondria isolated from control and Tim293XFLAG expressing cells were incubated with 0 . 2 mM DSP .", "Upon quenching of excess cross-linker , mitochondria were solubilised in SDS-containing buffer and subjected to immunoprecipitation using anti-FLAG resin .", "Cross-linked species were cleaved using the Laemmli buffer .", "Total ( 3% ) and Elution ( 100% ) fractions were analysed using SDS-PAGE and western blotting with the indicated antibodies .", "( C ) Mitochondria isolated from control and hTim10b3XFLAG expressing cells were treated as described for A and B . DOI: http://dx . doi . org/10 . 7554/eLife . 17463 . 018" ], [ "In this study , we have identified C19orf52 as a new subunit of the human TIM22 complex , and have retitled this protein Tim29 .", "While the yeast TIM22 complex and import pathway have been extensively characterised , the human TIM22 complex has remained ill defined .", "Conservation of members of the human TIM22 complex ( hTim22 and small TIM proteins ) based on sequence homology was originally published approximately 17 years ago ( Bauer et al . , 1999 ) .", "Advances and accessibility in techniques for purification of native complexes and mass spectrometry have now provided us with an opportunity to unravel the true subunit composition of the human complex .", "Our work brings the number of known subunits of the human TIM22 complex to five: hTim22 ( Sirrenberg et al . , 1996; Bauer et al . , 1999 ) , hTim9 , hTim10a , hTim10b ( Mühlenbein et al . , 2004; Gentle et al . , 2007; Bauer et al . , 1999 ) and Tim29 ( this study ) .", "Interestingly , our immunoprecipitations and mass spectrometry data for both hTim10b3XFLAG and C19orf523XFLAG did not reveal the presence of SDHC , suggesting the human protein is not a subunit of the human TIM22 complex , at least under the conditions in which we perform the immunoprecipitation experiments .", "Gebert et al . ( 2011 ) showed that the assembly of yeast Sdh3 at both Complex II and TIM22 is influenced by the formation of different sub-complexes with the highly homologous Sdh4 and Tim18 .", "The Sdh3-Sdh4 and Sdh3-Tim18 subcomplexes subsequently deliver the protein to either Complex II or TIM22 , respectively .", "It is feasible to suggest that the lack of Tim18 in human mitochondria could prevent the association of SDHC to the hTIM22 complex .", "Tim29 was a previously uncharacterised protein , however the MitoCarta 2 . 0 inventory indicated the protein was a mitochondrial protein with a wide tissue distribution ( Calvo et al . , 2016 ) .", "Mitochondrial subfractionation and carbonate extraction confirmed that Tim29 is an integral protein of the mitochondrial inner membrane .", "Given that there is only one predicted transmembrane region , we propose Tim29 is inserted into the mitochondrial inner membrane via this single transmembrane segment , with its amino-terminus facing the matrix and carboxyl-terminus exposed to the intermembrane space .", "Interestingly , we confirmed that targeting and proper sorting of Tim29 to the inner membrane relies on the first 89 amino acids of the protein .", "Although fluorescence microscopy suggested that constructs lacking the first 16 amino acids ( Tim29△16 and Tim29aa17-89 ) could be targeted to mitochondria , closer investigation using mitochondrial subfractionation and carbonate extraction reveals these proteins failed to sort to the inner membrane .", "Given this observation and that the knockdown of hTim22 did not influence the levels of Tim29 , the protein is most likely imported via the TIM23 complex , however this needs to be experimentally verified .", "Knockdown of Tim29 resulted in a reduction in the steady-state levels of ANT , but import and assembly kinetics of radiolabelled ANT were similar to control mitochondria .", "In contrast , hTim23 , which is also a substrate for the hTIM22 complex showed an assembly defect both in vitro and at the steady state level .", "Such a scenario has previously been reported for the yeast TIM22 complex , with deletion of Tim18 differentially affecting the import kinetics of the AAC and Tim23 ( Kerscher et al . , 2000; Koehler et al . , 2000 ) .", "It is possible that depletion of Tim29 affects the activity of the human TIM23 complex by a more direct mechanism , whereas the import of ANT only requires the TIM22 complex and the residual complex remaining following knockdown is enough to import and assemble the small amounts of radiolabelled protein used in the in vitro import assay .", "The depletion of Tim29 also caused a reduction in the levels of the TIM22 complex when assessed by BN-PAGE .", "Given that hTim22 is integrated into the membrane in mitochondria isolated from the Tim29 KD cells , we conclude that Tim29 is required for the assembly of hTim22 into the TIM22 complex .", "In addition to this role our data indicate that Tim29 facilitates cooperation between the TIM22 and TOM complexes , by directly interacting with hTom40 .", "Cooperation between the TOM and TIM23 complexes is well established in yeast cells ( Chacinska et al . , 2005 , 2003; Waegemann et al . , 2015; Mokranjac et al . , 2009; Popov-Čeleketić et al . , 2008; Chacinska et al . , 2010 ) , however a similar interaction between the TOM and TIM22 complex has not been reported thus far .", "We have captured an interaction between the TOM and TIM22 complexes in organello using both Tim29 and hTom22 as bait .", "Furthermore , using chemical crosslinking we narrowed down specific interactions between:", "( i ) Tim29 and hTim9;", "( ii ) Tim29 and hTim10b; hTim10b and hTim9; and Tim29 and hTom40 .", "We were puzzled to see an interaction between Tim29 and hTom40 , since in yeast TIM23 interacts with TOM via Tom22 , which exposes a domain into the intermembrane space ( Waegemann et al . , 2015; Chacinska et al . , 2010 ) .", "However , Shiota and colleagues ( 2015 ) recently showed that the intermembrane space chaperone , Tim10 interacts with an N-terminal region of Tom40 .", "This suggests that the N-terminal region of Tom40 extends through the β-barrel pore into the intermembrane space ( Shiota et al . , 2015 ) , and such a mechanism could be mediating the interaction between hTom40 and the intermembrane space localised region of Tim29 .", "In yeast the intermembrane space chaperones Tim9-Tim10 passage carrier precursors through the aqueous intermembrane space delivering them from TOM to TIM22 ( Chacinska et al . , 2009; Rehling et al . , 2004; Ryan et al . , 1999 ) .", "Why would a TOM-TIM22 interaction be necessary in human mitochondria ?", "The answer could lie with the nature of the small TIM chaperones within mammalian mitochondria .", "Early studies by Mühlenbein and colleagues ( 2004 ) suggested that human mitochondria lack soluble Tim9-Tim10 complexes , but rather these proteins are more tightly associated with the mitochondrial inner membrane .", "Thus , even though the function of the small TIM proteins has been conserved through evolution , the import of carrier proteins into mammalian mitochondria is efficient even in the absence of soluble Tim9-Tim10 complexes .", "Therefore , it is likely that other mechanisms have evolved that serve to prevent the hydrophobic carrier proteins from aggregating in the intermembrane space .", "A close association between the TOM and TIM22 complexes could be one such mechanism .", "Our data represents a major conceptual advance and challenges the common dogma that carrier proteins are transported across the mitochondrial intermembrane space via soluble intermediates .", "Further investigation is necessary to tease out the precise role of the TOM-TIM22 interaction and how it is influenced by the presence of precursor protein .", "In summary , by assessing the subunit composition of the human TIM22 complex , we have identified Tim29 and uncovered a novel interaction between the TIM22 and TOM complexes .", "This suggests that novel mechanisms are overseeing the import of carrier proteins into human mitochondria .", "Interestingly , Tim29 ( C19orf52 ) was recently shown to be up-regulated in primary hepatocellular carcinoma cells ( Xing et al . , 2015a , 2015b ) .", "Indeed , numerous human pathologies have been linked to protein import dysfunction ( Sokol et al . , 2014; Xu et al . , 2010; Sinha et al . , 2014; Di Fonzo et al . , 2009; Koehler et al . , 1999; Roesch et al . , 2002; Sankala et al . , 2011; Davey et al . , 2006 ) .", "Thus , examining protein import machineries and pathways in human cells is essential and will pave the way to deeper understanding of how mitochondrial protein import influences cell viability , physiology and ultimately human pathologies ." ], [ "Cell lines used in this work were Flp-IN T-REx 293 ( purchased from Thermo Fisher Scientific Australia; RRID:CVCL_U427 ) ; FreeStyle 293-F ( HEK293FS; purchased from ( Thermo Fisher Scientific; RRID:CVCL_D603 ) and HeLa cells ( RRID:CVCL_0030 ) ; kindly provided by Prof . Paul Gleeson , The University of Melbourne ) .", "All cell lines were mycoplasma negative based on screening using MycoAlert Mycoplasma Detection Kit ( Lonza ) .", "Cell lines were authenticated using short-tandem repeat ( STR ) typing using the Cell Line Authentication Service at the Garvan Institute of Medical Research ( Sydney , Australia ) .", "Cells were grown at 37°C and 5% CO2 in Dulbecco’s modified Eagle’s medium ( DMEM; Thermo Fisher Scientific ) supplemented with 5–10% ( v/v ) fetal bovine serum ( FBS; in vitro Technologies , Australia ) .", "Stable tetracycline-inducible cell lines ( Tim10b3XFLAG and Tim293XFLAG ) were generated using the Flp-IN T-REx System ( Thermo Fisher Scientific ) .", "Briefly , Flp-IN T-REx HEK293T cells were co-transfected with the plasmid pOG44 ( allowing expression of Flp-recombinase ) and the desired open reading frame cloned into pCDNA5 /FRT/TO ( modified to contain a 3X FLAG tag ) .", "We simultaneously generated a control cell line transfected with empty pCDNA5 /FRT/TO .", "Transfected cells were placed under selection with hygromycin at a concentration of 200 µg/ml .", "Cells were incubated until individual colonies appeared and these were selected and expanded and screened for protein expression by the addition of tetracycline at 1 µg/ml .", "Cells were cultured in with 5–10% ( v/v ) tetracycline-reduced FBS ( Clontech ) .", "For ectopic expression and generation of stable cells Lipofectamine 2000 was used according to the manufacturer’s guidelines ( Thermo Fischer Scientific ) .", "Custom siRNA oligonucleotides were purchased from Sigma-Adrich .", "The following sequences and concentrations were utilised: Tim29 ( 5’ GGCUCUUCGAUGAGAAGUA 3’ , 10 nM ) and hTim22 ( 5’ CCAUUGUGGGAGCCAUGUU 3’ , 10 nM ) .", "For RNA interference HEK293FS cells were ( 9 × 105 cells ) were transfected with DharmaFECT1 transfection reagent according to the manufacturer’s instructions ( Dharmacon ) .", "Cells were analysed after 72 or 96 hr post-transfection for protein knock-down .", "For immunofluorescence , cells grown on coverslips were fixed by incubation with 4% ( w/v ) paraformaldehyde solution ( Sigma-Aldrich ) for 10 min at room temperature and permeabilised by incubation with PBS containing 0 . 2% ( v/v ) Triton X-100 .", "This was followed by incubation with the desired primary antibody made up in 3% ( w/v ) bovine serum albumin , 0 . 2% ( v/v ) Triton X-100 in PBS for 1 hr .", "Following washing in 0 . 2% ( v/v ) TX-100 cells were incubated with secondary antibody anti-mouse Alexa Fluor 488 ( RRID:AB_2534069 ) or anti-rabbit Alexa Fluor 568 ( RRID:AB_2534078; Molecular Probes ) .", "For nuclear staining , cells were incubated with 0 . 01 mg/ml Hoechst 33258 ( Sigma-Aldrich ) in PBS for 5 min following incubation with secondary antibodies .", "Cells were analysed using Deltavision DV Elite Imaging System ( Applied Precision ) and then deconvolved images were analysed using ImageJ .", "Isolation of mitochondria from tissue culture cells was performed by differential centrifugation as described previously ( Johnston et al . , 2002 ) .", "For mitochondrial sub-fractionation experiments mitochondrial pellets ( 50 µg of mitochondrial protein ) were resuspended in the isolation buffer ( 20 mM HEPES-KOH pH 7 . 4 , 70 mM sucrose , 220 mM Mannitol , 1 mM EDTA ) , swelling buffer ( 10 mM HEPES-KOH pH 7 . 4 ) or solubilisation buffer ( 0 . 5% [v/v] Triton-X-100 ) .", "Samples were split and either left untreated or treated with proteinase K ( 50 µg/ml ) for 10 min on ice .", "For sodium carbonate extraction , mitochondrial pellets ( 50 µg ) were resuspended in freshly prepared Na2CO3 ( 100 mM , pH 11 or pH 12 ) .", "Samples were incubated on ice for 30 min with occasional mixing and subsequently centrifuged at 100 , 000 g for 30 min .", "Mitochondrial subfractionation and carbonate extraction samples were TCA precipitated and analysed by SDS- PAGE and immunoblotting .", "For crosslinking experiments , mitochondria ( 0 . 5–1 mg ) were resuspended in import buffer ( 20 mM HEPES-KOH pH 7 . 4 , 250 mM sucrose , 80 mM KOAc , 5 mM MgOAc , and 5 mM ATP ) and incubated with the amino-group-specific homobifunctional and cleavable crosslinker , DSP; dithiobis ( succinimidylpropionate ) ( ThermoFisher ) at a final concentration of 0 . 2 mM for 1 hr at 4°C .", "Crosslinking reactions were quenched using 100 mM Tris-Cl , pH 7 . 4 for 30 min at 4°C .", "Mitochondria were reisolated and solubilised in lysis buffer ( 20 mM Tris-Cl , pH 7 . 4 , 1 mM EDTA , 1% ( w/v ) SDS ) and boiled at 95°C for 5 min .", "Clarified samples were diluted with 1% Triton X-100-containing buffer ( 1% ( v/v ) Triton X-100 , 20 mM Tris-Cl pH 7 . 4 , 150 mM NaCl , 1X complete protease inhibitor ) and incubated with pre-equilibrated anti-FLAG resin for 1 hr at 4°C .", "Following washing specifically bound proteins were eluted in 2X Laemmli buffer .", "Open reading frames ( ORF ) encoding mitochondrial precursor proteins were cloned into pGEM4Z ( Promega ) .", "The desired ORFs were amplified by PCR using vector specific primers ( M13_FORWARD and M13_REVERSE ) and applied to in vitro transcription reactions using the mMessage SP6 transcription kit ( Ambion ) .", "mRNA was isolated by LiCl precipitation according to the manufacturer’s instructions and applied to in vitro translation reactions using rabbit reticulocyte lysate ( Promega ) in the presence of [35S]-methionine/cysteine ( Perkin-Elmer ) .", "Isolated mitochondria were incubated with translation products in import buffer ( 20 mM HEPES-KOH pH 7 . 4 , 250 mM sucrose , 80 mM KOAc , 5 mM MgOAc , 10 mM sodium succinate , 1 mM DTT and 5 mM ATP ) at 37°C for various times as indicated in the figure legends .", "Samples subjected to protease treatment were incubated on ice for 10 min with 50 μg/ml proteinase K ( Sigma-Aldrich ) , followed by the addition of 1 mM PMSF and incubation for 10 min on ice .", "For analysis of protein complexes by blue native electrophoresis and antibody-shift mitochondria were solubilised in 1% digitonin ( at 1 mg/mL ) and incubated with the desired antibody for 30 min on ice , prior to clarification and subsequent analysis by BN-PAGE .", "Following in vitro import samples were separated by SDS-PAGE and BN-PAGE and radiolabelled proteins were detected by digital autoradiography .", "Protein import assays were performed in triplicate unless indicated otherwise .", "[35S]-proteins were visualised using Typhoon PhosphorImage ( GE healthcare ) and were analysed using ImageJ software ( RRID:SCR_003070 ) .", "The intensity of relevant bands was determined by subtracting the background signal to give the true relative amount of protein ( assembled complexes for BN-PAGE analysis and imported proteins for SDS-PAGE analysis ) .", "Each specific time point was normalised against the value in control mitochondria with the longest import time point and were computed as a % of this control .", "Differences in the import of proteins between control ( scrambled siRNA ) and Tim29 or hTim22 knock-down mitochondria were examined using a paired two-tailed Student’s t-test on 2 d . f . based on the three log ratios ( control/knockdown ) of imported or assembled protein .", "Mitochondrial oxygen consumption was measured by high-resolution respirometry with an Oxygraph-2K oxygen electrode ( Oroboros Instruments , Innsbruck , Austria ) as previously described ( Lim et al . , 2015 ) .", "Intact cells were incubated in 2 mL of Dulbecco's Modified Eagle Medium ( DMEM , high glucose , pyruvate , Glutamax ) ( ThermoFisher ) supplemented with 5% v/v fetal bovine serum and 1x penicillin/streptomycin at 37°C .", "Basal oxygen consumption rates were recorded , followed by the addition of 10 μM carbonyl cyanide p-trifluoromethoxyphenylhydrazone ( FCCP ) to obtain maximal oxygen consumption rates .", "Non-mitochondrial oxygen consumption rates were determined by the addition of 5 μM antimycin A and subtracted from basal and maximal rates .", "Oxygen flux was calculated using DatLab software ( version 4 . 3 . 4 . 51 , Oroboros Instruments ) and expressed as pmol/s/mg of total cell protein .", "Each assay was performed in triplicate , with significant differences determined using a two-tailed Student’s t-test .", "Proliferation of control ( scrambled siRNA ) , hTim22 and Tim29 siRNA transfected cells was examined using the bromodeoxyuridine ( BrdU ) Cell Proliferation ELISA kit ( Abcam-ab126556 ) .", "BrdU reagent was added to cells 48 hr post siRNA transfection and incubated with cells for 24 hr .", "BrdU incorporation into newly synthesised DNA was measured by absorbance at 450 nm and 550 nm ( background ) using a FLUOstar OPTIMA microplate reader ( BMG LABTECH ) .", "Assays were performed in triplicate .", "Cell viability was determined using propidium iodide staining .", "Briefly , cells were harvested and washed and resuspended in FACS buffer ( 0 . 1% BSA diluted in PBS , 2 mM EDTA ) followed by incubation with 3 . 1 µg/mL of propidium iodide ( Sigma ) .", "Cells were screened by flow cytometry using Flowjo software ( RRID: SCR_008520 ) .", "Plots of the area of forward-scattered light ( FSC-A ) versus side-scattered light ( SSC-A ) were used as the cell gating strategy for identification of any changes in the scatter properties of the cells .", "Single cell events were identified using the height and width parameters in FSC and SSC .", "A dot plot using the area of propidium iodide ( PI-A ) was used to determine the relative proportion of viable and dead cells ( % ) .", "For Blue-native gel electrophoresis mitochondrial pellets were solubilised in ice-cold digitonin-containing buffer ( 1% [w/v] digitonin , 20 mM Tris , pH 7 . 0 , 0 . 1 mM EDTA , 50 mM NaCl , 10% [w/v] glycerol ) at 1 mg/mL and incubated on ice for 15–20 min .", "Samples were clarified by centrifugation at 16 , 000 g for 10 min at 4°C , upon which sample buffer ( 1/10th the volume- 5% [w/v] Coomassie brilliant blue G-250 , 100 mM Bis-Tris , pH 7 . 0 , 500 mM ε-amino-n-caproic acid ) was added .", "Samples were separated on 4–16% polyacrylamide gradient gels at 4°C .", "Thyroglobulin ( 669 kDa ) , Ferritin ( 440 kDa ) and Bovine Serum Albumin ( 132 and 66 kDa ) were used as molecular weight markers .", "SDS-PAGE was performed using gradient Tris-tricine gels as previously described ( Schägger and von Jagow , 1987 ) .", "For western blot analysis , gels were transferred to PVDF membranes .", "Primary antibodies included: anti-FLAG ( Sigma; F1804; RRID:AB_262044 ) , cytochrome c ( BD Biosciences; 556433; RRID:AB_396417 ) , anti-OPA1 ( BD Biosciences; 612606; RRID:AB_399888 ) , hTim23 ( BD Biosciences; 611223; RRID:AB_398755 ) , SDHA ( Abcam; ab14715; RRID:AB_301433 ) ; ANT3 ( Abcam; ab154007; RRID:AB_2619664 ) , glutamate carrier ( Abcam; ab137614; RRID:AB_2619663 ) , hTim22 ( Sigma; T8954; RRID:AB_1858017 ) , Tim29 ( Sigma; HPA041858; RRID:AB_10963429 ) ; Tim9 ( Abcam; ab57089; RRID:AB_945837 ) ; NDUFV1 ( Proteintech; 11238-1-AP , RRID: AB_2149040 ) ; NDUFV2 ( Proteintech; 15301-1-AP; RRID:AB_2149048 ) ; Tom70 ( ProteinTech 14528-1-AP; RRID:AB_2303727 ) ; COXIV ( Cell Signaling , 4850; RRID:AB_2085424 ) .", "Antibodies against human Bak , human Tom22 , human Tom40 , Mfn2 , NDUFAF2 and NDUFA9 were kindly provided by Prof . Michael Ryan ( Monash University , Australia ) .", "Standard immunoprecipitation experiments were performed with 0 . 5–1 mg mitochondria and analysed by SDS-PAGE and immunoblot analysis .", "For mass spectrometry ( MS ) experiments , 5 mg of mitochondria were used .", "Briefly , isolated mitochondria from control cells ( empty vector ) and cells expressing FLAG tagged proteins were solubilised in digitonin-containing buffer ( described above ) supplemented with 1X complete protease ( Roche ) at 2 mg/mL end over end on a rotary wheel for 30–60 min at 4°C .", "The mitochondrial lysate was cleared by centrifugation at 16 , 000 g at 4°C for 30 min and diluted in solubilisation buffer ( 20 mM Tris , pH 7 . 0 , 0 . 1 mM EDTA , 50 mM NaCl , 10% [w/v] glycerol , 1X complete protease inhibitor ) so that a final digitonin concentration of 0 . 1% was achieved .", "The cleared lysates were applied to pre-equilibrated anti-FLAG agarose ( Sigma-Aldrich ) and incubated at 4°C for 60 min under mild agitation .", "Unbound material was collected and the anti-FLAG resin and bound proteins were washed X 3 in digitonin-containing buffer ( 0 . 1% digitonin [w/v] , 20 mM Tris , pH 7 . 0 , 0 . 1 mM EDTA , 50 mM NaCl , 10% [w/v] glycerol , 1X complete protease inhibitor ) .", "Bound proteins were eluted with 0 . 2 M glycine , pH 2 . 5 .", "Glycine eluted fractions were electrophoretically separated using SDS-PAGE and proteins were visualised by staining with Coomassie Brilliant Blue stain .", "Gel lanes were cut into 10 x 2 mm bands using a scalpel blade and proteins were reduced , alkylated and trypsinised as described previously ( Keerthikumar et al . , 2015 ) .", "Briefly , the gel bands were subjected to reduction by 10 mM DTT ( Bio-Rad ) , alkylation by 25 mM iodoacetamide ( Sigma ) , tryptic digestion overnight with 150 ng of trypsin ( Promega ) .", "Subsequently , the tryptic peptides were further extracted using acetonitrile ( 50% w/v ) and 0 . 1% trifluoroacetic acid ( 0 . 1% ) .", "Extracted tryptic peptides were concentrated to ~10 μL by centrifugal lyophilisation and analysed by LC-MS/MS using LTQ Orbitrap Velos mass spectrometer ( Thermo Scientific ) fitted with nanoflow reversed-phase-HPLC ( Model 1200 , Agilent ) .", "MGF files were generated using MSConvert using peak picking .", "The MGF files were searched against the NCBI RefSeq database ( RRID:AB_2085424 ) in a target decoy fashion using MASCOT ( RRID:AB_2085424 v2 . 4 , Matrix Science , UK ) .", "Search parameters used were: fixed modification ( carbamidomethylation of cysteine; +57 Da ) , variable modifications ( oxidation of methionine; +16 Da ) , three missed tryptic cleavages , 20 ppm peptide mass tolerance and 0 . 6 Da fragment ion mass tolerance .", "Peptide identifications with mascot ion score greater than the identity score were deemed significant .", "The relative protein abundance between the samples was obtained by estimating the ratio of normalised spectral counts ( RSc ) as previously described ( Keerthikumar et al . , 2015 ) ." ] ]

[ "The TIM22 complex mediates the import of hydrophobic carrier proteins into the mitochondrial inner membrane .", "While the TIM22 machinery has been well characterised in yeast , the human complex remains poorly characterised .", "Here , we identify Tim29 ( C19orf52 ) as a novel , metazoan-specific subunit of the human TIM22 complex .", "The protein is integrated into the mitochondrial inner membrane with it’s C-terminus exposed to the intermembrane space .", "Tim29 is required for the stability of the TIM22 complex and functions in the assembly of hTim22 .", "Furthermore , Tim29 contacts the Translocase of the Outer Mitochondrial Membrane , TOM complex , enabling a mechanism for transport of hydrophobic carrier substrates across the aqueous intermembrane space .", "Identification of Tim29 highlights the significance of analysing mitochondrial import systems across phylogenetic boundaries , which can reveal novel components and mechanisms in higher organisms ." ]

[ "Mitochondria are like tiny bean-shaped “power stations” that provide our cells with the vast majority of the energy that they need .", "These structures , however , are not self-sufficient and instead rely on proteins and chemicals that are imported from elsewhere in the cell .", "Two layers of membrane enclose the mitochondria , and transporting proteins across the inner and outer membranes requires large molecular machines embedded within the membranes .", "One such complex , the TIM22 complex , organizes tunnel-like carrier proteins that in turn ferry chemicals across the inner membrane to fuel metabolism .", "The TIM22 complex is vitally important as it allows mitochondria to adapt their metabolism – that is , how and when they generate energy – to match the cell’s needs during development .", "Yet , while the TIM22 complex has been studied extensively in yeast , less is known about how it works in human cells .", "Now , Kang et al . have identified a new piece of the human equivalent of the TIM22 machinery , a protein called Tim29 , which helps to assemble the TIM22 complex in human cells .", "Experiments reveal that Tim29 also creates a link between human TIM22 and the TOM complex , a complex that serves as the general entry point through the outer mitochondrial membrane .", "Sequence analysis revealed that Tim29 is found in other animals , such as chimpanzees and cows , but not in yeast .", "This suggests that the mitochondrial machinery has changed during evolution .", "Kang et al . plan to further investigate how human carrier proteins reach the mitochondria , and exactly how Tim29 helps human TIM22 to cooperate with TOM .", "Overall , the discovery of Tim29 highlights the importance of looking at mitochondrial machinery across different species in the hope of revealing new components and mechanisms .", "A future challenge will be to determine how relevant these machines are in human development and diseases ." ]

[ "Introduction", "Results", "Discussion" ]

[ "biochemistry and chemical biology", "physics of living systems", "tools and resources" ]

[ [ "Membrane potential ( Vmem ) is an essential facet of cellular physiology .", "In electrically excitable cells , such as neurons and cardiomyocytes , voltage-gated ion channels enable rapid changes in membrane potential .", "These fast membrane potential changes , on the order of milliseconds to seconds , trigger release of neurotransmitters in neurons or contraction in myocytes .", "The resting membrane potentials of these cells , which change over longer timescales , affect their excitability .", "In non-electrically excitable cells , slower changes in Vmem—on the order of seconds to hours—are linked to a variety of fundamental cellular processes ( Abdul Kadir et al . , 2018 ) , including mitosis ( Cone and Cone , 1976 ) , cell cycle progression ( Huang and Jan , 2014 ) , and differentiation ( Tsuchiya and Okada , 1982 ) .", "Mounting lines of evidence point to the importance of electrochemical gradients in development , body patterning , and regeneration ( Levin , 2014 ) .", "Despite the importance of membrane potential to diverse processes over a range of time scales , the existing methods for recording Vmem are inadequate for characterizing distributions of Vmem states in a sample or studying gradual shifts in resting membrane potential ( Figure 1—source data 1 ) .", "Patch clamp electrophysiology remains the gold standard for recording cellular electrical parameters , but it is low throughput , highly invasive , and difficult to implement over extended time periods .", "Where reduced invasiveness or higher throughput analyses of Vmem are required , optical methods for detecting events involving Vmem changes ( e . g . whether an action potential occurred ) are often employed ( Huang et al . , 2006; McKeithan et al . , 2017; Zhang et al . , 2016 ) .", "However , optical approaches generally use fluorescence intensity values as a readout , which cannot report either the value of Vmem in millivolts ( ‘absolute Vmem’ ) or the millivolt amount by which Vmem changed ( Peterka et al . , 2011 ) .", "Variations in dye environment ( Ross and Reichardt , 1979 ) , dye loading , illumination intensity , fluorophore bleaching , and/or cellular morphology complicate fluorescence intensity measurements , making calibration and determination of absolute membrane potential difficult or impossible .", "This limitation restricts optical analysis to detection of acute Vmem changes , which can be analyzed without comparisons of Vmem between cells or over long timescales .", "One strategy to address these fluorescence intensity artifacts and quantify cellular parameters optically is ratio-based imaging .", "For Vmem specifically , ratio-based signals can be accessed either with a two-component system or with an electrochromic voltage sensitive dye , but neither strategy has enabled accurate absolute Vmem recordings .", "Two-component FRET-oxonol systems , with independent chromophores for ratio-based calibration , have seen limited success ( González and Tsien , 1997 ) , and they confer significant capacitive load on the cell ( Briggman et al . , 2010 ) .", "Further , their performance hinges on carefully tuned loading procedures of multiple lipophilic indicators ( Adams and Levin , 2012 ) , which can be challenging to reproduce across different samples and days .", "On the other hand , electrochromic probes report voltage as changes in excitation and emission wavelengths of a single chromophore ( Loew et al . , 1979 ) .", "While they benefit from simpler loading procedures , signals from electrochromic styryl dyes require normalization with an electrode on each cell of interest to determine absolute Vmem accurately ( Montana et al . , 1989; Zhang et al . , 1998; Bullen and Saggau , 1999 ) .", "As a result , ratiometric Vmem sensors cannot be used to optically quantify slow signals in the resting Vmem , which may be on the order of tens of millivolts .", "Indeed , ratiometric Vmem probes are most commonly applied to detect - rather than quantify - fast changes in Vmem ( Zhang et al . , 1998 ) , much like their single wavelength counterparts .", "An alternative approach to improved quantification in optical measurements is fluorescence lifetime ( τfl ) imaging ( FLIM ) , which measures the excited state lifetime of a population of fluorophores .", "Because fluorescence lifetime is an intrinsic property , FLIM can avoid many of the artifacts that confound extrinsic fluorescence intensity measurements , such as uneven dye loading , fluorophore bleaching , variations in illumination intensity , and detector sensitivity ( Berezin and Achilefu , 2010; Yellen and Mongeon , 2015 ) .", "If a fluorescent probe responds to the analyte of interest via changes in the lifetime of its excited state , there is the opportunity to use fluorescence lifetime to provide a more quantitative estimate of analyte parameters than can be achieved with fluorescence intensity alone .", "Although FLIM measurements can be affected by environmental factors such as temperature , ionic strength and local environment ( Berezin and Achilefu , 2010 ) , FLIM has been widely employed to record a number of biochemical and biophysical parameters , including intracellular Ca2+ concentration ( Zheng et al . , 2015 ) , viscosity ( Levitt et al . , 2009 ) , GTPase activity ( Harvey et al . , 2008 ) , kinase activity ( Lee et al . , 2009 ) , and redox state ( NADH/NAD+ ratio ) ( Blacker and Duchen , 2016 ) , among others ( Yellen and Mongeon , 2015 ) .", "Attempts to record absolute voltage with FLIM , however , have been limited in success ( Dumas and Stoltz , 2005; Hou et al . , 2014; Brinks et al . , 2015 ) .", "Previous work focused on genetically encoded voltage indicators ( GEVIs ) , which either possess complex relationships between τfl and voltage ( Hou et al . , 2014 ) or show low sensitivity to voltage in lifetime ( Brinks et al . , 2015 ) and require complex and technically challenging measurements of fast photochemical kinetics to estimate voltage ( Hou et al . , 2014 ) .", "Because of this poor voltage resolution , the fluorescence lifetimes of GEVIs cannot be used to detect most biologically relevant voltage changes , which are on the order of tens of millivolts .", "Fluorescent voltage indicators that use photoinduced electron transfer ( PeT ) as a voltage-sensing mechanism are promising candidates for a FLIM-based approach to optical Vmem quantification .", "Because PeT affects the nonradiative decay rate of the fluorophore excited state , it has been successfully translated from intensity to τfl imaging with a number of small molecule probes for Ca2+ ( Lakowicz et al . , 1992 ) .", "We previously established that VoltageFluor ( VF ) -type dyes transduce changes in cellular membrane potential to changes in fluorescence intensity and that the voltage response of VF dyes is consistent with a PeT-based response mechanism ( Miller et al . , 2012; Woodford et al . , 2015 ) .", "Changes in the transmembrane potential alter the rate of PeT ( Li , 2007; de Silva et al . , 1995 ) from an electron-rich aniline donor to a fluorescent reporter , thereby modulating the fluorescence intensity of VF dyes ( Miller et al . , 2012 ) ( Figure 1A , B ) .", "VoltageFluors also display low toxicity and rapid , linear responses to voltage .", "Here , we develop fluorescence lifetime imaging of VoltageFluor dyes ( VF-FLIM ) as a quantitative , all-optical approach for recording absolute membrane potential with single cell resolution .", "Using patch-clamp electrophysiology as a standard , we demonstrate that VF-FLIM reports absolute membrane potential in single trials with 10 to 23 mV accuracy ( root mean square deviation , RMSD; 15 s acquisition ) , depending on the cell line .", "In all cases tested , VF-FLIM tracks membrane potential changes with better than 5 mV accuracy ( RMSD ) .", "We benchmark VF-FLIM against previously reported optical absolute Vmem recording approaches and demonstrate resolution improvements of 8-fold over ratiometric strategies and 19-fold over other lifetime-based strategies .", "To highlight the increased throughput relative to manual patch-clamp electrophysiology , we document resting membrane potentials of thousands of cells .", "To our knowledge , this work represents the first broad view of the distribution of resting membrane potentials present in situ .", "VF-FLIM is limited to acquisition speeds on the order of seconds , but it is well-suited for studying gradual Vmem dynamics .", "Using VF-FLIM , we quantify and track the evolution of a 10–15 mV Vmem hyperpolarization over minutes following epidermal growth factor ( EGF ) stimulation of human carcinoma cells .", "Through pharmacological perturbations , we conclude that the voltage changes following EGF stimulation arise from activation of the calcium-activated potassium channel KCa3 . 1 .", "Our results show that fluorescence lifetime of VF dyes is a generalizable and effective approach for studying resting membrane potential in a range of cell lines ( Lakowicz et al . , 1992 ) ." ], [ "To characterize how the photoinduced electron transfer process affects fluorescence lifetime , we compared the τfl of the voltage-sensitive dye VF2 . 1 . Cl with its voltage-insensitive counterpart VF2 . 0 . Cl ( Figure 1B ) .", "We recorded the τfl of bath-applied VF dyes in HEK293T cells using time-correlated single-photon counting ( TCSPC ) FLIM ( Figure 1C–E , Scheme 1 ) .", "VF2 . 1 . Cl is localized to the plasma membrane and exhibits a biexponential τfl decay with decay constants of approximately 0 . 9 and 2 . 6 ns ( Figure 1—figure supplement 1 ) .", "For all subsequent analysis of VF2 . 1 . Cl lifetime , we refer to the weighted average τfl , which is approximately 1 . 6 ns in HEK293T cell membranes at rest .", "VF2 . 0 . Cl ( Figure 1B ) , which lacks the aniline substitution and is therefore voltage-insensitive ( Woodford et al . , 2015 ) , shows a τfl of 3 . 5 ns in cell membranes , which is similar to the lifetime of an unsubstituted fluorescein ( Magde et al . , 1999 ) ( Figure 1—source data 2 ) .", "We also examined VoltageFluor lifetimes at a variety of dye loading concentrations to test for concentration-dependent changes in dye lifetime , which have been reported for fluorescein derivatives ( Chen and Knutson , 1988 ) .", "Shortened VF lifetimes were observed at high dye concentrations ( Figure 1—figure supplement 2 ) ; all subsequent VF-FLIM studies were conducted at dye concentrations low enough to avoid this concentration-dependent change in lifetime .", "To assess the voltage dependence of VoltageFluor τfl , we controlled the plasma membrane potential of HEK293T cells with whole-cell voltage-clamp electrophysiology while simultaneously measuring the τfl of VF2 . 1 . Cl ( Figure 1C ) .", "Single-cell recordings show a linear τfl response to applied voltage steps , and individual measurements deviate minimally from the linear fit ( Figure 1F–H ) .", "VF2 . 1 . Cl τfl is reproducible across different cells at the same resting membrane potential , allowing determination of Vmem from τfl images taken without concurrent electrophysiology ( Figure 1I ) .", "Voltage-insensitive VF2 . 0 . Cl shows no τfl change in response to voltage ( Figure 1J , Figure 1—figure supplement 3 ) , consistent with a τfl change in VF2 . 1 . Cl arising from a voltage-dependent PeT process .", "In HEK293T cells , VF2 . 1 . Cl exhibits a sensitivity of 3 . 50 ± 0 . 08 ps/mV and a 0 mV lifetime of 1 . 77 ± 0 . 02 ns , corresponding to a fractional change in τfl ( Δτ/τ ) of 22 . 4 ± 0 . 4% per 100 mV .", "These values are in good agreement with the 27% ΔF/F intensity change per 100 mV originally observed for VF2 . 1 . Cl ( Miller et al . , 2012; Woodford et al . , 2015 ) .", "Because %ΔF/F is a fluorescence intensity-based metric , it cannot be used to measure absolute Vmem; however , agreement between %ΔF/F and %Δτ/τ is consistent with a PeT-based Vmem sensing mechanism in VFs .", "To estimate the voltage resolution of VF-FLIM , we analyzed the variability in successive measurements on the same cell ( intra-cell resolution ) and on different cells ( inter-cell resolution , see Materials and methods ) .", "We estimate that the resolution for tracking and quantifying voltage changes in a single HEK293T cell is 3 . 5 ± 0 . 4 mV ( intra-cell resolution , average RMSD from each electrophysiological calibration , Scheme 2 ) , whereas the resolution for single-trial determination of a particular HEK293T cell’s absolute Vmem is 19 mV ( inter-cell resolution , RMSD of each calibration slope to the average calibration , Scheme", "2 ) within a 15 s bandwidth .", "We compared the performance of VF-FLIM in HEK293T cells to that of two previously documented strategies for optical absolute Vmem determination .", "We first tested the voltage resolution of CAESR , the best previously reported GEVI for recording absolute Vmem with FLIM ( Brinks et al . , 2015 ) .", "Using simultaneous FLIM and voltage-clamp electrophysiology , we determined the relationship between τfl and Vmem for CAESR under one photon excitation ( Figure 1—figure supplement 4 ) .", "We recorded a sensitivity of −1 . 2 ± 0 . 1 ps/mV and a 0 mV lifetime of 2 . 0 ± 0 . 2 ns , which corresponds to a −6 . 1 ± 0 . 8% Δτ/τ per 100 mV ( mean ± SEM of 9 measurements ) , in agreement with the reported sensitivity of −0 . 9 ps/mV and 0 mV lifetime of 2 . 7 ns with 2 photon excitation ( Brinks et al . , 2015 ) .", "Relative to VF2 . 1 . Cl , CAESR displays 3-fold lower sensitivity ( −1 . 2 ps/mV vs 3 . 5 ps/mV in HEK293T cells ) and 7-fold higher voltage-independent variability in lifetime ( 0 . 46 ns vs 0 . 07 ns , standard deviation of the 0 mV lifetime measurement ) .", "For CAESR in HEK293T cells , we calculate a voltage resolution of 33 ± 7 mV for quantifying voltage changes on an individual cell ( intra-cell RMSD , compared to 3 . 5 mV for VF2 . 1 . Cl , see Materials and methods ) and resolution of 370 mV for determination of a particular cell’s absolute Vmem ( inter-cell RMSD , compared to 19 mV for VF2 . 1 . Cl ) .", "We also measured the absolute voltage resolution of the ratio-based sensor di-8-ANEPPS , which reports membrane potential by the wavelength of its excitation and emission spectra ( Loew et al . , 1979 ) .", "Ratio-based imaging can be achieved by comparing the fluorescence emission at different excitation wavelengths ( Zhang et al . , 1998 ) ; here , we used the ratio , R , of the blue-excited emission to the green-excited emission ( see Materials and methods ) .", "Via simultaneous ratio imaging and whole cell voltage clamp electrophysiology , we record a sensitivity of 0 . 0039 ± 0 . 0004 R per mV , with a y-intercept ( 0 mV ) R value of 1 . 8 ± 0 . 2 ( Figure 1—figure supplement 5; mean ± SEM of n = 16 HEK293T cells ) .", "R depends on the excitation and emission conditions used but should be relatively reproducible on a given microscope rig .", "To compare R from our system with previous work , we normalized all R values to the R value at 0 mV for each cell .", "Using the above data , we obtain a sensitivity of 0 . 0022 ± 0 . 0002 normalized R per mV , with a 0 mV normalized R of 1 . 02 ± 0 . 02 , in good agreement with reported values ( 0 . 0015 normalized R per mV ) ( Zhang et al . , 1998 ) .", "For analysis of voltage resolution , we compare VF-FLIM to the non-normalized R , since normalization requires an electrode-based measurement for every recording and is thus not a truly optical strategy .", "From the non-normalized di-8-ANEPPS R , we obtain an intra-cell resolution ( RMSD ) of 18 ± 3 mV ( 5-fold less accurate than VF-FLIM ) and an inter-cell resolution ( RMSD ) of 150 mV ( 8-fold less accurate than VF-FLIM ) .", "The sensitivities and resolutions of VF-FLIM , CAESR , and di-8-ANEPPS in HEK293T are tabulated in Figure 1—source data 3 .", "Because cellular resting membrane potentials and voltage changes ( e . g . action potentials ) are on the order of tens of millivolts , the resolution improvements achieved by VF-FLIM enable biologically relevant absolute Vmem recordings: impossible with previous approaches .", "To test the generalizability of VF-FLIM , we determined τfl-Vmem calibrations in four additional commonly used cell lines: A431 , CHO , MDA-MB-231 , and MCF-7 ( Figure 2 , Figure 2—figure supplement 1 , Figure 2—figure supplement 2 ) .", "We observe a linear τfl response in all cell lines tested .", "The slope ( voltage sensitivity ) and y-intercept ( 0 mV lifetime ) of the τfl-Vmem response varied slightly across cell lines , with average sensitivities of 3 . 1 to 3 . 7 ps/mV and average 0 mV lifetimes ranging from 1 . 74 to 1 . 87 ns .", "In all cell lines , we observed better voltage resolution for quantification of Vmem changes on a given cell versus comparisons of absolute Vmem between cells .", "Changes in voltage for a given cell could be quantified with resolutions at or better than 5 mV ( intra-cell resolution , Materials and methods ) .", "For absolute Vmem determination of a single cell , we observed voltage resolutions ranging from 10 to 23 mV ( inter-cell resolution , 15 s acquisition time , Figure 2—source data 1 ) .", "Statistically significant differences among the cell lines tested were observed for cellular τfl-Vmem calibrations in both the slope ( One-way ANOVA with Welch’s correction: F ( 4 , 23 . 07 ) =18 . 12 , p<0 . 0001 ) and average 0 mV lifetime ( One-way ANOVA: F ( 4 , 67 ) =14 . 43 , p<0 . 0001 ) .", "There were no statistically significant differences between A431 , CHO , and HEK293T cells ( p>0 . 05 , Games-Howell and Tukey-Kramer post hoc tests for the slope and 0 mV lifetime respectively ) .", "MDA-MB-231 and MCF-7 cells showed statistically significant variability from other cell lines in slope and/or 0 mV lifetime .", "To verify that VF-FLIM was robust in groups of cells in addition to the isolated , single cells generally used for patch clamp electrophysiology , we determined lifetime-voltage relationships for small groups of A431 cells ( Figure 2—figure supplement 3A–E ) .", "We found that calibrations made in small groups of cells are nearly identical to those obtained on individual cells , indicating that VF-FLIM only needs to be calibrated once for a given type of cell .", "For pairs or groups of three cells we recorded a sensitivity of 3 . 3 ± 0 . 2 ps/mV and a 0 mV lifetime of 1 . 78 ± 0 . 02 ns ( mean ± SEM of 7 cells ( 5 pairs and 2 groups of 3 ) ; values are for the entire group , not just the cell in contact with the electrode ) , which is similar to the sensitivity of 3 . 55 ± 0 . 08 ps/mV and 0 mV lifetime of 1 . 74 ± 0 . 02 ns we observe in single A431 cells .", "The slight reduction in sensitivity seen in cell groups is likely attributable to space clamp error , which prevents complete voltage clamp of the cell group ( Williams and Mitchell , 2008; Armstrong and Gilly , 1992 ) .", "Indeed , when we analyzed only the most responsive cell in the group ( in contact with the electrode ) , we obtained a slope of 3 . 7 ± 0 . 1 ps/mV and 0 mV lifetime of 1 . 79 ± 0 . 02 ns , in good agreement with the single cell data .", "The space clamp error can be clearly visualized in Figure 2—figure supplement 3E , where one cell in the group of 3 responded much less to the voltage command .", "To test whether VF-FLIM is also extensible to cells maintained with different culture conditions , we recorded lifetime-Vmem relationship in serum-starved A431 cells ( Figure 2—figure supplement 3F–K ) , obtaining an average sensitivity of 3 . 6 ± 0 . 1 ps/mV and a 0 mV lifetime of 1 . 76 ± 0 . 01 ns ( n = 7; two single cells , two pairs , 3 groups of 3 cells; values are average lifetime across the whole cell group ) , in excellent agreement with the values obtained for non-serum starved cells .", "We also tested for concentration-dependent changes in VF lifetime in all five cell lines and in serum starvation conditions .", "Similar to VF2 . 1 . Cl lifetime in HEK293T cells ( Figure 1—figure supplement 2 ) , we observed shortening of VF2 . 1 . Cl lifetimes beginning between 200 and 500 nM dye in all cases ( Figure 2—figure supplement 4 ) .", "All subsequent experiments were carried out at VF2 . 1 . Cl concentrations well below the regime where VF concentration-dependent lifetime changes were observed .", "The throughput of VF-FLIM enables cataloging of resting membrane potentials of thousands of cells in only a few hours of the experimenter’s time .", "We optically recorded resting membrane potential distributions for A431 , CHO , HEK293T , MCF-7 , and MDA-MB-231 cells using VF-FLIM ( Figure 3 , Figure 3—figure supplement 1 , Figure 3—figure supplement 2 ) .", "We report resting membrane potentials by cell group ( Materials and methods , Figure 1—figure supplement", "1 ) because adjacent cells in these cultures are electrically coupled to some degree via gap junctions ( Meşe et al . , 2007 ) .", "Each group of cells represents an independent sample for Vmem .", "In addition , the fluorescent signal originating from membranes of adjacent cells cannot be separated with a conventional optical microscope , so assignment of a region of membrane connecting multiple cells would be arbitrary .", "VF-FLIM images ( Figure 3 , Figure 3—figure supplement 1 , Figure 3—figure supplement", "2 ) contain spatially resolved voltage information , but caution should be employed in interpreting pixel to pixel differences in lifetime .", "Because VF-FLIM was calibrated here using the average plasma membrane τfl for each cell , optical Vmem should be interpreted per cell or cell group .", "Mean resting membrane potentials recorded by VF-FLIM range from −53 to −29 mV , depending on the cell line .", "These average Vmem values fall within the range reported in the literature for all of the cell lines we measured ( Figure 3—source data 1 ) .", "We also recorded resting membrane potentials in a high K+ buffer ( 120 mM K+ , ‘high K+ HBSS’ ) , where we observed a depolarization of 15 to 41 mV , bringing the mean Vmem up to −26 mV to +4 mV , again depending on the cell line .", "Although 120 mM extracellular K+ should be strongly depolarizing , it will not necessarily produce a membrane potential of 0 mV .", "Because few literature reports of electrophysiological measurements in 120 mM K+ exist as a point of comparison , we obtained a rough estimate of Vmem in 6 mM extracellular K+ and 120 mM extracellular K+ using the Goldman-Hodgkin-Katz ( GHK ) equation ( Hodgkin and Katz , 1949 ) .", "Under our imaging conditions and with a broad range of possible ion permeabilities and intracellular ion concentrations , the GHK equation allows Vmem ranging from −91 to −27 mV in 6 mM extracellular K+ and −25 to +2 mV in 120 mM extracellular K+ ( see Materials and methods ) .", "Recorded VF-FLIM values fall well within this allowed range .", "Notably , although the GHK equation can determine ranges of reasonable Vmem values , GHK-based Vmem results are approximate at best because of the difficulty in obtaining accurate values of permeabilities and intracellular ion concentrations for specific cell lines .", "Direct measurement of Vmem , rather than theoretical calculation , is required to obtain accurate values .", "We thought VF-FLIM was a promising method for elucidating the roles of membrane potential in non-excitable cell signaling .", "Specifically , we wondered whether VF-FLIM might be well-suited to dissect conflicting reports surrounding changes in membrane potential during EGF/EGF receptor ( EGFR ) -mediated signaling .", "Receptor tyrosine kinase ( RTK ) -mediated signaling is a canonical signaling paradigm for eukaryotic cells , transducing extracellular signals into changes in cellular state .", "Although the involvement of second messengers like Ca2+ , cyclic nucleotides , and lipids are well characterized , membrane potential dynamics and their associated roles in non-excitable cell signaling remain less well-defined .", "In particular , the activation of EGFR via EGF has variously been reported to be depolarizing ( Rothenberg et al . , 1982 ) , hyperpolarizing ( Pandiella et al . , 1989 ) , or electrically silent ( Moolenaar et al . , 1982; Moolenaar et al . , 1986 ) .", "We find that treatment of A431 cells with EGF results in a 15 mV hyperpolarization within 60–90 s in approximately 80% of cells ( Figure 4A–C , Figure 4—figure supplement 1 , Figure 4—figure supplement 2 ) , followed by a slow return to baseline within 15 min ( Figure 4D–F , Figure 4—figure supplements 3 and 0 second acquisitions ) .", "The voltage response to EGF is dose-dependent , with an EC50 of 90 ng/mL ( 14 nM ) ( Figure 4—figure supplement 4 ) .", "Vehicle-treated cells show very little τfl change ( Figure 4A–F ) .", "Identical experiments with voltage-insensitive VF2 . 0 . Cl ( Figure 4G–H , Figure 4—figure supplement 1 , Figure 4—figure supplement 3 , Figure 4—figure supplement 5 ) reveal little change in τfl upon EGF treatment , indicating the drop in τfl arises from membrane hyperpolarization .", "We observe the greatest hyperpolarization 1 to 3 min after treatment with EGF , which is abolished by inhibition of EGFR and ErbB2 tyrosine kinase activity with the covalent inhibitor canertinib ( Figure 4I–J , Figure 4—figure supplement 6 ) .", "Blockade of the EGFR kinase domain with gefitinib , a non-covalent inhibitor of EGFR , also results in a substantial decrease in the EGF-evoked hyperpolarization ( Figure 4I–J , Figure 4—figure supplement 6 ) .", "Together , these results indicate that A431 cells exhibit an EGF-induced hyperpolarization , which depends on the kinase activity of EGFR and persists on the timescale of minutes .", "Outward K+ currents could mediate EGF-induced hyperpolarization .", "Consistent with this hypothesis , dissipation of the K+ driving force by raising extracellular [K+] completely abolishes the typical hyperpolarizing response to EGF and instead results in a small depolarizing potential of approximately 3 mV ( Figure 5A , Figure 5—figure supplement 1B ) .", "Blockade of voltage-gated K+ channels ( Kv ) with 4-aminopyridine ( 4-AP ) prior to EGF treatment enhances the hyperpolarizing response to EGF ( Figure 5A and B , Figure 5—figure supplement 1C ) .", "In contrast , blockade of Ca2+-activated K+ channels ( KCa ) with charybdotoxin ( CTX ) results in a depolarizing potential of approximately 4 mV after exposure to EGF , similar to that observed with high extracellular [K+] ( Figure 5A and B , Figure 5—figure supplement 1D ) .", "TRAM-34 , a specific inhibitor of the intermediate-conductance Ca2+ activated potassium channel KCa3 . 1 ( Wulff et al . , 2000 ) , also abolishes EGF-induced hyperpolarization ( Figure 5A , Figure 5—figure supplement 1E ) .", "CTX treatment has little effect on the resting membrane potential , while TRAM-34 or 4-AP depolarizes cells by approximately 5–10 mV ( Figure 5—figure supplement 2 ) .", "To explore the effects of other components of the EGFR pathway on EGF-induced hyperpolarization , we perturbed intra- and extracellular Ca2+ concentrations during EGF stimulation .", "Reduction of extracellular Ca2+ concentration did not substantially alter the EGF response ( Figure 5A , Figure 5—figure supplement 1F ) .", "However , sequestration of intracellular Ca2+ with BAPTA-AM disrupts the hyperpolarization response .", "BAPTA-AM treated cells show a small , 4 mV depolarization in response to EGF treatment , similar to CTX-treated cells ( Figure 5A , Figure 5—figure supplement 1G ) .", "Perturbation of Ca2+ levels had little effect on the resting membrane potential ( Figure 5—figure supplement 2 ) .", "Introduction of wortmannin ( 1 μM ) to block downstream kinase activity has no effect on the membrane potential response to EGF , while orthovanadate addition ( Na3VO4 , 100 μM ) to block phosphatase activity results in a small increase in the hyperpolarizing response ( Figure 5A , Figure 5—figure supplement 1H–I ) .", "These results support a model for EGF-EGFR mediated hyperpolarization in which RTK activity of EGFR causes release of internal Ca2+ stores to in turn open KCa channels and hyperpolarize the cell ( Figure 5C ) ." ], [ "The key advantage of VF-FLIM over previously reported optical approaches is its superior Vmem resolution .", "Resolution can be interpreted as stability of the τfl-Vmem calibration over time and between cells .", "Any factors other than Vmem that change τfl decrease resolution .", "VF-FLIM exhibits a 19-fold improvement in inter-cell Vmem resolution over FLIM with the GEVI CAESR ( Brinks et al . , 2015 ) and a 8-fold improvement over di-8-ANEPPS excitation ratios ( Zhang et al . , 1998 ) .", "Although all optical strategies , including VF-FLIM , have worse Vmem resolution than modern electrophysiology , the greater throughput , improved spatial resolution , and reduced invasiveness of optical strategies make them a powerful complement to electrode-based recordings .", "The sources of variability that reduce resolution of optical Vmem measurements are manifold , but two major contributors are membrane specificity of the stain and the complexity of the lipid environment .", "Nonspecific staining is fluorescence signal from anywhere other than the plasma membrane , such as contaminating intracellular staining from poorly trafficked ( CAESR ) or internalized ( ANEPPS ) sensor .", "In contrast , exogenously loaded VF2 . 1 . Cl exhibits little fluorescence contribution from regions other than the plasma membrane .", "Secondly , membrane composition and dipole potential can vary between cells and cell lines , changing the local environment of the fluorescent indicator ( Wang , 2012; Brügger , 2014 ) .", "Styryl dyes like di-8-ANEPPS can respond to changes in dipole potential ( Zhang et al . , 1998; Gross et al . , 1994 ) , and VF dyes may be similarly sensitive to dipole potential .", "Additionally , fluorescence lifetime depends on certain environmental factors ( e . g . temperature , viscosity , ionic strength ) ( Berezin and Achilefu , 2010 ) , which may introduce variability .", "These parameters are usually determined by the biological system under study , and re-calibration is important if they change dramatically in an experiment .", "VF-FLIM , like all optical approaches , improves upon the spatial resolution of patch clamp electrophysiology .", "While VF-FLIM records the Vmem of an optically defined region of interest ( in this study a cell or cell group ) , electrophysiology records Vmem at an individual cell or part of a cell where the electrode makes contact , which may or may not reflect the Vmem of the entire cell or group .", "In this study , we interpret VF-FLIM at the whole cell level only , since that is the smallest unit in which the Vmem can be reliably calibrated by whole cell patch clamp electrophysiology .", "Intriguingly , there are differences in lifetime within some cells in VF-FLIM images at the pixel to pixel level .", "In small , mostly spherical cells under voltage clamp , one would expect uniform membrane potential ( Armstrong and Gilly , 1992 ) , so these subcellular differences are most likely noise in the measurement .", "We speculate that most of this pixel-to-pixel noise comes from variability in fitting the biexponential lifetime model .", "Lifetime estimates at each pixel are calculated from 20 to 100-fold fewer photons than the lifetime value for the entire ROI .", "These lower photon counts at the single pixel level produce Vmem estimates that are less precise than the Vmem estimate for the entire ROI .", "Collection of more photons at each pixel could likely reduce this noise but would require longer acquisition times .", "We also cannot fully rule out an alternative explanation that the observed subcellular variability is the result of local differences in membrane composition ( Gross et al . , 1994 ) .", "Vmem recordings in systems too large or too small for electrophysiological study could be an important application of VF-FLIM .", "Despite the improbability of Vmem compartmentalization in individual HEK293T cells , other cells with complex morphology and processes may display real , subcellular Vmem differences .", "In addition , delocalized Vmem patterns across tissues could in theory be stable ( Cervera et al . , 2016a ) and have been proposed to contribute to tissue development ( Levin , 2014 ) .", "One remaining challenge in expanding VF-FLIM to these areas is the requirement for an initial calibration with voltage clamp electrophysiology .", "Alternative ways to control Vmem , such as ionophores or optogenetic actuators ( Berndt et al . , 2009 ) , may prove useful in these systems .", "When applying VF-FLIM to tissues , the cellular specificity of the VF stain becomes a consideration , as the VF2 . 1 . Cl indicator used in this study labels all cell membranes efficiently .", "Looking ahead , recordings in tissue are an exciting area for future development of VF-FLIM , particularly in conjunction with cellular and sub-cellular strategies for targeting VF dyes ( Liu et al . , 2017; Grenier et al . , 2019 ) .", "To obtain absolute Vmem measurements with fluorescence lifetime , VF-FLIM sacrifices some of the temporal resolution of electrophysiology or intensity-based voltage imaging .", "VF-FLIM acquisition times are limited by the large numbers of photons needed per pixel in time-correlated single photon counting ( see Materials and methods ) .", "As a result , VF-FLIM in its current implementation can track Vmem events lasting longer than a few seconds .", "For ‘resting’ membrane potential or Vmem dynamics associated with cell growth or differentiation , this temporal resolution is likely sufficient .", "Nevertheless , in the future , we envision allying VF-FLIM with recently developed , faster lifetime imaging technology to enable optical quantification of more rapid Vmem responses ( Raspe et al . , 2015; Gao et al . , 2014 ) .", "Using the improved Vmem resolution and throughput of VF-FLIM , we optically documented resting membrane potential distributions in cultured cells to characterize the membrane potential state ( s ) present .", "The presence and significance of distinct Vmem states in cell populations is mostly uncharacterized due to the throughput limitations of patch-clamp electrophysiology , but some reports suggest that distinct Vmem states arise during the various phases of the cell cycle ( Ouadid-Ahidouch et al . , 2001; Wonderlin et al . , 1995 ) .", "Vmem histograms presented in this work appear more or less unimodal , showing no clear sign of cell cycle-related Vmem states ( Figure 3A , D; Figure 3—figure supplement 1A , D , G ) .", "We considered the possibility that VF-FLIM does not detect cell-cycle-related Vmem states because we report average Vmem across cell groups in cases where cells are in contact ( Figure 1—figure supplement 1 ) .", "This explanation is unlikely for two reasons .", "First , Vmem distributions for CHO cells appear unimodal , even though CHO cultures were mostly comprised of isolated cells under the conditions tested ( Figure 3D–F ) .", "Second , theoretical work suggests that dramatically different Vmem states in adjacent cells are unlikely , as electrical coupling often leads to equilibration of Vmem across the cell group ( Cervera et al . , 2016a; Cervera et al . , 2016b ) .", "Although we cannot rule out the possibility of poorly separated Vmem populations ( i . e . with a mean difference in voltage below our resolution limit ) , VF-FLIM both prompts and enables a re-examination of the notion that bi- or multimodal Vmem distributions exist in cultured cells .", "Furthermore , VF-FLIM represents an exciting opportunity to experimentally visualize theorized Vmem patterns in culture and in more complex tissues .", "Studies towards this end are ongoing in our laboratory .", "In the present study , we use VF-FLIM to provide the first cell-resolved , direct visualization of voltage changes induced by growth factor signaling .", "For long term Vmem recordings during growth-related processes , an optical approach is more attractive than an electrode-based one .", "Electrophysiology becomes increasingly challenging as time scale lengthens , especially if cells migrate , and washout of the cytosol with pipette solution can change the very signals under study ( Horn and Korn , 1992; Malinow and Tsien , 1990 ) .", "Previous attempts to electrophysiologically record Vmem in EGF-stimulated A431 cells were unsuccessful due to these technical challenges ( Pandiella et al . , 1989 ) .", "Because whole cell voltage-clamp electrophysiology was intractable , the Vmem response in EGF-stimulated A431 cells was addressed indirectly through model cell lines expressing EGFR exogenously ( Pandiella et al . , 1989 ) , bulk measurements on trypsinized cells in suspension ( Magni et al . , 1991 ) , or cell-attached single channel recordings ( Peppelenbosch et al . , 1991; Lückhoff and Clapham , 1994; Mozhayeva et al . , 1989 ) .", "By stably recording Vmem during EGF stimulation , VF-FLIM enables direct study of Vmem signaling in otherwise inaccessible pathways .", "In conjunction with physiological manipulations and pharmacological perturbations , we explore the molecular mechanisms underlying EGF-induced hyperpolarization .", "We find that signaling along the EGF-EGFR axis results in a robust hyperpolarizing current carried by K+ ions , passed by the Ca2+-activated K+ channel KCa3 . 1 , and mediated by intracellular Ca2+ ( Figure 5C ) .", "We achieve a complete loss of the hyperpolarizing response to EGF by altering the K+ driving force ( ‘High K+’ Figure 5A , Figure 5—figure supplement 1B ) , blocking calcium-activated K+ currents directly ( ‘CTX’ and ‘TRAM-34’ , Figure 5A , Figure 5—figure supplement 1D , E ) , or intercepting cytosolic Ca2+ ( ‘BAPTA-AM’ , Figure 5A , Figure 5—figure supplement 1G ) .", "These results , combined with transcriptomic evidence that KCa3 . 1 is the major KCa channel in A431 cells ( Thul et al . , 2017 ) , indicate that KCa3 . 1 mediates the observed hyperpolarization .", "Interestingly , under some conditions where K+-mediated hyperpolarization is blocked ( ‘CTX , ’ ‘high K+' , ‘BAPTA-AM’ ) , VF-FLIM reveals a small , secondary depolarizing current not visible during normal EGF stimulation .", "This current likely arises from initial Ca2+ entry into the cell , as previously observed during EGF signaling ( Pandiella et al . , 1987; Marquèze-Pouey et al . , 2014 ) .", "Although we have obtained direct and conclusive evidence of EGF-induced hyperpolarization in A431 cells , the interactions between this voltage change and downstream targets of EGFR remain incompletely characterized .", "Enhancing EGF signaling by blockade of cellular tyrosine phosphatases with orthovanadate ( Reddy et al . , 2016 ) correspondingly increases EGF-mediated hyperpolarization ( ‘Na3VO4’ Figure 5A , Figure 5—figure supplement 1H ) , but inhibition of downstream kinase activity appears to have little effect on hyperpolarization ( ‘wortmannin’ Figure 5A , Figure 5—figure supplement 1I ) .", "In the context of RTK signaling , Vmem may serve to modulate the driving force for external Ca2+ entry ( Huang and Jan , 2014; Yang and Brackenbury , 2013 ) and thereby act as a regulator of this canonical signaling ion .", "Alternatively , Vmem may play a more subtle biophysical role , such as potentiating lipid reorganization in the plasma membrane ( Zhou et al . , 2015 ) .", "Small changes in Vmem likely affect signaling pathways in ways that are currently completely unknown , but high throughput discovery of Vmem targets remains challenging .", "Combination of electrophysiology with single cell transcriptomics has begun to uncover relationships between Vmem and other cellular pathways in excitable cells ( Cadwell et al . , 2016 ) ; such approaches could be coupled to higher throughput VF-FLIM methods to explore pathways that interact with Vmem in non-excitable contexts .", "VF-FLIM represents a novel and general approach for interrogating the roles of membrane potential in fundamental cellular physiology .", "Future improvements to the voltage resolution could be made by use of more sensitive indicators , which may exhibit larger changes in fluorescence lifetime ( Woodford et al . , 2015 ) .", "VF-FLIM can be further expanded to include the entire color palette of PeT-based voltage indicators ( Huang et al . , 2015; Deal et al . , 2016 ) , allied with targeting methods to probe absolute membrane potential in heterogeneous cellular populations ( Liu et al . , 2017; Grenier et al . , 2019 ) , and coupled to high-speed imaging techniques for optical quantification of fast voltage events ( Raspe et al . , 2015; Gao et al . , 2014 ) ." ] ]

[ "All cells maintain ionic gradients across their plasma membranes , producing transmembrane potentials ( Vmem ) .", "Mounting evidence suggests a relationship between resting Vmem and the physiology of non-excitable cells with implications in diverse areas , including cancer , cellular differentiation , and body patterning .", "A lack of non-invasive methods to record absolute Vmem limits our understanding of this fundamental signal .", "To address this need , we developed a fluorescence lifetime-based approach ( VF-FLIM ) to visualize and optically quantify Vmem with single-cell resolution in mammalian cell culture .", "Using VF-FLIM , we report Vmem distributions over thousands of cells , a 100-fold improvement relative to electrophysiological approaches .", "In human carcinoma cells , we visualize the voltage response to growth factor stimulation , stably recording a 10–15 mV hyperpolarization over minutes .", "Using pharmacological inhibitors , we identify the source of the hyperpolarization as the Ca2+-activated K+ channel KCa3 . 1 .", "The ability to optically quantify absolute Vmem with cellular resolution will allow a re-examination of its signaling roles ." ]

[ "All living cells are like tiny batteries .", "As long as a cell is alive , it actively maintains a difference in electrical charge between its interior and exterior .", "This charge difference , or voltage , is called the membrane potential , and it is vital for our bodies to work properly .", "For example , fast changes in membrane potential control our heartbeat and underpin the electrical signals that brain cells use to communicate .", "Slower changes in membrane potential – ranging from minutes to days – may also play important roles in other organs .", "To understand how and why membrane potential is important in these contexts , we need methods to measure it accurately in individual cells .", "One way is to puncture cells with microscopic electrodes: this yields accurate results but damages the cells and can only measure one cell at a time .", "Alternative methods treat cells with special fluorescent dyes and then image them with a microscope .", "The dyes emit light in response to voltage variations: when the cells’ membrane potential changes , the dyes glow brighter .", "The changes in light intensity give an estimate of the size of the change in membrane potential .", "This allows many cells to be analyzed without harming them , but it is less accurate .", "Fluorescence lifetime refers to how long fluorescent dyes take to finish emitting light , and this phenomenon has already helped researchers to record a variety of processes in the cell .", "Lazzari-Dean et al . therefore wanted to use fluorescence lifetime to develop a better way of recording membrane potential .", "This method , called VF-FLIM , relied on measuring how long certain dyes took to finish emitting light at specific voltages , rather than how bright they were .", "Experiments using mammalian cells grown in the laboratory showed that the membrane potentials measured with VF-FLIM were similar to those recorded with electrodes , which represent the highest standard of accuracy .", "The new method was at least eight times more accurate than other techniques using fluorescent dyes .", "VF-FLIM could also measure many thousands of cells within a few hours , a hundred times faster than electrode-based methods .", "Finally , tests on human cancer cells revealed that VF-FLIM could detect that these cells go through gradual changes in membrane potential in response to growth signals .", "VF-FLIM is a new , non-invasive tool that can measure changes in membrane potential more quickly and accurately .", "This will help to better understand the many roles membrane potential could play in healthy and diseased cells ." ]

[ "Introduction", "Results and discussion", "Materials and methods" ]

[ "short report", "neuroscience" ]

[ [ "Association between events in time and space is a major mechanism for all animals , including humans , to learn about the world , which can potentially change their behavior in future circumstances .", "The most influential books concerning learning theory published early in the 20th century were Animal Intelligence ( 1911 ) , wherein Thorndike proposed that the rate of learning diminishes as the interval between response and discomfort/satisfaction is increased ( Thorndike , 1911 ) , and Conditioned Reflexes ( 1927 ) , wherein Pavlov highlighted that a connection is formed in the nervous system , not between a conditioned stimulus ( CS ) and unconditioned stimulus ( US ) separated in time , but between the sensory aftereffects , i . e . , trace of the CS , and the US ( Pavlov , 1927 ) .", "Thus , in essence , these theories mention that temporal contiguity has a vital influence on associative learning .", "The prevailing dogma for cellular mechanisms underlying associative learning is based on modifications of Hebb’s pioneering cell assembly theory ( 1949 ) which posits that ‘cells which fire together , wire together' , and thus the firing cells epitomize the internal representations of the two sensory events related in time ( Hebb , 1949; Miltner et al . , 1999; Nabavi et al . , 2014; Yiu et al . , 2014; Johansen et al . , 2014 ) .", "Indeed , it has been shown that at least in certain cases , selective facilitation of densely connected neurocircuits through excitability and/or synaptic plasticity , as a result of an experience , constitutes the basis for learning and memory ( Nabavi et al . , 2014; Yiu et al . , 2014; Johansen et al . , 2014 ) .", "Most of these studies have examined the cellular and molecular mechanisms underlying trace associative learning processes using classical conditioning paradigms in which the association takes place within the timescale of msec to sec ( Johansen et al . , 2014; Kitamura et al . , 2014; Beylin et al . , 2001 ) .", "However , evolution has produced other type ( s ) of associative learning , wherein the CS and US can be experienced several minutes to hours apart .", "For example , the inter-time-interval ( ITI ) between the CS ( novel taste ) and the US ( visceral information ) in the conditioned taste aversion ( CTA ) paradigm can last for hours ( Garcia et al . , 1955; 1985; Kalat and Rozin , 1971; 1973; Chambers , 1990; Rosenblum et al . , 1993; 1997; Yamamoto et al . , 1995; Hashikawa et al . , 2013; Adaikkan and Rosenblum , 2012; Stern et al . , 2013; Inberg et al . , 2013; Chinnakkaruppan et al . , 2014; Parkes et al . , 2014; Sano et al . , 2014 ) .", "Here , we attempted to understand the mechanisms that enable the taste memory trace to be associated with visceral information after such a long time ." ], [ "In order to evaluate the effect of the ITI between taste and malaise in CTA learning ( ITI-CTA ) quantitatively and qualitatively , we conducted a behavioral experiment , in which rats were presented with a novel taste ( 0 . 1% saccharin; CS ) and later were i . p . injected with lithium chloride ( 0 . 14 M LiCl; US ) at increasing time points ranging between 1 hr and 20 hr ( Figure 1A ) .", "In agreement with previous findings , we found that CTA was acquired after separating the taste and malaise stimuli for up to 8 hr but not 20 hr ( Figure 1B , Figure 1—source data 1 and Figure 1—figure supplement", "1 ) ( Kalat and Rozin , 1971; Kalat and Rozin , 1973; Rozin and Kalat , 1971; Revusky , 1971; Gutiérrez et al . , 2003; Koh et al . , 2009; Chinnakkaruppan et al . , 2014 ) .", "Interestingly , non-parametric cluster analysis revealed that there are two different clusters among 1–8 hr ITI-CTAs: 1–3 hr ( hereinafter short-trace ) and 4–8 hr ( hereinafter long-trace ) ITI-CTAs ( Figure 1B ) . 10 . 7554/eLife . 07582 . 003Figure 1 . Temporal boundaries of taste memory trace for the association with malaise in CTA .", "( A ) Schematic diagram of the experimental design .", "CS , 0 . 1% saccharin and US , 0 . 15M LiCl .", "( B ) Box-whisker plots showing Test1 results ( memory ) .", "1–8 hr ITI-CTA groups but not 20 hr ITI-CTA group showed significantly higher aversion index ( AI; a measure of CTA memory ) compared to control group .", "There are two different clusters among 1–8 hr ITI-CTA groups: short-trace ( 1–3 hr ITI-CTAs ) and long-trace ( 4–8 hr ITI-CTAs ) .", "( C ) 1–3 hr ITI-CTA groups exhibited a similar pattern of extinction and reached AI similar to control group by Test7 whereas 4–8 hr ITI-CTA groups extinguished CTA memory by Test4 .", "Dashed line denotes the mean AI of the control group upon Test1 .", "( D ) Box-whisker plots showing test1 results following ITI-CTA conditioning with weak US ( 0 . 025M LiCl ) .", "1 hr , 2 hr , 3 hr , 4 hr but not 5 hr ITI-CTA groups were significantly different from the control group .", "( E ) Top: schematic representation of the CTA conditioning and reverse conditioning trials .", "In reverse conditioning the novel taste was presented 1 h after the LiCl injection .", "Bottom: reverse conditioning group did not show CTA .", "Data in B and D are median and quartile range between 25% to 75% .", "Data in C and E are mean ± SEM , *p <0 . 05 , **p <0 . 01 , ***p <0 . 001 .", "n ≥ 5 . See also Figure 1—source data 1 and Figure 1—figure supplement 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 07582 . 00310 . 7554/eLife . 07582 . 004Figure 1—source data 1 . Statistical analysis of Figure 1 . Kruskal-Wallis non-parametric ANOVA ( 1B , 1E ) , cluster analysis ( 1B ) , Mann-Whitney test ( 1B ) and Friedman's non-parametric repeated measures ANOVA ( 1C ) and independent samples t-test ( 1F ) were conducted to analyse the group effect .", "DOI: http://dx . doi . org/10 . 7554/eLife . 07582 . 00410 . 7554/eLife . 07582 . 005Figure 1—figure supplement 1 . Attenuation of taste neophobia .", "( A ) The control group from the main Figure 1B , which exhibited taste aversion of about 36% upon the second taste encounter ( Test1 ) , demonstrated less aversion on the third taste encounter ( paired t-test between Test1 and Test2; T = 4 . 781 , p=0 . 001 ) .", "Upper panel depicts the experimental design .", "( B ) Rats demonstrated high aversion to a novel palatable taste upon sampling for the first time .", "This is referred to as neophobia , i . e . , reluctance to consume a novel taste due to lack of knowledge regarding the post-ingestive consequences .", "However , rats exhibited less aversion upon the second and third encounter , demonstrating that taste preference is an adaptive process .", "This phenomenon is called attenuation of neophobia .", "Each line represents a rat and a dotted line is a mean of the group ( one way ANOVA; F ( 1 . 427 , 14 . 273 ) = 27 . 925 , p=0 . 00003 .", "Bonferroni post hoc test , between Test1 and Test2 p=0 . 004; Test2 and Test3 p=0 . 009; and Test1 and Test3 p=0 . 0003 ) .", "( C ) As expected , the CTA control group upon Test1 ( i . e . second time consumption of the novel taste ) did not differ from Test2 of attenuation of neophobia group ( T ( 19 ) = 0 . 954 , p=0 . 352 ) , indicating that ingesting a taste only once is not sufficient to determine that the taste is completely safe to consume and thus rats need to ingest the same taste at least three times to form a memory that the taste is completely safe . DOI: http://dx . doi . org/10 . 7554/eLife . 07582 . 005 Consistent with the retrieval results , long-trace ( 4–8 hr ) CTA groups exhibited a similar faster CTA extinction pattern , whereas short-trace ( 1–3 hr ) CTAs exhibited a slower extinction .", "The long-trace CTAs were not different from an unpaired control group by extinction day4 , in comparison with day7 aversion index ( AI ) in short-trace CTAs ( Figure 1C and Figure 1—source data 1 ) .", "Reverse conditioning did not lead to taste aversion ( Figure 1E ) , which indicates the importance of temporal order between taste and nausea in CTA learning .", "The strength of an association can be affected by ITI between CS and US or by the strength of US .", "We thus conducted an ITI-CTA with a weak US ( 0 . 025M LiCl ) to test whether weaker CTA learning can be acquired when an association is made during the short- ( 1–3 hr ) but not the long-trace ( 4–8 hr ) intervals .", "We found that CTA was formed following the short-trace intervals , and interestingly , also following a 4 hr but not 5 hr trace interval .", "Together these results suggest that there is a shift in the associability of the taste memory trace at around 3–4 hr after exposure to the taste stimulus ( Figure 1D ) .", "The long ITI between CS and US in CTA is reasonable from physiological , ecological , and evolutionary perspectives .", "The minutes to hours process of digestion followed by absorption may dictate the animal to associate between the taste consumed and its delayed physiological consequences ( for more information see reviews , Gal-Ben-Ari and Rosenblum , 2012; Kong and Singh , 2008 ) .", "It is possible that depending on the ITI there are two taste memory traces ( as indicated by the behavioral readout ) : the first one is strong , but lasts for approximately 3 hr ( short-trace ) , whereas the second trace is weak , but lasts for up to 8 hr ( long-trace ) .", "This observation is further supported by the previous demonstration that micro-injection of the protein synthesis inhibitor , anisomycin , into the insular cortex ( IC ) within 3 hr but not 4 hr after the taste inhibits associative taste memory in the latent inhibition of CTA paradigm ( Merhav and Rosenblum , 2008 ) .", "It is therefore plausible to hypothesize that the underlying biological mechanism ( s ) of taste-nausea association may differ between short- and long-trace CTAs .", "Next , we set out to test this hypothesis .", "Given ample evidence that novel taste experience impacts the phospho-proteome in the IC ( see reviews , Adaikkan and Rosenblum , 2012; Gal-Ben-Ari and Rosenblum , 2012 ) and the idea that calcium calmodulin-dependent protein kinase II ( CaMKII ) has the potential to store information ( Lisman , 2014 and the references therein ) , we aimed to test the possibility that CaMKIIα , through auto-phosphorylation ( at T286; which can act as a molecular positive feedback loop ) , maintains the taste memory trace in the IC .", "Moreover , we were encouraged by the previous report that showed that novel taste consumption increases CaMKIIα expression in the IC ( Belelovsky et al . , 2005 ) .", "Therefore , we followed up this finding and expanded it by measuring the phosphorylation/expression levels of CaMKIIα in the IC for up to 8 hr after the consumption of novel taste .", "We subjected IC samples of rats exposed to either water or novel taste solution ( 0 . 1% saccharin ) to biochemical fractionation ( Stern et al . , 2013 ) , and examined the phosphorylation and expression levels of CaMKIIα in the crude synaptosomal fraction ( P2-fraction ) by Western-blotting analysis ( Figure 2A–C and Figure 2—figure supplement 1 ) .", "We replicated the finding from the previous report ( Figure 2—figure supplement 2 ) , that 15 min after novel taste consumption , CaMKIIα expression is increased in the IC .", "Interestingly , we observed that the T286 phosphorylation of CaMKIIα ( pT286CaMKIIα ) was increased in the P2-fraction 30 min following novel taste consumption , persisting for up to 3 hr ( 1 and 3 hr ) , but not 5 or 8 hr afterwards ( Figure 2D , E and Figure 2—figure supplement 1 , 2 ) . 10 . 7554/eLife . 07582 . 006Figure 2 . Novel taste experience induces CaMKIIα phosphorylation in the IC in an NMDAR-dependent manner .", "( A ) Experimental design depicting biochemical fractionation from the IC after behavioral training .", "( B ) Representative immunoblots of marker proteins for different fractions .", "( C ) Rats were sacrificed at the indicated time points after exposure to either water or novel taste solution .", "( D ) Representative pT286CaMKIIα , pT305CaMKIIα , and total CaMKIIα immunoblots from P2-fractions of water ( W ) and novel taste ( N ) groups are shown .", "( E ) Novel taste groups showed increased pT286CaMKIIα levels in the IC synaptosomal fraction at 0 . 5 , 1 , and 3 hr compared to water controls .", "5 and 8 hr groups showed no difference in pT286CaMKIIα levels between water and novel taste groups .", "( F , G )", "There was no difference in ( F ) pT305CaMKIIα and ( G ) total CaMKIIα levels between water and novel taste groups at any time point .", "( H ) Schematic representation of experimental design .", "Rats were injected with saline or MK801 30 min before they were exposed to water or novel taste , and 1 hr later were sacrificed and IC was extracted .", "( I ) Novel taste group injected with saline showed increased pT286CaMKIIα compared to water group .", "MK801-injected groups which received novel taste did not differ from the water group but expressed significantly less phosphorylated CaMKIIα ( pT286 ) than novel taste group injected with saline .", "( J , K )", "There was no difference in ( J ) pT305CaMKIIα and ( K ) total CaMKIIα levels between any groups .", "Data are mean ± SEM , *p<0 . 05 , **p<0 . 01 .", "n ≥ 6 . See also Figure 2—source data 1 and Figure 2—figure supplement 1–3 . DOI: http://dx . doi . org/10 . 7554/eLife . 07582 . 00610 . 7554/eLife . 07582 . 007Figure 2—source data 1 . Statistical analysis of Figure 2 . Independent samples t-test ( 2E , 2F , 2G ) and one way ANOVA ( 2I , 2J , 2K ) were conducted to analyse the group effect .", "DOI: http://dx . doi . org/10 . 7554/eLife . 07582 . 00710 . 7554/eLife . 07582 . 008Figure 2—figure supplement 1 . Uncropped immunoblots of main Figure 2 . ( A ) Immunoblots showing the expression patterns of GluA1 , pT286CaMKIIα , pT305CaMKIIα , CaMKIIα in H , P1 , P2 , P3 , LP1 , LP2 , and S3 fractions .", "Note the enrichment of pT286CaMKIIa in P2-fraction ( synaptosomal fraction ) .", "( B ) Thus , P2 fraction was further used for the expression analysis of the aforementioned proteins after the rats were exposed to either water or novel taste solution .", "All the uncropped original immunoblots for Figure 2D , E , and F are shown .", "W and N denote water and novel taste , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 07582 . 00810 . 7554/eLife . 07582 . 009Figure 2—figure supplement 2 . Total amount of CaMKIIα in the synaptoneurosomal fractions from IC is increased 15 min following novel taste learning .", "( A ) 25 min after the rats were exposed to water or novel taste , they were sacrificed , and immunoblot analysis was conducted using the P2 fraction .", "( B ) Consistent with a previous report ( Belelovsky et al . , 2005 ) , novel taste experience significantly increased total CaMKIIα compared to water controls ( T ( 10 ) = -2 . 273 , p=0 . 046 ) .", "( C , D )", "There was a non-significant trend in the novel taste group in pT286CaMKIIα ( T ( 10 ) = -1 . 471 , p= 0 . 17 ) and pT305CaMKIIα did not differ between water and novel taste groups ( T ( 10 ) = -0 . 134 , p=0 . 896 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 07582 . 00910 . 7554/eLife . 07582 . 010Figure 2—figure supplement 3 . Uncropped immunoblots of main Figure 2 . ( A ) Immunoblots of pT286CaMKIIα , pT305CaMKIIα , CaMKIIα , and β-Tubulin from P2-fraction for Figure 2I–K are shown . DOI: http://dx . doi . org/10 . 7554/eLife . 07582 . 010 There was no significant difference in T305 phosphorylation of CaMKIIα ( pT305CaMKIIα; a phosphorylation site which prevents calcium/calmodulin activation of CaMKII ) between water control and novel taste groups at any time point ( Figure 2D–G and Figure 2—figure supplement 1 , 2 ) .", "Intriguingly , the temporal dynamics of pT286CaMKIIα in the IC after the novel taste experience corresponds to the short-trace timescale , indicating a possible link between CaMKIIα and short-trace CTA learning .", "It is possible that NMDAR activation is upstream to CaMKIIα phosphorylation , because activation of NMDAR in the IC is crucial for taste-malaise association and NMDAR regulates CaMKIIα phosphorylation ( Rosenblum et al . , 1997; Ferreira et al . , 2002; Sanhueza et al . , 2011; Halt et al . , 2012; Parkes et al . , 2014 ) .", "Therefore , we examined if NMDAR activation is necessary for novel taste-dependent CaMKIIα activation .", "Indeed , novel taste experience-induced phosphorylation of T286CaMKIIα is NMDAR-dependent , since i . p . injection of the NMDAR antagonist , MK801 ( 0 . 2 mg/kg b . w ) , 30 min before novel taste learning precluded phosphorylation of T286CaMKIIα in the IC ( Figure 2H , I and Figure 2—figure supplement 3 ) .", "pT305CaMKIIα and total CaMKIIα levels did not differ among the water group injected with saline and the novel taste groups injected with either saline or MK801 ( Figure 2J , K and Figure 2—figure supplement 3 ) .", "Taste memory trace can persist for an association , and at the very same time can undergo sensory information processing to form incidental taste learning ( Chinnakkaruppan et al . , 2014; Rosenberg et al . , 2014 ) .", "Therefore , the correlation between the consumption of novel taste and NMDAR-dependent T286CaMKIIα phosphorylation can serve the taste memory trace for an association , and/or incidental taste memory .", "In order to dissociate between these possibilities , we investigated the role of NMDAR and CaMKIIα in non-associative incidental and associative CTA learning by micro-infusing the NMDAR antagonist APV ( 10 μg/1 μl/hemisphere ) or CaMKIIα inhibitor TatCN21 ( 0 . 3 nM/1 μl/ hemisphere ( Buard et al . , 2010 ) ) bilaterally into the IC .", "Consistent with the literature , our data reveal that NDMAR in the IC is dispensable for incidental taste learning but necessary for CTA learning ( Figure 3—figure supplement", "1 ) ( Rosenblum et al . , 1997; Barki-Harrington et al . , 2009; Parkes et al . , 2014 ) .", "We also found that CaMKIIα in the IC is dispensable for incidental taste learning but necessary for CTA learning ( Figure 3—figure supplement 1 , 2 ) .", "Given that NMDAR-dependent T286CaMKIIα phosphorylation in the IC is required specifically for CTA , we sought to test whether NMDAR-CaMKIIα signaling in the IC maintains the taste memory trace for the association with the US by infusing the respective antagonist or inhibitor into the IC at various time points between the CS and US .", "We micro-injected the NMDAR antagonist into the IC 25 min from the beginning of the taste consumption in 1 hr ITI-CTA conditioning with a weak US and observed an attenuated CTA memory , consistent with a previous report in which a strong US was administered ( Rosenblum et al . , 1997; but also see Ferreira et al . , 2002 ) ( Figure 3—figure supplement 3A ) .", "Intriguingly , we did not observe any effect when we made a similar manipulation in the IC 4 hr after the taste consumption in 5 hr ITI-CTA conditioning with a strong US ( Figure 3—figure supplement 3B; all the remaining experiments were done with a strong US ) .", "Next , we micro-injected CaMKIIα inhibitor TatCN21 or Tat control ( Tatcont ) into the IC 25 min from the beginning of the taste consumption in short-trace 3 hr ITI-CTA conditioning , and observed an attenuated CTA memory ( Figure 3B and Figure 3—source data 1 ) .", "We micro-injected TatCN21 into the IC 25 min ( i . e . short-trace timescale ) from the beginning of the taste consumption in the long-trace 5 h ITI-CTA conditioning , and also observed an attenuated CTA memory ( Figure 3C ) .", "However , consistent with the temporal dynamics of CaMKIIα phosphorylation in the IC , CaMKIIα inhibition in the IC 4 h after the taste consumption in the long-trace 5 hr ITI-CTA conditioning had no effect on CTA memory ( Figure 3D ) . 10 . 7554/eLife . 07582 . 011Figure 3 . The requirement of CaMKIIα in the IC for associative learning of CTA is a function of time .", "( A ) Outline of the experimental design .", "( B ) Infusion of CaMKIIα inhibitor TatCN21 into the IC 25 min after the taste consumption in 3 hr ITI-CTA conditioning attenuated the CTA memory .", "( C ) Infusion of CaMKIIα inhibitor TatCN21 into the IC 25 min after the taste consumption in 5 hr ITI-CTA conditioning attenuated the CTA memory .", "( D ) Infusion of CaMKIIα inhibitor TatCN21 into the IC 4 hr after the taste consumption in 5 hr ITI-CTA conditioning had no effect on CTA memory .", "( E ) TatCN21 micro-infusion 2 hr after the consumption of taste in 3 hr ITI-CTA training did not affect the CTA memory .", "Upper panel in B , C , D , and E depict the conditioning trial .", "The syringe represents microinfusion .", "( F ) A series of coronal sections from a representative rat brain , showing the cannula placement in the rostro-caudal planes ( lower panel ) and the corresponding coronal sections of rat brain atlas images ( upper panel ) .", "Abbreviations; AI-agranular insular cortex , DI-disgranular insular cortex , GI-granular insular cortex .", "Data are mean ± SEM , *p <0 . 05 .", "n ≥ 11 .", "See also Figure 3—source data 1 and Figure 3—figure supplement 1–4 . DOI: http://dx . doi . org/10 . 7554/eLife . 07582 . 01110 . 7554/eLife . 07582 . 012Figure 3—source data 1 . Statistical analysis of Figure 3 . Independent samples t-test was conducted to analyse the group effect .", "The differences between the variances of groups were corrected following Levene’s test for equality of variances .", "DOI: http://dx . doi . org/10 . 7554/eLife . 07582 . 01210 . 7554/eLife . 07582 . 013Figure 3—figure supplement 1 . NMDAR and CaMKIIα in the IC are required for associative but not for incidental taste learning . We investigated whether CaMKIIα and its upstream NMDAR in the IC are required for associative CTA and/or incidental taste learning .", "( A ) Infusion of CaMKIIα inhibitor TatCN21 into the IC 30 min before 1 hr ITI-CTA conditioning disrupted CTA memory .", "( B ) Infusion of NMDAR antagonist APV ( 10 μg/μl ) into the IC 30 min before 1 hr ITI-CTA conditioning disrupted CTA .", "( C ) TatCN21 infusion 30 min before novel taste consumption did not affect the incidental taste memory .", "( D ) APV infusion 30 min before novel taste consumption did not affect the incidental taste memory .", "Upper panels in all graphs depict the conditioning trial .", "Data are mean ± SEM , **p<0 . 01 . DOI: http://dx . doi . org/10 . 7554/eLife . 07582 . 01310 . 7554/eLife . 07582 . 014Figure 3—figure supplement 2 . The effect of CaMKIIα inhibitor on CTA learning is a function of time and concentration of TatCN21 . ( A ) Top: schematic representation of experimental design .", "Rats underwent CTA conditioning 8 days after TatCN21 microinjection into the IC .", "TatCN21 ( 0 . 3 nM/μl ) did not have an effect on CTA learning and memory ( n=Tatcont , 4; TatCN21 , 5; T ( 7 ) = 0 . 407 , p=0 . 696 ) .", "( B ) Upper panel shows the conditioning paradigm .", "Microinjection of low concentration of TatCN21 ( 0 . 05 nM/μl ) into the IC 30 min before 1 hr ITI-CTA conditioning did not have an effect on CTA learning ( n = Tatcont , 4; TatCN21 , 5; T ( 7 ) = 1 . 596 , p=0 . 154 ) .", "( C ) CaMKIIα inhibition 25 min after taste consumption did not have an effect on the non-associative form of incidental taste memory ( n = Tatcont , 14; TatCN21 ( 0 . 3 nM/μl ) , 14; T ( 20 . 97 ) = 0 . 669 , p=0 . 511 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 07582 . 01410 . 7554/eLife . 07582 . 015Figure 3—figure supplement 3 . NMDAR in the IC plays a time-dependent role in CTA learning .", "( A ) Microinjection of NMDAR antagonist , APV , into the IC 25 min after the consumption of novel taste in 1 hr ITI-CTA conditioning with US attenuated CTA memory ( T ( 16 ) = 2 . 046 , p=0 . 05 ) .", "( B ) APV micro-infusion 4 hr after the taste in 5 hr ITI-CTA conditioning did not affect the CTA memory ( T ( 15 ) = 1 . 069 , p=0 .", "302 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 07582 . 01510 . 7554/eLife . 07582 . 016Figure 3—figure supplement 4 . CaMKIIα in the IC does not affect the learned safety about the taste . In order to test the possibility that the effects produced by TatCN21 in Figures 3B , C are attributable to an increase in the short term learning about the safety of the taste rather than disruption in taste-LiCl association , we conducted a behavioral experiment in which rats were presented with a novel taste and at various time intervals ranging from 1–8 hr were tested for their preference to the pre-exposed taste .", "We later investigated whether short-term taste memory is amenable to perturbation of CaMKIIα in the IC .", "( A ) Outline of the behavioral experiment .", "Following habituation rats were exposed to novel taste and 1 , 3 , 5 , and 8 hr later they were subjected to a taste choice test and the aversion index was calculated .", "( B ) Interestingly , 5 and 8 hr groups exhibited less aversion and were significantly lower than both 1 hr and naïve preference ( NP ) groups ( Kruskal-Wallis ANOVA; n = 11 rats/group; X2 ( 4 , n = 55 ) = 25 . 162 , p<0 . 0001; Post-hoc test compared to NP group , 1 hr , p=0 . 884; 3 hr , p=0 . 199; 5 hr , p=0 . 018; 8 h , p=0 . 002 .", "Post-hoc test compared to 1 hr group; 3 hr , p=0 . 133; 5 hr , p=0 . 011; 8 hr , p=0 . 001 ) .", "( C ) The regression line between elapsed time after taste pre-exposure and aversion index , which is statistically significant , is shown ( n = 44 [naïve preference group was not included] , R = - 0 . 593 , p<0 . 001 ) .", "( D , E )", "The decreased AI to the pre-exposed taste as shown in A and B cannot be attributed to the difference in the fluid consumption as revealed by the negative relationship between saccharin and water consumption with the preference index ( MANOVA; Pillai's trace , general interaction; F ( 8 , 100 ) = 9 . 438 , p<0 . 001; Saccharin volume consumption , F ( 4 , 50 ) = 16 . 198 , p<0 . 0001; Post-hoc Tukey's HSD , compared to naïve preference group , 1 hr , p= 0 . 528; 3 hr , p<0 . 177; 5 hr , p=0 . 004; 8 hr , p<0 . 0001; compared to 1 hr group , 3 hr , p= 0 . 003; 5 hr , p<0 . 001; 8 hr , p<0 . 0001 .", "Water volume consumption , F ( 4 , 50 ) = 8 . 168 , p<0 . 0001; Post-hoc Tukey's HSD , compared to naïve preference group , 1 hr , p= 0 . 266; 3 hr , p=0 . 008; 5 hr , p=0 . 0001; 8 hr , p<0 . 0001; compared to 1 hr group , 3 hr , p=0 . 570; 5 hr , p=0 . 125; 8 hr , p=0 . 035 ) .", "( F ) Infusion of CaMKIIα inhibitor TatCN21 into the IC 30 min before taste consumption had no effect on the short term incidental taste memory ( t-test; n = Tatcont , 9 and TatCN21 , 8; T ( 15 ) = 0 . 388 , p=0 . 704 ) .", "Upper panel depicts the conditioning trial and the syringe represents microinfusion . DOI: http://dx . doi . org/10 . 7554/eLife . 07582 . 016 These results indicate that novel taste consumption induces NMDAR-dependent T286CaMKIIα phosphorylation in the IC , which is necessary for maintaining the short taste memory trace for the association with visceral information .", "If this is the case , then inhibiting CaMKII 2 hr after novel taste consumption in 3 hr ITI-CTA should still impede the CS-US association .", "To answer this , we performed a 3 hr ITI-CTA experiment in which we micro-infused CaMKIIα inhibitor TatCN21 2 hr after the taste consumption , and administered the LiCl 1 hr later .", "We observed a small non-significant effect ( Figure 3E ) .", "It is thus more likely that T286CaMKIIα phosphorylation in the IC is necessary for the development of the taste memory trace for the association and downstream target/s play a vital role in maintaining the taste memory trace for the CS-US association .", "CaMKII-dependent modulation of GluA1-containing AMPA receptors and GluN2B-containing NMDARs has been implicated in different forms of learning , memory and synaptic plasticity , and can induce changes in synaptic strength ( Hayashi et al . , 2000; Whitlock et al . , 2006; Sanhueza et al . , 2011 ) .", "Therefore , we investigated whether pT286CaMKIIα modulates GluA1 and/or GluN2B phosphorylation/expression in the IC after novel taste experience .", "Indeed , experiencing novel taste increased total GluA1 but not pS831GluA1 or pS1303GluN2B in the synaptosomal fraction in the IC 1 hr later ( Figure 4A and Figure 4—figure supplement 1 ) .", "Linear regression analysis revealed a positive correlation between pT286CaMKIIα and GluA1 expression in animals sampling novel taste ( Figure 4B and Figure 4—figure supplement 1 ) . 10 . 7554/eLife . 07582 . 017Figure 4 . The requirement of CaMKIIα-dependent GluA1 expression in the IC for the associative process of CTA is a function of time .", "( A ) 1 hr after novel taste consumption total GluA1 but not pS831GluA1 was increased in the P2-fraction .", "Upper panel shows the representative immunoblots .", "( B ) pT286CaMKIIα was positively correlated with GluA1 levels in the novel taste group but not in the water group .", "( C ) Upper panel depicts the experimental design .", "Novel taste-dependent increased GluA1 expression in the IC was precluded by TatCN21 microinjection into the IC .", "( D ) CNQX microinjection into the IC 1 hr after the taste consumption in 3 hr ITI-CTA conditioning attenuated the CTA memory .", "( E ) CNQX infusion into the IC 2 hr after the consumption of taste in 3 hr ITI-CTA training attenuated CTA memory .", "( F ) CNQX microinjection into the IC 1 hr after taste consumption in 5 hr ITI-CTA conditioning attenuated the CTA memory .", "( G ) CNQX application 4 hr after taste consumption in 5 hr ITI-CTA conditioning had no effect on CTA memory .", "Upper panels in D , E , F , and G depict the conditioning trial .", "Data are mean ± SEM , *p<0 . 05 , **p<0 . 01 .", "n ≥ 9 .", "See also Figure 4—source data 1 and Figure 4—figure supplement 1–5 . DOI: http://dx . doi . org/10 . 7554/eLife . 07582 . 01710 . 7554/eLife . 07582 . 018Figure 4—source data 1 . Statistical analysis of Figure 4 . Independent samples t-test ( 4A , 4C , 4D , 4E , 4F , 4G ) was conducted to analyse the group effect .", "Pearson’s correlation was used to analyse the association between the association between GluA1 and pT286CaMKIIα ( 4B ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 07582 . 01810 . 7554/eLife . 07582 . 019Figure 4—figure supplement 1 . Novel taste experience leads to an increase in Y1472 but not S1303 phosphorylation of GluN2B .", "( A ) Representative immunoblots of pY1472GluN2B , pS1303GluN2B , and GluN2B .", "Similarly to main Figure 3C , 1 hr after the rats were exposed to water or novel taste , they were sacrificed , and immunoblot analysis was conducted using the P2-fraction .", "( B ) Consistent with a previous report ( Barki-Harrington et al . , 2009 ) , novel taste experience significantly increased pY1472GluN2B compared to water controls ( T ( 10 ) = -2 . 319 , p=0 . 046 ) .", "( C ) Interestingly , however , pS1303GluN2B phosphorylation , which is known to be regulated by CaMKIIα , was not different from water and novel taste groups ( T ( 35 ) = -1 . 078 , p=0 . 288 ) .", "( D ) Also , total GluN2B was not different between water and novel taste groups ( T ( 35 ) = -0 . 748 , p=0 . 460 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 07582 . 01910 . 7554/eLife . 07582 . 020Figure 4—figure supplement 2 . Uncropped original immunoblots of main Figure 4 . ( A , B ) All the original ( A ) GluA1 and ( B ) pS831GluA1 immunoblots for Figure 4A are shown .", "The corresponding β-Tubulin immunoblots can be seen in Figure 2—figure supplement 1 .", "( C ) All original immunoblots of GluA1 and β-Tubulin for Figure 4C are shown .", "Water + Tatcont and novel taste + Tatcont samples from this experiment were combined to generate Figure 4B . DOI: http://dx . doi . org/10 . 7554/eLife . 07582 . 02010 . 7554/eLife . 07582 . 021Figure 4—figure supplement 3 . Novel taste experience-induced GluA1 in the IC is NMDAR-dependent .", "( A ) Outline of the experimental design .", "( B , C )", "Uncropped original immunoblots and ( C ) the quantification thereof .", "Novel taste group injected with saline showed increased GluA1 expression levels compared to water group .", "MK801 ( 0 . 2 mg per kg b . w . ) injected groups which received novel taste did not differ from the water group , but expressed significantly lower levels of GluA1 than novel taste group injected with saline , suggesting that NMDAR is upstream to increased GluA1 after the novel taste experience ( n = 14/group; one way ANOVA; F ( 2 , 41 ) = 6 . 132 , p=0 . 005; post-hoc LSD; water + saline Vs novel taste + saline p=0 . 002 , novel taste + saline Vs novel taste + MK801 p=0 . 009 and water + saline Vs novel taste + MK801 p=0 . 605 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 07582 . 02110 . 7554/eLife . 07582 . 022Figure 4—figure supplement 4 . AMPAR in the IC is dispensable for incidental taste learning . Micro-infusion of AMPAR antagonist CNQX 30 min before the taste learning did not affect the incidental form of the appetitive taste memory ( T ( 14 ) = 0 . 335 , p=0 . 743 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 07582 . 02210 . 7554/eLife . 07582 . 023Figure 4—figure supplement 5 . Schematic representation of the conceptual parallel taste-memory trace model .", "( A ) When an animal feels no negative visceral consequence after eating a novel taste , it forms a long term safe taste memory .", "However , when it encounters nausea , it forms an associative aversive taste memory ( CTA ) .", "The strength of the CS or the US or the time lapse between the two or other sensory cues at the time of learning can affect the formation of the internal representation .", "Here , we focused on the variable of ITI between the CS and US .", "( B ) In the present model we aim to explain the temporal constraints and biological mechanisms underlying trace-CTA .", "Using sodium saccharin as CS and 0 . 14M LiCl as strong US we found that the novel taste memory trace is generated after the consumption of novel taste , and lasts for approximately 8 hr to associate with its outcome .", "The associability of the taste memory trace changes at around 4 hr after the novel taste consumption with the US .", "Taken together , the behavioral readout with different ITIs and pharmacological manipulations of the NMDAR-CaMKIIα-GluA1 axis in the IC using different ITI-CTA training , we reason that there are two parallel taste memory traces with different underlying mechanisms: one that is robust but decays quickly ( strong-trace; solid blue line ) and the second trace , which is weak but lasts longer ( weak-trace; dashed blue line ) .", "X-axis depicts the time in hours between the taste and malaise .", "Y-axis indicates the strength of the associable taste memory trace .", "It is possible that the weak taste-memory trace is parallel to that of the strong taste-memory trace or independent of the strong trace .", "Given our findings that different pharmacological manipulations in the insular cortex ( APV , TatCN21 , CNQX ) affect the associability of the taste-memory trace when applied only during the strong-trace timescale but not in the weak taste-memory trace timescale ( longer than 3 hr ) , we suggest that the weak-taste memory trace is parallel to that of the strong one .", "( C ) Finally , we add the dimension of additional information registered during the association phase by additional brain areas .", "We propose a multiple memory systems/trace theory , which posits that CTA learning may engage multiple memory systems to take part in the long-trace associative process and that after experiencing the taste , the NMDA receptor-dependent CaMKIIα and GluA1 pathway in the insular cortex plays a critical role only during the short-trace , whereas NMDA receptors in the hippocampus play a crucial role as the time increases ( Chinnakkaruppan et al . , 2014 ) , for the association with the US .", "Note that the background information/experience exists right from the CS stimulus experience but arguably they accumulate more with time . DOI: http://dx . doi . org/10 . 7554/eLife . 07582 . 023 To examine whether pT286CaMKIIα is required for the increased GluA1 expression in the P2-fraction after the taste consumption , we micro-infused CaMKIIα inhibitor TatCN21 into the IC 30 min before novel taste exposure and measured GluA1 expression 1 hr later .", "TatCN21 application reduced novel taste experience-dependent GluA1 expression in the P2-fraction ( Figure 4C and Figure 4—figure supplement 2 ) .", "Moreover , NMDAR antagonist MK801 injection 30 min before the novel taste also reduced GluA1 expression ( Figure 4—figure supplement 3 ) , indicating that NMDAR- and CaMKIIα-dependent increased synaptic expression of GluA1 in the IC mediates short-trace taste memory for the association with the US .", "If indeed GluA1 in the IC mediates the taste memory trace for the association , we hypothesized that pharmacological inhibition of AMPAR following taste experience would interfere with the association with the US .", "Microinjection of AMPAR antagonist , CNQX , ( 1 μl/hemisphere; 3 nM/μl ) ( Tse et al . , 2011 ) into the IC 1 hr after the taste consumption in short-trace 3 hr ITI-CTA conditioning attenuated the CTA memory ( Figure 4D ) .", "Interestingly , CNQX micro-injection into the IC 2 hr after the taste consumption in 3 hr ITI-CTA learning also attenuated CTA memory ( Figure 4E ) .", "Furthermore , microinjection of CNQX into the IC 1 hr ( short-trace timescale ) after the taste consumption in long-trace 5 hr ITI-CTA conditioning also attenuated the CTA memory ( Figure 4F ) .", "However , in accordance with the timescale of short-trace and CaMKIIα-GluA1 activation , we found that CNQX micro-injection 4 hr after novel taste consumption in 5 hr ITI-CTA had no effect on CTA ( Figure 4G ) .", "It is intriguing that the inhibition of CaMKIIα or AMPAR in the IC during short-trace timescale attenuated short-trace CTA memory .", "In addition , inhibition of the CaMKIIα-GluA1 pathway during short-trace timescale also attenuated long-trace CTA .", "However , long-trace CTA was not affected when CaMKIIα-GluA1 pathway in the IC was left intact for 3 hr .", "Together , these data reveal that there are two parallel taste memory traces which are subserved by different mechanisms: one that is robust but decays quickly , and another which is weak but lasts longer .", "It is important to note that NMDAR-CaMKIIα-AMPAR in the IC are not necessary for incidental taste learning and memory , whereas muscarinic receptors are critical for incidental taste learning ( Ferreira et al . , 2002; Parkes et al . , 2014 ) .", "It is interesting that different molecular mechanisms take place in the same cortex , the insular cortex , to mediate associative and non-associative taste learning and memory .", "We should emphasize that different pharmacological manipulation ( APV , TatCN21 , CNQX ) in the IC when applied within 3 hr after the CS presentation only modifies but not completely erases the CTA memory .", "It is also noteworthy that several previous reports demonstrate that pharmacological perturbations in the IC during CTA conditioning only partially disrupt CTA memory , and that the disrupted CTA appears similar to long-trace CTA memory ( Rosenblum et al . , 1993; Rosenblum et al . , 1997; Berman et al . , 2000; Eisenberg et al . , 2003; Gutiérrez et al . , 2003; Barki-Harrington et al . , 2009; Inberg et al . , 2013; Stern et al . , 2013; Parkes et al . , 2014 ) .", "Thus , it is possible that over time , there is a transformation from the taste memory trace dominance ( i . e . short-trace ) and IC dependency to multiple memory traces ( i . e . during long-trace ) due to the multi-channeled background experience , e , g .", ", time , space , food digestion-dependent body physiology , etc .", "Such transformation , which involves multiple memory traces , for instance , episodic memory trace ( how long ago the taste was consumed and under what context ? ) , may result in a wider distribution and processing of the taste memory trace with the passage of time ( Kalat and Rozin , 1971; Revusky , 1971; Kalat and Rozin , 1973; Koh et al . , 2009; Chinnakkaruppan et al . , 2014 ) .", "Since the initial demonstration of CTA by Garcia ( 1955 ) , several studies have proposed that the reduced aversion index followed by the increased interval between the taste and malaise in CTA conditioning could be attributed to", "( i ) the learned safety about the taste ,", "( ii ) accumulating background interference , and", "( iii ) taste memory trace decay ( Kalat and Rozin , 1971; Rozin and Kalat , 1971; Revusky , 1971; Kalat and Rozin , 1973; Gutiérrez et al . , 2003; Koh et al . , 2009; Chinnakkaruppan et al . , 2014 ) .", "First , in support of the learned safety theory , and in line with the previous reports , we found that animals exhibit higher preference to the taste hours following the first-time experience ( Figure 3—figure supplement 4 ) ( Kalat and Rozin , 1973; Gutiérrez et al . , 2003 ) .", "Second , with regards to background interference theory , we propose that the multi-channeled background information can be viewed as episodic components and that , for example , the hippocampus is critical to assimilate the episodic component in CTA ( Chinnakkaruppan et al . , 2014; Koh et al . , 2009 ) .", "Third , in line with the trace decay theory , our findings demonstrate that the robust short-trace fades within 3 hr in the IC but the long-trace , which is weak , lasts longer .", "Moreover , the non-linear decay of the short trace suggests that it dominates over the long trace , so that and when the former fades , the latter is revealed ( Figure 4—figure supplement 5 ) .", "We propose a multiple memory trace theory , wherein we suggest that CTA learning may engage multiple memory systems to take part in the long-trace associative process and that after experiencing the taste , the IC plays a critical role during short-trace ( Figure 4—figure supplement 5 ) .", "Although multiple lines of evidence support that NMDAR-CaMKII-AMPAR signaling in the IC plays a crucial role in maintaining the short-trace for the association with the US , we do not rule out the possibility that other molecular mechanisms in the IC may participate as well in the long-trace CTA .", "For instance , protein acetylation , phosphorylation , and induction of proteins were observed in the IC many hours following novel taste consumption , and it is possible that these correlative molecular changes ( proteins synthesis and epigenetic regulation ) may contribute to the long-trace CTA ( Swank and Sweatt , 2001; Yefet et al . , 2006; Elkobi et al . , 2008; for reviews see Gal-Ben-Ari and Rosenblum , 2012 ) .", "Conceptually , on the one hand , our short-trace CTA complies with the commonly held assumption of neuroscience theories of associative learning that convergence of CS and US information onto particular cells/circuits leads to changes in synaptic strength at the synapses mediating the CS input to those commonly activated cells/circuit and that it underlies association formation , i . e . Hebbian plasticity ( NMDAR-CaMKII-AMPARs in the IC probably in co-ordination with the amygdala; Yasoshima et al . , 2000; Ferreira et al . , 2005; Hashikawa et al . , 2013 ) .", "On the other hand , the long-trace CTA challenges this assumption , and it is possible that because of the multi-channeled background information with an increasing time interval as discussed above , homeostatic plasticity may play a crucial role to encode multiple memory traces via coordinating several brain structures such as the IC , amygdala , hippocampus , prefrontal cortices in the long-trace CTA ( Vitureira et al . , 2012; Turrigiano , 2012; Pozo and Goda , 2010 ) .", "Overall , our data suggest that the neural mechanisms of associative learning can be an assimilation of multiple memory traces when the two relevant experiences are separated in time ." ], [ "The experimental subjects were rats ( Rattus Norvegicus; Wistar Hola and Sprague Dawley ) , obtained from Harlan ( Rehovot , Israel ) .", "They were maintained at the University of Haifa in a temperature controlled ( 22–24°C ) animal core facility under a 12 hr light/12 hr dark cycle ( light phase 7:00–19:00 ) .", "Experiments were conducted at least 7 days after the acclimatization to the facility when the body weight of the rats was 220-–350 g .", "Rats were group housed ( 4–6 rats ) in home cages with food and water ad libitum , and were individually housed before the start of the experiments .", "All the experiments were conducted during the light phase .", "All grouped data are presented as mean ± SEM .", "Comparisons between data of two independent groups were analyzed by unpaired Student's t test and the differences between the variances of groups were corrected following Levene’s test for equality of variances .", "Multiple group comparisons were assessed using one way analysis of variance ( ANOVA ) and repeated-measures ANOVA .", "Follow-up analyses were conducted using Fisher’s least significant difference , Bonferoni , and independent sample t-tests , when significant main effects or interactions were detected .", "Non-parametric Kruskal-Wallis test , Friedman's test , and Mann Whitney U test were conducted when the data were non-normally distributed .", "Pearson’s correlation was conducted to analyze the association between GluA1 and pT286CaMKIIα .", "All of the comparisons were conducted using two-tailed tests of significance .", "The null hypothesis was rejected at the p <0 . 05 level .", "Data analysis was performed using SPSS-version 19 ." ] ]

[ "Events separated in time are associatively learned in trace conditioning , recruiting more neuronal circuits and molecular mechanisms than in delay conditioning .", "However , it remains unknown whether a given sensory memory trace is being maintained as a unitary item to associate .", "Here , we used conditioned taste aversion learning in the rat model , wherein animals associate a novel taste with visceral nausea , and demonstrate that there are two parallel memory traces of a novel taste: a short-duration robust trace , lasting approximately 3 hr , and a parallel long-duration weak one , lasting up to 8 hr , and dependent on the strong trace for its formation .", "Moreover , only the early robust trace is maintained by a NMDAR-dependent CaMKII- AMPAR pathway in the insular cortex .", "These findings suggest that a memory trace undergoes rapid modifications , and that the mechanisms underlying trace associative learning differ when items in the memory are experienced at different time points ." ]

[ "The survival of animals , including us humans , depends on the ability to discriminate good food from bad .", "We would prefer eating a given taste if it did not cause any negative feelings after eating it for the first time; however , we would avoid eating that specific taste if it caused any digestive discomfort .", "This ability to connect sensory events that happen close in time is called associative learning .", "One longstanding theory of associative learning suggests that if the neurons that are activated by a taste fire at the same time as those that control nausea , the connections between the two groups of neurons are strengthened .", "This helps that particular taste to become associated with the feeling of illness .", "Animals can also link events that are separated in time – for example , they can become averse to a food even when its ill effects are felt several hours after eating it .", "An important question is how a new event ( such as a new food ) is internally represented and maintained for a certain time so that it associates with a response ( sickness ) that occurs much later .", "One method used to investigate associative learning is to feed rats a new food , and then later make them feel nauseous to measure how much this causes them to avoid the food in the future .", "The gustatory cortex is the part of the brain responsible for perceiving taste .", "Chinnakkaruppan and Rosenblum now use this experimental method to investigate the molecular mechanisms in the gustatory cortex that enable the internal representation ( or memory trace ) of a new taste to be associated with an unwell feeling that occurs much later .", "The results of the experiments show that rats will avoid food with a certain flavor if they feel unwell within eight hours of eating it .", "However , the response of the rats differs depending on when the rat becomes ill .", "Underpinning these behaviors is the formation of two parallel internal representations of the new taste: a short-term , robust trace that lasts for three hours; and a parallel , longer lasting , weaker trace that lasts for eight hours to associate the taste with its outcome .", "The weaker , longer-lasting memory trace only forms if the shorter , stronger trace also occurs .", "Chinnakkaruppan and Rosenblum found that forming the shorter , stronger memory requires the activity of a signaling pathway in the gustatory cortex that involves biochemical molecules called NMDAR-CaMKII-GluA1 .", "These molecules can increase the strength of signaling between neurons and are already implicated in learning and memory .", "The next challenge is to put this newly identified molecular mechanism within the relevant neural circuit in the gustatory cortex ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "microbiology and infectious disease", "genetics and genomics" ]

[ [ "Many eukaryotic genomes harbor endogenous viral elements ( EVEs ) ( Feschotte and Gilbert , 2012 ) .", "For retroviruses , integration as a provirus is an essential part of their replication cycles , but other viruses also occasionally endogenize , for instance with the help of cellular retroelements ( Holmes , 2011 ) .", "Some green algal genomes even contain giant EVEs of several hundred kilobase pairs ( kbp ) in length ( Moniruzzaman et al . , 2020 ) , but unlike prophages in bacteria and archaea , most eukaryotic EVEs are thought to be ‘genomic fossils’ and incapable of virion formation and horizontal transmission .", "However , some viral genes may be co-opted for various host functions ( Frank and Feschotte , 2017; Aswad and Katzourakis , 2012 ) .", "In recent years , the exploration of protist-infecting giant viruses has uncovered a novel class of associated smaller DNA viruses with diverse and unprecedented genome integration capabilities .", "Viruses of the family Lavidaviridae , commonly known as virophages , depend for their replication on giant DNA viruses of the family Mimiviridae and can parasitize them during coinfection of a suitable protist host ( La Scola et al . , 2008; Krupovic et al . , 2016a; Duponchel and Fischer , 2019 ) .", "A striking example is the virophage mavirus , which strongly inhibits virion synthesis of the lytic giant virus CroV during coinfection of the marine heterotrophic nanoflagellate Cafeteria sp .", "( Stramenopiles; Bicosoecida ) ( Fischer and Suttle , 2011; Fischer and Hackl , 2016 ) .", "Virophages possess 15–30 kbp long double-stranded ( ds ) DNA genomes of circular or linear topology that tend to have low GC-contents ( 27–51% ) ( Fischer , 2020 ) .", "A typical virophage genome encodes 20–30 proteins , including a major capsid protein ( MCP ) , a minor capsid or penton protein ( PEN ) , a DNA packaging ATPase , and a maturation cysteine protease ( PRO ) ( Krupovic et al . , 2016a ) .", "In addition to this conserved morphogenesis module , virophages encode DNA replication and integration proteins that were likely acquired independently in different virophage lineages ( Yutin et al . , 2013 ) .", "Viruses in the genus Mavirus contain a rve-family integrase ( rve-INT ) that is also found in retrotransposons and retroviruses , with close homologs among the eukaryotic Maverick/Polinton elements ( MPEs ) ( Fischer and Suttle , 2011 ) .", "MPEs were initially described as DNA transposons ( Pritham et al . , 2007; Kapitonov and Jurka , 2006 ) , but many of them carry the morphogenesis gene module and thus qualify as endogenous viruses ( Krupovic et al . , 2014 ) .", "Phylogenetic analysis suggests that mavirus-type virophages share a common ancestry with MPEs and the related Polinton-like viruses ( PLVs ) ( Fischer and Suttle , 2011; Yutin et al . , 2013 ) .", "We therefore tested the integration capacity of mavirus using the cultured protist Cafeteria burkhardae ( formerly Cafeteria roenbergensis; Fenchel and Patterson , 1988; Schoenle et al . , 2020 ) and found that mavirus integrates efficiently into the nuclear host genome ( Fischer and Hackl , 2016 ) .", "The resulting mavirus provirophages are transcriptionally silent unless the host cell is infected with CroV , which leads to reactivation and virion formation of mavirus .", "Newly produced virophage particles then inhibit CroV replication and increase host population survival during subsequent rounds of coinfection ( Fischer and Hackl , 2016 ) .", "The mutualistic Cafeteria-mavirus symbiosis may thus act as an adaptive defense system against lytic giant viruses ( Fischer and Hackl , 2016; Koonin and Krupovic , 2016 ) .", "The integrated state of mavirus is pivotal to the proposed defense scheme as it represents the host’s indirect antigenic memory of CroV ( Koonin and Krupovic , 2017 ) .", "We therefore investigated endogenous virophages to assess the prevalence and potential significance of virophage-mediated defense systems in natural protist populations .", "Here , we report that mavirus-like EVEs are common , diverse , and most likely active mobile genetic elements ( MGEs ) of C . burkhardae .", "Our results suggest an influential role of these viruses on the ecology and evolution of their bicosoecid hosts ." ], [ "In preparation of screening for endogenous virophages , we generated high-quality de novo genome assemblies of four cultured C . burkhardae strains ( Hackl et al . , 2020 ) .", "These strains , designated BVI , Cflag , E4-10P ( E4-10 ) , and RCC970-E3 ( RCC970 ) , were isolated from the Caribbean Sea in 2012 , the Northwest Atlantic in 1986 , the Northeast Pacific in 1989 , and the Southeast Pacific in 2004 , respectively .", "We sequenced their genomes using both short-read ( Illumina MiSeq ) and long-read ( Pacific Biosciences RSII ) technologies in order to produce assemblies that would resolve 20–30 kb long repetitive elements within the host genomic context .", "Each C . burkhardae genome assembly comprised of 34–36 megabase pairs with an average GC-content of 70% ( Hackl et al . , 2020 ) .", "To identify endogenous virophages , we combined sequence similarity searches against known virophage genomes with genomic screening for GC-content anomalies .", "The two approaches yielded redundant results and virophage elements were clearly discernible from eukaryotic genome regions based on their low ( 30–50% ) GC-content ( Figure 1A ) .", "Each element had at least one open reading frame ( ORF ) with a top blastp hit to a mavirus protein , with no elements bearing close resemblance to Sputnik or other virophages outside the genus Mavirus .", "In the four Cafeteria genomes combined , we found 138 endogenous mavirus-like elements ( EMALEs , Figure 1B and C; Figure 1—figure supplement 1 , Supplementary file 1 ) .", "Thirty-three of these elements were flanked by terminal inverted repeats ( TIRs ) and host DNA and can thus be considered full-length viral genomes .", "The remainder were partial virophage genomes that were located at contig ends or on short contigs .", "These cases arise from incomplete assembly rather than from biological truncations , since the assembly algorithm probably terminated due to the presence of multiple identical or highly similar EMALEs within the same host genome – a well-known issue for repetitive sequences ( Kolmogorov et al . , 2019 ) .", "With 55 elements , C . burkhardae strain BVI contained nearly twice as many EMALEs as any of the other strains , where we found 27–29 elements per genome ( Figure 1C , Supplementary file 1 ) .", "Compared to the total assembly length , EMALEs composed an estimated 0 . 7–1 . 8% of each host assembly ( Figure 1D ) .", "Contributions calculated from assemblies deviated only slightly ( 0 . 01–0 . 3% ) from read-based calculations .", "Therefore , the assemblies seem to provide a good representation of the actual contribution of EMALEs to the overall host genomes .", "From here on , we focus our analysis on the 33 complete EMALE genomes , which were 5 . 5–21 . 5 kb long with a median length of 19 . 8 kb , and TIRs that varied in length from 0 . 2 to 2 . 3 kb with a median of 0 . 9 kb ( Supplementary file 1 , Figure 3—figure supplement 1 ) .", "Their GC-contents ranged from 29 . 7% to 52 . 7% , excluding retrotransposon insertions where present .", "To classify EMALEs we used an all-versus-all DNA dot plot approach ( Figure 2 ) .", "It revealed two main blocks: The first block contained EMALEs with GC-contents of 29 . 7–38 . 5% ( median 35 . 3% ) , whereas EMALEs in the second block had GC-contents ranging from 47 . 2% to 52 . 7% ( median 49 . 3% ) .", "The C . burkhardae EMALEs can thus be roughly separated into low-GC and mid-GC groups .", "Based on the similarity patterns within each block , we further distinguish eight EMALE types , with low-GC EMALEs comprising types 1–4 and mid-GC EMALEs comprising types 5–8 ( Figure 2 ) .", "Representative genome diagrams for each EMALE type are shown in Figure 3 , for a schematic of all 33 complete EMALEs , see Figure 3—figure supplement 1 .", "According to this classification scheme , the reference mavirus strain Spezl falls within type 4 of the low-GC EMALEs ( Figures 2 and 3 , Figure 3—figure supplement 1 ) .", "Partial EMALEs were classified based on their sequence similarity to full-length type species ( Figure 2—figure supplement 1 ) .", "The codon and amino acid composition of EMALE genes clearly correlated with the overall GC-content of the EMALE genomes ( Figure 2—figure supplement 2 ) .", "For each encoded amino acid , we observed a strong shift toward synonymous codons reflecting the overall GC trend , and across amino acids , we observed a shift from those encoded by high-GC codons to those encoded by low-GC codons in low-GC EMALEs and vice versa .", "This uniform trend across all amino acids likely indicates that selection and evolutionary processes driving GC-content variation in these viruses act on the nucleotide level , rather than on the encoded proteins .", "With few exceptions , EMALEs are predicted to encode 17–21 proteins each .", "None of the encoding genes was found to contain introns .", "The virion morphogenesis module in EMALE types 1 and 3–7 consists of the canonical virophage core genes corresponding to MCP , PEN , ATPase , and PRO proteins .", "Type 2 EMALEs likely encode a different set of capsid genes as discussed below , and the truncated EMALE type 8 lacks recognizable morphogenesis genes .", "Another highly conserved gene in EMALE types 1 and 3–7 is MV14 , which is always found immediately upstream of the ATPase ( Figure 3 , Figure 3—figure supplement 1 ) and codes for a protein of unknown function that is part of the mavirus virion ( Born et al . , 2018 ) .", "MV14 is present in various metagenomic virophage sequences ( Paez-Espino et al . , 2019 ) and , based on synteny and protein localization , likely encodes an important virion component in members of the genus Mavirus .", "The replication/integration module consists of the rve-INT gene and at least one additional ORF coding for a primase/helicase and a DNA polymerase .", "Low-GC EMALEs encode a mavirus-related primase/helicase and protein-primed family B DNA polymerase ( pPolB ) ( Figure 3 , Figure 3—figure supplement 1 ) .", "Mid-GC EMALEs , on the other hand , lack the pPolB gene and feature a longer primase/helicase ORF that may include a DNA polymerase domain similar to the helicase-polymerase fusion genes described in PLVs ( Krupovic et al . , 2016b ) .", "Other mavirus genes frequently found in EMALEs include MV19 ( encoding a putative protease domain ) , and two genes of unknown function , MV08 and MV12 .", "Interestingly , all mid-GC EMALEs encode a predicted tyrosine recombinase ( YR ) in addition to the rve-INT and thus possess two predicted enzymes for genome integration .", "YRs have been found in other virophages and likely catalyze integration into giant virus genomes ( Desnues et al . , 2012; Yutin et al . , 2015 ) .", "Notable genes unique to one EMALE type include a putative DNA methylase and a ribonucleotide reductase small subunit gene found in EMALE07 .", "The Tlr6F protein encoded by EMALE types 1 + 2 is present in diverse MGEs , including other virophages , PLVs , and large DNA viruses of the phylum Nucleocytoviricota ( Koonin and Krupovic , 2017; Stough et al . , 2019 ) .", "In general , genes were syntenic between EMALEs of the same type , whereas gene order was poorly conserved among EMALEs of different types , with the following exceptions: MCP was always preceded by PEN , and ATPase was always preceded by MV14 , whereas the MV14-ATPase-PRO-PEN-MCP morphogenesis gene order as seen in mavirus was present only in EMALE types 4–7 .", "EMALE02 represents an interesting case , as it shares 6–7 kb of its 5’ part ( we chose the primase/helicase genes to mark the 5’ end of all EMALEs ) with EMALE01 , while the remaining 11 kb are not closely related to other EMALEs or virophages ( Figure 3—figure supplement 2 ) .", "Genes encoded in the latter region are mostly ORFans , with the exception of an MV12-like gene and divergent MCP and ATPase genes with remote similarity to PLVs ( Bellas and Sommaruga , 2021 ) and adintoviruses ( Starrett et al . , 2021 ) .", "EMALE02 may thus be the result of a recombination event that exchanged the canonical virophage morphogenesis module of EMALE01 with capsid genes of a PLV ( Figure 3 , dashed line ) .", "Overall , these observations support the notion that recombination and non-homologous gene replacement are important factors in virophage genome evolution ( Yutin et al . , 2013 ) .", "To validate our classification scheme for EMALEs and to place them in a phylogenetic context to other virophages , we used maximum likelihood reconstruction on the core proteins MCP , PEN , ATPase , and PRO , as well as on rve-INT ( Figure 4 ) .", "In the resulting phylogenetic trees , EMALE core proteins formed monophyletic clades with mavirus and related sequences from environmental samples , thus significantly expanding the known diversity of the genus Mavirus .", "The environmental sequences that clustered with EMALE core proteins include a single amplified genome ( SAG ) from an uncultured chrysophyte ( Castillo et al . , 2019 ) , the metagenomic Ace Lake Mavirus ( ALM ) ( Zhou et al . , 2013 ) , and four additional metagenomes that were identified in a global survey of virophage sequences ( Paez-Espino et al . , 2019 ) .", "The chrysophyte SAG is nearly identical to mavirus strain Spezl and indicates that the host range of mavirus extends beyond bicosoecids .", "The metagenomic sequences either clustered with one of the EMALE types , or branched separately from them , which suggests the existence of additional sub-groups ( e . g . M590M2_1006461 ) .", "Within the Mavirus clade , EMALEs of a given type were monophyletic for each of the four core proteins , which corroborates their dot plot-based classification .", "It is worth noting that although EMALEs of types 5 and 6 are largely syntenic ( Figure 3 , Figure 3—figure supplement 1 ) , they were clearly distinguishable in their phylogenetic signatures ( Figure 4 ) .", "A comparison of clade topologies revealed that even within the conserved morphogenesis module , individual proteins differed with regard to their neighboring clades , and low-GC and mid-GC EMALEs did not cluster separately from each other .", "These observations could suggest that the morphogenesis modules of different EMALE types diversified simultaneously and that adaptation of GC-content may occur rather quickly .", "In contrast , phylogenetic analysis of rve-INT proteins revealed separate clades for low-GC and mid-GC EMALEs ( Figure 4 ) .", "Each of these clades was affiliated with different cellular homologs that included MPEs and retroelements .", "Notably , the rve-INT genes of low-GC EMALEs were located near the 5’ end of the genomes , whereas in mid-GC EMALEs , they were located near the 3’ end ( Figure 3 , Figure 3—figure supplement 1 ) .", "These observations suggest that EMALEs encode two different rve-INT versions , one specific for low-GC EMALEs that co-occurs with the pPolB and a shorter primase/helicase ORF , and one specific for mid-GC EMALEs that co-occurs with a longer primase/helicase ORF .", "The two integrase versions may have been acquired independently , or one version could have replaced the other during EMALE evolution .", "Such non-homologous gene replacement appears to have taken place among the primase/helicase genes , too , as previously noted for virophages in general ( Yutin et al . , 2013 ) .", "EMALEs encode several different versions of primase/helicase genes with a degree of amino acid divergence that precluded their inclusion in a single multiple sequence alignment .", "The YR proteins encoded by EMALE types 5–8 formed a monophyletic clade and were part of a larger group of recombinases that included virophages from freshwater metagenomes , as well as microalgae and algal nucleocytoviruses ( Figure 4—figure supplement 1 ) .", "The four C . burkhardae strains displayed distinct EMALE signatures: strain BVI had the highest number of virophage elements with 13 complete and 42 partial EMALEs , whereas the other three strains had 6–7 complete and 20–22 partial EMALEs each ( Figure 1C , Supplementary file 1 ) .", "EMALE types 1 , 3 , 4 , 5 , and 6 were present in every host strain , EMALE07 was found in all strains except Cflag , and EMALE types 2 and 8 were detected in strains BVI and E4-10 only .", "We found no evidence for sequence-specific genome integration of EMALEs after inspecting the host DNA sequences that flanked EMALE integration sites , which confirms previous reports of mavirus integration ( Fischer and Hackl , 2016 ) .", "EMALEs were flanked by target site duplications ( TSDs ) that were predominantly 3–5 bp in length , although some were as short as 1 bp or as long as 9 bp ( Supplementary file 1 ) .", "By comparison , mavirus and MPEs generate 5–6 bp long TSDs upon integration ( Fischer and Hackl , 2016; Pritham et al . , 2007; Kapitonov and Jurka , 2006 ) .", "To assess whether homologous EMALEs were found in identical loci in closely related host genomes , we conducted sequence similarity searches with the flanking regions of each of the 33 fully resolved EMALEs .", "Whenever these searches returned a homologous full or partial EMALE with at least one matching host flank , we considered the EMALE locus to be conserved in these host strains .", "We found varying degrees of conservation , with examples shown in Figure 3—figure supplement 3 .", "In 11 cases , an EMALE insertion was conserved in at least two host strains ( Supplementary file 1 ) : three EMALE loci were shared by all four strains , four were shared by three strains , and another four were shared by two strains .", "Based on conserved EMALE loci , strains Cflag and RCC970 were most closely related with nine shared EMALE integrations , which is in line with phylogenetic and average nucleotide identity ( ANI ) analyses of these strains ( Hackl et al . , 2020 ) .", "The four C . burkhardae genomes have ANIs of >99% and thus appear to differ mostly based on their content of EMALEs and other MGEs .", "The most parsimonious scenario for the origin of EMALEs that are located in identical loci in different host strains is that they derived from a single integration event .", "For instance , EMALE03 BVI_101 is orthologous to Cflag_017C and RCC970_016A ( Figure 3—figure supplement 3C ) , which suggests that this element initially colonized the common ancestor of C . burkhardae strains BVI , Cflag , and RCC970 .", "Further cases of redundant EMALEs are Cflag_017B & RCC970_016B ( EMALE01 ) and BVI_029 & RCC970_095 ( EMALE06 ) .", "These elements may thus derive from relatively ancient integration events , whereas 18 of the 33 complete EMALEs represent integrations that were unique to a single host strain ( Supplementary file 1 ) .", "Strain BVI contained 10 of these 18 unique integrations , more than twice as many as any other strain .", "The genomic landscape around EMALE integration sites ranged from repeat-free flanking regions to complex host repeats ( Figure 3—figure supplement 4 ) .", "Of the 29 different integration sites represented by the 33 fully resolved EMALEs , 18 were located near repetitive host DNA ( within 10 kb from the insertion site ) .", "These repeats , in addition to EMALE TIRs , multiple copies of the same EMALE type , and the putative heterozygosity of EMALE insertions , occasionally caused assembly problems , as illustrated in Figure 3—figure supplement 3 .", "Next , we analyzed whether EMALE insertions interrupted coding sequences of the host .", "Fifteen integration sites were located within a predicted host gene ( 13 in exons , 2 in introns ) , four were found in predicted 3’ untranslated regions , and three were located in intergenic regions ( Supplementary file 1 ) .", "These data show that EMALE insertions may disrupt eukaryotic genes with potential negative consequences for the host .", "The apparent preference for integration in coding regions could be assembly related , driven by increased accessibility of euchromatin , or linked to host factors that could direct the rve-INT via its CHROMO domain ( Gao et al . , 2008 ) .", "Based on genomic features such as coding potential , ORF integrity , and host distribution , most EMALEs appear to be active MGEs .", "With the exception of EMALE08 and EMALE02 , all endogenous Cafeteria virophages encode the canonical morphogenesis gene module consisting of MCP , PEN , ATPase , PRO , as well as MV14 .", "EMALE02 likely encodes more distantly related capsid genes .", "Therefore , all EMALE types except EMALE08 should be autonomous for virion formation .", "In addition , all EMALEs contain at least one predicted enzyme for genome integration , an rve-INT in EMALE types 1–7 and a YR in EMALE types 5–8 .", "EMALEs thus encode the enzymatic repertoire for colonizing new host genomes .", "Finally , the high variability of EMALE integration loci among otherwise closely related host strains strongly argues for ongoing colonization of natural Cafeteria populations by virophages .", "The genomic similarity to mavirus implies that EMALEs may also depend on a giant virus for activation and horizontal transmission .", "Shared regulatory sequences in virophages and their respective giant viruses suggest that the molecular basis of virophage activation lies in the recognition of virophage gene promoters by giant virus encoded transcription factors ( Fischer and Suttle , 2011; Claverie and Abergel , 2009; Legendre et al . , 2010 ) .", "We therefore analyzed the 100 nt upstream regions of EMALE ORFs for conserved sequence motifs using MEME ( Bailey et al . , 2009 ) .", "For all type 4 EMALEs , which include mavirus , we recovered the previously described mavirus promotor motif ‘TCTA’ , flanked by AT-rich regions .", "This motif corresponds to the conserved late gene promoter in CroV ( Fischer and Suttle , 2011; Fischer et al . , 2010 ) , thus possibly indicating that all type 4 EMALEs could be reactivated by CroV or close relatives .", "EMALEs of other types lacked the ‘TCTA’ motif , but contained putative promoter sequences that may be compatible with different giant viruses ( Figure 3—figure supplement 5 ) .", "MGEs are prone to various decay processes including pseudogenization , recombination , and truncation .", "Among the 33 fully resolved EMALEs are three truncated elements: Cflag_215 and RCC970_122 ( both EMALE04 ) , and BVI_005 ( EMALE08 ) ( Figure 3—figure supplement 1 ) .", "Interestingly , even these shorter elements are flanked by TIRs , which must have regenerated after the truncation event .", "Whereas most EMALE ORFs appeared to be intact , as judged by comparison with homologous genes on syntenic elements , several EMALEs contained fragmented ORFs ( e . g . ATPase and PEN genes in EMALE04 BVI_055B , Figure 3 ) .", "To test whether premature stop codons may be the result of assembly artifacts , we amplified selected EMALEs by PCR and analyzed the products using Sanger sequencing .", "When we compared the Sanger assemblies with the Illumina/PacBio assemblies , we noticed that the latter contained several substitutions and indels .", "For example , the MCP gene of EMALE01 RCC970_016B was split into three ORFs in the Illumina/PacBio assembly , whereas a single ORF was present in the corresponding Sanger assembly ( Figure 3—figure supplement 6 ) .", "None of the Sanger assemblies confirmed fragmentation of conserved virophage genes , emphasizing the importance of independent sequence validation .", "However , since it was not possible to re-sequence all potential pseudogene locations , we cannot exclude that some EMALE genes may be fragmented .", "Overall , EMALE ORFs appeared to be intact and are thus likely to encode functional proteins .", "Strikingly , about one in four virophage elements was interrupted by a GC-rich sequence with a typical length of ~6 kb ( Figure 5A , Supplementary file 1 ) .", "We identified these insertions as retrotransposons of the Ngaro superfamily , within the DIRS order of retrotransposons ( Wicker et al . , 2007 ) .", "Ngaro retrotransposons feature split direct repeats with A1–[ORFs]–B1A2B2 structure ( Figure 5D ) , encode a YR instead of the rve-INT that is typical for retrotransposons , and are found in various eukaryotes from protists to vertebrates ( Poulter and Goodwin , 2005 ) .", "In the four C . burkhardae strains , we annotated 80 Ngaro elements , with 10–25 copies per strain ( Supplementary file 1 ) .", "In addition , we found isolated AB repeats scattered throughout the genome , which could have arisen from recombination of 5’ and 3’ repeats and are reminiscent of solo long terminal repeats of endogenous retroviruses ( Friedli and Trono , 2015 ) .", "Solo AB repeats were also occasionally present in EMALEs ( BVI_016 , BVI_115 , Cflag_024 , Cflag_214 , Cflag_215 ) .", "Dot plot analysis of concatenated Cafeteria Ngaro sequences revealed four distinct types that showed no similarity at the nucleotide level , but appeared to share the same coding potential ( Figure 5B and D ) .", "Based on synteny to previously described Ngaros , ORF1 may encode a Gag-like protein; ORF2 encodes predicted reverse transcriptase ( Pfam PF00078 ) and ribonuclease H domains ( Pfam PF17919 ) ; and ORF3 encodes a predicted YR ( Pfam PF00589 ) with the conserved His-X-X-Arg motif and catalytic Tyr residue .", "Ngaro YRs are related to putative transposons of bacteria and eukaryotes ( Figure 5—figure supplement 1 ) , but bear no sequence similarity to the EMALE-encoded YRs .", "Interestingly , the four Ngaro types in C . burkhardae differed with regard to their integration site preference .", "Whereas 91% of type 1 Ngaros were inserted in an EMALE , this was the case for only 14% and 8% of type 3 and type 4 Ngaros , respectively .", "At first glance , type 2 Ngaros were distributed evenly among viral and eukaryotic DNA ( 47% inside EMALEs , 53% outside EMALEs ) .", "However , several type 2 Ngaros were truncated at their 5’ end , featuring ~2 kb long deletions that covered ORF1 .", "All of these deletion variants , which we designate as type 2b , were located in host DNA , in contrast to 82% of the full-length type 2a Ngaros that were inserted in EMALEs ( Figure 5C and D ) .", "Similarly , most of the type 1 Ngaros that were inserted into eukaryotic chromatin also lacked ORF1 .", "Hence , it appears that ORF1 determines the integration site specificity of Ngaro retrotransposons .", "We did not observe any preference of host-genome-integrated Ngaros for regions with a GC-content lower than the host average .", "Out of 20 Ngaro insertion sites with analyzable EMALE flanking regions ( including partial EMALEs ) , 9 were located in intergenic regions , 7 in EMALE genes ( 1× primase/helicase , 1× pPolB , 1× MV12 , 4× MV19 ) , and 4 in TIRs ( Supplementary file 1 ) .", "Considering that intergenic regions comprise only 5–10% of an EMALE genome , we notice a significant bias ( p < 1e–5 , Fisher’s exact test ) toward Ngaro integration in intergenic EMALE DNA , which may be caused either by purifying selection of deleterious Ngaro insertions or by a higher preference for Ngaro integration into EMALE intergenic regions , e . g . due to their lower GC-content .", "A possible consequence of retrotransposon insertion is the loss of biological activity and subsequent pseudogenization .", "However , we found that Ngaro-containing EMALEs did not contain more fragmented genes than Ngaro-free EMALEs ( Figure 5—figure supplement 2 ) .", "Whereas the biological properties of Ngaro retrotransposons and their influence on host-virus-virophage dynamics remain to be explored , the EMALE-Ngaro interactions appear to be convoluted .", "For instance , an EMALE03 genome in strain Cflag is interrupted by two adjacent Ngaro1 insertions , while the EMALE itself is located inside an Ngaro4 element ( Figure 5—figure supplement 3 ) ." ], [ "Virophages represent a recently discovered family of eukaryotic dsDNA viruses that possess interesting genome integration properties and have potentially far-reaching eco-evolutionary consequences .", "Our genomic survey of the marine bicosoecid C . burkhardae revealed an unexpected abundance and diversity of endogenous virophages , with dozens of elements in a single host genome .", "Based on DNA dot plots and phylogenetic analysis , we distinguish eight different types of mavirus-related endogenous virophages .", "Similar to mavirus , these EMALEs could potentially reactivate and replicate in the presence of a compatible giant virus .", "Mavirus is proposed to act as an adaptive defense system against CroV in Cafeteria populations ( Fischer and Hackl , 2016; Koonin and Krupovic , 2016 ) , and our findings suggest that different types of EMALEs may respond to different giant viruses infecting Cafeteria .", "The assortment of endogenous virophages in a given host genome may thus reflect the giant virus infection history of that population ( Blanc et al . , 2015 ) .", "Some EMALEs are present in orthologous genomic loci in two or more host strains and likely date back to the common ancestor of these strains .", "However , at least half of the EMALE insertions are specific to a given host strain and may thus have been acquired relatively recently .", "Combined with the overall integrity of EMALEs and the conservation of integrase and capsid genes , these findings suggest that endogenous virophages in C . burkhardae are active MGEs .", "Similar to other MGEs , EMALEs are likely prone to various decay processes including pseudogenization , truncation , and recombination; however , the extent to which these mechanisms affect EMALE stability is currently unclear .", "Although nonsense mutations appear to be present in some EMALE genes , re-sequencing of selected genomic regions did not confirm pseudogene loci in those cases ( Figure 3—figure supplement 6 ) .", "We found three truncated EMALEs ( 5–15 kb long ) , but these are not to be confused with the 105 partial EMALEs that resulted from computational limitations during sequence assembly of these multi-copy elements .", "Recombination most likely created the EMALE02 variant , with no obvious signs of degradation .", "Based on our EMALE identification approach ( see Materials and methods ) , we are quite confident that we have not missed more severely degraded EMALEs or more divergent virophages , except perhaps for hypothetical elements lacking any recognizable sequence similarity to annotated virophages and possessing a GC-content similar to that of the host genome .", "Overall , these observations raise the question of how balance between new virophage integrations and loss of existing EMALEs is achieved to prevent overload of the host genome with mobile DNA .", "Possible explanations include loss by homologous recombination , for example , between TIRs of the same or similar EMALEs , excision of EMALEs via host- or EMALE-encoded integrases or endonucleases , or mechanisms to limit the uptake of new elements .", "Given that multiple mavirus genome integrations per host cell can be observed within a period of days ( Fischer and Hackl , 2016 ) and that virophage integration rates under natural infection conditions are likely to fall within similar timescales , we hypothesize that the majority of EMALE loss may occur before pseudogenization and degradation can take place .", "EMALEs would thus have relatively short residence times in the unicellular Cafeteria compared to germline EVEs of multicellular eukaryotes ( Barreat and Katzourakis , 2021 ) .", "Horizontal transmission of virophages in natural environments is likely limited by their requirement for two concurrent biological entities , namely a susceptible host cell infected with a permissive giant virus .", "Similar to temperate bacteriophages , persistence in the proviral state may thus be an essential survival strategy for virophages , as underscored by the abundance of EMALEs in C . burkhardae .", "Endogenous virophages may also be common in eukaryotes outside the order Bicosoecales , although a search in 2015 for provirophages in 1153 eukaryotic genomes found only one clear case in the chlorarachniophyte alga Bigelowiella natans ( Blanc et al . , 2015 ) .", "In contrast to rapidly increasing virophage reports from metagenomic sequence mining in recent years ( Paez-Espino et al . , 2019 ) , we propose that the discovery of host-integrated virophages is still hampered not only by sampling bias , but also by technical limitations .", "For instance , AT-rich mavirus DNA was severely underrepresented when we sequenced the genome of C . burkhardae strain E4-10M1 with the standard Illumina MiSeq protocol ( Fischer and Hackl , 2016 ) .", "Additional problems arise during binning and assembly procedures .", "Although our sequencing and assembly strategy was specifically tailored to endogenous virophages and resolved 33 EMALEs in their host genomic context , dozens of EMALEs were only partially assembled , some may contain assembly errors ( Figure 3—figure supplement 3 ) , and others may have been missed altogether .", "Advances in long-read sequencing technologies and assembly algorithms will likely alleviate such problems .", "Surprisingly , we found that EMALEs were frequently interrupted by Ngaro retrotransposons , which revealed an additional level of nested parasitism in this microbial system .", "Cafeteria genomes contain four distinct Ngaro types with different affinities for EMALEs .", "Deletion of ORF1 in type 1 and type 2 Ngaros coincides with a decreased occurrence of these retrotransposons in EMALEs ( Figure 5 ) .", "Syntenic ORFs in other Ngaros are predicted to encode a Gag-like structural protein ( Poulter and Butler , 2015 ) , and Gag proteins of several retroviruses have been linked to integration site specificity ( Lewinski et al . , 2006; Tobaly-Tapiero et al . , 2008 ) .", "The putative Gag proteins of Cafeteria Ngaros may thus influence whether retrotransposon insertion occurs in an EMALE or in eukaryotic chromatin .", "So far , retrotransposons have not been described for giant DNA viruses or virophages; however , pandoravirus genomes contain DNA transposons ( Sun et al . , 2015 ) , and a class of 7 kb long DNA MGEs called transpovirons interacts with the particles and genomes of Acanthamoeba-infecting mimiviruses and their virophages , apparently without affecting viral replication ( Desnues et al . , 2012; Jeudy et al . , 2020 ) .", "It remains to be studied whether Ngaro retrotransposons use reactivated virophages or giant viruses as vehicles for horizontal transmission , and what effect retrotransposon insertion in EMALEs has on the fitness of the virophage , the host cell , and their associated giant viruses .", "In conclusion , we show that endogenous virophage genomes are abundant and diverse in the marine heterotrophic protist C . burkhardae .", "These mavirus-like EVEs appear to be active and dynamic MGEs with significant potential to shape the genome evolution of their hosts .", "We present evidence for recombination and gene exchange within EMALEs , and a previously unknown affiliation between virophages and YR retrotransposons .", "Our findings imply an important role for EMALEs in the ecology and evolution of bicosoecids and are in line with the hypothesis that endogenous virophages provide adaptive defense against giant viruses ." ], [ "C . burkhardae strains BVI , Cflag , E4-10P , and RCC970-E3 were cultured , and their genomes sequenced and assembled as described previously ( Hackl et al . , 2020 ) .", "To identify endogenous virophages , we initially took a manual approach and searched for clearly visible drops in GC-content along contigs when visualized in Artemis Release 16 . 0 . 0 ( Rutherford et al . , 2000 ) .", "Based on our experience we suspected that integrated virophages would have a considerably lower GC-content than the host genomes .", "For those low-GC regions we then computed DNA dot plots with Gepard ( Krumsiek et al . , 2007 ) to detect TIRs , predicted ORFs with Artemis , manually curated them into gene calls , and also manually assigned functional annotations based on blastp searches against known virophages and public databases .", "This resulted in the identification of 33 manually curated , TIR-containing high-confidence ( ‘complete’ ) EMALEs .", "Partial EMALEs that could not be fully assembled and were thus located on contig termini were identified based on sequence similarity to annotated virophages and EMALEs using the BLAST suite .", "Their boundaries were determined based on GC-content or sequence similarity to already annotated TIRs .", "Partial EMALEs were annotated with prodigal-2 . 6 . 3 ( Hyatt et al . , 2010 ) .", "We pre-trained one gene model for mid-GC and one for high-GC EMALEs , and called genes on those genomes separately .", "Where present , we masked integrated Ngaro elements with ‘N’s prior to training and annotation because we observed in trial runs that automated gene prediction could not properly resolve the gene structure of the Ngaros and would produce fragmented and spurious gene calls for these regions .", "We also explored other gene prediction programs including PHANOTATE ( McNair et al . , 2019 ) and VGAS ( Zhang et al . , 2019 ) , but found pre-trained prodigal to produce results most consistent with manually curated expert annotations for EMALE genomes .", "Retrotransposon insertions were first noticed in GC-content graphs as 6–7 kb long GC-rich sequences that interrupted AT-rich EMALEs .", "DNA dot plot analysis of these regions showed split direct repeats , and conserved domain and tblastx searches predicted RNaseH , reverse transcriptase , and tyrosine recombinase domains .", "ORFs were annotated in Artemis based on the predicted conserved domains .", "These initially curated protein sequences and direct repeats were then used to seed blast searches against all four C . burkhardae genomes to identify additional Ngaro elements , which were inspected individually and annotated manually .", "To quantify how much of each host genome is comprised of EMALEs and Ngaros , we applied two complementary strategies:", "( i ) We compared the number of nucleotides annotated as EMALEs and Ngaros in the assemblies to the overall assembly sizes , and", "( ii ) we quantified the number of nucleotides in the PacBio reads that we could assign to either of the three fractions – host , EMALE , and Ngaro .", "The latter approach is less prone to assembly biases , such as overestimation of contributions for elements only present in one allele , or underestimation of contribution due to collapsed repeated copies , or elements not assembled because of low coverage .", "We aligned the PacBio reads to the assemblies using minimap2 v2 . 16 ( -x map-pb ) ( Li , 2018 ) and computed the coverage of the different genomic regions with samtools v1 . 9 ( Li et al . , 2009 ) .", "We did not consider Illumina data for the quantification due to an observed sequencing bias against low-GC regions that would lead to lower EMALE estimates .", "To analyze possible correlations between EMALE GC-content and the codon composition of their genes , we counted codons of all genes of complete EMALEs with a custom Perl script and visualized their distribution relative to their GC-content with a custom R script ." ] ]

[ "Virophages can parasitize giant DNA viruses and may provide adaptive anti-giant virus defense in unicellular eukaryotes .", "Under laboratory conditions , the virophage mavirus integrates into the nuclear genome of the marine flagellate Cafeteria burkhardae and reactivates upon superinfection with the giant virus CroV .", "In natural systems , however , the prevalence and diversity of host-virophage associations has not been systematically explored .", "Here , we report dozens of integrated virophages in four globally sampled C . burkhardae strains that constitute up to 2% of their host genomes .", "These endogenous mavirus-like elements ( EMALEs ) separated into eight types based on GC-content , nucleotide similarity , and coding potential and carried diverse promoter motifs implicating interactions with different giant viruses .", "Between host strains , some EMALE insertion loci were conserved indicating ancient integration events , whereas the majority of insertion sites were unique to a given host strain suggesting that EMALEs are active and mobile .", "Furthermore , we uncovered a unique association between EMALEs and a group of tyrosine recombinase retrotransposons , revealing yet another layer of parasitism in this nested microbial system .", "Our findings show that virophages are widespread and dynamic in wild Cafeteria populations , supporting their potential role in antiviral defense in protists ." ]

[ "Viruses exist in all ecosystems in vast numbers and infect many organisms .", "Some of them are harmful but others can protect the organisms they infect .", "For example , a group of viruses called virophages protect microscopic sea creatures called plankton from deadly infections by so-called giant viruses .", "In fact , virophages need plankton infected with giant viruses to survive because they use enzymes from the giant viruses to turn on their own genes .", "A virophage called mavirus integrates its genes into the DNA of a type of plankton called Cafeteria .", "It lays dormant in the DNA until a giant virus called CroV infects the plankton .", "This suggests that the mavirus may be a built-in defense against CroV infections and laboratory studies seem to confirm this .", "But whether wild Cafeteria also use these defenses is unknown .", "Hackl et al . show that virophages are common in the DNA of wild Cafeteria and that the two appear to have a mutually beneficial relationship .", "In the experiments , the researchers sequenced the genomes of four Cafeteria populations from the Atlantic and Pacific Oceans and looked for virophages in their DNA .", "Each of the four Cafeteria genomes contained dozens of virophages , which suggests that virophages are important to these plankton .", "This included several relatives of the mavirus and seven new virophages .", "Virophage genes were often interrupted by so called jumping genes , which may take advantage of the virophages the way the virophages use giant viruses to meet their own needs .", "The experiments show that virophages often co-exist with marine plankton from around the world and these relationships are likely beneficial .", "In fact , the experiments suggest that the virophages may have played an important role in the evolution of these plankton .", "Further studies may help scientists learn more about virus ecology and how viruses have shaped the evolution of other creatures ." ]

[ "Main text", "Materials and methods" ]

[ "short report", "genetics and genomics" ]

[ [ "Facioscapulohumeral muscular dystrophy ( FSHD ) is typically an adult-onset muscular dystrophy characterized by muscle weakness initially affecting the face ( facio ) , shoulders ( scapulo ) , and upper arms ( humeral ) .", "FSHD is caused by decreased epigenetic repression of the D4Z4 macrosatellite array in the subtelomeric region of chromosome 4q , due to either D4Z4 repeat contractions ( Lemmers et al . , 2010 ) or mutations affecting trans-acting epigenetic regulators of the D4Z4 repeat such as SMCHD1 ( Lemmers et al . , 2012 ) , which results in the misexpression of DUX4 mRNA in skeletal muscle and possibly other somatic tissues .", "DUX4 encodes a double homeobox transcription factor that activates germline genes and repetitive elements ( Geng et al . , 2012 ) and causes apoptosis and atrophic myotube formation when misexpressed in skeletal muscle ( Kowaljow et al . , 2007; Vanderplanck et al . , 2011; Wallace et al . , 2011; Mitsuhashi et al . , 2012 ) .", "DUX4 is expressed in only a small fraction of nuclei ( Snider et al . , 2010 ) , likely due to occasional ‘bursts’ of DUX4 expression .", "However , the mechanism ( s ) regulating DUX4 expression and toxicity remain incompletely understood .", "We previously ectopically expressed DUX4 in immortalized ( 54-1 ) and primary ( MB135 ) myoblasts and used RNA-seq to identify coding genes , repetitive elements , and non-coding RNAs induced by DUX4 ( Young et al . , 2013 ) .", "Further analysis of this data showed that DUX4 expression also resulted in the increased abundance of many coding RNA isoforms containing premature translation termination codons upstream of splice junctions .", "These isoforms , which are predicted substrates for degradation by nonsense-mediated decay ( NMD ) , were present at very low levels in control myoblasts .", "Following DUX4 expression , however , many such predicted NMD substrates increased in abundance and in many cases became the predominant mRNA product of the parent gene .", "For example , an isoform of the SRSF3 gene containing a well-characterized NMD-inducing cassette exon ( Lareau et al . , 2007; Ni et al . , 2007 ) was present at low levels prior to DUX4 expression but became the dominant isoform thereafter in both 54-1 and MB135 cells ( Figure 1A–B ) . 10 . 7554/eLife . 04996 . 003Figure 1 . DUX4 expression inhibits nonsense-mediated decay .", "( A ) RNA-seq read coverage of the SRSF3 gene in control and DUX4-expressing myoblasts .", "54-1 , immortalized myoblasts; MB135 , primary myoblasts .", "Purple shading , NMD-inducing cassette exon .", "Red stop sign , termination codon .", "( B ) Inclusion of the premature termination codon-containing cassette exon of SRSF3 illustrated in ( A ) .", "Error bars , 95% confidence intervals as estimated by MISO ( Katz et al . , 2010 ) .", "( C ) Relative levels of transcripts produced from NMD reporter plasmids encoding either premature termination codon-containing ( top ) or normal ( bottom ) β-globin ( Gl ) .", "Bar plot illustrates the ratio NMD ( + ) /NMD ( − ) of transcripts from the NMD ( + ) and NMD ( − ) constructs .", "( D ) Isoform ratios of predicted NMD substrates generated by cassette exon alternative splicing in control and DUX4-expressing myoblasts ( 54-1 cells ) .", "Red/blue , cassette exons exhibiting increases/decreases of ≥10% in isoform ratios for the isoforms that are predicted NMD substrates .", "( E ) Isoform ratios of mis-spliced isoforms of annotated constitutive splice junctions generated by abnormal 5′ and 3′ splice site recognition ( 54-1 cells ) .", "Color as in ( D ) .", "( F ) Global increases and decreases in relative levels of predicted NMD substrates generated by differential splicing .", "Annotated alternative splicing events are illustrated in upper panel , and alternative splicing and intron retention of annotated constitutively spliced junctions are illustrated in lower panel .", "Up/down arrows , percentages of predicted NMD substrates generated by alternative splicing exhibiting increases/decreases of ≥10% in isoform ratios in DUX4-expressing vs control cells .", "Enrichment for increased vs decreased levels of NMD substrates indicated in columns three and six . DOI: http://dx . doi . org/10 . 7554/eLife . 04996 . 00310 . 7554/eLife . 04996 . 004Figure 1—figure supplement 1 . DUX4-induced NMD inhibition is not a side effect of DUX4 toxicity .", "( A ) Poly-caspase activity ( red ) following transfection with a control siRNA or siRNA against TP53 40 hr after lentiviral infection .", "Box plot , percentage of nuclei with poly-caspase granules ( estimated by ImageJ; n = 8 fields ) .", "Whiskers , max and min over the fields .", "( B ) Isoform ratios of endogenously produced NMD-degraded isoforms of HNRNPD and SRSF2 .", "Error bars , standard deviation . DOI: http://dx . doi . org/10 . 7554/eLife . 04996 . 004 To determine whether increased levels of such normally degraded mRNAs were associated with reduced NMD efficiency , we used an exogenous reporter system .", "We transfected plasmids encoding either the wild-type β-globin open reading frame or β-globin with a premature termination codon that induces degradation by NMD ( Zhang et al . , 1998 ) .", "Relative levels of the β-globin NMD substrate were twofold higher in DUX4-expressing vs control myoblasts , indicating that NMD is indeed compromised by DUX4 ( Figure 1C ) .", "We then determined how reduced NMD efficiency affected global levels of predicted NMD substrates .", "Restricting to cassette exon splicing events where one isoform , but not both , was a predicted NMD substrate , we found that ∼13% of such predicted NMD substrates increased following DUX4 expression , while ∼1 . 6% decreased , in 54-1 cells ( Figure 1D ) .", "Impaired NMD also caused accumulation of aberrant mRNAs resulting from mis-splicing or incomplete splicing , which are common byproducts of the stochastic nature of the splicing process ( Weischenfeldt et al . , 2012 ) .", "We identified and quantified alternative splicing of annotated constitutive junctions , finding that ∼13% of such junctions exhibited increased aberrant splicing in DUX4-expressing vs control cells , while only ∼0 . 25% exhibited decreased aberrant splicing ( Figure 1E ) .", "The vast majority of these novel products of annotated constitutive junctions were present at very low or undetectable levels in control 54-1 myoblasts .", "We next extended this analysis to all classes of splicing events , including mis-splicing and intron retention of constitutive splice junctions .", "DUX4 expression caused increased levels of predicted NMD substrates for all classes of splicing events in both 54-1 and MB135 cells ( Figure 1F ) .", "These increases were generally more extreme in 54-1 than in MB135 cells , likely due to the ∼15-fold higher DUX4 expression achieved in 54-1 vs MB135 cells as well as the longer time period allowed for infection ( 48 hr vs 24 hr ) .", "High levels of NMD substrates in DUX4-expressing cells were not simply a side effect of DUX4-induced apoptosis .", "TP53 knock-down ( KD ) prevented apoptosis following DUX4 expression in normal myoblasts , confirming previous reports that DUX4 toxicity is p53-dependent ( Wallace et al . , 2011 ) .", "However , TP53 KD did not prevent DUX4-induced NMD inhibition ( Figure 1—figure supplement 1 ) .", "DUX4 could potentially inhibit NMD by transcriptionally repressing components of the NMD machinery .", "However , no UPF or SMG NMD factors exhibited decreased mRNA levels following DUX4 expression , and most were up-regulated by two- to fourfold ( Figure 2A ) .", "This expression pattern was reminiscent of a recent report that mRNA levels of most NMD factors increase following the knock-down of UPF1 , encoding a central component of the NMD machinery ( Huang et al . , 2011 ) .", "Therefore , we hypothesized that UPF1 mRNA and protein levels might be decoupled in DUX4-expressing cells .", "We measured levels of UPF1 , which was not transcriptionally up-regulated in DUX4-expressing cells , and UPF3B and SMG7 , which were transcriptionally up-regulated in response to DUX4 .", "UPF1 protein levels were markedly lower in DUX4-expressing myoblasts than in control myoblasts , as were SMG7 levels , although to a lesser extent .", "In contrast , UPF3B levels were unaffected by DUX4 expression ( Figure 2B ) . 10 . 7554/eLife . 04996 . 005Figure 2 . DUX4 destabilizes UPF1 via the proteasome .", "( A ) Relative mRNA levels of NMD factors in DUX4-expressing vs control myoblasts ( 54-1 cells ) .", "Red , up-regulation by ≥1 . 5-fold .", "( B ) Immunoblot for NMD factors UPF1 , SMG7 , and UPF3B in DUX4-expressing and control myoblasts ( 54-1 cells ) at 36 hr post-infection .", "H3 , histone H3 ( loading control ) .", "( C ) Immunoblot of total protein from a 36-hr time course of DUX4-expressing and control myoblasts ( 54-1 cells ) .", "H3 , histone H3 .", "Loading Control , a nonspecific band that serves as an additional loading control .", "( D ) Quantification of UPF1 protein level from the immunoblot presented in ( C ) , normalized to the nonspecific band that serves as a loading control .", "( E ) Relative levels of transcripts produced from the NMD ( + ) and NMD ( − ) β-globin reporter plasmids .", "( F ) Isoform ratios of endogenously produced NMD-degraded isoforms of SRSF2 and SRSF3 .", "Time course identical to ( C ) .", "Error bars , standard deviation .", "( G ) Immunoblot of total protein from DUX4-expressing and control myoblasts ( 54-1 cells ) treated with the proteasome inhibitor MG132 ( 10 µM; 8 hr treatment initiated 16 hr after infection with lentiviral expression constructs ) .", "Loading control H3 , histone 3 , has a long half-life ( Toyama et al . , 2013 ) .", "( H ) Quantification of UPF1 protein levels from three independent replicates of the immunoblot presented in ( G ) , normalized to the nonspecific band that serves as a loading control .", "Error bars , standard deviation . DOI: http://dx . doi . org/10 . 7554/eLife . 04996 . 005 To determine whether decreased UPF1 temporally correlates with DUX4-induced inefficient NMD , we conducted a time course following DUX4 expression in myoblasts .", "DUX4 was robustly detectable 12–14 hr after lentiviral infection , coincident with the beginning of a sharp decrease in UPF1 levels ( Figure 2C–D ) .", "SMG7 showed a more modest decrease through the time course , while UPF3B levels were relatively constant .", "NMD substrates produced from the β-globin reporter , as well as endogenously produced from the SRSF2 and SRSF3 genes , exhibited increased levels 12–14 hr after lentiviral expression ( Figure 2E–F ) .", "The close temporal coupling between DUX4 protein production , decreased UPF1 levels , and increased levels of both endogenous and exogenous NMD substrates suggests that insufficient levels of UPF1—and perhaps additional NMD machinery components such as SMG7—may contribute to inefficient NMD in DUX4-expressing cells .", "The rapid decrease in UPF1 levels that we observed suggested that DUX4 might trigger UPF1 degradation .", "To test this , we treated DUX4-expressing or control myoblasts with MG132 to inhibit the proteasome .", "MG132 treatment restored normal UPF1 levels in DUX4-expressing myoblasts , while UPF1 levels in control myoblasts were unaffected ( Figure 2G–H ) .", "As proteasome inhibition inhibits normal translation ( Cowan and Morley , 2004; Mazroui et al . , 2007 ) and therefore NMD , we were unable to test whether the restoration of normal UPF1 levels by proteasomal inhibition rescued NMD .", "However , the close temporal relationship between the onset of decreased UPF1 levels and increased NMD substrates strongly suggests that UPF1 degradation contributes to NMD inhibition in DUX4-expressing cells .", "Both DUX4 isoforms encoding the full-length protein contain a constitutively spliced intron within their 3′ UTRs , rendering them likely NMD substrates ( Figure 3A ) .", "To test this , we used cells isolated from FSHD1 ( 54-2 , which are isogenic to normal 54-1 cells but carry a contracted D4Z4 array ) and FSHD2 ( MB200 ) skeletal muscle ( Krom et al . , 2012; Schoenberg and Maquat , 2012; Young et al . , 2013 ) .", "We knocked down UFP1 in 54-2 and MB200 myoblasts to 24 . 3% and 32 . 4% of normal protein levels , respectively , and differentiated these myoblasts to myotubes to stimulate DUX4 transcription .", "DUX4 mRNA was expressed at approximately fourfold higher levels in UPF1 KD vs control KD myotubes , as was ZSCAN4 mRNA , which is transcriptionally activated by DUX4 ( Figure 3B–D ) . 10 . 7554/eLife . 04996 . 006Figure 3 . DUX4 mRNA is an endogenous NMD substrate .", "( A ) Schematic of the DUX4 mRNA .", "Intron 2 , constitutively spliced intron within the 3′ UTR .", "Black , coding sequence; purple , 5′ and 3′ untranslated regions ( UTRs ) .", "Red stop sign , termination codon .", "( B ) Immunoblot of total protein from FSHD1 ( 54-2 ) and FSHD2 ( MB200 ) myoblasts following transfection with a siRNA against UPF1 or a control non-targeting siRNA .", "α-tubulin , loading control .", "( C ) Levels of DUX4 mRNA following control or UPF1 knock-down , measured 2 days after the initiation of myogenesis .", "Error bars , standard deviation .", "( D ) Levels of ZSCAN4 mRNA following control or UPF1 knock-down , measured 2 days after the initiation of myogenesis .", "Error bars , standard deviation .", "( E ) Schematic of chimeric constructs encoding the β-globin opening reading frame ( cyan ) followed by the DUX4 3′ UTR ( purple ) containing ( top ) or lacking ( bottom ) the second intron of DUX4's 3′ UTR ( Intron 2 ) .", "( F ) Relative levels of transcripts from the Gl-DUX4-Intron2 ( + ) and Gl-DUX4-Intron2 ( − ) constructs following control or UFP1 KD in normal myoblasts ( 54-1 cells ) .", "For each construct , data are normalized such that the siControl treatment is 1 .", "Error bars , standard deviation .", "( G ) Immunofluorescence with an antibody against DUX4 following control or UPF1 knock-down , measured 2 days after the initiation of myogenesis in FSHD1 cells ( 54-2 ) , which was prior to significant fusion .", "Box plot , percentage of DUX4+ nuclei as estimated by ImageJ ( Fiji ) ; n = 8 fields .", "Whiskers , max and min over the fields . DOI: http://dx . doi . org/10 . 7554/eLife . 04996 . 006 We next sought to determine whether the intron-containing 3′ UTR of DUX4 contributed to the degradation of DUX4 mRNA by NMD .", "We created chimeric constructs containing the β-globin open reading frame followed by either the complete DUX4 3′ UTR or the DUX4 3′ UTR with the second intron removed ( Figure 3E ) .", "We focused on the constitutively spliced second intron within the 3′ UTR because it lies 100 nt downstream of the termination codon , and therefore it is predicted to trigger NMD .", "Transcripts from the chimeric construct containing the complete DUX4 3′ UTR increased twofold following UPF1 KD in normal myoblasts—a substantial but smaller increase than we observed for the endogenous DUX4 mRNA , perhaps due to the chimeric nature of the β-globin + DUX4 3′ UTR construct—while transcripts from the construct lacking the second intron of the DUX4 3′ UTR increased only 1 . 5-fold .", "We conclude that the second intron of the DUX4 3′ UTR is important for NMD-induced degradation of the DUX4 mRNA ( Figure 3F ) .", "DUX4 exhibits variegated expression in FSHD muscle cells , with only a few percent of nuclei detectable as DUX4+ ( Snider et al . , 2010 ) .", "Therefore , augmented DUX4 expression following UPF1 KD in myotubes could be due to increases in DUX4 mRNA in nuclei that are already DUX4+ and/or increases in the fraction of DUX4+ nuclei .", "Immunostaining of FSHD myotubes revealed that the fraction of DUX4+ nuclei increased from 0 . 3% to 2 . 1% following UPF1 KD , a substantial order-of-magnitude increase ( Figure 3G ) .", "Together , our data show that NMD is an endogenous suppressor of DUX4 mRNA levels that contributes to the very low and variegated expression of DUX4 , a characteristic feature of FSHD muscle cells .", "As DUX4 expression inhibits NMD and NMD degrades DUX4 mRNA , we hypothesized that DUX4 and the NMD pathway might participate in a double-negative feedback loop ( Figure 4A ) .", "This feedback loop predicts that DUX4 will indirectly stabilize its own mRNA by inhibiting NMD .", "To test this , we ectopically expressed DUX4 in FSHD1 and FSHD2 myotubes and measured levels of endogenously transcribed DUX4 mRNA .", "Ectopic DUX4 expression led to an approximately fivefold increase in endogenously transcribed DUX4 mRNA levels ( Figure 4B ) .", "We next tested whether DUX4's spliced 3′ UTR , which is important for NMD-mediated degradation of DUX4 mRNA , contributed to this increase .", "We transfected our chimeric β-globin + DUX4 3′ UTR reporters into normal myoblasts and ectopically expressed DUX4 .", "Levels of the NMD-susceptible construct containing the complete DUX4 3′ UTR increased 1 . 43-fold following ectopic DUX4 expression , while levels of the construct without the second intron of the DUX4 3′ UTR exhibited a more modest increase of 1 . 08-fold ( Figure 4C ) .", "As with the UPF1 KD experiments , the chimeric construct exhibited more modest effect sizes in these feedback loop experiments than we observed for the endogenous DUX4 mRNA itself . 10 . 7554/eLife . 04996 . 007Figure 4 . DUX4 and NMD form a feedback loop .", "( A ) Schematic of potential double-negative feedback loop between DUX4 and NMD , in which DUX4 inhibits NMD and NMD degrades DUX4 mRNA .", "( B ) Levels of endogenously transcribed DUX4 mRNA following control treatment or ectopic DUX4 expression , measured 2 days after the initiation of myogenesis in FSHD1 ( 54-2 ) and FSHD2 ( MB200 ) cells .", "( C ) Relative levels of transcripts from the Gl-DUX4-Intron2 ( + ) and Gl-DUX4-Intron2 ( − ) constructs following control treatment or ectopic DUX4 expression in normal myoblasts ( 54-1 cells ) .", "For each construct , data are normalized such that the siControl treatment is 1 .", "Error bars , standard deviation .", "( D ) Schematic of potential model of interactions between DUX4 and NMD in healthy ( top ) and FSHD ( bottom ) muscle cells .", "In healthy cells , DUX4 mRNA is efficiently degraded by NMD; in FSHD cells , DUX4 triggers proteolytic degradation of UPF1 and inhibits NMD , resulting in the accumulation of DUX4 mRNA and protein . DOI: http://dx . doi . org/10 . 7554/eLife . 04996 . 007 Together , our data demonstrate that the DUX4 3′ UTR targets DUX4 mRNA for NMD and that DUX4-mediated inhibition of NMD results in increased perdurance of the DUX4 mRNA as a possible mechanism of positive autoregulation ( Figure 4D ) .", "It is unclear whether NMD-mediated autoregulation is intrinsic to normal DUX4 function or instead an abnormal consequence of inappropriate DUX4 expression in skeletal muscle .", "However , it is interesting to consider that this mechanism might contribute to the spreading of DUX4 expression between adjacent nuclei in a muscle fiber .", "Because muscle fibers contain arrays of closely spaced nuclei , the expression of DUX4 mRNA from one nucleus will distribute protein to the surrounding nuclei and induce a region of NMD inhibition .", "If one of the surrounding nuclei subsequently expresses DUX4 , then that mRNA would be unusually stable due to locally inefficient NMD , thereby facilitating the spread of DUX4 mRNA and protein throughout the fiber .", "The close temporal coupling between the onset of DUX4 expression , decreases in UPF1 protein , and increases in NMD substrates ( Figure 2 ) strongly suggests that DUX4-mediated degradation of UPF1 contributes to DUX4-induced NMD inhibition .", "In the absence of a direct mechanistic link between UPF1 degradation and NMD inhibition , we were unable to determine whether insufficient UPF1 protein levels are primarily responsible for DUX4-induced NMD inhibition or instead merely one of the several contributing factors .", "Nonetheless , as we are unaware of other reports of physiological stimuli triggering rapid UPF1 protein degradation , our data suggest that UPF1 proteolysis constitutes a potential new regulator of cellular NMD efficiency .", "DUX4 may prove a useful system to gain insight into the biological relevance of this mechanism for altering NMD efficiency .", "We previously observed that many of the most up-regulated genes following DUX4 expression in normal myoblasts are involved in the ubiquitin–proteasome system , including numerous E3 ubiquitin ligases ( Geng et al . , 2012 ) .", "It is therefore tempting to speculate that DUX4-induced dysregulation of the ubiquitin–proteasome system is responsible for triggering UPF1 protein degradation .", "However , the precise mechanism by which DUX4 induces UPF1 proteolysis , and whether that mechanism is specific to the FSHD disease state , remains to be elucidated .", "DUX4-mediated inhibition of NMD may contribute to FSHD pathophysiology through both cell autonomous and non-cell autonomous mechanisms .", "The accumulation of abnormal RNAs may cause direct or indirect toxic effects in muscle cells due to intrinsic toxicity of abnormal RNAs or a stress response to the production of abnormal proteins .", "FSHD muscle is frequently characterized by a T-cell infiltrate ( Arahata et al . , 1995 ) , and it is possible that stabilized NMD substrates encode novel peptides with antigenic potential , contributing to an immune response ( Pastor et al . , 2010 ) .", "Production of antigenic peptides could potentially enable even a small fraction of DUX4+ nuclei to induce widespread pathology within a muscle fiber .", "Directly detecting or measuring DUX4-induced NMD inhibition in FSHD muscle biopsies or in bulk populations of cultured FSHD muscle cells is not feasible due to the low fraction of DUX4+ nuclei present at any given time in the absence of ectopic DUX4 expression .", "With DUX4+ nuclei constituting only 0 . 3% of the bulk population of cultured FSHD muscle cells ( Figure 3G ) , changes in the ratios of NMD and non-NMD isoforms in these DUX4+ nuclei are swamped by the normal levels expressed by the vast majority of DUX4− nuclei .", "Single-cell assays of NMD efficiency are likely required to demonstrate DUX4-induced NMD inhibition in unperturbed patient cells .", "However , future efforts to identify the downstream antigenic products or toxic effects of stabilized NMD substrates may prove fruitful even in a bulk cell population .", "Consistent with the idea that NMD inhibition may contribute to DUX4 toxicity in skeletal muscle , it is interesting to note that the degree of NMD inhibition induced by DUX4 is comparable to that observed in previous studies involving genetic ablation of components of the NMD machinery .", "For example , a recent study of mouse embryonic fibroblasts lacking Smg1 , which encodes a kinase responsible for phosphorylating UPF1 , found that 9% of predicted NMD substrates created by alternative splicing exhibited increased levels relative to wild-type cells ( McIlwain et al . , 2010 ) .", "In comparison , we found that 13% of such substrates were up-regulated following DUX4 expression ( Figure 1 ) , suggesting that DUX4-induced NMD inhibition causes profoundly abnormal RNA metabolism .", "As RNA toxicity is the major pathophysiologic mechanism in myotonic dystrophy , it is interesting to consider that RNA-mediated disease mechanisms may also have important roles in FSHD ." ], [ "FASTQ files for the DUX4 expression experiments were downloaded from the NCBI GEO database under accession number GSE45883 ( Young et al . , 2013 ) .", "The UCSC knownGene ( Meyer et al . , 2013 ) and Ensembl 71 ( Flicek et al . , 2013 ) genome annotations were merged to create a single genome annotation .", "Splicing event annotations from MISO v2 . 0 ( Katz et al . , 2010 ) were then added to this merged genome annotation .", "Constitutive splice junctions were defined as those for which neither the 5′ nor 3′ splice site was alternatively spliced in the UCSC knownGene annotation .", "Predicted NMD substrates were annotated by identifying isoforms containing premature termination codons >50 nt upstream of a splice junction .", "For the purposes of predicting NMD substrates , open reading frames were assigned based on UniProt annotations ( UniProt Consortium , 2012 ) when available , and Ensembl predicted reading frames when UniProt annotations were not available .", "For the purposes of RNA-seq read mapping , an additional annotation file consisting of all splice junctions annotated in the UCSC , Ensembl 71 , and MISO v2 . 0 annotations was created .", "This splice junction file was then with a list of all possible junctions between the annotated 5′ and 3′ splice sites of isoforms in the annotation ( to detect novel alternative splicing ) .", "Reads were mapped to the UCSC hg19 ( NCBI GRCh37 ) genome assembly .", "RSEM ( Li and Dewey , 2011 ) was modified to call Bowtie ( Langmead et al . , 2009 ) v1 . 0 . 0 with the -v 2 argument .", "RSEM was then called with the arguments --bowtie-m 100 --bowtie-chunkmbs 500 --calc-ci --output-genome-bam on the genome annotation .", "Read alignments with mapq scores of 0 and/or a splice junction overhang of less than 6 bp were then filtered out .", "Remaining unaligned reads were then aligned TopHat ( Trapnell et al . , 2009 ) v2 . 0 . 8b with the arguments --bowtie1 --read-mismatches 2 --read-edit-dist 2 --no-mixed --no-discordant --min-anchor-length 6 --splice-mismatches 0 --min-intron-length 10 --max-intron-length 1000000 --min-isoform-fraction 0 . 0 --no-novel-juncs --no-novel-indels --raw-juncs on the splice junction file ( --mate-inner-dist and --mate-std-dev were calculated by mapping to constitutive coding exons with MISO's exon_utils . py utility ) .", "Alignments produced by this call to TopHat were then filtered identically to the alignments produced by RSEM .", "Reads aligned by RSEM and TopHat were then merged to create BAM files of all aligned reads .", "Gene expression was quantified using RSEM as described above .", "Isoform ratios were quantified using two distinct methods .", "First , MISO was used to quantify isoform ratios for alternative splicing events contained in MISO's v2 . 0 annotations .", "Second , novel alternative splicing or intron retention of annotated constitutive splice junctions was quantified using reads crossing the 5′ or 3′ splice sites as previously described ( Hubert et al . , 2013 ) .", "Differentially spliced events were defined as those with at >20 identifying reads ( identifying reads support one or more , but not all , isoforms of a splicing event ) , a change in isoform ratio ≥10% , and a Bayes factor ≥5 ( computed with Wagenmakers's framework [Wagenmakers et al . , 2010] ) .", "We used previously described lentiviral constructs expressing full-length DUX4 or GFP as a control ( Geng et al . , 2012 ) .", "Lentiviral particles were generated by the FHCRC Core Center of Excellence in Hematology Vector Production Core .", "Viral particle number was estimated with the WPRE element within the viral vector .", "Myoblasts were transduced at a MOI of ∼15 in the presence of 8 µg/ml polybrene .", "At this MOI , >85% of myoblasts were DUX4+ or GFP+ .", "Unless otherwise noted , cells were collected for analysis 24 hr post-infection .", "Proliferating myoblasts were cultured in F-10-based growth media ( Gibco , Carlsbad , CA ) with 20% fetal bovine serum ( Gibco ) and 1% penicillin/streptomycin ( Gibco ) , supplemented with 10 ng/ml rhFGF ( Promega , Madison , WI ) and 1 µM dexamethasone ( Sigma , St . Louis , MO ) .", "Growth media was changed every other day , and proliferating myoblasts were cultured at ≤50% confluence .", "To initiate myogenic differentiation , myoblasts were switched to an F-10-based differentiation media containing 1% horse serum ( Gibco ) and 1% penicillin/streptomycin , supplemented with 10 µg/ml insulin ( Sigma ) and 10 µg/ml transferrin ( Sigma ) at 99% confluence .", "The β-globin NMD ( − ) and NMD ( + ) plasmids were previously published as pmCMV-Gl Norm and pmCMV-Gl 39Ter ( Zhang et al . , 1998 ) .", "Plasmid reporters were transfected with Lipofectamine 2000 ( Life Technologies , Carlsbad , CA ) , unless otherwise noted .", "To control for transfection efficiency , a control plasmid phCMV-MUP was co-transfected with the reporter as previously described ( Zhang et al . , 1998 ) .", "2 µg of reporter along with 500 ng of control plasmid was used for transfecting cells in a six-well format .", "To measure DUX4-induced changes in NMD efficiency , cells were infected with lentiviral DUX4 or GFP 24 hr after transfection of the NMD reporters .", "For the DUX4 time course experiments , the NMD ( − ) and NMD ( + ) reporters were transfected along with phCMV-MUP using the SuperFect reagent ( Qiagen , Valencia , CA ) , and the lentiviral transduction was performed 12 hr post-transfection .", "siRNAs against UPF1 ( Thermo Scientific , Waltham , MA , On-Target siRNA #J-011763-07 ) and TP53 ( Ambion , Silencer Select siRNA #4390824 ) , as well as the control siRNA ( Thermo Scientific ) , were transfected with Lipofectamine RNAiMAX ( Life Technologies ) .", "Cells were lysed with TRIzol ( Invitrogen , Carlsbad , CA ) and the RNA was extracted according to the manufacturer's instructions .", "RNA was subsequently cleaned up with Qiagen RNeasy columns , with on-column DNase digestion .", "1 µg of RNA was used for cDNA synthesis with Life Technologies SuperScriptIII First-Strand System .", "2% of this cDNA was used as a template for real-time qPCR with Life Technologies Power SYBR Green Master Mix .", "qPCR primer sequences are provided in Supplementary file 1 .", "Note that levels of endogenous DUX4 mRNA following ectopic DUX4 expression were measured with primers specific to the DUX4 mRNA's 3′ UTR ( the DUX4 lentiviral construct contained the coding sequence alone ) .", "To determine how UPF1 KD affected DUX4 expression , proliferating 54-2 or MB200 myoblasts were transfected with siUPF1 or siControl and switched to differentiation media 48 hr post-transfection .", "Protein extracts from the UPF1 KD experiments were generated by lysing cell pellets in protease inhibitor cOmplete ULTRA ( Roche , Switzerland ) containing RIPA buffer ( Cell Signaling Technology , Danvers , MA ) along with sonication .", "For the DUX4 time course and MG132 treatment , protein was extracted in parallel with the RNA from cells lysed in the TRIzol reagent .", "Protein pellets were resuspended in a sample buffer containing 5% SDS and 0 . 5 M unbuffered Tris base to ensure efficient solubilization .", "Protein concentrations were determined using the Bradford or BCA protein assay .", "5 µg of total protein was used for Western blotting .", "Antibodies used in this study are: anti-UPF1 ( Bethyl Laboratories , Montgomery , TX ) , anti-α-tubulin ( Sigma ) , anti-H3 ( Abcam , England ) , anti-UPF3B ( Bethyl Laboratories ) , anti-SMG7 ( Santa Cruz , Dallas , TX ) .", "HRP-conjugated ( Jackson ImmunoResearch , West Grove , PA ) secondary antibodies were used for protein detection in all experiments except for the time course and proteasome inhibitor studies ( Figure 2 ) .", "For the experiments reported in Figure 2 , immunoblotting was performed using the LICOR system with the Odyssey blocking buffer and IRDye-conjugated secondary antibodies ( LICOR , Lincoln , NE ) .", "Quantification was performed using ImageQuant software ( GE Healthcare , Cleveland , OH ) using the nonspecific band as a normalizer to account for differences in protein loading .", "Histone 3 served as an additional loading control , though its very high signal intensity made it an inappropriate normalizer for quantitative analyses .", "54-1 cells transduced with DUX4 or GFP lentivirus were treated 16 hr post-infection with 10 µM proteasome inhibitor MG132 ( Sigma ) .", "Samples were collected 8 hr after MG132 treatment , and UPF1 levels were estimated by immunoblotting .", "Histone H3 , which has a long half-life ( Toyama et al . , 2013 ) , was used as a loading control , in addition to the nonspecific band .", "Cells were permeabilized with PBS containing 0 . 5% Triton X-100 , rinsed in PBS , and blocked in 1% BSA .", "Primary antibody against DUX4 ( Abcam , ab124699 ) was diluted in blocking buffer at 1:500 , and secondary anti-Rabbit TRITC ( Jackson ImmunoResearch , 711-025-152 ) was diluted in blocking buffer at 1:400 .", "For assaying apoptosis in DUX4-cells , Image-iT LIVE Red Poly Caspases Detection Kit ( Life Technologies , I35101 ) was used .", "For both experiments , fluorescently labeled cells were then viewed under the ZEISS Axiophot fluorescence microscope .", "For each sample , pictures from eight random fields were taken .", "ImageJ ( Fiji ) was used for image analysis and quantification .", "The genomic locus of the DUX4 3′ UTR ( containing both introns ) was amplified from a genomic fragment harboring 2 . 5 D4Z4 repeats ( Gabriëls et al . , 1999 ) ( L42 clone; GenBank ID FJ439133 . 1 ) .", "The DUX4 3′ UTR lacking the second intron was amplified from cDNA isolated from differentiated 54-2 cells ( the first intron is frequently retained in DUX4 cDNA ) .", "The β-globin NMD ( − ) reporter backbone was linearized by forward and reverse PCR primers sitting downstream and upstream of the β-globin 3′ UTR ( primers listed in Supplementary file 1 ) .", "Amplicons of the DUX4 3′ UTR containing or lacking the second intron were flanked with sequences overlapping the linearized β-globin NMD ( − ) backbone lacking the β-globin 3′ UTR .", "The NEB Gibson Assembly Cloning Kit was used to insert the DUX4 3′ UTR fragments into the linearized β-globin NMD ( − ) backbone ( New England Biolabs , Ipswich , MA ) ." ] ]

[ "Facioscapulohumeral muscular dystrophy ( FSHD ) is a muscular dystrophy caused by inefficient epigenetic repression of the D4Z4 macrosatellite array and somatic expression of the DUX4 retrogene .", "DUX4 is a double homeobox transcription factor that is normally expressed in the testis and causes apoptosis and FSHD when misexpressed in skeletal muscle .", "The mechanism ( s ) of DUX4 toxicity in muscle is incompletely understood .", "We report that DUX4-triggered proteolytic degradation of UPF1 , a central component of the nonsense-mediated decay ( NMD ) machinery , is associated with profound NMD inhibition , resulting in global accumulation of RNAs normally degraded as NMD substrates .", "DUX4 mRNA is itself degraded by NMD , such that inhibition of NMD by DUX4 protein stabilizes DUX4 mRNA through a double-negative feedback loop in FSHD muscle cells .", "This feedback loop illustrates an unexpected mode of autoregulatory behavior of a transcription factor , is consistent with ‘bursts’ of DUX4 expression in FSHD muscle , and has implications for FSHD pathogenesis ." ]

[ "Genes are sequences of DNA that contain instructions for the cell that must be carefully controlled because it is not always appropriate or safe for these instructions to be followed .", "When genes are active , copies of the DNA are made using molecules of ribonucleic acid ( RNA ) and these can then be used as templates to make proteins .", "One way genes can be controlled is by adding small chemical tags that mark them out to be activated or deactivated , known as epigenetic control .", "The muscle disease facioscapulohumeral muscular dystrophy ( FSHD ) is caused by the loss of the chemical tags that normally keep certain genes switched off in many cell types .", "One of these genes is DUX4 , which in healthy males is normally only active in the testes , but in FSHD patients it is also active in other parts of the body .", "Another way to control genes is by nonsense-mediated decay , where incorrect or incomplete RNA molecules are destroyed before they can be used to make defective proteins .", "In this study , Feng et al . show that when DUX4 is activated following the failure of epigenetic control in FSHD patients , the effectiveness of nonsense-mediated decay is also reduced .", "This results in the build-up of incorrect RNA molecules inside muscle cells , which can harm the cell .", "In fact , 13% of the incorrect RNA molecules that are normally destroyed in cells were found at higher levels when DUX4 was active .", "To investigate how DUX4 could work , Feng et al . focused on another gene called UPF1 because cells without the protein encoded by this gene have similar defects in nonsense-mediated decay as cells with active DUX4 .", "No difference was found in how often the UPF1 gene is activated in FSHD cells and normal cells .", "However , the amount of the protein encoded by UPF1 was lower in cells with FSHD than in normal muscle cells .", "The experiments show that the protein encoded by UPF1 is broken down as a result of the activation of the DUX4 gene , leading to problems with nonsense-mediated decay , which may result in the worsening of FSHD symptoms .", "The twist in the tale is that DUX4 itself is also controlled by nonsense-mediated decay under normal circumstances .", "Therefore , in diseased cells , a failure in epigenetic control allows DUX4 to prevent its own destruction by tampering with nonsense-mediated decay .", "These findings offer new insights into the role of the DUX4 gene in FSHD .", "The next step is to test whether these defects in nonsense-mediated decay can explain any of the symptoms of FSHD , such as muscle inflammation ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "plant biology" ]

[ [ "To survive under fluctuating environmental conditions , all organisms have the ability to deal with various kinds of internal and external stresses .", "In animals , failure to adapt against disadvantageous stresses leads to various diseases such as cancer , and , in severe cases , cells perish ( Twayana and Ravanan , 2018 ) .", "To avoid such consequences , cells deploy mechanisms to temporarily arrest proliferation , thereby ensuring appropriate stress responses .", "For instance , the high osmolarity glycerol ( HOG ) MAP kinase ( MAPK ) cascade induces cell cycle arrest by inhibiting the transcription of cyclin genes in response to hyperosmotic stress ( Saito and Posas , 2012 ) .", "However , while such individual pathways inducing inhibition of cell proliferation have been well characterized , our understanding of the central module that is presumed to govern cell cycle arrest in response to different stresses is still limited .", "Due to their sessile lifestyle , plants also need to handle many kinds of abiotic and biotic stresses , and to adapt themselves to environmental conditions by activating particular signalling cascades .", "Various transcription factors that respond to abiotic stresses have been well studied , such as those belonging to the MYB , bZIP , drought-responsive element-binding protein ( DREB ) and DREB1s/C-repeat binding factor ( CBF ) families ( Vinocur and Altman , 2005; Umezawa et al . , 2006; Golldack et al . , 2011 ) .", "However , downstream signalling pathways that inhibit cell proliferation remain largely unknown; therefore , it remains to be established whether plants deploy central signalling pathway ( s ) that induce cell cycle arrest upon exposure to multiple stresses .", "DNA damage also arrests the cell cycle to allow DNA repair to occur before DNA replication or mitosis begins ( Harper and Elledge , 2007; Ciccia and Elledge , 2010; Hu et al . , 2016 ) .", "The programmed response to DNA damage is therefore crucial to maintain genome integrity .", "DNA damage is sensed by two kinases , ATAXIA TELANGIECTASIA MUTATED ( ATM ) and ATM AND RAD3-RELATED ( ATR ) ( Abraham , 2001; Garcia et al . , 2003; Culligan et al . , 2004 ) .", "ATM is activated by double-strand breaks ( DSBs ) , whereas ATR senses single-strand breaks ( SSBs ) and stalled replication forks ( Bensimon et al . , 2011; Flynn and Zou , 2011 ) .", "In animals , ATM phosphorylates the checkpoint kinase CHK2 , which stabilizes the transcription factor p53 and induces expression of the CDK inhibitor p21 , resulting in cell cycle arrest at G1/S phase ( Donjerkovic and Scott , 2000; Bartek and Lukas , 2001; Cheng and Chen , 2010 ) .", "CHK1 , which is phosphorylated by ATR , and CHK2 also activate the G2/M-phase checkpoint by phosphorylating CDC25 phosphatases ( Karlsson-Rosenthal and Millar , 2006; Perry and Kornbluth , 2007 ) .", "In contrast , while plants possess ATM and ATR , they lack functional orthologues for p21 , p53 , CDC25 or CHK1/2 .", "Instead , a plant-specific NAC-type transcription factor , SUPPRESSOR OF GAMMA RESPONSE 1 ( SOG1 ) , is phosphorylated and activated by ATM and ATR , and triggers DNA damage responses ( DDRs ) ( Yoshiyama et al . , 2009; Yoshiyama et al . , 2013; Sjogren et al . , 2015; Yoshiyama et al . , 2017 ) .", "A previous study demonstrated that in Arabidopsis , SOG1 regulates the expression of almost all genes induced by DSBs ( Yoshiyama et al . , 2009 ) , causes G2 arrest in the meristem , enhances the transition from cell division to endoreplication ( a repeated cycle of DNA replication without mitosis ) , and triggers stem cell death ( Furukawa et al . , 2010; Adachi et al . , 2011 ) .", "Expression of CDK inhibitors , such as SMR5 and SMR7 , is directly induced by SOG1 ( Yin et al . , 2014; Ogita et al . , 2018 ) ; however , their induction also occurs in DSB-tolerant Arabidopsis mutants , suggesting that it is not sufficient to arrest the cell cycle ( Chen et al . , 2017 ) .", "We previously demonstrated that simultaneous suppression of a set of G2/M-specific genes is accompanied by DNA damage-induced G2 arrest ( Adachi et al . , 2011 ) .", "G2/M-specific genes , such as those encoding mitotic cyclins , are controlled by three Myb repeat-containing transcription factors , called R1R2R3-Myb or MYB3R ( Ito , 2005 ) .", "Arabidopsis possesses five genes for MYB3R , MYB3R1 to MYB3R5 , among which MYB3R4 acts as a transcriptional activator ( Act-MYB ) , and MYB3R3 and MYB3R5 are transcriptional repressors ( Rep-MYB ) ( Haga et al . , 2007; Haga et al . , 2011; Kobayashi et al . , 2015 ) .", "MYB3R1 functions as both an activator and a repressor ( Kobayashi et al . , 2015 ) .", "MYB3Rs bind to target gene promoters via a cis-acting element called the mitosis-specific activator ( MSA ) element , and control the expression of G2/M-specific genes ( Kobayashi et al . , 2015 ) .", "We recently reported that Rep-MYBs are actively degraded via the ubiquitin-proteasome pathway , but become stabilized under DNA damage conditions , thereby suppressing G2/M-specific genes and causing G2 arrest ( Chen et al . , 2017 ) .", "Protein stability of Rep-MYBs is dependent on CDK activity: CDK phosphorylates the C-terminal domain of MYB3R3/5 and promotes their degradation .", "Therefore , we proposed a model in which DNA damage phosphorylates and activates SOG1 , and reduces CDK activities , probably by inducing CDK inhibitor genes , thereby stabilizing Rep-MYBs ( Chen et al . , 2017 ) .", "Since Rep-MYBs repress mitotic cyclin expression , their stabilization further decreases CDK activities , thus resulting in cell cycle arrest .", "A recent report also demonstrated that Rep-MYBs function as major repressors of cell cycle genes in the response to DNA damage ( Bourbousse et al . , 2018 ) .", "However , it remains unknown whether the initial reduction of CDK activity is sufficient to trigger Rep-MYB stabilization , or whether another signalling pathway is required to accumulate a sufficient amount of Rep-MYB for the suppression of numerous G2/M-specific genes .", "In this study , we show that the NAC-type transcription factors ANAC044 and ANAC085 , which are direct targets of SOG1 , play a crucial role in causing G2 arrest in response to DNA damage .", "In the absence of ANAC044 or ANAC085 , Rep-MYB does not accumulate and cannot inhibit G2/M progression , and the plants exhibit DNA damage tolerance .", "ANAC044 and ANAC085 are not required for the induction of genes for DNA repair proteins or CDK inhibitors , but rather are engaged in accumulation of Rep-MYBs .", "Our data show that they also participate in heat stress-induced inhibition of G2 progression , suggesting that an ANAC044- and ANAC085-mediated pathway plays a central role in orchestrating stress-induced G2 arrest by controlling Rep-MYB accumulation ." ], [ "Previous studies demonstrated that the SOG1 transcription factor controls cell cycle arrest and stem cell death ( Yoshiyama et al . , 2009; Furukawa et al . , 2010; Adachi et al . , 2011 ) .", "Therefore , it is conceivable that DNA damage-induced G2 arrest is controlled by signalling pathways downstream of SOG1 .", "Recently we identified 146 Arabidopsis genes that are directly targeted by SOG1 , among which were the NAC transcription factors ANAC044 and ANAC085 ( Ogita et al . , 2018 ) .", "Phylogenetic analysis of NAC transcription factors indicated that ANAC044 and ANAC085 are the closest relatives of SOG1 ( Figure 1A ) ; indeed , in the NAC domain , which is essential for DNA binding , their amino acid similarity to SOG1 is 72 . 0% for ANAC044 and 72 . 6% for ANAC085 .", "A striking difference is that the C-terminal regions carrying the domain needed for transcriptional regulation are shorter in ANAC044 and ANAC085 than in SOG1 .", "Moreover , five serine-glutamine ( SQ ) motifs , which are targets for phosphorylation by ATM and ATR , are present towards the C terminus of SOG1 , but missing in ANAC044 and ANAC085 ( Figure 1B ) .", "We therefore predicted that ANAC044 and ANAC085 exert distinct functions in the DDR .", "We first examined the transcriptional response of these NAC transcription factors to the DSB-inducing agent bleomycin .", "Five-day-old wild-type ( WT ) seedlings were transferred onto MS medium containing 0 . 6 µg/ml bleomycin , and quantitative real-time PCR ( qRT-PCR ) was conducted using total RNA isolated from root tips .", "The transcript levels of ANAC044 and ANAC085 increased soon after bleomycin treatment and reached maxima after 12 hr , indicating a rapid response to DSBs ( Figure 1—figure supplement 1A ) .", "We next generated promoter:GUS reporter lines and found that ProANAC085:GUS showed no GUS signal irrespective of bleomycin treatment , probably due to a lack of regulatory element ( s ) essential for transcriptional activation in the promoter region .", "On the other hand , in the ProANAC044:GUS line , GUS expression was elevated by bleomycin treatment in root tips and young leaves ( Figure 1—figure supplement 1B and C ) .", "Interestingly , we observed a patchy pattern of signals in the root tip , suggesting that ANAC044 is induced in cells at particular cell cycle stage ( s ) that are more sensitive to DSBs .", "We also tested other DNA-damaging agents , namely hydroxyurea ( HU ) , mitomycin C ( MMC ) and methyl methanesulfonate ( MMS ) .", "HU inhibits deoxyribonucleotide production , thereby causing DNA replication stress ( Wang and Liu , 2006; Saban and Bujak , 2009 ) .", "MMS is an alkylating agent that methylates guanine and adenine bases , resulting in base mispairing and replication blocks ( Beranek , 1990; Llorente et al . , 2008 ) .", "MMC generates interstrand cross-links on DNA ( Rink et al . , 1996 ) .", "Treatment with all three drugs again induced GUS expression in root tips and young leaves ( Figure 1—figure supplement 1B and C ) , suggesting that ANAC044 is associated with the DDR in actively dividing cells .", "To examine whether the transcriptional induction of ANAC044 and ANAC085 is mediated by SOG1 , we conducted qRT-PCR using RNA isolated from the sog1 knockout mutant sog1-101 ( Ogita et al . , 2018 ) .", "As shown in Figure 1—figure supplement 2A , bleomycin-induced expression of ANAC044 and ANAC085 was not observed in sog1-101 .", "A similar result was also obtained using the ProANAC044:GUS reporter gene , which did not show bleomycin-triggered GUS expression in the sog1-101 mutant background ( Figure 1—figure supplement 2B ) , indicating that SOG1 is required for the transcriptional induction .", "As described above , we had identified ANAC044 and ANAC085 as SOG1 target genes because their promoters displayed significant peaks in the ChIP-seq data of SOG1 ( Ogita et al . , 2018 ) .", "We searched for the SOG1-binding motif CTT ( N ) 7AAG in the promoter regions of ANAC044 and ANAC085 , and found CTTGGGGAGCAAG and CTTATTTACGAAG sequences , respectively ( Figure 1—figure supplement 2C ) .", "To reveal whether SOG1 binds to the identified promoter regions , we conducted ChIP-qPCR analysis .", "Using transgenic plants harbouring ProSOG1:SOG1-Myc , which can complement the sog1-1 mutation ( Yoshiyama et al . , 2013 ) , immunoprecipitation was performed with anti-Myc antibody , and chromatin fragments bound to SOG1-Myc were subjected to qPCR .", "The result showed that for both ANAC044 and ANAC085 , the promoter regions containing the SOG1-binding motifs were highly enriched when chromatin isolated from bleomycin-treated plants , but not from non-treated control , was used for immunoprecipitation ( Figure 1—figure supplement 2C ) .", "This strongly suggests that SOG1 binds to the ANAC044 and ANAC085 promoters under DNA damage conditions and induces their expression .", "DNA damage-dependent binding of SOG1 has also been demonstrated for promoters of other SOG1 target genes ( Yin et al . , 2014; Ogita et al . , 2018 ) .", "We then examined the involvement of ANAC044 and ANAC085 in the DDR using multiple mutant alleles .", "anac044-1 ( SAIL_1286D02 ) and anac044-2 ( GABI_968B05 ) have T-DNA insertions in the fourth and fifth exons of ANAC044 , respectively , and anac085-1 ( GABI_894G04 ) and anac085-2 ( SALK_208662 ) contain T-DNA fragments in the fourth exon of ANAC085 ( Figure 2—figure supplement 1A ) .", "Semi-quantitative RT-PCR showed that transcripts of ANAC044 in anac044-1 and anac044-2 , and ANAC085 in anac085-1 and anac085-2 , were undetectable using primers flanking the T-DNA insertion sites , indicating that all are null alleles ( Figure 2—figure supplement 1B ) .", "Five-day-old seedlings grown on MS medium were transferred onto a medium supplemented with or without bleomycin , HU , MMC or MMS , and grown for a further 5 days .", "Measurement of root growth showed that the two mutants for each of anac044 or anac085 were more tolerant than WT to all four DNA-damaging agents , and that root growth was almost the same between anac044 and anac085 ( Figure 2—figure supplement 2A ) .", "Moreover , the anac044-1 anac085-1 double mutant showed a similar tolerance to that of the single mutants ( Figure 2; Figure 2—figure supplement 3 ) .", "Previous studies demonstrated that sog1-1 was tolerant to gamma irradiation and bleomycin treatment , and was hypersensitive to HU ( Yoshiyama et al . , 2009; Hu et al . , 2015; Yoshiyama et al . , 2017 ) .", "We found that the tolerance to bleomycin and MMS was comparable between sog1-101 , anac044-1 anac085-1 and the triple mutant anac044-1 anac085-1 sog1-101 ( Figure 2—figure supplement 2B ) .", "Moreover , the triple mutant exhibited hypersensitivity to HU and MMC to a similar extent to that of sog1-101 ( Figure 2—figure supplement 2B ) , indicating that sog1-101 is epistatic to anac044-1 anac085-1 .", "These results suggest that ANAC044 and ANAC085 function downstream of SOG1 in DNA damage-induced root growth retardation .", "It was reported that DSBs induce cell death of stem cells and stele precursor cells in the root tip , and that SOG1 is involved in this response ( Furukawa et al . , 2010 ) .", "To reveal whether ANAC044 and ANAC085 are also required for DSB-induced stem cell death , five-day-old seedlings were treated with bleomycin for 24 hr , and root tips were observed .", "Propidium iodide ( PI ) -stained dead cells were visible in the stem cell and stele precursor cell populations in WT , while almost no cell death was observed in anac044-1 anac085-1 , sog1-101 or anac044-1 anac085-1 sog1-101 ( Figure 3A and B ) .", "This result suggests that ANAC044 and ANAC085 also participate in SOG1-mediated induction of stem cell death .", "Johnson et al . ( 2018 ) investigated the recovery of Arabidopsis roots after an acute dose of ionizing radiation ( 150 Gy ) .", "They found that the stem cell niche was regenerated through experiencing cell death , and that root growth recovered after about one week .", "However , the sog1 mutant underwent neither death nor regeneration of stem cells , leading to root growth arrest after long-term culture ( Johnson et al . , 2018 ) .", "This observation suggests that SOG1-dependent programmed cell death triggers regeneration of the stem cell niche , and is thus essential for the recovery of root growth .", "As described above , anac044-1 anac085-1 is also defective in DNA damage-induced stem cell death; therefore , we investigated root regeneration in anac044-1 anac085-1 after a one-day treatment with bleomycin .", "In WT roots , PI-stained dead cells derived from stem cells and stele precursor cells gradually decreased and almost disappeared within five days ( Figure 3C ) .", "In sog1-101 , stem cell death did not occur , although we observed sporadic cell death across the meristematic zone and distorted tissue organization ( Figure 3C ) , which were also described by Johnson et al . ( 2018 ) .", "On the other hand , anac044-1 anac085-1 displayed no cell death in the stem cell niche or in the meristematic zone , and cells were properly arranged in the root tip throughout 7 days after recovery ( Figure 3C ) .", "The anac044-1 anac085-1 sog1-101 triple mutant showed disorganized root tissues as observed in sog1-101 ( Figure 3C ) , again indicating epistasis of sog1-101 over anac044-1 anac085-1 .", "These results suggest that stem cells in sog1-101 and anac044-1 anac085-1 have different characteristics; namely , DNA-damaged stem cells in anac044-1 anac085-1 , but not sog1-101 , can function in producing transit-amplifying cells , which constitute the root meristem .", "SOG1-dependent , but ANAC044/085-independent , pathway ( s ) are probably required for preserving the integrity of stem cells that have been exposed to DNA damage .", "In Arabidopsis , DNA repair and recombination are known to be induced through SOG1 in response to DSBs ( Yoshiyama et al . , 2009 ) ; indeed , our recent study showed that SOG1 directly controls several DNA repair-related genes , such as RAD51 and BRCA1 ( Ogita et al . , 2018 ) .", "To investigate whether ANAC044 and ANAC085 participate in DNA repair , we examined the expression of DNA repair-related genes in the anac044 anac085 double mutant .", "Five-day-old seedlings were treated with bleomycin for 10 hr , at which time ANAC044 and ANAC085 are highly induced in WT ( Figure 1—figure supplement 1A ) , and total RNA was analysed using an Agilent Custom Microarray that covers all annotated Arabidopsis genes .", "Although the transcript levels of genes involved in non-homologous end-joining or mismatch excision repair did not change under our DNA damage conditions , several genes for homologous recombination ( HR ) ( BRCA1 , RAD51A , RAD51B , RAD54L and MND1 ) and nucleotide excision repair ( RPA1E ) were up-regulated in WT , but not in sog1-101 ( Figure 4A ) .", "In anac044 anac085 , the up-regulated genes were normally induced by bleomycin treatment ( Figure 4A ) , suggesting that neither ANAC044 nor ANAC085 is required for DSB-induced expression of DNA repair-related genes .", "To test the functional relevance of ANAC044 and ANAC085 to DNA repair , we performed an assay to detect HR-mediated DNA repair events using the beta-glucuronidase ( GUS ) recombination reporter line ( Swoboda et al . , 1994 ) .", "This assay uses parts of the GUS gene in directed orientation , which serve as a substrate for HR ( Figure 4B ) .", "In cells where HR occurs , the GUS gene is restored , and GUS activity can be visualized and scored as blue spots after histochemical staining ( Swoboda et al . , 1994 ) .", "Plants with the disrupted GUS gene were crossed with sog1-101 and anac044-1 anac085-1 , and blue spots were counted on leaves .", "Under normal growth conditions , sog1-101 displayed fewer blue spots than WT ( Figure 4B ) .", "Fifty grays of gamma irradiation increased the number of blue spots in both WT and sog1-101 , although the number was significantly lower in sog1-101 than in WT ( Figure 4B ) .", "These results indicate that HR is suppressed in the sog1-101 mutant .", "In contrast , no significant difference was observed in the number of blue spots between WT and anac044-1 anac085-1 irrespective of gamma irradiation ( Figure 4B ) , suggesting that ANAC044 and ANAC085 are not involved in HR .", "The above results indicate that ANAC044 and ANAC085 are required for the part of the DDR that is essential for root growth retardation and stem cell death , but not for DNA repair .", "One possibility is that they are engaged in cell cycle arrest , because the Arabidopsis myb3r3 and myb3r5 mutants , which cannot induce G2 arrest upon exposure to DNA damage , also show defects in root growth retardation and stem cell death ( Chen et al . , 2017 ) .", "To test this possibility , we first measured root meristem size by counting the number of cortical cells between the quiescent centre ( QC ) and the first elongated cell .", "Bleomycin treatment for 24 hr resulted in a 38% reduction in the meristem cell number in WT , while no significant reduction was observed in anac044-1 anac085-1 , sog1-101 or anac044-1 anac085-1 sog1-101 ( Figure 5A ) .", "This suggests that SOG1 , ANAC044 and ANAC085 control cell division in the meristem and/or the transition from cell division to endoreplication under DNA damage conditions .", "To clarify whether ANAC044 and ANAC085 are involved in DNA damage-induced G2 arrest , we conducted EdU incorporation experiments .", "Five-day-old roots were treated with or without bleomycin for 12 hr , and then incubated with EdU for 15 min .", "After EdU-labelled cells pass through the G2 phase , they are expected to show mitotic figures in the M phase ( Hayashi et al . , 2013 ) ; therefore , we counted the number of EdU-labelled cells with mitotic figures after 4 or 6 hr .", "In the absence of bleomycin , EdU-labelled cells with mitotic figures increased in WT , anac044 anac085 and sog1-101 after 4 hr ( Figure 5B ) , implying that EdU-labelled cells entered the M phase .", "On the other hand , bleomycin treatment significantly suppressed the increase of EdU-positive cells with mitotic figures in WT , indicating an impairment in G2/M progression , whereas an increase was observed in anac044 anac085 and sog1-101 irrespective of bleomycin treatment ( Figure 5B ) .", "This suggests that ANAC044 and ANAC085 as well as SOG1 are essential for inhibiting G2-to-M progression in response to DSBs .", "We previously reported that when Arabidopsis cultured cells were treated with the DSB inducer zeocin , many G2/M-specific genes were suppressed , thereby causing G2 arrest and endoreplication ( Adachi et al . , 2011 ) .", "To examine whether ANAC044 and ANAC085 are involved in repression of G2/M-specific genes , we measured the transcript levels of four MYB3R3 targets , KNOLLE , CYCB1;2 , EPS15 HOMOLOGY DOMAIN 2 ( EHD2 ) and PLEIADE/MAP65-3 ( Kobayashi et al . , 2015 ) , in root tips .", "When WT seedlings were treated with bleomycin , the mRNA levels decreased over time and were reduced by half after 24 hr ( Figure 6A ) .", "As reported previously , no reduction was observed in sog1-101 ( Yoshiyama et al . , 2009 ) , suggesting that SOG1 is essential to repress G2/M-specific genes .", "In the anac044-1 anac085-1 double mutant , the transcript levels decreased until 12 hr as in WT; however , they did not further decrease beyond 24 hr of bleomycin treatment ( Figure 6A ) .", "It is noteworthy that such an expression pattern is similar to that observed in myb3r3 and myb3r5 mutants , in which suppression of G2/M-specific genes is impaired at a later stage of the DDR ( Chen et al . , 2017 ) .", "It has been previously reported that the CDK inhibitors SMR5 and SMR7 are directly induced by SOG1 in response to DNA damage ( Yin et al . , 2014; Ogita et al . , 2018 ) .", "Some of the NAC-type transcription factors are known to form homo- or heterodimers to control their target genes ( Mitsuda et al . , 2004; Yamaguchi et al . , 2008; Gladman et al . , 2016 ) .", "Therefore , we examined whether ANAC044 and ANAC085 also participate in controlling the expression of SMR5 and SMR7 .", "Our qRT-PCR showed that bleomycin treatment rapidly induced the expression of SMR5 and SMR7 in both WT and anac044 anac085 ( Figure 6—figure supplement 1 ) , suggesting that ANAC044 or ANAC085 is not required for the immediate induction of CDK inhibitor genes .", "These data are consistent with the above-mentioned result that suppression of G2/M-specific genes was impaired only at a later stage of the DDR in anac044 anac085 ( Figure 6A ) .", "The repressor-type MYB3R transcription factors MYB3R3 and MYB3R5 function in repressing G2/M-specific genes ( Kobayashi et al . , 2015 ) .", "In the absence of DNA damage , they are actively degraded via the ubiquitin-proteasome pathway , while under DNA damage conditions , they accumulate to high levels and cause G2 arrest by inhibiting G2/M-specific gene expression ( Chen et al . , 2017 ) .", "To examine whether ANAC044 and ANAC085 are associated with the protein stability of repressor-type MYB3Rs , we observed the accumulation in roots of the MYB3R3-GFP fusion protein , which was expressed under the 1 . 3 kb MYB3R3 promoter .", "As reported previously , the GFP fluorescence was increased by bleomycin treatment in the root tip ( Chen et al . , 2017 ) , whereas no increase was observed in sog1-101 or anac044-1 anac085-1 ( Figure 6B and C ) .", "Immunoblot analysis using total protein from root tips also showed bleomycin-induced accumulation of MYB3R3-GFP in WT , but not in anac044-1 anac085-1 ( Figure 6D ) .", "Note that the MYB3R3 transcript level was unaltered by mutations in SOG1 , ANAC044 or ANAC085 ( Figure 6—figure supplement 1 ) .", "These results suggest that ANAC044 and ANAC085 as well as SOG1 are involved in DNA damage-induced accumulation of the repressor-type MYB3Rs .", "We have reported that expression of the activator-type MYB3R4 decreases in response to DNA damage at the mRNA level , and that this down-regulation requires SOG1 ( Chen et al . , 2017 ) .", "Our qRT-PCR showed that MYB3R4 transcripts were reduced in anac044 anac085 in a similar manner to that in WT ( Figure 6—figure supplement 1 ) , suggesting that ANAC044 and ANAC085 function in controlling the level of repressor-type , but not of activator-type , MYB3Rs .", "Our measurement of root growth showed that anac044 anac085 was as tolerant to bleomycin treatment as myb3r3-1 , a knockout mutant of MYB3R3 ( Chen et al . , 2017 ) ( Figure 6—figure supplement 2 ) .", "Moreover , the anac044 anac085 myb3r3-1 triple mutant also exhibited the same sensitivity to bleomycin ( Figure 6—figure supplement 2 ) , indicating that ANAC044/085 and MYB3R3 function in the same pathway .", "Taken together , our results suggest that ANAC044 and ANAC085 , which are induced by DNA damage-activated SOG1 , enhance protein accumulation of repressor-type MYB3Rs , thereby suppressing G2/M-specific genes and causing G2 arrest .", "To further examine the role of the ANAC044/085-mediated pathway in cell cycle arrest , we generated transgenic plants overexpressing ANAC044 fused to the glucocorticoid receptor , which is activated by dexamethasone ( Dex ) .", "We used two lines , of which #1 showed a higher expression of ANAC044-GR than #2 , for further analyses ( Figure 7A ) .", "Five-day-old seedlings grown on MS medium were transferred onto medium supplemented with or without 10 µM Dex and/or 0 . 6 µg/ml bleomycin , and root length was measured for 5 days .", "In the absence of bleomycin , the overexpression lines displayed similar root growth to WT irrespective of Dex treatment ( Figure 7B ) , suggesting that ANAC044 overexpression by itself does not inhibit cell division .", "However , in the presence of bleomycin , root growth was further retarded in the overexpression lines; #1 showed much slower root growth than #2 , which has lower ANAC044-GR expression ( Figure 7A and B; Figure 7—figure supplement 1 ) .", "These results clearly suggest that ANAC044 overexpression inhibits root growth under DNA damage conditions .", "Next , we conducted EdU incorporation experiments .", "In the absence of bleomycin , the number of EdU-positive cells with mitotic figures increased in both WT and the overexpression line #1 ( Figure 7C ) .", "However , in the presence of bleomycin , the increase of EdU-positive cells with mitotic figures was inhibited in WT , and more dramatically so in #1 ( Figure 7C ) .", "The expression of G2/M-specific genes was also severely suppressed in #1 compared to WT after bleomycin treatment ( Figure 7D ) .", "These results suggest that DSB-induced inhibition of G2/M progression is further enhanced by ANAC044 overexpression .", "The above results indicate that the ANAC044/085-mediated pathway plays a major role in DNA stress-induced cell cycle arrest .", "To examine whether the same signalling module also functions in other stress responses , we investigated the involvement of ANAC044 and ANAC085 in the response to heat and osmotic stresses , which are known to retard G2 and G1 phase progression , respectively , in maize roots ( Zhao et al . , 2014 ) .", "When transgenic Arabidopsis seedlings harbouring the cell cycle marker system Cytrap ( Yin et al . , 2014 ) were grown at higher temperature ( 37°C ) for 24 hr , root cells expressing the late G2/M marker CYCB1;1 increased significantly , while those expressing the S/G2 marker CDT1a were dramatically reduced ( Figure 8A and B ) .", "On the other hand , when the Cytrap line was treated with 400 mM mannitol for 24 hr , S/G2 phase cells decreased by half , and G1 phase cells , which are represented by the lack of expression of both CYCB1;1 and CDT1a markers , significantly increased ( Figure 8—figure supplement 1A and B ) .", "These results are consistent with the previous observation in maize ( Zhao et al . , 2014 ) .", "We found that the anac044 anac085 mutant exhibited the same sensitivity as WT to mannitol ( Figure 8—figure supplement 1C ) , and that the promoter activity of ANAC044 was not elevated by mannitol treatment ( Figure 8—figure supplement 1D ) .", "This suggests that ANAC044 and ANAC085 are not associated with the osmotic stress-induced inhibition of G1 progression; therefore , we focus on heat stress hereafter .", "anac044 anac085 seedlings grown on MS medium at 22°C for 5 days were incubated at 37°C for 24 hr , and root length was measured after transfer to 22°C .", "As shown in Figure 8C , root growth inhibition by heat stress was less evident in anac044 anac085 than in WT , indicating heat stress tolerance ( Figure 8—figure supplement 2 ) .", "A tolerance to heat stress was also observed in a reduction of cell number in the root meristem ( Figure 8D ) .", "sog1-101 exhibited the same sensitivity as WT ( Figure 8C; Figure 8—figure supplement 2 ) , suggesting that ANAC044 and ANAC085 inhibit root growth independently of SOG1 .", "We next examined the transcriptional response of ANAC044 and ANAC085 to heat treatment .", "qRT-PCR showed that the transcript levels of ANAC044 and ANAC085 were highly elevated by heat stress in WT , and to the same extent in sog1-101 ( Figure 8E ) .", "A similar result was also obtained using the ProANAC044:GUS reporter line ( Figure 8F ) .", "These results suggest that ANAC044 and ANAC085 perceive heat stress signals independently of the SOG1-mediated pathway , and that their induction leads to G2 arrest .", "The next question is whether ANAC044 and ANAC085 inhibit G2 phase progression by controlling protein accumulation of repressor-type MYB3Rs , as in the case of DNA stress .", "In the ProMYB3R3:MYB3R3-GFP reporter line , the GFP fluorescence was increased by incubation of seedlings at 37°C for 24 hr , but no such induction was observed in anac044 anac085 ( Figure 9A and B ) .", "This result was supported by immunoblotting ( Figure 9C ) .", "The MYB3R3 transcript level was elevated by heat treatment in both WT and anac044 anac085 ( Figure 9—figure supplement 1 ) , indicating that transcriptional induction does not affect protein abundance; rather , protein-level regulation is crucial in controlling MYB3R3 accumulation .", "Measurement of root growth and counting the meristematic cell number showed that myb3r3-1 and the anac044 anac085 myb3r3-1 triple mutant exhibited the same tolerance to heat stress as anac044 anac085 ( Figure 8D; Figure 9D; Figure 9—figure supplement 2 ) .", "These results suggest that ANAC044 and ANAC085 are required for MYB3R3 accumulation in response to heat stress , and that the ANAC044/085–MYB3R3/5 signalling pathway is a central module controlling G2 arrest in stress responses ." ], [ "In this study , we revealed that DNA damage-activated SOG1 causes cell cycle arrest through two other NAC-type transcription factors , ANAC044 and ANAC085 , which promote the accumulation of Rep-MYB proteins and suppress G2/M-specific genes , thereby inducing G2 arrest .", "We also tested heat and osmotic stresses that retard cell cycle progression in G2 and G1 phases , respectively .", "Although the anac044 anac085 mutant was as sensitive as WT to osmotic stress , it was tolerant to heat stress and defective in heat-induced accumulation of Rep-MYB .", "These data suggest that ANAC044 and ANAC085 play a crucial role in controlling cell cycle arrest at G2 , but not at the other phases , in response to stresses ( Figure 10 ) .", "Deployment of the ANAC044/085-mediated signalling module indicates that although eukaryotic cells commit to enter a new cell cycle during G1 ( Bertoli et al . , 2013 ) , plants have an additional critical point at G2 for deciding whether or not cells resume proliferation after escaping from stress .", "Indeed , DNA damage enhances the onset of endoreplication by inhibiting G2/M progression , thereby prohibiting resumption of the mitotic cell division ( Adachi et al . , 2011 ) .", "The deployment of such a module controlling G2 progression may be specific to plants , in which the induction of endoreplication is a sensible strategy to avoid cell death and enable continuous growth under stressful conditions ( Adachi et al . , 2011; De Veylder et al . , 2011; Radziejwoski et al . , 2011 ) .", "Since ANAC044 and ANAC085 are also involved in DNA damage-induced stem cell death ( Figure 3A ) , G2 arrest may be prerequisite for choosing between life and death of mitotic cells as well .", "Further studies will reveal how the ANAC044/085-mediated pathway is associated with the onset of endoreplication and cell death in response to stresses .", "Bourbousse et al . ( 2018 ) conducted transcriptomic analyses over a 24 hr time course after gamma irradiation and revealed that Rep-MYBs play a major role in repressing cell cycle-related genes in response to DSBs .", "We previously reported that Rep-MYBs are actively degraded through the ubiquitin-proteasome pathway , and that this degradation is triggered by CDK phosphorylation ( Chen et al . , 2017 ) .", "Under normal growth conditions , higher CDK activity enhances Rep-MYB degradation and maintains the expression of G2/M-specific genes; under DNA damage conditions , CDK activity is reduced by the induction and repression , respectively , of genes for CDK inhibitors and Act-MYB , resulting in stabilization of Rep-MYBs and suppression of G2/M-specific genes ( Chen et al . , 2017 ) .", "In the anac044 anac085 mutant , DNA damage up- and down-regulates the expression of CDK inhibitors ( SMR5 and SMR7 ) and MYB3R4 , respectively , to the same extent as in WT ( Figure 6—figure supplement 1 ) , suggesting that the initial reduction of CDK activities occurs normally .", "This idea is supported by the fact that transcript levels of G2/M-specific genes , which are induced by MYB3R4 ( Haga et al . , 2011 ) , decreased by 12 hr after bleomycin treatment as observed in WT ( Figure 6A ) .", "However , in anac044 anac085 , further reduction in G2/M-specific gene expression after 12 hr was suppressed ( Figure 6A ) , indicating that ANAC044 and ANAC085 stabilize Rep-MYBs at a later stage of the DDR by controlling factors other than CDK activity .", "One possibility is that ANAC044 and ANAC085 inhibit the expression , accumulation or activity of the degradation machinery for Rep-MYBs .", "Chen et al . ( 2017 ) and Kobayashi et al . ( 2015 ) reported that under normal growth conditions , MYB3R3 protein preferentially accumulates around early S phase , and that Rep-MYBs repress transcription of target genes along the cell cycle except for G2/M .", "Therefore , it is likely that ubiquitin E3 ligases such as SCF ( Skp-Cullin1-F-box ) mark Rep-MYBs by polyubiquitination for proteolysis during the cell cycle .", "A previous study showed that transcript levels of several F-box genes were reduced by gamma-irradiation ( Culligan et al . , 2006 ) ; therefore , such factors related to ubiquitin-mediated proteolysis may be under the control of ANAC044 and ANAC085 , thereby regulating the protein stability of Rep-MYBs under DNA damage conditions .", "We revealed that ANAC044 and ANAC085 are not required for induction of DNA repair-related genes , and that the frequency of HR was dramatically reduced in sog1-101 , but not in anac044 anac085 ( Figure 4 ) .", "This indicates that ANAC044 and ANAC085 are engaged in inhibiting cell division , whereas SOG1 is involved in DNA repair as well as cell cycle regulation ( Ogita et al . , 2018 ) .", "Johnson et al . ( 2018 ) reported that when the sog1 mutant is exposed to an acute dose of ionizing radiation , SOG1-independent cell death occurs in the root meristem , and root organization is severely impaired .", "We also observed a similar phenotype by transient treatment with bleomycin .", "It is noteworthy that unlike sog1 , anac044 anac085 displayed proper arrangement of root cells after recovery from bleomycin treatment ( Figure 3C ) .", "Therefore , although stem cell death is suppressed in both sog1 and anac044 anac085 , stem cells in sog1 , but not anac044 anac085 , may seriously suffer from DNA damage , thus failing to undergo mitotic cell division and to maintain root organization .", "It is likely that defects in activation of DNA repair make stem cells hypersensitive to DNA damage in the sog1 mutant .", "On the other hand , the phenotype of anac044 anac085 suggests that SOG1-mediated activation of DNA repair is sufficient to preserve stem cells , whereas stem cell death and regeneration of new stem cells are probably also important to preserve genome integrity in stem cells .", "In Arabidopsis , SOG1 is phosphorylated by ATM and ATR; ATM phosphorylates five serine-glutamine ( SQ ) motifs in the C-terminal region of SOG1 upon exposure to DSBs ( Yoshiyama et al . , 2013; Sjogren et al . , 2015; Yoshiyama et al . , 2017 ) .", "Such phosphorylation is essential for SOG1’s ability to bind to target promoters ( Ogita et al . , 2018 ) .", "In contrast , ANAC044 and ANAC085 lack SQ motifs , raising the possibility that they do not require phosphorylation of the SQ motif for their transcriptional function .", "Their involvement in cell cycle arrest in response to heat stress also indicates that ATM/ATR-mediated phosphorylation is not necessary for activation .", "Nonetheless , we found that ANAC044 overexpression by itself could not induce cell cycle arrest , and that DNA damage signals are required for ANAC044 function ( Figure 7 ) .", "This suggests that ANAC044/085 are phosphorylated by other kinase ( s ) or subjected to other modifications that are responsive to stresses .", "The DNA damage sensitivity of the anac044 anac085 double mutant was indistinguishable from that of the single mutants , implying that ANAC044 and ANAC085 work together in DDRs .", "Several NAC transcription factors are known to form homo- or heterodimers ( Mitsuda et al . , 2004; Yamaguchi et al . , 2008; Gladman et al . , 2016 ) , suggesting the possibility that ANAC044 and ANAC085 form dimers to act on target genes .", "This hypothesis is supported by our obsevation that ANAC044 overexpression further retarded G2 progression only in the presence of bleomycin , which induces ANAC085 .", "On the other hand , it is unlikely that ANAC044 and ANAC085 form heterodimers with SOG1 to control the SOG1 target genes , because transcript levels of SOG1 target genes were elevated by bleomycin treatment in anac044 anac085 , as they were in WT ( Figure 10—figure supplement 1 ) .", "However , it remains possible that ANAC044/085 interact with other NAC transcription factors including SOG1 , which are induced and/or activated by DNA damage , and control the expression of their target genes .", "Here we identified a core signalling module that controls G2 arrest in response to stresses .", "The promoter region of ANAC085 , but not ANAC044 , contains several consensus motifs of the heat shock element , which is recognized by heat shock transcription factors ( Guo et al . , 2008 ) .", "Moreover , our preliminary results show that ANAC044 is also up-regulated by cold and salt stresses ( Figure 10—figure supplement 2 ) .", "Therefore , we propose that ANAC044 and ANAC085 are induced by distinct stress factors , thereby functioning as a hub in transmitting environmental signals to the cell cycle machinery .", "Kobayashi et al . ( 2015 ) reported that Arabidopsis Rep-MYBs form a protein complex with retinoblastoma-related protein and the E2F transcription factor E2FC , thereby existing as a component of the DREAM/dREAM-like complex , which has been characterized in vertebrates and Drosophila ( Georlette et al . , 2007; Sadasivam and DeCaprio , 2013 ) .", "It is therefore likely that the ANAC044/ANAC085-mediated pathway also controls other subunits of the DREAM complex , thereby differentially controlling downstream genes in response to various stresses .", "Further studies will reveal whether vertebrate DREAM complexes are also involved in the transition from the mitotic to the quiescent state upon exposure to stresses , and how their activity is regulated to cope with distinct stresses ." ], [ "Arabidopsis thaliana ( ecotype Col-0 ) was used in this study .", "sog1-101 ( Ogita et al . , 2018 ) , ProSOG1:SOG1-Myc ( Yoshiyama et al . , 2013 ) , myb3r3-1 ( Chen et al . , 2017 ) , ProMYB3R3:MYB3R3-GFP ( Chen et al . , 2017 ) and the cell cycle marker system Cytrap ( Yin et al . , 2014 ) were described previously .", "anac044-1 ( SAIL_1286D02 ) , anac044-2 ( GABI_968B05 ) , anac085-1 ( GABI_894G04 ) and anac085-2 ( SALK_208662 ) were obtained from the Arabidopsis Biological Resource Center .", "To generate ProANAC044:GUS and ProANAC085:GUS , the 1 . 5 kb promoters of ANAC044 and ANAC085 were PCR-amplified from genomic DNA and cloned into the Gateway entry vector pDONR221 by BP reaction according to the manufacturer’s instructions ( Thermo Fisher Scientific ) .", "Primer sequences used for PCR are listed in Supplementary file 1 .", "An LR reaction was performed with the destination vector pGWB3 ( Nakagawa et al . , 2007 ) to generate a binary vector carrying the fusion construct with GUS .", "The construct was transferred into the Agrobacterium tumefaciens GV3101 strain harbouring the plasmid pMP90 ( Koncz and Schell , 1986 ) , and the obtained strain was used to generate stably transformed Arabidopsis by the floral dip transformation method ( Clough and Bent , 1998 ) .", "Plants were grown in Murashige and Skoog ( MS ) medium under continuous light conditions .", "For root growth analysis , five-day-old seedlings grown on MS plates were transferred to MS medium containing mannitol or the following DNA-damaging agents: bleomycin ( Wako ) , MMS ( Wako ) , MMC ( Nacalai Tesque ) or HU ( Nacalai Tesque ) .", "Seedlings were grown vertically , and the position of root tips was marked every 24 hr .", "Root growth was measured using ImageJ software by calculating the distance between successive marks along the root axis .", "Total RNA was extracted from Arabidopsis roots with a Plant Total RNA Mini Kit ( Favorgen ) .", "First-strand cDNAs were prepared from total RNA using ReverTra Ace ( Toyobo ) according to the manufacturer’s instructions .", "Quantitative RT-PCR was performed with a THUNDERBIRD SYBR qPCR Mix ( Toyobo ) with 100 nM primers and 0 . 1 µg of first-strand cDNA .", "PCR reactions were conducted with the Light Cycler 480 Real-Time PCR System ( Roche ) under the following conditions: 95°C for 5 min; 60 cycles at 95°C for 10 s , 60°C for 20 s and 72°C for 30 s .", "Primer sequences are listed in Supplementary file 1 .", "Seedlings were incubated in GUS staining solution [100 mM sodium phosphate , 1 mg/ml 5-bromo-4-chloro-3-indolyl ß-D-glucuronide , 0 . 5 mM ferricyanide and 0 . 5 mM ferrocyanide ( pH 7 . 4 ) ] at 37°C in the dark .", "The samples were cleared with a transparent solution [chloral hydrate , glycerol and water ( 8 g: 1 ml: 1 ml ) ] and observed under a light microscope , Axioskop 2 Plus ( Zeiss ) or SZX16 ( Olympus ) .", "ChIP was performed as described previously ( Gendrel et al . , 2005 ) .", "pSOG1:SOG1-MYC seeds were germinated in 100 ml of liquid MS medium , and cultured under continuous light at 23°C with gentle shaking ( 50 rpm ) .", "After a 2 week culture period , bleomycin was added to the medium to a final concentration of 0 . 6 µg/ml , and the seedlings were cultured for 12 hr .", "Chromatin bound to the SOG1-Myc fusion protein was precipitated with anti-Myc antibody ( Millipore ) .", "ChIP-qPCR was performed using immunoprecipitated DNA .", "Three independent ChIP experiments were conducted .", "To quantify the precipitated chromatin , gene-specific primers listed in Supplementary file 1 were used for real-time qPCR .", "PCR reactions were conducted with the Light Cycler 480 Real-Time PCR System ( Roche ) under the following conditions: 95°C for 5 min; 60 cycles at 95°C for 10 s , 60°C for 20 s and 72°C for 30 s .", "Microarray analysis was carried out as described previously ( Ogita et al . , 2018 ) .", "Five-day-old seedlings grown on MS plates were transferred onto MS medium containing 0 . 6 µm/ml bleomycin and grown for 10 hr .", "Total RNA was extracted from root tips with a Plant Total RNA Mini Kit ( Favorgen ) .", "Cyanine-3 ( Cy3 ) -labelled cDNA obtained from total RNA was hybridized to an Agilent-034592 Arabidopsis Custom Microarray .", "Slide scanning was performed using the Agilent DNA Microarray Scanner ( G2539A ver . C ) .", "The GUS direct-repeat recombination reporter line #1406 ( Gherbi et al . , 2001 ) was crossed with anac044-1 anac085-1 and sog1-101 .", "Plants homozygous for the reporter and the alleles of anac044-1 anac085-1 or sog1-101 were grown for two weeks , and irradiated with gamma rays ( 50 Gy ) .", "After a three-day cultivation , GUS staining was performed , and the number of blue spots was counted on leaves .", "Five-day-old seedlings were grown on MS plates supplemented with or without 0 . 6 µg/ml bleomycin for 12 hr .", "Seedlings were then transferred to liquid MS medium with or without bleomycin and with 20 µM EdU , followed by a 15 min incubation .", "After washing with MS medium , seedlings were transferred again to MS medium supplemented with or without bleomycin .", "EdU staining was conducted with a Click-iT Plus EdU Alexa Fluor 488 Imaging Kit ( Thermo Fisher Scientific ) according to the manufacturer’s instructions , and nuclei were stained with DAPI .", "Total protein extracted from root tips was separated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and blotted onto a polyvinylidene difluoride membrane ( Millipore ) .", "Immunoblotting was conducted with anti-GFP antibody ( A-6455 , Thermo Fisher Scientific ) or anti-histone H3 antibody ( ab1791 , Abcam ) at a dilution of 1:3000 or 1:2000 , respectively .", "Clarity Western ECL Substrate ( Bio-Rad Laboratories ) was used for detection ." ] ]

[ "Cell cycle arrest is an active response to stresses that enables organisms to survive under fluctuating environmental conditions .", "While signalling pathways that inhibit cell cycle progression have been elucidated , the putative core module orchestrating cell cycle arrest in response to various stresses is still elusive .", "Here we report that in Arabidopsis , the NAC-type transcription factors ANAC044 and ANAC085 are required for DNA damage-induced G2 arrest .", "Under genotoxic stress conditions , ANAC044 and ANAC085 enhance protein accumulation of the R1R2R3-type Myb transcription factor ( Rep-MYB ) , which represses G2/M-specific genes .", "ANAC044/ANAC085-dependent accumulation of Rep-MYB and cell cycle arrest are also observed in the response to heat stress that causes G2 arrest , but not to osmotic stress that retards G1 progression .", "These results suggest that plants deploy the ANAC044/ANAC085-mediated signalling module as a hub which perceives distinct stress signals and leads to G2 arrest ." ]

[ "During environmental stresses , such as high light or a drought , plants do not have the opportunity to up and leave .", "Instead , they need to buy time and energy by pausing their growth , which means stopping their cells from dividing .", "In this case , the cell cycle , a series of stages during which a cell prepares itself for division , must be halted .", "If the genetic information in cells is damaged , often under the influence of the environment , plants stop their cell cycle in the step just before division .", "However , it is still unclear how this process takes place , and which proteins participate in it .", "Researchers also do not know whether environmental stresses can directly trigger this mechanism .", "To investigate , Takahashi et al . conducted a series of genetic experiments on a common plant known as Arabidopsis thaliana , and they identified two proteins , ANAC044 and ANAC085 , which could stop the cell cycle when the genetic information is damaged .", "In particular , ANAC044 and ANAC085 worked by stabilizing other proteins that turn off certain genes that the cell needed to divide .", "Additional experiments showed that other types of stresses , such as heat , halted the cell cycle using the ANAC044 and ANAC085 pathway .", "This suggests that this mechanism may be a central ‘hub’ that responds to various stress signals from the environment to prevent cells from dividing .", "In the field , environmental stresses stop plants from growing , which reduces crop yields; ultimately , manipulating ANAC044 or ANAC085 might help to boost plant productivity even when external conditions fluctuate ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "biochemistry and chemical biology", "cell biology" ]

[ [ "Cytokinesis is the final stage of cell division when two daughter cells are physically separated .", "The process comprises several steps , including cleavage furrow ingression and abscission of the midbody—the thin intercellular bridge connecting the nascent daughter cells ( Caballe and Martin-Serrano , 2011; Mierzwa and Gerlich , 2014 ) .", "Midbody abscission occurs adjacent to the Flemming body , a central protein complex , and is mediated by the ESCRT pathway ( endosomal sorting complexes required for transport ) ( Carlton and Martin-Serrano , 2007; Morita et al . , 2007 ) .", "The ESCRT machinery performs topologically equivalent membrane fission events throughout the cell , including intraluminal vesicle formation at the multivesicular body , retroviral particle release , plasma membrane wound repair and abscission ( Hurley and Yang , 2008; Henne et al . , 2011; McCullough et al . , 2013; Jimenez et al . , 2014 ) .", "The core ESCRT machinery comprises five different factors or complexes: ALIX , ESCRT-I , ESCRT-II and ESCRT–III , and VPS4 .", "In most cases , location-specific adaptors initially recruit the early-acting ALIX and ESCRT-I/-II factors , which then recruit the late-acting ESCRT-III and VPS4 complexes to mediate membrane fission .", "In late stages of cytokinesis , the Flemming body protein CEP55 binds TSG101 ( ESCRT-I ) and ALIX , which leads to ESCRT-III subunit recruitment ( Carlton and Martin-Serrano , 2007; Morita et al . , 2007; Carlton et al . , 2008 ) .", "ESCRT-III-based helical filaments form on either side of the Flemming body and induce cortical constriction , thereby promoting abscission ( Elia et al . , 2011; Guizetti et al . , 2011 ) .", "In addition to driving membrane scission mechanically , ESCRT-III proteins recruit cytokinetic cofactors that contain MIT ( microtubule interacting and trafficking ) domains .", "MIT domains bind short motifs within the C-terminal tails of ESCRT-III proteins called MIMs ( MIT-interacting motifs ) , which become exposed and concentrated when the ESCRT-III proteins polymerize ( McCullough et al . , 2013 ) .", "MIT domain-containing proteins that participate in cytokinesis include the AAA-ATPase VPS4 and its activator LIP5 , which remodel and recycle the ESCRT-III subunits ( Babst et al . , 1998; Hurley and Yang , 2008; Lata et al . , 2008 ) , Spastin , an AAA-ATPase that severs microtubules , and MITD1 , a membrane-binding protein that stabilizes the intercellular bridge ( Yang et al . , 2008; Connell et al . , 2009; Skalicky et al . , 2012; Hadders et al . , 2012; Azmi et al . , 2006 ) .", "Evolutionarily conserved monitoring mechanisms regulate the proper timing of events during cytokinesis progression ( Agromayor and Martin-Serrano , 2013; Mierzwa and Gerlich , 2014 ) .", "When chromosomes persist within the midbody , the abscission checkpoint ( also known as NoCut ) inhibits abscission until chromatin has been cleared from the spindle midzone , thereby preventing aberrant segregation or cleavage furrow regression and tetraploidy ( Norden et al . , 2006; Mendoza et al . , 2009; Steigemann et al . , 2009 ) .", "Lagging chromosomes are sensed by the Aurora B kinase , which phosphorylates the ESCRT-III subunit CHMP4C to delay abscission ( Capalbo et al . , 2012; Carlton et al . , 2012 ) .", "Defective nuclear pore complex assembly also triggers Aurora B-dependent abscission delays ( Mackay et al . , 2010 ) .", "The full signaling cascade that connects nuclear pores to abscission is not yet clear , but ESCRT proteins themselves are involved in the surveillance and clearance of defective nuclear pore complex assembly intermediates in S . cerevisiae ( Webster et al . , 2014 ) .", "Tension forces applied by dividing cells on the midbody also regulate cytokinesis , with high-tension delaying abscission , and tension release triggering ESCRT-III assembly and membrane scission ( Lafaurie-Janvore et al . , 2013 ) .", "How these different physiological inputs converge to influence abscission timing is not understood .", "Here , we investigate the function of Unc-51-like kinase 3 ( ULK3 ) , a poorly characterized member of the ULK family of serine/threonine kinases that is predicted to contain tandem MIT domains ( Row et al . , 2007 ) .", "Live-cell imaging analysis revealed that ULK3 regulates abscission timing in response to lagging chromosomes , defects in nuclear pore complex assembly , and tension forces at the midbody .", "Furthermore , our biochemical and structural studies show that the ULK3 MIT domains bind tightly to IST1 , an ESCRT-III subunit required for cytokinesis ( Agromayor et al . , 2009; Bajorek et al . , 2009a ) .", "Finally , we show that ULK3 phosphorylates IST1 and other ESCRT-III proteins and that IST1 phosphorylation provides an essential inhibitory signal in the abscission checkpoint , thereby ensuring proper coordination of the final events in cell division ." ], [ "The predicted MIT domains in ULK3 suggested a novel mechanism of ESCRT regulation , and we , therefore , surveyed potential ULK3–ESCRT interactions using yeast two-hybrid ( Y2H ) experiments .", "ULK3 binding was observed for three ESCRT-III subunits: CHMP1A , CHMP1B , and CHMP2A , but not for other ESCRT complexes ( Figure 1—figure supplement 1A ) .", "These interactions were confirmed by co-immunoprecipitation of Myc-tagged ESCRT-III proteins from mixed 293T cell lysates that contained One-strep-flag ( OSF ) -tagged ULK3 ( Figure 1A , note interactions in lanes 2 , 4 , 6 , and 14 ) .", "This approach revealed that ULK3 also bound the ESCRT-III subunit IST1 ( lane 26 ) , an interaction not tested by Y2H because IST1 fusion constructs activated transcription non-specifically .", "Endogenous ULK3 also co-precipitated with overexpressed HA-tagged CHMP1A , CHMP1B , CHMP2A , or IST1 , but not with CHMP2B ( Figure 1B ) .", "Finally , endogenous IST1 was efficiently biotinylated in cells that expressed a biotin ligase BirA-ULK3 fusion protein , which promiscuously biotinylates proximal proteins ( Figure 1C , lane 4 , bottom panel ) ( Roux et al . , 2012 ) .", "Hence , ULK3 can interact with a specific subset of ESCRT-III proteins in cells . 10 . 7554/eLife . 06547 . 003Figure 1 . ULK3 binds ESCRT-III via tandem MIT domains .", "( A ) Lysates from 293T cells overexpressing Myc-endosomal sorting complexes required for transport ( ESCRT ) -III proteins were mixed with lysates from cells non-transfected ( − ) or overexpressing One-strep-flag ( OSF ) -Unc-51-like kinase 3 ( ULK3 ) ( + ) .", "OSF-ULK3 proteins were bound to streptactin resin and bound ESCRT-III proteins were detected with α-Myc antibody ( top ) .", "( B ) Lysates from 293T cells expressing HA-ESCRT-III were immunoprecipitated with α-HA antibody and co-precipitated endogenous ULK3 protein was detected by Western blot with α-ULK3 antibody .", "( C ) HeLa cells expressing ULK3 fused to the biotin protein ligase BirA-113G or unfused BirA were treated overnight with biotin .", "Vicinal biotinylated proteins were isolated with streptavidin-coated beads , and endogenous IST1 was found to be biotinylated , implying that it was in close proximity with ULK3 .", "‘Asterisks’ denote isolated BirA-Empty and BirA-ULK3 , respectively , on α-Avidin blot ( lanes 3 and 4 ) .", "Images shown for both lysate and pull-down samples were cropped from the same blot in all cases .", "( D ) Co-immunoprecipitation of Myc-IST1 with different ULK3 constructs .", "‘Asterisks’ denote phosphorylated IST1 species .", "( E ) Top: overlaid 15N-HSQC NMR spectra of 15N , 13C-IST1 ( residues 303–366 ) alone ( black ) or with 2 equivalents of ULK3 ( MIT ) 2 ( residues 277–449 ) ( red ) .", "Inset shows an expanded view of the boxed region in the spectrum .", "Bottom: individual IST1 backbone amide resonances perturbed ( 1 ) or not ( −1 ) upon addition of ULK3 ( MIT ) 2 .", "Ambiguous changes ( due to spectral overlap ) were scored as 0 .", "( F ) Fluorescence polarization ( FP ) -binding isotherms show interactions between different ULK3 MIT constructs corresponding to MIT2 ( residues 359–449 ) or both MIT domains ( ULK3 ( MIT ) 2 ) and fluorescently labeled IST1 peptides for MIT-interacting motifs ( MIM ) 1 , MIM2 , or both MIM1+MIM2 ( MIMs ) .", "Data points are averages ±SD of at least three separate measurements , and curves show fits to simple 1:1 binding models with dissociation constants .", "( G ) Crystal structure of the ULK3 MIT2-IST1 MIM1 complex .", "Inset shows key interacting side-chains ( in green ) with ULK3 M434 highlighted in red .", "Sequence alignment compares IST1 MIM1 to the consensus MIM1 sequence .", "See also Figure 1—figure supplement 1 and Figure 1—source data 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 06547 . 00310 . 7554/eLife . 06547 . 004Figure 1—source data 1 . Data Collection and Refinement Statistics for the ULK3 MIT2:IST1 MIM1 Complex . DOI: http://dx . doi . org/10 . 7554/eLife . 06547 . 00410 . 7554/eLife . 06547 . 005Figure 1—figure supplement 1 . ULK3 binds to ESCRT-III via tandem MIT domains .", "( A ) Y2H assays in which ULK3 fused to the VP16 activation domain was tested for interactions with the designated human components of ESCRT-I , ESCRT-II , ESCRT-III , and ESCRT-associated proteins fused to Gal4 DNA-binding domain .", "( B ) Overlay of the ULK3 MIT2 ( blue ) /IST1-MIM1 ( green ) structure with the structure of VPS4A ( red ) in complex with the MIM1 element of CHMP1A ( orange; PDB 2JQ9 ) .", "Structure-based sequence alignments of the IST1 and CHMP1A MIM1 sequences are shown below with residues within 4 Å of the MIT/MIM interface highlighted in red .", "( C ) Overlay of the ULK3 MIT2 ( blue ) structure with the structure of VPS4A ( red ) in complex with the MIM2 element of CHMP6 ( orange , PDB ID 2K3W ) .", "The overlay demonstrates that the N-terminal helix of ULK3 MIT2 would sterically preclude binding of a canonical MIM2 element .", "Structures in B and C were analyzed and generated using Chimera ( Pettersen et al . , 2004 ) .", "( D ) Co-precipitations from 293T cells co-transfected with HA-CHMP1A or HA-IST1 and GST-ULK3 WT , V338D , or M434D .", "An empty GST vector was used as a negative control .", "10% of the volume eluted from the beads was analyzed by Western blot with α-HA antibody to visualize bound proteins .", "Western blot with α-GST shows equivalent capture of GST fusion proteins , where empty GST bands ( lanes 1 and 5 ) were cropped from the same blot .", "( E ) Y2h assay in which CHMP1B WT and deletion and point mutants fused to Gal4 DNA-binding domains were tested for binding to ULK3 fused to the VP16 activation domain .", "Point mutant designations are as follows: M1 ( LSQ185-187/AAA , MIM1 element ) , M2 ( RLA188-190/AAA , MIM1 element ) , M3 ( RLR191-193/AAA , MIM1 element ) , M4 ( DQV194-196/AAA , MIM1 element ) , TAE/RRR ( T178A181E184/RRR , MIM3 element ) .", "Binding of all constructs to CHMP1A was used as a positive control .", "( F ) Y2H assay with ULK3 WT , V338D , or M434D fused to VP16 .", "Note that both mutations impair binding to CHMP1A and CHMP1B Gal4-fused proteins .", "( G ) In vitro kinase assay showing equivalent auto-phosphorylation activities for GST-ULK3 WT , GST-ULK3 V338D , and GST-ULK3 M434D .", "GST alone was used as a negative control; bands shown were cropped from the same blot .", "In ( A ) , ( E ) , and ( F ) data are represented as mean β-galactosidase activity ±SD from triplicate measurements of two separate experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 06547 . 005 Fluorescence polarization ( FP ) binding assays indicated that ULK3 interacted more tightly with IST1 ( KD ∼ 0 . 2 µM ) than with other ESCRT-III proteins ( KD ∼100 µM ) ( Figure 1F , and data not shown ) .", "We , therefore , focused on characterizing the IST1-ULK3 interaction .", "The IST1-binding site on ULK3 was mapped using co-precipitation experiments with Myc-tagged IST1 and different OSF-ULK3 deletion constructs .", "IST1 bound with similar efficiencies to full-length ULK3 ( residues 1–472 ) and to a minimal construct that spanned just the C-terminal tandem MIT domains ( residues 277–449 , hereafter denoted ULK3 ( MIT ) 2; Figure 1D , lanes 2 and 5 , respectively ) , but did not bind to the isolated ULK3 kinase domain ( residues 1–270; lane 3 ) .", "Thus , the ULK3 MIT domains form the primary IST1-binding site .", "As discussed below , co-expression of full-length ULK3 resulted in the appearance of lower mobility IST1 species , suggesting that the kinase can phosphorylate IST1 ( Figure 1D , lane 2 ) .", "NMR chemical shift perturbation experiments were used to map ULK3-binding sites on IST1 .", "IST1 contains two different MIT interaction motifs , termed MIM1 ( residues 352–363 ) and MIM2 ( residues 325–329 ) , that were included within a C-terminal IST1 peptide used in these experiments ( IST1303-366 ) ( Agromayor et al . , 2009; Bajorek et al . , 2009a ) .", "Both MIMs exhibited backbone amide proton chemical shift perturbations upon titration of 15N-labeled IST1303-366 with ULK3 ( MIT ) 2 , indicating that both IST1 MIMs can contact ULK3 ( MIT ) 2 ( Figure 1E ) , whereas the upstream IST1 residues 303–317 were not perturbed .", "To assess the binding contributions of each MIM and MIT element , we performed FP-binding experiments with fluorescently labeled peptides comprising either one or both of the IST1 MIM elements and one or both ULK3 MIT domains .", "The tightest interaction was observed for ULK3 ( MIT ) 2 binding to a construct that spanned both IST1 MIMs ( Figure 1F ) .", "However , nearly all of the binding energy was contributed by the IST1 MIM1 element , which bound ULK3 MIT2 .", "Consistent with these experiments , IST1316-366 binding to ULK3 ( MIT ) 2 was reduced more than 100-fold by a single point mutation in the MIM1-binding site of ULK3 MIT2 ( M434D ) , but not by an equivalent mutation in the MIM1-binding site of ULK3 MIT1 ( residues 277–358 , V338D mutant; <twofold , Figure 1F and Figure 1—figure supplement 1D ) .", "Hence , both IST1 MIM elements can contact both ULK3 MIT domains , but IST1 MIM1 binding to ULK3 MIT2 domain is the energetically dominant interaction .", "We determined the crystal structure of ULK3 MIT2 in complex with IST1 MIM1 to 1 . 4 Å resolution ( Figure 1G and Figure 1—source data 1 ) .", "ULK3 residues 372–446 form the characteristic MIT three-helix bundle , and the IST1 MIM1 helix binds parallel to MIT helix 3 in the groove between MIT helices 2 and 3 ( Figure 1G and Figure 1—figure supplement 1B ) .", "The N-terminus of the MIT domain ( residues 360–373 ) forms a short helix that packs in the groove connecting MIT helices 1 and 3 .", "This helix has not been seen in previous MIT domain structures and is expected to interfere sterically with the binding of canonical MIM2 ( and MIM3 ) elements ( Figure 1—figure supplement 1C ) .", "Core IST1 MIM1 residues make a series of interactions that are similar to those seen in other MIT-MIM1 complexes ( see Figure 1F magnification and Figure 1—figure supplement 1B ) ( Obita et al . , 2007; Stuchell-Brereton et al . , 2007; Hadders et al . , 2012; Guo and Xu , 2015 ) .", "The ULK3 M434 residue is central to this interface ( Figure 1G , red ) , which explains why the M434D mutation inhibits IST1 binding ( Figure 1F and Figure 1—figure supplement 1D ) .", "The interaction between ULK3 MIT2 and IST1 MIM1 ( KD = 0 . 77 µM ) is significantly tighter than many previously described MIT–MIM1 interactions ( KD ∼2–100 µM ) .", "The additional binding energy appears to be contributed by N-terminal IST1 MIM1 residues I350 and F352 , and by the central F359 residue , all of which make hydrophobic interactions that are absent in other MIM1-MIT complexes .", "Binding of the bulky hydrophobic IST1 MIM1 F352 and F359 residues is accommodated by complementary small ULK3 MIT2 interfacial residues A415 and G408 , respectively .", "Charged or polar residues are found in equivalent positions of other MIT domains such as VPS4A ( Figure 1—figure supplement 1B ) or ULK3 MIT1 .", "These interactions likely explain why IST1 MIM1 binds tightly and specifically to the ULK3 MIT2 domain .", "ULK3 binding also mapped to the MIM1 of CHMP1B as binding was inhibited by removal of CHMP1B MIM1 or by MIM1 point mutations ( Figure 1—figure supplement 1E , lanes 3–7 vs lane 2 ) , but not by a mutation in the more extended CHMP1B MIM3 binding site ( lane 8 ) ( Yang et al . , 2008 ) .", "However , unlike IST1 , the binding of CHMP1A and CHMP1B was reduced by point mutations in the MIM1-binding sites of either ULK3 MIT domain ( Figure 1—figure supplement 1D , lanes 1–4 and Figure 1—figure supplement 1F ) .", "Thus , the binding specificities of the CHMP1A/B and IST1 proteins differ , and our data indicate that CHMP1 MIM1 elements can bind either ULK3 MIT domain .", "We used siRNA depletion to test whether ULK3 participates in endosomal sorting or virus budding .", "Effects on ESCRT-dependent lysosomal targeting were tested using two established assays; down-regulation of MHC class I by the Kaposi sarcoma-associated herpesvirus ( KSHV ) K3 protein ( Hewitt et al . , 2002 ) , and degradation of Tetherin by the KSHV K5 protein ( Agromayor et al . , 2012 ) .", "ULK3 depletion did not affect either of these ESCRT-dependent degradation reactions , whereas control depletion of TSG101 inhibited both processes ( Figure 2—figure supplement 1A , B ) .", "Similarly , ULK3 depletion had no effect on ESCRT-dependent HIV-1 budding ( Figure 2—figure supplement 1C ) .", "We , therefore , conclude that ULK3 is not required for either of these ESCRT pathway functions .", "We next tested whether ULK3 functions in cytokinetic abscission using live imaging of HeLa mCherry-Tubulin cells .", "ULK3-depleted cells proceeded through mitosis normally , but resolved their midbodies faster than control cells , at rates comparable to the abnormally rapid abscission observed in CHMP4C-depleted cells ( Figure 2A and Figure 2—figure supplement 1D , Videos 1–3 ) ( Carlton et al . , 2012 ) .", "In addition , endogenous ULK3 protein was detected by immunofluorescence staining at the Flemming body , where it is positioned to function during cytokinetic abscission ( Figure 2B ) .", "Based on these results , we tested whether ULK3 was required for abscission delays induced by the abscission checkpoint .", "One event that triggers the abscission checkpoint is the presence of defectively assembled nuclear pore complexes ( Mackay et al . , 2010 ) .", "As expected , partial depletion of Nucleoporin 153 ( NUP153 ) induced abscission delays that increased the number of midbody-connected cells ( Figure 2C , lane 1 vs 2 ) .", "This phenotype was abrogated upon ULK3-depletion , despite comparable levels of NUP153 depletion ( lane 2 vs 4 ) .", "Active Aurora B ( Aurora B pT232 ) is known to localize to the midbody of dividing cells both in control or NUP153-depleted cells ( Mackay et al . , 2010 ) .", "Immunofluorescence imaging of cells treated as in Figure 2C revealed that ULK3 depletion did not alter the localization of phosphorylated Aurora B to the midbody in cells co-depleted of NUP153 , indicating that ULK3 likely functions downstream of Aurora B in the abscission checkpoint ( Figure 2—figure supplement 1E ) .", "These results were confirmed by live-cell imaging experiments , which demonstrated that ULK3 depletion abolished the 30-min abscission delay triggered by nuclear pore disruption ( Figure 2D ) .", "Furthermore , partial depletion of NUP153 in two different clonal ULK3-knockout cell lines , in which the endogenous ULK3 locus was removed using a lentiviral CRISPR-Cas9 system ( Ran et al . , 2013 ) , also failed to sustain the abscission checkpoint , as seen by a lack of an increase in midbody-connected cells when compared to non-targeting ( NT ) siRNA transfection , or control cell lines lacking guide RNAs against ULK3 ( Figure 2E , lanes 8 and 10 vs lanes 2 , 4 , 6 ) . 10 . 7554/eLife . 06547 . 006Figure 2 . ULK3 regulates abscission timing .", "( A ) Asynchronous cultures of HeLa mCherry-Tubulin cells were transfected with the specified siRNA .", "Midbody resolution times were calculated from three separate experiments ( mean times ±SD were non-targeting ( NT ) : 96 ± 36 min; CHMP4C: 74 ± 26 min; ULK3: 74 ± 31 min ) .", "( B ) HeLa cells were stained with Hoechst , α-Tubulin , and α-ULK3 antibody ( Santa Cruz ) .", "ULK3 was observed at the Flemming body in 79% of observed midbodies ( total n = 34 ) .", "Image shows a representative example of Flemming body localization , with the ULK3 signal in green , Tubulin in red , and nuclei in blue .", "Bar = 5 μm .", "Inset shows an expanded view of the midbody .", "( C and D )", "HeLa mCherry-Tubulin cells transfected with NT or ULK3 siRNA were transfected with Nucleoporin 153 ( NUP153 ) siRNA to trigger the abscission checkpoint .", "In ( C ) , cells were fixed and stained with α-Tubulin antibody to visualize midbody-arrested cells .", "Data are represented as a mean percentage of midbodies ±SD from six separate experiments .", "NUP153 depletion levels as mean percentages from three independent experiments were lane 1: 100% , 2: 67% , 3: 108% , 4: 70% .", "In ( D ) , midbody abscission times were analyzed ( mean times are 1: 86 ± 22 min; 2: 118 ± 50 min; 3: 70 ± 15 min; 4: 81 ± 25 min ) .", "( E ) Clonal HeLa cells stably expressing a lentiviral vector containing CRISPR-Cas9 with a guide RNA sequence targeting the ULK3 locus ( δULK3-1 or δULK3-2 ) or without guide RNA ( Controls ) were transfected with NT or NUP153 siRNA as above and stained with α-Tubulin antibody to visualize midbody-arrested cells .", "Data are represented as the mean percentage increase in midbody-connected cells compared to the percentage of midbodies in NT-treated cells for each cell line ±SD from four separate experiments .", "Mean percentages of NUP153 depletion levels compared to HeLa + NT siRNA ( lane 1 ) from four independent experiments were lane 1: 100% , 2: 52% , 3: 85% , 4: 52% , 5: 94% , 6: 51% , 7: 75% , 8: 48% , 9: 96% , 10: 48% .", "( F ) HeLa YFP-lamina-associated polypeptide 2β ( LAP2β ) expressing cells were transfected with NT and ULK3 siRNA , and resolution times of intercellular chromatin bridges were quantified in three separate experiments ( mean times are NT: 708 min; ULK3: 459 min ) .", "( G ) Analysis of tension-dependent modulation of abscission time , mean times: 1: 120 ± 53 min; 2: 73 ± 19 min; 3: 77 ± 41 min; 4: 69 ± 20 min ) .", "Data in ( A , D , F–G ) are represented as box plots showing median abscission times ( A , D , and G ) or LAP2β bridge resolution times ( F ) .", "Here and throughout , whiskers mark 5–95 percentiles , box edges represent the first and third quartiles , and red bars denote the median .", "n = total number of events counted per sample .", "Cell lysates in ( A , C , E , and F ) were examined by Western blot using indicated antibodies .", "See also Figure 2—figure supplements 1 and 2 , and Videos 1–11 . DOI: http://dx . doi . org/10 . 7554/eLife . 06547 . 00610 . 7554/eLife . 06547 . 007Figure 2—figure supplement 1 . ULK3 is not required for endosomal sorting or HIV-1 budding , but does regulate abscission timing .", "( A ) HeLa Kaposi sarcoma-associated herpesvirus ( KSHV ) K3 expressing cells were transfected with NT or two different siRNAs against ULK3 ( ULK3-1 and ULK3-4 ) .", "Cells were fixed and stained with a FITC-conjugated α-MHC-I antibody and analyzed by flow cytometry .", "TSG101 depletion was used as a positive control for disruption of ESCRT-mediated endosomal sorting .", "Cell lysates were analyzed by Western blot with α-ULK3 and α-TSG101 to verify knockdown of endogenous proteins .", "( B ) HT/THN-HA ( top ) and HT/THN-HA KSHV K5 ( bottom ) expressing cells were transfected with NT , TSG101 , or ULK3 siRNA .", "Cell lysates obtained 72 hr after initial transfection were analyzed by Western blot using α-HA and α-HSP90 antibodies .", "Data are represented as mean values of Tetherin-HA levels detected by Western blotting with infrared imaging ±SD from three separate experiments .", "( C ) 293T cells were co-transfected with an HIV-1 proviral pNL/HXB plasmid and the indicated siRNA .", "Infectious virion production was measured using β-galactosidase activity on infected TZM indicator cells after 48 hr .", "Mean infectivity values obtained in three separate experiments are represented as relative luminescence units ±SD .", "Cell lysates were analyzed by Western blot with α-Gag , α-ULK3 , α-TSG101 , and α-HSP90 antibodies .", "Extracellular virions were also analyzed with α-Gag and quantified by Western blotting with infrared imaging .", "Numbers below the blot represent the ratio of virion release calculated for each sample , compared to the NT siRNA-treated sample .", "( D ) Abscission dynamics for representative HeLa cells expressing mCherry-Tubulin treated with NT siRNA ( top ) or ULK3 siRNA ( middle and bottom ) .", "Times in minutes are shown for each frame and ‘arrowheads’ indicate the moment of midbody abscission .", "Panels correspond to Videos 1–3 , respectively .", "( E ) HeLa cells transfected with NT or ULK3 siRNA were transfected with NUP153 siRNA as in Figure 2C , fixed and stained with Hoechst ( in blue ) , α-Tubulin ( in red ) , and α-pT232 Aurora B ( in green ) antibodies to visualize midbody-arrested cells and active Aurora B , respectively .", "Insets show magnified views of midbodies .", "Scale bars = 10 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 06547 . 00710 . 7554/eLife . 06547 . 008Figure 2—figure supplement 2 . LAP2β-positive intercellular chromatin bridges resolve faster in ULK3-depleted cells .", "( A ) Selected frames from Videos 4 , 5 showing the resolution dynamics of intercellular chromatin bridges present in HeLa cells expressing YFP-LAP2β transfected with NT siRNA ( top ) or ULK3 siRNA ( bottom ) .", "Times in minutes are shown for each frame and ‘arrowheads’ indicate the moment of resolution of the LAP2β-positive chromatin bridges .", "( B ) Asynchronous HeLa cells doubly expressing YFP-LAP2β and mCherry-Tubulin transfected with NT or ULK3 siRNA were imaged live over 72 hr .", "For each condition , dividing cells were segregated between normal midbody-connected cells and chromatin bridge-containing cells , and further classified into cells achieving abscission ( bronze ) or cells showing failed abscission as seen by the regression of the cleavage furrow ( black ) ( NT normal n = 271; NT chromatin bridge n = 46; ULK3 normal n = 310; ULK3 chromatin bridge n = 49 ) .", "Dot plot in ( C ) shows resolution time of chromatin bridges from experiment ( B ) in three separate experiments .", "Red line indicates mean resolution times for NT siRNA: 950 min and ULK3 siRNA: 598 min , and n = total number of events counted per sample .", "**p = 0 . 0003 . DOI: http://dx . doi . org/10 . 7554/eLife . 06547 . 00810 . 7554/eLife . 06547 . 009Video 1 . Representative example of asynchronous HeLa mCherry-Tubulin cells treated with non-targeting ( NT ) siRNA . Midbody resolution is indicated with an arrow .", "Abscission time is 90 min .", "Related to Figure 2 and Figure 2—figure supplement 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 06547 . 00910 . 7554/eLife . 06547 . 010Video 2 . Representative example of asynchronous HeLa mCherry-Tubulin cells treated with ULK3 siRNA . Midbody resolution is indicated with an arrow .", "Abscission time is 60 min .", "Related to Figure 2 and Figure 2—figure supplement 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 06547 . 01010 . 7554/eLife . 06547 . 011Video 3 . Representative example of asynchronous HeLa mCherry-Tubulin cells treated with ULK3 siRNA . Midbody resolution is indicated with an arrow .", "Abscission time is 60 min .", "Related to Figure 2 and Figure 2—figure supplement 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 06547 . 011 Another trigger for the abscission checkpoint is the presence of lagging chromosomes within the midbody ( Norden et al . , 2006; Mendoza et al . , 2009; Steigemann et al . , 2009 ) , and we tested whether ULK3 was also required for this process .", "The presence of chromatin bridges within the midbody was monitored by time-lapse microscopy in cells expressing YFP-tagged lamina-associated polypeptide 2β ( LAP2β ) ( Steigemann et al . , 2009 ) .", "As described previously for CHMP4C-depleted cells ( Carlton et al . , 2012 ) , LAP2β bridges were also resolved more rapidly upon depletion of ULK3 ( Figure 2F and Figure 2—figure supplement 2A , Videos 4 , 5 ) .", "In control cells , persisting chromosome bridges were resolved in 680 min , whereas in ULK3-depleted cells , this resolution time was dramatically reduced to 300 min .", "Thus , our experiments demonstrate that ULK3 plays an essential role in supporting the Aurora B-dependent abscission checkpoint in response to either defective nuclear pore assembly or lagging chromosomes .", "Besides delaying abscission , Aurora B activation also prevents cleavage furrow regression in cells that contain chromosome bridges ( Steigemann et al . , 2009 ) .", "As expected , the frequency of furrow regression increased in cells with lagging chromosomes , but ULK3 depletion did not change this proportion ( Figure 2—figure supplement 2B , C , Videos 6 , 7 ) .", "This result indicates that ULK3 does not control furrow regression , but instead likely functions downstream of Aurora B in delaying abscission in response to chromatin bridges . 10 . 7554/eLife . 06547 . 012Video 4 . Representative example of asynchronous HeLa cells stably expressing YFP-LAP2β transfected with NT siRNA . Chromatin bridge resolution time is 510 min .", "Bridge resolution is indicated with an arrow .", "Related to Figure 2 and Figure 2—figure supplement 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 06547 . 01210 . 7554/eLife . 06547 . 013Video 5 . Representative example of asynchronous HeLa cells stably expressing YFP-LAP2β transfected with ULK3 siRNA . Chromatin bridge resolution time is 110 min .", "Bridge resolution is indicated with an arrow .", "Related to Figure 2 and Figure 2—figure supplement 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 06547 . 01310 . 7554/eLife . 06547 . 014Video 6 . Representative example of asynchronous HeLa cells stably expressing YFP-LAP2β and mCherry-Tubulin , containing an intercellular chromatin bridge that resolves in 850 min when the daughter cells faithfully divide . Bridge resolution is indicated with an arrow .", "Related to Figure 2—figure supplement 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 06547 . 01410 . 7554/eLife . 06547 . 015Video 7 . Representative example of asynchronous HeLa cells stably expressing YFP-LAP2β and mCherry-Tubulin , containing an intercellular chromatin bridge that does not resolve and the cleavage furrow regresses . Related to Figure 2—figure supplement 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 06547 . 015 A recent study revealed that the high-membrane tension of cells grown at low density delays abscission as compared to cells grown at high density ( Lafaurie-Janvore et al . , 2013 ) .", "We , therefore , asked whether ULK3 also plays a role in this largely unexplored regulatory mechanism .", "Abscission events from Figure 2A were stringently segregated into two extreme groups: those for isolated cells ( low density ) and those for closely packed cells ( high density ) .", "As reported previously , low-density cells showed significant abscission delays as compared to high-density cells ( Figure 2G ) .", "In contrast , ULK3-depleted cells showed similar rapid abscission times regardless of the cell density ( Figure 2G , lanes 3 and 4 , Videos 8–11 ) .", "Thus , ULK3 is also required to delay abscission in response to midbody tension . 10 . 7554/eLife . 06547 . 016Video 8 . Representative example of low-density asynchronous HeLa mCherry-Tubulin cells treated with NT siRNA . Midbody resolution is indicated with an arrow .", "Abscission time is 170 min .", "Related to Figure 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 06547 . 01610 . 7554/eLife . 06547 . 017Video 9 . Representative example of high-density asynchronous HeLa mCherry-Tubulin cells treated with NT siRNA . Midbody resolution is indicated with an arrow .", "Abscission time is 60 min .", "Related to Figure 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 06547 . 01710 . 7554/eLife . 06547 . 018Video 10 . Representative example of low-density asynchronous HeLa mCherry-Tubulin cells treated with ULK3 siRNA . Midbody resolution is indicated with an arrow .", "Abscission time is 60 min .", "Related to Figure 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 06547 . 01810 . 7554/eLife . 06547 . 019Video 11 . Representative example of high-density asynchronous HeLa mCherry-Tubulin cells treated with ULK3 siRNA . Midbody resolution is indicated with an arrow .", "Abscission time is 60 min .", "Related to Figure 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 06547 . 019 We next performed mutational analyses to test the requirements for ULK3 to function in the abscission checkpoint .", "The faster abscission in ULK3-depleted cells was restored to control levels by stable expression of an siRNA-resistant wild-type ULK3 construct ( ULK3R WT , Figure 3A , lane 3 ) , but not by expression of ULK3 proteins with inactivating mutations in the MIM1-binding sites of either ( or both ) MIT domains ( ULK3R V338D , ULK3R M434D and ULK3R V338D , M434D; Figure 3A , lanes 4–6 ) or with an inactivating mutation in the ATP-binding site ( K44H ) of the kinase domain ( Chan et al . , 2009 ) ( Figure 3E , lane 4 vs 3 ) .", "Similarly , stable overexpression of these MIT ( Figure 3B , Videos 12 ,", "13 ) or catalytic ( Figure 3F , lane 3 vs 2 , Video", "14 ) mutants did not change the timing of abscission , whereas ULK3R WT overexpression induced abscission delays ( Figure 3B , Videos 15–17 ) .", "The ULK3 MIT mutations did not alter ULK3 kinase activity ( Figure 1—figure supplement 1G ) , and we , therefore , attribute their effects in abscission specifically to reductions in ESCRT-III binding .", "The efficacy of the K44H mutation was confirmed by the lack of auto-phosphorylation activity of GST-ULK3 K44H in an in vitro kinase assay ( Figure 3C ) , and retention of ESCRT-III binding activity was confirmed by Y2H ( Figure 3D ) .", "Thus , ULK3 modulates midbody resolution timing through both its kinase activity and ESCRT-III interactions . 10 . 7554/eLife . 06547 . 020Figure 3 . ESCRT-III binding and kinase activity are required for ULK3 function .", "( A ) HeLa mCherry-Tubulin cells stably expressing empty vector or siRNA-resistant ULK3 ( ULK3R ) were transfected with the indicated siRNA .", "Midbody resolution times were calculated in three separate experiments ( mean times were 1: 95 ± 32 min; 2: 71 ± 25 min; 3: 115 ± 61 min; 4: 80 ± 38 min; 5: 77 ± 34 min; 6: 74 ± 31 min ) .", "( B ) HeLa mCherry-Tubulin cells expressing empty vector , ULK3 WT , V338D , or M434D were imaged live .", "Midbody resolution times were analyzed from three separate experiments ( mean times were 1: 81 ± 29 min; 2: 159 ± 88 min; 3: 73 ± 23 min; 4: 83 ± 37 min ) .", "( C ) In vitro kinase assay showing the auto-phosphorylation activity of GST-ULK3 WT and GST-ULK3 K44H .", "( D ) Yeast two-hybrid ( Y2H ) assay with ULK3 WT and ULK3 K44H fused to VP16 binding to ESCRT-III proteins fused to Gal4 .", "Data are represented as mean β-galactosidase activity ±SD from triplicate measurements of two separate experiments .", "( E ) HeLa mCherry-Tubulin cells expressing either empty vector or ULK3R constructs were transfected with the indicated siRNA .", "Midbody resolution times were analyzed in three independent experiments ( mean times were 1: 100 ± 42 min; 2: 77 ± 34 min; 3: 104 ± 57 min; 4: 74 ± 31 min ) .", "( F ) HeLa mCherry-Tubulin cells expressing empty vector , ULK3 WT , or K44H were imaged live .", "Midbody resolution times were calculated in four separate experiments ( mean times are 1: 122 ± 60 min; 2: 161 ± 73 min; 3: 113 ± 47 min ) .", "Cell lysates in ( A , B , E , and F ) were examined by Western blot using the indicated antibodies .", "See also Videos 12–17 . DOI: http://dx . doi . org/10 . 7554/eLife . 06547 . 02010 . 7554/eLife . 06547 . 024Video 12 . Representative example of asynchronous HeLa cells stably expressing mCherry-Tubulin and ULK3 V338D . Midbody resolution is indicated with an arrow .", "Abscission time is 80 min .", "Related to Figure 3 . DOI: http://dx . doi . org/10 . 7554/eLife . 06547 . 02410 . 7554/eLife . 06547 . 025Video 13 . Representative example of asynchronous HeLa cells stably expressing mCherry-Tubulin and ULK3 M434D . Midbody resolution is indicated with an arrow .", "Abscission time is 70 min .", "Related to Figure 3 . DOI: http://dx . doi . org/10 . 7554/eLife . 06547 . 02510 . 7554/eLife . 06547 . 026Video 14 . Representative example of asynchronous HeLa cells stably expressing mCherry-Tubulin and ULK3 K44H . Midbody resolution is indicated with an arrow .", "Abscission time is 80 min .", "Related to Figure 3 . DOI: http://dx . doi . org/10 . 7554/eLife . 06547 . 02610 . 7554/eLife . 06547 . 021Video 15 . Representative example of asynchronous HeLa cells stably expressing mCherry-Tubulin and empty vector . Midbody resolution is indicated with an arrow .", "Abscission time is 100 min .", "Related to Figure 3 . DOI: http://dx . doi . org/10 . 7554/eLife . 06547 . 02110 . 7554/eLife . 06547 . 022Video 16 . Representative example of asynchronous HeLa cells stably expressing mCherry-Tubulin and ULK3 WT . Midbody resolution is indicated with an arrow .", "Abscission time is 160 min .", "Related to Figure 3 . DOI: http://dx . doi . org/10 . 7554/eLife . 06547 . 02210 . 7554/eLife . 06547 . 023Video 17 . Representative example of asynchronous HeLa cells stably expressing mCherry-Tubulin and ULK3 WT . Midbody resolution is indicated with an arrow .", "Abscission time is 290 min .", "Related to Figure 3 . DOI: http://dx . doi . org/10 . 7554/eLife . 06547 . 023 The functional links with ESCRT-III subunits suggested that catalytically active ULK3 might regulate abscission timing by influencing ESCRT-III polymerization during cytokinesis .", "We tested this hypothesis by analyzing the midbody location of endogenous IST1 , which is essential for abscission ( Agromayor et al . , 2009; Bajorek et al . , 2009a ) .", "ULK3 overexpression altered IST1 midbody localization significantly , as indicated by a 1 . 5-fold increase in the proportion of IST1 found at the midbody rings and a marked ninefold increase in the proportion of midbodies containing a single ring of IST1 at the Flemming body ( Figure 4A ) .", "Importantly , HA-ULK3 was seen to co-localize with IST1 at this Flemming body ring ( in 42% of observed midbodies ) ( Figure 4B ) .", "Additionally , ULK3 and IST1 co-localized at midbody rings on both sides of the Flemming body ( 58% of cases ) ( Figure 4B ) , which could be a transient ULK3 localization rarely observed in steady-state conditions exacerbated in the overexpression context ( compare to endogenous staining of ULK3 in Figure 2B ) . 10 . 7554/eLife . 06547 . 027Figure 4 . Effects of ULK3 on IST1 localization at the midbody .", "( A ) HeLa mCherry-Tubulin cells stably expressing empty vector or ULK3 WT were stained with Hoechst and α-IST1 antibody .", "Cells connected by midbodies were classified by the localization pattern of IST1 as shown on the left panel , where Tubulin is red and IST1 is green .", "Bars = 1 μm .", "Data are represented as percentage of midbodies observed for each category from three separate experiments; n = total number of scored midbodies .", "Cells connected by chromatin bridges or multinucleated were excluded .", "( B and C )", "Panels show a representative example of Flemming body localization ( B ) or midbody rings ( C ) , where the IST1 signal ( middle left ) co-localizes with HA-ULK3 ( middle right ) in HeLa YFP-Tubulin ( left ) cells expressing HA-ULK3 stained with α-IST1 and α-HA antibodies .", "ULK3 and IST1 co-localization was observed in 100% of midbodies with Flemming body localization for IST1 ( n = 48 ) .", "Merged channels are shown on the right ( green: Tubulin , red: IST1 , yellow: HA , blue: nuclei ) .", "Magnifications of the midbody are shown for each channel .", "Bar = 5 μm .", "See also Figure 4—figure supplement 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 06547 . 02710 . 7554/eLife . 06547 . 028Figure 4—figure supplement 1 . Effects of ULK3 on the abscission machinery .", "( A ) HeLa cells stably expressing GFP-CHMP4B and empty vector , ULK3 WT , K44H , or M434D were fixed and stained with Hoechst , α-GFP , α-ULK3 , and α-Tubulin antibodies .", "Cells connected by midbodies were counted and classified by the localization pattern of GFP-CHMP4B as shown on the images on the right , where Tubulin is in red and GFP in green .", "Scale bars = 1 μm .", "Data are represented as percentage of midbodies observed for each category from three experiments; n is the total number of scored midbodies .", "Cells connected by chromatin bridges or multinucleated were excluded .", "( B ) Cells overexpressing ULK3 WT from ( A ) were stained with α-Tubulin and α-MKLP1 antibody , a marker of the Flemming body .", "Panel shows a representative midbody with a Flemming body ring , where the unusual GFP-CHMP4B pattern ( middle ) co-localizes with MKLP1 ( left ) .", "Merged channels are shown on the right ( red: Tubulin , green: GFP , white: MKLP1 ) .", "Scale bars = 5 μm .", "( C and D )", "Panels show co-localization of GFP and ULK3 signals in HeLa GFP-CHMP4B cells expressing ULK3 WT ( C ) or K44H ( D ) stained with Hoechst , α-ULK3 , and α-Tubulin antibodies .", "In C , top panels show a representative example of ULK3 WT co-localization with GFP-CHMP4B at the midbody rings ( 61% of observed midbodies ) and bottom panels an example of co-localization at the Flemming body ( 39% of midbodies , n = 33 ) .", "In C and D , column on the right shows merged channels ( red: Tubulin , green: GFP , purple: ULK3 , blue: nuclei ) .", "Scale bars = 5 μm .", "Magnifications of the midbody regions are shown for each channel ( B , C , and D ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 06547 . 028 As a second marker for ESCRT-III polymers , we investigated whether ULK3 overexpression altered the midbody localization of GFP-CHMP4B expressed at endogenous levels ( Jouvenet et al . , 2011 ) .", "As with IST1 , the proportion of midbodies exhibiting single Flemming body rings of GFP-CHMP4B also increased 20-fold upon ULK3 overexpression ( Figure 4—figure supplement 1A , B ) .", "In contrast , overexpression of either ESCRT-binding deficient ( M434D ) or kinase-defective ( K44H ) ULK3 had no effect on GFP-CHMP4B localization ( Figure 4—figure supplement 1A ) .", "Thus , the ability of ULK3 constructs to induce aberrant ESCRT-III localization correlates with their ability to delay abscission .", "Both WT and K44H ULK3 co-localized with GFP-CHMP4B polymers at the intercellular bridge ( Figure 4—figure supplement 1C , D ) , while only ULK3 WT co-localized with GFP-CHMP4B at the Flemming body ( 39% vs 61% of ULK3/GFP-CHMP4B at the midbody rings ) ( Figure 4—figure supplement 1C ) , more closely reflecting the Flemming body localization observed for endogenous ULK3 in a context without overexpression ( compare to Figure 2B ) .", "This observation suggests that ULK3 does not delay abscission by directly blocking or outcompeting ESCRT-III interactions required for polymerization .", "Instead , ULK3 apparently induces abscission delays and ESCRT-III mislocalization by phosphorylating substrates important for abscission completion .", "Taken together , these data show that catalytically active ULK3 changes the steady-state distribution of ESCRT-III proteins at the midbody , suggesting that the abscission delays induced by ULK3 are due , at least in part , to defective ESCRT-III polymerization .", "We next tested whether ESCRT-III subunits are phosphorylated by ULK3 .", "An in vitro kinase assay using recombinant ULK3 on immunoprecipitated HA-ESCRT-III proteins showed phosphorylation of CHMP1A , CHMP1B , CHMP2A , and IST1 ( Figure 5A , middle blot , lanes 2 , 3 , 4 , and 10 ) , but not other ESCRT-III subunits .", "Analogous phosphorylation events were recapitulated in cells overexpressing OSF-ULK3 and Myc-tagged ESCRT-III proteins .", "The enhanced separation of phosphorylated species by electrophoresis on Phos-tag SDS-PAGE gels revealed ULK3-dependent phosphorylation of IST1 , CHMP1A , CHMP1B , and CHMP2A , but not CHMP3 ( Figure 5B , lane 2 and Figure 5—figure supplement 1A , lanes 2 , 8 , 14 , and 20 ) .", "Phosphorylation was confirmed by sensitivity to calf intestinal phosphatase ( CIP ) treatment ( Figure 5B , lanes 4–6 and Figure 5—figure supplement 1A , lanes 4–6 , 10–12 , 16–18 , and 22–24 ) and by expressing the inactive ULK3 K44H protein as a control ( Figure 5B , lane 3 and Figure 5—figure supplement 1A , lanes 3 , 9 , and 15 ) .", "These data demonstrate that ULK3 ESCRT-III binding partners are also substrates for phosphorylation . 10 . 7554/eLife . 06547 . 029Figure 5 . Identification of ULK3 phosphorylation sites within IST1 . ( A ) In vitro kinase assay with recombinant ULK3 protein ( middle ) on immunoprecipitated HA-ESCRT-III proteins expressed in 293T cells .", "Western blot with α-HA antibody shows immunoprecipitated ESCRT-III proteins ( top ) .", "( B ) 293T cells co-transfected with Myc-IST1 and either empty vector , OSF-ULK3 WT or OSF-ULK3 K44H .", "Lysates were electrophoresed on a 10% Phos-tag gel to separate phosphorylated species ( denoted by ‘asterisk’ ) .", "Lysates were treated with calf intestinal phosphatase ( CIP , lanes 4–6 ) .", "( C ) Identified phosphorylation sites mapped onto the IST1 domain structure ( above ) and onto the crystal structure of the IST1 N-terminal domain ( below , PDB 3FRR ) 39 .", "( D ) HeLa cells stably expressing empty vector , siRNA-resistant IST1 ( IST1R ) WT , or IST1R 4SA were transfected with siRNA and treated overnight with DMSO or Nocodazole .", "( E ) HeLa cells were transfected with siRNA prior to overnight treatment with DMSO or Nocodazole .", "In ( D and E ) , cell lysates were resolved on 10% Phos-tag gels and analyzed by western blot , where ‘asterisks’ denote bands corresponding to IST1 phosphorylated species , sensitive ( * ) or insensitive ( ** ) to the 4SA mutation or ULK3/CHMP4C depletion .", "See also Figure 5—figure supplement 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 06547 . 02910 . 7554/eLife . 06547 . 030Figure 5—figure supplement 1 . ULK3 phosphorylation of ESCRT-III proteins .", "( A ) 293T cells were co-transfected with vectors expressing Myc-ESCRT-III proteins and either empty vector , OSF-ULK3 WT , or OSF-ULK3 K44H .", "Cell lysates were electrophoresed on 10% Phos-tag gels and blotted for the designated ESCRT-III proteins to reveal lower mobility phosphoproteins ( designated by ‘asterisks’ ) .", "Treatment with CIP confirmed that the mobility changes resulted from phosphorylation ( final three lanes in each blot ) .", "( B ) Overlay of ESI/MS spectra showing the intact mass of Myc-IST1 co-immunoprecipitated with either OSF-ULK3 WT ( red ) or the catalytically inactive OSF-ULK3 K139R protein together with CIP treatment ( black ) .", "Theoretical vs actual measured masses of phospho-IST1 species detected in this experiment are tabulated . DOI: http://dx . doi . org/10 . 7554/eLife . 06547 . 030 ULK3 phosphorylation of IST1 was characterized by mass spectrometry .", "To maximize IST1 phosphorylation , we co-overexpressed Myc-IST1 and OSF-ULK3 in 293T cells , affinity purified the IST1/ULK3 complexes , and determined the masses of intact IST1 proteins by electrospray ionization ( ESI ) mass-spectrometry .", "We observed four different IST1 species whose masses differed by ∼80 Da , the mass change corresponding to phosphorylation ( Figure 5—figure supplement 1B ) .", "The different masses corresponded to Myc-IST1 proteins with 1–4 phosphates ( and N-terminal acetylation ) .", "These assignments were confirmed by measuring the intact mass of Myc-IST1 co-expressed with a catalytically inactive ULK3 protein ( K139R ) ( Maloverjan et al . , 2010 ) and CIP treated ( Figure 5—figure supplement 1B ) .", "Phosphorylation sites were mapped by ESI/MS/MS analyses of IST1 peptides from tryptic and chymotryptic digests .", "These analyses confirmed that Myc-IST1 is N-terminally acetylated and identified four phosphorylated serine residues: S4 , S99 , S153 , and S214 .", "These sites are upstream of the IST1 MIM elements , either within the core ESCRT-III four-helix bundle ( Muziol et al . , 2006; Bajorek et al . , 2009b ) ( residues 1–189; S4 , S99 , and S153 ) or in a flexible region flanking the core ( S214; Figure 5C ) .", "We next compared the phosphorylation status of endogenous IST1 in cycling cells vs Nocodazole-arrested cells .", "Mitotic arrest was confirmed by the presence of Histone 3 phosphorylation ( P-H3 ) , a marker for mitotic chromosome condensation ( Hendzel et al . , 1997 ) ( Figure 5D ) .", "Nocodazole treatment induced accumulation of at least two different phosphorylated IST1 species ( Figure 5D ) .", "A similar banding pattern was observed when endogenous IST1 was depleted and replaced by an siRNA-resistant wild-type IST1 construct ( IST1R WT , lane 2 vs 4 ) .", "However , the lowest mobility IST1 species was eliminated when the four mapped phosphorylated serine residues were mutated to alanine ( IST1R 4SA , Figure 5D , lane 6 ) .", "The IST1R 4SA mutant still formed intermediate mobility species , which suggests that additional site ( s ) on IST1 may be phosphorylated by ULK3 or other mitotic kinases .", "Formation of the lowest mobility phosphorylated IST1 species was ULK3-dependent , as it was also eliminated by depletion of ULK3 from Nocodazole-treated cells ( Figure 5E , lane 5 vs 4 ) .", "Taken together , these results indicate that ULK3 phosphorylates endogenous IST1 during mitosis .", "To evaluate the functional consequences of IST1 phosphorylation by ULK3 , we tested the ability of the IST1R 4SA mutant to support the abscission checkpoint .", "These experiments revealed that NUP153 depletion failed to trigger the abscission checkpoint when endogenous IST1 was replaced with IST1R 4SA ( Figure 6A , lane 6 vs 2 ) , whereas the checkpoint was fully functional when IST1R WT was used in this assay ( Figure 6A , lane 4 vs 2 ) .", "Similarly , intercellular chromatin bridges in cells depleted of endogenous IST1 but expressing IST1R 4SA resolved significantly faster than chromatin bridges in control cells or cells that expressed IST1R WT ( Figure 6B ) .", "Importantly , both IST1R WT and IST1R 4SA were able to restore the abscission defects induced by IST1 depletion ( Figure 6C , lanes 3–4 vs lane 2 , and Figure 6D ) , indicating that IST1R 4SA is fully functional for cytokinesis under steady-state conditions .", "Similarly , IST1R 4SA retained its interactions with all known IST1-binding partners in Y2H assays ( Figure 6—figure supplement 1A ) .", "It was notable that the IST1R 4SA could also still support abscission delays when the checkpoint was induced by low-cell tension .", "Specifically , the effects on tension-mediated abscission regulation were similar when cells expressed IST1R WT or IST1R 4SA ( Figure 6—figure supplement 1B ) .", "Thus , the IST1R 4SA protein did not support the abscission checkpoint in response to chromatin within the midbody and nuclear pore disruption but retained other IST1 binding and abscission functions , including regulation of abscission timing in response to midbody tension .", "In contrast , a phosphomimetic IST1 mutant ( IST1R 4SE: S4E , S99E , S153E , S214E ) failed to restore abscission in IST1-depleted cells , as reflected by the accumulation of multinucleated and midbody-arrested cells ( Figure 6C , lane 5 and Figure 6D , bottom panel ) .", "Furthermore , live-cell imaging confirmed that even when cells expressing IST1R 4SE did complete abscission , they did so more slowly than cells expressing IST1R WT or 4SA ( Figure 6E , Videos 18–22 ) .", "Collectively , these experiments indicate that ULK3 phosphorylation of IST1 delays abscission as an essential component of the abscission checkpoint in the presence of chromatin bridges within the midbody or incomplete nuclear pore assembly . 10 . 7554/eLife . 06547 . 031Figure 6 . ULK3 phosphorylation of IST1 is required to sustain the abscission checkpoint and inhibits IST1 function in abscission .", "( A ) HeLa cells stably expressing empty vector , IST1R WT , or IST1R 4SA were co-transfected with the indicated siRNA and NUP153 siRNA to trigger the abscission checkpoint .", "Cells were fixed and stained with Hoechst and α-Tubulin antibody to visualize nuclei and midbodies , respectively .", "Data are represented as mean percentage of midbodies ±SD from four separate experiments , n = total number of scored midbodies .", "NUP153 depletion levels as a mean percentage quantified by Western blot from two independent experiments were lane 1: 100% , 2: 40% , 3: 85% , 4: 39% , 5: 120% , 6: 46% .", "( B ) HeLa YFP-LAP2β cells expressing empty vector , IST1R WT , or IST1R 4SA were transfected with NT or IST1 siRNA , and resolution times of intercellular chromatin bridges were quantified in two separate experiments ( mean times are 1: 906 min; 2: 965 min: 3: 638 min ) .", "( C and D )", "HeLa cells stably expressing empty vector , IST1R WT , IST1R 4SA , or IST1R 4SE were transfected with the indicated siRNA , fixed and stained with α-Tubulin antibody to visualize cytokinetic defects .", "Data in ( C ) are represented as mean percentage of midbody-arrested or multinucleated cells ±SD from three separate experiments .", "Microscopy images in ( D ) are representative examples from each sample in ( C ) , shown as merged images of Tubulin ( red ) and nuclei ( blue ) .", "‘Asterisks’ and ‘arrowheads’ denote multinucleated and midbody-connected cells , respectively .", "Bars = 10 μm .", "( E ) HeLa YFP-Tubulin cells stably expressing empty vector , IST1R WT , IST1R 4SA , or IST1R 4SE were transfected with the indicated siRNA .", "Live-cell imaging revealed midbody resolution times from two separate experiments ( mean times were 1: 97 ± 33 min; 2: 204 ± 95 min; 3: 103 min ±48; 4: 98 ± 38 min; 5: 145 ± 65 min ) .", "Cell lysates in ( A , B , C , and D ) were examined by Western blot using the indicated antibodies .", "( F ) Co-precipitation assay from 293T cells co-transfected with the indicated YFP and GST fusions .", "10% of the volume eluted from the beads was analyzed by Western blot with α-YFP antibody to visualize bound proteins and α-GST as a control for pull-down efficiency .", "α-YFP Western blot on input lysates ( top ) is representative for all experiments , and empty YFP band was cropped from the same blot .", "See also Figure 6—figure supplement 1 and Videos 18–22 . DOI: http://dx . doi . org/10 . 7554/eLife . 06547 . 03110 . 7554/eLife . 06547 . 032Figure 6—figure supplement 1 . The IST1 phosphorylation 4SA mutant retains IST1 binding and abscission functions .", "( A ) Y2H assays with IST1 WT and IST1 4SA fused to the VP16 activation domain showing binding to VPS4A , VPSP4B , CHMP1A , CHMP1B , LIP5 , and MITD1 proteins fused to the Gal4 DNA-binding domain .", "CHMP2A was used as a negative control .", "Data are mean β-galactosidase activity ±SD from two separate experiments .", "( B ) Analysis of tension-dependent modulation of abscission time from events in Figure 6D .", "Mean times from two independent experiments are: 1: 118 ± 38 min; 2: 68 ± 18 min; 3: 122 ± 54 min; 4: 66 ± 17 min; 5: 107 ± 43 min; 6: 65 ± 12 min ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 06547 . 03210 . 7554/eLife . 06547 . 033Video 18 . Representative example of asynchronous HeLa cells stably expressing YFP-Tubulin and empty vector transfected with NT siRNA . Midbody resolution is indicated with an arrow .", "Abscission time is 90 min .", "Related to Figure 6 . DOI: http://dx . doi . org/10 . 7554/eLife . 06547 . 03310 . 7554/eLife . 06547 . 034Video 19 . Representative example of asynchronous HeLa cells stably expressing YFP-Tubulin and empty vector transfected with IST1 siRNA . Midbody resolution is indicated with an arrow .", "Abscission time is 190 min .", "Related to Figure 6 . DOI: http://dx . doi . org/10 . 7554/eLife . 06547 . 03410 . 7554/eLife . 06547 . 035Video 20 . Representative example of asynchronous HeLa cells stably expressing YFP-Tubulin and siRNA-resistant IST1 ( IST1R ) WT transfected with IST1 siRNA . Midbody resolution is indicated with an arrow .", "Abscission time is 90 min .", "Related to Figure 6 . DOI: http://dx . doi . org/10 . 7554/eLife . 06547 . 03510 . 7554/eLife . 06547 . 036Video 21 . Representative example of asynchronous HeLa cells stably expressing YFP-Tubulin and IST1R 4SA transfected with IST1 siRNA . Midbody resolution is indicated with an arrow .", "Abscission time is 90 min .", "Related to Figure 6 . DOI: http://dx . doi . org/10 . 7554/eLife . 06547 . 03610 . 7554/eLife . 06547 . 037Video 22 . Representative example of asynchronous HeLa cells stably expressing YFP-Tubulin and IST1R 4SE transfected with IST1 siRNA . Midbody resolution is indicated with an arrow .", "Abscission time is 180 min .", "Related to Figure 6 . DOI: http://dx . doi . org/10 . 7554/eLife . 06547 . 037 We next used a co-precipitation approach to test whether phosphorylation altered the interactions of IST1 with its binding partners ( Figure 6F ) .", "These experiments revealed that the phosphomimetic mutations enhanced binding of YFP-IST1 4SE to both GST-VPS4 and GST-LIP5 as compared to YFP-IST1 WT and YFP-IST1 4SA ( Figure 6F , lanes 20 and 24 ) .", "In contrast , the interaction of YFP-IST1 4SE with GST-CHMP1B was unchanged ( Figure 6F , lane 12 ) , and the interaction with GST-CHMP2A remained negative .", "Thus , the enhanced binding effects of the IST1 phosphomimetic mutations were specific for VPS4 and its co-activator LIP5 .", "These results suggest that ULK3 phosphorylation of IST1 may delay abscission by regulating the interaction of IST1 with late-acting components of the ESCRT machinery .", "The phenotypic similarities between CHMP4C and ULK3 in regulating the abscission checkpoint suggested connections between these proteins .", "Consistent with this idea , CHMP4C depletion eliminated the IST1 hyperphosphorylation induced by Nocodazole treatment , mimicking the effects of ULK3 depletion ( Figure 5E , lane 6 ) .", "This observation reveals a requirement for CHMP4C in IST1 phosphorylation and suggests a functional link between ULK3 and CHMP4C .", "To examine whether ULK3 and CHMP4C function in the same regulatory pathway , we tested whether ULK3 is required for the abscission delays induced by overexpression of GFP-CHMP4C ( Carlton et al . , 2012 ) .", "GFP-CHMP4C overexpression increased the steady-state number of cells connected by midbodies , but ULK3 depletion reverted this phenotype to control levels ( Figure 7A , lane 5 vs 4 ) .", "Comparable effects were observed for cells treated with CHMP4C siRNA , which depleted both endogenous and exogenous CHMP4C ( lane 6 ) .", "Interestingly , ULK3 depletion did not alter GFP-CHMP4C localization within the central region of the midbody ( Figure 7B ) , suggesting that ULK3 is not required for transmission of the Aurora B signal that targets CHMP4C to the Flemming body ( Carlton et al . , 2012 ) .", "Endogenous ULK3 did co-localize with GFP-CHMP4C at the Flemming body , however , further supporting a functional connection between these proteins ( Figure 7C ) . 10 . 7554/eLife . 06547 . 038Figure 7 . ULK3 and CHMP4C are functionally interconnected within the abscission control pathway .", "( A ) HeLa cells expressing GFP-CHMP4C were transfected with NT , ULK3 , or CHMP4C siRNA , fixed and stained with Hoechst and α-Tubulin antibody to visualize multinucleated and cells connected by midbodies .", "Data are represented as mean percentage of midbody-arrested cells ±SD from three separate experiments .", "( B ) Confocal microscopy of HeLa GFP-CHMP4C cells treated as in ( A ) .", "Magnifications of the midbody are shown .", "Data are represented as mean percentage of CHMP4C positive midbodies ±SD from two separate experiments ( NT n = 110; ULK3 n = 43; CHMP4C n = 53 ) .", "Bars = 5 μm .", "( C ) HeLa GFP-CHMP4C cells were stained with α-ULK3 antibody .", "Panel shows a representative example of GFP-CHMP4C at the Flemming body co-localizing with endogenous ULK3 ( 94% of cases , n = 31 ) .", "Merged channels are shown on the right ( red: Tubulin , green: GFP , purple: ULK3 , blue: nuclei ) .", "Magnifications of the midbody are shown for each channel .", "Bar = 5 μm .", "( D ) HeLa GFP-CHMP4B or GFP-CHMP4C expressing cells were treated as in ( A ) and imaged live .", "Midbody resolution times were quantified in three separate experiments ( mean times were 1: 106 ± 52 min; 2: 180 ± 114 min; 3: 105 ± 47 min ) .", "( E ) HeLa cells expressing empty vector or ULK3 WT were transfected with the indicated siRNA , and midbody resolution times were scored in three separate experiments ( mean times were 1: 84 ± 34 min; 2: 136 ± 87 min; 3: 110 ± 79 min ) .", "n = total number of events analyzed per sample .", "( F ) HeLa cells expressing empty vector or ULK3 WT were treated with 1 μM DMSO or Aurora B inhibitor ( ZM447439 ) .", "Only cells at midbody stage were monitored and imaged live starting at the time of treatment .", "The time spent in abscission until midbody resolution was analyzed in two separate experiments ( mean times were 1: 100 ± 57 min: 2: 171 ± 122 min: 3: 53 ± 41 min: 4: 54 ± 41 min ) .", "( G ) HeLa cells were treated overnight with media containing DMSO or Nocodazole with or without 1 μM ZM44739 inhibitor .", "Cell lysates were resolved on 10% Phos-tag gels and analyzed by Western blot .", "‘Asterisks’ denote bands corresponding to IST1 phosphorylated species that were sensitive ( * ) or insensitive ( ** ) to Aurora B inhibition .", "( H ) HeLa cells stably expressing HA-CHMP4CR or HA-CHMP4CRδINS ( lacks residues 201–217 ) were transfected with NT or ULK3 siRNA prior to overnight treatment with media containing DMSO or Nocodazole .", "HA-CHMP4CR WT or δINS levels were quantified by infrared imaging; data are represented as mean percentage of phosphorylated CHMP4C ( P-CHMP4C , higher molecular weight band ) vs non-phosphorylated CHMP4C ( lower molecular weight ) ±SD from three separate experiments .", "Cell lysates in ( A , D-F ) were analyzed by Western blot with the indicated antibodies .", "See also Figure 7—figure supplement 1 and Videos 23–28 . DOI: http://dx . doi . org/10 . 7554/eLife . 06547 . 03810 . 7554/eLife . 06547 . 039Figure 7—figure supplement 1 . ULK3 phosphorylates CHMP4C .", "( A and B )", "Lysates from 293T cells expressing HA-CHMP4 constructs were immunoprecipitated with α-HA antibodies and subjected to in vitro kinase assays with recombinant ULK3 .", "Incorporated ATP γ32P was visualized by phosphorimaging ( upper panels ) , and CHMP4 proteins were detected by Western blotting ( lower panels ) .", "CHMP4C truncations used in ( B ) are described in the text .", "( C ) Same experiment as in Figure 5B and Figure 5—figure supplement 1A in which 293T cells were co-transfected with expression constructs for CHMP4C-Myc and either an empty vector or expression constructs for OSF-ULK3 or OSF-ULK3 K44H , and lysates were run on a 10% Phos-tag gel to separate phosphoproteins .", "The ‘asterisk’ denotes the band corresponding to phosphorylated CHMP4C .", "( D ) HeLa cells stably expressing siRNA-resistant HA-CHMP4CR or HA-CHMP4CRδINS ( lacks residues 201–217 ) constructs were treated overnight with media containing vehicle dimethyl sulfoxide ( DMSO ) or nocodazole to induce mitotic arrest .", "Cells were rinsed with PBS , released into complete DMEM , and lysed at the specified times ( 0 , 1 hr , 3 hr , 5 hr , and 7 hr ) .", "Cell lysates were examined by Western blot with α-HA , α-P-Histone 3 ( as a marker of mitotic entry ) and α-HSP90 antibodies . DOI: http://dx . doi . org/10 . 7554/eLife . 06547 . 039 The effects of ULK3 on GFP-CHMP4C-dependent abscission delays were confirmed by live imaging of cells co-expressing mCherry-Tubulin .", "In agreement with the fixed-cell data , ULK3 depletion reverted CHMP4C-induced delays in midbody resolution to control levels ( defined by cells expressing GFP-CHMP4B , Figure 7D , Videos 23–25 ) .", "In a reciprocal experiment , CHMP4C depletion attenuated abscission delays imposed by ULK3 overexpression , although in this case the effect was incomplete ( Figure 7E , Videos 26–28 ) .", "ULK3-induced abscission delays were abolished upon treatment of cells at midbody stage with an Aurora B inhibitor ( ZM447439 ) ( Ditchfield et al . , 2003 ) .", "ZM447439-treated cells rapidly underwent abscission , implying that Aurora B activity is required in order for ULK3 to delay abscission ( Figure 7F ) .", "These data indicate that ULK3 and CHMP4C act together in regulating abscission as part of the Aurora B-dependent abscission control pathway .", "The requirement for CHMP4C in abscission delay can be partially alleviated in cells overexpressing ULK3 , however , suggesting that excess ULK3 can overcome upstream CHMP4C functions in regulating midbody resolution . 10 . 7554/eLife . 06547 . 040Video 23 . Representative example of asynchronous HeLa cells stably expressing GFP-CHMP4B and mCherry-Tubulin transfected with NT siRNA . Midbody resolution is indicated with an arrow .", "Abscission time is 110 min .", "Related to Figure 7 . DOI: http://dx . doi . org/10 . 7554/eLife . 06547 . 04010 . 7554/eLife . 06547 . 041Video 24 . Representative example of asynchronous HeLa cells stably expressing GFP-CHMP4C and mCherry-Tubulin transfected with NT siRNA . Midbody resolution is indicated with an arrow .", "Abscission time is 140 min .", "Related to Figure 7 . DOI: http://dx . doi . org/10 . 7554/eLife . 06547 . 04110 . 7554/eLife . 06547 . 042Video 25 . Representative example of asynchronous HeLa cells stably expressing GFP-CHMP4C and mCherry-Tubulin transfected with ULK3 siRNA . Midbody resolution is indicated with an arrow .", "Abscission time is 80 min .", "Related to Figure 7 . DOI: http://dx . doi . org/10 . 7554/eLife . 06547 . 04210 . 7554/eLife . 06547 . 043Video 26 . Representative example of asynchronous HeLa cells stably expressing mCherry-Tubulin and empty vector transfected with NT siRNA . Midbody resolution is indicated with an arrow .", "Abscission time is 80 min .", "Related to Figure 7 . DOI: http://dx . doi . org/10 . 7554/eLife . 06547 . 04310 . 7554/eLife . 06547 . 044Video 27 . Representative example of asynchronous HeLa cells stably expressing mCherry-Tubulin and ULK3 transfected with NT siRNA . Midbody resolution is indicated with an arrow .", "Abscission time is 120 min .", "Related to Figure 7 . DOI: http://dx . doi . org/10 . 7554/eLife . 06547 . 04410 . 7554/eLife . 06547 . 045Video 28 . Representative example of asynchronous HeLa cells stably expressing mCherry-Tubulin and ULK3 transfected with CHMP4C siRNA . Midbody resolution is indicated with an arrow .", "Abscission time is 90 min .", "Related to Figure 7 . DOI: http://dx . doi . org/10 . 7554/eLife . 06547 . 045 We next assessed the effect of ULK3-mediated phosphorylation of IST1 and CHMP4C in the context of the Aurora B-dependent abscission pathway .", "As shown in Figure 7G , the lowest mobility IST1 species observed in mitotically arrested cells was eliminated upon treatment with Aurora B inhibitor ( lane 4 vs 3 ) , whereas the intermediate mobility species was not altered by this treatment .", "These effects are similar to those observed for the IST1R 4SA mutant or upon ULK3 depletion .", "These observations suggest that the low-mobility species is required for abscission checkpoint function and that additional site ( s ) on IST1 could be phosphorylated by ULK3 or other mitotic kinases in an Aurora B-independent manner .", "We , therefore , tested whether immunoprecipitated HA-tagged CHMP4 proteins could be phosphorylated by ULK3 in vitro .", "CHMP4C was phosphorylated by recombinant ULK3 in these experiments , whereas CHMP4A and CHMP4B were not ( Figure 7—figure supplement 1A ) .", "ULK3 phosphorylation was retained in a CHMP4C deletion mutant that lacked both the ALIX-binding site ( McCullough et al . , 2008 ) and the C-terminal insertion that is phosphorylated by Aurora B23 , 24 ( CHMP4C 1–200; Figure 7—figure supplement 1B , lane 3 ) .", "However , ULK3 phosphorylation was lost upon further deletion of the MIM2 element ( CHMP4C 1–184; Figure 7—figure supplement 1B , lane 4 ) .", "These results show that ULK3 and Aurora B target different residues in CHMP4C and indicate that ULK3-dependent phosphorylation requires the MIM2 region .", "In agreement with the in vitro data , CHMP4C phosphorylation was also observed in cells co-transfected with Myc-CHMP4C and OSF-ULK3 ( Figure 7—figure supplement 1C ) as was observed for other ULK3 ESCRT-III substrates .", "We have previously shown that CHMP4C is phosphorylated at the start of mitosis and dephosphorylated as mitosis progresses ( Carlton et al . , 2012 ) .", "This phosphorylation induces a mobility shift that can be followed by Western blot of cells expressing HA-CHMP4C at functional levels ( Carlton et al . , 2012 ) ( Figure 7—figure supplement 1D ) .", "Importantly , mitotic CHMP4C phosphorylation was also observed for a HA-CHMP4C mutant that lacked the Aurora B target residues ( CHMP4CδINS ) ( Figure 7—figure supplement 1D ) .", "Thus , Aurora B-independent phosphorylation of CHMP4C occurs during cell division .", "Quantitative Western blot revealed that mitotic arrest increased the fraction of phosphorylated CHMP4C ( P-CHMP4C ) to 81% vs 7% in asynchronous cells ( Figure 7H , lane 3 vs 1 ) .", "However , depletion of ULK3 reduced the mitotic fraction of P-CHMP4C to just 30% ( Figure 7H , lane 4 ) .", "These differences could not be attributed to gross changes in mitotic arrest because P-H3 levels remained constant upon ULK3 depletion .", "Similarly , the fraction of phosphorylated CHMP4CδINS increased in mitotic cells ( Figure 7H , lane 7 vs 3 ) , and phosphorylation levels were again reduced by ULK3 depletion , albeit not as dramatically as in the case of WT CHMP4C ( Figure 7H , lane 8 vs 7 and 4 ) .", "Hence , ULK3 contributes to mitotic CHMP4C phosphorylation , and ULK3-dependent phosphorylation and Aurora B-dependent phosphorylation of CHMP4C are separate events ." ], [ "Our study reveals that ULK3 is an essential component of the abscission checkpoint .", "ULK3 provides inhibitory signals to the abscission machinery that are sustained in response to defective nuclear pore complex assembly or the presence of lagging chromosomes within the midbody , thereby ensuring faithful cytokinesis progression .", "ULK3 is also the first identified component of a pathway that delays abscission in response to midbody tension .", "Both ULK3 kinase and ESCRT-III-binding activities are required for abscission regulation and our data support a model in which ULK3 phosphorylates ESCRT-III proteins , and thereby , delays the membrane cleavage step of abscission .", "Consistent with this model , we have identified a subset of ESCRT-III proteins , particularly IST1 , which are bound and phosphorylated by ULK3 .", "The MIT2 domain of ULK3 binds the MIM1 of IST1 with unusually high affinity .", "In contrast , both MIT domains contribute significantly to CHMP1A/B binding , suggesting that MIM1 elements of CHMP1 and IST1 proteins bind ULK3 in distinct ways .", "Both MIT domains contribute to ULK3 functions in the abscission checkpoint .", "These observations suggest that IST1 engages the ULK3 MIT2 domain , whereas the other MIT site is occupied by other ESCRT-III proteins and/or by yet unidentified binding partners .", "IST1 phosphorylation is required to mediate the abscission checkpoint and an IST1 phosphomimetic mutant failed to support abscission , indicating that phosphorylation inhibits at least one IST1 abscission function .", "We can envision different , non-exclusive mechanisms by which ULK3 phosphorylation of ESCRT-III proteins could delay abscission .", "In one model , ULK3 phosphorylation could directly prevent extension of ESCRT-III filaments to the abscission site either because phosphorylation stabilizes the auto-inhibited ESCRT-III conformation ( Muziol et al . , 2006; Lata et al . , 2008; Bajorek et al . , 2009b ) or inhibits essential interactions within the polymer .", "ESCRT-III phosphorylation could also modulate interactions with other components of the abscission machinery .", "Overexpression of catalytically active ULK3 induced both IST1 and CHMP4B to form single rings within the Flemming body , a phenotype rarely observed in control cells .", "Intriguingly , both endogenous ULK3 and functionally active CHMP4C also localize to the Flemming body ( Capalbo et al . , 2012; Carlton et al . , 2012 ) , suggesting that ULK3 may promote association of ESCRT-III subunits with abscission checkpoint regulators to form a cytokinetic inhibitory complex at the central region of the midbody .", "We also observed that phosphomimetic mutations enhanced IST1 interactions with the late-acting VPS4 protein and its activator LIP5 in cells .", "We speculate that these enhanced interactions are mediated by unidentified bridging factor ( s ) because the IST1 phosphorylation sites lie outside of the VPS4- and LIP5-binding sites .", "The Abscission/NoCut Checkpoint Regulator ( ANCHR ) protein delays abscission by retaining VPS4 at the Flemming body ( Thoresen et al . , 2014 ) , and it is , therefore , possible that phosphorylated IST1 may collaborate with ANCHR .", "Moreover , IST1 can also inhibit VPS4 ATPase activity by forming an inactive IST1-VPS4 heterodimer ( Dimaano et al . , 2008 ) , and phosphorylated IST1 could , therefore , delay abscission by inhibiting VPS4 ATPase activity or by inhibiting productive VPS4 recruitment by ESCRT-III proteins , including CHMP1 and IST1 , which help localize VPS4 to the midbody late in cytokinesis ( Agromayor et al . , 2009; Bajorek et al . , 2009a ) .", "We have shown that ULK3 acts in concert with Aurora B to regulate abscission through CHMP4C .", "We favor a model in which ULK3 is activated during mitosis and functions downstream of Aurora B and CHMP4C .", "This would explain why mitotic phosphorylation of IST1 requires ULK3 and CHMP4C and why ULK3 overexpression can partially overcome the requirement for CHMP4C in abscission delay .", "Given that ULK3 phosphorylates multiple ESCRT-III subunits , the initial phosphorylation of CHMP4C by Aurora B could be subsequently ‘amplified’ by ULK3 .", "Our data further show that ULK3 and Aurora B phosphorylate CHMP4C at different sites ( Carlton et al . , 2012 ) .", "This is consistent with the functional interdependence of ULK3 and CHMP4C in the abscission checkpoint and could contribute to creating a feedback loop that sustains the inhibitory signal .", "Mitotic phosphorylation of CHMP4C then decreases at ( or near ) the time of abscission , suggesting that a checkpoint-dependent phosphatase may override ULK3 activity and provide an ‘all clear’ signal to complete abscission .", "Finally , the recent identification of CHMP4C variants as risk factors for ovarian cancer suggests that abscission checkpoint defects may increase the tumorigenic potential of the daughter cells ( Pharoah et al . , 2013 ) .", "The identification of ULK3 as an essential component of the abscission checkpoint , thus , opens a new avenue in the emerging relationship between the abscission checkpoint and tumor formation ." ], [ "Yeast Y190 cells were co-transformed with 1 µg of each plasmid encoding the indicated proteins fused to the VP16 activation domain ( pHB18 ) or the Gal4 DNA-binding domain ( pGBKT7 ) .", "Co-transformants were selected on SD-Leu-Trp agar for 3 days at 30°C .", "Protein–protein interactions were determined by β-galactosidase activity in yeast extracts using chlorophenol red-β-D-galactopyranoside ( Roche , Switzerland ) as a substrate .", "HeLa and 293T cells were cultured in Dulbecco's Modified Eagle Medium ( DMEM ) containing 10% Fetal Calf serum ( FCS ) and gentamycin 20 μg/ml; ( no antibiotics were used to culture cells for experiments shown in Figures 1A , C , 5B , Figure 5—figure supplement 1 and Figure 7—figure supplement 1C ) .", "Stable cells lines were generated using MLV-based retroviruses as described previously ( Carlton and Martin-Serrano , 2007 ) and selected using puromycin ( 200 ng/ml ) or G418 ( 500 μg/ml ) .", "For live-cell imaging experiments , imaging was performed between 4 and 10 days after selection was added to the transduced cells .", "Polyethylenimine ( PEI; Polysciences , Germany ) or Lipofectamine 2000 ( Life Technologies , Carlsbad , CA ) was used to transfect plasmids into 293T and HeLa cells respectively , according to manufacturer's instructions .", "For all siRNA assays , cells were transfected twice with 100 nM of siRNA using Dharmafect-1 ( Dharmacon RNA Technologies , Lafayette , CO ) .", "For IST1 rescue experiments in Figure 6A , B , cells were transfected once with 100 nM of siRNA and fixed/imaged 12–16 hr after transfection .", "To trigger the abscission pathway by using partial depletion of NUP153 , cells were co-transfected with 10 nM of NUP153 siRNA ( diluted in NT siRNA ) .", "Two sets of guide RNA targeting the ULK3 locus were designed using the Zhang Lab website ( http://crispr . mit . edu ) and cloned into a lentiCRISPRv2 plasmid ( Addgene , Cambridge , MA ) ( Sanjana et al . , 2014 ) .", "The sequences for the two sets of guide oligos were ( quoted 5′ to 3′ ) set 1: CACCGCACGTACGCCACGGTGTACA and AAACTGTACACCGTGGCGTACGTGC; set 2: CACCGGGATCTCAATCTCCGTGAGG and AAACCCTCACGGAGATTGAGATCCC .", "Stable cell lines were generated by transfecting a 10-cm dish of 293T cells per construct , using 7 . 2 μg of lentiCRISPR , 7 . 2 μg p8 . 91 HIV Gag-pol , and 1 . 8 μg of VSV-G .", "48 hr post-transfection , released virions were collected and concentrated through a 20% sucrose gradient by ultracentrifugation at 28 , 000 rpm for 1 . 15 hr at 4°C .", "Pelleted virions were re-suspended in serum free media overnight and used to transduce HeLa cells in 6-well per plate by spinoculation for 2 hr .", "After 2 days , puromycin ( 100 ng/ml ) was used for selection and cells were maintained under selection until single cell clones were obtained by limiting dilution .", "After confirmation of efficient ULK3 knockout by Western blot , two δULK3 clones were selected and used for functional experiments .", "For the experiment shown in Figures 1A , 2 × 106 293T cells were seeded in 10-cm dishes and each dish was singly transfected 18–24 hr later with plasmids encoding Myc-tagged ESCRT-III proteins or OSF-ULK3 ( 6 µg/dish ) using PEI .", "To equalize expression levels , the following amounts of plasmids encoding Myc-tagged ESCRT-III proteins were transfected ( with empty vector used as necessary to bring the total to 10 µg ) : 10 µg each of CHMP2A-Myc , CHMP2B-Myc , CHMP4B-Myc , CHMP4C-Myc , and CHMP6-Myc; 5 µg of CHMP4A-Myc; 1 . 5 µg of CHMP1A-Myc and Myc-IST1; 0 . 25 µg of CHMP1B-Myc , CHMP3-Myc , CHMP5-Myc , and CHMP7-Myc .", "Cells were harvested 48 hr post-transfection and lysed in 50 mM Tris pH 7 . 2 , 150 mM NaCl , 0 . 5 mM MgCl2 , 5 mM β-mercaptoethanol ( BME ) , 0 . 2% Triton X-100 supplemented with DNAse I ( Roche ) , and protease inhibitors ( Sigma–Aldrich , St . Louis , MO ) .", "Lysates were clarified by centrifugation at 16 , 100×g for 10 min at 4°C .", "Lysates expressing ESCRT-III proteins were further diluted with lysate from untransfected cells to match expression levels and mixed with lysates prepared from cells expressing OSF-ULK3 .", "Mixed lysates were incubated overnight ( ∼18 hr ) at 4°C with 20 µl of pre-equilibrated streptactin sepharose resin ( IBA Lifesciences , Germany ) and washed once with wash buffer: 50 mM Tris , pH 7 . 2 , 350 mM NaCl , 5 mM BME , 0 . 5 mM MgCl2 , 0 . 2% Triton X-100 and three times with wash buffer containing 150 mM NaCl .", "After the final wash , the streptactin beads were aspirated to near dryness , and bound proteins were eluted by boiling in 20 µl of Laemmli sample buffer , resolved by SDS-PAGE , and examined by Western blotting .", "For the experiment shown in Figure 1C , 293T cells were seeded in a 6-well plate at 0 . 25 × 104 cells/well and co-transfected 18–24 hr later with 1 . 5 µg of the indicated plasmids .", "Cells were harvested 48 hr post-transfection , lysed , and clarified as described above .", "Cell lysates were incubated with 20 µl of streptactin resin for 2 hr at 4°C .", "The streptactin resin was washed four times in lysis buffer and aspirated to near dryness .", "Bound proteins were eluted by boiling in 20 µl of Laemmli sample buffer , resolved by SDS-PAGE , and examined by Western blotting .", "For co-precipitation experiments , 293T cells from 6-well plates were transfected with 1 µg of plasmids encoding the indicated pCAGGS/GST and pCR3 . 1-YFP fusion proteins .", "48 hr later , cells were lysed in 1 ml of lysis buffer ( 50 mM Tris , pH 7 . 4 , 150 mM NaCl , 5 mM EDTA , 5% glycerol , 1% Triton X-100 and a protease inhibitor cocktail [complete mini-EDTA free , Roche] ) .", "Clarified lysates were incubated with glutathione–sepharose beads ( Amersham Biosciences , Pittsburgh , PA ) for 3 hr at 4°C and washed three times with wash buffer ( 50 mM Tris , pH 7 . 4 , 150 mM NaCl , 5 mM EDTA , 5% glycerol , and 0 . 1% Triton X-100 ) .", "Bound proteins were eluted by boiling in 100 µl of Laemmli sample buffer , resolved by SDS-PAGE , and examined by Western blotting .", "4 . 5 × 106 293T cells were transfected with 20 µg of plasmids encoding HA-tagged proteins .", "After 48 hr , cells were lysed in 5 ml of lysis buffer ( 50 mM Tris , pH 7 . 4 , 150 mM NaCl , 5 mM EDTA , 5% glycerol , 1% Triton X-100 ) containing a protease inhibitor cocktail ( complete mini-EDTA free , Roche ) and a phosphatase inhibitor mixture ( PhosStop , Roche ) .", "After sonication , cleared lysates were incubated with 5 μg of monoclonal α-HA for 1 hr at 4°C , and then 40 μl of protein G-agarose for 2 hr at 4°C .", "Bead-bound complexes were washed four times in wash buffer ( 50 mM Tris , pH 7 . 4 , 150 mM NaCl , 5 mM EDTA , 5% glycerol , and 0 . 1% Triton X-100 ) , and bead-bound proteins were eluted by boiling in 80 µl of sample buffer , and examined by SDS-PAGE and Western blot .", "This approach is based on the fusion of ULK3 to the Escherichia coli modified biotin protein ligase BirA-113G , which can promiscuously biotinylate vicinal proteins in vivo that are later isolated with streptavidin-coated beads ( Roux et al . , 2012 ) .", "For these experiments , 2 . 5 × 106 HeLa cells stably expressing BirA-ULK3 were seeded in 10-cm dishes and treated overnight with 100 μM Biotin ( Sigma–Aldrich ) .", "Cells were dissociated with Phosphate Buffer Saline ( PBS ) 0 . 5 mM EDTA , washed once with 1X PBS , and lysed in 500 μl of lysis buffer ( 50 mM Tris , pH 7 . 4 , 500 mM NaCl , 5 mM EDTA , 0 . 4% SDS , 1 mM DTT and protease inhibitor cocktail ) .", "After a first pulse of sonication , Triton X-100 was added to a final concentration of 2% , samples were briefly sonicated again , and an equal volume of cold 50 mM Tris ( pH 7 . 4 ) was added prior to a final pulse of sonication .", "Lysates were cleared by centrifugation for 5 min and incubated with avidin–agarose beads ( Pierce , Rockford , IL ) for 3 hr at 4°C and washed three times at 25°C with lysis buffer .", "Bound proteins were eluted by boiling in 100 µl of Laemmli sample buffer , resolved by SDS-PAGE , and examined by Western blotting .", "Cell lysates or bead eluates were denatured in Laemmli buffer and resolved on polyacrylamide .", "Where indicated , Phos-tag ( Wako Chemicals , Japan ) and MnCl2 were added into 10% gels to induce mobility shifts in phosphorylated proteins , to final concentrations of 40 µM and 80 µM , respectively , for Figure 5B , Figure 5—figure supplement 1A and Figure 7—figure supplement 1C , or 25 µM and 50 µM , respectively , for Figures 5D , E , 7G .", "Proteins were transferred onto nitrocellulose or PVDF membranes and probed with the indicated antibody in 1% or 5% milk .", "IRDye-conjugated secondary antibodies ( Li-cor Biosciences , Lincoln , NE ) were imaged using an Odyssey infrared scanner ( Li-cor Biosciences ) .", "HRP-conjugated secondary antibodies ( Pierce ) were visualized using Amersham ECL Prime Western blotting detection reagent ( GE Healthcare , Pittsburgh , PA ) and ImageQuant LAS4000 system ( GE Healthcare ) .", "HA-tagged proteins were immunoprecipitated as described above .", "After four washes in wash buffer , bead-bound complexes were washed twice in wash buffer containing 0 . 5 M NaCl and twice in kinase assay buffer ( 50 mM Tris , pH 7 . 4 , 150 mM NaCl , 25 mM KCl , 5 mM MgCl2 , 0 . 02% Triton X-100 ) .", "Beads were resuspended in 40 μl of kinase assay buffer supplemented with 1 mM DTT .", "100 ng of recombinant His-ULK3 ( Abcam , UK ) was added on ice where required .", "2 . 5 μCi of ATP γ-32P was added and reactions were incubated with mixing at 30°C for 30 min .", "Reactions were terminated by addition of Laemmli buffer and boiling for 5 min .", "Lysates were then resolved by SDS-PAGE and transferred to nitrocellulose .", "Membranes were exposed to a storage phosphor screen , visualized using a Typhoon 9400 ( GE Healthcare ) and processed using ImageQuant .", "Subsequent Western blotting with polyclonal α-HA allowed visualization of immunoprecipitated proteins .", "Alternatively , when using ULK3 GST-fusion plasmids , 2 . 2 × 106 293T cells were co-transfected with 2 µg of the indicated GST plasmid .", "After 48 hr , cells were harvested and lysed in 2 ml of GST pull-down lysis buffer .", "Cleared lysates were incubated with 25 μl glutathione–sepharose beads ( Amersham Biosciences ) for 2 hr at 4°C and washed twice with wash buffer , twice with wash buffer/0 . 5 M NaCl , and twice with kinase assay buffer .", "Kinase assays proceeded as described above .", "IST1 ( residues 316–366 , expressed as a His-SUMO-fusion protein ) and GST-fusions of ULK3 ( MIT ) 2 ( residues 277–449 ) ( WT and V338D or M434D mutants ) and ULK3 MIT2 ( 359–449 ) were each expressed in 1 L cultures of BL21-Codon Plus ( DE3 ) RIPL cells ( Agilent , Santa Clara , CA ) in ZYP-5052 auto-induction media ( Studier , 2005 ) .", "For crystallographic studies , ULK3 ( MIT ) 2 was additionally expressed in 2 L auto-induction PA-5052 media containing selenomethionine ( SeMet ) ( Studier , 2005 ) .", "All purification steps were performed at 4°C .", "For GST-fusions , cells were resuspended in lysis buffer ( 40 ml/L of culture ) containing 50 mM Tris pH 8 . 0 , 150 mM NaCl , 0 . 5 mM EDTA , 2 mM DTT , 0 . 125% sodium deoxycholate supplemented with protease inhibitors ( aprotinin , leupeptin , pepstatin , and PMSF ) , lysozyme , and DNAse I ( Roche ) .", "Cells were lysed by sonication , and the cell lysate was clarified by centrifugation for 45 min at 32 , 000×g .", "Clarified lysate was incubated for 1–2 hr with 15 ml GST resin ( GE Healthcare ) equilibrated with binding buffer: 50 mM Tris , pH 8 . 0 , 150 mM NaCl , 2 mM DTT , and washed with 10 column volumes of binding buffer containing 500 mM NaCl and 10 column volumes of 150 mM NaCl-containing binding buffer .", "GST-affinity tags were removed from resin-bound ULK3 proteins by overnight incubation with PreScission Protease ( 0 . 3 mg ) in 40 ml of GST-binding buffer at 4°C .", "Cleaved proteins were collected from the column flow through and dialyzed against Q-sepharose-binding buffer ( 20 mM Tris , pH 8 . 0 , 50 mM NaCl , 2 mM DTT , 0 . 5 mM EDTA ) for further purification by Q-sepharose anion exchange chromatography ( GE Healthcare Life Sciences ) with a linear gradient from 50 to 500 mM NaCl .", "Fractions containing ULK3 were pooled and dialyzed against gel filtration buffer ( 25 mM Tris , pH 7 . 2 , 150 mM NaCl , 2 mM DTT , 0 . 5 mM EDTA ) and further purified by Superdex-75 size exclusion chromatography ( GE Healthcare Life Sciences ) .", "Typical protein yields were ∼15–20 mg/L culture in ZYP-5052 ( rich ) media and 2 . 5 mg/L culture in PA-5052 SeMet media .", "Cells expressing His-SUMO-IST1316-366 were lysed by sonication in buffer ( 40 ml/L of culture ) containing 50 mM Tris , pH 7 . 2 , 200 mM NaCl , 5 mM imidazole , 5 mM DTT , 0 . 5 mM EDTA , and 0 . 125% sodium deoxycholate , supplemented with lysozyme , protease inhibitors , and DNAse I ( Roche ) .", "Clarified cell lysate was incubated with 10 ml of cOmplete His-Tag purification resin ( Roche ) for 20 min , washed with 10 column volumes of wash buffer: 50 mM Tris , pH 7 . 2 , 500 mM NaCl , 5 mM Imidazole , 5 mM DTT , 0 . 5 mM EDTA followed by 10 column volumes of wash buffer containing 150 mM NaCl .", "The His-SUMO affinity tag was removed from resin bound IST1 peptide by overnight incubation with UPL1 protease ( 0 . 1 mg ) in 40 ml of the 150 mM NaCl wash buffer at 4°C .", "The cleaved IST1 peptide was collected from the column flow through and further purified by anion exchange and gel filtration chromatography as described above for ULK3 proteins .", "Typical IST1 peptide yields were 4 . 5 mg/L culture .", "Purified IST1 and ULK3 proteins contain non-native ‘GC’ and ‘GPHM’ residues at their N-termini , respectively .", "Masses of purified proteins were confirmed by ESI/MS as follows: IST1313-366 calculated = 5733 Da and experimental = 5732 Da; ULK3 ( MIT ) 2 calculated = 19 , 355 Da and experimental 19 , 355 Da; ULK3 ( MIT ) 2 M434D calculated = 19 , 399 Da and experimental = 19 , 399 Da; ULK3 ( MIT ) 2 V338D calculated = 19 , 371 Da and experimental = 19 , 371 Da; ULK3 MIT2 calculated = 10 , 535 Da , and experimental = 10 , 535 Da .", "Uniformly enriched 13C , 15N-IST1303-366 was expressed as a TEV-cleavable GST fusion protein in 1 L of M9 minimal medium containing 2 g each of 15N-ammonium chloride and 13C-glucose .", "Cells were lysed by sonication in 40 ml of lysis buffer: 50 mM Tris pH 8 . 0 , 100 mM NaCl , 1 mM DTT , 0 . 125% sodium deoxycholate , 0 . 1% Tween-20 in the presence of protease inhibitors ( aprotinin , leupeptin , pepstatin , and PMSF ) and lysozyme .", "Lysates were clarified as described above , and the supernatant was incubated with ∼15 ml of GST-sepharose resin ( GE Healthcare Life Sciences ) overnight at 4°C .", "The resin was washed with ∼10 column volumes of 20 mM Tris , pH 8 . 0 , 100 mM NaCl , 1 mM DTT , and bound protein was eluted in wash buffer containing 20 mM reduced l-glutathione .", "Eluted 15N , 13C-IST1303-366 was dialyzed against 20 mM Tris , pH 8 . 0 , 100 mM NaCl , 1 mM DTT in the presence of TEV protease ( at room temperature ) to remove the GST tag .", "Following TEV cleavage , the IST1 peptide was dialyzed into Q-sepharose buffer: 20 mM Tris pH 8 . 0 , 50 mM NaCl , 0 . 5 mM DTT , and purified by ion-exchange chromatography over a Q-sepharose column ( GE Healthcare Life Sciences ) using a linear gradient of 50–500 mM NaCl over ∼18 column volumes .", "Fractions containing 15N , 13C-IST1303-366 were exchanged into 20 mM sodium phosphate pH 6 . 2 , 25 mM NaCl , 0 . 1 mM EDTA , 0 . 1 mM NaN3 , 0 . 5 mM DTT , and 10% D2O for NMR spectroscopy .", "The yield of purified 15N , 13C-IST1303-366 peptide was ∼1 . 4 mg/L cell culture .", "The expected protein mass was confirmed by ESI/MS of an unlabeled sample: calculated = 7217 Da and experimental = 7216 Da .", "NMR data were recorded on a Varian INOVA 600 MHz spectrometer equipped with a cryogenic probe , processed using FELIX 2007 ( Felix NMR , Inc . ) , and analyzed using SPARKY3 ( Goddard and Kneller , 2001 ) , AutoAssign ( Zimmerman et al . , 1997 ) , and NMRViewJ ( OneMoon Scientific ) .", "IST1303-366 backbone resonances were assigned using standard triple resonance experiments ( Cavanagh , 2007 ) from a sample containing 0 . 4 mM uniformly 13C- and 15N-enriched IST1 in 20 mM sodium phosphate pH 6 . 2 , 25 mM NaCl , 0 . 1 mM EDTA , 0 . 1 mM NaN3 , 0 . 5 mM DTT , and 10% D2O .", "The chemical shifts for IST1 have been deposited in the Biological Magnetic Resonance Bank under accession number 25393 .", "Chemical shift perturbations induced by ULK3 binding to IST1 were identified by titrating 0 . 4 mM uniformly 13C- and 15N-enriched IST1 with increasing amounts of unlabeled ULK3 to final stoichiometries of ( IST1 : ULK3 ) : 0 , 0 . 25 , 0 . 5 , 1 . 0 , 1 . 1 , and 2 . 0 .", "The complex was in slow exchange on the NMR time scale .", "Chemical shifts were analyzed in NMRViewJ ( OneMoon Scientific ) , and IST1 resonances that were shifted more than ½ peak width in the bound complex were given a score of ‘+1’ .", "Unperturbed resonances were scored as ‘−1’ and resonances that could not be unambiguously assigned to either category owing to spectral overlap were scored as ‘0’ .", "IST1 MIMs peptide ( residues 316–366 , containing a non-native N-terminal ‘GC’ dipeptide ) was expressed and purified as a His-SUMO fusion protein as described above .", "MIM1 ( residues 344–366 , containing a non-native , N-terminal cysteine ) and MIM2 ( residues 316–343 , containing a non-native N-terminal ‘GC’ dipeptide ) peptides were synthesized , purified , and fluorescently labeled in the University of Utah Peptide Synthesis Core .", "Briefly , MIM1 and MIM2 peptides were synthesized on an ABI 433 synthesizer ( Applied Biosystems , Waltham , MA ) with a cysteine at the N-terminus using Fmoc solid phase technology , common protecting groups , and HBTU chemistry on an ABI 433 synthesizer ( Applied Biosystems ) .", "Both recombinant and synthesized peptides were purified by reversed phase chromatography using acetonitrile/water gradients with 0 . 1% trifluoroacetic acid in both solvents .", "Peptide-containing fractions were pooled and dried , then re-dissolved in DMSO .", "Fluorescent labeling was performed in DMSO at 4°C with approximately 1 . 3-fold molar excess of Oregon Green 488 maleimide ( Life Technologies/Molecular Probes 06 , 034 ) dissolved in a 1:1 solution of acetonitrile:DMSO .", "The reaction progress was monitored by HPLC , and labeled peptides were separated from free dye and residual unlabeled peptides using the same reversed phase conditions described above .", "Labeled peptide masses were measured by MALDI-TOF-MS at the University of Utah Mass Spectrometry Core facility: dye-labeled MIMs , calculated = 6196 Da and experimental = 6192 Da; dye-labeled MIM1 , calculated = 3298 Da and experimental = 3296 Da; and dye-labeled MIM2 , calculated = 3483 Da and experimental = 3480 Da .", "Confirmed peptide fractions were dried under vacuum , redissolved in water , and concentrations were calculated using the absorbance of Oregon Green 488 at 491 nm ( Extinction coefficient 83 , 000 cm−1 M−1 in 50 mM potassium phosphate , pH 9 ) .", "FP experiments were performed in 60-µl binding reactions in binding buffer: 25 mM Tris , pH 7 . 2 , 150 mM NaCl , 0 . 1 mg/mL Bovine Serum Albumin ( BSA ) , 0 . 01% Tween-20 , and 1 mM Dithiothreitol ( DTT ) using 250 pM fluor-labeled IST1 peptides and twofold dilutions of ULK3 proteins .", "FP was measured using a Tecan Infinite 200 plate reader with excitation at 485 nm and detection at 535 nm .", "Dissociation constants were calculated by fitting the increase in FP to a 1:1 binding equation using KaleidaGraph ( Synergy Software ) as described previously ( Skalicky et al . , 2012 ) .", "Each binding isotherm was measured at least three times independently , and mean KD values are reported ±SD .", "IST1 protein was mixed in a 1:1 molar ratio with native or SeMet-substituted ULK3 MIT2 protein to a final concentration of 10 mg/ml in 20 mM Tris , pH 8 . 0 , 100 mM NaCl , 2 mM DTT , and 3 mM NaN3 .", "ULK3:IST1 complexes were crystallized at 21°C in sitting drops by mixing equal volumes ( 2 μl ) of protein complex solution and precipitant solution ( 1 . 6–1 . 8 M ammonium sulfate , 0 . 1 M MES pH 6 . 4–6 . 7 , 0 . 01 M CoCl2 ) .", "Crystals were transferred to a cryoprotectant solution containing precipitant supplemented with 30% glycerol , looped , and flash frozen in liquid nitrogen prior to data collection .", "X-ray diffraction data were collected remotely ( Soltis et al . , 2008 ) at SSRL beamlines 11-1 and 12-2 .", "All data were processed using AutoXDS ( Gonzalez and Tsai , 2010; Kabsch , 2010a; 2010b ) at SSRL .", "Initial attempts to crystallize ULK3 ( MIT ) 2 ( residues 277–449 ) in complex with both IST1 MIMs ( residues 316–366 ) yielded crystals of a proteolytically truncated complex comprising ULK3 MIT2 and IST1 MIM1 fragments .", "The structure of this complex was determined using a data set derived from a crystal of SeMet-substituted ULK3 ( MIT ) 2 in complex with IST1 MIMs , in which both components had undergone proteolysis .", "The SeMet ULK3/IST1 complex was solved in space group P3121 ( a = 82 . 68 Å , b = 82 . 68 Å , c = 90 . 15 Å ) with single-wavelength anomalous dispersion data at 0 . 9791 Å ( 2 . 1 Å resolution , Figure 1—source data 1 ) .", "PHENIX ( Hybrid Structure Search; HySS ) ( Afonine et al . , 2012; Grosse-Kunstleve and Adams , 2003; McCoy et al . , 2004 ) was used to locate and refine the positions for 3 of 4 possible SeMet sites in ULK3 MIT2 .", "Phases were calculated and used to produce a 3 . 2 Å electron density map .", "Models for the three MIT molecules in the asymmetric unit were initially built into the electron density and the ULK3:IST1 models were rebuilt de novo in Coot ( Emsley et al . , 2010 ) and refined in PHENIX .", "The refined SeMet model was then used as an initial molecular replacement search model to determine structures from higher resolution native data sets ( Phaser in PHENIX ) .", "The protein termini were identified in the proteolyzed structure and used to design new constructs ( ULK3 MIT2: residues 359–449; IST1 MIM1 residues 344–366 ) for further crystallization .", "ULK3 MIT2 was expressed and purified as a recombinant protein ( described above ) , and IST1 MIM1 was synthesized by the University of Utah Peptide Synthesis Core .", "The complex of the shorter constructs crystallized in space group R32 ( a = 79 . 12 Å , b = 79 . 12 Å , c = 96 . 62 Å ) with a single ULK3 MIT2:IST1 MIM1 complex in the asymmetric unit and the crystal diffracted to 1 . 38 Å resolution ( Figure 1—source data 1 ) .", "This structure is described herein , and data collection and refinement statistics are provided in Figure 1—source data 1 .", "Structure coordinates have been deposited in the RCSB Protein Data Bank under PDB ID 4WZX .", "Chimera was used to analyze structures and generate figures ( Pettersen et al . , 2004 ) .", "For experiments shown in Figure 5B , Figure 5—figure supplement 1A , and Figure 7—figure supplement 1C , 293T cells were co-transfected with PEI and plasmids encoding Myc-tagged ESCRT-III proteins and OSF-ULK3 WT , or OSF-ULK3-K44H or empty vector .", "For IST1 , CHMP1A , CHMP1B , and CHMP3 experiments , transfections were performed in a 6-well plate seeded 18–24 hr earlier with 0 . 25 × 104 293T cells/well with the following plasmid amounts: 500 ng of OSF-ULK3 WT , 1 . 5 µg of OSF-ULK3 K44H , 1 . 5 µg Myc-IST1 , 1 µg CHMP1A-myc , 1 µg CHMP1B-myc , and 1 µg CHMP3-Myc .", "Each transfection was brought to a total of 3 µg DNA with empty vector .", "CHMP4C and CHMP2A experiments were performed using cell lysates from 10-cm dishes seeded at 2 . 2 × 106 and transfected 18–24 hr later .", "Transfections contained the following plasmid quantities: 6 µg CHMP4C-Myc , 6 µg CHMP1A-Myc and 3–4 µg OSF-ULK3 , 6 µg of OSF-ULK3 K44H , or 6 µg of empty vector .", "All transfections were brought to a total of 12 µg DNA with empty vector .", "Cells were harvested 48 hr post-transfection and lysed in buffer containing 50 mM Tris pH 7 . 2 , 150 mM NaCl , 5 mM BME , 0 . 2% Triton X-100 , 10 mM MgCl2 supplemented with DNAse I ( Roche ) and protease inhibitors ( Sigma–Aldrich MOP8340 ) .", "Cell lysates were clarified as described above .", "Where noted , 25 µl of each lysate was additionally treated with 40 units of CIP ( New England Biolabs , Ipswich , MA ) and incubated at 37°C for 1 hr .", "Gel samples were boiled in Laemmli sample buffer both pre-and post-CIP treatment , resolved by SDS-PAGE , and examined by Western blotting .", "Ten 10-cm dishes of 293T cells were co-transfected ( PEI ) with 6 µg of plasmids expressing OSF-ULK3 or OSF-ULK3 K139R ( kinase inactive Maloverjan et al . , 2010 ) , and 6 µg of Myc-IST1 .", "Transfected cells were harvested after 48 hr and lysed by sonication in 50 mM Tris pH 7 . 2 , 150 mM NaCl , 0 . 5 mM MgCl2 , 5 mM DTT ( or BME ) in the presence of DNase I ( Roche ) , Phos-Stop ( Roche ) , and mammalian protease inhibitors ( Sigma–Aldrich ) .", "Cell lysates were clarified by centrifugation at 16 , 100×g at 4°C for 10 min , and incubated with 75 µl of streptactin resin ( IBA-Lifesciences for 2 hr at 4°C .", "Bound ULK3/IST1 complexes were washed once with lysis buffer containing 500 mM NaCl , three times with lysis buffer ( 150 mM NaCl ) , and eluted with lysis buffer containing 10 mM d-desthiobiotin .", "The OSF-ULK3 K139R/IST1 elution was additionally incubated with CIP ( New England Biolabs , Ipswich , MA ) at 37°C for 1 hr .", "Samples were purified prior to mass spectrometry analysis using a C18 ZipTip ( Millipore , Temecula , CA ) to remove salts and other small molecule contaminants according to the manufacturer's instructions .", "Proteins were eluted from the ZipTip with three consecutive 0 . 75 µl washes of 70% methanol , 1% formic acid , and one 1 µl wash of 98% methanol , 1% formic acid , with 2 µl of 1% formic acid added prior to ESI/MS analysis .", "ESI/MS analyses of intact proteins were performed using a Quattro-II mass spectrometer ( Micromass , Inc . UK ) .", "The ZipTip eluant was infused into the instrument at 3 µl/min flow rate .", "Data were acquired with a cone voltage of 50 eV , spray voltage of 2 . 8 kV , and scanning from 600 to 1400 Da in 4 s .", "Scans were accumulated for ∼1 min .", "Spectra were combined and multiply charged molecular ions were deconvoluted into a molecular mass spectrum using MaxEnt 3 . 4 software ( Micromass , Inc . ) .", "To identify phosphorylation sites , co-purified IST1 and ULK3 proteins were digested in solution with a TPCK-modified trypsin and Lys-C protease mixture ( Promega , Madison , WI ) or chymotrypsin ( Princeton , Adelphia , NJ ) .", "Trypsin or chymotrypsin ( in 50 mM ammonium bicarbonate ) was added to the solution ( adjusted to pH 7 . 9 ) at a ratio of approximately 1:25 ( enzyme: protein ) .", "Samples were digested at 37°C overnight with trypsin or for 2–2 . 5 hr with chymotrypsin .", "Immobilized metal affinity chromatography ( IMAC ) was used to enrich phospho-peptides from the trypsin- and chymotrypsin-digested proteins .", "The IMAC procedure was performed using SwellGel gallium ( III ) chelated mini-spin columns ( Pierce ) , with minor modifications to the manufacturer's recommended procedure .", "Briefly , the trypsin- and chymotrypsin-digested proteins were acidified ( pH < 3 ) with 10% acetic acid .", "The samples were then incubated on the column for 15 min , washed twice with 50 µl of 0 . 1% acetic acid , twice with 50 µl of 0 . 1% acetic acid in 10% acetonitrile , and once with 75 µl of nanopure water .", "Phosphopeptides were eluted with two 20-µl volumes of 25 mM ammonium bicarbonate ( pH 9; adjusted with ammonium hydroxide ) , followed by 20 µl of 25 mM ammonium bicarbonate in 50% acetonitrile .", "All three eluant fractions were combined , dried down , and reconstituted in a solution of 5% acetonitrile with 0 . 1% formic immediately prior to nano-LC-MS/MS analysis .", "Both phospho-enriched and un-enriched peptides were analyzed using a nano-LC/MS/MS system equipped with a nano-HPLC pump ( 2D-ultra , Eksigent , Redwood City , CA ) and an ESI-LTQ-FT-ICR mass spectrometer equipped with a nanospray ion source ( ThermoElectron Corp . , Waltham , MA ) .", "Approximately 5 µl of peptide samples were injected onto a dC18 nanobore LC column ( 75 μm ID × 100 mm length , 3 µm particles , made in house , Atlantis , Waters Corp . , Milford , MA ) .", "Peptides were separated and eluted over a linear gradient of 5–96% acetonitrile in 0 . 1% formic acid with a constant total flow rate of 350 nl/min over 78 min .", "The LTQ-FT-ICR mass spectrometer was operated in the data-dependent acquisition mode with the ‘top 10’ most intense peaks observed in an FT primary scan selected for on-the-fly peptide fragmentation MS/MS acquisitions in the LTQ linear ion trap portion of the instrument .", "The LTQ linear ion trap was operated with the following parameters: precursor activation time 30 ms and activation Q at 0 . 25; collision energy at 35%; dynamic exclusion at low mass of 0 . 1 Da and high mass at 2 . 1 Da with one repeat count and 10 s duration .", "Spectra in the LTQ-FT-ICR were acquired from m/z 350 to 1400 Da at 50 , 000 resolving power with mass accuracies typically within 3 ppm mass error .", "Raw data files were processed with BioWorks software ( ThermoElectron Corp . ) to generate peak lists ( DTA ) .", "Resulting DTA files from each data acquisition file were searched to identify phosphopeptides against custom databases using the MASCOT search engine ( Matrix Science Ltd . ; version 2 . 2 . 7; in-house licensed ) .", "Molecular ions with +1 , +2 , or +3 charge states determined from an ESI-FTMS primary mass spectrum ( LTQ-FT instrument ) were typically considered .", "Searches for trypsin- or chymotrypsin-specific peptide cleavages allowed two missed cleavages , and a mass error tolerance of 5 ppm in the ESI-FT-ICR data and 0 . 5 Da for MS/MS ions .", "Peptide modifications included in the searches were oxidation on Met and/or phosphorylation on Ser , Thr , or Tyr residues .", "Identified peptides were accepted when the MASCOT ion score value exceeded 20 , mass errors were less than 5 ppm , and expect values were less than 1 .", "Peptide and phosphopeptide assignments were also manually validated .", "Sequence coverage of Myc-IST1 was 94% , with identified peptides encompassing 39 out of the 40 Ser and Thr residues .", "IST1 S4 , S99 , S153 , and S214 phosphorylations were identified in 1 , 2 , 4 , and 1 unique peptides , respectively .", "Phosphorylation sites were mapped onto the structure of IST1 ( PDB ID 3FRR ) ( Bajorek et al . , 2009b ) using PyMOL ( Version 1 . 3 , Schrödinger , LLC , New York , NY ) .", "Cells were incubated in 2 mM thymidine DMEM for 18 hr , washed with PBS , released into complete DMEM for 4 hr , and arrested in mitosis using DMEM containing 50 ng/ml nocodazole .", "Mitotic cells were collected the next day by shake-off , washed in PBS , and lysed in sample buffer .", "Asynchronous vehicle-treated ( DMSO ) cultures were lysed at equivalent time points .", "For siRNA treatments , cell synchronization was initiated 24 hr after the second transfection .", "Where indicated , the Aurora B inhibitor ZM447439 ( Santa Cruz Biotechnology , Dallas , TX ) was used at 1 μM final concentration .", "HeLa cells stably expressing KSHV-K3 ( KK3 ) were a kind gift from Prof Paul Lehner ( University of Cambridge , UK ) .", "HeLa KK3 cells ( Hewitt et al . , 2002 ) were seeded in a 12-well plate and transfected with siRNA ( final concentration 100 nM ) as described above .", "24 hr after the second siRNA transfection , cells were harvested with PBS 0 . 5 mM EDTA , washed once in cold PBS , and incubated with PBS containing 2% serum and a FITC-conjugated α-MHC-I antibody ( W6/32 clone , AbD Serotec ) for 1 hr at 4°C .", "After washing with PBS 2% serum , cells were resuspended and fixed in 4% paraformaldehyde .", "Surface MHC-I staining was analyzed by flow cytometry on a FACS-Calibur ( Becton–Dickinson , UK ) .", "HT1080/THN-HA and HT1080/THN-HA K5 have been previously described ( Pardieu et al . , 2010 ) .", "Cells were seeded in 12-well plates and transfected with siRNA ( final concentration 100 nM ) as described above .", "72 hr after the first siRNA transfection , cells were harvested with PBS 0 . 5 mM EDTA and resuspended in sample buffer .", "Total Tetherin-HA levels were analyzed by Western blot using α-HA and α-HSP90 antibodies , and visualized using Li-Cor secondary antibodies .", "293T cells were transfected with 100 nM of siRNA as described above .", "48 hr later , cells were co-transfected with an additional 100 nM of siRNA and 300 ng of HIV pNL/HXB provirus using Lipofectamine 2000 .", "After 48 hr , indicator HeLa-TZM-bl cells ( CD4+ , CXCR4+ , CCR5+ , HIV-1 LTR-LacZ ) were infected with 100 µl of harvested supernatant from 293T cells .", "After an additional 48 hr , β-galactosidase activity in cell lysates was measured using the chemiluminescent detection reagent Galacto-Star ( Applied Biosystems ) .", "In parallel , culture supernatants collected 48 hr after initial transfection were clarified by low-speed centrifugation , and virions were obtained through a 20% sucrose cushion ( 14 , 000 rpm , 2 hr ) .", "Viral protein contents in cells and particle lysates were analyzed by Western blot using α-Gag antibody .", "Cells were fixed with 4% PFA ( 20 min ) or 0 . 1% Tween-20 , 2% PFA in PBS ( 10 min ) , and 100% methanol ( 2 min ) .", "Cells were blocked with PBS 1% BSA or 3% FCS in PBS , stained with the indicated primary antibodies and Alexa 594 , 488 , or 647 conjugated secondary antibodies ( Invitrogen ) .", "Nuclei were visualized with Hoechst 33258 .", "Coverslips were mounted in Mowiol .", "Samples were imaged using an AOBS SP2 confocal microscope ( Leica , 60 × 1 . 4 N . A . oil-immersion objective ) .", "The AOTF was used to collect relevant narrow emission-λ windows for each fluorophore .", "Cells connected by midbodies were identified following tubulin staining , excluding multinucleated cells .", "For Figure 4 and Figure 4—figure supplement 1 , data were collected using an Eclipse Ti-E Inverted CSU-X1 Spinning Disk Confocal ( Nikon , Japan ) equipped with an Ixon3 EM-CCD camera ( Andor , UK ) .", "Images were acquired in series of 0 . 1 µm-spaced Z-stacks with a 100x objective .", "Deconvolution of 3D stacks was done using AutoQuant X3 Deconvolution Software ( Media Cybernetics , Rockville , MD ) ; projections were obtained using NIS-Elements Ar Microscope Imaging Software and merged using Photoshop .", "HeLa cells stably expressing YFP/mCherry-Tubulin and/or YFP-LAP2β were seeded on poly-L-lysine coated glass-bottomed 24-well plates ( MatTek ) and transfected with siRNA as specified .", "Except where indicated , 24 hr after the second transfection cells were imaged for 24 hr on a Nikon Ti-Eclipse wide-field inverted microscope ( Nikon 40 × 0 . 75 N . A . dry objective lens ) equipped with Perfect Focus system and housed in a 37°C chamber ( Solent Scientific , UK ) fed with 5% CO2 .", "Multiple fields of view were selected at various XY coordinates , where 3 slices were captured at a 1 . 25 μm Z-spacing .", "Images were acquired every 10 min using a CoolSnap HQ2 CCD camera ( Photometrics , Tucson , AZ ) , controlled by NIS-Elements software .", "Frame-by-frame analysis was performed within NIS-Elements , where abscission time was quantified as the period between midbody formation and severing .", "Midbody formation was scored as the first frame where two separate cells connected by a compacted bundle of tubulin and fully reformed nuclei were observed .", "YFP-LAP2β expressing cells were imaged for 72 hr , and chromatin bridge resolution time was scored as the time between nuclear envelope reassembly and bridge resolution .", "Intercellular bridges that regressed or left the field of view were excluded .", "For experiments with Aurora B inhibitor , cells at midbody stage were identified prior to treatment with 1 μM ZM447439 ( Santa Cruz Biotechnology ) , when imaging was initiated .", "Only cells at midbody stage where followed and time to abscission was scored following the same criteria as above .", "Selected TIF files were exported and assembled in Adobe Photoshop to generate movies with a 200-ms delay between each frame .", "All statistical significance was tested using the Mann–Whitney two-tailed U test: ***p < 0 . 0001 , **p = 0 . 0003 , *p < 0 . 05 , ns p > 0 . 1 ." ] ]

[ "The endosomal sorting complexes required for transport ( ESCRT ) machinery mediates the physical separation between daughter cells during cytokinetic abscission .", "This process is regulated by the abscission checkpoint , a genome protection mechanism that relies on Aurora B and the ESCRT-III subunit CHMP4C to delay abscission in response to chromosome missegregation .", "In this study , we show that Unc-51-like kinase 3 ( ULK3 ) phosphorylates and binds ESCRT-III subunits via tandem MIT domains , and thereby , delays abscission in response to lagging chromosomes , nuclear pore defects , and tension forces at the midbody .", "Our structural and biochemical studies reveal an unusually tight interaction between ULK3 and IST1 , an ESCRT-III subunit required for abscission .", "We also demonstrate that IST1 phosphorylation by ULK3 is an essential signal required to sustain the abscission checkpoint and that ULK3 and CHMP4C are functionally linked components of the timer that controls abscission in multiple physiological situations ." ]

[ "Our cells multiply by dividing into two .", "Many proteins are involved in this process , including a group called the ESCRT-III complex .", "This group is required to complete the final stage of cell division when the single membrane that surrounds the two new daughter cells separates .", "Before the cell divides , its DNA—which is packaged in structures called chromosomes—is copied , and the two sets of chromosomes are pulled to opposite ends of the cell .", "This ensures that each daughter cell will have a complete set of DNA .", "If the cell divides before the chromosomes have finished moving to opposite ends of the cell , the daughter cells may end up with the wrong number of chromosomes .", "This can lead to cancer or other diseases .", "To prevent this , cells have evolved a quality control system called the ‘abscission checkpoint’ , which delays cell division until the chromosomes have properly separated .", "Previous studies have shown that when the checkpoint is active , an ESCRT-III complex protein called CHMP4C is inactivated by an enzyme , which prevents the cell from dividing .", "Other signals that indicate that the new daughter cells are not yet ready to separate can also delay cell division , but it is not clear how those defects are detected by the checkpoint .", "Here , Caballe , Wenzel et al . found that a protein called ULK3 can bind to several proteins in the ESCRT-III complex , including one called IST1 .", "In doing so , ULK3 is able to delay cell division if the chromosomes have not finished separating , if there are defects in the nucleus of the cell , or if the cell is experiencing high levels of mechanical tension at the site where the membrane will separate .", "The experiments also show that ULK3 needs to bind to and regulate the activity of IST1 to sustain the abscission checkpoint , and that CHMP4C is required for this process .", "Caballe , Wenzel et al . 's findings reveal that ULK3 plays an essential role in controlling when a cell divides and imply that there may be additional proteins involved that release cells from the checkpoint delay imposed by ULK3 .", "The next challenges will be to identify these proteins and to understand how all checkpoint proteins work together to regulate cell division ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "ecology", "microbiology and infectious disease" ]

[ [ "Pelagic zones of the world's oceans seemingly constitute rather homogenous habitats , however , they feature enough spatial and temporal variation to support a large number of species with distinct niches .", "This phenomenon has been termed 'paradox of the plankton' by G . Evelyn Hutchinson ( Hutchinson , 1961 ) .", "Interactions within planktonic microbial communities are manifold and complex ( see Amin et al . , ( 2012 ) and Worden et al . ( 2015 ) for reviews ) .", "Still , planktonic microbial communities are simple in comparison to benthic or terrestrial soil communities and thus particularly suitable for the study of microbial community composition dynamics .", "In recent years , continuous biodiversity studies at long-term sampling stations have started to reveal discernible deterministic patterns within marine microbial plankton communities ( see Fuhrman et al . ( 2015 ) for a recent review ) .", "This is particularly true for less dynamic oligotrophic oceanic regions that are dominated by the members of the alphaproteobacterial Pelagibacteriaceae ( SAR11 clade ) and the cyanobacterial Prochlorococcaceae ( Prochlorococcus marinus ) .", "By contrast , more dynamic eutrophic coastal regions are subject to frequent system perturbations and thus seldom in a state of equilibrium .", "This can lead to apparently stochastic changes in bacterioplankton community composition .", "To capture recurrence of biodiversity patterns in such coastal areas , sampling must occur at the order of weekly to sub-weekly time scales over multiple years .", "Owing to the lack of such intensively sampled long-term time series data , our current understanding of the extent and predictability of recurring microbial biodiversity patterns for such marine habitats is still limited .", "A particularly important connection in the marine carbon cycle exists between marine microalgae as primary producers and heterotrophic bacteria that feed on algal biomass .", "Global photosynthetic carbon fixation is estimated to exceed 100 Gigatons yearly , of which marine algae contribute about half ( Falkowski et al . , 1998; Field et al . , 1998; Sarmento and Gasol , 2012 ) .", "Planktonic uni- to pluricellular algae such as diatoms , haptophytes , and autotrophic dinoflagellates are the most important marine primary producers .", "Diatoms alone are estimated to contribute 20–40% to global carbon fixation ( Nelson et al . , 1995; Mann , 1999; Armbrust , 2009 ) .", "Primary production by planktonic microalgae differs from primary production by sessile macroalgae or land plants as it is much less constant , but culminates in blooms that are often massive , as occurs worldwide during spring blooms from temperate to polar regions .", "These blooms are highly dynamic phenomena that are time-limited by nutrients , predator grazing and viral infections .", "Bloom termination results in a short-lived massive release of algal organic matter that is consumed by dedicated clades of heterotrophic bacterioplankton .", "This trophic connection leads to synchronized blooms of planktonic bacteria during phytoplankton blooms , as has been described in various studies ( Bell and Kuparinen , 1984; Niu et al . , 2011; Tada et al . , 2011; Teeling et al . , 2012; Yang et al . , 2015; Tan et al . , 2015 ) .", "The activities of these heterotrophic bacteria impact the proportion of algal biomass that is directly mineralized and released back into the atmosphere mostly as carbon dioxide , and the algae-derived biomass that sinks out to the bottom of the sea as carbonaceous particles .", "These are further remineralized by particle-associated bacteria while sinking and by benthic bacteria when reaching the sediment , even in the deep sea ( e . g . Ruff et al . , 2014 ) .", "The remainder is buried for a long time as kerogen and forms the basis for future oil and gas reservoirs .", "The ratio between bacterial mineralization and burial of algae-derived organic matter thus has a profound influence on the atmospheric carbon dioxide concentration ( Falkowski et al . , 1998 ) .", "However , the bulk of bacteria during phytoplankton blooms are free-living and not attached to particles or algae .", "These bacteria play a pivotal role in the mineralization of algae-derived non-particulate dissolved organic matter ( DOM ) .", "The bacterial clades that respond most to phytoplankton blooms belong to the classes Flavobacteriia ( phylum Bacteroidetes ) and Gammaproteobacteria , and the Roseobacter clade within class Alphaproteobacteria ( Buchan et al . , 2014 ) .", "This response is typically not uniform , but consists of a series of distinct clades that bloom one after another .", "In the year 2009 , we investigated the response of bacterioplankton to a diatom-dominated spring phytoplankton bloom in the German Bight ( Teeling et al . , 2012 ) .", "Within the free-living bacteria ( 0 . 2 to 3 µm ) we observed a swift succession of bacterial clades that were dominated by Flavobacteriia and Gammaproteobacteria , with consecutively blooming Ulvibacter ( Flavobacteriia ) , Formosa ( Flavobacteriia ) , Reinekea ( Gammaproteobacteria ) , Polaribacter ( Flavobacteriia ) genera and SAR92 ( Gammaproteobacteria ) as prominent clades .", "Using time-series metagenome and metaproteome analyses , we demonstrated that the substrate-spectra of some of these clades were notably distinct .", "The succession of bacterioplankton clades hence constituted a succession of distinct gene function repertoires , which suggests that changes in substrate availability over the course of the bloom were among the forces that shaped the bacterioplankton community .", "Dominance of bottom-up over top-down control is assumed to be characteristic for the initial phases of spring phytoplankton blooms .", "After winter , inorganic nutrients are aplenty , and the overall abundance of microbes is low .", "When suitable temperature and sunlight conditions are met in spring , algae and subsequently bacteria can enter an almost unrestricted proliferation .", "In contrast , predators such as flagellates , protists and zooplankton can only start proliferating when their food sources are available in larger numbers .", "Hence , top-down control by predation sets in only during later bloom phases .", "This situation is distinct from summer and fall phytoplankton blooms .", "Pronounced differences between blooming clades were found in the gene frequencies and protein expression profiles of transporters and carbohydrate-active enzymes ( CAZymes; [Cantarel et al . , 2009; Lombard et al . , 2014] ) , such as glycoside hydrolase ( GH ) , polysaccharide lyase ( PL ) , carbohydrate esterase ( CE ) , or carbohydrate-binding module ( CBM ) containing genes .", "The latter indicates a pronounced niche partitioning with respect to algal polysaccharide degradation .", "Marine algae produce large quantities of distinct polysaccharides , for example storage , cell matrix and cell wall constituents , or as part of extracellular transparent exopolymer particles ( TEP ) .", "It has been recently shown that in particular Flavobacteriales and Rhodobacterales respond to TEP availability ( Taylor et al . , 2014 ) .", "The diversity of algal polysaccharides is too high for a single bacterial species to harbor all the genes required for the complete degradation of all naturally occurring variants .", "Thus , polysaccharide-degrading bacteria specialize on dedicated subsets of polysaccharides , which is why the decomposition of algal polysaccharides during and after algal blooms is a concerted effort among distinct bacterial clades with distinct glycan niches ( e . g . Xing et al . , 2015 ) .", "In this study , we provide evidence that the succession of bacterioplankton clades that we reported for the 2009 North Sea spring phytoplankton bloom re-occurred during the spring blooms from 2010 to 2012 .", "We tested whether the bacterioplankton clades and their associated CAZyme repertoires differ from year to year or exhibit recurrent patterns .", "We analyzed spring bacterioplankton community composition via 16S rRNA catalyzed reporter deposition fluorescence in situ hybridization ( CARD-FISH ) and 16S rRNA gene tag sequencing , as well as gene function repertoires by deep metagenome sequencing .", "Our efforts have culminated into the as of yet highest resolved dataset capturing the response of planktonic bacteria to marine spring phytoplankton blooms and have allowed identification of recurring patterns that might ultimately lead to an explanatory model for bacterioplankton succession dynamics during spring algae blooms ." ], [ "The samples were taken at Helgoland Island about 40 km offshore in the southeastern North Sea in the German Bight at the station 'Kabeltonne' ( 54° 11 . 3' N , 7° 54 . 0' E; Figure 1 ) between the main island and the minor island , Düne ( German for 'dune' ) .", "Water depths at this site fluctuate from 6 to 10 m over the tidal cycle .", "During most of the year , a westerly current transports water from the English Channel alongside the Dutch and Frisian coast to Helgoland , but water around the island is also influenced by nutrient inputs from the rivers Weser and Elbe and from the northern North Sea ( Wiltshire et al . , 2010 ) .", "During the 2009 to 2012 study period , the lowest water temperatures were measured in mid to late February ( min . 2010: 1 . 1°C; max . 2009: 3 . 4°C ) , followed by a continuous increase until a peak in August ( min . 2011: 18 . 0°C; max . 2009: 18 . 7°C ) ( Supplementary file 1 ) . 10 . 7554/eLife . 11888 . 003Figure 1 . Location of Helgoland Island ( ca . 40 km offshore the northern German coastline ) and the long-term ecological research site 'Kabeltonne' ( red circle: 54° 11 . 3' N , 7° 54 . 0' E ) in the German Bight of the North Sea . DOI: http://dx . doi . org/10 . 7554/eLife . 11888 . 003 Spring phytoplankton blooms in the North Sea typically develop during March and reach highest intensities during April and May .", "The highest chlorophyll a concentrations are usually observed at the coastlines including the area around Helgoland Island ( Figure 2 ) .", "North Sea spring blooms are thus large-scale phenomena that are , however , influenced by local conditions , such as riverine inputs .", "At Helgoland island , spring phytoplankton blooms started around mid March when water temperatures surpassed 3 to 5°C ( Figure 3A–H; Supplementary file 1 ) .", "The diatoms Chaetoceros debilis and Chaetoceros minimus , Mediopyxis helysia , Rhizosolenia styliformis and Thalassiosira nordenskioeldii , the silicoflagellate Chattonella , the haptophyte Phaeocystis and dinoflagellates dominated these blooms in terms of cell numbers ( Figure 3I–L; Supplementary file 2 ) .", "Relative abundances of these algae varied in no apparent order during the observed blooms , and we have yet to understand the factors that determine these variations .", "The sizes of the dominant algae taxa are different , with Chaetoceros minimus and in particular Phaeocystis spp .", "featuring the smallest cells and Mediopyxis helysia and Rhizosolenia styliformis featuring the largest cells .", "Spherical Phaeocystis spp .", "cells for example have estimated biovolumes of ~50 to 250 µm3 , whereas elipsoid cylindrical Mediopyxis helysia cells have a biovolume of ~82 , 000 µm3 and for Rhizosolenia styliformis even a biovolume of ~282 , 000 µm3 has been reported ( Olenina , 2006; Loebl et al . , 2013 ) .", "Considering biomass , the blooms were largely dominated by the diatoms T . nordenskioeldii and M . helysia and the silicoflagellate Chattonella .", "Blooms of these three algae were bimodal in all years with dominance of first T . nordenskioeldii followed by Chattonella in 2009 ( Figure 3I ) , T . nordenskioeldii followed by M . helysia in 2010 ( Figure 3J ) , M . helysia followed by Chattonella in 2011 ( Figure 3K ) and a pronounced bimodal bloom of Chattonella species in 2012 ( Figure 3L ) .", "All blooms were accompanied by a notable decrease of silicate ( Figure 3A–D; Supplementary file 1 ) , which diatoms use for frustule formation ( see Yool and Tyrrell ( 2003 ) for the controlling effect of silicate on diatom abundance ) . 10 . 7554/eLife . 11888 . 004Figure 2 . Satellite chlorophyll a measurements . Data are shown for the southern North Sea for the months February to May ( monthly averages ) of the years 2009 to 2012 .", "Images were retrieved from the GlobColour website using the ‘extended Europe area’ at full resolution ( 1 km ) as merged products of weighted averages from the following sensors: MERIS , MODIS AQUA , SeaWIFS and VIIRS .", "See GlobColour website for details ( http://hermes . acri . fr ) .", "The position of Helgoland Island is indicated by a blue dot . DOI: http://dx . doi . org/10 . 7554/eLife . 11888 . 00410 . 7554/eLife . 11888 . 005Figure 3 . Physicochemical parameters , phytoplankton composition and bacterioplankton composition as assessed by CARD-FISH . Sampling: Surface seawater samples were taken at the North Sea island Helgoland between the main island and the minor island 'Düne' ( station 'Kabeltonne' , 54°11'03''N , 7°54'00''E ) using small research vessels ( http://www . awi . de/en/expedition/ships/more-ships . html ) and processed in the laboratory of the Biological Station Helgoland within less than two hours after sampling .", "Cells for microscopic visualization methods were first fixed by the addition of formaldehyde to sampled seawater , which was then filtered directly onto 0 . 2 µm pore sized filters .", "Physicochemical and phytoplankton data: Physicochemical parameters and phytoplankton data were assessed in subsurface water on a weekday basis as part of the Helgoland Roads LTER time series as described in Teeling et al . ( 2012 ) .", "The Helgoland Roads time series is accessible via the public database Pangaea ( http://www . pangaea . de ) and can be used to assess long-term changes of the North Sea pelagic ecosystem .", "Left-hand side legends correspond to ordinates on the left , and right-hand side legends to ordinates on the right .", "A-D: Physicochemical measurements including measurements of BBE Chl a ( chlorophyll a fluorescence by algal group analyzer sensor ) .", "Left ordinate: salinity [PSU] , silicate [µM] , nitrate [µM] , ammonium [µM] and chlorophyll a [mg/m3]; right ordinate: temperature [°C] and phosphate [µM] .", "E-H: Counts of the diatom groups .", "I-L: Microscopic cell counts of the most abundant algae genera ( red: diatoms; orange: dinoflagellates: green: haptophytes; blue: silicoflagellates ) .", "Algae with large cells and thus large biovolumes are depicted by bold solid lines and algae with small cells are represented by dotted lines .", "Rhizosolenia styliformis and Mediopyxis helysia feature large cells , whereas Chaetoceros minimus and in particular Phaeocystis species feature small cells .", "The latter typically reaches lengths of below 10 µm and Phaeocystis spp .", "biovolumes therefore typically are hundreds to thousands fold smaller than those of R . styliformis and M . helyisa cells ( Olenina , 2006; Loebl et al . , 2013 ) .", "Physicochemical data are summarized in Supplementary file 1 , and data on the major phytoplankton clades in Supplementary file 2 .", "Total cell counts and CARD-FISH of bacterioplankton: E-H: TCC ( total cell counts ) ; red triangles depict sampling of metagenomes .", "M-β: Recurrent bacterioplankton clades as assessed by CARD-FISH ( Catalyzed Reporter Deposition-Fluorescence in situ Hybridization ) with the following probes: M-P ( major bacterial groups ) : SAR11-486 and SAR11-441: alphaproteobacterial SAR11-clade; ROS537: alphaproteobacterial Roseobacter clade; GAM42a: Gammaproteobacteria; CF319a: Bacteroidetes .", "Q-T ( major Flavobacteriia clades ) : POL740: genus Polaribacter; FORM181A: genus Formosa; ULV995: genus Ulvibacter; VIS6-814: genus-level clade VIS6 within the family Cryomorphaceae-Owenweeksia; U-X ( major Gammaproteobacteria clades ) : REI731: genus Reinekea; BAL731: genus Balneatrix; ALT1413: families Alteromonadaceae and Colwelliaceae; SAR92-627: genus-level clade SAR92 .", "Y-β ( minor Bacteroidetes clades ) : FORM181B: species-specific for Formosa sp .", "Hel1_33_131; NS3a-840: NS3 marine group; NS5/VIS1-575: VIS1 genus-level clade within the NS5 marine group; NS9-664: NS9 marine group; CYT-734: Cytophagia clade Marinoscillum .", "Total and CARD-FISH cell counts are summarized in Supplementary file 3 and the corresponding probes in Supplementary file 4 . DOI: http://dx . doi . org/10 . 7554/eLife . 11888 . 005 Bloom maximum intensities decreased from 2009 to 2012 with chlorophyll a maxima ( measured by fluorescence ) reaching 28 mg m-3 ( day 82 ) , 18 mg m-3 ( day 125 ) , 15 mg m-3 ( day 116 ) , and 11 mg m-3 ( day 114 ) in each respective year ( Figure 3A–D; Supplementary file 1 ) .", "Maximum bacterioplankton cell counts were observed either close to or after the chlorophyll maxima ( Supplementary file 3 ) .", "In 2009 , the bacterioplankton peaked at 3 . 5x106 cells ml-1 ( day 118 ) , 36 days after the Chl a maximum .", "In 2010 , the peak in Chl a was broader and only four days ahead of the bacterioplankton peak abundance of 2 . 4x106 cells ml-1 ( day 129 ) .", "In 2011 , the Chl a peak was only two days ahead of the bacterioplankton peak abundance of 2 . 3x106 cells ml-1 ( day 118 ) , whereas it was nine days ahead in 2012 , where the bacterioplankton peaked at 3 . 2x106 cells ml-1 ( day 123 ) .", "DAPI and CARD-FISH cell staining ( Supplementary files 3 and 4 ) showed that SAR11 dominated the bacterioplankton community in winter , but with the onset of each spring bloom relative abundances of Bacteroidetes followed by Gammaproteobacteria increased and finally surpassed those of the SAR11 ( Figure 3M–P ) .", "Bacteroidetes reached higher maximum relative abundances than Gammaproteobacteria , 40% ( 2012 ) to 60% ( 2010 ) as compared to 10% ( 2012 ) to 30% ( 2010 ) , respectively .", "Bacteroidetes genera Polaribacter , Formosa , and VIS6 , a genus-level clade within the family Cryomorphaceae ( Gómez-Pereira et al . , 2010 ) , peaked each year with relative abundances well above 5% , reaching relative abundances of up to 25% sometimes within less than a week ( Figure 3Q–T ) .", "Ulvibacter reached similar peak abundances with the exception of 2012 , where this genus never surpassed 2% and ranged below 1% most of the time .", "Within Gammaproteobacteria the genus-level SAR92 clade responded notably in all years increasing from background levels below 1% to relative abundance of 8% to 16% .", "Members of the Alteromonadales families Alteromonadaceae and Colwelliaceae bloomed in three ( 2010: 8%; 2011: 6%; 2012: 7% ) , and genus Reinekea in two ( 2009: 16%; 2010: 5% ) of the years ( Figure 3U–X ) .", "Some less abundant , but nevertheless recurrent taxa included the genus Balneatrix within Gammaproteobacteria with relative abundances up to 2% ( Figure 3U–X ) .", "Minor recurring groups of Bacteroidetes ( Figure 3Y-β ) included the NS3a marine group ( 9% in 2009 and 6% in 2011 ) , the genus-level VIS1 clade within the NS5 marine group detected before the Chl a peaks of 2011 ( 7% ) and 2012 ( 5% ) , and the Cytophagia clade Marinoscillum that reached 1–3% abundance most years after initial blooms .", "We used complementary 16S rRNA gene tag sequencing for the detection of bacterioplankton clades that were not recovered by CARD-FISH probes ( Supplementary file 5 ) .", "Relative proportions of 16S tags from distinct clades correlated for the most part those inferred from CARD-FISH cell counts ( Figure 4A–P ) , but members of SAR11 were substantially underreported - a known limitation of the 806R primer used in V4 amplification for 2010 to 2012 samples ( Apprill et al . , 2015 ) .", "Additional abundant clades detected in the 16S amplicon data comprised the Flavobacteriia genus Tenacibaculum ( Figure 4U–X ) that bloomed in 2010 ( read frequencies of max . ~12% ) and 2011 ( max . ~5% ) .", "Within Gammaproteobacteria , clades with read frequencies ≥5% in at least one year comprised the genera Aeromonas , Glaciecola , Pseudoalteromonas , Pseudomonas , Psychrobacter and the SAR86 and ZD0405 clades ( Figure 4Q–T ) .", "Within the alphaproteobacterial Rhodobacteriaceae , high abundances of 'Candidatus Planktomarina temperata' ( DC5-80-3 lineage ) and the NAC11-7 clade were detected reaching 6–21% and 7–19% of the tag data , respectively ( Figure 4U–X ) .", "Also within Alphaproteobacteria the genus Sulfitobacter peaked with a read frequency of ~7% in 2010 ( Figure 4V ) , and within Betaproteobacteria the order Methylophilales ( dominated by OM43 clade members ) was detected with high relative abundances of up to ~10% before blooms , which decreased with bloom progression .", "Verrucomicrobia ( Figure 4U–X ) were detected with decreasing peak read frequencies of 7 . 7% ( 2010 ) , 5 . 0% ( 2011 ) and 2 . 9% ( 2012 ) .", "This decrease corresponds to decreasing bloom intensities , which supports a proposed role of Verrucomicrobia in polysaccharide decomposition ( e . g . Martinez-Garcia et al . , 2012 ) . 10 . 7554/eLife . 11888 . 006Figure 4 . Bacterioplankton diversity as assessed by 16S rRNA gene tag sequencing . Sampling: Surface seawater samples were taken at the North Sea island Helgoland between the main island and the minor island 'Düne' ( station 'Kabeltonne' , 54°11'03''N , 7°54'00''E ) using small research vessels ( http://www . awi . de/en/expedition/ships/more-ships . html ) and processed in the laboratory of the Biological Station Helgoland within less than two hours after sampling .", "Biomass of free-living bacteria for DNA extraction was harvested on 0 . 2 µm pore sized filters after pre-filtration with 10 µm and 3 µm pore sized filters to remove large debris and particle-associated bacteria .", "Biomass of the 0 . 2–3 µm bacterioplankton fraction was used for DNA extraction and subsequent 16S rRNA gene tag sequencing .", "16S rRNA gene tag sequencing: A total of 142 samples were collected for the years 2010 to 2012 .", "After DNA extraction , the V4 region of the 16S rRNA gene was amplified using the primers 515F ( 5' GTGCCAGCMGCCGCGGTAA 3' ) and 806R ( 5' GGACTACHVGGGTWTCTAAT 3' ) ( Caporaso et al . , 2011 ) .", "Sequencing was carried out on an Illumina ( San Diego , CA , USA ) MiSeq sequencer with and 2x250 bp chemistry .", "This dataset was complemented by 16S rRNA gene tags from 7 samples from our initial study on the 2009 spring bloom ( Teeling et al . , 2012 ) .", "DNA of these samples was amplified using the primers 314F ( 5’ CCTACGGGNGGCWGCAG 3' ) and 805R ( 5’ GACTACHVGGGTATCTAATCC 3' ) ( Herlemann et al . , 2011 ) and sequenced on the 454 FLX Ti platform .", "Data analysis: All tag data were analyzed using the SILVAngs pipeline with the SILVA ( Quast et al . , 2013 ) v119 database .", "The SAR92 clade was subsequently reclassified to comply with the recently released SILVA v123 , where the SAR92 no longer belong to the order Alteromonadales .", "The corresponding abundance data is summarized in Supplementary file 5 .", "Time points from days 50 to 160 were plotted for all years .", "Panel A-P depict data that are analogous to the CARD-FISH data presented in Figure 3 , with addition of the Flavobacteriia genus Tenacibaculum ( E-H ) .", "Panels Q-X show minor Gammaproteobacteria clades ( Q-T ) and Roseobacter clades together with miscellaneous other minor clades ( U-X ) that were not tested by CARD-FISH probes . DOI: http://dx . doi . org/10 . 7554/eLife . 11888 . 006 Within Bacteroidetes , Gammaproteobacteria and Rhodobacterales a total of eleven clades peaked during at least two of the four spring blooms with relative cell abundances or , for those clades that were not assessed by CARD-FISH , relative read frequencies ≥5% .", "These were six Flavobacteriia clades ( Formosa , Polaribacter , NS3a marine group , Tenacibaculum , Ulvibacter , VIS6 clade Cryomorphaceae ) , three Gammaproteobacteria clades ( Alteromonadaceae/Colwelliaceae , Reinekea and SAR92 ) , and two Roseobacter clades ( DC5-80-3 and NAC11-7 ) .", "Each year a succession was observed within the Flavobacteriia and Gammaproteobacteria clades .", "The succession in the Flavobacteriia was more pronounced than in the Gammaproteobacteria , but the sequence of clades varied .", "Spearman rank correlation analyses revealed that the abundances of the most prominent Flavobacteriia clades were for the most part correlated with multiple algae groups and physiochemical factors ( Supplementary file 6 ) .", "According to linear regression analyses , the strongest abiotic predictors were temperature , salinity , silicate and nitrate , and the strongest biotic predictors were Phaeocystis spp .", "haptophytes , Rhizosolenia spp .", ", Chaetoceros debilis , and Chaetoceros minimus diatoms and the silicoflagellate Chattonella ( Supplementary file 7 ) .", "It should be noted though that linear regressions were computed based on log-transformed abundance data and not algae volumes ( which were not measured ) .", "Thus , the influence of the rather small cell-sized algae such as Chaetoceros minimus is likely overestimated .", "Such limitations notwithstanding it is noteworthy that in no case a simple one-to-one relationship between specific algae and specific bacterioplankton groups was detected .", "The strongest significant ( p<0 . 05 ) correlations were obtained for the Ulvibacter clade that was positively correlated with diatoms and haptophytes and negatively correlated with silicoflagellates .", "Further results comprised an opposite trend for the VIS1 clade of the NS5 marine group , and a correlation of Polaribacter and Chattonella abundances ( see Supplementary file 6 for details ) .", "In total , 16 metagenomes of free-living bacterioplankton ( 0 . 2–3 µm ) were generated from time points before , during and after spring phytoplankton blooms , six during 2009 using the 454 FLX Ti platform that were published previously ( Teeling et al . , 2012 ) and ten during 2010–2012 using the Illumina HiSeq2000 platform .", "Most of the 454 ( 0 . 5–4 pico titer plates / metagenome ) and all of the Illumina metagenomes ( 1 lane / metagenome; 2x150 bp ) were deeply sequenced ( Supplementary file 8 ) with final assembled contigs of up to 96 kbp and 458 kbp , respectively .", "Taxonomic classification of the metagenome contigs resulted in identification of major bloom-associated clade sequence bins ( Supplementary file 9 ) , including Formosa , Polaribacter , the NS3a and NS5 marine groups , and Cryomorphaceae of the Flavobacteriia and Alteromonadales , Reinekea , Glaciecola and the SAR92 clade of the Gammaproteobacteria .", "Classification was poor , however , for Ulvibacter ( Flavobacteriia ) and Balneatrix ( Gammaproteobacteria ) , most likely since the only available reference genomes ( unidentified eubacterium SCB49; Balneatrix alpica ) were too distant from North Sea representatives .", "Clone libraries from 2009 ( Teeling et al . , 2012 ) indicated 16S rRNA similarities of only 94% and 91% , respectively , for these two clades .", "Other abundant clades comprised the betaproteobacterial Burkholderiales and Methylophilales ( including the OM43 clade ) , the alphaproteobacterial SAR116 and Roseobacter NAC11-7 clade , and the gammaproteobacterial SAR86 and ZD0405 clades .", "Lower abundant clades comprised , amongst others , the OM60 ( NOR5 ) group , the AEGEAN-169 group , and Sulfitobacter .", "We plotted contig GC contents versus coverage to evaluate our taxonomic classification , which in some cases allowed to assess the coherence of some of the clades ( Figure 5 ) .", "For example , Reinekea ( Figure 5D , H , I ) and the NS5 marine group ( Figure 5H , L , M , O ) were mostly represented by distinct clusters , whereas Polaribacter ( Figure 5D , H , I ) was almost always represented by at least two clusters indicating the presence of sub-populations .", "In general , the number of clusters increased from pre-bloom to mid-bloom situations and decreased slightly towards late bloom situations and notably towards post-bloom situations .", "This tendency was more evident in 2009 , the year with the highest bloom intensity and the largest number of metagenome samples spanning a broader timespan ( Figure 5A–F ) .", "It is noteworthy that high in situ abundance did not always correlate with good metagenome assemblies .", "SAR11 for example , while highly abundant in all metagenome datasets , yielded few large contigs , possibly due to population heterogeneity and presence of hyper-variable regions described in sequenced SAR11 genomes ( Wilhelm et al . , 2007 ) . 10 . 7554/eLife . 11888 . 007Figure 5 . Taxonomic classification of bacterioplankton metagenomesSampling: Surface seawater samples were taken at the North Sea island Helgoland between the main island and the minor island 'Düne' ( station 'Kabeltonne' , 54°11 . 03' N , 7°54 . 00' E ) and processed in the laboratory of the Biological Station Helgoland within less than two hours after sampling .", "Biomass of free-living bacteria was harvested on 0 . 2 µm pore sized filters after pre-filtration with 10 µm and 3 µm pore sized filters to remove large debris and particle-associated bacteria .", "Sequencing: Community DNA was extracted and sequenced; 2009 samples were sequenced on the 454 FLX Ti platform , and 2010-2012 samples on the Illumina HiSeq2000 platform ( 16 metagenomes in total ) .", "Reads were assembled using Newbler ( 2009 ) or a combination of SOAPdenovo and Newbler ( 2010-2012 ) and the resulting contigs were taxonomically classified ( Supplementary file 9 ) .", "Visualization: The resulting metagenome contigs are visualized as bubbles with radii that are proportional to their lengths and colors that indicate their predicted taxomomic affiliations .", "These bubbles are drawn in planes that are defined by the contig's GC contents and coverage values .", "Colors are restricted to selected abundant taxa ( see legend below ) to highlight distinct clusters , mostly from the Bacteroidetes , Alphaproteobacteria , Betaproteobacteria and Gammaproteobacteria .", "Likewise only contigs are shown that exceed a minimum length of 2750 bp for pyrosequencing data ( 2009 ) and 15 , 000 bp for llumina data ( 2010-2012 ) , respectively .", "Sparse contigs with very high coverage or GC contents below 20% or above 60% were also excluded from visualizations .", "The 16 metagenomes are shown arranged in order on yearly timescales that depict chlorophyll a contents as proxies for phytoplankton abundance .", "Metagenome sizes*: 2009-02-11: 49 . 1 Mbp / 2009-03-31: 44 . 9 Mbp / 2009-04-07: 52 . 7 Mbp / 2009-04-14: 96 . 0 Mbp / 2009-06-16: 29 . 8 Mbp / 2009-09-01: 79 . 2 Mbp 2010-03-03: 537 . 3 Mbp / 2010-04-08: 325 . 8 Mbp / 2010-05-04: 453 . 0 Mbp / 2010-05-18: 512 . 3 Mbp 2011-03-24: 629 . 1 Mbp / 2011-04-28: 541 . 8 Mbp / 2011-05-26: 604 . 0 Mbp 2012-03-08: 574 . 0 Mbp / 2012-04-16: 543 . 9 Mbp / 2012-05-10: 614 . 1 Mbp *sums of assembled basesDOI: http://dx . doi . org/10 . 7554/eLife . 11888 . 007 Analyses of functional genes identified in the bacterioplankton metagenomes revealed that increases in Flavobacteriia relative abundance during blooms was always accompanied by an increase in community-wide CAZyme gene frequency as well as an increase in the diversity of CAZyme families ( Figure 6A–C ) .", "As blooms subsided , CAZyme frequencies also declined .", "This was most pronounced for more densely sampled years in 2009 and 2010 .", "In 2012 the decline in CAZymes was not captured as the last metagenome sample was taken before the bloom decline ( Figure 6A , x-axis ) . 10 . 7554/eLife . 11888 . 008Figure 6 . Metagenome functional analyses: CAZyme , sulfatase and transporter gene frequencies . Sampling: Surface seawater samples were taken at the North Sea island Helgoland between the main island and the minor island Düne' ( station 'Kabeltonne' , 54°11'03''N , 7°54'00''E ) and processed in the laboratory of the Biological Station Helgoland within less than two hours after sampling .", "Biomass of free-living bacteria was harvested on 0 . 2 µm pore sized filters after pre-filtration with 10 µm and 3 µm pore sized filters to remove large debris and particle-associated bacteria .", "Sequencing: Community DNA was extracted and sequenced .", "2009 samples were sequenced on the 454 FLX Ti platform , and 2010–2012 samples on the Illumina HiSeq2000 platform ( 16 metagenomes in total ) .", "Reads were assembled using Newbler ( 2009 ) or a combination of SOAPdenovo and Newbler ( 2010–2012 ) and the resulting contigs were taxonomically classified ( Supplementary file 9 ) .", "Metagenome sizes*: 2009-02-11: 49 . 1 Mbp / 2009-03-31: 44 . 9 Mbp / 2009-04-07: 52 . 7 Mbp / 2009-04-14: 96 . 0 Mbp / 2009-06-16: 29 . 8 Mbp / 2009-09-01: 79 . 2 Mbp 2010-03-03: 537 . 3 Mbp / 2010-04-08: 325 . 8 Mbp / 2010-05-04: 453 . 0 Mbp / 2010-05-18: 512 . 3 Mbp 2011-03-24: 629 . 1 Mbp / 2011-04-28: 541 . 8 Mbp / 2011-05-26: 604 . 0 Mbp 2012-03-08: 574 . 0 Mbp / 2012-04-16: 543 . 9 Mbp / 2012-05-10: 614 . 1 Mbp *sums of assembled bases Data Analysis: CAZymes were predicted as consensus of searches against the CAZy , dbCAN and Pfam databases with custom E-value cutoffs ( Supplementary file 11 ) .", "Sulfatase and transporter genes were predicted based on HMMER searches against the Pfam databases with an E-value cutoff of E-5 .", "Gene frequencies were computed as [ ( sum of average coverage of target genes ) *100 / ( sum of average coverage of all genes ) ] .", "All dates in the graphs are in the format [yyyy-mm-dd] .", "DOI: http://dx . doi . org/10 . 7554/eLife . 11888 . 00810 . 7554/eLife . 11888 . 009Figure 6—figure supplement 1 . CAZyme repertoire within the family Cryomorphaceae ( Flavobacteriia ) at dates with high Cryomorphaceae abundances . GH and CBM frequencies were low compared to those of other abundant Flavobacteriia clades ( see Figure 6Q , S , T , U ) , indicating that these Cryomorphaceae might have a distinct ecophysiological niche in which polysaccharide degradation plays a lesser role . DOI: http://dx . doi . org/10 . 7554/eLife . 11888 . 00910 . 7554/eLife . 11888 . 010Figure 6—figure supplement 2 . CAZyme repertoire within the order Alteromonadales ( Gammaproteobacteria ) at dates with high abundances of Alteromonadales . As expected at the taxomomic level of order , the CAZyme repertoire was diverse .", "Nonetheless , the obtained pattern was relatively consistent across all four studied years and at some dates notably enriched in CAZymes that play a role during phytoplankton blooms such as GH13 and GH16DOI: http://dx . doi . org/10 . 7554/eLife . 11888 . 010 The 20 glycoside hydrolase families with the highest mean abundances during bloom dates were , in descending order , GH3 , 23 , 13 , 16 , 103 , 73 , 92 , 2 , 17 , 30 , 5 , 20 , 36 , 65 , 29 , 1 , 42 , 31 , 81 , and 18 ( Figure 6D ) .", "While most of these families comprise a diverse range of functions , they are indicative for the hydrolysis of certain glucans , xylans , mannans ( GH92 ) , cellulose ( GH5 ) , fucans ( GH29 ) , and peptidoglycan ( GH23 , 103 , 73 , and 18 ) .", "The abundant families GH3 , 16 , 17 and 5 comprise β-1 , 3-glucanases and the family GH30 β-1 , 6-glucanases .", "Both enzymes are involved in the cleavage of chrysolaminarin .", "Chrysolaminarin , a mostly linear β-1 , 3-glucan with occasional β-1 , 6 branches , is the storage polysaccharide in diatoms and thus one of the most abundant polysaccharides on Earth .", "The ten families of carbohydrate-binding modules with the highest mean abundances during blooms were , in descending order , CBM50 , 32 , 6 , 9 , 48 , 11 , 22 , 57 , 20 , and 4 ( Figure 6E ) .", "These CBM domains are indicative for the binding of peptidoglycan ( CBM50 ) , galactans ( CBM32 ) , glycogen ( CBM48 ) , β-1 , 3- and β-1 , 4-glucans ( CBM6 , 11 , 4 ) , xylan ( CBM6 , 9 , 22 , 4 ) , and cellulose ( CBM6 , 4 ) .", "This suggests a pronounced specialization of the bacterial community in the acquisition of storage polysaccharides ( β-1 , 3-glucans such as chrysolaminarin; α-1 , 4-glucans such as starch and glycogen ) and cell wall polysaccharides ( xylose , cellulose and peptidoglycan ) of both algae and bacteria .", "By contrast , CBMs for the binding of algal TEP , which consists predominantly of fucose- and rhamnose-rich anionic sulfated heteropolysaccharides ( Passow , 2002 ) , were only found in lower abundances , e . g . CBM47 ( fucose ) and CBM67 ( rhamnose ) .", "Among carbohydrate esterase families , CE11 , 4 , 1 , and 14 exhibited the highest mean abundances during blooms ( Figure 6F ) .", "The only known function for family CE11 is that of the UDP-3-O-acyl-N-acetylglucosamine deacetylase , an enzyme involved in lipid A biosynthesis .", "The second and third most abundant CE families 4 and 1 comprise a number of enzymatic functions including deacetylation of peptidoglycan , xylan , and chitin .", "Finally , the polysaccharide lyase families PL6 , 7 , 17 and 5 constituted the most abundant PL families during bloom dates ( Figure 6G ) .", "Known members of these PL families are all involved in the usage of alginate .", "However , gene frequencies of these PL families were an order of magnitude below those of abundant GHs , CBMs , and CEs , indicating that alginate degradation does not play a vital role in North Sea spring phytoplankton blooms .", "Alginate is a cell wall constituent in brown macroalgae , however , the microalgae that dominate North Sea spring blooms are devoid of alginate .", "For all of these GH , CBM , PL , and CE families , we observed remarkably similar gene frequency patterns during all four spring blooms , often with peaks in the same families ( Figure 6I–L ) .", "Alongside CAZymes , sulfatase and TonB-dependent transporter ( TBDT ) gene frequencies also increased during blooms while tripartite ATP-independent periplasmic ( TRAP ) transporter genes showed an almost opposite trend ( Figure 6H , M ) .", "A class-level analysis revealed that sulfatases and TBDT genes were predominantly present in Bacteroidetes , whereas TRAP transporters were mostly present in Alphaproteobacteria ( Figure 6R , W ) .", "The observed shifts in sulfatase , TBDT and TRAP transporter frequencies hence reflect the shifts in relative abundance between these two classes .", "This is in agreement with our study on the 2009 spring bloom ( Teeling et al . , 2012 ) and furthermore demonstrates recurrence of this phenomenon during four consecutive years .", "Class-level analyses of the most abundant GH and CBM families ( Figure 6N–P ) showed that Flavobacteriia not only contributed more total CAZymes to the microbial community than Alpha- and Gammaproteobacteria , but also exhibited a tighter coupling between GH and CBM genes with highly similar abundance profiles ( Figure 6N ) .", "The distribution of families was also more uneven in Flavobacteriia , indicative of a more pronounced substrate specialization compared to Alpha- and Gammaproteobacteria .", "GH92 ( mannosidase ) and GH16 ( including β-1 , 3-glucanase ) genes , for example , were predominantly found in Flavobacteriia , possibly indicating more readiness to decompose mannans and chrysolaminarin .", "Metagenome taxonomic classification provided sufficient data for analysis of CAZyme repertoires of the flavobacterial NS5 marine group , NS3a marine group , Formosa , Polaribacter , and Cryomorphaceae , and the gammaproteobacterial Alteromonadales and Reinekea clades ( Supplementary file 10 ) .", "For most of these clades , the analyses revealed fingerprint-like patterns , which corroborates the hypothesis that these clades have distinct glycan niches that are relatively stable across years .", "For example , the NS5 marine group ( Figure 6Q ) was rich in GH3 , 20 and 29 ( fucosidases ) , but notably devoid of GH92 ( mannosidases ) .", "By contrast , Formosa and Polaribacter clades ( Figure 6T , U ) contained higher abundances of GH92 genes .", "The Formosa CAZyme profile was also characterized by high proportions of GH16 , 17 , and 30 families , which all contain enzymes that can decompose chrysolaminarin .", "Polaribacter contained a broader set of CAZymes that included all families found in Formosa , however , Polaribacter was richer in GH13 ( e . g . α-amylase ) and poorer in GH92 ( mannosidases ) than Formosa .", "Likewise , the GH repertoires of the NS3a and NS5 marine groups were similar ( Figure 6Q , S ) , but the NS3a marine group was richer in GH13 and 32 and devoid of GH29 family fucosidases .", "The high number of CAZyme families in Polaribacter corroborated metagenome bin analyses that suggested a higher diversity within this clade .", "CAZyme gene frequencies were much lower in the Cryomorphaceae than in the other investigated Flavobacteriia clades with GH frequencies barely exceeding 0 . 5% ( Figure 6—figure supplement 1 ) .", "This suggests a different ecophysiological niche and a distinct role of the Cryomorphaceae during phytoplankton blooms .", "For Gammaproteobacteria , recurring patterns were detected for the prominent Alteromonadales and Reinekea clades .", "Alteromonadales contained some of the GH families that play important roles during phytoplankton blooms , such as GH13 and 16 , but were notably poor in or even devoid of others , such as GH29 and GH92 , respectively ( Figure 6—figure supplement 2 ) .", "In contrast to other prominent clades , we did not obtain sufficient metagenome sequences for Reinekea for all four years , but only for 2009 and 2010 ( Figure 6V ) .", "However , the Reinekea CAZyme patterns of 2009 and 2010 were well conserved with high proportions of GH23 and 13 , and CBM48 , 20 , 41 , 21 , and 25 .", "The GH23 family comprises peptidoglycan lyases and the GH13 family contains α-1 , 4-glucanases ( e . g . α-amylase ) .", "CBM48 , 20 , 41 , 21 , and 25 all bind α-1 , 4-glucans such as starch and glycogen , and the ubiquitous CBM50 contains peptidoglycan-binding members .", "These results are consistent with a glycan niche that involves decomposition of external α-1 , 4-glucans and possibly peptidoglycan ." ], [ "Bacterioplankton communities during spring phytoplankton blooms in the coastal North Sea undergo swift and dynamic composition changes and thus are difficult to investigate .", "Nonetheless , we found clades that recurrently reached high abundances among Flavobacteriia ( Formosa , Polaribacter , NS3a marine group , Ulvibacter , VIS6 clade Cryomorphaceae , Tenacibaculum ) , Gammaproteobacteria ( Alteromonadaceae/Colwelliaceae , SAR92 , Reinekea ) and Roseobacter clade Alphaproteobacteria ( DC5-80-3 , NAC11-7 ) .", "Recurrence was not only detectable on the taxonomic but also on the functional level with a highly predictable increase in TonB-dependent polysaccharide uptake systems and distinct CAZyme patterns .", "The niches of abundant bacterioplankton clades are more complex and manifold than the glycan niches that we explore in this study .", "CAZymes , however , have the advantage that they allow linking of gene repertoires and possible environmental functions in a way currently not feasible for other macromolecules such as proteins and lipids .", "Our results suggest that besides stochastic also deterministic effects influence phytoplankton-bacterioplankton coupling during blooms .", "They indicate that during spring phytoplankton blooms similar principles of resource partitioning and specialization are at play as within human gut microbiota that decompose fiber-rich plant material , albeit at a much larger scale .", "Rather the availability of substrates commonly occurring in microalgae than one-to-one interactions of particular phytoplankton and bacterioplankton species caused the succession of free-living bacterioplankton clades ." ], [ "Physicochemical parameters ( Supplementary file 1 ) and phytoplankton data ( Supplementary file 2 ) were assessed in subsurface water on a weekday basis as part of the Helgoland Roads LTER time series .", "Details on the acquisition of these data have been described previously ( Teeling et al . , 2012 ) .", "The Helgoland Roads time series is accessible via the public database Pangaea ( http://www . pangaea . de ) .", "Sampling of bacterioplankton was carried out as described previously ( Teeling et al . , 2012 ) .", "In brief , surface seawater samples were taken at the long-term ecological research station 'Kabeltonne' ( 54° 11 . 3' N , 7° 54 . 0' E ) at the North Sea island Helgoland using small research vessels ( http://www . awi . de/en/expedition/ships/more-ships . html ) and processed in the laboratory of the Biological Station Helgoland within less than two hours after sampling .", "Biomass of free-living bacteria for DNA extraction was harvested on 0 . 2 µm pore sized filters after pre-filtration with 10 µm and 3 µm pore sized filters to remove large debris and particle-associated bacteria .", "By contrast , cells for microscopic visualization methods were first fixed by the addition of formaldehyde to sampled seawater , which was then filtered directly onto 0 . 2 µm pore sized filters .", "All filters were stored at -80°C until further use .", "Assessment of absolute cell numbers and bacterioplankton community composition was carried out as described previously ( Thiele et al . , 2011 ) .", "To obtain total cell numbers , DNA of formaldehyde fixed cells filtered on 0 . 2 µm pore sized filters was stained with 4' , 6-diamidino-2-phenylindole ( DAPI ) .", "Fluorescently labeled cells were subsequently counted on filter sections using an epifluorescence microscope .", "Likewise , bacterioplankton community composition was assessed by catalyzed reporter deposition fluorescence in situ hybridization ( CARD-FISH ) of formaldehyde fixed cells on 0 . 2 µm pore sized filters .", "DAPI and CARD-FISH cell counts are summarized in Supplementary file 3 and the corresponding probes in Supplementary file 4 .", "Surface seawater samples were collected on bi-monthly to bi-weekly time scales from January 2010 to December 2012 at Helgoland roads .", "500 ml of each sample were subjected to fractionating filtration as described above using 10 , 3 and 0 . 2 µm pore size polycarbonate membrane filters ( Millipore , Schwalbach , Germany ) .", "DNA of the 0 . 2–3 µm fraction was extracted from filters as described previously ( Sapp et al . , 2007 ) and quantified using the Invitrogen ( Carlsbad , CA , USA ) Quant-iT PicoGreen dsDNA reagent as per manufacturer's instructions .", "Concentrations ranged from <1 to 20 µg DNA/ml .", "50 µl aliquots of each sample were pipetted into 96-well plates and sent to the Department of Energy ( DOE ) Joint Genome Institute ( JGI , Walnut Creek , CA , USA ) for amplification and sequencing as follows: Sample prep was done on a PerkinElmer ( Waltham , MA , USA ) Sciclone NGS G3 Liquid Handling Workstation capable of processing 96 plate-based samples in parallel , utilizing the 5 PRIME ( Gaithersburg , MD 20878 , USA ) HotMasterMix amplification kit and custom amplification primers targeting the V4 region of the 16S rRNA gene using 515F ( 5' GTGCCAGCMGCCGCGGTAA 3' ) and 806R ( 5' GGACTACHVGGGTWTCTAAT 3' ) ( Caporaso et al . , 2011 ) .", "Primers also contained Illumina adapter sequences and a barcode index .", "PCR reactions were set up in 75 µl with 1x HotMasterMix ( 5 PRIME ) with final concentrations of 0 . 4 µg/µl BSA and 0 . 2 µM of each primer .", "This volume was split into triplicate 25 µl reactions for independent amplification and then pooled to reduce PCR bias .", "Prepared amplicon libraries were normalized , multiplexed into a single pool per plate and quantified using the KAPA Biosystems ( Wilmington , MA , USA ) next-generation sequencing library qPCR kit on a Roche ( San Francisco , CA , USA ) LightCycler 480 .", "Libraries were sequenced on an Illumina ( San Diego , CA , USA ) MiSeq sequencer using the Reagent Kit v3 and 2x250 bp chemistry .", "The resulting sequences are available from the DOE-JGI GOLD database ( Reddy et al . , 2015 ) as part of the COGITO project ( Gp0056779 ) and from the NCBI short read archive ( SRA ) ( SRA278189 ) .", "Roche 454 16S rRNA gene tags from 2009 MIMAS ( Microbial Interactions in Marine Systems ) project ( Teeling et al . , 2012 ) were reanalyzed for comparison with the Illumina-based COGITO extension project from subsequent years 2010–2012 .", "The 2009 datasets was generated using the primers Bakt_314F ( 5' CCTACGGGNGGCWGCAG 3' ) and Bakt_805R ( 5’ GACTACHVGGGTATCTAATCC 3' ) ( Herlemann et al . , 2011 ) .", "The forward primers of both datasets target distinct , but the reverse primers target the same region .", "Hence , only those 454 reads sequenced from the 805 direction were reanalyzed for comparison .", "For 2010–2012 , raw MiSeq paired-end reads ( 2x250 bp ) were merged and filtered using illumina-utils ( https://github . com/meren/illumina-utils ) to retain only read pairs without mismatches in the overlapping regions .", "These high-quality Illumina tags and the 454 tags were then processed separately but with the same methods via the SILVAngs pipeline ( Quast et al . , 2013 ) , which includes additional quality filtering steps via alignment as well as length , ambiguity and homopolymer filters .", "Sequences were dereplicated at 100% identity and then globally clustered at 98% .", "Representative OTUs were classified to genus level against the SILVA ( Quast et al . , 2013 ) v119 database using BLAST with a similarity threshold = ( sequence identity + alignment coverage ) / 2 >=93% .", "The SAR92 clade was reclassified according to SILVA v123 .", "Reads were mapped against representative OTUs to obtain final abundance counts .", "For the purpose of this study , OTUs were collapsed based on shared taxonomy no higher than the genus level .", "For MIMAS samples , we retained a total of 110 , 995 454 reads across 7 samples with an average of 16 , 000 per sample .", "After SILVAngs quality filtering , 110 , 866 remained for clustering .", "6102 representative OTUs were identified and 107 , 708 total sequences were assigned to a relative in the database during classification within the 93% similarity threshold .", "The final abundance matrix collapsed on shared taxonomic classification contained 500 unique taxa .", "In total , 20 , 869 , 432 paired raw MiSeq reads were obtained across 142 samples from 2010–2012 COGITO samples .", "15 , 016 , 350 merged reads with no mismatches in the overlapping region were retained with an average of 106 , 000 per sample .", "Reads were randomly sub-sampled to 40 , 000 tags per sample to reduce computational demands .", "In total 6 , 120 , 000 tags were submitted to the SILVAngs pipeline .", "After additional quality filtering , 6 , 116 , 021 sequences were clustered at 98% and the resulting 935 , 006 representative OTUs were classified .", "A total of 5 , 676 , 259 sequences were assigned to a relative in the database within the 93% similarity threshold .", "The final abundance matrix collapsed on shared taxonomy no higher than the genus level contained 1995 unique taxa ( Supplementary file 5 ) .", "Total community DNA of 2009 samples ( 02/11/09; 03/31/09; 0407/09; 04/14/09; 06/16/09 ) was sequenced on the 454/Roche FLX Ti platform as described previously ( Teeling et al . , 2012 ) .", "Metagenome sequencing of 2010–12 samples ( 03/03/2010; 04/08/10; 05/04/10; 05/18/10; 03/24/11; 04/28/11; 05/26/11; 03/08/12; 04/16/12; 05/10/12 ) was performed at the DOE Joint Genome Institute on the Illumina HiSeq2000 platform .", "Libraries were created from 100 ng environmental DNA per sample that was sheared to 270 bp using a Covaris E210 ( Covaris , Woburn , MA , USA ) and size selected using SPRI beads ( Beckman Coulter , Indianapolis , IN , USA ) .", "The fragments were treated with end-repair , A-tailing , and ligation of Illumina compatible adapters ( IDT , Coralville , IA , USA ) using the KAPA-Illumina library creation kit .", "The libraries were quantified using KAPA Biosystem’s next-generation sequencing library qPCR kit and run on a Roche LightCycler 480 real-time PCR instrument .", "The quantified libraries were then prepared for sequencing on the Illumina HiSeq sequencing platform utilizing a TruSeq PE Cluster Kit v3 , and Illumina’s cBot instrument to generate a clustered flowcell for sequencing .", "Sequencing was performed on the Illumina HiSeq2000 sequencer using TruSeq SBS sequencing Kits , v3 , following a 2x150 bp indexed run recipe .", "Raw reads were screened against Illumina artifacts with kmer size of 28 , step size of 1 .", "Reads were subsequently trimmed from both ends using a minimum quality cutoff of 3; reads with three or more N's or with average quality score <Q20 were removed .", "In addition , reads <50 bp were removed .", "The remaining quality-filtered Illumina reads were assembled using SOAPdenovo v1 . 05 ( Luo et al . , 2012 ) at a range of kmers ( 81 , 85 , 89 , 93 , 97 , 101 ) with default settings ( options: -K 81 -p 32 -R -d 1 ) .", "Contigs generated by each assembly ( 6 total contig sets ) , were de-replicated using JGI in house Perl scripts .", "Contigs were then sorted into two pools based on length .", "Contigs <1800 bp were re-assembled using Newbler ( Life Technologies , Carlsbad , CA , USA ) in attempt to generate larger contigs ( options: -tr , -rip , -mi 98 , -ml 80 ) .", "Contigs >1800 bp as well as the contigs from the Newbler assembly were combined using minimus 2 ( options: -D MINID=98 -D OVERLAP=80 ) from the AMOS package ( http://sourceforge . net/projects/amos ) .", "Read depths were estimated based on read mapping with bbmap ( http://bio-bwa . sourceforge . net/ ) .", "The metagenome study information is available from the DOE-JGI GOLD database ( study: Gs0000079 ) .", "The unassembled reads are available from the NCBI SRA ( see Supplementary file 8 ) , and the assembled and annotated metagenome datasets from the IMG/M system ( Markowitz et al . , 2014 ) .", "The DOE-JGI MAP v . 4 annotation pipeline ( Huntemann et al . , 2015 ) was used for initial metagenome gene prediction and annotation .", "The annotated metagenomes were loaded in the IMG/M system as of mid 2014 , and subsequently imported into a GenDB v2 . 2 annotation system ( Meyer et al . , 2003 ) for taxonomic classification and data mining .", "All genes were searched against the NCBI non-redundant protein database ( as of June 17th , 2014 ) using USEARCH v6 . 1 . 544 ( Edgar , 2010 ) , against the Pfam v25 database ( Finn et al . , 2014 ) using HMMER v3 ( Punta et al . , 2012 ) , for signal peptides using SignalP v3 . 0 ( Nielsen et al . , 1999 ) and for transmembrane helices using TMHMM v2 . 0c ( Krogh et al . , 2001 ) .", "CAZymes were automatically annotated based on HMMER searches against the Pfam v25 and dbCAN ( Yin et al . , 2012 ) databases and BLAST ( Altschul et al . , 1990 ) searches against the CAZy database ( Cantarel et al . , 2009; Lombard et al . , 2014 ) using E-value cut-offs that were specifically adjusted for each CAZyme family ( Supplementary file 11 ) .", "Genes were only annotated as CAZymes when at least two of the search results were congruent , and CAZymes were only analyzed for contigs ≥500 bp .", "Taxonomic classification of the metagenome sequences into taxonomically coherent bins ( 'taxobins' ) was carried out with a modified version of the Taxometer approach described in ( Teeling et al . , 2012 ) .", "Taxometer consolidates predictions of a set of individual sequence classification tools into a consensus using a weighted assessment on seven selected ranks ( superkingdom , phylum , class , order , family , genus , species ) of the NCBI taxonomy ( http://www . ncbi . nlm . nih . gov/Taxonomy/ ) .", "We combined taxonomic information inferred from", "( i ) Pfam hits using the CARMA3 approach ( Gerlach and Stoye , 2011 ) ,", "( ii ) BLASTp hits using the KIRSTEN approach ( Teeling et al . , 2012; supp . data ) , and", "( iii ) mapping of quality-filtered ( illumina-utils; https://github . com/meren/illumina-utils/ ) Illumina reads to selected reference sequences .", "In contrast to the original Taxometer approach we omitted signature-based classification with Self-Organizing Maps and mapping of reads containing partial 16S rRNA gene sequences .", "The prediction tools that were used are outlined below: We used the HMMER-based module of CARMA3 ( not the BLAST-based module ) that infers taxonomy of sequences by post-processing genes with HMMER3 hits to the Pfam database .", "The basic principle is to apply a reciprocal search technique to reduce the number of identified matches and thereby to improve taxonomic classification quality .", "KIRSTEN ( Kinship Relationship Reestablishment ) infers taxonomy of sequences by post-processing BLASTp hits to the NCBI nr database by means of rank-based evaluations on all levels of the NCBI taxonomy with an increasing stringency from the superkingdom down to the species level .", "On each taxonomic level , all occurring taxa are weighted by the sum of their BLASTp bit scores .", "When the taxon with the highest weight exceeds an adjustable threshold , the process continues towards the next taxonomic level .", "The threshold increases with each taxonomic level , i . e . the algorithm becomes more critical while it progresses .", "For this study , we substituted BLASTp by the UBLAST module of USEARCH 6 . 1 ( Edgar , 2010 ) with an E-value cutoff of E-10 and maximum hit count of 500 .", "SMALT ( http://www . sanger . ac . uk/resources/software/smalt/ ) was used to map metagenome reads on a manually compiled set of 49 reference genomes and a streamlined version of the NCBI nr database .", "Both , the reference genomes and the sequences selected from the NCBI nr database were selected based on habitat-specific information .", "This was done manually for the reference genomes and automatically for the NCBI nr database as follows: Each of the hits from the UBLAST search during KIRSTEN analysis can be associated with multiple taxa , since in nr redundant sequences from different taxa are merged .", "We used this information to extract all taxa associated with a given hit , then combined the taxa of all hits and finally extracted all sequences of these taxa from Genbank .", "This way a sample-specific streamlined subset of Genbank was generated that greatly sped up the mapping process .", "Only metagenome reads with at least 95% identity to any sequence in the sample-specific sequence database were used .", "Reads were subsequently back-mapped to contigs .", "Since contigs consist of many reads , this way , contigs were associated with multiple taxonomic paths .", "Taxonomic paths resulting from classification with reference genomes and the habitat-specific streamlined nr were concatenated .", "Paths representing less than 1% of the reads of a given contig were discarded .", "Finally , Taxometer was used to combine all gene-based predictions from CARMA3 and KIRSTEN and the mapping results for each contig , and to infer a consensus taxonomy .", "Taxonomic predictions were possible for 94 . 6% of the contigs above 1 kbp .", "The results are summarized in Supplementary file 9 .", "The Spearman rank correlation test ( Supplementary file 6 ) was used to test for correlations between Bacteroidetes clade abundances and environmental variables ( chlorophyll a , temperature , salinity , silicate , phosphate , nitrate , nitrite , ammonia ) and phytoplankton abundances ( classes: diatoms , dinoflagellates , coccolithophorids , silicoflagellates , flagellates , ciliates , green algae; species: Mediophyxis helysia , Thalassiosira nordenskioeldii , Chaetoceros debilis and C . minimus , Rhizosolenia styliformis , Chattonella and Phaeocystis ) .", "Bacteroidetes and phytoplankton numbers were transformed to log-scale for better comparison .", "Linear regressions ( Supplementary file 7 ) were done using stepwise forward regression model by using the log transformed Bacteroidetes and phytoplankton abundances .", "All statistical analyses were performed using the software Sigma-Plot 12 ( SYSTAT , Santa Clara , CA , USA ) ." ] ]

[ "A process of global importance in carbon cycling is the remineralization of algae biomass by heterotrophic bacteria , most notably during massive marine algae blooms .", "Such blooms can trigger secondary blooms of planktonic bacteria that consist of swift successions of distinct bacterial clades , most prominently members of the Flavobacteriia , Gammaproteobacteria and the alphaproteobacterial Roseobacter clade .", "We investigated such successions during spring phytoplankton blooms in the southern North Sea ( German Bight ) for four consecutive years .", "Dense sampling and high-resolution taxonomic analyses allowed the detection of recurring patterns down to the genus level .", "Metagenome analyses also revealed recurrent patterns at the functional level , in particular with respect to algal polysaccharide degradation genes .", "We , therefore , hypothesize that even though there is substantial inter-annual variation between spring phytoplankton blooms , the accompanying succession of bacterial clades is largely governed by deterministic principles such as substrate-induced forcing ." ]

[ "Small algae in the world's oceans remove about as much carbon dioxide from the atmosphere as land plants .", "These algae do not grow continuously , but often surge in numbers during temporary blooms .", "Such blooms can be large enough to be seen from space by satellites .", "The lifespan of algae within such blooms is short , and when they die , marine bacteria feed on the remnants , which releases much of the stored carbon dioxide .", "Much of an algal cell consists of different types of polysaccharides .", "These large molecules are essentially made from sugars linked together .", "Polysaccharides are varied molecules and can contain many different sugars that can be linked in a number of different ways .", "During algae blooms bacteria proliferate that are specialized in the degradation of these polysaccharides .", "In 2012 , researchers reported how over the progression of an algae bloom different groups of marine bacteria bloomed in rapid succession .", "However , it remained unknown whether the same or different groups of bacteria respond to algae blooms at the same place from year to year , and whether or not these bacteria use the same enzymes to degrade the polysaccharides .", "Teeling , Fuchs et al . – who include many of the researchers from the 2012 study – now report on the analysis of a series of algae blooms that occurred in the southern North Sea between 2009 and 2012 .", "The analysis is based on samples collected every week during the spring seasons , and shows that certain groups of related bacteria , known as clades , became common during each bloom .", "Teeling , Fuchs et al . also found indications that the clades that repeatedly occurred had similar sets of genes for degrading algal polysaccharides , but that the sets were different between the clades .", "These data suggest that there is a specialized bacterial community that together can degrade the complex mixture of algal polysaccharides during blooms .", "This community reappears each year with an unexpectedly low level of variation .", "Since different species of algae made up the blooms in each year , this finding suggests that the major polysaccharides in these algae are similar or even identical .", "Future work will focus on the specific activities of bacterial enzymes that are needed to degrade polysaccharides during algae blooms .", "Study of these enzymes in the laboratory will help to resolve , which polysaccharides are attacked in which manner , and to ultimately help to identify the most abundant algal polysaccharides .", "This will improve our current understanding of the carbon cycle in the world’s oceans ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "neuroscience" ]

[ [ "Goal-directed actions aimed at obtaining a reward often involve exposure to an aversive event or punishment .", "For example , foraging for food in the wild may result in encountering a predator .", "In a causally and socially complex world , appropriate representation of punishment that lurks around during reward-seeking actions is critical for survival and optimal action selection .", "Deficits in this representation may be associated with detrimental behavioral patterns observed in impulsive behavior and addictive disorders while exaggerated representation of punishment risk may be linked to anxiety disorders ( Bechara et al . , 2000; Gillan et al . , 2016; Hartley and Phelps , 2012; Lee , 2013; Mineka et al . , 1998 ) .", "How is risk of punishment associated with reward-seeking actions represented by the brain ?", "To begin to address this question , we focused on the ventral tegmental area ( VTA ) and the medial prefrontal cortex ( mPFC ) .", "Neurons in the VTA including dopamine ( DA ) and non-dopamine neurons are critical components of the reward circuitry including reward-mediated actions ( Cohen et al . , 2012; Matsumoto et al . , 2016; Roesch et al . , 2007; Schultz , 1998; Tan et al . , 2012; van Zessen et al . , 2012; Wise , 2004; Wood et al . , 2017 ) .", "We , therefore , hypothesized that ensembles of VTA neurons represent risk of punishment associated with reward-guided behavior .", "Importance of VTA notwithstanding , the mPFC is also implicated in reward representation and reward-guided action selection ( Barraclough et al . , 2004; Buschman et al . , 2012; Kobayashi et al . , 2006; Powell and Redish , 2016; Rich and Shapiro , 2009 ) , as well as control of aversive and anxiety-like behavior ( Adhikari et al . , 2010; Kim et al . , 2017; Kumar et al . , 2014; Likhtik et al . , 2014; Park et al . , 2016; Ye et al . , 2016 ) .", "At the network level , the mPFC and VTA ( both DA and non-DA neurons ) send reciprocal projections to each other ( Berger et al . , 1976; Carr and Sesack , 2000a; 2000b ) .", "The VTA neurons projecting to mPFC have been shown to respond to stressful and anxiogenic drugs with a greater degree of sensitivity compared to the mesolimbic or mesostriatal projections ( Abercrombie et al . , 1989; Bradberry et al . , 1991; Moghaddam et al . , 1990; Thierry et al . , 1976 ) .", "Furthermore , photostimulation of the VTA DA input to the mPFC elicits anxiety-like behavior ( Gunaydin et al . , 2014; Lammel et al . , 2012 ) , suggesting a more causal role for this neural circuit in aversive behavior .", "Given this , we further hypothesized that the interaction between VTA and mPFC provides a dynamic representation of punishment risk during reward-seeking actions .", "To test these hypotheses , we first designed and validated a task that allowed us to assess reward-guided actions in the absence or presence of punishment risk in the same recording session .", "The latter criterion was critical because it allowed us to track the activity of the same ensembles of neurons as a function of punishment risk .", "The task was designed so that an instrumental action always procured a reward , but the same action probabilistically led to punishment with blockwise varying degrees of contingency .", "Thus , different blocks had varying risk of punishment associated with an action while action-reward contingency remained constant .", "We then recorded single unit activity and local field potentials ( LFP ) from the VTA and mPFC simultaneously during this task .", "The simultaneous recording allowed us to compare the inter- and intra-regional neural codes for punishment risk-based modulation of behavioral events .", "At the single unit level , we found that VTA and mPFC neurons encode risk of punishment and blockwise/trial-by-trial behavioral modulation during action execution .", "At the network level , we found that coherent theta oscillations synchronize the VTA and mPFC in a bottom-up direction , effectively phase-modulating the neuronal spike activity in the two regions during punishment-free actions .", "This oscillation-mediated neural synchrony declined as a function of punishment risk , suggesting that desynchronization of coordinated activity in these two regions signals punishment ." ], [ "Blockwise changes in punishment risk resulted in changes in behavior ( Figure 1a–b , Video 1 and 2 ) .", "Videos are representative examples of trials in block 1 ( Video", "1 ) and block 3 ( Video", "2 ) of the same animal .", "As clearly captured in Video 2 , animals do not merely freeze during RTs; they make incomplete nose pokes , which , importantly , are selectively observed during instrumental pokes but not during trough pokes to retrieve reward .", "The mean response time ( RT ) and time spent in immobility during RT markedly increased as a function of punishment ( Figure 1c; GLM repeated measures , F2 , 32 = 24 . 94 , p<0 . 001; GLM repeated measures , F2 , 32 = 22 . 44 , p<0 . 001 , respectively ) .", "These increases reflect a state anxiety but not diminished motivation to obtain reward as the latency for reward retrieval ( reward RT ) remained similar across blocks regardless of punishment ( Figure 1c inset; GLM repeated measures , F2 , 32 = 2 . 97 , p=0 . 07 ) .", "Punishment risk significantly increased the trial-to-trial variability in RT ( Figure 1—figure supplement 1a; GLM repeated measures , F2 , 32 = 13 . 89 , p<0 . 001 ) .", "This increase was associated with shock occurrences , since the RT varied as a function of the trial lag ( distance ) from the previous shock ( Figure 1—figure supplement 1b ) indicating that animals adjusted their behavior across trials even within the same block in the face of punishment risk .", "To examine whether the increase in RT reflected learning of action-punishment contingency or merely an immediate impact of shock , we compared RTs of early trials in block 2 and 3 , before the delivery of the 1st shock .", "Rats displayed a subtle but significant increase in RT in block 2 , and robust increase in block 3 before the first shock ( Figure 1—figure supplement 1c; GLM repeated measures , post hoc pairwise comparisons , p<0 . 001 ) , suggesting that there is learning of action-punishment contingency from previous experiences .", "During the no-shock control session , when animals performed the same number of trials and blocks in the absence of punishment , the mean RT and the variance RT did not differ across blocks ( Figure 1d , Figure 1—figure supplement 1d ) .", "The punishment-induced increase in RT suggested a transient anxiety-like state during block 3 .", "This was confirmed by the anxiolytic drug , diazepam ( 2 mg/kg ) , significantly attenuating the increase in RT of block 3 , compared with saline-pretreatment data ( Figure 1e; Repeated measures ANOVA , F4 , 48 = 3 . 27 , p=0 . 019; post hoc test , block 3 , p values < 0 . 01 ) .", "Diazepam or saline injected animals showed equivalent levels of reward RT ( Figure 1e inset; Repeated measures ANOVA , F4 , 48 = 0 . 34 , p=0 . 852; post hoc test , p values > 0 . 51 ) .", "During task performance , 167 mPFC and 102 VTA single units were recorded from histologically verified electrodes ( Figure 2—figure supplement 1 ) .", "For all single unit data analyses , we classified VTA units into putative dopamine ( DA , n = 55 ) and putative non-dopamine ( non-DA , n = 47 ) subtypes ( Figure 2—figure supplement 2 , Materials and methods ) .", "We first examined the trial-averaged neuronal activity of mPFC , VTA putative DA , and non-DA units to compare their general tuning properties during task events – cue onset , action , and reward delivery .", "Figure 2a shows peri-event neuronal activity averaged across all trials and blocks .", "The majority of VTA putative DA units displayed phasic excitatory responses at each task event as has been previously reported ( Schultz et al . , 1993 ) , whereas non-DA and mPFC units showed weaker and temporally diffuse responses ( Figure 2a–c; Repeated measures ANOVA , post-cue , F2 , 266 = 22 . 05; peri-action , F2 , 266 = 43 . 78; post-reward , F2 , 266 = 48 . 93 , p values < 0 . 001 ) .", "We then examined modulation of single neuronal activity across blocks ( Figure 2b–c ) .", "Some of the mPFC , VTA putative DA , and non-DA single units appeared to have modulated their peri-event firing rates across blocks as a function of punishment risk .", "Given this , we quantified individual neuronal representation of punishment using a percent explained variance ( ωPEV ) statistic ( Buschman et al . , 2012 ) .", "This analysis essentially quantifies how much of total variance in a neuron’s firing rate across trials can be explained by blockwise changes in punishment risk ( Materials and methods ) .", "Comparing the ωPEV statistics of the original spike trains with the ωPEV distribution of surrogate spike trains created by shuffling block labels ( Figure 2d–e , Materials and methods ) allowed us to identify punishment encoding units , i . e . units that responded to the same events ( cue , action , reward ) differently in different blocks .", "The most robust punishment encoding occurred during the peri-action epoch ( Figure 3 , Figure 3—figure supplement", "1 ) given that ωPEVs during this period was greater than that of the peri-cue or peri-reward epochs ( Figure 3a ) .", "There was a significant interaction between time and unit groups in the peri-action ωPEV ( Figure 3a; Repeated measures ANOVA , F38 , 5054 = 2 . 68 , p<0 . 001 ) , indicating distinct time-varying patterns in representation of punishment by different unit groups .", "Similar time-varying patterns were observed in the proportion of units representing punishment risk ( Figure 3b ) .", "To examine the time course of individual neuronal encoding in the peri-action epoch , we recalculated the peri-action ωPEV using a narrower moving window ( 50 ms width , 5 ms step ) to reveal time points of individual neuronal representation of punishment at a higher temporal resolution .", "Response of VTA putative DA neurons appeared to be concentrated specifically around the time of the action , whereas non-DA and mPFC units displayed temporally diffuse patterns ( Figure 3c ) .", "This result suggested that DA units may be involved in more precise signaling of action and punishment risk , whereas the mPFC and VTA non-DA units may represent more persistent effects of punishment on motivational/emotional states .", "Consistent with this function , substantial proportions of mPFC ( 31% ) and non-DA ( 32% ) units significantly modulated their baseline firing rates across blocks , suggesting that they represent punishment on a longer temporal scale ( Figure 3d ) .", "Fewer VTA putative DA units ( 14% ) modulated their baseline activity ( Figure 3d ) .", "Next we examined the direction of neuronal response ( excitation or inhibition ) as a function of punishment .", "Similar proportions of mPFC units encoded punishment with bidirectional modulation of activity ( Figure 4a ) , which resulted in the lack of net excitation or suppression of activity across blocks ( Figure 4b; Repeated measures ANOVA , F2 , 241 = 2 . 09 , p=0 . 126 ) .", "The majority of VTA putative DA units encoded punishment risk with an excitatory response ( Figure 4c ) specifically at the time of the action ( Figure 4d; Repeated measures ANOVA , F2 , 105 = 3 . 96 , p=0 . 022 ) , but not during other task events .", "Similarly , a greater number of non-DA punishment-encoding units displayed an excitatory modulation of activity ( Figure 4e ) with a trend toward a net excitatory effect of the block ( Figure 4f; Repeated measures ANOVA , F2 , 105 = 2 . 94 , p=0 . 057 ) .", "To distinguish neuronal encoding of punishment risk from other confounding factors that may cause block-dependent changes in neuronal activity ( e . g . , satiety , fatigue , or spontaneous drifting over time ) , we performed a control experiment recording from 126 mPFC and 57 VTA single units ( putative DA , n = 28; putative non-DA , n = 29 ) during performance of three consecutive punishment-free blocks .", "We observed a much weaker blockwise changes in firing rate indicating negligible impact of confounding factors on the neuronal encoding of punishment ( Figure 5 ) .", "Taken together , these data indicate that both VTA and mPFC single neurons convey information about punishment risk during reward-seeking behavior .", "Moreover , we observed distinct temporal and directional tuning properties from mPFC , VTA putative DA , and non-DA neuronal subpopulations .", "This heterogeneity may enable the VTA-mPFC circuit to represent diverse motivational/emotional aspects of punishment risk .", "Our behavioral data indicated remarkable trial-to-trial variability of RT in the face of punishment risk ( Figure 1—figure supplement 1 ) .", "To better understand the relationship between the behavioral and neural data , we examined whether , and to what extent , RT variability is related to mPFC and VTA neuronal activity .", "At the single neuron level , only a small fraction of single units exhibited significant correlation between firing rate and the RT across trials ( mPFC , 10 . 2%; VTA putative DA , 36 . 4%; VTA putative non-DA , 25 . 5% ) .", "This result indicated that inferring trial-by-trial behavior from single neuronal activity was limited .", "We , therefore , moved on to correlating single-trial RT with neuronal population activity .", "A conventional measure of the neuronal population activity is averaging across neurons , but the bi-directionality in the individual neuronal representation of risk ( Figure 4 ) undermined the feasibility of this approach .", "As an alternative approach , we used a neuronal population state space analysis that detected patterns of activity co-modulation in simultaneously recorded mPFC or VTA populations ( comprising of 10 or more units ) .", "Trial-by-trial neural population trajectories were extracted within this state space using a dimensionality reduction method Gaussian process factor analysis ( GPFA; Materials and methods ) ( Yu et al . , 2009 ) .", "Examining the correlation between the neural population trajectory and the RT across trials revealed that mPFC and VTA population trajectories track trial-to-trial variation in RT ( Figure 6 ) .", "Specifically , the deviation ( Euclidean distance ) of each trial’s neural trajectory from the block 1 mean trajectory was significantly correlated with the deviation of each trial’s RT from the block 1 mean RT .", "Simply put , the further each trial’s neural trajectory departed from the block 1 mean trajectory , the greater the RT appeared ( Figure 6 , Figure 6—figure supplements 1–2 ) .", "Significant correlations were observed in all four mPFC and three VTA populations comprising 10 to 25 single units subjected to this analysis ( Figure 6—figure supplements 1–2 ) .", "Next we looked to see if the neural population trajectories could track behavioral variation within each block .", "As our behavioral data indicated more pronounced trial-to-trial variability in RT in the riskiest block ( Figure 1—figure supplement 1 ) , it was of particular interest whether the increased behavioral variability was correlated with the neural population trajectories in this block .", "Significant correlations were observed specifically in the riskiest block in two of the four mPFC populations ( Figure 6—figure supplement 1 ) , indicating that the neural and behavioral correlation emerged as the behavioral variability arose along with risk of punishment .", "Similarly , significant correlations were detected in two of the three VTA populations in the riskiest block ( Figure 6—figure supplement 2 ) .", "Finally , we computed trial-by-trial trajectories of neuronal population activity recorded from the no-shock control sessions .", "Consistent with the lack of correlation observed in the absence of risk , there was no significant correlation between mPFC and VTA population activity and RT ( Figure 6—figure supplements 1–2 ) .", "These results indicate that distinct neuronal population activity track trial-to-trial variation of reward-seeking behavior in the presence , but not the absence , of punishment risk .", "At the neural network level , synchronous oscillations can provide temporal coordination among groups of neurons , which may subserve cognitive and affective functions ( Adhikari et al . , 2010; Buschman et al . , 2012; Fries , 2015; Karalis et al . , 2016a; Kim et al . , 2012; Likhtik et al . , 2014 ) .", "We , therefore , examined oscillation-mediated neural synchrony during reward-seeking behavior in the absence and presence of punishment risk .", "During punishment-free performance in block 1 , theta oscillations in the frequency band of 5 to 15 Hz emerged in mPFC and VTA before and during action execution ( Figure 7a–d ) .", "This oscillation was markedly reduced as a function of punishment risk in both regions ( Figure 7e–f ) .", "Of note , this change in theta oscillation observed before and during the action could not be caused by changes in animals’ motor activity because they all engaged in the action within this epoch .", "A significant interaction between punishment and frequency band was detected in LFP power during post-cue and pre-action time periods in both regions ( Repeated measures ANOVA , VTA , post-cue , F52 , 1092 = 5 . 11; pre-action , F52 , 1092 = 7 . 78 , p values < 0 . 001; mPFC , post-cue , F52 , 1872 = 1 . 60 , p=0 . 005; pre-action , F52 , 1872 = 2 . 29 , p<0 . 001 , Figure 7g–h ) .", "Theta oscillations appeared to be coherent between the two regions during the punishment-free action , but the coherence significantly decreased as a function of punishment ( Figure 7i ) .", "A significant interaction between punishment and frequency band was observed in LFP coherence during the pre-action period ( Repeated measures ANOVA , post-cue , F52 , 1092 = 0 . 93 , p=0 . 62; pre-action , F52 , 1092 = 2 . 45 , p<0 . 001 ) .", "To examine mutual influences ( directionality ) of LFP time series between the two regions , we quantified Granger causal influences ( GC ) in VTA-to-mPFC and mPFC-to-VTA directions ( Materials and methods ) .", "During punishment-free action in block 1 , the theta oscillation was driven by VTA , as mPFC was GC influenced by VTA significantly greater than the GC influence in the other direction ( Figure 7j ) .", "A significant interaction between directionality and frequency band was observed in GC coefficients in all blocks , indicating that the oscillation directionality varied across frequency bands ( Figure 7j; Repeated measures ANOVA , Block 1 , F25 , 700 = 2 . 05 , p=0 . 002; Block 2 , F25 , 700 = 2 . 38 , p<0 . 001; Block 3 , F25 , 700 = 5 . 59 , p<0 . 001 ) .", "Importantly post hoc analysis revealed significantly greater VTA-to-mPFC directionality in frequency bands including the theta band in block 1 , and the directionality became less apparent in blocks 2 and 3 ( Figure 7j , data not shown for block 2 ) .", "Taken together , these results suggest that the VTA-driven theta oscillation entrains the VTA-mPFC circuit during punishment-free action .", "Decline in this entrainment may represent punishment risk , since power , coherence , and directionality of the theta oscillation declined as a function of punishment risk .", "Analyses of no-shock control data revealed that these punishment-dependent changes in the VTA-mPFC theta oscillations do not occur in the absence of punishment ( Figure 7—figure supplement 1 ) .", "Synchronous oscillations can provide temporal coordination of spike activity between local and distant neurons and neuronal communication between coherently timed neuron groups ( Fries , 2015; Harris and Gordon , 2015 ) .", "The presence of such LFP-mediated spike timing coordination was examined by measuring phase-locking of the neuronal spike activity to local and inter-regional theta oscillations .", "Within each region , substantial proportion of single units ( 37% in VTA and 23% in mPFC ) showed significant phase-locking to local theta oscillation in the punishment-free block 1 .", "Consistent with the temporally specific increase in the theta spectral power ( Figure 7c–d ) , the phase synchrony arose during action from the baseline level in the phase-locked units in both regions ( Figure 8a and d; Signed-rank test , p values < 0 . 001 ) .", "Enhanced phase-locking , albeit to a lesser degree , was observed even in units that failed the Rayleigh’s test ( Figure 8a and d; Signed-rank test , p values < 0 . 001 ) , indicating widespread influence of the theta oscillation on local neuronal spike timing .", "The temporal relationship ( directionality ) between spike outputs and theta oscillation was examined using a time-lagged phase-locking analysis ( Likhtik et al . , 2014; Spellman et al . , 2015 ) .", "The spike-LFP phase-locking was recalculated using spike times shifted relative to the local or inter-regional LFP to infer the directionality in the LFP-spike interaction .", "We found that in block 1 greater proportions of units appeared to be phase-locked with negative time lags .", "The vast majority of phase-locked units ( VTA , 74%; mPFC , 67% ) had their maximum phase-locking values ( PLVs; Materials and methods ) with a negative lag ( Figure 8b and e; Signed-rank test , p values < 0 . 005 ) .", "These indicated an entrainment of spike timing by preceding cycles of the oscillation – that is , directionality from the theta oscillation to the spike activity .", "To examine the modulation of LFP-spike phase-locking by punishment , we compared PLVs across different blocks .", "A trend toward reduction in PLV was found in block 3 compared with block 1 in mPFC ( Figure 8c; Signed-rank test , p=0 . 077 ) , and a significant reduction was found in VTA ( Figure 8f; p=0 . 006 ) .", "Likewise , a trend toward reduction in the proportion of phase-locked units was observed in block 3 compared with block 1 in mPFC ( Figure 8b; Chi-square test , χ21 = 3 . 25 , p=0 . 071 ) , and a significant reduction was found in VTA ( Figure 8e; χ21 = 4 . 31 , p=0 . 038 ) .", "We next examined VTA putative DA and non-DA neuronal phase-locking separately .", "Greater fraction of putative DA units ( 45% ) appeared to be phase-locked compared with non-DA units ( 23% ) in block 1 ( Figure 8g; Chi-square test , χ21 = 5 . 04 , p=0 . 025 ) .", "The DA neuronal PLV in block1 significantly declined as a function of punishment contingency in block 2 and 3 ( Figure 8h; Signed-rank test , p values < 0 . 01 ) , whereas non-DA neuronal PLV did not differ across blocks ( p values > 0 . 43 ) .", "These indicated that the punishment risk-induced reduction in the VTA neuronal phase-locking was predominately due to the reduction in DA neuronal synchrony .", "Next we examined LFP-spike phase-locking between VTA and mPFC .", "Based on the Granger causal influence indicating VTA-to-mPFC directionality in theta oscillations , we anticipated a stronger mPFC neuronal synchrony to VTA than that of the other direction .", "Consistent with this , we found that a substantial proportion of mPFC units ( 31% ) were phase-locked to VTA theta oscillation in block 1 .", "A representative mPFC unit with significant phase-locking is shown in Figure 9a–b .", "The inter-regional spike-phase synchrony emerged during action compared to baseline ( Figure 9c , Signed-rank test , p values < 0 . 001 ) .", "We then examined directionality of the LFP-spike synchrony using the time-lagged phase-locking analysis .", "In block 1 , the majority of phase-locked units had their peak PLVs with a negative lag ( Figure 9d; Signed-rank test , p=0 . 066 ) .", "Likewise , greater proportions of phase-locked units were observed on negative lags ( Figure 9d , bottom ) .", "In addition , the mean PLV across negative time lags appeared to be greater than that of the positive lags ( Figure 9e; Signed-rank test , p=0 . 023 ) .", "These indicate that mPFC neurons are entrained by the preceding VTA theta oscillatory cycles suggesting VTA-to-mPFC directionality .", "When compared across blocks , the mPFC neuronal entrainment by the VTA theta oscillation declined as a function of punishment contingency ( Figure 9e–g , Signed-rank test , p=0 . 003 ) .", "As the degree of phase-locking diminished , the VTA-to-mPFC directionality also declined ( Figure 9d–e ) .", "Finally , we examined VTA neuronal phase-locking to the mPFC theta oscillation .", "The degree of VTA neuronal phase-locking to mPFC theta oscillation was weaker than the mPFC neuronal phase-locking to the VTA theta oscillation ( Wilcoxon Rank-sum test , p<0 . 001 ) , indicating VTA-to-mPFC directionality in the theta oscillation-mediated spike phase modulation ( Figure 9—figure supplement 1 ) .", "The PLVs did not differ across different blocks in both putative DA and non-DA units ( Figure 9—figure supplement 1 ) .", "Analyses of no-shock control data showed no change in neural synchrony across blocks in the absence of punishment ( Figure 9—figure supplements 2–3 ) .", "Collectively , these analyses indicate that a coherent theta oscillation transiently synchronizes the VTA-mPFC neural network during reward-guided actions and that this synchrony declines when there is a risk of punishment associated with those actions ( Figure 9h ) ." ], [ "Using a novel behavioral paradigm that assessed punishment risk during reward-guided behavior , we found that the majority of VTA and mPFC neurons respond to punishment risk by modulating their firing rates .", "Critically , this response was most pronounced at the time of the action as compared to other task events such as cue or reward delivery .", "This suggests that neurons in both structures preferentially encode risk of punishment during reward-seeking actions .", "Given the relatively low probability and modest experience of punishment in the present task , one may wonder to what extent animals were cognizant of the punishment contingency on the action .", "Our data suggest that the punishment based behavioral changes is due to both learning of action-punishment contingency and state-dependent effects of punishment .", "Learning of contingency is supported by data showing a selective increase in instrumental RTs but not in reward RTs .", "As shown in the video reproducing stereotypical risky actions , animals were timid/cautious at the time of instrumental actions and made many ‘incomplete’ nose pokes , which was not the case for nose pokes to the food trough .", "The latter is mechanically the same action to which punishment was never contingent on .", "These suggest that pronounced changes in neural activity within the peri-action epoch may reflect changes in action-punishment contingency .", "We also observed a correlation between behavior and neuronal responses as behavioral variability increased with risk of punishment suggesting that the direct impact of shock experiences may contribute , in part , to behavioral and neural changes irrespective of learned contingency .", "Punishment influences behavior , and neuronal representation associated with that behavior , on multiple timescales ( Cohen et al . , 2015; Somerville et al . , 2013 ) .", "On short timescales , real-time neural processing of punishment may be important to signal contingency of punishment on a specific event in order to promote rapid behavioral adaptation .", "Our data suggest that VTA DA neuronal signaling may be involved in this context .", "DA neurons displayed phasic excitatory responses tightly linked to each of the task events ( cue , action , and reward ) .", "Importantly , the DA neuronal encoding of punishment was concentrated around the time of the action compared with other task epochs , suggesting that DA neuronal signaling of punishment may primarily reflect the action-punishment contingency .", "On longer timescales , punishment can elicit persistent changes in motivational and emotional states – e . g . , changes in mood .", "We found that mPFC and VTA non-DA neurons display temporally diffuse encoding of punishment within the peri-action window .", "Likewise , many of the mPFC and non-DA neurons showed significant modulation of their baseline firing rates , suggesting that these neurons may encode punishment with persistent changes in activity .", "This sustained change in activity may be responsible for longer-lasting affective impact of punishment .", "Subpopulations of VTA putative DA , non-DA , and mPFC neurons responded to punishment risk by increasing or decreasing their peri-action firing rates .", "This is not surprising given that while punishment is an aversive event that should be avoided , it is highly salient and deserving prioritized attention .", "These aspects of punishment need to be represented for appropriate behavioral coping .", "In general , the direction of neuronal responses to appetitive vs aversive events is thought to carry information about motivational properties encoded by the neuronal activity .", "While heterogeneous response patterns have been widely observed in PFC neurons that respond to punishment ( Kobayashi et al . , 2006; Matsumoto et al . , 2007; Seo and Lee , 2009; Ye et al . , 2016 ) , there is some debate on whether the VTA DA neurons respond to punishment with excitatory or inhibitory responses ( Bromberg-Martin et al . , 2010; Schultz , 2016 ) .", "It has been demonstrated that reward prediction error ( RPE ) -coding DA neurons respond to appetitive ( better-than-predicted ) vs aversive ( worse-than-predicted ) events by excitation and inhibition , integrating information about appetitive and aversive events into a common currency of value ( Eshel et al . , 2016; Matsumoto et al . , 2016; Mileykovskiy and Morales , 2011; Mirenowicz and Schultz , 1996; Roitman et al . , 2008 ) .", "We found that a subset of VTA putative DA neurons conformed to this pattern .", "These neurons responded to punishment-free ( purely appetitive ) actions with phasic excitation , which decreased as a function of punishment risk .", "In contrast , a greater proportion of putative DA neurons showed excitatory responses to punishment; that is , they treated appetitive and aversive components in the same direction , and responded to actions prone to punishment with further excitation .", "We observed that excitatory representation of punishment risk was predominant among DA neurons , suggesting that risk of punishment is not simply encoded as reduced value of the action .", "Previous studies have reported that a subpopulation of putative DA neurons show similar excitatory responses to appetitive and aversive stimuli ( Brischoux et al . , 2009; Joshua et al . , 2008; Matsumoto and Hikosaka , 2009; Valenti et al . , 2011 ) .", "An excitatory DA neuronal encoding of aversive or neutral events has been interpreted as conveying motivational salience ( Bromberg-Martin et al . , 2010; Matsumoto and Hikosaka , 2009 ) , detection ( Nomoto et al . , 2010 ) , intensity ( Fiorillo et al . , 2013 ) of a sensory event , as well as generalization effect of rewarding stimuli ( Kobayashi and Schultz , 2014 ) .", "Combination of these factors may comprise the excitatory DA neuronal encoding of punishment-prone actions we observed .", "Furthermore , the DA neuronal encoding of aversion has been suggested to depend on animals’ behavioral state .", "A recent study demonstrated that DA neurons encoded aversive events with inhibition in a low reward context but with biphasic responses in a high reward context ( Matsumoto et al . , 2016 ) .", "In addition , different DA neural responses have been reported based on how animals responded to an aversive event .", "For example , DA concentration in the ventral striatum increased or decreased when rats displayed active avoidance or passive reaction ( freezing ) to shock-predicting cues ( Oleson et al . , 2012; Wenzel et al . , 2015 ) .", "These patterns of state dependency in DA neural responses to aversion are in line with our observation of excitatory encoding of punishment when risk was taken for reward in a highly rewarding context .", "Thus , our results support the view that DA neuronal signaling of aversion is not a uniform process and depends on behavioral states and reward availability in the environment .", "At the network level , we observed that coherent theta oscillation synchronizes VTA and mPFC specifically during punishment-free actions , effectively phase-modulating the neuronal spike activity in the two regions .", "Analyses of the temporal relationship indicated that the neural synchrony arose in the VTA-to-mPFC direction .", "That is , VTA-driven theta oscillation entrained mPFC LFPs and neuronal spike activity during punishment-free actions .", "The theta oscillation preferentially entrained putative DA neurons but much fewer non-DA neurons in the VTA .", "Considering the phasic excitatory responses of DA neurons during action , the theta oscillation-mediated neural synchrony may promote phase-coupling between VTA DA and mPFC neurons selectively during reward-seeking behavior when there is no risk of punishment .", "This phase-coupling diminished during punishment-prone actions .", "Thus , prediction of punishment can be inferred by dual alterations in the phase and the rate of the DA neurotransmission in the mPFC ( model in Figure 9h ) .", "Our data showing preferential DA ( vs non-DA ) neuronal phase-locking with VTA and mPFC theta oscillations supports the theoretical model suggesting that the VTA DA input may play a crucial role for cortical theta oscillations ( Buhusi and Meck , 2005 ) .", "In addition , our data is consistent with previous studies showing that inhibiting DA D1 receptors in mPFC diminishes theta oscillations ( Parker et al . , 2014 ) .", "Establishing the causality of VTA DA neuron driving mPFC theta oscillation , however , would require future experiments with pharmacological or optogenetic manipulation of specific cell types .", "While somewhat out of the scope of the present study , it is noteworthy that theta oscillations in the mPFC and/or VTA have been related to the hippocampal theta oscillation ( Hyman et al . , 2005; Jones and Wilson , 2005; Siapas et al . , 2005; Sirota et al . , 2008 ) .", "Local infusion of DA in mPFC enhances theta oscillations and coherence between mPFC and hippocampus ( Benchenane et al . , 2010 ) , while both mPFC and VTA neurons exhibit phase coherence to the hippocampal theta oscillation ( Fujisawa and Buzsáki , 2011 ) .", "Thus , the theta-mediated neural synchrony we observed in the VTA-mPFC circuit may be coupled with the hippocampal theta oscillation , including the possibility that the theta oscillation are led by the hippocampus and/or a third structure .", "Recent studies have reported slow ( 4 Hz ) oscillation in mPFC during fear-conditioned freezing in synchrony with other regions including the amygdala ( Dejean et al . , 2016; Karalis et al . , 2016; Likhtik et al . , 2014 ) .", "The distinct behavioral states associated with 4 Hz oscillations in these studies taken together with theta oscillations observed in our study may suggest that mPFC is entrained by different bands of oscillations in appetitive vs aversive states .", "However , the 4 Hz oscillation in the VTA-mPFC circuit has also been associated with working memory ( Fujisawa and Buzsáki , 2011 ) .", "The fast vs slow mPFC oscillations occurring in appetitive vs aversive states may arise in preferential synchrony with VTA or amygdala in appetitive vs aversive states , thereby the bottom-up information transfer from the two subcortical regions can be routed depending on the behavioral context .", "This scenario would predict that the theta-oscillation-mediated VTA-mPFC synchrony decreases in the presence of punishment while 4 Hz oscillation increases in the mPFC .", "Consistent with this , we observed that mPFC theta-oscillation-mediated synchrony significantly declined as a function of action-punishment contingency .", "We did not observe the emergence of 4 Hz oscillation presumably because , unlike fear conditioning paradigms , our task involved instrumental actions .", "Of note , entrainment of a neural circuit with varying frequency oscillations as a function of a task variable has been widely observed in sensory cortical circuits ( Bosman et al . , 2012; Jia et al . , 2013 ) .", "Such frequency modulation , along with the power modulation , could promote selection and binding of task-relevant neuronal ensembles to give rise to a functional neural network ( Fries , 2015 ) .", "Likewise , our data may reflect the rise and fall of coherent VTA and mPFC neuronal ensembles that may promote a flexible control of instrumental behavior as a function of punishment contingency .", "The neural synchrony mediated by different bands of oscillations in distinct behavioral states may also implicate neuron groups in the other connected structures such as nucleus accumbens , amygdala , hippocampus , and lateral habenula that are critical for appetitive and aversive behaviors .", "Mounting evidence suggests that distinct neuron groups within these regions selectively respond to appetitive vs aversive events and display different patterns of input-output connectivity ( Howe and Dombeck , 2016; Lammel et al . , 2012; Parker et al . , 2016; Roeper , 2013; Ye et al . , 2016 ) suggesting that projection specificity may be the foundation for selective recruitment of distinct neuron groups in distinct-valence experiences .", "The anatomical connectivity alone , however , may not be sufficient to bind the neuron groups tuned to appetitive or aversive events into a functional neural network in a timely manner .", "Therefore , the neural synchrony mediated by coherent oscillations , such as that demonstrated here , may play a key role for the rise and fall of the functional neural networks depending on the behavioral context .", "In conclusion , proper encoding of punishment risk and its contingency on actions and outcomes are fundamental to adaptive behavior and survival .", "Our data reveal dynamic coding schemes of the VTA-mPFC neural networks in representing risk of punishment and punishment-based modulation of rewarded actions ." ], [ "Male Long Evans rats weighing 300 ~ 400 g ( Harlan ) were singly housed on a 12 hr light/dark cycle ( lights on at 7 p . m . ) .", "All data were collected during the dark cycle .", "Microelectrode arrays were surgically implanted in ipsilateral mPFC and VTA ( N = 10 ) or bilateral mPFC ( N = 4 ) of isoflurane-anesthetized rats ( Figure 2—figure supplement 1 ) .", "All mPFC electrode arrays were placed in the prelimbic subregion of the mPFC .", "The following coordinates relative to the bregma were used: mPFC = AP +3 . 0 mm , ML 0 . 7 mm , DV 4 . 0 mm; VTA = AP −5 . 3 mm , ML 0 . 8 mm , DV 8 . 2 mm ( Paxinos and Watson , 1998 ) .", "Only the mPFC and VTA single units and field potentials simultaneously recorded were subjected to inter-regional network analyses .", "Behavioral training began after 1 week of postsurgical recovery .", "At the completion of all recordings , rats were anesthetized with 400 mg/kg chloral hydrate and perfused with saline and 10% buffered formalin .", "Coronal brain slices of mPFC and VTA were collected and cresyl-violet stained .", "Placements of electrode arrays were verified under a light microscope .", "All procedures were in accordance with the National Institute of Health’s Guide to the Care and Use of Laboratory Animals , and were approved by the University of Pittsburgh Institutional Animal Care and Use Committee .", "After the postsurgical recovery , rats were kept at 85% of their free-feeding weight on a restricted diet of 13 g food pellets a day with free access to water .", "In an operant chamber ( Coulbourn Instruments ) , rats were fully trained to make an instrumental nose poke to the cue port to receive a sugar pellet at the food trough located in the opposite side of the chamber on the fixed ratio schedule of one – that is , FR1 ( Figure 1a–b ) .", "The operant chamber was equipped with an infrared activity monitor ( Coulbourn Instruments ) which detected animals’ movement episodes .", "Time elapsed without any movements greater or equal to 1 s was scored as immobility and summed to compute the immobile RT ( Figure 1 ) .", "After completion of three FR1 sessions consisting of 150 trials in 60 mins , rats underwent a no-shock control recording session , in which they performed 150 trials of nose pokes divided into three blocks in the absence of punishment risk ( Figure 1d ) .", "Then rats were trained with the task consisting of three blocks with varying degrees of action-punishment contingency ( 50 trials per block ) .", "Each block was assigned an action-punishment contingency of 0 , 0 . 06 , or 0 . 1 – that is , the conditional probability of receiving an electrical foot shock ( 0 . 3 mA , 300 ms ) given an action .", "The action–reward contingency was kept at one across all training and recording sessions; that is , every nose poke procured a reward even in the shock trials .", "To minimize generalization of the action-punishment contingency across blocks , they were organized in an ascending shock probability order – Block1: 0 , Block2: 0 . 06 , Block3: 0 . 1 , interleaved with 2 min timeout between blocks .", "In block 2 and 3 of each session , 3 and 5 trials were pseudo-randomly selected and followed by an electrical foot shock .", "No explicit cue was provided on shock trials to keep the shock occurrence unpredictable .", "The cue onset only signaled initiation of a trial .", "Animals were informed of the block shift by the 2 min darkened timeout in between blocks .", "In addition , the first shock trial of block 2 and the first two shock trials of block 3 were randomly selected from the initial 5 trials of each block .", "Also , animals completed two sessions of this task before the recording session , thus the shock occurrence and the task design including the ascending punishment contingency were not novel to them at the time of the recording session .", "All training and recording sessions were terminated if not completed in 180 mins , and data from the completed sessions only were analyzed .", "Animals displayed stable behavioral performance overall without any sign of contextual fear conditioning , since they performed fearless in the safe block across all sessions .", "In addition , there was no evidence for habituation to the shock as they showed equivalent punishment-based behavioral changes across sessions .", "For the diazepam pretreatment experiment , a separate group of rats ( N = 9 ) were trained using abovementioned procedure , and they underwent three test sessions with intraperitoneal pretreatment of saline – diazepam ( 2 mg/kg , Hospira , Inc . ) – saline .", "After injection , animals were returned to their home cage for 10 min before they were placed in the operant chamber .", "Three days of washout period was allowed between sessions .", "Single-unit activity and local field potentials ( LFPs ) were recorded simultaneously using a pair of eight channel Teflon-insulated stainless steel 50 µm microwire arrays ( NB Laboratories ) .", "Unity-gain junction field effect transistor headstages were attached to a headstage cable and a motorized commutator nonrestrictive to the animals’ movement .", "Signals were amplified via a multichannel amplifier ( Plexon ) .", "Spikes were bandpass filtered between 220 Hz and 6 kHz , amplified ×500 , and digitized at 40 kHz .", "Single-unit activity was then digitally high-pass filtered at 300 Hz and LFP were low-pass filtered at 125 Hz .", "Continuous single-unit and LFP signals were stored for offline analysis .", "Single units were sorted using the Offline Sorter software ( Plexon ) .", "Only the single-units with a stable waveform throughout the recording session were further analyzed .", "If a unit presented a peak of activity at the time of the reference unit’s firing in the cross-correlogram , only either of the two was further analyzed .", "Single unit and LFP data analyses were conducted with Matlab ( MathWorks ) and SPSS statistical software ( IBM ) .", "For single unit data analyses , 1 ms binned spike count matrix of the peri-cue , action , and reward periods ( starting 2 s before each event and ending 2 s after each event ) were produced per unit .", "The baseline period was a 2 s time window beginning 2 . 5 s before the cue onset .", "For all neural data analyses , the trials with shock delivery ( three and five trials for block 2 and 3 , respectively ) were excluded as single-unit and LFP signals in these trials were affected by electrical artifacts during shock delivery .", "Parametric statistical tests were used for z-score normalized data and non-normalized data that are conventionally tested using a parametric test .", "Nonparametric approaches , such as conventional nonparametric tests or bootstrapping were used for a hypothesis test of data , of which statistical distribution is unknown , e . g . phase-locking values ( PLVs ) .", "For all tests , the Greenhouse–Geisser correction was applied as necessary due to violations of sphericity .", "All statistical tests were specified as two-sided .", "Multiple testing correction was applied for all tests including multiple comparisons using the Bonferroni correction ." ] ]

[ "Actions motivated by rewards are often associated with risk of punishment .", "Little is known about the neural representation of punishment risk during reward-seeking behavior .", "We modeled this circumstance in rats by designing a task where actions were consistently rewarded but probabilistically punished .", "Spike activity and local field potentials were recorded during task performance simultaneously from VTA and mPFC , two reciprocally connected regions implicated in reward-seeking and aversive behaviors .", "At the single unit level , we found that ensembles of putative dopamine and non-dopamine VTA neurons and mPFC neurons encode the relationship between action and punishment .", "At the network level , we found that coherent theta oscillations synchronize VTA and mPFC in a bottom-up direction , effectively phase-modulating the neuronal spike activity in the two regions during punishment-free actions .", "This synchrony declined as a function of punishment probability , suggesting that during reward-seeking actions , risk of punishment diminishes VTA-driven neural synchrony between the two regions ." ]

[ "When deciding what to do , we usually try to predict the likely outcomes of our actions .", "This helps us choose behaviors that will lead to positive outcomes , or rewards , and avoid those that will lead to negative outcomes , or punishments .", "But in practice , actions that offer the possibility of reward often involve varying degrees of risk .", "When animals forage for food , for example , they risk encountering a predator .", "In our complex social world , applying for a job or asking someone out on a date means risking rejection .", "Being able to weigh up the likelihood of positive and negative outcomes is vital for effective decision-making .", "Park and Moghaddam have now studied how the brain’s reward system takes account of possible negative outcomes .", "Rats learned to poke their noses into a window inside a testing box whenever a light came on , to earn a sugar reward .", "The rats completed three blocks of trials .", "During the first block , they received only rewards .", "But for a few trials during the second and third blocks , they also received a mild electric shock as well as their reward .", "Throughout the task , Park and Moghaddam monitored the activity of two regions of the brain that encode rewards , the ventral tegmental area ( or VTA for short ) and the medial prefrontal cortex .", "The VTA sits deep within the brain and produces the brain’s reward chemical , dopamine .", "The prefrontal cortex is at the front of the brain and helps support cognition .", "In the reward-only block of trials , neurons in the VTA synchronized their firing with neurons in the prefrontal cortex .", "In blocks two and three , where there was a risk of shock , this synchrony decreased .", "This suggests that the prefrontal cortex takes greater control of decision-making when an unpleasant outcome is possible .", "Consistently overestimating the risk of things going wrong will lead to anxiety .", "Underestimating the risks will lead to impulsivity and poor decision-making .", "Park and Moghaddam’s experiment offers a way to study the mechanisms underlying these processes in animals .", "The results also suggest that using scalp electrodes to track prefrontal cortex activity in patients could be helpful in clinical trials for anxiety or impulse-control disorders ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "evolutionary biology", "biochemistry and chemical biology" ]

[ [ "Biogenic amines are key intercellular signaling molecules that regulate physiology and behavior .", "In vertebrates , biogenic amines include adrenaline , serotonin , acetylcholine , histamine , and dopamine , and these biogenic amines can be detected by G Protein-Coupled Receptors ( GPCRs ) expressed on the plasma membrane of target cells .", "The biochemical and structural basis for biogenic amine recognition by GPCRs is well understood and involves an essential salt bridge between the ligand amino group and a highly conserved aspartic acid on the third transmembrane α-helix of the receptor ( Asp3 . 32; Ballesteros-Weinstein indexing ) ( Katritch et al . , 2013; Shi and Javitch , 2002 ) .", "Mutation of Asp3 . 32 in adrenaline , serotonin , acetylcholine , histamine , and dopamine receptors eliminates amine recognition ( Shi and Javitch , 2002; Surgand et al . , 2006 ) , and GPCRs that recognize amines through another motif have not been identified .", "Trace amine-associated receptors ( TAARs ) are a family of vertebrate GPCRs distantly related to biogenic amine receptors ( Liberles , 2015 ) .", "There are 15 TAARs in mouse , 6 in human , and 112 in zebrafish , and in these or related species , all TAARs except TAAR1 function as olfactory receptors ( Horowitz et al . , 2014; Hussain et al . , 2009; Liberles and Buck , 2006 ) .", "All human TAARs ( 6/6 ) , most mouse TAARs ( 13/15 ) , and some zebrafish TAARs ( 27/112 ) retain Asp3 . 32 , suggesting that many TAARs would recognize amines .", "High throughput chemical screens yielded ligands for TAAR1 ( Borowsky et al . , 2001; Bunzow et al . , 2001; Revel et al . , 2011 ) and many olfactory TAARs that retain Asp3 . 32 , including TAAR3 , TAAR4 , TAAR5 , TAAR7s , TAAR8s , TAAR9 , and TAAR13c ( Liberles and Buck , 2006; Ferrero et al . , 2012; Hussain et al . , 2013 ) .", "Each of these receptors detects different amines , some of which evoke innate olfactory behaviors ( Hussain et al . , 2013; Dewan et al . , 2013; Ferrero et al . , 2011; Li et al . , 2013; Li and Liberles , 2015 ) .", "In mouse , TAAR4 detects the aversive carnivore odor 2-phenylethylamine ( Ferrero et al . , 2011 ) , while TAAR5 detects the attractive and sexually dimorphic mouse odor trimethylamine ( Liberles and Buck , 2006; Li et al . , 2013 ) .", "Knockout mice lacking TAAR4 and TAAR5 lose behavioral responses to TAAR ligands identified in cell culture studies ( Dewan et al . , 2013; Li et al . , 2013 ) .", "The selectivity of olfactory TAARs for particular amine odors suggests structural variability within the ligand-binding pocket across the TAAR family .", "One clade of TAARs ( including TAAR1 , TAAR3 , and TAAR4 ) detects primary amines derived by amino acid decarboxylation , while a second clade ( including TAAR5 , TAAR7s , TAAR8s , and TAAR9 ) prefers tertiary amines ( Ferrero et al . , 2012 ) .", "Structural models and mutagenesis studies of TAAR1 , TAAR7e , and TAAR7f predicted that ligand binding occurs in the membrane plane , Asp3 . 32 forms a salt bridge to the ligand amino group , and other transmembrane residues provide a scaffold that supports ligand binding and enables selectivity ( Ferrero et al . , 2012; Reese et al . , 2014 ) .", "A third TAAR clade ( clade III TAARs ) arose in fish , and includes the majority ( 87/112 ) of zebrafish TAARs ( Hussain et al . , 2009 ) .", "Most clade III TAARs lack Asp3 . 32 ( 85/87 ) , and it has been proposed that these receptors may not detect amines ( Hussain et al . , 2009; Liberles , 2014 ) .", "However , ligands have not been identified for any of these receptors .", "More generally , ligands are known for only one zebrafish TAAR , TAAR13c , which retains Asp3 . 32 and detects the carrion odor cadaverine ( Hussain et al . , 2013 ) .", "Structure-activity analysis revealed that TAAR13c preferentially recognizes odd-chained , medium-sized diamines , and likely contains two distinct cation recognition sites within the ligand-binding pocket ( Hussain et al . , 2013 ) .", "However , the structural basis for diamine recognition by TAAR13c was unknown .", "Here , we find that TAAR13c recognizes cadaverine through two remote transmembrane aspartic acids , Asp3 . 32 and Asp5 . 42 .", "A TAAR13c variant with a charge-neutralizing mutation at Asp5 . 42 has an altered ligand preference for monoamines rather than diamines .", "We propose that Asp3 . 32 and Asp5 . 42 together form a general di-cation recognition motif , and note this motif to be present in several zebrafish , mouse , and human TAARs .", "Surprisingly , Asp5 . 42 is retained in 84 out of 85 zebrafish TAARs that lack Asp3 . 32 , raising the possibility that these receptors recognize amines positioned in a non-canonical inverted orientation with the amino group pointing towards transmembrane α-helix V . Using a high throughput chemical screening approach , we identified ligands for eleven additional zebrafish TAARs , and found that different receptors detect amines through salt bridge contacts at Asp3 . 32 and/or Asp5 . 42 .", "TAAR10a , TAAR10b , TAAR12h , and TAAR12i are Asp3 . 32-containing clade I TAARs that detect serotonin , tryptamine , 2-phenylethylamine , and 3-methoxytyramine , while TAAR16c , TAAR16e , and TAAR16f are Asp5 . 42-containing clade III TAARs that detect N-methylpiperidine , N , N-dimethylcyclohexylamine , and isoamylamine .", "TAAR13a , TAAR13d , TAAR13e , and TAAR14d have both Asp3 . 32 and Asp5 . 42 , and recognize the di-cationic molecules putrescine , histamine , and agmatine .", "Based on these findings , we propose that a large clade of olfactory GPCRs evolved a novel way to detect amines that involves a non-classical salt bridge to transmembrane α-helix V . Furthermore , new TAAR ligands include several biogenic amines for which other receptors were identified , including serotonin , histamine , and 2-phenylethylamine .", "These findings illustrate that different members of the GPCR superfamily can evolve divergent ligand binding pockets for recognition of the same agonist ." ], [ "To gain structural insights into diamine recognition by the cadaverine receptor TAAR13c , we generated a TAAR13c homology model ( Figure 1A ) based on the X-ray crystal structure of agonist-bound β2 adrenergic receptor ( Protein Data Bank Entry 4LDE ) ( Ring et al . , 2013 ) .", "The β2 adrenergic receptor is the closest homolog ( 33% amino acid identity ) of TAAR13c for which an active-state structure is currently available .", "The homology model of TAAR13c indicated a canonical fold of seven transmembrane α-helices with ligand bound at the core .", "Using the homology model , we performed computational docking of cadaverine to the orthosteric binding site .", "One cadaverine amino group of the docked ligand was located near Asp1123 . 32 , which corresponds to the highly conserved amine-contact site in biogenic amine receptors .", "The other cadaverine amino group was positioned at the opposing end of the ligand-binding pocket , 3 Å away from another aspartate , Asp2025 . 42 .", "The second amino group was also within 5 Å of side chains from Thr2035 . 43 and Ser2766 . 55 , Leu192 and Phe194 on extracellular loop 2 , and the backbone of Ser1995 . 39 ( additional nearby residues are depicted in Figure 1—figure supplement 1 ) .", "Based on its proximity and charge , Asp2025 . 42 was deemed a prime candidate to function as a counterion that forms a salt bridge to the second cadaverine amino group .", "Position 5 . 42 in several other GPCRs directly contacts ligands through hydrogen bonds , salt bridges , or van der Waals interactions ( Surgand et al . , 2006 ) , and our homology model suggests a role for this amino acid in ligand recognition by TAAR13c . 10 . 7554/eLife . 10441 . 003Figure 1 . Diamine recognition sites in the cadaverine receptor TAAR13c .", "( A ) Structural modeling of zebrafish TAAR13c bound to cadaverine ( yellow ) reveals two aspartates ( D1123 . 32 and D2025 . 42 ) with carboxylates ( red ) predicted to form salt bridges to ligand amino groups ( blue ) .", "( B ) Cartoon of zebrafish TAAR13c topology depicts the location of D1123 . 32 and D2025 . 42 .", "( C ) Charge-neutralizing mutation of D1123 . 32 and D2025 . 42 abrogates cadaverine responsiveness of zebrafish TAAR13c expressed in HEK-293 cells ( mean ± sem , n = 3 , **p<0 . 01 based on two-tailed unpaired Student's t-test ) .", "( D ) D202A5 . 42 mutation transforms TAAR13c from a diamine receptor into a monoamine receptor ( mean ± sem , n = 3 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 10441 . 00310 . 7554/eLife . 10441 . 004Figure 1—figure supplement 1 . Functional analysis and structural modeling of TAAR13c mutants .", "( A ) Structural modeling of TAAR13c bound to cadaverine ( yellow ) reveals residues within 5 Å of the amino group pointing towards transmembrane α-helix 5 .", "( B ) Charge-neutralizing mutation of Asp3 . 32 eliminates cadaverine responsiveness in TAAR13c .", "HEK-293 cells were co-transfected with Cre-Seap and the D112A3 . 32 mutant of TAAR13c , incubated with cadaverine or 3-methoxytyramine at various concentrations , and assayed for phosphatase activity ( mean ± sem , n = 3 ) .", "( C ) Structural modeling of wild type TAAR13c bound to cadaverine ( yellow ) and the TAAR13c D202A5 . 42 mutant bound to 3-methoxytyramine ( yellow ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 10441 . 004 We used an established reporter gene assay ( Liberles and Buck , 2006; Ferrero et al . , 2012 ) to query the response properties of TAAR13c variants with charge-neutralizing mutations at Asp3 . 32 ( TAAR13cD112A ) and Asp5 . 42 ( TAAR13cD202A ) .", "HEK-293 cells were co-transfected with plasmids encoding TAARs and a cAMP-dependent reporter gene encoding secreted alkaline phosphatase ( Cre-Seap ) .", "Transfected cells were exposed to test ligands , and assayed for reporter activity using a fluorescent phosphatase substrate .", "As previously published with this paradigm ( Hussain et al . , 2013 ) , cadaverine robustly activated TAAR13c , with a half maximal response occurring at ~10 μM .", "In contrast , cadaverine failed to activate either the TAAR13cD112A or TAAR13cD202A mutant at any concentration tested , up to 1 mM ( Figure 1 , Figure 1—figure supplement 1 ) .", "To ensure that mutation of Asp2025 . 42 did not impair protein folding or G protein coupling , we asked whether the mutant receptor could be activated by other ligands .", "We tested 50 chemicals for the ability to activate TAAR13cD202A , and identified the monoamine 3-methoxytyramine as an agonist ( Figure 1D ) .", "3-methoxytyramine failed to activate wild type TAAR13c at any concentration tested .", "Thus , charge-neutralizing mutation of Asp5 . 42 transformed TAAR13c from a diamine receptor into a monoamine receptor .", "The altered ligand-binding preference of the TAAR13cD202A mutant suggests that Asp5 . 42 contributes directly to the ligand-binding pocket , and homology modeling of TAAR13cD202A bound to 3-methoxytyramine ( Figure 1—figure supplement 1 ) supports a direct interaction between this site and ligand .", "Based on modeling and mutagenesis studies , we conclude that Asp5 . 42 in TAAR13c forms a salt bridge to a cadaverine amino group .", "We next asked whether Asp5 . 42 was present in other GPCRs , and might function more generally in amine detection .", "Biogenic amine receptors use Asp3 . 32 to contact primary amines ( Shi and Javitch , 2002; Surgand et al . , 2006 ) , and Asp5 . 42 is not present in any of 12 serotonin , 5 dopamine , 5 acetylcholine , or 9 adrenergic receptors in mouse ( Figure 2 ) .", "Asp3 . 32 is also observed in all 4 mouse histamine receptors , while Asp5 . 42 is observed in one ( histamine receptor H2 ) .", "Histamine is di-cationic at low pH , and the polar imidazole group of the histamine ligand is predicted to contact a transmembrane α-helix 5 asparagine in histamine receptor H1 ( Asn5 . 46 ) and transmembrane α-helix 5 anions in histamine receptors H2 ( Asp5 . 42 ) , H3 ( Glu5 . 46 ) , and H4 ( Glu5 . 46 ) ( Seifert et al . , 2013 ) . 10 . 7554/eLife . 10441 . 005Figure 2 . Asp5 . 42 is highly conserved in clade III TAARs .", "( A ) Cartoon depiction indicates the location of positions 3 . 32 and 5 . 42 in GPCRs .", "( B ) Bioinformatic analysis reveals the number of receptors with anions at positions 3 . 32 ( X ) or 5 . 42 ( Y ) across various biogenic receptor subfamilies in mouse , as well as TAARs in mouse , rat , humans , and zebrafish .", "Two mouse , rat , and human TAARs have Asp5 . 43 instead of Asp5 . 42 , and these are included in columns marked Y = Asp ( * ) .", "( C ) Phylogenetic analysis of the TAAR family in zebrafish ( solid lines ) , mice ( dashed lines ) , rats ( dashed lines ) , and humans ( dashed lines ) ; scale bar = 0 . 5 substitutions per site .", "TAARs containing only Asp3 . 32 ( blue ) , only Asp5 . 42 ( red ) , or both Asp3 . 32 and Asp5 . 42 ( green ) are depicted .", "Mammalian TAARs with Asp3 . 32 and Asp5 . 43 instead of Asp5 . 42 are green .", "Glu3 . 32-containing rodent TAARs and the one zebrafish TAAR , TAAR19f , that lacks both Asp3 . 32 and Asp5 . 42 are depicted in black . DOI: http://dx . doi . org/10 . 7554/eLife . 10441 . 00510 . 7554/eLife . 10441 . 006Figure 2—figure supplement 1 . The phylogenetic tree of tetrapod and fish TAARs .", "( A ) As in Figure 2 , bioinformatic analysis reveals the number of receptors with anions at positions 3 . 32 ( X ) or 5 . 42 ( Y ) .", "( B ) Phylogenetic analysis of the TAAR family in fish ( solid lines ) and tetrapods ( dashed lines ) ; scale bar = 0 . 8 substitutions per site .", "TAARs containing only Asp3 . 32 ( blue ) , only Asp5 . 42 ( red ) , both Asp3 . 32 and Asp5 . 42 ( green ) , and neither ( black ) are depicted . DOI: http://dx . doi . org/10 . 7554/eLife . 10441 . 00610 . 7554/eLife . 10441 . 007Figure 2—figure supplement 2 . Phylogenetic analysis of TAARs from different fish species . Using the phylogenetic tree in Figure 2—figure supplement 1B as a template , TAARs are highlighted from individual fish species indicated ( solid , color ) , as well as from zebrafish and tetrapods ( dashed , grey ) ; scale bar = 0 . 8 substitutions per site .", "TAARs containing only Asp3 . 32 ( blue ) , only Asp5 . 42 ( red ) , both Asp3 . 32 and Asp5 . 42 ( green ) , or neither ( black ) are depicted . DOI: http://dx . doi . org/10 . 7554/eLife . 10441 . 007 Most mammalian TAARs retain Asp3 . 32 , including 6/6 human TAARs , 13/15 mouse TAARs , and 15/17 rat TAARs .", "In mouse and rat , two TAAR7s have conservative changes of Asp3 . 32 to Glu3 . 32; the effect of this substitution is unknown as ligands have not been found for Glu3 . 32-containing TAARs .", "A small number of mammalian TAARs have an aspartate at position 5 . 43 including two in humans ( TAAR6 , TAAR8 ) , mice ( TAAR6 , TAAR8b ) , and rats ( TAAR6 , TAAR8a ) .", "Sequence alignments indicate that Asp5 . 43 of these receptors corresponds to Asp5 . 42 of TAAR13c , and Asp5 . 43 may be similarly positioned in the agonist-binding pocket .", "These mammalian TAARs are candidates to function as diamine receptors; however , ligands have not been identified for these receptors , perhaps due to difficulties in achieving their functional expression in heterologous cells .", "Mice lack a TAAR13c ortholog , yet a role for other TAARs in diamine recognition is supported by the finding that mice lacking all olfactory TAARs fail to avoid cadaverine odor ( Dewan et al . , 2013 ) .", "Analysis of the zebrafish TAAR repertoire surprisingly revealed that in contrast to mammalian TAARs and biogenic amine receptors , the vast majority of zebrafish TAARs ( 91/112 ) have an aspartate at position 5 . 42 .", "Some of these receptors ( 7/91 ) also retain Asp3 . 32 , including TAAR13c , while most ( 84/91 ) have lost Asp3 . 32 .", "In several other fish species such as medaka , stickleback , fugu , and salmon , most TAARs also contain Asp5 . 42 but lack Asp3 . 32 ( Figure 2—figure supplement 1 ) .", "Phylogenetic analysis indicates that TAARs containing Asp5 . 42 but not Asp3 . 32 are clade III TAARs found in teleosts , and constitute a large branch of the TAAR family tree ( Figure 2 , Figure 2—figure supplement 2 ) .", "Zebrafish lack clade II TAARs , and the teleost TAAR family evolved differently from mammalian TAARs by forming the large cohort of clade III TAARs .", "Several TAARs with both Asp5 . 42 and Asp3 . 32 reside between clade I TAARs and clade III TAARs phylogenetically ( including TAAR13s in zebrafish ) , suggesting that clade III TAARs derived from clade I TAARs by first gaining Asp5 . 42 and then losing Asp3 . 32 .", "Ligands for clade III TAARs were unknown , and since they lack the canonical amine recognition site , were proposed to detect chemicals other than amines ( Hussain et al . , 2009; Liberles , 2014 ) .", "However , the widespread retention of Asp5 . 42 in clade III TAARs lacking Asp3 . 32 raised the possibility that the corresponding 84 olfactory receptors in zebrafish indeed detect amines , but do so with ligands positioned in a non-classical orientation .", "We used the high throughput reporter gene assay to identify agonists for additional zebrafish TAARs ( Figure 3 , Figure 3—figure supplement 1 ) .", "Responses of 63 zebrafish TAARs were examined , including at least one representative from each TAAR subfamily in the zebrafish olfactory system ( TAAR10 to TAAR20 ) .", "Zebrafish TAARs were expressed as fusion proteins containing the N-terminal 20 amino acids of bovine rhodopsin , which enhances cell surface expression of some chemosensory receptors in heterologous cells ( Krautwurst et al . , 1998 ) .", "As above , HEK-293 cells or Hana3A cells ( Saito et al . , 2004 ) ( Hana3A cells express olfactory chaperones for promotion of receptor expression and were used for studies involving TAAR10b , TAAR12i , and TAAR16e ) were transfected with plasmids encoding zebrafish TAARs and Cre-Seap , incubated with test chemicals , and assayed for reporter phosphatase activity .", "TAAR ligand identification in heterologous cells has been extensively validated; TAAR ligands identified by this technique also activate TAAR-containing neurons in vivo ( Hussain et al . , 2013; Dewan et al . , 2013; Li et al . , 2013;Zhang , et al . , 2013 ) , and behavioral responses to identified TAAR ligands are lost in TAAR knockout mice ( Dewan et al . , 2013; Li et al . , 2013 ) .", "Here , we used the heterologous expression approach to identify the first ligands for zebrafish TAAR10a , TAAR10b , TAAR12h , TAAR12i , TAAR13a , TAAR13d , TAAR13e , TAAR14d , TAAR16c , TAAR16e , and TAAR16f . 10 . 7554/eLife . 10441 . 008Figure 3 . Identifying the first ligands for several zebrafish TAARs . HEK-293 cells were cotransfected with Cre-Seap and plasmids encoding zebrafish TAARs , incubated with test chemicals ( 100 μM ) , and assayed for phosphatase activity using a fluorescent substrate ( mean ± sem , n = 3 ) .", "Zebrafish TAAR10a and TAAR12h are clade I TAARs containing Asp3 . 32 but not Asp5 . 42 ( blue ) , zebrafish TAAR13a , TAAR13c , TAAR13d , and TAAR13e , and TAAR14d contain both Asp5 . 42 and Asp3 . 32 ( green ) , and zebrafish TAAR16c is a clade III TAAR containing Asp5 . 42 but not Asp3 . 32 ( red ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 10441 . 00810 . 7554/eLife . 10441 . 009Figure 3—figure supplement 1 . Functional expression of TAAR10b , TAAR12i , and TAAR16e in Hana3A cells . Hana3A cells were co-transfected with Cre-Seap and TAAR-encoding plasmids , incubated with ligands , and assayed for phosphatase activity ( mean ± sem , n = 6 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 10441 . 00910 . 7554/eLife . 10441 . 010Figure 3—figure supplement 2 . Structure-function studies of zebrafish clade I TAARs: TAAR10a and TAAR12h . HEK-293 cells were co-transfected with Cre-Seap and TAAR-encoding plasmids , incubated with ligands , and assayed for phosphatase activity . DOI: http://dx . doi . org/10 . 7554/eLife . 10441 . 010 Ligands for clade I TAARs .", "TAAR10a , TAAR10b , TAAR12h , and TAAR12i are clade I TAARs that retain Asp3 . 32 but lack Asp5 . 42 .", "TAAR10a sensitively detects serotonin ( EC50 = ~0 . 5 μM ) , has significantly reduced affinity for the related indole amines 5-methoxytryptamine ( EC50 = ~20 μM ) and tryptamine ( EC50 = ~70 μM ) , and does not detect other chemicals examined ( Figure 3—figure supplement 2 ) .", "The highest affinity ligand identified for TAAR12h was 2-phenylethylamine ( EC50 = ~0 . 3 μM ) , and TAAR12h also detects other alkyl amines with reduced sensitivity ( Figure 3—figure supplement 2 ) .", "TAAR10b and TAAR12i detect the primary amines tryptamine and 3-methoxytyramine respectively ( Figure 3—figure supplement 1 ) .", "Serotonin and 2-phenylethylamine are both biogenic amines that can be recognized by other GPCRs .", "Zebrafish lack an ortholog of mouse TAAR4 , which detects 2-phenylethylamine ( Ferrero et al . , 2011 ) , and mouse TAAR4 shares 41% identity with zebrafish TAAR12h .", "Likewise , zebrafish TAAR10a is distantly related to mouse serotonin receptors , sharing 27–35% identity .", "It is thought that the TAAR family derived from an ancestral duplicate of serotonin receptor subtype HTR4 ( Lindemann and Hoener , 2005 ) , and it is possible that either TAAR10a retains the ligand recognition properties of the ancestral TAAR or that serotonin responsiveness was lost and then re-gained in this lineage .", "Ligands for putative di-cation receptors .", "Seven zebrafish TAARs retain both Asp3 . 32 and Asp5 . 42 , including all five TAAR13s and two TAAR14s ( TAAR14c and TAAR14d ) .", "We previously found that zebrafish TAAR13c detects the diamine cadaverine ( Hussain et al . , 2013 ) , and now report that zebrafish TAAR13a , TAAR13d , TAAR13e , and TAAR14d also detect chemicals with two cations .", "TAAR13d prefers medium-chained alkyl diamines with highest affinity for putrescine ( EC50 = ~1 μM ) , and progressively reduced affinity for diamines with additional methylene groups ( Figure 4A ) .", "TAAR13d has ~3 , 000-fold reduced sensitivity for diaminopropane , which is only one methylene group shorter than putrescine , indicating a strict minimal agonist length .", "The differing preference of TAAR13c and TAAR13d for cadaverine and putrescine respectively is consistent with cross-adaptation studies indicating that separate olfactory receptors detect these two diamines ( Rolen , et al . , 2003 ) . 10 . 7554/eLife . 10441 . 011Figure 4 . Structure-activity studies of zebrafish TAARs .", "( A ) Zebrafish TAAR13d displays highest affinity for putrescine among tested ligands , and reduced affinity for longer or shorter diamines .", "( B ) Zebrafish TAAR16c recognizes several structurally related amines but not oxygen-containing analogs ( 1 mM ) .", "F: N-methylpiperidine; G: piperidine; H: N-methylpyrrolidine; I: pyrrolidine; J: tetrahydropyran; K: tetrahydrofuran .", "( C ) Dose-dependent responses of TAAR16c for N-methylpiperidine and tetrahydropyran .", "( D ) Zebrafish TAAR16f recognizes isoamylamine ( L ) but not isoamyl alcohol ( M ) at 1 mM . ( E ) Dose-dependent responses of TAAR16f for isoamylamine and isoamyl alcohol ( mean ± sem , n = 3 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 10441 . 01110 . 7554/eLife . 10441 . 012Figure 4—figure supplement 1 . Several TAARs detect amines but not amino acids . HEK-293 cells were co-transfected with Cre-Seap and TAAR-encoding plasmids , incubated with ligands , and assayed for phosphatase activity ( mean ± sem , n = 3 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 10441 . 01210 . 7554/eLife . 10441 . 013Figure 4—figure supplement 2 . Structure-function studies of the zebrafish clade III TAAR , TAAR16c . HEK-293 cells were co-transfected with Cre-Seap and plasmid encoding TAAR16c , incubated with ligands , and assayed for phosphatase activity ( mean ± sem , n = 3–6 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 10441 . 013 Histamine is an agonist for both TAAR13a ( EC50 = ~20 μM ) and TAAR13d ( EC50 = ~7 μM ) .", "TAAR13d recognizes other alkyl diamines of medium length ( see above ) , while TAAR13a is selective for histamine among tested ligands .", "TAAR13a and TAAR13d share only 18–28% identity with mouse histamine receptors , and the recognition of histamine by olfactory receptors is likely due to convergent evolution within the GPCR family .", "Agmatine also activates multiple TAARs , including TAAR13e with higher affinity ( EC50 = ~1 μM ) and TAAR14d with reduced affinity ( EC50 = ~100 μM ) .", "Agmatine is derived from arginine by decarboxylation , reminiscent of several other TAAR ligands that are biogenic amines similarly derived from natural amino acids ( Liberles , 2015 ) .", "TAARs that detect agmatine , histamine , serotonin , 2-phenylethylamine , or isoamylamine ( see below ) are not activated by the natural amino acids from which their ligands were derived ( Figure 4—figure supplement 1 ) .", "Ligands for clade III TAARs .", "Next , we found ligands for three clade III TAARs that lack Asp3 . 32 .", "Despite the absence of the classical amine recognition motif present in all known amine-detecting GPCRs , zebrafish TAAR16c , TAAR16e , and TAAR16f detect amines .", "Zebrafish TAAR16c detects N-methylpiperidine ( EC50 = ~10 μM ) , zebrafish TAAR16e detects N , N-dimethylcyclohexylamine ( EC50 = ~30 μM ) , and zebrafish TAAR16f detects isoamylamine ( EC50 = ~200 μM ) .", "Removing the amino group of TAAR16c and TAAR16f ligands abolished responses ( Figure 4 , Figure 4—figure supplement 2 ) .", "TAAR16c also detected N-methylpyrrolidine ( EC50 = ~70 μM ) , and the corresponding secondary amines piperidine and pyrrolidine were partial agonists .", "In contrast , oxygen-containing analogs , tetrahydropyran and tetrahydrofuran did not activate TAAR16c at any concentration tested , indicating that the amine is essential for receptor agonism .", "Likewise , TAAR16f failed to detect isoamyl alcohol , and thus TAAR16f agonism also required a ligand amine .", "These studies indicate that these clade III TAARs are in fact amine receptors , and that the ligand amino group is an essential moiety recognized by the receptor .", "We showed that Asp3 . 32 and Asp5 . 42 are essential for cadaverine detection by TAAR13c ( Figure 1 ) , and here asked if the corresponding residues are required for activation of other fish TAARs ( Figure 5 ) .", "Charge-neutralizing mutation of Asp3 . 32 in clade I TAARs , TAAR10a and TAAR12h , completely eliminated responses to serotonin and 2-phenylethylamine respectively , while charge-neutralizing mutation of Asp5 . 42 in clade III TAARs , TAAR16c and TAAR16f , completely eliminated responses to N-methylpiperidine and isoamylamine respectively .", "Similarly , mutation of either Asp3 . 32 or Asp5 . 42 in TAAR13a , TAAR13d , TAAR13e , and TAAR14d reduced or abolished responses to all di-cationic ligands .", "The asymmetric diamines histamine and agmatine could be positioned in either orientation , with the primary amine contacting either Asp3 . 32 or Asp5 . 42 .", "These mutagenesis studies reveal that clade I TAARs and clade III TAARs require different transmembrane aspartates for amine recognition . 10 . 7554/eLife . 10441 . 014Figure 5 . Dose-dependent responses of zebrafish TAARs and charge-neutralizing TAAR mutants .", "( A ) The identities of amino acids 3 . 32 and 5 . 42 in nine 'de-orphaned' zebrafish TAARs , as well as preferred ligands and corresponding EC50s , are depicted .", "( B ) Dose-dependent activation of TAARs and TAAR mutants by ligands indicated ( mean ± sem , n = 3 ) .", "Responses are shown in cells transfected with Cre-Seap alone ( black ) or together with wild type TAARs ( red ) , D3 . 32A mutant TAARs ( blue ) , and D5 . 42A mutant TAARs ( green ) .", "D3 . 32 is lacking in clade III TAARs ( TAAR16c , TAAR16f ) and D5 . 42 is lacking in clade I TAARs ( TAAR10a , TAAR12h ) , so the corresponding D3 . 32A and D5 . 42A mutants could not be generated . DOI: http://dx . doi . org/10 . 7554/eLife . 10441 . 014 Homology models of serotonin-bound TAAR10a and N-methylpiperidine-bound TAAR16c were generated ( Figure 6 ) , as described previously for cadaverine-bound TAAR13c ( Figure 1A ) .", "TAAR10a is a clade I TAAR and homology modeling indicated proximity of the serotonin amino group to Asp1173 . 32 .", "In contrast , TAAR16c is a clade III TAAR , and homology modeling indicated ligand inversion with the N-methylpiperidine amino group near Asp1925 . 42 .", "Inverting the ligand orientation in clade III TAARs would be expected to impact other ligand-receptor contacts; to test this model , we attempted to engineer such a ligand inversion in a canonical biogenic amine receptor , serotonin receptor 6 ( HTR6 ) .", "We analyzed the ligand preference of an HTR6 double mutant ( D3 . 32A; A5 . 42D ) in which the canonical amine recognition site at Asp3 . 32 was neutralized and a non-canonical amine recognition site at Asp5 . 42 was introduced .", "Ligand-identification studies revealed that the HTR6 double mutant no longer recognized serotonin , but instead recognized a different amine , ethanolamine ( Figure 6—figure supplement 1 ) .", "Re-orienting the ligand amine towards Asp5 . 42 presumably alters other receptor interactions dramatically , allowing Asp5 . 42-containing receptors to sample structurally distinct ligands .", "Together , these studies indicate that Asp5 . 42 forms a non-classical amine interaction site in GPCRs , and this site is present throughout a large clade of olfactory receptors . 10 . 7554/eLife . 10441 . 015Figure 6 . Modeling the structure and evolution of non-classical amine recognition .", "( A ) Structural modeling of zebrafish TAAR10a and TAAR16c bound to serotonin and N-methylpiperidine ( yellow ) respectively .", "( B ) A model for the birth of a large clade of olfactory receptors with non-classical amine recognition .", "We propose that clade III TAARs evolved a non-classical mode of amine recognition in two steps .", "First , an ancestral TAAR gained the ability to recognize diamines by acquiring Asp5 . 42 .", "Subsequently , a diamine-detecting TAAR lost the canonical amine recognition site , Asp3 . 32 , leading to non-classical amine recognition through a transmembrane α-helix V salt bridge .", "Extensive gene duplication and mutation expanded and diversified clade III TAARs , leading to a large clade of olfactory receptors with non-canonical amine-detection properties . DOI: http://dx . doi . org/10 . 7554/eLife . 10441 . 01510 . 7554/eLife . 10441 . 016Figure 6—figure supplement 1 . Re-engineering the amine contact site of HTR6 . HEK-293 cells were co-transfected with Cre-Seap and plasmids encoding HTR6 or an HTR6 double mutant ( D3 . 32A; A5 . 42D ) , incubated with ligands ( 0 , 10 or 100 μM ) , and assayed for phosphatase activity ( mean ± sem , n = 3 , *p < . 05 , **p < . 01 based on one-way ANOVA analysis and Dunnett’s test to compare controls and ligand-activated responses ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 10441 . 016" ], [ "Sensory receptors define the capacity of an organism to perceive its external environment .", "Olfactory and taste receptor families can rapidly evolve , with dramatic species-specific variations in repertoire size and function ( Nei et al . , 2008; Shi and Zhang , 2007 ) .", "In some lineages , receptor families can be reduced or die out , such as vomeronasal receptors in humans ( Liman , 2006; Zhang and Webb , 2003 ) or sweet receptors in carnivores ( Jiang et al . , 2012 ) , while in other lineages new receptor families can be born , such as formyl peptide receptors in rodents ( Liberles et al . , 2009; Rivière et al . , 2009 ) .", "Functional evolution of chemoreceptor families often involves a pattern of gene duplication and subsequent mutation ( Ferrero et al . , 2012 ) , although isolated functional transformations can also occur , such as taste receptor re-purposing in hummingbirds that led to a novel receptor for sugars found in nectar ( Baldwin et al . , 2014 ) .", "Fish are aquatic animals , and efficient detection of water-soluble odors such as amines is important for their ecological niche .", "Zebrafish generally use the same families of olfactory receptors as other vertebrates , including odorant receptors , vomeronasal receptors , and TAARs ( Kermen et al . , 2013 ) .", "The TAAR repertoire in mice and humans is relatively small , representing 1–2% of all main olfactory receptors .", "However , zebrafish have comparable numbers of TAARs ( 112 ) and odorant receptors ( 143 ) , and expansion of the teleost TAAR family suggests a relatively expanded role for TAARs in fish chemosensation .", "However , prior to this study , ligands were known for only one fish TAAR ( Hussain , et al . , 2013 ) .", "Moreover , the vast majority of zebrafish TAARs lack the canonical amine recognition motif , and the classes of ligands they might detect were unknown .", "Here , we show that almost all zebrafish TAARs ( 111/112 ) contain either a canonical amine-detection site on transmembrane α-helix III ( Asp3 . 32 ) , a non-canonical amine-detection site on transmembrane α-helix V ( Asp5 . 42 ) , or both .", "Thus , teleost TAARs likely represent a very large family of diverse amine receptors .", "Several zebrafish TAAR ligands are biogenic amines produced during tissue decomposition ( Shalaby , 1996 ) .", "Cadaverine , histamine , agmatine , putrescine , tryptamine , 2-phenylethylamine , and isoamylamine are derived in one step from the natural amino acids lysine , histidine , arginine , ornithine , tryptophan , phenylalanine , and leucine by microbe-mediated decarboxylation .", "Levels of each of these amines are measured in commercially sold fish as indicators of food quality and freshness ( Shalaby , 1996 ) ; for example , excessive histamine in consumed fish causes human scombroid poisoning ( Morrow et al . , 1991 ) .", "While some of these biogenic amines are aversive to mammals ( Dewan et al . , 2013; Ferrero et al . , 2011; Wisman and Shrira , 2015 ) , they are reported to evoke variable behaviors in different fish species .", "For example , agmatine , putrescine , and cadaverine increase feeding-associated pecking behavior in goldfish ( Rolen , et al . , 2003; Hara , 2006 ) while cadaverine and putrescine evoke avoidance behavior in zebrafish ( Hussain et al . , 2013 ) .", "Future studies are needed to examine the roles of each TAAR and its ligands in fish behavior .", "The evolution of divergent solutions to the problem of amine detection by GPCRs reveals the extensive landscape of change possible within the GPCR superfamily .", "Here , we propose a two-step model for the functional evolution of clade III TAARs that is based on phylogenetic analysis , ligand identification studies , structural modeling , and mutagenesis ( Figure 6 ) .", "First , an ancestral clade I TAAR gained Asp5 . 42 , and thus the ability to recognize di-cationic chemicals like diamines .", "Ligands were identified for five zebrafish TAARs that retain Asp3 . 32 and Asp5 . 42 , and both aspartates are important for ligand recognition in each receptor .", "Second , a diamine receptor lost Asp3 . 32 , leading to clade III TAARs that display non-classical monoamine recognition through a transmembrane α-helix V salt bridge to Asp5 . 42 .", "Flipping the amine orientation dramatically changes GPCR-ligand contacts , and presumably allows clade III TAARs to sample a distinct repertoire of structurally divergent amine odors .", "Once the new amine recognition motif was established , clade III TAARs dramatically expanded through a pattern of gene duplication and subsequent mutation .", "It seems likely that the massive expansion of this olfactory receptor clade increased the capacity of the fish olfactory system to detect amines , which are water-soluble and ecologically important stimuli for aquatic animals ." ], [ "Zebrafish Taar genes were cloned from zebrafish genomic DNA ( AB strain ) , and inserted into a modified pcDNA3 . 1- ( Invitrogen , Waltham , MA , USA ) vector containing a 5’ DNA extension encoding the first 20 amino acids of bovine rhodopsin followed by a cloning linker ( GCGGCCGCC ) .", "Sequencing Taar genes revealed several AB strain polymorphisms as compared with Tübingen strain-derived genomic sequences deposited at NCBI .", "Amino acid-altering polymorphisms identified were as follows: Taar10a ( NM_001082898 ) : A467G ( Ile to Val ) and G545A ( Val to Ile ) ; Taar10b ( NM_001082903 ) : A338G ( His to Arg ) , G496A ( Ala to Thr ) , G544A ( Val to Ile ) , and G775A ( Val to Met ) ; Taar12h ( NM_001082909 ) : A23G ( Ile to Val ) , T26C ( Tyr to His ) , C62T ( Pro to Ser ) , C285A ( Ser to Tyr ) , T743A ( Phe to Ile ) , C874T and G875A ( Gly to Ile ) ; Taar12i ( NM_001083085 ) : T23C ( Val to Ala ) , G313T , T314A ( Val to Tyr ) , and T583G ( Phe to Val ) ; Taar13a ( NM_001083102 ) : C427G ( Thr to Arg ) , T680A ( Asn to Lys ) , T803G ( Ile to Met ) , T882C ( Phe to Leu ) , and T948C ( Phe to Leu ) ; Taar13d ( NM_001083041 ) : A112G ( Ile to Val ) , A196C ( Thr to Pro ) , G382A ( Val to Ile ) , and G559A ( Val to Ile ) ; Taar13e ( NM_001083043 ) : A643G ( Ile to Val ) and G796A ( Val to Ile ) ; Taar16c ( XM_009307036 ) : A797C ( Asn to Thr ) ; Taar16e ( XM_009307037 ) : T103C ( Val to Ala ) , G711A , C712T ( Ala to Ile ) , and C844T ( Ala to Val ) ; Taar16f ( XM_009305637 ) : A23T ( Asn to Ile ) , A59G ( Asn to Ser ) , C190A ( His to Lys ) , A202T ( Thr to Ser ) , and A951C ( Leu to Phe ) .", "Htr6 was cloned from mouse brain cDNA and inserted into pcDNA3 . 1- .", "Taar mutants were generated by overlap extension PCR and the Htr6 mutant was generated using TagMaster site-directed mutagenesis kit ( GM Biosciences , Rockville , MD , USA ) .", "Reporter gene assays in HEK-293 cells or Hana3A cells ( Saito et al . , 2004 ) ( for studies involving TAAR10b , TAAR12i , and TAAR16e ) were performed as previously described ( Liberles and Buck , 2006; Ferrero et al . , 2012 ) with minor modifications .", "Modifications included use of tissue culture-treated 96 well plates ( BD Biosciences , San Jose , CA , USA ) pre-incubated with polylysine ( 250 ng per well ) ; transfection using Lipofectamine 2000 ( Invitrogen ) reagent according to manufacturer's protocols , and introduction of test stimuli ( see below ) four to six hours after transfection .", "To identify TAAR agonists , chemicals were simultaneously tested in mixtures ( 83 . 3 μM per chemical , DMEM ) .", "Mix 1 contained GABA , β-alanine , 2-aminopentane , benzylamine , butylamine , and cystamine; mix 2 contained dibutylamine , dimethylamine , N , N-dimethylcyclohexylamine , ethylamine , ethyleneamine , and ethanolamine; mix 3 contained hexylamine , isoamylamine , isobutylamine , isopropylamine , indole , and cadaverine; mix 4 contained methylamine , N-methylindole , N-methylpyrrolidine , N-methylpiperidine , pyrrolidine , and putrescine; mix 5 contained hexanal , ethyl butyrate , tryptamine , histamine , 2-phenylethylamine , and trimethylamine; mix 6 contained 2-methylbutylamine , cysteamine , 1-amino propan-2-ol , 3-methylthiopropylamine , and agmatine; mix 7 contained tyramine , N , N-dimethylethanolamine , octopamine , N , N-dimethylglycine , 5-hydroxyindole-3-acetic acid , and 3-methoxytyramine; mix 8 contained 5-methoxytryptamine , 4-methoxy phenethylamine , N , N-dimethylphenethylamine , 5-methoxy-N , N-dimethyltryptamine , N , N-dimethylaniline , N , N-dimethylisopropylamine; mix 9 contained 1-dimethylamino propanol , 2-dimethylaminoethanethiol , and 1- ( 2-aminoethyl ) -pyrrolidine; mix 10 contained 1 , 7-diaminoheptane , 1 , 8-diaminooctane , 1 , 6-diaminohexane , 1 , 10-diaminodecane , 1 , 3-diaminopropane; and mix 11 contained spermine , spermidine , dopamine , serotonin , and adenine .", "TAARs were tested with all 11 chemical mixtures , except TAAR14 , TAAR15 , and TAAR16 family members which were tested with mixes 1–10 .", "Amino acid sequences of 112 zebrafish TAARs , 15 mouse TAARs , 17 rat TAARs , 6 human TAARs , 12 biogenic amine receptors ( zebrafish HTR2B , HRH2 , and DRD2A; mouse HRH3 , DRD3 , HTR5 , DRD1A , and ADRB1; and rat HTR2A , HRH2 , DRD3 , and ADRB2 ) and 5 odorant receptors ( zebrafish OR103 and OR131 , mouse OR121 and OR446 , and rat ORI15 ) were aligned with MAFFT v7 . 017 ( Katoh , 2002 ) .", "For Figure 2—figure supplements 1 , 2 , published TAAR amino acid sequences from opossum , cow , chicken , frog , coelacanth , medaka , stickleback , tetraodon , fugu , Atlantic salmon , and elephant shark , as well as sea lamprey aminergic receptors , were analyzed ( Hussain et al . , 2009; Tessarolo et al . , 2014 ) .", "A phylogeny was inferred using a maximum likelihood approach implemented in RAxML ( RAxML v7 . 7 . 1 ) using the JTT + Γ model of codon substitution , empirical base frequencies , and the rapid bootstrapping technique ( 100 replicates ) ( Stamatakis et al . , 2008 ) .", "Nodes with support values < 50 were collapsed in the program TreeGraph 2 and trees were visualized using FigTree v1 . 3 . 1 ( Stöver and Müller , 2010 ) .", "The TAAR13c structural model was generated using SWISS-MODEL workspace ( Biasini et al . , 2014 ) ( http://swissmodel . expasy . org/interactive ) and based on the X-ray crystal structure of agonist-bound human β2 adrenergic receptor ( Protein Data Bank entry 4LDE ) ( Ring et al . , 2013 ) .", "Modeling involved an active-state template , in which agonist binding is favored .", "The sequence alignment for homology modeling was verified by inspection to confirm proper alignment of sequence landmarks including conserved sequence motifs ( DRY/DRH and NPxxY ) and transmembrane proline residues .", "Homology models for TAAR10a ( 37% amino acid identity ) and TAAR16c ( 30% amino acid identity ) were prepared similarly using the same template .", "Models of receptor point mutants were prepared by side chain substitution in previously prepared homology models and then subjected to energy minimization prior to ligand docking .", "Docking of ligands into homology models was conducted with the Schrödinger suite ( Schrodinger , 2015 ) using LigPrep version 3 . 4 and Glide version 6 . 7 .", "First , the receptor model was prepared for docking by adding protons and assigning charges to ionizable side chains .", "Asp3 . 32 and Asp5 . 42 were modeled in the deprotonated state expected at physiological pH . The resulting model was then subjected to energy minimization using the Schrödinger impref utility prior to docking .", "Ligands were docked to the orthosteric binding pocket using Glide Extra Precision ( Friesner et al . , 2006 ) , and figures were prepared in PyMol ( Schrodinger , 2010 ) ." ] ]

[ "Biogenic amines are important signaling molecules , and the structural basis for their recognition by G Protein-Coupled Receptors ( GPCRs ) is well understood .", "Amines are also potent odors , with some activating olfactory trace amine-associated receptors ( TAARs ) .", "Here , we report that teleost TAARs evolved a new way to recognize amines in a non-classical orientation .", "Chemical screens de-orphaned eleven zebrafish TAARs , with agonists including serotonin , histamine , tryptamine , 2-phenylethylamine , putrescine , and agmatine .", "Receptors from different clades contact ligands through aspartates on transmembrane α-helices III ( canonical Asp3 . 32 ) or V ( non-canonical Asp5 . 42 ) , and diamine receptors contain both aspartates .", "Non-classical monoamine recognition evolved in two steps: an ancestral TAAR acquired Asp5 . 42 , gaining diamine sensitivity , and subsequently lost Asp3 . 32 .", "Through this transformation , the fish olfactory system dramatically expanded its capacity to detect amines , ecologically significant aquatic odors .", "The evolution of a second , alternative solution for amine detection by olfactory receptors highlights the tremendous structural versatility intrinsic to GPCRs ." ]

[ "Many organisms make molecules called biogenic amines .", "These molecules , which include the human hormones adrenaline and histamine , have important roles in regulating the biology and behaviour of many animals .", "Some biogenic amines bind to receptor proteins called GPCRs on the surface of cells .", "Many drugs can affect the activity of GPCRs , so understanding how different GPCRs work is an important goal of the pharmaceutical industry .", "Like all proteins , GPCRs are made of chains of molecules called amino acids .", "The GPCRs that can detect biogenic amines use a particular amino acid named Asp3 . 32 , and when this amino acid is mutated , these GPCRs become unable to bind to their target amine .", "Trace amine-associated receptors ( TAARs ) are a type of GPCR that are found in many animals to detect odors .", "Most TAARs in mammals contain the Asp3 . 32 residue , and recognize amine odors .", "However , many fish TAARs do not contain Asp3 . 32 , and it was not clear what molecules these fish receptors detect .", "Here Li et al . find that these fish TAARs also recognize amines , and use a different amino acid called Asp5 . 42 .", "Also , some TAARs contain both Asp3 . 32 and Asp5 . 42 , and recognize chemicals with two amines named diamines .", "Some diamines that bind to TAARs are foul smelling odors; for example , cadaverine and putrescine are repulsive smells emitted by decomposing flesh .", "In total , the experiments identified amines that can bind to eleven zebrafish TAARs that previously had no odor partner .", "Li et al . propose that some fish TAARs lost the Asp3 . 32 during the course of evolution to leave the Asp5 . 42 as the main interaction site for amines .", "This change dramatically altered how these TAARs interact with amines , which probably expanded the number of different amines that fish can detect .", "These findings open up new ways to study how the fish brain processes information about its surroundings ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "neuroscience" ]

[ [ "In considering animal behavior and decision-making , it is exciting to consider the proposal ( Polani , 2009; Tishby and Polani , 2011 ) that animals may be guided by fundamental statistical quantities , such as the maximization of Shannon mutual information ( Cover and Thomas , 1991 ) .", "The advantage of mutual information as a measure is that its maximization encompasses optimization according to many other statistical measures , such as the peak height or the variance of the distribution .", "These other measures would give valid results only in certain contexts , such as for predominantly unimodal or Gaussian probability distributions underlying the decision variables .", "The fact that mutual information can be used with different types of probability distributions makes it possible to quantitatively compare the efficiency of behavioral decisions across species , sensory modalities , and tasks .", "Indeed , this idea has already yielded insights into diverse behaviors including human eye movements patterns ( Najemnik and Geisler , 2005 ) and animal navigation in a turbulent environment ( Vergassola et al . , 2007; Masson et al . , 2009 ) .", "Both these patterns of behavior can be accounted for by adapting a maximally informative search strategy to the appropriate behavioral context .", "This model allocates some actions to improving the estimate of the goal's position rather than directly moving the animal towards the goal ( Najemnik and Geisler , 2005; Vergassola et al . , 2007 ) .", "In these contexts , behavioral analyses have shown that strategies aimed at moving directly toward a goal are unable to explain key features of the animal's response .", "For example , humans sometimes make saccades to examine a region between , rather than directly at , the two likely locations for a target ( Najemnik and Geisler , 2005 ) .", "Similarly , birds and moths zigzag perpendicular to the wind direction to find the source of an odor plume ( Vergassola et al . , 2007 ) .", "Information-maximization ( ‘infotaxis’ ) is consistent with direct strategies such as chemotaxis in certain conditions .", "When the information content of the environment is very high , such as when chemical gradients can be tracked reliably , strategies based on information maximization converge to chemotaxis ( Vergassola et al . , 2007 ) .", "Thus , an information maximization approach can be viewed as a generalization of following direct sensory gradients to a broader and more challenging set of behavioral tasks .", "Among the multitude of decisions that animals make throughout the day , foraging for food is perhaps the most challenging and critical for survival .", "Interestingly , a number of species have been reported to spend more time in areas where they have recently observed food ( Karieva and Odell , 1987; Benedix , 1993 ) , suggesting that there might be an underlying logic to search that generalizes across species .", "Recent experimental studies have observed similar foraging patterns in the nematode Caenorhabditis elegans ( Hills et al . , 2004; Wakabayashi et al . , 2004; Gray et al . , 2005; Chalasani et al . , 2007 ) .", "After removal from food , the animal first performs an intense search around the area where it believes food is likely to be located ( Figure 1A ) .", "This period is characterized by an increased number of abrupt turns allowing the animal to stay in the proximal area ( Figure 1B ) and is termed ‘local search’ .", "After approximately 15 min , animals reduce their number of turns to a basal rate ( Figure 1B ) .", "This produces more extended trajectories ( Figure 1A ) and allows the animal to leave the proximal zone and explore a much larger area ( ‘global search’ ) .", "Although C . elegans is traditionally considered to be a chemotactic searcher ( Ferree and Lockery , 1999; Pierce-Shimomura et al . , 1999; Iino and Yoshida , 2009 ) , moving up or down chemical gradients to find the source of an odorant , in these conditions animals have no chemical gradient to follow .", "We set out to explore whether a single underlying strategy could explain the different aspects of food search behavior in this well-studied model . 10 . 7554/eLife . 04220 . 003Figure 1 . Transition between local and global search in C . elegans foraging trajectories following their removal from food .", "( A ) Animals search the local area by producing a large number of turns before abruptly transitioning to a global search .", "( B ) Across many animals , this transition is readily apparent in the mean turning rate .", "Standard error of the mean is shown as the lightly shaded region around the solid average line . DOI: http://dx . doi . org/10 . 7554/eLife . 04220 . 003" ], [ "Since C . elegans performs a food search even in the absence of a gradient ( Hills et al . , 2004; Wakabayashi et al . , 2004; Gray et al . , 2005 ) , they must have a prior belief about how food is distributed in the environment .", "For the sake of simplicity , we assume that the probability of finding food is initially distributed as a two-dimensional Gaussian distribution , which imposes the minimal structural constraint beyond the variance of the spatial distribution ( Jaynes , 2003 ) .", "When searching through this space , an animal using the maximally informative trajectory should move in the direction that maximizes its information about the location of food .", "This can be calculated by taking into account the probability that the nearby environment would emit food odor and then estimating the change in information the animal expects will result from any detection or non-detection events ( Vergassola et al . , 2007 ) .", "This means that even a non-detection of a food odorant is informative as it lessens the likelihood that food is nearby .", "Ultimately , the probability of detecting an odorant depends on the likelihood of food sources across the environment ( r→ ) .", "Information maximization can be analyzed with respect to this quantity , see ‘Materials and methods’ for details .", "Analyzing these solutions , we find that the maximally informative trajectories first take the searcher towards the peak in the maximum likelihood of food distribution ( Figure 2A ) and then follow an outward motion ( Figure 2B ) .", "This intensive search of a small area is qualitatively consistent with the local search performed by C . elegans . 10 . 7554/eLife . 04220 . 004Figure 2 . Maximally informative trajectories exhibit abrupt transitions between spiral-like and straight motion towards the boundary .", "( A ) Initial trajectories of the model head directly towards the peak probability of finding an odor source .", "( B ) After some period of time the model displays an abrupt transition in behavior from a spiral-like motion to a straight motion towards the boundary .", "( C ) The log probability that the food is elsewhere consistently increases as the search progresses .", "The transition between local ( spiral-like ) and global ( straight motion towards the boundary ) search occurs when this probability approaches 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 04220 . 004 Given an infinitely sized arena , this spiral-like motion would continue indefinitely ( Barbieri et al . , 2011 ) .", "However , searchers only have knowledge of a finite area ( the full extent of the area shown in Figure 2A–B ) .", "Further , it is not necessarily true that there will always be food near where it has been seen before .", "Thus , we have to allow for the possibility that food will not be in the nearby area .", "In mathematical terms , we allow for the probability that a food source is in the nearby area to deviate from 1 .", "Initially , this probability was set to be very close to one ( within numerical accuracy , deviation from 1 was ∼10−100 ) .", "However as the search progressed , and no odorants were detected , this probability decreased according to the Bayesian rule: ( 1 ) pt+1 ( A|n=0 ) =pt ( A ) P ( n=0|A ) P ( n=0 ) , where pt+1 ( A ) =pt+1 ( A|n=0 ) is the updated probability given that n = 0 odor detections were observed .", "The update rule in Equation ( 1 ) reflects the fact that , while the searcher at each step expects to detect a certain number of odorants , none are detected because the source is absent .", "While initially p0 ( A ) is set very close to 1 , it gradually decreased to zero .", "We found that allowing the probability to decrease during the search causes the local search to consistently end abruptly at locations that were very far from the boundaries of modeled area A ( Figure 2B ) .", "The abrupt transition occurred for any initial values of p0 ( A ) as long as it was not identically equal to 1 at the start of the search .", "[If p0 ( A ) =1 , then the probability to find food outside of the local area is zero and it will remain so even after the Bayesian update in Equation ( 1 ) ] .", "After the transition , the search trajectory would then follow a straight path to the boundary of the modeled area ( Figure 2A–B ) .", "These features of search trajectories are consistent with C . elegans transitioning between local and global search .", "One interesting feature of this maximally informative search strategy is the abruptness of the transition .", "Movement around the peak initial belief is followed by a sudden switch to motion away from it .", "The transition from local to global search corresponds , at least in the model , with the searcher's estimate that the probability that food is located elsewhere equals 1 ( Figure 2C ) .", "This indicates that qualitative changes in behavioral state arise due to beliefs that no longer reflect old information .", "As described above , in the maximally informative model , the transition between the local and global states of the search occurs abruptly .", "In experiments , the reduction in the number of turns appears to occur gradually ( Figure 1B ) .", "However this difference could be an artifact of the averaging of trajectories across multiple worms .", "In contrast , individual worm trajectories could still have sharply defined transitions , as can be seen in Figure 1A .", "To investigate the sharpness of this transition within individual worm trajectories , we applied a Hidden Markov model framework ( Abeles et al . , 1995; Seidemann et al . , 1996; Bishop , 2004; Jones et al . , 2007; Miller and Katz , 2010 ) to experimentally recorded trajectories .", "If segments of single-animal trajectories represent mixtures of states corresponding to local and global parts of the search , then the probability of observing global search patterns will increase gradually .", "However , analysis of experimental traces revealed a sharp transition between local and global search states on the order of a few minutes ( Figure 3A , B ) .", "Thus , the search trajectories both in experiment and theory exhibit a sharp transition between the local and global parts of the search . 10 . 7554/eLife . 04220 . 005Figure 3 . Sharp Transition between local and global phases of the search . We used a Hidden Markov model to estimate the probability that animal's behavior falls into one of two states .", "( A ) Example analysis based on a single trajectory shows fast ( several minutes ) switching time between the local and global phases of the search .", "( B ) The distribution of transition durations across a set of trajectories from different animals . DOI: http://dx . doi . org/10 . 7554/eLife . 04220 . 005 Next , we examined whether the infotaxis framework could quantitatively account for the distribution of worm search trajectories .", "The infotaxis model contains three independent parameters: the width of the initial prior probability distribution , filter length representing physical parameters , and how close the initial values for p0 ( A ) was set to 1 ( See ‘Materials and methods’ ) .", "Fitting these parameters of the infotaxis model , it is possible to quantitatively account for the experimental distribution of worm positions at the end of local search ( Figure 4A ) .", "Importantly , the same set values of these parameters adjusted to match the spatial distribution ( Figure 4A ) also produced ( without re-adjustment ) the cumulative distribution of local search duration and matched experimental measurements ( Figure 4B , two-sample Kolmogorov–Smirnov test , p = 0 . 45 ) .", "The conversion between the spatial axis in Figure 4A and the temporal scale in Figure 4B is set by the calculated value of the worm's speed ( ∼0 . 17 mm/s ) , and does not represent an adjustable parameter .", "Thus , the infotaxis model can quantitatively account for the properties of worm search behavior after removal from food . 10 . 7554/eLife . 04220 . 006Figure 4 . Infotaxis model quantitatively accounts for the worm trajectories .", "( A ) The distribution of worm displacements from an initial position at the end of the local search is non-Gaussian and can be fitted using the three parameters ( See ‘Materials and methods’ ) of the infotaxis model .", "( B ) The same set of parameters also accounts for the cumulative distribution for the local search duration across different individual worms . DOI: http://dx . doi . org/10 . 7554/eLife . 04220 . 00610 . 7554/eLife . 04220 . 007Figure 4—figure supplement 1 . Comparison of measured local search duration times with predictions based on chemotaxis and infotaxis models . The chemotaxis model was fit by measuring the maximum and steady-state turning rates of the animal and constraining the mean turning rate .", "The data ( blue dashed line ) and infotaxis ( red solid line ) are reproduced from Figure 4 . DOI: http://dx . doi . org/10 . 7554/eLife . 04220 . 007 One may wonder whether other search strategies could also account for food search behavior in worms .", "Among these , chemotaxis represents the most widely used and parsimonious model of animal behavior ( Brown and Berg , 1974; Pierce-Shimomura et al . , 1999 , 2005; Iino and Yoshida , 2009 ) .", "A searcher using this strategy would be expected to transiently increase its turning rate when removed from food due to a sudden , large change in food gradient .", "The subsequent decline in the number of turns would then be explained by adaptation to the low ( zero ) odorant concentration .", "Although this explanation seems plausible , it could not quantitatively account for three properties of foraging trajectories:", "( i ) the long duration of the local search ,", "( ii ) the rapid exit from the local search state , and", "( iii ) the inability of food concentration to influence local search .", "An explanation based on adaptation with a single time constant could be ruled out based on the juxtaposition between the relatively long duration of the local search with the fairly rapid transition between the local and global phases of the search .", "Adaptation with a slow time constant could explain the fairly substantial duration of the local search but not its sharp transition to a global search .", "On the other hand , adaptation with a short time constant could match the low number of turns during the global search but would underestimate the number of turns and the duration of the local phase of the search ( Figure 5A ) .", "Quantitatively , adjusting the adaptation time constant to match the observed durations of the local search phase produces trajectories with much broader transitions between local and global phases of the search than is observed experimentally ( Figure 3B , comparison between the solid line and histograms , see also Figure 4—figure supplement 1 ) . 10 . 7554/eLife . 04220 . 008Figure 5 . Foraging trajectories deviate from predictions of the chemotaxis model . The chemotaxis model explains the reduction in the average number of turns as adaptation to low odorant concentration .", "( A ) For different adaptation times , the predicted dynamics of turn rate can match either the slow decay in the beginning of the search or the small rate of turning at the end of the search , but not both .", "Black lines are predictions using adaptation while red shows experimental measurements .", "( B ) The average number of turns is unaffected by changes in food concentration ( black ) , in contrast to chemotaxis predictions ( grey bar ) and in agreement with the infotaxis predictions ( dashed line ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 04220 . 008 Perhaps a more striking illustration as to why chemotaxis does not fully describe the foraging trajectories comes from experiments where worms are transferred from patches of food of the same size but with different concentrations .", "The chemotaxis model makes predictions based on the change in odorant concentration .", "This change will be smaller for animals that are removed from patches with more diluted food .", "Therefore , the chemotaxis model in this case would predict that animals will make a smaller number of turns ( Figure 5B ) .", "In contrast , the infotaxis model makes predictions based not on the last odorant concentration that the animal experienced prior to its removal from food , but on the relative distribution of food in the environment .", "The spatial variance of this distribution is not affected by the dilution .", "Therefore , the infotaxis model would predict that the animals will make the same number of turns regardless of the bacteria concentration within the lawn , provided the lawns have the same size .", "This prediction was supported by our measurements ( Figure 5B ) .", "Overall , we have found that C . elegans behavior when removed from food cannot be explained as chemotaxis but is consistent with infotaxis .", "The results we have presented so far argue that the quantitative characteristics of animals' behavior match what would be expected for an optimal , maximally informative ( Vergassola et al . , 2007 ) or ( equivalently ) Bayesian ( Najemnik and Geisler , 2005 ) model of search .", "This model continuously updates the likelihood of a food source being present throughout the duration of search .", "At first glance , these calculations require the ability to maintain and update the corresponding “mental maps” of the environment .", "However , the animals could also approximate complex computations with empirically-tuned simple search heuristics that have only slightly smaller than maximal yields .", "Interestingly , as the search progresses , the log probability 1−pt ( A ) that the food is located elsewhere accumulates .", "Broader priors require more time to come to the conclusion that food is located elsewhere .", "When this probability reaches one , the local phase of the search ends and the global phase begins .", "The approximately linear increase in the log probability 1−pt ( A ) observed during most of the local search duration ( after the initial period of supra-linear increase , cf . Figure 2C ) suggests that the timing of the transition from the local to global search could be accounted for by a simple drift-diffusion model ( Bogacs et al . , 2006; Insabato et al . , 2006 ) .", "In our set-up the drift-diffusion model has effectively only one parameter—the drift rate .", "While in most applications drift-diffusion models are considered together with an adjustable threshold , here the lower threshold value is fixed to 0 because the dynamical variable represents probability .", "Similarly , the starting value of this probability ( which we set to be just under 1 ) also has relatively weak influence on the duration of local search .", "This is because the initial decrease in ln[1−pt ( A ) ] occurs supra-linearly before settling on the linear increase .", "The key property of the maximally informative foraging strategies is that they depend on the width of the initial ( ‘prior’ ) distribution of food in the environment .", "We find that changing the width of the distribution σ changes the rate at which the evidence that food is elsewhere accumulates ( Figure 6A ) .", "Adjusting the slope of the drift-diffusion model captures both the change in evidence-accumulation as well as the observed distribution of transition times from local to global search ( Figure 6B ) .", "Furthermore , the slope of the best-fitting drift-diffusion model scaled linearly with σ ( Figure 6C ) .", "These observations suggest that animals could empirically learn the appropriate slope for different distributions of food , and in this way perform nearly optimal foraging strategies with minimum computational effort .", "Notably , the distribution of local search duration times produced by the chemotaxis model show the opposite dependence on the width of the prior distribution compared to the infotaxis model ( Figure 6B ) . 10 . 7554/eLife . 04220 . 009Figure 6 . Drift-diffusion approximates maximally informative search across a range of conditions .", "( A ) The log probability that the food is located elsewhere has approximately linear dynamics , resembling a drift-diffusion decision variable .", "The drift rate ( slope ) decreases with the width of the prior distribution ( B ) The distribution of local search duration times can be approximated by a drift diffusion model for a range of conditions .", "The chemotaxis model predicts an opposite shift in the local search duration times between wide and narrow priors compared to the infotaxis predictions .", "In both panels ( A ) and ( B ) red and black curves correspond to wide and narrow priors , respectively .", "( C ) The drift rate increases linearly with the width σ of the prior distribution .", "( D ) The drift rate decreases linearly with filter width L ( E ) The drift rate depends primarily on the ratio L/ σ .", "( F ) Normalizing models with different filters by their prior distribution widths reveals a common strategy . DOI: http://dx . doi . org/10 . 7554/eLife . 04220 . 009 The maximally informative foraging trajectories are affected not only by the width of the prior distribution but also by odorant characteristics .", "For example , the diffusivity of odorant molecules affects the calculation of the likelihood of food source .", "Perhaps fortuitously for animals with small neural circuits , we found that the changes in diffusivity primarily affected the rate of increase ln[1−pt ( A ) ] , but the overall dynamics could still be described by the that drift-diffusion model ( Figure 6D ) .", "The slope of the drift-diffusion model increased approximately linearly ( Figure 6D ) with the spatial extent L of the diffusion filter ( Vergassola et al . , 2007 ) , see also Equation 3 in ‘Materials and methods’ .", "Notably , the drift rate depends primarily on the ratio σ/L ( Figure 6E–F ) .", "These results demonstrate that maximally informative foraging trajectories can be approximated by a simple drift-diffusion model across a range of behaviorally relevant conditions ." ], [ "The C . elegans neuroanatomy ( Gray et al . , 2005 ) suggests that multi-phase foraging strategies can be implemented at the neuronal level , even in simple nervous systems .", "Taking the two-phase foraging circuit ( Gray et al . , 2005 ) that we analyzed here as an example , one may hypothesize that the initial trigger for the start of the search is provided by sensory input , likely through the AWC sensory neuron ( Figure 7 ) .", "AWC neurons respond to a decrease in odorant concentration ( Chalasani et al . , 2007 ) .", "However , these responses are transient ( Chalasani et al . , 2007 ) and do not last long enough to account for the long duration of local search ( ∼15 min ) .", "Instead , we hypothesize that local search is maintained based on the responses of one of the interneurons .", "The gradual change in state of these neurons that receive sensory input , for example the AIB and AIZ interneurons , may encode the passage of time since the start of local search .", "Modulating the rate at which the internal state of a neuron changes allows the animal to adjust the duration of local search after sensing aspects of the environment such as how food is spatially distributed and how far the odorant molecules diffuse from this particular food source .", "We hypothesize that this modulation occurs through neuromodulatory signaling , which is known to be involved in local search behavior ( Hills et al . , 2004 ) .", "In summary , a neural circuit based on just a few neurons suffices to implement foraging strategies that approximate the maximally informative , but computationally intensive , decisions . 10 . 7554/eLife . 04220 . 010Figure 7 . A tentative neural model for near-optimal foraging . Maximally informative foraging can be approximated by a combination of local and global search phases .", "Responses of a sensory neuron initiate the start of the local search .", "The passage of time during the local search is encoded in the intracellular voltage of an interneuron .", "Finally , the duration of the local search can be modulated by the release of neuromodulators . DOI: http://dx . doi . org/10 . 7554/eLife . 04220 . 010 There are a wide range of possible foraging strategies that animals might follow .", "These include chemotaxis based on local sensory cues , different types of random walks ( Bartumeus et al . , 2002; Humphries et al . , 2010; Viswanathan et al . , 2011; Humphries et al . , 2012 ) , and computationally intensive models based on detailed memories of past experiences .", "This raises the question of whether the optimal foraging strategy is constrained not only by the physical environment but also by the computational complexity of its implementation ( Tishby and Polani , 2011 ) .", "One approach to this solution is provided by the conventional chemotaxis model ( Ferree and Lockery , 1999; Pierce-Shimomura et al . , 1999 ) .", "A chemotactic behavior can be implemented based on responses of just one sensory neuron .", "However , the resulting search strategies are driven directly by changes in the gradient and do not necessarily reflect the typical size of food patches .", "While infotaxis and chemotaxis strategies converge under conditions of smoothly varying gradients , this is not so in cases where transitions between patches are common .", "In fact , we found that chemotactic trajectories exhibited not only a much weaker dependence on the patch size compared to infotaxis trajectories but also predicted the opposite relationship between patch size and search duration ( Figure 6B ) .", "In addition , we found that worm and foraging trajectories were unaffected by the overall food concentration within the patch ( Figure 5B ) , in agreement with infotaxis but in contrast to chemotaxis predictions .", "The addition of interneurons to the circuit , as schematized in Figure 7 , makes it possible to dissociate the change in the gradient from the duration of local search .", "Thus , the modest increase in computational cost associated with the addition of interneurons allows for more flexible behavior than would be seen in a simple chemotaxis strategy .", "It has been noted that foraging strategies that maximize the mutual information about target locations do not always produce the maximal yield .", "Such situations have been observed in cases where the targets are mobile ( Agarwala et al . , 2012 ) .", "In this case , although the searcher knows precisely where the food is located at a given time , it might not be able to get to the food source before it moves again .", "Animals might counteract this problem with predictive coding , using foraging strategies that maximize information about the food source location at a sufficient time in the future ( Polani , 2009; Tishby and Polani , 2011; Bialek , 2013 ) .", "So long as the parameters of simpler models can be easily learned through experience , there are no barriers to implementing such strategies with a few neurons .", "Indeed , including predictive information may take the form of an increased rate of evidence accumulation in an infotaxis-like model .", "The fact that both infotaxis and drift-diffusion models can account for the properties of foraging trajectories does not take away from the stated goal that animal behavior is guided by information maximization .", "After all , the drift-diffusion models are fitted to parameters of infotaxis trajectories .", "These fits dictate how the animals should adjust search times depending on the typical widths of food patches in the environment .", "While the infotaxis model predicts that local search should last longer for wider food patches , the chemotaxis model makes the opposite prediction ( Figure 6 ) .", "At the same time , even to set parameters of drift-diffusion models , one would need to estimate the variance of the food distribution across space .", "This is quite a feat for such a small animal as C . elegans .", "Further , it might be possible that C . elegans are capable of adjusting their behavior based on higher-than-second moments of the probability distribution .", "Demonstrating this would require more fine-scale experiments to control differences in both size and shape of food patches from which the animals are removed .", "If such sensitivities are observed , they would implicate the involvement of more complicated circuits that could be mapped onto a single drift-diffusion model .", "Finally , it is worth noting that the local search of C . elegans exhibits striking similarities to other invertebrates , such as crabs ( Zeil , 1998 ) , bees ( Gould , 1986; Dyer , 1991 ) , and ants ( Wehner et al . , 2002 ) .", "In particular , the search patterns of desert ants that have been displaced on their return to the nest ( Wehner et al . , 2002 ) .", "When arriving near the presumed location of the nest , animals follow a spiral search pattern that is consistent with infotaxis trajectories ( Barbieri et al . , 2011 ) .", "Following large displacements , ants have great difficulties finding the nest with local search patterns and transition to a strategy that is reminiscent of the global search executed by C . elegans ( Wehner , 2003; Wehner et al . , 2006 ) .", "The large-scale foraging patterns in ants are difficult to study quantitatively because of the large areas involved and a few published foraging trajectories ( Wehner et al . , 2002 ) .", "Our results add to these by showing that invertebrates can integrate more abstract quantities than spatial position and operate directly on the probability that the food ( or nest ) is located elsewhere .", "Importantly , the animals do not need to perform information-theoretic calculations all of the time; instead they can set parameters of the approximating models through learning and experience .", "In summary , animals appear to guide their foraging behavior by searching for information .", "This simple behavioral rule is able to account for multiple search strategies , as well as the emergent transitions between them .", "While seemingly complex , this strategy can be easily implemented in a reduced neural system .", "We anticipate that this principle will prove useful as a general theory of search and decision-making in a wide range of contexts ." ], [ "C . elegans in the L4 larval stage were allowed to grow overnight on an agar plate containing a 100 μl circular patch of the E . coli OP50 strain ( OD600 = 0 . 4 ) .", "For testing , animals were moved to an agar observation plate without any food where they were corralled into a 1″ square by a filter paper soaked in 200 mM CuSO4 , which animals generally avoid .", "Moving an animal requires them to picked up using a metal object .", "These animals spend roughly 2 min moving forward before initiating their search .", "Worm movement was recorded for 30 min at three frames per second , and the first 2 min are ignored .", "Infotaxis trajectories were modeled using a 128 × 128 grid representing position and probability distribution of the food source in the environment .", "At each step , the anticipated change in entropy was computed taking into account two possibilities: observing or not observing odorant hits .", "Although the initial descriptions of the model separated odorant detection events according to the number of odorant hits , in our setup ( absent food source ) the computation of those probabilities was numerically unstable .", "This is the reason why we reduce the coding to binary , either ‘no hits’ or ‘a non-zero number of hits’ .", "As such , each change in entropy is calculated using the probability to receive a hit or the probability to receive zero hits .", "Computations are halted upon being within one space of the border or after no movement for 15 time steps .", "Trajectories are computed as in ( Vergassola et al . , 2007 ) .", "The probability of an odor source being located at location r0 after observing a trace of odor encounters is given by ( 2 ) Pt ( r0 ) =Lr0 ( Tt ) ∫Lx ( Tt ) dxor , alternately , Pt ( r0 ) =exp[−∫0tR ( r ( t' ) |r0 ) dt']∏i=1HR ( r ( ti ) |r0 ) ∫exp[−∫0tR ( r ( t' ) |x ) dt']∏i=1HR ( r ( ti ) |x ) dx where H is the number of hits observed during the trajectory at time ti .", "Here , Lr0 ( Tt ) is the likelihood of observing the trace Tt odor encounters from a source located at r0 .", "When a region is visited and the source is not found , that region has its probability set to 0 .", "R is the function representing the mean hit rate observed at location r if the source is at r0 .", "It has the following form: ( 3 ) R ( r|r0 ) =ρln ( Dτa ) K0 ( |r−r0|Dτ ) where a is the size of the searcher , ρ is the particle emission rate , D is the diffusivity of the particles , the particles have a finite lifetime τ , and K0 is the modified Bessel function of order 0 .", "The filter width L is defined as L=Dτ .", "During movement , the expected change in entropy when moving isΔS ( r→rj ) =Pt ( rj ) [−S]+[1−Pt ( rj ) ][ρ0 ( rj ) ΔS0+ρ1 ( rj ) ΔS1] where ρ0 represents the probability that 0 detections are made at rj during a timestep and ρ1 represents the probability that any detections are made .", "In this cases , the expected number of hits h ( rj ) =∫Pt ( rj ) R ( rj|r0 ) dr0 with the probability of hits following a Poisson law .", "In other words , ρ1= ( 1−pt ( A ) ) ( 1−e−h ) .", "During search , while a hit results in a change in the probability landscape of R ( r|r0 ) , no hits will update the prior by convolving it with exp ( −R ( r|r0 ) ) .", "The length scale of this filter is calculated by fitting it with an exponential function exp ( −x/L ) with an adjustable length scale L . The decision variable was modeled as an accumulating value with initial value set at −100 to represent the log-likelihood that food is elsewhere .", "Drift and diffusion parameters were extracted from the time series of infotaxis trajectories and decisions were simulated using the following equation: ( 4 ) dx=Adt+cdW .", "Here , x is the current evidence in favor of a decision .", "It grows with mean drift rate A and Gaussian noise dW is drawn with standard deviation s .", "Simulations were ended once the decision variable reached 0 ." ] ]

[ "Animals have evolved intricate search strategies to find new sources of food .", "Here , we analyze a complex food seeking behavior in the nematode Caenorhabditis elegans ( C . elegans ) to derive a general theory describing different searches .", "We show that C . elegans , like many other animals , uses a multi-stage search for food , where they initially explore a small area intensively ( ‘local search’ ) before switching to explore a much larger area ( ‘global search’ ) .", "We demonstrate that these search strategies as well as the transition between them can be quantitatively explained by a maximally informative search strategy , where the searcher seeks to continuously maximize information about the target .", "Although performing maximally informative search is computationally demanding , we show that a drift-diffusion model can approximate it successfully with just three neurons .", "Our study reveals how the maximally informative search strategy can be implemented and adopted to different search conditions ." ]

[ "How an animal forages for food can make the difference between life and death , and there are several different searching strategies that may be adopted .", "Foraging could be more productive if animals could take into account any of the patterns with which food is distributed in their environment , but how much could they measure and memorize ?", "Calhoun et al . show that a tiny worm called Caenorhabditis elegans can keep track of how its previous food finds were spread out , and uses this knowledge to optimize future searches for food .", "When C . elegans forages , it begins by performing an intensive search of where it believes food is likely to be found .", "This strategy , called ‘local search’ , is characterised by the worm making numerous sharp turns that keep it in its target search area .", "If the worm has not found food after 15 min , it abruptly switches its behavior to a so-called ‘global search’ strategy , which features fewer sharp turns and more forays into the surrounding area .", "C . elegans is often thought to follow the smell of a food source in order to locate it .", "While reliable on small scale , this strategy can prove problematic when the distribution of food is patchy .", "Calhoun et al . show that in extreme conditions , such as when food is completely removed , the animals determine where and for how long to persist with their search based on their knowledge of what was typical of their environment .", "Such a strategy is called infotaxis , which literally means ‘guided by information’ .", "While the neural circuits underpinning these behaviors remain to be found , Calhoun et al . propose a model that suggests that these circuits could be relatively simple , and made up of as few as three neurons ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "tools and resources", "neuroscience" ]

[ [ "Genetically-encoded calcium indicators ( GECIs ) enable non-invasive measurement of neuronal activity in vivo .", "Activity can be tracked across multiple spatial scales , from synapses ( Chen et al . , 2013b ) to populations of thousands of neurons ( Ahrens et al . , 2013; Peron et al . , 2015 ) .", "Neuronal dynamics can be probed over times of milliseconds ( Chen et al . , 2013a; O'Connor et al . , 2013; Li et al . , 2015 ) to months ( Huber et al . , 2012; Margolis et al . , 2012 ) .", "Green fluorescent protein ( GFP ) -based GECIs ( Tian et al . , 2009; Akerboom et al . , 2012; Ohkura et al . , 2012; Chen et al . , 2013b ) , such as GCaMP6 ( Chen et al . , 2013b ) , are widely used for imaging neural activity .", "GCaMP6 indicators exhibit excellent signal-to-noise ratio , allowing detection of single action potentials ( APs ) in many situations ( Chen et al . , 2013b; Peron et al . , 2015 ) .", "Applications of GCaMPs and other widely used GECIs are limited by their excitation and emission spectra .", "GCaMPs are difficult to use in transgenic animals that already express other GFP-based proteins .", "The blue excitation light used in standard wide-field microscopy can cause photodamage and is highly scattered in tissue .", "The green GCaMP emission is absorbed by blood ( Svoboda and Block , 1994 ) , which reduces the penetration depth of imaging in vertebrates in vivo .", "In addition , the GCaMP excitation spectrum overlaps with those of light-sensitive ion channels , including channelrhodopsin-2 ( ChR2 ) ( Nagel et al . , 2003 ) , which complicates the simultaneous use of green GECIs and optogenetics .", "Red-shifted GECIs thus promise three main advantages over GFP-based sensors: increased maximal imaging depth , parallel use of a red GECI with light-sensitive ion channels for all-optical neurophysiology experiments , and reduced photodamage .", "In addition , together with existing green GECIs , red GECIs allow simultaneous imaging of multiple components of neuronal circuitry .", "Current red GECIs share overall architecture with the GCaMP sensors .", "They are based on circularly permuted red fluorescent proteins ( RFPs ) , a calcium-binding protein ( calmodulin ) and a binding peptide ( M13 or ckkap ) .", "RCaMP1 ( Akerboom et al . , 2013 ) is derived from mRuby ( Kredel et al . , 2009 ) , whereas R-GECO ( Zhao et al . , 2011; Wu et al . , 2014 ) and R-CaMP2 ( Inoue et al . , 2015 ) are derived from mApple ( Shaner et al . , 2008 ) .", "R-GECO is more sensitive than RCaMP1 .", "However , mApple-based GECIs , such as R-GECO and R-CaMP2 , exhibit photoswitching when illuminated with blue light , causing a transient increase of red fluorescence that complicates their use in optogenetics ( Akerboom et al . , 2013; Wu et al . , 2013 ) .", "Here we performed large-scale structure-guided mutagenesis and neuron-based screening ( Wardill et al . , 2013 ) to develop improved red GECIs , starting with RCaMP1h ( Akerboom et al . , 2013 ) and R-GECO1 ( Zhao et al . , 2011 ) .", "We report on the mRuby-based jRCaMP1a and jRCaMP1b , and mApple-based jRGECO1a , all of which show several-fold improved sensitivity for detecting neural activity compared to their parent scaffolds ." ], [ "R-GECO1 and RCaMP1 are based on circularly permuted mApple ( Shaner et al . , 2008 ) and mRuby ( Kredel et al . , 2009 ) , respectively , fused to calmodulin ( CaM ) and the CaM-interacting M13 peptide ( Crivici and Ikura , 1995 ) .", "In the presence of calcium , CaM undergoes a conformational change and associates with M13 to form a complex proximal to the chromophore inside the RFP β-barrel ( Akerboom et al . , 2009; 2013 ) .", "The conformational change modifies the chromophore environment , modulating solvent access , chromophore pKa , absorption , and quantum yield , and altogether results in increased RFP brightness .", "Structure-guided mutagenesis and screening in a neuron-based assay have been successful in improving GCaMP sensitivity and kinetics ( Akerboom et al . , 2012; Chen et al . , 2013b ) .", "Here we applied a similar approach to red GECIs .", "We focused mutagenesis on the interfaces between the RFP and CaM , between CaM and M13 , and within CaM itself ( 78/442 and 87/451 amino acid positions were mutated to near saturation in RCaMP1h and R-GECO1 , respectively , see Supplementary files 1–2 ) ( Figure 1a ) .", "These regions are structurally homologous to the regions previously explored in the engineering of GCaMP6 ( Chen et al . , 2013b ) .", "Single-mutation variants ( 934 RCaMP1h; 689 R-GECO1 , Supplementary files 1–2 ) were tested in an automated neuronal assay ( Wardill et al . , 2013 ) ( Figure 1b–d ) .", "Dissociated rat hippocampal neurons in 96-well plates were transfected with plasmids expressing red GECI variants ( RCaMP1h variant or R-GECO1 variant ) .", "To normalize for expression level , the plasmid also expressed GFP that was localized to the nucleus .", "R-GECO1 and RCaMP1h were both expressed throughout the cytoplasm and nucleus ( data not shown ) .", "To sense calcium selectively in the cytoplasm we added a nuclear export sequence ( NES ) to the N-termini of R-GECO1 , RCaMP1h , and all their variants .", "As expected , addition of the NES restricted expression to the cytoplasm ( Figure 1b ) .", "R-CaMP2 was excluded from the nucleus without the NES . 10 . 7554/eLife . 12727 . 003Figure 1 . Mutagenesis and screening of jRCaMP1 and jRGECO1 in dissociated neurons .", "( a ) RCaMP1h and R-GECO1 structure and mutations introduced in jRCaMP1a , jRCaMP1b , and jRGECO1a .", "M13 peptide ( yellow ) , linker 1 ( gray ) , cpmRuby or cpmApple ( red ) , linker 2 ( gray ) , CaM ( blue ) .", "Mutation positions for jRCaMP1a ( green ) , jRCaMP1b ( cyan ) , jRGECO1a ( green ) .", "( b ) Schematic of the cultured neuron assay .", "Field electrodes ( gray , upper panel ) stimulate cultured neurons expressing a cytosolic red GECI variant and nuclear GFP .", "Changes in fluorescence are recorded ( lower panel ) and analyzed .", "An example response trace of a jRGECO1a-expressing neuron after 3 action potential ( AP ) stimulus is shown .", "( c ) Screening results for 855 R-GECO1 variants .", "Top , fluorescence changes in response to 1 action potential ( vertical bars , ΔF/F0 amplitudes; black bars , single R-GECO1 mutations and combinatorial mutations; red bars , R-CaMP2 left , jRGECO1a right ) .", "Middle , significance value for different AP stimuli ( color plot ) .", "Bottom , half decay times after 10 APs .", "Black line indicates R-GECO1 performance levels .", "( d ) Screening results for 1070 RCaMP1h variants .", "Top , fluorescence changes in response to 1 AP ( same order as in b; red bars , R-CaMP2 left , jRCaMP1a and jRCaMP1b right ) .", "Middle , significance value for different AP stimuli ( color plot ) .", "Bottom , half decay times after 10 APs .", "Black line indicates RCaMP1h performance levels . DOI: http://dx . doi . org/10 . 7554/eLife . 12727 . 003 A field electrode triggered trains of action potentials ( APs ) in all neurons within each well ( Materials and methods , [Wardill et al . , 2013] ) .", "Time-lapse images ( 800 μm x 800 μm fields of view; 35 Hz ) were acquired before , during and after stimulation .", "Fluorescence changes were extracted from single neurons to compute the sensitivity , dynamic range , and kinetics of the responses to various trains of APs .", "Individual red GECI variants were compared to the parent constructs and published GECIs , including R-CaMP2 ( Inoue et al . , 2015 ) , GCaMP6s , and GCaMP6f ( Chen et al . , 2013b ) .", "Numerous single mutations ( 353/934 RCaMP1h; 187/689 R-GECO1 , Supplementary files 1–2 ) improved sensitivity compared to the parent proteins ( higher ΔF/F0 amplitude in response to one AP; p<0 . 01 , Wilcoxon rank sum test ) ( Figure 1c , d ) .", "For example , T46W , I60N , and I60T exhibited enhanced sensitivity and accelerated kinetics compared to RCaMP1h .", "I109K had similar effects on R-GECO1 .", "M339F accelerated kinetics but did not affect response amplitude of R-GECO1 .", "Beneficial mutations were sorted based on improved response amplitudes ( in response to trains of 1 , 3 , and 10 APs ) and/or faster kinetics , without a significant reduction in the maximal fluorescence change elicited by 160 APs .", "Beneficial mutations were combined in a second round of mutagenesis ( 136 RCaMP1h and 166 R-GECO1 variants ) ( Figure 1c , d ) .", "Based on criteria similar to those outlined above , two new mRuby-based sensors , jRCaMP1a and jRCaMP1b , and one mApple-based sensor , jRGECO1a , were selected for in-depth analysis ( red bars in the histograms of Figure 1c , d ) .", "These sensors have similar absorption and emission spectra to each other and their parent constructs but they differ in sensitivity for detecting neural activity , kinetics , and other biophysical properties ( −Figure 2 , Figure 2—figure supplement 1–2 , Figure 2—source data 1 ) . 10 . 7554/eLife . 12727 . 004Figure 2 . jRGECO1 and jRCaMP1 performance in dissociated neurons .", "( a ) Average responses in response to one action potential ( AP ) for RCaMP1h ( 9479 neurons , 605 wells ) , R-GECO1 ( 8988 neurons , 539 wells ) , R-CaMP2 ( 265 neurons , 22 wells ) , jRGECO1a ( 383 neurons , 26 wells ) , jRCaMP1a ( 599 neurons , 38 wells ) , and jRCaMP1b ( 641 neurons , 31 wells ) .", "( b ) Same for 10 APs response .", "( c–f )", "Comparison of jRGECO1 and jRCaMP1 sensors and other red GECIs , as a function of number of APs ( color code as in", "a ) .", "( c ) Response amplitude , ΔF/F0 .", "( d ) Signal-to-noise ratio , SNR , defined as the fluorescence signal peak above baseline , divided by the signal standard deviation before the stimulation is given .", "( e ) Half decay time .", "( f ) Half rise time .", "Error bars correspond to s . e . m ( n=605 wells for RCaMP1h; 539 , R-GECO1; 22 , R-CaMP2; 38 , jRCaMP1a; 31 , jRCaMP1b; 26 , jRGECO1a ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 12727 . 00410 . 7554/eLife . 12727 . 005Figure 2—source data 1 . Biophysical properties of purified jRGECO1 and jRCaMP1 sensors . Summary of red GECI biophysical properties , mean ± s . d . , where indicated , for independently purified protein samples ( Materials and methods ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 12727 . 00510 . 7554/eLife . 12727 . 006Figure 2—figure supplement 1 . Absorption and emission spectra of red GECIs .", "( a ) One-photon excitation ( dashed lines ) and emission ( solid lines ) spectra for jRGECO1a ( left panel ) , jRCaMP1a ( middle ) , and jRCaMP1b ( right ) in Ca-free ( blue lines ) and Ca-saturated ( red lines ) states .", "( b ) Two-photon excitation spectra for jRGECO1a , jRCaMP1a , and jRCaMP1b , in Ca-free ( blue ) and Ca- saturated ( red ) states .", "Dashed green lines show the ratio between these two states . DOI: http://dx . doi . org/10 . 7554/eLife . 12727 . 00610 . 7554/eLife . 12727 . 007Figure 2—figure supplement 2 . Biophysical properties .", "( a ) Fluorescence quantum yield of red GECIs and red FPs ( all at 1070 nm ) , and GCaMP6s ( 940 nm ) .", "( b ) Peak two-photon molecular brightness ( Ca-bound state for GECIs ) of red GECIs , red FPs , and GCaMP6s ( note the different wavelengths used ) .", "mApple data in a and b is taken from Akerboom et al . , 2013 .", "Measurements were done in a purified protein assay ( Materials and methods ) .", "( c–d )", "One-photon bleaching curves of jRGECO1a ( blue ) , jRCaMP1a ( black ) , and jRCaMP1b ( red ) in Ca-free", "( c ) and Ca-saturated", "( d ) states .", "Note that Ca-free jRGECO1a bleaching is negligible , while ~40% of the Ca-saturated jRGECO1a molecules photobleach within few seconds .", "e–g , Two-photon bleaching curves of jRCaMP1a", "( e ) , jRGECO1a", "( f ) , and GCaMP6s", "( g ) in Ca-free and Ca-saturated states .", "h–i , Two-photon bleaching profile of Ca-saturated jRCaMP1a", "( h ) and jRGECO1a", "( i ) when excited with 2 different wavelengths while maintaining an identical SNR . DOI: http://dx . doi . org/10 . 7554/eLife . 12727 . 00710 . 7554/eLife . 12727 . 008Figure 2—figure supplement 3 . Photoswitching in purified protein assay . Fluorescence traces of purified Ca2+-free protein droplets ( red traces ) of jRCaMP1a ( left panel ) , jRGECO1a ( middle ) , and R-CaMP2 ( right ) constantly illuminated with 561 nm excitation light ( 40 mW/mm2 ) , and with 488 nm light pulses ( 3 . 2 mW/mm2 peak power , 50 ms duration , 0 . 2 Hz , 1 Hz , and 1 . 8 Hz in upper , middle , and lower rows respectively ) .", "For the mApple-based GECIs , jRGECO1a and R-CaMP2 , blue illumination induced a transient , calcium-independent increase in fluorescence intensity indicative of photoswitching , while jRCaMP1a does not photoswitch , but photobleaches . DOI: http://dx . doi . org/10 . 7554/eLife . 12727 . 00810 . 7554/eLife . 12727 . 009Figure 2—figure supplement 4 . jRCaMP1a is more compatible than jRGECO1a for simultaneous use with ChR2 .", "( a ) Fluorescence signal from cultured rat hippocampal neurons transfected with either jRGECO1a alone ( gray ) or jRGECO1a+ChR2-Venus ( blue ) following stimulation with blue light ( 33 mW , 10 ms pulses , 83 Hz , 200 μm X 200 μm FOV; Materials and methods ) stimulus pulses ( light blue bars ) .", "Inset , merged image of neuron expressing both jRGECO1a ( red ) and ChR2-Venus ( green ) .", "( b ) Same experiment as in a with cultured neurons expressing jRCaMP1a alone ( gray ) or jRCaMP1a+ChR2-Venus ( black ) .", "( c ) Peak ΔF/F0 values for 5 neurons expressing jRGECO1a+ChR2-Venus ( blue ) and 5 neurons expressing jRGECO1a alone ( gray ) as a function of blue stimulus light power .", "( d ) Peak ΔF/F0 for 5 neurons expressing jRCaMP1a+ChR2-Venus ( black ) and 5 neurons expressing jRCaMP1a alone ( gray ) as function of blue stimulus light power . DOI: http://dx . doi . org/10 . 7554/eLife . 12727 . 009 jRGECO1a is the most sensitive indicator , with 8 . 5-fold larger ΔF/F0 amplitude for 1AP stimuli and faster rise time than R-GECO1 ( Figure 2 ) .", "Decay time and maximal ΔF/F0 amplitude were similar to R-GECO1 .", "The improved amplitude and faster rise kinetics were associated with a tighter apparent calcium affinity ( Figure 2—source data 1 ) .", "jRGECO1a response amplitudes and kinetics are comparable to GCaMP6f for brief trains of 1–10 APs ( Chen et al . , 2013b ) .", "Similar to other mApple-based indicators , jRGECO1a exhibits photoswitching in response to blue light ( Figure 2—figure supplement 3 ) .", "jRCaMP1a and jRCaMP1b were also much improved compared to their parent sensors ( 24-fold and 13-fold improved sensitivity for detecting 1 AP stimuli ) ( Figure 2 ) .", "jRCaMP1a has higher sensitivity and slower decay kinetics than jRCaMP1b .", "jRCaMP1b has a larger dynamic range , without saturation in the range of 1–160 APs ( Figure 2c ) .", "Both jRCaMP1a and jRCaMP1b have higher calcium affinities than RCaMP1h ( Figure 2—source data 1 ) .", "However , jRCaMP1a has very limited dynamic range in cultured neurons and solution ( 160 ± 25% and 320 ± 10% respectively , mean ± s . d . ) .", "jRCaMP1a and jRCaMP1b are two-fold brighter than jRGECO1a in the calcium-bound state ( Figure 2—figure supplement 2 ) .", "Similar to RCaMP1h , jRCaMP1a and jRCaMP1b did not exhibit photoswitching ( Figure 2—figure supplement 3 ) .", "This allows independent photostimulation and imaging in neurons that co-express ChR2 and jRCaMP1a/b ( Figure 2—figure supplement 4 ) .", "We tested jRGECO1a , jRCaMP1a , jRCaMP1b , their parent indicators , and R-CaMP2 ( Inoue et al . , 2015 ) in the mouse primary visual cortex ( V1 ) in vivo ( Chen et al . , 2013b ) ( Figure 3a , Video 1 ) .", "The majority of V1 neurons can be driven to fire action potentials in response to drifting gratings ( Mrsic-Flogel et al . , 2007; Niell and Stryker , 2008 ) .", "V1 neurons were infected with adeno-associated virus ( AAV ) expressing one of the red GECI variants under the human synapsin1 promoter ( AAV-SYN1-red GECI variant ) and imaged 16–180 days later .", "Two-photon excitation was performed with a tunable ultrafast laser ( Insight DS+; Spectra-Physics ) running at 1040 nm or 1100 nm .", "L2/3 neurons showed red fluorescence in the neuronal cytoplasm .", "Visual stimuli consisted of moving gratings presented in eight directions to the contralateral eye ( Akerboom et al . , 2012; Chen et al . , 2013b ) .", "Regions of interest corresponding to single neurons revealed visual stimulus-evoked fluorescence transients that were stable across trials and tuned to stimulus orientation ( Figure 3b ) .", "Orientation tuning was similar for all constructs tested ( Figure 3—figure supplement 1 ) .", "Fluorescence transients tracked the dynamics of the sensory stimuli ( Figure 3b–d , Video 1 ) .", "mApple-based indicators tracked more faithfully than mRuby-based indicators because of their faster kinetics ( signal half-decay time after end of stimulus was 300 ± 22 ms for R-GECO1 , 175 cells; 390 ± 20 ms , jRGECO1a , 395 cells; 330 ± 16 ms , R-CaMP2 , 310 cells; 640 ± 30 ms , jRCaMP1a , 347 cells; 500 ± 45 ms , jRCaMP1b , 95 cells; activity of RCaMP1h expressing cells was to weak to be reliably characterized , mean ± s . e . m . , Materials and methods ) . 10 . 7554/eLife . 12727 . 010Figure 3 . jRGECO1a and jRCaMP1a and jRCaMP1b performance in the mouse primary visual cortex .", "( a ) Top , schematic of the experiment .", "Bottom , image of V1 L2/3 cells expressing jRGECO1a ( left ) , and the same field of view color-coded according to the neurons’ preferred orientation ( hue ) and response amplitude ( brightness ) .", "( b ) Example traces from three L2/3 neurons expressing jRGECO1a ( left ) and jRCaMP1a ( right ) .", "Single trials ( gray ) and averages of 5 trials ( blue and black for jRGECO1a and jRCaMP1a respectively ) are overlaid .", "Eight grating motion directions are indicated by arrows and shown above traces .", "The preferred stimulus is the direction evoking the largest response .", "jRGECO1a traces correspond to the cells indicated in panel a ( see also Video 1 ) .", "( c ) Average response of neurons to their preferred stimulus ( 175 cells , R-GECO1; 310 , R-CaMP2; 395 , jRGECO1a; 347 , jRCaMP1a; 95 , jRCaMP1b .", "n=4 mice for jRGECO1a and jRCaMP1a , n=3 mice for all other constructs .", "Panels c-f are based on the same data set .", "( d ) Fourier spectra normalized to the amplitude at 0 Hz for neurons driven with 1 Hz drifting gratings , transduced with RCaMP1h , R-GECO1 , R-CaMP2 , jRGECaMP1a , jRCaMP1b , and jRGECO1a .", "Inset , zoomed-in view of 1 Hz response amplitudes .", "( e ) Fraction of cells detected as responding to visual stimulus ( ANOVA test , p<0 . 01 ) when expressing different calcium indicators .", "This fraction was 8- and 6-fold higher for jRCaMP1a and jRCaMP1b compared to RCaMP1h , respectively , and 60% higher for jRGECO1a compared to R-GECO1 ( Wilcoxon rank sum test; * , p<0 . 05; ** , p<0 . 01; *** , p<0 . 001 ) .", "Error bars correspond to s . e . m ( 26 fields-of-view , RCaMP1h; 45 , jRCaMP1a; 31 , jRCaMP1b; 30 , R-GECO1; 40 , jRGECO1a; 33 , R-CaMP2; 23 , GCaMP6s; 29 , GCaMP6f )", "( f ) Distribution of ΔF/F amplitude for the preferred stimulus .", "A right-shifted curve , such as jRGECO1a vs . R-GECO1 or jRCaMP1a/b vs . jRCaMP1h , indicates enhancement of response amplitude ( 75 percentile values of 0 . 36 and 0 . 27 vs . 0 . 18 for jRCaMP1a and jRCaMP1b vs . RCaMP1h , and 0 . 66 vs . 0 . 38 for jRGECO1a vs . GCaMP6f , respectively ) .", "( 1210 cells , R-GECO1; 861 , RCaMP1h; 1733 , R-CaMP2; 1605 , jRGECO1a; 1981 , jRCaMP1a; 971 , jRCaMP1b; 907 , GCaMP6f; 672 , GCaMP6s ) , same colors as in e . DOI: http://dx . doi . org/10 . 7554/eLife . 12727 . 01010 . 7554/eLife . 12727 . 011Figure 3—figure supplement 1 . Comparison of orientation tuning in V1 neurons measured with different red GECIs . Distribution of orientation selectivity index ( OSI , 3 upper rows ) for all cells detected as responsive , measured using different GECIs .", "Bottom panel , mean ± s . d for all constructs show similar tuning properties ( n=238 cells , R-GECO1; 308 , jRGECO1a; 386 , R-CaMP2; 277 , jRCaMP1a; 141 , jRCaMP1b; 337 , GCaMP6s; 203 , GCaMP6f ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 12727 . 01110 . 7554/eLife . 12727 . 012Figure 3—figure supplement 2 . Long-term expression of red GECIs in mouse V1 .", "( a ) Example images of V1 L2/3 neurons after long term expression of red GECIs .", "( b ) Comparison of the proportion of responsive cells , orientation-tuned cells , and the ratio between them .", "Each time point corresponds to a different mouse .", "( c ) Distributions of peak ΔF/F0 for different animals imaged after different times of expression of the red GECI .", "No strong effect of expression time on peak response was detected .", "( d ) Half decay times of the fluorescence response for different animals with different expression time of the red GECI .", "No strong effect of expression time on the decay kinetics was seen .", "jRCaMP1a data for 16 days of expression is not shown because the number of cells eligible for this analysis was too small ( Materials and methods ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 12727 . 01210 . 7554/eLife . 12727 . 013Video 1 . jRGECO1a L2/3 functional imaging in the mouse V1 . The mouse was anesthetized and presented with moving gratings in eight directions to the contralateral eye .", "Gratings were presented for 4 s ( indicated by appearance of an arrowhead in the grating propagation direction ) followed by a 4 s of blank display .", "Field of view size was 250x250 μm2 , acquired at 15 Hz and filtered with a 5 frame moving average . DOI: http://dx . doi . org/10 . 7554/eLife . 12727 . 013 We compared the performance of red GECIs using standard metrics ( Chen et al . , 2013b ) .", "One measure of sensitivity is the fraction of neurons detected as responsive in the visual cortex ( Figure 3e ) .", "For jRCaMP1a and jRCaMP1b this fraction was 8- and 6-fold higher than for RCaMP1h ( p<10-6 , Wilcoxon rank-sum test ) , and comparable to GCaMP6f .", "For jRGECO1a , the fraction was 60% higher than for R-GECO1 ( p=0 . 012 ) , 40% higher than R-CaMP2 ( p=0 . 03 ) , 30% higher than GCaMP6f ( p=0 . 1 ) , but 40% lower than GCaMP6s ( p<10-4 ) .", "The mean ΔF/F0 at the preferred visual stimulus ( Materials and methods ) also showed enhanced sensitivity for jRCaMP1a and jRCaMP1b compared to RCaMP1h ( Figure 3f ) .", "jRGECO1a responses in vivo were larger than other red GECIs and GCaMP6f , but still smaller than GCaMP6s responses .", "Long-term and high expression of GCaMP was reported to affect neuronal health ( Tian et al . , 2009; Chen et al . , 2013b ) .", "GCaMP6 nuclear-filled cells show attenuated responses to sensory stimuli , slower decay times , and degraded orientation selectivity ( Tian et al . , 2009; Chen et al . , 2013b ) .", "We expressed jRGECO1a , jRCaMP1a , and jRCaMP1b for more than 130 days in mouse V1 .", "Unlike GCaMP indicators , nuclear filling was not observed ( Figure 3—figure supplement 2a ) , presumably due to the NES in the red GECI sequences .", "Red GECIs showed stable performance over time , although a small decrease of orientation selectivity was observed for jRCaMP1b after 180 days of expression ( Figure 3—figure supplement 2b–d ) .", "Interestingly , long-term expression with the tested red GECIs seems to be more stable than with GCaMP6s ( Chen et al . , 2013b ) .", "These data suggest that jRGECO1a and jRCaMP1a / jRCaMP1b can be used in experiments requiring long-term expression .", "The measured jRGECO1a and jRCaMP1a responses in the visual cortex were smaller than expected based on measurements in cultured neurons ( Figure 2a , Figure 4c ) and lower than those produced by GCaMP6s ( Chen et al . , 2013b ) .", "High-resolution microscopy of fixed brain tissue sections revealed bright fluorescent punctae in neurons labeled with jRGECO1a but not with jRCaMP1a/b .", "jRGECO1a punctae co-localized with LAMP-1 , a marker of lysosomes ( Figure 4—figure supplement 1a ) ( Katayama et al . , 2008 ) .", "Similar punctae were also found in cultured neurons , but were less numerous ( Wilcoxon rank sum test , p<0 . 001 , Figure 4—figure supplement 1b ) .", "When imaged in vivo , ROIs containing punctae had higher baseline fluorescence by 90% but lower peak ΔF/F0 by 20% compared to their surrounding soma ( 10 cells and 39 punctae , Figure 4—figure supplement 1c ) .", "This implies that accumulation of fluorescent , non-responsive jRGECO1a in lysosomes reduces the signal-to-noise ratio for in vivo imaging . 10 . 7554/eLife . 12727 . 014Figure 4 . Combined imaging and electrophysiology in the mouse visual cortex .", "( a ) Simultaneous fluorescence dynamics and spikes measured from jRGECO1a ( top , blue ) and jRCaMP1a ( bottom , black ) expressing neurons .", "The number of spikes for each burst is indicated below the trace ( single spikes are indicated by asterisks ) .", "Left inset , a jRCaMP1a expressing neuron with the recording pipette ( green ) .", "( b ) Zoomed-in view of bursts of action potentials ( corresponding to boxes in", "a ) .", "Top , jRGECO1a; bottom , jRCaMP1a .", "( c ) jRGECO1a fluorescence changes in response to 1 AP ( top , 199 spikes from 11 cells , n=6 mice ) , and jRCaMP1a fluorescence changes in response to 2 APs ( bottom , 65 spikes from 10 cells , n=5 mice ) .", "Blue ( top ) and black ( bottom ) lines are the median traces .", "( d ) Distribution of peak fluorescence change as a function of number of action potentials in a time bin ( jRGECO1a , blue boxes: 199 1AP events; 2 APs: 70 events within a 100 ms time bin; 3 APs: 29 , 125 ms; 4 APs: 34 , 150 ms; 5 APs: 35 , 175 ms; 6 APs: 22 , 200 ms; 7 APs:14 , 225 ms; 8 APs: 21 , 250 ms . jRCaMP1a , black boxes: 135 1 AP events; 2 APs: 65 , 150 ms; 3 APs: 71 , 200 ms; 4 APs: 52 , 250 ms; 5 APs: 33 , 300 ms; 6 APs: 20 , 350 ms; 7 APs: 14 , 350 ms; 8 APs: 11 , 350 ms ) .", "Each box corresponds to the 25th to 75th percentile of the distribution ( q1 and q3 respectively ) , whisker length is up to the extreme data point or 1 . 5l ( q3 - q1 ) .", "( e ) Receiver operating characteristic ( ROC ) curve for classifying 1 and 2 APs for jRGECO1a and jRCaMP1a ( jRGECO1a: 320 events of no AP firing within a 4 s bin , jRCaMP1a: 274 events of no AP firing within a 5 s bin , 1 AP and 2 APs data same as in", "d ) .", "( f ) Detection sensitivity index ( d’ ) as a function of number of spikes in a time bin ( same parameters as in d–e ) .", "( g ) Comparison of mean half rise ( left ) and decay ( right ) times of jRGECO1a and jRCaMP1a for 2 AP response .", "Error bars correspond to s . e . m . DOI: http://dx . doi . org/10 . 7554/eLife . 12727 . 01410 . 7554/eLife . 12727 . 015Figure 4—figure supplement 1 . jRGECO1a accumulates in lysosomes .", "( a ) Red GECI expression ( top , red ) and LAMP-1 immunostaining ( bottom , green ) of fixed tissue sections from mouse V1 .", "( b ) Distribution of the fraction of somatic area covered by protein aggregates for neurons expressing jRGECO1a .", "Neurons in V1 ( fixed tissue ) show more frequent jRGECO1a aggregates than cultured neurons ( Wilcoxon rank sum test , p<0 . 001; n=10 cultured cells; 14 , fixed tissue cells; 8 , fixed tissue LAMP-1 immunostained cells ) .", "( c ) Example traces from V1 L2/3 cell showing ΔF/F0 changes of somatic regions overlapping with protein aggregates ( red , black ) and regions excluding aggregates ( blue ) .", "Inset , image of the cell , with aggregates indicated by circles . DOI: http://dx . doi . org/10 . 7554/eLife . 12727 . 01510 . 7554/eLife . 12727 . 016Figure 4—figure supplement 2 . Red GECIs lack functional response at 900nm excitation . Functional responses of three example cells ( jRGECO1a , expressed in L4 V1 neurons ) at 1040 nm excitation ( left ) and 900 nm ( 25 vs . 1 cells were detected as responsive out of 50 cells in FOV; drifting grating stimuli , Materials and methods ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 12727 . 01610 . 7554/eLife . 12727 . 017Figure 4—figure supplement 3 . Complex spectral and functional characteristics of the red GECIs after long-term expression in vivo .", "( a ) Images of jRGECO1a ( left ) and jRCaMP1a ( right ) L2/3 neurons in vivo excited by 1040 nm ( top ) and 900 nm light ( bottom ) .", "Note the punctate fluorescence image in the bottom images .", "( b ) Emission spectra from fixed tissue cells of jRGEC1a ( top ) and jRCaMP1b ( bottom ) when illuminated with 900 nm and 1000 nm laser reveal a greenish emission band .", "( c ) Effect of long-term expression on accumulation of greenish species .", "Scatter plot of the ratio between jRGECO1a somatic signal detected at 900 nm excitation ( red and green channel summed ) and baseline fluorescence detected at 1040 nm excitation , and the peak somatic ΔF/F0 for drifting grating stimuli .", "Measurements were done 16 ( left ) and 132 ( right ) days after AAV injection .", "Red solid line shows the linear regression model , red dashed lines are 95% confidence interval; R2=0 . 09 for both datasets and slope of -9 . 2 and -10 respectively ( n=98 cells , left panel; n=274 cells , right panel; linear regression model , F test , p<0 . 005 ) .", "( d ) Distribution of the somatic fluorescence ratio between 900 nm and 1040 nm excitation ( same data as in", "c ) show an increase in both the ratio median and its range with longer expression time .", "Each box corresponds to the 25th to 75th percentile of the distribution ( q1 and q3 respectively ) , whisker length is up to the extreme data point or 1 . 5 ( q3 - q1 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 12727 . 017 We also detected green fluorescence ( 500–550 nm ) in the in vivo images of all red GECIs imaged in V1 .", "Green fluorescence was visible with 900 nm excitation , but not with 1040 nm excitation , suggesting that red GECIs partition into at least two species .", "The green fluorescence was distributed unevenly across the cytoplasm and does not report calcium ( Figure 4—figure supplement 2 , 3a–b ) .", "Indeed , jRGECO1a neurons with larger fractional fluorescence intensity at 900 nm showed lower response amplitudes .", "Longer expression times for jRGECO1a increased relative intensity at 900 nm and similarly correlated with lower peak ΔF/F0 ( Figure 4—figure supplement 3c–d , F test , p-value<0 . 002 ) .", "These data suggest that long-term expression in the mammalian brain can degrade sensitivity of the red GECIs .", "Imaging with red probes suffers less from scattering of excitation light and absorption of fluorescence compared with GFP-based sensors ( Figure 2—figure supplement 1 ) , which could allow deeper imaging in vivo ( Horton et al . , 2013 ) .", "To estimate how fluorescence decays with imaging depth we imaged apical dendrites from layer ( L ) 5 cells ( Figure 5a ) .", "We fitted an exponential decay to the fluorescence signal measured as a function of depth .", "Red GECI fluorescence decayed much more slowly ( 130 ± 30 μm; median ± s . d . , 19 dendrites , n=3 mice infected with RCaMP1h , R-GECO1 , or jRGECO1a ) than GCaMP6 fluorescence ( 75 ± 15 μm; median ± s . d . , 14 dendrites , n=2 mice infected with GCaMP6s or GCaMP6f; p<0 . 0001 , Wilcoxon rank sum test ) ( Figure 5b ) . 10 . 7554/eLife . 12727 . 018Figure 5 . Deep tissue imaging using red GECIs .", "( a ) Left , schematic of the measurement .", "L5 neuron apical dendrites were imaged at different depths ( FOVs 1-n ) .", "Right , RCaMP1h fluorescence from an L5 apical dendrite ( red dots ) as a function of imaging depth .", "For fixed excitation light the brightness decreases as a function of imaging depth because of scattering and absorption losses .", "The decay was characterized by fitting an exponential function to the signal ( solid black line ) .", "( b ) Exponential decay coefficients measured from dendrites expressing green ( GCaMP6s or GCaMP6f ) or red ( RCaMP1h , jRCaMP1a , and jRGECO1a ) GECIs .", "Red GECI signal decay coefficients were significantly longer than for green GECI ( Wilcoxon rank sum test , p<0 . 0001; 3 mice and 19 dendrites for red GECIs; 2 mice and 14 dendrites for green GECIs ) .", "( c ) L6 neurons , 850 μm under the pia .", "An NTSR1-cre mouse ( Gong et al . , 2007 ) was infected with FLEX-SYN1-NES-jRCaMP1a AAV .", "( d ) Example traces from three example L6 neurons .", "Single trials ( gray ) and averages of 5 trials ( black ) are overlaid .", "Eight grating motion directions are indicated by arrows and shown above traces . DOI: http://dx . doi . org/10 . 7554/eLife . 12727 . 018 This enhanced imaging depth enabled , for example , imaging deep in L6 .", "We infected L6 neurons by injecting AAV-SYN1-FLEX-jRCaMP1a virus into the visual cortex of NTSR1-cre ( a L6-specific marker ) mice ( Gong et al . , 2007 ) .", "Four weeks after infection we could detect orientation-tuned somatic transients in L6 cells ( down to 900 μm below the pia ) in response to visual stimuli ( Figure 5c , d ) .", "The spectral properties of red GECIs allow dual-color imaging together with green GCaMP6 indicators .", "To demonstrate dual-color functional imaging in spatially intermingled neuronal processes , a FLEX-SYN1-jRGECO1a AAV was injected into V1 of mouse lines expressing cre recombinase specifically in L5 neurons ( KJ18-cre [Gerfen et al . , 2013] ) and SYN1-GCaMP6s AAV was injected into cortical LM , which sends axons to V1 ( Andermann et al . , 2011; Oh et al . , 2014 ) .", "A single excitation source at 1000 nm was used to excite both indicators .", "Imaging was performed in L1 of V1 , where signals from overlapping axons and dendrites could be reliably detected ( Figure 6 , Video 2 ) .", "We note that bleaching of the red GECI was low when imaging relatively large cellular compartments , such as cell bodies or apical dendrites ( Video 1–2 ) , but significant bleaching was seen for thin axons .", "Larger compartments are more forgiving because of the dynamic balance between bleaching and diffusion of the imaged molecules . 10 . 7554/eLife . 12727 . 019Figure 6 . Dual color imaging in the mouse visual cortex .", "( a ) Two-photon action spectra of Ca- saturated GCaMP6s , jRCaMP1a , and jRGECO1a .", "Measurements were done on purified protein ( Materials and methods ) .", "( b ) Image of L5 apical dendrites ( red ) and LM axons ( green ) imaged in L1 ( 50 μm under the pia ) of KJ18-cre mice .", "( c ) ΔF/F0 traces of axonal ( green ) and dendritic ( red ) ROIs , as indicated in b ( Video 2 ) .", "( d ) Zoom-in corresponding to the dashed box in c . DOI: http://dx . doi . org/10 . 7554/eLife . 12727 . 01910 . 7554/eLife . 12727 . 020Video 2 . Dual-color imaging of axons and apical dendrites in L1 of the mouse V1 . jRGECO1a labeled apical L5 dendrites ( red ) , and GCaMP6s labeled axons from LM ( green ) were imaged in L1 of V1 ( left ) .", "ΔF/F0 traces from 4 ROIs ( Figure 6b ) are presented ( right ) .", "Field of view size was 40x40 μm2 acquired at 15 Hz and filtered with a 5 frame moving average . DOI: http://dx . doi . org/10 . 7554/eLife . 12727 . 020 We characterized the relationship between somatic fluorescence changes and spiking in L2/3 cells by combining imaging with loose-seal , cell-attached recordings ( Figure 4a , b ) .", "The visual stimulus was adjusted online so that a large range of instantaneous firing rates was recorded for each cell .", "Imaging was performed at high zoom to detect transients with the highest signal-to-noise ratio ( Peron et al . , 2015 ) .", "jRGECO1a fluorescence changes allowed relatively robust detection of activity ( Figure 4c , d ) .", "Single spikes were detected with 82% accuracy ( 5% false-positive rate , n=11 cells from 6 animals , 199 spikes ) ( Figure 4e , f ) .", "Two APs in a time bin of 100 ms were detected with 96% accuracy ( 5% false-positive rate , 70 events ) .", "Fluorescence changes of jRCaMP1a were smaller , with a detection rate for 1AP and 2AP events of 65% and 80% respectively ( 5% false-positive rate , n=10 cells from 5 animals , 135 and 65 events respectively ) .", "jRGECO1a showed higher ΔF/F0 amplitudes and better detection than jRCaMP1a over the entire range of AP bursts ( Figure 4d–f ) .", "jRGECO1a also had faster kinetics ( Figure 4g ) .", "Measured rise and decay time of jRGECO1a and jRCaMP1a were similar to GCaMP6f and GCaMP6s , respectively ( Chen et al . , 2013b ) .", "We next tested red GECIs in flies , zebrafish and worms .", "Red GECIs were expressed pan-neuronally in transgenic flies ( R57C10-Gal4 ) .", "Boutons were imaged in the larval neuromuscular junction ( NMJ ) after electrical stimulation ( Figure 7a , b; Materials and methods ) ( Hendel et al . , 2008 ) .", "Consistent with cultured neuron data , we saw a significant boost of single AP sensitivity in jRGECO1a , jRCaMP1a , and jRCaMP1b variants compared to their parent indicators ( p<0 . 008 for all comparisons; Wilcoxon rank sum test; Figure 7c , Figure 7—figure supplement 1–2 , Figure 7—source data 1–2 ) .", "Peak response amplitudes of jRGECO1a and jRCaMP1a outperform GCaMP6s in the 1–5 Hz stimulation range ( single AP ΔF/F0 amplitude 11 . 6 ± 0 . 9% , 8 . 6 ± 0 . 5% , and 4 . 5 ± 0 . 3% respectively , mean ± s . e . m; Figure 7c , Figure 7—figure supplement 3 , Figure 7—source data 3; 12 FOVs for jRGECO1a; 11 , jRCaMP1a; 12 , GCaMP6f; n=7 flies for all constructs ) .", "The decay kinetics of jRGECO1a ( half decay time at 160 Hz: 0 . 42 ± 0 . 02 s , mean ± s . e . m ) was similar to GCaMP6f ( 0 . 43 ± 0 . 01 s; Figure 7d , Figure 7—figure supplement 3 , Figure 7—source data 3 ) .", "The combination of high sensitivity and fast kinetics for jRGECO1a allows detection of individual spikes on top of the response envelope for stimuli up to 10 Hz ( Figure 7b , Figure 7—figure supplement 1 ) . 10 . 7554/eLife . 12727 . 021Figure 7 . Imaging activity in Drosophila larval NMJ boutouns with red GECIs .", "( a ) Schematic representation of Drosophila larval neuromuscular junction ( NMJ ) assay .", "Segmented motor nerve is electrically stimulated while optically imaging calcium responses in presynaptic boutons ( green arrows ) .", "( b ) Response transients ( mean ± s . e . m . ) to 5 Hz stimulation ( 2 s duration ) for several red and green GECIs .", "Response amplitudes were 4-fold and 60% higher for jRGECO1a than GCaMP6f and GCaMP6s respectively ( p<10–4 and p=0 . 01 , Wilcoxon rank sum test ) , jRCaMP1a response amplitude was 3-fold higher than GCaMP6f ( p=10–4 ) and similar to GCaMP6s ( 12 FOVs for jRGECO1a; 11 , jRCaMP1a; 13 , jRCaMP1b; 12 , GCaMP6s; 12 , GCaMP6f; n=7 flies for all constructs )", "( c ) Comparison of frequency tuned responses ( peak ΔF/F0 , mean ± s . e . m . ) of red and green GECIs for 1 , 5 , 10 , 20 , 40 , 80 and 160 Hz stimulation ( 2 s duration ) .", "jRGECO1a and jRCaMP1a response amplitudes were 2–3 fold higher than GCaMP6s and GCaMP6f for 1 Hz stimulus ( p<0 . 001 , Wilcoxon rank sum test ) , but lower for stimulus frequencies of 20 Hz and higher ( 12 FOVs for jRGECO1a; 10 , R-GECO1; 11 , jRCaMP1a; 13 , jRCaMP1b; 10 , RCaMP1h; 12 , GCaMP6s; 12 , GCaMP6f; n=5 flies for R-GECO1 , n=7 flies for all other constructs )", "( d ) Half decay time ( mean ± s . e . m . ) of red and green GECIs at 160 Hz stimulation ( same FOVs and flies as in c; *** , p<0 . 001 , Wilcoxon rank sum test ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 12727 . 02110 . 7554/eLife . 12727 . 022Figure 7—source data 1 . Summary of results shown in Figure 7—figure supplement 1 . Wilcoxon rank sum test is used for p-value . DOI: http://dx . doi . org/10 . 7554/eLife . 12727 . 02210 . 7554/eLife . 12727 . 023Figure 7—source data 2 . Summary of results shown in Figure 7—figure supplement 2 . Wilcoxon rank sum test is used for p-value . DOI: http://dx . doi . org/10 . 7554/eLife . 12727 . 02310 . 7554/eLife . 12727 . 024Figure 7—source data 3 . Summary of results shown in Figure 7—figure supplement 3 . Wilcoxon rank sum test is used for p-value . DOI: http://dx . doi . org/10 . 7554/eLife . 12727 . 02410 . 7554/eLife . 12727 . 025Figure 7—figure supplement 1 . Imaging activity in Drosophila larval NMJ boutons with jRGECO1a .", "( a ) Schematic of experimental setup .", "Epifluorescence and high magnification △F/F0 images of Type 1b boutons ( green arrowheads ) from muscle 13 ( segments A3-A5 ) , with image segmentation ROIs superimposed ( Materials and methods ) .", "( b ) Single trial and averaged fluorescence transients after 1 Hz stimulus for 2 s for R-GECO1 , jRGECO1a and jRGECO1b ( R-GECO1: 10 FOVs in 5 flies , 40 boutons; jRGECO1a: 12 FOVs in 7 flies , 48 boutons; jRGECO1b: 9 FOVs in 6 flies , 36 boutons . Same data set used for all other analyses ) .", "jRGECO1a performance was superior to jRGECO1b in the Drosophila NMJ and zebrafish trigeminal neuron; therefore jRGECO1b was not fully tested in other animal models .", "( c–d )", "Fourier spectra normalized to 0 Hz of fluorescence signals acquired during 5 Hz", "( c ) and 10 Hz", "( d ) stimulation .", "( e–f ) △F/F0", "( e ) and SNR", "( f ) traces ( mean ± s . e . m . ) recorded with 1 , 5 , 10 , 20 , 40 , 80 and 160 Hz stimulation for 2 s ( indicated by red curves at the bottom , amplitude not to scale ) .", "( g–h ) △F/F0", "( g ) and SNR", "( h ) traces ( mean ± s . e . m . ) recorded with 1 and 5 Hz stimulation for 2 s ( shown by red curves at the bottom , amplitude not to scale ) .", "( i–j )", "Comparison of △F/F0 traces ( mean ± s . e . m . ) of R-GECO1 and jRGECO1 variants with 1 , 5 , 10 Hz", "( i ) and 20 , 40 , 80 Hz", "( j ) stimulation for 2 s ( shown by red curves at the bottom , amplitude not to scale ) .", "( k–l )", "Comparison of frequency tuned averaged peak △F/F0", "( k ) and peak SNR", "( l ) ( mean ± s . e . m . ) of GCaMP6 variants , R-GECO1 and jRGECO1 variants , with 1 , 5 , 10 , 20 , 40 , 80 and 160 Hz stimulation .", "Note that vertical axes are log scale .", "( m ) Comparison of kinetics of GCaMP6 variants , R-GECO1 and jRGECO1 variants with 40 Hz stimulation .", "Horizontal axes are half rise time and vertical axes are half decay time . DOI: http://dx . doi . org/10 . 7554/eLife . 12727 . 02510 . 7554/eLife . 12727 . 026Figure 7—figure supplement 2 . Imaging activity in Drosophila larval NMJ boutons with jRCaMP1 constructs .", "( a ) Schematic of experimental setup .", "Epifluorescence and high magnification △F/F0 images of Type 1b boutons ( green arrowheads ) from muscle 13 ( segments A3-A5 ) , with image segmentation ROIs superimposed ( Materials and methods ) .", "( b ) Single trial and averaged fluorescence transients after 1 Hz stimulus for 2 s for R-GECO1 , jRCaMP1a and jRCaMP1b ( RCaMP1h: 10 FOVs in 7 flies , 40 boutons; jRCaMP1a: 11 FOVs in 7 flies , 44 boutons; jRCaMP1b: 13 FOVs in 7 flies , 52 boutons . Same data set used for all other analyses ) .", "( c-d ) △F/F0", "( c ) and SNR", "( d ) traces ( mean ± s . e . m . ) recorded with 1 , 5 , 10 , 20 , 40 , 80 and 160 Hz stimulation for 2 s ( shown by red curves at the bottom , amplitude not to scale ) .", "( e–f ) △F/F0", "( e ) and SNR", "( f ) traces ( mean ± s . e . m . ) recorded with 1 and 5 Hz stimulation for 2 s ( shown by red curves at the bottom , amplitude not to scale ) .", "( g–h ) , Comparison of △F/F0 traces ( mean ± s . e . m . ) of R-GECO1 and jRCaMP1 variants with 1 , 5 , 10 Hz", "( g ) and 20 , 40 , 80 Hz", "( h ) stimulation for 2 s ( shown by red curves at the bottom , amplitude not to scale ) .", "( i–j )", "Comparison of frequency tuned averaged peak △F/F0", "( i ) and peak SNR", "( j ) ( mean ± s . e . m . ) of RCaMP1h and jRCaMP1 variants , with 1 , 5 , 10 , 20 , 40 , 80 and 160 Hz stimulation .", "Note that vertical axes are log scale .", "( k ) Comparison of kinetics of RCaMP1h and jRCaMP1 variants with 40 Hz stimulation .", "Horizontal axes are half rise time and vertical axes are half decay time . DOI: http://dx . doi . org/10 . 7554/eLife . 12727 . 02610 . 7554/eLife . 12727 . 027Figure 7—figure supplement 3 . Comparing red and green GECI activity in Drosophila larval NMJ boutons .", "( a–b )", "Single trial and averaged fluorescence transients after 1 Hz", "( a ) and 5 Hz", "( b ) stimulus for 2 s for jRGECO1a , jRCaMP1a , jRCaMP1b , GCaMP6s and GCaMP6f ( jRGECO1a: 12 FOVs in 7 flies , 48 boutons; jRCaMP1a: 11 FOVs in 7 flies , 44 boutons; jRCaMP1b: 13 FOVs in 7 flies , 52 boutons; GCaMP6s: 12 FOVs in 7 flies , 48 boutons; GCaMP6f: 12 FOVs in 7 flies . Same data set used for all other analyses ) .", "( c ) Comparison of frequency tuned averaged peak △F/F0 ( mean ± s . e . m . ) of jRGECO1a , jRCaMP1 variants and GCaMP6 variants , with 1 , 5 , 10 , 20 , 40 , 80 and 160 Hz stimulation .", "Note that vertical axes are log scale .", "( d–e )", "Comparison of frequency tuned averaged half decay", "( d ) and half rise", "( e ) ( mean ± s . e . m . ) of jRGECO1a , jRCaMP1 variants and GCaMP6 variants , with 1 , 5 , 10 , 20 , 40 , 80 and 160 Hz stimulation . DOI: http://dx . doi . org/10 . 7554/eLife . 12727 . 027 Red GECIs were also expressed pan-neuronally in transgenic zebrafish under the elavl3/HuC promoter ( elavl3:GECI ) .", "Fish showing strong expression in the trigeminal ( Tg ) neurons 3–4 days post fertilization ( dpf ) were selected for imaging .", "Tg neurons are usually silent and fire one or few spikes in response to touch ( Douglass et al . , 2008 ) .", "Brief trains of electrical stimulation pulses ( 20 ms each; 1 , 5 , and 10 pulses at 20 Hz ) , which are known to stimulate Tg neurons ( Akerboom et al . , 2013 ) , were used to elicit responses in these cells and image calcium transients ( Figure 8a; widefield one-photon imaging , Materials and methods ) .", "Similar to results from cultured rat hippocampal neurons , mice , and Drosophila , jRGECO1a was the most sensitive red GECI tested in this assay: its response to one and five pulses outperformed all other GECIs , including GCaMP6f and GCaMP6s ( Figure 8b , c ) .", "For higher numbers of stimulus pulses the jRGECO1a response was partially saturated .", "jRCaMP1a and jRCaMP1b exhibited large improvements in sensitivity over their parent indicator , RCaMP1h , and also higher sensitivity than R-CaMP2 , with jRCaMP1b showing faster kinetics than jRCaMP1a ( Figure 8d ) . 10 . 7554/eLife . 12727 . 028Figure 8 . Imaging activity in the zebrafish trigeminal neurons with red GECIs .", "( a ) Schematic representation of zebrafish trigeminal neurons assay .", "Zebrafish larvae ( 3–4 days post fertilization ) were paralyzed , embedded in agarose , and stimulated with electrodes ( 20 ms pulses; 1 , 5 , and 10 pulses at 20 Hz; Materials and methods ) .", "( b ) Response transients to five-pulse stimulus ( mean ± s . e . m; n=5 fish for R-GECO1; 7 , jRGECO1a; 6 , R-CaMP2; 6 , RCaMP1h; 7 , jRCaMP1a; 6 , jRCaMP1b; 6 , GCaMP6s; 6 , GCaMP6f ) .", "Response amplitudes were 7- and 8- fold higher for jRCaMP1a and jRCaMP1b than RCaMP1h , respectively , and 4-fold higher for jRGECO1a than R-CaMP2 ( Wilcoxon rank-sum test , p<0 . 01 ) .", "( c ) Averaged peak ΔF/F0 ( same fish as in", "b ) in response to one , five , and ten pulses stimuli .", "( d ) Half decay time for different red and green GECIs ( 10 pulses stimulus , mean ± s . e . m . , same fish as in b; * , p<0 . 05; ** , p<0 . 01 , Wilcoxon rank sum test ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 12727 . 028 Finally , Red GECIs were tested in the ASH and AWC neurons of Caenorhabditis elegans , two sensory neurons with distinct graded signaling properties ( Akerboom et al . , 2012; Kato et al . , 2014 ) .", "ASH neurons , which respond to noxious stimuli with calcium increases , were exposed to high-osmolarity glycerol in one second pulses alternating with buffer ( Figure 9a , b ) .", "All tested GECIs exhibited an initial increase in fluorescence that was strongest for jRCaMP1b , jRCEGO1a , and R-GECO1 ( ΔF/F0 amplitude ~250% , Figure 9b ) , followed by modulation of the fluorescence signal and a slow decay .", "jRGECO1a and jRCaMP1b showed the best performance in tracking the 1 s stimuli ( Figure 9b , c ) , while RCaMP1h , jRCaMP1a , and R-GECO1 showed smaller signal modulations; the dim fluorescence of R-CaMP2 made it less useful .", "AWC neurons , which are tonically active and inhibited by odor , were exposed to a one minute pulse of 92 μM isoamyl alcohol alternating with buffer ( Figure 9d ) .", "jRGECO1a and jRCaMP1b robustly detected the three known components of the odor response: odor-induced calcium decreases , a calcium overshoot on odor removal , and a return to baseline ( Figure 9e ) .", "R-GECO1 and RCaMP1h , which have weaker affinity for calcium , did not detect the odor-induced calcium decrease well , whereas jRCaMP1a truncated the overshoot after odor removal ( Figure 9d , e ) and had the slowest kinetics ( Figure 9f ) .", "In summary , jRCaMP1b performed best in detection of graded calcium signals , whereas jRGECO1a had advantages in response speed . 10 . 7554/eLife . 12727 . 029Figure 9 . Imaging activity in the C . elegans ASH and AWC neurons with red GECIs .", "( a ) Schematic representation of the Caenorhabditis elegans imaging assay .", "A paralyzed animal is restrained in a microfluidic device with its nose exposed to a fluid channel .", "Delivery of stimulus ( S ) or buffer ( B ) is controlled indirectly by alternating side streams from control channels C1 and C2 .", "( b ) Mean fluorescence transients in ASH neurons in response to 1 s pulses of 1 M hyperosmotic glycerol ( mean across worms , n=10 worms for jRGECO1a; 8 , jRCaMP1a; 9 , jRCaMP1b; 8 , R-GECO1; 12 , R-CaMP2; 9 , RCaMP1h ) .", "( c ) Quantification of signal modulation across 1 s glycerol pulses ( mean peak to trough difference over all 2 s cycles , divided by the signal average fluorescence during the first 10 s in buffer ( mean ± s . e . m . , same data as in", "b ) .", "Asterisks represent a significant difference ( Wilcoxon rank sum test; * , p<0 . 05; ** , p<0 . 01; *** , p<0 . 001 ) .", "( d ) Mean fluorescence transients in AWC neurons in response to a one minute exposure to 92 µM isoamyl alcohol ( n=19 worms for jRGECO1a; 7 , jRCaMP1a; 8 , jRCaMP1b; 15 , R-GECO1; 6 , RcaMP1h ) .", "Epochs 1–3 are quantified in e and f .", "( e ) Comparison of fluorescence changes in AWC during epochs 1 ( odor addition ) , 2 ( odor removal ) and 3 ( return to baseline ) , same data as in d .", "Each box corresponds to the 25th to 75th percentile of the distribution ( q1 and q3 respectively ) , whisker length is up to the extreme data point or 1 . 5 l ( q3-q1 ) .", "( f ) Comparison of decay rates in AWC upon odor addition ( epoch 1 ) , same data as in d . DOI: http://dx . doi . org/10 . 7554/eLife . 12727 . 029" ], [ "GFP-based GECIs are well-established tools for studying neuronal activity across many model organisms .", "Current state-of-the-art green GECIs , such as GCaMP6 , have sufficient sensitivity to detect single action potentials in diverse cell types and even detect activity of single synapses .", "They can be targeted to specific cell types and subcellular compartments .", "Red GECIs can potentially be used in a similar manner with additional advantages .", "For example , they suffer less from tissue scattering and absorption .", "In addition , rhodopsin-based optogenetic tools have substantial absorption in the blue region of the wavelength spectrum , which overlaps with the excitation spectrum of GFP .", "Red GECIs can be imaged without exciting ChR2 .", "We developed red GECIs that rival best-of-class green GECIs in terms of sensitivity for detecting neural activity , thereby closing a significant performance gap between red and green GECIs .", "The new mApple-based jRGECO1a and the mRuby-based jRCaMP1a and jRCaMP1b are all improved several-fold compared to their parent indicators .", "jRGECO1a exhibits similar performance to the GCaMP6 indicators .", "jRCaMP1a and jRCaMP1b are less sensitive , but do not show photoswitching after illumination with blue light , making them suitable for experiments that combine calcium imaging and optogenetics .", "jRCaMP1a and jRCaMP1b are also slightly redder than jRGECO1a .", "This improved performance will enable a wide range of applications for red GECIs , for example , dual color imaging , where green and red GECIs are used in parallel to study the relation between two components of a neural circuit .", "Red GECIs will be useful for all-optical neurophysiology experiments , especially when there is spatial overlap between the stimulated and imaged parts of the neural circuit .", "The large spectral separation between ChR2 activation spectrum and the red GECI excitation spectrum ( Akerboom et al . , 2013 ) is preferable in situations when using a red-shifted light sensitive ion channel and a green GECI produces cross-talk ( Rickgauer et al . , 2014; Packer et al . , 2015 ) .", "The two-photon absorption spectra of red GECIs peak around 1040 nm , which overlaps with the output of cost-effective and powerful fiber lasers .", "Red GECIs still face challenges .", "First , all tested red GECIs show smaller ( 3- to 4-fold less ) maximal fluorescence changes upon calcium binding than the GCaMP6 indicators .", "This limits the overall dynamic range of red GECIs .", "Second , the relatively low absorption cross-section of the red GECIs ( Figure 2—figure supplement 1 ) further reduces the achievable signal-to-noise ratio for in vivo imaging .", "Third , red GECIs show complex intracellular behavior , such as accumulation in lysosomes ( jRGECO1a ) with long-term expression in the mouse brain .", "The presence of green fluorescence in all tested GECIs suggests the existence of multiple protein species , some of which do not produce functional signals ( Figure 4—figure supplement 2–3 ) .", "These issues seem to be related to the red fluorescent protein component of the sensor ( see , for example , Supplementary file 1 in Cai et al . , 2013 ) .", "Therefore , engineering red GECIs to produce less non-productive fluorescence might require replacing or re-engineering the RFP itself ( Moore et al . , 2012 ) , a direction that we plan to pursue in the future ." ], [ "GCaMP indicators are naturally excluded from the nucleus , presumably due to a cryptic nuclear exclusion sequence ( NES ) motif .", "This gives rise to the characteristic annular labeling .", "Both RCaMP and R-GECO are expressed throughout the cell , including the nucleus .", "GECI responses are larger and faster in the cytoplasm than in the nucleus ( unpublished data ) .", "We therefore included an NES motif to restrict expression to the cytoplasm .", "For R-CaMP2 , derived from R-CaMP1 . 07 ( Ohkura et al . , 2012; Inoue et al . , 2015 ) , it appears that the C-terminal F2A sequence functions as an NES and therefore no additional NES was added .", "Mutant red GECIs were made using mismatched oligonucleotides and cloned into an expression vector using gene assembly .", "The expression vector contained a human synapsin-1 ( SYN1 ) promoter for neuronal-specific expression , a nuclear export signal ( N-terminal MLQNELALKLAGLDINKTG , derived from the cAMP-dependent protein kinase inhibitor alpha subunit ) , a red GECI variant , an internal ribosome entry site , a nuclearly-targeted GFP , and a woodchuck hepatitis virus post-transcriptional regulatory element ( WPRE ) to augment expression .", "Amino acid positions are numbered in jRGECO1 and jRCaMP1 variants ( Supplementary files 1–2 ) starting from GGSHHHHHHGMASM . . . ( 1–14 . . . ) .", "NES positions are excluded from the numbering , to correspond with published numbering of the scaffolds .", "Red GECI variants were expressed after transfection by electroporation into rat primary hippocampal neurons ( P0 ) using the Lonza Nucleofector system ( P3 Primary Cell 96-well Kit , Lonza , Walkersville , MD ) .", "Transfected neurons were plated in glass-bottom 96-well plates ( MatTek , Ashland , MA ) and cultured as described ( Wardill et al . , 2013 ) .", "For stimulation and imaging , synaptic transmission was inhibited with glutamate and GABA receptor antagonists to make calcium changes solely dependent on opening of voltage sensitive calcium channels .", "APs ( 83 Hz ) were evoked by field stimulation as described ( Wardill et al . , 2013 ) , except that stimulation and imaging was in 96-well plates with modified electrodes .", "Illumination was provided by a warm white LED and TxRed ( 7mW at the sample plane , excitation: 540–580 nm; dichroic: 585 nm long-pass; emission: 593–668 nm ) and GFP ( excitation: 450–490 nm; dichroic: 495 nm long-pass; emission: 500–550 nm ) filter sets .", "Imaging conditions and analysis were similar to prior experiments ( Chen et al . , 2013b ) .", "For photoswitching measurement in jRGECO1a-expressing neurons ( Figure 2—figure supplement 4 ) , we added blue light stimulation ( up to 33 mW ) by using a blue LED and replacing the green excitation filter with a 561 nm shortpass filter ( Semrock , Rochester , NY ) .", "Sensors were purified , and calcium titrations were performed as described ( Chen et al . , 2013b ) .", "Red fluorescence was measured with excitation at 570 nm ( 5 nm bandpass ) and emission 600 nm ( 5 nm bandpass ) .", "For koff measurements , protein samples in 100 µM calcium were rapidly mixed with a solution of 10 mM EGTA in a stopped-flow device ( Applied Photophysics , UK ) coupled to a fluorometer with 560 nm excitation/580 nm emission filters ( 20 nm bandpass , Varian ) .", "Solutions were buffered with 50 mM MOPS pH 7 . 2 , 100 mM KCl at room temperature .", "Single exponential curves were fitted to the fluorescence decay .", "pKa was determined as described ( Chen et al . , 2013b ) .", "Chromophore extinction coefficients ( ε apo and ε sat ) were measured as described ( Akerboom et al . , 2012 ) .", "Values are mean ± standard deviation , except where indicated , for independently purified protein samples .", "Purified proteins were characterized in 30 mM MOPS , 100 mM KCl , pH 7 . 2 ( calcium calibration buffer C3008MP , ThermoFisher , Waltham , MA ) containing either 10 mM CaEGTA ( calcium buffer ) or 10 mM EGTA ( EGTA buffer ) .", "Absorption spectra were taken on a UV/VIS spectrometer ( Lambda 35 , PerkinElmer , Waltham , MA ) .", "Fluorescence emission and excitation spectra were measured on a LS-55 fluorimeter ( Perkin-Elmer ) with 5 nm slits , and excitation/emission wavelengths of 555 nm/600 nm .", "Absolute quantum yields were measured using a Quantaurus-QY integrating-sphere spectrometer ( model C11374 , Hamamatsu , Japan ) .", "Dim solutions ( e . g . jRGECO1a or GCaMP6s in EGTA buffer ) were not measurable on the Quantaurus , so relative quantum yields were determined from measured absorption in the UV/VIS spectrometer and integrated fluorescence measured in the fluorimeter , in comparison to the reference fluorophores mCherry ( measured absolute QY of 0 . 22 ) or fluorescein ( QY =0 . 93 in pH 9 . 5 borate buffer ) for either red or green GECIs .", "Extinction coefficients for RGECO variants were determined using the alkali denaturation method , assuming the denatured extinction coefficient for RGECO variants is the same as denatured mCherry , 37 , 000 M-1cm-1 at 455 nm at pH 13 ( Zhao et al . , 2011 ) .", "For RCaMP variants , the denatured absorption spectra is complicated by an absorbance peak at 383 nm that first appears with increasing pH at pH 11 . 5 , and is irreversibly formed .", "So for RCaMP variants we used fluorescence correlation spectroscopy ( FCS ) , described below , to quantify concentration by counting fluorophores ( Haustein and Schwille , 2003 ) , using mCherry as a concentration reference .", "FCS was performed on 10–100 nM protein solutions using 1060 nm excitation at low laser power ( 1–3 mW ) to determine the autocorrelation function G ( τ ) of the fluorescence signal .", "The number of fluorophores inside the beam volume is found from 1/G ( 0 ) .", "The beam volume was found from identical FCS measurements on the reference protein mCherry at known concentration .", "For consistency , jRGECO variants were measured using the two methods and results were similar .", "Measurements were carried out on aqueous droplets of purified protein isolated in octanol ( Kremers et al . , 2009 ) , and sandwiched between two coverslips .", "We used an epi-illumination microscope ( AxioImager , Zeiss , Germany ) with a 20x 0 . 8 NA air objective ( Zeiss ) .", "For photoswitching experiments co-linear laser excitation was provided to the microscope at 488 nm ( Sapphire 488 , Coherent , Santa Clara , CA ) and 561 nm ( Sapphire 561 , Coherent ) using a beam-combining dichroic , and computer-controlled shutters determined exposure sequence .", "Fluorescence collected by the objective passed through dichroic and bandpass filters ( Di02-R561 and 625/90 , Semrock ) , and was detected by a fiber-coupled avalanche photodiode ( SPCM-AQRH14 , Pacer , UK ) .", "Laser power was measured at the output of the objective , and laser beam area in the focal plane was imaged and measured using a CCD camera ( CoolSnap EZ , Photometrics , Tucson , AZ ) , in order to determine the laser intensity .", "Photobleaching experiments were done with the setup above and either 561 nm laser excitation ( 4 . 2 W/cm2 at sample ) or Hg-lamp excitation ( 550/25 bandpass , Semrock ) yielding 5 . 6 W/cm2 at the sample .", "Two-photon spectra and FCS were performed as previously described ( Akerboom et al . , 2013 ) .", "Briefly , protein solutions in coverslip-bottomed dishes were measured on an inverted microscope using a 60x 1 . 2 NA water-immersion objective ( Olympus ) .", "Near-infrared laser excitation over the range of 700–1500 nm was provided by either a Ti:sapphire laser ( Chameleon Ultra II , Coherent ) or an OPO ( Chameleon Compact OPO , Coherent ) .", "An overlap range 1000–1080 nm was used to connect the fluorescence spectra generated from the two different excitation sources .", "The Ti:sapphire beam was long-pass filtered ( 715/LP , Semrock ) to remove contaminating visible red spontaneous emission emanating from the Ti:sapphire laser cavity .", "A single dichroic mirror ( 675DCSPXR , Omega , Brattleboro , VT ) was used to bring the Ti:Sapphire or OPO beams to the objective .", "Fluorescence collected from protein samples , filtered by the dichroic mirror and a 720/SP short-pass and a 625/90 bandpass filter ( Semrock ) , was detected by an avalanche photodiode detector ( SPCM_AQRH-14 , Excelitas , Waltham MA ) .", "Output pulses from the detector were fed to an autocorrelator ( Flex03LQ , Correlator . com , Bridgewater , NJ ) , and the system operated under computer control with automated data acquisition .", "Two-photon action spectra were determined from excitation spectra of 1 micromolar protein solutions obtained at low laser power ( 0 . 5 mW ) , and comparing these to spectra measured under identical conditions for the reference dyes fluorescein and rhodamine B , for which we used published 2-photon cross section data ( Xu and Webb , 1996; Makarov et al . , 2008 ) to determine action spectra of the red GECIs .", "This setup was also used to perform FCS in order to measure chromophore concentration at low laser power , and two-photon peak brightness ( Mütze et al . , 2012 ) at high laser power .", "Peak brightness is the experimentally determined maximum rate of fluorescence per molecule for a given focusing geometry and is a measure of the brightness and photostability of a fluorophore under 2-photon excitation .", "To determine peak brightness , 20–100 nM solutions of protein in calcium buffer were illuminated at either 1070 nm or 1140 nm at increasing laser power , where at each power setting the mean fluorescence rate F and the mean number of fluorophores N in the laser beam volume were determined by FCS ( Mütze et al . , 2012 ) .", "The 2-photon peak brightness was then found from the maximum value of the quantity F/N as the laser power was increased .", "Two-photon bleaching measurements of green and red GECIs were acquired from isolated aqueous droplets of protein using a resonant-galvo scanning 2-photon microscope ( MPM-200 , Thorlabs , Newton , NJ ) .", "The microscope was equipped with a 40x 1 . 15 NA water immersion objective ( Nikon ) , a primary dichroic ( 680–1600 nm longpass filter , Thorlabs ) , and a secondary dichroic ( FF562-Di03 , Semrock ) with green ( 530/43 , Semrock ) and red ( 605/70 , Chroma , Bellows Falls , VT ) filters each followed by GaAsP PMTs ( H7422PA-40 , Hamamatsu ) .", "Laser excitation was at 1000 nm or 1070 nm for red GECIs , and 940nm for GCaMP6s .", "To completely bleach the protein droplets ( thickness , 5 μm ) repetitive z-stacks were taken .", "Beam scan area was 160 μm x 160 μm , and the scan rate was 8 frames/sec .", "Light intensity was kept constant across measurements ( Figure 2—figure supplement 2 ) .", "However , due to the use of different wavelengths , the laser spot size , and therefore the intensity profile , was slightly different across these measurements .", "Constructs used to produce AAV included pGP-AAV-SYN1-red GECI-WPRE and the cre-recombinase-activated construct pGP-AAV-FLEX-SYN1-red GECI-WPRE .", "Virus ( titer: ~0 . 7-3x1013 genomes/ml , R-CaMP2 promoter was CaMKII ) was slowly injected ( 25–30 nl over 5 min ) through a thinned skull into the primary visual cortex ( 1–2 injection sites at variable depths; 2 . 7 mm lateral and 0 . 2 mm anterior to lambda suture; 250 μm deep for L2/3 imaging , 250 μm and 450 μm for L4 imaging , 500 μm for L5 imaging , and 650 μm and 900 μm for L6 imaging ) .", "Sixteen to 180 days after virus injection , mice were anesthetized using isoflurane ( 3% for induction , 1 . 5–2% during surgery ) and a 2–3 mm circular craniotomy was made above V1 ( centered around the injection site ) .", "The craniotomy was covered by 1% agarose ( UltraPure Agarose , ThermoFisher ) .", "A round 3 mm glass coverslip ( #1 thickness , Warner Instruments , Hamden , CT ) was cemented to the skull to reduce motion of the exposed brain ( Huber et al . , 2012 ) .", "A custom titanium head post was fixed to the skull using black dental cement ( Contemporary Ortho-Jet , Land Dental , Wheeling , IL ) .", "For simultaneous imaging and cell-attached recording , the exposed brain was covered with a thick 1% agarose layer , and partially covered with a D-shape coverslip .", "The animal was then placed under a microscope on a warm blanket ( 37°C ) and kept anesthetized using 0 . 5% isoflurane and sedated with chlorprothixene ( 20–40 μl at 0 . 33 mg/ml , i . m . ) ( Niell and Stryker , 2008 ) .", "Imaging was performed with a custom-built 2-photon microscope equipped with a resonant scanner .", "The light source was an Insight DS Dual 120 femtosecond-pulse laser ( Spectra-Physics , Santa Clara , CA ) running at 1040 nm and 1100 nm for jRGECO1a and jRCaMP1 indicators , respectively .", "The objective was a 16x 0 . 8 NA water immersion lens ( Nikon , Japan ) .", "Images were acquired using ScanImage 5 ( vidriotechnologies . com ) ( Pologruto et al . , 2003 ) .", "For L2/3 , L4 , and L5 imaging , functional images ( 512x512 pixels , 250x250 μm2 ) were collected at 15 Hz .", "Typical laser powers were 15–60 , 30–75 , and 50–100 mW at the front aperture of the objective for L2/3 , L4 , and L5 imaging , respectively .", "For L6 imaging , functional images ( 512x512 pixels , 165x165 μm2 or 125x125 μm2 ) were collected at 5 Hz .", "Laser power was 100–150 mW at the front aperture of the objective lens .", "For dual-color imaging we used green and red detection channels ( 525/50 nm and 600/60 nm filters respectively , separated by a 565 nm LP dichroic mirror , Chroma ) .", "Bleedthrough of the red signal into the green channel was negligible , and penetration of green signal into the red channel was corrected by linear unmixing .", "Moving grating stimuli were generated using the Psychophysics Toolbox ( Brainard , 1997; Pelli , 1997 ) in MATLAB ( Mathworks , Natick , MA ) .", "Each stimulus trial consisted of a 4 s blank period ( uniform gray at mean luminance ) followed by a 4 s drifting sinusoidal grating ( 0 . 05 cycles per degree , 1 Hz temporal frequency ) .", "Eight drifting directions were used , separated by 45° , and 5 trials were recorded for each direction , giving a total of 40 stimulus trials per recording session ( 320 s recording time ) .", "The gratings were presented with an LCD monitor ( 30 x 40 cm ) , placed 25 cm in front of the center of the right eye of the mouse .", "The monitor subtended an angle of ± 38° horizontally and ± 31° vertically around the eye of the mouse .", "For experiments with cell-attached recording ( Figure 4 ) , pipette access required the use of a smaller LCD monitor ( 12 x 16 cm ) placed 10 cm in front of the right eye .", "During simultaneous imaging and electrophysiology , the optimal grating stimulus was repeatedly played , but the contrast of the stimulus grating was adjusted online to record variable spike rates .", "To minimize stimulus contamination in the red detection channel , a green colored plexiglass filter ( 500–555 nm FWHM ) was placed in front of the mouse eye .", "Data from mice expressing GECIs for 16 to 64 days were used for the following analysis ( Figure 3c–f ) .", "Mechanical drift in the imaging plane was corrected using the TurboReg plug-in in ImageJ ( Thévenaz et al . , 1998 ) .", "All remaining analyses were performed in MATLAB .", "Regions of interest ( ROIs ) corresponding to identifiable cell bodies were selected using a semi-automated algorithm ( Akerboom et al . , 2012; Chen et al . , 2013b ) .", "Depending on a neuron’s appearance , annular or circular ROIs were placed over the cytosolic region of each cell .", "The fluorescence time course was measured by averaging all pixels within the ROI , after correction for neuropil contamination ( Kerlin et al . , 2010 ) .", "The neuropil signal Fneuropil ( t ) surrounding each cell was measured by averaging the signal of all pixels within a 20 μm circular region from the cell center ( excluding all somata ) .", "The fluorescence signal of a cell body was estimated asFcell_true ( t ) = Fcell_measured ( t ) -", "r . Fneuropil ( t ) , with r = 0 . 7 .", "Neuropil correction was applied only to cells with baseline fluorescence ( F0 ) signal stronger than the surrounding neuropil signal by more than 3%; other cells ( approximately 15–20% ) were excluded from the analysis because F0 could not be reliably estimated .", "After neuropil correction , the ΔF/F0 of each trial was calculated as ( F-F0 ) /F0 , where F0 was averaged over a 1 s period immediately before the start of grating stimulation .", "Visually responsive neurons were defined as cells with ΔF/F0 >0 . 05 during at least one stimulus period , and using ANOVA across blank and eight direction periods ( p<0 . 01 ) .", "We calculated the decay time of fluorescence after the end of the preferred stimulus .", "For each cell we averaged responses from five trials; baseline fluorescence and standard deviation were calculated from 1 s before the start of the stimulus .", "Only cells with fluorescence response peaks with >4 times the standard deviation of the baseline during the last 1 s of the stimulus were analyzed .", "The time required for each trace to reach half of its peak value ( baseline fluorescence subtracted ) was calculated by linear interpolation .", "The same cells were used for plotting the average response showed in Figure 3c .", "Because each cell responded at slightly different times , depending on its receptive field structure , each trace was shifted to align maxima .", "For display purposes , traces were smoothed with a 3 sample moving average kernel .", "The orientation selectivity index ( OSI , Figure 3—figure supplement 2 ) was calculated for visually responsive cells ( Niell and Stryker , 2008 ) .", "First , the preferred orientation ( θpref ) of the cell was determined as the stimulus evoking the greatest response .", "The orientation tuning curve was constructed by measuring the mean ΔF/F0 , averaged over the stimulus period , for each orientation .", "We then fitted the tuning curve with the sum of two Gaussians centered on θpref and θpref+π/2 , both with width σ , amplitudes A1 and A2 , and a constant baseline B . The OSI was defined as OSI = ( Rpref -Rortho ) / ( Rpref+Rortho ) , where Rpref and Rortho are the response amplitudes at the preferred ( θpref ) and the orthogonal orientation ( θpref+π/2 ) respectively .", "In vivo cell-attached recordings were performed using glass pipettes ( ~7–12 MΩ ) filled with ( in mM ) : 125 NaCl , 5 KCl , 10 glucose , 10 HEPES , 2 CaCl2 , 2 MgSO4 , and 0 . 1 Alexa Fluor 488; pH 7 . 4 ) .", "Signals were amplified using an AxoPatch 200B amplifier ( Molecular Devices , Sunnyvale , CA ) , filtered at 5 kHz , and digitized at 10 kHz .", "Spikes were recorded using current clamp mode .", "The frame trigger pulses of ScanImage 5 were also recorded and used offline to synchronize individual frames to electrophysiological recordings .", "After establishment of a low-resistance seal ( 15–50 MΩ ) , the stimulus orientation was quickly optimized for individual neurons using recorded spikes .", "The optimal grating stimulus was repeated with variable contrast to acquire a range of spiking rates .", "Images ( 512x512 pixels , typically 40x40 μm2 ) were acquired at 30 Hz and 15 Hz for jRGECO1a and jRCaMP1a , respectively .", "Ring-shaped ROIs were placed over the cytosolic regions of the cells , and neuropil contamination was subtracted .", "To quantify the fidelity of single AP detection ( Figure 4e ) , we identified single AP events isolated from other APs by >1 s , and 2 APs within a time window of 100 and 150 ms for jRGECO1a and jRCaMP1a respectively , isolated from other APs by >1", "s . Noise traces were taken from periods without APs .", "F0 values were calculated as the average of 10 frames before the first AP , and peak ΔF/ F0 was calculated as the maximum of the ΔF/ F0 trace after the first spike firing , or the maximal ΔF/ F0 noise value for the noise traces .", "For d-prime calculation and receiver operating characteristic ( ROC ) curves , response templates for various APs were calculated by averaging response traces .", "Unit vectors were calculated from the templates by subtracting means and normalizing the vectors .", "The measured n-AP responses were projected on the unit vectors , where n indicates the number of APs in a time bin , as well as noise traces ( Chen et al . , 2013b ) .", "The scalar results of projecting the response and noise traces on the unitary response templates were used to calculate d-prime and ROC curves .", "Time windows in which the APs were fired varied with the number of spikes and the GECI ( jRGECO1a: 100 ms for 2APs and increasing by 25 ms for each additional AP , up to 250 ms for 8APs; jRCaMP1a: 150 ms for 2APs and increasing 50 ms for each additional AP , up to 350 ms for 6–8 APs ) .", "Time bins were chosen to reflect the accumulation of calcium signal for large number of APs ( longer for the slower jRCaMP1a/b , shorter for the faster jRGECO1a ) .", "Half rise and decay times were calculated from 2 AP responses .", "Only isolated responses ( without APs > 2 s before the recorded spikes ) were used .", "An exponential decay curve was fitted to each trace .", "Only traces with small fitting error were chosen ( 16 traces for both indicators ) .", "Mice were deeply anesthetized with isoflurane and transcardially perfused with 10 ml 1X Dulbecco’s phosphate-buffered saline ( DPBS , ThermoFisher ) , followed by 50 ml 4% paraformaldehyde in 0 . 1 M phosphate buffer .", "After perfusion , the brains were removed and post-fixed overnight at 4°C .", "The brains were embedded in 5% agarose in DPBS , and cut into 50 µm thick coronal sections with a vibratome ( Leica VT 1200S , Germany ) .", "Because DPBS contains saturating calcium ( 0 . 9 mM ) red GECI brightness is maximal .", "Sections were coverslipped with Vectashield mounting medium ( H-1400 , Vector laboratories ) .", "Confocal images ( LSM 710 , Zeiss ) were collected using a 20x 0 . 8 NA air objective .", "Emission spectra were characterized using a lambda scanning mode of the confocal microscope , and were corrected for background emission .", "For LAMP-1 immunostaining , brains were perfused , fixed ( 4 hr post-fixed , room temperature ) , and sectioned as described above .", "Sections were soaked and washed twice with DPBS , then blocked in blocking buffer ( 2% BSA , 0 . 4% Triton X-100 in DPBS ) for 1 hr at room temperature .", "Sections were then incubated in anti-LAMP-1 antibody conjugated to Alexa Fluor 488 ( clone 1D4B , diluted 1:200 in blocking buffer , Santa Cruz Biotechnology , Dallas , TX ) overnight at 4°C , and then washed 3 times with 0 . 1% Triton X-100 in DPBS , and washed again with DPBS .", "Finally , sections were mounted and coverslipped with Vectashield anti-fade mounting medium ( H-1500 , Vector laboratories , Burlingame , CA ) .", "We made w1118;; PBac{20XUAS-IVS-GECI-p10}VK00005 transgenic flies and crossed them with w1118;; R57C10-Gal4 in VK00020 , R57C10-Gal4 in VK00040 pan-neuronal driver line .", "Improved expression allows lower excitation light dosage and helps to prevent photobleaching .", "The NMJ assay is similar to the one used previously ( Chen et al . , 2013b ) .", "Briefly , actively crawling female 3rd instar larvae were dissected under minimum illumination intensity .", "Type Ib boutons on muscle 13 from segment A3-A5 were wide-field imaged in HL-6 saline while corresponding axons were electrically stimulated with a suction electrode driven by a customized stimulator .", "Temperature and pH were monitored during imaging .", "A mercury lamp ( X-CITE exacte , Excelitas ) light source was used for excitation and power of less than 5 mW at the objective front aperture was used .", "The light intensity was calibrated so that no significant photobleaching was detected during each trial .", "The filters for jRGECO1 and jRCaMP1 imaging were: excitation: 543/22 nm; dichroic: 562 nm; emission: 593/40 nm .", "Emitted light was collected on an EMCCD cooled to -70°C at 30 fps .", "Data were analyzed in MATLAB ( MathWorks ) .", "Mitfa−/− ( nacre ) zebrafish were maintained under standard conditions at 28°C and a 14:10 hr light:dark cycle .", "Embryos ( 1–2 cell stage ) were injected with 20 ng/μl DNA plasmids encoding the red GECI variants under the control of the ( near ) pan-neuronal elavl3/HuC promoter ( elavl3:GECI ) , and 40 ng/μl Tol2 transposase mRNA diluted in E3 medium with 0 . 025% Phenol Red .", "Three and four day post-fertilization embryos showing expression in isolated trigeminal neurons were treated with 1 mg/ml bath-applied α-bungarotoxin for 30 min to block tail movements .", "Larvae were mounted sideways in 1 . 5% low melting temperature agarose , and imaged using a microscope with a 20× , NA=1 objective lens ( Olympus , Japan ) and an sCMOS camera ( Hamamatsu Orca Flash 4 ) .", "Trains of 1 , 5 and 10 field stimuli ( 20 ms each; 20 Hz ) were applied using two mesh electrodes located inside the bath and a stimulator ( Grass SD9 , Warwick , RI ) .", "Stimulation voltage was calibrated to elicit an identifiable response to a single pulse in GCaMP6f expressing neurons .", "One spike in a trigeminal neuron has previously been shown to be sufficient to elicit tail movement ( Douglass et al . , 2008 ) .", "Image acquisition and stimulus control were handled using Hamamatsu HCImage ( 4 . 2 . 5 . 0 ) software .", "ROIs were selected manually , and data was analyzed using MATLAB ( MathWorks ) .", "Red GECIs were expressed in the C . elegans ASH and AWC sensory neurons under the sra-6 and str-2 promoters , respectively .", "Animals were restrained in custom-built microfluidic chambers ( Chronis et al . , 2007 ) in S Basal buffer ( Brenner , 1974 ) , and paralyzed with 1 mM tetramisole hydrochloride ( Sigma-Aldrich , St . Louis , MO ) during data acquisition to reduce movement .", "For ASH imaging , 1 M glycerol was delivered to the nose of the animal in alternating one second intervals for 4 min as in ( Kato et al . , 2014 ) .", "For AWC imaging , 92 μM isoamyl alcohol was delivered for one minute after one minute of exposure to buffer .", "Worms were illuminated with a solid-state lamp ( Lumencor SOLA-LE , Beaverton , OR , 560/40 nm excitation filter ) , and fluorescence images ( 630/75 nm emission filter ) were collected at 10 fps using a 40x , NA=1 . 4 objective ( Zeiss ) and an EMCCD camera ( Andor iXon3 , Metamorph Software , 250 x 140 pixels , covering 104 μm x 58 μm ) .", "DNA constructs , AAV particles and Drosophila with red GECIs variants were deposited for distribution at Addgene ( http://www . addgene . org ) , the University of Pennsylvania Vector Core ( http://www . med . upenn . edu/gtp/vectorcore ) and the Bloomington Drosophila Stock Center ( http://flystocks . bio . indiana . edu ) , respectively .", "Cell-attached recording and functional imaging data were deposited at CRCNS . org ( doi: 10 . 6080/K0W37T87 ) ." ] ]

[ "Genetically encoded calcium indicators ( GECIs ) allow measurement of activity in large populations of neurons and in small neuronal compartments , over times of milliseconds to months .", "Although GFP-based GECIs are widely used for in vivo neurophysiology , GECIs with red-shifted excitation and emission spectra have advantages for in vivo imaging because of reduced scattering and absorption in tissue , and a consequent reduction in phototoxicity .", "However , current red GECIs are inferior to the state-of-the-art GFP-based GCaMP6 indicators for detecting and quantifying neural activity .", "Here we present improved red GECIs based on mRuby ( jRCaMP1a , b ) and mApple ( jRGECO1a ) , with sensitivity comparable to GCaMP6 .", "We characterized the performance of the new red GECIs in cultured neurons and in mouse , Drosophila , zebrafish and C . elegans in vivo .", "Red GECIs facilitate deep-tissue imaging , dual-color imaging together with GFP-based reporters , and the use of optogenetics in combination with calcium imaging ." ]

[ "Neurons encode information with brief electrical pulses called spikes .", "Monitoring spikes in large populations of neurons is a powerful method for studying how networks of neurons process information and produce behavior .", "This activity can be detected using fluorescent protein indicators , or “probes” , which light up when neurons are active .", "The best existing probes produce green fluorescence .", "However , red fluorescent probes would allow us to see deeper into the brain , and could also be used with green probes to image the activity and interactions of different neuron types simultaneously .", "However , existing red fluorescent probes are not as good at detecting neural activity as green probes .", "By optimizing two existing red fluorescent proteins , Dana et al . have now produced two new red fluorescent probes , each with different advantages .", "The new protein indicators detect neural activity with high sensitivity and allow researchers to image previously unseen brain activity .", "Tests showed that the probes work in cultured neurons and allow imaging of the activity of neurons in mice , flies , fish and worms .", "History has shown that enhancing the techniques used to study biological processes can lead to fundamentally new insights .", "In the future , Dana et al . would therefore like to make even more sensitive protein indicators that will allow larger networks of neurons deeper in the brain to be imaged ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "chromosomes and gene expression", "evolutionary biology" ]

[ [ "Contingency is widespread in evolution , with chance historical changes dictating the repertoire of future possibilities ( Bershtein et al . , 2006; Bloom et al . , 2006; Blount et al . , 2012; Ortlund et al . , 2007; Sorrells et al . , 2015; Starr et al . , 2017 ) .", "Nevertheless , repeated evolutionary events in different lineages demonstrate that evolution is , at least in some instances , predictable .", "Repeatability is broadly referred to as convergent evolution , and when a trait arises repeatedly by a similar molecular mechanism , it is referred to as parallel evolution ( Stern , 2013 ) .", "Beginning with Darwin ( Darwin , 1883 ) , convergence has been taken as evidence for adaptation , but it can also be caused by drift within the constraints that arise from the properties of biological systems ( Losos , 2011 ) .", "Instances of repeated events can be thought of as natural experimental replicates for finding general principles that result in convergent evolution , and this information could be used to predict which evolutionary paths are most probable .", "Genomic studies have found sets of genes that underlie convergent evolution of a wide variety of traits from across the tree of life ( Bellott et al . , 2010; Denoeud et al . , 2014; Gallant et al . , 2014; Mycorrhizal Genomics Initiative Consortium et al . , 2015; Marvig et al . , 2015; McCutcheon et al . , 2009; Nagy et al . , 2014; Pfenning et al . , 2014; Sherwood et al . , 2014; Soria-Carrasco et al . , 2014 ) .", "Such sets of genes tend to evolve in functionally related groups and are controlled by cis-regulatory sequences for particular transcription regulators , forming a transcription network .", "One way that an entire network can be up- or down-regulated is through alterations in the expression of a ‘master’ regulator , propagating a new expression level to all of its downstream target genes ( Chan et al . , 2010; McGregor et al . , 2007; Rebeiz et al . , 2009; Reed et al . , 2011 ) .", "However , in many cases—instead of changing the expression pattern of a master regulator—each gene in the network independently acquires the same cis-regulatory sequence , requiring hundreds of mutations across the genome ( Booth et al . , 2010; Borneman et al . , 2007; Gasch et al . , 2004; Kasowski et al . , 2010; Lowe et al . , 2011; Paris et al . , 2013; Piasecki et al . , 2010; Schmidt et al . , 2010; Tanay et al . , 2005; Tuch et al . , 2008b ) .", "An outstanding question is how this process occurs .", "Several molecular mechanisms for gene-by-gene rewiring have been proposed ( Britten and Davidson , 1971; Tuch et al . , 2008b ) .", "Hitchhiking of a cis-regulatory site on a transposable element is a mechanism well-documented in plant and animal evolution , and can rapidly bring genes under new regulatory control ( Bourque et al . , 2008; Chuong et al . , 2016; Kunarso et al . , 2010; Lynch et al . , 2011; Schmidt et al . , 2012 ) .", "Alternatively , one transcription regulator can gain a protein-protein interaction with another , followed by the evolution of cis-regulatory sequences ( Baker et al . , 2012; Lynch et al . , 2008; Pérez et al . , 2014; Tsong et al . , 2006 ) .", "This latter mechanism is able , at least in principle , to rewire an entire set of genes at once upon evolution of the new protein-protein interaction; the individual gains of binding sites could occur secondarily .", "Nevertheless , many—if not most—examples of network rewiring seem to have occurred in the absence of evidence for either of these two mechanisms ( Borneman et al . , 2007; Gasch et al . , 2004; Kasowski et al . , 2010; Lowe et al . , 2011; Paris et al . , 2013; Piasecki et al . , 2010; Schmidt et al . , 2010; Tanay et al . , 2005; Tuch et al . , 2008a ) .", "Here , we studied an example of transcription network rewiring in which ~100 ribosomal protein genes gained binding sites for the Mcm1 transcription regulator , and we describe evidence supporting a new mechanism for the concerted gains .", "The gain of Mcm1 binding sites in the ribosomal protein genes occurred in parallel in two respects: ( 1 ) the cis-regulatory sites were gained upstream of a large proportion of the ribosomal proteins in each species; and ( 2 ) they were gained independently approximately 12 – 13 times during Ascomycete evolution .", "At each gene , several point mutations were probably necessary to produce a close match to the 16-basepair Mcm1 binding site , thus requiring several hundred mutations across the entire gene set .", "Based on results from a variety of experimental approaches , we argue that the gain of Mcm1 sites was potentiated in several different clades by the presence of an ancestral transcription regulator of the ribosomal protein genes , Rap1 .", "We demonstrate that Rap1 and Mcm1 have the intrinsic ability to cooperate in the activation of transcription , even when artificially introduced in species in which their sites are not found together .", "Biochemical and genetic experiments show that both regulators interact with the general transcription factor TFIID .", "We propose that the intrinsic , ancient ability of both proteins to interact with a common component of the general transcription machinery facilitated the repeated evolution of the Mcm1 sites in independent lineages ." ], [ "Previously , genome-wide chromatin immunoprecipitation and bioinformatics experiments revealed that Mcm1 cis-regulatory sites evolved independently at the ribosomal protein genes ( RPGs ) in several yeast lineages ( Tuch et al . , 2008a ) .", "To expand this analysis , we used 162 sequenced fungal genomes , identified RPG regulatory regions , and searched for cis-regulatory sequences for Mcm1 and 11 other transcription regulators that are known to regulate ribosomal components in at least one species ( Figure 1A , Figure 1—figure supplement 1 ) .", "Mcm1 sites were found highly enriched ( -log10 ( P ) > 6 ) at the RPGs in six different monophyletic groups including the clades represented by Kluyveromyces lactis , Candida glabrata , and Yarrowia lipolytica , as well as the individual species Kazachstania naganishii , Pachysolen tannophilus , and Arthrobotrys oligospora .", "Mcm1 sites were found moderately enriched ( 6 > -log10 ( P ) > 3 ) in the RPGs in seven additional lineages .", "The pattern of yeast lineages containing Mcm1 binding sites upstream of the RPGs could occur through three general scenarios: the Mcm1 binding sites were gained multiple times , they were present in an ancient ancestor and lost multiple times , or a combination of gains and losses .", "To estimate how many gains and losses occurred during the evolution of Mcm1 sites , we treated the presence of Mcm1 sites ( -log10 ( P ) > 3 ) as a discrete character and sampled 10 , 000 stochastic character maps from the posterior probability distribution of each of two models ( Bollback , 2006; Revell , 2012 ) ( Figure 1B ) .", "One model assumed gains and losses to be equal in probability and the other estimated them as two different rates .", "Under both models , the number of gains was substantial: an average of 13 . 4 and 12 . 0 for the equal and different rates models , respectively ( 95% highest posterior density interval of 12 – 14 and 7 – 17 gains , respectively ) .", "The number of losses differed between the models resulting in an average of 3 . 0 losses for the equal rates model and 17 . 0 for the different rates model .", "These models indicate that the number of times Mcm1 sites were gained in the Ascomycete evolution was high ( 12 – 13 on average ) whereas the number of losses is sensitive to the assumptions of the model .", "Three observations support the idea that the pattern of Mcm1 cis-regulatory sequences in the RPGs require independent gains for most of the monophyletic clades and species with Mcm1 sites .", "First , the gain model is more parsimonious because of the sparse distribution of clades with Mcm1 sites .", "This is still true when taking into account the ancient interspecies hybridization that occurred in two ancient members of Saccharomycetaceae ( Marcet-Houben and Gabaldón , 2015 ) because in most likely scenarios , the ancestors are concordant for the absence of Mcm1 sites .", "Second , other cis-regulatory sequences show similar patterns of evolution to that of Mcm1 .", "The Dot6/Tod6 and Rim101 motifs are also found upstream of the RPGs in several distantly related clades , although fewer clades than for Mcm1 ( Figure 1—figure supplement 1 ) .", "Other regulators show clear single gains in the common ancestor of all Saccharomycotina yeasts ( e . g . Rap1 ) , or Pezizomycotina and Saccharomycotina ( e . g . Cbf1 ) as well as losses in sparsely distributed individual clades and species .", "These results show that gains and losses of cis-regulatory sequences are common upstream of the RPGs , and that they can be distinguished from each other based on their distinct distributions among species ( Lavoie et al . , 2010; Tanay et al . , 2005 ) .", "Third , it was previously shown that the entire set of genes that Mcm1 regulates changes extensively over the timescale of Ascomycete evolution ( Tuch et al . , 2008a ) , suggesting that conservation from a distant ancestor would be a marked exception to this trend .", "To test whether the Mcm1 cis-regulatory sites we identified upstream of the RPGs are functional , we linked several full-length RPG upstream intergenic regions to the fluorescent reporter GFP .", "We chose RPL37 and RPS18 from Kl .", "lactis , each of which has Mcm1 binding sites and measured the expression of these reporters under nutrient-replete conditions .", "To test whether they contributed to expression , we scrambled the Mcm1 sites .", "In both cases , the scrambled site reduced expression of the reporter ( RPS18 p=0 . 017; RPL37 p=0 . 027; Welch’s t-test ) but left the cell-to-cell variability unchanged ( Figure 1C and D; Figure 1—figure supplement 2 ) .", "Given that there are many known regulators of ribosomal protein transcription , this demonstrates that Mcm1 plays a non-redundant role in activating these genes in Kl .", "lactis under conditions that require high expression of the translational machinery .", "Yeast ribosomes are composed of four ribosomal RNAs and 78 ribosomal proteins , assembled in equal stoichiometry ( Woolford and Baserga , 2013 ) .", "In rapidly growing cells , ribosomal protein transcripts are among the most highly expressed , and have short half-lives , leading to the estimation that approximately 50% of all RNA polymerase II initiation events occur at ribosomal proteins ( Warner , 1999 ) .", "These observations suggest that the expression of these genes is under strong selection as it plays a major role in energy expenditure in the cell .", "Given that the Mcm1 cis-regulatory sites increase RPG transcription levels during rapid growth , there are at least two plausible hypotheses for their appearance upstream of the RPGs .", "( 1 ) In those species that have acquired Mcm1 cis-regulatory sequences , the expression of the RPGs is higher than in species without these sequences .", "( 2 ) The gain of Mcm1 cis-regulatory sequences compensate for other cis-regulatory changes that lower expression of the genes , with no net gain in expression levels .", "This second hypothesis is plausible , in principle , because RPGs are known to lose regulator binding sites as well as gain them ( Ihmels et al . , 2005; Lavoie et al . , 2010; Tanay et al . , 2005; Tuch et al . , 2008a ) .", "To distinguish between these hypotheses , we examined directly whether the RPGs have a higher expression level in species that have acquired Mcm1 sites compared to those that have not .", "( We performed these experiments under conditions in which we have shown Mcm1 sites are functional , but we do not rule out different evolutionary scenarios under other environmental conditions ) .", "To accurately measure differences in RPG expression , we mated different species pairs to form interspecies hybrids; we then measured mRNA levels by RNA-seq and assigned each sequencing read to one genome or the other ( Figure 2A ) .", "Comparing the expression of orthologous genes in this way controls for differences in ‘trans-acting’ factors like transcription regulators and therefore reflects only differences caused by cis-regulatory changes ( Wittkopp et al . , 2004 ) .", "For this allelic expression experiment , we constructed hybrids between Kl .", "lactis and two other species , Kl .", "marxianus and Kl .", "wickerhamii .", "These two additional species are relatively closely related to Kl .", "lactis ( such that they can form interspecies hybrids ) , but have fewer Mcm1 sites at their RPGs ( Figure 2A ) , suggesting that Mcm1 binding site gains are ongoing in some lineages over this timescale .", "mRNA reads for each gene were normalized to genomic DNA reads to control for mappability and gene length ( see methods ) .", "Expression levels and differential allelic expression of genes in the two species’ genomes were reproducible between replicates ( Figure 2—figure supplement 1 ) .", "At a false discovery rate of 0 . 05 , we identified 2925 genes showing differential allelic expression out of a total of 4343 orthologs in the Kl .", "lactis × Kl .", "marxianus hybrid , and 3432 out of 4319 in the Kl .", "lactis × Kl .", "wickerhamii hybrid .", "To ask broadly whether RPGs have experienced concerted cis-regulatory evolution , in each hybrid we asked whether the RPGs were more likely to show differential allelic expression in one direction relative to all genes in the genome ( Figure 2B ) .", "In both hybrids , the RPGs showed evidence for cis-regulatory evolution ( hypergeometric test , p=2 . 36e-3 for Kl . lactis × Kl . marxianus hybrid; p=1 . 52e-6 for Kl . lactis × Kl . wickerhamii hybrid ) .", "We conclude that in these species , the expression of the ribosomal proteins has evolved directionally as a group through cis-regulatory changes .", "However , in both hybrids , the ribosomal protein genes were expressed , on average , lower in the Kl .", "lactis alleles than in alleles of either of the other species .", "This observation rules out the hypothesis that the gain of Mcm1 sites over this timescale was simply due to directional selection to increase expression of the ribosomal proteins as a whole ( see also Figure 2—figure supplement 2 ) .", "Instead , it favors the second hypothesis: ongoing evolution of Mcm1 sites in the Kluyveromyces clade compensates ( perhaps incompletely ) for other cis-regulatory changes that reduce RPG transcript levels .", "A compensatory model is consistent with the observation that the binding sites for other regulators ( including Fhl1 and Rrn7 ) show a reduction in the Kluyveromyces clade ( Figure 1—figure supplement 1 ) .", "We also revisited previously published yeast interspecies hybrid experiments performed with species from the Saccharomyces clade .", "Surprisingly , in all cases , the ribosomal proteins were among the sets of genes with reported directional cis-regulatory evolution ( Bullard et al . , 2010; Clowers et al . , 2015; Lee et al . , 2013; Martin et al . , 2012 ) .", "These observations demonstrate that RPG expression may frequently experience different evolutionary forces between closely related yeast species , and they are consistent with the high rate of gains and losses of cis-regulatory sequences that control RPG transcription .", "Although RPGs have experienced different selection pressures across different species , this observation does not explain why Mcm1 sites , as opposed to cis-regulatory sequences for many other transcription regulators , are repeatedly gained in the lineages we examined .", "One possibility is that Mcm1 expression changes under certain environmental conditions , conferring optimal RPG expression levels for the Kluyveromyces clade ( as well as the other clades that gained Mcm1 sites ) .", "The fact that Mcm1 is expressed across all yeast cell types and —in combination with dedicated regulators—controls many different genes that are part of different expression programs ( Tuch et al . , 2008a ) makes this possibility unlikely and we do not explicitly examine it here .", "We therefore investigated whether Mcm1 has other characteristics that allow it to gain cis-regulatory sites especially easily at the RPGs .", "We previously noted that , in Kl .", "lactis , the Mcm1 sites were gained a fixed distance away from the binding site for another transcription regulator , Rap1 , whose sites at the RPGs were ancestral to Kl .", "lactis and S . cerevisiae ( Tuch et al . , 2008a ) .", "By pooling data from additional species with Rap1-Mcm1 sites , we discovered that the spacing between Mcm1 and Rap1 sites was particularly precise with peaks at 54 , 65 , and 74 basepairs apart , favoring a configuration with the two proteins on the same side of the DNA helix ( Figure 3A–C ) .", "In order to understand whether the spacing between Mcm1 and Rap1 sites affects transcriptional activation , we moved a segment of the Kl .", "lactis RPS23 upstream region that contains the Rap1 and Mcm1 sites into a heterologous reporter containing a basal promoter from S . cerevisiae CYC1 ( Guarente and Ptashne , 1981 ) ( Figure 3D ) .", "This construct allowed us to study the Rap1 and Mcm1 cis-regulatory sequences independent of the additional regulators of the RPGs .", "We systematically varied the spacing between these two cis-regulatory sequences in 2 bp increments and found that the transcriptional output remained similar in most constructs ( Figure 3E ) .", "In these experiments the Mcm1 and Rap1 sites were both close to optimal , and we hypothesized that weaker sites would be more likely to reveal a spacing preference .", "To test this idea , we performed a second series of experiments with the Rap1 and Mcm1 cis-regulatory sequences from the Kl .", "lactis RPS17 gene .", "The binding sites from this gene are weaker matches to the Rap1 and Mcm1 position weight matrices ( log odds sum of 9 . 34 versus 12 . 48 for Rap1 and 7 . 24 versus 14 . 33 for Mcm1 ) .", "The transcriptional output of this second series of constructs showed sensitivity to spacing between the sites ( Figure 3E ) .", "We hypothesized that the observed sensitivity of transcriptional output to the strength and spacing of Rap1 and Mcm1 cis-regulatory sequences reflected cooperative activation of transcription .", "Deletion of the Mcm1 and Rap1 binding sites individually and in combination showed that the proteins KlMcm1 and KlRap1 activated transcription cooperatively in Kl .", "lactis: their combined expression was approximately four times the sum of their individual contributions ( Figure 3F; p=5 . 5e-4 , one sample t-test ) .", "One likely explanation for these observations is the evolution of a favorable protein-protein interaction between Rap1 and Mcm1 that occurred around the time that Mcm1 sites appeared at the RPGs in the Kluyveromyces and C . glabrata-Nakaseomyces clades ( Tuch et al . , 2008b ) , that is a ‘derived cooperativity’ model ( Figure 3G ) .", "To test this model , we placed the Kl .", "lactis Rap1-Mcm1 reporter into the genome of S . cerevisiae , a species that lacks Mcm1 sites at the RPGs .", "We found that ScRap1 and ScMcm1 activated expression of the reporter cooperatively ( p=3 . 9e-3 , one sample t-test ) , thereby demonstrating that these proteins have the capacity to work together even in a species where their binding sites did not evolve close proximity ( Figure 3H ) .", "This result rejects the derived cooperativity model and strongly suggests that the ability of Rap1 and Mcm1 to activate transcription cooperatively was ancestral , existing even in species that did not take advantage of it in regulating the RPGs .", "Having established that cooperativity of Rap1 and Mcm1 was likely ancestral to the monophyletic clade containing Kluyveromyces and Saccharomyces , we considered three possible mechanisms that could explain ancestral cooperativity: ( 1 ) Rap1 and Mcm1 could bind DNA cooperatively through an ancient , favorable protein-protein interaction , ( 2 ) they could bind nucleosomal DNA through cooperative displacement of histones , or ( 3 ) they could both bind a third transcription regulator resulting in cooperative transcriptional activation .", "To test the first possibility , we used a gel-mobility shift assay with a radiolabeled DNA sequence from the Kl .", "lactis RPS23 gene ( Figure 3D ) .", "We asked whether purified full-length S . cerevisiae and Kl .", "lactis proteins as well as cell lysates from three additional species bound to Rap1 and Mcm1 binding sites cooperatively .", "In all cases , each protein bound to the RPG promoter DNA independently and did not appear to increase the other’s affinity , indicating that Rap1 and Mcm1 do not , on their own , bind DNA cooperatively ( Figure 4A , Figure 4—figure supplement 1 ) .", "A second mechanism explaining cooperative activation is through nucleosome displacement .", "Many transcription regulators have the inherent property of competing for binding to DNA with nucleosomes , with some more effective than others ( Zaret and Carroll , 2011 ) .", "However , it is unlikely that cooperative nucleosome displacement is the primary mechanism for cooperative activation by Rap1 and Mcm1 at the RPGs .", "Although Rap1 can indeed bind nucleosomal Rap1 binding sites ( Koerber et al . , 2009; Rossetti et al . , 2001 ) , and displace nucleosomes ( Kubik et al . , 2015; Lickwar et al . , 2012; Platt et al . , 2013 ) , it remains bound at the RPGs even during stress conditions when the genes are repressed and show higher levels of nucleosome occupancy ( Bernstein et al . , 2004; Lee et al . , 2004 ) .", "Furthermore , a general mechanism for the cooperative assembly of all transcription regulators has little explanatory power for why cis-regulatory sites for Mcm1 were repeatedly gained across RPGs rather than sites for many of the ~250 other regulators coded in the yeast genome .", "We next considered the third possibility , namely that the cooperative transcriptional activation we observe for Rap1 and Mcm1 occurs through the interaction of both with a third factor that catalyzes a rate-limiting step in transcription activation ( Lin et al . , 1990 ) .", "In principle , a regulator could activate transcription through contacts with general transcription factors , Mediator , SAGA , various chromatin remodeling complexes , or RNA polymerase itself .", "To identify possible factors that might directly bind both Rap1 and Mcm1 , we searched the S . cerevisiae BioGRID database for common interaction targets of both proteins ( Oughtred et al . , 2016 ) .", "Two complexes , SWI/SNF and TFIID , fit this criterion .", "SWI/SNF is not required for ribosomal protein transcription ( Sudarsanam et al . , 2000 ) , so it is unlikely that binding to SWI/SNF plays a role in Rap1 and Mcm1 cooperativity at the RPGs .", "TFIID is a general transcription factor whose direct interaction with Rap1 is required for RPG transcription in S . cerevisiae ( Garbett et al . , 2007; Layer et al . , 2010; Mencía et al . , 2002; Papai et al . , 2010; Reja et al . , 2015 ) .", "Furthermore , TFIID activates transcription through contacts with RNA Polymerase II , and its binding to the promoter is a rate-limiting step in the activation of TFIID-dependent genes ( Wu and Chiang , 2001 ) , such as the RPGs .", "The mechanism of how activators increase transcription rate through contacts with TFIID is not entirely understood but occurs either through a structural rearrangement of the complex or simply by an increase in its occupancy on DNA ( Coleman et al . , 2017; Fuda et al . , 2009; Nogales et al . , 2017; Papai et al . , 2010; Sauer et al . , 1995a; Sauer et al . , 1995b ) .", "The interaction of Rap1 with TFIID has been extensively documented ( Garbett et al . , 2007 ) .", "Of particular importance is the interaction between the ‘activation domain’ of Rap1 and the Taf5 subunit of TFIID , as mutations that compromise this interaction strongly reduce ribosomal protein gene transcription ( Johnson and Weil , 2017 ) .", "To test directly whether Mcm1 also binds to TFIID ( as suggested by mass spectrometry-based analyses of TFIID and associated proteins [Sanders et al . , 2002] ) , we performed a Far-Western protein-protein binding assay using purified S . cerevisiae TFIID , ScMcm1 , and individual TFIID subunits .", "We found that Mcm1 binds directly to the Taf4 subunit ( Figure 4B , C ) ; using deletions of Taf4 , we further mapped the interaction to the N-terminal region of Taf4 ( Figure 4D , E ) .", "Although Rap1 also binds to Taf4 ( in addition to Taf5 and Taf12 ) , its target is in the C-terminus of this subunit , distinct from the Mcm1 interaction site .", "The finding that Rap1 and Mcm1 both interact with distinct domains of a common component of the transcription machinery , one whose assembly at the promoter is rate limiting , provides a simple explanation for their ability to activate transcription cooperatively .", "To test this idea explicitly , we performed a series of experiments in vivo .", "Because Rap1 is an essential gene , we took advantage of a version of Rap1 with altered DNA-binding specificity ( Rap1AS ) that binds to a non-natural cis-regulatory sequence and confers expression of a reporter ( Johnson and Weil , 2017 ) .", "Rap1AS could then be manipulated without compromising the function of the endogenous Rap1 .", "As shown in Figure 5A–C , Rap1AS shows cooperative transcriptional activation with Mcm1 .", "When the activation domain of Rap1AS was mutated by introducing seven point mutations ( Rap1AS7Ala ) its ability to activate transcription of a Rap1 reporter strongly decreased , but was not entirely eliminated ( Johnson and Weil , 2017 ) .", "When introduced in the presence of the Rap1-Mcm1 HIS3 reporter , these mutations strongly reduced growth on media containing 3-aminotriazole ( 3-AT ) , a competitive inhibitor of the HIS3 gene product ( Brennan and Struhl , 1980 ) , indicating that efficient expression requires an intact Rap1 activation domain ( Figure 5D–F ) .", "Taken together , these experiments indicate that the cooperative transcriptional activation by Rap1 and Mcm1 at the RPGs in these clades is due to both proteins interacting with TFIID , a component of the general transcriptional machinery .", "This idea explains how Mcm1—and not a random mixture of other regulatory proteins—came to be repeatedly gained at the ribosomal protein genes .", "We tested several evolutionary and molecular predictions of this model .", "One prediction is that Rap1 and Mcm1 cis-regulatory sequences would occur at the prescribed distance apart but located at other genes besides the RPGs .", "We searched a subset of hemiascomycete yeast genomes for Rap1-Mcm1 sites with similar spacing and orientation to that observed in the RPGs ( Figure 6—figure supplement 1 ) .", "Most species analyzed had only a few such genes of questionable significance , but Ka .", "naganishii had 33 , showing that Rap1 and Mcm1 sites can evolve at genes other than the RPGs .", "A second prediction of our model is that even a sub-optimal Mcm1 site would activate transcription of a regulatory region with an ancestral , strong Rap1 site .", "Because sub-optimal sites can evolve de novo with much higher probability than optimal sites ( Dermitzakis and Clark , 2002; Stone and Wray , 2001 ) , this property would increase the ease by which a large number of functional Mcm1 sites could arise during evolution .", "To test this prediction , we created a series of GFP reporter constructs with Mcm1 binding sites that drive different levels of expression ( Acton et al . , 1997 ) .", "We tested the expression level of each of these Mcm1 sites in the presence and absence of a neighboring Rap1 site in Kl .", "lactis ( Figure 6A ) .", "Consistent with the prediction , most of the sub-optimal Mcm1 sites displayed near wild-type levels of expression in the presence—but not in the absence—of neighboring Rap1 sites .", "Finally , we asked whether our conclusions are generalizable to other pairs of transcription factors besides Mcm1 and Rap1 .", "In particular , some of the clades identified in Figure 1A gained Mcm1 sites at RPGs that lack Rap1 sites .", "To address this question , we centered each RPG intergenic region on the best Mcm1 site and plotted the location of other RPG regulators relative to Mcm1 ( Figure 6B ) .", "This analysis revealed that , in some of these clades , Mcm1 sites were gained varying distances away from the pre-existing sites of other regulators ( Tbf1 , Rrn7 , and Fhl1 ) , suggesting that the evolutionary mechanism we identified with Rap1 and Mcm1 might be a generalizable to other instances of cis-regulatory evolution ( Figure 6C ) .", "Consistent with this idea , all three of these regulators are reported to interact ( directly or indirectly ) with TFIID ( Gavin et al . , 2002; Knutson et al . , 2014; Mallick and Whiteway , 2013; Zhong and Melcher , 2010 ) .", "In summary , the experiments we have presented describe a special relationship between Rap1 and Mcm1 by virtue of their interaction with different surfaces of the same rate-limiting component of transcription , TFIID .", "This relationship between Rap1 and Mcm1 is ancient , and in the next section , we discuss how this property can predispose transcription networks to evolve repeatedly along the same trajectory ." ], [ "Here we have investigated an example of parallel evolution where binding sites for a particular transcription regulator ( Mcm1 ) were gained in a large group of genes ( the ribosomal protein genes , RPGs ) .", "These gains occurred repeatedly in several independent fungal lineages .", "In three of these lineages , Mcm1 binding sites were gained a fixed distance from the sites for another transcription regulator , Rap1 , and we show that these newly acquired Mcm1 sites are required for full activation of the RPGs .", "We also show that Mcm1 and Rap1 cooperatively activate these genes .", "The direct interaction of both of these regulators with a common target , the general transcription factor TFIID , provides a plausible mechanism for this cooperative transcription activation .", "It also explains why the ability of Rap1 and Mcm1 to work together was ancestral to the more recent gains of Mcm1 sites adjacent to Rap1 sites at the RPGs .", "How do these observations account for the fact that Mcm1 sites ( as opposed to sites for other regulators ) were repeatedly gained in parallel next to the Rap1 site at the RPGs ?", "And how do they account for the distance constraints ?", "One common explanation for parallelism is a specific environmental adaptation that occurs through a similar molecular mechanism .", "However , the yeast species with Mcm1 sites at the RPGs are from diverse ecological niches ( e . g . plant leaves , mangrove sediment , the human body , soil ) and utilize different nutrient sources ( e . g . lactate , xylose , feline skin , nematode predation ) , defying a specific environmental adaptation explanation .", "Consistent with this view is our observation , based on analyzing interspecies hybrids , that a species in which the Mcm1 sites were gained at the RPGs does not express these genes at higher levels than related species that evolved fewer Mcm1 sites .", "The model that best fits all of our data holds that the parallel gains arose from the ease with which the functional Mcm1 sites ( and not the sites of other regulators ) appeared in evolution , rather than selective pressure for particular adaptation .", "Specifically , we propose that , in the Kluyveromyces-Saccharomyces ancestor ( before the parallel gains of Mcm1 sites ) Rap1 bound to the ribosomal protein genes and activated transcription through interactions with TFIID , as it does in extant species .", "We further propose that , in the ancestor , Mcm1 activated non-ribosomal genes by interaction with a second site on TFIID , as it does in the extant species S . cerevisiae .", "Any suboptimal Mcm1 site that arose by chance point mutation at a specified distance from a Rap1 site would immediately be functional ( Figure 7 ) because , as we show , even a weak Mcm1 DNA interaction would be stabilized by Mcm1’s intrinsic ability to directly bind TFIID ( see Figure 4C and 6A ) .", "In this way , even suboptimal cis-regulatory sequences ( which are much more likely to appear by chance than optimal sites [Dermitzakis and Clark , 2002; Stone and Wray , 2001] ) could form under selection .", "The appearance of Mcm1 sites likely occurred concomitantly with the gradual losses of other cis-regulatory sites in the RPGs; in other words , the Mcm1 cis-regulatory sites would fall under selection as other cis-regulatory sites deteriorated by mutation .", "In essence , we propose that the free energy gained from the intrinsic interaction between Mcm1 and TFIID would favor formation of new Mcm1 sites at the expense of pre-existing cis-regulatory sequences , particularly since the latter provide a larger target for inactivating mutations .", "This model accounts for why Mcm1 sites ( and not those of other transcription regulators ) were repeatedly gained at the RPGs and why the distance between Rap1 and Mcm1 sites is constrained in those species in which the gains occurred .", "Numerous experimental observations support this model and rule out alternative explanations: ( 1 ) in extant species , Rap1 and Mcm1 both interact with TFIID; ( 2 ) They interact with different parts of TFIID; ( 3 ) cooperative transcriptional activation by Mcm1 and Rap1 requires the activation domain of Rap1 , which is known to interact with TFIID; ( 4 ) the spacing between Rap1 and Mcm1 sites in the ribosomal protein genes places the proteins on the same side of the helix but at least 50 bp apart , consistent with a physical interaction with a large complex; ( 5 ) engineered suboptimal Mcm1 sites are functional as long as they are adjacent to Rap1 sites; ( 6 ) Mcm1 and Rap1 have the intrinsic ability to cooperate ( through interactions with TFIID ) even in a species where Mcm1 sites were not gained at the RPGs .", "We note that this model does not require any change in Rap1 or Mcm1 during the gains of Mcm1 sites at the RPGs .", "Presumably , Rap1 and Mcm1 activated many genes separately in the ancestor , thus preserving by stabilizing selection their ability to interact with TFIID .", "This idea is consistent with the observation that the two proteins were able to cooperate on artificial constructs introduced into S . cerevisiae even though their binding sites are not found together at the RPGs .", "Mutations that alter the function of transcription regulators ( for example , creating a new protein-protein interaction ) can be pleiotropic , decreasing the likelihood that they can arise without disrupting the proteins' ancestral functions ( Carroll , 2005; Stern and Orgogozo , 2008 ) .", "( We note that the transcriptional output was slightly more cooperative in Kl . lactis than in S . cerevisiae , leaving open the possibility that additional fine-scale evolutionary changes may have occurred in how these proteins interact . )", "According to our model , the ability of the two regulators to work together was ancestral , part of each protein’s intrinsic mechanism of transcriptional activation; therefore , their coupling at the RPGs would avoid such pleiotropic changes .", "How does this model account for the gains of Mcm1 sites observed in clades where Rap1 does not regulate the RPGs ?", "In these cases , Mcm1 cis-regulatory sequences also show preferred spacing relative to known regulators of the RPGs , specifically Tbf1 and Rrn7 , and we propose that the same type of cooperativity with TFIID can also account for these cases .", "These two regulators are reported to interact with TFIID ( Knutson et al . , 2014; Mallick and Whiteway , 2013 ) .", "Indeed , TFIID occupies the promoters of RPGs in human cell lines as well ( ENCODE Project Consortium et al . , 2012 ) , raising the possibility that TFIID is a conserved general activator of the RPGs across fungi and animals , while the specific transcription regulators that interact with TFIID simply interchange over this timescale .", "While our cooperative activation model provides an explanation for the parallel acquisition of Mcm1 cis-regulatory site evolution , selection must have operated to preserve the Mcm1 sites as they arose in the population .", "As described earlier , we favor a model where the gains of Mcm1 sites compensated for the degradation of other cis-regulatory sequences and thereby fell under selection .", "The allelic expression data ( Figure", "2 ) strongly supports this model for one clade , represented by Kluyveromyces species .", "However , it is also possible that , in other clades or over shorter timescales , the Mcm1 sites could have been gained due to selection for higher levels of RPG expression .", "The widespread differences in RPG expression revealed by the published interspecies hybrid experiments suggest that RPG expression may experience strong and shifting selection , helping to account for the surprising observation that transcriptional regulators that control the RPGs vary substantially across species ( Gasch et al . , 2004; Lavoie et al . , 2010; Mallick and Whiteway , 2013; Tanay et al . , 2005; Tuch et al . , 2008a; Zeevi et al . , 2014; Zeevi et al . , 2011 ) .", "We note that the intrinsic cooperativity model is sufficient to explain the gain of Mcm1 sites whether or not any change in selection occurs: if functional Mcm1 sites are relatively easy to form ( because of intrinsic cooperativity ) they will be favored over gains of other cis-regulatory sequences by mutational processes alone .", "Mutations creating Mcm1 sites and weakening other sites could occur sequentially in either order or simultaneously ( on the same haplotype ) , depending on the expression requirements of the RPG at that point in its evolutionary history .", "We feel that it is likely that most or all of the selection scenarios outlined above have occurred in at least one RPG at some point during the multiple and ongoing gains of Mcm1 sites over millions of years of fungal evolution .", "While the precise circumstances of each Mcm1 site gain are unknown , intrinsic cooperativity biases the RPGs as a whole toward gaining these sites .", "In conclusion , we have proposed a mechanism , supported by multiple lines of experimental evidence , to account for convergent regulatory evolution of a large set of genes .", "Although , on the surface , the parallel gains of Mcm1 sites at the ribosomal genes would seem to require a special evolutionary explanation , our model does not require an extraordinary mechanism beyond individual point mutations in the cis-regulatory region of each gene .", "However , the ancestral ability of the two key regulators to activate transcription simplifies the path to gaining these sites by producing a phenotypic output from even non-optimal sites .", "Mcm1 , because of its intrinsic ability to cooperate with Rap1 , can significantly activate transcription at the ribosomal proteins more easily than it would elsewhere in the genome; likewise , Mcm1 ( or another regulator that interacted with TFIID ) would be preferred at the RPGs over regulators that did not share this common direct protein interaction .", "Thus , the intrinsic cooperativity of Rap1 and Mcm1 ‘channels’ random mutations into functional Mcm1 cis-regulatory sequences , accounting for the observed parallel evolution .", "We speculate that gene activation through intrinsic cooperativity may be a general mechanism to explain the rapid and ubiquitous rewiring of transcription networks ." ], [ "Genomes were compiled from the Yeast Gene Order Browser ( YGOB ) ( Gordon et al . , 2011 ) , the Department of Energy Joint Genome Institute MycoCosm portal ( Grigoriev et al . , 2014 ) , and individual genome releases ( Supplementary file 3 ) .", "The Kl .", "wickerhamii , Kl .", "marxianus , and H . vinae genomes were annotated using the Yeast Gene Annotation Pipeline associated with YGOB .", "The annotations of genes and proteins in other genomes were obtained from the source of the genome sequence .", "RPGs were defined as any gene starting with ‘Rps’ or ‘Rpl’ in S . cerevisiae and were identified using the ortholog annotation in YGOB ( for species contained in this repository ) .", "These genes were identified in genomes from other sources through psi-blast in BLAST+ ( Camacho et al . , 2009 ) , based on their high conservation .", "The species tree was created as described ( Lohse et al . , 2010 ) .", "In total , 79 single-copy orthologs were chosen to create the species tree based on the ortholog mapping repositories YGOB and Fungal Orthogroups ( Byrne and Wolfe , 2005; Wapinski et al . , 2007 ) .", "They were aligned individually using MUSCLE with default parameters ( Edgar , 2004 ) .", "For species that were not included in these repositories , the corresponding ortholog was identified using psi-BLAST ( Altschul et al . , 1997 ) .", "Using the orthologs from every species , the genes were then re-aligned using MUSCLE with default parameters and concatenated into a single alignment .", "To calculate the tree topology , FastTree 2 . 1 . 8 was used with Blosum45 matrix , the JTT model with 20 rate categories , and two rounds of +NNI +SPR ( default parameters ) .", "Intergenic regions were extracted upstream of each gene using the python script intergenic . py .", "For scoring of potential transcription factor binding sites , motifs were obtained from the ScerTF database ( Spivak and Stormo , 2012 ) as position weight matrices .", "Each intergenic region ( in the ribosomal proteins or genome-wide , depending on the question ) was scored using the script TFBS_score . py by adding up the log likelihood values of each base at each position in the motif , forward and backward , and then repeating for each position in the intergenic region .", "It is important to note that the motifs from ScerTF are corrected for the GC-content of the S . cerevisiae genome ( or the genome ( s ) from which the matrix is derived ) , but not individually for the genomes of each species in other parts of the tree .", "The motif was left as-is instead of correcting for the GC-content in each genome , because the purpose of this scoring was to identify DNA sequences that are most similar to the Mcm1 binding site , not those that are most statistically enriched given the GC-content .", "Calculating enrichment of the binding site in ribosomal protein genes relative to the rest of the genome was done to take into account forces ( including , but not limited to GC-content ) that affect the prevalence of the motif genome-wide .", "After determining the conclusions were unchanged when using 500 , 1000 , and 1500 bp for the maximum length of an intergenic region , the length of 1000 bp was chosen .", "A cutoff was chosen for presence of the cis-regulatory site ( Figure 1—figure supplement 1 ) .", "Ribosomal protein gene intergenic regions were also screened for motifs that were not present or were more information-rich than the corresponding motif in the ScerTF database .", "This was done by querying the intergeneic regions in each species using MEME with the zero-or-one occurrence per sequence option .", "From these results the following motifs were chosen: Cbf1 from Spathaspora passalidarum , Rrn7 from Arxula adeninivorans , Tbf1 from Ascoidea rubescens and Schizosaccharomyces japonicus , Hmo1 from Lachancea thermotolerans and a widespread but previously unidentified motif from Botrytis cinerea .", "The Dot6 , Fhl1 , Rap1 , Mcm1 , Rim101 , Sfp1 , and Stb3 motifs were obtained from ScerTF .", "To estimate the number of gains and losses , species were categorized based on whether their RPGs contained Mcm1 sites ( -log10 ( P ) > 3 ) or not .", "We used phytools to simulate 10 , 000 stochastic character maps under the equal rates model ( ER ) and a different-rates model ( ARD ) assuming Mcm1 sites were a discrete character ( Bollback , 2006; Revell , 2012 ) .", "These trees were used to determine the number of gains and losses of Mcm1 sites under each model and find which ancestral nodes were likely to have Mcm1 sites at the RPGs and which were uncertain .", "Previous studies have indicated that many features of the RPGs are defined by their relative location to the Rap1 binding site ( Reja et al . , 2015 ) .", "To identify the relative locations , the best hit for the Rap1 site in each species was identified , then a cutoff was set ( approximately half of the maximum potential score for a given position weight matrix ) for the motif of the second transcription factor .", "The location of a binding site was defined as the midpoint of the motif .", "This analysis was carried out using the scripts TFBS_score . py and rel_locs_RPs . py .", "Genome-wide scoring of relative motif locations using rel_loc_RPs . py was used to identify additional genes beyond the RPGs that show a similar pattern of Rap1 and Mcm1 sites near to each other .", "Rap1 sites that faced toward the gene and had a score greater than 6 . 0 were identified , as were Mcm1 sites with a score above 6 . 0 .", "Then , genes that had both a forward-facing Rap1 site and an Mcm1 site between 52 and 78 bp downstream ( the spacing of most sites at the RPGs ) were identified .", "The order of Rap1 and Mcm1 cis-regulatory site appearance in evolution was inferred by the distribution of the sites in closely related species with available genome sequences .", "The GFP and HIS3 reporters used in K . lactis and S . cerevisiae have been previously described ( Garbett et al . , 2007; Mencía et al . , 2002; Sorrells et al . , 2015 ) .", "These reporters allow full intergenic regions to be cloned upstream of GFP or HIS3 , with the break between the original gene sequence and the reporter gene occurring at the start codon .", "A second version of the GFP reporter uses the CYC1 promoter from S . cerevisiae ( Guarente and Ptashne , 1981 ) with its upstream activation sequence replaced by two restriction enzyme sites .", "Different versions of these vectors were made to integrate into the K . lactis genome and into the S . cerevisiae genome .", "Plasmids and strains are reported in Supplementary files 1 and 2 .", "To make the full-length RPG GFP reporters , the wild-type intergenic regions were obtained by PCR with ExTaq ( Takara ) from genomic DNA , flanked by directional restriction enzyme sites for SacI and AgeI .", "These were cloned using a 2:1 ratio into pTS16 digested with the same restriction enzyme sites , and ligated using Fastlink ligase ( Epicentre ) to make pTS170 and pTS174 .", "To scramble Mcm1 and Rap1 binding sites , these sites were put into a text scrambler , then the resulting sequences were queried in ScerTF ( Spivak and Stormo , 2012 ) to see if they contained matches to any other known transcription factor binding site motifs .", "If not , they were used for further experiments .", "To make pTS171 , pTS175 , and pTS243-246 DNA sequences were synthesized containing scrambled Mcm1 , Rap1 sites , or both .", "The insert for pTS171 and pTS243-244 were cloned into pTS170 using the restriction enzymes SacI and Bsu36i .", "The insert for pTS175 and pTS245-246 was cloned into pTS174 using SacI and EcoRV .", "The vectors pTS176-179 contain the K . lactis RPS23 Rap1-Mcm1 operator upstream of the CYC1 promoter .", "These vectors were made by annealing oligos and ligating them in a 50:1 ratio into pTS26 digested with NotI and XhoI .", "The equivalent vectors for S . cerevisiae are pTS181-184 and were made by cloning into pTS180 with NotI and XhoI .", "The constructs testing the spacing between Rap1 and Mcm1 sites ( pTS189-203 and pTS209-224 ) were cloned using the same approach .", "For pTS189-203 the intervening sequence between the sites was partially duplicated for some constructs to increase the spacing .", "For pTS209-224 the endogenous spacing was 80 bp so the entire series was made with deletions starting immediately downstream from the Rap1 site .", "The reporters testing how weak Mcm1 sites cooperate with Rap1 were cloned by first adding a BamHI site along with a palindromic Mcm1 site ( Acton et al . , 1997 ) into the reporter containing the K . lactis RPS23 Rap1-Mcm1 operator to make pTS247 and pTS248 ( with a scrambled Rap1 site ) .", "Then variants of the palindromic site containing point mutations were cloned into these two vectors using BamHI and XhoI .", "For each variant , two point mutations were made to preserve the palindromic nature of the Mcm1 binding site ( Acton et al . , 1997 ) .", "These reporters were digested with KasI and HindIII and integrated into the K . lactis genome by transformation as previously described ( Kooistra et al . , 2004 ) and into the S . cerevisiae genome using a standard lithium-acetate transformation .", "Yeast were grown on non-selective media for 24 hr then replica plated onto plates containing at least 100 μg/mL Hygromycin B . For the spacing and weak Mcm1 reporter series , the reporters are enumerated in the plasmid list but not the strain list .", "This is because they were transformed into K . lactis , tested , then discarded due to their large numbers .", "For each reporter , four independent isolates were measured , but isolates where the full reporter had not integrated were discarded , resulting in 3 or four replicates per construct .", "The HIS3 reporters were generated by performing PCR on the equivalent GFP reporters to generate wild-type , Rap1AS , and scrambled versions of the RPS23 fragment containing Rap1 and Mcm1 binding sites with NcoI and SacII restriction sites on the ends .", "These fragments were cloned in a 3:1 ratio into similarly digested UASRap1WT-HIS3 reporter plasmid ( Garbett et al . , 2007; Mencía et al . , 2002 ) .", "These reporters were digested with SpeI and SalI and integrated into the S . cerevisiae genome via lithium-acetate transformation .", "Transformants were selected on media lacking TRP1 and integration at the correct locus was confirmed via PCR .", "K . lactis and S . cerevisiae reporters were grown overnight in 1 mL cultures in 96 well plates in synthetic complete media .", "The next day , cells were diluted into synthetic complete media to OD600 = 0 . 025 – 0 . 05 and grown for 3 hr .", "Cells were measured by flow cytometry on a BD LSR II between 3 hr and 4 hr after dilution .", "A total of 10 , 000 cells per strain were recorded .", "Cells were gated to exclude debris , and the mean fluorescence for each strain was used for comparing among different strains .", "For each reporter , three to four independent isolates were checked , as we were interested in large differences and standard deviations within samples were small .", "In the case that one of the isolates anomalously showed expression equivalent to background , while the other isolates showed similar but detectable fluorescence , the anomalous isolate was excluded from analysis .", "Experiments were performed a minimum of two times on different days .", "Strains were not blinded for data collection or analysis .", "HIS3 reporter expression in S . cerevisiae was scored by growth assays performed on three independent biological replicates .", "In these assays , S . cerevisiae were grown overnight to saturation and serially diluted 1:4 in sterile water in 96-well plates .", "These dilution series were spotted using a pinning tool ( Sigma ) onto Synthetic Complete ( SC ) media ( 0 . 67% ( w/v ) yeast nitrogen base without amino acids , 2% ( w/v ) dextrose , 0 . 2% ( w/v ) amino acid dropout mixture ) either with His ( +His , non-selective media ) or without His and with 3-aminotriazole ( -His + 3 AT , selective media ) .", "Plate images were acquired using the ChemiDoc MP Imager ( Bio-Rad ) and processed using ImageLab software ( Bio-Rad ) after 2 days of growth at 30˚C .", "To construct the interspecies hybrids for allelic expression measurements , multiple isolates of different species were mated together .", "Strains with complementary markers were grown on YEPD plates for 2 days , then mixed together in patches on 5% malt extract plates for 2 – 4 days .", "These patches were then observed under the microscope to check for zygotes and streaked out onto plates that select for mating products .", "Hybrids were tested by PCR for products that were species , and mating-type specific .", "Kl .", "lactis × Kl .", "dobzhanskii matings were attempted , but were unsuccessful , perhaps because the Kl .", "dobzhanskii isolate used was an a/α strain .", "Kl .", "lactis × L . kluyveri zygotes were observed when the two species were mixed with Kl .", "lactis alpha-factor , but no mating products were obtained .", "One cross of Kl .", "lactis and Kl .", "aestuarii produced zygotes and mating products , but the Kl .", "aestuarii isolate turned out to be an isolate of Kl .", "wickerhamii instead ( discovered upon genome-sequencing ) .", "In all , three Kl .", "lactis × Kl .", "wickerhamii matings , and one Kl .", "lactis × Kl .", "marxianus mating—each with three isolates—were obtained and carried forward for analysis .", "Kl .", "lactis × Kl .", "wickerhamii hybrids are yTS347 and yTS349 ( two matings between yLB13a and yLB122 ) , and yTS353 ( a mating between yLB72 and yLB66c ) .", "The Kl .", "lactis × Kl .", "marxianus mating was yTS352 ( a mating between yLB72 and CB63 ) .", "Sample sizes were determined by the number of samples in our sequencing kit and the number of isolates we recovered from matings .", "Both mRNA and genomic DNA were sequenced from the hybrids .", "Genomic DNA of one isolate of each of the Kl .", "lactis × Kl .", "wickerhamii hybrids ( yTS347 , yTS349 and yTS353 ) was sequenced , along with all three isolates of the Kl .", "lactis × Kl .", "marxianus hybrid , and one isolate of each of the parental strains .", "Cells were grown in 5 mL cultures overnight in YEPD .", "Genomic DNA was prepared using a standard ‘smash and grab’ protocol , where cells are lysed with phenol/choloroform/isoamyl alcohol and glass beads .", "DNA was precipitated twice and treated with RNase A , then sheared on a Diagenode Bioruptor for 2 × 10 min ( 30 s on 1 min off ) on medium intensity .", "Genomic DNA was prepared for sequencing using the NEBNext Ultra DNA Prep Kit for Illumina E7370 ( New England BioLabs ) .", "All three isolates of each of the four hybrids were prepared for mRNA sequencing .", "Cells were grown overnight , then diluted back into YEPD to OD600 = 0 . 2 and grown for 4 – 8 hr until they reached OD600 = 0 . 7 – 1 . 0 .", "The growth rate of the Kl .", "lactis ×Kl .", "wickerhamii hybrids was slower and more variable between isolates .", "At this point , cells were pelleted and frozen in liquid nitrogen .", "mRNA was extracted using the RiboPure kit AM1926 ( Applied Biosystems ) .", "Polyadenylated RNAs were selected using two rounds of the Oligotex mRNA Kit 70022 ( Qiagen ) .", "The samples were then concentrated using the RNA Clean and Concentrator-5 ( Zymo Research ) .", "Libraries were prepared using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina E7420 ( New England BioLabs ) .", "mRNA and library quality were assessed using a Bioanalyzer 2100 ( Agilent ) .", "Sequencing was performed at the University of California , San Francisco Center for Advanced Technology on an Illumina HiSeq 4000 .", "Raw sequencing data was checked for quality control using FastQC ( http://www . bioinformatics . babraham . ac . uk/projects/fastqc/ ) .", "Next , each of the genomic DNA isolates was aligned separately to each of four genomes: Kl .", "lactis , Kl .", "marxianus , Kl .", "wickerhamii , and Kl .", "aestuarii .", "In each case , reads uniquely mapped to the expected genome ( s ) and few reads mapped to other genomes .", "The strain yTS349 was previously thought to be a Kl .", "lactis ×Kl .", "aestuarii hybrid , but sequencing revealed it was in fact a Kl .", "lactis ×Kl .", "wickerhamii hybrid , and was treated as such for the analyses .", "The genomes for Kl .", "lactis , Kl .", "marxianus , and Kl .", "wickerhamii were annotated using the yeast genome annotation pipeline from YGOB to standardize the gene annotation and ortholog assignment .", "Kl .", "lactis is already included in YGOB .", "These files were converted to gff format using convert_YGAP_GFF . py and genes were extracted using pull_genes . py .", "Next , hybrid genomes were created in silico by concatenating fasta sequences of the genes of each species .", "mRNA and gDNA reads were aligned to the hybrid genomes on a computer cluster using the script aln_reads . py , which calls Bowtie 2 ( Langmead and Salzberg , 2012 ) .", "Default parameters were used , which allow mismatches in Bowtie 2 .", "However , reads that mapped equally well to multiple locations in the genome were removed after alignment using discard_multimapping . py .", "Because ribosomal proteins are highly conserved , they contain stretches of more than 50 bp that are identical between the orthologs belonging to each species in the hybrid .", "Thus , this filtering step is necessary to assure reads map uniquely to the ortholog from one species or the other .", "To quantify differential allelic expression , the reads aligning to each gene were counted using ASE_server . py .", "mRNA counts of the three Kl .", "lactis × Kl .", "marxianus replicates and seven of the nine Kl .", "lactis × Kl .", "wickerhamii hybrids that were sequenced were highly similar .", "The two other isolates showed that one of the genomes in the hybrid was present at a lower level than the other suggesting it had been lost from some of the cells ( although each of the two isolates lost a different parental genome ) .", "The seven reproducible isolates were then treated as replicates for the rest of the analysis .", "Genome sequencing also revealed that there were two copies of the Kl .", "marxianus genome in each of the Kl .", "lactis × Kl .", "marxianus hybrids , suggesting that our parental Kl .", "marxianus strain was a diploid .", "The mRNA read counts for each gene in each replicate were then normalized to the total reads in the experiment .", "Second , they were divided by the gDNA read counts from each gene , thus controlling for the effect of two Kl .", "marxianus genomes in the Kl .", "lactis × Kl .", "marxianus hybrids .", "( The gDNA read counts per gene were averaged across replicates for each hybrid , so all the replicates were divided by the same gDNA count ) .", "Finally , the Kl .", "lactis ortholog read count was divided by either the Kl .", "marxianus or the Kl .", "wickerhamii ortholog to get a differential allelic expression value for each ortholog pair in each replicate .", "To calculate significance of differential allelic expression , a two-sided one-sample t-test was used on the log2 ( differential allelic expression ) , across replicates ( three replicates in the case of the Kl . lactis × Kl . marxianus hybrid and seven in the case of the Kl . lactis × Kl . wickerhamii hybrid ) .", "The significance of each gene was calculated using the Benjamini-Hochberg procedure to control the false-discovery rate at 0 . 05 .", "To test for concerted differential allelic expression in the ribosomal proteins , as well as across all gene ontology ( GO ) terms , the geometric mean of each group of genes was calculated , then tested by the hypergeometric test to see if they were enriched for genes at least 1 . 1-fold up or down in Kl .", "lactis .", "Altering this fold cutoff had little effect on the results .", "These tests were carried out using the ASE_local . py script .", "His6-Rap1 ( S . cerevisaie ) used in main text gel shift experiments was expressed using a previously generated pET28a-Rap1 expression vector in Rosetta II DE3 E . coli and purified as described ( Johnson and Weil , 2017 ) .", "In brief , after inducing expression in E . coli cells grown to an OD600 of 0 . 5 – 1 for 4 hr with 1 mM IPTG at 37°C , cell pellets from 500 mL of culture were resuspended in 20 ml of Rap1 Lysis/Wash buffer ( 25 mM HEPES-NaOH ( pH 7 . 6 ) , 10% v/v glycerol , 300 mM NaCl , 0 . 01% v/v Nonidet P-40 , 1 mM Benzamidine , 0 . 2 mM PMSF ) and lysed via treatment with 1 mg/mL lysozyme and sonication .", "Lysate cleared via centrifugation was incubated with 2 . 5 mL Ni-NTA agarose ( Qiagen ) equilibrated with Rap1 Lysis/Wash buffer for 3 hr at 4°C to allow for His6-Rap1 protein binding .", "Following 3 washes with Rap1 Lysis/Wash buffer , Ni-NTA agarose-bound proteins were transferred to a disposable column and eluted using Rap1 Lysis/Wash buffer containing 200 mM Imidazole .", "To generate S . cerevisiae Mcm1 for the gel shift and Far Western protein-protein binding assays presented in the main text , S . cerevisiae MCM1 was cloned into a vector that would allow its expression and purification with an N-terminal MBP tag and PreScission protease cleavage site .", "Specifically , S . cerevisiae MCM1 was amplified from S . cerevisiae genomic DNA and cloned into a p425 GAL1 expression vector ( Mumberg et al . , 1994 ) containing an MBP-3C tag ( Feigerle and Weil , 2016 ) using the SpeI and XhoI restriction enzymes .", "MBP-3C-Mcm1 vector was expressed in yeast grown to an OD600 of ~3 in 1% w/v raffinose via induction with 2% w/v galactose for 3 hr at 30˚C .", "Yeast cell pellets obtained from 1L of culture were resuspended in 4 mL Mcm1 Lysis Buffer ( 20 mM HEPES-KOH ( pH 7 . 6 ) , 500 mM potassium acetate , 10% v/v glycerol , 0 . 5% v/v Nonidet P-40 , 1 mM DTT +1X protease inhibitors ( 0 . 1 mM PMSF , 1 mM Benzamidine HCl , 2 . 5 μg/mL aprotinin , 2 . 5 μg/mL leupeptin , 1 μg/mL pepstatin A ) ) .", "Cells were lysed via glass bead lysis .", "Soluble cell extract was obtained via centrifugation and mixed with 2 mL DE-52 resin pre-equilibrated with Mcm1 Lysis Buffer for 5 min at 4˚C .", "Flowthrough from the DE-52 purification was collected and diluted with 20 mM HEPES-KOH , 10% v/v glycerol , and protease inhibitors to reduce the concentration of potassium acetate and Nonidet P-40 to 200 mM and 0 . 2% v/v , respectively .", "Binding to 600 μl amylose resin was performed in batch for 2 hr at 4˚C .", "Amylose resin-bound proteins were transferred to a disposable column , washed with 10 column volumes of Mcm1 Wash Buffer ( 20 mM HEPES-KOH , 200 mM potassium acetate , 10% v/v glycerol , and protease inhibitors ) and eluted with 10 column buffers of Mcm1 Wash Buffer containing 10 mM maltose .", "For gel shift reactions , the N-terminal MBP tag was removed by incubating multiple 50 μl reactions each containing 12 pmol MBP-Mcm1 and 48 pmol lab-generated 3C protease for 30 min at 4˚C .", "Taf1-TAP TFIID used for Far Western protein-protein binding analyses was purified via a modified tandem affinity protocol as previously described ( Feigerle and Weil , 2016 ) .", "GST- , His6-Taf3 , His6-Taf4 and GST-Taf4 were expressed in E . coli and purified via chromatographic methods that varied upon each protein ( Layer et al . , 2010 ) .", "To generate material for the gel shift experiments in the supplement , the full-length Rap1-His6 protein from Kl .", "lactis was purified from E . coli .", "Rap1 was amplified from genomic DNA and cloned into the pLIC-H3 expression vector using XmaI and XhoI to make pTS207 .", "This protein was purified using a His6 tag as previously described ( Lohse et al . , 2013 ) .", "The full-length Mcm1-HA protein from Kl .", "lactis and S . cerevisiae was purified from S . cerevisiae .", "The genes were amplified from genomic DNA and cloned into p426 Gal1P-MCS ( ATCC 87331 ) using BamHI and HindIII to make pTS226 ( S . cerevisiae Mcm1 ) and pTS227 ( Kl . lactis Mcm1 ) .", "These plasmids were transformed into S . cerevisiae W303 for expression .", "Cells were grown 12 hr in SC –Leu at 30 ˚C , then in YPGL ( 1% yeast extract , 2% peptone , 3% glycerol , 2% lactate ) for 10 – 12 hr .", "Then cells were diluted into 1L YPGL to OD600 = 0 . 3 and grown for 4 – 5 hr until OD600 = 0 . 6 .", "Cells were induced with 2% galactose ( added from a 40% galactose stock ) for 1 hr .", "Then cells were pelleted , resuspended in an equal volume of lysis buffer , and pipetted into liquid nitrogen to make pellets .", "Cells were lysed in a Cryomill ( Retsch ) 6 times for 3 min at 30 Hz , refreezing in liquid nitrogen between cycles .", "Cell powder was thawed on ice and diluted to 4 mL/g frozen pellet with lysis buffer ( 100 mM Tris pH 8 . 0 , 1 mM EDTA , 10 mM fresh β-mercaptoethanol , 10% glycerol , 200 mM NaCl ) .", "Lysate was resuspended by pipetting , then cleared by centrifugation for 2 hr at 200 , 000 x g .", "Protein was bound to 250 μl of HA-7-agarose ( Sigma ) slurry per liter yeast culture for 1 hr at 4 ˚C .", "Lysate was applied to a Polyprep mini disposable gravity column ( Biorad ) and washed 4 times with 10 mL lysis buffer .", "The protein was eluted 4 times with one bed volume of 1 mg/mL HA peptide in lysis buffer after incubating 30 min on a tilt board at room temperature .", "The protein was stored in aliquots at −80 ˚C .", "Extract for gel shifts was prepared as previously described ( Baker et al . , 2011 ) .", "25 mL cultures of cells were grown to OD = 0 . 8 – 1 . 0 , and frozen at −80 ˚C .", "Cells were lysed in 300 μl extract gel shift buffer ( 100 mM Tris pH 8 . 0 , 200 mM NaCl , 1 mM EDTA , 10 mM MgCl2 , 10 mM β-mercaptoethanol , 20% glycerol , EDTA-free protease inhibitor cocktail ( Roche ) ) with 200 μl glass beads on a vortexer for 30 min .", "Lysate was cleared by centrifugation at 18 , 000 x g for 20 min and diluted for experiments .", "For the main text experiments , purified S . cerevisiae His6-Rap1 and Mcm1 were incubated either individually or in combination in increasing amounts as indicated in the figure legend .", "All binding reactions were performed using 10 fmol ( 7000 cpm ) of a 79 bp 32P-labeled fragment of the Kl .", "lactis RPS23 promoter containing the Rap1 and Mcm1 binding sites generated via PCR , EcoRI restriction enzyme digestion , and native PAGE purification .", "Binding reactions were performed in binding buffer ( 20 mM HEPES-KOH ( pH 7 . 6 ) , 10% v/v glycerol , 100 mM KCl , 0 . 1 mM EDTA , 1 mM DTT , 25 μg/μl BSA , 25 μg/μl Poly ( dG-dC ) ( double-stranded , alternating copolymer ) in a final volume of 20 μl .", "For competition reactions , binding was performed in the presence of 100-fold molar excess of cold Rap1 WT ( RWT ) or Rap1 scrambled ( Rsc ) sequences and/or the Mcm1 WT ( MWT ) or Mcm1 scrambled ( Msc ) .", "Reactions were allowed to proceed for 20 min at room temperature before loading onto 0 . 5X TBE-buffered ( 44 . 5 mM Tris , 44 . 5 mM Boric acid , 1 mM EDTA ( pH 8 . 0 ) ) 5% polyacrylamide gels and electrophoresed for 1 hr at 200V at room temperature .", "Gels were vacuum dried and 32P-DNA signals detected via K-screen imaging using a BioRad Pharos FX imager .", "Gel shift experiments in the supplemental material were performed as previously described ( Lohse et al . , 2010 ) .", "Binding reactions were carried out in 20 mM Tris pH 8 . 0 50 mM potassium acetate , 5% glycerol , 5 mM MgCl2 , 1 mM DTT , 0 . 5 μg/μl BSA , 25 μg/μl poly ( dI-dC ) ( Sigma ) .", "Purified proteins tested for direct interaction with Mcm1 in Far western protein-protein binding assays were separated on parallel 4 – 12% NuPAGE Bis-Tris polyacrylamide gels ( Life Technologies ) .", "0 . 5 pmol Taf1-TAP TFIID , 1 pmol His6-Taf3 , and 1 pmol His6-Taf4 were used in the assay to test for direct Mcm1 interaction with TFIID subunits .", "In the assay used to identify the Taf4 Mcm1 binding domain , ~0 . 4 pmol of each Taf4 form , 0 . 8 pmol His6-Taf3 , and 0 . 8 pmol GST were used .", "For each experiment , one gel was stained using Sypro Ruby protein stain ( Invitrogen ) to monitor protein integrity and amount .", "The other gel ( s ) were transferred to PVDF membranes pre-equilibrated in transfer buffer .", "Following transfer , PVDF membranes were incubated with renaturation buffer ( 20 mM HEPES-KOH pH 7 . 6 , 75 mM KCl , 25 mM MgCl2 , 0 . 25 mM EDTA , 0 . 05 v/v% Triton X-100 and 1 mM DTT freshly added ) for 90 min at 4˚C on a tiltboard .", "The PVDF membrane was then blocked using 5% non-fat milk in renaturation buffer for 30 min at room temperature on a tiltboard .", "The overlays were performed overnight with 10 nM MBP or 10 nM MBP-Mcm1 with 1% BSA ( Roche ) as a nonspecific competitor in renaturation buffer .", "Bound MBP- or MBP-Mcm1 was detected using a standard immunoblotting protocol ( primary antibody MBP ( NEB Catalog#E8032 ) , used at a dilution of 1:5 , 000 , secondary antibody horse anti-mouse IgG , HRP-linked ( Cell Signalling Catalog#7076 ) , used at a dilution of 1:5 , 000 ) .", "Detection of bound proteins was achieved via incubation with ECL ( GE ) and X-ray film .", "Interspecies hybrid expression data is available at the Gene Expression Omnibus ( GEO ) repository under accession number GSE108389 .", "Flow cytometry data is available at Flow Repository under accession numbers FR-FCM-ZYWS , FR-FCM-ZYWT , FR-FCM-ZYWU , FR-FCM-ZYWV , FR-FCM-ZYJZ , FR-FCM-ZYJY , and FR-FCM-ZYJ2 .", "Code used in computational analyses is available at doi . org/10 . 5281/zenodo . 1341284 ." ] ]

[ "Convergent evolutionary events in independent lineages provide an opportunity to understand why evolution favors certain outcomes over others .", "We studied such a case where a large set of genes—those coding for the ribosomal proteins—gained cis-regulatory sequences for a particular transcription regulator ( Mcm1 ) in independent fungal lineages .", "We present evidence that these gains occurred because Mcm1 shares a mechanism of transcriptional activation with an ancestral regulator of the ribosomal protein genes , Rap1 .", "Specifically , we show that Mcm1 and Rap1 have the inherent ability to cooperatively activate transcription through contacts with the general transcription factor TFIID .", "Because the two regulatory proteins share a common interaction partner , the presence of one ancestral cis-regulatory sequence can ‘channel’ random mutations into functional sites for the second regulator .", "At a genomic scale , this type of intrinsic cooperativity can account for a pattern of parallel evolution involving the fixation of hundreds of substitutions ." ]

[ "Sometimes evolution repeats itself .", "For example , independent butterfly species can evolve the same warning pattern to ward off predators .", "In many cases , the reason that a certain trait crops up again and again in parallel evolution is unknown .", "One example is from the evolution of fungi , where a particular DNA sequence appeared several times independently in a large range of genes in different fungus species .", "This DNA sequence binds to a protein called Mcm1 , which regulates nearby genes .", "Exactly why this DNA sequence has evolved in parallel so often in fungi has not been clear until now .", "Researchers want to find out what is so special about this DNA binding sequence for Mcm1 , as there are many other proteins that could do the same job .", "Now , Sorrells et al . investigated this further by testing whether a binding site for another protein Rap1 often found close by had a role to play .", "Experiments using 162 different fungus species showed that Mcm1 binding sites had evolved 12 times in parallel .", "Rap1 and Mcm1 did indeed turn out to work together to regulate nearby genes .", "The two proteins interact with a large protein complex critical for activating genes .", "As a result , Mcm1 binding sites are more likely to evolve and play a role in gene regulation in different species when they are located near Rap1 binding sites .", "This could explain why this particular DNA sequence has evolved in parallel so many times .", "The same principle may apply to other genetic sequences involved in parallel evolution .", "With this understanding , it could be possible to predict when and where this event might occur in the future in fungi .", "This could be particularly useful for working towards being able to predict and anticipate the evolution of drug-resistant fungal pathogens ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "biochemistry and chemical biology" ]

[ [ "Fatty acids play diverse cellular roles , including providing the hydrophobic tails of membrane phospholipids , energy storage in the form of triglycerides , and as cell signaling molecules .", "Aside from the import of exogenous fatty acids , mammalian cells use the well-known and well-studied cytoplasmic enzyme fatty acid synthase ( FASN ) to make palmitate , which is further modified to form the diverse array of cellular fatty acids ( Smith , 1994 ) .", "However , it is much less appreciated that mitochondria also harbor a spatially and genetically distinct fatty acid synthesis pathway ( mtFAS ) ( reviewed in Nowinski et al . , 2018 ) .", "In contrast to FASN , which is a very large protein that contains several domains and encompasses all of the enzymatic activities necessary for FAS condensed in a single polypeptide chain , the mtFAS pathway is comprised of at least six enzymes all encoded by separate genes .", "These enzymes catalyze sequential steps to achieve one cycle of two-carbon addition to a growing acyl chain ( Figure 1A ) .", "The nascent fatty acids are covalently attached to the mitochondrial acyl carrier protein ( ACP ) , which acts as a soluble scaffold upon which the acyl chains are built .", "In each cycle of the pathway , malonyl-CoA is converted to malonyl-ACP , which then undergoes a condensation reaction with the growing fatty acyl chain on ACP , extending the chain by two carbons and releasing a CO2 molecule .", "Thereafter , the remaining enzymes in the pathway must carry out a series of reduction and dehydration reactions to fully reduce the acyl chain to a saturated fatty acid , which is the substrate for further cycles of two-carbon addition .", "Among the several differences between mtFAS and cytoplasmic FAS , FASN exclusively produces palmitate , whereas mtFAS appears to have at least two major products .", "Although the human genes responsible for each step in the mtFAS pathway have been identified , and their ability to complement the orthologous mutants in yeast has been demonstrated ( Autio et al . , 2008; Chen et al . , 2009; Joshi et al . , 2003; Miinalainen et al . , 2003; Zhang et al . , 2005; Zhang et al . , 2003 ) , few loss-of-function studies have examined the consequences of mtFAS deficiency in mammalian systems .", "Inducible knockout of the mitochondrial malonyl CoA-acyl carrier protein transacylase ( Mcat ) using a Cre driver that expresses in most tissues in mice results in a severe phenotype characterized by weight loss , reduced muscle strength , and shortened lifespan despite the persistence of residual MCAT protein ( Smith et al . , 2012 ) .", "Knockout of the mitochondrial 2-enoyl thioester reductase , Mecr , is lethal in mice due to a placental defect ( Nair et al . , 2017 ) , while the inducible knockout of Mecr specifically in Purkinje cells leads to loss of this cell population and recapitulates many phenotypes of MePaN syndrome , the human disease caused by Mecr mutation ( Gorukmez et al . , 2019; Heimer et al . , 2016; Nair et al . , 2018 ) .", "Similarly , Mecr knockdown has been shown to inhibit the growth of hepatocellular carcinoma cells ( Cai et al . , 2019 ) .", "While these detrimental phenotypes are clear , the molecular consequences of mtFAS loss remain poorly understood .", "MtFAS currently has one known product: an eight-carbon saturated fatty acid , octanoate , that is subsequently converted to lipoic acid .", "This important cofactor is required for the catalytic activity of a number of mitochondrial enzymes , most notably pyruvate dehydrogenase and α-ketoglutarate dehydrogenase ( Brody et al . , 1997; Wada et al . , 1997 ) , but also branched chain amino acid dehydrogenase , the H protein of the glycine cleavage system , and 2-oxoadipate dehydrogenase ( reviewed in Solmonson and DeBerardinis , 2018 ) .", "This observation , made more than 20 years ago , has guided mtFAS-focused research for the past two decades .", "Studies have shown that loss of lipoic acid synthesis and/or failure to efficiently transfer lipoic acid to its target proteins is lethal in mice ( Ni et al . , 2019; Yi and Maeda , 2005 ) , and attributed other mitochondrial changes as downstream of lipoic acid synthesis ( Smith et al . , 2012 ) .", "However , though the mouse studies described above have demonstrated striking phenotypes that result from loss of mtFAS , they fail to distinguish between direct effects of loss of protein lipoylation versus loss of other mtFAS function ( s ) .", "Although lipoic acid unmistakably has important central functions in mitochondrial metabolism , there is reason to be skeptical that the sole function of mtFAS is the production of lipoic acid .", "In addition to the eight-carbon precursor for lipoic acid , it is clear that mtFAS also produces longer acyl chains of at least 14 carbons , yet the identity of these lipids and their cellular functions are uncertain ( Angerer et al . , 2017; Witkowski et al . , 2007 ) .", "Perhaps more surprisingly , these longer acyl chains appear to partially be maintained in attachment to ACP ( Angerer et al . , 2017 ) , further complicating the question of their cellular functions .", "We set out to systematically examine the cellular functions of the mtFAS pathway by mutating genes that encode three distinct steps in the pathway , Mcat , Oxsm , and Mecr .", "For comparison , we also engineered cells with loss of Lipt1 , the terminal enzyme in the production of lipoylated enzymes downstream of mtFAS .", "We found that several phenotypes resulting from loss of the mtFAS pathway are not related to lipoic acid , but must instead be due to loss of other products or functions of mtFAS .", "In particular , we find that loss of mtFAS , but not lipoic acid synthesis , leads to a profound impairment in the assembly of the mitochondrial electron transport chain machinery , with the attendant consequences on metabolism and cell behaviors .", "Our data therefore suggest that the mtFAS pathway acts as a key regulator of mitochondrial respiratory metabolism ." ], [ "The enzymes of mtFAS are ubiquitously expressed in mammalian tissues , with the highest expression in skeletal muscle and heart ( Triepels et al . , 1999 ) .", "Therefore , we chose cultured skeletal myoblasts as a model system in which to examine the role of this pathway .", "We employed a CRISPR/Cas9-based strategy to mutate three genes encoding enzymes in the mtFAS pathway , using two different guide RNA sequences per gene .", "We targeted Mcat , encoding the malonyl-CoA ACP transacylase , Oxsm , encoding the beta-ketoacyl synthase that condenses malonyl-ACP with the growing acyl chain , and Mecr , encoding the terminal reductase in each cycle of two-carbon unit addition ( Figure 1A ) .", "It is important to note that because mtFAS is a cycle that requires all enzymes for each stepwise two-carbon addition , loss of each individual enzyme blocks fatty acid synthesis at an early , albeit distinct step .", "Mcat mutants should be unable to attach any carbon to ACP , whereas Oxsm mutants should be able to produce malonyl-ACP , and Mecr mutants could at most build short four-carbon acyl chains .", "We observed roughly 70% editing efficiency for all six of the guides utilized ( based on T7E1 assays , Figure 1—figure supplement 1A ) .", "We found that transfection with Cas9 and sgRNAs targeting mtFAS genes led to smaller colonies at one-week post-single cell sorting relative to control guides ( Figure 1—figure supplement 1B ) .", "Many of the smallest clones stopped growing and/or did not survive expansion .", "After screening more than 100 of the surviving single cell clones , we failed to identify a single null mutant for any of the three genes targeted .", "This finding was not altogether unexpected , given that at least one other knockdown study concluded that NDUFAB1 , the mammalian mitochondrial ACP , is essential in HEK293T cells ( Feng et al . , 2009 ) .", "Indeed , The Broad Institute’s Depmap lists NDUFAB1 as a ‘common essential’ gene , and the mtFAS genes all display negative gene effects , indicating they are essential in at least some cell lines ( Broad , 2020 ) .", "Taken together , our results strongly support the conclusion that the mtFAS pathway is essential in C2C12 skeletal myoblasts .", "Although no complete null clones were generated , we were able to isolate several clonal cell lines with markedly decreased abundance of MCAT , OXSM , and MECR .", "These clones also had no detectable lipoylation of PDH and OGDH subunits ( DLAT and DLST , respectively ) in isolated mitochondrial fractions ( Figure 1B ) , and grew slowly relative to control clones ( Figure 1—figure supplement 1C ) .", "Growth of mtFAS mutant cells in both glucose and galactose , the latter of which requires mitochondrial respiration , was normalized by re-expression of the cognate mtFAS gene ( Figure 1C , Figure 1—figure supplement 1D and E ) .", "MECR has been reported to have dual localization to the cytoplasm in addition to mitochondria ( Kim et al . , 2014 ) , so we performed sub-cellular fractionation to assess these two MECR populations in our mutant cells .", "We observed an overall decrease in MECR expression that was most pronounced in the mitochondrial compartment ( Figure 1—figure supplement 1F ) .", "We thus moved forward with the characterization of these presumably hypomorphic cell lines .", "As mentioned previously , the canonical function of mtFAS is the production of octanoate , the eight-carbon precursor for lipoic acid synthesis .", "However , some have speculated that mtFAS might also generate fatty acids that contribute to phospholipid synthesis .", "Seminal studies found 3-hydroxymyristate as the predominant acyl modification on mitochondrial ACP in fungi ( Mikolajczyk and Brody , 1990; Schneider et al . , 1997 ) .", "A more recent effort to identify the predominant lipid species found a variety of medium and long chain acyl species , raising the possibility of multiple products in addition to octanoate ( Angerer et al . , 2017 ) .", "Although mtFAS mutant yeast display changes in steady-state lipids , the interpretation of this observation is confounded by other phenotypes of these cells ( Schneider et al . , 1995 ) .", "In the only study that examines the role of mtFAS in lipid synthesis in mammals , transient knockdown of ACP did not change the abundance of mitochondrial lipids; however , this does not exclude the possibility that cytoplasmic FAS can compensate for loss of mtFAS ( Clay et al . , 2016 ) .", "We therefore set out to test whether mtFAS contributes to cellular phospholipid pools in the presence and absence of the cytosolic fatty acid synthase , FASN .", "In wild type and Oxsm mutant cell lines , we stably expressed hairpins targeting FASN , which encodes the cytoplasmic fatty acid synthase , or a scrambled control ( Figure 1D ) .", "We observed no effect of FASN knockdown on protein lipoylation ( Figure 1D ) .", "We then fed the cells uniformly labeled 13C-glucose and observed labeling in cellular lipids to define the relative contributions of cytoplasmic FAS and mtFAS to various cellular lipid pools .", "By monitoring accumulation of the m+two isotopologue over time , we observed a dependence on FASN expression , but not mtFAS for the synthesis of new phospholipids ( shown are two representative PC species , Figure 1E and F ) .", "Higher order isotopologues from additional cycles of elongation by fatty acid synthesis ( e . g . m + 4 , m + 6 , m + 8 , and so on ) did not show labeling above background in this experiment .", "Similarly , fatty acid methyl esther analysis ( FAMES ) showed an effect of FASN knockdown , but not Oxsm mutation , on incorporation of U13C-glucose into c16:0 and c18:0 lipids , which confirmed that the m+2 labeling of phospholipids is likely in the fatty acid tails ( Figure 1—figure supplement 1G ) .", "These experiments demonstrate that longer acyl chains synthesized by mtFAS do not contribute to cellular fatty acid and phospholipid pools and likely perform some other function .", "Given the known and hypothesized functions of mtFAS , we next assayed mitochondrial function in the mtFAS mutant cells .", "We performed a standard Seahorse mitochondrial stress test in high glucose medium and found that the mtFAS mutant cells displayed a roughly 30–40% decrease in basal respiration rate compared with controls ( Figure 2A ) .", "More strikingly , the mtFAS mutants exhibited a lack of spare respiratory capacity , with FCCP-stimulated uncoupled respiration rates that were similar to the initially measured basal respiration ( Figure 2A ) .", "Interestingly , Oxsm and Mecr mutants exhibited an increased basal extracellular acidification rate , suggesting that these cell lines compensate for decreased respiration via increased glycolysis , whereas Mcat mutants do not ( Figure 2—figure supplement 1A ) .", "In agreement with the observed decrease in cellular respiration , mtFAS mutants also show a markedly decreased mitochondrial membrane potential , as measured by the ratio of MitoTracker Red to MitoTracker Green fluorescence ( Figure 2B , C and Figure 2—figure supplement 1B ) .", "Concurrent decreases in mitochondrial membrane potential and respiration can have several causes ranging from altered substrate utilization and TCA cycle activity to decreased expression and/or activity of electron transport chain ( ETC ) complexes .", "Therefore , we examined OXPHOS complex expression and assembly via blue-native PAGE analysis .", "Strikingly , fully assembled ETC complex I ( CI ) , complex II ( CII ) , and complex IV ( CIV ) were almost completely absent in mtFAS mutant mitochondria ( Figure 2D ) .", "Complex V was also decreased in abundance , albeit to a lesser magnitude than that seen for CI , CII , and CIV .", "In contrast , complex III ( CIII ) was relatively unaffected by loss of mtFAS , although CIII-containing supercomplexes ( SC ) were absent , likely resulting from loss of CI and CIV .", "Furthermore , while CIII abundance and assembly were relatively unaffected , its activity was reduced in each of the mtFAS-deficient cells ( Figure 2—figure supplement 1C ) .", "Altogether , these results depict a severe respiratory deficiency in mtFAS mutant cells , characterized by the striking loss of stably assembled OXPHOS complexes in mtFAS mutant mitochondria .", "Notably , this contrasts sharply with FASN , deletion of which does not affect fatty acid oxidation in muscle ( Funai et al . , 2013 ) .", "To further examine the mechanisms underlying loss of ETC complexes in mtFAS mutant cells , we performed an unbiased quantitative proteomics experiment on duplicate whole cell lysates from three mtFAS mutant clones ( one Oxsm and two Mecr mutants ) and two control clones .", "Overall , there was no general trend for the steady-state abundance of mitochondrial proteins , with 77 proteins being statistically more abundant and 34 proteins being decreased in abundance ( Figure 3A ) .", "We also observed that the majority of the proteins that comprise OXPHOS complexes were likewise similar in abundance between control and mtFAS mutant cells , including those in Complexes I , II and IV ( Figure 3A , B ) , despite the decrease in abundance of the completely assembled complexes by blue-native PAGE ( Figure 2D ) .", "However , a subset of eight OXPHOS proteins displayed significantly decreased abundance in both Oxsm and Mecr mutant cell lines compared to controls , including NDUFA6 , NDUFA12 , NDUFS4 , NDUFS6 , SDHB , COX5a , COX5b , and ATPIF1 ( Figure 3A , B ) .", "To test whether these changes were transcriptional , we performed RNAseq analysis on control and mtFAS mutant cells .", "However , we found no significant changes in the abundance of transcripts encoding ETC component proteins ( Figure 3C and Figure 3—figure supplement 1A–C ) , implying that the observed decrease in abundance of these proteins is likely due to post-translational regulation .", "The two proteins that were most decreased in abundance , NDUFA6 and SDHB ( Figure 3D , E ) , also stood out because of their relationships with the leucine-tyrosine-arginine motif ( LYRM ) protein family , a family of small proteins chiefly comprised of late-stage ETC assembly factors ( Angerer , 2015 ) .", "In many cases , such as for Complex II , LYRM proteins ( SDHAF1 and SDHAF3 ) bind to and facilitate the insertion of a target protein ( in this case SDHB ) in the final stage of ETC complex assembly ( Ghezzi et al . , 2009; Na et al . , 2014 ) .", "In other cases , such as for Complex I , the LYRM proteins NDUFA6 and NDUFB9 are actually stable subunits of the fully assembled complex ( Fiedorczuk et al . , 2016; Zhu et al . , 2016 ) .", "LYRM proteins are also found as a stable component of the NFS-containing iron-sulfur cluster biogenesis ( ISC ) complex ( LYRM4 ) as well as in an assembly intermediate of the mitochondrial ribosome ( AltMid51 ) ( Boniecki et al . , 2017; Brown et al . , 2017 ) .", "High-throughput interactomics studies in mammalian systems and targeted experiments in yeast have identified physical interactions between ACP and several LYRM proteins ( Floyd et al . , 2016; Huttlin et al . , 2015; Majmudar et al . , 2019; Van Vranken et al . , 2018 ) .", "Interestingly , the ACP protein found in complex with LYRM proteins appears often to have maintained its acyl modification , which is generated through the sequential actions of the mtFAS enzymes as described above ( Angerer et al . , 2014; Cory et al . , 2017; Runswick et al . , 1991; Van Vranken et al . , 2016 ) .", "Studies have shown that the acyl chain on ACP is intimately involved in these physical interactions , folding into the middle of the LYRM proteins , but the role of ACP acylation in its varied functions is mostly untested ( Angerer et al . , 2017; Boniecki et al . , 2017; Cory et al . , 2017; Fiedorczuk et al . , 2016; Zhu et al . , 2016 ) .", "In addition to the structurally verified ACP-LYRM interactions , three other LYRM family members with known target proteins have been suggested to interact with ACP based on high-throughput interactomics studies: SDHAF1 and SDHAF3 , discussed above , and LYRM7 , which mediates the addition of UQCRFS1 in the last step of complex III assembly ( Floyd et al . , 2016; Huttlin et al . , 2015; Na et al . , 2014; Sánchez et al . , 2013 ) .", "Thus , we hypothesized that the decreased abundance of SDHB ( Figure 3E ) could be explained by interaction of ACP with SDHAF1 and/or SDHAF3 .", "To verify whether ACP in fact interacts with these additional LYRM family members , we expressed epitope-tagged variants of ACP and each LYRM in cells and confirmed their physical interaction via co-immunoprecipitation and immunoblotting ( Figure 3—figure supplement 1D ) .", "Along with other published data ( Van Vranken et al . , 2018 ) , the finding that NDUFA6 , a known LYRM protein , and SDHB , the target of two known LYRM proteins , exhibit decreased abundance in mtFAS mutant cell lines supports the hypothesis that interaction with an acylated ACP is required for the complex assembly functions of these LYRM proteins .", "This finding prompted us to examine the abundance of other LYRM proteins and their targets in the mtFAS mutant cell lines .", "Indeed , LYRM4 and its target NFS1 were also significantly decreased in abundance in our whole cell proteomics dataset , which was confirmed by western blot , implying that LYRM4 also requires acylated ACP for the stability and function of itself and NFS1 ( Figure 3F–H ) .", "Interestingly , NDUFB9 , the other CI LYRM , and UQCRFS1 , a subunit of CIII and the target of LYRM7 , were not significantly decreased in abundance in mtFAS mutant cells ( Figure 3—figure supplement 1E , F ) , implying that either the interactions between ACP and these LYRM proteins is not dependent on acylation and mtFAS , or that other compensatory mechanisms are at play .", "We performed immunoblot analysis of UQCRFS1 and found that it was also decreased in abundance similarly to other LYR targets , perhaps explaining the observed decrease in CIII activity despite the partial maintenance of complex assembly ( Figure 3H ) .", "Finally , ACP has also been found to interact with an assembly intermediate of the mitochondrial ribosome through a novel LYRM protein called altMiD51 and its target , MALSU1 ( Brown et al . , 2017 ) .", "Brown et al . proposed that this interaction might negatively regulate mitochondrial translation because MALSU1 , altMiD51 , and ACP bind the large mitochondrial ribosomal subunit in a way that precludes small subunit binding , but the role of ACP acylation in this interaction is unclear ( Brown et al . , 2017; Dibley et al . , 2020; Rathore et al . , 2018 ) .", "Despite this proposed role of ACP in mitochondrial translation , we found that the abundance of mitochondrially encoded proteins was unchanged in the mtFAS mutant cell lines ( Figure 3—figure supplement 1G ) .", "Importantly , this includes MTCO1 and MTCO2 , two subunits of CIV , strongly implying that the diminution of CIV in mutant cells does not result from loss of mitochondrial translation .", "Interestingly , however , MALSU1 was significantly decreased in abundance ( Figure 3—figure supplement 1H ) , which raises the possibility that MALSU1 downregulation might be a compensatory adaptation that the cells make to avoid collapse of mitochondrial translation upon loss of mtFAS .", "The profound defects observed in mitochondrial respiration led us to examine mitochondrial and cellular metabolism more comprehensively in our mtFAS mutants .", "We first performed steady-state metabolomics analysis on control and mtFAS mutant cells .", "Purines were among the most significantly depleted metabolites in mtFAS mutants , but we observed no change in pyrimidines ( Figure 4—figure supplement 1A , B , all measured metabolites in Supplementary file 1 ) .", "Purine depletion may be a result of decreased activity of another lipoylated protein – the glycine cleavage H protein , which plays a role in purine biosynthesis ( Fujiwara et al . , 1991 ) .", "mtFAS mutants exhibited robust pyruvate accumulation , in agreement with the decreased lipoylation and reduced activity of PDH , but only a minor and statistically insignificant increase in intracellular lactate ( Figure 4A and Figure 4—figure supplement 1C ) .", "Similarly , no significant changes were observed in the abundance of glutamine or glutamate ( Figure 4—figure supplement 1D , E ) .", "In contrast , the TCA cycle intermediates citrate , fumarate , and malate all showed significantly decreased abundance ( Figure 4B–D ) .", "Aspartate , the synthesis of which requires the TCA cycle metabolite oxaloacetate and an electron acceptor such as the ETC ( Birsoy et al . , 2015; Sullivan et al . , 2015 ) , was strongly depleted in the mtFAS mutant cells ( Figure 4E ) .", "To further understand how TCA cycle metabolism is affected by impairment of mtFAS , we fed the cells uniformly labelled 13C-glutamine for 24 hr and assessed labeling of TCA cycle intermediates to monitor glutamine contribution to TCA cycle flux ( schematic in Figure 4F ) .", "This experiment led to three observations that were particularly interesting .", "First , glutamate cycling in the TCA cycle is decreased in the mtFAS mutant cells .", "We observed no differences in glutamine uptake in mutant cells compared to controls ( Figure 4—figure supplement 1F ) .", "However , in pre-steady state measurements we observed reduced labeling of glutamate in mtFAS mutants ( Figure 4—figure supplement 1G ) .", "Furthermore , after 24 hr of labeling , the mtFAS-deficient cells showed reduced fractional enrichments of the glutamate m+three and m+one isotopologues , which are synthesized on subsequent turns of the TCA cycle ( Figure 4G ) .", "In contrast , the m+five isotopologue contributes a larger fraction of the total pool ( Figure 4G ) .", "These data are consistent with decreased processivity of the TCA cycle in cells lacking mtFAS .", "Secondly , we observed that labeling of TCA cycle intermediates shows a clear distinction between succinate and later metabolites .", "Like glutamate , succinate labeling is initially slow ( Figure 4—figure supplement 1H ) .", "After 24 hr in 13C-glutamine , however , similar to what we observe for glutamate labeling , in control cells subsequent rounds of the TCA cycle cause m+two succinate to accumulate .", "In mtFAS mutant cells , m+two succinate labeling is reduced while m+four accumulates , indicating its decreased cycling in the TCA cycle ( Figure 4H ) .", "In contrast , m+4 labeling of fumarate , malate , and citrate are all markedly lower at short time points ( Figure 4—figure supplement 1I–K ) , and remain low after 24 hr ( Figure 4I–K ) , along with m+four aspartate , which is derived from m+four citrate ( Figure 4L ) .", "These data and the reduced abundances of fumarate and malate support a block of the TCA cycle at the succinate dehydrogenase ( SDH ) catalyzed step , and is in agreement with our data that demonstrate decreased steady state SDHB stability and a near-complete loss of fully assembled Complex II ( Figures 2D and 3H ) .", "Finally , the third observation we made from these data is that mtFAS mutants displayed significantly increased levels of m+five citrate ( Figure 4K ) , which is formed from the reductive carboxylation of α-ketoglutarate , and is the canonical marker of reductive carboxylation .", "Accordingly , we also saw significant increases in m+three fumarate , malate , and aspartate ( Figure 4I , J and L ) , which are formed from this pathway downstream of m+five citrate ( Mullen et al . , 2011 ) .", "Previous analysis of the reductive pathway concluded , as we observed here , that cells with ETC defects continue to produce succinate by the oxidative pathway , perhaps to generate reducing equivalents to drive reductive carboxylation ( Mullen et al . , 2014 ) .", "In summary , these data support the conclusion that oxidative cycling of TCA cycle intermediates is decreased overall , with a particular block at the SDH-catalyzed step .", "Furthermore , mtFAS mutants invoke reductive carboxylation to produce citrate , and reductive labeling of downstream metabolites is also increased .", "Reductive carboxylation is often seen in models of ETC inhibition or mutation ( Mullen et al . , 2011 ) and further supports the model that hypomorphic mtFAS mutant skeletal myoblasts display significant ETC dysfunction .", "The best-studied function of mtFAS is the biosynthesis of octanoate , the eight-carbon precursor of lipoic acid , but it also produces longer acyl chains .", "This raises the possibility that there are at least two independent mechanisms whereby mtFAS might act on mitochondrial function: one via lipoic acid synthesis and another in which these longer acyl chains facilitate the acyl-ACP dependent activation of LYRM proteins in ETC complex assembly .", "Therefore , we decided to determine whether the metabolic phenotypes observed in mtFAS mutant cells are also found in cells containing an intact mtFAS system , but lacking the ability to transfer lipoic acid to target proteins , including the 2-ketoacid dehydrogenases ( 2KADHs ) .", "We created mutant cell lines for Lipt1 , the gene that encodes the terminal enzyme in protein lipoylation ( Figure 5A ) .", "In these mutants , synthesis of octanoate and longer acyl chains by mtFAS should be unaffected , as Lipt1 catalyzes the terminal step in lipoic acid transfer to its target proteins downstream of mtFAS ( reviewed in Solmonson and DeBerardinis , 2018 ) .", "Similar to mtFAS mutants , Lipt1 mutant cells displayed undetectable protein lipoylation ( Figure 5A ) , with no change in the expression of MCAT , OXSM , and MECR proteins ( Figure 5—figure supplement 1A ) .", "The fact that mtFAS mutants and Lipt1 mutants both have drastically reduced protein lipoylation , below the limit of detection via western blotting , allows us to attribute any phenotypes observed in mtFAS mutants that are not phenocopied by Lipt1 mutants to non-lipoic acid products of mtFAS .", "Like mtFAS mutants , Lipt1 mutants display an analogous , albeit somewhat milder respiratory phenotype ( Figure 5B ) .", "However , in sharp contrast to the destabilization of ETC complexes observed in mtFAS mutants , Lipt1 mutants largely maintain an assembled ETC , with similar levels of CI , CII , and SC as in control cells ( Figure 5C ) .", "Notably , CV was more destabilized in Lipt1 mutants than in mtFAS mutants , suggesting a mtFAS-independent mode of CV regulation by LIPT1 outside of lipoic acid ( Figure 5C ) .", "Nevertheless , it appears that the mtFAS-dependent regulation of CI and CII is essentially independent of lipoic acid synthesis and therefore likely involves some other lipid product of mtFAS .", "Finally , we wanted to determine which portions of the metabolic phenotypes resulting from mtFAS loss are attributable to loss of protein lipoylation .", "We found that , in contrast to mtFAS mutants , Lipt1 mutants had normal abundance of TCA cycle metabolites ( Figure 5D ) .", "U13C-glutamine flux experiments showed that LIPT1 loss caused a reduction in oxidative labeling and at most a small increase in reductive labeling of fumarate and malate ( Figure 5E ) , consistent with its small effects on OCR and ETC assembly and with similar labeling effects in fibroblasts from LIPT1-deficient patients ( Ni et al . , 2019 ) .", "In all , our data strongly support a model in which mtFAS-dependent acyl chains facilitate acyl-ACP/LYRM interactions to support the biosynthesis of ETC complexes independent of lipoic acid .", "C2C12 myoblasts are muscle precursor cells that remain proliferative in culture unless stimulated to terminally differentiate .", "However , upon examination of the quantitative proteomics experiment described above , we noticed that the largest group of proteins with decreased abundance were all related to the terminal differentiation of muscle cells ( Figure 6A , Supplementary file 2 ) .", "Although some of these differences did not reach statistical significance , likely due to the low abundance of these proteins in proliferative myoblast cultures , we were nonetheless intrigued by this observation , which might imply a defect in myoblast differentiation .", "Indeed , when treated with differentiation medium for up to seven days , mtFAS mutant cell lines almost completely failed to differentiate , as monitored by the formation of multinucleated myotubes ( Figure 6B and Figure 6—figure supplement 1A ) .", "Muscle cell differentiation is controlled by a transcription factor cascade in which MyoD activates the transcription of Myogenin ( Myog ) , and Myog in turn activates the transcription of many terminal differentiation genes , such as myosin heavy chain ( MHC , encoded by Myh1 ) ( Zammit , 2017 ) .", "To further explore the effect of mtFAS mutation on differentiation , we immunoblotted for MyoD , Myog , and MHC in proliferative conditions ( day −1 ) and at 1 , 3 , and 7 days of culture in differentiation medium .", "Although MyoD expression was largely unaffected in mtFAS mutant cells , Myog induction appeared to be decreased or absent in mtFAS cells ( Figure 6C ) .", "Similarly , the Myog target protein MHC was almost undetectable in mtFAS mutant cells ( Figure 6C ) .", "To further explore the mechanism underlying impairment of Myog induction and muscle cell differentiation , we performed RNA-Seq analyses on cells grown at early time points in differentiation ( day −1 , day 0 , and day 1 ) .", "The results confirmed no difference in MyoD transcript expression , but showed both a delay and blunting of induction of the Myog transcript as well as those of its target genes such as Myh1 and myogenic differentiation genes generally ( Figure 6D , E and Figure 6—figure supplement 1B–D ) .", "Another Myog target , the transcription factor Mef2c , is also involved in the feed forward propagation of the myogenic program ( Ji et al . , 2009; Rogerson et al . , 2002 ) .", "Interestingly , in two of the three mtFAS mutant clones assayed , Mef2c transcripts were also strikingly down-regulated compared with control ( Figure 6D , E ) , which raises the possibility that it plays a particularly important role in the connection of mtFAS and mitochondrial metabolism with myocyte differentiation .", "Myog induction and myogenic differentiation requires extensive epigenetic remodeling ( Asp et al . , 2011 ) .", "Several cellular demethylases , including DNA demethylases and Jumonji histone demethylases use alpha-ketoglutarate ( αKG ) as a methyl acceptor , and the αKG to succinate ratio has been shown to affect demethylase activity ( Pavlova and Thompson , 2016; Soloveychik et al . , 2016 ) .", "Steady state metabolomics analysis of mtFAS mutant cell lines revealed a large increase in αKG as well as in the αKG/succinate ratio ( Figure 6F ) , suggesting a possible mechanism for the impairment of Myog induction and myocyte differentiation ." ], [ "Here we have shown that three genes in the mtFAS pathway ( Mcat , Oxsm , and Mecr ) are likely essential in cultured mammalian skeletal myoblasts .", "Hypomorphic mutants of these genes lead to loss of detectable protein lipoylation , without affecting structural phospholipids such as PC .", "Strikingly , mtFAS impairment results in a profound mitochondrial phenotype characterized by loss of assembled ETC complexes I , II , and IV .", "Quantitative proteomics analysis revealed the specific decreases in the abundance of two LYRM proteins , NDUFA6 , a subunit of CI , and LYRM4 , a member of the ISC complex , along with its target protein , NFS1 , and another LYRM target , SDHB .", "Importantly , the LYRM proteins that coordinate SDHB in CII assembly , SDHAF1 and SDHAF3 , were not identified in our proteomics dataset , however we infer that they are likely also decreased in abundance in mtFAS mutant cells , giving rise to the observed effect on SDHB .", "RNA-Seq analyses confirmed that the decreased abundance of these proteins is not a result of transcriptional down-regulation , but likely reflects post-translational destabilization .", "Our data together with that from multiple other eukaryotic model systems support a model in which this effect is mediated through the physical interaction of acyl-ACP with LYRM proteins , resulting in their acylation-dependent activation and functional assembly of ETC complexes ( Angerer et al . , 2017; Van Vranken et al . , 2018 ) .", "In contrast to mtFAS mutants , Lipt1 mutants can still synthesize acyl chains on ACP , but fail to transfer lipoate to its target proteins downstream of mtFAS .", "Thus , like mtFAS mutants , Lipt1 mutants lose detectable lipoylation of PDH and OGDH , and have impaired mitochondrial respiration .", "However , the acylation-dependent activation of LYRM proteins and ETC complex assembly appears to require lipid products of mtFAS other than lipoic acid , as although Lipt1 mutants lose protein lipoylation , they maintain mitochondrial ETC complexes I-IV .", "This finding is supported by studies in yeast that reached a similar conclusion based upon a suppressor mutation which can rescue OXPHOS in the absence of lipoic acid restoration ( Kursu et al . , 2013; Van Vranken et al . , 2018 ) .", "We also found that mtFAS mutants and Lipt1 mutants both undergo significantly decreased oxidative TCA cycling , however only mtFAS mutants appear to display decreased abundance of TCA cycle intermediates and robustly employ reductive carboxylation .", "We infer that these metabolic effects are likely downstream of loss of ETC complex function , as has been previously reported in the literature ( Mullen et al . , 2011 ) .", "As both mtFAS mutants and Lipt1 mutants have undetectable lipoylated PDH and OGDH , but only mtFAS mutants have impaired synthesis of longer acyl chains on ACP , these data suggest that it is these longer acyl chains that are important for the interaction of acyl-ACP and activation of LYRM proteins .", "These findings also have implications for how patients with mutations in the lipoic acid pathway vs . the mtFAS pathway are treated .", "It is important to note that some of the effects we observe on ETC assembly cannot be explained by the known interactions between LYRMs and acyl-ACP .", "For example , it is unclear how mtFAS impairment leads to the instability of Complex IV .", "One possibility is simply that CIV is less stable outside of its association with respiratory supercomplexes , which are almost completely absent in mtFAS mutant cells , likely as a result of Complex I loss .", "Alternatively , COX5A and COX5B are among the most affected ETC subunits by proteomics , and could be the targets of either a novel LYRM protein or another mtFAS-dependent mechanism .", "In mammals , the LYRM family includes at least eleven proteins but as this motif is degenerate and poorly defined , there are likely to be many that are still undiscovered .", "Of the currently annotated LYRM proteins , at least three ( LYRM1 , LYRM2 , and LYRM9 ) have no known molecular target , although LYRM2 has recently been implicated in CI assembly in addition to NDUFA6 and NDUFB9 ( Dibley et al . , 2020 ) .", "The extent to which these novel LYRM proteins require ACP acylation for function is also unknown .", "Similarly , further study into the difference ( s ) between LYRMs that depend on ACP acylation and those that do not is warranted .", "As mentioned above , we observed decreases in abundance of two LYRM proteins , NDUFA9 and LYRM4 , and can infer the destabilization of SDHAF1 and SDHAF3 from the decreased abundance of SDHB .", "Conversely , we did not observe destabilization of NDUFB9 and UQCRFS1 upon mtFAS mutation , implying that NDUFB9 and LYRM7 are not dependent on ACP acylation for their stability and functions , although orthologs in other eukaryotes seemed to be ( Angerer et al . , 2017; Van Vranken et al . , 2018 ) .", "However , it is possible that this effect could result from residual mtFAS activity , as complete knockout of the mtFAS pathway proved impossible in this system .", "Furthermore , at least in the case of UQCRFS1 and LYRM7 , the maintenance of CIII may have been clonally selected for as cells require CIII function for quinone oxidation and dihydroorotate dehydrogenase activity to make uridine ( Khutornenko et al . , 2014; Khutornenko et al . , 2010 ) .", "Therefore , cells that lack CIII are auxotrophic for orotate or uridine , and lack of these metabolites in the culture medium may have provided selective pressure for clones that employ some mechanism to escape the mtFAS-dependent regulation of CIII .", "In fact , it is tempting to speculate that assembly of CIII may be an essential function of mtFAS in mammalian cells .", "Finally , the question of why cells have evolutionarily maintained a parallel pathway for lipid synthesis in mitochondria remains interesting , especially given recent research demonstrating the transfer of lipids between endoplasmic reticulum , mitochondria , and various other organelles , implying that the answer cannot simply be compartmentalization ( Yang et al . , 2018 ) .", "Acetyl-CoA is the nearly universal fuel of mitochondrial oxidative metabolism , being the convergence point of the catabolism of carbohydrates , fatty acids , ketones and some amino acids .", "Acetyl-CoA is also the common substrate of mtFAS and the TCA cycle , where it is broken down to produce the reduced NADH and FADH2 that fuel the electron transport chain .", "Thus , the interaction of acyl-ACP with the LYRM proteins to facilitate ETC assembly has high potential to be a regulatory mechanism whereby acetyl-CoA regulates its own catabolism ( Kastaniotis et al . , 2004; Kursu et al . , 2013; Van Vranken et al . , 2018 ) .", "In other words , as acetyl-CoA is the substrate for mtFAS , it is required for ACP acylation , which in turn is required for interaction with and activation of LYRM proteins and ETC assembly .", "The assembled ETC catalyzes the oxidation of NADH and FADH2 , which drives the TCA cycle and consumption of acetyl-CoA .", "Thus , when acetyl-CoA is low , ACP acylation is diminished , ETC assembly is blunted , TCA cycling slows , and acetyl-CoA consumption falls .", "This hypothesis is supported by the observation that yeast with defects in mitochondrial pyruvate metabolism exhibit blunted ACP acylation and impaired ETC assembly ( Van Vranken et al . , 2018 ) .", "This elegant regulatory cycle provides cells with a potential mechanism to monitor acetyl-CoA substrate availability and adjust ETC complex levels accordingly , avoiding situations where the ETC sits idle in the absense of substrate , and reducing the attendant deleterious consequences , such as the production of reactive oxygen species .", "Similarly , it could potentially allow cells to ramp up oxidative capacity in settings of excess mitochondrial acetyl-CoA .", "Human mutations in genes encoding enzymes of the mtFAS pathway are rare , but continue to be discovered ( Gorukmez et al . , 2019; Heimer et al . , 2016; Li et al . , 2020 ) .", "Intriguingly , patients with Mecr mutations exhibit muscle weakness and we show herein that mtFAS mutation interferes with the normal differentiation of cultured myoblasts ( Figure 6 ) .", "The implications of these studies for the broader population remain to be seen , however .", "For example , many age-related human diseases exhibit evidence of decreased mitochondrial respiration , including frequent defects in OXPHOS complex assembly , suggesting the interesting possibility that alterations in mtFAS might contribute to mitochondrial impairments in human disease .", "The role ( s ) of mtFAS in aging and more prevalent diseases that also involve mitochondrial pathologies such as diabetes , heart disease , and cancer are essentially unstudied .", "One recent publication suggested that lipoylation of PDH and expression of Oxsm may be decreased in some tissues in diabetic mice ( Gao et al . , 2019 ) .", "This raises the intriguing prospect that augmenting mtFAS pathway function could ‘boost’ or rescue mitochondrial oxidative metabolism in settings where mitochondrial respiration is sub-optimal .", "Recent studies have shown enhancement of mitochondrial metabolism upon ACP overexpression , although the molecular mechanisms and dependence of these phenotypes on mtFAS are unclear ( Hou et al . , 2019; Zhang et al . , 2019 ) .", "It is therefore important to thoroughly examine how this pathway functions both in normal biology and in disease settings .", "Understanding the rate limiting steps in the cycle , its mode of regulation , and the mechanisms through which it can be manipulated could one day allow mtFAS to be harnessed therapeutically to build better mitochondria and treat metabolic disease ." ], [ "Unless otherwise indicated for specific experiments , C2C12 immortalized mouse skeletal myoblasts ( ATCC CRL-1772 , verification provided by ATCC , mycoplasma testing status: negative ) were grown in DMEM with 4 . 5 g/L glucose , glutamine , and sodium pyruvate ( Corning , 10–013-CV ) supplemented with 10% FBS ( Sigma , F0926 ) at 37°C in a humidified atmosphere containing 5% CO2 .", "To generate mtFAS knockout clones , two guides per gene were designed targeting exon 1 or exon 2 of the mouse Mcat , Oxsm , and Mecr genes ( see key resources table for sequences ) and cloned in the pLentiCRISPR v2 vector .", "Parental C2C12 cells were transfected with pLentiCRISPRv2_sgmtFAS or empty vector .", "Editing efficiency was assessed in the bulk transfected population by T7E1 assay .", "Briefly , DNA was isolated using QuickExtract solution ( Lucigen ) .", "Edited regions were PCR amplified with the primers listed in the key resources table .", "PCR products were denatured , reannealed , and digested with T7E1 endonuclease ( New England Biologicals ) , which cleaves mismatched DNA .", "Digests were run on a 2% agarose gel , visualized with SYBR safe DNA stain ( Invitrogen ) and percent editing was calculated as the sum of the cleaved fragments divided by the total DNA in each lane .", "To establish clonal lines , 48 hr after transfection , the cells were sorted based on GFP signal and plated in a 96-well plate to obtain single cell colonies .", "Colony number and size was assessed by brightfield microscopy .", "Clones were screened based on MCAT , OXSM , and MECR protein levels .", "Three Oxsm and Mecr mutant clones , three control clones , and two Mcat mutant clones were ultimately selected for further experiments .", "Quantification of single cell clone number and colony size was assessed 7 days after single cell sort .", "Small clones = <150 cells , Med = 150–500 cells , Large = >500 cells .", "For growth assays , cells were plated at 100 , 000 cells/plate in 10 cm dishes in 4 . 5 g/L glucose and counted every 24 hr using a BioRad TC20 automated cell counter .", "For rescue experiments , control , Mcat , Oxsm , and Mecr mutant clonal cell lines were infected with retrovirus harboring a control transfer plasmid ( pQXCIP mtDSRed , pCtrl ) or pQXCIP expressing the indicated mtFAS gene off the full CMV ( pQXCIP-CMV-Oxsm , pOxsm , pCMV-Mcat , pCMV-Mecr ) or Δ4 , 5 truncated CMV promoter ( pΔ4 , 5CMV-Mcat , pΔ4 , 5CMV- Mecr ) .", "mtFAS genes were cloned from mouse cDNA isolated from NIH3T3L1 cells .", "Stably infected cells were selected with 2 µg/mL puromycin for 7 days .", "10 , 000 cells/well were plated in 6-well or 12-well plates , and grown in normal growth medium containing 4 . 5 g/L glucose or 10 mM glutamate as indicated for 3 days ( glucose ) or 4 days ( galactose ) until control wells reached confluency .", "Plates were stained for 10 min in 2 . 5 mg/mL crystal violet in 20% methanol , washed 6x and allowed to dry , then imaged .", "HEK293T cells were transiently transfected with pLKO . 1 shFASN or scramble shRNA control along with the lentiviral packaging plasmids psPAX2 and pMD2 . G using polyethylenimine .", "48 hr after transfection , viral supernatant was collected , filtered through a 0 . 45 µm polyethersulfone membrane , and stored at 4°C .", "10 µg/mL Polybrene ( EMD Millipore , TR-1003-G ) was added and a 1:1 mixture of viral supernatant and fresh growth medium ( DMEM + 10%FBS ) was applied directly to Oxsm mutant cells , along with wild type C2C12 controls , and incubated for 16 hr at 37°C in a humidified incubator with 5% CO2 .", "Viral medium was discarded and replaced with fresh growth medium and cells were allowed to recover and expand for 24 hr .", "After the recovery period , stably infected cells were selected with 2 µg/mL puromycin for 1 week .", "Knockdown of FASN was confirmed via immunoblotting as described below .", "C2C12 myoblasts were grown in DMEM High Glucose ( Sigma , D5796 ) supplemented with 10% FBS ( Gemini Bio-Products #100–106 ) and penicillin/streptomycin ( HyClone ) at 37°C in a humidified atmosphere containing 5% CO2 .", "To establish LIPT1 mutant clones , two guides were designed targeting the coding region of the mouse Lipt1 gene and cloned in the px459 vector .", "Parental C2C12 cells were transfected with px459_sgLIPT1 or empty vector and three days later the cells were sorted based on GFP signal .", "GFP positive cells were subsequently plated to obtain single cell colonies and screened based on LIPT1 protein levels and lipoylation of pyruvate dehydrogenase ( PDH ) and 2-oxoglutarate dehydrogenase ( OGDH ) .", "Two Lipt1 mutant clones and one wild type clone were ultimately selected for further experiments .", "Cells were harvested by incubation with 0 . 25% trypsin-EDTA ( Gibco , 25200–072 ) , pelleted , washed once with ice cold sterile PBS ( Gibco , 10010–023 ) , and frozen at −80°C .", "Upon thawing , cells were resuspended in 1 mL CP-1 buffer ( 100 mM KCl , 50 mM Tris-HCl , 2 mM EGTA , pH 7 . 4 ) and mechanically lysed by 10 passes through a 27-gauge needle , and centrifuged at 700 x g to pellet unlysed cells and debris .", "Supernatant was moved to a new tube and centrifuged at 10 , 000 x g to pellet crude mitochondrial fraction .", "Post-mitochondrial supernatant ( PMS ) was saved for immunoblotting or discarded .", "Mitochondrial pellets were resuspended in a small volume of CP-1 buffer equal to approximate pellet volume and used in assays described below .", "Whole cell lysates were prepared by scraping cells directly into Ripa buffer ( 50 mM Tris-HCl , 1% NP-40 , 0 . 5% Na-Deoxycholate , 0 . 1% SDS , 150 mM NaCl , 2 mM EDTA ) supplemented with protease and phosphatase inhibitors ( Sigma Aldrich P8340 , Roche Molecular 04906845001 ) , incubated on ice for 45 min with vortexing every 5 min , and then centrifuged at 16 , 000 x g for 10 min at 4°C to remove insoluble material .", "Supernatant was saved as whole cell lysate ( WCL ) .", "WCL , PMS , and/or crude mitochondrial fractions were normalized for total protein content via BCA Assay ( Thermo Scientific 23225 ) .", "Samples were resolved by SDS-PAGE on Tris-glycine gels ( Invitrogen XP04205BOX ) and transferred to nitrocellulose or PVDF membranes .", "Immunoblotting was performed using the indicated primary antibodies which are listed in the key resources table according to the manufacturers’ recommendations , and analyzed by Licor Odyssey .", "Crude mitochondrial fractions were isolated and normalized for total protein content as described above .", "Blue Native ( BN ) PAGE was performed using the Invitrogen NativePAGE system .", "100 µg of mitochondria were pelleted at 10 , 000 x g for 10 min at 4°C and resuspended in 1x pink lysis buffer ( Invitrogen , BN20032 ) .", "Digitonin ( GoldBio D-180–2 . 5 ) was added to a final concentration of 1% mass/volume .", "Samples were incubated on ice for 15 min , then spun for 20 min at 20 , 000 x g .", "6 µL of NativePAGE sample buffer ( Invitrogen , BN20041 ) was added and 10 µL of sample was run on precast 3–12% NativePAGE gels ( Invitrogen , BN2011B × 10 ) with NativePAGE anode buffer ( Invitrogen , BN2001 ) and dark blue cathode buffer ( Invitrogen , BN2002 ) at 150 V for 1 hr then switched to light blue cathode buffer ( Invitrogen , BN2002 ) and run at 30 V overnight .", "Gels were subsequently transferred to PVDF at 100 V , washed with methanol , and blotted with the indicated primary antibodies which are listed in the key resources table according to the manufacturers’ recommendations .", "Secondary anti-mouse HRP antibody listed in the key resources table and SuperSignal West Femto Maximum Sensitivity Substrate ( Thermo , 34096 ) was used to visualize bands on film ( GeneMate , F-9024−8 × 10 ) .", "Quadruplicate biological replicates of mtFAS mutant and control cells were plated at 35% confluence in normal growth medium , then switched to labeling medium ( DMEM base ( Corning , 17–207-CV ) , supplemented with 25 mM U13C-glucose ( Cambridge Isotopes , CLM-1396 ) and 4 mM glutamine ( Sigma G7513 ) ) for 0 , 0 . 25 , 0 . 5 , or one population doubling time .", "Samples were harvested at 70% confluence via incubation with 0 . 25% trypsin ( Gibco , 25200–072 ) , and pelleted at 300 x g for 5 min at 4°C , flash frozen in liquid nitrogen , and stored at −80°C .", "Lipids were extracted from cell pellets in a randomized order using a solution of 225 µL methanol ( MeOH ) containing internal standards ( IS ) ( Avanti SPLASH LIPIDOMIX , 10 µL each/sample ) and 750 µL methyl tert-butyl ether ( MTBE ) ( Matyash et al . , 2008 ) .", "The samples were sonicated for 1 min , rested on ice for 1 hr , briefly vortexed every 15 min then 188 µL dd-H2O was added to induce phase separation .", "All solutions were pre-chilled on ice .", "The sample was then vortexed for 20 s , rested at room temperature for 10 min , and centrifuged at 3000 x g for 5 min at 4°C .", "The upper , organic phases were collected and evaporated to dryness under vacuum .", "Lipid samples were reconstituted in 250 µL isopropyl alcohol .", "Concurrently a process blank sample was brought forward as well as a pooled quality control ( QC ) sample , prepared by taking equal volumes ( ~10 µL per sample ) from each sample after final resuspension .", "Lipid extracts were separated on a Waters Acquity UPLC CSH C18 1 . 7 µm 2 . 1 × 100 mm column maintained at 65°C connected to an Agilent HiP 1290 Sampler , Agilent 1290 Infinity pump and Agilent 6530 Accurate Mass Q-TOF dual AJS-ESI mass spectrometer .", "For positive mode , the source gas temperature was set to 225°C , with a drying gas flow of 11 L/min , nebulizer pressure of 40 psig , sheath gas temp of 350°C and sheath gas flow of 11 L/min .", "VCap voltage was set at 3500 V , nozzle voltage 1000 V , fragmentor at 110 V , skimmer at 85 V and octopole RF peak at 750 V . For negative mode , the source gas temperature was set to 300°C , with a drying gas flow of 11 L/min , a nebulizer pressure of 30 psig , sheath gas temp of 350°C and sheath gas flow 11 L/min .", "VCap voltage was set at 3500 V , nozzle voltage 2000 V , fragmentor at 100 V , skimmer at 65 V and octopole RF peak at 750 V . Samples were analyzed in a randomized order in both positive and negative ionization modes in separate experiments acquiring with the scan range m/z 100–1700 .", "Mobile phase A consists of ACN:H2O ( 60:40 v/v ) in 10 mM ammonium formate and 0 . 1% formic acid , and mobile phase B consists of IPA:ACN:H2O ( 90:9:1 v/v ) in 10 mM ammonium formate and 0 . 1% formic acid .", "The chromatography gradient for both positive and negative modes starts at 15% mobile phase B then increases to 30% B over 2 . 4 min , it then increases to 48% B from 2 . 4 to 3 . 0 min , then increases to 82% B from 3 to 13 . 2 min , then increases to 99% B from 13 . 2 to 13 . 8 min where it was held until 16 . 7 min and then returned to the initial conditions and equilibrated for 5 min .", "Flow was 0 . 4 mL/min throughout , injection volume was 3 µL for positive and 10 µL negative mode .", "Tandem mass spectrometry was conducted using the same LC gradient at collision energy of 25 V . QC samples ( n = 8 ) and blanks ( n = 4 ) were injected throughout the sample queue and ensure the reliability of acquired data .", "Results from LC-MS experiments were collected using Agilent Mass Hunter ( MH ) Workstation and analyzed using the software packages MH Qual , MH Quant , and Lipid Annotator ( Agilent Technologies , Inc ) .", "Lipid extracts were saponified by an addition of 1 mL of 5% HCl in MeOH .", "Samples were vortexed for 15 seconds then placed into a sand bath heated at 80°C for 2 hours .", "The resulting methanolysis reaction products were extracted with hexanes ( 3 X 1 mL ) by vortexing for 15 seconds and then centrifuged for 4 minutes at 10 , 000 x g .", "Supernatants were transferred to new tubes and gently dried under a nitrogen stream at room temperature .", "Samples were resuspended in 200 µL EtOAc and transferred to GC-MS vials with insert .", "GC-MS analysis was performed with an Agilent 5977b GC-MS HES-MSD and an Agilent 7693A automatic liquid sampler .", "Samples were injected ( 1 µL ) 10:1 , with an inlet temperature of 250°C .", "The gas chromatograph had an initial temperature of 67°C for one minute followed by a 50°C/min ramp to 197°C , a second ramp of 10°C/minute up to 325°C began immediately with a hold time of 2 minutes .", "A 30-meter Agilent Zorbax DB-5MS with 10 m Duraguard capillary column was employed for chromatographic separation .", "Helium was used as the carrier gas at a rate of 1 mL/minute .", "A gain of 5 . 0 was used for data acquisition .", "High Efficiency Source and quadrupole temperatures were held at 275 °C and 190 °C , respectively .", "Target compounds were fed to the emass software created by the Rockwood group ( Rockwood and Haimi , 2006 ) .", "This software calculated masses and relative abundances for all isotopes observed so if for one compound only M2 was observed and another went out to M5 , emass would calculate M0-M2 for compound 1 and M0-M5 for compound 2 .", "Matrixes of the theoretical isotopic abundances and the measured isotopic abundances were created .", "The multiplicative inverse of each matrix was calculated and then the dot product of the theoretical inverse matrix and the measured inverse matrix was calculated .", "The data from one row of the resulting matrix ( all rows are identical ) is then sum normalized .", "In an unlabeled sample M0 will be measured as 1 and all other isotopes should be 0 ( standard Metabolomic Flux Analysis ) .", "CIII and CS activities were measured following the protocol published in Spinazzi et al . , 2012 .", "For CIII activity assay , 10 µg of purified mitochondria was mixed in 990 µL of the assay buffer ( 25 mM potassium phosphate ( pH 7 . 5 ) , 75 µM oxidized cytochrome c , 500 µM KCN , 100 µM EDTA ( pH 7 . 5 ) , 0 . 025% ( volume/volume ) Tween-20 ) .", "After the baseline reading at 550 nM for 2 min , the reaction was started by adding 10 µl of 10 mM decylubiquinol and the increasing absorbance at 550 nm was measured for the next 3 min .", "In parallel CIII-independent cytochrome c reduction was measured by adding antimycin A in a separate reaction mixture and subtracted from the reaction without antimycin A . CIII activity was calculated and expressed as nmole/min/mg .", "CS activity was measured by incubating 5 µg of purified mitochondria in 950 µL of the assay buffer ( 100 mM Tris ( pH 8 . 0 ) , 0 . 1% ( volume/volume ) Triton X-100 , 100 µM DTNB ( 5 , 5’-dithiobis ( 2-nitrobenzoic acid ) ) , 300 µM Acetyl CoA ) .", "After reading the baseline activity at 412 nm for 2 min , the reaction was started by adding 50 µL of 10 mM oxaloacetic acid ( OA ) and monitored the increase in the absorbance at 412 nm for 3 min .", "CS activity was calculated by subtracting the baseline increase at 412 nm from the increase after addition of OA and expressed as nmole/min/mg .", "Seahorse experiments were performed on a Seahorse XFe96 Analyzer .", "Cells from n = 2 clones ( Mcat mutants ) or n = 3 clones ( Oxsm , Mecr mutants and controls ) were plated in 8 wells of a 96-well seahorse plate at a density of 6 , 000 cells/well in DMEM + 10% FBS .", "The morning of the experiment , cells were washed 2x and then medium was replaced with DMEM ( Sigma D5030 ) supplemented with 25 mM glucose , 2 mM glutamine and 1 mM pyruvate , pH 7 . 4 .", "A standard mitochondrial stress test was performed using 1 µM oligomycin , 3 µM FCCP , and 0 . 5 µM Rotenone + 0 . 5 µM Antimycin A . Assay protocol was standard ( 3 measurements per phase , acute injection followed by 3 min mixing , 0 min waiting , and 3 min measuring ) .", "Data were normalized to total cellular protein per well ( Thermo BCA Kit cat . 23225 ) .", "Results were analyzed in WAVE software and processed through the XF Mito Stress Test Report .", "Cells were plated in chambered coverglass ( Fisher , 155409 ) and allowed to adhere overnight .", "Medium was changed and replaced with fresh DMEM + 10% FBS containing 25 nM MitoTracker Red CMXROS and 50 nM MitoTracker Green ( Invitrogen M7512 and M7514 ) .", "Cells were incubated with dyes for 30 min at 37°C in a humidified incubator with 5% CO2 , then washed twice and replaced with fresh medium , and imaged using a Zeiss AxioObserver Z1 fluorescence microscope ( Carl Zeiss ) equipped with 40x oil-immersion objective .", "Digital fluorescence and differential interference contrast ( DIC ) images were acquired using a monochrome digital camera ( AxioCam 506 , Carl Zeiss ) .", "Images were exported from Zeiss Zen software package and analyzed for fluorescence intensity using FiJi ImageJ .", "Images are representative of n ≥ 38 images from two clones imaged in two independent experiments .", "10 cm plates of HEK293T cells were transiently transfected with 10 µg of total DNA using Lipofectamine 2000 ( Invitrogen , 11668019 ) .", "DNA was made up of equal amounts of empty vector , pcDNA3 . 1 hNDUFAB1-Flag , and pcDNA3 . 1 LYRM-V5 constructs alone or in combination as indicated .", "48 hr after transfection , cells were collected by incubation with 0 . 25% trypsin , washed , and pelleted at 300 g .", "Crude mitochondria were isolated as described above and protein content was assessed via BCA assay .", "500 µg of crude isolated mitochondria were pelleted and resuspended in 500 µL of XWA buffer ( 20 mM HEPES , 10 mM KCl , 1 . 5 mM MgCl2 , 1 mM EDTA , 1 mM EGTA , 150 mM NaCl , 2 . 1 mg/mL NaF , 10 . 8 mg/mL β-glycerophosphate , pH 7 . 4 ) then lysed by incubation in 0 . 7% digitonin for 30 min rotating at 4°C .", "Insoluble material was removed by centrifugation at 20 , 000 g for 20 min , and supernatant was incubated with 20 µL anti-Flag agarose beads ( Sigma , F2426 ) for 2 hr rotating at 4°C .", "Beads were washed 3 times with XWA buffer and samples were eluted in 40 µL of 1x Lamelli buffer ( 10% glycerol , 50 mM Tris HCl , pH 6 . 8 , 1% SDS ) for 10 min at 65°C .", "Samples were loaded on Tris-Glycine gels , separated by SDS-PAGE , and immunoblotted for Flag and V5 as described above .", "Results are representative of three separate experiments .", "Duplicate biological samples of two control clones , two Oxsm mutant clones , and two Mecr mutant clones were grown in standard proliferative conditions to 70% confluence and harvested by incubation with 0 . 25% trypsin , pelleted at 300 g , washed with sterile PBS , flash frozen in liquid nitrogen and stored at −80°C .", "As only eleven samples could be run with the SL-TMT protocol , one Oxsm clone had only one sample analyzed and was ultimately omitted from the results .", "Seahorse experiments were performed on a Seahorse XFe96 Analyzer .", "Cells were plated at a density of 10 , 000 cells/well in DMEM + 10% FBS .", "The morning of the experiment , cells were washed 2x and then medium was replaced with Seahorse medium: DMEM ( Sigma D5030 ) supplemented with 10 mM glucose , 2 mM glutamine and 1 mM pyruvate , pH 7 . 4 .", "A standard mitochondrial stress test was performed using 2 μM oligomycin , 1 μM Carbonyl cyanide m-chlorophenyl hydrazone ( CCCP ) , and then 1 μM Antimycin A . Assay protocol was standard ( 3 measurements per phase , acute injection followed by 3 min mixing , 0 min waiting , and 3 min measuring ) .", "Data were normalized to 10 , 000 cells/well .", "Results were analyzed in WAVE software and processed through the XF Mito Stress Test Report .", "C2C12 cells were plated in a 60 mm plate at a density of 100 , 000 cells/plate .", "The following day , medium was changed to tracing medium: DMEM ( Sigma 5030 ) supplemented with 2 . 5 mM glucose , 4 mM [U-13C]glutamine , 1 mM pyruvate , and 5% dialyzed FBS .", "Cells were harvested 24 hr later by washing once with ice-cold normal saline and then quenched with 800 μL 80:20 methanol:water .", "Methanol lysates were then subjected to three rapid freeze-thaw cycle and then spun at 16 , 000 x g for 10 min at 4°C .", "The supernatants , with 1 μL d27-myristic acid added ( Sigma 68698 ) ( GCMS only ) as an internal control , were evaporated using a SpeedVac concentrator .", "A replicate plate was set up during tracing experiments to assess relative metabolite concentrations using LCMS and harvested as described above .", "Dried metabolites were reconstituted in 100 μl of 0 . 03% formic acid in analytical-grade water , vortexed and centrifuged to remove insoluble material .", "The supernatant was collected and subjected to targeted metabolomics analysis as described on an AB SCIEX QTRAP 5500 liquid chromatography/triple quadrupole mass spectrometer ( Applied Biosystems SCIEX ) ( Kim et al . , 2000 ) .", "The injection volume was 20 μl .", "Chromatogram review and peak area integration were performed using MultiQuant ( version 2 . 1 , Applied Biosystems SCIEX ) .", "The peak area for each detected metabolite was normalized against the total ion count of that sample .", "The supernatants , with 1 μL d27-myristic acid added as an internal control , were evaporated using a speed vac .", "Dried metabolites were re-suspended in 30 μL anhydrous pyridine with 10 mg/mL methoxyamine hydrochloride and incubated at room temperature overnight .", "The following morning , the samples were cooked at 70°C for 10–15 min and then centrifuged at 16 , 000 x g for 10 min .", "The supernatant was transferred to a pre-prepared GC/MS autoinjector vial containing 70 μL N- ( tert-butyldimethylsilyl ) -N-methyltrifluoroacetamide ( MTBSTFA ) derivatization reagent .", "The samples were incubated at 70°C for 1 hr following which aliquots of 1 μL were injected for analysis .", "Samples were analyzed using either an Agilent 6890 or 7890 gas chromatograph coupled to an Agilent 5973N or 5975C Mass Selective Detector , respectively .", "The observed distributions of mass isotopologues were corrected for natural abundance .", "MtFAS mutants and controls were seeded in 10 cm plates and grown in DMEM ( Corning , 10–013-CV ) supplemented with 10% FBS ( Sigma , F0926 ) to 70% confluency ( Day −1 ) .", "The next day at 95% confluency cells were switched to DMEM supplemented with 2% horse serum ( Thermo , 16050122 ) ( Day 0 ) .", "For the next 7 days , medium was changed and replaced with fresh DMEM + 2% horse serum every 24 hr .", "Samples were imaged via brightfield microscopy or harvested for immunoblotting or RNAseq experiments at the indicated time points after switching to differentiation medium .", "For flux lipidomics and metabolomics , mitoTracker quantification , and Seahorse cellular respiration , statistically significant differences were determined using GraphPad Prism 8 .", "Data were analyzed by one-way or two-way Anova as appropriate followed by Dunnett’s multiple comparison test ( when compared only to control ) or Tukey’s multiple comparison test ( when comparing all groups , such as for lipid isotopologue analysis ) .", "A p-value of 0 . 05 was considered to be statistically significant .", "For RNAseq and quantitative proteomics , analyses are described in the corresponding sections .", "All unique/stable reagents generated within this study are available from the corresponding author , Jared Rutter ( rutter@biochem . utah . edu ) , upon request without restriction .", "The data have been deposited to the ProteomeXchange Consortium via the PRIDE ( Perez-Riverol et al . , 2019 ) partner repository .", "The code used to process these data is available at Github https://github . com/j-berg/nowinski_2020 ( copy archived at https://github . com/elifesciences-publications/nowinski_2020 ) .", "Sequencing data have been deposited at the Gene Expression Omnibus ( http://www . ncbi . nlm . nih . gov/geo ) under accession number GSE148617 ." ] ]

[ "Cells harbor two systems for fatty acid synthesis , one in the cytoplasm ( catalyzed by fatty acid synthase , FASN ) and one in the mitochondria ( mtFAS ) .", "In contrast to FASN , mtFAS is poorly characterized , especially in higher eukaryotes , with the major product ( s ) , metabolic roles , and cellular function ( s ) being essentially unknown .", "Here we show that hypomorphic mtFAS mutant mouse skeletal myoblast cell lines display a severe loss of electron transport chain ( ETC ) complexes and exhibit compensatory metabolic activities including reductive carboxylation .", "This effect on ETC complexes appears to be independent of protein lipoylation , the best characterized function of mtFAS , as mutants lacking lipoylation have an intact ETC .", "Finally , mtFAS impairment blocks the differentiation of skeletal myoblasts in vitro .", "Together , these data suggest that ETC activity in mammals is profoundly controlled by mtFAS function , thereby connecting anabolic fatty acid synthesis with the oxidation of carbon fuels ." ]

[ "In human , plant and other eukaryotic cells , fats are an important source of energy and also play many other roles including waterproofing , thermal insulation and energy storage .", "Eukaryotic cells have two systems that make the building blocks of fats ( known as fatty acids ) and one of these systems , called the mtFAS pathway , operates in small compartments known as mitochondria .", "This pathway only has one known product , a small fat molecule called lipoic acid , which mitochondria attach to several enzymes to allow them to work properly .", "The main role of mitochondria is to break down fats and other molecules to release chemical energy that powers many processes in cells .", "They achieve this using large groups of proteins known as ETC complexes .", "To build these complexes , families of proteins known as ETC assembly factors carefully coordinate the assembly of many proteins and small molecules into specific structures .", "However , it remains unclear precisely how this process works .", "Here , Nowinski et al . used a gene editing technique to mutate the genes encoding three enzymes in the mtFAS pathway in mammalian cells .", "The experiments found that the mutant cells had fewer ETC complexes and seemed to be less able to break down fats and other molecules than ‘normal’ cells .", "Furthermore , a family of ETC assembly factors were less stable in the mutant cells .", "These findings suggest that the mtFAS pathway controls how mitochondria assemble ETC complexes .", "Further experiments indicated that lipoic acid is not involved in the assembly of ETC complexes and that the mtFAS pathway produces another , as yet unidentified , product that regulates this process , instead .", "MEPAN syndrome is a rare neurological disorder that leads to progressive loss of control of movement , slurred speech and impaired vision in children .", "Patients with this syndrome have genetic mutations affecting components of the mtFAS pathway , therefore , a better understanding of how the pathway works may help researchers develop new treatments in the future .", "More broadly , these findings will have important ramifications for many other situations in which the activity of ETC complexes in mitochondria is modified ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "neuroscience" ]

[ [ "The fossil record shows that by the middle Cambrian , camera-type eyes were already present in stem vertebrates ( Morris and Caron , 2014 ) , supporting the emerging concept that spatially resolved vision provided a major competitive advantage in those biota ( Paterson et al . , 2011 ) .", "Lampreys , the only surviving jawless vertebrates together with the related hagfish ( Heimberg et al . , 2010 ) , are a pivotal resource for gaining further insight into early vertebrate vision .", "In fact , their line diverged during the Cambrian ( ∼505 Ma [Erwin et al . , 2011] ) and they later remained remarkably stable .", "This is true both of their external morphology , as revealed by fossil specimens ( Janvier and Arsenault , 2002; Gess et al . , 2006; Janvier et al . , 2006; Chang et al . , 2014 ) , and of their anatomy , as demonstrated by primeval features such as the absence of bilateral limbs and of myelinated axons , and by their possession of the simplest nervous system among vertebrates .", "Adult lampreys have camera-type eyes with layered retinas containing all the major neuronal classes present in jawed vertebrates ( Lamb , 2013 ) and sending retinotopically organized projections to the tectum ( Jones et al . , 2009 ) , as well as a photosensory pineal organ ( Pu and Dowling , 1981 ) .", "Researchers have debated the rod or cone nature of lamprey retinal photoreceptors since the middle of the 19th century ( relevant literature reviewed by Walls , 1935; Govardovskii and Lychakov , 1984; Collin et al . , 2009 ) to ascertain whether , in vertebrates , cones pre-dated rods or vice versa .", "Current molecular genetic evidence indicates that modern rods evolved from an ancestral cone ( Okano et al . , 1992; Yokoyama , 2000; Lamb et al . , 2007; Kawamura and Tachibanaki , 2008; Shichida and Matsuyama , 2009 ) , implying that vision in near darkness is a relatively recent acquisition ( Lamb , 2013 ) and causing the point of contention to become that of the timing of rod evolutionary emergence .", "This advance must have occurred", "( i ) after the appearance of the precursor of rhodopsin and of other rod-specific phototransduction proteins isoforms and", "( ii ) before the initial diversification of extant jawed vertebrates ( ∼420 Ma; Erwin et al . , 2011 ) endowed with modern rods .", "Phylogenetic analysis of visual opsins constrains time bound i to have occurred anywhere between the divergence of ascidians ( ∼610 Ma; Erwin et al . , 2011 ) and that of the lamprey line ( ∼505 Ma; Erwin et al . , 2011 ) : the sea squirt Ciona intestinalis has only one jawed vertebrate-related visual opsin ( Kusakabe et al . , 2001 ) , while some lamprey species have all five major classes ( Yokoyama , 2000 ) including an Rh1 rhodopsin ortholog ( Pisani et al . , 2006 ) ( but see Collin et al . , 2003 ) .", "Recently , strong evidence has emerged indicating that these five opsin classes ( and the rod-specific molecular toolbox ) emerged in the context of two rounds of whole-genome duplication called ‘2R’ ( Kuraku et al . , 2009; Lagman et al . , 2013 ) .", "Furthermore , analysis of the whole sea lamprey genome suggests that the lamprey line diverged from the main vertebrate line shortly after 2R ( Smith et al . , 2013 ) .", "Therefore , unveiling the functional properties of lamprey photoreceptors may shed light on the evolution of dim-light vision in the critical time period following 2R ( Collin et al . , 2009; Lamb , 2013 ) .", "The two types of photoreceptors in the retina of Northern hemisphere lampreys have light-absorbing outer segments arranged in adjacent tiers ( Figure 1A ) : those of short photoreceptors ( SPs ) lie in an inner tier , while those of long photoreceptors ( LPs ) lie in an outer tier , next to the pigment epithelium .", "This nomenclature is based on the entire length of the photoreceptors that of the outer segments showing instead the reverse pattern .", "Importantly , SPs express an Rh1 rhodopsin ortholog ( Pisani et al . , 2006 ) and some of their phototransduction protein isoforms examined thus far clade with those of rods ( Muradov et al . , 2008 ) , but they also have molecular and morphological features of cones including outer segment discs that appear continuous with the plasma membrane ( Dickson and Graves , 1979 ) .", "Thus , while they retain archaic features of a cone progenitor , SPs are homologues of jawed vertebrate rods ( Lamb , 2013 ) .", "LPs , on the other hand , express an LWS red cone opsin and have a molecular fingerprint consistent with cones ( Muradov et al . , 2008 ) .", "Here , we examined single lamprey photoreceptors at the levels of their inner and outer segments using two different recording techniques that provide complementary information , to establish the extent to which SPs operate like jawed vertebrate rods .", "We found multiple striking similarities that , taken together , argue against convergent evolution , implying that middle Cambrian vertebrates possessed functionally advanced rod precursors . 10 . 7554/eLife . 07166 . 003Figure 1 . Signal processing in the inner segment of lamprey photoreceptors resembles that found in jawed vertebrates .", "( A ) Image of a live retinal slice showing the layered organization of lamprey photoreceptors: short photoreceptors ( SPs ) in an inner tier and long photoreceptors ( LPs ) in an outer tier .", "Scale bar 10 µm .", "( B ) Photoreceptors express the Ih current: membrane current of a SP in response to hyperpolarizing voltage clamp steps ( from a holding potential of −53 mV to −60/−67/−74/−81/−88/−95/−102/−109 mV and repolarization to −65 mV ) in control and during superfusion of the Ih blocker ZD7288 at 100 µM .", "Records are not averages .", "( C–F )", "Photovoltage responses reveal that SPs feed their signals into LPs .", "( C and D )", "Average responses to 520-nm flashes of a SP ( 0 . 5 , 1 . 6 , 5 . 4 , 15 , 45 , 136 , 398 , 1128 photons·µm−2 ) and a LP ( 16 , 51 , 170 , 469 , 1413 , 4314 , 12 , 597 , 38 , 847 , 77 , 695 ph·µm−2 ) .", "Insets show their outer segments ( scale bars 5 µm ) .", "( E and F )", "Response amplitudes to 520-nm ( green circles ) and 590-nm flashes ( orange circles with a dot ) of a SP and a LP .", "Fits are exponential saturation functions ( for the LP restricted to the first component: see text ) .", "In panel E , left and right ordinate values refer to left and right data sets , respectively , which were adjusted to match saturating amplitudes .", "In panel F such an adjustment could not be performed .", "Error bars are SEM .", "Action spectra templates for SPs and LPs are shown in Figure 1—figure supplement 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 07166 . 00310 . 7554/eLife . 07166 . 004Figure 1—figure supplement 1 . Predicted action spectra of SPs and LPs and their relative sensitivities at 520 and 590 nm .", "( A ) 11-cis-Retinal ( 11A1 ) based visual pigment templates ( Equation 1 in Govardovskii et al . , 2000 ) centered at the absorption peaks of SP ( 517 nm , green curve ) and LP outer segments ( 555 nm , orange curve ) determined previously by microspectrophotometry ( Govardovskii and Lychakov , 1984 ) .", "Small circles indicate , for each of the two photoreceptor types , the normalized sensitivities at our two stimulus wavelengths ( 520 nm , left vertical line; 590 nm , right vertical line ) .", "The predicted sensitivities ratios ( 520/590 nm ) match well with those found in our experiments ( see ‘Results’ ) .", "( B ) 9-cis-Retinal ( 9A1 ) based visual pigment template for regenerated LPs ( peak at 541 nm ) , obtained from that of red salamander cones ( Equation 3 and Table 2 in Makino et al . , 1999 ) by:", "( i ) assuming full replacement of the native chromophore and", "( ii ) introducing a slight hypsochromic shift ( 1 nm ) so as to match our experimentally determined sensitivities at 520 and 590 nm . DOI: http://dx . doi . org/10 . 7554/eLife . 07166 . 004" ], [ "First , we made perforated patch-clamp recordings from photoreceptor inner segments and found that the dark membrane potential was of −43 . 2 ± 0 . 7 mV for SPs ( n = 30 ) and −45 . 9 ± 1 . 1 mV for LPs ( n = 10 ) ( Table 1 ) ; these values are in line with those of jawed vertebrate rods and cones ( Cangiano et al . , 2012 ) .", "Input resistances were 518 ± 41 MΩ ( n = 8; SPs ) and 442 ± 68 MΩ ( n = 9; LPs ) .", "The membrane time constants , obtained by fitting single exponentials to the early rise of a current step response , were 31 . 9 ± 4 . 9 ms ( n = 8; SPs ) and 12 . 9 ± 1 . 3 ms ( n = 9; LPs ) ( p < 0 . 001 ) , equivalent to low-pass filtering with cut-off frequencies of ∼5 Hz for SPs and ∼12 Hz for LPs .", "Thus , the electrical properties of the inner segments of SPs seem adapted to process slower photocurrent changes than those of LPs .", "Both SPs ( n = 7 ) and LPs ( n = 2 ) expressed the hyperpolarization-activated current Ih , similarly to rods and cones ( Della Santina et al . , 2012 ) ; Ih was abolished by ZD7288 ( 100 µM , n = 1 SP; Figure 1B ) . 10 . 7554/eLife . 07166 . 010Table 1 . Electrophysiological parameters of SPs and LPs listed in the order they appear in the main textDOI: http://dx . doi . org/10 . 7554/eLife . 07166 . 010ParameterPatch clampSuction electrodeSPsLPsSPsVdark ( mV ) −43 . 2 ± 0 . 7 ( n = 30 ) −45 . 9 ± 1 . 1 ( n = 10 ) –IRmembrane ( MΩ ) 518 ± 41 ( n = 8 ) 442 ± 68 ( n = 9 ) –τmembrane ( ms ) 31 . 9 ± 4 . 9 ( n = 8 ) []a12 . 9 ± 1 . 3 ( n = 9 ) [***]a–Max response ( mv ) 3032–i1/2 ( 520 nm ph·µm−2 ) ; control149 ± 25 ( n = 10 ) []b1 . 9 × 105 ± 1 . 1 × 105 ( n = 7 ) []c–i1/2 ( 520 nm ph·µm−2 ) ; regenerated63 ± 11 ( n = 12 ) [**]b2385 ± 513 ( n = 7 ) [*]c–Sensitivity 520/590; control4 . 4 ± 0 . 9 ( n = 5 ) []d1 . 1 ± 0 . 04 ( n = 6 ) []e–Sensitivity 520/590; regenerated5 . 6 ± 1 . 0 ( n = 5 ) [n . s . ]d1 . 8 ± 0 . 1 ( n = 7 ) [**]e–Integration time , dim flash ( s ) ; control0 . 32 ± 0 . 05 ( n = 13 ) []f––Integration time , dim flash ( s ) ; regenerated0 . 81 ± 0 . 14 ( n = 9 ) [***]f–1 . 45 ± 0 . 10 ( n = 10 ) TTP at i1/2 ( s ) ; regenerated0 . 29 ± 0 . 04 ( n = 11 ) []g0 . 11 ± 0 . 007 ( n = 10 ) [***]g–τrec at i1/2 ( s ) ; regenerated1 . 05 ± 0 . 27 ( n = 11 ) []h0 . 12 ± 0 . 02 ( n = 10 ) [***]h–i1/2 ( λmax ph·µm−2 ) ; regenerated63 ± 11 ( n = 12 ) []i777 ± 167 ( n = 7 ) [***]i–Dim-flash sensitivity ( mV·ph−1·µm2 ) ; regenerated0 . 61 ± 0 . 17 ( n = 8 ) ––Dim-flash sensitivity ( %·ph−1·µm2 ) ; regenerated3 . 0 ± 0 . 6 ( n = 8 ) ––a ( pA ) ; regenerated––0 . 41 ± 0 . 04 ( n = 10 ) a% ( %·R* −1 ) ; regenerated2 . 6 ± 0 . 5 ( n = 8 ) –2 . 6 ± 0 . 3 ( n = 10 ) SNR; regenerated––1 . 5 ± 0 . 1 ( n = 10 ) Idark ( pA ) ; regenerated13 ± 3 ( n = 4 ) –16 ± 1 ( n = 10 ) Collecting area ( µm2·R*·ph−1 ) ; regenerated––0 . 83 ± 0 . 17 ( n = 10 ) Amplification constant ( s−2 ) ; regenerated––0 . 59 ± 0 . 09 ( n = 10 ) Values are given as ‘mean ± SEM ( sample size ) [statistical significance]identifier letter’; n . s . : not significant; *p < 0 . 05; **p < 0 . 01; ***p < 0 . 001; Vdark: dark membrane potential; IRmembrane: input resistance; τmembrane: membrane time constant; i1/2: half-maximal response flash strength; TTP: time-to-peak; τrec: decay time constant; a: absolute single photon response; a%: fractional single photon response; SNR: signal-to-noise ratio; Idark: dark current; SPs: short photoreceptors; LPs: long photoreceptors .", "Light stimulation evoked a hyperpolarization in both photoreceptors ( Figure 1C , D ) , with peak changes in membrane potential of up to 30 mV ( SPs ) and 32 mV ( LPs ) in response to saturating flashes .", "The amplitudes of the flash responses from SPs were described by exponential saturation functions ( Figure 1C , E ) .", "From the curves , we obtained a ratio of 4 . 4 ± 0 . 9 ( n = 5 ) for the sensitivities of these photoreceptors at 520 nm and 590 nm .", "This value is in reasonably good agreement with the ratio of 5 . 6 predicted by an 11A1 visual pigment template ( Govardovskii et al . , 2000 ) having a λmax of 517 nm ( Figure 1—figure supplement 1A ) , the absorbance maximum of SP outer segments found with microspectrophotometry ( Govardovskii and Lychakov , 1984 ) , and is thus consistent with the expression of an Rh1 visual pigment .", "For LPs , the flash responses displayed two components ( Figure 1D , F ) : the first component had kinetics , sensitivity , and spectral preference similar to SPs; the second component had faster kinetics , lower sensitivity , and a ratio of sensitivities at 520 nm and 590 nm of 1 . 1 ± 0 . 04 ( n = 6 ) .", "This ratio agrees with the value of 1 . 1 predicted by an 11A1 template ( Govardovskii et al . , 2000 ) , whose λmax is set at 555 nm ( Figure 1—figure supplement 1A ) , the absorbance maximum of LP outer segments ( Govardovskii and Lychakov , 1984 ) , consistent with their expression of an LWS pigment .", "It is likely that the first component of the flash response from LPs represents input from SPs , probably mediated by gap junctions; this arrangement would represent in lamprey retina an arrangement homologous to rod-cone coupling in jawed vertebrates ( Asteriti et al . , 2014 ) .", "In support of this interpretation , we observed with Lucifer Yellow injection thin telodendria emanating from the synaptic pole of the photoreceptors and extending laterally into the inner plexiform layer ( Figure 2 ) : the only known function of these processes in jawed vertebrates is that of forming inter-photoreceptor junctional contacts ( O'Brien et al . , 2012 ) .", "We attempted to uncouple these cells pharmacologically with MFA ( 100 µM ) or 2-APB ( 10–20 µM ) , known blockers of retinal gap junctions , but unfortunately these agents produced marked non-specific effects ( not shown ) . 10 . 7554/eLife . 07166 . 005Figure 2 . Lamprey photoreceptors extend telodendrial processes . An example of a lucifer yellow stain of a live SP showing two thin processes ( arrowheads ) extending laterally from the synaptic pole into the outer plexiform layer . DOI: http://dx . doi . org/10 . 7554/eLife . 07166 . 005 During their spawning run , lampreys do not feed and rely exclusively on stored reserves for several months , leading to a vitamin A deficiency ( Wald , 1942 ) that could hinder visual pigment regeneration .", "We thus wondered whether some of the visual pigment in our preparations might have been in a bleached state ( i . e . , devoid of its light-sensing chromophore ) .", "Clarifying this point was a crucial prerequisite to our subsequent assessment of single photon processing by SPs , for reasons explained in the rest of this paragraph .", "In jawed vertebrates , bleached rod and cone opsins constitutively activate the phototransductive cascade at a very low rate ( Cornwall and Fain , 1994; Cornwall et al . , 1995 ) .", "Due to this property in rods , in which pigment regeneration is much slower than in cones , bleaches of even a small fraction of the total pigment pool caused by bright light lead to a significant and long-lasting desensitization , which is much larger than what is expected from the simple decrease in light-sensitive visual pigment molecules ( Fain et al . , 2001; Lamb and Pugh , 2004 ) .", "Bleaching desensitization thus leads to a reduction in phototransduction gain , and therefore , in the single photon response amplitude .", "Assuming that lamprey opsins behave similarly to those of jawed vertebrates , the possible presence of bleached visual pigment in our experiments ( see above ) raises the possibility that SPs were desensitized relative to their full potential .", "To examine whether this was the case , we regenerated any bleached visual pigment molecules by superfusing the retinal slices in the recording chamber with the artificial analog 9-cis-Retinal ( 100 µM for 20–25 min ) .", "The sensitivity at 520 nm of two SPs , recorded both in control and during delivery of 9-cis-Retinal , increased by 2 . 0 and 2 . 5-fold ( Figure 3A ) .", "Moreover , sensitivity was higher ( p < 0 . 01 ) in regenerated than in control SPs: half-maximal response at 520 nm evoked with flashes ( i1/2 ) of 63 ± 11 photons·µm−2 ( n = 12; regenerated ) vs 149 ± 25 photons·µm−2 ( n = 10; control ) .", "Thus , some of the visual pigment molecules in SP outer segments were indeed bleached .", "The ratio of sensitivities at 520 and 590 nm did not differ significantly ( p = 0 . 42 ) between regenerated and control SPs: 5 . 6 ± 1 . 0 ( n = 5; regenerated ) vs 4 . 4 ± 0 . 9 ( n = 5; control ) .", "To test whether such bleaching was associated to desensitization , we examined dim-flash integration time ( Jones et al . , 1996 ) .", "In one SP , integration time increased by 2 . 6-fold after superfusion with 9-cis-Retinal and the same parameter was significantly higher ( p < 0 . 001 ) in regenerated than in control SPs ( Figure 3B ) : 0 . 81 ± 0 . 14 s ( n = 9; regenerated ) vs 0 . 32 ± 0 . 05 s ( n = 13; control ) .", "These results strongly suggest that SPs were in a state of bleaching desensitization . 10 . 7554/eLife . 07166 . 006Figure 3 . Visual pigment regeneration reveals the full sensitivity of photoreceptors in the upstream migrating river lamprey .", "( A ) Photovoltage response amplitudes to 520-nm flashes before ( gray circles ) and after visual pigment regeneration with 9-cis-Retinal ( red circles with a dot ) of a SP .", "Left and right ordinate values refer to left and right data sets , respectively , which were adjusted to match saturating amplitudes .", "( B ) Normalized-averaged-normalized dim-flash photovoltage responses in control ( n = 13 ) and regenerated SPs ( n = 9 ) , highlighting the difference in integration time .", "These records were obtained as follows:", "( i ) the average dim-flash response of each SP was normalized to its peak amplitude ( always below 2 mV ) ,", "( ii ) normalized responses were averaged across cells ,", "( iii ) the final average was normalized to its peak .", "Shaded areas show ±1 SEM .", "( C ) Photovoltage response amplitudes to 520-nm flashes before ( gray circles ) and after visual pigment regeneration with 9-cis-Retinal ( red circles with a dot ) of a LP .", "Responses to 590-nm flashes are also shown ( small empty circles; error bars are smaller than circle diameter ) .", "Error bars are SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 07166 . 006 Although secondary to the main goal of this analysis , we also tested the effect of 9-cis-Retinal on LPs .", "In one LP , the sensitivity at 520 nm increased by 22-fold after superfusion with 9-cis-Retinal ( Figure 3C; second , lower sensitivity component ) .", "Moreover , sensitivity was much higher ( p < 0 . 05 ) in regenerated than in control LPs: half-maximal response at 520 nm evoked with flashes ( i1/2 ) of 2385 ± 513 photons·µm−2 ( n = 7; regenerated ) vs 1 . 9 × 105 ± 1 . 1 × 105 photons·µm−2 ( n = 7; control; second , lower sensitivity component; see ‘Materials and methods’ for details on the uncertainty of this specific value ) .", "As expected for the incorporation of 9-cis-Retinal in a significant fraction of the LP visual pigment pool ( Makino et al . , 1999 ) , this increase in sensitivity was associated with a marked hypsochromic shift .", "Specifically , the ratio of sensitivities at 520 and 590 nm went from 1 . 1 to 1 . 7 in the single LP treated with 9-cis-Retinal ( Figure 3C , compare large and small circles ) and was significantly higher ( p < 0 . 01 ) in regenerated than in control LPs: 1 . 8 ± 0 . 1 ( n = 7; regenerated ) vs 1 . 1 ± 0 . 04 ( n = 6; control ) .", "To examine the properties of lamprey photoreceptors under fully dark-adapted conditions , all subsequent experiments were made on retinas pretreated with 9-cis-Retinal .", "After pigment regeneration , the photovoltage responses of SPs , recorded with patch clamp , remained markedly slower than those of LPs ( Figure 4A , B ) .", "To characterize the photoreceptors' kinetics , for each recorded cell we plotted time-to-peak ( TTP ) and decay time constant ( τrec ) as a function of flash strength normalized to its half-maximal value ( i1/2 ) ( Figure 4C , D ) .", "From linear fits to the data , we estimated the values of these parameters at i1/2: for SPs , the TTP was 0 . 29 ± 0 . 04 s ( n = 11 ) and the τrec was 1 . 05 ± 0 . 27 s ( n = 11 ) .", "In contrast , for LPs , the TTP was only 0 . 11 ± 0 . 007 s ( n = 10; p < 0 . 001 ) and the τrec only 0 . 12 ± 0 . 02 ( n = 10; p < 0 . 001 ) .", "Therefore , when compared at flash strengths eliciting responses of similar fractional amplitude , SPs were indeed slower than LPs .", "Importantly , while the estimates of TTP and τrec in LPs may have been influenced to some degree by the signals that are fed to them from SPs ( see above ) , the latter would have acted to reduce ( rather than increase ) the differences in kinetics between the two photoreceptors . 10 . 7554/eLife . 07166 . 007Figure 4 . SPs are markedly slower than LPs .", "( A and B )", "Average responses to 520-nm flashes of a SP ( 0 . 5 , 1 . 6 , 5 . 4 , 15 , 45 , 136 , 398 , 1128 photons·µm−2 ) and a LP ( 16 , 51 , 170 , 469 , 1413 , 4314 , 12 , 597 , 38 , 847 , 77 , 695 ph·µm−2 ) , both recorded with patch clamp after visual pigment regeneration with 9-cis-Retinal .", "( C and D )", "Plots of time-to-peak ( TTP ) and decay time constant ( τrec ) vs flash strength , normalized to its half-maximal value i1/2 , in regenerated SPs ( n = 11; empty circles ) and LPs ( n = 10; full circles ) .", "The data from each cell are connected by lines . DOI: http://dx . doi . org/10 . 7554/eLife . 07166 . 007 To compare the intrinsic light sensitivity of regenerated SPs and LPs , we first considered whether we should correct their half-maximal flash strengths ( i1/2 ) measured at 520 nm for:", "( i ) the position of the peaks of their action spectra ( λmax ) with respect to the stimulus wavelength and", "( ii ) the smaller quantum efficiency of pigment bound to 9-cis-Retinal ( about one third; Hubbard and Kropf , 1958; Hurley et al . , 1977 ) .", "Both factors have the effect of reducing the sensitivity displayed by the photoreceptor with respect to its maximum achievable level .", "For SPs , we made the conservative assumption that they incorporated only a negligible amount of 9-cis-Retinal , as suggested by their limited increase in sensitivity following regeneration combined with their expression of bleaching desensitization ( and supported by the non-significant change in their 520/590 nm sensitivity ratios ) .", "This implied that our 520-nm flashes essentially coincided with λmax ( 517 nm , see above ) and that no correction was necessary for their i1/2 of 63 ± 11 photons·µm−2 ( n = 12 ) .", "Given the conservative nature of the above assumption , this value provides a lower bound for the maximal sensitivity of fully dark-adapted SPs .", "For LPs , we made the equally conservative assumption that their entire visual pigment pool was replaced with 9-cis-Retinal .", "We then predicted their modified action spectrum ( Figure 1—figure supplement 1B ) by slightly adjusting two parameters of the 9A1 template for red cones of Makino et al . ( 1999 ) ( λmax_A0 from 508 to 507 nm and λmax_G1 from 567 to 566 nm; see their Table 2 ) so as to match our experimentally determined ratio of sensitivities at 520 and 590 nm of 1 . 8 ± 0 . 1 ( n = 7; see above ) .", "Taking into account the off-peak position of our flashes ( 520 nm ) relative to the λmax of this action spectrum ( 541 nm ) and the smaller quantum efficiency of regenerated pigment , the corrected i1/2 of LPs was 777 ± 167 photons·µm−2 ( n = 7 ) .", "Given the conservative initial assumption , this value provides an upper bound for the maximal sensitivity of fully dark-adapted LPs .", "The i1/2 of LPs was much higher than that of SPs ( p < 0 . 001 ) .", "Note that signals feeding from SPs into LPs would have acted to reduce ( rather than increase ) the differences in sensitivity between the two photoreceptors , leaving our conclusions unchanged .", "A crucial functional measure of the position of lamprey SPs with respect to the evolutionary transition from an ancestral cone to the modern rod is their performance in amplifying single photons ( Lamb , 2013 ) .", "Regenerated SPs were highly sensitive , with absolute and fractional dim-flash sensitivities in patch clamp of 0 . 61 ± 0 . 17 mV·photons−1·µm2 ( n = 8 ) and 3 . 0 ± 0 . 6%·photons−1·µm2 ( n = 8 ) .", "We obtained a first estimate of their fractional single photon response of 2 . 6 ± 0 . 5%·R* −1 ( n = 8 ) by dividing the fractional dim-flash sensitivity with a theoretical effective collecting area of 1 . 18 µm2·R*·photons−1 ( see ‘Materials and methods’ ) .", "A search for quantal responses using the patch-clamp technique proved inconclusive , as also observed in similar recordings of photoreceptors from those jawed vertebrates having extensively coupled rods ( Fain , 1975 ) .", "We thus performed suction electrode photocurrent recordings from the conical outer segments of SPs , in retinae pretreated with 9-cis-Retinal ( 100 µM for 20–25 min ) : this recording technique only measures the current flowing through the membrane enclosed in the pipette and is thus ideally suited to examine phototransduction in a given photoreceptor without an appreciable contribution of its electrically coupled neighbors ( Baylor et al . , 1979a ) .", "Under these conditions , responses to repeated delivery of dim flashes were highly variable in amplitude ( Figure 5A ) .", "We estimated the absolute amplitude of the single photon response ( a ) to be 0 . 41 ± 0 . 04 pA ( n = 10 ) , by dividing the increase in the time-dependent variance by the mean response for each SP ( Figure 5B ) ( Rieke and Baylor , 1998 ) ( for details on the use of variance analysis in single photon response estimation see the ‘Materials and methods’ ) .", "The normalized time-dependent squared mean responses and variance increases overlapped ( Figure 5B ) ( Rieke and Baylor , 1998 ) , consistent with the single photon responses being governed by Poisson statistics .", "The fractional amplitude of the single photon response ( a% ) , determined for each SP on the basis of its maximal response to a single saturating flash delivered prior to the dim flash trains , was 2 . 6 ± 0 . 3%·R* −1 ( n = 10 ) , in line with the independent estimate obtained with patch ( see above ) .", "Lastly , the signal-to-noise ratio ( SNR ) , determined for each SP as the ratio of a over the standard deviation of the biological component of dark noise measured between consecutive dim flashes ( 0 . 5–20 Hz ) , was 1 . 5 ± 0 . 1 ( n = 10 ) .", "Importantly , the values of a , a% , and SNR in SPs are within the range reported for jawed vertebrate rods ( Figure 6 ) .", "In these experiments , we:", "( i ) recorded only from intact outer segments ( Figure 1C , inset ) ,", "( ii ) observed similar dark currents ( maximum current change with a saturating flash ) with patch clamp ( 13 ± 3 pA , n = 4 ) and suction electrodes ( 16 ± 1 pA , n = 10 ) , and", "( iii ) measured similar collecting areas ( 0 . 83 ± 0 . 17 µm2·R*·photons−1 , n = 10; ratio of the squared mean response over the product of the variance increase and the flash strength [Rieke and Baylor , 1998] ) to theoretical prediction ( 1 . 18 µm2·R*·photons−1 ) .", "From this , it is highly likely that we had complete suction of the outer segment , and as such , we could make reliable estimates of a% and SNR . 10 . 7554/eLife . 07166 . 008Figure 5 . Suction electrode recordings of SP photocurrents in the single photon regime .", "( A ) Samples of dim-flash response trains recorded from the outer segments of 4 SPs ( SP1: flash strength = 1 . 64 photons·µm−2 , a = 0 . 25 pA; SP2: 3 . 26 ph·µm−2 , 0 . 43 pA; SP3: 1 . 64 ph·µm−2 , 0 . 56 pA; SP4: 3 . 26 ph µm−2 , 0 . 56·pA ) .", "( B ) Single photon response analysis from one SP ( SP3 in panel A ) , showing the mean response μ ( thin trace: gross mean response , dotted trace: mean dark current , thick trace: net mean response ) , time-dependent variance σ2 ( thin trace: gross variance , dotted trace: dark current variance , thick trace: net variance ) , normalized squared mean response norm μ2 ( thin trace ) and variance norm σ2 ( thick trace ) .", "Dashed lines indicate the current baseline or zero level .", "Dark current records were taken from the last 2 s preceding each flash and where baselined in the first 1 . 1 s , therefore , dark and net current records cover up to 0 . 9 s after the flash ( see ‘Materials and methods’ ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 07166 . 00810 . 7554/eLife . 07166 . 009Figure 6 . Lamprey SP single photon response parameters in the context of those of jawed vertebrate rods . River lamprey: this study; Jawed vertebrates: previous studies .", "( A ) Absolute amplitude of the single photon response ( a ) .", "( B ) Fractional amplitude of the single photon response ( a% ) .", "( C ) Signal-to-noise ratio ( SNR ) .", "Data points shown in the figure were obtained with suction pipette recordings of photoreceptor outer segments .", "Error bars for lamprey are SEM .", "Letters next to the data points correspond to the following references: a ( Palacios et al . , 1998 ) , b ( Ala-Laurila et al . , 2007 ) , c ( Baylor et al . , 1984 ) , d ( Baylor et al . , 1979a , 1979b ) , e ( Baylor et al . , 1980 ) , f ( Nakatani et al . , 1991 ) , g ( Robinson et al . , 1993 ) , h ( Field and Rieke , 2002b ) , i ( Field and Rieke , 2002a ) , j ( Okawa et al . , 2010 ) , k ( Mendez et al . , 2001 ) , l ( Burns et al . , 2002 ) , m ( Azevedo and Rieke , 2011 ) , n ( Krispel et al . , 2006 ) , o ( Luo and Yau , 2005 ) , p ( Makino et al . , 2004 ) , q ( Wen et al . , 2009 ) , r ( Gross and Burns , 2010 ) , s ( Palma et al . , 2001 ) , t ( Donner et al . , 1990 ) , u ( Vogalis et al . , 2011 ) , v ( Nikonov et al . , 2006 ) , w ( Rieke and Baylor , 2000 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 07166 . 009 The single photon response analysis made with suction pipettes allowed us to estimate an amplification constant of phototransduction ( Pugh and Lamb , 1993 ) in lamprey SPs of 0 . 59 ± 0 . 09 s−2 at 9–11°C ( n = 10 ) , which lies between that of the large amphibian rods at room temperature ( ∼0 . 1 s−2 ) and that of small mammalian rods at body temperature ( ∼8 s−2 ) ( Lamb and Pugh , 2006 ) .", "The integration time measured with suction electrodes was 1 . 45 ± 0 . 10 s ( n = 10 ) , somewhat higher than that obtained with patch clamp ( which also incorporates downstream processing in the inner segment ) .", "Taken together , the high-amplification constant and relatively long integration time are key contributors to SPs' single photon performance ." ], [ "We examined lamprey SPs and LPs and found that their general properties closely match those of jawed vertebrate rods and cones: dark membrane potentials of around −45 mV , hyperpolarizing responses of up to 30 mV upon illumination , and the expression of a prominent hyperpolarization-activated Ih current are all typical traits of jawed vertebrate photoreceptors and must therefore have been in place already by the middle Cambrian .", "A similar conclusion can be made for bleaching adaptation , which we confirmed to be present in SPs .", "In jawed vertebrates , this phenomenon is the result of a very low-constitutive activity of free opsin and occurs in both rods and cones exposed to bleaching lights ( Cornwall and Fain , 1994; Cornwall et al . , 1995; Fain et al . , 2001; Lamb and Pugh , 2004 ) .", "To assess whether SPs , the phylogenetic homologues of rods ( Pisani et al . , 2006; Muradov et al . , 2008; Lamb , 2013 ) , also operate like rods , we focused on multiple properties both at the level of single photon detection in the outer segment and of downstream signal processing in the inner segment .", "The rationale behind this approach is that , even if individual functional parameters of SPs and rods could have evolved independently towards a common present state ( convergent evolution ) , this becomes quite unlikely if multiple common features are observed .", "We find that SPs are exquisitely sensitive to light and feed their signals to the less sensitive but intrinsically faster LPs , similarly to the way rods feed their signals to cones via gap junctions in jawed vertebrates ( Asteriti et al . , 2014 ) .", "The likely anatomical substrate of this signal crossover is represented by the thin telodendrial processes that we observed to extend laterally from the synaptic pole of the photoreceptors .", "Telodendria are ubiquitous in jawed vertebrates and their only known function is to form gap junctional contacts with nearby photoreceptors ( O'Brien et al . , 2012 ) .", "While in mammals these processes are known to extend only from cone pedicles , in cold-blooded vertebrates they are also formed by rods ( Fadool , 2003 ) .", "Since rod-cone coupling is thought to provide a secondary route for rod signals when the high-gain output synapse of rods is saturated ( Attwell et al . , 1987 ) , the question arises of whether lamprey SPs have properties of synaptic transmission similar to those of rods and a dedicated postsynaptic circuitry for scotopic signaling .", "Furthermore , in mammalian photoreceptors , the Ih current has been proposed to assist rod-cone signaling via gap junctions ( Seeliger et al . , 2011 ) , a role it could also play in the lamprey retina given the presence of telodendria and SP–LP signal flow .", "The functional similarities between lamprey SPs and jawed vertebrate rods discussed above , extend to the efficient processing in the single photon regime .", "With regards to scotopic performance , we found that dark-adapted SPs approach the efficiency of jawed vertebrate rods , both in terms of absolute and fractional single photon response and of its SNR .", "Here , it is important to note that while the fractional single photon response a% and the SNR are potentially subject to overestimation due to the fact that their denominators ( the circulating current in darkness for a% and the biological dark noise for the SNR ) appear smaller if the outer segment is damaged or is not fully within the recording pipette ( see however the evidence above that these conditions were respected in our experiments ) , this issue does not exist when estimating the absolute amplitude a .", "Taken together , our findings raise the strong possibility that our last common ancestor in the middle Cambrian had already evolved scotopic vision .", "Recent evidence indicates that the lamprey line diverged soon after ( Smith et al . , 2013 ) the occurrence of two rounds of whole-genome duplication in stem vertebrates ( 2R ) ( Dehal and Boore , 2005; Putnam et al . , 2008 ) , which led to the diversification of the ancestral visual opsin and phototransduction protein repertoire ( Lagman et al . , 2013 ) .", "Therefore , dim-light vision appears to have been acquired rapidly once a dedicated set of genes became available for specializing a type of cone into the rod ( Okano et al . , 1992; Yokoyama , 2000; Lamb et al . , 2007; Kawamura and Tachibanaki , 2008; Shichida and Matsuyama , 2009 ) .", "Admittedly , one cannot definitively exclude that such performance was refined independently in the two branches following their split ∼505 Ma ( Erwin et al . , 2011 ) : to do so , one would need to resurrect ( Thornton , 2004 ) the full ancestral phototransductive cascade and then characterize its function .", "However , our data provide compelling support for the view that the evolution of the modern rod was already well under way at the time of divergence of the two branches:", "( i ) the similarities between SPs and rods are multiple and extend from phototransduction to downstream processing ,", "( ii ) the photoreceptors specialized for scotopic vision in lampreys and in jawed vertebrates derive from one and the same precursor ( and not from different ancestral cones ) ,", "( iii ) some of the phototransduction protein isoforms examined thus far in SPs clade with those of rods .", "This newly evolved ability to operate in dim light would have provided a significant advantage to early vertebrates faced with intense competition in a rapidly evolving ecological landscape ( Paterson et al . , 2011; Lacalli , 2012 ) .", "In coincidence with the submission of the present work , a study on the photoreceptors of a different species of lamprey was published ( Morshedian and Fain , 2015 ) , based on suction electrode recordings from outer segments .", "Although our study examines a broader range of photoreceptor properties , with both patch-clamp and suction electrode recordings , the two studies converge on the positive response to single photons ." ], [ "The inner segments of SPs and LPs lying close to the slice surface ( Figure 1A ) were visually targeted with 5–6 MOhm pipettes pulled with a P-97 ( Sutter Instruments , Novato , CA ) from borosilicate glass capillaries ( 1B120F-4 , WPI , Sarasota , FL ) and filled with a solution containing ( in mM ) 90 Kaspartate , 20 K2SO4 , 15 KCl , 10 NaCl , 5 K2Pipes , as well as 0 . 5 mg ml−1 Lucifer yellow , and corrected to a pH of 7 . 20 with KOH/HCl .", "The backfilling solution also contained 0 . 4 mg ml−1 Amphotericin B ( item no . 11636 , Cayman , Ann Arbor , MI ) pre-dissolved in dimethyl sulfoxide ( DMSO ) at 60 mg ml−1 .", "Based on an analysis of the liquid junction and Donnan potentials when using this solution and recording photoreceptors ( Cangiano et al . , 2012 ) , we report uncorrected values of membrane potential .", "Recordings were made with an Axopatch 1D amplifier , low-pass filtered at 500 Hz and acquired at 5 KHz with a Digidata 1320 and pClamp 9 software ( Molecular Devices , Sunnyvale , CA ) .", "The outer segment current of intact SPs lying close to the slice surface ( Figure 1C , inset ) was recorded with suction electrodes ( Baylor et al . , 1979a ) .", "Specifically , glass capillaries ( intraMARK , Blaubrand , Germany ) were pulled with a P-97 , broken to obtain even tips of 10–20 µm and heat polished to ∼4-µm inner diameter .", "Tips were silanized by dipping in Sigmacote solution ( SL2 , Sigma–Aldrich ) , followed by vigorous back suction in air .", "Finally , pipettes were rinsed and filled with filtered Ames' medium , for a resistance in the bath of 2–3 MOhm .", "Intrapipette pressure was controlled with a pneumatic system filled with light mineral oil ( 330779; Sigma–Aldrich ) using coarse and fine precision syringes ( 100 µL and 10 µL; Hamilton , Reno , NV ) actuated by micrometer heads .", "Recordings were made in voltage clamp ( holding voltage set at zero ) with the same apparatus described above , except that signals were low-pass filtered at 20 Hz before acquisition ( 4-pole Bessel filter ) .", "Full-field stimuli of unpolarized light were delivered by a green LED ( 520 nm; OD520; Optodiode Corp . , Newbury Park , CA ) or a yellow-orange LED ( 590 nm; APG2C3-590; Roithner LaserTechnik , Austria ) mounted beside the objective turret .", "LEDs were driven by current sources commanded through the analog outputs of a Digidata 1320A ( Axon Instruments , Foster City , CA ) .", "The power density reaching the recording chamber vs LED drive was measured separately with a calibrated low-power detector ( 1815-C/818-UV; Newport , Irvine , CA ) positioned at the recording chamber .", "Flash duration was in the range 1–27 ms . Consecutive bright flashes were delivered at intervals of 13 s between each other .", "The photon flux density reaching the photoreceptors was derived from the measured power density and was likely to be overestimated to varying degrees across recorded cells due to reflection at the air–water interface and absorption by the surrounding tissue .", "In patch-clamp recordings , outer segments were generally , but not strictly , oriented at right angles with respect to the direction of incident light .", "In suction electrode recordings , orthogonality was instead guaranteed by the pipette itself .", "Where specified , any bleached visual pigment was regenerated with the artificial chromophore analog 9-cis-Retinal .", "Stock solutions of 9-cis-Retinal ( R5754; Sigma–Aldrich ) in ethanol ( 100 mM ) were prepared in darkness and stored at −80°C .", "On the day of the experiment , an aliquot was thawed and diluted to a final concentration of 100 µM in Ames' medium integrated with 1% wt/vol fatty acid-free bovine serum albumin ( A8806; Sigma–Aldrich ) , an effective solubilizing and protective agent ( Li et al . , 1999 ) .", "This solution was delivered to the preparation directly in the recording chamber , without modifying flow rate or temperature , for 20–25 min followed by washout .", "A pharmacological blockade of gap junctions was attempted with meclofenamic acid ( MFA; M4531 , Sigma–Aldrich ) and 2-aminoethyl diphenylborinate ( 2-APB; D9754 , Sigma–Aldrich ) .", "The Ih current was blocked with 4-Ethylphenylamino-1 , 2-dimethyl-6-methylaminopyrimidinium chloride ( ZD7288; cat . no . 1000 , Tocris , United Kingdom ) .", "The effective collecting area of SPs was estimated according to the approximate relation ( Baylor et al . , 1979b ) : ( 1 ) Ac=2 . 303·V·f·α·Qisom , where V is the volume of their truncated conical outer segments , estimated at 102 µm3 from mean values of length ( 13 . 0 µm ) , basal diameter ( 4 . 5 µm ) , and apical diameter ( 1 . 6 µm ) measured in optical images acquired from live slices; f is a factor accounting for the dichroism of native opsin ( 1 for light incident along the outer segment axis and 0 . 5 for unpolarized light incident at right angles ) ; α is the specific axial pigment density at the maximum absorption wavelength , measured ( Govardovskii and Lychakov , 1984 ) in lamprey SPs at 0 . 015 µm−1 , a value similar to that of lower vertebrate rods containing rhodopsin and/or porphyropsin ( Harosi , 1975 ) ; Qisom is the quantum efficiency of photoisomerization , assumed to have the rhodopsin value of 0 . 67 R*·photons−1 .", "Note that any bleached visual pigment regenerated with 9-cis-Retinal would have the somewhat different absorption spectrum and quantum efficiency of isorhodopsin ( Hubbard and Kropf , 1958; Hurley et al . , 1977; Makino et al . , 1999 ) ; however , this contribution was neglected since the twofold–threefold increase in sensitivity displayed by SPs after delivery of 9-cis-Retinal could result from the regeneration of a small fraction of bleached pigment due to the phenomenon of bleaching desensitization ( e . g . , in larval tiger salamander rods , bleaching of ∼6% rhodopsin halves sensitivity [Jones et al . , 1996] ) .", "Data in the text and graphs are reported as mean and standard error of the mean .", "Statistical significance was assessed with the Mann–Whitney–Wilcoxon test .", "Integration time was defined as the integral of the dim-flash response divided by its peak amplitude .", "TTP was measured starting from the middle of the flash .", "Decay time constant ( τrec ) was estimated by fitting a single exponential to the second half of the flash response recovery phase ( i . e . , starting from the point when the response had recovered to about half of its peak amplitude ) .", "Exclusively for presentation purposes , the electrophysiological records shown in the figures were conditioned as follows:", "( i ) current-clamp patch recordings were ‘box car’ filtered with a running window of 20 ms ,", "( ii ) voltage-clamp suction pipette recordings were detrended ( Vogalis et al . , 2011 ) to remove slow fluctuations in the baseline current and digitally low-pass filtered at 2 Hz ." ] ]

[ "Vertebrates acquired dim-light vision when an ancestral cone evolved into the rod photoreceptor at an unknown stage preceding the last common ancestor of extant jawed vertebrates ( ∼420 million years ago Ma ) .", "The jawless lampreys provide a unique opportunity to constrain the timing of this advance , as their line diverged ∼505 Ma and later displayed high-morphological stability .", "We recorded with patch electrodes the inner segment photovoltages and with suction electrodes the outer segment photocurrents of Lampetra fluviatilis retinal photoreceptors .", "Several key functional features of jawed vertebrate rods are present in their phylogenetically homologous photoreceptors in lamprey: crucially , the efficient amplification of the effect of single photons , measured by multiple parameters , and the flow of rod signals into cones .", "These results make convergent evolution in the jawless and jawed vertebrate lines unlikely and indicate an early origin of rods , implying strong selective pressure toward dim-light vision in Cambrian ecosystems ." ]

[ "The eyes of humans and many other animals with backbones contain two different types of cells that can detect light , which are known as rod and cone cells .", "Rod cells are much more sensitive to light than cone cells .", "The rods allow us to see in dim light by amplifying weak light signals and transmitting information to other cells , including the cones themselves .", "It is thought that the rod cell evolved from the cone cell in the common ancestors of mammals , fish , and other animals with backbones and jaws at least 420 million years ago .", "Lampreys are jawless fish that diverged from the ancestors of jawed animals around 505 million years ago , in the middle of a period of great evolutionary innovation called the Cambrian .", "They have changed relatively little since that time so they provide a snapshot of what our ancestors' eyes might have been like back then .", "Like the rod and cone cells of jawed animals , the eyes of adult lampreys also have two types of photoreceptors .", "However , it was not clear whether the lamprey photoreceptor cells work in a similar way to rod and cone cells .", "Asteriti et al . collected lampreys in Sweden and France during their breeding season and used patch and suction electrodes to measure the activity of their photoreceptor cells .", "The experiments show that the short photoreceptor cells are more sensitive to light than the long photoreceptors and are able to amplify weak light signals .", "Also , the short photoreceptors send signals to the long photoreceptors in a similar way to how rod cells send information to cone cells .", "The similarities between lamprey photoreceptor cells and those of jawed animals support the idea that they have a common origin in evolutionary history .", "Therefore , Asteriti et al . conclude that the ability to see in low light evolved before these groups of animals diverged about 505 million years ago .", "The picture that emerges is one in which our remote ancestors inhabiting the Cambrian seas already possessed dim-light vision .", "This would have allowed them to colonize deep waters or to move at twilight , an adaptation suggestive of intense competition or predation from other life forms ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "chromosomes and gene expression" ]

[ [ "CpG islands ( CGIs ) are stretches of atypical genomic DNA sequences that mark most gene promoters ( Bird , 1986; Deaton and Bird , 2011 ) .", "Though individually unique in nucleotide sequence , mammalian CGIs share two features that often correlate but can vary independently: a G + C-rich base composition ( 65% vs 40% for the bulk genome ) and high density of the dinucleotide CpG ( 5–10-fold higher than the bulk genome ) .", "Whereas bulk genomic DNA is globally methylated , CGIs associated with promoters invariably lack DNA methylation .", "It has been proposed that CGIs function as generic platforms for gene regulation , probably via proteins that bind to CpG and influence chromatin modification ( Blackledge and Klose , 2011; Deaton and Bird , 2011 ) .", "This hypothesis has gained credence due to accumulating evidence that enrichment or depletion of specific chromatin marks at CGIs is linked to proteins that bind to unmethylated CpG .", "So far , all such proteins possess CXXC zinc finger domains that bind specifically to the CpG dyad in duplex DNA ( Lee et al . , 2001 ) .", "Cfp1 , for example , is a CXXC domain-containing component of the Set1/COMPASS complex ( Lee and Skalnik , 2005 ) , which generates H3K4 methylation , a signature chromatin mark at non-methylated CGIs ( Bernstein et al . , 2006; Guenther et al . , 2007 ) .", "Accordingly , Cfp1 is concentrated at CGIs as determined by chromatin immunoprecipitation ( ChIP ) and its absence is associated with reduced H3K4 methylation at many CGIs ( Thomson et al . , 2010; Clouaire et al . , 2012 ) .", "The H3K4 methyltransferases Mll1 and Mll2 are also CXXC proteins and each is found at CGIs in mouse embryonic stem cells ( ESCs ) as determined by ChIP-Seq ( Hu et al . , 2013; Denissov et al . , 2014 ) .", "Similarly the CXXC domain-containing proteins Kdm2a and Kdm2b are enriched at CGIs .", "Kdm2a is an H3K36 demethylase that contributes to depletion of H3K36me at CGI promoters ( Blackledge et al . , 2010 ) , whereas Kdm2b facilitates recruitment of the PRC1 complex to transcriptionally silent CGIs ( Farcas et al . , 2012; Wu et al . , 2013 ) .", "Previous studies have shown that CGI-like DNA sequences can impose an altered chromatin state in ESCs .", "Promoter-less CGI-like DNA sequences of invertebrate origin in mouse ES cells were initially shown to cause local enrichment of trimethylation of lysine 4 of histone H3 ( H3K4me3 ) and to recruit the CpG binding protein Cfp1 ( Thomson et al . , 2010 ) .", "Similar experiments using G + C-rich DNA derived from bacteria created chromatin marked by both H3K27me3 and H3K4me3 ( Mendenhall et al . , 2010 ) .", "Whereas H3K4me3 is characteristic of active promoters , H3K27me3 is associated with transcriptional repression via the polycomb complex .", "The coincidence of these two marks in so-called ‘bivalent’ chromatin is thought to be a feature of genes that are poised to become either active or silent during early development ( Azuara et al . , 2006; Bernstein et al . , 2006; Voigt et al . , 2013 ) .", "Many native CGIs in ESCs adopt a ‘bivalent’ chromatin structure when transcriptionally silent .", "Despite evidence that CXXC proteins play a role at CGIs , the hypothesis that CpG density is the critical determinant of CGI function has not been directly tested .", "Here we vary the DNA sequence composition of artificial promoter-less CGIs to assess the relative importance of G + C-richness and high CpG density .", "Our assay relies upon chromosomal integration of artificial CGI-like sequences into the genome of ESCs .", "We show that a bivalent chromatin configuration and absence of DNA methylation represent the default state of biologically inert CGI-like DNA sequences .", "While a high CpG density is essential , it is not sufficient to guarantee the bivalent chromatin structure , as CpG-rich DNA sequences with a G + C-poor average base composition do not acquire this chromatin signature .", "In fact A + T-rich , G + C poor insertions with a CpG frequency matching that of CGIs consistently become DNA methylated in ESCs , although the bivalent configuration can be restored if the dominant DNA methylation is removed .", "Our findings demonstrate that CpG-richness is essential for the formation of bivalent chromatin , whereas G + C-richness is required to exclude DNA methylation , which when present is dominant over the other chromatin marks ." ], [ "Comparison between CGIs and an equivalent number of sequences from the bulk genome shows that both CpG frequency and G + C richness are distinct in CGIs compared with bulk genomic DNA of mouse ( Figure 1A ) .", "A previous study showed that bacterial DNA with CGI-like features organised bivalent chromatin in ESCs ( Mendenhall et al . , 2010 ) .", "In order to verify and extend this result we introduced CGI-like DNA sequences of ∼1000 nucleotide pairs ( the average length of native CGIs ) into a bacterial artificial chromosome ( BAC ) containing a human ‘gene desert’ using recombineering ( See Figure 1—figure supplement 1 ) .", "A computer-generated CGI-like sequence ( Artificial CGI 1 ) was designed with a CpG frequency of one per ∼10 base pairs and a base composition of 65% G + C ( Figure 1A , Figure 1—figure supplement 2 and Figure 1—source data 1 ) .", "A second CGI-like sequence ( PuroGFP; Figure 1A , Figure 1—figure supplement 2 and Figure 1—source data 1 ) was of prokaryotic origin , being derived from a promoter-less bacterial puromycin gene adjacent to codon-optimised green fluorescent protein coding sequence ( Thomson et al . , 2010 ) .", "All constructs lack a promoter , allowing us to focus on the interaction between DNA sequence and chromatin modification without the complicating involvement of transcription .", "The gene desert regions flanking CGI-like sequences are intended to insulate against effects of the genomic and chromatin environment at different BAC integrations sites . 10 . 7554/eLife . 03397 . 003Figure 1 . A novel bivalent chromatin domain is formed at promoter-less artificial CGI-like sequences integrated within a gene desert in mouse ESCs .", "( A ) CpG frequency and G + C content of CGIs in the mouse genome ( blue circles ) and an equivalent number of equal-sized ( 1000 base pair ) random fragments of bulk genomic DNA ( red circles ) .", "( B ) Map of human gene desert 2 ( grey bars; Chr1:81 , 106 , 616-81 , 153 , 886 ) showing the integration site of the Artificial CGI-like construct ( purple box ) .", "Black boxes at the ends indicate bacterial BAC sequences .", "Black bars above indicate the position of Q-PCR amplicons ( not to scale ) .", "( C ) Representative anti-H3K4me3 and H3K27me3 ChIP profiles ( normalized to H3 ChIP ) and Suz12 ChIP profiles ( % Input; n = 3 ) for three independently transfected cell lines .", "Shaded box includes primers spanning the Artificial CGI .", "ChIP control amplicons are derived from the TSS of the active genes Sox2 ( S ) and GAPDH ( G ) ; the TSS of bivalent gene Hoxc8 ( H ) and an inconspicuous negative control region on mouse chromosome 15 ( − ) .", "Error bars indicate the standard deviation of PCR replicates .", "( D ) Bisulfite sequencing of the three cell lines shown in ( C ) .", "In the map above , blue strokes show CpGs in the CGI-like insert and the clear box indicates the bisulfite amplicon .", "Methylated and unmethylated CpGs are depicted as filled and open circles , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 03397 . 00310 . 7554/eLife . 03397 . 004Figure 1—source data 1 . Table showing properties of constructs used in these studies , including length , base composition and CpG frequency ( observed over expected CpG ratio; o/e ) of all tested constructs .", "Note that the o/e ratio takes the overall G + C content into account and therefore the High CpG/ Low G + C sequences have a high o/e ratio . DOI: http://dx . doi . org/10 . 7554/eLife . 03397 . 00410 . 7554/eLife . 03397 . 005Figure 1—figure supplement 1 . Bivalent chromatin at artificial CGI-like sequences in mouse ESCs . Schematic representation of the experimental protocol for insertion of artificial CGIs into the mouse ESC genome .", "A linearized plasmid containing the CGI like sequence , a selection cassette flanked by FRT sites , a single LoxP site and 5′ and 3′ homology regions were integrated into a human gene desert within a BAC via recombineering .", "The linearized BAC was then transfected into mouse ESCs .", "Colonies containing the BAC were screened for clones with low copy number integration and transfected with Flp to excise the selection cassette .", "Successful excision was confirmed by PCR and Southern blotting .", "Selected clones were used for analysis of chromatin modification and DNA methylation .", "( B ) Diagram above shows the CGI like sequence PuroGFP integrated into a human gene desert 1 ( mChr18:36 , 042 , 881-36 , 175 , 341 ) and the position of primer pairs used for ChIP ( not to scale ) .", "Representative H3K4me3 , H3K27me3 and Suz12 ChIP qPCR experiments ( n = 2 ) .", "The shaded box includes primers spanning the PuroGFP construct .", "( C ) Diagram above indicates the mouse ß-globin locus showing the integration site of the Artificial CGI 2 construct .", "Black bars indicate position of primers used for qPCR .", "H3K4me3 , H3K27me3 and Suz12 ChIP analysis are shown for a representative cell line ( data for 2 other cell lines not shown ) .", "Shading indicates primers spanning the Artificial CGI 2 .", "( D ) Bisulfite sequencing of the Artificial CGI 2 construct .", "In the map above , blue strokes show CpGs in the CGI-like insert and the clear box indicates the bisulfite amplicon .", "Methylated and unmethylated CpGs are depicted as filled and open circles , respectively .", "ChIP control amplicons as described in Figure 1C but including the TSS of the active gene ß-actin .", "Error bars indicate standard deviation of PCR replicates . DOI: http://dx . doi . org/10 . 7554/eLife . 03397 . 00510 . 7554/eLife . 03397 . 006Figure 1—figure supplement 2 . Synthetic DNA elements with different sequence properties .", "( A ) Sequence profiles of CGI-like constructs flanked on either side by 1 kb of human gene desert .", "Upper panels: % G + C plots; lower panels: CpGs-per-100 bp plots .", "X-axis length shows length in base pairs ( bp ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 03397 . 006 Three independently transfected stable ESC lines with low copy number random integrations of the BAC containing Artificial CGI 1 and one cell line with the PuroGFP CGI were selected for ChIP analysis ( See Figure 1—figure supplement 1 ) .", "As controls we monitored active genes ( Sox2 and Gapdh ) , which are marked by H3K4me3 , an endogenous bivalent gene ( Hoxc8 ) , which carries both H3K27me3 and H3K4me3 , and an inter-genic region of chromosome 15 ( m15 ) , which bears neither mark .", "The CGI-like insertions consistently generated a bivalent chromatin structure marked by H3K4me3 and H3K27me3 ( Figure 1B , C and Figure 1—figure supplement 1 ) .", "We refer to DNA sequence domains as bivalent using the convention that H3K4me3 and H3K27me3 marks coincide at a single integration site .", "It is possible that some cells in the population harbour an integrant marked by only one of these marks whereas other cells possess only the other mark .", "This configuration has not been detected at the few ESC bivalent domains tested so far .", "More likely is that the two marks are interspersed at a given bivalent sequence domain , though we did not test this experimentally ( See Voigt et al . , 2013 ) .", "The levels of H3K4me3 at the artificial CGI were similar to those at the endogenous bivalent gene Hoxc8 , whereas H3K4me3 levels in the DNA sequences flanking the CGI were unaffected by the insertion .", "H3K27me3 was less discrete , spreading variably into flanking regions of the human BAC , but Suz12 , a component of the PRC2 complex that deposits the H3K27me3 mark , was tightly localised to the CGI-like sequence in each case .", "We tested an unrelated artificial CGI-like sequence with similar overall sequence properties ( Artificial CGI 2; Figure 1—figure supplement 1 ) , this time integrated at a recombination cassette within the beta globin locus of mouse ESCs ( Lienert et al . , 2011 ) .", "Independent replicate stable transformant cell lines again showed consistent presence of a bivalent chromatin structure ( data not shown ) .", "The DNA methylation status of the integrated artificial CGIs was investigated by bisulfite sequencing .", "This showed that all the insertions reproducibly maintained a low level of DNA methylation ( Figure 1D; Figure 1—figure supplement 1 ) .", "DNA methylation levels at CGI-like sequences inserted into the gene desert ( ∼10% ) were consistently somewhat higher than in the cassette exchange system ( 0 . 5% ) and were somewhat higher than an endogenous control CGI in the same DNA samples ( Dlx5; ∼4–5% , data not shown ) .", "Despite this variability , it is evident that all of these artificial DNA sequences maintain a largely non-methylated status .", "To test whether these promoter-less artificial DNA sequences were transcriptionally inert , we performed ChIP with antibodies recognising three differentially phosphorylated forms of RNA polymerase II in replicate cell lines .", "None displayed a peak over the insert , whereas control active genes were RNA polymerase-positive as expected ( Figure 2A and Figure 2—figure supplement 1 ) .", "Consistent with the absence of RNA polymerase , we did not detect significant peaks of histone acetylation over the CGI-like insertions ( Figure 2B ) .", "In summary , data derived from three distinct promoter-less CGI-like sequences indicate that bivalent chromatin is the default state for transcriptionally inert CGI-like DNA in ESCs . 10 . 7554/eLife . 03397 . 007Figure 2 . H3K4me3 at a promoter-less artificial CGI forms independently of Cfp1 and RNA polymerase II .", "( A ) Map of gene desert 2 with integrated Artificial CGI-like construct labeled as in Figure 1B .", "Representative ChIP with an antibody specific for the N-terminus of RNA polymerase II for three independent cell lines ( % Input over IgG; n = 2 ) .", "( B ) Representative anti-H3K9/K14 acetylation ChIP profiles for three independently transfected cell lines normalized to H3 ChIP ( n = 2 ) .", "( C ) Mouse ES cells expressing GFP-tagged Cfp1 were transfected with Artificial CGI construct and bound Cpf1 was assayed by ChIP with anti-GFP antibodies in three independent cell lines ( n = 2 ) .", "( D ) Representative anti-H3K3me3 ChIP for three independent Cfp1−/− mouse ES cells transfected with the Artificial CGI construct ( n = 2 ) .", "Control ChIP amplicons are as in Figure 1C .", "Error bars indicate standard deviation of PCR replicates . DOI: http://dx . doi . org/10 . 7554/eLife . 03397 . 00710 . 7554/eLife . 03397 . 008Figure 2—figure supplement 1 . H3K4me3 at an artificial CGI independent of Cfp1 and RNA polymerase II .", "( A ) Scheme above shows gene desert 2 ( mChr1:81 , 106 , 616-81 , 153 , 886 ) with integration site of the Artificial CGI-like construct ( See Figure 1B ) .", "Representative ChIP with an antibody specific for the unphosphorylated C-terminus of RNA Pol II and the Serine 5 phosphorylated C-terminus of RNA Pol II for three independent cell lines ( n = 2 ) .", "Shaded bar indicates primers spanning the Artificial CGI .", "( B ) Undifferentiated mouse ES cells were cultured for 4 days without LIF and then another 4 days in the presence of retinoic acid ( RA ) .", "All panels photographed at 10× magnification .", "( C ) : H3K4me3 and H3K27me3 in ESCs vs neural precursor cells for two independent cell lines ( n = 2 ) .", "Error bars indicate standard deviation of PCR replicates . DOI: http://dx . doi . org/10 . 7554/eLife . 03397 . 008 We next asked whether the CXXC protein Cfp1 is enriched at the CGI-like sequences .", "To facilitate detection of Cfp1 , we introduced the BAC containing the artificial CGI into a transgenic cell line expressing a Cfp1-GFP fusion protein ( Denissov et al . , 2014 ) .", "Having verified that a bivalent domain was formed at the artificial CGI in these cells ( data not shown ) , ChIP was performed on three independent cell lines using an anti-GFP antibody .", "We consistently observed discrete enrichment of Cfp1 at the CGI-like insertion ( Figure 2C ) .", "To determine whether the formation of a bivalent domain at the inserted artificial CGI-like sequence was dependent on Cfp1 , the artificial CGI was introduced into Cfp1−/− mouse ES cells ( Carlone and Skalnik , 2001 ) and three independent lines were analysed by ChIP .", "H3K4me3 levels at the artificial CGI were clearly detectable in the Cfp1−/− cells , indicating that Cfp1 is not required for the formation of H3K4me3 levels at the bivalent domain ( Figure 2D ) .", "Depletion of Cfp1 was previously reported to preferentially cause a decrease of H3K4me3 at active genes without affecting non-productive genes ( Clouaire et al . , 2012 ) .", "In agreement with this finding , we observed reduced H3K4me3 at the active control gene Sox2 compared to wildtype cells ( Compare Figures 2D and 1C ) .", "We also followed the fate of chromatin modifications during differentiation of ESCs to neuronal progenitor cells ( Figure 2—figure supplement 1B ) and found a consistent drop in H3K4me3 accompanied by persistent or increased H3K27me3 ( Figure 2—figure supplement 1 ) .", "This transition from bivalency to H3K27me3 marking alone matches that at native CGI-associated genes that remain transcriptionally silent during differentiation ( Bernstein et al . , 2006 ) .", "Although G + C content and CpG frequency are related features , they can be varied independently ( Figure 1A ) .", "To establish the importance of these features for determination of bivalent chromatin , we varied CpG frequency and G + C content in 1000 base pair long artificial DNA sequences ( Figure 1—figure supplement 2 and Figure 1—source data 1 ) .", "An artificial CGI with a base composition similar to that of a normal CGI ( 65% G + C ) but with a low density of CpGs , similar to that of the bulk genome ( 1 CpG/100 bp ) , was designed ( Low CpG / High G + C ) .", "This Low CpG / High G + C sequence failed to create bivalent chromatin as neither H3K4me3 nor H3K27me3 was detected in three independent ESC lines ( Figure 3A ) .", "We note that the relative values of control and experimental data points are consistent between experiments although we observe variability in the absolute precipitation levels due to the use of different antibody suppliers between experiments over an extended time period .", "Our conclusion from this data is that a G + C-rich base composition alone is insufficient to recruit either H3K4me3 or H3K27me3 . 10 . 7554/eLife . 03397 . 009Figure 3 . High G + C content is not sufficient to create a bivalent chromatin domain . Map of gene desert 2 showing the integration site of the Low CpG / High G + C ( L-CpG / H-G + C ) construct labeled as in Figure 1B .", "Representative anti-H3K3me3 and H3K27me3 ChIP profiles ( normalized to H3 ) and Suz12 ChIP profiles ( % Input; n = 3 ) are shown for three independent transfected cell lines .", "Shaded bar includes primers spanning the Low CpG / High G + C construct .", "Control ChIP amplicons are as in Figure 1C .", "Error bars indicate standard deviation of PCR replicates . DOI: http://dx . doi . org/10 . 7554/eLife . 03397 . 009 This result raised the possibility that CpG frequency alone determines the chromatin state , with G + C content playing no role .", "To test this idea , we generated four different artificial DNA sequences that were CpG-rich to the same level as typical CGIs ( 10 CpGs/100 bp ) , but relatively A + T-rich in overall base composition ( three of 40% and one of 50% G + C on average; Figure 1—figure supplement 2 and Figure 1—source data 1 ) .", "Contrary to expectation , none of these insertions generated a focus of bivalent chromatin in multiple independent cell lines ( Figure 4A and Figure 4—figure supplement 1 ) .", "A potential explanation for this finding came from an analysis of DNA methylation status , which showed that in replicate cell lines the CGIs had all become densely methylated at CpGs ( Figure 4B and Figure 4—figure supplement 2 ) .", "The striking contrast between the consistent methylation-free status of three separate G + C-rich , CpG-rich integrants and the reproducible dense methylation of four unrelated A + T-rich , CpG-rich sequences of the same length indicates that base composition is a strong determinant of DNA methylation status .", "A plot of G + C-content against percentage CpG methylation showed a sharp transition between 50 and 60% G + C ( Figure 4D ) .", "Interestingly , a CGI-like insertion with a base composition of 55% G + C ( MeCP2-eGFP ) studied previously ( Thomson et al . , 2010 ) showed an intermediate DNA methylation level , suggesting that it lies on the transition point for triggering de novo methylation ( Figure 4D ) . 10 . 7554/eLife . 03397 . 010Figure 4 . CpG-rich DNA sequences on an A + T-rich background fail to form bivalent chromatin and reproducibly acquire DNA methylation .", "( A ) Above: Map of gene desert 2 indicating the integration site of the High CpG / Low G + C 1 ( H-CpG / L-G + C 1 ) construct as in Figure 1B .", "Representative anti-H3K4me3 , H3K27me3 and Suz12 ChIPs shown ( n = 3 ) for each of two independently transfected cell lines ( Third line not shown ) .", "The shaded bar includes primers spanning the High CpG / Low G + C 1 construct .", "( B ) Bisulfite sequence analysis of the two cell lines shown in ( A ) .", "Clear box indicates bisulfite amplicon .", "In the map above , blue strokes show CpGs in the CGI-like insert and the clear box indicates the bisulfite amplicon .", "Methylated and unmethylated CpGs are depicted as filled and open circles , respectively .", "( C ) The High CpG / Low G + C construct was integrated into Dnmt 3a−/− Dnmt 3b−/− double mutant mouse ES cells .", "Representative H3K4me3 , H3K27me3 and Suz12 ChIPs are shown ( n = 3 ) .", "Upper right panel shows bisulfite sequence analysis of a cell line containing the High CpG / Low G + C construct in Dnmt 3a/b −/− cells , presented as in panel ( B ) .", "( D ) The relationship between G + C content of constructs analysed in this study and their DNA methylation status .", "Data for Mecp2-eGFP and Nanog-PuroGFP refer to cell lines reported previously ( Thomson et al . , 2010 ) , but reanalyzed for this study .", "( E ) Diagrams depicting the influence of CGI sequence composition on chromatin structure .", "Upper panel: Sequences with high CpG frequency and high C + G content attract both H3K4 and H3K27 methyltransferase to establish bivalent chromatin domains and they remain unmethylated .", "SET1A/1B and MLL1/2 complexes contain CXXC domains that may target H3K4me3 to CGIs .", "The mechanism by which the PRC2 complex is targeted is unknown .", "Middle panel: Without CpGs , H3K4 and K27 methyltransferases are not recruited even when the DNA is G + C-rich .", "Lower panel: A + T-rich DNA fails to form a bivalent chromatin structure , even when the CpG density is high and is consistently subject to de novo methylation . DOI: http://dx . doi . org/10 . 7554/eLife . 03397 . 01010 . 7554/eLife . 03397 . 011Figure 4—figure supplement 1 . CpG-rich , A + T-rich DNA sequences do not form bivalent chromatin . s .", "( A and B )", "Above: diagrams ( See Figure 1B ) of gene desert 2 with ( A ) inserted A + T-rich , CpG-rich construct #2 ( H-CpG/L-G + C 2 ) and ( B ) medium-A + T , CpG-rich construct ( H-CpG/M-G + C ) .", "Below: representative anti-H3K3me3 and H3K27me3 ChIP normalized to H3 and Suz12 ChIP shown as enrichment over the negative control region ( n = 3 ) for two independently transfected cell lines for each construct .", "Shaded bars indicate primers spanning the CGI-like constructs .", "( C ) Scheme showing the mouse ß-globin locus ( See Figure 1—figure supplement 1 ) with integration site of the A + T-rich , CpG rich construct #3 ( H-CpG/L-G + C 3 ) construct .", "Black bars indicate position of primers used for Q-PCR .", "Representative H3K4me3 and H3K27me3 ChIP analyses ( data for cell lines 2 and 3 not shown ) .", "Shaded bar indicates primers spanning the H-CpG/L-G + C 3 construct . DOI: http://dx . doi . org/10 . 7554/eLife . 03397 . 01110 . 7554/eLife . 03397 . 012Figure 4—figure supplement 2 . DNA methylation at CpG-rich , A + T-rich DNA sequences blocks bivalent chromatin .", "( A ) Bisulfite sequence analysis of the H-CpG/L-G + C 2 and 3 and H-CpG/M-G + C constructs .", "Clear box indicates bisulfite amplicon and blue vertical lines show position of CpGs .", "Methylated CpGs are depicted as filled black circles , unmethylated CpGs as empty white circles .", "( B ) Bisulfite sequence analysis of IAP elements in wt vs Dnmt 3a/3b knockout mouse ES cells .", "( C ) Dnmt 3a/3b knockout cells can form bivalent chromatin when transfected with the Artificial CGI 1 ( see Figure 1C ) .", "Representative H3K3me3 and H3K27me3 ChIP normalized to H3 for 2 independent cell lines .", "( D ) Partial loss of DNA methylation causes increased H3K27me3 at the A + T-rich , CpG-rich CGI 1 , but does not show elevated H3K4me3 .", "Wt mouse ESCs were transfected and grown for 10 days in either normal medium or medium + 2i inhibitors .", "Representative H3K3me3 and H3K27me3 ChIP normalized to H3 shown ( second cell line not shown ) .", "( E ) Bisulfite sequencing of the H-CpG/L-G + C 1 after 10 days in medium +2i shows reduced DNA methylation at the inserted DNA . DOI: http://dx . doi . org/10 . 7554/eLife . 03397 . 01210 . 7554/eLife . 03397 . 013Figure 4—figure supplement 3 . CpG density and CGI length at bivalent CGIs correlate positively with H3K4me3 and H3K27me3 levels in mouse ESCs . Bivalent CGIs identified in published ChIPseq analyses ( Denissov et al . , 2014; Marks et al . , 2012 ) were divided into four equal bins based on length or CpG density and plotted against levels of H3K4me3 or H3K27me3 ( read counts ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 03397 . 013 The results indicate that DNA methylation is dominant over both H3K4me3 and H3K27me3 , as in its presence neither chromatin mark is observed at the artificial CGIs .", "To test this hypothesis , we asked if removal of DNA methylation could restore bivalent chromatin at A + T-rich CpG-rich sequences by using mutant ESCs lacking the de novo DNA methyltransferases Dnmt 3a and Dnmt 3b ( Okano et al . , 1999 ) , which display severe DNA hypomethylation ( Figure 4—figure supplement 2 ) .", "To ensure that histone modifying enzyme activities are not disrupted in the Dnmt 3a/3b double knock out cells , we generated stable cell lines with the artificial CGI-containing BAC and confirmed that a bivalent domain was observed over the insertion ( Figure 4—figure supplement 2 ) .", "When the High CpG /High A + T construct was introduced into Dnmt 3a/3b knock out cells it now remained unmethylated , and importantly , bivalent chromatin was detected at the inserted sequence ( Figure 4C ) .", "We noticed that H3K4me3 was reproducibly weaker than controls in Dnmt 3a/Dnmt 3b double mutant cells , whereas a robust H3K27me3 signal was obtained .", "This may indicate that A + T-rich DNA is less able to recruit H3K4me3 despite its high CpG density .", "The dominance of the polycomb-associated H3K27me3 mark at A + T-rich CGIs was also seen when DNA methylation was partially reduced by growing cells for 10 days in 2i medium , which enhances the pluripotent state .", "In line with previous reports ( Ficz et al . , 2013; Habibi et al . , 2013 ) , we found that the average level of genomic CpG methylation was reduced from ∼95% to ∼55% ( Figure 4—figure supplement 2 ) .", "Whereas cells grown in serum-containing medium lacked both the tested histone marks , 2i-grown cells displayed a strong increase in H3K27me3 without the appearance of noticeable H3K4me3 ( Figure 4—figure supplement 2 ) .", "We conclude that while CpG frequency is a key feature of CGIs that determines the signature chromatin marks at CGIs , A + T-richness confers an intrinsic susceptibility to de novo methylation that is dominant over these chromatin modifications ( Figure 4E ) .", "Our study assessed the role of G + C-richness vs CpG-richness on chromatin structure by experimentally varying these sequence parameters individually in synthetic DNA domains .", "The question arises: do equivalent atypical sequences exist naturally in the mouse genome and if so what is their chromatin modification status ?", "We searched first for G + C-rich ( ≥61% ) , CpG deficient ( ≤1/100 bp ) sequences of ≥500 bp in length and identified 1954 examples .", "None of these coincided with bivalent chromatin in ESCs , in agreement with our results using synthetic DNA .", "To determine if A + T-rich , CpG-rich sequences also exist naturally in mice , we divided the genome into windows of 100 bp and calculated the G + C content and CpG density of each .", "To ensure that we selected regions with profiles consistently above the thresholds ( as is the case for our artificial constructs ) rather than short CpG dense regions flanked by AT rich DNA , we subtracted windows with ≥50% GC content or <5 CpGs and searched for blocks of adjoining windows .", "Uniformly A + T-rich and CpG-rich DNA sequences of this kind were absent in the mouse genome .", "Even when the criteria were relaxed to include sequences of only 500 base pairs in length ( the approximate minimum size of CGIs ) , we found no examples .", "As A + T-rich regions are consistently DNA methylated , it seems likely that , in the absence of strong selection , the high mutation rate of 5-methylcytosine effectively resists CpG accumulation at these domains over evolutionary time ." ], [ "Epigenetic marks are often considered to be sensitive to both developmental and environmental signals .", "Our data reinforce the complementary view that the underlying genetic information , independent of nurture or developmental history , plays an important role in setting up CGI chromatin structures .", "In ESCs at least , each of the major CGI chromatin configurations is informed by DNA sequence .", "We find that the non-methylated state of CGIs depends on G + C-rich base composition and that both the signature histone marks H3K4me3 and H3K27me3 depend on a high density of the non-methylated CpG dyad .", "The importance of primary DNA sequence in determining bulk features of the epigenome have been suggested elsewhere .", "For example , DNA sequence motifs that are recognised by transcription factors strongly influence DNA methylation patterns ( Lienert et al . , 2011; Stadler et al . , 2011 ) and inter-individual variation in other epigenomic features maps to sites of human DNA sequence heterogeneity that are probably causal ( Kasowski et al . , 2013; Kilpinen et al . , 2013; McVicker et al . , 2013 ) .", "Our data show that even gross features of genomic DNA , such as base composition and the frequency of the 2-base pair sequence CpG , influence the epigenome .", "Do our findings reflect the relationship between naturally occurring DNA sequences and chromatin modification ?", "Almost all native CGIs in mouse ESCs are non-methylated at the DNA level and coincide with peaks of H3K4me3 , which is often seen as the signature histone mark of CGIs ( Bernstein et al . , 2006; Thomson et al . , 2010 ) .", "About one third of the H3K4me3-marked CGIs in mouse ES cells also carry H3K27me3 and are therefore defined as bivalent ( Ku et al . , 2008 ) .", "H3K27me3 is usually relatively dispersed compared with the discrete localisation of components of the PRC2 complex , which deposit this mark .", "It is estimated that at least 97% of peaks corresponding to the PRC2 component Ezh2 coincide with CGIs ( Ku et al . , 2008 ) .", "This has led to the suggestion that polycomb is targeted to G + C-rich or CpG-rich DNA ( Tanay et al . , 2007; Mendenhall et al . , 2010; Lynch et al . , 2012; Long et al . , 2013 ) .", "It was shown previously that CpG density within the non-methylated CGI fraction as a whole is proportional to the H3K4me3 ChIP-seq signal in both mouse and human cells ( Illingworth et al . , 2010 ) .", "We asked whether the same relationship holds true for bivalent CGIs and whether it also applies to H3K27me3 .", "Within a set of 2547 exclusively bivalent promoters from mouse ESCs ( Marks et al . , 2012; Denissov et al . , 2014 ) , we found that 92% coincide with CGIs .", "Within this bivalent group , CGI length and CpG density both correlate positively with H3K4me3 and also H3K27me3 levels ( Figure 4—figure supplement 3 ) .", "In contrast to typical CGI-like sequences , endogenous G + C-rich , CpG-poor sequences do not display a bivalent chromatin structure .", "A + T-rich , CpG-rich domains of CGI-like dimensions , however , are effectively absent from the mouse genome .", "The sum of available data argues strongly that a high abundance of the dinucleotide CpG is a key precondition for the formation of bivalent chromatin .", "A possible mechanism for recruitment of H3K4me3 involves DNA binding by H3K4 methyltransferases , each of which is associated with a CpG-binding CXXC domain .", "In Mll1 and Mll2 the DNA binding domains are within the SET-containing protein themselves ( Allen et al . , 2006; Cierpicki et al . , 2010 ) , whereas in the case of Set1/COMPASS the CXXC domain resides in the Cfp1 protein component of the multi-subunit complex ( Lee and Skalnik , 2005 ) .", "The H3K4me3 component of bivalent CGIs in ESCs depends on Mll2 ( Hu et al . , 2013; Denissov et al . , 2014 ) .", "Accordingly , we have found that bivalent CGIs form normally in Cfp1−/− ES cells .", "Since the binding of the CXXC domain to CpG is abolished by methylation of the cytosine moiety ( Lee et al . , 2001 ) , this may explain why DNA methylation prevents the formation of H3K4me3 even in the presence of high CpG densities .", "The mechanism responsible for recruiting PRC2 , which is the complex responsible for the establishment of H3K27me3 , remains uncertain , as no CpG binding component of PRC2 has yet been detected .", "( Thomson et al . , 2010; Hu et al . , 2013; Wu et al . , 2013 ) .", "A recent study , however , indicates that the CXXC-domain protein KDM2B can target the PRC1 complex to CGIs and recruit PRC2 secondarily , which would accord with our findings ( Blackledge et al . , 2014 ) .", "Bivalent chromatin has attracted attention due to the proposal that it represents a poised transcriptional state in pluripotent cells ( Bernstein et al . , 2006; Voigt et al . , 2013 ) .", "It is suggested that the poised state is resolved during differentiation as the affected gene becomes either transcriptionally activated or repressed .", "Recent evidence has clearly established that H3K27me and H3K4me can be present on the same nucleosome , albeit on different histone tails ( Voigt et al . , 2012 ) .", "The biological significance of bivalent chromatin has recently become less certain , however .", "Whereas silent CGI promoters in ESCs grown in serum usually exhibit a bivalent chromatin structure , this chromatin configuration is significantly reduced in 2i medium , which discourages differentiation and is thought to induce a more pronounced pluripotent state ( Marks et al . , 2012 ) .", "Also , cells in which the histone methyltransferase Mll2 is depleted lose H3K4me3 at hitherto bivalent CGIs , but this has no detectable effect on the induction kinetics of the associated genes upon differentiation ( Hu et al . , 2013 ) .", "The evidence remains inconclusive , but it raises the possibility that bivalency is not an essential precondition for gene activation during differentiation .", "Here we find that bivalency is not confined to poised developmental genes but is a default response to any CpG-rich DNA sequences , even when these are completely artificial .", "It is apparent from this that the bivalent chromatin structure is not reserved for developmentally important genes , but is a response to general features of local DNA sequence .", "Bulk mammalian DNA has a base composition of 40% G + C and is globally methylated at CpGs to an average level of ∼65% , whereas G + C-rich CGIs are usually DNA methylation-free .", "The transfection experiments reported here recapitulate this distinction as G + C-rich CGI-like DNA reproducibly resisted DNA methylation , whereas A + T-rich DNA reliably became densely methylated .", "Our findings raise the possibility that this broadly binary pattern of global DNA methylation may be determined by base composition .", "CpG density did not affect this susceptibility to methylation , which occurred even when CpG occurred at densities typical of endogenous CGIs .", "Two simple alternative explanations are possible: either A + T-rich DNA attracts de novo methylation , or G + C-richness excludes it , leaving the bulk genome to be methylated by default .", "Both Dnmt 3L and Dnmt 3A have the potential to be repelled by H3K4me3 ( Jia et al . , 2007; Ooi et al . , 2007 ) , suggesting that recruitment of H3K4 methyltransferases with CpG-binding CXXC domains protects CGIs against de novo methylation .", "We find , however , that A + T-rich CpG-rich sequences are reproducibly DNA methylated despite their ability to attract H3K4me3 when DNA methylation is absent .", "Since these sequences were initially non-methylated when transfected into cells , it may be that H3K4me3 alone is not sufficient to exclude CpG methylation from these regions .", "This would accord with our previous observation that a moderately G + C-rich artificial CGI when inserted into ES cells was extensively methylated despite the presence of H3K4me3 on non-methylated copies ( Thomson et al . , 2010 ) .", "Alternatively , A + T-rich CpG-rich sequences may attract H3K4me3 less robustly than G + C-rich CGIs , thereby allowing access to de novo DNA methyltransferases .", "The CGI phenomenon is conserved throughout the vertebrate lineage , but interestingly the G + C-richness characteristic of mammalian and bird CGIs is not seen in some vertebrate groups ( Long et al . , 2013 ) .", "Fish for example have non-methylated CGI-like sequences at promoters , but these are not markedly G + C-rich compared with the bulk genome ( Cross et al . , 1991; Long et al . , 2013 ) .", "Since A + T-rich sequences are susceptible to de novo DNA methylation in mammals , it follows that some vertebrate groups may rely on a different set of mechanisms to prevent methylation at G + C-poor CGIs .", "Identifying potential components that discriminate between mammalian CGIs and the bulk genome is a priority for future work ." ], [ "ES cells were grown in gelatinized dishes in Glasgow MEM ( Gibco , UK ) supplemented with 15% fetal bovine serum ( Hyclone; Fisher Scientific , UK ) , 1% sodium pyruvate , 1% non-essential amino acids , 0 . 1% β-mercaptoethanol , 100U/ml penicillin , 100 μg/ml streptomycin and leukemia inhibitory factor ( LIF ) .", "E14TG2a ES cells were used as wild-type ES cells .", "Cfp1−/− cells were a gift from David Skalnik and have been described previously ( Carlone et al . , 2005 ) .", "Dnmt 3a/3b double knock out mouse ESC ( DKOs ) were as described ( Okano et al . , 1999 ) .", "TC1-mES cells ( a gift from Dr Ann Dean ) with the hygromycin/thymidine kinase cassette in the β-globin locus ( Lienert et al . , 2011 ) were used for recombination-mediated cassette exchange .", "Cfp1-GFP tagged mouse ES cells have been described ( Denissov et al . , 2014 ) .", "This integrated BAC contains the whole Cfp1 gene with regulatory elements and GFP fused to the last codon of to create a C-terminal GFP tag .", "Custom Perl scripts were used to create random sequences with specific frequencies of CpG , G + C content and length ( https://github . com/swebb1/cpg_tools ) .", "Supplementary file 1 lists the CGI-like sequences used in this study .", "Artificial DNAs were synthesised ( GeneArt; Life Technologies , UK ) and cloned into a plasmid containing a selection cassette and homology arms for recombineering .", "CGI-constructs were introduced into human gene desert BACs by recombineering using the Red/ET system by Gene Bridges ( Germany ) .", "Briefly , bacteria containing the gene desert BAC of interest ( gene desert 1 mChr1:81 , 106 , 616-81 , 153 , 886 or gene desert 2 mChr18:36 , 042 , 881-36 , 175 , 341 ) were cultivated in LB medium plus chloramphenicol at 37°C o/n .", "On the next day 40 ng Red/ET plasmid were electroporated ( 1350 V , 10 μF , 600 Ohms ) into the cells containing the BAC and incubated in LB containing chloramphenicol and tetracycline at 30°C for at least 15hr .", "Next day 50 μl of 10% L-arabinose were added to induce the expression of the Red/ET recombination proteins and samples were incubated at 37°C for 1h .", "Cells were electroporated with 200–300 ng of linearized DNA containing the CGI-like sequence , a kanamycin selection cassette and homology arms .", "Cells were re-suspended in 1 ml of SOC medium and recovered for 1–2h at 37°C .", "Cells were plated on plates containing chloramphenicol and kanamycin .", "Plates were incubated at 37°C o/n .", "Colonies were screened by colony PCR and control digests for successful recombination .", "The linearized BAC containing the CGI-like constructs and a selection cassette was used for transfection of mESCs .", "Linearized BAC DNA ( 0 . 5–2 μg ) containing the CGI-like sequences and a selection cassette flanked by Frt sites was used to transfect 60% confluent mouse ESC growing in a 6-well plate using Lipofectamine LTX Plus ( Invitrogen , UK ) .", "DNA was made up with OptiMem ( Gibco , UK ) to 500 μl , 2 . 5 μl PLUS reagent were added and incubated for 5 min at RT .", "Afterwards 6 . 25 μl Lipofectamine were added , incubated for 30 min at RT and added to the ES cells .", "Cells were split in a range of different ratios ( 10–0 . 1% of transfected cells ) 24hr after transfection and plated onto 10 cm2 dishes .", "Next day , selection medium containing the appropriate antibiotic ( G418 250 μg/ml or Blasticidin 3 μg/ml ) was added and cells were grown until colonies were ready to be picked .", "Clones were analysed for incorporation of the constructs by PCR and 2–3 independent clones with low copy number integration were selected for the excision of the selection cassette .", "Circular plasmid ( 50 μg ) containing a eukaryotic expression cassette for Flp or Dre were added to 2 × 107 cells and electroporated at 250 V and 500 μF using a BioRad electroporator ( GenePulser Xcell; Biorad , UK ) .", "Cells were left to recover for 20 min at RT and seeded at different dilutions .", "Next day 0 . 8 μg/ml puromycin were added for 48hr and cells were cultured until colonies were big enough for picking .", "Successful excision was confirmed by Southern blotting .", "Artificial CGIs for insertion into the beta-Globin locus via recombination mediated cassette exchange were synthesised ( GeneArt ) with flanking inverted loxp sites and cloned into pBSIISK+ and electroporated into TC-1 ES cells carrying a Hygromycin/Thymidine Kinase double selection cassette in the beta-Globin locus ( Lienert et al . , 2011 ) .", "Cells ( 4 × 106 ) were pre-selected with hygromycin for 10 days , electroporated with 25 µg L1-artificial CGI-1L construct and 20 µg pCAGGS-Cre and selection for positive clones with 3 µm Ganciclovir was started 2 days after electroporation and continued for 8–10 days .", "Clones were tested for successful insertion of artificial CGIs by PCR screen and Southern Blot .", "For differentiation of mESC into neural precursors cells were plated ( 4 × 106 cells/dish ) in 15 ml EB medium ( ES cell medium with 10% FBS and no LIF ) .", "After 4 days in EB medium trans-retinoic acid ( Sigma , UK ) was added to start neuronal differentiation .", "Medium was changed every 2 days .", "On day 8 EBs were disrupted , trypsinized and used for formaldehyde crosslinking .", "Chemical crosslink of chromatin was performed for 10 min at room temperature by addition of formaldehyde to a final concentration of 1% .", "Crosslinking was stopped by addition of glycine to a final concentration of 0 . 125 mM .", "After 5 min incubation cells were washed twice with ice-cold 1× PBS .", "If acetylation was examined , sodium butyrate was added to the PBS .", "Cells were centrifuged for 5 min at 330×g at 4°C and washed once in wash buffer A ( 0 . 25% Triton X-100 , 10 mM EDTA pH 8 . 0 , 0 . 5 mM EGTA pH 7 . 5 , 10 mM , HEPES pH 7 . 5 ) and B ( 0 . 2M NaCl , 1 mM EDTA pH 8 . 0 , 0 . 5 mM EGTA pH 7 . 5 , 10 mM HEPES pH 7 . 5 ) .", "Each buffer contained PMSF to a final concentration of 1 μg/ml , 1× Proteinase Inhibitor Complete Mix ( Roche , UK ) and 10 mM sodium butyrate .", "Cells were re-suspended in lysis buffer ( 20% SDS , 0 . 5 M EDTA , 1 M Tris pH 8 . 1 , 1× complete protease inhibitors , 1 μg/ml PMSF , 10 mM sodium butyrate ) .", "Chromatin was sheared to 400–600 base pair fragments by sonication of the cell lysate using a twin Bioruptor ( Diagenode , Belgium ) for 15 cycles , 30 s on , 30 s off on high setting .", "The lysate was centrifuged for 10 min at 16 , 000×g at 4°C to collect cellular debris .", "Chromatin concentration was measured on a Nanodrop spectrophotometer .", "Chromatin ( 30 μg or 100 μg ) was used for ChIP with antibodies against histone modifications or other proteins .", "For the ChIP , chromatin was made up to 100 μl with lysis buffer and 900 μl of dilution buffer ( 20 mM Tris–HCl , pH 8 , 150 mM NaCl , 1% Triton X-100 , 1 mM EDTA ) were added .", "Antibodies were added as specified and samples were incubated o/n at 4°C on a rotating wheel .", "The next day debris was removed by centrifugation for 5 min at 16 , 000×g and 50 μl dynabeads protein G ( Life Technologies , UK ) were added to the supernatant of the IPs and samples were incubated on a rotating wheel for 2 hr at 4°C .", "IPs were washed 3× with 1 ml ice-cold wash buffer 1 ( 20 mM Tris–HCl , pH 8 , 150 mM NaCl , 1% Triton X-100 , 1 mM EDTA , 0 . 1% SDS ) , 2× with ice-cold wash buffer 2 ( 20 mM Tris–HCl , pH 8 , 500 mM NaCl , 1% Triton X-100 , 1 mM EDTA , 0 . 1% SDS ) and 1× with ice-cold TE .", "100 μl or 50 μl of freshly prepared 10% Chelex 100 Resin ( 100–200 mesh , BioRad ) were added to samples or input respectively .", "Samples were boiled for 12 min , cooled to RT .", "Proteinase K ( 2 μl of 20 mg/ml ) was added and incubated at 55°C for 30 min while shaking .", "After heating to 100°C , samples were spun down and 60 μl supernatant were transferred to fresh tubes .", "Each sample was made up to 300 μl total volume with 10 mM Tris pH 8 , 0 . 1 mM EDTA and assayed by q-PCR .", "Supplementary file 1 lists the qPCR primers used in this study .", "qPCR was used to assess enrichment of specific regions in ChIP samples and to determine the copy number of BAC DNA inserted into the mouse genome .", "qPCR reactions ( 10 μl ) contained SYBR Green SensiMix ( Bioline , UK ) , 250 nM primers and 3 µl ChIP DNA or 100 ng DNA for the determination of copy numbers .", "PCR was carried out using a Roche Lightcycler and cycling conditions were as follows; initial denaturation at 94°C for 10 min followed by 45 cycles of denaturation at 94°C for 10 s , primer annealing for 10 s and primer extension at 72°C for 15 s .", "Using the Roche Lightcycler software , SYBR Green fluorescence measurements were plotted relative to cycle number and the 2nd derivative maximum method was used to determine the cycle threshold values ( Ct ) for each sample .", "Values for duplicate of triplicate ChIP samples were calculated as %-input and copy number of artificial CGIs was assessed relative to Sox2 qPCR signal .", "α-H3K4me3 ( Abcam[UK]-8580 ) , α-H3K4me1 ( Abcam-8895 ) , α-H3K27me3 ( Millipore[UK]-07-449 ) , α-H3K9/K14ac ( Abcam 12 , 179 ) , α-H3 ( Abcam 1791 ) , α-SUZ12 ( Abcam 12 , 073-100 ) , α-RNA Pol II N20 ( Santa-Cruz[UK] 899 ) , α-RNA Pol II S5P ( Abcam 5131 ) , α-RNA Pol II unphosphorylated CTD ( Abcam 817 ) , α-GFP ( Chromotec [Germany] GFP-TRAP-A gta-20 ) , α-IgG ( Invitrogen 10500C ) .", "Bisulfite conversion of genomic DNA was carried out using the EpiTect Bisulfite Kit from Qiagen ( UK ) .", "The converted DNA was used for PCR amplification of regions of interest , Supplementary file 2 lists the CGI-like sequences used in this study .", "PCR products were gel-purified and cloned using the Stratagene blunt end cloning kit .", "Positive clones were sent for sequencing ." ] ]

[ "The mammalian genome is punctuated by CpG islands ( CGIs ) , which differ sharply from the bulk genome by being rich in G + C and the dinucleotide CpG .", "CGIs often include transcription initiation sites and display ‘active’ histone marks , notably histone H3 lysine 4 methylation .", "In embryonic stem cells ( ESCs ) some CGIs adopt a ‘bivalent’ chromatin state bearing simultaneous ‘active’ and ‘inactive’ chromatin marks .", "To determine whether CGI chromatin is developmentally programmed at specific genes or is imposed by shared features of CGI DNA , we integrated artificial CGI-like DNA sequences into the ESC genome .", "We found that bivalency is the default chromatin structure for CpG-rich , G + C-rich DNA .", "A high CpG density alone is not sufficient for this effect , as A + T-rich sequence settings invariably provoke de novo DNA methylation leading to loss of CGI signature chromatin .", "We conclude that both CpG-richness and G + C-richness are required for induction of signature chromatin structures at CGIs ." ]

[ "The building blocks of DNA are four molecules commonly named ‘A’ , ‘T’ , ‘C’ and ‘G’ .", "The order of these DNA letters in a gene contains the instructions to make specific proteins or other molecules .", "Other stretches of DNA contain codes that direct the cell's machinery to genes that need to be switched on or switched off .", "The start of a gene , for example , has a stretch of DNA called a promoter , which is where the molecular machinery that switches on the gene is assembled .", "A human cell can contain over two and half metres of DNA .", "To get this length to fit inside the cell , the DNA is wrapped tightly around proteins to form a structure called chromatin .", "However , this packing can make it difficult to access the right gene at the right time .", "As such , chromatin is often marked with small chemical tags that earmark which genes should be either activated or inactivated , and/or that cause the DNA to unpack .", "Most gene promoters contain a sequence of DNA with many Cs and Gs found one after the other , called a CpG island .", "Researchers have previously shown that the chromatin of CpG islands has two types of chemical markings—one that normally marks active genes , and another that often marks inactive genes .", "It was suggested that having both kinds of markings allows CpG islands to prime nearby genes , so that they are ready to be quickly switched on or off as the cell develops .", "However , the features of the DNA sequence in these CpG islands that are important for this process had not been directly tested .", "Wachter et al . have now inserted an artificial DNA sequence that included a CpG island into mouse stem cells .", "The chromatin around these CpG islands was readily marked with both activating and inactivating chemical marks .", "Furthermore , by changing the sequence of the artificial DNA , Wachter et al . revealed that these chemical marks were only added when the DNA sequences contained a lot of Cs followed by Gs .", "Other artificial sequences with lots of Cs and Gs , but where Gs were rarely found immediately after the Cs , had neither of the two chemical marks on the chromatin .", "This suggests that nearby genes would be harder to locate and activate as the cell grows and develops .", "On the other hand , when the DNA contained a lot of As and Ts , the chemical marks were added directly to the DNA ( rather than to the chromatin ) —and this prevented both the activating and the inactivating chemical marks being added to the chromatin .", "Now that the common features of CpG islands that influence chromatin are known , the next step is to find out how this is achieved .", "Further work will be needed to uncover which proteins in a cell interpret these DNA sequence such that nearby genes can be switched on or off ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "developmental biology", "immunology and inflammation" ]

[ [ "γδ T cells , together with αβ T cells and B cells , are the only cells in mammals capable of generating diverse antigenic receptors by somatic gene rearrangement , which enables them to specifically recognize and respond to a vast array of antigens .", "However , while the essential role of αβ T cells and B cells for the induction of long-lasting protective immune responses is undisputed , γδ T cells due to their low abundance in peripheral lymphoid organs have been often ignored by the scientific community ( Allison and Havran , 1991 ) .", "This has changed in the past decade , as it became evident that γδ T cells are important players not only during protective immune reactions ( Lockhart et al . , 2006 ) , but also in various models of autoimmune disease ( Petermann et al . , 2010; Cai et al . , 2011; Pantelyushin et al . , 2012 ) .", "Several features distinguish γδ T cells from their αβ counterparts ( reviewed in Vantourout and Hayday , 2013 ) .", "Most notably , γδ T cells recognize qualitatively different antigens .", "The scope of the potential antigens has not been revealed entirely , but known targets include non-classical MHC molecules ( Strid et al . , 2008 ) , phosophoantigens ( Constant et al . , 1994 ) as well as a discrete set of non-self proteins ( Zeng et al . , 2012 ) .", "The second main characteristic of γδ T cells is their ability for rapid production of pro-inflammatory cytokines such as INF-γ and IL-17 independent of TCR engagement ( Sutton et al . , 2009 ) .", "This feature has been suggested to be hardwired in developing γδ T cells already during thymic development , probably in dependency of self-antigen recognition ( Jensen et al . , 2008 ) .", "The surface molecule CD27 , which belongs to the tumor necrosis ( TNF ) receptor superfamily has been shown to distinguish IFN-γ-producing from IL-17-producing γδ T cells ( Ribot et al . , 2009 ) , as does the differential expression of the transcription factors T-bet and RORγt and certain chemokine receptors such as CCR6 ( Haas et al . , 2009 ) .", "Although the genetic diversity of the TCR γδ locus would theoretically allow an even higher number of different TCR specificities than for αβ T cells ( Bonneville et al . , 2010 ) , γδ T cells show a remarkable limitation in their TCR variability .", "Moreover , γδ T cells with a given TCR specificity often home to non-lymphoid organs , where the majority of mature γδ T cells can be found .", "The prime example of tissue homing-behavior is observed in dendritic epidermal T cells ( DETCs ) , which are found in the murine epidermis and almost exclusively express a clonotypic TCR consisting of the Vγ5 and Vδ1 chains ( Asarnow et al . , 1988 ) .", "Vγ5+ DETCs are among the very first T cells to develop during embryogenesis , populating the epidermis already around day 15/16 of fetal development ( Xiong et al . , 2004 ) .", "Also other subsets of γδ T cells , such as Vγ4+ γδ T cells ( mostly IL-17 producers ) , which migrate predominantly to the dermis and lung tissue , have been shown to develop only early in ontogeny ( Allison and Havran , 1991; Haas et al . , 2012; Prinz et al . , 2013 ) .", "The underpinning signaling pathways and precise developmental program as well as the mechanisms underlying the pre-determined cytokine profile of γδ T cells have been only partially resolved .", "One major step forward was the identification of Skint-1 ( Selection and upkeep of intraepithelial T cells-1 ) , which has an essential function for the development of Vγ5+ DETCs ( Lockhart et al . , 2006; Lewis et al . , 2006; Boyden et al . , 2008 ) .", "Expression of Skint-1 by medullary thymic epithelial cells ( mTECs ) ( Barbee et al . , 2011 ) leads to a signaling cascade in developing γδ thymocytes that induces the typical gene expression profile of DETCs , concomitant to their preferential homing to the skin ( Turchinovich and Hayday , 2011; Vantourout and Hayday , 2013 ) .", "On the other hand , the induction of the typical gene signature of IL-17-producing Vγ4+ γδ T cells depends on the function of the transcription factor Sox13 ( Gray et al . , 2013 ) , while the required receptors remain elusive .", "It has been known for some time that signaling pathways leading to activation of NFκB are essential for the function of various immune cell compartments ( reviewed in Li and Verma , 2002 ) .", "The so-called classical pathway of NFκB activation is induced mainly by molecular danger signals such as LPS or certain cytokines such as TNF or IL-1β .", "Non-canonical NFκB signaling , which requires the NFκB-inducing kinase ( hereafter referred to as NIK , the encoding gene being Map3k14 , ) ( Malinin et al . , 1997; Yin , 2001 ) is activated by a discrete subset of TNF family members , including CD40L , Lymphotoxin ( LT ) , BAFF ( B cell activating factor ) and RANKL ( Receptor activator of NFκB ligand ) .", "Signaling via the non-canonical pathway has been shown to fulfill roles in B cell survival ( Sen , 2006 ) , activation of T cells ( Matsumoto et al . , 2002; Ribot et al . , 2009 ) , maturation and function of dendritic cells ( DCs ) ( Hofmann et al . , 2011 ) as well as the induction of tolerance ( Zhu et al . , 2006; Bonneville et al . , 2010 ) , but it has not been studied in the context of γδ T cell biology in detail .", "Previous work has suggested that signaling via the CD70-CD27 axis ( which belongs to the tumor necrosis factor receptor superfamily ) can activate NFκB p52 processing ( Ribot et al . , 2010 ) .", "Also , LTβR signaling has been implicated in the differentiation program of bulk γδ thymocytes ( Silva-Santos , 2005 ) , and the NFκB family members RelA and RelB were shown to guide the development of IL-17-producing Vγ4+ γδ T cells ( Powolny-Budnicka et al . , 2011 ) .", "Furthermore , the genesis of invariant NKT cells was claimed to depend on signaling via NIK ( Elewaut et al . , 2003 ) .", "In the present study we set out to systematically analyze the impact of non-canonical NFκB signaling on the development and function of γδ T cells .", "In line with previous studies that were using knockout models of the TNF receptor family member RANKL ( Roberts et al . , 2012 ) we found that NIK-deficient mice showed a drastic loss of DETCs in the epidermis .", "Of note , in the absence of NIK most of the IL-17 production by splenic and lung-resident γδ T cells was lost , while their ability to express IFN-γ was not affected .", "Mechanistically , this was accompanied by a loss of Rorc and Sox13 expression in NIK-deficient CD27- γδ T cells , while in turn expression of Tbet and Egr3 was increased .", "While previous studies reported trans-conditioning of developing γδ T cell precursors by CD4+ thymocytes ( Silva-Santos , 2005; Powolny-Budnicka et al . , 2011 ) , our data suggest that NIK signaling specifically in thymic epithelium is essential for shaping the cytokine profile of the γδ T cell compartment ." ], [ "Previous studies have shown that the development of DETCs is partially dependent on signaling via the RANK-RANKL axis ( Roberts et al . , 2012 ) .", "In line with this , we observed a disturbed pool of DETCs in the epidermis of adult Map3k14-/- mice ( Yin , 2001 ) , with only 30-–50% of the γδ T cells present expressing the canonical Vγ5+ TCR ( Figure 1A ) .", "Since DETCs are among the very first T cells to develop in ontogeny and populate the epidermis already prior to birth , we analyzed the epidermis of mouse embryos at day 19 post conception .", "Whereas there was already a prominent population of Vγ5+ DETCs present in WT controls , DETCs were virtually absent in the skin of NIK-deficient embryos ( Figure 1B , C ) . 10 . 7554/eLife . 10087 . 003Figure 1 . In the absence of NIK , the development of DETCs is blocked in the embryonic thymus .", "( A ) Lymphocytes isolated from the epidermis of adult heterozygous control ( left panel ) and Map3k14-/- animals were analysed for the presence of Vγ5+ DETCs .", "Pregating is on live singlets and CD45+ CD11b- cells .", "( B ) Analysis of the epidermal γδ T cell compartment of heterozygous control ( upper panel ) and Map3k14-/- embryos ( day 19 post conception ) after pregating on live singlets and CD45+ CD11b- cells .", "( C ) Summary of the frequency of total γδ T cells as well as Vγ5+ cells within the γδ T cell gate .", "Data are mean +/- SD and are representative of two similar experiments .", "( D ) Analysis of developing Vγ5+γδ thymocytes in the thymi of E19 embryos .", "Flow Plots have been pregated on live singlets and CD45+ CD4- cells .", "Lower panel depicts the summary of the frequency of total γδ thymocytes as well as Vγ5+ cells within the γδ T cell gate in d19 embryonic thymi , and the median fluorescence intensity of the indicated markers .", "Data are mean +/- SD and representative of two similar experiments .", "( E ) Analysis of the expression level of CD45RB , CD122 , CD24 and CD62L on developing Vγ5+γδ thymocytes isolated from E19 embryonic thymi .", "Grey shaded histograms depict heterozygous controls , red histograms Map3k14-/- cells .", "Lower panel shows the summary for the frequency of positive cells for CD45RB and CD122 and the median fluorescence intensity of CD24 and CD62L , respectively .", "Data are mean +/- SD and are representative of two similar experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 10087 . 00310 . 7554/eLife . 10087 . 004Figure 1—figure supplement 1 . DETC development in NIK-deficient thymi at embryonic day 17 . ( A ) Analysis of developing Vγ5+ γδ thymocytes in E17 thymi .", "Flow Plots have been pregated on live singlets and CD45+ CD4- cells .", "Right panels depict the median fluorescence intensity of CD3 and Vγ5 expression .", "Data are mean +/- SD and representative of two similar experiments .", "( B ) Analysis of the expression level of CD45RB on developing Vγ5+ γδ thymocytes isolated from E17 embryonic thymi .", "Grey shaded histograms depict heterozygous controls , red histograms Map3k14-/- cells .", "Right panel shows the summary for the frequency of CD45RB positive cells .", "Data are mean +/- SD and are representative of two similar experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 10087 . 004 The absence of DETCs in the epidermis of Map3k14-/- embryos led us to speculate that NIK-deficient DETC precursors fail to develop in the embryonic thymus .", "To test this notion , we analyzed thymi from Map3k14-/- and heterozygous controls at embryonic day 19 for the presence of Vγ5+ thymocytes .", "Indeed , these cells were present in NIK-deficient thymi , albeit at reduced numbers and with a consistent reduction in staining intensity of the TCR ( Figure 1D ) .", "In order to assess the maturation status of the developing Vγ5+ thymocytes , we evaluated the expression level of various molecules that have been associated with normal DETC development , such as CD45RB , CD122 , CD24 and CD62L ( Lewis et al . , 2006 ) .", "The expected upregulation of CD45RB and CD122 , which is typical for developing DETCs was not found in Map3k14-/- embryos .", "In turn , the downregulation of CD24 and CD62L which normally coincides with DETC maturation was also reduced ( Figure 1E ) .", "Similar observations with respect to the expression of CD45RB were obtained during the analysis of thymi isolated from E17 embryos ( Figure 1—figure supplement 1 ) .", "Taken together , the loss of NIK abrogates normal development of DETC precursors in the embryonic thymus , corroborating previous findings using knockout animals for Tnfrsf11a ( Roberts et al . , 2012 ) .", "Based on the role of NIK in the formation of the epidermal DETC pool we assessed the contribution of non-canonical NFκB signaling to the development and function of other γδ T cell compartments in more detail .", "For comparison , throughout our study we included both targeted Map3k14-/- mice ( Yin , 2001 ) as well as aly/aly animals , which harbor a point mutation in NIK , expressing a dysfunctional protein ( Shinkura et al . , 1999 ) .", "Both in the spleen and the lung of these mutant animals , the total number of γδ T cells as well as the frequency of the two most prominent subclasses expressing the Vγ4+ and Vγ1 . 1+ TCR was unchanged .", "Also , the distribution of the CD27+ and CD27- subsets of γδ T cells was indistinguishable between Map3k14-/- and heterozygous control animals ( Figure 2A–C ) . 10 . 7554/eLife . 10087 . 005Figure 2 . In NIK-deficient mice , γδ T cells selectively lose their ability for production of IL-17 . ( A ) Flow cytometric analysis of the splenic γδ T cell compartment of heterozygous control ( upper panel ) and Map3k14-/- animals after pregating on live singlets and CD45+ CD11b- B220- cells .", "The distribution of Vγ1 . 1+ , Vγ4+ , and CD27- Vγ4+ γδ T cells is shown .", "( B ) Summary of two independent experiments .", "Data represent mean +/- SD of the indicated cell subsets .", "( C ) Analysis of the lung-resident γδ T cell compartment of heterozygous control ( upper panel ) and Map3k14-/- animals after pregating on live singlets and CD45+ CD11b- B220- cells .", "The distribution of Vγ1 . 1+ , Vγ6+ , and CD27- Vγ6+ γδ T cells is shown .", "( D ) Flow cytometric analysis of IFN-γ , IL-17 and TNF-α expression by peripheral γδ T cells after PMA/Ionomycin stimulation , isolated from the spleen of heterozygous control ( upper panel ) and Map3k14-/- animals ( lower panel ) .", "Left plots have been pregated on CD45+ CD11b- CD4- live singlets .", "( E ) Summary of the frequency of cytokine producing cells within the γδ T cell compartment .", "Data represent mean +/- SD .", "( F ) Flow cytometric analysis of IFN-γ and IL-17 expression by lung-resident γδ T cells isolated from the lung of heterozygous control ( upper panel ) and Map3k14-/- animals ( lower panel ) .", "Left plots have been pregated on CD45+ CD11b- CD4- live singlets .", "( G ) Summary of the frequency of cytokine producing cells within the lung-resident γδ T cell compartment .", "Data represent mean +/- SD .", "( H ) Flow cytometric analysis of IFN-γ and IL-17 expression by dermal γδ T cells isolated from the skin of heterozygous control and Map3k14-/- animals .", "Right panel depicts the frequency of cytokine producing cells within the dermal γδ T cell compartment .", "Data show mean +/- SD and are representative of at least three independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 10087 . 00510 . 7554/eLife . 10087 . 006Figure 2—figure supplement 1 . Ratio of Vγ4+ and Vγ6+ cells in the lung-resident IL-17-producing γδ T cell compartment .", "( A ) Flow cytometric analysis of Vγ4+ and Vγ6+ cells within IFN-γ and IL-17 expressing γδ T cells isolated from the lung of heterozygous control ( upper panel ) and Map3k14-/- animals ( lower panel ) .", "Left plots have been pregated on CD45+ CD11b- CD3+ TCRγδ+ live singlets .", "( B ) Summary of the frequency of Vγ4+ and Vγ6+ cells within the lung-resident γδ T cell compartment .", "Data represent mean +/- SD . DOI: http://dx . doi . org/10 . 7554/eLife . 10087 . 006 However , when we assessed the primary function of γδ T cells , which is the rapid production of pro-inflammatory cytokines , we noticed that after in vitro stimulation of bulk peripheral lymphoid γδ T cells their ability to express IL-17 was strongly diminished .", "In contrast , the frequency of both IFN-γ and TNF-α-producing γδ T cells was similar between Map3k14-/- and control animals ( Figure 2D , E ) .", "For lung-resident γδ T cells , which include Vγ6+ γδ T cells as one the most potent sources of IL-17 , we also observed a reduction in the ability of NIK-deficient cells for IL-17 secretion , but this was accompanied by a slight increase in the frequency of IFN-γ production ( Figure 2F , G ) .", "The ratio of Vγ4+ to Vγ6+ cells in the remaining fraction of IL-17-producing γδ T cells did not change ( Figure 2—figure supplement 1 ) .", "Similarly , for the steady state dermal γδ T cell compartment we observed that in the absence of NIK , γδ T cells were impaired in IL-17 expression ( Figure 2H ) .", "To reveal the underlying mechanism for the loss of IL-17 in γδ T cells , we analyzed the expression of Rorc and Il23r , which are both necessary for normal IL-17 production ( Cua and Tato , 2010; Turchinovich and Hayday , 2011 ) .", "To this end , we purified CD27- and CD27+ γδ T cells ( Figure 3A ) from Map3k14-/- and heterozygous control animals and analyzed gene expression by qPCR .", "While in controls , CD27- γδ T cells expressed high levels of Rorc and Il23r , the same molecules were drastically reduced in NIK-deficient CD27- γδ T cells ( Figure 3B ) , which was accompanied by an upregulation of Tbet .", "Furthermore , we assessed the expression of the transcription factors Sox13 and Egr3 , which have been proposed to be reciprocal regulators of γδ T17 cell development ( Turchinovich and Hayday , 2011; Gray et al . , 2013 ) .", "While the expression level of both genes was not affected in CD27+ γδ T cells , NIK-deficiency selectively in CD27- γδ T cells led to a loss of Sox13 and a gain of Egr3 expression ( Figure 3C ) .", "Collectively , these data indicate that NIK signaling is essential for RORγt and as well as Sox13 expression in CD27- γδ T cells , thereby conferring their full potential for production of IL-17 . 10 . 7554/eLife . 10087 . 007Figure 3 . NIK-deficient CD27- γδ T cells show reduced expression of Rorc and and Sox13 . ( A ) Gating strategy used for sorting of CD27+ and CD27-γδ T cells .", "Right plot depicts post-sort reanalysis , which routinely yielded purities >95% .", "( B ) Analysis of the expression level of Rorc , Il23r and Tbet mRNA in sorted CD27+ and CD27- γδ T cells isolated from the spleen of the indicated genotypes .", "Data represent mean +/- SD .", "( C ) Analysis of the expression level of Sox13 and Egr3 in sorted CD27+ and CD27- splenic γδ T cells isolated from the indicated genotypes .", "( D ) Backskin thickness after Aldara treatment of heterozygous control ( open triangles ) and Map3k14-/- animals ( closed squares ) .", "Data are representative of two independent experiments .", "( E ) IL17-A and IL17-F expression of dermal γδ T cell on day 6 after Aldara treatment . DOI: http://dx . doi . org/10 . 7554/eLife . 10087 . 007 In order to assess the functional impact of NIK-lesioned γδ T cells , we treated Map3k14-/- animals with Imiquimod , a well established system for the induction of psoriasis-like skin inflammation in mice ( van der Fits et al . , 2009; Cai et al . , 2011; Pantelyushin et al . , 2012 ) .", "After treatment with Aldara ( containing 5% Imiquimod ) , NIK-deficient animals displayed a slight , but not significant reduction of back skin thickness ( Figure 3D ) .", "Analysis of the dermal γδ T cell compartment not only revealed reduced numbers of γδ T cells ( data not shown ) , but also a significant reduction of both IL-17A and IL-17F expression by γδ T cells ( Figure 3E ) .", "This suggests that NIK-signaling also in an inflammatory setting is essential for full IL-17 production by γδ T cells , but that other cell types , such as ILCs ( Pantelyushin et al . , 2012 ) can compensate this functional impairment during Aldara induced skin inflammation .", "While our data and work from others ( Roberts et al . , 2012 ) clearly suggest a non-redundant role of non-canonical NFκB signaling in the development of DETCs , it has so far not been possible to determine whether NFκB signaling occurs in a γδ T cell intrinsic or extrinsic manner .", "To dissect the contribution of different cellular compartments in vivo , we generated a novel conditional mouse mutant that allows cell-type specific deletion of the Map3k14 gene using the Cre/LoxP system .", "Map3k14flox/wt animals were generated by gene targeting in embryonic stem ( ES ) cells , thereby inserting LoxP sites up- and downstream of Exon 4 and 6 of the Map3k14 gene locus , respectively ( Supp . Figure 1 ) .", "These mice were first crossed to a Deleter-Cre strain , followed by breeding the deleted locus to homozygosity .", "Examination of the resulting Map3k14del/del mice showed that these mice were lacking lymph nodes , suggesting that the conditional construct is working as expected ( Figure 4A ) .", "To further verify the phenotype of Map3k14del/del mice , we compared the cytokine production of peripheral γδ T cells to WT and Map3k14-/- animals , showing that Map3k14del/del mice phenocopy the full knockout ( Figure 4B ) .", "The same was true for the epidermal DETC pool ( Figure 4C ) . 10 . 7554/eLife . 10087 . 008Figure 4 . Conditional deletion of NIK in mTECs causes impaired DETC development .", "( A ) Map3k14flox/wt mice have been crossed to Deleter Cre animals for deletion of the Loxp flanked gene locus .", "After subsequent breeding to homozygosity , Map3k14del/del mice were analyzed for the presence of lymph nodes .", "Upper panel depicts heterozygous controls .", "( B ) Splenic γδ T cells from control , Map3k14-/- and Map3k14del/del mice were assessed for production of IL-17 ( left panel ) and IFN-γ ( right panel ) by intracellular cytokine staining .", "( C ) The epidermal γδ T cell compartment of control , Map3k14-/- and Map3k14del/del animals was assessed for the frequency of Vγ5+ DETCs .", "( D ) Lymphocytes isolated from the epidermis of adult control ( upper panel ) , Map3k14-/- and Ccl19-Cre+ Map3k14flox/flox animals were analysed for the presence of Vγ5+ DETCs .", "Pregating is on live singlets and CD45+ cells .", "Right panel depicts the summary of the frequency of total γδ T cells as well as Vγ5+ γδ T cells in the epidermis of Control , Map3k14-/- and Ccl19-Cre+ Map3k14flox/flox animals .", "Data represent mean +/- SD .", "( E ) Frequency of total γδ T cells in the skin of 12-–13 day old control littermates and Foxn1-Cre+ Map3k14flox/flox animals .", "Data represent mean +/- SD and is pooled from several independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 10087 . 00810 . 7554/eLife . 10087 . 009Figure 4—figure supplement 1 . Targeting strategy used for generation of the conditional Map3k14flox/wt strain .", "( A ) Schematic representation of the targeted Map3k14 gene locus showing the position of the Loxp Sites flanking exon 4-–6 .", "Abbreviations are denoted in the figure .", "( B ) Schematic representation of the Map3k14 locus after Cre-mediated recombination , leading to a frameshift and a stop codon in exon 7 . DOI: http://dx . doi . org/10 . 7554/eLife . 10087 . 009 As a next step , we assessed whether the DETC defect observed in complete NIK-knockout animals is due to the requirement for NIK signaling in thymic epithelial cells .", "To specifically delete NIK in medullary thymic epithelial cells ( mTECs ) , Map3k14flox/flox mice were crossed to Ccl19-Cre mice , which express Cre solely in lymph node stromal cells and mature medullary thymic epithelial cells ( Chai et al . , 2013; Onder et al . , 2015 ) .", "Ccl19-Cre+ Map3k14flox/flox animals phenocopied the aberrant DETC development observed in germline Map3k14-/- mice , with a loss of Vγ5 expression by DETCs ( Figure 4D ) .", "However , we observed some variation in the penetrance of the Ccl19-Cre transgene , with some litters presenting with a phenotype , while others did not , suggesting that the start of Cre expression driven by Ccl19 promotor sharply coincides with the start of DETC development in the embryo .", "Thus , we decided to utilize an additional strain targeting the thymic epithelium , namely Foxn1-Cre mice in which Cre is under control of the Foxn1 promotor and thus active in all thymic epithelial cells ( both cortical and medullary ) ( Gordon et al . , 2007 ) .", "Corroborating our findings , Foxn1-Cre+ Map3k14flox/flox showed a strong reduction in both frequency and number of skin-resident TCRγδhigh DETCs ( Figure 4E ) .", "To assess which cellular compartment requires NIK for the formation of IL-17 committed γδ T cells , we first deleted NIK specifically in CD27- γδ T cells using Rorc-Cre+ Map3k14flox/flox animals .", "Analysis of Fatemap Rorc-Cre+ R26-Stop-flox-YFP mice showed that around 90% of CD27- γδ T cells in the lung expressed yellow fluorescent protein ( YFP ) , indicating a good targeting efficiency of this cellular subset ( Figure 5A ) .", "However , lung-resident γδ T cells in Rorc-Cre+ Map3k14flox/flox mice expressed IL-17 at levels comparable to wildtype controls ( Figure 5B ) , suggesting that γδ T cell intrinsic signaling is dispensable for the commitment of IL-17-producing γδ T cells .", "To exclude a potential role of NIK signaling in the CD4+ αβ thymocyte compartment ( Powolny-Budnicka et al . , 2011 ) , we analyzed both peripheral and lung-resident γδ T cells in CD4-Cre+ Map3k14flox/flox mice .", "Their ability for IL-17 production was comparable to wildtype controls ( Figure 5C ) , demonstrating that also within the αβ thymocyte compartment NIK signaling is dispensable . 10 . 7554/eLife . 10087 . 010Figure 5 . Conditional deletion of NIK in TECs causes not only a loss of DETCs , but also IL-17-producing γδ T cells .", "( A ) γδ T cells were isolated from the lung of Rorc-Cre+ R26-Stop-flox YFP-mice and assessed for the frequency of YFP+ cells within the CD27-γδ T cell compartment .", "( B ) Lung-resident γδ T cells from adult control , Map3k14-/- and Rorc-Cre+ Map3k14flox/flox mice were assessed for production of IL-17 ( left panel ) and IFN-γ ( right panel ) by intracellular cytokine staining .", "( C ) Lung-resident γδ T cells from adult control and CD4-Cre+ Map3k14flox/flox mice were assessed for production of IL-17 ( left panel ) and IFN-γ ( right panel ) .", "( D ) Lung-resident as well as splenic γδ T cells from adult control and Ccl19-Cre+ Map3k14flox/flox mice were assessed for production of IL-17 and IFN-γ .", "Data is representative of three independent experiments .", "( E ) Frequency of IL-17-producing γδ T cells in the lung of 12-–13 day old control littermates and Foxn1-Cre+ Map3k14flox/flox animals , plots have been pregated on live CD45+ singlets , CD3+ TCRγδ+ cells .", "Right panels depict the summary of several independent experiments for cytokine production in the lung ( left ) and spleen ( right ) .", "Data are mean +/- SD . DOI: http://dx . doi . org/10 . 7554/eLife . 10087 . 010 Based on these results we speculated whether loss of NIK signaling in TECs will impact on the commitment of IL-17-producing γδ T cells .", "To this end , we analyzed the lung-resident γδ T cell compartment of both Ccl19-Cre+ Map3k14flox/flox and Foxn1-Cre+ Map3k14flox/flox mice .", "While γδ T cells isolated from the lung of Ccl19-Cre+ Map3k14flox/flox animals did not present with any abnormalities in cytokine production , γδ T cells isolated from P13 Foxn1-Cre+ Map3k14flox/flox mice showed significant defects in their ability for IL-17 production both in the spleen and lung ( Figure 5D ) , suggesting that indeed NIK signaling in thymic epithelial cells is needed for imprinting the cytokine commitment onto developing γδ T cells ." ], [ "In the past years , γδ T cells have attracted growing interest due to their unique features that place them at the interface of adaptive and innate immunity .", "Nevertheless , in contrast to αβ T cells , the signaling events underlying γδ T cell development and activation are much less understood ( reviewed in Prinz et al . , 2013 and Vantourout and Hayday , 2013 ) .", "Here , we have shown in vivo that NIK signaling in thymic epithelial cells is essential for the full function of two different γδ T cell populations , namely skin-resident DETCs as well as CD27- IL-17-producing γδ T cells .", "Previous work has suggested an involvement of the NFκB-inducing kinase in the differentiation of both αβ and γδ T cells ( Eshima et al . , 2014 ) .", "Contrary to the findings by Eshima and colleagues we could not observe a consistent overall reduction in the number of γδ T cells ( except the DETC pool ) , yet we observed functional impairment of CD27- IL-17-producing γδ T cells .", "This discrepancy might result from two mutually non-exclusive explanations: first , aly/aly mice as well as Map3k14-/- mice due to their complex phenotype ( including impairment of thymic negative selection ) are prone to develop some degree of tissue inflammation over time , which is accompanied by an expansion of both CD4+ and CD8+ cells ( Tsubata et al . , 1996; Ishimaru et al . , 2006; Zhu et al . , 2006 ) and could thereby induce a relative decrease in the proportion of γδ T cells .", "Second , it has recently been established that the γδ T cell compartment cannot be fully reconstituted using bone-marrow chimeric animals ( Gray et al . , 2011; Haas et al . , 2012 ) .", "Using a genetic model that allows the cell-type specific deletion of NIK avoids all these confounding phenotypes of mice in which the gene locus of NIK ( Map3k14 ) has been deleted in the germ-line .", "Two members of the TNFR family , namely CD40 and RANK , have been implicated both in the development of DETCs and full maturation of mTECs ( Hikosaka et al . , 2008; Akiyama et al . , 2008 ) .", "In embryogenesis , these events occur at similar time points post conception , and it seems that in the embryonic thymus , developing DETC precursors signal via the RANKL-RANK axis to immature mTECs , inducing their full maturation to Aire+ MHC-IIhigh mTECs ( Roberts et al . , 2012 ) .", "Hence , Tnfrsf11a-/- mice have reduced numbers of DETCs .", "Given that NIK is downstream of RANK ( Darnay et al . , 1999 ) , one would expect a similar phenotype in Map3k14-/- animals , but the DETC deficiency that we observed in newborn Map3k14-/- mice was much more pronounced than in Tnfrsf11a-/- animals , suggesting that during DETC development several upstream receptors converge in their signaling to NIK , making it a key pathway during thymic DETC development .", "Importantly , our data obtained using the conditional NIK-mutant strongly suggests that for normal function of the γδ T cell compartment , NIK signaling does not act γδ cell-intrinsically but in trans by its function in thymic epithelial cells .", "Conditional deletion of NIK solely in thymic epithelial cells was sufficient to copy the DETC phenotype seen in complete germ-line NIK-knockout animals .", "We believe this to be the first genetic in vivo demonstration that non-canonical NFκB signaling is not required in DETCs in a cell-intrinsic manner , but through its activity in TECs .", "The ability of CD27- γδ T cells to rapidly produce IL-17 even after being activated only by cytokines ( Sutton et al . , 2009; Ribot et al . , 2009 ) has been the focus of intense research .", "Only the neonatal thymic microenvironment appears to have the capacity to imprint a stable cytokine profile to γδ T cells , since the IL-17-producing γδ T cell subsets cannot be reconstituted in adult animals by bone marrow transfer ( Haas et al . , 2012 ) .", "Very recently , a hallmark paper has provided mechanistic insights into how this can be achieved: during embryonic development of certain γδ T cell subsets , the signaling threshold for activation of the TCR becomes markedly altered ( Wencker et al . , 2013 ) .", "Hence , mature γδ T cells do not necessarily rely on a TCR stimulation as “‘signal one”’ , although they can still respond to that ( Strid et al . , 2011 ) .", "However , while this intriguing mechanism ( Wencker et al . , 2013 ) explains the behavior of several γδ T cell subsets , the thymic cues orchestrating this hard-wiring of γδ T cells remain unclear .", "Previous work suggested either LTβR-dependent trans-conditioning by double positive thymocytes or γδ T cell intrinsic RelB activation ( Silva-Santos , 2005; Powolny-Budnicka et al . , 2011 ) .", "Further work by Silva-Santos and colleagues emphasized the role of TNF-receptor family members such as CD27 as well as NFκBp52 activation ( Ribot et al . , 2010 ) .", "However , the contribution of thymic stromal cells to these processes has not been experimentally addressed in vivo .", "Our data suggest that NFκB signaling via NIK seems to be dispensable both within CD27- γδ T cells as well as αβ T cells , at least for their ability for expression of pro-inflammatory IL-17 .", "Of note , deletion of NIK in all thymic epithelial cells using Foxn1-Cre phenocopied the complete NIK-knockout in terms of IL-17 expression by CD27-γδ T cells , while this was not the case using Ccl19-Cre .", "The latter has been reported to target only maturing medullary thymic epithelial cells ( Onder et al . , 2015 ) .", "This would suggest that the instruction of γδ T cells is mediated by early or junctional TECs , or that the timing of Ccl19-Cre-mediated recombination sharply coincides with the expression of the γδ T cell instructing program in TECs .", "Based on our observation that NIK signaling in the early thymic stroma has a profound and long-lasting effect on the maturation and cytokine profile of developing γδ T cells , further studies are required in order to precisely define the nature of γδ T cell instructing molecules , and which thymic epithelial cell type is required for delivery .", "While our data does not exclude a role for canonical-NFκB signaling via RelA ( Powolny-Budnicka et al . , 2011 ) , the impact of non-canonical signaling via NIK on the development of DETC precursors and IL-17-producing γδ T cells stems from signaling within the thymic stroma , for which the conditional NIK mutant is a unique tool for further studies .", "Future work will reveal whether NIK-deficient thymic stroma influences only γδ T cell development , or also the functional profile of the subsequently emerging αβ T cell compartment ." ], [ "C57Bl/6 ( wildtype ) mice were purchased from Jackson Laboratories and bred in-house at the Laboratory Animal Science Center ( LASC ) of the University of Zurich under specific pathogen-free ( SPF ) conditions .", "Map3k14-/- mice were kindly provided by Robert Schreiber ( Washington University School of Medicine , USA ) , backcrossed to C57Bl/6 background and bred in-house .", "Alymphoplasia mice ( here referred to as aly/aly ) were obtained from Clea Laboratories ( Japan ) and bred in-house .", "Map3k14flox/wt mice were generated in collaboration with the lab of Ari Waisman ( University of Mainz , Germany ) by TaconicArtemis .", "Briefly , a targeting vector containing the Loxp-flanked Exons 4-–6 of the Map3k14 loucs was introduced into embryonic stem ( ES ) cells , ES cell clones with homologous integration were selection and injected into blastocytes ( Figure 4—figure supplement 1 ) .", "Ccl19-Cre mice were generated in the lab of Burkhard Ludewig ( Institute of Immunobiology , St . Gallen , Switzerland ) by Lucas Onder ( Chai et al . , 2013 ) .", "Foxn1-Cre mice were purchased from Jackson ( Gordon et al . , 2007 ) .", "For the induction of psoriasis-like skin inflammation 55 mg of Aldara ( containing 5% of Imiquimod ) was applied to the shaved back skin of animals .", "Treatment of animals as well as measurement of skin thickness was done blinded .", "All animal experiments were approved by local authorities ( Swiss cantonal veterinary office Zurich , KVET license numbers 86/2012 , 70/2015 , 100/2015 and 68/2013 ) and performed in strict accordance with the corresponding license .", "Unless stated otherwise , ears were taken as the donor organ .", "Ears were split into dorsal and ventral sides using forceps , followed by incubation with Dispase ( 2 . 4 mg/ml , Gibco ) in HBSS with Ca2+/Mg2+ at 37°C for 90 min Epidermal sheets were removed from the dermis , cut into small pieces using scissors and further digested using Collagenase Type 4 ( 0 . 4 mg/ml , Sigma ) in HBSS with Ca2+/Mg2+ and 10% FCS at 37°C for 60-–90 min The resulting suspension was mechanically disrupted using 19 g needles and a syringe .", "After filtering , the cell suspension was used in downstream applications .", "Lungs were isolated , cut into small pieces using scissors and further digested using Collagenase D ( 0 . 5 mg/ml , Roche ) in RPMI with 2% FCS and 25 mM HEPES .", "The resulting suspension was mechanically disrupted using 19 g needles and a syringe , followed by filtering and downstream analysis .", "After setting up timed matings o/n , female mice were observed for signs of pregnancy , sacrificied on the indicated time points and embryos were isolated and euthanized by decapitation .", "The day after timed mating was considered day 0 .", "Embryonic thymi were collected using fine forceps and a stereo microscope ( Leica ) , and then dissociated by mechanical disruption using a cell strainer and a syringe pistil .", "Flow cytometric analysis was performed following standard methods ( reviewed in Perfetto et al . , 2004 ) .", "All flourochrome-conjugated antibodies used were obtained either from BD ( NJ , USA ) , BioLegend ( CA , USA ) or eBioscience ( CA , USA ) .", "In all stainings , dead cells were excluded using an Aqua or Near-IR Live/Dead fixable staining reagent ( Life Technologies , now Thermo Fisher Scientific , MA , USA / BioLegend ) , and doublets were excluded by FSC-A vs FSC-H gating .", "For intracellular cytokine staining , cells were incubated 4 hours in IMDM containing 10% FCS with PMA ( 50 ng/ml ) / Ionomycin ( 500 ng/ml ) and GolgiPlug ( Brefeldin A , BD ) .", "Cytofix/Cytoperm ( BD ) was used according to the manufacturers instructions , and Perm/Wash buffer was prepared in the lab ( PBS containing 0 . 5% Saponin and 5% BSA ) .", "Analysis was performed using a LSR II Fortessa ( special order research product , BD , equipped with 405 nm , 488 nm , 561 nm and 640 nm laser lines ) , cell sorting was carried out using a FACSAria III ( BD ) .", "Data analysis was performed using FlowJo V9 . x and 10 . 0 . x ( Treestar , OR , USA ) .", "Indicated cellular subsets were sort purified and collected in Trizol reagent .", "Afterwards , RNA was isolated using PureLink RNA Micro Kit ( Thermo Fisher Scientific ) following the manufacturers instructions .", "The eluted RNA was transcribed into cDNA using M-MLV reverse transcriptase ( Thermo Fisher Scientific ) or Superscript II ( Thermo Fisher Scientific ) and random primers .", "cDNA was diluted either 1:5 or 1:10 and used for qPCR with iTaq Universal SYBR Green Supermix ( BioRad , CA , USA ) and a BioRad C1000Touch / CFX384 real-time system according to the manufacturers instructions .", "The used primers ( intron-spanning ) were as follows: PolR2a ( 145 bp ) : CTGGTCCTTCGAATCCGCATC GCTCGATACCCTGCAGGGTCA Map3k14 ( 164 bp ) : CGAAACTGAGGACAACGAG CACACTGGAAGCCTGTCTG Skint-1 ( 143 bp ) : TTCAGATGGTCACAGCAAGC GAACCAGCGAATCTCCATGT Rorc ( 114 bp ) : CATATGCCTCTCTGACAGAC AAAAGAGGTTGGTGCGCTG Tbet: CAACAACCCCTTTGCCAAAG TCCCCCAAGCAGTTGACAGT Egr3: CAACGACATGGGCTCCATTC GGCCTTGATGGTCTCCAGTG Sox13: GCTTTACCTATTCAGCCCAT ACCTCTTCACCACAGGGG Unless stated otherwise , data is displayed as mean +/- standard deviation .", "Statistical calculations were performed using GraphPad Prism , and significance was calculated using an unpaired t-test unless stated otherwise ." ] ]

[ "γδ T cells contribute to first line immune defense , particularly through their ability for rapid production of proinflammatory cytokines .", "The cytokine profile of γδ T cells is hard-wired already during thymic development .", "Yet , the molecular pathways underlying this phenomenon are incompletely understood .", "Here we show that signaling via the NFκB-inducing kinase ( NIK ) is essential for the formation of a fully functional γδ T cell compartment .", "In the absence of NIK , development of Vγ5+ dendritic epidermal T cells ( DETCs ) was halted in the embryonic thymus , and impaired NIK function caused a selective loss of IL-17 expression by γδ T cells .", "Using a novel conditional mutant of NIK , we could show in vivo that NIK signaling in thymic epithelial cells is essential for the thymic hardwiring of γδ T cell cytokine production ." ]

[ "Our bodies are protected from infection and disease by several different types of immune cells .", "Gamma delta T cells are unusual in that they only make up a small proportion of the immune cells of the body , yet are present in many different animal species .", "These peculiar T cells are primarily found in the tissues that line the body ( such as the skin , lung and gut ) and are part of the first stage of the immune response that occurs when an invading microbe enters the body .", "Gamma delta T cells , like all other T cells , develop in an organ called the thymus , which is found in the chest .", "Although several complex signaling pathways have been identified that help specific immune cells to develop , there are still many open questions about them .", "It is also unclear if other cells in the thymus influence how gamma delta T cells develop .", "Mair et al . engineered mouse embryos that could not produce a signaling molecule known as NIK in certain subsets of their cells .", "This revealed that NIK is important for a structural cell in the thymus to instruct the early stages of gamma delta T cell development .", "However , gamma delta T cells themselves do not need to be able to produce NIK or the signaling pathway that it triggers .", "Further work will focus on discovering the exact way in which the structural cells of the thymus interact with gamma delta T cells .", "This will help us understand better how developing immune cells are ‘educated’ in the thymus so that they are able to work effectively in the adult ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "neuroscience", "genetics and genomics" ]

[ [ "Flight is an extraordinary biological trait that has only evolved in several kinds of animals ( e . g . insects , bats , and birds ) and is notably effective in searching for food and mates , finding habitats , defending against predators , and adapting to seasonal changes in environment ( Krause and Godin , 1996; Borgemeister et al . , 1997; Chapman et al . , 2010 ) .", "Despite these adaptive advantages , flight is one of the most intense and energy-demanding physiological processes , especially for insects that possess long-term flight capacity , such as locusts and butterflies ( Mentel et al . , 2003; Zhan et al . , 2014 ) .", "Long-term flight is usually defined as sustained flight for seasonal and long-range migration toward a distinct direction in populations ( Stefanescu et al . , 2013; Juhász et al . , 2021 ) .", "Metabolic rates in the flight muscle can increase by 20- to 100-fold during flight ( Harrison and Roberts , 2000; Suarez , 2000 ) .", "To meet the high energy demands of long-term flight , migratory insects have evolved a suite of adaptive physiological traits ( Arrese and Soulages , 2010 ) and exhibit highly efficient utilization of energy substrates , such as carbohydrates and lipids , in the flight muscle ( Canavoso et al . , 2003; Van der Horst and Rodenburg , 2010 ) .", "Compared to short-term flight , which exclusively employs carbohydrates as energy substrates , many physiological activities subsequently occur in the flight muscle during long-term flight , including carbohydrate/lipid metabolism transition , fatty acid transport , lipid oxidation , and lipid mobilization in the fat body .", "Clearly , complex and precise spatial and temporal regulation of energy metabolism is essential for long-term fight performance .", "However , the mechanisms underlying the highly efficient energy utilization associated with long-term flight have not been elucidated to date .", "A number of reports have emphasized the central roles played by neuropeptides in coordinating systematic energy metabolism during insect flight ( Gade , 1992; Lorenz and Gäde , 2009 ) .", "Most neuropeptides are produced by the central nervous system and perform distinct tasks through binding with their cognate G-protein-coupled receptors ( GPCRs ) ( Nässel , 2002 ) .", "By affecting energy metabolism either directly or indirectly , neuropeptides participate in a variety of biological events , such as flight , reproduction , diapause , and immune response ( Gäde and Marco , 2009; Sim and Denlinger , 2013; Ling et al . , 2017; Urbanski and Rosinski , 2018 ) .", "Furthermore , a single behavior or physiological process is usually controlled by multiple neuropeptides that play distinct roles in energy metabolism ( Waterson and Horvath , 2015; Toprak , 2020 ) .", "For example , adipokinetic hormone ( AKH ) has been demonstrated to be a conserved regulator of flight-related energy metabolism by promoting glycolysis and lipid mobilization in the fat body in different insect species ( Gäde et al . , 2006; Kaufmann and Brown , 2008 ) .", "Downstream signal transduction of AKH involved in lipid mobilization has been elucidated in insects ( Gäde and Auerswald , 2003 ) .", "In addition , other neuropeptides are also involved in either lipogenesis or lipolysis and play distinct roles in insect flight ( Toprak , 2020 ) .", "The migratory locust , Locusta migratoria , which possesses a notable long-term flight capacity , is one of the most destructive agricultural pests ( Wang and Kang , 2014 ) and has been employed as a useful study model for the neurohormonal regulation of flight-related energy metabolism ( Jutsum and Goldsworthy , 1976; Van der Horst and Rodenburg , 2010; Bullard et al . , 2017 ) .", "The locust displays strong adaption to long-distance flight at both the physiological and morphological levels , exhibiting clear expansion of the energy gene family and high plasticity of muscle metabolism ( Wang et al . , 2014 ) .", "In the locust , the patterns of energy metabolism display tissue-specific and time-dependent patterns during tethered flight .", "Usually , metabolism involved in long-term flight in insects contains two major phases: during the early stage of flight , carbohydrates in flight muscle and hemolymph significantly decrease , and lipid mobilization in the fat body gradually increases thereafter followed by utilization in the flight muscle during the prolonged flight phase ( Worm and Beenakkers , 1980; Wegener et al . , 1986 ) , indicating a clear transition in energy consumption from carbohydrates to lipids during long-term flight .", "Distinct neuropeptide and neurotransmitter have been demonstrated to modulate lipid mobilization and transport in the fat body ( Van der Horst , 2003 ) and carbohydrate catabolism in flight muscle at the beginning of flight ( Mentel et al . , 2003 ) , and the relevant regulatory mechanisms have also been uncovered .", "Nevertheless , neuroendocrine mechanisms underlying energy utilization associated with prolonged flight remain to be explored .", "In this study , we employed integrated multi-omics studies to screen potential neuropeptides involved in the long-term flight of locusts and elucidated relevant regulatory mechanisms by using the CRISPR/Cas9 method .", "Finally , we identified a novel flight modulator , the AKH/corazonin-related peptide ( ACP ) , which plays an important role in prolonged flight by facilitating lipid utilization in the flight muscle in locusts ." ], [ "Given that flight is a unique biological trait in adult locusts , we first performed comparative neuropeptidome analysis on the neuroendocrinal tissues brain ( Br ) and retrocerebral complex ( main endocrine tissues of the locust , RC ) between final instar nymphs ( 5th ) and adult locusts through high-resolution and high-sensitivity MS ( LTQ-Orbitrap Elite ) to identify neuropeptides possibly related to flight .", "In total , 201 and 362 nonredundant peptides ( including both mature neuropeptides and their potential degradation products ) derived from 37 neuropeptide precursors were identified in Br and RC , respectively ( Supplementary file 1 ) .", "Tissue-specific analysis showed that neuropeptides from 20 precursors were considerably more abundant in the RC , whereas neuropeptides from 16 precursors were more abundant in the Br .", "The GPB5-derived peptides showed similar abundance levels in Br and RC ( Figure 1—figure supplement 1 ) .", "The abundant levels of neuropeptides in Br and RC between 5th-instar nymphs and adult locusts were further compared by a label-free quantitative strategy .", "Compared to 5th-instar nymphs , there were 20 and 18 upregulated neuropeptides in the Br and RC of adult locusts , respectively ( Figure 1—figure supplement 2 ) , and 10 neuropeptides displayed significantly higher abundance ( Log2FC > 1 . 5 ) in either Br or RC of adult locusts ( Figure 1A and B ) .", "To validate whether these neuropeptides were closely related to flight activity , we examined the expression levels of the precursor genes of these ten neuropeptides in either Br or RC ( depending on their tissue-specific expression patterns , Figure 1—figure supplement 3 ) after 1 h-sustained flight .", "The mRNA levels of four neuropeptide precursor genes , namely , AKH/corazonin-related peptide ( ACP ) , adipokinetic hormone ( AKH2 ) , NPF1 , and GPB5 , changed significantly after flying treatment .", "Among these genes , three ( ACP , AKH2 , and NPF1 ) exhibited clearly increased expression levels , whereas GPB5 displayed decreased expression levels after 1 h- of sustained flight ( Figure 1C and Figure 1—figure supplement 4 ) .", "To explore whether these four neuropeptides are involved in the modulation of flight performance in locusts , we performed gene knockdown by RNA interference ( RNAi ) for each gene in adult locusts .", "Of these four genes , only the ACP and AKH2 RNAi treatments exhibited significant effects on the flight activity of adult locusts , although the expression levels of all neuropeptide genes were successfully downregulated ( Figure 1—figure supplement 5 ) .", "Compared with the control , total flight time and total flight distance decreased by more than 50% after the knockdown of ACP or AKH2 ( Figure 1D and E ) .", "However , the average flight velocity and maximum flight velocity of the locusts did not significantly change ( Figure 1—figure supplement 6A and B ) .", "AKH family members have been determined to play conserved roles in flight activity by promoting the mobilization of lipids and carbohydrates stored in the fat body of locusts ( Van der Horst , 2003 ) .", "However , to date , few studies have investigated ACP functions in insects .", "Thus , we then validated the regulatory role of ACP in flight activity by injecting synthetic ACP peptide in adult locusts .", "After peptide injection , the locusts exhibited significantly enhanced total flight time and total flight distance , whereas average and maximum flight velocity were unaffected ( Figure 1—figure supplement 7 ) .", "These results confirm the essential regulatory role of ACP peptide in locust flight ability .", "The ACP precursor gene exhibited a brain-specific expression pattern ( Figure 2A ) .", "In addition , the mRNA level of ACP did not significantly change until sustained flight for 1 h , with a 100% increase being observed at this time point ( Figure 2B ) , implying that the ACP peptide plays major roles in facilitating long-term flight in locusts .", "To further explore the functional roles played by ACP in long-term flight , we generated an ACP mutant line using a CRISPR/Cas9-mediated gene editing system .", "We injected Cas9 protein and a gRNA targeting the second exon of the ACP gene into eggs < 2 h after they were laid ( Figure 2C ) .", "The ACP gRNA-induced mutation at high efficiency in the G0 generation , as well as their progeny ( G1 ) ( Figure 2—figure supplement 1A , Supplementary file 2 ) .", "We finally successfully obtained a heritable homozygous ACP mutant line with a 13 bp deletion modification ( ACP13/13 , referred to as ACP-/- in the following text ) by performing a series of crossing experiments ( Figure 2D and Figure 2—figure supplement 1B ) .", "The ACP-/- locusts were predicted to produce a frameshift precursor unable to give rise to mature ACP peptide .", "Through immunohistochemistry analysis , we detected strong signals for ACP peptide in the neurons of the par intercerebralis and bilateral forebrain of wild-type locusts ( WT ) ( Figure 2E and Figure 2—figure supplement 2 ) , whereas the fluorescence signal was not observed in the brain of ACP-/- locusts ( Figure 2E ) , which further confirmed the successful construction of ACP mutants of the migratory locust .", "In comparison with WT locusts , no significant difference in the survival rate of either females or males was observed ( Figure 2—figure supplement 3 ) , whereas ACP-/- locusts had a larger body size ( Figure 2F ) .", "Next , we compared the flight performance of ACP mutants and WT locusts during the 60 min sustained tethered flight test .", "Within the first 15 min , there was no significant difference in total flight distance and total flight time between female ACP mutants and WT locusts .", "However , for the 60 min tethered flight test , both females and males of ACP mutants displayed significantly shorter flight times and flight distances compared with the WT locusts ( Figure 2G and H ) .", "The average flight velocity and maximum flight velocity did not show any changes between ACP mutants and WT locusts in either 15 min or 60 min flight tests ( Figure 2I and Figure 2—figure supplement 4 ) .", "These results indicated that the neuropeptide ACP was involved in modulating long-term flight in locusts .", "Distinct receptors play essential roles in mediating the functions of neuropeptides , and tissue-specific expression patterns of neuropeptide receptors have been proposed to be sources of critical information for exploring the regulatory mechanisms of neuropeptides ( Garcia et al . , 2015; Nässel and Vanden Broeck , 2016 ) .", "Therefore , we further identified the ACP receptor by homolog searching the genome and transcriptome databases .", "Phylogenetic analysis showed that the putative locust ACPR was closely related to ACP receptors from other insects but was evolutionarily divergent from its structure-related neuropeptide receptors , AKH receptors and corazonin receptors ( Figure 3A ) .", "Compared to other organs tested , ACPR was highly expressed in the fat body and flight muscle of adult locusts ( Figure 3B ) .", "To validate the role of ACPR in regulating long-term flight , we generated an ACPR mutant locust line using the CRISPR/Cas9 system .", "Combined injection of designed ACPR gRNA and Cas nine protein induced multiple kinds of mutations around the target sequence in the G0 generation ( Figure 3C and Supplementary file 3 ) .", "Using the crossing strategy similar to ACP mutant line construction , we successfully obtained a homozygous ACPR mutant line with a 13 bp deletion ( ACPR13/13 ) .", "The ACPR13/13 locusts were predicted to produce truncated protein that losses the last four transmembrane domains ( Figure 3D ) .", "Compared with the WT locusts , both ACPR female and male mutants showed significantly reduced flight time and flight distance during the 60 min tethered flight test ( Figure 3E and F ) , although intense immunostaining signal of ACP peptide was detected in the brain of ACPR mutants ( Figure 3—figure supplement 1 ) .", "However , no significant changes in average flight velocity and maximum flight velocity were observed between ACPR mutants and WT locusts ( Figure 3—figure supplement 2 ) .", "The flight phenotypes caused by ACPR knockout was similar to that observed in ACP mutants , supporting that the essential role of ACP peptide system in modulating long-term flight in locusts .", "Based on the tissue-specific expression pattern of ACPR , we hypothesized that fat body and flight muscle may be the main tissues targeted by ACP to participate in flight regulation .", "We then performed comparative transcriptome analysis of the fat body and flight muscle tissues in ACP-/- and WT locusts .", "The results showed that the number of differentially expressed genes ( DEGs ) in flight muscle was greater than that in fat body ( 520 in flight muscle and 318 in fat body , Log2 FC >1 , FDR < 0 . 05 , RPKM > 0 . 5 ) .", "For ACP-/- locusts , there were 212 upregulated and 308 downregulated genes in the flight muscle and 200 upregulated and 118 downregulated genes in the fat body ( Figure 4—figure supplement 1 ) .", "The fat body and flight muscle had more tissue-specific DEGs and fewer overlapping DEGs after knockout of the ACP gene ( Figure 4A ) .", "Several pathways associated with energy metabolism were significantly changed in the flight muscle of ACP-/- locusts ( -Log2 ( P value ) >10 , enriched gene number >15 ) , including oxidation phosphorylation , fatty acid degradation , valine , leucine and isoleucine degradation , cardiac muscle contraction , and fatty acid metabolism .", "However , only a small number of genes were enriched in the KEGG analysis of the fat body ( -Log2 ( P value ) <10 , gene number <10 ) ( Figure 4B ) , indicating that the gene expression profiles of the flight muscle were more strongly affected by ACP knockout .", "The expression levels of 4 genes responsible for fatty acid transport , 10 genes involved in beta-oxidation , and 19 genes associated with mitochondrial energy metabolism were clearly downregulated in the flight muscle of the ACP-/- locust ( Figure 4C ) .", "A fatty acid binding protein ( FABP ) , as the most highly expressed gene ( Log10 ( mean expression ) >4 ) , exhibited an expression decrease of more than 80% ( log2FC = −2 . 43 ) in ACP-/- locusts ( Figure 4D ) .", "Reduced expression levels of FABP in the flight muscle of ACP-/-locusts were further confirmed by qPCR and western blot analyses ( Figure 4E and F ) .", "We also validated decreased mRNA levels of eight other genes involved in fatty acid transport and beta-oxidation in the flight muscle of ACP mutants ( Figure 4—figure supplement 2A ) .", "Reduced expression of representative genes that participate in mitochondrial fatty acid transport ( CPT2 ) and beta-oxidation ( ACDM ) was also confirmed at the protein level ( Figure 4—figure supplement 2B ) .", "Moreover , we found that the expression levels of genes related to fatty acid transport and oxidation were strongly enhanced in the flight muscle upon ACP peptide injection as well as 1 h-sustained flight of WT locusts ( Figure 4—figure supplement 3 ) , implying that the ACP peptide may facilitate long-term flight by promoting fatty acid utilization .", "Given that FABP serves as a primary transporter for fatty acid translocation through the aqueous cytosol to mitochondria , where the beta-oxidation process takes place ( Haunerland and Spener , 2004 ) , we further assessed whether the change in FABP could affect the expression levels of beta-oxidation-related genes .", "RNAi knockdown of FABP resulted in a 99 . 3% decrease in FABP mRNA levels ( Figure 4—figure supplement", "4 ) and clearly suppressed the expression levels of multiple beta-oxidation-related genes , including CROT , CPT2 , ACDM , ACADS , ACADSB , ECH-6 , ACAT1 , and CRAT ( Figure 3G ) .", "Taken together , these results indicated that the lipid metabolism pathway was significantly suppressed in the flight muscle of ACP-/- locusts and that FABP may serve as an important molecular target of ACP peptide signaling .", "Based on the above results , we hypothesized that ACP knockout may affect energy utilization in the flight muscle of locust adults .", "To verify this possibility , we performed comparative metabolome analysis between ACP-/- and WT flight muscles by ultra-high-performance liquid chromatography-high resolution mass spectrometry ( UPLC-HRMS ) .", "In total , we identified 881 metabolites with confidence from all twelve samples ( Figure 5—figure supplement 1 ) .", "Locust samples of WT and ACP-/- can be clearly separated by unsupervised principal component analysis ( PCA ) and orthogonal projection to latent structures–discriminant analysis ( OPLS-DA ) ( Figure 5A and Figure 5—figure supplement 2 ) .", "The overall metabolite distribution was considerably more intense in WT samples than in ACP-/- samples ( Figure 5A ) , suggesting reduced general metabolic activity in the flight muscle of ACP-/- locusts .", "Statistically , we obtained 204 downregulated metabolites and 31 upregulated metabolites in ACP-/- samples ( Student’s t-test , p<0 . 05 ) ( Figure 5B ) .", "The differential metabolites identified between the two groups included many triacylglycerols ( TG ) , glycerophosphatides ( PC , PE , PG , LysoPC , LysoPE ) , amino acids , carbohydrates , acylcarnitine , and nucleotides ( Figure 5C and Figure 5—figure supplement 3 ) .", "Clustering analysis demonstrated that eight medium/long-chain acylcarnitines ( AcCa18:1 , AcCa16:0 , AcCa18:0 , AcCa16:1 , AcCa14:1 , AcCa14:0 , AcCa17:1 , AcCa16:2 ) were present at significantly reduced levels in the flight muscle of ACP-/- locusts .", "Meanwhile , several coenzymes involved in mitochondrial oxidative phosphorylation were decreased , such as coenzyme Q9 , coenzyme Q10 , nicotinamide ribotide , and flavin mononucleotide ( Figure 5D ) .", "These data indicated that acyl carnitine-dependent fatty acid transport to the mitochondrion and subsequent metabolism were notably decreased in the flight muscle of ACP mutants .", "We further evaluated beta-oxidation status between WT and ACP-/- samples by determining the relative amounts of two end metabolites of beta-oxidation , acetyl-CoA and NADH .", "Relative amounts of acetyl-CoA and NADH in the ACP-/- samples significantly decreased by 77% and 65% , respectively , relative to the levels observed in WT locusts ( Figure 5E and F ) .", "Instead , the injection of ACP peptide could significantly enhance the relative amounts of acetyl-CoA and NADH in the flight muscle of WT locusts ( Figure 5—figure supplement 4A ) .", "However , upon 1 h-tethered fight , the relative content of acetyl-CoA did not change but the NADH level was decreased ( Figure 5—figure supplement 4B ) .", "As a key intermediate metabolite of energy metabolism , acetyl-CoA is primarily generated from fatty acid oxidation and oxidative decarboxylation of pyruvate , which is an important product of glycolysis ( Rui , 2014 ) .", "We next assessed whether the glycolysis process was affected by ACP knockout by measuring the pyruvate amount .", "We observed that the relative level of pyruvate showed no significant difference between WT and ACP-/- samples ( Figure 5G ) .", "To confirm the changes in energy metabolism in ACP-/- locusts , we also determined the abundance of citric acid , a representative metabolite of the tricarboxylic acid ( TCA ) cycle produced from acetyl-CoA ( Koubaa et al . , 2013 ) .", "The relative level of citric acid in the WT samples was 2 . 82-fold higher than that in ACP-/- samples ( Figure 5H ) .", "Therefore , by combined analysis of gene expressions and metabolite contents , we inferred that the downregulation of genes and metabolites involved in lipid transport and beta-oxidation in the flight muscle contributes to the deteriorated flight ability of ACP-/- mutant ( Figure 5I ) .", "We further verified whether FABP acts as a key downstream molecular target of ACP to regulate lipid metabolism in flight muscle during the long-term flight of locusts .", "First , we carried out a 60 min-tethered flight test after performing gene knockdown of FABP in locust adults .", "Compared to dsGFP-injected locusts , the total flight duration and total flight distance did not change within the first 15 min but were significantly decreased after 60 min of flight in dsFABP-injected locusts ( Figure 6A and B ) .", "The average and maximus flight velocity did not change significantly , although a declining tendency was observed after FABP knockdown ( Figure 6—figure supplement 1 ) .", "Next , we measured the amount of acetyl-CoA and NADH because they indicate beta-oxidation status after dsFABP treatments .", "Compared to the dsGFP control , the relative levels of acetyl-CoA and NADH were strongly reduced by 70% and 44% in the dsFABP-injected samples ( Figure 6C and D ) , respectively .", "To demonstrate the key role played by FABP in mediating the effect of ACP on lipid metabolism in flight muscle , we performed a molecular rescue experiment by combined peptide injection and gene knockdown in ACP-/- locusts .", "When ACP peptide was injected in ACP-/- locusts , the expression levels of eight key beta-oxidation genes significantly increased in the flight muscle , whereas this stimulatory effect of ACP injection was remarkably abolished by FABP knockdown ( Figure 6E ) .", "The knockdown of FABP also alleviated the upregulated amount of acetyl-CoA and NADH in the flight muscle ( Figure 6F and G ) .", "In addition , the enhancement of the contents of multiple medium/long-chain acylcarnitines induced by ACP peptide administration also disappeared after FABP gene silencing ( Figure 6H ) .", "In particular , the impaired prolonged flight performance in ACP-/- locusts , including reductions in both total flight duration and flight distance , could be efficiently recovered by ACP peptide injection .", "Moreover , this recovered flight activity induced by the ACP peptide was clearly blocked by dsFABP treatments ( Figure 6I ) .", "Taken together , these results indicated that FABP acts as a key component of ACP signaling , regulating lipid metabolism of the flight muscle during long-term flight in locusts ." ], [ "Through peptidome analysis , we obtained a lot of non-abundant neuropeptides as well as their potential degradation products produced by 37 precursors in the main neuroendocrine tissues , including brain and retrocerebral complex .", "The neuropeptides detected here including most of previously identified peptides ( Clynen and Schoofs , 2009 ) .", "Many of the peptides were abundant at adult stage , indicating their distinct roles in adult-related biology .", "However , several neuropeptides ( e . g . AST-C , inotocin , and AKH4 ) identified from previous peptidomic study and transcriptome data-based prediction , were not found in the current study .", "The absence of these neuropeptides in the neuropeptidomic analysis may thanks to their low abundance in tested samples , relative short half-life period , unsuitable chromatographic condition or data acquisition setting .", "Different sample collection methods as well as multiple mass spectrometry methods may be helpful for systematically identification of all neuropeptides in future .", "We also found that the neuropeptides displayed apparently tissue-specific distributions , with either brain- or retrocerebral complex-specific distribution in the locust .", "By comparing our results with previously peptidome study ( Clynen and Schoofs , 2009 ) , we found that most of the neuropeptides show similar tissue distribution in the two studies , except for sulfakinin and PVK .", "The discrepancy between the two studies may be attributed to different sample collection strategies .", "In the present study , the whole retrocerebral complex of mature adults was used for peptidomic analysis , in contrast to the study of Clynen and Schoofs , 2009 who analyzed the organs of the retrocerebral complex of immature adults separately .", "Our results suggested that the ACP peptide acts as a neuroendocrine hormone to regulate locust flight capacity .", "To the best of our knowledge , this study is the first to clearly demonstrate the biological function of ACP in insects .", "In fact , the ACP peptide was initially isolated from the storage lobes of the CC of migratory locusts and was named locust hypertrehalosemic hormone ( Lom-HrTH ) because of its activity in the induction of hemolymph trehalose levels in cockroaches but not in locusts ( Siegert , 1999 ) .", "Further MS analysis shows that the peptide is highly distributed in CC , hypocerebral ganglion , frontal ganglion , protocerebrum , pars intercerebralis , tritocerebrum , as well as thoracic ganglia of immature Africa migratory locust ( Clynen and Schoofs , 2009 ) .", "However , the biological significance of ACP has not been described in detail .", "Our results show that the ACP precursor gene in the brain displays strong transcription responses to prolonged flight .", "The regulatory roles played by ACP in locust flight are clearly supported by tethered flight experiments after knockdown and knockout of its precursor gene , as well as reduced extended flight ability of ACPR mutants .", "Based on ours and previous finding , ACP mature peptide is abundantly detected in the CC of locusts , indicating that ACP may be synthesized in neurosecretory cells of the brain and transported to the storage lobe of the CC via nervi corporis cardiaci I and/or II ( Hekimi and O'Shea , 1987 ) .", "Thus , the ACP peptide may be involved in locust flight through secretion into the circulation , to modulate distinct physiological activities in target tissues , such as the flight muscle and fat body , where its receptor is expressed .", "The ACP peptide is an insect structural intermediate of corazonin and AKH hormones , all of which belong to the vertebrate gonadotropin-releasing hormone ( GnRH ) family ( Hansen et al . , 2010 ) .", "ACP and its cognate receptor have been found in various insects , although they are selectively lost in several insect species , such as the fruit fly ( Diptera ) , the honey bee ( Hymenoptera ) , the pea aphid ( Hemiptera ) , and the body louse ( Phthiraptera ) ( Hansen et al . , 2010 ) .", "The role played by ACP in energy metabolism has been suggested in several insect species; for example , this gene has been suggested to regulate hemolymph carbohydrate and lipid levels in Gryllus bimaculatus ( Zhou et al . , 2018 ) and to play a possible role in glycogen hydrolysis in Platypleura capensis ( Gäde and Janssens , 1994 ) .", "Thus , ACP may play species-specific roles in modifying metabolic activity in insects .", "Furthermore , it appears that all three GnRH neuropeptides ( AKH , ACP , and corazonin ) are related to energy embolism to some extent ( Andreatta et al . , 2020 ) , although they have distinct tissue or cellular distributions and do not share overlapping detailed biological roles ( Patel et al . , 2014 ) .", "Our results demonstrate that ACP modulates long-term flight by primarily affecting lipid transport and utilization in the flight muscle of locusts .", "This finding is strongly supported by a significant decrease in the levels of genes ( e . g . FABP , CPT2 , CRAT , ACDs , ECHs , HADHs , ACATs ) and metabolites ( carnitine and acylcarnitine ) related to lipid transport and beta-oxidation ( Rubiogozalbo et al . , 2004 ) in the flight muscle of ACP mutant locusts , as well as the enhanced metabolism-related gene expressions and fatty acid oxidation activity in WT locusts upon ACP peptide administration .", "Integrating energy metabolism related to long-term flight is a complex and multistep physiological process ( Auerswald and Gäde , 2006 ) .", "Generally , the initial flight primarily consumes carbohydrates as an energy substrate , whereas subsequent prolonged flight depends largely on highly efficient lipid utilization as an energy supply ( van der Horst et al . , 1993; Van der Horst and Rodenburg , 2010 ) .", "We show that ACP strongly affects lipid transport and oxidation , not carbohydrate metabolism , in the locust .", "The changes in lipid metabolism in the flight muscle upon ACP manipulations ( both gene knockout and peptide injection ) are closely in line with the alteration of long-term flight performance in the parallel treatments , demonstrating the distinct functional roles played by ACP during long-term flight .", "Despite the inconsistent effects on beta-oxidation products in the flight muscle upon ACP peptide injection ( stimulatory effect ) and sustained flight ( partially inhibitory effect ) , both two treatments could significantly enhance the expressions levels of gene related to lipid utilization in the flight muscle , implying that the ACP peptide may facilitate lipid utilization in response to prolonged flight .", "The decreased beta-oxidation products may reflect rapid energy utilization in subsequent mitochondrial metabolism during sustained flight .", "We showed that FABP serves as a key molecular target mediating the regulatory effects of ACP on lipid metabolism during locust long-term flight .", "Although multiple FABP family members are predicted in the locust genome ( Wang et al . , 2014 ) , the FABP identified in this study is specifically expressed in the flight muscle of adult locusts with notably high abundance ( Haunerland et al . , 1992 ) .", "The mediating role played by FABP in ACP-controlled flight-related lipid metabolism was further confirmed by knockdown and rescue experiments at the molecular , metabolic , and behavioral levels .", "The significant role of FABP in prolonged flight has also been reported in the desert locust ( Rajapakse et al . , 2019 ) , indicating that FABP plays a conserved role in lipid metabolism and long-term flight in locust species .", "FABP has been characterized as an evolutionarily conserved fatty acid carrier that plays essential roles in lipid utilization by affecting mitochondrial beta-oxidation ( Luxon , 1993; Luxon et al . , 1997; Binas and Erol , 2007 ) .", "Usually , tissues with high fatty acid oxidative capacities possess more FABP than those that use carbohydrates as an energy source .", "The locust flight muscle is structurally and functionally closely related to the mammalian heart muscle , which also depends mostly on fatty acids to fuel its continuous contractions ( Neely and Morgan , 1974 ) .", "Therefore , FABP is suggested to be equally important for insect flight muscle and mammalian heart muscle .", "It has been shown that FABP gene expression in muscle can be upregulated by sustained flying ( Chen and Haunerland , 1994 ) and extended physical exercise ( Lammers et al . , 2012 ) .", "However , the regulatory mechanisms underlying FABP transcription by upstream molecules have not been fully characterized .", "The regulation of FABP expression by ACP during long-term locust flight thus presents a typical case showing the modulation of FABP expression by neuroendocrine factors and may help to elucidate the common molecular mechanisms that participate in the modulation of muscle FABP expression in different species .", "Further studies are warranted to decipher signaling pathways mediating the regulatory effect of ACP on FABP expression , and the results of this research may help to elucidate the precise metabolic mechanisms regulating high energy-demanding activities .", "The central roles played by neuropeptides in glycogen hydrolysis and lipid mobilization have been extensively demonstrated in various insect species ( Arrese and Soulages , 2010; Andreatta et al . , 2020 ) .", "However , few studies have examined the regulatory mechanism underlying flight-related lipid utilization in flight muscle .", "The transition of substrate utilization from carbohydrates to lipids in the flight muscle has been proposed to be crucial for maintaining long-term flight ( Van der Horst and Rodenburg , 2010 ) .", "Glycolysis in flight muscle during early-stage flight is controlled by octopamine ( Mentel et al . , 2003 ) .", "In comparison , lipid mobilization and glycogen hydrolysis in the fat body during prolonged flight are directly modulated by AKH , whose receptor primarily localizes in the fat body ( Van der Horst , 2003 ) .", "The expression levels of all three AKH genes strongly increase upon sustained flight in the locust ( Bogerd et al . , 1995 ) .", "However , enhanced expression after sustained flight was observed only for AKH2 in our experiments .", "The difference between the two studies may be attributed to the different sample collection strategy and detection methods used retrocerebral complex for expression analysis examined by qPCR in our study , whereas only CC tissue for expression analysis detected by norther blot in previous work .", "A strong reduction in lipid metabolites , such as acylcarnitines , acyl-CoA , NADH , triglycerides , and phosphoglycerides , was observed in the flight muscle of ACP mutants , whereas pyruvic acid generated from glycolysis did not change , suggesting that ACP primarily affects lipid metabolism , rather than glycolysis , in the flight muscle of locusts .", "We infer that these three flight-related regulators have functional differentiation in either a temporally or spatially dependent manner in the modulation of flight metabolism .", "A coordinated regulatory network involving AKH , ACP , and octopamine is thus proposed to modulate the cooperation of substrate mobilization , transport , and utilization in different tissues during long-term flight .", "Here , we also revealed that knockdown of either ACP or AKH2 induced similarly suppressed effects on locust flight performance .", "Further work is warranted to investigate the potential interaction among these neuroendocrine factors in energy regulation associated with flight activity .", "Through phenotype examination , we also observed a larger body size in ACP mutants .", "Usually , the body size of locusts is stable after adult eclosion thanks to its hard exoskeleton .", "Therefore , the effects of ACP on body size could not be assessed through RNAi of the gene after adult eclosion in the current study .", "It has been suggested that the growth state for an organism can be negatively affected by other physiological traits , such as locomotion , reproduction , or life span ( Lee et al . , 2010 ) .", "Therefore , the increased body size of ACP mutants may be attributed to the continuous metabolism changes associated with trade-off effects between flight activity and body growth .", "Similarly , the loss of function of the AKH peptide results in adult-onset obesity in Drosophila ( Gáliková et al . , 2015 ) .", "These findings may reflect a common role played by ACP and AKH in governing the energy balance of insects .", "It will be an interesting work to explore the molecular and metabolic basis for body size determination on the basis of established ACP mutant locust line .", "In summary , we demonstrate that the ACP peptide acts as a novel neuroendocrine regulator controlling lipid transport and utilization associated with long-term flight in locusts .", "The ACP-FABP axis involved in long-term flight may serve as an effective molecular target for the prevention of locust plagues and may provide insights into metabolic hemostasis related to sustained locomotion ." ], [ "Locusts used in these experiments were obtained from a colony maintained at the Institute of Zoology , Chinese Academy of Sciences , Beijing , China .", "Both nymphs ( 300–400 insects per cage ) and adult locusts ( ~100 per cage ) were reared under a 14:10 light:dark photocycle regime at 30 ± 2°C .", "The locusts were fed with fresh wheat seedlings and bran ( Hou et al . , 2017 ) .", "For age definition , adult locusts between 0 and 12 hafter molting were referred as day 0 post-adult eclosion ( PAE 0 day ) .", "For sample preparation , the brain tissues containing only the protocerebrum , deuterocerebrum , and tritocerebrum and the retrocerebral complex ( including corpora cardiaca , corpora allata , hypocerebral ganglion , and small neuronal structures ) from 35 fifth-instar female nymphs ( the third day after molting ) and 35 mature female adults ( PAE 10 days ) were carefully microdissected and frozen in liquid nitrogen .", "All samples were stored at −80°C .", "Three independent biological replicates were prepared for each sample .", "Tissues were homogenized in 200 μl lysis buffer ( methanol/ddH2O/formic acid = 90/9/1 ) through sonication on ice followed by centrifugation at 12 , 000 × rpm for 20 min at 4°C to remove insoluble fractions .", "The supernatants were ultrafiltered with a 10 kDa ultrafiltration column and freeze-dried for peptide collection .", "The peptide pellets were resuspended in 20 μl 0 . 1% formic acid prior to MS analysis ( Han et al . , 2015 ) .", "LC-MS/MS analysis was performed on an Easy-nLC 1000 ( Thermo Fisher Scientific , Bremen , Germany ) coupled LTQ-Orbitrap Elite ( Thermo Fisher Scientific ) hybrid mass spectrometer .", "Peptides were separated on a column packed with 2 μm C18 ( 100 Å , 75 μm x 50 cm , Thermo Fisher Scientific ) using a 130 min gradient from 3–30% acetonitrile ( 0 . 1% formic acid ) with a flow of 250 nL/min .", "The eluted neuropeptides were injected into the mass spectrometer via a nano-ESI source ( Thermo Fisher Scientific ) .", "Ion signals were collected in a data-dependent mode and run with the following settings: full scan resolution at 70 , 000 , automatic gain control ( AGC ) target 3E6; maximum inject time ( MIT ) 20 ms; scan range m/z 300–1800; MS/MS scans resolution at 17 , 500; AGC target 1E5; MIT 60 ms; isolation window 2 m/z; normalized collision energy 27; loop count 10; charge exclusion: unassigned , 1 , 8 , >8; peptide match: preferred; exclude isotopes: on; dynamic exclusion: 30 s; dynamic exclusion with a repeated count:", "1 . The MS/MS data were acquired in raw files using Xcalibur software ( version 2 . 2 , Thermo Fisher Scientific ) .", "The extracted MS/MS spectra were searched against a composite database of Locust migratoria ( 3286 protein sequences , download from NCBI , 2019 ) and a protein database ( containing 17 , 307 protein sequences , http://www . locustmine . org:8080/locustmine ) ( Yang et al . , 2019 ) using in-house PEAKS software ( version 7 . 0 , Bioinformatics Solutions , Waterloo , Canada ) .", "The database search parameters: parent ion mass tolerance is 15 ppm , and fragment ion mass tolerance is 0 . 05 Da; enzyme specificity , none .", "The following modifications were applied: C-terminal amidation ( A , −0 . 98 ) and pyroglutamination from Q ( P , −17 . 03 ) , maximum missed cleavages per peptide: 2 , and maximum allowed variable PTM per peptide:", "2 . A fusion target and decoy approach was used to for the estimation of the false discovery rate ( FDR ) and controlled at ≤1 . 0% at the peptide level .", "Identified neuropeptides were further validated by comparison with predicted neuropeptide precursors and previous neuropeptidome analysis in the locust ( Clynen and Schoofs , 2009; Hou et al . , 2015 ) .", "Relative quantification of the neuropeptidome was performed by the label-free approach in the PEAKS Q module ( Han et al . , 2015 ) .", "Feature detection was performed separately on each sample by using the expectation-maximization algorithm .", "The features of the same peptide from different samples were reliably aligned together using a high-performance retention time alignment algorithm .", "The identification results were chosen to attach as the last step of the label-free quantification .", "Peptides were determined to be significantly changed between different samples if Student’s t-test yielded p-values<0 . 01 .", "Total RNA of experimental samples was isolated using TRIzol reagent ( Invitrogen ) .", "RNA quantification and reverse transcription were performed as previously described ( Hou et al . , 2017 ) .", "Transcript levels of target genes were detected by SYBR Green kit on a LightCycler 480 instrument according to the manufacturer’s instructions ( Roche ) .", "RP49 was used as an internal reference .", "Dissociation curves were determined for each gene to confirm unique amplification .", "At least four biological replicates were performed for gene expression level analysis .", "The primers are shown in Supplementary file", "4 . Tissues including the brain containing only the protocerebrum , deuterocerebrum , and tritocerebrum , thoracic ganglion , retrocerebral complex , fat body , flight muscle , and ovary ( or testis ) of adult locusts at PAE 7 days were dissected and frozen in liquid nitrogen .", "All samples were stored at −80°C .", "Four independent biological replicates were prepared for each sample .", "Tissues of six to eight individuals were collected for each replicate .", "RNA extraction and qPCR analysis were performed as described above .", "Double-stranded RNAs of target genes were synthesized using the T7 RiboMAXTM Expression RNAi system ( Promega , USA ) .", "The dsRNA was first injected into the hemolymph of adult locusts at day one post-adult eclosion ( PAE 1 day , 6 μg/locust ) .", "A second injection was performed at PAE 4 days .", "dsGFP was used as the control .", "Flight performance was measured 3 days after the second injection ( PAE 7 days ) .", "The flight activities of individual locusts were measured by using a computer‐aided flight‐mill device modified from previous studies ( Beerwinkle et al . , 1995 ) .", "The arm length of the fight‐mill is 12 cm , and the flight circumference is 0 . 75 m .", "One plastic rod forms the upper , free‐turning body of the flight mill that supports the mill arm and serves as a bearing on the pivot pin .", "The locust was tethered at the end of the lightweight arm , which allowed it to fly along the cycle derived from the arm .", "The interruption of an infrared beam by a rotating arm generated an electrical signal that was recorded by the computer .", "Flight was induced by a fan placed above the flight mill ( blow for 2 s with an interval of 30 s , 1 . 5 m/s wind speed ) .", "During the assay , four flight parameters were obtained for each locust , including total flight distance and duration and average flight velocity and maximum flight velocity .", "The flight mill device was positioned in a room under a photoperiod of 14:10 ( L:D ) h at 30 ± 1°C .", "Locusts that did not flight in this assay were excluded .", "At least 20 locusts were used in each treatment .", "Individuals were randomly allocated into experimental group and control group , and no restricted randomization was applied .", "For sample preparation of flight treatment , the locusts were forced to sustain flight for 15 , 30 , 45 , and 60 min , respectively .", "Insects that stopped flying were artificially stimulated to continue flight .", "Brains of tested locusts were collected , rapidly frozen in liquid nitrogen and stored at −80°C .", "Four independent biological replicates were prepared for each time point .", "Tissues of six to eight individuals were collected for each replicate .", "Individuals who could not finish sustained flight were discarded .", "The establishment of mutant locusts using the CRISPR/Cas9 system was performed as previously described ( Li et al . , 2016 ) .", "The gRNAs containing 20 bases adjacent to a PAM sequence for both ACP and ACPR were designed using the CasOT tool .", "The gRNAs were synthesized using the GeneArt Precision gRNA Synthesis Kit ( ThermoFisher , A29377 ) .", "In brief , a 13 . 8 nl mixture of purified Cas9 protein ( Invitrogen , A36496 , Massachusetts , USA ) and guide RNA of target genes ( final concentrations: 300 and 150 ng/μl , respectively ) was injected into the newly collected embryos ( 2 hr after production ) using a microinjector .", "The injected embryos were then placed in a 30°C incubator until the nymphs hatched .", "The hatched nymphs were reared as described above .", "For genotype analysis , part of the middle foot of each adult locust was collected and lysed with 45 μl NAOH buffer ( 50 mM ) at 95°C for 30 min and then neutralized by adding 5 μl Tris-HCl ( 1 M , pH = 8 . 0 ) .", "The supernatant ( 3 μl ) was used as the PCR template to amplify the targeted DNA fragment .", "Primers for gRNA synthesis were shown in Supplementary file", "4 . The brains of adult locusts were fixed in 4% paraformaldehyde overnight .", "After being washed with 1 × PBS buffer , the samples were blocked with 5% BSA for 1 h and then incubated with affinity-purified polyclonal rabbit antibody against ACP ( produced by ABclone , China , 1: 200 ) at 4°C for 24 h .", "Alexa Fluor-488 goat anti-rabbit IgG ( Cat . A-11008 , 1: 500; Life Technologies ) was used as the secondary antibody .", "Fluorescence was detected using an LSM 710 confocal laser-scanning microscope ( Zeiss ) .", "Negative controls were imaged under the same detection conditions as positive staining .", "Adults of WT and ACP-/- locusts at PAE 1 day were reared under a 14:10 light/dark cycle at 30°C .", "The numbers of dead insects were assessed every day .", "The survival curves were drawn using GraphPad Prism five software .", "Differences in the survival rate of females or males between WT and ACP mutant locusts were compared by using the log-rank ( Mantel-Cox ) test method .", "At least 50 individuals were assayed in each group to determine the survival rate .", "The protein sequences of ACPR from Tribolium castaneum and Aedes aegypti were used as seed sequences to search their homologs in the locust genome and transcriptome database using the tblastn algorithm .", "Three kinds of structure-related neuropeptide receptor proteins , adipokinetic hormone receptor ( AKHR ) , ACPR , and corazonin receptor ( CRZR ) , from locusts and several representative insect species were used to construct their phylogenetic relationship by using MEGA software ( Tamura et al . , 2011 ) .", "Total proteins from flight muscles of WT and ACP-/- locusts were extracted using TRIzol reagent , as previously described ( Hou et al . , 2019 ) .", "The protein extracts ( 50 μg ) were electrophoresed on 4–20% Biofuraw precast gels ( Tanon , China ) and then transferred to polyvinylidene difluoride ( PVDF ) membranes ( Millipore ) .", "The membrane was incubated with polyclonal antibody against target protein ( anti-FABP , developed by ABclone , China , 1:5000; anti-CPT2 , Abcam , 1:1000; anti-ACDM , Abcam , 1:1000 ) .", "Goat anti-rabbit IgG ( EASYBIO , 1:5000 ) was used as the secondary antibody .", "Polyclonal antibody against tubulin was used as an internal control .", "Protein bands were detected by chemiluminescence ( ECL kit , Thermo Scientific ) .", "The flight muscle and fat body tissues were dissected from WT and ACP-/- female locusts under resting state at PAE 7 days .", "Total RNA of these samples with three biological replicates was extracted using TRIzol ( Invitrogen ) and treated with DNase I following the manufacturer’s instructions .", "RNA quality was assessed using an Agilent 2100 Bioanalyzer ( Agilent ) to verify RNA integrity .", "cDNA libraries were prepared according to Illumina’s protocols .", "The adaptor sequences in the raw sequencing data were filtered using Trimmomatic-0 . 30 .", "Clean reads were mapped to the locust genome sequence using Tophat software .", "The number of total reads was normalized by multiple normalization factors .", "Transcript levels were calculated using the reads per kb million mapped ( RPKM ) reads criteria .", "The differences between the test and control groups were based on P values with false discovery rate ( FDR ) correction .", "Differentially expressed genes with FDR < 0 . 1 , Log2 ( FC ) >1 , and RPKM > 0 . 5 in each comparison were enriched .", "The raw sequence data reported in this paper have been deposited in the Genome Sequence Archive ( Genomics , Proteomics & Bioinformatics 2017 ) in National Genomics Data Center , Beijing Institute of Genomics ( China National Center for Bioinformation ) , Chinese Academy of Sciences , under accession number CRA003348 that are publicly accessible at https://bigd . big . ac . cn/gsa .", "The flight muscle was dissected from WT and ACP-/- female locusts under resting state at PAE 7 days .", "The metabolomic profile analysis contained extraction , separation , and detection of metabolites together with metabolomic data processing .", "For metabolite extraction , 20 mg flight muscle was homogenized in 600 μl cold extraction buffer ( Vmethanol/VddH2O , 8/2 ) .", "The 300 μl homogenate was transferred to a new tube containing 900 μl methyl tert-butyl ether ( MTBE ) and 250 μl ddH2O .", "After 10 min of shaking , the mixture was centrifuged at 13 , 000 × g at 4°C for 10 min .", "Lipid extract in the upper layer and polar metabolites in the lower layer were collected for further freeze drying .", "Lipid pellets and polar metabolites were thus resuspended using acetonitrile/isopropanol and acetonitrile/ddH2O for subsequent chromatographic separation , respectively .", "For polar extract separation , untargeted metabolomics analysis was conducted on an Ultimate 3000 ultra-high-performance liquid chromatograph coupled with a Q Exactive quadrupole-Orbitrap high-resolution mass spectrometer UPLC-HRMS system ( Thermo Scientific , USA ) .", "The polar metabolome extracts were profiled on reversed-phase chromatographic separation with positive and negative ionization detection , respectively .", "Metabolites were separated by using an Acquity HSS C18 column ( Waters Co . , USA , 2 . 1 × 100 mm ) and eluted by 0 . 1% formate/water and acetonitrile using linear gradient ramping from 2% organic mobile phase to 98% in 10 min .", "Furthermore , other mobile phases consisting of water and ammonium acetonitrile/methanol both containing ammonium bicarbonate buffer salt were employed to elute metabolites separated on an AcquityTM BEH C18 column ( Waters Co . , USA , 1 . 7 μm , 2 . 1 × 100 mm ) , the gradient was used as follows: 0 min 2% organic phase ramped to 100% in 10 min , and another 5 min was used for column washing and equilibrating .", "The flow rate , injection volume and column temperature were all set at the same conditions , 0 . 4 ml/min , 5 μl and 50°C , respectively .", "The quadrupole-Orbitrap mass spectrometer was operated under identical ionization parameters with a heated electrospray ionization source except ionization voltage including sheath gas 45 arb , aux gas 10 arb , heater temperature 355°C , capillary temperature 320°C and S-Lens RF level 55% .", "The metabolome extracts were profiled with full scan mode under 70 , 000 FWHM resolution with AGC 1E6 and 200 ms max injection time .", "A 70 ~ 1000 m/z scan range was acquired .", "QC samples were repeatedly injected into the acquired Top 10 data-dependent MS2 spectra ( full scan-ddMS2 ) for comprehensive metabolite and lipid structural annotation .", "The 17 , 500 FWHM resolution settings were used for full MS/MS data acquisition .", "Apex trigger , dynamic exclusion and isotope exclusion were turned on , and the precursor isolation window was set at 1 . 0 Da .", "Stepped normalized collision energy was employed for collision-induced disassociation of metabolites using ultrapure nitrogen as the fragmentation gas .", "All the data were acquired in profile format .", "The chromatographic separation of untargeted lipidomics was performed under positive and negative ionization detection modes , respectively , as described above .", "An Accucore C30 core-shell column was utilized for lipid molecule separation at 50°C , which was eluted with 60% acetonitrile in water ( A ) and 10% acetonitrile in isopropanol ( B ) , with both containing 10 mM ammonium formate and 0 . 1% formate .", "The separation gradient was optimized as follows: initial 10% B ramping to 50% in 5 min and further increasing to 100% in 23 min , the other 7 min for column washing and equilibration using 0 . 3 mL/min flowrate .", "The ionized lipid molecules were detected using the same parameters as previously described .", "Lipid extracts ( 300–2000 m/z ) were profiled with the same parameters as the metabolome used .", "Lipids were structurally identified by acquiring data-dependent MS2 spectra , and the key settings included 70 , 000 FWHM full scan resolution , 17 , 500 FWHM MS/MS resolution , loop count 10 , AGC target 3e6 , maximum injection time 200 ms and 80 ms for full scan and MS/MS , respectively , and dynamic exclusion 8 s .", "Stepped normalized collision energy 25% + 40% and 35% were employed for positive and negative mode after optimization .", "The full scan and data-dependent MS2 metabolic profile data were further processed with Compound Discoverer software for comprehensive component extraction .", "The polar metabolites were structurally annotated by searching acquired MS2 against a local proprietary iPhenome SMOL high resolution .", "The MS/MS spectrum library was created using authentic standards , the NIST 17 Tandem MS/MS library ( National Institute of Standards and Technology ) , the local version MoNA ( MassBank of North America ) , and the mzCloud library ( Thermo Scientific , USA ) .", "In addition , the exact m/z of MS1 spectra was searched against a local KEGG and HMDB metabolite chemical database .", "For metabolite identification or structural annotation , the mass accuracy of the precursor within ±5 ppm was a prerequisite; meanwhile , isotopic information including at least 1 isotope within 10 ppm and a fit score of a relative isotopic abundance pattern of 70% were introduced to confirm the chemical formula in addition to the exact mass .", "Furthermore , retention time information as well as high resolution MS/MS spectra similarity was employed to strictly confirm the structural annotation of metabolites .", "The area under curve values were extracted as quantitative information of metabolites with XCalibur Quan Browser information , and all peak area data for the annotated metabolites were exported into Excel software for trimming and organization before statistical analysis ( Microsoft , USA ) .", "On the other hand , untargeted lipidomics data were processed with LipidSearch software , including peak picking and lipid identification .", "The acquired MS2 spectra were searched against in silico predicted spectra of various compounds , including phospholipids , neutral glycerolipids , spingolipids , neutral glycosphingolipids , glycosphingolipids , steroids , and fatty esters .", "The mass accuracies for the precursor and MS/MS product ion searches were 5 ppm and 5 mDa , respectively .", "The MS/MS similarity score threshold was set at", "5 . The potential ionization adducts include hydrogen , sodium , and ammonium for positive ion and hydrogen loss , as well as formate and acetate adducts for negative mode .", "Lipid identification was strictly manually checked and investigated one-by-one to eliminate false positives chiefly based on peak shaking , adduct ion behavior , fragmentation pattern , and chromatographic behavior .", "The metabolome and lipidome data derived from different measurements were normalized to the sample weight used prior to further processing .", "Then , the resultant quantitative information from the foregoing methods was merged , and those detected with multiple methods were excluded to guarantee the uniqueness of metabolites and lipids .", "Log10 was then transformed for final statistical analysis .", "Principal component analysis was conducted with SIMCA-P software ( Umetrics , Sweden ) , and other univariate analyses , including independent sample t-test and p value FDR adjustment , as well as metabolic pathway analysis , were conducted on the MetaboAnalyst website .", "For measurement of acetyl-CoA , NADH , pyruvate , and citric acid , tissues ( 20–40 mg ) of 3–4 individuals were dissected out and homogenized in distinct extraction buffer .", "The extraction solution was centrifuged at 10 , 000 × g at 4°C for 10 min .", "Of these supernatants , 6 μl was aspirated for protein determination , and the rest was deproteinized with a 10 kDa molecular weight cutoff spin filter to remove proteins prior to the reaction according to the manufacturer’s procedures .", "Intracellular acetyl-CoA levels were measured by the acetyl-CoA assay kit ( Sigma , MAK039 ) following the manufacturer’s instructions .", "A 50 μl sample was added to the reaction mixture and incubated for 10 min at 37°C .", "Fluorescence intensity ( λexcitation = 535/λ emission = 587 nm ) was measured for acetyl-CoA detection .", "The NADH content was measured using the NAD/NADH Assay Kit ( Abcam , ab65348 ) .", "Samples were incubated at 60°C for 30 min to remove NAD+ .", "The 20 μl sample was added to the reaction mixture and incubated for 1 h- at room temperature .", "The absorbance at 450 nm was measured for NADH detection .", "Pyruvate was detected by using the Pyruvate Colorimetric/Fluorometric Assay Kit ( BioVison , K609 ) according to the manufacturer’s instructions .", "The 20 μl sample was added into the reaction mix and incubated at room temperature for 30 min .", "The absorbance at 570 nm was measured in a microplate reader .", "Citric acid was detected using the citric acid content detection kit ( Solarbio , BC2150 ) following the manufacturer’s instructions .", "A 50 μl sample was used in the reaction .", "The absorbance at OD545 nm was detected for citric acid measurement using SpectraMax Plus 384 .", "For all metabolite measurements , the background was corrected by subtracting the blank standard value from all readings .", "All data were normalized to the protein concentration , which was measured by using the BCA method .", "At least five biological replicates were performed for each treatment .", "For both WT locusts and ACP-/- mutants , commercially synthetic ACP peptide ( 20 pmol , 2 μL , ABclone ) was injected into the hemolymph of female ACP-/- adults every two days beginning at PAE 1 day .", "To validate the involvement of FABP in the regulation of energy metabolism and flight activity by ACP peptide , metabolites in muscles and flight performance were measured in ACP-/- locusts injected with ACP peptide or combined with injection of ACP peptide and dsFABP .", "For ACP peptide and dsFABP dual treatments , dsFABP ( 6 μg/μL ) was mixed with ACP peptide ( 40 pmol ) and injected into the ACP-/- adults at PAE 3 days .", "Tissue collection and flight performances of tested insects were both conducted at PAE 7 days .", "For measurement of acyl carnitine , muscle tissues ( 20 ± 0 . 5 mg ) were homogenized in 600 μL prechilled methanol-water ( 8/2 , v:v ) solution containing deuterium-labeled internal standards ( 200 ng/mL AcCa ( 16:0 ) -D3 , 20 ng/mL AcCa ( 12:0 ) -D3 , 10 ng/mL AcCa ( 8:0 ) -D3 , 200 ng/mL AcCa ( 2:0 ) -D3 , 50 ng/mL Carnitine-D9 ) .", "The homogenates were vortexed and centrifuged at 13 , 000 × g for 10 min at 4°C .", "250 μL aliquots of supernatant were transferred into another EP tubes and dried in vacuum using the CentriVap Concentration Systems ( Labconco Corporation , USA ) .", "The extracts were dissolved in 150 μL methanol-water ( 5:5 , v:v ) for further chromatography analysis .", "Statistical methods for omic analysis were performed as described above .", "The data that do not meet normal distribution was excluded for the analysis of gene expression , biochemistry assay , and flight activity .", "All data are presented as the mean ± SEM and statistically analyzed using GraphPad Prism five software .", "Two-tailed unpaired student’s t-test was used for two-group comparisons , and one-way ANOVA followed by Tukey’s post hoc test was used for multigroup comparisons .", "Differences were considered statistically significant at p<0 . 05 ." ] ]

[ "Long-term flight depends heavily on intensive energy metabolism in animals; however , the neuroendocrine mechanisms underlying efficient substrate utilization remain elusive .", "Here , we report that the adipokinetic hormone/corazonin-related peptide ( ACP ) can facilitate muscle lipid utilization in a famous long-term migratory flighting species , Locusta migratoria .", "By peptidomic analysis and RNAi screening , we identified brain-derived ACP as a key flight-related neuropeptide .", "ACP gene expression increased notably upon sustained flight .", "CRISPR/Cas9-mediated knockout of ACP gene and ACP receptor gene ( ACPR ) significantly abated prolonged flight of locusts .", "Transcriptomic and metabolomic analyses further revealed that genes and metabolites involved in fatty acid transport and oxidation were notably downregulated in the flight muscle of ACP mutants .", "Finally , we demonstrated that a fatty-acid-binding protein ( FABP ) mediated the effects of ACP in regulating muscle lipid metabolism during long-term flight in locusts .", "Our results elucidated a previously undescribed neuroendocrine mechanism underlying efficient energy utilization associated with long-term flight ." ]

[ "Flight allows insects to find food or seek a better environment .", "Some insects have developed the ability of ‘long-term flight’ , which allows them to make continuous journeys over large distances .", "For example , one locust species regularly crosses the Red Sea which is up to 300 km wide – a spectacular feat for insects only a few inches long .", "However , flight is an energy-intensive activity , and insects’ muscles need the right sort of chemical fuel to work properly .", "Previous work has shown that this ‘fuel consumption’ is highly dynamic and happens in two stages .", "First , immediately after take-off , the muscles rapidly consume carbohydrates ( sugars ) ; then , during the prolonged phase of the flight , muscles switch to exclusively consume lipids ( fats ) .", "How the flight muscles ‘know’ when to start using fats for energy remains largely unclear .", "It has been suggested that this switch may involve hormone-like chemicals made in the brain called neuroendocrine peptides .", "Hou et al . therefore set out to test this hypothesis , using the locust species Locusta migratoria as a representative migratory insect .", "Initial experiments used an abundance detection technique to determine which of the neuroendocrine peptides were active in adult locusts .", "Further analysis , looking specifically at locusts that had just been flying , revealed that the gene for a peptide called ACP became much more active after one hour of continuous flight .", "Further evidence that the ACP hormone could indeed be helping to power long-term flight came from locusts with a mutated , ‘switched-off’ version of the gene .", "These insects could only fly for half the time , and half the distance , compared to locusts that did not have mutations in the gene for ACP .", "Biochemical studies of the ACP mutant locusts confirmed that their flight muscle cells could not transport and break down fatty acids normally .", "These experiments also showed that ACP was acting through a type of carrier protein called FABP , which is present in many different insects and normally ‘ferries’ lipids to the places they are needed .", "These findings shed new light on the biological mechanisms that control long-term flight in migratory insects .", "The ability to move over long distances is key to the outbreak of locust plagues , which in turn cause widespread crop damage around the world .", "Hou et al . therefore hope that this knowledge will one day help develop effective strategies for locust pest control ." ]

[ "Introduction", "Results and discussion", "Materials and methods" ]

[ "evolutionary biology", "microbiology and infectious disease" ]

[ [ "Microsporidia are a highly successful group of strict intracellular eukaryotic parasites that infect a broad range of animal hosts , including humans and economically important species of fish , honeybees , and silkworms ( Stentiford et al . , 2016; Vávra and Lukeš , 2013 ) .", "Microsporidia can only complete their life cycle inside infected eukaryotic cells .", "In the external environment they exist only as resistant thick-walled spores .", "New infections are then initiated by spore germination followed by transit of the parasite through a unique polar tube infection apparatus into a eukaryotic host cell ( Vávra and Lukeš , 2013 ) .", "As a result of this lifestyle , Microsporidia have undergone dramatic genomic and cellular streamlining including the complete loss of biosynthetic pathways for de novo synthesis of purine and pyrimidine nucleotides ( Dean et al . , 2016; Dean et al . , 2018; Heinz et al . , 2014; Heinz et al . , 2012 ) .", "At the organelle level , reduction is dramatically illustrated by their minimal mitochondria ( called mitosomes ) which have lost the organelle genome and the capacity to generate ATP ( Freibert et al . , 2017; Goldberg et al . , 2008; Williams et al . , 2002 ) .", "While glycolysis is conserved in some , but not all ( Wiredu Boakye et al . , 2017 ) Microsporidia , it appears to be mainly active in spores and is not used during intracellular growth and replication ( Dolgikh et al . , 2011; Heinz et al . , 2012; Williams et al . , 2014 ) .", "The loss of indigenous pathways for making ATP and nucleotides means that Microsporidia are now entirely dependent upon the host cells they infect for the energy , cofactors and nucleic acid building blocks that they need to complete their life cycle ( Dean et al . , 2016; Nakjang et al . , 2013 ) .", "Hence parasite transport proteins must play critical roles in servicing the energetic and metabolic demands imposed by parasite growth and replication , but there is currently little functional data for Microsporidia transporters ( Dean et al . , 2016; Dean et al . , 2014 ) .", "Microsporidia have far fewer ( ~2000–3500 ) protein-encoding genes than free-living eukaryotes , providing strong evidence that gene loss , particularly for metabolism , is a pervasive feature of their evolution ( Cuomo et al . , 2012; Nakjang et al . , 2013 ) .", "Genes for candidate surface-located transporters that have been retained against this background of gene loss , are likely to be important for supporting parasite growth and replication ( Heinz et al . , 2012; Nakjang et al . , 2013 ) .", "We previously identified 10 gene families encoding candidate surface transport proteins that are conserved in all published Microsporidia genomes ( Heinz et al . , 2012; Nakjang et al . , 2013 ) .", "The conserved families include the experimentally characterised nucleotide transporters ( NTT ) that are expressed on the cell surface of intracellular Microsporidia where they can import host purine nucleotides , including ATP , GTP and NAD+ ( Dean et al . , 2016; Dean et al . , 2014; Dean et al . , 2018; Tsaousis et al . , 2008 ) .", "Intriguingly , the NTT transporters did not transport pyrimidine nucleotides in these experiments ( Dean et al . , 2018; Heinz et al . , 2014 ) , even though genome analyses suggest that Microsporidia can no longer make these substrates for themselves ( Dean et al . , 2016; Dean et al . , 2018; Heinz et al . , 2014; Tsaousis et al . , 2008 ) .", "These data suggest that additional transporters must exist to supply Microsporidia with the pyrimidines that they need to grow and replicate .", "In the present study , we have characterised a family of Microsporidia Major Facilitator Superfamily ( MFS ) transport proteins which were discovered in Nematocida spp .", "( Cuomo et al . , 2012 ) and subsequently shown to be present in all Microsporidia for which genomes are available ( Cuomo et al . , 2012; Dean et al . , 2018; Heinz et al . , 2014; Heinz et al . , 2012; Nakjang et al . , 2013; Watson et al . , 2015 ) .", "Some of the Nematocida proteins share a Pfam domain ( PF03825 ) with the NupG transporter of Escherichia coli ( Xie et al . , 2004 ) , suggesting ( Cuomo et al . , 2012 ) that , like NupG , they might be purine and pyrimidine nucleoside transporters , potentially solving the parasite pyrimidine deficit discussed above .", "To test this hypothesis and to investigate the evolution and role ( s ) of these MFS transporters in Microsporidia , we characterised the expression , cellular location and functional characteristics of the homologous proteins from Trachipleistophora hominis ( Heinz et al . , 2012; Nakjang et al . , 2013; Watson et al . , 2015 ) , a model species that can be maintained in cell culture and was originally isolated from an HIV/AIDS patient ( Field et al . , 1996 ) .", "Our data provide no evidence that the T . hominis proteins can transport the pyrimidine nucleoside uridine , a known substrate for NupG ( Xie et al . , 2004 ) , or the pyrimidine nucleotides CTP or UTP , but demonstrate that they do transport the purine nucleotides ATP and GTP .", "These data reveal that T . hominis , and potentially other Microsporidia , have at least two distinct transport systems for importing the ATP and GTP from infected host cells , that they need to complete their intracellular lifecycles ." ], [ "We used BlastP to search for sequences related to the Microsporidia conserved protein family # c_456 ( Nakjang et al . , 2013 ) which contains the Nematocida spp .", "putative NupG-like nucleoside/H+ symporter ( Cuomo et al . , 2012 ) , in the genomes of Microsporidia and their endoparasitic relatives among the Rozellomycota ( Corsaro et al . , 2016 ) , including species of Rozella ( James et al . , 2013 ) , Mitosporidium ( Haag et al . , 2014 ) and Amphiamblys , ( Mikhailov et al . , 2017 ) .", "Recent phylogenetic analyses suggest that Rozellomycota is a paraphyletic group containing a ‘core Microsporidia’ clade ( Corsaro et al . , 2016 ) , although others have argued for a different taxonomy with an expanded definition for the Microsporidia ( Bass et al . , 2018 ) .", "The core Microsporidia ( the term we use here ) contains all the species traditionally classified as Microsporidia and which share diagnostic features of the group , including a coiled polar tube , a polaroplast , and the absence of a mitochondrial genome ( Corsaro et al . , 2016; Mikhailov et al . , 2017 ) .", "A total of 63 members of the protein family c_456 were identified among the Rozellomycota and core Microsporidia ( Figure 1—source data 1 ) .", "Phylogenetic analysis including homologues from prokaryotes and eukaryotes recovered the Microsporidia transporters as a separate clade with homologues from Mitosporidum , Amphiamblys and Rozella ( Figure 1 , Figure 1—figure supplements 1 and 2 ) ; providing evidence that the genes were present in the common ancestor of these species .", "Homologues of the Microsporidia proteins were also detected in other eukaryotes including free-living fungi , Oomycetes , Metazoa , Euglenozoa , Alveolata and Stramenopiles .", "The broad distribution of the genes across major lineages of eukaryote including free-living species suggests that this family of transport proteins may be ancestral among eukaryotes .", "In particular , the tree topology provides no compelling support for an origin of the Microsporidia genes through LGT from prokaryotes as previously suggested ( Cuomo et al . , 2012 ) , and there is no evidence from the tree for a specific relationship to the E . coli NupG and XapB ( Nakjang et al . , 2013; Xie et al . , 2004 ) transporters ( Figure 1—figure supplements 1 and 2 ) .", "Detailed phylogenetic analysis identified two clades ( A and B ) of Microsporidia proteins ( Nakjang et al . , 2013 ) originating from a gene duplication in the common ancestor of the core Microsporidia and some Rozellomycota ( Figure 1 , Figure 1—figure supplement 1 ) .", "Clade A contains at least one copy from every Microsporidia genome sampled .", "Clade B contains sequences from most Microsporidia apart from the Encephalitozoon/Nosema/Ordospora and Enterocytozoon/Vittaforma lineages , which appear to have lost the genes for these transporters ( Figure 1 ) .", "The majority of the Microsporidia sequences from clade A contain an indel ( insertion or deletion ) between the 7th and 8th transmembrane domains ( TMD ) compared to other proteins ( Nakjang et al . , 2013 ) ( Figure 1—figure supplement 3 ) .", "The indel is between 16 to 38 residues long ( Figure 1—figure supplement", "3 ) apart from one of the paralogues from Anncaliia algerae which has lost most of the indel ( Figure 1—figure supplement 3 ) .", "The sequences from Rozella , Mitosporidium and Amphiamblys at the base of clade A do not possess the indel ( Figure 1—figure supplement 3 ) .", "Clades A and B both contain evidence for further lineage-specific duplications affecting some Microsporidia ( Figure 1 ) .", "For example , in clade A , T . hominis and some other microsporidian genomes encode only a single gene , whereas others have multiple paralogues ( Figure 1 , Figure 1—source data 1 ) .", "In clade B , T . hominis has three paralogues , whereas other species have only a single gene or appear to have lost their clade B homologue altogether ( Figure 1 ) .", "This type of lineage-specific gene duplication is a feature of the evolution of other Microsporidia transporters ( Nakjang et al . , 2013 ) providing the raw material ( new genes ) for functional divergence among paralogues ( Dean et al . , 2018; Heinz et al . , 2014 ) .", "We used the four T . hominis sequences and related proteins from Microsporidia and Rozellomycota as search queries in HMMer searches against the Pfam domain database ( Figure 1—source data 1 ) .", "The Microsporidia members of clade A , one sequence from Mitosporidium daphniae and four from Rozella allomycis matched the MFS profile PF03825 found in nucleoside/H+ symporters including E . coli NupG .", "This profile did not match any of the other transporter homologues , including the proteins from Amphiamblys sp .", "( Figure 1—source data 1 ) .", "Microsporidia sequences from clade B were characterised by matches to the more general MFS transporter profiles MFS_1 ( PF07690 ) and MFS_2 ( PF13347 ) ( Figure 1—source data 1 ) .", "The alignment for the indel in sequences from clade A did not generate a significant hit ( all e-values ≥ 13 ) in a HHPred profile-profile search of gene families in the protein domain databases ( data not shown ) .", "The Microsporidia MFS transporters are predicted to have 12 alpha helical TMD , with both termini facing the cytoplasmic side ( Figure 1—figure supplement 4 ) .", "This is a typical conformation ( Reddy et al . , 2012 ) as previously reported for the E . coli NupG and XapB nucleoside/H+ symporters ( Nørholm and Dandanell , 2001; Vaziri et al . , 2013; Xie et al . , 2004 ) and is consistent with the Microsporidia proteins being functional MFS transporters .", "In summary , all the Microsporidia proteins are members of the major facilitator family ( MFS ) of transport proteins , but there is no compelling evidence for a particularly close relationship to NupG , or strong indication from trees or bioinformatics concerning their substrates or transport mechanisms .", "In the rest of the manuscript we refer to the four T . hominis transporters as T . hominis ThMFS1-4 with the following locus tags and uniprot accessions ( Heinz et al . , 2012 ) : in clade A , ThMFS1: THOM_0963 - L7J × 55; in clade B , ThMFS2: THOM_1192 - L7J × 19; ThMFS3: THOM_1681 - L7JVD0; ThMFS4: THOM_3170 - L7JT12 .", "We used RNA-Seq and measured transcript abundance ( expressed as transcripts per million , TPM ) at six time points representing different stages in the parasite lifecycle , during a synchronised intracellular infection of rabbit kidney ( RK13 ) cells by T . hominis ( Dean et al . , 2018 ) .", "ThMFS1 mRNA was characterised by a relatively high abundance at the first time point ( 3 hr ) followed by a gradual decrease at 14 , 22 and 40 hr ( Figure 2A ) and then a dramatic increase after 40 hr during spore formation ( 70 hr ) and maturation ( 96 hr ) , when it was the most abundant ThMFS mRNA ( peaking with a mean value of 363 TPM , Figure 2A ) .", "A previous RNA-Seq analysis of a sample taken at a late time point in a T . hominis infection of RK13 cells , also identified ThMFS1 as the most abundant transcript among the four ThMFS genes ( Watson et al . , 2015 ) .", "By contrast , the expression profile of ThMFS3 mRNA peaked during the early stages of infection at the 3 hr post-infection time-point ( 238 TPM ) , and gradually declined to about 50% of this level by the 22 hr time point ( Figure 2A ) .", "ThMFS2 and ThMFS4 were both characterised by significantly lower levels of transcript abundance throughout the infection , with mean levels at all time points below 19 TPM .", "Relative expression profiles for ThMFS1-4 ( Figure 2B ) further emphasise the contrasting levels of transcripts for the different transporters during infection .", "Published RNA-Seq data for other Microsporidia ( Cuomo et al . , 2012; Desjardins et al . , 2015; Grisdale et al . , 2013 ) show similar patterns in the expression of paralogous MFS-like genes ( Figure 2—figure supplement 1 ) .", "For example , the Vavraia culicis orthologue of ThMFS1 ( Figure 1 ) shows its highest expression during spore formation ( Figure 2—figure supplement 1 ) and the V . culicis orthologues for ThMFS2 and ThMFS4 ( Figure 1 ) have the lowest levels of transcription ( Figure 2—figure supplement 1 ) .", "Based upon the branch lengths in the phylogenetic tree ( Figure 1 ) , ThMFS2 and ThMFS4 are evolving much faster than ThMFS3 ( Figure 1 ) and ThMFS2 and ThMFS4 also have the highest number of single-nucleotide polymorphisms ( SNPs ) among the ThMFS homologues ( Figure 2—source data 1 ) .", "Previous work ( Dean et al . , 2018 ) on the T . hominis NTT ( ThNTT ) demonstrated a similar reciprocal relationship between the degree of ThNTT sequence conservation ( expressed as tree branch lengths ) and expression levels , with the fastest evolving ThNTT paralogues showing the lowest expression levels .", "Comparing ThMFS and ThNTT ( Dean et al . , 2018 ) transcript abundance indicated that ThMFS1 and ThMFS3 are characterised by higher levels of expression than ThNTT1-3 throughout the time course of the experiments ( Figure 2—source data 2 ) .", "Notably , ThNTT4 is characterised by the highest level of transcripts among all ThMFS and ThNTT transporters , with mean TPM values of >1000 across six different time points of the synchronised infection ( Figure 2—source data", "2 ) ( Dean et al . , 2018 ) .", "We made anti-peptide ( Figure 2—figure supplement", "3 ) rabbit antibodies for each of the four T . hominis MFS transporters and used them in immunofluorescence assays ( IFA ) of infected cells grown on slides and sampled at different time points ( Figure 2C ) .", "Each slide was co-incubated with rabbit antisera from one of the ThMFS ( red ) and rat antisera to T . hominis mitochondrial Hsp70 ( ThmitHsp70 ) ( green ) , with the latter labelling parasite mitosomes and acting as a control for the fixation and permeabilization protocol ( Freibert et al . , 2017; Goldberg et al . , 2008 ) .", "DAPI ( 4' , 6-diamidino-2-phenylindole ) ( blue ) was also added to stain parasite and host nuclear DNA ( Figure 2C ) .", "During intracellular infection ( Field et al . , 1996 ) the surface plasma membrane of T . hominis is exposed to the host cell cytosol or to the lumen of an intracellular compartment called a sporophorous vesicle ( SPOV ) ( Cali and Tokvarian , 2014 ) .", "In the infective spore stage , the plasma membrane is protected from the external environment by a thick spore coat ( Vávra and Lukeš , 2013 ) .", "The antisera to ThMFS1 and ThMFS3 gave clear and specific labelling of the surface of parasites consistent with a plasma membrane location ( Heinz et al . , 2014 ) in samples taken during the time course of infection ( Figure 2C ) .", "By contrast , no parasite specific IFA signals were detected for the antisera to ThMFS2 and ThMFS4 at any stage of the infection cycle , despite observing consistent labelling of the mitosomes and nuclei of parasites ( Figure 2—figure supplement 4 ) .", "The reasons for the lack of ThMFS2 and ThMFS4 signals are not known but the expression levels of these two genes were much lower than for ThMFS1 and ThMFS3 ( Figure 2A ) , so it is possible that the antibodies we used were insufficiently sensitive to detect expression in our model system or the proteins were not expressed under the experimental conditions used .", "Consistent with these possibilities , the antisera for ThMFS2 and ThMFS4 gave no parasite-specific signal in western blot analyses on total protein extracts from T . hominis infected RK13 cells and T . hominis spores , in contrast to the strong signals for antisera to ThMFS1 and ThMFS3 ( Figure 2—figure supplement 5 ) .", "The antisera for ThMFS2 and ThMFS4 also generated relatively weaker signals against the peptides used as antigens in dot blots suggesting the antibodies may be ineffective ( Figure 2—figure supplement 6 ) .", "The antisera for ThMFS1 and ThMFS3 showed different patterns of parasite labelling during the infection ( Figure 2C ) , suggesting that they have evolved functional differences following gene duplication , as previously shown for the paralogous NTT transporters of T . hominis ( Dean et al . , 2018 ) .", "We detected no labelling of the parasite surface by the antisera to ThMFS1 in replicate slides at 3 hr post-infection , despite transcript levels for the gene being relatively high at this point ( Figure 2A ) and clear labelling of mitosomes and nuclei on the same slides ( Figure 2C ) .", "The ThMFS1 antisera gave strong labelling of the surface of parasites at 14 hr , 22 hr and 40 hr post-infection ( Figure 2C ) .", "At 70 hr and 96 hr post-infection , strong signal was located in the SPOV membrane surrounding groups of T . hominis ( Figure 2C ) sporonts and spores ( Hollister et al . , 1996; Watson et al . , 2015 ) .", "This suggests that ThMFS1 has a role in supporting parasite development within the SPOV .", "The spores inside the SPOV were not labelled with either of the antibodies or by DAPI , suggesting that the developing spore coat may exclude access of these reagents .", "A lack of signal from spores in these types of experiments has previously been observed using antibodies to the ThNTT transporters and to several mitosomal proteins ( Dean et al . , 2018; Freibert et al . , 2017; Goldberg et al . , 2008 ) .", "Proteomics data for purified T . hominis spores ( Heinz et al . , 2012 ) has shown , however , that ThMFS1 is present in spores .", "We detected strong parasite surface labelling by antisera to ThMFS3 from 3 hr up to 40 hr post-infection , but no labelling by antisera to ThMFS3 of either parasites or SPOV membrane in slides for 70 hr and 96 hr ( Figure 2C ) .", "The absence of signal for ThMFS3 on the surface of mature vegetative cells at 70 hr appears to be stage specific because fields of highly infected RK13 cells that contain a mix of mature and early stage parasites ( through reinfection ) , show strongly labelled smaller cells ( Figure 2—figure supplement 7 ) .", "Labelling of small cells characteristic of earlier time points , with antisera for ThMFS1 and ThMFS3 at 96 hr post-infection , are also consistent with the initiation of new infections from germinating newly differentiated spores ( Figure 2—figure supplement 8 ) .", "Consistent with the absence in IFA of a detectable signal for ThMFS3 in the later stages of parasite development , ThMFS3 was not detected in proteomics data for purified T . hominis spores ( Heinz et al . , 2012 ) .", "Upon their discovery , it was suggested ( Cuomo et al . , 2012 ) that the Nematocida homologues of ThMFS transporters might be purine and pyrimidine nucleoside transporters like the E . coli NupG transporter .", "NupG has a broad specificity for nucleosides ( Nørholm and Dandanell , 2001; Patching et al . , 2005; Vaziri et al . , 2013; Xie et al . , 2004 ) including the pyrimidine nucleoside uridine ( Nørholm and Dandanell , 2001; Xie et al . , 2004 ) .", "We used heterologous expression in E . coli strains ( Dean et al . , 2018; Heinz et al . , 2014; Tsaousis et al . , 2008 ) to test if the ThMFS1-4 proteins could transport radiolabelled uridine , using the E . coli NupG transporter as a positive control .", "Because expression of eukaryotic proteins in bacteria can be improved by codon optimisation in the bacterial host ( Gustafsson et al . , 2012 ) we tested expression of native and E . coli codon-optimised ThMFS in different E . coli strains .", "The E . coli strain GD1333 , which lacks the two endogenous nucleoside transporters NupG and NupC ( Nørholm and Dandanell , 2001 ) , was used as the expression host in these assays ( Figure 3A ) .", "In contrast to the positive control expressing recombinant NupG , none of the ThMFS proteins transported [14C]-uridine above background levels for the empty vector control ( ptrc99a ) ( Figure 3A ) .", "These data suggest that uridine is not a substrate for transport by ThMFS1-4 .", "We recently characterised the substrate specificities and evolution of a family of Microsporidia nucleotide transport proteins ( NTT , Pfam profile PF03219 ) that import ATP and GTP for parasite growth and replication inside infected host cells ( Dean et al . , 2018; Heinz et al . , 2014 ) .", "These transporters ( family # c_336 in Nakjang et al . , 2013 ) are also members of the MFS protein superfamily ( clan CL0015; Dean et al . , 2018 ) .", "To investigate if the structural similarity between the ThMFS and NTT transporters is reflected in their transport properties , we tested if the ThMFS proteins could transport radiolabelled purine and pyrimidine nucleotides when expressed in E . coli ( Figure 3B and C ) .", "All four ThMFS transported ATP above background , whereas E . coli expressing NupG did not transport ATP ( Figure 3B ) .", "Further experiments demonstrated that all four ThMFS proteins can also transport radiolabelled-GTP , but not radiolabelled-CTP or UTP ( Figure 3C ) .", "Consistent with the specific uptake of [α32P]-ATP and [α32P]-GTP , uptake of both substrates by E . coli cells expressing ThMFS transporters was time-dependent ( Figure 4 ) .", "Our data demonstrate that ThMFS1-4 can transport purine and thus their substrate specificity overlaps with that of the previously characterised Microsporidia NTT nucleotide transporters ( Dean et al . , 2018; Heinz et al . , 2014 ) .", "The lack of a positive control in these assays makes it difficult to interpret the negative results for transport of the two tested pyrimidines .", "The E . coli NupG transporter is a H+ symporter that uses a proton gradient to drive the transport of uridine and other nucleosides ( Xie et al . , 2004 ) .", "We have previously shown that some Microsporidia NTTs have evolved into symporters ( Dean et al . , 2018 ) capable of mediating the net import of nucleotides .", "To test if nucleotide uptake by ThMFS proteins is also proton dependent , we investigated [α32P]-ATP and [α32P]-GTP uptake by ThMFS1 and ThMFS3 in the presence of the protonophore carbonyl cyanide m-chlorophenyl hydrazone ( CCCP ) .", "ThMFS1 and ThMFS3 are the most abundant transcripts in RNA-Seq ( Figure 2A ) and both are detected on the cell surface of actively growing parasites ( Figure 2C ) .", "The addition of CCCP had no effect on nucleotide uptake by E . coli expressing ThMFS1 or ThMFS3 ( Figure 5 ) , suggesting that they are not H+-symporters .", "By contrast , CCCP did inhibit import of nucleotides by Protochlamydia amoebophila PamNTT5 , a known H+-symporter of GTP ( Haferkamp et al . , 2006 ) ( Figure 5 ) .", "The nucleotide transport activity of some Microsporidia NTT ( e . g . ThNTT4 ) were previously shown to be sensitive to CCCP treatment in the same expression system ( Dean et al . , 2018 ) .", "Previous studies have shown that Microsporidia lack the genes needed to make primary metabolites including nucleotides , and that they have a limited capacity to make their own energy ( Dean et al . , 2016; Williams et al . , 2014 ) .", "This raises the question of how these intracellular parasites obtain the enormous amounts of ATP and other nucleotides that they need to support their rapid growth and replication .", "For example , it is suggested that it takes at least 109 ATPs ( Phillips and Milo , 2009 ) to make a single E . coli , and the ATP requirement to make the larger cells of Microsporidia is likely to be considerably higher .", "Previous work suggests that Microsporidia can use surface-located NTT nucleotide transporters to import purine nucleotides including ATP and GTP during their intracellular growth ( Dean et al . , 2016; Dean et al . , 2018; Tsaousis et al . , 2008 ) .", "In the present study , we show that Microsporidia have a second set of MFS transporters that they can potentially use to supplement their energy and nucleotide budget .", "Thus , all four T . hominis ThMFS transporters were able to transport ATP and GTP when expressed in E . coli ( Figure 3C ) , and the two most highly expressed proteins ( ThMFS1 and ThMFS3 ) are present on the surface of parasites ( along with the NTTs; Dean et al . , 2018 ) when they are most actively growing inside infected rabbit kidney cells ( Figure 2C ) .", "The functional relevance of the transporters ThMFS2 and ThMFS4 is unclear as we were unable to detect any evidence of their protein expression in our model system .", "It was originally suggested that the Nematocida homologues of the T . hominis ThMFS transporters might have similar transport properties to the E . coli MFS NupG ( Cuomo et al . , 2012 ) .", "NupG is an MFS H+ symporter that uses the proton gradient to drive transport of purine and pyrimidine nucleosides ( Xie et al . , 2004 ) .", "However , as we show in the present study , the levels of shared sequence similarity between NupG and Microsporidia sequences are generally low .", "Moreover , characterised members of the MFS superfamily that includes the Microsporidia sequences and NupG , transport a broad spectrum of ions and solutes using a variety of different mechanisms ( Yan , 2015 ) .", "This diversity makes it difficult to make reliable functional inferences based upon sequence similarity ( Finn et al . , 2016; Saier et al . , 2016; Yan , 2015 ) .", "This is exemplified by recently published data ( Dean et al . , 2018 ) for Microsporidia NTT transporters where substrate and mechanism vary among closely related paralogues ( Dean et al . , 2018 ) .", "In this study , we show that ThMFS1-4 do not transport uridine , a known pyrimidine nucleoside substrate of NupG ( Xie et al . , 2004 ) , and that ThMFS1 and ThMFS3 , are not inhibited by CCCP , a classic inhibitor of H+ symporters like NupG ( Xie et al . , 2004 ) .", "Moreover , although T . hominis has a putative uridine kinase ( Heinz et al . , 2012 ) , most Microsporidia genomes appear to lack the kinases needed to utilise uridine and other nucleosides in their metabolism ( Dean et al . , 2016 ) , even if they could import them .", "In recent years , a number of endoparasitic microbial eukaryotes related to Microsporidia have been identified and their genomes sequenced ( Galindo et al . , 2018; Haag et al . , 2014; James et al . , 2013; Mikhailov et al . , 2017; Quandt et al . , 2017 ) .", "Phylogenomic analyses suggest that these taxa form a monophyletic sister group to the true fungi , for which the name Rozellomycota has been suggested ( Corsaro et al . , 2016 ) , and that core Microsporidia form a distinct clade within Rozellomycota ( Galindo et al . , 2018; Mikhailov et al . , 2017; Quandt et al . , 2017 ) .", "Like the Microsporidia , many of the Rozellomycota lack genes for making nucleotides de novo and some have a limited capacity for making their own ATP ( Dean et al . , 2016; Dean et al . , 2018; Galindo et al . , 2018; James et al . , 2013; Mikhailov et al . , 2017; Quandt et al . , 2017 ) .", "This suggests that they all depend to some degree upon host energy and nucleotides for their own intracellular growth and replication .", "Recent work has shown that Rozella and Microsporidia possess horizontally acquired nucleotide ( NTT ) transport proteins that they can use to import host ATP , and in the case of Microsporidia other purine nucleotides and NAD+ ( Dean et al . , 2018 ) .", "The topology of the NTT tree ( Dean et al . , 2018 ) suggests that NTT transporters were acquired by lateral gene transfer into the common ancestor of Rozella and Microsporidia and subsequently vertically inherited ( Dean et al . , 2018; Major et al . , 2017 ) .", "Analysis of the draft genomes of Amphiamblys sp .", "( Mikhailov et al . , 2017 ) , Metchnikovella incurvata ( Galindo et al . , 2018 ) , Mitosporidium daphnia ( Haag et al . , 2014 ) and Paramicrosporidium saccamoebae ( Quandt et al . , 2017 ) , suggest that they lack NTT genes .", "Based upon the topology of published trees ( Dean et al . , 2018; Galindo et al . , 2018; James et al . , 2013; Mikhailov et al . , 2017; Quandt et al . , 2017 ) which place these species as internal branches of the Rozellomycota , it appears that they have lost NTT genes during their independent evolution .", "By contrast , all these species contain multiple homologues of the ThMFS transporters ( Figure 1 , Figure 1—source data", "2 ) suggesting that these transporters provide an alternative and hitherto unrecognised way for endoparasites to exploit the host nucleotide pool ." ], [ "Homologues of the Microsporidia proteins from the MFS family c_456 in Nakjang et al . ( 2013 ) were collected by performing BlastP searches against the NCBI non-redundant protein ( nr ) database .", "Escherichia coli NupG and annotated ( PF03825 ) sequences at the Pfam database ( Finn et al . , 2016 ) , as well as selected sequences from previously published phylogenies ( James et al . , 2013; Nakjang et al . , 2013; Xie et al . , 2004 ) served as initial queries .", "A HMMer search based upon the sampled MFS proteins was performed against the Pfam profiles using the Pfam server ( Finn et al . , 2016 ) .", "For profile-profile searches we took advantage of the HHPred server ( Söding et al . , 2005 ) .", "Alignments for sequence comparisons and phylogenetic analyses were generated within SEAVIEW ( Gouy et al . , 2010 ) using MUSCLE ( Edgar , 2004 ) and trimmed with TrimAl using the setting ‘gappyout’ ( Capella-Gutiérrez et al . , 2009 ) leading to a total of 477 aligned residues for the 63 species Rozellomycota dataset and 464 residues for the broader taxonomic dataset .", "Phylogenies were inferred with Maximum likelihood using the LG+C60 model in IQ-TREE ( Nguyen et al . , 2015 ) with 1000 ultrafast bootstrap replicates ( Hoang et al . , 2018 ) .", "[U-14C] uridine ( 539 mCi/mmol ) was purchased from Perkin Elmer .", "α32P-labelled ATP GTP , CTP , and UTP ( 3000 Ci/mmol or 800 Ci/mmol ) were obtained from Hartmann Analytic or Perkin Elmer .", "Cold nucleotides and nucleosides were obtained from Sigma Aldrich and prepared according to the manufacturer’s instructions .", "The protonophore CCCP was from Sigma Aldrich .", "Microsporidia were grown in rabbit kidney ( RK13 ) host cells at 33°C in complete DMEM as described previously ( Dean et al . , 2018; Goldberg et al . , 2008; Heinz et al . , 2014; Heinz et al . , 2012 ) .", "The RK13 rabbit kidney cell line is positive for the bovine viral diarrhea virus ( BVDV ) , obtained from the ATCC: ATCC CCL-37 and Mycoplasma free .", "The parasite T . hominis was originally obtained from Prof . Liz Canning ( Williams et al . , 2002 ) .", "To initiate the synchronised infection by T . hominis , non-infected RK13 cells grown on 150 cm2 round tissue culture dishes were incubated with freshly purified spores as previously described ( Watson et al . , 2015 ) before processing for RNA-Seq analysis as described ( Dean et al . , 2018; Watson et al . , 2015 ) .", "Briefly , 2 hr after the addition of T . hominis spores to the RK13 cells monolayer , the host cell monolayer was extensively washed with PBS in order to remove spores , followed by the addition of fresh culture medium .", "Two tissue culture dishes were processed independently for RNA purification , sequencing and mRNA quantification ( Watson et al . , 2015 ) at six different time points post-infection: 3 hr , 14 hr , 22 hr , 40 hr , 70 hr and 96 hr ( Dean et al . , 2018 ) .", "Previous studies ( in triplicate ) indicated that the variation between independent replicates for this experimental design was low ( Watson et al . , 2015 ) .", "Library preparation and sequencing were carried out using the Illumina stranded preparation kit and run on two lanes of an Illumina HiSeq 2500 , which produced 27 . 5 M paired-end reads from both the RK13 host cells and T . hominis transcripts .", "CutAdapt ( Martin , 2011 ) was used to remove adapter sequences and low quality 3’ sequence regions ( set at the q −20 threshold ) .", "Two approaches were used to quantify transcripts in the resulting dataset .", "Trimmed reads were mapped to the T . hominis genome using TopHat2 ( Kim et al . , 2013 ) and transcripts assembled and quantified using Cufflinks ( Trapnell et al . , 2012 ) as previously described ( Dean et al . , 2018; Watson et al . , 2015 ) .", "Transcript abundances were presented in FPKM ( Fragments Per Kilobase per Million mapped reads ) ( Figure 2—source data 2 ) ( Dean et al . , 2018; Watson et al . , 2015 ) .", "In a second approach ( presented in Figure 2A , Figure 2—source data 2 ) , predicted T . hominis transcripts were quantified by pseudoalignment of reads using kallisto ( Bray et al . , 2016 ) .", "The results were then analysed using sleuth ( Pimentel et al . , 2017 ) with transcript abundances presented as Transcripts Per Kilobase per million mapped reads ( TPM ) ( Wagner et al . , 2012 ) .", "The RNA-Seq data were submitted to GenBank and listed at the NCBI under the existing BioProject PRJNA278775 with the BioSample accession numbers SAMN11265032-SAMN11265043 ( one accession number for each of the two samples per time point post infection ) .", "Pairs of peptides were selected from hydrophilic segments of the ThMFS1-4 amino acid sequences and used to generate custom-made antisera targeting each of the four ThMFS1-4 candidate transporters ( Figure 2—figure supplement 6 ) .", "Anti-peptides antisera were produced in rabbits by BioGenes GmbH ( Germany ) and commercially affinity purified against the peptide antigens .", "Two rabbits for each pair of peptides were processed in parallel .", "To test the antisera specificity for their respective peptides , we used western blots with 300 ng of peptide directly spotted onto nitrocellulose membranes , affinity purified antibodies were diluted 1:1000 and secondary goat anti-rabbit antisera conjugated to HRP were diluted 1:10000 ( Figure 2—figure supplement 6 ) .", "Western blot detection was processed with a chemiluminescent substrate ( Thermo Scientific - Pierce ECL ) following the manufacturer’s instructions .", "Image development was processed with a Biorad gel imager ChemiDoc XRS+ with images taken every 10 s .", "The same T . hominis infected RK13 cells used for the synchronised infection experiments ( see RNA-Seq section ) were also grown on 13 mm glass coverslips and then fixed in methanol/acetone ( 50:50 at −20°C for at least 10 min ) ( Dean et al . , 2018; Goldberg et al . , 2008; Heinz et al . , 2014; Tsaousis et al . , 2008 ) at the different time points post-infection .", "Rat antisera against ThmtHSP70 ( locus tag THOM_3057 ) were used to label T . hominis mitosomes and both host and parasite nuclei were stained with DAPI , as previously described ( Dean et al . , 2018; Freibert et al . , 2017; Goldberg et al . , 2008; Heinz et al . , 2014; Tsaousis et al . , 2008 ) .", "Dilutions of antisera were as follows: anti-ThmtHSP70 1:200; anti-ThMFS1-4 1:50 .", "For anti-ThMFS2 and anti-ThMFS4 1:10 and 1:2 dilutions were also tested but no IFA signal was observed in any of the tested conditions .", "For ThMFS1 and ThMFS3 both rabbit antisera gave parasite-specific IFA signals .", "Blocking and antibody incubations were performed in 5% milk prepared from skimmed milk powder in PBS , with PBS used for washing steps .", "Cells were stained with DAPI ( Molecular Probes ) and mounted in Vectashield Hard set ( VectorLabs ) .", "Peptide competition assays were performed with the addition of solubilized pairs of peptides in a 200-fold molar excess compared to the affinity-purified antibody .", "The IgG molar mass was considered as 150 , 000 Daltons , and the molar masses of the blocking peptides were taken into consideration for the calculations .", "For ThMFS1 , the final antibody concentration was 5 . 2 μg/ml and the concentrations of peptide 1 and peptide 2 were 13 . 6 μg/ml and 11 . 2 μg/ml , respectively .", "For ThMFS3 , the final antibody concentration was 3 . 3 μg/ml and the concentrations of peptide 1 and peptide 2 were 8 . 3 μg/ml and 8 . 5 μg/ml , respectively .", "As expected , the control mitosomal ThmitHsp70 signal was not affected by any of these conditions ( Figure 2—figure supplement 9 ) .", "Images were taken with a Zeiss Axioimager II epifluorescence microscope using a 63x objective lens and images processed with ImageJ ( Schneider et al . , 2012 ) .", "Quantification of the ThMFS antibody fluorescence signal was determined for fields including intracellular stages of the parasite .", "Axiovision software and Image J[Fiji] with the Bioimport plugin was used to determine relative fluorescence for each specific antibody including the mitosome-specific antibody to mitHsp70 .", "Background signal was determined in a similar manner and subtracted from the specific signal .", "For total protein extracts , confluent monolayers of either T . hominis-infected or non-infected RK13 cells were washed three times with PBS , and lysed with ice cold 2% SDS-PBS lysis buffer containing protease inhibitor cocktail ( Sigma P8340 ) , PMSF ( Sigma ) , MgCl2 ( 0 . 6 mM final concentration ) , and Benzonase ( 25U , Novagen ) .", "T . hominis spores were purified from the culture medium from T . hominis infected RK13 cultures using Percoll ( Sigma ) density gradient centrifugation .", "Purified spores were lysed by boiling for 10 min in 2% SDS-PBS lysis buffer with protease inhibitors .", "The protein concentration of the samples for Western blotting was determined with a BCA assay ( Pierce BCA Protein Assay Kit ) .", "The samples were boiled for 5 min in Laemmli loading buffer and the indicated amount of total protein extracts were loaded per lane for SDS–PAGE .", "To estimate the size of the detected proteins and to monitor protein migration and transfer we used the PageRuler prestained protein ladder , 10 to 180 kDa ( ThermoFisher Scientific ) .", "Western blot analysis used the indicated affinity purified rabbit antibodies ( diluted 1:1000 ) in combination with HRP-conjugated secondary goat anti-rabbit antisera ( diluted 1:10000 ) ( Sigma ) .", "Image development was processed using a Biorad gel imager ChemiDoc XRS+ .", "The four native ThMFS genes were PCR amplified from purified genomic DNA and cloned into the pET-16b expression vector .", "Sequences of cloned genes were verified and submitted to GenBank when distinct from the previously deposited genome sequence-derived ORF ( Heinz et al . , 2012 ) .", "These have the following accession numbers: ThMFS2_native: MH824667 , ThMFS3_native: MH824668 .", "Codon-optimized genes for expression in E . coli that contained a C-terminal 2x HA-tag were produced synthetically ( GeneArt , Thermo Fisher Scientific ) and then cloned into the ptrc99a plasmid .", "These sequences have the following GenBank accession numbers: ThMFS1_synthetic: MH824663; ThMFS2_synthetic: MH824664; ThMFS3_synthetic: MH824665; ThMFS4_synthetic: MH824666 .", "Proteins were expressed as described previously for Microsporidian NTTs ( Dean et al . , 2018; Heinz et al . , 2014; Tsaousis et al . , 2008 ) .", "T7-based constructs were used for expression in E . coli Rosetta2 ( DE3 ) pLysS and trc-based constructs were expressed in E . coli GD1333 ( kindly provided by Prof . Gert Dandanell ) ( Nørholm and Dandanell , 2001 ) .", "Briefly , a starting culture from a single colony was grown in LB at 37°C overnight and used to inoculate a 50 ml culture grown at 37°C until an OD600 of 0 . 4–0 . 6 .", "Protein expression was induced by addition of 1 mM IPTG and the culture was incubated for and additional 16 hr at 18°C .", "Cells were harvested at 5000 x g for 10 min and were washed twice with PBS to remove residual medium .", "The cells were re-suspended in PBS with the OD600 adjusted to 5/ml .", "Cultures were kept in PBS at room temperature until used for uptake assays .", "Transport of nucleosides and nucleotides was assayed using 0 . 5 µM or 2 µM of radiolabelled substrate , as described in the main text/Figures .", "α32P-radioisotopes were present at 1–2 µCi/ml and used as described ( Audia and Winkler , 2006 ) , and [U-14C] isotopes were used at 2 . 5–5 mCi/mmol as described ( Nørholm and Dandanell , 2001 ) .", "To test if a proton gradient affected transport by E . coli cell expressing the ThMFS1 and ThMFS3 transporters , we added 250 µM of CCCP to dissipate the H+ gradient across the bacterial membranes prior to transport assays ( Dean et al . , 2018; Haferkamp et al . , 2006 ) ." ] ]

[ "Plasma membrane-located transport proteins are key adaptations for obligate intracellular Microsporidia parasites , because they can use them to steal host metabolites the parasites need to grow and replicate .", "However , despite their importance , the functions and substrate specificities of most Microsporidia transporters are unknown .", "Here , we provide functional data for a family of transporters conserved in all microsporidian genomes and also in the genomes of related endoparasites .", "The universal retention among otherwise highly reduced genomes indicates an important role for these transporters for intracellular parasites .", "Using Trachipleistophora hominis , a Microsporidia isolated from an HIV/AIDS patient , as our experimental model , we show that the proteins are ATP and GTP transporters located on the surface of parasites during their intracellular growth and replication .", "Our work identifies a new route for the acquisition of essential energy and nucleotides for a major group of intracellular parasites that infect most animal species including humans ." ]

[ "Microsporidia are a group of microscopic parasites that spend part of their lives inside the cells of a broad range of animal hosts , including humans .", "These parasites are considered to be related to fungi , some of which also live within the cells of other species and are known as fungal endoparasites .", "One of the shared characteristics of these parasites is that they cannot make nucleotides , molecules that are both the main source of energy of the cell and also the building blocks of DNA .", "Instead , they take nucleotides , or the materials needed to make nucleotides , from their host cells .", "Once Microsporidia have depleted a host cell , they turn into spores that can survive outside the host until they invade a new cell , starting the cycle anew .", "Microsporidia have proteins on their surface , including nucleotide transporter family proteins ( NTT ) , that enable them to import nucleotides from their host into themselves .", "Although most fungal endoparasites are also thought to steal nucleotides from their hosts , many do not have NTT proteins , raising the question of how they import the nucleotides .", "A group of proteins called the Major Facilitator Superfamily ( MFS ) consists of proteins that were thought to transport the materials cells need to make nucleotides ( which are also called nucleotide precursors ) .", "Members of this family are found throughout Microsporidia and related fungal endoparasites .", "These proteins could explain how fungal endoparasites take nucleotides from their hosts .", "To test this hypothesis , Major et al . infected mammalian cells with Microsporidia and then checked where two MFS proteins were located during infection .", "This showed that the proteins were on the surface of the endoparasites , implying that they could be nucleotide precursor transporters .", "Next , Major et al . genetically modified Escherichia coli bacteria so they would produce MFS proteins , and showed that the proteins could transport two types of nucleotides .", "Together these results show that MFS proteins could be responsible for nucleotide transport in fungal endoparasites .", "In addition to humans , Microsporidia and related fungal endoparasites infect a wide range of animals , including pollinating insects , which have ecological and economic importance .", "Given that Microsporidia can only survive if they take nucleotides from their hosts , knowing more about the proteins that import the nucleotides could lead to new cures for Microsporidia infections ." ]

[ "Introduction", "Results", "Discussion", "Material and methods" ]

[ "plant biology", "developmental biology" ]

[ [ "Developmental systems strikingly produce regular patterns and analysis of eukaryote development has classically been focused on regularities as the main source of information to understand these complex systems .", "However , it is becoming increasingly evident that intrinsic molecular noise is an inherent property of biological systems ( Elowitz et al . , 2002; Kupiec , 1997; Lander , 2011 ) .", "This noise can be buffered , e . g . ( Okabe-Oho et al . , 2009 ) , but can also theoretically propagate through scales and generate patterning disorders e . g . ( Itoh et al . , 2000 ) .", "In this case , disorders observed during development could be informative not only on the origin of noise but also on the underlying developmental mechanisms that propagate the noise .", "Here we address this question theoretically using phyllotaxis , the remarkably regular geometric organization of plant aerial organs ( such as leaves and flowers ) along the stem , as a model system ( Appendix section 1 ) .", "Phyllotaxis primarily arises at the shoot apical meristem , a specialized tissue containing a stem cell niche and located at the tip of growing shoots .", "Rooted in early works of pioneers such as ( Bonnet , 1754; Braun , 1831; Bravais and Bravais , 1837 ) and after decades of research , the idea that phyllotactic patterns emerge from simple physical or bio-chemical lateral inhibitions between successive organs produced at the meristem has become largely prevalent , ( Adler et al . , 1997; Jean , 1995; Kuhlemeier , 2007; Pennybacker et al . , 2015; Reinhardt , 2005 ) .", "Microscopic observations and modeling led to propose that this self-organizing process relies on five basic principles:", "i ) organs can form only close to the tip of growing shoots ,", "ii ) no organ can form at the very tip ,", "iii ) pre-existing organs prevent the formation of new organs in their vicinity ( Hofmeister , 1868 ) , forming altogether an inhibitory field that covers the organogenetic zone ,", "iv ) due to growth , organs are progressively moved away from the organogenetic zone ,", "v ) a new organ is formed as soon as the influence of the inhibitory field produced by the existing organs fades away at the growing tip , ( Schoute , 1913; Snow and Snow , 1962; Turing , 1952 ) .", "Computer simulations were used to analyze the dynamical properties of an inhibitory field model relying on these assumptions ( Mitchison , 1977; Thornley , 1975; Veen and Lindenmayer , 1977; Young , 1978 ) and many others after them , including ( Chapman and Perry , 1987; Douady and Couder , 1996a; Green et al . , 1996; Meinhardt , 2003; Schwabe and Clewer , 1984; Smith et al . , 2006b ) .", "In a detailed computational analysis ( Douady and Couder , 1996a; 1996b; 1996c ) , Douady and Couder demonstrated the ability of such models to recapitulate a wide variety of phyllotactic patterns in a parsimonious way and that these patterns are under the control of a simple geometric parameter corresponding to the ratio between the radius of organ inhibitory fields and the radius of the central zone ( area at the very tip where no organ can form ) .", "This modeling framework thus provides a deterministic theory of self-organizing patterns in the meristem characterized by a global geometric parameter , capturing macroscopic symmetries and orders emerging from lateral inhibitions , e . g . ( Adler , 1975; Atela , 2011; Newell et al . , 2008; Smith et al . , 2006b ) .", "In the sequel , we will refer to this widely accepted view as the classical model of phyllotaxis .", "In recent years , plausible molecular interpretations of the abstract concepts underlying the classical model have been proposed .", "They mainly rely on distribution in the meristem of the plant hormone auxin , a central morphogenetic regulator .", "Auxin is actively transported at the meristem surface , notably by both PIN-FORMED1 ( PIN1 ) polar efflux carriers and non-polar influx carriers ( AUX/LAX family ) .", "These transporters form a dynamic network that permanently reconfigures and that periodically accumulates auxin at specific locations on the meristem flanks ( the organogenetic domain ) , initiating organ primordia ( Reinhardt et al . , 2003 ) .", "By attracting auxin , the growing primordium depletes auxin in its vicinity , thus preventing organ formation in this region .", "This mechanism is now thought to be at the origin of the predicted inhibitory fields in the meristem ( Barbier de Reuille et al . , 2006; Brunoud et al . , 2012; Jönsson et al . , 2006; Smith et al . , 2006a; Stoma et al . , 2008 ) .", "The range of this inhibition corresponds to one of the two key parameters of the classical model: as primordia get away from the tip , inhibition is relaxed and auxin can accumulate again to initiate new primordia .", "For the second key parameter , i . e . the size of the apical domain in which no organ can form , it has been suggested that the very tip of the meristem contains significant quantities of auxin but is actually insensitive to auxin due to a down-regulation of the effectors of transcriptional auxin signaling ( Barbier de Reuille et al . , 2006; Vernoux et al . , 2011 ) .", "A low auxin sensitivity then participates in blocking organ initiation in the central domain ( where the stem cells are located ) of the meristem .", "These molecular insights support the hypothesized structure of the classical model .", "Comparatively , little attention has been paid as of today to disorders in phyllotaxis ( Jean , 2009; Jeune and Barabé , 2004 ) .", "However , in the recent years , the presence of irregularities in phyllotactic patterns has been repetitively observed in various genetic backgrounds ( Besnard et al . , 2014; Couder , 1998; Douady and Couder , 1996b; Guédon et al . , 2013; Itoh et al . , 2000; Landrein et al . , 2015; Leyser and Furner , 1992; Mirabet et al . , 2012; Peaucelle et al . , 2011; Prasad et al . , 2011; Refahi et al . , 2011 ) , suggesting that phyllotaxis has a non-deterministic component .", "In some cases , the departure from any known regular pattern is so strong that plant phyllotaxis is considered random , e . g . ( Itoh et al . , 2000 ) .", "Recently , strong disorders have been observed and quantified in spiral patterns of Arabidopsis thaliana wild-type and arabidopsis histidine phosphotransfer protein ( ahp6 ) mutants ( Besnard et al . , 2014 ) , Figure 1 .", "Surprisingly , a structure could be found in these disorders that corresponds to either isolated or series of permutations in the order of lateral organs along the stem when taking a perfect spiral with divergence angle 137 . 5° ( golden angle ϕ ) as a reference .", "Live-imaging of meristems showed that organs are sometimes co-initiated in the meristem , leading randomly to post-meristematic organ order permutations in around half of the cases .", "This phenomenon showed that , while the azimuthal directions of the organs are highly robust , the time between consecutive organ initiation ( or plastochron ) is variable , Figure 1G .", "Taken together , these observations called for revisiting in depth models of phyllotaxis to account for disorders in this self-organizing developmental system . 10 . 7554/eLife . 14093 . 003Figure 1 . Irregularity in phyllotaxis patterns .", "( A ) wild type inflorescence of Arabidopsis thaliana showing regular spiral phyllotaxis .", "( B ) aph6 mutant inflorescence showing an irregular phyllotaxis: both the azimuthal angles and the distances between consecutive organs are largely affected .", "( C1 )", "Organ initiation in the wild type: the size of organs is well hierarchized , initiations spaced by regular time intervals .", "( C2 )", "Organ initiation in the ahp6 mutant: several organs may have similar sizes , suggesting that they were initiated simultaneously in the meristem ( co-initiations ) .", "( D ) A typical sequence of divergence angles in the WT: the angle is mainly close to ( ≈137° ) with possible exceptions ( M-Shaped pattern ) .", "( E ) In ahp6 , a typical sequence embeds more perturbations involving typically permutations of 2 or 3 organs .", "( F–I )", "Frequency histogram of divergence angle: wild type ( F ) ; ahp6 mutant ( G ) ; WS-4 , long days ( H ) ; WS-4 short days - long days ( I ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 14093 . 003 Here , we show that the same disorders , the permutations , occur in various plant species , suggesting noisy plastochrons are a characteristic of phyllotactic systems at the origin of pattern disorders .", "In addition , we demonstrate that inhibitory fields pre-specify a number of organogenesis sites , suggesting noise on inhibition perception as the most likely origin of disorders .", "Building on this observation , we developed a stochastic model of organ initiation that is fully local and relies on a stochastic modeling of cell responses to inhibitory fields .", "Our stochastic model fully and precisely captures the observed dynamics of organogenesis at the meristem , recapitulating both regular and irregular phyllotactic patterns .", "We show that the stochastic model also makes quantitative predictions on the nature of the perturbations that may arise due to different genetic and growth manipulations .", "Most importantly , we demonstrate that disorders in phyllotactic patterns instruct us on the parameters governing the dynamics of phyllotaxis .", "Disorders can thus provide access to the biological watermarks corresponding to the parameter values of this self-organizing system , providing a striking example where disorders inform on mechanisms driving the dynamics of developmental systems ." ], [ "As permutations have been notably reported in Arabidopsis ( Besnard et al . , 2014; Guédon et al . , 2013; Landrein et al . , 2015; Refahi et al . , 2011 ) and in sunflower ( Couder , 1998 ) , we sampled a variety of unrelated species in the wild and searched for permutations .", "We could easily find permutations in several other Brassicaceae showing spiral phyllotaxis as well as in either monocotyledonous or dicotyledonous species from more distant families such as Asparagaceae , Sapindaceae or Araliaceae ( Figure 2 ) ( Appendix section 1 ) .", "As suggested by the results on Arabidopsis , these observations raise the possibility that these organ permutations result from a noise on the plastochron and that such perturbation could be a common feature of phyllotactic systems that occurs in meristems with different geometries .", "In addition this disorder is probably under complex genetic control as different unrelated genetic modifications ( including the Arabidopsis ahp6 mutant ) can modulate its intensity . 10 . 7554/eLife . 14093 . 004Figure 2 . Permutations can be observed in various species with spiral phyllotaxis . A schema in the bottom right corner of each image indicates the rank and azimuthal directions of the lateral branches .", "The first number ( in yellow , also displayed on the picture ) indicates the approximate azimuthal angle as a multiple of the plant’s divergence angle ( most of the times close to 137° or 99° ) .", "The second number ( in red ) corresponds to the rank of the branch on the main stem .", "( A ) Brassica napus ( Inflorescence ) ( B ) Muscari comosum ( Inflorescence ) ( C ) Alliara petiolata ( D ) Aesculus hippocastanum ( Inflorescence ) ( E ) Hedera Helix ( F ) Cotinus Dummeri . DOI: http://dx . doi . org/10 . 7554/eLife . 14093 . 004 To understand how the timing of organ initiation could be affected during meristem growth , we first analyzed the relative stability of inhibitory field minima in the classical deterministic model .", "For this , we implemented a computational version of the classical model based on ( Douady and Couder , 1996b ) ( Appendix section 2 ) .", "Primordia are created on the meristem surface at the periphery of the meristem central zone , at a fixed distance R from the meristem center .", "Once created , primordia drift away radially from the central zone at a speed proportional to their distance from the meristem center .", "As soon as created , a primordium q of radius r0 , generates an inhibitory field , E ( q ) in its neighborhood such that at a point x , at a distance d ( q , x ) >r0 , the inhibition due to q decreases with the distance to q:E ( q ) ( x ) = ( r0d ( q , x ) ) s , where s is a geometric stiffness parameter ( E ( q ) is often regarded as an inhibitory energy emitted by primordium q , e . g . Douady and Couder , 1996b ) .", "As a result , at any moment and for any point x of the meristem surface , the existing primordia create altogether a cumulated inhibition E ( x ) that is the sum of the individual primordium contributions: E ( x ) =∑q∈QE ( q ) ( x ) , where Q denotes the set of all preexisting primordia .", "High inhibition levels on the peripheral circle prevent the initiation of new primordia at corresponding locations .", "However , as the preexisting primordia are moving away from the central zone during growth , the inhibitory level tends to decrease at each point of the peripheral circle .", "A new primordium initiates where and when the inhibitory field is under a predefined threshold on the peripheral circle .", "We then performed a systematic analysis of the inhibition profiles and their dynamics along the peripheral circle and observed the following properties ( Figure 3 , Video 1 , 2 ) : In addition , we noticed that depending on time , the difference in inhibition level between consecutive local minima may markedly vary .", "In some cases this difference is so small that a biological noise may lead the biological system to perceive the ordering between two or more local minima differently from the ordering of the actual inhibition levels .", "Such errors would lead to initiate several primordia together or to change the temporal order of their initiation , thus inducing perturbations in the sequence of divergence angle .", "Also , as suggested by property 3 , the number of primordia initiation events affected by these errors would decrease with the Γ parameter and would thus depend on the geometry of the meristem . 10 . 7554/eLife . 14093 . 005Figure 3 . Properties of the inhibition profiles in the classical model and effect of a forced perturbation on divergence angles and plastochrons .", "( A ) Inhibition variation ( logarithmic scale ) along the peripheral circle and its global and local minima for a control parameter Γ1 = 0 . 975 .", "Ek−E1 is the difference in inhibition levels between the kth local minimum and the global minimum .", "The angular distances between the global minimum and the kth primordiu inhibition m are multiple of the canonical angle ( α=137° ) .", "( B ) Similar inhibition profile for a control parameter Γ2 = 0 . 675 < Γ1 .", "The difference Ek−E1 in inhibition levels is higher than in A . ( C ) Variation of the distance E2−E1 between the global minimum of the inhibition landscape and the local minimum with closest inhibition level ( i . e . the second local minimum ) , as a function of Γ .", "( D ) Inhibition profile just before an initiation at azimuth 80° and ( E ) just after .", "( F ) Variation of inhibition profile in time .", "As the inhibition levels of local minima decrease , their angular position does not change significantly , even if new primordia are created ( peak qn ) , color code: dark red for low inhibition and dark blue for high inhibition values .", "( G ) Sequence divergence angles between initiations simulated with the classical model ( control parameter Γ1 ) .", "At some point in time ( red arrow ) , the choice of the next initiation is forced to occur at the 2th local minimum instead of the global minimum .", "After the forcing , the divergence angle makes a typical M-shaped pattern and returns immediately to the α baseline .", "( H ) Corresponding plastochrons: the forcing ( red arrow ) induces a longer perturbation of the time laps between consecutive organs . DOI: http://dx . doi . org/10 . 7554/eLife . 14093 . 00510 . 7554/eLife . 14093 . 006Figure 3—figure supplement 1 . Divergence angle of a series of simulations of the classical model with control parameter Γ1=0 . 975 and for which the choice of the jth local minimum ( instead of the global minimum , i . e . j=1 ) has been forced at a given time-point ( red arrow ) .", "After the forcing , the reaction of the classical model is observed .", "1 . divergence angles ( left column )", "2 . corresponding plastochrons ( right column ) ( A , B ) j=2 .", "( C , D ) j=3 .", "( E , F ) j=3 and j=2 are imposed in this order . DOI: http://dx . doi . org/10 . 7554/eLife . 14093 . 00610 . 7554/eLife . 14093 . 007Video 1 . Temporal variation of the inhibitory profile around the central zone in the classical model for a large value of the parameter Γ .", "The number of inhibition mimima is stable ( 3 ) in time .", "When the absolute minimum reaches the initiation threshold ( here E = 0 ) , a primordium is created that instantaneously creates a strong inhibition locally , which suddenly increases the inhibition level at its location .", "Between initiations , local minima regularly decrease in intensity due to the fact that growth is moving existing primordia away from the center .", "This movement is accompanied by a slight drift in position common to all primordia ( here to the right ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 14093 . 00710 . 7554/eLife . 14093 . 008Video 2 . Temporal variation of the inhibitory profile around the central zone in the classical model for a small value of the parameter Γ .", "The dynamics is similar to that of small Γ except that the number of local minima of inhibition is higher ( here 5 ) and that the distance between two consecutive minima is lower . DOI: http://dx . doi . org/10 . 7554/eLife . 14093 . 008 To investigate this possibility , we started to induce a perturbation in a stationary spiral pattern by forcing at a given time the system to initiate a primordium at the site of second local minimum instead of that of the global minimum ( i . e . at an angle 2ϕ , Figure 3G , red arrow ) .", "The system was then left free to self-organize .", "We observed that the next primordium was always initiated at the site of the original global minimum , resulting in a divergence angle −ϕ and a quasi-null plastochron ( Figure 3H ) .", "The system was then able to recover from the perturbation by initiating the next primordium at the originally expected site ( i . e . with a divergence angle 2ϕ ) , with a long plastochron , leading to a M-shaped pattern ( Figure 3G , Video 3 ) .", "The rest of the divergence angle sequence was then not affected and remained at a value close to ϕ while long oscillatory perturbations were observed on plastochrons ( Figure 3H ) .", "Stronger perturbations induced by forcing various other local minima to initiate instead of the global minimum ( Figure 3—figure supplement 1 and Appendix section 6 . 6 for related control simulation experiments ) similarly demonstrated that the system spontaneously makes a short distorted pattern and then returns to the normal ϕ baseline in every case .", "We concluded that", "i ) a noise in the perception of the local primordium order does not propagate far in the divergence angle sequence and that angle specification in the classical model patterning system is highly robust to perturbations in local minima initiation ordering and", "ii ) however , the plastochron itself is affected during a much longer time span . 10 . 7554/eLife . 14093 . 009Video 3 . Temporal variation of the inhibitory profile around the central zone in the stochastic model for a small value of the parameter Γ .", "Here , due to stochasticity , global minimum is not always the one that triggers an initiation .", "The dynamics of the divergence angle and of the plastochron are shown in the bottom graphs to interpret the model's initiations based on the inhibition levels . DOI: http://dx . doi . org/10 . 7554/eLife . 14093 . 009 Together , these results suggest that time and space in primordium initiation are largely decoupled in inhibitory field-driven self-organization as locations of primordia are strongly pre-specified and relatively stable , while plastochrons are not .", "This observation is in line with the observations from live-imaging of Arabidopsis shoot apical meristem that demonstrated that despite variability in the plastochron ( with almost 30% of organs co-initiated ) the specification of initiation sites was extremely robust ( Besnard et al . , 2014 ) .", "Our previous results suggest that high variability in the timing of organ initiation could result from the joint effect of noise in the perception of inhibitory fields and of the decoupling between space and time in this self-organizing system .", "We therefore decided to revisit the inhibitory field models to integrate locality and stochasticity as central components in the patterning system .", "For this , we kept from the classical deterministic model the assumptions related to the movement of primordia at the meristem through growth and to the definition of inhibitory fields .", "However , we completely reformulated the way primordia are initiated as local stochastic processes .", "At any time t , the K cells that make up the periphery of the central zone potentially may take the identity of a primordium , depending on the local value of the inhibitory field ( signaling ) in each cell .", "We assume that this switch in cell identity does not depend on a threshold effect as in the classical model but , rather , depends on the cellular perception of the inhibitory signal which , in essence , is stochastic ( Eldar and Elowitz , 2010 ) .", "We thus assumed that each cell k reads out the local inhibitory field value , Ek ( t ) , and switches its state to primordium identity with a probability that", "( i ) depends on the level of inhibition Ek ( t ) ;", "( ii ) is proportional to the amount of time δt the cell is exposed to this level of signaling ( Gillespie , 1976; 1977 ) : ( 1 ) P ( Xk ( t , δt ) =1 ) =λ ( Ek ( t ) ) δt , where Xk ( t , δt ) denotes the number of primordia initiated at cell k in the time interval [t , t+δt] ( Xk ( t , δt ) will typically have a value 0 or 1 ) , and λ is a rate parameter that depends on the local inhibition value Ek ( t ) at cell k and that can be interpreted as a temporal density of initiation .", "To express the influence of inhibitory fields on the probability of initiation , the dependence of λ on the local inhibition Ek ( t ) must respect a number of general constraints:", "i ) λ must be a decreasing function of the inhibition as the higher the inhibition level at one site , the lower the probability to observe an initiation at this site during δt;", "ii ) for small δt , for P ( Xk ( t , δt ) =1 ) to be a probability , we must have 0≤λ ( Ek ( t ) ) δt≤1;", "iii ) the ratio of the probabilities to trigger an initiation at two different sites is a function of the difference in inhibition levels between these sites .", "Under all these constraints , the rate parameter takes the following form ( see Appendix section 3 for a detailed derivation of the model ) : ( 2 ) λ ( Ek ( t ) ) =e−β ( Ek ( t ) −E∗ ) , where E∗ is a parameter controlling the sensitivity of the system to inhibition , and β is a parameter controlling the ability of the system to discriminate between inhibition levels , i . e . to respond differently to close inhibition levels ( acuity ) .", "Therefore , for each cell k , the probability to initiate a primordium during a small time interval δt can be expressed as: ( 3 ) P ( Xk ( t , δt ) =1 ) =e−β ( Ek ( t ) −E∗ ) δt .", "If we now assume that the probabilities to observe an initiation at a site k in disjoint time intervals are independent , the process described by Equation 3 is known as a non-homogeneous Poisson process of intensity λ ( Ek ( t ) ) ( e . g . Ross , 2014 ) .", "Therefore for each cell k of the periphery , our model assumes that the probability to initiate a primordium is a non-homogeneous Poisson process , whose parameter is regulated by the local level of the inhibitory field at that site .", "This stochastic formulation of the model at the level of cells , called SMPmicro ( Stochastic Model of Phyllotaxis at microscopic level ) , makes it possible to develop the calculus of different key quantities or properties of the system .", "For example , if we assume that recruitments of cells for organ initiation are stochastically independent from each other ( the probability to draw an initiation at a site k only depends on the value of the local parameters at site k but not on what may be drawn at other places ) , then we can estimate the expected number of cells independently recruited for organ initiation during the timespan δt .", "Let us denote X ( t , δt ) the number of cells initiated along the peripheral circle , and for a given time t and a small time span δt , X ( t , δt ) =∑k=1KXk ( t , δt ) .", "Its expectation is simply the sum of the expectations of the individual independent Poisson processes: ( 4 ) E ( X ( t , δt ) ) =∑k=1KE ( Xk ( t , δt ) ) =∑k=1KP ( Xk ( t , δt ) =1 ) =δt∑k=1Ke−β ( Ek ( t ) −E∗ ) , therefore giving us an estimate of the expected number of peripheral cells initiated during time δt and for the inhibition profile E ( t ) .", "So far we have considered peripheral cells as independent sites that may independently switch to primordium identity with a probability that depends on their local level of inhibition .", "As a local inhibition valley may span over several cells , one might expect that the probability to trigger a primordium initiation in a valley is increased by the fact that several founder cells can potentially contribute to this initiation during time δt .", "To formalize this , we then upscaled our stochastic model at the level of valleys where a stochastic process is now attached to each local minimum l instead of to each cell k .", "This upscaled model is called SMPmacro .", "The idea is that the stochastic processes of all the cells k spanned by a local inhibition valley l sum up and together define a stochastic process Nl that a primordium is initiated in l at a higher level .", "Being the sum of independent Poisson processes , this upscaled process is also a Poisson process with intensity Λl=∑k∈Klλk ( Ek ( t ) ) , where k varies over the set of cells spanned by the valley of the l th local minimum and indexed by Kl .", "If L denotes the number of local minima , then the expected number of primordia initiations during a small time laps δt is thus: ( 5 ) E ( Nl ( t , δt ) ) =δt∑l=1LΛl ( t ) .", "Therefore , at microscopic scale , SMPmicro couples", "i ) a deterministic part inherited from the classical model and related to the geometry and the dynamics of the fields and", "ii ) a new stochastic part related to the perception of this inhibitory field , i . e . representing signal perception capacities .", "It relies on the assumption that the perception sites , corresponding to the cells surrounding the central zone periphery , are stochastically independent of each other .", "Decisions regarding primordium initiation are taken in a cell-autonomous manner , thus reflecting more realistically the outcome of the initiation signaling pathway in each cell .", "At a macroscopic level , in each inhibition valley several cells may trigger primordium initiation .", "The probability to trigger an initiation increases in SMPmacro with the size of the valley when more than one founder cell are likely to contribute to initiation .", "A variant of this upscaled model consists of defining the probability for a valley to initiate a primordium by the probability of the cell with lowest level of inhibition in this valley .", "In this variant , called SMPmacro-max , Λl=maxk∈Kl λk ( Ek ( t ) ) .", "To study the emergent properties of this system , we implemented a computational version of SMPmacro-max and tested its sensitivity to parameter changes .", "In addition to the geometrical parameters of the classical model , two new parameters , β and E∗ now reflect the ability of the system to perceive the inhibitory signal from the fields ( or equivalently the initiation signal ) .", "As expected from a phyllotaxis model , the stochastic model is able to produce both spiral and whorl modes , from either imposed initial distributions of organs ( Figure 4A–C ) , or random starting points ( Appendix section 6 . 1 ) .", "However , the great majority of the sequences of divergence angle generated for different values of Γ=r0/R displayed divergence angle perturbations ( i . e . angles different from those predicted by the classical model with the same Γ ) of the type observed in Figure 4D–E .", "As suggested by their typical distributions ( Figure 4F–H left ) , these perturbations correspond to permutations very similar to those observed on real plants in previous studies ( Besnard et al . , 2014; Landrein et al . , 2015 ) with the appearance of secondary modes at multiples of 137 , Figure 1F–I .", "The amplitude of these secondary modes is correlated with the amount of perturbations in the sequences . 10 . 7554/eLife . 14093 . 010Figure 4 . Patterns generated by the stochastic model .", "( A ) The model generates spiral patterns ( in ( A–C ) , up: sequence of simulated divergence angles , down: corresponding plastochrons ) .", "( B–C ) and whorled patterns .", "( D ) Simple M-shaped permutations simulated by the stochastic model ( β = 10 . 0 , E∗ = 1 . 4 , Γ = 0 . 625 ) .", "( E ) More complex simulated permutations involving 2- and 3-permutations ( β = 10 . 0 , E∗= 1 . 4 , Γ = 0 . 9 ) .", "The permutations are here: [4 , 2 , 3] , [14 , 13 , 12] , [16 , 15] , [19 , 18] .", "( F ) Typical histogram of simulated divergence angles and corresponding plastochron distribution for β = 11 . 0 , E∗ =1 . 2 , Γ= 0 . 8 .", "( G ) Histogram of simulated divergence angles and corresponding plastochron distribution for β = 9 . 0 , E∗ = 1 . 2 , Γ = 0 . 8 ( H ) Histogram of simulated divergence angles and corresponding plastochron distribution for β = 9 . 0 , E∗ = 1 . 2 , Γ = 0 . 625 . DOI: http://dx . doi . org/10 . 7554/eLife . 14093 . 010 To complete this analysis , we looked at plastochron distributions in the simulated sequences ( Figure 4F , G , H right ) .", "In all cases , distributions were displaying a single mode largely spread along the x-axis .", "Interestingly , the more sequences were perturbed , the more negatively skewed the distributions , showing thus a higher occurrence of short plastochrons ( Figure 4H ) .", "This is reminiscent of the observation of co-initiations in growing meristems associated with perturbed phyllotaxis ( Besnard et al . , 2014; Landrein et al . , 2015 ) .", "The stochastic model is thus able to produce perturbed sequences with realistic series of divergence angles and corresponding realistic distributions of plastochrons .", "We then aimed to quantitatively assess the complexity level of permutations as a proxy for plastochron noise .", "For this we focused on spiral phyllotaxis modes and used two measures: the density of permuted organs respectively involved in 2 and 3-permutations π2=2 . σ2/Σ and π3=3 . σ3/Σ where σ2 and σ3 are respectively the number of 2- and 3-permutations in the sequence and Σ the total number of organs in the sequence .", "The quantities σ2 and σ3 were estimated a posteriori from simulated sequences using the algorithm described in ( Refahi et al . , 2011 ) .", "The total density of permuted organs involved either in 2- or 3 permutations is denoted by π=π2+π3 .", "We first explored the intensity of permutations of different natures ( 2- and 3-permutations ) in simulated phyllotaxis sequences .", "For this , we carried out simulations for a range of values for each parameter Γ , β , E∗ ( Figure 5—source data 1 ) .", "These values can be regarded as predictions of the model for each triplet ( Γ , β , E∗ ) .", "Remarkably , the simulations show that the values of π , π2 and π3 are not linearly related to each other .", "To make this relationship explicit , we plotted the proportion of organs involved in 2-permutations π2 as a function of the total proportion of perturbed organs π ( Figure 5 ) .", "Surprisingly , the points , when put together on a graph , were organized in a narrow crescent showing a convex curve-like relationship between π and π2 , revealing a remarkable property of the stochastic model: the more perturbations there are in the simulated sequences , the higher the proportion of 3-permutations , and this independently of the model parameters .", "The fact that this non-linear relationship emerges from a fairly large sampling of the parameter space suggests that it can be considered as a key observable property characterizing the model’s underlying structure .", "We tested this by gathering all measured values of π2 and π3 published in the literature for Arabidopsis ( Besnard et al . , 2014; Landrein et al . , 2015 ) and plotted the corresponding points on the original graph showing the model’s simulations ( Figure 5 , red crosses ) .", "The measured points fall within the range of predicted values and show that the measured values follow the same non-linear variation as the one predicted by the model: the larger the total percentage of permutations , the larger the proportion of 3-permutations in the sequences .", "This confirms a first prediction from the stochastic model and indicates that disorder complexity increases non-linearly with the frequency of disorders . 10 . 7554/eLife . 14093 . 011Figure 5 . Intensity of 2-permutations as a function of the total amount of perturbations . As the perturbation intensity π increases , the percentage of 2-permutations decreases in a non-linear way to the benefit of more complex 3-permutations .", "The diagonal line denotes the first bisector .", "In red: values of 2- and 3-permutations observed in different mutants and ecotypes of Arabidopsis thaliana ( Besnard et al . , 2014; Landrein et al . , 2015 and this study ) placed on the plot of values predicted by the stochastic model . DOI: http://dx . doi . org/10 . 7554/eLife . 14093 . 01110 . 7554/eLife . 14093 . 012Figure 5—source data 1 . Source files for simulated permutation intensities . This file contains a table showing the variation of permutation intensities with model parameters .", "We ran simulations using the SMPmacro-max model for different parameter values where the local minima of inhibition profile indicate the potential initiation sites ( for this , the inhibitory field values were estimated at 360 sampling points regularly distributed around the periphery of the central zone ) .", "We then used the combinatorial model ( Refahi et al . , 2011 ) to detect permutations in the simulated sequences .", "The model has mainly three parameters , β , E∗ and Γ .", "For each parameter value of β , E∗ and Γ , we run sixty simulations .", "Each simulation generated a sequence of 25 divergence angles .", "We then analyzed the sequences using the combinatorial model to detect permutation patterns . DOI: http://dx . doi . org/10 . 7554/eLife . 14093 . 012 Based on our simulations , we then investigated the variations of these perturbations as a function of the model parameters Γ , β .", "E∗ and s .", "As a general trend , for a value of s fixed to 3 as in ( Douady and Couder , 1996b ) , we noticed that for a given value of Γ , an increase in β was roughly counteracted in terms of permutation intensity by a decrease in E∗ .", "Likewise , an increase of Γ was canceled by a decrease in β .", "We gathered all these observations in one graph and plotted the global amount of perturbation π in a sequence as a function of a combination of the three original model parameters and reflecting the observed trend: ΓP=ΓβE∗ .", "In the resulting graph ( Figure 6A ) , each point corresponds to a particular instance of the three model parameters .", "The points form a narrow and decreasing band associating a small set of possible perturbation intensities π with each value of ΓP .", "For combinations of the three model parameters leading to a small ΓP , the perturbations can affect up to 50% of the organs whereas for high values of ΓP , there may be no perturbation at all .", "Interestingly , ΓP being a combination of the three elementary model parameters , plants having identical geometrical parameter Γ may show substantially different intensities of perturbations if their perception for different values of parameters β , E∗ .", "Consequently , since the intensity of perturbation in the system is more simply reflected by ΓP than by values of Γ , β , E∗ taken independently , ΓP can be considered to be a control parameter for perturbations . 10 . 7554/eLife . 14093 . 013Figure 6 . Key parameters controlling phyllotaxis phenotypes in the stochastic model . Phyllotaxis sequences were simulated for a range of values of each parameter β , E∗ , Γ .", "Each point in the graph corresponds to a particular triplet of parameter values and represents the average value over 60 simulated sequences for this triplet .", "( A ) Global amount of perturbation π as a function of the new control parameter ΓP=ΓβE∗ .", "( B ) Divergence angle α as a function of the control parameter Γ of the classical model on the Fibonacci branch .", "( C ) Divergence angle α as a function of the new control parameter ΓD=Γ1β1/6E∗1/2 on the Fibonacci branch ( here , we assume s = 3 , see Appendix 1—figure 6 for more details ) .", "( D ) Plastochron T as a function of control parameter of the classical model Γ .", "( E ) Plastochron T as a function of the new control parameter ΓD .", "( F ) Parastichy modes ( i , j ) identified in simulated sequences as a function of ΓD .", "Modes ( i , j ) are represented by a point i+j .", "The main modes ( 1 , 2 ) , ( 2 , 3 ) … correspond to well marked steps .", "( Figure 5—source data 1 ) DOI: http://dx . doi . org/10 . 7554/eLife . 14093 . 01310 . 7554/eLife . 14093 . 014Figure 6—figure supplement 1 . New control parameter ΓD for divergence angle and plastochrons . Each graph is made up of points that correspond to different values of the parameters Γ , β , E∗ of the stochastic model .", "Left column: different trials to define a control parameter for divergence angles α .", "Right column: different tries to define a control parameter for the plastochrons .", "For the parameter ΓD=Γ1 ( β1/3E∗ ) 1/2 , both clouds of points collapse on a single curve ( we assume here that s = 3 , see Appendix 1—figure 6 for more details ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 14093 . 014 To further investigate the structure of the stochastic model , we then studied how the usual observable quantities of a phyllotaxis system , i . e . divergence angles and plastochrons , depend on the model parameters Γ , β , E∗ and s .", "In the classical deterministic model , divergence angles are a function of a unique control parameter Γ=r0/R ( Douady and Couder , 1996b ) , meaning that the same divergence angle can be obtained in the model for different couples of r0 and R provided that their ratio is unchanged .", "We thus checked whether Γ could also serve as the control parameter for the divergence angles and plastochrons in the stochastic model .", "For this , we simulated various spiral phyllotaxis sequences ( from the Fibonacci branch where divergence angles are close to 137° ) by varying Γ , β , E∗ and estimated the corresponding divergence angle α and plastochron T , Figure 6B , D .", "We observed that , although the point clouds evoke the corresponding curves in the standard deterministic model ( Appendix 1—figure 2 ) , significantly different values of the divergence angles ( or plastochrons ) can be observed for many particular values of Γ .", "This phenomenon suggests that Γ is not a satisfactory control parameter in the stochastic model: for a given value of Γ , varying β or E∗ can significantly modify the divergence angle or the plastochron .", "We therefore tested various combinations of these parameters ( Figure 6—figure supplement 1 ) , and finally found that for ΓD=Γs/31β1/6E∗1/2 both cloud of points collapse into a single curves ( Figure 6B–C , D–E and Appendix section 6 . 3 ) .", "For this definition of ΓD , each value of the control parameter can be associated with quasi-unique divergence angle and plastochron , i . e . defines a precise observable state of the system .", "We then checked whether parastichies were also controlled by ΓD .", "For this , we computed the parastichies ( i , j ) corresponding to each simulated sequence and plotted the sum , i+j , as a function of ΓD ( Figure 6F ) .", "The resulting points were arranged on a stepwise curve , where each step corresponds to a Fibonacci mode: ( 1 , 2 ) , ( 2 , 3 ) , ( 3 , 5 ) , ( 5 , 8 ) .", "Each value of ΓD thus defines a precise value of the mode .", "We concluded that ΓD can be considered as a second control parameter of the stochastic model , relating the system’s state to the observable variables α and T through a unique combination of the system’s parameters .", "According to the stochastic model , a particular phyllotaxis system is characterized by a particular set of values of the parameters Γ , β , E∗ .", "Upon growth , a specific spatio-temporal dynamics emerges that is characterized by observable variables: divergence angle , plastochron and frequency of permutations .", "In the current state of our knowledge and measuring means , the parameters Γ , β , E∗ are not directly observable .", "Therefore , we investigated what can be learned about them from the observable variables .", "For this , we can use the relationships established above between the control parameters ΓP and ΓD and the observable variables α , T , π2 , π3 , .", ". . .", "For a genotype G , we have: ( 6 ) ΓP=ΓβE∗ΓD=Γs/31β1/6E∗1/2 Using the characteristic curves of Figure 6C , E , both measurements of α and T give possible estimates for ΓD .", "In a consistent model , these estimates should be compatible .", "Similarly , an estimate of ΓP can be derived from the observation of π .", "In the case of the WT for example , ΓD is estimated to be in [0 . 425 , 0 . 5] and ΓP in [8 . 30 , 11 . 49] according to the observed value of the plastochron , divergence angle and permutation intensity .", "This value is itself derived from the measurement of the plastochron ratio ρ ( Appendix section 4 . 1 . 3 ) .", "Based on estimated values of ΓD and ΓP , equations 6 define a system of 2 equations and 3 unknowns ( s was fixed to 3 in the simulations ) .", "This system is underdetermined and does not allow us to identify exactly the values of Γ , β , E∗ .", "However , being underdetermined in one dimension only , this system plays the role of a generalized control parameter: a given value of the generalized control parameter ( ΓD , ΓP ) determines the parameters Γ , β , E∗ up to one degree of freedom .", "If one of the parameters is given , then the others are automatically determined according to equation 6 .", "Several recent works demonstrated that mutations or changes in growth conditions could alter phyllotaxis and disorder patterns .", "Our model predicts correlations between main observable phyllotaxis variables .", "According to the model , plastochron positively correlates with ΓD ( Figure 6E ) .", "If signaling is not altered by the experimental setup ( β and E∗ are unchanged ) then ΓD positively correlates with Γ=r0/R ( Equation 6 ) .", "Therefore , the model predicts that , like in the classical model , plastochron positively correlates with r0 ( size of the primordia inhibitory fields ) and negatively correlates with R ( size of the central zone ) .", "However , the model makes also in this case the new prediction that plastochron negatively correlates with the frequency of observed permutations ( Figure 6A ) .", "A series of recent observations support this prediction .", "By changing growth conditions ( plants first grown in different day-length conditions and then in identical conditions ) or by using different accessions or mutants with markedly different meristem sizes from that of the wild type ( Landrein et al . , 2015 ) , changes in the size of the meristem could be induced .", "The authors hypothesized that this change affected the size of the central zone only and not the size of primordia inhibitory fields .", "Corresponding changes in Γ were observed to positively correlate with the frequency of organ permutations and negatively correlate with plastochron , as predicted by our model .", "In addition , the stochastic model makes it possible to quantitatively estimate the changes in central zone sizes from the measured phyllotaxis disorders with an error less than 5% ( Appendix section 4 . 3 ) .", "In a previous study on ahp6 mutants , Besnard et al . ( 2014 ) showed that the frequency of disorders could markedly augment while the size of meristems did not significantly change like in ( Landrein et al . , 2015 ) .", "As discussed above , this change in disorder intensity could theoretically be due to an alteration of initiation perception in the mutant .", "However , the stochastic model suggests that it is not the case .", "Indeed , we re-analyzed plastochrons of the mutants and could observe that , although the change is limited , mutant plastochrons are significantly smaller than those of the wild type ( Appendix section 4 . 2 ) .", "According to Figure 6E , this means that ΓD is reduced in the mutant .", "If this reduction was due to an increase in either β or E∗ , then according to the model , one should expect a corresponding increase of ΓP ( Equation 6 ) and , thus , a decrease of disorders ( Figure 6A ) .", "On the contrary a significant increase of disorders was actually observed , suggesting that perception is not altered and that , rather , a decrease in Γ=r0/R could be the source of variation .", "Since the size R of the meristems did not change , the model suggests that ahp6 is altered in the size r0 of the primordia inhibitory fields .", "In both the previous analysis and in related works ( Besnard et al . , 2014; Guédon et al . , 2013; Landrein et al . , 2015; Refahi et al . , 2011 ) , the permutation detection was restricted to 2- and 3-permutations .", "However , the stochastic model potentially predicts the existence of higher order permutations , i . e . 4- and 5-permutations , in Arabidopsis thaliana especially for small values of the control parameter ΓP .", "Following this prediction , we revisited the measured divergence angles in ( Landrein et al . , 2015 ) on the WS4 mutant grown in standard conditions ( π2= 29 . 45% , π3= 14 . 91% ) and for which 7 . 7% of angles were left unexplained when seeking for 2- and 3-permutations ( Figure 7A ) .", "When higher order permutation are allowed in the detection algorithm ( Appendix sections 4 . 1 and 5 ) , most of the unexplained angles for WS4 can be interpreted as being part of 4- and even 5-permutations ( Figure 7B ) . 10 . 7554/eLife . 14093 . 015Figure 7 . Detection of higher-order permutations in WS4 . The detection algorithm ( see ref [7] for details ) searches plausible angle values , i . e . values within the 99% percentile given the Gaussian like distributions fitted in Figure 1 , such that the overall sequence is n-admissible , i . e . composed of permuted blocks of length at most n .", "( A ) When only 2- and 3-permutations are allowed , some angles in the sequences cannot be explained by ( i . e . are not plausible assuming ) permutations ( the blue line of successfully interpreted angles is interrupted ) .", "( B ) Allowing higher order permutations allows to interpret all the observe angles as stemming from 2- , 3- 4- and 5-permutations ( the blue line covers the whole signal ) .", "Organs indexes involved in permutations: [3 , 2] , [5 , 8 , 6 , 4 , 7] , [13 , 12] , [16 , 14 , 17 , 15] , [19 , 18] , [22 , 21] , [24 , 25 , 23] , [27 , 26] , [30 , 29] , [32 , 31] , [35 , 34] , [39 , 40 , 38] , [43 , 42] , [46 , 45] , [48 , 49 , 47] , [52 , 50 , 53 , 51] .", "DOI: http://dx . doi . org/10 . 7554/eLife . 14093 . 015 Previous observations ( Landrein et al . , 2015 ) point to the existence of positive correlations between meristem size and intensities of perturbations .", "As discussed above , the stochastic model explains this: if a mutation , or a change in growth conditions only affects the geometry of the system Γ=r0/R , then ΓD and ΓP are both affected in the same sense ( Equation 6 ) .", "However , it also allows predicting new observable facts .", "Assume that a mutation or a change in growth conditions affects the ability of the plant to perceive initiation signals without modifying the geometry of the system , i . e . β and/or E∗ are modified while Γ is left unchanged .", "Then , according to equation 6 , this induces opposite variations in ΓD and ΓP that can be detected with the observed variables .", "For example , assuming that a mutation decreases the sensitivity of the system to the initiation signal ( decrease of E∗ ) , ΓP is decreased while ΓD is increased .", "The decrease of ΓP induces an increase of the perturbation intensity π while the increase of ΓD induces an increase of the plastochron .", "The model thus predicts that , in such a case , it is possible to expect an augmentation in the disorder correlated with a decrease in organ initiation frequency .", "To date , such a fact has not yet been observed and constitutes a testable prediction of our model ." ], [ "While the stochastic model is able to reproduce the major spiral and whorl phyllotaxis patterns , stochasticity induces alterations in the patterning process mainly affecting the plastochron i . e . the timing of organ initiation .", "These alterations take the form of permutations of the order of organ initiation in the meristem .", "If the plastochron is small as frequently observed in real sequences of permuted organs , permutations in the model can be considered equivalent to co-initiations that have been identified in the Arabidopsis meristem as the main source of permutations observed on the inflorescence stem .", "Less frequently , simulated permutations can have longer plastochrons .", "In this case , they can be interpreted as true permutations of the order of organ initiation in the meristem , consistently with the low frequency of such meristematic permutations observed also in Arabidopsis ( Besnard et al . , 2014 ) .", "These results are in line with a previous attempt at introducing stochasticity in the classical deterministic model that could also induce permutations ( Mirabet et al . , 2012 ) .", "However in this latter work only a limited frequency of defects could be induced ( even when the noise was fixed at high levels ) , while requiring time discretization and post-meristematic randomization of organ order when more than one organ initiation were detected in the same simulation time step .", "By contrast , the capacity of the stochastic model to reproduce faithfully perturbed sequences as observed in Arabidopsis indicates that the model captures accurately the dynamics of the phyllotactic system .", "Taken with the fact that permutations are observed in a variety of species and genotypes from a given species , our theoretical results identify noise on the plastochron as a common characteristic of phyllotaxis systems that may generate disorders in these developmental systems .", "It is important to note that our work points at stochasticity in signaling mechanisms allowing perception of inhibitory fields as the most likely origin of this developmental noise but does not entirely rule out the idea that other phenomenon might contribute ( see Appendix section 6 ) .", "A major contribution of stochasticity in signaling is supported by the robustness of the model to changes in different assumptions and parameters .", "However we have observed that spatial discretization for example can modify the frequency of permutations in the model , although this effect is limited ( see Appendix section 6 . 2 ) .", "A possible interpretation is that changes in the size of cells could also contribute to a certain point to noise on the plastochron , an idea that could be further explored .", "Importantly , phyllotaxis dynamics in the stochastic model relies not only on the geometry of the inhibitory fields captured by the Γ parameter as in previous deterministic models ( Douady and Couder , 1996b; Richards , 1951 ) ( Figure 8B ) , but also on two new parameters E∗ and β ( Figure 8C ) .", "E∗ and β describe respectively the sensitivity of cells to the inhibitory signal and the acuity of their perception , i . e . their capacity to differentiate close signal values .", "Our work thus suggests that a robust self-organization of a 3D developmental system driven by lateral inhibition depends both on the geometr of inhibitory fields in a tissue but also on the signaling capacities of cells in tissues .", "These theoretical observations are consistent with the key role of signaling in phyllotaxis ( Vernoux et al . , 2011 ) , and with the setting of patterning dynamics in animal systems , downstream of morphogenetic signals ( Kutejova et al . , 2009 ) .", "This pinpoints the interplay between global information provided by signal distribution and local interpretation of the information as a general principle for patterning emergence .", "In addition , we predict that due to pre-specification of initiation sites , noise in phyllotaxis is expected mainly on the timing of patterning .", "This might explain the selection through evolution of genetic mechanisms , such as the one recently described implicating the AHP6 protein ( Besnard et al . , 2014 ) , to diminish noise on plastochron and disorders in phyllotaxis .", "In biological systems , disorders are frequently viewed as a result of biological or environmental noise that mainly alters systems function or development .", "It is in this sense for instance that noise on phyllotaxis patterns had previously been analyzed ( Itoh et al . , 2000; Jeune and Barabé , 2006; Peaucelle et al . , 2008 ) .", "Here we show that biological noise at microscopic scale may be revealed at macroscopic scale in the form of organ disorders , the permutations .", "Our stochastic model of phyllotaxis suggests that these disorders bear information on the more profound , hidden variables that control the phyllotaxis patterning .", "Much like digital watermarks that represent a copyright or any information to be hidden in images or audio signals , the actual variables ( Γ , β , E∗ ) representing the state of the phyllotaxis system are not directly apparent in the plant phenotype ( i . e . in the sequence of lateral organ angles and its dynamics ) .", "However , by scrutinizing carefully the image or , here , the phyllotaxis pattern and their perturbations with adequate decoding algorithms , it is possible to reveal the hidden information that was “watermarked” in the original signal .", "In this way , permutations together with divergence angle α and plastochron T reveal key information about the state of the system that has produced them , as their knowledge drastically reduces the set of possible Γ , β and E∗ values .", "Reciprocally , any experimental alteration of these values modifies the biological watermark .", "Our model suggests that this change is reflected in macroscopic alterations of the phyllotaxis patterns that convey information about their possible molecular origin .", "To illustrate this , we used our stochastic model to confirm that changes in permutation frequencies due to changes in growth conditions are most likely explained by a specific modulation of Γ , as previously proposed ( Landrein et al . , 2015 ) ( Appendix section 4 . 1 . 5 ) .", "Such a biological watermarking also suggests a different interpretation of the function of AHP6 in phyllotaxis ( Besnard et al . , 2014 ) .", "Movement of AHP6 from organs has been proposed to generate secondary inhibitory fields that filter co-initiation at the meristem , decreasing the frequency of permutations .", "Based on permutation modifications , our theoretical framework suggests that AHP6 effect on the phyllotactic system does not need to be viewed as an additional specific mechanism acting on plastochron robustness but could be simply interpreted as a mechanism increasing Γ slightly .", "As no differences in auxin-based inhibitory fields could be detected between ahp6 mutant and wild-type plants ( Besnard et al . , 2014 ) ( Appendix section 4 . 2 ) , our model thus leads to a vision with composite inhibitory fields resulting at least from the combined effect of auxin-based and AHP6-based subfields .", "Conversely , this predicts that inhibitory fields in general cannot be explained only by auxin-based mechanisms as previously proposed .", "Combined with data on divergence angles and on the plastochron , we further predict that the phyllotactic disorders could be used to identify mutants affected in biological mechanisms that controls β and/or E* .", "The model indicates that such mutations would have an opposite effect on frequency of permutations and on plastochron .", "Mutants behaving accordingly would allow not only to test this prediction of the model but also to dissect the molecular mechanisms at work .", "Precise and automated quantifications of the permutation , divergence angles and plastochron would allow for screening for such mutants and should become feasible with the fast development of phenotyping tools ( Dhondt et al . , 2013; Granier and Vile , 2014 ) .", "Our model only takes into account stochasticity in the perception of inhibitory fields by cells and is based on two biologically plausible assumptions: that this perception is mostly cell autonomous and that it only depends on the local level of the inhibitory signal .", "This provides a reasonable abstraction of local stochastic fluctuations in", "i ) hormonal concentrations related to inhibition produced by each primordium", "ii ) in the activity of the signal transduction pathway leading to initium creation .", "The detailed molecular mechanisms controlling organ initiation are for the moment only partially known .", "However the capacity of the stochastic model to capture accurately phyllotaxis suggests that it also captures plausible emergent properties of the underlying molecular mechanisms .", "This model thus not only provides a framework to understand the dynamics of patterning in the meristem but also the properties of the signaling mechanisms that process the different signals involved .", "Note also that the predictive capacities of our model suggest that noise on perception could be the most influential source of noise in the system .", "However demonstrating this would require further exploration of other potential sources of stochasticity acting at different scales , such as growth variations , spatial discretization of the peripheral zone ( to account for the real size of plant cells ) , in order to assess their relative contribution to disorders .", "Moreover , similarly to the classical deterministic model of phyllotaxis , our stochastic model does not explicitly account for the cascade of molecular processes that participate to the establishment of new inhibitory fields at the location of incipient primordia .", "This might limit the ability of these models to fully capture the dynamics of the self-organization of the system .", "To do so , more mechanistic versions of this stochastic model could be developed in the future , combining more detailed cellular models of hormone-based fields , e . g . ( Jönsson et al . , 2006; Smith et al . , 2006a; Stoma et al . , 2008 ) , and stochastic perception of these hormonal signals in 2D or 3D models with cell resolution .", "Heterogeneity of biological systems at all scale has attracted an ever-growing attention in the recent years ( Oates , 2011 ) .", "Deterministic models do not account for the high variability that can be observed in systems behaviors , indicating that they fail to capture some key characteristics of biological systems ( Wilkinson , 2009 ) .", "While more demanding computationally , stochastic models are required in such cases , e . g . ( Greese et al . , 2014; Uyttewaal et al . , 2012; Wennekamp et al . , 2013 ) , and our work illustrates how dynamic stochastic modeling can help understanding quantitatively self-organization and more broadly patterning in higher eukaryotes ." ], [ "Based on the classical model of phyllotaxis ( Appendix section 2 ) , a complete and formal presentation of the stochastic model is described in the Appendix section", "3 . In particular , it is shown how the exponential form of the intensity law can be derived from basic model assumptions and how different observable quantities can be expressed using the model parameters .", "A computational version of the stochastic model SMPmacro-max was implemented in Python programming language using Numpy and SciPy .", "Similarly to ( Douady and Couder , 1996b ) , unless otherwise stated , the stiffness parameter was fixed in all simulations to s = 3 . The non-homogeneous Poisson process was simulated using the algorithm described in ( Ross , 2012 ) .", "A pseudo-code version of the stochastic model algorithm is given in the Appendix section 3 . 2 .", "To estimate the value of the different variables characterizing phyllotaxis α , T and π , π2 , π3 , etc . in either simulated or observed sequences , we used a method based on the algorithm developed in ( Refahi et al . , 2011 ) and described in the Appendix section", "4 . As this algorithm is central to the identification of permutations , we additionally tested its ability to detect correctly permutations on synthetic data in which known permutation patterns were introduced ( Appendix section 5 ) .", "Results show that the algorithm is able to detect permutations with a success rate of 98% on average .", "The parameter space of the stochastic model was explored by varying values of Γ , β and E∗ .", "60 stochastic runs have been made for each 3-tuple of the parameter values .", "Each simulation run generated a sequence of 25 divergence angles and corresponding plastochrons .", "The different observable variables have been extracted from these simulations .", "Results are reported in Tables 1 and 2 of the Figure 5—source data 1 .", "The models describing the different non-linear relationships between the observable variables and the control parameters ΓD and ΓP were fitted with Gauss-Newton non-linear least-squares method ( Bates and Watts , 2007 ) .", "Approximate 95%-prediction bands of the response variables were computed by assuming random errors of the models independent and identically normally distributed ." ] ]

[ "Exploration of developmental mechanisms classically relies on analysis of pattern regularities .", "Whether disorders induced by biological noise may carry information on building principles of developmental systems is an important debated question .", "Here , we addressed theoretically this question using phyllotaxis , the geometric arrangement of plant aerial organs , as a model system .", "Phyllotaxis arises from reiterative organogenesis driven by lateral inhibitions at the shoot apex .", "Motivated by recurrent observations of disorders in phyllotaxis patterns , we revisited in depth the classical deterministic view of phyllotaxis .", "We developed a stochastic model of primordia initiation at the shoot apex , integrating locality and stochasticity in the patterning system .", "This stochastic model recapitulates phyllotactic patterns , both regular and irregular , and makes quantitative predictions on the nature of disorders arising from noise .", "We further show that disorders in phyllotaxis instruct us on the parameters governing phyllotaxis dynamics , thus that disorders can reveal biological watermarks of developmental systems ." ]

[ "Plants grow throughout their lifetime , forming new flowers and leaves at the tips of their stems through a patterning process called phyllotaxis , which occurs in spirals for a vast number of plant species .", "The classical view suggests that the positioning of each new leaf or flower bud at the tip of a growing stem is based on a small set of principles .", "This includes the idea that buds produce inhibitory signals that prevent other buds from forming too close to each other .", "When computational models of phyllotaxis follow these ‘deterministic’ principles , they are able to recreate the spiral pattern the buds form on a growing stem .", "In real plants , however , the spiral pattern is not always perfect .", "The observed disturbances in the pattern are believed to reflect the presence of random fluctuations – regarded as noise – in phyllotaxis .", "Here , using numerical simulations , Refahi et al . noticed that the patterns of inhibitory signals in a shoot tip pre-determine the locations of several competing sites where buds could form in a robust manner .", "However , random fluctuations in the way cells perceive these inhibitory signals could greatly disturb the timing of organ formation and affect phyllotaxis patterns .", "Building on this , Refahi et al . created a new computational model of bud patterning that takes into account some randomness in how cells perceive the inhibitory signals released by existing buds .", "The model can accurately recreate the classical spiral patterns of buds and also produces occasional disrupted patterns that are similar to those seen in real plants .", "Unexpectedly , Refahi et al . show that these ‘errors’ reveal key information about how the signals that control phyllotaxis might work .", "These findings open up new avenues of research into the role of noise in phyllotaxis .", "The model can be used to predict how altering the activities of genes or varying plant growth conditions might disturb this patterning process .", "Furthermore , the work highlights how the structure of disturbances in a biological system can shed new light on how the system works ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "cell biology", "immunology and inflammation" ]

[ [ "There is an ongoing debate as to whether allergic disease is a risk factor for cancer or whether it is protective , with recent studies indicating that the relationship is complex and site specific ( Arana et al . , 2010; Wedgeworth et al . , 2011; Hwang et al . , 2012 ) .", "Several epidemiological studies have suggested that atopic dermatitis ( AD; eczema ) , hives , allergies to animal fur , and certain food ingredients are inversely associated with cancers of tissues that provide an interface with the external environment , such as the skin ( Jensen-Jarolim et al . , 2008; Sherman et al . , 2008 ) .", "In a recent large-scale analysis , the risk of nonmelanoma skin cancer was reduced in patients who had both allergic rhinitis and asthma ( Hwang et al . , 2012 ) .", "It is challenging to draw firm conclusions from the epidemiological data , in part because of the relapsing and remitting nature of AD and the potential cancer modulatory effects of treatments that are used to manage AD .", "Therefore , we sought to examine the link between AD and cancer susceptibility using a mouse model of AD in which the primary defect is in the epidermal barrier that is normally protective against pathogens .", "The skin barrier is formed by terminally differentiated keratinocytes in the outermost layers of the epidermis , known as the cornified layers or stratum corneum .", "In cornified keratinocytes , the plasma membrane is replaced with a layer of highly insoluble , transglutaminase cross-linked proteins with covalently attached lipids , known as the cornified envelope ( Candi et al . , 2005 ) .", "Involucrin , envoplakin , and periplakin are the first proteins to be cross-linked by transglutaminase-1 and create the protein scaffold for the attachment of lipids on which the cornified envelope assembles ( Rice and Green , 1977; Simon and Green , 1984; Ruhrberg et al . , 1996 , 1997 ) .", "Mice triply deficient in envoplakin , periplakin , and involucrin ( EPI−/− mice ) have a defective epidermal barrier and exhibit a reduction in epidermal γδTCR+ CD3+ cells ( dendritic epidermal T cells; DETCs ) and infiltration of CD4+ T cells into the dermis ( Sevilla et al . , 2007 ) .", "In contrast , the individual and double knockouts for envoplakin , periplakin , and involucrin do not have observable barrier defects or an altered immune infiltrate ( Sevilla et al . , 2007 ) .", "The skin barrier phenotype of EPI−/− mice shares similarity with the flaky tail mouse , which has nonsense mutations in the keratin filament associated protein filaggrin and the transmembrane protein Tmem79 , which is a component of lamellar granules ( Fallon et al . , 2009; Sasaki et al . , 2013; Saunders et al . , 2013 ) , the latter resulting in defective stratum corneum formation .", "Filaggrin mutations in humans are linked to ichthyosis vulgaris ( dry , flaky skin ) and increased risk of AD ( Palmer et al . , 2006 , 2007; Brown and McLean , 2012 ) , while Tmem79 mutations are found in some patients with AD ( Saunders et al . , 2013 ) .", "To test the potential association between a defective skin barrier , AD and cancer , we have subjected EPI−/− mice to the classic two-stage chemical carcinogenesis protocol ( Perez-Losada and Balmain , 2003 ) .", "A single exposure to the polycyclic aromatic hydrocarbon 7 , 12-dimethylbenz ( a ) anthracene ( DMBA ) induces oncogenic mutations in HRas ( initiation ) .", "Repeated applications of the promoting agent 12-O-tetradecanoylphorbol-13-acetate ( TPA ) ( promotion ) allow initiated , mutagenized cells to clonally expand and form benign tumours called papillomas , some of which progress to malignant squamous cell carcinomas ( SCCs ) ( Abel et al . , 2009 ) .", "While most human skin SCCs are associated with UV , rather than chemical , carcinogenesis , 10% do have Ras gene mutations ( Lopez-Pajares et al . , 2013 ) , and DMBA/TPA carcinogenesis has contributed substantially to our understanding of the steps of tumour development in human skin ( Hirst and Balmain , 2004 ) ." ], [ "To determine whether lack of Evpl , Ppl and Ivl influences sensitivity to tumour formation , we compared DMBA/TPA skin carcinogenesis ( Abel et al . , 2009 ) in mice lacking all three genes ( EPI−/− ) and wild-type ( WT ) mice on the same genetic background .", "Since EPI−/− mice were on a mixed Sv129 and C57Bl/6 background , F2 crosses between WT Sv129 and C57Bl/6J mice were used to generate the WT controls for all experiments .", "The data from one experiment are presented in Figure 1 .", "A second , independent , carcinogenesis experiment gave similar results ( data not shown ) .", "Mice that received DMBA or TPA alone did not develop tumours . 10 . 7554/eLife . 01888 . 003Figure 1 . Chemical carcinogenesis in EPI−/− and WT mice .", "( A ) Average number of papillomas per mouse .", "( B ) % mice with one or more papilloma .", "( C ) Average number of SCCs per mouse .", "( D ) % mice with one or more SCC .", "( A and C )", "Data are means ± SEM .", "( E ) Back skin , papillomas ( pap ) and SCCs from WT mice were immunostained for Envoplakin , Periplakin , or Involucrin ( red ) and DAPI ( blue ) .", "Dotted line indicates basement membrane .", "Scale bars: 100 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 01888 . 003 The first papillomas emerged in both groups 5–6 weeks after the start of TPA promotion , and the maximum number of papillomas was reached by 15–16 weeks ( Figure 1A ) .", "The average number of papillomas per mouse was approximately sixfold lower in EPI−/− than WT mice ( Figure 1A; week 15 , p=0 . 0007; week 57 , p=0 . 0001 ) .", "The incidence of papilloma formation was also lower in EPI−/− mice ( Figure 1B ) .", "Over 95% of WT mice had at least one papilloma 14 weeks after the start of promotion , whereas 50% of EPI−/− mice were tumour-free at the end of the experiment ( p=0 . 0063 ) .", "Thus combined deletion of envoplakin , periplakin , and involucrin decreased epidermal susceptibility to chemically induced benign tumours .", "As expected from the genetic background of the mice ( Woodworth et al . , 2004 ) , only a small subset of papillomas progressed to invasive squamous cell carcinomas ( SCCs ) ( Figure 1C ) .", "There was no difference in SCC burden or incidence between EPI−/− and WT mice ( Figure 1C , D ) .", "However , since EPI−/− mice had fewer papillomas , the SCC conversion frequency was higher than in WT mice .", "We observed a downregulation of envoplakin , periplakin , and involucrin in WT papillomas and SCCs ( Figure 1E ) , leading us to speculate that the increased risk of SCC conversion in EPI−/− papillomas may be linked to the lack of two desmosomal proteins ( Thiery and Chopin , 1999 ) .", "No metastases were observed in any mice ( data not shown ) .", "To determine whether the resistance of EPI−/− mice to papilloma formation was due to a reduced DMBA response , we examined responses to a single topical application of carcinogen .", "Skin was analysed 24 hr after one application of DMBA in 4 mice per genotype .", "DMBA binds to the aryl hydrocarbon receptor ( AhR ) , leading to upregulation of the cytochrome P-450 enzymes Cyp1a1 and Cyp1b1 , which convert DMBA into the active metabolite that causes HRas mutations ( Gao et al . , 2005 ) .", "There were no differences in expression of AhR , the AhR repressor ( AhRR ) , Cyp1a1 and Cyp1b1 between WT and EPI−/− epidermis , either in the steady state or after application of DMBA ( Figure 2A ) .", "Langerhans cells resident in the epidermis metabolise DMBA to the active form and as a result DMBA/TPA treated Langerhans cell-deficient mice do not develop papillomas ( Modi et al . , 2012 ) .", "However , EPI−/− and WT epidermis contained similar numbers of CD207+ Langerhans cells ( Figure 2B ) .", "We conclude that DMBA was activated to a similar extent whether or not the epidermal cornified envelope was defective . 10 . 7554/eLife . 01888 . 004Figure 2 . Response of EPI−/− and WT mice to DMBA .", "( A ) Q-PCR of enzymes responsible for DMBA uptake and metabolic activation .", "( B ) Number of CD207+ Langerhans cells per mm2 ear epidermis .", "( C–E )", "Number of epidermal cells that scored positive for γH2AX ( C ) , PH3 ( D ) or active caspase-3 ( E ) per cm .", "7-cm skin was analysed per marker .", "( F ) Q-PCR of anti- and pro-apoptotic genes .", "( A–F )", "Data in all histograms are means ± SEM of at least three mice per genotype .", "( G ) Hras codon 61 mutations in 4 SCCs per genotype were quantified by Q-PCR of genomic DNA . DOI: http://dx . doi . org/10 . 7554/eLife . 01888 . 004 We next analysed epidermal protective responses to carcinogen-induced DNA damage ( DNA repair and induction of apoptosis ) .", "Phosphorylated Histone H2A variant H2AX ( γH2AX ) is recruited to sites of dsDNA damage and subsequent breaks , and is upregulated on DMBA treatment .", "The proportion of epidermal cells that expressed γH2AX was similar in EPI−/− and WT epidermis , whether or not the skin had been treated with DMBA ( Figure 2C ) .", "The proliferative response of keratinocytes to DMBA was comparable in both genotypes , as assessed by the proportion of phospho-Histone 3 ( PH3 ) positive cells ( Figure 2D ) .", "Furthermore , topical application of DMBA resulted in a similar apoptotic response , as detected by cleaved caspase-3-positive cells and quantitation of mRNA levels for a panel of apoptotic and anti-apoptotic genes ( Figure 2E , F ) .", "Levels of the DMBA ‘signature’ Hras ( codon 61 ) mutation ( Modi et al . , 2012 ) were the same in mRNA extracted from SCCs of WT or EPI−/− mice ( Figure 2G ) .", "We did not detect mutations in exons 4 , 5 , 8 and 9 of p53 in WT or EPI−/− tumours ( data not shown ) .", "We conclude that differences in DMBA uptake and activation or activity did not account for the resistance of EPI−/− mice to chemical carcinogenesis .", "Since WT and EPI−/− mice did not differ in DMBA responsiveness , we next investigated whether their response to the promotion phase of the two-stage carcinogenesis protocol was aberrant .", "Mice received three topical applications of TPA ( or vehicle control ) , which corresponds to 1 week of promotion , and skin was collected 48 hr after the last treatment .", "EPI−/− mice exhibited an exacerbated response to TPA ( Figure 3A ) .", "Their skin was reddened , dry and scaly , whereas that of WT mice was not ( data not shown ) .", "Histological analysis revealed increased hyperkeratosis ( thickened stratum corneum ) and parakeratosis ( retention of nuclei in cornified cells ) ( Figure 3B , C ) , consistent with defective desquamation ( Sevilla et al . , 2007 ) .", "Widening of intercellular spaces in the epidermal basal layer ( spongiosis ) was extensive in EPI−/− but not WT skin ( Figure 3A ) .", "Epidermal thickness , measured as the distance from the basal to the upper granular layer , was greater in EPI−/− mice treated with TPA or DMBA/TPA than in WT mice , and this correlated with a greater number of suprabasal layers ( Figure 3A , D ) .", "The numbers of keratinocytes with dsDNA breaks and active caspase-3 were similar in EPI−/− and WT epidermis ( Figure 3F , G ) . 10 . 7554/eLife . 01888 . 005Figure 3 . Keratinocyte responses to TPA treatment .", "( A ) H&E stained skin sections of mice treated three times with acetone or TPA .", "Asterisk: neutrophil containing pustule; white arrow: spongiosis; black arrow: parakeratosis .", "% hyperkeratotic ( B ) and parakeratotic ( C ) stratum corneum ( 8 cm skin analysed per condition ) .", "( D ) Epidermal thickness in μm ( 8 cm skin analysed per condition ) .", "( E–G )", "Number of epidermal cells positively labeled for PH3 ( E ) , γH2AX ( F ) and active caspase-3 ( G ) per cm skin ( 7 cm skin analysed per condition ) .", "( H ) Number of BrdU positive cells in basal and uppermost two granular layers per cm epidermis of mice treated three times with TPA and injected with BrdU 24 hr before harvesting .", "Data from all graphs represent means ± SEM from at least four mice per genotype .", "Scale bar: 100 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 01888 . 005 The number of mitotically active basal cells , measured by PhosphoHistone3 staining , was similar in TPA-treated EPI−/− and WT epidermis , whether or not the skin was pre-treated with DMBA ( Figure 3E ) .", "To examine whether the transit time through the suprabasal layers was altered , mice were injected with BrdU 24hr prior to sacrifice and the position of labelled cells was quantified .", "The numbers of BrdU positive cells in the basal layer and outermost granular layers of EPI−/− and WT epidermis were not significantly different ( Figure 3H ) .", "Since EPI−/− epidermis contained more suprabasal layers , the transit time of keratinocytes through the epidermis was faster in EPI−/− than WT epidermis .", "These observations are consistent with a model whereby precocious differentiation can act as a tumour suppressive mechanism in skin ( Guinea-Viniegra et al . , 2012 ) .", "We conclude that , in contrast to the DMBA response , the response of epidermal keratinocytes to TPA was markedly different in EPI−/− and WT mice .", "Because inflammation is a crucial factor in skin tumour development ( Johansson et al . , 2008; Zamarron and Chen , 2011 ) and EPI−/− skin has an abnormal number of DETCs and CD4+CD3+ lymphocytes ( Sevilla et al . , 2007 ) , we examined whether the inflammatory response to short term TPA treatment was altered in skin with a defective epidermal barrier .", "Consistent with our previous observations ( Sevilla et al . , 2007 ) , vehicle-treated EPI−/− skin had a reduced number of epidermal γδTCR+CD3+ lymphocytes ( DETCs ) ( Figure 4A ) and EPI−/− γδ T cells exhibited a more rounded morphology , indicative of activation ( Figure 4I; Strid et al . , 2008 ) .", "On TPA treatment , the number of DETCs in both EPI−/− and WT epidermis decreased ( Figure 4A ) .", "In contrast , TPA treatment stimulated recruitment of CD4+CD3+ lymphocytes into the skin of EPI−/− mice to a greater extent than in WT mice ( Figure 4B , J ) .", "On TPA treatment , CD4+ T cells infiltrated EPI−/− but not WT epidermis ( Figure 4J ) .", "Infiltration of granulocytes of the neutrophil family ( Cd11b+LY6G+ ) is a typical sign of chronic skin inflammation , where they accumulate in pustules ( Figure 4D , E ) .", "The number of pustules was significantly higher in TPA-treated EPI−/− compared with WT skin ( Figure 4E ) . 10 . 7554/eLife . 01888 . 006Figure 4 . Immune cell responses to TPA treatment .", "( A–C and E–H )", "Quantitation of specific T lymphocyte populations ( A–C ) , pustules ( E ) , mast cells ( F ) , eosinophils ( G ) , and macrophages ( H ) per mm epidermis or mm2 dermis .", "Data in all histograms are means ± SEM of at least three mice per genotype .", "( D , I , J )", "Skin sections of mice treated three times with acetone ( I ) or TPA ( D and J ) and labelled for CD11b ( green , D ) , Ly6G ( red , D ) , CD3 ( red I , green J ) , γδTCR ( green I ) or CD4 ( red , J ) with DAPI nuclear counterstain ( blue ) .", "White asterisk: neutrophil pustule .", "Insets in I and J are higher magnification views of boxed areas .", "Scale bars: 100 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 01888 . 006 TPA treated EPI−/− skin showed a pronounced infiltration of dermal mast cells and eosinophils ( Figure 4F , G ) , but not macrophages ( F4/80+Cd11b+ cells ) ( Figure 4H ) .", "Similar differences in immune cell recruitment in EPI−/− and WT skin were observed when mice were treated with DMBA prior to TPA ( data not shown ) .", "Pustule formation and dermal infiltration of mast cells , eosinophils and CD4+ T cells are hallmarks of the acute phase of atopic dermatitis in humans .", "Thus the response of EPI−/− skin to TPA resembled the acute phase of the human disease and involved recruitment of multiple leukocyte subsets .", "Keratinocytes communicate with different immune cell populations by production of an array of cytokines and chemokines , collectively known as the ‘epimmunome’ ( Swamy et al . , 2010 ) .", "Tissue damage triggers upregulation of keratinocyte ‘stress-associated’ genes such as Rae-1 ( Gasser et al . , 2005 ) and H60 ( Whang et al . , 2009 ) .", "These stress antigens engage the immunoreceptor NKG2D expressed by cells of the innate ( NK cells and DETC ) and adaptive ( activated CD8+ T cells , NKT cells ) immune system , triggering a lymphoid stress-surveillance response ( Hayday , 2009 ) .", "To investigate how a defective epidermal barrier could lead to an enhanced inflammatory response to TPA , we compared cytokine and chemokine expression in epidermis and dermis of WT and EPI−/− mice .", "We examined markers of innate , type 1 ( cellular ) , type 2 ( humoral ) and type 17 ( anti-microbial ) immunity .", "In EPI−/− epidermis treated with acetone , transcripts related to innate immunity ( in particular RAGE , S100A8 , S100A9 and IL-1β ) were higher than in WT , and these differences were maintained when transcript levels were upregulated by TPA treatment ( Figure 5A ) .", "Type 1 cytokine transcripts were expressed at similar levels in WT and EPI−/− epidermis and dermis and were not upregulated to the same extent as other types of cytokines by TPA ( Figure 5A ) .", "There was a striking upregulation of type 2 cytokines , including IL-4 , IL-6 , IL-13 , IL-33 and thymic stromal lymphopoietin ( TSLP ) , in epidermis and dermis of EPI−/− TPA-treated mice; this was not observed to a similar extent in WT skin .", "Type 17 transcripts were upregulated by TPA in epidermis of both genotypes , but were higher in EPI−/− epidermis ( Figure 5A ) .", "Increased transcript levels of a number of chemokines , in particular Cxcl-5 and GM-CSF , were found in TPA treated EPI−/− vs WT skin , in agreement with the increased dermal infiltration of neutrophils and eosinophils .", "We conclude that the immune response to TPA in EPI−/− skin correlated with expression of pro-inflammatory genes with a type 2/type 17 profile . 10 . 7554/eLife . 01888 . 007Figure 5 . Stress signals , cytokine , and chemokine production in EPI−/− and WT skin .", "( A ) Heatmap of mRNA levels relative to GAPDH from epidermis and dermis .", "Each value represents the mean of data obtained from four mice .", "( B and C )", "Serum levels of IgE ( B ) and TSLP ( C ) determined by ELISA .", "( D–F )", "Q-PCR of indicated mRNAs in epidermis .", "All histograms represent mean ± SEM ( N = 5 untreated , N = 7 acetone , N = 4 DMBA , N = 10 TPA treated mice per genotype ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 01888 . 007 Consistent with the type 2 response ( Figure 5A ) and mast cell influx ( Figure 4F ) , serum IgE levels were 3 . 4-fold higher in EPI−/− than WT mice ( Figure 5B ) .", "There was also a higher level of TSLP in EPI−/− serum ( Figure 5C ) .", "TSLP is mainly produced by keratinocytes and is associated with increased resistance to skin carcinogenesis ( Kuramoto et al . , 2002; Aberg et al . , 2008; Demehri et al . , 2008 , 2012; Di Piazza et al . , 2012 ) .", "TSLP levels are elevated when Notch signalling in skin is impaired ( Demehri et al . , 2008; Dumortier et al . , 2010 ) .", "However , EPI−/− and WT epidermis expressed comparable levels of Notch1 , 2 and 3 , Notch ligand Jag1 and Notch target genes Hey-1 , Hes-1 and Hes-5 , with or without TPA treatment ( Figure 5D ) .", "Thus , upregulation of TSLP is associated with barrier defects rather than being a direct consequence of altered Notch signalling ( Demehri et al . , 2008; Dumortier et al . , 2010 ) .", "These results suggested that lymphoid stress-surveillance of tissue damage could underlie the exacerbated inflammatory response to TPA exposure in EPI−/− mice .", "We therefore measured expression of the keratinocyte ‘stress-associated’ genes Rae-1 and H60 ( Figure 5E , F ) .", "Rae-1 and H60 levels were significantly higher in EPI−/− than WT epidermis under steady-state conditions .", "Both were strongly upregulated 24 hr after DMBA treatment , H60 to a greater extent in EPI−/− mice .", "The effects of TPA treatment on Rae-1 and H60 expression were less pronounced .", "We conclude that the defective barrier of EPI−/− mice results in elevated expression of keratinocyte stress-associated genes that could prime them for an exacerbated atopic response to TPA .", "To investigate the mechanism by which TPA provoked an exacerbated atopic response in EPI−/− epidermis , we used several different inhibitory strategies .", "Treatment with the anti-inflammatory drug dexamethasone ( DXM ) markedly reduced epidermal thickening and dermal cellularity ( Figure 6A , B ) .", "DXM also prevented the selective increase in spleen size observed in TPA-treated EPI−/− mice ( Figure 6F ) . 10 . 7554/eLife . 01888 . 008Figure 6 . Blocking strategies to revert the atopic response to TPA treatment .", "( A–E )", "H&E stained skin sections of WT and EPI−/− mice painted with TPA and injected with IgG ( A ) , Dexamethasone ( B ) , α-CD4 antibody ( C ) , α-Ly6G antibody ( D ) or α-IL4 ( E ) .", "Scale bars: 100 μm .", "( F ) Spleen mass of mice treated with acetone , TPA or TPA + Dexamethasone as % of body weight .", "Data are means ± SEM from 12 ( acetone , TPA ) or 3 ( TPA + DXM ) mice per genotype .", "( G ) Numbers of granulocytes per μl blood in TPA treated mice injected with IgG or α-Ly6G antibody .", "( H ) Quantification of serum levels of IL-4 in EPI−/− mice treated with IL-4 or control antibodies .", "( I and J )", "Effects of anti-IL-4 on epidermal thickness ( μm ) ( 8 cm skin analysed per condition ) ( I ) and dermal CD4 + T cells ( J ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 01888 . 008 Since tumour resistance of Notch deficient skin is attributable mainly to CD4+ T cells ( Demehri et al . , 2012 ) , we examined the effect of intraperitoneal injections of blocking CD4 antibodies .", "In vivo CD4 depletion was confirmed by quantifying CD4+ cells in the spleen ( data not shown ) .", "Specific CD4 targeting resulted in augmented skin hyperplasia and dermal inflammation in TPA-treated WT mice , but had no effect on EPI−/− skin ( Figure 6C ) .", "The immunosuppressive role of CD4+ cells in WT skin may be attributable to the regulatory subset of CD4+ cells ( CD4+CD25+Foxp3+ ) ( Teige et al . , 2009 ) .", "There was a marked increase in neutrophil infiltration in EPI−/− TPA-treated skin compared to WT ( Figure 4D , E ) .", "However , when circulating neutrophils were depleted by anti-LY6G injection ( Figure 6G ) , there was no effect on epidermal hyperplasia and dermal inflammation ( Figure 6D ) .", "We also targeted IL-4 to evaluate whether inhibition of a generalised type 2 immune response could normalize the effects of TPA in EPI−/− skin ( Figure 6E ) .", "We confirmed a decrease in IL-4 serum levels by ELISA ( Figure 6H ) .", "Inhibition of IL-4 reduced the number of CD4+ cells in the dermis ( Figure 6J ) , as expected , but had no effect on epidermal thickness ( Figure 6I ) nor on the number of mast cells , eosinophils or epidermal γδ T cells ( data not shown ) .", "Taken together , these observations indicate that the exacerbated TPA response in EPI−/− skin involves multiple immune cell types and cannot be normalized by inhibiting one specific leukocyte subset .", "To analyse the role of the Rae-1/NKG2D/TSLP axis in the exacerbated atopic response of EPI−/− skin to TPA , we interfered with the epidermal lymphoid stress surveillance response by in vivo antibody blockade of TSLP or NKG2D ( Figure 7A ) .", "We confirmed effective inhibition of TSLP by ELISA ( Figure 7B ) . 10 . 7554/eLife . 01888 . 009Figure 7 . TSLP and NKG2D inhibition reduce epidermal responses to TPA .", "( A ) H&E stained sections of skin from TPA-treated mice injected with IgG , anti-TSLP , or anti-NKG2D antibodies .", "( B ) Quantification of serum levels of TSLP in WT and EPI−/− mice treated with TSLP or control antibodies .", "( C and D )", "Quantification of PH3+ cells ( C ) and epidermal thickness ( μm ) ( D ) .", "( E and F )", "Number of BrdU positive cells in basal ( E ) and uppermost two granular ( F ) epidermal layers in mice treated with TPA and the indicated antibodies .", "( G and H ) % hyperkeratotic ( G ) and parakeratotic ( H ) stratum corneum .", "( C–D and E–F ) 7-cm skin analysed per condition . DOI: http://dx . doi . org/10 . 7554/eLife . 01888 . 009 Neutralization of TSLP completely abrogated the atopic dermatitis phenotype in TPA treated EPI−/− mice ( Figure 7A ) .", "Anti-NKG2D also reduced the atopic phenotype , albeit to a lesser extent ( Figures 7A and 8 ) .", "TPA-induced epidermal thickening was significantly reduced in TSLP or NKG2D suppressive conditions , without any differential effect on the number of PH3+ basal layer cells ( Figure 7A , C , D ) .", "TSLP treatment reduced the transit time of BrdU + cells from the basal to the outermost granular layers since although the epidermis was thinner the proportion of labelled granular cells was unaffected ( Figure 7E , F ) .", "There was a slight reduction in hyperkeratosis , and parakeratosis was clearly decreased ( Figure 7G , H ) .", "Targeting the Rae-1/NKG2D/TSLP axis also reduced the number of dermal CD4+CD3+ lymphocytes , mast cells , eosinophils , and epidermal pustules in EPI−/− mice ( Figure 8A–D ) .", "Furthermore , under TSLP suppressive conditions the number of epidermal DETCs was increased in EPI−/− skin ( Figure 8E ) .", "Macrophage numbers were unaffected by TSLP or NKG2D blockade ( data not shown ) . 10 . 7554/eLife . 01888 . 010Figure 8 . Effects of TSLP and NKG2D inhibition and quantitation of cytokine levels in SCCs .", "( A–E )", "CD4+ T cells ( A ) , mast cells ( B ) , eosinophils ( C ) , pustules ( D ) , and γδ T cells ( E ) per mm epidermis or mm2 dermis .", "Data are means ± SEM from at least three mice per genotype .", "( F and G )", "Serum ( F ) and whole skin ( G ) protein levels of the cytokines indicated .", "Data are means ± SEM from 6 IgG and 3 α-TSLP-treated mice .", "( H and I )", "Q-PCR of mRNAs indicated in SCCs .", "Data are means ± SEM of 4 SCCs per genotype .", "( J ) Serum levels of TSLP in EPI−/− mice bearing papillomas smaller than 2 mm2 or at least one papilloma larger than 2 mm2 .", "Data are means ± SEM of at least three mice per group . DOI: http://dx . doi . org/10 . 7554/eLife . 01888 . 010 To examine whether the anti-inflammatory effects of blocking TSLP were mediated by down-regulating epidermal expression of inflammatory mediators ( Figure 5A ) , we measured expression of representative type 1 and type 2 cytokines .", "TPA treatment induced a marked and significant release of type 2 cytokines ( IL-4 , IL-6 ) in the skin and serum of EPI−/− mice , and this was diminished by systemic administration of anti-TSLP antibodies ( Figure 8F , G ) .", "In contrast , there was no effect on the type 1 cytokines , TNFα and IFNγ .", "Thus , the inflammation-suppressive effects of systemic TSLP inhibition were mediated at the level of both local and systemic suppression of inflammatory mediator production .", "EPI−/− SCCs expressed higher TSLP levels than WT SCC ( Figure 8I ) , whereas there were no significant differences in IL-4 and IL-17 ( Figure 8H ) .", "In addition , EPI−/− mice bearing papillomas larger than 2 mm2 had higher levels of systemic TSLP than EPI−/− mice with smaller papillomas ( Figure 8J ) .", "This indicates that when tumours do arise in an atopic environment , TSLP may have a tumour growth-promoting role .", "This is in contrast to the reported ability of TSLP to shrink tumours in Notch-deficient skin ( Demehri et al . , 2012 ) , but in agreement with the potent tumorigenic effect of TSLP in breast and pancreatic cancers ( De Monte et al . , 2011; Pedroza-Gonzalez et al . , 2011 ) .", "We conclude that targeting the Rae1/NKG2D/TSLP axis reverts the atopic phenotype observed in EPI−/− mice upon TPA treatment by interfering with the crosstalk between keratinocytes and immune cells ." ], [ "Mice deficient in Evpl , Ppl and Ivl exhibited a striking resistance to benign tumour formation .", "This was not due to a difference in DMBA-induced initiation , indicating that the tumour-protective effect was not mediated by cell-autonomous changes associated with acquisition of oncogenic mutations .", "Rather , short-term TPA treatment exacerbated atopic dermatitis in EPI−/− mice , resulting in a phenotype that shared key hallmarks of the acute phase of the disease in humans ( Bieber , 2010 ) ( Figure 9 ) .", "These hallmarks included epidermal acanthosis , spongiosis , hyperkeratosis , and parakeratosis , systemic type 2 cell expansion with a dramatic accumulation of CD4+ T cells , neutrophils , eosinophils and dermal mast cells , and elevation of serum IgE and TSLP levels . 10 . 7554/eLife . 01888 . 011Figure 9 . Model of the role of an epidermal barrier defect in tumour protection . EPI−/− mice lack three cornified envelope proteins , resulting in a defective epidermal barrier .", "Topical application of DMBA induces H-Ras mutations , as in wild-type mice .", "Topical TPA treatment elicits an exaggerated atopic response , characterized by altered keratinocyte differentiation and an enhanced inflammatory response .", "Epidermal production of TSLP and activation of the ligand-NKG2D pathway on immune cells are proposed to contribute to tumour protection . DOI: http://dx . doi . org/10 . 7554/eLife . 01888 . 011 The enhanced thickening of TPA-treated EPI−/− epidermis reflected an increase in the number of suprabasal , differentiated cell layers and defective desquamation .", "Proliferation in the basal layer was similar to wild type , but the transit time of cells through the epidermis was faster .", "These changes in differentiation have previously been shown to be tumour-suppressive in oncogene-driven skin carcinogenesis ( Guinea-Viniegra et al . , 2012 ) , consistent with our observation that EPI−/− mice are resistant to developing papillomas .", "Upon TPA treatment EPI−/− defective keratinocytes released high levels of chemokines and type 2-type 17 cytokines , including TSLP .", "This correlated with recruitment of innate immune cells , including mast cells , granulocytes ( both neutrophils and eosinophils ) and adaptive immune cells , such as CD4+ T cells , which could potentially play a role in immuno-editing of nascent papillomas .", "Defective epidermal differentiation in concert with activation of innate and adaptive immune responses is known to create the first line of defence against physical , chemical , and toxic assaults in the skin ( Milstone , 2004; Sherman et al . , 2008 ) .", "Key players in the crosstalk between keratinocytes and leukocytes are DETCs , since they orchestrate the inflammatory response to pathogen-derived and environmental signals ( King et al . , 1999; Moore et al . , 2000 ) .", "DETCs are already in an activated state in untreated EPI−/− epidermis , as demonstrated by their round shape and reduced number ( Sevilla et al . , 2007 ) .", "This is most likely a consequence of increased expression of keratinocyte stress signals ( Rae-1 and H60 ) , which would be responsible for establishing the observed Th2 immune-surveillance response .", "We propose that this pre-activated , ‘alarmed’ condition enables EPI−/− skin to react more promptly and strongly to TPA treatment than WT skin .", "In contrast to EPI−/− mice , in mice that overexpress activin βA in the epidermis downregulation of DETCs only occurs after DMBA/TPA treatment and is linked to increased skin carcinogenesis ( Antsiferova et al . , 2011 ) .", "The exaggerated TPA response in EPI−/− mice resulted in increased numbers of CD4+ T cells , neutrophils , mast cells , and eosinophils , and many of these leukocyte populations have been shown to be able to exert tumour protective effects , which could potentially bypass the function of γδ T cells .", "Targeting the Rae-1/NKG2D/TSLP axis , by blocking TSLP or NKG2D , suppressed the crosstalk between keratinocytes and immune cells and abrogated the atopic phenotype of TPA-treated EPI−/− skin , as seen by normalisation of epidermal differentiation and inflammation .", "In mice , epidermal overexpression of TSLP results in an atopic dermatitis phenotype characterized by epidermal hyperproliferation , acanthosis , spongiosis , and hyperkeratosis , as well as mast cell infiltration in the dermis ( Yoo et al . , 2005 ) .", "In humans , TSLP is implicated in the pathogenesis of both atopic dermatitis and asthma ( Leonard , 2002; Al-Shami et al . , 2005; Zhou et al . , 2005; Huston and Liu , 2006; Liu , 2006 ) .", "TSLP has potent anti-tumour activity in skin carcinogenesis ( Demehri et al . , 2012; Di Piazza et al . , 2012 ) .", "Consistent with this , TPA-treated EPI−/− skin produced higher levels of TSLP than WT skin , and was protected from chemically induced papilloma formation .", "NKG2D ligands have been shown to be upregulated in many inflammatory lesions and can act as an axis of immunoregulation ( Strid et al . , 2008 ) .", "Our findings demonstrate that the proposed role for Rae-1 and NKG2D in establishment of the lymphoid stress-surveillance response and consequent atopic phenotype also applies to the situation in which there is a defective epidermal barrier .", "Although two-stage chemical skin carcinogenesis has been used extensively to elucidate the role of individual components of the immune system ( specific cell types , cytokines and signalling molecules ) , ours is the first mouse model to link the lack of structural proteins and consequent defective epidermal barrier to cancer susceptibility .", "Epidemiological studies of the association between allergic disease and cancer risk have given conflicting results , in part because of the difficulty of factoring in immune suppressive treatments for the disease and in part because the association can be positive or negative according to cancer type ( Arana et al . , 2010; Van Hemelrijck et al . , 2010; Wiemels et al . , 2011; Jensen et al . , 2012; Kaae et al . , 2013 ) .", "Our study provides a mechanistic framework for further analysis of the link between atopic dermatitis and cancer incidence ." ], [ "Mice deficient for envoplakin , periplakin , and involucrin ( EPI−/− ) were generated as previously described ( Sevilla et al . , 2007 ) by interbreeding single knockout mice for envoplakin ( Maatta et al . , 2001 ) , involucrin ( Djian et al . , 2000 ) , and periplakin ( Aho et al . , 2004 ) .", "Single knockout mice were generated and maintained on a variety of genetic backgrounds and as a result there was a potential contribution of Sv129 , C57BL/6 , BALB/c and nu/nu to EPI−/− animals .", "Since DMBA/TPA sensitivity is affected by genetic background ( Hennings et al . , 1993; Woodworth et al . , 2004 ) , DNA of 4 EPI−/− mice ( each from a different breeding pair ) was subjected to genome scanning by the Jackson Laboratory ( Bar Harbor , ME , USA ) .", "Single nucleotide polymorphisms ( SNPs ) were used to determine the relative contributions of different genetic backgrounds .", "EPI−/− mice were primarily Sv129 ( 40 . 98% , ±1 . 52 ) and C57Bl/6 ( 51 . 39% , ±1 . 62 ) , with a small contribution of BALB/c ( 4 . 7% , ±1 . 24 ) .", "The nu/nu mutation was not detected .", "Based on these results , the F2 generation of crosses between Sv129 and C57Bl/6J mice was used as the WT control ( 51 . 82% , ±3 . 35 Sv129; 49 . 92% , ±1 . 98 C57Bl/6 ) for all experiments .", "Chemical carcinogenesis experiments were performed as previously described ( Abel et al . , 2009 ) .", "All experiments were carried out under the terms of a UK Government Home Office project licence , with local ethical approval .", "The back skin of 7-week-old female mice was shaved with electric clippers .", "3 days later animals that did not show signs of hair regrowth received a topical application of 100 nmol ( 25 μg ) DMBA ( Sigma–Aldrich ) in 200 μl acetone , followed by three times weekly applications of 6 nmol ( 3 . 7 μg ) of TPA ( Sigma-Aldrich , Dorset , UK ) in 200 μl acetone for 15 weeks .", "As controls , mice ( 5–10 animals/group ) were subjected to the same protocol , but substituting DMBA or TPA with acetone .", "Papillomas and SCCs were recorded once a week for up to 57 weeks after the start of promotion .", "Recordings were made by an observer who was ‘blinded’ to the experimental groups .", "1 hr before culling , mice were injected intraperitoneally with 50 mg/kg bodyweight 5-bromo-2'-deoxyuridine BrdU ( Sigma-Aldrich ) .", "In some experiments , age and gender matched mice received 1 application of DMBA and/or 3 applications of TPA on alternating days .", "Skin was harvested 24 hr after DMBA painting or 48 hr after the last TPA treatment .", "For paraffin sections , tissue was fixed in 10% neutral buffered formalin for 24 hr and embedded in paraffin after dehydration .", "For frozen sections , tissue was submerged in OCT embedding matrix ( Raymond A Lamb , UK ) and frozen on dry ice .", "4–6 μm ( paraffin or frozen ) sections were prepared and collected onto glass slides .", "Quantitation of the proportion of the epidermis with a parakeratotic or hyperkeratotic stratum corneum was performed on hematoxylin-eosin stained paraffin sections .", "A minimum epidermal length of 7 cm was analysed from ≥4 mice per condition .", "Parakeratosis was defined as retention of nuclei in the cornified layers .", "Hyperkeratosis was defined as abnormal thickening of the stratum corneum relative to wild type , untreated stratum corneum .", "BrdU , PH3 , and Caspase-3 were detected in formalin-fixed paraffin-embedded sections .", "Sections were dewaxed and rehydrated on an automated Leica ST5020 , then treated for antigen-retrieval and incubated with primary antibody at the indicated dilution .", "Secondary antibodies were diluted 1:250 and probed using the Bond Intense R Detection kit ( Leica Microsystems; Wetzlar , Germany ) .", "Sections were scanned and analysed using the Ariol SL-50 system ( Applied Imaging Corp . , San Jose ) .", "Prior to immunofluorescence staining , frozen sections were air-dried for 10 min at room temperature and fixed in 4% PFA/PBS pH 7 . 4 for 10 min .", "Sections were blocked with 2% BSA , 0 . 02% fish skin gelatin , and 10% goat serum for 1 hr in PBS at room temperature .", "Sections were incubated in unconjugated or fluorochrome-conjugated primary antibody and DAPI overnight at 4°C , washed in PBS and then mounted using DAKO mounting reagent ( DAKO ) .", "AlexaFluor 488- or 633-conjugated goat anti-rabbit or anti-mouse IgG ( Invitrogen ) were diluted 1:1000 and used for detection of unconjugated primary antibodies .", "The following species-specific antibodies conjugated with Alexafluor 488 or 555 or 647 were used at a dilution of 1:100: anti-CD3 ( clone 17 . A2; BDPharmingen ) , anti-CD4 ( clone RM4-5; eBioscience ) , anti-CD8α ( clone 53-6 . 7; BioLegend ) , anti-γδ TCR ( clone GL3; BDPharmingen ) , anti-F4/80 ( clone BM8; eBioscience ) , anti-Ly6G ( clone 1A8; BDPharmingen ) , anti-CD207 ( clone eBioRMUL . 2; eBioscience ) and anti-CD11b ( clone M1/70; eBioscience ) .", "In-house produced antibodies against periplakin , envoplakin and involucrin were also used ( Ruhrberg et al . , 1996 , 1997; Sevilla et al . , 2007 ) .", "The following species-specific unconjugated antibodies were used at the dilutions stated: anti-PH3 ( Upstate rabbit polyclonal , catalogue number 06-570 , 1:500 ) , anti-BrdU ( Abcam sheep polyclonal , ab1893 , 1:500 ) , anti-caspase-3 ( Cell Signaling rabbit monoclonal , catalogue number 9664 , 1:100 ) and anti-γH2AX ( 05-636; 1;10; Millipore0 ) .", "Mast cells were detected with toluidine blue , and eosinophils were detected by Congo red staining .", "Hematoxylin and eosin–stained sections of skin and tumours were graded in a blinded manner by two experts in mouse skin pathology , as described previously ( Owens and Watt , 2001 ) .", "When quantifying the number of cells that were positive for a particular marker , at least 6 fields per treated skin or tumour section were analysed .", "RNA was extracted from whole skin , tumours or epidermis that had been scraped from skin following heat treatment for 60 s at 56°C in PBS +10 mM EDTA or from cultured keratinocytes using the RNeasy mini kit ( Qiagen , UK ) .", "RNA was quantified using a ND-100 NanoDrop spectrophotometer ( NanoDrop Technologies ) .", "cDNA was prepared using a Superscript III First-Strand Synthesis Supermix for qRT-PCR kit ( Invitrogen ) according to the manufacturers' instructions .", "Quantitative RT-PCR was carried out using designed primers and fast SYBR Green or Taqman probes with Taqman Fast Universal PCR Master Mix ( Applied Biosystems ) and run on an ABI Prism 7900HT Sequence detection system ( Applied Biosystems ) .", "The RT-PCR was run for 40 cycles and specificity of the reactions was determined by subsequent melting curve analysis .", "Quantitation was based on ΔCt calculations using the SDS analysis software and all samples were compared against mouse GAPDH as a house keeping control .", "Matrix visualization was performed using the online Matrix2PNG tool ( Pavlidis and Noble , 2003 ) .", "Primers and TaqMan probes are listed in Supplementary file 1 .", "Genomic DNA was isolated from SCCs using the NucleoSpin kit ( Macherey–Nagel ) as per the manufacturer's protocol .", "PCR amplification of HRas and p53 exons of interest was performed on 100 ng of DNA using primers described previously ( Niemann et al . , 2007 ) .", "PCR products were purified and sequenced .", "To quantify the number of DMBA-induced codon-61 Hras CAA->CTA mutations , mutant-specific primers were used as previously described ( Modi et al . , 2012 ) .", "Obtained Ct values were normalised against genomic GAPDH ( gGAPDH ) .", "Mice were anesthetized with Isoflurane ( Anaesthesia , UK ) and then blood for differential white blood cell counts was collected by cardiac puncture in the presence of EDTA ( 1 . 75 mg of EDTA dihydrate solution per ml of blood ) to prevent coagulation .", "For plasma protein analysis , blood was allowed to clot at room temperature for at least 1 hr .", "After centrifugation at 14 , 000 rpm for 15 min at 4°C the serum was collected , aliquoted , and kept at −80°C until analysis .", "Whole skin lysates and serum were collected , snap-frozen in liquid nitrogen and kept at −80°C prior to analysis .", "The Cytometric Bead Array Cytokine Flex Kit ( BD ) was used following the manufacturer's instructions .", "Samples were run on a LSR-II flow cytometer and the data were analysed with BD Cytometric Bead Array software .", "Cytokine production was determined as picograms per ml serum or mg of whole skin lysate .", "Epidermal sheets were prepared from mouse ears that had been split into dorsal and ventral sides and floated dermal side down for 2 hr at 37°C in 20 mM EDTA .", "Epidermal sheets were gently lifted from the dermis , washed in PBS and fixed in cold acetone for 20 min at −20°C .", "After washing in PBS , epidermal sheets were incubated for 1 hr at room temperature with 2% ( g/vol ) BSA in PBS and stained overnight at 4°C with CD207 Alexa 647 conjugated antibody ( eBioscience , clone eBioRMUL . 2 ) and DAPI .", "After extensive washing , epidermal sheets were mounted on slides with DAKO mounting medium and visualized by confocal microscopy .", "Functional grade purified antibodies against CD4 ( 16-0042 , clone RM4-5 and clone GK1 . 5 , rat IgG2bK; eBioscience ) , TSLP ( MAB555 , clone 152614; R&D System , 16-5491 , clone eBio28F12; eBioscience ) , NKG2D ( catalogue number BE0111; BioXCell ) , LY6G ( clone 1A8; BioXCell ) , IL-4 ( catalogue number BE0045; BioXCell ) , and appropriate isotype controls ( BioXCell Rat IgG2a , catalogue number BE0089 for TSLP and Ly6G; Rat IgG2b , catalogue number BE0090 for CD4; hamster IgG catalogue number BE0091 for NKG2D; Rat IgG1 , catalogue number BE0088 for IL-4 ) were injected intraperitoneally at a dose of 0 . 1 mg/mouse .", "Dexamethasone 21-phosphate disodium salt ( DXM ) ( D1159; Sigma ) was injected at 10 mg/kg mouse body weight , the same volume of PBS being injected in control mice .", "TSLP ( Quantikine mouse TSLP kit , R&D Systems ) , IL-4 ( ELISA IL-4 Ready-set-go , eBiosciences ) , and IgE ( Bethyl Laboratory ) immunoassays were performed on mouse serum according to the manufacturer's instructions .", "Whole skin protein lysates were prepared by mechanical lysis using ceramic beads ( Precellys ) in NP-40 buffer ( 50 mM Tris–HCl , 150 mM NaCl , 1% NP-40 , pH . 7 . 4 ) with complete protease inhibitor cocktail ( Roche , Burgess Hill , UK ) .", "Tissue was homogenized twice at 1600 rpm for 50 s , then centrifuged for 20 min 200 µl aliquots were snap frozen and kept at −80C .", "Statistical analyses were performed using GraphPad Prism ( GraphPad Software ) .", "The statistical significance of differences between most experimental groups was determined with a two-tailed Student's t-test for unpaired data or Fisher's exact test .", "For analysis of differences between WT and EPI−/− mouse tumour burden Mann Whitney testing was performed .", "For analysis of differences between WT and EPI−/− mouse tumour incidence a test of difference in proportions was used .", "p-values are indicated with: * 0 . 01<p<0 . 05 , ** 0 . 01<p<0 . 001 , *** 0 . 0001<p<0 . 001 , ****p<0 . 0001 ." ] ]

[ "Atopic dermatitis can result from loss of structural proteins in the outermost epidermal layers , leading to a defective epidermal barrier .", "To test whether this influences tumour formation , we chemically induced tumours in EPI−/− mice , which lack three barrier proteins—Envoplakin , Periplakin , and Involucrin .", "EPI−/− mice were highly resistant to developing benign tumours when treated with 7 , 12-dimethylbenz ( a ) anthracene ( DMBA ) and 12-O-tetradecanoylphorbol-13-acetate ( TPA ) .", "The DMBA response was normal , but EPI−/− skin exhibited an exaggerated atopic response to TPA , characterised by abnormal epidermal differentiation , a complex immune infiltrate and elevated serum thymic stromal lymphopoietin ( TSLP ) .", "The exacerbated TPA response could be normalised by blocking TSLP or the immunoreceptor NKG2D but not CD4+ T cells .", "We conclude that atopy is protective against skin cancer in our experimental model and that the mechanism involves keratinocytes communicating with cells of the immune system via signalling elements that normally protect against environmental assaults ." ]

[ "Skin cancer is a common and growing problem—according to the World Health Organization , skin cancers account for one in every three cancers diagnosed world wide .", "There is some evidence from epidemiological studies that patients with certain allergies might be protected against cancer and , in particular , that the allergic skin condition atopic dermatitis is associated with reduced levels of various skin cancers .", "However , it is difficult to know if this reduction is due to the atopic dermatitis itself or to the drugs used to treat this allergy .", "Genetically engineered mice that are lacking three proteins that are involved in the formation of the cornified envelope—the protective layer that replaces the normal plasma membrane in the cells of the outermost skin layers—can be used to study atopic dermatitis .", "These ‘triple knockout mice’ have a defective epidermal barrier and altered levels of immune T-cells in the skin .", "Now Cipolat et al . have investigated whether defects in the epidermal barrier protect against skin cancer .", "Knockout mice and wild-type mice were treated with two chemicals: DMBA , which causes mutations in a gene called HRas , and TPA , which promotes the formation of tumours from cells that contain HRas mutations .", "After about 16 weeks almost all of the wild-type mice had at least one benign tumour , whereas half of the knockout mice had no tumours .", "Overall , the average number of benign tumours per mouse was six times higher in the wild-type mice .", "This shows that the mutations that cause the epidermal barrier defects in knockout mice also protect them against the tumours caused by the combined effects of DMBA and TPA .", "Cipolat et al . then compared how the mice responded to DMBA or TPA alone .", "The knockout mice and the wild-type mice responded to DMBA in the same way; however , the knockout mice showed an exaggerated response to TPA , including a strong inflammatory reaction .", "This response comprised the production of higher levels of various proteins that are involved in communications between skin cells and the immune system .", "Cipolat et al . propose that the immune reaction caused by this exaggerated response could help to prevent tumour formation by eliminating tumour-forming cells in the skin ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "biochemistry and chemical biology", "cell biology" ]

[ [ "Protein folding homeostasis ( proteostasis ) is achieved by balancing the rate of production , folding and protein degradation .", "Proteostasis is strongly influenced by the phosphorylation state of serine 51 of the α subunit of eukaryotic translation initiation factor 2 ( eIF2α ) ( Sonenberg and Hinnebusch , 2009 ) .", "Diverse stress conditions activate kinases that phosphorylate eIF2α , resulting in attenuated rates of translation initiation of most mRNAs and increasing translation of a small group of mRNAs with special 5′ untranslated regions ( Hinnebusch , 2014 ) .", "The latter encode potent transcription factors such as ATF4 that couple eIF2α phosphorylation to the Integrated Stress Response ( ISR ) ( Harding et al . , 2003 ) , a transcriptional and translational program that adapts cells to stress and participates in diverse biological processes such as memory , immunity and metabolism ( Baird and Wek , 2012 ) .", "Signalling in the ISR is terminated by eIF2α-P dephosphorylation .", "This requires the presence of a regulatory subunit , PPP1R15 , to direct the catalytic , PP1 subunit , to its specific substrate .", "Two mammalian genes encode PPP1R15 regulatory subunits .", "Ppp1r15b ( or CReP ) encodes a constitutively expressed regulatory subunit ( Jousse et al . , 2003 ) , whereas Ppp1r15a ( or GADD34 ) encodes an ISR inducible regulatory subunit that contributes to a negative feed-back loop operative in the ISR ( Brush et al . , 2003; Ma and Hendershot , 2003; Novoa et al . , 2001 , 2003 ) .", "A minimal rate of eIF2α-P dephosphorylation is an essential cellular function , as reflected in the severe phenotypes of Ppp1r15b mutation or deletion and in the very early lethality of compound Ppp1r15a;b deficient mice ( Abdulkarim et al . , 2015; Harding et al . , 2009 ) .", "Interestingly , whilst deletion of the inducible Ppp1r15a gene results in sluggish recovery of protein synthesis during the waning phase of stress ( Kojima et al . , 2003; Novoa et al . , 2003 ) , mice lacking any PPP1R15A-directed eIF2α-P dephosphorylation ( homozygous Ppp1r15atm1Dron ) are superficially indistinguishable from wildtype .", "Moreover , when challenged with tunicamycin , which causes unfolded protein stress in the endoplasmic reticulum by inhibiting N-linked glycosylation , homozygous Ppp1r15atm1Dron mice and cultured cells derived from them are relatively resistant to the toxin’s lethal effects ( Marciniak et al . , 2004; Reid et al . , 2016 ) .", "This feature is plausibly attributed to sustained activity of the ISR in the Ppp1r15a mutant mice , which favours proteostasis by limiting the production of unfolded proteins under stress conditions ( Boyce et al . , 2005; Han et al . , 2013 ) .", "The proteostasis-promoting features of interfering with PPP1R15A-mediated eIF2α-P dephosphorylation are also played out in the context of certain disease models associated with protein misfolding and proteotoxicity .", "Both the neuropathic phenotype associated with Schwann cell expression of a mutant misfolding-prone myelin constituent , P0S63∆ , and a mutant superoxide dismutase expressed in motor neurones are ameliorated by a concomitant dephosphorylation-defective Ppp1r15atm1Dron mutation ( D'Antonio et al . , 2013; Wang et al . , 2014 ) , and similar amelioration of inflammatory-mediated central nervous system demyelination is observed in the Ppp1r15atm1Dron mice ( Lin et al . , 2008 ) .", "These features have led to an interest in the therapeutic potential of targeting PPP1R15-mediated eIF2α-P dephosphorylation with small molecule inhibitors .", "Early work led to discovery of salubrinal , a small molecule that increases levels of eIF2α-P and retards its dephosphorylation .", "However , salubrinal is only known to work in vivo and its mechanism of action remains unclear ( Boyce et al . , 2005 ) .", "Limitations of in vitro assays for substrate-specific PPP1R15-mediated eIF2α-P dephosphorylation ( see below ) have all but precluded a biochemical approach to the problem , but a cell based search for proteostasis regulators suggested that the α2 adrenergic blocker Guanabenz , [ ( o , o-dichlorobenzylidene ) amino]guanidine , might exert its beneficial effects on proteotoxicity by interfering with eIF2α-P dephosphorylation ( Tsaytler et al . , 2011 ) .", "This theme was extended further by the discovery of Sephin1 , [ ( o-chlorobenzylidene ) amino]guanidine , that had lost its α2 adrenergic blocking activity but retained its proteostasis promoting properties ( Das et al . , 2015 ) .", "Importantly , biochemical characterization of Guanabenz and Sephin1 suggested that both disrupt the complex between PPP1R15A and PP1 , providing strong support for a mechanism of action that involves interfering with PPP1R15A-mediated eIF2α-P dephosphorylation ( Das et al . , 2015 ) , Figure 1C therein ) . 10 . 7554/eLife . 26109 . 003Figure 1 . A tripartite assay for human PPP1R15A-dependent eIF2α-P dephosphorylation .", "( A ) Cartoon representation of human PPP1R15A ( GADD34 ) .", "The minimal C-terminal peptide required for eIF2α-P dephosphorylation is outlined ( ‘active fragment’ ) and key residues in the PP1 and G-actin binding regions are annotated .", "( B ) Alignment of C-terminal active fragments of mammalian PPP1R15A and PPP1R15B ( CREP ) using ClustalX .", "Grey highlighted residues represent conserved or highly similar residues .", "Red asterisks highlight key residues that are analysed in further detail .", "( C ) Image of Coomassie-stained PhosTag-SDS-PAGE on which phosphorylated ( eIF2αP ) and non-phosphorylated ( eIF2α0 ) forms of eIF2α from 20 min dephosphorylation reactions were resolved .", "The composition of the reaction , PPP1R15A533-624 [80 nM] , PPP1R15A325-636 [400 nM] , PP1c [12 . 5 nM] and G-actin [750 nM] are noted above and the fraction of dephosphorylated eIF2α is noted underneath each lane ( %dP ) .", "The migration of molecular weight markers and G-actin are noted ( the signal from the PPP1R15A and PP1c is undetectable on these gels ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 26109 . 003 Genetic analysis reveals that a regulatory PPP1R15 subunit is essential for eIF2α-P dephosphorylation in vivo , and over-expression of either PPP1R15A or PPP1R15B or merely their conserved C-terminal portion , is sufficient to deregulate eIF2α-P dephosphorylation in vivo and inhibit the ISR ( Brown et al . , 1997; Brush et al . , 2003; Jousse et al . , 2003; Novoa et al . , 2001 ) .", "Though PPP1R15 regulatory subunits stably bind the catalytic subunit ( PP1 ) , the resulting binary complex is devoid of specificity towards eIF2α-P .", "However , G-actin joins the PPP1R15-PP1 binary complex as an ancillary subunit to form a ternary complex endowed with substrate-specific eIF2α-P directed phosphatase activity , both in cells ( Chambers et al . , 2015 ) and when constituted with pure components in vitro ( Chen et al . , 2015 ) .", "To explore in detail the mechanism of action of the [ ( o-chlorobenzylidene ) amino]guanidines we reconstructed PPP1R15A-PP1-G-actin-mediated eIF2α-P dephosphorylation in vitro with pure components .", "Using mutants that interfere with complex formation and function we established the correlation of enzymatic activity with the kinetic parameters of complex formation to develop an assay responsive to the stability of the core PPP1R15-PP1 binary interaction; the proposed target of Guanabenz and Sephin1 .", "The results of our inquiry , reported on below , question the role of destabilization of the eIF2α-P directed holophosphatase in the proteostatic effects of these compounds ." ], [ "PPP1R15A/GADD34 is a protein of >600 residues , but only the C-terminal 70 residues are necessary for substrate-specific dephosphorylation of eIF2α-P ( Figure 1A ) .", "This active fragment is also the most conserved segment of the protein; both between homologues of PPP1R15A and with the paralogous PPP1R15B ( Figure 1B ) .", "This region of the protein is natively unfolded ( Yu et al . , 2004 ) , attaining its structure upon binding the PP1 catalytic and the G-actin ancillary subunits ( Chen et al . , 2015; Choy et al . , 2015 ) .", "For structural studies we found it convenient to co-express PPP1R15 and PP1 in bacteria ( Chen et al . , 2015 ) .", "However , the fixed 1:1 stoichiometry of the two subunits imparted by co-expression is unsuited to a detailed examination of the bimolecular affinities involved in complex formation or to the design of an assay sensitive to the stability of PPP1R15A-PP1 complex .", "To circumvent this limitation , we incorporated a highly soluble maltose-binding protein ( MBP ) moiety C-terminal to the natively-unstructured human PP1R15A active fragment .", "When expressed in E . coli as a fusion protein with a cleavable N-terminal glutathione S-transferase ( GST ) tag , GST-PPP1R15A-MBP remained soluble and when added as a purified protein in vitro ( after cleavage of the GST ) , imparted eIF2α-P dephosphorylation activity to reactions containing purified PP1 and G-actin ( Figure 1C , left panel ) .", "Moreover , the solubilizing MBP tag enabled recovery not only of a human PPP1R15A active fragment ( residues 533–624 ) but also a much larger N-terminally extended fragment ( residues 325–636 ) .", "The minimal active fragment and the much longer N-terminally-extended PPP1R15A had similar activity in this assay ( Figure 1C ) .", "Nonetheless the ability to purify a soluble , N-terminally extended PPP1R15A regulatory subunit expanded the possibilities to study more physiological models of PPP1R15A-mediated eIF2α-P dephosphorylation ( a point we shall return to below ) .", "We quantified the dependence of eIF2α-P dephosphorylation rates on both the concentration of the regulatory human PPP1R15A subunit ( EC50 = 7 nM ) and on the ancillary G-actin subunit ( EC50 = 13 nM ) ( Figure 2 ) .", "The latter values agreed with our previous measurements of G-actin’s stimulation of enzymatic activity ( in an assay using the murine PPP1R15A ) ( Chen et al . , 2015 ) , whereas the EC50 of human PPP1R15A was within an order of magnitude of the affinity of human PPP1R15A for PP1 , as measured by isothermal titration calorimetry ( Choy et al . , 2015 ) ( see below ) . 10 . 7554/eLife . 26109 . 004Figure 2 . eIF2α-P dephosphorylation kinetics as a function of human PPP1R15A533-624 and G-actin concentration .", "( A ) Schema of the human PPP1R15A533-624 construct used .", "The C-terminal Maltose Binding Protein ( MBP ) component , which stabilizes the fusion protein , is noted .", "( B ) Upper panel .", "Coomassie-stained PhosTag-SDS-PAGE tracking the dephosphorylation of eIF2αP to eIF2α0 in 20 min dephosphorylation reactions constituted with eIF2αP [2 µM] , PP1 [0 . 625 nM] , G-actin [1 . 5 µM] and an escalating concentration of PPP1R15A533-624 .", "Shown is a representative of three independent experiments performed .", "Lower panel: Semi-log10 plot of the initial velocity of eIF2αP dephosphorylation as a function of PPP1R15A533-624 concentration derived from three repeats ( one shown above ) .", "The EC50 for PPP1R15A533-624 was calculated using the agonist fitting function on GraphPad Prism V7 .", "( C ) Upper panel .", "As in ‘B’ but dephosphorylation of eIF2αP to eIF2α0 was carried out in the presence of a fixed concentration of PPP1R15A533-624 [50 nM] and an escalating concentration of G-actin .", "Shown is a representative of two independent experiments performed .", "Lower panel: Semi-log10 plot of initial velocity as a function of G-actin concentration derived from two repeats ( one shown above ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 26109 . 004 Previous studies have identified mutations in PPP1R15 that abolish substrate-specific dephosphorylation in vitro and block PPP1R15’s ability to repress the ISR , when expressed in vivo .", "The human PPP1R15AV556E mutation alters a key residue , part of the RVxF motif involved in binding of diverse regulatory subunits to PP1 ( Egloff et al . , 1997 ) ; its presence abolished all PPP1R15A-mediated eIF2α-P dephosphorylation ( Figure 3A ) .", "Two previously-identified mutations in the C-terminal extension of PPP1R15A - the portion that interacts with the G-actin ancillary subunit ( human PPP1R15AW582A and PPP1R15AF592A ) ( Chen et al . , 2015 ) - also abolished all PPP1R15A-mediated eIF2α-P dephosphorylation ( Figure 3B and C ) .", "These findings establish the dependence of the tripartite assay described above on features known to be important for PPP1R15 function . 10 . 7554/eLife . 26109 . 005Figure 3 . eIF2α-P dephosphorylation by ternary complexes constituted with human PPP1R15A ( 533-624 ) V556E , PPP1R15A ( 533-624 ) W582A or PPP1R15A ( 533-624 ) F592A .", "( A ) Coomassie-stained PhosTag-SDS-PAGE tracking the dephosphorylation of eIF2αP to eIF2α0 as in Figure 2 above , but with PP1[32 nM] , G-actin [400 nM] and an escalating concentration of mutant human PPP1R15A ( 533-624 ) V556E .", "Shown is a representative of three independent experiments performed .", "The position of the mutation is provided in the schema above .", "The plot of initial velocity as a function of PPP1R15A ( 533-624 ) V556E derived from three repeats ( one shown ) is below the SDS-PAGE image .", "( B ) As in ‘A’ above but using human PPP1R15A ( 533-624 ) W582A and G-actin [3 . 7 µM] ( Note: only the highest concentration of PPP1R15A was repeated three times ) .", "( C ) As in ‘A’ above but using human PPP1R15A ( 533-624 ) F592A and G-actin [3 . 7 µM] ( Note: only the highest concentration of PPP1R15A was repeated three times ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 26109 . 005 A fourth mutation tested affects a residue whose counterpart in human PPP1R15BR658C results in a syndromatic form of diabetes mellitus .", "Consistent with the destabilizing effect of this mutation on PP1 binding ( Abdulkarim et al . , 2015 ) , its presence in human PPP1R15AR578A resulted in a ~4 fold increase in EC50 for eIF2α-P dephosphorylation ( Figure 4A ) .", "The mutation also affected the maximal stimulation afforded by human PPP1R15AR578A , as even at saturating concentrations of regulatory subunit , eIF2α-P dephosphorylation reactions assembled with the mutant were three times slower than those assembled with the wildtype ( Figure 4B ) .", "The human PPP1R15AR578A mutation does not appear to have a major effect on the stability of the G-actin containing ternary complex , as the EC50 for G-actin ( 20 nM ) was relatively unaffected ( Figure 4C ) . 10 . 7554/eLife . 26109 . 006Figure 4 . eIF2α-P dephosphorylation by ternary complexes constituted with human PPP1R15A ( 533-624 ) R578A .", "( A ) Coomassie-stained PhosTag-SDS-PAGE tracking the dephosphorylation of eIF2αP in a 20 min reaction , as in Figure 2 and 3 above , but with an escalating concentration of mutant human PPP1R15A ( 533-624 ) R578A .", "Shown is a representative of three independent experiments performed .", "The position of the mutation is provided in the schema above the gel .", "The plot of initial velocity as a function of PPP1R15A ( 533-624 ) R578A derived from three repeats ( one shown ) is below the SDS-PAGE image .", "The EC50 for PPP1R15A ( 533-624 ) R578A was calculated using the agonist fitting curve in GraphPad Prism V7 .", "( B ) Time-course of eIF2αP dephosphorylation using a low concentration of PP1 [0 . 625 nM] , saturating concentrations of G-actin [400 nM] , and wildtype [100 nM] or mutant human PPP1R15A ( 533-624 ) R578A [100 nM in one assay and 200 nM in the two other assays] .", "Shown is a representative of three independent experiments performed .", "Below the gel is a plot of the fraction of substrate dephosphorylated as a function of time derived from three repeats ( one shown ) .", "The slope of the reaction was derived by fitting the data to a linear model in GraphPad Prism V7 .", "( C ) As in ‘A’ above but with saturating concentration of PPP1R15A ( 533-624 ) R578A [100 nM] and escalating concentration of G-actin .", "Shown is a representative of three independent experiments performed .", "The plot of initial velocity as a function of G-actin derived from three repeats ( one shown ) is below the SDS-PAGE image .", "The EC50 for G-actin was calculated using the agonist fitting curve in GraphPad Prism V7 . DOI: http://dx . doi . org/10 . 7554/eLife . 26109 . 006 The features of the human PPP1R15AR578A mutant noted above suggest that the tripartite assay is sensitive not only to the affinity of the three components for one another but also to subtle structural features of the holophosphatase .", "To explore this issue further , we used Bio-Layer Interferometry ( BLI ) ( Abdiche et al . , 2008 ) to measure directly the affinity of the three components of the holophosphatase for each other .", "PPP1R15A533-624 was biotinylated on a single lysine residue of an AviTag ( Fairhead and Howarth , 2015 ) added between the cleavable GST tag and PPP1R15A peptide ( Figure 5A ) and the biotinylated protein was immobilized on a streptavidin-derivatized BLI biosensor tip .", "The biotinylated PPP1R15A533-624 ligand showed a robust 1:1 bimolecular interaction , with pure PP1 , yielding a koff = 0 . 21 ± 0 . 01 min−1 and a Kd = 20 ± 0 . 61 nM ( Figure 5B ) .", "The higher affinity of PPP1R15 for PP1 observed here , compared to isothermal titration calorimetry ( ITC ) measurements of Choy et al . ( 2015 ) ( Kd = 62 ± 14 nM ) might reflect the contribution of contacts made by residues C-terminal to PPP1R15AL567 , which are present in the construct used here , but absent from the one used in the ITC measurements ( Choy et al . , 2015 ) .", "Cooperativity provided by G-actin ( present in the enzymatic assay , but absent from the BLI experiment ) and steric hindrance from probe components might have contributed to the 3–5 fold lower value of the PPP1R15A EC50 for eIF2α-P dephosphorylation in the enzymatic assay ( 7 nM , Figure 2B ) compared to the Kd observed by BLI . 10 . 7554/eLife . 26109 . 007Figure 5 . Affinity of the components of the tripartite holophosphatase for one another analysed by Bio-Layer Interferometry ( BLI ) .", "( A ) Schema of the biotinylated human PPP1R15A533-624 immobilized onto the BLI biosensor tip .", "( B ) Plot of Bio-Layer Interferometry ( BLI ) signal as a function of time in a representative experiment ( repeated three times ) in which immobilized PPP1R15A533-624 was reacted with PP1 [40 nM] in solution ( blue trace ) .", "The fitting curve using ‘association then dissociation’ model in GraphPad Prism V7 is shown in red .", "Vertical dashed line marks the beginning of the dissociation phase .", "Table summarizes kinetic parameters extracted from fitting curves of three repeats of the experiment shown in left panel ( mean ± standard deviation ) .", "( C ) As in ‘B’ above , but the immobilized PPP1R15A533-624 was first exposed to PP1 [200 nM] , before being exposed to a solution of both PP1 [200 nM] and G-actin [400 nM] .", "Shown is a representative of an experiment repeated three times . DOI: http://dx . doi . org/10 . 7554/eLife . 26109 . 007 To gauge the affinity of the ancillary G-actin subunit for the complex , we first assembled a binary complex between the biotinylated PPP1R15A533-624 ligand ( described above ) and a saturating concentration of PP1 and then measured the BLI signal induced by addition of G-actin .", "A robust association-dissociation signal was observed with purified G-actin ( koff = 2 . 84 ± 0 . 11 min−1 and Kd = 151 ± 14 . 3 nM ) ( Figure 5C ) .", "The human PPP1R15AV556E mutation , affecting the RVxF motif , abolished all measureable association with PP1 , but had no effect on the kinetics of G-actin binding .", "Conversely , the human PPP1R15AF592A mutation markedly enfeebled G-actin binding but had no effect on PP1 binding ( Figure 6A and B ) .", "Together , these observations confirm the ability of PPP1R15A to engage PP1 and G-actin independently , via the N- and C-terminal parts of its active portion .", "Despite their strong detrimental effects on enzymatic activity ( Figures 3C and 4 ) , neither the PPP1R15AR578A nor the PPP1R15AW582A mutations had a major effect on the kinetics of PP1 or G-actin binding ( Figure 6 ) .", "Together , these observations suggest that the tripartite enzymatic assay is sensitive both to mutations that grossly interfere with complex stability ( V556E and F592A ) and to mutations that more subtly affect the structure of the complex ( R578A and W582A ) . 10 . 7554/eLife . 26109 . 008Figure 6 . The effect of human PPP1R15A mutations on the affinity of the components of the tripartite holophosphatase for one another analysed by Bio-Layer Interferometry ( BLI ) .", "( A ) Plot of Bio-Layer Interferometry ( BLI ) signal as a function of time in a representative experiment ( repeated three times ) in which immobilized wildtype and indicated mutant PPP1R15A533-624 proteins were reacted with PP1 [40 nM] in solution ( thick traces ) .", "The fitting curve using ‘association then dissociation’ model in GraphPad Prism V7 is shown in thin red line .", "Vertical dashed line marks the beginning of the dissociation phase .", "Table summarizes kinetic parameters extracted from fitting curves of three repeats of the experiment shown in left panel ( mean ± standard deviation ) .", "( B ) As in ‘A’ above , but the immobilized wildtype and mutant PPP1R15A533-624 probes were first reacted with PP1 [200 nM] , before being exposed to a solution of both PP1 [200 nM] and G-actin [400 nM] .", "Shown is a representative of an experiment repeated three times . DOI: http://dx . doi . org/10 . 7554/eLife . 26109 . 008 Das and colleagues previously reported that addition of 50 µM Sephin1 to tissue culture media disrupts the PPP1R15A-PP1 complex recovered from cells ( Das et al . , 2015 ) .", "To determine if these observations correlate with an effect of Sephin1 on the complex formed in vitro between PPP1R15A533-624 and PP1 , we sourced Sephin1 and confirmed its purity and identity by reverse phase HPLC and mass spectrometry ( Figure 7A ) .", "When added to the BLI assay at a concentration of 50 µM ( before exposure to PP1 ) , Sephin1 had no measureable effect on either the association or dissociation phase of the assay ( Figure 7B ) . 10 . 7554/eLife . 26109 . 009Figure 7 . Sephin1’s effect on PP1-human PPP1R15A association analysed by Bio-Layer Interferometry ( BLI ) .", "( A ) From left to right: Absorbance trace ( at 254 nm ) of Sephin1 resolved by reverse-phase HPLC and mass spectra of the minor early ( blue ) peak eluting at 1 . 14 min and the major later eluting ( green peak ) at 1 . 83 min .", "The predicted structure , chemical formula and exact mass of Sephin1 , are provided for reference .", "( B ) Plot of BLI signal as a function of time of a representative experiment ( repeated three times ) performed with immobilized PPP1R15A533-624 ( Ligand ) and PP1 [40 nM] ( Analyte ) .", "Where indicated , the analyte was mixed with Sephin1 [50 µM ) which was then present during the binding phase of the experiment . ( C ) As in ‘B’ above but with biotinylated PP1 as the ligand and PPP1R15A533-624 as the analyte , in the absence or presence of Sephin1 . ( D ) As in ‘B’ above but with biotinylated PP1 as the ligand and PPP1R15A325-636 as the analyte , in the absence or presence of Sephin1 . ( E ) Table summarizing data extracted from fitting curves of three repeats of the experiments shown above ( mean ± standard deviation ) . DOI: http://dx . doi . org/10 . 7554/eLife . 26109 . 009 A biotinylated N-terminally extended PPP1R15A325-636 , corresponding to the construct studied by Das and colleagues , proved unsuited as a ligand in the BLI experiment . To circumvent this problem we biotinylated PP1 and exploited it as a BLI ligand . Addition of either the minimal active fragment , human PPP1R15A533-624 , or the longer human PPP1R15A325-636 , gave rise to a robust BLI signal but addition of Sephin1 affected neither the association nor dissociation phase of the experiment ( Figure 7C and D ) . The kinetics of the bimolecular PP1-PPP1R15A interaction were reproducibly different when one or the other was used as a ligand ( summarized in Figure 7E ) . These may reflect different distorting effect of other elements of the BLI biosensor on the kinetics of dissociation when PP1 or PPP1R15A were used as ligands , and/or a contribution of the N-terminal repeats of PPP1R15A to its interactions with PP1 , as suggested previously ( Brush and Shenolikar , 2008 ) . However , reproducible inertness in all three assays lends confidence to the conclusion that in this experimental system 50 µM Sephin1 does not directly interfere with assembly or stability of the PPP1R15A-PP1 complex . Next we sought to examine the effect of Sephin1 on the in vitro dephosphorylation activity of a tripartite eIF2α-P holophosphatase assembled with the N-terminally extended PPP1R15A325-636 ( corresponding to the construct studied by Das and colleagues ) . As Sephin1 is proposed to inhibit eIF2α-P dephosphorylation by disrupting the binding of PPP1R15A to PP1 , we sought to incorporate the PPP1R15A325-636 component at or below its EC50 , thereby maximizing the prospects of detecting an inhibitory effect . Addition of purified PPP1R15A325-636 to PP1 and G-actin accelerated eIF2α-P dephosphorylation with an EC50 of 5–10 nM ( Figure 8A ) . However , Sephin1 had no effect on the dephosphorylation reaction ( Figure 8B ) , whilst tautomycin readily inhibited the reaction ( IC50 = 2 . 4 nM ) confirming the sensitivity of the assay to a known inhibitor ( Figure 8C ) . These observations were also confirmed in an assay set up with the corresponding murine PPP1R15A273-657 ( Figure 8—figure supplement 1A and B ) . The related compound Guanabenz also proved inert , even when added to the enzymatic assay at the high concentration of 50 µM ( Figure 8D ) . Salubrinal , added at 12 µM ( higher concentrations led to conspicuous precipitation ) had a mild but highly reproducible inhibitory effect ( Figure 8E , inhibition = 22% ± 2 . 045 , unpaired t test , p<0 . 0001 , n = 6 ) . The weakness of salubrinal’s inhibitory effect and the compound’s tendency to precipitate at higher concentrations in the assay buffer frustrated our efforts to establish if inhibition was specific to the eIF2α-P directed ternary complex . Nonetheless these observations showcase the sensitivity of our assay to even weak inhibitors and strengthen the conclusion regarding Sephin1’s inertness in the same assay . 10 . 7554/eLife . 26109 . 010Figure 8 . Sephin1’s effect on the eIF2α-P dephosphorylation activity of the human PP1-PPP1R15A-G-actin holophosphatase in vitro . ( A ) Coomassie-stained PhosTag-SDS-PAGE tracking the dephosphorylation of eIF2αP in 20 min reactions constituted with PP1 [0 . 625 nM] , G-actin [1 . 5 µM] and an escalating concentration of human PPP1R15A325-636 .", "Shown is a representative of three independent experiments performed .", "A schema of the human PPP1R15A325-635 construct is shown above the gel .", "A semi-log10 plot of the initial velocity of eIF2αP dephosphorylation as a function of PPP1R15A325-636 concentration derived from three repeats of the experiment is shown below .", "The EC50 for PPP1R15A325-636 was calculated using agonist fitting function on GraphPad Prism V7 .", "( B ) As in ‘A’ above , but in the presence of a fixed concentration of PPP1R15A325-636 below the EC50 [2 nM] and escalating concentrations of Sephin1 .", "Shown is a representative of the two independent experiments performed .", "Plot contains data from the two repeats .", "( C ) As in ‘B’ above , but in the presence of an escalating concentrations of the PP1 active site inhibitor tautomycin ( Tau ) .", "Shown is a representative of the two independent experiments performed .", "Plot contains data from the two repeats .", "( D ) As above , triplicate reactions of eIF2α-P dephosphorylation conducted in the absence or presence of Sephin1 or the related compound , Guanabenz .", "( E ) As in ‘D’ using Sephin1 , salubrinal or tautomycin .", "Shown is a representative experiment , ( of two repeats ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 26109 . 01010 . 7554/eLife . 26109 . 011Figure 8—figure supplement 1 . Sephin1’s effect on the eIF2α-P dephosphorylation activity of the mouse PP1-PPP1R15A-G-actin holophosphatase in vitro .", "( A ) Coomassie-stained PhosTag-SDS-PAGE tracking the dephosphorylation of eIF2αP in 20 min reactions constituted with mouse PPP1R15A273-657 and escalating concentrations of Sephin1 .", "A schema of the mouse PPP1R15A273-657 construct is shown above the gel and a semi-log10 plot of the initial velocity as a function of Sephin1 concentration is shown below .", "( B ) As in ‘A’ above , but in the presence of an escalating concentrations of the PP1 active site inhibitor tautomycin ( Tau ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 26109 . 011 Sephin1’s role as a proteostasis promoting agent was explored in cultured CHO-K1 cells containing reporters for both the ISR ( CHOP::GFP ) ( Novoa et al . , 2001 ) and the branch of the endoplasmic reticulum unfolded protein response ( UPR ) mediated by IRE1 ( XBP1s::Turquoise ) ( Iwawaki et al . , 2004; Sekine et al . , 2016 ) .", "Previous studies have emphasized the dominance of translational recovery in the physiological action of PPP1R15A , such that Ppp1r15aKO attenuates both the burden of protein misfolding ( Marciniak et al . , 2004 ) and the response to it ( Reid et al . , 2016 ) .", "In keeping with these ideas and with the findings of Das and colleagues ( Das et al . , 2015 ) , Sephin1 attenuated the activity of both UPR pathways in cells exposed to tunicamycin; an inhibitor of N-linked glycosylation , that promotes misfolding of newly-synthesized proteins ( Figure 9A ) .", "Though observed only over a narrow concentration range of tunicamycin ( Figure 9—figure supplement 1A ) and at relatively high concentrations of the drug ( Figure 9—figure supplement 1B ) , Sephin1’s effects in this assay can be reconciled with a mechanism involving a net reduction of protein synthesis; as suggested by Das and colleagues .", "Sephin1 also inhibited induction of the ISR in response to histidinol ( Figure 9B ) , an agent that interferes with tRNA charging and thereby activates the eIF2α kinase GCN2 ( Zhang et al . , 2002 ) without affecting protein folding . 10 . 7554/eLife . 26109 . 012Figure 9 . Sephin1 broadly attenuates the ER stress response in cultured CHO cells .", "( A ) Two-dimensional plot of the fluorescence signals derived from CHO cells stably transduced with both a CHOP::GFP reporter ( on the horizontal axis , Ex: 488 nm/ Em 530 ± 30 nm; reflecting mostly ISR activity ) and a XBP1::Turquoise reporter ( on the vertical axis , Ex: 405 nm/ Em 450 ± 50 nm; reflecting IRE1α activity ) analysed by flow cytometry .", "Color-coded signals from untreated cells ( blue ) or cells exposed to a low concentration of tunicamycin ( 0 . 2 µg/mL; 20 hr ) alone ( green ) or together with Sephin1 ( 50 µM , red ) are superimposed .", "Histograms of the distribution of the two reporter signals in the three cell populations are plotted on the corresponding axis and the mean ± CV ( coefficient of variation ) of the fluorescence intensity of the two reporters is depicted in the bar diagram to the right .", "( B ) As in ‘A’ above , but the cells were exposed to histidinol , an ISR inducer that does not promote unfolded protein stress in the ER and does not activate the XBP1::Turquoise reporter .", "Shown is one of three independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 26109 . 01210 . 7554/eLife . 26109 . 013Figure 9—figure supplement 1 .", "Concentration-dependence of the response of cultured cells to tunicamycin and Sephin1 . ( A ) Histograms of distribution of the fluorescence intensity of the CHOP:GFP reporter ( left panel ) and XBP1s:turquoise reporter ( right panel ) in populations of untreated CHO cells and cells exposed for 20 hr to the indicated concentration of tunicamycin in the absence and presence of Sephin1 ( 50 µM ) analysed by flow cytometry .", "( B ) As in ‘A’ above but the cells were either untreated or exposed to a fixed concentration of tunicamycin ( 0 . 2 µg/mL ) and varying concentrations of Sephin1 . DOI: http://dx . doi . org/10 . 7554/eLife . 26109 . 013 To probe deeper into this matter , we exploited an in vivo assay that monitors eIF2α-P dephosphorylation in cells .", "In this kinase shut-off experiment ( Chambers et al . , 2015 ) ( Figure 10A ) , cultured cells are first exposed to a brief , 30 min pulse of thapsigargin , which rapidly activates the eIF2α kinase PERK and builds levels of eIF2α-P , and then exposed to a PERK kinase inhibitor ( GSK260414A ) .", "The resulting decay in the eIF2α-P signal reflects its dephosphorylation .", "To minimize the contribution of other kinases to the eIF2α-P signal , the experiment was performed in cells lacking GCN2 ( Chambers et al . , 2015 ) , which we inactivated in the CHO-K1 cells by CRISPR-Cas9 gene editing .", "Normally , the dephosphorylation of eIF2α-P is a rapid process , complete in 60 min ( Figure 10B , lanes 2–6 ) .", "It was markedly delayed by inclusion of jasplakinolide ( Figure 10B lanes 7–10 ) , which depletes the pool of G-actin ( by promoting its oligomerization ) , thereby depriving the PPP1R15 subunits of an essential co-factor , as observed previously ( Chambers et al . , 2015 ) . 10 . 7554/eLife . 26109 . 014Figure 10 . eIF2αP dephosphorylation in untreated and Sephin1-treated cells .", "( A ) Schema of the kinase shut-off experiment used to evaluate the decay of the eIF2αP signal in cells .", "Thapsigargin ( 300 nM ) was added at t = −30 min to the media to activate PERK kinase and induce eIF2α phosphorylation .", "Sephin1 ( 50 µM ) was introduced either at t = −30 min ( alongside thapsigargin , in the experiment shown in panel B below and in Figure 10—figure supplement 1 panel C ) or at t = −300 min ( Figure 10—figure supplement 1 panel D ) .", "A PERK kinase inhibitor , PERKi/GSK260414A ( 2 µM ) , was added at t = 0 to visualize eIF2αP dephosphorylation at specified times .", "( B ) Immunoblot of the time-dependent changes in the eIF2αP signal of compound-mutant Ppp1r15bKO; Gcn2KO CHO-K1 cells ( clone #1 ) treated as in ‘A’ .", "Where indicated , the cells were additionally exposed to Sephin1 ( 50 µM ) or the actin-polymerizing agent Jasplakinolide ( 1 µM ) , which inhibits eIF2αP-dephosphorylation by sequestering G-actin .", "The immunoblot of eIF2α ( lower panel ) serves as a loading control .", "Shown is a representative experiment repeated five times .", "( C ) Plot of the eIF2αP signal ( normalised to the value at t = 0 of the vehicle only ( DMSO ) sample ) as a function of time derived from five independent experiments .", "The data have been fitted to an exponential decay curve ( grey solid line for the vehicle and blue solid line for the Sephin1-treated sample ) .", "The exponential decay rate and the R2 of the fit are indicated . DOI: http://dx . doi . org/10 . 7554/eLife . 26109 . 01410 . 7554/eLife . 26109 . 015Figure 10—figure supplement 1 . Analysis of Sephin1 in a different Ppp1r15b mutant cell line .", "( A ) Schema of the procedure to create dual reporter ( CHOP::GFP , XBP1s::Turquoise ) compound mutant Gcn2KO;Ppp1r15bKO CHO-K1 cells using the CRISPR-Cas9 system .", "( B ) Schema of the mutant alleles .", "The two coding exons of wildtype hamster Ppp1r15b are denoted as is the region encoding the PP1-binding RVxF motif ( KVTF , in PPP1R15B ) and the positions of the guide RNAs used to direct the Cas9-mediated double strand breaks .", "( C ) Two-dimensional plot and histograms of the fluorescent signal of the CHOP::GFP and XBP1s::Turquoise reporters in compound Ppp1r15bKO; Gcn2KO CHO-K1 cells ( clone #2 ) .", "Where indicated , the cells were exposed to a low concentration of tunicamycin ( 0 . 2 µg/mL; 20 hr ) alone or together with Sephin1 ( 50 µM ) .", "The mean ± CV ( coefficient of variation ) of the fluorescence intensity of the two reporters is displayed in the bar diagram .", "Shown is one of three independent experiments .", "( D ) As in ‘Figure 10B' but using Ppp1r15bKO; Gcn2KO ( clone #2 ) . ( E ) As in ‘D’ above , but Sephin1 pretreatment was extended to 5 hr , before the kinase shut-off procedure , and continued throughout .", "( F ) As in ‘D’ above , but substituting tunicamycin ( 2 . 5 µg/mL; 2 hr ) for thapsigargin as the PERK inducer .", "Shown is a representative experiment ( of two repeats ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 26109 . 015 Together , PPP1R15A and PPP1R15B account for the bulk of eIF2α-P dephosphorylation activity of mammalian cells ( Harding et al . , 2009 ) , but their relative contribution to the process in any given circumstance is unknown .", "Therefore , to adapt this assay to measure Sephin1’s effect on PPP1R15A-mediated eIF2α-P dephosphorylation , it was essential to inactivate the gene encoding PPP1R15B , leaving PPP1R15A as the sole regulatory subunit of the eIF2α-P phosphatase .", "CRISPR-Cas9 mediated gene editing was used to create two different GCN2KO; Ppp1r15bKO compound-mutant CHO-K1 cells ( Figure 10—figure supplement 1A and B ) .", "As expected , the GCN2KO; Ppp1r15bKO compound-mutant CHO-K1 cells retained their responsiveness to Sephin1 ( Figure 10—figure supplement 1C ) .", "However , in this experimental system dependent solely on PPP1R15A , the time-dependent decline of the eIF2α-P signal ( fitted to an exponential decay curve ) yielded a time constant of 0 . 23 min−1 for the untreated and 0 . 19 min−1 for the Sephin1 treated sample , an insignificant difference ( Figure 10B lanes 11–15 , Figure 10—figure supplement 1D and E and Figure 10C ) .", "To mimic conditions used in the flow cytometry experiments ( and those used by Das et al . , 2015 ) kinase shut-off experiments were carried out on tunicamycin-treated cells ( Figure 10—figure supplement 1F ) .", "eIF2α-P dephosphorylation proceeded rapidly in tunicamycin-treated cells .", "However , Sephin1 had no inhibitory effect on the rate of dephosphorylation .", "This experiment reveals that under conditions in which Sephin1 exerts its proteostasis-promoting activities , it does not affect rates of eIF2a-P dephosphorylation .", "To follow up on this matter , both copies of the gene encoding PPP1R15A were inactivated by CRISPR-Cas9 in the reporter containing CHO-K1 cells ( Figure 11—figure supplement 1A and B ) .", "Inactivation was confirmed by loss of the PPP1R15A signal in immunoblot of lysates from stressed mutant cells ( Figure 11A ) .", "Sephin1 retained its ability to attenuate the response of both the XBP1s::Turquoise and the CHOP::GFP reporter in tunicamycin treated PPP1R15A null cells ( Figure 11B ) .", "Similar observations were made in regard to the effect of Sephin1 in histidinol-treated cells ( Figure 11C ) .", "These observations suggest that Sephin1 also exerts its proteostatic effect ( s ) in cells lacking PPP1R15A . 10 . 7554/eLife . 26109 . 016Figure 11 . Cells lacking PPP1R15A remain responsive to Sephin1 . ( A ) Immunoblot of endogenous PPP1R15A recovered by immunoprecipitation ( using an anti-PPP1R15A antibody conjugated to Protein A Sepharose ) from untreated and tunicamycin exposed parental cells and cells from two different Ppp1r15aKO CHO-K1 clones .", "The position of PPP1R15A is indicated and the immunoglobulin heavy-chain is marked with an asterisks .", "The immunoblot of eIF2α ( lower panel ) serves as a loading control for the content of cellular protein in the lysates .", "( B ) Two-dimensional plot and histograms of the fluorescent signal of the CHOP::GFP and XBP1s::Turquoise reporters in the two Ppp1r15aKO CHO-K1 clones .", "Where indicated , the cells were exposed to a low concentration of tunicamycin ( 0 . 2 µg/mL; 20 hr ) alone or together with Sephin1 ( 50 µM ) .", "The mean ± CV ( coefficient of variation ) of the fluorescence intensity of the two reporters in each of the two clones is displayed in the bar diagram .", "Shown is one of three independent experiments .", "( C ) As in ‘C’ above , but cells were exposed to histidinol .", "Shown is one of three independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 26109 . 01610 . 7554/eLife . 26109 . 017Figure 11—figure supplement 1 .", "Mutant Ppp1r15a alleles .", "( A ) Schema of the procedure to create dual reporter ( CHOP::GFP , XBP1s::Turquoise ) Ppp1r15aKO CHO-K1 cells using the CRISPR-Cas9 system .", "( B ) Schema of the mutant alleles .", "The two coding exons of wildtype hamster Ppp1r15a are denoted as is the region encoding the crucial RVxF motif ( KVHF , in PPP1R15A ) and the positions of the guide RNAs used to direct the Cas9-mediated double strand breaks . DOI: http://dx . doi . org/10 . 7554/eLife . 26109 . 017 Next , we used CRISPR-Cas9-mediated homologous recombination to introduce a site-specific mutation into the Eif2s1 locus , to encode an ISR-blocking eIF2αS51Amutation in the endogenous gene ( Figure 12A and B ) .", "Surprisingly , Sephin1 retained its ability to attenuate the XBP1s::Turquoise reporter in tunicamycin-treated eIF2αS51Amutant cells ( Figure 12C ) .", "As CHOP activation is highly dependent on the ISR ( Harding et al . , 2000 ) , activity of the CHOP::GFP reporter was strongly attenuated in mutant eIF2αS51Acells .", "Nonetheless , it is notable that residual activation of the reporter by tunicamycin ( likely a consequence of ATF6 action at the CHOP promoter [Yoshida et al . , 2000] ) , was also attenuated by Sephin1 ( Figure 12C ) .", "These observations bring into question the primacy of the ISR in Sephin1’s mechanism of action . 10 . 7554/eLife . 26109 . 018Figure 12 . ISR-deficient Eif2s1S51A ( eIF2αS51A ) cells retain their responsiveness to Sephin1 . ( A ) Schematic representation of procedure used to create dual reporter ( CHOP::GFP , XBP1s::Turquoise ) Eif2s1S51A ( eIF2αS51A ) CHO-K1 cells using CRISPR-Cas9 system .", "( B ) Immunoblot of CHO-K1 cell lysates using anti- eIF2αP ( upper panel ) , anti- eIF2α ( middle panel ) and anti-BiP ( lower panel ) antibodies .", "Two-fold more cell lysate was loaded onto lanes 5 and 6 to compensate for the lower eIF2α content of the haploid mutant Eif2s1S51A cells .", "( C ) Two-dimensional plot and histograms of the fluorescent signal of the CHOP::GFP and XBP1s::Turquoise reporters in the Eif2s1S51A CHO-K1 cells .", "Where indicated , the cells were exposed to a low concentration of tunicamycin ( 0 . 2 µg/mL; 20 hr ) alone or together with Sephin1 ( 50 µM ) .", "The mean ± CV ( coefficient of variation ) of the fluorescence intensity of the two reporters in each of the two clones is displayed in the bar diagram .", "Shown is representative experiment of two independent experiments performed .", "Note the blunted expression of CHOP::GFP wrought by the ISR-defect imposed by the Eif2s1S51A mutation . DOI: http://dx . doi . org/10 . 7554/eLife . 26109 . 018" ], [ "The role of the eIF2α-P-dependent ISR in defending against unfolded protein stress is well supported by genetic and pharmacological experiments ( Baird and Wek , 2012; Ron and Harding , 2007 ) .", "By retarding its dephosphorylation , the primary consequence of eliminating PPP1R15A is to prolong the duration of the eIF2α-P signal in stress response scenarios ( Kojima et al . , 2003; Novoa et al . , 2003 ) and to alter the repertoire of mRNA translation ( Reid et al . , 2016 ) .", "Therefore , the finding that cells and mice lacking PPP1R15A are relatively resistant to pharmacological and genetic models associated with unfolded protein stress in the endoplasmic reticulum ( ER stress ) has engendered a specific interest in targeting the PPP1R15A-containing phosphatase complex for inhibition , as a means for accessing the therapeutic potential of enhanced ISR signalling .", "Sephin1 and Guanabenz , compounds previously proposed to exert their proteostatic effects by disrupting the essential PP1-PPP1R15A complex and inhibiting eIF2α-P dephosphorylation ( Das et al . , 2015 ) are found here to have no effect in in vitro enzymatic assays dependent on the formation of a PP1-PPP1R15A complex .", "Sephin1 likewise proved inert in a Bio-Layer Interferometry assay that measured directly the affinity of PPP1R15A and PP1 for one another .", "Furthermore , we find that Sephin1 does not interfere with eIF2α-P dephosphorylation in cells ( as measured by a kinase shut-off experiment ) and that Sephin1 retains its ability to attenuate the impact of a challenge to proteostasis even in cells lacking PPP1R15A , or in ISR-defective Eifs1S51A cells .", "These observations suggest that the previously-reported attenuation of the recovery of PP1 in complex with PPP1R15A , when both were purified from lysates of cells treated with Sephin1 was unlikely to be a direct consequence of Sephin1 interference with complex formation or of destabilization by Sephin1 of a pre-existing complex and also call into question the importance of any indirect disruption of the PP1-PPP1R15A complex that may occur in vivo and remain undetected by our assays .", "Our findings , questioning whether Sephin1 attains its proteostatic activity by inhibiting the PPP1R15A-containing eIF2α-P directed holophosphatase , and similar concerns raised by the Peti and Shenolikar labs ( Choy et al . , 2015 ) , do nothing to diminish the attractiveness of PPP1R15A inhibition as a potential means for defending proteostasis .", "Similarly , there is nothing in our study to question the beneficial effects reported for Sephin1 in mouse models of neurodegeneration ( Das et al . , 2015 ) nor we do not challenge the enhanced susceptibility of Ppp1r15bKO cells to the [ ( o-chlorobenzylidene ) amino]guanidine , Guanabenz ( Tsaytler et al . , 2011 ) .", "However , our findings that Sephin1 exerts its effects in CHO-K1 cells lacking PPP1R15A or in ISR-defective Eifs1S51A cells raise doubts as to whether these phenomena were attained via inhibition of PPP1R15A or indeed modulation of the ISR .", "Crystal structures of the PPP1R15 ( A or B ) -PP1 and the related PNUTS/PPP1R10-PP1 and spinophilin/PPP1R9B-PP1 complexes reveal that both the residues corresponding to human PPP1R15AV556 ( of the RVxF motif ) and the conserved arginine ( human PPP1R15AR578 ) insert deeply into the surface of the PP1 subunit ( Chen et al . , 2015; Choy et al . , 2014 , 2015 ) .", "The contrast between the dramatic effect of the PPP1R15AV556E mutation and the more modest effect of the human PPP1R15AR578A mutation on the PPP1R15A-PP1 complex may reflect a role for the former early in the pathway to complex assembly ( a process that can be thought of as PPP1R15A folding on the surface of PP1 ) .", "It is therefore possible that inhibitors of the eIF2α-P holophosphatase might disrupt complex assembly , without affecting the stability or activity of a preformed complex .", "However , Sephin1 is unlikely to belong to such a category , as it failed to exert an inhibitory effect on enzymatic activity or on the BLI signal even when added to pure PPP1R15A , before addition of PP1 and G-actin .", "But other compounds that remain to be found might selectively disrupt the assembly of the PPP1R15A-PP1 complex by binding to and stabilizing an intermediate step in its formation .", "Similarly instructive is the human PPP1R15AW582A mutation , which eliminates all detectable selectivity of PPP1R15-holophosphatases for eIF2α-P ( Chen et al . , 2015 ) ( and Figure 3B here ) without affecting the kinetics of the bimolecular association of PPP1R15A with PP1 or G-actin .", "These features suggest that the side chain of W582 - a residue conserved throughout the PPP1R15 family - may have a special role in aligning PP1 and G-actin to form a composite surface with affinity for the substrate , without contributing measurably to the stability of the tripartite holophosphatase .", "That the side chain of single tryptophan residue can bias the complex towards activity ( without affecting its stability ) , suggests the possibility that small molecules might access this allosteric mechanism and bias the tripartite holophosphatase towards or away from enzymatic activity without the need to disrupt an extensive protein-protein interface .", "Specific cellular dephosphorylation events are notoriously difficult to target with small molecules ( Sakoff and McCluskey , 2004 ) .", "Here we presented evidence that the perception of Sephin1 as a milestone in overcoming that challenge may need re-thinking .", "However , features of the PPP1R15A-PP1-G-actin holophosphatase noted above suggest ways in which eIF2α-P dephosphorylation might indeed be selectively targeted by small molecules .", "In vitro assays for selective eIF2α-P dephosphorylation , such as the one described here , might prove useful in discovery of small molecules with such a mechanism of action ." ], [ "Diverse cloning techniques were used to create the bacterial and mammalian expression vectors listed in Table 1 .", "This table contains information about lab number , name , description and reference for each plasmid used . 10 . 7554/eLife . 26109 . 019Table 1 . Plasmids used . DOI: http://dx . doi . org/10 . 7554/eLife . 26109 . 019Lab numberLab nameDescriptionReferenceUK105eIF2a-NM_pET30aHis6-tagged mouse eIF2a 1–185 pET-30a ( + ) \"PMID 15341733UK168PerkKD-pGEX4T-1Bacterial expression plasmid for mouse PERK kinase domainPMID 9930704UK622PGV_PP1G_1–323_V1Bacterial expression plasmid forfull-length PP1 phosphatase catalytic domainPMID 25774600UK1359pSpCas9 ( BB ) −2A-GFPMammalian expression of GFP-tagged Cas9 and single guide RNA to introduce double strand breaks ( Addgene 48138 ) PMID 24157548UK1367pSpCas9 ( BB ) −2A-PuroMammalian expression of Puror-tagged Cas9 and single guide RNA to introduce double strand breaks ( Addgene 48138 ) PMID 24157549UK1497CHO_EIF2K4_guideA*_pSpCas9 ( BB ) −2A-PuroPuro-tagged CRISPR for targeting human CHO GCN2 ( EIF2K4 ) geneThis paperUK1507CHO_Eif2s1_guideC_pSpCas9 ( BB ) −2A-PuroA single guide gRNA plasmid for eIF2a ( EIF2S1 ) locusThis paperUK1599CHO_PPP1R15A_guide1_pSpCas9 ( BB ) −2A-GFPA single guide gRNA plasmid for GADD34 locusThis paperUK1600CHO_PPP1R15A_guide2_pSpCas9 ( BB ) −2A-GFPA single guide gRNA plasmid for GADD34 locusThis paperUK1610pSpCas9 ( BB ) −2A-mCherry_V2modified pSpCas9 ( BB ) −2A vector to express mCherry together with guide RNA and Cas9This paperUK1645GST_Myd116_273–657_malE_pGEX_TEVBacterial expression of GST-mouse GADD34 273–657 -MBPThis paperUK1677huPPP1R15A_325_636_malE_pGEX_TEVBacterial expression of GST-human GADD34- MBPThis paperUK1881EcBirA_WT_pGEX_TEV ( MP1 ) Bacterial expression of fastidious E . coli BirA biotin ligase ( R118 intact ) This paperUK1897mPP1G_1–323_pGEX_TEV_AviTag ( MP2 ) Bacterial expression GST_TEV_AviTag_FL mPP1G with non-tempaled C-term LEThis paperUK1920huPPP1R15A_533_624_malE_pGEX_TEV_AviTag ( MP1 ) Bacterial-expression plasmid for N-tern AviTagged human GADD34 533–624This paperUK1921huPPP1R15A_325_636_malE_pGEX_TEV_AviTag ( MP4 ) Bacterial-expression plasmid for N-tern AviTagged human GADD34 325–624This paperUK1992huPPP1R15A_I596A_533_624_malE_pGEX_TEV_AviTagBacterial-expression plasmid for N-term AviTagged human GADD34 533–624 , I596A mutationThis paperUK1993huPPP1R15A_V556E_R578A_533_624_malE_pGEX_TEV_AviTagBacterial-expression plasmid for N-term AviTagged human GADD34 533–624 , v556e R578A mutationThis paperUK1994huPPP1R15A_V556E_533_624_malE_pGEX_TEV_AviTagBacterial-expression plasmid for N-term AviTagged human GADD34 533–624 , v556e mutationThis paperUK1995huPPP1R15A_F592A_533_624_malE_pGEX_TEV_AviTagBacterial-expression plasmid for N-term AviTagged human GADD34 533–624 , F592A mutationThis paperUK2081CHO_PPP1R15B_guideA_pSpCas9 ( BB ) −2A-mCherry_V2guide targeting cgPPP1R15B ( hamster CReP ) gene 5' end of exon1This paperUK2082CHO_PPP1R15B_guideB_pSpCas9 ( BB ) −2A-mCherry_V2guide targeting cgPPP1R15B ( hamster CReP ) gene 3' end of exon 1This paper Actin was purified from rabbit muscle according to ( Pardee and Spudich , 1982 ) as modified by ( Chen et al . , 2015 ) .", "Expression plasmids for PPP1R15A ( GADD34 ) variants contained ampicillin resistance marker , N-terminal GST tag and C-terminal maltose binding protein ( MBP ) tag ( UK1677 , UK1920 ) ( Table 1 ) .", "They were transformed into BL21 T7 Express lysY/Iq E . coli ( C3013 , New England Biolabs ) and colonies that grew in LB-ampicillin plates ( 100 μg/ml ampicillin ) were used to create a saturated over-night culture .", "This saturated culture was used to inoculate 2–4 Litres of LB media supplemented with 100 μg/ml ampicillin .", "The cultures were incubated at 37°C until OD600 = 0 . 6–0 . 8 .", "At this point , they were induced with 1 mM Isopropyl β-D-1-thiogalactopyranoside ( IPTG ) and cultured for 20 more hours at 18°C .", "It was followed by a centrifugation step to pellet bacteria and resuspension of the ice-cold pellets in 3–4 pellet volumes of lysis buffer ( 50 mM Tris pH 7 . 4 , 500 mM NaCl , 1 mM MnCl2 , 1 mM MgCl2 , 1 mM tris ( 2-carboxyethyl ) phosphine ( TCEP ) , 100 μM phenylmethylsulfonyl fluoride ( PMSF ) , 20 mTIU/ ml aprotinin , 2 μM leupeptin , and 2 μg/ml pepstatin in 10% glycerol ) .", "An EmulsiFlex-C3 homogenizer ( Avestin , Inc , Ottawa , Ontario ) was used to lyse the bacteria , which were then clarified in a JA-25 . 50 rotor ( Beckman Coulter ) at 33 , 000×g for 30 min at 4°C .", "These suspensions were bound to pre-equilibrated glutathione sepharose 4B beads ( 17-0756-05 , GE Healthcare ) for 1–2 hr at 4°C .", "Beads were transferred to a 10 mL column after being batch-washed with 20 bed volumes of lysis buffer .", "Proteins were eluted in glutathione elution buffer ( 50 mM Tris pH 7 . 4 , 100 mM NaCl , 40 mM glutathione , 0 . 5 mM MnCl2 , 0 . 5 mM TCEP , 10% glycerol ) , and cleaved with Tobacco Etch Virus protease ( TEV ) ( 12 . 5 μg TEV protease/mg protein ) overnight at 4°C to remove the N-terminal GST tag .", "Cleaved proteins were bound to amylose beads ( E8021S , New England Biolabs ) for 1–2 hr at 4°C .", "Twenty/thirty bed volumes of lysis buffer were used to batch-wash the amylose beads , which were transferred to a 10 mL column and eluted with HEPES buffer ( 20 mM HEPES , 100 mM NaCl , 0 . 2 mM CaCl2 , 0 . 2 mM ATP , 0 . 2 mM TCEP , 0 . 5 mM MnCl2 , 100 μM PMSF , 20 mTIU/ ml aprotonin , 2 μM leupeptin , and 2 μg/ml pepstatin ) and 10 mM maltose .", "PP1 ( UK622 ) ( Table 1 ) was purified as above , with the following modifications: LB media cultures were supplemented with MnCl2 , after TEV cleavage proteins were buffer exchanged using a 2 mL desalting column in HEPES buffer and re-bound to glutathione sepharose 4B beads to remove free GST tag .", "Phosphorylated eIF2α was encoded by an expression plasmid containing N-terminal His-Tag and kanamycin resistance marker ( UK105 ) ( Table 1 ) .", "BL21 T7 Express lysY/Iq E . coli were co-transformed with this plasmid and a GST-Tagged PERK plasmid carrying ampicillin resistance marker ( UK168 ) ( Table 1 ) .", "Colonies that grew in ampicillin ( 100 μg/ml ) and kanamycin ( 50 μg/ml ) LB-plates were used to create a saturated over-night cultured with which 2L of ampicillin and kanamycin LB were inoculated .", "Growth , induction and purification was as described for PPP1R15A , with the following changes: beads used were Ni-NTA ( 30230 , Qiagen ) to bind His-tag , lysis buffer contained 20 mM imidazole and elution buffer contained 500 mM imidazole instead of glutathione .", "This protein did not require TEV cleavage but an additional size exclusion chromatography step was included .", "A Superdex S200 ( GE Healthcare ) was used to gel filter the protein in 25 mM Tris , 100 mM NaCl , 0 . 1 mM EDTA , 1 mM DTT and 10% glycerol buffer .", "All proteins were snap frozen and kept at −80°C in small aliquots .", "Final concentration of proteins was calculated from UV absorbance at 280 nm measurements in Nanodrop ( Thermo Scientific , UK ) and based on their extinction coefficient values predicted by MacVector .", "Biotin ( B1595 , Thermo Scientific ) was added to the AviTagged specified proteins ( encoded by UK1897 , 1920 , 1921 , 1992 , 1993 , 1994 , 1995 ) ( Table 1 ) using BirA .", "BirA was amplified by PCR reaction from E . coli genomic DNA and inserted into an expression vector containing N-terminal GST ( TEV cleavable ) and ampicilin resistance marker ( UK1881 ) ( Table 1 ) .", "This protein was purified following standard GST-tagged protocols , eluted in glutathione elution buffer , aliquoted and stored in this buffer .", "All proteins were biotinylated and its biotinylation was checked as described ( Fairhead and Howarth , 2015 ) Proteins in glutathione elution buffer were biotinylated and buffer exchanged into a HEPES buffer to remove excess of biotin that would interfere with the Bio-Layer Interferometry measurements .", "Drugs used: Sephin1 ( EN300-195090 , Enamine ) , tautomycin ( 5805551 , Calbiochem ) , Guanabenz ( D6270 , Sigma-Aldrich ) , salubrinal ( Sal003 , S4451 , Sigma-Aldrich ) Dephosphorylation reactions were conducted as described ( Chen et al . , 2015 ) .", "In summary , reactions were conducted by combining the different proteins for 20 min at 30°C whilst shaking at 500 rpm and were stopped by addition of Laemmli buffer .", "A fraction of the reactions were loaded into PhosTag SDS-PAGE , stained using Coomasie and scanned .", "ImageJ ( NIH ) was used to quantify signal intensity .", "Enzyme velocity , V was measured at substrate concentrations well below the enzyme’s Km and in samples with less than 25% substrate depletion .", "Under these conditions , the instantaneous velocity ( i . e . rate of substrate conversion to product per molecule of enzyme ) is proportional to instantaneous substrate concentration and the equivalent velocity is obtained with the equation below , derived from the integrated rate equation for first order kinetics:Vi=ln[S]0[S]f∗[S]0Δt∗[ENZ] Where Vi is the initial velocity ( the instantaneous velocity at t = 0 , with the dimensions of 1/t ) , [s]0 and [s]f are , respectively , the substrate concentrations at the beginning and end of the reaction , Δt is the time interval of the reaction and [ENZ] is the concentration of enzyme .", "The ‘agonist fitting’ or ‘inhibitor fitting’ functions of GraphPad Prism V7 ( RRID: SCR_002798 ) were used to analyze the effects of varying concentrations of reaction components on velocity .", "Proteins were diluted in HEPES buffer at the specified concentrations .", "Two-hundred microliters of each diluted protein preparation was placed in a 96 well plate ( 655209 , Greiner ) .", "Streptavidin sensors ( 18–5019 , ForteBio ) were hydrated in this buffer for 2–5 min before the binding assay was performed .", "The plate was placed in the ForteBio Octet RED96 System for data acquisition which was performed at 25° C at a constant orbital flow of 600 rpm .", "The binding assays consisted of the measurement of change in layer thickness ( in nanometres ) during a series of sequential steps .", "The sensor was equilibrated in the buffer ( 240 s ) and the ligand ( biotyninated protein ) was loaded on the sensor .", "Ligand attachment to the sensor was checked by immersion of the sensor in buffer after loading ( 400–2000 s ) .", "Finally , association and dissociation of the proteins studied was analysed by soaking the sensor in analyte solutions and buffer , respectively .", "The duration of the ligand loading on the sensor was set to a specific time ( 600 s ) or a specific value ( 2 nm displacement ) depending on the experiment performed .", "The duration of the association and dissociation of the analyte to the ligand , was adjusted in order to capture bindings that had not reached equilibrium phase .", "Data analysis were performed using GraphPad V7 ( RRID: SCR_002798 ) and curves were fitted to a receptor binding kinetics association then dissociation built-in model .", "CHO cells were plated in six well plate at 3·105 cells/well density .", "Next day , they were treated for 20 hr with specified compounds .", "They were washed twice with PBS and suspended in PBS 4 mM EDTA to be evaluated by flow cytometry .", "Flow cytometry data were analyzed using FlowJo ( FlowJo , LLC , RRID: SCR_008520 ) and GraphPad-Prism V7 ( RRID: SCR_002798 ) was used to create bar graphs .", "Replicates of flow cytometry experiments were analysed using Stata 14 StataCorp .", "( 2015 . Stata Statistical Software: Release 14 . College Station , TX: StataCorp LP , RRID: SCR_012763 ) .", "The interaction between treatments and genotypes in the different repeats was modeled using linear regression .", "The model was used to test whether the effect of different treatments differed between the genotypes , allowing for different mean values of CHOP::GFP and XBP1s::Turquoise on each repeat .", "The analysis showed non-significant differences between genotypes .", "However , for each cell type , there were significant differences ( p<0 . 02 ) between untreated cells versus stressed cells and also between the latter and cells co-treated with Sephin1 .", "Antibodies used: rabbit anti-PPP1R15A ( 10449–1-AP , ProteinTech , RRID: AB_2168724 ) , rabbit anti-eIF2α-P ( ab32157 , Abcam , RRID: AB_732117 ) , chicken anti-BiP ( Avezov et al . , 2013 ) , mouse anti-eIF2α ( Scorsone et al . , 1987 ) Drugs used: tunicamycin ( T2250 , Melford ) , thapsigargin ( 586005 , Calbiochem ) , L-Histidinol ( 228830010 , Acros Organics ) , PERKi ( Gift from GSK , GSK2606414A ) Sephin1 ( EN300-195090 , Enamine ) CHO-K1 cells were plated in 10 cm dishes until they reached 80% confluency , at which point they were treated with 2 . 5 μg/mL of tunicamycin or DMSO ( vehicle ) for 7 hr .", "Cells were washed twice with ice-cold PBS , scraped in presence of PBS with 1 mM EDTA and centrifuged at 376 g for 5 min at 4°C ( 5424 R , Eppendorf ) .", "Four pellet-volume of harvest buffer ( 10 mM HEPES pH 7 . 9 , 50 mM NaCl , 0 . 1 mM EDTA , 0 . 5% Triton , 0 . 5 M Sucrose , 1 mM DTT , 4 μg/mL Aprotinin , 1 mM PMSF , 2 μg/mL Pepstatin , 17 . 5 mM β-Glycerophosphate , 10 mM Tetrasodium Pyrophosphate , 100 mM NaF ) was used to lyse the cells .", "After 5–10 min of incubation on ice , samples were clarified at 21130 g for 15 min at 4°C ( 5424 R , Eppendorf ) .", "Protein quantification of the clarified supernatants was performed using Bradford method .", "For immunoprecipitation , 15 μL Protein A-Sepharose beads ( Zymed , 10–1042 ) per sample where preincubated with anti-PPP1R15A antibody .", "Equal amounts of protein extract ( 800 μg ) were incubated with the beads over night rotating at 4°C .", "After four washes with 1 mL of TBS , 20 μL of 2X Laemmli loading buffer were added to the samples .", "Once incubated at 70°C , same volumes were loaded into a 10% SDS-PAGE gel and transferred to a PVDF membrane .", "The experimental procedure was adapted from ( Chambers et al . , 2015 ) .", "Briefly , CHO cells ( Gcn2-/-; Ppp1r15b-/- ) were plated in 10 cm dishes at 40% confluency .", "Sixteen-twenty hours later , fresh media was added and cells were incubated for 2 hr .", "Sephin1 ( 50 μM ) or DMSO was added to the media for either 30 min or 5 hr before application of thapsigargin ( 300 nM for 30 min ) or tunicamycin ( 2 . 5 µg/mL for 2 hr ) to induce stress by activation of PERK kinase .", "GSK2606414A [2 µM] was added to inhibit PERK .", "The PP1R15A-PP1-dependent decay of the eIF2α-P signal ( by its dephosphorylation ) was tracked by stopping the reaction at different time points by addition of ice-cold PBS .", "eIF2α-P and total eIF2α were detected by immunoblot .", "ImageJ ( NIH ) was used to quantify signal intensity and one phase decay model was used ( GraphPad-Prism V7 , RRID: SCR_002798 ) to analyse the rate decay of eIF2αP dephosphorylation .", "A Shimadzu UFLCXR system coupled to an Applied Biosystems API2000 mass spectrometer was used .", "Column: Phenomenex Gemini-NX , 3 μm , 110 Å , C18 , 50 × 2 mm ( at 40°C ) .", "Mobile phase: solvent A: 0 . 1% formic acid in water; solvent B: 0 . 1% formic acid in acetonitrile .", "Gradient: pre-equilibration for 1 min at 5% solvent B in solvent A; then linear gradient 5–98% solvent B over 2 min , 98% B for 2 min , 98–5% B over 0 . 5 min , then 5% B for 1 min .", "Flow rate: 0 . 5 mL/min .", "Detector: UV detection at 254 nm ( channel 1 ) , 220 nm ( channel 2 ) .", "Mass spectrometer: positive ion mode ." ] ]

[ "Dephosphorylation of translation initiation factor 2 ( eIF2α ) terminates signalling in the mammalian integrated stress response ( ISR ) and has emerged as a promising target for modifying the course of protein misfolding diseases .", "The [ ( o-chlorobenzylidene ) amino]guanidines ( Guanabenz and Sephin1 ) have been proposed to exert protective effects against misfolding by interfering with eIF2α-P dephosphorylation through selective disruption of a PP1-PPP1R15A holophosphatase complex .", "Surprisingly , they proved inert in vitro affecting neither stability of the PP1-PPP1R15A complex nor substrate-specific dephosphorylation .", "Furthermore , eIF2α-P dephosphorylation , assessed by a kinase shut-off experiment , progressed normally in Sephin1-treated cells .", "Consistent with its role in defending proteostasis , Sephin1 attenuated the IRE1 branch of the endoplasmic reticulum unfolded protein response .", "However , repression was noted in both wildtype and Ppp1r15a deleted cells and in cells rendered ISR-deficient by CRISPR editing of the Eif2s1 locus to encode a non-phosphorylatable eIF2α ( eIF2αS51A ) .", "These findings challenge the view that [ ( o-chlorobenzylidene ) amino]guanidines restore proteostasis by interfering with eIF2α-P dephosphorylation ." ]

[ "Most drugs work by tweaking the way that cells are regulated .", "Adding or removing a phosphate group from proteins regulates many cellular decisions .", "There are known drugs that bind to and inhibit the enzymes that add phosphate to proteins , thereby controlling various aspects of cell behaviour .", "However , drug developers have been far less successful in finding drugs that inhibit phosphatases , the enzymes that remove phosphate from proteins .", "Genetically modified mice can be used as ‘models’ to investigate human diseases .", "In 2015 a drug called Sephin1 was reported to suppress neurodegeneration in a group of these mice by inhibiting a particular phosphatase .", "The phosphatase is made of three component proteins that come together to create the active enzyme .", "Sephin1 was reported to disrupt the association between two of these three components .", "This discovery was met with excitement; both for its potential therapeutic implications in humans and as an important “first” in pharmacology .", "To understand how Sephin1 and a related drug , Guanabenz , work at the molecular level , Crespillo-Casado et al . reconstructed in a test tube the phosphatase that Sephin1 and Guanabenz were reported to inhibit .", "To examine the effects the drugs have on the phosphatase , Crespillo-Casado et al . developed assays to measure the association between the components that make up the phosphatase .", "Further assays measured the removal of phosphate from the phosphatase’s target , a protein called eIF2α .", "The results of the assays show that Sephin1 did not affect the coming together of the components that make up the active phosphatase .", "The drug also did not inhibit the removal of phosphate from eIF2α in the test tube .", "To extend these findings Crespillo-Casado et al . exposed cells to Sephin1 and observed features that are consistent with the drug’s reported ability to supress neurodegeneration .", "However , these features were also observed both in cells lacking the phosphatase that Sephin1 was reported to inhibit and in cells in which eIF2α never acquired a phosphate in the first place .", "The findings presented by Crespillo-Casado et al . do not challenge Sephin1’s role in supressing neurodegeneration , but do question its ability to do so by inhibiting the phosphatase that dephosphorylates eIF2α .", "This knowledge will be useful to drug developers and those interested in molecular mechanisms of drug action .", "For those researchers who are interested in Sephin1 , further work is needed to discover alternative molecular mechanisms by which it suppresses neurodegeneration .", "And for those researchers who are interested in eIF2α dephosphorylation , there is a need to look further for inhibitors of this process , as Sephin1 is unlikely to serve in that role ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "tools and resources", "neuroscience" ]

[ [ "The survival of an organism depends on its ability to detect and respond appropriately to its environment .", "Afferent neurons innervating the skin provide sensory information to guide and refine behavior ( Seymour , 2019; Zimmerman et al . , 2014 ) .", "Cutaneous stimuli are used to study a wide range of neurobiological mechanisms since neurons densely innervating skin function to provide diverse information as the body interfaces with its immediate environment .", "These afferents maintain the integrity of the body by recruiting rapid sensorimotor responses , optimize movement through feedback loops , provide teaching signals that drive learning , and update internal models of the environment through higher-order perceptual and cognitive processes ( Barik et al . , 2018; Brecht , 2017; Corder et al . , 2019; de Haan and Dijkerman , 2020; Haggard et al . , 2013; Huang et al . , 2019; Petersen , 2019; Seymour , 2019 ) .", "Damaging stimuli , for example , evoke rapid motor responses to minimize immediate harm and generate pain that motivates longer-term behavioral changes .", "Compared to visual , olfactory , and auditory stimuli , somatosensory inputs are challenging to deliver in awake unrestrained mammals .", "This is due to the nature of stimuli that require contact and the diversity of stimulus features encoded by afferents that innervate skin .", "Cutaneous afferent neurons are functionally and genetically heterogeneous , displaying differential tuning , spike thresholds , adaptation rates , and conduction velocities ( Abraira and Ginty , 2013; Dubin and Patapoutian , 2010; Gatto et al . , 2019; Häring et al . , 2018 ) .", "The arborization of their peripheral terminals can delineate spatial and temporal dimensions of the stimulus ( Pruszynski and Johansson , 2014 ) , particularly once many inputs are integrated by the central nervous system ( Prescott et al . , 2014 ) .", "Cutaneous stimulation in freely moving mice often requires the experimenter to manually touch or approach the skin .", "This results in inaccurate timing , duration , and localization of stimuli .", "The close proximity of the experimenter can cause observer-induced changes in animal behavior ( Sorge et al . , 2014 ) .", "Stimuli also activate a mixture of sensory neuron populations .", "For example , intense stimuli can co-activate fast-conducting low-threshold afferents that encode innocuous stimuli simultaneously with more slowly conducting high-threshold afferents ( Wang et al . , 2018 ) .", "The latter are nociceptors that trigger fast protective behaviors and pain .", "Consequently , mixed cutaneous inputs recruit cells , circuits , and behaviors that are not specific to the neural mechanism under study .", "A way to control genetically defined afferent populations is to introduce opsins into these afferents and optogenetically stimulate them through the skin ( Abdo et al . , 2019; Arcourt et al . , 2017; Barik et al . , 2018; Beaudry et al . , 2017; Browne et al . , 2017; Daou et al . , 2013; Iyer et al . , 2014 ) .", "However , these methods in their current form do not fully exploit the properties of light .", "The behaviors that are evoked by cutaneous stimuli are also typically measured with limited and often subjective means .", "Manual scoring introduces unnecessary experimenter bias and omits key features of behavior .", "Behavioral assays have traditionally focused on a snapshot of the stimulated body part rather than dynamics of behavior involving the body as a whole ( Gatto et al . , 2019 ) .", "Recent advances in machine vision and markerless pose estimation have enabled the dissection of animal behavioral sequences ( Mathis et al . , 2018; Pereira et al . , 2019; Wiltschko et al . , 2015 ) .", "However , these have not been adapted to study behavioral outputs relating to specific cutaneous inputs .", "Here we developed an approach to project precise optogenetic stimuli onto the skin of freely behaving mice ( Figure 1A ) .", "The strategy elicits time-locked individual action potentials in genetically targeted afferents innervating a small stimulation field targeted to the skin .", "Stimuli can be delivered remotely as predefined microscale patterns , lines , or moving points .", "The utility of the system was demonstrated by precisely stimulating nociceptors , or Aβ low threshold mechanoreceptors ( LTMRs ) , in freely behaving mice to map behavioral outputs at high speed .", "We provide an analysis toolkit that quantifies the millisecond-timescale dynamics of behavioral responses using machine vision methods .", "We dissect discrete behavioral components of local paw responses , head orienting and body repositioning behaviors , and determine how these specific behavioral components relate to precise somatosensory inputs ." ], [ "The design of the optical strategy had eight criteria: ( 1 ) that somatosensory stimuli are delivered non-invasively without touching or approaching the mice; ( 2 ) localization of stimuli are spatially precise and accurate ( <10 μm ) ; ( 3 ) freely moving mice can be targeted anywhere within a relatively large ( 400 cm2 ) arena; ( 4 ) stimuli can be controlled with a computer interface from outside the behavior room; ( 5 ) stimulation patterns , lines , and points are generated by rapidly scanning the stimuli between predefined locations; ( 6 ) stimulation size can be controlled down to ≥150 μm diameter; ( 7 ) stimuli are temporally precise to control individual action potentials using sub-millisecond time-locked pulses; and ( 8 ) behavioral responses are recorded at high speed at the stimulated site and across the whole body simultaneously .", "An optical system was assembled to meet these specific criteria ( Figure 1B and C ) .", "The stimulation path uses two mirror galvanometers to remotely target the laser stimulation to any location on a large glass stimulation floor .", "A series of lenses expands the beam and then focuses it down to 0 . 018 mm2 ( 150 μm beam diameter ) at the surface of this floor .", "This was defocused to provide a range of calibrated stimulation spot sizes up to 2 . 307 mm2 , with separable increments that were stable over long periods of time ( Figure 1—figure supplement 1A ) .", "The optical power density could be kept equal between these different stimulation spot sizes .", "The glass floor was far ( 400 mm ) from the galvanometers , resulting in a maximum focal length variability of <1 . 5% ( see Materials and methods ) .", "This design yielded a spatial targeting resolution of 6 . 2 μm while minimizing variability in laser stimulation spot sizes across the large stimulation plane ( coefficient of variation ≤0 . 1 , Figure 1—figure supplement 1B ) .", "The beam ellipticity was 74 . 3% ± 14 . 3% ( median± MAD , range of 36–99% ) for all spot sizes .", "The optical power was uniform across the stimulation plane ( Figure 1—figure supplement 1C ) .", "The galvanometers allow rapid small angle step ( 300 µs ) responses to scan the laser beam between adjacent positions and shape stimulation patterns using brief laser pulses ( diode laser rise and fall time: 2 . 5 ns ) .", "Custom software ( see Materials and methods ) was developed to remotely control the laser stimulation position , trigger laser pulses , synchronize galvanometer jumps , and trigger the camera acquisition ( Figure 1—figure supplement 2 ) .", "The camera acquisition path was used to manually target the location of the laser stimulation pulse ( s ) ; the path was descanned through the galvanometers so that the alignment between the laser and camera is fixed ( Figure 1B ) .", "The camera feed is displayed in the user interface and enables the operator to use this image to target the laser to the desired location .", "High signal-to-noise recordings were obtained using near-infrared frustrated total internal reflection ( NIR-FTIR ) in the glass stimulation floor ( Roberson , D . P . et al . , manuscript submitted ) .", "If a medium ( skin , hair , tail , etc . ) is within a few hundred microns of the glass , it causes reflection of the evanescent wave and this signal decreases non-linearly with distance from the glass such that very minor movements of the paw can be detected .", "The acquisition camera acquired the NIR-FTIR signal in high-speed ( up to 1000 frames/s ) with a pixel size of 110 μm .", "A second camera was used to record the entire arena and capture behaviors involving the whole body before and after stimulation .", "Offline quantification was carried out using custom analysis code combined with markerless tracking tools ( Mathis et al . , 2018 ) .", "To validate the strategy , we first crossed Trpv1-IRES-Cre ( TRPV1Cre ) and R26-CAG-LSL-ChR2-tdTomato mice to obtain a line ( TRPV1Cre::ChR2 ) in which ChR2 is selectively expressed in a broad class of nociceptors innervating glabrous skin ( Browne et al . , 2017 ) .", "These mice were allowed to freely explore individual chambers placed on the stimulation plane .", "When mice were idle ( still and awake ) , a time-locked laser pulse was targeted to the hind paw .", "Stimuli could be controlled remotely from outside the behavior room .", "We recorded paw withdrawal dynamics with millisecond resolution .", "For example , a single , small 1 ms laser pulse initiated a behavioral response at 29 ms , progressing to complete removal of the hind paw from the glass floor just 5 ms later ( Figure 2A and Figure 2—video 1 ) .", "The stimulus used for this protocol was S6 , 0 . 577 mm2 in area , which corresponds to less than 1% of the glabrous paw area and highlights the sensitivity of the nociceptive system .", "Motion energy , individual pixel latencies , and response dynamics could be extracted from these high-speed recordings ( Figure 2B and C ) .", "We probed multiple sites across the plantar surface and digits and found that the hind paw heel gave the most robust responses ( Figure 2—figure supplement 1 ) .", "This region was targeted in all subsequent experiments .", "Littermates that did not express the Cre recombinase allele confirmed that the laser stimulation did not produce non-specific responses .", "These mice did not show any behavioral responses , even with the largest stimuli ( spot size S8 , 30 ms pulse , Figure 2—figure supplement 2 ) .", "We next provide some examples of the utility of the strategy by examining the relationship between nociceptive input and protective behaviors .", "Fast protective withdrawal behaviors can be triggered by the first action potential arriving at the spinal cord from cutaneous nociceptors .", "A brief optogenetic stimulus generates just a single-action potential in each nociceptor activated ( Browne et al . , 2017 ) .", "This is due to the rapid closing rate of ChR2 relative to the longer minimal interspike interval of nociceptors .", "The same transient optogenetic stimulus ( Browne et al . , 2017 ) , or a pinprick stimulus ( Arcourt et al . , 2017 ) , initiates behavior before a second action potential would have time to arrive at the spinal cord .", "That the first action potential can drive protective behaviors places constraints on how stimulus intensity can be encoded , suggesting that the total population of nociceptors firing a single-action potential can provide information as a \"Boolean array . \"", "The consequences of this have not been investigated previously as precise control of specific nociceptive input had not been possible .", "We predicted that the relative number of nociceptors firing a single-action potential determines the features of the behavioral response .", "Varying the pulse duration with nanosecond precision influences the probability of each nociceptor generating a single-action potential within the stimulation site .", "A pulse as short as 300 μs elicited behavioral responses but with relatively low probability ( Figure 2D ) .", "This probability increased with pulse duration until it approached unity , closely matching the on-kinetics of the ChR2 used ( τ = 1 . 9 ms; Lin , 2011 ) .", "We next controlled the spatial , rather than temporal , properties of the stimulation in two further experiments .", "Firstly , we find that the total area of stimulated skin determines the behavioral response probability , such that the larger the nociceptive input the larger the response probability ( Figure 2E ) .", "Secondly , we generated different stimulation patterns .", "We find that sub-threshold stimulations are additive ( Figure 2F ) .", "Specifically , seven spatially displaced small sub-threshold stimulations could reproduce the response probability of a single large stimulation that was approximately seven times their size .", "This could not be achieved by repeated application of the small stimulations to the same site ( Figure 2F ) .", "Time-locking the stimulus enabled us to examine the hind paw responses with high temporal resolution .", "The nociceptive input size influenced the behavioral response latency: for example , a 3 ms pulse resulted in response latencies of 27 ± 1 ms , 30 ± 2 ms , 33 ± 5 ms , and 112 ± 46 ms for spot sizes S8 , S7 , S6 , and S5 , respectively ( Figure 3A and B ) .", "The shorter latencies are consistent with medium-conduction velocity Aδ-fibers that arrive at the spinal cord before slower C-fiber action potentials ( >35 ms ) ( Browne et al . , 2017 ) .", "The rank order of response latencies follows the nociceptive input size for both pulse durations , and they fit well with log-log regressions ( 3 ms pulse R2 = 0 . 87 , 1 ms pulse R2 = 0 . 90 ) .", "Once a hind limb motor response was initiated , it developed rapidly , lifting from the glass with rise times that show the vigor of the motor response was also dependent on nociceptive input size ( Figure 3C ) .", "These responses , in >65% of cases , proceeded to full withdrawal .", "However , in a fraction of trials the paw moved but did not withdraw ( Figure 3D ) , highlighting the sensitivity of the acquisition system .", "Even the smallest of nociceptive inputs still produced a large fraction of full withdrawal responses , despite decreases in response probability ( Figure 3E ) .", "The fraction of full withdrawal responses increased with the size of nociceptive input .", "The onset latency of both full and partial responses decreased as nociceptive input increased ( Figure 3F ) .", "Pain-related responses are not limited to the affected limb but involve simultaneous movement of other parts of the body ( Blivis et al . , 2017; Browne et al . , 2017 ) .", "These non-local behaviors theoretically serve several protective purposes: to investigate and identify the potential source of danger , move the entire body away from this danger , attend to the affected area of the body ( Huang et al . , 2019 ) and to maintain balance ( Sherrington , 1910 ) .", "Whole-body movements were quantified as motion energy ( Figure 4—figure supplement 1A ) and high-speed recordings show this initiated with a mean response latency of 30 ± 1 ms , with the first movement bout displaying a mean duration of 136 ± 14 ms ( 80 trials from 10 mice ) ( Figure 4—figure supplement 2 ) .", "The magnitude of whole-body movement increased with the stimulation spot size ( Figure 4—figure supplement 1B ) .", "Peak motion energy had a lognormal relationship with nociceptive input size ( R2 = 0 . 99 ) .", "This indicates that global behaviors are also proportional to the relative size of the nociceptive input; the recruited nociceptors firing a single-action potential ( Figure 4—figure supplement 1B ) .", "Most behaviors arise from the complex coordination of discrete body parts , which can be tracked individually .", "To dissect specific components of these behaviors , we implemented DeepLabCut ( Mathis et al . , 2018 ) by training a network using frames from the high-speed ( 400 frames/s ) videos to track 18 user-defined body parts across the mouse ( for details , refer to Materials and methods , Global behaviors during optogenetic stimulation ) .", "The high-speed video recordings of stimulation trials were analyzed using this network .", "Specific nociceptive input at the hind paw ( S8 , 2 . 307 mm2 , 10 ms pulse ) causes behavior that initiates simultaneously across the body .", "Inspection of the movements of each body part relative to the baseline pose ( Figure 4A ) shows fast outward movement of the stimulated and contralateral hind paws , and concomitant initiation of head orientation ( two example responses in Figure 4B ) .", "Based on these observations , we examined the behavioral trajectories in the first 115 ms across the population of 80 trials .", "The first three principal components ( PCs ) were fit using six body part x and y values at 115 ms after the stimulus onset .", "These PCs explain 88 . 8% of the variance ( 50 . 4 , 26 . 5 , and 11 . 9% for PC1 , PC2 , and PC3 , respectively ) .", "PC1 is dominated by hind paw translation , PC2 by head and body movement , and PC3 by head orientation ( Figure 4C ) .", "Projecting the entire time course onto these same PCs can explain 78 . 1% of the variance ( 37 . 1 , 24 . 3 , and 16 . 7% for PC1 , PC2 , and PC3 , respectively ) .", "The response trajectories revealed that movements occur largely in same direction within PC space with a circular standard deviation of 52 . 9° ( Figure 4D and E ) .", "Shuffling body parts on each trial gave non-directional trajectories with a circular standard deviation of 126 . 8° ( Figure 4—figure supplement 3 ) .", "Behavioral trajectories also show that the response magnitude in PC space can be partly explained by initial PC1 and PC2 values ( Figure 4F and G ) .", "This suggests that the initial pose influences these fast behavioral responses .", "Examining specific features of these behaviors over a slightly longer period ( 300 ms ) provides further insights .", "Displacement of each body part relative to their baseline position reveals the response timing , extent , and coordination ( Figure 4H ) .", "The stimulated paw started moving at 29 ± 1 ms , the contralateral hind paw at 34 ± 4 ms , and the nose at 33 ± 2 ms ( 80 trials from 10 mice ) .", "With this intense stimulus , only in 6% of trials did the hind paws or single body parts move alone , although the magnitude of the head movement varied between trials .", "The distance traveled by the nose positively correlates with the distance for the stimulated paw ( Pearson’s r = 0 . 64 , n = 80 trials from 10 mice ) .", "Examining the relative distance between the nose and stimulated hind paw shows a reliably short latency ( Figure 4I ) , indicating that these responses are driven by Aδ-nociceptor input rather than more slowly conducting C-fibers .", "A diversity of responses was observed: the head and stimulated paw move closer together in some trials and in others moved further apart ( Figure 4I and J ) .", "This could result from the head moving towards or away from the stimulated paw but also the stimulated paw moving backwards as the body rotates .", "Indeed , consistent with initial observations ( Figure 4A and B ) and principal component analysis ( PCA; Figure 4C–G ) , we find that the head selectively and rapidly orients to the stimulated side ( Figure 4K ) .", "The presence of head orientation suggests that a brief nociceptive input can rapidly generate a coordinated spatially organized behavioral response .", "This is likely integral to protective pain-related behaviors and might function to gather sensory information about the stimulus or its consequences , and potentially provides coping strategies .", "Protective behaviors can be statistically categorized ( Abdus-Saboor et al . , 2019 ) and computational discrimination of high-speed hind paw responses used as a score of pain ( Jones et al . , 2020 ) .", "We have shown that the analysis can easily be customized to incorporate computational tools that facilitate quantification and reveal insights into complex behavioral responses .", "The vesicular glutamate transporter-1 ( Vglut1 ) is a known marker of Aβ-LTMRs ( Alvarez et al . , 2004; Brumovsky et al . , 2007 ) .", "To demonstrate the utility of the system in the broader context of somatosensation , we crossed Slc17a7-IRES2-Cre-D ( Vglut1Cre ) mice with R26-CAG-LSL-ChR2-tdTomato mice to generate a line ( Vglut1Cre::ChR2 ) that express ChR2 in LTMRs ( Harris et al . , 2014 ) .", "A recent detailed anatomical and physiological characterization of Vglut1Cre::ChR2 mice further confirmed that in DRG neurons , ChR2 is restricted to broad class of myelinated Aβ-LTMRs ( Chamessian et al . , 2019 ) .", "Here , we find that a single 3 ms stimulus ( S7 = 1 . 155 mm2 ) precisely delivered to the hind paw of these mice rarely elicited hind paw responses ( mean paw withdrawal probability = 0 . 10 ± 0 . 03 SEM , 99 trials from n = 11 mice ) , with the earliest response occurring at 206 ms after stimulation ( Figure 5A and B ) , which is an order of magnitude slower than we observed in TRPV1Cre::ChR2 mice ( fastest response: 19 ms ) .", "Trains of five pulses , however , frequently elicited responses , showing mean paw withdrawal probabilities of 0 . 31 ± 0 . 09 ( SEM , 108 trials from n = 12 mice ) for 5 Hz and 0 . 40 ± 0 . 10 ( SEM , 117 trials from n = 12 mice ) for 10 Hz trains ( Figure 5C ) .", "Increasing stimulation frequency to 20 Hz did not result in higher withdrawal probabilities , which may reflect ChR2 desensitization , rather than a physiological process ( Lin , 2011 ) .", "While the responses at first seem to be frequency-dependent ( Figure 5D , left ) , inspection of recordings indicated that these occurred after the second or third pulse in most trials , regardless of stimulation frequency ( Figure 5A ) .", "We find that the response distributions superimpose when withdrawal latencies are normalized to the interstimulus interval ( pulse-matched latencies in Figure 5D , right ) .", "This observation suggests that response probability is likely driven by pulse summation , rather than by stimulation frequency .", "Indeed , we find that the probabilities and latencies can be explained by the probability sum rule , using the values for a single pulse to predict the values for five pulses ( Figure 5C and D ) .", "The magnitude of whole-body motion was not altered by increasing frequencies ( Figure 5—figure supplement 1 ) .", "In contrast to the TRPV1Cre::ChR2 line , whole-body behaviors in response to optogenetic stimulation of Vglut1Cre::ChR2 mice were subtle: visual inspection of high-speed whole-body behavior videos revealed that responses were mostly limited to small hind paw lifts or shifts towards the center of the body in cases where the stimulated paw was initially further away from the body .", "In most instances , these movements did not disturb balance or alter the animal’s posture .", "Interestingly , we observed that whisking and , to a lesser extent , circular movements of the upheld forepaws would precede hind paw responses and initiate as early as the first pulse , even in trials that would not proceed to withdrawal .", "We speculate that mice may perceive the stimulation early on , but only act on this after a delay ." ], [ "We describe a strategy for remote , precise , dynamic somatosensory input and behavioral mapping in awake unrestrained mice .", "The approach can remotely deliver spatiotemporally accurate optogenetic stimuli to the skin with predefined size , geometry , duration , timing , and location , while simultaneously monitoring behavior in the millisecond timescale .", "Microscale optogenetic stimulation can be used to simulate patterns , edges , and moving points on the skin .", "Responses to these precisely defined points and patterns can be mapped using machine vision approaches .", "The design is modular , for example , additional lasers for multicolor optogenetic control or naturalistic infrared stimuli can be added and complementary machine vision analysis approaches readily implemented .", "As an example , we combine this with DeepLabCut ( Mathis et al . , 2018 ) , for markerless tracking of individual body parts to further dissect specific components of whole-body responses .", "We validated the system in two transgenic mouse lines , providing optical control of broad-class Aδ and C-nociceptors , and Aβ-LTMRs .", "Advances in transcriptional profiling have identified a vast array of genetically defined primary afferent neuron populations involved in specific aspects of temperature , mechanical , and itch sensation ( Usoskin et al . , 2015 ) .", "Selective activation of these populations is expected to recruit a specific combination of downstream cells and circuits depending on their function .", "For example , nociceptive input generates immediate sensorimotor responses and also pain that acts as a teaching signal .", "This strategy can be thus combined with techniques to modify genes , manipulate cells and neural circuits , and record neural activity in freely behaving mice to probe these mechanisms ( Boyden et al . , 2005; Kim et al . , 2017 ) .", "We provide approaches to map behavioral responses to defined afferent inputs across the spectrum of somatosensory modalities ( Browne et al . , 2017; Huang et al . , 2019 ) .", "We find that the probabilistic recruitment of nociceptors determines the behavioral response probability , latency , and magnitude .", "We propose that the aggregate number of first action potentials arriving from nociceptors to the spinal cord can be utilized to optimize the timing and extent of rapid protective responses .", "These first action potentials could be summated by spinal neurons so that appropriate behaviors are selected based on thresholds .", "Resultant fast behaviors are diverse but include coordinated head orientation and body repositioning that depends on the initial pose .", "In contrast , responses to optogenetic activation of Aβ-LTMRs occurred with slower onset , lower probability , and resulted in more subtle whole-body movements .", "Using a fixed number of pulses , we find that responses from multiple Aβ-LTMR inputs can be explained by the sum rule of probabilities rather than frequency-dependence ( Chamessian et al . , 2019 ) .", "This does not , however , rule out the tuning of responses to more spatially or temporally complex stimuli .", "We used broad-class Cre driver lines to selectively stimulate either nociceptors or Aβ-LTMRs , and it is possible that their respective subpopulations exploit a diversity of coding strategies .", "This optical approach can reveal how such subpopulation and their specific downstream circuits guide behavior .", "In summary , we have developed a strategy to precisely control afferents in the skin without touching or approaching them by projecting light to optogenetically generate somatosensory input in patterns , lines , or points .", "This is carried out non-invasively in awake freely behaving mice in a way that is remote yet precise .", "Remote control of temporally and spatially precise input addresses the many limitations of manually applied contact stimuli .", "The timing , extent , directionality , and coordination of resultant millisecond-timescale behavioral responses can be investigated computationally with specific sensory inputs .", "This provides a way to map behavioral responses , circuits , and cells recruited by defined afferent inputs and dissect the neural basis of processes associated with pain and touch .", "This strategy thus enables the investigation of sensorimotor , perceptual , cognitive , and motivational processes that guide and shape behavior in health and disease ." ], [ "Optical elements , optomechanical components , mirror galvanometers , the diode laser , LEDs , controllers , machine vision cameras , and structural parts for the optical platform are listed in the table in Supplementary file 1 .", "These components were assembled on an aluminum breadboard as shown in the Solidworks rendering in Figure 1C .", "The laser was aligned to the center of all lenses and exiting the midpoint of the mirror galvanometer housing aperture when the mirrors were set to the center of their working range .", "A series of lenses ( L1–L3 ) expanded the beam before focusing it on to the glass stimulation plane , on which mice are placed during experiments .", "The glass stimulation platform was constructed of 5-mm-thick borosilicate glass framed by aluminum extrusions .", "NIR-FTIR was achieved by embedding an infrared LED ribbon inside the aluminum frame adjacent to the glass edges ( Roberson , D . P . et al . , manuscript submitted ) .", "The non-rotating L1 lens housing was calibrated to obtain eight defined laser spot sizes , ranging from 0 . 0185 mm2 to 2 . 307 mm2 , by translating this lens along the beam path at set points to defocus the laser spot at the 200 mm × 200 mm stimulation plane .", "The beam size can be altered manually using this rotating lens tube per design , but this is modular and could be altered by the user .", "To ensure a relatively flat field in the stimulation plane , the galvanometer housing aperture was placed at a distance of 400 mm from its center .", "In this configuration , the corners of the stimulation plane were at a distance of 424 mm from the galvanometer housing aperture and variability of the focal length was below 1 . 5% .", "Optical power density was kept constant by altering the laser power according to the laser spot area .", "Neutral density ( ND ) filters were used so that the power at the laser aperture was above a minimum working value ( ≥8 mW ) and to minimize potential changes in the beam profile at the stimulation plane .", "The laser and mirror galvanometers were controlled through a multifunction DAQ ( National Instruments , USB-6211 ) using custom software written in LabVIEW .", "The software displays the NIR-FTIR camera feed , whose path through the mirror galvanometers is shared with the laser beam , so that they are always in alignment with one another .", "Computationally adjusting mirror galvanometer angles causes identical shifts in both the descanned NIR-FTIR image field of view and intended laser stimulation site , so that the laser can be targeted to user-identified locations .", "Shaped stimulation patterns were achieved by programmatically scaling the mirror galvanometer angles to the glass stimulation plane using a calibration grid array ( Thorlabs , R1L3S3P ) .", "The timings of laser pulse trains were synchronized with the mirror galvanometers to computationally implement predefined shapes and lines using small angle steps that could be as short as 300 µs .", "The custom software also synchronized image acquisition from the two cameras , so that time-locked high-speed local paw responses were recorded ( camera 1: 160 pixels × 160 pixels , 250–1000 frames/s depending on the experiment ) .", "Time-locked global whole-body responses were recorded above video-frame rate ( camera 2: 664 pixels × 660 pixels , 40 frames/s ) or at high speed ( camera 2: 560 pixels × 540 pixels , 400 frames/s ) across the entire stimulation platform .", "To calibrate the L1 lens housing and ensure consistency of laser spot sizes across the glass stimulation platform , we designed a 13 . 90 ± 0 . 05 mm thick aluminum alignment mask .", "This flat aluminum mask was used to replace the glass stimulation platform and was combined with custom acrylic plates that align the aperture of a rotating scanning-slit optical beam profiler ( Thorlabs , BP209-VIS/M ) to nine defined coordinates at different locations covering the stimulation plane .", "The laser power was set to a value that approximates powers used in behavioral experiments ( 40 mW ) .", "The laser power was then attenuated with an ND filter to match the operating range of the beam profiler .", "Using Thorlabs Beam Software , Gaussian fits were used to determine x-axis and y-axis 1/e2 diameters and ellipticities for each laser spot size over three replicates at all nine coordinates .", "The averages of replicates were used to calculate the area of the eight different laser spot sizes that were measured in each of the nine coordinates ( Figure 1—figure supplement 1A ) and then fitted with a two-dimensional polynomial equation in MATLAB to create heatmaps ( Figure 1—figure supplement 1 B ) .", "The average values over the nine coordinates were defined for each laser spot size: S1 = 0 . 0185 mm2 , S2 = 0 . 0416 mm2 , S3 = 0 . 0898 mm2 , S4 = 0 . 176 mm2 , S5 = 0 . 308 mm2 , S6 = 0 . 577 mm2 , S7 = 1 . 155 mm2 , S8 = 2 . 307 mm2 .", "These measurements were repeated 6 months after extensive use of the optical system to ensure stability over time ( Figure 1—figure supplement 1A ) .", "In addition , the uniformity of laser power was assessed by measuring optical power at five positions of the experimental platform with a power meter ( Thorlabs , PM100D ) ( Figure 1—figure supplement 1C ) .", "Experiments were performed using mice on a C57BL/6j background .", "Targeted expression of ChR2-tdTomato in broad-class cutaneous nociceptors was achieved by breeding mice homozygous for Cre-dependent ChR2 ( H134R ) -tdTomato at the Rosa26 locus ( RRID:IMSR_JAX:012567 , R26-CAG-LSL-hChR2 ( H134R ) -tdTomato , Ai27D; Madisen et al . , 2012 ) with mice that have Cre recombinase inserted downstream of the Trpv1 gene in one allele ( RRID:IMSR_JAX:017769 , Trpv1-IRES-Cre , TRPV1Cre; Cavanaugh et al . , 2011 ) .", "Aβ-LTMRs were selectively stimulated by breeding homozygous Ai27D mice with mice in which Cre recombinase is targeted to cells expressing the vesicular glutamate transporter 1 ( RRID:IMSR_JAX: 023527 , Slc17a7-IRES2-Cre-D , Vglut1Cre; Harris et al . , 2014 ) .", "Resultant mice were heterozygous for both transgenes and were housed with control littermates that do not encode Cre recombinase but do encode Cre-dependent ChR2-tdTomato .", "Adult ( 2–4 months old ) male and female mice were used in experiments .", "Mice were given ad libitum access to food and water and were housed in 21°C ± 2°C , 55% relative humidity and a 12 hr light:12 hr dark cycle .", "Experiments were carried out on at least two separate cohorts of mice , each cohort contained 4–6 mice .", "Experiments were spaced by at least one day in the case where the same cohort of mice was used in different experiments .", "All animal procedures were approved by University College London ethical review committees and conformed to UK Home Office regulations .", "Prior to the first experimental day , mice underwent two habituation sessions during which each mouse was individually placed in a plexiglass chamber ( 100 mm × 100 mm , 130 mm tall ) on a mesh wire floor for 1 hr , then on a glass platform for another hour .", "On the experimental day , mice were again placed on the mesh floor for 1 hr , then up to six mice were transferred to six enclosures ( 95 mm × 60 mm , 75 mm tall ) positioned on the 200 mm × 200 mm glass stimulation platform .", "Mice were allowed to settle down and care was taken to stimulate mice that were calm , still , and awake in an ‘idle’ state .", "The laser was remotely targeted to the hind paw glabrous skin using the descanned NIR-FTIR image feed .", "The laser spot size was manually set using the calibrated L1 housing , while laser power and neutral density filters were used to achieve a power density of 40 mW/mm2 regardless of spot size .", "The software was then employed to trigger a laser pulse of defined duration ( between 100 μs and 30 ms ) and simultaneously acquire high-speed ( 1000 , 500 , or 250 frames/s depending on experiment ) NIR-FTIR recordings of the stimulated paw , as well as a global view of the mice with a second camera ( 400 frames/s or 40 frames/s ) ( Figure 1C ) .", "Recordings of stimulations of TRPV1Cre::ChR2 mice were 1500 ms in duration , with the laser pulse initiated at 500 ms . For each stimulation protocol , six pulses , three on each hind paw , spaced by at least 1 min were delivered to eight mice , split into two cohorts .", "For experiments involving Vglut1Cre::ChR2 mice , we used a single stimulation spot size ( S7 = 1 . 155 mm2 ) and duration ( 3 ms ) .", "In addition to the single-pulse stimulation , these mice received a train of five pulses applied at 5 , 10 , or 20 Hz .", "The recording time for each trial was extended to 2000 ms to accommodate for the longer stimulation period .", "For each protocol , Vglut1Cre::ChR2 mice were stimulated in 10 trials , split equally between the two hind paws .", "Data was collected from 12 Vglut1Cre::ChR2 mice and 8 littermate controls lacking Cre recombinase split into five cohorts .", "In all experiments , the behavioral withdrawal of the stimulated hind paw was also manually recorded by the experimenter .", "TRPV1Cre::ChR2 mice were stimulated on the heel of the hind paw with each of the following protocols: ( 1 ) a single 1 ms pulse with spot size S7 ( 1 . 155 mm2 ) ; ( 2 ) a single 1 ms pulse with spot size S4 ( 0 . 176 mm2 ) ; ( 3 ) seven 1 ms pulses with spot size S4 , superimposed on the same stimulation site and spaced by 500 μs intervals; ( 4 ) seven 1 ms pulses with spot size S4 , spaced by 500 μs intervals and spatially displacing stimuli with 0 . 3791 mm jumps such as to draw a small hexagon; ( 5 ) seven 1 ms pulses with spot size S4 , spaced by 500 μs intervals and spatially displacing stimuli with 0 . 5687 mm jumps such as to draw a hexagon expanded by 50% compared to the previous shape; and ( 6 ) seven 1 ms pulses with spot size S4 , spaced by 500 μs intervals and spatially displacing stimuli with 0 . 3791 mm jumps such as to draw a straight line .", "The power density of the stimulations was kept constant at 40 mW/mm2 as before .", "Seven mice , split into two cohorts , received 10 stimulations per protocol ( five on each hind paw ) after a baseline epoch of 500 ms . An additional cohort of four littermates lacking Cre recombinase were stimulated in the same way and served as negative controls .", "Finally , three TRPV1Cre::ChR2 mice were stimulated ( spot size S8 , 10 ms pulse duration ) with a single pulse adjacent to the hind paw , five times on each side , in order to control for potential off-target effects .", "The NIR-FTIR signal was recorded at 500 frames/s .", "To obtain recordings optimized for markerless tracking with DeepLabCut , a single acrylic chamber ( 100 mm × 100 mm , 150 mm tall ) was centered on the glass stimulation platform of the system .", "Rapid movements were recorded at 400 frames/s using a below-view camera ( FLIR , BFS-U3-04S2M-CS ) .", "Two white and two infrared LED panels illuminated the sides of the behavioral chamber in order to optimize lighting for these short exposure times and achieve high contrast images .", "NIR-FTIR was not used in this configuration .", "TRPV1Cre::ChR2 mice received between 10 and 20 single-shot laser pulse stimulations of 10 ms each , at least 1 min apart and equally split between right and left hind paw and using spot size S8 ( 2 . 31 mm2 ) .", "The first 10 trials that exceeded quality control were used ( see Markerless tracking of millisecond-timescale global behaviors , Data processing ) .", "Each trial consisted of a 500 ms baseline and 4000 ms after-stimulus recording epoch .", "High-speed NIR-FTIR recordings were saved as uncompressed AVI files .", "A Python script was implemented in Fiji to verify the integrity of the high-speed NIR-FTIR recordings and extract average 8-bit intensity values from all frames within a circular region of interest on the stimulation site ( 60 pixels diameter ) .", "This output was then fed into RStudio to calculate the average intensity and associated standard deviation of the baseline recording ( first 500 ms ) .", "A hind paw response was defined as a drop of intensity equal to or below the mean of the baseline minus five times its standard deviation .", "Paw response latency was defined as time between the start of the pulse and the time at which a hind paw response was first detected .", "For purposes of quality control , only recordings with a baseline NIR-FTIR intensity mean ≥ 3 and a standard deviation/mean of the baseline ratio ≥23 were retained for analysis .", "Another criterion was that response latencies are not 10 ms or shorter since this would be too short to be generated by the stimulus itself .", "Only one trial out of 2369 trials did not meet this criterion ( spot size S6 , 1 ms pulse , 8 ms response latency ) .", "In addition to this two-step workflow using Fiji/Python to process AVI files and then RStudio to analyze the resulting output , alternative code was written in Python 3 , which combines both steps and also computes individual pixel latencies and motion energy using NumPy and Pandas packages .", "A median filter ( radius = 2 pixels ) was applied to the NIR-FTIR recordings used to create the representative time series in Figure 2A and Figure 2—video 1 .", "For raster plots of hind paw response dynamics in Figure 4A , NIR-FTIR intensity values were normalized to the average baseline value .", "For the patterned stimulation experiments in Figure 2F and Vglut1Cre::ChR2 experiments in Figure 5A–D , trials were analyzed as stated to compute local response probabilities , but an additional rule was introduced to further minimize the risk of false positives .", "A response required the signal to fall by 20% and exceed a threshold of four times the standard deviation of baseline .", "Compared to the performance of an experimenter manually processing the videos with Fiji , the automated analysis pipeline was substantially faster for similar accuracy .", "For example , it took an experimenter two working days to analyze 127 videos , whereas the Fiji/Python pipeline generated the identical output within 90 s .", "Videos of the entire stimulation platform were cropped into individual mouse chambers ( 200 × 315 pixels ) and then analyzed using RStudio to quantify the amount of whole-body movements , including those stemming from the response of the stimulated limb , herein referred to as global behavior ( GB ) .", "GB was approximated as the binarized motion energy: the summed number of pixels changing by more than five 8-bit values between two subsequent frames ( Pixel Change ) .", "Briefly , for each pixeln ( n = 63 , 000 pixels/frame ) , the 8-bit value at a given frame ( Fn ) was subtracted from the corresponding pixeln at the previous frame ( Fn-1 ) .", "If the resulting absolute value was ≤5 , 0 would be assigned to the pixel .", "If the absolute resulting value was >5 , 1 would be assigned to the pixel .", "The threshold was chosen to discard background noise from the recording .", "The pixel binary values were then summed for each frame pair to obtain binarized motion energy .", "Normalized binarized motion energy was calculated by subtracting each post-stimulus frame binarized motion energy from the average baseline binarized motion energy .", "As an alternative to this analysis strategy , we have developed code in Python that processes the video files and calculates motion energy .", "The peak normalized binarized motion energy was determined and only trials displaying a peak response ≥5 standard deviations of the baseline mean were retained for further analysis and plotting .", "For TRPV1Cre::ChR2 mice , the analysis was restricted to a time window of 100 ms after stimulus onset ( first three frame pairs proceeding the stimulus frame ) to enable time-locking to the stimulus .", "Between 41 and 47 videos from eight mice were analyzed per spot size .", "For experiments with Vglut1Cre::ChR2 mice , the peak normalized binary motion energy exceeding five standard deviations of the baseline mean was determined for the entire 1 . 5 s recording epoch proceeding stimulus onset .", "Between 51 and 80 trials from 11 to 12 mice were analyzed per stimulation frequency .", "GB was analyzed within a 1 ms time frame following stimulation by computing the binarized motion energy relative to a baseline reference frame 5 ms prior to stimulation as described above .", "Here , the threshold for pixel change was set to seven 8-bit values .", "The binarized motion energy ( sum of pixel binaries ) of a given frame was normalized to the total number of pixels within that frame after removing those frames that had been affected by the stimulation laser pulse .", "The global response latency of movement initiation was determined as the time when binarized motion energy was greater than 10 times the standard deviation at baseline .", "Termination of movement was determined as the time point when binarized motion energy returned below 10 times standard deviation from baseline following the first movement bout .", "Data was analyzed in RStudio 1 . 2 . 5019 , Python 3 . 6 . 8 , ImageJ/Fiji 2 . 0 . 0 and Prism 7 and visualized using Seaborn , Prism 7 , and Adobe Illustrator 24 . 0 .", "In all experiments , repeated measurements were taken from multiple mice .", "Paw responses to patterned stimulation were reported as mean probabilities ± standard error of the mean ( SEM ) and analyzed using Friedman’s non-parametric test for within-subject repeated measures followed by Dunn’s signed-rank test for multiple comparisons ( Figure 2F ) .", "In this experiment , one of the seven TRPV1Cre::ChR2 mice was removed from the dataset because it displayed saturating responses to Protocol 3 preventing comparison of values across a dynamic range .", "Response latencies , response rise times , and response durations were computed using a hierarchical bootstrap procedure ( Saravanan et al . , 2020 ) modified to acquire bootstrap estimates of the median with balanced resampling .", "Briefly , mice are sampled with replacement for the number of times that there are mice .", "For each mouse within this sample , its trials were sampled with replacement , but the number of selected trials was balanced , ensuring each mouse contributes equally to the number of trials in the sample .", "The median was taken for this resampled population and this entire process was repeated 10 , 000 times .", "Bootstrap estimates from 1000 simulated experiments show that an additional 1 . 6–3 . 1% of values fall within 1% of the population median for seven mice with between 2 and 6 responses .", "Values provided are the mean bootstrap estimate of the median ± the standard error of this estimate .", "The median bias was small due to the resampled population size from hierarchically nested data and only moderate distribution skew .", "Global peak motion energy ( Figure 4B ) was examined in a similar way , except the mean of resampled populations was used as it represents a better estimator of the population mean .", "In this case , we report the mean bootstrap estimate of the mean ± the standard error of this estimate .", "Pearson’s correlation coefficients were determined to compare maximum distances moved from baseline for each body part ( Figure 4F ) .", "Experimental units and n values are indicated in the figure legends ." ] ]

[ "Somatosensory stimuli guide and shape behavior , from immediate protective reflexes to longer-term learning and higher-order processes related to pain and touch .", "However , somatosensory inputs are challenging to control in awake mammals due to the diversity and nature of contact stimuli .", "Application of cutaneous stimuli is currently limited to relatively imprecise methods as well as subjective behavioral measures .", "The strategy we present here overcomes these difficulties , achieving ‘remote touch’ with spatiotemporally precise and dynamic optogenetic stimulation by projecting light to a small defined area of skin .", "We mapped behavioral responses in freely behaving mice with specific nociceptor and low-threshold mechanoreceptor inputs .", "In nociceptors , sparse recruitment of single-action potentials shapes rapid protective pain-related behaviors , including coordinated head orientation and body repositioning that depend on the initial body pose .", "In contrast , activation of low-threshold mechanoreceptors elicited slow-onset behaviors and more subtle whole-body behaviors .", "The strategy can be used to define specific behavioral repertoires , examine the timing and nature of reflexes , and dissect sensory , motor , cognitive , and motivational processes guiding behavior ." ]

[ "To safely navigate their world , animals need to be able to tell apart a gentle touch from an eye-watering pinch , detect cold water or sense the throbbing pain stemming from an infected cut .", "These ‘somatic’ sensations are relayed through thousands of nerve endings embedded in the skin and other tissues .", "Yet the neurological mechanisms that underpin these abilities are complex and still poorly understood .", "Indeed , these nerve endings can be stimulated by extreme temperatures , harmful chemicals , friction or even internal signals such as inflammation .", "One event can also recruit many different types of endings: a cut for example , will involve responses to mechanical pressure , tissue damage and local immune response .", "To disentangle these different actors and how they affect behavior , scientists need to develop approaches that allow them to deliver specific stimuli with increased precision , and to monitor the impact on an animal .", "To achieve this goal , Schorscher-Petcu et al . used mice in which blue light could trigger specific types of nerve endings .", "For instance , depending on the genetic background of the animals , a laser could either activate nerve endings involved in pain or gentle touch .", "Crucially , this could be done from a distance by beaming light with exquisite precision onto the paws of the mice without physically touching or disturbing the animals .", "How the mice responded could then be observed without any interference .", "Their behavior was analyzed using a combination of high-speed videos , computer-driven recording systems , and machine learning .", "This revealed subtle changes in behavior that had not been detected before , spotting microscopic movements of the stimulated paw and mapping simultaneous whole-body movements such as changes in posture or head orientation .", "The approach therefore allows scientists to assess the impact of touch , pain or temperature sensation in freely behaving mice .", "It could also be harnessed to develop much needed treatments against chronic pain ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "microbiology and infectious disease" ]

[ [ "The expression of many bacterial genes is controlled by elements in the 5´ untranslated regions ( UTRs ) of mRNAs .", "Changes in the secondary structures of these cis-acting RNA elements lead to altered expression of the associated gene ( s ) by modulating accessibility of ribosomes to sites of translation initiation , accessibility of RNases , or premature transcription termination .", "The RNA secondary structure changes can occur in response to temperature ( RNA thermometers ) , translation of small upstream open reading frames ( uORFs ) , or the binding of trans-acting factors such as metabolites ( riboswitches ) , tRNAs , RNA-binding proteins such as CsrA , or small base-pairing RNAs ( sRNAs ) ( reviewed in Breaker , 2018; Kreuzer and Henkin , 2018; Loh et al . , 2018; Orr et al . , 2020; Romeo and Babitzke , 2019; Storz et al . , 2011 ) .", "Some of the regulatory events in 5´ UTRs are associated with premature transcription termination , which occurs by one of two mechanisms: intrinsic ( Rho-independent ) or Rho-dependent ( reviewed in Roberts , 2019 ) .", "Intrinsic termination requires only RNA polymerase and an RNA hairpin followed by a U-rich tract in the nascent RNA .", "Rho-dependent termination requires the loading of the hexameric Rho protein complex onto nascent , untranslated RNA at Rho utilization ( Rut ) sites that are typically C-rich , G-poor , and unstructured sequences ( reviewed in Mitra et al . , 2017 ) .", "Rho translocates along the RNA until the protein catches RNA polymerase and promotes transcription termination , typically between 100 and 200 nt downstream of the Rut site , leading to 3´ ends that are processed by 3´ to 5´exonucleases ( Dar and Sorek , 2018b; Wang et al . , 2019 ) .", "Several studies have sought to identify sites of transcription termination across the E . coli genome by sequencing RNA 3´ ends or by mapping the distribution of transcribing RNA polymerase ( Dar and Sorek , 2018b; Ju et al . , 2019; Peters et al . , 2012; Peters et al . , 2009; Yan et al . , 2018 ) .", "The vast majority of identified termination sites are in 3´ UTRs .", "In some studies , termination was compared in cells grown with/without the Rho inhibitor , bicyclomycin ( BCM ) , facilitating the identification of Rho termination sites ( Dar and Sorek , 2018b; Ju et al . , 2019; Peters et al . , 2012 ) .", "These data provided evidence for Rho termination of mRNAs in 3´ UTRs and spurious transcripts initiated within genes .", "While termination within 5´ UTRs was noted in some of these studies , extensive global characterization of premature termination has not been performed .", "Many of the uncharacterized 3´ ends in 5´ UTRs or open reading frame ( ORF ) -internal regions are likely to be the result of regulatory events , given that riboswitches ( Bastet et al . , 2017; Hollands et al . , 2012 ) , attenuators ( Ben-Zvi et al . , 2019; Gall et al . , 2016; Herrero Del Valle et al . , 2020; Konan and Yanofsky , 1997; Kriner and Groisman , 2015 ) , RNA-binding proteins ( Baniulyte et al . , 2017; Figueroa-Bossi et al . , 2014 ) , and sRNAs ( Bossi et al . , 2012; Sedlyarova et al . , 2016 ) have all been implicated in affecting premature Rho termination events .", "In addition to being the product of a regulatory event , RNA fragments generated by premature termination or RNase cleavage themselves can have functions as regulatory sRNAs .", "sRNAs commonly base pair with trans-encoded mRNAs , frequently with the assistance of the RNA chaperone protein Hfq , resulting in changes in the stability or translation of the target mRNA ( reviewed in Hör et al . , 2020 ) .", "Most sRNAs characterized to date are transcribed independent of other genes or are processed from mRNA 3´ UTRs , though a few 5´ UTR-derived sRNAs have been reported ( reviewed in Adams and Storz , 2020 ) .", "RNA fragments entirely internal to coding sequences ( Dar and Sorek , 2018a ) also have been suggested to function as regulators , though this has not been tested .", "While sRNAs generally base pair with mRNA targets , a few small transcripts have been shown to have roles as competing endogenous RNAs ( ceRNAs ) also known as ‘sponges’ , which base pair primarily with sRNAs , targeting the sRNAs for degradation or blocking their interactions with mRNA targets ( reviewed in Denham , 2020; Figueroa-Bossi and Bossi , 2018; Grüll and Massé , 2019 ) .", "To systematically identify new regulatory elements in E . coli , we globally mapped RNA 3´ ends , and specifically characterized those ends in 5´ UTRs and ORF-internal regions .", "We compared this 3´ end dataset with another dataset where BCM treatment was used to identify sites of Rho termination .", "Using these approaches , we detected hundreds of RNA 3´ ends within 5´ UTRs and internal to ORFs , likely generated by premature transcription termination or RNase processing .", "We propose the majority of these 3´ ends are the consequence of regulatory events , and we document regulation for multiple examples .", "For instance , we show 3´ ends are associated with the translation of uORFs , or result from the binding of some sRNAs to mRNA 5´ UTRs .", "Furthermore , we demonstrate that RNA fragments generated by premature transcription termination and from within coding sequences function as independent sRNA regulators; one as part of an autoregulatory loop and another that connects cell division to the cell envelope stress response .", "These findings reveal extensive and diverse regulation through premature transcription termination and RNase processing of mRNAs , which can lead to the generation of RNA by-products with independent functions ." ], [ "Two independent cultures of wild-type E . coli MG1655 ( WT ) were grown to OD600 ~0 . 4 in rich ( LB ) medium , OD600 ~2 . 0 in LB , and OD600 ~0 . 4 in minimal ( M63 ) glucose medium .", "Total RNA was isolated and analyzed using modified RNAtag-seq ( Shishkin et al . , 2015 ) ( total RNA-seq ) and 3´ end ( Term-seq ) protocols ( Dar et al . , 2016; Figure 1—figure supplement 1 ) .", "The replicate total RNA-seq and Term-seq datasets were highly correlated ( Figure 1—figure supplement 2A ) .", "Using the Term-seq data , we curated a list of dominant RNA 3´ ends ( Supplementary file 1 ) .", "The total numbers of identified 3´ ends were 1175 and 882 for cells grown in LB to OD600 ~0 . 4 or 2 . 0 , respectively , and 1053 for cells grown in M63 glucose to OD600 ~0 . 4 ( Figure 1—figure supplement 2B ) .", "The detected 3´ ends were further subclassified ( see Materials and methods for details ) according to their locations relative to annotated genes ( Figure 1A ) .", "3´ ends that could not be assigned to one unique category were counted in multiple categories .", "For cells grown to OD600 ~0 . 4 in LB , this analysis revealed that , while 23% of 3´ ends mapped <50 bp downstream of an annotated gene ( primary 3´ ends ) , hundreds ( 58% ) were classified as orphan and internal 3´ ends , many mapping upstream of , and within ORFs ( Figure 1B ) .", "We compared the detected 3´ ends to those identified by three other RNA-seq based studies ( Dar and Sorek , 2018b; Ju et al . , 2019; Yan et al . , 2018 ) .", "Note that the number of overlapping 3´ ends differs depending on the direction of the comparison ( is non-commutative ) because multiple 3´ ends from one dataset can be close to a single 3´ end from the other dataset .", "While there was significant overlap between the 3´ ends identified in our work and those identified in each of the other studies ( Figure 1—figure supplement 2C , E , F; hypergeometric test p<2 . 2e−16 in all three cases ) , the majority were unique to our study .", "Given that a previous E . coli Term-seq study only reported 3´ ends downstream of annotated genes ( Dar and Sorek , 2018b ) , we used our 3´ end calling algorithm to re-analyze the sequencing data from this study ( Figure 1—figure supplement 2D ) .", "Again , there was significant overlap ( hypergeometric test p<2 . 2e−16 ) , but 51% were unique to our study .", "Here , we focused on , and characterized 3´ ends mapping upstream of , and internal to , coding sequences , most of which were not detected in the other RNA-seq studies .", "Concurrently , we mapped instances of Rho-dependent transcription termination .", "One E . coli MG1655 culture grown to OD600 ~0 . 5 in LB was split with half left untreated and the other half treated with 100 µg/ml BCM for 15 min .", "Total RNA was isolated and analyzed using a DirectRNA-seq protocol ( Ozsolak and Milos , 2011; Figure 1—figure supplement 1 ) , a method optimized for sequencing very short reads ( 20–30 nt ) directly from RNA , an advantage for examining the effect of Rho on the generation of small RNA fragments .", "Using these data , we calculated a ‘Rho score’ for each genomic position by comparing DirectRNA-seq coverage in 800 nt windows upstream and downstream in the treated ( +BCM ) and untreated ( -BCM ) samples ( see Materials and methods ) .", "This ratio reflects the degree of transcriptional Rho readthrough in the +BCM cells , where a score of >2 . 0 is indicative of at least a two-fold increase in readthrough in the +BCM compared to the -BCM sample .", "1078 genomic regions were putatively associated with a Rho termination event ( Supplementary file 2 ) .", "We note that these genomic positions are likely closer to processed RNA 3´ ends than the termination sites , since Rho-terminated transcripts typically are processed by 3´ to 5´ exonucleases ( Dar and Sorek , 2018b; Wang et al . , 2019 ) .", "Hence , we refer to the identified genomic locations as ‘Rho termination regions’ .", "As for 3´ ends mapped by Term-seq , we classified Rho termination regions by their position relative to annotated genes ( Figure 1C ) .", "3´ ends antisense to annotated genes represented the largest category ( 51 . 5% ) , consistent with significant Rho termination of antisense RNAs ( Peters et al . , 2012 ) .", "Only a small percentage ( 4 . 5% ) of the Rho termination regions are <50 nt downstream of an annotated gene ( primary ) , likely because Rho loading and termination typically requires >50 nt untranslated RNA ( reviewed in Mitra et al . , 2017 ) .", "As for the 3´end-mapping by Term-seq , many Rho termination regions ( 44% ) were classified as orphan and internal , frequently mapping upstream of , or within ORF sequences .", "The C:G nucleotide usage was calculated for the putative Rho termination regions ( Supplementary file 2 ) , as well as for a control group of randomly selected genomic coordinates .", "Relative to the control group , there was a higher local C:G ratio within 200 nt of the 3´ ends associated with Rho termination regions ( Figure 1D ) , consistent with enrichment for C-rich , G-poor sequences attributed to Rut sites ( reviewed in Mitra et al . , 2017 ) .", "We also compared the Rho termination regions ( Supplementary file", "2 ) to the sites of Rho termination reported in three previous genome-wide studies ( Dar and Sorek , 2018b; Ju et al . , 2019; Peters et al . , 2012; Figure 1—figure supplement 2G ) .", "Again , there was significant overlap with each of the three previous studies ( hypergeometric test p<2 . 2e−16 in all cases ) , though many ( 43% ) of the putative Rho termination regions we identified are >500 bp away from any of the previously identified Rho termination regions ( Dar and Sorek , 2018b; Ju et al . , 2019; Peters et al . , 2012 ) .", "The large sets of Rho termination regions that differ between the studies suggest that much remains to be learned about Rho-dependent termination .", "The comparison of our Term-seq LB 0 . 4 dataset ( Supplementary file", "1 ) with our DirectRNA-seq LB 0 . 5 dataset ( Supplementary file", "2 ) revealed that there was significant overlap ( hypergeometric test p<2 . 2e−16 ) , with 34 . 6% of the Term-seq 3´ ends within 500 nt of a Rho termination region ( Figure 1—figure supplement 2H ) .", "This suggests that these 3´ ends are associated with Rho-terminated transcripts .", "Many 3´ ends identified by Term-seq mapped upstream or internal to annotated mRNAs ( Supplementary file 1 ) .", "We specifically focused on these 3´ ends ( see Materials and methods ) , since we hypothesized they could be reflective of regulatory events .", "Our analysis identified 665 and 507 3´ ends for the LB 0 . 4 and LB 2 . 0 samples , respectively , and 580 3´ ends for the M63 0 . 4 sample ( Supplementary file 3 ) .", "Among the 3´ ends in 5´ UTRs , several correspond to sites of known cis-acting RNA regulation ( annotated in Supplementary file 3 ) .", "These include mRNAs that previously have been shown to be regulated by premature Rho-dependent termination , such as the riboswitch-regulated genes thiM ( Bastet et al . , 2017; Figure 2—figure supplement 1A ) , mgtA ( Hollands et al . , 2012 ) , ribB ( Hollands et al . , 2012 ) and lysC ( Bastet et al . , 2017 ) , and the translationally-repressed genes ilvX ( Lawther and Hatfield , 1980 ) and topAI ( Baniulyte and Wade , 2019 ) .", "We also noticed that even some 5´ UTRs where regulation has only been reported to be at the level of translation , such as the RNA thermometer upstream of rpoH ( Morita et al . , 1999a; Morita et al . , 1999b ) and at ribosomal protein operons ( reviewed in Zengel and Lindahl , 1994 ) , harbored defined 3´ ends in 5´ UTRs .", "For the LB 0 . 4 Term-seq dataset , for which the growth conditions were most similar to those of the DirectRNA-seq dataset , we determined whether each 3´ end was associated with a detected Rho termination event .", "Significant Rho scores are listed in Supplementary file 3 ( see Materials and methods for details ) .", "It should be noted that some genes containing 3´ ends with significant Rho scores in Supplementary file 3 are absent from Supplementary file 2 , because the genomic position with the highest Rho score in that region is located in a neighboring gene/region , and thus given a different gene designation .", "We also used a previously described quantitative model to score putative intrinsic terminators , where a score >3 . 0 is predictive of intrinsic termination ( Chen et al . , 2013 ) .", "Based on these analyses for the 3´ ends for the LB 0 . 4 dataset in Supplementary file 3 , we predict that 20% have secondary structures and sequences consistent with intrinsic termination ( intrinsic terminator score ≥3 . 0 ) , 11% have detectable Rho termination , and the remaining 3´ ends are likely the result of RNA processing .", "However , the number of Rho termination sites could be an underestimate , because inhibition of Rho leads to extensive readthrough transcription from very strongly transcribed genes that can mask overlapping transcripts , and 3´ ends generated by Rho termination are typically unstable ( Dar and Sorek , 2018b; Wang et al . , 2019 ) .", "Some 3´ends also may be generated by a combination of mechanisms .", "Nonetheless , overall , our data suggest that modulation of premature transcription termination in 5´ UTRs or ORFs is a widespread regulatory mechanism .", "Several genes harboring Rho-dependent 3´ ends within the 5´ UTR or ORF have not been previously described as being regulated by Rho but are associated with characterized cis-acting RNA regulators .", "Examples are the sugE ( gdx ) and moaA genes , which are preceded by the guanidine II riboswitch ( Sherlock et al . , 2017 ) and molybdenum cofactor riboswitch ( Regulski et al . , 2008 ) , respectively .", "A browser image of the RNA-seq data for the sugE locus in the LB 0 . 4 condition documents a predominant 3´ end 76 nt downstream the sugE transcription start site ( TSS ) ( Figure 2A ) .", "This region was associated with significant readthrough in the +BCM DirectRNA-seq sample and a 3´ end Rho score of 3 . 7 ( Supplementary file 3 ) , strongly suggesting that the riboswitch impacts Rho-dependent premature termination , as is the case for other riboswitches .", "While the involvement of 5´ UTRs in sugE and moaA regulation was known , most of the genes for which we found 3´ ends in 5´ UTRs or ORFs have not been reported to have RNA-mediated regulation ( Supplementary file 3 ) .", "In some cases , such as the mdtJI mRNA , encoding a spermidine efflux pump , we observed a novel 3´ end that is clearly Rho-dependent ( Figure 2B ) with a Rho score of 2 . 3 .", "In other cases , such as ispU ( uppS ) , encoding the undecaprenyl pyrophosphate synthase , a novel 3´ end was observed with no readthrough upon Rho inhibition ( Figure 2C; Rho score of 0 . 7 ) .", "Rather , this 3´ end had an intrinsic terminator score ( Chen et al . , 2013 ) of 14 . 1 ( Supplementary file 3 ) , and we predicted a 6 bp stem-loop followed by eight U residues , consistent with intrinsic termination .", "To further test whether genes associated with 3´ ends in 5´ UTRs or ORFs are indeed regulated by premature Rho-dependent transcription termination , we generated lacZ transcriptional reporter fusions using the entire 5´ UTR and ORF for 27 genes ( Supplementary files 2 and 3 ) arbitrarily chosen for a range of calculated Rho scores and possible regulation .", "All constructs had the same constitutive promoter .", "β-galactosidase activity was assayed in the context of a WT E . coli background or a mutant strain with an R66S substitution in Rho ( rhoR66S ) , which disrupts the primary RNA-binding site ( Baniulyte et al . , 2017; Bastet et al . , 2017; Martinez et al . , 1996 ) .", "A fusion to thiM , which harbors a 5´ UTR Rho-dependent terminator ( Bastet et al . , 2017 ) , exhibited significantly higher levels of β-galactosidase activity in the Rho mutant strain , indicative of a disruption in Rho-dependent termination ( Figure 2—figure supplement 1B ) .", "Northern analysis identified an RNA consistent with the 3´ end identified by Term-seq ( Figure 2—figure supplement 1C ) , though we noted the abundance of this 5´ RNA fragment and extent of readthrough in the Rho R66S mutant varied with growth phase .", "Among the other constructs assayed , the expression of 14 fusions was >2-fold higher in rhoR66S compared to WT cells ( Figure 2D and Figure 2—figure supplement 1D ) , consistent with Rho termination in the 5´ UTR or ORF for sugE , cfa , cyaA , mdtJ , add , cspB , cspG , moaA , pyrG , yhaM , ydjL , yhiI , ytfL , and yajO .", "The effect of the Rho mutation on the eptB , chiP , and crp-yhfK fusions was intermediate ( 1 . 6- to 1 . 8-fold ) ( Figure 2D ) , while the fusions to ispU ( Figure 2D ) , as well as mnmG , rpsJ , argT , srkA , and trmL ( Figure 2—figure supplement 1D ) , displayed similar levels of β-galactosidase activity ( 1 . 0- to 1 . 4-fold ) for rhoR66S compared to WT cells .", "These assays support the notion that the 3´ end observed in the ispU 5´ UTR ( Figure 2C ) is generated by intrinsic termination .", "It is unclear why we did not detect evidence for Rho termination of mnmG , rpsJ , argT , srkA or trmL , despite these genes having significant Rho scores .", "Interestingly , fusions to the ompA , yebO , glpF , and rimP 5´ UTR and ORF had decreased expression in the Rho mutant background ( Figure 2—figure supplement 1D ) .", "This may be a consequence of additional levels of regulation or indirect effects of Rho inhibition .", "The ompA and glpF genes were not associated with Rho termination by DirectRNA-seq .", "Transcriptional lacZ reporter gene fusions to only the 5´ UTR ( i . e . without the ORF ) were also generated for seven genes , to distinguish between Rho termination in the ORF and in the 5´ UTR .", "The effect of rhoR66S was eliminated for the shorter cyaA and eptB fusions ( Figure 2E ) , suggesting that Rho-dependent termination occurs within the coding sequence of these genes .", "Regulation of Rho termination in 5´ UTRs is probably associated with the accessibility of Rut sequences , whereas Rho termination within coding sequences is probably associated with regulated translation initiation ( reviewed in Kriner et al . , 2016 ) , with translational repression indirectly leading to Rho termination .", "Finally , using RNA extracted from WT and rhoR66S strains grown to OD600 ~0 . 4 and 2 . 0 in LB , northern analysis was performed with probes for the 5´ UTRs of sugE , cfa , cyaA , speA , mdtJ , eptB and ispU ( Figure 2F ) as well as with probes for the coding sequences of cfa , cyaA , speA and mdtJ ( Figure 2—figure supplement 1E ) .", "For all of the mRNAs , we detected 5´ fragments that likely correspond to the 3´ ends detected by Term-seq ( indicated with an asterisk ) ( Supplementary file 3 ) .", "For sugE and cfa , however , the dominant band on the northern blot was not necessarily the most dominant 3´ end sequenced using Term-seq .", "The enrichment for the 5´ fragments as well as longer transcripts likely corresponding to the full-length sugE , cfa , cyaA , speA and mdtJ mRNAs ( indicated with an arrow ) in the rhoR66S samples reflect transcriptional readthrough in the mutant background .", "This is consistent with Rho-dependent termination as seen for the lacZ fusions .", "The effect of rhoR66S on eptB was intermediate , while no effect was observed for ispU .", "For all of the Rho-terminated genes , we were surprised to observe the significant increase in the levels of short transcripts , as seen most strikingly for speA .", "This suggests that the detected 3´ ends can be generated by RNA processing from both Rho-terminated and full-length transcripts , with the increased abundance of longer transcripts in the rhoR66S samples leading to higher levels of the processed product .", "We also noted that the effects of the rho mutation varied under the different growth conditions tested , as is most apparent for cfa and cyaA .", "Collectively , these data validate premature termination in 5´ UTRs and , in several cases , suggest complex regulation .", "The mdtJI mRNA , encoding a spermidine exporter , has a long 5´ UTR ( TSS located 278 nt from the start codon ) ( Figure 3A ) and a 3´ end that mapped six nt into the ORF ( Figure 2B ) .", "Additionally , the transcript is subject to premature Rho termination ( Figure 2D–F , Figure 2—figure supplement 1E ) .", "The levels of the mdtJI transcript were previously reported to increase in response to high levels of spermidine , a polyamine ( Higashi et al . , 2008a ) , though a mechanism for this regulation was not described .", "Polyamines play important roles in RNA-mediated regulation , reported to cause structural changes to the 5´ UTR of the oppA mRNA ( Higashi et al . , 2008b; Yoshida et al . , 1999 ) and induce ribosome stalling at a uORF in the 5´ UTR of the speFL mRNA ( Ben-Zvi et al . , 2019; Herrero Del Valle et al . , 2020 ) .", "In light of these studies , we examined the effect of spermidine on mdtJI mRNA levels .", "Total RNA was extracted from cells grown in LB medium with or without 10 mM spermidine at either neutral or high pH ( spermidine and other polyamines have a stronger negative effect on cell growth at high pH Yohannes et al . , 2005 ) .", "Northern analysis of these samples probed for the mdtJI mRNA revealed a ~280 nt transcript , consistent with the 3´ end detected by Term-seq ( Supplementary file 3 ) , that was susceptible to readthrough upon the addition of spermidine for cells grown at high pH ( Figure 3B ) .", "We therefore hypothesized that spermidine inhibits premature Rho termination of the mdtJI mRNA .", "Closer inspection of the mdtJI 5´ UTR revealed a putative upstream small ORF ( uORF ) of 34 codons with the stop codon 106 nt upstream of the mdtJ AUG ( Figure 3A ) .", "Translation of this uORF was previously detected by ribosome profiling , with expression of the corresponding small protein ( YdgV ) documented by western blot analysis ( Weaver et al . , 2019 ) .", "To independently verify translation of the uORF , the coding sequence was translationally fused to lacZ together with the upstream sequence and native mdtUJI promoter .", "Robust β-galactosidase activity was detected for cells carrying the fusion ( Figure 3C ) .", "By contrast , no β-galactosidase activity was observed for cells carrying an equivalent fusion with the uORF start codon mutated ( ATG→ACG ) , supporting the conclusion that the uORF ( ydgV ) , here renamed mdtU , is indeed translated .", "To investigate the role of mdtU in spermidine-mediated regulation of mdtUJI , the mdtU start codon mutation ( ATG→ACG ) was introduced on the chromosome of a strain where a 3XFLAG tag was translationally fused to C-terminus of MdtJ .", "Northern and western blot analysis of strains encoding mdtUJ-3XFLAG-I showed mRNA and protein levels were strongly induced by spermidine , which was abolished in the strain with the mdtU start codon mutation ( Figure 3D ) .", "We suggest that translation of MdtU is critical for spermidine-mediated expression of MdtJ .", "Northern analysis was also carried out to determine if Rho termination in the mdtJI 5´ UTR impacts the induction by spermidine .", "In the rhoR66S strain , the spermidine-dependent increase in full-length mdtUJI mRNA levels was substantially reduced relative to WT cells ( Figure 3E ) .", "However , inhibition of Rho did not completely abolish the stimulatory effect of spermidine on mdtUJI mRNA levels , perhaps because growth in spermidine and high pH may increase transcription initiation of mdtUJI .", "Together , these data support the model that spermidine , Rho , and translation of the mdtU uORF affect the levels of MdtJI and hence spermidine transport , though the mechanisms deserve further study .", "A screen for uORFs upstream of mdtJ orthologs in other gammaproteobacterial species showed that MdtU , particularly the C-terminal region , is conserved in at least 17 genera ( Figure 3—figure supplement 1 ) .", "This suggests that mdtU-dependent regulation of mdtUJI expression is a conserved process that may depend on the sequence of the MdtU C-terminus .", "The presence of 3´ ends downstream of putative uORFs could be a way to identify new uORF-dependent regulatory sequences .", "A search for 3´ ends located <200 nt downstream of experimentally validated , but uncharacterized potential uORFs ( Hemm et al . , 2008; VanOrsdel et al . , 2018; Weaver et al . , 2019 ) revealed nine examples: ybgV-gltA , yhiY-yhiI , ykiE-insA-7 , yliM-ompX , ymdG-putP , ymiC-acnA , yqgB-speA , and ytgA-iptF ( Supplementary file 3 ) , consistent with this suggestion .", "The vast majority of characterized regulatory binding sites for sRNAs are in 5´ UTRs , and we observed several RNA 3´ ends in 5´ UTRs near positions of documented sRNA base pairing ( Supplementary file 3 ) .", "For instance , the eptB , ompA and chiP mRNAs , which are targets of MgrR ( Moon and Gottesman , 2009 ) , MicA ( Udekwu et al . , 2005 ) and ChiX ( Figueroa-Bossi et al . , 2009 ) , respectively , all had 3´ ends directly downstream of the sequences involved in sRNA base pairing ( Figure 4A and Figure 4—figure supplement 1 ) .", "The presence of RNA 3´ ends at these positions suggested that stable 5´ mRNA fragments could be generated or perhaps protected by sRNA-mediated regulation .", "Indeed , 5´ transcripts for eptB and chiP were previously detected by total RNA-seq ( Dar and Sorek , 2018a ) , and the chiP 5´ transcript was reported to accumulate in the absence of the 3´-to-5´ phosphorolytic exoribonuclease PNPase ( Cameron et al . , 2019 ) .", "To examine how sRNAs impacted the 5´ derived mRNA fragments of eptB , ompA , and chiP , we used northern analysis to examine the consequences of deleting or overexpressing the cognate sRNA gene .", "Two oligonucleotide probes , one within the coding sequence ( downstream of the 3´ end identified using Term-seq ) and one within the 5´ fragment , were used to determine the relative levels of the full-length mRNAs ( Figure 4B ) and 5´ fragments ( Figure 4C ) .", "As expected , given the known sRNA-mediated downregulation of eptB , ompA and chiP , the levels of the target mRNAs were elevated in the sRNA deletion background compared to the WT strain and decreased with sRNA overexpression ( Figure 4B ) .", "Some other RNA species were detected for eptB ( ~400 nt ) , ompA ( <3000 nt ) , and chiP ( ~200–500 nt ) , but these did not match the expected sizes for the mRNAs , and may be degradation and/or readthrough products .", "For the 5´ UTR region of eptB , there was a reciprocal effect of the ∆mgrR background , with a strong decrease in the abundance of a ~140 nt band ( Figure 4C ) .", "Given that we observed a moderate effect of Rho mutation on eptB expression ( Figure 2D , F ) , we speculate that MgrR base pairing both promotes Rho termination and protects the resultant RNA from exonucleases .", "For the 5´ UTR region of ompA , which showed only modest de-repression in the ∆micA strain , there was a decrease in the abundance of an ~120 nt fragment in the deletion strain ( Figure 4C ) , and the levels of this fragment increased upon MicA overexpression .", "These effects are consistent with MicA sRNA-directed cleavage of the ompA mRNA generating the fragment , or the sRNA base pairing protecting the 3´ end from exonucleolytic processing .", "The effect of the ∆chiX background on the chiP 5´ fragment was strikingly different .", "Instead of decreasing , there was a large increase in the levels of an ~90 nt RNA ( Figure 4C ) .", "Given the strong signal detected for this transcript , we hypothesized that this RNA might have an independent role as a regulatory RNA .", "To test the hypothesis that 5´ UTR transcripts with defined bands have independent functions as sRNAs , we carried out further studies on the ~90 nt chiP 5´ UTR transcript ( Figure 4C ) , which we renamed ChiZ , and the 81 and 60 nt ispU 5´ UTR transcripts ( likely expressed from two TSSs with a shared 3´ end , Figure 2C and F ) , denoted IspZ ( Figure 5A ) .", "To obtain more information about the expression of these putative sRNAs , we performed northern analysis using the same RNA analyzed in the Term-seq experiment ( Figure 5B ) .", "Distinct bands were detected for the two 5´ UTRs , consistent with the generation of stable RNAs with predicted stems protecting the ends ( Figure 5—figure supplement 1A–B ) .", "While ChiZ was most abundant in cells grown to exponential phase in LB , IspZ was abundant in LB and M63 glucose medium in both exponential and stationary phase .", "Since many sRNA levels are negatively affected by the lack of the RNA chaperone Hfq ( reviewed in Hör et al . , 2020 ) , we also conducted northern analysis using RNA extracted from WT or Δhfq cells across growth in LB ( Figure 5B ) .", "Similar to other base-pairing sRNAs , and consistent with Hfq binding , ChiZ abundance was low in the ∆hfq background .", "IspZ levels were only slightly affected by the absence of Hfq , even though this RNA is bound by Hfq ( Melamed et al . , 2020 ) .", "Given that the binding of the sRNA ChiX to the mRNA chiP ( containing ChiZ ) increases Rho-mediated regulation of chiP ( Bossi et al . , 2012 ) , we tested the role of Rho on ChiZ levels ( Figure 6A ) .", "The effects of the rhoR66S mutant were dependent on growth phase , with a decrease in ChiZ for cells grown in LB to OD600 ~0 . 4 , when ChiZ levels are highest .", "In contrast , IspZ levels were not affected by the rhoR66S mutant ( Figure 2F ) , and IspZ is likely subject to intrinsic termination as stated previously .", "Given their association with Hfq , we tested the independent functions of ChiZ and IspZ as base-pairing sRNAs .", "Since the region of chiP encoding ChiZ has been documented to be a target for base pairing with the sRNA ChiX through compensatory mutations ( Figueroa-Bossi et al . , 2009; Overgaard et al . , 2009 ) , we postulated that ChiZ reciprocally regulates ChiX , sponging its base-pairing activity .", "To test this possibility , we assayed the effects of ChiZ overexpression .", "As for chromosomally-encoded ChiZ ( Figure 5B ) , longer transcripts were observed for plasmid-encoded ChiZ , likely due to readthrough , but only the levels of the 90 nt ChiZ band were strongly reduced in the ∆hfq mutant ( Figure 5—figure supplement 1C ) .", "Upon ChiZ overexpression in the WT background , we observed increased levels of the chiP mRNA , with a reciprocal change in ChiX levels ( Figure 6B ) .", "The levels of the chiP mRNA overall were higher in the Δhfq mutant background , but we no longer observed an increase upon ChiZ overexpression , likely due to the instabilities of ChiX and ChiZ .", "We also observed upregulation of a PBAD-chiP-lacZ chromosomal translational fusion ( Schu et al . , 2015 ) upon ChiZ overexpression in a WT but not a ∆chiX background ( Figure 6C ) .", "These observations support a novel sRNA regulatory network in which an mRNA ( chiP ) that is the target of an sRNA ( ChiX ) produces an RNA fragment ( ChiZ ) that reciprocally sponges the sRNA ( ChiX ) ( Figure 6D ) .", "We expected IspZ also might function as a base-pairing sRNA , and thus searched for potential targets identified by RIL-seq ( Melamed et al . , 2020; Melamed et al . , 2016 ) , an approach where RNAs in proximity on an RNA-binding protein are identified by co-immunoprecipitation , ligation , and sequencing of the chimeras .", "The predominant target for IspZ in these datasets is the oxidative stress-induced sRNA OxyS ( Altuvia et al . , 1997 ) .", "This observation led us to test whether IspZ might act as a sponge of OxyS .", "As for chromosomally-encoded IspZ ( Figure 5 ) , and in contrast to ChiZ , little readthrough and no effect of ∆hfq was observed for plasmid-expressed IspZ ( Figure 5—figure supplement 1D ) .", "We examined the effect of IspZ overexpression on OxyS levels in cells treated with 0 . 2 mM hydrogen peroxide , a condition known to induce OxyS expression ( Altuvia et al . , 1997 ) .", "High levels of IspZ were associated with slightly lower OxyS levels , in line with sponging activity ( Figure 6E ) .", "To obtain evidence for direct base pairing between IspZ and OxyS , IspZ was mutated on the overexpression plasmid ( IspZ-M1 ) , and compensatory mutations were introduced into the chromosomal copy of OxyS ( OxyS-M1 ) .", "Consistent with the predicted pairing ( Figure 6F ) , IspZ-mediated down-regulation was eliminated with IspZ-M1 or OxyS-M1 alone but was restored when both mutations were present ( Figure 6G ) .", "We also noted examples of abundant 3´ ends internal to ORFs ( Supplementary file 3 ) , downstream of nearby 5´ ends previously identified by dRNA-seq ( Thomason et al . , 2015 ) , and associated with a strong signal in total RNA-seq ( Figure 7 ) .", "A previous study inferred from total RNA-seq data that some sRNAs might be derived from sequences internal to ORFs ( Dar and Sorek , 2018a ) .", "To test whether we could detect defined transcripts for these internal ( int ) signals , we selected candidate RNAs derived from the ftsI ( renamed FtsO ) , aceK , rlmD , mglC , and ampG ORFs for further investigation ( Figure 7A ) .", "Analysis of dRNA-seq data ( Thomason et al . , 2015 ) suggested that the FtsO , aceK int and ampG int 5´ ends likely are generated by RNase processing of the overlapping mRNA , whereas rlmD int and mglC int likely are transcribed from promoters internal to the overlapping ORFs .", "In nearly all cases , the RNA 3´ ends are not predicted to be due to Rho-dependent transcription termination events ( Supplementary file 3 ) , strongly suggesting they are generated by RNase processing or , for aceK int , intrinsic termination ( intrinsic terminator score = 5 . 9 , Supplementary file 3 ) .", "Northern analysis was performed for these RNAs using the same RNA analyzed in the Term-seq experiment ( Figure 7B ) ; distinct bands were detected for all the RNAs tested .", "While FtsO and ampG int were relatively abundant under all growth conditions tested , mglC int and rlmD int were only expressed in cells grown in LB , and aceK int was most abundant in LB at OD600 ~ 2 . 0 .", "We also conducted northern analysis using RNA extracted from WT or Δhfq cells across growth ( Figure 7B ) .", "The aceK int transcript was strongly dependent upon hfq , whereas the other RNAs were unaffected by hfq deletion , though all five transcripts have been reported to co-immunoprecipitate with Hfq ( Melamed et al . , 2020; Melamed et al . , 2016 ) .", "Additionally , the ORF-internal sRNAs are found in chimeras with other putative mRNA and sRNA targets in the Hfq RIL-seq datasets ( Melamed et al . , 2020; Melamed et al . , 2016 ) : FtsO and RybB , aceK int and ompF , rlmD int and MicA , mglC int and ArcZ , ampG int and CyaR .", "Significant chimeras also were detected between aceK int and gatY in the RIL-seq data set for the ProQ RNA chaperone ( Melamed et al . , 2020 ) .", "Collectively , these data suggest these ORF-internal transcripts have independent regulatory functions .", "To test for the suggested regulatory function , we focused on FtsO , which is encoded internal to the coding sequence of the essential cell division protein FtsI , and exhibited high levels across growth ( Figure 7B ) .", "The predominant target for FtsO in the RIL-seq datasets was the sRNA RybB ( Figure 8A ) followed by the sRNA CpxQ ( Melamed et al . , 2020 ) .", "The Hfq-mediated FtsO-RybB interaction was also detected in an independent CLASH dataset ( Iosub et al . , 2020 ) .", "We hypothesized that FtsO functions as a sponge for RybB and CpxQ , which are induced by misfolded outer membrane proteins and inner membrane proteins , respectively , and down-regulate the corresponding classes of proteins ( reviewed in Hör et al . , 2020 ) .", "This model was tested by overexpressing FtsO in WT or Δhfq cells grown to exponential and/or stationary phase ( 150 and 360 min after subculturing ) .", "For the 360 min time point when the levels of both RybB and CpxQ are highest , a reduction was observed for both sRNAs in cells overexpressing FtsO ( Figure 8B ) .", "As reported previously , the levels of RybB and CpxQ are significantly lower in the ∆hfq strain , though some FtsO-dependent downregulation of RybB is still detected .", "Interestingly , we also observed that RybB overexpression had the reciprocal effect of decreasing FtsO levels , though only at the 360 min timepoint ( Figure 8—figure supplement 1A ) .", "To confirm the direct interaction between FtsO and RybB , we mutated three nucleotides in the site of predicted base pairing ( Figure 8C ) on the plasmid copy of ftsO .", "This mutation ( FtsO-M3 ) abolished the effect of FtsO overexpression on RybB levels ( Figure 8D ) .", "The repressive effect was similarly abolished by mutating the predicted site of base pairing in the chromosomal copy of rybB but was restored by combining the complementary mutations in ftsO and rybB ( Figure 8D ) .", "To test the downstream effect of sponging RybB , we examined the levels of the known RybB mRNA-target ompC ( Johansen et al . , 2006 ) compared the effects of RybB to the effects of the previously characterized RybB sRNA sponge , RbsZ ( Melamed et al . , 2020; Figure 8—figure supplement 1B ) .", "Both FtsO and RbsZ overexpression led to a decrease in RybB levels and a concomitant increase in ompC mRNA levels , though the effect of RbsZ was stronger .", "With both sRNA sponges , the level of OmpC protein was not obviously changed , likely because of the high levels of this protein .", "Finally , we mutated the chromosomal copy of ftsO to introduce the same nucleotide substitutions ( Figure 8C ) that are silent with respect to the FtsI amino acid sequence .", "WT cells and cells carrying these mutations were treated with ethanol , causing outer membrane stress , which is known to induce RybB ( Peschek et al . , 2019 ) .", "In both strains , a transient increase in RybB levels was observed 5 min after ethanol addition ( Figure 8E ) .", "In WT cells , RybB levels then were decreased for 30 min after ethanol treatment .", "By contrast , in cells with mutant ftsO , RybB levels again increased at 20 min following ethanol treatment .", "This effect was also observed in a second experiment ( Figure 8—figure supplement 1C ) that documented higher RybB levels up to 60 min following ethanol treatment in ftsO-M3 cells .", "We also assessed the consequences of FtsO sponging RybB on the levels of ompC for the same RNA samples .", "In both experiments , the ompC mRNA levels decreased after cells were treated with ethanol but for the ftsO-M3 mutant strain there was a further decrease at the 20 min time point ( Figure 8E ) .", "Along with RbsZ and the 3´ETSleuZ tRNA fragment ( Lalaouna et al . , 2015 ) , FtsO is the third documented sponge of RybB .", "It is possible the other sponges mask some of the effects of FtsO .", "The conservation of ftsO was examined by aligning ftsI orthologs across 18 species of gammaproteobacteria , revealing a striking degree of ftsO conservation ( 91% average identity ) at ftsI wobble positions compared to the entire ftsI mRNA wobble positions ( 67% average identity ) ( Figure 8—figure supplement 2A ) .", "The region of base pairing parallels the conservation of the RybB seed sequence and is predicted to be within an unstructured region of FtsO ( Figure 8—figure supplement 2B and C ) .", "Collectively , our data suggest that FtsO base pairing with RybB is conserved , and lowers RybB levels and activity following outer membrane stress ( Figure 8F ) .", "The work demonstrates stable regulatory sRNAs can be derived from sequences internal to ORFs such that the same DNA sequence encodes two different functional molecules ." ], [ "Through our transcriptome-wide mapping of 3´ ends and Rho-dependent termination , we uncovered extensive RNA-mediated regulation and sRNA regulators encoded by 5´ UTRs and internal to ORFs .", "Other studies have previously identified RNA 3´ ends and regions of Rho-dependent termination in E . coli ( Dar and Sorek , 2018b; Ju et al . , 2019; Peters et al . , 2012; Yan et al . , 2018 ) .", "While there was considerable overlap between our work and these prior studies , there were also substantial differences .", "For example , reporter gene fusion data and northern analysis supported Rho termination of sugE , cfa , cyaA , speA , and mdtJ , of which , only sugE was detected in the previous genome-wide surveys ( Dar and Sorek , 2018b; Ju et al . , 2019; Yan et al . , 2018 ) .", "Some of the differences between studies can be attributed to differences in the E . coli strains , growth conditions , and methods used .", "Indeed , we previously found that small methodological differences have a large impact on the identification of mapped TSSs ( Thomason et al . , 2015 ) .", "Like any RNA-seq method , a few 3´ ends also could be due to RNA degradation during library preparation .", "Nevertheless , our follow-up experiments confirmed the biological relevance of several 3´ ends in 5´ UTRs and internal to ORFs .", "Given that strain and growth conditions used for our Term-seq and total RNA-seq match those of the previous dRNA-seq analysis ( Thomason et al . , 2015 ) in which we identified TSSs and 5´ processed ends , the combined sets represent a valuable resource for examining the E . coli transcriptome ( see Materials and methods for links to interactive browsers ) .", "The majority of the 3´ ends that we identified were classified as ‘internal’ or ‘orphan’ , most of which map within 5´ UTRs or internal to ORFs , and a significant number of which are predicted to be generated by premature transcription termination .", "This notion of widespread premature transcription termination has been underappreciated in other studies that detected RNA 3´ ends and Rho-mediated termination .", "It is generally not possible to identify the exact position of Rho termination due to post-transcriptional RNA processing .", "Nevertheless , our reporter assays showed that in most cases tested , Rho termination could be localized to the 5´ UTR , suggesting that modulation of Rut accessibility in 5´ UTRs could be a common mechanism of regulation .", "Presuming that many premature termination events are regulatory , we documented and characterized examples of novel , diverse regulatory events for several of the 3´ ends .", "Undoubtedly , additional unique regulatory mechanisms exist for many of the other 3´ ends .", "We propose that the identification of 3´ ends in 5´ UTRs and ORFs is an effective approach to discover novel regulatory elements .", "Classically , these regulators , such as riboswitches and attenuators , have been identified by serendipity , studies of individual genes , or searches for conserved RNA structures ( reviewed in Breaker , 2018 ) , but these approaches may miss regulatory RNA elements if the function of the downstream gene is unknown or the region is not broadly conserved .", "Given that Term-seq is a sensitive , relatively unbiased , and genome-wide approach , it is another means of obtaining evidence for regulation in 5´ UTRs .", "As an example , the Term-seq data showed that transcripts from the 5´ UTR of the E . coli glycerol facilitator glpF have different 3´ ends under LB and M63 growth conditions , which could be due to uncharacterized regulation .", "3´ end-mapping applied to E . coli grown under other conditions or other bacterial species should lead to the characterization of many more regulators , particularly in organisms such as the Lyme disease pathogen Borrelia burgdorferi ( Adams et al . , 2017 ) that lack any known cis-acting RNA elements .", "Consistent with broad applicability , Term-seq previously led to the identification of known riboregulators in Bacillus subtilis and Enterococcus faecalis , and a novel attenuator in Listeria monocytogenes ( Dar et al . , 2016 ) .", "A number of 3´ ends in 5´ UTRs and ORFs were found to be associated with uORFs .", "Our characterization of the mdtU uORF suggests that regulation of premature mdtJI transcription termination occurs in response to ribosome stalling induced by polyamines .", "Two recent studies showed that the polyamine ornithine can stall ribosomes immediately upstream of the stop codon of the speFL uORF , affecting Rho binding and the secondary structure of the speFL mRNA ( Ben-Zvi et al . , 2019; Herrero Del Valle et al . , 2020 ) .", "Strikingly , conservation of MdtU is strongest at the C-terminus , and overlaps a region of the mdtU RNA that is predicted to base pair with the mdtJI ribosome binding site .", "Thus , a mechanism similar to the one found for speFL may regulate mdtJI induction by spermidine , a hypothesis that deserves further study , together with other examples where 3´ ends are located downstream of uORFs .", "We also documented three instances of 3´ ends that localized a short distance downstream of known trans-acting sRNA base-pairing sites .", "These 3´ ends could be generated by endonuclease processing as a result of sRNA base pairing , or could be due to protection against exonucleases as a result of sRNA pairing .", "An examination of sRNA base-pairing sites predicted by RIL-seq points toward other instances of this type of regulation .", "For example , Term-seq identified 3´ ends immediately downstream of the predicted sRNA base-pairing regions for the uncharacterized mRNA-sRNA interactions rbsD-ArcZ , dctA-MgrR , and yebO-CyaR detected by RIL-seq chimeras ( Melamed et al . , 2020 ) .", "In all these instances , the 3´ ends could be a result of the sRNA regulatory effect , and in some cases , may result in the formation of a new sRNA , as we observed for ChiZ .", "In general , our data further illustrate the complex regulation that occurs once transcription has initiated .", "Previous studies have shown that intergenic regions and mRNA 3´ UTRs are major sources of regulatory sRNAs , with a few characterized examples of sRNAs derived from 5´ UTRs , and no characterized ORF-internal sRNAs ( reviewed in Adams and Storz , 2020 ) .", "Our data document that 5´ UTRs and ORFs can indeed encode functional base-pairing sRNAs .", "However , our work also raises important questions , including the mechanisms by which 5´ UTR-derived and ORF-internal sRNAs are generated .", "Given that sRNAs derived from 5´ UTRs only require the generation of a new 3´ RNA end ( likely sharing a TSS with their cognate mRNA ) , and are not usually constrained by codon sequences , they could evolve rapidly .", "We documented the formation of several 5´ UTR fragments by cis-regulatory events .", "These by-products of regulation could obtain independent regulatory functions , as has been reported for a few riboswitches and attenuators ( DebRoy et al . , 2014; Melior et al . , 2019; Mellin et al . , 2014 ) .", "The sRNA 3´ end can be formed by intrinsic termination or Rho-dependent termination and/or processing .", "Importantly , for the downstream mRNA to be expressed , there needs to be some transcriptional readthrough .", "RNA structure predictions strongly suggest the IspZ 3´ end is generated by intrinsic termination ( Supplementary file 3 ) for which we observed very little readthrough .", "In contrast , the ChiZ 3´ end is generated by Rho-dependent termination with significant readthrough that would allow chiP expression .", "The mechanisms that drive formation of either 5´-derived sRNAs or the corresponding full-length mRNAs could be regulated and are an interesting topic for future work .", "Less is known about both the 5´ and 3´ ends of the ORF-internal sRNAs .", "The ends might be generated by ORF-internal promoters , termination , or RNase processing .", "In cases where one or both sRNA ends are generated by processing , this is presumably coupled with down-regulation of the overlapping mRNA .", "Strikingly , the number of sequencing reads for FtsO is orders of magnitude higher than that for the ftsI mRNA .", "How and when FtsO is produced are interesting questions for future studies .", "It is possible that the ftsI mRNA is protected from cleavage by ribosomes during cell division such that FtsO is only generated in the absence of mRNA translation .", "Other coding sequence-derived sRNAs , such as the putative regulator internal to mglC , likely have their own TSS .", "In some cases , such as rlmD int and ampG int , a transcript originating from a TSS internal to the cognate mRNA could be processed to form the 5´ end of the sRNA .", "These observations underscore how the interplay of transcription initiation , transcription termination , and RNase processing leads to many short transcripts that have the potential to evolve independent regulatory functions .", "We identified and characterized three sRNA sponges that have 3´ ends either in 5´ UTRs or internal to the coding sequence .", "The first example , ChiZ-ChiX-chiP , represents a novel reciprocal autoregulatory loop .", "ChiZ is generated from the chiP 5´ UTR encompassing the site of pairing with the ChiX sRNA .", "We found ChiZ is formed by Rho-dependent termination , and in the absence of ChiX base pairing with chiP .", "When cells utilize chitobiose as a carbon source and ChiX levels are naturally low , there are higher levels of chiP ( Overgaard et al . , 2009 ) and likely also higher levels of ChiZ .", "In this model , when chitooligosaccharides need to be imported , ChiZ prevents ChiX from base pairing , and promotes degradation of ChiX .", "When metabolic needs shift , the levels of ChiZ could decrease , allowing ChiX to regulate chiP and other targets .", "This may work in competition , conjunction , or at separate times from the chbBC intergenic mRNA sequence , which also sponges and promotes decay of ChiX ( Overgaard et al . , 2009 ) .", "It will be interesting to see if other 5´ UTRs and sRNAs form similar autoregulatory loops , since 5´ UTRs are enriched for sRNA pairing sites , and we have shown that sRNA pairing is associated with distinct small transcripts from 5´ UTRs .", "In a second example , we characterized IspZ , which is generated from the 5´ UTR of ispU ( uppS ) , encoding the synthase for undecaprenyl pyrophosphate ( UPP ) , a lipid carrier for bacterial cell wall carbohydrates ( Apfel et al . , 1999 ) .", "We suggest IspZ may connect cell wall remodeling with the oxidative stress response .", "Cellular levels of toxic reactive oxygen species are increased when cell wall synthesis is blocked , and oxidative damage impedes ispU-related cell wall growth ( Kawai et al . , 2015 ) .", "Thus , IspZ downregulation of the hydrogen peroxide-induced sRNA OxyS may dampen the oxidative response at a time when the response might be detrimental .", "While small transcripts from within coding sequences have been noted previously ( Dar and Sorek , 2018a; Reppas et al . , 2006 ) , and a homolog of FtsO ( STnc475 ) has been detected for Salmonella enterica ( Smirnov et al . , 2016 ) , we are the first to document a regulatory role for a bacterial ORF-internal sRNA .", "FtsO was found to base pair with , and negatively regulate the membrane stress response sRNA , RybB .", "The ftsI mRNA encodes a low-abundance but essential penicillin-binding protein that is localized to the inner membrane at the division site and cell pole ( Weiss et al . , 1997 ) .", "The cell may need to alter its response to membrane stress during the division cycle when many membrane components are needed , and we suggest FtsO could facilitate crosstalk between cell division and membrane stress by regulating RybB activity .", "Intriguingly , we observed the greatest effect of ethanol addition on RybB levels in the ΔftsO background at 20 min , which is also the doubling time for E . coli MG1655 .", "While it is reasonable to assume that regulatory sRNAs encoded by intragenic sequences are rare , due to the challenge of encoding two functions in one region of DNA , we think it is likely that other ORF-internal sRNAs have function .", "Our work has significantly increased the number of sRNAs documented to modulate the activities of other sRNAs by sponging their activities , as found for ChiZ , or affecting their levels , as shown for IspZ and FtsO .", "The findings raise other questions including how many more short transcripts generated by termination or processing have regulatory functions .", "It also is intriguing that some abundant sRNAs are subject to regulation by multiple sponges , including ChiX , which is regulated by ChiZ and the chbBC intergenic region ( Overgaard et al . , 2009 ) , and RybB , which is regulated by FtsO , RbsZ ( Melamed et al . , 2020 ) and the 3´ETSleuZ tRNA fragment ( Lalaouna et al . , 2015 ) .", "Finally , little is known about how the activities and levels of the sponges themselves are regulated .", "The levels of some , but not others , are influenced by Hfq binding .", "A number appear to be constitutively expressed , such that target sRNA levels must increase to overcome the effects of the sponges .", "Further identification and characterization of RNA fragments generated by premature termination or processing , detected by mapping the 5´ and 3´ ends of bacterial transcriptomes , will help elucidate the effects of regulatory RNAs .", "Our datasets point to a plethora of potential cis- and trans-acting regulatory elements in 5´ UTRs and ORF-internal regions , providing a valuable resource for further studies of gene regulation ." ], [ "Derivatives of E . coli K12 MG1655 ( WT ) were used for all experimental studies .", "All strains , plasmids and oligonucleotides used are listed in Supplementary file 4 .", "Engineered mutations and plasmid inserts were verified by sequencing .", "The E . coli strains carrying mdtJ-3XFLAG were engineered using the FRUIT method ( Stringer et al . , 2012 ) .", "Briefly , a 3XFLAG-thyA-3XFLAG tag/selection marker was PCR-amplified with primers JW9000 + JW9001 and recombineered into strain AMD061 and then counter-selected for thyA loss due to recombination of the FLAG tags , resulting in the intermediate strain YY18 .", "This intermediate was used to create the mdtU start codon mutant ( ATG→ACG ) by recombineering the PCR-amplified thyA marker ( using primers JW10309 + JW10310 ) into the mdtU gene and then replacing the thyA marker with the mdtU mutation ( recombineering a PCR-amplified product made using primers JW10311 - JW10314 ) .", "The native thyA locus was restored as described previously ( Stringer et al . , 2012 ) .", "This resulted in the wild-type mdtU mdtJ-3XFLAG ( YY20 ) or the mdtU start codon ( ATG→ACG ) mutant mdtJ-3XFLAG ( AMD742 ) strain .", "The ΔmgrR::kan ( GSO769 ) , ΔmicA::kan ( GSO157 ) , and ΔchiX::kan ( GSO169 ) deletions ( Hobbs et al . , 2010 ) were transduced into MG1655 ( GSO982 ) by P1 transduction , resulting in GSO993 , GSO994 and GSO995 respectively .", "The oxyS-M1::kan ( GSO996 ) and rybB-M3::kan ( GSO997 ) strains were constructed by PCR-amplifying the kanR sequence in pKD4 ( Datsenko and Wanner , 2000 ) using primers PA313 + PA314 ( oxyS-M1 ) or PA218 + PA219 ( rybB-M3 ) and recombineering the product ( Datsenko and Wanner , 2000; Yu et al . , 2000 ) into the chromosome of E . coli NM400 ( kind gift of Nadim Majdalani ) .", "The ftsO-M3::kan ( GSO999 ) strain was constructed by first transforming a temperature sensitive ftsI Y380D mutant ( PA215 ) ( Dai et al . , 1993 ) , with pKD46 ( Datsenko and Wanner , 2000 ) .", "This strain was electroporated with an ftsO-M3 PCR product ( amplified from the ftsO-M3 geneblock ( Supplementary file 4 ) with primers PA216 + PA217 ) in the presence of 20 mM L-arabinose to induce the λ recombinase on pKD46 .", "Colonies were selected by plating at 45 °C .", "Colony PCRs ( using primers PA216 + PA217 ) and sequencing was performed to check for repair of the ftsI Y380D mutation and simultaneous ftsO-M3 incorporation ( GSO999 ) .", "A colony without the ftsO-M3 change was kept as a wild-type control ( GSO998 ) .", "All sRNA mutant alleles were subsequently transferred into E . coli MG1655 ( GSO982 ) by P1 transduction .", "The pMM1 β-galactosidase transcriptional reporter fusion plasmid was constructed by PCR-amplifying the high expression promoter KAB-TTTG ( Burr et al . , 2000 ) from pJTW064 ( Stringer et al . , 2014 ) using primers JW10252 + JW10253 , and the DNA was ligated into pAMD-BA-lacZ plasmid ( Stringer et al . , 2014 ) digested with the NsiI and HindIII restriction enzymes .", "Either the entire 5´ UTR and annotated ORF region , or 5´ UTR region alone for selected genes was PCR-amplified using the oligonucleotides listed in Supplementary file 4 , and cloned into the pMM1 vector , cut with NsiI and NheI restriction enzymes , using the NEBuilder HiFi kit ( NEB ) .", "The mdtU-lacZ translational fusions were constructed by PCR-amplifying the mdtU gene from either a WT ( E . coli MG1655 ) genomic template or mdtU start codon mutant ( ATG→ACG ) template ( AMD742 ) using primers JW7269 + JW8934 .", "These PCR products were subsequently ligated into the pAMD-BA-lacZ plasmid ( Stringer et al . , 2014 ) , cut with SphI and HindIII restriction enzymes , using the NEBuilder HiFi kit ( NEB ) .", "sRNAs were over expressed using the pBRplac plasmid ( Guillier and Gottesman , 2006 ) .", "sRNA sequences were PCR-amplified using the oligonucleotides listed in Supplementary file 4 , digested with AatII and EcoRI , and cloned into pBRplac digested with the same restriction enzymes .", "The ChiZ overexpression construct was engineered using the NEBuilder kit ( NEB ) , according to the manufacturer’s instructions with primers PA311 + PA312 and LW043 + LW044 and the pBRplac-lacI derivative , pNM46 ( kind gift of Nadim Majdalani ) .", "Bacterial strains standardly were grown with shaking at 250 rpm at 37 °C in either LB rich medium or M63 minimal medium supplemented with 0 . 2% glucose and 0 . 001% vitamin B1 .", "Ampicillin ( 100 µg/ml ) , kanamycin ( 30 µg/ml ) , chloramphenicol ( 30 µg/ml ) and/or IPTG ( 1 mM ) were added where appropriate .", "Unless indicated otherwise , overnight cultures were diluted to an OD600 of 0 . 05 and grown to the indicated OD600 or time point .", "E . coli cells corresponding to the equivalent of 10 OD600 were collected by centrifugation , washed once with 1X PBS ( 1 . 54 M NaCl , 10 . 6 mM KH2PO4 , 56 . 0 mM Na2HPO4 , pH 7 . 4 ) and pellets snap frozen in liquid N2 .", "RNA was isolated using TRIzol ( Thermo Fisher Scientific ) exactly as described previously ( Melamed et al . , 2020 ) .", "RNA was resuspended in 20–50 µl DEPC H2O and quantified using a NanoDrop ( Thermo Fisher Scientific ) .", "Two biological replicates of E . coli MG1655 ( GSO988 ) were diluted 1:500 from an LB overnight culture in either LB or M63 glucose media .", "Cells were collected at an OD600 ~0 . 4 and 2 . 0 for LB and an OD600 ~0 . 4 for M63 grown cultures .", "RNA was extracted as described above and analyzed using an Agilent 4200 TapeStation System to check the quality .", "Any contaminating DNA in the samples was removed by treating 15 µg of RNA with 10 U of DNase I ( Roche ) for 15 min at 37 °C in the presence of 80 U of recombinant RNase inhibitor ( Takara Bio ) .", "Next , RNA was purified by mixing the sample with an equal volume of phenol stabilized:chloroform:isoamyl alcohol ( 25:24:1 ) and centrifugation at maximum speed in Heavy Phase Lock Gel tubes ( 5 PRIME ) .", "A volume of chloroform , equal to the original sample volume , was added to the same Heavy Phase Lock Gel tubes and spun again .", "The aqueous layer was removed and ethanol precipitated in the presence of 15 µg GlycoBlue ( Ambion ) .", "RNA pellets were reconstituted in 10 µl DEPC H2O and analyzed using an Agilent 4200 TapeStation System to ensure DNase-treated RNA was at high quality .", "Term-seq libraries were prepared using a modified version of the RNAtag-seq methodology ( Shishkin et al . , 2015 ) , based on the previously published Term-seq methodology ( Dar et al . , 2016 ) .", "1 . 5 µg of DNA-free RNA was first ligated at the 3´ end with 150 µM barcoded oligonucleotide adapters which were 5´phosphorylated and dideoxycytidine 3´ terminated ( Supplementary file 4 ) .", "RNA and 3´ adapters were incubated at 22 °C for 2 . 5 hr with 51 U of T4 RNA Ligase I ( NEB ) and 12 U of recombinant RNase inhibitor ( Takara Bio ) in 1X T4 RNA Ligase Buffer ( NEB ) , 9% DMSO , 20% PEG 8000 , and 1 mM ATP .", "3´ ligated RNA was cleaned by incubating with 2 . 5X volume of RNAClean XP beads ( Beckman Coulter ) and 1 . 5X volume of isopropanol for 15 min , before separation on a magnetic rack .", "Bead-bound RNA was washed with 80% ethanol , air dried , and resuspended in DEPC H2O .", "RNA-containing-supernatants were removed and the same RNAClean XP bead cleanup protocol was repeated , with a final DEPC H2O elution of 9 . 5 µl .", "RNA was fragmented by incubating 9 µl of cleaned-up RNA with 1X Fragmentation Reagent ( Invitrogen ) for 2 min at 72 °C , followed by an addition of 1X Stop Solution ( Invitrogen ) .", "Samples were stored on ice following individual fragmentation of each sample .", "Fragmented-RNA was pooled together and cleaned using the RNA Clean and Concentrator-5 kit ( Zymo ) according to the manufacturer’s instructions .", "Library construction continued following the bacterial-sRNA adapted , RNAtag-seq methodology starting at the rRNA removal step ( Melamed et al . , 2018 ) .", "Term-seq RNA libraries were analyzed on a Qubit 3 Fluorometer ( Thermo Fisher Scientific ) and an Agilent 4200 TapeStation System prior to paired-end sequencing using the HiSeq 2500 system ( Illumina ) .", "Raw sequence reads were processed using lcdb-wf ( lcdb . github . io/lcdb-wf/ ) according to the following steps .", "Raw sequence reads were trimmed with cutadapt 1 . 18 ( Martin , 2011 ) to remove any adapters while performing light quality trimming with parameters ‘-a AGATCGGAAGAGC -q 20 –minimum-length = 25 . ’ Sequencing library quality was assessed with fastqc v0 . 11 . 8 with default parameters .", "The presence of common sequencing contaminants was evaluated with fastq_screen v0 . 11 . 3 with parameters ‘–subset 100000 –aligner bowtie2 . ’ Trimmed reads were mapped to the E . coli reference genome ( MG1655 NC_000913 . 3 ) using BWA-MEM .", "Multimapping reads were filtered using samtools ( Li et al . , 2009 ) .", "Uniquely aligned reads were then mapped to gene features using subread featureCounts v1 . 6 . 2 with default parameters .", "BedGraph files were generated using deepTools ( Ramírez et al . , 2016 ) on reads from each strand separately .", "An initial set of termination peaks was called per sample on the bedGraph files from uniquely aligned reads using a novel signal processing approach combined with a statistically-informed method of combining multiple replicates .", "Briefly , the scipy . signal Python package was used to call peaks on each replicate in a manner which handled high , sharp peaks as found in Term-seq data , using the scipy . signal . find_peaks function with a width of ( 1 , None ) , a prominence of ( None , None ) , and a relative height of 0 . 75 .", "Peaks for each replicate were then combined using the IDR framework ( Landt et al . , 2012 ) into a set of peaks that were reproducible ( both strong and consistent ) across replicates .", "The code for this can be found at https://github . com/NICHD-BSPC/termseq-peaks Adams , 2020 ( copy archived at swh:1:rev:8bb48d2a034b22a312a7e848e0f8694a284a8324 ) and can be used in general for Term-seq peak-calling in other bacteria .", "Termination peaks were subsequently curated according to the following criteria .", "The single-bp peak coordinate was set to the strongest signal nucleotide within the boundary of the initial broader peak using multiBigWigSummary from deepTools 3 . 1 . 3 .", "The most downstream position , relative to the peak orientation , was chosen when several positions were equally strong .", "Scores from peaks within a distance of up to 100 bp were assessed to select the peak with the highest score among the cluster for further analysis .", "These curated peaks were used for all analysis herein ( Supplementary file 1 ) .", "Total RNA-seq was performed using the same RNA that was used for the Term-seq library preparations .", "Total RNA-seq library construction was carried out based on the RNAtag-seq methodology ( Shishkin et al . , 2015 ) , which was adapted to capture bacterial sRNAs ( Melamed et al . , 2018 ) .", "Total RNA-seq RNA libraries were sequenced as for Term-seq .", "Total RNA-seq data processing followed the same procedures as Term-seq data analysis for QC , adaptor removal and sequencing read mapping .", "One culture of E . coli MG1655 cells ( GSO989 ) was grown in LB to an OD600 ~0 . 5 and the culture was split and half was treated with 100 μg/ml of BCM ( gift from Max Gottesman ) for 15 min .", "Total RNA was isolated from 1 . 5 ml of untreated and BCM-treated cultures using the hot-phenol RNA extraction method followed by ethanol precipitation as described previously ( Stringer et al . , 2014 ) .", "Genomic DNA was removed by treating 8 μg of total RNA with 4 U of Turbo DNase ( Invitrogen ) for 45 min at 37 °C .", "DNA-free RNA was purified using phenol:chloroform:isoamyl alcohol and ethanol precipitation as described previously ( Stringer et al . , 2014 ) .", "rRNA was removed using a Ribo-Zero ( Bacteria ) kit ( Epicenter ) according to the manufacturer’s instructions .", "The RNA libraries were prepared and processed at the Helicos BioSciences facility where poly-A tails and a 3´-dATP block were added to make the RNA suitable for direct sequencing on the HeliScope Single-Molecule Sequencer ( Ozsolak and Milos , 2011 ) .", "CLC Genomics Workbench ( v8 . 5 . 1 ) was used to align DirectRNA-sequencing reads from untreated and BCM-treated samples to the MG1655 NC_000913 . 3 sense and reverse-complemented genome to properly identify the position of the first mapped 3´ end nucleotide .", "Mapping parameters were set to default , except for ‘Length fraction’ and ‘Similarity fraction’ , which were set to 0 . 7 and 0 . 9 , respectively .", "Quality scores were not generated by the HeliScope Sequencer; arbitrary quality scores were added to each read in fasta files to fit import requirements for CLC Genomics Workbench , but they were ignored when mapping .", "The read count and position of sequenced transcript 3´ ends were used for further analysis .", "The approximate Rho-dependent transcription termination sites were predicted by identifying the locations of transcriptional readthrough in the BCM-treated sample .", "The read counts in 800 nt regions upstream ( BCM_us ) and downstream ( BCM_ds ) of each position were compared to read counts from the same positions in the untreated sample ( Untreated_us , Untreated_ds ) .", "We then used a Fisher’s exact test to compare the downstream:upstream read count ratios for untreated and BCM-treated samples , and we calculated a ratio of ratios: ( R ( BCM/untreated ) = BCM_ds/BCM_usUntreated_ds/Untreated_us ) .", "We refer to the p-value from the Fisher’s exact test as the ‘significance score’ , and we refer to the R ( BCM/Untreated ) ratio as the ‘Rho score’ ( Supplementary file 2 ) .", "Putative Rho termination regions were those genome coordinates with a positive Rho score and a significance score <1e−4 .", "Only the position with the highest Rho significance score within an 800 nt window upstream and downstream is reported in Supplementary file", "2 . Note that Rho scores and significance scores listed in Supplementary file 3 were calculated for specific positions matching the dominant Term-seq 3´ ends; there may be nearby positions with a lower significance score and/or a higher Rho score .", "The intersect function of Bedtools 2 . 28 . 0 ( Quinlan and Hall , 2010 ) , ran via pybedtools v0 . 8 . 0 ( Dale et al . , 2011 ) , was used to assign each peak to one or more classes: Primary ( 3´ peaks located on the same strand either within 50 bp downstream of the 3´ end of an annotated mRNA ORF , tRNA , rRNA or sRNA with the highest score ) , Antisense ( 3´ peaks located on the opposite strand of an annotated mRNA ORF , tRNA , rRNA or sRNA within 50 bp of its start and end coordinates ) , Internal ( 3´ peaks located on the same strand within an annotated mRNA ORF , tRNA , rRNA or sRNA coordinates , excluding the 3´ end coordinate ) and Orphan ( 3´ peak not falling in any of the previous classes ) .", "3´ ends were also categorized according to their position relative to mRNA 5´ UTRs and internal mRNA regions ( Supplementary file 3 ) .", "Any 3´ end ( Supplementary file 1 ) that was located within a region of 200 bp upstream of an annotated start codon to the stop codon were extracted and further analyzed .", "To remove any 3´ ends that likely belonged to an upstream gene in the same direction , TSS data ( Thomason et al . , 2015 ) obtained using the same growth conditions and E . coli strain as Term-seq was considered .", "All these 3´ ends were examined for the first upstream feature ( either a TSS or an ORF stop codon ) .", "Any 3´ end where the first upstream feature was a stop codon was eliminated , unless there was also a TSS ≤200 bp upstream the 3´ end or that upstream feature was the stop codon of an annotated ‘leader peptide’ on the EcoCyc E . coli database ( mgtL , speFL , hisL , ivbL , ilvL , idlP , leuL , pheL , pheM , pyrL , rhoL , rseD , thrL , tnaC , trpL , uof ) .", "Any 3´ end where a TSS was only 20 bp or less upstream was also eliminated .", "This resulted in the 3´ end coordinates in Supplementary file", "3 . For the LB 0 . 4 condition , 3´ ends were given a Rho score from Direct-RNA-seq ( as described above ) and an intrinsic terminator score ( with a custom script as defined in Chen et al . , 2013 ) .", "uORFs for which synthesis was detected by western analysis and/or translational reporter fusions ( Hemm et al . , 2008; VanOrsdel et al . , 2018; Weaver et al . , 2019 ) , sRNAs for which synthesis was detected by northern analysis ( this study ) and other characterized RNA regulators were noted for the LB 0 . 4 condition .", "As detailed in Figure 1—figure supplement 2 , we compared the 3´ ends identified by Term-seq with 3´ ends identified in other studies , and we compared Rho termination regions with putative sites of Rho termination identified in other studies .", "Specifically , we determined how many of the positions in our list of genome coordinates are within a given distance threshold of a genome coordinate from another study .", "We also performed the reciprocal comparison .", "Note that the two numbers may differ if two coordinates in one study are close to one coordinate in the other; hence , we include both numbers in the Venn diagrams in Figure 1—figure supplement", "2 . To determine the extent of overlap between lists we calculated the proportion of the genome that is within the given distance threshold of each genome coordinate in a single list .", "We then divided this number by double the genome size ( doubled to account for the two possible strands ) , to determine the frequency with which a randomly selected genomic position would overlap with a genome coordinate in that list .", "We assessed the statistical significance of the overlap between lists of genome coordinates by using a hypergeometric test to compare the number of overlapping positions and the frequency expected by chance .", "For example , in Figure 1—figure supplements 2C , 19 of the 296 3´ ends in the LB 0 . 4 dataset are within 10 nt of a 3´ end in the Dar and Sorek dataset .", "We determined how many genomic positions are within 10 nt of a position in the Dar and Sorek dataset ( 22899 positions ) .", "The input parameters for the hypergeometric test were 219 ( number of successes ) , 296 ( sample size ) , 22899 ( number of successes in the population ) , and 9283304 ( population size; twice the genome length , to account for the two possible orientations ) .", "The p values are reported as <2 . 2e−16 since that is the machine epsilon for 64-bit double precision values in R and Python .", "Rho transcriptional and MdtU translational reporter assays were performed as previously described ( Baniulyte et al . , 2017 ) .", "Briefly , the pMM1constructs ( Supplementary file 4 ) were assayed in MG1655ΔlacZ ( AMD054 ) and MG1655ΔlacZ rhoR66S ( GB4 ) backgrounds .", "Three separate colonies were grown overnight in LB with 30 μg/ml chloramphenicol , diluted 1:100 in the same medium , and grown to a final OD600 ~ 0 . 4–0 . 6 at 37 °C .", "Cells were lysed in Z buffer ( 0 . 06 M Na2HPO4 , 0 . 04 M NaH2PO4 , 0 . 01 M KCl , 0 . 001 M MgSO4 ) , supplemented with β-mercaptoethanol ( 50 mM final concentration ) , sodium dodecyl sulfate ( 0 . 001% final concentration ) , and chloroform .", "Assays were initiated by adding 2-nitrophenyl β-D-galactopyranoside and stopped by adding Na2CO3 .", "All assays were done at room temperature .", "The OD600 and A420 of the cultures were measured using a Jenway 6305 spectrophotometer .", "The translational chiP-lacZ fusions ( DJS2979 and DJS2991 ) were assayed as above , with the following changes .", "Three separate colonies were grown overnight in LB with 100 μg/ml ampicillin , diluted to an OD600 of 0 . 05 in the same medium supplemented with 0 . 2% arabinose and 1 mM IPTG , and grown for 150 min ( OD600 ~ 1 . 5 ) at 37 °C .", "Reactions were performed at 28 °C and the OD600 and A420 of the cultures were measured using an Ultrospec 3300 pro spectrophotometer ( Amersham Biosciences ) .", "For all experiments , β-galactosidase activity was calculated as ( 1000 x A420 ) / ( OD600 x Vml x timemin ) .", "Northern blots were performed using total RNA exactly as described previously ( Melamed et al . , 2020 ) .", "For small RNAs , 5 μg of RNA were fractionated on 8% polyacrylamide urea gels containing 6 M urea ( 1:4 mix of Ureagel Complete to Ureagel-8 ( National Diagnostics ) with 0 . 08% ammonium persulfate ) and transferred to a Zeta-Probe GT membrane ( Bio-Rad ) .", "For longer RNAs , 10 μg of RNA were fractionated on a 2% NuSieve 3:1 agarose ( Lonza ) , 1X MOPS , 2% formaldehyde gel and transferred to a Zeta-Probe GT membrane ( Bio-Rad ) via capillary action overnight .", "For both types of blots , the RNA was crosslinked to the membranes by UV irradiation .", "RiboRuler High Range and Low Range RNA ladders ( Thermo Fisher Scientific ) were marked by UV-shadowing .", "Membranes were blocked in ULTRAhyb-Oligo Hybridization Buffer ( Ambion ) and hybridized with 5´ 32P-end labeled oligonucleotides probes ( listed in Supplementary file 4 ) .", "After an overnight incubation , the membranes were rinsed with 2X SSC/0 . 1% SDS and 0 . 2X SSC/0 . 1% SDS prior to exposure on film .", "Blots were stripped by two 7 min incubations in boiling 0 . 2% SDS followed by two 7 min incubations in boiling water .", "Immunoblot analysis was performed as described previously with minor changes ( Zhang et al . , 2002 ) .", "Samples were separated on a Mini-PROTEAN TGX 5–20% Tris-Glycine gel ( Bio-Rad ) and transferred to a nitrocellulose membrane ( Thermo Fisher Scientific ) .", "Membranes were blocked in 1X TBST containing 5% milk , probed with a 1:2000 dilution of monoclonal α-FLAG-HRP ( Sigma ) or a 1:500 dilution of polyclonal α-OmpC follwed by a 1:1000 dilution of peroxidase labeled α-rabbit and developed with SuperSignal West Pico PLUS Chemiluminescent Substrate ( Thermo Fisher Scientific ) on a Bio-Rad ChemiDoc MP Imaging System .", "The processed RNA-seq data from this study are available online via UCSC genome browser at the following links:" ] ]

[ "Many bacterial genes are regulated by RNA elements in their 5´ untranslated regions ( UTRs ) .", "However , the full complement of these elements is not known even in the model bacterium Escherichia coli .", "Using complementary RNA-sequencing approaches , we detected large numbers of 3´ ends in 5´ UTRs and open reading frames ( ORFs ) , suggesting extensive regulation by premature transcription termination .", "We documented regulation for multiple transcripts , including spermidine induction involving Rho and translation of an upstream ORF for an mRNA encoding a spermidine efflux pump .", "In addition to discovering novel sites of regulation , we detected short , stable RNA fragments derived from 5´ UTRs and sequences internal to ORFs .", "Characterization of three of these transcripts , including an RNA internal to an essential cell division gene , revealed that they have independent functions as sRNA sponges .", "Thus , these data uncover an abundance of cis- and trans-acting RNA regulators in bacterial 5´ UTRs and internal to ORFs ." ]

[ "In most organisms , specific segments of a cell’s genetic information are copied to form single-stranded molecules of various sizes and purposes .", "Each of these RNA molecules , as they are known , is constructed as a chain that starts at the 5´ end and terminates at the 3´ end .", "Certain RNAs carry the information present in a gene , which provides the instructions that a cell needs to build proteins .", "Some , however , are ‘non-coding’ and instead act to fine-tune the activity of other RNAs .", "These regulatory RNAs can be separate from the RNAs they control , or they can be embedded in the very sequences they regulate; new evidence also shows that certain regulatory RNAs can act in both ways .", "Many regulatory RNAs are yet to be catalogued , even in simple , well-studied species such as the bacterium Escherichia coli .", "Here , Adams et al . aimed to better characterize the regulatory RNAs present in E . coli by mapping out the 3´ ends of every RNA molecule in the bacterium .", "This revealed many new regulatory RNAs and offered insights into where these sequences are located .", "For instance , the results show that several of these RNAs were embedded within RNA produced from larger genes .", "Some were nested in coding RNAs , and were parts of a longer RNA sequence that is adjacent to the protein coding segment .", "Others , however , were present within the instructions that code for a protein .", "The work by Adams et al . reveals that regulatory RNAs can be located in unexpected places , and provides a method for identifying them .", "This can be applied to other types of bacteria , in particular in species with few known RNA regulators ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "cell biology", "immunology and inflammation" ]

[ [ "Blood-borne naïve lymphocytes migrate along the fibroblastic reticular cell ( FRC ) network in lymph nodes ( LNs ) ( von Andrian and Mempel , 2003; Miyasaka and Tanaka , 2004; Girard et al . , 2012 ) .", "B cells then migrate into LN follicles , whereas T cells remain in the paracortex and migrate continually along the FRC network ( Bajénoff et al . , 2006 ) .", "This intranodal migration provides critical opportunities for T cells to encounter cognate antigen-presenting dendritic cells .", "Two-photon microscopic analysis has shown that naïve T cells crawl along the FRC network in an apparently random pattern of motion , at an average velocity of 10–15 μm per minute ( Miller et al . , 2002; Okada and Cyster , 2007; Worbs et al . , 2007 ) .", "FRCs promote intranodal T-cell motility by signaling naïve lymphocytes with CCL21/CCL19 via CCR7 , thus activating the small GTPase Rac ( Okada and Cyster , 2007; Worbs et al . , 2007; Faroudi et al . , 2010; Huang et al . , 2007 ) , although CCR7 signaling only partially account for the interstitial T cell motility ( Okada and Cyster , 2007; Huang et al . , 2007 ) .", "LPA is a bioactive lysophospholipid produced both extracellularly and intracellularly .", "Extracellularly produced LPA is involved in such diverse biological functions as vascular remodeling and cell growth , survival , and migration ( Choi et al . , 2010; Yanagida et al . , 2013 ) .", "Intracellularly produced LPA is an intermediate in the synthesis of triglycerides and glycerophospholipids , and thought to act as a 'housekeeper' inside the cell ( Mills and Moolenaar , 2003 ) .", "Extracellular LPA is predominantly produced by autotaxin ( ATX , also referred to as ENPP2 [ectonucleotide pyrophosphatase/phosphodiesterase family member 2] ) , an ectoenzyme that was originally identified as a tumor-cell motility-enhancing factor ( Stracke et al . , 1992 ) .", "ATX is a lysophospholipase D that produces LPA by hydrolyzing lysophosphatidylcholine ( LPC ) ( Okudaira et al . , 2010; Moolenaar and Perrakis , 2011 ) .", "We and others have reported that ATX is strongly expressed in HEV endothelial cells ( ECs ) , and that ATX regulates lymphocyte migration into the LN parenchyma ( Kanda et al . , 2008; Nakasaki et al . , 2008; Umemoto et al . , 2011 ) .", "We also demonstrated that LPA enhances lymphocyte detachment from ECs and promotes lymphocyte transmigration across the HEV basal lamina , at least in part by acting on HEV ECs ( Bai et al . , 2013 ) .", "LPA also acts on naïve T cells to induce chemokinesis and cell polarization ( Kanda et al . , 2008; Katakai et al . , 2014; Zhang et al . , 2012 ) and transmigration ( Zhang et al . , 2012 ) .", "While a study using pharmacological inhibitors revealed that ATX/LPA promotes intranodal lymphocyte motility in an ex vivo LN explant model ( Katakai et al . , 2014 ) , the physiological significance of the ATX/LPA axis in interstitial lymphocyte migration remains unknown .", "To date , six LPA receptors ( LPA1–LPA6 ) have been identified .", "LPA receptors couple to multiple G proteins , including Gi , G12/13 , Gq , and Gs , and upon ligand binding , these G proteins activate diverse intracellular signaling components including Rho and Rac .", "Although LPA2 has recently been reported to play a role in intranodal T-cell migration ( Knowlden et al . , 2014 ) , it remains unclear how LPA2-mediated signaling affects interstitial T-cell motility and whether LPA is the prime activating ligand .", "Leukocyte migration in a confined environment is regulated at least partly by cell contraction ( Lämmermann et al . , 2008 ) .", "Jacobelli et al . ( 2010 ) reported that a contractile protein , myosin IIA , is required for T-cell amoeboid motility in confined environments such as LNs ( Jacobelli et al . , 2010 ) .", "Myosin IIA cross-links actin , thus limiting surface adhesion and allowing T cells to exert contractile force .", "Myosin IIA’s activity is regulated by RhoA and Rho-associated protein kinase ( ROCK ) signaling .", "While the cell-extrinsic factor ( s ) that regulate myosin II’s activity during T-cell migration in a confined environment have been poorly defined , a recent study using zebrafish germ progenitor cells showed that LPA induces cell polarization in a ROCK-myosin II-dependent manner , which enables rapid cell migration in a confined environment ( Ruprecht et al . , 2015 ) .", "In this study , by using mice conditionally deficient for the LPA-generating enzyme ATX in FRCs and those deficient in LPA2 , we demonstrated that bioactive LPA species are produced by FRCs in an ATX-dependent manner and that LPA acts locally on LPA2 on T cells .", "This LPA2-mediated signaling activates the RhoA-ROCK-myosin II pathway and promotes confinement-optimized interstitial T-cell migration .", "The FRC-derived LPA thus serves as a cell-extrinsic factor that optimizes T-cell movement through the densely packed LN reticular network , to fine-tune T-cell trafficking ." ], [ "Although CCR7 ligands are reported to stimulate intranodal T-cell motility ( Okada and Cyster , 2007; Worbs et al . , 2007; Huang et al . , 2007 ) , they are not sufficient to account for effective T-cell migration in LNs .", "Given that ATX , which generates the motogenic lysophospholipid , LPA , is expressed in HEV ECs to modulate lymphocyte motility ( Nakasaki et al . , 2008; Bai et al . , 2013 ) , we speculated that ATX and its product LPA are also expressed in other stromal cell subsets in the LN parenchyma and may control intranodal lymphocyte migration .", "Indeed , a recent paper showed that ATX is expressed in CCL21+ CD31- stromal cells in LNs ( Katakai et al . , 2014 ) .", "We therefore subdivided the CD45- LN stromal cells into four stromal subsets ( Figure 1A ) .", "As shown in Figure 1B , Enpp2/Autotaxin was readily detected in GP38+ CD31- FRCs as well as GP38- CD31+ blood endothelial cells ( ECs ) , with negligible expression in lymphatic ECs and double-negative cells .", "Electron microscopic analysis confirmed that ATX was expressed in the FRCs surrounding collagen fiber bundles ( Figure 1C ) .", "Interestingly , analyses using Ccl19-Cre x R26-EYFP mice , which constitutively express yellow fluorescent protein ( YFP ) in FRCs ( Chai et al . , 2013 ) , revealed that Enpp2 was selectively expressed in LN FRCs but not splenic FRCs ( Figure 1D ) .", "This Enpp2 expression was apparently dependent on LTβR signaling , because blocking LTβR signaling significantly reduced expression of Enpp2 , Cxcl13 , and Ccl19 , but not Icam1 , in FRCs ( Figure 1E ) .", "This blockade also downregulated transcription of HEV marker genes such as Glycam1 and Ccl21 in BECs ( Figure 1E ) , consistent with a previous report ( Browning et al . , 2005 ) .", "These results confirm that similarly to HEV ECs , FRCs constitutively express the LPA-generating enzyme ATX , which is maintained at least in part by LTβR signaling . 10 . 7554/eLife . 10561 . 003Figure 1 . FRCs and vascular ECs express autotaxin in an LTβR-signaling-dependent manner .", "( A ) Representative dot plot of the CD45- stromal cells in LNs .", "CD45- cells were divided by CD31 and gp38 expression into FRCs ( gp38+ CD31- ) , lymphatic endothelial cells ( LECs; gp38+ CD31+ ) , blood endothelial cells ( BEC; gp38-CD31+ ) , and double-negative cells .", "Numbers indicate the frequency of cells in each gate .", "( B ) Enpp2/Autotaxin expression in stromal LN fractions , analyzed by quantitative RT-PCR .", "( C ) ATX expression examined by immunoelectron microscopy; arrow indicates positive signals in an FRC .", "C: collagen fibers; L: lymphocytes .", "Bar , 1 μm .", "( D ) Enpp2 in CD45- EYFP+ cells in LNs and the spleen of Ccl19-Cre x R26-EYFP mice .", "( E ) Effect of LTβR signaling on Enpp2 expression in FRCs and BECs .", "LTβR signaling was blocked by injecting 100 μg of recombinant LTβR-Fc or an isotype control into adult mice intraperitoneally weekly for 4 weeks .", "The FRCs and BECs were then sorted , and the indicated gene expressions were analyzed by quantitative PCR .", "Data are representative of three ( A , B , D ) or two ( C , E ) independent experiments ( n = 3 per group ) .", "Differences between groups were evaluated by Student’s t-test .", "*P < 0 . 05; **P < 0 . 005; ***P < 0 . 0005 . DOI: http://dx . doi . org/10 . 7554/eLife . 10561 . 003 To verify that LPA is generated in situ by FRC-derived ATX , we crossed Enpp2fl/fl mice and Ccl19-Cre mice to generate Ccl19-Cre Enpp2fl/fl mice that lacked ATX expression specifically in the FRCs .", "As expected , in the Ccl19-Cre Enpp2fl/fl mice , Enpp2 was completely lost in the FRCs but not in the BECs , whereas Ccl21 and GP38 expression was comparable between these strains ( Figure 2A , Figure 2—figure supplement 1 ) .", "The frequency of FRCs in stromal cells also appeared to be uncompromised by the deficiency of Enpp2 in FRCs ( Figure 2—figure supplement 1 ) .", "We then compared LPA production in the LN of these mice using imaging mass spectrometry ( IMS ) .", "To this end , we first injected fluorescein-conjugated dextran , which labels lymphatics and the medulla , into the footpad , and LPA ( 18:0 ) , LPA ( 18:1 ) , LPA ( 18:2 ) , and LPA ( 20:4 ) were then visualized in LN sections .", "As shown in Figure 2B , signals corresponding to LPA ( 18:0 ) were widely distributed in the LN .", "The signals were comparable in intensity and frequency in Enpp2fl/fl and Ccl19-Cre Enpp2fl/fl mice; this LPA species appears to be produced mainly within the cell ( Aoki , J; unpublished observation ) independently of ATX ( Yukiura et al . , 2011 , Nishimasu , et al . , 2011 ) .", "In sharp contrast , signals corresponding to LPA ( 18:1 ) , LPA ( 18:2 ) , and LPA ( 20:4 ) , the major species produced extracellularly by ATX ( Yukiura et al . , 2011 ) , were predominantly observed in the paracortex both close to and at a distance from HEVs , but only marginally in the medulla .", "These signals were substantially decreased in the cortex of Ccl19-Cre Enpp2fl/fl as compared with Enpp2fl/fl mice ( Figure 2B ) . 10 . 7554/eLife . 10561 . 004Figure 2 . Multiple LPA species are produced in the LN parenchyma by FRCs .", "( A ) Enpp2 and Ccl21 mRNA expression in LN stromal cells in Ccl19-Cre Enpp2fl/fl or Enpp2fl/fl mice .", "See also analysis of stromal cell populations in those mice in Figure 2—figure supplement", "1 . ( B ) LPA-species distribution in the LNs of Ccl19-Cre Enpp2fl/fl and Enpp2fl/fl mice .", "Fluorescein-conjugated dextran ( pseudocolored in yellow ) was injected into the footpads to visualize the lymphatics , and draining LNs were collected 10 min after the injection .", "Signals corresponding to LPA ( 18:0 ) ( ion transition from m/z 437 to 153 ) , LPA ( 18:1 ) ( from m/z 435 to 153 ) , LPA ( 18:2 ) ( from m/z 433 to 153 ) , and LPA ( 20:4 ) ( from m/z 457 to 153 ) were visualized by IMS analysis ( magenta ) and overlapped with MECA-79 staining ( blue ) on LN serial sections .", "LPA signals located within 50 μm of an HEV are circled by dotted lines and signals more than 50 μm away from an HEV are indicated by arrows for each of the LPA ( 18:1 ) , LPA ( 18:2 ) , and LPA ( 20:4 ) species .", "( C ) The frequency of LPA signals by distance from an HEV , and the median distance from an HEV .", "Data are representative of three ( A ) or two ( B , C ) independent experiments .", "Differences between groups were evaluated by Student’s t-test .", "*P < 0 . 05 , **P < 0 . 005 , ***P < 0 . 0005 .", "Bars , 100 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 10561 . 00410 . 7554/eLife . 10561 . 005Figure 2—figure supplement 1 . Stromal cell populations in Enpp2fl/fl and Ccl19-Cre Enpp2fl/fl mice . The frequency of FRCs , BECs , LECs and double negative cells in Enpp2fl/fl and Ccl19-Cre Enpp2fl/fl mice was analyzed by flow cytometry ( n = 3 per group ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 10561 . 00510 . 7554/eLife . 10561 . 006Video 1 . FRC-specific ATX ablation reduced the velocity of T cells in LNs . Intravital two-photon microscopy was used to analyze the intranodal migration of eGFP-expressing CD4+ T cells ( green ) 15 hr after the cells were injected into Enpp2fl/fl ( left ) or Ccl19-Cre Enpp2fl/fl mice ( right ) .", "Data shown are representative of three independent experiments .", "Bars , 50 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 10561 . 006 To verify that the cortical LPA signals associated with non-HEV structures were derived from FRCs , we next mapped the LPA signals relative to HEVs in Enpp2fl/fl mice and Ccl19-Cre Enpp2fl/fl mice by measuring the distance between individual signals and the nearest HEV .", "As shown in Figure 2C , the frequency of LPA ( 18:1 ) , LPA ( 18:2 ) , and LPA ( 20:4 ) signals within 50 μm of an HEV did not differ significantly between Enpp2fl/fl and Ccl19-Cre Enpp2fl/fl mice .", "However , the frequency of relatively distant signals ( more than 50 μm ) decreased substantially when ATX was ablated in FRCs .", "Hence , the median distance between LPA signals and HEVs was significantly reduced in Ccl19-Cre Enpp2fl/fl compared with Enpp2fl/fl mice , consistent with the idea that distant LPA signals were associated with FRCs .", "Together with the observations showing robust expression of ATX in FRCs , these findings indicate that the cortical LPA signals not associated with HEVs are mainly produced by FRCs in an ATX-dependent manner .", "To understand the role of FRC-derived LPA in regulating intranodal T-cell migration , we next examined the CD4+ T-cell interstitial migration in LNs by intravital two-photon microscopy .", "To this end , CD4+ T cells from WT mice expressing a transgene encoding enhanced GFP ( eGFP ) were injected intravenously into Enpp2fl/fl and Ccl19-Cre Enpp2fl/fl mice , and the intranodal T-cell migration in popliteal LNs ( PLNs ) was imaged 15–25 hr later ( Video 1 ) .", "As shown in Figure 3A , B , CD4+ T-cell movement and displacement from the original location in the PLN was substantially restricted in Ccl19-Cre Enpp2fl/fl compared with Enpp2fl/fl mice .", "The median T-cell velocity was also lower in Ccl19-Cre Enpp2fl/fl than in Enpp2fl/fl mice ( Figure 3C , Figure 3—figure supplement 1 ) .", "Measurement of the mean displacement and the motility coefficient , which represents the volume in which an average cell scans per unit time ( Sumen et al . , 2004 ) , also indicated that T-cell motility was impaired in the LN parenchyma of Ccl19-Cre Enpp2fl/fl mice ( Figure 3D , E ) , whereas the directionality of the intranodal T-cell movement was comparable in these mouse groups ( Figure 3—figure supplement 2 ) , supporting the hypothesis that FRC-derived LPA is required for efficient intranodal T-cell migration . 10 . 7554/eLife . 10561 . 007Figure 3 . FRC-specific ATX ablation attenuates intranodal T-cell migration . Intranodal T-cell migration in Enpp2fl/fl mice and Ccl19-Cre Enpp2fl/fl mice , analyzed by intravital two-photon microscopy .", "Intranodal T-cell migration in the PLN was analyzed 15–25 hr after injecting eGFP-expressing CD4+ T cells into Ccl19-Cre Enpp2fl/fl or Enpp2fl/fl mice .", "( A ) Automated tracking of CD4+ T-cell migration ( upper panels ) , and images rotated to display the z-dimension of the volume ( lower ) .", "Trajectories of 50 randomly chosen cells are displayed as color-coded tracks to show movement over time , from blue ( start of imaging ) to red ( end of imaging ) .", "( B ) Translated tracking with a common origin .", "( C-E )", "Analysis of T cell motility .", "See also Figure 3—figure supplement", "2 . ( C ) Average cell velocity .", "Each dot represents the average velocity of an individual cell , and bars indicate the median .", "Pooled data are shown in Figure 3—figure supplement", "1 . ( D ) Mean displacement plot .", "( E ) Motility coefficients , calculated from the slope of the regression line of the mean displacement plot as x2/6t , where x is the displacement at time t .", "Data represent the mean ± SD ( C ) or mean ± SEM ( D , E ) .", "Data are representative of three independent experiments .", "Differences between groups were evaluated by Student’s t-test .", "*P < 0 . 05 , **P < 0 . 005 , ***P < 0 . 0005 .", "Bars , 100 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 10561 . 00710 . 7554/eLife . 10561 . 008Figure 3—figure supplement 1 . Pooled data of WT CD4+ T-cell velocity in Enpp2fl/fl and Ccl19-Cre Enpp2fl/fl LNs . WT CD4+ T cells were injected into Enpp2fl/fl or Ccl19-Cre Enpp2fl/fl mice , and their intranodal migration was analyzed by by two-photon microscopy .", "The data were pooled from two independent experiments .", "Differences between groups were evaluated by Student’s t-test .", "***P < 0 . 0005 . DOI: http://dx . doi . org/10 . 7554/eLife . 10561 . 00810 . 7554/eLife . 10561 . 009Figure 3—figure supplement 2 . Directionality of WT CD4+ T-cell movement in Enpp2fl/fl and Ccl19-Cre Enpp2fl/fl LNs . Directionality was calculated by dividing cells’ net displacement by the total path length .", "The data were pooled from two independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 10561 . 009 To understand how LPA regulates intranodal T-cell migration , we analyzed the LPA-receptor expression in lymphocytes .", "As shown in Figure 4A , CD4+ T cells , CD8+ T cells , and B cells expressed different levels of Lpar2 , Lpar5 , and Lpar6 , encoding LPA2 , LPA5 and LPA6 respectively , with B cells expressing relatively high levels of Lpar2 compared to T cells . 10 . 7554/eLife . 10561 . 010Figure 4 . Lpar2 deficiency causes T cells to be retained in LNs .", "( A ) LPA-receptor expression in lymphocyte subsets: CD4+ T , CD8+T , B cells and whole LNs were analyzed by real-time RT-PCR; expression levels were normalized to Gapdh .", "Data shown are representative of three individual experiments .", "( B–D )", "Migration of LPA receptor–deficient lymphocytes into secondary lymphoid tissues .", "Splenocytes from eGFP-expressing mice and from Lpar2-/- , Lpar5-/- , or Lpar6-/- mice were mixed in equal numbers , labeled with biotin , and injected intravenously into WT recipients .", "After 1 . 5 hr , secondary lymphoid tissues were collected from the recipient mice and donor-derived lymphocytes were detected with flow cytometry .", "( B ) Ratio of LPA receptor-deficient ( Lpar2-/- , Lpar5-/- or Lpar6-/- ) cells to eGFP-expressing ( WT ) cells that migrated into the spleen , peripheral LN , and MLN after adoptive transfer into WT recipient mice .", "( C ) Representative dot plots of donor cells ( before transfer ) and biotinylated cells in LNs of recipient mice ( after transfer ) .", "( D ) Lpar2-/- lymphocyte subpopulations that migrated into the spleen , peripheral LN , and MLN of recipient mice .", "Data shown for adoptive transfer experiments using Lpar2-/- , Lpar5-/- and Lpar6-/- lymphocytes are representative of five , two , and three independent experiments , respectively ( n=4 mice per group ) .", "( E , F )", "Localization of adoptively transferred WT and Lpar2-/- CD4+T cells in LNs .", "DiD-labeled CD4+ T cells from WT mice ( 1 x 107 cells , pseudocolored in green ) were mixed equally with CMTMR-labeled Lpar2-/- CD4+ T cells ( red ) and injected intravenously into WT recipient mice; 90 min later , Alexa Fluor 488-conjugated MECA-79 mAb ( pseudocolored in blue ) was injected intravenously to visualize HEVs .", "( E ) LNs were collected and analyzed by confocal microscopy .", "( F ) The distance that CD4+ T cells migrated from HEVs was measured , and the ratio of Lpar2-/- to WT T cells was calculated .", "Data represent mean ± SD from 3 mice .", "Data are representative of two ( E , F ) independent experiments .", "( G ) Ratio of Lpar2-/- T cells to WT T cells retained in LNs when lymphocyte entry into LNs was blocked .", "CFSE-labeled CD45 . 1+ WT and CD45 . 2+Lpar2-/- CD4+ T cells were intravenously injected into CD45 . 2+ WT mice .", "Two hours later , anti-L-selectin antibody ( MEL-14 ) was injected ( T = 0 hr ) .", "At T=0 hr and T=23 hr , the total cell number in LNs ( left ) and the frequency of donor-derived cells ( right ) were measured .", "Data are pooled from two independent experiments .", "Data represent the mean ± SD ( A , B , D , F , G ) .", "Differences between groups were evaluated by one-way ANOVA ( B , D , G [right] ) or Student’s t-test ( G [left] ) .", "*P < 0 . 05 , **P < 0 . 005 , ***P < 0 . 0005 .", "Bars: 100 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 10561 . 010 We next examined whether LPA-receptor deficiency in lymphocytes causes abnormal migration into LNs , as seen in myosin IIA–deficient T cells ( Jacobelli et al . , 2010 ) .", "Splenocytes from eGFP-expressing mice and from LPA receptor-deficient mice were biotinylated , mixed in equivalent numbers , and injected intravenously into WT recipients , and lymphocytes that migrated into the LNs and spleen were counted 1 . 5 hr later .", "As shown in Figure 4B , Lpar5-/- and Lpar6-/- lymphocytes migrated into the LN and spleen at levels comparable to WT lymphocytes .", "In contrast , there were more Lpar2-/- than WT lymphocytes in the LNs but not the spleen ( Figure 4B , C ) .", "Among the migrating cells , this increase was evident for CD4+ T cells and CD8+ T cells but not B cells ( Figure 4D ) .", "A similar increase was also observed when purified CD4+ T cells were used ( Figure 4G ) .", "These results suggest two possibilities for the role of LPA2: one is that LPA2 negatively regulates the entry of T cells into LNs across HEVs , and the other is that LPA2 promotes intranodal T-cell motility and hence their egress from the LNs .", "To test these hypotheses , we co-injected differentially labeled WT and Lpar2-/- T cells and examined their intranodal localization after 90 min by whole-mount LN analysis .", "Among the donor T cells in the LN parenchyma , Lpar2-/- and WT T cells localized in the vicinity of HEVs ( within 50 μm distance ) at comparable levels ( Figure 4E and F ) , indicating that LPA2 plays a minor role in T cell entry into LNs across HEVs .", "By contrast , Lpar2-/- T cells localized more frequently in the area distant ( more than 50 μm away ) from HEVs compared with WT T cells ( Figure 4E and F ) , consistent with the hypothesis that LPA promotes intranodal T cell migration via LPA2 , and hence , Lpar2-deficient T cells were retained in the LN parenchyma .", "To directly assess the involvement of the LPA2 signaling in T-cell egress from LNs , we blocked the T cell entry by injecting anti-L-selectin antibody MEL-14 intravenously ( T = 0 hr ) , and measured the number of WT and Lpar2-/- CD4+ T cells residing in the LNs 23 hr after the injection .", "As shown in Figure 4G , the blockade of T cell entry ( T = 23 hr ) resulted in reduction of the cellularity in LNs and increased the ratio of Lpar2-/- T cells to WT cells compared to that before the injection .", "These results indicate that retention of Lpar2-deficient T cells was seen even after blockade of lymphocyte ingress to LNs and are compatible with the hypothesis that Lpar2-deficiency leads to T cell retention in the LN parenchyma without affecting lymphocyte migration into LNs .", "To verify that LPA2-mediated signaling is required for intranodal T-cell motility , we next compared the intranodal migration of Lpar2-deficient and WT T cells .", "We adoptively transferred CD4+ T cells from Lpar2-/- mice and WT mice and used intravital two-photon microscopy to compare the behavior of these cells in the PLN of the recipient mice .", "Preliminary experiments indicated that the lymphocyte labeling used for in vivo cell tracking did not affect cell motility under the experimental conditions ( unpublished observation ) .", "Compared to WT T cells , the Lpar2-/- T cells moved noticeably more slowly and had comparatively shorter track lengths and less displacement from their original position per unit of time ( Figure 5A , Figure 5—figure supplement 1 , Video 2 ) .", "Lpar2-/- T cells had a 13% lower average velocity ( Figure 5B ) , a 45% lower mean square displacement ( Figure 5C ) , and a 70% lower motility coefficient than WT T cells ( Figure 5D ) , although the directionality of T-cell motion was comparable in these cell types ( Figure 5—figure supplement 2 ) .", "Results using explanted LNs were compatible with those from intravital imaging analysis ( unpublished observation ) . 10 . 7554/eLife . 10561 . 011Figure 5 . Lpar2 deficiency attenuates intranodal T-cell migration .", "( A–D )", "Analysis of the intranodal migration of WT and Lpar2-/- CD4+ T cells in the PLN of WT mice by intravital two-photon microscopy .", "CD4+ T cells from eGFP-expressing mice ( green , 5 x 106 cells ) mixed with an equal number of CMTMR-labeled Lpar2-/- CD4+ T cells ( red ) were injected into WT mice; the transferred cells' internodal migration was analyzed by intravital two-photon microscopy 15–25 hr later ( Figure 5—figure supplement 1 ) .", "( A ) Translated tracking of CD4+ T-cell migration with a common origin .", "( B-D )", "Analysis of T cell motility .", "See also Figure 5—figure supplement", "2 . ( B ) Average cell velocity .", "Each dot represents the average velocity of an individual cell; bars indicate the median .", "( C ) Mean displacement plot .", "( D ) Motility coefficient .", "( E–G )", "The intranodal migration of WT and Lpar2-/- CD4+ T cells in the PLN of Ccl19-Cre Enpp2fl/fl or Enpp2fl/fl mice .", "Lpar2-/- CD4+ T cells and WT CD4+ T cells were mixed in equivalent numbers and injected into Enpp2fl/fl ( E ) or Ccl19-Cre Enpp2fl/fl mice ( F ) .", "The mean displacement ( E , F ) and motility coefficient ( G ) are shown .", "( H–J )", "The intranodal migration of WT and Lpar2-/- CD4+ T cells in the presence of anti-LFA-1 blocking antibody .", "( H ) Average cell velocity .", "( I ) Mean displacement plot .", "( J ) Motility coefficient .", "Data are representative of at least three ( A–D , H–J ) or , two ( E–G ) independent experiments .", "Pooled data of two photon microscopic analyses are shown in Figure 5—figure supplement", "3 . Data represent the mean ± SD ( B , H ) or mean ± SEM ( C–G , I , J ) .", "Differences between groups were evaluated by Student’s t-test ( B , D , G , J ) or one-way ANOVA ( H ) .", "*P < 0 . 05 , **P < 0 . 005 , ***P < 0 . 0005 .", "Bars: 100 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 10561 . 01110 . 7554/eLife . 10561 . 012Figure 5—figure supplement 1 . Tracking of WT and Lpar2-/- T cells in popliteal LN using intravital two-photon microscopy . CD4+ T cells from eGFP-expressing mice ( green , 5 x 106 cells ) mixed with an equal number of CMTMR-labeled Lpar2-/- CD4+ T cells ( red ) were injected into WT mice; the transferred cells' intranodal migration was analyzed by intravital two-photon microscopy 15–25 hr later .", "Secondary harmonic signals are indicated in blue .", "The T-cell migration was tracked by Imaris software . DOI: http://dx . doi . org/10 . 7554/eLife . 10561 . 01210 . 7554/eLife . 10561 . 013Figure 5—figure supplement 2 . Directionality of WT and Lpar2-/- CD4+ T-cell movement in WT LNs . Directionality was calculated by dividing cells’ net displacement by the total path length .", "The data were pooled from three independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 10561 . 01310 . 7554/eLife . 10561 . 014Figure 5—figure supplement 3 . Pooled data of cell behaviors analyzed by intravital two-photon microscopy .", "( A , B )", "Motility of WT and Lpar2-/- CD4+ T cells in Enpp2fl/fl or Ccl19-Cre Enpp2fl/fl LNs .", "Mean displacement plot ( A ) and motility coefficient ( B ) are shown .", "( C–E ) .", "Motility of WT and Lpar2-/- CD4+ T cells in WT LNs before and after the injection of anti-LFA-1 antibody .", "Velocity ( C ) , mean displacement plot ( D ) and motility coefficient ( E ) are shown .", "The data were pooled from two ( A , B ) or three ( C–E ) independent experiments .", "Differences between groups were evaluated by one-way ANOVA ( B , C , and E ) .", "*P < 0 . 05 , **P < 0 . 005 , ***P < 0 . 0005 . DOI: http://dx . doi . org/10 . 7554/eLife . 10561 . 01410 . 7554/eLife . 10561 . 015Video 2 . Lpar2 deficiency attenuated the intranodal T-cell motility . Intravital two-photon microscopy of the intranodal migration of eGFP-expressing CD4+ T cells ( green ) and CMTMR-labeled Lpar2-/- CD4+ T cells ( red ) in PLNs 15 hr after the cells were injected into WT mice .", "Data shown are representative of three independent experiments .", "Bar , 100 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 10561 . 015 We next examined the importance of the LPA2 receptor in the FRC-derived LPA-dependent T-cell motility in the LN parenchyma .", "WT and Lpar2-/- CD4+ T cells were mixed and injected intravenously into Enpp2fl/fl or Ccl19-Cre Enpp2fl/fl mice , and the migration of these cells within the LN was monitored .", "In Enpp2fl/fl mice , Lpar2-/- T cells had significantly less intranodal motility than WT cells ( Figure 5E , G and Figure 5—figure supplement 3A , B ) .", "On the other hand , a reduced intranodal motility of Lpar2-/- T cells was not observed in Ccl19-Cre Enpp2fl/fl mice , which lack ATX/LPA in the FRCs ( Figure 5F , G and Figure 5—figure supplement 3A , B ) .", "These results indicate that intranodal T-cell motility is regulated via LPA2 on T cells and that the LPA2-mediated T-cell motility requires FRC-derived LPA .", "Because LFA-1 regulates high-speed intranodal T cell migration ( Katakai et al . , 2013; Woolf et al . , 2007 ) , we examined integrin dependency in the LPA2-mediated intranodal T-cell migration by transferring the WT and Lpar2-/- T cells and monitoring the cell migration before and after injecting anti-LFA-1 antibody ( M17/4 ) .", "As shown in Figure 5H–J and Figure 5—figure supplement 3C–E , the blockade of the interaction between LFA-1 and ICAM-1 significantly attenuated WT T cell motility as reported previously ( Katakai et al . , 2013; Woolf et al . , 2007 ) .", "Of note , even in the absence of the LFA-1/ICAM-1 interaction , Lpar2 deficiency reduced T-cell motility , and the extent of reduction in T-cell velocity ( 19 . 9 ± 5 . 5% ) and motility coefficient ( 43 . 5 ± 16 . 8% ) was comparable to that observed in the presence of the integrin interaction ( 15 . 8 ± 4 . 4% and 43 . 0 ± 13 . 6% reduction in velocity and motility coefficient , respectively ) ( Figure 5H–J and Figure 5—figure supplement 3C–E ) .", "These results support the hypothesis that LPA/LPA2 regulates T-cell motility at least partly in an integrin-independent manner .", "LPA is reported to promote T-cell migration in a two-dimensional ( 2D ) environment by inducing chemokinesis ( Katakai et al . , 2014; Zhang et al . , 2012; Knowlden et al . , 2014 ) .", "We therefore investigated the motility of WT and Lpar2-deficient CD4+ T cells in a 2D environment by time-lapse microscopy .", "Untreated or LPA-pretreated CD4+ T cells were applied to one side of an EZ-Taxiscan chamber coated with ICAM-1 , and the migration of cells toward CCL21 placed on the other side of the chamber was monitored in real time .", "As shown in Figure 6A , B , WT T cells efficiently migrated toward CCL21 in response to LPA in a time-dependent manner .", "Compared to untreated WT T cells , LPA-sensitized WT T cells had a higher velocity and a smaller mean turning angle ( Figure 6C , D ) .", "In contrast , LPA treatment did not enhance the migration of Lpar2-/- T cells toward CCL21 . 10 . 7554/eLife . 10561 . 016Figure 6 . LPA/LPA2-mediated signaling promotes T-cell migration on a 2D surface . CD4+ T cells from WT or Lpar2-/- mice were left untreated or were treated with LPA ( 1 μM ) and immediately loaded on one side of an EZ-Taxiscan chamber , and medium containing CCL21 ( 100 ng ) was applied on the other side .", "( A ) Cell migration on a surface coated with ICAM-1-Fc was monitored at 1-min intervals .", "( B ) Cells that migrated toward the CCL21-containing contra-wells were counted , and the ( C ) average cell velocity and ( D ) turning angle were calculated .", "Bars represent the median .", "Differences between groups were evaluated by Student’s t-test .", "*P < 0 . 05 , **P < 0 . 005 . DOI: http://dx . doi . org/10 . 7554/eLife . 10561 . 016 We next examined whether LPA also regulates T-cell migration via LPA2 in a three-dimensional ( 3D ) environment , in which leukocyte migration largely depends on actomyosin contraction ( Lämmermann and Germain , 2014 ) .", "Transwell analyses have shown that lymphocyte migration across a filter with a 3-μm pore diameter depends on ROCK/myosin II signaling , while migration across a 5-μm pore does not ( Soriano et al . , 2011 ) .", "Since LPA induces chemokinesis ( Kanda et al . , 2008; Katakai et al . , 2014 ) , we added LPA to lymphocytes placed in the upper chamber of a Transwell apparatus with a 3-μm pore diameter filter and counted the cells that migrated to the lower well .", "As shown in Figure 7A , LPA sensitization enhanced the migration of CD4+ T cells , and this enhancement was strongly inhibited by the ROCK inhibitor Y27632 ( Figure 7A ) , or the myosin II ATPase inhibitor blebbistatin ( Figure 7B ) , but not by pertussis toxin ( PTX ) , which inhibits Gαi signaling ( Figure 7A ) .", "At 1 μM , LPA prominently induced chemokinesis in WT CD4+T cells , consistent with previous reports ( Kanda et al . , 2008; Katakai et al . , 2014 ) ( Figure 7C ) , but this effect was not observed in Lpar2-/- CD4+ T cells ( Figure 7C ) .", "As shown in Figure 7D and E , LPA induced RhoA activation in WT but not Lpar2-/- CD4+ T cells .", "Reminiscent of the findings of Soriano et al for ROCK/myosin II signaling ( Soriano et al . , 2011 ) , LPA noticeably enhanced the CCL21-dependent migration of T cells across a 3-μm- but not 5-μm-pore diameter filter ( Figure 7F ) , and that Lpar2 deficiency abolished the LPA-induced enhancement ( Figure 7G ) . 10 . 7554/eLife . 10561 . 017Figure 7 . LPA/LPA2 axis regulates T-cell migration in a Rho/ROCK/myosin II-dependent manner .", "( A , B )", "The involvement of ROCK-myosin II signaling in LPA-enhanced cell migration across narrow pores .", "Lymphocytes were pretreated with the indicated concentrations of blebbistatin ( Bleb ) , Y27632 ( 10 μM ) , or PTX ( 100 ng/ml ) , and were added with LPA to the upper chamber of a Transwell apparatus with a 3-μm-pore filter .", "After 2 hr , the migrated CD4+ T cells in the lower chambers were counted by flow cytometry .", "( C ) Role of LPA2 in the LPA-induced chemokinesis of CD4+ T cells .", "WT or Lpar2-/- lymphocytes were added with various concentrations of LPA to the upper chamber .", "( D ) Role of LPA2 in the LPA-induced activation of RhoA .", "WT and Lpar2-/- T cells were incubated with 1 μM LPA for the periods indicated , and were lysed immediately thereafter .", "RhoA-GTP in the lysates was pulled down using Rhotekin and was detected with an anti-RhoA antibody .", "( E ) The relative band intensities of LPA-treated WT and Lpar2-/- T cells , normalized to untreated WT and Lpar2-/- T cells , respectively .", "( F ) Effect of LPA on CD4+ T cell migration through Transwell membranes with a pore diameter of 3 or 5 μm .", "WT lymphocytes pretreated with or without LPA ( 1 μM ) were applied to the upper chamber , and CCL21 ( 200 ng/ml ) was added to the lower chamber .", "( G ) Role of LPA2 in LPA-induced enhancement of CD4+ T cell chemotaxis toward CCL21 .", "WT or Lpar2-/- lymphocytes were added with LPA ( 1 μM ) to the upper chamber and CCL21 ( 200 ng/ml ) was added to the lower chamber .", "Data are representative of two ( A , B ) or at least three ( C–G ) independent experiments ( n = 3 per group ) .", "Data were evaluated by one-way ANOVA ( A–C , F , G ) or Student’s t-test ( E ) and represent the mean ± SD .", "*P < 0 . 05 , **P < 0 . 005 . DOI: http://dx . doi . org/10 . 7554/eLife . 10561 . 017 The myosin II signaling allows T cells to make selective contacts with the adhesive substrates , enabling rapid movement ( Jacobelli et al . , 2010 ) .", "We thus measured cell surface adhesion area in WT and Lpar2-/- T cells by total internal reflection fluorescence ( TIRF ) microscopy ( Jacobelli et al . , 2010 ) .", "As shown in Figure 8A , B , and D , LPA limited T-cell adhesion area on the fibronectin- or ICAM-1-coated substrate in WT T cells , which was inhibited by treatment of cells with blebbistatin , but this effect was not observed in Lpar2-/- T cells , suggesting that Lpar2-/- T cells over-adhere to substrate coated with or without LFA-1 ligand .", "Consistently , the LPA/LPA2 signaling induced T-cell elongation in a myosin II-dependent manner , allowing swift cell movement ( Figure 8A , C , E ) . 10 . 7554/eLife . 10561 . 018Figure 8 . LPA/LPA2 axis limits T cell-adhesion area on adhesive substrates .", "( A–C )", "Role of the LPA/LPA2 axis in T cell adhesion and morphology .", "CMFDA-labeled T cells were incubated on a fibronectin- ( A–C ) or ICAM-1- ( D , E ) coated slide glass , pretreated with or without blebbistatin ( 50 μM ) , and stimulated with LPA ( 1 μM ) .", "The adhesion area of CD4+ cells was assessed by TIRF microscopy ( green ) ( A-B , D ) .", "Aspect ratio ( cell length/cell breadth ) and circularity ( 4 x π x area/perimeter2 ) ( C , E ) were assessed by bright-field images .", "Bars: 5 μm .", "Data are pooled from two independent experiments .", "Differences between groups were evaluated by one-way ANOVA .", "***P < 0 . 0005 . DOI: http://dx . doi . org/10 . 7554/eLife . 10561 . 018 We finally analyzed LPA's effect on lymphocyte migration in a more complex confined 3D environment using 3D collagen gel ( Figure 9A ) .", "T cells were placed in type I collagen gel , with LPA placed on one side of the gel and CCL21 plus LPA ( 5 μM ) on the other .", "WT T cells efficiently migrated toward CCL21 in the presence of LPA , with increased displacement ( Figure 9B ) and velocity ( Figure 9C ) compared with cells stimulated with CCL21 alone , whereas the directionality of T cell movement in the gel was comparable in the presence or absence of LPA ( Figure 9—figure supplement 1 ) .", "The LPA’s effect was abrogated by treatment of the cells with Y27632 or blebbistatin ( Figure 9D ) .", "In Lpar2-/- T cells , CCL21 did not enhance motility in the presence of LPA ( Figure 9B , C ) .", "These results collectively indicate that in confined 3D environments , the LPA-LPA2 axis regulates T-cell motility in a manner dependent on Rho , ROCK , and actomyosin . 10 . 7554/eLife . 10561 . 019Figure 9 . LPA/LPA2 axis promotes T-cell motility in a confined 3D environment .", "( A ) Illustration of the 3D migration assay .", "The cell suspension was mixed with collagen gel ( 1 . 6 mg/ml ) .", "CCL21 ( 5 μg/ml ) was loaded into one side of the gel , and LPA ( 5 μM ) was applied to both sides .", "The migration of eGFP-expressing CD4+ T cells or their Lpar2-/- counterparts was recorded for 120 min by time-lapse microscopy .", "( B ) Manual tracking of T cell motility .", "( C ) T cell velocity .", "See also the directionality of T cell movement in Figure 9—figure supplement", "1 . ( D ) Involvement of myosin II/ ROCK in LPA-induced T cell motility .", "After eGFP-expressing CD4+ T cells and WT counterparts were treated with vehicle and indicated inhibitors , respectively , they were mixed with the collagen gel and monitored for cell migration .", "Data are representative of two independent experiments ( B , C ) and pooled from two independent experiments ( D ) .", "Differences between groups were evaluated by Student’s t-test ( C ) or one-way ANOVA ( D ) .", "*P < 0 . 05 , **P < 0 . 005 , ***P < 0 . 0005 . DOI: http://dx . doi . org/10 . 7554/eLife . 10561 . 01910 . 7554/eLife . 10561 . 020Figure 9—figure supplement 1 . Effect of LPA on the directionality of T-cell movement in 3D collagen gel . WT and Lpar2-/- CD4+ T cells were mixed with collagen gel ( 1 . 6 mg/ml ) .", "After applying CCL21 into one side of the gel , the T-cell migration in the collagen gel in the presence or absence of LPA was monitored by time-lapse microscopy .", "Directionality of cell migration was calculated as ( net displacement ) / ( total path length ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 10561 . 020 Taken together , these results strongly suggest that the LPA2 signaling modulates T cell motility in the confined environments at least partly in an integrin-independent manner and may also act on cell adhesion to prevent over-adhesion to the substrates .", "LPA produced by FRCs is essential for this LPA2-dependent T-cell motility in densely packed LN parenchyma ." ], [ "LN tissue is densely packed with several types of cells , including lymphocytes , dendritic cells , and mesenchymal stromal cells .", "Lymphocyte migration in such confined environments is influenced by a delicate balance between actin polymerization/cell adhesion and actomyosin contraction/cell de-adhesion .", "In this study , we demonstrated that LPA is generated ATX-dependently by FRCs in the LN parenchyma , and that the LPA/LPA2-mediated signal activates Rho/myosin II in T cells .", "LPA2 signaling enhances T-cell migration in confined 3D collagen matrices and promotes interstitial T-cell migration in LNs in a manner dependent on FRC-derived LPA .", "These results strongly suggest that the LPA-LPA2 axis critically regulates the confinement-optimized T-cell motility in LNs .", "We previously reported that ATX and LPA are produced by HEV ECs and pericytes ( Bai et al . , 2013 ) .", "In the present study , we found that ATX is also generated by LN FRCs , in agreement with another recent study ( Katakai et al . , 2014 ) , and that multiple LPA species are present in the LN parenchyma; in particular , we showed that LPA ( 18:1 ) , LPA ( 18:2 ) , and LPA ( 20:4 ) are produced in the paracortex in a manner dependent on FRC-derived ATX .", "The enzyme ATX has substrate specificity for LPCs with different acyl-chain lengths and saturations .", "Recombinant mouse ATX prefers LPC species with relatively short , unsaturated acyl chains , and produces LPA ( 18:2 ) , LPA ( 16:0 ) , LPA ( 20:4 ) , and LPA ( 18:1 ) but not LPA ( 18:0 ) ( Yukiura et al . , 2011 ) .", "Consistent with this observation , crystal structural analysis revealed that ATX accommodates LPA ( 18:1 ) and LPA ( 18:3 ) but not LPA ( 18:0 ) in a stable conformation in its hydrophobic pocket , accounting for ATX's preferences for the number of unsaturated bonds of LPC ( 18:0 < 18:1 < 18:3 ) ( Nishimasu et al . , 2011 ) .", "While LPA ( 18:0 ) signals were detected in the LN parenchyma , they were ubiquitously found in other tissues independently of ATX expression; these signals are likely to represent intracellularly produced LPA , an intermediate in the synthesis of triglycerides and glycerophospholipids inside the cell ( Mills and Moolenaar , 2003 ) , which is relatively inert in activating LPA receptors ( Bandoh et al . , 2000 ) .", "Taken together , our findings indicate that multiple biologically relevant LPA species ( 18:1 , 18:2 , 20:4 ) are produced by FRCs , which may in turn act locally on LPA receptors expressed by adjacent lymphocytes .", "Lymphocyte morphology and migration modes adjust according to the microenvironmental context .", "T-cell motility on a 2D surface requires integrin-mediated adhesive forces and detachment from the integrin substrates , whereas the 3D T-cell motility in LNs only partially requires LFA-1-mediated interactions ( Woolf et al . , 2007 ) .", "Jacobelli et al . ( Jacobelli et al . , 2010; Jacobelli et al . , 2009 ) reported that lymphocytes are not restricted to adhesion-dependent motility in 3D environments , and that myosin IIA controls T-cell motility modes—including amoeboid motility , which is dependent on the degree of confinement of 3D environment , and prevents excessive adhesions on non-integrin substrates .", "In our in vitro model , LPA limited T-cell adhesion area on the fibronectin- and ICAM-1-coated substrate , and promoted chemokine-induced T-cell migration across 3-μm but not 5-μm filter pores and also in a confined collagen gel via LPA2 signaling .", "This enhanced migration was inhibited by the ROCK inhibitor Y27632 or the myosin II ATPase inhibitor blebbistatin .", "In vivo , the LPA/LPA2 axis promoted intranodal T-cell migration even in the absence of interaction between LFA-1 and its ligand .", "These findings indicate that LPA acts on LPA2 to enhance T-cell motility in chemokine-rich , confined environments at least partly independently of integrin-mediated adhesion or de-adhesion , and this enhancement depends mainly on ROCK/myosin IIA activity .", "The importance of LPA2 in lymphocyte motility is suggested in recent findings by Knowlden et al ( Knowlden et al . , 2014 ) indicating that Lpar2 deficiency in lymphocytes is associated with inefficient lymphocyte movement within LNs , although it was not shown how LPA2-signaling affects lymphocyte migration and what kind of cells provide the ligand for LPA2 .", "On the other hand , we showed that LPA activated the Rho GTPase in T cells via LPA2 , promoting chemokine-induced T-cell migration across 3-μm- but not 5-μm-pore filters in vitro .", "We further demonstrated that FRC-derived LPA signaled T cells through LPA2 to sustain optimal T-cell motility in the LN parenchyma .", "Thus , LPA appears to serve as a cell-extrinsic factor regulating myosin II activity in CD4+ T cells , and the LPA2/ROCK/myosin II signaling pathway is critical for T-cell migration in the confined LN parenchymal environment .", "In addition , we showed that transferred Lpar2-/- T cells accumulated in LNs more abundantly than WT counterparts 1 . 5–2 hr after the cell transfer ( Figure 4 ) , whereas Knowlden et al showed that Lpar2-deficiency did not affect T-cell trafficking to LNs significantly 42 hr after cell transfer ( Knowlden et al . , 2014 ) .", "It has been shown previously that intravenously injected CD4+ T cells swiftly migrate into LNs and a majority of them reside there for only about 12 hr ( Mandl et al . , 2012 ) before they enter the circulation .", "Therefore , at the time point used by Knowlden et al ( Knowlden et al . , 2014 ) , labeled T cells in LNs might have included those that have re-entered into LNs .", "We and others previously reported that LPA locally produced by HEV ECs regulates constitutive lymphocyte transmigration across HEVs ( Bai et al . , 2013; Zhang et al . , 2012 ) .", "This raises the question of whether LPA produced in the HEV area also affects T-cell motility in the LN parenchyma .", "In this regard , we are currently investigating the effect of EC-specific ATX ablation on lymphocyte behaviors within LNs but do not yet have a definitive answer .", "Concerning the question of whether LPA produced in the LN parenchyma would affect T-cell extravasation from the HEVs , we found comparable lymphocyte migration across HEVs in Enpp2fl/fl and Ccl19-Cre Enpp2fl/fl mice ( unpublished observation ) .", "Given that Ccl19-Cre mice express transgenes in FRCs and in the mesenchymal cells ensheathing HEV ECs , but not in HEV ECs ( Chai et al . , 2013 ) , and that biologically relevant LPA species were produced near HEVs at comparable levels in Ccl19-Cre Enpp2fl/fl mice and Enpp2fl/fl mice ( Figure 2B , C ) , it appears that ATX produced outside HEVs plays a minor or dispensable role in the lymphocyte transmigration across HEVs .", "While B cells expressed high levels of Lpar2 ( Figure 4A ) , adoptively transferred Lpar2-deficient B cells were not retained in the LN ( Figure 4D ) .", "Since B cells , like T cells , showed chemokinesis in response to LPA in an LPA2-dependent manner ( unpublished observation ) , B cells may possess functional downstream elements for LPA2-mediated signals .", "However , whether their intranodal motility depends on motogenic factor ( s ) other than LPA requires further investigation .", "Naïve T cells continuously search for antigen-bearing dendritic cells in the FRC-rich paracortex , and their random migration creates critical opportunities to encounter cognate antigens .", "It is possible that LPA promotes immune responses by increasing the T cells' chances of encountering dendritic cells .", "Once activated by TCR stimulation , T cells reduce their migration speed and S1P1 expression , and thus gain time to proliferate and differentiate into effector cells within the LN ( Schwab and Cyster , 2007 ) .", "Interestingly , TCR activation is reported to downregulate LPA2expression ( Knowlden et al . , 2014; Huang et al . , 2002 ) , which might reduce the velocity of the T cells and allow them to achieve proper activation .", "Taken together , our work offers direct evidence that multiple biologically relevant LPA species produced by FRCs in an ATX-dependent manner act locally on LPA2 on T cells .", "This LPA2-mediated signaling activates the RhoA-ROCK-myosin II pathway and promotes confinement-optimized interstitial T-cell migration in LNs at least partly in an integrin-independent manner .", "Thus , the LPA-LPA2 axis is an attractive pharmacological target for novel strategies to control immune responses ." ], [ "C57BL/6J mice were obtained from Japan SLC .", "Beta-actin-eGFP mice were a gift from Dr . Masaru Okabe ( Research Institute of Microbial Diseases , Osaka University ) ( Okabe et al . , 1997 ) .", "Lpar2-/- mice and Enpp2-flox mice were provided by Dr . Jerold Chun ( the Scripps Research Institute ) ( Contos et al . , 2002 ) and Dr . Woulter H . Moolenaar ( The Netherlands Cancer Institute ) ( van Meeteren et al . , 2006 ) , respectively , to one of us ( JA ) .", "The Ccl19-Cre mice were described previously ( Chai et al . , 2013 ) .", "The Lpar5-/- and Lpar6-/- mice will be described elsewhere .", "B6 . 129X1-Gt ( ROSA ) 26Sortm1Hjf ( R26-YFP ) mice and CD45 . 1 mice were purchased from the Jackson Laboratory ( Bar Harbor , ME ) .", "All mice were housed at the Institute of Experimental Animal Sciences at Osaka University Medical School or Central Animal Laboratory at University of Turku .", "Animal experiments at Osaka University followed protocols approved by the Ethics Review Committee for Animal Experimentation of Osaka University Graduate School of Medicine .", "The experiments performed in Finland were approved by the Ethical Committee for Animal Experimentation in Finland and , they were done in accordance with the rules and regulations of the Finnish Act on Animal Experimentation ( 62/200 ) .", "Anti-PNAd ( MECA-79 ) mAb was purified using a size-exclusion column with size-exclusion resin ( Toyopearl TSK HW55; Tosoh , Japan ) from the ascites of mice inoculated with the hybridoma .", "Purified MECA-79 was labeled with the Alexa Fluor 488 Protein Labeling Kit ( Thermo Fisher Scientific , Waltham , MA ) .", "APC anti-CD4 , Pacific Blue anti-CD8 , APC-Cy7 anti-B220 , APC anti-gp38 , and biotin anti-CD31 mAbs were purchased from Biolegend ( San Diego , CA ) .", "Anti-L-selectin mAb ( MEL-14 ) was generated from the hybridoma , and anti-integrin LFA-1 ( αL ) mAb ( M17/4 ) was purchased from Bio X cell ( Lebanon , NH ) .", "Streptavidin-BD Horizon V500 was obtained from BD Biosciences ( San Jose , CA ) , and LPA ( 18:1 ) and blebbistatin were from Sigma-Aldrich ( St . Louis , MO ) .", "Mouse CCL21 and mouse ICAM-1-Fc were from R&D Systems ( Minneapolis , MN ) .", "FRCs and BECs were isolated as described previously ( Fletcher et al . , 2011 ) .", "Cells were incubated with PE anti-CD45 , APC anti-gp38 , and biotin anti-CD31 mAbs , followed by streptavidin-BD Horizon V500 .", "Stromal-cell populations were sorted by FACSAria ( BD Biosciences ) .", "Total RNA was isolated using RNeasy ( Qiagen , Germany ) , and single-strand cDNA was synthesized using M-MLV reverse transcriptase ( Promega , Fitchburg , WI ) with a random primer .", "PCR was performed using the Go Taq qPCR system ( Promega ) at 95°C for 10 min followed by 40 cycles at 95°C for 15 s and 60°C for 1 min .", "The PCR primer pairs are described in Supplementary file 1 .", "ATX localization was examined by immuno-transmission electron microscopy as previously described ( Bai et al . , 2013 ) .", "Cryosections were incubated with a guinea pig–derived polyclonal antibody against mouse ATX .", "The LTβR-Fc expression vector , in which LTβR's extracellular domain was fused to the Fc region of human IgG1 , was a gift from Dr . Atsushi Togawa ( Yoshida et al . , 2002 ) ( Fukuoka University Hospital ) .", "LTβR-Fc was expressed in 293T cells and purified by a HiTrap Protein A column ( GE Healthcare , UK ) .", "To block LTβR signaling , LTβR-Fc or control human IgG Fc ( 100 μg each ) was injected intraperitoneally into mice ( 8–12 weeks old ) weekly for 4 weeks as described previously ( Browning et al . , 2005 ) .", "IMS was performed as previously described ( Bai et al . , 2013 ) , with minor modifications .", "Briefly , fluorescein-conjugated dextran ( 40 kDa ) ( Thermo Fisher Scientific ) was injected into the foreleg footpads to visualize LN structures , and 10 min later , brachial LNs were collected and snap-frozen in liquid nitrogen .", "Cryosections ( 8-μm thick ) were cut and transferred onto glass slides coated with Indium Tin Oxide ( Resistance , 20 ohm; Matsunami , Japan ) .", "The sections were washed with 0 . 03% formate to remove endogenous salts and coated with matrix compound ( 10 mg/ml 9-aminoacridine dissolved in 70% ethanol ) .", "IMS analysis was performed with a MALDI-QIT-TOF-mass spectrometer ( iMscope; Shimadzu , Japan ) equipped with a 355-nm Nd:YAG laser .", "Ions at m/z 437 , 435 , 433 and 457 , which include ions of LPA ( 18:0 ) , LPA ( 18:1 ) , LPA ( 18:2 ) , and LPA ( 20:4 ) respectively , were fragmented by collision-induced dissociation , and the ions produced were analyzed .", "The ion at m/z 153 corresponded to the LPA-specific fragment ion .", "MS/MS imaging analysis was performed at a raster scan pitch of 50 μm .", "Image reconstruction of the LPA signals was performed with BioMap Software ( Novartis , Switzerland ) .", "Images of fluorescein-dextran in the same section were acquired before IMS analysis .", "For immunohistochemical analyses , serial sections were stained with Alexa Fluor 594-conjugated MECA-79 and Hoechst 33342 , and examined by confocal microscopy ( LSM710; Zeiss , Germany ) .", "Distances between LPA signals and MECA-79+ HEVs were measured with ImageJ software .", "Intravital two-photon microscopy was performed as described previously ( Liou et al . , 2012 ) .", "Briefly , CD4+ T cells were isolated by negative selection kits ( Stemcell Tech , Canada or Miltenyi Biotec , Germany ) from the LNs and spleen of eGFP-expressing and Lpar2-/- mice .", "Lpar2-/- CD4+ T cells were labeled with 5 μM CMTMR ( Thermo Fisher Scientific ) , mixed with an equivalent number of WT CD4+ T cells , and injected into recipient mice ( 5 x 106 cells/mouse ) ; 15–25 hr later , the mice were anesthetized using isoflurane admixed with O2 .", "The animals were placed on a custom-built preparation stage , the right hind leg was immobilized , and microdissection tweezers were used to expose the PLNs without jeopardizing the blood vessels and afferent lymphatic vessels .", "Sterile PBS at 37°C was applied to the stage to submerge the LNs , and the temperature was maintained at 37°C using a heating pad with a feedback probe .", "Two-photon laser-scanning microscopy was performed with an upright Leica TCS SP5 equipped with a 20x water immersion objective ( HCX APO , N . A . 1 . 0; Leica , Germany ) and a MaiTai Ti:sapphire-pulsed laser ( Spectra-Physics , Santa Clara , CA ) set at 880 nm for two-photon excitation .", "To generate time-lapse series , stacks of more than 30 x-y sections spaced at 2–3 μm were acquired every 20–30 s in resonance mode .", "For evaluating integrin-dependency in LPA-induced T-cell motility , 300 μg of anti-LFA-1 antibody was intravenously injected , and 1 hr later , cell migration was monitored again .", "Imaris software ( Bitplane , UK ) was used to track cells in 3D and measure the cells’ velocity and x , y , z coordinates .", "Mean displacement plots were calculated from the x , y , z coordinates using Excel ( Microsoft , Redmond , WA ) as described previously ( Sumen et al . , 2004 ) .", "The motility coefficient ( M ) was calculated from the mean displacement plot as M = D2/6t , in which D is displacement and t is time , using 10-min tracks .", "The directionality of cells’ motion was calculated by dividing cells’ net displacement by the total path length , using over 10-min tracks ( Sumen et al . , 2004 ) .", "Lymphocyte migration was assayed by flow cytometry as previously described ( Bai et al . , 2009 ) .", "Splenocytes from eGFP-expressing mice and LPA receptor–deficient mice were mixed in equal numbers and incubated with Sulfo-NHS-LC-biotin reagent ( 80 μg/ml ) ( Thermo Fisher Scientific ) for 30 min ( Nolte et al . , 2004 ) .", "The biotin-labeled cells were injected intravenously into recipient mice ( 2 x 107 cells/mouse ) , and the LNs and spleen were harvested 1 . 5 hr later .", "The cell suspension was incubated with APC anti-CD4 , Pacific Blue anti-CD8 , APC-Cy7 anti-B220 mAbs , and streptavidin-PE .", "The frequencies of T and B cells in GFP+ and GFP- populations of biotinylated cells were analyzed by flow cytometry ( FACSCanto , BD Biosciences ) .", "Lymphocyte migration was assayed by whole-mount microscopy as described previously ( Kanemitsu et al . , 2005 ) .", "Briefly , WT and Lpar2-/- CD4+ T cells ( L-selectin+ CD44- naïve T cells in CD4+ T cells of WT and Lpar2-/- mice were 79 . 7 ± 10 . 7% and 78 . 7 ± 9 . 1% , respectively ) were isolated by negative immunomagnetic cell sorting ( Miltenyi Biotec ) and labeled with 5 μM DiD and 5 μM CMTMR ( Thermo Fisher Scientific ) , respectively .", "The cells were mixed in equal numbers ( 1 x 107 cells ) and injected intravenously into WT mice; after 1 . 5 hr , MECA-79 mAb was injected to label the luminal HEV surface , and inguinal LNs were isolated .", "The LNs were fixed with 4% paraformaldehyde in phosphate buffer and incubated with sucrose ( 30% ) .", "Immunofluorescence signals were observed with a confocal laser-scanning microscope ( FV1000-D , Olympus , Japan ) .", "The distance between T cells and the nearest HEV was measured using Imaris software .", "T cell retention was assessed as described before with minor modifications ( Pham et al . , 2008 ) .", "CD45 . 1+ WT and CD45 . 2+Lpar2-/- CD4+ T cells were isolated by negative sorting , mixed in equal numbers ( 1 x 107 cells ) , and labeled with 2 μM of CFSE ( Thermo Fisher Scientific ) .", "The cells were injected intravenously into recipient WT mice; 2 hr later , the LNs and spleen were collected from half of the recipient mice , and 100 μg of anti-L-selectin antibody ( MEL-14 ) was intraperitoneally injected into the rest of the recipient mice; 23 hr later , tissues were harvested from the antibody-treated mice .", "The frequency of donor-derived T cells was measured by flow cytometry .", "An EZ-Taxiscan chamber ( Effector Cell Institute , Japan ) was used for time-lapse chemotaxis assays as described previously ( Bai et al . , 2009 ) .", "Negatively sorted CD4+ T cells were incubated in serum-free medium for 30 min at 37°C .", "Cells were suspended in LPA ( 1 μM ) -containing or control buffer ( 0 . 1% BSA/RPMI1640 ) and loaded into microchamber wells ( 4-μm deep ) , each holding a cover glass precoated with ICAM-1 ( 5 μg/ml ) , and 100 ng of CCL21 was applied to the contra-wells .", "Phase-contrast images of migrating cells were acquired at 1-min intervals for 1 hr at 37°C .", "Cells that migrated toward the contra-wells were counted from the images , and 30 migrating cells were tracked using the ImageJ ( NIH ) manual tracking plug-in .", "ImageJ software was also used to measure the cells’ velocity and x , y coordinates .", "Turning angles were calculated with Excel using the cells’ x , y coordinates .", "LN cells were incubated in serum-free medium ( 0 . 1% BSA/RPMI1640 ) for 30 min at 37°C .", "The cells ( 1 x 106 cells ) were added in the presence or absence of LPA to the upper chamber of a Transwell apparatus ( pore size , 3 or 5 μm; Corning , Corning , NY ) and allowed to migrate for 2 hr at 37°C .", "In some experiments , CCL21 ( 200 ng/ml ) was added to the lower chamber .", "Migrated cells were counted by flow cytometry using 6 . 0-μm Fluoscribe microspheres ( Polysciences , Warrington , PA ) .", "FCS and fatty acid–free BSA were incubated with activated charcoal ( Wako , Japan ) to remove all lipid fractions .", "The spleen and LNs were collected , and a single-cell suspension was prepared in RPMI1640 containing 1% FCS .", "T cells were negatively sorted and starved in serum-free medium ( 0 . 1% BSA/RPMI1640 ) for 2 hr at 37°C .", "Next , cells ( 1 x 107 ) were stimulated with 1 μM LPA at 37°C for 2 or 5 min .", "The cells were collected by centrifugation and immediately dissolved in lysis buffer .", "The GTP-bound form of RhoA in the lysate was pulled down with Rhotekin and detected by western blotting using the RhoA Activation Assay Biochem Kit ( Cytoskeleton , Denver , CO ) .", "Splenocytes were labeled with 2 μM CMFDA ( Thermo Fisher Scientific ) and incubated with biotin-conjugated anti-CD4 mAb .", "The cells were applied onto human fibronectin ( 0 . 002% , Sigma-Aldrich ) or mouse ICAM-1-Fc ( 5 μg/ml ) -coated glass slides and starved in serum-free medium ( 0 . 1% BSA/RPMI ) for 2 . 5 hr at 37°C .", "The cells were then cultured in the presence or absence of 50 μM blebbistatin for 15 min , followed by stimulation with LPA ( 1 μM ) for 30 min .", "After unbound cells were gently washed off , adherent cells were fixed with paraformaldehyde and CD4+ cells were stained by applying Alexa Flour 594-conjugated streptavidin .", "Surface adhesion zones of CD4+ T cells to the substrate were measured as CMFDA+ area by total internal reflection fluorescence ( TIRF ) microscopy ( Jacobelli et al . , 2010 ) .", "TIRF images were acquired with Olympus IX71 equipped with a 100x oil immersion objective ( PlanApo; Olympus ) , 488 nm blue laser ( FITEL HPU50101; Furukawa Electric , Japan ) and an electron multiplier CCD camera ( C9100; Hamamatsu , Japan ) .", "The laser and camera were controlled by Metamorph software ( Molecular Devices , Sunnyvale , CA ) .", "The circularity was calculated as ( 4 x π x area ) /perimeter2 .", "The value should range from 0 to 1 , and value 1 represents most circular .", "The aspect ratio was calculated as a ratio of cell length to cell breadth .", "These morphology parameters were analyzed with brightfield images by using ImageJ software .", "3D migration assays were performed with μ-Slide Chemotaxis3D ( Ibidi , Germany ) according to the manufacturer's protocol .", "Negatively sorted CD4+ T cells were incubated in serum-free medium for 1 hr at 37ºC .", "In some experiments , the cells were treated by 50 μM blebbistatin or 10 μM Y27632 .", "Cell suspensions were mixed with collagen type I ( Advanced Biomatrix , San Diego , CA ) and incubated for 1 hr at 37°C to allow collagen polymerization .", "The final cell and gel concentrations were 9 x 106 cells/ml and 1 . 6 mg/ml , respectively .", "CCL21 ( 5 μg/ml ) and LPA ( 5 μM ) were loaded into one side of the reservoir , and LPA ( 5 μM ) was applied to the other side .", "Fluorescent and phase-contrast images of migrating cells were acquired at 1-min intervals for 3 hr at 37°C using an inverted microscope ( IX-81 , Olympus ) , and 30 migrating cells were manually tracked and their velocity measured using ImageJ software .", "Differences between groups were evaluated using Prism software ( GraphPad Software , La Jolla , CA ) by Student's t-test for single comparisons or one-way ANOVA for multiple comparisons , followed by post-hoc Tukey tests .", "A P value < 0 . 05 was considered significant .", "Data are presented as mean ± SD unless otherwise described ." ] ]

[ "Lymph nodes ( LNs ) are highly confined environments with a cell-dense three-dimensional meshwork , in which lymphocyte migration is regulated by intracellular contractile proteins .", "However , the molecular cues directing intranodal cell migration remain poorly characterized .", "Here we demonstrate that lysophosphatidic acid ( LPA ) produced by LN fibroblastic reticular cells ( FRCs ) acts locally to LPA2 to induce T-cell motility .", "In vivo , either specific ablation of LPA-producing ectoenzyme autotaxin in FRCs or LPA2 deficiency in T cells markedly decreased intranodal T cell motility , and FRC-derived LPA critically affected the LPA2-dependent T-cell motility .", "In vitro , LPA activated the small GTPase RhoA in T cells and limited T-cell adhesion to the underlying substrate via LPA2 .", "The LPA-LPA2 axis also enhanced T-cell migration through narrow pores in a three-dimensional environment , in a ROCK-myosin II-dependent manner .", "These results strongly suggest that FRC-derived LPA serves as a cell-extrinsic factor that optimizes T-cell movement through the densely packed LN reticular network ." ]

[ "Small organs called lymph nodes are found throughout the body and help to filter out harmful particles and cells .", "Lymph nodes are packed with different types of immune cells , such as the T-cells that play a number of roles in detecting and destroying bacteria , viruses and other disease-causing microbes .", "Within the lymph node , T-cells crawl along a meshwork made up of cells called fibroblastic reticular cells .", "The T-cells appear to move in random patterns , but the signals that drive this movement remain ill-defined .", "Now , Takeda et al . reveal that a lipid called lysophosphatidic acid ( LPA ) , which is produced by the fibroblastic reticular cells , is responsible for regulating how T-cells move around inside the lymph nodes .", "T-cells are able to detect LPA via certain receptor proteins on their surface .", "Takeda et al . engineered mice that were either unable to produce a particular LPA receptor on their T-cells , or that produced less LPA than normal .", "The T-cells of these mice moved around less than T-cells in normal mice .", "Further experiments revealed that LPA signaling also affects the signaling pathway that alters how well the T-cells stick to nearby surfaces .", "This suggests that LPA helps to optimize T-cell movement to allow the cells to navigate the small spaces found between the fibroblastic reticular cells .", "In the future , targeting the processes involved in LPA signaling could help to develop new treatments for disorders of the immune system ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "chromosomes and gene expression" ]

[ [ "The genomic DNA of eukaryotes is packaged into chromatin , which regulates all DNA transactions including transcription , replication and repair .", "The basic subunit of chromatin is the nucleosome , which is made up of 147 bp of DNA wrapped around a core octamer of histone proteins .", "Proteins that regulate chromatin dynamics are frequently mutated in cancer , and recently mutations in the histone genes themselves have been discovered ( Schwartzentruber et al . , 2012; Wu et al . , 2012; Behjati et al . , 2013 ) .", "One such mutant is the Glycine 34 to Arginine ( G34R ) mutant of histone H3 . 3 , identified as a frequent somatic mutation in pediatric high-grade cortical glioma ( pHGG ) .", "H3 . 3 is a non-canonical H3 variant , whose deposition is not linked to replication and which can accumulate in post-mitotic cells and in areas of high transcriptional activity ( Skene and Henikoff , 2013 ) .", "Notably , G34R mutations are only found in H3 . 3 , primarily in one of two H3 . 3 genes ( H3F3A ) and not in the replication coupled H3 proteins H3 . 1 and H3 . 2 , which are encoded by a total of 13 genes .", "G34R mutations are commonly found in tumors additionally mutant for the tumor suppressor p53 and ATRX , a chromatin remodeler involved specifically in nucleosomal deposition of the H3 . 3 variant histone at telomeres and pericentric heterochromatin ( Goldberg et al . , 2010; Lewis et al . , 2010; Drané et al . , 2010 ) .", "While p53 and ATRX mutations may contribute to the genomic instability exemplified by these gliomas , the specific role of the G34R H3 . 3 mutant in disease remains a mystery .", "One published study showed G34R nucleosomes had reduced K36me2/3 on the same H3 . 3 tail ( Lewis et al . , 2013 ) , suggesting a potential role in regulating H3K36 methylation in gliomas .", "Furthermore , mutations in SETD2 , the enzyme that performs H3K36 di- to tri-methylation in mammals ( Edmunds et al . , 2008 ) , have also frequently been identified in pHGG ( Fontebasso et al . , 2013 ) .", "However , SETD2 mutations are not found in conjunction with histone mutations , suggesting the importance of disruption of the H3K36me3/ SETD2 axis in cortical pHGG .", "To gain biological insight into the role of the G34R mutant , we engineered fission yeast to express either wild-type or G34R mutant histone H3 .", "In fission yeast , a single enzyme , Set2 , performs all methylation on H3K36 ( Morris et al . , 2005 ) .", "We hypothesized that H3-G34R mutation may reduce Set2 function and influence important chromatin-templated processes such as DNA transcription , replication and repair , which are highly conserved between fission yeast and human .", "Here , we show that histone H3-G34R mutation specifically reduced tri-methylation , but not di-methylation of H3K36 , and caused a decrease in H3K36 acetylation .", "However , G34R mutants displayed different phenotypes to set2Δ , suggesting that defective H3K36 modification may not be the sole defect in G34R .", "H3-G34R mutants showed chromosome instability and sensitivities to DNA damaging agents which are distinct from cells lacking Set2 .", "H3-G34R mutants had defects in HR-directed repair , which may be attributed to a delay in DNA repair dynamics at compromised replication forks .", "Together , our work provides valuable insight to the potential role of H3-G34R mutations in pediatric high-grade gliomas ." ], [ "In mammalian cells , ectopic expression of H3 . 3G34R reduced K36me2/3 methylation on the mutant histone tail ( Lewis et al . , 2013 ) .", "Five enzymes can methylate H3K36 in mammals , with only SETD2 responsible for H3K36me3 ( Edmunds et al . , 2008 ) .", "In fission yeast , a single enzyme ( Set2 ) performs all methylation on H3K36 ( Morris et al . , 2005 ) .", "We began our investigation by determining if H3-G34R mutation reduces Set2 H3K36 methyltransferase activity .", "Because of the proximity of G34R to K36 , we tested whether antibodies can specifically recognize K36 methylation states in the G34R mutant .", "Using di- or tri-methylated K36 H3 peptides as targets , we identified two anti-K36me3 antibodies that were minimally affected by G34R mutation , and one antibody against K36me2 whose binding was weakened ~10 fold by the G34R mutation ( Figure 1b , Figure 1—figure supplement 1f ) .", "Figure 1—figure supplement 1f–g serve to demonstrate reproducibility of the H3K36me3 methylation pattern with an alternate H3K36me3-specific antibody .", "Using these antibodies , we found that H3K36me3 was markedly reduced in H3-G34R compared to H3-WT cell extracts , but that H3K36me2 levels were unchanged ( Figure 1c , Figure 1—figure supplement 1g ) .", "As we observed that binding of the H3K36me2 antibody is reduced ~10 fold on G34R peptides , these results suggest that K36me2 is elevated in H3-G34R cells .", "Thus Set2 function is altered in H3-G34R cells , with specific reduction of H3K36me3 and retention/ accumulation of H3K36me2 .", "Since endogenously tagged Set2-FLAG protein levels were similar in H3-WT and H3-G34R cells ( Figure 1—figure supplement 1h ) , the reduction in H3K36me3 is not caused by a loss of set2+ expression in H3-G34R cells .", "Additionally , chromatin immunoprecipitation ( ChIP ) analysis revealed that Set2-FLAG recruitment to chromatin was not reduced in H3-G34R cells ( Figure 1d ) , suggesting that the H3-G34R mutation hinders Set2 activity but not access to K36 .", "We therefore asked if we could force the generation of H3K36me3 in H3-G34R cells by overexpression of the Set2 methyltransferase ( Figure 1—figure supplement 1i ) .", "Western analysis of extracts prepared from cells that overexpress Set2 showed that pSet2-FLAG restored K36me2 and me3 in H3-WT set2Δ strains but was able to generate only low levels of H3K36me3 in both H3-G34R and H3-G34R set2Δ cells ( Figure 1e ) .", "Thus the H3-G34R mutation reduces Set2 activity on the H3 tail , leading to a specific reduction in tri-methylation of H3K36 , and this effect cannot be bypassed by overexpression of Set2 .", "In addition to methylation of H3K36 , this residue is also regulated by acetylation ( Morris et al . , 2007 ) .", "To address whether H3-G34R impedes acetylation at H3K36 , we used quantitative mass spectrometry to perform targeted analysis of acetylation of tails of histone H3 and H4 in histones acid extracted from H3-WT , H3-G34R and set2Δ strains ( Figure 1f ) ( Kuo and Andrews , 2013 ) .", "Calibration was performed to monitor the elution time of the acetylated and propionylated tryptic peptides and transitions were created to study acetylation of H3-WT and H3-G34R as well as the H4 tails ( see Supplementary files 1 and 2 and Materials and methods ) .", "These studies showed acetylation of K36 on histone H3 was greatly reduced in H3-G34R strains and somewhat reduced in set2Δ .", "Additionally , there was a slight upregulation of H3K18 acetylation and possibly enhanced K27 acetylation in H3-G34R , although K27 acetylation in H3-WT was highly variable .", "set2Δ cells showed more widespread changes , with a slight reduction of H4 K16 acetylation , some induction of H3K9 and K18 acetylation and a marked increase in H3K14 acetylation .", "In summary , H3-G34R cells showed a focal pattern of chromatin changes centered on inefficient tri-methylation and reduced acetylation of histone H3K36 , whereas set2Δ effects were more widespread .", "H3K36me3 and H3K36 acetylation have been shown to be cell cycle regulated ( Li et al . , 2013; Pai et al . , 2014 ) , with K36me3 accumulating in G1 and early-mid S phase .", "We assessed the consequence of H3-G34R mutation on K36me3 accumulation at several stages of the cell cycle , using cells synchronized in G1 , S , G2 and M phases ( Figure 1—figure supplement 2 ) .", "Although levels of H3K36me3 were reduced in H3-G34R cells compared to H3-WT at all stages of the cell cycle ( Figure 1—figure supplement 2a , b , c ) , H3K36me3 did accumulate in both H3-WT and H3-G34R cells synchronized in G1 , indicative that the G34R mutation does not override cell cycle control of Set2 function .", "Additionally , this experiment also demonstrates that H3K36me2 levels are higher throughout the cell cycle in H3-G34R compared to H3-WT cells .", "Post-translational modifications of H3K36 are important for transcriptional control , raising the possibility of transcriptional disruption in H3-G34R cells .", "Cells that lack the H3K36 methyltransferase Set2 or that express a mutant of Set2 that allows only di-methylation of K36 show a similar widespread transcriptional upregulation in fission yeast ( Suzuki et al . , 2016; Matsuda et al . , 2015 ) , suggesting that H3K36me2 is not sufficient for silencing .", "Since H3-G34R cells exhibit reduced H3K36me3 and possibly enhanced K36me2 , as well as a reduction in H3K36Ac , we asked how H3-G34R mutation influences transcriptional control .", "In genome-wide RNA-Seq analyses , set2Δ had many transcripts with altered regulation ( 753 genes were up and 149 genes downregulated ) ( Figure 2a , b ) in an organism with ~5100 protein coding genes and ~1500 non coding RNAs ( Supplementary file 3 ) .", "Unexpectedly , the H3-G34R mutant showed more mild transcriptional effects ( repression of 69 genes and upregulation of 172 genes ) .", "This difference in gene regulation may stem from additional chromatin changes such as increased H3K14Ac in set2Δ . 10 . 7554/eLife . 27406 . 006Figure 2 . H3-G34R mutants show a distinct transcriptional profile to set2Δ cells . Schematic of genes that are", "( a ) differentially-regulated , or", "( b ) similarly down-regulated or up-regulated comparing either H3-G34R or set2Δ cells with H3-WT cells in RNA-seq analyses .", "Triplicate biological replicates were analyzed with cut-offs of 1 . 5 fold differences in expression , and false discovery rates of 5% .", "( c ) RNA-seq profiles for chromosomes I , II , and III comparing Log fold change ratios for H3-G34R/H3-WT or set2Δ/H3 WT plotted against chromosome coordinates .", "( d ) Zoomed-in regions of RHS Chr I ( 5 . 3 Mb-end , top ) and LHS Chr II ( first 300 Kb , bottom ) as depicted in the boxed regions in", "( c ) showing log FC data for individual biological replicates .", "( e ) RT-PCR validation of fah1+ and grt1+ expression relative to adh1+ expression .", "( f ) Density plots of RNA seq reads for sense ( black ) and antisense transcripts ( blue ) against log fold change ratios for set2Δ/H3 WT ( left ) and H3-G34R/H3 WT ( right ) .", "Please see Figure 2—figure supplement 1 for analysis of heterochromatic loci , and Supplementary file 3 for full RNA-seq analysis results . DOI: http://dx . doi . org/10 . 7554/eLife . 27406 . 00610 . 7554/eLife . 27406 . 007Figure 2—figure supplement 1 . Heterochromatin integrity is maintained in H3-G34R cells .", "( a ) Real-time ( RT ) PCR analysis of pericentromeric imr , dg , and dh expression as well as the subtelomeric tlh expression in the indicated strains .", "Transcripts were compared relative to adh1+ expression .", "These domains are silenced in WT cells , so clr4Δ cells that lack heterochromatin represent a positive control .", "Error bars represent SEM from 2 independent experiments .", "( b ) Chromatin immunoprecipitation ( ChIP ) of H3K9me2 and", "( c ) Swi6 in the indicated cells at either the dh or tlh loci relative to adh1+ .", "Error bars represent SEM from 5 biological replicates .", "( d ) ChIP analysis of the Cnp1 protein to central core sequence cnt2 , with ChIP enrichment depicted relative to input material .", "Data represents 6 biological replicates with error bars representing standard deviation . DOI: http://dx . doi . org/10 . 7554/eLife . 27406 . 007 143 genes were deregulated in both H3-G34R and set2Δ cells: 97 were coordinately regulated , with 9 genes downregulated , and 88 genes upregulated in both set2Δ and H3-G34R ( Figure 2b ) .", "In most other cases ( 44/46 ) , transcripts accumulated in set2Δ but were repressed in H3-G34R .", "As can be seen on chromosome-wide plots ( Figure 2c , d ) , transcripts that are differentially regulated between H3-G34R and set2Δ map to regions adjacent to subtelomeric regions of chromosome 1 and 2 ( ST or sub-subtelomeric domains [Matsuda et al . , 2015; Buchanan et al . , 2009] ) , which are not heterochromatic and are normally expressed at low levels ( Mata et al . , 2002 ) .", "We confirmed gene regulation trends for 2 genes from ST domains by real time qPCR ( Figure 2e ) , and additionally show that transcript levels for these genes in the double mutant , H3-G34R set2Δ , are similar to set2Δ cells .", "Thus H3K36me2 is perhaps critical for the repression in ST domains seen in H3-G34R cells .", "Intriguingly , these domains have been recently shown to associate with Shugoshin ( Sgo2 ) to form highly condensed subtelomeric ‘knob’ regions which are transcriptionally silenced and delayed for replication ( Matsuda et al . , 2015; Tashiro et al . , 2016 ) .", "Set2 has also been shown in both S . cerevisiae and S . pombe to repress antisense transcription ( Nicolas et al . , 2007; Shim et al . , 2012; Venkatesh et al . , 2016 ) .", "Focusing on just these antisense transcripts , of which there are 635 in fission yeast , we found that , compared to H3-WT cells , 318 were differentially regulated in set2Δ , and 62 in H3-G34R mutants , with the vast majority being upregulated ( 313 and 57 , respectively ) ( Figure 2f ) .", "47 antisense transcripts were targeted by both Set2 and H3-G34R , and 43 of these accumulated in both mutants .", "Therefore H3-G34R cells , similar to set2Δ cells , upregulate antisense transcripts , indicative that trimethylation of K36 may repress antisense transcription .", "In conclusion , interpretation of the transcriptional profiling results is complex .", "There is some overlap with Set2-mediated control as shown by similar regulation of some sense and some antisense genes , but also differences as evidenced by opposite regulation of transcription with sub-subtelomeric domains .", "We conclude that suppression of antisense gene transcription appears linked to trimethylation of K36 , as it is reduced in both H3-G34R and set2Δ , and that repression of sub-subtelomeric domains may be linked to dimethylation of K36 in H3-G34R , as it is abolished in set2Δ or compound set2Δ H3-G34R mutants that lack dimethylation of K36 .", "RNA-Seq provides information on unique transcripts .", "To determine whether constitutive heterochromatin that forms on repetitive sequences was affected by H3-G34R mutation , we monitored transcripts from pericentromeric loci and the subtelomeric tlh genes in H3-WT and H3-G34R cells ( Figure 2—figure supplement 1a ) , with clr4Δ serving as a control for loss of heterochromatin ( Ekwall et al . , 1996; Allshire et al . , 1995 ) .", "Transcript levels were similar in H3-G34R and H3-WT , suggesting that heterochromatin is intact in H3-G34R mutants , whereas set2Δ showed a slight upregulation in subtelomeric tlh transcripts .", "Additionally we monitored H3K9me2 , a conserved heterochromatin mark , and the Swi6HP1 protein which binds to that mark ( Nakayama et al . , 2001; Bannister et al . , 2001 ) .", "As shown in Figure 2—figure supplement 1b and c , H3-G34R mutation had no impact on H3K9 methylation or Swi6HP1 recruitment to heterochromatic regions ( Ekwall et al . , 1995 ) .", "ChIP against Cnp1 ( Castillo et al . , 2007 ) , the fission yeast CENP-A protein that underlies and supports kinetochore assembly , showed no change in Cnp1 recruitment to centromeric central core sequences in H3-G34R mutant cells ( Figure 2—figure supplement 1d ) .", "Together , the lack of apparent effect on kinetochore or heterochromatic structures indicates that the architecture of centromeric and telomeric heterochromatin is intact in H3-G34R cells .", "We next tested genomic stability in H3-G34R mutants by monitoring the frequency of chromosome loss using a non-essential minichromosome Ch16 ( Niwa et al . , 1989 ) .", "As expected , H3-WT cells showed low levels of loss of Ch16 ( 0 . 9% cells ) ( Mellone et al . , 2003 ) and cells lacking the heterochromatin protein Swi6HP1 displayed high frequencies of Ch16 loss ( 6 . 4% , Figure 3a ) .", "Interestingly , we found that H3-G34R cells lost minichromosomes at an elevated frequency ( 3 . 5% ) , while minichromosome loss in set2Δ was not significantly upregulated ( 1 . 6% ) .", "We further tested for specific chromosome segregation defects in H3-G34R strains by monitoring frequencies of lagging chromosomes on late anaphase spindles , using anti-tubulin antibodies to stain the spindle , and DAPI to stain DNA ( Figure 3b ) ( Ekwall et al . , 1995 ) .", "Counting only late anaphase cells ( spindle >10 microns ) , we found that chromosomes mis-segregated in 7 . 4% of H3-G34R cells compared to 0 . 9% in H3-WT , 1 . 3% in set2Δ and 25 . 6% in clr4Δ cells that lack heterochromatin .", "Furthermore , in cells synchronized using an nda3-KM311 early mitotic block and release ( Hiraoka et al . , 1984 ) , and stained for tubulin and DNA , ~38% of H3-G34R cells showed stretching of DNA or aberrantly positioned DNA on mid to late anaphase spindles compared with ~14% of H3-WT cells ( Figure 3c ) .", "Together , these data show that the H3-G34R cells exhibit elevated chromosomal instability , which is independent of heterochromatin or kinetochore defects .", "The nature of the chromosome segregation defects in H3-G34R cells is suggestive of a defect in DNA condensation or resolution of sister chromatids after DNA replication ( Saka et al . , 1994 ) . 10 . 7554/eLife . 27406 . 008Figure 3 . H3-G34R cells exhibit genomic instability .", "( a ) Frequency of cells that lose the non-essential minichromosome Ch16 in H3-WT , H3-G34R , set2Δ and swi6Δ cells .", "Asterisk indicates significant difference from H3-WT ( p<0 . 05 ) .", "Mean ± SEM from 4 experiments shown .", "( b ) Frequency of late anaphase cells that show a lagging chromosome in H3-WT , G34R , set2Δ , and clr4Δ .", "Mean ± SEM from 2 experiments shown .", "G34R and clr4Δ have small p values ( 0 . 1 ) although not significantly different from WT", "( c ) Frequency of cells with chromosome segregation defects in H3-WT and H3-G34R cells .", "Cells were synchronized using nda3-KM311 and chromosome segregation phenotypes were scored in cells with different spindle lengths .", "Data are represented as mean ± SD from 2 biological replicates .", "Right panels depict representative images of", "( b ) normal and lagging or", "( c ) ‘trailing’ chromosomes ( DAPI = DNA; FITC = tubulin ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 27406 . 008 Since H3-G34R cells show increased chromosomal instability , and are reduced in histone H3 K36 acetylation and tri-methylation , which are modifications that are linked to DNA damage responses ( Pai et al . , 2014; Pfister et al . , 2014; Jha and Strahl , 2014; Li et al . , 2013 ) , we tested H3-G34R strains for sensitivity to DNA damaging agents .", "Cells were plated on media containing hydroxyurea ( HU; a ribonucleotide reductase inhibitor that depletes dNTPs ) , camptothecin ( CPT; an agent that blocks topoisomerase 1 ligase activity ) , or methyl methanesulfonate ( MMS; an alkylating agent ) ( Figure 4a ) .", "At the concentrations used , each of these agents predominantly affects DNA replication ( HU and MMS ) , or requires DNA replication to inflict DNA damage ( CPT ) .", "We found that chronic exposure to each genotoxin resulted in significant sensitivity of H3-G34R cells compared with H3-WT cells .", "Conversely , when H3-G34R and H3-WT cells were exposed to gamma irradiation ( γIR ) or bleomycin , two replication-independent genotoxins , H3-G34R mutants showed no sensitivity , whereas set2Δ cells were sensitive to γIR and bleomycin ( Figure 4b and", "c ) as seen previously ( Pai et al . , 2014 ) .", "Thus H3-G34R , but not set2Δ cells appear selectively sensitive to agents that generate DNA replication stress . 10 . 7554/eLife . 27406 . 009Figure 4 . H3-G34R mutants show DNA damage sensitivity upon replication stress .", "( a ) 5-fold serial dilutions showing the effect of methyl methanesulfonate ( MMS ) , camptothecin ( CPT ) , and hydroxyurea ( HU ) on the indicated strains .", "( b ) Effect of γ−irradiation ( IR ) exposure on viability of H3-WT , H3-G34R , set2Δ , and rad51Δ cells .", "Data represent mean ± SEM from 2 biological replicate experiments .", "( c ) Serial dilution assay showing the effect of bleomycin on the indicated strains .", "( d ) Serial dilution assay showing the genotoxin sensitivity of H3-WT , H3-G34R , H3-WT set2Δ , and H3-G34R set2Δ cells upon MMS , CPT , or HU treatment .", "( e ) Genotoxin sensitivity of indicated strains containing either an empty vector ( EV ) or a vector overexpressing Set2-3xFLAG ( pSet2 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 27406 . 009 To further address the relationship of the H3-G34R mutant with Set2 , we tested genetic interactions between set2Δ and H3-G34R .", "In the absence of damage , there were no synthetic growth defects in the set2Δ H3-G34R double mutant .", "set2Δ were more sensitive than H3-G34R to MMS and CPT , but showed similar sensitivity to H3-G34R on HU .", "Additive growth defects were seen for set2Δ H3-G34R double mutants on CPT and MMS , but not with HU ( Figure 4d ) .", "Repair of damage from CPT or MMS-induced lesions likely involves the use of Holliday junctions , whereas recovery from HU-induced stress generally does not ( Branzei and Foiani , 2008 ) .", "The enhanced sensitivity of double mutants to CPT and MMS may indicate that set2Δ and H3-G34R mutants are defective in distinct aspects of HR-mediated repair requiring assembly of Holliday junctions .", "We further asked whether overexpression of Set2 could compensate for the DNA damage sensitivities of H3-G34R cells .", "Whereas pSet2-FLAG overexpression fully compensated for set2Δ growth defects on damaging agents , no growth advantage was conferred on H3-G34R cells ( Figure 4e ) .", "However , since Set2 overexpression cannot compensate for H3K36me3 in H3-G34R cells ( Figure 1e ) , it is unclear whether the DNA damage sensitivity of H3-G34R cells is linked to the lack of efficient tri-methylation on K36 .", "We have shown that H3-G34R mutants are sensitive to chronic exposure to replication stress .", "DNA damage checkpoint signaling is conserved in fission yeast ( Figure 5a ) ( Melo and Toczyski , 2002 ) .", "Following DNA damage , the upstream sensor kinases Rad3 ( ATR ) and Tel1 ( ATM ) phosphorylate histone H2A on a C-terminal serine to generate γH2A and activate the downstream DNA damage checkpoint kinases Chk1 ( Capasso et al . , 2002 ) and the replication checkpoint kinase Cds1 ( Lindsay et al . , 1998 ) . 10 . 7554/eLife . 27406 . 010Figure 5 . H3-G34R cells have intact DNA replication checkpoints .", "( a ) Scheme depicting major kinases involved in checkpoint control and in brackets , their mammalian homologs .", "( b ) Western blot analysis of γ-H2A phosphorylation in WT , G34R , and set2Δ cells .", "Cells were collected ±4 hr treatment with 15 mM HU or 0 . 05% MMS .", "Total H2A serves as a loading control .", "( c ) Western blot showing Chk1-HA activation .", "Chk1-HA shows phosphorylation-dependent mobility shift in 0 . 05% MMS treated H3-WT and H3-G34R cells .", "Cells with all three H3/H4 genes ( 3xH3 ) serve as control .", "Tubulin is used as a loading control .", "( d ) RPA ( Rad11-GFP ) immunofluorescence of indicated strains in either untreated conditions or after 6 hr release following 4 hr of 11 mM HU treatment .", "( e ) Immunofluorescence imaging of DAPI stained cells for analysis of checkpoint-dependent cell elongation after treatment with 11 mM HU for 4 hr .", "Combinations of checkpoint mutants ( cds1Δ , chk1Δ , or cds1Δ chk1Δ ) with either H3-WT or H3-G34R mutations were assessed . DOI: http://dx . doi . org/10 . 7554/eLife . 27406 . 010 To address whether DNA damage checkpoint signaling is functional in H3-G34R cells , we first monitored phosphorylation of H2A ( γH2A ) .", "Western analyses of cells treated with HU or MMS showed no differences in γH2A phosphorylation levels in H3-G34R or set2Δ strains compared to H3-WT , indicative that the function of upstream kinases is not perturbed in H3-G34R cells ( Figure 5b ) .", "Next , we monitored phosphorylation-related mobility shifts of Chk1-HA and found that Chk1 was efficiently activated by MMS treatment in H3-G34R cells , suggesting that DNA damage checkpoint signaling was normal ( Figure 5c ) ( Walworth and Bernards , 1996 ) .", "To assess Cds1 replication checkpoint function , we performed HU treatment of H3-G34R cells since , in cds1Δ cells , prolonged DNA synthesis occurs both during HU treatment and after release .", "This causes an accumulation of single stranded DNA ( ssDNA ) that can be visualized as large foci of replication protein A ( RPA ) ( Sabatinos et al . , 2012 ) .", "Cells were arrested in HU , and RPA foci monitored 6 hr post-release .", "In contrast to the ‘flares’ of RPA seen in cds1Δ after release , H3-G34R showed no accumulation of RPA foci ( Figure 5d ) , supporting that the Cds1 pathway is functional in H3-G34R mutants .", "Finally , we tested H3-G34R in combination with checkpoint mutants for their ability to arrest after a prolonged exposure to genotoxin ( HU ) , which can be monitored by elongation of the cells as an indication of proper checkpoint arrest .", "Control cells lacking both checkpoints ( cds1Δ chk1Δ ) failed to arrest after HU treatment , as indicated by their short length and entry into lethal mitoses with incompletely replicated chromosomes ( Figure 5e ) ( Lindsay et al . , 1998 ) .", "Cells lacking either cds1 or chk1 elongated properly , likely arrested by compensation of the other kinase .", "H3-G34R cds1Δ and H3-G34R chk1Δ double mutants also arrested normally as elongated cells , suggesting that both checkpoints are intact in H3-G34R ( Figure 5e ) .", "In summary , DNA damage checkpoint signaling appears to be functional in H3-G34R cells .", "Thus far , we have shown that H3-G34R mutants exhibit genomic instability that does not involve disruption of heterochromatin ( Figure 3 ) , that they are sensitive to DNA replication stress ( Figure 4 ) , and have functional DNA damage checkpoint signaling ( Figure 5 ) .", "We next assessed DNA damage repair pathways in H3-G34R cells ( Figure 6a ) .", "First we asked how cells recover from replication stress .", "After HU treatment , completion of DNA replication occurs without the need for Origin Replication Complex ( ORC ) activity .", "However , some mutants that are sensitive to replication stress , such as a mutant of the PTIP homolog ( Cho et al . , 2003 ) , brc1Δ , require ORC to recover from HU arrest ( Bass et al . , 2012 ) .", "We tested whether H3-G34R cells depend on ORC to recover from HU , by use of a temperature sensitive allele in ORC , orp1-4 .", "Cells were synchronized with HU at 25°C , and released at either 25° or 36°C for 4 hr prior to plating ( Figure 6b ) .", "As expected , any orp1-4 cells that are released to 36°C die ( green and purple bars ) , whereas orp1-4 H3-WT cells that are HU arrested and released at 25°C survive .", "Intriguingly , unlike orp1-4 brc1Δ which die due to severe mitotic defects ( Bass et al . , 2012 ) ( red bars , Figure 6b ) , we found that orp1-4 H3-G34R survive .", "Thus , H3-G34R cells do not rely on ORC to recover from HU-imposed replication stress . 10 . 7554/eLife . 27406 . 011Figure 6 . H3-G34R cells show defects in homologous recombination pathways .", "( a ) Scheme depicting DNA damage repair pathways .", "( b ) Cells of indicated genotypes were cultured at 25°C in the presence or absence of 11 mM HU for 6 . 5 hr and then washed and either kept at 25°C ( permissive ) or shifted to 36°C ( restrictive ) for 4 hr .", "Cells were then plated at 25°C to determine cell viability .", "Numbers were normalized to untreated 25°C control values for each genotype , and results were averaged from 2 independent biological replicates .", "( c–e )", "Serial 5-fold dilution assays of H3-WT or H3-G34R cells and double mutants with", "( c ) PRR pathway mutants rad18Δ or ubc13Δ ,", "( d ) NHEJ mutant ku70Δ or", "( e ) HR mutant rad51Δ plated on media ± MMS treatment .", "( f ) Homologous recombination assay based on correction of leu1-32 mutation by HR .", "Cells of indicated genotypes were transformed with a leu1 gene fragment to measure HR , or plasmid to measure transformation efficiency .", "Relative HR efficiency is shown as 100% for H3-WT , and results are averaged from 6 independent experiments with error bars representing SEM .", "Black asterisks reflect significant differences with H3-WT cells ( p<0 . 05 ) , and red asterisks , significant differences from both H3-G34R and set2Δ ( p<0 . 01 ) .", "See also Figure 6—figure supplement 1 .", "( g ) Percentage of cells that form foci of either RPA ( Rad11-GFP ) , Rad52-GFP , or Rad51 before or after treatment with 0 . 05% MMS for 4 hr .", "Immunostaining with GFP antibodies was used for monitoring RPA and Rad52 while anti-Rad51 antibodies were used to detect Rad51 .", "Cells were counted from 2 independent experiments with errors representing SEM .", "See also images in Figure 6—figure supplements 1 and 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 27406 . 01110 . 7554/eLife . 27406 . 012Figure 6—figure supplement 1 . Characterization of H3-G34R and set2Δ cells in homologous recombination-directed repair .", "( a ) Schematic illustrating the experimental set up for the HR efficiency assay ( see Figure 6f ) .", "All cells contained a point mutant in leu1 gene ( leu1-32 ) and were transformed with either plasmid DNA to assess transformation efficiency , or a PCR-generated fragment of the leu1+ gene .", "Following transformation , colonies that grew on PMG-leu plates were counted since they had undergone HR-directed repair to correct the leu1 gene .", "HR efficiency was calculated relative to the plasmid transformation efficiency .", "( b ) Efficiency of HR in WT and set2Δ cells with either normal histone complement ( 3xH3 ) or sole copy histone complement .", "Error bars represent SEM using 2 biological replicates .", "( c ) Efficiency of HR in WT or set2Δ or ku70Δ cells with normal histone complement ( 3xH3 ) .", "set2Δ cells tested were from ( 1 ) Partridge lab or ( 2 ) set2ΔKAN or ( 3 ) set2Δura4+ from Humphrey lab ( Pai et al . , 2014 ) .", "Error bars represent SEM using 2–4 biological replicates . DOI: http://dx . doi . org/10 . 7554/eLife . 27406 . 01210 . 7554/eLife . 27406 . 013Figure 6—figure supplement 2 . FACS analysis of cdc10-M17 arrested and released cells ( G1 ) on the left , or mitotically arrested and released cells ( nda3-km311 ) on right . DOI: http://dx . doi . org/10 . 7554/eLife . 27406 . 01310 . 7554/eLife . 27406 . 014Figure 6—figure supplement 3 . Localization of homologous recombination-directed repair proteins in H3-G34R cells .", "( a ) Representative images showing foci formation of RPA ( Rad11-GFP ) or Rad52-YFP in either H3-WT or H3-G34R backgrounds .", "Cells were either treated in the presence ( right ) or absence ( left ) of 0 . 05% MMS for 4 hr prior to imaging .", "Experiment was repeated twice .", "( b ) Western blot analysis depicting the total levels of Rad51 and Rad52 in either H3-WT or H3-G34R cells before and after treatment with 0 . 05% MMS for 4 hr .", "Cells lacking rad51 or containing the rad52-YFP allele serve as antibody-specificity controls .", "Blots were reprobed for total H2A which serves as a loading control .", "One example is shown of duplicate experiments .", "( c ) Representative images depicting foci formation and co-localization of RPA ( Rad11-GFP ) and Rad52-YFP in either H3-WT or H3-G34R backgrounds before ( left ) or after treatment with 0 . 05% MMS for 4 hr ( right ) .", "Errors represent the SEM from two independent experiments .", "( d ) RPA localization during mitosis .", "cdc10-m17 cells carrying Rad11-GFP ( RPA-GFP ) in H3-G34R background were arrested in G1 and released at 25°C before fixation .", "Representative cells from 150 min after release with ultrafine anaphase bridges labeled by RPA-GFP are shown . DOI: http://dx . doi . org/10 . 7554/eLife . 27406 . 014 Next , we systematically mutated genes that are key to well defined DNA repair pathways in H3-G34R and H3-WT cells .", "We mutated genes for post-replication repair ( PRR ) , non-homologous end joining ( NHEJ ) , and homologous recombination ( HR ) in H3-WT and H3-G34R cells ( Figure 6c–e ) .", "To test PRR , we generated combination mutants of H3-G34R with either rad18 ( important for trans-lesion synthesis ) or ubc13 ( important for template switching ) ( Branzei et al . , 2008 ) .", "H3-G34R mutants with either of these mutations show synergistic sensitivity to MMS , suggesting that the PRR pathways are functional in H3-G34R cells ( Figure 6c ) .", "Additionally we monitored epistasis with a mutant in the NHEJ pathway .", "Combination mutants of H3-G34R were generated with mutants in Ku70 , a subunit of the Ku complex responsible for recognizing double strand breaks ( DSBs ) ( Manolis et al . , 2001 ) .", "H3-G34R showed synergistic defects with ku70Δ when assessed on MMS compared with either single mutant , indicating that NHEJ is functional in H3-G34R cells ( Figure 6d ) .", "Furthermore , we assessed the genotoxin sensitivity of H3-G34R in combination with a mutant necessary for HR-directed repair .", "Combination mutants were generated with rad51Δ ( Rad51 encodes a strand exchange protein that forms a filament on DNA and facilitates the search for homology ) .", "In contrast to mutations with PRR or NHEJ proteins , H3-G34R rad51Δ double mutants showed epistasis with single mutants when plated on MMS ( Figure 6e ) .", "This suggests that H3-G34R cells have a defective HR pathway .", "To extend this analysis , we developed an assay to directly monitor the frequency of HR-directed repair ( Figure 6—figure supplement 1a ) .", "We transformed leu1-32 mutant cells ( that bear a single nucleotide mutation in leu1 that renders cells auxotrophic for leucine ) with a 576 bp fragment of wild type leu1+ and scored leu1+ transformants that can only arise by HR , normalizing for transformation efficiencies of strains using plasmid DNA ( Figure 6f ) .", "As expected , rad51Δ cells were very defective with HR rates of only ~3% of H3-WT cells .", "H3-G34R and set2Δ strains were also defective ( ~35% efficiency ) and the double H3-G34R set2Δ mutant showed an even stronger defect , with a 90% drop in HR efficiency .", "The reduction in HR in set2Δ was not due to the genetic background , as we obtained similar results when set2Δ was assessed in a 3xH3/H4 genetic background ( Figure 6—figure supplement 1b ) .", "Thus both H3-G34R and set2Δ reduce HR efficiency but via distinct mechanisms , as additive defects are seen in the set2Δ H3-G34R double mutant .", "We note that our finding of an HR defect in set2Δ differs from the previously published result of enhanced HR and defective NHEJ in 3xH3/H4 set2Δ cells as described in Pai et al . , 2014 .", "Their study utilized an engineered non-essential minichromosome with an HO break site .", "The authors monitored four different markers for loss , and interpret combinations of marker loss as NHEJ-repair , gene conversion , loss of heterozygosity and loss of the chromosome .", "However , due to the nature of the assay , there are many possible causes of apparent marker loss which may cloud interpretation .", "Our simple and direct approach to measure HR relies on a site specific gain of marker function at a defined locus .", "We note that we have just assessed HR at one site , and it is quite feasible that distinct outcomes may be obtained by assessing HR at different genomic regions .", "We sought to determine if the difference in interpretation of Set2 function was indeed caused by the different assays or by strain differences between the set2Δ backgrounds .", "We performed the transformation-based HR assay in the set2Δ strains used in Pai et al . , 2014 , and in NHEJ defective ku70Δ cells ( Figure 6—figure supplement 1c ) , and found that all set2Δ strains tested showed a defect in HR , whereas HR efficiency was strongly enhanced in ku70Δ , as HR is now the sole method of DNA break repair available .", "We conclude that the difference in interpretation of whether set2Δ cells are deficient in HR lies with the use of distinct assays .", "In fission yeast , NHEJ is restricted to G1 , while HR operates in S and G2 phases of the cell cycle ( Ferreira and Cooper , 2004 ) .", "One possible cause of the decreased HR in H3-G34R cells and the dependence of H3-G34R cells on NHEJ for viability ( Figure 6d ) is an alteration in the cell cycle , with an extension of G1 .", "We measured the generation time during exponential growth and found that H3-G34R had an extended doubling time of 201 min ( ±4 min ) compared to 180 min ( ±10 min ) for H3-WT , and 194 min ( ±8 min ) for set2Δ in rich media at 32°C .", "To determine if the growth delay of H3-G34R mutants was linked to a particular cell cycle stage , we monitored cell cycle kinetics using FACS sorting of cells synchronized in G1 or in metaphase and then released back into the cycle at 25°C .", "As shown in Figure 6—figure supplement 2 , cdc10-M17 ( G1 ) arrested and released cells begin DNA synthesis at 40 min after release .", "H3-WT cells have completed DNA synthesis by 80–100 min , whereas S phase is prolonged in H3-G34R cells and not completed until 140–160 min after release .", "It is difficult to draw conclusions from the nda3-km311 arrest and release experiment since fission yeast do not undergo cytokinesis until G1 , which complicates analysis of G1 cells .", "From these data we can surmise that H3-G34R cells spend longer in S phase when released from a G1/S block .", "Next we asked whether there were defects in localization of HR proteins .", "We evaluated foci formation of RPA , Rad51 , and Rad52 ( Figure 6g and Figure 6—figure supplement 3a ) .", "Consistent with enhanced replication stress in H3-G34R cells , H3-G34R cells showed increased numbers of RPA and Rad52 foci ( Bass et al . , 2012 ) .", "As expected , all cells showed increased foci for all three proteins following MMS treatment .", "Surprisingly however , fewer MMS-treated H3-G34R cells showed Rad51 and Rad52 foci compared with H3-WT and set2Δ .", "The reduction in foci was not linked to a change in protein expression as Rad51 and Rad52 protein levels were similar in MMS treated H3-WT and H3-G34R cells ( Figure 6—figure supplement 3b ) .", "Additionally , Rad52 foci co-localized with RPA , suggesting that Rad52 foci occur at sites of DNA damage ( Figure 6—figure supplement 3c ) .", "In summary , H3-G34R cells exhibit elevated marks of replicative stress , and on MMS-damage , have diminished foci formation for Rad51 and Rad52 , which may be linked to the HR defect seen in H3-G34R cells .", "We questioned whether the chromosome segregation defects in H3-G34R may be related to delayed resolution of replicative intermediates .", "To address this , we monitored RPA localization in anaphase cells following cdc10 block and release , and found that H3-G34R cells exhibit anaphase bridges , with RPA marking ultrafine bridges between DAPI staining masses ( Figure 6—figure supplement 3d ) .", "We are unable to determine the frequency of these bridges in anaphase cells since attempts to co-stain the spindle hampered visualization of bridges , but they were easy to identify in synchronized populations suggesting that they are frequent events .", "This supports that chromosome segregation defects in H3-G34R cells are linked to the delayed resolution of replication intermediates .", "HR-directed repair is often utilized to restart replication forks following stress .", "Because we observed a defect in HR , and a defect in Rad51 and Rad52 foci formation in MMS-treated H3-G34R cells , we speculated whether this was due to a delay in Rad51/Rad52 filament formation , and thus a defect in replication restart .", "To address whether the kinetics of recovery were affected , cells were arrested with HU , washed , and then counted at intervals following release ( Figure 7a ) .", "Checkpoint-defective cds1Δ cells do not recover from the arrest .", "While H3-G34R do delay proliferation for longer than H3-WT ( 3 hr rather than 2 hr ) , the proliferation rates at later time points appear similar .", "To directly monitor the rate of DNA replication , we arrested cells with HU and on release , monitored de novo DNA synthesis by EdU incorporation , gating on cells that had fully incorporated EdU ( Figure 7b ) .", "Interestingly , de novo DNA synthesis is initially delayed in H3-G34R cells but at later time points after HU release , the rates of DNA synthesis appear similar in H3-WT and H3-G34R cells . 10 . 7554/eLife . 27406 . 015Figure 7 . H3-G34R mutants exhibit delayed recovery from replicative stress .", "( a ) Cell proliferation of H3-WT , H3-G34R , and cds1Δ cells was analyzed after synchronization in 11 mM HU for 4 hr .", "Cells were counted at 30 min intervals following release .", "Results represent average of 2 independent experiments , with error bars reflecting SEM ( H3-WT and H3-G34R ) and a single cds1Δ experiment .", "( b ) Measure of EdU labeling of newly synthesized DNA .", "Cells were modified to incorporate nucleotide analogs .", "WT and G34R cells were synchronized in 11 mM HU for 4 hr and released into fresh media containing EdU and collected and ethanol fixed over a time course .", "EdU was labeled by ClickIT with Alexa Fluor 488 and samples were analyzed by FACS , gating on cells that fully incorporated EdU .", "Assay was performed twice , and each sample was read three times .", "Data from one experiment is shown , with values representing means of triplicate reads with background from control samples subtracted .", "Error bars represent SD .", "( c–d )", "Time course to analyze", "( c ) γH2A by western blotting or", "( d ) Rad52 ( Rad52-GFP ) foci formation in H3-WT and H3-G34R cells during treatment with 0 . 05% MMS and recovery .", "For", "( d ) , error bars represent SEM for 2 independent experiments , and the time course for γH2A westerns was repeated twice . DOI: http://dx . doi . org/10 . 7554/eLife . 27406 . 015 To further evaluate the kinetics of DNA damage response , we monitored γH2A phosphorylation ( Figure 7c ) and counted cells that bear Rad52 foci ( Figure 7d ) during MMS treatment and recovery .", "MMS treatment led to a rapid induction of γH2A signal in H3-WT cells , such that at 45 min treatment , maximal signal was achieved which was retained during treatment .", "Surprisingly , γH2A signal accumulated more slowly in H3-G34R cells , and peaked at 45 min into the recovery period , at higher levels than seen in H3-WT cells .", "On recovery from MMS , γH2A remained intensely phosphorylated at later time points in H3-G34R cells compared with H3-WT cells ( see Figure 7c ) .", "Similar to γH2A , Rad52 foci accumulated faster in MMS treated H3-WT cells than H3-G34R , even though more Rad52 foci were present in untreated H3-G34R cells than in H3-WT ( Figure 7d ) .", "During recovery , H3-WT cells with Rad52 foci dropped to ~50% by 180 mins while Rad52 foci continued to accumulate in H3-G34R , peaking at 90 min following release , before dropping .", "These data suggest that the kinetics of DNA damage response are different between H3-WT and H3-G34R cells , with H3-G34R mutants showing a slower induction of damage response , as well as accumulation of recombination proteins at later timepoints following damage , which may contribute to the HR-mediated repair defect in H3-G34R mutants ." ], [ "In this study , we generated single copy histone mutants in fission yeast with the aim of studying the behavior of the H3-G34R mutant .", "This system allows a detailed examination of the possible consequences of this histone mutant , divorced from the complexity of mammalian histone regulation .", "Using this system , we show that H3-G34R mutants have focal histone modification changes , with reduced tri-methylation and acetylation , but retention of di-methylation on the nearby K36 residue on histone H3 .", "These results are consistent with some perturbation of Set2 function and we demonstrate that this defect cannot be overridden by overexpression of Set2 as , even under these conditions , H3-G34R cells fail to accumulate normal levels of H3K36me3 .", "Furthermore , the transcriptional consequence of H3-G34R mutation is limited , with fewer genes affected than in set2Δ .", "Indeed for genes whose transcription is altered in both set2Δ and H3-G34R , only some are coordinately regulated .", "Notably , these include antisense genes , which suggests that trimethylation of H3K36 is important for suppression of antisense gene transcription .", "Interestingly , genes showing discordant regulation map to specific chromosomal loci- the ST regions .", "Transcriptional upregulation in ST domains in set2Δ is likely a direct consequence of loss of Set2 function on H3K36 , since the histone H3 K36A mutant shows similar upregulation of expression of ST domains ( Matsuda et al . , 2015 ) .", "Of interest , ST regions have recently been shown to be the most highly condensed regions of the genome , forming ‘knobs’ , with condensation linked to Set2 function ( Matsuda et al . , 2015 ) , but the biological implications of knob formation are not yet known .", "Perhaps the most consequential findings from this work are the effects of H3-G34R mutation on genome stability .", "We see evidence for enhanced chromosome loss and for defects in chromosome segregation , with accumulation of cells that have trailing or ‘stretched’ chromosomes .", "We find that RPA coats ultrafine anaphase bridges in mitotic H3-G34R cells , marking DNA that links chromosome masses , which is indicative of unresolved replication problems .", "These data are consistent with our finding that H3-G34R cells are sensitive to replication stress , and that HR pathways are defective in these cells .", "These phenotypes are unlikely to be due to transcriptional deregulation in H3-G34R cells since transcription of DNA damage genes , under non-perturbed conditions , is unaltered .", "Instead , as summarized in Figure 8 , these findings may derive from the altered kinetics of signal response to damage in H3-G34R cells , with slowed accumulation of γH2A and delayed formation of Rad52 foci , features which may also contribute to the delayed replication of H3-G34R cells during recovery from replication stress .", "H3-G34R mutation may thus impair HR-mediated repair of DNA damage induced DSBs . 10 . 7554/eLife . 27406 . 016Figure 8 . Model illustrating how HR defects in H3-G34R mutants may compromise genomic integrity . Model illustrates the key defects in H3-G34R cells and how these may contribute to genomic instability .", "Replication forks provide an endogenous source of DNA damage , as they are fragile structures that are prone to stalling and collapsing .", "( left )", "To cope with replication stress , cells activate the checkpoint signaling pathway by sensing ssDNA via Rad3 .", "Rad3 interacts with ssDNA through RPA and phosphorylates H2A , which helps to transduce the checkpoint response to downstream kinases .", "For lesions that require HR-directed repair , nucleosome removal and subsequent 5’ to 3’ DNA resection exposes ssDNA which is initially coated with RPA , and then replaced by Rad51 , facilitated by the Rad52 protein .", "The search for a homologous template can begin once sufficient Rad51-ssDNA filament has been formed and strand invasion of the Rad51-ssDNA filament generates Holiday junctions which need to be resolved prior to cell division .", "( right )", "In H3-G34R cells , HR is defective .", "Checkpoint signaling is functional , but appears delayed with slower accumulation of γH2A possibly due to impaired nucleosome removal and delayed resection , which would limit the amount of Rad3 on RPA/DNA .", "Retention of γH2A indicates a defect in nucleosome removal and resection which in turn , could account for the delayed Rad52 foci formation in H3-G34R cells and likely delayed Holiday junction resolution following repair .", "H3-G34R cells show elevated chromosome segregation defects without defects in heterochromatin or kinetochore chromatin , suggestive that a delayed resolution of HR products contributes to genomic instability . DOI: http://dx . doi . org/10 . 7554/eLife . 27406 . 016 How might H3-G34R mutation elicit these effects ?", "One possibility may be via modulation of H3K36 methylation signal .", "However this seems unlikely given that cells that lack Set2 show no defects in chromosome segregation , and the HR defects that we uncovered in set2Δ cells are not epistatic to HR defects in H3-G34R cells .", "Another alternative is that the altered kinetics for γH2A phosphorylation and Rad52 foci formation during MMS treatment and recovery are indicative of chromatin defects in the H3-G34R cells .", "H2A phosphorylation occurs within the C-terminal tail of H2A , which projects out from the DNA and occupies a territory close to the N-terminal tail of H3 within the nucleosome structure ( Du and Briggs , 2010 ) .", "Incorporation of an additional basic charge in the H3 tail close to where the H3 tail exits the H3 core domain may have conformational consequences and the H3-G34R tail may modulate kinase activity on the C-terminal H2A tail ( Figure 8 ) .", "However , checkpoint signaling is clearly functional in H3-G34R cells , even if it is delayed ( Figure 5 ) .", "Once damage has occurred , H3-G34R cells retain γH2A for longer than H3-WT ( Figure 7c ) .", "It is thought that , in order for γH2A to be dephosphorylated , nucleosomes must first be removed from chromatin where they can be acted upon by phosphatases in free nuclear pools ( Keogh et al . , 2006; Jablonowski et al . , 2015 ) .", "Retention of γH2A may stem from a defect in efficient chromatin remodeling , thereby preventing timely repair .", "In support of this , an H2A mutant that mimics γH2A shows decreased interaction with the chromatin remodeler Fun30 ( Eapen et al . , 2012 ) .", "H3-G34R cells show defects in HR-directed repair .", "Nucleosome removal from damaged chromatin facilitates resection and subsequent coating by RPA .", "After sufficient resection of DNA , Rad52 helps to facilitate the exchange from RPA-coated DNA to a Rad51-coated filament , to allow for homology searching and HR repair .", "The presence of a γH2A mimetic can inhibit the rate of resection ( Eapen et al . , 2012 ) .", "Here , we show that H3-G34R mutants are specifically deficient for HR ( Figure 6e ) with prolonged retention of γH2A and delayed Rad52 foci formation ( Figure 7c , d ) .", "It is possible that H3-G34R cells are defective in timely nucleosome removal and resection ( see Figure 8 ) .", "Following this model , delayed HR-mediated repair in H3-G34R cells , or delayed resolution of replicative intermediates in undamaged H3-G34R cells , may allow accumulation of unresolved Holliday junctions prior to mitosis , and contribute to the chromosome segregation defects shown in this study ( Figure 3 and Figure 6—figure supplement 3d ) .", "We note that consistent with our findings , clearance of γH2A . X foci in human cells that lack H3K36me3 due to deletion of SETD2 is delayed ( Hacker et al . , 2016 ) , and such cells are defective for HR and exhibit genomic instability ( Pfister et al . , 2014 ) .", "Clearly an important consideration is that our data was obtained in an organism where the whole population of histone H3 is mutant .", "In pHGG , only one allele of four that encodes H3 . 3 protein is affected in cells with 13 other genes that encode wild type H3 . 1 and H3 . 2 proteins .", "Unlike the H3K27M mutation that dominantly blocks PRC2 activity , G34R mutation does not dominantly affect K36 methylation ( Lewis et al . , 2013; Zhang et al . , 2017 ) .", "Instead , we suggest that the H3 . 3 G34R mutant may be greatly enriched at specific chromosomal loci .", "H3 . 3 is deposited into regions of constitutive heterochromatin including repetitive telomeric and pericentromeric domains , and at regions of high transcriptional activity through the activity of specialized chaperones ( reviewed in Kallappagoudar et al . , 2015 ) .", "These sites of H3 . 3 deposition are already subject to replicative stress .", "As cells traverse S phase , the replicative delay incurred by the presence of G34R mutant H3 . 3 at these sites and impaired resolution of replication intermediates could greatly enhance genomic instability and offer a potent source of genomic perturbations to facilitate tumor development ( Macheret and Halazonetis , 2015 ) ." ], [ "Histone mutant strains were generated as described ( Mellone et al . , 2003 ) .", "Gene mutagenesis used standard PCR-based procedures , and strains are listed in Supplementary file", "4 . All crosses used random spore analysis with nutritional/ drug selection and PCR verification ( including verification of loss of additional H3/H4 genes ) and sequencing of H3 . 2 allele .", "Two independent clones for each genotype were used in nearly all experiments , and all experiments were performed at least twice .", "Fission yeast were maintained on rich ( YES ) , or pombe minimal with glutamate ( PMG ) media with appropriate supplements ( Moreno et al . , 1991 ) .", "PMG is Edinburgh minimal medium with glutamate .", "pSet2-3xFLAG vector for overexpression of Set2 ( JP 2595 ) was generated by amplification of an endogenously tagged Set2 strain ( PY 9101 ) using primers JPO-4159 and JPO-4160 , and cloned into pREP41 ( Basi et al . , 1993 ) using Clontech in-fusion cloning kit .", "JPO-4159: TTTGTTAAATCATATGTCGACATGCAGACGGCATCATCTCT , JPO-4160: ACCCGGGGATCCTCTAGAACCTACAGGAAAGAGTTACTCAAGAATAAGAAT Histones were purified following a previously described purification protocol with some modification ( Sinha et al . , 2010 ) .", "A 150 mL culture was inoculated to a density of 1 . 4 × 106 cells/mL in 4X YES media from a starter culture grown overnight in 4X YES at 25°C .", "The cells were grown to a density of 3 . 6 × 107 cells/mL and harvested by spinning at 3000 RPM for five minutes in a benchtop centrifuge .", "Cells were washed with H2O containing 10 mM sodium butyrate .", "The cells were then washed with NIB buffer ( 250 mM sucrose , 20 mM HEPES pH 7 . 5 , 60 mM KCl , 15 mM NaCl , 5 mM MgCl2 , 1 mM CaCl2 , 0 . 8% Triton X 100 , 0 . 5 mM spermine , 2 . 5 mM spermidine , 10 mM sodium butyrate , 1 mM PMSF , and Sigma yeast protease inhibitor ) .", "The pellet was frozen on dry ice and stored at −80°C .", "For lysis , the pellet was resuspended in 2 mL of NIB and transferred to a bead beater tube along with chilled acid washed glass beads .", "The sample and beads were frozen on dry ice and bead beaten for a total of 10 min at max power .", "The sample was collected by ‘piggy backing’ into a 50 mL Oak Ridge tube at 3000 RPM at 4°C in a benchtop centrifuge .", "The samples were then pelleted by centrifuging at 20 , 000 RPM for 10 min at 4°C in a Beckman Avanti centrifuge J-30I centrifuge using a JA25 . 50 rotor .", "The supernatant was discarded and the pellet was washed in 15 mL of NIB .", "The pellet was then resuspended in 10 mL of 0 . 4N H2SO4 and sonicated for one minute at max power before incubation for two hours on a rotating wheel at 4°C .", "The sample was pelleted by centrifuging at 20 , 000 RPM at 4°C for 10 min and the supernatant was transferred to a new tube along with 5 mL of 5% buffer G ( 5% guanidine HCl and 100 mM potassium phosphate buffer pH 6 . 8 ) where the pH was adjusted to 6 . 8 using 5N KOH .", "0 . 5 mL of Bio-Rex pre-equilibrated in 5% buffer G was added to the sample and incubated at room temperature with rotation overnight .", "The resin was then washed 2X with 20 mL of 5% buffer G and incubated with 3 mL of 40% buffer G ( 40% guanidine HCl and 100 mM potassium phosphate buffer pH 6 . 8 ) for one hour at room temperature to elute the bound protein .", "Buffer exchange and concentration was performed against 5% acetonitrile with 0 . 1% TFA to a final volume of 150 µl and the sample was stored at −80°C .", "Protein concentration was measured and 10 µg of sample was run on a gel for quality control .", "1 . PFGE .", "( Figure 1—figure supplement 1–1e )", "Cells were washed in ice-cold Stop Buffer ( 150 mM NaCl , 50 mM NaF , 10 mM EDTA , 1 mM NaN3 ) prior to processing for PFGE as described previously ( Andrews et al . , 2005 ) .", "Samples were run on 0 . 8% chromosome-grade agarose ( Bio-Rad ) Tris-acetate-EDTA gels for 72 hr with a pulse time of 1800 s at 2 V/cm and an angle of 100° .", "2 . Minichromosome loss .", "( Figure 3a ) .", "The minichromosome ( Ch16 ) ( Niwa et al . , 1989 ) bears an ade6-216 allele which can complement the ade6-210 allele present within the strain background .", "Loss of Ch16 causes loss of complementation of function of ade6+ and accumulation of red pigment when cells are grown on limiting adenine media .", "Strains containing Ch16 were grown in PMG –Leu ( to maintain Ch16 ) at 32°C to a density of 5 × 106 cells/mL .", "Cells were diluted in PMG ( no additives ) to a final concentration of 5−10 × 103 cells/mL .", "Cells were plated onto PMG agar supplemented with amino acids and nucleobases with limiting ( 10% normal concentration ) adenine , incubated at 25°C for 5 days and then transferred to 4°C to let red color develop .", "Ch16 loss frequencies were calculated by counting half sectored colonies ( and those with >50% red but <100% red ) and dividing by the total number of white , white sectored and less than half red colonies , excluding red colonies from the analysis as they have lost the minichromosome before plating .", "Results represent data from multiple ( 2-4 ) independent cultures of cells , and 5−10 × 103 colonies were scored for each strain .", "3 . Lagging chromosome analysis .", "( Figure 3b ) .", "Chromosome missegregation frequencies were obtained as previously described ( Mellone et al . , 2003 ) .", "220 late anaphase cells were scored for presence of lagging chromosomes for each genotype , using two independent cultures of strains .", "4 . Mitotic arrest and release ( Figure 3c ) .", "nda3-km311 mutant ( Hiraoka et al . , 1984 ) in H3-WT and H3-G34R backgrounds was used to synchronize cells in mitotic phase .", "Cells were grown in YES to 5 × 106 cells/ml at 32°C , then were filtered to rapidly collect cells and transferred to media at 18°C for 8 hr .", "Cells were released to warm media at 32°C and aliquots fixed at 15 min intervals in 3 . 7% paraformaldehyde for tubulin and DAPI staining as described in lagging chromosome analysis method .", "1 . Denatured extracts in 2xSB .", "Whole cell extracts ( WCE ) were made as published previously ( Alper et al . , 2013 ) .", "These were used for Figures 1c , e , 5b and 7c , Figure 1—figure supplement 1b , g , h , i , Figure 1—figure supplement 2a , Figure 6—figure supplement 3b .", "2 . Native WCE and chromatin enrichment ( NIB buffer ) .", "Cell lysates were fractionated using a method modified from ( Ekwall et al . , 1997; Sinha et al . , 2010 ) .", "Briefly , cell pellets from 50 ml cultures in YES were washed with NIB [0 . 25 M sucrose , 60 mM KCl , 15 mM NaCl , 5 mM MgCl2 , 1 mM CaCl2 , 2 . 5 mM spermidine , 20 mM HEPES , 10 mM N-butyric acid , 0 . 8% Triton X-100 ( w/v ) with 1:1000 protease inhibitors ( Sigma ( P8215 ) ) and 1 mM phenylmethylsulfonyl fluoride ( PMSF ) ] and resuspended in 1 ml NIB .", "Bead beating was performed for 3 min in presence of 500 ul glass beads ( Sigma , G8772 ) .", "After removal of beads , 500 ul of lysate was taken as ‘total’ extract .", "The rest was centrifuged at 13 , 000 rpm for 15 min at 4°C .", "Supernatant was saved as ‘soluble’ fraction .", "Pellet was washed three times in 1 ml NIB buffer , and resuspended in 500 ul NIB buffer as ‘chromatin enriched’ fraction .", "Samples were heated with sample buffer prior to PAGE and western .", "This extract type was used for Figure 1—figure supplement 1c .", "3 . Denatured extracts in Urea ( Figure 5c ) Previously published protocols were used for Chk1-HA western blot ( O'Connell et al . , 1997; Bass et al . , 2012 ) .", "25 ml cultures of strains were treated ±0 . 05% MMS for 1 hr .", "MMS was inactivated with freshly made 5% sodium thiosulphate .", "Cells were disrupted with glass beads ( Sigma , G8772 ) using a bead beater and extracted into urea lysis buffer ( 8 M urea , 100 mM NaH2PO4 , 10 mM Tris pH 8 , 60 mM β-glycerophosphate , 1% Triton X-100 with protease inhibitor cocktail ( Sigma P8215 ) and 1 mM PMSF ) .", "The extract was cleared by centrifugation at 13 , 000 rpm for 10 min , and the supernatant was boiled in SDS sample buffer .", "4 . Antibodies used .", "Anti H3 ( active motif 39163 , lot no . 26311003 ) .", "Anti-H4 ( millipore 05–858 lot no . 2020541 ) .", "Anti-HA ( Roche 12CA5 ) .", "Anti-GFP ( Abcam ab290 ) , Anti-FLAG M2 monoclonal ( Sigma F1804 ) , anti-tubulin ( TAT1 kind gift from Keith Gull ) ( Woods et al . , 1989 ) .", "Anti-H3K36me2: Abcam ab9049 , Anti-H3K36me3: Abcam ab9050 , Cell signaling technology 4909 .", "Anti-Rad51 ( Bioacademia 63001 ) .", "Anti γH2A ( Abcam ab15083 , lot no . GR284225-2 ) .", "Anti H2A ( active motif 39235 , lot no . 03108001 ) .", "Western blot quantification used Image Studio Lite from LiCor .", "Equal sized squares were drawn using the software , which automatically assigns signal intensity for each band after subtracting background signal .", "The signal intensities were used to quantify the images , relative to signal intensity for total H3 bands .", "5 . Peptide sequences used for assessment of K36 methyl Abs ( Figure 1 , and Figure 1—figure supplement 1 ) are listed in Supplementary file", "1 . Dot blots used a 10-fold serial dilution series with 2 ul spots of 1 mM , 0 . 1 mM , 0 . 01 mM and 0 . 001 mM peptides spotted on activated PVDF 0 . 2 micron membrane .", "Spots were air dried for 1 . 5 hr , blocked in 5% BSA in PBST at RT for 1 hr , incubated with primary Ab for 1 hr at RT , washed with PBST , incubated with HRP-conjugated anti-rabbit secondary Ab for 30 min , washed with PBST and then developed with enhanced chemiluminescence and images captured by LiCor imaging .", "Anti-H3 K36 methylation Abs are listed above and peptide loading was verified by ponceau staining .", "ChIP assays ( Figure 1d , Figure 2—figure supplement 1b , c ) were performed as described previously ( Alper et al . , 2013 ) .", "Antibodies for H3K9me2: ( Abcam ab1220 ) and Swi6: ( Thermo Scientific PA 1–497 ) .", "Real time PCR was used to quantify enrichment at heterochromatic loci relative to adh1+ .", "Set2-3xFLAG ChIP used Anti-FLAG: ( Sigma F1804 ) and monitored Set2 association with act1+ or clr4+ loci as a ratio of signal from input DNA .", "For Cnp1 ChIPs ( Figure 2—figure supplement 1d ) , 10 ul anti-Cnp1 antiserum ( Kniola et al . , 2001 ) was used per ChIP .", "Crosslinks were reversed by heating at 100°C for 12 min in the presence of 100 ul Chelex-100 resin ( 10% slurry in dH2O; BioRad ) , followed by treatment with Proteinase K ( 2 . 5 mg/ml ) 30 min at 55°C with shaking and heat-inactivation of Proteinase K ( 10 min , 100°C ) .", "DNA was recovered and used in qPCR analysis .", "Primer sequences used for q-PCR are listed in Supplementary file", "5 . RNA seq studies ( Figure 2 ) : Hot phenol extraction was used to prepare the RNA ( Leeds et al . , 1991 ) .", "Cultures were grown overnight in YES at 25°C to a density of 2 . 5 × 106 cells/ml in a 25 mL culture .", "The cells were pelleted by centrifugation and washed in DEPC H2O .", "The pellet was resuspended in 750 ul TES Buffer [50 mM Tris-HCl pH 7 . 5 , 10 mM EDTA , 100 mM NaCl , 0 . 5% SDS made in DEPC H2O] along with an equal volume of 5:1 phenol:chloroform pH 4 . 7 and incubated at 65°C for one hour with vortexing for 10 s every 10 min .", "The samples were then cooled on ice and centrifuged for five minutes at 13 , 000 RPM .", "The aqueous phase was transferred to a 2 mL phase lock tube and an additional phenol:chloroform extraction was performed .", "After centrifugation , the aqueous phase was transferred to a new tube and an equal volume of chloroform was added .", "To precipitate the RNA , the aqueous phase was transferred to a 2 mL microcentrifuge tube and three volumes of ice cold ethanol and 3M NaOAc pH 5 . 2 were added and the samples were kept at −20°C to precipitate the RNA .", "The next day , samples were centrifuged at 14 , 000 RPM and 4°C for 15 min .", "The pellet was washed with ice cold 70% ethanol and air dried for 30 min .", "A Turbo DNAse ( Ambion ) reaction was set up with 100 ug of RNA in a 150 ul reaction containing 5 ul of Turbo DNAse .", "The reaction was incubated at 37°C for 30 min and another 5 ul of Turbo DNAse was added and incubated for an additional 30 min prior to the removal of the DNAse using 50 ul of inactivation beads .", "An RNeasy Mini kit ( Sigma ) was used to further clean up and concentrate the RNA which was eluted in a final volume of 30 ul of DEPC H2O .", "RNA-seq was performed by the St . Jude Hartwell Center .", "RNA was quantified using a Quant-iT assay ( Life Technology ) .", "The quality was checked by 2100 Bioanalyzer RNA 6000 Nano assay ( Agilent ) or LabChip RNA Pico Sensitivity assay ( PerkElmer ) before library generation .", "Libraries were prepared from 2 ug of RNA .", "Ribosomal RNA was removed from the samples using Ribo-Zero Gold rRNA Removal Kit ( Yeast ) following manufacturer instructions ( Illumina ) .", "Libraries were prepared using the TruSeq Strand Total RNA Library Prep Kit , beginning at Elution 2 – Fragment – Prime step immediately preceding cDNA synthesis according to the manufacturer instructions ( Illumina ) with the following modifications; the 94 C Elution 2 – Fragment – Prime incubation was reduced to five minutes and the PCR was reduced to 11 cycles .", "Libraries were quantified using the Quant-iT PicoGreen dsDNA assay ( Life Technologies ) or Kapa Library Quantification kit ( Kapa Biosystems ) .", "One hundred cycle paired end sequencing was performed on an Illumina HiSeq 2500 .", "Three biological replicates were used for each strain analyzed .", "The total RNA was sequenced using stranded protocol with 2 × 100 bp setting .", "The paired end reads were mapped to S . pombe ( ftp://ftp . ebi . ac . uk/pub/databases/pombase/pombe/Chromosome_Dumps/Schizosaccharomyces_pombe . ASM294v2 . 30 . dna . genome . fa ) genome using STAR .", "Reads counts for each gene were counted using HTSEQ .", "Raw counts were TMM and quantile normalized and differentially expressed genes were analyzed using limma/Bioconductor .", "LogFC was produced from the limma/voom packages ( Law et al . , 2014 ) in R Bioconductor .", "The log read count was fit to a linear model of the mean-variance trend using the voom package in R and differentially tested with limma producing the logFC ratios , which were plotted by chromosome location in Figure 2c and tabulated in Supplementary file", "3 . RNA-seq data has been deposited at GEO .", "Accession no .", "GSE96842 .", "Antisense gene transcription data was analyzed using LogFC of 1 . 5 and p<0 . 05 for triplicate samples , and is tabulated in Supplementary file", "3 . Real-time Q-PCR was performed on random primed cDNA generated from two independent RNA preps for each strain as previously described ( Debeauchamp et al . , 2008; Partridge et al . , 2007 ) using primers ( Supplementary file 5 ) that had been tested for linear amplification parameters , and working within the Ct range of linear amplification and using an Eppendorf Mastercycler RealPlex2 machine .", "Transcript levels were normalized to adh1+ transcripts .", "1 . DAPI staining for cell imaging ( Figure 5e ) .", "Cells were grown in PMG complete media ± 11 mM HU for 4 hr at 30°C .", "Cell were fixed in 3 . 7% paraformaldehyde and stained with DAPI as published previously ( Bass et al . , 2012 ) .", "2 . Monitoring markers of DNA damage ( Figure 6g and Figure 6—figure supplement 3a , c ) .", "Cells were grown in YES ±0 . 05% MMS for 4 hr at 30°C .", "Cells expressing Rad52–YFP ( Rad22-YFP ) or Rad11–GFP ( RPA ) in H3-WT and H3-G34R backgrounds were fixed with 70% ethanol or with 3% formaldehyde .", "For co-localization , H3-WT and H3-G34R cells co-expressing Rad52–RFP and Rad11-GFP were fixed with 0 . 5% formaldehyde .", "Rad51 ( Rhp51 ) was detected by indirect immunofluorescence microscopy as described previously ( Bass et al . , 2012 ) using cells fixed with 3% paraformaldehyde .", "Anti-Rhp51 antibody ( Thermo scientific Pierce TM PA1-4968 ) was used at a 1:400 dilution , and detected with FITC-coupled anti-rabbit IgG antibodies at a 1:100 dilution .", "S129 phosphorylated histone H2A ( γH2A ) was detected with a phosphorylation-specific antibody ( Abcam ab15083 ) at 1:100 dilution using cells fixed in 1 . 6% formaldehyde , 0 . 2% glutaraldehyde .", "Two independent experiments were performed for each analysis and 300 cells were counted for each genotype and condition .", "3 . RPA flare experiment ( Figure 5d ) .", "Cells expressing Rad11–GFP ( RPA ) were cultured in PMG complete medium to 5 × 106 cells/ml at 30°C ( asynch sample ) , or were treated with 11 mM HU for 4 hr .", "Cells were washed by filtration , resuspended in fresh medium and grown at 30°C for 6 hr ( 6 hr release sample ) .", "Cells were fixed with 70% ethanol prior to imaging .", "4 . RPA localization during mitosis ( Figure 6—figure supplement 3d ) .", "cdc10-m17 cells expressing Rad11-GFP ( RPA ) were cultured in YES medium at 25°C , shifted to the restrictive temperature of 36°C for 4 hr and then released to 25°C .", "Cells were collected at 30 min intervals , fixed with 3% formaldehyde , and a portion were processed for IF with anti-tubulin antibodies to determine the best time point for capture of late anaphase cells .", "The remaining cells were DAPI stained and imaged for DAPI and GFP fluorescence .", "( Figure 1—figure supplement 2 , Figure 6—figure supplement 2 ) .", "Cells collected at the indicated time points were fixed in ice-cold 70% ethanol .", "Cells were washed twice in 20 mM EDTA and incubated with 100 ug/mL RNase A ( Amresco E866 ) overnight at 36C .", "Prior to analysis , 2 uM of Sytox Green DNA stain ( Invitrogen S34860 ) was added to each sample containing 20 mM EDTA .", "Flow cytometry was performed on a BD LSR Fortessa cytometer .", "( Figure 1—figure supplements 1d , 4a , c–e and 6c–e ) .", "Five-fold serial dilution assays were performed using exponentially growing cells and were spotted on agar plates with 1 . 2 × 104 cells in the First spot .", "Plates were incubated at indicated temperatures .", "For chronic exposure assay , cells were grown to a density of 5 × 106 cells/ml .", "A fivefold serial dilution was spotted onto PMG complete agar plates ± HU , on YES agar plates with DMSO or DMSO and CPT , and on YES agar plates ± MMS .", "Plates were photographed after 4–5 days incubation at 30°C ( CPT and MMS ) , or 6–7 days at 25°C ( HU ) .", "All experiments were repeated at least twice .", "( Figure 4b ) .", "Cells at a density of 5 × 106 cells/ml were irradiated using a Cobalt source , and 100 ul samples were taken following increments of 200 Gy exposure ( Pai et al . , 2014 ) .", "Cells were plated on 6 YES plates for each assay condition , and colonies scored following incubation at 32°C for 4 days .", "The experiment was performed twice to obtain average viability of treated versus untreated cells , and error bars represent the SEM .", "Protocols were adapted from a previously published protocol ( Sheedy et al . , 2005 ) .", "HU synchronization and release ( Figure 7a ) : cells were cultured in PMG complete medium to 5 × 106 cells/ml at 30°C and were treated with 11 mM HU for 4 hr .", "Cells were washed by filtration , resuspended in fresh medium and grown at 30°C .", "Samples were taken at appropriate intervals and counted three times .", "The experiment was repeated twice and average cell numbers were plotted ± SEM .", "HU synchronization and release of orp1-4 strains ( Figure 6b ) : Cells were grown at 25°C in PMG complete medium and treated ±11 mM HU for 6 . 5 hr at 25°C .", "Cells were washed by filtration , resuspended in fresh medium and incubated at 25 or 36°C for 4 hr before aliquots were plated and incubated at 25°C .", "Duplicate experiments were performed and data represents mean ± SEM .", "HU synchronization and EdU labeling ( Figure 7b ) : This experiment was performed using strains modified to incorporate nucleotide analogs ( Hodson et al . , 2003a2003 ) , and according to the procedure detailed in ( Sabatinos and Forsburg , 2015 ) .", "Cells were blocked in 11 mM HU for 4 hr , washed by filtration , resuspended in fresh media containing 10 mM EdU and collected at 15 or 30 min intervals over a 4 hr timecourse , and fixed with 70% ethanol .", "Controls included incorporating strains that were not exposed to EdU , and a non-incorporating strain that was exposed to EdU .", "EdU was labeled by use of a ClickIT Alexa Fluor 488 kit ( Thermoscientific ) , and data was collected by FACS analysis on FITC channel .", "Gating was on cells with fully incorporated EdU .", "Assay was performed twice , and each sample was read three times .", "Data from one experiment is shown , with values representing means of triplicate reads with background from control samples subtracted .", "Error bars represent SD .", "( Figure 6f , Figure 6—figure supplement 1a , b , c ) : We transformed leu1-32 mutant cells ( that bear a single nucleotide mutation in leu1 that renders cells auxotrophic for leucine ) with a 576 bp fragment of wild type leu1+ and scored leu1+ transformants that can only arise by homologous recombination of the wild type leu1 fragment into the mutant allele .", "The rates of HR were normalized by calculating transformation efficiencies of the different strains using leu1+ plasmid DNA .", "Sequence analysis of ~40 independent leu1+ recombinants from different genetic backgrounds confirmed that repair always occurred at the leu1 chromosomal locus .", "Non-parametric statistical methods were applied .", "To compare two independent groups , Wilcoxon-Mann-Whitney ( WMW ) exact tests were used .", "Sign test , a non-parametric version of the one sample t-test , was used for cases when data were normalized .", "A significance level of 0 . 05 was used without adjusting for multiplicity .", "All p-values reported were two-sided ." ] ]

[ "Recurrent somatic mutations of H3F3A in aggressive pediatric high-grade gliomas generate K27M or G34R/V mutant histone H3 . 3 .", "H3 . 3-G34R/V mutants are common in tumors with mutations in p53 and ATRX , an H3 . 3-specific chromatin remodeler .", "To gain insight into the role of H3-G34R , we generated fission yeast that express only the mutant histone H3 .", "H3-G34R specifically reduces H3K36 tri-methylation and H3K36 acetylation , and mutants show partial transcriptional overlap with set2 deletions .", "H3-G34R mutants exhibit genomic instability and increased replication stress , including slowed replication fork restart , although DNA replication checkpoints are functional .", "H3-G34R mutants are defective for DNA damage repair by homologous recombination ( HR ) , and have altered HR protein dynamics in both damaged and untreated cells .", "These data suggest H3-G34R slows resolution of HR-mediated repair and that unresolved replication intermediates impair chromosome segregation .", "This analysis of H3-G34R mutant fission yeast provides mechanistic insight into how G34R mutation may promote genomic instability in glioma ." ]

[ "Children suffering from a brain cancer called high-grade glioma rarely recover because there are no therapies that can effectively target this disease .", "Recently , mutations in a gene that encodes a protein called histone H3 were found in children’s glioma cells .", "Histone proteins bind to DNA to help package it into cells .", "They help to protect the DNA from damage , control when genes are switched on or off , and influence how the DNA is copied when cells are preparing to divide to produce two daughter cells .", "However , little was known about why one of the histone H3 mutations is associated with high-grade gliomas .", "Humans and other animals have many different versions of the histone H3 protein , which makes it difficult to study a mutation that only affects one particular version .", "Therefore Yadav et al . used fission yeast – which they engineered to only contain one form of histone H3 – to study what effect the mutation has on cells .", "The experiments show that fission yeast cells with the mutant form of histone H3 took longer to replicate their DNA and were more likely to die when exposed to chemicals that interfere with DNA duplication .", "Furthermore , the mutant cells were slower at repairing certain types of damage to DNA and were more likely to go on to divide before DNA duplication and repair were completed .", "As a result , the daughter cells produced were more likely to have gained or lost whole chunks of their DNA .", "This phenomenon is known as chromosomal instability and is often seen in cases of high-grade glioma in children .", "The findings of Yadav et al . give new insight into how a specific mutation affecting the histone H3 protein may act in cells .", "Future experiments will need to confirm whether this mutation also has a similar effect on children’s glioma cells , which may help to provide new options for treating this cancer ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "developmental biology" ]

[ [ "A range of disorders from isolated organ defects like blindness or nephronophthisis to multi-system disorders like Joubert ( JS ) , Bardet–Biedl , or Meckel–Gruber syndromes are correlated with mutations in genes involved in formation or stability of the primary cilium ( Goetz and Anderson , 2010; Waters and Beales , 2011; Brown and Witman , 2014 ) .", "JS is characterized by a distinctive midbrain–hindbrain malformation , named the ‘molar tooth sign’ on brain magnetic resonance imaging , and clinically by developmental delay , oculomotor apraxia and hypotonia .", "Currently , 25 genes are known to cause JS when mutated in a bi-allelic or X-linked fashion ( Akizu et al . , 2014; Beck et al . , 2014; Romani et al . , 2014 ) .", "Most of the encoded proteins from these genes localize to the primary cilium or are involved in ciliary-related transport and commonly result in defective ciliation in patient cells or in animal models ( Singla et al . , 2010; Valente et al . , 2013; Akizu et al . , 2014 ) .", "Importantly , still about half of cases studied by exome sequencing remain genetically unsolved , suggesting many as yet unidentified causes ( Akizu et al . , 2014 ) .", "Although traditional homozygosity mapping or exome sequencing has uncovered many genes for these conditions , these approaches may fall short for genes under strong selective pressure or for genes in which homozygous loss-of-function mutations are embryonic lethal .", "One approach to identify new human disease genes is to intersect cell biological , genomic , or protein interaction data in order to prioritize candidates for closer inspection .", "For instance , a protein interaction network derived from genes previously implicated in the ciliopathies identified mutations in TCTN2 in JS patients ( Sang et al . , 2011 ) .", "Similarly , comparing gene content from species with and without cilia led to identification of BBS5 in Bardet–Biedl syndrome patients ( Li et al . , 2004 ) .", "There have been few systematic approaches towards characterization of genes required for ciliogenesis .", "A small interfering RNA ( siRNA ) screen of 7784 pharmacologically relevant genes identified 36 positive and 13 negative ciliogenesis modulators ( Kim et al . , 2010 ) , and a study of 815 ‘kinome’ genes identified 9 candidates affecting ciliary signaling ( Evangelista et al . , 2008 ) , but neither study was genome-wide .", "A recent phylogenetic co-occurrence study identified 206 core cilia components ( Dey et al . , 2015 ) , but no link with disease was shown .", "Given defective ciliogenesis in patient cells , we reasoned that a genome-wide siRNA screen to identify ciliogenesis factors could help prioritize candidates , especially for families in which traditional exome-sequencing approaches have not yet yielded a cause .", "One of the caveats of screening for such genes is that ciliogenesis is intimately linked with mitosis ( Kim et al . , 2011; Plotnikova et al . , 2012 ) , and thus , genes arresting the cell cycle prior to ciliogenesis might be inadvertently flagged as affecting ciliogenesis .", "Recent live cell cycle imaging markers make it possible to separately flag cell cycle genes , which could greatly increase the specificity of ciliogenesis screens .", "Our focus was to identify novel genes involved in JS , by applying a functional genomics approach , then intersecting the data with a cohort of unsolved exome-sequencing results from JS patients .", "We conducted a high-throughput genome-wide siRNA knockdown study for 18 , 045 human genes in a ‘two-color’ cell line engineered to report ciliary-localized EGFP and cells in G2/M phase using mCherry-tagged Geminin .", "A range of cellular features were measured for all genes , and compared with a positive and negative training set , resulting in a prioritized list of 591 ciliary candidates .", "This list was used to prioritize variants from 145 JS patients on whom exome sequencing had not revealed a cause .", "We identified deleterious variants KIAA0586 in a total of 15 families .", "This gene was previously missed by exome sequencing , most likely due to a high-carrier frequency of a common allele in a predominantly compound heterozygous inheritance , thus , precluding a homozygosity mapping approach or filtering focused on rare variants .", "Together with a lethal phenotype in other species ( Bangs et al . , 2011 ) , the data suggest that humans may have redundancy or compensation that preclude lethality or that the KIAA0586 mutations only partially inactivate protein function .", "The results also support a cell-based screening approach to complement exome sequencing in human mutation identification ." ], [ "The ciliated stable cell line , human telomerase reverse transcriptase ( hTERT ) -retinal pigment epithelial 1 ( RPE1 ) Smo-EGFP ( Kim et al . , 2010 ) , in which Smoothened–tagged EGFP is stably integrated in the polarized human RPE1 cells , reliably reports a single primary cilium upon serum withdrawal in 60–80% of cells .", "This line was stably transfected with mCherry-tagged Geminin ( Sakaue-Sawano et al . , 2008 ) , a nuclear marker for S/G2/M cell cycle phases , to produce the Smo-EGFP-mCherry-Geminin/hTERT-RPE1 ( SEMG ) line , enabling differential analysis of ciliogenesis as a function of the cell cycle .", "Cells lacking a cilium ( i . e . , absent ciliary-localized EGFP fluorescence ) were divided into those in G2/M phase ( should normally not display a cilium ) and those in G0/G1 ( most should display a cilium; Figure 1A , B ) .", "The incorporation of mCherry-Geminin increased the specificity of the screen by filtering siRNAs leading to cell cycle arrest as the primary reason for absent cilia . 10 . 7554/eLife . 06602 . 003Figure 1 . Schematic representation , validation and enrichment of genome-wide siRNA cell screen for machine learning approach .", "( A ) High-content small interfering RNA ( siRNA ) cell-based screen using reverse transfection of the library in media containing serum for 72 hr , followed by 24 hr serum starvation , fixation and DAPI staining .", "Subsequent fluorescent imaging and algorithmic analysis performed for all pooled siRNAs .", "To assess ciliary candidates for the positive training , we used SYSCILIA gold standard ( SCGSv1 ) and for the negative training the human metabolome database ( HMDB 3 . 0 ) as well as a manually curated housekeeping gene data set .", "FDR , false discovery rate .", "( B ) Segmentation algorithm for cytoplasm and cilia detection: ( 1 ) detected nuclei from DAPI channel , ( 2 ) nuclear automated segmentation , ( 3 ) cell outline automated using cytoplasm_detection_D of the program Acapella , and ( 4 ) cilia automated detection and segmentation .", "Images have been modified for illustration purposes .", "Scale bar: 10 μm .", "( C ) Representative images of serum-starved SEMG cells without siRNA showing basal ciliation ( small green rods in EGFP channel ) .", "Red ( mCherry ) marks cells in S/G2/M phase of the cycle , green ( EGFP ) marks cilia , blue ( DAPI ) marks nuclei .", "siRNAs used as positive controls: KIF3A interferes with ciliation but not cell cycle .", "ACTR3 shows increased length of cilia ( Kim et al . , 2010 ) .", "CRNKL1 implicated in cell cycle progression ( Zhang et al . , 1991 ) and showed increased mCherry nuclei and reduced ciliation .", "Scale bar: 10 μm .", "( D ) Receiver operating characteristic ( ROC ) for the classifier , which used features from three data sources .", "Dashed line: theoretical random classifier .", "( E ) Precision-recall curve for the final classifier .", "( F ) Median value ( red center bar ) and interquartile ranges ( blue box ) box plot of the classifier scores for the corresponding number of supporting number of evidences ( NOEs ) in Cildb and the genes used as negative and positive training examples .", "The indicated contrasts were found significant ( * ) with a highest value of p < 1 . 03 × 10−4 ( one-tailed Wilcoxon's Rank sum test ) .", "( G ) Same as ( F ) , limited to the NOEs from humans only .", "The indicated contrasts were found significant ( * ) with a highest value of p < 1 . 43 × 10−10 ( one-tailed Wilcoxon's Rank sum test ) .", "See Figure 1—figure supplement 1 , 2 for the prediction score on the gold standard and candidates as well as the visible improvement of the ROC curve and precision–recall curve . DOI: http://dx . doi . org/10 . 7554/eLife . 06602 . 00310 . 7554/eLife . 06602 . 004Figure 1—figure supplement 1 . Prediction score on Gold standard and Gold standard candidates .", "( A–C )", "Box plot reporting median value ( red center bar ) and interquartile ranges ( blue box ) of the classifier scores for gold standard positive and negative genes ( out of bag performance , that is , for every gene the score excludes trees where the gene was used for training ) , also included are boxes for a set of ciliopathy candidate genes ( SYSCILIA candidate genes ) and genes not annotated to be ciliopathy related ( Unknown ) , which were not used in the training .", "( A ) Classifier based on cilia siRNA screen features only .", "( B ) Classifier based on cilia siRNA screen and centriole siRNA screen features only .", "( C ) Classifier including all siRNA and GTex project expression signature based features .", "In all cases , the median value for positive set or candidate genes differed significantly from the negative set or unknown set of genes ( One-tailed Wilcoxon rank sum test ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 06602 . 00410 . 7554/eLife . 06602 . 005Figure 1—figure supplement 2 . Visible improvement of ROC curve and precision-recall curve .", "( A ) ROC for classifiers trained on different partitions of the feature space ( blue: final set , magenta: excluding centriole biogenesis siRNA based features , red: including only features from the whole genome siRNA screen performed in this study ) .", "The dashed black line corresponds to a theoretical random classifier .", "( B ) As in A but showing precision-recall curve for each classifier . DOI: http://dx . doi . org/10 . 7554/eLife . 06602 . 005 Using this approach , we first optimized seeding density , serum withdrawal conditions , and imaging parameters using a siRNA positive control for cilia ( i . e . , no known effect on the cell cycle but blocking ciliogenesis ) of KIF3A , and for cell cycle ( i . e . , no direct effect on ciliogenesis but traps cells in G2/M phase of the cell cycle or the effect described above ) of ACTR3 and CRNKL1 , and verified reporters were robust ( Figure 1C ) .", "We conducted a high-throughput siRNA knockdown study for 18 , 045 genes of the human genome performed in duplicate , using 4–5 unique pooled siRNAs per gene .", "After siRNA transfection , ciliation was induced by serum starvation , then fixed and imaged in 384-well plates in three channels ( see ‘Materials and methods’ ) .", "18 non-overlapping cellular features reflecting nuclear , cytoplasm and ciliary state , combined into 31 parameters ( Supplementary file 1 ) , yielding 559 , 395 values across the screen ( Supplementary file 2 ) .", "The rationale of our whole-genome siRNA screen with SEMG cells was to obtain data allowing for identification of genes as potential candidates as a cause of JS by using a supervised learning approach .", "We trained a Random Forest classifier using known ‘ciliary genes’ as a positive training set , derived from the SYSCILIA consortium gold standard ( SCGSv1 ) composed of 303 confirmed factors ( van Dam et al . , 2013 ) .", "The negative set incorporated genes not involved in any currently known ciliary processes and included 5445 genes annotated in the human metabolome database ( HMDB 3 . 0 ) ( Wishart et al . , 2013 ) , as well as a manually curated set of 666 housekeeping genes .", "To ensure accurate annotation of gene sets used in the classifier training , all genes were cross checked with Cildb V3 . 0 , a database of ‘ciliary genes’ ( i . e . , genes with presumed ciliary function ) based on high-throughput studies across multiple species ( Arnaiz et al . , 2009 , 2014 ) .", "Based on this resource , we removed genes with conflicting annotation from both the positive and negative sets , leaving a final list of high-confidence positive ( n = 244 ) and negative ( n = 1802 ) cilia candidates .", "We evaluate the performance of the trained classifier on cilia candidates from the SCGSv1 , which included an additional list of 419 ciliary gene candidates , not used to train the classifier .", "Of these , 21% were flagged by the classifier as likely ciliary .", "Furthermore , there was significant enrichment compared to the negative set of metabolomics and housekeeping genes not included in the classifier training set ( p < 1 . 08 × 10−25 , one-tailed Wilcoxon rank sum ) .", "Next , classifier performance was evaluated by examination of the area under the receiver operating characteristic-curve ( AUC ) .", "Along with both replicates of the whole-genome siRNA screen , we included data from a siRNA screen designed to identify regulators of centriole biogenesis ( Balestra et al . , 2013 ) and gene expression signatures derived from the Genotype-Tissue expression ( GTEx ) tissue specific RNAseq data ( Figure 1D , E , Figure 1—figure supplement 1 , Figure 1—figure supplement", "2 ) ( GTEx Consortium , 2013 ) .", "Of the 16 , 431 genes screened in all three data sets , the classifier predicted 1299 genes ( 7 . 9% ) as likely ciliary , which we call the CILIOGENESIS database ( Ciliary List of Candidate Genes using an siRNA Strategy , Supplementary file 3A , B ) .", "We also define a high-confidence subset of 591 ciliary genes by controlling for the false discovery rate ( FDR < 0 . 1 ) , which is estimated based on the classifier score and training set labels calculated .", "This high-confidence list includes many established ciliopathy genes such as TTC26 , CEP83 , IFT88 , and SPATA7 , as well as 14 of 25 known JS causative genes .", "Of the remaining JS causative genes , two others were included when FDR scores were loosened to 0 . 21 and 0 . 25 .", "The remaining eight other JS causative genes ( 32% ) were all found well above the genome-wide median classifier score ( lowest ranked gene observed at 58th percentile ) , but not in the top list , possibly as a result of their activity outside the cilium .", "Of the high-confidence genes included in the CILIOGENESIS database , 26% were previously included in the SCGCv1 , yielding 438 novel candidates .", "Cildb is a multispecies knowledge base constructed through integration of high-throughput screens aimed at identifying ciliary or ciliary-related genes .", "Cildb outputs two integers for each gene in the knowledge base , referring to independent experimental ‘number of evidences’ ( NOEs , i . e . , publications ) indicating ciliary association , with one for NOE in human studies and one for NOE in ‘any species’ .", "We compared gene-specific classifier score ( excluding any genes used in training ) with the Cildb NOE output .", "Significant positive trends were observed when comparing to increasing NOE in both the multi-species and human-only sets ( Jonckheere–Terpstra test , see methods , p < 3 . 04 × 10−29 and p < 6 . 50 × 10−42 , Figure 1F , G ) .", "Moreover , we also observed a significant difference when comparing scores in any of the NOE bins to the zero NOE bin in both the multi-species and human sets ( p < 1 . 03 × 10−4 and p < 1 . 43 × 10−10 , respectively , one-tailed Wilcoxon rank sum ) .", "To identify possible candidates for ciliopathies , we performed a gene ontology ( GO ) -term enrichment analysis on the high-confidence gene list , with functional annotation clustering using DAVID ( Huang da et al . , 2009a; Huang da et al . , 2009b ) .", "We used a GO-enrichment cutoff of FDR <0 . 05 ( Benjamini–Hocheberg test ) .", "To ascertain the novelty of genes included in the CILIOGENESIS data set , we excluded SCGSv1 genes used in the training , leaving 1 , 177 genes .", "GO enrichment resulted in several significant terms including non-membrane bound organelle , microtubule cytoskeleton/centrosome , spermatogenesis , and microtubule cytoskeleton organization demonstrating an agreement with previous annotations for cilia associations ( Supplementary file 3C ) .", "The involvement of ciliary processes in the CILIOGENESIS data set was supported by MsigDB analysis showing gene enrichment among others for the recruitment of mitotic centrosome proteins and complexes , microtubule/cytoskeleton and centrosome ( Supplementary file 3D , E ) ( Subramanian et al . , 2005 ) .", "Enrichment validation suggested that the CILIOGENESIS data set may be enriched for ciliopathy disease genes .", "Previous whole-exome sequencing in 287 cases of JS left ∼50% without a genetic explanation ( Akizu et al . , 2014 ) , suggesting additional causes remain to be identified .", "Of these , 75% displayed parental consanguinity , suggesting that causative variants might be homozygous .", "In about half of the remaining cases , sequencing on at least one parent was available , enabling phasing of identified alleles .", "From these 145 individuals , we tabulated 5485 variants containing 2348 homozygous variants and 3137 potentially compounds heterozygous variant pairs .", "We prioritized variants occurring within the coding region and canonical splice sites of any of the 591 CILIOGENESIS genes , and identified 179 variants including 106 homozygous and 73 potentially compound heterozygous variant pairs , or a 96 . 7% reduction in variants to be considered .", "Collectively , variants were identified in 112 of the 591 CILIOGENESIS genes , respectively .", "The only gene with more than two families displaying variants was KIAA0586 , prompting further analysis .", "KIAA0586 ( i . e . , the orthologue of chicken and mouse Talpid3 ) is composed of 34 exons with at least six major transcripts ( Figure 2A ) .", "From these 145 sequenced probands ( written informed consent provided ) , there were four displaying putative compound heterozygous and two displaying homozygous potentially deleterious variants .", "Interestingly , in each of the four compound heterozygous probands , there was a shared frameshift mutation , ( chr14:58899157del; c . 428del , p . Arg143Lysfs*4 ) , which we refer to as M1 ( mutation 1 ) .", "Each of the four carried a single additional potentially deleterious variant , including mutations in a canonical acceptor splice site ( chr14:58915212G>A; c . 1120+1G>A , p . Thr323Hisfs*3; M2 ) , a canonical donor splice site ( chr14:58923419G>C; c . 1413-1G>C; p . Phe472Alafs*5; M3 ) , and a missense affecting the start codon of two transcripts ( chr14:58896138T>C; c . 293T>C; p . Met98Thr; M4; T1; or c . 2T>C; p . Met1 ? ; T4-T5 , where T refers to transcript number ) .", "Implementing an algorithm to identify copy number variants from exome-sequencing data ( Fromer et al . , 2012 ) , we additionally identified a deletion of 15 . 5 kilobases ( Kb ) spanning exon 10–17 ( chr14: ? _58923420_58938997_ ? del; c . 1413- ? _2793+ ? del; p . ? ; M5 ) in one patient .", "These mutations were all confirmed with Sanger sequencing or quantitative PCR , and all segregated according a strict recessive mode of inheritance in all available family members ( Figure 2B , Figure 2—figure supplement 1 , Figure 2—figure supplement 2 ) .", "We conclude that compound heterozygous variants in KIAA0586 contribute to JS .", "Each patient carrying the M1 mutation had a demonstrable second mutation on the other allele , suggesting a recessive mode of inheritance . 10 . 7554/eLife . 06602 . 006Figure 2 . Pedigrees and schematic representation of KIAA0586 . ( A ) Genomic structure and mRNA transcripts of KIAA0586 .", "Transcript 1 ( T1 ) : full-length isoform with 34 exons .", "T2–T4 have different initiation sites , lack exon 5 , and T3 lacks exon 14 .", "T5 starts at the same position as T4 and incorporates exon 6 .", "The shortest transcript ( T6 ) initiates in exon 7 , lacks exon 32 and 33 , and terminates using an alternative exon , which is not incorporated in the other transcripts .", "Gray boxes represent alternative exons .", "UTR's are represented by half-height boxes .", "The location of the mutations is indicated by M1–M7 .", "( B ) Pedigrees of the Joubert syndrome ( JS ) families with ancestries of USA ( MTI-233 and MTI-103 ) , Mexico ( MTI-165 ) , Turkey ( MTI-1944 and COR354 ) , and Syria ( MTI-505 ) , respectively , demonstrating the segregation of the compound heterozygous mutations in non-consanguineous families and homozygous mutations in consanguineous families .", "Inferred genotype is italicized .", "M , mutation; T , transcript .", "See Figure 2—figure supplement 1 for the chromatograms of the mutations in KIAA0586 .", "Figure 2—figure supplement 2 shows the results of the quantitative PCR confirming the large heterozygous mutation in MTI-1944 . DOI: http://dx . doi . org/10 . 7554/eLife . 06602 . 00610 . 7554/eLife . 06602 . 007Figure 2—figure supplement 1 . Chromatograms of mutations in the KIAA0586 gene . The chromatograms of the mutations in identified in KIAA0586 of individuals with JS .", "A ) M1 , B ) M2 , C ) M3 , D ) M4 , E ) M6 , F ) M7 . DOI: http://dx . doi . org/10 . 7554/eLife . 06602 . 00710 . 7554/eLife . 06602 . 008Figure 2—figure supplement 2 . Quantitative PCR confirmed heterozygous mutation in MTI-1944 . Using quantitative PCR on genomic DNA of the large deletion with unknown specific boundaries was confirmed to segregate in MTI-1944 .", "By analyzing two primer sets outside the presumed heterozygous deletions spanning exon 12 to 20 and two within the deletion absence of approximately half the product in the mother and affected child was shown .", "Input of genomic DNA was normalized against GAPDH .", "C , control; F , father; M , Mother; A , affected child . DOI: http://dx . doi . org/10 . 7554/eLife . 06602 . 008 Two consanguineous families each showed a homozygous mutation in KIAA0586 .", "One was predicted to alter splicing in a constitutively incorporated exon ( c . 2414-1G>C; p . ? ; M6 ) .", "The other was a single base-pair deletion ( c . 74del; p . Lys25Argfs*6; M7 ) , in an exon incorporated into only three of the six annotated transcripts , all of which are ubiquitously expressed .", "We conclude that homozygous mutations in KIAA0586 can also contribute to JS .", "The common frameshift variant M1 was identified in all four families with compound heterozygous mutations .", "Evaluation of M1 in the Exome Variant Server ( NHLBI GO Exome Sequencing Project ( ESP ) , Seattle , WA , URL: http://evs . gs . washington . edu/EVS/ [May , 2015] ) identified in 25/7 , 757 European American alleles and 3/3511 African American alleles , all in a heterozygous state , presumably all in healthy individuals .", "Exome Aggregation Consortium ( ExAC , Cambridge , MA , URL: http://exac . broadinstitute . org [May , 2015] ) showed an overall frequency of 244/120 , 680 M1 alleles .", "Combining these with the 1000 Genomes data suggests an allele frequency of 0 . 0036 in the general population .", "We conclude that M1 is a relatively common allele in the general population , found in about 1/300 individuals .", "The M1 variant was found in individual of varying ancestry , but we cannot exclude a common founder mutation .", "We speculated that M1 was likely to represent a common mutation among JS patients .", "Thus , we screened an additional cohort of 163 classical JS patients with a proven ‘molar tooth sign’ collected primarily from Mediterranean regions .", "The M1 allele was surprisingly identified in 17 of 326 alleles ( 5 . 21% ) , of which one was homozygous ( Figure 3 individual NG2872 ) .", "Ethnically matched Mediterranean controls showed 2/536 M1 alleles ( 0 . 37% , p < 0 . 0001 , odds ratio 13 . 51 ) .", "In the remaining 15 individuals , we attempted comprehensive Sanger sequencing of the entire KIAA0586 transcript , eventually identifying a pathogenic variant in eight individuals ( 57% ) , all leading to predicted splice , stop or frameshift changes , again consistent with recessive inheritance .", "In the other seven JS patients , a second mutation was not yet identified ( Table 1 , Table 1—source data 1 ) .", "Although it is possible that one or more of these individuals carries M1 by chance , it is most likely that a second mutation exists , not yet uncovered . 10 . 7554/eLife . 06602 . 009Figure 3 . MRI scans from patients with KIAA0586 mutations . Magnetic resonance imaging ( MRI ) in a healthy individual and patients with KIAA0586 mutations showing thickened and mal-oriented superior cerebellar peduncle ( upper , red ‘arrowheads’ ) , deepened interpeduncular fossa and constituting the ‘molar tooth sign’ ( red circle ) .", "In COR-354-2-3 , the molar tooth sign was very mild , possibly due to suboptimal image averaging .", "Figure 3—figure supplement 1 shows the imaging phenotype of affected JS individual MTI-1944-2-1 . DOI: http://dx . doi . org/10 . 7554/eLife . 06602 . 00910 . 7554/eLife . 06602 . 010Figure 3—figure supplement 1 . Imaging phenotype of affected JS individual MTI-1944-2-1 with KIAA0586 mutations . MRI of individual MTI-1944 affected by KIAA0586 mutations causing JS .", "For the affected individuals , the diagnosis of JS was confirmed by the deepened interpeduncular fossa and abnormal superior cerebellar peduncles , showing the ‘molar tooth sign’ ( red circle ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 06602 . 01010 . 7554/eLife . 06602 . 011Table 1 . All alleles identified in KIAA0586 causative for Joubert syndromeDOI: http://dx . doi . org/10 . 7554/eLife . 06602 . 01110 . 7554/eLife . 06602 . 012Table 1—source data 1 . Chromatograms of mutations in the KIAA0586 gene identified in the additional cohort of Mediterranean individuals with Joubert syndrome . DOI: http://dx . doi . org/10 . 7554/eLife . 06602 . 012Allele 1 ( based on T1 ) Allele 2 ( based on T1 ) Patient IDGenotypeGenomicDNAProteinGenomicDNAProteinMTI-233M1/M2g . 58899157delc . 428delp . Arg143Lysfs*4g . 58915212G>Ac . 1120+1G>Ap . Thr323Hisfs*3MTI-103M1/M3g . 58899157delc . 428delp . Arg143Lysfs*4g . 58923419G>Cc . 1413-1G>Cp . Arg472Serfs*2MTI-165M1/M4g . 58899157delc . 428delp . Arg143Lysfs*4g . 58896138T>Cc . 2T>C ( based on T4-T5 ) p . Met1 ?", "( based on T4-T5 ) MTI-1944M1/M5g . 58899157delc . 428delp . Arg143Lysfs*4g .", "? _58923420_58938997_ ?", "delc . 1413- ?", "_2793+ ?", "delp .", "? MTI-505M6/M6g . 58934452G>Cc . 2414-1G>Cp .", "? g . 58934452G>Cc . 2414-1G>Cp .", "? COR354M7/M7g . 58895020delc . 74delp . Lys25Argfs*6g . 58895020delc . 74delp . Lys25Argfs*6Mediterranean cohort analysisNG2872M1/M1g . 58899157delc . 428delp . Arg143Lysfs*4g . 58899157delc . 428delp . Arg143Lysfs*4NG4158M1/M8g . 58899157delc . 428delp . Arg143Lysfs*4g . 58909503C>Tc . 649C>Tp . Gln217*NG2326M1/M9g . 58899157delc . 428delp . Arg143Lysfs*4g . 58910790_58910791delc . 863_864delp . Gln288Argfs*7NG1776M1/M9g . 58899157delc . 428delp . Arg143Lysfs*4g . 58910790_58910791delc . 863_864delp . Gln288Argfs*7NG3928M1/M10g . 58899157delc . 428delp . Arg143Lysfs*4g . 58915097C>Tc . 1006C>Tp . Gln336*NG2458M1/M11g . 58899157delc . 428delp . Arg143Lysfs*4g . 58924613_58924616delinsAAAc . 1658_1661delinsAAAp . Val553Glufs*79NG2286M1/M12g . 58899157delc . 428delp . Arg143Lysfs*4g . 58925263G>Ac . 1815G>Ap .", "= / p .", "? NG1485M1/M13g . 58899157delc . 428delp . Arg143Lysfs*4g . 58927869C>Tc . 2209C>Tp . Arg737*NG3758M1/M14g . 58899157delc . 428delp . Arg143Lysfs*4g . 58953883delc . 3462delp . Gly1155Glufs*40M; mutation; T; transcript .", "Table 1—Source data 1 shows chromatograms belonging to the identified mutations in the Mediterranean cohort .", "To evaluate the effect of predicted splicing mutations in KIAA0586 , we generated mRNA from cultured fibroblasts of an affected and unaffected member of family MTI-233 and MTI-103 , displaying an M1 compounded with a splice mutation ( M2 or M3 , respectively ) .", "Sanger sequencing of poly-A primed mRNA showed that the mutation M2 led to the skipping of exon 9 and mutation M3 led to utilization of a cryptic splice acceptor located 16 bp downstream ( i . e . , 3′ ) , resulting in a frameshifted transcript ( Figure 2—figure supplement 1B , C ) , suggesting partial or complete loss-of-function .", "Loss-of-function mutations in Talpid3 result in a short-rib polydactyly-like phenotype in chicken and mouse , with a vascular defect and early lethality , all attributable due to defective ciliogenesis ( Bangs et al . , 2011; Davey et al . , 2014 ) .", "Our patients presented classical features of JS including the MTI of varying severity ( Figure 3 , Figure 3—figure supplement 1 ) , without lethality or demonstrable excessive fetal wasting in affected families .", "Most cases displayed hypotonia , ataxia , developmental delay , and intellectual disability without skeletal or limb malformations .", "Breathing abnormalities , seizures , macrocephaly , and ophthalmological defects were found in a subset of the cases ( Supplementary file 4A ) .", "The affected child of MTI-165 passed away at the age of 18 months from apnea , and no imaging was available .", "The results support the involvement of KIAA0586 in the pathogenesis of JS .", "RT-PCR analysis with primers spanning various transcripts showed ubiquitous KIAA0586 expression in various tissues ( Figure 4—figure supplement 1A ) .", "To determine the effect of mutations on KIAA0586 protein level , we analyzed patient fibroblasts of family MTI-103 and MTI-233 by Western analysis using a KIAA0586-specific antibody ( Kobayashi et al . , 2014 ) .", "The level of KIAA0586 protein in patient samples was below detection , whereas both carriers showed reduced but detectable expression compared with control ( Figure 4 ) .", "In human RPE1 cells transfected with KIAA0586 siRNA , we documented reduced protein levels , supporting antibody specificity . 10 . 7554/eLife . 06602 . 013Figure 4 . Absent KIAA0586 protein in patient fibroblasts . Immunoblot analysis of KIAA0586 in fibroblasts from family MTI-103 and MTI-233 .", "Lysates from RPE1 cells transfected with scrambled or KIAA0586 siRNA were used as control .", "M , unaffected carrier ( mother ) ; A , affected child .", "RPE1 , retinal pigment epithelial-1 cell line .", "Figure 4—figure supplement 1A represents an expression analysis of the KIAA0586 gene . DOI: http://dx . doi . org/10 . 7554/eLife . 06602 . 01310 . 7554/eLife . 06602 . 014Figure 4—figure supplement 1 . Expression analysis of the KIAA0586 gene . RT-PCR analysis showing differential expression levels of the KIAA0586 transcripts amongst various ciliated and non-ciliated tissues was observed .", "Co , colon; Ce , cerebellum; K , kidney; L , liver; MQ , MilliQ; T , testis; T1-T5 transcript number corresponding to Figure 2A . DOI: http://dx . doi . org/10 . 7554/eLife . 06602 . 014" ], [ "Here , we identify KIAA0586 mutations in JS using a combination of cell-based screening and exome sequencing .", "By training of a classifier to prioritize ciliary candidate genes based upon shared loss-of-function phenotypes , we generated a data set we called CILIOGENESIS consisting of 591 prioritized genes .", "Intersecting these genes with WES data of genetically unexplained JS individuals led to the discovery of mutations in KIAA0586 , which we found to be a relatively common cause ( i . e . , about 5% ) in unsolved JS cases .", "In patient cells , there was undetectable KIAA0586 protein supporting its role in JS pathogenesis .", "It remains to be determined whether mutations in KIAA0586 can lead to other ciliopathies like Meckel–Gruber syndrome or nephronophthisis , which are often allelic to JS .", "Our siRNA screen incorporated several improvements over previously published but similar screens .", "As the first genome-wide siRNA high-content screen for defective ciliogenesis , we evaluated nearly each of the annotated human genes with at least four siRNAs per gene .", "Second , we incorporated a specific cell phase marker , mCherry-Geminin , to exclude false-positives that might result from cell cycle defects .", "Third , we incorporated a machine learning approach with positive and negative training sets , which enhanced the predictability of measured cellular features as they relate to ciliogenesis .", "It is noteworthy that including features in the classifier from multiple sources , while improving performance of the classifier , caused a reduction from 18 , 045 targets to 16 , 431 targets due to missing values ( n = 798 targets lost by the biogenesis siRNA screen; n = 786 targets lost by the GTEx RNAseq data ) .", "It is possible that some ciliary factors were not correctly classified as such due to incomplete data in these comparative screens .", "Inevitability , our machine learning approach will be biased towards currently known ciliary factors , and as more knowledge is gained , the power of such approaches will improve .", "Even by combining the CILIOGENESIS data set with exomes from 145 individuals identified only a single recurrently mutated gene , leaving the majority of families still unexplained ( Akizu et al . , 2014 ) .", "This observation leads us to postulate that there are probably few commonly mutated genes remaining to be discovered in JS .", "Our siRNA screen is probably underpowered to detect JS genes primarily involved in effects like signaling through Sonic hedgehog or Wnt pathways .", "Gene set enrichment analysis of the true positive SCGCv1 genes ( SCGCv1 genes ranked within the CILIOGENESIS data set genes ) with MsigDB ( Subramanian et al . , 2005 ) showed enrichment for cytoskeletal genes as expected ( Supplementary file 3F ) , whereas analysis on false negative genes ( SCGCv1 genes ranked outside the CILIOGENESIS data set genes ) showed significant enrichment for photoreceptor cell maintenance , sensory perception , Sonic hedgehog pathway , and post-chaperonin tubulin folding pathway ( Supplementary file 3G ) .", "This suggests that the CILIOGENESIS data set may be enriched for genes involved in the process of ciliogenesis , whereas genes involved in signaling functions are less likely to be detected .", "Moreover , this is in agreement with analysis of candidate targets involved in Hedgehog signaling from screens described in literature for which we observe no enrichment in the CILIOGENESIS data set ( Supplementary file 3A , B ) ( Evangelista et al . , 2008; Jacob et al . , 2011 ) .", "It is possible that extending the CILIOGENESIS data set to include factors regulating the ciliary responsiveness to Hedgehog or Wnt activators or suppressors could further improve sensitivity .", "Talpid3 participates in the earliest stages of ciliation , including centriolar satellite dispersal and plasma membrane docking of the basal body ( Davey et al . , 2014; Kobayashi et al . , 2014 ) .", "Although Cep290 and Talpid3 share some similarities in ciliary phenotypes , there are distinct cellular functions ( Kobayashi et al . , 2014 ) .", "Talpid3 forms a ring-like structure at the distal end of both centrioles and is involved in the initiation of ciliary vesicle formation and docking , whereas Cep290 functions in the maturation of these vesicles .", "Moreover , Talpid3 is localized asymmetrically in mother and daughter centrioles and is crucial for limiting the levels of Cep120 at the mother centriole ( Wu et al . , 2014 ) .", "In Talpid3 mutant mouse embryos , centrosomes fail to dock at the plasma membrane and cilia are absent in various tissues ( Yin et al . , 2009 ) , associated with embryonic lethality .", "KIAA0586 might have been identified as mutated in JS even without the CILIOGENESIS data set , but was missed , probably for several reasons .", "First , the difference in names of the human and mouse genes made it difficult to link the two in automated curation of exome variants .", "Second , the majority of mutations were compound heterozygous , precluding homozygosity mapping analysis .", "Third , the higher frequency of the common allele M1 in the general population reduced its priority as a candidate , since the rarest alleles are prioritized over common alleles .", "Thus , we foresee the CILIOGENESIS data set and other orthogonal approaches as potentially beneficial in gene discovery .", "The 1/300 calculated carrier frequency of M1 in the population is comparable to the deep intronic founder mutation of ∼1/500 ( c . 2991+1655A>G ) in CEP290 as the most common cause of Leber congenital amaurosis in Caucasians , but less than the ∼1/100 in TMEM216 as a cause for JS in the Ashkenazi population ( den Hollander et al . , 2006; Valente et al . , 2010 ) .", "Of the 15 patients with heterozygous M1 in the Mediterranean cohort , we identified a second truncating allele in KIAA0586 in 57% , and the remaining are still under investigation for non-coding or deletion mutations .", "We screened a cohort of 800 individuals with nephronophthisis with retinopathy , and found four carrying the M1 mutation , close to the predicted 0 . 0036 expected carrier frequency and no convincing second mutations were documented in this cohort .", "Thus , it remains to be determined if KIAA0586 mutations are associated with other ciliopathy phenotypes or can lead to embryonic lethality .", "Because the mutations affect only exons incorporated in a subset of transcripts or affect splicing ( which can be leaky ) and because of embryonic lethality in mouse and chick with homozygous null mutations , we speculate that humans surviving with KIAA0586 mutations may retain partial function .", "The M4 allele was predicted to cause loss of the initiator methionine in transcript T4 and T5 , potentially leaving other transcripts intact .", "M4 was encountered in public sequence databases ESP and ExAC with a frequency of 0 . 002 ( 322/132 , 340 alleles ) , including three homozygous cases with unknown health status .", "The M7 allele affects three of six transcripts , while no protein was detected on Western blot from patient cells .", "It will be important to model these alleles or check for complementation of two null alleles with the patient alleles ." ], [ "hTERT-transformed RPE1 cells were cultured in DMEM/F12 medium supplemented with 10% fetal bovine serum ( FBS ) , under standard conditions ( 37°C , 5% CO2 ) .", "Plasmid DNAs harboring mouse Smo-EGFP and mCherry-Geminin ( 1–110aa ) fusion genes were transfected to hTERT-RPE1 cells and the stable cell line; Smo-EGFP-mCherry-Geminin/hTERT-RPE1 ( SEMG ) was established by G418 selection .", "To induce ciliogenesis , the cells were serum starved on serum-free DMEM/F12 media for 24–48 hr prior to fixation ." ] ]

[ "Defective primary ciliogenesis or cilium stability forms the basis of human ciliopathies , including Joubert syndrome ( JS ) , with defective cerebellar vermis development .", "We performed a high-content genome-wide small interfering RNA ( siRNA ) screen to identify genes regulating ciliogenesis as candidates for JS .", "We analyzed results with a supervised-learning approach , using SYSCILIA gold standard , Cildb3 . 0 , a centriole siRNA screen and the GTex project , identifying 591 likely candidates .", "Intersection of this data with whole exome results from 145 individuals with unexplained JS identified six families with predominantly compound heterozygous mutations in KIAA0586 .", "A c . 428del base deletion in 0 . 1% of the general population was found in trans with a second mutation in an additional set of 9 of 163 unexplained JS patients .", "KIAA0586 is an orthologue of chick Talpid3 , required for ciliogenesis and Sonic hedgehog signaling .", "Our results uncover a relatively high frequency cause for JS and contribute a list of candidates for future gene discoveries in ciliopathies ." ]

[ "Joubert syndrome is a rare disorder that affects the brain and causes physical , mental , and sometimes visual impairments .", "In individuals with this condition , two parts of the brain called the cerebellar vermis and the brainstem do not develop properly .", "This is thought to be due to defects in the development and maintenance of tiny hair-like structures called cilia , which are found on the surface of cells .", "Currently , mutations in 25 different genes are known to be able to cause Joubert syndrome .", "However , these mutations only account for around 50% of the cases that have been studied , and the ‘unexplained’ cases suggest that mutations in other genes may also cause the disease .", "Here , Roosing et al . used a technique called a ‘genome-wide siRNA screen’ to identify other genes regulating the formation of cilia that might also be connected with Joubert syndrome .", "This approach identified almost 600 candidate genes .", "The data from the screen were combined with gene sequence data from 145 individuals with unexplained Joubert syndrome .", "Roosing et al . found that individuals with Joubert syndrome from 15 different families had mutations in a gene called KIAA0586 .", "In chickens and mice , this gene—known as Talpid3—is required for the formation of cilia .", "Roosing et al . 's findings reveal a new gene that is involved in Joubert syndrome and also provides a list of candidate genes for future studies of other conditions caused by defects in the formation of cilia .", "The next challenges are to find out what causes the remaining unexplained cases of the disease and to understand what roles the genes identified in this study play in cilia ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "immunology and inflammation", "cancer biology" ]

[ [ "Microglia are the resident macrophages of the brain ( for review see [Kettenmann et al . , 2011; Casano and Peri , 2015] ) .", "Microglia are derived from primitive macrophages that invade the brain during development ( Ginhoux et al . , 2010; Schulz et al . , 2012; Herbomel et al . , 2001 ) .", "Due to the neuronal environment , they finally differentiate to microglia and build up a resident population that is almost evenly dispersed throughout the central nervous system .", "Microglia fulfil a tremendous repertoire of functions in the brain .", "In addition to their classical immune functions , numerous studies have described roles for microglia in brain development , vessel patterning , synaptic pruning , the regulation of neuronal activity and even the influence of certain animal behaviors ( Casano and Peri , 2015 ) .", "Despite the variety of beneficial functions in the brain , microglia can also act detrimentally during certain pathologies .", "Brain tumors represent probably the most severe example of harmful microglial functions .", "Microglia and infiltrating macrophages have been shown to infiltrate high-grade gliomas and can account for up to 30% of the tumoral mass ( Graeber et al . , 2002; Badie and Schartner , 2001; Li and Graeber , 2012; Yang et al . , 2010; Coniglio and Segall , 2013 ) .", "Instead of anti-tumoural activity , they display pro-tumoural functions and promote tumor growth .", "Macrophages and microglia have been shown to promote tumor cell proliferation and invasiveness , to modify the extracellular matrix , to induce angiogenesis and to induce an immunosuppressive environment promoting tumor growth ( Zhai et al . , 2011; Zhang et al . , 2012; Markovic et al . , 2005; Komohara et al . , 2008; Pyonteck et al . , 2013; Wu et al . , 2010; Hambardzumyan et al . , 2016; Wang et al . , 2013; Ellert-Miklaszewska et al . , 2013 ) .", "Interestingly , these pro-tumoural functions seem to be independent of the tumor grade , as they have also been described for low-grade gliomas ( Daginakatte and Gutmann , 2007; Daginakatte et al . , 2008; Simmons et al . , 2011 ) .", "However , the earliest responses of macrophages and microglia to cancerous cells in the brain have not been addressed so far .", "It is not known when macrophages and microglia respond to oncogenic transformations in the brain and which signals are involved .", "Furthermore , we do not know how these initial responses physically appear .", "Are aberrant oncogenic cells initially removed by macrophages and microglia ?", "Do pro-tumoural activities of macrophages and microglia develop only at later tumor stages ?", "Or is the behavior of macrophages and microglia already co-opted during the early oncogenic stages in a way that they promote tumor growth ?", "Understanding these early events is crucial as it is tempting to speculate that similar responses of macrophages and microglia occur during tumor recurrence .", "Thus , identifying the underlying mechanisms is a first step toward the development of new therapeutic strategies that target macrophages and microglia within brain tumors .", "To address the early responses of macrophages and microglia to oncogenic alterations in the brain , we made use of the larval zebrafish model .", "We and others have already demonstrated that the zebrafish is a powerful tool to study microglia ( Peri and Nüsslein-Volhard , 2008; Sieger et al . , 2012; Xu et al . , 2016; Oosterhof et al . , 2017; Svahn et al . , 2013; Shiau et al . , 2013; Shen et al . , 2016 ) .", "Besides the genetic and optical advantages of the zebrafish larvae , the small size of the zebrafish brain allows imaging of the entire microglial network and its responses to pathological events .", "Furthermore , recent work has highlighted the suitability of the larval zebrafish model to investigate brain tumor growth ( Ju et al . , 2014; Mayrhofer et al . , 2017; Shin et al . , 2012; Ju et al . , 2015; Lal et al . , 2012; Kitambi et al . , 2014; Geiger et al . , 2008; Lally et al . , 2007 ) ; Yang et al . , 2013; Jung et al . , 2013; Hamilton et al . ( 2016 ) ; Eden et al . , 2015; Welker et al . , 2016 ) .", "Here , we combined these advantages to study the responses of macrophages and microglia to early pre-neoplastic cells in the brain .", "By overexpression of human dominant active AKT1 in neural cells , we induced abnormal cellular growth and morphology , increased proliferation and early differentiation , mimicking the earliest stages of brain tumor growth .", "Importantly , we detected an immediate response of macrophages and microglia to these oncogenic events in the brain .", "This included a significant increase in macrophage and microglia cell numbers , which was caused by the infiltration of peripheral macrophages and mediated by Sdf1b released from oncogenic cells to activate the CXCR4b receptor on macrophages .", "Furthermore , macrophages and microglia showed highly dynamic cellular interactions with oncogenic cells .", "Finally , by reducing the number of macrophages and microglia , we showed that these cells actively promote proliferation of the pre-neoplastic cells in the brain ." ], [ "To investigate the response of macrophages and microglia to early pre-neoplastic cells , we overexpressed a dominant active version of the human AKT1 gene in the larval zebrafish brain ( Ramaswamy et al . , 1999 ) .", "AKT1 is a downstream serine-threonine kinase in the RTK/PTEN/PI3K pathway which is highly activated in the majority of human glioblastomas ( McLendon and Cancer Genome Atlas Research Network , 2008 ) .", "Human AKT1 has been reported previously to induce malignant transformations in various neural cell types in zebrafish leading to glioma growth ( Mayrhofer et al . , 2017; Jung et al . , 2013 ) .", "To achieve expression in the nervous system , we expressed AKT1 under the neural-specific beta tubulin ( NBT ) promoter using a dominant active version of the LexPR transcriptional activator system ( ΔLexPR ) ( Emelyanov and Parinov , 2008; Mazaheri et al . , 2014 ) .", "The NBT promoter is active in neural cells throughout zebrafish development ( Peri and Nüsslein-Volhard , 2008 ) .", "We either injected a lexOP:AKT1-lexOP:tagRFP construct into embryos of NBT:ΔLexPR:lexOP-pA ( NBT:ΔLexPR ) transgenic fish or co-injected an NBT:∆lexPR-lexOP-pA driver plasmid together with a lexOP:AKT1-lexOP:tagRFP construct to achieve AKT1 expression ( Figure 1A ) .", "In both scenarios , the NBT promoter drives expression of the transcriptional activator LexPR in neural cells which activates transcription of genes downstream the lexOP promoter sequence .", "These injections gave rise to mosaic expression of the oncogene within the larval nervous system ( visualised through RFP expression of the construct ) ( Figure 1 ) .", "During the course of development , AKT1 expression-induced morphological transformations resulting in larger cells with an abnormal morphology which was not observed upon injection of a lexOP:tagRFP control construct ( Figure 1B ) .", "Immunohistochemistry using an anti-AKT1 antibody revealed strong expression of the human AKT1 protein upon lexOP:AKT1-lexOP:tagRFP construct injection ( Figure 1C ) .", "To test for differentiation in AKT1 overexpressing cells , we performed immunohistochemistry for the differentiation marker Synaptophysin , which has been shown to be expressed in CNS tumors with neuronal differentiation as well as in neuroendocrine tumors ( Wiedenmann et al . , 1986 ) .", "Indeed , expression of AKT1 led to an early onset of expression of Synaptophysin , which was not observed in controls ( Figure 1D ) .", "To test for differentiation toward the glial lineage in AKT1-positive cells we stained for the glial cell marker GFAP .", "GFAP was neither detected in AKT1-expressing cells nor in RFP control cells at 8 dpf ( Figure 1E ) .", "Intriguingly , oncogenic alterations have been previously shown to induce dedifferentiation of neuronal cells ( Friedmann-Morvinski et al . , 2012 ) , thus we wondered if AKT1 overexpression is capable of inducing dedifferentiation in zebrafish neural cells .", "Interestingly , immunohistochemistry for the stem cell marker Sox2 revealed a subset of AKT1 cells which were positive for Sox2 while none of the RFP control cells appeared to be positive for Sox2 ( Figure 1F ) .", "Furthermore , the number of proliferating AKT1-positive cells was significantly increased compared to control RFP cells ( assessed by immunostaining for proliferating cell nuclear antigen ( PCNA ) ; Figure 1G ) .", "Finally , the oncogene expression led to significant impairment in survival with only 35 . 6% ( 62/174 larvae ) surviving until 30 days post-fertilization ( dpf ) upon AKT1 overexpression compared to 90 . 8% ( 118/130 larvae ) in controls ( Figure 1H ) .", "Examination of 20 AKT1 expressing fish at 1 month of age showed persistent expression of the RFP-tag in the brain , with variable mosaicism ( Figure 1—figure supplement 1A–H ) .", "Some of the RFP expressing cells showed neuronal features , including RFP-labeled axons and dendrites ( Figure 1—figure supplement 1D ) , while 1/3 of the brains showed large clones of RFP-expressing cells , which grew deeply in the brain parenchyma , apparently without disrupting the brain architecture .", "Immunostaining of these areas with a panel of antisera ( PCNA , AKT1 , GFAP and Synaptophysin ) showed disrupted cell architecture , with increased mitotic figures , as well as positive staining for AKT1 , GFAP and Synaptophysin , suggesting abnormal growth and differentiation , suggestive of brain tumors with mixed neuronal and glial components ( Figure 1—figure supplement 1I–N ) .", "The heterogeneous population of cells detected within these tumors is most probably a result of the early dedifferentiation events that we observed ( Figure 1F ) .", "This is in line with previous observations in rodent models , showing that dedifferentiated cells are capable of generating a NSC or progenitor state which gives rise to heterogeneous populations observed in mature tumors ( Friedmann-Morvinski et al . , 2012 ) .", "In summary , these results confirm that human AKT1 has the capacity to induce pre-neoplastic alterations in the larval zebrafish brain .", "Microglia numbers have been shown to be increased in brain tumors ( Badie and Schartner , 2001; Graeber et al . , 2002; Li and Graeber , 2012; Bowman et al . , 2016; Chen et al . , 2012 ) .", "However , it is unclear at which stage of tumor growth microglia numbers start to increase .", "To test if overexpression of AKT1 leads to an increase in microglial numbers , we used the 4C4 antibody that specifically labels microglia precursors and microglia in zebrafish ( Becker and Becker , 2001; Ohnmacht et al . , 2016 ) and quantified microglia numbers at 8 dpf ( Figure 2A , B ) .", "As shown previously , AKT1 positive cells were observed to be highly proliferative at 8 dpf , with subsets of cells positive for Synaptophysin and others for Sox2 , while GFAP-positive cells were not detected ( Figure 1D , E , F , G ) .", "Interestingly , at this early time point we observed a significant increase in microglia numbers in AKT1-positive brains when compared to controls ( Figure 2B ) .", "Furthermore , overexpression of AKT1 impacted on microglial morphology .", "Under normal physiological conditions , microglia show a highly ramified phenotype with several fine processes that are constantly extended and retracted to survey their environment ( Nimmerjahn et al . , 2005 ) .", "In response to a pathological insult in the brain , the microglia retract their processes and assume an amoeboid morphology which reflects their activation ( Karperien et al . , 2013 ) .", "This change in morphology leads to a change in the ratio of the microglia cell surface to microglia cell volume , which can be used as a read out for their activation ( Gyoneva et al . , 2014 ) ( Figure 2Ai–iii ) .", "Based on these morphological criteria , we analyzed the microglia in AKT1 overexpressing brains and compared to control brains at 8 dpf ( Figure 2A , C ) .", "This analysis showed a significantly higher percentage of amoeboid ( activated ) microglia in AKT1 overexpressing brains compared to control brains ( Figure 2C ) .", "In summary , these results show that early oncogenic events lead to an immediate response of the microglial network which is reflected in higher numbers and increased activation of microglia .", "To analyze if microglia respond directly to AKT1 positive cells , we performed confocal live imaging using the mpeg1:EGFP transgenic zebrafish , in which all macrophages including microglia can be visualized and tracked ( Ellett et al . , 2011 ) .", "Injection of lexOP:AKT1-lexOP:tagRFP or lexOP:tagRFP constructs into oocytes of double transgenic mpeg1:EGFP/NBT:ΔLexPR fish allowed us to follow microglial responses to oncogenic cells or control cells in high temporal and spatial resolution .", "In control embryos , microglia showed the typical ramified morphology , and cell surface contacts between microglia and control RFP cells were only occasionally observed and of short durations ( Figure 3A , Video 1 , n = 7 samples analyzed ) .", "In line with our previous observations based on 4C4 immunohistochemistry ( Figure 2 ) , the density of microglia within AKT1-expressing brains was high and most microglial cells showed an amoeboid morphology ( Figure 3B , Video 2 , n = 9 samples analyzed ) .", "Interestingly , microglia were observed to stay within close vicinity to AKT1 positive cells throughout the duration of the experiment and most microglial cells were in direct surface contact with the oncogenic cells .", "These surface contacts were long lasting and consisted of different types .", "Microglia were observed to flatten their surfaces against the surfaces of AKT1-positive cells and then moving their surfaces around the surfaces of AKT1 cells for hours ( Figure 3B , arrowheads , Video 2 ) .", "Other microglial cells showed interactions with several AKT1-positive cells by constantly expanding and retracting their processes upon contact with surfaces of AKT1 positive cells ( Figure 3B , arrows , Video 2 ) .", "Importantly , many of these interactions were observed for several hours without any disruption .", "Thus , based on the persistence of these interactions it is unlikely that these were random contacts simply enforced by the high density of microglial and AKT1-positive cells .", "To address this further , we injected lower amounts of the lexOP:AKT1-lexOP:tagRFP construct into oocytes of double transgenic mpeg1:EGFP/NBT:ΔLexPR fish and screened for fish with lower numbers and isolated AKT1-positive cells .", "In these larvae , microglia were frequently seen to interact with AKT1-positive cells ( Figure 3C , D Video 3 and 4 , n = 6 samples analyzed ) .", "As observed previously , interactions lasted for several hours and consisted of different types from flattening of microglial surfaces against surfaces of AKT1-positive cells ( Figure 3C ) to constant extensions and retractions of processes toward AKT1 cells ( Figure 3D ) .", "Thus , microglia are pro-actively interacting with AKT1-positive cells .", "Importantly , the observed interactions were not ‘anti-tumoral’ as we did not observe phagocytosis of AKT1-expressing cells .", "Thus , changes induced by AKT1 overexpression stimulate direct cellular interactions with microglia .", "To address if the increased numbers of microglia in AKT1 positive brains were caused by proliferation of resident microglia , we performed co-immunostainings for microglia ( 4C4 ) and the proliferation marker PCNA .", "Interestingly , we did not detect a difference in the number of PCNA positive microglia when comparing AKT1-positive brains to control brains ( not shown ) .", "Thus , we speculated that the presence of pre-neoplastic cells in the brain might cause an infiltration of peripheral macrophages , which then differentiate into 4C4-positive microglia-like cells .", "To address this question , we performed an L-plastin staining to identify all leukocytes in the brain ( Feng et al . , 2010 ) .", "Through the co-labeling of L-plastin and 4C4 , macrophages and neutrophils ( L-plastin+/4C4- ) can be distinguished from microglia ( L-plastin+/4C4+ ) .", "Indeed , we detected significantly higher numbers of L-plastin+/4C4- cells within the brain parenchyma upon AKT1 overexpression at 8 dpf , which resemble either newly infiltrated macrophages or neutrophils ( Figure 4A , Bi ) .", "To test if neutrophils contributed to the higher number of L-plastin+/4C4- cells we performed AKT1 overexpression in transgenic mpx:GFP zebrafish in which neutrophils express GFP under the neutrophil-specific myeloperoxidase promoter ( Renshaw et al . , 2006 ) .", "This experiment showed no infiltration of neutrophils upon AKT1 overexpression ( data not shown ) , thus infiltrated L-plastin+/4C4- cells were solely macrophages .", "In line with the previous results on increased microglial numbers , we detected a significant increase in the total amount of all L-plastin+ cells following the overexpression of AKT1 compared to age-matched controls ( Figure 4A , Biii ) .", "Within this population of L-plastin+ cells , the majority of cells were positive for 4C4 ( Figure 4Bii ) .", "As we did not detect proliferation of resident microglia , we hypothesized that infiltrated macrophages differentiated into microglia-like cells , leading to the higher numbers of 4C4-positive cells in AKT1-positive brains .", "If this hypothesis was true , then we should be able to detect an earlier time point when macrophages have just entered the brain but not differentiated to 4C4-positive cells yet .", "To test this , we performed L-plastin and 4C4 immunostainings at 3 dpf in AKT1-positive brains .", "Importantly , at 3 dpf we detected a 4 . 5-fold increase in the number of L-plastin+/4C4- cells in AKT1 positive brains compared to controls ( Figure 4Ci ) .", "However , numbers for 4C4-positive microglia were similar to controls ( Figure 4Cii ) .", "Thus , these L-plastin+/4C4- cells represented newly infiltrated macrophages .", "As numbers of 4C4+ cells were only increased at later time points ( Figure 4Bii ) we conclude that these infiltrated macrophages differentiated into microglia like ( 4C4+ ) cells over time .", "To visualize these infiltration and differentiation events , we made use of a double transgenic model and overexpressed AKT1 in p2ry12:p2ry12-GFP/mpeg1:mCherry zebrafish ( Ellett et al . , 2011; Sieger et al . , 2012 ) .", "In these zebrafish , all macrophages ( including microglia ) are positive for mCherry and microglia can be identified based on their additional P2ry12-GFP expression .", "To achieve AKT1 overexpression , we performed co-injections of the NBT:ΔLexPR driver plasmid and a lexOP:AKT1-lexOP:tagBFP expression plasmid .", "Intriguingly , we detected p2ry12-GFP-/mCherry+ macrophages at the dorsal periphery of AKT1-positive brains ( Figure 4—figure supplement 1 ) at 5 . 5 dpf .", "Eventually , some of these macrophages infiltrated the brain parenchyma and started expressing GFP over time , showing their differentiation toward a p2ry12-GFP+/mCherry+ microglia-like cell ( Figure 4—figure supplement 1 , arrowheads , Video 5 ) .", "These infiltration and differentiation events were not observed in control larvae ( not shown ) , which is in line with previously published data showing that developmental colonization of the brain by primitive macrophages is happening before 4 . 5 dpf ( Casano et al . , 2016 ) .", "Importantly , similar observations have been made recently in a rodent glioma model where infiltrating monocytes take on a microglia-like identity ( Chen et al . , 2017 ) .", "In conclusion , these results show that early oncogenic events lead to a significant increase in the macrophage and microglia cell population in the brain .", "We have shown that activation of AKT1 in neural cells leads to an increase in the macrophage and microglia cell population .", "To address the underlying mechanism , we focused on the chemokine receptor Cxcr4 as its role in the recruitment of tumor supportive macrophages has been shown previously ( Beider et al . , 2014; Boimel et al . , 2012; Hughes et al . , 2015; Arnò et al . , 2014 ) .", "To test a putative role for Cxcr4 in our model , we made use of the zebrafish cxcr4b-/- mutant ( Haas and Gilmour , 2006 ) .", "To achieve overexpression of AKT1 in the cxcr4b-/- mutant , we performed co-injections of the NBT:ΔLexPR driver plasmid and the lexOP:AKT1-lexOP:tagRFP expression plasmid into embryos of the mutant ( Figure 5A ) .", "As in cxcr4b wild-type larvae , these injections resulted in a mosaic expression of the oncogene within the larval nervous system ( Figure 5B ) .", "AKT1 expression induced morphological transformations resulting in larger cells with an abnormal morphology and expression of the human AKT1 protein in the cxcr4b-/- mutant ( Figure 5B ) .", "In line with this , we detected an early onset of expression of the differentiation marker Synaptophysin ( Figure 5C ) .", "Thus , overexpression of AKT1 in the cxcr4b-/- mutant induces alterations as observed in wild-type larvae .", "However , overexpression of AKT1 in the cxcr4b-/- mutant did not lead to an increase in microglia numbers compared to overexpression of AKT1 in wild-type larvae ( Figure 5D ) .", "Notably , microglia numbers were similar in cxcr4b-/- controls and wild-type controls , showing that Cxcr4b signaling is not needed for the normal developmental population of the brain by microglia ( Figure 5D ) .", "Thus , Cxcr4b signaling is either required for the infiltration of macrophages upon AKT1 overexpression or for their differentiation into 4C4-positive microglia-like cells .", "To address this question , we performed a co-labeling of L-plastin and 4C4 upon AKT1 overexpression in the cxcr4b-/- mutant .", "Intriguingly , the number of newly infiltrated macrophages ( L-plastin+/4C4- ) did not change upon AKT1 overexpression in the cxcr4b-/- mutant ( Figure 5Ei ) .", "In line with this , we did not detect an increase in the total number of all L-plastin+ cells upon AKT1 overexpression in the cxcr4b-/- mutant ( Figure 5Eii ) .", "Importantly , cxcr4b-/- mutant zebrafish showed similar numbers of peripheral macrophages as wild-type zebrafish ( Figure 5—figure supplement 1 ) , thus the lack of infiltration of L-plastin+ cells was not due to a general deficiency in peripheral macrophages .", "Furthermore , macrophages in cxcr4b-/- mutant embryos showed normal recruitment and function during mycobacterial infections in a recent study ( Torraca et al . , 2017 ) .", "Thus , Cxcr4b signaling is specifically required for the infiltration of macrophages upon AKT1 overexpression in the brain .", "To address if the lower numbers of microglia upon AKT1 overexpression in the cxcr4b-/- mutant impacted on the oncogenic cells , we analyzed their proliferation rates based on immunostainings for PCNA .", "Interestingly , while no observable impact on proliferating control-RFP cells was detected ( Figure 5F ) , we detected an almost 50% reduced proliferation rate of AKT1-positive cells in the cxcr4b-/- mutant compared to the wild-type larvae following overexpression of AKT1 ( Figure 5F ) .", "These results show that Cxcr4b signaling is required for the infiltration of macrophages and subsequent increase in 4C4-positive microglia numbers in AKT1 positive brains .", "In addition , these data imply an impact of macrophages and microglia on the proliferation rate of pre-neoplastic cells .", "Alternatively , Cxcr4b might have a direct role in AKT1 positive cells and might be necessary for increased proliferation .", "To test if Cxcr4b signaling acts cell autonomously in macrophages and microglia or AKT1 positive neural cells , we performed cell specific rescue experiments in the cxcr4b-/- mutant .", "To rescue Cxcr4b expression in macrophages/microglia , we made use of the macrophage/microglia-specific mpeg1 promoter and injected a mpeg1:cxcr4b construct into embryos of cxcr4b-/- mutant fish in parallel to overexpression of AKT1 ( Figure 6A ) .", "This led to a transient , mosaic expression of Cxcr4b on macrophages and microglia , thus reflecting a partial rescue ( Figure 6B ) .", "To test the alternative that Cxcr4b expression is needed in the AKT1-positive neural cells , we performed a specific rescue of Cxcr4b in these cells .", "This was achieved by co-injection of a lexOP:cxcr4b plasmid in addition to a lexOP:AKT1-lexOP:tagRFP plasmid together with the NBT:ΔLexPR driver plasmid into the mutant background ( Figure 6A ) .", "Again , this resulted in a transient , mosaic expression of Cxcr4b in oncogenic cells reflecting a partial rescue ( Figure 6B ) .", "Upon cell-specific rescue of Cxcr4b in macrophages/microglia or AKT1-positive neural cells , microglia numbers in rescued larvae were analyzed .", "Interestingly , the rescue of Cxcr4b in macrophages/microglia led to an increase in 4C4 microglia numbers upon AKT1 overexpression in the cxcr4b-/- mutant ( Figure 6C ) .", "On the contrary , the specific rescue of Cxcr4b expression in AKT1 positive neural cells did not lead to increased microglia numbers ( Figure 6C ) .", "These results show that Cxcr4b function is needed cell autonomously in macrophages and microglia for an increase in microglial numbers induced by AKT1-overexpressing cells .", "To further address a putative role of Cxcr4b in AKT1-positive neural cells , we analyzed the proliferation rate of AKT1-positive cells in the two specific rescue scenarios .", "Interestingly , while the Cxcr4b rescue in AKT1-positive neural cells did not increase their proliferation rates ( Figure 6D ) , the specific rescue of Cxcr4b expression in macrophages and microglia led to a 70% increase in proliferation rates of AKT1-positive cells ( Figure 6D ) .", "Thus , Cxcr4b signaling is not required in AKT1-positive neural cells to increase their proliferation rates .", "Importantly , these results imply a role for microglia in inducing proliferation in early pre-neoplastic cells .", "The Cxcr4b receptor can be activated by its cognate ligands Sdf1a ( Cxcl12a ) and Sdf1b ( Cxcl12b ) and the noncognate ligand macrophage migration inhibitory factor ( Mif ) .", "To address which of these ligands is responsible for Cxcr4b activation upon AKT1 overexpression , we isolated and sorted AKT1-RFP-positive cells and control cells from larval brains by FACS and performed qPCR for the respective ligands .", "While sdf1a expression levels did not show differences to control cells we detected a three-fold increase in mif expression levels and an over 10-fold increase in sdf1b expression levels in AKT1-positive neural cells compared to control cells ( Figure 7A ) .", "To further test a putative role for Sdf1b in Cxcr4b induced microglial population increase , we performed Sdf1b overexpression in larval brains in the absence of AKT1 overexpression .", "Injection of a lexOP:sdf1b-lexOP:tagRFP construct into NBT:ΔLexPR transgenic fish led to significantly increased microglia numbers compared to controls ( Figure 7B ) .", "Thus , higher levels of Sdf1b are sufficient to induce an increase in microglia numbers .", "To test if Sdf1b activity was solely mediated via Cxcr4b activation , we performed Sdf1b overexpression in the cxcr4b-/- mutant and quantified microglia numbers .", "Indeed , in the cxcr4b-/- mutant background , Sdf1b overexpression did not impact on microglia numbers ( Figure 7B ) .", "Thus , high levels of Sdf1b produced by AKT1 positive cells lead to increased microglia numbers via Cxcr4b activation .", "We have shown that Cxcr4b signaling functions cell autonomously in macrophages and microglia during early stages of AKT1 activation in the brain .", "To further assess the impact of macrophages and microglia on AKT1-positive cells , we decided to inhibit microglial function independently of Cxcr4b signaling .", "We treated AKT1 overexpressing larvae with the immunosuppressant Dexamethasone ( DEX ) which has previously been shown to inhibit macrophages and microglia upon spinal cord injury and brain inflammation in zebrafish ( Ohnmacht et al . , 2016; Kyritsis et al . , 2012 ) .", "Furthermore , to specifically deplete macrophages and microglia , we treated AKT1-expressing larvae with the CSF-1R inhibitor Ki20227 that has previously been shown to deplete macrophages and to reduce tumor growth in a rodent melanoma model ( Tham et al . , 2015; Ohno et al . , 2006 ) .", "Treatment with DEX or Ki20227 had a direct impact on microglia in control and AKT1-positive larvae , leading to significantly reduced microglia numbers in controls and AKT1 larvae , respectively ( Figure 8Ai ) .", "Furthermore , while proliferation rates of neural cells remained constant in controls ( Figure 8Aii ) , DEX and Ki20227-treated AKT1-positive larvae showed a more than 50% reduction in the number of proliferating AKT1 cells compared to DMSO controls ( Figure 8Aii ) .", "Finally , to directly address the impact of macrophages and microglia on AKT1-positive cells without any pharmacological interference , we made use of the zebrafish irf8-/- mutant ( Shiau et al . , 2015 ) .", "Interferon regulatory factor 8 ( Irf8 ) is vital for macrophage development in mammals and in teleosts .", "The irf8 null mutant ( irf8-/- ) zebrafish was characterized to lack macrophages up to around 6 dpf , with recovery from 7 dpf while microglia were absent in the brain until 31 dpf ( Shiau et al . , 2015 ) .", "In line with published observations , we did not detect 4C4 positive microglial cells in irf8-/- larvae at 8dpf ( Figure 8Bi ) .", "Interestingly , upon AKT1 overexpression in irf8-/- larvae , we detected a small population of 4C4-positive cells ( Figure 8Bi ) .", "These cells were probably derived from the population of macrophages that recover in irf8-/- larvae from 7 dpf , which then infiltrated into the brain due to AKT1 overexpression and differentiated into 4C4+ cells .", "Importantly , the number of 4C4-positive cells was 80% lower in irf8-/- larvae compared to wild-type larvae upon AKT1 overexpression ( Figure 8Bi ) , which allowed us to address the impact on the proliferation of AKT1-positive cells .", "While proliferation rates of neural cells remained constant in irf8-/- controls ( Figure 8Bii ) , we detected an almost 60% reduction in proliferation of AKT1-positive cells compared to AKT1 positive cells in wild-type larvae ( Figure 8Bii ) .", "In summary , these experiments show that macrophages and microglia promote proliferation of AKT1-positive cells from the earliest stages of brain tumor growth ." ], [ "In this study , we addressed the responses of macrophages and microglia to the earliest events of brain tumor growth .", "Several elegant studies have shown that macrophages and microglia infiltrate brain tumors and promote their growth ( for review see ( Hambardzumyan et al . , 2016 ) ) .", "However , so far it was unclear when myeloid cells first respond to oncogenic events in the brain .", "We made use of the larval zebrafish model to address the earliest stages of tumor induction due to activation of oncogenes .", "By overexpressing a dominant active version of the human AKT1 gene , we induced cellular alterations in the larval zebrafish brain .", "These were reflected in abnormal cellular morphology , increased proliferation and an early onset of Synaptophysin expression .", "Intriguingly , we detected an immediate response of macrophages and microglia to AKT1-induced cellular alterations .", "By using a combination of mutant and transgenic zebrafish lines , as well as immunohistochemistry for macrophages and microglia , we showed an immediate increase in the population of macrophage and microglia cells .", "This increase was the result of infiltrating macrophages that differentiated into microglia-like cells as observed by expression of the 4C4 antigen .", "These results are in line with recent data from rodent models investigating the contribution of macrophages and microglia to the myeloid cell population within gliomas .", "Based on multiple models of murine brain malignancy and genetic lineage tracing , Bowman et al . showed that infiltrating macrophages are abundant in primary and metastatic brain tumors ( Bowman et al . , 2016 ) .", "A further study using genetically engineered mouse models to distinguish monocytes/macrophages from microglia showed that 85% of the tumor-associated macrophages ( TAMs ) within the glioma were infiltrated monocytes/macrophages ( Chen et al . , 2017 ) .", "Furthermore , the authors concluded that infiltrating monocytes transitioned to macrophages and microglia-like cells ( Chen et al . , 2017 ) .", "The increased number of 4C4 positive cells detected in our model might reflect these microglia-like cells .", "This is further supported by our live imaging experiments showing that infiltrating macrophages start expressing p2ry12 , which has been shown to be microglia specific across different species ( Crotti and Ransohoff , 2016 ) .", "However , further markers will be required to test if these cells are microglia-like or fully differentiated microglial cells .", "Interestingly , while the CCL2/CCR2 signaling axis seems to be the main attractant for monocytes at the later stages of glioma growth , we show that Sdf1b/Cxcr4b signaling is responsible for macrophage infiltration during the initial stages of oncogene activation in the brain .", "Importantly , a role for SDF1/CXCR4 signaling in macrophage attraction has been described before in rodent models of breast cancer and tumor relapse after chemotherapy ( Hughes et al . , 2015; Boimel et al . , 2012 ) .", "Our data show that the pre-neoplastic AKT1 cells produce Sdf1b , which is in line with previous studies showing that AKT1 is involved in activating the expression of SDF1 ( Furue et al . , 2017; Conley-LaComb et al . , 2013 ) .", "Other studies revealed an AKT-mediated induction of the transcription factor specificity protein 1 ( Sp1 ) and Sp1-binding sites were shown to be functional within the human SDF1 promoter ( Gómez-Villafuertes et al . , 2015; Pore et al . , 2004; García-Moruja et al . , 2005 ) .", "Thus , AKT1-induced Sdf1b expression might be mediated via Sp1 .", "Furthermore , we show that Sdf1b produced by the pre-neoplastic cells acts specifically on macrophages and microglia but is not required in an autocrine fashion during these early stages .", "By using a zebrafish mutant for cxcr4b , we showed that Cxcr4b signaling is required for macrophage infiltration upon AKT1 expression in the brain .", "Only the specific rescue of Cxcr4b in macrophages and microglia led to increased microglial numbers in the mutant background while a specific rescue in oncogenic cells did not alter the phenotype .", "Importantly , we also detected a significant decrease in the number of proliferating AKT1 cells in the cxcr4b-/- mutant background , which could not be rescued by Cxcr4b expression in these cells .", "Thus , Sdf1b/Cxcr4b signaling is not required for proliferation of AKT1-expressing cells in an autocrine fashion during initial stages of oncogenic activation .", "This might highlight another difference between the early and late stages of tumor growth , as autocrine SDF1/CXCR4 signaling has been shown to be involved in proliferation of various glioma cell lines ( for review see ( Gagliardi et al . , 2014 ) ) .", "Intriguingly , rescuing Cxcr4b expression in macrophages and microglia in the cxcr4b-/- mutant background led to a significant increase in proliferation of AKT1-positive cells , suggesting a role for microglia in promoting proliferation of pre-neoplastic cells .", "To further address this , we depleted macrophages and microglia independently of Cxcr4b using the immunosuppressant Dexamethasone , the specific CSF-1R inhibitor Ki20227 and the zebrafish irf8-/- mutant .", "Importantly , we observed a significant decrease in the number of proliferating AKT1 cells upon macrophage and microglia depletion .", "Thus , macrophages and microglia promote proliferation of pre-neoplastic cells in the brain .", "Microglia have been shown to release stress-inducible protein 1 ( STI1 ) for example , which increased proliferation of glioma cells in vitro and in vivo ( Carvalho da Fonseca et al . , 2014 ) .", "Furthermore , co-culture studies showed that release of TGF-β by microglia-stimulated glioma growth ( Wesolowska et al . , 2008 ) .", "Whether the same signals are released by microglia to promote proliferation during early stages of transformation needs to be addressed in future studies .", "Importantly , the fact that microglia promote the growth of pre-neoplastic cells rather than inhibiting their growth is further supported by the live imaging experiments conducted in this study .", "As macrophages and microglia are professional phagocytes , they are able to phagocytose cells that are detrimental to the body .", "However , within the time series acquired to monitor the behavior of macrophages/microglia toward AKT1-positive cells , we did not observe phagocytosis events .", "Macrophages/microglia infiltrated areas with high numbers of oncogenic cells and showed continuous interactions with these cells .", "Interestingly , similar observations were made in rodent and zebrafish live imaging models showing direct cellular interactions between microglia and glioma cells ( Resende , 2016; Hamilton et al . , 2016; Bayerl et al . , 2016; Ricard et al . , 2016 ) .", "Our study highlights that signals stimulating these cellular interactions are produced early upon activation of an oncogene and seem to persist to later stages of tumor growth .", "However , neither the identity of these signals nor the impact of these interactions on tumor cells is known yet .", "As microglia have been shown to directly interact with highly active neurons with increased Ca2+ levels ( Li et al . , 2012 ) , it is tempting to speculate that Ca2+ levels might be increased in AKT1-positive cells resulting in ATP/ADP release to promote the observed interactions with microglia .", "Identification of the signals mediating these interactions is of high priority now to address the functional significance of these cellular contacts .", "In summary , we show for the first time that macrophage and microglia are activated during the earliest stages of oncogene activation in the brain and develop their pro-tumoural activity immediately by promoting proliferation of pre-neoplastic cells .", "It is tempting to speculate that similar interactions occur during tumor recurrence , thus inhibiting the underlying signals might offer additional therapeutic options ." ], [ "Zebrafish were housed in a purpose-built zebrafish facility , in the Queen’s Medical Research Institute , maintained by the University of Edinburgh Biological Resources .", "All zebrafish larvae were kept at 28°C on a 14 hr light/10 hr dark photoperiod .", "Embryos were obtained by natural spawning from adult Tg ( mpeg1:EGFP ) gl22 referred to as mpeg1:EGFP , Tg ( mpeg1:mCherry ) gl23 referred to as mpeg1:mCherry ( Ellett et al . , 2011 ) wild type ( AB ) , irf8st95/st95 referred to as irf8-/- ( Shiau et al . , 2015 ) , TgBAC ( p2ry12:p2ry12-GFP ) hdb3 referred to as p2ry12:p2ry12-GFP ( Sieger et al . , 2012 ) and cxcr4t26035/t26035 referred to as cxcr4b-/- ( Haas and Gilmour , 2006 ) zebrafish strains .", "Tg ( XIa . Tubb:LEXPR ) Ed7 referred to as NBT:∆lexPR-lexOP-pA ( NBT:∆lexPR ) transgenic fish were newly generated using Tol2-mediated transgenesis as described before ( Kwan et al . , 2007 ) .", "∆lexPR ( Mid Entry vector; Gateway Invitrogen ) , a constitutively active form of the inducible LexPR system ( Emelyanov and Parinov , 2008; Mazaheri et al . , 2014 ) was placed under control of the neural-specific beta tubulin ( NBT promoter ( 5’ Entry vector; Gateway Invitrogen ) .", "Embryos were raised at 28 . 5°C in embryo medium ( E3 ) and treated with 200 μM 1-phenyl 2-thiourea ( PTU ) ( Sigma ) from the end of the first day of development for the duration of the experiment to inhibit pigmentation .", "Animal experimentation was approved by the ethical review committee of the University of Edinburgh and the Home Office , in accordance with the Scientific Procedure Act 1986 .", "To achieve transient expression of AKT1 , zebrafish embryos were injected at the 1 cell stage .", "Approximately 2 nL of plasmid DNA ( 30 ng/µL ) containing Tol2 capped mRNA ( 20 ng/µL ) and 0 . 2% phenol red were injected into the eggs of NBT:∆lexPR-lexOP-pA fish .", "To obtain AKT1 expression , Tol2-pDEST-lexOP:AKT1-lexOP:tagRFP or Tol2-pDEST-lexOP:AKT1-lexOP:tagBFP was injected .", "To obtain control RFP or BFP expression Tol2-pDEST-lexOP:tagRFP-pA or Tol2-pDEST-lexOP:tagBFP-pA were injected respectively .", "In embryos absent of the transgenic NBT promoter , a Tol2-pDEST-NBT:∆lexPR-lexOP-pA ( 20 ng/µL ) plasmid was co-injected to drive AKT1 expression in neural cells .", "Larvae were screened at 2 days post-fertilization ( dpf ) for positive transgene expression and selected for the required experiments .", "To rescue Cxcr4b expression in macrophages or neural cells in the cxcr4b-/- mutant fish , a cell specific rescue construct was injected in addition to the NBT driver and lexOP:tagRFP-pA/lexOP:AKT1-lexOP:tagRFP constructs .", "Cxcr4b expression was recovered in macrophages through the mpeg1 promoter ( ‘Macrophage’ rescue ) via a mpeg1:cxcr4b-pA construct .", "The expression of cxcr4b in neural cells ( ‘Neural’ rescue ) was rescued through the lexOP:cxcr4b-pA construct .", "Whole mount immunostaining of samples was performed as previously described ( Astell and Sieger , 2017 ) .", "Briefly , larvae were fixed in 4% PFA/1% DMSO at room temperature for 2 hr , followed by a number of washes in PBStx ( 0 . 2% Triton X-100 in 0 . 01 M PBS ) , and blocked in 1% goat serum blocking buffer ( 1% normal goat serum , 1% DMSO , 1% BSA , 0 . 7% Triton X-100 in 0 . 01 M PBS ) for 2 hr prior to incubation with primary antibodies overnight at 4°C .", "Primary antibodies used were mouse anti-4C4 ( 1:50 ) ( courtesy of Becker Laboratory , University of Edinburgh ) , rabbit anti-AKT1 ( 1:100 ) ( PA5-29169 , Fisher Scientific ) , rabbit anti-Cxcr4b ( 1:100 ) ( ab111053 , abcam ) , rabbit anti-GFAP ( 1:500 ) ( courtesy of Becker Laboratory , University of Edinburgh ) , rabbit anti-L-plastin ( 1:500 ) ( courtesy of Feng Laboratory , University of Edinburgh ) , rabbit anti-Mfap4 ( 1:500 ) ( courtesy of Becker Laboratory , University of Edinburgh ) , rabbit anti-PCNA ( 1:300 ) ( ab18197 , abcam ) , rabbit anti-SOX2 ( 1:200 ) ( ab97959 , abcam ) , and rabbit anti-Synaptophysin ( 1:100 ) ( ab32594 , abcam ) .", "A number of washes in PBStx was carried out before samples were subsequently incubated in conjugated secondary antibodies ( goat anti-mouse Alexa Fluor 488 [1:200]; goat anti-mouse Alexa Fluor 647 [1:200]; goat anti-rabbit Alexa Fluor 488 [1:200]; goat anti-rabbit Alexa Fluor 647 [1:200] ) ( Life Technologies ) overnight at 4°C to reveal primary antibody localizations .", "Samples were washed following secondary antibody incubation and kept in 70% glycerol at 4°C until final mounting in 1 . 5% low melting point agarose ( Life Technologies ) in E3 for image acquisition .", "Whole brain immuno-fluorescent images were acquired using confocal laser scanning microscopy ( Zeiss LSM710 and LSM780; 20x/0 . 8 objective; 2 . 30 µm intervals; 488- , 543- , and 633 nm laser lines ) .", "Whole mount in situ hybridization was done following the protocol described by Thisse and Thisse ( Thisse and Thisse , 2008 ) using a digoxygenin labeled mpeg1 RNA probe .", "Live imaging of zebrafish larvae was performed as previously described ( Hamilton et al . , 2016; Sieger et al . , 2012 ) ; samples were mounted dorsal side down in 1 . 5% low melting point agarose ( Life Technologies ) , in glass-bottom dishes ( MatTek ) filled with E3 containing 0 . 2 mg/mL Tricaine ( MS222 , Sigma ) .", "Single time-point live images were acquired through confocal imaging ( Zeiss LSM710; 20x/0 . 8 objective; 2 . 30 µm intervals; 405- , 488- , and 543 nm laser lines ) .", "To investigate direct interactions between oncogene-expressing cells and microglia , time-lapse imaging was carried out on a spinning disk confocal microscope ( Andor iQ3; 20x/0 . 75 and W40x/1 . 15 objectives; 1 . 5–2 µm z-intervals; 405- , 488- , and 543 nm laser lines ) .", "All time-lapse acquisitions were carried out in temperature-controlled climate chambers set to 28°C for 10–18 hr .", "Twenty zebrafish , injected as previously described to obtain transient expression of AKT1 in neural cells , were used at 1 month of age to assess the presence of proliferative lesions .", "The brains were observed and photographed immediately upon removal under a fluorescent stereomicroscope and fixed in 4% PFA in PBS for 12 hr .", "After sucrose treatment and OCT embedding , 12 um thick sections were cut and collected , to allow staining of adjacent sections for different antibodies .", "Immunostaining was performed as previously described ( Mayrhofer et al . , 2017 ) .", "Sections were counterstained with DAPI and observed through confocal imaging ( Zeiss LSM710; 40x/1 . 3 objective ) .", "Analyses of all images were conducted using Imaris ( Bitplane , Zurich , Switzerland ) .", "For the quantification of 4C4+ and/or L-plastin+ cells , only cells within the brain ( telencephalon , tectum , and cerebellum ) were counted for each sample using the ‘Spots’ function tool in Imaris 8 . 0 . 2 .", "To determine 4C4+/L-plastin+ cells , the ‘Coloc’ function in Imaris was used to generate an independent channel for cells that were double-positive .", "To quantify proliferation rates , the number of PCNA+/RFP+ cells were counted in relation to the total number of RFP+ cells and the averaged value expressed as measure of percentage proliferation .", "To assess and quantify microglial morphology , we used the surface rendering tool in Imaris 8 . 0 . 2 .", "This allowed segmentation of individual cells in 3D .", "To assess their morphological activation we calculated the ratio of the cellular surface and cellular volume of individual cells as previously described ( Gyoneva et al . , 2014 ) .", "Microglia with a ratio smaller than 0 . 8 were classified as activated .", "For cell isolation , anaesthetized 8 dpf zebrafish larvae were transferred into ice cold E3 containing 0 . 2 mg/mL Tricaine .", "The heads were removed using surgical micro-scissors and then collected in ice cold medium A ( HBSS 1X , 15 mM Hepes and 25 mM Glucose ( Life technologies ) ) .", "On ice , heads were manually homogenized using a 1 mL glass dounce until the cell suspension was homogenous .", "Subsequently , the cell suspension was run through a 40 μm cell strainer and transferred into a 1 . 5-mL Eppendorf tube .", "Cells were centrifuged at 300 g for 10 min at 4°C .", "The pellet was re-suspended in 22% percoll solution overlaid by ice cold PBS , followed by centrifugation at 950 g without brake for 30 min at 4°C .", "The pellet was washed twice with medium A supplemented with 2% normal goat serum and centrifuged at 300 g for 10 min at 4°C .", "Cells were re-suspended in ice cold medium A - 2% normal goat serum then transferred to FACS tubes through 35 μm cell strainer cap , immediately followed by FACS sorting using a FACSAria II ( Becton Dickinson ) .", "DAPI was added to label and exclude dead cells .", "Total RNA extraction from the sorted cells of interest was performed using the Qiagen RNeasy Plus Micro kit ( Qiagen ) and the RNA sample concentration was determined using the Quant-IT Ribogreen RNA assay kit ( Invitrogen ) .", "For quantification of gene expression , cDNA synthesis following RNA isolation was carried out using the SuperScript® III First-Strand Synthesis System ( Invitrogen ) .", "Quantitative PCR ( Q-PCR ) amplifications were performed in duplicates in a 20 μL reaction volume containing SsoAdvanced Universal SYBR Green Supermix ( Bio-Rad ) using a LightCycler 96 Real-Time PCR System ( Roche ) .", "The PCR protocol used was: Initial denaturation step of 5 min at 95°C , and 45 cycles of 10 s at 95°C , 20 s at 56°C , and 20 s at 72°C .", "Primers used were: ß-actin forward: 5'-CACTGAGGCTCCCCTGAATCCC-3' , ß-actin reverse: 5'-CGTACAGAGAGA GCACAGCCTGG-3'; mif forward: 5'-AAAGACTCGGTTCCGGCG-3' , mif reverse: 5'-CACACGGGTCTCCTTTTCCC-3'; sdf1a forward: 5'-CGCCAT TCATGCACCGATTT-3' , sdf1a reverse: 5'-TGACTTGGAAGGGGCAGTTG-3' , sdf1b forward: 5'-AGCAAAGTAGTAGCGCTGGTG-3' , and sdf1b reverse: 5'-TCTCTCGGATGCTCCGTTG-3' .", "Melting curve analysis was used to ensure primer specificity .", "For Q-PCR analysis , the threshold cycle ( Ct ) values for each gene were normalized to expression levels of ß-actin and relative quantification of gene expression determined with the comparative Ct ( ΔΔCt ) method using the LightCycler® 96 Software ( Roche ) .", "Q-PCR analysis was repeated three times for each gene .", "To suppress the immune response , larvae were treated with dexamethasone ( DEX , Sigma-Aldrich ) .", "Briefly , larvae were incubated in 100 µM DEX/1% DMSO from 3 dpf until the end of the experiment .", "To deplete macrophages and microglia , larvae were treated with the CSF-1R inhibitor Ki20227 ( Tocris ) at 25 µM/1% DMSO from 3 dpf until the end of the experiment .", "To obtain experimental controls , age-matched samples were incubated in 1% DMSO .", "Media were changed daily until the respective experimental endpoints at 8 dpf .", "All experiments were performed in at least two replicates and ‘n’ indicates the total number of larvae used .", "All measured data were analyzed ( StatPlus , AnalystSoft Inc . ) .", "Two-tailed Student’s t-tests were performed between two experimental groups and one-way ANOVA with Bonferroni’s post-hoc tests performed for comparisons between multiple experimental groups .", "Statistical values of p<0 . 05 were considered to be significant .", "All graphs were plotted in Prism 6 . 1 ( GraphPad Software ) and values presented as population means ( ±SEM ) ." ] ]

[ "It is now clear that microglia and macrophages are present in brain tumors , but whether or how they affect initiation and development of tumors is not known .", "Exploiting the advantages of the zebrafish ( Danio rerio ) model , we showed that macrophages and microglia respond immediately upon oncogene activation in the brain .", "Overexpression of human AKT1 within neural cells of larval zebrafish led to a significant increase in the macrophage and microglia populations .", "By using a combination of transgenic and mutant zebrafish lines , we showed that this increase was caused by the infiltration of peripheral macrophages into the brain mediated via Sdf1b-Cxcr4b signaling .", "Intriguingly , confocal live imaging reveals highly dynamic interactions between macrophages/microglia and pre-neoplastic cells , which do not result in phagocytosis of pre-neoplastic cells .", "Finally , depletion of macrophages and microglia resulted in a significant reduction of oncogenic cell proliferation .", "Thus , macrophages and microglia show tumor promoting functions already during the earliest stages of the developing tumor microenvironment ." ]

[ "Brain tumors can be aggressive , difficult to treat and are often incurable .", "Removing brain tumors by surgery can be challenging because the tumor cells infiltrate into the healthy tissue .", "Brain tumors grow in close physical contact with other cells , such as cells of the immune system .", "This includes cells called macrophages and microglia , which normally defend us against injuries and infections .", "However , instead of acting against the tumor as one might expect , macrophages and microglia actually support the growth of brain tumors .", "It is not clear how and when during the development of a brain tumor the macrophages and microglia start helping the tumor cells to grow .", "Previous studies in this area have focused on these cell types found in advance brain tumors .", "Chia et al . have now looked at the earliest stages of tumor development , monitoring how macrophages and microglia respond to cancer cells .", "To mimic human brain tumors , Chia et al . expressed a cancer-promoting version of a human protein in nerve cells of zebrafish larvae .", "These cells started behaving like early tumor cells .", "Live microscopy revealed that the tumor cells attract macrophages and microglia into their area , and that they also activate the immune cells .", "Biochemical experiments showed that the early tumor cells make and release a protein called Sdf1 .", "Macrophages and microglia sense Sdf1 in the environment with a protein called Cxcr4 on their cell surfaces .", "When the gene for Cxcr4 was deleted in the zebrafish , the macrophages and microglia were not recruited into the developing tumors .", "When macrophages and microglia were depleted from the zebrafish larvae , the nerve cells with the mutant protein grew less well , supporting the idea that the immune cells enhance the development of early tumors .", "A better understanding of how tumor cells and immune cells interact in the brain may help in the search for new anti-cancer drugs .", "Furthermore , the way in which macrophages and microglia are recruited to the tumor cells could be similar when tumors return after treatment .", "Future studies will test this hypothesis and , if it proves true , interfering with the macrophage and microglia response might delay the relapse of tumors ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "cell biology", "cancer biology" ]

[ [ "The urokinase-type plasminogen activator receptor ( uPAR ) is a central player in a complex signaling network implicated in a variety of remodeling processes , both physiological and pathological , ranging from embryo implantation to wound healing and tumor progression ( Boonstra et al . , 2011; Ferraris and Sidenius , 2013; Smith and Marshall , 2010 ) .", "uPAR is a glycosylphosphatidylinositol ( GPI ) -anchored protein and hence lacks intrinsic signaling capacity .", "Instead , uPAR acts by binding two major ligands , namely the protease urokinase plasminogen activator ( uPA ) and the extracellular matrix ( ECM ) protein vitronectin ( Ferraris and Sidenius , 2013; Madsen et al . , 2007; Smith and Marshall , 2010 ) .", "Through uPA binding , uPAR localizes plasmin generation to the cell surface and thereby promotes pericellular proteolysis and ECM degradation ( Ferraris and Sidenius , 2013; Smith and Marshall , 2010 ) .", "In addition , through vitronectin binding and functional interactions with integrins and growth factor receptors , uPAR activates intracellular signaling pathways leading to cytoskeletal reorganization , enhanced cell adhesion and motility and other features of tissue remodeling and cell transformation ( Ferraris et al . , 2014; Madsen et al . , 2007; Smith and Marshall , 2010 ) .", "As such , uPAR is a master regulator of extracellular proteolysis , cell motility and invasion .", "uPAR expression is elevated during inflammation and in many human cancers , where it often correlates with poor prognosis , supporting the view that tumor cells hijack the uPAR signaling system to enhance malignancy ( Boonstra et al . , 2011; Ferraris and Sidenius , 2013; Smith and Marshall , 2010 ) .", "Increased uPAR expression in solid tumors and the corresponding activated stroma is being evaluated by PET-imaging for patient stratification ( Persson et al . , 2015 ) .", "It has long been known that full-length uPAR is released from the plasma membrane resulting in a soluble form ( suPAR ) ( Pedersen et al . , 1993; Ploug et al . , 1992 ) , which is detectable in body fluids and considered a marker of disease severity in cancer and other life-threatening disorders ( Haupt et al . , 2012; Hayek et al . , 2016; Shariat et al . , 2007; Sidenius et al . , 2000; Stephens et al . , 1999 ) .", "Circulating suPAR is derived from activated immune and inflammatory cells ( Ferraris and Sidenius , 2013; Smith and Marshall , 2010 ) , and also from circulating tumor cells ( Mustjoki et al . , 2000 ) .", "Locally produced suPAR might function as a ligand scavenger to confer negative feedback on uPAR ( Smith and Marshall , 2010 ) .", "In addition , both uPAR and suPAR can undergo proteolytic fragmentation by uPA and other proteases , possibly leading to new signaling activities ( Montuori and Ragno , 2009 ) .", "Yet , despite decades of research , the mechanism of uPAR release and its physiological implications have been elusive .", "A GPI-specific phospholipase D ( GPI-PLD ) ( Scallon et al . , 1991 ) has often been assumed to mediate the shedding of GPI-anchored proteins , but this unique PLD does not function on native membranes ( Mann et al . , 2004 ) .", "A possible clue to the mechanism of uPAR release comes from recent studies showing that a member of the glycerophosphodiester phosphodiesterase ( GDPD/GDE ) family ( Corda et al . , 2014 ) , termed GDE2 , promotes neuronal differentiation by cleaving select GPI-anchored proteins , notably a Notch ligand regulator and heparan sulfate proteoglycans ( glypicans ) ( Matas-Rico et al . , 2016; Matas-Rico et al . , 2017; Park et al . , 2013 ) .", "GDE2 , along with GDE3 and GDE6 , belongs to a GDE subfamily characterized by six-transmembrane-domain proteins with a conserved catalytic ectodomain ( Figure 1A ) ( Corda et al . , 2009; Matas-Rico et al . , 2016 ) .", "GDE2’s close relative , GDE3 , accelerates osteoblast differentiation through an unidentified mechanism ( Corda et al . , 2009; Yanaka et al . , 2003 ) , while the function of GDE6 is unknown .", "Here we identify GDE3 as the first mammalian GPI-specific phospholipase C ( GPI-PLC ) that cleaves and sheds uPAR with consequent loss of uPAR activities in both HEK293 and breast cancer cells ." ], [ "We set out to determine whether uPAR can be released by any of the three related GDE family members , GDE2 , GDE3 and GDE6 .", "When expressed at relatively low levels in HEK293 cells , human GDE2 and GDE3 ( HA-tagged ) localized to distinct microdomains at the plasma membrane , possibly representing clustered lipid rafts where GPI-anchored proteins normally reside ( Maeda and Kinoshita , 2011 ) , as well as to filopodia-like extensions ( Matas-Rico et al . , 2016 ) ( Figure 1—figure supplement 1A , B ) .", "By contrast , human GDE6 was mainly detected in intracellular compartments and therefore was not further tested ( Figure 1—figure supplement 1A ) .", "To assess GDE activity , we generated stable uPAR-expressing HEK293 cells ( HEK-uPAR cells ) , expressed GDE2 and GDE3 and examined the appearance of suPAR in the medium , using bacterial phospholipase C ( PI-PLC ) as a positive control ( Matas-Rico et al . , 2016 ) .", "Strikingly , uPAR was readily released into the medium by GDE3 and PI-PLC , but not by GDE2 ( Figure 1B ) .", "GDE3 competed with exogenous PI-PLC to deplete uPAR , since PI-PLC was much less efficient in GDE3-overexpressing than in control HEK-uPAR cells ( Figure 1—figure supplement 1C ) .", "Mutating putative active-site residue H229 , corresponding to H233 in GDE2 ( Matas-Rico et al . , 2016 ) , abolished GDE3 activity without affecting its membrane localization ( Figure 1C; Figure 1—figure supplement 1D ) .", "Furthermore , a transmembrane version of uPAR ( uPAR-TM ) lacking the GPI moiety ( Cunningham et al . , 2003 ) was resistant to GDE3 attack , consistent with GDE3 acting through GPI-anchor hydrolysis ( Figure 1—figure supplement 1E ) .", "Flow cytometry analysis of GDE3-overexpressing cells confirmed decreased uPAR levels in the plasma membrane ( Figure 1D ) , while TIRF microscopy revealed substantial uPAR loss from the ventral cell surface ( Figure 1E ) .", "The selectivity of GDE3 versus GDE2 towards uPAR cleavage is striking .", "In an attempt to understand the structural basis of this selectivity , we constructed homology-based models of the globular α/β barrel GDPD domains , using I-TASSER ( Yang et al . , 2015 ) .", "We reasoned that the catalytic domains must recognize not only the PI lipid moiety at the membrane-water interface , but also the attached protein .", "While GDE2 and GDE3 have a similar putative GPI-binding groove leading to the active site , they show striking differences in their surface charge distribution , particularly at the putative substrate interaction surface ( Figure 1F ) .", "It therefore seems likely that surface properties are a major determinant of selective substrate recognition by GDEs , a notion that should be validated by future structural studies .", "We asked whether GDE3 attacks uPAR in cis ( same cell ) or in trans ( adjacent cell ) , or both .", "By mixing GDE3-expressing cells ( lacking uPAR ) with uPAR-expressing cells ( lacking GDE3 ) , GDE3-expressing cells failed to shed uPAR from the GDE3-deficient cell population ( Figure 2A ) .", "Thus , GDE3 acts in cis , attacking uPAR on the same plasma membrane , not on adjacent cells .", "To determine whether GDE3 acts as a phospholipase in GPI-anchor cleavage , we used Triton X-114 partitioning and liquid chromatography-mass spectrometry ( LC-MS ) .", "Triton X-114 partitioning revealed that suPAR did not contain lipid moieties , as it was not detected in the detergent phase ( Figure 2B ) .", "Next , suPAR was immunoprecipitated from the medium of GDE3-expressing cells and treated with nitrous acid ( HONO ) to cleave the glucosamine-inositol linkage in the GPI core ( Figure 2C ) .", "Subsequent analysis by LC-MS revealed that the acid-treated suPAR samples contained inositol 1-phosphate ( Figure 2D ) .", "This result defines GDE3 as the first mammalian GPI-specific phospholipase C ( GPI-PLC ) .", "When compared to uPAR-deficient cells , HEK-uPAR cells showed markedly increased cell adhesion , loss of intercellular contacts and enhanced spreading with prominent lamellipodia formation on vitronectin , but not on fibronectin ( Figure 3A ) , typical features of a Rac-driven motile phenotype , in agreement with previous studies ( Kjøller and Hall , 2001; Madsen et al . , 2007 ) .", "Cell spreading coincided with activation of focal adhesion kinase ( FAK ) , indicative of integrin activation ( Figure 3B ) .", "Strikingly , expression of GDE3 largely abolished the uPAR-induced phenotypes and cellular responses ( Figure 3A–F ) .", "Catalytically dead GDE3 ( H229A ) had no effect , neither had overexpressed GDE2 ( Figure 3F and results not shown ) .", "Thus , by releasing uPAR from the plasma membrane , GDE3 suppresses the vitronectin-dependent activities of uPAR .", "Treatment of diverse cell types with suPAR-enriched conditioned media from HEK293 cells did not evoke detectable cellular responses , supporting the notion that suPAR is biologically inactive , at least under cell culture conditions .", "We next assessed the impact of GDE3 on endogenous uPAR activity in MDA-MB-231 triple-negative breast cancer cells .", "These cells express relatively high levels of uPAR ( Figure 4A ) and its ligand uPA ( LeBeau et al . , 2013 ) , thus forming an autocrine signaling loop .", "Expression of GDE3 ( encoded by GDPD2 ) is relatively low in breast cancer lines ( n = 51 ) , including MDA-MB-231 cells ( Barretina et al . , 2012 ) ( Figure 4B ) .", "GDE3 expression in MDA-MB-231 cells led to a modest loss of uPAR from the plasma membrane as shown by flow cytometry ( Figure 4C ) .", "To determine how GDE3 expression affects localized uPAR levels at the basolateral plasma membrane , we used confocal and dual-color super-resolution microscopy in TIRF mode .", "Strikingly , basolateral membrane microdomains containing wild-type GDE3 showed little or no colocalization with endogenous uPAR .", "In marked contrast , catalytically dead GDE3 ( H229A ) clearly colocalized with uPAR in those membrane domains ( Figure 4D ) .", "Quantification of co-localization was performed on multiple cells ( Figure 4D , right panel ) .", "These results strongly suggest that active GDE3 depletes uPAR levels from distinct domains at the basolateral membrane .", "Consistent with this , CRISPR-based knockout of GDE3 resulted in increased basolateral uPAR levels when the cells were plated on vitronectin ( Figure 4E , F; Figure 4—figure supplement 1 ) .", "Wild-type MDA-MB-231 cells adopted a motile phenotype on vitronectin , as evidenced by increased cell spreading with marked lamellipodia formation ( Figure 5A–C ) , strongly reminiscent of a uPAR-regulated phenotype .", "Overexpressed GDE3 abolished the vitronectin-dependent phenotype of MBD-MB-231 cells ( Figure 5A–C ) .", "Very similar effects of GDE3 overexpression were observed in another uPAR-positive breast cancer cell line ( triple-negative Hs578T cells ) ( Figure 5—figure supplement 1 ) .", "Of note , no effects were observed upon GDE2 overexpression in these cells ( data not shown ) .", "To confirm that GDE3 acts through uPAR attack , we expressed non-cleavable uPAR-TM to compete out endogenous uPAR , and found that the GDE3-induced reduction of cell spreading on vitronectin was largely inhibited ( Figure 5B ) .", "Furthermore , shRNA-mediated knockdown uPAR ( Figure 5D ) gave rise to the same phenotype as GDE3 overexpression , namely reduced cell adhesion , spreading and lamellipodia formation on vitronectin ( Figure 5E , F , G ) .", "In long-term assays , wild-type MDA-MB-231 cells showed marked scattering , indicative of increased cell motility with loss of intercellular contacts ( Figure 6A ) .", "Again , GDE3 overexpression mimicked uPAR depletion either in greatly reducing both cell motility and clonogenic potential , using either shRNA-mediated knockdown ( Figure 6A , B ) or CRISPR-mediated knockout of uPAR ( Figure 6—figure supplement 1 ) .", "Having shown that GDE3 suppresses the non-proteolytic activities of uPAR , we next examined how GDE3 affects uPAR-driven proteolytic matrix degradation by MDA-MB-231 cells .", "Also in this cell system , GDE3 overexpression mimicked uPAR silicencing in inhibiting the degradation of a gelatin matrix ( mixed with vitronectin ) in the presence of serum ( Figure 6C , D ) ( Figure 6—figure supplement 1 ) .", "On the basis of these results , we conclude that GDE3 attenuates the transformed phenotype of uPAR-positive breast cancer cells through loss of functional uPAR .", "When injected into the mammary fat pads of female nude mice , GDE3-overexpressing MDA-MB-231 cells showed diminished tumor growth over time , when compared to empty vector-expressing cells ( Figure 7A ) , consistent with the cell-based data .", "However , the full implications of GDE3 expression on uPAR-dependent tumor growth remain to explored in further detail .", "Finally , in patients , high expression of GDPD2 was found to correlate with prolonged relapse-free survival in breast cancer , particularly in triple-negative ( basal-like ) subtype patients ( N = 618 ) ( Figure 7B ) .", "No such correlation was found for GDE2 ( encoded by GDPD5; not shown ) .", "This suggests that GDE3/GDPD5 may serve as a marker of clinical outcome in breast cancer ." ], [ "GPI-anchoring is a compIex post-translational modification that anchors select proteins in the outer leaflet of the plasma membrane .", "Despite decades of research , the biological significance of GPI anchors has long remained a mystery ( Kinoshita and Fujita , 2016; Paulick and Bertozzi , 2008 ) .", "Some GPI-anchored proteins are released from their anchor and detected in body fluids , implying involvement of one or more endogenous GPI-specific hydrolases .", "Recent studies have advanced the field by showing that cleavage and shedding of certain GPI-anchored proteins is mediated by a cell-intrinsic transmembrane glycerophosphodiesterase , termed GDE2 ( or GDPD5 ) , thereby promoting neuronal differentiation through multiple signaling pathways ( Matas-Rico et al . , 2016; Matas-Rico et al . , 2017; Park et al . , 2013 ) .", "In this study , we focused on the shedding of GPI-anchored uPAR because of its regulatory role in multiple cellular and ( patho ) physiological activities , while soluble uPAR is considered a biomarker of various human pathologies .", "Here we report that GDE3 functions as a long-sought GPI-specific PLC that releases uPAR from its anchor .", "By contrast , its homologue GDE2 failed to release uPAR .", "As a consequence of GDE3 action , uPAR loses its vitronectin-dependent and matrix-degrading activities , when assayed in HEK293-uPAR and triple-negative breast cancer cells that express both uPAR and uPA .", "Importantly , loss of uPAR expression by GDE3 was found to be restricted to certain microdomains at the basolateral plasma membrane , where signal transduction is likely to take place .", "Thus , by acting as a GPI-specific PLC towards uPAR , GDE3 is a negative regulator of the uPAR signaling network ( Figure 7C ) that includes uPAR’s proteolytic and non-proteolytic activities .", "Consistent with this , GDE3 overexpression in uPA/uPAR-positive MDA-MB-231 breast cancer cells slowed tumor progression in a xenograft mouse model .", "Although statistically significant , the inhibitory effect of GDE3 overexpression on tumor growth was not dramatic , which should not come as a surprise since MDA-MBA-31 cells express the strongly oncogenic mutant K-RAS protein , which tends to override the regulation of numerous signaling pathways .", "Yet , this finding adds to the relevance of GPI-specific phospholipases in slowing tumor progression .", "Furthermore , high GDE3 expression was found to correlate with increased survival probability in triple-negative breast cancer patients .", "Interestingly , our previous work revealed a similar association between overexpression of GDE2 and positive clinical outcome in neuroblastoma patients , which appears attributable to GDE2-induced glypican shedding ( Matas-Rico et al . , 2016 ) .", "The present patient survival analysis should be interpreted with caution , however , since involvement of uPAR release remains to be formally proven .", "Furthermore , we cannot rule out that GDE3 may cleave additional GPI-anchored substrates whose functional loss could contribute to positive clinical outcome .", "The present results predict that , depending on its expression levels , GDE3 may downregulate normal uPAR-dependent remodeling processes .", "Indeed , upregulated GDE3 accelerates osteoblast differentiation ( Corda et al . , 2009; Yanaka et al . , 2003 ) in a manner resembling the uPAR knockout phenotype ( Furlan et al . , 2007 ) .", "Furthermore , a striking >200 fold upregulation of GDE3/GDPD2 is observed during blastocyst formation ( Munch et al . , 2016 ) , implicating GDE3 in the invasion of pre-implantation embryos , a process in which the uPA/uPAR signaling network has been implicated ( Multhaupt et al . , 1994; Pierleoni et al . , 1998 ) .", "Although correlative , these results support the view that GDE3 is upregulated to downregulate uPAR activity in vivo .", "The present findings also suggest that circulating full-length suPAR should be regarded as a marker of GDE3 activity , not necessarily reflecting uPAR expression levels .", "It will now be important to determine how GDE3 expression and activity are regulated and , furthermore , to explore the substrate selectivity of the respective GDEs in further detail .", "Homology modeling revealed striking differences in electrostatic surface properties of GDE2 versus GDE3 , suggesting that protein-protein interactions may determine substrate recognition by these GDE family members .", "Specific GPI-anchor modifications ( Kinoshita and Fujita , 2016; Paulick and Bertozzi , 2008 ) could also determine the sensitivity of GPI-anchored proteins to GDE attack .", "Finally , when regarded in a broader context , the present and previous findings ( Matas-Rico et al . , 2016; Matas-Rico et al . , 2017; Park et al . , 2013 ) support the view that vertebrate GDEs , notably GDE2 and GDE3 , have evolved to modulate key signaling pathways and alter cell behavior through selective GPI-anchor cleavage ." ], [ "HEK293 , MDA-MB-231 and Hs578T cells were obtained from the ATCC and grown in Dulbecco’s modified Eagle’s medium ( DMEM ) supplemented with 10% fetal bovine serum ( FBS ) and antibiotics at 37°C under 5% CO2 .", "Original MDA-MB-231 cells were pathogen tested using the ImpactI test ( Idexx Bioresearch , Westbrook , ME , USA ) and were negative for all pathogens tested .", "All cell lines were routinely tested negative for mycoplasma contamination .", "Antibodies used: anti-mCh and anti-GFP , home-made; anti-Flag , M2 , anti-Vinculin and β-Actin ( AC-15 ) from Sigma; anti-uPAR ( MAB807 ) from R&D systems; anti-uPAR ( 13F6 ) ( Zhao et al . , 2015 ) ; anti-FAK ( pTyr397 ) from Thermo Fisher .", "Vitronectin , fibronectin , inositol 1-phosphate ( dipotassium salt ) and inositol were purchased from Sigma-Aldrich .", "B . cereus PI-PLC was from Molecular Probes .", "Phalloidin red ( actin-stain 647 phalloidin ) and green ( actin-stain 488 phalloidin ) were from Cytoskeleton .", "GM6001 was from Millipore .", "Research Source Identifiers: MDA-MB-231 cells RRID:CVCL_0062; Hs578T cells RRID:CVCL_0332; Antibodies: Flag M2 RRID:AB_259529; Anti Vinculin RRID:AB_10746313; Anti actin RRID:AB_2223210; uPAR RRID:AB_2165463 .", "GDE2 cDNA was subcloned as described ( Matas-Rico et al . , 2016 ) .", "GDE3 cDNA was amplified by PCR and subcloned into a pcDNA3-HA plasmid using AflII/HpaI ( PCR product ) and AflII/EcoRV ( plasmid ) restriction sites .", "GDE3 was recloned into a pcDNA3 ( -mCherry ) construct by PCR amplification with restriction sites PmI1/Xba1 , followed by vector digestion using EcoRV/Xba1 .", "GDE6 transcript variant X7 ( GDPD4; NCBI: XM_011544834 . 1 ) was cloned into pcDNA5_FRT_TO_puro ( provided by dr . Geert Kops , University Medical Center Utrecht ) .", "All constructs were epitope- tagged on the C-terminus unless otherwise stated .", "Mutant GDE3 ( H229A ) was generated by amplification with oligos containing the mutation , followed by Dpn1 digestion of the template .", "The viral plasmids ( pBABE-GDE3-mCh , pBABE-uPAR-GFP ) were constructed by subcloning the GDE3-pcDNA3 and uPAR-GFP-pEGFP-N1 into a pBABE plasmid .", "GDE3-mCherry-pcDNA3 was cut using PmeI followed by digestion of the pBABE backbone with SnaBI .", "uPAR-GFP-pEGFP-N1 was cut with BglII and HpaI and ligated into the pBABE vector , digested with BAMHI and SnaB1 .", "Constructs uPAR-GFP , uPAR-FLAG and non-cleavable uPAR-TM were previously described ( Caiolfa et al . , 2007; Cunningham et al . , 2003 ) .", "Transmembrane-anchored uPAR-TM was constructed by substituting the GPI-anchoring sequence of uPAR ( aa 274–313 ) with the transmembrane region ( aa 614–653 ) of the human epidermal growth factor receptor ( EGFR ) , as described ( Cunningham et al . , 2003 ) .", "Cells stably expressing uPAR-GFP or GDE3-mCherry were generated using retroviral transduction and subsequent selection with puromycin .", "Transient transfections were done using the calcium phosphate protocol or XtremeGene 9 agent ( Roche ) .", "Stable uPAR knockdown in MDA-MB-231 cells was achieved using shRNAs in a lentiviral pLKO vector; five shRNAs from three RC human shRNA library were tested: TRCN0000052637 , TRCN0000052636 , TRCN0000052634 , TRCN0000052633 and TRCN0000052635 .", "The latter two were used for experiments; sequences: CCGGCCCATGAATCAATGTC TGGTACTCGAGTACCAGACATTGATTCATGGGTTTTTG and CGGGCTTGAAGA TCACCAGCCTTACTCGAGTAAGGCTGGTGATCTTCAAGCTTTTTG , respectively .", "For virus production , HEK293T cells were transiently transfected using calcium phosphate , and virus particles were collected 48 hr thereafter .", "uPAR knockdown cells were selected in medium containing 2 μg/ml puromycin .", "uPAR and GDE3 knockout cell lines were generated using CRISPR/Cas9 genome editing .", "CRISPR sequences were designed targeting uPAR ( PLAUR; exon 2; 5’- CATGCAGTGTAAGACCAACG-3’ and 5’-CCAGGGCGCACTCTTCCACA-3’ ) or GDE3 ( GDPD2; exon 2; 5’-AGGATGCAAACCAGCAAGG-3’ ) and cloned into pX330 ( Cong et al . , 2013 ) .", "MDA-MB-231 cells were transfected with the exon-specific pX330 plasmids in addition to a plasmid containing a guide RNA to the Danio Rerio TIA gene ( 5’- GGTATGTCGGGAACCTCTCC -3’ ) and a cassette of a 2A sequence followed by a BlastR gene , flanked by two TIA target sites .", "Co-transfection results in infrequent integration of the BlastR gene at the targeted genomic locus by NHEJ , as previously described ( Blomen et al . , 2015 ) .", "Successful integration of the cassette renders cells resistant to blasticidin .", "Three days following transfection , the culture medium was supplemented with blasticidin ( 25 μg/ml ) .", "Surviving colonies were clonally expanded , screened for cassette integration and indels into the query gene by PCR ( GDE3; 5’- TATGAATCCTGCCCGAAAAG-3’ and 5’-AGAGCAGGCCAAACCAGATA-3’ ) or by western blot analysis of the target protein ( uPAR ) .", "To determine the inositol phosphate content of cleaved uPAR , suPAR was immuno-precipitated from HEK293 cell conditioned medium using anti-GFP beads ( ChromoTek ) .", "To remove inositol phosphate from suPAR , the beads were treated with 0 . 1M acetate buffer ( pH 3 . 5 ) and subsequently with 0 . 5M NaNO2 or 0 . 5M NaCl ( Control ) for 3 hr as previously described ( Mehlert and Ferguson , 2009 ) .", "Inositol phosphate-containing samples were preprocessed by adding methanol to a final concentration of 70% and shaken at 1000 RPM at room temperature for 10 min .", "Following centrifugation ( 20 , 400 x g at 4°C for 10 min ) , the supernatant was evaporated to dryness in a Speedvac at room temperature .", "The dried extracts were reconstituted in 50 mM ammonium acetate ( pH 8 . 0 ) , centrifuged ( 20 , 400 x g at 4°C for 10 min ) and transferred to autosampler vials .", "Liquid chromatography ( LC ) was performed using a Dionex Ultimate 3000 RSLCnano system ( Thermo Fisher Scientific ) .", "A volume of 5 μl was injected on a Zorbax HILIC PLUS column ( 150 × 0 . 5 mm , 3 . 0 μm particles ) maintained at 30°C .", "Elution was performed using a gradient: ( 0–5 min , 20% B; 5–45 min 20–100% B; 45–50 min 100% B; 50–50 . 1 min 20% B; 50 . 1–60 min 20% B ) of 100% acetonitrile ( mobile phase A ) and 50 mM ammonium acetate adjusted to pH 8 . 0 with ammonium hydroxide ( mobile phase B ) at a flow rate of 15 μl/min .", "Inositol 1-phosphate was detected with an LTQ-Orbitrap Discovery mass spectrometer ( Thermo Fisher Scientific ) operated in negative ionization mode scanning from m/z 258 to 260 with a resolution of 30 , 000 FWHM .", "Electrospray ionization was performed with a capillary temperature set at 300°C and the sheath , auxiliary and sweep gas flow set at 17 , 13 and 1 arbitrary units ( AU ) , respectively .", "Setting for Ion guiding optics were: Source voltage: 2 . 4 kV , capillary voltage: −18 V , Tube Lens: −83 V , Skimmer Offset: 0 V , Multipole 00 Offset: 5 V , Lens 0: 5 V , Multipole 0 Offset: 5 . 5 V , Lens 1: 11 V , Gate Lens Offset: 68 V , Multipole 1 Offset: 11 . 5 V , Front Lens: 5 . 5 V . Data acquisition was performed using Xcalibur software ( Thermo Fisher Scientific ) .", "Reference inositol phosphate ( Sigma ) was used to determine the retention on the Zorbax HILIC plus column .", "After applying Xcalibur’s build-in smoothing algorithm ( Boxcar , 7 ) , extracted ion chromatograms ( m/z 259 . 02–259 . 03 ) were used to semi-quantitatively determine inositol phosphate levels .", "48-wells plates were coated overnight at 4°C with fibronectin ( 10 μg/ml ) or vitronectin ( 5 μg/ml ) , or left untreated .", "Thereafter , plates were blocked for 2 hr at 37°C using 0 . 5% BSA in PBS .", "Cells were washed and harvested in serum-free DMEM supplemented with 0 . 1% BSA .", "Equal numbers of cells were seeded and allowed to adhere for 1 hr .", "Non-adherent cells were washed away using PBS .", "Attached cells were fixed with 4% paraformaldehyde ( PFA ) for 10 min , followed by washing and staining with Crystal violet ( 5 mg/ml in 2% ethanol ) for 10 min .", "After extensive washing , cells were dried and lysed in 2% SDS for 30 min .", "Quantification was done by measuring absorbance at 570 nm using a plate reader .", "For cell-matrix contact area and lamellipodia measurements , coverslips were coated overnight with fibronectin or vitronectin and washed twice with PBS .", "Cells were trypsinized , washed and resuspended in DMEM and left to adhere and spread for 4 hr .", "After fixation ( 4% PFA ) and F-actin staining with phalloidin , images were taken using confocal microscopy .", "Cell and lamellipodia area was quantified using an ImageJ macro .", "Cell scattering was determined as described ( LeBeau et al . , 2013 ) .", "In brief , single MDA-MB-231 cells were allowed to grow out as colonies , and the area covered by the scattered colonies ( colony size ) was measured at 6 days after plating .", "For measuring colony outgrowth , 500 cells were plated and colonies were counted after 14 days .", "Coverslips were coated with OG-labelled gelatin ( InVitrogen ) supplemented with vitronectin ( 5 μg/ml ) .", "About 100 . 000 cells per coverslip were seeded in DMEM with 10% FCS .", "After 20 hr , cells were washed , fixed with 4% PFM and stained with phalloidin-Alexa647 ( InVitrogen ) .", "Gelatin degradation was determined from confocal images of >15 randomly automatically chosen fields of view per coverslip ( testing at least two coverslips/condition on two separate days: total four coverslips per condition ) .", "The images were randomized and the area of degradation was normalized to the total area of cells or to the number of cells .", "Total RNA was isolated using the GeneJET purification kit ( Fermentas ) .", "cDNA was synthesized by reverse transcription from 2 μg RNA with oligodT 15 primers and SSII RT enzyme ( Invitrogen ) .", "Relative qPCR was measured on a 7500 Fast System ( Applied Biosystems ) as follows: 95°C for 2 min followed by 40 cycles at 95°C for 15 s followed by 60°C for 1 min . 200 nM forward and reverse primers , 16 μl SYBR Green Supermix ( Applied Biosystems ) and diluted cDNA were used in the final reaction mixture .", "GAPDH was used as reference gene and milliQ was used as negative control .", "Normalized expression was calculated following the equation NE = 2 ( Ct target-Ct reference ) .", "Primers used: GDE3 , forward TCAGCAGGACCACGAATGTA , reverse GCTGCAGCTTCCTCCAATAG; uPAR , forward AATGGCCGCCAGTGTTACAG , reverse CAGGAGACATCAATGTGGTTC; Cyclophilin , forward CATCTGCACTGCCAAGACTGA , reverse TTGCCAAACACCACATGCTT .", "For RT-PCR 25 ng cDNA was used in a RT-PCR reaction using GoTaq ( Promega ) .", "Primer sequences GAPDH forward 5’-CCATGTTCGTCATGGGTGT-3’ , GAPDH reverse 5’-CCAGGGGTGCTAAGCAGTT-3’ , GDE3 forward 1 5’-TGTTTGAGACTGATGTGATGGTC-3’ , GDE3 reverse 1 5’-TTCGGGTTGGGAATACAGAG-3’ For Western blotting , cells were washed with cold PBS , lysed in RIPA buffer supplemented with protease inhibitors and spun down .", "Protein concentration was measured using a BCA protein assay kit ( Pierce ) and LDS sample buffer ( NuPAGE , Invitrogen ) was added to the lysate or directly to the medium .", "Equal amounts were loaded on SDS-PAGE pre-cast gradient gels ( 4–12% Nu-Page Bis-Tris , Invitrogen ) followed by transfer to nitrocellulose membrane .", "Non-specific protein binding was blocked by 5% skimmed milk in TBST; primary antibodies were incubated overnight at 4°C in TBST with 2% skimmed milk .", "Secondary antibodies conjugated to horseradish peroxidase ( DAKO , Glostrup , Denmark ) were incubated for 1 hr at room temperature; proteins were detected using ECL Western blot reagent .", "HEK293 cells transiently transfected with GDE2 , GDE3 or empty plasmid , were plated on PEI-coated 6-well plates .", "After 24 hr complete medium was replaced with 1 ml serum free medium , 24 hr thereafter the conditioned medium was collected .", "2% pre-condensed Triton X-114 was added to ice-cold conditioned medium and phases were separated as previously described ( Doering et al . , 2001 ) .", "The top aqueous phase and the bottom detergent phase were analyzed by SDS-PAGE and Western blotting .", "Cells cultured on 24 mm , #1 , 5 coverslips were washed and fixed with 4% PFA , permeabilized with 0 . 1% Triton X-100 and blocked with 2% BSA for 1 hr .", "Incubation with primary antibodies was done for 1 hr followed by incubation with Alexa-conjugated antibodies or Phalloidin for 45 min at room temperature .", "For confocal microscopy , cells were washed with PBS , mounted with Immnuno-MountTM ( Thermo Scientific ) and visualized on a LEICA TCS-SP5 confocal microscopy ( 63 x objective ) .", "Super-resolution imaging was done using a SR-GSD Leica microscope equipped with an oxygen scavenging system , as previously described ( Matas-Rico et al . , 2016 ) .", "In short , 15000 frames were taken in TIRF mode , at 10 ms exposure time .", "After post image analysis , movies were analyzed and corrected using the ImageJ plugin Thunderstorm ( http://imagej . nih . gov/ij/ ) followed by correction with an ImageJ macro using the plugin Image Stabilizer .", "For Total Internal Reflection ( TIRF ) microscopy , HEK293 cells stably expressing UPAR-GFP and transiently transfected with GDE3-mCherry were imaged using a Leica AM TIRF MC microscope with a HCX PL APO 63x , 1 . 47 NA oil immersion lens .", "Excitation was at 488 and 561 nm and detection of fluorescence emission was by a GR filter cube ( Leica ) .", "Before each experiment , automatic laser alignment was carried out and TIRF penetration depth was set to 200 nm .", "Data were acquired at 500 ms frame rate .", "Basolateral uPAR patches were visualized using confocal microscopy and the area was quantified using an ImageJ macro that measured the uPAR patches based on fluorescence intensity .", "HEK293 cells stably expressing uPAR-GFP were left untreated , treated with PI-PLC or transiently transfected with GDE3-mCherry .", "MDA-MB-231 cells were stably transfected with GDE3-mCherry .", "Cells were trypsinized , blocked in 2%BSA and stained with rabbit anti-GFP primary antibody or mouse anti-uPAR ( 13F6 ) antibody followed by AlexaFluor-647 coupled anti-rabbit secondary antibody .", "Cells were analyzed using a BD LSR Fortessa flow cytometer .", "Equal groups ( n = 16 ) of eight-week-old female NMRI nude mice were injected subcutaneously into the mammary fat pads .", "Prior to injection , GDE3-expressing or control MDA-MB-231 cells ( 5 × 105 ) were suspended in an equal volume of cold phosphate buffered saline and diluted 1:1 with Matrigel .", "The tumor growth was monitored 3 times a week for 9 weeks by caliper measurements .", "The tumor volumes were calculated with the formula: 0 , 5 x length x width¬2 and statistical analysis of the tumor growth was done using a multiple t-test corrected for multiple comparison .", "A P value < 0 . 05 was considered statistically different .", "The mouse experiments were approved by the Animal Ethics Committee of the Netherlands Cancer Institute ( protocol number: 30100 2015 407 appendix 1 WP 6061 ) .", "For all single comparisons , a two-tailed unpaired Student’s t-test was used; for multiple comparisons , an ordinary ANOVA with Tukey’s test was used .", "A P value < 0 . 05 was considered statistically significant .", "Error bars shown in the bar diagrams were calculated as the standard error of the mean ( SEM ) ; whiskers in the box plots depict 95% confidence intervals ." ] ]

[ "The urokinase receptor ( uPAR ) is a glycosylphosphatidylinositol ( GPI ) -anchored protein that promotes tissue remodeling , tumor cell adhesion , migration and invasion .", "uPAR mediates degradation of the extracellular matrix through protease recruitment and enhances cell adhesion , migration and signaling through vitronectin binding and interactions with integrins .", "Full-length uPAR is released from the cell surface , but the mechanism and significance of uPAR shedding remain obscure .", "Here we identify transmembrane glycerophosphodiesterase GDE3 as a GPI-specific phospholipase C that cleaves and releases uPAR with consequent loss of function , whereas its homologue GDE2 fails to attack uPAR .", "GDE3 overexpression depletes uPAR from distinct basolateral membrane domains in breast cancer cells , resulting in a less transformed phenotype , it slows tumor growth in a xenograft model and correlates with prolonged survival in patients .", "Our results establish GDE3 as a negative regulator of the uPAR signaling network and , furthermore , highlight GPI-anchor hydrolysis as a cell-intrinsic mechanism to alter cell behavior ." ]

[ "Every process in the body , from how cells divide to how they move around , is tightly regulated .", "For example , cells only migrate when they receive the correct signals from their environment .", "These signals are recognised by receptor proteins that sit on the cell surface and connect the outside signal with the cell’s response .", "However , in cancer cells , these processes are out of control , which is why cancer cells can grow very quickly or spread to many different parts of the body .", "One important receptor protein is the urokinase receptor , which helps to reorganize the tissue , for example , when wounds heal , but also enables cancer cells to grow and spread .", "A special feature of urokinase receptor is the way it is connected to the cell surface , namely through a molecule that acts as an anchor , called the GPI anchor .", "The urokinase receptor and some other GPI-anchored proteins can be released from their anchor .", "However , until now it was not clear why and how the urokinase receptor is released from cells , or how losing the receptor affects the cell .", "Now , van Veen , Matas-Rico et al . studied breast cancer cells , and discovered that an enzyme called GDE3 cuts the urokinase receptor off its GPI anchor to release the receptor from the cells .", "However , when breast cancer cells shed the urokinase receptor , they also lost the receptor from the cell surface in specific areas .", "As a result , the receptor could not work anymore .", "When breast cancer cells were experimentally modified to produce high levels of GDE3 , the cancer cells became less mobile and aggressive .", "Van Veen , Matas-Rico et al . then implanted ‘normal’ breast cancer cells , and breast cancer cells with extra GDE3 into mice , and observed that the tumors of mice with additional GDE3 grew less quickly .", "Moreover , breast cancer patients with high levels of GDE3 tend to live longer than patients with low levels of GDE3 .", "These results suggest that the enzyme GDE3 can suppress tumor growth .", "These findings uncover a new way how cells can alter their behavior , namely by cleaving GPI anchors at the cell surface .", "Future experiments will need to address how GDE3 itself is controlled , and if it releases other GPI-anchored proteins from cells .", "Once we know how to increase GDE3 activity in tumor cells , the new knowledge could one day lead to therapies to help patients with cancer ." ]

[ "Introduction", "Results and discussion", "Materials and methods" ]

[ "ecology", "tools and resources", "genetics and genomics" ]

[ [ "Over the past two decades , our collective understanding of microbial diversity has been profoundly expanded by cultivation-independent molecular methods ( Pace , 1997; Whitman et al . , 1998; Rappé and Giovannoni , 2003; DeLong , 2009; Hanson et al . , 2012 ) .", "It is now widely recognized that interconnected microbial communities drive matter and energy transformations in natural and engineered ecosystems ( Falkowski et al . , 2008 ) , while also contributing to health and disease states in multicellular hosts ( Clemente et al . , 2012 ) .", "Concomitant with this changing worldview is a growing awareness that viruses modulate microbial interaction networks and long-term evolution with resulting feedbacks on ecosystem functions and services ( Suttle , 2007; Rodriguez-Valera et al . , 2009; Forterre and Prangishvili , 2013; Hurwitz et al . , 2013; Brum et al . , 2014; Brum and Sullivan , 2015 ) .", "However , our understanding of viral diversity and virus–host interactions remains a major bottleneck in the development of predictive ecosystem models and unifying eco-evolutionary theories .", "This is because the lack of a universal marker gene for viruses hinders environmental survey capabilities , while the number of isolate viral genomes in databases remains limited: for comparison , more than 25 , 000 bacterial and archaeal host genomes are available in NCBI RefSeq ( January 2015 ) , whereas only 1 , 531 of their viruses were entirely sequenced and most ( 86% ) of these derive from only 3 of 61 known host phyla ( Roux et al . , 2015a ) .", "Thus , although advances in high-throughput sequencing expand the bounds of viral sequence space , these data sets are dominated by uncharacterized sequences ( usually 60–95% ) , termed ‘viral dark matter’ ( Reyes et al . , 2012; Youle et al . , 2012; Mizuno et al . , 2013; Brum and Sullivan , 2015 ) .", "In the absence of closely related isolates , viral genes and genomes remain unlinked to hosts , which greatly limits ecological and evolutionary inferences .", "Alternatively , viral sequence space can be explored in a known host context by revealing putative viral sequences hidden in microbial genomes .", "Such signal was first analyzed through annotation of prophages—viral genomes integrated in microbial genomes .", "Numerous tools exist to automatically detect prophages ( Fouts , 2006; Lima-Mendez et al . , 2008a; Zhou et al . , 2011; Akhter et al . , 2012 ) , so prophage diversity and abundance are relatively well studied ( Casjens , 2003; Canchaya et al . , 2004 ) .", "Early estimations , when only a few hundred bacterial genomes were available , suggested that prophages are common ( 62% of bacterial genomes tested contained at least one ) , existing as intact and functional forms or in varying degrees of decay ( Casjens , 2003 ) .", "Given that tens of thousands more microbial genomes are now publicly available , it is expected that many new prophages and other viral sequences remain to be discovered .", "Further , other viral signals might be prevalent in modern microbial genomic data sets .", "First , certain types of prophage do not integrate into the host genome .", "These ‘extrachromosomal prophages’ ( also termed ‘plasmid prophage’ ) exist outside the microbial chromosome until induced to undergo lytic replication .", "These have been known to occur for decades ( e . g . , coliphage P1 , Sternberg and Austin , 1981 ) , though their abundance in nature is unknown .", "Second , some phages can enter a ‘chronic’ cycle , in which they replicate in the cell outside of the host chromosome , and produce virions that are extruded without killing their host ( Abedon , 2009; Rakonjac et al . , 2011 ) .", "Third , a phage ‘carrier state’ has been observed , in which a lytic phage is maintained and multiplied within a cultivated host population without measurable effect on cell growth ( Bastías et al . , 2010 ) .", "This phenomenon is thought to arise due to the presence of both resistant and sensitive cells that frequently transition between these two states .", "Sometimes also termed ‘partial resistance’ , such states that enable the coexistence of phage and host in culture have now been observed in different systems ( Vibrio , Escherichia coli , Salmonella , Flavobacterium ) , and are linked to slight decreases in growth rate or cell concentration but no host cell clearing as would be observed for ‘typical’ lytic viruses ( i . e . , plaque formation ) , thus could go unnoticed in a microbial cell culture ( Fischer et al . , 2004; Carey-Smith et al . , 2006; Middelboe et al . , 2009 ) .", "All three of these lesser studied types of infection would result in the assembly of viral sequences outside of the main host chromosome in a microbial genome sequencing project and could be a new type of viral signal in modern microbial genomic data sets due to deep sequencing and public release of draft ( i . e . , not completely assembled ) genomic sequences .", "Finally , single amplified genome ( SAG ) data sets , sourced from anonymously sorted , amplified , and sequenced cells , are especially valuable for accessing the vast majority of environmental microbes that remain uncultivated in the lab ( Rinke et al . , 2013; Kashtan et al . , 2014 ) .", "Single-cell amplified genomes can reveal viral sequences directly linked to uncultivated hosts ( Yoon et al . , 2011; Roux et al . , 2014; Labonté et al . , 2015 ) .", "When combined with metagenomic sequences , these data provide information on population dynamics , lineage-specific viral-induced mortality rates , relative ratios of prophages and current lytic infections , as well as putative links between viral infection and host metabolic state ( Roux et al . , 2014; Labonté et al . , 2015 ) .", "Thus , as microbial genomic data sets evolve from complete genomes to fragmented draft and single-cell genomes , new windows into viral diversity and virus–host interactions are opened .", "Here , we applied a recently developed and automated virus discovery pipeline , VirSorter ( Roux et al . , 2015a ) , to mine the viral signal from 14 , 977 publicly available bacterial and archaeal genomic data sets .", "This identified 12 , 498 high-confidence viral sequences with known hosts , ∼10-fold more than in the RefSeqVirus database , that we then used to expand our understanding of viral diversity and virus–host interactions ." ], [ "VirSorter is designed to predict bacterial and archaeal virus sequences in isolate or single-cell draft genomes , as well as complete genomes ( Roux et al . , 2015a ) .", "Briefly , VirSorter identifies viral sequences through", "( i ) statistical enrichment in viral gene content , using a reference database composed of viral genomes of archaeal and bacterial viruses from RefSeq ( hereafter named RefSeqABVir for ‘RefSeq Archaea and Bacteria Viruses’ ) and assembled from viral metagenomes ( database ‘Viromes’ in VirSorter ) , or", "( ii ) a combination of viral ‘hallmark’ gene ( s ) that code for virion-related functions such as major capsid proteins or terminases ( Koonin et al . , 2006; Roux et al . , 2014 ) , and at least one viral-like genomic feature: statistical depletion in genes with a hit in the PFAM database , statistical enrichment in uncharacterized genes , short genes , or strand bias ( i . e . , consecutive genes which tend to be coded on the same strand ) .", "Applied to 14 , 977 publicly available microbial genomes ( Figure 1—source data 1 ) , VirSorter identified 12 , 498 high-confidence viral sequences representing either long genome fragments ( >10 kb when linear ) or complete genomes ( contigs detected as circular ) .", "These viral sequences were found in 5492 of the microbial genomes ( ∼30% ) .", "Simply scanning the identified viruses for novel hosts extended the host range of common viral families to now include several recently described phyla like Caldiserica ( formerly known as candidate phylum OP5 ) , Marinimicrobia ( SAR406 also known as Marine Group A ) , or Omnitrophica ( OP3 ) , in addition to other understudied groups such as Poribacteria , Nitrospinae , Cloacimonetes ( WWE1 ) , and Chloroflexi-type SAR202 ( Figure 1 , Figure 1—source data 2 , Figure 1—source data 3 ) .", "Uncovering the first viruses infecting these major microbial groups is critical given that many candidate phyla are abundant in understudied ecosystems and play substantial roles in coupled biogeochemical cycling ( Wright et al . , 2012; Wrighton et al . , 2012; Castelle et al . , 2013; Kamke et al . , 2013; Rinke et al . , 2013; Allers et al . , 2013b; Emerson et al . , 2015 ) . 10 . 7554/eLife . 08490 . 003Figure 1 . Distribution of viral sequences from the VirSorter curated data set across the bacterial and archaeal phylogeny . For each bacteria or archaea phylum ( or phylum-level group ) , corresponding viruses in RefSeq ( gray ) and VirSorter curated data set ( red ) are indicated with circles proportional to the number of sequences available .", "Groups for which no viruses were available in RefSeq are highlighted in black . DOI: http://dx . doi . org/10 . 7554/eLife . 08490 . 00310 . 7554/eLife . 08490 . 004Figure 1—source data 1 . List of data sets mined for viral signal . Bacterial and archaeal genomes searched with VirSorter for viral sequences originated from NCBI Refseq and WGS , as well as the Microbial Dark Matter data set ( MDM , Rinke et al . , 2013 ) and the SUP05 SAGs data set ( Roux et al . , 2014 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 08490 . 00410 . 7554/eLife . 08490 . 005Figure 1—source data 2 . New virus–host associations detected in VirSorter sequences . The star ( * ) marks the questionable detection of an Inoviridae genome in a Caldiserica SAG , which could originate from another bacterium contaminating MDA reagents ( see ‘Materials and methods’ ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 08490 . 00510 . 7554/eLife . 08490 . 006Figure 1—source data 3 . Summary table of VirSorter data set sequences . All sequences currently identified as plasmids on NCBI and which did not display any viral gene in the automatic annotation from NCBI are gathered at the bottom of the table and highlighted in orange .", "‘Detection tag’ column indicates how the sequence was detected as viral by VirSorter: ‘hallmark’ for the presence of viral hallmark gene ( s ) , ‘refseq’ for an enrichment in bacterial and archalea virus genes , ‘noncaudo’ for an enrichment in non-Caudovirales genes , and ‘vdb’ for an enrichment in virome-like genes . DOI: http://dx . doi . org/10 . 7554/eLife . 08490 . 00610 . 7554/eLife . 08490 . 007Figure 1—figure supplement 1 . Viral diversity in the VirSorter data set . The best BLAST hits of predicted proteins along each sequence ( i . e . , within 75% of the best BLAST hit for this sequence ) were used in a Lowest Common Ancestor affiliation ( here displayed at the family level ) .", "‘Unclassified Caudovirales’ gathers viruses only affiliated to the Caudovirales level without confident affiliation to the Myo- , Sipho- , or Podoviridae .", "The number and percentage of sequences affiliated is indicated next to each family . DOI: http://dx . doi . org/10 . 7554/eLife . 08490 . 00710 . 7554/eLife . 08490 . 008Figure 1—figure supplement 2 . Genome map comparison ( A ) and recruitment plot ( B ) of Bacteroidia virus sequences from a putative new order . Replication-associated , Relaxase , and hypothetical proteins are depicted in blue , orange , and gray respectively .", "The recruitment plot includes two viromes from human feces samples from two different studies ( Human gut assembly , Minot et al . , 2012 , and Human feces , Kim et al . , 2011 ) .", "Identity percentage is based on a blastn between virome contigs and the reference genome . DOI: http://dx . doi . org/10 . 7554/eLife . 08490 . 008 BLAST-based family-level affiliations suggested that 90 . 45% of these 12 , 498 sequences correspond to Caudovirales , 6 . 82% to ssDNA viruses ( predominantly Inoviridae family ) , and 2 . 73% could not be confidently assigned ( Figure 1—figure supplement 1 ) .", "Among the unassigned group , 7 sequences lacked any hit to a viral reference genome .", "These 7 short ( 4 . 1 kb ) near-identical circular contigs from Bacteroides draft genomes were detected as viral based on sequence similarity with human gut viromes , but contained two genes associated with plasmid replication ( Figure 1—figure supplement 2A ) .", "This could suggest a plasmid origin , but the high and even coverage of these genomes across several CsCl-purified viromes from different studies ( Kim et al . , 2011; Minot et al . , 2012 ) suggests that they are derived from encapsidated particles typical of viruses ( Figure 1—figure supplement 2B ) .", "If confirmed , these sequences would represent the first complete genomes for an entirely new viral order .", "To better determine relationships between viral genomes and host range , we next built a network based on shared gene content to quantify genetic relatedness between the 12 , 498 sequences identified with VirSorter and the 1 , 240 taxonomically curated genomes available in RefSeqABVir ( Figure 2—figure supplement 1 and see ‘Materials and methods’ ) .", "Despite the absence of a universal marker gene , a long history of organizing viral sequence space through genome-to-genome comparison exists using either gene content ( Rohwer and Edwards , 2002; Lima-Mendez et al . , 2008b ) or nucleotide composition ( Sims and Jun , 2009; Labonté and Suttle , 2013 ) .", "We used MCL ( Markov Cluster Algorithm ) based on the number of shared genes between sequence pairs as it had been previously shown to accurately recapitulate taxonomic relationships in the Caudovirales , which dominated our data set ( Enright et al . , 2002; Lima-Mendez et al . , 2008b ) .", "Most ( 99 . 3% of 12 , 498 ) sequences affiliated to one of 614 virus clusters ( VCs ) , of which 535 contained at least one complete genome or large genomic fragment ( >30 kb ) , and approximately half ( 271 of 535 VCs ) included RefSeqABVir sequences ( Figure 2A , Figure 2—source data 1 ) .", "Those VCs with RefSeqABVir sequences provided the opportunity to evaluate whether a VC corresponded to any particular taxonomic level of ICTV classification .", "Of 43 RefSeq-curated viral genera , 27 have all their sequences in the same VC , 12 were spread across two VCs , and 4 were spread across >2 VCs—these latter genera included the Spouna-like viruses ( 3 VCs ) , N4-like viruses ( 4 VCs ) , Lambda-like viruses ( 9 VCs ) , and Inoviruses ( 11 VCs ) .", "Consistent with previous applications of this method , VCs identified in this analysis were thus approximately equivalent to a RefSeq-curated viral genus ( Lima-Mendez et al . , 2008b ) . 10 . 7554/eLife . 08490 . 009Figure 2 . Degree of novelty of viruses detected in VirSorter curated data set .", "( A ) Viral clusters ( VCs ) are considered as putative new genera when including at least one sequence larger than 30 kb , circular , or known to be a complete genome ( from RefSeq ) .", "These putative genera were considered as ‘new’ when the VC did not include any RefSeq sequence , and ‘known’ otherwise .", "( B ) The proportion of new VCs ( containing no RefSeqABVir ) , VCs with only one RefSeqABVir sequence , and VCs with more than one RefSeqABVir sequence is displayed for host classes associated with more than 10 virl sequences .", "Only ‘putative genera’ VCs were considered ( i . e . , clusters containing a RefSeqABVir genome , a circular sequence , or a sequence with more than 30 predicted genes ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 08490 . 00910 . 7554/eLife . 08490 . 010Figure 2—source data 1 . Summary table of virus clusters ( VCs ) .", "Cluster affiliation is based on the combination of BLAST-based taxonomic affiliation of its members .", "For VCs with more than 10 proteins , those composed only of VirSorter sequences are highlighted in green and those with only one sequence from RefSeqABVir are marked in blue .", "Cases where sequences affiliated to both ssDNA and dsDNA viruses are clustered together are highlighted in red .", "‘Detection tags’ lists the different detection tags for the cluster members , with ‘NCBI_RefSeq’ for complete genomes from the RefSeq database .", "These NCBI RefSeq sequences are counted as ‘complete’ in the ‘type of sequences’ column . DOI: http://dx . doi . org/10 . 7554/eLife . 08490 . 01010 . 7554/eLife . 08490 . 011Figure 2—figure supplement 1 . Structure of viral sequence space sampled in VirSorter data set . Network of virus clusters ( VCs ) based on gene content comparison between viral genome sequences from RefSeqABVir and VirSorter data set .", "VCs including only VirSorter sequences are highlighted with a black outline .", "The size of nodes is proportional to the number of sequences in the cluster and the color of the node corresponds to the BLAST-based affiliation ( at the family level ) of its members when consistent ( i . e . , agreement between >75% of the cluster members , otherwise clusters are indicated as ‘unaffiliated’ ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 08490 . 01110 . 7554/eLife . 08490 . 012Figure 2—figure supplement 2 . Benchmarks used to determine the best value for inflation and significance thresholds for virus clustering . For each pair of values ( inflation and significance threshold ) , the genome network was computed and its overall shape evaluated with ICCC ( intra-cluster clustering coefficient ) .", "The chosen values are highlighted in green in the table and with a star on the associated plot . DOI: http://dx . doi . org/10 . 7554/eLife . 08490 . 012 Given this level of taxonomic resolution and ignoring the 79 VCs that lacked large ( >30 kb ) genome sequences , we identified a total of 264 new candidate viral genera ( i . e . , 264 VCs with no sequences from RefSeqABVir , Figure 2—source data 1 ) .", "These 264 candidate genera were derived from both understudied and well-studied hosts ( e . g . , Gammaproteobacteria and Bacilli , Figure 2B ) and included 5 of the 30 highest-membership VCs ( Figure 2—source data 1 ) , which confirms that our knowledge of viral diversity is limited even in well-studied hosts and with prevalent viruses .", "Of the 12 , 498 sequences , 5 , 232 were prophages ( i . e . , a viral genome integrated into a microbial contig ) and 1 , 756 were either complete ( circularized ) or large ( >30 kb ) genome fragments assembled outside of the host chromosome ( i . e . , no microbial gene was detected on the contig , Figure 3A , Figure 1—source data 1 ) . 10 . 7554/eLife . 08490 . 013Figure 3 . Extrachromosomal prophages in VirSorter curated data set and improvement in virome affiliation .", "( A ) The distribution of VirSorter curated data set as ‘integrated’ ( i . e . , prophages integrated in the host chromosome ) , ‘extrachromosomal’ ( i . e . , >30 kb or circular sequences with no microbial genes ) , or ‘undetermined’ ( <30 kb linear with no microbial genes ) is indicated for each host class with at least five VirSorter curated data set sequences .", "The number of sequences associated with each host class in indicated above the histogram .", "( B ) Improvement in the proportion of affiliated genes from viromes with VirSorter data set .", "Predicted genes from the Pacific Ocean Viromes ( Hurwitz and Sullivan , 2013 ) , Tara Ocean Viromes ( Brum et al . , 2015 ) , and Human Gut Viromes ( Minot et al . , 2012 ) were compared to RefSeqVirus ( May 2015 ) and the VirSorter data set ( BLASTp , threshold of 50 on bit score and 0 . 001 on e-value ) .", "Predicted proteins affiliated to VirSorter ( in blue ) did not display any significant similarity to a RefSeq sequence . DOI: http://dx . doi . org/10 . 7554/eLife . 08490 . 01310 . 7554/eLife . 08490 . 014Figure 3—figure supplement 1 . Contig map of a putative new extrachromosomal prophage . Contig Spirochaetia_gi_359585655 represent a complete genome ( the contig was detected as circular ) from a new genus ( affiliated to a VC with no RefSeqABVir sequence ) .", "Functional affiliation of predicted genes is indicated on the map , with notably two genes ( ParA/ParB ) indicative of extrachromosomal prophages , as well as two genes ( in orange ) affiliated to the ACR_tran efflux pump family , of which some members are involved in antiobiotic resistance phenotypes .", "This contig belongs to the virus cluster VC_61 , composed of 35 new putative extrachromosomal prophages from different Spirochetes genomes . DOI: http://dx . doi . org/10 . 7554/eLife . 08490 . 014 To estimate how often a large ( >30 kb ) genome fragment could be an integrated prophage and not capture the microbial gene content , we simulated the process for 22 different prophage-containing bacterial genomes ‘sequenced’ ( in silico ) at coverage of 5 , 25 , 50 , 75 , and 100× ( see ‘Material and methods’ ) .", "These analyses suggested that only 2 . 3% of large ( >30 kb ) prophage-originating contigs lacked any identifiable microbial genes .", "Thus , these latter 1 , 756 sequences must largely be extrachromosomal sequences and so represent a unique data source for quantifying the prevalence of under-studied viral infection modes including chronic infections , lytic viruses , or extrachromosomal prophages .", "Although we identified no clear sequence-based marker for the first two infection types , we could conservatively estimate the fraction of extrachromosomal prophages by identifying plasmid partition genes ( ParA and ParB , Davis et al . , 1992; Saint Girons et al . , 2000; see Figure 3—figure supplement 1 for an example of a putative extrachromosomal prophage displaying ParA-ParB genes ) .", "These genes were significantly more abundant in the 1 , 756 circular and large genome fragments than in the rest of the data set ( 13% vs 1% , respectively; poisson test p-value < 10−05 ) .", "Thus , at least 13% of these sequences appear to be bona fide extrachromosomal prophages , whereas the others might be lytic viruses in ‘carrier’ states , chronic infections , or extrachromosomal prophages without detectable ParA/ParB genes .", "Beyond this glimpse into under-studied viral infection modes , these new reference genomes are likely to help improve taxonomic affiliation for the ‘viral dark matter’ in viromes .", "To quantify this , we added these sequences to the RefSeqABVir database and assigned taxonomy to predicted genes in three large-scale virome data sets available .", "We found that the VirSorter curated data set improved affiliation by 32 and 40% , respectively , in the marine Pacific Ocean Viromes ( POV ) ( Hurwitz and Sullivan , 2013 ) and Tara Oceans Viromes ( TOV ) ( Brum et al . , 2015 ) data sets , and more than doubled the number of affiliated genes in human gut viromes ( Minot et al . , 2012 , Figure 3B ) .", "This particularly strong improvement in the human gut virome affiliation is presumably due to enterobacteria being abundant among current publicly available microbial genomes .", "Finally , both the detection of non-integrated viral genomes and the improved virome affiliation suggest that the VirSorter curated data set includes not only integrated prophage data , but also viruses actively infecting these microbes ( i . e . , not integrated in the host chromosome and producing virions ) with under-studied infection modes .", "Examination of the VCs network beyond classification revealed additional higher order patterns .", "First , bacterial and archaeal viruses clustered separately in >99% of VCs; the exception ( VC_89 ) included a single and unique ( Garrett et al . , 2010 ) archaeal virus ( Hyperthermophilic Archaeal Virus 2 , NC_014321 ) that clustered with 21 bacterial viruses , presumably due to poor archaeal virus representation .", "Second , >95% of these VCs contained exclusively one nucleic acid type ( e . g . , DNA or RNA , and dsDNA or ssDNA , Figure 2—figure supplement 1 ) , although RNA viral representation is low because only RefSeq-curated families Cystoviridae and Leviviridae were available ( no RNA viruses were detected with VirSorter , presumably because DNA-based data sets were analyzed ) .", "The 15 VCs including both ssDNA and dsDNA viral genomes are either associated with archaeal viruses for which composite ssDNA/dsDNA genomes were already described ( 2 VCs; Sencilo et al . , 2012 ) or more surprisingly with ssDNA Inoviridae , which clustered with Caudovirales in 13 VCs ( Figure 2—source data 1 ) .", "For 9 of these 13 Inoviridae–Caudovirales VCs , some of the sequences were wrongly affiliated due to genes shared by both viral families such as integrases , exonucleases , and replication-associated proteins .", "Two other VCs corresponded to prophage sequences that include genes similar to Inoviridae and Caudovirales and could actually be two different viruses integrated at the same genome location .", "However , the 2 remaining mixed VCs ( VC_128 and VC_215 ) include sequences displaying a mix of Caudovirales and Inoviridae genes ( VC_215 sequences also included Corticoviridae genes ) .", "We posit that these might represent new composite genomes beyond the ones already described for archaea viruses ( Sencilo et al . , 2012 ) and the recently discovered RNA–DNA chimeric viruses ( Diemer and Stedman , 2012; Roux et al . , 2013; Krupovic et al . , 2015 ) .", "We next evaluated the scale and range of viral co-infection , a phenomenon critical to viral genome evolution and thought to blur this vertical gene inheritance signal used to classify genomes into VCs .", "Indeed , the fact that super-infection of prophage-containing bacteria would provide genomic proximity for gene acquisition via illegitimate recombination was posited more than a decade ago ( Mosig , 1998 ) .", "However , viral co-infection rates remain unconstrained with the only data for natural systems derived from a single large-scale single-cell genomic data set where ∼35% of infected cells contained multiple viruses ( Roux et al . , 2014 ) .", "Here , in the 5492 microbial genomes with detectable viral signal , nearly half ( 2445 ) contained more than one detectable virus ( Figure 4 ) .", "Most ( ∼82% ) of these co-infections involved multiple Caudovirales , as previously observed ( Casjens , 2003 ) , and likely provides mechanism for viral gene exchange and may be more common in some phages displaying rampant mosaicism ( e . g . , the Siphoviridae , Hendrix et al . , 1999 ) than others .", "The second most commonly observed co-infections ( 9% ) occurred between ssDNA Inoviridae and dsDNA Caudovirales ( Figure 4 ) .", "These genomes represented the mixed VCs from the network analyses and putative new composite genomes described above .", "Mechanistically , Inoviridae might be more prone to such co-infection due to their long infection cycle whereby they extrude their filamentous virions without killing their host ( Rakonjac et al . , 2011 ) , with a dsDNA replication stage ( Salim et al . , 2008 ) that could increase genomic exchanges with co-infecting dsDNA viruses . 10 . 7554/eLife . 08490 . 015Figure 4 . Scale and range of co-infection .", "( A ) Number of different viral sequences detected by host genome .", "Numbers are based on the set of microbial genomes with at least one viral sequence detected ( 5492 genomes ) .", "( B ) Affiliation of viruses involved in multiple infections of the same host .", "Affiliations are deduced from best BLAST hits alongside the viral sequences , as in Figure 1 .", "Co-infections involving dsDNA and ssDNA viruses are highlighted in bold . DOI: http://dx . doi . org/10 . 7554/eLife . 08490 . 015 Together , these findings suggest that genome-based network analyses could be used to identify novel viruses , as well as to infer host domain ( archaeal or bacterial , >99% accuracy ) and nucleic acid type ( ssDNA or dsDNA , >95% accuracy ) .", "Evolutionarily , we posit that while co-infection by multiple viruses appears common , the consistency of so many VCs with ICTV taxonomy suggests that most phage genomes harbor a largely vertically inherited core gene set as detected for marine T4-like populations ( Ignacio-Espinoza and Sullivan , 2012; Marston et al . , 2012; Deng et al . , 2014 ) rather than the rampant mosaicism paradigm largely derived from Siphoviridae genomes ( Hendrix et al . , 1999 ) .", "While data remain limited to a subset of the known microbial phyla , it might be that viral infection modes influence the tempo of their genome evolution .", "Specifically , we posit that horizontal gene transfer is more prevalent in phages that occupy host cells longer due to lysogenic or chronic infection stages and/or infect densely packed hosts ( e . g . , biofilms or clumped life stages ) as these parameters would increase the probability of co-infection .", "Perhaps then , at least for more highly lytic viral groups , genome-based clustering approaches can now be leveraged for their taxonomic predictive value as suggested over a decade ago ( Rohwer and Edwards , 2002 ) .", "Beyond charting diversity and taxonomic affiliation of viral sequence space , the VirSorter data set provided a unique opportunity to explore virus–host interactions .", "Beyond the above-noted expansion of viruses to novel hosts , we next examined these patterns on a global scale by constructing a virus–host interaction network based on database-available taxa .", "When considering viral diversity at the genus level , the network displays a modular topology ( Figure 5 and Figure 5—figure supplement 1 ) .", "Such modularity in virus–host interaction networks suggests that hosts are specifically associated with particular viruses ( Weitz et al . , 2012 ) , probably reflecting long-term coevolution between microbial hosts and their viruses .", "Such modular structure was expected , but not observed in previous virus–host interaction network studies , likely due to the short phylogenetic distances between hosts evaluated in available data sets ( Flores et al . , 2011 ) .", "The modular network presented here derives from a data set spanning 18 phyla across bacterial and archaeal domains .", "These results confirmed the prediction that ‘at macroevolutionary scales , host–phage interaction matrices should be typified by a modular structure’ ( Flores et al . , 2011 ) , as also had been observed across 215 phage types against 286 host types of unknown diversity ( Flores et al . , 2013 ) . 10 . 7554/eLife . 08490 . 016Figure 5 . Virus–host network between virus clusters and host classes ( matrix visualization ) .", "A cell in the matrix is colored when at least one virus from a virus cluster ( VC , rows ) was retrieved in a genome from a host class ( columns ) .", "This virus–host network is detected as significantly modular by lp-Brim ( modularity Q = 0 . 45; the same index computed from 99 randomly permuted matrices ranged from 0 . 02 to 0 . 17 , with an average of 0 . 08 ) .", "The different modules are highlighted in color , with inter-module links in gray .", "Virus clusters are identified by their number and their family-level affiliation ( based on BLAST-based affiliation of the cluster members ) is indicated next to each cluster when available ( virus clusters with inconsistent members affiliation are considered as ‘unclassified’ , affiliations are spread along the x-axis for spacing purpose ) .", "Host phylum and class are indicated for each host column , with domains indicated above the corresponding hosts . DOI: http://dx . doi . org/10 . 7554/eLife . 08490 . 01610 . 7554/eLife . 08490 . 017Figure 5—figure supplement 1 . Virus–host network between virus clusters and host classes ( network visualization ) .", "An edge is displayed between a virus cluster ( VC ) and a host class when at least one virus from this cluster was retrieved in a genome from the host class .", "This network is detected as significantly modular by lp-Brim ( modularity Q = 0 . 45; the same index computed from 99 randomly permuted matrices ranged from 0 . 02 to 0 . 17 , with an average of 0 . 08 ) .", "The different modules are highlighted in color , with inter-module links in gray .", "VCs are identified by their number and their family-level affiliation ( based on BLAST-based affiliation of the cluster members ) is indicated below each cluster when available ( VCs with inconsistent members affiliation are considered as ‘unclassified’ ) .", "Host phylum and class are indicated for each host node , with phyla ( when multiple class from the same phylum are included in the network ) and domains indicated above the corresponding host nodes . DOI: http://dx . doi . org/10 . 7554/eLife . 08490 . 017 Finally , given the number of virus–host linkages revealed by VirSorter , we evaluated the adaptation of viral genome composition within the host milieu—an idea practiced in the literature with limited genomic information ( Pride et al . , 2006; Carbone , 2008; Cardinale and Duffy , 2011 ) .", "To this end , we computed the distance between viral and microbial genomes in terms of mono- , di- , tri- , tetra-nucleotide frequency and codon usage , and compared the distances between the virus and its host vs non-hosts in the data set .", "Every metric tested displayed a smaller distance between viruses and their hosts than with non-host genomes , with tetranucleotide frequency ( TNF ) maximizing the host to non-host distances ( Figure 6 ) . 10 . 7554/eLife . 08490 . 018Figure 6 . Adaptation of viral genome composition and codon usage to the host genome . K–S distances between distributions of virus–host distances and virus–non-host distances for each metrics ( in color ) and different subsets of the viral sequences ( all sequences , by type , and by taxonomy ) .", "Only families with more than 5 genomes are displayed ( although it should be noted that the VirSorter data set includes only 6 Microviridae sequences ) .", "The number of sequences in each category is indicated in brackets .", "Distributions used to compute distances are displayed in Figure 6—figure supplement 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 08490 . 01810 . 7554/eLife . 08490 . 019Figure 6—figure supplement 1 . ( A ) K–S distances between distributions of virus–host distances and virus–non-host distances for each metrics ( in color ) and different subsets of the viral sequences ( based on the number of tRNA genes detected ) .", "The number of sequences in each category is indicated below the number of tRNA .", "( B ) Distribution of k-mer distances between viral and cellular genomes and codon usage adaptation index for host , host genus , host family , and non-host ( different order ) genomes .", "For each viral genome , the distance to the host is displayed , as well as 10 randomly taken distances to genomes from each category and different subsets of the viral sequences ( by taxonomy on the left column , and by number of tRNA genes on the rigth column ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 08490 . 01910 . 7554/eLife . 08490 . 020Figure 6—figure supplement 2 . Distance between k-mer frequency vectors of virus genome subsamples and host genomes for Caudovirales . Viral genomes ( 1000 ) were randomly sub-sampled at different sizes ( from 2000 to 20 , 000 bp ) .", "Only Caudovirales genomes were selected for this subsample analysis .", "For each size of k-mer , the result of a linear regression of distance between host or non-host and viral subsample size is indicated .", "The same distances for the Microviridae and Inoviridae ( taken from Figure 6A ) are indicated for comparison , and associated with the size of the reference genome of each group ( Enterobacteria phage phiX174 and Enterobacteria phage M13 ) .", "For clarity's sake , the almost-identical values for 2-mer , 3-mer , and 4-mer for Microviridae are slightly horizontally shifted . DOI: http://dx . doi . org/10 . 7554/eLife . 08490 . 020 Among dsDNA viruses , host-correlated genome composition patterns were robust across integrated prophages and extrachromosomal genomes ( i . e . , viral sequences assembled outside of the main host chromosome , Figure 6A , Figure6—figure supplement 1 ) .", "Our expectations were that prophages would be largely optimized towards the genome of their host , but that genome composition of the extrachromosomal category would be less correlated .", "Particularly , as cyanophage host range breadth scales with the number of tRNA genes encoded by the virus ( Enav et al . , 2012 ) , we expected that genome composition of viral genomes with many tRNA genes might have poor correlation to that of their host genomes , assuming that the viral-encoded tRNA genes could compensate for codon mismatches across hosts .", "However , these latter expectations were not met as viral and host genome composition correlations were strong regardless of the number of viral-encoded tRNA genes ( Figure 6—figure supplement 1 ) , which suggests that host-optimized viral genome composition may be beneficial even when the virus encodes its own tRNA genes .", "Among ssDNA viruses , nucleotide composition of viral genomes was also correlated to host genomes , but less so than for dsDNA viruses ( Figure 6A ) .", "This contrasts with a previous analysis of 500 phage genomes that did not detect any difference between dsDNA and ssDNA genomes adaptations to their host's genome ( Cardinale and Duffy , 2011 ) .", "One ssDNA viral group , the Microviridae , had a reduced signal for genome composition metrics except for codon usage where its signal was comparable to that of the dsDNA viruses ( Figure 6A ) .", "Although this could indicate a bias linked to the small genome size of these viruses ( around 5 kb ) , dsDNA viruses' genomes subsampled to similar sizes displayed a minimal signal loss ( Figure 6—figure supplement 2 ) , which suggests other mechanisms may be driving this lower genome composition adaptation in Microviridae .", "Another ssDNA group , the Inoviridae had reduced genome composition and codon usage adaptation signals .", "Again , because Inoviridae release virions without killing their hosts , it is possible that the virus is exposed to host resources over a much longer time interval , lowering the selection pressure toward transcription and translation speed and efficiency , which is the main mechanism thought to drive genome composition and codon usage adaptation of viral genomes ( Cardinale and Duffy , 2011 ) .", "Pragmatically , to assess whether this signal could be used to predict the host of a new virus , we calculated the distance based on TNF vectors between each VirSorter curated data set sequence and the 14 , 977 microbial genomes .", "The taxonomy of the microbial genome with the lowest distance to the viral sequence ( i . e . , the predicted host ) was then compared to the taxonomy of the actual host ( i . e . , the genomic data set in which the viral sequence was identified ) .", "When the host database included all host genomes , this host prediction was 99% accurate at both the family and genus level for virus–host TNF distances lower than 4 . 10−04 , 88%/51% ( family/genus level ) for TNF distances ranging between 4 . 10−04 and 1 . 10−03 , and 70%/37% for distances greater than 10−03 ( Table 1 ) .", "When genomes from the actual host species are excluded , the accuracy of host prediction drops slightly ( 95% , 83%/30% , 67%/30% for the same distance ranges ) , and even more when all genomes from the host genus are excluded ( 70% and 37% at the family level , no correct genus could be predicted in that case , and only one distance lower than 4 . 10−04 was observed , Table 1 ) .", "Hence , TNF comparison provides a promising in silico approach to link new viral genomes to hosts at different levels of accuracy within the taxonomic hierarchy when the suitable host reference genome is available . 10 . 7554/eLife . 08490 . 021Table 1 . Accuracy of host prediction based on distance ( d ) between tetranucleotide frequencies of viral and microbial genomesDOI: http://dx . doi . org/10 . 7554/eLife . 08490 . 021PredictedHost orderHost familyHost genusCorrectRatio ( % ) CorrectRatio ( % ) CorrectRatio ( % ) All reference sequences d < 4 × 10−04989798 . 989798 . 989798 . 98 4 × 10−04 ≤ d < 1 × 10−0310 , 173936192 . 02897188 . 18526151 . 72 1 × 10−03 ≤ d2508187274 . 64175770 . 0691736 . 56Host species excluded d < 4 × 10−04212095 . 242095 . 242095 . 24 4 × 10−04 ≤ d < 1 × 10−0310 , 003906790 . 64837283 . 69299229 . 91 1 × 10−03 ≤ d2755198171 . 91184066 . 7981829 . 69Host genus excluded d < 4 × 10−04100 . 0000 . 0000 . 00 4 × 10−04 ≤ d < 1 × 10−039085730380 . 39618168 . 0400 . 00 1 × 10−03 ≤ d3693176847 . 87138837 . 5800 . 00For each viral genome , the order , family , and genus of its host were predicted from the taxonomy of the closest microbial genome ( based on the mean absolute difference between tetranucleotide frequency vectors ) and compared to the order , family , and genus of the actual host ( i . e . , the taxonomy of the genome with which the virus was identified ) .", "These predictions were computed with", "( i ) all microbial genomes ,", "( ii ) excluding specifically all genomes from the host species , and", "( iii ) excluding all genomes from the host genus .", "Cases with over 75% of prediction accuracy are highlighted in gray .", "As evidenced by the improvement in virome taxonomic affiliation ( Figure 3B ) , VirSorter curated data set should represent a useful reference data set for future virome studies .", "This data set also likely harbor novel biology beyond the global patterns of viral diversity and virus–host interactions presented in this manuscript , to be revealed through analyses targeted toward specific viral or host subgroups .", "To facilitate these follow-up studies , VirSorter curated data set is made available through two complementary websites: MetaVir and iVirus .", "MetaVir ( project ‘VirSorter’ , data set ‘VirSorter curated data set’ ) provides an automatic annotation of each sequence , with multiple visualization tools to explore and compare genome maps , as well as multiple ways of searching the data ( by host , by phage affiliation , by taxonomic or functional affiliation of predicted genes , etc ) and extract a specific subset of interest ( these tools are under the tab ‘Contig maps’ ) .", "Nucleotide sequences from the VirSorter curated data set are also hosted at iVirus , alongside the viral clusters annotation and network ( as cytoscape-ready text files ) , the virus–host matrix , and the complete list of viral sequence predictions in the 14 , 977 archaeal and bacterial genomic data sets including the category 3 predictions that are not in VirSorter curated data set ( http://mirrors . iplantcollaborative . org/browse/iplant/home/shared/ivirus/VirSorter_curated_dataset ) .", "Finally , a summary of the sequences and clusters is provided as Figure 1—source data 1 and Figure 2—source data 1 , and a Data Dryad package including all annotated genbank files from the VirSorter curated data set is available ( http://dx . doi . org/10 . 5061/dryad . b8226; Roux et al . , 2015b ) .", "While recent advances in high-throughput sequencing and viral metagenomics continue to expand the bounds of viral sequence space ( e . g . , Reyes et al . , 2012; Mizuno et al . , 2013; Brum and Sullivan , 2015 ) , such viruses are typically unlinked to cognate hosts , severely limiting ecological and evolutionary inferences .", "Concurrently , emerging methods provide new virus–host linkage capabilities , but do not scale well with increasing data set size and complexity ( e . g . , Andersson and Banfield , 2008; Tadmor et al . , 2011; Allers et al . , 2013a; Deng et al . , 2014 ) .", "Here , the mining of publicly available microbial genomic data proved to be a useful complement to these approaches as it enables the exploration of host-linked viral diversity .", "The resulting viral sequences hidden in microbial genomes represent a powerful data set , increasing the number of known , host-linked viruses by an order of magnitude , with analyses of these data elucidating viral dark matter in ocean and human gut viromes , as well as augmenting our understanding of viral taxonomy , viral genome evolution , and virus–host interactions on multiple fronts .", "While this current VirSorter data set remains limited by the cultivation bias inherent in the publicly available complete and draft microbial genomes , such bias will progressively be eliminated as SAGs are used to better map microbial dark matter ( e . g . , Rinke et al . , 2013 ) .", "Such a drastically improved map of the virosphere , together with advances in experimental approaches and theory ( Brum and Sullivan , 2015 ) , will help reveal the eco-evolutionary forces shaping virus–host interactions across diverse ecosystems and eventually shift our inference capability from observation to prediction ." ], [ "A total of 14 , 977 bacterial and archaeal genomes ( complete and draft ) included in RefSeq and WGS databases ( Pruitt et al . , 2009 ) were downloaded from the NCBI ftp website in March 2014 ( RefSeq release 64 ) .", "The 264 new candidate phyla ( ‘Microbial Dark Matter’ ) SAGs' ( Rinke et al . , 2013 ) raw reads were downloaded from the JGI portal page and assembled with SPAdes Genome Assembler ( Bankevich et al . , 2012 ) ( default parameters ) .", "Finally , 127 SUP05 SAGs that we previously analyzed manually were added to the cellular genome pool ( Roux et al . , 2014 ) .", "This data set included 4240 complete genomes and 10 , 547 draft genomes ( as there is no clear annotation of a genome as ‘draft’ or ‘complete’ at the NCBI , we identified as ‘draft’ genomes all genome projects including more than 5 different sequences , to avoid considering genomes split into different chromosomes or including one or several plasmids as ‘draft’ ) .", "Genomes were processed with VirSorter ( Roux et al . , 2015a ) separately for each class ( except for Cyanobacteria , SUP05 SAGs , and the Microbial Dark Matter data set that were all processed together ) , first using the RefSeqABVir database , and then using the Viromes database , yielding 89 , 301 total predicted viral sequences .", "Among these , 938 correspond to Enterobacteria phage PhiX174 , which is used for quality control during Illumina sequencing , and were thus discarded .", "We focused on a subset of the putative viral sequences extracted from RefSeq , WGS and the Microbial Dark Matter and SUP05 SAGs ( 89 , 301 sequences ) , and targeted the active prophages and lytic virus signatures .", "To this end , we discarded all predictions lacking a viral hallmark gene or a viral gene enrichment ( i . e . , category 3 predictions , Roux et al . , 2015a ) , and all prophage detections displaying viral gene enrichment only and lacking viral hallmark genes , as these are likely defective prophages for which boundaries are difficult to predict in silico and that often include bacterial genes .", "We next removed all linear sequences shorter than 10 kb except for sequences detected with the non-Caudovirales score where a threshold of 5 kb was used , as these viruses can frequently have genomes smaller than 10 kb .", "We also discarded all circular contigs ( which should represent complete genomes ) smaller than 3 kb as these are likely short repeat regions ( the smallest known genome for a bacteria or archaea virus is ∼5 kb ) .", "The resulting 13 , 391 sequences were then manually curated to remove false positives .", "These false positives corresponded to defective prophages ( wherein most are expected to be smaller than 10 kb ) , plasmid-like sequences , GTA gene clusters , and low complexity regions .", "In addition , this manual curation step allowed us to adjust the boundaries of some prophage predictions and/or modify the prophage vs complete viral contig automatic prediction .", "Consequently , 892 sequences were discarded ( false-positive rate of 6 . 7% ) , leaving 12 , 498 curated sequences .", "Among these , 7266 sequences were entirely viral ( thus potentially represent lytic , chronic , or extrachromosomal lysogenic infections assembled in draft genomes ) , and 5232 were prophages ( viral-like regions detected within a cellular genome fragment ) .", "Among the sequences detected as entirely viral , 6 were tagged in the NCBI database as bacteriophages , and 108 as plasmids .", "Viruses and plasmids can be difficult to distinguish , as gene exchange is known to occur between these two types of mobile genetic elements ( Leplae et al . , 2010 ) .", "Here , 84 out of these 108 ‘plasmid’ sequences displayed conclusive evidence of a viral origin as they contained viral hallmark genes ( coding for terminase large subunits or major capsid proteins ) and originated from draft unpublished genomes , hence likely to have been named ‘plasmid’ because they formed extrachromosomal circular assembly ( see e . g . , sequence gi:383080718 available at RefSeq ) .", "The 24 others were more ambiguous ( highlighted in orange in Figure 1—source data 1 ) since the automatic annotation from NCBI did not display any viral-like gene , yet these sequences all displayed statistical viral-like gene enrichment , and as such were maintained in the VirSorter curated data set .", "Finally , one additional ambiguous sequence , considered as entirely viral by VirSorter , was detected in the Caldiserica SAG ( Caldiserica_bacterium_sp_JGI_0000059-M03_ID_3757 ) .", "Even though this sequence looks indeed like a complete Inoviridae genome , it displayed a high level of similarity ( 99% identity ) to the complete genome of Delftia acidovorans SPH-1 ( gi:160361034 , from coordinates 2300885 to 2307389 ) .", "Such high similarity with another virus is suspicious , as well as the fact that the matching genome is Delftia , a bacterium known to contaminate some MDA reagents .", "This sequence was maintained in the VirSorter data set as there is no definite proof of the contamination , but the existence of a Caldiserica-infecting Inoviridae should be considered as uncertain until further evidence is available ( and is displayed as such in Figure 2—source data 2 ) .", "The pool of 450 , 047 proteins predicted from the 12 , 498 viral sequences was clustered with all proteins from RefSeq and the viral metagenomes ( i . e . , sequences from the Viromes database ) with MCL based on reciprocal best BLAST hit ( threshold of 50 on score and 0 . 001 on e-value , Enright et al . , 2002 ) .", "Most of these sequences ( 423 , 618 ) could be included in 22 , 460 protein clusters ( PCs ) .", "About a third ( 7742 ) of these PCs also contained sequences from the RefSeqABVir database , and the remainder formed new PCs .", "This protein clustering was then used to cluster genomes as in Lima-Mendez et al . ( Enright et al . , 2002; Lima-Mendez et al . , 2008b ) .", "Briefly , the number of shared PCs between each pair of sequences ( either RefSeq or VirSorter ) is computed , and a significance value is deduced by comparing it to an expected number of shared PCs ( modeled with a hypergeometric formula taking into account the number of genes of both sequences ) .", "We used ICCC ( intracluster clustering coefficient , which estimates cluster homogeneity by measuring around each node how many of its neighbors are part of the same cluster ) to determine the best inflation value ( from 1 . 5 to 5 by 0 . 25 increments ) and significance threshold ( i . e . , which minimum significance was required to draw an edge between two sequences , from 1 to 50 ) .", "As expected , the number of VCs formed increased with inflation and with significance .", "ICCC was clearly higher with the lowest threshold in significance ( sig ≥ 1 ) , regardless of the inflation value used .", "For the lowest significance threshold , ICCC increased with inflation , usually with a first small peak around 2 and plateau around", "4 . These different values of inflation did not have a major impact on the clustering though , as 95–99% of pairs of sequences were clustered similarly using inflations values of 2 . 75 , 3 , 3 . 25 , 3 . 5 , 3 . 75 , 4 , 4 . 25 , 4 . 5 , 4 . 75 , or", "5 . We eventually settled for the combination yielding the highest ICCC: a significance threshold of 1 and inflation of 4 ( Figure 2—figure supplement 2 ) .", "Taxonomic affiliation of sequences was based on hits to the RefSeqABVir database .", "Each profile in the database was first affiliated based on the origin of its members , with a 75% majority rule: at each taxonomic level , a profile is affiliated to a taxon if more than 75% of the profile sequences are affiliated to this taxon .", "Then , for each of the 12 , 498 viral sequences identified by VirSorter , a set of relevant hits was selected:", "( i ) first the profile with the best hit across all genes along the sequence , and", "( ii ) the best hit from other genes with a score close to this ‘absolute’ best hit in the sequence ( >75% of the score of the first best hit ) .", "The sequence was then affiliated to the Lowest Common Ancestor ( LCA ) of this set of relevant hits .", "Hence , a predicted protein will only be affiliated if pointing toward sequences or profiles typical of a viral group , and a sequence detected by VirSorter will only be affiliated if its best hits are consistent .", "Functional affiliation for each PC was based on the comparison of its members ( predicted proteins ) with PFAM ( v . 27 , threshold of 50 on score ) .", "VCs were affiliated based on its members affiliations if >75% were consistent .", "For the detection of new genera in the VCs , we chose to ignoring the 79 VCs that lacked large ( >30 kb ) genome sequences .", "This 30 kb threshold is conservative as it avoids considering short genome fragments as new genera but would also overlook small non-circular viral genomes ( such as some Tectiviridae ) .", "However , because the latter comprise a minority ( ∼0 . 1% of 12 , 498 sequences ) of the VirSorter data set ( Figure 2 ) , we chose to retain the larger , more conservative threshold .", "The 7 short circular sequences from Bacteroidia only detected with the Viromes database ( gi 319430465 , 298484481 , 329959038 , 423221334 , 423242675 , 423298785 , 345651594 ) were targeted for further examination .", "Hits to PFAM domains could be found on two proteins: a relaxase ( PF03432 . 9 , score ∼170 ) , and one replication initiator protein ( PF01051 . 16 , score ∼80 ) .", "Genome organization was compared with Easyfig ( Sullivan et al . , 2011 ) after aligning all genomes to the same starting point ( one base before the start of the Rep-domain protein ) .", "Recruitment plots of virome contigs ( extracted from Kim et al . , 2011; Minot et al . , 2012 ) were generated with ggplot2 and based on blastn comparison .", "The virus–host network was assessed considering only VCs with more than 10 sequences .", "Hosts were grouped at the class level .", "The modularity Q value of the virus–host matrix was computed with the lp-BRIM module in R software ( https://github . com/tpoisot/lp-brim ) .", "The virus–host matrix had a modularity of 0 . 45 .", "The same index computed from 99 randomly permuted matrices ranged from 0 . 02 to 0 . 17 , with an average of 0 . 07 .", "Co-infection was defined as the detection of several distinct viruses in one genome project ( one complete genome or one SAG ) .", "However , different viral contigs in a single draft genome could also originate from a single viral genome mis-assembled in several different contigs .", "This will be especially true for Caudovirales that are the most detected viruses as well as the ones with the largest genomes .", "To limit the over-estimation of co-infection due to mis-assembled Caudovirales genomes , co-infection was only considered in the cases where multiple copies of the large subunit of the terminase were detected , because this gene is present in single copy in Caudovirales genomes , and usually detected even in new viruses ( due to a high level of sequence conservation ) .", "Relative frequencies of k-mers ( mono- , di- , tri- , and tetra-nucleotide ) were computed with Jellyfish ( Marçais and Kingsford , 2011 ) for every VirSorter sequence and every bacterial and archaeal genome initially mined .", "Mean absolute error ( i . e . average of absolute differences ) between k-mer frequency vectors were then computed with an in-house perl script for each pair of VirSorter sequence and cellular genome , and used as a distance metric between viruses and putative hosts .", "For each VirSorter sequence , a set of distances that included its host ( i . e . , the genome with which the sequence was initially associated ) alongside 10 randomly selected sequences from the same genus , the same family , and a different order than the host were factored into in the distance distribution ( Figure 6—figure supplement 1 ) .", "Codon usage adaptation was evaluated with cusp and cai from the European Molecular Biology Open Software Suite ( EMBOSS , Rice and Longden , 2000 ) .", "First , codon usage bias of each bacterial and archaeal genome was computed .", "Then , the codon usage adaptation index ( cai ) was calculated for each gene between VirSorter sequences and cellular genomes .", "The global distribution displays the average ( across genes ) adaptation index for each VirSorter sequence and ( as for the k-mer distances ) a subset of cellular genomes including its host and 10 randomly selected sequences from respectively the same genus , the same family , and a different order than the actual host .", "Function-specific codon usage bias was based on the gene-by-gene adaptation between each VirSorter sequence and its host .", "For each category studied , the distance between distribution of distances to host genome ( in red on Figure 5—figure supplement 1 ) and distribution of distances to non-host genomes ( in purple on Figure 5—figure supplement 1 ) was evaluated with a Kolmogorov–Smirnov ( K–S ) statistic .", "The codon usage adaptation indexes for the different functional categories were compared to the ‘other functions’ values with a Wilcoxon signed-rank test to detect categories with statistically different averages .", "Both statistics were computed with R software .", "To evaluate the effect of small genome size on distance between k-mer frequencies , a sub-sample of 1000 Caudovirales was randomly taken at different sizes ( from 2000 to 20 , 000 bp ) , and the same procedure as for complete sequences was used to determine the distance between host and non-host distributions of k-mer distances .", "Even though the signal was slightly less strong for shorter fragments , this simulation indicates that genome size is not the only factor that could explain such low viral–host genome adaptation for ssDNA viruses .", "The prediction of the host taxonomy for each viral sequence was based on the microbial genome with the lowest tetramer frequency distance to the viral sequence .", "A prediction was considered as ‘correct’ when this closest microbial genome taxonomy was the same as the original genome in which the viral sequence was detected .", "This prediction was computed using", "( i ) all microbial genomes ,", "( ii ) only genomes from a different species than the actual host ( i . e . , the genome in which the viral sequence was originally detected ) , and", "( iii ) only genomes from a different genus than the actual host .", "Protein sequences predicted from the POV ( Hurwitz and Sullivan , 2013 ) , TOV ( Brum et al . , 2015 ) , and human gut viromes ( Minot et al . , 2012 ) data sets were compared to RefSeqABVir ( Jan . 2014 ) using BLAST ( blastp , threshold of 50 on bit score and 0 . 001 on e-value ) .", "Those proteins that did not affiliate at >50 bit score and <0 . 001 e-value thresholds were considered ‘unclassified’ and then used as queries in a secondary BLAST ( blastp with the same thresholds ) against the predicted proteins from the VirSorter curated data set .", "Any unclassified proteins matching the VirSorter data set better were considered newly affiliated .", "To evaluate the efficiency of prophage assembly , we simulated genome sequencing from 23 bacterial genomes with identified prophages ( NC_000907 NC_000913 NC_000964 NC_002570 NC_002655 NC_002662 NC_002695 NC_002935 NC_003030 NC_003212 NC_003295 NC_003366 NC_003997 NC_004070 NC_004307 NC_004310 NC_004431 NC_004557 NC_004567 NC_004668 NC_004722 NC_005085 NC_005362 ) .", "NeSSM ( Jia et al . , 2013 ) was used to simulated HiSeq Illumina reads ( 100 bp paired-ends ) with a coverage of the prophage region varying between 5× , 25× , 50× , 75× , and 100× .", "Reads were then assembled with Idba_ud ( Peng et al . , 2012 ) , and viral contigs were predicted with VirSorter ( Roux et al . , 2015a ) .", "On the 481 contigs larger than 30 kb detected as viral by VirSorter , 11 were considered as ‘entirely viral’ even though these originated from integrated prophages , resulting in a ‘false-positive’ ratio of integrated prophages wrongly considered as extrachromosomal viral genomes of 2 . 3% for contigs of 30 kb and more .", "As could be expected , this same ‘false-positive’ ratio was higher for smaller contigs ( 12 . 06% for contigs <20 kb , and 22 . 81% for contigs <10 kb ) , so that we considered the origin of these small contigs as ‘undetermined’ , since they may come from integrated prophages or extrachromosomal genomes .", "All scripts used in this study are available on the TMPL wiki as a zip package: http://tmpl . arizona . edu/dokuwiki/doku . php ?", "id=bioinformatics:scripts:vsb and on github: http://github . com/simroux/virsorter-curated-dataset-scripts-package ." ] ]

[ "The ecological importance of viruses is now widely recognized , yet our limited knowledge of viral sequence space and virus–host interactions precludes accurate prediction of their roles and impacts .", "In this study , we mined publicly available bacterial and archaeal genomic data sets to identify 12 , 498 high-confidence viral genomes linked to their microbial hosts .", "These data augment public data sets 10-fold , provide first viral sequences for 13 new bacterial phyla including ecologically abundant phyla , and help taxonomically identify 7–38% of ‘unknown’ sequence space in viromes .", "Genome- and network-based classification was largely consistent with accepted viral taxonomy and suggested that", "( i ) 264 new viral genera were identified ( doubling known genera ) and", "( ii ) cross-taxon genomic recombination is limited .", "Further analyses provided empirical data on extrachromosomal prophages and coinfection prevalences , as well as evaluation of in silico virus–host linkage predictions .", "Together these findings illustrate the value of mining viral signal from microbial genomes ." ]

[ "Viruses are infectious particles that can only multiply inside the cells of microbes and other organisms .", "Little is known about the genetic differences between virus particles ( so-called ‘genetic diversity’ ) , especially compared to what we know about the diversity of bacteria , archaea , and other single-celled microbes .", "This lack of knowledge hampers our understanding of the role viruses play in the evolution of microbial communities and their associated ecosystems .", "Studying the genetics of the viruses in these communities is challenging .", "There is no single ‘marker’ gene that can be used to identify all viruses in environmental samples .", "Also , many of the fragments of viral genomes that have been identified have not yet been linked to their host microbes .", "Many viruses integrate their genome into the DNA of their host cell , and there are computational tools available that exploit this ability to identify viruses and link them to their host .", "However , other viruses can live and multiply inside cells without integrating their genome into the host's DNA .", "Earlier in 2015 , researchers developed a new computational tool called VirSorter that can predict virus genome sequences within the DNA extracted from microbes .", "VirSorter identifies viral genome sequences based on the presence of ‘hallmark’ genes that encode for components found in many virus particles , together with a reference database of genomes from many viruses .", "Now , Roux et al . —including some of the researchers from the earlier work—use VirSorter to predict viral DNA from publicly available bacteria and archaea genome data .", "The study identifies over 12 , 000 viral genomes and links them to their microbial hosts .", "These data increase the number of viral genome sequences that are publically available by a factor of ten and identify the first viruses associated with 13 new types of bacteria , which include species that are abundant in particular environments .", "It is possible for several different viruses to infect a single cell at the same time .", "Some viruses are known to be able to exchange DNA , and if this happens frequently in other viruses , it could have a big impact on how viruses evolve .", "Roux et al . 's findings suggest that although it is common for several different viruses to infect the same cell , it is relatively rare for these viruses to exchange genetic material .", "Roux et al . 's findings demonstrate the value of searching publicly available microbial genome data for fragments of viral genomes .", "These new viral genomes will serve as a useful resource for researchers as they explore the communities of viruses and microbes in natural environments , the human body and in industrial processes ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "cell biology", "neuroscience" ]

[ [ "Most membrane and secreted proteins are N-glycosylated during their synthesis and processing in the secretory pathway ( Moremen et al . , 2012 ) .", "During this process , as nascent proteins emerge in the lumen of the endoplasmic reticulum ( ER ) , a mannose-rich precursor is first transferred en bloc to specific aspargine residues .", "These immature 'core-glycans' are then trimmed down and modified by the sequential addition of diverse monosaccharides as proteins exit the ER and progress through the Golgi apparatus before they are sent to their final destination ( Moremen et al . , 2012; Aebi et al . , 2010 ) .", "This sequential and combinatorial modification results in a huge potential diversity of N-glycans and regulates virtually every aspect of membrane protein biology , in particular protein folding , trafficking , stability , ligand-binding and interaction with the extracellular matrix ( Moremen et al . , 2012; Aebi et al . , 2010; Scott and Panin , 2014; Miller and Aricescu , 2014 ) .", "Consequently , congenital N-glycosylation defects , especially in the brain , result in severe and often lethal developmental disorders ( Cylwik et al . , 2013 ) .", "Although the primary organelles of the secretory pathway were first described in neurons ( Golgi , 1989; Nissl , 1903 ) , little is known about the N-glycosylation of neuronal membrane proteins .", "Numerous mRNAs encoding surface and secreted proteins are localized to dendrites ( Cajigas et al . , 2012 ) .", "Yet , how dendritic secretory cargo is processed after local synthesis is still debated .", "In mammals , neuronal dendrites contain ER , ER-exit sites and ER-Golgi intermediate compartments ( ERGIC ) and occasionally Golgi outposts , but , for the most part lack canonical Golgi membranes ( Torre and Steward , 1996; Gardiol et al . , 1999; Krijnse-Locker et al . , 1995; Horton and Ehlers , 2003; Hanus and Ehlers , 2008; Cui-Wang et al . , 2012; Hanus et al . , 2014 ) .", "It is thus conceivable that , depending on their synthesis in the soma or dendrites , nascent cargo visits distinct sets of secretory subcompartments , and hence acquire specific types of N-glycans .", "For example , it is believed that nascent neurotransmitter receptors may follow multiple and specific secretory itineraries ( Jeyifous et al . , 2009 ) , but whether this impacts their glycosylation is unknown .", "Intriguingly , Concanavalin A ( ConA ) , a mannose-binding lectin , has been widely used to block the desensitization of plasma-membrane localized AMPA and kainate glutamate receptors ( Reiner and Isacoff , 2014; Hoffman et al . , 1998; Evans and Usherwood , 1985 ) .", "From a cell-biological perspective , this is puzzling as core-glycosylated ( mannose-rich ) N-glycans are typically not found at the cell-surface ( Moremen et al . , 2012; Aebi et al . , 2010; Grieve and Rabouille , 2011 ) .", "Indeed , a specific sensitivity to the glycosidase EndoH ( which cleaves mannose-rich glycans ) is commonly used to identify intracellular immature membrane proteins in total cellular lysates ( Shi et al . , 2010; Greger et al . , 2002; Tomita et al . , 2003; Sans et al . , 2001; Tucholski et al . , 2013a ) .", "Here we demonstrate that hundreds of neuronal surface membrane proteins are indeed core-glycosylated , resulting in the neuronal membrane displaying atypically high and activity-dependent levels of ConA-reactive species .", "We found that while N-glycosylation is generally required for the proper expression of membrane proteins at the neuronal surface , 'immature' core-glycosylated proteins are sufficient to sustain dendritic development and synaptic transmission , indicating that these proteins are fully functional .", "Focusing on candidate neurotransmitter receptors and auxiliary subunits , we show that core-glycosylated proteins access the cell-surface in a Golgi-independent manner indicating a bypass or a hypo-function of the Golgi apparatus .", "This atypical N-glycosylation results in an accelerated turnover of membrane proteins and modulates synaptic signaling , revealing a novel mechanism controlling membrane protein homeostasis and function in morphologically complex cells such as neurons ." ], [ "Their specific binding and sensitivity to distinct lectins and glycosidases distinguishes 3 basic types of N-glycans on membrane proteins: core-glycosylated ( or 'immature' ) , hybrid and complex ( Figure 1A; and see Materials and methods for details ) ( Moremen et al . , 2012; Scott and Panin , 2014; Zielinska et al . , 2010 ) .", "To compare the surface levels of these three generic N-glycan types in neurons to non-neuronal cells , we labeled mixed ( neuron-glia ) hippocampal cultures and 3 commonly used cell-lines ( COS7 , BHK and CHO ) with different lectin biotin-conjugates under non-permeabilizing conditions .", "As expected , all cell types examined ( including neurons and glia ) displayed both hybrid and complex N-glycans ( labeled by RCA and WGA , respectively ) ( Figure 1B ) .", "Surprisingly , however , mature neurons ( Figure 1—figure supplement 1 ) also displayed a high level of core-glycosylated N-glycans ( strongly labeled by ConA and GNA ) ( Figure 1B ) .", "This observation was reinforced by the comparison of neurons and several other cell types: only neurons showed a prominent surface labeling by ConA and GNA , confirming high levels of core-glycosylated proteins at the neuronal plasma membrane ( Figure 1C ) . 10 . 7554/eLife . 20609 . 003Figure 1 . Core-glycosylated proteins are abundant at the neuronal surface .", "( A ) The binding to specific lectins and the sensitivity to distinct glycosydases distinguish different N-glycans .", "Hybrid and complex N-glycans detected at the plasma membrane ( PM ) and the Golgi apparatus ( Golgi ) bind ricin agglutinin ( RCA ) and wheat germ agglutinin ( WGA ) , respectively , and are hydrolyzed by PNGase .", "In contrast , core-glycosylated N-glycans are usually specifically localized to the endoplasmic reticulum ( ER ) , bind concanavalin A and galanthus nivalis agglutinin ( GNA ) and are hydrolyzed by both PNGase and EndoH .", "( B ) Cultured hippocampal neurons ( DIV 40 ) after surface labeling with ConA , GNA , WGA ( red ) and RCA ( green ) to identify core-glycosylated ( ConA , GNA ) , complex ( WGA ) and hybrid ( RCA ) glycans , respectively .", "In iii , 'n' and 'g' mark neurons and glial cells , respectively .", "Note the high reactivity of the neuronal surface to ConA , WGA , GNA and RCA ( arrows ) .", "In contrast , in iii note the low and high reactivity of glial cells to GNA and RCA , respectively , resulting in a 'green-only' appearance ( arrowheads ) .", "Scale bars 25 µm .", "( C ) Surface levels of ConA , GNA , RCA and WGA reactive glycans in fibroblasts ( COS , white; BHK , light grey; and CHO , dark grey ) and in neurons ( green ) .", "Mean ± SEM .", "N=27 to 132 cells in 2 experiments .", "*p<0 . 05; **p<0 . 01 , ***p<1x10−4; Kruskal-Wallis’s multiple comparison test . DOI: http://dx . doi . org/10 . 7554/eLife . 20609 . 00310 . 7554/eLife . 20609 . 004Figure 1—figure supplement 1 . Viable hippocampal neurons can be maintained in culture for more than two months . Labeling of dendrites ( MAP2 , green ) , synapses ( bassoon , red ) and nuclei ( DAPI , blue ) in DIV28 and DIV70 neurons documenting the viability of DIV 40 and older neurons as in Figure 1 .", "Scale bar 10 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 20609 . 00410 . 7554/eLife . 20609 . 005Figure 1—figure supplement 2 . Blocking the maturation of core-glycans with kifunensine increases the surface levels of ConA- and GNA-reactive glycans but decreases RCA- and WGA-reactive glycans .", "( A ) Simplified representation of the N-glycosylation pathway and site of action of kifunensine ( Kf ) .", "( B ) Surface levels of ConA- , GNA- , RCA- and WGA-reactive glycans in neurons ( DIV 14–18 ) treated with Kf ( 5 μM ) for two or three days .", "Shown are values in neurons treated with Kf normalized to control values ( log 2 scale ) .", "Kf increases ConA and GNA levels but decreases RCA and WGA .", "N=53–81 cells in 2 experiments for each drug .", "***p<1x10−4; Dunn’s multiple comparison test . DOI: http://dx . doi . org/10 . 7554/eLife . 20609 . 00510 . 7554/eLife . 20609 . 006Figure 1—figure supplement 3 . Intracellular ConA- , RCA- and WGA-reactive glycans are localized in distinct organelles in neurons . Images ( single confocal planes ) of neurons ( DIV 28 ) after labeling with ConA or WGA ( red ) , RCA ( green ) , DAPI ( blue ) and anti-MAP2 antibodies ( cyan ) .", "ConA labels structures that have the morphological characteristics of endoplasmic reticulum ( ER , arrows ) .", "In contrast , WGA labels the nuclear envelope ( NE , crossed arrow ) and Golgi-like compartments ( arrowhead ) .", "RCA labels pleiomorphic structures ( arrowheads ) , which are distinct from ConA-reactive structures but partly colocalize with some of theWGA-reactive compartments ( other than the NE ) .", "Scale bar 10 or 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 20609 . 00610 . 7554/eLife . 20609 . 007Figure 1—figure supplement 4 . ConA- , RCA- and WGA-reactive glycans are localized in distinct organelles and display contrasting sensitivities to EndoH and PNGase in COS cells .", "( A ) Subcellular distribution ( maximal intensity projections of confocal stacks ) and ( B ) average levels of ConA- , RCA- and WGA-reactive glycans in COS 7 cells in control conditions ( light grey ) or after glycan removal by EndoH ( darker grey ) or PNGAse ( darkest grey ) .", "ConA labels the endoplasmic reticulum ( ER , arrows in A ) and to a lesser extent the Golgi apparatus ( GA , arrowheads in A ) .", "Pretreatment with PNGase or EndoH ( only shown for Endo H in A ) strongly reduces the labeling of the ER , resulting in a marked decrease of total ConA levels ( B ) .", "RCA and WGA label the GA ( RCA and WGA , arrowheads in A ) and the nuclear envelope ( WGA , arrows in A ) .", "EndoH has no effect on the intensity of the labeling with these two lectins ( compare RCA and WGA labeling in control cells and in cells treated with EndoH , respectively , and see B ) .", "In contrast , PNGase strongly reduces the labeling of the GA ( arrowheads in A ) , resulting in decreased levels of RCA- and WGA-reactive glycans .", "Note that the quantification in B was done without fluorescence thresholding , partly explaining the relatively high background signal remaining after enzymatic digestion .", "In A , scale bar = 10 μm .", "Shown in B is mean ± SEM , N=19–36 cells .", "***p<1x10−4; Dunn’s multiple comparison test . DOI: http://dx . doi . org/10 . 7554/eLife . 20609 . 007 To confirm the specificity of the labeling , neurons were labeled with the same lectins after treatment with Kifunensine ( Kf ) , an inhibitor of ER and Golgi type I mannosidases , which prevents the maturation of core-glycans into hybrid and complex glycans ( Tulsiani et al . , 1982 ) .", "As expected , Kf increased the surface levels of ConA and GNA-reactive glycans and reduced the surface levels of RCA and WGA-reactive glycans ( Figure 1—figure supplement 2 ) , thus confirming the presence of core-glycosylated proteins on the neuronal surface .", "To verify that intracellular proteins were indeed localized in the expected secretory compartment , we labeled neurons with ConA , RCA and WGA under permeabilizing conditions together with anti-MAP2 antibodies to visualize the somatodendritic compartment .", "As expected , ConA marked ER-like structures ( Cui-Wang et al . , 2012 ) while RCA and WGA labeled distinct Golgi-like membranes and , for WGA , the nuclear envelope ( EN ) ( Figure 1—figure supplement 3 ) .", "We also validated the labeling of these intracellular compartments in COS7 cells , where the clear morphology of secretory organelles allows one to unambiguously identify various intracellular structures , and obtained similar results ( Figure 1—figure supplement 4 ) .", "Altogether , these data thus indicate that the atypical abundance of ConA-reactive species at the neuronal surface is indeed due to core-glycosylated proteins .", "How abundant is the core-glycosylation of neuronal membrane proteins ?", "To compare directly the relative expression of the different glycans at the neuronal cell surface , we fractioned surface and intracellular proteins by affinity purification after surface biotinylation ( Figure 2A and Figure 2—figure supplement 1 ) and quantified the levels of ConA , RCA and WGA reactive-glycans in these two fractions by far-western blotting ( see Materials and methods ) .", "We again found , both in cultured neurons and brain tissue , that core-glycans ( ConA-reactive species ) were surprisingly abundant at the neuronal surface ( Figure 2B ) .", "Control experiments in total neuronal extracts treated with PNGase and EndoH showed that only ConA-reactive species were sensitive to EndoH , thus confirming that these were core-glycosylated N-glycans ( Figure 2—figure supplement 2 ) .", "Interestingly , quantification of the relative surface expression of ConA , RCA and WGA reactive species showed that , in neurons , core-glycosylated proteins were expressed at relative levels comparable to conventional 'mature' N-glycans ( Figure 2C ) . 10 . 7554/eLife . 20609 . 008Figure 2 . Core-glycosylated proteins are expressed at the neuronal surface at levels similar to hybrid and complex N-glycosylated proteins .", "( A ) Isolation of surface ion channel ( GluA1 ) versus cytoplasmic proteins ( RhoA ) after surface biotinylation and purification with varying amounts of NHS-SS-biotin and streptavidin-beads .", "( B ) Surface ( S ) and intracellular ( I ) proteins prepared from neurons , COS7 cells and acute brain slices after labeling ( far western blots ) with ConA , RCA and WGA .", "For each condition , 15 and 5 μg of proteins were loaded for S and I fractions , respectively .", "Note the high levels of ConA reactive species in cultured neurons and in brain slices .", "( C ) Relative surface expression ( surface / total signal ) of ConA , RCA and WGA reactive species in cultured neurons ( green ) and different non-neuronal cell types ( COS , white; BHK , light grey; CHO , darker grey; L-cells , black ) showing that the high surface expression of core N-glycans is specifically observed in neurons while other cell types show low levels of core glycosylation but higher levels of hybrid and complex glycosylation .", "Mean ± SEM and individual data points .", "N=4 in 2 experiments .", "* p<0 . 05; ** p<0 . 01 , Dunn’s multiple comparison test .", "( D ) Relative surface expression of ConA- and RCA-reactive glycans in neurons from one to nine weeks in vitro , showing stable levels of surface core-glycosylation and a developmental increase in the surface expression of hybrid glycans .", "N= 27–28 dishes in 3 independent experiments .", "***p<1x10−4; Pearson’s r test . DOI: http://dx . doi . org/10 . 7554/eLife . 20609 . 00810 . 7554/eLife . 20609 . 009Figure 2—figure supplement 1 . The neuronal plasma membrane is intact after surface biotinylation . Intrinsic fluorescence ( single confocal plane image ) of pHluo-TM ( green ) , a transmembrane protein marker that selectively exhibits fluorescence at the plasma membrane ( and to a lesser extent the endoplasmic reticulum ) , and streptavidin-Alexa647 fluorescence ( red ) after surface biotinylation of 15 DIV neurons , showing the selective biotinylation and the integrity of the neuronal surface ( arrows ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 20609 . 00910 . 7554/eLife . 20609 . 010Figure 2—figure supplement 2 . ConA- , RCA- and WGA-reactive glycans assessed by far-western blotting display distinct and specific sensitivities to PNGAse and EndoH . Total neuronal extracts labeled with ConA , RCA or WGA after or without pretreatment with PNGase or EndoH showing the specificity of the labeling .", "In contrast to ConA reactivity that is abolished by either EndoH or PNGase treatment , RCA and WGA reactivities are only abolished by PNGase . DOI: http://dx . doi . org/10 . 7554/eLife . 20609 . 010 Where are core-glycosylated proteins localized in the neuronal plasma membrane ?", "To address this , we imunolabeled synapses with the presynaptic marker protein bassoon after surface labeling with ConA .", "Core N-glycans were distributed throughout the entire neuronal surface but were particularly abundant at synapses , notably in dendritic spines ( Figure 3A ) .", "To examine whether synaptic proteins themselves displayed core-glycosylation profiles , we assessed the glycosylation status of a cast of key synaptic proteins using their electrophoretic profiles after digestion by PNGase and EndoH , to identify core-glycosylated ( EndoH-sensitive ) proteins among the family of N-glycosylated ( PNGase-sensitive ) surface molecules ( Figure 3B ) .", "We found that a substantial fraction of purified surface GABAA receptor or AMPA-type and NMDA-type glutamate receptor subunits were core-glycosylated ( EndoH-sensitive ) ( Figure 3C ) .", "More specifically , these proteins were either entirely core-glycosylated ( e . g . GluN1 and GABAAR β3 ) , partially core-glycosylated ( i . e . displayed both mature and core-glycosylated glycans on the same polypeptide chain , such as GluA1 , and GluN2B ) or were expressed as a mixture of proteins with either core-glycosylated or mature N-glycans ( e . g . GluA2 , GluA4 ) .", "We note that while there are few reports of core-glycosylated sugars on functional proteins ( Hirayama et al . , 2016 ) , these are typically associated with a few , select glycosylation sites thought to be inaccessible to Golgi enzymes .", "In contrast , here we show that all of the glycosylated sites of GluN1 ( an obligatory subunit of NMDA receptors with 10 predicted N-glycosylation sites ) , GluA2 and GluA3 ( 4 predicted sites ) are core-glycosylated .", "In contrast , TARP γ8 , an AMPA receptor auxiliary protein that is thought to be co-trafficked with nascent receptors in the secretory pathway ( Tomita et al . , 2003 ) was completely insensitive to EndoH ( Figure 3C ) as is expected for a typical mature surface glycoprotein in non-neuronal cells ( Figure 3—figure supplement 1 ) .", "Similarly , the synaptic adhesion protein Neuroligin 1 ( NLG1 , Figure 3C ) was also completely insensitive to EndoH .", "These results strongly indicate that synaptic proteins and receptors are processed and trafficked to the cell-surface through distinct mechanisms / secretory routes: a classical pathway that generates glycoproteins with mature N-glycans ( e . g . TARP γ8 and NLG1 ) , which in some instances are detected together with core-glycosylated sites on the same polypeptide ( e . g . GluA1 ) , as well as an unconventional route that generates proteins with only core-glycosylated profiles ( e . g . GluN1 and GABAAR β3 ) . 10 . 7554/eLife . 20609 . 011Figure 3 . A number of key neuronal surface proteins display unconventional N-glycosylation profiles .", "( A–C )", "Synaptic localization of ConA reactive species and core-glycosylation of surface excitatory neurotransmitter receptors .", "( A ) Fluorescent micrographs of cultured hippocampal neurons ( DIV 26 ) after labeling of surface core-glycosylated proteins ( ConA , red ) and presynaptic terminals ( bassoon immunoreactivity , green , shown together with MAP2-IR in blue ) showing the presence of core-glycosylated proteins at synapses .", "Scale bar 5 μm .", "( B , C )", "Purification scheme ( B ) and migration profiles ( C , immunoblots ) of a group of surface glutamate receptors and associated proteins after PNGase and EndoH treatment .", "Note the complete EndoH sensitivity of GluN1 and GluA1 , the partial sensitivity of GluA2 and GluA4 and the lack of sensitivity of TARP γ8 and neuroligin 1 ( NLG1 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 20609 . 01110 . 7554/eLife . 20609 . 012Figure 3—figure supplement 1 . Standard mature surface N-glycans are typically insensitive to EndoH . 9AB ) Immunoblots ( A ) and relative surface expression ( B , surface / total protein ) of thermosensitive vesicular stomatitis virus glycoprotein ( VSVG-GFP ) ( Presley et al . , 1997 ) in COS 7 cells at various times after release ( 39–32°C shift ) of a temperature-dependent blockade of VSVG exit from the endoplasmic reticulum .", "Note the progressive accumulation of VSVG at the cell surface at permissive temperature ( 32°C ) , but not after a temperature-dependent blockade ( 23°C ) [ ( Matlin and Simons , 1983 ) ] of post-Golgi transport .", "( C ) Contrasting sensitivity to EndoH of surface VSVG at an early ( 20 min ) or later ( 120 min ) phase after ER-exit release .", "Note the complete lack of sensitivity of VSVG as mature proteins accumulate at the cell-surface . DOI: http://dx . doi . org/10 . 7554/eLife . 20609 . 012 While both binding to ConA and digestion by EndoH rely on a similar biochemical substrate - a sufficient number of mannose residues in a given N-glycan – these two reagents are typically used experimentally to probe molecules that should reside in different cellular compartments: ConA is used to block the desensitization of AMPA and kainate receptors on the surface ( Reiner and Isacoff , 2014; Hoffman et al . , 1998; Evans and Usherwood , 1985 ) and EndoH is used to mark immature intracellular proteins in total cellular extracts ( Tucholski et al . , 2013a; Rouach et al . , 2005 ) .", "To assess whether ConA and EndoH might interact with the same proteins in neurons , we devised a two-round purification strategy to assess the EndoH-sensitivity of surface proteins interacting with ConA ( Figure 4A ) .", "Consistent with their respective electrophoretic profiles after deglycosylation ( Figure 3C ) , GluN1 and GluA2 were purified based on their interaction with ConA , whereas TARP γ8 was not detected ( Figure 4B ) .", "As expected , the binding of surface GluN1 and GluA2 to ConA was abolished by competition with free mannose ( Figure 4C ) .", "Finally we observed that prior digestion with EndoH also blocked GluA2 and GluN1 binding to ConA ( Figure 4D ) , thus demonstrating that in hippocampal neurons , the binding of surface membrane proteins to ConA and their sensitivity to EndoH are equivalent .", "Thus EndoH-sensitive proteins – proteins that are classically regarded as intracellular and immature - are abundant at the neuronal surface . 10 . 7554/eLife . 20609 . 013Figure 4 . Hundreds of surface neuronal membrane proteins are core-glycosylated . Purification scheme ( A ) and immunoblots ( B–D ) showing the isolation of core-glycosylated N-glycans of the neuronal plasma membrane .", "( B ) Presence of GluA2 and GluN1 , but not TARP γ8 , among proteins binding to ConA ( vol . in µL ) .", "The lane 'S' shows GluA2 , GluN1 and TARP γ8 immunoreactivity in the surface fraction that was used for the experiment .", "( C ) Incubation of surface proteins with ConA in the presence of mannose-BSA ( vol . in µL ) abolishes GluA2 binding to ConA .", "( D ) Bound and unbound fractions after purification with ConA in control conditions or after protein digestion with EndoH .", "Core-glycan removal with EndoH abolishes GluA2 and GluN1 binding to ConA .", "( E , F )", "Mass spectrometry of proteins purified as in A . ( E ) Schemes of a synapse and a growth cone highlighting examples of core-glycosylated surface neuronal proteins of particular physiological relevance for synaptic function and neuron development .", "( F ) Fold enrichment of N-glycoproteins included in the indicated pathways in core-glycosylated surface proteins versus total proteins .", "Highlighted in blue and in purple are proteins of particular relevance for neuronal functions or autoimmune diseases and cancer , respectively .", "*p<0 . 05 , **p<0 . 01 , ***p<1x10−4 , hypergeometric test . DOI: http://dx . doi . org/10 . 7554/eLife . 20609 . 01310 . 7554/eLife . 20609 . 014Figure 4—figure supplement 1 . Evaluation of mass spectrometry data part 1 . Enrichment of peptides from proteins in the A ( w/o EndoH ) and B ( w EndoH ) sets as a function of repeatability across experiments and technical replicates ( see Materials and methods ) .", "( A ) Identification of high confidence core-glycosylated proteins .", "Plot of normalized peptide intensities ( label free quantification ) in samples without ( 'A' , target core-glycosylated ) or with prior treatment with Endo H ( 'B' , background ) as a function of detection reproducibility ( intra class correlation ) across multiple technical replicates and independent experiments .", "Shown in colors are the various groups detected after hierarchical clustering .", "Note the clear separation of peptides close to y=0 ( equally distributed in A and B ) and y=1 ( only found in A ) as reproducibility increases .", "Shown in red are peptides with the highest enrichment and reproducibility which were used to identify 'core-glycosylated' proteins .", "( B–C )", "Retrospective analysis of all the peptides originating from proteins identified in high-confidence core-glycosylated group .", "( B ) Core-glycosylated protein peptides ( red ) versus all the other peptides ( grey ) , plotted as in A . ( C ) Distribution ( cumulative probability ) of core-glycosylated protein peptides ( red ) versus all the other peptides ( grey ) showing the more reliable occurrence of core-glycosylated proteins and their sensitivity to EndoH .", "KS , Kolmogorov-Smirnov’s test . DOI: http://dx . doi . org/10 . 7554/eLife . 20609 . 01410 . 7554/eLife . 20609 . 015Figure 4—figure supplement 2 . Evaluation of mass spectrometry data part 2 . ( A ) Enrichment for secreted and transmembrane proteins among identified surface core-glycosylated proteins .", "Relative frequencies ( solid bars ) of soluble ( intracellular or secreted ) , transmembrane and predicted N-glycoproteins among 'total' hippocampal proteins and 'core-glycosylated' surface proteins .", "Shown in blue ( thin line ) is the statistical significance ( -log p value ) of the difference between the two groups for the indicated category .", "( B ) Fold enrichment of core-glycosylated proteins with annotations associated to the indicated subcellular compartments , and statistical significance ( -log p value ) .", "Note the enrichment of cell-surface , secreted , lysosomal and endoplasmic reticulum proteins , but not Golgi proteins , among the core-glycosylated proteins .", "***p<1x10−4; Hypergeometric test . DOI: http://dx . doi . org/10 . 7554/eLife . 20609 . 015 How many surface proteins fall in this category and how important are they for neuronal function ?", "To determine whether core-glycosylation is limited to a small set of neuronal surface proteins or represents a more widespread phenomenon , we used high-resolution mass spectrometry to identify neuronal surface proteins that interact with ConA ( Figure 4A , E and F ) .", "To increase the specificity of our detection and obtain a high-confidence dataset , we used a label-free quantification ( LFQ ) approach to identify proteins that were consistently detected across experiments and whose binding to ConA was impaired by EndoH .", "In brief , plasma membrane proteins were first isolated after surface biotinylation and affinity purified with ConA , after ( 'background' control group ) or without prior treatment with EndoH ( target 'core-glycosylated' group ) .", "Purified proteins were fragmented with proteases and the resulting peptides were separated by HPLC , ionized and subjected to LFQ and fingerprinting by mass spectrometry .", "Surface core-glycosylated proteins were identified by focusing the analysis on peptides that showed the highest reproducibility and enrichment in the target group ( see Materials and methods and Figure 4—figure supplement 1 ) .", "As expected , the resulting protein dataset was markedly enriched for secreted and plasma membrane transmembrane proteins with predicted N-glycosylation sites ( Figure 4—figure supplement 2 ) .", "To our surprise , we found that hundreds of transmembrane or secreted neuronal proteins were core-glycosylated ( n=227 protein groups , 647 protein IDs; Supplementary file 1A–1C ) , including major ionotropic and metabotropic glutamate and GABA receptor subunits , synaptic adhesion proteins , neurotrophin receptors and voltage-gated ion channels ( Figure 4E ) .", "Protein pathway and gene ontology analysis of our dataset compared to a control dataset ( a proteome obtained from total neuronal lysates ) ( Supplementary file 1D ) showed that multiple functional classes of glycoproteins related to axon guidance , synaptic transmission , synaptic plasticity and addiction were significantly overrepresented among the family of coreglycosylated surface proteins ( Figure 4F and Supplementary file 1E ) .", "Of note , multiple terms associated with autoimmune diseases and cancer were also overrepresented in our dataset ( Figure 4F and Supplementary file 1E ) .", "As with any affinity purification procedure , we cannot exclude the possibility that some false positive proteins are present owing to their strong interaction with surface proteins .", "We note , however , that we isolated surface AMPA receptor subunits from TARP γ8 based on their distinct glycosylation profiles ( Figure 4B ) , even though these two proteins that are typically found in the same macromolecular complexes ( Schwenk et al . , 2014 ) , thus suggesting a high level of specificity .", "Taken together , the above data indicate that a large number of neuronal plasma membrane proteins display unconventional N-glycosylation profiles that are typically expressed at low levels in the plasma membrane of heterologous cells , suggesting that core-glycosylation plays a major physiological role in neurons .", "During neuronal development , the extension and elaboration of dendrites and axons places a high demand on neuronal secretory function to provide membrane , and thus an equally high demand on protein glycosylation .", "To address whether the presence of core-glycans at the neuronal membrane is specific to a particular developmental stage , we examined the relative surface expression of core and hybrid N-glycans throughout neuronal and synaptic development ( over a nine week time-course ) .", "We found that while the surface expression of core-glycans remains constant , the surface expression of hybrid glycans progressively increases as neurons mature ( Figure 2D ) , documenting a development-dependent regulation of neuron surface N-glycosylation .", "We next examined the dependence of dendritic development on N-glycans by treatment with tunicamycin ( Tm ) , a drug that completely blocks all N-glycosylation by preventing the transfer of the N-glycan precursor to target proteins in the ER ( Figure 5A ) .", "Dendritic growth and complexity were assessed by performing a Sholl analysis ( Figure 5C ) or by quantifying the total dendritic length and number of tips ( Figure 5D ) .", "As shown in Figure 5B , we found that dendritic growth was severely impaired by a global blockade of N-glycosylation .", "To distinguish the contributions of mature vs . immature glycans to dendritic growth we used swainsonine ( Sw ) , a selective blocker of Golgi type-II mannosidase Man2b1 and Man2b2 ( Tulsiani et al . , 1982 ) , the enzymes that convert EndoH-sensitive proteins into EndoH-resistant species ( a prerequisite for their subsequent maturation ) , and two other mannosidase inhibitors: kifunensine ( Kf ) and deoxymannojirimycin ( DMJ ) which inhibit ER or Golgi mannosidases acting upstream of Man2b1/Man2b2 ( Herscovics , 1999 ) ( Figure 5A ) .", "Surprisingly , we found that the immature N-glycans that remain following treatment with Kf , DMJ or Sw were sufficient to initiate and maintain dendritic growth ( Figure 5C–D and Figure 5—figure supplement 1 ) .", "To verify that these drugs had the expected effects on the surface expression of specific N-glycan subtypes , we assessed the surface expression of core , hybrid and complex glycans by far-western blotting ( Figure 5—figure supplement 2 ) .", "As expected , Kf , DMJ and Sw increased the surface levels of core-glycans while decreasing the levels of hybrid and , at least for Kf and DMJ , complex glycans .", "Thus , while N-glycosylation is necessary for dendritic development and maintenance , 'immature' N-glycans are sufficient to sustain these processes , which indicates that core-glycosylated proteins on the neuronal membrane surface are fully functional . 10 . 7554/eLife . 20609 . 016Figure 5 . Mature N-glycans are not required for normal dendritic development .", "( A ) Simplified representation of the N-glycosylation pathway and site of action of the glycosylation inhibitors tunicamycin ( Tm ) , kifunensine ( Kf ) , 1-deoxymannojirimycin ( DMJ ) and swainsonine ( Sw ) .", "( B–C )", "Camera lucida drawings ( B ) and/or Sholl analysis ( C ) of the dendrites of hippocampal neurons ( DIV 11 ) after a three day exposure to vehicle ( DMSO , Ct ) , Tm ( 1 . 2 µM ) , Kf ( 5 μM ) , DMJ ( 75 μM ) or Sw ( 88 μM ) .", "( D ) Total dendritic length ( left panel ) and branch tip numbers ( right panel ) in neurons treated with vehicle ( Ct ) , Tm , Kf , DMJ or SW .", "Mean ± SEM .", "N=31 to 137 cells in 2–6 independent experiments .", "***p<1x10−4; Kruskal-Wallis/Dunn’s multiple comparison test .", "In B–D , note that while Tm strongly impairs dendritic growth and branching , blocking the maturation of N-glycans in the ER or the GA has no detectable effect on dendritic development . DOI: http://dx . doi . org/10 . 7554/eLife . 20609 . 01610 . 7554/eLife . 20609 . 017Figure 5—figure supplement 1 . Other examples of dendrites after treatment with the drugs described in Figure 5A . DOI: http://dx . doi . org/10 . 7554/eLife . 20609 . 01710 . 7554/eLife . 20609 . 018Figure 5—figure supplement 2 . The lack of effect of specific N-glycosylation inhibitors on dendritic development is not due to a lack of activity or to non-specific effects on the glycosylation pathways .", "( A ) Blots and ( B ) surface levels of immature/core-glycans ( ConA ) , hybrid glycans ( RCA ) and complex/mature glycans ( WGA ) in neurons ( DIV 7–10 ) treated for three days with Tm , Kf , DMJ or Sw as in Figure 5 .", "Core-glycans are decreased by Tm but are increased by Kf and , to a lesser extent , DMJ and Sw .", "In stark contrast , hybrid glycans are decreased by the 4 drugs .", "Mature N-glycans are decreased by Tm , Kf and DMJ , but slightly increased by Sw .", "N=8–12 dishes in 4–6 experiments .", "*p<0 . 05; **p<0 . 01 , ***p<1x10−4; Dunn’s multiple comparison test . DOI: http://dx . doi . org/10 . 7554/eLife . 20609 . 018 As stated above , the trimming of the N-glycan mannose-core and the resulting loss of glycoprotein sensitivity to EndoH occurs in the Golgi apparatus ( Moremen et al . , 2012 ) , raising the possibility that the neuronal core-glycosylated proteins that we detect on the plasma membrane may be trafficked to the cell-surface via a Golgi-independent mechanism .", "To address this , we used Brefeldin A ( BFA ) – a drug that is commonly used to disrupt the Golgi apparatus ( Klausner et al . , 1992 ) ( Figure 6A ) – and examined its effect on the surface expression of nascent proteins .", "To do so , neurons were pre-treated for with BFA ( 2 . 5 μg/mL; 1 hr ) , and metabolically labeled by bio-orthogonal non-canonical amino acid tagging ( BONCAT ) with azido-homo-alanine ( AHA ) for 120–150 min ( Dieterich et al . , 2006 ) , or as a negative control methionine ( Figure 6—figure supplement 1 ) .", "Surface and intracellular proteins were then separated and quantified after surface-biotinylation and immunobloting ( Figure 6B and C ) .", "As a positive control , similar experiments were performed in COS 7 cells .", "In neurons , BFA had no detectable effect on the levels of nascent surface proteins and slightly decreased intracellular nascent proteins ( Figure 6B ) .", "In contrast in COS cells , BFA had no effect on intracellular proteins but markedly reduced the levels of nascent proteins at the plasma membrane ( Figure 6B ) , as also observed for other cell types ( Davis and Mecham , 1996 ) .", "Thus , while BFA strongly reduced secretory trafficking in COS 7 cells , it had no detectable effect on the accumulation of nascent proteins at the neuronal surface under these experimental conditions ( Figure 6C ) .", "We cannot rule out that BFA impairs secretory trafficking in neurons but that our method is not sensitive enough to detect this effect .", "Yet , our results indicate that secretory trafficking in neurons is markedly less dependent on the Golgi apparatus ( GA ) than in other cell types , providing an explanation for the atypical prominence of core-glycosylated proteins at the neuronal surface . 10 . 7554/eLife . 20609 . 019Figure 6 . Unconventional secretory processing of core-glycosylated surface proteins .", "( A ) GalT-GFP expression in neurons showing the morphology of the somatic Golgi apparatus after a 2 hr long exposure to vehicle ( DMSO; control , Ct ) or 2 . 5 μg/mL brefeldin A ( BFA ) .", "Scale bar 5 μm .", "( B–C )", "Immunoblots ( B ) and surface or intracellular levels ( C ) of nascent proteins metabolically labeled for 120 to 150 min by bioorthogonal non-canonical aminoacid tagging ( BONCAT ) in COS cells and in neurons ( DIV 25–26 ) in the absence ( control or Ct ) or in the presence of 2 . 5 μg/mL BFA .", "Note the strong effect of BFA on the surface expression of nascent proteins in COS cells and the relative lack of effect in neurons .", "N=5 to 7 dishes in 2–3 experiments .", "*p<0 . 05 , **p<0 . 01; Dunn’s multiple comparison test .", "See also Figure 6—figure supplement 1 .", "( D ) Immunoblot of surface GluN1 , GluA2 and TARP γ8 after a 6–7 hr exposure to vehicle ( Ct ) or BFA , showing the reduced surface levels of TARP γ8 , GluA2 and to a lesser extent GluN1 to the cell surface after disruption of the Golgi apparatus .", "( E ) Sensitivity to BFA ( BFA / control ) as a function of core-glycosylation levels ( core-glycosylated fraction / total ) .", "Note the selective effect of BFA on mature proteoglycans ( low core glycosylation / total ratio ) .", "Mean ± SEM .", "N=10–11 in 4 experiments .", "*p<0 . 05 , ANOVA/Tukey’s multicomparison test . DOI: http://dx . doi . org/10 . 7554/eLife . 20609 . 01910 . 7554/eLife . 20609 . 020Figure 6—figure supplement 1 . Nascent neuronal intracellular and surface proteins ( BONCAT ) after a 5-hr metabolic labeling with AHA or Methionine ( Met ) in the presence or absence of BFA . DIV19 neurons .", "Note the absence of signal in neurons incubated with Methionine ( which for surface proteins demonstrates the absence of residual biotin after elution from streptavidin beads during their purifcation ) and the lack of detectable effect of BFA on the levels of surface nascent proteins . DOI: http://dx . doi . org/10 . 7554/eLife . 20609 . 020 Do the complex and atypical ( i . e . core-glycosylated ) glycosylation profiles of candidate proteins reflect processing and lack of processing by the GA , respectively ?", "To address this , we chose GluN1 , GluA2 and TARP γ8 because of their physiological relevance and their respective complete , mixed ( co-existence of EndoH sensitive and insensitive species ) or absent core-glycosylation ( Figure 3C ) .", "We found that exposure to BFA ( 5 μg/mL; 6–7 hr ) significantly reduced the surface expression of TARP γ8 ( Figure 6D , E ) – as expected for a typical mature N-glycan ( Figure 3C ) .", "The surface expression of GluA2 was also reduced by BFA , albeit to a lesser extent ( Figure 6E ) .", "In contrast , the surface expression of GluN1 - a protein whose full surface complement is core-glycosylated – was insensitive to Golgi disruption ( Figure 6E ) .", "Interestingly , the sensitivity to EndoH of these proteins was inversely correlated with their sensitivity to Golgi-disruption ( Figure 6E ) .", "Importantly , as proteins with a faster turnover can be expected to respond more quickly to disruption of their biosynthetic pathway , protein stability must be taken into account in interpreting these results .", "We note , however , that previous studies have shown that GluA2 has a slower turnover ( t1/2 1 . 95 days ) than GluN1 ( t1/2 1 . 61 days ) ( Hanus and Schuman , 2013 ) , which indicates that the differential sensitivity of GluA2 and GluN1 to BFA cannot be accounted for by differences in stability .", "The stability of TARP γ8 is not known at present .", "Thus , these data suggest that the surface expression of core-glycosylated glutamate ionotropic receptors is indeed due to a Golgi-independent secretory processing .", "Further supporting this view , our protein annotation analysis of the nascent proteins identified by mass spectrometry showed a significant enrichment for ER proteins , but not Golgi proteins , among the surface core-glycosylated proteins ( Figure 4—figure supplement 2 ) .", "Together with the relative paucity of canonical Golgi membranes as compared to other components of the secretory pathway in dendrites ( Hanus and Ehlers , 2008; Hanus et al . , 2014 ) , the abundance of core-glycosylated proteins at the neuronal surface suggests that Golgi-by pass is surprisingly common for neuronal membrane proteins ( Torre and Steward , 1996 ) .", "The coexistence of two forms of GluA2 on the cell surface – one form that presents only standard N-glycans and one form that presents only unconventional glycans – suggests that complex glycosylation profiles are not required for the expression of AMPA receptors on the cell-surface .", "To determine whether this was indeed the case , we used the glycosylation inhibitors described above to determine which glycosylation step ( s ) are required for the surface expression of GluA2 .", "We found that whereas blocking N-glycosylation entirely with Tm markedly reduced GluA2 surface expression ( Figure 7—figure supplement 1 ) , blocking the maturation of core-glycans with Kf , DMJ or Sw had no detectable effect on GluA2 surface levels , despite clear effects ( at least for Kf and Sw ) on the glycosylation profile of the protein ( Figure 7—figure supplement 1 ) .", "Thus , the delivery of GluA2 to the neuronal surface requires N-glycosylation but does not require processing by Golgi glycosylation enzymes .", "To determine more generally the dependence of synaptic receptors on mature glycans , we examined whether Kf impaired synaptic transmission using patch-clamp recording of spontaneous excitatory miniature postsynaptic AMPA/kainate currents .", "We found that blocking the maturation of core-glycans in the ER for 48 hr had no detectable effect on the frequency or the amplitude of synaptic currents ( Figure 7A , B ) , thus strengthening the view that mature N-glycans are largely dispensable for synaptic transmission . 10 . 7554/eLife . 20609 . 021Figure 7 . Blocking the maturation of N-glycosylation in the ER does not impair synaptic transmission and modulates the properties of postsynaptic AMPA receptors .", "( A ) Example traces of miniature excitatory postsynaptic currents ( mEPSCs ) and ( B ) average mEPSC amplitude and frequency and input resistance ( Mean ± SEM ) in control ( Ct ) neurons ( DIV 20–21 ) and in neurons exposed to Kf ( 48 hr prior to recording ) showing the lack of effect of Kf on synaptic transmission .", "N = 10 and 7 cells for Ct and Kf , respectively , in 3 independent experiments .", "NS , not significant , Mann-Whitney’s test .", "( C ) Pictures of a synaptic marker ( PSD-mCh , red ) and a calcium indicator ( GCaMP6 , green , DIV14 neuron ) before and after local glutamate uncaging at the position indicated by a white box ( arrow ) .", "( D ) Representative calcium traces and ( E ) average responses ( peak amplitude normalized to baseline ) induced by repetitive glutamate uncaging in individual dendrites ( 14 DIV neurons ) before and after inhibition of AMPA receptors with CNQX .", "Note the complete block of the calcium signal by CNQX .", "N= 5 in 2 experiments .", "**p<10–1; Mann-Whitney’s test .", "( F ) Example calcium trace , ( G ) average responses ± SEM ( normalized to peak amplitude ) and ( H ) time-to-peak and plateau decay in control neurons or in neurons exposed to Kf ( 48 hr prior to recording ) .", "In G and H , note the faster onset of postsynaptic responses in neurons treated with Kf .", "N= 29–31 ( time to peak ) or 22 ( plateau decay ) in 3 experiments .", "**p<10–1; Mann-Whitney’s test . DOI: http://dx . doi . org/10 . 7554/eLife . 20609 . 02110 . 7554/eLife . 20609 . 022Figure 7—figure supplement 1 . Core-glycosylation regulates GluA2 surface expression . Effects of ER ( TM , Kf , DMJ ) or Golgi ( DMJ , Sw ) N-glycosylation inhibitors ( see Figure 5A ) on the glycosylation profile and surface expression of GluA2 .", "( A ) GluA2 before or after digestion with EndoH in control neurons ( Ct ) and in neurons ( DIV 24–27 ) exposed to Kf for two days , showing the effect of Kf on the glycosylation profile of surface proteins .", "Note the decreased intensity of the EndoH-insensitive ( upper ) band and the converse increased intensity of the EndoH-sensitive ( lower ) band .", "( B ) Average surface levels of these proteins in neurons after a two-days-exposure to DMSO ( control , Ct ) , tunicamycin ( Tm , 1 , 2 μM ) , Kifunensine ( Kf , 5 μM ) , 1-deoxymannojirimycin ( DMJ , 75 μM ) or swainsonine ( Sw , 88 μM ) .", "Tm reduces the surface expression of GluA2 .", "In contrast , Kf , DMJ and SW have no detectable effect on the surface expression of the protein .", "Mean ± SEM and individual data points; N=4–8 samples from 2–5 independent experiments .", "*p<0 . 05; Kruskal-Wallis/Dunn’s multiple comparison test . DOI: http://dx . doi . org/10 . 7554/eLife . 20609 . 02210 . 7554/eLife . 20609 . 023Figure 7—figure supplement 2 . Decay plateau ( fraction of peak amplitude ) as a function of time to peak ( s ) for synapses in control neurons ( Ct , blue ) or in neurons treated with Kf ( pink ) .", "Note higher proportions of synapses with short time to peak and rapid decay in Kf .", "N=22 synapses in 3 experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 20609 . 023 Do core-glycosylated proteins have specific functional properties ?", "As a first step towards addressing this , we determined whether Kf , which inhibits processing beyond core-glycans , alters postsynaptic signaling by combining local glutamate uncaging and calcium imaging .", "We focused on AMPA-dependent signaling by monitoring postsynaptic calcium responses elicited by repetitive glutamate uncaging ( ~25 stimuli at ~2 . 5 Hz ) in the presence of AP5 ( an NMDA receptor blocker ) ( Figure 7C–G ) .", "This stimulation induced strong postsynaptic responses , which were completely blocked by the AMPA/kainate receptor blocker CNQX ( Figure 7D , E ) .", "In blind recordings and analyses , we compared the responses of Kf-treated ( for 48 hr prior to recording ) and control neurons and discovered that , on average , the Kf-treated neurons exhibited responses with a shorter time to peak and a slight trend towards a larger decay ( Figure 7F; Figure 7—figure supplement 2 ) .", "It will be important for future studies to address how these parameters are mechanistically linked to increased core-glycosylation as multiple proteins including voltage-dependent calcium channels , calcium pumps and binding proteins ( Rose and Konnerth , 2001 ) might be involve in shaping the intracellular calcium responses induced by AMPA receptor activation and postsynaptic depolarization .", "Nevertheless , our results show that core-glycosylation is sufficient to maintain normal synaptic transmission and may regulate the kinetics and magnitude of postsynaptic signaling .", "In native AMPA-type glutamate receptor complexes , the inclusion of the GluA2 subunit prevents the direct permeation by calcium ( Isaac et al . , 2007 ) .", "Its presence or absence in synaptic receptor complexes is highly regulated , notably by protein synthesis ( Mameli et al . , 2007 ) .", "We thus took advantage of the co-existence of standard ( mature N-glycans ) and core-glycosylated GluA2 subunits at the neuronal surface to directly compare the turnover of two distinct glycosylated forms of a well-studied and functionally important synaptic protein .", "Because the overall lifetime of a protein is a key determinant of how fast and by which mechanisms its levels can be tuned in a compartment specific manner ( Hanus and Schuman , 2013; O'Leary et al . , 2013 ) , we compared the stability of core-glycosylated and mature glycosylated GluA2 with a chase assay after surface biotinylation ( Figure 8A–C ) .", "The two forms of the protein were quantified by immunoblotting after separation by treatment with EndoH .", "Consistent with values measured for GluA1 in spinal cord neurons ( Mammen et al . , 1997 ) , the overall stability of GluA2 ( core + standard glycosylated forms ) was on the order of tens of hours ( 15 . 8 ± 1 . 5 hr ) ( Figure 8B ) .", "However , the core-glycosylated pool of GluA2 exhibited a substantially shorter half-life than the standard form of the protein ( 3 . 4 versus 21 . 1 hr , respectively ) ( Figure 8C ) , demonstrating an accelerated turnover of the core-glycosylated form of the surface receptor . 10 . 7554/eLife . 20609 . 024Figure 8 . The core-glycosylation of surface proteins may accelerate their turnover and is regulated by synaptic activity .", "( A ) Immunoblot of biotinylated-GluA2 at various chase-times ( hours ) after surface biotinylation .", "Surface proteins were deglycosylated with EndoH to allow the distinction of mature versus core-glycosylated receptors .", "( B ) Decay of total biotinylated GluA2 levels ( log scale , n=2–4 experiments ) .", "( C ) Decay of mature versus core-glycosylated GluA2 .", "Mean ± SEM; N=6–8 in 4 experiments .", "Note the shorter lifetime of core-glycosylated receptors .", "Protein half-lifes are indicated in hours .", "*p<0 . 05 , **p<0 . 01 , ANOVA/Tukey’s multiple comparison test .", "( D–E )", "Total and relative surface expression ( surf . / total ) of GluA2 ( D ) or ConA-reactive glycans ( E ) after treatment with bicuculline ( bic ) or CNQX plus AP5 ( CNQX + AP5 ) .", "Bic and CNQX + AP5 levels normalized to control values .", "Mean ± SEM .", "N=7 in 4 experiments .", "*p<0 . 05 , **p<0 . 01 , Mann-Whitney test . DOI: http://dx . doi . org/10 . 7554/eLife . 20609 . 024 GluA2 surface expression is also regulated during homeostatic synaptic scaling ( Gainey et al . , 2009 ) .", "We thus determined whether modulation of synaptic activity also impacts the surface expression of core-glycosylated N-glycans .", "Neuronal activity was either increased by blocking inhibitory synaptic transmission with the GABAA receptor blocker bicuculline ( 20 μM ) or reduced with the ionotropic glutamate receptor blockers CNQX and AP5 ( 50 μM ) for 20 hr and then the levels of ConA-reactive species among surface and intracellular proteins were analyzed by far western blotting of total surface proteins .", "As expected ( Gainey et al . , 2009 ) , we found that decreased synaptic activity increased GluA2 surface expression ( Figure 8D ) .", "This was associated with an increase in the surface expression of total ConA-reactive ( core-glycosylated ) N-glycans ( Figure 8E ) , thus indicating an activity-dependent regulation of core-glycosylated membrane protein trafficking ." ], [ "It has been previously observed that , compared to other hexose-types , protein mannosylation was particularly pronounced in dendrites ( Torre and Steward , 1996; Villanueva and Steward , 2001 ) .", "Yet , it remained unclear whether mannose-rich N-glycosylated proteins are trafficked to the neuronal-surface .", "Here , we report that such 'immature' glycoproteins are abundant at the plasma membrane .", "We show that numerous synaptic adhesion proteins , surface neurotransmitter receptors , voltage-dependent ion channels and growth factor receptors present in the plasma membrane are core-glycosylated , thus displaying ( mannose-rich ) N-glycosylation patterns previously associated with nascent ion channels in the early secretory pathway ( Greger et al . , 2002; Tomita et al . , 2003; Sans et al . , 2001; Rouach et al . , 2005 ) .", "While it is commonly believed that the maturation of N-glycans in the Golgi apparatus is required for the genesis of functional surface membrane proteins , we found that mature N-glycans are largely dispensable for proper dendritic development and spontaneous synaptic transmission .", "Interestingly , the core-glycosylation of surface proteins is regulated by synaptic activity and , as seen for GluA2 , increases protein turnover .", "Altogether , these results point towards an important physiological role for core-glycosylation in neurons .", "Although AMPA receptor subunits and TARPs are thought to be co-assembled and co-trafficked in the secretory pathway ( Zheng et al . , 2015 ) , our results show that these proteins display distinct glycosylation profiles and distinct sensitivities to BFA .", "This indicates that these proteins are processed by distinct mechanisms and likely use distinct secretory routes ( Jeyifous et al . , 2009 ) to reach the plasma membrane .", "Intriguingly , the complete and selective loss of 'mature' N-glycosylated GluA2/3 in hippocampal neurons of TARP γ8 knockout mice results in a relatively modest decrease ( 34% ) in AMPA excitatory postsynaptic potential amplitude ( Rouach et al . , 2005 ) .", "This suggests that core-glycosylated receptors , which account for 33% of total surface receptors , mediate 66% of synaptic transmission , which represents a relative contribution to synaptic currents ( and an enrichment at synapses ) that is ~4-fold higher .", "The present study thus challenges the notion that the core-glycosylated GluA2/3 subunits that remain unaltered in the TARP γ8 knockout are retained in the ER , and rather indicate that these receptors are localized at synapses and are functional both in wild type and in TARP γ8 knockout mice .", "Similarly , AMPA , kainate and GABAA receptors display abnormally increased or decreased EndoH sensitivities in schizophrenic patients ( Tucholski et al . , 2013a , 2013b; Mueller et al . , 2014 ) .", "Our results question the interpretation that this merely reflects altered intracellular levels of immature receptors and rather suggest that these glycosylation defects impact functional synaptic receptors present on the plasma membrane .", "Although dendritic Golgi outposts can be found in some neurons , most dendrites contain early secretory compartments ( i . e . ER and ERGICs ) but lack generic Golgi membranes ( Hanus and Ehlers , 2008; Hanus et al . , 2014 ) .", "We previously showed that multiple postsynaptic signaling pathways control the local processing of nascent secretory cargo in dendrites ( Cui-Wang et al . , 2012; Hanus et al . , 2014 ) .", "However , it remained unclear whether and how nascent membrane proteins could be N-glycosylated in segments of dendrites lacking Golgi compartments .", "Thus far , few mammalian proteins have been shown to be trafficked to the plasma membrane via unconventional secretory processing ( USP ) ( Grieve and Rabouille , 2011 ) .", "The atypical prevalence of core-glycosylated proteins at the neuronal surface that we report here indicates that USP ( e . g . Golgi by-pass or hypo-function of Golgi enzymes ) is a much more widespread phenomenon than initially anticipated , and allows neurons to modulate the properties of key membrane proteins .", "The correlation that we observed between the glycosylation status and the turnover of GluA2 is particularly interesting in this regard ( Hanus and Schuman , 2013 ) .", "The lifetime of a protein has a critical impact on its regulation in space and time .", "For example , a synaptic protein that is stable for multiple days has the time to potentially explore multiple synapses and go through several rounds of internalization and recycling before being degraded ( Ehlers , 2000 ) .", "It is thus hard to imagine how the local dendritic synthesis of such a protein could lead to rapid and localized changes of synaptic composition and properties .", "On the other hand , a protein that is much less stable will likely be more efficiently regulated by a fine-tuning of its local synthesis and degradation .", "It is thus conceivable that the unconventional glycosylation of key proteins and the resulting decrease of their lifetime may tune the spatial length-scale over which local protein synthesis may functionalize synapses ( Govindarajan et al . , 2011 ) .", "In the recent years , our group and others have implemented and developed new tools and strategies to assess the distribution and translation of mRNAs ( Cajigas et al . , 2012; Buxbaum et al . , 2014; Wu et al . , 2016 ) , the site of synthesis and the redistribution of specific nascent proteins ( tom Dieck , 2015 ) , and their dynamics in dendritic secretory organelles ( Cui-Wang et al . , 2012; Hanus et al . , 2014 ) .", "Here , we show that the glycosylation status of functional neuronal proteins is the result of distinct post-translational processing mechanisms that likely reflect the availability of secretory machinery in the different subcellular compartments that support protein synthesis .", "It will thus be interesting for future studies to investigate to what extent synthesis and secretory processing in the soma versus specific segments of dendrites determine protein glycosylation and hence their dynamics and function .", "Previous studies in cancer cells have shown that surface proteins may be internalized , trafficked to the GA for further glycan maturation and sent back to the plasma membrane ( Snider and Rogers , 1986 ) .", "We cannot exclude that a similar mechanism exists in neurons and may convert core-glycosylated AMPA receptors into mature proteins .", "We note however that core-glycosylated GluA2 represents ~one-third of the GluA2 – an abundant neuronal protein – at the steady state .", "Owing to the paucity of dendritic Golgi membranes compared to endosomal compartments ( Ehlers , 2000; Cooney et al . , 2002 ) , we find it unlikely that the large fraction of neuronal proteins identified here visit the Golgi following internalization .", "It will thus be interesting to investigate whether the rapid turnover of core-glycosylated synaptic receptors is due to a faster degradation or to another mechanism .", "Given the prevalence of N-glycosylation in the brain ( Zielinska et al . , 2010 ) , and its influence on membrane protein folding , trafficking , ligand-binding and ion channel conductivity ( Moremen et al . , 2012; Scott and Panin , 2014 ) , it is clear that the atypical glycosylation of a large number of neuronal surface proteins is physiologically meaningful .", "In a broader context , it is worth noting that N-glycosylation is dysregulated in numerous human pathologies , and in particular in various cancers .", "Most notably , an increased branching of complex N-glycans is typically associated with a poor prognosis for breast and colon cancers in humans ( Fernandes et al . , 1991; Seelentag et al . , 1998 ) .", "Consistently , studies in mice show that Golgi-associated glycosyltransferases such as N-GlcNac , sialyl- and fucosyl-transferases are instrumental to tumor invasiveness ( Granovsky et al . , 2000; Tsui et al . , 2008 ) .", "The prevalence of core-glycosylated surface proteins in neurons may thus provide important insights on how N-glycans terminal branching is regulated and can be opposed .", "The immunological isolation of the brain likely plays a permissive role in surface proteins acquiring atypical glycosylation patterns in neurons , as those may otherwise trigger an immunological response .", "Indeed , multiple lines of evidence suggest that altered glycosylation profiles are important drivers of autoimmune diseases ( Rabinovich et al . , 2012; Maverakis et al . , 2015 ) .", "The enrichment of proteins involved in such pathologies among the core-glycoproteins that we identified thus indicates that investigating core-glycosylation in neurons may provide important cues on autoimmunity ." ], [ "Dissociated hippocampal neurons and cell lines ( COS 7 , BHK , CHO , L cells; obtained from the American Type Culture Collection ) were prepared and maintained essentially as previously described ( Cui-Wang et al . , 2012; Hanus et al . , 2014 ) .", "Neurons were maintained for up to two months in vitro ( Figure 1—figure supplement 1 ) .", "Lack of cell line contamination with mycoplasma was checked by PCR ( eMyco detection kit , Intron Biotechnology ) .", "Acute hippocampal slices were prepared from three week-old Sprague Dawley rats and the CA1 area carefully microdissected by hand as previously described ( Cajigas et al . , 2012 ) .", "The VSVG-GFP , GalT-GFP , PSD95-mCh and pHluo-TM plasmids were described previously ( Cui-Wang et al . , 2012 ) .", "GCaMP6-S ( Chen et al . , 2013 ) was purchased from Addgene ( plasmid 40753 ) .", "COS 7 and neurons were transfected with Extreme Gene 9 or Lipofectamine 2000 ( Life Technologies ) , respectively , according to the manufacturer's instructions .", "All drugs were used in the neuron maintenance medium .", "BFA ( Sigma or Tocris ) , bicuculline , 6-cyano-7-nitroquinoxaline-2 , 3-dione ( CNQX ) and amino-5-phosphonovaleric acid ( AP5 ) ( Tocris ) were used at final concentrations of 5 μg/mL , 20 μM , 50 μM , 50 μM , respectively .", "Tunicamycin ( Tm ) , Kifunensine ( Kf ) , deoxymannojirimycin ( DMJ ) and swainsonine ( Sw ) ( Tocris ) were used at final concentrations of 1 , 2 μM , 5 μM , 75 or 100 μM and 88 μM , respectively .", "Peptide-N-Glycosidase F ( PNGase , New England Biolab ) and Endoglycosidase H ( EndoHf , NEB ) were used according to the manufacturer's instructions .", "In brief , proteins were denaturated in PBS supplemented with 1% Triton , ~0 . 6% SDS and 50 mM DTT for 15 min at 75°C , and diluted ( ~1 . 5 fold ) in sodium phosphate ( 50 mM pH 5 . 5 final ) or sodium citrate buffer ( 50 mM pH 7 . 5 ) plus NP40 ( or triton , ~1% final ) for PNGase and EndoHf , respectively .", "Proteins were typically digested with 1000 ( PNGase ) or 3000 ( EndoHf ) units/ug protein at 37°C overnight .", "The following lectins ( fluorescein or biotin conjugates ) were used for cytochemistry ( CC ) or far-western blotting ( FWB ) at the indicated final concentrations .", "Concanavalin A ( biotin-ConA , Sigma; CC , 0 . 33 μg/mL; FWB , 1 μg/mL ) , galanthus nivalis agglutinin ( biotin-GNA , Galab; CC , 1 μg/mL ) , RCA120-fluorescein ( RCA120-FITC , Vector laboratories; CC , 0 . 7 μg/mL ) and RCA ( biotin-RCA , Vector laboratories; FWB , 1 μg/mL ) , wheat germ agglutinin ( biotin-WGA , Sigma; CC 0 . 4 μg/mL; FWB 1 μg/mL ) , Streptavidine Alexa647 ( Life Technologies , CC , 1 μg/mL ) , IRDye-streptavidin ( Licor , FWB , 1:15 , 000 ) .", "The following antibodies were used for immunocytochemistry ( ICC ) or immunoblotting ( IB ) at the indicated dilutions .", "Mouse anti-βactin ( Sigma , IB , 1:10 , 000 ) , mouse anti-bassoon ( Enzo Life , ICC , 1:1000 ) , rabbit anti-biotin ( Cell signaling , ICC , 1:1000 ) , rabbit anti-cacng8/stargazin ( Millipore , IB , 1:1000 ) , rabbit anti-GABAA receptor β3 subunit ( Synaptic Systems , IB , 1:750 ) , rabbit anti-GABAA receptor γ2 subunit ( Synaptic Systems , IB , 1:1000 ) , chicken anti-GFP ( Aves Labs , IB , 1:5000 ) , rabbit anti-GluA1 ( Abcam , IB , 1: 1000 ) , rabbit anti-GluA2 ( Abcam , IB , 1:2000 ) , rabbit anti-GluA4 ( Synaptic Systems , IB , 1:1000 ) , mouse anti-GluN1 ( BD Pharmingen , IB , 1:1000 ) , rabbit anti-GluN2A ( Millipore , IB , 1:1000 ) , rabbit anti-GluN2B ( Millipore , IB , 1:1000 ) , guinea pig anti-MAP2 ( SYSY , ICC , 1:2000 ) , mouse anti-MAP2 ( Sigma , ICC , 1:1000 ) , mouse anti-Neuroligin 1 ( Neuromab , IB , 1:200 ) , IRDye secondary antibodies ( Li-Cor , IB , 1:15 , 000 ) , goat anti-guinea pig-Alexa 647 ( Life Technologies , ICC , 1:750 ) , goat anti-mouse ( GAM ) -RRX ( Jackson Laboratory , ICC , 1:1000 ) , goat anti-rabbit-RRX and GAM-FITC ( Jackson Laboratory , ICC , 1:800 ) .", "For labeling of surface glycoproteins , cells were rinsed in ACSF ( 119 mM NaCl , 2 . 5 mM KCl , 1 . 3 mM MgSO4 , 2 . 5 mM CaCl2 , 1 . 0 mM NaH2PO4 , 26 . 2 mM NaHCO3 , and 11 . 0 mM glucose ) or in Hibernate A without phenol red ( Brain Bits ) and incubated with lectin biotin conjugates diluted in ACSF or Hibernate A , for 10 min at room temperature .", "After washes , cells were fixed in 4% PFA , blocked in PBS supplemented with 1% fish skin gelatin ( Sigma ) for 15 min and incubated for 30 min with streptavidin-Alexa647 in the same buffer .", "For co-immunolabeling , cells were incubated live with ConA-biotin , fixed and permeabilized in 0 . 2% Triton in PBS for 15 min , blocked in a buffer containing 10% goat serum ( Life Technologies ) and 3% fish skin gelatin for 30 min and incubated with primary ( anti-bassoon , anti-biotin and anti-MAP2 ) and secondary antibodies diluted in a 1:2 dilution of the same blocking buffer .", "Confocal imaging was performed with a 40x 1 . 4 NA objective on Zeiss LSM780 or LSM880 laser point scanning confocal microscopes .", "Cell average fluorescence was quantified in Metamorph ( Molecular Devices ) using Z-stack maximal intensity projections .", "Surface biotinylation was performed essentially as described previously ( Cui-Wang et al . , 2012; Hanus et al . , 2014; Ehlers , 2000 ) .", "In brief , cells were rinsed in ACSF or E4 buffer ( 120 mM NaCl , 3 mM KCl , 15 mM glucose , 10 mM HEPES , 2 mM CaCl2 , 2 mM MgCl2 CaCl2 ) and incubated with 0 . 8 to 1 mg/mL NHS-SS-biotin ( Thermo ) in the same buffer for 7 min at room temperature .", "Cells were then rinsed and quenched in ACSF or E4 supplemented with 10–20 mM L-lysine , scraped in PBS supplemented with L-lysine and , when appropriate , protease inhibitors ( Calbiochem ) .", "Cell pellets harvested after centrifugation were then either stored at −80°C or directly lyzed .", "Pulse chase experiments were performed essentially as described previously ( Cui-Wang et al . , 2012; Mammen et al . , 1997 ) .", "In brief , cells were biotinylated , washed in ACSF supplemented with 0 . 1% BSA , and put back in fresh ( cell lines ) or conditioned ( neurons ) maintenance medium and kept at 37°C for varying times .", "Slices were surface-biotinylated in oxygenated ACSF at 4°C for 20 min , quenched in L-lysine at 4°C for 10 min , homogenized and then lyzed as descried above .", "Surface ( biotinylated ) proteins were eluted from streptavidin agarose or magnetic beads ( Thermo ) by reduction of S-S-biotin with 50 mM DTT ( in PBS supplemented with ~1% Triton and ~0 . 6% SDS ) for 15 min at 75°C , resulting in the complete removal of biotin from surface proteins ( Figure 4B and Figure 6—figure supplement 1 ) .", "Immunoblotting was performed essentially as described previously using far-red fluorescent dyes and a Licor Odyssey scanner ( Cui-Wang et al . , 2012 ) .", "For far-western blotting , lectin biotin-conjugates , antibodies and streptavidin were diluted in PBS-Tween without the addition of blocking agents .", "Protein levels in bands of interest were quantified in Image Studio ( Licor ) or ImageJ ( NIH ) .", "Known amounts of proteins were separated into a surface ( biotinylated ) and an intracellular fraction ( remaining supernatant ) and processed in parallel for immunoblotting or far western blotting .", "The total fluorescent intensity of individual bands ( immunoblotting ) or smear of proteins ( far western blotting with lectins , or BONCAT , e . g . between ~250 and ~ 70–25KDa ) was then quantified and protein relative surface expression calculated as follows: with S , I the total fluorescence intensity and 1/a , 1/b sample dilution factors in surface and intracellular fractions , protein relative surface expression was calculated as the ratio: S / ( S + bI/a ) .", "Surface ( biotinylated ) proteins were eluted from streptavidin-agarose beads at 70°C with 50 mM DTT for 15 min and incubated in the absence ( group A ) or in the presence ( group B – background ) of EndoHf ( NEB ) overnight at 37°C .", "EndoHf was then heat-inactivated at 80°C for 25 min .", "Proteins were then incubated with ConA biotin-conjugate in PBS supplemented with 1% Triton x100 and 0 . 1% SDS for 3 hr at 4°C in the absence , or , for control experiments , in the presence of BSA-mannose ( Vector laboratories ) and purified with high-capacity streptavidin-agarose or streptavidin-magnetic beads ( Thermo ) .", "For each sample group A ( target group: surface proteins binding to ConA without prior treatment with EndoH ) and B ( background group: surface proteins binding to ConA after treatment with EndoH ) , 2 independent biological replicates ( surface proteome preparations from separate primary neuron preparations ) , 2 experimental replicates ( replicate affinity purifications and digestions for each surface preparation ) and 3 to 4 technical replicates ( replicate LC-MS runs on identical peptide preparations ) were analysed , so a total of 13–14 replicates per sample .", "Proteins were incubated in 6 M urea , 2 mM DTT , alkylated using 5 mM iodoacetamide and sequentially digested overnight using LysC ( 1:15 protease:protein ratio ) .", "After bringing the urea concentration to a final concentration of 3 M , the samples were incubated with Trypsin ( 1:15 ) overnight .", "The crude peptide mixtures were purified using c18-ZipTips ( Millipore ) , dried using a SpeedVac and stored at −80°C .", "Four sets of proteolytic fragments ( two technical replicates for 2 independent neuronal preps ) were loaded 3–4 time each on reverse phase HPLC columns ( trapping column: particle size <2 µm , C18 , L=20 mm; analytical column: particle size <2 µm , C18 , L = 50 cm; ThermoFisher Scientific ) using a nano-UPLC device ( Dionex Ultimate 3000 RSLC , ThermoFisher Scientifc ) , and eluted in binary solvent gradients ( buffer I: 5% acetonitrile , 95% water , 0 . 1% formic acid; buffer II: 80% acetonitrile , 20% water , 0 . 1% formic acid ) .", "Typically , gradients were ramped from 5% to 55% buffer II within 200 min at flow rates of 300 nl/min .", "Peptides eluting from the column were ionised 'online' using a FlexIonSource ( Thermo ) and analyzed in a hybrid ion trap mass spectrometer ( Orbitrap Elite , ThermoFisher Scientific ) .", "Sequence information was obtained by computer-controlled , data-dependent switching to MS2 mode ( TOP15 , FT-IT-mode ) using collision energies based on mass and charge state of the candidate ions .", "Proteins were identified by matching the derived mass lists against a NCBI or 'refseq' database ( downloaded from http://www . ncbi . nlm . nih . gov ) on a local Mascot server ( Matrix Sciences , United Kingdom ) .", "In general , a mass tolerance of 2 ppm for parent ions and 0 . 5 Da for fragment spectra , two missed cleavages and oxidation of methionine as a variable modification and carbamidomethylation of cysteine as a fixed modification were selected as matching parameters in the search program .", "For quantitative evaluation , the dataset was processed using the MaxQuant software package , Perseus and custom scripts in Matlab ( https://github . molgen . mpg . de/MPIBR-Bioinformatics/AtypicalNeuroNGlycans ) .", "We then used a high-stringency label-free quantification ( LFQ ) approach to identify proteins that were significantly enriched in samples A ( target , core-glycosylated ) over samples B ( i . e . whose binding to ConA was impaired by EndoH and thus represent “background ) .", "The efficiency of EndoH was high but not absolute , resulting in B samples consisting of missing values and low-intensity peptides .", "Although protein abundance was clearly higher in A than in B , this bias ( zero values for B sample peptides ) complicated the use of average peptide intensities as in a typical LFQ approach .", "We thus developed a peptide based-strategy and sorted peptides according to their repeatability across experiments ( intraclass correlation ( ICC ) index between biological replicates x ICC index between technical replicates ) and their enrichment in A or B: ( [average intensity in A – average intensity in B] / max intensity in A or B ) .", "Peptides were then separated by hierarchical clustering based on Euclidian distance ( 0 . 2 threshold in the distance matrix ) .", "Peptides with the highest enrichment and repeatability were clearly separated as a 'natural' cluster ( Figure 4—figure supplement 1A ) , and were used to define our high-confidence 'core-glycosylated' proteins: resulting in the identification of 227 protein groups and 647 protein IDs ( Supplementary files 1A and 1C ) .", "As expected , the retrospective analysis of all the peptides that corresponded to these proteins showed a higher enrichment in A and a higher repeatability than the rest of the 'background' proteins ( Figure 4—figure supplement 1B and C ) .", "As an additional validation , we crossed referenced these proteins with those identified using a standard LFQ approach .", "Here , we selected proteins based on peptides that were detected in at least 10 out of 13–14 'A' replicates , and whose average peptide intensity was at least 1 . 3 times higher ( Kruskal Wallis test ) in A than in B samples .", "88 . 4% of the protein groups that we identified with our peptide-based strategy were also detected by this approach ( Supplementary file 1B ) .", "To compare our dataset to the full hippocampal proteome , we selected proteins identified by both mass spectrometry ( Sharma et al . , 2015 ) and mRNA deep sequencing in mouse hippocampi ( You et al . , 2015 ) , yielding 19 , 690 proteins .", "Likely owing to the double purification procedure that was used to purify surface core-glycosylated proteins , a few proteins present in our A and/or B sets were not found among these proteins and were thus added to the later list to generate our final input dataset ( Supplementary file 1D ) .", "Protein topology , N-glycosylation sites and subcellular localization were defined with Signal IP and TMHMM ( Sprenger et al . , 2008 ) , NetNGlyc1 . 0 ( http://www . cbs . dtu . dk/services/NetNGlyc/ ) and CELLO ( Yu et al . , 2006 ) , respectively .", "For gene pathway and ontology analyses ( Figure 4 and Supplementary file 1E ) , overrepresented protein functional families were determined using the Kyoto Encyclopedia of Genes and Genomes’ ( KEGG ) pathway database ( Kanehisa et al . , 2012 ) or Gene Ontology annotation ( Ashburner et al . , 2000 ) using full protein lists , or subclasses determined according to expected topology ( soluble intracellular versus secreted + transmembrane proteins ) or predicted N-glycosylation sites .", "ConA binds with high affinity to terminal α-linked mannoses , thus preferentially to core glycans but may , in theory , also bind to hybrid N-glycans .", "In this study , we can clearly distinguish between these two types of N-glycans: as exemplified in Figures 1A and 5A , hybrid N-glycans are typically recognized by RCA .", "However , as shown in Figure 2—figure supplement 2 , RCA-binding proteins are EndoH insensitive in hippocampal neurons , in contrast to ConA binding proteins that are EndoH sensitive .", "Further , as shown in Figure 1—figure supplement 2 and Figure 5—figure supplement 1 , RCA-reactive proteins respond completely differently from ConA-reactive species to Kf , DMJ and Sw .", "We cannot exclude that rare hybrid and yet unrecognized glycans that are Endo-H sensitive and selectively bind ConA but not RCA may be found in neurons .", "Yet , our data strongly indicate that core-glycans are atypically abundant at the neuronal surface .", "Hippocampal neurons were transfected with GFP at DIV7 and were treated with vehicle ( control , DMSO ) , Tm , Kf , DMJ , Sw at DIV8 , fixed at DIV11 and immunolabeled for the somatodendritic marker protein MAP2 .", "Dendrite morphology was assessed basically as previously described ( Cui-Wang et al . , 2012 ) using the Simple Neurite Tracer plugin ( Longair et al . , 2011; Ferreira et al . , 2014 ) in ImageJ/Fiji ( NIH ) .", "The experimenter was blind to the experimental conditions tested both during image acquisition and analysis .", "Cells were preincubated for 1 hr with 2 . 5 ug/mL BFA ( or vehicle , DMSO ) and then metabolically labeled with 4 mM azido-homo-alanine ( AHA ) ( tom Dieck , 2015 ) for 120 to 150 min ( or for control for 5 hr with 4 mM AHA versus methionine , Figure 6—figure supplement 1 ) in Neurobasal without methionine supplemented with glutamax and B27 .", "Cells were then surface biotinylated and surface ( S ) and intracellular proteins ( I ) separated as described above .", "S and I proteins ( 50 μg protein/lysate equivalent ) were then precipitated in cold acetone .", "Precipitated samples were resuspended in 120 μL of PBS ( pH 7 . 8 ) supplemented with 0 . 07% SDS and 0 . 1% triton and protease inhibitors , cleaned on desalting columns ( PD Spin trap G25 ) and were then biotinylated by azide-alkyne cycloaddition ( 'CLICK' reaction ) as previously described ( tom Dieck , 2015 ) , immunobloted and detected with an anti-biotin antibody .", "DIV10 neurons were transfected with PSD-mCh and GCaMP6 as described above and treated with Kf for 48 hr .", "Neurons were then washed and monitored at 37°C in standard E4 medium ( 150 mM NaCl , 3 mM KCl , 15 mM glucose , 10 mM HEPES , pH 7 . 4 ) supplemented with 3 mM CaCl2 , 0 . 5 mM MgCl2 and 50 μM AP5 and 2 . 5 mM MNI-glutamate ( Tocris ) .", "Confocal imaging was performed using a 60x 1 . 4 NA objective on a Zeiss Observer Z1 inverted microscope equipped with a CSUX1 spinning disk unit ( Yokugawa , Inc ) , an EM-CCD camera ( Evolve 512 , Photometrics ) , and a custom diode-laser illumination module ( 3I Intelligent Imaging Innovations , Inc ) .", "MNI-glutamate was uncaged by local irradiation at diffraction limited spots with a tunable 2-photon laser set at 720 nm ( Chameleon Ultra , Coherent ) coupled to a high speed x , y scanner ( Vector ) .", "Dendrites were stimulated at individual synapses identified by PSD-mCh fluorescence .", "Imaging was done in alternance with 26 uncaging pulses delivered at 2 . 6 Hz .", "The peak amplitude and time to peak of stimulated calcium responses ( GCaMP6 fluorescence ) were determined after normalization to baseline by detection of fluorescence maxima .", "Responses decay and plateau were calculated after normalizing responses to peak values and fitting of the resulting decay plots with a mono-exponential function using Prism ( GraphPad ) .", "The experimentalist was blind to experimental conditions during data acquisition and analysis .", "Whole-cell recordings were performed in DIV 20–21 neurons after a ~48 hr exposure to Kf .", "Neurons were held at −70 mV in voltage clamp and mEPSCs were recorded for at least 10 min using an Axopatch 200B amplifier .", "The extracellular solution contained ( in mM ) 140 NaCl , 3 KCl , 10 HEPES , 2 CaCl2 , 1 MgSO4 , 15 glucose , 0 . 002 TTX , 0 . 04 bicuculline and 0 . 05 AP5 ( pH 7 . 4 ) .", "Recording pipettes , with resistances between 3–8 MΩ , were filled with a solution containing ( in mM ) 120 Potassium gluconate , 20 KCl , 10 HEPES , 2 MgCl2 , 0 . 1 EGTA , 2 Na2-ATP and 0 . 4 Na2-GTP ( 300 mOsm/l , pH 7 . 2 ) .", "Data were analyzed offline with a template-matching algorithm ( Guzman et al . , 2014 ) using Stimfit ( Guzman et al . , 2014 ) or in Matlab using a custom script .", "Selected mEPSC events were individually screened with an amplitude threshold of >5 pA and an exponential decay .", "The experimenter was blind to the experimental conditions tested during both acquisition and analysis .", "One outlier cell in the control group had a mEPSC frequency close to 4 standard deviations higher than average and was excluded from the data set .", "Data are presented as means ± SEM unless otherwise indicated .", "The number of measured values and independent experiments used for quantification are indicated in the text or in the figure legends .", "Mann Whitney non-parametric test was used to compare two means .", "Data normality was assessed with Shapiro Wilk’s or Kolmogorov Smirnov’s tests .", "When data passed normality test , one-way ANOVAs and post hoc Tukey or Sidak’s multicomparison tests were performed to determine whether significant differences existed among all or preselected pairs of means ( i . e . control versus BFA ) across multiple conditions ( i . e . N>2 ) .", "Otherwise , multiple comparisons were assessed with Kruskal-Wallis and Dunn’s multiple comparison tests .", "Hypergeometric test was used to assess the significance of relative frequencies in total or subpopulation of proteins ( e . g . core-glycosylated versus total proteins ) .", "See Statistical reporting in Supplementary file 1F ." ] ]

[ "N-glycosylation – the sequential addition of complex sugars to adhesion proteins , neurotransmitter receptors , ion channels and secreted trophic factors as they progress through the endoplasmic reticulum and the Golgi apparatus – is one of the most frequent protein modifications .", "In mammals , most organ-specific N-glycosylation events occur in the brain .", "Yet , little is known about the nature , function and regulation of N-glycosylation in neurons .", "Using imaging , quantitative immunoblotting and mass spectrometry , we show that hundreds of neuronal surface membrane proteins are core-glycosylated , resulting in the neuronal membrane displaying surprisingly high levels of glycosylation profiles that are classically associated with immature intracellular proteins .", "We report that while N-glycosylation is generally required for dendritic development and glutamate receptor surface expression , core-glycosylated proteins are sufficient to sustain these processes , and are thus functional .", "This atypical glycosylation of surface neuronal proteins can be attributed to a bypass or a hypo-function of the Golgi apparatus .", "Core-glycosylation is regulated by synaptic activity , modulates synaptic signaling and accelerates the turnover of GluA2-containing glutamate receptors , revealing a novel mechanism that controls the composition and sensing properties of the neuronal membrane ." ]

[ "Information is carried around the nervous system by cells called neurons .", "The ability of neurons to communicate with each other relies on many proteins that are found on the surfaces of the cells .", "Like in all animal cells , surface proteins are made inside the cell in a compartment called the endoplasmic reticulum .", "During this process , one or several complex sugar molecules are usually added to newly made proteins .", "These sugar molecules are then modified as the proteins leave the endoplasmic reticulum and pass through another compartment called the Golgi apparatus on the way to the cell membrane .", "The precise number and structure of the sugar molecules attached to the protein define its glycosylation profile .", "Neurons receive information from other neurons at branch-like structures called dendrites , which trigger electrical signals that travel through the rest of the cell .", "To directly control how dendrites generate these signals , neurons make surface proteins locally in dendrites .", "However , while the endoplasmic reticulum is found all over the neuron , including in the dendrites , the Golgi apparatus is generally only present in the main cell body .", "It is not known how surface proteins are made in the dendrites or how the proteins’ glycosylation profiles are altered in the absence of a Golgi apparatus .", "Hanus et al . used microscopy and biochemical techniques to study the glycosylation profiles of surface proteins in rat neurons .", "The experiments revealed that immature glycosylation profiles are found on hundreds of different proteins that have been transported to the cell surface .", "This includes many proteins that are needed to transmit electrical signals between neurons .", "Next , Hanus et al . selectively blocked the modification of sugar molecules on proteins in the Golgi apparatus .", "This showed that dendrites are able to form and work properly even if surface proteins have primarily immature glycosylation profiles .", "Further experiments suggest that immaturely glycosylated proteins might travel to the surface of neurons using a different route that bypasses the Golgi apparatus .", "The next step will be to investigate exactly how these proteins are delivered to the surface and how they influence the way neurons behave ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "ecology", "microbiology and infectious disease" ]

[ [ "Sourdough bread is a globally distributed fermented food that is made using a microbial community of yeasts and bacteria .", "The sourdough microbiome is maintained in a starter that is used to inoculate dough for bread production ( Figure 1A ) .", "Yeasts , lactic acid bacteria ( LAB ) , and acetic acid bacteria ( AAB ) in the starter produce CO2 that leavens the bread .", "Microbial activities including the production of organic acids and extracellular enzymes also impact bread flavor , texture , shelf-stability , and nutrition ( Arendt et al . , 2007; De Vuyst et al . , 2016; Gobbetti et al . , 2014; Hansen and Schieberle , 2005; Salim-ur-Rehman et al . , 2006 ) .", "Starters can be generated de novo by fermenting flour and water or acquired as established starters from community members or commercial sources .", "Home-scale fermentation of sourdough is an ancient and historically important practice ( Cappelle et al . , 2013 ) that experienced a cultural resurgence during the COVID-19 pandemic ( Easterbrook-Smith , 2020 ) .", "Despite being an economically and culturally significant microbiome , a comprehensive survey of sourdough starter microbial communities has not yet been conducted .", "Previous studies have primarily focused on starters from regions within Europe ( De Vuyst et al . , 2014; Gänzle and Ripari , 2016; Hammes et al . , 2005; Minervini et al . , 2014 ) and the diversity of sourdough starters in North America is poorly characterized ( Kline and Sugihara , 1971; Sugihara et al . , 1971 ) .", "Most previous studies have applied a range of culture-based techniques to characterize sourdough microbial diversity ( De Vuyst et al . , 2014; Gänzle and Ripari , 2016 ) making it difficult to understand distributions of sourdough bacterial and fungal taxa due to the variability and biases in these approaches .", "Sourdough starters are maintained in many households , but these starters have generally been overlooked in previous studies which have focused on large bakeries and industrial settings .", "Household starters are likely distinct from those found in bakeries due to a greater heterogeneity in environments , production practices , and ingredients .", "Two major factors , geographic location and maintenance practices , are often invoked as major drivers of sourdough biodiversity .", "Sourdough communities in the same region may be similar in composition due to restricted dispersal of microbes or in response to regional microclimates .", "Producers often tout their breads’ distinct regional properties , crediting the environment for unique bread characteristics .", "There is some evidence of local geographic structure of sourdough microbes ( Liu et al . , 2018; Scheirlinck et al . , 2007b ) , but biogeographic patterns of sourdough diversity have not been quantified at a continental-scale .", "Likewise , experimental evidence from individual sourdoughs suggests that abiotic conditions ( process parameters including differences in starter maintenance techniques , ingredients , and environmental conditions ) and microbial interactions can impact starter community structure ( De Vuyst et al . , 2014; Gänzle and Ripari , 2016; Minervini et al . , 2014; Ripari et al . , 2016; Van Kerrebroeck et al . , 2017 ) , but the relative importance of these processes across diverse starters is unknown .", "Through the collaborative power of a global network of community scientists , we collected 500 sourdough starters from across the world with dense sampling of the United States ( United States = 429; Canada = 29; Europe n = 24; Australia and New Zealand = 17; and Thailand = 1; Figure 1B ) to comprehensively characterize the microbial diversity of household sourdough starters .", "These starters varied greatly in their reported ages and maintenance histories ( Figure 1C–G ) .", "Through both cultivation-dependent and cultivation-independent methods , we revealed the ecological distributions of widespread sourdough yeasts and bacteria .", "By analyzing a broad suite of starter metadata , our intensive sampling identified the roles of geography and process parameters in shaping starter diversity .", "Using synthetic sourdough communities , we identified a dynamic network of species interactions within sourdough microbiomes that helps explain the distributions of major yeasts and bacteria .", "We also determined linkages between sourdough starter microbial diversity and baking-relevant functions including the rate of dough rise and volatile organic compound ( VOC ) production .", "This study is the first to combine a large-scale survey of sourdough starter microbial diversity with quantitative analysis of the factors that shape the composition and function of starter microbiomes ." ], [ "We first identified the microbial communities of sourdough starters by 16S and ITS rRNA gene amplicon sequencing of samples that were shipped to us and frozen upon arrival .", "When considering both fermentation-relevant microbes ( yeasts , LAB , and AAB ) as well as other microbial taxa , each starter sample contained a median of seven bacterial and 35 fungal amplicon sequence variants ( ASVs ) .", "LAB ( order: Lactobacillales ) and AAB ( order: Rhodospirillales ) together comprised over 97% of bacterial reads ( per sample mean ) , with yeasts ( order: Saccharomycetales ) comprising over 70% of fungal reads ( Figure 2—figure supplement 1 , Figure 2—source data 1 , 2 ) .", "The other fungi and bacteria detected were common indoor and outdoor molds , plant pathogens , and plant endophytes as well as microbes associated with human skin , drinking water , and soil .", "Unless otherwise indicated , we did not include these environmental microbes in our further analyses because of their limited roles in sourdough fermentation .", "Sourdough communities exhibited consistent patterns of strong species dominance or co-occurrence ( Figure 2A ) .", "Many communities were dominated by a single yeast and/or bacterial species with a median of three LAB/AAB and one yeast per starter ( Figure 2A-Figure 2—figure supplement 2 ) .", "For example , Saccharomyces cerevisiae accounted for >50% of fungal ITS reads in 77% of samples .", "The LAB L . sanfranciscensis was the dominant bacterium in most sourdoughs where it occurred and was negatively associated with the widespread L . plantarum and L . brevis ( p<0 . 001; Figure 2A , Figure 2—figure supplement 1 , Figure 2—source data 3 ) .", "The LAB Lactobacillus plantarum and L . brevis were the most commonly observed pair of co-occurring taxa ( in 177 of 500 starters , p<0 . 001; Figure 2A-Figure 2—figure supplement 3 ) .", "Interactions predicted in the literature , including L . sanfranciscensis:Kazachstania humilis co-occurrence ( Brandt et al . , 2004; De Vuyst et al . , 2016 ) and L . sanfranciscensis:S .", "cerevisiae co-exclusion ( Gobbetti et al . , 1994 ) , were supported by in situ patterns of diversity ( L . sanfranciscensis:K . humilis p<0 . 01 , L . sanfranciscensis:S . cerevisiae p=0 . 01 ) .", "One striking pattern across our dataset was the highly variable abundance of AAB across individual starters .", "These bacteria have been reported in sourdough ( Minervini et al . , 2014; Ripari et al . , 2016 ) , but are generally understudied as indicated by their almost complete absence in many key reviews of sourdough microbial diversity ( De Vuyst et al . , 2014; Gänzle and Ripari , 2016; Van Kerrebroeck et al . , 2017 ) .", "In our sample set , 147 starters contained AAB ( >1% relative abundance ) including Acetobacter , Gluconobacter , or Komagataeibacter species ( Figure 2A , Figure 2—source data 2 ) .", "AAB require specialized culture conditions ( Kim et al . , 2019 ) and cultivation biases in previous studies ( De Vuyst et al . , 2014; Gänzle and Ripari , 2016; Van Kerrebroeck et al . , 2017 ) may explain their widespread omission from our understanding of sourdough biodiversity .", "We first examined whether sourdough starter community composition was correlated with geographic distance between starters using a distance-decay analysis .", "Across the continental U . S . where we had the highest sample density , taxonomic composition was not correlated with geographic distance ( Mantel ρ = 0 . 0 , p>0 . 05 for both LAB/AAB and yeasts ) .", "At the global-scale , yeast taxonomic composition was weakly predicted by geography ( Mantel ρ = 0 . 07 , p<0 . 01 ) .", "The geographic signal was stronger when all fungal ASVs were included ( Mantel ρ = 0 . 23 , p≤0 . 001 globally ) , potentially due to differences in the non-yeast fungi ( molds , plant pathogens ) found within local ingredients and/or environments ( Barberán et al . , 2015 ) .", "While differences in the overall composition of sourdough starter communities were not correlated with geographic distance between starters , species of fermentation-relevant bacteria or yeast may be enriched in some geographic regions due to dispersal or production processes .", "To determine if there are sourdough microbial species which are restricted to particular regions of the U . S . , we used k-means clustering to group samples at two scales: k = 4 ( larger regions , Figure 2B ) and k = 15 ( smaller geographic regions , Figure 2C ) .", "Next , we identified indicator taxa that were significantly enriched in these regions .", "Indicator species analysis detects individual taxa that are enriched under particular conditions , where indicator strength ranges from 0 to 1 .", "An indicator strength above 0 . 25 is traditionally classified as a species that is strongly associated with a condition ( Dufrêne and Legendre , 1997; Gebert et al . , 2018 ) .", "We detected several taxa enriched within regions of the continental U . S . , although indicator strength for each of these was weak ( ≤0 . 2; Figure 2B–C ) .", "Collectively , the distance-decay and indicator species analyses demonstrate a limited role of geography in structuring the taxonomic diversity of sourdough microbial communities ( Figure 2B–C-Figure 2—figure supplement 4 ) .", "We next tested whether 33 other types of metadata collected for each starter could predict the observed composition of starters; these factors included age of starter , storage location , feed frequency , grain input , home characteristics , and climatic factors ( Figure 2D–E; Figure 2—source data 4 ) .", "Together , these predictors accounted for less than 10% of the variation in community composition for both bacterial and fungal communities in both the U . S . and global datasets ( PERMANOVA R2 of all bacterial ASVs = 8 . 3% and R2 of all fungal ASVs = 7 . 5% of the overall variation in Bray-Curtis dissimilarities for the global dataset; R2 bacteria = 9 . 0% and fungi = 5 . 0% for the U . S . ; Figure 2D–E , Figure 2—source data 5 ) .", "Some fermentation-relevant taxa were enriched under particular conditions ( Figure 2D–E , Figure 2—source data 6 ) .", "For example , younger starters were often dominated by the LAB L . plantarum ( indicator strength ( IS ) = 0 . 238 , p<0 . 001 ) and L . brevis ( IS = 0 . 254 , p<0 . 001 ) , while older starters often contained L . sanfranciscensis ( IS = 0 . 043 , p=0 . 01 ) and P . parvulus ( IS = 0 . 222 , p<0 . 001; Figure 2D , Figure 2—source data 6 ) .", "Previous studies have not found strong associations between flour type or other fermentation practices and yeast species present ( De Vuyst et al . , 2014; Vrancken et al . , 2010 ) .", "In our study , S . cerevisiae was weakly associated with starters whose grain base was whole wheat ( IS = 0 . 157 , p=0 . 04 ) .", "Most of the other fungal indicator species were non-yeast molds and plant endophytes , which were enriched under particular climatic conditions ( Figure 2E , Figure 2—source data 6 ) .", "No AAB taxa were enriched under any particular fermentation practice or climatic condition .", "The history and origins of sourdough starters may also explain the distribution of some widespread microbial species .", "Sourdough bakers can either begin their starters de novo from flour and water or obtain an established starter from a business or individual .", "The LAB L . brevis was associated with de novo starters ( IS = 0 . 206 , p=0 . 04 ) .", "There were 73 starters in our collection that were originally acquired by home bakers from a bakery or other commercial source and L . sanfranciscensis was abundant in these commercial starters ( IS = 0 . 267 , p=0 . 04 ) .", "This suggests that L . sanfranciscensis thrives under commercial production conditions and has been widely distributed among bakers , where it persists in home fermentations .", "We next determined whether individual growth rate and/or biotic interactions among taxa could help to explain distributions of sourdough species ( Friedman et al . , 2017; Vega and Gore , 2018 ) .", "Whereas previous studies have focused on single pairs of interacting sourdough microbes ( De Vuyst and Neysens , 2005; Gobbetti et al . , 1994; Sieuwerts et al . , 2018 ) , we chose eight isolates: four LAB and four yeasts , representing the most frequent yeasts and bacteria in sourdough that also displayed strong positive and/or negative patterns of co-occurrence ( Figure 3A , Figure 2—source data 2 , 3 ) .", "We did not include AAB in these interaction experiments because they were not significantly associated with other microbial taxa in the amplicon dataset .", "To determine the growth ability of each species alone , we measured colony forming units at the end of six 48 hr transfers in a liquid , cereal-based fermentation medium ( Figure 3A ) .", "To determine competitive ability , we serially passaged 1:1 mixtures of each pair of the eight microbial species through this medium for six 48 hr transfers and assessed the relative abundance of each isolate in each pair ( Figure 3A–B-Figure 3—figure supplement 1 ) .", "We determined whether each of the eight species could co-persist in pairwise competitions , where co-persistence was defined as both isolates being present at >1% relative abundance after the six transfers .", "Taxa that are able to reach high cell densities in the sourdough environment may be able to outcompete many other sourdough species .", "When comparing the growth of each isolate alone over six transfers to its ability to persist in pairwise competitions , we found a significant positive relationship ( Spearman’s ρ = 0 . 81 , p<0 . 05 , Figure 3C ) .", "For example , the LAB L . brevis is able to reach high densities when grown alone in our synthetic sourdough environment and is also able to persist when paired with all seven competing LAB and yeast species .", "In contrast , the LAB L . sanfranciscensis has one of the lowest densities when grown alone and is only able to persist when grown with the yeast K . humilis .", "We did not detect a significant correlation between the ability to persist in pairwise competitions and frequency of each taxon across the amplicon sequencing dataset ( Spearman’s ρ = 0 . 39 , p>0 . 05 , Figure 3D ) , or between growth alone and frequency in the amplicon sequencing dataset ( Spearman’s ρ = 0 . 57 , p>0 . 05 ) .", "To test how well specific interactions among yeast and LAB detected in sourdoughs could be recapitulated in our in vitro system , we compared significant co-occurrence patterns inferred from amplicon sequence data ( positive or negative associations ) , with synthetic co-persistence patterns from the competition experiments .", "Of the 16 significant associations detected in the amplicon dataset , most ( 14 out of 16 ) were within-kingdom interactions ( yeast:yeast and bacteria:bacteria ) and only two were cross-kingdom interactions: a negative pattern of co-occurrence between Saccharomyces cerevisiae and Lactobacillus sanfranciscensis , and a positive pattern of co-occurrence between Kazachstania servazzii and Pediococcus damnosus .", "In the competition experiment , yeasts and bacteria co-persisted with each other in half of all pairings ( 8 of 16 ) , and within-kingdom ( yeast:yeast or bacteria:bacteria ) species pairs co-persisted in 3 of 12 pairings ( Figure 3B and E ) .", "Most species pairs ( 20 of 28 ) did not show significant positive or negative associations in the amplicon dataset .", "For the eight significant co-occurrence interactions detected in the amplicon sequence dataset , seven out of eight were recapitulated in our synthetic communities ( Figure 3E; p<0 . 05 ) .", "The consistency in directionality between pairwise co-occurrence patterns observed between the 500 sourdough starters and in vitro suggests robust microbial interactions in sourdough despite differences in environmental conditions and fermentation practices .", "To determine how variation in starters’ community composition impacts their functional attributes , we selected a subset of 40 starters that spanned the spectrum of sourdough diversity ( Figure 2A ) .", "We measured two baking-relevant functions: emissions of volatile organic compounds ( VOCs ) , which can impact baked sourdough bread aromas ( Pétel et al . , 2017 ) , and leavening ( measured as rate of dough rise ) , which can impact bread structural properties ( Arendt et al . , 2007 ) .", "Across all samples , 123 volatile compounds were detected by GC/MS including well-known sourdough compounds 3-methyl-1-butanol , ethyl alcohol , acetic acid , and ethyl acetate ( median number of compounds detected per starter = 85; Figure 4—figure supplement 1 ) .", "Sensory analysis yielded 14 dominant notes across the 40 starters , including yeasty , vinegar/acetic acid/acetic sour , green apple , fermented sour , and ethyl acetate/solventy ( Figure 4—figure supplement 3 , Figure 4—source data 2 ) .", "The source of the starter inoculum explained most of the variation in VOC profile dissimilarities ( PERMANOVA R2 = 91% and p≤0 . 001 ) , differences in maximum dough height ( adjusted R2 = 22% , ANOVA p<0 . 05 ) , and dough rise rates ( adjusted R2 = 42% , ANOVA p<0 . 001; Figure 4—figure supplements 1–2 and Video 1 ) .", "This demonstrates wide variation in functional capacities across the 40 starters and low variation across experimental replicates within one starter .", "Starter community composition did not correlate with the dominant sensory note assigned by the sensory panel ( PERMANOVA R2 = 37% , p=0 . 16 ) .", "While total bacterial and yeast species richness was not significantly correlated with VOC richness ( Spearman’s ρ = −0 . 09 , p>0 . 05 ) , starter microbial community composition was significantly correlated with VOC profiles ( Bray-Curtis dissimilarity for community composition and VOC profiles; Mantel ρ = 0 . 19 , p≤0 . 01 ) .", "When we determined whether VOC variation was driven by particular sourdough species , only the Acetobacter malorum spp .", "group emerged as significant ( ρ = 0 . 528 , FDR-corrected p<0 . 05; Figure 4—source data 1 ) .", "More generally , total % AAB explained was strongly correlated with variation in VOCs ( Mantel ρ = 0 . 73 , p<0 . 001; Figure 4 ) and sensory notes ( Kruskal-Wallis p<0 . 05; Figure 4 ) , with the differences most strongly tied to the vinegar note compared to green apple and fermented sour notes ( Dunn test p=0 . 04 and 0 . 06 respectively; Figure 4—figure supplement 3 ) .", "The total relative abundance of AAB was also negatively correlated with the rate of dough rise ( ρ = −0 . 51 , p<0 . 001; Figure 4 ) , which may be explained by the production of compounds such as acetic acid and inhibition of yeast or LAB growth in sourdough starters .", "The combined functional datasets highlight that variation in AAB abundances is a key driver of functional diversity across our sourdough collection ." ], [ "This study presents the first intercontinental atlas of sourdough starter microbial communities .", "Our unprecedented scale of sampling demonstrates how sourdough maintenance and acquisition practices as well as microbial interactions can impact the biodiversity of starters .", "We also reveal novel structure-function linkages in this ancient fermented food .", "In combination with thousands of years of traditional knowledge about how to make good bread , these results provide possible management strategies for manipulating starter diversity .", "Contradicting widespread beliefs about the regionality of sourdough microbiomes ( e . g . the famous ‘San Francisco sourdough’ ) , our comprehensive sampling demonstrates that geographic location does not determine sourdough microbial composition .", "Previous studies using limited sampling have suggested that sourdough starters can vary across geographic regions ( Liu et al . , 2018; Scheirlinck et al . , 2007b ) , but we are unaware of other studies that have rigorously explored sourdough microbiome biogeographic patterns in a distance-decay framework .", "The limited role of geography in explaining sourdough diversity may be driven by the widespread movement of starters across large geographic distances through starter sharing or commercial distribution .", "Flour , a major potential source of microbes in de novo starters ( Minervini et al . , 2015; Reese et al . , 2020 ) , is also moved across large spatial scales .", "This geographic homogenization of starter and flour microbes likely swamps out any regional differences in potential yeasts or bacteria that can disperse into starters .", "Biogeographic patterns of sourdough microbes may exist at finer-scale resolutions than those used in the current study .", "We used amplicon sequencing to assess taxonomic diversity of sourdough because we were interested in species-level distributions of sourdough microbes .", "We acknowledge that intraspecific strain diversity plays important roles in many fermented foods ( El Khoury et al . , 2017; Niccum et al . , 2020; Walsh et al . , 2017 ) , that microbial strains may have unique biogeographic patterns ( Gayevskiy and Goddard , 2012 ) , and that amplicon sequencing does not typically have the resolution to fully capture strain-level diversity ( Turaev and Rattei , 2016 ) .", "However , previous studies of other ecosystem types using similar amplicon sequencing approaches and at similar spatial scales have found strong distance-decay relationships of both fungal and bacterial communities ( Finkel et al . , 2012; Ma et al . , 2017; Talbot et al . , 2014 ) .", "Future work using shotgun metagenomic sequencing or whole-genome sequencing of widespread sourdough microbes may reveal more nuanced biogeographic patterns in sourdough biodiversity .", "Relative to bakeries , home producers likely follow less strict and sustained regimens for starter maintenance; they may vary techniques over time or keep imperfect records in regard to reported metadata .", "Still , process parameters explained some of the variation we detected through amplicon sequencing , with individual taxa being significantly associated with certain parameters .", "For example , L . sanfranciscensis was prevalent in older starters and starters purchased by participants from a business , which supports the hypothesis that it is selected for by bakers over generations of sourdough production ( Gänzle and Ripari , 2016 ) .", "Future studies that explicitly test how starter composition changes over time and across geographies in home fermentations are needed to better understand selection and stability in these ecosystems .", "The concordance between our amplicon sequencing and competition experiment suggest that commonly or uncommonly observed species pairs may be due to complementary or competitive inter-species interactions .", "An important caveat is that the single representative isolates used in these experiments do not capture strain-level genomic and metabolic diversity , which has been shown to produce different competitive outcomes among and between strains of these sourdough taxa ( Rogalski et al . , 2020 ) .", "Additionally , the pairwise interaction assay that we used does not capture the potential for interactions between three or more species .", "Though pairwise competitions have been shown to be predictive of higher-order interactions in simple microbial ecosystems ( Friedman et al . , 2017 ) , outcomes of interactions in more complex synthetic sourdough communities with three or more species may differ from what we observed here .", "Future studies that use ‘leave-one-out’ approaches can identify potential roles of multidimensional microbial interactions in sourdough microbiomes .", "We observed many striking microbial interactions in our synthetic system that were not predicted by sequencing .", "For example , W . anomalus was a strong competitor in vitro in this and other studies ( Daniel et al . , 2011; Vrancken et al . , 2010 ) , but it is not frequently found across the starters we sampled and co-occurred with other yeast species in the sourdoughs that were sequenced .", "This work adds to an existing body of evidence suggesting that W . anomalus is strongly competitive in sourdough , but may be dispersal limited , and/or that environmental conditions or multispecies interactions in home sourdough starters mitigate its competitive performance in situ .", "Our integrative functional analysis of starter microbiomes highlights the disproportionate effects of AAB in shaping the sensory and leavening properties of sourdough .", "These bacteria have been historically underappreciated in most studies of sourdough microbes ( De Vuyst et al . , 2014; Gänzle and Ripari , 2016; Van Kerrebroeck et al . , 2017 ) .", "The limited use of media that can selectively grow AAB and the lack of metagenomic approaches in previous studies are two potential explanations for the underestimation of AAB abundances in the literature .", "Based on our findings , we argue that the relative abundance of this group should be considered a key factor in predicting the functional attributes of sourdough .", "However , the influence of AAB on sensory properties and dough rise is not straightforward and the correlation between percentage of AAB and vinegar sensory properties as well as slower dough rise rates was not seen in all samples .", "Further studies are needed to understand how VOCs produced by AAB contribute to the quality of baked sourdough bread .", "Sourdough starters exemplify a unique type of microbial community: a top-down engineered microbiome that is stably maintained for many years .", "As our co-occurrence analysis and synthetic community validation revealed , these simple countertop microbial ecosystems provide ample opportunities to identify processes that structure microbiomes .", "Further experimental approaches that manipulate other dimensions of sourdough , including phages , genomic heterogeneity , and evolutionary dynamics , will continue to uncover mechanisms of microbiome assembly in this ancient fermented food ." ], [ "Sourdough starters were submitted by community scientists participating as part of the Sourdough Project ( http://robdunnlab . com/projects/sourdough/ ) .", "Community scientists were recruited through web sites , social media , and email campaigns worldwide January-March 2017 .", "They were directed to an online Informed Consent form approved by the North Carolina State University’s Human Research Committee ( IRB Approval Reference #10590 ) .", "Each participant first answered an extensive online questionnaire consisting of 40 questions related to the source , history , maintenance , and use of their sourdough starter .", "Upon completion , participants were assigned a unique ID .", "Participants were instructed to triple-bag ~4 oz of their freshly fed sourdough starter in a new resealable plastic bag , label the bag with their participant ID , and then ship it to Tufts University .", "In order to preserve participant confidentiality , samples were reassigned a new Sample ID number upon arrival .", "Each starter sample was subdivided into two subsamples: ( 1 ) 1 mL was transferred to a 1 . 5 mL tube and stored at −80°C until samples could be processed for DNA sequencing , ( 2 ) glycerol stocks ( 15% glycerol ) were made of each sample by combining equal parts sample and 30% glycerol and stored at −80°C for competition and VOC analyses .", "In total , we received , processed , and sequenced 560 sourdough starter samples with completed surveys from participants .", "After quality filtering and rarefying amplicon datasets ( see below ) , 500 were retained .", "To characterize the bacterial and fungal communities of sourdough starters , we followed previously described molecular marker gene sequencing protocols ( Ramirez et al . , 2014; Oliverio et al . , 2017 ) .", "In brief , we extracted DNA from 2 mL sub-samples using a Qiagen PowerSoil DNA extraction kit , and then amplified extracted DNA with barcoded primers to enable multiplexed sequencing in duplicate , using the 515 f/806 r for bacteria and ITS1f/ITS2 for fungi .", "Amplicon concentrations were normalized and sequenced on the Illumina MiSeq platform at the University of Colorado Next Generation Sequencing Facility with 2 × 150 and 2 × 250 bp paired-end chemistry for bacterial and fungal sequencing , respectively .", "We sequenced multiple DNA extractions and PCR negative controls to check for contamination .", "Raw sequences were processed with the DADA2 pipeline ( Callahan et al . , 2016 ) .", "The DADA2 pipeline detects ASVs as opposed to clustering sequences by percent sequence similarity .", "Briefly , sequences with N’s were removed prior to primer removal with Cutadapt ( Martin , 2011 ) .", "Then sequences were quality filtered .", "For the bacterial sequence data , we used the following parameters: truncLen = 150 for forward reads and 140 for reverse reads , maxEE = 1 , and truncQ = 11 and for the fungal data we used the following parameters: minLen = 50 , maxEE = 2 , truncQ = 2 and maxN = 0 .", "The parameters are different for 16S and ITS due to the variable nature of the ITS region .", "After quality filtering , sequence variants were inferred with the DADA2 algorithm , and then we merged paired-end reads .", "We removed chimeras and taxonomy was determined using the Silva database for bacteria ( Quast et al . , 2013 ) and the UNITE database ( Nilsson et al . , 2019 ) for fungi .", "We then filtered out reads assigned to either chloroplasts or mitochondria from the bacterial taxa table , along with reads that were unassigned at the phylum level .", "Likewise , we filtered out fungal reads unassigned at the phylum or class levels .", "We then rarefied the ASV tables to 1260 bacterial reads per sample and 4000 fungal reads per sample and converted to percent relative abundances for downstream analyses .", "We retained 500 sourdough samples for analyses ( e . g . those samples for which we obtained both bacterial and fungal amplicon data ) .", "To obtain high-quality taxonomic species assignments for LAB and AAB and to facilitate comparisons to longer 16S rRNA sequences of isolates , we built phylogenetic trees for both bacterial groups ( Figure 2A-Figure 2—figure supplement 1 ) .", "We included high-quality ( ≥1200 bp ) isolate sequences for both groups from the ribosomal database project ( Cole et al . , 2014 ) .", "In order to preserve continuity between our analysis and previously published studies of sourdough ecology , we refer to the Lactobacillus species by their traditional names rather than recently proposed taxonomic reassignments ( Zheng et al . , 2020 ) .", "To obtain assignments , we created alignments with MUSCLE ( Edgar , 2004 ) and then built phylogenetic trees with raxml-HPC ( Stamatakis , 2014 ) with the GTR-GAMMA model .", "We used a patristic distance of 0 . 97 to assign species taxonomic labels ( Pommier et al . , 2009 ) .", "ASVs that clustered with two closely related reference sequences were assigned both names .", "ASVs that are clustered with more than two reference sequences were named by their cluster number , except when reference sequences in a cluster are documented to be closely related , as in the case for the L . plantarum/L .", "pentosus/L .", "fabifermentans spp .", "group ( Mao et al . , 2015 ) , which is referred to as the L . plantarum spp .", "group or L . plantarum; the L . casei/L .", "paracasei/L .", "zeae/L .", "rhamnosus spp .", "group ( Dobson et al . , 2004 ) referred to as the L . casei spp .", "group; the L . crustorum/L .", "mindensis/L .", "farcisminis spp .", "group ( Scheirlinck et al . , 2007a ) , referred to as the L . crustorum spp .", "group; the A . lambici/A .", "lovaniensis/A .", "okinawensis/A .", "syzygii/A .", "ghanensis/A .", "fabarum spp .", "group ( Iino et al . , 2012; Spitaels et al . , 2014 ) , which is referred to as the A . lovaniensis spp .", "group; the A . orientalis/A .", "farinalis/A .", "malorum/A .", "cerevisiae/A .", "persici/A .", "cibinongensis spp .", "group ( Li et al . , 2014 ) , referred to as the A . malorum spp .", "group; and the Gluconobacter wanchernii/G .", "jabonicus/G .", "thailandicus/G .", "cerinus/G .", "nephelii spp .", "goup ( Matsutani et al . , 2014 ) , referred to as the G . frateurii spp .", "group .", "We also characterized 40 sourdough starters used for functional analyses ( dough rise , VOCs , and sensory notes ) via shotgun metagenomic sequencing in order to confirm that the experimental communities revived from glycerol stocks were similar to the original samples .", "We prepared the samples for shotgun metagenomic sequencing as per Oliverio et al . , 2020 and samples were sequenced on the Illumina NextSeq platform with 2 × 150 bp chemistry at the University of Colorado Next Generation Sequencing Facility .", "We filtered raw reads with Sickle ( Joshi and Fass , 2011 ) with the specified parameters -q 50 and -I 20 .", "On average , each sample consisted of ~3 . 7 million paired-end reads after quality filtering .", "We classified the taxonomy of metagenomic reads with Kaiju , using the RefSeq database ( Menzel et al . , 2016 ) .", "Eight individual isolates of abundant yeasts and bacteria from amplicon data ( four yeasts and four bacteria ) were isolated from glycerol stocks which were plated onto Lactobacilli MRS agar ( Criterion ) or Yeast Potato Dextrose ( YPD ) .", "The yeasts S . cerevisiae , W . anomalus , K . humilis , and K . servazii as well as the LAB L . sanfranciscensis , L . brevis , L . paramilentarius , and L . plantarum were chosen because of their abundance ( within the top five bacteria and yeast in amplicon sequencing , respectively; mean relative abundance ) .", "The eight isolates were sourced from eight different starters in our collection .", "Identities were confirmed using Sanger sequencing of the ITS region for yeast using the primer ITS1f ( Gardes and Bruns , 1993 ) and ITS4 ( White et al . , 1990 ) and 16s regions of bacteria using the primers 27f ( Lane , 1991 ) and 1492r ( Turner et al . , 1999 ) .", "Sanger sequences of the four bacterial isolates were included in the tree used for ASV classification and all clustered with their respective presumed identities’ reference taxa ( Figure 2A-Figure 2—figure supplement 1 ) .", "For growth and competition assays , a liquid cereal-based fermentation medium ( CBFM ) was made to approximate the dough environment similar to a previously described approach ( Charalampopoulos et al . , 2002 ) .", "To make CBFM , 100% whole wheat and all-purpose flour were combined in equal proportion ( 1:1 by mass ) .", "The flour mixture was suspended in room temperature ( 24°C ) deionized water ( 1:9 flour:water by mass ) in 500 ml plastic conical centrifuge bottles by shaking for 1 min .", "This mixture was immediately centrifuged ( Beckman GS-6 Series ) at x3000 rpm for 30 min ( 24°C ) and the pellet was discarded .", "The CBFM was then filtered to exclude microbial cells through Falcon disposable filter funnels ( 0 . 20 µm pore size ) .", "To quantify growth of each yeast and LAB alone , we standardized input inocula to 2 , 000 CFUs per 10 µL .", "Inocula were standardized by diluting 15% glycerol stocks ( stored at −80°C ) that had a known concentration of CFUs with 1X phosphate buffered saline ( PBS ) .", "We inoculated 10 µL of each species into 190 µL of CBFM in individual wells of a 96-well plate ( n = 5 ) .", "After cultures grew statically for 48 hr , cultures were homogenized and then transferred 10 µL of the culture into 190 µL of fresh CBFM .", "We repeated these transfers a total of six times .", "All incubations were kept at 24°C throughout the duration of the experiment .", "Total abundance of each species was determined using CFU plating described below .", "For competition experiments , yeasts and bacteria were inoculated into wells of a 96-well plate in a fully factorial pairwise design from frozen glycerol stocks ( glycerol stocks prepared as described for growth assays ) .", "For each inoculum , frozen stocks were plated and counted on either MRS or YPD and standardized to 1 , 000 CFUs per 5 µL .", "All reciprocal pairs of each standardized inoculum ( 5 µL of each member of the pair ) were added to 190 µL of CBFM , for a total of 200 µL .", "After 48 hr of growth at room temperature , cultures were homogenized and 10 µL of each culture was transferred to 190 µL of fresh CBFM .", "All pairs were replicated five times .", "A few replicates were lost due to unexpected contamination ( Figure 3—figure supplement 1 ) .", "For both growth and competition assays , the number of replicates was chosen based on pilot experiments that demonstrated the extent of variability across replicates .", "All replicates were plated and relative abundance of the interacting species was determined after transfers one , three and six .", "Yeast:yeast pairs were plated on Chromagar Candida plates ( CHROMagar ) at a 10−4 dilution to differentiate species based on colony morphology , with the exception of pairs containing Wickerhamomyces anomalus , which were differentiated on YPD .", "Bacteria:bacteria pairs were plated at 10−5 dilutions on MRS where species could be differentiated based on colony morphology .", "Yeast:bacteria pairs were spot-plated ( 5 µL of each dilution ) on selective media at full to 10−5 dilutions to quantify CFUs .", "Selective media were YPD plus chloramphenicol ( 50 mg/L ) to select for yeast and MRS plus natamycin ( 21 . 6 mg/L ) to select for bacteria .", "Individual isolates for our experiment were determined to have ‘persisted’ if they were above the detection limit of 1/100th of the total population at the end of the experiment ( transfer six ) .", "Co-persistence was defined as both isolates being detectable at that threshold .", "To test how distinct sourdough community structures impacted functions , we revived frozen glycerol stocks of 40 starters in a standard flour medium ( see medium preparation description below ) using a common garden approach .", "These 40 starters spanned the diversity we encountered in sequencing ( Figure 2A ) ; we limited our functional analysis to 40 starters due to practical constraints in annotating VOC data .", "Rather than directly using frozen starters which were shipped to us from community scientists , we first grew up samples in a ‘pre-inoculum’ that was used to inoculate doughs for functional analysis .", "We did this to ensure that all cultures were at comparable growth stages prior to inoculation .", "The flour used was the same mixture used for CBFM ( 100% whole wheat and all-purpose flour combined in equal proportion 1:1 by mass ) , but it was prepared differently in order to approximate the moisture content of dough .", "Flour was autoclaved on a gravity cycle for 20 min to reduce microbial load .", "Glycerol-stocked communities ( 200 µL ) were added to 1 . 8 mL sterile distilled water and 2 g autoclaved flour in three replicate communities for each starter pre-inoculum ( n = 3 captured the variability we encountered in pilot experiments ) .", "Pre-inoculum was mixed with flour and sterile water using a sterile wooden dowel until no dry flour was visible .", "The mixture was briefly centrifuged in a 15 mL culture tube ( to remove dough stuck to the side of the tube walls ) and then incubated for 72 hr at room temperature .", "Inoculum was created by mixing sterile water ( 4 mL ) and pre-inoculum ( ~4 g ) and was vortexed to homogenize .", "Autoclaved flour ( 2 g ) , water ( 1600 µL ) , and inoculum ( 400 µL ) were added to 15 mL falcon tubes and mixed using sterile wooden dowels and then briefly centrifuged to remove from the sides of tubes .", "To confirm that the microbial community that formed in these experimental sourdoughs ( grown from the 40 starters selected for functional analyses ) resembled the sourdough starters from which they originated , we compared microbial community composition between shotgun metagenomic data collected from experimental sourdoughs and the corresponding amplicon sequence data from the original sourdoughs .", "We calculated Bray-Curtis dissimilarities for the 40 samples from: ( 1 ) the initial amplicon data and ( 2 ) 16S taxonomic annotations from the shotgun metagenomic data from the experimental samples .", "We then compared the two dissimilarity matrices with a Mantel test ( based on Spearman rank correlation and 999 permutations ) .", "We also correlated relative abundances of some of the most dominant taxa including L . sanfranciscensis , L . brevis , L . plantarum , P . damnosus , and also the total percent of AAB detected in each sample .", "Initial and experimental communities were similar in terms of their overall bacterial composition ( Mantel ρ = 0 . 55 , p≤0 . 001 ) and abundances of common taxa ( L . sanfranciscensis ρ = 0 . 64 , p≤0 . 001; L . brevis ρ = 0 . 50 , p≤0 . 001; L . plantarum ρ = 0 . 70 , p≤0 . 001; P . damnosus ρ = 0 . 47 , ≤0 . 001; and % total AAB ρ = 0 . 75 , p≤0 . 001 ) .", "Dough rise was recorded with a Canon EOS 70D DSLR camera set to image every two minutes for 36 hr .", "The camera was centered at the vertical and horizontal midpoint of each dough batch .", "Timelapse photos were compiled , cropped , and enhanced for contrast using Adobe AfterEffects .", "The top of every dough was tracked through each frame in the MatLab-based digitizing program DLTdv5 ( Hedrick , 2008 ) .", "The height of each culture tube ( Th ) was measured in pixels in Adobe Photoshop .", "Changes in dough height ( ∆X ) were normalized to account for distance from the camera and differences in starting height using the following formula: ∆X = ( X-Xº ) /Th .", "Distances relative to tube length were converted to absolute distance ( mm/hr ) by multiplying values by the dimensions of culture tubes ( 103 mm ) .", "Before fitting curves , data was truncated to cut instances where dough fell by more than 5 percent of its maximum height .", "For each replicate , a logistic growth curve was fitted , and growth rate ( r ) was calculated with the R package Growthcurver ( Sprouffske and Wagner , 2016 ) , with a goodness of fit cutoff for r values of p≤0 . 01 .", "Sourdough starter volatiles were collected using headspace sorptive extraction , which is an equilibrium-driven sample enrichment technique employing a polydimethylsiloxane coated magnetic stir bar ( commercially known as Twister , Gerstel ) .", "Stir bars ( 10 mm long x 0 . 5 mm thick ) were suspended in the headspace of each sample using a magnet on the outside of the sample tube cap to suspend the stir bar above the sample .", "Three replicates of each community were sampled for 24 hr ( beginning 12 hr after inoculation ) .", "After collection , the stir bars were removed and spiked with 1 μL of 10 ppm ethylbenzene-d10 , which was used as an internal standard ( Restek ) .", "Relative peak areas ( analyte/internal standard ) served to measure relative concentrations of each compound in the sample .", "Sterile deionized water and autoclaved flour were analyzed to measure chemical interferences from background samples .", "Compounds in the starters measured at concentrations less than or equal to concentrations in the water/flour samples were eliminated from the data .", "Stir bars were thermally desorbed into the GC column in the gas chromatograph/mass spectrometer ( GC/MS , Agilent models 7890A/5975C ) .", "The instrument was equipped with an automated multi-purpose sampler ( Gerstel ) that transferred stir bars to the thermal desorber ( TDU ) /programmable temperature vaporization inlet ( CIS ) .", "The TDU ( Gerstel ) provided transfer of the VOCs from the stir bar to the CIS by heating it from 40°C ( 0 . 70 min ) to 275°C ( 3 min ) at 600 °C/min , using 50 mL/min of helium .", "After 0 . 1 min the CIS , operating in solvent vent mode , was heated from −100°C to 275°C ( 5 min ) at 12 °C/s .", "The GC column ( 30 m x 250 μm x 0 . 25 μm HP5-MS , Agilent ) was heated from 40°C ( 1 min ) to 280°C at 5 °C/min with 1 . 2 mL/min of constant helium flow .", "MS operating conditions were: 40 to 350 m/z at 10 scans/s , 70 eV electron impact source energy , with ion source and quadrupole temperatures of 230°C and 150°C , respectively .", "The retention index ( RI ) for each compound in the sample was calculated based on a standard mixture of C7 to C30 n-alkanes ( Sigma-Aldrich , St . Louis , MO ) .", "Approximately 300 reference standards were purchased from Sigma-Aldrich , Fisher Scientific , Alfa Aesar ( Ward Hill , MA ) , TCI ( Tokyo , Japan ) , Acros Organics ( Pittsburgh , PA ) and MP Biomedicals ( Santa Ana , CA ) to confirm compound identity .", "Detailed descriptions of data analysis procedures have been previously described ( Kfoury et al . , 2018; Robbat et al . , 2017 ) .", "Briefly , Ion Analytics ( Gerstel ) data analysis software was used to analyze the data .", "Peak identification was based on the match between sample and reference compound RI and mass spectrum ( positive identification ) and between sample and compounds found in NIST05/17 , Adams Essential Oil Library , and the literature ( tentative identification ) .", "In instances where no identification was possible , a numerical identifier was assigned such that the data can be used in other metabolomic studies and to capture knowledge of elution and mass spectral profiles should these compounds become important enough to warrant independent collection and analysis .", "Compound identity is assigned as follows .", "First , peak scans were required to be constant for five or more consecutive scans ( ≤20% difference ) .", "Second , the level of scan-to-scan variance ( SSV ) had to be ≤5 .", "The SSV represents the relative error of each scan compared to all other mass spectrum scans in the peak .", "The smaller the difference , i . e . , the closer the SSV is to zero , the better the MS agreement .", "Third , the Q-value was set to ≥93 .", "The Q-value is an integer between 1 and 100; it measures the total ratio deviation of the absolute value calculated by dividing the difference between the expected and observed ion ratios by the expected ion ratio , then multiplied by 100 for each ion across the peak .", "The closer the value is to 100 , the higher the certainty of accuracy between sample and reference compound spectrum ( positive ) or sample and database/library spectrum ( tentative identification ) .", "Finally , the Q-ratio represents the ratio of the molecular ion intensity to confirmatory ion intensities across the peak; it also must be ≤20% .", "When all of the individual criteria are met , they form a single criterion of acceptance and the software assigns a compound name or numerical identifier .", "There were two sets of sensory descriptors that were used in our study: those that were provided by individuals at the time of sample collection ( Figure 1; Figure 2—source data 4 ) , and those that a trained sensory panel used during the functional analysis of a subset of sourdough starters ( Figure 4—source data 2 ) .", "We used a simplified system with the larger set of study participants .", "Moreover , our trained sensory panel was able to distinguish additional sensory notes that we did not anticipate when we sent out the survey to participants .", "For the initial survey , the aroma characteristics were guided by a list of aroma characteristics commonly used when discussing aroma notes in maltose-based fermented products with consumers .", "The list was reduced and condensed for survey purposes .", "For example , ‘medicinal’ was used to combine the descriptors ‘phenolic antiseptic’ and ‘solvent nail polish , ’ and the term ‘rose’ was translated to the more general ‘floral . ’ The descriptor ‘musty’ was additionally included to account for the ‘farmyard’ aroma and the potential contribution of VOCs from filamentous fungal growth .", "For our detailed functional analysis of the subset of 40 starter samples , sourdough starter aromas were assessed after 36 hr of incubation by a trained and certified descriptive sensory analysis panel from the Tufts University Sensory and Science Center .", "Samples were coded , randomized , and served blind to the panel one at a time .", "The panel used modified flavor profile analysis to measure the intensity and characteristics of the aroma in each sample ( Hootman and Keane , 1992 ) , resulting in primary ( dominant ) and secondary notes for each sample ( Figure 4—source data 2 ) .", "All statistical analyses were performed in the R environment ( ‘R Core Team , 2019 , ” 2012 ) .", "To assess and visualize overall similarity in sample composition ( Figure 2A ) , we hierarchically clustered samples based on pairwise distances in Bray-Curtis dissimilarities ( method = ward . d2 ) , as implemented in hclust .", "Bacteria including LAB and AAB were weighted equally to fungi ( yeast only ) .", "To test whether geographic location explained any of the variation in observed microbial composition , we ran Mantel tests ( method = Spearman with 999 permutations ) comparing bacterial and fungal community dissimilarities ( Bray-Curtis ) to geographic distances ( calculated with the Haversine distance from the R package geosphere ) .", "We evaluated this relationship for the whole dataset ( n = 500 ) and using continental US samples only ( n = 424 ) .", "We also compared overall fungal to bacterial dissimilarities with Mantel tests for the global and US-only datasets ( Figure 2—figure supplement 4 ) .", "To assess whether abiotic factors including process parameters ( data collected from participants in initial survey ) and climatic variables ( obtained from latitude/longitude of sample submission ) were correlated with any of the observed patterns in microbial composition , we performed PERMANOVA tests on bacterial and fungal dissimilarities , including 33 potential predictors ( Figure 2D–E; Figure 2—source data 4 ) : grain base inputs , other process attributes ( starter origin , starter input , storage location , number of feeds per month , frequency opened per week , container material , container lid ) ; water source; sensory notes described by participant; pet presence; and location-based climatic variables obtained from latitude/longitude reported ( mean annual temperature , maximum temperature , minimum temperature , precipitation , and net primary productivity ) .", "We tested pairwise correlations between all continuous predictor variables .", "Absolute Pearson correlation values were <0 . 6 , except for the following pairs: maximum temperature and temperature ( 0 . 8 ) , maximum temperature and seasonality ( −0 . 9 ) , filtered water and tap water ( −0 . 7 ) .", "Final PERMANOVA models only included significant predictors of community composition ( Figure 2D–E; Figure 2—source data 5 ) .", "Some survey responses were not completed by all participants and we excluded those missing predictors from our models ( thus , n = 426 samples for all models ) .", "For those variables found to explain a significant portion of the variation in overall microbial community composition ( for either fungal or bacterial communities ) , we tested whether particular taxa were enriched under specific conditions ( e . g . ‘indicator taxa’; Figure 2D–E ) .", "To test this with continuous variables ( for example , starter age or mean annual temperature of starter location ) we used Spearman rank correlations to compare to the relative abundances of particular taxa ( ASVs ) .", "For categorical variables ( for example , whether the starter grain base included rye flour ‘grain base rye’ or the starter storage location ) we used indicator correlation indices ( r ) values as implemented in the indicspecies package ( De Caceres et al . , 2016 ) .", "For all comparisons , we only included taxa that were detected in ≥10% of samples , and we considered taxa to be indicators if the false-discovery rate ( fdr ) corrected p-value was ≤0 . 05 .", "To determine if there are sourdough microbial species that might be restricted to particular regions of the U . S . ( Figure 2B–C ) , we used k-means clustering to group samples at two scales: k = 4 ( larger regions ) and k = 15 ( smaller geographic regions ) and identified indicator taxa that were significantly ( <0 . 05 ) enriched in these regions ( Gebert et al . , 2018 ) .", "Positive and negative co-occurrence interactions ( Figure 3F; Figure 2A-Figure 2—figure supplement 3 ) were detected using the R package Cooccur ( Griffith et al . , 2016 ) , which uses a probabilistic approach to determine whether , given their abundance in data , species occur more or less often than is expected by chance .", "Data were transformed to presence-absence of assigned species taxonomy of LAB , AAB , and yeast above a one percent within-sample threshold .", "Additionally , only species interactions that were predicted to co-occur more than once were considered .", "To control for multiple comparisons and minimize false positives , Bonferroni-corrected p values are reported .", "A Monte Carlo simulation was used to determine the likelihood that seven out of eight synthetic co-persistence tests would match our co-occurrence data .", "We constructed a matrix with the same distribution as that of our significant ( p≤0 . 05 ) positive and negative co-occurrence interactions; of 16 significant interactions , eight were positive .", "We randomly drew from that matrix ( n = 10 , 000 ) to determine the likelihood that the prediction from our synthetic system- that is , that at least seven of a series of eight interactions , would be correct ( four negative and four positive interactions , representing the eight interactions observed in our experimental system ) .", "For two functional outputs , dough rise rate and VOC profiles ( Figure 4—figure supplements 1–2 ) , we first assessed how similar starters were from the same initial inoculum .", "For VOC profiles , we ran a PERMANOVA to assess how much variation in VOC profiles ( represented as Bray-Curtis dissimilarities ) was explained by initial starter inoculum .", "For dough rise , we ran an ANOVA to assess how differences in observed dough rise rates were predicted again , by initial starter inoculum .", "We then assessed whether overall community composition ( of yeast , LAB , or AAB ) predicted the composition of VOCs , using mean values within replicates from the same initial inoculum ( thus , n = 40 ) for both VOC profiles and dough rise rates .", "We used Mantel tests to compare community dissimilarities in VOCs to LAB , AAB and yeast dissimilarities , and also to the Euclidean distances in total % AAB across samples .", "We also assessed whether any particular taxa were driving differences in the overall VOC composition with Spearman’s correlations between taxa relative abundances and the two non-metric multidimensional scaling ( NMDS ) axes representing dissimilarities in VOC compositions .", "Likewise , for dough rise we assessed whether any particular taxa were driving differences in the observed rates via Spearman’s correlations .", "For both , taxa were considered significantly correlated if the p-value≤0 . 05 ( fdr-corrected ) .", "Sensory analysis yielded 14 dominant notes across the 40 starters , including yeasty ( n = 8 ) , vinegar/acetic acid/acetic sour ( n = 9 ) , green apple ( n = 6 ) , fermented sour ( n = 5 ) , ethyl acetate/solventy ( n = 3 ) , and n = 1 sample for alcoholic sour , bready , cheesy , fermented apple , fruity sour , sweaty , toasted corn chips , veggie sulfide , and winey sour .", "To test whether the dominant sensory note observed from the Tufts Expert Sensory Panel was predicted by % total AAB , we ran a Kruskal-Wallis test with the dominant note as the predictor and used a Dunn test to assess significance of pairwise comparisons .", "For the sensory analyses ( Figure 4—figure supplement 3 ) , we only tested notes that were dominant in ≥ .", "5 samples , thus n = 26 samples rather than 40 .", "We included only the first note reported by the expert sensory panel for our analyses , as this represented the most dominant note ." ] ]

[ "Humans have relied on sourdough starter microbial communities to make leavened bread for thousands of years , but only a small fraction of global sourdough biodiversity has been characterized .", "Working with a community-scientist network of bread bakers , we determined the microbial diversity of 500 sourdough starters from four continents .", "In sharp contrast with widespread assumptions , we found little evidence for biogeographic patterns in starter communities .", "Strong co-occurrence patterns observed in situ and recreated in vitro demonstrate that microbial interactions shape sourdough community structure .", "Variation in dough rise rates and aromas were largely explained by acetic acid bacteria , a mostly overlooked group of sourdough microbes .", "Our study reveals the extent of microbial diversity in an ancient fermented food across diverse cultural and geographic backgrounds ." ]

[ "Sourdough bread is an ancient fermented food that has sustained humans around the world for thousands of years .", "It is made from a sourdough ‘starter culture’ which is maintained , portioned , and shared among bread bakers around the world .", "The starter culture contains a community of microbes made up of yeasts and bacteria , which ferment the carbohydrates in flour and produce the carbon dioxide gas that makes the bread dough rise before baking .", "The different acids and enzymes produced by the microbial culture affect the bread’s flavor , texture and shelf life .", "However , for such a dependable staple , sourdough bread cultures and the mixture of microbes they contain have scarcely been characterized .", "Previous studies have looked at the composition of starter cultures from regions within Europe .", "But there has never been a comprehensive study of how the microbial diversity of sourdough starters varies across and between continents .", "To investigate this , Landis , Oliverio et al . used genetic sequencing to characterize the microbial communities of sourdough starters from the homes of 500 bread bakers in North America , Europe and Australasia .", "Bread makers often think their bread’s unique qualities are due to the local environment of where the sourdough starter was made .", "However , Landis , Oliverio et al . found that geographical location did not correlate with the diversity of the starter cultures studied .", "The data revealed that a group of microbes called acetic acid bacteria , which had been overlooked in past research , were relatively common in starter cultures .", "Moreover , starters with a greater abundance of this group of bacteria produced bread with a strong vinegar aroma and caused dough to rise at a slower rate .", "This research demonstrates which species of bacteria and yeast are most commonly found in sourdough starters , and suggests geographical location has little influence on the microbial diversity of these cultures .", "Instead , the diversity of microbes likely depends more on how the starter culture was made and how it is maintained over time ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "structural biology and molecular biophysics" ]

[ [ "Regulation of transcriptional activity is essential for every biological process .", "Common mechanisms for regulation include post-translational modifications and/or cooperativity among multiple activators and repressors ( Banerjee and Zhang , 2003 ) .", "Some transcription factors contain multiple regulatory sites for either post-translational modifications ( Meek and Anderson , 2009 ) or binding partners ( Cantor and Orkin , 2002 ) , and their activity is thus tuned by the combined action of these components .", "Recent studies have revealed a high degree of intrinsic disorder in transcription factors , indicating that the inherent dynamical behavior harbored by these structures is critical for these regulatory events to take place ( Li et al . , 2017; Liu et al . , 2006; Minezaki et al . , 2006 ) .", "Our developing understanding suggests that the intrinsically disordered domains in transcription factors may provide a multivalent platform for the recruitment of regulatory binding partners ( Currie et al . , 2017; Shammas , 2017 ) .", "While the functional consequence of multivalent binding to an intrinsically disordered region has been described for a few transcription systems ( Dyson and Wright , 2016; Uversky et al . , 2009 ) , it remains unclear for the vast majority of cases .", "Human ASCIZ ( ATMIN-Substrate Chk-Interacting Zn2+ finger ) is an 88 kDa protein that has recently been identified as a transcription factor for the hub protein , LC8 ( dynein light chain 8 ) ( Jurado et al . , 2012a ) .", "Mice with mutations in ASCIZ that prevent LC8 transcription die in late embryogenesis and exhibit serious developmental defects in kidneys and lungs ( Goggolidou et al . , 2014a; Goggolidou et al . , 2014b; Jurado et al . , 2010 ) .", "Drosophila ASCIZ knockouts die in early embryogenesis and localized knockdowns using RNAi show mitotic defects ( Zaytseva et al . , 2014 ) .", "Mutant phenotypes in Drosophila , developing mouse B lymphocytes , and cultured cells are rescued by ectopic overexpression of LC8 , demonstrating that the observed defects of ASCIZ knockouts are due to ASCIZ regulation of LC8 expression ( Goggolidou et al . , 2014b; Jurado et al . , 2012b; Zaytseva et al . , 2014 ) .", "In addition , it has recently been shown that a conditional knockout of LC8 almost perfectly copies the corresponding phenotypes of ASCIZ knockouts in mouse B cell development and B cell lymphomagenesis ( King et al . , 2017; Wong et al . , 2016 ) .", "LC8 is a highly conserved 20 . 6 kDa protein homodimer ( 10 . 3 kDa monomer ) that facilitates self-association of its primarily disordered partners ( Barbar , 2008; Barbar and Nyarko , 2014; Clark et al . , 2015 ) ( Figure 1a ) .", "LC8 binding is associated with a range of cellular processes , from cell division to apoptosis , underscoring LC8’s essential role as a regulatory hub ( Dunsch et al . , 2012; Puthalakath et al . , 1999 ) .", "LC8 preferentially binds to a 10-amino acid motif in intrinsically disordered regions ( IDRs ) containing highly conserved TQT residues at positions 7–9 ( Barbar , 2008; Rapali et al . , 2011b ) ( Figure 1b ) .", "In complex with LC8 , the otherwise intrinsically disordered motif adopts a ß-strand conformation ( Benison et al . , 2007; Liang et al . , 1999 ) ( Figure 1a , b ) .", "Analysis of the 11 crystal structures of LC8 bound to short peptides containing the motif explains why the TQT residues are essential for binding: the Q is involved in interactions with both LC8 subunits in the dimer , while both T’s are fully buried and thus evolutionarily constrained .", "In these interactions , TQT acts as the motif anchor while the other seven highly variable motif residues modulate affinity , as described in the anchored flexibility model of LC8 motif recognition ( Clark et al . , 2016 ) .", "LC8 binds one motif in each of its two symmetrical binding grooves ( Figure 1a ) , creating an IDP duplex that serves as a bivalent scaffold .", "This scaffold aids in higher order complex assembly by promoting binding of other proteins , including additional LC8 dimers ( Figure 1c ) , and enhancing self-association and oligomerization processes that often involve coiled-coil formation ( Hall et al . , 2009; Kidane et al . , 2013 ) .", "In recent years , the number of experimentally characterized LC8 partners has risen to more than 40 , and prediction methods indicate that dozens more may specifically bind LC8 ( Rapali et al . , 2011b ) .", "Gaining insight into how LC8 interacts with partner proteins , and how LC8 levels in the cell are balanced , is therefore paramount to understanding the regulation of many cellular processes .", "A distinctive feature of ASCIZ is the high number of LC8 recognition motifs within its C-terminal domain .", "Although some LC8 partners have multiple recognition motifs ( Dunsch et al . , 2012; Fejtova et al . , 2009; Gupta et al . , 2012; Stelter et al . , 2007 ) ( Nucleoporin Nup159 has 5 , Chica and Bassoon each has 3 ) , human ASCIZ contains 11 functional LC8 binding sites ( Rapali et al . , 2011a ) , the most by far of any partner protein identified to date .", "This enrichment in LC8-binding sites is conserved throughout the animal kingdom , underscoring the importance of multiple motifs in ASCIZ function ( Figure 1d ) .", "Cell culture transcription assays demonstrate that ASCIZ regulates LC8 transcription via a system of negative autoregulation , for which the mechanism is not well understood .", "Disruption of the ASCIZ/LC8 interaction via mutation of the TQT sites results in an increased level of LC8 transcription , while overexpression of LC8 decreases transcription ( Jurado et al . , 2012a ) .", "This observation led to the hypothesis that ASCIZ acts as a sensor for cellular LC8 and regulates LC8 transcription levels according to cellular needs ( Jurado et al . , 2012a ) .", "As LC8 expression levels vary among tissue types ( Chintapalli et al . , 2007 ) and LC8 overexpression enhances the survival and proliferation of breast cancer cells in culture ( Vadlamudi et al . , 2004 ) , regulation of LC8 levels is critical for cellular health and homeostasis .", "However , while high levels of LC8 inhibit ASCIZ transcriptional activity ( Jurado et al . , 2012a ) , it is not known how this activity is controlled at the molecular level nor the requirement for multiple binding sites .", "In this work , we use a combination of biophysical , structural , and molecular biology tools to explore the relationship between ASCIZ multivalency and LC8 transcription .", "We show that human and Drosophila ASCIZ bind to multiple LC8 dimers simultaneously in both a positively and negatively cooperative fashion , enabling the formation of a dynamic equilibrium of complexes , of which low occupancy intermediates are highly populated .", "We propose that this dynamic ensemble of complexes is important for transcriptional regulation and validate the main aspects of our hypothesis via transcriptional assays with human ASCIZ .", "These observations support a novel model of autoregulation , whereby ASCIZ engages in a dynamic equilibrium of multivalent interactions that tune the level of ASCIZ transcriptional activity ." ], [ "The 45 kDa Drosophila ASCIZ protein , dASCIZ , is predicted to contain four Zn-finger motifs at the N-terminus followed by a 243-amino acid long region of intrinsic disorder .", "The disordered region has six predicted LC8 recognition sites identified by a canonical TQT motif ( Figure 2a , dark blue bars ) : QT1 ( residues 251–262 ) , QT2 ( 274–285 ) , QT4 ( 323–334 ) , QT5 ( 340–351 ) , QT6 ( 354–365 ) , and QT7 ( 374–385 ) .", "QT3 ( 285–296 ) lacks the TQT residues but is identified experimentally as an LC8 recognition site in this work ( below ) .", "Purification of full-length dASCIZ is impeded by poor expression levels and insolubility , and therefore we designed and produced constructs corresponding to the zinc finger domain , dZnF ( residues 1–156 , red bar Figure 2a ) , and the LC8-binding domain , dLBD ( residues 241–388 , Figure 2a ) .", "Sedimentation velocity analysis of the dZnF and dLBD ( Figure 2b ) indicates that each is a monomer in solution with molecular weights of 17 . 4 kDa and 18 kDa , respectively ( theoretical MW 17 . 6 kDa and 17 kDa ) .", "The CD spectrum of dZnF shows a large negative ellipticity at 208 nm and a small negative ellipticity at 222 nm ( Figure 2c , red ) indicative of a mix of alpha helices and loops , similar to CD spectra of other ZnF proteins ( Ezomo et al . , 2010 ) .", "The dLBD CD spectrum has a large negative ellipticity at 200 nm , indicating that it is primarily disordered ( Figure 2c , blue ) .", "From 5D NMR experiments , backbone assignments for 90% of the 148 residues in dLBD were obtained ( Figure 2d ) .", "A high level of disorder in dLBD is revealed by the limited amide proton chemical shift dispersion in 15N HSQC spectra ( Figure 2d ) , and a lack of secondary structure preference is further supported by small ΔCα-ΔCβ chemical shift differences from random coil values ( Figure 2e ) .", "Together these data demonstrate that dASCIZ contains an N-terminal structured domain as well as a long intrinsically disordered domain , and constructs of each domain are monomeric in solution .", "Local dynamics of dLBD were assessed by NMR measurement of the 15N longitudinal ( R1 ) , transverse ( R2 ) relaxation , and 1H-15N heteronuclear NOEs .", "R2/R1 values range from 1 . 5 to 5 . 3 with an average of 3 . 3 ( Figure 2f ) .", "Relatively higher R2/R1 values for residues 321–363 suggest motional restriction in this region .", "Heteronuclear NOE values measured at 10°C are very low overall , with values ranging from −0 . 1 to 0 . 3 , but are also slightly higher for residues 321–363 ( Figure 2g ) .", "Together , the R2/R1 and heteronuclear NOE values imply that dLBD is highly flexible with slight motional restriction in its C-terminal half .", "The difference in flexibility between the N-terminus and C-terminus was validated by generating shorter constructs of the dLBD that include the first three ( QT1-3 ) , two internal sets of three ( QT2-4 and QT4-6 ) , and last four binding sites ( QT4-7 ) ( Figure 3a ) .", "All constructs are of a similar size , varying from 68 to 84 residues in length .", "Circular dichroism demonstrates that the C-terminal constructs , QT4-6 and QT4-7 , are slightly more ordered than N-terminal constructs , QT1-3 and QT2-4 ( Figure 2—figure supplement 1a ) .", "Size-exclusion chromatography supports this result , as the QT4-6 and QT4-7 constructs elute later , indicating that they are more compact than the N-terminal constructs ( Figure 2—figure supplement 1b ) .", "Isothermal titration calorimetry ( ITC ) experiments on dASCIZ constructs were applied to identify the number of recognition motifs and provide estimates of their overall binding affinity for LC8 .", "A range of constructs containing three to seven recognition motifs were tested for their binding to LC8 and all display a single binding step ( Figure 3 ) .", "Thus the measured Kd and stoichiometry are ‘effective’ values that present an overall simplified picture of a much more complicated complex assembly process .", "The full length dLBD binds LC8 with a dLBD:LC8 stoichiometry of 1:7 ( two chains of dLBD for 7 LC8 dimers ) and an overall Kd of 1 . 4 μM ( Figure 3a , Table 1 ) , suggesting that an additional non-TQT ASCIZ site binds LC8 .", "A plausible candidate is a TMT motif corresponding to residues 285–296 ( designated QT3 in Figure 3a ) .", "To confirm the functionality of this motif , ITC binding of LC8 was measured for constructs QT1-3 , QT2-4 , QT4-6 , and QT4-7 ( Figure 3b ) .", "QT1-3 and QT2-4 contain the TMT binding motif , and both bind LC8 with a stoichiometry of 3 , demonstrating that this TMT motif is the seventh LC8 recognition site ( Figure 3b , Table 1 ) .", "Each of the other two constructs bind LC8 with the stoichiometry expected from the number of TQT binding motifs .", "Interestingly , construct QT4-6 , containing recognition sites 4–6 , binds LC8 with a Kd of 1 . 0 μM , a slightly higher overall affinity than the full dLBD construct .", "QT4-7 , on the other hand , binds LC8 with a 1 . 6 μM affinity and is slightly more entropically disfavored than either QT4-6 or dLBD ( Table 1 ) .", "The overall Kd values of QT1-3 , QT2-4 , and QT4-7 , are 2 . 4 μM , 4 . 1 μM , and 1 . 6 μM , respectively .", "Given the small differences in binding affinity among the shorter dLBD constructs , we asked if any individual site binds LC8 with higher affinity than all others .", "A series of 14–15 amino acid peptides were synthesized , each corresponding to one of the seven recognition motifs , and their LC8 affinity was measured ( Figure 3c , Table 2 ) .", "The Kd values for all peptides indicate surprisingly weak affinity .", "QT3p interaction with LC8 is not even detected at 30 μM LC8 .", "Intriguingly , the QT4-6 construct has a slightly higher binding affinity than the other constructs , while the QT4 , QT5 , and QT6 peptides display among the lowest affinity as individual peptides .", "These results indicate that positive cooperativity enhances LC8 binding to neighboring recognition motifs .", "Since ITC experiments on the full length dLBD and smaller constructs identify 7 LC8 recognition sites , we sought to establish the size of the dLBD:LC8 complex at varying LC8 concentrations by analytical ultracentrifugation .", "The dLBD was titrated with increasing concentrations of LC8 and complex formation was assessed at dLBD:LC8 molar ratios 1:1 , 1:3 , 1:6 , and 1:10 .", "Plots of the continuous size distribution , c ( S ) , vs . sedimentation coefficient ( Figure 4a ) show that titration at sub-saturating concentrations of LC8 results in a broad peak that is likely an equilibrium mixture of complexes with varying LC8 occupancy in exchange with each other and with free dLBD .", "At a saturating concentration of LC8 ( 1:10 ratio ) , a high occupancy complex whose size ( 7 . 5 s ) approximately corresponds to a fully bound complex ( 197 kDa ) is clearly evident .", "Contrary to expectations , a low stoichiometry complex ( 5 s , approx . 114 kDa ) is even more highly populated .", "A high frictional ratio ( f/f0 ~ 1 . 6 ) indicates an elongated complex , and the molecular weight of this low occupancy complex corresponds roughly to a 1:3 complex , although its broadness , and the approximate nature of the molecular weight determination , indicate that multiple species are present .", "It is likely that the low occupancy peak , which is roughly twice the intensity of the high occupancy peak , is a heterogeneous mixture of 1:2 , 1:3 and 1:4 complexes .", "In summary , while high occupancy complexes are evident in AUC profiles , consistent with the ITC results , these complexes are in equilibrium with many smaller sub-saturated species , the most populated of which is a mixture of 1:2-1:4 complexes of dLBD:LC8 .", "The low occupancy complexes are favored , relative to higher occupancy complexes , even in samples having a large excess of LC8 .", "The presence of stable , low occupancy complexes is supported by small angle X-ray scattering ( SAXS ) data .", "A sample composed of dLBD and a large excess of LC8 was injected into an in-line size-exclusion chromatography system and X-ray scattering data were collected for the largest peak .", "Guinier analysis of the data indicates a monodisperse sample suitable for further analysis ( Figure 4—figure supplement 1a ) .", "The distance distribution function suggests a moderately compact structure for dLBD:LC8 complexes , with Dmax = 240 Å , and a molecular weight of roughly 110 kDa , confirming the presence of an unresolved mixture of dLBD:LC8 complexes with stoichiometries ranging from 1:2-1:4 ( Figure 4—figure supplement 1b ) .", "Additionally , a Kratky plot of the scattering data indicates that the dLBD:LC8 complex is a mix of globular domains and intrinsically disordered chains , consistent with low occupancy complex structures ( Figure 4—figure supplement 1c ) .", "Native gel electrophoresis titration of dLBD with LC8 corroborates the presence of a mixture of complexes ( Figure 4b ) .", "When unbound , dLBD and LC8 each migrate as a single band .", "When LC8 is added to dLBD at a 1:1 molar ratio , the band for free LC8 disappears , and the complexes formed migrate above LC8 , as a diffuse band likely corresponding to two dLBD chains bound to two or more LC8 dimers ( since some free dLBD persists ) .", "As the molar ratio is increased , the diffuse upper band becomes a dark smear , free LC8 accumulates in a pronounced dark band , and the free dLBD band disappears .", "We think the most likely explanation is that the decreasing mobility of the upper edge of the smear indicates increasing sizes of the complexes formed .", "Unbound LC8 is clearly visible at ratios ≥ 1:4 , indicating that a pool of free LC8 accumulates even at conditions well below LC8 saturation of dLBD .", "We similarly assessed the gel mobility of complexes formed by LC8 and the LC8-binding domain of human ASCIZ ( hLBD ) , which contains 11 TQT motifs ( Rapali et al . , 2011a ) ( Figure 1d ) .", "Very similar behavior is observed for hLBD , although the gel mobility of hLBD bands is much lower than the mobility of dLBD bands due to molecular sieving of the much larger hLBD ( 53 kDa ) compared to dLBD ( 17 kDa ) .", "Molecular sieving is the dominate effect on hLBD mobility as both hLBD and dLBD are disordered and highly extended and have a pI of ~4 .", "As LC8 is added to hLBD , the free hLBD band disappears and a lower mobility smear becomes increasingly evident .", "Decreasing mobility of the upper smear indicates increasing sizes of the complexes formed , while the appearance of a bands migrating the same as free LC8 at ratios ≥ 1:4 indicates the presence of a pool of free LC8 well below LC8 saturation of hLBD .", "Together the gel titration data for dLBD and hLBD suggest that hLBD:LC8 complexes form a dynamic ensemble with varying levels of LC8 occupancy in which lower occupancy forms are favored .", "Titration of the hLBD with LC8 by size-exclusion chromatography ( Figure 4d ) supports our interpretation of the native gel experiments in Figure 4c .", "Here , the amount of hLBD is held constant , and increases in peak intensity are due to effects on hLBD complexes from increasing LC8 .", "Both LMW and HMW complexes form at the first titration point and increase in population as more LC8 is added .", "A low occupancy intermediate , labeled LMW , is clearly evident at the lowest molar ratio of 1:3 , and persists at the same elution volume even at the highest molar ratios .", "Higher occupancy species , HMW , are also apparent at a ratio of 1:3 and become increasingly distinct as LC8 is increased .", "The peak corresponding to excess LC8 is discernable at a ratio of 1:7 , and steadily increases as LC8 is added .", "Taken together , the data in Figure 4 are consistent with the explanation that a low occupancy form of the hLBD:LC8 complex is favored even with a large excess of LC8 .", "A minor population of high occupancy complexes , present even at the lowest molar ratio , increases with increasing LC8 and is in equilibrium with the LMW species and with free LC8 , and therefore with each other .", "Both Drosophila and human LBD exhibit this dynamic behavior , suggesting it is a conserved feature of the ASCIZ:LC8 interaction .", "In order to visualize the various oligomeric states of ASCIZ-LC8 , we analyzed electron microscopy data of dLBD and hLBD under saturating concentrations of LC8 .", "As a positive control , and for validation of EM conditions , similar experiments were carried out with complexes of Nucleoporin159 ( Nup159 ) , another intrinsically disordered protein with multiple LC8 binding sites ( Stelter et al . , 2007 ) .", "Nup159:LC8 complexes were clearly visualized as a linear array of 5 stacked densities of LC8 , as previously reported ( Stelter et al . , 2007 ) ( data not shown ) , and consistent with the conclusions of Nup159:LC8 biophysical solution experiments ( Nyarko et al . , 2013 ) .", "In contrast , despite the similar overall affinity ( Nup159-LC8 Kd = 2 . 9 μM ) ( Nyarko et al . , 2013 ) , in negative stain images of dLBD:LC8 and hLBD:LC8 complexes , the vast majority of complex species appear dissociated on the grid ( Figure 5a , arrow heads ) .", "However , a few observable complexes could be clearly resolved from raw micrographs , identified as linear stacks of punctate densities , akin of beads on a string ( Figure 5a , squares ) , similar to images of Nup159 bound to LC8 ( Stelter et al . , 2007 ) .", "Furthermore , although dLBD contains seven and hLBD 11 LC8 binding sites , the vast majority of complexes observed by EM appeared to be of low LC8 occupancy .", "Reference-free two-dimensional ( 2D ) classification routines were carried out on datasets of ~2000 single particle images of dLBD:LC8 complexes and ~1000 particles of hLBD:LC8 complexes extracted from ~300 and 200 micrographs , respectively .", "These produced 2D projection averages for dLBD:LC8 and hLBD:LC8 oligomers displaying complexes formed with 2–4 stacked densities , corresponding to LC8 dimers , deduced from the dimensions of the averaged bead-like densities ( ~4 nm diameter ) ( Benison et al . , 2007; Stelter et al . , 2007 ) .", "Complexes with three or more LC8 dimers displayed significant conformational flexibility in 2D class averages ( Figure 5b ) and in the single-particle images ( Figure 5a and Figure 5—figure supplement 1 ) .", "The extent of conformational variability is consistent with ~10–20 Å spacing measured between LC8 densities , and the intrinsic flexibility of the IDP duplex chain separating the neighboring LC8 TQT recognition motifs .", "The formation of higher-order oligomers appeared relatively rare in comparison to the low occupancy complexes .", "The scarcity of higher order complexes , coupled with the intrinsic conformational heterogeneity , precluded our ability to obtain 2D class averages of the high-occupancy complexes .", "To overcome this limitation , statistical analysis describing the distribution of oligomeric states was obtained by hand-selection and classification from single-molecule images ( Figure 5—figure supplement 1 ) .", "Both dLBD:LC8 and hLBD:LC8 complexes form an ensemble of structures , displaying an exponential distribution with low-occupancy states ( i . e . 2–4 stacked LC8 dimers ) being most abundant ( Figure 5c , d ) .", "Density corresponding to the IDP duplex chain cannot be resolved by negative stain EM , therefore complexes formed with a single LC8 dimer were not included in this analysis , as they could not be distinguished from unbound LC8 dimers .", "Together , this analysis shows dLBD and hLBD form dynamic assemblies with LC8 that favor low occupancy states ( Figure 5d ) .", "Although uncommon , high-occupancy and fully-formed complexes of dLBD:LC8 ( 1:7 ratio ) could be identified from the raw single particle images ( Figure 5—figure supplement 1 ) , further confirming the stoichiometry obtained by our ITC studies .", "For the hLBD:LC8 dataset , complexes containing as many as 7–9 LC8 dimers could be distinguished from the single particle image data , while higher-order complexes containing 10–11 LC8 dimers were either not distinguishable or were simply absent under the limiting concentrations required for negative stain EM specimen preparation .", "Nevertheless , the remarkable similarity in distribution of oligomeric species formed by dLBD and hLBD , obtained under similar binding conditions , is consistent with the nearly equivalent overall LC8 affinity determined by ITC , and suggests that a conserved mechanism of negative cooperativity is used by ASCIZ to regulate the formation and distribution of higher-order LC8 assemblies .", "If dLBD and LC8 form stable intermediate complexes with excess LC8 , which of the seven recognition sites in Drosophila ASCIZ are preferentially bound ?", "Notably , the linear assembly pattern of LC8 dimers observed by negative stain EM suggests an ordered ( or quasi-ordered ) sequence of assembly , apparently favoring neighboring TQT sites .", "However , the location of LC8 binding sites could not be resolved in these experiments .", "Therefore , to examine interactions between individual motifs and LC8 in the context of the full dLBD , we turned to NMR .", "As unlabeled LC8 is titrated into solutions of 15N-13C- labeled dLBD , changes in NH peak intensities can be measured in 3D HNCO spectra recorded for dLBD:LC8 molar ratios of 1:0 . 25 , 1:1 , 1:2 , 1:5 , 1:8 .", "As LC8 concentration increases , a corresponding decrease in dLBD peak intensity is observed ( Figure 6a ) .", "At a molar ratio of 1:5 , less than 10% of the original peak intensity remains at all seven LC8 binding sites .", "At a ratio of 1:8 , all dLBD peaks completely disappear except for peaks corresponding to eight N-terminal residues ( 241-248 ) , indicating that all TQT sites have been occupied to some degree .", "The absence of peaks for bound dLBD is attributed to line broadening associated with intermediate exchange processes and/or faster transverse relaxation as a result of increased complex size .", "Therefore , we consider a decrease in peak intensity as a measure of increased complex formation .", "Notably , peaks at the C-terminal half of the protein , QT4-7 , decrease more quickly than peaks at the N-terminal half , implying that LC8 preferentially occupies these motifs .", "To confirm this observation and to obtain titration information for the missing residues in this region , we performed similar experiments on the smaller QT2-4 and QT4-6 constructs .", "QT2-4 was chosen to further validate LC8 binding to the QT3 motif that as an individual peptide showed weak binding by ITC , and QT4-6 was chosen because it has the highest LC8 binding affinity ( Figure 3b ) .", "Further , the two constructs share the QT4 motif , allowing us to assess its affinity in two sequence contexts .", "Due to the smaller number of peaks for shorter constructs , HSQC spectra have a sufficiently high resolution to render an HNCO-based titration unnecessary .", "Unlabeled LC8 was titrated into 15N-labeled QT2-4 or QT4-6 and changes in peak intensity were analyzed ( Figure 6b , c ) .", "As with dLBD , there is a gradual decrease in peak intensity as more LC8 is added to QT2-4 or to QT4-6 at molar ratios: 1:0 . 25 , 1:1 , 1:2 , 1:3 , and 1:4 .", "Significantly , peak intensities in QT4-6 decrease at lower LC8 ratio than in QT2-4 , confirming the trend we observe in full-length dLBD .", "In QT4-6 , nearly all peaks in the motif region disappear at a ratio of 1:2 , while ~30% of peak intensity remains in QT2-4 .", "Furthermore , NMR titration of QT2-4 with LC8 confirms that QT3 is an LC8 binding motif .", "The peaks corresponding to QT3 decrease in intensity at the same rate as peaks corresponding to the QT2 and QT4 motifs .", "The peaks in the linker region ( residues 305–320 ) decrease more slowly , indicating that they are not interacting with LC8 , but merely experiencing the effects of a larger correlation time .", "Plots of the average peak intensity ( I/I0 ) for each 10-amino acid motif in dLBD , and in each of QT2-4 and QT4-6 constructs clearly show the dichotomy in the pattern of peak attenuation ( Figure 6d–g ) .", "In dLBD , the first three ( Figure 6d ) show a different titration pattern and weaker binding than the last four motifs ( Figure 6e ) .", "This dichotomy is replicated in separate plots of the 3 QT motifs in each of the constructs QT2-4 and QT4-6; the average I/I0 of motifs in QT2-4 ( Figure 6f ) drops to 0 . 2 at LC8 molar ratio 1:2 , twice that observed for motifs in QT4-6 ( Figure 6g ) which reach the same I/I0 at molar ratio 1:1 .", "The apparently higher LC8 affinity of motifs 4–6 ( Figure 6e , g ) , relative to motifs 2–4 ( Figure 6d , f ) is consistent with our ITC experiments ( Figure 3a ) .", "The QT4 motif , common to both constructs , has a different rate of peak disappearance in each construct , suggesting that motif environment , not local sequence , determines its affinity .", "We conclude that the recognition motifs QT4-QT7 are the sites favored in stable low occupancy complexes .", "In summary , the data in Figure 6 showing peak attenuation across the whole sequence , even at low LC8 ratios , suggest population of an ensemble with all LC8 sites occupied to varying degree , but with clear preference for the C-terminal motifs .", "Preferential binding of the C-terminal motifs is additionally supported by ITC results , which show that LC8 binds to the QT4-6 construct with slightly higher affinity than the full-length dLBD ( Figure 3a , b ) .", "NMR dynamics experiments also demonstrate that residues in the C-terminal motifs are slightly more ordered in comparison to the N-terminal motifs ( Figure 2f–g , Figure 2—figure supplement 1a ) , which may explain the tighter LC8 binding to this region .", "To investigate how the number of bound LC8 molecules affects the transcriptional activity of ASCIZ , we turned our attention to the human protein whose transcriptional activity can be assayed in cell culture using an ASCIZ knockout mouse embryonic fibroblast cell line ( Jurado et al . , 2012a ) .", "Human ASCIZ has eleven LC8 recognition motifs that we have numbered 1 to 11 ( Figure 7a ) .", "To prevent LC8 binding to specific ASCIZ motifs , TQT recognition motifs were mutated to AAA .", "Five human ASCIZ mutant constructs were generated: AAA1-4 with an AAA replacement at each TQT motifs 1–4 , and similarly named mutant constructs of AAA8-11; AAA5-11; AAA1-4 , 8–11; and AAA-all .", "To confirm loss of LC8 binding in vitro for each TQT when replaced with AAA , identical mutations were made in ASCIZ hLBD constructs , named in the same fashion .", "To completely eliminate LC8 binding in the AAA-all construct , it was necessary to also mutate three SQT/VQT motifs in addition to the 11 TQT motifs identified in our ITC experiments and in pepscan experiments ( Figure 7a , gray bars ) ( Rapali et al . , 2011a ) .", "This suggests that human ASCIZ may contain additional binding motifs , as seen with the TMT motif in Drosophila ASCIZ .", "The effective affinity and stoichiometry of binding of LC8 to WT ASCIZ hLBD and each mutant hLBD was determined ( Figure 7b , and Table 3 ) .", "WT hLBD binds LC8 with an hLBD:LC8 ratio of 1:11 ( two chains of ASCIZ to 11 LC8 dimers ) , and an overall Kd value of 0 . 9 μM ( Table 3 ) .", "The four hLBD mutants each binds LC8 with the expected stoichiometry for the number of intact LC8 recognition motifs and with overall affinities in the range of 0 . 7–4 . 4 μM .", "To assess the impact of these mutations on ASCIZ transcriptional activity , luciferase reporter assays were carried out using immortalized ASCIZ knockout mouse embryonic fibroblasts transiently transfected with the full-length WT or a mutant ASCIZ gene and a plasmid containing the LC8 promoter .", "The measured luciferase activity was normalized against Renilla luciferase ( Figure 7c ) .", "Empty vector and the ΔZnF construct showed limited transcriptional activity compared to ASCIZ constructs .", "Most significantly , transcriptional activity of the AAA mutants can be ranked to form an activity gradient ( Figure 7c ) notable for a clear inverse relationship between their transcriptional activity and their affinity for LC8 .", "While the differences between each construct are very small , the overall trend supports the hypothesis that affinity and transcriptional activity are correlated .", "The construct with the highest affinity for LC8 by ITC , AAA8-11 , exhibits equal or slightly lower transcriptional activity than WT ASCIZ , while the construct with the lowest affinity for LC8 , AAA1-4 , 8–11 , has 2 . 5x the activity of WT ASCIZ .", "The correlation between transcriptional activity and affinity for LC8 is also shown in Figure 7d .", "As the number of available binding sites decreases and Kd correspondingly increases , transcriptional activity also increases .", "One exception to this trend is the AAA-all construct with zero functional LC8 binding sites ( AAA-all ) , which shows equal or somewhat lower activity than the construct with three intact sites ( AAA1-4 , 8–11 ) .", "As both the ZnF and LBDs of ASCIZ are monomeric in the absence of LC8 , a plausible explanation for this effect could be that dimerization of ASCIZ by a minimal number of LC8 molecules is required for optimal binding to the DYNLL1 gene promoter , or the binding of dimeric transcription co-activators to ASCIZ .", "In summary , the data in Figure 7 indicate that , in general , ASCIZ transcriptional activity appears to vary inversely with the number of LC8 recognition motifs and with binding affinity , and that fine tuning within this trend depends on which motifs are occupied and their specific dissociation constants ." ], [ "ASCIZ has three structural and functional features that together set it apart from other multivalent transcription factors .", "( 1 ) ASCIZ has an exceptionally long intrinsically disordered C-terminal domain compared to other intrinsically disordered transcription factors .", "Human ASCIZ contains an intrinsically disordered domain that is 600 amino acids long , or 73% of its overall sequence .", "The well-studied transcription factor p53 , by contrast , is 40% disordered and the disordered regions are dispersed through three different regions of the protein rather than being concentrated on one terminus ( Laptenko et al . , 2016 ) .", "Intrinsically disordered regions longer than 50 amino acids are considered to be of significant size for eukaryotic transcription factors ( Liu et al . , 2006 ) .", "As intrinsic disorder is proposed to play an important role in regulating function ( Shammas , 2017 ) , it is possible that the length of the disordered domain enables a larger diversity of functions .", "Indeed , recent work has shown that the length of the intrinsically disordered domain can control transcriptional activity through ‘energetic frustration’ , wherein opposing energetic couplings mediate the overall activity ( Li et al . , 2017 ) .", "( 2 ) ASCIZ interactions with LC8 display both positive and negative cooperativity that together create a dynamic equilibrium of stable , low occupancy ASCIZ-LC8 complexes .", "ASCIZ binding to LC8 forms an IDP duplex scaffold ( Clark et al . , 2015 ) onto which other copies of LC8 or other dimeric partners can bind with higher affinity .", "The first two to four recognition motifs bind to LC8 with positive cooperativity , as evidenced by ITC experiments that show an enhancement in binding affinity from the presence of neighboring motifs ( Figure 3 , Table 1 ) .", "Negative cooperativity regulates the formation and distribution of higher-order LC8 assemblies , as shown by the dominance of low occupancy complexes at saturating concentrations of LC8 ( Figures 4 and 5 ) .", "Negative cooperativity observed between low occupancy complexes and the fully occupied complex suggests that when the concentration of LC8 exceeds the buffering capacity of the low occupancy intermediates , the fully occupied complex is formed to switch off transcription .", "A distribution of low occupancy dynamic complexes is a conserved feature of the ASCIZ:LC8 interaction .", "Evidence for a dynamic ASCIZ:LC8 ensemble comes from a combination of AUC , gel filtration , native gel electrophoresis , and negative stain electron microscopy data ( Figures 4–5 , Figure 4—figure supplement 1 ) .", "For both Drosophila and human LBD constructs , addition of excess LC8 results in formation of stable low molecular weight ( LMW ) complexes and a minor population of high molecular weight complexes ( Figures 4 and 5 ) .", "Negative stain electron microscopy experiments show an exponential distribution of complexes , with assemblies containing 2–4 stacked copies of LC8 clearly visualized in 2D projection averages and suggesting a high degree of flexibility within the duplex IDP linkers persists upon complex formation ( Figure 5 ) .", "Multivalency and intrinsic disorder in ASCIZ’s LC8 binding domain enable this dynamic ensemble of low occupancy complexes .", "Many proteins utilize multiple binding sites within intrinsically disordered regions for regulation , complex formation , and a multitude of other functions ( Cortese et al . , 2008; Uversky , 2015 ) .", "In some examples , multiple binding sites serve as a scaffold to bring proteins together ( Cortese et al . , 2008 ) , while in others , they modulate phase transitions that lead to the formation of bimolecular condensates ( Banani et al . , 2017; Banani et al . , 2016 ) .", "The diversity of these examples highlights the importance of multivalency and intrinsically disordered regions in protein function and regulation .", "It is of note that although ASCIZ’s multiple binding sites are similar to those that lead to phase transitions in other systems ( Li et al . , 2012 ) , we did not detect this behavior in vitro .", "However , ASCIZ puncta formation has been observed in cell culture in response to treatment with MMS , a DNA methylation agent ( Jurado et al . , 2012a; Jurado et al . , 2010 ) , ( McNees et al . , 2005 ) .", "These puncta do not form in the absence of LC8 , indicating that LC8 binding to multiple recognition motifs is necessary for foci formation .", "Comparison of ASCIZ with another LC8 multivalent binding partner , Nup159 , underscores the uniqueness of the ASCIZ-LC8 assembly .", "While three other multivalent LC8 binding partners with more than two recognition motifs are known to exist ( Dunsch et al . , 2012; Fejtova et al . , 2009; Gupta et al . , 2012; Stelter et al . , 2007 ) , the role of multiple sites has only been characterized for ASCIZ ( this work ) and Nup159 ( Nyarko et al . , 2013 ) .", "Nup159 cooperatively binds five LC8 dimers and forms a relatively stable complex readily visible by electron microscopy and 2D classification analysis ( Stelter et al . , 2007 ) .", "As Nup159 has a slightly lower affinity for LC8 than ASCIZ ( 2 . 9 μM vs . 0 . 9 μM , respectively ( Nyarko et al . , 2013 ) , the difference between the Nup159 grids with uniformly stacked structures and the ASCIZ grids with sparse and heterogeneous structures of 2–4 stacked LC8 dimers is intriguing .", "This difference in structural heterogeneity can be attributed to the higher flexibility or short life time of ASCIZ:LC8 complexes relative to Nup159:LC8 complexes , while the scarcity of high-occupancy states is consistent with a unique mode of negative cooperativity .", "Given the different function of Nup159 ( in nuclear pore assembly ) versus ASCIZ:LC8 complexes ( transcription regulation ) , the data suggest that the dynamic properties and unique mechanism of assembly that is conserved in LC8-ASCIZ complexes may reflect an important feature required for autoregulation of LC8 transcription .", "( 3 ) ASCIZ regulates its transcriptional activity by binding multiple copies of its gene product , LC8 .", "Cell culture based transcription assays demonstrate that ASCIZ affinity for LC8 is negatively correlated with transcriptional activity ( Figure 7 ) .", "Although the differences between each construct are very small , we see an obvious trend where fewer occupied LC8 binding motifs lead to increased ASCIZ transcriptional activity , and vice versa .", "The results of this assay suggest that LC8 concentration could fine-tune ASCIZ activity in a cellular environment by shifting the population of complexes towards higher or lower occupancy states .", "A great example of multisite regulation is the E26 transformation-specific transcription factor ( Ets-1 ) , which tunes its transcriptional activity through multisite phosphorylation of its serine-rich domain .", "Phosphorylation of the serine rich region occludes the DNA-binding interface and stabilizes its helical inhibitory module , inhibiting Ets-1 DNA binding ~20 fold ( Desjardins et al . , 2014; Lee et al . , 2008; Pufall et al . , 2005 ) .", "Similarly , the function of p53 is modulated by over 50 posttranslational modifications that are proposed to be interdependent ( Meek and Anderson , 2009 ) .", "Phosphorylation of specific p53 residues prevents binding to the inhibitory protein HDM2 , while increasing binding to the activating proteins CREB-binding protein ( CBP ) and p300 ( Ferreon et al . , 2009 ) .", "p53 affinity for CBP/p300 depends on the extent of p53 phosphorylation; successive phosphorylation events increase p53 affinity for the TAZ1 , TAZ2 , and KIX domains of CBP/p300 ( Lee et al . , 2010; Teufel et al . , 2009 ) .", "p53 also binds to a multitude of other proteins that regulate its activity ( Beckerman and Prives , 2010 ) .", "While many other transcription factors tune their activity through multisite regulation , we could find no other examples besides ASCIZ where binding to multiple copies of their gene product modulates activity; yet , the prevalence of IDP domains in transcription factors indicates that such mechanisms are likely to be widespread and studies such as these are becoming more tractable with the integrated approaches used here .", "Based on our experimental data , we have developed a model of ASCIZ transcriptional regulation that illustrates the relationship between transcriptional activity ( red arrow ) and LC8 concentration ( blue arrow ) ( Figure 8 ) .", "As the cellular level of LC8 ( blue dimers ) increases , the number of LC8 molecules bound to ASCIZ also increases .", "The ASCIZ-LC8 complex primarily exists as a dynamic equilibrium of different complex stoichiometries and degrees of disorder ( center brackets ) .", "We propose that this low occupancy conformational ensemble is important for maintaining a basal level of LC8 transcription .", "It acts as a buffer for changes in the concentration of LC8 and fine-tunes transcription levels according to cellular needs .", "A change in LC8 cellular concentration would shift this dynamic equilibrium to a higher or lower occupancy state without dramatically altering the level of transcription .", "All 11 ( or 7 ) binding sites are therefore occupied in the heterogeneous mixture of complexes , as is demonstrated by NMR titration ( Figure 6 ) , and participate in maintaining a homeostatic concentration of LC8 .", "Thus , it is ensured that a high concentration of LC8 does not drastically decrease LC8 production , but rather shifts it to a lower level .", "As LC8 is a hub protein that interacts with >40 protein partners , and is predicted to bind to 100 additional proteins from a diverse selection of cellular pathways ( Rapali et al . , 2011b ) , maintaining a constant level of its transcription is essential ." ], [ "Studies were carried out using constructs from human ASCIZ ( Uniprot O43313 ) as well as Drosophila ASCIZ ( dASCIZ ) ( Uniprot Q9VZU1 ) which , with its fewer recognition motifs , smaller size , and available mutant phenotypes , is a tractable model of the human ASCIZ .", "Constructs of the dASCIZ zinc finger domain ( ZnF ) and the LC8 binding domain ( dLBD ) were generated by cloning residues 1–156 or 241–388 , respectively , of Drosophila ASCIZ into the pET2Zt2-1a vector .", "The constructs were expressed in frame with a hexahistidine tag , Protein A solubility tag , and cleavage site for the tobacco etch virus ( TEV ) enzyme .", "Shorter constructs of the dLBD were generated by cloning residues 241–324 ( QT1-3 ) , 271–341 ( QT2-4 ) , 299–376 ( QT4-6 ) , and 321–388 ( QT4-7 ) into the pET2Zt2-1a vector .", "The human LC8 binding domain ( hLBD ) construct was generated by cloning human ASCIZ ( Uniprot O43313 ) residues 362–823 into the pET24d vector ( Novagen ) and expressing the construct in frame with a hexahistidine tag and TEV cleavage site .", "For the five human ASCIZ mutants , ASCIZ AAA1-4 , AAA8-11 , AAA5-11 , AAA1-4 , 8–11 , and AAA-all , residues 7–9 of the LC8 binding motif ( usually the residues TQT ) , were mutated to AAA to prevent binding .", "Mutations were performed using either the QuikChange Lightening Mutagenesis Kit ( Agilent ) or by synthesizing short constructs ( 300–350 bp ) containing the desired mutations and using Gibson Assembly ( New England Biosciences , Ipswich ) to insert them into the LC8-binding domain ( hLBD ) gene ( residues 362–823 of human ASCIZ ) .", "All constructs were transformed into Escherichia coli Rosetta DE3 cells and expressed at 37°C in LB or minimal autoinduction media with 12C or 13C glycerol and 15NH4Cl as the sole carbon and nitrogen sources , respectively .", "Recombinant protein expression was induced with 0 . 4 mM IPTG ( for LB cultures ) and growth continued at 25°C for 16 hr .", "Cells were harvested and purified under denaturing conditions using TALON His-Tag Purification protocol ( Clontech ) .", "The solubility tag and/or hexahistidine tag were cleaved by TEV protease and the protein was further purified using strong anion exchange chromatography ( Bio-Rad , Hercules , California ) followed by gel filtration on a SuperdexTM 75 gel filtration column ( GE Health ) .", "The purity of the recombinant proteins , as assessed by SDS-polyacrylamide gels , was >95% .", "The pure proteins were stored at 4°C and used within 1 week .", "LC8 was prepared as previously described ( Barbar et al . , 2001 ) .", "Peptides corresponding to the seven putative recognition sequences from dASCIZ were commercially synthesized: YMSSQKLDMETQTEE ( QT1p ) , YLAPLLRDIETQTPD ( QT2p ) , YTPDTRGDIGTMTDD ( QT3p ) , DLQTSAHMYTQTCD ( QT4p ) , EELGLSHIQTQTHW ( QT5p ) , WPDGLYNTQHTQTCD ( QT6p ) , and EPDNFQSTCTQTRW ( QT7p ) ( GenScript , Piscataway , NJ ) .", "Non-native amino acids ( underlined in Table 2 ) were added to the N-terminus to enhance solubility or concentration determination by UV absorbance at 280 nm .", "Binding thermodynamics of the ASCIZ and dASCIZ construct/peptide-LC8 interactions were obtained at 25°C with a VP-ITC microcalorimeter ( Microcal , Westborough , MA ) .", "The binding buffer was composed of 50 mM sodium phosphate , 50 mM sodium chloride , 1 mM sodium azide , 5 mM β-mercaptoethanol , pH 7 . 5 .", "Protein concentrations were determined by absorbance measurement at 280 nm .", "Extinction coefficients for each construct are as follows .", "LC8 = 14 , 565 M−1cm−1 , dLBD = 17 , 085 M−1cm−1 , LBD = 15 , 470 M−1cm−1 , QT1−3 = 2 , 980 M−1cm−1 , QT2−4 = 2 , 980 M−1cm−1 , QT4−6 = 10 , 095 M−1cm−1 , QT4−7 = 14 , 105 M−1cm−1 .", "dLBD was placed in the reaction cell at a concentration of 8 μM and titrated with LC8 at a concentration of 800 μM .", "For binding of dASCIZ constructs QT1-3 , QT2-4 , QT4-6 , and QT4-7 , 10 μM of construct was titrated with 400 μM LC8 .", "For interactions with synthetic peptide , peptides were dissolved in binding buffer to a final concentration of 300 μM and then added to LC8 at a concentration of 30 μM in the reaction cell .", "ASCIZ hLBD and mutant hLBD constructs were placed in the reaction cell at a concentration of 9–16 μM and titrated with 900 μM LC8 .", "Peak areas were integrated and data were fit to a single-site binding model in Origin 7 . 0 from which the stoichiometry ( N ) , dissociation constant ( Kd ) , and the change in enthalpy ( ΔH ) , and entropy ( ΔS ) were obtained .", "Reported data are the average of two or more independent experiments .", "As the binding-model fit was very good and data were reproducible , error was determined based on a 5% uncertainty in protein concentration calculations .", "CD experiments were conducted on a Jasco720 spectropolarimeter in a 1 mm cell .", "For the spectrum of dLBD and smaller constructs , ten scans were averaged at a concentration of 30 μM in a buffer composed of 10 mM sodium phosphate , pH 7 . 5 , at 25°C and 10°C .", "For the ZnF , ten scans were averaged at a concentration of 25 μM in a buffer composed of 10 mM sodium phosphate , 200 mM sodium sulfate , 50 μM zinc sulfate , pH 7 . 5 , at 10°C , 25°C , and 35°C .", "Sedimentation velocity experiments for the titration of dLBD and LC8 were performed in a Beckman Coulter Model XL-I analytical ultracentrifuge equipped with UV/Vis scanning optics .", "Reference ( 400 μL binding buffer; 50 mM sodium phosphate , 50 mM sodium chloride , 1 mM sodium azide , 5 mM TCEP , pH 7 . 5 ) and sample ( 380 μL ) solutions were loaded into 12 mm double-sector cells with quartz windows and the cells were then mounted in an An-50 Ti 8-hole rotor .", "LC8 was prepared at a concentration of 15 µM while the concentration of dLBD was varied from 15 to 1 . 5 µM .", "Proteins were centrifuged at 50 , 000 rpm at 20°C , and radial absorbance data were collected at appropriate wavelengths in continuous mode every 5 min without averaging .", "Data were fit to a continuous size-distribution [c ( S ) ] model using the program SEDFIT ( Schuck , 2000 ) .", "The partial specific volume of the proteins , buffer density , and buffer viscosity were computed using the program SEDNTERP ( Hayes and Philo , 1995 ) .", "Sedimentation velocity experiments for the ZnF domain were performed on a Beckman ProteomeLab XL-A/XL-I analytical ultracentrifuge in a buffer composed of 50 mM sodium phosphate , 200 mM sodium chloride , 0 . 4 mM zinc sulfate , 1 mM sodium azide , 2 mM TCEP , pH 7 . 0 .", "The sample was centrifuged at 40 , 000 rpm at 20°C for 7 hr and absorbance data were collected at 286 nm .", "Data were fit to a continuous size-distribution [c ( s ) ] model using the program SEDPHAT ( Vistica et al . , 2004 ) .", "NMR measurements were collected at 10°C , using 300–350 μM isotopically ( 13C/15N or 15N ) labeled dLBD in a buffer at pH 6 . 5 composed of 10 mM sodium phosphate , 10 mM sodium chloride , 1 mM sodium azide , 10 mM β-mercaptoethanol , a protease inhibitor mixture ( Roche Applied Science , Madison , WI ) , and 2–2 dimethylsilapentane-5-sulfonic acid for 1H chemical shifts referencing .", "Data for backbone assignments were collected on a Bruker Avance 850 MHz spectrometer equipped with a cryoprobe .", "Five-dimensional HN ( CA ) CONH and HabCabCONH experiments ( Kazimierczuk et al . , 2010; Motáčková et al . , 2010; Nováček et al . , 2011 ) and a three-dimensional HNCO experiment were acquired with non-uniform sampling of the indirectly detected dimensions and used for sequential assignment of 13C-15N- dLBD .", "Interaction of unlabeled LC8 and 13C-15N labeled dLBD was characterized by collecting three-dimensional BEST-TROSY-HNCO spectra at multiple molar ratios of LC8 , 1: 0 . 25 ( dLBD: LC8 ) , 1:1 , 1:2 , 1:5 , and 1:8 .", "For the interaction of unlabeled LC8 with 15N-labeled QT2-4 ( residues 271–341 ) or QT4-6 ( residues 321–376 ) , two-dimensional BEST-TROSY-HSQC spectra were collected at the molar ratios ( QT2-4/QT4-6:LC8 ) 1:0 . 25 , 1:1 , 1:2 , 1:3 , and 1:4 .", "NMR titration data were analyzed and plotted by measuring peak volumes using Sparky and averaging over each 10 amino acid QT motif .", "HNCO-based R1 relaxation measurements experiments were recorded with relaxation delay times ranging from 11 . 2 to 2352 ms , and the R2 relaxation data were acquired using relaxation delays ranging from 14 . 4 to 259 ms . Sixteen total R1 or R2 experiments were recorded , including six duplicate experiments for error determination .", "Curve fitting was performed using the rate analysis script Sparky2Rate and the program Curvefit ( A . G . Palmer , Columbia University ) .", "Steady-state 1H−15N heteronuclear NOEs were acquired using 6 s total saturation time .", "Error bars were determined from the intensities of the baseline noise using the formula σ/ ( NOE ) = [ ( σIsat/Isat ) 2 + ( σIunsat/Iunsat ) 2]1/2 , where Isat and σIsat correspond to the intensity of the peak and its baseline noise .", "All two-dimensional spectra and the three-dimensional HNCO spectra were processed using TopSpin ( Bruker Biosciences; RRID:SCR_014227 ) , and the non- uniformly sampled five-dimensional HN ( CA ) CONH and HabCabCONH spectra were processed with Sparse Multidimensional Fourier Transform ( Kazimierczuk et al . , 2009; Stanek and Koźmiński , 2010 ) , ( the software for data processing is available online at the Warsaw University Laboratory ( nmr . cent3 . uw . edu . pl/software ) ) .", "All spectra were analyzed with the graphical NMR assignment and integration software NMRFAM-Sparky ( RRID:SCR_014228 ) .", "hLBD at a concentration of 30 μM was incubated with 600 μM LC8 at various molar ratios: ( hLBD:LC8 ) 1:3 , 1:5 , 1:7 , 1:9 , 1:11 , and 1:13 .", "The complex was loaded on a Superdex 200 analytical column ( GE healthcare , Wauwatosa , WI ) in binding buffer: 50 mM sodium phosphate , 50 mM NaCl , 5 mM β-mercaptoethanol , 1 mM sodium azide , pH 7 . 5 .", "100 or 200 μl of protein samples were injected at a flow rate of 0 . 5 ml/min at room temperature and samples were monitored by UV absorption at 280 nm .", "For native gel electrophoresis titrations , dLBD or hLBD and LC8 were incubated at the molar ratios listed above and run on a 10% polyacrylamide gel at a constant 10 mAmps for 5–7 hr .", "Small-angle X-ray scattering experiments were conducted at the ESRF BioSAXS beamline BM29 ( Pernot et al . , 2013 ) in Grenoble , France .", "dLBD and LC8 samples were purified as described above and dialyzed into binding buffer ( 50 mM sodium phosphate , 50 mM sodium chloride , 10 mM beta-mercaptoethanol , 1 mM sodium azide , pH 7 . 5 ) before SAXS measurements .", "30 μl of dLBD:LC8 complex ( 1:8 molar ratio ) at five different concentrations for each sample ( and buffer ) were exposed to X-rays and scattering data collected using the robotic sample handling available at the beamline .", "10 individual frames were collected for every exposure , each 2 s in duration using the Pilatus 1M detector ( Dectris , Switzerland ) .", "Individual frames were processed automatically and independently within the EDNA framework , yielding individual radially averaged curves of normalized intensity versus scattering angle s = 4πSinθ/λ .", "Additional data reduction within EDNA utilizes the automatic data processing tools of EMBL-Hamburg ATSAS package ( M . V . K . Petoukhov , P . V . ; Kikhney A . G . ; Svergun D . I . , 2007 ) , to combine timeframes , excluding any data points affected by aggregation induced by radiation damage , yielding the average scattering curve for each exposure series .", "Matched buffer measurements taken before and after every sample were averaged and used for background subtraction .", "Merging of separate concentrations and further analysis steps were performed manually using the tools of the ATSAS package ( M . V . K . Petoukhov , P . V . ; Kikhney A . G . ; Svergun D . I . , 2007 ) ( RRID:SCR_015648 ) .", "The forward scattering I ( 0 ) radius of gyration , Rg were calculated from the Guinier approximation ( A . , 1938 ) , the hydrated particle volume was computed using the Porod invariant ( Porod , 1982 ) and the maximum particle size Dmax , was determined from the pair distribution function computed by GNOM ( Svergun , 1992 ) using PRIMUS .", "Electron microscopy ( EM ) studies were conducted using dLBD and hLBD incubated with a molar excess of LC8 , and the formed complexes were negatively stained for contrast enhancement using established protocols ( Myers et al . , 2017 ) .", "Briefly , dLBD ( 50 nM ) was mixed with LC8 at a molar ratio 1:8 , and human hLBD peptide ( 50 nm ) was mixed with LC8 at a molar ratio of 1:13 , in EM buffer containing 20 mM Tris , pH 7 . 5 , 50 mM NaCl , 10 mM BME and 1 mM NaN3 .", "A 3 μl drop of sample was applied to a glow-discharged continuous carbon coated EM specimen grid ( 400 mesh Cu grid , Ted Pella , Redding , CA ) .", "Excess protein was removed by blotting with filter paper and washing the grid two times with EM buffer .", "The specimen was then stained with freshly prepared 0 . 75% ( wt vol−1 ) uranyl formate ( SPI-Chem , West Chester , PA ) .", "Negatively stained specimens were visualized on a 120 kV TEM ( iCorr , FEI , Hillsboro , OR ) at a nominal magnification of 49 , 000x at the specimen level .", "Digital micrographs were recorded on a 2K × 2K CCD camera ( FEI Eagle ) with a calibrated pixel size of 4 . 37 Å pixel−1 and a defocus of 2 . 0–3 . 5 μm .", "For the dLBD-LC8 specimen , a total of 2574 single particle images were extracted from ~300 micrographs , and for hLBD-LC8 , 1234 particles were extracted from ~200 micrographs .", "Complexes with clear oligomeric structure could be identified and were manually-selected using EMAN2 ( Tang et al . , 2007 ) .", "Single particle images were extracted with a box size of 160 × 160 pixels and CTF-corrected ( phase-flipped ) in EMAN2 .", "Reference-free 2D class averages were generated in EMAN2 and RELION 2 . 0 ( Scheres , 2012 ) using CTF-corrected and high-pass filtered image datasets .", "Statistical analysis of oligomeric composition was performed by counting the number of subunits identified from single particle images and classifying them manually as 2 – 7mers ( dLBD:LC8 complexes ) or 2 – 11mers ( hLBD:LC8 complexes ) ( Figure 5—figure supplement 1 ) .", "Particles that could not be confidently assigned were discarded , leaving 2334 oligomers assigned for the dLBD:LC8 and 967 for hLBD:LC8 datasets .", "Complexes containing only a single LC8 dimer could not be distinguished from unbound LC8 particles , and were not included in our analysis .", "As a positive control , Nucleoporin159 ( Nup159 ) in complex with LC8 was also prepared for negative stain EM under similar conditions to the dLBD/hLBD samples , and as previously described ( Stelter et al . , 2007 ) ( not shown ) .", "As a negative control , we prepared EM grids with LC8 alone and dLBD/hLBD alone .", "No oligomeric structures ( i . e . beads on a string ) were observed in these images ( not shown ) .", "To measure transcriptional activity of ASCIZ mutants , six ASCIZ constructs were cloned into the pEGFP vector ( Clontech ) : WT ASCIZ ( 1-823 ) , ΔZnF ( 230-823 ) , ASCIZ AAA1-4 , ASCIZ AAA8-11 , ASCIZ AAA5-11 , and ASCIZ AAA1-4 , 8–11 .", "Approximately 2 kbp of the Dynll1 promoter was cloned into the pGL3 vector ( Promega , Madison , WI ) upstream of the firefly luciferase gene as previously described ( Jurado et al . , 2012a ) .", "Using FuGENE 6 ( Promega ) , immortalized ASCIZ knockout mouse embryonic fibroblasts ( MEFs ) ( Jurado et al . , 2010 ) were co-transfected with ASCIZ constructs , the Dynll1 promoter , and a pRL-CMV vector containing Renilla luciferase for normalization of firefly/luciferase ratios .", "The authenticity of the ASCIZ knockout MEF cell line was verified by PCR genotyping of the ASCIZ locus .", "The cell line tested to be free from mycoplasma contamination using a MycoAlert mycoplasma detection kit ( Lonza , Switzerland ) .", "24 hr after transfection , cells were transferred to 96-well plates and incubated overnight before determining reporter gene activities using the dual-luciferase reporter assay kit ( Promega ) and a Polarstar Optima ( BMG Labtechnologies , Germany ) instrument .", "For assessment of protein expression levels , human U2OS cells were transfected with ASCIZ constructs using FuGENE six and were probed with ASCIZ antibody ( McNees et al . , 2005 ) .", "The chemical shifts for dLBD ASCIZ have been deposited in the Biological Magnetic Resonance Data Bank under accession code 27412 ." ] ]

[ "The transcription factor ASCIZ ( ATMIN , ZNF822 ) has an unusually high number of recognition motifs for the product of its main target gene , the hub protein LC8 ( DYNLL1 ) .", "Using a combination of biophysical methods , structural analysis by NMR and electron microscopy , and cellular transcription assays , we developed a model that proposes a concerted role of intrinsic disorder and multiple LC8 binding events in regulating LC8 transcription .", "We demonstrate that the long intrinsically disordered C-terminal domain of ASCIZ binds LC8 to form a dynamic ensemble of complexes with a gradient of transcriptional activity that is inversely proportional to LC8 occupancy .", "The preference for low occupancy complexes at saturating LC8 concentrations with both human and Drosophila ASCIZ indicates that negative cooperativity is an important feature of ASCIZ-LC8 interactions .", "The prevalence of intrinsic disorder and multivalency among transcription factors suggests that formation of heterogeneous , dynamic complexes is a widespread mechanism for tuning transcriptional regulation ." ]

[ "Proteins help to regulate almost every process in the body , and come in various forms , sizes and purposes .", "Cells contain thousands of different proteins , but not every protein is needed at all times .", "To create new proteins , the information on a gene first needs to be transcribed into RNA ( template molecules of the DNA ) in a process known as transcription .", "A complex machinery inside the cell then uses the copy as a template to assemble the protein .", "So-called transcription factors ( also proteins ) can switch the copying process on or off by binding to the start point of a gene .", "They can act alone or in complex with other proteins .", "The transcription factor called ASCIZ , for example , helps to regulate the production of a protein called LC8 .", "LC8 attaches to more than 100 different proteins and plays an important role in many cell processes .", "Therefore , fine-tuning its production is essential .", "The shape of a protein is critical to its purpose .", "Like most proteins , transcription factors are made up of chains of amino acids that fold into a specific three-dimensional ( 3D ) structurewith a region that recognizes and binds to a specific DNA sequence .", "But many transcription factors also contain flexible , ‘disordered’ regions that do not fold into a rigid 3D shape .", "These may help to control the activity of genes , but their exact role is unclear .", "ASCIZ contains an exceptionally long , disordered region that has multiple positions for binding LC8 along its chain .", "Previous research has shown that ASCIZ binds to the LC8 gene and increases transcription to produce more LC8 proteins .", "Once the protein levels are high enough , LC8 is thought to bind to the disordered region of ASCIZ and switch off transcription .", "Human ASCIZ proteins have 11 binding sites for LC8 molecules , while fruit flies have seven .", "Until now it was not clear why so many different binding sites exist .", "To address this question , Clark et al . combined biophysical , structural and molecular biology techniques to analyze proteins from humans and fruit flies and to test their role in human cells .", "This revealed that LC8 and ASCIZ form a dynamic mixture of complexes , instead of a single fully-occupied complex .", "As the number of LC8 molecules bound to ASCIZ increased , the rate of transcription dropped .", "However , all of the binding sites were rarely fully occupied .", "Instead , three to four attached LC8 molecules seemed to be sufficient to ensure that LC8 levels remain balanced .", "When the number of LC8 molecules exceeded this value , the attachment rate for additional LC8 slowed down .", "So , even when there was an excess of LC8 , most of the human ASCIZ binding sites were only partially filled .", "This way , the production of LC8 proteins was slowed , rather than fully shut down .", "As a result , the cells were able to fine-tune the transcription rate of LC8 and maintain a stable and balanced pool of these proteins .", "This work suggests that disordered regions on transcription factors could help to keep cellular systems steady in the face of changing conditions .", "In the future , the combination of methods used here could reveal new information about other proteins with disordered regions ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "neuroscience" ]

[ [ "Social behaviors are known to be affected by persistent internal states ( Anderson , 2016; Berridge , 2004; Lorenz and Leyhausen , 1973 ) .", "These states correspond with levels of arousal or drive , and can impact whether and how individuals interact , with consequences for mating decisions and reproduction ( Chen and Hong , 2018; Kennedy et al . , 2014; Stowers and Liberles , 2016 ) .", "The neural mechanisms underlying arousal states remain largely unknown .", "In male flies , a small population of male-specific neurons ( P1 ) that express the sex-specific transcription factors Fruitless and Doublesex ( Auer and Benton , 2016 ) , drive both male-aggression and male-mating behaviors ( Hoopfer et al . , 2015; Koganezawa et al . , 2016; von Philipsborn et al . , 2011 ) .", "Brief optogenetic activation of P1 neurons drives both persistent song production in solitary males and persistent aggression upon introduction of another male , both over minutes ( Bath et al . , 2014; Hoopfer et al . , 2015; Inagaki et al . , 2014 ) .", "While P1 activation is sufficient for eliciting the persistent behavioral phenotypes , other groups of neurons , such as pCd ( also called pC3 [Rideout et al . , 2010] ) , are involved in maintaining the persistent state ( Jung et al . , 2020; Zhang et al . , 2019 ) - these neurons are not synaptically coupled to P1 , and the circuit architecture that mediates persistence remains unresolved .", "P1 neurons also receive dopaminergic input that affects mating drive over longer timescales of hours ( Zhang et al . , 2016 ) , suggesting neuromodulation is also important for persistent changes in behavior driven by P1 .", "Work on P1 in flies bears some similarity to work in mice .", "In male mice , optogenetic activation of SF1 ( steroidogenic factor 1 ) expressing neurons in the dorsomedial part of the ventromedial hypothalamus ( VMHdmSF1 ) drive multiple defensive behaviors that outlast the stimulation by up to 1 min ( Kunwar et al . , 2015; Wang et al . , 2015 ) .", "In addition , activity of VMHdmSF1 neurons can persist for over a minute following the presentation of a fear stimulus .", "Computational modeling work suggests a mix of recurrent circuitry and neuromodulation underlie the persistence ( Kennedy et al . , 2020 ) , but this has not yet been tested .", "We know little about the persistence of social behaviors in females across taxa .", "P1 neurons are a subset of the larger Doublesex+ pC1 neural subset ( Kimura et al . , 2008 ) .", "While Drosophila females lack P1 neurons , they have Doublesex+ pC1 neurons ( Rideout et al . , 2010; Robinett et al . , 2010; Zhou et al . , 2014 ) , including a subset that are female-specific ( Wu et al . , 2019 ) .", "Activation of pC1 neurons affects receptivity toward males ( Wang et al . , 2020b; Zhou et al . , 2014 ) , drives chasing of males ( Rezával et al . , 2016; Wu et al . , 2019 ) , and aggressive behaviors toward females ( Palavicino-Maggio et al . , 2019; Schretter et al . , 2020 ) .", "These data suggest that female pC1 neurons can drive an arousal state , similar to male P1 neurons , but whether female pC1 neurons can drive persistent changes in behavior and persistent neural activity , has not yet been investigated .", "Importantly , a complete electron microscopic volume is currently available for the entire adult female brain ( Zheng et al . , 2018 ) , making it possible to link brain activity to complete wiring diagrams in females .", "Direct measurements of synaptic connectivity can determine whether recurrent neural networks , known to be important for persistent neural activity lasting for seconds ( Aksay et al . , 2007 ) , also underlie minutes-long persistent activity and changes in behavior , as has been recently proposed for male mice and flies ( Jung et al . , 2020; Kennedy et al . , 2020 ) .", "Here , we show that pC1 activation drives persistent changes in female behavior for minutes following stimulus offset , and we identify the subset of pC1 neurons ( called pC1d/e [Wang et al . , 2020a]; also referred to as pC1-Alpha [Wu et al . , 2019] ) that affects the persistent aggressive and male-like behaviors .", "A companion study Schretter et al . , 2020 demonstrates that pC1d , but not pC1e , drives female aggressive behaviors .", "By leveraging the automated segmentation of an electron microscopic volume of the female brain ( Dorkenwald et al . , 2020; Zheng et al . , 2018 ) , we map all inputs and outputs of both pC1d and pC1e and find a strong recurrent neural network with Fruitless+ aIPg neurons .", "Using pan-neuronal calcium imaging , we find that pC1d/e activation can elicit persistent activity for minutes among multiple cell types .", "The persistent activity is present in Doublesex+ and Fruitless+ neurons , including pC1 neurons themselves .", "We thus link minutes-long persistent neural activity and behavior with reciprocal connectivity in the female brain ." ], [ "To investigate the neural basis of a persistent internal state in the female brain , we focused on pC1 neurons , one of eight Doublesex-expressing cell types in the central brain ( Figure 1A; Kimura et al . , 2015 ) .", "We used an intersection between two driver lines ( Dsx-GAL4 and R71G01-LexA , hereafter referred to as pC1-Int; see Fly genotype table for list of genotypes used in this study ) , to label pC1 neurons , as done previously ( Rezával et al . , 2016; Zhou et al . , 2014 ) – prior work shows this line labels , per hemisphere , ~6 neurons , all of which project to the lateral junction ( Zhou et al . , 2014 ) .", "We tracked male and female body parts ( head and thorax ) in addition to recording all sounds ( Figure 1B , Figure 1—figure supplement 1A , Video 1; see Materials and methods for details on song segmentation and tracking of flies on a non-homogenous background ) .", "Silencing pC1-Int neurons in females affects receptivity Zhou et al . , 2014; we corroborated these results ( Figure 1C ) and additionally showed that silencing pC1-Int neurons diminished responses to male song ( Figure 1D ) .", "Persistent changes in the behavioral state of Drosophila males have been studied by examination of social behaviors following optogenetic activation of P1 neurons ( Hoopfer et al . , 2015; Jung et al . , 2020 ) .", "We activated pC1-Int in a solitary virgin female for 5 min , followed by a variable delay period , after which a virgin male was introduced to examine female behaviors in the context of courtship ( Figure 1E ) - there was no optogenetic activation following the first 5 min .", "The activity of stimulated neurons should decay during the variable delay period ( d0 ( 0 min delay ) , d3 ( 3 min delay ) , or d6 ( 6 min delay ) ) – below , we test this explicitly with neural imaging , and also examine shorter activation periods .", "This paradigm therefore allowed us to examine the effects of differing levels of persistent activity on behavior .", "Experimental flies were fed all-trans-retinal ( ATR ) , which is required for ReaChR ( red-shifted Channelrhodopsin ) function in flies ( Inagaki et al . , 2014 ) .", "Control flies shared the same genotype but were not fed ATR .", "We found that activation of pC1-Int neurons induces a persistent effect on female receptivity and responses to male song , but that this effect diminishes with a delay period between neural activation and introduction of a male .", "pC1-Int-activated females copulated significantly faster than controls in the d0 condition , with reduced copulations following a delay between optogenetic activation and introduction of a male ( Figure 1F ) .", "About 75% of the flies copulated within 5 min in the d0 and d3 conditions , compared with fewer than 50% in the control and d6 groups ( Figure 1F , inset ) .", "Another set of Dsx+ neurons called pCd1 ( Figure 1A ) was also shown to control female receptivity ( Zhou et al . , 2014 ) and , additionally , to enable P1-induced persistent activity in males ( Jung et al . , 2020 ) .", "While we confirmed that pCd1 silencing in females reduced receptivity ( Figure 1—figure supplement 1B ) , we found that 5 min pCd1 activation had no persistent effect on female receptivity ( Figure 1—figure supplement 1C ) .", "Activating pC1-Int also produced a persistent effect on responses to male song , overall with the strongest effect at the d0 delay ( Figure 1G ) - d0 females , in comparison with controls , accelerated more in response to all song elements , behaving like unreceptive females ( Coen et al . , 2014 ) .", "This effect was not due to changes in wild-type male song structure for the d0 condition , although male song bouts were shorter and less frequent in the d3 and d6 conditions ( Figure 1—figure supplement 1D ) , possibly due to the strong effect of pC1-Int activation on male-female interactions at longer delays , as shown below .", "Because at d0 , females are both hyper-receptive ( mate quickly ) but also accelerate in response to song , we suspect that different subsets of pC1 neurons may control these two effects .", "To quantify other behaviors elicited by pC1-Int activation , we used an unsupervised approach , and first decomposed male and female movements and interactions into 17 parameters ( Calhoun et al . , 2019; Figure 2A ) .", "We then used a Support-Vector Machine ( SVM ) framework ( Cortes and Vapnik , 1995; Cristianini and Shawe-Taylor , 2000 ) to find the weights that best classify single frames as belonging to sessions of control vs experimental groups ( all delay conditions , d0-d6 ) .", "We found that the weights of 8 out of 17 parameters were significantly different from zero ( Figure 2B and Figure 2—figure supplement 1A ) , with the strongest weight being fmAngle , defined as the degrees the female needs to turn in order to point toward the male centroid .", "The weight of fmAngle is negative because this parameter is smaller in the experimental flies compared with controls , indicating that pC1-Int activated females spend more time facing the male ( Figure 2—figure supplement 1B ) .", "When separated by experimental condition , the SVM classifier performed best on the d3 condition versus control ( Figure 2C ) , indicating that male-female movements and interactions are most distinct following this delay .", "Next , we clustered individual video frames based on the values of the eight parameters identified as most important by the SVM ( Figure 2—figure supplement 1A , asterisks ) , and found that the largest seven clusters accounted for over 90% of all frames ( Figure 2D , inset ) .", "The weights of the eight parameters were different for each cluster ( Figure 2—figure supplement 1C ) , representing different behaviors .", "Five of the clusters describe behaviors that are the same or reduced following pC1-Int activation ( clusters 1 and 3 , male chasing female , and 5–7 , increased male-female distance ) .", "Two clusters , however , describe behaviors that occur with higher probability following pC1-Int activation ( clusters 2 and 4; Figure 2D ) .", "Cluster 2 is characterized by small fmAngle and small mfAngle ( indicating that the male and female are facing each other ) , decreased male-female distance , and large fmFV ( indicating that the female is close to the male and moving in his direction ) .", "Cluster 4 is characterized by small fmAngle and large mfAngle ( indicating that the female is behind the male ) , decreased male-female distance , and large fmFV ( indicating that the female is moving in the direction of the male ) .", "Based on the weight values , and verified by inspection of the videos following clustering ( Video 2 ) , we termed cluster 2 ‘female shoving’ and cluster 4 ‘female chasing’ .", "For both female shoving and female chasing , the amount of each behavior was highest in the d3 condition relative to control ( Figure 2D ) .", "We then used JAABA ( Kabra et al . , 2013 ) to train a classifier to recognize epochs ( groups of video frames ) of female chasing and female shoving in the data ( Figure 2—figure supplement 1D; see Materials and methods ) .", "This analysis confirmed that the amount of female shoving and chasing was greatest in the d3 condition relative to control ( Figure 2E ) , in addition to revealing that the duration of female chasing and shoving bouts was longer ( Figure 2—figure supplement 1E ) .", "In many cases females transitioned directly between shoving and chasing , and the conditioned probability ( transition probability , given that a transition occurred ) for shoving →chasing was more than double at the d3 condition compared to the d0 and d6 conditions .", "By examining shoving and chasing probabilities over time ( Figure 2F ) , following the introduction of a male ( t = 0 ) , we found that both behaviors persisted for as long as 30 min in the d3 condition ( Figure 2F ) , but not in the d0 and d6 conditions , suggesting a complex interaction between neural activity in the female brain at the time of introduction of the male and feedback or social cues .", "While the percent of time females spent shoving or chasing in the first two minutes after the male was introduced was similar in the d0 and d3 conditions ( 19 . 7/21 . 3% for shoving , 2 . 8/3 . 1% for chasing ) , shoving and chasing probabilities rose over time in the d3 , but not in the d0 condition .", "In the d6 condition , shoving probability was comparable to the probability in the d0-d3 conditions in the first two minutes ( 15% ) , but decayed to control level after 6 min .", "The transition into ‘chasing’ from ‘shoving’ or from ‘other’ ( no shoving and no chasing ) epochs also peaked at d3 ( Figure 2—figure supplement 2 ) .", "In all cases , we only examined female behaviors prior to copulation .", "We also activated another Dsx+ cell type , pCd1 , which is part of a circuit that drives persistent behavior in the male brain ( Jung et al . , 2020 ) , but observed neither shoving nor chasing following 5 min of pCd1 activation in females ( Figure 2—figure supplement 2A ) .", "This suggests that the persistent behavioral effects we observe are specific to pC1 activation .", "Manual inspection of the videos identified several additional behaviors produced by females following pC1-Int activation ( Figure 2—figure supplement 3 ) .", "These include ‘female approaching’ , ‘circling’ , ‘head-butting’ , and ‘female wing extension’ ( Figure 2—figure supplement 3A–D; Videos 2 and 3 ) .", "We found that some of these behaviors were coupled; for example , ‘circling’ was often preceded by ‘female shoving’ ( Figure 2—figure supplement 3B , inset and Video 3 ) and ‘female wing extension’ was often coincident with ‘female chasing’ ( Figure 2—figure supplement 3E and Video 2 ) , similar to male behavior during courtship ( although we did not observe sounds from the females that resembled male courtship song ( Figure 2—figure supplement 3D ) ) .", "Our automated behavioral classifier did not find these behaviors because we only tracked the head and thorax of each fly , which did not provide enough information to automatically identify these behaviors , or to keep accurate track of identities during behaviors in which the male and female often overlap ( e . g . , during ‘circling’ ) .", "In sum , we found that for minutes following pC1-Int activation , females produced a variety of behaviors directed at the male .", "Some of these appear aggressive , such as shoving and head-butting ( Nilsen et al . , 2004; Palavicino-Maggio et al . , 2019; Schretter et al . , 2020 ) , while others resemble male courtship behaviors , such as chasing and unilateral wing extension ( albeit without song ) .", "These behaviors typically peaked in the d3 condition , where they remained high after male introduction .", "In contrast , the effect on female receptivity and female responses to male song , were both strongest in the d0 condition , ruling out the possibility that the effect on female receptivity is an indirect consequence of modified male courtship behavior in response to changes in female behaviors .", "Below we identify the pC1 cell types that drive persistent shoving and chasing , determine that they are part of a recurrent neural network using a new EM connectomic resource , and demonstrate that they can drive persistent neural activity on timescales similar to behavior .", "We propose three possible circuit configurations to explain our behavioral results ( Figure 3A ) .", "In the first configuration , the same set of pC1 neurons activate different downstream circuits , each one controlling a different behavior .", "The differences in the temporal dynamics arise downstream of pC1 .", "In the second configuration , three non-overlapping subsets of pC1 neurons control the different behaviors .", "In the third configuration , one pC1 subset controls female receptivity ( that peaks at d0 ) , and another set controls chasing and shoving ( both peaking at d3 ) .", "The second and third models assume some functional heterogeneity in the pC1 population .", "To evaluate these circuit models , we examined the behavioral consequences of activating distinct subsets of pC1 neurons .", "To define pC1 cell types , we used automated reconstruction of neurons in an EM volume of a female brain ( FAFB; Zheng et al . , 2018 ) ; neuron segmentation and reconstruction were accomplished using a novel platform for visualization and proofreading called FlyWire ( Dorkenwald et al . , 2020 ) .", "We examined the morphologies of neurons that send projections to the lateral junction through a thin neuronal bundle , as pC1 neurons are known to ( Figure 1A , red arrow; Figure 3B , red arrow; see also Deutsch et al . , 2019 and Zhou et al . , 2014 ) .", "We systematically checked all cell segments that pass through a cross-section in the pC1 bundle ( Figure 3—figure supplement 1A ) and excluded neurons that do not project to the lateral junction ( Figure 3—figure supplement 1B–D ) , as all pC1 cells characterized so far project to the lateral junction ( Kimura et al . , 2015; Rezával et al . , 2016; Wu et al . , 2019; Zhou et al . , 2014 ) .", "We found five pC1 cells per hemisphere ( Supplementary file 1 ) , consistent with the cell types found from manual tracing in the same EM volume ( Wang et al . , 2020a ) , although with differences in some projections ( Figure 3C; one cell/hemisphere for pC1a-e was also found in the hemibrain , a second EM dataset of the adult female brain [Scheffer et al . , 2020] ) .", "We used genetic intersections to label two non-overlapping pC1 subpopulations .", "pC1-A labels a single pC1d and a single pC1e neuron in each hemisphere ( Wu et al . , 2019; same as line pC1dSS3 [Schretter et al . , 2020] ) and no cells in the VNC ( Figure 3D and Figure 3—figure supplement 2; 2 ± 0 , n = 7 ) .", "The second intersection ( pC1-S , a split-Gal4 intersection between Dsx . DBD Pavlou et al . , 2016 and R71G01 . AD; Figure 3D and Fly genotype table ) does not label pC1d or pC1e cells , as evidenced by absence of the medial projections of pC1d/e neurons ( Figure 3C–D ) .", "The pC1-S line labels 7 . 7 ± 4 pC1 cells per hemisphere in the brain , and no cells in the VNC ( Figure 3—figure supplement 2C ) .", "All cells in the pC1-S line project to the lateral junction ( Figure 3D ) .", "Next , we tested if activation of these two non-overlapping pC1 sub-populations drives persistent behavioral phenotypes .", "Activation of either line pC1-A or pC1-S did not affect female receptivity ( Figure 4A , D ) , but activation of pC1-A drove persistent shoving and chasing ( Figure 4B–C ) , while activation of pC1-S did not ( Figure 4E ) .", "These results are consistent with model 3 ( Figure 3A ) , in which female receptivity and female shoving/chasing are driven by different populations , and also consistent with prior work , showing that 10 min of thermogenetic pC1-A activation drives persistent chasing in females ( Wu et al . , 2019 ) .", "pC1d/e neurons are female-specific ( Wu et al . , 2019 ) , and prior work reveals that pC1 neurons in females , but not males , respond to courtship song ( Deutsch et al . , 2019 ) .", "Using in vivo whole-cell patch clamp recordings of neurons labeled in the pC1-A line , we found that pC1d/e neurons in virgin females depolarize in response to features in conspecific courtship song ( Figure 4—figure supplement 1 ) .", "This finding indicates that pC1d/e neurons can be activated during courtship by male song .", "When examining the fraction of time spent shoving or chasing across the three conditions ( control , d0 , and d3 ) to compare results of activating neurons in pC1-A and pC1-Int lines ( Figure 4F ) , we found that levels of female chasing are increased with pC1-A activation ( relative to pC1-Int ) at d3 .", "The levels of shoving are slightly decreased at d0 , but this effect was not significant .", "This suggests that pC1 cell types in the pC1-Int driver , other than pC1d/e ( and other than the cells in the pC1-S driver ) , have a modulatory effect on the shoving and chasing behaviors , at distinct timepoints .", "pC1-Int , but not pC1-A , activation drives persistent female receptivity ( Figure 1F vs Figure 4A ) , indicating that pC1-Int cells other than pC1d/e drive female receptivity ( although high copulation rates in control flies could mask a potential effect of pC1-A on female receptivity ) .", "We hypothesized that the same pC1-Int cells that drive female receptivity , also affect the probability of shoving and chasing .", "If true , we would expect a different rate of shoving and chasing in receptive and non-receptive females .", "Indeed , we found that following pC1-Int activation , females that eventually copulated ( receptive ) showed a slightly higher level of shoving compared with females that did not eventually copulate ( unreceptive ) , although this effect was not statistically significant ( Figure 4G ) .", "In contrast , receptive females showed a strong and significant reduction in chasing ( Figure 4H ) .", "Taken together , our results suggest that cells within the pC1-Int line that control receptivity modulate the amount of female chasing ( and possibly also shoving ) ( Figure 4I ) .", "We also tested shorter activation durations and found robust persistent shoving and chasing following 2 min activation , but reduced persistent shoving and chasing following 30 s activation ( Figure 4J ) .", "Below we address how minutes-long pC1d/e activation affects neural activity .", "The pC1-A line ( same as pC1dSS3 [Schretter et al . , 2020] ) includes two cells per hemisphere , one pC1d and one pC1e cell .", "Schretter et al . , 2020 have demonstarted that optogenetic activation of genetic lines that contain pC1d , but not pC1e , drive female aggressive behaviors , such as those we refer to here as ‘shoving’ .", "We therefore first focused on pC1d , and mapped the major inputs and outputs , searching for circuit motifs that could account for pC1d’s ability to drive a persistent behavioral state in females .", "We used automated reconstruction of all neurons within an EM volume of an entire adult female brain called FAFB ( Zheng et al . , 2018; Dorkenwald et al . , 2020 ) .", "This volume contains both brain hemispheres , enabling complete reconstruction of pC1d cells that send projections across the midline ( Figure 3C ) .", "Focusing on a single pC1d cell ( Figure 5A ) we first manually detected synaptic connections ( Figure 5—figure supplement 1A; see Materials and methods ) , as done previously for other circuits in FAFB ( Felsenberg et al . , 2018; Sayin et al . , 2019; Zheng et al . , 2018 ) - while our manual detection process was unbiased , in that we looked for synapses between pC1d and any other overlapping segment in FlyWire ( see Materials and methods ) , we only sampled a subset of all pC1d synapses ( see below for analysis based on automated synapse detection ) .", "After proofreading the pC1d cell and its input and output cells , and excluding neurons with weak connections ( using three synapses as a threshold , see Materials and methods ) , we counted a total of 417 presynaptic and 421 postsynaptic sites ( Figure 5B , Video 4 ) .", "We sorted all pC1d synaptic partners by cell type , based on morphology , and examined the distribution of synapses by type for input ( presynaptic partners ) and output ( postsynaptic partners ) neurons separately ( Figure 5C ) .", "The three output types with the largest number of synapses with pC1d share a common morphology: all pass through a single neurite bundle ( Figure 5D–E , Figure 5—figure supplement 1B–C and Video 5 ) and all send projections to the ring ( Figure 5A ) , including dense projections in the lateral junction ( Figure 5E ) .", "The top matches ( using NBLAST [Costa et al . , 2016] ) for all three types were sexually dimorphic Fru+ neurons called aIP-g ( Figure 5E and Figure 5—figure supplement 1B , see Cachero et al . , 2010 ) .", "FlyWire cells that share the aIP-g morphology were sorted into three types , aIPg-a , aIPg-b , and aIPg-c , based on the three separate bundles through which their projections pass ( Figure 5E ) .", "According to our manual synapse detection , aIPg-a cells have 131 sites postsynaptic to pC1d and only five presynaptic sites , while aIPg-b , c cells have stronger reciprocal connections with pC1d , with an output:input ratio of ~1:1 ( 38:39 ) for aIPg-b , and 2 . 8:1 ( 39:14 ) for aIPg-c .", "FlyWire provides a mapping of the publicly available , automatically detected synapses from Buhmann et al . , 2019; see Materials and methods for details .", "We re-evaluated synaptic partners using automatic detection , and focused on cells with strong connections with pC1d using two criteria: ( 1 ) minimum of six synapses , ( 2 ) the cell belongs to a cell type ( based on morphology ) with at least one cell with 15 synapses or more with pC1d .", "Consistent with manual detection , we found that aIPg-a cells are postsynaptic to pC1d , while aIPg-b , c are reciprocally connected to pC1d ( Figure 5F ) .", "Some cells from the aIPg-b , c groups are also interconnected ( Figure 5G ) .", "Interestingly , the most interconnected cells within the aIPg-b group were also the ones that are reciprocally connected to pC1d ( Figure 5G , red lines ) .", "We also examined synaptic connectivity between pC1d and aIPg cells in a second EM database that consists of a portion of the adult female brain ( the ‘hemibrain’ [Scheffer et al . , 2020] ) , and found a set of 13 neurons identified as aIPg ( also evaluated in [Schretter et al . , 2020] ) , compared with 39 neurons we identified as aIPg in FlyWire .", "Twelve of these aIPg cells ( denoted as types aIPg1-3 in the hemibrain ) share the aIPg-b morphology ( Figure 5—figure supplement 1D ) , and are synaptically connected to pC1d ( excluding one connection with less than three synapses ) .", "One ( aIPg4 ) shares the aIPg-c morphology .", "Consistent with our results in FlyWire , pC1d in the hemibrain has more presynaptic sites than postsynaptic sites with aIPg-b ( hemibrain aIPg1-3 ) cells , and aIPg-b neurons form many recurrent connections with each other ( Figure 5H ) .", "Note that while our classification of aIPg cell types was based on morphology alone , the classification in the hemibrain is based on both morphology and connectivity .", "In the hemibrain v1 . 1 , we found additional cells that match ( morphologically ) neurons we term aIPg-a and aIPg-c – these neurons are called SMP555/556 and SMP558 , respectively .", "Finally , in FlyWire , we found that pC1d forms connections with other Dsx+ cells , including direct connections with pMN1 ( DNp13 ) and pMN2 ( vpoDN ) ( Figure 5I ) , other pC1 cells ( Figure 5J ) , and pC2-like cells with similar morphology to Fru+ pIP5 cells ( Figure 5K , Video 6 and Supplementary file 1 ) .", "These results indicate that pC1d may serve as a hub within the central brain for Dsx+ and Fru+ neurons ( Figure 5L ) .", "Using automated synapse detection in FlyWire , we examined all the major inputs and outputs of both pC1d ( Figure 6A ) and pC1e ( Figure 6—figure supplement 1 ) , as our driver line pC1-A labeled both neurons in each hemisphere ( see full list of inputs and outputs and public links to FlyWire neurons in Supplementary file 1 ) .", "aIPg-b neurons have the most reciprocal connections with pC1d , and the three strongest output types of pC1d are aIPg-a ( 588 synapses ) , aIPg-c ( 300 synapses ) , and aIPg-b ( 232 synapses ) .", "The pC1d output:input ratio with aIPg ( pC1d-to-aIPg:aIPg-to-pC1d ) was 588:0 for aIPg-a , 2 . 6:1 for aIPg-b and 14 . 3:1 for aIPg-c ( Figure 5F ) – consistent with the results obtained by manual detection , showing that aIPg-b has the most ‘balanced’ reciprocal connectivity with pC1d .", "For pC1e ( Figure 6—figure supplement 1 ) , of the top inputs and outputs , the only reciprocal connections were with aIPg-b .", "Shared inputs between pC1d and pC1e also include pC1a , a cell previously shown to control female receptivity ( Wang et al . , 2020b ) .", "Shared outputs include aIPg-a , c , and cell types that share the hemibrain morphology of SIP024 and LAL003 .", "Some of the top connections of pC1d were cross-hemispheric neurons or neurons with synapses contralateral to pC1d ( Figure 6B ) – these neurons and connections fall outside of the volume of the hemibrain .", "However , several of the top inputs and outputs could be identified in the hemibrain , and we have indicated the corresponding cell type name where we could find matches ( Figure 6 and Figure 6—figure supplement 1 ) .", "Seven of the aIPg cells that are pC1d synaptic partners , also synapse with pC1e ( Figure 6—figure supplement 1 ) .", "As recurrent connectivity between neurons is known to support persistent neural activity ( Goldman-Rakic , 1995; Major and Tank , 2004; Zylberberg and Strowbridge , 2017 ) , we next examined whether activating pC1d/e could drive long-lasting changes in brain activity , with a spatial distribution that matches the pC1d or pC1e connectomes .", "Persistent neural activity is defined as activity that continues after a triggering stimulus ends ( Zylberberg and Strowbridge , 2017 ) .", "To relate our findings above to persistent neural activity , we first activated either neurons in the pC1-A or pC1-S lines using 5 min of optogenetic stimulation ( similar to behavioral experiments , see Materials and methods ) , and imaged responding cells via GCaMP6s expressed pan-neuronally ( Figure 7A ) .", "To compare activity across flies and to map activity onto a reference atlas , we used a recently developed pipeline for two-photon volumetric calcium imaging , motion correction , registration , and region-of-interest ( ROI ) segmentation ( Pacheco et al . , 2021 ) , and scanned the entirety ( in the anterior-posterior axis ) of the dorsal half of a single brain hemisphere ( either left or right ) in each fly ( Figure 7A ) – we mirrored all activity onto one hemisphere for display .", "We measured neuronal activity during the 5 min of optogenetic activation in addition to 9 . 5 min following activation offset ( Video 7 ) , and found that out of 47 , 882 ROIs with activity segmented across 28 brains and all genotypes ( Figure 7B; see Materials and methods ) , 4254 ROIs showed significant responses to optogenetic stimulation ( Figure 7C; Ft1 > 3σ0 , σ0 = standard deviation of activity during baseline ) .", "We then clustered these ROIs based on response patterns ( Figure 7D ) , which revealed four types of responses .", "Transient responses - ROIs with elevated activity during the optogenetic stimulus ( t1 ) , but not following the stimulus ( t2 ) - could be grouped into two clusters ( response types 3 and 4 ) .", "The other two types showed sustained activity lasting at least 5 min after the optogenetic stimulus offset ( response types 1 and 2; Ft2 > 3σ0 , see Materials and methods ) .", "The temporal dynamics of persistent neural activity , continuing to at least 10 min following stimulation , is consistent with our observation of female shoving and chasing of a male introduced 6 min after stimulation offset ( Figure 2E–F ) .", "In addition , response type 2 could be split into two clusters ( Figure 6D , right ) based on response temporal dynamics .", "While response type 1 had low spatial consistency across animals , response types 2–4 showed higher spatial consistency , and the spatial distribution of ROIs differed between controls , pC1-S , and pC1-A activated flies ( Figure 7E ) .", "Activation of pC1d/e neurons ( in line pC1-A ) drove persistent activity ( response type", "2 ) in more than 30% of the imaged flies , and in 24 . 7 times more voxels than in controls , and 6 . 8 times more voxels compared with pC1-S activation .", "Making use of neuropil segmentation of an in vivo brain atlas to which all ROIs were registered ( Pacheco et al . , 2021 ) , we evaluated the distribution of pC1d/e-elicited activity by brain neuropil ( Ito et al . , 2014 ) .", "Persistent activity ( response type", "2 ) was clustered in the posterior-dorsal portion of the brain spanning the Superior Medial , Lateral and Intermediate Protocerebrum ( SMP , SLP , SIP ) , the Anterior Optic Tubercle ( AOTU ) , and the Inferior and Superior Clamp ( ICL and SCL; Figure 7F and Figure 7—figure supplement 1A ) ; these brain regions contain a large number of projections from sexually dimorphic neurons expressing either Doublesex or Fruitless ( Rideout et al . , 2010; Yu et al . , 2010 ) .", "We found that 61% ( 4717/7759 ) of all the presynaptic terminals and 85% ( 3283/3848 ) of all the postsynaptic terminals for the group of 11 aIPg1-3 ( all share the aIPg-b morphology ) cells in hemibrain v1 . 1 are in these six areas , with the SMP , SIP , and AOTU being the most dominant input and output regions for the aIPg1-3 cells .", "Our behavioral results indicated that female brain state must differ between the d0 , d3 , and d6 conditions ( Figure 2F and Figure 4B–C ) – we therefore quantified neural activity at these specific time points ( 0 , 3 min , and 6 min ) following optogenetic activation .", "In the neuropils with highest activity , we found that responses were highest immediately following stimulation ( t = 0 ) and decayed significantly by t = 3 min , and further still by t = 6 min ( Figure 7G; Figure 7—figure supplement 1B ) .", "In order to measure the overlap of pC1-elicited activity with Dsx+ neurons , we generated anatomical labels for the lateral protocerebral complex ( LPC ) , a diffuse brain area to which all Dsx+ neurons send their projections , and also for all major groups of Dsx+ somas ( pC1 , pC2l , pCd1 , and pCd2 ) within the in vivo brain atlas ( Figure 7F , see Materials and methods ) .", "We found that ROIs with persistent activity ( response type", "2 ) overlap with the LPC , in addition to the regions occupied by pC1 somas , and to a lesser extent with regions occupied by pC2 , pCd1 , and pCd2 somas , suggesting that Dsx+ pC1 neurons carry persistent activity .", "We also looked for overlap between ROIs with persistent activity and the projections of individual aIPg neurons , all registered into the same reference brain ( Figure 7H; see Materials and methods ) .", "While persistent activity ( response type 2; Figure 7D–E ) spans only 4 . 3% of the central brain ( Figure 7E; see Materials and methods ) , we found response type 2 activity in 20 . 14% ( union of the overlap ) of the voxels that include aIPg-a/b/c example cell ( one of each type ) from FlyWire ( Figure 7H ) .", "In addition , activity driven by 5 min of activation was not aberrant .", "DF/F values following 5 min optogenetic activation ( data from Figure 7C ) fell within the distribution of DF/F values observed in separate experiments ( Pacheco et al . , 2021 ) in which activity was driven by auditory stimuli rather than optogenetic activation ( Figure 7—figure supplement 1C ) .", "We also examined persistent activity following a shorter activation period of 2 min , and found ROIs with persistent activity ( response Type 2; Figure 7I–J ) .", "The persistent activity following 2 min activation was weaker in this condition compared to 5 min activation , suggesting that persistent activity scales with the activation period .", "We next expressed GCaMP6s in only Dsx+ neurons , to confirm the specific Dsx+ cells with persistent neural activity – this is possible because Dsx+ somas are clustered by cell type ( Figure 1A ) .", "We activated pC1d/e neurons for 5 min ( Figure 8A and Video 8 ) and recorded activity in 273 cells ( ROIs drawn manually ) across 16 flies .", "We examined the responses during ( t1 ) and after ( t2 ) optogenetic stimulation ( same as for the pan-neuronal dataset ) , and compared these responses to controls in which pC1d/e neurons were not activated ( n = 11 flies , 192 ROIs; See Fly genotype table for full genotypes; Figure 8B ) .", "A number of Dsx+ pC1 cells showed strong persistent activity ( Figure 8B; same definition as for the pan-neuronal screening , Ft2 > 3σ0 ) following optogenetic activation .", "We observed some heterogeneity in responses across the pC1 cells ( Figure 8B ) , with some cells showing faster decay than others following stimulus offset , consistent with the two clusters underlying response type 2 ( Figure 7D , green and brown ) .", "We did not observe persistent activity in any non-pC1 Dsx-expressing cell types ( Figure 8—figure supplement 1 ) , including pC2 neurons or pCd1 neurons , previously shown to be necessary for P1-induced persistent activity in males ( Jung et al . , 2020; Zhang et al . , 2019 ) .", "Last , using the same methodology , we examined neural activity in single Fru+ cells following pC1d/e activation , by expressing GCaMP6s via the Fru-LexA driver ( Figure 8C , top ) .", "We found persistent activity in two group of cells , denoted as ‘Group 1’ and ‘Group 2’ ( Figure 8C , bottom ) , that often lasted over a minute following activation ( Figure 8D–E ) – ROIs drawn manually for individual cells within each group .", "By comparing the location of Fru+ cell bodies with persistent activity with the position of single Fru+ cells and to the location of pC1/pC2/aIPg cells in FlyWire ( Figure 8—figure supplement 2 ) .", "We conclude that Group 2 includes pC1 neurons , while Group 1 likely includes pC2/pIP5 neurons and possibly also aIP-g cells .", "The persistent activity is most likely not in Dsx+/Fru+ pC2 neurons given our observations that Dsx+ pC2 cells do not show persistent activity following pC1d/e activation , but could be in pIP-e ( also called pIP5 in the hemibrain ) .", "This is consistent with our analysis in FlyWire , showing that pIP5 is both pre- and post-synaptic to pC1d .", "In sum , our pan-neuronal imaging reveals that female brain state is different at 0 and 3 min following activation , providing an explanation for the differences in behaviors produced following introduction of a male at these different delays .", "In addition , by clustering response types , we were able to map pC1d/e-driven persistent neural activity to brain regions containing both Dsx+ neurons and Fru+ aIPg neurons , and with follow-up experiments showed that several Dsx+ pC1 neurons as well as Fru+ putative pC1 and aIPg cells contain persistent neural activity .", "This is consistent with the recurrent circuit architecture we found using EM reconstruction ( Figures 5–6 ) ." ], [ "We used unsupervised methods to identify the most prominent behaviors ( beyond receptivity and responses to courtship song ) produced following activation of pC1 neurons in virgin females - these include behaviors that resemble male courtship ( female chasing the male ) and aggression ( female shoving the male ) ( Figure 2 ) .", "Both behaviors are not typically observed in mature virgin females interacting with a male; this suggests that sensory cues from the virgin male do not inhibit these aberrant behaviors , but rather may enhance the persistent effects of pC1 activation ( Figure 2F ) , most likely via visual inputs to aIPg neurons ( Schretter et al . , 2020 ) .", "pC1 neurons also drive aggressive behaviors toward females during stimulation ( Palavicino-Maggio et al . , 2019; Schretter et al . , 2020 ) , but whether the quality of aggression generated toward males versus females is similar remains to be determined .", "As one of our manually scored behaviors , ‘female approaching’ ( Figure 2—figure supplement 3A ) , begins from a distance greater than four body lengths from the male fly ( a distance at which it may be difficult to discern male from female [Borst , 2009] ) and often ends with shoving or circling ( see Video 3 ) , we hypothesize that pC1 activation most likely drives persistent behaviors toward another fly , and not specifically a male or female fly , consistent with ( Schretter et al . , 2020 ) .", "What is the role of female aggression ?", "Female aggression , whether toward males or females , has been previously reported across model systems ( Huhman et al . , 2003; Stockley and Bro-Jørgensen , 2011; Woodley et al . , 2000 ) .", "In Drosophila , female-female fights over food source are strongly stimulated by the receipt of sperm at mating ( Bath et al . , 2017 ) , and include both patterns that are common with male aggression ( such as shoving and fencing ) and female-only patterns ( Nilsen et al . , 2004 ) .", "Female-male aggression was reported in the context of rejecting behavior in mated , immature , or older females ( Cook and Connolly , 1973 ) .", "The behavioral changes in our study do not mimic those in a mated female , as we also observe that pC1 activation drives enhanced receptivity .", "Although we have not confirmed which pC1 cell types control receptivity , our work reveals a separation: pC1d/e neurons are sufficient to drive persistent shoving/chasing , but do not have a persistent effect on female receptivity ( Figure 4A–C ) , while separate pC1 neurons that control receptivity modulate the pathways that control chasing and aggression ( Figure 4G–I ) .", "Recent work reveals that pC1a neurons are modulated by the sex peptide receptor pathway , such that following mating ( when sex peptide is transferred ) , pC1a neurons are inhibited ( Wang et al . , 2020b ) .", "Because we find that pC1a provides direct input to pC1d and pC1e neurons ( Figure 6A , Table 1; see also Schretter et al . , 2020 ) , we speculate that pC1a provides this receptivity information .", "Interestingly , work in male flies suggests a separation in pC1 subsets that control courtship versus aggression ( Koganezawa et al . , 2016 ) , with reciprocal inhibitory influences between persistent courtship and aggression , following pC1 activation ( Hoopfer et al . , 2015 ) .", "Although the phenotypes are sex-specific ( male singing vs female receptivity; male tussling vs female shoving ) , and the pC1 subsets driving these behaviors are sex-specific ( P1 in males , pC1d/e in females ) , this suggests some common architecture .", "Ultimately , comparing the connectomes of male and female brains , combined with functional studies , should elucidate both similarities and differences .", "Because courtship interactions unfold over many minutes ( Coen et al . , 2014 ) , we postulate that the changes in brain state we observed following pC1d/e activation may occur naturally as females receive continual drive to pC1 neurons .", "Our connectomic analyses reveal inputs to pC1d/e neurons from AVLP cells ( Figure 6; Figure 6—figure supplement 1 ) .", "The AVLP contains multiple auditory cells ( Baker et al . , 2020 ) - this is consistent with our patch clamp recordings of pC1d/e neurons ( Figure 4—figure supplement 1 ) , showing auditory activity , and also with prior work on auditory responses in female , but not in male pC1 neurons ( Deutsch et al . , 2019 ) .", "Thus , during natural courtship interactions , male song should drive pC1d/e neurons – in combination with other inputs , this may shift behaviors from receptive ones toward chasing and shoving .", "FlyWire enabled a systematic search for all the synaptic partners of a single pC1d and pC1e cell .", "Using manual and automatic synapse detection ( Buhmann et al . , 2019 ) , we found that pC1d is reciprocally connected with aIPg-b and aIPg-c cells and that aIPg-b and aIPg-c cells are also interconnected – pC1e also shows reciprocal connectivity with aIPg-b neurons .", "We also identified pC1d and aIPg-c cells in a separate EM volume of an adult female brain , the hemibrain ( Scheffer et al . , 2020 ) , and found similar results ( see Schretter et al . , 2020 for a thorough analysis of the pC1d connectome in the hemibrain ) .", "Because FlyWire is based on the FAFB dataset of the entire adult female brain ( Zheng et al . , 2018 ) , our search for synaptic partners of pC1d and pC1e could completely cover both hemispheres , showing that , for example , pC1d is reciprocally connected to itself in the contralateral hemisphere , and is presynaptic to some contralateral aIPg cells .", "Comparisons between connectivity diagrams in the two female EM volumes of FAFB ( segmented in FlyWire ) and the hemibrain will continue to be of value for future studies .", "Synaptically recurrent neural networks have been proposed to contribute to persistent neural activity lasting for seconds , and to underlie processes like accumulation of evidence and working memory ( Aksay et al . , 2007; Barak and Tsodyks , 2007; Major and Tank , 2004; Mante et al . , 2013; Seung , 1996; Wang , 2008; Zylberberg and Strowbridge , 2017 ) .", "For example , in Drosophila , recurrent excitatory loops in the ellipsoid body of the central complex contribute to stabilization of a heading navigation signal over timescales of seconds ( Turner-Evans et al . , 2020 ) .", "Internal states underlying social behaviors , however , persist on much longer timescales of minutes to hours ( Chen and Hong , 2018 ) .", "Neuromodulation , via hormones or peptides , is thought to support longer timescales of persistence ( Adolphs and Anderson , 2018; Bargmann , 2012; Marder , 2012; Zelikowsky et al . , 2018 ) .", "Our work now links a strongly recurrent neural network ( pC1d/e to aIPg ) to both minutes-long persistent neural activity and minutes-long changes in behavior .", "Work from Schretter et al . , 2020 demonstrates that pC1d , pC1e , and aIPg neurons are all cholinergic , suggesting these neurons make up an excitatory neural network .", "While our study found persistent changes in behavior following pC1d/e activation , the Schretter et al . , 2020 study did not – this discrepancy could arise from a number of methodological reasons , including differences in the optogenetic activator , timescale of activation , or in behavioral protocols .", "Nonetheless , Schretter et al . , 2020 demonstrate that aIPg-b activation drives persistent female aggression following activation , and that increasing stimulation of aIPg-b , increases the amount of persistent aggression .", "This is consistent with a model in which strong activation of pC1d/e neurons recruits aIPg-b neurons ( via their excitatory interconnections ) to drive , at least , persistent shoving .", "Work in male flies has proposed a recurrent circuit motif including pCd neurons that contributes to persistent aggressive behaviors ( Jung et al . , 2020 ) or even longer term changes in mating drive ( driven by male-specific P1 neurons , but without mapping the synaptic connections ) .", "Our studies of pCd , revealed that , in females , it does not contribute to the persistent arousal state ( Figure 2—figure supplement 2 ) , nor is it persistently active following pC1d/e activation ( Figure 8—figure supplement 1 ) .", "In males , activating P1 neurons for 5 s was enough to induce some persistent activity in pCd cells ( Jung et al . , 2020 ) .", "We did not measure neural responses to pC1d/e stimulation shorter than 2 min , and so whether shorter activation periods drive some persistent neural activity in females remains open .", "While pC1d/e neurons are female-specific , aIPg neurons are present in both males and females – it will be interesting to determine what role the aIPg neurons play in male brains and whether they represent a shared component of persistence in the two sexes .", "Finally , it is important to point out that recent EM connectomic work in Drosophila using FAFB ( via manual tracing ) has revealed a number of recurrent or reciprocal circuits throughout the brain ( Dolan et al . , 2018 , Sayin et al . , 2019; Turner-Evans et al . , 2020 ) .", "Half of the outputs of pC1d are the aIPg neurons , and many of these neurons ( several aIPg-b and aIPg-c cells ) are strong inputs .", "Our identification of a recurrent circuit motif containing pC1d links it to the persistent neural activity and changes in behavior we observe following activation , especially in Fru+ somas .", "This is an important first step toward determining how recurrent neural networks contribute to such long timescales of persistence ." ], [ "Figure panelGenotypeAdditional information1AUAS-2xEGFP; Dsx-Gal4Immunostaining1C-Dw;R71G01-LexA/8xLexAop2-FLP; Dsx-Gal4/UAS > STOP > TNTExpress TNT in pC1-Int1S1Bw;R41A01-LexA/8xLexAop2-FLP; Dsx-Gal4/UAS > STOP > TNTExpress TNT in pCd1 neurons1C-D , 1S1Bw;+/8xLexAop2-FLP;Dsx-Gal4/UAS > STOP > TNTControl for TNT expression in pC1-Int and in pCd11 F-G , 2 , 4 F-H , 1S1D , 2S1 , 2S2B-C , 2S3w;R71G01-LexA/8xLexAop2-FLP; Dsx-Gal4/10xUAS > STOP > ReachRExpress ReachR in pC1-Int neurons; ATR+ experimental , ATR- control1S1C , 2S2Aw;R41A01-LexA/8xLexAop2-FLP; Dsx-Gal4/10xUAS > STOP > ReachRExpress ReachR in pCd1 neurons; ATR+ experimental , ATR- control4D-Ew;R71G01 . AD/+;DSX . DBD/UAS-ReachRExpress ReachR in ‘pC1-s’ neurons ( behavioral experiment ) ; ATR+ for experimental , ATR- for control4A-C , F , Jw;VT25602 . AD/+;VT2064 . DBD/10xUAS-ReachRExpress ReachR in ‘pC1-A’ neurons ( behavioral experiment ) ; ATR+ for experimental , ATR- for controls .", "pC1-A is similar to pC1dSS3 in Schretter et al . , 2020 . 4S1w;UAS-2xEGFP/+;VT25602-Gal4/+Express GFP in pC1d/e neurons for patch clamp recordings3D ( top ) , 3S2Cw;R71G01 . AD/UAS-10x-IVS-myr::GFP;Dsx . DBD/+Immunostaining ( pC1-S ) 3D ( bottom ) , 3S2Bw;VT25602 . AD/UAS-10x-IVS-myr::GFP;VT2064 . DBD/+Immunostaining ( pC1-A ) 3S2AR71G01-LexA/8xLexAop2-FLP; Dsx-Gal4/UAS > STOP > myrGFPImmunostaining ( pC1-Int ) 7B-E , 7S1Aw+NorpA[36] , 20xUAS- csChrimson . mVenus; R71G01 . AD/R57C10-LexA; Dsx . DBD/8xLexAop-mCD8tdTomato , 13xLexAop-GCaMP6spC1-S activation , measure pan-neuronal Ca response; ATR+7B-J , 7S1A-Bw+NorpA[36] , 20xUAS-csChrimson . mVenus; VT25602 . AD/R57C10-LexA; VT2064 . DBD/8xLexAop-mCD8tdTomato , 13xLexAop-GCaMP6spC1-A activation , measure pan-neuronal Ca response; ATR+7B-E , I-J , 7S1Aw+NorpA[36] , 20xUAS-csChrimson . mVenus; R57C10-LexA/CyO;8xLexAop-mCD8tdTomato , 13xLexAop-GCaMP6s/TM6B , tbControl; ATR+8A-B , 8S110xUAS-Chrimson . tdTomato , 13LexAop2-GCaMP6S/W+NorpA[36] , 20xUAS-csChrimson . mVenus; VT25602 . AD/CyO;Dsx-LexA/TV2064 . DBDpC1-A activation , measure Ca response in Dsx+ neurons; ATR+8A-B , 8S110xUAS-Chrimson . tdTomato , 13LexAop2-GCaMP6s/+; Sp/CyO;Dsx-LexA/TM6B , tbControl; ATR+8C-E , 8S110xUAS-Chrimson . tdTomato , 13LexAop-GCaMP6s/+; VT25602 . AD/CyO; Fru-LexA/VT2064 . DBDpC1-A activation , measure Ca response in fru+ neurons; ATR+8C-E10xUAS-Chrimson . tdTomato , 13LexAop-GCaMP6s; Sp/CyO;Fru-LexA/Tm6TbControl; ATR+ All flies were raised at 25°C on standard medium in a 12 hr light/12 hr dark cycle at 60% relative humidity .", "Female flies used for optogenetic experiments were fed with food that contained all-trans-retinal ( Sigma R2500-100MG; ATR concentration is 1 mM ) for a minimum of 3 days post-eclosion .", "Control flies were raised on regular fly food after eclosion .", "Both experimental and control female flies used for optogenetic experiments , were reared post-eclosion in dark blue acrylic boxes ( acrylic available from McMaster-Carr , #8505K92 ) .", "The full details on genotypes for the flies used in this study and their source are in Key resources table and in Fly genotype table .", "All behavioral experiments were carried out using a behavioral chamber ( diameter ~25 mm ) tiled with 16 microphones ( NR23158 , Knowles Electronics; Figure 1B and Videos 1–3 ) , and connected to a custom amplifier ( Arthur et al . , 2013 ) .", "Audio signals were recorded at 10 KHz , and the fly song was segmented as previously described ( Arthur et al . , 2013; Coen et al . , 2014 ) .", "A point gray camera ( FL3-U3-13Y3M; 1280 × 960 ) was used to record fly behavior from a top view ( see Figure 1B , S1A ) at 60 frames per second using custom written software in Python and saved as compressed videos ( H . 264 ) .", "Virgin females ( see Fly genotype table for genotype used in each experiment ) and wild-type NM91 virgin males ( both males and females were 3–7 days old ) were used for all behavioral experiments .", "For inactivation experiments ( Figure 1C–D , Figure 1—figure supplement 1B ) , a male and a female were introduced into the behavioral chamber simultaneously .", "For activation experiments , a female was positioned in the behavioral chamber , and red light ( a ring of 6 LEDs , 627 nm , LuxeonStar ) was then delivered at 1 . 1 mw/mm2 ( ±5% across the chamber ) at 100 Hz ( 50% duty cycle ) for 5 min ( Figure 1E ) or for 2 min ( Figure 4J ) .", "Following stimulus offset , a male was introduced with either no delay ( d0 ) , or after a 3 or 6 min delay ( d3 , d6 ) .", "We collected data from the time the male was introduced ( t = 0 ) until 30 min or when the flies copulated , whichever came first .", "We chose to use ReachR ( Inagaki et al . , 2014 ) for activation experiments rather than using the more sensitive red shifted channelrhodopsin csChrimson ( Klapoetke et al . , 2014 ) to minimize optogenetic activation due to background light .", "The percent of flies that copulated as a function of time ( Figure 1C , F , Figure 1—figure supplement 1B–C ) was calculated from the time the male was introduced ( t = 0 ) and until t = 30 min .", "Each video frame was analyzed by first finding the fly centroid , then detecting the location of the thorax and head which formed the heading vector for each fly .", "Having microphones at the chamber bottom results in a highly inhomogeneous background ( Figure 1—figure supplement 1A ) posing a major challenge for accurate tracking .", "The centroid was found in one of two ways that yielded similar results .", "In the first method , the inhomogeneous background of the video was found by taking the median across all frames .", "Because the animals move throughout the video , finding the median pixel usually does not contain any pixels containing an animal’s body .", "However , as animals occasionally sit for long periods , they can become part of the background .", "To avoid this , we divided the video into 10 shorter videos of equal length and found the median 'frame' ( median set of pixel values ) for each sub-video .", "We then created a median frame by computing the median across these medians .", "Each video frame then had this background subtracted to identify pixels that were potentially part of each fly .", "These pixels were smoothed by a series of operations using the OpenCV Python package and then thresholded .", "Using OpenCV , we identified all contours surrounding collections of pixels and any smaller or larger than some predefined threshold ( less than half the size of a typical ‘fly’ or more than twice its size ) were discarded .", "The remaining pixels were then clustered via k-means .", "The number of clusters were iteratively increased until the compactness of each cluster reached some threshold .", "The least-compact clusters were discarded , and the remaining pixels were clustered again with k-means with k = 2 to identify the two clusters corresponding to the animals .", "These clusters were then fit with an ellipse to identify the centroid of each animal .", "In the second method , we trained a deep convolutional network to detect all instances of individual body parts ( head , thorax ) within each frame using a modified version of LEAP ( Pereira et al . , 2019 , or SLEAP [Pereira et al . , 2020] https://sleap . ai/; 544 labeled frames were used for training; Figure 1—figure supplement 1Ai-ii and Video 1 ) .", "Using the same software and neural network architecture , a separate network was then trained to group these detections together with the correct animals by inferring part affinity fields ( Figure 1—figure supplement 1Aiii; Cao et al . , 2017 ) .", "This enabled estimation of the vector that represents fly heading for both flies ( Figure 1—figure supplement 1Aiv ) .", "Seventeen parameters were extracted for each frame based on the tracking of male and female centroid and heading ( Figure 2A ) , describing either the female movements ( fFV – female forward velocity; fLS – female lateral speed; fFA/fLA – female forward/lateral acceleration; fRS – female rotational speed ) , male movements ( similar to female movements – mFV/mLS/mFA/mLA/mRS ) , or male-female interaction ( mfDist – male-female distance; fmAngle – female heading relative to the female-male axis , mfAngle – male heading relative to male-female axis; fmFV or fmLS – female speed in the male direction or in the perpendicular axis; mfFV or mfLS – male speed in the female direction or perpendicular ) .", "Using 17 parameters for each frame , we trained binary support vector machine ( SVM ) linear classifiers to find the parameters ( dimensions ) that best separate the groups .", "We first trained classifiers that separate between frames that belong to experimental flies ( class 1 , pC1-Int activated , either one condition - d0/d3/d6 or all groups together , d0-d6 ) , and controls ( class 2 ) .", "We trained 90 classifiers , randomly choosing a set of 3000 frames from each class ( ‘training set’; non-overlapping - the same frame was never used in two classifiers; increasing the number of frames beyond 3000 did not increase performance ) .", "We used the MATLAB R2019b procedure fitcsvm ( MathWorks , Natick , MA ) , with a linear kernel .", "We then used a separate set of 30 , 000 frames per class for each classifier ( ‘validation set; the same frame was never used twice , either between classifiers or between sets ) to test the performance of each classifier ( fraction of frames correctly classified ) .", "We then choose the 30 best-performing classifiers ( Figure 2B for control vs d0-d6 ) .", "We used a third set of frames for each classifier ( 30 , 000 frames/class , again – with no overlap with other sets ) to measure the performance of each classifier .", "The MATLAB function predict was used to find the SVM-predicted class for each frame in the validation or train set .", "Performance was calculated as the percent of frames correctly classified ( Figure 2C ) .", "For each weight ( out of the 17; control vs d0-d6 ) , we looked at the distribution coming from the 30 independent classifiers , and tested whether the mean was significantly different than zero ( Figure 2—figure supplement 1A ) .", "We used a two-sample t-test to measure the probability that the mean weight associated with each parameter is different from zero ( Figure 2—figure supplement 1A ) .", "We found 8 out of the 17 parameters to be highly significant ( *p<0 . 0001 ) .", "The eight most significant parameters found by the SVM classifier ( see previous section ) were used for classification .", "We took the same number of frames from each group ( control/d0/d3/d6 ) - 357 , 997 frames ( 99 . 4 min ) per group , corresponding to the number of frames in the smallest group ( d6 ) .", "We repeated the clustering 30 times ( Figure 2D , black dots ) , each time selecting 99 . 4 min of data from each one of the other groups ( d0 , d3 , d6 , control ) randomly ( with replacements – the same frame could be used in multiple repeats ) , therefore having >1 . 4 million frames for clustering on each repeat .", "The sets are not independent ( overlapping frames between repeats ) and no statistical test was performed over the repeats .", "After z-scoring each parameter ( over all the frames in a given repeat ) , k-means clustering was performed ( using MATLAB function kmeans ) , allowing 20 clusters and a maximum of 500 iterations ( other parameters set to default ) .", "We found that the first seven largest clusters ( cluster size being the number of frames in the cluster ) capture 90 . 4% of the frames , averaged over repeats ( Figure 2D , inset ) .", "To match clusters between repeats ( for each cluster number in repeat 1 , find the corresponding cluster number in repeats 2–30 ) , we used the smallest distance between clusters , by calculating the mean square error over the weights ( the variability in weight size across repeats is shown in Figure 2—figure supplement 1C ) .", "The Janelia Automatic Animal Behavior Annotator ( JAABA; http://jaaba . sourceforge . net/; Kabra et al . , 2013 ) was used to detect epochs of ‘female shoving’ and ‘female chasing’ .", "Two independent classifiers ( ‘shoving classifier’ , ‘chasing classifier’ ) were trained , one for each behavior .", "We used the automatic segmentations to find examples for shoving and chasing epochs , used as a first step in training each classifier .", "We then added example epochs ( positive and negative examples are used for each classifier ) , in an iterative manner ( using examples where the classifiers made wrong predictions ) .", "Altogether we used 24 , 222 frames ( 6 . 7 min ) to train the ‘shoving classifier’ , and 11 , 941 frames ( 3 . 3 min ) for the ‘chasing classifier’ .", "The classifiers were based on the 17 parameters defined above ( denoted as ‘per-frame’ features ) , as well as on ‘window features’ ( ‘mean’ , ‘min’ , ‘max’ , ‘change’ , ‘std’ , ‘diff_neighbor_mean’ , ‘diff_neighbor_min’ , ‘diff_neighbor_max’ , ‘zscore_neighbors’ with a window radius of 10 and default ‘windows parameters’ ) , therefore taking into account longer timescales for classification , rather than the single frames we used for SVM classification and k-means clustering ( see Figure 2—figure supplement 1C for comparison ) .", "We cross validated each classifier before applying the classification on all the data , using the cross-validation procedure available in JAABA package ( with default parameters ) .", "A total of 94 . 2% of the frames annotated by the user as shoving were correctly classified as shoving , while 92 . 8% of the frames annotated as no-shoving were classified as no-shoving .", "For the ‘chasing classifier’ , we got 96% and 90 . 8% success in classifying chasing and no-chasing .", "The trained shoving classifier was used to annotate each frame as belonging or not belonging to ‘female shoving’ epoch , and the trained chasing classifier was used independently to classify each frame as belonging or not-belonging to a ‘female chasing’ epoch .", "The same classifiers were used for all experimental conditions ( 5 min or 2 min activation duration , d0/d3/d6 delay conditions ) .", "We annotated a subset of the data manually ( pC1-Int , d0-d6 ) , to confirm our automatic behavior detection , as well as in search for more rare events , or events that are not captured due to tracking issues .", "Three behaviors were annotated by two observers: female shoving , female chasing and circling ( Figure 2—figure supplement 3A–C ) .", "The two observers annotated different sets of movies , while a small subset ( n = 5 movies ) were annotated by the two observers and we confirmed that both detected three behaviors ( shoving , chasing , circling ) similarly .", "Female circling was not detected by our automated procedures for two reasons .", "First , during circling male and female bodies often overlap , causing large errors in heading detection .", "Second , these events are relatively sparse .", "One observer also detected three other rare behaviors: head butting , female mounting ( Figure 2—figure supplement 3C , Video", "3 ) and wing extension ( Figure 2—figure supplement 3D–E , Video 2 ) .", "Statistical analysis was performed using MATLAB ( Mathworks , Natick , MA ) procedures , and corrected for multiple comparisons using the Bonferroni correction when appropriate .", "The details on the statistical test used are listed under the Results section and the Figure legends .", "Black lines between two groups indicate a statistically significant difference between the groups after multiple comparison correction , while a red line indicates that the difference is statistically significant only when multiple comparisons test is not used .", "To test for significant differences in copulation rate , we used Cox’s proportional hazards regression model , using the MATLAB procedure coxphfit .", "‘Censoring’ was used to account for the fact that some flies copulated within the 30 min time window ( after which the experiment was terminated ) , while others did not .", "The correlation between female velocity and male song ( Figure 1D , G ) was done as previously described ( Clemens et al . , 2015 ) .", "Briefly , female absolute speed and male song were averaged over 1 min windows .", "In each window , we calculated the mean value of female ( absolute ) speed , bout amount ( the total amount of song in the window ) , bout number ( the number of song bouts in the window ) , and bout duration ( the mean bout duration in the window ) .", "Then , for each condition , we calculated the correlation between female speed and male song by pooling all windows for a given group together .", "The MATLAB procedure corr was used to calculate the Pearson correlation , and one way analysis of covariance ( ANOCOVA ) was used to compare the slopes ( x , y being the male song and the female speed ) between groups using aoctool ( MATLAB ) .", "The 30 SVM ( Support Vector Machine ) classifiers ( Figure 2B–C , Figure 2—figure supplement 1C ) were trained using non-overlapping sets of frames and are therefore considered independent .", "One-sample t-test was used to calculate a test decision for the null hypothesis that the 30 weight values ( for a given parameter ) come from a normal distribution with a mean of zero ( and unknown variance ) .", "For each parameter , -log ( P ) is shown , and a vertical dashed indicates p<10−4 ( Figure 2—figure supplement 2C ) .", "Flies were dissected in S2 insect medium ( Sigma #S0146 ) .", "Dissected brains were moved through six wells ( 12ηl/well ) containing a fixation solution 4% paraformaldehyde , Electron microscopy sciences #15713 in PBT ( 0 . 3% Triton in PBSX1; Triton X-100 Sigma Aldrich #X100; PBS - Cellgro #21–040 ) , before sitting for 30 min on a rotator at room temperature .", "Following fixation , brains were moved through six wells containing PBT , 15 min in each well .", "Then , brains were transferred through four wells containing a blocking solution ( 5% Goat Serum in PBT; Life Technologies #16210–064 ) , and sitting in the last well for 30 min .", "Brains were then moved to a solution containing primary antibodies ( see below ) and then incubated for two nights at 4°C ( sealed and light protected ) .", "After eight washes ( 20 min per wash ) in PBT , brains were incubated overnight with secondary antibodies .", "After eight washed ( 20 min each ) in PBT , brains were placed on a slide ( Fisher Scientific #12-550-15 ) , between two zero numbered coverslips used as spacers ( Fisher Scientific 12-540B ) and under a coverslip ( Fisher Scientific #12-542B ) , and Vectashield ( Vector Laboratories ) was applied .", "Nail polish was used to seal around the center coverslip edges , and brains were stored in dark at 4°C overnight to harden , before imaging .", "See Key Resources Table for the list of antibodies used in this study .", "Imaging was done using a Leica confocal microscope ( TCS SP8 X ) .", "Figure 1A was modified from Deutsch et al . , 2019 .", "Neurons in a complete EM volume of an adult female brain ( Zheng et al . , 2018 ) were automatically reconstructed in FlyWire ( flywire . ai , [Dorkenwald et al . , 2020] ) .", "Within FlyWire , we first searched for reconstructed segments that match the morphology of known pC1 cells .", "We used anatomical landmarks to find the bundle that projects dorsally from pC1 cells bodies ( Figure 1A , red arrow ) .", "We then looked at two cross-sections of this bundle in each hemisphere ( Figure 3B and Figure 3—figure supplement", "1 ) and scanned systematically all the segments that pass through this bundle .", "Based on known morphology of female pC1 cells ( Deutsch et al . , 2019; Kimura et al . , 2015; Zhou et al . , 2014 ) , we defined cells as pC1 when they crossed through the pC1 bundle , and also projected to the lateral junction ( Figure 1A ) .", "Similarly , pC2l cells were found by looking through the pC2l bundle ( see Deutsch et al . , 2019; we refer to these cells as pC2 below ) .", "The aIPg cells were first found by searching for neurons synaptically connected to pC1d ( see below ) , and other aIPg cells were found by systematically exploring a cross section within the aIPg bundle ( Figure 6E and Figure 5—figure supplement 1 ) .", "pMN1 and pMN2 were found when mapping the pC1d synaptic partners , and then named pMN1 and pMN2 based on their morphology ( Deutsch et al . , 2019; Kimura et al . , 2015 ) .", "pC1 and aIPg cells were sorted manually into subtypes based on morphology ( Figure 3C and Figure 5—figure supplement 1B ) .", "Proofreading of a neuron was performed using the tools available in FlyWire ( flywire . ai , [Dorkenwald et al . , 2020] ) .", "In short , this process has two parts: ( 1 ) removing ( ‘splitting’ ) parts that do not belong to the cell ( ‘mergers’ ) , such as parts of glia or parts of other neurons ( for example , when detecting two cell bodies in one segment ) , and ( 2 ) adding missing parts ( ‘merging’ ) .", "We had an average of 5 . 4 splits and 10 . 7 merges per neuron , and proofreading a single cell took 43 min on average ( we measured the proofreading time for a subset of the cells we proofread ) .", "Proofreading was complete when no additional obvious mergers were found , and we couldn’t identify missing parts at the edge of any processes .", "In some cases , the known morphology of the cell ( e . g . pMN1 or pMN2 ) or the existence of other cells with similar morphology ( in the same or the other hemisphere ) were used to verify that no major processes were missing .", "Sorting cells into types was done manually , based on their morphology .", "pC1 was divided into five subtypes , and aIPg were divided into three subtypes .", "Assigning names to known neurons we found in the EM volume was done solely based on morphology .", "It is possible , that in some cases ( e . g . for pC1 , pC2 , or aIPg cells ) , some of the neurons we found are not actually Dsx+ or Fru+ cells .", "Additional work is needed to compare LM based and EM based morphologies , and to classify cell types based both on morphology and connectivity ( Scheffer et al . , 2020 ) .", "Finding cell types in hemibrain version 1 . 1 with shared morphology was done for the major inputs and outputs of pC1d/e manually .", "We mapped all the direct inputs and outputs of a single pC1d neuron ( Figure 3C ) by manually detecting pre- and postsynaptic partners for this cell .", "After proofreading the cell ( see details above ) , we looked systematically , branch by branch , for synaptic partners based on previously defined criteria .", "For a contact to be defined as a chemical synapse , it had to meet three conditions: ( 1 ) the presence of a synaptic cleft between the pre- and postsynaptic cells , ( 2 ) presynaptic active zone with vesicles near the contact point , and ( 3 ) one of two ( or both ) must exist: a presynaptic T-bar adjacent to the cleft ( Fouquet et al . , 2009 ) at the presynaptic terminal or a postsynaptic density ( PSD , [Ziff , 1997] ) .", "In flies , PSDs are variable , and are often unclear or absent ( Prokop and Meinertzhagen , 2006 ) .", "Typically , we observed T-bars rather than PSDs , as a T-bar is easier to identify .", "Our criteria was slightly more conservative than the one used in Zheng et al . , 2018 , possibly leading to less false positives ( wrongly assigned synapses ) , and more false negatives ( missing synapses ) .", "On top of using a conservative criterion , synapses were missed for several other reasons , including missed detections by the manual observers ( see comparison to automatic detection below ) and signal to noise issues in the dataset .", "Once a synapse was detected , we then looked for the post-synaptic partner .", "Around 10% of the inputs to pC1d and about 60% of the outputs were short segments ( ‘twigs’ ) , that we could not connect to backbones in order to identify or proofread the connected neuron .", "The twigs were not restricted to a specific part of the pC1d cell , and we therefore believe that they do not impose a bias on the distribution of pC1d connections , though it is possible that specific output types ( e . g . cells with thinner processes ) are less likely to be detected .", "Following the detection of pC1d synaptic partners , we mapped the inputs and outputs to pC1d in three steps .", "First , we manually proofread the input and output segments .", "Second , we eliminated cells that connect to pC1d with less than three synapses , to reduce the number of potential false positives , and to focus on stronger connections .", "We ended up having 78 input and 52 output cells .", "Third , we sorted cells manually into cell types based on morphology .", "Some cells were classified based on known morphology from light microscopy ( pMN1 , pMN2 , pC1 , pC2 , aIPg ) .", "In order to look for connections between pC1 and pC2 cells ( the largest sets of Dsx+ neurons ) in an unbiased way ( not focusing on specific types or individual pC1 or pC2 ) , we first identified and proofread pC1 and pC2 cells .", "Synaptic connections between individual cells of pC1 or pC2 type were detected by manually inspecting the volume plane by plane .", "Once a pair of segments that came within proximity of one another was detected , we zoomed and looked for synaptic connections based on the criteria defined above .", "We also used automatic detection of synapses in FAFB .", "FlyWire provides a mapping of the publicly available , automatically detected synapses from Buhmann et al . , 2019 .", "We estimated the reliability of synapse detection by manually testing all the synapsis between pC1d ( presynaptic ) and two different output cells ( a single aIPg-b and a single pC2-like ) .", "We found 61 . 7% ( 71/115 ) of the tested synapses to be true positives according to our criteria , 24 . 3% ( 28/115 ) redundant ( two coordinates for the same synapse ) and 13 . 9% false positives .", "By looking at pairwise distances between presynaptic coordinates , and using a minimum threshold of 150 nm , we were able to remove 27/28 of the redundant synapses , while eliminating only three true synapses , thus ending up having 85% ( 98/115 ) true positives .", "We therefore used this threshold whenever using the Buhmann detector .", "We detected all the inputs and outputs of a single pC1d ( FlyWire coordinates 145817 , 41367 , 5139 ) and a single pC1e ( FlyWire coordinates 142741 , 44980 , 4908 ) cells .", "Cells were included in the count only if they followed the following two criteria: ( 1 ) Connected to the inspected pC1d or pC1e with six synapses or more and ( 2 ) is part of a cell type ( based on morphology , using manual classification ) that includes at least one cell with strong connection ( defined as 15 synapses or more ) with pC1d or pC1e .", "Only synapses that belong to these cells were counted ( see Table 1; Figure 5F; Figure 6; Figure 6—figure supplement 1 ) .", "Finding the best match in the single-clone dataset FlyCircuit for a given EM segment was done in two steps .", "First , an .", "swc file was generated for a given segment ( using the automatically segmented cells rather than the proofread ones for technical reasons ) .", "Second , we performed an NBLAST search ( Costa et al . , 2016 ) either online ( http://nblast . virtualflybrain . org:8080/NBLAST_on-the-fly/ ) or using ‘natverse’ , an R package for neuroanatomical data analysis ( Bates et al . , 2020 ) .", "For visualization purposes , we first created mesh files ( . obj ) for proofread neurons , and then used either Meshlab ( http://www . meshlab . net/ ) to create images , or Blender ( https://www . blender . org/ ) to create movies ( see support/FAQ in https://flywire . ai/ for instructions on creating . swc and . obj files ) .", "We imaged brain activity following pC1 optogenetic activation ( though the microscope objective ) under a two-photon custom made microscope ( Pacheco et al . , 2021 ) in females , using the calcium indicator GCaMP6s ( Chen et al . , 2013 ) .", "Both GCaMP6s and the structural marker tdTomato ( Shaner et al . , 2004 ) were expressed pan-neuronally in blind flies ( NorpA[36] mutant ) using the nsyb enhancer ( Bussell et al . , 2014 ) .", "For pC1 activation , we used the same temporal pattern as the one used in the behavioral experiments: 5 or 2 min of light on , at 100 Hz and 50% duty cycle .", "Imaging started 5 min before stimulus onset , where baseline activity was measured , and lasted 9 . 5 min after stimulus offset for whole-brain imaging and 30 min after stimulus offset for doublesex imaging .", "While the red shifted channelrhodopsin ReachR was used for behavior experiments to minimize optogenetic activation by background light , we used Chrimson for two-photon imaging for two reasons .", "First , to minimize the amount of bleed-through from the optogenetic activation light to the green photomultiplier tube ( PMT ) .", "For this reason we also choose a longer wavelength of 700 nm ( M700L4 , Thorlabs , Newton , NJ with a band pass filter FF01-708/75-25 , Semrock , Rochester , NY ) that is well separated from the range of light that cross the green PMT entrance filter ( Semrock FF01-593/40-25 ) .", "Second , we wanted to minimize the amount of light needed for neuronal activation by using a more sensitive effector , to reduce the amount of heat accumulating in the brain during imaging ( on top of the heating caused by the two-photon laser , whose power was limited to 15 mW ) .", "We are aware of possible differences in pC1 activation level between the behavioral and imaging experiments .", "Based on existing literature , we tried to choose an activation level for the imaging experiments that will roughly match the activation induced during behavior .", "In order to match the activation level between the behavioral experiments ( ReachR , 627 nm light , intact fly ) and the imaging experiments ( csChrimson , 700 nm light , cuticle removed above the fly brain ) we used data from existing literature .", "By comparing the amount of light needed for driving proboscis extension reflex ( PER ) in 100% of adult flies in Inagaki et al . , 2014 ( ReachR , 1 . 1 mW/mm2 , 627 nm ) to the level of light used to saturate PER score in Klapoetke et al . , 2014 ( CsChrimson , 0 . 07 mW/mm2 , 720 nm ) , taking into account the different duty cycles used in the two studies and given the penetration rate through the cuticle ( Based on Inagaki et al . , 2014; Figure 1A , around 6% of the light penetrates at 627 nm ) , we choose a light intensity of 0 . 013 mW/mm2 .", "A volume of ~307 × 307 × 200 µm3 from the dorsal part of the central brain was scanned at 0 . 1 Hz ( 1 . 4 × 1 . 2 × 2 µm3 voxel size ) , covering a complete dorsal quadrant ( full anterior-posterior axis of the central brain ) which represents about 58 . 02 ± 3 . 97% of the whole hemisphere ( mean ± SD , n = 28 animals ) .", "Volumetric data was processed as described in Pacheco et al . , 2021 .", "In brief , tdTomato signal was used to motion-correct volumetric time-series of GCaMP6s signal in XYZ axis ( using the NoRMCorre algorithm [Pnevmatikakis and Giovannucci , 2017] ) .", "Volumes were spatially resampled to have isotropic XY voxel size of 1 . 2 × 1 . 2 × 2 µm3 ( bilinear interpolation on X and Y axes ) , and temporally resampled to correct for different slice timing across planes of the same volume , and to align timestamps of volumes relative to the start of the optogenetic stimulation ( linear interpolation ) .", "Next , the GCaMP6s signal was 3D-ROI segmented using the Constrained Nonnegative Matrix Factorization ( CNMF ) algorithm to obtain temporal traces and spatial footprints per segmented ROI as implemented in CaImAn ( Giovannucci et al . , 2019; Pacheco et al . , 2021 ) .", "In this algorithm , each ROI is defined as a contiguous set of pixels within the field of view that are correlated in time , these ROIs are initialized around locations with maximum variance across time .", "Spatially overlapping ROIs with correlated activity are merged ( correlation coefficient >0 . 9 ) ; therefore , ROIs could have different sizes .", "Code to perform these processing steps are available at https://github . com/murthylab/FlyCaImAn ( Pacheco et al . , 2021 ) .", "In addition , to further remove residual motion artifacts from the GCaMP6s signal , in particular slow drift over tens of minutes , we performed independent component analysis ( ICA ) on the tdtomato ( Ftdtomato ) and GCaMP6s ( FGCAMP ) signal for each ROI independently , similar to Scholz et al . , 2018 .", "To remove opto-related artifact bleeding through the red channel , Ftdtomato was linearly interpolated from 20 s before stimulus onset to 20 after stimulus offset ( to ignore opto-related artifact bleeding through the red channel ) and random noise ( from normal distribution centered at 0 ) added to interpolated timepoints .", "Ftdtomato was then smoothed ( moving average with a window of 50 s ) , and ICA was used ( rica function implemented in MATLAB ) to extract background and signal components from Ftdtomato and FGCAMP .", "Independent component highly correlated to Ftdtomato ( absolute correlation coefficient >0 . 9 ) was considered the background component ( ICABackground ( Ftdtomato , FGCAMP ) ) , whereas the other component considered the signal component ( ICASignal ( Ftdtomato , FGCAMP ) ) .", "Sign of ICASignal ( Ftdtomato , FGCAMP ) was corrected using the sign of the correlation between ICASignal ( Ftdtomato , FGCAMP ) and FGCAMP .", "For ROIs extracted from pan-neuronal data , we report calcium signals as ICASignal ( Ftdtomato , FGCAMP ) as shown in Figure 5B .", "We defined responsive ROIs as ROIs with a mean activity during optogenetic stimulation ( Ft1 ) higher than 3σo ( σo - standard deviation of activity during baseline ) .", "We then split ROIs into transiently and persistently active units using the mean activity after optogenetic stimulation ( Ft2 , from stimulus offset to 5 min after stimulus offset ) , transient ROIs had Ft2 ≤ 3σo , while persistent ROIs had Ft2 > 3σo .", "To evaluate the diversity of these coarse activity types , we hierarchically clustered transient and persistent responses ( we evaluated the number of clusters these response types split into using the consensus across Calinski-Harabasz , Silhouette , Gap , and Davies-Bouldin criteria ) , obtaining two clusters of transient responses and two clusters of persistent responses ( Figure 5C–D ) .", "For recordings of Dsx+ cell types , we imaged pC1 , pC2 , pCd1 , and pCd2 cells ( 1–2 groups at a time ) , located in the dorsal side of the central brain , at a speed of 0 . 5–0 . 25 Hz ( 0 . 5 × 0 . 5 × 1 µm3 - 2 . 5 × 2 . 5 × 1 µm3 voxel size ) .", "Similarly , for recordings of Fru+ cell types we imaged Fru+ cells ( both within the same field of view ) , located in the dorsal side of the central brain , at a speed of 0 . 5 Hz ( 1 . 2 × 1 . 2 × 2 µm3 voxel size ) .", "Volumetric time-series of GCaMP6s signal was motion-corrected in the XYZ axes using the NoRMCorre algorithm ( Pnevmatikakis and Giovannucci , 2017 ) , and temporally resampled to correct for different slice timing across planes of the same volume , and to align timestamps of volumes relative to the start of the optogenetic stimulation ( linear interpolation ) .", "Dsx+ and Fru+ somas were manually segmented by finding the center and edge of each cell body stack by stack ( Deutsch et al . , 2019 ) .", "Stimulus design , recording techniques , and analysis methods are described in detail in Clemens et al . , 2015 .", "Briefly , virgin female flies ( 1–2 days old ) were mounted and dissected as described previously , and the cell bodies were accessed from the posterior surface of the head and visualized by expressing GFP in pC1-A neurons ( see Fly genotype table ) – 1 cell was recorded per fly .", "Raw data were further analyzed using MATLAB ( Mathworks ) .", "To construct the IPI , sine frequency , and intensity tuning curves , we calculated the integral voltage in response to each stimulus , after subtracting the baseline voltage .", "Each response was then normalized by the duration of the stimulus .", "To compare across individuals , tuning curves were normalized to peak at 1 . 0 ." ] ]

[ "Sustained changes in mood or action require persistent changes in neural activity , but it has been difficult to identify the neural circuit mechanisms that underlie persistent activity and contribute to long-lasting changes in behavior .", "Here , we show that a subset of Doublesex+ pC1 neurons in the Drosophila female brain , called pC1d/e , can drive minutes-long changes in female behavior in the presence of males .", "Using automated reconstruction of a volume electron microscopic ( EM ) image of the female brain , we map all inputs and outputs to both pC1d and pC1e .", "This reveals strong recurrent connectivity between , in particular , pC1d/e neurons and a specific subset of Fruitless+ neurons called aIPg .", "We additionally find that pC1d/e activation drives long-lasting persistent neural activity in brain areas and cells overlapping with the pC1d/e neural network , including both Doublesex+ and Fruitless+ neurons .", "Our work thus links minutes-long persistent changes in behavior with persistent neural activity and recurrent circuit architecture in the female brain ." ]

[ "Long-term mental states such as arousal and mood variations rely on persistent changes in the activity of certain neural circuits which have been difficult to identify .", "For instance , in male fruit flies , the activation of a particular circuit containing ‘P1 neurons’ can escalate aggressive and mating behaviors .", "However , less is known about the neural networks that underlie arousal in female flies .", "A group of female-specific , ‘pC1 neurons’ similar to P1 neurons could play this role , but it was unclear whether it could drive lasting changes in female fly behavior .", "To investigate this question , Deutsch et al . stimulated or shut down pC1 circuits in female flies , and then recorded the insects’ interactions with male flies .", "Stimulation was accomplished using optogenetics , a technique which allows researchers to precisely control the activity of specially modified light-sensitive neurons .", "Silencing pC1 neurons in female flies diminished their interest in male partners and their suitor’s courtship songs .", "Activating these neural circuits made the females more receptive to males; it also triggered long-lasting aggressive behaviors not typically observed in virgin females , such as shoving and chasing .", "Deutsch et al . then identified the brain cells that pC1 neurons connect to , discovering that these neurons are part of an interconnected circuit also formed of aIPg neurons – a population of fly brain cells that shows sex differences and is linked to female aggression .", "The brains of females were then imaged as pC1 neurons were switched on , revealing a persistent activity which outlasted the activation in circuits containing both pC1 and aIPg neurons .", "Thus , these results link neural circuit architecture to long lasting changes in neural activity , and ultimately , in behavior .", "Future experiments can build on these results to determine how this circuit is activated during natural social interactions ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "research communication", "neuroscience" ]

[ [ "Conflict between groups of people can profoundly change their demographic , economic and cultural outlook .", "Across human history , intergroup conflict functioned as a critical change agent and selection pressure that may have shaped the biological preparedness for in-group oriented cooperation and self-sacrifice on the one hand , and out-group hostility and aggression on the other hand ( Bar-Tal et al . , 2007; Boyd and Richerson , 1982; Choi and Bowles , 2007; De Dreu et al . , 2010; Macfarlan et al . , 2014 ) .", "Out-group hostility and aggression serves to subordinate and exploit rivaling out-groups ( henceforth out-group attack ) , and/or to defend the in-group against such hostility from neighboring groups ( henceforth in-group defense ) ( De Dreu et al . , 2016a; Halevy et al . , 2008; Rusch , 2014a; Rusch , 2014b; Lopez , 2017; Wrangham , 2018 ) .", "Recent work has showed that individuals are more strongly motivated to contribute to defend one’s own group than to attack out-groups and , importantly , that out-group attacks frequently fail because individual contributions to out-group attacks are poorly coordinated ( De Dreu et al . , 2016a ) .", "Indeed , attacking rivals who are expected to have strong defenses more likely results in failure and waste , than attacking groups expected to be weak and defenseless ( De Dreu et al . , 2016b; Grossman and Kim , 2002; Goeree et al . , 2003 ) .", "Thus , to succeed in intergroup competition and conflict , group members not only need to contribute to their group’s competitive strength , but they also need to coordinate within their group the intensity and timing of attacks .", "How group members coordinate behavior into effective joint actions during intergroup competition and conflict remains poorly understood ( De Dreu et al . , 2016a ) and we lack insight into the underlying neurobiological mechanism .", "Here , we target the neuro-hormone oxytocin as a neurobiological mechanism underlying within-group coordination of out-group attack in dynamic intergroup contests .", "Studies in ethnography and anthropology have shown that small groups preparing for intergroup conflict build cohesion and commitment through social bonding routines and rituals .", "Groups selectively invite friends to join a raid ( Glowacki et al . , 2016 ) , bond like family ( Macfarlan et al . , 2014 ) , and engage in cultural rituals that simulate self-sacrifice , cooperation and coordination ( Xygalatas et al . , 2013 ) .", "Neuroendocrine studies ( Burkett et al . , 2016; Carter , 2014; Ma et al . , 2016b; Samuni et al . , 2017 ) have linked these and related practices to the release of oxytocin , a nine-amino acid neuropeptide .", "In turn , intranasal administration of oxytocin has been shown to modulate neural responses in brain regions involved in threat detection and reward processing ( Ma et al . , 2016b; Paloyelis et al . , 2016; Wang et al . , 2017; Liu et al . , 2019 ) .", "Moreover , in both non-human and human primates , elevated levels of oxytocin via intranasal administration have been linked to a range of cognitive and behavioral effects including within-group conformity , trust , affiliation , and cooperation ( Arueti et al . , 2013; Aydogan et al . , 2017; De Dreu et al . , 2010; Madden and Clutton-Brock , 2011; Rilling et al . , 2012; Samuni et al . , 2017; Stallen et al . , 2012; Yan et al . , 2018 ) .", "Whereas these work together point to oxytocin as a possible neurobiological mechanism underlying group coordination , three issues remain unclear .", "First , we lack empirical evidence for the possibility that oxytocin promotes group-level coordination of collective action in general , and during intergroup competition and conflict in particular .", "Second , we poorly understand how group members coordinate collective action during intergroup competition and conflict , and whether oxytocin can directly influence coordination and/or the strategy group members use to coordinate .", "Third , we do not know whether oxytocin differentially modulates tacit coordination within groups when collective contributions are focused on attacking one’s rival , versus defending the in-group against possible attacks by one’s rival .", "To address these issues , we examined the role of oxytocin in 80 interactive , multi-round contests between three-person attacker and three-person defender groups .", "Group members on each side made individual contributions to their group pool ( aimed at attacking the other side , or at defending against such possible attacks ) .", "We provided individuals with real-time feedback on group investments and success after each contest round , which allowed group members to learn and adapt to their rival’s past investments , and use the history of play as a focal point to coordinate their future attacks and defenses .", "Indeed , when lacking explicit coordination mechanisms such as a leader or decision-making protocols ( De Dreu et al . , 2016a; Gavrilets and Fortunato , 2014; Hermalin , 1998 ) , groups use social norms and focal points to tacitly coordinate collective action ( Halevy and Chou , 2014; Schelling , 1960 ) .", "In multi-round intergroup contests , the rival’s history of play can serve as a focal point for groups to coordinate their contributions to attack and/or defend ( De Dreu et al . , 2016b ) .", "Thus , when attacking out-groups , group members may coordinate their contributions on their rival’s historical level of defense , and attack when historical defense is low and the target appears vulnerable; and not attack when historical defense is high and the target appears strong and difficult to beat .", "We expected effects of oxytocin to be stronger during out-group attack than during in-group defense .", "In-group defense is first and foremost an adaptation to the attackers’ ( past ) aggression and is typically well-coordinated—individuals fight towards the same goal of self-preservation and group survival .", "Although there is some evidence that oxytocin may increase protective aggression ( for a review see De Dreu and Kret , 2016 ) , administering oxytocin may contribute little to the relatively high baseline levels of contribution and coordination during in-group defense .", "In contrast , out-group attacks are typically less strong , more variable , and less well-coordinated ( De Dreu et al . , 2016a ) , leaving more room for oxytocin administration to influence behavior .", "We anticipated two possibilities for the effect of oxytocin .", "On the one hand , oxytocin has been linked to prosociality ( Madden and Clutton-Brock , 2011; Rilling et al . , 2012; Liu et al . , 2019 ) , empathy ( Hurlemann et al . , 2010; Bartz et al . , 2010 ) and consolation ( Burkett et al . , 2016 ) .", "This line of research would lead us to anticipate that oxytocin may enable individuals in attacker groups to coordinate on a peaceful no-attack strategy that is independent of the rivals’ history of defense .", "On the other hand , oxytocin has been shown to enhance in-group conformity ( Aydogan et al . , 2017; Stallen et al . , 2012; De Dreu and Kret , 2016 ) , increase behavioral coordination and neural synchronization within pairs of individuals performing a joint task ( Arueti et al . , 2013; Mu et al . , 2016 ) , enhance facial mimicry , motor imitation , and neural responses linked to action-intention mirroring ( Korb et al . , 2016; De Coster et al . , 2014; Kret and De Dreu , 2017; Levy et al . , 2016 ) , and improve memory and learning from feedback ( Striepens et al . , 2012; Guastella et al . , 2008; Ma et al . , 2016a ) .", "These functionalities alone and in combination may facilitate coordination on social norms and shared focal points that emerge during group interaction , suggesting that oxytocin may facilitate the use of rival’s history , enable individuals in attacker groups to coordinate better on the level and timing of their attacks , and to efficiently appropriate resources from their out-group .", "Evidence for this possibility would be consistent with earlier findings that oxytocin shifts the focus from self-interest to in-group interests ( De Dreu and Kret , 2016 ) , limits trust and cooperation to in-group members ( De Dreu et al . , 2010; Ma et al . , 2015; Ten Velden et al . , 2017 ) , and promotes aggression toward threatening rivals ( Madden and Clutton-Brock , 2011; Samuni et al . , 2017; Striepens et al . , 2012; Ne'eman et al . , 2016 ) ." ], [ "We examined these possibilities using a dynamic , fully incentivized Intergroup Attacker-Defender Contest game ( De Dreu et al . , 2016a ) .", "The IADC is an all-pay contest ( Abbink et al . , 2010; Dechenaux et al . , 2015 ) involving six individuals randomly assigned to a three-person attacker and a three-person defender group .", "In the IADC game , 3 attackers made individual contributions to the group pool to subordinate the other group and increase gains through victory ( but always keep the remaining resources ) , and 3 defenders contributed to their group pool to defend rival’s attack and protect against loss and defeat ( defenders ‘survive’ from left with 0 only if defense succeeds ) .", "We created 80 IADC sessions ( with N = 480 healthy males ) ; in 40 ( 40 ) sessions , participants received intranasal oxytocin ( matching placebo ) ( Figure 1 ) .", "Each IADC session involved two blocks of 15 investment rounds with real-time feedback in between rounds .", "For each investment round ( Table 1 ) , each individual received an initial endowment of 20 MUs ( Monetary Units ) .", "Each individual decided the amount ( Ii , 0 ≤ Ii ≤ 20 ) to the group’s pool G ( 0 ≤ G ≤ 60 , GAttacker = IAttacker-1 + IAttacker-2 + IAttacker-3 , GDefender = IDefender-1 + IDefender-2 + IDefender-3 ) .", "Contributions were wasted , but when GAttacker ≤ GDefender , attackers failed and defenders survived and all six individuals kept their remaining endowment ( leftovers , 20 – Ii ) .", "However , when GAttacker > GDefender , defenders failed and left with 0 .", "The attacker group won and took away defender group’s remaining MU ( spoils from winning , 60 – GDefender ) , which were divided equally among three attacker group members ( each attacker member received: ( 60 – GDefender ) /3 ) and added to their remaining endowments ( 20 – IAttacker-i ) .", "Thus , contributions in the attacker group ( GAttacker ) and in the defender group ( GDefender ) reflect the contribution level to out-group attack and to in-group defense , respectively .", "In one 15-round block , individuals made their decisions individually and simultaneously without communication ( i . e . Simultaneous decision-making block ) .", "In the other 15-round block ( order counterbalanced ) , individuals within groups made their decisions in sequence: one randomly drawn member made his decision first , followed by the second randomly selected member who was informed about the first member’s contribution , and then the third and final member made his decision ( i . e . Sequential decision-making block , De Dreu et al . , 2016a; Gavrilets and Fortunato , 2014; Figure 2 ) .", "The sequential decision-making protocol acts as a ‘coordination device’ that facilitates behavioral coordination within groups ( De Dreu et al . , 2016a; Gavrilets and Fortunato , 2014; Hermalin , 1998 ) .", "This treatment thus provides a benchmark to compare groups in the simultaneous decision-protocol who lack an explicit coordination mechanism and have to find other means—such as a shared social norm or the rival’s past defense — to coordinate individual contributions into effective joint action ( De Dreu et al . , 2016a ) .", "We operationalized within-group coordination as behavioral alignment of contribution to the group , indicated by the variance in contributions to group fighting , with lower group-level variance reflecting better coordination .", "We expected that effects of oxytocin on group coordination would emerge especially under simultaneous rather than sequential decision-making .", "Before examining within-group coordination , we examined treatment effects on contributions to group pool .", "Individual contributions were averaged across the three members within each three-person group , and submitted to a 2 ( Treatment: Oxytocin vs . Placebo ) × 2 ( Role: Attack vs . Defense ) × 2 ( Procedure: Simultaneous vs . Sequential ) × 15 ( Rounds ) mixed-model Analysis of Variance ( ANOVA ) with Treatment between-sessions .", "Individuals contributed more under sequential than simultaneous decision-making ( M ± SE = 6 . 89 ± 0 . 20 vs . 6 . 47 ± 0 . 24; F ( 1 , 78 ) = 5 . 109 , p = 0 . 027 , η2 = 0 . 061 ) , and somewhat less when given oxytocin rather than placebo ( M ± SE = 6 . 28 ± 0 . 27 vs . 7 . 08 ± 0 . 30; F ( 1 , 78 ) = 3 . 719 , p = 0 . 057 , η2 = 0 . 046; marginal significance ) .", "Figure 3A showed higher contributions to in-group defense than to out-group attack , especially in earlier rounds ( Role , F ( 1 , 78 ) = 287 . 903 , p < 0 . 001; η2 = 0 . 787; Role × Round , F ( 14 , 65 ) = 4 . 529 , p < 0 . 001 , η2 = 0 . 494 ) .", "Treatment effects also emerged when we examined the number of non-contributors .", "There were more non-contributors in attacker compared to defender groups ( M ± SE = 20 . 23 ± 0 . 90 vs . 4 . 25 ± 0 . 44; F ( 1 , 78 ) = 408 . 489 , p < 0 . 001; η2 = 0 . 840 ) , and more non-contributors in groups given oxytocin than placebo ( M ± SE = 13 . 46 ± 0 . 90 vs . 11 . 01 ± 0 . 75; F ( 1 , 78 ) = 4 . 345 , p = 0 . 040; η2 = 0 . 053 ) .", "Crucially , oxytocin increased the number of non-contributors in attacker groups but not in defender groups ( Role × Treatment , F ( 1 , 78 ) = 5 . 043 , p = 0 . 028 , η2 = 0 . 061 , Figure 3B ) .", "This Role × Treatment effect is especially true when decisions were made simultaneously ( F ( 1 , 78 ) = 5 . 712 , p = 0 . 019 , η2 = 0 . 068 ) but less so when decisions were made sequentially ( F ( 1 , 78 ) = 2 . 143 , p = 0 . 147; η2 = 0 . 027 ) .", "We then examined participants’ decision time when deciding to ( not ) contribute , we showed that individuals in attacker groups made their decisions to not contribute faster than to contribute ( F ( 1 , 78 ) =137 . 679 , p < 0 . 001 , η2 = 0 . 641 ) .", "Oxytocin increased the speed with which individuals in attacker groups decided to not contribute ( Treatment × Contribute: F ( 1 , 77 ) = 4 . 857 , p = 0 . 031; η2 = 0 . 059 , Figure 4 ) .", "Next , we analyzed group-level coordination operationalized as the within-group variance in contributions , with lower variance indicating stronger coordination ( De Dreu et al . , 2016a ) .", "First , there was a Procedure × Role interaction ( F ( 1 , 78 ) = 101 . 978 , p < 0 . 001 , η2 = 0 . 567 ) showing that sequential decision-protocol facilitated coordination for attack ( F ( 1 , 78 ) = 77 . 852 , p < 0 . 001 , η2 = 0 . 500 ) and , unexpectedly , reduced coordination for defense ( F ( 1 , 78 ) = 39 . 268 , p < 0 . 001 , η2 = 0 . 335 ) .", "Importantly , as predicted , oxytocin facilitated group-level coordination ( i . e . reduced within-group variance in contributions ) of out-group attack but not of in-group defense , especially in earlier rounds ( Role × Treatment × Round , F ( 14 , 1092 ) = 1 . 753 , p = 0 . 041 , η2 = 0 . 022; Figure 5A; Figure 5—figure supplement 1 for similar results of another index of coordination ) .", "To examine what strategy attackers given oxytocin coordinated on , we first examined the contest outcomes .", "As noted , oxytocin may enable groups to coordinate on a peaceful ‘no-attack strategy’ , in which case we should find ( 1 ) lower victories in attacker groups given oxytocin rather than placebo , ( 2 ) the oxytocin effect on the number of non-contributing attackers should not differ when attacks succeed or fail , and ( 3 ) no effect of oxytocin on tracking the rival’s defense history .", "This was not the case .", "First , the number of victories was similar in attacker groups given oxytocin ( M ± SE: 24 . 8 ± 1 . 8% ) and placebo ( M ± SE: 26 . 6 ± 1 . 6% ) , ( F ( 1 , 78 ) =0 . 572 , p=0 . 452; η2 = 0 . 007 ) .", "Thus , rather than making groups coordinate on a peaceful no-attack strategy , oxytocin may enable groups to coordinate on attacking at the right moment with the right force .", "Indeed , analyses of the spoils and leftovers showed that groups given oxytocin rather than placebo had higher leftovers when attacks failed ( t ( 78 ) = 2 . 609 , p=0 . 011 , Cohen’s d = 0 . 581 , 95% Confidence Interval ( CI ) , 0 . 232 to 1 . 76 , Figure 5B ) and somewhat higher spoils when attacks were successful ( t ( 74 ) = 1 . 819 , p=0 . 073 , Cohen’s d = 0 . 419 , 95% CI , −0 . 086 to 1 . 888 , marginal significance , Figure 5C ) .", "To illustrate the increased efficiency of attack under oxytocin , attackers’ contribution and payment under the four conditions ( i . e . Simultaneous/Sequential x Oxytocin/Placebo ) were plotted in Figure 5D using a bootstrapping technique ( Davison and Hinkley , 1977 ) .", "Specifically , a bootstrapped dataset with sample size N = 40 was resampled with replacement separately for each of the four conditions .", "The mean contribution and payment of the bootstrapped sample was then calculated and saved as a new data point .", "For each condition , this procedure was repeated for 1000 times to represent the population information .", "As illustrated in Figure 5D , oxytocin decreased contributions to attack ( a shift toward less contribution ) but increased payment ( a shift toward more payment ) .", "The distribution of attacker groups under oxytocin and placebo in a space defined by the two vectors ( contribution and payment ) indicates a clear separation of oxytocin and placebo treatments ( especially when decisions were made simultaneously , that is black dots vs . grey dots ) .", "Second , we conducted an ANOVA on the average number of non-contributing attackers , with Treatment ( Oxytocin vs . Placebo ) as a between-subjects factor , Procedure ( Simultaneous vs . Sequential ) and Success ( Success vs . Failure ) as within-subjects factors .", "There were more non-contributing attackers when attack failed than succeeded ( Failure vs . Success: M ± SE = 1 . 68 ± 0 . 60 vs . 0 . 37 ± 0 . 05 , F ( 1 , 74 ) = 636 . 941 , p < 0 . 001; η2 = 0 . 891 ) .", "Interestingly , we found a significant Treatment × Success interaction on the number of non-contributing attackers ( F ( 1 , 74 ) = 6 . 345 , p = 0 . 014; η2 = 0 . 075 , Figure 5E ) : Oxytocin increased the number of non-contributing attackers only in failed rounds ( Oxytocin vs . Placebo: M ± SE = 1 . 84 ± 0 . 09 vs . 1 . 52 ± 0 . 09; F ( 1 , 74 ) = 7 . 036; p = 0 . 010; η2 = 0 . 083 ) but not in successful attacks ( Oxytocin vs . Placebo: M ± SE = 0 . 34 ± 0 . 06 vs . 0 . 40 ± 0 . 06; F ( 1 , 74 ) = 0 . 448; p = 0 . 505; η2 = 0 . 006 ) .", "Third , we examined how attacker groups collectively identified when to attack by creating a past defense parameter α ( average defender group’s investment in the last two rounds , that is ( Dj-1+Dj-2 ) /2 on round", "j ) and regressed attacker group’s investments onto α ( attack increased when defender groups were vulnerable rather than strong , as indicated by α approaching -1 ) .", "It showed that attacker groups given oxytocin tracked their rival’s past defense and attacked especially when defenders appeared more rather than less vulnerable ( i . e . attack regressed negatively on rival’s historical defense ) .", "Specifically , when decisions were made simultaneously , attack regressed more strongly on α when groups received oxytocin ( M ± SE: -0 . 30 ± 0 . 05 ) rather than placebo ( M ± SE: -0 . 042 ± 0 . 098; t ( 78 ) = -2 . 334; p = 0 . 022 , Cohen’s d = -0 . 522 , 95% CI , -0 . 482 to -0 . 038 ) , and under oxytocin but not placebo , the regression on α was also stronger in simultaneous rather than sequential decision-making ( Treatment × Procedure interaction , F ( 1 , 78 ) = 8 . 312 , p = 0 . 005 , η2 = 0 . 097 , Figure 6A ) .", "Combined , results suggest that groups given oxytocin created more spoils from winning and had higher leftovers when attacks failed because they better coordinated attack at the right time and with the proper force .", "Indeed , when decisions were made simultaneously , the more strongly attacker groups relied on tracking parameter α , the lower their within-group variance when contributing ( r = 0 . 281 , p=0 . 012 , Figure 6B ) , and the lower within-group variance when contributing , the higher the attacker’s spoils when winning the conflict ( r = −0 . 328 , p=0 . 004 , Figure 6C ) .", "Indirect mediation analyses confirmed that the attacker’s higher spoils under oxytocin was mediated by", "( i ) increased tracking of defender group’s past investments and ( ii ) concomitant increased within-group coordination ( indirect effect = 0 . 147 , SE = 0 . 103; 95% CI , 0 . 019 to 0 . 511 , Figure 6D ) ." ], [ "To be victorious in intergroup conflict , group members not only need to contribute to their group’s fighting capacity .", "They also need to coordinate collective action so that they attack when their rival is expected to be weak , and avoid wasting resources on attacking tough defenders .", "Here we found , using a dynamic intergroup contest between attackers and defenders , that those groups who tracked their defender’s history of play and coordinated their attacks of weak rather than strong defenders wasted less resources on failed attacks and enjoyed greater spoils when winning .", "In addition , we uncovered that oxytocin serves as a neurobiological mechanism underlying such well-timed and coordinated attacks .", "Specifically , we found that oxytocin enabled individuals within attacker groups to converge their individual contributions on each other more , to collectively refrain from attacking apparently strong defenders , and effectively attacking weak defenders .", "These findings emerged when groups lack an explicit coordination device; providing attacker groups with a sequential decision-making protocol as an explicit coordination device substituted oxytocin-induced tacit coordination .", "Our finding that oxytocin enables within-group coordination of contributions to out-group attacks resonates with two heretofore disconnected sets of findings—that small groups of warriors engage in social bonding and cultural rites ( Glowacki et al . , 2016; Macfarlan et al . , 2014; Schelling , 1960; Wilson and Wilson , 2007; Xygalatas et al . , 2013 ) , and that social bonding and synchronized action can trigger the release of oxytocin ( Burkett et al . , 2016; Carter , 2014; Samuni et al . , 2017 ) .", "Combined with the current results , it suggests that oxytocin might be a potential neurobiological mechanism through which social bonding and performing coordinated rituals can help groups to better coordinate their attacks .", "Our findings furthermore suggest that such oxytocin-mediated within-group coordination can be substituted by institutional arrangements , such as a sequential decision-making protocol .", "We speculate that related institutional arrangements , like appointing a leader or having open communication channels can similarly obviate the need for bonding rituals and/or oxytocin to make out-group attack well-coordinated and successful .", "Neither oxytocin nor the sequential decision-protocol contributed to within-group coordination in defender groups .", "One possibility is that in-group defense is tacitly well-coordinated because of the stronger alignment of individual interests within defender groups .", "After all , when defender groups survive an enemy attack , those who did not contribute to in-group defense come out relatively wealthy .", "But when in-group defense fails , all group members lose regardless of whether they contributed or not .", "As a result of this stronger common fate in defender groups , individuals contribute spontaneously and tacitly coordinate well on avoiding collective defeat .", "Exogenous enhancers , whether at the neurobiological or institutional level , appear not necessary .", "Previous studies provided evidence for the role of oxytocin in intergroup interaction: It can increase the positive view and benign approach of the in-group and has been linked to subtle forms of intergroup discrimination ( De Dreu et al . , 2010; De Dreu and Kret , 2016; Ma et al . , 2015; Stallen et al . , 2012; Ten Velden et al . , 2017 ) .", "Without exception , this early evidence was limited to individual-level decision-making , and did not clearly distinguish the distinct motives for contributing to in-group efficiency and out-group hostility .", "It remained unknown whether and how oxytocin differentially affects attack and defense behavior during intergroup conflict .", "Using a dynamic attack-defense contest we revealed selective effects of oxytocin on group attacking .", "As such , the present study provides a new perspective on oxytocin in intergroup conflict by highlighting its functionality for strategic attack ( rather than defense ) .", "In doing so , we also obtained first-time evidence that in-group coordination for collective action can be tracked to evolutionary preserved neurobiological factors .", "Our study has two potential limitations .", "First , it involved only Chinese males as study participants .", "Whereas we cannot exclude specific cultural effects , findings for the comparison between out-group attack and in-group defense and between sequential and simultaneous decision-making protocols resonate with findings obtained in Western culture , with both male and female participants ( De Dreu et al . , 2016a ) .", "This generates confidence in the generality of the behavioral effects for attack/defense and decision protocols .", "Furthermore , recent work on the role of oxytocin in in-group cooperation suggests similar effects for both male and female participants ( Ten Velden et al . , 2017 ) .", "Thus , and given the absence of strong counter-evidence , we cautiously conclude that current findings may generalize across cultural contexts and apply to male as well as female participants .", "Second , it is unclear how exogenous administration of oxytocin operates at the neurophysiological level .", "Whereas some have questioned whether intranasal administration of oxytocin can have a direct impact on brain and behavior ( Leng and Ludwig , 2016 ) , recent studies in rodents ( Neumann et al . , 2013; Tanaka et al . , 2018 ) , rhesus macaques ( Lee et al . , 2018; Modi et al . , 2014 ) and in humans ( Paloyelis et al . , 2016 ) collectively suggest that intranasal oxytocin elevates brain-level presence of oxytocin , and impacts behavioral decisions through neural networks involved in threat detection and reward processing ( Carter , 2014; Hurlemann et al . , 2010; Ma et al . , 2016b; Rilling et al . , 2012; Stallen et al . , 2012; Wang et al . , 2017; Liu et al . , 2019 ) .", "In addition , there is some evidence that higher levels of oxytocin in saliva , blood , or urine relate to in-group affiliation and cooperation ( Madden and Clutton-Brock , 2011; Samuni et al . , 2017 ) and intergroup discrimination ( Levy et al . , 2016 ) .", "Nevertheless , new research is needed to uncover the neurophysiological pathways through which intranasal oxytocin impact human cognition and behavior , and how findings on intranasal oxytocin relate to endogenous oxytocin measured from saliva , urine , or blood samples .", "Intergroup competition and conflict shape the economic and cultural outlook of groups and societies , and interferes with individual life-trajectories .", "Success and survival in times of intergroup competition and conflict depend on the extent to which individuals make personally costly contributions and , as shown here , on their ability to coordinate their contributions when attacking out-groups , or defending against threatening out-groups .", "Both institutional arrangements , such as leading-by-example , and neurobiological mechanisms , such as oxytocin , facilitate behavioral coordination and the effective exploitation of out-group rivals .", "Indeed , as shown here , providing group members with oxytocin or an explicit coordination device enables them to waste less on failed attacks , and to earn more from their victories ." ], [ "We recruited 486 healthy males , mostly science and engineering students , as paid volunteers .", "One IADC session ( N = 6 ) was dropped from data analysis because of technical failure .", "Data from 480 participants ( 80 IADC sessions , age 18–29 years; M ± SE = 20 . 28±0 . 09 years ) were included in the final data analysis .", "The data analysis on the current study was conducted on a 6-person-group level , thus we conducted sample size estimation by G*Power to determine the number of groups sufficient to detect a reliable effect .", "Based on an estimated average small-to-medium effect size of oxytocin effect on social behaviors ( d = 0 . 28 , Walum et al . , 2016 ) , 80 6-person groups were needed to detect a significant effect ( α = 0 . 05 , β = 0 . 85 , ANOVA: repeated measures , within-between interaction , G-Power , Faul et al . , 2009 ) .", "All participants were healthy and had normal or corrected-to-normal vision and no history of neurological or psychiatric disorders .", "Those who majored in psychology or economics or participated in any other drug study in recent weeks were excluded from participation .", "Participants were instructed to refrain from smoking or drinking ( except water ) for 2 hr before the experiment .", "The experiment involved no deception , and participants were paid for their presence for the experiment ( i . e . $10 show-up fee ) plus their average earnings in two randomly selected IADC rounds .", "All participants provided written informed consent to participate after the experimental procedures had been fully explained , and were acknowledged their right to withdraw at any time during the study .", "All experimental procedures adhered to the standards set by the Declaration of Helsinki and were approved by the Institutional Review Board at the State Key Laboratory of Cognitive Neuroscience and Learning , Beijing Normal University , Beijing , China ( protocol number: ICBIR_A_0107_001 ) .", "Participants were randomly assigned to the intranasal administration of oxytocin or placebo in a double-blind placebo-controlled between-subjects design ( Figure 1 ) .", "For each IADC session , six strangers were invited to the lab at the same time and randomly assigned to six individual cubicles within the same room .", "Upon arrival , participants first completed questionnaires that measured current mood , empathic capacity , prosocial personality , impulsiveness , subjective socio-economic status , and cooperative personality .", "Participants then self-administered oxytocin or placebo .", "After 35 min , participants were given instructions for the IADC game and completed two practice rounds .", "When they also passed a comprehension check , they played 15 simultaneous rounds and 15 sequential rounds of IADC investments ( order of simultaneous and sequential blocks was counterbalanced across sessions , Figure 2 ) .", "All the experimental instructions used neutral language ( e . g . contribution was labeled investment; defense and attack were avoided , and groups were labeled as group A or B ) .", "Finally , participants filled out a post-survey for mood measurement and manipulation check .", "The attacker and defender groups under oxytocin or placebo did not differ in demographic information , mood change , and prosocial-related traits ( Supplementary file 1 , Table 1A and B ) .", "The procedure of oxytocin and placebo administration was similar to previous work that showed oxytocin effects on decision-making behaviors or in-group favoritism ( De Dreu et al . , 2010; Ma et al . , 2015; Rilling et al . , 2012; Yan et al . , 2018 ) .", "A single intranasal dose of 24 IU oxytocin or placebo ( containing the active ingredients except for the neuropeptide ) was self-administered by nasal spray about 35 min before the experimental task under experimenter supervision .", "A 24 IU dosage is the most commonly used dosage in oxytocin literature ( Wang et al . , 2017 ) and recently shown as having more pronounced effects ( compared with 12 or 48 IU dose of oxytocin ) on behavioral and neural responses ( Spengler et al . , 2017 ) .", "The spray was administered to participants three times , and each administration consisted of one inhalation of 4 IU into each nostril .", "Six participants in the same IADC session were assigned to the same treatment ( oxytocin or placebo ) , so as to avoid potential influence of oxytocin to placebo between individuals .", "Data were aggregated to the group level , with Role ( Attacker vs . Defender ) , Procedure ( Simultaneous vs . Sequential ) and Round as within-group variables , and Treatment ( Oxytocin vs . Placebo ) as a between-group factor .", "Analyses were performed on", "( i ) contribution ( the averaged investment of each round , range: 0–20 ) ,", "( ii ) the number of non-contributors ( the number of group members making a 0 contribution across a 15-round block , range: 0–45 ) ,", "( iii ) variance ( within-group variance of each round ) ,", "( iv ) success rate for attack ( range 0–100% ) ,", "( v ) the attackers’ leftovers when losing a conflict ,", "( vi ) the attackers’ spoils when winning a contest and , finally ,", "( vii ) inter-group tracking .", "The within-group variance is calculated for each decision round , thus reveals the group coordination of contribution at each round , with lower variance indicating stronger coordination .", "In addition , to complement and validate these analyses , we analyzed", "( viii ) decision time for investment decisions ( log 10-transformed decision time ) , and", "( ix ) within-group coordination as reflected in the Intra-class correlation coefficient .", "The IADC is an all-pay contest with a single mixed-strategy Nash equilibrium ( Abbink et al . , 2010; Dechenaux et al . , 2015 ) .", "With two three-person groups , each member assumed to have risk-neutral preferences and having a discretionary resource of e = 20 MU to invest from , the IADC game has unique mixed-strategy Nash equilibrium with out-group attack ( in-group defense ) expected to average 10 . 15 ( 9 . 77 ) across rounds , and attackers ( defenders ) should win ( survive ) 32 . 45% ( 67 . 55% ) of the contest rounds ( De Dreu et al . , 2016a ) .", "Across the 15 rounds of simultaneous decision-making under placebo , both out-group attack and in-group defense fell below the Nash-equilibrium ( t ( 39 ) = −11 . 30 , p < 0 . 001 and t ( 39 ) = −4 . 18 , p < 0 . 001 ) .", "Attackers defeated defenders in 22 . 08% ( SE = 2 . 2% ) of their attacks , which is below the Nash success-rate ( t ( 39 ) = −4 . 734 , p < 0 . 001 ) .", "Oxytocin did not influence deviations from rationality ( attack: t ( 39 ) = −15 . 78 , p<0 . 001; defense: t ( 39 ) = −6 . 022 , p<0 . 001; success-rate: t ( 39 ) = −6 . 615 , p < 0 . 001 ) .", "We showed that oxytocin increases the number of non-contributors in attacker groups .", "To further reveal how oxytocin influenced the non-contributing decisions , we examined participants’ decision time by calculating the response time separately for the round that participants decided to or not to contribute .", "This analysis was conducted only on the attacker group because ( 1 ) there were very few rounds ( 9% ) in which defenders decided not to contribute; ( 2 ) oxytocin selectively influenced the number of non-contributors in attacker groups .", "Since the distribution of decision times is heavily right skewed , linear regression is not appropriate .", "Similar to our previous study ( Ma et al . , 2015 ) , we first log 10-transformed decision times in all the analyses that involved decision times .", "The log 10-transformed decision time was submitted into a 2 ( Contribute: Yes vs . No ) × 2 ( Treatment: Oxytocin vs . Placebo ) ANOVA for the attacker group .", "The decisions of which the response time exceeded 180 seconds were excluded in final data analysis ( 0 . 51% of the decisions , due to network problem during the experiment ) .", "To complement and cross-validate the results for within-group variance as an indicator of within-group coordination , we computed another related index — the intra-class correlation .", "The intraclass correlation ( ICC , De Dreu et al . , 2016a; LeBreton and Senter , 2008 ) operates on a data structured as groups , rather than data structured as paired observations .", "ICC describes the amount of statistical interdependence within a group ( group cohesion ) , reflects how strongly individuals’ contributions in all rounds in the same group resemble each other , that is how similar group members are in their contributions to the group pool across rounds .", "Higher ICC values in essence mean group members are more similar to each other in the contributions made to their group pool .", "A Treatment × Role × Procedure ANOVA showed effects for Role ( F ( 1 , 78 ) = 43 . 090 , p < 0 . 001 , η2= 0 . 356 ) , Procedure ( F ( 1 , 78 ) = 166 . 199 , p < 0 . 001 , η2 = 0 . 681 ) and for the Role x Procedure interaction ( F ( 1 , 78 ) = 147 . 586 , p < 0 . 001 , η2 = 0 . 654 ) .", "Fitting the results for within-group variance reported in the Main Text ( Figure 5A ) , results further showed that oxytocin increased attacker groups’ ICC under simultaneous decision-making ( t ( 78 ) = 2 . 057 , p = 0 . 043 , Cohen’s d = 0 . 460 , Figure 5—figure supplement 1; not under sequential block: t ( 78 ) = -0 . 179 , p = 0 . 859 , Cohen’s d = 0 . 040 ) , but did not influence defender groups’ ICC ( simultaneous: t ( 78 ) = 0 . 485 , p = 0 . 629 , Cohen’s d = 0 . 108; sequential: t ( 78 ) = 0 . 389 , p = 0 . 698 , Cohen’s d = 0 . 087 ) .", "To test whether attacker groups made their contributions based on tracking of their rival’s historical level of defense , we built a multiple linear regression of attacker groups’ average contribution on round j ( referred as Aj , with j range from 3 to 15 ) as a function of average level of defense of last rounds ( calculated as ( Dj-1 +Djj-2 ) /2 , regression weight referred as α ) .", "The regression weight α was Fisher’s z transformed for statistical analysis .", "Similar to a previous study ( De Dreu et al . , 2016b ) , we also included another parameter: defense change of the last and before-last rounds , calculated as ( Dj-1 - Dj-2 ) , regression weight referred as β .", "However , the analysis on β failed to show significant main effects of Treatment/Procedure or their interaction ( ps >0 . 1 ) .", "To complement the analysis of attacker groups , we also examined whether and how defender groups tracked the historical level of attack in their rivals .", "This showed that defender groups relied more on α to track attacker groups under simultaneous ( relative to sequential ) decision-making .", "The main effect of Treatment and its interaction with Procedure were not significant ( ps >0 . 05 ) .", "Mediation analysis .", "We performed formal mediation analyses to examine through which route oxytocin increased attacker group’s spoils from winning a conflict .", "Two potential mediators were included in the mediation model: one is tracking coefficient ( α ) the other is within-group variance .", "Four different regression models were constructed , as shown below: ( 1 ) Y=β11X+β10 ( 2 ) M1−β21X+β20 ( 3 ) M1=β31X+β32MI+B30 ( 4 ) Y=β41X+β42MI+B43M2+B40 In these models , X is the independent variable ( Treatment , dummy-coded , 0 for placebo and one for oxytocin ) , M1 is the first mediator ( the weight for attacker group’s tracking of the historical defense , the tracking coefficient , α ) , M2 is a second mediator ( attacker group’s within-group variance ) , and Y is the dependent variable ( attacker groups’ spoils from winning a conflict , reported in the Main Text , and DV with the sum of attacker groups’ spoils from winning a conflict and leftovers from losing a conflict reported in the SI ) .", "A resampling method known as bootstrapping was used to test the direct and indirect path .", "Bootstrapping is a nonparametric approach to effect-size estimation and hypothesis testing that is increasingly recommended for many types of analyses , including mediation ( Mackinnon et al . , 2004; Shrout and Bolger , 2002 ) .", "Rather than imposing questionable distributional assumptions , bootstrapping generates an empirical approximation of the sampling distribution of a statistic by repeated random resampling from the available data , and uses this distribution to calculate p-values and construct confidence intervals ( 5000 resamples were taken for these analyses ) .", "Moreover , this procedure supplies superior CIs that are bias-corrected and accelerated ( Preacher et al . , 2007 ) .", "Results are summarized in Figure 6D , Figure 6—figure supplement 1 , Supplementary file 1 , Table 1C and D . As can be seen , multistep mediational analysis showed that the oxytocin effect on increasing attacker group’s spoils from winning the conflict plus leftovers from losing the conflict was mediated by its effect on increasing tracking of defenders’ history so as to increase within-group coordination ." ] ]

[ "Intergroup conflict contributes to human discrimination and violence , but persists because individuals make costly contributions to their group’s fighting capacity .", "Yet how group members effectively coordinate their contributions during intergroup conflict remains poorly understood .", "Here we examine the role of oxytocin for ( the coordination of ) contributions to group attack or defense in a multi-round , real-time feedback economic contest .", "In a double-blind placebo-controlled study with N=480 males in an Intergroup Attacker-Defender contest game , we found that oxytocin reduced contributions to attack and over time increased attacker’s within-group coordination of contributions .", "However , rather than becoming peaceful , attackers given oxytocin better tracked their rival’s historical defense and coordinated their contributions into well-timed and hence more profitable attacks .", "Our results reveal coordination of contributions as a critical component of successful attacks and subscribe to the possibility that oxytocin enables individuals to contribute to in-group efficiency and prosperity even when doing so implies outsiders are excluded or harmed .", "Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review .", "The Reviewing Editor's assessment is that all the issues have been addressed ( see decision letter ) ." ]

[ "Conflict between groups is a recurring theme in human history .", "We tend to form social bonds with others who share the same characteristics as ourselves , whether that is nationality , ethnicity , or supporting the same football team .", "Individuals that belong to the same group as us comprise our ‘in-group’ .", "All other individuals make up our ‘out-groups’ .", "Competition and conflict with out-groups – from benign sporting rivalry to warfare – has a key role in shaping human cultures and societies .", "Such conflict often requires individuals to act in ways that harm their own self-interests .", "It also requires them to coordinate their actions with other members of their in-group .", "How does our biology drive this behavior ?", "When small groups prepare for conflict with other groups , they often perform social bonding routines and rituals .", "These trigger the brain to release a hormone called oxytocin into the bloodstream .", "Known as the ‘love hormone’ , oxytocin helps promote pair bonding as well as social bonding with in-group members .", "Studies in both humans and monkeys show that boosting oxytocin levels artificially via a nasal spray makes individuals more trusting and cooperative .", "But Zhang et al . now show that the ‘love hormone’ also helps individuals launch more coordinated ‘attacks’ on out-groups .", "In a study involving a multi-round economic contest game between groups of ‘attackers’ and ‘defenders’ , oxytocin did not make attackers less aggressive .", "Instead it enabled them to better coordinate their attacks .", "Each contest game involved three attackers individually contributing money to a group pool to outbid the other group and win more money , and three defenders making similar contributions to their own group pool to defend it against the rivals’ attacks and protect themselves from losing all their money .", "Attackers who used an oxytocin nasal spray were better at tracking their rivals' defensive strategies than attackers whose nasal spray contained a placebo .", "Under the influence of oxytocin , the attackers timed their strikes to occur when their rivals were vulnerable .", "Over time , the oxytocin users became better at coordinating their behavior with other members of their in-group .", "This resulted in more earnings .", "Success – and even survival – in intergroup conflicts depends on how willing individuals are to make contributions that incur a personal cost .", "They also depend on how well individuals coordinate their contributions .", "Social strategies , such as leading by example , and neurobiological mechanisms such as oxytocin can both help achieve the coordination needed to exploit out-group rivals ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "plant biology", "evolutionary biology" ]

[ [ "Plant chemical defenses play a central role in the coevolutionary arms race with herbivorous insects .", "In response to diverse environmental challenges , plants have evolved a plethora of structurally diverse organic compounds with repellent , antinutritive , or toxic properties ( Fraenkel , 1959; Mithöfer and Boland , 2012 ) .", "Chemical defenses can impose barriers to consumption by herbivores , but in parallel may favor the evolution of specialized herbivores that can tolerate or disable these defenses ( Cornell and Hawkins , 2003 ) .", "Chemical diversity is likely evolving in response to a multitude of plant-herbivore interactions ( Salazar et al . , 2018 ) , and community-level phytochemical diversity may be a key driver of niche segregation and insect community dynamics ( Richards et al . , 2015; Sedio et al . , 2017 ) .", "For individual plants , the production of diverse mixtures of chemicals is often considered advantageous ( Romeo , 1996; Firn and Jones , 2003; Gershenzon et al . , 2012; Forbey et al . , 2013; Richards et al . , 2016 ) .", "For example , different chemicals may target distinct herbivores ( Iason et al . , 2011; Richards et al . , 2015 ) , or may act synergistically to increase overall toxicity of a plant ( Steppuhn and Baldwin , 2007 ) .", "However , metabolic constraints can limit the extent of phytochemical diversity within individual plants ( Firn and Jones , 2003 ) .", "Most defensive metabolites originate from a small group of precursor compounds and conserved biosynthetic pathways , which are modified in a hierarchical process into diverse , species-specific end products ( Moore et al . , 2014 ) .", "As constraints are likely strongest for the early stages of these pathways , related plant species commonly share the same functional ‘classes’ of defensive chemicals ( Wink , 2003 ) , but vary considerably in the number of compounds within each class ( Fahey et al . , 2001; Rasmann and Agrawal , 2011 ) .", "Functional conservatism in defensive chemicals among related plants should facilitate host expansion and the evolution of tolerance in herbivores ( Cornell and Hawkins , 2003 ) , as specialized resistance mechanisms against one type of compound are more likely to be effective against structurally similar than structurally dissimilar compounds .", "This may result in a seemingly paradoxical scenario , wherein well-defended plants are nonetheless attacked by a diverse community of specialized herbivores ( Agrawal , 2005; Bidart-Bouzat and Kliebenstein , 2008 ) .", "For example , most plants in the Brassicaceae produce glucosinolates as their primary defense , which upon activation by myrosinase ( thioglucoside glucohydrolase ) enzymes upon leaf damage become potent repellents of many herbivores ( Fahey et al . , 2001 ) .", "However , despite the potency of this defense system and the large diversity of glucosinolates produced by the Brassicaceae , several specialized herbivores have evolved strategies to overcome this defense , enabling them to consume most Brassicaceae and even to sequester glucosinolates for their own defense against predators ( Müller , 2009; Winde and Wittstock , 2011 ) .", "Plants may occasionally overcome the constraints on functional diversification and gain the ability to produce new classes of defensive chemicals as a ‘second line of defense’ ( Feeny , 1977 ) .", "Although this phenomenon is likely widespread across the plant kingdom , it has most commonly been reported from the well-studied Brassicaceae .", "In addition to producing evolutionarily ancestral glucosinolates , plants in this family have gained the ability to produce saponins in Barbarea vulgaris ( Shinoda et al . , 2002 ) , alkaloids in Cochlearia officinalis ( Brock et al . , 2006 ) , cucurbitacins in Iberis spp .", "( Nielsen , 1978b ) , alliarinoside in Alliaria petiolata ( Frisch and Møller , 2012 ) , and cardenolides in the genus Erysimum ( Makarevich et al . , 1994 ) .", "These recently-evolved chemical defenses with modes of action distinct from glucosinolates have likely allowed the plants to escape attack from specialized , glucosinolate-adapted herbivores ( Nielsen , 1978b; Dimock et al . , 1991; Haribal and Renwick , 2001; Shinoda et al . , 2002 ) .", "Gains of novel defenses are expected to result in a release from selective pressures imposed by specialized antagonists , and thus may represent key steps in herbivore-plant coevolution that lead to rapid phylogenetic diversification ( Weber and Agrawal , 2014 ) .", "The production of cardenolides by species in the genus Erysimum is one of the longest- and best-studied examples of an evolutionarily recent gain of a novel chemical defense ( Jaretzky and Wilcke , 1932; Nagata et al . , 1957; Singh and Rastogi , 1970; Makarevich et al . , 1994 ) .", "Cardenolides are a type of cardiac glycoside , which act as allosteric inhibitors of Na+/K+-ATPase , an essential membrane ion transporter that is expressed ubiquitously in animal cells ( Agrawal et al . , 2012 ) .", "Cardiac glycosides are produced by plants in approximately sixty genera belonging to twelve plant families , and several cardiac glycoside-producing plants are known for their toxicity or medicinal uses ( Agrawal et al . , 2012; Züst et al . , 2018 ) .", "Erysimum is a species-rich genus consisting of diploid and polyploid species with diverse morphologies , growth habits , and ecological niches ( Al-Shehbaz , 1988; Polatschek and Snogerup , 2002; Al-Shehbaz , 2010; Gómez et al . , 2015 ) .", "Of the Erysimum species evaluated to date , all produce some of the novel cardenolide defenses ( Makarevich et al . , 1994 ) .", "Previous phylogenetic studies suggest a recent and rapid diversification of the genus , with most species divergence occurring within the last 2–3 million years ( Gómez et al . , 2014; Moazzeni et al . , 2014 ) , resulting in 150 to 350 extant species ( Polatschek and Snogerup , 2002; Al-Shehbaz , 2010 ) .", "The large uncertainty in species number reflects taxonomic challenges in this genus , which includes many species that readily hybridize , as well as cryptic species with near-identical morphology ( Abdelaziz et al . , 2011 ) .", "In most Erysimum species , cardenolides appear to have enabled an escape from at least some glucosinolate-adapted specialist herbivores .", "Cardenolides in Erysimum act as oviposition and feeding deterrents for different pierid butterflies ( Chew , 1975; Chew , 1977; Wiklund et al . , 1978; Renwick et al . , 1989; Dimock et al . , 1991 ) , and several glucosinolate-adapted beetles ( Phaedon spp . and Phyllotreta spp . ) were deterred from feeding by dietary cardenolides at levels commonly found in Erysimum ( Nielsen , 1978a; Nielsen , 1978b ) .", "Nonetheless , Erysimum plants are still attacked by a range of herbivores and seed predators , including some mammals and several glucosinolate-adapted aphids , true bugs , and lepidopteran larvae ( Gómez , 2005; Züst et al . , 2018 ) .", "These herbivores likely rely on general detoxification mechanisms for tolerance of the novel defense , while to date there are no reports of de novo gains of specialized cardenolide resistance in Erysimum herbivores .", "However , the gain of the novel defense may have facilitated host shifts in at least one cardenolide-adapted herbivore: in addition to its main host Digitalis purpurea ( Plantaginaceae ) , the seed-feeding bug Horvathiolus superbus ( Lygaeinae ) commonly feeds on seeds of E . crepidifolium , and is able to sequester cardenolides from both sources ( Georg Petschenka , personal observations ) .", "The gain of a novel chemical defense makes the genus Erysimum an excellent model system to study the causes and consequences of phytochemical diversification ( Züst et al . , 2018 ) .", "While an increasing number of studies are beginning to describe taxon-wide patterns of chemical diversity in plants ( e . g . , Richards et al . , 2015; Sedio et al . , 2017; Salazar et al . , 2018 ) , the Erysimum system is unique in combining two classes of plant metabolites with primarily defensive function – although a broader role of glucosinolates is increasingly recognized ( e . g . , Katz et al . , 2015 ) .", "The system thus is ideally suited to evaluate the evolutionary consequences of co-expressing two functionally distinct but potentially redundant defenses .", "Here , we present a high-quality genome sequence assembly and annotation for the short-lived annual E . cheiranthoides as an important resource for future molecular studies in this system .", "Furthermore , we present a new phylogeny for 48 species constructed from transcriptome sequences ( Figure 1 ) , corresponding to 10–30% of species in the genus Erysimum .", "We combine this phylogeny with a characterization of the full diversity of glucosinolates and cardenolides in leaves to evaluate macroevolutionary patterns in the evolution of phytochemical diversity across the genus .", "We complemented the characterization of defensive phenotypes by quantifying glucosinolate-activating myrosinase activity , inhibition of animal Na+/K+-ATPase by leaf extracts , and defense inducibility in response to exogenous application of jasmonic acid ( JA ) .", "By assessing co-variation of diversity , abundance and inducibility of ancestral and novel defenses , we provide evidence that these two classes of defense metabolites evolved in response to different selective pressures and appear to have specific , non-redundant functions ." ], [ "A total of 39 . 5 Gb of PacBio sequences with an average read length of 10 , 603 bp were assembled into 1087 contigs with an N50 of 1 . 5 Mbp ( Table 1 ) .", "Hi-C scaffolding oriented 98 . 5% of the assembly into eight large scaffolds representing pseudomolecules ( Table 1 , Figure 2—figure supplement 1 ) , while 216 small contigs remained unanchored .", "The final assembly ( v1 . 2 ) had a total length of 174 . 5 Mbp , representing 86% of the estimated genome size of E . cheiranthoides and capturing 99% of the BUSCO gene set ( Table 1 , Figure 2—figure supplement 2 ) .", "Sequences were deposited under GenBank project ID PRJNA563696 and additionally are provided at www . erysimum . org .", "A total of 29 , 947 gene models were predicted and captured 98% of the BUSCO gene set ( Figure 2—figure supplement 3 ) .", "In the presumed centromere regions of each chromosome , genic sequences were less abundant , whereas repeat sequences were more common ( Figure 2A ) .", "Repetitive sequences constituted approximately 29% of the genome ( Supplementary file 2 ) .", "Long terminal repeat retrotransposons ( LTR-RT ) made up the largest proportion of the repeats identified ( Figure 2—figure supplement 4 ) .", "Among these , repeats in the Gypsy superfamily constituted the largest fraction of the genome ( Supplementary file 2 ) .", "The majority of the LTR elements appeared to be relatively young , with most having estimated insertion times of less than 1 MYA ( Figure 2—figure supplement 5 ) .", "Synteny analysis showed evidence of several chromosomal fusions and fissions between the eight chromosomes of E . cheiranthoides and the five chromosomes of Arabidopsis ( Figure 2B ) .", "Three aliphatic glucosinolates – glucoiberverin ( 3-methylthiopropyl glucosinolate ) , glucoiberin ( 3-methylsulfinylpropyl glucosinolate ) , and glucocheirolin ( 3-methylsulfonylpropyl glucosinolate ) – have been reported as the main glucosinolates in E . cheiranthoides ( Cole , 1976; Huang et al . , 1993 ) .", "We confirmed their dominance in E . cheiranthoides var .", "Elbtalaue , but also identified additional aliphatic and indole glucosinolates at lower concentrations .", "By making use of the known glucosinolate biosynthetic genes from Arabidopsis ( Hull et al . , 2000; Mikkelsen et al . , 2000; Bak and Feyereisen , 2001; Bak et al . , 2001; Kliebenstein et al . , 2001b; Kroymann et al . , 2001; Reintanz et al . , 2001; Chen et al . , 2003; Kroymann et al . , 2003; Naur et al . , 2003; Grubb et al . , 2004; Mikkelsen et al . , 2004; Piotrowski et al . , 2004; Textor et al . , 2004; Nozawa et al . , 2005; Klein et al . , 2006; Schuster et al . , 2006; Hansen et al . , 2007; Textor et al . , 2007; Hansen et al . , 2008; Knill et al . , 2008; Li et al . , 2008; Farquharson , 2009; Geu-Flores et al . , 2009; Gigolashvili et al . , 2009; He et al . , 2009; Klein and Papenbrock , 2009; Knill et al . , 2009; Pfalz et al . , 2009; Sawada et al . , 2009; He et al . , 2010; Geu-Flores et al . , 2011; He et al . , 2011; Pfalz et al . , 2011; Lächler et al . , 2015; Kong et al . , 2016; Pfalz et al . , 2016 ) , BLASTn comparisons to the E . cheiranthoides genome , and creating phylogenetic trees to compare nucleotide coding sequences of Arabidopsis , Brassica , and E . cheiranthoides , we identified homologs of genes encoding both indole ( Figure 3 , Figure 3—figure supplements 1–7 ) and aliphatic ( Figure 4—figure supplements 1–8 ) glucosinolate biosynthetic enzymes .", "Homologs of all genes of the biosynthetic pathway for glucobrassicin ( indol-3-ylmethyl glucosinolate ) and its 4-hydroxy and 4-methoxy derivatives were present in E . cheiranthoides ( Figure 3 ) .", "Consistent with the absence of neoglucobrassicin ( 1-methoxy-indol-3-ylmethyl glucosinolate ) in E . cheiranthoides var .", "Elbtalaue , we did not find homologs of the Arabidopsis genes encoding the biosynthesis of this compound .", "Genes encoding the complete biosynthetic pathway of the E . cheiranthoides aliphatic glucosinolates glucoiberverin , glucoiberin , glucoerucin ( 4-methylthiobutyl glucosinolate ) , and glucoraphanin ( 4-methylsulfinylbutyl glucosinolate ) were present in the genome ( Figure 4 , Figure 4—figure supplements 1–6 ) .", "Because the E . cheiranthoides methylsulfonyl glucosinolates glucocheirolin , glucoerysolin ( 4-methylsulfonylbutyl glucosinolate ) , and 3-hydroxy-4-methylsulfonylbutyl glucosinolate are not present in Arabidopsis , genes encoding their biosynthesis are unknown and could not be identified as part of this study .", "The E . cheiranthoides genome contains genes with similarity to Arabidopsis ALKENYL HYDROXALKYL PRODUCING ( AOP2 and AOP3 ) , and 3-BUTENYL GLUCOSINOLATE 2-HYDROXYLASE ( GS-OH ) ( Figure 4—figure supplements 7 and 8 ) .", "However , the apparent absence of sinigrin ( 2-propenyl glucosinolate ) , 2-hydroxypropyl glucosinolate , progoitrin ( 2-hydroxy-3-butenyl glucosinolate ) , and 4-hydroxybutyl glucosinolate in E . cheiranthoides ( Figure 4 ) , suggests that the encoded enzymes of these genes have other functions .", "CYP79A2 , which functions in the biosynthesis of benzyl glucosinolates that are present in very small amounts in seeds of Arabidopsis ecotype Columbia ( Wittstock and Halkier , 2000 ) , has a homolog in the E . cheiranthoides genome ( Figure 3—figure supplement 1 ) .", "Although we did not observe benzyl glucosinolates in E . cheiranthoides , this lack of detection could be due to assay sensitivity or not testing all tissue types .", "Homologs of the Arabidopsis CYP79C1 and CYP79C2 genes , which have unknown functions but are hypothesized to be involved in glucosinolate biosynthesis ( Halkier and Gershenzon , 2006 ) , are present in the E . cheiranthoides genome ( Figure 3—figure supplement 1 ) .", "CYP79D2 from cassava catalyzed the formation of valine- and isoleucine-derived glucosinolates in Arabidopsis ( Mikkelsen and Halkier , 2003 ) , yet no CYP79D genes appear to be present in E . cheiranthoides ( Figure 3—figure supplement 1 ) .", "Additionally , there was no clear E . cheiranthoides homolog of GLUCORAPHASATIN SYNTHASE 1 ( GRS1 , Figure 4—figure supplement 9 ) , a 2-oxoglutarate-dependent dioxygenase that contributes to glucoraphasatin ( 4-methylthio-3-butenyl glucosinolate ) biosynthesis in R . sativus ( Kakizaki et al . , 2017 ) .", "In response to insect feeding or pathogen infection , glucosinolates are activated by myrosinase enzymes ( Halkier and Gershenzon , 2006 ) .", "Between-gene phylogenetic comparisons revealed that homologs of known Arabidopsis myrosinases , the main foliar myrosinases TGG1 and TGG2 ( Xue et al . , 1995; Barth and Jander , 2006 ) , root-expressed TGG4 and TGG5 ( Andersson et al . , 2009 ) , and likely pseudogenes TGG3 and TGG6 ( Rask et al . , 2000; Zhang et al . , 2002 ) , were also present in the E . cheiranthoides genome ( Figure 4—figure supplement 10 ) .", "Additionally , we found homologs of the more distantly related Arabidopsis myrosinases PEN2 ( Bednarek et al . , 2009; Clay et al . , 2009 ) and PYK10 ( Sherameti et al . , 2008; Nakano et al . , 2017 ) .", "In Arabidopsis , protein products of epithiospecifier protein ( ESP ) , epithiospecifier modifier ( ESM ) , and nitrile specifier protein ( NSP ) direct glucosinolate breakdown into nitriles , thiocyanates , or isothiocyanates ( Lambrix et al . , 2001; Burow et al . , 2006; Zhang et al . , 2006 ) .", "Although we did not measure glucosinolate breakdown in Erysimum , we did find ESP , ESM , and NSP homologs in the E . cheiranthoides genome ( Figure 4—figure supplements 11 and 12 ) .", "Therefore , the pathway of glucosinolate activation appears to be largely conserved between Arabidopsis and E . cheiranthoides .", "Assemblies of transcriptomes from 48 Erysimum species ( including E . cheiranthoides ) had N50 values ranging from 574 to 2 , 160 bp ( Supplementary file 3 ) .", "Transcriptome assemblies contained completed genes from 54–94% of the BUSCO set and coding sequence lengths were generally shorter on average than the E . cheiranthoides coding sequence lengths ( Supplementary file 3 ) .", "Transcriptome sequences were deposited under GenBank project ID PRJNA563696 and at www . erysimum . org .", "The large number of orthologous gene sequences identified among the E . cheiranthoides genome and the 48 transcriptomes resulted in an ASTRAL species tree with high posterior probabilities for most nodes ( Figure 5—figure supplement 1 , Supplementary file 4 ) .", "To determine divergence times among the 48 species , we generated a chronogram using a concatenated ExaML species tree with branch length information ( Figure 5 ) .", "While we relied on published estimates to constrain ages of several internal nodes , our analysis aligns well with a recent , rapid radiation of the species included in our study within the last 2–4 Mya ( Figure 5 ) .", "The concatenated ExaML species tree and the ASTRAL species tree shared overall similar topologies , but very short internal branch lengths on both trees indicated high levels of gene tree discordance .", "We further dissected this discordance by assessing support of the main topology of the ASTRAL species tree ( Figure 5—figure supplement", "1 ) using quartet scores , which compare the main tree topology relative to its first and second alternative topology .", "For most nodes in the ingroup , each topology had quartet scores near the minimum value of 1/3 ( Supplementary file 4 ) , indicating that the possible gene trees were present in almost equal frequency for each topology .", "For the ExaML tree , we assessed discordance at each node using concordance factors , which are the proportion of gene trees that agree with the main topology ( Figure 5 ) .", "Again , most nodes in the ingroup had very low support for the main topology ( <5% of gene tree agreement; Figure 5 , Supplementary file 5 ) .", "This suggests that many internal branches had lengths near 0 , indicating polytomies that could not be resolved even with the extensive sampling of gene sequences from transcriptome data .", "These high levels of discordance were likely caused by frequent polyploidization ( Figure 5 ) , incomplete lineage sorting , and high degrees of hybridization .", "A high prevalence of hybridization was further indicated by a high frequency of gamma scores ( hybridization proportion ) between 0 . 3 and 0 . 7 across all ingroup taxa ( Figure 5—figure supplement", "2 ) recovered in the HyDe analysis ( Blischak et al . , 2018 ) .", "Despite extensive levels of discordance and low agreement of individual gene trees with the species trees , the main topologies of the ExaML and ASTRAL species trees revealed geographic clades that matched the generally limited native species ranges .", "The three Mediterranean annual species E . incanum ( INC ) , E . repandum ( REP ) , and E . wilczekianum ( WIC ) formed a well-supported monophyletic sister clade to all other sequenced species ( Figure 5 , Figure 5—figure supplement 1 ) .", "The only other annual in the set of sampled species , E . cheiranthoides ( ECE ) , was part of a weakly-supported clade ( high posterior probability but low concordance ) , comprised of several perennial species from Greece and central Europe , including the widespread ornamental E . cheiri ( CHR ) .", "Species from the Iberian peninsula/Morocco , North America , and Iran formed additional , weakly-supported clades conserved between species trees ( Figure 5 ) , while another clade of Turkish and Greek Erysimum species was only monophyletic in the ASTRAL species tree ( Figure 5—figure supplement 1 ) .", "The clear geographic structure in the main topologies of the species trees was confirmed by a strong correlation between the cophenetic and geographic distance matrices for the subset of 43 species with geographic information ( Mantel test , p<0 . 001 ) .", "Across the 48 Erysimum species , we identified 25 candidate glucosinolate compounds with distinct molecular masses and HPLC retention times ( Supplementary file 6 ) .", "Of these , 24 compounds could be assigned to known glucosinolate structures with high certainty .", "The last remaining compound appeared to be an unknown isomer of glucocheirolin .", "Individual Erysimum species produced between 5 and 18 glucosinolates ( Figure 6A ) , and total glucosinolate concentrations were highly variable among species ( Figure 6B ) .", "The ploidy level of species explained a significant fraction of total variation in the number of glucosinolates produced ( F4 , 38 = 4 . 63 , p=0 . 004 ) , with hexaploid species producing the highest number of compounds ( Figure 6—figure supplement 1 ) .", "However , neither the number of distinct glucosinolate compounds nor their total concentrations exhibited a phylogenetic signal , and related species were less similar than expected under a model of Brownian motion ( Table 2 ) .", "Clustering species by dissimilarities in glucosinolate profiles mostly resulted in chemotype groups corresponding to known underlying biosynthetic genes , although support for individual species clusters in the chemogram was variable ( Figure 7 ) .", "The majority of all species produced glucoiberin as the primary glucosinolate .", "Of these , approximately half also produced sinigrin as a second dominant glucosinolate compound .", "Further chemotypic subdivision , related to the production of glucocheirolin and 2-hydroxypropyl glucosinolate , appeared to be present but only had relatively weak statistical support .", "However , eight species clearly differed from these general patterns .", "The species E . allionii ( ALI ) , E . rhaeticum ( RHA ) , and E . scoparium ( SCO ) mostly lacked glucosinolates with 3-carbon side-chains , but instead accumulated glucosinolates with 4- , 5- and 6-carbon side-chains .", "The two closely related species E . odoratum ( ODO ) and E . witmannii ( WIT ) predominantly accumulated indole glucosinolates , while E . collinum ( COL ) , E . pulchellum ( PUL ) , and accession ER2 predominantly produced glucoerypestrin ( 3-methoxycarbonylpropyl glucosinolate ) , a glucosinolate that is exclusively found within Erysimum ( Fahey et al . , 2001 ) .", "Similar to the lack of phylogenetic signal for compound numbers and concentrations , dissimilarity in glucosinolate profiles was unrelated to phylogenetic relatedness ( Mantel test , p=0 . 331 ) , and neither of the first two principal coordinates of the glucosinolate dissimilarity matrix showed a significant phylogenetic signal ( Table 2 ) .", "The lack of phylogenetic signal was visualized by optimizing vertical matching of tips between the ExaML species tree and the glucosinolate chemogram ( Figure 5—figure supplement 3A ) .", "For five species pairs , the closest phylogenetic neighbor was also the most chemically similar species , but in general , close relatives more often belonged to chemically distant species clusters .", "Finally , reconstruction of the ancestral states for total glucosinolate content and the first principal coordinate of the glucosinolate dissimilarity matrix suggests that both traits likely originated at intermediate levels and repeatedly evolved towards opposite extremes in closely related species ( Figure 5—figure supplement 4 ) .", "As glucosinolates require activation by myrosinase enzymes upon tissue damage by herbivores , myrosinase activity in leaf tissue determines the rate at which toxins are released .", "We quantified myrosinase activity of Erysimum leaf extracts and found it to be highly variable among species ( Figure 6C ) .", "After grouping species into nine chemotypes defined by chemical dissimilarity and the production of characteristic glucosinolate compounds ( Figure 7C ) , we found that myrosinase activity significantly differed among these chemotypes ( Figure 8 , F8 , 33 = 8 . 31 , p<0 . 001 ) .", "Chemotypes that predominantly accumulated methylsulfonyl glucosinolates , hydroxy glucosinolates , or indole glucosinolates had low to negligible activity against the assayed glucosinolate sinigrin .", "It is important to note that sinigrin is an alkenyl glucosinolate and Erysimum myrosinases targeting other , structurally dissimilar glucosinolates may not effectively cleave sinigrin .", "After chemotype differences were accounted for , myrosinase activity was marginally related to total glucosinolate concentrations ( F1 , 33 = 3 . 60 , p=0 . 067 ) .", "Similar to other glucosinolate traits , uncorrected myrosinase activity exhibited no phylogenetic signal ( Table 2 ) .", "With the exception of E . collinum ( COL ) , which only contained trace amounts of cardenolides in leaves , all Erysimum species contained diverse mixtures of cardenolide compounds and accumulated considerable amounts of cardenolides ( Figure 6D–E ) .", "The ploidy level of species again explained a significant fraction of the total variation in the number of cardenolides ( F4 , 38 = 3 . 47 , p=0 . 016 ) , with hexaploid species producing the highest average number of compounds ( Figure 6—figure supplement 1 ) .", "To obtain an estimate of biological activity and evaluate quantification from total MS ion counts , we used an established assay that quantifies cardenolide concentrations from specific inhibition of animal Na+/K+-ATPase by crude Erysimum leaf extracts .", "We found generally strong enzymatic inhibition , with leaves of Erysimum species on average containing an equivalent of 5 . 72 ± 0 . 12 µg mg−1 ( ±1 SE ) of the reference cardenolide ouabain .", "Despite only producing trace amounts of cardenolides , E . collinum ( COL ) extracts caused significantly stronger inhibition than the Brassicaceae control plant , S . arvensis ( Figure 6—figure supplement 2 ) .", "Overall , quantification of cardenolide concentrations by Na+/K+-ATPase inhibition was highly correlated with the total MS ion count ( Figure 6—figure supplement 2 , r = 0 . 95 , p<0 . 001 ) .", "Thus , the use of ion count data for cross-species comparisons was appropriate for this purpose .", "Both the total numbers of compounds and the total abundances exhibited a strong phylogenetic signal ( Table 2 ) , indicating that closely related species shared similar cardenolide traits .", "Cardenolide diversity was considerably higher than that of glucosinolates , with a total of 97 distinguishable candidate cardenolide compounds identified across the 48 Erysimum species ( two compounds were later excluded , leaving 95 compounds; Supplementary file 7 ) .", "Of these , 46 compounds had distinct molecular masses and mass fragments , while the remaining compounds likely were isomers , sharing a molecular mass with other compounds but having distinct HPLC retention times .", "The 95 putative cardenolides comprised nine distinct genins ( Figure 9 , Figure 9—figure supplement 1 ) , the majority of which were glycosylated with digitoxose , deoxy hexoses , xylose , or glucose moieties .", "Only digitoxigenin and cannogenol accumulated as free genins , while all other compounds occurred as either mono- or di-glycosides .", "A likely major source of isomeric cardenolide compounds was thus the incorporation of different deoxy hexoses of equivalent mass , such as rhamnose , fucose , or gulomethylose .", "A subset of compounds had molecular masses that were heavier by 42 . 011 m/z than known mono- or di-glycoside cardenolides .", "Such a gain in mass corresponds to the gain of an acetyl-group , and mass fragmentation patterns indicated that these compounds were acetylated on the first sugar moiety ( Supplementary file 7 ) .", "Out of the nine detected genins , six had previously been described from Erysimum species ( Makarevich et al . , 1994 ) .", "In addition , we identified three previously undescribed mass features with fragmentation patterns characteristic of cardenolide genins ( Figure 9—figure supplement 1 ) .", "Of these three , one matched an acetylated cannogenol , one matched formylated cannogenol , and one matched formylated nigrescigenin , assuming acetylation/formylation of a free OH-group on the precursor molecule .", "Formyl adducts can sometimes be formed during LC-MS due to the addition of formic acid to solvents , although this is less common with positive ionization .", "To exclude the possibility that these were technical artefacts , we analyzed a subset of extracts by LC-MS without the addition of formic acid and found both formyl-genins at comparable concentrations ( Figure 9—figure supplement 2 ) .", "We therefore assume that all three novel structures are natural variants of cardenolides produced by Erysimum plants , even though we currently lack final structural elucidation .", "Clustering of species by dissimilarities in cardenolide profiles revealed fewer obvious species clusters in the chemogram than for glucosinolates , and particularly higher-level species clusters had only weak statistical support ( Figure 10 ) .", "A clear exception to this was a species cluster that included E . cheiranthoides ( ECE ) and E . sylvestre ( SYL ) , which were characterized by a chemotype lacking several otherwise common cannogenol- and strophanthidin-glycosides , while accumulating unique digitoxigenin-glycosides .", "A second major cluster was visually apparent , yet not statistically significant , and separated groups of species that did or did not produce glycosides of the newly discovered putative formyl-nigrescigenin ( Figure 10 ) .", "Similarity in cardenolide profiles among species was strongly correlated with phylogenetic relatedness ( Mantel test , p<0 . 001 ) , and the first two principal coordinates of the Bray-Curtis dissimilarity matrix exhibited strong phylogenetic signals ( Table 2 ) .", "Closely related species were therefore not only more similar in their total cardenolide concentrations , but also had more similar cardenolide profiles than expected by chance .", "These results were again visualized by optimizing vertical matching of tips between the ExaML species tree and the cardenolide chemogram ( Figure 5—figure supplement 3B ) .", "For twelve species pairs ( half of all species in our phylogeny ) , the closest phylogenetic neighbor was also the most chemically similar species , and phylogenetically related species more commonly belonged to chemically similar species clusters , indicated by a significantly lower total length of tip links compared to what was observed for glucosinolates ( Figure 5—figure supplement 3B ) .", "Reconstruction of the ancestral states for total cardenolide content suggests that trait values likely originated at low total concentrations , and increased to intermediate levels in the North American and Spanish/Moroccan clades , and independently to very high levels in the species pair of E . horizontale and E . crepidifolium ( Figure 5—figure supplement 4 ) .", "For the first principal coordinate of cardenolide dissimilarity , trait values originated at intermediate values , but sub-clades more commonly evolved towards shared chemical profiles than was the case for glucosinolates ( Figure 5—figure supplement 4 ) .", "Similarity in glucosinolate and cardenolide chemical profiles of the 48 species was not correlated ( Mantel test , p=0 . 171 ) , and neither the number of compounds ( PGLS: F1 , 46 = 0 . 09 , p=0 . 771 ) nor their total concentrations ( PGLS: F1 , 46 = 0 . 51 , p=0 . 478 ) were correlated between compound classes .", "Tip-specific estimates of speciation rates were not correlated with the number of glucosinolate compounds produced by a species , regardless of speciation rate metric or statistical method used ( Table 3 ) .", "In contrast , we found a significantly positive correlation between the node density ( ND ) measure and the number of cardenolide compounds , while for the alternate equal split ( ES ) measure the correlation was marginally significant for the simulation-based method only ( Table 3 ) .", "Given the correlation coefficients of the simulation-based method , variation in the number of cardenolide compounds thus explained 17–28% of the total variation in speciation rate .", "Variation in total glucosinolate or cardenolide concentrations was not correlated with speciation rates ( Table 3 ) .", "Foliar application of JA was expected to stimulate accumulation of defensive compounds in plant leaves and among the 30 tested species , glucosinolate levels responded positively to JA , with the majority of species increasing their foliar glucosinolate concentration ( Figure 11 ) .", "However , the glucosinolate inducibility of a species was independent of constitutive glucosinolate levels ( PGLS: F1 , 28 = 0 . 17 , p=0 . 680 ) .", "By contrast , the majority of species exhibited lower cardenolide levels in response to JA , resulting in lack of inducibility across species ( Figure 11 ) .", "The species E . crepidifolium ( CRE ) heavily influenced inducibility patterns , as it not only had three times higher constitutive concentrations of cardenolides than any other Erysimum species , but also markedly increased both glucosinolate and cardenolide concentrations in response to JA treatment ( Figure 11 ) .", "When this outlier species was removed , inducibility ( or suppression ) of foliar cardenolides was not correlated with constitutive cardenolide levels ( PGLS: F1 , 27 = 0 . 20 , p=0 . 657 ) , and inducibilities of glucosinolates and cardenolides were likewise not correlated with each other ( PGLS: F1 , 27 = 0 . 36 , p=0 . 551 ) ." ], [ "The genus Erysimum poses considerable phylogenetic challenges , with its evolutionary recent radiation resulting in a large number of hybridizing species and high prevalence of polyploidization ( Polatschek and Snogerup , 2002; Marhold and Lihová , 2006; Al-Shehbaz , 2010 ) .", "Both previous partial phylogenies of the genus , constructed from internal transcribed spacer ( ITS ) or chloroplast sequences , consequently struggled to resolve polytomies among species ( Gómez et al . , 2014; Moazzeni et al . , 2014 ) .", "Here , we attempted to construct a better-resolved phylogeny from 9868 orthologous genes extracted from transcriptome sequences .", "However , while our species tree provided good posterior probabilities for all nodes , it also revealed very high levels of gene discordance .", "Several internal nodes of our species tree topology were supported by less than 1% of all gene trees , and only the most recent branching events were supported by more than 10% of gene trees .", "High levels of discordance , likely driven by introgression and incomplete lineage sorting , are common during ongoing species radiations , with many recent plant examples reporting similar findings ( Novikova et al . , 2016; Pease et al . , 2016; Copetti et al . , 2017; Wu et al . , 2018 ) .", "The abundance of polyploid species in our phylogeny ( at least 21 out of 48 , Figure 5 ) may have further exacerbated levels of discordance .", "Specifically , if these are allopolyploid rather than autopolyploid species , discordance could be introduced by our methodological approach for gene selection , which randomly retained only a single copy for each identified orthologous gene with multiple copies .", "This same problem could also have inflated the estimation of hybridization by our HyDe analysis , although a high rate of gene flow is likely , at least among geographically close species ( Abdelaziz et al . , 2014 ) .", "More fundamentally , the high levels of gene discordance also highlight the limitations of simple bifurcating species trees to represent the significantly more complicated network of splits and reticulate evolutionary events that is likely the true history of the genus Erysimum ( Marhold and Lihová , 2006 ) .", "However , while we have been unsuccessful in reconstructing the exact evolutionary history of the genus , we nevertheless believe our species tree captures key aspects of its phylogenetic relationships , particularly in respect to closely related species and geographic clades .", "In our species tree , the three annual species , E . repandum , E . incanum , and E . wilczekianum grouped together as a well-supported monophyletic clade sister to all other Erysimum species .", "These species co-occur geographically with several perennial Erysimum species , but they are largely isolated by non-overlapping flowering times .", "Further separate clades were present for species from Iran , North America , and a combined clade of species from Spain , Morocco , and the Canary Islands , while the remaining species grouped into four central and eastern European clades .", "Within the Spanish clade , species from southeastern Spain exhibited closer relatedness to Moroccan species than to species from northeastern or northwestern Spain , loosely matching more fine-scale evaluations of species relatedness in this region ( Abdelaziz et al . , 2014 ) .", "Therefore , even though none of these clades were supported by high gene concordance factors , the main topology of our species tree captured an apparently meaningful pattern of closely related species commonly occurring in close geographic proximity .", "Clustering of species by dissimilarities in glucosinolate profiles revealed distinct groups of chemically similar species , largely corresponding to nine chemotypes determined by the predicted function of few major-effect glucosinolate genes .", "However , we found no phylogenetic signal for chemical similarity , compound number , or total concentrations of glucosinolates ( Blomberg’s K < 1 for all traits ) .", "Closely related species thus appear to be less likely to share similar glucosinolate chemotypes .", "In a comparative study of 30 Strepthanthus species , Cacho et al . ( 2015 ) reported similarly low values for Blomberg’s K for most glucosinolate traits , suggesting that this may be a general pattern in glucosinolate diversity .", "In contrast , clustering of species by dissimilarities in cardenolide profiles revealed fewer distinct groups of chemically similar species , suggesting that the considerably more complex cardenolide chemotypes may be controlled by many minor-effect genes .", "However , we found strong phylogenetic signals for chemical similarity , compound numbers , and total concentrations of cardenolides ( Blomberg’s K > 1 ) .", "Closely related species were therefore more likely to share similar cardenolide chemotypes .", "Given the high levels of gene discordance in our phylogeny , it seems probable that at least the glucosinolate results may have been affected by hemiplasy , i . e . , a phenomenon where a trait is determined by genes whose topologies does not match the species tree ( Avise and Robinson , 2008; Pease et al . , 2016; Wu et al . , 2018 ) .", "Hemiplasy may result in the overestimation of a trait’s evolutionary rate ( Mendes et al . , 2018 ) , and results must be evaluated with caution .", "However , even though trait evolution within Erysimum almost certainly has been significantly more complex than can be represented by a simple bifurcating species tree ( Novikova et al . , 2016; Pease et al . , 2016; Wu et al . , 2018 ) , we believe our results remain robust in three key points .", "First , geographically close Erysimum species tend to be more closely related , likely because geographic proximity may facilitate gene flow .", "Indeed , geographic signatures were present in both previously published phylogenies ( Gómez et al . , 2014; Moazzeni et al . , 2014 ) as well as in our tree .", "Second , geographically close , related species share similar cardenolide but not glucosinolate phenotypes .", "Third , these distinct patterns in chemical classes indicate distinct evolutionary mechanisms for glucosinolate and cardenolide defenses , with a small number of major-effect glucosinolate genes evolving independently of a putatively larger set of minor-effect cardenolide genes .", "Despite vast morphological differences among sampled Erysimum species , the diversity in glucosinolate profiles across species was relatively limited , with a total of 25 different glucosinolate compounds detected .", "Even though this diversity may be further amplified by enzymes that direct glucosinolates into different toxic products upon activation ( not measured here; Lambrix et al . , 2001; Burow et al . , 2006; Zhang et al . , 2006 ) , intraspecific glucosinolate diversity of Arabidopsis alone is significantly higher , with more than 30 compounds reported across a range of accessions ( Kliebenstein et al . , 2001a ) .", "In contrast , an evaluation of 30 Streptanthus species revealed a similarly low total number of 35 glucosinolate compounds ( Cacho et al . , 2015 ) .", "The intraspecific diversity of Arabidopsis may therefore not be typical for all plants of the Brassicaceae .", "Additional broadly comparative studies of glucosinolate diversity in other Brassicaceae species are needed to provide a reliable ‘baseline’ for glucosinolate diversity .", "Importantly , we intentionally ignored intraspecific chemical diversity as we lacked genetically diverse seed material for most species .", "However , a preliminary screening of multiple E . cheiranthoides accessions suggests that there is little to no variation in glucosinolate profiles within this one species , while there is considerable variation in cardenolide profiles ( T . Züst , unpublished data ) .", "The majority of Erysimum species produced glucoiberin as their main glucosinolate .", "Aliphatic glucosinolates such as glucoiberin are derived from methionine in a process that involves elongation and modification of a variable side-chain ( Halkier and Gershenzon , 2006 ) , and in this context the 3-carbon glucosinolate glucoiberin is one of the least biosynthetically complex glucosinolates .", "However , the potential to produce additional aliphatic glucosinolates with longer side chains clearly exists in the genus , as 4- , 5- , and 6-carbon glucosinolates with more complex modifications were scattered across the phylogeny .", "A few species produced glucosinolates that are not found in Arabidopsis , including a sub-class of aliphatic glucosinolates , the methylsulfonyl glucosinolates .", "The homolog of GS-OH , which in Arabidopsis forms 2-hydroxy-but-3-enyl glucosinolate from 3-butenyl glucosinolate , does not have a clear function in E . cheiranthoides due to the lack of alkenyl glucosinolates .", "It is therefore possible that the GS-OH homolog in E . cheiranthoides may code for the unknown enzyme that hydroxylates 4-methylsulfonylbutyl glucosinolate to form 3-hydroxy-4-methylsulfonylbutyl glucosinolate ( Figure 4 , Figure 4—figure supplement 8 ) .", "Methylsulfonyl glucosinolates are found in several Brassicaceae genera ( Fahey et al . , 2001 ) , and glucocheirolin , the most abundant methylsulfonyl glucosinolate in Erysimum species , is only a weak egg-laying stimulant for the cabbage white butterfly ( Pieris rapae ) , compared to other glucosinolates ( Huang et al . , 1993 ) .", "Methylsulfonyl glucosinolates may thus represent a plant response to specialist herbivores that use plant defenses as host-finding cues .", "The species E . pulchellum ( PUL ) and E . collinum ( COL ) from Turkey and Iran , respectively , accumulated glucoerypestrin as their main glucosinolate compound .", "This compound was first described in E . rupestre [syn . E . pulchellum , ( Polatschek , 2011 ) ] by Kjær et al . ( 1957 ) and to date has been found exclusively in plants of the genus Erysimum ( Fahey et al . , 2001 ) .", "Radioactive labeling experiments indicated that glucoerypestrin is derived from a dicarboxylic amino acid , possibly 2-amino-5-methoxycarbonyl-pentanoic acid ( Chisholm , 1973 ) .", "Modification of the amino acid side chain during methionine-derived aliphatic glucosinolate biosynthesis as a pathway to glucoerypestrin is less likely , due to the lower specific incorporation of 14C-labeled methionine compared to 14C-labeled dicarboxylic acids into this compound ( Chisholm , 1973 ) .", "In any case , the gain of glucoerypestrin represents yet another evolutionary novelty in the Erysimum genus , but its relative toxicity and the adaptive benefits of its production have yet to be elucidated .", "Myrosinase activity levels differed among glucosinolate chemotypes , and activity was positively correlated with glucosinolate abundance in plants when controlling for glucosinolate chemotype .", "Erysimum species that predominantly produced indole glucosinolates or 4-methylsulfinyl glucosinolates had negligible myrosinase activity against the assayed aliphatic glucosinolate sinigrin .", "Indole glucosinolates can be activated by PEN2 – a thioglucosidase that is more specific for indole glucosinolates ( Bednarek et al . , 2009; Clay et al . , 2009 ) – or even break down in the absence of plant-derived myrosinase ( Kim et al . , 2008 ) .", "The negligible activity in these species could therefore indicate the existence of selective pressures to tailor myrosinase expression to the type and concentrations of glucosinolates that are produced .", "Mirroring results for glucosinolate defenses , myrosinase activity was not more similar among related species , suggesting that the two components of glucosinolate defense evolve in concert .", "We detected considerable amounts of the evolutionarily novel cardenolide defense in 47 out of 48 Erysimum species or accessions .", "Among the 95 likely cardenolide compounds detected , at least 22 had not been described previously in Erysimum .", "This metabolic diversity had three main sources: modification of the genin core structure , variation of the glycoside chain , or isomeric variation ( e . g . , through the incorporation of different isomeric sugars ) .", "Structural variation in cardenolides affects the relative inhibition of Na+/K+-ATPase ( Dzimiri et al . , 1987; Petschenka et al . , 2018 ) and physiochemical properties such as lipophilicity , which play an important role in uptake and metabolism of plant metabolites by insects ( Duffey , 1980 ) .", "Individual Erysimum species produced between 15 and 50 different cardenolide compounds , and the comparison of quantification by total mass ion counts vs . quantification by inhibition of Na+/K+-ATPase revealed highly similar results .", "While both methods of quantification are only approximate , this correlation at least provides no obvious indication of vast differences in Na+/K+-ATPase inhibitory activity among Erysimum cardenolides .", "The metabolic pathways involved in the biosynthesis and modification of cardenolides have yet to be elucidated ( Kreis and Müller-Uri , 2010; Züst et al . , 2018 ) .", "Here , we propose a pathway for the modification of digitoxigenin , commonly assumed to be the least biosynthetically complex cardenolide ( Kreis and Müller-Uri , 2010 ) , into the eight structurally more complex genins found within Erysimum ( Figure 9 ) .", "Variation in glycoside chains is likely mediated by glycosyltransferases that act on the different genins .", "In the Brassicaceae genus Barbarea , plants produce saponin glycosides as an evolutionary novel defense , and a significant proportion of glycoside diversity in this system has been linked to the action of a small set of UDP glycosyltransferases ( Erthmann et al . , 2018 ) .", "Similarly , through the joint action of genin-modifying enzymes and glycosyltransferases , a relatively small set of enzymes and corresponding genes could generate the vast cardenolide diversity found in the Erysimum genus .", "The identification and manipulation of these genes in different Erysimum species will make it possible to test the adaptive benefits of this structural diversity .", "On average , leaves of Erysimum species contained cardenolides equivalent to 6 µg ouabain per mg dry leaf weight ( estimated from Na+/K+-ATPase inhibition ) , placing them slightly above most species of the well-studied cardenolide-producing genus Asclepias ( Rasmann and Agrawal , 2011 ) .", "However , two species , E . collinum ( COL ) and E . crepidifolium ( CRE ) , were clear outliers in terms of cardenolide content ( Figure 6D–E ) .", "The almost complete absence of cardenolides in E . collinum ( COL ) , which clustered phylogenetically with two other Middle Eastern species producing average concentrations of these compounds ( E . crassipes [CSS] and E . crassicaule [CRA] , Figure 5 ) , likely represents a secondary loss of this trait in the course of evolution .", "This species also accumulated an evolutionary novel glucosinolate , glucoerypestrin ( see above ) , which may have resulted in a shift in selective pressures that led to the loss of potentially costly cardenolide production .", "Conversely , E . crepidifolium ( CRE ) had cardenolide concentrations more than three times higher than any other tested Erysimum species ( Figure 6E ) .", "This is consistent with the highly toxic nature of this species , which has the German vernacular name ‘Gänsesterbe’ ( geese death ) and has been associated with mortality in geese that consume the plant .", "Whereas most species did not induce cardenolide accumulation in response to JA , E . crepidifolium ( CRE ) had a significant 48% increase .", "While not as extreme , this observation is similar to the results of Munkert et al . ( 2014 ) , who reported a three-fold increase in cardenolide levels of E . crepidifolium in response to a high dose of methyl jasmonate .", "Plants use conserved transcriptional networks to continuously integrate signals from their environment and optimize allocation of resources to growth and defense ( Havko et al . , 2016 ) .", "Thus , while these networks commonly govern hardwired responses ( e . g . , an attenuation of growth upon activation of JA signaling ) , they may nevertheless be altered by mutations at key nodes of the network ( Campos et al . , 2016 ) .", "Given this relative flexibility in signaling networks , it is perhaps not surprising that the evolutionary novel cardenolides have been integrated into the defense signaling of Erysimum species to variable degrees .", "Investigating gene expression changes in the inducible E . crepidifolium relative to the non-inducing E . cheiranthoides may therefore provide valuable insights into the molecular regulation of this defense .", "The study of the speciose genus Erysimum , which has two co-expressed chemical defense classes , revealed largely independent evolution of the ancestral and the novel defense .", "With no evidence for trade-offs between the structurally and biosynthetically unrelated defenses , the diversity , abundance , and inducibility of each class of defenses appears to be evolving independently in response to the unique selective environment of each individual species .", "The evolutionarily recent gain of novel cardenolides has resulted in a system in which no known specific adaptations to cardenolides have yet evolved de novo in insect herbivores , although general adaptations to toxic food may still allow herbivores to consume the plants .", "Erysimum is thus an excellent model system for phytochemical diversification , as it facilitates the study of coevolutionary adaptations in real time .", "Our current work provides the foundation for a more mechanistic evaluation of these processes , which promises to greatly improve our understanding of the role of phytochemical diversity in plant-insect interactions ." ], [ "The genus Erysimum is distributed across the northern hemisphere , with the center of diversity stretching from the Mediterranean Basin into Central Asia , and a smaller number of species centered in western North America ( Moazzeni et al . , 2014 ) .", "Seeds of Erysimum species spanning a range of distributions in Europe and Western North America were collected in their native habitats or obtained from botanical gardens and commercial seed suppliers ( Figure 1 , Supplementary file 1 ) .", "Ploidy levels of species were inferred from literature reports to test for the effect of ploidy on chemical diversity ( Supplementary file 1 ) .", "For seeds obtained from botanical gardens , we generally used species names as provided by the supplier .", "As an exception , seeds of E . collinum ( COL ) had originally been designated as E . passgalense , but these species names are now considered to be synonymous ( German , 2014 ) .", "Furthermore , plants of four seed batches did not exhibit the expected phenotypes and likely were the result of seed mislabeling by the suppliers .", "We nonetheless included these plants for transcriptome sequencing , but refer to them as accessions ER1 , ER2 , ER3 , and ER4 ( Supplementary file 1 ) .", "For genome sequencing of E . cheiranthoides , seeds that were collected in 2015 by H . Christier from a natural population in the Elbe River floodplain ( Germany , Figure 1 ) were planted in a greenhouse in one-liter pots in Cornell mix ( by weight 56% peat moss , 35% vermiculite , 4% lime , 4% Osmocote slow-release fertilizer [Scotts , Marysville , OH] , and 1% Unimix [Scotts] ) .", "This lineage , which we have designated ‘Elbtalaue’ , was propagated by self-pollination and single-seed descent for six generations prior to further experiments .", "Sixth-generation var .", "Elbtalaue seeds were submitted to the Arabidopsis Biological Resource Center ( https://abrc . osu . edu ) under accession number CS29250 and to the National Plant Germplasm System ( https://www . ars-grin . gov/npgs/ ) under accession number PI 691911 .", "For transcriptome sequencing and metabolomic analyses of Erysimum species , subsets of the full species pool were grown in three separate experiments in 2016 and 2017 .", "While some species were included in all three experiments , others could only be grown once due to limited seed availability or germination .", "To maximize germination success , seeds were placed on water agar ( 1% ) in Petri dishes and cold-stratified for two weeks .", "After stratification , Petri dishes were moved to a growth chamber set to 24 °C day / 22°C night at a 16:8 hr photoperiod .", "Viable seeds germinated within 3–10 days of placement in the growth chamber .", "As soon as cotyledons had fully extended , we transplanted the seedlings into 10 × 10 cm plastic pots filled with a mixture of peat-based germination soil ( Seedlingsubstrat , Klasmann-Deilmann GmbH , Geeste , Germany ) , field soil , sand , and vermiculite at a ratio of 6:3:1:5 .", "Plants were moved to a climate-controlled greenhouse set to 24 °C day / 16°C night and 60% RH with natural light and supplemented artificial light set to a 14:10 hr photoperiod .", "Plants were watered as needed throughout the experiments , and fertilized with a single application of 0 . 1 L of fertilizer solution ( N:P:K 8:8:6 , 160 ppm N ) three weeks after transplanting .", "DNA sequencing for genome assembly and RNA sequencing for annotation were conducted with samples prepared from sixth-generation inbred E . cheiranthoides var .", "Elbtalaue .", "High molecular weight genomic DNA was extracted from the leaves of a single E . cheiranthoides plant using Wizard Genomic DNA Purification Kit ( Promega , Madison WI , USA ) .", "The quantity and quality of genomic DNA was assessed using a Qubit three fluorometer ( Thermo Fisher , Waltham , MA , USA ) and a Bioanalyzer DNA12000 kit ( Agilent , Santa Clara , CA , USA ) .", "Twelve µg of non-sheared DNA were used to prepare the SMRTbell library , and the size-selection of 15–50 kb was performed on Sage BluePippin ( Sage Science , Beverly , MA , USA ) following manufacturer's instructions ( Pacific Biosciences , Menlo Park , CA , USA ) and as described previously ( Chen et al . , 2019 ) .", "PacBio sequencing was performed by the Sequencing and Genomic Technologies Core of the Duke Center for Genomic and Computational Biology ( Durham , NC , USA ) .", "For genome polishing , one DNA library was prepared using the PCR-free TruSeq DNA sample preparation kit following the manufacturer’s instructions ( Illumina , San Diego , CA ) , and sequenced on an Illumina MiSeq instrument ( paired-end 2 × 250 bp ) at the Cornell University Biotechnology Resource Center ( Ithaca , NY ) .", "The transcriptome of sixth-generation inbred E . cheiranthoides var .", "Elbtalaue plants was sequenced using both PacBio ( Iso-Seq ) and Illumina sequencing methods .", "Total RNA was isolated from stems , flowers , buds , pods , young and mature leaves of five plants ( siblings of the plant used for genome sequencing ) using the SV Total RNA Isolation Kit with on-column DNAse I treatment ( Promega , Madison , WI , USA ) .", "The RNA quantity and quality were assessed by RIN ( RNA Integrity Number ) using a 2100 Bioanalyzer ( Agilent Technologies , Santa Clara , CA ) .", "The samples with a RIN value of >7 were pooled across all six tissue types .", "One µg of the pooled total RNA was used for the Iso-Seq following the manufacturer’s instructions ( Iso-Seq ) .", "The library preparation and sequencing were performed by Sequencing and Genomic Technologies Core of the Duke Center for Genomic and Computational Biology ( Durham , NC , USA ) .", "For Illumina sequencing , 2 μg of purified pooled total RNA from three replicates was used for the preparation of strand-specific RNAseq libraries with 14 cycles of final amplification ( Zhong et al . , 2011 ) .", "The purified libraries were multiplexed and sequenced with 101 bp paired-end read length in two-lanes on an Illumina HiSeq2500 instrument ( Illumina , San Diego , CA ) at the Cornell University Biotechnology Resource Center ( Ithaca , NY ) .", "For Hi-C scaffolding , 500 mg of E . cheiranthoides leaf tissue was flash-frozen and sent to Phase Genomics ( Phase Genomics Inc Seattle , WA , USA ) .", "PacBio sequences from the genome of E . cheiranthoides were assembled using Falcon ( Chin et al . , 2016 ) .", "The assembly was polished using Arrow from SMRT Analysis v2 . 3 . 0 ( https://www . pacb . com/products-and-services/analytical-software/smrt-analysis/ ) with PacBio reads , and then assembled into chromosome-scale scaffolds using Hi-C methods by Phase Genomics ( Seattle , WA , USA ) .", "Scaffolding gaps were filled with PBJelly v13 . 10 ( English et al . , 2012 ) using PacBio reads followed by three rounds of Pilon v1 . 23 correction ( Walker et al . , 2014 ) with 9 Gbp of Illumina paired-end 2 × 150 reads .", "BUSCO v3 ( Waterhouse et al . , 2018 ) metrics were used to assess the quality of the genome assemblies .", "For gene model prediction , de novo repeats were predicted using RepeatModeler v1 . 0 . 11 ( Smit , AFA , Hubley , R . RepeatModeler Open-1 . 0 . 2008–2015 http://www . repeatmasker . org ) , known protein domains were removed from this set based on identity to UniProt ( Boutet et al . , 2007 ) with the ProtExcluder . pl script from the ProtExcluder v1 . 2 package ( Campbell et al . , 2014 ) , and the output was then used with RepeatMasker v4-0-8 ( Smit , AFA , Hubley , R and Green , P . RepeatMasker Open-4 . 0 . 2013–2015 http://www . repeatmasker . org ) in conjunction with the Repbase library .", "For gene prediction , RNA-seq reads were mapped to the genome with hisat2 v2 . 1 . 0 ( Kim et al . , 2015 ) .", "Portcullis v1 . 1 . 2 ( Mapleson et al . , 2018 ) and Mikado v1 . 2 . 2 ( Venturini et al . , 2018 ) were used to filter the resulting bam files and make first-pass gene predictions .", "PacBio IsoSeq data were corrected using the Iso-Seq classify + cluster pipeline ( Gordon et al . , 2015 ) .", "Augustus v3 . 2 ( Stanke et al . , 2008 ) and Snap v2 . 37 . 4ubuntu0 . 1 ( Korf , 2004 ) were trained and then implemented through the Maker pipeline v2 . 31 . 10 ( Cantarel et al . , 2008 ) with Iso-Seq , proteins from Swiss-Prot , and processed RNA-seq added as evidence .", "Functional annotation was performed with BLAST v2 . 7 . 1+ ( Altschul et al . , 1990 ) and InterProScan v . 5 . 36–75 . 0 ( Jones et al . , 2014 ) .", "The genome of E . cheiranthoides was analyzed for LTR retrotransposons using LTRharvest ( Ellinghaus et al . , 2008 ) , included in GenomeTools v1 . 5 . 10 , with the parameters -seqids yes -minlenltr 100 -maxlenltr 5000 -mindistltr 1000 -motif TGCA -motifmis 1 -maxdistltr 15000 -similar 85 -mintsd 4 -maxtsd 6 -vic 10 -seed 20 -overlaps best .", "The genome was also analyzed using LTR_FINDER v1 . 07 ( Xu and Wang , 2007 ) with parameters -D 15000 -d 1000 -L 5000 -l 100 -p 20 -C -M 0 . 85 -w 0 .", "The results from LTRharvest and LTR_FINDER were then passed as inputs to LTR_retriever v2 . 0 ( Ou and Jiang , 2018 ) using default parameters , including a neutral mutation rate set at 1 . 3 × 10−8 .", "Using the LTR_retriever repeat library , the genome was masked with RepeatMasker v4 . 0 . 7 , and additional repetitive elements were identified de novo in the genome using RepeatModeler .", "These repeats were used with blastx v2 . 7 . 1+ ( Altschul et al . , 1990 ) against the Uniprot and Dfam libraries and protein-coding sequences were excluded using the ProtExcluder . pl script from the ProtExcluder v1 . 2 package ( Campbell et al . , 2014 ) .", "The masked genome was then re-masked with RepeatMasker , with the repeat library obtained from RepeatModeler .", "Coverage percentages for repeat types were obtained using the fam_coverage . pl and fam_summary . pl scripts , which are included with LTR_retriever .", "All percentages were calculated based on the total length of the assembly .", "A circular representation of the E . cheiranthoides genome was made with Circos v0 . 69–6 ( Krzywinski et al . , 2009 ) .", "Gene and repeat densities were calculated by generating 1 Mb windows and by calculating percent coverage for the features using bedtools coverage v2 . 26 . 0 ( Quinlan and Hall , 2010 ) .", "The coverage values from the repeat library and the genome annotation were calculated independently of each other .", "Similarly , the total percentage of genic sequence for the genome was also calculated using bedtools genomecov v2 . 26 . 0 .", "The gene and repeat percentages for the 1 Mb windows were then plotted as histogram tracks in Circos .", "For analysis of synteny between E . cheiranthoides and Arabidopsis thaliana ( Arabidopsis; TAIR10; www . arabidopsis . org ) , the genome sequences were aligned using NUCmer , from MUMmer v3 . 23 ( Kurtz et al . , 2004 ) with the parameters --maxgap=500 --mincluster=100 .", "The alignments were filtered with delta-filter -r -q -i 90 -l 1000 and coordinates of aligned segments were extracted with show-coords .", "The extracted coordinates were then used as the links input for Circos .", "Glucosinolate and myrosinase genes in E . cheiranthoides were annotated based on homology to known Arabidopsis pathway genes in the Plant Metabolic Network database ( https://pmn . plantcyc . org ) ( Schläpfer et al . , 2017 ) , as well as genes from Brassica rapa ( Wang et al . , 2011 ) , Raphanus sativus ( Kakizaki et al . , 2017 ) , and Manihot esculenta ( Mikkelsen and Halkier , 2003 ) .", "Coding sequences for the Arabidopsis and Brassica glucosinolate and myrosinase genes were obtained from GenBank ( https://www . ncbi . nlm . nih . gov/genbank/ ) , The Arabidopsis Information Resource ( www . arabidopsis . org ) , and the Brassica Database ( brassicadb . org/brad/glucoGene . php ) .", "Identified genes were used in a BLASTn query against the E . cheiranthoides coding sequence dataset .", "Coding sequences from E . cheiranthoides , Arabidopsis , and Brassica were used to construct maximum likelihood phylogenetic trees with 1000 bootstraps using MEGA version 7 ( Kumar et al . , 2016 ) .", "Nucleotide sequences were aligned using MUSCLE , and the best model for each tree was selected based on neighbor-joining trees using the maximum likelihood method with gaps and missing data treated as a partial deletion with a 95% site coverage cut-off .", "To generate the large number of gene sequences required for a well-resolved phylogeny , we sequenced the foliar transcriptomes of 48 Erysimum species or accessions , including a first-generation inbred E . cheiranthoides var .", "Elbtalaue .", "Transcriptomes were generated from pooled leaf material of several individuals collected in the same experiment .", "Five species were sequenced from plants in experiment 2016 , 18 species from experiment 2017–1 , and 25 species from experiment 2017–2 ( Supplementary file 1 ) .", "Leaf material was harvested 5–7 weeks after plants were transplanted into soil .", "To average environmental and individual effects on RNA expression , we pooled leaf material from 2 to 5 individual plants from one or two time points ( separated by 1–2 weeks ) to create a single pooled RNA sample per species ( see Supplementary file 1 for details ) .", "For large-leaved species , we collected approximately 50 mg of fresh plant material from each harvested plant using a heat-sterilized hole punch ( 0 . 5 cm diameter ) .", "For smaller-leaved species , we collected an equivalent amount of material by harvesting multiple whole leaves .", "All leaf tissue was immediately snap frozen in liquid nitrogen and stored at −80°C until further processing .", "For sample pooling , we combined leaf material of individual plants belonging to the same species in a mortar under liquid nitrogen and ground all material to a fine powder .", "We then weighed out 50–100 mg of frozen pooled powder for each species .", "We extracted RNA from pooled leaf material using the RNeasy Plant Mini Kit ( Qiagen AG , Hombrechtikon , Switzerland ) , including a step for on-column DNase digestion , and following the manufacturer’s instructions .", "The purified total RNA was dissolved in 50 µL RNase-free water , split into three aliquots , and stored at −80°C until further processing .", "Assessment of RNA quality , library preparation , and sequencing were all performed by the Next Generation Sequencing Platform of the University of Bern ( Bern , Switzerland ) .", "RNA quality was assessed in one aliquot per extract using a Fragment Analyzer ( Model CE12 , Agilent Technologies , Santa Clara , USA ) , and samples with low RIN scores ( <7 ) were re-extracted and assessed again for quality .", "RNA libraries for TruSeq Stranded mRNA ( Illumina , San Diego , USA ) were assembled for each species and multiplexed in groups of eight , using unique index combinations ( Illumina , 2017 ) .", "Groups of eight multiplexed libraries were run together in single lanes ( for a total of six lanes ) of an Illumina HiSeq 3000 sequencer using 150 bp paired-end reads .", "RNA-seq data were cleaned with fastq-mcf v1 . 04 . 636 ( https://github . com/ExpressionAnalysis/ea-utils/blob/wiki/FastqMcf . md ) using the following parameters: quality = 20 , minimum read length = 50 .", "Filtered reads were assembled using Trinity v2 . 4 . 0 ( Haas et al . , 2013 ) .", "The longest ORF was determined using TransDecoder v5 . 5 . 0 ( https://github . com/TransDecoder ) .", "BUSCO v2 ( Waterhouse et al . , 2018 ) was run with lineage Embryophyta to assess gene representation and Orthofinder v2 . 3 . 1 ( Emms and Kelly , 2015 ) was used to cluster proteins from all 48 transcriptomes into orthogroups .", "To construct a phylogenetic tree , we translated the assembled transcriptomes using TransDecoder v5 . 5 . 0 .", "We then followed the Genome-Guided Phylo-Transcriptomics Pipeline ( Washburn et al . , 2017 ) to infer orthologous genes using synteny between genomes of E . cheiranthoides and Arabidopsis ( TAIR10 ) .", "Briefly , we obtained 26 , 830 orthologs between E . cheiranthoides and Arabidopsis through the syntenic blocks that were identified by SynMap from CoGe ( https://genomevolution . org/coge/SynMap . pl ) .", "Sequences for each of the 48 Erysimum species and Arabidopsis ( TAIR10_pep_20101214; www . arabidopsis . org ) ( total of 49 samples ) were annotated using protein sequences of the orthologs using blastp v2 . 7 . 1 with an e-value <10−4 and identity >85% .", "After annotation , single copy genes and one copy of repetitive genes were kept if they were present in more than 39 ( >80% of 49 ) species .", "As a potential caveat , this approach would result in single copies from one parent of allopolyploid species to be retained randomly .", "In total , we recovered 11 , 890 genes , 9868 of which had orthologs with Arabidopsis .", "Each of these 9868 genes was aligned using MAFFT v7 . 394 ( Katoh et al . , 2002 ) , and cleaned using Phyutility v2 . 2 . 6 ( Smith and Dunn , 2008 ) with the parameter -clean 0 . 3 .", "Maximum-likelihood tree estimation for each gene was constructed in RAxML v8 . 2 . 8 ( Stamatakis , 2014 ) using the PROTCATWAG model with 100 bootstrap replicates .", "Finally , species tree inference was performed with these 9868 gene trees as input , using ASTRAL-III v5 . 6 . 3 ( Zhang et al . , 2018 ) .", "In this species tree we observed very short internal branch lengths , which generally indicates high levels of gene tree discordance .", "We therefore additionally ran ASTRAL-III with the full annotation option ( -t 2 ) for information on quartet support , total number of quartet trees in gene trees , and local posterior probabilities for the main topology and first and second alternative topologies .", "We additionally concatenated the cleaned alignments of the 9868 genes using the Genome-Guided Phylo-Transcriptomics Pipeline concatenate_matrices . py script ( Washburn et al . , 2017 ) to generate a completely random starting tree with RAxML v8 . 2 . 8 , again using the PROTCATWAG model .", "Finally , we ran ExaML ( Kozlov et al . , 2015 ) to generate a concatenated phylogeny with parameters: -a -B 100 m GAMMA -s , and then evaluated the phylogeny with ExaML using -f E -m GAMMA and calculated gene concordance factors for each branch of the species tree using IQ-Tree v2 ( Minh et al . , 2020 ) .", "Using the ExaML concatenated phylogeny with branch length information , we dated the Erysimum radiation using treePL v1 . 0 ( Smith and O’Meara , 2012 ) .", "We constrained four nodes using inferred dates from two published studies .", "First , we constrained the node of the divergence between Erysimum species and Arabidopsis using a range of 11–22 Mya , determined by Cardinal-McTeague et al . ( 2016 ) .", "This range had been estimated using four fossil taxa and 151 extant taxa across the Brassicales , of which Erysimum is a member .", "Next , we constrained three additional nodes using dates determined by Moazzeni et al . ( 2014 ) based on a molecular clock approach with a published rate of substitution [5 × 10−9 substitutions per site per year , ( Kay et al . , 2006 ) . We constrained the node of the Erysimum clade to 2 . 33–5 . 2 Mya , the West European clade to 0 . 5–2 . 0 Mya , and the North American clade to 0 . 7–1 . 65 Mya . To screen for evidence of hybridization we used HyDe v0 . 4 . 3 ( Blischak et al . , 2018 ) which tests sets of triples across the whole phylogeny . As HyDe requires the input file as a nucleotide alignment , we pulled the same set 9868 genes recovered during orthology determination from the original de novo assembly of transcriptomes . We then aligned and cleaned each gene sequence separately using MAFFT v7 . 394 ( Katoh et al . , 2002 ) , and Phyutility v2 . 2 . 6 ( Smith and Dunn , 2008 ) with the parameter -clean 0 . 3 . A final concatenated alignment , used as input , of the 9868 genes was generated using the Genome-Guided Phylo-Transcriptomics Pipeline concatenate_matrices . py script ( Washburn et al . , 2017 ) . We harvested leaf material for targeted metabolomic analysis of defense compounds from the same plants as used for transcriptome sequencing , one week after leaves for RNA extraction had been harvested . In each of the three experiments , we collected several leaves from 1 to 5 plants per species , and immediately snap froze the harvested leaves in liquid nitrogen . While most plant samples were screened for constitutive levels of chemical defenses only , we quantified inducibility of chemical defenses in a subset of 30 species with sufficient replication ( eight or more plants ) in the third experiment ( 2017–2 ) . For these species , half of all plants were randomly assigned to the induction treatment and given a foliar spray of JA one week prior to harvest . Plants were sprayed with 2–3 mL of a 0 . 5 mM JA solution ( Cayman Chemical , MI , USA ) in 2% ethanol until all leaves were evenly covered in droplets on both sides . Control plants were sprayed with an equivalent amount of 2% ethanol solution . Harvested frozen plant material was lyophilized to dryness and ground to a fine powder . We weighed out 10 mg leaf powder per sample into separate tubes and added 1 mL of 70% MeOH extraction solvent . Samples were extracted by adding three 3 mm ceramic beads to each tube and shaking tubes on a Retsch MM400 ball mill three times for 3 min at 30 Hz . We centrifuged samples at 18 , 000 x g and transferred 0 . 9 mL of the supernatant to a new tube . Samples were centrifuged again , and 0 . 8 mL of the final supernatant was transferred to an HPLC vial for analysis by high-resolution mass spectrometry . We analyzed extracts of individual plants ( all experiments ) or of multiple pooled individuals per species and induction treatment ( experiment 2017–2 only ) on an Acquity UHPLC system coupled to a Xevo G2-XS QTOF mass spectrometer with electrospray ionization ( Waters , Milford MA , USA ) . Due to large differences in the physiochemical properties between glucosinolates and cardenolides , each plant extract was analyzed in two different modes to optimize detection of each compound class . For glucosinolates , extracts were separated on a Waters Acquity charged surface hybrid ( CSH ) C18 100 × 2 . 1 mm column with 1 . 7 µm pore size , fitted with a CSH guard column . The column was maintained at 40°C and injections of 1 µl were eluted at a constant flow rate of 0 . 4 mL/min with a gradient of 0 . 1% formic acid in water ( A ) and 0 . 1% formic acid in acetonitrile ( B ) as follows: 0–6 min from 2% to 45% B , 6–6 . 5 min from 45% to 100% B , followed by a 2 min wash phase at 100% B , and 2 min reconditioning at 2% B . For cardenolides , extracts were either separated using an equivalent method , or an optimized method using a Waters Cortecs C18 150 × 2 . 1 mm column with 2 . 7 µm pore size , fitted with a Cortecs C18 guard column . The column was maintained at 40°C and injections were eluted at a constant flow rate of 0 . 4 mL/min with a gradient of 0 . 1% formic acid in water ( A ) and 0 . 1% formic acid in acetonitrile ( B ) as follows: 0–10 min from 5% to 40% B , 10–15 min from 40% to 100% B , followed by a 2 . 5 min wash phase at 100% B , and 2 . 5 min reconditioning at 5% B . Compounds were ionized in negative mode for glucosinolate analysis and in positive mode for cardenolide analysis . In both modes , ion data were acquired over an m/z range of 50 to 1200 Da in MSE mode using alternating scans of 0 . 15 s at low collision energy of 6 eV and 0 . 15 s at high collision energy ramped from 10 to 40 eV . For both positive and negative modes , the electrospray capillary voltage was set to 2 kV and the cone voltage was set to 20 V . The source temperature was maintained at 140°C and the desolvation gas temperature at 400°C . The desolvation gas flow was set to 1000 L/h , and argon was used as a collision gas . The mobile phase was diverted to waste during the wash and reconditioning phase at the end of each gradient . Accurate mass measurements were obtained by infusing a solution of leucine-enkephalin at 200 ng/mL at a flow rate of 10 µL/min through the LockSpray probe . Glucosinolates consist of a β-D-glucopyranose residue linked via a sulfur atom to a ( Z ) -N-hydroximinosulfate ester and a variable R group ( Halkier and Gershenzon , 2006 ) . We identified candidate glucosinolate compounds from negative scan data by the exact molecular mass of glucosinolates known to occur in Erysimum and related species ( Huang et al . , 1993; Fahey et al . , 2001 ) . In addition , we screened all negative scan data for characteristic glucosinolate mass fragments to identify additional candidate compounds ( Cataldi et al . , 2010 ) . For mass features with multiple possible identifications , we inferred the most likely compound identity from relative HPLC retention times and the presence of biosynthetically related compounds in the same sample . We confirmed our identifications using commercial standards for glucoiberin , glucocheirolin ( both Phytolab GmbH ) , and sinigrin ( Sigma-Aldrich ) , as well as by comparison to extracts of Arabidopsis accessions with known glucosinolate profiles . Compound abundances of all glucosinolates were quantified by integrating ion intensities of the [M-H]- adducts using QuanLynx in the MassLynx software ( v4 . 1 , Waters ) .", "All cardenolides share a highly conserved structure consisting of a steroid core ( 5β , 14β-androstane-3β14-diol ) linked to a five-membered lactone ring , which as a unit ( the genin ) mediates the specific binding of cardenolides to Na+/K+- ATPase ( Dzimiri et al . , 1987 ) .", "While cardenolide genins are sufficient to inhibit Na+/K+- ATPase function , genins are commonly glycosylated or modified by hydroxylation on the steroid moiety to change the physiochemical properties and binding affinity of compounds ( Dzimiri et al . , 1987; Petschenka et al . , 2018 ) .", "We obtained commercial standards for the abundant Erysimum cardenolides erysimoside and helveticoside ( Sigma-Aldrich ) , allowing us to identify these compounds through comparison of retention times and mass fragmentation patterns .", "Additional cardenolide compounds were tentatively identified from characteristic LC-MS fragmentation patterns .", "Sachdev-Gupta et al . ( 1990 ) and Sachdev-Gupta et al . ( 1993 ) reported fragmentation patterns for glycosides of strophanthidin , digitoxigenin , and cannogenol from E . cheiranthoides .", "Their results highlight the propensity of cardenolides to fragment at glycosidic bonds , with genin masses in particular being a prominent feature of cardenolide mass spectra .", "Additionally , cardenolide genins exhibit further fragmentation related to the loss of OH-groups from the steroidal core structure .", "We confirmed these rules of fragmentation for our mass spectrometry system using commercial standards of strophanthidin and digitoxigenin ( Sigma-Aldrich ) .", "Importantly , while fragments were most abundant under high-energy conditions ( MSE ) , they were still apparent under standard MS conditions , likely due to in-source fragmentation .", "Characteristic fragmentation allowed us to identify candidate cardenolide compounds in a genin-guided approach , where the presence of characteristic genin fragments in a chromatographic peak indicated the likely presence of a cardenolide molecule .", "We then identified the parental mass of these chromatographic peaks from the presence of paired mass features separated by 21 . 98 m/z , corresponding to the [M+H]+ and [M+Na]+ adducts of the intact molecule .", "For di-glycosidic cardenolides , additional fragments corresponding to the loss of the outer sugar moiety allowed us to determine the mass and order of sugar moieties in the linear glycoside chain of the molecule .", "We screened our data for the presence of glycosides of strophanthidin , digitoxigenin , and cannogenol , and additional genins known to occur in Erysimum species ( Makarevich et al . , 1994 ) .", "Multiple cardenolide genins can share the same molecular structure and may not be distinguished by mass spectrometry alone .", "Thus , all genin identifications are tentative and based on previous literature reports .", "We screened LC-MS data from two experiments ( Phylo16 and Phylo17-2 ) to generate a list of cardenolide compounds .", "Cardenolide data of experiment Phylo17-1 could not be used due to technical problems with the LC-MS analysis in positive mode .", "Relative compound abundances were quantified by integrating the ion intensities of the [M+H]+ or the [M+Na]+ adduct , whichever was more abundant for a given compound across all samples .", "In the third experiment , we added hydrocortisone ( Sigma-Aldrich ) to each sample as an internal standard , but between-sample variation ( technical noise ) was negligible compared to between-species variation .", "For glucosinolate and cardenolide data separately , we averaged raw ion counts for each compound across experiments to yield a single chemical profile per compound class and species .", "We thereby intentionally ignored potential intraspecific variation in chemical diversity , as a lack of multiple seed origins or accessions for most species did not allow us to appropriately quantify this dimension of complexity .", "Raw ion counts were standardized by the dry sample weight , possible dilution of samples , and internal standard concentrations ( where available ) .", "For pooled samples , ion counts were standardized by the average dry weight calculated from all samples that contributed to a pool .", "The full set of standardized compound ion counts was then analyzed using linear mixed effects models ( package nlme v3 . 1–137 in R v3 . 5 . 3 ) .", "Because standardized ion counts still had a heavily skewed distribution , we applied a log ( +0 . 1 ) transformation to all values .", "Log-transformed ion counts were modelled treating experiment as a fixed effect , and a species-by-compound identifier as the main random effect .", "Nested within the main random effect , we fitted a species-by-compound-by-experiment identifier as a second random effect to account for the difference of pooled or individual samples among experiments .", "The fixed effect of this model thus captures the overall differences in compound ion counts between experiments , while the main random effect captures the average deviation from an overall compound mean for each compound in each species .", "We extracted the overall compound mean and the main random effects from these models , providing us with average ion counts for each compound in each species on the log-scale .", "Negative values on the log-scale were set to zero as they would correspond to values below the limit of reliable detection of the LC-MS on the normal scale .", "Although all cardenolides target the same enzyme in animal cells , structural variation among different cardenolides can significantly influence binding affinity and thus affect toxicity ( Dzimiri et al . , 1987; Petschenka et al . , 2018 ) .", "Cardenolide quantification from LC-MS mass signal intensity does not capture such differences in biological activity , and furthermore may be challenging due to compound-specific response factors and narrow ranges of signal linearity .", "To evaluate whether total ion counts are an appropriate and biologically relevant measure for between-species comparisons of defense levels , we therefore quantified cardenolide concentrations by a separate method ( Züst et al . , 2019 ) .", "For the subset of plants in the 2016 experiment , we measured the biological activity of leaf extracts on the Na+/K+-ATPase from the cerebral cortex of pigs ( Sus scrofa , Sigma-Aldrich , MO , USA ) using an in vitro assay introduced by Klauck and Luckner ( 1995 ) and adapted by Petschenka et al . ( 2013 ) .", "This colorimetric assay measures Na+/K+-ATPase activity from phosphate released during ATP consumption , and can be used to quantify relative enzymatic inhibition by cardenolide-containing plant extracts .", "Briefly , we tested the inhibitory effect of each plant extract at four concentrations to estimate the sigmoid enzyme inhibition function from which we could determine the cardenolide content of the extract relative to a standard curve for ouabain ( Sigma Aldrich , MO , USA ) .", "We dried a 100 µL aliquot of each extract used for metabolomic analyses at 45°C on a vacuum concentrator ( SpeedVac , Labconco , MO , USA ) .", "Dried residues were dissolved in 200 µL 10% DMSO in water , and further diluted 1:5 , 1:50 , and 1:500 using 10% DMSO .", "To quantify potential non-specific enzymatic inhibition that could occur at high concentrations of plant extracts , we also included control extracts from Sinapis arvensis leaves ( a non-cardenolide producing species of the Brassicaceae ) in these assays .", "Assays were carried out in 96-well microplate format .", "Reactions were started by adding 80 μL of a reaction mix containing 0 . 0015 units of porcine Na+/K+-ATPase to 20 μL of leaf extracts in 10% DMSO , to achieve final well concentrations ( in 100 μL ) of 100 mM NaCl , 20 mM KCl , 4 mM MgCl2 , 50 mM imidazol , and 2 . 5 mM ATP at pH 7 . 4 .", "To control for coloration of leaf extracts , we replicated each reaction on the same 96-well plate using a buffered background mix with identical composition as the reaction mix but lacking KCl , resulting in inactive Na+/K+-ATPases .", "Plates were incubated at 37°C for 20 min , after which enzymatic reactions were stopped by addition of 100 μL sodium dodecyl sulfate ( SDS , 10% plus 0 . 05% Antifoam A ) to each well .", "Inorganic phosphate released from enzymatically hydrolyzed ATP was quantified photometrically at 700 nm following the method described by Taussky and Shorr ( 1953 ) .", "Absorbance values of reactions were corrected by their respective backgrounds , and sigmoid dose-response curves were fitted to corrected absorbances using a non-linear mixed effects model with a 4-parameter logistic function in the statistical software R ( function nlme with SSfpl in package nlme v3 . 1–137 ) .", "Absorbance= A+ ( B-A ) 1+exmid-xscal The absorbance values at four dilutions x are thus used to estimate the upper ( A , fully active enzyme ) and lower ( B , fully inhibited enzyme ) asymptotes , the dilution value xmid at which 50% inhibition is achieved , and a shape parameter scal .", "In order to estimate four parameters from four absorbance values per extract , the scal parameter was fixed for all extracts and changed iteratively to optimize overall model fit , judged by AIC .", "Individual plant extracts were treated as random effects to account for lack of independence within extract dilution series .", "For each extract we estimated xmid from the average model fit and the extract-specific random deviate .", "Using a calibration curve made with ouabain ranging from 10−3 to 10−8 M that was included on each 96-well plate , we then estimated the concentration of the undiluted sample in ouabain equivalents , i . e . , the amount of ouabain required to achieve equivalent inhibition .", "For the subset of plants in experiment 2017–2 , we extracted the total amounts of soluble myrosinases from leaf tissue and quantified their activity as an important component of the glucosinolate defense system of these species .", "At the time of harvest of metabolomic samples , we collected an additional set of leaf disks from each plant , corresponding to approximately 50 mg fresh weight .", "After determination of exact fresh weight , samples were flash frozen in liquid nitrogen and stored at −80°C until enzyme activity measurements .", "Following the protocol of Travers-Martin et al . ( 2008 ) , frozen leaf material was ground and extracted in Tris-EDTA buffer ( 200 mM Tris , 10 mM EDTA , pH 5 . 5 ) and internal glucosinolates were removed by rinsing the extracts over a DEAE Sephadex A25 column ( Sigma-Aldrich ) .", "Myrosinase activities were determined by adding sinigrin to plant extracts and monitoring the enzymatic release of glucose from its activation .", "Control reactions with sinigrin-free buffer were used to correct for plant-derived glucose .", "All samples were measured in duplicate and mean values related to a glucose calibration curve , measured also in duplicate .", "Reactions were carried out in 96-well plates and concentrations of released glucose were measured by adding a mix of glucose oxidase , peroxidase , 4-aminoantipyrine and phenol as color reagent to each well and measuring the kinetics for 45 min at room temperature in a microplate photometer ( Multiskan EX , Thermo Electron , China ) at 492 nm .", "To quantify chemical similarity among species , we performed separate cluster analyses on the glucosinolate and cardenolide profile data averaged across the three experiments .", "For each species , the log-transformed average ion counts of all compounds were converted to proportions ( all compounds produced by a species summing to 1 ) .", "From this proportional data we then calculated pairwise Bray-Curtis dissimilarities for all species pairs using function vegdist in the R package vegan v2 . 5–4 ( Oksanen et al . , 2019 ) .", "We incorporated vegdist as a custom distance function for pvclust in the R package pvclust v2 . 0 ( Suzuki and Shimodaira , 2014 ) , which performs multiscale bootstrap resampling for cluster analyses .", "We constructed chemical dendrograms ( chemograms ) of glucosinolate and cardenolide profile similarities by fitting hierarchical clustering models ( Ward’s D ) and estimated support for individual species clusters from 10 , 000 permutations .", "To test whether similarity in glucosinolate and cardenolide profiles was correlated with each other and with phylogenetic relatedness , we performed Mantel tests [function mantel . test in R package ape v5 . 0 , ( Paradis and Schliep , 2019 ) with 1000 permutations to compare Bray-Curtis dissimilarity matrices or the cophenetic distance matrix extracted for the ultrametric ExaML species tree .", "We visualized a potential correlation between the ExaML species tree and each chemogram by optimizing vertical matching of tree tips using function cophylo in the R package phytools v0 . 6–60 ( Revell , 2012 ) .", "In addition , we performed principal coordinate analyses ( PCoA ) on Bray-Curtis dissimilarity matrices of glucosinolate and cardenolide data using function pcoa in R package ape v5 . 0 and extracted the first two principal coordinates for each defense trait to test for phylogenetic signal .", "We evaluated a prevalence of phylogenetic signal in chemical defense traits , myrosinase activity , and principal coordinates for both chemical dissimilarity matrices using Blomberg’s K ( Blomberg et al . , 2003 ) .", "K is close to zero for traits lacking phylogenetic signal; it approaches one if trait similarity among related species matches a Brownian motion model of evolution , and it can be >1 if similarity is even higher than expected under a Brownian motion model .", "We estimated K for all traits using function phylosig in the phytools package and the ultrametric ExaML species tree .", "Additionally , we reconstructed the ancestral states for total glucosinolate and cardenolide content , and for the first principal coordinates of both chemical dissimilarity matrices using maximum-likelihood function fastAnc in the phytools package , which models the evolution of continuous traits using Brownian motion .", "We tested for correlations among plant traits using phylogenetic generalized least squares ( PGLS , function gls in the nlme package ) with a Brownian correlation structure inferred from the ultrametric ExaML species tree .", "As variation in traits can impact the propensity for evolutionary diversification , we also tested for correlations between trait variation across the tips of the ExaML phylogeny and tip-specific estimates of speciation rates ( tip-rate correlation , TRC ) ( Harvey and Rabosky , 2018 ) .", "Tip-specific speciation rates were estimated as node density ( ND ) ( Freckleton et al . , 2008 ) and as inverse equal split ( ES ) measures ( Jetz et al . , 2012 ) , where ND captures splitting dynamics over the entire history of a lineage leading to a tip , while ES is weighted towards branching patterns nearer the tips ( Harvey and Rabosky , 2018 ) .", "We tested for significant correlations between speciation rate measures and chemical traits using standard PGLS models , as well as a more robust simulation-based method ( Harvey and Rabosky , 2018 ) , thereby performing four tests for each of four chemical plant traits .", "To estimate the likelihood of false positives in this approach , we performed all four tests on 1000 sets of randomly generated plant traits and quantified the number of traits with more than one significant test .", "Finally , we evaluated the evidence for geographic signal in phylogenetic relatedness by performing a Mantel test to compare the cophenetic distance matrix to the pairwise geographic distances for all species with known collection locations ." ] ]

[ "Phytochemical diversity is thought to result from coevolutionary cycles as specialization in herbivores imposes diversifying selection on plant chemical defenses .", "Plants in the speciose genus Erysimum ( Brassicaceae ) produce both ancestral glucosinolates and evolutionarily novel cardenolides as defenses .", "Here we test macroevolutionary hypotheses on co-expression , co-regulation , and diversification of these potentially redundant defenses across this genus .", "We sequenced and assembled the genome of E . cheiranthoides and foliar transcriptomes of 47 additional Erysimum species to construct a phylogeny from 9868 orthologous genes , revealing several geographic clades but also high levels of gene discordance .", "Concentrations , inducibility , and diversity of the two defenses varied independently among species , with no evidence for trade-offs .", "Closely related , geographically co-occurring species shared similar cardenolide traits , but not glucosinolate traits , likely as a result of specific selective pressures acting on each defense .", "Ancestral and novel chemical defenses in Erysimum thus appear to provide complementary rather than redundant functions ." ]

[ "Plants are often attacked by insects and other herbivores .", "As a result , they have evolved to defend themselves by producing many different chemicals that are toxic to these pests .", "As producing each chemical costs energy , individual plants often only produce one type of chemical that is targeted towards their main herbivore .", "Related species of plants often use the same type of chemical defense so , if a particular herbivore gains the ability to cope with this chemical , it may rapidly become an important pest for the whole plant family .", "To escape this threat , some plants have gained the ability to produce more than one type of chemical defense .", "Wallflowers , for example , are a group of plants in the mustard family that produce two types of toxic chemicals: mustard oils , which are common in most plants in this family; and cardenolides , which are an innovation of the wallflowers , and which are otherwise found only in distantly related plants such as foxglove and milkweed .", "The combination of these two chemical defenses within the same plant may have allowed the wallflowers to escape attacks from their main herbivores and may explain why the number of wallflower species rapidly increased within the last two million years .", "Züst et al . have now studied the diversity of mustard oils and cardenolides present in many different species of wallflower .", "This analysis revealed that almost all of the tested wallflower species produced high amounts of both chemical defenses , while only one species lacked the ability to produce cardenolides .", "The levels of mustard oils had no relation to the levels of cardenolides in the tested species , which suggests that the regulation of these two defenses is not linked .", "Furthermore , Züst et al . found that closely related wallflower species produced more similar cardenolides , but less similar mustard oils , to each other .", "This suggests that mustard oils and cardenolides have evolved independently in wallflowers and have distinct roles in the defense against different herbivores .", "The evolution of insect resistance to pesticides and other toxins is an important concern for agriculture .", "Applying multiple toxins to crops at the same time is an important strategy to slow the evolution of resistance in the pests .", "The findings of Züst et al . describe a system in which plants have naturally evolved an equivalent strategy to escape their main herbivores .", "Understanding how plants produce multiple chemical defenses , and the costs involved , may help efforts to breed crop species that are more resistant to herbivores and require fewer applications of pesticides ." ]

[ "Introduction", "Results and discussion", "Methods" ]

[ "evolutionary biology", "short report", "computational and systems biology" ]

[ [ "In our previous work ( Hose et al . , 2015 ) , we reported from a genome-sequencing survey that aneuploidy was frequently observed in wild strains of S . cerevisiae , and that 10–30% of amplified genes , depending on the strain and affected chromosome , show lower-than-expected expression in naturally aneuploid isolates compared to isogenic euploid controls .", "We argued that this group is enriched for genes whose expression is regulated by one or more modes of gene-dosage compensation , which we validated at several genes .", "Importantly , the group of genes we identified shows non-random associations , including enrichment for specific functional groups , enrichment for genes that are toxic when over-expressed in the lab strain , and a higher rate of gene copy-number variation ( CNV ) despite higher constraint on expression variation in wild isolates of yeast .", "A main point of our paper was that dosage compensation at select genes has an important evolutionary effect , by buffering expression against CNV in wild strains .", "Torres et al . take issue with the conclusions in our manuscript , arguing that", "a ) wild strains of yeast are not tolerant of aneuploidy and our results emerge as an artifact of culturing wild strains in the lab , and", "b ) errors in our statistical analysis explain the reduced expression of amplified genes and instead there are no genes subject to dosage-responsive control .", "We disagree with these assertions and provide additional computational analysis that support our original claims ." ], [ "Torres et al . argue that the wild strains analyzed in our study are not tolerant of aneuploidy , since the chromosome-wide average of the relative Illumina read depth measured for each amplified gene is not precisely 1 . 0 , 1 . 5 , or 2 . 0-fold higher than the euploid control ( see Torres et al . Methods ) .", "They take this to reflect heterogeneous populations in which cells in the culture have lost the aneuploidy .", "However , this is not valid for several reasons .", "Due to technical biases in Illumina sequencing , it is highly unlikely that the mean value of relative gene copy number across whole chromosomes is a precise integer .", "Indeed , the plots shown by Torres et al . indicate the expected spread in relative read depth across the amplified chromosomes – similar to the spread in read counts of the euploid chromosomes – with mean values very close to the relative DNA abundance we reported .", "Furthermore , some genes on the chromosomes are not amplified ( particularly those near the telomeres [Hose et al . , 2015] ) , which can also slightly reduce the mean value away from a precise integer .", "Regardless of the precise mean copy number values , there can be no doubt from the figures presented by Torres et al . that for the strains we analyzed in our original paper , the vast majority of cells in each culture were aneuploid .", "We point out that several of the chromosomes and strains highlighted by Torres et al . ( Figure 1D-F and Figure 2F-H ) were not presented in our manuscript or used in any of our analyses ( see Hose et al . Figure 1A for strains and chromosomes used in our study ) .", "It is true that some chromosome amplifications , namely in sake strains , are variable ( appearing or disappearing ) across replicates , and thus these chromosomes ( Torres et al . Figure 1F and 2F ) were not considered as part of this work .", "The karyotype of these sake strains may indeed be somewhat ‘unstable’ at the culture level; however , the random appearance of extra chromosomes across replicates again suggests a low fitness cost to aneuploidy and an observable rate of mitotic errors ( Zhu et al . , 2014 ) .", "Nonetheless , the vast majority of aneuploidies we reported are relatively stable and maintained at high frequencies over many generations and in the absence of any selection .", "Instead , our data show that the wild strains we studied are tolerant of aneuploidy , and that it is the laboratory W303 strain that is highly aberrant .", "1 ) By conservative estimation , 30% of the strains we sequenced are aneuploid – these strains were identified in an unbiased sequencing survey in which aneuploidy was not generated or selected for .", "2 ) The aneuploid strains we studied show little growth reduction compared to isogenic euploid strains , both for naturally aneuploid isolates and strains for which we artificially generated aneuploidy ( Hose et al . , 2015 ) .", "( In cases where we cited the specific growth rate , we always verified that aneuploidy remained at the end of the experiment by relative qPCR . )", "Thus , aneuploidy tolerance is not due to unusual adaptation in the lab .", "In contrast , the tetrasomic W303_Chr12-4n strain – carrying a chromosome reported to be one of the least toxic in this background ( Sheltzer et al . , 2012 ) – has a 70% reduction in growth rate compared to its isogenic control ( Hose et al . Figure 5B ) .", "3 ) While extra chromosomes can be lost stochastically , it generally took >200 generations of growth to detect significant chromosome loss in the wild-strain cultures we analyzed .", "W303_Chr12-4n cultures reproducibly lose the extra chromosome in one culture passaging ( ~20 generations ) .", "The extreme fitness defect incurred by the aneuploid W303 strain explains the rapid emergence of cells that lose the extra chromosome , since the euploid W303 grows nearly twice as fast and rapidly takes over the culture .", "4 ) Aneuploid W303 cells show aberrant gene expression and strong activation of the environmental stress response ( ESR , [Gasch et al . , 2000] ) , regardless of the chromosome amplified ( Sheltzer et al . , 2012; Torres et al . , 2007 ) .", "But as we published , naturally aneuploid strains simply do not activate the ESR ( Hose et al . Figure 2A ) , indicating that they are not experiencing significant stress compared to their euploid controls .", "Taken together , these results strongly support our conclusion that aneuploidy is relatively well tolerated in wild isolates of S . cerevisiae , at least for the strains and chromosomes we studied , but not in W303 as previously published ( Sheltzer et al . , 2012; Torres et al . , 2007; 2010 ) .", "It is certainly possible that some wild strains will not tolerate aneuploidy , and likely that some chromosome amplifications are more problematic than others , regardless of strain background .", "The prevalence of aneuploidy in our original study is consistent with other reports of aneuploidy in wild isolates ( Tan et al . , 2013; Strope et al . , 2015; Sirr et al . , 2015; Muller and McCusker , 2009 ) and certainly industrial strains ( Bakalinsky and Snow , 1990; Bond et al . , 2004; Hadfield et al . , 1995 ) .", "Aneuploidy commonly emerges in response to selective pressure ( Mulla et al . , 2014 ) ; interestingly , a recent study by Filteau et al . showed that aneuploidy was relatively frequent in a wild strain , but not a laboratory strain , subjected to the same selection conditions ( Filteau et al . , 2015 ) .", "W303 harbors six auxotrophies , several of which affect but do not entirely explain the intolerance of Chr12 amplification ( unpublished ) .", "Thus , while many nice studies have been done with the W303 laboratory strain , we caution against using it ( or any single strain ) as the sole representative of S . cerevisiae ( Gasch et al . , 2016 ) .", "To examine expression effects in naturally aneuploid strains , we measured mRNA and DNA abundance in aneuploid isolates compared to closely related or isogenic euploid controls .", "We did three sets of analyses in Hose et al . We first surveyed expression in six naturally aneuploid strains compared to paired euploid relatives , in biological duplicate .", "Pooling data across the strains identified 838 out of 2 , 204 amplified genes ( 38% over all genes considered in the six strains ) whose relative mRNA abundance was reproducibly lower than the relative DNA abundance measured in the strain pairs ( Hose et al . Figure 4A ) .", "The reduced expression could be because of a response to the aneuploidy , a response to the increased gene copy number , or due to heritable polymorphisms that reduce expression – we emphasize the presentation in Hose et al . that these genes should not be taken as dosage compensated from this analysis alone .", "To distinguish the above possibilities , we performed two analyses on isogenic strain pairs , in which the only difference between strains was chromosome copy number .", "First , we measured mRNA abundance ( in biological duplicate ) for three aneuploid strains and their isogenic euploids and compared relative mRNA abundance to relative DNA abundance measured in those strain pairs ( outlined in more detail below ) .", "This identified 163 of 882 genes ( or 18% of the total set of genes assessed across these three strains ) with lower-than-expected expression in aneuploid cells ( Hose et al . Figure 4B and Supplementary File 3 ) .", "Second , we generated isogenic strain panels for two other naturally aneuploid strains , in which diploid cells carried two , three , or four copies of the amplified chromosome .", "Using a mixture-of-linear regressions ( MLR ) model , we defined genes whose relative mRNA abundance did not increase proportionately to relative DNA abundance measured in the aneuploid versus isogenic euploid strains .", "The MLR analysis identified 172 genes in this class out of 773 genes ( or 22% of the genes assessed in these two strain panels , Hose et al . , Table 1 , Class 3a ) .", "We then combined the gene groups from the two analyses of isogenic strain sets for downstream analysis .", "Because the paired strain analysis was less stringent ( in part because only duplicates were analyzed ) , we required that genes be identified in both Figure 4B ( isogenic strain pairs ) and Figure 4A ( non-isogenic strain pairs ) .", "This left 73 genes whose expression was lower-than-expected in four biological measurements from isogenic strain pairs plus the 172 genes identified by the more sensitive MLR analysis from the isogenic strain panels , for a combined total of 245 genes out of 1655 ( 15% ) total genes assessed in the two analyses of isogenic strains .", "In Hose et al . , we cited that 10–30% of genes , depending on strain and chromosome , met our criteria .", "There was an error in the abstract of the published article that we request to be changed , where '>30%' should have read 'up to 30%' of genes may be subjected to dosage compensation .", "We regret the error , but point out that the correct values are cited clearly throughout the manuscript . 10 . 7554/eLife . 14409 . 003Table 1 . The center ( mean ) of log2 distributions for mRNA or DNA ratios measured across all unamplified genes in isogenic aneuploid-euploid strain comparisons are shown .", "mRNA data from each strain was normalized by either ‘spike-in’ of Sz .", "pombe cells to the S . cerevisiae cell collections or by reads-per-kb per million mapped reads ( RPKM ) of S . cerevisiae genes only .", "Normalized data were then compared across strain pairs to provide a log2 ratio of relative mRNA or DNA abundance for each gene . DOI: http://dx . doi . org/10 . 7554/eLife . 14409 . 003Mean log2 mRNA ratios ( spike-in ) Mean log2 mRNA ratios ( RPKM ) Mean log2 DNA ratios ( RPKM ) 1 SD used for thresholdT73_Chr8-4n vs -2n rep10 . 08−0 . 019−0 . 0720 . 197T73_Chr8-4n vs -2n rep20 . 102−0 . 020n . a .", "YJM428_Chr16-4n vs -2n rep1−0 . 401−0 . 037−0 . 1210 . 168YJM428_Chr16-4n vs -2n rep2−0 . 4570 . 004n . a .", "YPS163_Chr8-2n vs -1n rep1n . a .", "*-0 . 1460 . 0450 . 242YPS163_Chr8-2n vs -1n rep2n . a .", "*−0 . 135n . a .", "NCYC110_Chr8-3n vs -2n rep50 . 014−0 . 005n . a .", "NCYC110_Chr8-4n vs -2n rep50 . 2730 . 186n . a .", "NCYC110_Chr8-4n vs -2n rep1n . a . 0 . 058−0 . 076NCYC110_Chr8-4n vs -2n rep2n . a . 0 . 011−0 . 108NCYC110_Chr8-4n vs -2n rep3n . a . 0 . 078n . a .", "YPS1009_Chr12-4n vs -2n rep1n . a . 0 . 0140 . 003YPS1009_Chr12-4n vs -2n rep2n . a .", "-0 . 094−0 . 229YPS1009_Chr12-4n vs -2n rep3n . a .", "−0 . 184n . a .", "*We attempted spike-in normalization for haploid YPS163-disomic ( ‘2n’ ) and -monosomic ( ‘1n’ ) strains but were unable to accurately count cells due to differences in flocculation across aneuploid-euploid strains .", "As described in Hose et al . ( 2015 ) , RPKM normalization produced data that agreed as well or better across replicates compared to spike-in normalization and in the case of YJM428_Chr16-4n agreed better with qPCR measurements of Chr16 abundance in the culture ( not shown ) .", "Note data from NCYC110 replicate ( rep ) 5 were not used in the analysis but were generated during the Hose et al . manuscript revision stage for normalization controls .", "The SD of DNA ratios on the affected chromosome that were subtracted from gene-level measurements of relative DNA abundance ( i . e . to generate the gene-specific thresholds , see text ) are shown for reference where relevant .", "Torres et al . disagree with our analysis methods , stating that", "i ) the data were misnormalized ,", "ii ) the thresholds used were not valid ,", "iii ) the mixture of linear regressions ( MLR ) model was inappropriate , and", "iv ) we did not correct for false discovery .", "They further propose that expression differences in aneuploid strains fit a normal distribution across the transcriptome , which they take as a null model for no dosage compensation .", "Below , we briefly address each of these points in turn .", "First , as published in our original manuscript , several normalization methods were compared to ensure accuracy , including RPKM , RPKM excluding the amplified chromosomes , and the most accurate method of normalizing based on the number of collected cells .", "The latter is done by doping a fixed number of Schizosaccharomyces pombe cells relative to carefully counted S . cerevisiae cells in the collection , such that the S . cerevisiae sequencing reads can be scaled according to the known distribution of Sz .", "pombe reads across the samples ( see Hose et al . for specifics ) .", "This is the most appropriate method of normalization , since it makes no assumptions about the data; however , it is particularly challenging for wild yeast that are often flocculent and difficult to count .", "Nonetheless , for several of the strains we analyzed the data normalized by Sz .", "pombe ‘spike-in’ agreed as well with the same data normalized by RPKM as did biological replicates normalized by RPKM ( Table 1 ) .", "( The exception was YJM428_Chr16 for which the spike-in normalization was clearly off based on comparing calculated Chr16 relative abundance to qPCR-measured Chr16 relative abundance from the same culture pairs , not shown ) .", "Data values were otherwise similar for different normalization methods , indicating that RPKM is a valid approach ( Hose et al . , 2015 ) .", "Importantly , for our final analysis the DNA and RNA samples were normalized with the identical RPKM procedure – this produced global data centers ( i . e . mean log2 values across all measurements ) that were very similar for both mRNA and DNA measurements , indicating that the data were normalized in a comparable manner .", "Thus , there is no evidence that misnormalization of the data dramatically skewed our results .", "Second , the description of our analysis methods presented by Torres et al . does not recapitulate what was done in our manuscript .", "For isogenic strain pairs shown in Hose et al . Figure 4B , we generated biological duplicate RNA-seq replicates and single DNA-seq samples , where DNA and one RNA sample were taken from the same culture , for both the aneuploid and euploid strains .", "For each replicate , we measured the relative mRNA abundance for each gene in the aneuploid versus euploid strain , as well as the relative DNA abundance for that gene in the aneuploid versus euploid strain .", "We then defined a gene-specific threshold to identify genes with lower-than-expected mRNA abundance: we took the relative DNA abundance measured for a given gene , minus one SD of the chromosome-wide mean of relative DNA abundances ( Table 1 ) .", "We then identified genes whose relative mRNA abundance in the aneuploid versus euploid strain was lower than the gene-specific cutoff in both biological mRNA replicates .", "Comparing measured mRNA ratios to measured DNA ratios ( as opposed to theoretical DNA ratios ) is critical to capture systematic and stochastic variation in the measurements .", "Our thresholding method allowed us to account for gene-specific biases in sequencing counts while incorporating measurement noise ( and minimizing sequencing costs ) .", "Importantly , to meet our criteria for downstream analysis , a gene must have also met these criteria in the comparable analysis of non-isogenic strain pairs ( Hose et al . Figure 4A ) .", "Thus , each gene had to be expressed below the threshold in four biological replicates .", "We note that it would be inappropriate to analyze the mean gene-level values from small numbers of replicates , which was not done in our analyses .", "In attempt to estimate the false discovery rate ( FDR ) , we performed random permutations on the data ( see Methods ) .", "We estimate the FDR for this analysis to be below 15% .", "Torres et al . argue that our approach for selecting thresholds is not valid , citing the high standard deviation ( SD ) for mRNA abundance levels ( RPKM ) across the transcriptome ( Torres et al . Figure 8B ) .", "However , we did not use abundance values , but rather relative abundance across isogenic strains , and thus the SD of RPKM values across all transcripts is not relevant to our analysis .", "What is relevant is the SD of replicate mRNA abundance ratios compared to the SD of the relative DNA values used to define the threshold applied to replicates .", "The average SD of the replicate mRNA ratios for each gene ranged from 0 . 12–0 . 3 , for amplified genes and for unamplified genes .", "This was the same range as the SDs of the relative DNA abundance ratios ( 0 . 17–0 . 24 ) used to define the gene-specific thresholds ( Table 1 ) , which were applied to four biological measurements of relative mRNA abundance .", "Torres et al . attempt to estimate the false discovery rate of our method on permuted data; while their methods are not entirely clear , they identify hundreds more genes on randomized data than we did on real data , and the SD values cited in their Table 2 are not close to our values , suggesting critical differences in methods .", "In a second analysis , we generated two sets of isogenic strain panels in which diploid cells carried two , three , or four copies of a chromosome .", "Triplicate mRNA and duplicate DNA measurements were generated for each strain in each panel , and the data were fit using a sensitive mixture of linear regressions ( MLR ) model , which did not use the same cutoff approach to call genes .", "In fitting the MLR model , we did not average the replicates to reduce data to three points in a scatterplot per gene – such an approach could be adversely affected by outliers .", "Instead , we used all replicate measurements on all genes simultaneously , contrary to the assertion in Torres et al . The model takes into account variation in the replicate mRNA measurements ( see Hose et al . for details ) .", "For simplicity , we classified genes based on their maximum posterior probability , and no cutoff on the magnitude of the expression effect was used – indeed , we showed in the paper that many genes have only partially reduced expression ( Hose et al . Figure 4 and 6 ) .", "Though it was not a focus of the original report , the MLR mixture model allows for a conditional FDR assessment , following the approach in Newton et al . ( 2004 ) .", "The 142 genes called Class 3A in the YPS1009 panel correspond to 17% FDR; the 30 genes called Class 3A in the NCYC110 panel correspond to 5% FDR by this method .", "An independent check on MLR computations comes by using ordinary linear regression on the same input data , and using the regression to test the null hypothesis ( per gene ) of no deviation from proportional expression ( i . e . the null is intercept=0 and slope=1 ) .", "Under normal theory , the likelihood ratio test statistic has a chi-squared distribution on 2 degrees of freedom .", "An estimate of the proportion of null genes comes by processing the collection of chi-square p-values through Storey's q-value calculator ( Storey , 2003 ) .", "For both strain panels , these null proportion estimates are remarkably close to the Class 1 proportion estimates from MLR ( in the YPS1009 panel: 14% by q-value and 15% by MLR; in the NCYC110 panel: 7% by q-value and 8% by MLR , both among the set of genes showing linear relationships on the log scale mRNA versus DNA ) .", "The likelihood ratio approach does not offer a direct classification of non-null genes , in contrast to the favored MLR approach used in Hose et al . , but does provide a check on inferences about the breath of putative-dosage compensation effects .", "In summary , the two methods we used to identify genes expressed lower-than-expected in isogenic aneuploid versus euploid strains identified 245 genes for downstream analysis .", "This amounts to 10 to 30% of genes depending on the strain and affected chromosome , or 15% of all genes assessed , at an FDR between 5 and 17% .", "Torres et al . perform their own analysis to identify potentially dosage compensated genes , focusing on global averages , correlations , and distributions across the chromosomes .", "They argue that the distribution of expression effects across each aneuploid strain versus its isogenic euploid fits a normal distribution , in which there is a similar number of genes with higher expression on the affected chromosome as genes with lower expression .", "The authors cite that this is the null expectation in the absence of any genes subject to dosage compensation .", "While such a global approach would capture chromosome-wide dosage compensation as seen in sex chromosomes , we argue that this approach will miss many individual genes potentially subject to dosage control .", "As published in Hose et al . , there are genes on and off the amplified chromosome that have higher expression in the aneuploid strains .", "The MLR analysis reported nearly the same number of amplified genes with higher expression as with reduced expression ( Hose et al . Table 1 ) .", "We regret the magenta coloring to highlight amplified genes in Figure 4 , which was added during the revisions to make a separate point and thus the genes were not selected with the same criteria as genes with lower expression .", "Nonetheless , as we describe in Hose et al . , the two tails of the distribution are enriched for different functional groups: whereas amplified genes with lower-than-expected expression are enriched for particular functional groups ( see below ) and for genes that are toxic when over-expressed in the lab strain , the set of amplified genes with higher-than-expected expression is enriched for genes encoding membrane and cell-surface proteins .", "As we cited , the higher-than-expected expression of amplified genes ( and of genes on unamplified chromosomes ) is likely an indirect response to the known influence that ploidy has on cell size/shape and flocculence ( Wu et al . , 2010 ) .", "Therefore , we do not believe that the distribution across all genes can be used to select individual genes subject to dosage compensation , nor did we focus on global averages or correlations .", "Instead , we identified individual genes that have the lower-than-expected expression phenotype and analyzed the pooled set to explore enriched features , as described below .", "A major point of our original manuscript that was not addressed by Torres et al . is that the genes we identified through the above analyses are strikingly enriched for unique patterns .", "The set of genes with lower-than-expected expression in aneuploid strains is strongly enriched for certain functional groups , including genes known to feedback on their own expression ( see more below ) , genes that are toxic when over-expressed , and genes that are under higher expression constraint but display elevated copy number variation ( CNV ) in wild populations ( Hose et al . , 2015 ) .", "Such non-random associations are unlikely to occur if the gene selection was truly random and driven by noisy data .", "We ruled out the possibility that these genes reflect a common , indirect response to aneuploidy .", "However , it is true that a subset of the genes we identified could be responding transcriptionally to the particular chromosome amplified , rather than the gene’s copy number per se .", "We attempted to minimize these effects in our original downstream analysis by pooling amplified genes from five aneuploid strains and across three amplified chromosomes .", "Here , we provide an additional series of controls to show that the trends seen for the gene group we identified cannot be explained as indirect transcriptional responses to aneuploidy .", "As presented by Torres et al . , chromosome-specific responses to aneuploidy should affect groups of functionally related genes , whether or not the genes are amplified .", "In an attempt to remove amplified genes that may be a part of such indirect responses , we first identified unamplified genes in each aneuploid strain with significantly reduced expression compared to its isogenic euploid ( FDR 0 . 01 ) and identified enriched functional groups ( see Methods ) .", "We then removed from consideration all amplified genes in that strain belonging to any of those strain-specific functional groups .", "( Note: we consider cytosolic and mitochondrial translation factors as separate groups ) .", "This analysis removed ~30% of the genes classified as potentially dosage compensated in the original analysis; the majority of these were linked to mitochondrial function and is consistent with the common mitochondrial response we reported ( even though , interestingly , the removed genes were not affected in multiple aneuploid strains ) .", "We note that our procedure may remove genes that are legitimately dosage compensated , especially those that show only partial compensation , which could cause indirect effects on unamplified genes in the same functional category ( e . g . partial compensation of a transcriptional repressor ) .", "The revised set of amplified genes with lower-than-expected expression remains enriched for functional groups that cannot be explained by common or chromosome-specific responses , including cytosolic RPs ( p=6e-7 ) , other proteins involved in ribosome biogenesis ( p=5e-4 ) , and an overlapping gene set linked to translation ( p=3e-6 ) .", "RPs are known to feedback on their own expression ( Warner et al . , 1985; Vilardell and Warner , 1997; Dabeva and Warner , 1987; Dean et al . , 1981; Tsay et al . , 1988 ) , and we validated the trend at two genes ( Hose et al . , 2015 ) .", "The revised group is also weakly enriched for sequence-specific DNA binding proteins ( p=0 . 006 ) , including several transcription factors that bind their own promoters and/or regulate their own expression ( including , Hap1 , Stb5 , Bdf1 , and Rsc30 [Venters et al . , 2011; Deckert et al . , 1995; Hon et al . , 2005; Denby et al . , 2012] ) .", "Importantly , these enrichments remained significant when genes from each chromosome were held out ( p<0 . 007 , except for one case where DNA binding protein enrichment p=0 . 04 ) , showing that the result is not skewed by one particular aneuploidy .", "As cited in Hose et al . , these categories are not enriched among unamplified genes whose expression is affected by the common aneuploidy response ( Hose et al . , 2015 ) .", "Thus , our analysis is clearly enriching for genes subject to dosage compensation .", "All of the evolutionary signatures we reported in the original manuscript remain statistically significant on this reduced gene group ( Figure 1 ) .", "The revised gene set enriched for dosage-compensated genes displays significantly higher levels of CNV in S . cerevisiae populations , compared to all genes and compared to amplified genes with proportionately elevated expression ( Figure 1A ) .", "They also have significantly higher CNV buffering scores ( Figure 1B , see Methods and ( Hose et al . , 2015 ) ) , indicating a higher level of CNV despite increased expression constraint .", "If these trends are driven by genes subject to repression or indirect effects of aneuploidy , we would expect to see the same patterns for unamplified genes that show reduced expression compared to euploid controls .", "But this is not the case: unamplified genes with reduced expression ( identified either by edgeR or by the identical methods used to call dosage-compensated genes ) show no increase in CNV propensity or buffering scores compared to the control group .", "The trends for the affected amplified genes remain if we define genes using a more stringent threshold to call affected genes , if we remove from the analysis all genes belonging to any of the enriched functional groups , or if we separately hold out each amplified chromosome from the analysis ( see Methods ) .", "A final prediction is that genes with larger effect sizes in terms of the level of compensation should have more striking evolutionary patterns , and this is indeed the case ( Figure 1 ) .", "Therefore , the results we report cannot be explained as a secondary transcriptional response to the amplified chromosome – we believe that the group is legitimately enriched for genes subject to dosage control and that dosage compensation has an important effect on genome evolution . 10 . 7554/eLife . 14409 . 004Figure 1 . Gene sets enriched for dosage-compensated genes show unique signatures . Gene sets and the numbers contained within them are indicated by the key .", "The revised list of amplifed genes with lower-than-expected expression were partitioned into thirds , based on genes with the greatest ( top third ) or smallest ( bottom third ) reduction in expresison compared to expectation .", "( A ) The fraction of genes in each group for which at least three of 103 strains showed gene amplification .", "( B ) The distribution of Buffering Scores for genes with CNV .", "Here , Buffering Score represents the number of 103 strains with a gene duplication divided by expression constraint ( Vg/Vm , see Hose et al . for details ) .", "Higher values indicate a higher propensity for CNV despite expression constraint .", "The orange line indicates the median value for amplified genes with proportionate expression as a reference point .", "Asterisks indicate statistical significance ( p<0 . 035 ) compared to the amplified genes with proportionate expression .", "In some cases , the trends were consistent but not significant ( likely owing to small sample sets ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 14409 . 004 Our original intention in Hose et al . was not to claim that dosage compensation is ‘widespread’ in S . cerevisiae , but rather that it exists at a significant number of genes and has an important role in evolution .", "We certainly agree with Torres et al . that dosage compensation does not function at most amplified genes , as seen for sex chromosomes in other organisms .", "Our goal was not to define individual genes subject to dosage compensation , but rather to investigate the group – we caution that no single gene from our lists should be taken as dosage compensated without orthogonal evidence .", "In the end , the revised gene set we identified here at the highest confidence encompasses 157 ( 13% ) genes out of 1 , 243 interrogated across all the isogenic strain sets .", "It is true that some of the genes we identified , particularly those with only subtly reduced expression patterns , are false positive calls from our analysis .", "However , the number of genes we identified is inline with the 14% of dosage compensated genes reported by Springer and colleagues ( Springer et al . , 2010 ) ( using GFP reporters for 730 genes interrogated ) and is consistent with ( but on the lower end ) our original report of 10–30% .", "Future work will undoubtedly clarify the precise number and identity of genes subject to dosage compensation , as well as the genetic basis for phenotypic differences in aneuploidy tolerance in laboratory versus wild strains ." ], [ "In the process of this work , we verified our original analyses to define gene groups .", "We estimated the FDR of our thresholding method as follows:", "1 ) We calculated the log2 ratio of mRNA in aneuploid versus euploid cells , minus the corresponding log2 ratio of relative DNA abundance for that gene minus 1SD of the chromosome-wide mean of relative DNA values .", "A negative value indicates that the relative mRNA expression was below the relative DNA copy number minus 1SD .", "2 ) These difference values were randomly permuted with regard to the gene labels , within each of two replicates in both the isogenic and non-isogenic strain pairs .", "3 ) We calculated the number of genes for which all four of the randomized data values were negative , over 10 , 000 iterations .", "The FDR ( average number of genes identified from 10 , 000 iterations on random data divided by the number of genes identified in real data ) was calculated to be below 15 . 0% .", "We note that some fraction of true positives will meet the threshold in randomized trials .", "We also generated a revised list of putatively dosage compensated genes as follows: Unamplified genes with significant expression differences in each strain were identified using edgeR ( Robinson et al . , 2010 ) comparing RPKM values in aneuploid versus isogenic diploid strains .", "Biological duplicate RNA-seq data were analyzed for T73_Chr8-4n versus T73_Chr8-2n , YJM428_Chr16-4n versus YJM428_Chr16-2n , and YPS163_Chr8-disomic versus YPS163_Chr8-monosomic; biological triplicates were analyzed for YPS1009_Chr12-4n versus YPS1009_Chr12-2n and for NCYC110_Chr8-4n versus NCYC110_Chr8-2n ( see Hose et al . for all other details ) .", "Unamplified genes with an FDR <0 . 01 ( Robinson et al . , 2010 ) and a negative mean log2 ( fold-change ) in aneuploid versus euploid expression were taken as 'lower expressed' .", "Functional enrichment was done on the set of lower-expressed unamplified genes for each strain separately , using the program FunSpec ( Robinson et al . , 2002 ) and taking p<0 . 0004 as significant .", "Amplified genes from that strain belonging to any of the strain-specific enriched functional categories ( excluding those based on cellular localization ) were removed from the original list of putatively dosage compensated genes .", "Note that we considered cytosolic and mitochondrial RPs and translation factors as separate groups .", "One of the strains ( YJM428_Chr16-4n ) had a substantial secondary response at unamplified genes , amounting to two thirds of the 1 , 456 lower-than-expressed unamplified genes pooled across the isogenic strain pairs – the effected genes from YJM428_Chr16-4n were admittedly enriched for translation factors and rRNA biogenesis genes , causing the removal of amplified Chr16 genes in those categories from the list of affected genes .", "Nonetheless , the functional categories described in the text remained significant for the remaining 157 genes with reduced expression in aneuploid strains .", "For analysis in Figure 1 , the genes were ranked and partitioned into thirds based on the magnitude of expression reduction ( ‘effect size’ , where ‘top third’ represents amplified genes with the strongest reduction in expression ) , taken as the average relative mRNA ratio for the isogenic aneuploid and euploid strains compared to the relative DNA ratio between those strains .", "As a separate approach to call putatively dosage compensated genes , we used the same methods described for Figure 4B of Hose et al . , except that we selected genes whose relative mRNA values were less than the relative DNA ratio measured for that gene minus 3 SD of the chromosome-wide mean in that strain .", "A gene had to be below that gene-specific threshold in all ( two or three where relevant ) biological replicates of isogenic strain pairs .", "This identified 56 genes across the five strains .", "We applied an identical method to identify non-amplified genes with reduced expression; for this , we used the same SD as applied for amplified genes ( although this was generally very similar to the SD for all genes on unamplified chromosomes ) .", "This identified a total of 454 unamplified genes with lower expression ( ‘3SD’ in Figure 1A ) .", "We plotted the fraction of genes for which at least three of 103 strains previously analyzed had evidence of gene amplification ( see Hose et al . for details ) .", "Trends were similar when plotting the fraction of genes in which at least two strains displayed CNV .", "For simplicity , the buffering score plotted in Figure 1 represents the number of 103 strains with CNV divided by the expression constraint score Vg/Vm ( see Hose et al . for details ) .", "We performed a variety of other controls , ensuring that trends were the same when each chromosome was held out separately , when genes belonging to any functional group were held out , and using different cutoffs for CNV .", "The trends were consistent in all cases ( although not always statistically significant owing to small datasets in some cases ) ." ] ]

[ "In our prior work by Hose et al . , we performed a genome-sequencing survey and reported that aneuploidy was frequently observed in wild strains of S . cerevisiae .", "We also profiled transcriptome abundance in naturally aneuploid isolates compared to isogenic euploid controls and found that 10–30% of amplified genes , depending on the strain and affected chromosome , show lower-than-expected expression compared to gene copy number .", "In Hose et al . , we argued that this gene group is enriched for genes subject to one or more modes of dosage compensation , where mRNA abundance is decreased in response to higher dosage of that gene .", "A recent manuscript by Torres et al . refutes our prior work .", "Here , we provide a response to Torres et al . , along with additional analysis and controls to support our original conclusions .", "We maintain that aneuploidy is well tolerated in the wild strains of S . cerevisiae that we studied and that the group of genes enriched for those subject to dosage compensation show unique evolutionary signatures ." ]

[ "Cells package their DNA into structures called chromosomes .", "Sometimes when a cell divides , it fails to allocate the right number of chromosomes to each new cell and so they end up with too many or too few chromosomes .", "The extra copies of the genes on an additional chromosome can be harmful to the cells , because the levels of the proteins encoded by those genes may rise abnormally .", "Some organisms counteract the harmful effect of having additional chromosomes through a process called dosage compensation .", "Proteins are produced using genetic information via two steps: first a gene’s DNA sequence is copied into a molecule of RNA , which is then translated into a protein .", "Dosage compensation can inactivate single genes or whole chromosomes via various means to ensure that the levels of RNA expressed remain normal , even in the presence of extra genes .", "In 2015 , researchers from the University of Wisconsin-Madison reported that dosage compensation occurs in wild strains of budding yeast and effectively protects against the harmful effects of having extra chromosomes .", "However , these findings conflicted with earlier studies of laboratory strains of this yeast , and earlier in 2016 , other researchers re-analysed the previous study’s data and challenged its findings .", "Now , Gasch et al . – who conducted the work reported in 2015 – provide additional controls and computational experiments that support their original analysis .", "The latest analysis confirmed that the genes identified in the first study are indeed commonly duplicated in wild yeast populations , yet the expression of these genes remains controlled .", "This is consistent with a model of dosage compensation , for at least some of duplicated genes .", "Gasch et al . believe that part of the difference in interpretation of the data relates to perspective .", "The challenging researchers tested to see if there was a mechanism of dosage compensation that acted across entire chromosomes , which is known to occur in the case of sex chromosomes in mammals .", "Gasch et al . on the other hand took a different approach and looked to identify effects at the level of individual genes .", "Together , the analyses show that , while there is no evidence for a widespread mechanism , the expression of a select set of genes in wild yeast is consistent with gene-specific dosage compensation .", "Future work will now undoubtedly test the mechanisms behind the gene-specific effects , and explore why wild yeast strains are more tolerant to extra chromosomes than laboratory strains ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "biochemistry and chemical biology", "cell biology", "tools and resources" ]

[ [ "Many cellular proteins function under the control of biological regulatory systems .", "Protein–protein interactions ( PPIs ) comprise part of the biological regulation system for proteins .", "Besides PPIs , biological protein function is post-translationally promoted by multiple modifications such as complex formation , phosphorylation , and ubiquitination .", "Therefore , it is very important to understand how proteins interact with target proteins .", "The identification of partner proteins has been carried out using several technologies such as the yeast two-hybrid system ( Zhao et al . , 2017; Li et al . , 2016 ) , mass spectrometry analysis after immunoprecipitation ( Ohshiro et al . , 2010; Han et al . , 2015 ) , and cell-free-based protein arrays that we have previously described ( Nemoto et al . , 2017; Takahashi et al . , 2016 ) .", "These methods provide many critical findings .", "As intracellular proteins are regulated by quite complicated systems , such as signaling transduction cascades , the use of multiple technologies can strongly promote our understanding of cellular protein regulation .", "Proximity-labelling technology has been widely used to identify partner proteins ( Chang et al . , 2017 ) .", "As proximity labelling is thought to detect proteins that are very close together , it is expected to obtain more precise information about interacting proteins ( Kim et al . , 2014 ) .", "Currently , EMARS ( enzyme-mediated activation of radical sources ) ( Honke and Kotani , 2012; Kotani et al . , 2008 ) , APEX ( engineered ascorbate peroxidase ) ( Martell et al . , 2012; James et al . , 2019 ) , and BioID ( the proximity-dependent biotin identification ) Choi-Rhee et al . , 2004; Roux et al . , 2012 have been developed as proximity-labelling technologies .", "EMARS and APEX methods produce H2O2 as a label ( Kotani et al . , 2008; Martell et al . , 2012 ) .", "They can rapidly label protein , but H2O2 is highly cytotoxic ( Halliwell et al . , 2000 ) .", "At present , proximity biotinylation is based on the Escherichia coli enzyme , BirA .", "BioID ( proximity-dependent biotin identification ) was first reported in 2004 , and its main improvement was the single BirA mutation at R118G ( BirA* ) ( Choi-Rhee et al . , 2004 ) .", "BioID generally has promiscuous activity and releases highly reactive and short-lived biotinoyl-5′-AMP .", "Released biotinoyl-5′-AMP modifies proximal proteins ( within a distance of 10 nm ) ( Kim et al . , 2014 ) .", "BioID can be used by expressing the BioID-fusion protein and adding biotin .", "In cells expressing BioID-fusion bait protein , proteins with which the bait protein interacts are biotinylated and can be comprehensively analyzed using precipitation with streptavidin followed by mass spectrometry ( Roux et al . , 2012 ) .", "BioID can easily analyze the protein interactome in mild conditions .", "However , BioID takes a long time ( >16 hr ) and requires a high biotin concentration to biotinylate interacting proteins .", "Therefore , it cannot easily detect short-term interactions and is difficult to use in vivo .", "Second , BioID was improved using R118S and 13 mutations via yeast-surface display; this yielded TurboID ( Branon et al . , 2018 ) .", "TurboID has extremely high activity and can biotinylate proteins in only ten minutes .", "However , TurboID caused non-specific biotinylation and cell toxicity when labeling times were increased and biotin concentrations were high ( Branon et al . , 2018 ) .", "In addition , a small BioID enzyme from Aquifex aeolicus was reported as BioID2 ( Kim et al . , 2016 ) .", "BiolD , TurboID , and BiolD2 are excellent enzymes , and they offer some improvements for the proximity biotinylation of cellular target proteins .", "Further improvement of BirA enzymes is an important goal that would enhance the convenience of proximity biotinylation in cells .", "Evolutionary protein engineering using metagenome data have recently been used to improve enzymes ( Nakano and Asano , 2015; Nakano et al . , 2018; Nakano et al . , 2019 ) .", "Here , we newly designed five ancestral BirA enzymes using an ancestral enzyme reconstruction algorithm and a large genome dataset .", "The combination of ancestral reconstruction and site-directed mutagenesis has provided a newly useful BirA enzyme , AirID ( ancestral BirA for proximity-dependent biotin identification ) , which functions in proximity biotinylation in vitro and in cells .", "Although the sequence similarity between BioID and AirID is 82% , AirID showed high biotinylation activity against interacting proteins .", "Our results indicate that AirID is a useful enzyme for analyzing protein–protein interactions in vitro and in cells ." ], [ "BioID and TurboID were designed on the basis of the biotin ligase BirA from E . coli .", "Using a different approach , we attempted to reconstruct the ancestral BirA sequence .", "Five ancestral sequences were obtained using the following process .", "A comprehensive and curated sequence library was prepared querying the Blastp web server and using a custom Python script ( Source code 1 ) , which exhibited more than 30% sequence identity with E . coli BirA ( EU08004 . 1 ) .", "Next , further curation approaches were applied to the library as shown in previous studies ( Nakano et al . , 2018; Nakano et al . , 2019 ) .", "The procedure consists of the following steps:", "1 ) preparation of sequence pairs consisting of one of the submitted BirA sequences and one sequence ( total 1275 genes ) in the library ,", "2 ) sequence alignment of the all pairs , and", "3 ) selection of sequences bearing ‘key residues’ ( Figure 1A ) .", "In detail , we prepared the following four combinations of the key 26th , 124th , 171th , and 297th residues to classify the library: Ala , Val , Val , Ala ( pattern 1 , AVVA ) ; Ala , Phe , Val , Ala ( pattern 2 , AFVA ) ; Ala , His , Leu , Ala ( pattern 3 , AHLA ) ; and Gly , Phe , Val , Ala ( pattern 4 , GFVA ) ( Figure 1A ) .", "After the selection , we classified the library as follows: the library could be divided into 17 , 9 , 9 , or 66 genes depending on whether the key residues consisted of pattern 1 , 2 , 3 , or 4 , respectively ( Figure 1 ) .", "Utilizing each of the classified genes , we designed four artificial sequences using the ancestral sequence reconstruction ( ASR ) method ( Supplementary file 1 ) .", "The designed sequences were named on the basis of the patterns; the sequences were classified using the patterns 1 to 4 , which we refer to as AVVA , AFVA , AHLA , and GFVA , respectively ( Figure 1A ) .", "Furthermore , we added an ‘all’ BirA enzyme from the common ancestor of AVVA , AFVA , and GFVA .", "BirA enzymes of AVVA , AFVA , AHLA , and GFVA shared similarity with those in the Shewanella genus , the Frischella and Glliamella genera , the Thiobacillus and Betaproteobacteria genera , and multiple genera , respectively .", "When the AVVA , AFVA , AHLA , GFVA , and 'all' amino-acid sequences were compared to the sequence E . coli BirA they showed 45% , 58% , 42% , 82% , and 73% similarity , respectively , and the region including the active site ( 107–134 amino acids ) was the same sequence throughout .", "On the basis of the amino-acid sequences discussed above , five DNA templates for AVVA , AFVA , AHLA , GFVA , and 'all' were prepared using artificial DNA synthesis .", "To convert the designed proteins into DNA sequences , we used the codon usage profile from the plant Arabidopsis , the average genic AT content of which is nearly 50% ( Arabidopsis Genome Initiative , 2000 ) .", "All ancestral BirA genes were fused an N-terminal AGIA tag , because this is a highly sensitive tag based on a rabbit monoclonal antibody ( Yano et al . , 2016 ) .", "We synthesized these ancestral BirA proteins ( AncBirAs ) using a wheat cell-free protein production system to investigate their enzymatic potentials ( Sawasaki et al . , 2002 ) .", "Biotin ligase activity was subsequently checked as all ancestral BirA proteins were obtained as a soluble form ( Figure 1B ) .", "A His-bls-FLAG-GST protein with a N-terminal biotinylation site ( bls ) of GLNDIFEAQKIEWHE for E . coli BirA ( EcBirA ) was used as a substrate .", "Three ancestral BirA proteins—AFVA , GFVA , and 'all'—showed activity against the bls sequence ( Figure 1C ) .", "GFVA had the greatest activity , similar to that of EcBirA , whereas AVVA and AHLA did not have activity .", "In all of the designed ancestral BirA proteins , an arginine residue corresponding to R118 of EcBirA was conserved in an active site for biotinylation ( Supplementary file 1 ) .", "Because EcBirA gained proximity biotinylation activity as a result of the R118G mutation known as BioID ( Choi-Rhee et al . , 2004 ) , each R residue in the five genes was substituted with glycine ( RG mutants ) .", "To compare the proximity biotinylation activity among these genotypes , wild-type or RG mutant BirA gene was N-terminally fused to the p53 gene .", "The resulting BirA-p53 proteins were synthesized using the cell-free system before mixing with FLAG-GST ( FG ) -MDM2 , because an interaction between p53 and MDM2 has been widely observed ( Momand et al . , 1992; Michael and Oren , 2003 ) .", "Immunoblotting revealed BirA-p53 biotinylation in EcBirA-RG ( BioID ) , AVVA-WT , AVVA-RG , AFVA-RG , and GFVA-RG ( Figure 1D ) .", "FG-MDM2 proximity biotinylation was detected under these conditions in three ancestral BirA-RG mutants—AVVA-RG , AFVA-RG , and GFVA-RG—indicating that they are candidate enzymes for proximity biotinylation .", "AVVA-RG showed both the highest activity of proximity biotinylation and extra biotinylations in the lower size region ( ‘non-specific biotinylation’ in Figure 1D ) when compared to the three ancestral BirA-RG mutants .", "GFVA-RG indicated the highest biotinylation activity for a specific peptide ( Figure 1C ) and the lowest extra proximity biotinylation .", "According to these results , we focused on two enzymes , AVVA and GFVA , for further analysis .", "TurboID was recently reported as an improved BioID enzyme ( Branon et al . , 2018 ) .", "As TurboID has an R118S mutation ( RS mutant ) that increases the activity of proximity biotinylation , we made RS mutants of the two ancestral BirA enzymes and compared their proximity biotinylation activities in vitro and in cells .", "An interaction between N-terminal AGIA-BirA-fusion p53 ( AGIA-BirA-p53 ) and FG-MDM2 was used to validate proximity biotinylation ability in vitro .", "Incubation time , biotinylation temperature , and biotin concentration were investigated as conditions for proximity biotinylation .", "Consequently , TurboID , AVVA-RG , AVVA-RS , and GFVA-RS showed higher proximity biotinylation activity after 3 hr than did BioID with a 16-hr incubation ( Figure 2A ) .", "The GFVA enzyme with a RS mutation dramatically increased the activity of proximity biotinylation to RelA ( GFVA-RS in Figure 2A and Figure 2—figure supplement 1 ) , but proximity biotinylation was almost the same in AVVA-RG and -RS .", "AVVA-RG , AVVA-RS , and GFVA-RS showed high activity of proximity biotinylation at temperatures above 16 °C ( Figure 2—figure supplement 1A ) .", "AVVA-RG , AVVA-RS , and GFVA-RS showed high proximity biotinylation activity at biotin concentrations greater than 0 . 5 µM ( Figure 2—figure supplement 1B ) .", "On the basis of these results , three BirA enzymes—AVVA-RG , AVVA-RS , and GFVA-RS—were used for further analysis .", "We used IκBα and RelA to validate the proximity biotinylation ability of these three enzymes in other protein–protein interactions because the IκBα–RelA interaction has been widely observed ( Beg et al . , 1992; Baeuerle and Baltimore , 1988 ) .", "As in the analysis of the p53–MDM2 interaction , N-terminal AGIA-BirA-fusion IκBα ( AGIA-BirA-IκBα ) and FLAG-GST-RelA ( FG-RelA ) sequences were constructed .", "FG-MDM2 was used as a negative control .", "To compare the abilities of the different enzymes directly , the reactions of all enzymes were carried out under the same conditions .", "After co-incubating AGIA-BirA-IκBα and FG-RelA , AVVA-RS or GFVA-RS , high RelA biotinylation was indicated ( Figure 2B ) .", "FG-MDM2 biotinylation by AGIA-BirA-IκBα was not observed .", "Next , the proximity biotinylation ability of these three enzymes was validated in cells .", "MDM2 dramatically degrades p53 protein in cells ( Michael and Oren , 2003 ) , so a CS mutant ( MDM2 ( CS ) ) lacking E3 ubiquitin ligase activity was used for this assay .", "In addition , GFP ( green fluorescent protein ) was terminally fused to MDM2 ( GFP-MDM2 ( CS ) ) because the mobility size of this fusion protein on SDS-PAGE is very similar to that of BirA-fusion p53 and MDM2 .", "AGIA-BirA-p53 fusions were transiently expressed in HEK293T cells with or without GFP-MDM2 ( CS ) , and they were compared with or without biotin supplement .", "GFVA-RS showed higher biotinylation of FG-MDM2 than did other enzymes under conditions without biotin supplementation ( left panel in Figure 2—figure supplement 2 ) .", "Furthermore , GFVA-RS also indicated biotinylation of FG-MDM2 under biotin supplementation conditions ( right panel ) .", "Taken together , these results indicated that an ancestral BirA with GFVA-RS is a good enzyme for analyzing protein–protein interactions both in vitro and in cells .", "Thus , we selected GFVA-RS , and we called this AirID ( ancestral BirA for proximity-dependent biotin identification , an homage to BioID and TurboID ) .", "Before utilizing AirID for various applications , we assessed AirID for the two activities self-biotinylation and 5'-biotinyl-AMP production , because these activities indicate proximity biotinylation .", "It is known that the p53 protein makes a homo multimer ( Friedman et al . , 1993; Delphin et al . , 1994 ) .", "Each enzyme alone or the p53-fusion form was used to investigate the two activities in BioID , TurboID , and AirID .", "BioID and TurboID showed self biotinylation , and TurboID had the highest activity ( Figure 2—figure supplement 3 ) .", "AirID did not have the activity , indicating that AirID does not self-biotinylate .", "As TurboID was selected as an enzyme by screening the yeast-surface display that showed the highest self-biotinylation activity ( Branon et al . , 2018 ) , the highest activity from TurboID is reasonable .", "The lack of self-biotinylation activity in AirID may be caused by a property of AirID as the enzyme or by a lack of accessible lysine residues on AirID .", "We next investigated the ability of the AirID enzyme to produce biotinoyl-5′-AMP .", "His-tagged TurboID , GFVA-RG , and AirID proteins were produced in an E . coli system and purified using nickel sepharose beads .", "Highly purified enzymes were obtained ( Figure 2—figure supplement 4A ) and biotinylation of TurboID was found , indicating that TurboID biotinylated itself in E . coli cells .", "As shown in Figure 2—figure supplement 3 , self-biotinylation of AirID was not observed .", "Furthermore , to investigate the biotinylation ability of AirID at the biotin ligation site ( bls ) of E . coli BirA , purified His-tag and bls fusion FLAG-GST protein ( His-bls-FLAG-GST ) was used as a substrate .", "AirID and GFVA-RG biotinylated His-bls-FLAG-GST ( Figure 2—figure supplement 4B ) , but TurboID did not do so .", "Radio-isotope-labelled ATP [32P-α-ATP] was used according to a previous report ( Henke and Cronan , 2014 ) to detect biotinoyl-5′-AMP production by the enzymes .", "The ATP concentration reported by this assay was very low ( final 1 µM ) because of the use of labelled ATP .", "AirID and GFVA-RG produced biotinoyl-5′-AMP , and this activity was decreased by supplementing with His-bls-FLAG-GST ( Figure 2C ) .", "AMP was increased at the same time , indicating that biotinoyl-5′-AMP is produced by these enzymes and that AMP is released after biotinylation of His-bls-FLAG-GST .", "Biotinoyl-5′-AMP formation was shown as AirID > GFVA-RG > TurboID by comparing the enzymes under these conditions ( Figure 2—figure supplement 4C ) , suggesting that AirID has higher biotinoyl-5′-AMP formation than TurboID under low ATP conditions .", "We compared the optimal conditions for proximity biotinylation in cells between BioID , TurboID , and AirID .", "As a model of proximity biotinylation , AGIA-BirA-fusion p53 and FG-MDM2 ( CS ) were co-expressed in cells .", "Biotin concentration and biotinylation time were investigated as the variable conditions for proximity biotinylation .", "Consequently , AirID and TurboID biotinylated MDM2 at biotin concentrations higher than 0 . 5 µM within 3 hr and 1 hr in cells , respectively ( Figure 2—figure supplement 5A and B ) .", "Although TurboID-p53 dramatically increases biotinylation of high molecular weight products with long incubations of >6 hr with >5 µM biotin , AirID-p53 showed similar results from 3 to 24 hr and with 0 . 5–50 µM biotin supplementation in culture medium .", "This indicates that the AirID-fusion protein could function in a wide variety of conditions .", "Furthermore , PPI dependency was examined between AirID-IκBα and TurboID-IκBα .", "GFP and either AGIA-AirID-IκBα or AGIA-TurboID-IκBα were coexpressed in HEK293T cells .", "Next , biotinylation after 1 , 3 , 6 , and 24 hr incubation with 5 µM of biotin supplementation was analyzed using a streptavidin-pull down assay .", "Each protein was detected using each specific antibody .", "As shown in the input sample ( left panel in Figure 2D ) , expression of both fusion enzymes was at nearly the same level ( IB: AGIA ) .", "TurboID showed much higher biotinylation in whole lysates than did AirID .", "In the pull-down assay , both enzymes biotinylated endogenous RelA at all points .", "After 1 hr of incubation , biotinylation of co-expressed GFP was found in TurboID-IκBα , and continuous tubulin biotinylation was also carried out after 3 hr ( right panel in Figure 2D ) .", "They found no AirID-IκBα biotinylation , although AirID was incubated for 24 hr with biotin .", "These results indicated that AirID has high PPI dependency .", "We used AirID for various in vitro applications .", "It has been widely known that p53–MDM2 interaction is inhibited by nutlin-3 ( Vassilev et al . , 2004 ) .", "Nutlin-3 was used to investigate whether AirID can be used to validate an inhibitor of PPI .", "Immunoblotting revealed that nutlin-3 inhibited FG-MDM2 biotinylation by AGIA-AirID-p53 ( Figure 3A ) .", "As we used AlphaScreen technology for drug screening of PPI in previous reports ( Uematsu et al . , 2018; Nemoto et al . , 2018; Nomura et al . , 2019; Yamanaka et al . , 2020 ) , we used it to detect the biotinylation of drug-dependent PPI inhibition ( Figure 3B ) .", "FG-MDM2 biotinylation by AGIA-AirID-p53 interaction was also detected ( 0 µM in Figure 3C ) using AlphaScreen technology , and the signal was decreased by supplementing with nutlin-3 ( >10 µM ) .", "These results indicate that AirID can detect PPI inhibition by the drug .", "Thalidomide and its derivatives such as pomalidomide bind to cereblon ( CRBN ) ( Ito et al . , 2010; Lopez-Girona et al . , 2012 ) and degrade target proteins such as IKZF1 ( Lu et al . , 2014; Krönke et al . , 2014 ) and SALL4 ( Matyskiela et al . , 2018 ) in cells .", "These small chemical compounds are known as molecular glue ( Fischer et al . , 2016 ) .", "As the CRBN-YW/AA mutant loses thalidomide binding ability ( Ito et al . , 2010 ) , it could not interact with IKZF1 and SALL4 proteins .", "To investigate whether AirID detects a drug-dependent PPI in vitro , CRBN–IKZF1 and CRBN–SALL4 interactions were analyzed with or without pomalidomide .", "Biotinylations of FG-IKZF1 and FG-SALL4 by AGIA-AirID-CRBN ( WT ) were increased by supplementing with pomalidomide ( Figure 3D ) .", "However , biotinylations were not found in CRBN-YW/AA .", "These results indicate that AirID can detect a drug-dependent PPI in vitro .", "Furthermore , the biotinylations of neosubstrates by AirID-CRBN with pomalidomide were investigated in cells .", "Myc-tag fusion IKZF1 ( myc-IKZF1 ) and myc-SALL4 were transiently co-expressed with AGIA-AirID-CRBN ( WT ) or AGIA-AirID-CRBN-YW/AA in CRBN-knockout HEK293T cells .", "By supplementing with 5 µM biotin , biotinylations of both myc-IKZF1 and myc-SALL4 were detected by expressing AGIA-AirID-CRBN ( WT ) ( Figure 3—figure supplement 1 ) .", "They were not observed in co-expression with AGIA-AirID-CRBN-YW/AA .", "These results indicate that AirID can detect a drug-dependent PPI in cells .", "CRBN is involved in the Cullin-4 complex consisting of DDB1 , RBX1 , and CUL4 ( Fischer et al . , 2014 ) .", "To investigate whether AirID detects proteins in a multiple complex , AirID-CRBN was mixed in with the complex members .", "Biotinylations of DDB1 , CUL4 , and RBX1 by AGIA-AirID-CRBN were observed ( Figure 3E ) , but AGIA-AirID was not biotinylated .", "This indicated that AirID can detect PPI in a multiple protein complex .", "The Flowering locus T ( FT ) protein , known as the flowering hormone florigen in plants , induces the differentiation of flowering with Flowering locus D ( FD ) protein , which has a bZip DNA-binding domain ( Abe et al . , 2005 ) .", "FT–FD interaction in the floral meristem has been thought to be an important event for flowering development ( Jaeger et al . , 2006 ) .", "To investigate whether the FT–FD interaction detects biotinylation of AirID , FT and FD genes in Arabidopsis were selected to co-synthesize FT-AirID and AGIA-FD proteins using by the wheat cell-free system with 500 nM biotin .", "As a negative control , E . coli dihydrofolate reductase ( DHFR ) was synthesized with FT-AirID .", "Under these conditions , FT-AirID biotinylated AGIA-FD , but AGIA-DHFR biotinylation was not observed ( Figure 3—figure supplement 2 ) .", "This co-translational condition reaction was incubated for 16 hr at 16 °C with biotin , indicating that AirID-dependent biotinylation functions in co-translational conditions based on the cell-free system .", "Taken together , these results indicate that the AirID enzyme is useful for biochemical analysis of PPI .", "We next analyzed the cellular localization of AirID and cellular biotinylation by AirID .", "The p53 protein is known to localize mainly to the nucleus ( Shaulsky et al . , 1990; Rotter et al . , 1983 ) .", "AGIA-AirID alone or AGIA-AirID-p53 was transiently expressed in HEK293T cells .", "Fluorescent streptavidin was found in whole cells by supplementing AirID expression cells with biotin ( 50 µM in Figure 4A ) .", "In AirID-p53 expression cells , the fluorescence was mainly observed in the nucleus , and it was at the same level for cells exposed to either 5 µM or 50 µM biotin concentration ( Figure 4A ) .", "Cellular fractions from cytosol and nuclei were isolated to confirm the cellular localization by immunostaining .", "These fractionations indicated that AirID and AirID-p53 were mainly found in the cytosol or nucleus , respectively ( Figure 4B ) .", "This suggested that AirID-fusion protein localization is dependent on fusion protein features .", "We investigated whether proteins that were biotinylated by AirID have native function .", "Because MDM2 has been known to induce p53 degradation via the ubiquitin-proteasome system in cells ( Michael and Oren , 2003 ) and AirID-p53 provided both self and MDM2 biotinylation ( Figure 2A ) , AGIA-AirID-p53 and GFP-MDM2 were transiently co-expressed in cells .", "Treatment with proteasome inhibitor MG132 inhibited AirID-p53 degradation , but degradation was extremely decreased without the treatment ( Figure 4C ) .", "The inactive MDM2 form , FG-MDM2 ( CS ) , also did not promote AirID-p53 ( Figure 2A ) , indicating that AirID-p53 degradation is carried out by GFP-MDM2 .", "In addition , MG132 treatment biotinylated AirID-p53 under biotin supplementation conditions .", "These results indicated that biotinylated MDM2 works as a E3 ligase for biotinylated AirID-p53 .", "RelA was selected to investigate the transactivation activity of the biotinylated transcription factor because RelA has transactivation activity for the NF-κB promoter ( Ganchi et al . , 1992 ) .", "Two types of expression plasmids , AGIA-AirID-RelA and AGIA-RelA , were constructed .", "Each plasmid was transiently transfected in HEK293 cells with a NF-κB promoter-luciferase plasmid .", "Biotin supplementation induced biotinylation of AGIA-AirID-RelA , but AGIA-RelA biotinylation was not found ( Figure 4D ) .", "The luciferase assay revealed that the transactivation activity of AirID-RelA was nearly the same for AGIA-RelA , AGIA-AirID-RelA , and biotinylated AGIA-AirID-RelA , indicating that biotinylated RelA functions as a normal transcription factor .", "TurboID almost completely inhibits HEK293T cell growth under 50 µM biotin supplementation conditions ( Branon et al . , 2018 ) .", "HEK293T cells that stably expressed AirID or AirID-IκBα were constructed using a lentivirus system to investigate whether AirID affects HEK293T cell viability .", "In the stable cells expressing AirID-IκBα , RelA biotinylation was clearly found by supplementing with >50 µM for 6 hr .", "It was not found in cells in which AirID was stably expressed ( Figure 4—figure supplement 1 ) .", "The cell growth of both cell types was not fully inhibited by 50 µM biotin supplementation ( Figure 4E ) , indicating that AirID does not affect cell viability under 50 µM biotin supplementation conditions .", "It was demonstrated that TurboID showed cytotoxicity within 48 hr with 50 µM biotin ( Branon et al . , 2018 ) .", "Therefore , AirID- or TurboID-expressing cells were cultured with or without biotin for 48 hr and then the viability of them was analyzed .", "Compared with control ( Mock ) , the viability of TurboID-expressing cell was significantly decreased with 50 µM biotin , but the viability of AirID-expressing cells was not significantly affected ( Figure 4—figure supplement 2 ) .", "We investigated whether AirID could biotinylate dependently interacting endogenous proteins in cells .", "Streptavidin-conjugated beads were used as in a previous report to recover biotinylated endogenous proteins from cell lysates ( Van Itallie et al . , 2013 ) .", "AGIA-AirID-IκBα was transiently expressed in HEK293T cells under different biotin concentration conditions .", "Streptavidin-pull down assay of the cell lysates was carried out , and the biotinylated endogenous proteins were detected using immunoblotting with each specific antibody .", "Endogenous RelA protein was biotinylated without supplemental biotin by transiently expressing AGIA-AirID-IκBα in cells ( IB: RelA , 0 µM biotin concentration in Figure 5A ) .", "Biotin supplementation enhanced biotinylations of p50 or p105 , which are known IκBα interactors ( IB: p50/p105 and p50 , 5 µM or 10 µM biotin concentration ) .", "These biotinylations were not found for AGIA-AirID alone .", "As the IκBα protein interacts with RelA , this result illustrated that biotinylated AGIA-AirID-IκBα may bring endogenous RelA without biotinylation .", "To confirm this , immunoprecipitation using a specific antibody recognizing endogenous RelA was performed in severe conditions after proteins were denatured with 1% SDS .", "Biotinylation of endogenous RelA recovered by immunoprecipitation was observed as a result ( Figure 5B ) .", "Using the same lysates , the streptavidin-pull down assay recovered RelA protein , indicating that RelA biotinylation depends on AGIA-AirID-IκBα .", "These results showed IκBα-interaction-dependent biotinylation by AirID .", "As in vitro protein biotinylation involving in a CRL4AirID-CRBN complex was found ( Figure 3E ) , we investigated whether proteins in the CRL4 complex were biotinylated by AirID-fusion CRBN in cells .", "AGIA-AirID-CRBN was transiently expressed in HEK293T cells , and cell lysates were pulled down using streptavidin beads .", "Biotinylation of CUL4 and RBX1 was found after supplementing with 5 µM biotin ( Figure 5C ) , but DDB1 biotinylation was not found .", "Taken together , these results indicate that the biotinylation assay using the AirID-fusion target is a useful tool for analyzing PPI in the cell .", "Since BioID has been widely used to identify PPI in cells using mass spectrometry ( MS ) analysis ( Ikeda and Freeman , 2019 ) , we also analyzed biotinylated proteins using LC-MS/MS in the cells stably expressing AirID alone or AirID-IκBα that were used in Figure 4E .", "The flowchart for the analysis of biotinylated peptides is shown in Figure 6A .", "The cells were treated with 50 µM biotin for 6 hr .", "Proteins were digested using trypsin after cell lysis .", "Biotinylated peptides were captured using Tamavidin2-Rev .", "Tamavidin2-Rev can bind to biotin-labelled substances and can release them under high concentrations of free biotin ( Takakura et al . , 2013 ) .", "The biotinylated peptides were eluted using 2 mM biotin , and the eluted peptides were analyzed using LC-MS/MS .", "The biotinylation by free biotinoyl-5′-AMP occurs on lysine ( Lys ) residues on proteins ( Choi-Rhee et al . , 2004 ) .", "Trypsin digests Lys or arginine ( Arg ) , but it cannot cleave modified Lys in the same way as it does biotinylated Lys ( Bheda et al . , 2012 ) .", "These features show that an eluted biotinylated peptide has a single biotin , indicating that the direct determination of biotinylated peptide provides a biotinylation site on the peptide .", "Using this method , we found 12 biotinylated peptides that were present in AirID-IκBα-expressing cells at levels that were more than five times higher than those in cells expressing only AirID ( Figure 6B and C , Figure 6—source data 1 ) .", "In the top five peptides , three biotinylated peptides were derived from RelA proteins ( Figure 6C ) , indicating that AirID-IκBα could accurately biotinylate a major partner RelA protein in the cells .", "Furthermore , we investigated whether AirID-dependent biotinylation occurs in a specific region .", "Comparison of amino-acid sequences among the top 20 biotinylated peptides showed no similarity except for a single Lys residue ( Figure 6—figure supplement 1 ) , suggesting that the proximate biotinylation by AirID happens on the Lys residue but does not have a preferred sequence .", "In Figure 5A , the streptavidin pull down clearly showed biotinylation of the endogenous RelA protein in transiently AirID-IκBα expressing cells .", "We assessed whether an AirID-IκBα-dependent biotinylation of RelA could be detected using LC-MS/MS in transiently AirID-IκBα expressing cells .", "A flowchart for the analysis of biotinylated peptides using transiently expressing cells is shown in Figure 6D .", "As expected , the top biotinylated peptide was RelA ( Figure 6E ) , as in stably expressing cells .", "Taken together , these results suggest that detection of AirID-dependent biotinylation using LC-MS/MS is useful for PPI analysis in cells ." ], [ "Here , we used an algorithm of ancestral enzyme reconstruction using a large genome dataset , and we investigated five ancestral BirA enzymes .", "Finally , we combined biochemical experiments and RS mutations to create AirID with high PPI proximity biotinylation .", "Classical evolutionary protein engineering used random mutations to improve the activity ( Branon et al . , 2018 ) .", "Therefore , the sequence similarity is extremely high because random mutations cannot provide dynamic sequence changes .", "However , sequence similarity between E . coli BirA and ancestral BirA was between 40% to 80% , indicating that a computational approach using large genome datasets can more dynamically design enzyme sequences .", "As another aspect to this approach , the BirA active region ( 115GRGRRG121 ) ( Kwon and Beckett , 2000 ) was conserved , and RG and RS mutations introduced into ancestral BirA enzymes ( Figures 1 and 2 ) .", "In the present direction of computational protein evolution , dynamic changes to the backbone region of protein enzymes with a conserved active pocket would be acceptable .", "Further accumulation of knowledge about the enzyme function would be required to change the enzyme active region dynamically .", "When we looked at BioID ( BirA* ) , TurboID , and AirID , the proximal biotinylation activity of BioID ( BirA* ) was considerably lower than that of TurboID and AirID ( Figure 2A and Figure 2—figure supplement 1 ) .", "By contrast , TurboID showed the highest proximate biotinylation activity in vitro and in cells ( Figure 2A and Figure 2—figure supplement 1 ) .", "This enzyme could be used for biotinylation within one hour ( Branon et al . , 2018 ) .", "However , the highest activity from TurboID provided extra biotinylation on unexpected proteins , such as like GFP or tubulin , in cells that were treated for a long incubation of more than six hours and higher biotin concentrations ( such as 50 µM biotin ) ( Figure 2D ) .", "In the first report describing TurboID , it was used as a biotin-labelling enzyme rather than as a proximal biotinylation enzyme for PPI ( Branon et al . , 2018 ) .", "If it was used analyze PPI , TurboID would show the best performance under limiting conditions , such as a short treatment ( 1 hr ) in cells .", "In the case of AirID , GFP and tubulin biotinylations were not observed in the same conditions as those catalyzed by TurboID ( Figure 2D ) .", "Streptavidin-pull down assay and LC-MS/MS analysis also indicated that AirID-fusion proteins were able to biotinylate each well-known interactor accurately in the transient- and stable-expression cells ( Figures 5 and 6 ) .", "The formation of biotinoyl-5′-AMP was greater for AirID than for TurboID in low ATP concentrations ( 1 µM ) ( Figure 2—figure supplement 4C ) , and it prefers lower concentrations of biotin ( with 5 µM biotin or without biotin supplement ) ( Figure 2—figure supplement 2 ) .", "In addition , analysis of biotinylation sites from LC-MS/MS showed that AirID biotinylation happened with no special sequence preference on a proximate Lys residue ( Figure 6—figure supplement 1 ) .", "Taken together , our AirID is expected to enhance PPI-dependent biotinylation accuracy , suggesting that AirID is suitable for PPI analysis in cells .", "Inhibition of MDM2–p53 interaction by nutline-3 was detected using AirID biotinylation ( Figure 3A and C ) , and several pomalidomide-dependent interactions between CRBN and neosubstrates were also detected by AirID biotinylation ( Figure 3D ) .", "These results indicate that AirID-dependent biotinylation would be useful for PPI analysis using chemical compounds .", "Furthermore , in vivo proximity biotinylation using BioID has been performed in many studies because the identification of in vivo partner proteins of target proteins is key for understanding biological functions ( Odeh et al . , 2018; Motani and Kosako , 2018 ) , and it has uncovered new PPIs .", "Stable expression of AirID-IκBα did not induce cell-growth inhibition even under biotin-supplementation conditions ( Figure 4E ) , suggesting that AirID-fusion protein expression would have very low toxicity .", "Therefore , AirID could also be used for in vivo screening for protein interactors of a target protein .", "In conclusion , AirID is a novel enzyme providing proximity biotinylation for PPI analysis ." ], [ "The BirA homologous sequences , classified into four groups using the key residues , were aligned using MAFFT software 2 ( Katoh et al . , 2002 ) .", "Each of the aligned sequences was analyzed using MEGA6 software 3 , and the phylogenetic tree was generated using the maximum likelihood method ( Tamura et al . , 2013 ) .", "Aligned sequences and phylogenetic tree data were submitted to FastML ( Ashkenazy et al . , 2012 ) .", "The JTT empirical model was adopted for analysis .", "Finally , we obtained four ancestral BirA forms named AVVA , AFVA , AHLA , and GFVA .", "Furthermore , we applied the three designed sequences ( AVVA , AFVA , and GFVA ) and an identical procedure to design another ancestral BirA called ‘all’ .", "All of the five designed sequences are shown in Supplementary file 1 .", "BioID ( BirA* ) , TurboID , or ancestral BirAs ( AncBirAs ) were cloned into pcDNA3 . 1-AGIA or pEU-AGIA vectors using the BamH1 and Not1 restriction sites .", "AncBirAs-fused p53 , IκBα or CRBN were cloned into pcDNA3 . 1-AGIA or pEU-AGIA vectors using BamH1 , Kpn1 , and Not1 .", "Mutants of AncBirAs were generated using site-directed mutagenesis with a PrimeSTAR mutagenesis basal kit ( Takara ) .", "MDM2 and RelA were cloned into pEU-FLAG-GST or pcDNA6 . 2-GFP vectors using the Gateway cloning system ( Thermo Fisher Scientific ) .", "Lentivirus-based AGIA-tagged AirID and AirID-IκBα plasmids were generated using restriction enzyme digestion of a CS II-CMV-MCS-IRES2-Bsd vector .", "For E . coli expression , TurboID , GFVA-R118G , or AirID was cloned into the pET30a-His vector using an In-Fusion HD Cloning Kit ( Takara ) .", "HEK293T cells ( purchased from RIKEN RCB , Tsukuba , Japan , catalog number RCB2202 ) were incubated at 37 °C and 5% CO2 in Dulbecco’s Modified Eagle Medium ( DMEM ) ( wako ) supplemented with 10% fetal bovine serum ( Biosera ) and antibiotics ( 100 units/mL penicillin and 100 µg/mL streptomycin ) ( Thermo ) .", "We confirmed that the cell line was free of mycoplasma contamination .", "Lentiviruses expressing AGIA-AirID and AGIA-AirID-IκBα were generated by transfection using PEI MAX - Transfection Grade Linear Polyethylenimine Hydrochloride ( Polyscience ) .", "After transmission of the transgene , a pool of HEK293T cells that were resistant to Blasticidin S ( 10 μg/mL ) ( Invitrogen ) was generated and used in subsequent experiments .", "In vitro transcription and wheat cell-free protein synthesis were performed using the WEPRO1240 expression kit ( Cell-Free Sciences ) .", "A transcript was made from each of the DNA templates mentioned above using SP6 RNA polymerase .", "The translation reaction was performed using the WEPRO1240 expression kit ( Cell-Free Sciences ) .", "For biotin labelling , 1 μl of BirA or of the ancestral BirAs produced by the wheat cell-free expression system were added to the bottom layer , and 500 nM ( final concentration ) of D-biotin ( Nacalai Tesque ) was added to both upper and bottom layers as described previously ( Sawasaki et al . , 2008 ) .", "The aliquots were used for expression analysis and functional characterization .", "1 mL of synthesized His-bls-FLAG-GST was mixed with Glutathione Sepharose 4B ( GE Healthcare ) and rotated for 3 hr at 4 °C .", "The mixture was washed with PBS .", "Proteins were eluted in 100 µL fractions with elution buffer ( 50 mM Tris-HCl [pH8 . 0] , 10 mM reduced glutathione ) .", "Protein was subjected to SDS-PAGE and CBB staining to determine purity .", "To purify TurboID , GFVA-R118G , and AirID proteins , the genes encoding them were inserted into pET30a and transformed into E . coli strain BL21 .", "The E . coli cells were grown at 37 °C in LB medium to an OD600 of 0 . 6 and induced by adding IPTG to 1 mM for an additional 6 hr at 37 °C .", "Cells were centrifuged and resuspended in lysis buffer ( 20 mM sodium phosphate , 300 mM NaCl , 10 mM imidazole ) .", "The cells were lysed using sonication , and the lysates were centrifuged .", "The supernatants were added to Ni Sepharose High Performance ( GE Healthcare ) and incubated for 3 hr at 4 °C .", "The mixture was washed with three column volumes of wash buffer ( 20 mM sodium phosphate , 300 mM NaCl , 50 mM imidazole ) .", "Proteins were eluted in 500 µL fractions with elution buffer ( 20 mM sodium phosphate , 300 mM NaCl , 500 mM imidazole ) .", "Fractions were dialyzed against PBS .", "Proteins were subjected to SDS-PAGE and CBB staining to determine purity .", "HEK293T cells were transfected with various plasmids using PEI MAX ( Polyscience ) .", "Immunoblotting was performed according to standard protocols .", "Briefly , proteins in whole-cell lysates were separated using SDS-polyacrylamide gel electrophoresis ( SDS-PAGE ) and transferred onto a PVDF membrane using semi-dry blotting .", "After blocking with 5% milk/TBST or Blocking one ( Nakalai Tesque ) , the membrane was incubated with the appropriate primary antibodies followed by a horseradish peroxidase ( HRP ) -conjugated secondary antibody .", "In vitro biotinylation assays were performed .", "Briefly , 5 µL of each synthesized protein was mixed and incubated at 26 °C for 1 hr .", "Biotin was added , and the biotinylation reaction was performed in a total volume of 15 µL .", "After the reaction , biotinylated proteins were analyzed using SDS-PAGE and immunoblotting .", "In cell biotinylation assays were also performed .", "Briefly , each BirA or BirA fused gene and substrate gene were transfected into HEK293T .", "At the same or each time , biotin was added and cells were lysed using SDS sample buffer ( 125 mM Tris-HCl [pH 6 . 8] , 4% SDS , 20% glycerol , 0 . 01% BPB , 10% 2-mercaptoethanol ) 24 hr after transfection .", "Whole cell lysates were analyzed using SDS-PAGE and immunoblotting .", "A streptavidin pull-down was performed to analyze biotinylated proteins .", "In vitro reaction mixtures after biotinylation were diluted with wash buffer ( 50 mM Tris-HCl [pH7 . 5] , 1% TritonX-100 , 150 mM NaCl ) , and 1% SDS was added to stop the reaction .", "After biotinylation , cells were lysed using RIPA buffer ( 50 mM Tris-HCl [pH 8 . 0] , 150 mM NaCl , 0 . 5% sodium deoxycholate , 0 . 1% SDS , 1% NP-40 ) including protease inhibitor cocktail and sonication .", "Lysates were centrifuged at 15 , 000 rpm at 4 °C for 10 min .", "SDS was added to supernatants to 1% .", "In vitro reaction mixtures or cell lysates were mixed with equilibrated Streptavidin Sepharose High Performance ( GE Healthcare ) and rotated at 4 °C for 1 hr .", "After flow-through was removed , beads were washed three times using wash buffer ( 50 mM Tris-HCl [pH7 . 5] , 1% TritonX-100 , 150 mM NaCl ) , and beads were boiled into SDS sample buffer .", "Boiled solution was analyzed using SDS-PAGE and immunoblotting .", "Synthesized FG-MDM2 and Nutlin-3 were mixed and incubated for 30 min at 26 °C .", "AGIA-AirID-p53 was added to the mixture and incubated for 1 hr at 26 °C .", "In addition , biotin was added to the reaction mixture to 500 nM and incubated for 3 hr at 26 °C .", "Inhibition was examined using the AlphaScreen IgG ( Protein A ) detection kit ( Perkin Elmer ) and immunoblotting .", "Briefly , for AlphaScreen , 10 μL of detection mixture containing 100 mM Tris-HCl ( pH 8 . 0 ) , 0 . 1% Tween 20 , 100 mM NaCl , 10 ng anti-FLAG antibody ( Sigma ) , 1 mg/mL BSA , 0 . 1 μL streptavidin-coated donor beads , and 0 . 1 μL protein A-conjugated acceptor beads were added to each well of a 384-well Optiplate before incubation at 26 °C for 1 hr .", "Luminescence was detected using the AlphaScreen detection program with an EnVision device ( PerkinElmer ) .", "For immunoblotting , solutions were boiled in SDS sample buffer .", "The boiled solution was analyzed using SDS-PAGE and immunoblotting .", "Cells after biotinylation were lysed with RIPA buffer and sonication for immunoprecipitation .", "Lysates were centrifuged and SDS was added to supernatants to denature proteins .", "Their solutions were diluted 10-fold .", "After 2 µg of the indicated antibodies were bound to either protein A or protein G Dynabeads ( Thermo Fisher Scientific ) for 30 min at room temperature , the beads were incubated with cell lysates diluted overnight at 4 °C .", "The immunocomplexes were boiled in SDS sample buffer after washing three times with PBS .", "The boiled solution was analyzed using SDS-PAGE and immunoblotting .", "Cells were seeded into 96-well plates at a density of 0 . 25 × 104 cells/well and treated with 50 µM biotin after 24 hr .", "Cell viability was determined using the MTS assay with a CellTiter96 Aqueous One Solution Cell Proliferation Assay kit ( Promega ) .", "In brief , 20 μL of the MTS reagent was added into each well , and the cells were incubated at 37 °C for 1 hr .", "The absorbance was detected at 490 nm ( reference: 650 nm ) with a Microplate Reader ( SpectraMaxM3 Multi-Mode Microplate Reader; Molecular Devices ) .", "Cells were seeded into 96-well plates at a density of 0 . 25 × 104 cells/well and transfected after 24 hr .", "After 2 days , cell viability was determined using CellTiter-Glo Luminescent Cell Viability Assay Cell system ( Promega ) .", "In brief , the CellTiter-Glo reagent was added into each well , and the cells were incubated at room temperature for 10 min .", "The luminescence was detected with a Microplate Reader ( GloMax Discover Microplate Reader ) .", "The assays contained 50 mM Tris-HCl buffer ( pH 8 . 0 ) , 5 . 5 mM MgCl2 , 100 mM KCl , 0 . 1 mM TCEP , 1 µM ATP including [α-32P]ATP , 25 µM biotin , 2 µM BirA and with or without 1 µM His-bls-FLAG-GST for a total reaction mixture of 10 µl ( Henke and Cronan , 2014 ) .", "The reaction mixtures were incubated at 37 °C for 30 min .", "A portion of each reaction mixture ( 1 µl ) was spotted onto cellulose thin-layer chromatography ( TLC ) plates and developed in isobutyric acid-NH4OH-water ( 66∶1∶33 ) ( Prakash and Eisenberg , 1979 ) .", "The thin-layer chromatograms were exposed to a phosphorimaging screen and visualized using a Typhoon FLA-3000 ( GE Healthcare ) .", "HEK293T cells were seeded onto a 24-well plate .", "Next day , cells were transfected , and biotin was added at the same time or each time .", "Subcellular fractionation was performed 24 hr after transfection using a ProteoExtract Subcellular Proteome Extraction kit ( Merck ) according to the protocol .", "Cells were fixed with 4% paraformaldehyde in phosphate-buffered saline ( PBS ) for 15 min at room temperature before permeabilizing with 0 . 5% Triton X-100 in PBS for 15 min .", "Cells were incubated with a primary antibody overnight at 4 °C after blocking with 0 . 5% CS in TBST for 1 hr .", "After washing with TBST , cells were incubated with the appropriate Alexa Flour 488- and/or 555-conjugated secondary antibody and streptavidin for 1 hr at room temperature .", "Nuclei were counterstained with 4 , 6-diamidino-2-phenylindole .", "After washing with TBST , coverslips were mounted with anti-fade .", "Preparation of cell lysates and reverse transcription were performed using SuperPrep II Cell Lysis and RT Kit for qPCR ( TOYOBO ) .", "Real-time PCR was carried out using KOD SYBR qPCR Mix ( TOYOBO ) .", "qRT-PCR primers used were as follows; TNF-α: 5′- CAGCCTCTTCTCCTTCCTGAT ( forward ) , 5′-GCCAGAGGGCTGATTAGAGAGA ( reverse ) ; GAPDH: 5′-AGCAACAGGGTGGTGGAC ( forward ) , 5′- GTGTGGTGGGGGACTGAG ( reverse ) .", "The proximity-dependent biotin identification method using AirID was performed according to a previous report ( Kim et al . , 2016 ) .", "Briefly , confluent HEK293T cells stably expressing AirID or AirID-IκBα fused at the N-terminus with an AGIA tag in a 6 cm dish were incubated with 50 μM biotin for 6 hr before harvesting using ice-cold PBS .", "Cell pellets were lysed and digested with trypsin .", "The digested peptides were incubated with Tamavidin2-Rev magnetic beads ( FUJIFILM ) before eluting with 2 mM biotin .", "Detailed procedures will be described elsewhere ( Motani K and Kosako H , in preparation ) .", "LC-MS/MS analysis of the resulting peptides was performed on an EASY-nLC 1200 UHPLC connected to a Q Exactive Plus mass spectrometer using a nanoelectrospray ion source ( Thermo Fisher Scientific ) .", "The peptides were separated on a 75-µm inner diameter ×150 mm C18 reverse-phase column ( Nikkyo Technos ) with a linear gradient from 4–28% acetonitrile for 0–40 min followed by an increase to 80% acetonitrile during 40–50 min .", "The mass spectrometer was operated in a data-dependent acquisition mode with a top 10 MS/MS method .", "MS1 spectra were measured with a resolution of 70 , 000 , an AGC target of 1 × 106 , and a mass range from 350 to 1500 m/z .", "HCD MS/MS spectra were acquired at a resolution of 17 , 500 , an AGC target of 5 × 104 , an isolation window of 2 . 0 m/z , a maximum injection time of 60 ms , and a normalized collision energy of 27 .", "Dynamic exclusion was set to 10 s .", "Raw data were directly analyzed against the SwissProt database restricted to Homo sapiens using Proteome Discoverer version 2 . 3 ( Thermo Fisher Scientific ) for identification and label-free precursor ion quantification .", "The search parameters were as follows:", "( a ) trypsin as an enzyme with up to two missed cleavages;", "( b ) precursor mass tolerance of 10 ppm;", "( c ) fragment mass tolerance of 0 . 02 Da;", "( d ) carbamidomethylation of cysteine as a fixed modification; and", "( e ) protein N-terminal acetylation , methionine oxidation , and lysine biotinylation as variable modifications .", "Peptides were filtered at a false-discovery rate of 1% using the percolator node .", "Normalization was performed such that the total sum of abundance values for each sample over all peptides was the same .", "Significant changes were analyzed using a one-way or two-way ANOVA followed by Tukey’s post-hoc test using Graph Pad Prism eight software ( GraphPad , Inc ) .", "For all tests , a P value of less than 0 . 05 was considered statistically significant ." ] ]

[ "Proximity biotinylation based on Escherichia coli BirA enzymes such as BioID ( BirA* ) and TurboID is a key technology for identifying proteins that interact with a target protein in a cell or organism .", "However , there have been some improvements in the enzymes that are used for that purpose .", "Here , we demonstrate a novel BirA enzyme , AirID ( ancestral BirA for proximity-dependent biotin identification ) , which was designed de novo using an ancestral enzyme reconstruction algorithm and metagenome data .", "AirID-fusion proteins such as AirID-p53 or AirID-IκBα indicated biotinylation of MDM2 or RelA , respectively , in vitro and in cells , respectively .", "AirID-CRBN showed the pomalidomide-dependent biotinylation of IKZF1 and SALL4 in vitro .", "AirID-CRBN biotinylated the endogenous CUL4 and RBX1 in the CRL4CRBN complex based on the streptavidin pull-down assay .", "LC-MS/MS analysis of cells that were stably expressing AirID-IκBα showed top-level biotinylation of RelA proteins .", "These results indicate that AirID is a novel enzyme for analyzing protein–protein interactions ." ]

[ "Proteins in a cell need to interact with each other to perform the many tasks required for organisms to thrive .", "A technique called proximity biotinylation helps scientists to pinpoint the identity of the proteins that partner together .", "It relies on attaching an enzyme ( either BioID or TurboID ) to a protein of interest; when a partner protein comes in close contact with this construct , the enzyme can attach a chemical tag called biotin to it .", "The tagged proteins can then be identified , revealing which molecules interact with the protein of interest .", "Although BioID and TurboID are useful tools , they have some limitations .", "Experiments using BioID take more than 16 hours to complete and require high levels of biotin to be added to the cells .", "TurboID is more active than BioID and is able to label proteins within ten minutes .", "However , under certain conditions , it is also more likely to be toxic for the cell , or to make mistakes and tag proteins that do not interact with the protein of interest .", "To address these issues , Kido et al . developed AirID , a new enzyme for proximity biotinylation .", "Experiments were then conducted to test how well AirID would perform , using proteins of interest whose partners were already known .", "These confirm that AirID was able to label partner proteins in human cells; compared with TurboID , it was also less likely to mistakenly tag non-partners or to kill the cells , even over long periods .", "The results by Kido et al . demonstrate that AirID is suitable for proximity biotinylation experiments in cells .", "Unlike BioID and TurboID , the enzyme may also have the potential to be used for long-lasting experiments in living organisms , since it is less toxic for cells over time ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "chromosomes and gene expression" ]

[ [ "Most eukaryotic mRNAs are polyadenylated at their 3′ ends in a process associated with transcriptional termination .", "In the nucleus , these poly ( A ) tails can facilitate mRNA nucleocytoplasmic export ( Kühn and Wahle , 2004 ) , whereas in the cytoplasm , they serve as molecular timers for mRNA decay , with their lengths becoming progressively shorter by deadenylation , which eventually leads to mRNA de-capping and turnover ( Chen and Shyu , 2011; Eisen et al . , 2020; Goldstrohm and Wickens , 2008 ) .", "The length of a poly ( A ) tail can also influence mRNA translational efficiency ( TE ) .", "Pioneering studies in maturing oocytes and early embryos show that lengthening of poly ( A ) tails through cytoplasmic polyadenylation is critical for regulating gene expression during these early stages of animal development ( Richter , 1999; Sallés et al . , 1994; Sheets et al . , 1995 ) .", "Results from these and other single-gene studies in oocytes and early embryos had led to the notion that the length of a poly ( A ) tail generally correlates with TE ( Eckmann et al . , 2011; Weill et al . , 2012 ) .", "More recent transcriptome-wide studies confirm a strong global relationship between tail length and TE in oocytes and early embryos ( Eichhorn et al . , 2016; Lim et al . , 2016; Subtelny et al . , 2014 ) .", "However , in fish , frogs , and flies , this correlation diminishes near the time of gastrulation , and coupling between poly ( A ) -tail length and TE is essentially nonexistent in post-embryonic systems ( Eichhorn et al . , 2016; J . -E . Park et al . , 2016; Subtelny et al . , 2014 ) .", "Thus , these global analyses reveal a developmental transition in how translation is regulated ( Subtelny et al . , 2014 ) , which closely follows the long-known maternal-to-zygotic transition in transcriptional control .", "The existence of this transition in translational control brings to the fore mechanistic questions as to how coupling between poly ( A ) -tail length and TE is established in oocytes and early embryos and why this coupling disappears later in development .", "Cytoplasmic poly ( A ) -binding proteins ( PABPCs ) are highly conserved RNA-binding proteins in eukaryotes ( Mangus et al . , 2003 ) .", "Although Saccharomyces cerevisiae has only one PABPC ( Pab1p ) , most animals contain multiple paralogs that have spatially and temporally varied expression patterns ( Smith et al . , 2014; Wigington et al . , 2014 ) .", "PABPCs have high affinity to poly ( A ) sequences in vitro ( Kd ~5 nM for A25 ) and require at least 12 As for efficient binding ( Kühn and Wahle , 2004 ) .", "Binding of PABPCs to mRNA poly ( A ) tails can enhance translation , but the mechanism of this enhancement is unclear .", "One model posits that the mRNA forms a closed-loop structure mediated by the association of the eukaryotic translation initiation factor eIF4G ( a scaffolding protein ) with both PABPC and the cap-binding protein eIF4E ( Hinnebusch , 2014; Thompson and Gilbert , 2017; Wells et al . , 1998 ) .", "This association is proposed to stabilize the interaction between eIF4E and the mRNA 5′ cap and facilitate recruitment and/or recycling of ribosomes to increase translation initiation ( Kahvejian et al . , 2001 ) .", "However , despite direct visualization of loop-like assemblies both within some cells and in an in vitro reconstituted system ( Christensen et al . , 1987; Wells et al . , 1998 ) , results of several studies have questioned the universality of this model among different mRNAs and biological systems ( Adivarahan et al . , 2018; Amrani et al . , 2008; Costello et al . , 2015; Rissland et al . , 2017; Thompson and Gilbert , 2017 ) .", "PABPCs can also influence mRNA stability , as shown in yeast .", "Genetic ablation of yeast Pab1p is lethal and causes lengthening of steady-state poly ( A ) -tail lengths ( Sachs and Davis , 1989 ) , which is attributed to pre-mature mRNA decapping and compromised deadenylation ( Caponigro and Parker , 1995 ) .", "Both yeast and mammalian PABPCs can interact with two mRNA deadenylation complexes PAN2-PAN3 and CCR4-NOT , and either promote or inhibit their activities in vitro ( Schäfer et al . , 2019; Uchida et al . , 2004; Webster et al . , 2018; Yi et al . , 2018 ) .", "Because mRNA decay is coupled to deadenylation ( Decker and Parker , 1993; Eisen et al . , 2020 ) , the deadenylation-stimulatory effects of PABPC would accelerate the demise of bound mRNAs , which contrasts to other studies suggesting PABPC protects mRNAs from degradation in cell extracts ( Bernstein et al . , 1989; Wang et al . , 1999 ) .", "The dichotomous and potentially conflicting functions of metazoan PABPC examined in vitro raise the question of the extents to which PABPC might influence mRNA poly ( A ) -tail length and stability in metazoan cells .", "PABPCs are generally thought to coat mRNA poly ( A ) tails in the cytoplasm ( Kühn and Wahle , 2004; Mangus et al . , 2003 ) .", "However , the stoichiometry between PABPC and poly ( A ) sites might vary in different biological contexts ( Cosson et al . , 2002; Voeltz et al . , 2001 ) , and it is unclear whether this potentially variable stoichiometry might impact gene regulation in cells .", "Moreover , the possibility that PABPC might influence protein synthesis by affecting either mRNA stability or TE can complicate analysis of its molecular functions in different biological systems , leaving its mechanistic roles poorly understood .", "Here , we uncover mechanistic requirements for coupling between poly ( A ) -tail length and TE observed in oocytes and early embryos , showing that this coupling and the subsequent uncoupling observed later in development rely on a context-dependent switch in the function of PABPCs ." ], [ "To assay the influence of poly ( A ) -tail length on TE , we used an in vitro translation extract made from stage VI Xenopus laevis oocytes , where cytoplasmic polyadenylation leads to translational activation of the c-mos , cdk2 and some cyclin mRNAs ( Richter and Lasko , 2011 ) .", "Into this extract we added Nanoluc luciferase ( Nluc ) reporter mRNAs with either a short ( 29 nt ) or a long ( 139 nt ) poly ( A ) tail ( Figure 1A ) .", "These mRNAs were made by in vitro transcription from DNA templates that encoded the mRNA body followed by the poly ( A ) tail as well as the hepatitis delta virus ( HDV ) self-cleaving ribozyme , which cleaved during in vitro transcription to generate not only a defined 3′ end at the desired poly ( A ) -tail length but also a 2′−3′-cyclic phosphate designed to inhibit undesired lengthening or shortening of the tail ( Avis et al . , 2012 ) .", "When added to the frog oocyte extracts together with a firefly luciferase ( Fluc ) mRNA , used to normalize for overall translation activity , the long-tailed reporter was translated substantially better than was the short-tailed reporter ( Figure 1B ) .", "In contrast , the same reporter mRNAs were translated nearly equally well in rabbit reticulocyte lysate , a post-embryonic differentiated system for which no coupling between tail length and TE was expected ( Subtelny et al . , 2014; Figure 1B ) .", "Similar results were observed for an analogous pair of Renilla luciferase reporter mRNAs ( Figure 1—figure supplement 2A ) .", "In both the oocyte and reticulocyte systems , the reporter mRNAs were stable with no detectable changes to their tail lengths ( Figure 1—figure supplement 1A–C ) .", "Thus , the large difference in luciferase signal observed between the short- and long-tailed reporters in the oocyte extract was attributable to a difference in TE .", "These results showed that the causal relationship between longer poly ( A ) -tail length and greater TE observed for some maturation-specific mRNAs in frog oocytes ( Sheets et al . , 1995; Stebbins-Boaz and Richter , 1994 ) is not unique to those mRNAs , and indicated that frog oocyte extracts provide a system for probing the mechanism that couples tail length to TE .", "When considering the potential mechanisms for reading out tail length and promoting translation , a role for PABPC seemed plausible .", "For instance , translation might be sensitive to the number of PABPC molecules associated with an mRNA .", "In one mechanistic possibility , PABPC might be in excess over its binding sites within tails , such that tails are coated with the protein , as is generally thought to occur ( Kühn and Wahle , 2004; Mangus et al . , 2003 ) , in which case , mRNAs with longer tails might be detected as those able to bind more PABPC molecules .", "At another mechanistic extreme , PABPC might be limiting , such that mRNAs compete with each other for PABPC binding , in which case , those with long poly ( A ) -tail lengths would compete more effectively and thereby preferentially benefit from any enhancement in TE that PABPC binding confers .", "To distinguish between these possibilities , we increased available PABPC in our oocyte extracts , reasoning that if PABPC were already coating the tails , adding more would have little effect , whereas if PABPC were limiting , adding more would diminish the competition for PABPC binding and thereby reduce the difference in TE observed between short- and long-tailed mRNAs .", "Accordingly , we purified recombinant Xenopus PABPC1 to near homogeneity ( Figure 1—figure supplement 1D ) and examined its influence when added to the in vitro translation extract derived from stage VI oocytes .", "As more PABPC1 was added , translation of the short-tailed reporter increased , with little change in translation of the long-tailed reporter , whereas adding equivalent amount of eGFP had little impact on translation of either reporter ( Figure 1C ) .", "This concentration-dependent diminution of coupling between tail-length and TE strongly supported the hypothesis that limiting PAPBC is required for strong coupling .", "To investigate whether this requirement of limiting PABPC was restricted to our in vitro extracts or whether it also applied to living oocytes , we performed serial-injection experiments in oocytes .", "Stage VI frog oocytes were first injected with either PABPC1 mRNA or a control , and after waiting 24 hr to allow PABPC1 protein to accumulate ( Figure 1—figure supplement 1E ) , oocytes were injected with the reporter mRNAs and assayed for luciferase activity ( Figure 1D ) .", "Whereas injecting the control mRNA , eGFP , had no more influence than injecting water , injecting PABPC1 mRNA significantly reduced the extent to which poly ( A ) -tail length and TE were coupled ( Figure 1E ) .", "Similar results were observed for an analogous pair of Renilla luciferase reporter mRNAs or when injecting ePAB mRNA rather than PABPC1 mRNA ( Figure 1—figure supplement 2B ) .", "Reporter poly ( A ) -tail lengths did not change over the course of the experiment ( Figure 1—figure supplement 1F ) , which indicated that the increased relative translation of the short-tailed reporter mRNA was not due to elongated poly ( A ) tails .", "Introducing additional PABPC into frog oocytes specifically improved translation of the short-tailed reporter while having little effect on translation of either the long-tailed reporter or reporters for which tails were replaced with either a stem-loop from the 3′ end of a histone mRNA ( Ling et al . , 2002 ) or a triple-helix from the 3′ end of the Malat1 non-coding RNA ( Wilusz et al . , 2012; Figure 1F ) .", "The observation that mRNAs required a tail to benefit from added PABPC indicated that the effects of adding PABPC were mediated in cis through tail-bound PABPC molecules , and were direct and not some secondary consequence of altering translation .", "Moreover , the observation that PABPC had little effect on translation of long-tailed mRNAs suggested that these mRNAs competed for the limiting endogenous PABPC so effectively that binding of additional PABPC imparted no detectable additional benefit to their translation .", "Introducing PABPC1 ( M161A ) , which encodes a PABPC1 mutant that is unable to bind eIF4G ( Groft and Burley , 2002 ) , also diminished coupling but did so by repressing translation of the long-tailed reporter .", "The reduced translation of the long-tailed reporter was presumably due to a dominant-negative effect of replacing functional endogenous PABPC molecules with defective ones .", "The observation that the long-tailed reporter was preferentially affected agreed with our conclusion that endogenous PABPC was limiting and preferentially binding to long-tailed mRNAs .", "The idea that the M161A mutant was unable to enhance translation in frog oocytes implied that the ability for PABPC to bind eIF4G and form the closed-loop structure is important for enhancing translation in this context ( Wakiyama et al . , 2000 ) .", "In summary , our results with reporters in oocytes and oocyte extracts confirmed both the positive effect of PABPC on translation and the causal relationship between poly ( A ) -tail length and TE in these systems .", "Moreover , these results revealed that strong coupling between poly ( A ) -tail length and TE requires limiting PABPC .", "To examine the global effect of increasing PABPC on the translational regulatory regime acting in the oocyte , we monitored the relationship between tail length and TE for endogenous mRNAs of the oocytes .", "As expected from results of single-gene experiments in frog oocytes ( Figure 1E; Sheets et al . , 1995; Stebbins-Boaz and Richter , 1994 ) and the strong coupling between poly ( A ) -tail length and TE observed in both frog embryos and fly oocytes ( Eichhorn et al . , 2016; Lim et al . , 2016; Subtelny et al . , 2014 ) , we found that poly ( A ) -tail length correlated strongly with TE in stage VI frog oocytes ( Figure 2A ) .", "Overexpressing either PABPC1 or ePAB in these oocytes significantly diminished the coupling , with the Spearman correlation ( Rs ) for the relationship between tail length and TE dropping from 0 . 62 to 0 . 36 and 0 . 38 , respectively ( Figure 2A , both p = 0 , modified Dunn and Clark’s z-test [Diedenhofen and Musch , 2015] ) .", "In contrast , overexpressing eGFP had no significant impact on the coupling ( p = 0 . 11 ) , which indicated that this transcriptome-wide effect was a result of additional PABPC protein rather than a non-specific effect of adding more mRNA .", "Accompanying the reduced coupling observed upon PABPC overexpression was a significant relative increase of TE for short-tailed mRNAs , an effect not observed in eGFP-expressing oocytes ( Figure 2—figure supplement 1A ) .", "This TE increase was not accompanied by corresponding lengthening of poly ( A ) tails ( Figure 2—figure supplement 1B ) , implying that tail-length changes did not cause these relative TE changes .", "To make comparisons of absolute TE changes , we repeated the ePAB-overexpression experiment but omitted rRNA depletion during sequencing library construction , thereby allowing us to normalize TE using mitochondrial mRNAs ( Iwasaki et al . , 2016 ) , which were otherwise depleted by Illumina Ribo-Zero kits ( Figure 2—figure supplement 1C ) .", "In this experiment , we also injected oocytes with a short-tailed Nluc mRNA reporter and a long-tailed Fluc mRNA reporter and monitored their absolute TE changes together with those of endogenous mRNAs .", "Most endogenous mRNAs had greater absolute TE in ePAB-overexpressing oocytes compared to eGFP-expressing control oocytes ( Figure 2B ) .", "This result was consistent with 35S metabolic-labeling experiments showing that overexpression of PABPC1 but not eGFP significantly increased global protein synthesis in oocytes ( Figure 2C–D ) .", "Moreover , the magnitude of the TE increase conferred by ePAB-overexpression negatively correlated with tail length , which showed that translation of short-tailed mRNAs improved substantially more than that of long-tailed mRNAs ( Figure 2E ) , as observed for our co-injected reporters .", "Indeed , adding ePAB had essentially no overall effect on TE of endogenous mRNAs with the longest tails ( median TE fold change = 1 . 06 for the 54 mRNAs with median tail lengths > 80 nt ) , as observed for our long-tailed reporters .", "The preferential improvement of TEs for short-tailed mRNAs led to not only an overall shift in TE but also narrowing of the TE distribution ( Figure 2F ) to more closely resemble the distributions observed in cells in which poly ( A ) -tail length and TE are not coupled ( Subtelny et al . , 2014 ) .", "These results supported the hypothesis that increasing PABPC in oocytes increases the opportunity for short-tailed mRNAs to bind a PABPC molecule , thereby promoting translation .", "Overall , the results of our global analyses of mRNAs in frog oocytes agreed with those of reporter assays , thereby extending to endogenous mRNAs support for the conclusion that limiting PABPC plays a critical role in conferring strong coupling between poly ( A ) -tail length and TE .", "Our global analysis examining the relationship between poly ( A ) -tail length and TE of endogenous mRNAs in oocytes differed from our reporter assays in that the comparison was made between mRNAs of different genes , which can be confounded by features other than tail length that vary between these mRNAs .", "To overcome this issue , we developed a high-throughput method for comparing effects on different tail-length isoforms from each gene .", "This approach for intragenic analyses , called PAL-TRAP ( Poly ( A ) tail-Length profiling following Translating Ribosome Affinity Purification ) , resembled other TRAP approaches in that ribosomes were sparsely tagged such that their immunoprecipitation ( IP ) preferentially isolated mRNA isoforms associated with more ribosomes , which were inferred to be more highly translated ( Chen and Dickman , 2017; Heiman et al . , 2008 ) .", "In a system in which poly ( A ) -tail length and TE were coupled , longer-tail mRNAs were expected to be associated with more ribosomes and therefore enriched in the eluate ( Figure 3A ) , whereas in an uncoupled system , longer-tail mRNAs were not expected to be enriched in the eluate .", "To implement PAL-TRAP , we first injected stage VI frog oocytes with an mRNA encoding C-terminal HA-tagged RPL3 ( Chen and Dickman , 2017 ) and allowed time for RPL3-HA protein expression and incorporation into ribosomes ( Figure 3B ) .", "As a control , HA-tagged eGFP was expressed in separate oocytes .", "To examine the requirement of limiting PABPC for coupling between poly ( A ) -tail length and TE , we then injected oocytes with mRNAs that expressed either eGFP or ePAB .", "Confirming that RPL3-HA was incorporated into functional ribosomes , HA-IP from RPL3-HA-expressing oocytes enriched for proteins from both ribosomal subunits , cytoplasmic mRNAs , and ePAB , whereas HA-IP from eGFP-HA-expressing oocytes did not ( Figure 3—figure supplement 1A–C ) .", "In control oocytes expressing eGFP , longer-tail mRNAs were enriched in the eluate compared to the input ( median tail lengths 41 and 36 nt , respectively ) , which reflected the coupling between tail length and TE ( Figure 3C ) .", "Overexpression of ePAB reduced this enrichment for longer-tail mRNAs in the eluate compared to the input ( median tail lengths 37 and 35 nt , respectively ) , as expected if limiting PABPC was required for this coupling ( Figure 3C ) .", "We also analyzed the flowthrough fractions from these pulldown experiments .", "For both eGFP- and ePAB-expressing oocytes , the cumulative distribution of poly ( A ) -tail lengths from the flowthrough was nearly identical to that from the input ( Figure 3—figure supplement 1D ) , as expected when considering that only a small fraction of input was depleted by HA-beads .", "These analyses indicated that our methods were able to capture small differences in tail-length distributions .", "When analyzing , for each gene , mRNAs with different tail-length isoforms , mRNAs from most genes ( 84 . 4% ) had longer median poly ( A ) -tail lengths in the eluate than in the input ( Figure 3D–E ) , implying that for mRNAs from most genes , long-tailed isoforms were translated more efficiently than short-tailed ones .", "Although the median of the median tail-length differences was moderate ( +2 . 5 nt ) , 28 . 3% mRNAs had a ≥ 5 nt longer median tail length in the eluate ( Figure 3E ) .", "In contrast , when input and flowthrough were compared , the median tail-length differences centered on 0 nt , and mRNAs from only 5 . 4% of genes had a ≥ 5 nt longer median tail length in one of the samples , as expected when considering that only a small fraction of input was depleted by HA-beads ( Figure 3—figure supplement 1E–F ) .", "Overexpressing ePAB reduced the number of genes with long-tailed isoforms enriched in the eluate and shifted the distribution of median tail-length differences closer to 0 nt ( Figure 3D–E ) , as expected if coupling between poly ( A ) -tail length and TE diminished .", "In these PAL-TRAP experiments , a mixture of Rluc reporter mRNA molecules with different poly ( A ) -tail lengths was co-injected with mRNAs that expressed either eGFP or ePAB .", "In eGFP-expressing oocytes , longer-tail Rluc isoforms were highly enriched in the eluate compared to the input , whereas in ePAB-overexpressing oocytes , this difference diminished dramatically ( Figure 3F ) .", "As expected , Nluc reporter mRNA , which was added to the lysate as a spike-in during HA pulldown , was not significantly enriched in longer-tail species in the eluate , regardless of the treatment , which indicated that the changes observed for Rluc mRNA reflected changes occurring in the oocyte , prior to lysis ( Figure 3F ) .", "Although endogenous mRNAs from some genes , such as btg4 . S , also underwent large changes in median tail differences in response to ePAB-overexpression ( Figure 3F ) , changes were typically smaller for endogenous mRNAs ( Figure 3E ) , which was at least partly attributable to the narrower range of initial tail length isoforms for endogenous mRNAs compared to the injected Rluc mRNA .", "Taken together , the PAL-TRAP results revealed intragenic coupling between poly ( A ) -tail length and TE for endogenous mRNAs as well as reporters in stage VI frog oocytes .", "Moreover , these PAL-TRAP analyses showed that as observed both in our reporter assays and in our global intergenic comparisons , substantial coupling between tail length and TE requires limiting PABPC .", "Having established the necessity of limiting PABPC for coupling , we investigated if it could also be sufficient , that is , whether limiting PABPC could confer coupling between poly ( A ) -tail length and TE in cells in which these features were normally uncoupled .", "To this end , we knocked down PABPCs in HeLa cells .", "Based on available mRNA-seq and mass spectrometry results ( Nagaraj et al . , 2011 ) , PABPC1 and PABPC4 are the two major PABPC paralogs in HeLa cells , with PABPC1 four times more abundant than PABPC4 and the two together accounting for >95% of all PABPC in HeLa cells .", "Consistent with the idea that PABPC is not normally limiting in uncoupled systems , PABPC1 is estimated to be present in threefold excess over the poly ( A ) sites in HeLa cells ( Görlach et al . , 1994 ) .", "To reduce PABPC to limiting levels , we used siRNAs to reduce either PABPC1 or PABPC4 alone or both PABPC1 and PABPC4 by >90% ( Figure 4—figure supplement 1A ) and examined the relationship between median tail lengths and TE .", "Knocking down PABPC4 alone had little impact on the coupling ( Figure 4—figure supplement 1B ) , consistent with the inference that PABPC4 constitutes less than 20% of the total PABPC protein .", "Although correlation between median poly ( A ) -tail length and TE gradually increased as more PABPC was depleted , it reached an Rs of only 0 . 18 ( p<10–19 ) in double-knockdown cells ( Figure 4—figure supplement 1B ) , which was much weaker than that observed in frog oocytes ( Figure 2A ) and frog and fish early embryos ( Subtelny et al . , 2014 ) .", "Minor Rs increases were observed in other mouse and human post-embryonic cell lines in which PABPC was depleted , but strong coupling between poly ( A ) -tail length and TE was not established , with no Rs values exceeding 0 . 3 ( data not shown ) .", "Thus , other conditions in addition to limiting PABPC must also be met to confer strong coupling between poly ( A ) -length and TE .", "A striking consequence of depleting PABPC in HeLa cells was a sharp increase in median poly ( A ) -tail lengths , which for HeLa mRNAs increased an average of 17 and 39 nt in PABPC1- and double-knockdown cells , respectively ( Figure 4A ) .", "Substantial changes were also observed in the distributions of global poly ( A ) -tail length , which showed that mRNAs with tails ranging from 10 to 50 nt were >2-fold depleted in the PABPC1-knockdown cells and that mRNAs with tails ranging from 10 to 135 nt were 2- to 20-fold depleted in the double-knockdown cells ( Figure 4B ) .", "Similar results were obtained in NIH3T3 cells ( Figure 4—figure supplement 2A–B ) .", "In contrast , loss of short-tailed mRNAs was not observed for mitochondria-encoded mRNAs , as expected for these mRNAs that never encounter PABPC ( Figure 4A–B , Figure 4—figure supplement 2A–B ) , and examination of internal standards and replicates confirmed that the loss of short-tailed cytoplasmic mRNAs was not attributable to inaccurate or variable measurements ( Figure 4—figure supplement 2C–D ) .", "Moreover , knocking down the minor isoform ( PABPC4 ) alone did not have a similar effect ( Figure 4—figure supplement 2E ) , suggesting that the tail-length changes observed for cytoplasmic mRNAs were a consequence of limiting PABPC .", "We also used northern blots to examine effects of PABPC depletion on tail-length distributions of mRNAs from individual genes and found that the results corresponded well to those observed by tail-length sequencing .", "Both northern blots and sequencing showed strong depletion of short-tailed isoforms for a cytoplasmic mRNA ( GAPDH ) after PABPC knockdown but no substantial change in the tail-length distribution of a mitochondrial mRNA ( MT-CYB ) ( Figure 4C ) .", "Using sequencing data to examine the intragenic tail-length distributions of cytoplasmic mRNA from each of more than five thousand other genes revealed findings resembling those observed for GAPDH ( Figure 4D–E ) .", "After knocking down PABPC1 , the reduction in short-tailed mRNAs was typically most severe for mRNA isoforms with tail lengths of ~25 nt , and in the double knockdown , this dip at ~25 nt became more pronounced , with reductions extending to all but the longest-tail isoforms ( Figure 4D–E ) .", "Indeed , for more than half of the genes examined , ≥2-fold reductions extended to isoforms with tails as long as 135 nt ( Figure 4D–E ) .", "Similar results were observed when examining tail-length distributions in the NIH3T3 dataset ( Figure 4—figure supplement 2B ) , and when examining individual tail-length distributions for mRNAs of each of the top-expressed nuclear genes but not when examining those for mRNAs of mitochondrial genes ( Figure 4—figure supplement 2F–G ) .", "These results , which showed that mRNAs with short tails were preferentially destabilized when PABPC was depleted provided genetic loss-of-function evidence that PABPC stabilizes mRNAs in mammalian cell lines .", "Although genetic evidence for the role of PABPC in mRNA stability has been reported in yeast ( Caponigro and Parker , 1995; Coller et al . , 1998 ) , this function had not been established in mammalian cells .", "Although in principle the mRNA destabilization that we observed upon PABPC-depletion might have been indirect , two lines of evidence support the conclusion that this destabilization was a direct consequence of the loss of PABPC binding to poly ( A ) tails .", "First , destabilization preferentially occurred for short-tailed mRNAs , which were expected to be the least successful at competing for binding under conditions of limiting PABPC .", "Second , destabilization sharply diminished at tail lengths of 10–15 nt , which corresponded to the 12 nt poly ( A ) sequence reported to be the minimal length that can be bound by PABPC1 with high affinity ( Kühn and Pieler , 1996 ) .", "Indeed , the modest loss observed for mRNAs with very short poly ( A ) tails ( Figure 4D–E ) suggested that even in control cells that had abundant PABPC , mRNAs with tails shorter than 12 nt were poorly bound by PABPCs , and thus PABPC depletion did not substantially influence their abundance .", "A recent study observed similar poly ( A ) tail-length changes in PABPC1-depleted cells but attributed these changes to impaired deadenylation ( Yi et al . , 2018 ) .", "Because PABPC can promote deadenylation in vitro ( Schäfer et al . , 2019; Uchida et al . , 2004; Webster et al . , 2018; Yi et al . , 2018 ) , it is conceivable that the loss of PABPC would slow deadenylation , thereby increasing mRNA median tail lengths , as observed in Pab1-knockout yeast ( Caponigro and Parker , 1995 ) .", "However , our analyses , which had the benefit of quantitative tail standards that enabled measurement of absolute abundance changes , revealed little added accumulation of long-tailed isoforms in PABPC-depleted cells ( Figure 4B–E , Figure 4—figure supplements 2B , F ) , indicating that a deadenylation defect was not the major cause for the perturbed tail-length distributions .", "Moreover , we found that mRNA half-life values , as determined by metabolic labeling , reduced significantly when PABPC was knocked down ( Figure 4—figure supplement 3A–C ) , which concurred with the conclusion that PABPC knockdown destabilized short-tailed mRNA isoforms and argued against the previous assertion that PABPC knockdown impaired deadenylation , in that impaired deadenylation would have lengthened mRNA half-lives .", "Taken together , these results show that PABPC binding stabilizes mRNAs of cultured mammalian cells; if PABPC becomes limiting in these cells , the short-tailed mRNAs become destabilized , presumably because they compete less effectively for PABPC .", "Most importantly , the destabilization of mRNAs that competed poorly for PABPC binding helps explain why limiting PABPC was insufficient to cause strong coupling in mammalian cell lines , in that strong coupling between tail length and TE would be difficult to establish in a regulatory regime in which short-tailed mRNA molecules that lack PABPC binding are degraded rather than translated less efficiently .", "Thus , these results identify a second mechanistic requirement for strong coupling between tail length and TE: In addition to limiting PABPC , strong coupling requires metabolic stability of the mRNAs that compete poorly for PABPC binding .", "The identification of this second requirement for strong coupling brought to the fore the question of why mRNAs that competed poorly for limiting PABPC were destabilized .", "To explore the possible mechanisms , we searched for perturbations that could restore stability of short-tailed mRNAs in HeLa cells undergoing PABPC knockdown , monitoring tail-length distributions of endogenous GAPDH using northern blots .", "As a positive control , expressing an siRNA-resistant PABPC1 restored stability of short-tailed species , as did frog ePAB , which further illustrated functional conservation of PABPC from different species and developmental stages ( Figure 5A ) .", "Interestingly , a PABPC1 variant with substitutions that disrupt its interaction with eIF4G ( Chorghade et al . , 2017 ) also restored the stability of short-tailed species , implying that the classical closed loop is not necessary for PABPC1 to protect short-tailed mRNAs from degradation .", "Because PABPC has been implicated in inhibiting mRNA terminal uridylation in vitro ( Lim et al . , 2014 ) and in cells ( Yi et al . , 2018 ) , and terminal uridylation has been linked to mRNA decay ( Lim et al . , 2014; Rissland and Norbury , 2009 ) , we asked if terminal uridylation contributed to the loss of short-tailed mRNAs .", "Knocking down both TUT4 and TUT7 in PABPC-depleted cells partially restored short-tailed GAPDH mRNAs ( Figure 5B ) .", "Similar results were observed in HCT116 cells , in which we tagged endogenous PABPC1 with an auxin-inducible degron ( AID ) and induced depletion by adding indole-3-acetic acid ( IAA , a form of auxin ) ( Figure 5C; Natsume et al . , 2016 ) .", "To examine the global rescue of short-tailed mRNAs and at the same time monitor mRNA terminal uridylation levels , we modified our tail-length sequencing protocol by including in the adaptor-ligation step a splint oligonucleotide designed to accommodate tails with a 3′ terminal U ( Eisen et al . , 2020 ) .", "Knockdown of PABPC1 alone significantly increased the terminal uridylation levels across essentially all tail-length isoforms ( Figure 5D–E ) , consistent with a previous report ( Yi et al . , 2018 ) .", "Knockdown of PABPC4 in addition to PABPC1 further increased uridylation of mRNA isoforms with longer tail lengths ( Figure 5E ) .", "Knockdown of TUT4 and TUT7 in PABPC-depleted cells brought terminal uridylation of all tail-length isoforms to background levels ( Figure 5D–E ) and , more importantly , preferentially rescued shorter-tail isoforms , thereby decreasing median tail lengths ( Figure 5F ) .", "These results were consistent with those of our northern assays , and together , our results indicated that in these mammalian cells , limiting PABPC makes short- and medium-tailed mRNA isoforms that poorly compete for PABPC binding more susceptible to terminal uridylation , thereby accelerating their decay .", "Having found that destabilization of short-tailed mRNAs dampened coupling between poly ( A ) -tail length and TE in PABPC-depleted mammalian cells , a key question remained regarding how mRNA TE , if not influenced strongly by poly ( A ) -tail length , was affected in these cells .", "To answer this question , we conducted global profiling of HeLa cells 48 hr after siRNA transfection , a time point at which PABPC1 and PABPC4 knockdowns were substantial but secondary effects were presumably not yet too severe ( Figure 6—figure supplement 1A ) .", "We again implemented RNA-seq and ribosome-profiling protocols that enabled absolute TE comparison by using mitochondrial mRNAs for normalization ( Rooijers et al . , 2013 ) .", "Surprisingly , near-complete depletion of PABPCs had no detectable effect on global mRNA TEs ( Figure 6A–B ) .", "Although the protein synthesis rate in double-knockdown cells was reduced to 75 . 7% of that observed in control cells , as measured by averaging ribosome-footprint changes observed for the 9697 analyzed genes ( Figure 6A–B ) , which agreed with results of a global puromycin-based translation assay ( 78 . 7% ) ( Figure 6C ) , this reduced protein synthesis was fully explained by the decrease in mRNA levels , as indicated by a distribution of TE changes that centered near zero ( Figure 6A–B ) .", "Examination of our ribosome-footprinting data revealed no upregulation of other PABPC paralogs , although the TE of PABPC1 mRNA increased 2 . 5-fold ( Figure 6A ) , consistent with its known autoinhibitory translational control ( Bag and Wu , 1996 ) .", "Results of polysome profiling confirmed those of our sequencing-based methods , in that the reduction in translation output , as measured by the height of polysome peaks in double-knockdown cells , was attributable to an overall decrease of mRNA levels rather than to decreased TEs that would otherwise cause a shift of mRNA distribution from heavy to light fractions ( Figure 6—figure supplement 1B ) .", "Together , these results indicated that PABPC depletion in HeLa cells had negligible effect on mRNA TE .", "The slow dynamics of siRNA-mediated knockdown , which dictated the relatively late 48 hr time point for our global measurements , might have prevented detection of any TE changes that happened earlier , before a new steady state had been reached .", "To examine this possibility , we monitored the dynamics of tail-length , mRNA-abundance , and translation changes soon after PABPC depletion , using the HCT116 PABPC1-AID degron cell line , in which PABPC1 was rapidly and efficiently depleted after adding IAA ( 85% within 30 min , >99% within 1 hr , Figure 6D ) .", "Because PABPC1 is the primary PABPC isoform in HCT116 cells , depletion of PABPC1 alone caused substantial destabilization of shorter-tail mRNAs 1 hr after IAA addition , and this destabilization further increased after 3 hr ( Figure 6E ) .", "Accompanying the loss of shorter-tail mRNAs was a corresponding reduction of mRNA abundance for most genes ( Figure 6F , Figure 6—figure supplement 1C ) .", "Importantly , ribosome footprints declined in lockstep with mRNA abundance , leading to median TE changes that centered near zero over the entire course of PABPC1 depletion ( Figure 6F , Figure 6—figure supplement 1C ) .", "Thus , as PABPC became limiting , mRNAs that lost PABPC had no detectable reduction in TE before they were destabilized .", "These data from the PABPC degron line allowed us to examine whether some coupling between tail length and TE might have occurred very soon after PABPC depletion—during the time window in which PABPC had become limiting but short-tailed mRNAs had only started to degrade .", "However , no coupling between tail length and TE was detected over the course of rapid PABPC depletion ( Figure 6—figure supplement 2 ) .", "Thus , in this context in which two conditions for strong coupling were satisfied ( i . e . PABPC was limiting and short-tailed mRNAs were largely intact ) , no coupling was observed , presumably because coupling also requires a regulatory regime in which PABPC enhances translation .", "These results show that in contrast to mRNAs of frog oocytes and presumably those of other coupled systems , mRNAs of HeLa and HCT116 cells do not require PABPC for efficient translation , which explains why poly ( A ) -tail lengths were not able to strongly influence TE after we reduced PABPC of these cells to limiting levels .", "Thus , these results identify a third mechanistic requirement for coupling between poly ( A ) -tail lengths and TE: coupling requires a regulatory regime in which PABPC affects mRNA translation ." ], [ "We find that three fundamental molecular conditions must be met for cells to use poly ( A ) -tail lengths to effectively regulate TE .", "First , PABPC must be limiting compared to the number of poly ( A ) sites available for binding .", "Under this condition , short-tailed mRNA isoforms that poorly compete for PABPC are less likely to have PABPC bound to their 3′ ends ( Figure 7 ) .", "To the extent that PABPC is not bound , these isoforms lose the translation-activating capability of PABPC observed in coupled systems .", "Cooperative binding of adjacent PAPBC molecules ( Lin et al . , 2012; Schäfer et al . , 2019 ) would further enhance partitioning of limiting PABPC away from short-tailed mRNAs and onto longer-tailed mRNAs .", "When additional PABPC is introduced into a coupled system , short-tailed mRNAs benefit more from PABPC binding than long-tailed ones , which are more likely to already possess the number of PABPC molecules required for more efficient translation .", "The requirement of limiting PABPC for coupling TE to poly ( A ) -tail length raises the question of the stoichiometry between PABPC and its sites in the poly ( A ) tails and whether this stoichiometry changes during the embryonic switch of gene-regulatory regimes .", "Our sequencing results indicated that the mRNAs of a stage VI frog oocyte have ~2 . 8×1011 PABPC sites ( ~7×1010 sites per 1 µg total RNA , ~4 µg total RNA per oocyte ) , which concurred with the previous estimate of 2 ~ 3×1011 sites per oocyte ( Sagata et al . , 1980 ) .", "The amount of PABPC ( including both ePAB and PABPC1 ) in frog oocytes has been estimated at either 1 × 1011 ( Cosson et al . , 2002 ) or 1 . 4 × 1011 ( Peuchen et al . , 2017; Smits et al . , 2014 ) molecules per oocyte .", "Our results showing that PABPC activity is limiting in frog oocytes , agreed with these estimates that imply that PABPC levels are sufficient to bind no more than half of the available PABPC sites .", "Poly ( A ) -site occupancy would be even lower if some PABPC proteins were sequestered from the mRNA pool or in an inactive form , which could be conferred by factors that either bind to or post-translationally modify PABPC to affect its ability to either bind poly ( A ) tails or promote translation ( Brook et al . , 2012; Khaleghpour et al . , 2001 ) .", "Until stage 15 , developing frog embryos maintain the number of PABPC sites at a level resembling that of oocytes ( Sagata et al . , 1980 ) .", "In contrast , the total amount of PABPC molecules increases significantly , nearly tripling by stage 12 ( Peshkin et al . , 2019 ) —the stage at which the coupling between tail length and TE starts to disappear ( Subtelny et al . , 2014 ) .", "This increased PABPC would shift the stoichiometry toward PABPC being less limiting .", "Importantly , dysregulation of this tightly controlled stoichiometry not only disrupts the normal gene-regulatory regime but also can cause severe consequences during oocyte maturation and embryonic development ( Gorgoni et al . , 2011; Wormington et al . , 1996 ) .", "In our overexpression experiment , we increased the level of PABPC1 to >6 times its endogenous level ( Figure 1—figure supplement 1E ) .", "When considering that the ratio between endogenous PABPC1 and ePAB is about 1:3 ( Wühr et al . , 2014 ) , the estimated increase in overall PABPC level was >2 . 5-fold , which might have been sufficient to saturate the poly ( A ) sites .", "Despite this potential saturation and diminished coupling between poly ( A ) -tail length and TE , coupling was not completely lost ( Figures 1E , 2A and 3C–E ) .", "To the extent that poly ( A ) sites were saturated , the residual coupling suggests that oocytes might have a mechanism for counting bound PABC molecules that enables some coupling to persist even after PABPC overexpression saturates the poly ( A ) sites .", "One way to achieve this counting would be for a critical PABPC-interacting factor to also be limiting such that long-tailed mRNAs , with their higher number of bound PABPC molecules would more effectively compete for binding to this limiting factor .", "A top candidate for such a factor is eIF4G .", "Indeed , when eIF4G was overexpressed together with PABPC1 in frog oocytes , the coupling between poly ( A ) -tail length and TE was further reduced ( Figure 1—figure supplement 2C ) .", "The idea that PABPC interactions with limiting eIF4G might favor translation of long-tailed mRNAs in oocytes also helps to explain why overexpressing the PABPC1 M161A mutant was most detrimental to translation from the long-tailed reporter ( Figure 1E ) .", "Another way that some coupling could persist even after overexpressing saturating levels of PABPC is through sequestration of some mRNAs away from the translation machinery .", "One likely location for such sequestration would be germ granules , which have been implicated in regulating mRNA translation in oocytes of diverse animal species ( Voronina et al . , 2011 ) .", "This mechanism might reinforce coupling between poly ( A ) -tail length and TE , perhaps by selectively sequestering short-tailed mRNAs from the active translation pool .", "The second condition required for strong coupling between poly ( A ) -tail length and TE is the survival of short-tailed mRNAs under conditions in which PABPC is limiting ( Figure 7 ) .", "This condition was not met in the post-embryonic mammalian cell lines we examined .", "When PABPC was depleted in these uncoupled systems , many mRNA molecules , particularly short-tailed ones that presumably competed poorly for the remaining PABPC , were degraded .", "The preferential loss of mRNAs not bound by PABPC reduced the range of tail lengths , which correspondingly reduced the range of TEs that could potentially be imparted by coupling between tail length and TE .", "This reduced range also presumably reduced the ability to detect coupling , although some ability was expected to be retained , as indicated by an analysis in which data from a coupled system was sampled to match the more restricted tail-length distribution of an uncoupled system ( Subtelny et al . , 2014 ) .", "More importantly , the loss of mRNAs not bound by PABPC reduced the number of PABPC-binding sites , thereby reducing the extent to which these sites were in excess over PABPC and thus reducing coupling between tail length and TE .", "In oocytes and early embryos , mRNA decapping is uncoupled from deadenylation ( Gillian-Daniel et al . , 1998 ) , which helps explain why short-tailed mRNAs survive in these systems despite our finding that they have limiting PABPC activity .", "We suggest two nonexclusive mechanistic explanations for this unusual decoupling of decapping from deadenylation .", "First , oocytes and early embryos have relatively low expression of decapping enzymes ( Ma et al . , 2013; Peshkin et al . , 2019 ) .", "Second , mRNA terminal uridylation activity is very low in frog oocytes ( Figure 5—figure supplement 1; Chang et al . , 2018 ) , and it remains low throughout early embryonic development and only starts to increase dramatically after zygotic genome activation ( Chang et al . , 2018 ) .", "This developmental delay of terminal uridylation might help ensure the survival of short-tailed mRNAs when PABPC is limiting and thereby enable strong coupling between poly ( A ) -tail length and TE in oocytes and early embryos .", "Later in development , short-tailed mRNAs are protected from terminal uridylation by saturating PABPC , which helps explain why deletion of TUT4 and TUT7 in mouse somatic cells has little impact on mRNA abundance ( Morgan et al . , 2017 ) .", "The third condition required for coupling between tail length and TE is that PABPC must have the ability to influence TE of bound mRNAs ( Figure 7 ) .", "In the coupled system of frog oocytes , increasing PABPC levels substantially improved TE of nearly all mRNAs ( Figure 2B–E ) .", "In contrast , in uncoupled systems such as HeLa and HCT116 cells , severe depletion of PABPC , such that short- and medium-tailed mRNAs were markedly destabilized , had no consistent impact on mRNA TE ( Figure 6B and F ) .", "We suspect that the differential effect of PABPC on TE observed in coupled and uncoupled systems is related to the divergent levels of basal translation initiation observed between these systems .", "Indeed , the overall translation measured by polysome profiles in oocytes and early embryos is much lower than that observed in either later developmental stages ( Woodland , 1974 ) or post-embryonic mammalian cell lines ( Figure 3—figure supplement 1A , Figure 6—figure supplement 1B ) , which provides the opportunity for a translation-activating effect of PABPC to be more prominent in the coupled systems .", "Our results showing that PABPCs , while playing a crucial role in protecting mRNA from premature decay , have minimal contribution to translation in post-embryonic mammalian cell lines might seem to contradict the well-accepted function of PABPC as a translational activator .", "However , many previous studies that established the role for PABPC in promoting translation were carried out in frog oocytes or early embryonic systems ( Smith et al . , 2014 ) , where we found PABPC to globally enhance TE .", "Other previous studies were conducted in vitro , with mixed results: in reconstituted systems , PABPC is dispensable for translation initiation ( Mitchell et al . , 2010 ) , and in rabbit reticulocyte lysates , PABPC has a minimal effect on translation ( Hinton et al . , 2007 ) , whereas in some other cell extracts , PABPC activates translation ( Kahvejian et al . , 2005; Tarun and Sachs , 1995 ) .", "Additional experiments will be required to determine whether this discrepancy between results we obtained from living cells and those obtained in some post-embryonic cell extracts are attributable to differences between cell types or to differences between cellular cytoplasm and in vitro extracts—perhaps imparted by dilution of translational components in extracts .", "Such studies will need to differentiate between the translation-activation and mRNA-stabilization activities of PABPC .", "In the meantime , it is helpful to know that PABPC stabilizes mRNAs of post-embryonic metazoan cells and that the two activities of PABPC can be context dependent , such that in frog oocytes PABPC strongly activates translation and has no effect on mRNA stability , whereas in mammalian cell lines it stabilizes mRNAs and has no detectable effect on TE .", "The dual potential of PABPC in stabilizing mRNA and promoting translation bestows PABPC with distinct and context-dependent roles in regulating protein synthesis .", "In oocytes and early embryos , the lack of mRNA transcription and degradation leaves differential TE as the primary option for modulating protein synthesis .", "Our work indicates that in this context limiting PABPC proteins bind primarily to mRNAs with longer poly ( A ) tails and activate their translation .", "In contrast , in post-embryonic mammalian cell lines , where transcription , mRNA degradation , and translation are each operating at high efficiency , PABPCs protect mRNAs from pre-mature decay , enabling them to contribute the proper amount of protein during their lifetimes , but without any additional enhancement of TE .", "These context-dependent activities are not only crucial for understanding how coupling between tail length and TE is established in oocytes and early embryos and why it is lost later in development , they also provide mechanistic insight into the effects of the many posttranscriptional regulatory phenomena that alter poly ( A ) -tail lengths .", "For example , during miRNA-mediated repression , the Argonaute–miRNA complex binds target mRNAs and recruits factors that displace PABPC from the poly ( A ) tail and accelerate tail shortening ( Fabian et al . , 2009; Giraldez et al . , 2005; Moretti et al . , 2012; Rissland et al . , 2017; Wu et al . , 2006 ) .", "In early zebrafish embryos , these effects are expected to disadvantage target mRNAs at competing for limited PABPC , which in this context would reduce their TE without changing their stability , thereby explaining why miRNAs primarily cause translational repression in these early embryos ( Bazzini et al . , 2012; Subtelny et al . , 2014 ) .", "However , in later embryonic development as well as in post-embryonic mammalian cells , displacement of PABPC and accelerated tail shortening would reduce PABPC binding to target mRNAs , which in this context would reduce their stability without changing their TE , thereby explaining why miRNAs primarily cause mRNA destabilization in these cells ( Bazzini et al . , 2012; Guo et al . , 2010; Subtelny et al . , 2014 ) .", "How PABPC promotes translation remains an enigma , although the closed-loop model has offered a sound mechanistic explanation .", "The interaction between eIF4G and PABPC is well characterized and provides a physical link connecting both ends of the mRNA , and some studies are able to catch a glimpse of possible circular structures of mRNAs in fixed tissues ( Christensen et al . , 1987 ) or in vitro ( Wells et al . , 1998 ) .", "However , recent single-molecule imaging studies in mammalian cells question the widespread existence of mRNA closed-loop structures ( Adivarahan et al . , 2018; Koch et al . , 2020 ) .", "Our finding that depletion of PABPC had minimal impact on TE in mammalian cell lines supports a model in which pervasive eIF4G–PABPC-associated looping of mRNAs is generally lacking in uncoupled systems .", "This idea is consistent with the finding that the interaction between eIF4G and PABPC is dispensable in yeast ( E . -H . Park et al . , 2011 ) and HEK293 cells ( Adivarahan et al . , 2018 ) , both of which are uncoupled systems ( Subtelny et al . , 2014 ) .", "In contrast , the eIF4G–PABPC interaction is critical during frog oocyte maturation ( Wakiyama et al . , 2000 ) , a process that relies on coupling between poly ( A ) -tail length and TE ( Richter and Lasko , 2011 ) , and our results showed that increasing PABPC had a global effect on upregulating TE in frog oocytes .", "Moreover , translation of a long-tailed reporter was substantially repressed when the PABPC M161A mutant was overexpressed in frog oocytes ( Figure 1E ) .", "Thus , the closed-loop structure , if it exists , might be more prevalent in coupled systems , such as oocytes or early embryos .", "Our experiments examined only a few systems that inherently possessed either all or none of the three molecular conditions that we found to be required for strong coupling between tail length and TE , that is , limiting PABPC activity , stabilization of mRNAs lacking a bound PABPC , and PABPC-sensitive translation .", "Whether these conditions are broadly applicable to many other systems remains to be investigated .", "For example , some cells might fall in between the two extremes , processing one or two of these conditions but not all three .", "The results of our experiments in which we removed one of the conditions from frog oocytes or imposed one or two of the conditions in post-embryonic mammalian cells , predict that such cells that inherently fall between the two extremes have minimal if any coupling .", "Indeed , the concept that multiple conditions must be met before strong coupling can be established helps to explain why coupling between tail length and TE has been so infrequently detected outside the gene regulatory regime operating in oocytes and early embryos ( Subtelny et al . , 2014 ) ." ], [ "All DNA plasmids were assembled by restriction-free cloning unless explained otherwise ( Unger et al . , 2010 ) .", "Site-directed mutagenesis was also carried out with this method .", "For plasmids used for mammalian cell transfection , human PABPC1 ( from HeLa cell cDNA ) and X . laevis ePAB ( from oocyte cDNA ) coding sequences were cloned into pcDNA5/FRT/TO ( Thermo Fisher , V652020 ) .", "For siRNA-resistant human PABPC1 , silent mutations were introduced at D107 , K108 , S109 , I110 , D111 , N112 , V131 , C132 , D249 , E250 , N252 , and G253 .", "Additional substitutions were made at I110L , D111E , D117E , A121G , G139A , Y140F , T147S , and R166K to disrupt the interaction between PABPC1 and eIF4G ( Chorghade et al . , 2017 ) .", "For siRNA-resistant X . laevis ePAB , silent mutations were introduced at C128 , K129 , V130 , V131 , T249 , E250 , and N252 .", "Sequences of oligos used for mutagenesis are provided in Supplementary file", "1 . A list of plasmids used in this study is provided in Supplementary file", "2 . Plasmids and their sequence information will be available at Addgene .", "Plasmids for preparing DNA templates for in vitro transcription were assembled using the pGEM-11Zf ( + ) ( Promega ) backbone , inserting the appropriate sequence segments after the T7 promoter .", "HDV ribozyme sequence was obtained from the plasmid p2RZ ( Avis et al . , 2012 ) .", "X . laevis PABPC1 ( pabpc1 . S ) , ePAB ( pabpc1l . L ) , and RPL3 ( rpl3 . L ) coding sequences were amplified from cDNA generated from X . laevis oocytes .", "Renilla ( Rluc ) and firefly luciferase ( Fluc ) coding sequences were obtained from pIS2 and pIS0 , respectively ( Farh et al . , 2005 ) .", "NanoLuc ( Nluc ) coding sequence was obtained from pNL1 . 1 . TK ( Promega ) .", "X . laevis β-globin 5′- and 3′-UTR sequences were obtained from pT7TS ( Addgene #17091 ) .", "Mouse Malat1 3′ sequence was obtained from the Comp . 25 mutant plasmid ( Wilusz et al . , 2012 ) .", "Rluc and Fluc reporters contained 5′- and 3′-UTR sequences inherited from the pGEM-11Zf ( + ) backbone , whereas Nluc reporters had the X . laevis β-globin 5′- and 3′-UTR sequences .", "Fragments containing variable poly ( A ) lengths were put in desired plasmids after all other DNA fragments were assembled , also using restriction-free cloning , except that C3040H competent cells ( NEB ) were used to amplify the assembled plasmids .", "Because long homopolymers tend to become shorter or get lost when plasmids are propagated in E . coli , individual clones were selected and checked by PCR and Sanger sequencing to confirm the desired length of each poly ( A ) region .", "These plasmid preparations were then used to generate templates for in vitro transcription without further propagation in E . coli .", "For generating DNA templates for in vitro transcription , PCR reactions were carried out using KAPA HiFi HotStart PCR Kit , with a common 5′ primer upstream of the T7 promoter and different 3′ primers .", "For making RNAs ending in defined lengths of poly ( A ) sequence , a primer 300 ~ 600 nt downstream of the HDV cleavage site was used to facilitate separating the 5′ cleavage product from the 3′ cleavage product and the uncleaved transcript .", "For making RNAs not ending in defined lengths of poly ( A ) sequence , a primer pairing to the end of the desired 3′ UTR sequence was used .", "All DNA templates for in vitro transcription were purified on agarose gels using the QIAquick Gel Extraction Kit ( QIAGEN ) .", "Sequences of oligos used for generating DNA templates are provided in Supplementary file", "1 . In vitro transcription was carried out with T7 RNA polymerase purified in house and used at 3 . 2 ng/µl final concentration in a standard 50 µl reaction containing 40 mM Tris pH 8 . 0 , 21 mM MgCl2 , 2 mM Spermidine ( Sigma ) , 1 mM dithiothreitol ( DTT , GoldBio ) , 5 mM NTP ( buffered ATP , UTP , CTP , GTP mix , Thermo Fisher ) , 0 . 1 units yeast inorganic pyrophosphatase ( NEB ) , 40 units SUPERase•In ( Thermo Fisher ) , and 1 µg DNA template .", "After incubation at 37°C for 2 hr , 2 units of TURBO DNase ( Thermo Fisher ) were added , followed by another 20 min incubation at 37°C .", "For constructs with the HDV ribozyme , thermal cycling was performed to enhance HDV cleavage ( 65°C for 90 s and 37°C for 5 min , three cycles , 50 µl of reaction per tube ) .", "Before gel loading , 2 µl 0 . 5M EDTA pH 8 . 0 and 50 µl 2x RNA Gel Loading Dye ( Thermo Fisher , R0641 ) were added to all in vitro transcription reactions , regardless of whether the HDV cleavage step was performed or not .", "After incubation at 65°C for 5 min , RNAs were separated on 5% acrylamide denaturing gels ( National Diagnostics , EC-829 ) .", "Desired RNA bands were identified by UV-shadowing , excised , macerated and eluted in 300 mM NaCl at 23°C overnight on a rotator .", "The gel pieces were removed using Spin-X columns ( Corning 8160 ) , and RNAs were precipitated with isopropanol and resuspended in water for downstream reactions .", "Capping of RNAs was carried out with the Vaccinia Capping System ( NEB , M2080S ) following the manufacturer’s protocol .", "RNAs were then purified by phenol/chloroform extraction and ethanol precipitation .", "Water-resuspended RNAs were applied to Micro Bio-Spin columns ( Bio-Rad , 7326250 ) for desalting .", "All RNAs were checked for integrity by visualizing on formaldehyde-agarose denaturing gels before being stored at –80°C .", "X . laevis ovaries were obtained from Nasco ( LM00935 ) .", "Ovaries were broken down to 2–3 cm pieces with tweezers and incubated in calcium-omitted OR-2 buffer ( 82 . 5 mM NaCl , 2 . 5 mM KCl , 1 mM MgCl2 , 1 mM Na2HPO4 , 5 mM HEPES , pH 7 . 5 ) with 0 . 2% ( w/v ) collagenase A ( Roche , 11088793001 ) on a rocker at 23°C for ~3 hr , at which point most oocytes were defolliculated .", "The oocytes were then washed extensively with calcium-omitted OR-2 buffer followed by complete OR-2 buffer ( with 1 mM CaCl2 ) .", "Stage V and VI oocytes were separated from the rest with a ~ 0 . 8 mm diameter mesh sieve .", "For injection experiments , healthy stage VI oocytes were hand-picked and incubated in complete OR-2 buffer at 18°C overnight ( >16 hr ) for recovery before injection .", "When preparing oocyte extracts , the bulk stage V and VI oocytes ( 500–1 , 000 ) were washed three times with ample oocyte extraction buffer ( 10 mM HEPES pH 7 . 5 , 10 mM sodium acetate , 1 mM magnesium acetate and 2 mM DTT ) .", "After removing all excess buffer , oocytes were centrifuged at 20 , 000 g for 20 min .", "The middle layer containing the extract was collected with a pipette and transferred to a new tube .", "The collected extract was centrifuged at 20 , 000 g for 10 min , and the middle layer was again collected , avoiding the top lipid layer and the bottom insoluble layer as much as possible .", "The extract was passed through a 0 . 45 µm filter , then aliquoted and stored at –80°C .", "The concentration of the oocyte extract was measured using the Bradford assay ( Thermo Fisher , 23236 ) and was usually at 35–45 mg/ml .", "Five hundred µl rabbit reticulocyte lysate obtained from Promega ( L4151 , untreated ) was supplemented with 1 µl 12 . 5 mM Hemin ( Sigma , 51280 ) , 25 µl 1 M HEPES pH 7 . 5 , and 2 . 5 µl 10 mg/ml yeast tRNA ( Sigma , 10109495001 ) .", "In vitro translation reactions were carried out with 5 µl of either supplemented rabbit reticulocyte lysate or oocyte extract in total volume of 20 µl containing 10 µM amino acid mix ( Promega , L4461 ) , 0 . 5 units SUPERase•In , 10 mM creatine phosphate ( Roche 10621714001 ) , 200 µg/ml creatine kinase ( Roche , 10127566001 ) , 20 mM HEPES pH 7 . 5 , 75 mM potassium acetate , 1 . 5 mM magnesium acetate , and 1 fmol/µl Rluc or Nluc reporter RNA with indicated poly ( A ) tails .", "When using Rluc reporters , 2 . 5 fmol/µl Fluc RNA with a 120 nt poly ( A ) region followed by a mutant mouse Malat1 3′ end was included in each reaction for use as a normalization control .", "When using Nluc reporters , 2 . 5 fmol/µl Fluc RNA with a histone mRNA 3′-end stem-loop was included in each reaction for use as a normalization control .", "When examining the effects of adding purified proteins , the reaction also included 2 µl of the indicated amount of either X . laevis PABPC1 , eGFP , or buffer G ( 20 mM Tris pH 7 . 5 , 150 mM NaCl , 5% glycerol , 5 mM DTT ) .", "X . laevis PABPC1 ( pabpc1 . S ) coding sequence was amplified from cDNA generated from X . laevis oocytes .", "eGFP was obtained from the plasmid pCS2+-eGFP ( Chen et al . , 2017 ) .", "All coding sequences were cloned into pET28a ( Novagen ) vector by restriction-free cloning .", "The recombinant protein carried a hexahistidine tag at its C terminus .", "The plasmid was transformed into E . coli BL21 ( DE3 ) Star cells .", "After growing cells at 37°C to an optical density ( OD600 ) of 0 . 6 , expression of recombinant protein was induced with 0 . 5 mM isopropyl β-D-thiogalactoside ( GoldBio ) .", "For purification of PABPC1 , the cells continued to grow at 18°C for 16 hr , after which they were collected by centrifugation , resuspended in 10 volumes of lysis buffer ( 20 mM Tris pH 7 . 5 , 2 M NaCl , 5% ( v/v ) glycerol and 5 mM 2-mercaptoethanol ) and lysed by sonication .", "Lysate was cleared by centrifugation at 25 , 000 g for 30 min and incubated with Ni-NTA agarose ( Qiagen , 30210 ) at 4°C for 1 hr ( 0 . 5 ml resin per 50 ml supernatant ) .", "The resin was washed with 20 resin volumes of buffer H ( 20 mM Tris pH 7 . 5 , 2 M NaCl , 5% glycerol , 5 mM 2-mercaptoethanol and 10 mM imidazole pH 7 . 5 ) and then with 20 resin volumes of buffer L ( 20 mM Tris pH 7 . 5 , 150 mM NaCl , 5% glycerol , 5 mM 2-mercaptoethanol and 20 mM imidazole pH 7 . 5 ) .", "Proteins were eluted with buffer E ( 20 mM Tris pH 7 . 5 , 150 mM NaCl , 5% glycerol , 5 mM 2-mercaptoethanol and 200 mM imidazole pH 7 . 5 ) .", "The eluate was diluted 1:9 with buffer CL ( 20 mM Tris pH 7 . 5 , 25 mM NaCl , 5% glycerol , 5 mM DTT ) and loaded onto a Mono S column ( GE Healthcare ) .", "Bound proteins were eluted by linear NaCl gradient with buffer CL and buffer CH ( 20 mM Tris pH 7 . 5 , 500 mM NaCl , 5% glycerol , 5 mM DTT ) .", "Fractions from the desired peak were pooled , concentrated with an Amicon filter unit ( Millipore , UFC805024 ) and applied to a Superdex 200 column ( GE Healthcare ) in buffer G ( 20 mM Tris pH 7 . 5 , 150 mM NaCl , 5% glycerol , 5 mM DTT ) .", "Fractions from the desired peak were pooled , concentrated with an Amicon filter unit , flash-frozen in liquid nitrogen and stored at −80°C .", "eGFP protein was purified similarly , except all buffers had 150 mM NaCl , and the cation-exchange step was omitted .", "Healthy X . laevis stage VI oocytes that had recovered from defolliculation were selected for injection .", "When examining the effects of expressing additional PABPC , either water or the indicated amount of capped mRNA ( 0 . 25–2 pmol/µl ) coding for either eGFP , frog PABPC1 , frog PABPC1 ( M161A ) , frog ePAB , or human eIF4G was injected into oocytes in a volume of 2–8 nl per oocyte ( PLI-100 Plus Pico-Injector , Harvard Apparatus ) at 23°C .", "Injected oocytes were incubated in complete OR-2 buffer at 23°C for 20 hr , after which they were either lysed for making sequencing libraries or co-injected with an Fluc reporter mRNA with a histone mRNA 3′-end stem-loop ( 20 fmol/µl , used for normalization ) , and either an Rluc reporter mRNA with indicated poly ( A ) -tail length ( 10 fmol/µl ) or an Nluc reporter mRNA with indicated poly ( A ) -tail length ( 10 fmol/µl ) in a total volume of 2 nl per oocyte , except in Figure 1F , where only 2 nl Nluc reporter mRNAs ( 10 fmol/µl ) with indicated 3′ ends were injected .", "These oocytes were incubated in complete OR-2 buffer at 23°C for 6 hr before lysis for dual luciferase assays .", "For PAL-TRAP , oocytes were injected with mRNA ( 1 pmol/µl ) coding for either X . laevis RPL3-HA or eGFP-HA at a volume of 4 nl per oocyte .", "After incubation in complete OR-2 buffer at 23°C for 1 day , these oocytes were injected again with a mixture of mRNA coding for either eGFP or ePAB ( 1 pmol/µl ) and a population of Rluc reporter mRNAs with different poly ( A ) -tail lengths ( 0 , 30 , 63 , 98 , and 120 nt , equimolar ratio; combined concentration , 166 . 5 fmol/µl ) in a volume of 4 nl per oocyte .", "After incubation in complete OR-2 buffer at 23°C for another day , oocytes were collected and lysed for PAL-TRAP analysis .", "Five to 10 oocytes were lysed by vigorous shaking and pipetting in Passive Lysis Buffer ( Promega , E1980 ) , using 20 µl per oocyte .", "Lysates were cleared by centrifugation at 5000 g for 5 min at 4°C , after which 20 µl of each supernatant was transferred to a 96-well microplate and equilibrated to room temperature .", "Luciferase assays were carried out with Dual-Luciferase Reporter Assay System ( Promega , E1980 ) in a Veritas Microplate Luminometer according to the manufacturer’s protocol , regardless of whether Nluc or Rluc was used as the reporter .", "Total RNA ( 1–10 µg ) was separated on an agarose-formaldehyde gel ( Mansour and Pestov , 2013 ) and transferred to a nylon membrane using the Whatman Nytran SuPerCharge ( SPC ) TurboBlotter system ( Sigma , WHA10416300 ) .", "After overnight transfer , RNA was crosslinked to the membrane using a UV Stratalinker 2400 at wavelength 254 nm for a total of 1200 µJ .", "For probing Rluc and 18S RNAs , membranes were pre-incubated with ULTRAhyb Ultrasensitive Hybridization Buffer ( Thermo Fisher , AM8670 ) at 68°C under rotation for 1 hr and then hybridized under the same conditions overnight with DNA probes , which were body-labeled with [α-32P]dCTP ( PerkinElmer ) using a Random Primer DNA Labeling Kit ( TaKaRa , 6045 ) according to the manufacturer’s protocol .", "The templates for the labeling reactions were DNA fragments generated by PCR with gene-specific primers .", "After probe hybridization , membranes were washed two times ( 5 min each ) with a low-stringency buffer ( 2X SSC and 0 . 1% SDS ) at 68°C with rotation , and two times ( 15 min each ) with high-stringency buffer ( 0 . 1X SSC and 0 . 1% SDS ) at 68°C with rotation .", "For probing endogenous mRNAs , membranes were hybridized to radiolabeled gene-specific DNA oligonucleotide probes in ULTRAhyb-Oligo buffer ( Thermo Fisher , AM8663 ) overnight at 42°C .", "The DNA probes were labeled with T4 PNK ( NEB , M0201S ) and [γ-32P]ATP ( PerkinElmer ) .", "Following hybridization , membranes were washed three times ( 20 min each ) with a wash buffer ( 2X SSC and 0 . 5% SDS ) at 42°C with rotation .", "The blots were then analyzed using a Typhoon FLA 7000 phosphor-imager ( GE Healthcare Life Sciences ) .", "Before probing for a second mRNA on the same blot , the blots were stripped three times ( 20 min each ) in a boiling stripping buffer ( 0 . 04% SDS ) with gentle shaking and then checked for any residual radioactivity by extended phosphorimaging .", "Sequences of oligos used for northern blots are provided in Supplementary file", "1 . Total RNA ( 1–10 µg ) was mixed with a DNA oligo ( 100 pmol , Supplementary file 1 ) complimentary to a segment of the 3′-UTR in water in a volume of 15 µl .", "In some experiments , two DNA oligos , each complementary to a different mRNA , were added together in one reaction .", "After incubating the RNA–DNA mix at 85°C for 5 min , the temperature was gradually lowered to 42°C ( 0 . 1°C per s ) , and then 2 µl 10x Hybridase Buffer ( 500 mM HEPES pH 7 . 5 , 1 M NaCl , 100 mM MgCl2 ) , 0 . 5 µl Hybridase ( Lucigen , H39500 , five units/µl ) and 2 . 5 µl water were added to the mix .", "After incubation at 42°C for 20 min , nucleic acids were extracted with phenol/chloroform , precipitated with ethanol , resuspended in 1x RNA Gel Loading Dye ( Thermo Fisher , R0641 ) , and resolved on an 8% acrylamide denaturing gel ( National Diagnostics , EC-829 ) .", "RNA was transferred onto Hybond-NX membranes ( GE Healthcare , RPN303T ) using a Trans-Blot SD Semi-Dry Transfer Cell ( Bio-Rad ) .", "Membranes were incubated at 60°C for 1 hr with EDC ( 1-ethyl-3- ( 3-dimethylaminopropyl ) carbodiimide hydrochloride ) , Thermo Fisher , 22981 diluted in 0 . 17 M 1-methylimidazole pH 8 . 0 to chemically crosslink 5′ phosphates to the membrane .", "Blots were hybridized to radiolabeled DNA oligonucleotide probes in ULTRAhyb-Oligo buffer ( Thermo Fisher , AM8663 ) overnight at 42°C .", "The DNA probes were complimentary to the 3′-UTR regions immediately adjacent to the poly ( A ) tails and were labeled with T4 PNK ( NEB , M0201S ) and [γ-32P]ATP ( PerkinElmer ) .", "Following hybridization , membranes were washed three times ( 20 min each ) with a wash buffer ( 2X SSC and 0 . 5% SDS ) at 42°C with rotation .", "The blots were then analyzed using a Typhoon FLA 7000 phosphor-imager .", "Before probing for a second RNA on the same blot , the blots were stripped as described for conventional northern blots .", "Sequences of oligos used for RNase H northern blots are provided in Supplementary file", "1 . Samples for western blots were boiled in NuPAGE LDS Sample Buffer ( Thermo Fisher , NP0007 ) , resolved on NuPAGE 4–12% Bis-Tris protein gels ( Thermo Fisher , NP0323BOX ) , and transferred to 0 . 2 µm PVDF membranes ( Thermo Fisher , LC2002 ) with a Mini Gel Tank ( Thermo Fisher , A25977 ) , according to the manufacturer’s protocol .", "Membranes were then blocked with 5% ( w/v ) non-fat dry milk in TBS buffer ( 20 mM Tris pH 7 . 5 , 150 mM NaCl ) for 30 min .", "Primary antibodies were diluted at 1:1000 in TBST buffer ( 20 mM Tris pH 7 . 5 , 150 mM NaCl , 0 . 1% ( v/v ) Tween 20 ) and incubated with the blot at 4°C overnight .", "Secondary antibodies were diluted at 1:10 , 000 in TBST buffer and incubated with the blot at 23°C for 1 hr .", "Blots were analyzed using an Odyssey Clx machine ( LI-COR ) and the Image Studio software ( LI-COR ) .", "For total protein detection , Revert 700 Total Protein Stain Kits ( LI-COR , 926–11010 ) were used before incubation with primary antibodies , according to the manufacturer’s protocol .", "Total protein levels were quantified with the ImageQuant TL software ( GE Healthcare Life Sciences ) .", "Individual protein levels were quantified with the ImageStudio software ( LI-COR , v5 . 2 . 5 ) .", "Groups of five oocytes were incubated with 0 . 5 mCi/ml EasyTag EXPRESS 35S protein labeling mix ( PerkinElmer , NEG772007MC ) in 100 µl complete OR-2 buffer at 23°C for 1 hr .", "For a control , a group of oocytes was pretreated with 100 µg/ml cycloheximide in 100 µl complete OR-2 buffer for 5 min before adding the labeling mix .", "After labeling , buffers were removed and oocytes were washed with 1 ml complete OR-2 buffer .", "After removing all residual wash buffer , 100 µl ice cold lysis buffer ( 20 mM HEPES pH 7 . 5 , 100 mM KCl , 5 mM MgCl2 , 1% ( v/v ) Triton X-100 , 1x Halt proteinase inhibitor cocktail ( Thermo Fisher , 78429 ) , 2 mM DTT ) was added to each group , and oocytes were lysed by vigorous shaking and pipetting .", "Lysates were cleared by centrifuging at 5 , 000 g at 4°C for 5 min , and supernatants were boiled in NuPAGE LDS Sample Buffer and resolved by SDS-PAGE .", "Gels were dried and analyzed using the Typhoon FLA 7000 phosphor-imager and the ImageQuant TL software ( GE Healthcare Life Sciences ) .", "Injected oocytes ( ~100 ) were washed once with complete OR-2 buffer and three times with 1 ml ice-cold buffer RL ( 20 mM HEPES pH 7 . 5 , 100 mM KCl , 5 mM MgCl2 , 1% ( v/v ) Triton X-100 , 100 µg/ml cycloheximide , 20 units/ml SUPERase•In , cOmplete protease inhibitor cocktail [Sigma , 11836170001 , 1 tablet per 10 ml buffer] ) .", "After removing all wash buffer , oocytes were lysed in buffer RL ( 10 µl/oocyte ) by vigorous shaking and pipetting .", "Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min .", "A portion of each supernatant ( 5% ) was saved as the input for western analysis and RNA isolation .", "The remaining supernatant was incubated with anti-HA magnetic beads ( Thermo Fisher , 88837 ) using 5 µl slurry per 100 µl supernatant .", "A population of Nluc RNAs with varied poly ( A ) -tail lengths ( 29 , 63 , and 139 nt , equimolar ratio ) were spiked in at 5 ng per 100 µl supernatant , and the bead mixture was incubated at 4°C for 1 hr with end-to-end rotation .", "Beads were then immobilized using a magnetic stand , and the supernatant was removed , with a portion ( 5% ) saved as the flowthrough for western blot and RNA isolation .", "Beads were washed four times with 1 ml buffer RL and resuspended in 110 µl buffer RL , 10 µl of which was taken for western blot and the remainder was used for RNA isolation .", "RNA was isolated with 10 volumes of Tri Reagent ( Thermo Fisher , AM9738 ) according to the manufacturer’s protocol .", "One µg purified RNA was incubated at 37°C for 30 min with 10 units of T4 PNK ( NEB , M0201S ) in PNK buffer containing 20 units SUPERase•In in a 25 µl reaction to remove 2′−3′-cyclic phosphates of reporter RNAs .", "Two PAL-seq v3 libraries were made from each input and eluate sample , and after monitoring reproducibility , data from each pair of replicates were merged during analyses , whereas only one library was made from the each flowthrough sample .", "Lysate preparation and centrifugation were performed the same as for ribosome profiling , except that no nuclease was added prior to centrifugation .", "RNA was purified using 3 volumes of Trizol LS ( Thermo Fisher , 10296010 ) according to manufacturer’s protocol and proteins were extracted by chloroform-methanol precipitation .", "All mammalian cells were cultured at 37°C with 5% CO2 .", "HeLa cells were obtained from the Bartel lab ( Nam et al . , 2014 ) and cultured in DMEM ( VWR , 45000–304 ) with 10% FBS ( TaKaRa , 631106 ) .", "HeLa RPL3-3xHA ( sC262-4 ) cells and their parental cells , a Flp-In T-Rex HeLa cell line that had an OsTIR1 gene obtained from Andrew Holland , were cultured in DMEM with 10% FBS .", "HCT116 OsTIR1 cells , obtained from the Kanemaki lab ( Natsume et al . , 2016 ) , and their derivative HCT116 PABPC1-AID ( sC152-C16 ) cells were cultured in McCoy’s 5A media ( Thermo Fisher , 16600082 ) supplemented with 10% FBS , 2 mM L-glutamine ( Thermo Fisher , 25030081 ) .", "HCT116 PABPC1-AID ( sC278-C2 ) cells were cultured as HCT116 OsTIR1 cells but supplemented with 600 µg/ml G418 ( Thermo Fisher , 10131027 ) .", "NIH3T3 cells were obtained from the Bartel lab ( Eisen et al . , 2020 ) and cultured in DMEM with 10% BCS ( Sigma , 12133C ) .", "ZF4 cells were obtained from ATCC ( CRL-2050 ) and cultured in DMEM F-12 media ( ATCC , 30–2066 ) and 10% FBS , at 28°C with 5% CO2 .", "Mycoplasma testing was performed and no contamination was observed .", "For siRNA transfection , cells were plated at 2 × 106 cells per 10 cm plate , cultured for 12 hr , and transfected with 15 pmol total siRNAs and 45 µl Lipofectamine RNAiMAX ( Thermo Fisher , 13778500 ) in 1 ml Opti-MEM media ( Thermo Fisher , 31985062 ) according to manufacturer’s protocol .", "Cells were split 1:4 onto new plates 24 hr after transfection and then collected for analysis 48 hr ( Figure 4—figure supplement 2A-B , Figure 4—figure supplement 3 , Figures 5–6 , Figure 6—figure supplement 1 ) or 72 hr ( Figure 4 , Figure 4—figure supplement 1 and Figure 4—figure supplement 2C–G ) after transfection .", "For rescue of PABPC-knockdown , cells were plated at 0 . 6 × 106 cells per well in 6-well plates , and transfected with 2 . 5 pmol total siRNAs and 7 . 5 µl Lipofectamine RNAiMAX in 250 µl Opti-MEM media .", "After 24 hr , cells were transfected with 1 µg DNA and 3 µl FuGENE HD ( Promega , E2311 ) in 150 µl Opti-MEM media according to manufacturer’s protocol , and collected for analysis 40 hr after DNA transfection .", "For other DNA plasmid transfections , cells were plated at 0 . 6 × 106 cells per well in 6-well plates , cultured for 24 hr , transfected with 1 µg DNA and 3 µl FuGENE HD ( Promega , E2311 ) in 150 µl Opti-MEM media according to manufacturer’s protocol .", "siRNAs were all purchased from Dharmacon , including siControl ( D-001810-10-05 ) , human siPABPC1 ( L-019598-00-0005 ) , human siPABPC4 ( L-011528-01-0005 ) , mouse siPabpc1 ( L-060385-00-0005 ) , human siTUT4 ( L-021797-01-0005 ) , and human siTUT7 ( L-026009-00-0005 ) .", "Plasmids and their sequence information will be available at Addgene .", "Assays were performed as described ( Schmidt et al . , 2009 ) .", "Puromycin ( Thermo Fisher , A1113802 ) was added to cell culture media at final concentration 1 µg/ml .", "Cells were incubated at 37°C for 10 min before harvest .", "For a control , cycloheximide was added to the media at final concentration 100 µg/ml 5 min before the addition of puromycin .", "AID was introduced at the C terminus of PABPC1 using Cas9-mediated genome engineering in HCT116 OsTIR cells .", "The 5′ and 3′ homology arms ( ~500 nt ) flanking the stop codon of PABPC1 were amplified from genomic DNA of HCT116 OsTIR1 cells and cloned into a donor plasmid ( kindly provided by Iain Cheeseman ) .", "The AID coding sequence followed by the T2A peptide and mCherry , whose sequences were obtained from pMK1221 ( Addgene , #84220 ) , was inserted immediately before the PABPC1 stop codon .", "The PAM region targeted by Cas9 on the donor plasmid was mutated .", "The Cas9 guide RNA was cloned into pX330-BFP , as described ( McKinley and Cheeseman , 2014 ) .", "Both the donor and the Cas9 plasmids were co-transfected into HCT116 OsTIR cells at 0 . 5 µg each in a six-well format .", "After 72 hr , single cells with strong mCherry signal were sorted with flow cytometry into 96-well plates .", "Clones were expanded and genotyped by PCR and western blot .", "Only clones that were homozygous for AID integration were retained .", "One clonal line ( sC152-C16 ) was used for results shown in Figure 5C .", "Six hr after siRNA transfection , 1 µg/ml doxycycline was added to induce OsTIR1 .", "After another 18 hr , indole-3-acetic acid ( IAA , GoldBio ) was dissolved in ethanol and added to cells at a concentration of 0 . 5 mM .", "Twenty-four hr after IAA addition , cells were harvested for analysis .", "Sequences of oligos used for making the donor and the Cas9 plasmids are provided in Supplementary file", "1 . IAA-induced depletion of PABPC1 in the PABPC1-AID cell line ( sC152-C16 ) was relatively slow and generally required more than 12 hr for 90% depletion .", "To achieve faster depletion dynamics , more copies of OsTIR1 were introduced .", "OsTIR1 coding sequences were obtained from pBabe Puro osTIR1-9Myc ( Addgene #80074 ) and subcloned into PiggyBac-dCas9-Tet1 ( Liu et al . , 2016 ) ( kindly provided by Shawn Liu ) , replacing the dCas9 coding sequence .", "This plasmid was co-transfected with piggyBac Transposase expression vector ( System Biosciences , PB210PA-1 ) into the PABPC1-AID cell line ( sC152-C16 ) .", "48 hr after transfection , cells were selected in 600 µg/ml G418 for 10 d .", "Single clones were expanded and checked for OsTIR1 expression .", "The clone with the highest OsTIR1 expression ( sC278-C2 ) was picked for IAA-induced PABPC1-AID degradation .", "To minimize background degradation , cells were incubated with 1 µg/ml doxycycline and 0 . 2 mM auxinole ( Aobious , AOB8812 ) , an inhibitor of OsTIR1 ( Yesbolatova et al . , 2019 ) for 6 hr , after which fresh media with 1 µg/ml doxycycline and 0 . 5 mM IAA were added .", "Cells were collected after 0 . 5 , 1 , and 3 hr of induction .", "For the non-induced condition , fresh media with 1 µg/ml doxycycline but no IAA were added , and cells were collected after 1 hr .", "Plasmids used for making the PABPC1-AID cell lines and their sequence information will be available at Addgene .", "To prepare lysate from mammalian cells , cycloheximide ( CHX ) was added to each plate at final concentration 100 µg/ml .", "Plates were immediately moved to a cold room and culture media were removed .", "Cells were washed twice with ice-cold PBS supplemented with 100 µg/ml CHX .", "After the last wash , 1 ml buffer RLL ( 20 mM HEPES pH 7 . 5 , 100 mM KCl , 5 mM MgCl2 , 1% ( v/v ) Triton X-100 , 100 µg/ml cycloheximide , 500 unit/ml RNasin Plus [Promega , N2615] , 2 mM DTT , cOmplete protease inhibitor cocktail [Sigma , 11836170001 , 1 tablet per 10 ml buffer] ) was added to each 15 cm plate , and cells were scraped off and incubated on ice for 10 min .", "The resulting lysates were passed through a 26-gauge needle six times and cleared by centrifugation at 1300 g at 4°C for 10 min .", "To prepare lysate from frog oocytes , injected oocytes were washed once with complete OR-2 buffer and three times with buffer RLL .", "After removing all wash buffer , oocytes were lysed in buffer RLL ( 10 µl/oocyte ) by vigorous shaking and pipetting .", "Lysates were cleared by centrifugation at 5000 g at 4°C for 10 min .", "One tenth of each cleared lysate was added to 10 volumes of Tri Reagent for total-RNA preparation , following the manufacturer’s protocol .", "Another small portion ( ~10 µl ) was taken for western analysis .", "The remainder was aliquoted , flash frozen in liquid nitrogen , and stored at –80°C .", "For ribosome profiling , RNase I ( Thermo Fisher , AM2294 ) was added to lysate , using 30 units per OD260 unit for lysate from mammalian cells and 10 units per OD260 unit for lysate from frog oocytes .", "After incubation at 23°C for 30 min , lysates were loaded onto a 10–50% sucrose gradient ( 20 mM HEPES pH 7 . 5 , 100 mM KCl , 5 mM MgCl2 , 10–50% ( w/v ) sucrose , 100 µg/ml cycloheximide , 20 units/ml SUPERase•In , 2 mM DTT ) , centrifuged in a Beckman ultracentrifuge with SW41Ti rotor at 36 , 000 rpm for 2 hr , and then fractioned on a BioComp gradient fractionator .", "Fractions corresponding to 80S ribosomes were collected , except in experiments that generated data for Figure 6A and B , in which fractions corresponding to 40S , 60S , and 80S ribosomes were collected so as to avoid loss of the smaller-sized mitochondrial ribosomes ( Rooijers et al . , 2013 ) .", "Collected fractions were concentrated with an Amicon filter unit ( Millipore , UFC810024 ) , and ribosome-protected RNA fragments were released in a buffer containing 20 mM HEPES pH 7 . 5 , 100 mM KCl , 5 mM MgCl2 , 2 mM EDTA , 20 units/ml SUPERase•In , 2 mM DTT .", "Released RNA fragments were incubated with 0 . 2 mg/ml Proteinase K ( Thermo Fisher , AM2548 ) in the presence of 1% SDS at 42°C for 20 min , and then purified by phenol/chloroform extraction and isopropanol precipitation .", "Sequencing libraries were prepared as described previously ( Subtelny et al . , 2014 ) , except in experiments that generated data for Figure 6A and B , in which different size markers were used to isolate RNA fragments from a larger size range ( 20–40 nt in the first gel ) so as to avoid loss of mitochondrial ribosome-protected fragments ( Rooijers et al . , 2013 ) .", "For matched RNA-seq , rRNAs were either depleted with the RiboZero Gold Kit ( Illumina , Figure 2A , Figure 2—figure supplement 1A-B , and Figure 4—figure supplement 1B ) , depleted with the NEBNext rRNA Depletion Kit ( Human/Mouse/Rat , NEB , E6310L , Figure 6 and Figure 6—figure supplement 1–2 ) , or not depleted ( Figure 2B , E and F , and Figure 2—figure supplement 1C ) .", "Because the RiboZero Gold Kit depletes mitochondrial mRNAs ( Figure 2—figure supplement 1C ) , mitochondrial mRNAs were not used for normalization of data generated with this kit .", "Total RNA ( with rRNA depleted or not depleted ) was fragmented by incubating at 95°C for 20 min in RNA Fragmentation buffer ( 2 mM EDTA , 12 mM Na2CO3 , 88 mM NaHCO3 ) and then ethanol precipitated .", "RNA fragments were size-selected and sequenced in parallel with ribosome-protected fragments .", "A detailed protocol for ribosome profiling is available at http://bartellab . wi . mit . edu/protocols . html .", "Sequencing was performed on an Illumina HiSeq 2500 , with standard runs of either 40 or 50 cycles .", "Reads were trimmed at both ends to removed adapter sequences using Cutadapt ( v1 . 18 ) ( Martin , 2011 ) and then mapped to their respective genome reference using STAR ( v2 . 4 . 2a ) with the parameters ‘--runMode alignReads --outFilterMultimapNmax 1 --outReadsUnmapped Fastx --outFilterType BySJout --outSAMattributes All --outSAMtype BAM SortedByCoordinate’ .", "Mapped reads were counted for each gene with htseq-count ( 0 . 11 . 0 ) .", "For both ribosome profiling and RNA-seq , only reads that uniquely mapped to coding regions of annotated genes ( excluding the first 15 codons and the last five codons ) were included in downstream analyses .", "For TE analyses , an expression cutoff of 30 RNA-seq reads was applied for each gene , with no cutoff for ribosome-footprint reads .", "Normalizations of RNA-seq and ribosome-footprint reads were performed with DESeq2 ( Love et al . , 2014 ) , considering reads for all genes passing the cutoff , except when absolute TE comparisons were made between two samples—in which case , RNA-seq and ribosome-footprints data were normalized by only considering reads from mitochondrial genes using software DESeq2 ( Love et al . , 2014 ) .", "When only relative TEs were considered , TEs were manually centered at", "0 . Our implementation of TAIL-seq ( Eisen et al . , 2020 ) resembled that of mTAIL-seq ( Lim et al . , 2016 ) in that it used splint ligation to append the 3′ adapter , as in PAL-seq v1 ( Subtelny et al . , 2014 ) .", "Total RNA ( 1–30 µg ) was mixed with two sets of tail-length standards ( 0 . 1 ng per 1 µg total RNA for each set ) ( Subtelny et al . , 2014 ) , and trace 5′-radiolabeled marker RNAs ( Eisen et al . , 2020 ) , which were used to evaluate tail-length measurements and 3′ ligation efficiency , respectively .", "These RNAs were ligated to a 3′ adapter in a 64 µl reaction containing 1 . 5 µM 3′ adapter , 1 . 25 µM 3′ splint oligo , 0 . 2 mM ATP , one unit/µl RNasin Plus ( Promega , N2615 ) , 10 mM MgCl2 , 1x T4 RNA Ligase two reaction buffer ( NEB , M0239S ) , and 0 . 5 units/µl T4 RNA Ligase 2 ( NEB , M0239S ) , with the enzyme added after the other components had been mixed and incubated at 23°C for 5 min .", "The ligation reaction was incubated at 18°C for 18 hr .", "Small portions ( 2 µl ) were removed at the start and end of the reaction for examining the ligation efficiency .", "After ligation , RNA was extracted with phenol/chloroform , precipitated with ethanol , resuspended in 10 µl water , and mixed with 90 µl 1x RNA Sequencing Buffer from the RNase T1 kit ( Thermo Fisher , AM2283 ) .", "After incubation at 50°C for 5 min and on ice for 5 min , 1 µl RNase T1 ( one unit/µl , Thermo Fisher , AM2283 ) was added , and the reaction was incubated at 23°C for 15 min , followed by phenol/chloroform extraction and precipitation with the Precipitation/Inactivation Buffer in the RNase T1 kit .", "RNA was resuspended in 12 µl RNA Gel Loading Dye and RNA fragments ranging between 100 and 760 nt were purified on an 8% acrylamide denaturing gel , captured on streptavidin beads , 5′ phosphorylated , and ligated to an equal mix of four phased 5′ adapters as described ( Eisen et al . , 2020 ) .", "cDNA was generated using SuperScript III ( Thermo Fisher , 18080044 ) with DNA primers containing barcodes used for multiplexing , eluted from beads by base hydrolysis of RNA and resolved on 6% urea-acrylamide denaturing gels as described ( Subtelny et al . , 2014 ) , except that cDNA fragments with sizes between 150 nt and 760 nt were selected .", "cDNAs were sequenced directly using a HiSeq 2500 machine in a paired-end 50-by-250 run in normal mode with a v3 kit as described ( Eisen et al . , 2020 ) .", "Poly ( A ) -tail lengths shown in Figure 2 , Figure 2—figure supplement 1 , Figure 4 , Figure 4—figure supplement 1 and Figure 4—figure supplement 2C–G were measured with this method .", "Sequences of adapters and other oligos used for TAIL-seq are provided in Supplementary file", "1 . Because TAIL-seq appears to require an 8-lane flow cell ( Eisen et al . , 2020 ) and thus requires many samples for efficient implementation , we stopped using this method and switched to PAL-seq .", "PAL-seq v3 differs from PAL-seq v2 ( Eisen et al . , 2020 ) in two major ways: ( 1 ) Barcodes were embedded in the 3′ adapters so that different samples could be pooled for downstream stages of library construction .", "( 2 ) Sequencing was performed with cDNA rather than PCR-amplified cDNA .", "Each reaction was set up in a total volume of 20 µl by mixing total RNA ( 0 . 3–3 µg ) with two sets of tail-length standards ( 0 . 1 ng of each set per 1 µg total RNA ) ( Subtelny et al . , 2014 ) , two splint DNA oligos ( 55 pmol A-splint and 2 . 75 pmol U-splint , which allowed polyadenylated RNAs with a terminal uridine to be more efficiently captured ) and one barcoded 3′ adapter ( 50 pmol ) .", "Poly ( A ) -selected mRNA from zebrafish ZF4 cell line ( 0 . 1 ng per µg total RNA ) was also added to mammalian RNA samples to enable additional assessment of tail-length measurement reproducibility .", "The RNA–DNA mix was incubated at 65°C for 5 min , and the temperature was gradually lowered to 16°C ( 0 . 1°C per sec ) before other component were added to a volume of 30 µl containing 0 . 2 mM ATP , one unit/µl RNasin Plus , 10 mM MgCl2 , 1x T4 RNA Ligase 2 reaction buffer and 0 . 5 units/µl T4 RNA Ligase", "2 . After incubation at 16°C for 16 hr , EDTA ( 1 µl , 0 . 5 M , pH 8 . 0 ) was added to stop ligation and samples to be sequenced together ( with different barcodes ) were combined , phenol/chloroform extracted , and precipitated with ethanol .", "Ligated RNA was resuspended in 20 µl water , mixed with 180 µl RNase T1 buffer ( 20 mM sodium citrate pH 5 . 0 , 1 mM EDTA , 7 M urea ) , heated to 50°C for 5 min , and chilled on ice for 5 min , before adding 1 . 6 µl RNase T1 ( one unit/µl , Thermo Fisher , AM2283 ) .", "After incubating the reaction at 23°C for 30 min , RNA was extracted with phenol/chloroform and precipitated using the Precipitation/Inactivation Buffer in the RNase T1 kit .", "Fragments between 150 and 760 nt were gel-purified , selected on beads , 5′-end phosphorylated , ligated to a 5′ adapter , and used for cDNA synthesis in the same way as in our implementation of TAIL-seq , except one of the four phased 5′ adapters was used and cDNAs with lengths between 200 and 760 nt were selected .", "Sequencing was performed similarly as in PAL-seq v2 ( Eisen et al . , 2020 ) but with some differences: ( 1 ) 1 fmol cDNA rather than PCR-amplified cDNA was used per lane .", "( 2 ) A single primer was used to obtain two reads .", "( 3 ) After cluster generation and sequencing-primer hybridization , and before extension of the primer through the poly ( A ) -tail region using the Klenow fragment , 16 dark cycles were performed in order to extend the sequencing primer past the barcode and constant regions of the 3′ adapters as well as past two nucleotides corresponding to the RNA 3′ termini .", "( 4 ) 40 cycles of standard sequencing-by-synthesis were performed to yield the first sequencing read ( read 1 ) .", "( 5 ) After obtaining read 1 , the flow cell was stripped , the same sequencing primer was annealed , and 260 cycles of standard sequencing-by-synthesis were performed to read the barcode ( six nt , which required seven cycles ) , a constant segment of the 3′ adapter ( eight nt , which required only seven cycles because of an extra cycle in the barcode region ) , and the sequence of the RNA , beginning at its 3′ terminus , which revealed whether the RNA had a terminal uridine and provided information used to measure the length of the poly ( A ) tail .", "Poly ( A ) -tail lengths shown in Figure 3 , Figure 3—figure supplement 1 , Figure 4—figure supplement 2A–B and Figure 5 were measured with this method .", "Sequences of adapters and other oligos used for PAL-Seq v3 are provided in Supplementary file", "1 . We found that in PAL-seq v3 , some barcoded 3′ adapters gave rise to much lower numbers of mapped reads , possibly due to low ligation efficiency .", "Therefore , we developed the PAL-seq v4 protocol , in which we made several changes .", "The 3′-ligation reaction was essentially the same , except that one 3′ adapter was used for all samples .", "After the ligation , each sample was processed separately rather than mixed .", "Ligated RNA was resuspended in 10 µl water and fragmented in a 100 µl rather than 200 µl volume , and fragments with sizes between 130 and 760 nt were then gel purified .", "For reverse transcription , different primers containing the multiplexing barcode sequences as well as a region with five random nucleotides was used .", "After cDNA elution from the beads , half of each sample was saved at –20°C for future use .", "cDNA samples to be sequenced on the same lane in a flow cell were combined , ethanol-precipitated and gel-purified as in PAL-seq v3 , except cDNA fragments with sizes between 190 nt and 800 nt were selected .", "Sequencing cluster generation was performed similarly to PAL-seq v3 , using 0 . 3 fmol cDNA mixture for each lane .", "Read 1 started with 12 cycles of standard sequencing-by-synthesis that first sequenced the 5-nt random region ( used to call clusters ) and then sequenced the 6-nt barcode region ( which required seven cycles ) with a custom primer .", "The flow cell was stripped , a second sequencing primer annealed , and two dark cycles were performed in order to extend this primer past the two nucleotides corresponding to the RNA 3′ termini .", "The custom extension of the primer through the poly ( A ) tail region with Klenow was then performed , as in PAL-seq v2 ( Eisen et al . , 2020 ) and v3 , followed by 40 cycles of standard sequencing-by-synthesis to complete the read 1 , which was generated using two sequencing primers and had a total length of 52 nt ( 12 cycles before and 40 cycles after the Klenow reaction ) .", "The flow cell was then stripped , and the second sequencing primer was used for 255 cycles of standard sequencing-by-synthesis to generate read", "2 . Poly ( A ) tail lengths shown in Figure 6E and Figure 6—figure supplement 2 were measured with this method .", "Sequences of adapters and other oligos used for PAL-Seq v4 are provided in Supplementary file", "1 . Both human ( release 25 , GRCh38 . p7 , primary assembly ) and mouse ( release 10 , GRCh38 . p4 , primary assembly ) genomic sequences were downloaded from the GENCODE website .", "Sequences of mitochondrial pseudogenes ( Supplementary file 4 ) in human and mouse genomes were masked to avoid losing mitochondrial mRNA reads due to multi-mapping .", "X . laevis genomic sequences were downloaded from the Xenbase website ( v9 . 1 assembly , repeat masked ) , and scaffolds were removed so that only chromosomal sequences would be considered .", "The X . laevis mitochondrial genomic sequence was obtained from NCBI website ( NC_001573 . 1 ) and appended to the X . laevis genome .", "Both human ( release 25 , GRCh38 . p7 , main annotation ) and mouse ( release 10 , GRCh38 . p4 , primary assembly ) gene annotations were downloaded from the GENCODE website .", "X . laevis gene annotations ( v9 . 1 assembly , v1 . 8 . 3 . 2 primary transcript ) were downloaded from the Xenbase website , and only chromosomal annotations were used .", "X . laevis mitochondrial gene annotations were curated based on the information obtained from the NCBI website ( NC_001573 . 1 ) and appended .", "For all species , annotations for protein-coding genes were extracted , and for each gene , the isoform with the longest open reading frame ( ORF ) was selected to represent that gene .", "In cases in which multiple isoforms had ORFs of the same length , the isoform with the longest transcript was selected as the reference annotation .", "For TAIL-seq and PAL-seq analyses , the databases were supplemented with sequences and annotations of tail-length standards , eGFP , Rluc , Fluc , and Nluc .", "For ribosome profiling and matched RNA-seq analyses , the databases were supplemented with coding sequences and annotations of eGFP , Rluc , Fluc , and Nluc .", "For RNA-seq analyses used to determine half-lives , the databases were supplemented with coding sequences and annotations of AcGFP ( Eisen et al . , 2020 ) , Rluc , and Fluc .", "All supplemented sequences are provided in Supplementary file 3 .", "Each PAL-seq tag corresponding to an mRNA provides the site of cleavage and polyadenylation , which enabled annotation of mRNA 3′-end isoforms .", "Uniquely mapped tags from all PAL-seq datasets in the same cell type ( either HeLa or frog oocyte ) were merged .", "RNA-seq reads generated for comparison to ribosome profiling were similarly merged .", "Software HOMER ( Heinz et al . , 2010 ) was used to call peaks by using the merged RNA-seq data as the background and the merged PAL-seq data as the signal , with the following parameters ‘-style factor -o auto -strand separate -fdr 0 . 001 -ntagThreshold 50 -fragLength 40 -size 40 -inputFragLength 30 -center’ .", "The peaks that intersected with annotated protein exons were retained as unique mRNA 3′ ends .", "The annotated 3′ ends are provided in Supplementary file 5 .", "Phased constant sequences at the 5′ end and poly ( A ) sequences at the 3′ end of read one were removed with a custom script ( Eisen et al . , 2020 ) .", "The trimmed sequences were mapped to reference genomes with STAR ( v2 . 4 . 2a ) with the parameters ‘--runMode alignReads --outFilterMultimapNmax 1 --outReadsUnmapped Fastx --outFilterType BySJout --outSAMattributes All --outSAMtype BAM SortedByCoordinate’ .", "Mapped sequences were intersected with sequences of protein-coding genes by bedtools ( v2 . 26 . 0 ) with the parameters ‘intersect -wa -wb -bed -s’ , retaining only those sequences that were assigned to a single gene .", "Remaining clusters were then filtered , requiring at least five combined N and T bases in the first 6 nucleotides of read", "2 . For each library , 1% of the filtered read clusters ( but no more than 50 , 000 and no less than 5000 ) were randomly picked as the training set for determining the Hidden Markov Model .", "For each Illumina sequencing cluster , average intensities of each channel from position 15–50 of read one were used to normalize intensities of each channel in read", "2 . Then a T-signal value for each cycle of read two in each cluster was calculated by dividing normalized intensity from T channel by the sum of normalized intensities from the other three channels .", "If the T-signal was 0 for a specific cycle , the T-signal values from neighboring cycles ( up to 10 , minimum 5 ) were averaged to infer the value for that cycle .", "If a cluster had more than five cycles with a read 2 T-signal value of 0 , the cluster was discarded .", "A five-state mixed Gaussian Hidden Markov Model ( from python ghmm package ) was then used to decode the sequence of states that occurred in read", "2 . It consisted of an initiation state ( state 0 ) , a strong poly ( A ) -tail state ( state 1 ) , a weak poly ( A ) -tail state ( state 2 ) , a weak non-poly ( A ) -tail state ( state 3 ) and a strong non-poly ( A ) -tail state ( state 4 ) .", "All reads started in state 0 , and all states were only allowed to go forward ( from 0 to 4 ) .", "The model was initialized with the following transition probability matrix ( from state in row to state in column ) :0 . 040 . 930 . 020 . 010 . 000 . 000 . 940 . 030 . 020 . 010 . 000 . 000 . 500 . 400 . 100 . 000 . 000 . 000 . 600 . 400 . 000 . 000 . 000 . 001 . 00 The emission matrix for the mixed population one was initialized with ( states in row , mean , variance and population fraction in column ) :100 . 001 . 001 . 001 . 501 . 500 . 951 . 501 . 500 . 751 . 501 . 500 . 501 . 501 . 500 . 25 The emission matrix for the mixed population two was initialized with ( states in row , mean , variance and population fraction in column ) :0 . 001 . 001 . 00-1 . 001 . 500 . 05-1 . 001 . 500 . 25-1 . 001 . 500 . 50-1 . 001 . 500 . 75 After model initialization , all clusters from the training set were used to perform unsupervised training , and then the trained model was used to decode the sequence of states for all retained clusters .", "For each cluster , the poly ( A ) tail length was determined by summing the number of states in state 1 and", "2 . Each cluster assigned to a specific gene by read one was considered as a poly ( A ) tag .", "When evaluating tail-length distributions of mRNAs from individual genes , only results from genes with at least 100 tags were considered .", "Note that the HiSeq 2500 machine in high-throughput mode raises the laser intensities after 50 cycles in read 2 , causing irregular T-signal at this position and lower-than-expected state transition predicted by the Hidden Markov Model .", "This led to a mild depletion of poly ( A ) tags with tail lengths called at 50 nt , but it did not affect results and conclusions made from overall tail-length distributions .", "For normalization of poly ( A ) tags among samples , DESeq2 ( Love et al . , 2014 ) was used with all spike-in tail-length standards to obtain the scaling factor for each dataset .", "When analyzing median tail lengths , only genes with poly ( A ) tag counts exceeding an indicated cutoff were included in the analyses .", "For PAL-seq v3 , read one sequences were mapped to reference genomes with STAR ( v2 . 4 . 2a ) with the parameters ‘--outFilterMultimapNmax 1 --outFilterType BySJout --outSAMattributes All --outSAMtype BAM SortedByCoordinate’ .", "Mapped sequences were intersected with annotations for protein-coding genes by bedtools ( v2 . 26 . 0 ) with the parameters ‘intersect -wa -wb -bed -S’ , retaining only those tags for which the read one sequence was assigned to a single gene or to an mRNA isoform , when mRNA 3′-end annotations were used .", "Remaining clusters were then filtered by first removing the first 14 bases of read 2 ( which corresponded to a constant region and the barcode region ) and then requiring at least five combined N and T bases within the next 6 nucleotides of read", "2 . ( This filtering step was skipped for analyses of terminal uridylation in Figure 5 and Figure 5—figure supplement 1 ) .", "For each library , 1% of the filtered read clusters ( but no more than 50 , 000 and no less than 5000 ) were randomly picked as the training set for determining the Hidden Markov Model .", "The sequence of poly ( A ) states was determined similarly as for TAIL-seq , except a single Gaussian Hidden Markov Model was used , and the emission matrix was initialized with ( states in row , mean and variance in column ) :100 . 01 . 02 . 00 . 51 . 00 . 5-1 . 00 . 5-2 . 00 . 5 For normalization of poly ( A ) tags among samples , DESeq2 ( Love et al . , 2014 ) was used with all spike-in tail-length standards to obtain the scaling factor for each dataset .", "When analyzing median tail lengths , only mRNA isoforms with poly ( A ) tag counts exceeding an indicated cutoff were included in the analyses .", "Data analyses for PAL-seq v4 were the same as for PAL-seq v3 , except before mapping , the first 12 nt of read one were trimmed to remove the random region and the barcode region , and no nucleotides of read two were trimmed .", "Note that the HiSeq 2500 machine in rapid-run mode raises the laser intensities after 101 cycles in read 2 of a PAL-seq v4 run , causing irregular T-signal at this position and lower-than-expected state transition predicted by the Hidden Markov Model .", "This led to a mild depletion of poly ( A ) tags with tail lengths called at 101 nt , but it did not affect results and conclusions made from overall tail-length distributions .", "A variant HeLa cell line ( sC262-4 ) was used for data shown in Figure 5D–F and Figure 5—figure supplement", "1 . This Flp-In T-Rex HeLa cell line had an OsTIR1 gene knocked-in .", "An RPL3-3xHA gene was inserted using the Flp-In ( Thermo Fisher ) system and a single clone was picked after selection with 400 µg/ml Hygromycin B ( Thermo Fisher 10687010 ) for 10 days .", "We have no reason to suspect that the unique features of this line affected any results shown .", "Data were processed as in PAL-seq v3 , except that mapped reads were intersected with HeLa mRNA 3′-end annotations ( see Annotation of mRNA 3′-end isoforms ) .", "All poly ( A ) tags with poly ( A ) -tail lengths ≥ 2 nt were used when examining the presence of U nucleotides near the ends of poly ( A ) tails .", "Forty-eight hr after siRNA transfection , HeLa cells were incubated with pre-warmed fresh media with 400 µM 5-ethynyl uridine ( 5EU , Jena Biosciences ) for 1 , 2 , 4 , and 8 hr .", "Cells were removed from plates by treatment with trypsin , washed once with ice cold PBS and lysed with 200 µl ice cold buffer RL .", "Lysates were cleared by centrifuging at 1300 g for 5 min .", "Supernatants were transferred to 2 ml Tri Reagent , and total RNA was prepared according to the manufacturer’s protocol .", "Biotinylation of 5EU-labeled RNA and purification of biotinylated RNA were performed at described ( Eisen et al . , 2020 ) .", "RNA-seq libraries were prepared from purified 5EU-labeled RNA and from total RNA with the NEXTflex Rapid Directional mRNA-seq Kit ( Bioo Scientific , 5138–10 ) .", "Sequencing was performed on an Illumina HiSeq 2500 with a standard 40-cycle run .", "Sequencing reads were mapped with STAR ( v2 . 4 . 2a ) with parameters ‘--runMode alignReads --outFilterMultimapNmax 1 --outReadsUnmapped Fastx --outFilterType BySJout --outSAMattributes All --outSAMtype BAM SortedByCoordinate’ .", "Exon-mapped reads were counted for each gene with htseq-count ( 0 . 11 . 0 ) .", "Relative 5EU-labeled mRNA levels for each gene at each time point were obtained by normalizing read counts based on the counts for a 5EU-containing GFP RNA standard that had been spiked into each sample prior to 5EU biotinylation ( Eisen et al . , 2020 ) .", "The steady-state mRNA levels of each gene were measured as the average of normalized read counts obtained from sequencing the input RNA for the 1 and 8 hr time points .", "A total of 987 genes with values that differed by >2-fold at these two time points were excluded from analysis .", "The nls package in R was used to fit the following equation for each gene:y=C+log21-e-kx-t0 , where x is the labelling time in hours ( using 9999 h as the labeling time for the steady-state data point ) , y is log2 value of the normalized read count ( level of labeled RNA ) , and t0 is the time offset .", "k is the decay constant , which was used to determine the half-life t1/2 using , t1/2=ln⁡2k , and C is a coefficient determined by both k and the synthesis rate S , such that , C=log2Sk .", "To fit t0 for each condition , the value of t0 was varied from 0 . 05 to 0 . 7 , with an interval of 0 . 05 , and the value that gave the smallest mean square-loss of y when fitting to data from all genes was used .", "When fitting to results for each gene , C was bound by ( 0 , Inf ) , and k was bound by ( 0 , Inf ) .", "C was initialized with max ( y ) , and k was initialized with 0 . 23 .", "Genes without a converged fit ( 27 of 9644 , 17 of 9646 , and 12 of 9728 in analysis of the siControl , siPABPC1 , and siPABPC1&4 samples , respectively ) were omitted from down-stream analyses .", "mRNA half-life values are reported in Figure 4—figure supplement 3—source data", "1 . Graphs were generated and statistical analyses were performed using R ( R Development Core Team , 2013 ) .", "Statistical parameters including the value of n , statistical test , and statistical significance ( p value ) are reported in the figures or their legends .", "No statistical methods were used to predetermine sample size .", "Statistical tests for correlations ( between nonoverlapping dependent groups ) were performed based on a published method ( Silver and Hittner , 2004 ) using R package cocor ( Diedenhofen and Musch , 2015 ) .", "For luciferase assays of injected oocytes , each replicate refers to a group of 7–10 oocytes .", "For 35S labeling of injected oocytes , each replicate refers to a group of 5–8 oocytes .", "For the puromycin-based translation assay , each replicate refers to a separate transfection experiment .", "Raw and processed data from sequencing were deposited in the GEO database ( GSE166544 ) .", "TAIL-seq and PAL-seq analyses were performed using a custom script written in Python 2 . 7 and available at https://github . com/coffeebond/PAL-seq , ( copy archived at swh:1:rev:b0aa1ba99cc89ce2080069b50cd441a7718b7b03 , Xiang , 2021a , Xiang , 2021b ) ." ] ]

[ "In animal oocytes and early embryos , mRNA poly ( A ) -tail length strongly influences translational efficiency ( TE ) , but later in development this coupling between tail length and TE disappears .", "Here , we elucidate how this coupling is first established and why it disappears .", "Overexpressing cytoplasmic poly ( A ) -binding protein ( PABPC ) in Xenopus oocytes specifically improved translation of short-tailed mRNAs , thereby diminishing coupling between tail length and TE .", "Thus , strong coupling requires limiting PABPC , implying that in coupled systems longer-tail mRNAs better compete for limiting PABPC .", "In addition to expressing excess PABPC , post-embryonic mammalian cell lines had two other properties that prevented strong coupling: terminal-uridylation-dependent destabilization of mRNAs lacking bound PABPC , and a regulatory regime wherein PABPC contributes minimally to TE .", "Thus , these results revealed three fundamental mechanistic requirements for coupling and defined the context-dependent functions for PABPC , which promotes TE but not mRNA stability in coupled systems and mRNA stability but not TE in uncoupled systems ." ]

[ "Cells are microscopic biological factories that are constantly creating new proteins .", "To do so , a cell must first convert its master genetic blueprint , the DNA , into strands of messenger RNA or mRNA .", "These strands are subsequently translated to make proteins .", "Cells have two ways to adjust the number of proteins they generate so they do not produce too many or too few: by changing how many mRNA molecules are available for translation , and by regulating how efficiently they translate these mRNA molecules into proteins .", "In animals , both unfertilized eggs and early-stage embryos lack the ability to create or destroy mRNAs , and consequently cannot adjust the number of mRNA molecules available for translation .", "These cells can therefore only regulate how efficiently each mRNA is translated .", "They do this by changing the length of the so-called poly ( A ) tail at the end of each mRNA molecule , which is made up of a long stretch of repeating adenosine nucleotides .", "The mRNAs with longer poly ( A ) tails are translated more efficiently than those with shorter poly ( A ) tails .", "However , this difference disappears in older embryos , when both long and short poly ( A ) tails are translated with equal efficiency , and it is largely unknown why .", "To find out more , Xiang and Bartel studied frog eggs , and discovered that artificially raising levels of a protein that binds poly ( A ) tails , also known as PABPC , improved the translation of short-tailed mRNAs to create a situation in which both short- and long-tailed mRNAs were translated with near-equal efficiency .", "This suggested that short- and long-tailed mRNAs compete for limited amounts of the translation-enhancing PABPC , and that long-tailed mRNAs are better at it than short-tailed mRNAs .", "Further investigation revealed that eggs also had to establish the right conditions for PABPC to enhance translation and had to protect mRNAs not associated with PABPC from being destroyed before they could be translated .", "Overall , Xiang and Bartel found that in eggs and early embryos , PABPC and poly ( A ) tails enhanced the translation of mRNAs but did not influence their stability , whereas later in development , they enhanced mRNA stability but not translation .", "This research provides new insights into how protein production is controlled at different stages of animal development , from unfertilized eggs to older embryos .", "Understanding how this process is regulated during normal development is crucial for gaining insights into how it can become dysfunctional and cause disease .", "These findings may therefore have important implications for research into areas such as infertility , reproductive medicine and rare genetic diseases ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "neuroscience" ]

[ [ "Understanding how neural circuits process information requires knowing not only the neuroanatomical connectivity and electrophysiological properties of individual neurons , but also the organization of synapses between specific neuronal types .", "Knowledge of these basic organizational principles , as well as the mechanisms for how they are achieved in a complex circuit , represents a critical goal for neuroscience .", "Historically , neuromuscular junctions have been model synapses for mechanistic studies because of their ease of access ( Keshishian et al . , 1996; Sanes and Lichtman , 1999 ) .", "However , neuromuscular circuits display less complexity compared to neural circuits in the central nervous system ( CNS ) .", "Serial-section electron microscopy ( EM ) has offered remarkable resolution in studying CNS synapses in the context of circuits , including the entire Caenorhabditis elegans nervous system ( White et al . , 1986 ) , the mouse olfactory bulb ( Hinds and Hinds , 1976a , 1976b ) , retina ( Briggman et al . , 2011; Helmstaedter et al . , 2013 ) , and visual cortex ( Bock et al . , 2011 ) , and the Drosophila visual system ( Meinertzhagen and O'Neil , 1991; Takemura et al . , 2013 ) .", "However , these approaches are labor intensive and not easily amenable to analysis following genetic perturbation , at varying developmental stages , or with intact samples .", "Indeed , even for well-known synaptic organizers like Neurexin and Neuroligin ( Chia et al . , 2013 ) , studies investigating their function were rarely carried out in an intact system with a resolution sufficient to reveal their detailed functions in synaptogenesis between defined neuronal types .", "As such , there is a need to examine CNS synapses in the intact brain , at the level of light microscopy in a system with sufficient complexity to reflect features of the CNS , but tractable enough to dissect connectivity and function using genetic tools .", "Current work in Drosophila has sought to fulfill this need ( Kremer et al . , 2010; Christiansen et al . , 2011; Berger-Muller et al . , 2013; Chen et al . , 2014 ) .", "The Drosophila olfactory system contains complex circuits that transform chemosensory input to the regulation of behavioral output .", "Olfactory information is received by first-order olfactory receptor neurons ( ORNs ) and is transmitted by ORN axons to the antennal lobe , the first olfactory processing center in the brain .", "There , the information is conveyed to the dendrites of second-order projection neurons ( PNs ) through class-specific ORN–PN synapses .", "PN axons carry signals to higher-order brain centers ( Vosshall and Stocker , 2007 ) .", "The local interneurons ( LNs ) innervate the antennal lobe extensively with their dendrites ( Chou et al . , 2010 ) and are involved in transforming ORN input to PN output ( Wilson , 2013 ) .", "These three groups of neurons have extensive interconnectivity and indeed , the antennal lobe has emerged as a model neural circuit for investigating the general principles of information processing ( Wang , 2012; Wilson , 2013; Twick et al . , 2014 ) .", "While many antennal lobe neurons have been examined electrophysiologically ( Wilson , 2013 ) , there has been little analysis of their synaptic architecture: how many connections are made , where they are made in relation to other neurons in the circuit , and what molecules are required for their organization .", "Electron microscopy has been used to probe different genetic conditions ( Acebes and Ferrus , 2001; Acebes et al . , 2011 ) but could not definitively assign glomerular identity or cell type for individual synapses , highlighting the need for structural analysis at the level of light microscopy in three dimensions .", "Knowledge about synaptic organization is critical to understand how sensory information is received , processed , and relayed by this circuit .", "Thus , the fly olfactory circuit represents an excellent opportunity to assess synaptic organization in an intact system at the resolution of individual , defined neuronal classes , linking structure to physiology and function .", "Here , we utilized an approach where presynaptic active zone and postsynaptic neurotransmitter receptor proteins were tagged with fluorescent molecules to highlight and quantitatively describe the synaptic organization of the Drosophila antennal lobe .", "We identified distinct rules that govern the number , density , and spatial organization of olfactory synapses for multiple classes of neurons .", "To understand how these rules are implemented at the molecular level , we investigated the function of the Teneurins , evolutionarily conserved type II transmembrane proteins recently shown to regulate synaptic partner matching and neuromuscular synaptic organization ( Hong et al . , 2012; Mosca et al . , 2012 ) .", "We show that presynaptic Ten-a and postsynaptic Ten-m regulate central synapse number and active zone structure .", "These represent the first in vivo functions of Teneurins in organizing CNS synapses , which could contribute to their association with brain disorders such as bipolar disorder ." ], [ "To visualize synapses in vivo in genetically identifiable cells , we explored strategies to label active zones , sites of presynaptic neurotransmitter release ( Zhai and Bellen , 2004 ) .", "In Drosophila , the primary unit of active zone architecture is an electron dense structure called the T-bar , a specialized formation that promotes vesicle docking and release ( Wichmann and Sigrist , 2010 ) present at peripheral ( Jia et al . , 1993 ) and central synapses ( Meinertzhagen and O'Neil , 1991; Prokop and Meinertzhagen , 2006 ) .", "The major structural components of the T-bar include Bruchpilot ( Brp ) and Rim-binding protein ( Wagh et al . , 2006; Liu et al . , 2011 ) , with Brp encoding the ‘table-top’ of the T-bar and Rim-binding protein comprising the ‘pedestal’ ( Figure 1A ) .", "Widely used monoclonal antibodies ( nc82 ) against Brp ( Blanco Redondo et al . , 2013 ) label synaptic neuropil and discrete puncta corresponding to active zones ( Wagh et al . , 2006 ) .", "However , these antibodies cannot distinguish synapses formed by a given class of neurons in complex CNS circuits .", "Therefore , to visualize active zones in genetically identifiable cells , we sought to label Brp with the GAL4/UAS system ( Brand and Perrimon , 1993 ) . 10 . 7554/eLife . 03726 . 003Figure 1 . Measuring CNS synapses using Bruchpilot-Short .", "( A ) Diagram comparing full-length Bruchpilot and Bruchpilot-Short .", "Numbers denote amino acids and orange represents coiled coil regions ( after Wagh et al . , 2006 ) .", "The positions of Brp and Brp-Short in relation to known active zone proteins are also shown in a simplified format ( after Liu et al . , 2011 ) .", "( B–D )", "High magnification confocal stacks of a single DA1 glomerulus with putative synapses labeled by Brp-Short ( B ) , neurites labeled by mCD8-GFP ( C ) , and the merge ( D ) .", "Insets show higher magnification of a single optical section to demonstrate the punctate nature of Brp-Short-mStraw .", "( E–G )", "Screenshots of three-dimensional renderings show conversion of Brp-Short into ‘Spots’ ( E ) and mCD8-GFP into a volumetric surface rendering ( F ) .", "For ( F ) and ( G ) , the transparency of the surface rendering is at 60% to highlight internal structure and make Brp-Short puncta visible .", "( H ) Quantification of Brp-Short puncta ( red , left axis ) and neurite volume ( black , right axis ) in males and females for five ORN classes .", "Statistical comparisons between males and females of a single genotype were done by student's t test .", "Significance across all genotypes for density was assessed with a one-way ANOVA and corrected for multiple comparisons by a posthoc Tukey's multiple comparisons test .", "***p < 0 . 001 .", "In all cases , n ≥ 9 brains , 18 antennal lobes for each genotype .", "( I ) Quantification of synaptic density ( Brp-Short puncta/volume of neurites ) in males ( blue ) and females ( pink ) for five ORN classes .", "Despite considerable differences in Brp-Short puncta number , all classes of ORNs display nearly identical synaptic densities .", "Scale bar = 5 µm for all images . DOI: http://dx . doi . org/10 . 7554/eLife . 03726 . 00310 . 7554/eLife . 03726 . 004Figure 1—figure supplement 1 . Validation of Brp-Short .", "( A ) Representative high magnification confocal single sections of Or67d-positive ORNs innervating the DA1 glomerulus expressing Brp-Short-mStraw and DSyd1-EGFP and stained with antibodies against mStraw ( red ) and GFP ( green ) .", "Note the significant overlap between Brp-Short and DSyd-1 , a known presynaptic marker .", "( B ) Representative high magnification confocal single sections of Or67d-positive ORNs innervating the DA1 glomerulus expressing Brp-Short-mStraw and mCD8-GFP in a control animal .", "( C ) Representative high magnification confocal single sections of Or67d-positive ORNs innervating the DA1 glomerulus expressing Brp-Short-mStraw , mCD8-GFP and an RNAi transgene against Brp .", "As endogenous Brp has been impaired , Brp-Short-mStraw is greatly diminished at the synapse .", "Scale bar = 5 μm .", "( D ) A single electron dense region as imaged by immunoelectron microscopy in an animal expressing Brp-Short-EGFP in all ORNs .", "5-nm Nanogold dots ( arrowheads ) decorate electron-dense structures characteristic of active zones following immunostaining against the GFP epitope , suggesting that Brp-Short localizes to endogenous active zones .", "Scale bar = 25 nm . DOI: http://dx . doi . org/10 . 7554/eLife . 03726 . 00410 . 7554/eLife . 03726 . 005Figure 1—figure supplement 2 . Additional examples of the Brp-Short assay . Representative high magnification confocal z-stack images of ORNs innervating the VA1d ( Or88a-GAL4; [A] ) , VA1lm ( Or47b-GAL4; [B] ) , DL4 ( AM29-GAL4; [C] ) , and DM6 ( AM29-GAL4; [D] ) glomeruli expressing Brp-Short-mStraw and stained with antibodies to mStraw ( red ) and N-Cadherin ( blue ) .", "In all cases , puncta are clearly visible; these glomeruli were quantified in Figure 1H .", "Scale bar = 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 03726 . 005 Full-length Brp is a 1740 amino acid protein that can aggregate upon ectopic overexpression ( Wagh et al . , 2006 ) , limiting its use as a labeling strategy .", "However , Bruchpilot-Short ( Brp-Short ) , a nonfunctional 754-residue portion of Brp ( Figure 1A ) , localizes to endogenous sites of full-length Brp ( Schmid et al . , 2008; Fouquet et al . , 2009 ) without deleterious effects .", "Thus , under the control of a binary expression system , Brp-Short can act as a reporter of endogenous active zones without disrupting morphology or function ( Schmid et al . , 2008; Fouquet et al . , 2009; Kremer et al . , 2010 ) .", "This strategy has been employed at neuromuscular synapses ( Schmid et al . , 2008; Fouquet et al . , 2009 ) , in the mushroom body ( Kremer et al . , 2010; Christiansen et al . , 2011 ) , and in the visual system ( Berger-Muller et al . , 2013 ) .", "We utilized Brp-Short to quantitatively measure the number of active zones , a key element of understanding synaptic organization .", "We expressed an mStrawberry-tagged version ( Brp-Short-mStraw: Owald et al . , 2010 ) in Or67d ORNs that innervate the DA1 glomerulus .", "Coexpression with a known presynaptic protein , DSyd-1 , revealed extensive colocalization ( Figure 1—figure supplement 1 , panel A ) , suggesting Brp-Short-mStraw correctly localizes to synapses .", "Further , RNAi against a portion of endogenous Brp not encoded by the Brp-Short transgene in those ORNs resulted in a loss of Brp-Short signal at ORN presynapses ( Figure 1—figure supplement 1 , panels B–C ) , though Brp-Short was still detectable in the cell body .", "This supports the hypothesis that Brp-Short requires endogenous Brp for proper localization to synapses; alternatively , Brp-Short could require endogenous Brp for proper trafficking to axon terminals .", "Finally , to confirm the veracity of the synaptic signal , we performed immuno-electron microscopy ( immuno-EM ) using antibodies to the GFP-tag ( the mStraw tag is not amenable to immuno-EM ) of a Brp-Short-GFP transgene ( Schmid et al . , 2008 ) expressed in all ORNs via pebbled-GAL4 ( Sweeney et al . , 2007 ) .", "With pebbled-GAL4 , Brp-Short expression and localization was similar to that observed for ORN-class-specific GAL4 drivers ( data not shown ) .", "While immuno-EM cannot be utilized as a quantitative strategy for determining the number of active zones ( as the conditions needed to preserve the GFP epitope necessarily reduce the resolution of the technique to clearly discern all synaptic densities ) , it can be used to verify that Brp-Short localizes to electron-dense regions that are consistent with synaptic active zones .", "Indeed , 5-nm gold particles were observed to decorate putative active zone profiles ( Figure 1—figure supplement 1 , panel D ) in ultrathin sections of fly antennal lobes .", "90 . 1% of Nano-Gold dots colocalized with synaptic densities ( 675 5-nm Nano-Gold particles out of 749 particles observed ) .", "Combined , these experiments demonstrate that Brp-Short localization is consistent with active zones in the fly CNS .", "To determine the number of active zones produced by ORN axons that project to a given glomerulus , we expressed Brp-Short-mStraw in various ORN classes ( Figure 1B–D , Figure 1—figure supplement", "2 ) together with UAS-mCD8-GFP ( Lee and Luo , 1999 ) as a general neurite label .", "Single optical sections ( Figure 1B–D , insets ) revealed the punctate nature of the Brp-Short-mStraw signal .", "To quantify the number of Brp-Short puncta in these ORNs , we imaged individual glomeruli using high magnification confocal microscopy followed by image deconvolution and 3-D rendering ( Video 1; see ‘Materials and methods’ ) .", "Image processing allowed us to calculate the number of Brp-Short puncta ( Figure 1B , E ) as ‘Spots’ and the neurite volume by surface rendering of the membrane marker mCD8-GFP ( Figure 1C , F ) .", "The numbers of Brp-Short puncta exhibited low variability for multiple classes of ORNs ( Figure 1H , Figure 1—figure supplement 2 ) , suggesting that neuronal classes form stereotyped numbers of active zones across animals .", "While extensive EM reconstruction is not available for the antennal lobe ( and thus , absolute counts of olfactory synapses in each glomerulus for comparison ) , the recent use of labeled Brp in the Drosophila visual system indicated that one Brp punctum is equivalent to one active zone ( Chen et al . , 2014 ) .", "Therefore , it is likely that the Brp-Short label largely reflects actual active zone number . 10 . 7554/eLife . 03726 . 006Video 1 . Three-dimensional rendering of a Brp-Short and mCD8-GFP in a DA1 glomerulus .", "( related to Figure", "1 ) Animation of a three-dimensional rendering of the glomerulus shown in Figure 1B–D .", "Each channel is highlighted and the transformation to either Spots ( for Brp-Short puncta ) or a surface rendering to measure neurite volume ( for mCD8-GFP staining ) is shown .", "High magnification of the rendered glomerulus reveals the distribution of Brp-Short puncta within the glomerulus . DOI: http://dx . doi . org/10 . 7554/eLife . 03726 . 006 In examining five classes of ORNs , we found that ORN synapse number , as indicated by the number of Brp-Short puncta , scaled with ORN axon volume ( Figure 1H ) , which itself scaled with glomerular size .", "Moreover , the assay revealed sex-specific differences in synapse number for two glomeruli , DA1 and VA1lm , that coincide with known sex-specific differences in glomerular volume ( Stockinger et al . , 2005 ) .", "Remarkably , despite differences in synapse number and ORN neurite volume , all observed ORN classes had a constant synaptic density of 0 . 56 Brp-Short puncta per µm3 ( Figure 1I ) .", "These data suggested the existence of an organizational mechanism that controls synaptic density .", "In all , our results validated the use of Brp-Short as a quantifiable presynaptic active zone label in central neurons and revealed quantitative parameters that govern the synaptic contacts of ORNs: a stereotyped number of connections for each neuronal class from animal to animal and a constant synaptic density regardless of ORN class .", "In the olfactory system , individual ORNs of a particular class ( as denoted by their odorant receptor expression ) project to an identifiable glomerulus ( Vosshall and Stocker , 2007 ) .", "In terms of their projection pattern ( Jefferis and Hummel , 2006 ) and physiological function ( Wilson , 2013 ) , individual ORNs can largely be treated equivalently .", "However , it is unknown whether each neuron contributes an equal number of synapses to the aggregate , stereotyped average or whether there is heterogeneity in synapse number at the level of individual neurons .", "Different results would suggest distinct rules for how individual neurons determine how many connections to form .", "To address this , we utilized MARCM ( Lee and Luo , 1999 ) to label the synapses and membranes of individual ( or small subsets of ) ORNs that innervate two glomeruli , DL4 and DM6 .", "Most frequently , we obtained 2–3 cell clones ( Figure 2A–B ) , but we observed occurrences of single-cell clones ( Figure 2—figure supplement 1 , panels A–B ) and large clones ( Figure 2—figure supplement 1 , panels C–D ) that encompassed the entire glomerulus .", "Single neuron clones were readily discernible in confocal stacks; cell numbers could not be precisely assigned to larger than 2–3 cell clones , however , due to axon fasciculation .", "For all clones ( n = 100 for DL4 , 99 for DM6 ) except the largest 7 for DL4 and 9 for DM6 , we quantified the number of Brp-Short puncta and the volume of the mCD8-GFP-labeled neurites as above ( Figure 1 ) , and plotted the distribution of puncta number across all clones in histograms .", "For both DL4 ( Figure 2C ) and DM6 ( Figure 2D ) , we observed that as clone size increased , the number of Brp-Short puncta appeared to increase in discrete steps of a similar size , suggesting that each individual neuron contributes an equal number of synapses to the glomerular average .", "If each neuron contributed a widely different number of synapses to the average , we would have observed a more continuous distribution . 10 . 7554/eLife . 03726 . 007Figure 2 . Clonal analysis of ORN synapse number . Representative high magnification confocal stacks of small clones of DL4 ORNs ( A ) and DM6 ORNs ( B ) expressing Brp-Short-mStraw ( red ) and mCD8-GFP ( green ) .", "( C ) Frequency histogram of Brp-Short puncta in DL4 clones ( n = 93 clones from 64 animals ) .", "( D ) Frequency histogram of Brp-Short puncta in DM6 clones ( n = 90 clones from 64 animals ) .", "Both graphs exhibit notable periodicity , suggesting that each neuron contributes approximately the same number of synapses .", "Colored dashed lines indicate Gaussian fits for individual peaks .", "Solid black lines represent the best-fit sum of Gaussian relationship for each dataset .", "( E ) Quantification of Brp-Short puncta from identified single neuron clones to DL4 ( n = 16 clones ) or DM6 ( n = 17 clones ) .", "( F ) Quantification of synapse density from all clones .", "In both DL4 and DM6 , the clonal average density is identical to the class average density .", "In ( E ) and ( F ) , data represent mean ± SEM .", "Scale bar = 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 03726 . 00710 . 7554/eLife . 03726 . 008Figure 2—source data 1 . Table of Gaussian curve-fitting data . Table of statistical values for three potential sum of Gaussian fits each for the DL4 and DM6 MARCM datasets . DOI: http://dx . doi . org/10 . 7554/eLife . 03726 . 00810 . 7554/eLife . 03726 . 009Figure 2—source data 2 . Table of resampling approach . Table of simulation data for three distributions of the DL4 and DM6 MARCM data sets and the probabilities of obtaining simulated data more evenly distributed than observed . DOI: http://dx . doi . org/10 . 7554/eLife . 03726 . 00910 . 7554/eLife . 03726 . 010Figure 2—figure supplement 1 . Additional DL4 and DM6 ORN MARCM clones . Representative high magnification confocal stacks of single cell clones of DL4 ORNs ( A ) and DM6 ORNs ( B ) and large clones of DL4 ORNs ( C ) and DM6 ORNs ( D ) expressing Brp-Short-mStraw and mCD8-GFP and stained for antibodies against mStraw ( red ) , GFP ( green ) , and N-Cadherin ( blue ) .", "As previously observed ( Joo et al . , 2013 ) , single cell ORN clones have a low signal-to-noise ratio while discerning larger clones is more evident .", "In A–B , the glomeruli are outlined ( dashed line ) .", "Scale bar = 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 03726 . 010 To statistically analyze the distributions for DL4 and DM6 ORNs , we determined their best-fit relationships using the first four peaks for both data sets , which had the most observations .", "Each individual peak could be reliably fit using a single Gaussian relationship ( r2 > 0 . 98 , Figure 2C–D , colored dashed lines ) , and the entire set of peaks could be fit by a sum of Gaussians ( see Figure 2—source data 1; ‘Materials and methods’ ) .", "The DL4 relationship was best fit by a 4-term sum of Gaussians ( Figure 2C , solid black line ) with a value of 19 . 13 for the apex of the first peak , corresponding to the ‘single cell’ contribution of active zone puncta .", "This is in close agreement with our original data of 19 . 75 ± 0 . 68 puncta for single cell clones ( Figure 2E ) .", "The DL6 relationship was best fit by a 5-term sum of Gaussians ( Figure 2D , solid black line ) , with a value of 28 . 62 active zone puncta as the ‘single cell’ contribution , which is again in strong agreement with our original data of 29 . 06 ± 0 . 95 puncta for single cell clones ( Figure 2E ) .", "Thus , in both cases , a sum of Gaussians can accurately represent the observed peaks and predict single ‘quantum’ contributions similar to those observed for single cell clones .", "These ‘quantum’ values are consistent with the ‘step size’ of Brp-Short puncta measured in clones with increasing size ( Figure 2C–E ) .", "Further , in comparing DL4 and DM6 , the difference in the number of puncta per single cell clone ( Figure 2E ) is consistent with the numerical relationship between the aggregate glomerular averages ( Figure 1H ) .", "All observed clones also displayed a nearly identical synaptic density ( Figure 2F ) regardless of their size , consistent with measurements in whole glomeruli .", "To further test whether each ORN contributes the same number of synapses to the aggregate , we examined the spacing of the peaks .", "If the peaks were perfectly evenly spaced , the standard deviation in the distances ( S ) between each peak would be 0 .", "Using the individual Gaussian relationships ( colored dashed lines ) , we calculated an S value of 0 . 235 for DL4 and 2 . 26 for DM6 .", "While visual assessment suggested uniformity , we used these values to more rigorously assess this .", "We used a resampling approach and calculated an S value for each simulation to test the null hypothesis that there is no periodic relationship between the peaks .", "A lower S value than that which was observed means that the simulation resulted in peaks that were more evenly spaced .", "Figure 2—source data 2 details these results .", "For distributions of DL4 or DM6 , there was a low probability ( <0 . 00048 or <0 . 05 , respectively ) of seeing peaks as evenly distributed as that observed .", "Thus , we reject the null hypothesis of no quantal relationship for both DL4 and DM6 .", "In all , this approach demonstrates that the peaks have equal spacing in between , further supporting our assertion that each ORN contributes an equivalent number of active zones to the aggregate and that each class of ORNs has a characteristic ‘step size’ consistent with its aggregate average .", "To better understand CNS synaptic organization , it is important to know not only how many connections are formed , but also where they form .", "Brp-Short allowed us to examine the spatial organization of active zones of any specific neuronal class within individual glomeruli .", "As an approximation of the complement of active zones for the DA1 glomerulus , we examined its component ORNs , PNs , and LNs .", "Using GAL4 drivers for the ORNs ( Or67d-GAL4 ) , PNs ( Mz19-GAL4 ) , and an LN line that includes most ipsilateral LNs ( NP3056-GAL4 ) , all of which innervate the DA1 glomerulus , we examined the localization of their synapses .", "For all classes , Brp-Short puncta colocalized with endogenous Brp staining ( Figure 3—figure supplement 1 , panels A–C ) recognized by an antibody whose epitope is not contained within Brp-Short ( Wagh et al . , 2006 ) , demonstrating that Brp-Short recognizes endogenous active zones .", "These three classes of neurons , when combined , provide 3342 ± 99 Brp-Short puncta per glomerulus .", "When the endogenous number of active zones is quantified using an antibody to endogenous Brp using the same image-processing strategy , DA1 contains 3849 ± 80 active zones ( Figure 3—figure supplement 1D ) .", "This suggests that the majority of synapses in the DA1 glomerulus are indeed made by Or67d-GAL4+ ORNs , Mz19-GAL4+ PNs , and NP3056-GAL4+ LNs; the discrepancy may be contributed by synapses made by additional neurons , such as other classes of local interneurons ( Chou et al . , 2010 ) , atypical projection neurons ( Lai et al . , 2008 ) , or potentially peptidergic neurons ( Ignell et al . , 2009; Carlsson et al . , 2010 ) , or may represent a slight underestimation by the Brp-Short assay .", "Using single optical sections within the DA1 glomerulus , we observed two levels of organization: ( 1 ) the location of neurites and Brp-Short puncta with respect to the glomerulus ( the subglomerular distribution ) , and ( 2 ) Brp-Short puncta with respect to their own neurites for each class of neurons ( the subcellular distribution ) .", "Of the three classes of neurons observed , ORNs contribute the majority of the Brp-Short puncta ( 1632 ± 22 ) and were widely distributed within the glomerulus ( Figure 3A , D ) , though with distinct voids ( Figure 3D , asterisk ) where the glomerulus lacked ORN neurites and Brp-Short puncta .", "PNs were the second major contributors of Brp-Short puncta within the glomerulus ( 1182 ± 24 ) .", "These connections likely synapse onto PN and LN dendrites , as shown by electrophysiology ( Kazama and Wilson , 2009 ) , and are analogous to the dendrodendritic synapses between secondary dendrites of mitral cells and olfactory bulb interneurons in the vertebrate olfactory bulb ( Urban and Arevian , 2009 ) .", "These Brp-Short puncta showed a more even distribution throughout the glomerulus ( Figure 3B , E ) , with smaller voids than those observed for ORNs despite reduced overall density .", "Finally , local interneurons ( LNs ) contribute the fewest Brp-Short puncta ( 528 ± 54 ) of the three classes .", "The LNs accessed by the NP3056-GAL4 line make up the majority of ipsilateral LNs including most or all pan-glomerular projections ( Chou et al . , 2010 ) , but may not be representative of all classes of LNs .", "Within the glomerulus , LN distribution was the most restricted ( Figure 3C , F ) .", "When examined at equivalent optical sections , these projections mostly filled the gaps in the ORN projection pattern , consistent with previously observed results for neurites ( Hummel and Zipursky , 2004 ) . 10 . 7554/eLife . 03726 . 011Figure 3 . Antennal lobe neurons display distinct subglomerular architecture .", "( A–C )", "Single , high-magnification confocal optical sections through the center of the DA1 glomerulus in three animals where ORNs ( A ) , PNs ( B ) , or LNs ( C ) are expressing Brp-Short .", "A different color denotes the synapses of each class .", "( D ) Single , high magnification confocal optical sections through the center of the DA1 glomerulus where ORNs express Brp-Short-mStraw ( red ) and mCD8-GFP ( green ) .", "Brp-Short clusters ( arrowheads ) are visible , along with regions of neurite devoid of Brp-Short puncta ( arrow ) and voids where ORNs do not project ( asterisk ) .", "( E ) Single , high magnification confocal optical sections through the center of the DA1 glomerulus where PNs are expressing Brp-Short-mStraw ( red ) and mCD8-GFP ( green ) .", "Regions of PN neurite without Brp-Short puncta are visible ( arrow ) as well as small voids lacking Brp-Short puncta and neurite projections ( asterisk ) .", "( F ) Single , high magnification confocal optical sections through the center of the DA1 glomerulus where LNs are expressing Brp-Short-mStraw ( red ) and mCD8-GFP ( green ) .", "Brp-Short puncta are largely distributed evenly throughout neurites .", "Images in panels D–F ( from three animals ) demonstrate distinct patterns of synapse localization and distribution within the glomerulus .", "Dashed lines denote the glomerulus in ( C ) and ( F ) as defined by N-Cad staining ( not shown ) .", "Scale bar = 5 μm .", "( G–I )", "Cumulative frequency histograms of the nearest neighbor distance between Brp-Short puncta in ORNs ( G ) , PNs ( H ) , and LNs ( I ) .", "The average is indicated ( μ ) and the Cluster % of puncta with an NND ≤ 0 . 75 μm .", "Gray traces represent individual glomeruli .", "Red traces represent the aggregate average .", "In all cases , n ≥ 10 animals , 19 antennal lobes , and 400 ( I ) , 800 ( H ) , or 1400 ( G ) individual puncta . DOI: http://dx . doi . org/10 . 7554/eLife . 03726 . 01110 . 7554/eLife . 03726 . 012Figure 3—figure supplement 1 . Comparison of Brp-Short to endogenous Brp .", "( A ) Representative high magnification confocal z-stack of the DA1 glomerulus in animals with an Or67d-mCD8-GFP transgene to mark its borders and stained with antibodies against GFP ( red ) and endogenous Brp ( green ) .", "( B–D )", "Representative high magnification confocal single sections of Or67d-positive ORNs ( B ) , Mz19-positive PNs ( C ) , and NP3056-positive LNs ( D ) innervating the DA1 glomerulus , each expressing Brp-Short-mStraw and stained with antibodies against mStraw ( red ) and endogenous Brp ( green ) .", "Each image was taken from different animals at a roughly equivalent Z-plane .", "In all cases , Brp-Short-mStraw puncta are coincident with endogenous Brp puncta .", "Endogenous Brp puncta that are not Brp-Short-mStraw positive are likely from other neurons not expressing the Brp-Short-mStraw transgene .", "Dashed lines indicate the glomerular boundary as defined by N-Cad staining ( not shown ) .", "( D ) Quantification of Brp-Short puncta from ORNs ( green ) , PNs ( blue ) , and LNs ( magenta ) , the sum of ORNs + PNs + LNs , and the number of endogenous Brp puncta from the DA1 glomerulus ( black ) .", "In all cases , data represent mean ± SEM and n ≥ 7 animals , 14 antennal lobes .", "Scale bar = 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 03726 . 012 With respect to their own neurites , Brp-Short puncta in ORNs were not evenly distributed: distinct sections of neurite lack Brp-Short puncta ( Figure 3D , arrow ) while other sections had large clusters ( Figure 3D , arrowheads ) of Brp-Short puncta ( Figure 3D ) .", "To quantify this distribution , we conducted a nearest neighbor distance ( NND ) analysis for Brp-Short puncta in each class of neurons , which has been informative in understanding spatial relationships between synapses ( Bleckert et al . , 2013 ) .", "We found that all three types of neurons had distinct mean NND values ( Figure 3G–I ) : ORNs had the closest puncta and LNs the furthest with PNs falling in between the two .", "To quantify the clusters observed , we defined clustered puncta as having an NND of 0 . 75 μm or less ( an NND of 0 . 6 μm corresponds to directly touching ) .", "For ORNs , these large clusters amounted to 17% of puncta that fell within this range ( Figure 3G ) .", "PNs lacked these large clusters , but had some smaller clusters , as well as extended regions of neuronal membrane devoid of Brp-Short puncta ( Figure 3E ) .", "Indeed , only 7 . 9% of PN puncta were clustered by this metric ( Figure 3H ) .", "Within LN neurites , Brp-Short puncta also displayed significant clustering ( 15%; Figure 3I ) .", "However , unlike ORNs and PNs , no extended regions of membrane were observed without synaptic puncta .", "This suggests a more even distribution for the remaining puncta , which is supported by the higher value for NND ( Figure 3I ) and consistent with previous imaging using a synaptic vesicle marker ( Chou et al . , 2010 ) .", "Overall , these results show distinct synaptic architecture , with respect to the glomerulus and the neurites themselves , made by three different types of olfactory neurons .", "Moreover , distinct parameters can be defined for each class in terms of how puncta are clustered and the minimum distance between puncta .", "Though some distances may be underestimated , these facets may represent additional rules that govern synaptic organization in the antennal lobe .", "Having utilized Brp-Short to determine aspects of presynaptic organization in olfactory neurons ( Figures 1–3 ) , we next sought to investigate the molecular mechanisms that may underlie these rules .", "We began with genetic perturbations of candidate molecules , the Teneurins .", "Teneurins are type II transmembrane proteins involved in neuronal development ( Young and Leamey , 2009 ) and are disrupted in human neurological disorders ( Aldahmesh et al . , 2012; Psychiatric GWAS Consortium Bipolar Disorder Working Group , 2011 ) .", "Recently , we demonstrated that the Drosophila Teneurins , Ten-m and Ten-a , regulate synaptic partner matching in the olfactory and neuromuscular systems via homophilic interaction between elevated Teneurin levels ( Hong et al . , 2012; Mosca et al . , 2012 ) .", "Basal levels of the Teneurins interact heterophilically at NMJ synapses to organize connections ( Mosca et al . , 2012 ) .", "However , due to the absence of techniques for quantitatively examining central synapses in vivo , a role for the Teneurins in organizing central synapses has not been established .", "Due to the similarity in partner matching mechanisms between central and peripheral nervous systems , and the fact that both Ten-a and Ten-m are expressed at basal levels in all glomeruli ( Hong et al . , 2012 ) , we hypothesized that the CNS may use Teneurins similarly to the PNS for synaptic organization .", "Specifically , we hypothesized that olfactory neurons would utilize basal levels of Ten-a and Ten-m to control synaptic number .", "To test this hypothesis , we used Brp-Short to quantify active zone number in Or47b-positive ORNs innervating the VA1lm glomerulus .", "Ten-a is expressed at a basal level in this glomerulus , and therefore does not directly affect synaptic partner matching ( Hong et al . , 2012 ) .", "Comparing wild-type ( Figure 4A ) and ten-a null mutant flies ( Figure 4B ) , we found that Or47b ORNs in the ten-a mutant ( Figure 4B ) had 23% fewer Brp-Short puncta ( Figure 4D ) .", "Notably , loss of ten-a did not alter ORN axon volume ( Figure 4E ) , despite affecting glomerular morphology ( Figure 4B ) , which is due to the loss of ten-a in neurons of the nearby DA1 glomerulus ( Hong et al . , 2012 ) .", "As such , synaptic density was similarly reduced ( Figure 4F ) .", "To ensure that changes in glomerular morphology were not related to changes in synapse number , we also examined ORNs that innervate the DL4 glomerulus ( Figure 4—figure supplement 1 ) .", "In ten-a mutants , glomerular morphology was unchanged in 87% of animals examined , but all animals displayed a similar reduction in synapse number and density without affecting the axon volume ( Figure 4—figure supplement 1 , panels C–E ) .", "Interestingly , at the NMJ , Teneurin perturbations reduced active zone and synaptic bouton numbers , but did not alter active zone density ( TM , unpublished observations ) .", "However , in ORNs , neurite volume was unchanged , so active zone density was reduced , suggesting a difference in the specific consequence of Teneurin perturbation in the CNS compared with the NMJ . 10 . 7554/eLife . 03726 . 013Figure 4 . Presynaptic Ten-a is required for proper ORN synapse number and density . Representative high-magnification confocal stacks of Brp-Short puncta in VA1lm ORNs stained with antibodies against Brp-Short-mStraw ( red ) and N-Cadherin ( blue ) in control ( A ) , ten-a null mutants ( B ) and ten-a null mutants where a Ten-a transgene is expressed in this specific class of ORNs using Or47b-GAL4 ( C ) .", "( D ) Quantification of Brp-Short puncta in the above conditions as well as following presynaptic RNAi against ten-a , presynaptic RNAi against ten-m , or in ten-a null mutants where a Ten-a transgene is expressed in 2/3 of the PNs , including those innervated by Or47b-positive ORNs .", "( E ) Quantification of ORN neurite volume in the same genotypes .", "n . s . = not significant .", "( F ) Quantification of ORN synaptic density in the same genotypes .", "Significance was assessed with a one-way ANOVA and corrected for multiple comparisons by a posthoc Tukey's multiple comparisons test .", "**p < 0 . 01 , ***p < 0 . 001 .", "Unless otherwise noted , significance is compared to control .", "In all cases , the ten-a mutant and ten-a ORN RNAi phenotypes are statistically indistinguishable and , in the ten-a mutant , presynaptic Ten-a expression rescues the observed phenotypes .", "The disrupted glomerular shape is due to partner matching errors in the ten-a mutant ( Hong et al . , 2012 ) , which is not rescued due to the late onset of Or47b-GAL4 relative to the partner matching process .", "Dashed lines denote the glomeruli as delineated by Brp-Short label .", "In all cases , data represent mean ± SEM and n ≥ 7 animals , 14 antennal lobes .", "Scale bar = 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 03726 . 01310 . 7554/eLife . 03726 . 014Figure 4—figure supplement 1 . Representative images for additional genetic manipulations from Figure 4 . Representative high-magnification confocal stacks of Brp-Short puncta in VA1lm ORNs stained with antibodies against Brp-Short-mStraw ( red ) and N-Cadherin ( blue ) in control ( A ) , flies expressing ten-m RNAi in Or47b-positive ORNs ( B ) , flies expressing ten-a RNAi in Or47b-positive ORNs ( C ) , and ten-a null mutants where a Ten-a transgene is expressed in 2/3 of the PNs of the antennal lobe using GH146-QF ( D ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 03726 . 01410 . 7554/eLife . 03726 . 015Figure 4—figure supplement 2 . ten-a phenotypes in DL4 , a glomerulus with no morphology defects .", "( A ) Representative high magnification confocal stacks of ORNs innervating the DL4 glomerulus in control animals expressing Brp-Short-mStraw and mCD8-GFP and stained with antibodies to mStraw ( red ) , GFP ( green ) , and N-Cadherin ( blue ) .", "( B ) Representative high magnification confocal z-stacks of ORNs innervating the DL4 glomerulus in ten-a null mutant animals expressing Brp-Short-mStraw and mCD8-GFP and stained with antibodies to mStraw ( red ) , GFP ( green ) , and N-Cadherin ( blue ) .", "Morphology is unaffected but synapse number is reduced .", "( C ) Quantification of Brp-Short-mStraw puncta in control and ten-a mutant animals .", "( D ) Quantification of ORN volume in control and ten-a mutant animals .", "( E ) Quantification of Brp-Short-mStraw puncta density in control and ten-a mutant animals .", "In all cases , data represent mean ± SEM and n ≥ 7 animals , 14 antennal lobes .", "***p < 0 . 0001 .", "Scale bar = 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 03726 . 015 As ten-a mutants lack Ten-a in ORNs and PNs , we next sought to determine where Ten-a was required to regulate normal synapse number using tissue-specific rescue and RNAi experiments .", "Restoring Ten-a expression to the Or47b ORNs in the ten-a mutant ( Figure 4C ) , but not to the VA1lm PNs that are innervated by Or47b ORNs , rescued the reduction in synapse number and density ( Figure 4D , F ) .", "When Ten-a was restored to Or47b-positive ORNs , defects in glomerular morphology persisted despite rescue of synaptic phenotypes ( compare Figure 4C–A ) , as partner matching occurs earlier than synaptogenesis ( and before Or47b-GAL4 turns on ) .", "Further consistent with a presynaptic role for Ten-a , knockdown of Ten-a in ORNs by RNAi also reduced the number and density of synapses similarly to the mutant ( Figure 4D , F ) .", "Here , glomerular morphology was unaffected as VA1lm is already a ‘Ten-a low’ glomerulus and does not directly require Ten-a for partner matching .", "Interestingly , knockdown of ten-m in the Or47b ORNs did not result in any synaptic phenotype ( Figure 4D–F ) , demonstrating specificity for ten-a .", "Thus , Ten-a is required presynaptically for proper synapse number and density , likely in a partially redundant fashion with additional molecules .", "Previous data and our Brp-Short analyses suggest that the assay is largely reflective of actual synapse number in neurons .", "However , we sought to confirm these results using a method independent of confocal microscopy or the Brp-Short label .", "We compared synapse number in ultrathin EM sections of wild-type and ten-a mutant flies ( Figure 5A–B ) .", "Quantification of all synapses in a glomerulus ( and thus a direct comparison to the Brp-Short assay ) would require serial reconstruction of that entire glomerulus by EM .", "However , if the Brp-Short assay accurately reflects changes in synapse number following genetic perturbation , we reasoned that this should be evident in the change in the number of T-bar profiles in individual sections at the EM level .", "Indeed , quantification of T-bar profiles showed a 27% reduction in the number of T-bars ( Figure 5I ) .", "This is consistent with the results from the Brp-Short assay ( Figure 4D ) , which shows a 23% reduction .", "The slight difference may reflect an under-report of the phenotype by our confocal-level analysis or may reflect the fact that the EM analysis does not differentiate between ORN , PN , and LN presynapses while the Brp-Short assay does .", "The largely similar magnitude of synapse reduction in EM and confocal studies supports the validity of the Brp-Short assay , and further substantiates that Ten-a is required for proper synapse number in the CNS . 10 . 7554/eLife . 03726 . 016Figure 5 . Ultrastructural analysis of active zones in ten-a mutants . Representative transmission electron microscopy ( TEM ) images of active zones in control ( A ) and ten-a null mutant antennal lobes ( B ) .", "T-bar profiles are visible in both genotypes , though reduced in number in the ten-a mutant .", "Scale bar = 500 nm .", "( C ) High magnification TEM of a single active zone in a control animal .", "Note the normal T-bar morphology .", "( D–H )", "High magnification TEM images of active zones in ten-a mutants .", "In some cases , normal ( D ) morphology is observed .", "In the majority of cases , defects including multiple T-bars ( E ) , misshapen T-bars ( F ) , detached T-bars ( G ) , and continuous electron dense material ( H ) are evident .", "Scale bar = 100 nm .", "( I ) Quantification of T-bars per unit perimeter of measured antennal lobe terminals .", "All terminals were measured , including ORNs , PNs , and LNs .", "ten-a mutants display a 27% reduction from control animals .", "Data represent mean ± SEM .", "( J ) Distribution of T-bar defects as a percentage of the total T-bars .", "Here , n = 1103 T-bar per unit perimeter measurements from three animals for control and n = 847 T-bar per unit perimeter measurements from three animals for ten-a . DOI: http://dx . doi . org/10 . 7554/eLife . 03726 . 016 At neuromuscular synapses , Teneurins are required for proper T-bar structure .", "When Teneurins are absent , T-bars display defects consistent with failed synaptogenesis ( Mosca et al . , 2012 ) .", "To determine whether Ten-a was also required in olfactory neurons for T-bar structure , we examined individual T-bar morphology in ultrathin sections of wild-type and ten-a antennal lobes .", "In wild-type animals , T-bar morphology was nearly invariant ( Figure 5C ) , with infrequent occurrences of misshapen or multiple T-bars ( Figure 5J ) .", "In ten-a mutants , 44% of T-bar profiles appeared normal ( Figure 5D , J ) .", "The remaining 56% displayed notable structural defects including continuous , multiple T-bars ( Figure 5E ) , misshapen T-bars ( Figure 5F ) , and T-bars that were detached from the synaptic membrane ( Figure 5G ) .", "These defects resembled those observed at ten-a mutant NMJs ( Mosca et al . , 2012 ) .", "We also observed continuous , electron-dense material resembling undivided T-bars ( Figure 5H ) that was not found previously .", "Thus , Ten-a is required not just for the normal number of active zones , but also for their proper structure in ways both consistent with and beyond that of previous studies .", "Specifically , ten-a may be required for the maturation of synapses , as misshapen and undivided T-bars resemble immature synapses ( Owald et al . , 2010 ) .", "Teneurins can regulate cytoskeletal architecture ( Nunes et al . , 2005; Morck et al . , 2010; Suzuki et al . , 2014 ) and function at the Drosophila NMJ by organizing spectrin , a cytoskeletal component ( Mosca et al . , 2012 ) .", "However , it is unknown whether this function of the Teneurins is conserved in the CNS .", "To further probe the mechanism by which Teneurins regulate central synapse number and to determine whether cytoskeletal regulation is involved , we compared levels of spectrin staining in the antennal lobe between wild-type and ten-a mutant flies .", "We found that spectrin immunofluorescence in the antennal lobe was reduced in the ten-a mutant compared to wild-type ( Figure 6A–B ) while N-Cadherin , a marker of synaptic neuropil , was unaffected ( Figure 6F ) .", "This was observed for α- and β-spectrin ( Figure 6F , Figure 6—figure supplement 1 , panels A–D ) , which function as a heterotetramer ( Machnicka et al . , 2014 ) . 10 . 7554/eLife . 03726 . 017Figure 6 . Ten-a and spectrin function together for normal synapse number .", "( A–B )", "Representative single confocal optical sections taken at equivalent positions of antennal lobes stained for α-spectrin and N-Cadherin in control ( A ) and ten-a mutant ( B ) adults .", "In ten-a mutants , α-spectrin staining is reduced compared to control .", "The disrupted morphology of the antennal lobe itself is due to partner matching errors evident in the ten-a mutant ( Hong et al . , 2012 ) .", "Scale bar = 20 μm .", "( C–E )", "Representative confocal Z-stack images of the ORNs in the VA1lm glomerulus expressing Brp-Short in control animals ( C ) , or animals expressing dsRNA against α-spectrin ( D ) or β-spectrin ( E ) , and stained with antibodies against Brp-Short ( red ) and N-Cadherin ( blue ) .", "( F ) Quantification of α-spectrin ( green ) , β-spectrin ( red ) , and N-Cadherin ( blue ) immunofluorescence in control and ten-a mutants .", "***p < 0 . 001 .", "( G ) Quantification of Brp-Short puncta in the noted genotypes .", "In all cases , similar reductions in puncta number are observed .", "Moreover , the genetic perturbations do not enhance each other , suggesting function in the same pathway .", "Significance was assessed with a one-way ANOVA and corrected for multiple comparisons by a posthoc Tukey's multiple comparisons test .", "***p < 0 . 001 ( compared with control ) .", "In all cases , data represent mean ± SEM and n ≥ 10 animals , 19 antennal lobes .", "Scale bar = 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 03726 . 01710 . 7554/eLife . 03726 . 018Figure 6—figure supplement 1 . ten-a regulates spectrin levels and spectrin regulates synapse number . Representative confocal single optical sections of control ( A ) and ten-a mutant ( B ) antennal lobes stained with antibodies to β-spectrin ( red ) and N-Cadherin ( blue ) .", "Note the reduction ( quantified in Figure 6F ) of staining in the ten-a mutant .", "Scale bar = 20 μm .", "( C–E )", "Representative high magnification confocal z-stacks of Or67d ORNs expressing Brp-Short-mStraw in control animals ( C ) and animals co-expressing dsRNA against α-spectrin ( D ) or β-spectrin ( E ) and stained with antibodies against mStraw ( red ) and N-Cadherin ( blue ) .", "Glomerular morphology is unaffected by spectrin dsRNA but synapse number is reduced .", "Scale bar = 5 μm .", "( F ) Quantification of Brp-Short puncta in the genotypes from C–E .", "***p < 0 . 0001 .", "In all cases , data represent mean ± SEM and n ≥ 10 animals , 19 antennal lobes . DOI: http://dx . doi . org/10 . 7554/eLife . 03726 . 018 To test whether proper spectrin level is required for synapse number , we used ORN-specific knockdown of either α- or β-spectrin and measured Brp-Short puncta .", "In both cases ( Figure 6D–E ) , RNAi in Or47b-positive ORNs resulted in a 17–18% reduction in Brp-Short puncta ( Figure 6G ) .", "This effect , though slightly weaker , phenocopied the reduction seen in the ten-a mutant .", "We observed a similar effect in another ORN class , the Or67d-positive neurons ( Figure 6—figure supplement 1 ) , further supporting a role for spectrin in regulating olfactory synapse number .", "Moreover , the ten-a phenotype was not enhanced by concurrent RNAi knockdown of α-spectrin in ORNs ( Figure 6G ) , suggesting that Ten-a and α-spectrin function in the same pathway to regulate olfactory synapse number .", "A functional synapse consists of a presynaptic neurotransmitter release site and a postsynaptic neurotransmitter receptor cluster ( Figure 1A ) .", "Therefore , critical parameters of synaptic organization within a circuit not only include the location and number of presynaptic active zones , but also postsynaptic receptor clusters .", "Therefore , we also examined the organization of postsynapses .", "Given that ORNs are cholinergic ( Wilson , 2013 ) , an ideal labeling strategy would image postsynaptic acetylcholine receptors .", "We selected the Dα7 acetylcholine receptor subunit because it is endogenously expressed in the antennal lobe ( Fayyazuddin et al . , 2006 ) and it has been used to examine organization in the mushroom body , a higher olfactory center ( Leiss et al . , 2009a , 2009b; Kremer et al . , 2010; Christiansen et al . , 2011 ) .", "We used a GFP-tagged Dα7 transgene ( Leiss et al . , 2009b ) under the control of the GAL4/UAS system ( Figure 7A ) to visualize postsynapses in vivo .", "Expression of Dα7-GFP in PNs revealed distinct puncta , possibly corresponding to acetylcholine receptor ( AChR ) clusters ( Figure 7B ) .", "These puncta were apposed to endogenous Brp puncta , as revealed by nc82 staining ( Supplement 1 to Figure 7 ) , consistent with these puncta representing bona fide synapses .", "To examine AChR clusters in PNs , we co-expressed Dα7-GFP with mtdT as a general neurite label ( Figure 7B ) .", "As such , the approach is analogous to our Brp-Short assay and yielded similar results , enabling a quantitative assessment of the number ( Figure 7C ) and density ( Figure 7D ) of AChR clusters . 10 . 7554/eLife . 03726 . 019Figure 7 . Measuring postsynaptic acetylcholine receptor clusters with Dα7-GFP .", "( A ) Diagram of the GFP-tagged Dα7 acetylcholine receptor subunit used for AChR visualization .", "( B ) High magnification confocal z-stack images of PNs in the DA1 and VA1d glomeruli expressing Dα7-GFP and mtdT , stained with antibodies against GFP ( green ) , mtdT ( red ) , and endogenous Brp ( blue ) .", "Patches of mtdT labeling represent ascending PN axons within the plane of the glomerulus .", "Insets show a high magnification single optical section demonstrating the punctate nature of Dα7-GFP .", "( C ) Representative high magnification single optical sections of Mz19-positive PNs in the DA1 glomerulus expressing Dα7-GFP and stained with antibodies against GFP ( green ) and endogenous Brp ( red ) .", "The majority of GFP-positive puncta are apposed to or colocalized with endogenous Brp ( yellow ) , consistent with their association with bona fide active zones .", "Insets show high magnification of a single optical section where asterisks denote apposed Dα7 puncta .", "Brp puncta without apposition likely belong to synapses not labeled by the PN-GAL4 driver .", "Scale bar = 5 μm ( 2 μm for inset ) .", "( D ) Representative high magnification single optical sections of Mz19-positive PNs and Or67d-positive ORNs in the DA1 glomerulus expressing Dα7-GFP in the PNs and Brp-Short-mStraw in the ORNs using two binary expression systems .", "Most ORN active zones ( labeled by Brp-Short , red ) are apposed to or colocalized ( yellow ) with PN Dα7 puncta ( green ) , further supporting that these puncta label bona fide synaptic contacts .", "Insets show high magnification of a single optical section where asterisks denote apposed Brp-Short::Dα7 pairs .", "Brp puncta without apposition likely belong to ORN synapses with neurons other than PNs and Dα7 puncta without apposition likely correspond to PN postsynaptic sites apposed to synaptic contacts from neurons other than ORNs .", "( E ) Quantification of Dα7 AChR puncta ( green , left axis ) and neurite volume ( black , right axis ) in the DA1 and VA1d glomeruli of both male and female adult flies .", "Statistical comparisons between males and females of a single genotype were done by student's t test .", "***p < 0 . 001 .", "( F ) Quantification of AChR density in male ( blue ) and female ( pink ) adults based on the data from ( C ) .", "Significance was assessed with a one-way ANOVA and corrected for multiple comparisons by a posthoc Tukey's multiple comparisons test .", "*p < 0 . 05 .", "n . s . = not significant .", "In all cases , data represent mean ± SEM and n ≥ 11 animals , 21 antennal lobes .", "Scale bar = 5 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 03726 . 019 As we are limited to genetically accessible PN subsets , we focused on identifying organizational parameters in the PNs that innervate the DA1 and VA1d glomeruli via the Mz19-GAL4 driver ( Ito et al . , 1998 ) .", "As with ORN presynapses , the assay revealed that the number of AChR puncta scales with glomerular size ( Figure 7E ) .", "Further , known sex-specific differences in DA1 , as seen in glomerular volume ( Stockinger et al . , 2005 ) and in ORN synapses ( Figure 1 ) , were also observed ( Figure 7E ) .", "The differences between the Brp-Short and AChR assays for the DA1 ( 1632 ± 22 for Brp-Short vs 2007 ± 48 for Dα7 ) and VA1d ( 1107 ± 18 for Brp-Short vs 1762 ± 38 for Dα7 ) glomeruli may reflect the fact that the Brp-Short assay does not distinguish ORN synapses onto PNs and LNs , and the Dα7-GFP assay does not distinguish synapses from ORNs and LNs onto PNs .", "As these values are less than twofold different , this is consistent with the majority of synapses labeled being ORN to PN synapses ( Kazama and Wilson , 2009; Wilson , 2013 ) .", "The similarity between the numbers of endogenous Brp and Brp-Short puncta suggests that Brp-Short is a more accurate estimator of absolute synapse number .", "The larger number of AChRs detected in each glomerulus may reflect an overestimation associated with full-length Dα7 overexpression or that these are postsynaptic not just to cholinergic ORNs , but also other excitatory neurons such as local interneurons ( Chou et al . , 2010 ) or PN-PN chemical synapses ( Ng et al . , 2002; Wilson et al . , 2004 ) .", "Calculation of AChR puncta density in PNs revealed subtle but significant differences across different glomeruli .", "In the VA1d glomerulus , the densities were identical between males and females ( Figure 7F ) .", "However , these were different from AChR puncta densities in the DA1 glomerulus .", "There was a modest but significant difference between both male and female AChR densities in DA1 and between both DA1 AChR densities and the shared VA1d AChR density ( Figure 7F ) .", "Unlike ORNs , where the Brp-Short density was identical across different classes of neurons , PNs can have different densities between distinct glomeruli and even between sexes for the same glomerulus .", "Thus , the parameters that govern presynaptic density may differ from those that govern postsynaptic density in the same glomerulus .", "To further examine if the Teneurins regulate postsynaptic acetylcholine receptor number and density , we utilized the Dα7-GFP assay to determine the effect of Teneurin perturbation on AChR puncta number .", "We examined PNs in DA1 and VA1d ( Figure 8A ) and counted the AChR puncta of both glomeruli together as one measurement , as partner matching defects following Teneurin perturbation make it difficult to differentiate between the two glomeruli ( Hong et al . , 2012 ) .", "In ten-a mutants ( Figure 8B ) , the number of AChR clusters in these glomeruli was decreased by 23% , compared to wild type ( Figure 8D ) .", "This is consistent with results from the Brp-Short assay .", "Moreover , PN neurite volume was unaffected ( Figure 8E ) , so AChR puncta density was similarly reduced ( Figure 8F ) .", "Thus , two independent assays , both pre- and postsynaptic , show the same phenotypes , demonstrating a clear effect of ten-a loss on synapse organization in olfactory neurons . 10 . 7554/eLife . 03726 . 020Figure 8 . Teneurins function to regulate acetylcholine receptor number .", "( A–C )", "Representative high magnification confocal z-stack images of DA1 and VA1d PNs expressing Dα7-GFP in control animals ( A ) , ten-a mutants ( B ) , or animals expressing RNAi against ten-m in the same PNs ( C ) , and stained with antibodies against GFP ( green ) and N-Cadherin ( blue ) .", "DA1 and VA1d glomeruli are outlined together ( white dashed line ) and were determined using the accompanying mtdT label ( not shown ) .", "Due to partner matching defects in the ten-a mutant that prevent clear delineation between the two glomeruli , the combined count was compared across genotypes .", "( D ) Quantification of Dα7-GFP puncta in the noted genotypes .", "( E ) Quantification of PN neurite volume in all genotypes .", "( F ) Quantification of AChR density in all genotypes .", "All observed phenotypes are statistically indistinguishable .", "Significance was assessed with a one-way ANOVA and corrected for multiple comparisons by a posthoc Tukey's multiple comparisons test .", "***p < 0 . 001 in comparison with control .", "n . s . = not significant .", "In all cases , data represent mean ± SEM and n ≥ 8 animals , 16 antennal lobes .", "Scale bar = 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 03726 . 020 At the NMJ , presynaptic Ten-a functions largely in a transsynaptic , heterophilic complex with postsynaptic Ten-m to regulate synapse organization ( Mosca et al . , 2012 ) .", "Ten-a functions presynaptically in ORNs to ensure proper synapse number ( Figure 4 ) .", "Thus , we hypothesized that the loss of Ten-m in postsynaptic PNs should result in a similar phenotype .", "As the ten-m mutant is larval lethal ( Zheng et al . , 2011 ) , we expressed a previously validated transgenic RNAi line against ten-m ( Hong et al . , 2012; Mosca et al . , 2012 ) in Mz19 PNs and quantified AChR puncta number using the Dα7 assay ( Figure 8C ) .", "ten-m knockdown phenocopied the ten-a phenotype ( Figure 8D ) .", "As above , PN neurite volume was unaffected ( Figure 8E ) , leading to a concomitant decrease in AChR puncta density that also phenocopied the ten-a phenotype ( Figure 8F ) .", "Further , this reduction was not enhanced by knocking down ten-m in PNs of a ten-a null mutant ( Figure 8D–F ) , suggesting that the two function in the same genetic pathway .", "Though genetic evidence suggests that Ten-m functions with Ten-a transsynaptically to regulate synapse number , it could conceivably regulate AChR number independently of how Ten-a regulates active zone number .", "Thus , we sought to more explicitly test a transsynaptic role of Ten-m in regulating proper active zone number .", "Due to the availability of genetic access to both the presynaptic ORNs and the postsynaptic PNs , we examined the DA1 glomerulus .", "Further , DA1 is a ten-m low glomerulus ( Hong et al . , 2012 ) , allowing us to specifically test the role of the basal level of Ten-m .", "Using the GAL4 and QF systems , we simultaneously labeled Or67d-positive ORNs with QUAS Brp-Short-mStraw using Or67d-QF ( Liang et al . , 2013 ) and expressed transgenic RNAi against ten-m in DA1 PNs using Mz19-GAL4 ( Figure 9A ) .", "Control animals displayed a similar number of Brp-Short puncta as the UAS construct ( Figure 9B , D ) , demonstrating the validity of this reagent .", "Postsynaptic ten-m knockdown , however , reduced the number of presynaptic Brp-Short puncta by 20% ( Figure 9C , D ) .", "This reduction is similar to the proportion by which ten-m RNAi reduced AChR number ( Figure 8D ) and also to the proportion by which loss of ten-a reduced Brp-Short number ( Figure 4D ) .", "This suggests that Ten-m is the postsynaptic partner of Ten-a in regulating presynaptic active zone number , and that the presynaptic Ten-a/postsynaptic Ten-m interaction that regulates NMJ synapses is conserved in olfactory synapses . 10 . 7554/eLife . 03726 . 021Figure 9 . Ten-m regulates presynaptic active zone number transsynaptically .", "( A ) Diagram of the antennal lobe with the DA1 glomerulus outlined showing the experimental design .", "Or67d-positive ORN axon terminals that innervate DA1 are outlined in black; their active zones are labeled by Or67d-QF-driven QUAS-Brp-Short-mStraw .", "Mz19-GAL4-positive PNs that project dendrites to DA1 express UAS-RNAi against ten-m in experimental animals .", "( B–C )", "Representative high magnification confocal z-stack images of DA1 ORNs expressing Brp-Short in control animals ( B ) or in animals concurrently expressing ten-m RNAi in the DA1 and VA1d PNs ( C ) and stained with antibodies against mStraw ( red ) and N-Cadherin ( blue ) .", "( D ) Quantification of Brp-Short puncta in the noted genotypes .", "Controls 1 and 2 represent Brp-Sh puncta assayed by GAL4/UAS ( as in Figure", "1 ) and QF/QUAS binary system binary system , respectively , and are not significantly different , as assayed by student's t test ( n . s . ) .", "Significance of ten-m PN RNAi was assayed by student's t test between it and Control 2 .", "***p < 0 . 001 .", "In all cases , data represent mean ± SEM and n ≥ 5 animals , 10 antennal lobes .", "Scale bar = 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 03726 . 021" ], [ "In Drosophila , previous approaches to studying central synapses used tagged synaptic vesicle proteins ( Ito et al . , 1998; Robinson et al . , 2002 ) to reveal putative synaptic sites .", "While consistent with synapses , they could also stain non-synaptic , trafficking vesicles .", "We utilized a structural active zone component , Brp ( Wagh et al . , 2006 ) .", "By expressing a truncated Brp transgene , Brp-Short ( Schmid et al . , 2008 ) , using the GAL4/UAS system , this approach can label synapses in any neurons with genetic access .", "Brp-Short expression requires endogenous Brp for proper localization ( Figure 1—figure supplement 1 ) and does not alter function or morphology ( Fouquet et al . , 2009; Kremer et al . , 2010 ) .", "Thus , it accurately reports endogenous active zones .", "Recently , an elegant technique , STaR , was developed , which tags an additional , BAC-sourced copy of Brp with an epitope tag whose expression is conditional upon FLP-recombinase-mediated excision of an intervening stop codon ( Chen et al . , 2014 ) .", "An important advantage of STaR is that Brp expression is controlled by its endogenous promoter , thus guarding against mislocalization of Brp or perturbation of synaptic function due to overexpression .", "A caveat of Brp-Short is that it is controlled by GAL4 and thus the levels may be different from the endogenous level .", "While Brp-Short overexpression does not interfere with synaptic function , care must be taken not to overexpress it to a level that saturates the active zone localization machinery when utilized in new cell types .", "The advantages of Brp-Short over STaR are", "( a ) that it does not require a cell type-specific FLP transgene , which is not as widely available as GAL4 lines , or a BAC-bearing copy of Brp , and", "( b ) that it can be co-expressed with UAS-transgenes for rescue or RNAi for perturbation experiments , although STaR can also achieve this aspect by using a cell-type-specific GAL4 and an extra UAS-FLP transgene .", "To examine putative postsynaptic acetylcholine receptor clusters , we use a GFP-tagged subunit , Dα7 , to study cholinergic synapses in the antennal lobe .", "This transgene has been used to examine synaptic organization ( Leiss et al . , 2009a , 2009b; Kremer et al . , 2010; Christiansen et al . , 2011 ) .", "Though false positives can be associated with full-length protein overexpression , our observation of endogenous Brp puncta apposed to these Dα7-GFP puncta suggests that these receptors are properly localized to endogenous synapses .", "This assay complements the Brp-Short presynaptic assay and can be adapted for other tagged postsynaptic receptor transgenes .", "Though considerable advances have been made in our understanding of the wiring specificity ( Hong and Luo , 2014 ) and physiological properties ( Wilson , 2013 ) of the Drosophila olfactory circuit in the antennal lobe , little is known about its synaptic organization .", "As this system has emerged as a model neural network , a detailed mapping of synaptic organizational principles is integral towards advancing the study of circuit dynamics .", "Indeed , the juxtaposition of distinct types of synapses between multiple neuronal classes in the antennal lobe provides a model to study complex synaptic interactions compared to a neuromuscular synapse that features only two synaptic partners: the motoneuron and the muscle .", "We utilized the Brp-Short and Dα7 assays to probe how synapses in ORNs , PNs , and LNs are organized in the antennal lobe with respect to their number , density , and location .", "This work offers the first comprehensive information on ( 1 ) the number of active zones made by each ORN within a glomerulus , ( 2 ) the stereotypy of synapse numbers between those individual ORNs , ( 3 ) the prominence of PN presynaptic inputs within a glomerulus , suggesting a robust feedback mechanism , and ( 4 ) the relatively small contribution of LN active zones to the antennal lobe circuit .", "Our analyses suggest distinct rules that govern the synaptic organization of antennal lobe neurons .", "ORNs , the primary input neurons of the olfactory system , are diverse in their olfactory receptor expression , ligand specificity , and glomerular targeting specificity ( Liang and Luo , 2010 ) ; we now show that they also differ in the absolute synapse number ( Figure 1 ) .", "However , despite such differences , all five classes we examined ( DA1 , VA1d , VA1lm , DL4 , and DM6 ORNs ) have identical synaptic densities , suggesting that this represents a general rule for other ORN classes .", "This may further suggest that the primary job of the ORN is to convey information from the environment into the system as faithfully as possible , that all information is treated equally at this level , and that weighting computations occur downstream in the brain ( Masse et al . , 2009; Wilson , 2013 ) .", "Indeed , our analyses indicate that each ORN makes an equivalent , discrete number of synapses within a given glomerulus with little variation ( Figure 2 ) , further supporting this hypothesis .", "Interestingly , the density of postsynaptic receptors differs between glomeruli and even within the same glomerulus between sexes ( Figure 7 ) .", "This variation can be due to technical caveats , such as Mz19-GAL4 does not label all PNs that innervate DA1 and VA1d ( Jefferis et al . , 2001; Liang et al . , 2013 ) , or that Dα7-GFP clusters do not reflect the absolute number of AChRs .", "As the relative numbers still show these differences , an interesting possibility suggested by these results is that postsynaptic PN AChRs already reflect a transformed olfactory representation compared to output synapses of ORNs .", "The difference in PN AChR density as compared to the constant density of ORN active zones suggests that different classes of PNs may modulate how information is received by regulating the number of acetylcholine receptors .", "This can thus contribute to the transformation of olfactory representation by antennal lobe neurons ( Wilson , 2013 ) .", "Fine-scale analysis of synapse localization within projections of ORNs , LNs , and PNs ( Figure 3 ) suggested that these three types of neurons differ in their subglomerular organization .", "While occupying the vast majority of territory throughout the entire glomerulus , ORN processes and synapses leave distinct voids .", "A significant proportion of LNs form synapses in these voids , likely to other LNs or PNs .", "However , there is also overlap between LN and ORN processes and synapses , consistent with physiologically characterized ORN → LN and LN → ORN synapses ( Olsen et al . , 2007 , 2010; Root et al . , 2007; Kazama and Wilson , 2008 , 2009; Olsen and Wilson , 2008; Root et al . , 2008; Huang et al . , 2010; Yaksi and Wilson , 2010; Root et al . , 2011; Wilson , 2011 , 2013 ) .", "Within their respective neurites , ORNs and PNs display uneven distributions and synaptic clusters to varying degrees while active zones in LNs are more evenly distributed throughout their processes .", "These characterizations contribute to the growing repertoire of studies seeking to understand synaptic organization in Drosophila olfactory circuits ( Murthy et al . , 2008; Caron et al . , 2013; Fisek and Wilson , 2014 ) , adding synapse-level imaging to physiological techniques .", "Finally , the existence of synaptic organization parameters detailing number and location suggests molecular mechanisms designed to enforce those rules , both at cellular and circuit levels .", "Indeed , such analysis represents an integral part of neuronal circuit analysis , as recent work on retinal neurons has shown that accounting for synapse position is a critical aspect of modeling connectivity ( Kim et al . , 2014 ) .", "Our data demonstrate that the Teneurins , a family of transsynaptic adhesion molecules , regulate one of these synaptic organizational paradigms: synapse number .", "Beyond molecules like RPTPs ( Takahashi and Craig , 2013 ) and Wnts ( Park and Shen , 2012 ) , there is little known conservation between the organization mechanisms of central synapses and the NMJ .", "In vertebrates , previous studies have identified a number of synaptogenic signaling and cell adhesion molecules in the CNS ( Gerrow and El-Husseini , 2006; Missler et al . , 2012 ) , but in many cases , their roles at the NMJ are either minimal or unknown .", "Likewise , the roles of pathways including Rapsyn , Dok7 , MuSK , and Tid1 , which are well established at the NMJ ( Wu et al . , 2010 ) , have no well-established roles at CNS synapses .", "Among the identified central synaptogenic molecules , no master controller of synapses , like Agrin , has been discovered ( Shen and Scheiffele , 2010 ) .", "In the mammalian CNS , synaptic adhesion molecules like Neurexin and Neuroligin have demonstrated organizational roles ( Sudhof , 2008 ) but their role ( if any ) at the NMJ is largely unknown .", "In Drosophila , considerable work has been done at the NMJ to understand synapse formation and organization ( Menon et al . , 2013 ) .", "For example , neuromuscular Neurexin and Neuroligin regulate synaptic development and assembly ( Li et al . , 2007; Banovic et al . , 2010; Sun et al . , 2011; Chen et al . , 2012; Mosca et al . , 2012; Owald et al . , 2012 ) , but remain ( as with many other identified molecules ) largely untested in the CNS due to the absence of techniques for doing so .", "Our approaches have now enabled such an examination , uncovering a strongly conserved synaptic organization function of the Teneurins from PNS to CNS .", "Recent work has shown that these evolutionarily conserved proteins are involved in synaptic partner matching between neurons in the Drosophila olfactory system and between muscles and motoneurons at the NMJ ( Hong et al . , 2012; Mosca et al . , 2012 ) .", "As there are marked differences between central and peripheral synapses ( like the NMJ ) , it is further unclear whether the mechanisms would be conserved or if they would be wholly different .", "Here we find , as assayed by Brp-Short ( Figure 4 ) , ultrastructural ( Figure 5 ) , and Dα7 ( Figure 8 ) analyses , that Teneurins in olfactory neurons are required for normal synapse number .", "Teneurin perturbation also reduces synaptic density , a parameter that is highly invariable for ORNs under normal conditions ( Figure 1I ) .", "We determined that presynaptic Ten-a in ORNs ( Figure 4 ) likely functions with postsynaptic Ten-m in PNs ( Figures 8 , 9 ) to regulate levels of the spectrin cytoskeleton ( Figure 6 ) .", "Our evidence is consistent with spectrin and the Teneurins functioning in the same genetic pathway to regulate synapse organization and density .", "The perturbation of active zone number in ORNs by knocking down ten-m in PNs further suggests that Teneurins regulate synapse number and organization in the CNS via a transsynaptic mechanism .", "This highlights further conservation between central and peripheral synapse organization in the use of Teneurins .", "There are , however , some differences between the CNS and the PNS regarding the Teneurins .", "While presynaptic ten-m has a minor role in synaptic organization at the NMJ ( Mosca et al . , 2012 ) , our data suggests the lack of such a role in the CNS ( Figure 4D ) .", "Thus , these different systems may also use the Teneurins differently .", "Mammalian Teneurins organize the visual system ( Leamey et al . , 2007; Suzuki et al . , 2012; Antinucci et al . , 2013; Young et al . , 2013 ) and Ten-2 can serve as a ligand for Latrophilin and localize to synapses in cultured neurons ( Silva et al . , 2011; Boucard et al . , 2014 ) .", "However , as proper synaptic function is impaired in many neuropsychiatric disorders ( Guilmatre et al . , 2014 ) and human Teneurin-4 is associated with increased susceptibility to bipolar disorder ( Psychiatric GWAS Consortium Bipolar Disorder Working Group , 2011 ) , understanding how the Teneurins regulate central synapses is a question with clinical relevance .", "Studies in vivo will be important to determine how mammalian Teneurins regulate synaptic organization and whether the different Teneurins can have specific roles at synapses .", "In summary , our results demonstrate a role for the Teneurins in regulating the number of central synapses and highlight mechanistic conservation between peripheral and central synapse formation .", "Moreover , the fact that ORNs can be mistargeted but still have the correct number of synapses suggests that target choice and synapse organization can be biologically separable , even when they employ the same molecules ." ], [ "The following GAL4 driver lines were used to restrict expression to individual classes of neurons: AM29-GAL4 ( DL4 and DM6 ORNs; Endo et al . , 2007 ) , Or47b-GAL4 ( VA1lm ORNs; Vosshall et al . , 2000 ) , Or67d-GAL4 ( DA1 ORNs; Kurtovic et al . , 2007 ) , Or88a-GAL4 ( VA1d ORNs; Vosshall et al . , 2000 ) , Mz19-GAL4 ( DA1 , DC3 , and VA1d PNs; Jefferis et al . , 2004 ) , Pebbled-GAL4 ( all ORNs; Sweeney et al . , 2007 ) , LN5 ( NP3056 ) -GAL4 ( ∼50 pan-glomerular LNs; Chou et al . , 2010 ) .", "The GH146-QF line ( Potter et al . , 2010 ) was used to genetically access 2/3 of all PNs .", "The following transgenes were used for labeling or genetic manipulation: UAS-Brp-Short-mStraw ( Fouquet et al . , 2009 ) , UAS-Brp-Short-EGFP ( Schmid et al . , 2008 ) , UAS-Dα7-GFP ( Leiss et al . , 2009b ) , UAS-3xHA-mtdT ( Potter et al . , 2010 ) , UAS-mCD8-GFP ( Lee and Luo , 1999 ) , UAS-DSyd1-EGFP ( Owald et al . , 2010 ) , UAS-α-spectrin-dsRNA ( Pielage et al . , 2005 ) , UAS-β-spectrin-dsRNA ( Pielage et al . , 2005 ) , UAS-Ten-a ( Mosca et al . , 2012 ) , UAS-Brp-IR-104422 ( Vienna Drosophila RNAi Center ) , UAS-Dcr2 ( Dietzl et al . , 2007 ) , UAS-ten-a-RNAi-V32482 , UAS-ten-m-RNAi-V51173 ( Hong et al . , 2012; Mosca et al . , 2012 ) , QUAS-mCD8-GFP ( Potter et al . , 2010 ) , QUAS-Ten-a ( Hong et al . , 2012 ) , and QUAS-Brp-Short-mStraw ( see below ) .", "The Df ( X ) ten-a line was used as a ten-a null mutant ( Hong et al . , 2012 ) .", "Brp-D3-mStraw ( Brp-Short-mStraw ) was amplified by PCR from the pTWmStraw-Brp-D3 vector ( Owald et al . , 2010; a kind gift of Stephan Sigrist ) using the forward primer 5ʹ-CACCATGGGAACTAGTGAC-3ʹ and the reverse primer 5ʹ-CTAGCTTACGTCACG-3ʹ with a CACC appended to the 5ʹ end of the forward primer for subcloning into the Gateway ( Invitrogen , Carlsbad , CA ) pENTR/D-TOPO vector .", "Following a BP reaction to produce pENTR-Brp-Short-mStraw , this plasmid was recombined into the destination vector pQUAST-attB-Gateway ( Mosca et al . , 2012; a kind gift of Vincenzo Favaloro ) using LR clonase .", "The resulting pQUAST-attB-Brp-Short-mStraw vector was transformed into the ΦC31 landing site 86Fb on the third chromosome using standard methods .", "hsFLP MARCM analyses were conducted as described ( Lee and Luo , 1999; Joo et al . , 2013 ) with the following modification: to obtain small DL4 and DM6 ORN clones , animals were heat-shocked between 48 hr and 72 hr after egg-laying at 37°C for 20 min .", "Adult brains were dissected at 10 days post-eclosion and fixed , stained , and processed as previously described ( Wu and Luo , 2006 ) .", "The following antibodies were used: rat anti-N-Cadherin [DN-EX#8; 1:40 , Developmental Studies Hybridoma Bank ( DSHB ) ] , mouse anti-Brp ( NC82; 1:40 , DSHB ) , rabbit anti-dsRed ( Living Colors DsRed Polyclonal Antibody , 1:250 , Clontech ) , chicken anti-GFP ( GFP-1020; 1:1000 , Aves Lab ) , mouse anti-α-spectrin ( 3A9; 1:50 , DSHB ) , rabbit anti-β-spectrin ( 1:500 , gift of R Dubreuil ) .", "Alexa488- , Alexa568- , and Alexa647-conjugated secondary antibodies were used at a concentration of 1:250 ( Invitrogen , Carlsbad , CA ) .", "Samples were mounted in SlowFade Gold Antifade medium ( Life Technologies , Grand Island , NY ) by bridge mount under #1 . 5 cover slips .", "Images were obtained on a Zeiss LSM510 Meta confocal laser-scanning microscope ( Carl Zeiss , Oberkochen , Germany ) using a 63× 1 . 4 NA PlanApo lens at an optical zoom of 3× .", "Images were centered on the glomerulus in question and the Z-boundaries set according to the confines of the synaptic label ( Brp-Short-mStraw or Dα7-GFP ) .", "System gain was set to minimize saturation but maintain a high signal-to-noise ratio .", "Selected raw image stacks are available at http://web . stanford . edu/group/luolab/ALsynapse . shtml .", "Raw image stacks for quantification were first deconvolved using AutoQuant X3 software ( Media Cybernetics , Rockville , MD ) on a custom-built computer ( Digital Storm , Fremont , CA ) .", "Specifically , the images were deconvolved using 10 iterations with settings of medium noise , and a blind point spread function .", "Following deconvolution , images were visually inspected to ensure that striping , ringing , or discontinuity artifacts were not introduced .", "Further , each image was visually examined to ensure that the borders of puncta were sharpened by the deconvolution and that the membrane staining remained continuous .", "Following , images were imported into Imaris 7 . 4 . 1 ( Bitplane AG , South Windsor , CT ) for quantification .", "Though deconvolution improved the visual quality of the images , it was dispensable for accurate quantification of Brp-Short or Dα7 puncta .", "The ‘Spots’ function was used to quantify Brp-Short or Dα7 puncta in Imaris .", "A region of interest was drawn around the glomerulus being quantified .", "For individually labeled glomeruli , the standard Imaris box was sufficient to mark the region of interest .", "Where multiple glomeruli were labeled and a single glomerulus was quantified , a freehand region of interest was drawn in each section using the neuropil staining as a guideline and a 3D region extrapolated by Imaris .", "The spot size ( puncta diameter ) was set at 0 . 6 μm for Brp-Short and 0 . 8 μm for Dα7 , as determined by direct measurement of images and previous studies ( Kremer et al . , 2010 ) .", "Background subtraction was selected .", "Following automatic detection of puncta , the threshold was manually adjusted ( usually within 5% among different samples ) with visual inspection to ensure that all puncta were identified , minimal background staining or noise was recognized , and that individual puncta were not double-counted .", "In all , the resultant count was recorded as the number of puncta .", "The accuracy was assessed by comparing puncta detection figures to glomeruli that were counted by hand following 3D projection .", "In all cases , there was less than 5% difference , supporting the accuracy of the semi-automatic quantification method .", "In male Or67d-GAL4 samples that did not express the synaptic label ( normally have ∼1500 puncta ) , but were processed identically using primary and secondary antibodies , the number of puncta recognized by this method was <30 , suggesting that the ‘false positive’ rate was low .", "To ensure that user-introduced noise in threshold adjustment did not introduce significant bias , each image was quantified a second time , at a later time , without access to the prior quantification .", "If the difference exceeded 5% , the sample was discarded .", "Further , if the averages of an entire genotype were significantly different from each other ( as determined by student's t test ) , the entire cohort was discarded and the experiment repeated .", "For the nearest neighbor distance ( NND ) analysis , the ‘DistMin’ was calculated in Imaris using the ‘Spots to Spots Closest Distance XTension’ plug-in for Matlab .", "Cumulative frequency histograms were compiled in Prism 6 . 04 ( GraphPad Software , San Diego , CA ) .", "As each puncta is ∼0 . 6 μm in diameter , the minimum NND should be 0 . 6 .", "Indeed , few measurements were seen below 0 . 6 .", "Thus , ‘clustering’ was defined as puncta with an NND of less than 25% of the diameter .", "The ‘% Cluster’ was calculated by expressing the number of puncta with an NND value ≤0 . 75 divided by the total number of puncta .", "To obtain neurite volume based on mCD8-GFP or mtdT labeling , the ‘Surfaces’ function was used .", "Regions of interest were drawn as above .", "Background subtraction was set to 3 μm and the smoothing to 0 . 2 μm; these settings optimally reflected actual neurite labeling .", "Automatic detection with a 10e5 threshold was then used to remove background , detecting only the glomerulus in question .", "For smaller objects ( MARCM clones ) , a 10e1 threshold was used and only the specific objects that directly corresponded to mCD8-GFP labeling were selected and counted .", "Volume was then calculated by Imaris .", "Density was then expressed as the number of Brp-Short puncta divided by the neurite volume .", "For images involving a neurite co-stain , the Brp-Short-mStraw channel was masked in Imaris so that only the puncta within the neurite were identified and counted ( not background puncta resulting from secondary antibodies , or image noise ) .", "Images were processed using Photoshop CS4 ( Adobe , San Jose , CA ) and levels or contrast adjusted identically for all cases of comparison .", "For cases of immunofluorescence comparison , brains were stained in parallel , imaged on the same slide in the same session , and processed identically .", "Statistical and graphical analyses were completed in Excel ( Microsoft , Redmond , WA ) , Prism 6 . 04 ( GraphPad Software , San Diego , CA ) , and Matlab ( Mathworks , Natick , MA ) .", "Adult brains were dissected in 0 . 1 M cacodylate buffer and fixed at 4°C for 3 hr in 2 . 5% paraformaldehyde , 5% glutaraldehyde ( vol/vol ) , and 0 . 06% picric acid ( vol/vol ) in 0 . 1 M cacodylate buffer .", "They were then washed three times for 20 min each in 0 . 1 M cacodylate buffer on ice and post-fixed with 2% osmium tetroxide for 1 hr at 4°C .", "Dehydration , infiltration with resin , and staining with uranyl acetate were all done according to standard protocols .", "Antennal lobe regions were identified in thick section following 1% toluidine blue in 1% sodium borate staining and ultrathin sections taken from those regions .", "Sections were imaged on a JEM-1400 ( JEOL , Tokyo , Japan ) transmission electron microscope at X3 , 000 to X20 , 000 magnification .", "For immuno-EM , adult brains were dissected in 0 . 1 M sodium phosphate buffer and fixed in modified Zamboni's fixative ( 4% paraformaldehyde , 1 . 6% glutaraldehyde , 0 . 2% saturated picric acid in 0 . 1 M sodium phosphate buffer at pH 7 . 4 ) as described ( Kolodziejczyk et al . , 2008 ) .", "Brains were then washed in PBS , processed similarly as above , and sectioned .", "Sections were then blocked in PBST containing 0 . 05% Tween-20 , 0 . 5% ( wt/vol ) ovalbumin , and 0 . 5% ( wt/vol ) BSA .", "Sections were incubated overnight at 4°C in rabbit anti-GFP at 1:300 ( MBL International , Woburn , MA ) .", "Following , grids were washed in PBST and incubated in 5 nm gold-conjugated goat anti-rabbit secondary antibody ( Ted Pella , Redding , CA ) at 1:50 for 1 hr at RT .", "Grids were then washed in PBST , incubated in 8% glutaraldehyde for 15 min , washed again in water , and contrast stained with 2% uranyl acetate followed by lead citrate .", "Ultrastructural analysis was done as previously described ( Watts et al . , 2004; Mosca et al . , 2012 ) and T-bar defects quantified using consecutive sections .", "For pairwise comparisons , student's t test was used to assess statistical significance .", "For multiple comparisons , a one-way ANOVA was done and corrected for multiple comparisons with posthoc Tukey's multiple comparisons tests done between all genotypes .", "Analysis was done in Prism 6 . 0 . 4 ( Graphpad Software , San Diego , CA ) .", "For synapse contribution by individual ORNs , GraphPad Prism was used to fit individual Gaussian relationships to each separate peak for the ORN MARCM data ( Figure 2C–D , colored dashed lines ) .", "The Curve Fitting Tool in Matlab ( Mathworks , Natick , MA ) was used to determine the best-fit curves that corresponded to the aggregate data set ( Figure 2C–D , solid black line ) .", "In each case , multiple strong candidate relationships that recognized the first four peaks were identified using r2 and root mean square error ( RMSE ) values .", "To determine which candidate best fit the data , they were then compared using two posthoc tests: corrected Akaike's Information Criteria ( AICc ) and the F-test .", "AICc is advantageous as it penalizes distributions with more parameters , avoiding potential over-fitting , and is useful with binned data .", "The F-test was used to test specific null hypotheses between the models .", "To examine the distribution of the individual peaks ( i . e . , the ‘quantal’ nature ) , the standard deviation in the distances between the peaks ( S ) was calculated .", "To determine the probability of such an S value occurring , the aggregate DL4 and DM6 data were individually fit to a Poisson distribution or a Gaussian distribution using Matlab .", "Matlab was then used to generate 50 , 000 simulations selecting four peaks that fit those distributions .", "Also , a uniform probability distribution was used to select four peaks between 0 and the upper bound of the DL4 and DM6 data sets , respectively .", "In all , 300 , 000 simulations were conducted , and the number of events resulting in an S value less than that observed used to determine a probability of obtaining a more ordered set of four peaks than the observed data .", "In some cases , UAS-mCD8-GFP or UAS-3xHA-mtdT was included in the genotype ( but not shown in the image ) to ensure an equivalent number of constructs between control and mutant or rescue conditions .", "Figure 1: ( B–D ) w; UAS-Brp-Short-mStraw/UAS-mCD8-GFP; Or67d-GAL4 [Nr-1]/+; + .", "( H–I )", "DL4 and DM6 = w; AM29-GAL4/UAS-Brp-Short-mStraw , UAS-mCD8-GFP; +; + .", "DA1 = w; UAS-Brp-Short-mStraw/UAS-mCD8-GFP; Or67d-GAL4 [Nr-1]/+; + .", "VA1d = w , Or88a-GAL4/+ or Y; UAS-mCD8-GFP , UAS-Brp-Short-mStraw/+; +; + .", "VA1lm = w; Or47b-GAL4/UAS-Brp-Short-mStraw , UAS-mCD8-GFP; +; + .", "Figure 1—figure supplement 1: ( A ) w; UAS-Brp-Short-mStraw/+; Or67d-GAL4 [Nr-1]/UAS-DSyd1-EGFP; + .", "( B ) w , UAS-Dcr2; UAS-mCD8-GFP/UAS-Brp-Short-mStraw; Or67d-GAL4 [Nr-1]/+; + .", "( C ) w; UAS-mCD8-GFP , UAS-Brp-Short-mStraw/UAS-Brp-IR-v104422; Or67d-GAL4 [Nr-1]/+; + .", "( D ) w , pebbled-GAL4/+; +; UAS-Brp-Short-GFP/+; + .", "Figure 1—figure supplement 2: ( A ) w , Or88a-GAL4/+ or Y; UAS-Brp-Short-mStraw/+; +; + .", "( B ) w; Or47b-GAL4/UAS-Brp-Short-mStraw; +; + .", "( C and D ) w; AM29-GAL4/UAS-Brp-Short-mStraw; +; + .", "Figure 2: ( A–F ) hsFLP122 , UAS-mCD8-GFP; AM29-GAL4/UAS-Brp-Short-mStraw; FRT 2A/FRT2A , tubP-GAL80; + .", "Figure 2—figure supplement 1: ( A–L ) hsFLP122 , UAS-mCD8-GFP; AM29-GAL4/UAS-Brp-Short-mStraw; FRT 2A/FRT2A , tubP-GAL80; + .", "Figure 3: ( A ) w; UAS-Brp-Short-mStraw/+; Or67d-GAL4 [Nr-1]/+; + .", "( B ) w; Mz19-GAL4/UAS-Brp-Short-mStraw; +; + .", "( C ) w; UAS-Brp-Short-mStraw/+; NP3056-GAL4/+; + .", "( D , G ) w; UAS-Brp-Short-mStraw/UAS-mCD8-GFP; Or67d-GAL4 [Nr-1]/+; + .", "( E , H ) w; Mz19-GAL4 , UAS-mCD8-GFP/UAS-Brp-Short-mStraw; +; + .", "( F , I ) w; UAS-Brp-Short-mStraw/UAS-mCD8-GFP; NP3056-GAL4/+; + .", "Figure 3—figure supplement 1: ( A ) w; UAS-Brp-Short-mStraw , UAS-mCD8-GFP/+; Or67d-GAL4 [Nr-1]/+; + .", "( B ) w; Mz19-GAL4/UAS-Brp-Short-mStraw , UAS-mCD8-GFP; +; + .", "( C ) w; UAS-Brp-Short-mStraw , UAS-mCD8-GFP/+; NP3056-GAL4/+; + .", "Figure 4: ( A ) w; Or47b-GAL4/UAS-Brp-Short-mStraw; UAS-mCD8-GFP/+; + .", "( B ) w , Df ( X ) ten-a; Or47b-GAL4/UAS-Brp-Short-mStraw; +; + .", "( C ) w , Df ( X ) ten-a; Or47b-GAL4/UAS-Brp-Short-mStraw; UAS-Ten-a/+; + .", "( D–F )", "Control as in ( A ) .", "ten-a as in ( B ) .", "ten-a ORN RNAi = w , UAS-Dcr2; Or47b-GAL4/UAS-Brp-Short-mStraw; UAS-ten-a-IR-v32482/+; + .", "ten-a + Ten-a ORN as in C . ten-m ORN RNAi = w , UAS-Dcr2; Or47b-GAL4/UAS-Brp-Short-mStraw; UAS-ten-m-RNAi-V51173/+; + .", "ten-a + Ten-a PN = w , Df ( X ) ten-a; Or47b-GAL4/UAS-Brp-Short-mStraw; GH146-QF , QUAS-mCD8-GFP/QUAS-Ten-a; + .", "Figure 4—figure supplement 1: ( A ) w; Or47b-GAL4/UAS-Brp-Short-mStraw; UAS-mCD8-GFP/+; + .", "( B ) w , UAS-Dcr2; Or47b-GAL4/UAS-Brp-Short-mStraw; UAS-ten-m-RNAi-V51173/+; + .", "( C ) w , UAS-Dcr2; Or47b-GAL4/UAS-Brp-Short-mStraw; UAS-ten-a-IR-v32482/+; + .", "( D ) w , Df ( X ) ten-a; Or47b-GAL4/UAS-Brp-Short-mStraw; GH146-QF , QUAS-mCD8-GFP/QUAS-Ten-a; + .", "Figure 4—figure supplement 2: ( A–C ) w; UAS-Brp-Short-mStraw , UAS-mCD8-GFP/AM29-GAL4; +; + .", "( D–F ) w , Df ( X ) ten-a; UAS-Brp-Short-mStraw , UAS-mCD8-GFP/AM29-GAL4; +; + .", "Figure 5: Control = Canton S . ten-a = Df ( X ) ten-a; +; +; + .", "Figure 6: ( A ) Canton S . ( B ) Df ( X ) ten-a; +; +; + .", "( C ) w; Or47b-GAL4 , UAS-Brp-Short-mStraw/UAS-mCD8-GFP; UAS-mCD8-GFP/+; + .", "( D ) w; Or47b-GAL4 , UAS-Brp-Short-mStraw/UAS-α-spectrin-dsRNA; UAS-α-spectrin-dsRNA/+; + .", "( E ) w; Or47b-GAL4 , UAS-Brp-Short-mStraw/UAS-β-spectrin-dsRNA; UAS-β-spectrin-dsRNA/+; + .", "( F ) Control as in ( A ) and ten-a as in ( B ) .", "( G ) Control as in ( C ) , α-spec ORN RNAi as in ( D ) , β-spec ORN RNAi as in ( E ) , ten-a = w , Df ( X ) ten-a; Or47b-GAL4/UAS-Brp-Short-mStraw; +; + .", "ten-a + α-spec RNAi = w , Df ( X ) ten-a; Or47b-GAL4 , UAS-Brp-Short-mStraw/UAS-α-spectrin-dsRNA; UAS-α-spectrin-dsRNA/+; + .", "Figure 6—figure supplement 1: ( A ) Canton S . ( B ) Df ( X ) ten-a; +; +; + .", "( C ) w; UAS-Brp-Short-mStraw , UAS-mCD8-GFP/+; Or67d-GAL4 [Nr-1] , UAS-mCD8-GFP/+; + .", "( D ) w; UAS-Brp-Short-mStraw/UAS-α-spectrin-dsRNA; Or67d-GAL4 [Nr-1]/UAS-α-spectrin-dsRNA; + .", "( E ) w; UAS-Brp-Short-mStraw/UAS-β-spectrin-dsRNA; Or67d-GAL4 [Nr-1]/UAS-β-spectrin-dsRNA; + .", "( F ) Genotypes as in ( C ) , ( D ) , and ( E ) .", "Figure 7: ( B , E–F ) w; Mz19-GAL4 , UAS-3xHA-mtdT/+; UAS-Dα7-EGFP/+; + .", "( C ) w; Mz19-GAL4/+; UAS-Dα7-GFP/+; + .", "( D ) w , Or67d-QF; Mz19-GAL4/+; UAS-Dα7-GFP/QUAS-Brp-Short-mStraw; + .", "Figure 8: ( A ) w , UAS-Dcr2; Mz19-GAL4 , UAS-3xHA-mtdT/+; UAS-Dα7-GFP/+; + .", "( B ) w , Df ( X ) ten-a; Mz19-GAL4 , UAS-3xHA-mtdT/+; UAS-Dα7-GFP/+; + .", "( C ) w , UAS-Dcr2; Mz19-GAL4 , UAS-3xHA-mtdT/+ , UAS-Dα7-GFP/UAS-ten-m-IR; + .", "( D–F )", "Control as in ( A ) .", "ten-a as in ( B ) .", "ten-m PN RNAi as in C . ten-a −/− + ten-m PN RNAi = w , Df ( X ) ten-a; Mz19-GAL4 , UAS-3xHA-mtdT/UAS-Dcr2; UAS-Dα7-GFP/UAS-ten-m-IR; + .", "Figure 9: ( B ) w , Or67d-QF; +; QUAS-Brp-Short-mStraw/+; + .", "( C ) w , Or67d-QF; Mz19-GAL4/+; QUAS-Brp-Short-mStraw/UAS-ten-m-RNAi-V51173; + .", "( D ) Control ( UAS ) = w; UAS-Brp-Short-mStraw/+; Or67d-GAL4 [Nr-1]/+; + .", "Other genotypes as in B–C ." ] ]

[ "Understanding information flow through neuronal circuits requires knowledge of their synaptic organization .", "In this study , we utilized fluorescent pre- and postsynaptic markers to map synaptic organization in the Drosophila antennal lobe , the first olfactory processing center .", "Olfactory receptor neurons ( ORNs ) produce a constant synaptic density across different glomeruli .", "Each ORN within a class contributes nearly identical active zone number .", "Active zones from ORNs , projection neurons ( PNs ) , and local interneurons have distinct subglomerular and subcellular distributions .", "The correct number of ORN active zones and PN acetylcholine receptor clusters requires the Teneurins , conserved transmembrane proteins involved in neuromuscular synapse organization and synaptic partner matching .", "Ten-a acts in ORNs to organize presynaptic active zones via the spectrin cytoskeleton .", "Ten-m acts in PNs autonomously to regulate acetylcholine receptor cluster number and transsynaptically to regulate ORN active zone number .", "These studies advanced our ability to assess synaptic architecture in complex CNS circuits and their underlying molecular mechanisms ." ]

[ "Just as progress in science relies on researchers communicating their findings to other people working in their field , our bodies rely on neurons being able to communicate with other neurons .", "This is where structures called synapses come in: synapses allow signals to be passed from one neuron to another .", "Neurons and synapses process information by forming circuits in the brain , but relatively little is known about how synapses develop or how they are organized within circuits .", "Mosca and Luo have now examined a neural circuit in the fruit fly ( Drosophila ) that receives sensory information about smells in the environment , and then converts this information to signals which can be understood by other parts of the brain .", "This particular circuit has previously been identified as a good model of how the brain processes information .", "Mosca and Luo found that the synapses in this circuit were organized according to specific ‘rules’ that determined factors such as the quantity and location of synapses at different points in the circuit .", "Additionally , it was found that the successful development of synapses required the involvement of two members of a family of proteins called the Teneurins: this family of proteins is involved in a variety of neurodevelopmental processes .", "Teneurins have been implicated in bipolar disorder , and malfunctioning synapses are thought to be associated with a number of other mental health conditions , so the results of Mosca and Luo could lead to a better understanding of these conditions ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "neuroscience" ]

[ [ "You ask a friend about the tilt of a canvas that is perfectly horizontal and she says that the top right corner is up .", "What causes her inaccuracy ?", "One possibility is that her sensory representation is biased and she perceives the canvas tilted .", "Another possibility , however , is that under uncertainty about the orientation of the canvas , she prefers to choose up over down .", "This situation exemplifies a major problem in the study of perception: perceptual decisions not only depend on the sensory evidence , but also on decisional components ( Green and Swets , 1966; Gold and Ding , 2013 ) .", "When a decision maker needs to select an action from a set of alternatives , the tendency to choose one alternative over the others is known as choice bias ( Gold and Ding , 2013 ) .", "Choice biases occur in perceptual tasks even in simple scenarios like the one described above , in which the stimuli carry similar levels of signal relative to a neutral point ( Newsome and Paré , 1988; Mareschal and Clifford , 2012; Jazayeri and Movshon , 2007; Milner et al . , 1992; Tadin et al . , 2003 ) .", "Choice biases are extensively studied in relation to how current decisions are influenced by previous decisions , what is known as choice history biases ( Abrahamyan et al . , 2016; Akaishi et al . , 2014; Fründ et al . , 2014; Urai et al . , 2017; Fischer and Whitney , 2014; Braun et al . , 2018; St John-Saaltink et al . , 2016; Fritsche et al . , 2017; Hermoso-Mendizabal et al . , 2019 ) .", "Depending on the perceptual scenario , people tend to repeat their previous choice ( Akaishi et al . , 2014; Braun et al . , 2018 ) , alternate between choices ( Fritsche et al . , 2017 ) or idiosyncratically repeat or alternate ( Urai et al . , 2017; Abrahamyan et al . , 2016 ) .", "Whether choice history biases reflect a bias in the sensory evidence or in the decision process is under debate ( Akaishi et al . , 2014; Fischer and Whitney , 2014; St John-Saaltink et al . , 2016; Fritsche et al . , 2017 ) .", "Unlike choice history biases , some choice biases are related to the overall idiosyncratic tendency to choose one alternative over the others , not conditioned by the previous choices .", "We will refer to these as global choice biases .", "The existence of these biases is acknowledged ( Gold and Ding , 2013; Kingdom and Prins , 2016; Morgan et al . , 2012; García-Pérez and Alcalá-Quintana , 2013; Peters et al . , 2016 ) and they are included in current models of perceptual decision making ( Abrahamyan et al . , 2016; Akaishi et al . , 2014; Urai et al . , 2017; Braun et al . , 2018; Hermoso-Mendizabal et al . , 2019 ) , but whether they reflect sensory or decisional processes has not been , to our knowledge , assessed ( we searched in Google Scholar several times , last one on February 2019 , the following keywords: choice biases , response biases , motor biases , perceptual biases and sensory biases; we identified the relevant articles and searched within the references cited; we also tracked the articles that cited the relevant articles ) .", "The problem to identify their origin is that , in standard perceptual paradigms , the tendency to choose one alternative more often is consistent with a biased sensory representation , but also with a bias in the decision process ( García-Pérez and Alcalá-Quintana , 2013; Gold and Ding , 2013 ) .", "Here , to disentangle the contribution of sensory and decisional processes to global choice bias , we measured choice behavior in a simple common perceptual discrimination task with two symmetric alternatives , but under two different task instructions .", "The instructions varied the response mapping between perception and the category of the alternatives ." ], [ "On a given trial of the symmetric task ( the model for the asymmetric task is described in Supplementary Models ) a grating with orientation θi that could be clockwise or counterclockwise , is presented and the participant decides whether its orientation is consistent with clockwise acw or counterclockwise accw orientation .", "A simple standard signal detection theory ( SDT ) model assumes that: ( 1 ) the sensory evidence s associated to the stimulus is a random variable normally distributed with variance σ2 centered on μcw when θi is clockwise and centered on μccw when θi is counterclockwise; ( 2 ) to make choices , the participant sets a criterion β , computes the log likelihood ratio log⁡Λ of s under the hypothesis that μ=μcw and the hypothesis that μ=μccw ( 1 ) log⁡Λ ( s ) =log⁡ ( p ( s|μ=μcw ) p ( s|μ=μccw ) ) =log⁡ ( ( 2πσ2 ) −1/2e− ( s−μcw ) 22σ2 ( 2πσ2 ) −1/2e− ( s−μccw ) 22σ2 ) and chooses the action acw when log⁡Λ ( s ) >β .", "In standard SDT models the origin and scale of the sensory evidence is arbitrary ( Wickens , 2001; Green and Swets , 1966 ) .", "A typical choice is ( Wickens , 2001; Green and Swets , 1966 ) ( 2 ) μcw=d′2μccw=−d′2σ=1which results in log⁡Λ ( s ) =d′s .", "That is , one can use directly the evidence as a decision variable and choose acw when s>c=β/d′ ( Wickens , 2001; Green and Swets , 1966 ) .", "The probability of choosing clockwise when θi is clockwise is ( 3 ) P ( a=acw;μ=μcw ) =12π∫c∞e− ( s−μcw ) 22ds=Φ ( μcw−c ) where Φ is the standard cumulative normal function .", "To relate this probability to the magnitude of the stimulus , this standard SDT could be extended to include how the stimulus is transduced into sensory evidence ( García-Pérez and Alcalá-Quintana , 2013; Schneider and Bavelier , 2003; García-Pérez and Peli , 2014; Schneider and Komlos , 2008 ) .", "Adding the transduction provides a meaningful origin and scale to the sensory evidence .", "Assuming that the transduction is linear ( 4 ) μcw=aθi+bthe probability of choosing clockwise as a function of θi becomes ( 5 ) P ( a=acw;θi ) =Φ ( aθi+b−c ) =Φ ( θi− ( δD−δS ) σ ) Where ( 6 ) δS=ba=sensorybiasδD=ca=decisionalbiasσ=1awhich corresponds to a psychometric function centered on δD−δS with slope given by σ .", "Consider the trials in which the grating is presented in orientations around the horizontal orientation and the reference is on the right ( the reasoning that follows also holds for vertical orientations ) .", "If the orientation of the grating can take M different values , each value is presented n times and ki is the number of times that the participant pressed the down key ( clockwise ) , then the probability density function that defines statistical model for these trials is ( 7 ) f ( k1 , … , kM;δS , δD , σ ) =∏i=1M ( nki ) Φ ( θi− ( δD−δS ) σ ) ki ( 1−Φ ( θi− ( δD−δS ) σ ) ) n−ki Given that f depends on δD−δS , it is not possible to distinguish sensory from decisional biases ( Witt et al . , 2015; Schneider and Komlos , 2008; García-Pérez and Alcalá-Quintana , 2013 ) .", "Consider now the trials for the left reference .", "A criterion c associated to a bias to choose responses consistent with clockwise orientation for the right reference corresponds to a criterion −c for the left reference .", "The statistical model that incorporates responses for the two locations of the reference is ( 8 ) f ( k1 , … , k2M;δS , δD , σ ) =∏i=1M ( nki ) ( 1−ΦR ) n−ki∏i=M+12M ( nki ) ΦLki ( 1−ΦL ) n−kiwhere ( 9 ) ΦR=Φ ( θi− ( δD−δS ) σ ) ΦL=Φ ( θi− ( −δD−δS ) σ ) and the index i larger than M refers to the trials for the left reference .", "Now , by fitting two psychometric functions conjointly , δD and δs are not degenerated and could be estimated .", "This is the exact model that we fitted for 17 of the 34 groups ( 17 participants that discriminate orientations around the horizontal and the vertical orientation in Experiment 1 ) .", "For the rest of the groups , we also fitted this basic model , but adding some extra features that significantly improved the quality of the fit ( using likelihood ratio tests , see Materials and methods ) .", "The first feature is related to the variability of the sensory evidence σ .", "The basic model considers that this variability does not depend on where the participants imagined the reference , which results in the two psychometric functions having the same slope .", "We found this to be the case in 28 of the 34 fits , but in six groups we found that considering different variabilities ( σ1 and σ2 ) for the two locations of the reference was better .", "It might be that orienting attention to different parts of the visual field might change the uncertainty in the sensory evidence for some participants .", "The other feature that we added consisted in adding lapses , which are responses that are incorrect independently of the sensory evidence ( Kingdom and Prins , 2016; Gold and Ding , 2013 ) .", "This might occur , for example , when the participant misses the stimulus because of blinking or a loss of attention .", "To incorporate lapses into the basic model , ΦR need to be replaced by λ1+ ( 1−λ1−λ2 ) ΦR and ΦL by λ1′+ ( 1−λ1′−λ2′ ) ΦL ( Kingdom and Prins , 2016 ) .", "From the 34 groups , adding lapses improved the fit in 14 cases .", "From those , three fits required the four lapse parameters , 4 fits required two lapse parameters ( one for each psychometric function , λ1=λ2 ; λ1′=λ2′ ) and 7 fits required one lapse rate ( λ1=λ2=λ1′=λ2′ ) .", "Importantly , including a lapse rate parameter λ∗ placed on the right asymptote for the right reference ( 10 ) λ1+λ∗+ ( 1−λ1−λ1−λ∗ ) ΦRand placed on the left asymptote for the left reference ( 11 ) λ2+ ( 1−λ2−λ2−λ∗ ) ΦLdid not improve significantly the fit for any group , which indicates that decisional biases cannot be explained by a tendency of participants to select one alternative completely independently of the sensory evidence .", "Perceptual tasks with two symmetric alternatives have been also modeled using a high threshold model , called the indecision model ( García-Pérez and Alcalá-Quintana , 2013; García-Pérez and Peli , 2014 ) .", "This model is similar to the SDT model described , but divides the sensory axis in three regions delimited by c1 and c2 .", "When the sensory evidence is lower than c1 the participant chooses accw and when is larger than c2 , chooses acw .", "Critically , when the sensory evidence lies between c1 and c2—called interval of uncertainty—the model assumes that the observer is undecided and guesses the response ( choosing accw with probability ξ ) .", "The probability of choosing clockwise when θi is clockwise and the reference is on the right is ( 12 ) P ( a=acw;μ=μcw ) =12π∫c2∞e− ( s−μcw ) 22ds+ξ12π∫c1c2e− ( s−μcw ) 22ds=Φ ( μcw−c2 ) +ξ ( Φ ( c2−μcw ) −Φ ( c1−μcw ) ) For the left reference , assuming that c1 and c2 do not change , ξ should be replaced by 1−ξ .", "We fitted this model to the 19 groups with significant decisional biases and found that for all of them ( except for participant eight for the vertical condition in Experiment", "1 ) the SDT model was better using the Akaike information criterion ( Burnham and Anderson , 2004 ) .", "Given that the indecision model has two parameters more than the SDT model ( c is replaced by c1 and c2 and ξ is introduced ) , we also compared the SDT model to a simplified version of the indecision model with symmetric boundaries c1=−c2 ( García-Pérez and Alcalá-Quintana , 2013; García-Pérez and Peli , 2014 ) .", "In this case , the SDT model was better for all the groups ." ], [ "We showed that , in a simple discrimination task with two symmetric alternatives , most people exhibit idiosyncratic global choice biases .", "Changing the stimulus-response mapping and testing a task with two asymmetric alternatives , we found that these biases reflect biases in the sensory evidence and in the decisional process .", "Specifically , about half of the participants showed biases consistent with only a sensory origin and about half of the participant biases with a contribution of sensory and decisional biases .", "We also found that a very few participants exhibited biases consistent with only a decisional origin .", "Across participants , the magnitude of the sensory bias was about three times as larger as the magnitude of the decisional bias .", "Our findings suggest that if a person has a bias to report that a perfectly horizontal canvas is tilted towards a given direction , it is likely that the largest contribution to the bias has a sensory origin .", "To our knowledge , these idiosyncratic sensory biases have not been linked to asymmetries in the neural representation of the stimulus .", "Possible asymmetries include a difference in the number of neurons tuned to clockwise and counterclockwise orientation if the decoding is of the population-vector type or a difference in the gain of neurons tuned to clockwise and counterclockwise orientation for more general decoding schemes ( Dayan and Abbott , 2001; Schwartz et al . , 2007 ) .", "More recently , it has been proposed that the asymmetries could also emerge from the dynamics of competing neural networks ( Lebovich et al . , 2018 ) .", "Importantly , for these asymmetries to bias perception , the downstream decoding mechanisms should have not been calibrated with the environmental property to compensate the asymmetries ( decoding ambiguity; Schwartz et al . , 2007 ) .", "An SDT model , in which the sensory biases were included as an intercept term in a linear transduction of the stimuli ( García-Pérez and Alcalá-Quintana , 2013; Schneider and Bavelier , 2003; García-Pérez and Peli , 2014; Schneider and Komlos , 2008 ) and decisional biases correspond to a shift in the criterion , fitted well the choice behavior of the participants .", "A model in which participants have a tendency to choose one alternative independently of the magnitude of the sensory evidence did not fit well the data; it rather predicts an asymmetry on the lapse rate across locations of the reference that was not observed .", "Finally , a model ( García-Pérez and Alcalá-Quintana , 2013; García-Pérez and Peli , 2014 ) in which participants guess when the sensory evidence lies within some uncertainty range was not parsimonious: first , to fit the data for the participants with pure sensory biases , the model needs that participants , when uncertain , guess the two alternatives equally often; second , for the participants with decisional biases , we found that the SDT model provided a better fit .", "Perceptual discrimination with two symmetric alternatives are often regarded as Type 1 tasks , tasks for which the responses could be designated as correct or incorrect ( Kingdom and Prins , 2016 ) .", "If a stimulus has positive signal ( for example , rightward motion ) relative to a neutral point ( no net motion ) , but the decision-maker chooses the alternative consistent with negative signal ( leftward motion ) , the response is considered an error .", "In this case , a psychometric function of the proportion of correct responses as a function of the unsigned signal is often fitted , and precision is estimated as the amount of signal that is required to reach a certain level of correct responses .", "Our results , however , suggest that in some cases a positive signal might be perceived consistently as a negative signal ( sensory bias ) .", "Consequently , it might be inappropriate to consider these responses as errors and , in case feedback is given , provide a negative reward .", "Our results suggest , thus , that perceptual discrimination tasks with two symmetric alternatives might be better regarded as Type 2 tasks , tasks for which there are not correct and incorrect responses ( Kingdom and Prins , 2016; Gold and Ding , 2013 ) .", "Accordingly , the psychometric function that should be fitted is the proportion of times that a category was selected ( for example , rightward motion ) as a function of the signed signal , and precision should be estimated using the slope of the psychometric function ( Gold and Ding , 2013 ) .", "Discrimination tasks with two symmetric alternatives are commonly used to assess how perception is affected by contextual cues ( Carrasco et al . , 2004; Schwartz et al . , 2007 ) , but in some scenarios it is unclear whether the context influences perception or biases decisions ( Carrasco , 2011; García-Pérez and Alcalá-Quintana , 2013; Morgan et al . , 2012; Störmer et al . , 2009; Schneider , 2011; Anton-Erxleben et al . , 2010; Jogan and Stocker , 2012; Mather and Sharman , 2015 ) .", "Our results indicate that , even when the symmetry of the task is not broken by the context , half of the participants exhibit decisional biases .", "This suggests that to reliably estimate sensory biases in the presence of contextual cues , it might be better to use manipulations of the stimulus-response mapping like the one that we used or tasks for which the potential contribution of decisional biases is reduced or eliminated ( Jogan and Stocker , 2012; Patten and Clifford , 2015; Morgan , 2014 ) .", "The perceptual discrimination task with two symmetric alternatives that we have used is also known as the method of single stimuli ( Morgan et al . , 2012 ) In this method , the decision-maker is asked to categorize the signal presented against an internal absolute reference that corresponds to a natural neutral point that the decision-maker possibly has learnt during her life ( Morgan et al . , 2012 ) —verticality or horizontality in our case .", "The sensory biases that we have measured are deviations from this internal reference .", "In the non-symmetric version of this task , the Yes-No task ( Green and Swets , 1966; Kingdom and Prins , 2016; Wickens , 2001 ) , the decision-maker decides , for example , whether a dim light is present or absent .", "For this task , decision-makers have also idiosyncratic tendencies to report that the signal is present or absent ( Green and Swets , 1966 ) .", "Given that , in this task , there is no natural neutral point , it is harder to assess whether these tendencies reflect differences in the strength that each decision-maker perceives or differences in how conservatively they report that the signal is present or absent ( Jin and Glickfeld , 2018 ) .", "Another popular task to assess perception is the two alternative forced choice task ( Green and Swets , 1966; Wickens , 2001; Kingdom and Prins , 2016 ) .", "In this task , two stimuli are presented in a random order—for example , a vertical grating and a grating slightly tilted clockwise—and the decision-maker decides in which interval the grating was more tilted clockwise .", "This task assesses the precision to discriminate similar orientations , but cannot measure sensory biases from verticality: a bias of the decision-maker to perceive orientations clockwise will affect the stimuli presented in the two intervals in the same direction without affecting the selection of the interval with the more tilted stimulus .", "To facilitate that participants internalize two different associations between perception and the responses , we asked them to compare the orientation of the stimulus to a reference imagined in two different locations .", "Given that the reference was not physically present , we think , however , that it is likely that participants were performing judgments of absolute orientation relative to an internal point of horizontality ( or verticality ) .", "As we found evidence that the sensory biases were consistent with a global perceptual rotation , if participants were performing judgments of relative orientation between the stimulus and the reference—instead of judgments of absolute orientation—then sensory biases should not be expected , as both the stimulus and the reference should be biased in the same direction .", "We found a good agreement between the sensory biases estimated from the symmetric and the asymmetric task .", "We also found a good agreement between tasks when the biases were estimated taking into account only one localization of the reference , and thus for which it was not possible to assess the contribution of sensory and decisional biases ( see legend for Figure 3A ) .", "This is expected given the large contribution of sensory biases to the total magnitude of the biases .", "This agreement across tasks contrasts with the disagreement found between the analogous tasks in the temporal domain .", "In the temporal domain , the analogous to the symmetric task is the temporal order judgment .", "Given , for example , an auditory and a visual event presented at some asynchrony , the task is to report whether the auditory or the visual event is perceived first .", "The location parameter of the psychometric fit for the proportion of ‘auditory perceived first’ responses as a function of the asynchrony provides a measure of the bias .", "The asymmetric task is the simultaneity judgment , in which is necessary to report whether the auditory and the visual event were perceived simultaneously .", "In this case , the bias is estimated as the asynchrony for which the proportion of simultaneous responses is maximum .", "It has been shown that the biases estimated from these two tasks do not agree ( Love et al . , 2013; Linares and Holcombe , 2014; van Eijk et al . , 2008 ) , which suggests that decision making for time perception might be more affected by decisional biases ( Linares and Holcombe , 2014; Shore et al . , 2001; Schneider and Bavelier , 2003 ) ." ], [ "The study was approved by the ethical committee of the University of Barcelona and followed the requirements of the Helsinki convention .", "The participants , who did not know the hypothesis of the experiments , provided written consent to perform the experiments .", "Twenty-one participants were recruited for Experiment 1 and sixteen for Experiment 2 .", "Stimuli were generated using PsychoPy ( Peirce , 2007 ) , displayed on a Sony G520 CRT screen ( 40 cm width and 30 cm height; 60 Hz refresh rate ) and viewed from a distance of 57 cm in a dark room .", "They consisted in a Gabor patch ( standard deviation ( sd ) of the Gaussian envelope: 1 . 33 degrees of visual angle ( dva ) ; maximum luminance: 79 cd/m2 ) and a red Gaussian blob ( sd: 0 . 1 dva; maximum luminance: 19 cd/m2 ) on top of it that participants were asked to fixate during the experiment .", "Stimuli were presented against a uniform circular grey background ( diameter: 25 dva; luminance: 43 cd/m2 ) that was displayed in a black background ( luminance: 0 . 2 cd/m2 ) .", "The verticality of the Gabor was calibrated using a pendulum .", "Before the experiments , to facilitate the understanding of the instructions , a reference ( a green gaussian luminance profile; sd: 0 . 1 dva; maximum luminance: 29 cd/m2 ) was displayed for 5 to 10 trials at the corresponding cardinal location at a distance of 6 dva from the center of the fixation point .", "The data and the code to do the statistical analysis and create the figures is available at https://github . com/danilinares/2018LinaresAguilarLopezmoliner ( Linares , 2019; copy archived at https://github . com/elifesciences-publications/2018LinaresAguilarLopezmoliner ) .", "The model fitting , goodness of fit and model selection ( likelihood ratio test and Akaike information criterion ) was performed using the R package quickspy ( Linares and López-Moliner , 2016 ) , which under the development version allows fitting several psychometric functions conjointly ." ] ]

[ "The contribution of sensory and decisional processes to perceptual decision making is still unclear , even in simple perceptual tasks .", "When decision makers need to select an action from a set of balanced alternatives , any tendency to choose one alternative more often—choice bias—is consistent with a bias in the sensory evidence , but also with a preference to select that alternative independently of the sensory evidence .", "To decouple sensory from decisional biases , here we asked humans to perform a simple perceptual discrimination task with two symmetric alternatives under two different task instructions .", "The instructions varied the response mapping between perception and the category of the alternatives .", "We found that from 32 participants , 30 exhibited sensory biases and 15 decisional biases .", "The decisional biases were consistent with a criterion change in a simple signal detection theory model .", "Perceptual decision making , thus , even in simple scenarios , is affected by sensory and decisional choice biases ." ]

[ "Imagine that every day , you split a chocolate bar into two and offer one half to your friend .", "Even though you take care to divide the bar into equal pieces , your friend nearly always chooses the left half .", "Why is that ?", "One possibility is that sensory bias in her visual system makes her perceive the left half of the bar to be larger than the right .", "But it is also possible that she does not see any difference between the two halves .", "Instead she simply decides to pick the left half because she prefers doing so .", "The above example illustrates a key problem in studying perception .", "When asked to make a decision where there is no obviously correct answer such as deciding whether a painting is hanging perfectly straight people typically respond one way more often than the other .", "But does this response bias reflect biased perception or biased decision making ?", "Linares et al . have designed an experiment to tease apart these alternatives .", "Healthy volunteers had to decide whether gratings were tilted slightly upward or slightly downward .", "Almost all volunteers showed biases in their choice behavior in one of the two directions .", "To decouple sensory biases from ‘decisional’ biases , the volunteers had to press a particular key to select ‘upward’ on some trials , but ‘downward’ on others .", "This would not affect responding if the volunteers showed a decisional bias to press a key .", "But it would affect responding if the volunteers showed a sensory bias .", "The results revealed that both sensory and decisional biases influenced the volunteers’ choice behavior .", "However , sensory biases were more common .", "People diagnosed with psychiatric disorders like schizophrenia often respond differently on perceptual tasks compared to healthy volunteers .", "Future studies should investigate whether this difference results from altered perception or altered decision making .", "This information could help narrow down the neural circuits affected by these disorders ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "cell biology" ]

[ [ "Regulation of protein abundance within a cell is of fundamental importance to homeostasis , cell identity and responsiveness to changes in external environment .", "Many layers of regulatory control have evolved to fine-tune gene expression to meet cellular needs , including transcriptional regulation , cis-acting genomic elements , epigenetic structures , and translational efficiency .", "Selective control of mRNA translation at the level of the ribosome is emerging as important for protein dynamics within the cell; however , the regulatory mechanisms remain unclear ( Sonenberg and Hinnebusch , 2009 ) .", "Protein synthesis is the most energetically-demanding process for a cell to perform and , therefore , mRNA translation is closely coupled to mitochondrial function ( Morita et al . , 2013 ) .", "Mitochondria are fundamental cellular components of eukaryotes , generating the majority of cellular ATP .", "The human mitochondrial genome contains only 37 genes , of which 13 are subunits of respiratory complexes , 22 encode mitochondrial tRNAs , and a further two encode rRNA .", "Whilst the mitochondrion translates the 13 protein-coding genes of its own genome using a bespoke mitochondrial apparatus , the majority of the genes required for a functional mitochondrion are encoded in the cell nuclear genome and are , therefore , dependent on the cytosolic translational apparatus .", "This relationship implies a fundamental role for the eukaryotic 80S ribosome in control of mitochondrial abundance and function .", "Indeed , mitochondrial content and function varies between tissues and cell-types , as well as within a tissue , in response to external and internal stimuli including energy demand , oxidative stress and cellular signals ( Palmer et al . , 2011 ) .", "For example , red blood cells contain no mitochondria whereas in cardiomyocytes mitochondria occupy 30% of cell volume to meet the exceptionally high ATP demand ( Piquereau et al . , 2013 ) .", "Mitochondrial dysfunction has been linked to a wide range of cardiac disorders , often due to the aberrant production of ROS , and attendant cell death ( Kanaan and Harper , 2017; Ott et al . , 2007 ) .", "BUD23 ( previously known as WBSCR22 ) was initially identified as a putative methyltransferase implicated in tumour metastases ( Nakazawa et al . , 2011 ) .", "Its expression is responsive to diverse inflammatory and cancer pathologies ( Jangani et al . , 2014 ) , and reduction of BUD23 expression can affect cellular response to glucocorticoid and alter histone methylation ( Jangani et al . , 2014 ) .", "However , BUD23 actions were diverse , and a unifying mechanism of action was elusive .", "More recent studies identified ribosomal RNA as the preferred substrate of BUD23 ( Haag et al . , 2015; White et al . , 2008; Zorbas et al . , 2015 ) .", "There is little understanding of the physiological role of BUD23 in a mammalian context although it is one of the genes deleted in Williams-Beuren syndrome ( WBS ) .", "WBS patients have a complex phenotype with prominent neurological and morphological features .", "In addition metabolic pathologies exist including diabetes and obesity ( Morris , 1993 ) .", "The contribution of individual genes within the 22-gene interval to the human phenotype has not been determined , but the neurological and metabolic features suggest a bioenergetic contribution .", "BUD23 is a ribosomal RNA methyltransferase which imparts a methyl mark on a key guanosine residue ( forming N7-methylguanosine ) located between the E and P site of the small ribosomal subunit that has been mapped to residue G1575 of yeast 18S rRNA and G1639 of human 18S rRNA ( Haag et al . , 2015; Õunap et al . , 2013; White et al . , 2008; Zorbas et al . , 2015 ) .", "BUD23 protein is found in both the nucleus and the cytoplasm ( Õunap et al . , 2015 ) , and its depletion leads to a nuclear accumulation of 18SE-pre-RNA ( Haag et al . , 2015 ) .", "In both yeast and human cells the methyltransferase catalytic activity of BUD23 is not required for the processing of 18S pre-RNA , or the synthesis of 40S subunits , indicating that BUD23 has an additional role , distinct from its methyltransferase activity ( Haag et al . , 2015; Zorbas et al . , 2015 ) .", "In human cell lines it has been suggested that some 18S rRNA lacks the m7-G1639 mark , which may indicate a selective role in ribosome function ( Haag et al . , 2015 ) .", "BUD23 stability requires interaction with TRMT112 ( TRM112 in yeast ) , an obligate binding partner , which is known to stabilise four client methyltransferase enzymes , all involved in the generation of the translational apparatus ( Bourgeois et al . , 2017; Létoquart et al . , 2014 ) .", "The binding of TRMT112 with these four methyltransferases is competitive , which results in tight regulation of BUD23 protein concentration and enzymatic function at the level of protein stability .", "Here we identify a novel mechanism linking the energy-demanding process of protein translation to mitochondrial dynamics through BUD23 .", "We examine the role of BUD23 in ribosome function and discover that BUD23 preferentially promotes the selection of mRNA species with low GC-content 5’UTRs .", "We also identify a role for BUD23 in mitochondrial transcript translation which impacts mitochondrial function in vitro .", "We go on to examine the role of BUD23 in a murine in vivo system and discover that the production of mitochondrial proteins is dependent on BUD23 .", "Finally , we examine the role of BUD23 in the mitochondrially-rich and energetically-demanding cardiac tissue and discover a cardiomyopathy phenotype leading to premature death .", "These discoveries identify BUD23 as essential for mammalian mitochondrial function , with implications for human mitochondrial disease and cardiomyopathy ." ], [ "We previously identified pleiotropic actions of BUD23 in airway epithelial cells , and attributed these to an epigenetic effect ( Jangani et al . , 2014 ) .", "The discovery that BUD23 modifies ribosomal RNA rather than histone proteins prompted re-examination of the BUD23 role in physiology .", "To identify candidate pathways affected by BUD23 we depleted expression in human airway epithelial cells ( Figure 1A , B ) .", "This intervention caused a small but statistically significant decrease in cell proliferation ( Figure 1C ) , which is similar to the anti-proliferative effects caused by BUD23 deletion in yeast ( White et al . , 2008 ) .", "To investigate comprehensively the role of BUD23 in ribosome function we performed polysome profiling .", "The 48 hr knockdown of BUD23 resulted in a reduction of the 40S subunit peak , and a concomitant increase in the 60/80S peak ( Figure 1D , E ) .", "There was , however , little change in the profile of the polysome fractions at this early time-point following transient BUD23 knockdown , allowing us to investigate the changes in mRNA substrate selection in the intact polysomes .", "To test the global efficiency of the translational apparatus in cells lacking BUD23 , we used 35-S-methionine incorporation for 1 hr ( Figure 1—figure supplement 1 ) .", "This demonstrated little overall impact on global protein translation rate , confirming that the identified polysomes were essentially competent at this time-point , despite the impact on 40S maturation .", "In order to determine if BUD23-loss differentially impacted the translation of a specific subset of mRNAs , we profiled the translational efficiency ( TE ) of individual mRNA species within the polysome profiles .", "To do this , the polysome fractions were divided into ‘heavy’ ( more than three ribosomes ) or ‘light’ ( less than three ribosomes ) and pooled , prior to RNA extraction and sequencing .", "This defined a total of 14 , 527 transcripts , from which the relative proportion of each individual transcript in the heavy compared to the light fraction was calculated to give a translation efficiency ( TE ) score .", "Average TE scores across the three replicates for each condition ( + /- BUD23 ) were then plotted against each other ( Figure 1F ) .", "This revealed that the most efficiently translated transcripts in the control samples ( more heavily loaded with ribosomes ) , were most significantly reduced in the BUD23 knockdown condition .", "To examine the identity of the most highly affected transcripts , a threshold of >1 or <-1 difference in the relative TE was set ( i . e . TE doubled or halved ) , and applied to Figure 1F , shown as trend lines .", "There were 650 transcripts with a TE ratio <-1 and 95 transcripts where the TE ratio >1 after BUD23 depletion; indicating the marked loss of high efficiency mRNA translation .", "The 650 transcripts for which TE was reduced ( TE ratio <-1 after BUD23 depletion ) constituted a highly connected network , with a significantly higher number of protein-protein interactions than would be expected from a random dataset of similar size , and an interaction analysis ( PPI ) enrichment p-value<1e−16 .", "Gene set analysis of these transcripts revealed enrichment for genes involved in RNA processing , metabolic processes and organelle organisation among other high energy demand processes ( Figure 1—figure supplement 1 ) .", "Surprisingly , the TE of mitochondrially-encoded transcripts was observed to be particularly strongly down-regulated ( Figure 1G ) .", "In fact , all mitochondrial transcripts had a negative change in TE after BUD23 loss , with approximately 50% reduction in TE .", "To investigate the mechanism underlying the BUD23 dependent selection of transcripts we examined specific mRNA features associated with translational efficiency .", "Given the known influence of 5’UTR features on translation efficiency , we reasoned that specific features of the 5’UTR may be particularly important for BUD23 action .", "A number of well-characterised 5’UTR features impact mRNA translation , including length , secondary structure , GC content , and specific motifs including the TOP sequence ( Gandin et al . , 2016; Meijer and Thomas , 2002 ) .", "We saw no enrichment for TOP sequence content in the differentially translated mRNA species ( Figure 1—figure supplement 2 ) , and no correlation between change in TE score and 5’UTR length was observed ( Figure 1H ) .", "We also noted that transcripts with shorter total lengths were not more likely to shift from the heavy to the light fraction ( Figure 1—figure supplement 3 ) .", "There was , however , a highly significant correlation between change in TE score and GC content ( Pearson’s correlation value of −0 . 1337 , p-value<2 . 2e−16 ) ( Figure 1I ) .", "This identifies an intersection between mRNA transcript 5’UTR GC content and BUD23 ribosome maturation .", "There was no difference in the distribution of 5’UTR GC in mitochondrial-related transcripts relative to all transcripts detected in the polysome analysis ( Figure 1—figure supplement 3 ) .", "Our studies reveal the consequences for the ribosome of BUD23 action and point to an impact on the cellular proteome .", "Therefore , to investigate the downstream consequences of BUD23 loss we examined the cellular proteome by LC-MS/MS .", "BUD23 knockdown downregulated 83 proteins and upregulated 64 , out of a total of 3255 identified ( Figure 2—source data 1 ) .", "When tested using PANTHER GO cellular component ontology analysis , upregulated proteins were over-represented for components of the large ribosomal subunit ( 14/64 proteins ) , whilst downregulated proteins were over-represented for components of the small ribosomal subunit ( 22/83 proteins ) , ( Figure 2A , Figure 2—figure supplement 1 ) .", "This imbalance in the relative abundance of ribosomal proteins , recapitulates the observation in the polysome profiles and confirms a major role for BUD23 in the maturation of the protein translation apparatus of the cell .", "Interestingly , TRMT112 protein , an obligate partner for BUD23 , was also significantly down-regulated with BUD23 knock-down , which may indicate a reciprocal stabilising interaction .", "Ontology analysis of the proteomics dataset was used to predict the functional consequences of BUD23 reduction ( PANTHER GO biological processes ) .", "Over-representation analysis of the up-regulated proteins overlapped significantly with the ‘protein translation’ .", "Analysis of the down-regulated proteins again showed a statistically significant over-representation for proteins associated with GO biological terms including ‘translation’ , as well as ‘rRNA metabolic process’ and ‘protein metabolic process’ which support a major effect on the translational apparatus ( Figure 2B ) .", "Unexpectedly , there was also over-representation within the down-regulated proteins for the terms ‘Mitochondrion organization’ and ‘Cellular component biogenesis’ , suggesting a consequential impact on mitochondrial function , a major destination for new protein synthesis in the cell .", "Because the TE of mitochondrial transcripts was down-regulated and mitochondrial organisation emerged from the proteome analysis , specific assays of mitochondrial activity were performed .", "Depletion of BUD23 with any of three , independent siRNAs resulted in a > 50% reduction in oxidative phosphorylation ( OXPHOS ) and around a 25% reduction in Electron Transfer Capacity ( ETC ) ( Figure 2C , D , E ) but no observable difference in leak respiration ( LEAK ) , relative to control ( Figure 2—figure supplement 2 ) .", "Moreover , no alteration in glycolytic capacity was observed indicating specificity for mitochondrial energetic pathways ( Figure 2F ) .", "We also observed a reduction in mitochondrial mRNA abundance with BUD23 knockdown , but no change in mitochondrial genome copy number , implying a functional defect , rather than loss of mitochondrial mass ( Figure 2G , H , I ) , although these measurements were performed soon after siRNA knockdown , and so a later impact on mitochondrial mass resulting from prolonged BUD23 loss cannot be excluded .", "To examine whether this mitochondrial transcription defect was specific to BUD23 deficiency , or part of a more general mechanism resulting from 40S/60S imbalance , we performed siRNA-mediated knockdowns of other known ribosome biogenesis factors , LTV1 and RIOK2 , as well as the ribosomal small subunit protein RPS27A ( also reported to result in ribosomal subunit imbalance ) ( Sloan et al . , 2019 ) .", "Depletion of LTV1 , RIOK2 and RPS27A all resulted in significant decrease in 18S/28S ratio relative to control siRNA , indicating a similar 40S/60S imbalance as observed with BUD23 knockdown ( Figure 2G , Figure 2—figure supplement 2 ) .", "LTV1 knockdown resulted in a similar decrease in mitochondrial transcript expression as observed in BUD23 knockdown ( Figure 2H ) .", "However , RPS27A and RIOK2 knockdown-induced ribosome imbalance did not affect mitochondrial transcript expression ( Figure 2H , Figure 2—figure supplement 2 ) .", "This indicates that disruption of 40S subunit biogenesis is not by itself sufficient to result in mitochondrial dysfunction .", "To check that BUD23 was not regulating mitochondria directly , we blotted for the protein after sub-cellular fractionation .", "BUD23 was found to be abundant in the cytosolic fraction but was not detected in the mitochondrial fraction after proteinase K digestion ( Figure 2J ) .", "This suggests the mechanism of BUD23 action does not occur within the mitochondria .", "Furthermore , TRMT112 , the obligate BUD23 binding partner , was also abundant in the cytosol , but barely detectable within mitochondria ( Figure 2J ) .", "Together these data indicate that BUD23-dependent mitochondrial regulation is likely to be a downstream consequence of effects on ribosome composition , function , and cellular protein repertoire , rather than a direct effect of BUD23 protein within the mitochondria .", "To investigate the physiological impact of BUD23 we generated Bud23 null mice .", "We introduced a frameshift deletion within the Bud23 gene , which resulted in a null allele ( Figure 3A ) .", "However , upon further breeding of heterozygous mice a clear deviation from the expected Mendelian ratio was observed in the resultant offspring .", "Out of 74 pups born , 39 were found to be wild-type for the Bud23 gene , 35 were heterozygous , and zero were homozygous for the null allele ( Figure 3B ) , indicating embryonic lethality in Bud23-null mice .", "This is in contrast to reports in yeast where BUD23 was shown to be non-essential for life ( White et al . , 2008 ) .", "Furthermore , it is notable that approximately half the expected heterozygote birth rate was seen , implying an additional haplo-insufficiency developmental death rate .", "Accordingly , we analysed embryos at day E10 . 5 and detected Mendelian ratios of wild-type , and heterozygous animals , but again no homozygous null embryos were seen ( Figure 3B ) , suggesting total Bud23 loss is incompatible with embryogenesis , but haploinsufficiency results in fetal death later in development .", "Surprisingly , surviving adult heterozygous mice showed no difference compared to wildtype littermate controls in body weight , lean body weight or body fat between 10 and 30 weeks of age ( Figure 3C ) .", "There was also no significant change in energy expenditure or respiratory exchange ratio in Bud23+/- mice relative to WT littermate controls ( Figure 3—figure supplement 1 ) , suggesting compensatory mechanisms to permit survival .", "To circumvent embryonic lethality , we generated a floxed Bud23 allele mouse , allowing post-natal tissue-specific KO ( Figure 3A ) .", "Mice homozygous for this floxed Bud23 ( Bud23fl/fl ) allele showed no observable differences from wild-type littermates .", "To confirm the embryonic lethality phenotype using an independent genetic approach , we crossed these mice with a Cre-deleter mouse line ( Hprt-cre ) ( Nichol et al . , 2011 ) to globally delete Bud23 alleles .", "Bud23+/- heterozygous crosses also failed to generate any homozygous null offspring ( 0 out of 28 cre positive offspring ) , confirming the observation in the global knockout mouse line ( Figure 3D ) .", "We targeted Bud23 disruption to skeletal and cardiac muscle using Muscle Creatine Kinase ( Mck ) -cre as the driver ( Whitnall et al . , 2008 ) .", "Cardiac muscle relies heavily on mitochondrial production of ATP in post-natal life , but less so during development , so offering us the chance to analyse BUD23 impact on a highly mitochondrially-dependent tissue .", "Bud23fl/fl Mck-Cre+/- mice exhibited Cre-mediated deletion of exon 7 of the Bud23 gene in cardiac tissue , with a consequent reduction in BUD23 protein levels ( Figure 3E , F ) .", "Loss of BUD23 in cardiac muscle resulted in sudden death between the age of 28–35 days ( Figure 4A ) .", "Littermate control mice expressing Bud23fl/flMck-Cre-/- or Bud23fl/wt Mck-Cre+/- ( i . e . mice retaining at least one functional copy of the Bud23 allele ) were viable , fertile and did not die prematurely ( Figure 4A ) .", "Mouse tissues from this line were therefore subsequently harvested at 26 days of age .", "A significant reduction in the 18S/28S RNA ratio , consistent with the in vitro data using siRNA knockdown was observed at this time point ( Figure 4B ) .", "Proteomic analysis of cardiac tissue recovered from 26 day old Bud23fl/flMck-Cre+/- mice and littermate controls ( Bud23fl/flMck-Cre-/- ) revealed a significant decrease in protein content per cell with loss of BUD23 ( Figure 4C ) , with clear separation by genotype ( Figure 4—figure supplement 1 ) .", "When detected proteins were annotated by sub-cellular compartment , mitochondria were found to be subject to the largest loss of protein content , with small reciprocal increases in the other compartments ( Figure 4D ) , again identifying mitochondria as especially sensitive to BUD23 action .", "Of the 2047 proteins identified by mass spectroscopy , 347 were found to be significantly up-regulated and 442 were found to be significantly down-regulated in the BUD23 deficient condition ( Figure 4E , Figure 4—source data 1 ) .", "Whilst an approximately equivalent number of down and up regulated proteins was identified globally , when this list was refined to mitochondrial proteins only 20 were up-regulated compared to 220 down-regulated ( 50% of all down-regulated proteins ) ( Figure 4F ) .", "By contrast , the up-regulated dataset was enriched for translational ( 60S proteins ) and proteasomal proteins ( Figure 4—figure supplement 1 ) .", "Whilst correlation between change in protein abundance in BUD23-deficient heart tissue and change in mRNA TE of the corresponding transcripts in the BUD23 siRNA treated A549 cells was poor overall ( Figure 4—figure supplement 2 ) , it is striking that mitochondria are affected to a great extent in both models .", "These observations show a strong selectivity for impaired expression of mitochondrial proteins as a result of BUD23 deficiency .", "Within the set of mitochondrial proteins , we found that electron transport chain complexes I , IV and V ( ATP synthase ) were most strongly reduced in response to loss of BUD23 ( Figure 4—figure supplement 3 ) .", "Furthermore , ontology analysis of all the significantly down-regulated proteins identified three main clusters of proteins: mitochondrial proteins , mitochondrial ribosomal proteins and proteins involved in energy metabolism , indicating a functional mechanism linking between the BUD23 dependent ribosomal defect , through reduced mitochondrial protein expression to result in disruption of energy metabolic pathways ( Figure 4G ) .", "Therefore , BUD23 actions on the ribosome result in down-regulation of core mitochondrial proteins involved in ATP synthesis .", "Given the observation of preferential dysregulation of mitochondrial proteins , we hypothesised that mitochondrial function was likely to be compromised in BUD23-deficient cardiac tissue .", "Mitochondrial DNA copy number was reduced in BUD23 deficient cardiac tissue ( Figure 5A ) .", "Functional analysis of mitochondrial function in cardiac homogenates using the Oroboros microrespirometer system revealed that mitochondrial OXPHOS and ETC were both significantly reduced in mice deficient for BUD23 , while LEAK respiration was unaffected ( Figure 5B ) .", "The reduction in OXPHOS led to a lower respiratory control ratio ( RCR ) indicating that mitochondria from mice deficient for BUD23 were less efficient at producing ATP .", "These effects were apparent regardless of which substrate was used for electron donation .", "In addition to reduced mitochondrial capacity , mice deficient for BUD23 had lower citrate synthase activity , indicating a reduction in cardiac mitochondrial density ( Figure 5C ) , which accords with the measured loss of mitochondrial genome copy number ( Figure 5A ) .", "To test whether haplo-insufficiency of BUD23 protein was enough to impair mitochondrial function we also tested heart homogenates from Bud23+/- mice .", "These were found to have no significant difference in mitochondrial respiratory capacity across any mitochondrial state ( LEAK , OXPHOS , ETS ) , whether normalised to citrate synthase activity or protein content ( Figure 5—figure supplement 1 ) .", "It was , however , noted that , compared to wild-types , Bud23+/- mice did exhibit significantly higher citrate synthase activities , indicating greater mitochondrial density .", "This may result from increased mitochondrial biogenesis to compensate for defective oxidative phosphorylation function and may partly explain the reduced viability in Bud23+/- mice .", "Interestingly , the impact of BUD23 loss on mitochondrial function appears to be sexually-dimorphic .", "When mitochondrial respiration was normalised to citrate synthase , male heterozygous ( HZ ) mice exhibited a deficiency in OXPHOS and ETS relative to wildtype , whereas female mice did not ( Figure 5—figure supplement 1 ) .", "This observation indicates a more severe phenotype in BUD23 deficient males , with further derangement of individual components of the electron transport chain .", "Electron microscopy ( EM ) analysis revealed that despite decreased mitochondrial protein abundance and reduced mitochondrial function in the BUD23 cardiac tissue , the individual mitochondria appear to be formed normally with an equivalent number of cristae compared to the wildtype .", "The intermyofibrillar mitochondria were typically arranged in a highly ordered pattern in the wildtype cardiac tissue , however , there was marked disorganisation in the BUD23-deficient cardiac tissue ( Figure 5D ) .", "In addition , we identified the presence of numerous spherical electron dense inclusion bodies within the mitochondria of BUD23 deficient cardiac cells ( Figure 5D , yellow arrows ) .", "Similar electron dense structures have been previously reported in myocardial mitochondria , which may accumulate in response to increased cellular bioenergetic demand ( Jacob et al . , 1994 ) .", "The observation of these structures here may be consistent with attempted mitochondrial adaptation to energetic-demand challenge .", "A frequent cellular adaptation to impaired oxidative phosphorylation is a switch to glycolytic ATP generation , and so we measured rate-limiting glycolytic gene expression ( Figure 5E ) .", "We identified a significant induction of glucokinase ( GCK ) , and a trend to induction of phosphofructokinase ( PKM ) and hexokinase ( HK2 ) supporting such an adaptive switch to glycolysis .", "A further adaptation predicted was an induction in mitochondrial biogenesis programmes .", "We identified a significant induction in PGC1a and PGC1b , but no change in TFAM1 expression ( Figure 5E ) .", "Taken together these results point to some of the expected cellular responses to loss of oxidative phosphorylation , but it was surprising that the changes were so few .", "This implies that additional cellular injury may be proceeding , which acts to limit successful adaptation .", "Impaired mitochondrial function may result in overproduction of reactive oxygen species , which can trigger the apoptotic cascade and lead to cell death .", "Such a progression may explain the distorted and thinned ventricular walls observed in the cardiac Bud23-null mice .", "We found induction of NRF2 ( Figure 5E ) , a key sentinel gene controlling cellular responses to reactive oxygen species stress , which led us to measure reactive oxygen species directly , and found that these were significantly higher in hearts lacking BUD23 expression ( Figure 5F ) , identifying a burden of increased reactive oxygen species in BUD23 deficient cardiomyocytes resulting from the action of deranged mitochondria .", "Deficient mitochondrial generation of ATP in cardiac tissue is a key cause of cardiomyopathy .", "Given the strong mitochondrial deficiencies observed in BUD23-deficient cardiac tissue , we predicted that the cardiomyopathy was the likely cause of sudden death .", "Histological examination of hearts from Bud23fl/fl Mck-Cre+/- ( BUD23-deficient ) mice showed significant enlargement , ventricle dilatation and decreased ventricle wall thickness compared to littermate controls at 26 days old ( Figure 6A–D ) .", "Littermate control mice expressing Bud23fl/flMck-Cre-/- or Bud23fl/wt Mck-Cre+/- ( i . e . mice retaining at least one functional copy of the Bud23 allele ) were viable , fertile and did not die prematurely ( Figure 3A ) .", "Echocardiogram analysis of Bud23fl/fl Mck-Cre+/-mice at 26–28 days old showed that cardiac BUD23-deficiency resulted in systolic dysfunction indicated by significantly dilated left ventricle ( sD ) , as well as significantly decreased fractional shortening and relative wall thickness ( Figure 6E , Figure 6—source data 1 ) .", ") .", "There was , however , no observed pulmonary oedema or liver oedema ( Figure 6—figure supplement 1 , ) .", "Further analysis of the heart proteomics data from Figure 4 showed an increase in a number of markers of fibrosis , including Col4a2 , CTGF , Galectin-3 and others ( Figure 6—figure supplement 1 ) .", "Taken together , these observations indicate that Bud23fl/fl Mck-Cre+/- mice exhibit the early stages of heart failure around 26–28 days of age .", "Electrocardiogram analysis of the same mice at the same age revealed significantly shorter QRS and corrected QT ( QTc ) intervals but no change in heart rate ( Figure 6F; Figure 6—source data 1 ) .", ") ." ], [ "BUD23 is tightly conserved through evolution and has recently emerged as a ribosomal RNA methyltransferase , although its physiological role in mammals is unexplored .", "It is one of the genes deleted in Williams-Beuren Syndrome , a complex multi-gene deletion syndrome with neurological , and energy homeostatic features , although themediating role of individual genes to the phenotype has not been determined ( Morris , 1993 ) .", "We now identify BUD23 as playing a major role in regulating the translational efficiency of specific transcripts through its role in ribosome maturation .", "Indeed , translation of low 5’UTR GC-content mRNA species were particularly dependent on BUD23 expression , in contrast to the picture seen with simple ribosome deficiency in which transcripts with short/unstructured 5’UTRs were seen to be most affected ( Khajuria et al . , 2018 ) .", "Furthermore , we identified a surprising role for BUD23 in maintaining mitochondrial oxidative phosphorylation capacity .", "As protein translation is tightly coupled to ATP demand this raised the possibility that BUD23-dependent ribosomal maturation is critical to link the two processes in living cells and tissues ( Morita et al . , 2013 ) .", "Indeed , BUD23 loss greatly impaired mitochondrial ATP generation , both in vitro and in vivo .", "In mice , cardiomyocyte loss of BUD23 greatly impaired mitochondrial ATP generation leading to dilated cardiomyopathy and premature death , and global loss of BUD23 resulted in embryo-lethality .", "BUD23 is thought to perform two functions in the generation of the translational apparatus: firstly , in the processing of pre-18S RNA into its mature form , and secondly imparting a m7-G methyl mark on a key residue .", "As the catalytic activity of BUD23 is not required for efficient pre-18S RNA processing , these two functions appear to be independent of each other ( Haag et al . , 2015; Zorbas et al . , 2015 ) .", "It is notable that both the BUD23 imparted m7-G methyl mark and structurally complex 5’UTRs are characteristic features of translation specific to Eukaryotes .", "We speculate the co-evolution of this regulatory 18S methyl mark with the need for more complex regulation of differential translation .", "Further work should focus on delineating the effect of BUD23-dependent 18S maturation , from the role of the methylation mark .", "We used proteomics to examine the relative changes in protein abundance which result from targeted loss of BUD23 in cardiomyocytes .", "This revealed a selective loss of mitochondrial proteins , explaining the observed reduction in mitochondrial capacity .", "As BUD23 appears to have no direct role within mitochondria we propose that the mitochondrial phenotype results from impaired translation of nuclear-encoded genes with a role in mitochondrial homeostasis .", "Indeed , we found that the translational efficiency of a number of mitochondria-targeted as well as mitochondrial regulating proteins were BUD23-dependent .", "There are many potential candidates linking the change of translational efficiency to the mitochondrial phenotype .", "Mammalian target of rapamycin ( mTOR ) , for example , is known to regulate mitochondrial activity and biogenesis by regulation of translation ( Morita et al . , 2013 ) and , interestingly , BUD23 loss resulted in impaired translation of mTOR itself .", "We also observed a marked decrease in the translational efficiency of mitochondrially-encoded transcripts .", "We initially generated and characterised a frame shift-generating null allele mouse .", "The homozygote null animals were all lost prior to embryonic day e10 . 5 and we were unable to identify a cause but considered that a major defect in a mitochondrially–dependent organ such as heart may be a plausible mechanism .", "We moved , therefore , to a conditional allele mouse , using which we confirmed the developmental lethality of the null allele by crossing with a global deleter-cre strain ( Hprt-cre ) .", "Using the conditional allele mice with a muscle Cre driver we were able to profile the impact of BUD23 in a physiological role .", "Loss of BUD23 in heart and skeletal muscle resulted in early death from cardiac failure .", "This striking phenotype was accompanied by morphological changes including cardiac dilatation , ventricular wall thinning , and impaired ventricular contractility .", "There was evidence for an attempted switch to glycolytic ATP generation in the hearts , with induction of rate-limiting glycolytic enzyme genes; and also adaptation to conditions of oxidative stress with induction of NRF2 .", "Mitochondrial dysfunction frequently imposes a burden of oxidative stress on affected cells , which can result in apoptosis , thereby explaining the loss of cardiac muscle seen .", "Detailed ultrastructure analysis of cardiac mitochondria revealed no major loss of mitochondrial volume , but there was a striking additional feature seen within the mitochondria: electron-dense , spheroid inclusions .", "These were not observed in control hearts , and are not typically seen in mitochondria from individuals with mitochondrial myopathies , but have been reported before under conditions of extreme ATP demand , and to result from bilayer budding from the inner cristae , to generate additional ATP generating surface area ( Jacob et al . , 1994; Somlyo et al . , 1974 ) .", "Therefore , we propose this may be a further adaptation to the impaired oxidative phosphorylation function within the mitochondria .", "There is a growing recognition that transcriptional control mechanisms are insufficient to explain differentiated cell function .", "Recent advances have identified differential translation of mRNA species dependent on 5’UTR features as an important control mechanism , potentially coupling global changes in protein translation with appropriate upregulation of mitochondrial ATP generation to meet demand 2 .", "BUD23 , a highly conserved ribosomal RNA methyltransferase which lies within a multigene interval , deleted as a cause of Williams-Beuren syndrome , plays a specific role in highly ATP-hungry cells , promoting translation of mitochondrial proteins , and impacting on oxidative phosphorylation .", "This function likely contributes to aspects of the human phenotype , in particular neurological dysfunction , glucose intolerance and obesity .", "Complete global loss of Bud23 is embryonic lethal , and early post-natal death follows deletion of Bud23 in the heart .", "The cardiac phenotype is severe , with marked mitochondrial dysfunction , and evidence of compensatory changes towards glycolytic ATP generation , and adaptation to oxidative stress .", "Therefore , BUD23 plays a critical role in coupling protein translation to mitochondrial function , with implications for mitochondrial diseases , and cardiomyopathies ." ], [ "All mice were routinely housed in 12:12 light/dark ( L:D ) cycles with ad libitum access to food and water .", "All experiments were carried out in accordance with the Animals ( Scientific Procedures ) Act 1986 ( UK ) under Home Office protocol number 70/8768 and P3A97F3D1 .", "In studies utilizing conditional targeted mice , littermate controls ( floxed/floxed ) were used as control; these mice carried no copies of the Cre or iCre .", "Genotyping was performed on all experimental animals .", "To assess body composition , whole body , fat mass and lean body mass was assessed using the EchoMRI system ( Echo Medical Systems ) .", "To assess metabolic gas exchange , mice were individually housed in indirect calorimetry cages ( CLAMS , Columbus Instruments ) .", "Mice were acclimatised to the cages for 48 hr prior to recording data .", "Measurements of O2 consumption and CO2 production were recorded every 10 min for >72 hr .", "RER was derived from these measures ( VCO2/VO2 ) , as was energy expenditure ( 3 . 815∗VO2 + 1 . 232∗VCO2 ) .", "In order to conditionally KO the Bud23 gene by Cre recombinase expression we generated a transgenic mouse line with the critical exon seven flanked by LoxP sites .", "Exon seven has an unequal splicing phase , meaning its Cre mediated excision would lead to a frame shift and KO of the Bud23 gene .", "This exon is also flanked by large introns giving sufficient space to insert LoxP sites without perturbing normal gene splicing and regulation .", "We used CRISPR ( clustered regularly interspaced short palindromic repeats ) -Cas9 in the mouse zygote to integrate these LoxP sites .", "Briefly , sgRNA targeting this genomic region , and with minimal off target potential , were identified ( sgRNA-1 GGCATTGGGCCTACTTAAAG-GGG , sgRNA-2 AGTTGAAGGGTTCCATAATG-AGG , sgRNA-3 CTTTACAGCCCAAGACCACT-TGG ) and selected for use .", "sgRNA was synthesised in vitro , using protocols developed by Bassett et al . ( 2013 ) .", "Forward primers of the form GAAATTAATACGACTCACTATA GGN18-20GTTTTAGAGCTAGAAATAGC ( where GGN18-20 is the sgRNA sequence ) for each guide were combined with the universal reverse primer , AAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC and used in a PCR reaction with high fidelity polymerase ( Phusion , NEB ) .", "200 ng of the resulting amplicon were used overnight in an in vitro transcription reaction ( HiScribe , NEB , as per manufacturer’s instructions ) , before purification ( Megaclear , Ambion ) and quantification by nanodrop .", "A double stranded DNA ( dsDNA ) repair template was designed to", "( i ) incorporate loxP sites , with unique restriction sites for screening purposes , 300 bp upstream and 200 bp downstream of exon seven respectively and", "( ii ) integrate silent shield mutations for all three sgRNA to prevent cutting of the repair template and", "( iii ) contain 1000 bp 5’ and 3’ homology arms to the target region .", "The above template was synthesised in a pUC57 vector ( Genscript , US ) , the linear 5’homology-loxP-exon7-loxP-3’homology fragment excised from the vector by restriction digest , gel extracted ( Bioline ) and further cleaned prior to microinjection ( Monarch PCR purification kit , NEB ) .", "An injection mix of the three sgRNA ( 40 ng/ul each ) , Cas9 mRNA ( 100 ng/ul ) and the dsDNA repair template ( 10 ng/ul ) was prepared and directly microinjected into B6D2F1 ( Envigo ) zygote pronuclei using standard protocols .", "Zygotes were cultured overnight and the resulting 2 cell embryos surgically implanted into the oviduct of day 0 . 5 post-coitum pseudopregnant mice .", "Potential founder mice were screened by PCR and digest .", "Animals positive for both 5’ and 3’ LoxP integration had the region fully verified by pCR-Blunt cloning followed by Sanger sequencing .", "two founder mice were identified and then back-crossed to C57BL/6J wild-type mice to assess germline penetrance .", "Additionally , the described CRISPR targeting strategy resulted in deletion of critical exon 7 , a global Bud23 KO line was generated from this founder animal .", "DNA was extracted from ear snip or tail tip using REDExtract-N-Amp tissue PCR kit ( Sigma ) .", "Primer sequences and PCR reaction conditions are listed in the key resources table and the primer sequences supplementary file .", "PCR products were resolved on 1 . 5% agarose gels .", "STR authenticated A549 cells were obtained from the European Collection of Cell Cultures ( ECACC Cat no: 86012804 ) and maintained in culture , incubated at 37°C in humidified air , 5% v/v CO2 .", "Cultured cells were tested regularly for mycoplasma contamination .", "A549 cells were cultured in DMEM growth medium ( Sigma , D6546 ) , supplemented with 2 mM L-Glutamine ( Sigma , G7513 ) , 10% foetal calf serum ( FCS ) ( Sigma , F96665 ) .", "A549 cells were plated at 1 × 10^6 cells in a 10 cm cell culture dish and transfected with BUD23 specific or non-targeting control siRNA ( S41529 , S41530 , S41531 , negative control 1 , negative control 2 , Ambion Silencer Select ) using Dharmafect DF-1 according to the manufacturer’s guidelines .", "A549 cell pellets were lysed in M-PER mammalian protein extraction reagent ( Thermo Scientific ) .", "Whole mouse hearts were dissected , quartered and washed in ice cold PBS to remove blood before disruption of the tissue in 1x TBS supplemented with protease inhibitor cocktail using a TissueRuptor ( Qiagen ) .", "SDS was added to a final concentration of 4% ( w/v ) prior to sonication .", "Samples were then reduced with 100 mM DTT and heated for 3 min at 95°C .", "Samples were prepared for label-free quantification in accordance with published protocols ( Hernandez-Valladares et al . , 2016 ) .", "Digested samples were analysed by LC-MS/MS using an UltiMate 3000 Rapid Separation LC ( RSLC , Dionex Corporation , Sunnyvale , CA ) coupled to a Q Exactive HF ( Thermo Fisher Scientific , Waltham , MA ) mass spectrometer using a stepped normalized collision energy centered at 28 .", "A549 peptide mixtures were separated using a multistep gradient from 95% A ( 0 . 1% FA in water ) and 5% B ( 0 . 1% FA in acetonitrile ) to 7% B at 1 min , 18% B at 58 min , 27% B in 72 min and 60% B at 74 min at 300 nL min−1 , using a 75 mm x 250 μm i . d . 1 . 7 μM CSH M-Class C18 , analytical column ( Waters ) , for a final run time of 90 min .", "Cardiac samples were separated using a gradient from 5% B to 7% B at 1 min , 18% B at 81 . 5 min , 27% B at 102 min and 60% B at 104 min , with a final run time of 120 min .", "The top 12 precursors were selected for fragmentation automatically by data dependant analysis during each cycle .", "Ionization potential was set at 1900V with a survey scan window of 300–1750 m/z .", "MS1 used a resolution of 120 , 000 with a target ion intensity of 3e6 .", "Higher-energy collisional dissociation was used .", "MS2 was set at a resolution of 60 , 000 with a target ion intensity of 2e5 .", "Maximum injection times for MS1 and MS2 were 20 ms and 110 ms respectively .", "Peptides were dynamically excluded for 15 s after one occurrence .", "Mass spectra were analysed using MaxQuant version 1 . 6 . 0 . 16 ( Cox and Mann , 2008 ) , searching against either the Uniprot Mus musculus database ( accessed 21/12/2017 ) or Homo sapiens database ( accessed 12/03/2017 ) using the native Andromeda search engine .", "Match between runs was selected with a retention time of 1 min , using default parameters for all other settings .", "MS1 search tolerance was set at 20ppm , and MS2 at 4 . 5ppm .", "Modifications were set as Carbamidomethyl ( C ) for fixed , and Oxidation ( M ) and Acetyl ( Protein N-term ) variable .", "A minimum of 1 spectra was required for identification , while a minimum of 2 spectra were required for quantification .", "Resulting data was processed using Perseus version 1 . 6 . 0 . 7 ( Tyanova et al . , 2016 ) and R statistical software ( Wickham , 2009; R Development Core Team , 2017 ) .", "All proteins identified by site only , as a potential contaminant , or in the decoy reverse database were excluded .", "Data was then filtered to exclude proteins with more than 3 ‘zero-values’ across all groups .", "All proteins assigned to the GO term ‘blood microparticle’ were removed as well .", "LFQ values were log ( 2 ) transformed and missing values imputated in Perseus using a normal distribution ( Lazar et al . , 2016 ) .", "The width of the new distribution was 0 . 3 standard deviations of the data , and was shifted downwards by 1 . 8 standard deviations of the data .", "A total of 6 . 68% of values were imputated .", "Significance was determined using a T-test with a permutation based FDR ( FDR < 0 . 05 , s0 = 0 . 1 ) .", "s0 represents the relative importance of the difference between the means , with non-zero s0 values taking fold change , and not only p-value , into account .", "Estimations of copy numbers were performed using the proteomic ruler plug-in version 0 . 1 . 6 ( Wiśniewski et al . , 2014 ) .", "Proteins were then mapped to subcellular localisations to determine percentage protein mass by organelle according to published protocols using the HeLa spatial proteome database ( Doll et al . , 2017; Itzhak et al . , 2016 ) .", "Interaction networks were built using String 10 online software ( Szklarczyk et al . , 2017 ) using seven data sources ( textmining , experiments , databases , co-expression , neighborhood , gene fusion co-occurrence ) and imported into Cytoscape ( Shannon et al . , 2003 ) .", "Network edges represent confidence .", "A minimum interaction score of 0 . 4 ( medium confidence ) was required for network inclusion .", "Enrichment analysis was performed using PANTHER ( Mi et al . , 2017 ) .", "Lists of proteins were exported for the identified GO terms and then manually coloured using Cytoscape for visualization .", "Transthoracic echocardiography ( TTE ) was performed using a Vevo 770 High-Resolution Imaging System ( Visualsonics Inc , Toronto , Canada ) and a 30MHz probe .", "Mice were lightly anesthetized with 1–1 . 5% isoflurane via facemask , maintaining the heart rate at approximately 450 beats/min .", "Views were taken in planes that approximated the parasternal short-axis view and the apical long-axis view .", "M-mode tracings were used to determine left ventricular end-diastolic diameter ( LVDD ) and end-systolic diameter ( LVSD ) , posterior wall thickness in diastole ( LVPWD ) and systole ( LVPWS ) , and interventricular septum thickness in diastole ( IVSD ) and systole ( IVSS ) over three cardiac cycles .", "The analysis was performed blinded to animal details .", "LV fractional shortening ( FS ) was calculated using the formula FS = [ ( LVDD - LVSD ) / ( LVDD ) ] x 100 .", "Relative wall thickness ( RWT ) was calculated using the equation RWT = ( IVDD + LVPWD ) /LVDD .", "Mice were lightly anesthetized with 1–1 . 5% isoflurane via facemask .", "The body temperature was maintained around 37°C using a heat pad .", "The lead II ECG was recorded using Power Lab/4SP system ( Adinstruments ) from needle electrodes inserted subcutaneously into the left and right forelimbs and the right hindlimb .", "The signal was acquired for about 5 min using Chart 7 software ( Adinstruments ) .", "During offline analysis , the 5 min recording was examined for unusually shaped P , QRS , or ‘T’ waves and for time-varying phenomenon ( e . g . irregularities in interval durations ) .", "Ectopic or abnormal beats are noted .", "A representative 10–15 s segment of the recording was averaged to obtain the signal averaged ECG to calculate Heart rate , RR interval , P wave duration , PR interval , QRS , JT and QT durations .", "QT duration was corrected ( QTc ) using the Bazett's formula ( Bazett , 1997 ) .", "Polysome profiles were prepared according to the protocols previously published in: ( Gandin et al . , 2014 ) .", "Two conditions were profiled , BUD23 siRNA and Control siRNA , with an n of 3 samples per condition .", "Fractions were collected from each sample and RNA was purified using the Trizol extraction method .", "RNA from fractions containing three or less ribosomes were pooled ( ‘Low translational efficiency ) and RNA from fractions containing more than three ribosomes ( ‘high efficiency’ ) were pooled .", "Sub-polysomal fractions were similarly pooled .", "RNA sequencing was used to quantify the expression of RNA species in each of the pooled samples .", "Translational efficiency ( TE ) was defined as the relative abundance of a transcript in the high and low efficiency pooled fractions .", "A TE score for each transcript was calculated by averaging the ratio derived from each of the three replicates in each condition .", "TE was plotted for the two conditions using R software .", "Translational efficiency ( TE ) was then compared to both the length and GC content of the 5' UTR region .", "5' UTRs were defined using the TxDb . Hsapiens . UCSC . hg38 . knownGene ( Bioconductor Core Team and Bioconductor Package Maintainer , 2016 ) and org . Hs . eg . db ( Carlson , 2018 ) R packages .", "Sequences of the regions were extracted using the twoBitToFa program from the BLAT suite ( Kent , 2002 ) and then GC content was calculated using the seqinr R package ( Charif and Lobry , 2007 ) .", "The Pearson correlation coefficients , and their relative significances , were calculated using cor . test function from the stats R package ( R Development Core Team , 2017 ) .", "Live cell metabolic assays were performed on A549 cells pre-treated with either BUD23 siRNA or control siRNA using a Seahorse XFe 96 analyser ( Agilent ) .", "Mitochondrial stress tests was performed according to the manufacturer’s instructions ( Agilent ) .", "2M Oligomycin ( OA ) , 1M FCCP and 0 . 5M rotenone/antimycin A ( AA+R ) were used for all conditions .", "A549 cells were plated into Seahorse XF96 plates at 160 , 000 cells per well , utilising 16 wells per condition .", "Cell density was normalised using SRB assay .", "Experiments were performed in triplicate .", "Of the three distinct experimental preparations available ( isolated mitochondria , permeabilized fibers , and tissue homogenates ) , we utilized cardiac homogenates for mitochondrial assessment ( see Goo et al . , 2013 for advantages of this method ) .", "Ventricular tissue ( ~20 mg ) was weighed and transferred to 1 . 5 ml of ice-cold MiRO5 medium ( in mM: EGTA 0 . 5 , MgCl2 1 . 4 , taurine 20 , KH2P04 10 , HEPES 20 , BSA 1% , K-MES 60 mM , sucrose 110 mM , pH 7 . 1 , adjusted with 5 N KOH ) .", "Tissue was homogenized for 3 s in 1 s bursts with a tissue homogenizer and loaded immediately ( 40 µg/ml ) into an Oroboros Oxygraph 2 k high resolution respirometry system ( Oroboros Instruments , Innsbruck , Austria ) for measurement of mitochondrial respiration combined with the Fluorescence-Sensor Green of the O2k-Fluo LED2-Module for H2O2 measurement ( see Makrecka-Kuka et al . , 2015 for full details ) .", "Two identical respiration chambers ( chamber A and chamber B ) held at the same temperature were run in parallel for each experimental run .", "All measurements of respiration rates were carried out at 37°C .", "Oxygen electrodes were calibrated daily with air-saturated respiration solution Zero calibrations were achieved by injecting yeast into the experimental chambers .", "Oxygen solubility in the assay medium was calculated as described previously ( Lienig and Forstner , 1984 ) .", "H2O2 flux was measured simultaneously with respiration in the O2k-Fluorometer using the H2O2-sensitive probe Amplex UltraRed ( 10 μM ) with 1 U/mL horse radish peroxidase ( HRP ) and 5 U/mL superoxide dismutase ( SOD ) .", "AmR in the presence of H2O2 is catalysed by HRP to the fluorescent product resorufin .", "Calibrations were performed with two sequential injections of H2O2 at 0 . 1 μM steps .", "Three parameters are commonly used to assess mitochondrial function ( for reviews , see Brand and Nicholls , 2011; Pesta and Gnaiger , 2012 ) .", "Firstly , the capacity for oxidative phosphorylation ( OXPHOS ) is the respiratory capacity of mitochondria in the ADP-activated state of oxidative phosphorylation ( saturating concentrations of ADP , inorganic phosphate , oxygen , and defined substrates ) .", "Secondly , LEAK respiration rate represents mitochondrial respiration that occurs in the absence of ATP generation , mainly to compensate for proton leak across the mitochondrial inner membrane .", "Lastly , the respiratory electron transfer-pathway capacity ( ETC ) is mitochondrial respiration in the noncoupled state in the presence an uncoupler; this induces maximum oxygen flux through the electron transport chain .", "The Respiratory Control Ratio ( RCR , calculated here as OXPHOS/LEAK ) provides a measure of the degree of coupling between oxidation and phosphorylation , or in other words , the efficiency of mitochondrial ATP production .", "OXPHOS , LEAK and ETS were measured in the presence of Complex I substrates pyruvate and malate ( electron transfer through Complexes I-IV ) or Complex I+II substrates ( addition of succinate ) .", "Additionally , respiratory flux with electron transfer through Complex IV alone was measured via the addition of the electron donor tetramethyl-phenylene-diamine ( TMPD ) .", "The protocol used to measure these parameters was adapted from Pesta and Gnaiger ( 2012 ) .", "Briefly , pyruvate ( 5 mmol l −1 ) , malate ( 0 . 25 mmol l−1 ) and glutamate ( 10 mmol l−1 ) are added as carbon substrates and to spark the citric acid cycle .", "Under these conditions , mitochondria are in LEAK respiration with CI substrates in the absence of adenylates .", "OXPHOS with CI substrates was achieved through addition of saturating levels of ADP ( 2 mM l−1 ) .", "Following steady-state conditions , succinate ( 10 mmol l−1 ) was added to achieve OXPHOS with CI+CII substrates .", "To uncouple respiration and achieve ETS with CI+CII substrates , carbonyl cyanide 4-trifluoromethoxyphenylhydrazone ( FCCP ) was carefully titrated to a maximum concentration of 0 . 25 μmol l−1 .", "Rotenone was then added to achieve ETS with CII substrates and antimycin A ( 5 μmol l−1 ) was given to block Complex III and measure background non-mitochondrial residual oxygen consumption ( ROX ) .", "OXPHOS through Complex IV alone was assessed by adding the electron donor TMPD ( 0 . 5 mmol l−1 ) .", "To avoid oxidation of TMPD , ascorbate ( 2 mMol l−1 ) was added prior to TMPD injection .", "The citrate synthase activity of cardiac homogenates were measured in frozen homogenates .", "Briefly , maximal activity ( Vmax ) was determined with a spectrophotometer at 37°C with a Synergy HTX spectrophotometer ( BioTek , UK ) in assay buffer; 50 mM TRIS-HCl , pH 8 . 0 .", "Citrate synthase activity was monitored in the presence or absence of oxaloacetate by the appearance of 5-thio-2-nitrobenzoic acid as a result of the reaction of free acetyl-CoA with 5 , 5’-dithiobis ( 2-nitrobenzoic acid ) at 412 nm over a 10 min incubation period ( assay buffer with 0 . 5 oxaloacetate , 0 . 3 mM acetyl-CoA , 0 . 15 mM 5 , 5-dithiobis-2-nitrobenzoic acid ) .", "Extinction coefficients were empirically determined to quantify Vmax values .", "Enzyme activities were normalised to total soluble protein , which was quantified according to Bradford ( Bradford , 1976 ) .", "Total cell protein was isolated from cells using protein extraction buffer ( 50 mM Tris , 150 mM NaCl , 1% TritonX-100 , supplemented with protease inhibitors ) .", "Total protein was isolated from snap-frozen tissue using RIPA buffer supplemented with protease inhibitor cocktail after disruption using Lysing Matrix D tubes ( MP Bio ) .", "All experiments were performed according to current UK Home Office regulations and under approval of the relevant University of Manchester ( Manchester , UK ) local ethics committee .", "Six mice were used for this study ( n = 3 control , n = 3 Bud 23 KO ) .", "Immediately after euthanasia , hearts were harvested and small portions ( cubes with sides smaller than 0 . 5 mm ) of right and left ventricle were immersion fixed in 2 . 5% glutaraldehyde and 2% paraformaldehyde in 100 mM sodium cacodylate buffer ( pH 7 . 2 ) overnight .", "Specimen preparation was performed , with small modifications , according to the Ellisman protocol ( Holcomb et al . , 2013 ) .", "Briefly , after washings in sodium cacodylate , samples were sequentially stained in: 2% osmium tetroxide and 1 . 5% potassium ferrocyanide in 100 mM sodium cacodylate for 1 hr; 1% aqueous thiocarbohydrazide for 20 min; 2% aqueous osmium for 30 min; 1% aqueous uranyl acetate overnight and Walton’s lead aspartate for 30 min the following morning at 60°C .", "Samples were washed 3 times for 10 min in double distilled water after each staining step .", "Staining was followed by dehydration in ethanol ascending series ( 50% , 70% , 90% , 100% ) and infiltration with TAAB 812 hard resin mixed with propylene oxide .", "After embedding , resin blocks were cured at 70° C for 40 hr .", "Plastic blocks were sectioned at 80 nm thickness using a Leica UC6 ultramicrotome and sections were imaged using a FEI Tecnai12 Biotwin operated at 100kV .", "Images were analysed using Fiji ( Schindelin et al . , 2012 ) .", "Fractionation experiments were performed by differential centrifugation as previously described previously ( Rorbach et al . , 2014 ) .", "Unless otherwise stated in the figure legend , parametric data was analysed by ANOVA with a Dunnett’s multiple comparisons test .", "Non-parametric data was analysed using a Mann-Whitney test .", "RNAseq data was analysed using edgeR ( Robinson et al . , 2010 ) .", "For Mass Spectrometry data analysis please refer to the dedicated section ." ] ]

[ "Efficient mitochondrial function is required in tissues with high energy demand such as the heart , and mitochondrial dysfunction is associated with cardiovascular disease .", "Expression of mitochondrial proteins is tightly regulated in response to internal and external stimuli .", "Here we identify a novel mechanism regulating mitochondrial content and function , through BUD23-dependent ribosome generation .", "BUD23 was required for ribosome maturation , normal 18S/28S stoichiometry and modulated the translation of mitochondrial transcripts in human A549 cells .", "Deletion of Bud23 in murine cardiomyocytes reduced mitochondrial content and function , leading to severe cardiomyopathy and death .", "We discovered that BUD23 selectively promotes ribosomal interaction with low GC-content 5’UTRs .", "Taken together we identify a critical role for BUD23 in bioenergetics gene expression , by promoting efficient translation of mRNA transcripts with low 5’UTR GC content .", "BUD23 emerges as essential to mouse development , and to postnatal cardiac function ." ]

[ "Cells need to make proteins to survive , so they have protein-making machines called ribosomes .", "Ribosomes are themselves made out of proteins and RNA ( a molecule similar to DNA ) , and they are assembled by other proteins that bring ribosomal components together and modify them until the ribosomes are functional .", "Mitochondria are compartments in the cell that are in charge of providing it with energy .", "To do this they require several proteins produced by the ribosomes .", "If not enough mitochondrial proteins are made , mitochondria cannot provide enough energy for the cell to survive .", "One of the proteins involved in modifying ribosomes so they are functional is called BUD23 .", "People with certain diseases , such as Williams-Beuren syndrome , do not make enough BUD23; but it was unknown what specific effects resulted from a loss of BUD23 .", "To answer this question , Baxter et al . first genetically removed BUD23 from human cells , and then checked what happened to protein production .", "They found that ribosomes in human cells with no BUD23 were different than in normal cells , and that cells without BUD23 produced different proteins , which did not always perform their roles correctly .", "Proteins in the mitochondria are one of the main groups affected by the absence of BUD23 .", "To determine what effects these modified mitochondrial proteins would have in an animal , Baxter et al . genetically modified mice so that they no longer produced BUD23 .", "These mice developed heart problems caused by their mitochondria not working correctly and being unable to provide the energy the heart cells needed , eventually leading to heart failure .", "Heart problems are common in people with Williams-Beuren syndrome .", "Many diseases arise when a person’s mitochondria do not work properly , but it is often unclear why .", "These experiments suggest that low levels of BUD23 or faulty ribosomes may be causing mitochondria to work poorly in some of these diseases , which could lead to the development of new therapies ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "ecology", "neuroscience" ]

[ [ "The ability of an animal to change the speed and direction of movement , defined as maneuverability ( Dudley , 2002 ) , can determine its success at avoiding predators , obtaining food , and performing other behaviors that determine the margin between life and death ( Webb , 1976; Hedenström and Rosén , 2001; Walker et al . , 2005 ) .", "Most biomechanical research on birds has focused on either brief ( e . g . , take off ) or steady state movements ( e . g . , forward flight ) that can be studied most readily in the laboratory .", "Maneuverability is therefore one of the most important but least understood aspects of animal locomotion .", "Warrick and coworkers ( Warrick et al . , 1988; Warrick and Dial , 1998 ) proposed that there are both intrinsic and facultative influences on maneuvering performance .", "For animals that perform powered flight , intrinsic maneuverability is defined by the physical limitations imposed by morphology ( Norberg and Rayner , 1987 ) , but excess muscle capacity should allow them to facultatively overcome the costs of suboptimal morphology , achieving higher levels of performance by sacrificing efficiency .", "Although compelling , this hypothesis has never been tested explicitly .", "Wing morphology is defined using measures of size ( e . g . , area or length ) and non-dimensional measures of shape ( e . g . , aspect ratio ) .", "Wing area and aspect ratio have significant and well known effects on the aerodynamics of flight in animals ( Pennycuick , 1975; Kruyt et al . , 2014; 2015 ) , and should affect maneuvering performance .", "Wing morphology influences flight efficiency ( Feinsinger and Chaplin , 1975 ) , ecological roles ( Feinsinger , 1976; Feinsinger and Colwell , 1978; Warrick , 1998 ) and competitive ability ( Feinsinger and Chaplin , 1975; Feinsinger and Colwell , 1978; Feinsinger et al . , 1979; Altshuler , 2006 ) .", "Because these previous studies focused on species and gender comparisons , less is known about how individual variation in wing morphology influences performance , especially with respect to maneuverability .", "One complication is that different wing sizes and shapes can be favored depending on the specific maneuver performed , e . g . , yaw versus banked turns .", "Given the diversity of flight behaviors , it is unclear if the requirements for maneuvering exert strong selection on wing morphology .", "Muscle capacity affects the maximum aerodynamic force a flying animal can produce .", "Aerodynamic force can be directed for performing maneuvers that require greater output than the minimum requirements for flight .", "Excess muscle capacity can also be used to compensate for anatomical or spatial constraints on wing movement ( Warrick , 1998 ) .", "Muscle output of hummingbirds has been quantified in several ways including oxygen consumption to determine metabolic input , wingbeat kinematics to estimate mechanical power output , and electromyography ( EMG ) to measure myoelectric input .", "Considering hovering flight as the point of comparison , forward flight at the fastest speeds recorded in a wind tunnel requires about 20% more metabolic ( Clark and Dudley , 2010 ) and myoelectric input ( Tobalske et al . , 2010 ) .", "Maximum sustained hovering performance has been studied by experimentally lowering air density to the lowest values in which birds are still able to hover .", "These experiments revealed that hovering in hypodense air requires ~40% higher mechanical power output ( Chai and Dudley , 1995 ) and ~60% higher spatial recruitment of muscle fibers , as measured by the spike amplitude of the electromyogram recordings ( Altshuler et al . , 2010b ) , in comparison to hovering in normal air .", "By far the most expensive flight behavior studied to date in hummingbirds is maximum load lifting , which requires 200–400% more mechanical power output ( Chai et al . , 1997; Chai and Millard , 1997; Altshuler et al . , 2010a ) , about 200% more spatial recruitment ( EMG spike amplitude ) , and 150% more temporal recruitment ( EMG spike frequency ) ( Altshuler et al . , 2010b ) compared to hovering .", "Maximum load lifting is a transient behavior that uses the bird’s natural escape response to measure burst power output .", "Thus , it is not surprising that this assay provides the maximum muscle capacity that has been measured in hummingbirds .", "It is particularly useful for quantifying variation among and within species in burst muscle capacity .", "Studies using the load lifting assay have revealed that maximum burst muscle capacity is related to hummingbird evolutionary ecology .", "Altshuler and coworkers ( Altshuler et al . , 2004b; Altshuler , 2006 ) demonstrated that ecological role is more strongly related to load lifting ability than morphological parameters such as wing loading .", "Load lifting ability is also associated with species- and gender-specific competitive ability at different elevations .", "Altshuler ( Altshuler , 2006 ) suggested that the relationship between maximum muscle capacity and competitive ability may be mediated through maneuvering performance .", "Unconstrained maneuvering performance of birds , including hummingbirds , has recently been quantified in the field without individual identification ( Shelton et al . , 2014; Sholtis et al . , 2015 ) .", "Although field studies are valuable for quantifying average species performance , individual identification and large sample sizes are required to examine sources of within-species variation .", "Here , we studied the free-flight maneuvering performance of Anna's hummingbirds ( Calypte anna ) in a large flight chamber ( Video 1 ) .", "Flight maneuvers in a chamber are not expected to be the same as outdoors , and may have lower velocities and accelerations .", "The benefit of this approach is that a large number of measurements from the same individuals can be combined with other data to examine how variation in the observed maneuvers is influenced by individual morphology and muscle capacity . 10 . 7554/eLife . 11159 . 003Video 1 . The multi-camera , automated tracking system filming two hummingbirds in the flight arena at 200 frames per second . Continuously tracked sequences are assigned an object number ( from 0 to 4 over this sequence ) .", "Body position and orientation are calculated and reprojected onto the video of four cameras .", "The videos are saved using a compression algorithm that only records the sections of the image that are moving ( Straw et al . 2011 ) .", "Thus , birds disappear from the video when they land and stop moving .", "The trajectory shown in Figure 1 is taken from the bird labeled #2 and begins at 5 . 1 seconds and ends at 8 . 05 seconds . DOI: http://dx . doi . org/10 . 7554/eLife . 11159 . 003 We used a high-throughput computational approach to record the flight performance of 20 individuals alone and in the presence of a competitor .", "Flight trajectories were parsed into a set of performance metrics based on body position and orientation .", "The first goal of our study was to determine if voluntary maneuvering performance is repeatable within individuals .", "Repeatability of maneuvering performance can arise either through a strong influence of fixed traits such as morphology and anatomy , or through other consistent influences , such as motivation .", "We expect that repeatable measurements will be most useful for our second goal , determining how variation in maneuverability among individuals is influenced by natural variation in morphology and muscle capacity .", "This also required measuring morphological traits and maximum burst performance for each individual .", "Our third goal was to determine how motivation state induced by the presence of a competitor influenced maneuvering performance .", "To address this question we compared flight trials with and without competitors ." ], [ "The first stage of analysis was estimating instantaneous velocities , accelerations , and headings from the raw tracking data ( Figure 1—figure supplement 1 ) .", "Translational velocity and acceleration were calculated by taking the first and second derivatives of an interpolation spline fit to the body position data ( splev and splrep functions , Scientific Python ) .", "The velocities and accelerations were split into vertical and horizontal components .", "The body orientation vector was represented in spherical coordinates as azimuth and pitch angles .", "We took the first derivatives to obtain azimuth and pitch velocities .", "Because the video tracking system did not allow a measurement of body roll , we decided to use a global coordinate system instead of a body axis-centered coordinate system .", "In our frame of reference , pitch is a global measure defined relative to the horizontal plane .", "Heading was calculated as the instantaneous direction of the horizontal translation velocity , and the heading velocity was calculated as the derivative of heading . 10 . 7554/eLife . 11159 . 004Figure 1 . A multi-camera , automated tracking system extracted hummingbird body position ( blue circle ) and orientation ( red line ) from solo and competitive flights . The trajectory shown for one bird", "( a ) is also shown in Video 1 ( see Figure 1—figure supplement 1 for time series of position , velocity , and acceleration values ) .", "Stereotyped maneuvers were classified in each trajectory", "( b ) and between one and five performance metrics were calculated from each maneuver .", "Maneuvers within a trajectory may be overlapping ( e . g . #4 , 5 , 6 ) .", "The trajectory presented in b is a top down ( x-y projection ) view of the trajectory shown in a .", "Body position and orientation were smoothed with an extended Kalman filter ( c , d ) .", "The effects of four different sets of smoothing parameters are presented for an arcing turn ( maneuver #9 in", "b ) and an upward acceleration ( maneuver #1 in", "b ) .", "Shown here are the unsmoothed position and orientation ( black trace and text ) , the chosen levels of smoothing ( blue ) , a lower level of smoothing ( green; 0 . 1 x Rpos; 0 . 1 x Rori ) , and a higher level of smoothing ( red; 10 x Rpos; 10 x Rori ) .", "The chosen smoothing parameters for body position were determined by tracking multiple dropped objects and calibrating the Z-axis acceleration to gravity .", "The chosen smoothing parameters for body orientation were determined by re-projecting the body axis vector onto the video .", "The higher and lower levels of smoothing for body position presented in this figure were both deemed too extreme , when re-projected onto the video .", "However , the level of smoothing for body orientation had minimal effect on the average yaw velocity . DOI: http://dx . doi . org/10 . 7554/eLife . 11159 . 00410 . 7554/eLife . 11159 . 005Figure 1—figure supplement 1 . The representative trajectory from Figure 1 and Video 1 displayed through time . The upper three panels provide the position , velocity , and acceleration values .", "The lower three panels provide the orientation vector , orientation angle and rotation velocity values .", "The maneuvers extracted for this sequence are given by thick black lines below the traces . DOI: http://dx . doi . org/10 . 7554/eLife . 11159 . 005 We then used the velocity , acceleration , and orientation data to search for a series of ten stereotyped maneuvers that were independent of time and distance scales ( Figure 1b ) .", "Five of the maneuvers were sequences defined by changes in translational velocity: 1 ) 3D accelerations , 2 ) horizontal accelerations , 3 ) horizontal decelerations , 4 ) vertical upward accelerations , and 5 ) vertical downward accelerations .", "Three maneuvers were sequences defined by changes in rotation: 6 ) pitch-up rotations , 7 ) pitch-down rotations , and 8 ) yaw turns .", "Two of the maneuvers were defined as turns with translational components: 9 ) arcing turns and 10 ) pitch-roll turns .", "These ten maneuvers are not meant to be mutually exclusive , exhaustive , or to divide the entire filming session into a set of discrete behaviors , but are instead intended to extract simple measurements that can be used as an assay for maneuvering performance .", "The search criteria for the maneuvers are given in Table 1 . Because we assume that a new maneuver must involve a change in velocity , the first search parameter was to find sequences bounded by velocity maxima and minima , or vice versa .", "We next describe the additional search parameters and the performance metrics used to quantify each maneuver . 10 . 7554/eLife . 11159 . 006Table 1 . Search parameters for the ten maneuvers analyzed in the study .", "The definitions , units , and symbols for the 14 related performance metrics are also provided . DOI: http://dx . doi . org/10 . 7554/eLife . 11159 . 006ManeuverSearch parametersPerformance metricUnitsSymbol3D accelerationStart: velocity xyz minimumEnd: velocity xyz maximumDistance xyz > 25 cmMaximum velocitym/sVelmaxHorizontal accelerationStart: velocity xy minimumEnd: velocity xy maximumDistance xy > 25 cmDistance z < 10 cmMaximum acceleration xym/s2AccHormaxHorizontal decelerationStart: velocity xy maximumEnd: velocity xy minimumDistance xy > 25 cmDistance z < 10 cmMaximum deceleration xym/s2AccDecmaxVertical upwards accelerationStart: velocity z minimumEnd: velocity z maximumDistance z > 25 cmMaximum acceleration zm/s2AccVUmaxVertical downwards accelerationStart: velocity z maximumEnd: velocity z minimumDistance z > 25 cmMaximum acceleration zm/s2AccVDmaxPitch-up rotationStart: pitch minimumEnd: pitch maximumDegrees rotated > 45 degDistance xyz < 10 cmAverage pitch velocityrev/sPitchUvel , avgPitch-down rotationStart: pitch maximumEnd: pitch minimumDegrees rotated > 45 degDistance xyz < 10 cmAverage pitch velocityrev/sPitchDvel , avgYaw turnStart: velocity yaw = 0 deg/sEnd: velocity yaw = 0 deg/sDegrees rotated > 90 degPitch maximum < 75 degDistance xyz < 10 cmAverage yaw velocityrev/sYawvel , avgArcing turnStart: Δ heading velocity > 0 . 25 rev/sEnd Δ heading velocity < 0 . 25 rev/sVelocity xy min > 50 cm/sDistance xy > 25 cmDistance z < 10 cmAverage xy velocity*radius*Centripetal acceleration*m/smm/s2Arcvel , avgArcradArccent , maxPitch roll turnStart: velocity maximumEnd: velocity maximumPitch maximum > 75 degDistance xy before velocityMin > 12 . 5 cmDistance xy after velocity Min < 12 . 5 cmDistance z < 10 cmtime†degrees turned†sdegPRTtimePRTdeg*for a 25 cm segment centered at the sharpest point of the turn†for a 25 cm segment centered at the minimum velocity xyz The five translational maneuvers were defined using velocity minima and maxima , and only sequences with at least 25 cm of travel were analyzed .", "The 3D acceleration maneuvers started from a velocity minimum and ended with a velocity maximum .", "The performance metric calculated for these maneuvers was the maximum translational velocity ( Velmax ) .", "The horizontal acceleration maneuvers were bounded by horizontal velocity minima and maxima , and were constrained to no more than 10 cm of vertical distance traveled .", "The performance metric calculated for these maneuvers was the maximum horizontal acceleration ( AccHormax ) .", "The horizontal deceleration maneuvers and the corresponding performance metric , maximum horizontal deceleration ( DecHormax ) , were bounded by horizontal velocity maxima and minima .", "The vertical upward acceleration and vertical downward acceleration maneuvers were bounded by vertical velocity minima and maxima .", "The performance metrics calculated from these maneuvers were , respectively , maximum upward ( AccVUmax ) and maximum downward ( AccVDmax ) accelerations .", "All translational accelerations and decelerations were expressed as positive values , so that higher values represent a higher level of performance .", "We defined three rotational maneuvers: pitch-up rotations , pitch-down rotations , and yaw turns .", "These sequences were bounded by the zero-crossings of the azimuthal and pitch velocities .", "In contrast to translational maneuvers , which were defined by the maxima and the minima of the velocities , the rotational maneuvers begin and end with changes in rotational velocity direction .", "Thus , the performance metrics calculated from these rotational maneuvers were the average rotational velocities over the whole maneuver instead of maximum accelerations or decelerations .", "An additional constraint common to all three rotational maneuvers is that the linear distance traveled was less than 10 cm .", "We chose 10 cm as a general cutoff here and elsewhere because this value is close to the body length of a bird and the wing span at mid-downstroke , thus providing a good threshold for distinguishing translational motion .", "The pitch-up and pitch-down maneuvers were defined as having continuous pitch velocity in the upward or downward direction , respectively .", "Only maneuvers with a total pitch rotation greater than 45° were analyzed .", "From these maneuvers we calculated either the average pitch-up ( PitchUvel , avg ) or pitch-down ( PitchDvel , avg ) velocity as performance metrics .", "Defining yaw turns is challenging because hummingbirds fly with an upright body posture .", "When the body posture is near vertical , azimuthal rotation is implemented by rolling about the body axis , but when the body posture is near horizontal , azimuthal rotation is implemented by yawing the body axis .", "We therefore define yaw turns as azimuthal changes in direction when the body pitch angle is below 75° .", "An additional constraint specific to yaw turns was a requirement for at least 90° change in azimuth .", "From these trajectories we measured the average yaw velocity ( Yawvel , avg ) as the performance metric .", "In addition to five translational and three rotational maneuvers , we also considered two maneuvers that are complex turns with translational components .", "Arcing turn maneuvers were defined as sequences with a heading velocity > 90°/sec , a minimum total translational velocity > 0 . 5 m/s , a total distance traveled > 25 cm , and a vertical distance traveled < 10 cm .", "These search parameters reliably extract arcing turns that occur in the horizontal plane .", "To compare arcing turns of different shapes and scales we clipped the trajectories to a length of 25 cm centered at the sharpest point of the turn .", "From the clipped trajectory we analyzed three performance metrics , average velocity ( Arcvel , avg ) , radius ( Arcrad ) , and the maximum centripetal acceleration ( Arccent , max ) .", "The latter two were calculated using the following equations: Arcrad=Arcdistance traveledΔHeadingrad Arccent , max=Arcvel , avg2Arcrad Pitch-roll turn maneuvers have been described in hummingbirds and are characterized by the following sequence:", "a ) deceleration ,", "b ) increase in pitch to near vertical ,", "c ) azimuthal rotation by rolling the body , and", "d ) acceleration in a new direction ( Clark , 2011 ) .", "These maneuvers were identified by searching for sequences of deceleration followed by acceleration with a maximum pitch > 75° .", "Just as we did for the yaw turns , we assume that above a pitch angle of 75° , the rotation is primarily dominated by a body axis roll , even if there may be a slight yawing component .", "For this reason , we maintain the established 'pitch-roll' terminology to describe these types of turns .", "These sequences were clipped to a linear distance of 25 cm centered on the point of the lowest translational velocity .", "Only clipped sequences in which the total vertical displacement was less than 10 cm were analyzed .", "The performance metrics for pitch-roll turns were the time taken ( PRTtime ) and the degrees turned ( PRTdeg ) .", "Arcing turns and pitch-roll turns are two different mechanisms for generating a change in heading with no overlap in our data set by definition ( Table 1 ) .", "We analyzed how morphology , burst capacity , and competitor presence influenced the relative use of these two turns .", "The pitch-roll percent ( PRT% ) was defined as the number of pitch-roll turns divided by total the number of arcing and pitch-roll turns extracted from each trial .", "Descriptive statistics for morphology and load lifting are provided in Table 2 . A large sample of values was obtained for each maneuvering performance metric ( Table 3 ) .", "Figure 2 shows the distributions of trial means for all performance metrics . 10 . 7554/eLife . 11159 . 007Table 2 . Wing morphology and load lifting performance of male Anna’s hummingbirds ( n = 20 individuals ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 11159 . 007TraitMeanRangeWing length50 . 97 mm[45 . 76 , 55 . 45]Wing area 1355 mm2[1051 , 1653]Wing aspect ratio7 . 73[7 . 13 , 8 . 46]Body mass4 . 64 g[4 . 09 , 5 . 61]Mass of weights lifted5 . 93 g[4 . 00 , 7 . 24]10 . 7554/eLife . 11159 . 008Table 3 . Descriptive statistics and sample sizes for maneuvering performance .", "Grand mean values were calculated by first taking the mean of each bird’s trial averages ( i . e . , the bird means ) , and then taking the mean of the bird means ( n = 20 birds in 20 solo trials and 16 paired competition trials ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 11159 . 008ManeuverabilityPerformance metric# TrajectoriesGrand mean[Range of means]Linear accelerationsVelmax71 , 0072 . 22 m/s[1 . 20 , 2 . 94]AccHormax47 , 2876 . 30 m/s2[2 . 96 , 8 . 83]DecHormax51 , 2456 . 67 m/s2[9 . 03 , 3 . 45]AccVUmax6 , 9353 . 78 m/s2[2 . 98 , 4 . 67]AccVDmax9 , 2843 . 58 m/s2[4 . 69 , 2 . 68]Rotational velocitiesPitchUvel , avg6 , 0851 . 13 rev/s[0 . 91 , 1 . 34]PitchDvel , avg14 , 8071 . 00 rev/s[1 . 19 , 0 . 78]Yawvel , avg12 , 6601 . 52 rev/s[1 . 32 , 1 . 75]Complex turnsPitch-rollPRTdeg17 , 133133 . 3 º[34 . 9 , 162 . 7]PRTtime17 , 1330 . 47 s[0 . 38 , 0 . 60]ArcingArcrad6 . 9450 . 48 m[0 . 14 , 0 . 70]Arcvel , avg6 , 9451 . 57 m/s[0 . 80 , 2 . 26]Arccent , max6 , 9456 . 59 m/s2[3 . 42 , 10 . 80]Use of turnsPRT% 24 , 0780 . 69[0 . 39 , 0 . 87]10 . 7554/eLife . 11159 . 009Figure 2 . Distributions of mean performance metric values for n = 52 bird-trial combinations . Only PRTdeg has statistically significant outliers .", "See Figure 2—figure supplement 1 for distributions of residuals from the best-fit model in each case .", "Note that two statistical outliers were omitted from the analysis of PRTdeg . DOI: http://dx . doi . org/10 . 7554/eLife . 11159 . 00910 . 7554/eLife . 11159 . 010Figure 2—figure supplement 1 . Distributions of residuals from the best-fit model for each performance metric . Note that two statistical outliers were omitted from the analysis of PRTdeg . DOI: http://dx . doi . org/10 . 7554/eLife . 11159 . 010 All performance metrics based on total and horizontal linear accelerations and complex turns were highly repeatable , with >80% of the variation in these metrics attributable to differences among individuals ( Figure 3 ) .", "The rotational performance metrics and the percent of turns that were pitch-roll turns were moderately repeatable , with 40–70% of the variation in these metrics attributable to among-individual differences .", "The vertical accelerations were not repeatable , as the 95% confidence intervals for repeatability of these metrics overlapped zero . 10 . 7554/eLife . 11159 . 011Figure 3 . Most maneuvering performance metrics are highly repeatable . Values > 70% are considered to have high repeatability , 40–70% moderate repeatability , and < 40% low repeatability .", "A metric is considered not repeatable if its 95% confidence intervals overlap zero . DOI: http://dx . doi . org/10 . 7554/eLife . 11159 . 011 The best-supported models for each maneuvering performance metric are given in Table 4 .", "Burst muscle capacity was an important predictor for most of the maneuvering performance metrics .", "Birds that lifted more weight ( accounting for their wing morphology ) tended to accelerate and decelerate faster , and they tended to perform maneuvers with higher velocity ( Figure 4 ) .", "However , burst muscle capacity was not an important determinant of vertical acceleration and deceleration , as candidate models including burst performance as a predictor were not supported .", "Birds that lifted more weight also executed pitch-up and pitch-down maneuvers with higher rotational velocities .", "Burst capacity was not a strong determinant of yaw performance .", "Although yaw velocity was somewhat positively related to burst capacity ( Figure 4 ) , candidate models of yaw velocity that included burst as a predictor were not well supported . 10 . 7554/eLife . 11159 . 012Table 4 . Maneuvering performance in relation to burst performance , wing morphology , and competitor presence ( n = 20 birds in 20 solo trials and 16 paired competition trials ) .", "Standardized beta coefficients and R2GLMM ( m ) values are reported for either the best-fit model , or , if there was support for more than one model , the average of supported models .", "The standardized beta coefficient is a measure of effect size that can be compared among predictors in the same model .", "Relative importance is a measure of the weight of evidence in favor of a predictor on a scale from 0–1 , and is reported for burst capacity and wing morphology variables as these alone were subject to model selection .", "Marginal R2GLMM ( m ) provides a measure of the combined explanatory power of fixed effects of interest ( competitor presence , burst muscle capacity , and wing morphology effects combined ) .", "Details of all candidate models are provided in Supplementary file 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 11159 . 012ModelSupport forFixed effectsStd beta coef [95% CI]Relative importanceR2GLMM ( m ) Burst+ morphology+ competitorVelmaxburstcompetitor presencemassburstwing lengthwing aspect ratioexperiment ( CA1 ) experiment ( CA2 ) days post-capture–0 . 04 [–0 . 18 , 0 . 10]0 . 10 [–0 . 01 , 0 . 22]0 . 09 [0 . 00 , 0 . 18]–0 . 08 [–0 . 22 , 0 . 06]0 . 10 [–0 . 07 , 0 . 28]1 . 01 [0 . 59 , 1 . 42]1 . 06 [0 . 68 , 1 . 43]–0 . 07 [–0 . 24 , 0 . 11]----1 . 000 . 250 . 26------0 . 28AccHormaxburst + competitioncompetitor presencemassburstexperiment ( CA1 ) experiment ( CA2 ) days post-capture–0 . 46 [–0 . 82 , –0 . 11]0 . 20 [–0 . 28 , 0 . 69]0 . 39 [0 . 00 , 0 . 77]4 . 01 [2 . 46 , 5 . 56]3 . 68 [2 . 72 , 4 . 64]–0 . 39 [–1 . 09 , 0 . 32]----1 . 00------0 . 18DecHormaxburst + competitioncompetitor presencemassburstexperiment ( CA1 ) experiment ( CA2 ) days post-capture–0 . 47 [–0 . 78 , –0 . 16]0 . 31 [–0 . 13 , 0 . 74]0 . 41 [0 . 06 , 0 . 76]3 . 86 [2 . 47 , 5 . 25]3 . 64 [2 . 76 , 4 . 51]–0 . 24 [–0 . 88 , 0 . 39]----1 . 00------0 . 19AccVUmaxintercept-onlyNANANA0 ( NA ) AccVDmaxintercept-onlyNANANA0 ( NA ) PitchUvel , avgburstcompetitor presencemassburstexperiment ( CA1 ) experiment ( CA2 ) 0 . 02 [–0 . 02 , 0 . 06]0 . 00 [–0 . 04 , 0 . 04]0 . 03 [–0 . 01 , 0 . 07]0 . 14 [0 . 06 , 0 . 23]0 . 13 [0 . 03 , 0 . 23]----1 . 00----0 . 10PitchDvel , avgcompetition+ burstcompetitor presencemassburstwing lengthwing aspect ratioexperiment ( CA1 ) experiment ( CA2 ) 0 . 06 [0 . 01 , 0 . 10]0 . 01 [–0 . 04 , 0 . 05]0 . 03 [–0 . 01 , 0 . 08]0 . 04 [–0 . 03 , 0 . 12]–0 . 04 [–0 . 13 , 0 . 05]0 . 19 [0 . 03 , 0 . 34]0 . 22 [0 . 03 , 0 . 41]----0 . 660 . 370 . 28----0 . 18Yawvel , avgintercept-onlyNANANA0 ( NA ) PRTdegintercept-onlyNANANA0 ( NA ) PRTtimeburstcompetitor presencemassburstwing lengthwing aspect ratioexperiment ( CA1 ) experiment ( CA2 ) 0 . 00 [–0 . 01 , 0 . 01]–0 . 01 [–0 . 03 , 0 . 00]–0 . 02 [–0 . 03 , 0 . 00]–0 . 01 [–0 . 03 , 0 . 01]0 . 01 [–0 . 01 , 0 . 04]–0 . 08 [–0 . 12 , –0 . 03]–0 . 11 [–0 . 16 , –0 . 05]–--1 . 000 . 210 . 23–--0 . 29Arcradburstcompetitor presencemassburstwing aspect ratioexperiment ( CA1 ) experiment ( CA2 ) –0 . 02 [–0 . 07 , 0 . 03]0 . 01 [–0 . 03 , 0 . 06]0 . 06 [0 . 01 , 0 . 10]–0 . 06 [–0 . 15 , 0 . 03]0 . 25 [0 . 12 , 0 . 37]0 . 29 [0 . 06 , 0 . 52]----1 . 000 . 44–--0 . 22Arcvel , avgburstcompetitor presencemassburstexperiment ( CA1 ) experiment ( CA2 ) days post-capture–0 . 01 [–0 . 09 , 0 . 08]0 . 03 [–0 . 06 , 0 . 12]0 . 11 [0 . 04 , 0 . 19]0 . 89 [0 . 59 , 1 . 19]0 . 74 [0 . 56 , 0 . 92]–0 . 06 [–0 . 20 , 0 . 08]––1 . 00–----0 . 18Acccent , maxwing shapecompetitor presencemasswing aspect ratioexperiment ( CA1 ) experiment ( CA2 ) days post-capture0 . 29 [–0 . 37 , 0 . 94]–0 . 20 [–0 . 74 , 0 . 34]1 . 09 [0 . 19 , 1 . 99]5 . 93 [4 . 02 , 7 . 84]0 . 85 [–1 . 59 , 3 . 28]–1 . 76 [–2 . 62 , –0 . 90]----1 . 00------0 . 36PRT% wing shape + competition+ burst + wing sizecompetitor presencemassburstwing lengthwing aspect ratioexperiment ( CA1 ) experiment ( CA2 ) –0 . 14 [–0 . 19 , –0 . 09]0 . 00 [–0 . 04 , 0 . 05]0 . 04 [0 . 00 , 0 . 09]–0 . 06 [–0 . 13 , 0 . 01]–0 . 16 [–0 . 24 , –0 . 07]0 . 17 [–0 . 03 , 0 . 36]0 . 44 [0 . 19 , 0 . 69]----1 . 000 . 611 . 00----0 . 2710 . 7554/eLife . 11159 . 013Figure 4 . Burst muscle capacity was associated with most maneuvering performance metrics . Each panel shows partial residuals for a performance metric ( y-axis ) in relation to burst muscle capacity ( x-axis ) for the most supported candidate model with burst capacity as a predictor .", "Partial residual values ( y-axis ) account for the other fixed effects in that model .", "Lines show model predictions assuming the median value of continuous predictors , and averaging across experiments and levels of competitor presence .", "Prediction lines are dashed for metrics where burst performance was not present in any of the supported models .", "Color is used to denote data points from the same bird ( online version only ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 11159 . 013 Burst muscle capacity was also associated with some , but not all maneuvering performance metrics related to complex turns .", "Birds that lifted more weight for their wing morphology tended to execute faster , larger radius arcing turns ( Figure 4 ) .", "However , the centripetal acceleration of arcing turns was not associated with burst capacity .", "Hummingbirds with higher load lifting capacity executed pitch-roll turns in less time .", "Burst capacity was not a strong determinant of heading change during pitch-roll turns .", "Lastly , birds with higher burst muscle capacity used pitch-roll turns for proportionately more of their heading changes .", "Wing morphology , specifically the aspect ratio , was an important predictor for two performance metrics: centripetal acceleration and the percent of direction changes that were pitch-roll turns ( Figure 5 ) .", "Hummingbirds with long , narrow wings tended to perform arcing turns with higher centripetal accelerations , relative to birds with short , wide wings .", "Birds with higher aspect ratio wings also used proportionately more arcing turns than birds with low aspect ratio wings . 10 . 7554/eLife . 11159 . 014Figure 5 . Aspect ratio was associated with two maneuvering performance metrics . Each panel shows partial residual performance ( y-axis ) in relation to wing aspect ratio ( x-axis ) from a best-fit model that identified aspect ratio as an important predictor .", "Note that the partial residuals for PRT% in", "( b ) go above 1 because PRT% was modeled as a normally-distributed ( Gaussian ) variable .", "All other features as in Figure 4 . DOI: http://dx . doi . org/10 . 7554/eLife . 11159 . 014 Body mass was included in candidate models 1–7 because we had anticipated that body mass would have a strong influence on variation in maneuvering performance .", "However , for every performance metric in Table 4 , the coefficient estimate for body mass had confidence intervals that broadly overlapped zero .", "We did not detect a substantial effect of competitor presence on many of the performance metrics ( Table 4 ) .", "Two metrics , horizontal acceleration and deceleration , were affected , but in the direction opposite to what we predicted .", "Specifically , birds performed maneuvers with lower acceleration ( –0 . 46 m/s2 difference on average ) and lower deceleration ( –0 . 47 m/s2 ) in the presence of a competitor , relative to solo flight ( Figure 6a , b ) .", "One metric , pitch-down velocity ( Figure 6c ) , did increase during competition as predicted ( 0 . 06 rev/s difference on average ) .", "We had no prediction for how competition would influence the relative use of pitch-roll and arcing turns , but found that birds used proportionately more arcing turns in the presence of a competitor ( Figure 6d ) .", "Specifically , 35% of direction changes were arcing turns on average ( and 65% pitch-roll ) when a competitor was present , whereas during solo flight , only 23% of direction changes were arcing turns ( and 77% pitch-roll ) on average . 10 . 7554/eLife . 11159 . 015Figure 6 . Competitor presence was associated with four maneuvering performance metrics . Each panel shows residual performance ( y-axis ) in relation to competitor presence from a best-fit model where competitor presence had a detected effect .", "All other features as in Figure 4 . DOI: http://dx . doi . org/10 . 7554/eLife . 11159 . 015" ], [ "We collected a large number of free flight measurements for each of 20 individual hummingbirds to examine the biomechanical determinants of maneuverability .", "Other studies have measured elements of maneuvering performance of hummingbirds in the field ( Clark , 2009; Sholtis et al . , 2015 ) and documented the maximum velocities , accelerations , and rotations obtained during specific maneuvers .", "Our values for velocity and acceleration are considerably lower than either of the field studies , likely because of cage size .", "However , the benefit of using a flight chamber is that it allowed us to evaluate the relative contributions of different factors to the performance we observed .", "We found that hummingbirds maneuvered with highly repeatable performance ( Figure 3 ) .", "Maximum weight lifted during load lifting trials predicted most of the performance metrics that we measured , independent of a bird’s wing size and shape , such that birds with higher burst muscle capacity flew faster , had higher horizontal accelerations , faster rotations , and higher performance during complex turns ( Figure 4 ) .", "Aspect ratio predicted only two performance metrics , such that birds with higher aspect ratio wings performed turns with higher centripetal acceleration and a greater percentage of arcing turns ( Figure 5 ) .", "When flying in the presence of a competitor , hummingbirds used faster pitch velocities , although they used slower horizontal accelerations and decelerations .", "During competition trials birds also increased the proportion of arcing turns used ( Figure 6 ) .", "Collectively , these results suggest that burst muscle capacity is a much more important predictor of flight maneuverability than within-species variation in body mass , wing morphology , and competition with conspecifics .", "Why were body mass and wing size not associated with maneuvering performance ?", "Wing morphology has well-known physical affects on flight performance: aspect ratio predicts aerodynamic efficiency , wing area is directly proportional to aerodynamic force , and wing length is a strong predictor of wingbeat frequency .", "All of these morphological traits , along with body mass , could affect maneuverability in flight , either individually or in combination .", "For example , wing loading ( the ratio of body mass to wing area or to area swept by the wings ) was initially thought to be a key predictor of hummingbird flight performance and behavioral ecology ( Feinsinger and Chaplin , 1975; Feinsinger , 1976; Feinsinger and Colwell , 1978; Feinsinger et al . , 1979 ) .", "However , in our analysis the hypothesis that wing size and body mass together determine maneuvering performance was not supported for any performance metric ( see Supplementary file 1 ) .", "We found it especially surprising that only wing shape ( and not wing size ) predicted maneuvering performance .", "It is possible that other morphological traits may determine maneuvering performance , or that subtle relationships may have gone undetected , because our analysis was limited to 20 individuals of a single species .", "It would be informative to expand this analysis to other species with potentially greater within-species variation in wing morphology , and to assess maneuverability across different hummingbird species with divergent morphologies .", "Almost all of the performance metrics were highly repeatable , which indicates a potential role for intrinsic influences of wing morphology in determining maneuverability .", "However , aspect ratio was the only morphological parameter that predicted performance , and only for a limited set of maneuvers .", "Aspect ratio is a key determinant in wing efficiency for fixed wings , such as during gliding ( Pennycuick , 1983 ) , and it has recently been demonstrated that higher aspect ratio wings correspond to higher power factors in the revolving wings of hummingbirds ( Kruyt et al . , 2014 ) .", "We found that aspect ratio had a strong effect on the few performance metrics that it predicted , but did not affect most features of maneuvering performance .", "This suggests a limited role for aerodynamic efficiency in many features of maneuvering .", "Burst muscle capacity predicted most of the performance metrics we considered , independently of any association with wing size or shape .", "Load lifting is measured as a transient escape maneuver that is likely anaerobic and performed inefficiently .", "All hummingbirds reach maximum load lifting performance at a geometric limit set by the amplitude of the wings: wing stroke amplitude cannot extend much past 180° without the two wings interfering with each other physically and aerodynamically ( Chai and Dudley , 1995; Chai et al . , 1997; Chai and Millard , 1997; Altshuler and Dudley , 2003 ) .", "Maximum load lifting also elicits a substantial increase in wingbeat frequency as a constant fraction of baseline wingbeat frequency ( Altshuler and Dudley , 2003 ) .", "Thus , maximum load lifting performance involves brief increases in muscle strain and muscle velocity to physically imposed limits .", "The capacity to increase muscle strain and velocity has previously been shown to influence foraging behavior and competitive ability ( Altshuler , 2006 ) .", "The results of the current study demonstrate that it also underlies multiple features of maneuvering performance .", "The two performance metrics that were not repeatable are vertical accelerations and decelerations , which were expected to be important based on previous observations of hummingbird competitive interactions ( Altshuler , 2006 ) and mating displays ( Clark , 2009 ) .", "Moreover , vertical performance was not well predicted by morphology , burst capacity , or competitor presence in this study .", "The dimensions of our experimental chamber likely influenced our observations of vertical performance .", "Hummingbirds in captivity tend to fly near the top of their cages , and the vertical dimension of the chamber ( 1 . 5 m ) may have limited vertical movement .", "Male hummingbirds are extremely aggressive towards conspecifics ( Kodric-Brown and Brown , 1978; Carpenter et al . , 1983 ) and other species of hummingbirds ( Stiles and Wolf , 1970; Wolf et al . , 1976 ) .", "The most territorial species will vigorously defend territories ( Carpenter et al . , 1983 ) and lekking sites ( Rico-Guevara and Araya-Salas , 2015 ) .", "In staged competition studies , paired hummingbirds will also establish and defend territories ( Tiebout , 1993 ) .", "We originally intended to use competition to elicit high levels of flight activity and maneuvering performance in territorial male Anna's hummingbirds ( Stiles , 1982 ) .", "However , we found that competitor presence affected only a small number of the maneuvering performance metrics that we measured .", "Pitch-down velocity increased with competition whereas horizontal acceleration and deceleration actually decreased .", "We do not know why these three metrics ( in addition to PRT%; see below ) were strongly affected by competition or why they were affected in the directions observed .", "However , there are several possible causes for why competitor presence did not affect the other metrics:", "1 ) we were unable to elicit a high level of competition or territoriality;", "2 ) the birds may have worked out dominance without the aggressive interactions normally seen outdoors; and/or", "3 ) the interactions required to establish dominance may have been very brief ( Maynard Smith , 1974 ) such that they comprised only a minuscule sample of the maneuvers we analyzed .", "This experiment was not designed to study the effects of maneuvering performance on competitive success , although this represents an important topic for future investigation .", "Laboratory performance tests do not always reflect field behavior ( Irschick , 2003 ) and outdoor studies of maneuvering performance will be important for understanding the role of maneuverability in competitive interactions .", "Recent advances in video tracking ( Theriault et al . , 2014; Shelton et al . , 2014 ) should make it possible to track individuals for multiple measurements .", "The most substantial result of competitor presence was the increase in the use of arcing over pitch-roll turns .", "These two types of turns represent different strategies for changing direction that differ in duration and amount of heading change .", "Arcing turns require less time but are used for smaller heading changes , whereas pitch-roll turns are longer but can be used to change heading by 180° ( Figure 7 ) .", "Given that hummingbird agonistic interactions can involve direct contact and stabbing with bills ( Tiebout , 1993; Clark and Russell , 2012; Rico-Guevara and Araya-Salas , 2015 ) , slow turns in place could make a bird more vulnerable during competition . 10 . 7554/eLife . 11159 . 016Figure 7 . Arcing and pitch-roll turns are two classes of complex maneuver that differ in turn magnitude and duration . Representative examples of arcing", "( a ) and pitch-roll", "( b ) turns are depicted from the above perspective .", "Arcing turns ( Arc; orange ) and pitch-roll turns ( PRT; green ) differed in the degrees turned", "( c ) and elapsed time", "( d ) .", "Circles represent bird-trial means ( n = 52 ) with grand means indicated with black lines .", "Histograms for the pooled dataset of all maneuvers are given on the right .", "The outliers for degrees turned in pitch-roll turns were included when calculating the grand means but not in the model analyses ( Table 4 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 11159 . 016 The relative use of arcing and pitch-roll turns was the only metric in our study that was influenced by all of morphology , burst muscle capacity , and competitor presence .", "The minimum radius of an arcing turn is limited by the maximum centripetal acceleration that a bird can generate while maintaining lift .", "The speed of a pitch-roll turn is limited by the ability to decelerate and then accelerate .", "Birds with higher wing aspect ratio may have preferred arcing turns because they were able to generate higher centripetal accelerations .", "Birds with higher burst muscle capacity may have favored pitch-roll turns because they had higher accelerating and decelerating performance .", "These observations suggest the hypothesis that high aspect ratio and high burst capacity enhance maneuverability .", "This hypothesis could be evaluated by comparing hummingbird species that differ in wing shape , foraging strategy , and burst capacity ( Altshuler et al . , 2004b; 2010a; Altshuler , 2006; Kruyt et al . , 2014 ) .", "By constraining hummingbirds to fly in a large chamber we were able to track and measure a large sample of maneuvers attributed to individuals with known morphological traits and burst performance .", "A major contribution of our study is the development of an assay of free flight maneuvering performance based on large numbers of stereotyped movements .", "Using this method , we identify several performance metrics that were highly repeatable across trials for individual hummingbirds , strongly correlated with individual morphological and physiological characteristics , and largely uninfluenced by the added motivation of a conspecific competitor .", "This approach to measuring maneuverability will be useful for future studies comparing maneuvering performance across different experimental manipulations , geographic ranges , or ecological , morphological , and phylogenetic groups ." ], [ "We captured and filmed 20 adult male Anna's hummingbirds ( Calypte anna ) at the University of California , Riverside ( eight birds in July-October 2009; four birds in January-March 2010 ) and the University of British Columbia ( eight birds in December 2013-April 2014 ) .", "The hummingbirds were housed in individual cages and fed ad libitum with a solution of artificial nectar ( Nektar-Plus , Nekton , Pforzheim , Germany ) and sucrose .", "The flight arenas were large rectangular cages ( 3 x 1 . 5 x 1 . 5", "m ) built with an aluminum frame and had either garden mesh ( California ) or clear acrylic ( British Columbia ) side panels .", "The cages contained multiple perches and a single feeder hung from the roof of the cage .", "Before the first trial , each bird was allowed to acclimate to the flight arena and learn where the perches and the feeder were located .", "The trials began once the birds were actively exploring the cage and consistently visiting the feeder and both perches .", "At this point , we recorded high-speed video of a two-hour solo trial for each bird .", "Following solo trials ( between 0–23 days later ) , birds were paired and filmed for another two hours in competition trials .", "One bird in each pair was marked with a small square of retro-reflective tape placed between the shoulder blades for identification .", "The birds filmed in British Columbia had one competition trial and the birds in California had two competition trials .", "In the latter case , the second trial consisted of previously unknown opponents that were chosen randomly from the remaining pool .", "The competition trials involved chases , displacements , and aerial displays , but very little contact .", "Regardless , we monitored the competition trials to ensure that no birds were harmed or excluded from the feeder .", "Following each round of solo and competition trials , we performed asymptotic load lifting experiments using the techniques described in Chai et al . ( Chai et al . , 1997 ) , and subsequently used in other studies estimating maximum burst power output ( Chai and Millard , 1997; Altshuler et al . , 2004a; 2010b; Altshuler , 2006 ) .", "Here , we use the mass of maximum number of beads lifted by each individual as a measure of burst performance .", "Immediately following load lifting , we weighed the birds and photographed both wings in an outstretched position against white paper with a reference scale ( Chai and Dudley , 1995 ) .", "We oriented the wing image and divided it into pixel wide strips representing the wing chords at each value of wing radius .", "Values for aspect ratio , wing area , and wing length were then calculated based on equations in Ellington ( Ellington , 1984 ) .", "We considered wing area and wing length as two potential measures of wing size , but these traits were highly correlated in our dataset ( R2 = 0 . 85 , p < 0 . 0001 , n = 20 ) .", "Because these two traits did not vary independently in our relatively small sample of 20 hummingbirds , we could not consider them independently .", "We therefore selected wing length as the more robust measure of wing size , because unlike area , wing length is less prone to measurement error as a result of variation in feather overlap when wings are positioned for measurements .", "We verified that our results were consistent when using wing area instead of length , and thus these two traits should be considered interchangeable as measures of wing size in this study .", "Because both wing morphology and muscle capacity may influence burst performance , we used burst performance controlled statistically for wing morphology as a measure of burst muscle capacity .", "Further details are provided below in the Statistical analysis section .", "All procedures were conducted under approval of the Institutional Animal Care and Use Committee at the University of California , Riverside and the Animal Care Committee at the University of British Columbia .", "We used an automated tracking system to measure both body position and orientation of flying birds in three dimensions ( Video 1 ) .", "A complete description of the tracking algorithm and hardware components is provided in ( Straw et al . , 2011 ) .", "The core algorithms were written in Python ( Python Software Foundation , 2012 ) , and are available via github ( PyMVG: https://github . com/strawlab/pymvg; adskalman: https://github . com/astraw/adskalman; MultiCamSelfCal: https://github . com/strawlab/MultiCamSelfCal/ ) .", "We adapted this system for recording hummingbird solo and competitive flight trajectories with four or five digital cameras ( GE680 , Allied Vision Technologies , Burnaby , Canada ) .", "The cameras were mounted on the ceiling and recorded at 640 x 480 pixel resolution at 200 frames per second ( Figure 1a ) .", "We calibrated the filming volume by moving a single light-emitting diode throughout the arena to acquire data for an automated self calibration algorithm ( Svoboda et al . , 2005 ) .", "This algorithm provides a relative calibration ( non-linear warping distortion parameters and 3x4 camera calibration matrices ) across all cameras .", "This calibration is brought into absolute terms ( the scale , rotation , and translation are found ) by matching a manually measured 3D model of the flight arena with reconstructed image coordinates using the ‘estsimt’ function of the MultiCamSelfCal toolbox ( Svoboda et al . , 2005 ) .", "To minimize the effect of errors in the 3D tracking , we used a forward/reverse non-causal Kalman filter ( Rauch–Tung–Striebel smoother ) applied to the online state estimate of position and velocity from the realtime Kalman filter .", "The smoothing parameters were chosen so that seven traces of a tracked , falling object yielded an average peak acceleration of 9 . 8 m/s2 .", "The process covariance matrix we used is: Qpos=σ2×T3300T22000T3300T22000T3300T22T2200T000T2200T000T2200T where σ2 is 0 . 01 and T is the interval between frames ( 0 . 005 s ) .", "The observation covariance matrix we used is: Rpos=0 . 0001440000 . 0001440000 . 000144 Figure 1c and d show examples of two trajectories with plots of the unsmoothed data , the data smoothed with Qpos and Rpos , and the effects of two different smoothing parameters ( Rpos x 10 , Rposx 0 . 1 ) .", "Following establishment of the 3D trajectories , the tracking system assigned 3D body orientation vectors to each bird in each frame based on 2D estimates of the long axis of the body .", "Body orientation was estimated using an algorithm that fit orientations to the body axis in each 2D image .", "Each sequence of five consecutive images cropped around the bird was aligned at the optical center of intensity .", "Averaging these images effectively eliminated the wings and emphasized the body .", "Orientation was estimated by calculating the covariance matrix of the image luminance and then computing the eigensystem of this covariance matrix .", "The eigenvector associated with the largest eigenvalue was taken as the orientation .", "Orientation vector assignments were also smoothed with a Kalman filter using more restrictive smoothing parameters than were used to smooth body position .", "To determine appropriate smoothing parameters we replotted the smoothed body orientation vectors onto a sample of videos , and visually chose the ones that provided the best fit .", "The process covariance matrix used for body orientation ( Qori ) is the same as the process covariance matrix used for body position ( Qpos ) and the observation covariance matrix used is: Rori=0 . 000001440000 . 000001440000 . 00000144 Once the body orientations were calculated we used a dynamic programming algorithm to decide which end of the vector was the head and which end was the tail .", "The direction of the head was chosen based on the direction of the previous orientation , the direction of travel , and the vertical up direction .", "For each frame ( n ) , the 'cost' associated with the two possible orientations ( Ori→ , -Ori→ ) were calculated: CostOri=Speed ( Ori→n⋅Vel→modOri→nVel→mod ) + ( 1−Speed ) ( Ori→n⋅Up→Ori→nUp→ ) + ( 1−Speed ) ( Ori→n⋅Ori→n−1Ori→nOri→n−1 ) Cost−Ori=Speed ( −Ori→n⋅Vel→modOri→nVel→mod ) + ( 1−Speed ) ( −Ori→n⋅Up→Ori→nUp→ ) + ( 1−Speed ) ( −Ori→n⋅Ori→n−1Ori→nOri→n−1 ) where Ori→ is the body vector , Vel→mod is the modified velocity vector tipped up 15º towards the vertical direction .", "Up→ is the vertical direction vector , Ori→n−1 is the orientation during the previous frame , and if the magnitude of the velocity is greater than 0 . 5m/s: Speed=Vel→ otherwise: Speed=0 . 5 This approach accounted for the tendency of hummingbirds to fly forwards and with an upright posture , but allowed for exceptions in the case of backwards flight , inversions , and dives , particularly if these occurred at low speeds .", "The magnitudes of calculated accelerations and , to a lesser extent , velocities derived from position data were influenced by the specific smoothing parameters .", "Examples of maneuvers with different smoothing parameters and their effects on the calculated performance metrics are given in Figure 1c and d .", "This influence of smoothing parameters is a well known limitation of video tracking ( Walker , 1998 ) .", "Thus , although acceleration values are comparable within a study , caution must be applied when comparing the magnitude of acceleration values among studies differing in camera frame rate , filming volume , calibrations , and smoothing parameters ( Walker , 1998 ) .", "For our final performance metrics we used instantaneous body orientation and orientation velocity , but not orientation acceleration .", "The automated tracking system extracted the 3D coordinates of multiple flying animals and saved each trajectory as a separate object ( Video 1 ) .", "An object began when the tracking system detected new movement and ended when either the object stopped moving , the error in the 3D reprojection grew too large , or multiple objects came within 2 cm of each other .", "In our experiments tracking hummingbird flight led to two problems in determining distinct objects .", "The first is that very stable hovering can be misidentified as perching .", "For example , as a bird went into an extended hovering bout , such as at a feeder , the tracking system detected the cessation of movement and ended the trajectory .", "Conversely , when the bird perched at the end of a flight or in between two flights , especially if it continued to move its head or fluff its feathers , the tracking system treated the bird as moving and continued the trajectory .", "Because our study focused on identifying and analyzing relatively long , moving trajectories , these types of errors did not cause problems .", "The second challenge concerned identification of birds during close encounters in competition trials .", "When two tracked objects became close to each other , even if they did not physically touch , the tracking system could not accurately distinguish them .", "We used a conservative solution and terminated the trajectories whenever two birds came close enough that the tracked objects merged .", "Birds were later identified manually by a team of digitizers who viewed the videos and assigned each object number to either the marked or unmarked bird .", "The automated digitization produced a small number of extreme tracking errors , which we did not want to unduly influence statistical analyses .", "We accordingly removed values >5 SDs more extreme than the mean for each performance metric .", "The trimmed values comprised only 0–0 . 31% of the original pooled sample size for each metric .", "We next calculated the mean of each performance metric for each bird-trial combination ( n= 52 means; 20 birds in 20 solo trials and 16 paired competition trials ) .", "All statistical analyses were performed on these bird-trial means using R 3 . 1 . 1 ( R Development Core Team , 2014 ) , and the data used for the analysis are available online ( Segre et al . , 2015 ) .", "Repeatability , or the intra-class correlation coefficient ( ICC ) , is defined as the proportion of variation that is attributable to differences among individuals ( Nakagawa and Schielzeth , 2010 ) .", "We estimated repeatability for each performance metric from an intercept-only mixed effects model that included estimates of the population intercept ( i . e . , the grand mean ) as well as an individual intercept for each bird ( Nakagawa and Schielzeth , 2010 ) .", "Such a model has two variance components , the variance of the random intercept values ( variance among individuals ) and a residual variance associated with the error term .", "Repeatability is the variance among individuals divided by the total variance ( Nakagawa and Schielzeth , 2010 ) .", "We used parametric bootstrapping with 5000 iterations to obtain confidence intervals for these repeatability estimates via the bootMer function in the lme4 ( v1 . 1 . 7 ) package .", "Because our second question involved evaluating several possible scenarios for the influence of morphology and burst performance on maneuverability , we used an information-theoretic approach to multi-model inference ( Burnham and Anderson , 2010 ) .", "Unlike dichotomous null hypothesis testing , this approach quantifies support for multiple hypotheses , and it avoids the problem of eliminating potentially important predictors when two or more alternative models are equally well supported .", "The output for interpretation includes the effect size and relative importance of each predictor , and there are no null hypotheses or P values associated with this approach .", "As a measure of effect size we report the standardized partial regression coefficient , std β , for each predictor , which can be used to compare their independent associations with a given response variable .", "Unstandardized regression coefficients corresponding to units of the predictor variables are provided in Supplementary file 1 .", "We also examined associations between burst performance , wing size , and wing shape because our load lifting assay may have incorporated effects of wing morphology as well as muscle capacity .", "The mass of weights lifted during load lifting was not significantly associated with wing length in our sample of 20 individuals ( p = 0 . 87 ) , however , it was negatively associated with wing aspect ratio ( p = 0 . 04 ) controlling for site .", "Thus in our model analyses we used residual burst performance controlling for wing aspect ratio and site as a measure of burst muscle capacity independent of a bird’s wing morphology .", "We considered eight candidate mixed-effects models that could plausibly explain variation in each maneuvering performance metric ( Table 5 ) .", "All candidate models included an individual intercept for each bird ( the random intercept term ) and were fit using the nlme ( v 3 . 1–117 ) package ( Zuur et al . , 2009 ) .", "The intercept-only model included an estimate of the population intercept ( grand mean ) and random intercept terms , but no fixed effects .", "Other candidate models are listed in Table 5 .", "All models except the intercept-only model included the fixed effects of competitor presence , body mass , and experiment to account for potential effects of these factors .", "Experiment had three levels , one for each round of trials ( California 2009 , 2010 , British Columbia 2014 ) to account for differences such as location , time of year , and filming conditions . 10 . 7554/eLife . 11159 . 017Table 5 . Candidate models of maneuvering performance .", "All models include an intercept as well as a random effect of bird identity to account for repeated measures of individuals . DOI: http://dx . doi . org/10 . 7554/eLife . 11159 . 017ModelFixed effectsDescription1 . Solo/comp + experiment + body mass + wing lengthWing size2 . Solo/comp + experiment + body mass + wing aspect ratioWing shape3 . Solo/comp + experiment + body mass + wing length + wing aspect ratioWing size & shape4 . Solo/comp + experiment + body mass + weight liftedBurst power5 . Solo/comp + experiment + body mass + weight lifted + wing lengthBurst power & wing size6 . Solo/comp + experiment + body mass + weight lifted + wing aspect ratioBurst power & wing shape7 . Solo/comp + experiment + body mass + weight lifted + wing length + wing aspect ratioBurst power , wing size & shape8 . Intercept-only*Candidate models 1-7 also include a fixed effect of days post-capture for the following metrics: Velmax , AccHormax , DecHormax , Arcvel , avg , and Arccent , max Two issues arose in the preliminary examination of data .", "The first issue was that five of the performance metrics were significantly influenced by the number of days a bird had been in captivity .", "We therefore included an additional fixed effect of the number of days since capture when analyzing these five metrics ( Table 5 ) .", "The second issue was that one of the metrics , the heading change in pitch-roll turns ( PRTdeg ) , had three values that were significant outliers ( Grubb’s test , all G > 3 . 09 , all p<0 . 03; Figure 7 ) .", "We determined that these three statistical outliers were not errors in the tracking system but were instead derived from one individual that used pitch-roll turns to make small heading changes , unlike the other birds .", "We omitted these outliers from the analysis of heading change in pitch-roll turns to ensure that all fitted Gaussian models met the required assumptions , with no other outliers or problems of skew or heteroskedasticity .", "The best-fit model for heading change in pitch-roll turns was the intercept-only model regardless of whether the outliers were included .", "To quantify the variance explained by the fixed effects of interest in each model , we calculated the marginal R2GLMM", "( m ) using the r . squaredGLMM function in the MuMIn ( v1 . 10 . 5 ) package ( Nakagawa and Schielzeth , 2013 ) .", "This measure does not have all the properties of a traditional coefficient of determination , but like R2 it ranges from 0 to 1 , and it is an appropriate estimate of the variance explained by the fixed effects in a mixed model .", "We removed the effect of experiment and the number of days post-capture when calculating R2GLMM", "( m ) , because these were not effects of interest .", "Thus , R2GLMM", "( m ) provides a measure of the variance explained by the other supported fixed effect variables .", "We evaluated the support for different models using the Akaike information criterion ( AICc ) adjusted for small sample sizes .", "This was calculated using the MuMIn ( v 1 . 10 . 5 ) package with maximum likelihood estimation .", "We defined the group of supported models as those with a difference in AICc < 2 from the best-fit model for each performance metric .", "If no other models came within 2 AICc units of the best-fit model , we present effect size measures , their confidence intervals , and R2GLMM", "( m ) for only that model .", "Otherwise , we present averages of all supported models .", "Details of all candidate models are provided in Supplementary file 1 .", "Our third question concerned the influence of competitor presence on the performance metrics .", "If the confidence interval for the coefficient estimate of competitor presence excluded zero , we examined the magnitude and direction of that effect .", "Positive coefficient estimates indicate that performance was higher during competitive flights , whereas negative coefficients indicate that performance was lower in the presence of a competitor ." ] ]

[ "Despite recent advances in the study of animal flight , the biomechanical determinants of maneuverability are poorly understood .", "It is thought that maneuverability may be influenced by intrinsic body mass and wing morphology , and by physiological muscle capacity , but this hypothesis has not yet been evaluated because it requires tracking a large number of free flight maneuvers from known individuals .", "We used an automated tracking system to record flight sequences from 20 Anna's hummingbirds flying solo and in competition in a large chamber .", "We found that burst muscle capacity predicted most performance metrics .", "Hummingbirds with higher burst capacity flew with faster velocities , accelerations , and rotations , and they used more demanding complex turns .", "In contrast , body mass did not predict variation in maneuvering performance , and wing morphology predicted only the use of arcing turns and high centripetal accelerations .", "Collectively , our results indicate that burst muscle capacity is a key predictor of maneuverability ." ]

[ "The ability of an animal to maneuver can determine its success at avoiding predators , catching prey , and outperforming its competitors .", "However , little is known about the characteristics that determine maneuverability .", "Why are some individuals more maneuverable than others ?", "To investigate this question , Segre et al . used an automated video tracking system to track male Anna's hummingbirds as they flew around a large chamber .", "These tracks were then compared with the physical characteristics of the birds to see which , if any , affect the birds’ maneuverability .", "This revealed that body size did not affect how well the birds could maneuver .", "Instead , the muscle capacity of the birds – their ability to generate force rapidly – determined how well the birds performed most types of movement .", "Birds with higher muscle capacity flew faster , had faster accelerations and decelerations , could rotate their bodies more quickly , and performed more demanding and complex turns .", "Segre et al . also found that wing shape is important for a type of maneuver called an arcing turn .", "Hummingbirds with a more slender wing shape were able to execute more demanding arcing turns involving higher accelerations , and they used arcing turns more often than birds with wider wings .", "Future research will aim to determine whether these relationships are also found in other species of birds ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "structural biology and molecular biophysics" ]

[ [ "In most cells , the bulk of ATP , the principal source of cellular energy , is synthesized by ATP synthase .", "This molecular generator couples ion flow across membranes with the addition of inorganic phosphate ( Pi ) to ADP thereby generating ATP ( Iino and Noji , 2013; Stewart et al . , 2014 ) .", "Most bacteria , including Escherichia coli have only one type of rotary ATPase , referred to as F-type ATPase .", "Like the analogous complexes in other kingdoms , it is based on two reversible motors , termed F1 and Fo ( Negrin et al . , 1980 ) , connected by central and peripheral stalks ( Wilkens and Capaldi , 1998a ) ( Figure 1 ) .", "The Fo motor spans the membrane converting the potential energy of the proton motive force ( pmf ) into rotation of the central stalk that in turn drives conformational changes in the F1 catalytic sites . 10 . 7554/eLife . 21598 . 003Figure 1 . Schematic illustration showing the arrangement of subunits in E . coli F-ATPase . Subunits α in red , β in yellow , γ in blue , ε in green , c in grey , a in orange , b in magenta or pink , and δ in teal .", "The proton path and ATP synthesis are labeled accordingly . DOI: http://dx . doi . org/10 . 7554/eLife . 21598 . 003 The Fo motor is constructed from subunits a , b and c ( Figure 1 ) .", "Subunit c assembles into a ring , thought , in E . coli , to have decameric stoichiometry ( Jiang et al . , 2001; Ballhausen et al . , 2009; Düser et al . , 2009; Ishmukhametov et al . , 2010 ) , whereas subunits a and b associate to form a helical bundle adjacent to this ring .", "Recent sub nanometer electron cryo-microscopy ( cryo-EM ) reconstructions of F-type ( Allegretti et al . , 2015; Zhou et al . , 2015; Kühlbrandt and Davies , 2016; Hahn et al . , 2016 ) and the analogous V- and A-type ATPases ( Zhao et al . , 2015; Schep et al . , 2016 ) as well as a low-resolution crystal structure of Paracoccus denitrificans F-ATPase ( Morales-Rios et al . , 2015 ) are consistent with a two half-channel mechanism for the generation of rotation within the membrane ( Vik and Antonio , 1994; Junge et al . , 1997 ) .", "All structures confirm that a four-helix bundle of subunit a , inclined by 20–30˚ to the membrane plane , forms a crucial structural component .", "In this mechanism , protons from the bacterial periplasm access a conserved negatively charged carboxylate in subunit c ( Asp61 in E . coli [Hoppe et al . , 1982] ) through an aqueous half channel at the subunit a/c interface ( Steed and Fillingame , 2008 ) .", "Neutralizing this carboxylate enables the c-ring to rotate within the hydrophobic membrane and to access a second aqueous half channel that opens to the cytoplasm into which the protons are released ( Pogoryelov et al . , 2010 ) .", "A conserved arginine residue in helix-4 of subunit a ( Arg210 in E . coli ) prevents the c-ring rotating in the opposite direction and short-circuiting of the system ( Lightowlers et al . , 1987; Cain and Simoni , 1989; Mitome et al . , 2010 ) .", "The sequential binding of protons in combination with thermal fluctuations generates rotation within the complex in a manner akin to a turbine ( Oster and Wang , 1999; Pogoryelov et al . , 2010; Aksimentiev et al . , 2004 ) .", "The torque generated in the Fo motor is then transferred to the F1 motor by the central shaft consisting of subunits γ and ε ( Wilkens et al . , 1995 ) .", "The N- and C-termini of subunit γ form a curved coiled-coil that extends into the central cavity of F1 .", "The F1 motor is the chemical generator in which ATP is synthesized .", "The motor comprises a ring of three heterodimers , each containing an active site at the interface of subunits α and β .", "Within the F1 motor , each αβ dimer has a different conformation at any point in time and can be either empty , bound to ADP and Pi , or bound to ATP ( open , half-closed , closed ) ( Abrahams et al . , 1994; Yoshida et al . , 2001 ) .", "These different catalytic states relate to the position of the curved coiled-coil of subunit γ in the central stalk , which drives the conformational changes associated with catalysis .", "To enable the central stalk to rotate relative to the F1 αβ heterodimers , the Fo and F1 motors need to be coupled .", "This coupling is mediated by the peripheral stalk that is constructed from subunits b and δ ( Figure 1 ) .", "Subunit b forms an amphipathic homodimeric coiled-coil that spans the periphery of the complex linking subunit a with subunit α , whereas subunit δ provides additional coupling of the C-termini of the b and α subunits ( Carbajo et al . , 2005; Wilkens et al . , 2005 ) .", "The bacterial F-ATPase can also function in reverse , employing ATP hydrolysis to generate a proton gradient across the membrane when needed ( von Ballmoos et al . , 2008 ) .", "In vivo , subunit ε is believed to change conformation in an ATP-dependent manner to prevent rotation of the complex ( Capaldi et al . , 1992; Rodgers and Wilce , 2000; Yagi et al . , 2007; Imamura et al . , 2009 ) thereby conserving ATP when its concentration is low .", "This regulatory function is mediated by the C-terminal domain of subunit ε ( εCTD ) that , when not bound to ATP , opens to an extended conformation and inserts into the αβ heterodimers .", "The crystal structures of both the E . coli and Bacillus PS3 F1 motors in this autoinhibited state show the εCTD intercalating into the αβ heterodimers ( Cingolani and Duncan , 2011; Shirakihara et al . , 2015 ) .", "However , in each structure , the F1 motor had been captured in a different conformation ( E . coli – half-closed , closed , open [Cingolani and Duncan , 2011] and Bacillus PS3 – open , closed , open [Shirakihara et al . , 2015] ) which could either relate to inter species differences or crystal contacts and crystallization conditions .", "The crystal structure of the F-ATPase from P . denitrificans ( Morales-Rios et al . , 2015 ) , that is closely related to E . coli ( 38% sequence identity over all subunits ) , shows a similar overall architecture to bovine F1Fo ATP synthase as well as to the main features of A/V ATPases .", "However , it is inhibited by the ζ-protein rather than by subunit ε , which is generally employed by bacterial F-ATPases for this purpose .", "Moreover , this crystal structure shows only one conformation of the rotary catalytic cycle .", "Here , we present three cryo-EM maps along with molecular models of E . coli F-ATPase in its autoinhibited state , determined to resolutions of 6 . 9 , 7 . 8 , and 8 . 5 Å .", "In all three reconstructions , the εCTD is in an extended conformation , stabilizing an overall F1 motor conformation similar to that seen in the thermophilic Bacillus PS3 F1 ATPase structure .", "Density for the peripheral stalk extends the entire length of the complex and its coiled-coil bifurcates towards the N-terminus to enter the membrane as two separate helices that clamp the a subunit to the c ring .", "Moreover , our maps allowed us to interpret the complete F1-delta interface , showing the three α subunit N-termini in distinct orientations .", "Each map also confirmed the c-ring stoichiometry to be decameric , which to date has been only characterized by crosslinking and single molecule analyses .", "We used our maps in combination with published crosslinking and mutagenesis information to generate a molecular model of the complex in three states .", "These models provide crucial structural information on a key complex that extends our understanding of the mechanism of rotary ATPases in general , together with information on the bacterial ATP synthase , which is seen as an important antimicrobial target in organisms related to E . coli such as Mycobacterium tuberculosis ( Ahmad et al . , 2013 ) ." ], [ "Cysteine-free E . coli F-ATPase , as described in Ishmukhametov et al . ( 2005 ) where all 10 cysteines were replaced with alanines and a His-tag introduced on the β subunit , was solubilized in digitonin detergent and purified as described in the Materials and methods .", "This procedure provided pure protein ( Figure 2—figure supplement 1 ) capable of ATP hydrolysis-driven proton pumping upon reconstitution into proteoliposomes ( Figure 2—figure supplement 1 ) .", "N , N`-dicyclohexylcarbodiimide ( DCCD ) , a compound which selectively modifies Asp61 of subunit c at 50 µM ( Pogoryelov et al . , 2010 ) completely abolished proton pumping ( Figure 2—figure supplement 1B ) and inhibited 90% of ATPase activity of isolated protein ( Figure 2—figure supplement 1C ) .", "Such inhibition indicates coupling between the F1 and Fo motors ( Cook et al . , 2003; Peskova and Nakamoto , 2000; Tsunoda et al . , 2000 ) .", "Protein was further examined by cryo-EM without addition of nucleotides .", "395 , 140 particles were picked , of which 216 , 711 were used in refinement .", "Three different conformations of the complex were identified using 3D classification in RELION ( Scheres , 2012 ) .", "The particles in each subset were then refined to generate sub-nanometre reconstructions , to a resolution of 6 . 9 , 7 . 8 and 8 . 5 Å ( Figure 2A–C , and Figure 2—figure supplements 2 and 3 ) .", "In these three conformations , the central stalk was progressively rotated 120° relative to the peripheral stalk . 10 . 7554/eLife . 21598 . 004Figure 2 . The three states of the autoinhibited E . coli F-ATPase .", "( A–B )", "Cryo-EM maps shown as surface representation , states 1 , 2 and 3 , respectively , resulting from rotation of the central stalk by 120° .", "( D–F )", "Molecular models built into the cryo-EM maps shown as cartoon representation .", "Subunits α in red , β in yellow , γ in blue , ε in green , c in grey , a in orange , b in magenta or pink and δ in teal . DOI: http://dx . doi . org/10 . 7554/eLife . 21598 . 00410 . 7554/eLife . 21598 . 005Figure 2—source data 1 . Data collection and image processing statistics . DOI: http://dx . doi . org/10 . 7554/eLife . 21598 . 00510 . 7554/eLife . 21598 . 006Figure 2—figure supplement 1 . Characterization of E . coli F1Fo ATP synthase used for cryo-EM .", "( A ) SDS page gel showing protein purity .", "Lane1: SeeBlue Plus2 marker .", "Lane 2: E . coli F-ATPase .", "( B ) Inhibition of ATP-hydrolysis-driven proton pumping by F1Fo in the presence of and without 50 µM DCCD .", "The protein was reconstituted into proteoliposomes and assayed for ACMA quenching as described in the Materials and methods .", "Red arrow marks addition of ATP , blue arrows indicate addition of the uncoupler FCCP .", "( C ) Inhibition of ATP hydrolysis by isolated F1Fo in the presence of 50 µM DCCD .", "The protein was inhibited with and without 50 µM DCCD and assayed with the ATP regenerating system as described in Materials and methods . DOI: http://dx . doi . org/10 . 7554/eLife . 21598 . 00610 . 7554/eLife . 21598 . 007Figure 2—figure supplement 2 . cryoEM analysis .", "( A ) Representative micrograph with picked particles , scale bar = 500 Å .", "( B ) 25 highest populated classes from round 1 2D classification , scale bar = 100 Å .", "( C ) Gold standard FSC plots of States 1 , 2 and 3 . ( D ) Orientation distribution of particles in the State one reconstruction . DOI: http://dx . doi . org/10 . 7554/eLife . 21598 . 00710 . 7554/eLife . 21598 . 008Figure 2—figure supplement 3 . Flowchart describing cryoEM data analysis . DOI: http://dx . doi . org/10 . 7554/eLife . 21598 . 00810 . 7554/eLife . 21598 . 009Figure 2—figure supplement 4 . Examples of the electron density map of State 1 , to highlight strengths and weaknesses .", "( A ) Extra density at the N-terminus of subunit β shows 6xHis-tag .", "( B ) Helical register visible in the density for the peripheral stalk .", "( C ) β barrel of the ε subunit .", "( D ) Poor density for the c-ring . DOI: http://dx . doi . org/10 . 7554/eLife . 21598 . 00910 . 7554/eLife . 21598 . 010Figure 2—figure supplement 5 . Local resolution map of State 1 . ( A ) Overall complex .", "( B ) Slice through F1 .", "( C ) Slice through FO .", "Dashed lines show area of cross section and scale on right shows color/resolution relationship .", "Made with ResMap ( Kucukelbir et al . , 2014 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 21598 . 01010 . 7554/eLife . 21598 . 011Figure 2—figure supplement 6 . Quality of the models built into the state one cryoEM map . The model presented is a mosaic refined model created by docking crystal , NMR , homology models and de novo built sections .", "The colors represent the origin of these models prior to refinement .", "Crystal structures in blue , NMR models in cyan , homology model using a crystal structure in green , modeled using other EM structure as guide in yellow and built into the map in red . DOI: http://dx . doi . org/10 . 7554/eLife . 21598 . 01110 . 7554/eLife . 21598 . 012Figure 2—figure supplement 7 . Position of the natural cysteines in E . coli F1Fo . CryoEM map of state one containing built model .", "Yellow spheres depict the positions of Cys-Ala mutants in the cysteine-free sample used . DOI: http://dx . doi . org/10 . 7554/eLife . 21598 . 01210 . 7554/eLife . 21598 . 013Figure 2—figure supplement 8 . Transmembrane architecture of ( A ) E . coli , ( B ) P . denitrificans , ( C ) Y . lipolytica . Unassigned subunits of P . denitrificans shown in cyan and subunit 8 of Y . lipolytica shown in pink . DOI: http://dx . doi . org/10 . 7554/eLife . 21598 . 01310 . 7554/eLife . 21598 . 014Figure 2—figure supplement 9 . Comparison of peripheral stalk position between the three states; diagrams on left depict part of complex that each state is superposed to . State 1 , 2 and 3 in red , green and blue , respectively .", "The F1 has been removed for clarity .", "( A–C )", "Structures superposed to the a subunit .", "( D–F )", "Structures superposed to the delta subunit .", "Arrows and triangle show distance displaced between states .", "Circles highlight possible hinge/swivel regions . DOI: http://dx . doi . org/10 . 7554/eLife . 21598 . 01410 . 7554/eLife . 21598 . 015Figure 2—figure supplement 10 . FSC curves showing the effects of masking on the refined map , with the gold-standard , corrected FSC curve ( black ) , FSC of the unmasked map ( green ) , FSC of the masked map ( blue ) , and FSC of the phase-randomized masked map ( red ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 21598 . 015 Even though the resolution of the reconstructions varied throughout the complex , it was sufficient to resolve individual helices .", "Additional density of the N-terminal His-tag of the β subunit , as well as helical and β sheet patterns observed in parts of the map in the F1 motor region illustrate the high quality of the maps , with the c-ring density being poorest ( Figure 2—figure supplement 4 ) .", "Local resolution estimates showed the region corresponding to the F1 motor to be of highest quality , the Fo motor with moderate detail and , the detergent micelle being clearly the worst region of the map ( Figure 2—figure supplement 5 ) .", "Docking of high-resolution crystal and NMR models of different components into the maps followed by manual building and refinement enabled virtually complete molecular models of the three different states to be built ( Figure 2D–F ) , with varying quality of the docked structures as indicated in Figure 2—figure supplement 6 . The positions of the Cys-Ala mutants are depicted in Figure 2—figure supplement 7 . The cryo-EM maps provided novel insights into the architecture and function of the E . coli F-ATPase .", "Thus , although its overall architecture was similar to that of F-ATPase from P . denitrificans ( Morales-Rios et al . , 2015 ) and F1Fo ATP synthases from Bos Taurus ( Zhou et al . , 2015 ) , Yarrowia lipolytica ( Hahn et al . , 2016 ) and Polytomella ( Allegretti et al . , 2015 ) , with the catalytic F1 motor attached to a proton powered membrane Fo motor and single central and peripheral stalks , differences in the individual motors and peripheral stalk were apparent .", "Comparison with the membrane-embedded motors from other sub nanometre cryo-EM maps indicated the E . coli F-ATPase had a simpler stator architecture , containing only seven helices in the a and b subunits rather than the eight seen in mitochondrial F-type ATP synthase ( Figure 2—figure supplement 8 ) , consistent with labeling approaches ( Wada et al . , 1999 ) .", "This difference suggested that the extra helices present in other rotary ATPase subtypes could have additional functions such as the dimerization seen in mitochondria ( Hahn et al . , 2016 ) .", "A movie generated by interpolation between the three states ( Video 1 ) indicated that the F1 motor rocks or wobbles during the catalytic cycle ( Kinosita et al . , 2000; Stewart et al . , 2012 ) as previously predicted , although of course the structures described here do not represent the complex in its uninhibited active synthesizing form .", "Two pivot points , one near the peripheral stalk/Fo interface ( ~bArg36 ) ( Welch et al . , 2008 ) and one near the peripheral stalk/F1 interface ( ~bGln106 ) , enabled the stalk to accommodate this eccentric movement of F1 ( Figure 2—figure supplement 9 ) . 10 . 7554/eLife . 21598 . 016Video 1 . Interpolation between States 1 , 3 and 2 to simulate ATP synthesis by E . coli F-ATPase . A and B are rotated 90° about the y-axis . DOI: http://dx . doi . org/10 . 7554/eLife . 21598 . 016 The maps showed a long right-handed coiled-coil dimer generated by the two b subunits of the peripheral stalk together with the globular δ subunit that anchors them to the catalytic head ( Figure 3A ) .", "The quality of the map was sufficient to enable almost the entire of the δ subunit to be built as a polyalanine model ( Figure 2D–F ) , whereas previous structural information was limited to the N-terminal domain ( Wilkens et al . , 2005 ) .", "Interestingly , the peripheral stalk contacted all three α subunits via their N-terminal helices , but did so asymmetrically employing three different interfaces with each α subunit ( Figure 4 ) .", "Although the resolution of the map was insufficient to assign the precise interface , the binding of the peripheral stalk to three anchor points in different geometries would provide a molecular key that would result in the δ subunit binding in a single orientation across the top of the symmetrical αβ heterodimers . 10 . 7554/eLife . 21598 . 017Figure 3 . The peripheral and central stalks of E . coli F-ATPase .", "( A ) The peripheral stalk is comprised of a globular head ( subunit δ in teal ) and a homodimeric coiled-coil ( subunits b in pink and magenta ) that bifurcates at the membrane interface to brace subunit a ( orange ) .", "( B ) The εCTD is in an extended conformation , inhibiting the enzyme from rotating .", "The arrow depicts the extended vs closed conformation of subunit ε .", "DOI: http://dx . doi . org/10 . 7554/eLife . 21598 . 01710 . 7554/eLife . 21598 . 018Figure 3—figure supplement 1 . Fitting of the of the autoinhibited E . coli F1-ATPase crystal structure ( pdb 3oaa ) into the State one cryoEM map of E . coli F-ATPase .", "( A and B ) 3oaa rigid body fitted into the cryoEM map .", "β1 needs substantial movement to fit the density well .", "( C and D )", "Final refined model of the E . coli F1-ATPase shows all subunits fitting well . DOI: http://dx . doi . org/10 . 7554/eLife . 21598 . 01810 . 7554/eLife . 21598 . 019Figure 3—figure supplement 2 . Stimulation of ATP hydrolase activity of isolated F1Fo by 0 . 4% LDAO . ATPase activity of the protein was registered with the ATP regenerating system as described in Materials and methods .", "Red arrow marks addition of the protein .", "Blue arrow marks addition of LDAO . DOI: http://dx . doi . org/10 . 7554/eLife . 21598 . 01910 . 7554/eLife . 21598 . 020Figure 4 . Subunit δ and peripheral stalk attachments to the α subunits . Top panel; left , the segmented cryoEM map viewed from the side and right , viewed from above with the orientation of views 1 , 2 and 3 depicted .", "Bottom panel; detailed views of the three attachment points labeled 1 , 2 and 3 , with δ in teal , b in pink and magenta and α in red . DOI: http://dx . doi . org/10 . 7554/eLife . 21598 . 020 The b subunits formed a homodimeric coiled-coil that spaned almost the entire complex ( 212 of 232 Å ) with their N-termini bifurcating just above the membrane to generate two separate helices within the membrane ( Figure 3A ) .", "This was unexpected , albeit reminiscent of the yeast F-type ATP synthase dimer ( Figure 2—figure supplement 8C ) , where subunit eight is an evolutionary derivative of the bacterial b subunit ( Hahn et al . , 2016 ) .", "Furthermore , NMR analysis of the transmembrane domain of E . coli F-ATPase b subunit ( Dmitriev et al . , 1999 ) showed a helical structure that was interrupted by a rigid 20° bend at residues 23–26 that result in a structure consistent with the b subunit bifurcation .", "All three reconstructions showed the complex in its autoinhibited state , with clear density for the εCTD extending deep into the central cavity of the F1 enzyme ( Figure 3B ) .", "Fitting of the E . coli α3β3γε crystal structure ( Cingolani and Duncan , 2011 ) into our cryo-EM maps showed that the β1 subunit had adopted a different more open conformation ( Figure 3—figure supplement 1 ) .", "In the above crystal structure , the εCTD contacts more subunits in F1 ( α1 , α2 , β1 , β2 and γ ) compared to our cryo-EM reconstructions , where it contacted fewer subunits ( α1 , α2 , β2 and γ ) .", "The conformation of our cryo-EM structure was more similar to that seen in the Bacillus PS3 structure ( Shirakihara et al . , 2015 ) , where it is proposed to be in an ‘open , closed , open’ conformation ( Figure 5 ) . 10 . 7554/eLife . 21598 . 021Figure 5 . Autoinhibted E . coli F1-ATPase conformation . The αβ hetrodimers of state 1 as viewed from the membrane with the peripheral stalk to the left of the figure .", "The ‘open , closed , open’ conformation of the F1 motor is labeled and the positions of nucleotides are shown as blue surfaces . DOI: http://dx . doi . org/10 . 7554/eLife . 21598 . 021 To ascertain the enzymatic state of the F1 motor , we generated a difference density map between the cryo-EM density and our built model .", "Remarkably , clear peaks were seen at the nucleotide binding pockets , indicating that the non-catalytic binding sites in subunit α contained nucleotide , as well as the ‘closed’ αβ heterodimer ( Figure 5 ) .", "These were modeled as ATP and ADP respectively based on known orientations of these nucleotides from high-resolution crystal structures .", "Interestingly , this did not correspond to the nucleotide binding states of either the E . coli ( Cingolani and Duncan , 2011 ) or the PS3 crystal structures ( Shirakihara et al . , 2015 ) .", "Single particle analysis revealed highly uniform homogeneity of the protein preparation , where 100% of F1Fo molecules observed demonstrated the extended conformation of εCTD .", "Our structural data were supported by an enzymatic assay ( Figure 3—figure supplement 2 ) , where the extent of ATP hydrolysis inhibition of the protein by subunit ε was tested with N , N-dimethyldodecylamine N-oxide ( LDAO ) , a well-known activator of the subunit ε inhibited protein .", "LDAO used at 0 . 4% ( weight/volume ) concentration has been shown to stimulate ATP hydrolysis by the E coli protein 3–4 times ( Dunn et al . , 1990; Peskova and Nakamoto , 2000 ) , including earlier studies ( Ishmukhametov et al . , 2008 , 2016 ) on the same cysteine-free F1Fo construct using the same batch of LDAO .", "Prior to addition of LDAO , the hydrolysis rate was 0 . 75 µmol ATP/min/mg protein but surprisingly in presence of 0 . 4% LDAO it was stimulated ~13 times .", "To our best knowledge , such a high LDAO stimulation of ATP hydrolysis by E . coli F1Fo was not described in the literature before and is consistent with our single particle data .", "Density in Fo defined the overall architecture of the membrane-embedded motor together with two invaginations of the detergent micelle that have previously been proposed to facilitate proton translocation ( Allegretti et al . , 2015; Kühlbrandt and Davies , 2016 ) ( Figure 6—figure supplement 1 ) .", "While the overall density of the c-ring was relatively weak , 10 peaks of density were clearly present when viewed from above ( Figure 6—figure supplement 2 ) , confirming the stoichiometry of the c-ring to be decameric in E . coli F-ATPase ( Jiang et al . , 2001; Ballhausen et al . , 2009; Düser et al . , 2009; Ishmukhametov et al . , 2010 ) .", "Furthermore , density inside the c-ring corroborates data suggesting it to be filled with phospholipids ( Oberfeld et al . , 2006 ) .", "By combining the helical density from the cryo-EM maps ( Figure 6A ) , with models previously suggested for the related bovine subunit , together with crosslinking data and transmembrane topography prediction for the E . coli F-ATPase ( Jiang and Fillingame , 1998; Valiyaveetil and Fillingame , 1998; Moore and Fillingame , 2008; Wada et al . , 1999 ) , it was possible to build a molecular model of the a subunit ( Figure 6B ) .", "The crosslinks mapped to two clusters ( Figure 6—figure supplement 3 ) , allowing a likely sequence register for the model to be proposed .", "This was consistent with the two half channel hypothesis , placing Arg210 of subunit a adjacent to Asp61 of the c-ring ( Figure 6B ) .", "Interestingly , density for the c subunit is clearest adjacent to Arg210 of subunit a suggesting this area to be well ordered ( Figure 6—figure supplement 4 ) . 10 . 7554/eLife . 21598 . 022Figure 6 . The E . coli F-ATPase subunit a and the suggested path of proton translocation .", "( A ) Density map of subunit a , shown as orange surface viewed from the c-ring .", "Grey outline depicts invaginations of the detergent micelle , with arrows showing possible proton path .", "( B ) Cartoon representation of subunit a with a horizontal stripe to depict the position of Asp61 on the c-ring ( red where Asp61 would be bound to a proton and blue when bound to Arg210 ) .", "Functional mutants labeled as follows; essential arginine in blue , substitution with Arg210 resulting in functional complex in yellow , mutation to arginine resulting in a dysfunctional complex in teal and residues that are aqueous accessible in red .", "Solid arrows show a possible proton path via two ‘half’ channels and dashed arrows show the path when bound to Asp61 of the c-ring and rotating . DOI: http://dx . doi . org/10 . 7554/eLife . 21598 . 02210 . 7554/eLife . 21598 . 023Figure 6—figure supplement 1 . Aqueous cavities of the E . coli FO motor . Left: overall map , with cross-section and viewpoints highlighted .", "( a ) and", "( b ) : cross-sections to show invaginations ( red arrows ) .", "( c ) and", "( d ) : views from cytoplasm and periplasm to show invaginations ( within red circles ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 21598 . 02310 . 7554/eLife . 21598 . 024Figure 6—figure supplement 2 . View of the State two map from F1 to show c-ring stoichiometry ( numbered ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 21598 . 02410 . 7554/eLife . 21598 . 025Figure 6—figure supplement 3 . Crosslinks of the E . coli FO motor . Yellow lines depict distances between Cα atoms known to crosslink when double cysteine mutants are introduced .", "( A–C ) intramolecular crosslinks identified in subunit a .", "The sequence was mapped to minimize the distance of these crosslinks , with them localizing to two groups .", "( D–F ) intermolecular crosslinks added to model show that the docked sequence maps well to the c-ring , but not as well to the b subunit . DOI: http://dx . doi . org/10 . 7554/eLife . 21598 . 02510 . 7554/eLife . 21598 . 026Figure 6—figure supplement 4 . Strong density near Arg210 . Arg210 of subunit a shown as blue stick , subunit a shown as orange cartoon , c-ring shown as grey cartoon and State one density shown as white surface . DOI: http://dx . doi . org/10 . 7554/eLife . 21598 . 02610 . 7554/eLife . 21598 . 027Figure 6—figure supplement 5 . Functional mutants of E . coli F-ATPase subunit a . As in main text Figure 6B , but with residues labeled and referenced ( superscript number ) .", "( 1 . Cain and Simoni , 1986; 2 . Lightowlers et al . , 1987; 3 . Lightowlers et al . , 1988; 4 . Cain and Simoni , 1989; 5 . Hatch et al . , 1995; 6 . Angevine and Fillingame , 2003; 7 . Angevine et al . , 2003 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 21598 . 027" ], [ "The three models and maps were deposited in the pdb and emDB with codes 5T4O , EMD-8357 ( State 1 ) , 5 T4P , EMD-8358 ( State 2 ) , 5T4Q and EMD-8359 ( State 3 ) ." ], [ "A cysteine-free version of E . coli F-ATPase cloned in plasmid pFV2 and expressed in E . coli DK8 strain was used ( Ishmukhametov et al . , 2005 ) .", "Cells were grown at 37°C in LB medium supplemented with 100 μg/ml ampicillin , for 4–5 hr .", "The harvested cells were resuspended in lysis buffer containing 50 mM Tris/Cl pH 8 . 0 , 100 mM NaCl , 5 mM MgCl2 , 0 . 1 mM EDTA , 2 . 5% glycerol and 1 μg/ml DNase I and processed by one pass in French press at 20 kPSI .", "Cellular debris was removed by centrifuging at 7700 × g for 15 min , and the membranes were collected by ultracentrifugation at 100 , 000 × g for 1 hr .", "The ATP synthase complex was extracted from membranes at 4°C for 1 hr by resuspending the pellet in extraction buffer consisting of 20 mM Tris/Cl , pH 8 . 0 , 300 mM NaCl , 2 mM MgCl2 , 100 mM sucrose , 20 mM imidazole , 10% glycerol , 4 mM digitonin and EDTA-free protease inhibitor tablets ( Roche ) .", "The complex was then purified by binding on Talon resin ( Clontech ) and eluted in 150 mM imidazole .", "The protein was further purified and sugars removed by size exclusion chromatography on a 16/60 Superose six column equilibrated in a buffer containing 20 mM Tris/Cl pH 8 . 0 , 100 mM NaCl , 4 mM digitonin and 2 mM MgCl2 .", "The purified protein was then concentrated to 2 mg/ml for cryo-EM .", "Seventy microgram of F1Fo was reconstituted into extrusion-preformed 100 nm soybean phosphatidylcholine liposomes exactly as descried ( Ishmukhametov et al . , 2016 ) .", "Proton pumping by proteoliposomes was studied using quenching of a pH sensitive fluorescent probe 9-Amino-6-Chloro-2-Methoxyacridine ( ACMA ) exactly as described ( Ishmukhametov et al . , 2016 ) .", "The assay was performed with 100 µl of proteoliposomes in 2-ml cuvettes .", "The reaction was started with 0 . 25 mM ATP and stopped by 2 µM of the uncoupler FCCP .", "ATP hydrolase activity and its stimulation by LDAO was measured with ATP regenerating system using 5 µg of the protein with 1 mM ATP exactly as described ( Ishmukhametov et al . , 2016 ) .", "DCCD inhibition of proteoliposomes was done as described ( Ishmukhametov et al . , 2016 ) .", "DCCD inhibition of pure F1Fo was done as described ( Ishmukhametov et al . , 2005 ) , with the following modification .", "Ten microgram of the protein was incubated in 1 ml buffer A ( 50 mM MES , pH 6 . 4 , 100 mM KCl , 1 mM MgCl2 ) with 50 µM DCCD for 30 min at room temperature .", "Control sample contained 1% ethanol instead of DCCD .", "Reaction was started by mixing the inhibited protein with 1 ml of buffer A containing all the components of ATP regenerating system .", "All the functional experiments presented here were repeated two to three times using the protein isolated by MS and shipped to RI at liquid N2 temperature .", "Results of typical experiments are shown .", "Aliquots of 4 μl of purified E . coli F-ATPase at a concentration of 3 . 58 μM were placed on glow-discharged holey carbon grids ( Quantifoils Copper R2/2 , 200 Mesh ) .", "Grids were blotted for 2 s and flash-frozen in liquid ethane using an FEI Vitrobot Mark IV .", "Grids were transferred to an FEI Titan Krios transmission electron microscope that was operating at 300 kV .", "Images were recorded automatically using the FEI EPU software , yielding a pixel size of 1 . 4 Å .", "A dose rate of 29 electrons ( spread over 20 frames ) per Å2 per second , and an exposure time of 2 s were used on the Falcon-II detector .", "8640 movies were collected .", "MotionCorr ( Li et al . , 2013 ) was used to correct local beam-induced motion and to align resulting frames .", "Defocus and astigmatism values were estimated using CTFFIND4 ( Rohou and Grigorieff , 2015 ) , and 252 micrographs were excluded due to drift or excessive ice contamination .", "1208 particles were manually picked and subjected to 2D classification to generate templates for autopicking in RELION ( Scheres , 2012 ) .", "The automatically picked micrographs were manually inspected to remove false positives , finally yielding 395 , 140 particles .", "These particles then underwent two rounds of 2D classification to generate 22 classes with 311 , 887 particles .", "The final particles were classified into four 3D classes using a previously generated model from a low-resolution data set of the same sample ( unpublished ) , low-pass filtered to 60 Å .", "The resolution was estimated using Fourier Shell Correlation ( FSC = 0 . 143 , gold-standard ) .", "Three of the four classes containing 104 , 510 ( State 1 ) , 67 , 829 ( State", "2 ) and 53 , 587 ( State", "3 ) particles were movie-refined and post-processed in RELION producing maps at 7 . 4 , 7 . 8 , 8 . 5 Å , respectively ( Figure 2—figure supplement 10 ) .", "State 1 was further processed using masked classification ( Bai et al . , 2015 ) with residual signal subtraction with a mask created by removing parts of the detergent micelle .", "Three out of the four classes from this classification containing 95 , 345 particles were combined and refined to generate the final 6 . 9 Å map .", "Figure 2—figure supplement 3 is a summary of these methods , shown as a flowchart .", "Local resolution of different parts of the complex was estimated using RELION and ResMap ( Kucukelbir et al . , 2014 ) .", "Crystal and NMR structures of subunits from E . coli ( αβγε - 3oaa [Cingolani and Duncan , 2011] , δ - 2a7u [Wilkens et al . , 2005] , b - 1b9u [Dmitriev et al . , 1999] , 1l2p [Del Rizzo et al . , 2002] and 2khk [Priya et al . , 2009] ) and related organisms ( c - 3u2f [Symersky et al . , 2012] and a - 5fik [Zhou et al . , 2015] ) were rigid body docked into the highest resolution cryo-EM map and the side chains ‘pruned’ to Cα .", "The sequence was mapped to subunit a using crosslinks as restraints .", "Subsequent manual model building and refinement was performed with Coot ( Emsley et al . , 2010 ) , Phenix ( Adams et al . , 2010 ) and Refmac ( Murshudov et al . , 2011 ) ( excluding subunit c due to weak density ) , with crosslinks again used as external restraints ( a summary of the types of models used to build the initial model can be found in Figure 2—figure supplement 6 ) .", "Nucleotide occupancy was determined by first building the model without any nucleotide present , and then segmenting the map and selecting any density with 15% overlap with atoms and deleting this density .", "Nucleotide was subsequently docked into this difference density using the known positions from previous structures .", "Once a complete model was built of the highest resolution map , this was docked and refined to the other two maps to create three models .", "The three models and maps were deposited in the pdb and emDataBank with codes 5T4O , EMD-8357 ( State 1 ) , 5 T4P , EMD-8358 ( State 2 ) , 5T4Q and EMD-8359 ( State 3 ) .", "Data statistics shown in Figure 2—source data 1 ." ] ]

[ "A molecular model that provides a framework for interpreting the wealth of functional information obtained on the E . coli F-ATP synthase has been generated using cryo-electron microscopy .", "Three different states that relate to rotation of the enzyme were observed , with the central stalk’s ε subunit in an extended autoinhibitory conformation in all three states .", "The Fo motor comprises of seven transmembrane helices and a decameric c-ring and invaginations on either side of the membrane indicate the entry and exit channels for protons .", "The proton translocating subunit contains near parallel helices inclined by ~30° to the membrane , a feature now synonymous with rotary ATPases .", "For the first time in this rotary ATPase subtype , the peripheral stalk is resolved over its entire length of the complex , revealing the F1 attachment points and a coiled-coil that bifurcates toward the membrane with its helices separating to embrace subunit a from two sides ." ]

[ "ATP synthase is a biological motor that produces a molecule called adenosine tri-phosphate ( ATP for short ) , which acts as the major store of chemical energy in cells .", "A single molecule of ATP contains three phosphate groups: the cell can remove one of these phosphates to make a molecule called adenosine di-phosphate ( ADP ) and release energy to drive a variety of biological processes .", "ATP synthase sits in the membranes that separate cell compartments or form barriers around cells .", "When cells break down food they transport hydrogen ions across these membranes so that each side of the membrane has a different level ( or “concentration” ) of hydrogen ions .", "Movement of hydrogen ions from an area with a high concentration to a low concentration causes ATP synthase to rotate like a turbine .", "This rotation of the enzyme results in ATP synthase adding a phosphate group to ADP to make a new molecule of ATP .", "In certain conditions cells need to switch off the ATP synthase and this is done by changing the shape of the central shaft in a process called autoinhibition , which blocks the rotation .", "The ATP synthase from a bacterium known as E . coli – which is commonly found in the human gut –has been used as a model to study how this biological motor works .", "However , since the precise details of the three-dimensional structure of ATP synthase have remained unclear it has been difficult to interpret the results of these studies .", "Sobti et al . used a technique called Cryo-electron microscopy to investigate the structure of ATP synthase from E . coli .", "This made it possible to develop a three-dimensional model of the ATP synthase in its autoinhibited form .", "The structural data could also be split into three distinct shapes that relate to dwell points in the rotation of the motor where the rotation has been inhibited .", "These models further our understanding of ATP synthases and provide a template to understand the findings of previous studies .", "Further work will be needed to understand this essential biological process at the atomic level in both its inhibited and uninhibited form .", "This will reveal the inner workings of a marvel of the natural world and may also lead to the discovery of new antibiotics against related bacteria that cause diseases in humans ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "cell biology", "neuroscience" ]

[ [ "Axons allow long-range bidirectional communication in neurons .", "They carry action potentials along their plasma membrane from dendrites and cell body to presynaptic terminals; and they transport cell components and signaling complexes using motor proteins , anterogradely and retrogradely .", "Another potential route for communication along axons is endoplasmic reticulum ( ER ) ; axons possess a network of ER tubules , which appears physically continuous both locally ( Tsukita and Ishikawa , 1976; Villegas et al . , 2014 ) and with ER throughout the neuron ( Lindsey and Ellisman , 1985; Terasaki et al . , 1994 ) .", "The physical continuity of ER and its ensuing potential for long-distance communication has been likened to a ‘neuron within a neuron’ ( Berridge , 1998 ) .", "Axonal ER appears mostly tubular and smooth , with some cisternae ( Tsukita and Ishikawa , 1976; Lindsey and Ellisman , 1985; Villegas et al . , 2014 ) .", "Some rough ER is likely to be present too: numerous mRNAs are found in both growing and mature axons ( Zivraj et al . , 2010; Shigeoka et al . , 2016 ) , and local axonal translation can occur in response to injury ( Ben-Yaakov et al . , 2012; Perry et al . , 2012 ) .", "However , rough ER sheets ( Tsukita and Ishikawa , 1976; Villegas et al . , 2014 ) , and markers of protein export and folding that are characteristic of rough ER ( Röper , 2007; O'Sullivan et al . , 2012 ) , are relatively sparse in mature axons .", "Axonal ER therefore likely has major roles other than protein export; these could include lipid biosynthesis ( Tidhar and Futerman , 2013; Vance , 2015 ) , calcium homeostasis and signaling ( Ross , 2012 ) , and coordination of organelle physiology ( Phillips and Voeltz , 2016 ) .", "The continuity of the ER network suggests that some of these roles might have long-range as well as local functions; indeed , an ER-dependent propagating calcium wave is seen after axotomy of Caenorhabditis elegans or mammalian dorsal root ganglion neurons ( Ghosh-Roy et al . , 2010; Cho et al . , 2013 ) .", "A strong hint of the importance of ER in axons is found in Hereditary Spastic Paraplegia ( HSP ) , a group of axon degeneration disorders characterized by progressive spasticity and weakness of the lower limbs ( Blackstone et al . , 2011; Blackstone , 2012 ) .", "Mutations affecting spastin , atlastin-1 , reticulon-2 , REEP1 and REEP2 account for most cases of autosomal dominant ‘pure’ HSP ( Hazan et al . , 1999; Zhao et al . , 2001; Züchner et al . , 2006; Montenegro et al . , 2012; Esteves et al . , 2014 ) .", "These proteins share a common feature of one or two hydrophobic hairpin-loops inserted in the ER membrane , promoting ER membrane curvature in a process termed hydrophobic wedging ( Voeltz et al . , 2006 ) .", "Proteins of the REEP and reticulon families localize preferentially to tubular or smooth ER , and their loss results in disruption of ER tubular organization ( Shibata et al . , 2006; Voeltz et al . , 2006; Park et al . , 2010; Shibata et al . , 2010 ) ; they may also contribute to modeling of rough ER sheets by stabilizing their curved edges ( Shibata et al . , 2009 ) .", "What is the link between ER modeling and axon structure and function ?", "HSP-causing mutations often appear to cause loss of protein expression or function ( Beetz et al . , 2013; Novarino et al . , 2014 ) , and the ability of hairpin-loop proteins to form homomeric and heteromeric complexes ( Shibata et al . , 2008 ) allows some point mutations to have dominant negative effects ( Züchner et al . , 2006; Beetz et al . , 2012 ) .", "Therefore loss of normal ER modeling appears to compromise axon maintenance and function .", "Given the roles of hairpin-loop proteins in ER modeling , we aimed to test the model that hairpin-loop-containing HSP proteins organize the axonal ER network .", "Since reticulon and REEP family proteins are redundantly required for most peripheral ER tubules in yeast ( Voeltz et al . , 2006 ) , we focus on the requirement for these two families in axons .", "We previously showed that knockdown of the Drosophila reticulon Rtnl1 causes expansion of epidermal ER sheets , and partial loss of smooth ER marker from distal but not proximal motor axons ( O'Sullivan et al . , 2012 ) .", "Here we show that REEP proteins have similar roles .", "We also show that simultaneous loss of reticulon and REEP family members leads to a range of axonal ER phenotypes , including a reduced network with fewer and larger tubules , and occasional gaps in the network .", "Our work implicates hairpin-loop-containing HSP proteins as important players in the axonal ER network , and suggests further models for how the network is organized ." ], [ "The reticulon and REEP families of double-hairpin-containing proteins are collectively responsible for formation or maintenance of most peripheral ER tubules in yeast ( Voeltz et al . , 2006 ) .", "We previously showed that the Drosophila reticulon ortholog Rtnl1 was strongly localized in axons , and that its knockdown caused partial loss of a smooth ER marker in posterior larval segmental axons ( O'Sullivan et al . , 2012 ) .", "To test the roles of Drosophila REEP proteins in axonal ER localization , we first dissected the ortholog relationships between the six Drosophila and six human REEP proteins .", "Multiple sequence alignment of mammalian and Drosophila REEP protein sequences suggested that CG42678 was the single Drosophila ortholog of mammalian REEP1-REEP4 ( Figure 1A ) .", "CG42678 has previously been designated Reep1 ( http://flybase . org/reports/FBgn0261564 . html ) , but we propose the name ReepA to reflect its orthology to the four mammalian genes REEP1-REEP4 .", "Mammalian REEP5 and REEP6 appeared to share two Drosophila orthologs , CG8331 and CG4960 ( Figure 1 ) .", "We designated CG8331 as ReepB because of its widespread expression ( www . flyatlas . org; Chintapalli et al . , 2007 ) and slower rate of evolutionary sequence divergence ( Supplementary file 1 ) .", "We excluded CG4690 and three additional Drosophila REEP genes from further study , since their expression was restricted to testes and larval fat body ( www . flyatlas . org; Chintapalli et al . , 2007 ) , and their faster rate of evolutionary sequence divergence ( Supplementary file 1 , and reflected in longer branch lengths in Figure 1A ) , suggesting poorly conserved function . 10 . 7554/eLife . 23882 . 003Figure 1 . Drosophila Rtnl1 and REEP genes and products .", "( A ) A dendrogram based on ClustalW sequence alignment of Drosophila and human REEP proteins shows two branches corresponding to human REEP1-4 ( CG42678 ) and human REEP5-6 .", "Broken lines represent Drosophila REEP proteins that are evolving rapidly ( Supplementary file 1 ) , reflected by longer branch lengths .", "Sequences used are NP_075063 . 1 , NP_057690 . 2 , NP_001001330 . 1 , NP_079508 . 2 , NP_005660 . 4 , NP_612402 . 1 , NP_726266 . 1 , NP_610936 . 2 , NP_651429 . 1 , NP_611831 . 2 , NP_572730 . 1 , NP_726366 . 1 .", "( B ) Rtnl1 genomic and transcript map , showing the region deleted in Rtnl1− by excision of P-element NP7026 ( Wakefield and Tear , 2006; Figure 1—figure supplement 1 ) , the RHD domain ( Pfam 02453 ) and its coordinates in protein isoform G , the Rtnl1::YFP exon trap insertion CPTI001291 ( green triangle ) , and the fragment targeted by GD RNAi 7866 .", "Map and coordinates are from the Drosophila Genome Browser ( www . flybase . org , version R6 . 04 ) , here and in subsequent panels; light regions in transcripts represent coding regions , dark shaded regions represent untranslated regions .", "( C ) ReepA genomic and transcript map showing the region deleted in ReepA− by excision of P-element CB-0501–3 , the position of the DP1 domain ( Pfam 03134 ) and its coordinates in protein isoforms H and J . GFP insertion sites for ReepA1::GFP , ReepA2::GFP and ReepA3::GFP fusions are shown with green triangles .", "( D ) ReepB genomic and transcript map showing the region deleted in ReepB− by excision of P-element EY05130 , the position of the DP1 domain ( Pfam 03134 ) and its coordinates in protein isoforms A and D ) .", "( E–I )", "Confocal sections showing localization of ReepA::GFP isoforms and ReepB::GFP .", "( E ) Overlap of ReepA1::GFP and ReepB::GFP with anti-KDEL labeling in larval epidermal cells .", "To facilitate display of weaker ReepA1::GFP , the GFP channel in wild-type control ( WT ) and ReepA::GFP images has been brightened four times as much as for ReepB::GFP .", "( F ) Expression of ReepA3::GFP and ReepB::GFP in third instar ventral nerve cord .", "The GFP channels for WT and ReepA::GFP have been brightened by twice as much as for ReepB::GFP .", "VNCs are outlined with yellow dashed lines .", "Arrowheads show ReepB::GFP extending into peripheral nerves .", "( G ) A single confocal section of a peripheral nerve , showing ReepB::GFP localized continuously along its length .", "( H ) Double labeling of an NMJ for ReepB::GFP and the mainly postsynaptic marker Dlg , showing ReepB::GFP in an axon emerging from nerve bundles ( arrowhead ) to extend to the NMJ , as well as in axons traversing the muscle surface ( arrow ) ; note the dispersed ReepB::GFP staining also in the underlying muscle .", "( I ) Double labeling of ReepA::GFP and ReepB::GFP lines for GFP and Dlg ( mainly postsynaptic ) shows presynaptic expression of ReepB::GFP .", "( J ) GFP expression in a wildtype negative control ( WT ) , or from Rtnl1::GFP expressed in two closely apposed motor neurons by m12-GAL4 .", "Scale bars 10 µm , except F , 20 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 23882 . 00310 . 7554/eLife . 23882 . 004Figure 1—figure supplement 1 . Molecular lesions in Rtnl11 , ReepA541 and ReepB48 . BLASTN searches of the Drosophila genome , using the sequences of ( A ) Rtnl11 , ( B ) ReepA541 and ( C ) ReepB48 mutations as queries , show the coordinates of each molecular lesion in the genome , as gaps in alignments between mutant and genomic sequences .", "The ReepA541 and ReepB48 sequences highlighted in yellow show P element footprints left at the excision sites . DOI: http://dx . doi . org/10 . 7554/eLife . 23882 . 004 A Rtnl1::YFP exon trap , Rtnl1CPTI001291 ( Figure 1B ) was previously shown to localize to ER , including in axons ( O'Sullivan et al . , 2012 ) .", "To study localization of ReepA and ReepB , we recombineered C-terminal GFP-tagged versions of these ( Figure 1C , D ) using P[acman] genomic clones ( Venken et al . , 2009 ) .", "For ReepA , we generated EGFP fusions at three different C-termini ( Figure 1C ) , that we called ReepA1::GFP ( for protein isoforms D , E , G ) , ReepA2::GFP ( for protein isoforms H , I , J , K ) and ReepA3::GFP ( for protein isoforms L , R , S ) .", "In epidermal cells , we detected weak expression of ReepA1::GFP , but not of the other ReepA::GFP fusions; ReepB::GFP showed stronger expression still , and both fusions overlapped with an ER marker ( KDEL; Figure 1E ) , similar to REEP proteins in other organisms ( Voeltz et al . , 2006; Shibata et al . , 2008; Park et al . , 2010 ) .", "ReepB::GFP was also more strongly expressed in third instar larval CNS than ReepA3::GFP , which was the only ReepA::GFP fusion that we detected there ( Figure 1F ) .", "ReepB::GFP , but no ReepA::GFP fusion , was also detected in segmental nerves ( Figure 1F , G ) , and in individual axons emerging from nerve bundles leading to the NMJ ( Figure 1H ) .", "ReepB::GFP , but none of the ReepA::GFP fusions , localized as a mostly continual structure along the length of presynaptic terminals of neuromuscular junctions ( NMJs ) ( Figure 1H , I ) .", "We did not have a UAS-ReepB construct available , but UAS-Rtnl1::GFP ( Rao et al . , 2016 ) expressed in two adjacent motor neurons using m12-GAL4 ( Xiong et al . , 2010 ) also showed strong axonal localization .", "We therefore conclude that ReepB::GFP localizes in axonal and presynaptic ER , similar to Rtnl1::YFP ( O'Sullivan et al . , 2012 ) , but that none of the ReepA::GFP fusions is detectable in axonal or presynaptic ER .", "An Rtnl1 loss-of-function mutant , Rtnl11 ( Wakefield and Tear , 2006 ) , hereafter referred to as Rtnl1− , lacks the hydrophobic hairpin loop domain ( RHD region ) that induces ER membrane curvature ( Figure 1B; Figure 1—figure supplement 1 ) .", "We also used P-transposase-mediated imprecise excision to generate ReepA− and ReepB− mutants , both of which lack most of the curvature-mediating DP1 hairpin domains ( Figure 1B , C; Figure 1—figure supplement 1 ) .", "First , we asked whether Drosophila reticulon and REEP proteins contribute to ER network organization in third instar larval epidermal cells; Rtnl1 knockdown leads to a more diffuse ER network organization and expansion of ER sheets in these cells , compared to wild-type controls ( O'Sullivan et al . , 2012 ) .", "Rtnl1− mutant larvae also showed loss of ER network organization ( Figure 2A ) .", "Intensity of KDEL labeling along a line from the nucleus to cell periphery displayed fluctuating intensity , reflecting the reticular distribution of KDEL in wild-type larvae , but less fluctuation in Rtnl1− larvae; overall levels of KDEL remained unchanged ( Figure 2A ) .", "Similarly , loss of both ReepA and ReepB , but not loss of either gene alone , made KDEL levels in larval epidermal cells fluctuate less than in controls .", "Mean intensity of KDEL staining was decreased in all ReepA and ReepB mutant genotypes , and fluctuation in intensity was increased in ReepB− larvae ( Figure 2B ) .", "Since confocal analysis suggests altered organization of ER in epidermal cells , but does not have sufficient resolution to reveal the details of how it is altered , we performed electron microscopy of epidermal cells .", "This revealed that ReepA− ReepB− double mutant epidermal cells showed longer ribosome studded sheet ER profiles , compared to controls , and to ReepA− and ReepB− single mutants ( Figure 2C ) .", "This mutant phenotype is similar to , although less severe than loss of Rtnl1 ( O'Sullivan et al . , 2012 ) .", "ReepA− ReepB− mutants also showed increased ER stress in epidermal cells ( Figure 2D ) but not in CNS ( Figure 2E ) .", "Therefore , Rtnl1 and REEP proteins shape the ER network in Drosophila , and their loss disrupts ER organization , causing longer ER sheet profiles . 10 . 7554/eLife . 23882 . 005Figure 2 . Rtnl1− mutants and ReepA− ReepB− double mutants show disrupted ER organization in epidermal cells .", "( A ) KDEL distribution in third instar larval epidermal cells appears diffuse in Rtnl1− larvae compared to a more reticular staining in wild-type ( WT ) larvae .", "Micrographs show 1 . 5 µm z-projections of confocal sections .", "KDEL intensity along yellow lines from nuclear envelope to the cell periphery shows less fluctuation in Rtnl1− ( blue dotted line on line graph ) than in WT larvae ( black line on line graph ) .", "Fluctuation of intensity is quantified using the local normalized variance of intensity ( variance/mean for rolling 10-pixel windows ) over a 12 µm line for each cell .", "n = 61 epidermal cells from 12 larvae , 3–5 cells from each larva , from 3 independent experiments .", "( B ) KDEL distribution in third instar larval epidermal cells shows less spatial fluctuation in ReepA− ReepB− double mutant larvae compared to ReepA+ ( WT ) , and ReepA− and ReepB− single mutant larvae .", "This is confirmed by quantification as in A ) ; overall KDEL intensity is reduced in all ReepA and ReepB mutant genotypes .", "n = 45–61 epidermal cells in total from 12 to 16 different larvae , 3–5 cells from each larva , from 6 independent experiments .", "Intensity datapoints are experimentwise averages of multiple larvae , compared by paired t-tests , since larva-wise data were too significantly different to pool across experiments in this case .", "( C ) Electron micrographs show increased ER sheet profile length in ReepA− ReepB− double mutant third instar larval epidermal cells compared to single ReepA− and ReepB− mutants and controls .", "Arrowheads , ER sheets; M , mitochondria .", "( n = 56–58 cells in total from three independent larvae , 18–19 cells from each . ( D , E )", "ReepA− ReepB− double mutant larvae have an increased ER stress response compared to a ReepA+ ( WT ) control , measured by Xbp1::GFP expression , in larval epidermis ( D ) but not in neuronal cell bodies ( E ) .", "Graphs show quantification of Xbp1::GFP staining intensity relative to controls .", "( n = 12–15 larvae ) .", "All graphs show individual datapoints and mean ±SEM .", "Occasional outlier datapoints off the top of the scale are omitted from graphs but included in statistical analyses .", "ns , p>0 . 05; *p<0 . 02; **p<0 . 005; ***p<0 . 0003; ****p<0 . 0001 , two-tailed Student’s t-test .", "Scale bars: A , B , D , E , 10 µm; C , 0 . 5 µm ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 23882 . 005 To understand the roles of reticulon and REEPs in axonal ER organization , we labeled axonal ER by expressing Acsl::myc ( O'Sullivan et al . , 2012 ) in two adjacent motor neurons using m12-GAL4 ( Xiong et al . , 2010 ) .", "Loss of Rtnl1 caused partial loss of Acsl::myc from posterior ( segment A6 ) axons , but not from anterior axons ( segment A2 ) ; it also caused Acsl::myc staining to appear more irregular in posterior but not anterior axons , reflected in a higher coefficient of variation ( SD/mean ) of Acsl::myc staining intensity along the length of posterior axons lacking Rtnl1 , compared to wild-type ( Figure 3A ) .", "These Rtnl1 loss-of-function phenotypes were found either on targeted knockdown of Rtnl1 in these motor axons or in Rtnl1− mutant larvae , and the Rtnl1− mutant phenotypes could be partially rescued by one copy of an Rtnl1Pacman genomic clone ( Figure 3A ) . 10 . 7554/eLife . 23882 . 006Figure 3 . Loss of either Rtnl1 or ReepB leads to partial loss of smooth ER marker from distal motor axons .", "( A ) Effects of Rtnl1 loss on ER , visualized by Acsl::myc expressed in two adjacent motor axons using m12-GAL4 .", "Images show effects of Rtnl1 knockdown , Rtnl1− mutation , rescue of Rtnl1− by Rtnl1Pacman and respective ReepA+ control ( WT ) axons .", "Graphs show mean staining intensity ( top graphs ) or coefficient of variation of intensity ( bottom graphs ) as a measurement of staining variability along the length of each axon .", "Rtnl1 loss leads to partial loss of Acsl::myc in posterior but not anterior axons ( top graphs ) , and to some disorganization of posterior axonal ER seen by increased coefficient of variation of Acsl::myc staining intensity .", "There is partial rescue of Rtnl1 phenotypes by one copy of a Rtnl1Pacman genomic clone .", "n = 15–20 larvae per genotype pooled from 5 independent experiments for RNAi; n = 24–36 larvae per genotype from 10 to 12 independent experiments for Rtnl1− mutant .", "Graphs show individual datapoints with mean ±SEM; ns , p>0 . 05; *p<0 . 03; **p<0 . 005; two-tailed unpaired Student’s t-test; two-tailed paired Student’s t-test was used when larva-wise data were too significantly different across experiments ( p<0 . 05 , ANOVA ) to pool .", "( B ) Effects of ReepA or ReepB loss on axonal ER .", "ReepB knockdown or ReepB− mutation , but not ReepA mutation , causes partial loss of smooth ER marker Acsl::myc in posterior but not in anterior motor axons .", "The ReepB− phenotype can be mostly rescued by one copy of a genomic ReepB::GFP clone .", "A ReepA− ReepB− double mutant shows a similar phenotype to a ReepB− single mutant .", "ReepA− ReepB− double mutant , but not single ReepA− or ReepB− mutants , show increased coefficient of variation of Acsl::myc staining levels in posterior axons; n = 7–47 larvae from 4 to 11 independent experiments .", "All graphs show individual datapoints with mean ±SEM; occasional outlier datapoints off the top of the scale are omitted from graphs but included in statistical analyses .", "ns , p>0 . 05; *p<0 . 04 **p<0 . 01 .", "ReepB RNAi was analyzed using two-tailed unpaired Student’s t-tests; multiple comparisons of ReepA and ReepB mutant genotypes were analyzed by ANOVA followed by post-hoc Tukey HSD tests .", "Scale bars , 10 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 23882 . 006 ReepA− mutants showed no loss of Acsl::myc from axons .", "Similar to Rtnl1 loss of function , ReepB− mutants showed partial loss of Acsl::myc from posterior but not anterior motor axons ( Figure 3B ) ; this phenotype was partially rescued with one copy of a genomic ReepB::GFP clone , and was also observed on ReepB knockdown in m12-GAL4-expressing neurons ( Figure 3B ) .", "A ReepA− ReepB− double mutant showed loss of Acsl::myc from posterior axons , that was similar to that seen in ReepB− mutants ( Figure 3B ) .", "ReepA− ReepB− double mutant larvae , but not ReepA− single mutants , showed an increased coefficient of variation of Acsl::myc staining intensity in posterior but not in anterior axons; ReepB mutant and knockdown axons both showed a slight increase in coefficient of variation in posterior axons , although this was not significant for ReepB mutants ( Figure 3B ) .", "In summary , loss of either Rtnl1 or at least ReepB alters axonal ER distribution in posterior motor axons , therefore supporting roles for these two protein families in axonal ER organization; in contrast loss of ReepA has either subtle or no effects .", "Since loss of both reticulon and REEP families in yeast removes most peripheral ER tubules ( Voeltz et al . , 2006 ) , we tested whether loss of both protein families in Drosophila might have similarly severe effects on axonal ER .", "Rtnl1− ReepA− ReepB− triple mutants were viable and fertile as adults , but survived poorly beyond two weeks of adulthood , compared to 4–5 weeks for wild-type adults .", "Triple mutant larvae showed increased fluctuation of Acsl::myc staining intensity along motor axons compared to wild-type , mainly in middle parts ( segment A4 and A5 ) of longer axons .", "At its most extreme , this manifested as fragmentation of Acsl::myc labeling , which was never seen in wild-type axons ( Figure 4A , B ) .", "When gaps in labeling were found in the central regions of axons , labeling in the anterior and posterior parts of the same axons was usually continuous ( Figure 4A ) .", "Double labeling of plasma membrane ( CD4::tdGFP ) and axonal ER ( Acsl::myc ) showed no effect on axonal plasma membrane in Rtnl1− ReepA− ReepB− triple mutant compared to control axons ( Figure 4—figure supplement 1 ) , implying that the phenotype was limited to ER and did not affect axon integrity .", "To compare genotypes and labels , we quantified irregular labeling along a 45 µm stretch of axon traversing the larval A4/A5 region in two ways: first we measured gaps in labeling , defined as intensity below a threshold that could consistently distinguish labeled axons above background labeling in the nerve; and second , we quantified the coefficient of variation of labeling intensity along the axon ( Figure 4C , D ) .", "Rtnl1 loss-of-function axons , but not ReepA− ReepB− mutant axons , also showed a mild ER fragmentation phenotype ( Figure 4A , B ) .", "Rtnl1 ( RNAi ) ReepA− ReepB− larvae also showed a fragmentation phenotype similar to Rtnl1− ReepA− ReepB− , suggesting that loss of Rtnl1 is essential for it ( Figure 4B ) .", "Rtnl1− ReepB− double mutant axons also showed a similar phenotype to Rtnl1− ReepA− ReepB− triple mutants .", "Therefore , loss of Rtnl1 causes a mild irregular organization of axonal ER , and this phenotype is exacerbated by loss of ReepB , with no detectable contribution of ReepA loss; and loss of both Reep proteins has no apparent effect . 10 . 7554/eLife . 23882 . 007Figure 4 . Loss of hairpin proteins leads to discontinuity of axonal ER staining .", "( A ) Rtnl1− larvae and Rtnl1− ReepA− ReepB− triple mutant larvae sometimes show fragmented axonal ER labeling in the middle parts ( segment A5 ) of long motor axons that express Acsl::myc under control of m12-GAL4 .", "Anterior ( segment A2 ) and posterior ( segment A6 ) portions of the same motor axons show continuous ER labeling .", "Arrowheads show gaps in Acsl::myc staining; brighter versions of the same images show gaps in staining in mutants but not wild-type ( WT ) , even when brightness of remaining staining is saturating .", "( B ) Panels show three examples of the range of Acsl::myc distributions found in the middle parts of long motor axons of each genotype of ReepA+ ( WT ) , Rtnl1− , Rtnl1− rescued with a Rtnl1Pacman construct , ReepA− ReepB− and Rtnl1− ReepB− double mutants , Rtnl1− ReepA− ReepB− triple mutant , and Rtnl1 ( RNAi ) ReepA− ReepB− larvae .", "A variety of phenotypes , from continuous to fragmented Acsl::myc labeling , are found in genotypes that lack Rtnl1 , and tend to be more severe in genotypes that also lack ReepB .", "Insets show examples of gaps in ER continuity ( arrowheads ) at higher magnification .", "Scale bars 10 µm , and 5 µm in higher zoom images .", "( C ) Percentages of a 45 µm length in the middle ( A4/A5 segment ) of each axon that lacks Acsl::myc staining , using an intensity threshold of 20 on a scale of 0–255 .", "Individual axons are plotted , together with median and interquartile range; comparisons use Mann-Whitney U-tests .", "All genotypes that lack Rtnl1 show more gaps than WT; Rtnl1− ReepA− ReepB− triple mutants do not differ from Rtnl1 ( RNAi ) ReepA− ReepB− or Rtnl1− ReepB− double mutants , but are significantly more severe than Rtnl1− single mutants .", "( D ) The coefficient of variation of Acsl::myc labeling in middle axon portions is increased in genotypes lacking Rtnl1 , relative to controls .", "Graphs show individual datapoints with mean ±SEM; occasional outlier datapoints off the top of the scale are omitted from graphs but included in statistical analyses .", "Comparison between Rtnl1− and Rtnl1− ReepA− ReepB− mutant genotypes was analyzed by two-tailed Student’s t-test , other multiple comparisons by ANOVA followed by Dunnett’s T3 test .", "ns , p>0 . 05; *p<0 . 04; **p<0 . 01; ***p<0 . 0005; ****p<0 . 0001; individual axons are plotted from 3 to 20 independent experiments , each with 2–3 different larvae for each genotype . DOI: http://dx . doi . org/10 . 7554/eLife . 23882 . 00710 . 7554/eLife . 23882 . 008Figure 4—figure supplement 1 . Plasma membrane integrity is not affected in Rtnl1− ReepA− ReepB− triple mutant larvae ) .", "Coefficient of variation for plasma membrane labeling along axon length is unchanged in Rtnl1− ReepA− ReepB− triple mutant compared to control ( WT ) axons , whereas the ER to plasma membrane ratio of the coefficient of variation is increased .", "Higher zoom images of the boxed areas show gaps ( yellow arrowheads ) and brightly labeled axonal ER accumulations .", "Graphs show mean ±SEM; *p<0 . 02 , two-tailed Student’s t-test; n = 5 different larvae from 3 independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 23882 . 008 To confirm the physical continuity of ER in normal axons , and that gaps in Acsl::myc staining in Rtnl1− ReepA− ReepB− triple mutant axons reflect physical gaps in the ER network , we also visualized ER using fluorescent protein markers .", "The lipase CG9186::GFP ( Thiel et al . , 2013 ) , Rtnl1::GFP ( Rao et al . , 2016 ) and tdTomato::Sec61β ( Summerville et al . , 2016 ) all localize to ER , and all showed continuous labeling in wild-type ( ReepA+ ) axons ( Figure 5A; Figure 5—figure supplement 1A ) .", "However , CG9186::GFP showed occasional gaps in triple mutant axons ( Figure 5A ) , with at least one gap per larva visible by confocal microscopy in 6/17 larvae .", "Since confocal imaging might not reveal all physical discontinuities , and since gaps in marker distribution might not mean gaps in ER distribution , we probed the physical continuity of CG9186::GFP labeling using fluorescence recovery after photobleaching ( FRAP ) .", "After bleaching a 12 µm length of axon in either wild-type axons or in Rtnl1− ReepA− ReepB− triple mutant axons lacking gaps , we observed rapid recovery of fluorescence from both ends of the bleached region ( Figure 5; Figure 5—figure supplement 1B; Video 1 ) , suggesting no physical barrier to CG9186::GFP diffusion .", "The kinetics of recovery were similar between wildtype and triple mutant axons ( Figure 5B–D ) .", "We also performed FRAP on regions next to gaps in CG9186::GFP labeling in triple mutant axons ( Figure 5A ) ; here we observed good recovery of fluorescence from the end of the bleached region opposite the gap , but no recovery across the gap ( Figure 5; Figure 5—figure supplement 1C; Videos 2–4 ) , suggesting that gaps in CG9186::GFP labelling were also physical barriers to diffusion of CG9186::GFP , and hence gaps in the ER network . 10 . 7554/eLife . 23882 . 009Figure 5 . Live imaging of ER in wild type and hairpin mutant axons .", "( A ) Representative FRAP assay images from ReepA+ ( WT ) or Rtnl1− ReepA− ReepB− triple mutant axons in which ER was visualized using CG9186::GFP expression driven in two motor axons by m12-GAL4 .", "One triple mutant axon shows a gap in labeling ( arrowheads ) .", "Regions of interest ( 12 µm , between dashed lines ) were photobleached .", "Fluorescence was visualized before photobleaching ( ‘pre’ ) , and during recovery over 200 s .", "A kymograph was generated for each axon indicated by an arrow in the top panel .", "Most areas of intense and less intense ER labeling remain stable over time ( e . g . black arrows below kymographs ) ; occasional movements of ER features are indicated by white arrows in kymographs .", "( B ) Relative fluorescence intensities within the photobleached region were plotted ( mean ±SEM ) during recovery for wild-type axons or for triple mutant axons lacking ER gaps .", "( C–D )", "Quantification of half recovery time ( C ) and rate constant ( D ) for wild-type and triple mutant axons lacking gaps , with median and interquartile range .", "Data in B–D are from seven recordings from 4 wild-type larvae , and 20 recordings from 14 mutant larvae .", "ns , p>0 . 05; Mann-Whitney U test .", "Scale bars , 5 µm .", "( E ) Representative kymographs from time-lapse recording of CG9186::GFP in unbleached single ReepA+ ( WT ) or Rtnl1− ReepA− ReepB− triple mutant axons lacking gaps .", "The left panel shows retrograde movement of a ~ 3 µm length of ER labeling ( arrows ) ; the right panel shows anterograde movement of a ~ 5 µm stretch of ER labeling ( arrows ) .", "Retrograde movements of labeled puncta are seen in both panels ( arrowheads ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 23882 . 00910 . 7554/eLife . 23882 . 010Figure 5—figure supplement 1 . Continuity , stability and movement of fluorescent ER labeling in wild-type ( WT ) and Rtnl1− ReepA− ReepB− triple mutant axons .", "( A ) Time series and kymographs of two closely apposed motor axons expressing either Rtnl1::GFP or Sec61bβ::tdTomato ER markers under control of m12-GAL4 .", "Images show continuity of ER staining in axons , and kymographs show stability of most ER features over the acquisition time .", "( B ) Kymographs showing fluorescence recovery after photobleaching in two ReepA+ ( WT ) axons , or in two Rtnl1− ReepA− ReepB− triple mutant axons lacking ER gaps .", "Recovery proceeds from both ends of the bleached region ( dotted lines ) .", "Most ER features seen before bleaching ( top line of kymographs ) can also be seen after recovery , implying that recovery proceeds by protein diffusion , not by movement of ER structures .", "( C ) Kymographs showing fluorescence recovery after photobleaching immediately distal to gaps in ER staining , in four Rtnl1− ReepA− ReepB− triple mutant axons .", "Recovery proceeds only from the distal end of the bleached region ( dotted lines ) opposite the gap , but not across the proximal gap .", "While most ER gaps and many other features are stable , one axon shows a retrogradely moving particle ( white arrowhead ) , and one shows anterograde movement of ER that closes one gap and opens another more proximally ( white asterisk ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 23882 . 01010 . 7554/eLife . 23882 . 011Video 1 . Representative time-lapse microscopy of FRAP analysis in wild-type axons . Wild-type ( WT ) axons expressing CG9186::GFP under control of m12-GAL4 were photobleached as described in Figure 5A .", "The video shows 2 s of pre-bleach images and 200 s of post-bleach images at one frame every 5 s .", "The bleached area is shown with a rectangle in the bleaching frame . DOI: http://dx . doi . org/10 . 7554/eLife . 23882 . 01110 . 7554/eLife . 23882 . 012Video 2 . Representative time-lapse microscopy of FRAP analysis in Rtnl1− ReepA− ReepB− triple mutant axons . Rtnl1− ReepA− ReepB− triple mutant axons expressing CG9186::GFP were photobleached and imaged as in Video 1 , in a region immediately distal to a gap ( between white arrows ) in ER labeling in one axon . DOI: http://dx . doi . org/10 . 7554/eLife . 23882 . 01210 . 7554/eLife . 23882 . 013Video 3 . FRAP analysis in Rtnl1− ReepA− ReepB− triple mutant axons showing a CG9186::GFP labeled particle passing retrogradely across the gap region . Rtnl1− ReepA− ReepB− triple mutant axons expressing CG9186::GFP were photobleached and imaged immediately distal to an ER gap as in Video", "2 . A labeled particle ( white arrow ) can be seen moving retrogradely across the ER gap ( white arrowhead ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 23882 . 01310 . 7554/eLife . 23882 . 014Video 4 . FRAP analysis in Rtnl1− ReepA− ReepB− triple mutant axons showing dynamic ER gap generation . Rtnl1− ReepA− ReepB− triple mutant axons expressing CG9186::GFP were photobleached and imaged immediately distal to an ER gap as in Video", "2 . Anterograde movement of a 5 µm length of ER simultaneously closes up a gap distal to it ( first white arrow ) and opens up a new gap ( second white arrow ) proximal to it .", "A kymograph from this preparation is shown in Figure 5—figure supplement 1C . DOI: http://dx . doi . org/10 . 7554/eLife . 23882 . 014 During live imaging , we also followed the stability of the ER network in both wild-type and mutant axons .", "Features such as higher or lower levels of CG9186::GFP labelling ( which could reflect variation in the numbers of tubules , or the presence of structures like cisternae ) were mostly stable throughout a 200 s acquisition period ( Figure 5A , E; Figure 5—figure supplement 1 ) .", "However , we also observed some dynamic features , including anterograde or retrograde movement of more brightly labeled regions ( Figure 5A , E ) perhaps representing movement of ER tubules detached from the ER network , and retrograde movement of labeled puncta ( Figure 5E; Figure 5—figure supplement 1C; Video 3 ) , at around 0 . 3 µm/s .", "While most gaps in ER labeling in triple mutants were stable over 200 s ( 7 out of 8 gaps imaged , including Videos 2–3 ) , in one case a gap was closed up by anterograde movement of a 5 µm length of ER proximal to the gap , which simultaneously opened up a new gap proximal to the moving section ( Figure 5—figure supplement 1C; Video 4 ) .", "In one case a labeled punctum moved retrogradely across an ER gap without pausing ( Figure 5—figure supplement 1C ) , implying that microtubule-based transport was intact .", "Therefore , live imaging and photobleaching of CG9186::GFP both suggest that ER is normally continuous in axons , that loss of reticulon and REEP proteins leads to occasional gaps in ER labeling that represent physical breaks of ER continuity , and that ER network organization in axons shows both static and dynamic features .", "We also tested for possible defects in axon transport by staining for abnormal accumulation of the synaptic vesicle protein CSP in axons .", "Rtnl1− larvae and Rtnl1− ReepA− ReepB− triple mutant larvae showed large accumulations of CSP in many peripheral nerves .", "The large accumulations of CSP in Rtnl1− larvae could be rescued by two copies of a Rtnl1Pacman genomic clone ( Figure 6 ) . 10 . 7554/eLife . 23882 . 015Figure 6 . Loss of Rtnl1 causes mild accumulation of synaptic vesicles in axons .", "( A ) Peripheral nerves of Rtnl1− and Rtnl1− ReepA− ReepB− triple mutant larvae show larger accumulations of synaptic vesicle protein CSP ( e . g . yellow arrows ) , and smaller elongated CSP puncta ( e . g . yellow arrowheads ) .", "In contrast , control larvae ( WT ) and ReepA− ReepB− double mutant larvae show an even distribution of small round CSP puncta .", "CSP accumulations are not significantly bigger in Rtnl1− ReepA− ReepB− triple mutants than in Rtnl1− mutants .", "The CSP accumulations in Rtnl1− larvae can be rescued by two copies of a Rtnl1Pacman genomic clone .", "All axons shown are crossing abdominal segment A2 .", "( B ) Graph shows mean ±SEM; n = 16–92 axons from 8 to 46 larvae , from 3 different experiments .", "ns , p>0 . 05; **p<0 . 006; ****p<0 . 0001 , two-tailed Student’s t-test .", "Scale bar , 10 µm ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 23882 . 015 To better understand wild-type axonal ER organization , and the mutant phenotypes seen in confocal microscopy , we performed electron microscopy ( EM ) on 60-nm-thick serial sections of third instar peripheral nerves .", "Peripheral nerves contain both motor and sensory axons , arranged in fascicles , and wrapped in three main classes of glial cell ( Stork et al . , 2008; Matzat et al . , 2015 ) .", "We used ROTO staining ( Tapia et al . , 2012; Terasaki et al . , 2013 ) to preferentially highlight cellular membranes including ER .", "Wild-type larvae showed a network of ER tubules , in every axon that could be observed ( Figure 7A left; serial sections in Video 5 ) .", "Tubule outer diameter averaged around 40 nm ( Figure 7B , C ) , and axons contained an average of around 1 . 6 ER tubules in each cross-section ( Figure 7D , E ) .", "Reconstruction ( Figure 7F left; Supplementary file 2 ) showed a tubular network with multiple branches , some dead ends , and continuity along nearly every axon sectioned .", "ER tubules often showed proximity to mitochondria or plasma membrane ( Figure 7G left ) .", "We also found occasional structures resembling small patches of ER sheets with an adjoining cisterna ( Figure 7H ) , continuous with the tubular ER network ( Video 6 ) , at a frequency averaging around one per 20 µm per axon . 10 . 7554/eLife . 23882 . 016Figure 7 . Loss of hairpin proteins leads to fewer but enlarged ER tubules in larval peripheral nerve axons .", "( A ) EMs of peripheral nerve axons from wild-type ReepA+ ( WT , left ) or Rtnl1− ReepA− ReepB− triple mutant ( right ) larvae .", "Arrowheads indicate ER tubules ( seen as continuous structures in serial sections in Videos 5 and 7 ) .", "Triple mutant axons show enlarged ER tubules , sometimes with a clear lumen ( arrows ) , seldom observed in wild-type .", "Quantification of ER tubule diameter ( B , C ) and tubules per axon cross-section ( D , E ) in control and mutant larvae .", "Data from individual ER tubules or axons are shown in B and D , averaged larval values , mean ±SEM in C and E . ( F ) 3D reconstruction of a 4 . 5 µm axon segment from wild-type ( left ) or mutant ( right ) peripheral nerves , generated from 75 serial 60 nm sections , showing ER ( green ) , mitochondria ( magenta ) and plasma membrane ( gray ) .", "Interactive versions of the reconstructions are in Supplementary files 2 and", "3 . ( G ) Electron micrographs of peripheral nerve axons from wild-type ( ReepA+ , left ) or Rtnl1− ReepA− ReepB− triple mutant ( right ) larvae , showing proximity of ER to mitochondria ( arrowheads ) or plasma membrane ( arrows ) .", "( H ) Representative EM of short ER sheet ( arrowhead ) and cisterna ( asterisk ) from a wild-type larva; further sections in Video 6 .", "( I ) section of an axonal swelling from a wild-type larva showing mitochondria ( m ) , vesicles ( v ) , and a large clear cisterna ( c ) ; further sections in Video 8 .", "( J ) Serial EM sections show ER discontinuity in two mutant axons: axon 1 lacks ER tubules in sections z6-z14 and axon 3 in sections z1-z11; neighboring axons ( e . g . axon 2 ) show a continuous ER network .", "( K ) 3D reconstruction of a 4 . 5 µm axon segment from mutant peripheral nerves , generated from 75 serial 60 nm sections , showing multiple gaps ( indicated by arrows ) .", "ER is in green and plasma membrane in gray .", "Raw EM data and an interactive version of the reconstruction are in Video 9 and Supplementary file 4 , respectively .", "( L ) Frequency of axons with gaps in the 4 . 5 µm lengths analyzed .", "ER gap length ( M ) , numbers of ER gaps per µm ( N ) , and proportion of axon length with gaps ( O ) in affected axons .", "In M–O , top graphs show data from individual ER gaps ( M ) or individual axons ( N–O ) , with second and third quartiles and 5th and 95th percentiles; bottom graphs show averaged larval values , mean ±SEM .", "In all graphs , ns p>0 . 05; *p<0 . 04; **p<0 . 003; ****p<0 . 0001; Mann-Whitney U test for B , D , top graphs in M–O; two-tailed Student’s T test for C , E , L , bottom graphs in M–O .", "Scale bars , 500 nm . DOI: http://dx . doi . org/10 . 7554/eLife . 23882 . 01610 . 7554/eLife . 23882 . 017Video 5 . Serial EM sections of a wild-type peripheral nerve , showing continuity of tubular membrane structures through multiple sections . The sixth section in the series is shown in Figure 7A ( left ) .", "ER tubules were identified as darkly stained structures present for multiple sections .", "See Figure 7A for annotations . DOI: http://dx . doi . org/10 . 7554/eLife . 23882 . 01710 . 7554/eLife . 23882 . 018Video 6 . Serial EM sections of a sheet-like ER structure in a wild-type axon , shown in Figure 7H . Arrows indicate short ER sheets and associated cisternae , present across multiple sections .", "Note that these structures usually appear continuous with tubules in adjacent sections . DOI: http://dx . doi . org/10 . 7554/eLife . 23882 . 018 If Rtnl1− ReepA− ReepB− triple mutant larvae have less ER membrane curvature , we would expect them to have larger ER tubules , fewer tubules per section , loss of tubules , or a combination of these .", "EM revealed all these mutant phenotypes to varying degrees ( Figure 7 ) .", "ER tubule diameter was increased to around 60 nm ( Figure 7B , C ) , allowing a lumen to be seen in some tubules ( Figure 7A right; Video 7 ) that was rarely seen in wild-type ( Figure 7A left; Video 5 ) , and most triple mutant axons exhibited only a single ER tubule ( Figure 7D , E ) .", "Reconstructions ( Figure 7F right; Supplementary file 3 ) showed a less extensive ER network in mutant axons .", "Frequent contacts of ER with mitochondria and plasma membrane were found in both wild-type and Rtnl1− ReepA− ReepB− mutant axons ( Figure 7G ) .", "Both wild-type and mutant axons showed swellings containing mitochondria and clusters of vesicles resembling synaptic vesicles ( Figure 7I; Video 8 ) .", "We did not detect large swellings with synaptic vesicle materials similar to those seen with confocal microscopy ( Figure 6 ) , but this may reflect the much shorter lengths of nerve examined by EM , around 5 µm compared to around 50 µm in confocal . 10 . 7554/eLife . 23882 . 019Video 7 . Serial EM sections of a Rtnl1− ReepA− ReepB− peripheral nerve showing continuity of tubular membrane structures through multiple sections . The sixth section in the series is shown in Figure 7A ( right ) .", "ER tubules were identified as darkly stained structures present for multiple sections .", "See Figure 7A for annotations . DOI: http://dx . doi . org/10 . 7554/eLife . 23882 . 01910 . 7554/eLife . 23882 . 020Video 8 . Serial EM sections of a wild-type axonal swelling shown in Figure 7I . An axon with a swelling is highlighted in the first frame .", "Sections show accumulated mitochondria , vesicles , and a large clear cisterna , as labeled in Figure 7I . DOI: http://dx . doi . org/10 . 7554/eLife . 23882 . 020 Serial EM sections also revealed variable fragmentation of ER in Rtnl1− ReepA− ReepB− mutant axons ( Figure 7J , K ) , consistent with that seen using confocal microscopy ( Figure 4 ) .", "Some discontinuity of the ER network was observed in about 10% of wild-type or Rtnl1− ReepA− ReepB− mutant axons ( Figure 7L ) .", "However , gaps in Rtnl1− ReepA− ReepB− mutant axons ( Figure 7J , K; Video 9; Supplementary file 4 ) were longer ( Figure 7M ) , and slightly more numerous ( but not significantly when averaged across larvae; Figure 7N ) than in wild-type axons , resulting in nearly a four-fold increase in the length of affected axons that lacked ER tubules ( Figure 7O ) . 10 . 7554/eLife . 23882 . 021Video 9 . Serial EM sections of a 4 . 5 µm segment of a Rtnl1− ReepA− ReepB− mutant axon with disrupted continuity of ER , used for 3D reconstruction in Figure 7K . An axon with gaps in its ER network is highlighted in the first frame .", "Continuous ER tubules were identified as the presence of signals at the same position for three or more sections .", "Given the varying brightness and contrast of EM sections , faint staining that coincided with a tubule signal in adjacent sections was also considered as an ER tubule .", "Complete loss of ER tubules from three or more sections was defined as a gap . DOI: http://dx . doi . org/10 . 7554/eLife . 23882 . 021 Rtnl1− ReepA− ReepB− mutant peripheral nerves also showed glial cell phenotypes .", "Wild-type peripheral nerves are surrounded by an outer perineurial glial cell , and just beneath this a subperineurial glial cell; axons or axon fascicles are wrapped imperfectly by a wrapping glia cell ( Stork et al . , 2008; Matzat et al . , 2015 ) .", "All glial classes , but particularly subperineurial glia , showed a trend towards increased ER sheet profile length compared to control cells ( Figure 8A–I ) , similar to Rtnl1 knockdown ( O'Sullivan et al . , 2012 ) or ReepA− ReepB− double mutant ( Figure 2 ) epidermal cells .", "Triple mutant wrapping glia also displayed more extensive wrapping , sometimes completely ensheathing axons , which was rarely observed in control nerve sections ( Figure 8J–M; Figure 8—figure supplement 1 ) . 10 . 7554/eLife . 23882 . 022Figure 8 . Loss of reticulon and REEP proteins leads to ER disorganization in glial cells and hyper-wrapping of peripheral axons .", "( A–B )", "EMs of peripheral nerve sections from ReepA+ ( WT ) ( A ) or Rtnl1− ReepA− ReepB− triple mutant ( B ) larvae .", "Perineurial , subperineurial , and wrapping glial cells are shaded green , blue , and magenta , respectively .", "( C–H )", "Higher magnification images of perineurial ( C , F ) , subperineurial ( D , G ) , and wrapping ( E , H ) glia from wild-type ( C–E ) or mutant ( F–H ) nerve sections , showing ER tubules ( white arrows; confirmed as tubules by presence in adjacent sections ) and sheets ( white arrowheads ) .", "Note the longer ER sheet profiles and fewer ER tubules in the subperineurial ( G ) and wrapping glial cells ( H ) of mutant nerves .", "Asterisks show mitochondria; yellow arrowheads show glial plasma membrane , identified by its continuity .", "( I ) ER sheet profile length in control and mutant glial cells from 3 wild-type and 4 mutant larvae .", "Top graphs represent all individual sheet profiles , with second and third quartiles and 5th and 95th percentiles; bottom graphs show averaged larval values , showing mean ±SEM .", "Two-way ANOVA showed a significant effect of genotype ( p<0 . 002 ) but not glial class ( p>0 . 3 ) on ER sheet length , with no interaction between factors ( p>0 . 3 ) .", "( J , K )", "Sketches of wild-type ( J ) and mutant ( K ) wrapping glial cells , showing excess processes in the triple mutant .", "Asterisks indicate completely wrapped one-axon or two-axon fascicles , rarely seen in wild-type nerve sections .", "More examples of each phenotype are in Figure 8—figure supplement 1 .", "( L–M )", "Quantification of wrapping glial membrane profile length per nerve cross-section ( L ) and percentage of axons that are wrapped individually or as two-axon fascicles ( M ) in wild-type and mutant nerve sections ( mean ±SEM ) .", "ns , p>0 . 05; *p<0 . 05; ***p<0 . 001; ****p<0 . 0001 .", "Mann-Whitney U test ( I ) , top graphs ) ; two-tailed Student’s t-test ( I ) , bottom graphs; L , M , ) .", "Scale bars , 1 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 23882 . 02210 . 7554/eLife . 23882 . 023Figure 8—figure supplement 1 . Sketches of wrapping glia in wild-type ( ReepA+ ) and Rtnl1− ReepA− ReepB− mutant peripheral nerves , similar to those in Figure 8J , K . Sketches of wild-type ( ReepA+ , upper ) and mutant ( lower ) wrapping glial cells were abstracted from cross-sections of peripheral nerves .", "Excess formation of processes were observed in triple mutants . DOI: http://dx . doi . org/10 . 7554/eLife . 23882 . 023" ], [ "The existence of a tubular axonal ER network has been known for decades .", "Nevertheless , the cellular mechanisms that organize a compartment that is usually distributed throughout cells , along the great lengths of axons , are until now largely unknown .", "The finding that several causative genes for the axon degenerative disease HSP encode ER modeling proteins , suggests a link between ER modeling and axon function or maintenance , and provides candidate proteins that may be instrumental in structure and function of the axon ER network .", "These candidates include several hairpin-loop-containing HSP proteins , of the spastin , atlastin , reticulon , REEP , and Arl6IP1 families , that influence ER structure in situations including yeast , mammalian cultured cells , and neuronal cell bodies in vivo ( Shibata et al . , 2006; Voeltz et al . , 2006; Hu et al . , 2008; Shibata et al . , 2009; Park et al . , 2010; Shibata et al . , 2010 ) .", "The HSP-related protein families that model ER , and some other proteins that interact with them , share a common feature of one or two intramembrane hairpin loops that can insert into the cytosolic face of the ER membrane , thereby recognizing or inducing curvature .", "This property makes the reticulon and REEP ( DP1 ) families together responsible for most peripheral ER tubules in yeast , and contribute to the curved edges of ER sheets ( Voeltz et al . , 2006; Hu et al . , 2008 ) .", "The latter property may explain the expansion of ER sheets in Drosophila lacking the reticulon Rtnl1 ( O'Sullivan et al . , 2012 ) and in REEP1 homozygous mutant mice ( Beetz et al . , 2013 ) .", "Given this background , we set out to test how far the reticulon and REEP families contribute to axonal ER organization .", "REEP1 homozygous mutant mice were not previously tested for effects on axonal ER , although knockdown of Drosophila Rtnl1 led to partial loss of smooth ER marker in distal motor axons ( O'Sullivan et al . , 2012 ) .", "Here we build on this work by analyzing mutants of all the widely expressed and highest conserved members of the reticulon and REEP families in Drosophila: Rtnl1 , an ortholog of all four human reticulons ( O'Sullivan et al . , 2012 ) ; ReepA , an ortholog of human REEP1-REEP4; and ReepB , an ortholog of human REEP5-REEP6 .", "We monitored phenotypes of these mutants , singly and in combination , by confocal microscopy of axonal ER markers in small numbers of motor neurons , live imaging and photobleaching of ER , and EM using membrane-specific staining .", "Confocal microscopy revealed a partial loss of ER marker in distal but not in more proximal motor axons in Rtnl1 and in ReepB mutants .", "Although the only REEP genes identified as causative for HSP are REEP1 and REEP2 , loss of their ortholog ReepA had at most only mild effects on axonal ER ( Figures 3 and 4 ) .", "The stronger axonal ER phenotypes of ReepB or Rtnl1 loss of function , compared to ReepA− mutants , is consistent with the higher levels of ReepB and Rtnl1 expression , judged by transcriptomics ( www . flyatlas . com ) , and the detection of ReepB and Rtnl1 but not ReepA fusions in peripheral nerves ( Figure 1F , G; O'Sullivan et al . , 2012 ) or individual axons ( Figure 1H , J ) .", "The partial loss of distal axonal ER marker in both mutants and motor-neuron knockdowns , of either Rtnl1 or ReepB ( Figure 3; O'Sullivan et al . , 2012 ) , and qualitatively similar ER fragmentation phenotypes in Rtnl1 ReepA ReepB triple loss-of-function genotypes , obtained using either Rtnl1 mutants or motor-neuron knockdown ( Figure 4 ) , suggest that the axonal ER phenotypes are cell autonomous in motor neurons .", "Given the joint and partly redundant requirement of the reticulon and REEP families for ER tubule formation in yeast ( Voeltz et al . , 2006 ) , we tested whether this was also true for axonal ER .", "Flies lacking Rtnl1 , ReepA and ReepB – probably equivalent to mammals lacking all four reticulons and all six REEPs , and homozygous viable – indeed showed more extreme ER phenotypes than axons lacking either Rtnl1 or ReepA and ReepB alone ( Figures 3 and 4 ) .", "However , only a fraction of mutant axons showed severe fragmentation , and even affected axons still had continuous labeling of axons with ER marker through much of their length .", "ER fragmentation in the middle parts of long axons might be a consequence of axon expansion during larval growth , in which the somatic and presynaptic ends of the axon are gradually pulled apart , with insufficient ER-modeling proteins to maintain the expanding tubular network throughout the axoplasm .", "Therefore reticulon and REEP proteins are present in axons and have roles in ER organization there – but since triple mutant axons still mostly possess ER , there must be additional proteins required too .", "These might be found among the increasing number of other HSP genes that encode ER proteins with possible hairpins , such as Arl6IP1/SPG61 , which affects ER organization ( Novarino et al . , 2014; Yamamoto et al . , 2014; Fowler and O'Sullivan , 2016 ) , or C19orf12/SPG43 ( Landouré et al . , 2013 ) .", "The variable nature of the triple mutant fragmentation phenotype might reflect stochastic variation in the amounts of such proteins , the amount of ER present , external factors like physical stresses during larval movement , or dynamic fluctuations in the local levels and connections of ER tubules .", "The tubular ER network is highly dynamic in non-neuronal cells ( Nixon-Abell et al . , 2016; Valm et al . , 2017 ) and in axons our live imaging suggests dynamic features superimposed on a structure that is largely stable over the 1–2 min of imaging ( Figure 5 ) .", "EM examination of wild-type axonal ultrastructure revealed that ER tubules were effectively ubiquitous in Drosophila peripheral nerve sections , as seen previously in mammalian neurons ( Tsukita and Ishikawa , 1976; Villegas et al . , 2014 ) , albeit with fewer tubules , presumably reflecting the smaller diameters of the axons examined here .", "Reconstruction over several µm showed a continuous network of ER tubules in most axons examined , in agreement with the ER continuity found in neurons by lipid dye labeling ( Terasaki et al . , 1994 ) , and recently in reconstructions of serial sections from focused ion beam SEM ( Wu et al . , 2017 ) .", "However , in a few axons , we found short lengths of axon with no detectable ER ( Figure 7J–O ) .", "There could be several reasons for this: Terasaki et al . ( 1994 ) only assessed continuity in dendrites , cell body and proximal axon; some of the gaps we observe in EM could be short transient gaps in a dynamic network; some of the apparent gaps could be ‘thin ER’ observed using higher-resolution focused-ion-beam SEM ( Wu et al . , 2017 ) , but that might be missed using our approach; larval axons with low diameters might be intrinsically more susceptible to occasional gaps in the ER network , than wider axons with more tubules; and we might occasionally miss an ER tubule due to weaker staining , or close proximity to other structures like plasma membrane .", "We also observed occasional small ER sheet-like structures in wild-type axons ( Figure 7H; Video 6 ) .", "Although we do not see ribosomes on these , this could be due to lack of staining by the ROTO protocol .", "As discussed above , rough ER and translation are relatively sparse in axons , but low levels of rough ER are possible , and consistent with the occasional sheet structures observed here .", "EM also showed phenotypes consistent with loss of ER membrane curvature in Rtnl1− ReepA− ReepB− triple mutant axons ( Figure 7 ) .", "Mutant axons had ER tubules of larger diameter , fewer tubules per axon cross-section , and consistent with our confocal data ( Figure 4 ) , longer gaps in the ER network than wild-type , although most parts of most mutant axons examined still had a continuous ER network ( Figure 7 ) .", "These mutant phenotypes could potentially have physiological consequences .", "Larger tubules could potentially store and release more calcium than thinner ones , while the reduced network could make the role of the ER in calcium buffering or release more localized .", "The less extensive ER network in mutants might also reduce the amount of contact between ER and other organelles , with consequences for calcium and lipid homeostasis that require these contacts , or for regulation of mitochondrial fission ( Friedman et al . , 2011 ) – although the continuing proximity of ER to mitochondria and plasma membrane in mutants means that any effects are presumably quantitative rather than qualitative .", "The reduced curvature of ER membrane in mutants might also influence their protein composition , since many membrane proteins have mechanisms for recognizing differential membrane curvature ( Antonny , 2011 ) .", "The occasional lack of continuity could prevent propagation of ER-dependent Ca2+ signals like those seen in injured mammalian sensory neurons ( Cho et al . , 2013 ) ; it could also cause local impairments in Ca2+ or lipid homeostasis that could lead to local transport inhibition , as is the case for mitochondrial transport ( Wang and Schwarz , 2009 ) , although lack of ER continuity does not appear to directly prevent axon transport ( Figure 5—figure supplement 1C; Video 3 ) .", "Sporadic lack of ER continuity might explain the preferential sensitivity of distal longer axons to HSPs , since these would be more likely to suffer from a gap in ER continuity to the cell body , compared to proximal or shorter axons .", "In this model , disease-causing alleles in single hairpin-encoding genes could promote degeneration in distal motor axons by increasing the probability of such gaps , dependent on factors such as age , axon length or diameter , and ER tubule density and dynamics .", "The apparent ubiquity of ER in axons , the extent of its continuity over long distances , and the preferential susceptibility of distal longer axons to mutations that affect ER-modeling proteins , all point to important physiological roles of this compartment and of its continuity .", "In this work we have begun to reveal the mechanisms that determine its organization .", "We have shown roles for two protein families that contain HSP disease gene products , in influencing the shape of individual tubules and the axonal ER network , with potential physiological consequences that would also be affected by mutations in these genes .", "Understanding the consequences of axonal ER structural defects for ER dynamics and axonal physiology , both in the genotypes we have described here , and in other genotypes that might also affect axonal ER organization , will provide models for the potential physiological defects in HSP and other axon degeneration diseases ." ], [ "ReepA541 ( referred to as ReepA− ) , and ReepB48 ( referred as ReepB− ) mutants were generated by imprecise excision of P elements CB-0501–3 ( RRID:DGGR_123207 ) and EY05130 ( RRID:BDSC_16636 ) shown in Figure 1 .", "A precise excision generated in these experiments , ReepA+C591 ( referred to as ReepA+ ) was used as a genetic background control where feasible .", "Rtnl11 , referred as Rtnl1− , was a gift from G . Tear ( Wakefield and Tear , 2006; FlyBase ID FBal0246222 ) .", "Rtnl1− , ReepA− and ReepB− recombinants were generated by meiotic recombination on the second chromosome , and recombinants were screened using PCR primers ( Supplementary file", "5 ) to diagnose wild-type or mutant alleles of all three genes .", "Mutant and wild-type stocks were frequently genotyped to ensure that experimental flies were not contaminated .", "For knockdown experiments , either UAS-Rtnl1-RNAi line 7866 ( construct GD900 , which has no predicted off-targets; FlyBase ID FBti0098310 ) , or the w1118 control stock , 60000 ( both obtained from the Vienna Drosophila RNAi Center , www . vdrc . at ) , or the UAS-ReepB-RNAi line 8331 R-3 ( National Institute of Genetics Fly Stock Center , Japan; FlyBase ID FBal0275953 ) was crossed with UAS-Dcr2; CyO/If; m12-GAL4 , UAS-Acsl::myc .", "UAS-Dcr2 ( RRID:BDSC_24648; Dietzl et al . , 2007 ) was also present for knockdown in Rtnl1 ( RNAi ) ReepA− ReepB− larvae .", "Other fly stocks used were P{UAS-Acsl . 715 . MycC}3 ( RRID:BDSC_32330; Zhang et al . , 2009 ) , PBac{681 . P . FSVS-1}Rtnl1CPTI001291 ( RRID:DGGR_115146; Wakefield and Tear , 2006 ) , m12-GAL4 ( Xiong et al . , 2010; RRID:BDSC_2702 ) , UAS-Rtnl1::GFP ( Rao et al . , 2016 ) , {UAS-Xbp1 . EGFP . LG}4 ( RRID:BDSC_39719; Ryoo et al . , 2007 ) and UAS-tdTomato::Sec61β ( RRID:BDSC_64746; Summerville et al . , 2016 ) .", "A second-chromosome insertion of P{UAS-CG9186::GFP} ( Thiel et al . , 2013 ) was mobilized onto the third chromosome using the P transposase source P{∆2–3}99B ( Robertson et al . , 1988; RRID:BDSC_3612 ) , and expressed using m12-GAL4 in either a ReepA+ or an Rtnl1− ReepA− ReepB− mutant second chromosome background .", "For rescue of Rtnl11 we generated transgenic flies carrying P[acman] clone CH322-124P15 inserted at attP2 on chromosome 3 ( Bloomington stock 25710 ) , referred to as Rtnl1Pacman ( Venken et al . , 2009 ) .", "For C-terminal EGFP-LAP-tagging of ReepA and ReepB we used recombineering with the P[acman] system with minor modifications ( Venken et al . , 2009 ) , utilizing the CH322-97D15 ( ReepA ) and CH322-16N11 ( ReepB ) BAC clones ( BPRC; http://bacpac . chori . org ) .", "An EGFP-LAP-tagging cassette was amplified from R6Kamp-LAP ( GFP ) ( Poser et al . , 2008 ) using primers ( Supplementary file", "5 ) with 50 bp homology to the corresponding Reep clone and around 20 bp of homology to the tagging cassette , and used to transform the recombineering E . coli SW102 strain .", "Correct clones were verified at every step by PCR and/or restriction digestion .", "DNA for Drosophila transformation was extracted using a PureLink HiPure Maxiprep kit ( Invitrogen ) , and injected into y w M ( eGFP , vasa-integrase , dmRFP ) ZH-2A; M ( attP ) ZH-51D or y w M ( eGFP , vasa-integrase , dmRFP ) ZH-2A; M ( attP ) ZH-86Fb ( RRID:BDSC_24483 or RRID:BDSC_24749 , respectively ) at the Department of Genetics embryo injection facility , University of Cambridge , UK .", "Transformant lines were screened for the presence of the insert by PCR and GFP fluorescence .", "The primers used are described in Supplementary file 5 .", "BLAST sequence searches were used to define genome coordinates of P-element excisions , and Pfam domain coordinates in coding regions , and compare protein divergence rates .", "They were performed at the National Center for Biotechnology Information ( www . ncbi . nlm . nih . gov ) .", "REEP dendrograms were drawn from a ClustalW alignment ( Larkin et al . , 2007 ) using the neighbor-joining algorithm in MEGA 5 . 05 ( Tamura et al . , 2011 ) .", "Third instar larvae were dissected in chilled Ca2+-free HL3 solution ( Stewart et al . , 1994 ) , and fixed for 30 min in PBS with 4% formaldehyde .", "Dissected Drosophila preparations were permeabilized in PBS containing 0 . 3% Triton X-100 ( PBT ) at room temperature , and blocked in PBT with 4% bovine serum albumin for 30 min at room temperature .", "Primary antibodies were: Csp ( 6D6 , RRID:AB_10013286 , 1:50; Zinsmaier et al . , 1994 ) , Dlg ( 4F3 , RRID:AB_2314321 , 1:100; Parnas et al . , 2001 ) , ( both from the Developmental Studies Hybridoma Bank , Iowa , USA ) , GFP ( Ab6556 , RRID:AB_305564 , 1:600 Abcam , UK ) , HRP ( P97899 , RRID:AB_2314650 , 1:300 , Sigma ) , KDEL ( Ab50601 , RRID:AB_880636 , 1:25 , Abcam , UK ) , myc ( 2272 , RRID:AB_331667 , 1:25 , Cell Signaling , USA ) .", "Fixed preparations were mounted in Vectashield ( Vector Laboratories , USA , RRID:AB_2336789 ) , and images were collected using EZ-C1 acquisition software ( Nikon ) on a Nikon Eclipse C1si confocal microscope ( Nikon Instruments , UK ) .", "Images were captured using 10x/0 . 30NA , or a 60x/1 . 4NA oil objective .", "Confocal images were analyzed blind to genotype using ImageJ ( Schneider et al . , 2012 ) .", "Images of entire epidermal cells were obtained as z-projections of three consecutive sections .", "Using the line tool of ImageJ a 12 µm line was drawn from the nuclear envelope towards the periphery of each cell analyzed .", "Pixel intensity along the line was recorded in an Excel file .", "Local variance of intensity was calculated by dividing the rolling variance of the intensity ( in 10-pixel windows ) , by the rolling mean intensity , all along the line .", "Proximal ( anterior ) axons were imaged from segment A2 , middle images were from the end of segment A4 and A5 , distal ( posterior ) axons were imaged from segment A6 of third instar larvae .", "Mean gray intensity for single-axon images was measured by drawing a 45 µm line , either along both M12-GAL4-expressing axons ( where they could not be separated ) , or along the most strongly labeled axon ( where they appeared as separate axons ) , and quantifying gray intensity ( 0–255 ) by ImageJ; occasional images with saturated pixels were excluded from analysis after blinding .", "Coefficient of variation was calculated by dividing the standard deviation of staining intensity by the mean; occasional images with faint staining throughout the axon were excluded from analysis after blinding .", "Gaps were defined as regions where staining intensity was less than 20 ( out of 255 ) , after background subtraction .", "FRAP experiments were performed on a Nikon Eclipse C1si confocal microscope ( Nikon Instruments , UK ) , using a 20 mW Argon laser and a 40× 0 . 8 N . A . water dipping objective .", "Third instar larvae were dissected and incubated in chilled Ca2+-free HL3 solution ( Stewart et al . , 1994 ) .", "Time-lapse images were acquired on a single focal plane every 0 . 5 or 1 s for 40 loops .", "For FRAP , a defined region of interest ( 12 × 4 µm ) was photobleached at full laser power ( 488 nm ) for two iterations at a scan speed of 0 . 5 frame/s .", "Postbleaching images were acquired on a single focal plane at 15% laser power once every 5 s for 200 s .", "Experiments were completed within 20 min after dissection .", "Kymographs were generated for a hand-drawn line selection along the axon using the Multiple Kymograph plugin in Fiji ( Schindelin et al . , 2012 ) .", "Average fluorescence intensity of each axon in each frame was measured by creating a line selection along the axon .", "After subtracting background ( average intensity in a nearby non-GFP-expressing region within the segmental nerve ) , the intensity of the bleached region was normalized to the average intensity cross the axon length in the same frame .", "Then the data were further normalized by taking the prebleaching intensity as 100% and bleach intensity as 0 .", "Normalized postbleaching data were plotted and fitted to a single exponential function to calculate rate constant ( k ) and half time ( t1/2 ) : I ( t ) =A ( 1-e−kt ) , in which I ( t ) represents the fluorescence intensity at time point t , A the highest postbleaching intensity .", "For epidermal cell EM , larvae were prepared and fixed as described by O'Sullivan et al . ( 2012 ) .", "Transverse sections were cut on a Leica Ultracut UCT ultra-microtome at 70 nm , using a diamond knife , and contrasted with uranyl acetate and lead citrate ( for epidermal cells ) .", "Sections were viewed using a Tecnai G2 electron microscope operated at 120 kV , and an AMT XR60B camera running the Deben software in the Cambridge Advanced Imaging Centre , School of Biology , University of Cambridge .", "For EM of peripheral nerves , we used a ROTO protocol ( Tapia et al . , 2012; Terasaki et al . , 2013 ) to highlight membranes .", "Third instar larvae were dissected in HL3 solution and fixed in 0 . 05 M sodium cacodylate ( pH 7 . 4 ) containing 4% formaldehyde , 2% vacuum distilled glutaraldehyde , and 0 . 2% CaCl2 ) , at 4°C for 6 hr .", "Larvae were dissected as for confocal analysis , but leaving overlying organs such as gut and fat body attached , to reduce loss of peripheral nerves during processing .", "Preparations were then washed 3 times for 10 min each at 4°C using cold cacodylate buffer with 2 mM CaCl2 .", "A solution of 3% potassium ferricyanide in 0 . 3 M cacodylate buffer with 4 mM CaCl2 was mixed with an equal volume of 4% aqueous osmium tetroxide; larval preparations were incubated in this solution at 4°C for 1–12 hr , then rinsed with deionized water at room temperature 5 times for 3 min each .", "Thiocarbohydrazide solution was prepared by adding 0 . 1 g thiocarbohydrazide to 10 ml deionized water , kept in a 60°C oven in a secondary embedding pot for 1 hr , swirled every 10 min to facilitate dissolution , and filtered through two 9 cm filter papers just before use .", "Larval preparations were incubated in thiocarbohydrazide solution for 20–30 min at room temperature and covered with foil to protect from light .", "Then they were rinsed with deionized water at room temperature 5 times for 3 min each , incubated in 2% osmium tetroxide for 30–60 min at room temperature , and rinsed with deionized water at room temperature 5 times for 3 min each .", "Preparations were incubated in 1% uranyl acetate ( maleate-buffered to pH 5 . 5 ) at 4°C overnight and rinsed with deionized water at room temperature 5 times for 3 min each .", "Then they were incubated in lead aspartate solution ( 0 . 66 g lead nitrate dissolved in 100 ml 0 . 03 M aspartic acid , pH adjusted to 5 . 5 with 1 M KOH ) at 60°C for 30 min and rinsed with deionized water at room temperature 5 times for 3 min .", "Then they were dehydrated twice with 50% , 70% , 90% and 100% ethanol , twice with dried ethanol , twice with dried acetone and twice with dry acetonitrile .", "Preparations were incubated in 50/50 acetonitrile/Quetol 651 overnight at room temperature , three times for 24 hr each in Quetol epoxy resin 651 ( Agar Scientific , Stansted , UK ) and three times for 24 hr each in Quetolepoxy resin 651 with BDMA ( dimethylbenzylamine ) .", "They were then incubated at 60°C for at least 48 hr .", "Serial 60-nm-thick transverse sections were cut in the larval abdominal region , visualized using scanning EM , and images were aligned for analysis of serial sections and reconstruction , as described by Terasaki et al . ( 2013 ) .", "To quantify axonal ER tubule diameter , non-axonal staining was removed manually , and ER tubule profiles were identified based on the local threshold in a single cross-section , and the presence of signals at the same position in adjacent sections .", "The minimum Feret diameter of each tubule was measured using ImageJ Fiji ( https://fiji . sc ) via the Analyze Particles command .", "ER numbers per axon were counted manually for all axons detected in the nerve .", "3D reconstruction was carried out using the Fiji TrakEM2 plug-in .", "To quantify gaps in the tubule network , each axon was analyzed throughout the entire stack of sections .", "To allow for occasional lightly stained or blurred sections , only complete loss of ER tubules from three or more consecutive sections in an axon was defined as a gap .", "Continuous ER tubules were identified as the presence of signals at the same position for three or more consecutive sections .", "Given the varying brightness and contrast of EM sections , faint staining that coincided with a tubule signal in adjacent sections was also considered as an ER tubule .", "Color shading and sketches drawn in Figure 8 were processed in Adobe Photoshop CS6 .", "For quantification of glial ER sheet length , individual ER sheet profiles were measured using Fiji via the line tool and Measure command .", "Wrapped axons were defined as one-axon or two-axon fascicles which were completely wrapped by glial cells and isolated from other neighboring axons .", "Statistical analyses were performed in GraphPad Prism 6 or IBM SPSS 22 .", "Data were analyzed by two-tailed Student’s t-tests or ANOVA followed by post-hoc tests ( for comparison of datapoints that were means of raw measurements and hence expected to be normally distributed ) , or by Mann-Whitney U tests ( for data that were not normally distributed ) .", "Multiple comparisons after ANOVA were performed by a Tukey HSD test when equal variances were found , or otherwise by Dunnett’s T3 test .", "Bar graphs and scatter plots show mean ±SEM; box plots show median with interquartile range , and the 5% and 95% percentiles as whiskers .", "Sample sizes are reported in figures .", "No outliers were excluded from analysis after quantification; data were only excluded from analysis if they were unanalyzable , i . e . when confocal images were too faint or saturated , and EM images lacked clearly identifiable plasma or ER membranes .", "Most data were analyzed and presented as individual axon or larval datapoints , unpaired between genotypes .", "For two comparisons where ANOVA showed significant ( p<0 . 05 ) experiment-to-experiment differences within a genotype ( Figures 2B and 3A ) , data were averaged within each independent experiment to produce experiment-wise datapoints , which were compared using two-tailed paired t-tests .", "P levels are indicated as ns p>0 . 05 , *p<0 . 05 , **p<0 . 01 , ***p<0 . 001 , or ****p<0 . 0001 , except where indicated otherwise , when criteria could be defined more stringently ." ] ]

[ "Axons contain a smooth tubular endoplasmic reticulum ( ER ) network that is thought to be continuous with ER throughout the neuron; the mechanisms that form this axonal network are unknown .", "Mutations affecting reticulon or REEP proteins , with intramembrane hairpin domains that model ER membranes , cause an axon degenerative disease , hereditary spastic paraplegia ( HSP ) .", "We show that Drosophila axons have a dynamic axonal ER network , which these proteins help to model .", "Loss of HSP hairpin proteins causes ER sheet expansion , partial loss of ER from distal motor axons , and occasional discontinuities in axonal ER .", "Ultrastructural analysis reveals an extensive ER network in axons , which shows larger and fewer tubules in larvae that lack reticulon and REEP proteins , consistent with loss of membrane curvature .", "Therefore HSP hairpin-containing proteins are required for shaping and continuity of axonal ER , thus suggesting roles for ER modeling in axon maintenance and function ." ]

[ "The way we move – from simple motions like reaching out to grab something , to playing the piano or dancing – is coordinated in our brain .", "These processes involve many regions and steps , in which nerve cells transport signals along projections known as axons .", "Axons rely on sophisticated ‘engineering’ to work properly over long distances and are vulnerable to diseases that disrupt their engineering .", "For example , in genetic diseases called ‘hereditary spastic paraplegias’ , damages to the ‘distal’ end of axons – the end furthest from the nerve cell body – cause paralysis of the lower body .", "Axons have several internal structures that make sure everything works properly .", "One of these structures is the endoplasmic reticulum , which is a network of tubular membranes that runs lengthwise along the axon .", "It is known that spastic paraplegias are sometimes caused by mutations affecting proteins that help to build and shape the endoplasmic reticulum , for example , the proteins of the reticulon and REEP families .", "However , until now it was not known how the ER forms its network in the axons and if this is influenced by these proteins .", "To see whether reticulons and REEPs affect the shape of the endoplasmic reticulum , Yalçιn et al . used healthy fruit fly larvae , and genetically modified ones that lacked the proteins .", "The results show that in healthy flies , the tubular network runs continuously along the axons .", "When either reticulon or REEP proteins were removed , the distal axons contained less endoplasmic reticulum .", "In mutant fly larvae that lacked both protein families , the endoplasmic reticulum was more interrupted and contained more gaps than in normal larvae .", "Using high-magnification electron microscopy confirmed these findings , and showed that the tubules of the endoplasmic reticulum in mutant axons were larger , but fewer .", "A next step will be to test whether these mutations also affect how the axons work and communicate over long distances .", "A better knowledge of the role of the endoplasmic reticulum in axons will help us to understand how damages to it could affect hereditary spastic paraplegias and other degenerative conditions ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "developmental biology", "neuroscience" ]

[ [ "Inherited retinal diseases ( IRDs ) encompass a group of genetically linked retinopathies characterized by progressive photoreceptor death ( Duncan et al . , 2018 ) .", "IRDs lead to irreversible vision loss , for which treatment strategies are limited .", "Retinitis pigmentosa ( RP ) , the most common IRD with approximately 1 . 5–2 . 5 million RP patients a worldwide ( Dias et al . , 2018; Hartong et al . , 2006; Verbakel et al . , 2018 ) , is characterized by early onset night blindness , gradual loss of visual field , and eventual loss of central vision ( Ferrari et al . , 2011; Hamel , 2006 ) .", "The initial pathological feature of RP is selective rod photoreceptor cell death causing night blindness , which is then followed by loss of cone photoreceptors and eventual full blindness ( Léveillard et al . , 2014 ) .", "Mutations in more than 70 genes have been linked to RP ( Dias et al . , 2018; https://sph . uth . edu/retnet/ ) .", "How these mutations affect gene function or initiate aberrant photoreceptor cell loss is largely unknown .", "Given the genetic diversity , pan-disease therapeutics are highly desirable .", "Accordingly , the purpose of our study was to identify compounds capable of promoting rod cell survival in multiple RP models and across species .", "As RP progression is relatively protracted , and even small numbers of surviving rod photoreceptors can preserve cone photoreceptor function ( Guadagni et al . , 2015; Hartong et al . , 2006; Punzo et al . , 2012 ) , pharmacological interventions aimed at slowing rod photoreceptor death are sought ( Duncan et al . , 2018; Wubben et al . , 2019 ) .", "However , currently there are no effective therapies for promoting rod photoreceptor survival .", "As a means of discovering new pharmacological treatments , target-directed high-throughput screening ( HTS ) approaches have been highly successful in identifying compounds that bind to and/or modulate disease-implicated molecules .", "However , many promising leads fail during late-stage animal model testing or clinical trials ( Munos , 2009; Sams-Dodd , 2013; Scannell et al . , 2012 ) .", "This trend has renewed interest in phenotypic drug discovery ( PDD ) , a complementary approach where drug effects are evaluated in cells or living disease models ( Bickle , 2010; Lee et al . , 2012; Swinney , 2013 ) .", "A number of first-in-class drugs were recently discovered using PDD ( Eder et al . , 2014; Swinney and Anthony , 2011; Swinney , 2013 ) .", "To expand opportunity on this front , we developed a PDD platform enabling quantitative HTS ( qHTS; Inglese et al . , 2006 ) in zebrafish ( Walker et al . , 2012; Wang et al . , 2015a; White et al . , 2016 ) .", "Zebrafish offer several distinct advantages as a retinal disease modeling system ( Angueyra and Kindt , 2018; Richardson et al . , 2017 ) .", "First , the structure of the zebrafish retina is similar to the other vertebrates ( Angueyra and Kindt , 2018; Richardson et al . , 2017; Schmitt and Dowling , 1999 ) .", "In particular , the zebrafish retina is ‘cone rich’ like the human retina .", "Second , about 70% of human genes have at least one ortholog in zebrafish ( Howe et al . , 2013 ) .", "Moreover , all RP-associated genes listed in RetNet ( https://sph . uth . edu/retnet/ ) have conserved zebrafish orthologs .", "Third , the zebrafish retinal system develops quickly being fairly mature by day five of development ( Brockerhoff et al . , 1995; Moyano et al . , 2013; Schmitt and Dowling , 1999 ) .", "Fourth , zebrafish are amendable to large-scale chemical screening due to their high fecundity rate , small size , and ease of visualizing and quantifying a variety of phenotypes ( Mathias et al . , 2012; Zon and Peterson , 2005 ) .", "To streamline such screens , we developed a high-throughput plate reader-based method for quantifying reporter gene expression in vivo ( ‘ARQiv’; Walker et al . , 2012 ) .", "Recently , we adapted the ARQiv system to human stem-cell-derived retinal organoids ( Vergara et al . , 2017 ) to enable cross-species PDD .", "To realize full throughput potential , ARQiv was combined with robotics-based automation to create ‘ARQiv-HTS’ ( Wang et al . , 2015b; White et al . , 2016 ) .", "Here , to identify neuroprotective compounds promoting rod photoreceptor survival , ARQiv-HTS was used to perform a large-scale chemical screen in a transgenic zebrafish model enabling inducible rod photoreceptor cell death ( Walker et al . , 2012; White et al . , 2017 ) .", "In these fish , YFP-NTR ( a yellow fluorescent protein-nitroreductase bacterial enzyme fusion protein ) is selectively expressed in rod photoreceptors .", "NTR expression enables inducible ablation of rod cells upon exposure to prodrug substrates , such as metronidazole ( Mtz ) , which are thought to cause apoptosis through activated metabolites that cause DNA damage ( Curado et al . , 2007; White and Mumm , 2013 ) .", "Close to 3000 largely human-approved drugs were tested across six concentrations ( i . e . using qHTS principles , Inglese et al . , 2006 ) in more than 350 , 000 zebrafish larvae .", "Statistically , 113 hits were identified as hits and 42 of the top performing compounds advanced through confirmation and orthogonal assays .", "Eleven compounds passed all secondary tests and moved forward as lead drug candidates .", "Validation tests in an orthogonal series of mouse RP models followed , reasoning that drugs effective across species and/or independent of disease mutation may translate better clinically .", "All leads were screened in primary mouse photoreceptor cell cultures and a subset of leads was evaluated in retinal explants from the retinal degeneration 1 ( Pde6brd1 , hereafter rd1 ) mutant mouse model of RP .", "Finally , one of three top-performing leads was tested in retinal degeneration 10 mutant retinas in vivo ( Pde6brd10 , hereafter rd10 ) .", "An analysis of potential mechanisms of action ( MOA ) suggested both shared and complementary targets/pathways .", "The role of Poly ( ADP-ribose ) polymerase ( PARP ) , the most common shared target , was evaluated using chemical inhibition , genetic targeting , and western blot analysis .", "The results implicated PARP as a key mediator of NTR/Mtz-mediated rod cell ablation , and PARP1 inhibition as a shared MOA for a subset of leads .", "As PARP-dependent cell death pathways are implicated in the etiology of Parkinson’s disease ( Kam et al . , 2018 ) , RP ( Power et al . , 2020 ) , and other neurodegenerative disorders ( Fan et al . , 2017; Fatokun et al . , 2014 ) , NTR/Mtz-mediated cell ablation may serve as an inducible and titratable methodology for modeling neurodegenerative disease .", "To interrogate complementary MOA , lead compounds were tested in pairs in mouse photoreceptor cell culture assays and in zebrafish .", "Intriguingly , enhanced survival effects were common in mouse photoreceptor cell cultures , while additive and even synergistic effects were evident in zebrafish for the majority of pairs tested .", "Taken together , our results suggest drugs providing neuroprotective effects across diverse RP models , between species , and/or through complementary MOA , will provide promising new therapeutic opportunities for IRD/RP patients ." ], [ "We initiated our study using a robotics-automated large-scale in vivo screening system to identify compounds that promoted rod photoreceptor survival in a transgenic zebrafish line enabling targeted ablation of rod photoreceptors , Tg ( rho:YFP-Eco . NfsB ) gmc500 , hereafter , rho:YFP-NTR ( Walker et al . , 2012; White et al . , 2017 ) .", "In this line , a 3 . 7 kb rhodopsin ( rho ) promoter fragment ( Hamaoka et al . , 2002 ) drives transgene expression exclusively in rod photoreceptor cells ( Figure 1A ) .", "The transgene is a fusion protein linking a yellow fluorescent protein ( YFP ) reporter to a nitroreductase prodrug converting enzyme ( NTR , encoded by the nfsB gene from Escherichia coli ) .", "NTR expression enables pro-drug inducible targeted cell ablation ( Curado et al . , 2007; Pisharath et al . , 2007 ) .", "Exposing rho:YFP-NTR fish to the prodrug metronidazole ( Mtz ) leads to the selective death of rod photoreceptors and concomitant loss of YFP ( Figure 1A–C ) , physiologically mimicking the onset of RP ( Hamel , 2006 ) .", "An immunohistological analysis of rod and cone photoreceptor markers was performed on 7 days post-fertilization ( dpf ) zebrafish retinal sections to test if Mtz-induced ablation was specific to rod cells .", "In non-ablated controls , rod outer segment labeling was well correlated with YFP expression ( Figure 1—figure supplement 1A , B; -Mtz , arrows ) .", "In Mtz-treated retinas , rod outer segment labeling and YFP expression were markedly diminished ( Figure 1—figure supplement 1A , B; +Mtz ) .", "Cone photoreceptor labeling showed no overlap with YFP expression , and cone cell labeling appeared similar in non-ablated and Mtz-treated retinas ( Figure 1—figure supplement 1C ) , suggesting cone cells were not affected by Mtz exposure or by acute rod cell loss .", "Prior studies suggest NTR/Mtz-mediated ablation occurs by apoptosis ( Chang et al . , 1993; Doonan et al . , 2003; Portera-Cailliau et al . , 1994; Zeiss et al . , 2004 ) and loss of rods in RP has also been linked to apoptosis ( Chang et al . , 1993; Doonan et al . , 2003; Portera-Cailliau et al . , 1994; Zeiss et al . , 2004 ) .", "Thus , we reasoned rho:YFP-NTR fish could be used to identify compounds that protected rods from apoptosis .", "To identify neuroprotective compounds with this model ( i . e . drugs that sustain YFP expression after Mtz exposure ) , we used our established plate reader-based ARQiv-HTS assay ( Figure 1D; Walker et al . , 2012; White et al . , 2016 ) .", "We first determined optimal conditions for inducing rod cell loss while maintaining larval health in a 96-well plate format .", "Major aspects of retinal cytogenesis are largely complete by five dpf in zebrafish ( Schmitt and Dowling , 1999; Stenkamp , 2011 ) .", "Reporter expression in rho:YFP-NTR larvae has also stabilized by this time point ( Unal Eroglu et al . , 2018 ) , consistent with rho expression ( Raymond et al . , 1995 ) .", "We therefore chose 5 dpf to initiate Mtz-induced rod cell ablation .", "We previously determined that rod cell loss reached a nadir at 7 dpf following a 24 hr pulse of 10 mM Mtz at 5 dpf ( Walker et al . , 2012 ) .", "We reasoned that a 48 hr Mtz exposure initiating at 5 dpf would maximize the signal window to test for neuroprotective effects .", "Concluding the experiment by 7 dpf also avoids challenges associated with feeding , as zebrafish can subsist on their yolk sac up to that time point ( Hernandez et al . , 2018; Jardine and Litvak , 2003 ) .", "However , 10 mM Mtz treatments extending beyond 24 hr become increasingly toxic ( Mathias et al . , 2014 ) and removing Mtz from microtiter plates after a 24 hr pulse could not be easily automated .", "We therefore sought a 48 hr Mtz treatment regimen sufficient for inducing maximal rod cell loss by 7 dpf that showed no evidence of general toxicity .", "Five concentrations of Mtz were tested across a twofold dilution series from 10 mM to 625 μM .", "YFP reporter signals were quantified daily from 5 to 8 dpf using ARQiv .", "Changes in YFP levels were calculated as percentages normalized to non-ablated YFP controls .", "The data showed concentration-dependent reductions in YFP with maximal loss observed at 7 dpf ( Figure 1B ) .", "Mtz exposures at 0 . 625 , 1 . 25 , 2 . 5 , 5 , and 10 mM led to a 55 , 79 , 87 , 87 , and 83% decrease in YFP detection , respectively ( Supplementary file 1a ) .", "Although no lethality was observed for any condition , signs of distress were evident for 10 mM Mtz exposures ( i . e . reduced motility ) .", "At ≤5 mM Mtz , however , no signs of stress were observed .", "As 2 . 5 mM Mtz was the lowest concentration producing maximal loss of YFP detection , and confocal imaging verified YFP loss following a 2-day ( 5–7 dpf ) 2 . 5 mM Mtz treatment ( Figure 1C ) , this condition was selected as the treatment regimen for the large-scale screen .", "Previously established power analysis methods using ablated and non-ablated controls ( White et al . , 2016 ) determined that a sample size of nine larvae was sufficient to detect a 50% neuroprotective effect .", "For ease of dispensing , microtiter plate formatting , and to account for larval dispensing errors , we increased the sample size to 16 larvae per condition for the primary screen .", "We also reasoned that this would also allow us to detect subtler neuroprotective effects .", "The strictly standardized mean difference quality control ( SSMD QC ) score was 1 . 67 , indicating the assay was of sufficient quality to justify a large-scale screening effort ( Zhang , 2011 ) .", "To establish a positive control , we tested 17 compounds and one compound ‘cocktail’ previously implicated as neuroprotectants in mammalian RP models ( Supplementary file 2a ) .", "Unfortunately , none sustained YFP expression at the concentrations tested ( 4 μM to 125 nM ) .", "However , none have proven effective in RP patients either , suggesting a lack of conservation of neuroprotective effects across species .", "Our screen was designed to address this issue by testing for neuroprotective effects of these compounds across species and between different RP models .", "Fortunately , a compound identified as a retinal cell neuroprotectant by the Zack lab ( manuscript in preparation ) did show-dose-dependent effects on YFP levels .", "This compound was therefore used as a positive control ( POS ) for assay performance throughout the large-scale screen .", "The Johns Hopkins Drug Library ( JHDL ) was chosen for the large-scale screen .", "The JHDL is comprised of nearly 3000 compounds , most being human-approved drugs ( Shim and Liu , 2014 ) .", "To minimize false discovery rates , all compounds were tested using qHTS principles ( Inglese et al . , 2006 ) – that is , across six concentrations ( 4 μM - 125 nM ) using a twofold dilution series .", "The screen largely followed published ARQiv-HTS methodologies ( Wang et al . , 2015b; White et al . , 2016 ) , with additional assay-specific details ( Figure 1D , steps 1–8 ) .", "In all , 2934 compounds were screened and more than 350 , 000 transgenic zebrafish larvae evaluated .", "Real-time data analysis was performed as previously detailed ( White et al . , 2016 ) to generate: ( 1 ) a plot of YFP signal levels , ( 2 ) a plot of SSMD scores across all tested concentrations , ( 3 ) a signal intensity heat map of each plate , and ( 4 ) an SSMD score table ( Figure 1D , step 7 ) .", "Compounds producing SSMD scores of ≥one were considered potential hits and flagged for visual inspection to assess fluorescence and general morphology using a stereo fluorescence microscope .", "This step facilitated elimination of false-positive compounds producing aberrant fluorescence due to larval toxicity ( six drugs ) or compound autofluorescence ( 27 drugs; Figure 1C , step 8; Supplementary file 2b ) .", "Additionally , this allowed visual confirmation of sustained YFP expression within the retina .", "At the conclusion of the primary screen , 113 compounds were identified as hits ( Supplementary file 2c ) .", "Hit compounds were classified according to the highest SSMD score achieved across all concentrations tested , and whether concentration-dependent effects were observed .", "SSMD scores suggested one drug produced a strong effect ( SSMD of 2–3 ) ; three had semi-strong effects ( 1 . 645–2 ) , 21 showed moderate effects ( 1 . 28–1 . 645 ) , and 88 had fairly moderate effects ( 1–1 . 28 ) ( Supplementary file 2c ) .", "Forty-two drugs showed concentration-dependent effects , while 71 exhibited discontinuous or singular concentration effects .", "‘On label’ mechanism of action ( MOA ) information available for the 113 hits implicated more than 50 targets/pathways in rod cell neuroprotection ( Supplementary file 2d ) .", "We next performed a series of confirmatory and orthogonal assays to evaluate a subset of 42 hit compounds prioritized by SSMD score , dose-response profile , and/or implicated MOA ( Supplementary file 2c , highlighted compounds ) .", "Having extensive MOA data is a key advantage of testing human-approved compounds which we leveraged in a previous large-scale zebrafish PDD screen ( Wang et al . , 2015b ) .", "Similarly here , as studies have suggested inflammation plays a key role in retinal degeneration and regeneration ( Hollyfield et al . , 2008; Mitchell et al . , 2018; White et al . , 2017; Yoshida et al . , 2013 ) , hits implicated as modulators of inflammatory signaling were included as part of the prioritization scheme .", "In addition , several compounds that did not produce concentration-dependent effects were selected to test whether this criterion was useful in predicting reproducibility .", "All compounds were obtained from new sources to ensure reagent authenticity .", "To confirm survival promoting effects , three repeats of the primary screening assay ( Figure 2A ) were conducted , but using a wider concentration range to account for differences in reagent quality ( from 100 μM to 1 . 28 nM , fivefold dilution series ) .", "If toxicity was observed at higher concentrations , dilution series were initiated at 10 µM or 1 µM .", "Using this strategy , 11 of the 42 prioritized hit compounds were confirmed as lead candidates ( 26% ) .", "Estimated effects on rod cell survival – that is , YFP signal levels relative to +Mtz/0 . 1% DMSO controls – ranged from 38 to 9% ( Figure 2B; lead drug candidate names , abbreviations , PubChem CID , and chemical structures are provided in Table 1 ) .", "We next asked whether there was a correlation between SSMD scores and/or concentration-dependent effects and confirmation rates .", "Among 19 selected hit compounds with higher SSMD scores ( ≥1 . 3 ) , seven ( 37% ) were confirmed; among 23 with lower SSMD scores ( 1–1 . 28 ) , four ( 17% ) were confirmed .", "Of 27 selected hit compounds with a concentration-dependent trend , eight ( 30% ) were confirmed .", "Conversely , of 15 compounds that did not show a concentration-dependent trend , 3 ( 20% ) were confirmed .", "Among the 11 confirmed leads , 8 ( 73% ) showed dose-dependent effects and 7 ( 64% ) had higher SSMD scores ( >1 . 28 ) .", "These results suggest that prioritizing hit compounds by both relative SSMD scores and dose-dependent trends provides predictive value for confirming activity , consistent with qHTS principles ( Inglese et al . , 2006 ) .", "As rod cell death is induced by NTR-mediated reduction of the prodrug Mtz in our model , it is possible that some lead compounds simply suppressed NTR enzymatic activity .", "To test this , NTR activity was evaluated in the presence of each lead compound by assaying the reduction kinetics of the prodrug CB1954 in vitro ( Prosser et al . , 2010 ) .", "To ensure any potential for NTR inhibition was accounted for , all compounds were tested at 300 μM ( except MIC and CHL which precipitated at higher concentrations and were tested at 50 µM ) that is , ~100-fold greater than neuroprotective concentrations .", "Compounds were deemed potential inhibitors if NTR activity was less than 75% of the NTR alone control .", "Seven compounds showed no evidence of NTR inhibition by this criterion , but four did: warfarin ( WAR ) , ciclopirox olamine ( CPO ) , calcimycin ( CAL ) , and sulindac ( SUL; Figure 2—figure supplement 1A , B ) .", "However , IC50 measures ranged from 150 µM ( for CPO ) to 350 µM ( for SUL , Figure 2—figure supplement 1B ) , approximately one to two orders of magnitude higher than observed neuroprotective concentrations ( i . e . 0 . 4–20 µM ) .", "When NTR reduction of Mtz was tested for WAR , CPO , and CAL at 300 μM , similarly weak inhibitory effects were observed ( Figure 2—figure supplement 1B ) .", "The differences in concentrations between neuroprotective and NTR inhibitory activities diminish the likelihood that these leads act directly on NTR .", "To test this further , lead compounds were assayed for neuroprotective effects in NTR-independent mouse RP models ( see below ) .", "To control for the possibility that lead candidates promoted rod photoreceptor development , rather than neuroprotection , YFP levels were quantified in rho:YFP-NTR larvae exposed solely to lead drugs from 5 to 7 dpf ( Figure 2—figure supplement 2A ) .", "Retinoic acid ( RA , 1 . 25 μM ) was used as a positive control as it promotes rod fates during development in zebrafish ( Hyatt et al . , 1996 ) .", "RA-treated fish displayed statistically significant increases in YFP levels compared to untreated controls ( Figure 2—figure supplement 2B ) .", "In contrast , none of the retinas treated with lead compounds exhibited increased YFP expression , suggesting they do not promote rod photoreceptor cell fate .", "In fact , three lead compounds cloxyquin ( CLO ) , cortexolone ( COR ) and CPO reduced YFP signals relative to the untreated control ( Figure 2—figure supplement 2B , asterisks ) , suggesting negative effects on rod cell development .", "It is well known that the zebrafish retina regenerates ( Gorsuch and Hyde , 2014; Lenkowski and Raymond , 2014; Wan and Goldman , 2016 ) .", "Therefore , to determine whether lead compounds acted by stimulating regeneration , we used a previously described ARQiv assay designed to detect changes in rod cell replacement kinetics ( Walker et al . , 2012; White et al . , 2017 ) .", "Briefly , rho:YFP-NTR larvae were first treated with 10 mM Mtz at 5 dpf for 24 hr to induce rod cell loss .", "At 6 dpf , Mtz was washed out and larvae were treated with lead compounds at concentrations corresponding to maximal neuroprotective effects and YFP levels quantified at 9 dpf ( Figure 2—figure supplement 3A ) .", "Dexamethasone , which accelerates rod cell regeneration kinetics ( White et al . , 2017 ) , was used as a positive control .", "The results showed that none of the compounds increased rod cell regeneration rates ( Figure 2—figure supplement 3B ) , while four compounds , dihydroartemisinin ( DHA ) , CLO , CPO and MIC inhibited regeneration .", "These data suggest that lead compounds do not increase YFP levels by promoting rod cell regeneration .", "To test if lead compounds simply increased YFP signal intensity , rather than promoted rod cell survival , intravital microscopy was used to image Mtz-treated retinas ±lead compounds and non-ablated ( -Mtz ) controls .", "Mtz-treated retinas had reduced numbers of YFP-NTR-expressing rods ( Figure 3 , +Mtz ) .", "Conversely , rods in control retinas displayed robust YFP signal throughout the retina and elongated morphologies suggestive of healthy outer segments ( Figure 3 , -Mtz ) .", "In retinas exposed to Mtz and lead compounds , YFP signal loss was attenuated and rods typically displayed elongated morphologies ( Figure 3 ) .", "However , for some compounds , cells appeared rounded , suggesting degeneration was not fully inhibited ( e . g . miconazole , MIC; Figure 3 ) .", "To confirm lead compounds increased rod cell survival , confocal stacks of YFP-expressing rods were 3D-rendered and fluorescence volume quantified using Imaris software-based automated image analysis ( White et al . , 2017 ) .", "The data showed a statistically significant increase in YFP volumes for all drug-treated groups ( Figure 3—figure supplement 1 ) , confirming that lead compounds promoted increased rod cell numbers and/or preserved outer segment morphology .", "To further investigate if lead compounds preserved rod outer segments , we used a newly developed zebrafish transgenic line , Tg ( rho:GAP-YFP-2A-nfsB_Vv F70A/F108Y ) jh405 ( hereafter rho:YFP-NTR2 . 0 ) .", "In this line , rods co-express membrane-tagged YFP and an improved NTR ( ‘NTR 2 . 0’ ) .", "The membrane-tagged YFP facilitates improved imaging of rod outer segments , while NTR 2 . 0 enables cell ablation at a reduced concentration of Mtz ( e . g . 10 µM versus 2 . 5 mM; Sharrock et al . , 2020 ) .", "To assess if lead compounds preserved outer segment morphologies , intravital confocal imaging was performed as above but using the rho:YFP-NTR2 . 0 line and 10 µM Mtz treatments .", "Qualitative image analysis suggested the majority , six of seven tested lead compounds , preserved rod outer segments ( Figure 3—figure supplement 2A ) .", "Reasoning that preservation of outer segments would equate to increased rod cell size , relative to rounded morphologies of dead or stressed cells , average rod cell sizes were calculated as the total YFP volume divided by the number of rod cells per each 3D confocal stack ( i . e . average YFP volume per cell ) .", "Compared to rod ablated controls , five of seven lead compounds tested promoted statistically significant increases in rod cell volumes ( Figure 3—figure supplement 2B ) .", "These data suggest some lead compounds were able to maintain rod outer segment morphology , thus potentially maintaining rod cell function .", "To identify new therapeutics for RP patients , we reasoned compounds producing neuroprotective effects across fish and mammalian RP models would likely target conserved mechanisms , and thus be more likely to translate successfully in the clinic .", "We therefore tested the efficacy of lead compounds in a series of mouse models of retinal photoreceptor degeneration .", "First , we tested compounds for the capacity to protect mouse primary photoreceptor cells from stressor-induced cell death in culture .", "Photoreceptors were isolated from postnatal day four ( P4 ) QRX mice , a transgenic line in which GFP expression is restricted to photoreceptors ( Wang et al . , 2004 ) , and grown as previously described ( Fuller et al . , 2014 ) .", "To induce photoreceptor cell death , thapsigargin ( 0 . 25 µM ) or tunicamycin ( 0 . 6 µg/mL ) were added .", "These ‘stressor’ compounds deplete endoplasmic reticulum ( ER ) calcium levels ( Thastrup et al . , 1990 ) and inhibit protein glycosylation ( Fliesler et al . , 1984 ) , respectively ( Lai et al . , 2007b ) .", "In turn , they elicit an unfolded protein response ( UPR ) ( Wang et al . , 2015b ) and related ER stress ( Oslowski and Urano , 2011; Zhang et al . , 2014 ) , both implicated in the etiology of RP ( Griciuc et al . , 2011; Rana et al . , 2014 ) .", "To test for survival effects , all eleven lead compounds were screened at seven concentrations across a threefold dilution series ( from 30 µM to 40 nM ) .", "After 48 hr in stressor ± lead compounds , cells were imaged using an automated high-content screening system .", "Photoreceptor survival was assessed by automated quantification of GFP-expressing cells ( Figure 4A ) .", "Nine of 11 lead compounds , proved protective in the thapsigargin-induced cell death assay ( Figure 4B ) .", "MIC , CLO and SUL also protected photoreceptors from tunicamycin-induced cell death ( Figure 4—figure supplement 1 ) .", "Thus , nine of 11 lead compounds promoted the survival of mouse primary photoreceptor cells in at least one stressor-induced cell death assay , and three were neuroprotective in both assays .", "We next tested lead compounds for survival effects in rd1 mouse retinal explant cultures .", "The rd1 mouse model of RP exhibits early onset rod cell degeneration caused by a mutation in the Pde6b gene ( Danciger et al . , 1990; Pittler and Baehr , 1991; Sidman and Green , 1965 ) , which is an ortholog of the human RP-associated PDE6B ( Khramtsov et al . , 1993; McLaughlin et al . , 1993 ) .", "In rd1 mice , photoreceptor degeneration begins around P10 and progresses rapidly .", "By P21 , only a single row of photoreceptor cells remain in the outer nuclear layer ( ONL; Tansley , 1951 ) making it an excellent system for screening potential neuroprotectants ( Beeson et al . , 2016 ) .", "Here , retinal explants from P10 rd1 mice were isolated and cultured ex vivo ( Bandyopadhyay and Rohrer , 2010 ) .", "Eight lead compounds , all but CAL , SUL , and Zinc pyrithione ( ZPT ) , were tested for survival effects at three concentrations across a fivefold dilution series centered on the concentration most effective in fish RP models .", "After 11 days in culture , explants were fixed , stained and the number of photoreceptor rows counted ( Figure 5A ) .", "Neuroprotective effects were defined as a concentration-dependent increase in the number of photoreceptor rows remaining in the ONL relative to untreated controls ( p≤0 . 05 ) .", "An average of 1 . 2 ±0 . 19 rows of photoreceptors remained in the ONL of control explants .", "Three lead drugs , CPO , DHA , and ART , increased the number of surviving photoreceptor layers ( Figure 5B ) .", "Representative images of control ( DMSO and POS ) and lead-treated explants demonstrate photoreceptor layer protection ( Figure 5C ) .", "CPO , DHA , and ART thus promoted rod cell survival in the fish RP model and two mouse cell culture RP models .", "However , high concentration CPO treatments ( 15 µM ) led to disruption of retinal histology due to induction of proliferation in the inner nuclear layer ( INL ) and ONL .", "Therefore , as DHA is the active metabolite of ART-related compounds , we proceeded with testing DHA in an in vivo mouse RP model .", "To test the potential of DHA as a ‘pan-disease’ therapeutic , that is , whether it was effective across multiple genetic RP models , it was tested for photoreceptor survival effects in the rd10 mouse model of RP ( Pde6brd10 ) .", "The rd10 line was selected for in vivo experiments due to its slower rate of photoreceptor degeneration relative to other rd mutants , thus allowing a prolonged window for pharmacological intervention .", "DHA was selected based on its superior performance in rd1 retinal explant assays ( Figure 5 ) and because it is the active metabolite of a second lead drug candidate , ART , recently shown to protect photoreceptors from light damage ( Lu and Xie , 2019 ) .", "To create a long-release formulation , DHA was encapsulated in PLGA polymers ( Poly ( D , L-lactide-co-glycolide ) ) .", "Release kinetics assays in PBS/0 . 1% DMSO in vitro suggested DHA would reach maximal concentrations after 30 days , and remain stable for at least 20 days thereafter ( Figure 6A ) .", "We noted that release was minimal in the absence of DMSO , but were reluctant to include DMSO for injections due to the potential for deleterious effects on the retina ( Tsai et al . , 2009 ) .", "PLGA-DHA was injected into the vitreous of one eye of rd10 mice at P14 , with the contralateral eye serving as a vehicle injection control .", "At P32 , 18 days after injection , and when DHA levels were predicted to reach 80% of maximal concentration based on in vitro releases kinetics , rd10 mouse eyes were processed for immunohistochemistry ( Figure 6B ) and ONL thickness quantified ( Figure 6C ) .", "Despite initial promising results in pilot assays , no reproducible neuroprotective effects were observed .", "It is possible that in the absence of DMSO release kinetics may have been limiting in vivo .", "We plan to address this possibility in future studies .", "Previously , we used ‘on label’ information to explore MOA of hit compounds identified during an in vivo phenotypic screen of the JHDL ( Wang et al . , 2015b ) .", "Recently , we have become interested in additional advantages afforded by HTS-based MOA data and whole-organism phenotypic screening , such as polypharmacology ( Dar et al . , 2012; Rennekamp and Peterson , 2015; Rihel et al . , 2010 ) .", "Accordingly , we applied a target-agnostic MOA analysis process by evaluating lead compound performance in HTS and ultra-HTS studies archived on PubChem ( https://pubchem . ncbi . nlm . nih . gov/ ) .", "Many lead compounds exhibited shared target/pathway activities , suggesting in-common MOA ( Table 2 ) .", "Among shared targets , Tyrosyl-DNA Phosphodiesterase 1 ( TDP1 ) , a DNA repair enzyme , was the most popular ( eight leads ) .", "To test whether TDP1 inhibition promoted rod photoreceptor survival , we tested three TDP1 inhibitors in rod:YFP-NTR fish: paromomycin ( PM ) , thiostrepton ( ThS ) , and methyl-3 , 4-dephostatin ( MD ) ( Huang et al . , 2011; Liao et al . , 2006 ) .", "Both PM and ThS showed neuroprotective effects ( Table 3 , Figure 7 ) .", "This result was surprising given that NTR reduction of Mtz is thought to cause DNA damage-induced cell death ( Curado et al . , 2007 ) .", "Thus , inhibition of a DNA repair enzyme would be expected to enhance , not inhibit , NTR/Mtz-mediated cell death .", "However , an alternative means of disrupting TDP1 activity is by inhibiting Poly ( ADP-ribose ) Polymerases ( PARPs ) .", "Indeed , in a comprehensive 400 , 000 compound qHTS assay designed to identify TDP1 inhibitors , all five hits turned out to be PARP inhibitors ( Murai et al . , 2014 ) .", "PARPs also mediate DNA repair but , interestingly , hyperactivation of PARP1 leads to a specific form of DNA damage-induced cell death , termed parthanatos ( Fatokun et al . , 2014; Wang et al . , 2016 ) .", "PARP is also involved in a cGMP-dependent cell death pathway implicated in RP ( Power et al . , 2020 ) .", "We therefore assayed PARP inhibitors for the capacity to promote rod cell survival in rod:YFP-NTR fish .", "All eight PARP inhibitors tested had neuroprotective activity , ranging from 9% to 20% ( Figure 7 ) .", "To account for other cell death mechanisms implicated in neurodegeneration , we tested an inhibitor of necroptosis ( necrostatin-1; NEC ) , and three inhibitors of apoptosis ( Ac-DEVD-CHO , CASI , and CASVII , Table 3 ) .", "Surprisingly , NEC promoted rod cell survival , while the apoptosis inhibitors did not ( Figure 7 , Table 3 ) .", "Interestingly , paired testing of PARP and necroptosis inhibitors resulted in additive effects ( Figure 7—figure supplement 1 , Supplementary file 1b ) , suggesting either differential responses to NTR/Mtz-induced DNA damage among rod cells , or that inhibiting both pathways keeps cells from activating the other mechanism of cell death when only one pathway is blocked .", "To further dissect cell death pathways mediating NTR/Mtz-induced rod cell ablation , we used a ‘redundant’ CRISPR/Cas9 gene targeting approach ( i . e . four guide RNA targeting ) , which enables phenotyping in injected zebrafish embryos/larvae ( Wu et al . , 2018 ) .", "Key factors for apoptosis ( casp3a and casp3b ) , PARP-dependent cell death pathways parthanatos and/or cGMP-dependent cell death ( parp1 ) , and necroptosis ( ripk1l ) were targeted .", "In addition , tdp1 was targeted as a DNA repair control .", "For each targeted gene , four gRNAs and Cas9 protein were co-injected into one-cell stage embryos .", "Injected and uninjected ( control ) larvae were treated ±2 . 5 mM Mtz at five dpf and rod-YFP levels quantified at six dpf by ARQiv .", "For Mtz-ablated larvae , knockdown of parp1 markedly increased rod cell survival ( 39% compared to 20% in the uninjected control , p=4E-12 ) , and ripk1l knockdown modestly protected rods ( 26% compared to 22% in the uninjected control , p=0 . 05 ) .", "However , no improvement in survival was observed for either casp3a/b or tdp1 knockdown ( Figure 8A ) .", "In no Mtz controls , knockdown of parp1 , ripk1l or tdp1 had no effect on rod cell numbers .", "Conversely , casp3a/3b double knockdown increased rod cell numbers ( Figure 8B ) suggesting inhibition of developmental apoptosis and confirming efficacy of casp3a/3b knockdown .", "Confocal imaging confirmed parp1 knockdown effects on rod cell survival and casp3a/3b knockdown effects on rod cell development ( Figure 8—figure supplement 1A ) .", "Reduced gene expression was verified by qPCR ( Figure 8—figure supplement 1B , Supplementary file 3 ) .", "These results are consistent with the NTR/Mtz system eliciting DNA damage-associated cell death mediated by parp1 , that is , parthanatos , in rod cells .", "To further test for activation of PARP signaling during NTR/Mtz-induced rod cell death , accumulation of polymerized poly ( ADP-ribose ) ( PAR ) , a downstream effector of PARP1 activation ( Kam et al . , 2018 ) , was analyzed by western blot .", "five dpf rho:YFP-NTR larvae were treated ±2 . 5 mM Mtz and protein samples collected from ~30 fish at 3 , 6 , 12 , 24 , and 48 hr post-Mtz treatment initiation .", "Western blots showed clear accumulation of PAR in Mtz-treated fish at 24 and 48 hr post-Mtz ( Figure 8—figure supplement 1C ) which was verified quantitatively ( Figure 8—figure supplement 1D ) .", "Collectively , results of cell death pathway analyses are concordant across chemical inhibition , genetic knockdown , and biochemical assays , strongly suggesting NTR/Mtz-induced cell ablation is mediated by PARP-dependent cell death ( e . g . parthanatos , cGMP-dependent cell death ) , a target/pathway implicated across multiple mammalian models of RP ( Arango-Gonzalez et al . , 2014 ) .", "To determine if lead compound effects were mediated by inhibition of PARP signaling , parp1 expression was knocked down prior to testing for effects on rod cell survival in rho:YFP-NTR larvae .", "Four leads predicted to be PARP inhibitors ( MIC , CLO , CPO , DHA ) , one predicted to be a non-PARP inhibitor ( WAR ) , and a known PARP inhibitor control ( BMN ) were tested .", "Two lead compounds , MIC and DHA and BMN ( the PARP inhibitor control ) produced no statistically significant enhanced neuroprotective effects over parp1 knockdown .", "Conversely , WAR , CLO and CPO increased rod cell survival in parp1 knockdown fish ( Figure 9A ) .", "qPCR confirmed mRNA knockdown ( Figure 9B ) and controls showed no effects of parp1 knockdown on rod cell development ( Figure 9C , -Mtz ) and enhanced rod cell survival upon Mtz treatment ( Figure 9C , +Mtz ) .", "These data confirm that PARP inhibition mediates the neuroprotective effects of some lead compounds ( e . g . MIC and DHA ) while others are PARP-independent .", "The latter result suggests lead compounds may act via complementary neuroprotective mechanisms .", "Combined , these findings are consistent with rod cell loss in our fish RP model occurring , at least partially , by PARP-dependent cell death .", "PubChem data also suggested potential complementary MOA , that is , multiple independent targets across lead compounds ( Table 2 ) .", "We therefore hypothesized that combining lead compounds may produce enhanced , additive , or even synergistic effects .", "To investigate this , combinatorial lead drug treatments were tested in primary mouse photoreceptor cell cultures .", "As above , photoreceptors were isolated from QRX mouse retinas and used in stressor-treated primary cell culture assays .", "All 11 lead compounds were tested at a selected concentration individually and in pairs and the survival of GFP-labeled photoreceptors analyzed by high-content imaging and automated cell counting .", "The results showed no wholly additive effects , that is , survival percentages equaling or excelling the summed effect of individual compound assays .", "However , enhanced effects – i . e . , better median survival than for either compound alone – were observed for 30 of 55 pairs in thapsigargin-treated cultures and for 20 of 55 pairs in tunicamycin-treated cultures ( Figure 4—figure supplement 2 ) .", "Finally , seven lead compounds were tested alone and in pairs in rho:YFP-NTR zebrafish using optimal effective concentrations .", "Pairs producing survival effects equal to or greater than the sum of their individual values ( ±10% ) were considered additive .", "By this criterion , 10 of 19 viable pairs ( two pairs proved lethal ) exhibited additive effects ( Figure 10 , bolded; Supplementary file 1c ) and three pairs produced supra-additive effects ( i . e . ≥25% greater than summed effects ) suggesting possible synergy ( Figure 10 , asterisks ) .", "The maximal average rod cell survival effect was 58% ( WAR + CPO ) .", "Several compounds showed broadly additive effects , for example , WAR was additive with all six drugs and COR with four of five pairs ( one pair proving lethal ) .", "Additive effects suggest multiple signaling pathways are involved in NTR/Mtz-induced photoreceptor degeneration , thus that combinatorial drug regimens may be required to achieve maximal therapeutic benefits for RP patients ." ], [ "Identifying effective neuroprotective therapies for RP and other IRDs stands as a critical unmet need for the field ( Duncan et al . , 2018; Wubben et al . , 2019 ) .", "Although , neurotrophic factors , anti-apoptotic agents , nutritional supplements , and antioxidants have shown neuroprotective effects in animal models of RP ( Dias et al . , 2018 ) .", "Unfortunately , these reagents have produced , at best , only limited benefits for patients to date and , for some , mild improvements are offset by adverse side effects associated with long-term use ( Dias et al . , 2018 ) .", "For example , ciliary neurotrophic factor ( CNTF ) was shown to be effective in protecting photoreceptors in mouse ( Cayouette et al . , 1998 ) , dog ( Tao et al . , 2002 ) , and chicken ( Fuhrmann et al . , 2003 ) models of retinal degeneration .", "However , CNTF failed to improve either visual acuity or field sensitivity in short- and long-term RP clinical trials ( Birch et al . , 2016; Ciliary Neurotrophic Factor Retinitis Pigmentosa Study Groups et al . , 2013; Argus II Study Group et al . , 2015 ) .", "Clinical trials of Vitamin A in combination with Vitamin E ( Berson et al . , 1993 ) , docosahexaenoic acid ( Berson et al . , 2004 ) , lutein ( Berson et al . , 2010 ) , or valproic acid ( Birch et al . , 2018 ) were reported to produce some benefits for RP patients , but only in subpopulations , and some of these studies have been controversial ( Massof and Finkelstein , 1993 ) .", "Our strategy for addressing this challenge was to: ( 1 ) scale up the number of compounds tested directly in complex living disease models and ( 2 ) perform a cross-species screening cascade that starts with small animal models amenable to HTS and proceeds to mammalian models .", "We hypothesize that compounds producing beneficial outcomes across evolutionarily diverse species will target conserved MOA and thus stand a higher chance of successful translation .", "As a generalized strategy , large-scale drug discovery screens using small animal models are showing increasing promise across multiple disease paradigms ( Cagan et al . , 2019; Cully , 2019; Kitcher et al . , 2019; MacRae and Peterson , 2015 ) .", "Here , using a large-scale in vivo drug screening platform ( Figure 1 ) , we tested 2934 largely human-approved compounds for neuroprotective effects across six concentrations in >350 , 000 larval zebrafish models of RP .", "The primary screen implicated 113 compounds as neuroprotectants ( Supplementary file 2c ) .", "Confirmatory repeats and a series of four orthogonal assays validated 11 of 42 prioritized hit compounds in protecting zebrafish rod photoreceptors from cell death ( Figures 2 and 3 , Figure 2—figure supplements 1–3 , Figure 3—figure supplement 1 and Table 1 ) .", "Importantly , investigations of lead compound MOA , led to the discovery that NTR/Mtz-mediated rod cell death appears to proceed through alternative cell death pathways ( Figure 8 ) recently linked to photoreceptor degeneration in mammalian models of RP ( Arango-Gonzalez et al . , 2014; Paquet-Durand et al . , 2007; Power et al . , 2020; Sancho-Pelluz et al . , 2008; Tolone et al . , 2019 ) .", "A summary schematic of the entire screening cascade is provided in Figure 11 .", "Confirmatory tests showed survival effects of lead compounds varied from 9 to 38% ( Figure 2 ) , which might suggest limited therapeutic potential .", "However , even small numbers of surviving rod cells can have a significant impact on cone photoreceptor survival ( Guadagni et al . , 2015; Hartong et al . , 2006; Punzo et al . , 2012 ) .", "This effect is mediated by the rod-derived cone viability factor ( RdCVF ) ( Léveillard et al . , 2004 ) which stimulates glucose metabolism to promote cone survival ( Aït-Ali et al . , 2015 ) .", "Therefore , as cone cells provide high-acuity daytime vision , protecting small numbers of rod cell has significant therapeutic potential for RP patients .", "To test this further , lead compounds will need to be tested for the ability to prolong cone survival and function in rod-cone degeneration models .", "Visualization by in vivo confocal imaging showed variations in surviving cell morphologies across lead compounds ( Figure 3 and Figure 3—figure supplement 2 ) , suggesting .", "variations in the extent of rod cell protection that could impact function .", "In particular , maintenance of the outer segment , a specialized primary cilium in which phototransduction occurs , is necessary for photoreceptor function .", "Volumetric quantification of surviving rod cells further suggested lead compounds differ in their capacity to maintain outer segments .", "Visual function tests will need to be performed to test this possibility and thus to prioritize lead compounds for in vivo testing in mammalian RP models .", "To test conservation of neuroprotective effects across species , lead compounds were evaluated in three mouse IRD/RP models .", "Nine of 11 leads were confirmed as neuroprotectants in primary photoreceptor cell cultures ( Figure 4 ) .", "Three of eight leads assayed using rd1 retinal explant cultures were also validated ( Figure 5 ) ; two being active in both paradigms , DHA and ART .", "We chose the rd10 RP model for in vivo testing because it undergoes a slower rate of rod cell loss than rd1; spanning from approximately P16 to P35 ( Chang et al . , 2007; Gargini et al . , 2007 ) .", "DHA was the most promising compound for these tests as it had shown strong effects across assays and was amenable to a long-term release formulation designed to sustain drug action over weeks to months ( PLGA-DHA , Figure 6A ) .", "In addition , DHA is the active metabolite of artemisinin , another of our cross-species confirmed leads that was shown to have neuroprotective activity in rat models of stress-induced neuronal damage and light-induced photoreceptor degeneration ( Yan et al . , 2017 ) .", "Unfortunately , we did not observe increased photoreceptor survival in rd10 retinas injected with PLGA encapsulated DHA .", "A potential issue that needs to be resolved is the release kinetics of encapsulated DHA in vivo .", "DHA has poor water solubility , therefore in vitro release kinetics were obtained in the presence of 0 . 1% DMSO .", "However , because intravitreal injection of DMSO can have deleterious effects ( Tsai et al . , 2009 ) we avoided this potential complication .", "To assess DHA release in vivo , pharmacokinetic assays such as high-performance liquid chromatography ( Ayalasomayajula and Kompella , 2004; Shen et al . , 2014 ) will be used to quantify lead compound concentrations in the retina for future tests .", "Another possible explanation is that rd1 and rd10 models have differential responses to DHA .", "This has been reported for the histone-deacetylase inhibitor valproic acid , which shows opposing effects in rd1 ( neuroprotective ) and rd10 ( deleterious ) mice ( Mitton et al . , 2014 ) and across four different frog models of RP ( Vent-Schmidt et al . , 2017 ) .", "In addition valproic acid has produced inconsistent results in clinical trials with RP patients ( Chen et al . , 2019; Todd and Zelinka , 2017; Totan et al . , 2017 ) .", "These results emphasize the need for a more thorough understanding of IRD and RP disease mechanisms and downstream cell pathways to support the development of both personalized and pan-disease therapeutics .", "Numerous IRD/RP-linked mutations have been identified ( Dias et al . , 2018; https://sph . uth . edu/retnet/ ) implicating an array of disease mechanisms ( Dharmat et al . , 2020 ) .", "However , cell death pathways common across different IRD/RP patient subpopulations may provide pan-disease targets for neuroprotective therapies .", "Apoptosis has long been thought to be the mechanism by which rod photoreceptors die in IRD/RP ( Chang et al . , 1993; Doonan et al . , 2003; Portera-Cailliau et al . , 1994; Zeiss et al . , 2004 ) .", "However , these reports relied on terminal deoxynucleotidyl transferase dUTP nick end labeling ( TUNEL ) , which does not distinguish apoptosis from other types of cell death ( Ansari et al . , 1993; Charriaut-Marlangue and Ben-Ari , 1995; Grasl-Kraupp et al . , 1995; Dmitrieva and Burg , 2007; Kanoh et al . , 1999; Nishiyama et al . , 1996 ) .", "More recent evaluations of multiple apoptosis-related markers ( e . g . BAX , cytochrome c , caspase-9 , cleaved caspase-3 ) suggest apoptosis occurs in only a minority of RP models ( Arango-Gonzalez et al . , 2014; Sancho-Pelluz et al . , 2008 ) .", "Moreover , inhibition of apoptosis does not block cell death in many mouse photoreceptor degeneration models ( Hamann et al . , 2009; Yoshizawa et al . , 2002 ) , suggesting other pathways may mediate rod and/or cone cell death in retinal degenerative disease .", "Recently , non-apoptotic cell death mechanisms have been implicated in IRD/RP .", "In a comprehensive biochemical analysis of 10 mammalian RP models—involving mutations in cnga3 , cngb1 , pde6a , pde6b , pde6c , prph2 , rho , and rpe65—non-apoptotic cell death signatures were found to be common across all models tested ( Arango-Gonzalez et al . , 2014 ) .", "Conversely , definitive apoptotic markers were found only for the S334ter ( rho ) rat model .", "Shared features included activation of poly ( ADP-ribose ) polymerase ( PARP ) , histone deacetylase ( HDAC ) , and calpain , as well as accumulation of cyclic guanosine monophosphate ( cGMP ) and poly ( ADP-ribose ) ( PAR ) .", "For PARP , chemical inhibitors and a knock out line provided further confirmation ( Jiao et al . , 2016; Paquet-Durand et al . , 2007; Sahaboglu et al . , 2017; Sahaboglu et al . , 2016; Sahaboglu et al . , 2010 ) .", "Prior reports had suggested the NTR/Mtz system elicits caspase-3 activation and apoptotic cell death ( Chen et al . , 2011 ) .", "Initially , we had used this as a rationale for pursuing a neuroprotective screen with the rho:YFP-NTR line- however , the recent reports outlined above suggested apoptosis may have limited relevance to IRD/RP .", "Interestingly , when we tested cell death processes implicated in photoreceptor degeneration directly , an inhibitor of apoptosis ( BEL ) did not promote rod cell survival , whereas inhibition of necroptosis ( NEC ) and PARP were neuroprotective in our fish RP model ( Figure 7 , Table 3 ) .", "Serendipitously , an exploration of shared lead compound MOA helped to clarify cell death mechanism ( s ) mediating NTR/Mtz-induced rod cell ablation .", "To explore molecular MOA of our lead compounds , we searched bioactivity data from prior HTS and ultra HTS assays ( PubChem ) .", "The results suggested both shared and independent MOA .", "The most common shared target was TDP1 ( eight of 11 lead compounds; Table 2 ) .", "An initial test confirmed two of three TDP1 inhibitors tested , though neuroprotective activity was relatively weak ( ~10% survival; Figure 7 ) .", "TDP1 is a DNA repair enzyme that repairs topoisomerase I-induced DNA damage ( Dexheimer et al . , 2008; El-Khamisy , 2011 ) .", "Interestingly , a qHTS cell-based screen of 400 , 000 compounds for inhibitors of human TDP1 found that all five confirmed compounds actually inhibited PARP activity not TDP1 ( Murai et al . , 2014 ) .", "This is consistent with findings showing that TDP1 acts in conjunction with PARP1 ( Das et al . , 2014; Lebedeva et al . , 2015 ) , thus PARP inhibition can indirectly affect TDP1 activity .", "Combined with the results discussed above , we were motivated to test whether PARP inhibition was protective against NTR/Mtz-induced rod cell death .", "All eight PARP inhibitors tested promoted rod cell survival in our fish RP model ( Figure 7 and Table 3 ) .", "CRISPR/Cas9-based knockdown of parp1 resulted in a statistically significant reduction of Mtz-induced rod cell death ( Figure 8A ) .", "Moreover , PAR polymer , an indicator of PARP activation , accumulated in Mtz-treated fish ( Figure 8—figure supplement 1C–D ) .", "Collectively , these results suggest PARP activation plays an important role in NTR/Mtz-induced rod cell death .", "PARP1 overactivation initiates a caspase-independent form of DNA damage-induced cell death , termed parthanatos ( Fan et al . , 2017 ) .", "Parthanatos has been strongly implicated in the etiology of Parkinson’s disease ( Kam et al . , 2018 ) and in a variety of neurodegenerative conditions as well ( Fan et al . , 2017; Fatokun et al . , 2014 ) , including RP/IRDs ( Power et al . , 2020 ) .", "Importantly , PARP is also a key component of the cGMP-dependent cell death pathway which has been linked to photoreceptor degeneration ( Iribarne and Masai , 2017; Tolone et al . , 2019; Power et al . , 2020 ) .", "Thus , the NTR/Mtz-mediated cell ablation system may provide a novel approach for modeling neurodegenerative diseases associated with PARP-dependent cell death that is both inducible and titratable .", "To investigate cell death more broadly , we also explored the roles of necroptosis , apoptosis and tdp1 signaling in the zebrafish-inducible RP model .", "Interestingly , inhibiting necroptosis but not apoptosis promoted rod cell survival in rho:YFP-NTR fish ( Figure 7 and Table 3 ) .", "Consistent with this , CRISPR/Cas9-based knockdown of ripk1l protected rods , but casp3a/3b knockdown did not ( Figure 8 ) .", "Necroptosis is primarily associated with secondary cone cell death in RP models ( Murakami et al . , 2015; Murakami et al . , 2012; Yang et al . , 2017 ) but has also been implicated in rod cell death in IRBP mutant RP models ( Sato et al . , 2013 ) and/or may damage photoreceptors indirectly via necroptotic microglia signaling ( Huang et al . , 2018 ) .", "Combined , these results suggest that necroptosis , PARP1-dependent parthanatos , and/or c-GMP-dependent cell death mediate NTR/Mtz-induced rod cell ablation .", "The potential relevance of these alternative cell death pathways to heritable photoreceptor degeneration may explain the relatively high rate of validation we observed in cross-species tests of lead compounds .", "To evaluate whether lead compound effects were mediated by inhibition of PARP signaling , a subset of lead compounds and a PARP inhibitor control ( BMN ) were analyzed for survival effects in parp1 knockdown fish .", "Among four leads tested that were implicated as parp1 inhibitors , two ( MIC and DHA ) were no longer protective when parp1 expression was suppressed ( Figure 9 ) , suggesting parp1 inhibition mediated their neuroprotective effects .", "PARP1 has also been shown to have a role in stem cell reprogramming ( Chiou et al . , 2013; Doege et al . , 2012; Weber et al . , 2013 ) .", "PARP inhibition might therefore block Müller glia dedifferentiation , diverting injury-induced activity from a regenerative to neuroprotective program ( Bringmann et al . , 2009 ) .", "Finally , MOA analyses also implicated additional , potentially complementary , lead targets ( Table 2 ) .", "Compounds acting through independent targets/pathways have the potential to produce additive or even synergistic effects .", "This possibility was confirmed for 10 of 19 paired lead compounds tested in rho:YFP-NTR fish ( Figure 9 , Supplementary file 1c ) .", "Analogous tests in mouse photoreceptor cell cultures showed a trend toward enhanced survival effects of paired leads but no fully additive effects ( Figure 4—figure supplement 2 ) .", "This discrepancy could be due to cell culture assays lacking in vivo complexity , such as signals requiring intact tissue or interactions between cell types or with other tissues .", "This result highlights a potential advantage of whole organism phenotypic drug screening: the ability to identify compounds/conditions that would be missed in more simplified assay systems .", "That combinatorial assays could be performed efficiently and rapidly also exemplifies key advantages the zebrafish system affords phenotypic drug discovery , for example , versatility and low cost .", "In summary , we identified 11 lead compounds promoting rod cell survival in an inducible zebrafish RP model using a large-scale in vivo phenotypic drug discovery platform .", "Nine lead compounds were also effective as neuroprotectants in primary mouse photoreceptor cell cultures and three promoted photoreceptor survival in rd1 mouse retinal explants .", "MOA studies indicated a subset of lead compounds may protect rod cells by inhibiting PARP-dependent cell death pathways and/or necroptosis .", "Combinatorial assays in fish showed additive and even synergistic effects , suggesting some lead compounds target independent neuroprotective pathways ( summarized in Figure 11 ) .", "We hypothesize that compounds producing beneficial outcomes across diverse animal disease models likely target highly conserved MOA and may therefore stand an increased likelihood of successfully translating to the clinic .", "Further , our data suggest polypharmacological targeting of complementary neuroprotective mechanisms has the potential to maximize therapeutic benefits for IRD/RP patients ." ], [ "All animal studies described herein were performed in accordance with both the Association for Research in Vision and Ophthalmology ( ARVO ) statement on the ‘Use of Animals in Ophthalmic and Vision Research’ and the National Institutes of Health ( NIH ) Office of Laboratory Animal Welfare ( OLAW ) policies regarding studies conducted in vertebrate species .", "Animal protocols were approved by the Animal Care and Use Committees of the Johns Hopkins University School of Medicine ( protocol # FI19M489 and #MO20M253 ) and Medical University of South Carolina ( protocol #2018–00399 ) .", "Zebrafish were maintained using established temperature and light cycle conditions ( 28 . 5°C , 14 hr of light/10 hr of dark ) .", "A previously generated zebrafish transgenic line , Tg ( rho:YFP-Eco . NfsB ) gmc500 ( hereafter , rho:YFP-NTR ) expresses a fusion protein of enhanced yellow fluorescent protein ( YFP ) and a bacterial nitroreductase ( NTR ) enzyme ( encoded by the E . coli nfsB gene ) selectively in rod photoreceptors ( Walker et al . , 2012 ) .", "When rho:YFP-NTR fish are exposed to the prodrug metronidazole ( Mtz ) , NTR reduces Mtz to a DNA damage-inducing cytotoxic derivative , resulting in the death of NTR-expressing cells ( Chen et al . , 2011; Curado et al . , 2007 ) .", "A newly generated transgenic line , Tg ( rho:GAP-YFP-2A-nfsB_Vv F70A/F108Y ) jh405 ( hereafter rho:YFP-NTR2 . 0 ) expresses a membrane tagged YFP and an improved version of NTR which enables induction of cell death at much lower concentrations of Mtz ( Sharrock et al . , 2020 ) .", "These lines were propagated in a pigmentation mutant with reduced iridophore numbers , roya9 ( roy ) , to facilitate YFP reporter signal detection in vivo .", "Non-transgenic roy fish were used to define reporter signal cutoff values for fluorescent microplate reader assays .", "For each large-scale drug screening assays , 6000–12 , 000 eggs were collected by group breeding ~300 adult rho:YFP-NTR fish ( White et al . , 2016 ) .", "rho:YFP-NTR larvae were treated with 2 . 5 mM Mtz or 0 . 1% DMSO at 5 dpf for 2 days ( 10 fish per condition ) , and collected at 7 dpf for immunostaining using previously reported methods ( Unal Eroglu et al . , 2018 ) .", "Briefly , larvae were fixed in 4% PFA at 4°C overnight , infiltrated with 30% sucrose , and embedded in OCT .", "Cryosections of 10 µm thickness were made for immunofluorescence staining .", "Each section was blocked with 1x PBS containing 0 . 5% Triton X-100% and 5% goat serum for one hour followed by primary antibody staining overnight .", "The next day , each section was washed and stained with the secondary antibody for 2 hr .", "All slides were mounted and underwent confocal imaging .", "The primary antibodies used in this study were zpr-1 ( anti-Arrestin3a; 1:200 dilution; from ZIRC ) , 1d1 ( anti-Rhodopsin; 1:50; gift from Dr . James M . Fadool ) and 4C12 ( uncharacterized rod cell antigen , 1:100 dilution; gift from Dr . James M . Fadool ) .", "The secondary antibody used was goat anti-mouse Alexa 647 ( 1:1000; Invitrogen ) .", "rho:YFP-NTR larvae were separated into six groups of ~23 larvae per group .", "Each group was treated with varying Mtz concentrations ( 10 , 5 , 2 . 5 , 1 . 25 , 0 . 625 , 0 mM ) , from 5 to 8 days post-fertilization ( dpf ) .", "YFP signals were quantified daily by fluorescence microplate reader ( TECAN Infinite M1000 PRO; excitation 514 nm , bandwidth 5 nm; emission 538 nm , bandwidth 10 nm ) to track changes in rod photoreceptor cell numbers relative to Mtz concentration ( Walker et al . , 2012; White et al . , 2016 ) .", "Two technical repeats were performed and results aggregated across experiments by normalizing to non-ablated controls .", "To calculate sample size ( N ) and evaluate assay quality , two 96-well plates of larvae were treated with 2 . 5 mM Mtz/0 . 1% DMSO ( ablated ‘+Mtz’ control ) or 0 . 1% DMSO only ( non-ablated ‘-Mtz’ control ) from 5 to 7 dpf ( 196 fish per condition ) .", "YFP signals were then quantified at seven dpf ( due to the large sample size , a single technical replicate was performed ) .", "Power calculations were used to determine sample sizes across a range of error rates and effect sizes ( White et al . , 2016 ) .", "This analysis suggested a sample size of nine per condition tested would sufficiently minimize false-discoveries ( i . e . type I and type II error rates of 0 . 05 and 0 . 05 , respectively ) and account for drug effects reaching 50% of signal window of the positive ( non-ablated ) control .", "However , to account for plating errors and other confounding variables , we chose to increase the sample size to 16 .", "A schematic of the primary screening process is presented in Figure 1C .", "For the primary screen , 2934 compounds from John Hopkins Drug Library ( JHDL ) were screened across six concentrations ( 4 μM-125 nM using a twofold dilution series ) .", "The JHDL consists of ~2200 drugs approved for use in humans ( e . g . FDA approved ) with the remainder approved for clinical trials ( Chong et al . , 2006 ) .", "The ARQiv screening process has been detailed previously ( White et al . , 2016 ) and was adapted here for large-scale quantification of YFP-expressing rod photoreceptors ( Walker et al . , 2012; White et al . , 2016 ) .", "rho:YFP-NTR embryos were collected and raised in zebrafish E3 embryo media ( 5 mM NaCl; 0 . 17 mM KCl; 0 . 33 mM CaCl; 0 . 33 mM MgSO4 ) .", "At 16 hr post fertilization ( hpf ) , N-phenylthiourea ( PTU ) was added to E3 media ( E3/PTU ) at a final concentration of 0 . 2 mM to promote ocular transparency by inhibiting melanosome maturation in the retinal pigment epithelium .", "At 4 dpf , visual screens were performed to remove larvae with abnormal morphology or low YFP expression levels .", "Stock drug and DMSO ( negative control ) solutions were automatically dispensed and diluted across a 96-well plate containing E3/PTU using a robotic liquid handling system ( Hudson Robotics ) .", "At 5 dpf , a COPAS-XL ( Complex Object Parametric Analyzer and Sorter , Union Biometrica ) was used to dispense single larvae into individual wells containing either drug or DMSO; the final DMSO concentration was 0 . 1% across all conditions .", "After a 4 hr pre-exposure to test drugs or DMSO alone , larvae were treated with 2 . 5 mM Mtz to induce rod photoreceptor death .", "Larvae were maintained under these conditions for 2 days until 7n dpf and then anesthetized with clove oil ( 50 ppm final concentration ) ; YFP signals were measured as described above .", "Larvae exposed to 2 . 5 mM Mtz/0 . 1% DMSO without any tested drug served as controls ( ‘+Mtz’ ) for maximal rod cell ablation .", "Larvae treated solely with 0 . 1% DMSO served as non-ablated controls ( ‘-Mtz’ ) to calculate maximal YFP signal levels .", "Non-transgenic larvae were used to establish a signal cutoff value , as previously described ( White et al . , 2016 ) .", "A customized R code program was applied for real-time data analysis of compound performance relative to controls , including dose-response curves and strictly standardized mean difference ( SSMD ) scores ( https://github . com/mummlab/ARQiv2; Ding and Zhang , 2021 ) .", "Compound concentrations producing a SSMD score ≥1 were considered potential ‘hits’ and evaluated visually using fluorescence microscopy to eliminate non-specific fluorescence; e . g . , dead larvae or autofluorescent compounds .", "After the initial screen , 42 top-performing ‘hit’ compounds were selected for confirmatory and orthogonal assays .", "Hit drugs were obtained from new sources and tested across a wider range of concentrations ( fivefold dilution series , eight total concentrations , n=30 fish/condition ) .", "Based on the toxicity profile of each drug , the starting concentration was either 100 , 10 , or 1 μM .", "For validation assays , YFP signals were measured by fluorescence microplate reader with at least three experimental repeats conducted and SSMD scores calculated .", "All experimental results were normalized and pooled to calculate effect sizes , confidence intervals , sample sizes ( N ) and p-values .", "p Values were calculated using Student’s t-test followed by Bonferroni correction for multiple comparisons resulting in an adjusted significance level of 0 . 005 ( α=0 . 005 ) .", "For in vivo confocal imaging assays , five dpf rho:YFP-NTR larvae were treated with drug plus 2 . 5 mM Mtz/0 . 1% DMSO ( ablated +Mtz controls ) , or 0 . 1% DMSO ( non-ablated -Mtz controls ) for 48 hr and processed for intravital imaging at 7 dpf .", "Similar treatment strategy was applied to five dpf rho:YFP-NTR2 . 0 larvae except the Mtz treatment was reduced to 10 μM .", "All compounds were tested at the maximal effective concentration in 0 . 1% DMSO .", "Larvae from each group were anesthetized in 0 . 016% tricaine and embedded on their sides in 1% low melt agarose gel .", "An Olympus Fluoview FV1000 confocal microscope with a 20x water immersion objective ( 0 . 95 NA ) was used to collect 30–40 z-stack images of YFP-expressing rod cells across the whole retina at 4 μm intervals .", "These image slices were stacked into a single maximal intensity projection image using Image J . To provide greater detail of rod photoreceptor morphology , a region in the dorsal-nasal quadrant was imaged using a 60× water immersion objective ( 1 . 10 NA ) at 4 . 18 μm intervals .", "Three contiguous image slices were then stacked into a single maximal intensity projection image using Image J . As a means of assessing rod photoreceptor survival , intravital confocal retinal images were collected of YFP-expressing rod cells per condition and processed for volumetric quantification of YFP signal volume using automated image analysis software ( Imaris 3 . 9 . 1; see White et al . , 2017 ) .", "Briefly , YFP-expressing rod cells volumes were automatically rendered using the same parameters across all treatment groups with the sum of all volumes used to estimate rod photoreceptor numbers per each retina .", "For images from rho:YFP-NTR2 . 0 larvae , the sum of rod cell YFP volumes in each retina was normalized to the number of YFP-expressing cells to estimate the average rod photoreceptor cell size per each condition .", "The average number of cells quantified per retina was 18 . 52 , with sample sizes ranging from 9 to 19 fish per condition .", "The eleven lead compounds were tested to eliminate false-positives that directly inhibited E . coli NTR enzymatic activity ( from the nfsB_Ec gene ) using an established anticancer prodrug ( CB1954 ) reduction assay ( Prosser et al . , 2010 ) .", "The highest soluble concentration of the test drug ( up to 300 μM maximum ) or 0 . 1% DMSO ( rate control ) was added to a master mix of 250 μM CB1954/1 μM NTR/250 μM NADPH/10 mM Tris ( pH 7 . 0 ) to determine any inhibitory effect of the compounds on NTR-mediated reduction of CB1954 .", "Reactions were conducted in 100 μl volume in a 96-well plate format .", "CB1954 reduction kinetics were assayed at 420 nm unless the test compound exhibited confounding absorbance at 420 nm , in which case NADPH depletion was monitored at 340 nm .", "The reaction rates for CB1954 reduction in the presence of tested compounds were compared to the DMSO control .", "Compounds with NTR activity <75% of controls were deemed potentially inhibitory and IC50 values ( the concentration required to restrict NTR activity to 50% of the DMSO control ) determined .", "Compounds were also evaluated for the ability to inhibit NTR-mediated reduction of Mtz .", "For this assay , the highest soluble concentration of the test drug ( up to 300 μM maximum ) or DMSO ( rate control ) was added to a master mix of 500 μM Mtz/17 μM NTR/100 μM NADPH/0 . 55 μM glucose dehydrogenase/5 mM glucose/50 mM sodium phosphate buffer ( pH 7 . 0; the glucose dehydrogenase used for cofactor regeneration , maintaining a steady-state of NADPH to allow Mtz reduction to be monitored directly at 340 nm without interference from NADPH oxidation ) .", "Reactions were conducted in 100 μl volume and assayed at 340 nm using 1 mm quartz cuvettes ( Rich et al . , 2018 ) .", "IC50 values for potentially inhibitory compounds were measured as with CB1954 .", "All kinetic assays were conducted in triplicate with a single purified batch of NfsB_Ec protein .", "To evaluate whether lead compounds promoted rod photoreceptor development , rho:YFP-NTR larvae were handled as described for the primary screen with the following exceptions: at five dpf , larvae were exposed solely to test compounds; larvae were not treated with Mtz .", "YFP reporter signals were quantified by fluorescence microplate reader at 7 dpf and compared to non-ablated ‘-Mtz’ controls treated only with 0 . 1% DMSO .", "Sample sizes ranged from 58 to 88 across at least two experimental repeats .", "To determine whether lead compounds stimulated retinal regeneration , rho:YFP-NTR larvae were handled as described for the primary screen with the following exceptions: at 5 dpf , larvae were incubated with either 10 mM Mtz or 0 . 1% DMSO for 24 hr .", "At 6 dpf , larvae were then placed in new 0 . 1% DMSO/E3/PTU media containing test compounds ( or DMSO alone ) for 3 days .", "YFP signal intensity was measured at 9 dpf .", "Sample sizes ranged from 31 to 83 across at least two experimental repeats .", "A transgenic mouse line with an IRES-GFP cassette integrated at the QRX locus ( QRX-IRES-EGFP ) has GFP reporter expression restricted to retinal photoreceptors , and was used here to isolate primary photoreceptor cells .", "Mice were housed with 12 hr light/12 hr dark cycles , at 22°C , 30–70% relative humidity and food/water ad libitum .", "Primary mouse retinal photoreceptor cells were isolated and prepared for culture as previously described ( Fuller et al . , 2014 ) .", "Briefly , murine retinas were isolated at postnatal day four ( P4 ) .", "Retinal tissue was dissociated into a single-cell suspension by incubating whole tissue in activated papain in Hibernate-E without Calcium ( BrainBits ) for 15 min at 37°C .", "Cells were resuspended in culture media ( Neurobasal , 2% B-27 , 0 . 5 mM L-Glutamine and 1x final Penicillin/streptomycin; all Life Technologies ) and seeded onto poly-D-lysine coated 384 well tissue culture plates .", "To test lead compounds for survival effects , thapsigargin and tunicamycin ( Sigma ) and were used as stressor compounds to induce photoreceptor cell death .", "Stressors and test compounds were added at the time of primary cell seeding .", "Eleven lead compounds were tested at seven concentrations across a threefold dilution series ( from 30 µM to 40 nM ) .", "For paired tests , each compound was applied at a single concentration , WAR ( 3 . 33 µM ) , CPO ( 0 . 04 µM ) , DHA ( 1 . 11 µM ) , ART ( 10 µM ) , ZPT ( 0 . 12 µM ) , CAL ( 0 . 04 µM ) , COR ( 3 . 33 µM ) , CHL ( 3 . 33 µM ) , SUL ( 3 . 33 µM ) , MIC ( 10 µM ) , and CLO ( 1 . 11 µM ) .", "After a 48 hr treatment , cells were stained with Hoescht and ethidium homodimer; nine field images were acquired via an automated imager ( Cellomics Vti; ThermoFisher ) using a 20x objective and images analyzed .", "Photoreceptor number and the percent of photoreceptor to retinal cell population per well was determined by automated quantification of the number of live Hoechst 33342-stained , GFP-expressing cells using a custom algorithm ( Neuronal Profiling software package; ThermoFisher ) .", "A minimum of two experimental repeats was performed for each test with individual assays consisting of six biological replicates per condition tested .", "Mice were generated from retinal degeneration 1 ( Pde6brd1 , hereafter rd1 ) breeding pairs ( gift from Dr . Debora Farber; UCLA ) and housed under a 12:12 light:dark cycle with access to food and water ad libitum .", "All chemicals used for organ cultures were tissue culture grade and purchased from Invitrogen unless otherwise noted .", "Cultures of retina with attached retinal pigment epithelium ( RPE ) were grown according to published protocols ( Ogilvie et al . , 1999; Pinzón-Duarte et al . , 2000; Rohrer and Ogilvie , 2003 ) with modifications ( Bandyopadhyay and Rohrer , 2010 ) .", "P10 pups were decapitated and heads were rinsed in 70% ethanol; whole eyes were dissected and placed in ice-cold Hanks balanced salt solution plus glucose ( 6 . 5 g/L ) .", "Eyes were first incubated in 1 mL of high-glucose Hanks balanced salt solution/0 . 5 mg/ml proteinase K ( 37°C , 7 min ) and then in Neurobasal medium ( Life Technologies ) plus 10% fetal calf serum to stop enzymatic activity .", "The retina with attached RPE was dissected free from the choroid and sclera after removing the anterior chamber , lens and vitreous .", "Relaxing cuts were made to flatten the tissue prior to transfer to the upper compartment of a Costar Transwell chamber ( RPE layer faced-down ) using a drop of Neurobasal medium .", "Neurobasal media with B-27 supplement ( Life Technologies ) was placed in the lower compartment .", "For each of the cohorts , three to fourindividual P10 retina/RPE explants were placed in culture .", "The cultures were kept in an incubator ( 5% CO2 , balanced air , 100% humidity , 37°C ) and the lower compartment media changed every 2 days ( neither antimitotics nor antibiotics were required ) .", "Test compounds were added to the culture media and refreshed every 2 days for 11 days .", "At completion , explants were fixed in 4% PFA , sectioned 14 µm thick , and stained with toluidine blue ( Bandyopadhyay and Rohrer , 2010 ) .", "For each culture , 10 counts of rows of photoreceptors were performed in the center of the explant; the average of this cell counts provides the mean for each retina , with the average of all retinas providing the mean ± SEM per culture condition .", "Cell counts were compared by repeated measure ANOVA to assess dose-response treatment effects followed by a Bonferroni posthoc analysis to determine if differences between control and lead compounds compared across the entire dose range were statistically significant .", "An ANOVA across groups but within a particular dose , using a Bonferroni/Dunn test to reduce type I errors , was used to determine if lead compounds promoted a statistically significantly increase in cell numbers compared to control media at low , middle , and/or high concentrations .", "PLGA-DHA microparticles were prepared using a single emulsion solvent evaporation method .", "Briefly , 200 mg PLGA ( Resomer RG 502 hr , Poly ( D ) , L-lactide-co-glycolide; Evonik Corporation ) was dissolved in 1 mL of dichloromethane ( DCM , Sigma-Aldrich ) , and mixing with 40 mg DHA ( TCI , Tokyo , Japan ) dissolved in 0 . 125 ml dimethyl sulfoxide ( DMSO , Sigma-Aldrich ) .", "The mixture was homogenized ( L4RT , Silverson Machines ) at 7000 RPM for 1 min .", "The homogenized mixture was then poured into a solution containing 1% polyvinyl alcohol ( 25 kDa , 88% hydrolys , Polysciences , Warrington , PA ) in water under continuous stirring .", "Particles were hardened by allowing solvent to evaporate while stirring at room temperature for 2 hr .", "Particles were collected via centrifugation ( International Equipment Co ) at 1000 x g for 5 min , and washed three times with HyPure cell culture grade water ( endotoxin-free , HyClone , Logan , UT ) and re-collected by centrifugation three times .", "The washed particles were then lyophilized and stored frozen until use .", "Microparticles were resuspended at the desired concentration prior to injection in a sodium hyaluronate solution ( Healon ) diluted fivefold with endotoxin-free HyPure water at the desired concentration prior to injection .", "Particle size distribution was determined using a Coulter Multisizer 4 ( Beckman Coulter , Inc , Miami , FL ) .", "Particles were resuspended in double distilled water and added dropwise to 100 ml of ISOTON II solution until the coincidence of the particles was between 8% and 10% .", "At least 100 , 000 particles were sized for each batch of particles to determine the mean particle size and size distribution .", "To determine the drug loading , microparticles were dissolved in DMSO and the total drug content was calculated by measuring the UV absorbance at 289 nm after react with NaOH in triplicate ( C . -S . Lai et al . , 2007a ) .", "The average microparticle size was 14 . 2 ± 1 . 9 µm and the DHA loading was 20 . 3% ± 0 . 7 ( w/w ) across three experimental replicates .", "Release kinetics were obtained by resuspending microparticles in 1 ml phosphate buffered saline ( PBS containing 0 . 1% DMSO , pH 7 . 4 ) and incubating at 37°C on a platform shaker ( 140 RPM ) .", "Supernatant was collected at predetermined intervals by centrifugation at 2000 x g for 5 min .", "Drug-containing supernatant was collected and particles were resuspended in 1 ml of fresh 1xPBS containing 0 . 1% DMSO .", "DHA concentration in the collected supernatant was assayed via absorbance at 289 nm in triplicate for each sample ( Figure 4—figure supplement 1 ) .", "B6 . CXB1-Pde6brd10/J animals ( Jackson Laboratories stock #004297 ) were maintained as homozygotes .", "On P14 , animals were anesthetized with ketamine/xylazine ( 115 mg/kg and 5 mg/kg , respectively ) , and placed on a heating pad to maintain body temperature throughout injection and recovery .", "Glass needles were pulled and a bore size of approximately 75 µm was made to allow for the movement of the microspheres into and out of the needle .", "A PLI-100 picospritzer ( Harvard Apparatus ) was used for intravitreal injections with settings of 350 ms injection time at 30 psi , which yielded the injection of approximately 500 nL .", "One eye was randomly selected for injection with vehicle and the other was used for microsphere injections .", "Following injection , GenTeal gel ( Novartis ) was applied to the eyes to prevent corneal drying .", "Animals were monitored until they were awake and moving normally .", "A total of 17 mice was used across three technical repeats .", "Eighteen days post-injection , animals were anesthetized with isoflurane and decapitated; the eyes were removed and placed in 4% PFA for 30 min .", "After removing the cornea and lens , the eye cups were placed back into fixative for 2 hr on ice .", "After thorough washing with 1× PBS , eyecups were soaked in 15% sucrose ( w/v ) in 1× PBS for 3 hr , then incubated in 30% sucrose ( w/v ) /1× PBS overnight .", "Samples were embedded in OCT compound ( TissueTek ) and frozen on dry ice .", "Cryosections of 12–14 µm thickness were cut on a Leica CM3050S cryostat and were mounted on Superfrost Plus slides ( Fisher Scientific ) .", "Slides were rehydrated with 1× PBS and then incubated in blocking buffer ( 10% Normal donkey serum/ 0 . 3% Triton X-100/1× PBS ) for 2 hr , and were then incubated for 30 hr at 4°C with primary antibodies diluted in blocking buffer .", "Sections were washed in 1× PBS and incubated for 2 hr at room temperature with secondary antibodies diluted in blocking buffer .", "Following additional washes , sections were incubated with DAPI and mounted with FluoroGel ( Electron Microscopy Sciences ) .", "Sections were examined using a Zeiss Axioplan two epifluorescence microscope fitted with an Axioscope camera .", "Following acquisition with a 40× air objective , images were analyzed in ImageJ using the measure tool to examine Outer Nuclear Layer ( ONL ) thickness .", "Four to 5 measurements were taken at 75 µm intervals for 200–500 µm both superior and inferior from the optic nerve head .", "The measured values from multiple sections were averaged for each eye .", "Data were analyzed and statistical tests were performed in GraphPad Prism 7 .", "An Olympus FV1000 confocal microscope was also used to image sections stained with multiple dyes .", "To evaluate tdp1 and parp1 as a potential shared target across multiple leads , as well as cell death pathways implicated in photoreceptor degeneration , eight PARP inhibitors , one necroptosis inhibitor , three apoptosis inhibitors , and three Tdp1 inhibitors were selected for testing using the confirmation screening protocol ( Figure 2A ) .", "Based on the toxicity profile of each inhibitor , upper concentrations were adjusted to 64 , 32 , or 8 µM and a twofold dilution series was used to test a total of 10 concentrations for rod cell survival effects in rho:YFP-NTR larvae .", "Sample sizes varied from 27 to 173 across two to six experimental replicates for each tested compound .", "YFP signal levels were measured by fluorescence microplate reader ( i . e . ARQiv assay ) .", "Results across conditions were normalized to non-ablated controls and pooled across experimental repeats to calculate effect sizes , confidence intervals , and p-values .", "P values were calculated by performing Student’s t-tests .", "Bonferroni correction for multiple comparisons resulted in an adjusted significance level of 0 . 003 ( α=0 . 003 ) .", "Note: assessments of TDP1 inhibitors followed the fivefold dilution series protocol used for secondary confirmation assays described above .", "For paired inhibitor tests , three concentrations of BMN ( parp inhibitor ) and three concentrations of NEC ( necroptosis inhibitor ) were combined .", "YFP quantification and statistical analysis of rod cell survival in Mtz-treated rho:YFP-NTR fish were performed as above .", "Five genes , parp1 , ripk1l , casp3a , casp3b , tdp1 , were selected for CRISPR/Cas9-based knockdown in rho:YFP-NTR zebrafish using an established ‘redundant targeting’ methodology ( Wu et al . , 2018 ) .", "Four sgRNAs were designed to target each gene .", "The oligonucleotide for generating each sgRNA was either those suggested by Wu et al . , 2018 , Table S2 or designed using CRISPRscan ( https://www . crisprscan . org , Supplementary file 3; Moreno-Mateos et al . , 2015 ) .", "Preparation of sgRNAs followed the published method ( Wu et al . , 2018 ) .", "Briefly , four sgRNAs of each gene were in vitro transcribed using the HiScribe T7 High Yield RNA Synthesis kit ( New England BioLabs ) and purified using the NEB Monarch RNA Cleanup kit .", "A mixture of four sgRNAs ( 1 ng in total ) and Cas9 protein ( 2 . 5 µM , IDT ) was injected into rho:YFP-NTR embryos at the one-cell stage for targeting parp1 , ripk1l and tdp1 .", "Four sgRNAs each for casp3a and casp3b were pooled to co-target both genes .", "Injected and uninjected embryos were treated ±Mtz ( 2 . 5 mM ) at 5 dpf .", "Rod cell survival was evaluated by fluorescence microplate reader-based quantification of YFP at 6 dpf .", "Student’s t-test was used to compare rod survival between injected and uninjected ( control ) fish .", "Each gene knockdown experiment was conducted in triplicate or quadruplicate .", "A subset of lead compounds ( and a PARP inhibitor control , BMN ) was also tested in parp1 knockdown fish to determine if Parp1 inhibition was required for survival effects .", "Leads were applied to larvae one hour before Mtz administration and YFP quantification performed as above .", "Each drug test was conducted in duplicate or triplicate .", "To calculate p-values relative to +Mtz controls , Student’s t-tests were performed .", "Bonferroni correction for multiple comparisons resulted in an adjusted significance level of 0 . 006 ( α=0 . 006 ) .", "To quantify gene expression levels in rho:YFP-NTR larvae ±CRISPR/Cas9-based gene knockdown , 16–24 larvae from each condition were pooled for mRNA extraction at six dpf .", "RNA was purified using NEB Monarch RNA Cleanup kit and reverse transcribed to cDNA using qScript cDNA synthesis kit ( QuantaBio ) according to the instruction from the manufacturer .", "Quantitative PCR was conducted using designed primers ( Supplementary file", "3 ) and PowerUp SYBR Green Master Mix ( ABI ) in QuantaStudio ( ABI ) .", "Delta delta CT analysis was performed to calculate relative fold change in gene expression levels between knockdown larvae and controls .", "Each experiment was performed in triplicate or quadruplicate .", "To further evaluate Parp-dependent signaling downstream of Mtz treatments in rho:YFP-NTR larvae , PAR accumulation , a vetted measure of Parp activity , was quantified by western blot following the published method ( Kam et al . , 2018 ) .", "Briefly , ~30 rho:YFP-NTR fish were treated ±2 . 5 mM Mtz at five dpf were collected at 3 , 6 , 12 , 24 , 48 hr post treatment ( hpt ) .", "Larvae were homogenized and lysed using RIPA buffer ( Sigma ) supplemented with protease inhibitors ( Roche ) .", "Protein concentrations were quantified using Pierce BCA kit ( ThermoScientific ) following the manufacturer’s instruction .", "Protein samples were separated on Tris-Glycine gel and then transferred onto nitrocellulose membranes .", "The membrane was blocked with 5% non-fat milk in TBST ( Tris-buffered saline with 0 . 1% Tween 20 ) at room temperature for 1 hr and then blotted with the primary antibody , anti-human-PAR ( 1:2000 , Dowson lab reagent ) , at 4°C overnight .", "After rinsing in TBST for three times , the secondary antibody , anti-Human IgG-HRP ( 1:10 , 000 , Abcam ) was applied for 1 hr at room temperature .", "Anti-beta-actin-HRP antibody ( 1:20 , 000 ) was used as a loading control .", "Immunolabeled bands were visualized by ECL substrate and quantified in Fiji .", "24 hr post-Mtz PAR accumulation assays were performed in triplicate .", "Mann-Whitney U test was performed and a p-value of ≤0 . 05 used to indicate statistical significance .", "To test for additive neuroprotective effects in zebrafish , seven lead compounds were tested at optimal concentrations either individually or in pairs using the primary screen protocol .", "All experimental results were normalized to controls and pooled per compound tested to calculate effect sizes , confidence intervals , and p-values using Student’s t-test .", "Bonferroni correction for multiple comparisons resulted in an adjusted significance level of 0 . 002 ( α=0 . 002 ) .", "Sample sizes for each paired condition varied from 86 to 160 across at least four experimental replicates .", "For the primary screening , under the R environment , a customized ARQiv data analysis package was used to calculate sample size ( n ) , quality control strictly standardized mean difference ( SSMD ) and SSMD scores as previously described ( White et al . , 2016 ) .", "The following results of each drug were derived: ( 1 ) a plot of signal to background ratio at all tested concentrations , ( 2 ) a plot and a table of SSMD scores , and ( 3 ) a signal intensity heat map of each drug plate ( 96-well plate view ) .", "To combine and analyze the data from different experiments , data normalization was conducted by ( Si -X-neg ) / ( X- Pos -X-neg ) .", "Si is the signal of ith reading , X-Pos is mean of positive controls , and X-Neg is mean of negative controls .", "To compare between experimental conditions and controls , Student’s t-tests were performed followed by Bonferroni correction for multiple comparisons ( α/n ) .", "Relative effects , 95% confidence intervals and p-values were calculated .", "For mouse photoreceptor cell culture assays and volumetric confocal image analyses , adjusted p-values were calculated by performing Mann-Whitney U tests followed by false discovery rate correction for multiple comparisons ." ] ]

[ "Retinitis pigmentosa ( RP ) and associated inherited retinal diseases ( IRDs ) are caused by rod photoreceptor degeneration , necessitating therapeutics promoting rod photoreceptor survival .", "To address this , we tested compounds for neuroprotective effects in multiple zebrafish and mouse RP models , reasoning drugs effective across species and/or independent of disease mutation may translate better clinically .", "We first performed a large-scale phenotypic drug screen for compounds promoting rod cell survival in a larval zebrafish model of inducible RP .", "We tested 2934 compounds , mostly human-approved drugs , across six concentrations , resulting in 113 compounds being identified as hits .", "Secondary tests of 42 high-priority hits confirmed eleven lead candidates .", "Leads were then evaluated in a series of mouse RP models in an effort to identify compounds effective across species and RP models , that is , potential pan-disease therapeutics .", "Nine of 11 leads exhibited neuroprotective effects in mouse primary photoreceptor cultures , and three promoted photoreceptor survival in mouse rd1 retinal explants .", "Both shared and complementary mechanisms of action were implicated across leads .", "Shared target tests implicated parp1-dependent cell death in our zebrafish RP model .", "Complementation tests revealed enhanced and additive/synergistic neuroprotective effects of paired drug combinations in mouse photoreceptor cultures and zebrafish , respectively .", "These results highlight the value of cross-species/multi-model phenotypic drug discovery and suggest combinatorial drug therapies may provide enhanced therapeutic benefits for RP patients ." ]

[ "Photoreceptors are the cells responsible for vision .", "They are part of the retina: the light-sensing tissue at the back of the eye .", "They come in two types: rods and cones .", "Rods specialise in night vision , while cones specialise in daytime colour vision .", "The death of these cells can cause a disease , called retinitis pigmentosa , that leads to vision loss .", "Symptoms often start in childhood with a gradual loss of night vision .", "Later on , loss of cone photoreceptors can lead to total blindness .", "Unfortunately , there are no treatments available that protect photoreceptor cells from dying .", "Research has identified drugs that can protect photoreceptors in animal models , but these drugs have failed in humans .", "The classic way to look for new treatments is to find drugs that target molecules implicated in a disease , and then test them to see if they are effective .", "Unfortunately , many drugs identified in this way fail in later stages of testing , either because they are ineffective , or because they have unacceptable side effects .", "One way to reverse this trend is to first test whether a drug is effective at curing a disease in animals , and later determining what it does at a molecular level .", "This could reveal whether drugs can protect photoreceptors before research to discover their molecular targets begins .", "Tests like this across different species could maximise the chances of finding a drug that works in humans , because if a drug works in several species , it is more likely to have shared target molecules across species .", "Applying this reasoning , Zhang et al . tested around 3 , 000 drug candidates for treating retinitis pigmentosa in a strain of zebrafish that undergoes photoreceptor degeneration similar to the human disease .", "Most of these drug candidates already have approval for use in humans , meaning that if they were found to be effective for treating retinitis pigmentosa , they could be fast-tracked for use in people .", "Zhang et al . found three compounds that helped photoreceptors survive both in zebrafish and in retinas grown in the laboratory derived from a mouse strain with degeneration similar to retinitis pigmentosa .", "Tests to find out how these three compounds worked at the molecular level revealed that they interfered with a protein that can trigger cell death .", "The tests also found other promising compounds , many of which offered increased protection when combined in pairs .", "Worldwide there are between 1 . 5 and 2 . 5 million people with retinitis pigmentosa .", "With this disease , loss of vision happens slowly , so identifying drugs that could slow or stop the process could help many people .", "These results suggest that placing animal testing earlier in the drug discovery process could complement traditional target-based methods .", "The compounds identified here , and the information about how they work , could expand potential treatment research .", "The next step in this research is to test whether the drugs identified by Zhang et al . protect mammals other than mice from the degeneration seen in retinitis pigmentosa ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "developmental biology", "genetics and genomics" ]

[ [ "Much research has been done to understand the cell biology underlying the events of directed cell migration; however , how the events are encoded in the genome and how gene regulatory networks ( GRNs ) control this process are works in progress .", "Cell migration is the directed movement of a single cell or a collective group of cells through the early embryo or the adult body .", "Many different cell types undergo chemotaxis-dependent directed migration during development , including primordial germ cells ( PGCs ) , Drosophila border cells , the zebrafish posterior lateral line , Drosophila tracheal cells , vertebrate neural crest cells , and vertebrate anterior mesoderm ( Reig et al . , 2014 ) .", "The ability for cells to undergo directed migration towards a target location requires the use of different signal transduction mechanisms to remodel their actin cytoskeleton in a directed fashion such that they extend filopodia , lamellipodia , and blebs to create a polarized leading edge .", "In the sea urchin , a near complete developmental GRN describes the specification of endomesoderm ( McClay , 2011; Peter and Davidson , 2011 ) .", "Studies of this specification network have made the sea urchin a viable model for extending the study of how GRNs can explain control of complex cell behaviors ( Saunders and McClay , 2014 ) .", "The migration of the sea urchin small micromeres serves as a powerful experimental model for connecting the genomic regulatory control of morphogenesis to an upstream GRN .", "Small micromere cells arise from an asymmetric cleavage of the micromeres at the embryonic fifth cleavage ( Figure 1A ) .", "These cells divide once within the vegetal plate to produce eight cells ( Pehrson and Cohen , 1986 ) .", "The eight small micromeres migrate along with the growing archenteron during gastrulation until they reach the animal pole ( Yajima and Wessel , 2012; Campanale et al . , 2014 ) .", "Post-migration , the small micromeres incorporate into the coelomic pouches , which are found on either side of the developing esophagus ( Hyman , 1955; Pehrson and Cohen , 1986; Luo et al . , 2012 ) . 10 . 7554/eLife . 08827 . 003Figure 1 . Small micromere movements during gastrulation .", "( A ) The small micromeres arise at the vegetal pole at the asymmetric fifth cleavage from the micromere lineage ( red ) .", "During gastrulation , they remain at the tip of the gut .", "They migrate through the top of the blastocoel and enter the posterior half of the coelomic pouches by prism stage .", "( B ) To study small micromere movements and migration at a higher resolution , membrane-GFP-labeled micromeres were transplanted to a H2b-RFP-labeled host .", "( C ) Small micromeres actively changed shape throughout gastrulation ( extend filopodia and lamellipodia ) until they reach the tip of the archenteron .", "Skeletogenic lineages are labeled as ‘Skel’ , and small micromeres are labeled as ‘SM’ .", "See also , Video 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 08827 . 003 The coelomic pouches , a mesodermal sub-type , appear at the tip of the forming archenteron .", "Their specification is initiated early in development by Delta/Notch signaling ( Sherwood and McClay , 1999; Sweet et al . , 2002 ) .", "During gastrulation , several other mesodermal cell types undergo epithelial-to-mesenchymal transitions ( EMTs ) into the blastocoel where they take on different roles in the embryo .", "The mesodermal cell sheet remaining at the tip of the archenteron at the end of gastrulation forms the two coelomic pouches on either side of the foregut ( Figure 1A ) .", "Only those small micromeres that reach the left coelomic pouch , which will become the future adult rudiment , will survive until adulthood .", "During metamorphosis of the indirect developing sea urchin , the embryonic small micromeres incorporate into the adult rudiment's left somatocoel , which will later give rise to the gonads of the adult animal and possibly other tissues ( Hyman , 1955 ) .", "Previous research has suggested that the small micromeres contribute to the adult PGCs; however , it has not been shown directly whether the small micromeres contribute to additional adult tissues or whether the only source of the adult PGCs are the small micromeres ( Pehrson and Cohen , 1986; Voronina et al . , 2008; Juliano et al . , 2010a; Juliano et al . , 2010b; Yajima and Wessel , 2010; Yajima and Wessel , 2012; Wessel et al . , 2014 ) .", "The possibility remains that the small micromeres go on to produce multiple cell types including , but not limited to PGCs ( Yajima and Wessel , 2015 ) .", "Recent publications tracked small micromeres as they moved from the vegetal pole to the coelomic pouches ( Yajima and Wessel , 2012; Campanale et al . , 2014 ) .", "In Yajima et al . , it was observed that during gut invagination , the small micromeres did not change position relative to the adjacent mesoderm cells of the advancing archenteron .", "It was concluded that once they reach the tip of the archenteron , the small micromeres must actively migrate to the left and right coelomic pouches ( Yajima and Wessel , 2012 ) .", "Seemingly contradictory evidence from Campanale et al . described an active migration throughout gastrulation and post-gastrulation as they make their way to the coelomic pouch ( Campanale et al . , 2014 ) .", "While both studies conclude that there is an active migration post-gastrulation , we clarified whether the small micromeres acquired their active movement before or after gastrulation .", "When removed from their endogenous location and placed ectopically , we find that the small micromeres are autonomous and active while migrating throughout gastrulation to make it ‘home’ to the coelomic pouch .", "Their active motility during gastrulation is essential for the ectopic small micromeres to undergo directed ‘homing’ migration to the coelomic pouch .", "In order to understand this homing ability at a systems level , we took advantage of the robust homing nature of the small micromeres to determine the GRN at work during their migration .", "Using a homing assay to quantitatively score the ability of the coelomic pouch cells to direct movement of the small micromeres , we constructed a GRN that governs effector genes directly responsible for the homing mechanism .", "We find that a specific subcircuit composed of Pax6 , Six3 , Six1/2 , Eya , and Dach1 that is canonically found in retinal development has been deployed for this distinct developmental process .", "Here , we describe a GRN for homing behavior and uncover a striking similarity of our morphogenesis GRN to the retinal determination gene network ( RDGN ) ." ], [ "The mechanisms by which the small micromeres make their way to the coelomic pouch is a topic of debate: one paper cites an active migratory event while another a passive translocation during gastrulation ( Yajima and Wessel , 2012; Campanale et al . , 2014 ) .", "We followed the migratory process at a higher cellular resolution using time-lapse to resolve the seemingly contradictory data on the movement of small micromeres during gastrulation .", "Small micromeres were fluorescently labeled , so they could be uniquely followed throughout gastrulation allowing a finer analysis of their behavior than if those cells were not specifically identified ( Figure 1B and Video 1 ) .", "Host embryos were labeled by injection of histone2B-RFP mRNA and micromere-donor embryos with a membrane-GFP mRNA .", "At the 16-cell stage , one donor fluorescent micromere was transplanted to the vegetal pole where it replaced a surgically removed micromere ( Figure 1B ) .", "Each fourth cleavage micromere gives rise to two lineages: the small micromere ( the proposed primordial germ cell ) and the large micromere ( the skeletogenic mesoderm ) .", "The transplant surgery was performed at the 16-cell stage to ensure the survival of the small micromere daughters for two reasons .", "First , it assured that the small micromeres ( which arise at fifth cleavage by an asymmetric division of the micromere ) were not damaged by separation from the large micromeres during micromanipulation since they had not yet been born .", "Second , when the small micromeres appeared , their only initial connection to the embryo was via the spindle remnant to the large micromere—this attachment to the large micromere was crucial for the incorporation of the transplant into the host embryo .", "Both reasons for surgically transplanting the micromere ensured the re-establishment and survival of a high percentage of small micromeres in the host embryo ( Table 1 shows scoring of transplant efficacy ) .", "At the beginning of gastrulation , this recombinant approach resulted in 8 fluorescent large micromere progeny and 2 fluorescent small micromeres ( Figure 1C and Video 1 ) .", "It was easy to distinguish the donor small micromere progeny from the large micromere progeny when the large micromeres fused with non-fluorescent host mesoderm cells to form the skeletal syncytium thereby greatly diluting their fluorescence ( Figure 1C and Video 1 ) . 10 . 7554/eLife . 08827 . 004Video 1 . Movement of small micromeres throughout gastrulation . Membrane-GFP labeled micromeres were transplanted to a H2b-RFP-labeled host .", "An image was acquired every 3 min for 6 hr starting at 12 hr post fertilization .", "Videos were projected from multiple z-stacks ( every 3 μm , spanning the blastocoel of the embryo ) using SoftWorx for DeltaVision .", "AVIs were then rotated and translated ( due to swimming embryos ) in FIJI .", "Frame rate = 5 frames per second . DOI: http://dx . doi . org/10 . 7554/eLife . 08827 . 00410 . 7554/eLife . 08827 . 005Table 1 . Transplant efficacy for microsurgeriesDOI: http://dx . doi . org/10 . 7554/eLife . 08827 . 005MicrosurgerySuccessful transplants ( % ) Failed transplants ( % ) Ctrl host/Ctrl micromeres ( Figure 5 ) 51 ± 949 ± 9Ctrl host/MO micromeres ( Figure 5 ) 40 ± 2060 ± 20MO host/Ctrl micromeres ( Figure 5 ) 48 ± 1752 ± 17 Chimeric embryos made in this manner were examined over six hours of development to follow cell behavior .", "As seen in Figure 1C and Video 1 , throughout invagination of the archenteron , the small micromeres were highly active .", "They underwent many cell shape changes , blebbing , and pseudopodia extensions as was also seen by Campanale et al . They extended filopodia and lamellipodia through the basal lamina surrounding the archenteron .", "Nevertheless , they retained an adhesion and were not displaced relative to the adjacent invaginating epithelial cells ( Figure 1C and Video 1 , 12 hr 00 min–18 hr 00 min ) .", "When gastrulation was completed at 20 hpf ( early prism stage ) , small micromere behavior changed in tandem with a change in the basal lamina .", "At a time corresponding to the end of gastrulation , laminin immunostaining indicated that the basal lamina was undergoing remodeling , as there were large gaps in the laminin component of the basement membrane at the tip of the archenteron ( Figure 2A ) .", "Through those gaps , the small micromeres delaminated ( Figure 2A ) , moved to the blastocoelar side of the remodeling basal lamina , and then actively migrated to the posterior end of the left or right coelomic pouch where they were then re-enveloped with a basal lamina layer by 2 dpf ( Figure 2B ) .", "The migratory behavior of the small micromeres suggested that they underwent an EMT at the beginning of their migratory phase over the archenteron and to their coelomic pouch .", "These cells de-adhered , breached a basal lamina , changed shape , actively migrated within the blastocoel , and then rejoined an epithelium , demonstrating a number of EMT , and perhaps mesenchymal–epithelial transition ( MET ) , properties . 10 . 7554/eLife . 08827 . 006Figure 2 . Laminin remodeling at the tip of the archenteron facilitates migration of the small micromeres to the posterior end of the coelomic pouch .", "( A ) Once the micromeres reach the tip of the gut , they undergo an epithelial–mesenchymal transition ( EMT ) .", "By immunostaining , we see that laminin ( red ) , at the time , is reduced as the small micromeres ( SM , green ) breach the top of the gut .", "( B ) Once they reach the posterior coelomic pouch , laminin ( red ) surrounds the small micromeres ( green ) and NSM to encapsulate the coelomic pouch ( yellow dashed circle ) .", "( C , D )", "Ectopically placed small micromeres underwent an EMT coincident with the endogenous EMT event of the skeletogenic cells .", "Laminin ( red ) is absent both at the site of skeletogenic cell ingression ( indicated by white arrows at the site of ingression ) and at the site of ectopic small micromere ( SM , green ) ingression ( indicated by a yellow arrow ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 08827 . 006 In many embryonic systems , cells home , or migrate in a directed fashion , from their place of origin to a distant target site .", "Since that movement is directed , in each case there must be a mechanism to provide guidance ( Richardson and Lehmann , 2010; Yajima and Wessel , 2012; Campanale et al . , 2014; Reig et al . , 2014 ) .", "Knowing that the small micromeres were actively changing cell shape then moving to a new location at the end of gastrulation , we tested to see if small micromeres would undergo a directed migration to the coelomic pouch when placed in an ectopic location .", "Normally , small micromeres start their trajectory at the vegetal pole , which is the most posterior location in the embryo and terminate in the coelomic pouches near the anterior end of the embryo .", "Accordingly , small micromeres were placed ectopically at varying locations along the anterior/posterior axis of 16- to 60-cell staged embryos , and the frequency of their ability to find their way to the coelomic pouch at 2 days post fertilization ( dpf ) was scored ( Figure 3 ) .", "Micromeres placed at the vegetal pole served as a control since that is their endogenous starting location .", "Donors placed in the endogenous location homed 86 . 6% ( n = 213 ) of the time ( Figure 3A ) .", "13 . 4% of observed cases that were considered ‘no homing’ were instances where we could not find fluorescent small micromeres in the host embryo's coelomic pouch .", "Those that did not make it to the coelomic pouch became lost in the blastocoelar space , which we scored as ‘no homing’ .", "Micromeres were then placed ectopically between the Veg1 and Veg2 tier and in the animal half between the An1 and An2 tier of presumptive ectodermal cells ( scored together as equator ) , at the animal pole , or in the blastocoel .", "No matter where they were placed , the vast majority of small micromeres transplanted to ectopic positions anywhere in the embryo made it home ( Equator = 93 . 7% ( n = 74 ) , Animal Pole = 78 . 6% ( n = 11 ) , Blastocoel = 78 . 6% ( n = 11 ) ) ( Figure 3B–D ) .", "Interestingly , the majority of transplants were scored to be in the left coelomic pouch after homing suggesting a survival mechanism to ensure the proposed primordial germ cells make it onto adulthood to contribute to the gamete pool . 10 . 7554/eLife . 08827 . 007Figure 3 . Ectopically placed micromeres are able to find their way to the coelomic pouches via a directed homing mechanism from any ectopic location .", "( A ) The endogenous , vegetal pole , ( B ) the equator ( p < 0 . 004 ) , ( C ) the animal pole ( p < 0 . 004 ) , or ( D ) inside of the blastocoel ( p < 0 . 004 ) .", "Illustrations on the left designate their ectopic placement ( ectopic micromere = green , host embryo = red ) .", "Yellow dashed lines indicate the location of the whole coelomic pouch with the ectopically placed micromeres labeled in green .", "Graphs depict the percentage of embryos scored with the given ability to home ( Yes vs No ) , ‘n’ equals the number of embryos scored with the phenotype , and p-values were calculated using a χ2 test .", "‘**’ denotes a statistically significant ( p < 0 . 05 ) p-value . DOI: http://dx . doi . org/10 . 7554/eLife . 08827 . 00710 . 7554/eLife . 08827 . 008Figure 3—figure supplement 1 . Ectopic small micromeres arrive at the coelomic pouches independently . Small micromeres were ectopically placed as fourth division micromeres .", "The ectopic skeletogenic cells joined the endogenous skeletogenic cells while the ectopic small micromeres reached the coelomic pouch independently of the endogenous small micromeres ( vegetally placed ) no matter their starting location , either the equator ( A , B ) or the animal pole ( C , D ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 08827 . 008 Next , we wanted to see how the small micromeres homed .", "Did they move from the ectopic position within the plane of the epithelium , or did they ingress from the ectopic location into the blastocoel and find the coelomic pouch from there ?", "If they did ingress into the blastocoel , were they first attracted to the endogenous small micromeres before migrating to the coelomic pouch mesoderm , or did the small micromeres autonomously move to the coelomic pouches ?", "Could it be that the ectopically placed small micromeres precociously ingressed through the basement membrane and thus headed directly to their final destination ?", "To see when and where the ectopic small micromeres found their way home , we transplanted differentially labeled micromeres to two different locations ( one at the animal pole [Figure 3—figure supplement 1A] and one at the equator [Figure 3—figure supplement 1B] ) to host embryos .", "We then assayed at intervals up to 24 hpf and saw that the ectopically placed micromeres arrived independently of the endogenous micromeres ( Figure 3—figure supplement 1A , B ) .", "The transplanted small micromeres remained at the ectopic site until the primary mesenchyme cells ( PMCs ) ingressed .", "At that time ( 9–10 hpf ) , the ectopic small micromeres also ingressed into the blastocoel ( Figure 2C , D ) .", "Because the ectopic small micromeres ingressed at the time of skeletogenic cell ingression , we wondered whether isolated small micromeres could ingress on their own .", "By ectopically transplanting just 32-cell stage small micromeres ( as opposed to the usual 16-cell stage micromere , which would result in large and small micromere cells ) ( Figure 2C ) , we observed the small micromeres autonomously invade through the laminin layer independent of large micromeres .", "The ectopic small micromeres breached the laminin layer ( Figure 2D , yellow arrow ) at the same time as the PMC EMT ( Figure 2D , white arrows ) suggesting that the small micromeres create the hole through which they breach and invade , even though the endogenous small micromeres actually breach the basement membrane about 6–8 hr later when they leave the tip of the archenteron .", "Once we learned that the ectopically placed small micromeres home to the coelomic pouch from any location in the embryo , we next investigated whether the signal to which they were responding was coming from the coelomic pouch mesoderm .", "The question was whether the small micromeres home directly to the coelomic pouch region or indirectly .", "First , to ask if the mesoderm is the lineage to which the small micromeres home , we knocked out Delta , a signal necessary for mesodermal specification ( Sherwood and McClay , 1999; Sweet et al . , 2002 ) .", "The lack of mesoderm , indeed , had an effect on homing .", "In the experiment , Delta was knocked down in host embryos and control ( unperturbed ) micromeres were placed ectopically at the equator of those embryos ( Figure 4A ) .", "The small micromeres did not home to any significant location in the embryo ( coelomic pouches were non-existent in the KD ) and became lost in the blastocoelar space as is seen in cases we deemed ‘no homing’ 65% of the time ( n = 23 , p < 0 . 0001 ) ( Figure 4C ) .", "35% of the time , there was an incomplete knockdown of the Delta signal resulting in a coelomic pouch and the small micromeres homed to that location .", "Further investigation of the cell types and sub-territories within the coelomic pouch allowed us to elaborate on the genomic control underpinning the homing mechanism . 10 . 7554/eLife . 08827 . 009Figure 4 . Specification of the non-skeletogenic mesoderm ( NSM ) by Delta signaling is required for homing . Control micromeres labeled with membrane-GFP were ectopically transplanted to either a control TMR ( red ) host or a Delta–MO TMR ( red ) host ( A ) .", "When control micromeres were transplanted to a control host , homing of the small micromeres occurs 100% ( n = 9 ) of the time ( B ) .", "When NSM specification was blocked in a host embryo using a delta morpholino , homing of transplanted control micromeres was significantly reduced and only homed 53% of the time ( n = 23 , p < 0 . 0001 ) ( C , white arrows ) .", "( D ) The graph depicts the percentage of embryos ( Control Host vs Delta Host ) seen with the given ability to home ( Black = Homing vs Gray = No Homing ) , ‘n’ equals the number of embryos scored in each case , and p-values were calculated using a χ2 test .", "‘**’ denotes a statistically significant ( p < 0 . 05 ) p-value . DOI: http://dx . doi . org/10 . 7554/eLife . 08827 . 009 Next , we wanted to know which transcription factors within each territory of the final coelomic pouch ( aboral or oral mesoderm ) were upstream of the homing mechanism .", "We surveyed a list of known coelomic pouch and small micromere transcription factors , and genes found to maintain expression levels during homing were included in our list of potential upstream regulators .", "The genes further investigated were ( 1 ) oral coelomic pouch transcription factors FoxC , FoxF , PitX2; ( 2 ) aboral coelomic pouch transcription factors Dach1 , Pax6 , SoxE , Six3 , Six1/2 , and transcriptional co-activator Eya; and ( 3 ) small micromere transcription factors FoxC and Pitx2 .", "Accession numbers are found in Table 2 . 10 . 7554/eLife . 08827 . 010Table 2 . Plasmids used in this studyDOI: http://dx . doi . org/10 . 7554/eLife . 08827 . 010PlasmidFragmentAccession numberDach1Full CDSKR181947DeltaFull CDSKR181946EyaFull CDSKR181945FoxCFull CDSKR181944FoxFFull CDSKR181943Pax6Full CDSKR181942PitX2Full CDSKR181941Six3Full CDSKR181940SoxEFull CDSKR181939 The experiment was to transplant a labeled micromere to a differentially labeled host embryo and assay the small micromeres' ability to home in either of the two perturbed scenarios: either with a morpholino-perturbed host embryo or morpholino-perturbed micromere transplant .", "For each transcription factor , we transplanted control micromeres to control hosts ( a positive control for homing ) , perturbed micromeres transplanted to control hosts , and control micromeres transplanted to perturbed hosts .", "Transplants were done at the 16-cell stage and scored at two days post-fertilization .", "Transplant efficacy was also scored and can be found in Table 1 .", "Only those transplants containing small micromere were scored for homing or no homing .", "Failed transplants were cases where no small micromeres were observed in the embryo .", "One transcription factor , Forkhead family member FoxC , was found to affect small micromere homing when only knocked down in the micromere .", "FoxC is expressed in both the oral coelomic pouch mesoderm and the small micromeres; however , only when it was perturbed in the small micromere did it affect homing , and only 26% ( n = 19 , p < 0 . 001 ) of the time did the small micromeres successfully make it to the coelomic pouch ( Figure 5A ) . 10 . 7554/eLife . 08827 . 011Figure 5 . Homing is controlled by upstream transcription factors . By selectively knocking down specific transcription factors in either an ectopically placed micromere or the host embryo , transcription factors for the homing were identified .", "( A ) FoxC , a transcription factor expressed in the small micromeres and oral mesoderm was necessary for homing of the small micromere ( n = 19 , p < 0 . 001 ) , but had no inhibitory effect on homing when knocked down in just the mesoderm ( n = 13 ) .", "( B ) Dach1 , an aboral mesoderm transcription factor , affected homing when knocked down in the mesoderm ( n = 10 , p < 0 . 0001 ) but did not affect homing when perturbed in the small micromere ( n = 13 ) .", "( C ) Pax6 , affected homing when knocked down in the mesoderm ( n = 10 , p < 0 . 004 ) but had no effect on homing when perturbed in the small micromere ( n = 14 ) .", "( D ) Aboral mesoderm transcription factor , Six3 affected homing when knocked down in the mesoderm ( n = 9 , p < 0 . 00009 ) but had no effect homing when perturbed only in the small micromere ( n = 9 ) .", "( E ) Aboral transcriptional co-activator , Eya , affected homing when knocked down in the mesoderm ( n = 7 , p < 0 . 002 ) but did not affect homing when perturbed in the small micromeres ( n = 8 ) .", "( F ) Aboral transcription factor , Six1/2 , affected homing when knocked down in the mesoderm , as well ( n = 17 , p < 0 . 02 ) and did not affect homing when knocked down in the micromere ( n = 11 ) .", "Yellow circles indicate the location of the coelomic pouch .", "White arrows indicate ‘lost’ micromeres .", "The graph depicts the percentage of embryos ( Control Host vs MO micromere vs MO Host ) seen with the given ability to home ( Black-Homing vs Gray-No Homing ) , ‘n’ equals the number of embryos scored in each case p-values were calculated using a χ2 test .", "‘**’ denotes a statistically significant ( p < 0 . 05 ) p-value . DOI: http://dx . doi . org/10 . 7554/eLife . 08827 . 01110 . 7554/eLife . 08827 . 012Figure 5—figure supplement 1 . Coelomic pouch transcription factors that do not affect homing . Transcription factors FoxF ( A ) , SoxE ( B ) , and PitX2 ( C ) are not seen to affect homing when they are perturbed in either the small micromeres or the NSM .", "The graph depicts the percentage of embryos ( Control Host vs Delta Host ) seen with the given ability to home ( Black = Homing vs Gray = No Homing ) , ‘n’ equals the number of embryos scored in each case , and p-values were calculated using a χ2 test .", "No p-values were deemed significant . DOI: http://dx . doi . org/10 . 7554/eLife . 08827 . 01210 . 7554/eLife . 08827 . 013Figure 5—figure supplement 2 . Whole embryo perturbation of coelomic pouch transcription factors assayed for vasa expression . Embryos injected with a Control-MO and stained for vasa mRNA expression was seen to appropriately segregate small micromeres in the left and right coelomic pouches ( A ) .", "Embryos perturbed for Delta ( B ) , FoxC ( C ) , Dach1 ( D ) , Six3 ( E ) , Six1/2 ( F ) , Eya ( G ) , and Pax6 ( H ) were assayed for vasa expression in the coelomic pouches to test whether migration defects were seen in endogenous knockdown situations .", "Small micromeres were seen to properly segregate to coelomic pouches , or in the case of Delta to the side of the archenteron ( since there are no coelomic pouches in a Delta–MO ) in each perturbation . DOI: http://dx . doi . org/10 . 7554/eLife . 08827 . 013 The first coelomic pouch transcription factor we found to be involved in homing was a Ski/Sno family member , Dachshund1 .", "When control micromeres were transplanted to a Dachshund1 morpholino perturbed host , the control small micromeres were only able to home in 10% ( n = 10 , p < 0 . 0001 ) of scored cases ( Figure 5B ) .", "In contrast , when Dach1 was knocked down in the small micromeres only , and the host embryo was a control , homing ability was scored as successful in 77% of cases .", "We concluded that Dach1 was necessary in the targeted tissue for homing to occur .", "Next , we found the paired box family transcription factor Pax6 was upstream of the small micromeres' ability to home .", "When control micromeres were transplanted to a Pax6 knockdown host , the small micromeres were only able to home 40% ( n = 10 , p < 0 . 004 ) of the time ( Figure 5C ) .", "We then found the six family transcription factor , Six3 , to regulate homing .", "When control micromeres were transplanted to a Six3 knockdown host , the small micromeres were able to home 11% ( n = 9 , p < 0 . 00009 ) of scored cases ( Figure 5D ) .", "Next , we saw transcriptional co-activator and tyrosine phosphatase , Eya , to exhibit an effect on homing .", "When control micromeres were transplanted to an Eya knockdown host , small micromeres were able to home 28 . 5% of the time ( n = 7 , p < 0 . 002 ) ( Figure 5E ) .", "Finally , we found another six family transcription factor , Six1/2 , to affect homing .", "Control micromeres transplanted to a Six1/2 knockdown host were able to home 65% ( n = 17 , p < 0 . 02 ) of scored cases ( Figure 5F ) .", "This effect is modest , but statistically significant relative to controls within that experiment .", "Transcription factors FoxF , SoxE , FoxC , and Pitx2 did not appear necessary for homing when perturbed in the coelomic pouch mesoderm ( Figure 5—figure supplement 1 ) .", "It is important to note that when the same knockdowns are stained for endogenous vasa expression ( labeling the small micromeres ) , small micromeres are still able to reach the coelomic pouches ( Figure 5—figure supplement 2 ) .", "We observe that the control endogenous small micromeres are within the coelomic pouches in tight clusters ( Figure 5—figure supplement 2A ) while the knockdowns exhibit various levels of disorganization ( Figure 5—figure supplement 2B–H ) .", "Since the endogenous small micromeres normally home over a very short distance , the knockdown small micromeres , as might be expected , remain close to the coelomic pouches—they do not disperse from that vicinity , but they do not tightly cluster in the correct location .", "Also , at this time , there is prevalent cell rearrangement at the tip of the archenteron that leads to the fusion of the oral ectoderm and foregut endoderm ( unpublished observations ) .", "Thus of the eleven genes screened for homing behavior , only six were necessary for homing .", "We next wanted to better understand how the five coelomic pouch mesodermal transcription factors scored as necessary for homing might relate in the network circuit that runs in that tissue .", "Each of the five transcription factors involved in controlling homing is expressed in the aboral coelomic pouch .", "Luo and Su showed that transcription factors Dach1 , Eya , Six1/2 , and Pax6 are co-localized in the aboral coelomic pouch ( Luo et al . , 2012 ) .", "Six3 was found to be expressed in the aboral mesoderm early in development , and by double fluorescent in situ hybridization , we see six3 to also overlap with aboral marker , eya ( Figure 6B ) ( Wei et al . , 2011 ) .", "Six3 was seen to be tightly apposed with vasa expression but is exclusive from small micromeres .", "Also , six3 expression is distinct from the oral , myosin , domain of expression ( Figure 6A , C ) .", "Expression domains within the coelomic pouch are illustrated in Figure 6D .", "Luo and Su also noted that transcription factors Dach1 and FoxC are expressed in the oral coelomic pouch ( Luo et al . , 2012 ) .", "Transcription factor FoxC , however , is expressed in both the oral coelomic pouch and the small micromeres at the time of small micromere homing ( Luo et al . , 2012 ) . 10 . 7554/eLife . 08827 . 014Figure 6 . Expression domains within the coelomic pouch . Double fluorescent in situ hybridization of six3 with vasa shows a tight apposition of their expression domains but a lack of co-localization ( A–A′′′′ ) .", "Z projection of six3 and vasa is seen in ( A ) , and individual Z sections are seen from aboral to oral most locations in the embryo in ( A′–A′′′′ ) .", "Insets on panels A–A′′′′ show a zoomed perspective of just the left coelomic pouch apposed expression of six3 and vasa .", "six3 and eya were seen to overlap in the aboral coelomic pouch in both the Z projection ( B ) and Z sections ( B′′ and B′′′ ) but not in Z sections ( B′ or B′′′′ ) .", "myosin expression was seen to be in the most oral expression domain of the coelomic pouch , distinct of six3 ( C–C′′′′ ) .", "An illustration demonstrating the expression domains observed in the coelomic pouch from our data and the data of Luo and Su , 2012 is displayed in ( D ) from both the lateral and oral views . DOI: http://dx . doi . org/10 . 7554/eLife . 08827 . 01410 . 7554/eLife . 08827 . 015Figure 6—figure supplement 1 . Spatial and temporal expression of homing genes by whole mount in situ hybridization . Dach1 , dachshund , is first detected early in development .", "It becomes spatially restricted around mesenchyme blastula to the coelomic pouch mesoderm ( A3 ) .", "During gastrulation , it is found throughout and at the tip of the archenteron in the presumptive coelomic pouch cells ( A5-8 ) and is maintained in that tissue and gut throughout coelomic pouch coalescence with the small micromeres at the pluteus stage ( A7-8 ) .", "six3's earliest detectable expression is at early blastula ( B1 ) .", "It is found in the animal pole region , and expression in the coelomic pouch mesoderm begins around mesenchyme blastula ( B3 ) .", "Throughout gastrulation , six3 is found in the apical plate domain and the future coelomic pouch cells ( B3-4 ) .", "As was seen in Wei 2009 , the six3 apical domain of expression is responsible for giving rise to neural fates .", "Post-gastrulation , six3 is expressed in both coelomic pouches ( B8 ) .", "Six1/2 is seen to be expressed in the NSM beginning at mesenchyme blastula ( C3 ) .", "It maintains expression in the NSM throughout early development and is in the coelomic pouches by early pluteus ( C7 ) .", "Genes activated at mid-gastrula stage at the tip of the archenteron and endure throughout gastrulation include foxc and eya .", "mRNA expression is detected in the coelomic pouch mesoderm at the tip of the archenteron and end up in the coelomic pouches .", "eya begins expression at mid-gastrula in the aboral coelomic pouch at the tip of the archenteron ( D4 ) .", "eya expression by 32 hpf is found in the left coelomic pouch ( D8 ) .", "pax6 expression begins at mid to late gastrula stage in the aboral coelomic pouch at the tip of the archenteron , roughly at the time of ectopic small micromere homing ( E5 ) , and by 32 hpf is found in the left coelomic pouch and two lateral patches of ectoderm presumed to be neural in fate ( E8 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 08827 . 015 To determine the order of expression of the transcription factors that could potentially act in a homing subcircuit , we first performed whole mount in situ hybridization on all five homing genes ( dach1 , pax6 , eya , six1/2 , and six3 ) at eight time points ranging from pre-hatched blastula ( before small micromere migration along the archenteron ) to early pluteus ( after the small micromeres and coelomic pouches have coalesced ) with a 2- to 4-hr step-time to establish relative timing of expression ( Figure 6—figure supplement 1 ) .", "By surveying this gene set , we conclude that their co-localization in the coelomic pouches and temporal expression patterns suggest that these transcription factors may be interacting ( diagram , Figure 6D ) .", "To ask how or if these genes interact in a gene network to drive the morphogenetic cassette , we undertook a perturbation analysis to build a homing GRN upstream of the morphogenetic movement .", "Each transcription factor was perturbed and the effect of that perturbation on the others was examined .", "When Dach1 was perturbed using a morpholino specific to the start of translation , we saw by in situ hybridization an increased expression of dach1 ( indicating self repression ) .", "Knowing that dach1 is expressed in the endoderm earlier in development from our time course data , we concluded that the upregulation seen in the gut was a result of dach1 self-repression not only in the gut but also later in the coelomic pouch mesoderm expression .", "A down-regulation of six3 and a down-regulation of eya transcripts was also seen in the Dach1 perturbation .", "These data indicate that Dach1 positively regulates six3 and eya and inhibits the overexpression of itself by repression ( Figure 7 ) . 10 . 7554/eLife . 08827 . 016Figure 7 . Perturbation analysis unveils regulatory linkages in a homing GRN . Perturbations using a Dach1 morpholino ( MO ) showed Dach1 to be upstream of dach1 , six3 , and eya .", "A Six3-MO caused a downregulation of eya and pax6 .", "Pertubations with an Eya-MO showed Eya to be upstream of six3 , six1/2 , and eya .", "Six1/2 is seen to be upstream of six3 , eya , and six1/2 .", "Finally , a Pax6-MO caused a downregulation of six3 , eya , and pax6 .", "Arrows represent up or downregulation seen for each panel .", "‘n’ equals the total number of embryos scored , and the adjacent percentage designates the percent of embryos scored with the shown effect .", "Morpholino morphology phenotypes at 24 hpf and 48 hpf are presented in Figure 7—figure supplement 1 .", "Unpublished morpholino data has been validated using a second , distinct morpholino ( see Figure 7—figure supplement 2 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 08827 . 01610 . 7554/eLife . 08827 . 017Figure 7—figure supplement 1 . Morpholino phenotypes . Morpholino morphologies and general health for all morpholinos used were assayed at 24 and 48 hpf .", "Each morpholino was imaged at two time points and multiple focal planes .", "GFP images are shown as proof of injection .", "Since many of the morphants display a short arm phenotype at 2 dpf , specific focus on their forming skeleton was focused at 24 hpf . DOI: http://dx . doi . org/10 . 7554/eLife . 08827 . 01710 . 7554/eLife . 08827 . 018Figure 7—figure supplement 2 . Morpholino effects seen in this paper that have not previously been validated were confirmed using a second morpholino designed in a location distinct to the site of the first morpholino .", "( A ) Using the same Ctrl-MO as was used in Figure 5 , 73% of the time , eya mRNA expression is present in the pattern shown .", "( B ) The second morpholino designed to the translation start site of Eya , matched results of the first morpholino with eya mRNA downregulated in 96% of cases .", "( C ) The second Pax6 morpholino , also designed to block translation , also matched the perturbation effect of the first morpholino in that 68% of the embryos scored were downregulated for eya mRNA .", "N is equal to the number of embryos scored . DOI: http://dx . doi . org/10 . 7554/eLife . 08827 . 018 When Six3 was perturbed using a start site morpholino , we saw a self up-regulation of six3 but only in the apical expression domain , a down-regulation of eya , and a down-regulation of pax6 transcripts .", "We conclude from these data that Six3 positively regulates eya and pax6 in the coelomic pouch mesoderm .", "Perturbing Eya in the same way showed a down-regulation of six3 , six1/2 , and a self-down-regulation of eya .", "Knowing that Eya is a transcriptional co-activator of Six1/2 , Eya must positively regulate itself , six3 , and six1/2 by co-acting with Six1/2 .", "Next , Six1/2 was perturbed , and , as expected , regulatory interactions found reflected those found in the Eya knockdowns indicating that Eya and Six1/2 do , in fact , act synergistically as has been seen before ( Heanue et al . , 1999; Materna et al . , 2013 ) .", "Finally , Pax6 perturbation caused down-regulation of six3 , eya , and itself .", "Pax6 , therefore , positively regulates six3 , eya , and itself ( Figure 7 ) .", "When the network was laid out in a Biotapestry model , the subcircuit controlling the homing mechanism was readily discerned as a coherent feed-forward loop composed of multiple feedback loops that sustain its robust nature ( Figure 8A ) ( Longabaugh et al . , 2005 ) .", "It also displayed a striking resemblance to another circuit well known in development: the RDGN .", "The Pax-Six-Eya-Dach RDGN is necessary for specification of retinal cell types in many systems ( Purcell et al . , 2005; Kozmik et al . , 2007; Kumar , 2009; Salzer et al . , 2010; Weasner and Kumar , 2012 ) .", "The same five factors contribute to a coherent feed-forward circuit for Drosophila eye specification ( Kumar , 2009 ) ( Figure 8B ) .", "Few linkages between the eye and small micromere homing circuits are different ( Figure 8 ) . 10 . 7554/eLife . 08827 . 019Figure 8 . Homing GRN subcircuit shows striking resemblance to Drosophila RDGN .", "( A ) The perturbation analysis was mapped as a Biotapestry network model .", "( B ) A retinal gene network subcircuit extracted from Drosophila ( Kumar , 2009 ) shows a very similar circuit with few regulatory linkage changes in comparison . DOI: http://dx . doi . org/10 . 7554/eLife . 08827 . 01910 . 7554/eLife . 08827 . 020Figure 8—figure supplement 1 . The RDGN subcircuit is conserved throughout evolution . Similar putative GRN models were deduced based on publications in systems also known to utilize RDGN components and assembled in Biotapestry: ( A ) Demosponge canal systems , ( B ) vertebrate skeletal myogenesis , and ( C ) the mouse otic placode . DOI: http://dx . doi . org/10 . 7554/eLife . 08827 . 020" ], [ "The data from the chimera experiments show that normally small micromeres retain an adhesion to adjacent cells during archenteron extension .", "When they arrive at the tip of the archenteron , they then leave the epithelium using an EMT through a laminin-containing basement membrane , migrate to the posterior end of the coelomic pouch , and transition back to an epithelium as they coalesce with the coelomic pouch .", "Ectopically placed small micromeres behave differently from the endogenous small micromeres in that they fail to retain epithelial adhesions through gastrulation and instead go through an EMT at the time PMCs ingress .", "The fact that the ectopically placed small micromeres lose adhesion and go through EMT independent of the timing of archenteron tip formation implies that there must be a non-autonomous cue to keep the endogenous small micromeres in place during gastrulation .", "Without that local cue , the small micromeres actively home from the beginning of gastrulation as seen with the behavior of ectopically placed small micromeres .", "Further , there must be a non-autonomous signal that is recognized by small micromeres when they reach the tip of the forming archenteron that cues them to initiate the EMT and migration .", "The behavior of the ectopic cells suggest that the migratory capacity can be activated at the beginning of gastrulation , but since they normally do not move from the epithelium until at the tip of the archenteron , another event may stimulate them to complete their journey to the coelomic pouches .", "Observations of ectopically placed small micromeres also reveal that the cells undergo directed cell migration to get to the coelomic pouch mesoderm .", "The cells find their way to the coelomic pouch no matter their starting location .", "In other systems cells become polarized in response to a chemoattractant signal at the leading edge of the cell while the remainder of the cell is cued to receive inhibitory or repulsion signals responsible for increasing its sensitivity to its target location ( as reviewed by Richardson and Lehmann , 2010; Reig et al . , 2014 ) .", "Small micromeres appear to take a fairly direct route to their eventual target no matter where they are initially placed .", "Since the normal route involves movement through part of the blastocoel , and since ectopic small micromeres also use the blastocoelar route , the putative homing signal likely is produced earlier than the end of gastrulation since the ectopic small micromeres move through the blastocoel to the correct target well before gastrulation is completed .", "At present , it is only speculation that there is a chemoattraction mechanism at play .", "If it were chemoattraction , the cue must work at the individual cell level since single small micromeres respond to the cue when ectopically placed .", "The transcriptional regulation upstream of that putative signal offers approaches for its identification .", "Potentially , as an alternative hypothesis , the coelomic pouch NSM could be drawing the small micromeres to their final site by filopodia , since it is known that these cells as well as the small micromeres extend filopodia ( Hardin , 1988; Campanale et al . , 2014 ) .", "These hypotheses will need to be explored further .", "The goal of determining upstream transcriptional regulation of the homing process requires connection of the aforementioned network ( Figure 8A ) to chemoattraction mechanisms ( both attractant and repulsion ) directly responsible for the directing migration .", "In addition , other subcircuits are necessary to drive the EMT , for reception of the putative signal , and for directed movement in response .", "Transcriptional control of directed cell migration is not well studied at a systems level .", "There are a few examples , however , that suggest the way these systems work .", "Findings in the tunicate , Ciona intestinalis , describe the distinct transcriptional regulation of a cellular module that drives migration of heart precursors .", "The heart GRN drives membrane protrusions and RhoGTPase signaling , tail retraction , adhesion , and polarity ( Christiaen et al . , 2008 ) .", "One important feature of that study was that the cells constitutively expressed many of the factors required for these cell behaviors .", "It only took one or two factors to be under direct control of the developmental GRN to regulate the timing , directionality , onset , and termination of the heart cell precursors' journey .", "In the sea urchin , five different GRN subcircuits were shown to drive five different cellular components of an EMT ( Saunders and McClay , 2014 ) .", "It is highly likely , like Ciona heart cells , that the sea urchin developmental GRN will control directly expression of only a fraction of the proteins used for de-adhesion , motility , invasion of the basement membrane , altered cell polarity , and cell shape changes , all components of an EMT , while many other proteins in those processes will be constitutively expressed .", "Using small micromere homing as an assay , we were able to begin to unravel the intricacy of GRN subcircuits that control cell migration to a target site .", "This work thus demonstrates how morphogenesis can be controlled by conserved specification GRN subcircuits .", "The small micromere homing example reveals something else in morphogenetic movement control .", "We tend to think of the result of the movement: the small micromeres become associated with the aboral coelomic pouch through homing .", "Yet , to accomplish that feat , both the cells that home and the target cells must enact a series of coordinated , but independent processes .", "Here , we found a set of genes that control the production of the putative homing signal .", "Being that the subcircuit is of coherent feed-forward topology , we suspect it will be found at the most distal part of a larger GRN; therefore , it will act downstream of the specification circuitry of the signal-producing cell type .", "Connecting that upstream subcircuit to the downstream signal ( s ) will provide a tool for explaining one component of the homing mechanism that we know to be transcriptionally controlled .", "Recognizable RDGN components are deployed in a variety of developmental processes in different organisms .", "After pooling published data from three different fields of study ( demosponge canal systems , vertebrate skeletal muscle , and mouse otic placodes ) , we were able to construct putative GRN models ( Figure 8—figure supplement 1 ) .", "Although each respective subcircuit is embedded in a larger specification GRN , when extracted as a subcircuit , it appears that the RDGN feed-forward topology has been deeply conserved ( Torres et al . , 1996; Heanue et al . , 1999; Relaix and Buckingham , 1999; Xu et al . , 1999; Ridgeway and Skerjanc , 2001; Kardon et al . , 2002; Laclef et al . , 2003; Riley and Phillips , 2003; Zheng , 2003; Ozaki , 2004; Silver , 2004; Relaix et al . , 2013; Rivera et al . , 2013 ) .", "Only now that we have been able to make a complete RDGN subcircuit in the sea urchin , can we hypothesize about what evolutionary mechanism may be at play .", "With the same transcription factors involved in a similar feed-forward process in such a variety of tissues , we hypothesize that the RDGN subcircuit is ancient and has been repeatedly deployed as a unit to provide feed-forward control .", "If this subcircuit is redeployed throughout evolution as a unit , as seems to be the case given the frequency of its presence , the mechanism that keeps it intact is unknown .", "Evolving as a modular unit must mean that the subcircuit serves as an indispensible or important component in a network full of modular components .", "The assembly of the coherent feed-forward circuit , once established , appears to remain intact in separate lineages for the same reason: that circuit must be critical for the GRNs in which it is embedded .", "Its presence raises the question of whether there are other ancient or more recent subcircuits that have evolved as modules .", "Since analysis of developmental GRNs is a relatively young field , it is possible that a number of ancient modular subcircuits evolve as units to assist in the diversification of cell types and morphogenesis .", "One obvious outcome of this mode of inheritance would be a contribution to robustness in developmental mechanisms .", "And perhaps once a subcircuit is particularly well integrated and suited for a given type of regulatory function , each of its members ( in our case , transcription factors ) might act as a constraint on the others because any reduction in integration would be less adaptive .", "What is intriguing is how ancient the RDGN must be .", "It is a circuit that is used because it efficiently provides an excellent feed-forward circuit that stabilizes the process to which it is attached .", "It is best studied in retinal determination but its ancestral role is unknown and could equally well be directed cell migration ." ], [ "Adult Lytechinus variegatus were obtained from Reeftopia ( Key West , FL , United States ) or the Duke University Marine Lab ( Beaufort , NC , United States ) .", "Gametes were obtained by injection of 0 . 5 M KCl into the adult coelom .", "Embryos were cultured at 23°C in artificial seawater ( ASW ) .", "The full-length coding sequences for NSM transcription factors , Delta , and RDGN genes were obtained by designing primers against a transcriptome data set .", "Nucleotide sequences were annotated and deposited on GenBank ( Accession Numbers: Table 2 ) .", "Morpholino antisense oligonucleotides were designed against the start site of translation by GeneTools .", "Morpholino sequences and concentrations are as listed in Table 3 .", "Morpholinos were diluted in molecular-grade H20 and either FITC or TMR injectable dyes .", "Morpholino experiments were carried out on three biological triplicates , cultured at 23°C , and assayed by in situ hybridization .", "Phenotypes of morpholinos at 24 hpf and 48 hpf were imaged to show the overall health and morphology of knockdowns ( Figure 7—figure supplement 1 ) .", "Published morpholinos listed in Table 3 were verified using appropriate controls explained in the citation referenced .", "Two morpholinos were assayed to test the specificity of Six3 , FoxF , and FoxC .", "Appropriate efficacy controls were also carried out previously for Six3 , FoxF , FoxC , Delta , Six1/2 , and Dach1 as is cited in Table 3 .", "Previously unpublished morpholinos were validated by ordering a second , distinct morpholino to the start of translation or 5′ UTR and were verified using in situ hybridization ( Figure 7—figure supplement 2 ) .", "mRNA for injection was transcribed in vivo using Ambion mMessage mMachine .", "Concentrations for mRNA injections: 300 ng/μl Histone2B-GFP , 500 ng/μl Histone2B-RFP , 500 ng/μl membrane-RFP , and 300 ng/μl membrane-GFP .", "For injection , mRNAs were diluted in 20% glycerol in diethylpyrocarbonate-treated H2O . 10 . 7554/eLife . 08827 . 021Table 3 . Morpholino anti-sense oligonucleotide sequences used in this studyDOI: http://dx . doi . org/10 . 7554/eLife . 08827 . 021MASOMASO sequenceMorpholino typeWorking concentrationLvPax6-MO1GTTGACCTGGCATAGCAGCATTTACTranslation blocking1 . 2 mMLvPax6-MO2CATGTCCCCGTGACCCATAGTTTTCTranslation blocking0 . 3 mMLvEya-MO1GCGCTGAAGCTATTTGACATGCTGTTranslation blocking0 . 75 mMLvEya-MO2GTTGAAACCTGTTTGACTGTAGGCCTranslation blocking1 mMLvSix3-MOATGTTTCCGACTCCGTCCAAACCATTranslation blocking ( Wei et al . , 2009 ) 0 . 75 mMLvSix1/2-MOCCCAAGTCCGTGGCAAGGATAAGATTranslation blocking ( Ransick and Davidson , 2012 ) 0 . 5 mMLvDach1-MOAGTAGGCGGTGGACTTCCCATTTTCTranslation blocking ( Peter and Davidson , 2011 ) 0 . 5 mMLvFoxC-MOTGAAGCGTACATTGGCATGGATGTTTranslation blocking ( Andrikou et al . , 2015 ) 0 . 75 mMLvDelta-MOGTGCAGCCGATTCGTTATTCCTTTTranslation blocking ( Sweet et al . , 2002 ) 0 . 4 mMLvFoxF-MOTCTAATTGAGTCATCTGGAGAGTGTTranslation blocking ( Andrikou et al . , 2015 ) 0 . 75 mMLvSoxE-MOGCTCTAAACTCTCAGGGCTACTCATTranslation blocking0 . 75 mMLvPitX2-MOACTGGTTCATCGCTGCTGATTAATTTranslation blocking0 . 6 mMControl-MOCCTCTTACCTCAGTTACAATTTATATranslation blockingExptDependent At 2 . 5 hpf , one micromere from an injected donor embryo was transplanted onto a differentially injected 16-cell host embryo in the place of one discarded host micromere or at an ectopic location .", "Microsurgery was performed with fine glass needles and Narishige micromanipulators .", "Detailed methods of injections and transplants were followed as previously described ( Logan et al . , 1999 ) .", "Transplant efficacy was scored in Table 1 .", "At 11 hpf , embryos were de-ciliated in 2× hypertonic artificial seawater , mounted in 1× artificial seawater containing 10 μM p-methoxy-phenyl isoxazoline ( Semenova et al . , 2011 ) on a slide coated in 1% protamine sulfate and sealed with a combination of Vasoline , lanolin , and paraffinwax ( also known as V . A . L . A . P . ) .", "Images were acquired using Coolsnap high-resolution CCD camera on the DeltaVision Elite with 40×/0 . 65–1 . 35 Oil UAPO40X0I3/340 DIC objective .", "Images were collected at 3-min intervals beginning at 12 hpf and ending at 18 hpf or later and projected as videos using SoftWorx for DeltaVision .", "Embryos were fixed in 4% paraformaldehyde for 10 min , washed in PBST , blocked in 4% normal goat serum ( in PBST ) for 45 min , and incubated in polyclonal anti-Laminin antibody ( 1:250 ) ( ab11575; Abcam [Cambridge , MA , United States] ) and anti-GFP ( 1:2000 ) ( ab13970; Abcam ) overnight at 4°C .", "Embryos were then washed in PBST , incubated in a 1:200 dilution of either Cy2- or Cy3-conjugated secondary antibody , and finally , stained with Hoechst's ( 1:1000 ) ( Molecular Probes [Eugene , OR , United States] ) to label all nuclei .", "Images were acquired using Zeiss LSM 510 upright confocal with a 40×/1 . 4 Oil Plan-Apochromat objective .", "Z-slices were spaced 1 . 0 μm apart spanning the diameter of the embryo .", "RNA in situ hybridization was performed using Digoxigenin-11-UTP-labeled probes as previously described ( McIntyre et al . , 2013 ) .", "Briefly , embryos were fixed in 4% paraformaldehyde overnight at 4°C , washed with ASW , and stored in methanol at −20°C .", "RNA probes were synthesized in vitro and used at 1 ng/μl .", "Hybridization took place at 60–65°C .", "Probes were visualized using alkaline phosphatase-conjugated anti-DIG antibody ( 1:1500 , Roche [Indianapolis , IN , United States] ) .", "Finally , color was developed using NBT/BCIP ( Roche ) .", "For double fluorescent in situ hybridization , a second probe ( labeled with Fluorescein-12-UTP ) was hybridized .", "Expression was visualized using the Tyramide Signal Amplification system ( TSA-plus kit , Perkin Elmer [Waltham , MA , United States] ) .", "Embryos were visualized with a Zeiss Axioplan2 upright microscope .", "GraphPad software was used to analyze a 2 × 2 contingency table with rows corresponding to the condition ( controls and knockdown micromeres or controls and knockdown hosts ) and columns corresponding to outcomes ( ‘homing’ or ‘no homing’ ) .", "Chi-squared was then calculated , and a p-value was declared significant at a value of less than 0 . 05 ." ] ]

[ "Gene regulatory networks ( GRNs ) provide a systems-level orchestration of an organism's genome encoded anatomy .", "As biological networks are revealed , they continue to answer many questions including knowledge of how GRNs control morphogenetic movements and how GRNs evolve .", "The migration of the small micromeres to the coelomic pouches in the sea urchin embryo provides an exceptional model for understanding the genomic regulatory control of morphogenesis .", "An assay using the robust homing potential of these cells reveals a ‘coherent feed-forward’ transcriptional subcircuit composed of Pax6 , Six3 , Six1/2 , Eya , and Dach1 that is responsible for the directed homing mechanism of these multipotent progenitors .", "The linkages of that circuit are strikingly similar to a circuit involved in retinal specification in Drosophila suggesting that systems-level tasks can be highly conserved even though the tasks drive unrelated processes in different animals ." ]

[ "Within an animal embryo , groups of cells tend to move , or migrate , between different areas before they form into tissues and organs .", "These cell migrations are regulated by hundreds of genes , which must be expressed at the right time and in the right place .", "Cells use proteins called transcription factors to regulate the expression of genes .", "These proteins work together in circuit board-like networks called gene regulatory networks in order to drive different aspects of development , including cell migration .", "The sea urchin is a useful model organism to study how animals develop .", "This is because these marine animals express many of the same genes as humans , but they can be easily manipulated and studied in the laboratory .", "In a developing sea urchin embryo , cells called the small micromeres move towards one end of animal and get incorporated into a pocket-like structure known as the coelomic pouch .", "From this pouch , these cells mature and eventually contribute to the adult germ cells ( the precursors to the sperm and eggs ) .", "Martik and McClay have now analyzed how small micromeres make their way to their final location in the coelomic pouch .", "Micromeres were labeled with a dye that fluoresces green so that they could be tracked under a microscope .", "This revealed that , like other moving cells , micromeres actively change their shape as they migrate .", "Furthermore , when micromeres were experimentally moved to abnormal locations in the sea urchin embryo , they were still able to actively home in on the coelomic pouch no matter their starting location .", "Martik and McClay then identified five transcription factors expressed in the coelomic pouch in the sea urchin that are involved in this homing activity .", "Reducing the expression of any of these transcription factors was enough to hinder the ability of the micromeres to find their way to the coelomic pouch .", "Further experiments and analysis then revealed that these five transcription factors work together in a sub-circuit , which is in turn embedded in a larger gene regulatory network .", "This sub-circuit that drives cell migration is unexpectedly similar to another circuit in the fruit fly Drosophila .", "Intriguingly , the sub-circuit in the fly controls eye development , which is unrelated to cell homing and migration .", "These observations raise the possibility that this circuit has been conserved as a unit over millions of years of evolution and redeployed in new networks under completely different circumstances .", "The data also suggest the possibility that additional conserved sub-circuits will be identified as more systems are analyzed in detail ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "biochemistry and chemical biology" ]

[ [ "Numerous biological events are orchestrated epigenetically upon defining cellular fates ( Atlasi and Stunnenberg , 2017; Berdasco and Esteller , 2019 ) .", "Among the key epigenetic regulators are protein methyltransferases ( PMTs ) , which can render downstream signals by modifying specific Arg or Lys residues of their substrates with S-adenosyl-L-methionine ( SAM ) as a methyl donor cofactor ( Luo , 2018 ) .", "Significant efforts have been made to identify the PMT-dependent epigenetic cues that are dysregulated or addicted under specific disease settings such as cancer ( Berdasco and Esteller , 2019 ) .", "Many PMTs are implicated as vulnerable targets against cancer malignancy ( Kaniskan et al . , 2018; Luo , 2018 ) .", "The pro-cancerous mechanism of these PMTs can be attributed to their methyltransferase activities , which act individually or in combination to upregulate oncogenes , downregulate tumor suppressors , and maintain cancer-cell-addicted homeostasis ( Berdasco and Esteller , 2019; Blanc and Richard , 2017 ) .", "Pharmacological inhibition of these epigenetic events thus presents promising anti-cancer strategies ( Berdasco and Esteller , 2019 ) , as exemplified by the development of the clinical inhibitors of DOT1L ( Bernt et al . , 2011; Daigle et al . , 2011 ) , EZH2 ( Kim et al . , 2013; Konze et al . , 2013; McCabe et al . , 2012; Qi et al . , 2012; Qi et al . , 2017 ) , and PRMT5 ( Bonday et al . , 2018; Chan-Penebre et al . , 2015 ) .", "Protein arginine methyltransferases ( PRMTs ) act on their substrates to yield three different forms of methylated arginine: asymmetric dimethylarginine ( ADMA ) , symmetric dimethylarginine ( SDMA ) , and monomethylarginine ( MMA ) , which are the terminal products of Type I , II and III PRMTs , respectively ( Blanc and Richard , 2017; Yang and Bedford , 2013 ) .", "Among the important Type I PRMTs is CARM1 ( PRMT4 ) , which regulates multiple aspects of transcription by methylating diverse targets including RNAPII , SRC3 , C/EBPβ , PAX3/7 , SOX2/9 , RUNX1 , Notch1 , p300 , CBP , p/CIP , Med12 , and BAF155 ( Blanc and Richard , 2017; Hein et al . , 2015; Vu et al . , 2013; Wang et al . , 2015; Wang et al . , 2014a; Yang and Bedford , 2013 ) .", "The physiological function of CARM1 has been linked to the differentiation and maturation of embryonic stem cells to form immune cells , adipocytes , chondrocytes , myocytes , and lung tissues ( Blanc and Richard , 2017; Yang and Bedford , 2013 ) .", "The requirement of CARM1 is implicated in multiple cancers , with its methyltransferase activity particularly addicted by hematopoietic malignancies and metastatic breast cancer ( Drew et al . , 2017; Greenblatt et al . , 2018; Nakayama et al . , 2018; Wang et al . , 2014a ) .", "Our prior efforts using in vivo mouse and in vitro cell models uncovered the role of CARM1 in promoting breast cancer metastasis ( Wang et al . , 2014a ) .", "Mechanistically , CARM1 methylates Arg1064 of BAF155 and thus facilitates the recruitment of the BAF155-containing SWI/SNF complex to a specific subset of gene loci that are essential for breast cancer metastasis .", "CARM1 thus emerges as a novel anti-cancer target ( Wang et al . , 2014a ) .", "Although this cancer relevance inspired the development of CARM1 inhibitors ( Kaniskan et al . , 2018; Scheer et al . , 2019 ) , many small-molecule CARM1 inhibitors lack target selectivity or cellular activity ( Kaniskan et al . , 2018 ) , two essential criteria of chemical probes ( Frye , 2010 ) .", "To the best of our knowledge , EZM2302 ( Drew et al . , 2017; Greenblatt et al . , 2018 ) , TP-064 ( Nakayama et al . , 2018 ) and SKI-73 ( 6a in this work , www . thesgc . org/chemical-probes/SKI-73 ) , which were developed by Epizyme , Takeda/SGC ( Structural Genomic Consortium ) , and our team , respectively , are the only selective and cell-active CARM1 chemical probes .", "EZM2302 and TP-064 were developed from conventional small-molecule scaffolds occupying the substrate-binding pocket of CARM1 ( Drew et al . , 2017; Greenblatt et al . , 2018; Nakayama et al . , 2018 ) .", "The potential utility of EZM2302 and TP-064 is implicated by their selective anti-proliferative effects on hematopoietic cancer cells , in particular multiple myeloma cells ( Drew et al . , 2017; Greenblatt et al . , 2018; Nakayama et al . , 2018 ) .", "However , definitive molecular mechanisms of the CARM1 addiction in these contexts remain elusive ( Greenblatt et al . , 2018 ) .", "Here , we report the characterization and novel utility of SKI-73 , a chemical probe of CARM1 with pro-drug properties .", "SKI-73 ( 6a in this work ) can readily penetrate cell membranes and then be processed into two active CARM1 inhibitors that contain 6′−homosinefungin ( HSF ) as their core scaffold ( Scheer et al . , 2019; Wu et al . , 2016 ) .", "Notably , the two inhibitors can accumulate inside cells to remarkably high concentrations and for a prolonged period .", "The potency , selectivity , modes of action , on-target engagement , and off-target effects of these compounds were characterized with multiple orthogonal assays in vitro and under cellular settings .", "The pharmacological inhibition of CARM1 by SKI-73 ( 6a ) recapitulates the anti-invasion effect of the genetic perturbation of CARM1 .", "In the context of cellular heterogeneity , we developed a cell-cycle-aware algorithm for single-cell RNA-seq ( scRNA-seq ) analysis and dissected the invasion-prone subset of breast cancer cells that is sensitive to SKI-73 ( 6a ) treatment .", "Our scRNA-seq analysis provides the unprecedented insight that pharmacological inhibition of CARM1 alters epigenetic plasticity and suppresses invasion by suppressing the most invasive subpopulation of breast cancer cells ." ], [ "Upon developing cofactor-competitive PMT inhibitors ( Wu et al . , 2016; Zheng et al . , 2012 ) , we tailored the SAM analog sinefungin ( Figure 1a ) around its 6′-amino moiety to potentially engage CARM1’s substrate-binding pocket .", "6′-homosinefungin ( HSF , i . e . , 1 ) , a sinefungin analog with the insertion of 6′−methylene moiety , was discovered for its general high affinity to Type I PRMTs ( Figure 1a , b , Figure 1—figure supplement 1 , Supplementary file 1-Table A ) .", "As a SAM mimic , 1 binds to the Type I PRMTs ( namely PRMT1 , CARM1 , PRMT6 and PRMT8 ) with IC50 of 13‒300 nM ( Figure 1a , c , Supplementary file 1-Table A ) .", "Its relative affinity to Type I PRMTs aligns with that of the SAM mimics SAH and SNF ( around 20-fold lower IC50 of 1 versus SAH and SNF , Figure 1a , c , Supplementary file 1-Table A ) .", "This observation argues that 1 retains the structural features of SAH and SNF to engage PRMTs and meanwhile leverages its 6′-methyleneamine group for additional interaction .", "To further explore the 6′-region of HSF , we synthesized HSF derivatives from the same precursor 3 ( Figure 1b , Figure 1—figure supplement 2 ) , by further expanding the 6′-methylene amine moiety with different substituents .", "The HSF derivative 2a ( Figure 1b ) was identified for its preferential binding to CARM1 with IC50 = 30 ± 3 nM and >10 fold selectivity over other seven human PRMTs and 26 methyltransferases of other classes ( Figure 1c , Supplementary file 1-Table A ) .", "The structural difference between 2a and 1 ( Figure 1b ) suggests that the N-benzyl substituent enables 2a to engage CARM1 through a distinct mechanism ( see results below ) .", "With 2a as a lead , we then explored its α-amino carboxylate moiety with different amides from the common precursor three and then the intermediate 4 ( Figure 1—figure supplement 2 ) , which led to the discovery of 5a .", "This engagement of CARM1 with 2a is expected to be largely maintained by 5a .", "Here , 5a shows an IC50 of 43 ± 7 nM against CARM1 and a >10-fold selectivity over the panel of 33 diverse methyltransferases ( Figure 1c , Supplementary file 1-Table A ) .", "In comparison , the negative control compounds 2b ( Bn-SNF ) ( Zheng et al . , 2012 ) and 5b ( Figure 1b , Figure 1—figure supplement 3 ) , which differ from 2a and 5a only by the 6′-methylene group , poorly inhibit CARM1 ( IC50 = 22 ± 1 µM and 1 . 91 ± 0 . 03 µM ) ( Figure 1c , Supplementary file 1-Table A ) .", "The dramatic increase of the potency of 2a and 5a in contrast to 2b and 5b supports an essential role of the 6′-methylene moiety upon binding CARM1 .", "Distinguished from the SAM mimics SAH , SNF and 1 as nonspecific PMT inhibitors , 2a and 5a were developed as potent and selective SAM analogs .", "With 2a and 5a characterized as CARM1 inhibitors , we leveraged orthogonal in vitro assays to explore their modes of interaction ( Figure 2a ) .", "To examine whether 2a and 5a are SAM- or substrate-competitive , CARM1 inhibition by 2a and 5a was assessed in the presence of various concentrations of SAM cofactor and H3 peptide substrate ( Figure 2b , c ) .", "IC50 values of 2a and 5a showed a linear positive correlation with SAM concentrations , as expected for SAM-competitive inhibitors ( Daigle et al . , 2011; Luo , 2018; Zheng et al . , 2012 ) .", "The Kd values of 2a and 5a ( Kd , 2a = 17 ± 8 nM; Kd , 5a = 9 ± 5 nM ) were extrapolated from the y-axis intercepts upon fitting the equation IC50 = [SAM]×Kd/Km , SAM+Kd ( Figure 2b ) ( Segel , 1993 ) .", "Km , SAM of 0 . 21 ± 0 . 09 µM and 0 . 28 ± 0 . 14 µM ( an averaged Km , SAM = 0 . 25 µM ) for competition with 2a and 5a , respectively , can also be derived through the ratio of the y-axis intercepts to the slopes ( Figure 2b and Materials and methods ) ( Segel , 1993 ) .", "By contrast , the presence of the H3 peptide substrate had negligible effect on the binding of 2a and 5a , indicating their substrate-noncompetitive character ( Figure 2c ) .", "The SAM analogs 2a and 5a were thus characterized as SAM-competitive , substrate-noncompetitive inhibitors of CARM1 .", "For the direct binding of 2a and 5a to CARM1 , the CARM1-binding kinetics of 2a and 5a were examined using surface plasmon resonance ( SPR ) ( Figure 2d ) .", "The SPR signal progression of 2a and 5a fits with a biphasic rather than a mono-phasic binding mode , with the lower Ki1 , 2a = 0 . 06 ± 0 . 02 µM , Ki1 , 5a = 0 . 10 ± 0 . 01 µM , and the higher Ki2 , 5b = 0 . 54 ± 0 . 07 µM , Ki2 , 2a = 0 . 4 ± 0 . 1 µM , probably because of the multi-phase binding kinetics of 2a and 5a ( Figure 2d ) .", "To cross validate the binding of 2a and 5a to CARM1 , we conducted an in vitro thermal shift assay , for which ligand binding is expected to increase CARM1’s thermal stability ( Blum et al . , 2014 ) .", "The binding of 2a and 5a ( at 5 μM concentration ) increased the melting temperature ( Tm ) of CARM1 by 4 . 4°C and 6 . 5°C , respectively ( Figure 2e , Tm , 2a = 44 . 2 ± 0 . 4 °C and Tm , 5a = 46 . 3 ± 0 . 3 °C versus Tm , DMSO = 39 . 8 ± 0 . 3 °C as control ) .", "By contrast , the binding of SAM and 1 show much reduced effects on Tm of CARM1 ( Figure 2e , Tm , SAM = 40 . 1 ± 0 . 3 °C and Tm , 1 = 42 . 8 ± 0 . 4 °C versus Tm , DMSO = 39 . 8 ± 0 . 3 °C ) .", "Therefore , although the affinities of 1 , 2a and 5a to CARM1 are comparable ( IC50 = 13‒43 nM , Figure 1c ) , their well-separated effects on Tm suggest that these inhibitors engage CARM1 differentially ( see results below ) .", "The two orthogonal biochemical assays thus verified the tight binding of 2a and 5a with CARM1 .", "To further seek a structural rationale for 5a and 2a for CARM1 inhibition , we solved the X-ray structure of CARM1 in complex with 5a with resolution of 2 . 00 Å and modeled the CARM1 binding of 2a ( Figure 3 , Materials and methods ) .", "The overall topology of the CARM1–5a complex is indistinguishable with the V-shaped subunit of the CARM1 dimer in complex with SNF and 1 ( details in the next section ) , which is typical of the Rossmann fold of Class I methyltransferases ( Figure 3a , b , Table", "1 ) ( Luo , 2018 ) .", "However , 5a adopts a noncanonical pose with its 6′-N-benzyl moiety in a binding pocket that used to be occupied by the α-amino carboxylate moiety of canonical ligands such as SAH , SNF and 1 ( Figures 3c and 4 ) , while the α-amino methoxyphenethyl amide moiety of 5a protrudes into the substrate-binding pocket ( Boriack-Sjodin et al . , 2016; Sack et al . , 2011 ) .", "This noncanonical mode is consistent with the SAM-competitive character of 5a ( Figure 2b ) .", "In contrast to the noncanonical mode , Arg168 in the CARM1–5a complex adopts an alternative orientation ( two possible configurations ) , accompanied by an altered conformation of Glu257 , to accommodate the 6′-N-benzyl moiety of 5a ( Figure 3d ) .", "The α-amino amide moiety of 5a also engages CARM1 through the combined outcomes of a hydrogen-bond network and hydrophobic interactions with nearby resides ( Figure 3e ) .", "Interestingly , the overlaid structures of CARM1 in complex with 5a and a substrate peptide implicate a steric clash and thus a potential for binding competition between 5a and a CARM1 substrate ( Figure 3f ) .", "However , the apparent substrate-noncompetitive character of 5a ( Figure 2c ) suggests that this steric clash might be avoided if there is no significant energy penalty when the substrate Arg adopts alternative conformation ( s ) .", "The binding mode of the CARM1–2a complex was modeled via molecular docking followed by molecular dynamics ( MD ) simulation ( Materials and methods ) .", "Here , we uncovered two distinct poses of 2a ( Binding Pose 1/2 or BP1/2 , Figure 3g , Figure 3—figure supplement 1 ) .", "BP1 was characterized by the direct interaction between the α-amino carboxylate moiety of 2a and the guanidinium of Arg168 , whereas BP2 features a titled orientation of Arg168 to accommodate the 6′-N-benzyl moiety of 2a ( Figure 3g , Figure 3—figure supplement 1 ) .", "The BP1 and BP2 of 2a closely resemble those of 1 and 5a , respectively , in terms of the orientations of Arg168 and the α-amino carboxylate moiety of the ligands .", "When the same modeling protocol was applied to the CARM1–SNF complex , only the canonical pose was identified ( Materials and methods ) .", "Energy calculation indicated that both BP1 and BP2 are stable with comparable binding free energies .", "Interestingly , the side chain configurations of His414 in both BP1 and BP2 are different from those in the CARM1–5a complex and the CARM1–SNF complex ( Figure 3g ) .", "Collectively , 5a and 2a , though structurally related to the SAM analogs 1 and SNF , engage CARM1 via distinct modes of interaction .", "Upon comparing the CARM1 structure in complex with 5a and 2a , we observed the additional hydrogen-bond and hydrophobic interactions of 5a that involve its α-amino amide moiety ( Figure 3e ) .", "Interestingly , these interactions do not increase but rather decrease the affinity of 5a to CARM1 by two-fold ( Kd , 2a = 17 ± 8 nM versus Kd , 5a = 9 ± 5 nM , Figure 2b ) .", "By contrast , there is a significant 10-fold increase of affinity between 2b and 5b ( Figure 1c , Supplementary file 1-Table A ) .", "These observations suggest that , although 5b facilitates CARM1’s engagement better than 2b via the former’s α-amino amide moiety , such an effect is dispensed with in the presence of the 6′-methylene ( N-benzyl ) amine moiety of 5a and 2a .", "Given the tight CARM1 binding by 1 , we solved the X-ray structure of CARM1 in complex with 1 ( HSF ) with a resolution of 2 . 00 Å ( Figure 4 ) .", "The overall folding of the CARM1–1 complex ( PDB: 4IKP ) is similar to those of 1 in complex with SNF and SAH ( PDB: 2Y1X , 2Y1W ) with a V-shape subunit in a dimer of dimers ( Figure 4a , Table", "2 ) ( Sack et al . , 2011 ) .", "However , the CARM1–1 complex is distinct for multiple configurations of its ligand and interactions via its 6′-methyleneamine moiety ( Figure 4b–d , Figure 4—figure supplement 1 ) .", "In the CARM1–1 complex , 1 can adopt four alternative configurations ( Configuration I in Chains B , D; Configuration II in Chain C; Configuration III and IV in Chain A ) accompanied with the structural accommodation of the adjacent residues and water hydrogen bonds ( Figure 4b–d , Supplementary file 1-Table B–D ) .", "By contrast , only a single configuration of the ligands was observed in SAH- or SNF-bound CARM1 ( Figure 4c , d , PDB: 2Y1X , 2Y1W ) ( Sack et al . , 2011 ) .", "Detailed structural comparison of CARM1 in complex with SNF , SAH and 1 further revealed that 1 maintains common interactions observed in the CARM1–SNF and CARM1–SAH complexes with several exceptions ( Figure 4c , d , Supplementary file 1-Table B–D ) .", "Most noticeably , the CARM1–1 complex gains the strong hydrogen bonds via the 6′-methyleneamine moiety of the ligand with", "( i ) the backbone carbonyl of CARM1’s Glu258 ( Configuration I , II in Chains B , C , D and Configuration III in Chain A ) or", "( ii ) the side chain of Tyr154 ( Configuration IV in Chain A ) , together with several less conserved water hydrogen bonds ( Figure 4c , d , Supplementary file 1-Table B–D ) .", "By contrast , the 6′-amine of SNF in the CARM1–SNF complex forms weaker hydrogen bonds with Glu258 and may fewer water hydrogen bonds ( Figure 4c , Supplementary file 1-Table B , C , PDB: 2Y1W ) .", "Comparable interactions are completely absent from the CARM1–SAH complex ( Figure 4d , Supplementary file 1-Table B–D , PDB: 2Y1X ) .", "The desired hydrogen-bond networks of the 6′-methyleneamine moiety of 1 with CARM1 , which are present in the CARM1-1 complex but absent from the CARM1–SNF and CARM1–SAH complexes , can rationalize the significant decrease of IC50 from SNF and SAH to 1 .", "Another key difference among CARM1–1 , CARM1–SNF and CARM1–SAH complexes lies in the region around the carboxylate moiety of these ligands .", "In Chains A and C of the CARM1–1 complex , the carboxylate moiety of the ligand forms an ionic bond with Arg169 and a hydrogen bond with Gln160 ( Figure 4b , c , d , Supplementary file 1-Table B , C ) .", "Such interactions are absent from CARM1–SNF and CARM1–SAH complexes ( Figure 4d ) .", "By contrast , in Chains B and D of the CARM1–1 complex , the same carboxylate moiety forms the ionic bonds with Arg169 and a water hydrogen bond ( Figure 4b , c , d , Supplementary file 1-Table B , C ) .", "To accommodate the latter conformation , the Gln160 residue flips toward the 3′-ribosyl hydroxyl moiety of 1 to form a new hydrogen bond ( Figure 4b , c , Supplementary file 1-Table B , C ) .", "Similar interaction patterns can also be found in the CARM1–SNF and CARM1–SAH complexes ( Figure 4 , Supplementary file 1-Table B–D ) .", "With regards to the rest of the CARM1–ligand interactions , the CARM1 complexes with 1 , SAH and SNF are nearly identical except for slightly altered water hydrogen bonds ( Figure 4 , Supplementary file 1-Table S2–S4 ) .", "Here , the α-amino moiety of these ligands forms hydrogen bonds with the carbonyl backbone of Gly193 , as well as two water hydrogen bonds; their 2′ , 3′-ribosyl hydroxyl groups form two hydrogen bonds with the side chain of CARM1’s Glu215; adenine’s N1 and N6 form the hydrogen bonds with Asn243 and Glu244/Ser272 , respectively; and the adenine ring of these ligands is buried within a hydrophobic pocket .", "By contrast , there are fewer conserved water hydrogen bonds , such as those involved with the carboxylate and 3′-ribosyl hydroxyl moieties of 1 in Chain A of the CARM1–1 complex ( Figure 4 , Supplementary file 1-Table C ) .", "By contrast , adenine-N7 in the CARM1–SNF and CARM1–SAH complexes forms water hydrogen bonds bridged to Ser272 , which are absent from the CARM1–1 complex ( Figure 4 , Supplementary file 1-Table C , D ) .", "Collectively , the general high affinity of 1 , SNF and SAH ( Figure 4 , Supplementary file 1-Table B–D ) arises from the combined hydrophilic and hydrophobic interactions of these ligands with CARM1 .", "However , in comparison with SNF and SAH , 1 gains the extra interactions via its 6′-methyleneamine moiety ( Figure 4c , d , Supplementary file 1-Table B–D ) .", "In addition , 1 adopts the canonical pose with its α-amino carboxylate moiety interacting with Arg168 , which is similar to that of SNF and SAH but different from the noncanonical pose of 2a and 5a , upon binding CARM1 ( Figure 4d ) .", "Although the in vitro characterization demonstrated the potency and selectivity of 2a and 5a against CARM1 , we anticipated their poor membrane permeability as observed for structurally related analogs such as SAH and SNF ( Figure 1a ) ( Boriack-Sjodin et al . , 2016; Sack et al . , 2011 ) .", "The lack of membrane penetration is probably due to their primary amine moiety , which has pKa of ~10 and is fully protonated at a physiological pH of 7 . 4 .", "Given the essential roles of the 9′−amine moiety of 2a and 5a in CARM1 binding ( Figure 3e ) , we envisioned overcoming the membrane permeability issue through a pro-drug strategy by cloaking this amine moiety with a redox-triggered trimethyl-locked quinone butanoate moiety ( TML , Figure 5a ) ( Levine and Raines , 2012 ) .", "We thus prepared 6a and its control compound 6b by derivatizing 5a and 5b with the TML moiety ( Figure 1b , Figure 1—figure supplements 1 and 2 ) .", "To assess the cellular activity of 6a , we relied on our prior knowledge that CARM1 methylates the Arg1064 of BAF155 , a core component of the SWI/SNF chromatin-remodeling complex , and CARM1 knockout abolishes this posttranslational modification in MCF-7 cells ( Wang et al . , 2014a ) .", "Treatment of MCF-7 cells with 10 µM of 6a fully suppressed this methylation mark , whereas treatment with 2a and 5a did not affect this mark ( Figure 5b ) .", "We thus demonstrated the prodrug-like cellular activity of 6a .", "To further evaluate 6a as a chemical probe against CARM1 , we quantified the efficiency by which 6a engages CARM1 in a cellular context and thus suppresses the CARM1-dependent invasion by breast cancer cells .", "Because of the pro-drug character of 6a and its control compound 6b , we first developed quantitative LC-MS/MS methods to examine their cellular fates for CARM1 engagement ( see Materials and methods ) .", "Upon treatment of MDA-MB-231 cells with 6a , we observed its time- and dose-dependent intracellular accumulation ( Figure 5c , Figure 5—figure supplements 1 and 2 ) .", "We anticipated the conversion of the pro-drug 6a into 5a , but a striking finding is that 6a can also be readily processed into 2a inside cells ( Figure 5c , Figure 5—figure supplements 1 and 2 ) .", "Remarkably , >100 μM of 2a can be accumulated inside cells for 2 days after 6 hr treatment with a single dose of 5‒10 µM 6a .", "This observation probably reflects a slow efflux and thus effective intracellular retention of 2a due to its polar α-amino acid zwitterion moiety .", "Given that cellular CARM1 inhibition is involved with multiple species ( 2a , 5a and 6a ) in competition with SAM , we modeled the ligand occupancy of cellular CARM1 on the basis of their Kd values ( Kd , 2a=17 nM , Kd , 5a=9 nM , Kd , 6a=0 . 28 µM and Kd , SAM≈Km , SAM=0 . 25 µM ) and MS-quantified intracellular concentrations ( Figure 5d , Figure 5—figure supplement 3 , Equations 5–7 , see Materials and methods ) .", "The SAM cofactor , whose intracellular concentration was determined to be 89 ± 16 µM ( Figure 5c , d ) , is expected to occupy >99 . 5% CARM1 with residual <0 . 5% as the apo-enzyme under a native setting .", "With single doses of 6a of 2 . 5‒10 µM , the combined CARM1 occupancy by 2a , 5a and their pro-drug precursor 6a rapidly reached the plateau of >95% within 6 hr , and was maintained at this level for at least 48 hr ( Figure 5e , Figure 5—figure supplements 1 and 2 ) .", "Notably , treatment with 6a concentrations as low as 0 . 5 µM is sufficient to reach 60% target engagement within 10 hr and to maintain this occupancy for 48 hr ( Figure 5e , Figure 5—figure supplements 1 and 2 ) .", "The time- and dose-dependent progression of the CARM1 occupancy by these ligands thus provides quantitative guidance upon the treatment of MDA-MB-231 cells with 6a .", "Given that 2a is the predominant metabolic product of 6a within cells ( Figure 5c , e ) and also shows certain affinity to SMYD2 ( ~10 fold higher IC50 in comparison with CARM1 , Figure 1c , Supplementary file 1-Table A ) , we evaluated SMYD2 engagement of 2a for its potential off-target effect .", "In a similar manner to that described for the ligand occupancy of cellular CARM1 , we modeled the occupancy of cellular SMYD2 by 2a on the basis of Kd , 2a=150 nM and Kd , SAM=60 nM for SMYD2 ( Figure 5e , Figure 5—figure supplement 4 , Materials and methods ) .", "Largely because of the high affinity of 2a to SAM and thus a 37-fold larger Kd , 2a/Kd , SAM ratio of SMYD2 relative to CARM1 ( Kd , 2a/Kd , SAM of 2 . 5 and 0 . 068 for SMYD2 and CARM1 , respectively ) , the occupancy of SMYD2 by 2a is below 20% ( Figure 5e , Figure 5—figure supplement 4 ) under the efficacy doses of 6a ( see cellular data for 6a below ) .", "We were thus less concerned about the SMYD2-associated off-target effect under our assay conditions .", "The metabolic stability of 6a was also evaluated with a microsomal stability assay ( Figure 5c , e , Figure 5—figure supplement 5 , see Materials and methods ) .", "In the presence of rat liver microsomes , 6a showed decent stability with 24% residual 6a after one-hour incubation .", "Here , the conversion of 6a into 5a accounted for 40% of the microsome-processed 6a; no production of 2a was detected .", "Such observation suggests that NQO1 , the putative enzyme candidate to reduce the TML moiety in 6a or 6b , is present in microsomes as well as in tumor cells ( Dias et al . , 2018; Huang et al . , 2016 ) .", "By contrast , peptidase enzymes that are expected to process 5a into 2a are absent from microsomes but rich in tumor cells .", "We then conducted a cellular thermal shift assay ( CETSA ) , in which ligand binding is expected to increase CARM1’s thermal stability in a cellular context ( Jafari et al . , 2014 ) .", "Our data showed that the treatment of MDA-MB-231 cells with 6a but with not the control compound 6b increased cellular Tm and thus the thermal stability of CARM1 by 4 . 3 ± 0 . 6°C ( Figure 5f ) .", "The distinct effect of 6a in contrast to 6b on the cellular Tm of CARM1 aligns well with the 4 . 1‒6 . 2°C difference in the in vitro Tm of CARM1 upon binding 2a and 5a versus SAM ( Figure 2e ) .", "Here , 6b can penetrate cell membranes and be processed into 5b and 2b in a similar manner as 6a ( Figure 5—figure supplement 6 ) .", "These observations thus present the cellular evidence to show that CARM1 engages 2a and 5a .", "To further characterize 6a as a CARM1 chemical probe , we examined the Arg1064 methylation of BAF155 and the Arg455/Arg460 methylation of PABP1 , two well-characterized cellular methylation marks of CARM1 , upon treating MDA-MB-231 cells with 6a ( Lee and Bedford , 2002; Wang et al . , 2014a ) .", "These methylation marks can be fully suppressed by 6a in a dose-dependent manner ( Figure 6a ) .", "The resultant EC50 values of 0 . 45‒0 . 75 µM ( Figure 6b ) are well correlated with the modeled 60% cellular occupancy of CARM1 upon treatment with 0 . 5 µM 6a for 48 hr ( Figure 5e ) .", "By contrast , the treatment of the negative control compound 6b showed no effect on these methylation marks ( Figure 6a ) .", "We therefore demonstrated the robust use of 6a ( SKI-73 ) as a CARM1 chemical probe and of 6b ( SKI-73N ) as its control compound .", "After demonstrating the utility of SKI-73 ( 6a ) as a chemical probe for CARM1 , we examined whether chemical inhibition of CARM1 can recapitulate biological outcomes that are associated with CARM1 knockout ( CARM1-KO ) ( Wang et al . , 2014a ) .", "Our prior work showed that CARM1’s methyltransferase activity is required for invasion of MDA-MB-231 cells ( Wang et al . , 2014a ) .", "We thus conducted a matrigel invasion assay with MDA-MB-231 cells in the presence of 6a .", "Relative to the control treatment with DMSO , treatment with SKI-73 ( 6a ) but not its negative control compound SKI-73N ( 6b ) suppressed the invasion of MDA-MB-231 cells ( EC50 = 1 . 3 µM ) ( Figure 6c , d ) .", "The treatment with ≥10 µM 6a produced the maximal 80% suppression of the invasion by MDA-MB-231 relative to the DMSO control , which is comparable with the phenotype of CARM1-KO ( Figure 6e ) .", "Critically , no further inhibition by 6a on the invasiveness was observed upon 6a treatment ( in comparison with the treatment with DMSO or 6b treatment ) of MDA-MB-231 CARM1-KO cells ( Figure 6e ) .", "Notably , treatment with 6a and 6b under the current condition has no apparent impact on the proliferation of parental or CARM1-KO MDA-MB-231 cells ( Figure 6—figure supplement 1 ) , consistent with the intact proliferation upon treatment with other CARM1 chemical probes ( Drew et al . , 2017; Greenblatt et al . , 2018; Nakayama et al . , 2018 ) .", "These results suggest that SKI-73 ( 6a ) and CARM1 knockout perturb the common , proliferation-independent biological process and then suppresses 80% of the invasiveness of MDA-MB-231 cells .", "We thus characterized SKI-73 ( 6a ) as a chemical probe that can be used to interrogate the CARM1-dependent invasion of breast cancer cells .", "Because of the advancement of scRNA-seq technology , stunning subpopulation heterogeneity has been uncovered even for well-defined cellular types ( Tanay and Regev , 2017 ) .", "In the context of tumor metastasis , including its initial invasion step , epigenetic plasticity is required to allow a small subset of tumor cells to adapt distinct transcriptional cues for neo-properties ( Chatterjee et al . , 2018; Flavahan et al . , 2017; Wu et al . , 2019 ) .", "To explore the feasibility of dissecting the CARM1-dependent , invasion-prone subset of MDA-MB-231 breast cancer cells , we formulated a cell-cycle-aware algorithm of scRNA-seq analysis and dissected those subpopulations that were sensitive to CARM1 perturbation ( Figure 7a , see Materials and methods ) .", "Here we conducted 10 × Genomics droplet-based scRNA-seq of 3232 , 3583 and 4099 individual cells ( a total of 10 , 914 cells ) exposed to 48 hr treatment with SKI-73 ( 6a ) , SKI-73N ( 6b ) and DMSO , respectively .", "Guided by Silhouette analysis , cell-cycle-associated transcripts were identified as dominant signatures of subpopulations ( Figure 7—figure supplements 1–18 ) .", "These signatures naturally exist for proliferative cells and are not expected to be specific for the invasive phenotype .", "To dissect the subpopulation-associated transcriptomic signatures of invasive cells , we included one additional layer for hierarchical clustering by first classifying the individual cells into G0/G1 , S , and G2/M stages ( 6885 , 1520 and 2509 cells , respectively ) ( Figure 7—figure supplement 6 , Supplementary file 1-Table E ) , and then conducted the unsupervised clustering within each cell-cycle-aware subset ( Figure 7b , Figure 7—figure supplements 19–30 , Supplementary file 1-Table E ) .", "To resolve the subpopulations without redundant clustering , we developed an entropy analysis method and relied on the Fisher Exact test ( see Materials and methods ) .", "The optimal scores of the combined methods were implemented to determine the numbers of cluster for each subset ( Figure 7b , Figure 7—figure supplements 20 , 24 and 28 ) ( Butler et al . , 2018 ) .", "The cell-cycle-aware algorithm allowed the clustering of these cells according to the three cell cycle stages under the three treatment conditions and resulted in 21 , 7 and 6 subpopulations in G0/G1 , S , and G2/M phases , respectively ( Figure 7b , Figure 7—figure supplements 21 , 25 and 29 , Supplementary file 1-Tables F–H ) .", "Notably , the 48 hr treatments with SKI-73 ( 6a ) or SKI-73N ( 6b ) had no effect on the cell cycle , as indicated by their comparable cell-cycle distribution patterns ( Figure 7—figure supplement 6; Supplementary file 1-Table E ) , a finding that is consistent with intact proliferation after all three treatments ( Figure 6—figure supplement 1 ) .", "With the 21 , 7 and 6 subpopulations clustered into the G0/G1 , S , and G2/M stages , respectively , we then conducted population analysis , comparing SKI-73 ( 6a ) and SKI-73N ( 6b ) against DMSO ( Figure 7c , Figure 7—figure supplements 31 and 32 , Supplementary file 1-Table F–H ) .", "These subpopulations can be readily classified into five distinct categories according to their cell-cycle-aware responses to SKI-73 ( 6a ) and SKI-73N ( 6b ) treatment: commonly resistant/emerging/depleted versus differentially depleted/emerging ( SKI-73/SKI-73N- or 6a/6b-specific ) ( Figure 7c , Figure 7—figure supplements 31 and 32 , Supplementary file 1-Table F–H ) .", "Here , we are particularly interested in the SKI-73 ( 6a ) -specific depleted subpopulations ( Subpopulation 0/2/8/11/13/14/17/19 of G0/G1-phase cells and 3 of S-phase cells ) as the potential invasion-associated subpopulations , given their sensitivity to SKI-73 ( 6a ) but not its control compound SKI-73N ( 6b ) .", "The subpopulations that remain unchanged after the treatment with SKI-73 ( 6a ) or SKI-73N ( 6b ) ( Subpopulation 1/7/12/15 of G0/G1-phase cells; 2/4/5 of S-phase cells; 1 of G2/M-phase cells ) were defined as the common resistant subset .", "SKI-73 ( 6a ) -specific emerging subpopulations ( Subpopulation 3/4/5/6/16 of G0/G1-phase cells; 6 of S-phase cells; 4 of G2/M-phase cells ) are expected to be suppressed by CARM1 but emerge upon its inhibition .", "The remaining subpopulations are either associated with the effects of the small-molecule scaffold of SKI-73 ( 6a ) /SKI-73N ( 6b ) ( commonly emerging Subpopulation 9 of G0/G1-phase cells , 0/5 of G2/M-phase cells; commonly depleted Subpopulation 2/3 of G2/M-phase cells ) or with SKI-73N ( 6b ) -specific effects ( differentially depleted Subpopulation 10/18 of G0/G1-phase cells , 0 of S-phase cells; differentially emerging Subpopulation 20 of G0/G1-phase cells ) .", "Interestingly , scRNA-seq analysis of CARM1-KO cells ( in comparison with SKI-73 ( 6a ) -treated cells ) suggests that CARM1 knockout has more profound effects on the overall landscape of epigenetic plasticity ( Figure 7d , Figure 7—figure supplement 33 ) .", "Collectively , the chemical probe SKI-73 ( 6a ) alters the epigenetic plasticity of MDA-MB-231 breast cancer cells via the combined effects of SKI-73 ( 6a ) ’s molecular scaffold and specific inhibition of CARM1’s methyltransferase activity .", "Given that SKI-73 ( 6a ) has no effect on the cell cycle and proliferation of MDA-MB-231 cells under the current treatment dose and duration , we envision that the invasion capability of MDA-MB-231 cells mainly arises from an invasion-prone subset , 80% of which is depleted by SKI-73 ( 6a ) treatment ( Figure 6c–e ) .", "We thus focused on Subpopulations 0/2/8/11/13/14/17/19 of G0/G1-phase cells and Subpopulation 3 of S-phase cells: in total nine depleted subpopulations specific for SKI-73 ( 6a ) ( Figure 7c , Figure 7—figure supplements 31 and 32 , Supplementary file 1-Table F–H ) .", "To identify invasion-prone subpopulation ( s ) among these candidates , we compared the transcriptional signature ( s ) of these subpopulations with those of cells that freshly invaded through Matrigel within 16 hr .", "Strikingly , in comparison with the highly heterogenous scRNA-seq signature of the parental MDA-MB-231 cells , the freshly harvested invasive cells ( 3793 cells for scRNA-seq ) are relatively homogeneous , with their subpopulations mainly determined by the cell-cycle-related transcriptomic signatures ( Figure 7d , Figure 7—figure supplements 33–37 ) .", "We then classified the freshly harvested invasive cells into G0/G1 , S and G2/M stages ( Figure 7—figure supplements 38–40 , Supplementary file 1-Table E ) .", "Through correlation analysis comparing the invasive cells and the subpopulations within each cell-cycle stage ( Figure 7d , Figure 7—figure supplements 39–42 ) , we revealed the subsets whose transcriptional signatures closely relate to those of the invasive cells , including Subpopulations 6/7/8/9/14 in G0/G1-phase cells , 0/3 in S-phase cells and 1/2 of G2/M-phase cells ( Supplementary file 1-Table F–K ) .", "In the context of population analysis for the nine SKI-73 ( 6a ) -specific depleted subpopulations , Subpopulations 8/14 of G0/G1-phase cells and Subpopulation 3 in S-phase are putative invasion-prone candidates .", "Subpopulation 8 of G0/G1-phase cells is the most sensitive and the only subpopulation that can be depleted by around 80% with SKI-73 ( 6a ) treatment ( Figure 7c ) .", "Given the ~80% suppression and ~20% residual invasion capability upon SKI-73 ( 6a ) treatment , we argue that the invasive phenotype of MDA-MB-231 cells predominantly arises from Subpopulation 8 G0/G1-phase cells , which only accounts for ~8% of the parental cells in G0/G1 phase ( ~5% without cell-cycle awareness ) .", "Differential expression analysis further revealed the single-cell transcriptional signatures of metastasis-implicated genes ( e . g . MORC4 , S100A2 , RPL39 , IFI27 , ARF6 , CHD11 , SDPR and KRT18 ) that are specific for the G0/G1-phase Subpopulation 8 and invasive cells but not for other G0/G1-phase invasion-prone candidates such as Subpopulation 6/7/9/14 ( Figure 7e , Figure 7—figure supplement 43 , Supplementary file 1-Table L ) .", "The remaining cells of G0/G1-phase Subpopulation 8 after SKI-73 ( 6a ) treatment ( Figure 7c , d ) together with others ( subpopulation-6/7/9/14 in G0/G1-phase cells , 0/3 in S-phase cells and 1/2 of G2/M-phase cells , Figure 7—figure supplements 31 , 32 , 41 , 42 ) may account for the 20% residual invasion capacity .", "In the context of SKI-73 ( 6a ) -specific depletion of G0/G1-phase subpopulations , there are SKI-73 ( 6a ) -specific emerging G0/G1-phase subpopulations: Subpopulation 3/4/5/6/16 ( Figure 7c ) .", "Population analysis of G0/G1-phase cells further revealed that Subpopulations 4 and 16 account for 90% of the emerging subset upon SKI-73 ( 6a ) treatment ( Supplementary file 1-Table F ) .", "The transcriptional signatures and probably the associated invasion capability of Subpopulations 4 and 16 are dramatically different from those of the freshly harvested invasive cells and the bulk population of the parental cells , including the invasion-prone Subpopulation 8 ( Figure 7b , d ) .", "Collectively , either CARM1 knockout or CARM1 inhibition with SKI-73 ( 6a ) alters the epigenetic plasticity in a proliferation-independent manner by replacing the most invasion-prone subpopulation with the non-invasive subpopulation ( s ) to suppress the invasive phenotype ( Figure 7f ) ." ], [ "On the basis of a novel small-molecule scaffold , 6′-homosinefungin ( HSF ) , SKI-73 ( 6a ) was developed as a pro-drug-like chemical probe for CARM1 by cloaking the 9′-amine moiety of 5a with the TML moiety .", "SKI-73N ( 6b ) was developed as a control compound for SKI-73 ( 6a ) .", "The inhibitory activity of SKI-73 ( 6a ) against CARM1 was demonstrated by the ability of SKI-73 ( 6a ) but not SKI-73N ( 6b ) to abolish the cellular methylation marks of CARM1: the Arg1064 methylation of BAF155 and the Arg455/Arg460 methylation of PABP1 ( Lee and Bedford , 2002; Wang et al . , 2014a ) .", "The ready intracellular cleavage of TML is expected for the conversion of SKI-73 and SKI-73N ( 6a and 6b ) into 5a and 5b , respectively , but it is remarkable that SKI-73 and SKI-73N ( 6a and 6b ) can also be efficiently processed into 2a and 2b inside cells .", "In comparison , 6a showed decent metabolic stability with no production of 2a in the presence of microsomes .", "Here , 2a and 5a are presented as potent and selective CARM1 inhibitors , whereas their control compounds 2b and 5b interact poorly with CARM1 .", "Competitive assays with the SAM cofactor and the peptide substrate showed that 2a and 5a act on CARM1 in a SAM-competitive and substrate-noncompetitive manner .", "The SAM-competitive mode is consistent with the ligand–complex structures of CARM1 , in which the SAM binding site is occupied by 2a and 5a .", "Strikingly , as revealed by their ligand–CARM1 complex structures , 2a and 5a engage CARM1 through noncanonical modes , with their 6′-N-benzyl moieties in the binding pocket that is otherwise occupied by the α-amino carboxylate moiety of conventional SAM analogs such as SAH , SNF and 1 .", "This observation is consistent with the 4 . 1‒6 . 5 °C increase in the in vitro and cellular Tm of CARM1 upon binding 2a and 5a , which contrasts with the smaller Tm changes with SAM as a ligand .", "The distinct modes of interaction of CARM1 with 2a and 5a ( Figure 3c , g ) also rationalize the CARM1 selectivity of the two SAM analogs over other methyltransferases , including closely related PRMT homologs .", "Through mathematic modeling using the inputs of the LC-MS/MS-quantified intracellular concentrations and CARM1-binding constants of relevant HSF derivatives and the SAM cofactor , we concluded that high intracellular concentrations of 5a and 2a , and thus efficient CARM1 occupancy , can be achieved rapidly and maintained for several days with a single low dose of SKI-73 ( 6a ) .", "By contrast , the occupancy by 5a and 2a of SMYD2 , the next likely engaged target , is below 20% with the efficacious doses of 6a that affect cell invasion .", "The polar α-amino acid zwitterion moiety of 2a and the polar α-amino moiety of 5a probably account for their accumulation and retention inside cells .", "To the best of our knowledge , EZM2302 , TP-064 , and SKI-73 ( also 6a in this work , www . thesgc . org/chemical-probes/SKI-73 ) and their derivatives are the only selective and cell-active CARM1 inhibitors ( Drew et al . , 2017; Nakayama et al . , 2018 ) .", "Although the potency , selectivity , on-target engagement and potential off-target effects associated with these compounds have been examined in vitro and in cellular contexts as chemical probes , EZM2302 , TP-064 , and SKI-73 ( 6a ) are differentiated by their molecular scaffolds and modes of interaction with CARM1 ( www . thesgc . org/chemical-probes/SKI-73 ) ( Drew et al . , 2017; Nakayama et al . , 2018 ) .", "SKI-73 ( 6a ) is a cofactor analog inhibitor embedding a N6'-homosinefungin moiety to engage the SAM binding site of CARM1 in a cofactor-competitive , substrate-noncompetitive manner; EZM2302 and TP-064 occupy the substrate-binding pocket of CARM1 in a SAH-uncompetitive or SAM-noncompetitive manner ( Drew et al . , 2017; Nakayama et al . , 2018 ) .", "In particular , the prodrug property of SKI-73 ( 6a ) allows its ready cellular uptake , followed by rapid conversion into its active forms inside cells .", "The prolonged intracellular CARM1 inhibition further distinguishes SKI-73 ( 6a ) from EZM2302 and TP-064 .", "With SKI-73 ( 6a ) as a CARM1 chemical probe and SKI-73N ( 6b ) as a control compound , we showed that pharmacological inhibition of CARM1 with SKI-73 ( 6a ) , but not SKI-73N ( 6b ) , suppressed 80% of the invasion capability of MDA-MB-231 cells .", "By contrast , the pharmacological inhibition of CARM1 with SKI-73 ( 6a ) had no effect on the proliferation of MDA-MB-231 cells .", "This result is consistent with the lack of anti-proliferation activities of the other two CARM1 chemical probes , EZM2302 and TP-064 , against breast cancer cell lines ( Drew et al . , 2017; Nakayama et al . , 2018 ) .", "The anti-invasion efficiency of SKI-73 ( 6a ) is in good agreement with the intracellular occupancy and the resulting abolition of several methylation marks of CARM1 upon treatment with SKI-73 ( 6a ) .", "Our prior work showed that the methyltransferase activity of CARM1 is required for breast cancer metastasis ( Wang et al . , 2014a ) .", "Among the diverse cellular substrates of CARM1 ( Blanc and Richard , 2017 ) , BAF155—a key component of the SWI/SNF chromatin-remodeling complex–is essential for the invasion of MDA-MB-231 cells ( Wang et al . , 2014a ) .", "Mechanistically , the CARM1-mediated Arg1064 methylation of BAF155 facilitates the recruitment of the SWI/SNF chromatin-remodeling complex to a specific subset of gene loci ( Wang et al . , 2014a ) .", "Replacement of the native CARM1 with its catalytically dead mutant or with an Arg-to-Lys point mutation at the Arg1064 methylation site of BAF155 is sufficient to abolish the invasive capability of breast cancer cells ( Wang et al . , 2014a ) .", "CARM1 inhibition with SKI-73 ( 6a ) , but not with its control compound SKI-73N ( 6b ) , recapitulates the anti-invasion phenotype associated with the genetic perturbation of CARM1 .", "More importantly , there is no additive effect upon combining CARM1-KO with SKI-73 ( 6a ) treatment , underlying the fact that the two orthogonal approaches target the commonly shared pathway ( s ) that are essential for the invasion of breast cancer cells .", "In comparison to SKI-73 ( 6a ) , the CARM1 inhibitors EZM2302 and TP-064 demonstrated anti-proliferation effects on hematopoietic cancer cells , in particular multiple myeloma ( Drew et al . , 2017; Greenblatt et al . , 2018; Nakayama et al . , 2018 ) .", "Mechanistically , genetic perturbation of CARM1 in the context of leukemia impairs cell-cycle progression , promotes myeloid differentiation , and ultimately induces apoptosis , probably by targeting pathways of proliferation and cell-cycle progression , that is , E2F- , MYC- , and mTOR-regulated processes ( Greenblatt et al . , 2018 ) .", "In comparison , CARM1 inhibition with EZM2302 led to a slightly different phenotype , which includes reduction of RNA stability , E2F target downregulation , and induction of a p53 response signature for senescence .", "( Greenblatt et al . , 2018 ) .", "Collectively , the effects of CARM1 chemical probes are highly context-dependent , with SKI-73 ( 6a ) having different uses in impairing the invasiveness of breast cancer cells , while TP-064 and EZM2302 have uses in preventing the proliferation of hematopoietic cancer cells .", "Given the increased awareness of epigenetic plasticity ( Flavahan et al . , 2017 ) , we employed the scRNA-seq approach to examine MDA-MB-231 cells and their responses to chemical and genetic perturbation with CARM1 .", "Because of the lack of a prior reference to define subpopulations of MDA-MB-231 cells , we developed a cell-cycle-aware algorithm to cluster the subpopulations with a resolution that was able to dissect subtle changes upon treatment with SKI-73 ( 6a ) versus its control compound SKI-73N ( 6b ) in each cell-cycle stage .", "Guided by Silhouette analysis , the population entropy analysis and the Fisher Exact test , >10 , 000 MDA-MB-231 breast cancer cells were classified on the basis of their cell-cycle stages and then clustered into 34 subpopulations .", "With further annotation of these subpopulations according to their different responses to treatment with SKI-73 ( 6a ) versus SKI-73N ( 6b ) , we readily dissected the subpopulations that were altered in a SKI-73 ( 6a ) -specific ( CARM1-dependent ) manner and then identified subsets with transcriptional signatures that are similar to that of the freshly isolated invasive cells .", "Quantitative analysis of SKI-73 ( 6a ) -depleted subpopulations further revealed the most invasion-prone subpopulation , which accounts for only 5% of the total population but at least 80% of the invasive capability of the parental cells .", "Collectively , we propose a model in which MDA-MB-231 cells consist of subpopulations , with their epigenetic plasticity ( Figure 7f ) determined by multiple factors including the CARM1-involved BAF155 methylation ( Wang et al . , 2014a ) .", "SKI-73 ( 6a ) inhibits the methyltransferase activity of CARM1 , the Arg1064 methylation of BAF155 , and thus the target genes associated with the methylated BAF155 .", "These effects alter the cellular epigenetic landscape by affecting certain subpopulations of MDA-MB-231 cells without any apparent effect on cell cycle and proliferation .", "In the context of the invasion phenotype of MDA-MB-231 cells , the subset of invasion-prone cells is significantly suppressed upon the treatment with SKI-73 ( 6a ) .", "Essential components that are used to dissect the invasion-prone population in this CARM1-dependent epigenetic plasticity model are the scRNA-seq analysis of sufficient MDA-MB-231 cells ( >10 , 000 cells here ) , the utility of the freshly isolated invasive cells as the reference , the timing and duration of treatment , and the use of SKI-73N ( 6b ) and DMSO as controls .", "Interestingly , although the invasion-prone subpopulation is also abolished in the CARM1-KO strain , CARM1-KO reshapes the epigenetic plasticity in a much more profound manner , significantly reducing the subpopulation heterogeneity of MDA-MB-231 cells .", "The distinct outcomes for the pharmacological and genetic perturbation could be due to their different modes of action: short-term treatment with SKI-73 ( 6a ) versus long-term clonal expansion of CARM1-KO cells .", "The pharmacological inhibition captures the immediate response , whereas the genetic perturbation reports long-term and potential resistant outcomes .", "This work thus presents a new paradigm to understand cancer metastasis in the context of epigenetic plasticity and provides guidance for similar analyses in broader contexts: other cell lines , patient-derived xenograft samples , and in vivo mouse models of breast cancer ." ], [ "SAM- and substrate-dependent IC50 values were determined with the radiometric filter paper assay as described above except in the presence of the varied concentrations of the SAM cofactor and the peptide substrate .", "The IC50 values of 2a and 5a were obtained in the presence of 0 . 09 , 0 . 18 , 0 . 37 , 0 . 75 , 1 . 50 , 2 . 25 , 3 . 00 , 5 . 62 , and 7 . 50 µM [3H-Me]-SAM or 0 . 3 , 1 . 5 , 7 . 5 , 15 , 30 , and 50 µM H3 peptide ( aa 1–40 ) .", "The resultant IC50 values were plotted as a function of the concentrations of SAM and the peptide substrate using GraphPad Prism Software .", "Given the SAM-competitive character of 2a and 5a , their SAM-dependent IC50 values were then fitted according to Equation", "1 . Here [SAM] is the concentration of SAM , Km , SAM is the Michaelis-Menten constant , and Kd is the dissociation constant of the CARM1 inhibitor 2a or 5a .", "Kd , Kd/Km , SAM and Km , SAM can therefore be obtained through the intercept of the y-axis , the slope , and their ratio , respectively , upon fitting Equation", "1 . Given that 6a showed a much higher IC50 ( 1 . 1 ± 0 . 1 µM , Figure 5—figure supplement 3 ) , the Kd value of 6a ( Kd , 6a = 0 . 32 µM ) was directly obtained from the IC50 value , the Km , SAM derived above , and the assayed SAM concentration according to Equation 1 . ( 1 ) IC50=[SAM]×kd/km , SAM+kd Full-length CARM1 was used for SPR assay .", "DNA fragment encoding the full-length CARM1 was cloned into pFB-N-flag-LIC donor plasmid .", "The resulting plasmid was transformed into DH10Bac-competent E . coli cells ( Invitrogen ) and a recombinant Bacmid DNA was purified , followed by a recombinant baculovirus generation in Sf9 insect cells .", "Sf9 cells grown in HyQ SFX insect serum-free medium ( ThermoScientific ) were infected with 10 mL of P3 viral stocks per 1 L of suspension cell culture and incubated at 27°C using a platform shaker set at 150 revolutions per minute .", "The cells were collected when viability dropped to 70–80% ( ~72 hr after infection ) .", "Harvested cells were re-suspended in PBS , 1X protease inhibitor cocktail ( 100X protease inhibitor stock in 70% ethanol containing 0 . 25 mg/mL aprotinin , 0 . 25 mg/ml leupeptin , 0 . 25 mg/mL pepstatin A and 0 . 25 mg/mL E-64 ) and 2X Roche complete EDTA-free protease inhibitor cocktail tablet .", "The cells were lysed chemically by rotating 30 min with NP40 ( final concentration of 0 . 6% ) and 50 U/mL benzonase nuclease ( Sigma ) , 2 mM 2-mercaptoethanol and 10% glycerol followed by sonication at frequency of 7 ( 10′‘on/10′‘off ) for 2 min ( Sonicator 3000 , Misoni ) .", "The crude extract was clarified by high-speed centrifugation ( 60 min at 36 , 000 × g at 4°C ) by Beckman Coulter centrifuge .", "The recombinant protein was purified by incubating the cleared lysate with anti-FLAG M2 affinity agarose gel ( Sigma , Cat # A2220 ) and then rotating for 3 hr , followed by washing with 10 CV TBS ( 50 mM Tris-HCl , 150 mM NaCl , pH 7 . 4 ) containing 2 mM 2-mercaptoethanol , 1X protease inhibitor cocktail ( 100X protease inhibitor stock in 70% ethanol containing 0 . 25 mg/mL aprotinin , 0 . 25 mg/mL leupeptin , 0 . 25 mg/mL pepstatin A and 0 . 25 mg/mL E-64 ) and 1X Roche complete EDTA-free protease inhibitor cocktail tablet .", "The recombinant protein was eluted by competitive elution with a solution containing 100 µg/mL FLAG peptide ( Sigma , Catalog # F4799 ) in 20 mM Tris pH:7 . 4 , 150 mM NaCl , 5% glycerol , 3 mM 2-mercaptoethanol .", "Quality of CARM1 ( >95% ) was determined by SDS–PAGE .", "The protein was then concentrated , flash frozen with liquid nitrogen , and stored at −80°C for future use .", "SPR analysis was performed using a Biacore T200 ( GE Health Sciences Inc ) at 25°C .", "Approximately 5500 response units of CARM1 ( amino acids 1–608 ) were amino-coupled onto a CM5 chip in one flow cell according to the protocol of the manufacturer .", "Another flow cell was left empty for reference subtraction .", "SPR analysis was conducted in the HBS-EP buffer ( 20 mM HEPES pH 7 . 4 , 150 mM NaCl , 3 mM EDTA , 0 . 05% Tween-20 ) containing 2% ( v/v ) DMSO .", "The stock solutions of five concentrations of compound 2a ( 24 . 7 , 74 . 1 , 222 , 667 and 2000 nM ) and four concentrations of 5a ( 6 . 2 , 18 . 5 , 55 . 6 and 167 nM ) were prepared by serial dilution .", "Binding kinetic experiments were performed with single cycle kinetics with the contact time of 60 s and off time of 300 s in a flow rate of 30 µL/min .", "To facilitate complete dissociation of compound for the next cycle , a regeneration step ( 300 s , 40 µL/min of buffer ) , a period of stabilization ( 120 s ) and two blank cycles were included between each cycle .", "Kinetic curve fittings were carried out with a 1:1 binding model or a heterogeneous ligand model using Biacore T200 Evaluation software ( GE Health Sciences Inc ) .", "TSA was performed as described previously ( Blum et al . , 2014; Niesen et al . , 2007 ) to examine the melting temperature ( Tm ) of CARM1 in the presence or absence of ligands .", "For each measurement ( triplicate of each data point ) , the assay solution containing 50 mM HEPES-HCl ( pH = 8 . 0 ) , Tween-20 0 . 005% ( v/v ) , 1 mM TCEP , 0 . 5 μM CARM1 , and 5 μM ligand ( SAM , 1 , 2a or 5a ) was mixed with 5 × SYPRO Orange Protein Gel Stain stock ( Sigma Aldrich ) in a 96-well PCR plate .", "The mixture was equilibrated at 25°C in dark for 5 min and then loaded onto Bio-Rad CFX96 Real-Time PCR Detection System .", "The fluorescence readouts were recorded upon increasing the heating temperature from 25°C to 100°C at a rate of 0 . 2 °C/s .", "The raw data of the fluorescence readouts versus the temperatures were exported with CFX software and processed as the percentage of the fluorescent signal normalized between the lowest readout of 0% and the highest readout of 100% within the 25‒100°C region .", "The melting curves were plotted as the normalized fluorescence ( % ) versus the heating temperature and fit with a sigmoid curve with GraphPad Prism .", "The Tm corresponds to the temperature with the 50% relative fluorescent signal in the sigmoidal curve .", "A DNA fragment encoding the methyltransferase domain of human CARM1 ( residues 140–480 ) was cloned into a baculovirus expression vector pFBOH-MHL ( http://www . thesgc . org/sites/default/files/toronto_vectors/pFBOH-MHL . pdf ) .", "The protein was expressed in Sf9 cells as an N-terminal 6 × His tag fusion protein and purified by metal chelating affinity chromatography ( TALON resin , Clontech , Mountain View , CA , USA ) followed by size-exclusion chromatography ( Superdex 200 , GE Healthcare ) .", "Pooled fractions containing CARM1 were subjected to the treatment of tobacco etch virus to remove the 6 × His tag .", "The protein was purified to homogeneity by ion-exchange chromatography .", "Purified CARM1 ( 6 . 5 mg/mL ) was crystallized with the sitting drop vapor diffusion method at 20°C .", "For the CARM1–1 complex , CARM1 was mixed with 1 at a 1:5 molar ratio ( protein:ligand ) and crystallized with the sitting drop vapor diffusion method at 20°C by mixing 1 µL of the protein solution with 1 µL of the reservoir solution containing 20% PEG3350 and 0 . 2 M diammonium tartrate .", "X-ray diffraction data for the CARM1–1 complex were collected at 100 K at beam line 23ID-B of Advanced Photon Source ( APS ) , Argonne National Laboratory .", "Data sets were processed using the HKL-2000 suite ( Otwinowski and Minor , 1997 ) .", "The structure of the CARM1–1 complex was solved by molecular replacement using MOLREP ( Otwinowski and Minor , 1997 ) with the PDB entry 2V74 as the search template .", "REFMAC ( Emsley and Cowtan , 2004; Murshudov et al . , 1997 ) was used for the structure refinement .", "Graphics program COOT was used for model building and visualization .", "Crystal diffraction data and refinement statistics for the structure are displayed in Table", "2 . To further confirm the electronic densities of the ligand , the total omission electron density map was calculated using SFCHECK from CCP4suite and contoured at 1 . 0 sigma ( Figure 4—figure supplement 1 ) ( Emsley and Cowtan , 2004; Murshudov et al . , 1997 ) .", "For the CARM1-5a complex , CARM1 was crystallized with the sitting drop vapor diffusion method at 20°C by mixing 1 µL of protein solution with 1 µL of the reservoir solution containing 25% PEGG3350 , 0 . 1M ammonium sulfate and 0 . 1 M Hepes ( pH = 7 . 5 ) .", "The compound 5a ( 0 . 2 μL of 10 μM in DMSO ) was added to the drops with apo crystals and incubated overnight .", "X-ray diffraction data for the CARM1-5a complex were collected at 100 K at beamline 24ID-E of Advanced Photon Source ( APS ) , Argonne National Laboratory .", "Data were processed using the HKL-3000 suite ( Minor et al . , 2006 ) .", "The structure was isomorphous to PDB entry 4IKP , which was used as a starting model .", "REFMAC ( Murshudov et al . , 1997 ) was used for structure refinement .", "Geometric restraints for compound refinement were prepared with GRADE v . 1 . 102 developed at Global Phasing Ltd . ( Cambridge , UK ) .", "The COOT graphics program ( Emsley et al . , 2010 ) was used for model building and visualization , and MOLPROBITY ( Williams et al . , 2018 ) was used for structure validation .", "To further confirm the electronic densities of the ligand , the total omission electron density map was calculated and contoured at 2 . 5 sigma ( Figure 3b ) ( Emsley and Cowtan , 2004; Murshudov et al . , 1997 ) .", "The ligands were docked into the binding site of CARM1 using the induced-fit docking ( IFD ) protocol ( Sherman et al . , 2006 ) implemented in the Schrodinger suite ( release 2016–4 ) .", "The poses for SNF and 2a were selected according to the IFD scores .", "Specifically , the results identified two distinct poses for 2a with similar scores but only one pose for SNF .", "The poses were then further relaxed by all-atom , explicit solvent molecular dynamics ( MD ) simulations .", "Herein , CARM1 models in complex with the two ligands were placed into explicit water boxes .", "A simple point charge ( SPC ) water model ( Berendsen et al . , 1981 ) was used to solvate the system , charges were neutralized , and 0 . 15 M NaCl was added .", "The total system size was ∼50 , 000 atoms .", "Desmond MD systems ( D . E . Shaw Research , New York , NY ) with OPLS3 force field ( Harder et al . , 2016 ) were used .", "The system was initially minimized and equilibrated with restraints on the ligand heavy atoms and protein backbone atoms , followed by production runs with all atoms unrestrained .", "The isothermal-isobaric ensemble was used with constant temperature ( 310 K ) maintained with Langevin dynamics and 1 atm constant pressure achieved using the hybrid Nose-Hoover Langevin piston method ( Feller et al . , 1995 ) on a flexible periodic cell .", "For each CARM1–ligand complex , a 600 ns trajectory was collected .", "MCF-7 and MDA-MB-231 ( parental and CARM1-KO ) cell lines ( Wang et al . , 2014a ) were used after their quality was confirmed with STR profile and standard mycoplasma contamination testing .", "Using the standard working curves generated above , the concentration ( CA ) of each analyte ( 6a , 5a , 2a , and SAM ) in the MeOH extraction of cell lysates was obtained through the ratio ( PA/PIS ) of the mass peak areas of each analyte ( PA ) versus each internal standard ( PIS ) , and the concentration of the internal standard ( CIS ) according to Equation", "2 . For 6a , 5a , and 2a under each assay condition , three similar CA values ( CA-6b , CA-5b , and CA-2b ) were obtained with 6b , 5b and 2b as the internal standards , respectively .", "An average concentration of the analyte ( C¯A ) was obtained on the basis of the three concentrations weighted by the mass peak areas of the three internal standards ( PIS , 6b , PIS , 5b and PIS , 2b ) according to Equation 3: ( 3 ) C¯A=CA−6b×PIS−6bPIS−6b+PIS−5b+PIS−2b+CA−5b×PIS−5bPIS−6b+PIS−5b+PIS−2b+CA−2b×PIS−2bPIS−6b+PIS−5b+PIS−2b On the basis of the C¯A values of 6a , 5a , and 2a ( C¯A , 6a , C¯A , 5a and C¯A , 2a ) in the MeOH extraction of cell lysates , the intracellular concentrations of the analyte 6a , 5a , and 2a ( Cintra , 6a Cintra , 5a and Cintra , 2a ) were calculated according to Equation 4 .", "Here C¯A , analyte is the weighted average of the three concentrations in the MeOH extraction , N is the cell number , and V is the mean volume of cells ( µL ) .", "The mean volume of MDA-MB-231 cells is 1 . 3 × 10−6 µL/cell as reported previously ( Coulter et al . , 2012 ) .", "The cell number ( N ) was determined with a hemocytometer .", "The concentration of SAM ( CA , SAM ) in the MeOH extraction was obtained according to Equation 2 , in which ‘X’ is the ratio ( PA , SAM/PIS , CD3-SAM ) of the mass peak areas of SAM ( PA , SAM ) versus the internal standard CD3-SAM ( PIS , CD3-SAM ) and ‘Y’ is the ratio ( CA , SAM/CIS , CD3-SAM ) of the concentrations of SAM versus CD3-SAM in the MeOH extraction .", "Given the identical LC-MS properties of SAM and CD3-SAM , CA , SAM was obtained solely on the basis of the working curve with CD3-SAM as the internal standard .", "The intracellular concentration of SAM ( Cintra , SAM ) was then calculated using Equation 4 , in which N is the cell number and V is the mean volume of MDA-MB-231 cells ( 1 . 3 × 10−6 µL/cell ) .", "( 4 ) Cinter , analyte=C¯A , analyte×40μLN×V Similar experimental procedures were carried out to quantify the intracellular concentrations of 6b , 5b , 2b and SAM except that 6b , 5b , and 2b were the analytes and their structurally related 6a , 5a and 2a were used as the internal standards with their CIS values of 0 . 125 µM , 0 . 125 µM , and 0 . 0125 µM , respectively .", "For each analyte ( 6b , 5b , and 2b ) , three working curves ( Figure 5c , e , Figure 5—figure supplement 2 ) were generated to cover the concentration range of 3 . 9 nM‒18 . 0 µM of the analyte ( CA , 6b , CA , 5b and CA , 2b ) with 6a , 5a and 2a ( CIS-6a , CIS-5a , and CIS-2a ) as the LC-MS/MS internal standards , respectively .", "In a manner similar to that described above for the ligand occupancy of CARM1 , the SMYD2 occupancy by 2a was modeled with Kd , SAM = 60 nM , Kd , 2a = 150 nM and Equation 6 .", "Here , the Kd , SAM value was reported previously upon developing the activity assay of SMYD2 ( Sweis et al . , 2015 ) .", "The Kd , 2a value was obtained according to IC50 = [SAM]×Kd/Kd , SAM+Kd , in which [SAM]=Kd , SAM = 60 nM for the IC50 assay and IC50 = 0 . 3 µM was obtained ( Supplementary file 1-Table A ) .", "The terms of SMYD2 occupancy by exogenous ligands were ignored given their low concentrations and low affinity relative to that of 2a ( Supplementary file 1-Table A ) .", "Potential in vivo clearance of 6a was evaluated in the presence of liver microsomes as previously reported ( Hansen et al . , 2012 ) .", "Briefly , 6a was dissolved into 50 mM potassium phosphate buffer ( pH 7 . 4 ) to a final concentration of 100 μM .", "To a 0 . 5 mL Eppendorf vial containing ice-cold potassium phosphate buffer ( pH 7 . 4 ) , we added 5 μL of the 100 μM stock solution of 6a , 3 μL of a freshly prepared NADPH solution ( 5 mM , Sigma-Aldrich , N7785 ) , and 1 . 33 μL of freshly thawed rat liver microsomes ( Sigma-Aldrich , M9066 ) to yield assay samples with a final volume of 50 μL .", "The resulting mixture was immediately vortexed and incubated in a 37°C shaker .", "At the time interval of 0 , 20 , 40 , and 60 min in triplicates , respective assay samples were quenched by adding 50 μL of ice-cold methanol , vigorously vortexed , and centrifuged with 5 , 000 g at 4°C for 20 min .", "From the methanol-quenched sample , 50 μL of supernatant was collected and mixed with 50 μL of 0 . 1% ( v/v ) TFA/MeOH solution containing 0 . 125 µM 6b , 0 . 125 µM 5b , and 16 µM 2b as the internal standards .", "The resulting mixture was transferred into a 96-well plate ( Agilent , 5042–1386 ) and stored at −20°C for LC-MS/MS analysis .", "The microsomal stability of 6a was evaluated via LC-MS/MS quantification of the residual 6a and of two potential microsome-processed products , 5a and 2a , upon quantification of intracellular concentrations of 6a , 5a and 2a ( as described above ) .", "The amount of 6a was plotted as the percentage of the residual concentration at each time point against its initial concentration at 0 min with GraphPad Prism Software .", "Potential production of 5a and 2a from 6a was examined in parallel .", "Interestingly , despite robust accumulation of 5a , no 2a was identified with the detection threshold of 0 . 02 µM .", "For negative controls , 3 μL of 50 mM potassium phosphate buffer ( pH 7 . 4 ) replaced the freshly prepared NADPH solution; two assay samples at the time interval of 0 and 60 min were collected .", "Under the current microsomal condition , the concentration of 6a remained the same ( 10 ± 0 . 7 μM ) and there was no production of 5a or 2a .", "MDA-MB-231 cells and MCF-7 parental and CARM1-KO ( Wang et al . , 2014a ) cells were maintained in DMEM ( Gibco , Gaithersburg , MD ) medium containing 10% FBS ( Gibco ) .", "These cells were then treated with compounds or with DMSO for 48 hr .", "The resultant cells were washed twice in phosphate-buffered saline ( PBS ) and the samples were sonicated in ice-cold RIPA buffer ( Thermo , Waltham , MA ) .", "The lysates were centrifuged ( 15 , 000 g ) at 4°C for 15 min .", "The supernatants were kept with the total protein amount determined with Bradford protein assay ( BioRad , Hercules , CA ) .", "After quantification , 50 μg of protein from each sample was loaded onto 6% SDS-PAGE and transferred onto a nitrocellulose membrane ( PALL , Port Washington , NY ) .", "For the Arg1064 methylation mark of BAF155 , the blots were blocked in 5% non-fat milk for 1 hr and incubated with anti-me-BAF155 ( Cancer Cell , 2014 ) , anti-BAF155 ( 1:1000; Santa Cruz Biotechnology , Dallas , TX ) , anti-CARM1 ( 1:1000; Genemed Synthesis , San Antonio , TX ) , and anti-β-actin ( 1:20000; Sigma-Aldrich , St . Louis , MO ) overnight at 4°C .", "After washing three times in Tris-buffered saline with Tween 20 ( TBST ) , the blots were incubated with HRP-conjugated secondary antibody ( 1:3000; Jackson ImmunoResearch , West Grove , PA ) .", "After washing blots with TBST , the membranes were developed using SuperSignal West Pico ECL solution ( Thermo ) .", "For the Arg455/Arg460 methylation mark of PABP1 , anti-me-PABP1 ( 1:1000 ) and anti-PABP1 ( 1:1000 ) antibodies ( Genemed Synthesis , San Antonio , TX ) were used instead .", "Here the antibodies against CARM1 , PABP1 , and me-PABP1 were custom generated by Genemed Synthesis ( San Antonio , TX ) ( Zeng et al . , 2013 ) .", "The density of the protein bands was quantified using ImageJ software ( NIH , Bethesda , MD ) .", "The EC50 values were obtained by fitting the methylation percentage ( % ) of BAF155 or PABP1 against the concentrations of the inhibitor using a sigmoidal equation with GraphPad Prism Software .", "CETSA was performed as described previously ( Jafari et al . , 2014 ) to examine the intracellular engagement of 6a or 6b with CARM1 .", "Briefly , 2 . 0 × 106 MDA-MB-231 cells were incubated with 15 μM 6a or 6b and DMSO for 48 hr .", "The harvested cells were re-suspended in PBS buffer and divided into eight aliquots ( 30 µL/aliquot ) , and then heat-shocked at various temperatures ( 49 . 1 , 54 . 6 , 57 . 0 , 59 . 5 , 61 . 8 , 63 . 9 , 65 . 6 , and 67 . 0°C ) for 3 min with a Bio-Rad CFX96 Real-Time PCR Detection Instrument ( using a temperature gradient of 49‒67°C ) .", "The heat-shocked cells were then lysed with the freeze-thaw method with a liquid nitrogen bath followed by a 25°C water bath for five cycles .", "The cell lysate was centrifuged at 4°C at 18 , 000 g for 20 min .", "The resultant supernatant containing the soluble protein fraction was collected and loaded onto an SDS-PAGE gel ( 20 µL ) .", "Western blotting of CARM1 was performed with anti-CARM1 antibody ( Cell Signaling Technology , C31G9 ) .", "For each sample , the band intensity of CARM1 was quantified with ImageJ .", "The band intensity of CARM1 was normalized to the band intensity at 49 . 1°C ( the lowest heat-shock temperature ) .", "Melting curves were obtained by plotting the normalized band intensity against the heat-shock temperatures and fit with a Boltzmann sigmoidal equation in GraphPad Prism .", "The melting temperatures ( Tm ) of 6a , 6b , or DMSO were obtained as the heat-shock temperatures that correspond to the 50% normalized band intensity in the fitted sigmoidal curves .", "MDA-MB-231 parental and CARM1-KO cells ( Wang et al . , 2014a ) were maintained in DMEM ( Gibco , Gaithersburg , MD ) medium containing 10% FBS ( Gibco ) .", "Cell invasion assays were performed using 8 . 0 µm pore size Transwell inserts ( Greiner Bio-One , Kremsmünster , Austria ) .", "MDA-MB-231 parental and CARM1-KO ( Cancer Cell , 2014 ) cells were harvested with trypsin/EDTA and washed twice with serum-free DMEM ( Gibco ) .", "2 × 105 cells in 0 . 2 mL serum-free DMEM ( Gibco ) were seeded onto the upper chamber , which was pre-coated with a thin layer of 40 uL of 2 mg/mL Matrigel ( Corning , NY , USA ) for 2 hr incubation at 37°C .", "To the lower chamber , we added 0 . 6 mL DMEM containing 10% FBS ( GIBCO ) and compounds or DMSO .", "After 16 hr in the 37°C incubator , the cells on the inner side of the upper chamber together with the Matrigel layer were removed using cotton tips .", "The residual invasive cells in the outer side of the upper chamber were fixed in 3 . 7% formaldehyde ( a weight percentage ) at an ambient temperature ( 22°C ) for 2 min and 100% methanol for 20 min , and then stained for 15 min with a solution containing 1% crystal violet and 2% ethanol in 100 mM borate buffer ( pH 9 . 0 ) .", "The number of invasive cells was counted under a microscope by taking five independent fields .", "Relative cell invasion was determined by the number of the invasive cells normalized to the total number of cells adhering to 0 . 8 µm transwell filters .", "The EC50 was obtained with GraphPad Prism Software upon fitting Equation 8 , in which ‘%Inhibition’ is the percentage of the inhibition of invasiveness , ‘Maximal Inhibition%’ is the percentage of the maximal inhibition of invasiveness , and [Inhibitor] is the concentration of the inhibitor .", "( 8 ) Inhibition%=MaximalInhibition%×[Inhibitor][Inhibitor]+EC50 To examine proliferation of MDA-MB-231 cells , 5000 cells of parental and CARM1-KO cells were seeded in a 96-well plate and incubated in 37°C overnight .", "These cells were treated with various doses ( 0 . 0001‒10 μM ) of SKI-73 or SKI-73N ( 6a or 6b ) in DMSO and incubated for 72 hr .", "An MTT assay was then performed to examine viability with DMSO-treated cells as the control .", "The relative viability of compound-treated cells versus DMSO-treated parent cells were plotted against the concentrations of SKI-73 and SKI-73N ( 6a and 6b ) .", "Cell preparation for scRNA-seq .", "10 × Genomics droplet-based scRNA-seq was implemented to characterize five types of MDA-MB-231 cells: MDA-MB-231 cells treated with DMSO , SKI-73 ( 6a ) and SKI-73N ( 6b ) , MDA-MB-231 cells that freshly invaded through Matrigel , and CARM1-KO MDA-MB-231 cells .", "For the first three samples , MDA-MB-231 cells were treated with DMSO , 10 μM SKI-73 ( 6a ) , and 10 μM SKI-73N ( 6b ) for 48 hr .", "The resulting cells were trypsinized at 37°C for 3 min .", "The harvested cells were washed twice with 1 × PBS ( phosphate-buffered saline ) containing 0 . 04% ( volume% ) bovine serum albumin ( BSA ) , gently dispersed to dissociate cells , and then filtered through a cell strainer ( 100 μm Nylon mesh , Fisherbrand ) to obtain single-cell suspensions for scRNA-seq .", "For the CARM1-KO sample , the cells were trypsinized at 37°C for 3 min , washed twice with 1 × PBS containing 0 . 04% BSA , and filtered through a 100 μm Nylon-mesh cell strainer ( Fisherbrand ) to obtain single-cell suspensions for scRNA-seq .", "To collect the cells that freshly invaded through Matrigel , the conditions described above for cell invasion assays were applied to allow approximately 5% of the 1 × 107 seeded MDA-MB-231 cells to invade through Matrigel .", "Briefly , ten Transwell inserts with a diameter of 24 mm and a pore size of 8 . 0 μm were pre-coated with a thin layer of 54 μL of 2 mg/mL Matrigel ( Corning ) .", "1 × 106 MDA-MB-231 cells in 1 . 0 mL serum-free DMEM ( Gibco ) were seeded into the upper chamber of each Matrigel-coated Transwell insert .", "2 . 0 mL DMEM containing 10% FBS ( Gibco ) was added into the lower chambers .", "After 16 hr incubation , the cells on the inner side of the upper chamber together with the Matrigel layer were removed using cotton tips .", "The cells that freshly invaded–those attached on the outer side of the upper chamber–were subjected to 3 min trypsin digestion at 37°C for detachment .", "The resulting cells were washed twice with 1 × PBS containing 0 . 04% BSA , gently dispersed to dissociate cells , and filtered through a 100 μm Nylon-mesh cell strainer ( Fisherbrand ) to obtain single-cell suspensions for scRNA-seq .", "The scRNA-seq libraries were prepared following the user guide manual ( CG00052 Rev E ) provided by the 10 × Genomics and Chromium Single Cell 3' Reagent Kit ( v2 ) .", "Briefly , samples containing approximately 8700 cells ( 93–97% viability ) were encapsulated in microfluidic droplets at a dilution of 66–70 cells/µL , which resulted in 4369–5457 recovered single-cells per sample with a multiplet rate ~3 . 9% .", "The resultant emulsion droplets were then broken and barcoded cDNA was purified with DynaBeads , followed by 12-cycles of PCR-amplification: −98 °C for 180 s , 12× ( 98°C for 15 s , 67°C for 20 s , 72°C for 60 s ) , and 72°C for 60 s .", "The 50 ng of PCR-amplified barcoded-cDNA was fragmented with the reagents provided in the kit and purified by SPRI beads with an averaged fragment size of 600 bp .", "The DNA library was then ligated to the sequencing adapter followed by indexing PCR: −98 °C for 45 s; 12× ( 98°C for 20 s , 54°C for 30 s , 72°C for 20 s ) , and 72°C for 60 s .", "The resulting DNA library was double-size purified ( 0 . 6–0 . 8× ) with SPRI beads and sequenced on Illumina NovaSeq platform ( R1 – 26 cycles , i7 – eight cycles , R2 – 96 cycles ) resulting in 70–79 million reads per sample with average reads per single-cell being 8075–10 , 342 and average reads per transcript 1 . 11–1 . 15 .", "The fastq files containing the transcriptome and barcoding metadata were demultiplexed using the SEquence Quality Control ( SEQC ) pipeline ( http:github . com/ambrosejcarr/seqc . git ) resulting in around 8000 UMIs per one cell .", "The table of UMI counts was used as the input and Seurat package v . 2 . 3 . 4 ( Butler et al . , 2018 ) was applied for scRNA-seq analysis .", "Here , the raw UMI counts were normalized per cell by dividing the total number of UMIs in each individual cell , multiplying by a scale factor of 10 , 000 and transforming into natural logarithm values .", "Cells with 1000~5000 genes and <20% mitochondrial RNA transcripts were kept for further analysis .", "Dimensionality reduction was carried out by selecting a set of highly variable genes on the basis of the average expression and dispersion per gene .", "The set of genes was used for principle component analysis ( PCA ) .", "Top principle components were then chosen for cell clustering analysis and t-SNE projection .", "Regression was performed to remove cell–cell variation in gene expression driven by the UMI number , mitochondrial gene content and ribosomal gene content using ‘ScaleData’ function in Seurat package ( Nestorowa et al . , 2016 ) .", "Clusters of cells were identified by clustering algorithm based on a shared nearest neighbor ( SNN ) modularity optimization that was included in Seurat package v . 2 . 3 . 4 ( Butler et al . , 2018 ) .", "Fisher’s Exact Test was implemented to evaluate the agreement of the clusters with the three cell origins ( DMSO- , SKI-73 ( 6a ) - or SKI-73N ( 6b ) -treated cells ) using R package ‘fisher . test’: http://mathworld . wolfram . com/FishersExactTest . html ( Mehta and Patel , 1983 ) .", "Because of the significant computation cost of Fisher’s Exact Test , the cell population of each Fisher’s Exact Test was down-sampled to 150 cells and this process was repeated for 100 times to cover a majority of the cell population .", "The p-value of Fisher’s Exact Test was then computed by Monte Carlo simulation .", "Means and standard errors of p-values were calculated and reported as the outputs of Fisher’s Exact Test .", "Three algorithmic scoring systems were used over a range of the resolution parameter that sets the corresponding ‘granularity’ of clustering , with higher values indicating a greater number of clusters .", "Here , silhouette analysis was applied to determine the number of clusters in an unsupervised manner , without awareness of the three cell origins ( DMSO- , SKI-73 ( 6a ) - or SKI-73N ( 6b ) -treated cells ) .", "The entropy scoring and Fisher’s Exact Test were implemented to evaluate the biological meaning of the clustering , using the minimal cluster number to resolve the cells between the three treatment conditions maximally .", "Given the awareness of the three cell origins , the minimal number of clusters with the maximal resolution of cell origin guided by the entropy scoring and Fisher’s Exact Test is expected within the 1~3-fold range of the optimized number of clusters guided by Silhouette analysis .", "Fisher’s Exact Test was used as the primary scoring method to determine the efficiency of clustering , given its higher resolution .", "‘dj , i’ ( ‘dDMSO , i’ , ‘dSKI-73N , i’ and ‘dSKI-73 , i’ in Equation 9 are defined as the fraction of the cells with the ‘j’ origin ( j = 1 , 2 or three for the treatment with DMSO , SKI-73N/6b and SKI-73/6a , respectively ) within the ‘i’ subpopulation ( i = 0‒n for the clustered subpopulation ) .", "‘di , total’ is defined as the fraction of the cells of the ‘i’ subpopulation within the total cell population in each cell-cycle stage .", "‘d ( j , i ) , total =djj , i ×di , total’ represents the fraction of the cells with the ‘j’ origin ( DMSO , SKI-73/6a , and SKI-73N/6b ) and the ‘i’ subpopulation within the total cell population in each cell-cycle stage .", "For a specific subpopulation ‘i’ , there are three ‘d ( j , i ) , total’ values ( d ( DMSO , i ) , total , d ( SKI-73N , i ) , total and d ( SKI-73 , i ) , total ) for the treatments with DMSO , SKI-73N ( 6b ) and SKI-73 ( 6a ) , respectively .", "The alteration of subpopulations is defined as the ratios of d ( SKI-73N , i ) , total/d ( DMSO , i ) , total or d ( SKI-73 , i ) , total/d ( DMSO , i ) , total fall out of the range of 1 . 0 ± 0 . 2 .", "Population analysis was conducted by classifying the SKI-73N/SKI-73 ( 6a/6b ) -treated subpopulations into the following five categories: commonly resistant ( 0 . 8 < ‘d ( SKI-73N , i ) , total/d ( DMSO , i ) , total’ and ‘d ( SKI-73 , i ) , total/d ( DMSO , i ) , total’<1 . 2 ) , commonly emerging ( ‘d ( SKI-73N , i ) , total/d ( DMSO , i ) , total’ and ‘d ( SKI-73 , i ) , total/d ( DMSO , i ) , total’≥1 . 2 ) , commonly depleted ( ‘d ( SKI-73N , i ) , total/d ( DMSO , i ) , total’ and ‘d ( SKI-73 , i ) , total/d ( DMSO , i ) , total’≤0 . 8; ∣‘d ( SKI-73N , i ) , total/d ( DMSO , i ) , total’−‘d ( SKI-73 , i ) , total/d ( DMSO , i ) , total’∣<0 . 15 ) , differentially emerging ( either ‘d ( SKI-73N , i ) , total/d ( DMSO , i ) , total’ or ‘d ( SKI-73 , i ) , total/d ( DMSO , i ) , total’>1 . 2 ) , and differentially depleted ( ‘d ( SKI-73N , i ) , total/d ( DMSO , i ) , total’ or ‘d ( SKI-73 , i ) , total/d ( DMSO , i ) , total’<0 . 8; ∣‘d ( SKI-73N , i ) , total/d ( DMSO , i ) , total’−‘d ( SKI-73 , i ) , total/d ( DMSO , i ) , total’∣>0 . 15 ) .", "Although additional combinations of differentially altered subpopulations could be possible , only those defined above were found under our treatment conditions .", "In each cell cycle ( G0/G1 , S and G2/M ) of the cells treated with DMSO , SKI-73 ( 6a ) or SKI-73N ( 6b ) and ‘invasion cells’ , correlation analysis of subpopulations was conducted with ‘BuildClusterTree’ function in the Seurat package ( https://rdrr . io/cran/Seurat/man/BuildClusterTree . html ) ( Nestorowa et al . , 2016 ) .", "The phylogenetic trees were constructed by averaging gene expressions across all cells in each subpopulation and then calculating distance on the basis of expressions averaged between different subpopulations .", "Differentially expressed genes were identified by comparing two groups of cells using the Wilcox rank sumtest with the ‘FindMarkers’ function in the Seurat package ( Nestorowa et al . , 2016 ) .", "In particular , the ‘invasion cells’ and their most correlated clusters ( which were revealed in the correlation analysis ) were selected as the ‘high’ group; the remaining remotely related clusters were selected as the ‘low’ group .", "The differential expression analysis was then performed by comparing cells in the ‘high’ group with the cells in the ‘low’ group .", "For the G0/G1-phase cells , the ‘invasion cells’ and Subpopulation 6 , 7 , 8 , 9 , 14 were selected as the ‘high’ group; Subpopulation 0 , 1 , 2 , 3 , 4 , 5 , 10 , 11 , 12 , 13 , 15 , 16 , 17 , 18 , 19 , 20 were selected as the ‘low’ group .", "For the G2/M-phase cells , the ‘invasion cells’ and Subpopulations 1 , 2 were selected as the ‘high’ group; and Subpopulations 0 , 3 , 4 , 5 were selected as the ‘low’ group .", "For the S-phase cells , the ‘invasion cells’ and Subpopulations 0 , 3 were selected as the ‘high’ group; and Subpopulations 1 , 2 , 4 , 5 , 6 were selected as the ‘low’ group .", "Differentially expressed genes were ranked according to the average log2 fold change ‘avg_logFC’ and the adjusted p-values ‘p_val_adj’ with the Seurat package .", "Top upregulated and downregulated genes were chosen by setting a cutoff on their ‘avg_logFC’ values ( >0 . 25 or <−0 . 25 ) .", "Then , curated genes with potential functional relevance to cancer malignancy ( 30 upregulated and 10 downregulated genes ) were selected as representative genes for generating heat map plots using the ‘DoHeatmap’ function in the Seurat package ." ] ]

[ "CARM1 is a cancer-relevant protein arginine methyltransferase that regulates many aspects of transcription .", "Its pharmacological inhibition is a promising anti-cancer strategy .", "Here SKI-73 ( 6a in this work ) is presented as a CARM1 chemical probe with pro-drug properties .", "SKI-73 ( 6a ) can rapidly penetrate cell membranes and then be processed into active inhibitors , which are retained intracellularly with 10-fold enrichment for several days .", "These compounds were characterized for their potency , selectivity , modes of action , and on-target engagement .", "SKI-73 ( 6a ) recapitulates the effect of CARM1 knockout against breast cancer cell invasion .", "Single-cell RNA-seq analysis revealed that the SKI-73 ( 6a ) -associated reduction of invasiveness acts by altering epigenetic plasticity and suppressing the invasion-prone subpopulation .", "Interestingly , SKI-73 ( 6a ) and CARM1 knockout alter the epigenetic plasticity with remarkable difference , suggesting distinct modes of action for small-molecule and genetic perturbations .", "We therefore discovered a CARM1-addiction mechanism of cancer metastasis and developed a chemical probe to target this process ." ]

[ "Drugs that are small molecules have the potential to block the individual proteins that drive the spread of cancer , but their design is a challenge .", "This is because they need to get inside the cell and find their target without binding to other proteins on the way .", "However , small molecule drugs often have an electric charge , which makes it hard for them to cross the cell membrane .", "Additionally , most proteins are not completely unique , making it harder for the drugs to find the correct target .", "CARM1 is a protein that plays a role in the spread of breast cancer cells , and scientists are currently looking for a small molecule that will inhibit its action .", "The group of enzymes that CARM1 belongs to act by taking a small chemical group , called a methyl group , from a molecule called SAM , and transferring it to proteins that switch genes on and off .", "In the case of CARM1 , this changes cell behavior by turning on genes involved in cell movement .", "Genetically modifying cells so they will not produce any CARM1 stops the spread of breast cancer cells , but developing a drug with the same effects has proved difficult .", "Existing drugs that can inhibit CARM1 in a test tube struggle to get inside cells and to distinguish between CARM1 and its related enzymes .", "Now , Cai et al . have modified and tested a CARM1 inhibitor to address these problems , and find out how these small molecules work .", "At its core , the inhibitor has a structure very similar to a SAM molecule , so it can fit into the SAM binding pocket of CARM1 and its related enzymes .", "To stop the inhibitor from binding to other proteins , Cai et al . made small changes to its structure until it only interacted with CARM1 . Then , to get the inhibitor inside breast cancer cells , Cai et al . cloaked its charged area with a chemical shield , allowing it to cross the cell membrane .", "Inside the cell , the chemical shield broke away , allowing the inhibitor to attach to CARM1 .", "Analysis of cells showed that this inhibition only affected the cancer cells most likely to spread .", "Blocking CARM1 switched off genes involved in cell movement and stopped cancer cells from travelling through 3D gels .", "This work is a step towards making a drug that can block CARM1 in cancer cells , but there is still further work to be done .", "The next stages will be to test whether the new inhibitor works in other types of cancer cells , in living animals , and in human patient samples ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "structural biology and molecular biophysics", "microbiology and infectious disease" ]

[ [ "The outer membrane ( OM ) of Gram-negative bacteria is a permeability barrier to antibiotics and other cytotoxic agents , such as detergents ( Nikaido , 2003 ) .", "A key feature of the OM is its distinctive asymmetrical bilayer populated with lipopolysaccharide ( LPS ) in the outer leaflet and phospholipids in the inner leaflet ( Funahara and Nikaido , 1980; Kamio and Nikaido , 1976 ) .", "Lateral interactions between LPS molecules mediated by divalent cations on the surface and packing of hydrocarbon chains in the membrane impede the passage of both hydrophilic and large hydrophobic molecules ( Nikaido , 2003 ) .", "LPS is composed of a conserved lipid A molecule , a core oligosaccharide , and a variable O-antigen glycan .", "Lipid A is synthesized in the cytoplasm where the core oligosaccharide is attached to form core-LPS ( Whitfield and Trent , 2014 ) .", "This molecule is flipped into the periplasmic space by the inner membrane ATPase MsbA ( Ho et al . , 2018; Mi et al . , 2017; Zhou et al . , 1998 ) .", "The O-antigen is appended onto core-LPS in the periplasm and LPS is transported to the outer leaflet of the OM by the LPS transport ( Lpt ) system ( Abeyrathne et al . , 2005; Han et al . , 2012; Okuda et al . , 2016 ) .", "In E . coli , all 7 components of the Lpt pathway , LptABCDEFG , are essential and shuttle LPS molecules from the IM across the periplasm to the OM ( Okuda et al . , 2016; Okuda et al . , 2012; Ruiz et al . , 2008; Wu et al . , 2006 ) .", "LptD is a β-barrel outer membrane protein ( OMP ) responsible for the final unidirectional insertion of LPS into the OM outer leaflet ( Botos et al . , 2016; Gu et al . , 2015; Li et al . , 2015 ) .", "The LptD β-barrel is comprised of 26 anti-parallel β-strands with a proposed lateral gate formed where the first and last β-strands interact ( Dong et al . , 2014; Qiao et al . , 2014 ) .", "Like other Gram-negative β-barrel OMPs , LptD possess long extracellular , environmentally-exposed loops and short periplasmic loops connecting adjacent strands ( Franklin and Slusky , 2018; Rollauer et al . , 2015; Schulz , 2002 ) .", "The roles of the extracellular loops ( ECLs ) in folding or activity of LptD have not been determined .", "LptD forms an obligate interaction with the OM lipoprotein LptE ( Chimalakonda et al . , 2011; Freinkman et al . , 2011 ) .", "An N-terminal jelly roll domain is located on the periplasmic side of the β-barrel below the lateral gate .", "Structural , biochemical , and genetic analyses suggest that the hydrophobic lipid A portion of LPS is embedded in the hydrophobic jelly roll domain .", "The presumed model for LPS transport across the OM posits that lipid A is inserted into the hydrophobic OM through a lateral gate of LptD ( Botos et al . , 2016; Gu et al . , 2015; Li et al . , 2015 ) .", "By an unknown mechanism , the polar sugars of the LPS core oligosaccharide and O-antigen are presumably moved through the LptD β-barrel lumen and flipped to the exterior of the cell .", "Due to its essential role in OM biogenesis , high conservation among Gram-negative bacteria , and exposure to the extracellular environment , LptD represents a potential new antibacterial target .", "Genetic experiments show that crosslinking LPS to LptD in the jelly roll domain or the lateral gate , or introduction of disulfide bonds that disrupt the proposed LPS path through LptD , inhibit growth ( Gu et al . , 2015; Li et al . , 2015 ) .", "Indeed , two antibacterial peptides targeting LptD have been described , both presumably targeting the periplasmic jelly roll domain ( Srinivas et al . , 2010; Vetterli et al . , 2018 ) .", "Critical roles for ECLs in OMP function have been described ( Browning et al . , 2013; Rigel et al . , 2013 ) .", "For example , removal of the LptD extracellular loop 4 ( L4 ) , imp4213 , is known to disrupt the OM barrier leading to sensitization to antibiotics ( Sampson et al . , 1989 ) .", "However , the roles of the extracellular LptD loops remain largely underexplored due to a lack of robust tools to systematically assess structure-function relationships in the context of a native OM .", "Monoclonal antibodies ( mAbs ) represent useful tools to probe protein structure and function because they typically have high target specificity and affinity .", "Accordingly , here we pursued four diverse strategies to produce thousands of mAbs that bound to LptD .", "The breadth and depth of mAb discovery that we report here is unusual , especially for an integral membrane protein , and also takes advantage of an efficient and novel targeted boost-and-sort α-OMP discovery approach recently reported for another OMP , BamA ( Vij et al . , 2018 ) .", "Characterization of the α-LptD mAbs discovered herein allowed us to map the environmentally-exposed surfaces of LptD and to systematically explore the structure-function relationship of the large ECLs of this essential OMP within its native OM environment .", "Through the construction and characterization of LptD loop deletion strains , we identified non-exposed loops that are critical for LptD function under defined conditions , which are all inaccessible to antibody interference .", "Our data provide unique insights into the structure and function of LptD and suggest that non-antibody approaches for interfering with the function of this essential OMP in E . coli should be prioritized to develop novel antibiotics ." ], [ "To probe the functional relevance of the ECLs of the essential E . coli OMP LptD , we set out to discover α-LptD antibodies to these surface-exposed structures .", "We generated a diverse mAb library against E . coli LptD by running four independent discovery campaigns .", "Specifically , we immunized ( 1 ) rats with cyclic and linear peptides derived from extracellular LptD loop sequences , ( 2 ) rats and ( 3 ) mice with purified LptDE protein in detergent ( n-octyl-β-D-glucopyranoside ) and non-detergent polymer ( amphipol A8-35 ) , and ( 4 ) rats with a targeted boost-and-sort strategy that uses whole bacterial cell immunizations followed by immune boosts with purified LptDE protein ( Figure 1A and Figure 1B ) .", "Within each campaign , the adjuvants and boosting strategies were varied to increase potential antibody diversity ( see Materials and methods for details ) .", "Cumulatively , the discovery campaigns generated more than 7000 hybridomas and more than 3 , 000 ELISA+ LptD-specific mAbs ( Figure 1C ) .", "In total , 774 ELISA+ α-LptD mAbs from the peptide and protein rat immunizations , 952 ELISA+ α-LptD mAbs from the mouse immunizations , and 1494 α-LptD mAbs from the targeted boost-and-sort immunizations were purified ( Figure 1C ) .", "To explore epitope coverage and accessibility of the α-LptD ELISA+ mAbs from three of the antibody discovery campaigns , we first measured their extracellular binding by a fluorescence-assisted cell sorting ( FACS ) -based assay to two strains of E . coli ( Figure 2A ) .", "The first strain was a laboratory E . coli strain , K-12 , that produces an intact core-LPS but no O-antigen .", "The second strain was an E . coli ΔwaaD mutant , which produces a truncated form of LPS on the cell surface ( Kneidinger et al . , 2002 ) .", "This truncation of LPS increases the exposed epitopes accessible to mAbs ( Bentley and Klebba , 1988; Storek et al . , 2018 ) .", "Depending on the immunization campaign , 9–30% of the ELISA+ α-LptD mAbs were FACS+ on E . coli ΔwaaD , while <1% of the same antibodies were FACS+ on E . coli K-12 ( Figure 2A ) .", "The E . coli K-12 FACS+ α-LptD mAbs were a subset of all the FACS+ mAbs , binding equally well to E . coli K-12 and E . coli ΔwaaD cells ( Figure 2—figure supplement 1 ) , and were discovered in both mouse and rat immunization campaigns .", "These results reinforce the finding that LPS presents a significant barrier to accessing surface epitopes on E . coli ( Bentley and Klebba , 1988; Storek et al . , 2018 ) and suggest that the ECLs of OMPs may be masked or intimately associated with the core sugars of the LPS .", "The diversity of epitopes bound by α-LptD mAbs in our library was next assessed by monitoring binding to LptD from different species .", "ELISAs were performed with 134 FACS+ and 233 FACS- α-LptD mAbs using purified LptDE protein from two closely related Enterobacteriaceae species , Klebsiella pneumoniae and Enterobacter cloacae , which share 82% and 84% overall protein identity and 71% and 76% identify between the ECLs only when compared with E . coli LptD , respectively ( Figure 2B and Figure 2—figure supplement 2A ) .", "The majority of FACS+ ( 61% ) and FACS- ( 69% ) α-LptD mAbs bound only E . coli LptDE by ELISA ( Figure 2C ) .", "This result was not surprising considering that all protein immunization campaigns utilized only purified E . coli LptDE protein .", "Analysis of the surface-exposed portion of LptD predicted from experimental x-ray crystallographic structural models identified regions of >5 amino acids that were conserved among all three Enterobacteriaceae species , the size of a typical hot spot of residues that contribute to antibody binding ( Stave and Lindpaintner , 2013 ) ( Figure 2B and Figure 2—figure supplement 2B ) .", "Indeed , we identified 10 ( 7% ) α-LptD mAbs that bound to LptDE from all three species and 42 that bound either E . coli plus K . pneumoniae ( 22% ) or E . coli plus E . cloacae ( 9% ) LptDE ( Figure 2C ) .", "Thus , our immunization campaigns using E . coli LptD-derived peptides and E . coli LptDE protein yielded diverse antibodies that bound to both unique and conserved extracellular accessible ( i . e . , FACS+ ) and extracellular-inaccessible or periplasmic ( i . e . , FACS- ) epitopes .", "The differences in cross-species reactivity of the α-LptD mAbs suggested that the FACS+ antibodies bound multiple surface-exposed LptD epitopes .", "To determine if these antibodies bound unique extracellular epitopes , we tested α-LptD mAbs with the highest FACS+ signal for their abilities to compete with each other for binding to purified E . coli LptDE .", "Every possible FACS+ pair of α-LptD mAbs was screened for the ability to either bind LptDE protein simultaneously ( i . e . , different epitopes ) or to compete for binding ( i . e . , same or nearby epitopes ) using a SPR-based binding assay ( Abdiche et al . , 2017 ) .", "From these results , we constructed a surface epitope map for the FACS+ α-LptD mAbs ( Figure 2D ) .", "We identified two major epitope bins and a possible third bin with limited overlap ( Figure 2D ) .", "When the species cross-reactivity data were considered , most three-species cross-reactive α-LptD mAbs were tightly clustered , while those that bound only E . coli LptDE formed a distinct cluster ( Figure 2D ) .", "There were some outliers to these trends with the dual-species binders and E . coli-only binders binning with the cross-reactive mAbs suggesting distinct , but overlapping epitopes .", "Thus , the α-LptD mAbs we discovered covered multiple distinct and overlapping extracellular LptD epitopes .", "In order to map the extracellular epitopes of the α-LptD mAbs in a native asymmetrical OM environment , we first generated and characterized a panel of lptD mutants each lacking one ECL .", "This strain panel also allowed us to assess the essentiality of each ECL in strains with different OM compositions .", "We constructed 13 lptD mutants , each lacking the region encoding one of the 13 ECLs ( Figure 3A ) .", "These loop deletions were based on available x-ray crystal structures of LptD and were constructed as complete loop deletions without replacing the removed sequence .", "In the case of loop 4 ( L4 ) , which encompassed the classical imp4213 allele ( Sampson et al . , 1989 ) and in the case of loop 8 , which is an extensive loop that interacts with LptE in the LptD lumen ( Freinkman et al . , 2011 ) , we only removed a portion of the loop .", "Each lptD loop mutant was expressed in a lptD-conditional E . coli strain .", "In the presence of arabinose , which induces a chromosomal wild-type lptD , all strains grew ( Figure 3B ) .", "In the absence of arabinose , all lptD loop mutants supported growth in the K-12 strain with the exception of loop 2 ( L2 ) removal , which showed reduced growth ( Figure 3B ) .", "In all cases when growth was observed , the levels of the LptD loop deletion protein produced were similar to the protein level of wild-type LptD ( Figure 3—figure supplement 1A ) .", "Thus , LptD is highly tolerable to genetic manipulation of its ECLs .", "When the lptD loop mutants were expressed in an lptD-conditional E . coli ΔwaaD strain , which produces LPS with a truncated core oligosaccharide , the observed loop requirements were different .", "In the E . coli ΔwaaD background , LptD lacking either loop 2 or loop 10 were unable to support bacterial growth on solid growth agar ( Figure 3B ) .", "The inability of LptD lacking L10 ( LptDΔL10 ) to support growth of the E . coli ΔwaaD strain suggested that this ECL is important for LptD folding or activity in this strain .", "In contrast , the LptDΔL10 variant was produced and did support growth of E . coli K-12 ( Figure 3B and Figure 3—figure supplement 1A ) , indicating that LptDΔL10 was folded and sufficiently functional in the strain producing core-LPS but not the minimal LPS of E . coli ΔwaaD strain .", "To determine if the other LptD loops had differential activities in these two strains , we monitored the growth of all of the LptD loop variants in both the E . coli K-12 and E . coli ΔwaaD strain backgrounds over time in liquid growth media .", "In the presence of arabinose , which induces a chromosomal wild-type lptD , all strains had similar growth patterns ( Figure 3—figure supplement 1C ) .", "In the absence of arabinose , the E . coli K-12 strain producing LptDΔL10 grew , but at a decreased rate , and the strain with LptDΔL2 exhibited a dramatically prolonged lag phase and severe growth phenotype ( Figure 3C and Supplementary file 1 ) .", "In contrast , E . coli ΔwaaD expressing LptDΔL10 did not grow at all , but expression of LptDΔL2 supported growth in liquid media ( Figure 3D and Supplementary file 1 ) .", "Thus , L2 and L10 are important for LptD folding , activity , interaction with the β-barrel assembly machinery component BamA , or a combination of these .", "Also , the LptDΔL10 loop deletion mutant was better tolerated in E . coli K-12 compared to the LPS-truncated ΔwaaD strain while in liquid growth media the LptDΔL2 mutant is better tolerated in the E . coli ΔwaaD strain .", "This result is consistent with the synthetic lethality of other mutations that affect OM biogenesis , specifically bam101 , ΔbamB , ΔsurA , and ΔlpxL , in LPS-truncated E . coli strain backgrounds ( Klein et al . , 2009; Storek et al . , 2019 ) highlighting the redundancy that exists to maintain this important barrier structure .", "The lptDΔL4 mutant used in this study encompassed the imp4213 allele .", "This particular loop mutant is viable but increases membrane permeability and sensitivity to OM-excluded antibiotics ( Sampson et al . , 1989 ) .", "We tested whether the strains producing other LptD loop mutants had increased membrane permeability by measuring sensitivities to rifampicin and vancomycin , two antibiotics excluded by the Gram-negative bacteria OM .", "The minimal inhibitory concentrations ( MICs ) of both rifampicin and vancomycin were lower for strains producing LptDΔL4 , as expected , and also for strains producing LptDΔL2 , LptDΔL8 , and LptDΔL10 indicating an OM defect ( Table 1 ) .", "Increases in OM permeability were observed in both E . coli K-12 and E . coli ΔwaaD strain backgrounds .", "These results suggest that extracellular L2 , L4 , L8 and L10 are important for LptD function and potentially influence LptD folding , interaction with BamA , or activity .", "We utilized our lptD loop deletion panel to map the α-LptD mAb surface coverage in a native E . coli membrane environment .", "We characterized binding of 52 FACS+ α-LptD mAbs to the E . coli ΔwaaD loop deletion panel .", "In the absence of arabinose driving expression of the chromosomal wild-type lptD , production of each mutant LptD was detected and growth of the E . coli ΔwaaD conditional lptD strain was supported by all LptD loop deletion mutants with the exception of L10 , so this construct was not tested in this assay ( Figure 3B and Figure 3—figure supplement 1B ) .", "The mAbs in the panel were confirmed to bind to an E . coli ΔwaaD strain expressing wild-type lptD ( Figure 4A ) .", "We expected that if an extracellular LptD loop was critical for binding , removing the loop would reduce α-LptD mAb binding .", "The lack of binding by any particular mAb to a LptD loop mutant could be due to removal of the mAb binding site , in part or in full , or a structural change that alters the mAb binding site when a loop is removed .", "The majority of the α-LptD mAbs could be categorized into one of 7 general binding patterns based on the mutants they were unable to bind with multiple representatives in each grouping: ( 1 ) Loop 4 ( L4 ) , ( 2 ) Loop 6/7 ( L6/L7 ) , ( 3 ) Loop 6/7/8 ( L6/L7/L8 ) , ( 4 ) Loop 8/9 ( L8/L9 ) , ( 5 ) Loop 9 ( L9 ) , ( 6 ) Loop 11 ( L11 ) , and ( 7 ) Loop 13 ( L13 ) ( Figure 4B and Figure 4—figure supplement 1 ) .", "Thus , our immunization campaigns produced antibodies that allow systematic probing of accessible ELCs around the LptD structure .", "In some cases , for example , a representative E . coli K-12 FACS+ α-LptD mAb ( Figure 2—figure supplement 1B ) , no loop mutant dramatically altered antibody binding .", "This pattern suggests a surface-exposed epitope was present that was not altered in these particular loop mutants or the mAb bound a composite epitope that was not sufficiently disrupted by loss of a portion of the epitope encompassed by one individual loop deletion ( Figure 4B ) .", "This could be especially true for L8 as the deletion encompassed only part of the loop that , based on structural models , is likely to occupy the LptD lumen while additional exposed portions were left unchanged ( Figure 4C ) .", "For every loop deletion , at least one FACS+ α-LptD mAb had reduced cell binding by >90% .", "The notable exceptions were L1 , L2 , L5 and L12 , but mAbs could be found that exhibited >80% decreased binding for L1 , L5 and L12 .", "Comparing these data with the species cross-reactivity and in vitro binding competition epitope map provided a complete picture of the mAb accessible surface of LptD .", "Specifically , a majority of the two- and three-species cross-reactive α-LptD mAbs were dependent upon L8 , L9 , or both for binding ( Figure 2B , Figure 2D , Figure 2—figure supplement 2 , Figure 4B , and Figure 4—figure supplement 1 ) .", "The cross-reactive mAbs 5A3 and 3D4 exhibited slightly different binding requirements , L6/L8/L9/L11 and L7/L8 , respectively , consistent with these mAbs recognizing different epitopes in our in vitro binning experiment ( Figure 2D ) .", "Additionally , E . coli-specific α-LptD mAbs , but not the three-species cross-reactive mAbs , were defective for binding upon removal of L3 , L7 , or L13 ( Figure 2B , Figure 2D , Figure 2—figure supplement 2 , Figure 4B , and Figure 4—figure supplement 1 ) .", "These α-LptD mAbs and knowledge of their binding sites are valuable tools for probing the accessible LptD surface and suggest that LptD can tolerate extensive alteration to its extracellular surface with little or no effect on folding or function .", "Because distinct antibody discovery approaches were utilized , we were also able to evaluate the potential correlation of LptD targeting with regard to the immunization campaign .", "Partitioning the mAbs with respect to the host animal used for immunization highlighted a trend for L6 , L7 , and L8 to be critical from the rat immunization campaign , while mAbs from the mouse immunization campaign tended to yield FACS+ antibodies that required L8 and L9 .", "Because these animals are likely not tolerized to the foreign bacterial LptD protein , this difference could reflect differences in the types of binding sites presented for rat versus mouse antibodies .", "Thus , even within these immunization campaigns , diverse binding profiles to the loop mutants were seen ( Figure 4B and Figure 4—figure supplement 1 ) .", "A potential application of antibodies raised against extracellularly-exposed bacterial proteins is as direct-acting antibacterial molecules ( LaRocca et al . , 2009; Storek et al . , 2018 ) .", "Disruption of L2 or L10 resulted in growth defects for E . coli ΔwaaD ( Figure 3B ) , however , we did not identify any α-LptD mAbs that bound L2 , and both L2 and L10 appear to be inaccessible in available structural models of LptD ( Figure 4C ) ( Botos et al . , 2016; Dong et al . , 2014; Qiao et al . , 2014 ) .", "Indeed , when we screened our entire antibody catalog using growth inhibition as a readout for mAb activity , we did not identify any α-LptD mAbs able to inhibit growth of E . coli K-12 or E . coli ΔwaaD by >50% ( Table 2 and Figure 4—figure supplement 2 ) .", "Combined with our LptD loop deletion analysis , the lack of ability to identify an inhibitory α-LptD mAb suggests that LptD may be specifically evolved to withstand extracellular modulation by antibodies while protecting critical structural elements of LptD function ." ], [ "The OM is a critical feature of Gram-negative bacteria and presents a barrier to the discovery of new antibiotics ( Lewis , 2013 ) .", "There are two essential OMPs embedded in the OM and involved in construction of the OM barrier .", "BamA is part of the β-barrel assembly machine that folds and inserts β-barrel OMPs , including BamA and LptD , into the OM ( Konovalova et al . , 2017; Ricci and Silhavy , 2012 ) .", "LptD is the last protein in the Lpt pathway that establishes and maintains OM asymmetry by inserting LPS exclusively into the outer leaflet of the OM ( Okuda et al . , 2016 ) .", "Despite the importance of these β-barrel OMPs , understanding of how their structures contributes to their folding and function is still incomplete .", "Similar to other Gram-negative β-barrel OMPs , both BamA and LptD possess short periplasmic loops but long , variable , and environmentally-exposed ECLs ( Franklin and Slusky , 2018; Rollauer et al . , 2015; Schulz , 2002 ) .", "A deeper examination of the roles of these ECLs in a native OM context could contribute to a greater understanding of how they contribute to the structure and function of OMPs in general .", "To systematically interrogate the LptD ECLs , we performed four antibody discovery campaigns to generate thousands of antibodies targeting LptD to probe structure-function relationships of this essential OMP in its native OM environment .", "The unusual breadth and depth of our approach represents a powerful way to probe the accessible surfaces of the ECLs of LptD and to tease apart the changes that accompany LPS insertion by LptD in the native OM environment of the bacterial cell .", "By characterizing hundreds of these α-LptD mAbs , we discovered that extensive portions of the extracellular surface of LptD are dispensable in E . coli and critical features of LptD are likely occluded and protected by non-essential loops ( Figure 5 ) .", "Little is known about the role and dynamics of the ECLs of LptD in the function of LPS insertion into the OM and there are few available tools to study this process .", "In an effort to probe as many extracellular epitopes as possible , we generated a large , diverse α-LptD mAb library by undertaking multiple immunization campaigns utilizing different antigens , adjuvants , and hosts .", "Varying antigens systematically presented LptD in increasingly complex arrangements to potentially capture different available epitopes .", "First , peptide immunizations utilized both linear peptides derived from conserved and non-conserved LptD ECL sequences and cyclic variants in an effort to constrain particular conformations .", "Next , purified full-length LptDE immunizations aimed to capture a more native state of the ECLs .", "Because the matrix in which the protein is reconstituted can alter the conformational flexibility and exposure of membrane-proximal epitopes , we utilized multiple reconstitution matrices ( i . e . , detergents and amphipols ) .", "Finally , using an approach that we have recently leveraged to find rare , inhibitory α-BamA antibodies , a targeted boost-and-sort strategy , we immunized rats with sub-lethal E . coli infections and then boosted them with purified LptDE protein to enrich for any LptD antibody response ( Vij et al . , 2018 ) .", "Taken together , our effort to maximize the epitope coverage in our α-LptD mAb library led to distinct clusters of antibodies that possessed differing abilities to cross-react with LptD species variants , required different ECLs for binding , and exhibited varying levels of access to LptD through the protective LPS layer .", "Although the full breadth of antibody diversity in our library required the multiple immunization approaches described here , we propose a streamlined workflow for future OMP efforts .", "An accessible approach is to immunize SD rats with recombinant purified OMP either with or without whole bacterial cells ( strategies 3 and 4 , Figure 1 ) .", "Both of these workflows returned the highest percentages of FACS+ antibodies coupled with recognition of diverse epitopes ( Figure 2 , Figure 4B , and Figure 4—figure supplement 1 ) .", "Given the ease of manipulating bacterial strains , it could even be possible to steer immunizations towards particular ECLs by initially immunizing with a ΔECL-OMP strain followed by boosting with recombinant protein .", "Other scaffolds or antibodies from different host species might be able to recognize distinct epitopes on the exposed portions of LptD .", "For example , recognition of partially buried epitopes could be achieved using species , such as rabbits , chickens , llamas , or camels , that contain longer CDRH3s that are better suited to reach into cavities on a particular antigen ( Lavinder et al . , 2014; Muyldermans and Smider , 2016 ) .", "In the case of the mouse and rat antibodies described here , we found that seven different LptD ECLs , out of a total of 13 , could affect binding of the mAbs and the remaining six loops are likely partially or fully buried based on models from crystal structures ( Botos et al . , 2016; Dong et al . , 2014; Qiao et al . , 2014 ) ( Figure 5 ) .", "When this α-LptD mAb campaign is combined with of our α-BamA mAb effort ( Storek et al . , 2018; Vij et al . , 2018 ) , we now have the ability to probe the structure-function relationships of the only two essential OMPs in their native OM environment in E . coli at a level not previously possible .", "Importantly , we note the antibody discovery methods that we describe herein are efficient , robust , and a streamlined approach can be readily applied within a standard laboratory setting .", "We have observed that in LptD there appear to be differential requirements for particular ECLs , L2 and L10 , depending on the structure of LPS , specifically wild-type LPS versus the truncated ΔwaaD LPS .", "Notably , BamA appears to be defective in the highly fluid OM of an LPS-truncated strain grown under low salt and high temperature conditions ( Storek et al . , 2018 ) .", "This is manifest by an increased sensitivity to a recently discovered inhibitory α-BamA antibody MAB1 and a requirement for elevated BamA levels .", "In these cases , the defects can be suppressed by altering the conditions to increase membrane rigidity ( Storek et al . , 2019 ) .", "The LPS-dependent requirement for LptD loops suggests that effects of the OM structure on OMPs might be more general .", "Among the 13 extracellular LptD loops , both L2 and L10 are short and lack large insertions or deletions across LptD homologs when compared to the longer loops in highly divergent LptD proteins ( Figure 5—figure supplement 1A ) .", "One possible reason for this is that these loops are uniquely positioned to make interactions with the LPS in the OM to allow for proper folding or functioning of this essential OMP .", "Although these interpretations are speculative and complicated by the fact that LPS is the substrate for LptD , it is tempting to hypothesize that the structure and function of OMPs evolved to be maximal when embedded in the constrained , liquid crystalline , OM matrix ( Paracini et al . , 2018 ) and altering the nature of this membrane leads to defects .", "Thus , membrane environment is a critical consideration when interpreting biochemical and structural data of LptD and other OMPs in detergent micelles and must be considered when attempting to design or discover inhibitors of these essential processes in Gram-negative bacteria .", "Genetic , biochemical , and structural data support a model for LPS insertion into the OM whereby the acyl chains of lipid A are inserted into the hydrophobic bilayer through a lateral gate created between the first and last strands of the LptD β-barrel ( Botos et al . , 2016; Dong et al . , 2014; Qiao et al . , 2014 ) .", "It is less clear how the polar sugars of the core oligosaccharide and O-antigen emerge from the periplasm to the cell exterior as it would be unfavorable for them to pass directly through the membrane .", "Presumably the sugars must pass through the LptD transmembrane β-barrel lumen , which is also occupied by an essential lipoprotein , LptE , and then through a proposed opening in the surface-exposed cap of LptD .", "Thus , consistent with prior molecular dynamic simulations , there are likely major structural surface re-arrangements in LptD during LPS insertion ( Dong et al . , 2014 ) .", "Given this model , we hypothesized that an antibody could have interfered with this process in a number of ways .", "For example , a mAb could staple the lateral gate in either a closed or open conformation , prevent the movements associated with expelling the sugars to the outside , or allosterically alter the protein so that LPS movement is thwarted .", "It is informative , therefore , that none of our identified α-LptD mAbs were able to inhibit the activity of LptD .", "One explanation is that binding to a single epitope is insufficient to disrupt the required movements of LptD .", "Alternatively , the positions on LptD critical for these presumed structural changes might be inaccessible to mAbs .", "Indeed , few or no α-LptD mAbs bound to the extracellular LptD loops that were found genetically to be important for viability or OM maintenance under specific conditions .", "Consistent with this possibility , in crystal structures of LptDE , the two loops we found most critical were occluded by other parts of the protein: L2 by L3 , and L10 by L9 and L11 ( Dong et al . , 2014; Qiao et al . , 2014 ) .", "By burying critical regions , LptD appears to avoid direct interference by antibodies , providing bacteria an evolutionary advantage in evading functional antagonism by the host immune system .", "Accordingly , the antibody-accessible LptD loops also are the most diverse in sequence , suggesting that these regions may be subject to selective pressures from the immune system ( Botos et al . , 2016 ) ( Figure 5—figure supplement 1B ) .", "Moreover , the proximity of a particular epitope to the membrane can sterically or electrostatically block binding .", "However , in rare cases , recognition can be achieved via stabilizing hydrophobic residues in an antibody CDR that transiently interact with the membrane , as observed for several α-HIV or α-GPCR antibodies ( Ishchenko et al . , 2017; Scherer et al . , 2010 ) .", "It remains to be seen whether immobilizing these loops in LptD , for example , with a small molecule or peptide macrocycle that can access these positions , would inhibit LptD activity .", "While our observations make direct interference with LptD function by antibodies unlikely , they do not rule out the potential value of LptD as a vaccine candidate ( Zha et al . , 2016 ) .", "Overall , our study represents an exhaustive α-LptD antibody discovery campaign , and potentially the most exhaustive on any membrane protein described to date .", "For the first time , we have been able to map essential and non-essential structural features of this intriguing and important potential antibiotic target .", "We have observed extreme tolerability of the ECLs to interference , suggesting that the architecture of the exposed ECLs of LptD play a role in protecting critical functions of this essential OMP .", "More generally , we provide a robust template for future efforts to dissect the structure-function relationships of complex membrane proteins from bacteria and mammals in their native environments , and a method to rapidly assess the therapeutic potential of antibody targeting ." ], [ "Luria-Bertani broth ( ThermoFisher Scientific 12795027 ) and Mueller Hinton II cation-adjusted broth ( BBL 212322 ) was prepared according to manufacturer's instructions .", "Bacterial cultures were grown at 37°C .", "When appropriate , media was supplemented with kanamycin ( 50 μg/mL ) , carbenicillin ( 50 μg/mL ) , chloramphenicol ( 12 . 5 μg/mL ) , hygromycin ( 200 μg/mL ) , gentamicin ( 10 μg/mL ) and arabinose ( 0 . 2% vol/vol ) .", "Bacterial strains and relevant primers are listed in Supplementary file", "2 . Kanamycin deletion-insertion mutations of waaD was obtained from the Keio collection ( Baba et al . , 2006 ) .", "Briefly , pKD4 or pKD3 was amplified with primers containing 50 bp nucleotide homology extensions ( Supplementary file 2 ) to the gene of interest .", "The linear product was transformed into the appropriate background strain containing pSIM18 ( Chan et al . , 2007 ) recovered for 4 hr and selected on media containing 50 μg/mL kanamycin or 12 . 5 μg/mL chloramphenicol as appropriate .", "Mutations were confirmed by PCR .", "To make sequential mutants , the antibiotic marker was flipped out using pCP20 ( Datsenko and Wanner , 2000 ) .", "The conditional lptD strain , ΔlptD::PBAD-lptD , was created by inserting PBAD-lptD at the attB site in MG1655 followed by deletion of the native copy of lptD ( Datsenko and Wanner , 2000; Diederich et al . , 1992 ) .", "Briefly , lptD was cloned into pBAD24 using standard methods .", "PBAD-lptD was amplified from pBAD24-lptD and sub-cloned into pLDR9 .", "pLDR9-PBAD-lptD was digested with XbaI , ligated , and transformed into MG1655 expressing pLDR8 .", "PCR and DNA sequencing confirmed insertion of PBAD-lptD at the attB site .", "After integration of PBAD-lptD , the native copy of lptD was deleted using λ Red recombination as described above .", "In the absence of arabinose , the conditional DlptD::PBAD-lptD strain did not grow .", "To obtain a conditional lptD strain truncated for LPS , waaD was deleted using λ Red recombination .", "To clone the loop mutants into the lptD conditional strains , pLMG18 was modified to replace the original antibiotic cassette providing chloramphenicol resistance with a gentamicin resistance cassette using Gibson Assembly .", "The lptD gene and mutant constructs were codon optimized and ordered as gBlocks ( Integrated DNA technologies ) .", "The genes were amplified and cloned into pLMG18gm using Gibson Assembly .", "Clones were confirmed by sequencing .", "The E . coli LptDE expression plasmid was constructed by cloning E . coli LptD ( amino acids 1–784 ) and E . coli LptE ( amino acids 1–193 ) into pCDFDuet1 vector at the first and second multiple cloning sites .", "An AviTag followed by an 8x histidine tag was inserted into N-terminal of E . coli LptD .", "The expression plasmid was expressed in E . coli C41 ( DE3 ) .", "When the OD600 reached 0 . 9 , cells were induced with 0 . 5 mM isopropyl-β-D-1 thiogalactopyranoside ( IPTG ) overnight at 18°C .", "Cells were harvested by centrifugation at 4500 rpm for 15 min and the cell pellets were resuspended in lysis buffer ( 50 mM Tris , pH 8 . 0 , 200 mM NaCl , 1x complete protease inhibitor mixture ( Roche ) ) .", "The resuspended cells were disrupted by passing through a microfluidizer at 10 , 000 psi three times .", "The cell lysate was added with 2% Zwittergent 3–14 and rocked overnight at 4°C .", "The suspension was ultracentrifuged ( 45Ti rotor , 40 , 000 rpm ) for 1 hr at 4°C .", "The supernatant was incubated with pre-equilibrated Ni-NTA resins ( Qiagen ) and rocked for 1 hr at 4°C .", "The resins were washed by the buffer containing 50 mM Tris , pH 8 . 0 , 200 mM NaCl , 1% Zwittergent 3–14 , 15 mM imidazole , 1x complete protease inhibitor mixture and eluted with the buffer consisting of 50 mM Tris , pH 8 . 0 , 200 mM NaCl , 1% Zwittergent 3–14 , 300 mM imidazole and 1x complete protease inhibitor mixture .", "The eluate was concentrated and purified by a Superdex 200 16/60 column ( GE Healthcare ) using 20 mM HEPES , pH 8 . 0 , 100 mM NaCl , 1 . 5% n-Octyl-β-D-Glucopyranoside ( OG; Anatrace ) , 1x complete protease inhibitor mixture as the running buffer .", "The obtained LptD/E protein was diluted 4-fold into the buffer of 20 mM HEPES , pH 8 . 0 , 1 . 5% OG and further polished using Q HP anion exchange 5 ml column ( GE Healthcare ) .", "20 mM HEPES , pH 8 . 0 , 25 mM NaCl , 1 . 5% OG was used as the start buffer and elution was achieved with linear gradient of 0 . 025–1 M NaCl .", "When protein was required in amphipol , LptD/E protein was incubated with biotinylated amphipol ( Anatrace ) overnight at 4°C and then purified over a Superdex 200 16/60 column in a buffer consisting of 20 mM HEPES , pH 8 . 0 , 100 mM NaCl .", "Peptide pools are listed in Supplementary file", "3 . Peptides were designed based on sequence conservation , surface exposure and loop location .", "When LptDE protein was required for ELISA or antigen-based sorting , an in vitro biotinylation reaction using BirA enzyme targeting the N-terminal Avi tag was first carried out according to the manufacturer's suggestions ( Avidity ) ; LptDE was then rerun over the Superdex 200 ( 26/60 ) in buffer E as described above in order to remove free biotin and other reaction components .", "When protein was required in amphipol ( either non-biotinylated or biotinylated ) , LptDE at 1 mg/mL in buffer E was incubated with a stock solution of 2 mg/mL A8-35 amphipol ( Anatrace ) at 22°C for 1 hr and then applied over a Superdex 200 ( 26/60 ) column in buffer F ( 50 mM Tris pH 8 , 100 mM NaCl ) .", "Protein reconstituted in A8-35 amphipol for immunization was concentrated to 1 mg/mL using a centrifugal device ( 10 K MWKO; Millipore ) .", "When PE-labeled protein was required for antigen-specific sorting , LptDE at 1 mg/mL ( biotinyated and reconstituted in A8-35 amphipol in buffer F ) was mixed 1:1 ( vol/vol ) with PE-streptavidin ( Jackson ImmunoResearch ) reconstituted in buffer F; the PE-streptavidin-biotin-LptDE-amphipol complex was incubated at 22°C for at least 15 min prior to use .", "All animal study designs for the mouse and rat immunizations were reviewed and approved by the Genentech Institutional Animal Care and Use Committee prior to the start of this work .", "All animal work was performed in accordance with relevant guidelines and regulations .", "LptD peptide immunization campaign immunized Balb/c mice ( Charles River Laboratories , Hollister , CA ) with cyclic and linear LptD peptide pools ( Supplementary file 3 ) .", "The initial immunization contained 100 μl Complete Freund’s Adjuvant ( CFA ) and subsequent dosing included 100 μl Incomplete Freund’s Adjuvant ( IFA ) ( Sigma ) .", "pAbs were purified by Protein A and assayed by ELISA , FACS , and inhibition of bacterial growth as described below .", "Hybridoma fusions were performed as previously described and supernatants were screened for protein binding by ELISA ( Hazen et al . , 2014 ) .", "All ELISA positive clones were purified and screened for inhibition of bacterial growth .", "LptDE protein immunization campaign immunized Balb/c mice ( Charles River Laboratories , Hollister , CA ) with detergent-solubilized or amphipol-reconstituted E . coli LptDE protein and either CFA or Ribi adjuvants ( Sigma ) .", "pAbs were purified and screened as described above .", "LptDE protein immunization campaign immunized Sprague Dawley rats ( Charles River Laboratories , Hollister , CA ) with detergent-solubilized or amphipol-reconstituted E . coli LptDE protein and either CFA or Ribi adjuvants ( Sigma ) .", "pAbs were purified and screened as described above .", "Bacterial immunization campaign immunized Sprague Dawley rats ( Charles River Laboratories , Hollister , CA ) with E . coli K-12 cells in PBS ( 107–109 colony forming units via intravenous injection ) .", "The rats were boosted four times with E . coli K-12 cells followed by two protein boosts with 10 μg E . coli LptDE protein in amphipol .", "To generate monoclonal antibodies , hybridoma fusions were performed as previously described except with a myeloma partner SP2ab that enables surface display of IgG cell ( Hazen et al . , 2014; Price et al . , 2009 ) .", "After HAT selection in ClonaCell-HY Medium C ( StemCell Technologies ) for 4 days , hybridomas were stained with a cocktail of FITC-conjugated anti-rat IgG1/IgG2a/IgG2b mAbs ( 1:100 dilution; Bethyl Laboratories ) and PE-conjugated LptDE protein antigen ( 5 μg/μl ) .", "Samples were sorted on a FACSAria II cell sorter ( BD Biosciences ) .", "Gating strategy to identify hybridoma cells was first based on size ( FSC/SSC ) .", "After dead-cell exclusion with Propidium iodide ( Sigma-Aldrich P-4864 ) , IgG +LptDE + single cell hybridomas were sorted into 96-well tissue-culture plates containing 200 μl of ClonaCell-HY Medium E ( StemCell Technologies ) .", "Profiles were analyzed by FlowJo v . 9 . 7 . 7 software .", "Sorted cells were cultured for seven days .", "Hybridomas were cultured using a previously described semi-automated high throughput process for hybridoma culturing and antibody purification ( Vij et al . , 2018 ) .", "Epitope bins were determined by 96 × 96 array-based SPR imaging system ( Carterra , USA ) using classical sandwich method .", "Purified antibodies were diluted at 20 μg/ml in 10 mM sodium acetate buffer pH 4 . 5 .", "Using amine coupling , antibodies were directly immobilized onto a SPR sensorprism CMD 200 M chip ( XanTec Bioanalytics , Germany ) using a Continuous Flow Microspotter ( Carterra , USA ) to create an array of 96 antibodies .", "For antibody binning , printed chip was loaded on IBIS MX96 SPRi ( Carterra USA ) and E . coli LptDE protein , diluted to 50 nM in 1 . 5% bOG HBS-P buffer , was injected over the chip at 25°C followed by a second injection of purified antibody , diluted at 20 μg/ml in 1 . 5% bOG HBS-P buffer , to make a sandwich .", "The epitope binning data were processed using Carterra binning software tool .", "Epitope binning experiments were performed as part of our high throughput antibody screening workflow and are purely descriptive .", "The test strain was grown to log phase in MHB II supplemented with 0 . 002% Tween-80 , diluted to a final OD600 0 . 01 in sterile round-bottom 96-well plates ( Costar ) .", "Antibodies were added at 10 μg/mL , incubated statically at 37°C and monitored for bacterial growth after 4 hr of static incubation .", "A plate reader at OD600 measured optical density of bacterial growth after shaking the plate for 25 s .", "Bacterial inhibition was calculated by subtracting the OD600 value from the media control , followed by dividing that value by the no antibody control OD600 value .", "Antibody activity experiments were performed as part of our high throughput antibody screening workflow and are purely descriptive .", "Antibodies were screened by capture ELISA .", "Briefly , 50 µl of biotinylated LptDE protein , diluted in assay buffer ( PBS + 1 . 5% OG +0 . 5% BSA ) was added to streptavidin coated 384 well plates ( Thermo Scientific USA , Cat # 15504 ) and incubated at RT for 1 hr , while shaking .", "The plates were washed 3X with wash buffer ( PBS + 1 . 5% OG ) .", "50 µl of supernatants or purified antibody , neat or diluted in assay buffer , were added to the wells and incubated at RT for 1 hr , while shaking .", "The plates were washed 3X with wash buffer .", "The captured antibody was detected with goat anti-rat HRP or goal anti-mouse HRP secondary antibody as appropriate ( 50 μl per well diluted at 1:10K in assay buffer , Bethyl Laboratories USA , Cat # A110-236P ) .", "The plates were incubated at RT for 1 hr , washed 3X with wash buffer and 50 µl of substrate , TMB solution ( Surmodics , USA Cat # TMBW-1000 ) , was added to each well .", "The plates were incubated for 5 min at RT followed by addition of 50 μl of TMB stop solution ( Surmodics , USA Cat # LPSP-1000 ) .", "Plates were read at 630 nm .", "ELISA assays were performed as part of our high throughput antibody screening workflow and are purely descriptive .", "Bacterial cells were grown to log phase , pelleted , re-suspended in ice-cold PBS with 1% BSA , and normalized to an OD600 of 0 . 5 .", "Bacterial suspensions were diluted 1:2 . 5 in ice-cold PBS , 1% BSA and 2 × 106 CFU were incubated with 10 µg/ml mAb at 4°C for 1 hr under gentle agitation .", "Bacterial cells were then washed three times in ice-cold PBS , 1% BSA and labeled using Alexa 488 anti-mouse IgG ( H + L ) ( 1:1000; Invitrogen ) or Alexa 488 anti-rat IgG ( H + L ) ( 1:1000; Invitrogen ) for 30 min 4°C .", "After three washes in ice-cold PBS , bacterial cells were fixed by adding one vol .", "of 2% w/v paraformaldehyde in PBS .", "Samples were run on a BD FACSCalibur and data from 10 , 000 events were analyzed using FlowJo software .", "FACS experiments were performed as part of our high-throughput antibody screening workflow and are purely descriptive .", "An isotype matched antibody was used as a negative control .", "Bacterial cells were grown to log phase , normalized to OD600 , and pelleted .", "Samples were resuspended in 1x LDS sample buffer ( ThermoFisher Scientific ) and boiled for 5 min prior to loading on a 4–12% Bis-Tris SDS-PAGE gel .", "Proteins were transferred onto cellulose membranes using the iBlot 2 Dry Blotting System ( ThermoFisher Scientific ) .", "Membranes were blocked for 1 hr in Blocking Buffer ( TBS containing 5% nonfat milk and 0 . 05% Tween-20 ) , washed , then incubated either overnight at 4°C or room temperature for 1 hr with the following primary Abs: human α-LptD 3D11 ( 1 μg/mL , Genentech ) and rabbit α-GroEL ( 1:25 , 000 , Enzo ) .", "Appropriate HRP-linked secondary antibodies ( GE Healthcare ) were diluted 1:20 , 000 in TBST and incubated with the membrane for 1 hr at RT .", "Blots were developed using ECL Prime Western Blotting Detection Reagent ( Amersham ) .", "The displayed Western blot experiment shows a single biological replicate .", "Growth curves were performed in biological triplicate .", "Bacterial strains were grown overnight on LB agar containing gentamicin and arabinose .", "Cells were scraped from the plate into fresh media .", "OD600 was measured and subsequently diluted to 0 . 001 ( OD600 ) .", "100 μl transferred to a 96-well plate ( Corning ) and monitored for growth by measuring OD600 ( EnVision Multimode Plate Reader , PerkinElmer ) .", "Bacterial growth curves were analyzed by determining the doubling time ( dt ) during exponential growth phase and compared via the unpaired Student’s t test using Prism 6 . 0 ( GraphPad Software ) ( Supplementary file 1 ) .", "The Bonferroni correction was applied to control for multiple comparisons .", "Minimum inhibitory concentration ( MIC ) assays were performed by inoculating 105 colony forming units into medium with only test antibiotics ( rifampicin and vancomycin ) .", "Biological duplicates were performed .", "MIC defined as the lowest concentration of drug to inhibit bacterial growth ." ] ]

[ "Outer membrane proteins ( OMPs ) in Gram-negative bacteria dictate permeability of metabolites , antibiotics , and toxins .", "Elucidating the structure-function relationships governing OMPs within native membrane environments remains challenging .", "We constructed a diverse library of >3000 monoclonal antibodies to assess the roles of extracellular loops ( ECLs ) in LptD , an essential OMP that inserts lipopolysaccharide into the outer membrane of Escherichia coli .", "Epitope binning and mapping experiments with LptD-loop-deletion mutants demonstrated that 7 of the 13 ECLs are targeted by antibodies .", "Only ECLs inaccessible to antibodies were required for the structure or function of LptD .", "Our results suggest that antibody-accessible loops evolved to protect key extracellular regions of LptD , but are themselves dispensable .", "Supporting this hypothesis , no α-LptD antibody interfered with essential functions of LptD .", "Our experimental workflow enables structure-function studies of OMPs in native cellular environments , provides unexpected insight into LptD , and presents a method to assess the therapeutic potential of antibody targeting ." ]

[ "The overuse and misuse of antibiotics has led to the rise of multi-drug resistant bacteria which threaten global public health .", "Antibiotics interfere with essential processes in bacteria so they are unable to divide or survive , but over time , the microbes have found ways to become immune to the drugs .", "New antibiotics are now desperately needed .", "Gram-negative bacteria are wrapped in an outer membrane made of large molecules called lipopolysaccharides .", "This structure is an extra barrier to molecules ( such as drugs ) that try to enter the cell , but it could also hold new targets for antibiotics to exploit .", "A protein called LptD is embedded in the outer membrane , where it inserts new lipopolysaccharides .", "It is critical for bacteria to grow and survive , and is a relatively new potential target for antibiotic development .", "The protein has a number of ‘extracellular loops’ that extend into the environment , but their roles in the structure and the activity of LptD are still largely unknown .", "This is partly due to a lack of tools to investigate these elements .", "In response , Storek et al . built a library of over 3 , 000 custom antibodies , which are small Y-shaped proteins that can each recognise a specific portion in one of the extracellular loops and potentially incapacitate LptD .", "The antibodies were used to target LptD in its native environment , when it is embedded in the bacteria .", "In parallel , mutant bacteria were created in which the loops were genetically removed one by one to assess their importance for LptD activity .", "The experiments revealed that although the antibodies could target most extracellular loops , they could not target the few loops that were essential for LptD to work properly .", "This suggests that antibody-accessible loops are expendable and that these structures could serve to shield other regions of LptD which are critical for survival .", "The findings will help to prioritise research that develops other approaches to inhibit LptD .", "Finally , the antibody workflow designed by Storek et al . can serve as a road map to study other membrane proteins in their native cellular environment ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "developmental biology", "neuroscience" ]

[ [ "Intermediate neural progenitor cells ( INPs ) play a critical role in increasing the brain size and complexity .", "Transient amplification of INPs dramatically boosts the neural output from neural stem cells ( NSCs ) ( Kriegstein and Alvarez-Buylla , 2009; Florio and Huttner , 2014 ) .", "Recent studies in developing human brains as well as other mammalian brains suggest that an expansion of the number of transiently amplifying INPs , the outer sub-ventricular zone radial glia-like cells ( oRGs ) , likely contributes to the increased cortical size and complexity in humans and other gyrencephalic animals ( Fietz et al . , 2010; Hansen et al . , 2010; Lui et al . , 2011; Wang et al . , 2011 ) .", "On the other hand , accumulating body of evidence suggests that brain tumors could originate from dedifferentiation and unrestricted proliferation of INPs ( Holland et al . , 2000; Dai et al . , 2001; Walton et al . , 2009; Persson et al . , 2010; Zong et al . , 2012 ) .", "Therefore , it is fundamentally important to understand how the generation and proliferation of INPs are regulated .", "The recently discovered type II neuroblasts ( NBs , the Drosophila NSCs ) in developing Drosophila larval brains provide an excellent model system for studying mechanisms regulating the generation and proliferation of INPs ( Bello et al . , 2008; Boone and Doe , 2008; Bowman et al . , 2008 ) .", "There are 8 type II NBs in each brain lobe .", "Like mammalian NSCs , Drosophila type II NBs produce neurons and glia indirectly by generating INPs .", "Individual INPs undergo 4–6 rounds of asymmetric divisions to produce a new INP to self-renew and a ganglion mother cell ( GMC ) , which divides terminally to produce neurons and/or glia ( Bayraktar et al . , 2010; Viktorin et al . , 2011; Yang et al . , 2013 ) .", "Meanwhile , individual INPs produce distinct types of neurons by sequentially expressing a set of distinct transcription factors to specify the identity of their progeny ( Bayraktar and Doe , 2013; Wang et al . , 2014 ) .", "Through self-renewing divisions , INPs not only amplify the number but also increase the diversity of neural progeny generated from type II NBs .", "Therefore , the neurogenesis pattern in type II NB lineages is remarkably similar to that in mammalian brains and the Drosophila INPs are functionally analogous to mammalian INPs , particularly oRGs .", "The generation of INPs in type II NB lineages involves multiple steps ( Bello et al . , 2008; Boone and Doe , 2008; Bowman et al . , 2008 ) .", "Newly generated INPs are immature and do not express any NB markers , such as the proneural protein Asense ( Ase ) or the bHLH protein Deadpan ( Dpn ) , except for Miranda ( Mira ) .", "The Ase− immature INPs first turn on the expression of Ase to become Ase+ immature INPs .", "Ase+ immature INPs then further differentiate to become mature INPs , which express both Ase and Dpn .", "INPs do not divide until they are fully mature .", "The maturation of INPs requires Numb , the NHL family protein Brain tumor ( Brat ) , the transcription factor Earmuff ( Erm ) , as well as the BAP and Histone deacetylase 3 ( HDAC3 ) chromatin remodeling complexes ( Bowman et al . , 2008; Weng et al . , 2010; Eroglu et al . , 2014; Koe et al . , 2014 ) .", "Both Numb and Brat are segregated into Ase− immature INPs during the division of type II NBs to prevent them from dedifferentiating into NB fate , but they function through independent pathways .", "Numb inhibits Notch activity in Ase− immature INPs , whereas Brat likely antagonizes the activity of the EGR family transcription factor Klumpfuss ( Klu ) and Armadillo/β-Catenin in Ase− immature INPs ( Bowman et al . , 2008; Komori et al . , 2014 ) .", "Erm functions together with BAP and HDAC3 chromatin remodeling complexes after Brat and Numb to further restrict the developmental potential of INPs by attenuating the response of INPs to self-renewal factors such as Klu and Dpn ( Janssens et al . , 2014; Koe et al . , 2014 ) .", "In addition , the BAP chromatin remodeling complex limits the self-renewal of INPs by activating the expression of Prdm protein Hamlet ( Eroglu et al . , 2014 ) .", "In the absence of Numb , Brat , Erm , or chromatin remodeling complexes , INPs dedifferentiate into type II NBs and initiate tumorigenic overproliferation ( Bowman et al . , 2008; Weng et al . , 2010; Eroglu et al . , 2014; Koe et al . , 2014 ) .", "Therefore , these proteins are critical to prevent dedifferentiation of INPs .", "However , despite the significant progress on elucidating mechanisms that promote maturation and prevent dedifferentiation of INPs in the past few years , much less is known about why only type II NBs produce self-renewing INPs but not the type I NBs , which produce neurons by generating terminally dividing GMCs .", "One major difference between INPs and GMCs is that INPs divide to self-renew whereas GMCs divide terminally ( Bello et al . , 2008; Boone and Doe , 2008; Bowman et al . , 2008; Yang et al . , 2013 ) .", "Therefore , in addition to avoiding dedifferentiation and unrestricted tumorigenic overproliferation , INPs need to overcome another challenge–to avoid over-differentiation and cell cycle exit–in order to maintain their progenitor state and self-renewal while they differentiate to mature and undergo self-renewing divisions .", "Type II NBs and newly born Ase− immature INPs differ from type I NBs and GMCs by the lack of the expression of Ase and the homeodomain protein Prospero ( Pros ) ( Bello et al . , 2008; Boone and Doe , 2008; Bowman et al . , 2008 ) .", "In type I NB lineages , Pros is expressed in the cytoplasm in the NBs and translocates to the nucleus in GMCs to promote differentiation and cell cycle exit by inhibiting NB self-renewing genes and activating neural differentiation genes ( Li and Vaessin , 2000; Choksi et al . , 2006 ) .", "In type II NB lineages , Pros is not expressed in the NB or Ase− immature INPs .", "In Ase+ immature INPs and mature INPs , Pros is expressed at low levels in the cytoplasm ( Bello et al . , 2008; Boone and Doe , 2008; Bowman et al . , 2008 ) .", "It has been demonstrated that the lack of Ase and Pros in type II NBs and Ase− immature INPs is essential for the generation of self-renewing INPs in type II NB lineages .", "Forced expression of Ase or Pros in type II NBs and their progeny is sufficient to eliminate INPs although removing Ase or Pros in type I NBs does not change the identity of type I NBs or induce the generation of INPs ( Bowman et al . , 2008; Bayraktar et al . , 2010; Zhu et al . , 2012 ) .", "Our recent studies demonstrated that the Ets family transcription factor Pointed P1 ( PntP1 ) suppresses Ase in type II NBs and is required for the generation of INPs ( Zhu et al . , 2011 ) .", "However , mechanisms that prevent premature differentiation of INPs and/or inhibit Pros expression in type II NBs and immature INPs are not known .", "In this study , we investigate the role of the Sp family transcription factor Buttonhead ( Btd ) in type II NB lineage development .", "Btd is a homolog of mammalian Sp8 ( Treichel et al . , 2003; Estella and Mann , 2010 ) .", "In developing mammalian brains , Sp8 is expressed in neural progenitor cells to regulate forebrain patterning and interneuron development ( Griesel et al . , 2006; Waclaw et al . , 2006; Sahara et al . , 2007; Li et al . , 2011 ) .", "In Drosophila embryos , Btd is required for the formation of specific head segments and NB formation ( Wimmer et al . , 1993; Younossi-Hartenstein et al . , 1997 ) .", "In addition , Sp8/Btd also promotes the growth of limbs and other appendages ( Estella et al . , 2003; Treichel et al . , 2003; Kawakami et al . , 2004; Estella and Mann , 2010 ) .", "In this study , we report that Btd is expressed in type II NB lineages to prevent premature differentiation of INPs into GMCs by suppressing Pros expression in immature INPs .", "We also demonstrate that PntP1 and Btd function cooperatively to specify type II NB lineages and promote the generation of INPs ." ], [ "Our recent studies demonstrated that the Ets family transcription factor PntP1 is specifically expressed in type II NB lineages to promote the generation of INPs .", "However , although forced expression of PntP1 suppress Ase expression in nearly all type I NB lineages , it induces the generation of INP-like cells only in a subset of type I NBs ( Zhu et al . , 2011 ) .", "Therefore , it is likely that other protein ( s ) may function together with PntP1 to specify type II NB lineages and promote the generation of INPs .", "A recent functional genomic study showed that in addition to PntP1 , there are other nine genes that are highly expressed in brain tumors derived from type II NB lineages ( Carney et al . , 2012 ) .", "We wondered whether any of these genes could function together with PntP1 to promote INP generation .", "To test this idea , we first examined how knockdown of these genes would affect INP generation in type II NB lineages .", "A normal type II NB lineage contains 2–3 Ase− immature INPs , 2–3 Ase+ immature INP , and about 20–30 ( 26 . 9 ± 4 . 1 , mean ± SD ) Ase+ Dpn+ mature INPs ( Figure 1A–A′ , C–C′ , G ) .", "Interestingly , RNAi knockdown of Btd using the type II NB lineage-specific pntP1-GAL4 ( named as GAL414−94 previously ) ( Zhu et al . , 2011 ) as a driver led to a complete elimination of mature INPs in about 50% of type II NB lineages ( Figure 1B–B′ , G–H ) .", "Instead , only a few ( 3 . 7 ± 1 . 2 ) Ase+ Dpn− cells were observed next to the Ase− immature INPs ( Figure 1B–B′ ) .", "However , type II NBs remain Ase− as normal type II NBs ( Figure 1B–B′ ) , suggesting that the identity of the type II NBs was not affected by Btd RNAi knockdown . 10 . 7554/eLife . 03596 . 003Figure 1 . Loss of Btd eliminates mature INPs in type II NB lineages .", "( A–A′ , C–C′ )", "Wild-type type II NB lineages in a third instar larval brain .", "mCD8-GFP driven by pntP1-GAL4 labels all type II NB lineages ( A–A′ ) or a single type II NB clone ( C–C′ ) .", "Ase− immature INPs , Ase+ immature INPs , and mature INPs are indicated by open arrows , solid arrows , and arrowheads , respectively .", "( B–B′ , D–F′ )", "Btd RNAi knockdown type II NB lineages ( B–B′ ) or type II NB clones homozygous mutant for btdXG81 ( D–E′ ) or btdXA ( F–F′ ) in 3rd instar larval brains produce Ase− immature INPs ( open arrows ) and a few Ase+ daughter cells ( arrows ) but no mature INPs .", "Only 3 out of total 8 type II NB lineages are shown in ( A–A′ ) and ( B–B′ ) .", "In this and all other figures , asterisks indicate type II NBs and scale bars equal to 20 µm .", "Dpn staining alone shows the NB and mature INPs .", "( G–H )", "Quantifications of the number of mature INPs ( G ) and the percentage of type II NB lineages with mature INPs ( H ) in the wild type , Btd RNAi knockdown , and btd mutant type II NB lineages .", "The numbers on top of each bar are the numbers of type II NB lineages analyzed except for the numbers for the wt and btd RNAi in ( H ) , which are the number of brain lobes examined .", "The mean and stdev for btdXG81 and btdXA in ( H ) are calculated by bootstrapping .", "**p < 0 . 01 , *p < 0 . 05 ( Student t test ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 03596 . 00310 . 7554/eLife . 03596 . 004Figure 1—figure supplement 1 . Expression of mouse Sp8 ( mSp8 ) .", "( A–A′′ ) and Drosophila Btd ( B–B′′ ) rescues the loss of mature INPs in btd mutant type II NB clones .", "Multiple mature INPs are observed in btd mutant type II NB clone that expresses UAS-mSp8 ( A–A′′ ) or UAS-btd ( B–B′′ ) .", "Type II NBs , Ase− immature INPs , Ase+ immature INPs , and mature INPs are indicated by asterisks , open arrows , solid arrows , and arrowheads , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 03596 . 004 To confirm that the loss of INPs indeed results from the knockdown of Btd rather than off-target effects of UAS-Btd RNAi , we generated btd mutant type II NB clones using two loss-of-function alleles , btdXA and btdXG81 ( Wimmer et al . , 1993; Estella and Mann , 2010 ) .", "Consistent with the Btd RNAi knockdown , all btdXG81 mutant and 90% of btdXA mutant type II NB clones failed to generate any mature INPs except for 4–6 Ase+ Dpn− cells ( Figure 1D–F′ , G–H ) .", "Moreover , about 40% of btd mutant type II NBs ectopically express Ase , making them appear as type I NB lineages ( Figure 1E ) .", "The loss of INPs resulting from the Btd RNAi knockdown and btd loss-of-function mutations suggests that Btd is required for the generation of INPs .", "Remarkably , the loss of INPs in btd mutant clones can be similarly rescued by the expression of mouse Sp8 or Drosophila Btd ( Figure 1—figure supplement 1 ) , suggesting that mammalian Sp8 could have a conserved role in promoting the generation of transient amplifying INPs .", "Since the loss of mature INPs occurred even when Ase was not ectopically expressed in btd mutant type II NBs , the loss of INPs is not primarily due to the ectopic Ase expression or transformation of type II NBs into type I NBs .", "Therefore , we first focused our phenotypic analyses on lineages without the ectopic Ase expression in the NB .", "We also used the btdXG81 allele for further mutant phenotypic analyses below , given that btdXG81 shows slightly stronger phenotypes than btdXA .", "Why does the loss of Btd lead to the elimination of mature INPs ?", "When mature INPs are eliminated in the absence of Btd , the type II NBs without the ectopic Ase expression still produce Ase− immature INPs and a few Ase+ Dpn− daughter cells .", "In normal type II NB lineages , Ase+ Dpn− cells can be either Ase+ immature INPs or GMCs .", "Therefore , three possible scenarios could happen when mature INPs are eliminated in the absence of Btd:", "1 ) Ase− immature INPs differentiate into GMCs instead of Ase+ immature INPs;", "2 ) Ase− immature INPs differentiate into Ase+ immature INPs , which then directly differentiate into neurons/glia without further dividing;", "3 ) Ase− immature INPs differentiate into Ase+ immature INPs , which in turn differentiate into terminally dividing GMCs .", "To distinguish these possibilities , we first wanted to determine if Ase− immature INPs still differentiate into Ase+ immature INP in the absence of Btd by examining the expression of INP specific marker R9D11-CD4-tdTomato and progenitor marker Miranda ( Mira ) in the Ase+ cells next to the Ase− immature INPs .", "R9D11-CD4-tdTomato utilizes a DNA fragment R9D11 from the erm promoter to drive the expression of CD4-tdTomato ( Han et al . , 2011 ) .", "In normal type II NB lineages , R9D11-CD4-tdTomato is first turned on in Ase+ immature INPs and becomes stronger as INPs mature ( Figure 2A–A′ ) , which is similar to R9D11-mCD8-GFP ( Zhu et al . , 2011 ) .", "Mira is expressed in all NBs as well as INPs but not ( or very weakly ) in GMCs ( Figure 2—–figure supplement 1 ) .", "In Btd RNAi knockdown type II NB lineages without mature INPs , we found that R9D11-CD4-tdTomato was expressed in Ase+ daughter cells next to the Ase− immature INPs but its overall expression was much weaker than that in normal type II NB lineages ( Figure 2B–B′ , I ) .", "Consistently , Mira is also expressed in those Ase+ cells next to the Ase− immature INPs in the Btd RNAi knockdown type II NB lineages ( Figure 2—figure supplement 1 ) .", "The expression of R9D11-CD4-tdTomato and Mira suggests that Ase− immature INPs still differentiate into Ase+ immature INP in the absence of Btd as in wild-type type II NB lineages ( Figure 2J ) . 10 . 7554/eLife . 03596 . 005Figure 2 . Loss of Btd results in ectopic nuclear Pros in immature INPs and premature differentiation of Ase+ immature INPs into GMCs .", "( A–A′ )", "R9D11-CD4-tdTomato is expressed in Ase+ immature INPs ( solid arrows ) and mature INPs ( arrowheads ) but not in Ase− immature INPs ( open arrows ) in a wild-type type II NB lineage .", "( B–B′ )", "R9D11-CD4-tdTomato remains expressed in Ase+ daughter cells ( solide arrows ) next to the Ase− immature INPs ( open arrows ) when mature INPs are eliminated by Btd RNAi knockdown .", "( C–C′ ) pH3 is not detected in Ase+ immature INPs ( solid arrows ) in a wild-type type II NB lineage .", "( D–E′ ) pH3 is expressed in Ase+ daughter cells ( solid arrows ) that are the furthest from the Ase− immature INPs ( open arrows ) in a Btd RNAi knockdown type II NB lineage without mature INPs ( D–D′ ) or btd mutant type II NB lineages ( E–E′ ) .", "( F–F′ )", "Nuclear Pros is not expressed in Ase− immature INPs ( open arrows ) in a wild-type type II NB lineage .", "( G-H′ )", "Nuclear Pros is ectopically expressed in both Ase− immature INPs ( open arrows ) and Ase+ cells ( solid arrows ) in Btd RNAi knockdown ( G–G′ ) or btd mutant ( H–H′ ) type II NB lineages .", "( I ) Quantifications of relative overall expression levels of R9D11-CD4-tdTomato in wild-type ( A–A′ ) and Btd RNAi knockdown ( B–B′ ) type II NB lineages .", "( J ) A diagram of neurogenesis patterns in type II NB lineages in the presence or absence of Btd . DOI: http://dx . doi . org/10 . 7554/eLife . 03596 . 00510 . 7554/eLife . 03596 . 006Figure 2—figure supplement 1 . Mira expression in Btd RNAi knockdown type II NB clone .", "( A–A′ )", "Mira is expressed in INPs ( yellow arrows ) in a wild-type type II NB lineage and forms a basal crescent ( white arrows ) at metaphase .", "( B–B′ )", "In a Btd RNAi knockdown type II NB lineage without mature INPs , Mira is expressed in Ase+ daughter cells ( yellow arrows ) next to the Ase− immature INP ( open arrows ) but does not form a basal crescent at metaphase in the dividing Ase+ cell ( white arrows ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 03596 . 006 Next we asked if Ase+ immature INPs differentiate into neurons/glia directly or GMCs in the absence of Btd .", "GMCs express both Ase and nuclear Pros and divide terminally but do not form a Mira crescent while dividing .", "If Ase+ immature INPs directly differentiate into neurons/glia , then all the Ase+ daughter cells should be Ase+ immature INPs and none of them should be dividing .", "In contrast , if Ase+ immature INPs differentiate into GMCs , then some Ase+ daughter cells will become mitotically active and express nuclear Pros but will not form a Mira crescent at the metaphase or telophase .", "Immunostaining with the mitotic marker phospho-histone 3 ( pH3 ) showed that unlike Ase+ immature INPs , which never become pH3-positive ( Figure 2C–C′ ) , some Ase+ daughter cells generated in both Btd RNAi knockdown type II NB lineages without mature INPs ( Figure 2D–D′ ) and btd mutant type II NB lineages became mitotically active ( Figure 2E–E′ ) .", "However , the pH3 positive cells were always the furthest from the Ase− immature INPs among the Ase+ daughter cells ( Figure 2D–D′ ) , suggesting that the Ase+ daughter cells divide terminally like GMCs .", "Consistently , unlike in mature INPs , which form a Mira crescent at metaphase ( Figure 2—figure supplement 1 ) , we did not observe any Mira crescents in the Ase+ daughter cells at metaphase ( Figure 2—figure supplement 1 ) .", "The terminal division and the lack of the Mira crescent strongly argue that Ase+ immature INPs differentiate into GMCs in the absence of Btd .", "To further confirm that late immature INPs differentiate into GMCs in the absence of Btd , we then examined the expression of nuclear Pros in the Ase+ daughter cells .", "Nuclear Pros is a cell fate determinant of GMCs .", "In normal type II NBs lineages , Pros is expressed in the cytoplasm of Ase+ immature INPs and mature INPs and in the nucleus of GMCs and post-mitotic neurons , but not in type II NBs or Ase− immature INPs ( Figure 2F–F′ ) .", "If Ase+ immature INPs differentiate into GMCs in the absence of Btd , we expected that some of the Ase+ daughter cells express nuclear Pros .", "Interestingly , immunostaining of Pros showed that nuclear Pros was expressed not only in all Ase+ daughter cells but also in Ase− immature INPs generated in Btd RNAi knockdown or btd mutant type II NB lineages ( Figure 2G–H′ ) .", "Given that Pros promotes cell cycle exit and GMC differentiation and that forced expression of Pros is sufficient to eliminate INPs in type II NB lineages ( Li and Vaessin , 2000; Choksi et al . , 2006; Bayraktar et al . , 2010 ) , the ectopic expression of nuclear Pros in immature INPs resulting from the loss of Btd very likely promotes the premature differentiation of Ase+ immature INPs into GMCs and cell cycle exit , leading to the loss of mature INPs ( Figure 2J ) .", "These results also reveal that it is Btd that is responsible for the suppression of Pros in immature INPs .", "To determine if the ectopic expression of nuclear Pros in immature INPs is indeed responsible for the elimination of mature INPs in the absence of Btd , we next examined if reducing Pros expression was able to rescue the elimination of INPs in Btd RNAi knockdown or btd mutant type II NB lineages .", "To reduce Pros expression , we either removed one wild-type copy of pros or knocked down Pros by RNAi in type II NB lineages .", "Remarkably , the elimination of mature INPs resulting from the Btd RNAi knockdown was nearly fully rescued even just by removing one wild-type copy of pros ( Figure 3A–D′ , I–J ) .", "Unlike Btd RNAi knockdown in wild-type background , which resulted in a completely elimination of mature INPs in about 50% of type II NB lineages ( Figure 1B–B′ , G–H , Figure 3B–B′ , I–J ) , knockdown of Btd in pros17 or pros10419 heterozygous mutant animals no longer led to an obvious loss of mature INPs ( Figure 3D–D′ , I–J , and data not shown ) , although type II NB lineages develop normally in pros17 or pros10419 heterozygous mutant animals ( Figure 3C–C′ , I–J , and data not shown ) .", "Similarly , the loss of INPs in btd mutant clones was also largely rescued when btd mutant type II NB clones were generated in pros17 heterozygous mutant background ( Figure 3—figure supplement 1 ) . 10 . 7554/eLife . 03596 . 007Figure 3 . Reducing Pros rescues the elimination of INPs resulting from the loss of Btd .", "( A–D′ )", "The loss of INPs resulting from Btd RNAi knockdown is rescued in pros17 heterozygous mutant background .", "Only two lineages are shown in each brain .", "( A–A′ )", "Wild-type type II NB lineages have multiple mature INPs .", "( B–B′ )", "Btd RNAi knockdown causes a loss of mature INPs .", "( C–C′ )", "Type II NB lineages in pros17 heterozygous mutant larvae produce a similar number of mature INPs as in wild-type larvae .", "( D–D′ )", "Btd RNAi knockdown no long leads to the loss of mature INPs in pros17 heterozygous mutant type II NB lineages .", "( E–H′ )", "Pros RNAi knockdown rescues the loss of INPs in btd mutant type II NB clones .", "( E–E′ )", "A wild-type type II NB clone has multiple mature INPs .", "( F–F′ )", "A btd mutant type II NB clone contains no mature INPs .", "( G–G′ )", "Pros RNAi knockdown causes overproliferation of mature INPs in a type II NB clone .", "( H–H′ )", "Pros RNAi knockdown rescues the loss of mature INPs in a btd mutant type II clone .", "Arrowheads point to mature INPs in all images .", "( I–L )", "Quantifications of the number of mature INPs ( I–K ) and the percentage of lineages with mature INPs ( J–L ) for the rescue of Btd RNAi knockdown phenotypes in pros17/+ larvae ( I–J ) or the rescue of btd mutant phenotypes by Pros RNAi knockdown ( K–L ) .", "The samples sizes on top of each bar represent the number of type II NB lineages ( I , K , L ) or the number of brain lobes ( J ) .", "The mean and stdev in ( L ) are calculated by bootstrapping .", "**p < 0 . 01 , *p < 0 . 05 ( Student t test ) .", "NS: not significant . DOI: http://dx . doi . org/10 . 7554/eLife . 03596 . 00710 . 7554/eLife . 03596 . 008Figure 3—figure supplement 1 . Reducing Pros rescues the elimination of INPs resulting from the loss of Btd .", "( A–D′ )", "The loss of INPs resulting from Btd RNAi knockdown is rescued by Pros RNAi knockdown .", "( A–A′ )", "Wide-type type II lineages labeled with mCD8-GFP driven by insc-GAL4 have multiple mature INPs .", "( B–B′ )", "Btd RNAi knockdown driven by insc-GAL4 eliminates mature INPs in a subset of type II NB lineages .", "( C–C′ )", "RNAi knockdown of Pros results in overproliferation of mature INPs .", "( D–D′ )", "Simultaneous knockdown of Btd and Pros leads to a similar overproliferation of mature INPs in type II NB lineages as Pros RNAi knockdown alone .", "( E–H′ )", "The loss of mature INPs in btd mutant clones is rescued in pros17 heterozygous mutant larvae .", "( E–E′ )", "A wild-type type II NB clone has multiple mature INPs .", "( F–F′ )", "A btd mutant type II NB clone contains no mature INPs .", "( G–G′ ) pros17 heterozygous mutant type II NB clone contains multiple mature INPs as the wild-type type II NB clone ( A–A′ ) .", "( H–H′ )", "A btd mutant type II NB clone generated in pros17 heterozygous mutant larvae has multiple mature INPs .", "Asterisks indicate NBs and arrowheads point to mature INPs in all images .", "( I–L )", "Quantifications of the number of mature INPs ( I–K ) or the percentage of type II NB lineages with mature INPs ( J–L ) for the rescue of Btd RNAi knockdown phenotypes by Pros RNAi knockdown ( I–J ) or the rescue of btd mutant phenotypes in pros17 heterozygous mutant larvae ( K–L ) .", "The sample size on top of each bar represents the number of lineages ( I , K , L ) or the number of brain lobes ( J ) .", "The mean and stdev in ( L ) are calculated by bootstrapping .", "** , p < 0 . 01; NS: not significant . DOI: http://dx . doi . org/10 . 7554/eLife . 03596 . 008 Consistent with the rescue in pros heterozygous mutant animals , Pros RNAi knockdown also rescued the loss of INPs resulting from the loss of Btd ( Figure 3E–H′ , K–L , Figure 3—figure supplement 1 ) .", "Pros RNAi knockdown led to overproliferation of mature INPs as observed in pros mutant type II NB clones ( Figure 3G–G′ , K , Figure 3—figure supplement", "1 ) ( Bowman et al . , 2008 ) .", "When Pros was knocked down in btd mutant type II NB clones , mature INPs were rescued in all btd mutant clones ( Figure 3H–H′ , L ) .", "In about 70% of btd mutant type II NB clones , Pros RNAi knockdown led to a similar mature INP overproliferation as in wild-type clones .", "In other 30% of btd mutant clones , Pros RNAi knockdown partially or fully rescued mature INPs without causing the overproliferation of mature INPs .", "Similarly , Pros RNAi knockdown also rescued the loss of mature INPs resulting from Btd RNAi knockdown ( Figure 3—figure supplement 1 ) .", "Results from these rescue experiments demonstrate that the ectopic nuclear Pros in immature INPs is indeed responsible for the loss of mature INPs .", "Interestingly , removing one wild-type copy of pros or knocking down Pros not only rescued the loss of mature INPs but also suppressed the ectopic Ase expression in all btd mutant type II NBs ( Figure 3H , Figure 3—figure supplement 1 ) , suggesting that the ectopic Ase expression in btd mutant type II NBs also results from the ectopic expression of nuclear Pros in immature INPs .", "Our results showed that Btd is required to suppress Pros in Ase− immature INPs .", "We next asked if Btd is also required to partially suppress Pros at later stages of INP development .", "In normal type II NB lineages , Pros is absent in Ase− immature INPs but is expressed at low levels in the cytoplasm of Ase+ immature INPs and mature INPs ( Bello et al . , 2008; Boone and Doe , 2008; Bowman et al . , 2008 ) .", "Maintaining the expression of Pros at low levels is essential for the self-renewal of INPs ( Bayraktar et al . , 2010 ) .", "However , the complete elimination of mature INPs makes it difficult to assess the role of Btd in mature INPs .", "Therefore , we used erm-GAL4 ( III ) and erm-GAL4 ( II ) to knock down Btd .", "Both erm-GAL4 ( III ) and erm-GAL4 ( II ) are expressed in Ase+ immature INPs and mature INPs , whereas erm-GAL4 ( II ) is also expressed in Ase− immature INPs except for the newly born Ase− immature INPs ( Xiao et al . , 2012 ) .", "However , knockdown of Btd using either erm-GAL4 ( III ) ( Figure 4A–B′′ , E ) or erm-GAL4 ( II ) ( Figure 4C–D′′ , F ) did not result in any obvious loss of mature INPs in type II NB lineages .", "In line with these RNAi knockdown results , we were able to recover multicellular btd mutant INP clones that were comparable to wild-type INP clones ( Figure 4—figure supplement", "1 ) while we generated btd mutant type II NB clones , indicating that btd mutant INPs were still able to divide multiple rounds like wild-type INPs and did not prematurely differentiate into GMCs .", "These data suggest that Btd likely suppresses Pros expression only in newly born Ase− immature INPs but not in immature INPs at later developmental stages or mature INPs . 10 . 7554/eLife . 03596 . 009Figure 4 . Knockdown of Btd in immature or mature INPs by erm-GAL4 lines does not lead to the loss of mature INPs .", "( A–A′′ , C–C′′ )", "Wild-type type II NB lineages are labeled with mCD8-GFP driven by erm-GAL4 ( III ) ( A–A′′ ) or erm-GAL4 ( II ) ( C–C′′ ) .", "( B–B′′ , D–D′′ )", "Knockdown of Btd in Ase+ immature INPs and mature INPs by erm-GAL4 ( III ) ( B–B′′ ) or in Ase− immature INPs as well as Ase+ immature INP and mature INPs by erm-GAL4 ( II ) ( D–D′′ ) does not cause a reduction of the number of mature INPs .", "Only two lineages are shown in each brain .", "( E–F )", "Quantifications of the number of mature INPs in type II NB lineages in which Btd is knocked down by erm-GAL4 ( III ) ( E ) or erm-GAL4 ( II ) ( F ) .", "NS: not significant . DOI: http://dx . doi . org/10 . 7554/eLife . 03596 . 00910 . 7554/eLife . 03596 . 010Figure 4—figure supplement 1 . Btd likely does not function in mature INPs .", "( A ) A wild-type INP clone with 4 post-mitotic cells .", "( B ) A btd mutant INP clone with 6 post-mitotic cells .", "INP clones labeled with mCD8-GFP are outlined by dashed circles .", "INP clones are identified as clones that have more than two cells but no NBs . DOI: http://dx . doi . org/10 . 7554/eLife . 03596 . 010 Our btd mutant MARCM analyses showed that in addition to the loss of INPs , Ase was ectopically expressed in about 40% of btd mutant type II NBs .", "We showed previously that PntP1 is expressed in type II NBs as well as Ase− and Ase+ immature INPs ( Figure 5A–A′′ ) ( Zhu et al . , 2011 ) .", "Inhibiting PntP1 activity results in ectopic Ase expression in type II NBs and elimination of INPs ( Zhu et al . , 2011 ) .", "Therefore , we wondered if PntP1 expression was reduced or even lost in btd mutant type II NBs .", "Immunostaining of PntP1 showed that PntP1 was expressed at reduced levels in most btd mutant type II NB lineages without the ectopic Ase expression ( Figure 5B–B′ ) .", "The reduction is about 10% in the NBs and 50% in the immature INPs ( Figure 5E–F ) .", "In those btd mutant clones , PntP1 was also detected in the Ase+ daughter cells next to the Ase− immature INPs ( Figure 5B–B′ ) , providing additional evidence to support that Ase− immature INPs still differentiate into Ase+ immature INPs in the absence of Btd .", "However , in btd mutant type II NB lineages with the ectopic Ase expression in the NB , PntP1 was largely abolished in both the NBs and their progeny ( Figure 5C–C′ , E–F ) .", "The correlation of the ectopic Ase expression in the NB and the severe reduction or loss of PntP1 suggesting that the ectopic Ase expression in btd mutant type II NBs could result from the severe reduction or loss of PntP1 expression . 10 . 7554/eLife . 03596 . 011Figure 5 . PntP1 expression is reduced in btd mutant type II NB clones .", "( A–A′′ )", "PntP1 is expressed in the NB ( * ) , Ase− immature INPs ( open arrows ) , as well as Ase+ immature INPs ( solid arrows ) in a wild-type type II NB clone .", "( B–B′′ )", "PntP1 expression is much lower in a btd mutant type II NB clone without the ectopic Ase expression in the NB than that in a neighboring btd heterozygous type II NB lineage .", "The reduction is particularly obvious in the Ase− immature INPs ( open arrows ) .", "Note that PntP1 remains expressed in Ase+ daughter cells ( arrows ) next to the Ase− immature INPs .", "( C–C′′ )", "PntP1 expression is largely abolished in a btd mutant type II NB clone with the ectopic Ase expression in the NB .", "In a neighboring btd heterozygous type II NB lineages , PntP1 is still detected in the NBs ( * ) , Ase- immature INPs ( open arrows ) and Ase+ immature INPs ( arrows ) .", "( D–D′′ )", "Knocking down Pros restores the expression of PntP1 in the NB ( * ) , Ase− immature INPs ( open arrows ) , and Ase+ immature INPs ( arrows ) in a btd mutant clone to levels comparable to those in a neighboring btd heterozygous type II NB lineage .", "Wild-type ( A–A′′ ) or btd mutant type II NB clones ( B–D′′ ) are outlined by dashed lines and neighboring btd heterozygous type II NB lineages ( B–D′′ ) are marked with dotted lines .", "( E–F )", "Quantifications of PntP1 expression levels in type II NBs ( E ) and Ase− immature INPs ( F ) in btd mutant type II NB clones relative to neighboring type II NB lineages in the same brains .", "**p < 0 . 01; NS: not significant . DOI: http://dx . doi . org/10 . 7554/eLife . 03596 . 01110 . 7554/eLife . 03596 . 012Figure 5—figure supplement 1 . The reduction of PntP1 is unlikely responsible for the loss of INPs in btd mutant type II NB clones .", "( A–A′′ )", "A btd mutant type II NB clone has similar expression levels of PntP1 as a neighboring btd heterozygous mutant type II NB lineage but fails to produce mature INPs .", "( B–B′′ )", "Expressing UAS-PntP1 does not rescue the loss of mature INPs in a btd mutant clone , even though the PntP1 expression in the btd mutant clone is much higher than that in a neighboring btd heterozygous mutant type II NB lineage .", "( C–C′′ )", "Nuclear Pros remains ectopically expressed in Ase− immature INPs and Ase+ daughter cells in a btd mutant type II NB clone expressing UAS-PntP1 .", "( D–D′′ )", "Mature INPs are partially rescued by UAS-PntP1 in a btd mutant type II NB clone .", "In all images , btd mutant clones are outlined by dashed lines and their neighboring btd heterozygous mutant type II NB lineages are marked by dotted lines .", "Open arrows: Ase− immature INPs; solid arrows: Ase+ immature INPs; arrowheads: mature INPs . DOI: http://dx . doi . org/10 . 7554/eLife . 03596 . 012 To determine if the reduction/loss of PntP1 is responsible for the ectopic Ase expression and/or the loss of INPs in btd mutant type II NB lineages , we examined if restoring PntP1 expression was sufficient to suppress ectopic Ase expression and/or rescue the loss of INPs resulting from the loss of Btd by expressing UAS-pntP1 in btd mutant type II NB clones .", "Our results showed that expressing UAS-pntP1 resulted in higher expression of PntP1 in btd mutant type II NB clones than that in neighboring btd heterozygous mutant type II NB lineages and suppressed the ectopic Ase expression in all btd mutant type II NBs ( n = 12 ) ( Figure 5—figure supplement 1 ) .", "However , unlike reducing Pros expression , which rescued mature INPs in nearly all btd mutant type II NB clones ( Figure 3 , Figure 3—figure supplement 1 ) , expressing UAS-pntP1 failed to rescue mature INPs or suppress the ectopic nuclear Pros in Ase− immature INPs or Ase+ daughter cells in the majority of btd mutant clones ( Figure 5—figure supplement 1 ) .", "Only in 3 out of total 10 btd mutant clones expressing UAS-PntP1 , we observed that mature INPs were partially rescued to 9 . 3 ± 3 . 2 per lineages ( Figure 5—figure supplement 1 ) , which is still much fewer than the number of mature INPs ( 20–30 per lineages ) in normal type II NB lineages .", "Consistent with the inability of UAS-pntP1 to fully rescue the loss of mature INPs , we found occasionally that btd mutant clones that did not show an obvious reduction of PntP1 in either the NBs or early immature INPs still failed to generate any mature INPs ( Figure 5—figure supplement 1 ) .", "Therefore , these results demonstrate that the severe reduction/loss of PntP1 accounts for the ectopic Ase expression in btd mutant type II NBs but is not the primary reason for the loss of mature INPs .", "Given that reducing the expression of Pros suppressed the ectopic Ase expression in btd mutant type II NBs , we then asked if reducing Pros expression could also rescue the reduction/loss of PntP1 in btd mutant type II NB clones .", "Indeed , consistent with the suppression of the ectopic Ase expression by Pros RNAi knockdown , PntP1 expression in both the NBs and immature INPs returned to normal levels when the loss of INPs was rescued by Pros RNAi knockdown in btd mutant type II NB clones ( Figure 5D–F ) .", "These results suggest that the reduction/loss of PntP1 in btd mutant type II NB clones is due to the ectopic Pros expression in immature INPs .", "However , given that ectopic nuclear Pros is only observed in immature INPs but not in the NBs in btd mutant type II NB clones , the reduction/loss of PntP1 and the subsequent ectopic Ase expression in the NB is most likely a secondary effect of the ectopic nuclear Pros expression in immature INPs .", "Our Btd loss of function analyses demonstrated that Btd is critical for the generation of INPs in type II NBs lineages .", "We next examined if Btd is only expressed type II NB lineages .", "Since Btd antibodies are not available and our in situ hybridization signals of btd mRNAs in the central brain were barely detectable ( data now shown ) , we used the btd-GAL4 as a reporter for btd expression .", "btd-GAL4 is a GAL4 enhancer trap line , in which the GAL4 transgene is inserted at 753bp upstream of the transcription start site of btd ( Estella et al . , 2003 ) .", "btd-GAL4 shows similar expression patterns as endogenous Btd in ventral imaginal discs ( Estella et al . , 2003 ) .", "We found that mCD8-GFP driven by the btd-GAL4 is expressed in all type II NB lineages but not type I NB lineages on the dorsal side of larval brains ( Figure 6A–A′ ) .", "In type II NB lineages , the expression of mCD8-GFP driven by btd-GAL4 is detected in the NB but becomes much stronger in immature INPs next to the NBs ( Figure 6A ) .", "The expression of mCD8-GFP then becomes progressively weak in cells away from the NBs and is barely detectable in some mature INPs distal from the NB ( Figure 6A–A′ ) .", "The expression pattern of btd-GAL4 in type II NB lineages is similar to that of pntP1-GAL4 ( e . g . Figure 1A–A′ ) and is consistent with our results that Btd mainly functions in immature INPs . 10 . 7554/eLife . 03596 . 013Figure 6 . Btd is expressed in type II NB lineages and a subset of type I NB lineages .", "( A–A′ ) mCD8-GFP driven by btd-Gal4 is expressed in all type II NB lineages ( outlined by dashed lines ) but not type I NB lineages ( e . g . arrows ) on the dorsal side of a 3rd instar larval brain .", "The expression of mCD8-GFP becomes progressively weak in cell away from the NB .", "Some mature INPs ( e . g . arrowheads ) distant from the NB have no obvious expression of mCD8-GFP .", "Only seven out of total eight type II NB lineages are shown in this particular focal plane .", "( B–B′ )", "Two type I NB lineages are labeled by mCD8-GFP driven by btd-GAL4 on the ventral side of a 3rd instar larval brain .", "( C–C′ ) mCD8-GFP driven by btd-Gal4 labels a subset of type I NB lineages ( e . g . arrows ) in the ventral nerve cord ( VNC ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 03596 . 01310 . 7554/eLife . 03596 . 014Figure 6—figure supplement 1 . The GAL4 insertion in the btd-GAL4 line does not affect type II NB lineage development .", "( A–A′′ )", "A wild-type type II NB clone contains multiple mature INP .", "( B–B′′ )", "A btd-GAL4 mutant type II NB clone has a similar number of mature INPs as wild-type type II NB clone .", "( C ) Quantifications of the number of mature INPs in wild-type and btd-GAL4 mutant type II NB clones .", "Arrowheads: mature INPs; NS: not significant . DOI: http://dx . doi . org/10 . 7554/eLife . 03596 . 01410 . 7554/eLife . 03596 . 015Figure 6—figure supplement 2 . The loss of INP phenotype resulting from Btd RNAi knockdown is rescued by the expression of mouse Sp8 ( mSp8 ) or Drosophila Btd .", "( A–A′′ )", "Btd RNAi knockdown driven by btd-GAL4 completely eliminates mature INPs in all type II NB lineages in a 3rd instar larval brain .", "( B–B′′ )", "The expression of UAS-mSp8 driven by btd-GAL4 fully rescues the loss of mature INPs in all type II NB lineages resulting from Btd RNAi knockdown .", "( C–C′′ )", "The expression of UAS-btd driven by btd-GAL4 partially rescues the loss of mature INPs in type II NB lineages resulting from Btd RNAi knockdown .", "Arrowheads point to mature INPs in all images .", "( D–E )", "Quantifications of the percentage of type II NB lineages with mature INPs ( D ) and the number of mature INPs ( E ) in 3rd instar larvae with indicated genotypes .", "Sample sizes represent the number of brain lobes ( D ) or the number of type II NB lineages ( E ) .", "* , p < 0 . 05; ** , p < 0 . 01; NS: not significant . DOI: http://dx . doi . org/10 . 7554/eLife . 03596 . 015 In addition to type II NB lineages , mCD8-GFP driven by btd-GAL4 is also expressed in two type I NB lineages on the ventral side of larval brains as well as about 31 type I NB lineages in the ventral nerve cord ( VNC ) ( Figure 6B–C′ ) .", "In those type I NB lineages , mCD8-GFP driven by btd-GAL4 is expressed in the NBs , GMCs , and newly born neurons .", "The expression pattern of btd-GAL4 in single type I NB lineages is similar to those of other NB GAL4 lines such as insc-GAL4 ( e . g . Figure 7D ) , suggesting that Btd likely functions in the NB in these type I NB lineages . 10 . 7554/eLife . 03596 . 016Figure 7 . Btd and PntP1 function cooperatively to promote the generation of INPs .", "( A–C′ )", "Ectopic expression of PntP1 consistently promotes the generation of INP-like cells in Btd-positive type I NB lineages in larval brains .", "( A–A′ )", "A wild-type type I NB lineage labeled by btd-GAL4 has no INP-like cells .", "( B–C′ )", "The ectopic expression of PntP1 driven by btd-GAL4 suppresses Ase in the NB ( * ) and induces the generation of Ase+ Dpn+ INP-like cells ( arrowheads ) ( B–B′ ) , which also express INP-specific marker R9D11-CD4-tdTomato ( C–C′ ) .", "( D–E′ )", "The expression of PntP1 driven by tub-GAL4 suppresses Ase in both wild-type ( D ) and btd mutant ( E ) type I NBs ( * ) but only induced the generation of INP-like cells in the wild-type type I NB clone ( D–D′ ) but not in the btd mutant clone ( E–E′ ) .", "( F–I′ ) insc-GAL4 drives the expression of UAS-mCD8-GFP alone ( F–F′ ) or together with UAS-btd ( G–G′ ) , UAS-pntP1 ( H–H′ ) , or UAS-btd plus UAS-pntP1 ( I–I′ ) in type I NB lineages .", "Images are from the ventral side of larval brains , where only type I NB lineages are observed in wild-type animals ( F–F′ ) .", "The expression of UAS-btd ( G–G′ ) or UAS-pntP1 ( H–H′ ) alone promotes the generation of INP-like cells only in small subset of type I NB lineages ( dashed circles ) .", "The expression of Btd only suppresses/reduces Ase expression in type I NBs ( arrows ) that produce INP-like cells ( G–G′ ) but not in other type I NBs ( arrowheads ) , where PntP1 suppresses Ase in nearly all type I NBs ( e . g . arrows ) regardless of the generation of INP-like cells ( H–H′ ) .", "Co-expression of Btd and PntP1 promotes the generation of INP-like cells nearly in all type I NB lineages ( dashed circles ) ( I–I′ ) .", "( J–J′ )", "Quantifications of the percentage of type I NB lineages that produce INP-like cells in larvae with indicated genotypes .", "The number on top of each bar represents the number of brain lobes except for numbers for the expression of UAS-pntP1 driven by tub-GAL4 , which are the number of clones .", "The mean and stdev for the percentage of wild-type or btd mutant clones expressing UAS-pntP1 driven by tub-GAL4 are calculated by bootstrapping .", "**p < 0 . 01 . DOI: http://dx . doi . org/10 . 7554/eLife . 03596 . 01610 . 7554/eLife . 03596 . 017Figure 7—figure supplement 1 . Btd and PntP1 function cooperatively to induce the generation of INP-like cells in type I NB lineages in the VNC .", "( A–A′ )", "Type I NB lineages labeled by mCD8-GFP driven by btd-GAL4 in the VNCs have no INPs ( e . g . dashed circles ) .", "( B–B′ )", "Expression of PntP1 driven by btd-GAL4 induces the generation of INP-like cells ( small Dpn+ cells ) in nearly all Btd-positive lineages ( dashed circles ) .", "( C–C′ )", "Type I NB lineages labeled by mCD8-GFP driven by insc-GAL4 in the VNC have no INPs .", "( D–D′ )", "Expression of UAS-btd driven by insc-GAL4 neither suppresses Ase in the NB nor induces the generation of INP-like cells in type I NB lineages ( e . g . dashed circles ) in the VNC .", "( E–E′ )", "Expression of UAS-pntP1 driven by insc-GAL4 suppresses Ase in nearly all NBs ( * ) and induces the generation of INP-like cells in a subset of type I NB lineages ( dashed circles ) in the VNC .", "( F–F′ )", "Co-expression of UAS-btd and UAS-pntP1 driven by insc-GAL4 induces INP-like cells in almost all type I NB lineages ( dashed circles ) in the VNC .", "( G ) Quantifications of the percentage of type I NB lineages with INP-like cells in brain lobes with indicated genotypes .", "**p < 0 . 01 . DOI: http://dx . doi . org/10 . 7554/eLife . 03596 . 01710 . 7554/eLife . 03596 . 018Figure 7—figure supplement 2 . Overexpression of Btd promotes the generation of INP-like cells .", "( A–A′′ )", "INP specific marker R9D11-CD4-tdTomato is not expressed in any type I NB lineages ( e . g . arrows ) on the ventral side of a larval brain .", "( B–B′′ )", "Overexpression of Btd induces the generation of INP-like cells in a subset of type I NB lineages on the ventral side of a larval brain as indicated by the expression of R9D11-CD4-tdTomato ( e . g . arrows ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 03596 . 018 In order to determine if the btd-GAL4 reflects the endogenous Btd expression pattern in the central brain , we tried to rescue the btd loss-of-function phenotypes by expressing UAS-mSp8 or UAS-btd driven by btd-GAL4 .", "However , the GAL4 insertion in the btd-GAL4 line does not affect the type II NB lineage development although it causes a lethal mutation of btd ( Figure 6—figure supplement 1 ) .", "Therefore , we tried to rescue Btd RNAi knockdown phenotypes by the expression of UAS-mSp8 or UAS-btd driven by btd-GAL4 instead .", "The expression of UAS-btd RNAi driven by btd-GAL4 completely eliminated mature INPs in nearly all type II NB lineages ( Figure 6—figure supplement 2 ) , which was much stronger than the phenotype of Btd RNAi knockdown driven by pntP1-GAL4 .", "As expected , the expression of UAS-mSp8 driven by btd-GAL4 fully rescued the loss of INPs resulting from Btd RNAi knockdown in all type II NB lineages ( Figure 6—figure supplement 2 ) .", "Similarly , the expression of UAS-btd driven by btd-GAL4 partially rescued the loss of INPs in about 67% of lineages ( Figure 6—figure supplement 2 ) .", "The incomplete rescue by UAS-btd is likely because UAS-btd contains the sequence targeted by UAS-btd RNAi .", "The rescue of Btd RNAi knockdown phenotypes by the expression of UAS-mSp8 and UAS-btd driven by btd-GAL4 together with the strong loss of INP phenotypes in all btd mutant type II NB clones strongly argue that btd-GAL4 expression is likely consistent with the endogenous Btd expression pattern in the central brain .", "Our previous studies showed that ectopic expression of PntP1 could induce the generation of INP-like cells in more type I NB lineages in VNCs than in larval brains ( Figure 7H–H′ , J , Figure 7—figure supplement", "1 ) ( Zhu et al . , 2011 ) .", "Since Btd is expressed in much more type I NB lineages in VNCs than in larval brains and Btd is required to prevent the premature differentiation of INPs , we wondered if ectopic PntP1-induced generation of INP-like cells requires Btd activity and occurs mostly in Btd-positive type I NB lineages .", "To test this idea , we examined if co-expression of PntP1 and Btd in type I NB lineages was sufficient to induce the generation of INPs and if the ectopic PntP1-induced generation of INP-like cells would be impaired in the absence of Btd .", "To coexpress PntP1 and Btd in type I NB lineages , we used either btd-GAL4 to drive the expression of UAS-pntP1 in Btd-positive type I NB lineages or insc-GAL4 to drive the expression of UAS-pntP1 and UAS-btd simultaneously in all type I NB lineages .", "INP-like cells were identified by their expression of Ase and Dpn as well as INP-specific marker R9D11-CD4-tdTomato .", "Since the GAL4 insertion in the btd-GAL4 line causes a lethal mutation in btd ( Estella et al . , 2003 ) , we examined the phenotype of the expression of UAS-pntP1 driven by btd-GAL4 only in btd-GAL4 heterozygous female larvae .", "As shown in Figure 6A–A′ , type II NB lineages in btd-GAL4 heterozygous mutant larvae are indistinguishable from those in wild-type animals ( e . g . Figure 1A–A′ ) .", "Furthermore , as mentioned above , btd-GAL4 homozygous mutant type II NB clones develop normally ( Figure 6—figure supplement 1 ) .", "Therefore , the generation of INPs is not affected in the btd-GAL4 line .", "Interestingly , ectopic expression of PntP1 using btd-GAL4 as a driver induced the generation of INP-like cells in about 90% Btd-positive type I NB lineages in both larval brains ( Figure 7A–C′ , J ) and VNCs ( Figure 7—figure supplement 1 ) .", "Consistently , the co-expression of UAS-pntP1 and UAS-btd driven by insc-GAL4 induced INP-like cells in about 95% of type I NB lineages in both larval brains and VNCs ( Figure 7F–F′ , I–I′ , J , Figure 7—figure supplement 1 ) .", "In contrast , the expression of UAS-pntP1 alone driven by insc-GAL4 only induced INP-like cells in about 10% and 46% of type I NB lineages in larval brains ( Figure 7H–H′ , J ) and VNCs ( Figure 7—figure supplement 1 ) , respectively , although ectopic PntP1 expression suppressed Ase in nearly all type I NBs ( Figure 7H , Figure 7—figure supplement 1 ) .", "The expression of UAS-btd alone neither suppressed Ase nor induced the generation of INP-like cells in VNCs ( Figure 7—figure supplement 1 ) , whereas in larval brains , the expression of UAS-btd driven by insc-GAL4 suppressed/reduced the expression of Ase in the NB and promoted the generation of INP-like cells in about 20% of type I NB lineages ( Figure 7G–G′ , J ) .", "These ectopic INP-like cells induced by the expression of UAS-btd also expressed INP-specific marker R9D11-CD4-tdTomato ( Figure 7—figure supplement 2 ) .", "These results indicate that the generation of INP-like cells induced by the ectopic PntP1 expression requires Btd activity , whereas the expression of either PntP1 or Btd alone has limited ability to induce the generation of INP-like cells in type I NB lineages .", "To further confirm if Btd is required for ectopic PntP1 to induce the generation of INP-like cells , we then examined if the generation of INP-like cells induced by ectopic PntP1 expression would be impaired in the absence of Btd .", "To this end , we expressed UAS-pntP1 in wild-type or btd mutant type I NB clones in VNCs in that there are more Btd-positive type I NB lineages and the expression of UAS-pntP1 can induce INP-like cells in much more type I NB lineages in VNCs than in larval brains .", "Consistent with the induction of INP-like cells in nearly all type I NB lineages when PntP1 and Btd were coexpressed , the efficiency of PntP1 to induce the generation of INP-like cell was drastically reduced in the absence of Btd .", "Our results showed that the expression of UAS-pntP1 driven by tub-GAL4 could have induced the generation of INP-like cells in about 50% of wild-type type I NB clones but not in btd mutant type I NB clones , although the expression of PntP1 equally suppressed the expression of Ase in both wild-type and btd mutant type I NBs ( Figure 7D–E′ , J ) .", "These results provide additional evidence to support that only in the presence of Btd could PntP1 induce the generation of INP-like cells in type I NB lineages .", "Taken together , these results suggest that the generation of INPs requires the cooperative action of PntP1 and Btd .", "Thus this study together with our previous work ( Zhu et al . , 2011 ) identified two key factors , PntP1 and Btd , a combination of which is sufficient to specify type II NB lineages and promote INP generation ." ], [ "The most striking phenotype resulting from the loss of Btd is the elimination of mature INPs .", "In addition , about 40% of btd mutant type II NB lineages ectopically express Ase in the NB and become type I-like NB lineages .", "However , although forced expression of Ase in type II NBs is sufficient to eliminate INPs in type II NB lineages ( Bowman et al . , 2008; Zhu et al . , 2012 ) , the loss of INPs is obviously not primarily due to the ectopic Ase expression or the transformation of type II NB lineages into type I-like NB lineage in that the loss of mature INPs occurs independently of the ectopic Ase expression in most btd mutant or Btd RNAi knockdown type II NB lineages .", "Instead , we demonstrate that the loss of mature INPs in the absence of Btd is due to the premature differentiation of Ase+ immature INPs into GMCs .", "We show that in Btd RNAi knockdown or btd mutant type II NB lineages without the ectopic Ase expression , Ase− immature INPs differentiate into Ase+ immature INPs normally as indicated by the expression of R9D11-mCD8-GFP , Mira , as well as PntP1 in Ase+ daughter cells next to the Ase− immature INPs .", "However , instead of differentiating into mature INPs , we argue that Ase+ immature INPs prematurely differentiate into GMCs based on the following two pieces of evidence .", "First , Ase+ daughter cells eventually undergo terminal divisions as indicated by the positive pH3 staining and the position of the pH3-positive cells .", "Second , unlike mature INPs , the dividing Ase+ daughter cells do not form basal Mira crescent at metaphase .", "The terminal division and the lack of Mira crescent during the division are two unique features that distinguish GMCs from INPs in addition to the expression of nuclear Pros ( Ikeshima-Kataoka et al . , 1997; Matsuzaki et al . , 1998; Schuldt et al . , 1998 ) .", "Therefore , the elimination of mature INPs resulting from the loss of Btd is due to the premature differentiation of Ase+ immature INPs into GMCs .", "Why does the loss of Btd lead to premature differentiation of INPs ?", "Our results show that the loss of Btd results in a reduction or loss of PntP1 in type II NBs and immature INPs as well as ectopic expression of Pros in early immature INPs .", "Our previous studies show that PntP1 suppresses Ase in type II NBs and that inhibiting PntP1 activity leads to ectopic expression of Ase in type II NBs and elimination of INPs ( Zhu et al . , 2011 ) .", "Given that the ectopic Ase expression in btd mutant type II NBs is closely associated with the severe reduction or complete loss of PntP1 and that expression of UAS-pntP1 largely suppresses the ectopic Ase expression in btd mutant type II NBs , the severe reduction or loss of PntP1 most likely accounts for the ectopic Ase expression in btd mutant type II NBs .", "However , although the loss of PntP1 could lead to the loss of INPs , we provide several lines of evidence to demonstrate that the elimination of INPs in btd mutant or Btd RNAi knockdown type II NB lineages is primarily due to the ectopic activation of Pros in immature INPs rather than the reduction or loss of PntP1 .", "First , ectopic nuclear Pros is consistently expressed in Ase− immature INPs when mature INPs are eliminated .", "Second , the loss of mature INPs can be fully rescued by Pros RNAi knockdown or even just by removing one wild-type copy of pros .", "Third , Pros RNAi knockdown also rescues the reduction of PntP1 and suppresses the ectopic Ase expression in btd mutant type II NBs .", "In contrast , the expression of UAS-pntP1 fails to rescue mature INPs in most btd mutant type II NB lineages although it largely suppresses the ectopic Ase expression in the NBs .", "Furthermore , the complete elimination of mature INPs is also observed occasionally in btd mutant type II NB lineages without the reduction of PntP1 .", "Therefore , the elimination of mature INPs resulting from the loss of Btd is primarily due to the ectopic Pros expression , which likely promotes premature differentiation of INPs into GMCs and cell cycle exit .", "The severe reduction or loss of PntP1 is responsible for the ectopic Ase expression in btd mutant type II NBs and is more likely a secondary effect due to the ectopic Pros expression and/or the loss of INPs .", "INPs and/or other progeny may provide feedback signals to the NBs as has been demonstrated in other systems ( Yoon et al . , 2008; Hsu et al . , 2014 ) .", "The ectopic expression of Pros in Ase− immature INPs resulting from the loss of Btd suggests that Btd is critical for suppressing Pros expression in Ase− immature INPs .", "Btd was known as a head gap gene .", "It has been suggested that gap factors act largely as transcriptional repressors ( Schroeder et al . , 2004 ) .", "Btd could directly suppress Pros by binding to the pros promoter as a transcriptional repressor .", "Alternatively , Btd could suppress Pros indirectly by regulating the expression or antagonizing the activity of factor ( s ) that activate ( s ) pros expression .", "Our results show that ectopic/overly expression of Btd in type I NB lineages or mature INPs does not lead to overproliferation of type I NBs as observed in pros mutant type I NB lineages .", "Instead , ectopic expression of Btd promotes the generation of INP-like cells from type I NBs and transforms some type I NB lineages into type II-like NB lineages .", "Therefore , it is more likely that Btd suppresses Pros indirectly by regulating the expression or antagonizing the activity of pros activator ( s ) .", "Previous studies have suggested that Ase , Daughterless , Numb , and Erm could activate pros expression ( Reddy and Rodrigues , 1999; Southall and Brand , 2009; Weng et al . , 2010; Yasugi et al . , 2014 ) .", "Since Ase and R9D11-Cd4-tdTomato , which are under the control of erm promoter , are not expressed in Ase− immature INPs in the absence of Btd , it is unlikely that they are involved in the activation of pros in immature INPs .", "It would be interesting to investigate in the future if Numb or Daughterless could activate pros in immature INPs in the absence of Btd .", "In this study , we provided several lines of evidence to demonstrate that Btd and PntP1 function cooperatively to specify type II NB lineages and promote the generation of INPs .", "Results from this study as well as our previous study ( Zhu et al . , 2011 ) show that ectopic expression of UAS-pntP1 or UAS-btd alone can only promote the generation of INP-like cells in a subset of type I NB lineage , whereas ectopic expression of UAS-pntP1 in Btd-positive type I NB lineages or co-expression of UAS-btd and UAS-pntP1 can promote the generation of INP-like cells in nearly all type I NB lineages and transforms all these lineages into type II-like NB lineages .", "Consistently , the ability of PntP1 to promote the generation of INP-like cells in btd mutant type I NB lineages is largely impaired .", "These results suggest that the specification of type II NB lineages and the generation of INPs requires both PntP1 and Btd and that the combinatorial PntP1 and Btd is sufficient to promote the generation of INPs .", "We propose that PntP1 and Btd function cooperatively but through different mechanisms to promote INP generation .", "PntP1 is responsible for the suppression of Ase in type II NBs .", "Meanwhile , PntP1 must be regulating the expression of other unknown target gene ( s ) that are/is essential for the generation of INPs , such as specification of immature INPs , because loss of Ase is not sufficient to promote the generation of INP-like cells in any type I NB lineages .", "Btd likely acts after PntP1 to mainly prevent premature differentiation of INPs into GMCs by indirectly suppressing pros in immature INPs .", "The role of Btd in suppressing Ase in type II NBs is minimal if there is any because unlike PntP1 , which suppresses ase in nearly all type I NBs when it is ectopically expressed , overexpression of Btd only suppresses Ase in a small subset of type I NBs that produce INP-like cells in larval brains .", "Furthermore , Ase is expressed in Btd+ type I NBs , indicating that Btd does not suppress Ase in type I NBs when it is expressed at normal levels .", "Studies in mammals as well as in Drosophila suggest that the Btd/Sp8 could function downstream of Wnt signaling to regulate the expression of Fgf8 as well as Distal-less ( Dll ) and Headcase ( Hdc ) during the forebrain patterning as well as limb development ( Estella et al . , 2003; Treichel et al . , 2003; Kawakami et al . , 2004; Sahara et al . , 2007; Estella and Mann , 2010 ) .", "However , inhibiting Wnt signaling alone in type II NB lineages does not have any obvious phenotypes ( Komori et al . , 2014 ) , indicating that Btd unlikely functions downstream of Wnt signaling in type II NB lineages .", "Whether Fgf8 , Dll , or Hdc could function downstream of Btd to regulate INP generation remains to be investigated in the future .", "In mammals , the Btd homolog Sp8 plays important roles in brain development .", "In the developing mouse forebrain , Sp8 is expressed in cortical progenitors in a mediolateral gradient across the ventricular zone as well as in the lateral ganglionic eminence ( LGE ) and medial ganglionic eminence ( MGE ) ( Sahara et al . , 2007; Zembrzycki et al . , 2007 ) .", "In developing human brains , Sp8 is abundantly expressed in the ventricular zone and the outer sub-ventricular zone where RGs and oRGs reside ( Ma et al . , 2013 ) .", "In addition to its roles in interneuron development and the patterning of developing mammalian brains and spinal cords ( Griesel et al . , 2006; Waclaw et al . , 2006; Sahara et al . , 2007; Li et al . , 2011 ) , it was also shown that the loss of Sp8 led to the reduction of the progenitor pool ( Zembrzycki et al . , 2007 ) .", "Our results show that mammalian Sp8 can rescue the loss of mature INPs resulting from the loss of Btd in Drosophila , suggesting that Btd/Sp8 could have conserved functions across different species .", "It would be interesting to investigate if Sp8 has similar roles in promoting the generation of transient amplifying INPs , such as oRGs , in developing mammalian brains ." ], [ "For btd loss-of-function analyses , yw btdXA FRT19A/FM7c and yw btdXG81 FRT19A/FM7c ( Wimmer et al . , 1993; Estella and Mann , 2010 ) were used for generating btd mutant clones and UAS-btd RNAi ( #29453; Bloomington Drosophila stock Center , Bloomington , Indiana ) for Btd RNAi knockdown .", "Type II NB lineage-specific pntP1-GAL4 ( also named as GAL414−94 ) ( Zhu et al . , 2011 ) and erm-GAL4 ( II ) or ( III ) ( Pfeiffer et al . , 2008; Xiao et al . , 2012 ) were used to drive the expression of UAS-transgenes in type II NB lineages or in immature as well as mature INPs , respectively .", "insc-Gal4 ( Gal41407 inserted in inscuteable promoter ) ( Luo et al . , 1994 ) was used to drive UAS-transgenes in all NB lineages .", "UAS-mCD8-GFP driven by Btd-GAL4 was utilized as reporter for btd expression .", "The R9D11-CD4-tdTomato transgenic line ( Han et al . , 2011 ) was used for labeling Ase+ immature INPs and mature INPs .", "Other fly stocks include: hs-Flpase tub-GAL80 FRT19A; UAS-mCD8-GFP; pntP1-GAL4 for generating type II NB clones; pros17/TM6 Tb , pros10419/Tm3 Sb , and UAS-pros RNAi ( #26745; Bloomington stock ) for rescuing Btd loss-of-function phenotypes .", "For RNAi knockdown analyses of Btd and Pros , larvae were raised at 29°C to increase the expression of UAS-RNAi transgenes after hatching .", "Furthermore , UAS-Dcr2 was expressed together with UAS-RNAi transgenes to enhance the efficiency of RNAi knockdown .", "For clonal analyses , MARCM ( Lee and Luo , 1999 ) clones were induced by 1 hr heat shock at 38°C for 1 day after larval hatching .", "Larval brains were dissected at third instar larval stages for the examination of phenotypes .", "Larval brains were dissected , fixed , and stained as described before ( Lee and Luo , 1999 ) .", "Primary antibodies used in this study include: rabbit anti-Mira ( 1:500 ) , guinea pig anti-Ase ( 1:5000 ) , rabbit anti-Dpn ( 1:500 ) ( a gift from Y . N . Jan ) , rat anti-mCD8 ( Life Technologies , Grand Island , New York , 1:100 ) , mouse anti-Pros ( Developmental Studies Hybridoma Bank , Iowa City , Iowa , 1:20 ) , mouse monoclonal anti-α-tubulin ( Sigma , St . Louis , Missouri , 1:1000 ) , rabbit anti-dsRed ( Clontech , Mountain View , California , 1:500 ) , rabbit anti-PntP1 ( 1:500 , a gift from JS Skeath ) .", "Secondary antibodies conjugated to Cy2 , Cy3 , Cy5 , or DyLight 647 ( Jackson ImmunoResearch , West Grove , Pennsylvania ) were used at 1:100 , 1:500 , or 1:500 , respectively .", "Images were taken with a Zeiss LSM510 confocal microscopy and processed with Adobe Photoshop .", "For quantifications of the number of mature INPs or the percentage of type II NB lineages with mature INPs , we focus on the medial group of type II NB lineages ( lineages DM1–DM6 ) .", "Two-tailed t-tests were used for statistics analyses ." ] ]

[ "Intermediate neural progenitor cells ( INPs ) need to avoid differentiation and cell cycle exit while maintaining restricted developmental potential , but mechanisms preventing differentiation and cell cycle exit of INPs are not well understood .", "In this study , we report that the Drosophila homolog of mammalian Sp8 transcription factor Buttonhead ( Btd ) prevents premature differentiation and cell cycle exit of INPs in Drosophila larval type II neuroblast ( NB ) lineages .", "We show that the loss of Btd leads to elimination of mature INPs due to premature differentiation of INPs into terminally dividing ganglion mother cells .", "We provide evidence to demonstrate that Btd prevents the premature differentiation by suppressing the expression of the homeodomain protein Prospero in immature INPs .", "We further show that Btd functions cooperatively with the Ets transcription factor Pointed P1 to promote the generation of INPs .", "Thus , our work reveals a critical mechanism that prevents premature differentiation and cell cycle exit of Drosophila INPs ." ]

[ "Whereas the majority of cells in the brain are unable to divide to produce new cells , neural stem cells can divide numerous times and have the potential to become many different types of brain cells .", "However , in between these two extremes there is another group of cells called neural progenitors .", "These cells can give rise to multiple types of neurons but , in contrast to stem cells , they can undergo only a limited number of divisions .", "Many of the molecular mechanisms by which stem cells give rise to progenitors are similar in mammals and in the fruit fly Drosophila .", "In the brains of fruit fly larvae , neural stem cells called neuroblasts give rise to ‘intermediate neural progenitors' , each of which can divide between four and six times . Every division generates a replacement intermediate progenitor and a cell called a GMC , which divides one last time to produce two brain cells . Intermediate progenitors must be tightly regulated to ensure that they undergo an appropriate number of divisions: too few divisions will result in a shortage of cells , disrupting brain development , whereas too many divisions will result in the formation of tumors . Now , using Drosophila brains in the laboratory , Xie et al . —and , independently , Komori et al . —have shown that a protein called ‘Buttonhead’ is responsible for maintaining this balance .", "Xie et al . show that deletion of the gene for Buttonhead gene caused the progenitor cells to become GMCs before they had undergone the correct number of divisions .", "Further experiments revealed that Buttonhead prevents this problem by suppressing a protein called Prospero .", "The mammalian equivalent of Buttonhead—a protein called Sp8—can substitute for Buttonhead in Drosophila neural progenitors , suggesting that the observed mechanisms may also apply to mammals .", "Further work is required to test this possibility directly and to examine the involvement of Sp8 in brain development and tumor formation ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "structural biology and molecular biophysics" ]

[ [ "Mediator is a complex of at least 21 subunits , with a mass of greater than 1 million Daltons , conserved from yeast to humans ( reviewed in Kornberg , 2005; Conaway and Conaway , 2011; Ansari and Morse , 2013 ) .", "Genetic studies have shown that Mediator is essential for RNA polymerase II ( pol II ) transcription and required for positive and negative regulation of transcription .", "Biochemical studies have confirmed the importance of Mediator for both basal and regulated transcription , and have demonstrated interactions of Mediator with pol II , with general transcription factors , and with transcriptional activator proteins .", "Through these interactions , Mediator is thought to promote assembly of the entire machinery for pol II transcription .", "Electron microscopy ( EM ) has shown a division of Mediator in three modules , termed Head , Middle , and Tail ( Asturias et al . , 1999 ) .", "EM studies further showed that Mediator structure is conserved from yeast to humans , that Mediator is compact when free in solution , and that a major structural rearrangement occurs upon interaction with pol II , which is surrounded by the Head and Middle modules in the so-called ‘holoenzyme’ ( Asturias et al . , 1999; Dotson et al . , 2000 ) .", "Details of subunit interaction networks within Mediator modules have come from co-expression studies ( Koh et al . , 1998; Lee et al . , 1998; Kang et al . , 2001; Koschubs et al . , 2010 ) and from Mediator-specific ( Guglielmi et al . , 2004 ) and genome-wide ( Uetz et al . , 2000; Ito et al . , 2001 ) two-hybrid approaches .", "The currently accepted subunit assignments are Med 6-8-11-17 ( Srb4 ) -18 ( Srb5 ) -20 ( Srb2 ) -22 ( Srb6 ) in the Head module , Med 1-4-7-9 ( Cse2 ) -10 ( Nut2 ) -19 ( Rox3 ) -21 ( Sbr7 ) -31 ( Soh1 ) in the Middle module , and Med 2-3 ( Pgd1 ) -5 ( Nut1 ) -14 ( Rgr1 ) -15 ( Gal11 ) -16 ( Sin4 ) in the Tail module ( old naming convention in parentheses ) .", "X-ray crystal structures of Head modules from Saccharomyces cerevisiae and Schizosaccharomyces pombe have been determined , with ( Robinson et al . , 2012 ) and without ( Imasaki et al . , 2011; Lariviere et al . , 2012 ) the carboxy-terminal domain ( CTD ) of the largest pol II subunit , Rpb1 , bound to a conserved site on the surface .", "CTD interaction with Head and Middle modules plays an important role in the association of Mediator with pol II and the pre-initiation complex .", "Beyond the X-ray crystallography of the Head module , structural information on Mediator is limited .", "Only small portions of the Middle ( Baumli et al . , 2005; Koschubs et al . , 2009 ) and Tail modules ( Bontems et al . , 2011; Eletsky et al . , 2011; Vojnic et al . , 2011 ) have been solved at near atomic resolution .", "EM at low resolution with subunit-labeling has given an approximate idea of the spatial organization of the modules ( Tsai et al . , 2014; Wang et al . , 2014 ) , and an architectural model for a subcomplex of the Middle module ( lacking Med 1 and 19 ) has been proposed on the basis of cross-linking data and homology modeling ( Lariviere et al . , 2013 ) .", "Recently , reconstituted yeast Head and Middle Mediator modules were visualized by EM in a complex with pol II and a subset of general transcription factors ( Plaschka et al . , 2015 ) .", "Mediator is representative of a large number of macromolecular complexes that are refractory to classical high-resolution structural approaches , due to low native abundance and to conformational and compositional heterogeneity .", "For the study of such systems , data from chemical cross-linking and mass spectrometry can provide multi-protein interaction maps at residue-level resolution ( Murakami et al . , 2013; Erzberger et al . , 2014; Shi et al . , 2014 ) .", "The approach is particularly effective when combined with other structural information , such as electron density maps from EM ( Murakami et al . , 2013 ) and subunit structures from X-ray crystallography ( Robinson et al . , 2012 ) .", "To construct structural models of macromolecular assemblies by combining such diverse types of information , the open-source Integrative Modeling Platform ( IMP ) program was developed ( http://integrativemodeling . org ) ( Russel et al . , 2012 ) .", "IMP translates the data into spatial restraints , computes an ensemble of models by satisfying these restraints as well as possible , and finally assesses the ensemble against the data used or not used in its construction ( Schneidman-Duhovny et al . , 2014 ) .", "IMP has been successfully applied to a number of protein complexes ( Lasker et al . , 2012; Erzberger et al . , 2014; Shi et al . , 2014 ) .", "Here we determined the molecular architecture of Mediator by integrative structure determination , based on chemical cross-links , X-ray crystal structures , homology models , and a cryo-EM electron density map , with the use of IMP .", "Possible configurations of the Mediator subunits were exhaustively sampled to identify those that best satisfied the experimental restraints .", "The resulting model was validated by re-computing it with random subsets of chemical cross-links ( i . e . , ‘jackknifing’ ) ( Brunger et al . , 1993 ) as well as by comparison with known protein structures and protein interaction networks ( Guglielmi et al . , 2004 ) .", "The Mediator model will serve as a basis for studies of Mediator interaction with pol II , general transcription factors , gene activators , and gene repressors ." ], [ "Mediator was isolated from yeast as originally described ( Kim et al . , 1994 ) in the form of a complex with pol II ( holoenzyme ) .", "The complex is not only a functionally relevant form of Mediator , but is also more soluble than free Mediator under the conditions used for lysine directed cross-linking .", "We employed a double affinity-tagging strategy ( Figure 1—figure supplement", "1 ) and obtained 20-fold more stoichiometric holoenzyme than from previous purification procedures .", "The native complex was stable , persisting throughout purification , whereas a complex formed from separately isolated Mediator and pol II was disrupted by the same purification procedures .", "Cross-linking and mass spectrometry were performed on the native holoenzyme , with a mixture of D12-labelled and unlabeled BS3 cross-linking reagents , or with unlabeled BS3 and enrichment of cross-linked trypsin fragments by gel filtration .", "The BS3 reagent bridges free-amine moieties at surface lysines and N-termini with Cα spacing within approximately 25 Å .", "Cross-links were assigned with the Protein Prospector platform and a refined scoring function ( Trnka et al . , 2014 ) .", "The 320 , 767 initial product ion spectra were searched against a database containing the sequences of the yeast transcriptional machinery as well as 520 randomized decoy sequences , identifying 20 , 388 potential cross-linked spectra ( Figure 1A ) .", "Classification of these matches and the application of quality filters resulted in the identification of 3196 holoenzyme cross-linked spectra at a false discovery rate ( FDR , fraction of decoy hits above score cutoff ) of 0 . 7% ( Figure 1A ) .", "These spectra defined 402 unique pairs of linked amino acid residues ( ‘cross-links’ ) with a FDR of 4 . 1% ( Figure 1—source data 1 , Figure 1—figure supplement 2 , Supplementary file 1 ) .", "The majority ( 260 cross-linked fragments ) were between Mediator proteins , 124 were between pol II subunits , and 18 were between Mediator and pol II . 10 . 7554/eLife . 08719 . 003Figure 1 . Holoenzyme cross-linking and modeling results and methodology .", "( A ) Cross-links were identified by searching MS2 product ion spectra against concatenated target and decoy databases containing 52 Saccharomyces cerevisiae transcription proteins +520 sequence randomized versions , respectively .", "Confidence in the spectral assignment is represented by an support vector machine ( SVM ) classifier ( Trnka et al . , 2014 ) .", "The distribution of the target and decoy spectral matches in relation to the acceptance threshold ( red line ) is shown with an overall spectral false discovery rate ( FDR ) of 0 . 7% .", "( B ) Mapping identified cross-linked spectra onto regions of known structure such as the Mediator Head module and RNA polymerase II yields distance distributions that reflect the accuracy of cross-link identification .", "The Cα violation distance of 35 Å is indicated with a dashed line .", "( C ) Demonstration of RNA pol II structural recapitulation from a random starting configuration ( top panel ) of individual pol II subunits represented as rigid bead models and restrained by cross-links ( green links ) and electron microscopy ( EM ) density map ( mesh ) .", "A representation of the converged modeling solution is presented alongside the 12-subunit pol II X-ray structure ( PDB: 1WCM ) for reference .", "( D ) Schematic representation of the Mediator complex cross-linking network .", "Mediator subunits are colored according to their location within the Head , Middle and Tail modules .", "All Inter-subunit cross-links and selected intra-subunit cross-links from the current study as well from ( Lariviere et al . , 2013 ) are represented , colored according to their origin . DOI: http://dx . doi . org/10 . 7554/eLife . 08719 . 00310 . 7554/eLife . 08719 . 004Figure 1—source data 1 . Categorization of cross-links with respect to module and data source . Numbers of unique cross-linked residue pairs ( ‘cross-links’ ) and cross-linked spectral matches used for integrative modeling of Mediator apo-complex ( ‘Total Mediator’ ) as well as total number of holoenzyme cross-links identified in this study ( ‘Total [this study]’ ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 08719 . 00410 . 7554/eLife . 08719 . 005Figure 1—figure supplement 1 . Purification of Native S . cerevisiae Holoenzyme complex .", "( A ) Schematic of the affinity capture and chromatographic fractionation of S . cerevisiae holoenzyme complex from whole-cell extract using a double-affinity tag and stepwise cleavage protocol .", "In the procedure , a mixture of holoenzyme and free Mediator and RNA Pol II are affinity captured , with release of free Mediator through TEV cleavage ( panel B ) and subsequent release of holoenzyme and free Mediator upon 3C protease cleavage ( panel C ) .", "Following the enrichment of Mediator-bound pol II using ion-exchange chromatography , the holoenzyme was purified to homogeneity using size-exclusion chromatography ( panel D ) .", "Mass spectrometry analysis confirmed the presence of all 21 Mediator and 12 pol II subunits ( panel E ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 08719 . 00510 . 7554/eLife . 08719 . 006Figure 1—figure supplement 2 . Annotated product ion spectra for selected cross-linked peptides . HCD product ion spectra of BS3 cross-linked Mediator tryptic peptides classified by Protein Prospector .", "Spectra representing highly confident ( SVM score 5 . 1 ) to less confident ( SVM score 0 . 0 ) classifications are shown .", "The modified lysine position is denoted with a star .", "N-terminal acetylation and carbamidomethylation of cysteine residues ( Cam ) are denoted .", "P , PL , and PLK ions refer to ions formed from dissociation of the cross-linking reagent to lysine bond ( Trnka et al . , 2014 ) .", "The full list of cross-linked Mediator peptides may be viewed online using search key = qzpxihtngx at: http://prospector2 . ucsf . edu/prospector/cgi-bin/msform . cgi ?", "form=msviewer . DOI: http://dx . doi . org/10 . 7554/eLife . 08719 . 006 The pol II component of the holoenzyme provided an internal control , enabling validation of the cross-link dataset by two approaches .", "First , the X-ray crystal structure of pol II and also that of the Mediator Head module could be used to assess the validity of cross-links , as 130 of the 402 high confidence cross-links were between residues in these structures .", "Only five of these 130 cross-links occurred between residues further apart than the 35 Å threshold ( Figure 1B ) .", "This number of violations was therefore consistent with the FDR of 4 . 1% , estimated from the number of decoy database hits .", "Second , we constructed an integrative model of pol II based on the cross-link dataset , a cryo-EM electron density map ( EMD-1883 , 20 . 9 Å resolution using FSC 0 . 5 ) , and X-ray crystal structures of the pol II subunits , using IMP .", "The X-ray crystal structures of the subunits were represented as independent rigid bodies , and the regions not observed in the structure were represented by flexible strings of beads .", "A low resolution EM map was used for comparison with the EM data available for Mediator ( see below ) .", "Starting from scrambled configurations , the positions and orientations of the pol II subunits were optimized with a Monte Carlo procedure ( which applies a large number of random movements guided by the input data ) , resulting in configurations that satisfied the input data well .", "This ensemble of good-scoring solutions recapitulated the known architecture of pol II ( Figure 1C ) .", "The average root-mean-square deviation ( RMSD ) of the solutions with respect to the crystal structure of pol II was 14 Å .", "All subunits were correctly placed , except for Rpb8 and Rpb9 , whose locations were undetermined , due to a lack of cross-link restraints .", "We augmented our Mediator cross-link dataset with 38 Mediator Middle module cross-links from a dataset collected on a recombinant preparation of S . cerevisiae Middle module containing 6 subunits ( Lariviere et al . , 2013 ) .", "The 38 cross-links were selected according to our minimal length requirement of four amino acid residues per cross-linked peptide; three cross-links were common to both datasets .", "This modest overlap is likely due to the use of a different chemical cross-linker , as well as to the presence of additional subunits in our full native Middle module .", "The complete dataset of 294 inter-subunit cross-links defined an interaction map of 19 of the 21 Mediator proteins ( Figure 1D ) .", "The two subunits for which there were no inter-subunit cross-links have been crystallized in complexes with other Mediator subunits: Med6 as part of the Head module ( Robinson et al . , 2012 ) , and Med31 in a complex with the N-terminal region of Med7 ( Koschubs et al . , 2009 ) .", "Both proteins were present in our native holoenzyme preparations , as shown by SDS PAGE and mass spectrometry ( Figure 1—figure supplement 1 ) .", "Two subunits , Med17 ( Head module ) and Med14 ( Tail module ) showed patterns of cross-links split between two or more different Mediator modules .", "The majority of Med17 ( residues 123–687 ) formed an extensive cross-link network with other subunits of the Head module , as expected from the X-ray crystal structure ( Robinson et al . , 2012 ) .", "The N-terminal region of Med17 ( 1–122 ) , not observed in the Head module crystal structure , formed many cross-links with Med7 and Med21 , subunits of the Middle module ( Figure 1D ) .", "Similarly , Med14 , a presumptive Tail module subunit , formed cross-links to other Tail module proteins ( Med2 and 15 ) , but almost exclusively in its C-terminal region ( residues 712–1082 ) , while the N-terminal region and the majority of the protein ( residues 1–711 ) cross-linked instead to Middle module subunits ( Med4 , Med9 , Med10 , and Med21 ) .", "The N-terminal regions of Med17 and Med14 not only extended into the Middle module but also formed many cross-links with each other .", "For these reasons , we redefine the Mediator modules to include the N-terminal domains of Med17 ( residues 1–122 ) and Med14 ( residues 1–711 ) in the Middle module rather than the Head and Tail modules ( Figure 1D , Med14 and Med17 colored by module localization ) .", "Although cross-linking was performed on the holoenzyme , about 90% of the cross-links in our dataset were within Mediator modules ( intra-modular ) or within pol II .", "Due to the paucity of cross-links between Mediator and pol II , and because a cryo-EM map at sufficient resolution for molecular modeling was only available for Mediator without pol II , modeling calculations were performed for Mediator alone .", "The four-step modeling ( ‘integrative structure determination’ ) procedure ( Figure 2—figure supplement", "1 ) entailed: ( 1 ) gathering data; ( 2 ) representing and translating the data into spatial restraints; ( 3 ) sampling the conformational space and identifying good scoring solutions; and ( 4 ) analyzing and assessing the ensemble of solutions .", "All IMP input files , scripts , and output models for this study are available at http://salilab . org/mediator/ .", "In step 1 , the data comprised our holoenzyme cross-link dataset , X-ray crystal structures of some subunits and subunit regions , homology models for some subunits , and the highest resolution cryo-EM map of free Mediator available ( Tsai et al . , 2014 ) .", "In step 2 , we constructed a set of 21 Mediator subunit model representations ( Figure 2—figure supplement 2 ) .", "Atomic models from X-ray crystallography or homology models were represented by rigid bodies ( Figure 2—figure supplement 2 , blue subunit shading ) , and unmodeled subunit regions were represented as flexible strings of beads ( Figure 2—figure supplement 2 , yellow subunit shading ) .", "The entire Head module was represented as a rigid body ( based on the X-ray structure PDB 4GWP ) , with the exception of the unmodeled N-terminus of Med17 ( 1–181 ) , the last 103 residues of Med6 , and other missing regions , all of which were represented as flexible strings of beads .", "Manual docking and the mapping of Med 8 , 18 , and 20 onto 2-D EM projections previously supported a single docking solution for the Head module in the EM density ( Tsai et al . , 2014 ) .", "We performed an exhaustive docking calculation , comparing all possible Head module locations with a cross-correlation scoring function ( Materials and methods ) .", "We found a docking solution that is consistent with the solution proposed previously , with a normalized cross-correlation coefficient approximately 40% better than the next best solution .", "We modeled the Mediator complex with the Head module fixed in a single position corresponding to the highest scoring docking solution .", "Other regions defined by atomic models were Med7C-Med21 and Med7N-Med31 , represented by rigid bodies based on their X-ray structures ( Baumli et al . , 2005; Koschubs et al . , 2009 ) , and Med4-Med9 , based on a homology model ( Lariviere et al . , 2013 ) .", "In addition , we created a homology model of the first 540 residues of Med16 , also in a rigid body representation , based on a seven-bladed β-propeller fold ( proposed and justified below ) .", "The EM map was divided into segments to reflect the modular organization of the complex .", "Since the Head module was docked and fixed in a portion of the density map , the remaining density was segmented into regions corresponding to the Middle and Tail modules on the basis of previous approximate localization of Middle and Tail module subunits within two distinct regions of the EM map ( Tsai et al . , 2014; Wang et al . , 2014 ) .", "During modeling , each Middle or Tail subunit was restrained to the corresponding Middle or Tail EM density .", "EM spatial restraints were based on a Gaussian decomposition of the electron density ( Materials and methods ) .", "The cross-linking data was encoded in a Bayesian scoring function ( Erzberger et al . , 2014; Shi et al . , 2014 ) .", "The scoring function allowed the independent optimization of the relative weights of arbitrary subsets of the cross-linking data .", "We split the cross-link dataset into two subsets composed of inter-module ( 8% of the total ) and intra-module ( 92% of the total ) cross-links ( Figure 1—source data 1 ) .", "In step 3 , we conducted a large number of independent modeling runs .", "The positions and orientations of rigid bodies and flexible strings of beads were repeatedly perturbed in an effort to satisfy the scoring function consisting of the excluded volume , sequence connectivity , EM , and cross-linking restraints .", "A total of 165 , 523 Mediator model configurations were produced .", "The 500 best-scoring models ( solutions ) were grouped on the basis of RMSD into four clusters ( C1-4 , Figure 2—figure supplement 3 ) , the minimum number required to fully represent the main structural differences between the best scoring models .", "To confirm that we had sampled conformational space sufficiently to reach model convergence , we compared two independent halves of the solutions to each other and to the entire set , showing they were all similar to one another ( Figure 2—figure supplement 4 ) .", "The solutions satisfied all excluded volume , sequence connectivity , and EM restraints .", "Intra-module cross-links were satisfied to a high degree ( approximately 95% ) , and inter-modular cross-links to a much lesser degree ( about 10% ) ( Figure 2—figure supplement 3 ) .", "In fact , the Bayesian scoring function automatically assigned a low scoring weight to the inter-modular cross-links , pointing to an inconsistency of the inter-modular cross-links in the holoenzyme dataset with the EM density map of the free Mediator .", "The satisfaction of intra-modular cross-links suggests that the arrangement of Mediator subunits within modules of the holoenzyme is similar to that in the free Mediator , whereas the inconsistency of the inter-modular cross-links with the EM map suggests that motions of the modules relative to one another occur upon association with pol II in the holoenzyme .", "Indeed , EM studies have indicated large motions about hinges between the modules , opening the compact structure of free Mediator for wrapping around pol II in the holoenzyme ( Asturias et al . , 1999; Davis et al . , 2002 ) .", "Based on these observations , the inter-modular cross-links were not considered in our analysis of the free Mediator .", "The four clusters of best-scoring solutions differed in two respects .", "Cluster 3 , which represented a minor subset of best-scoring models ( ∼8% ) , differed from the other clusters by an inversion of orientation of the Middle module .", "This cluster was disregarded because the inverted orientation was inconsistent with prior EM localization experiments ( Tsai et al . , 2014 ) and because of significantly worse intra-module cross-link violation statistics ( Figure 2—figure supplement 3D–E ) .", "The remaining three clusters were virtually identical in the locations of all subunits except for Med5 , Med15 , and Med16 in the Tail module ( Figure 2—figure supplement 3C , G ) .", "Only Cluster 1 showed an arrangement of these three subunits consistent with previous EM localization ( Figure 2—figure supplement 3H ) .", "Cluster 1 also showed the best cross-link satisfaction statistics and had the best average score .", "Cluster 1 was therefore taken to represent the true Mediator subunit architecture .", "The overall precision of our integrative model , computed as the average RMSD of the solutions in Cluster 1 with respect to the cluster center , is 19 Å .", "The precision of the Middle and Tail modules is 24 and 33 Å , respectively .", "These values represent the average fluctuations of the individual residues or beads in 3-D space across the ensemble of solutions comprising the highest scoring cluster .", "Precision defined in this way is not directly comparable to the resolution of an EM map , which refers to the uncertainty of the overall electron density , without regard to the placement of components within the density .", "Although the precision of the Tail module is lower than that of the Middle or Head modules , it is sufficient to position the Tail subunits within the Tail EM density as features with high cross-link density , and consequently high local precision values ( such as the Middle-Tail junction ) , provide strong spatial restraints .", "The overall precision of the Mediator model sufficed to determine the positions and orientations of all Mediator subunits , and 20–40 residue beads could be localized with precisions of 10–50 Å ( Figure 2—figure supplement 5 ) , depending on the density of cross-linking restraints .", "We represented the cluster of solutions by a localization density ( Figure 2 ) , defined as the probability of observing a particular subunit at a specific point in space in the cluster of solutions .", "Because the model is three-dimensional , it depicts the contact surfaces between subunits , with residue-level resolution at many points where cross-links are formed between them .", "This contact information is especially rich and reliable in regions where multiple cross-links are formed and where contacts occur in multiple models in the cluster , as depicted by the darkest boxes in the domain interaction map ( Figure 3 ) .", "Some cross-links anticipated from the domain interaction map were not observed , most likely because cross-linking depends on the occurrence of unprotonated , appropriately oriented , solvent–accessible pairs of lysine residues , and on the formation of tryptic peptides with physicochemical properties amenable to liquid chromatography and mass spectrometry . 10 . 7554/eLife . 08719 . 007Figure 2 . Mediator complex architecture .", "( A ) Mediator subunit localization density map colored by individual subunit .", "( B ) Mediator localization density map ( solid grey ) calculated from the highest scoring model cluster and shown at a threshold level ( T = 0 . 1 ) that most closely matches the volume of the EM density map used as 3D spatial restraint during modeling ( EMD-2634 , T = 0 . 35: Blue mesh ) .", "The position of the three Mediator modules ( Head , Middle , and Tail ) is indicated .", "( C ) Schematics of the architecture of the Mediator complex obtained from the localization density analysis .", "Med14 and Med17-NTD are also schematically represented by splines .", "( D ) Protein–protein interactions derived from published Yeast-two hybrid , immunoprecipitation and sub-complex isolation data ( Uetz et al . , 2000; Ito et al . , 2001; Kang et al . , 2001; Guglielmi et al . , 2004; Zhang et al . , 2004; Beve et al . , 2005; Koschubs et al . , 2010 ) .", "The data is represented by a graph superposed on the EM density map of the complex ( Tsai et al . , 2014 ) .", "Nodes are Mediator subunits , the edges are the observed proteomic interactions , and the green circles are isolated sub-complexes .", "Green and red edges are interactions that are in agreement and in disagreement with the Mediator model , respectively .", "( E ) Localization by labeling ( blue triangle ) and domain deletion ( areas encircled by light red closed lines ) mapped on the EM density map ( Tsai et al . , 2014; Wang et al . , 2014 ) .", "Straight light-red lines represent domain deletion assays that split the complex . DOI: http://dx . doi . org/10 . 7554/eLife . 08719 . 00710 . 7554/eLife . 08719 . 008Figure 2—figure supplement 1 . Schematic of integrative structure determination highlighting the individual data inputs and the four stages in our approach . DOI: http://dx . doi . org/10 . 7554/eLife . 08719 . 00810 . 7554/eLife . 08719 . 009Figure 2—figure supplement 2 . Input Model Representations . Individual Mediator subunit model representations are grouped according by membership within the Head , Middle , and Tail modules .", "For each subunit , a schematic representation of the model substructure is shown below the corresponding bead representation .", "The subunit substructure is mapped onto the protein sequence and coloured according to atomic model coverage ( blue fill ) or bead structure ( yellow ) where each bead in the subunit model is represented by a single box in the schematic representation .", "The multi-protein complex structures from which most subunit atomic models are derived are represented on the right . DOI: http://dx . doi . org/10 . 7554/eLife . 08719 . 00910 . 7554/eLife . 08719 . 010Figure 2—figure supplement 3 . Cluster analysis of the solution ensemble .", "( A ) Number of satisfied intra-module cross-links plotted against the score for all 165 , 523 models .", "The vertical brown line represents the score threshold used to select the best scoring 500 models .", "( B ) RMSD distance matrix for the 500 best-scoring models .", "The distance matrix is represented by a heat map , where the bin color is proportional to the RMSD distance of the corresponding solution pair .", "( C ) Difference between the four clusters represented by a hierarchical graph .", "Clusters 1 , 2 , and 4 have the middle module oriented in the ‘normal’ , expected orientation ( i . e . , Med1 and Med19 are at the bottom and at the top of the module , respectively ) .", "The three clusters differ in the arrangement of the tail subunits .", "Cluster 3 has the middle module oriented in a ‘flipped’ orientation ( i . e . , Med1 and Med19 are at the top and at the bottom of the module , respectively ) .", "( D ) Detailed statistics of cross-link satisfaction for the whole dataset ( top ) , intra-module cross links ( middle ) , and inter-module cross-links ( bottom ) .", "( E ) Student t-test analysis of the intra-module cross-link violation distance .", "( F ) Precision of the four clusters .", "( G ) Statistical properties of the four clusters .", "( H ) Agreement between subunit localization in Cluster 1 ( left ) and the interpretation of localization data mapped on the EM density map of the Tail module ( right ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 08719 . 01010 . 7554/eLife . 08719 . 011Figure 2—figure supplement 4 . Exhaustiveness of sampling and robustness of cross-link data .", "( A ) Comparison of localization density maps calculated from the ensemble of solutions ( 500 best-scoring models ) for the entire sample of models , the first half , the second half , and for jackknifing modeling runs ( where 10% of cross-links were randomly removed ) .", "( B ) Comparison of localization density maps calculated for Cluster 1 for the entire sample of models , the first half , the second half , and for jackknifing runs .", "( C , D , E )", "Precision of Cluster 1 solutions ( diagonal values ) and average RMSD between Cluster 1 solutions ( off-diagonal values ) computed for the four ensembles , considering the whole Mediator complex , the Middle module , and the Tail module . DOI: http://dx . doi . org/10 . 7554/eLife . 08719 . 01110 . 7554/eLife . 08719 . 012Figure 2—figure supplement 5 . Representation of subunit position precision .", "( A ) Root-mean-square fluctuation ( RMSF ) plots for each subunit of the Middle and Tail modules show how precisely the residues of all proteins were localized within the best cluster of models .", "For each subunit , RMSF values are mapped onto a stick representation of their centroid model as a color gradient with low and high values of RMSF colored blue and red , respectively .", "Each subunit model is annotated with several residue numbers for reference .", "( B ) Precision plots show the relative localization precision of each subunit of the Mediator complex , grouped by membership in the Head , Middle , and Tail domains .", "The high localization precision of Head module subunits is due to the Head module X-ray structure being fixed at a single position in the EM map .", "Within the Middle module , the subunits comprising the backbone scaffold ( Med7-21-4-9 ) are all precisely localized . DOI: http://dx . doi . org/10 . 7554/eLife . 08719 . 01210 . 7554/eLife . 08719 . 013Figure 3 . Mediator subunit domain interaction matrix . Average domain–domain contact map calculated for the cluster of best-scoring solutions .", "Long sequences are divided into domains of 200 residues .", "The intensity of the color for each box is proportional to the fraction of models for which the contact between corresponding domains is formed .", "Two domains are in contact when the surface of the beads of one domain is within 10 Å from the surface of any of the beads of the other domain . DOI: http://dx . doi . org/10 . 7554/eLife . 08719 . 013 The Mediator model ( Figure 2A ) fits the EM map used as a modeling restraint ( Figure 2B and Figure 7—figure supplement 1 ) , satisfied most of the cross-link restraints ( Figure 2—figure supplement 3 ) , and was consistent with almost all data from previous subunit interaction and subunit localization studies ( Figure 2C–E ) .", "The model explains subunit interactions deduced from co-expression trials ( Kang et al . , 2001; Beve et al . , 2005; Koschubs et al . , 2010 ) , pulldowns/immunoprecipitation ( Kang et al . , 2001; Zhang et al . , 2004 ) , and yeast two-hybrid analyses ( Uetz et al . , 2000; Ito et al . , 2001; Guglielmi et al . , 2004 ) ( Figure 2D , green lines ) .", "There were only three discrepancies ( Figure 2D , red lines ) , corresponding to the Med3-Med21 , Med1-Med7 , and Med1-Med5 interactions .", "The Med3-Med21 interaction , from two-hybrid data , is clearly spurious , because the proteins are found in different Mediator modules ( Head and Tail ) and our model , consistent with an EM localization study ( Tsai et al . , 2014 ) , places the two subunits more than 100 Å apart .", "A second subunit interaction that is unsupported by our model , between the C-terminal domain of Med1 and Med5 , also comes from two-hybrid data ( Guglielmi et al . , 2004 ) .", "Although it also relates proteins located in different modules ( Middle and Tail ) , the two proteins are less than 30 Å apart in our model .", "The holoenzyme cross-link dataset includes one cross-link of Med5 ( Tail ) to Med1 ( Middle ) and two cross-links of Med5 ( Tail ) to Med15 ( Tail ) .", "Because the Med1-Med5 cross-links are inter-modular , they were assigned lower weights by our scoring function and were not satisfied in the final model .", "It is possible that the interactions occur in alternative conformational states , where Med1 is in close proximity to Med5 .", "A third subunit interaction that is unsupported by our model , between Med1-Med7 , comes from immunoprecipitation data ( Kang et al . , 2001 ) , and cannot be ruled out , because the N-terminus of Med1 is in the vicinity of the 16 C-terminal residues of Med7 , which are unmodeled in the crystal structure and not well localized in our study ( Figure 2—figure supplement 5 ) .", "A lack of lysine residues in the 16 C-terminal residues of Med7 might explain why this putative interaction was not detected by cross-linking .", "Our Mediator model was also validated by comparison with results of previous EM studies that mapped subunit terminal tags or subunit deletions onto 2-D class averages ( Tsai et al . , 2014; Wang et al . , 2014 ) .", "Our detailed 3-D model was consistent with all 2-D mapping results ( compare Figure 2C , E ) .", "It also confirms a proposal that a Med7C-Med21-Med4-Med9 tetramer serves as a structural backbone within the central portion of the Middle module ( Lariviere et al . , 2013 ) ( Figure 4 ) . 10 . 7554/eLife . 08719 . 014Figure 4 . Architecture of the Mediator Middle module .", "( A ) Blow-out views of the Middle module subunit localization maps with the first view and coloring identical to Figure 2A and the second view related by a 180° rotation around the y-axis .", "( B ) Average contact maps calculated for the cluster of best scoring solutions .", "Each square is a contact map calculated between a given pair of Middle module proteins with border length proportional to the length of corresponding subunit sequences .", "The grey-shaded areas indicate observed interactions , with the grey-scale proportional to the fraction of models observing the corresponding interaction .", "The colored circles are observed cross-links , where the green and orange colors represent respectively satisfied and violated cross-links for the cluster center ( i . e . , the solution that has the minimal root-mean-square deviation ( RMSD ) from all the other solutions in the cluster ) .", "( C ) Top view of Middle module with individual panels showing the full localization map of each Middle subunit within its surrounding density , made semi-transparent for visual clarity .", "In the central portion of the Middle module the end-to-end stacking of Med7C-21 ( PDB 1YKH ) and Med4-9 ( Homology Model [Lariviere et al . , 2013] ) heterodimers forms a backbone scaffold ( top panel ) , upon which Med10 and Med19 associate at the Med7C-21 extreme ( left panels ) while Med1 associates closely with the N-terminal portions of Med4-9 ( bottom right panel ) .", "The Med7N-31 complex ( PDB 3FBI ) is located proximal to the Med7C-21 heterodimer and the unmodeled carboxy-terminal domain ( CTD ) of Med4 ( top panel ) .", "The centrally located Med17 NTD ( bottom panel ) is wedged between the Med7C-21-4-9 backbone and Med14 , which forms contacts with Med10 and Med21 at its extreme NTD , while the bulk of its density localizes to Med4-9 and Med1 ( top right panel ) .", "Subunit localization density and ribbon models are colored according to Figure 2A . DOI: http://dx . doi . org/10 . 7554/eLife . 08719 . 014 The proposed structure of the Med7C-Med21-Med4-Med9 tetramer was based on a set of 40 cross-links , an X-ray crystal structure of Med7C-Med21 , and Med4-Med9 modeled as a structural homolog of Med7C-Med21 ( Lariviere et al . , 2013 ) .", "Med7C and Med21 form a four-helix bundle , proposed to stack end-to-end on a four-helix bundle of Med4 and Med9 , in a manner analogous to the packing observed in Med7C-21 crystals .", "In our modeling , we did not impose end-to-end stacking , but rather represented the Med7C-21 crystal structure and the Med4-Med9 homology model as two distinct rigid bodies .", "In our final Mediator model , the ends of the two four-helix bundles are in close proximity ( Figure 4B , C ) , although without enforcing end-to-end helical stacking .", "Satisfaction of the EM restraints in other regions of the Middle module appears to require a distortion of the putative end-to-end interaction ( Figure 4C ) , and many solutions displayed a twist of one four-helix bundle with respect to the other , reflecting the asymmetrical nature of the Middle module EM density .", "The Med7C-Med21-Med4-Med9 tetramer provides a structural scaffold for the association of all remaining Middle module subunits ( Figure 4B , C ) .", "The Middle module can be subdivided into two halves: Med7C-Med21 , with interacting proteins Med10 , Med19 , and Med31; and Med4-Med9 , with interacting protein Med1 .", "The only two proteins that interact extensively with both halves of the Middle module are Med14 and Med17 , which perform special architectural roles in the Mediator complex , discussed in more detail below .", "A Med7N-Med31 heterodimer , connected to the helical Med7C domain by an unstructured 27 amino acid residue linker , is localized to a distinct protrusion within the central portion of the EM map .", "The flexible C-terminal domain of Med4 is localized to the same region .", "The Med4-9 half of the Middle module is defined by a close association of the two large subunits Med1 and Med14 with opposite faces of the helical backbone .", "The N-terminus of Med1 forms an extensive interaction surface with Med4-9 ( Figures 3 , 4 ) , with eight cross-links between residues in the first 418 residues of Med1 and residues of Med4-9 located on one face of the N-terminal four-helix bundle ( Figures 1D , 4B and Supplementary file 1 ) .", "This high density of cross-links leads to a precise localization of the first half of Med1 , with Cluster 1 showing low root-mean-square fluctuations ( RMSFs ) for residues within this portion of the protein .", "In contrast , the C-terminus of Med1 lacks cross-links ( Figures 1D , 4B ) and is restrained only by the EM density , resulting in a lower precision of the model and higher RMSF values ( Figure 2—figure supplement 5 ) .", "The region of the Tail module that abuts the Middle module is a rich subunit interaction hub , composed of residues from the N-termini of Med2 and Med3 and the C-termini of Med15 and Med16 ( Figures 3 , 5 , 6C ) .", "The C-terminus of Med14 also contributes to the structure of this region , consistent with the finding that a C-terminal deletion of Med14 results in the loss of all Tail subunits from Mediator ( Li et al . , 1995 ) .", "The Med2 and Med3 NTDs are modeled with relatively high precision ( Figure 2—figure supplement 5 ) , and structural similarity searches with these NTDs show a number of high similarity hits to coiled-coil proteins , in particular to multiple chains within the highly elongated Fibrinogen trimeric coiled-coil structure ( Figure 5—source data 1 ) .", "We extended this analysis to calculate the odds that each of these NTDs forms a coiled-coil structure , with the use of two independent prediction servers ( Figure 5D ) .", "We found a good correspondence between regions of similarity to coiled-coil proteins and regions predicted with high confidence ( p > 0 . 8 ) to form coiled-coil structures .", "These analyses , together with the co-localization of these domains in our Mediator model and the previous observation that Med2 and Med3 can be isolated as a heterodimer ( Beve et al . , 2005 ) , suggest that the N-termini of Med2 and 3 form a coiled-coil motif in the Middle-Tail contact region . 10 . 7554/eLife . 08719 . 015Figure 5 . Architecture of the Mediator Tail module .", "( A ) Subunit localization within the Tail module .", "For each Tail subunit , the corresponding localization density is shown within a semi-transparent Tail density ( grey ) and colored according to Figure 2A .", "( B ) Average contact maps calculated for the cluster of best-scoring solutions and represented as described in Figure 4B .", "( C ) β-propeller model of the Med16 NTD .", "The location of 7 WD domains within the N-terminus of Med16 , predicted by MSA analysis ( Bourbon , 2008 ) , is shown in the context of the predicted Med16 N-term secondary structure ( bottom panel ) .", "Similarity searches identified a high-confidence match to the 7-bladed β-propeller of the S . cerevisiae vesicle coat protein Sec31 ( PDB 2PM9 ) .", "( D ) Schematic showing the predicted coiled-coil regions of three interacting Tail proteins , Med2 , Med3 and Med15 .", "The co-localization of the Med2/3 N-termini and Med15 C-term ( panel A ) supports the formation of a coiled-coil at this tail locus . DOI: http://dx . doi . org/10 . 7554/eLife . 08719 . 01510 . 7554/eLife . 08719 . 016Figure 5—source data 1 . HHpred comparative modeling results for Tail module proteins . DOI: http://dx . doi . org/10 . 7554/eLife . 08719 . 01610 . 7554/eLife . 08719 . 017Figure 6 . Novel structural insights into Mediator complex architecture and module connectivity .", "( A ) Subunit localization density for Med17 ( blue ) reveals the extension of the unmodeled N-terminal domain to contact Middle module subunits .", "( Left A panel )", "The N-terminus of the Head subunit Med17 ( blue density and beads ) forms an extensive cross-linking network ( green links ) within the Middle module , acting as an inter-modular bridge ( see right A panel and Supplementary file 1 for detailed cross-link information ) .", "Head and Middle module ribbon models are colored light grey and according to their subunit color in Figure 2A , respectively , and unmodeled residues cross-linked to Med17 are represented with light grey beads .", "Only Med17 beads with cross-links are shown for clarity and coordinates are derived from the full-complex centroid model .", "( Right A panel )", "A detailed stick representation of the cross-linking network shown in the left panel .", "( B ) Subunit localization density for Med14 ( salmon ) highlights its unique function as a Mediator scaffold protein spanning ∼220 Å to connect all three modules .", "( Left B panel )", "The N-terminus of Med14 forms an extensive cross-linking network across the full Middle module .", "The structural elements of Med14 and other subunits are represented as in left panel of ( A ) .", "( Right B panel )", "A detailed stick representation of the cross-linking network shown in left panel .", "( C ) The localization of the Med14 CTD to the Middle-Tail junction is observed through a pair of cross-links to a Tail protein located in the region ( Left C panel ) .", "( Right C panel )", "A detailed stick representation of the subunit localization and cross-linking network within the Middle-Tail junction .", "( D ) Subunit localization density for the subunits localized in the central portion of the Tail module .", "( Left D panel )", "Bead representation of the cross-links involving the N- and C-terminal portions of Med16 ( yellow ) with Med5 ( brown ) and Med15 ( green ) , respectively .", "The Med16 N-terminal β-propeller is centrally located in the Tail module and forms extensive interactions with Med5 .", "The Med16 CTD links Med16-Med5 with the remainder of the Tail .", "( Right D panel )", "A detailed stick representation of the cross-linking network shown in left panel .", "Coordinates for both panels of ( C ) and ( D ) are derived for the Tail module centroid model . DOI: http://dx . doi . org/10 . 7554/eLife . 08719 . 017 Med15 is co-localized in our Mediator model with Med2 and Med3 and can be isolated with Med2 and Med3 in a trimeric complex ( Zhang et al . , 2004 ) .", "Central residues of Med15 exhibit homology to Fibrinogen ( Figure 5—source data 1 ) , and may participate in a coiled-coil structure , but it is the C-terminal domain of Med15 ( 850–1081 ) that interacts with Med2 and Med3 in the Middle-Tail contact region .", "The Med15 CTD also interacts with the Med16 CTD , apparently stabilizing the association of Med16 within the Tail , and that of Med5 as well , because truncation of the Med16 CTD results in the dissociation of both Med16 and Med5 from the Mediator ( Tsai et al . , 2014 ) .", "Previous sequence analysis of Mediator subunits identified seven WD repeats in the N-terminal domain of Med16 ( Figure 5C ) ( Bourbon , 2008 ) .", "We extended this analysis and identified a number of high-confidence hits to seven-bladed β-propeller structures .", "The most complete match to a full β-propeller was to Sec31 , a component of the S . cerevisiae coat protein complex ( COPII ) involved in ER vesicle transport ( Figure 5C and Figure 5—source data 1 ) .", "We used a homology model , in which the first 540 residues of Med16 were mapped onto the Sec31 β-propeller structure , in our modeling of the Tail module architecture ( Figure 2—figure supplement 2 ) .", "Two intra-protein cross-links of Med16 could be mapped onto the homology model without violation ( Supplementary file 1 ) .", "In our model , the Med16 β-propeller was located in a central position at the base of the Tail module , making extensive contacts with Med5 ( Figure 6D ) .", "The central location of Med16 within the Tail module and a large interaction surface with Med5 are consistent with the finding that the entire Tail module is destabilized and lost in Mediator preparations from a strain from which the MED16 gene was deleted ( Li et al . , 1995 ) .", "In contrast , Med5 occupies a terminal position within the Tail module , and the MED5 gene can be deleted without disrupting the remainder of the module ( Tsai et al . , 2014 ) .", "Although the majority of Med5 occupies a single location within the Tail module , the N-domain of Med5 follows a path around the outside edge of the Med16 β-propeller domain before interacting with Med15 at a more internal locus ( Figure 6D ) .", "This interaction is consistent with an earlier finding that Med5 can form a tetrameric complex with the Med2-Med3-Med15 trimer in the absence of Med16 ( Beve et al . , 2005 ) .", "Especially noteworthy in our Mediator model are the special roles performed by Med17 and Med14 .", "An integral component of the Mediator Head module , Med17 is of particular interest for a temperature sensitive allele ( srb4-138ts ) in which all pol II-dependent transcription is abolished at the restrictive temperature ( Thompson and Young , 1995; Holstege et al . , 1998 ) .", "N-terminal truncations of Med17 in S . cerevisiae are inviable ( data not shown ) .", "The N-terminal 181 residues of Med17 are disordered in Mediator Head module crystals .", "Our Mediator model reveals a central role of the Med17 N-terminal domain in the connection between the Head and Middle modules .", "The Med17 NTD is strongly localized at a central site in the Middle module , wedged between Med7-Med21-Med4-Med9 backbone density on one side and Med14 density on the other ( Figures 2 , 4 ) .", "The entire path of Med17 from this Middle interaction site to its first modeled residue in the Head module is clearly resolved in the model ( Figure 6A ) .", "The essential Med14 subunit was originally identified as a repressor protein in yeast ( Sakai et al . , 1990 ) .", "Reports of the location of Med14 in the Mediator have been contradictory .", "Med14 truncations uncouple the Tail module , suggesting that Med14 is primarily a Middle module protein ( Li et al . , 1995 ) , but in yeast strains in which the Middle module has been disrupted by subunit deletion , Med14 remains associated with the Tail ( Baidoobonso et al . , 2007 ) .", "Furthermore , Med14 is required along with reconstituted Head and Middle modules for basal transcription in vitro in Mediator-depleted cell extracts ( Cevher et al . , 2014 ) .", "Med14 formed cross-links with components of the reconstituted Head and Middle modules , as well as with Med17 ( Cevher et al . , 2014 ) .", "In our Mediator model , Med14 makes extensive contacts with proteins from all three modules , and is the only Mediator subunit that does so ( Figure 1D ) .", "It spans almost the entire Mediator complex , extending a distance of about 220 Å from N-terminal interactions with Med10-Med19 at the tip of the Middle module to C-terminal interactions at the Middle-Tail contact region ( Figure 6B , C ) .", "Med14 residues 264–600 interact with the Med4-Med9 four-helix bundle ( Figures 3 , 4 , 6B ) .", "Med14 residues 247–350 participate in bridging the Head and Middle modules through multiple contacts with the Med17 NTD .", "Additional interactions with the Head module proteins are suggested by contacts of Med14 residues 601–1082 with Med20 and the C-terminal region of Med17 ( residues 401–687 ) ( Figure 3 ) .", "Plaschka et al . ( 2015 ) recently reported a 9 . 7 Å structure of a yeast ‘core initiation complex’ , comprising the Mediator Head module , a minimal Middle module , pol II , a nucleic acid scaffold , and the general transcription factors TBP , TFIIB and TFIIF , obtained by cryo-EM and chemical cross-linking ( Plaschka et al , 2015 ) .", "The Mediator portion of the cryo-EM map represents only about 50% of the mass of Mediator , as the ‘core initiation complex’ does not include the 480 kDa Tail module or the Middle module protein Med1 .", "The structure shows the ‘neck’ of the Head module binding to pol II near subunits Rpb 4 and Rpb7 , with the ‘mobile jaw’ regions of Med 18 and Med20 extending to contact the TFIIB-binding site and subunits Rpb 3 , 10 , 11 , and 12 .", "Cross-links between subunits within the Head ( 30 unique cross-links ) and Middle ( 71 unique cross-links ) modules are in complete agreement with our three-dimensional architectural model of the free Mediator complex ( Figure 7—figure supplement 1B ) .", "Docking our Mediator model to the EM map of Plaschka et al . ( 2015 ) resulted in a holoenzyme model that was also largely consistent with Mediator to polymerase cross-links ( 12 non-redundant and within the modeled sequence ) identified by Plaschka et al . ( 2015 ) ( Figure 7A , C ) .", "However , a set of 17 Mediator to polymerase cross-links from our study of holoenzyme showed agreement only in the region where the Head module contacts Rpb4 and Rpb7 ( Figure 7B ) .", "Cross-links between the Tail and Middle modules to pol II in our study were not consistent with the map of Plaschka et al . ( 2015 ) ( Figure 7C ) .", "As mentioned above , cross-links between Mediator modules in our study were also poorly satisfied by the EM density for free Mediator used in our integrative modeling .", "Together , these findings suggest a different conformation of the Mediator-polymerase holoenzyme in the presence of the Tail module ( not included in the map of Plaschka et al . ( 2015 ) ) and in the absence of the nucleic acid scaffold and general factors .", "Our results point to a similar contact site between the Head module and pol II to that in the model of the ‘core initiation complex’ , but suggest that a conformational change in the Mediator brings the Tail module in contact with pol II near the DNA binding cleft of pol II in the holoenzyme , a suggestion that is consistent with our own preliminary cryo-EM data on the holoenzyme ( P Robinson , unpublished ) ( Figure 7D ) .", "Such conformational dynamics are in keeping with the idea that interaction between Mediator and pol II in the absence of other factors is largely determined by interactions between the Rpb1 CTD and the neck of the Head module ( Robinson et al . , 2012 ) . 10 . 7554/eLife . 08719 . 018Figure 7 . Holoenzyme cross-linking data indicate different conformational states between the holoenzyme and the core Mediator initiation complex .", "( A ) Docking of our Mediator model to the EM density of Plaschka et al . ( 2015 ) provides a subunit architectural map that is highly consistent with the 12 Mediator to pol II cross-links of Plaschka et al . ( 2015 ) ( non-redundant and within modeled sequence ) , collected on a core initiation complex containing the Mediator Head and Middle modules with pol II , nucleic acid scaffold and the general factors TBP , TFIIB and TFIIF .", "( B ) In contrast , the 17 equivalent Mediator to pol II cross-links of the current study are largely inconsistent with the position of Mediator modules found in the core initiation complex .", "( C ) Distributions of cross-link Cα distances from the two datasets measured in the context of the docked Mediator model .", "( D ) The pattern of holoenzyme cross-links implies a major conformational rearrangement in the presence of the Tail module and absence of DNA scaffold and general transcription factors . DOI: http://dx . doi . org/10 . 7554/eLife . 08719 . 01810 . 7554/eLife . 08719 . 019Figure 7—figure supplement 1 . Further validation of the Mediator model . Agreement between the structural model of the Mediator complex and the data for the core initiation complex collected by Plaschka et al . ( 2015 ) .", "( A ) Comparison between our model ( left ) , the EM map of the apo-mediator complex ( center ) ( Tsai et al . , 2014 ) , and the Mediator Head-Middle EM density from the core initiation complex ( right ) ( Plaschka et al . , 2015 ) .", "( B ) Agreement between our model and the CX-MS data from the core initiation complex .", "The histogram is obtained from cross-linked residue–residue distances calculated on our model .", "In the inset , the box plots are calculated on the inter-module , intra-head module , and intra-middle module cross-links .", "The solid box represents the interquartile range of the distance distribution .", "The median is represented by the red line , and the cross-link distances are represented with green crosses .", "To make the comparison more stringent , we removed distances of intra-molecular cross-links calculated on residues pairs that were less than 50 residues apart in the sequence . DOI: http://dx . doi . org/10 . 7554/eLife . 08719 . 019" ], [ "Through integration of data from diverse sources , we have arrived at a 3-D model of Mediator , which explains virtually all previous findings , and which provides a complete picture , including internal details , of the complex .", "One source of data was cross-linking , performed on a native holoenzyme , comprising pol II and Mediator , for several reasons: Mediator structure was thereby analyzed in a more functionally relevant state; the native holoenzyme , isolated from yeast , was much more soluble and stable than a reconstituted holoenzyme formed by mixing pol II and Mediator in vitro; and the X-ray crystal structure of the pol II component of the holoenzyme provided an internal control for the modeling process .", "We restricted our analysis to the internal organization of the three Mediator modules , because their relationship to one another is believed to differ between free Mediator and the holoenzyme , and free Mediator was the source of our EM information .", "Notable features of our 3-D Mediator model include the following:The definition of the three Mediator modules .", "Whereas Med17 and Med14 were previously classified as Head and Tail proteins , the N-terminal domains of both proteins are now seen to form part of the Middle module .", "The two NTDs not only interact with core components of the Middle module but also with each other . The special roles of Med17 and Med14 in the architecture of the entire Mediator .", "Med17 connects Head and Middle modules , while Med14 extends the entire length of Mediator , connecting all three modules .", "The path of the NTD of Med17 , not revealed in X-ray crystal structures of the Head module , can now be followed from a site of interaction in the Middle module to its connection with the body of the protein in the Head module . The complete 3-D arrangement of subunits of the Middle module .", "A Med7-Med21-Med4-Med9 tetramer is seen to form the core of the module , with a cap of Med10-Med19 at one end and a cap of Med1 at the other . The complete 3-D arrangement of components of the Tail module .", "Of particular note are the extensive interactions of the NTDs of Med2 and Med3 and the CTDs of Med 15 and Med16 , which form the region of the Tail module that contacts the Middle module , and which is important for Mediator-activator protein interaction .", "The Med2-Med3-Med15 triad is a binding target of multiple yeast transcriptional activator proteins , including Gcn4 and Gal4 ( Lee et al . , 1999; Myers et al . , 1999; Zhang et al . , 2004 ) .", "Yeast harboring a Med3Δ Med15Δ double knockout exhibits decreased levels of Mediator and of transcription pre-initiation complexes at inducible gene promoters ( Ansari et al . , 2012 ) .", "Secondary structural features of Tail module proteins .", "The NTDs of Med2 and Med3 interact in a coiled-coil motif in the Middle-Tail junction .", "A 7-bladed β-propeller encompassing the N-terminal half of Med16 interacts extensively with Med5 at the base of the Tail .", "Both proteins play roles in transcriptional repression , and expression analysis in yeast Tail mutants has established a functional link between them ( van de Peppel et al . , 2005 ) .", "The 3-D Mediator model not only unifies and resolves discrepancies in the extensive literature on Mediator organization , but also forms a starting point for future investigation .", "High-resolution structures can be docked and additional structural information incorporated in the model , using the same integrative approach we applied here ( Russel et al . , 2012 ) .", "Interactions with pol II , general transcription factors , and transcriptional regulatory proteins can be mapped to the surface .", "Changes associated with activity or due to mutation can be identified and their functional significance understood .", "Our Mediator model could be docked into the Mediator portion of the EM map of Plaschka et al . ( 2015 ) , as association with pol II appears to cause little change in the structure and relative position of the Head and Middle Mediator modules compared to those in the free Mediator EM map used as restraint in our modeling study ( Tsai et al . , 2014 ) ( Figure 7—figure supplement 1A ) .", "Our docked model provides a more detailed map of the locations of Middle module subunits across the pol II surface , and therefore allows a more complete interpretation of the Mediator portion of the map of Plaschka et al . ( 2015 ) .", "Our model provides a structural basis for the Mediator to pol II cross-links reported by Plaschka et al . ( 2015 ) , which can be mapped with precision onto both the Mediator and pol II surfaces to yield a structurally consistent set of cross-link distances ( Figure 7A , C ) .", "Furthermore , mapping novel Middle module cross-links from Plaschka et al . ( 2015 ) onto our Mediator model , we find additional strong support for the validity of our subunit localizations ( Figure 7—figure supplement 1B ) .", "Also as mentioned above , the cross-links between Mediator modules that we obtained with holoenzyme are inconsistent with the result of docking our Mediator model into the map of Plaschka et al . ( 2015 ) ( Figure 7B , C ) .", "We found multiple cross-links between the Tail module and the region of pol II around the active center cleft , whereas docking our Mediator model into the map of Plaschka et al . ( 2015 ) places the Tail module on the opposite side of pol II .", "We have noted the Mediator conformational change required to satisfy our Tail module-pol II cross-links ( Figure 7D ) .", "Tail module interactions with pol II in the vicinity of the active center cleft may be relevant to the role of Tail module subunits Med14 , 15 and 16 in transcriptional repression at multiple loci in yeast ( Covitz et al . , 1994; Sussel et al . , 1995 ) .", "The success of the integrative modeling approach in the derivation of Middle and Tail module structures warrants application to the issues raised here and to other challenging problems .", "It is of immediate interest to apply IMP to the holoenzyme with an expanded cross-link dataset and a high resolution holoenzyme EM map .", "Further studies may be directed towards a complete RNA polymerase II pre-initiation complex including Mediator , and to additional biological particles of great size and complexity ." ], [ "The S . cerevisiae Holoenzyme was purified as an intact complex using a yeast strain in which both endogenous Mediator and RNA Pol II were affinity tagged .", "To achieve double tagging the following genetic manipulations were performed in the CB010 background ( Matα pep4::HIS3 prb1::LEU2 prc1::HISG can1 ade2 trp1 ura3 his3 leu2–3 leu2–112 ) .", "First , the N-terminus of the essential Mediator Head module subunit , Med17p , was TAP-tagged using the N-terminus tagging cassette , including Kluyveromyces lactis TRP1 marker gene , amplified from vector pBS1761 as described previously ( Puig et al . , 2001 ) .", "The MED17 gene was returned to endogenous promoter regulation following Cre-mediated LoxP recombination driven from pSH47 ( Ura+ ) .", "The C-terminus of the largest RNA Pol II subunit , Rpb1p , was tagged with a custom protein G affinity tag with 3C PreScission protease cleavage site .", "The vector for C-terminal protein G tag cassette amplification [pCEMM-CTAP ( KanMX ) ] was first generated by sub-cloning a 1633 bp fragment containing the Tn903 KanMX marker gene with flanking LoxP sites from pUG6 between the XhoI ( 597 ) and NotI ( 616 ) sites of mammalian expression vector pCEMM-CTAP ( SG ) , positioned immediately C-terminal to the protein G domain sequence ( Forward primer 5′-3′: actgtcactgaatagctcgaggaacgcggccgccagctgaagcttcgtacg–Reverse primer 5′-3′: ggcggaatttacgtagcggccgcccgcggccgcataggccactagtggatctg ) .", "The Rpb1 C-terminal protein G tagging cassette including the protein G and KanMX elements was PCR amplified from pCEMM-CTAP ( KanMX ) using primers with flanking sequence including 45 bp complementary to Rpb1 genomic DNA immediately upstream/downstream of the STOP codon and sequence encoding the 3C PreScission cleavage site ( 5′-3′: ctggaagttctgttccaaggtcca ) .", "The endogenous yeast complex was purified from whole-cell extracts using an affinity capture method described previously with minor modifications ( Robinson et al . , 2012 ) .", "Double-tagged CB010 were grown in 200L YPAD to 9 . 0 A600 and 3 . 0 Kg cells were lysed by continuous-flow bead beating in A25 buffer ( 25 mM ammonium sulfate , 100 mM HEPES pH 7 . 8 , 1 mM EDTA , 5% glycerol , 5 mM DTT , and 1× protease inhibitor cocktail [0 . 6 µM leupeptin hemisulfate , 2 µM pepstatin A , 1 mM PMSF and 2 . 1 mM benzamidine hydrochloride] ) with 25 µg/ml RNAse A ( Qiagen , Hilden , Germany ) .", "The cell debris was pelleted by centrifugation at 12 , 250×g for 1 hr at 4°C and the supernatant made up to 300 mM ammonium sulfate with the addition of cold 3 . 9 M ammonium sulfate and gentle stirring .", "Nucleic acids were depleted from the lysate by adding 500 ml DEAE ( GE healthcare , Little Chalfont , UK ) pre-equilibrated in A300 buffer .", "After 30 min of stirring the DEAE was pelleted by centrifugation ( 12 , 250×g , 30 min at 4°C ) and the supernatant loaded onto an IgG column before washing in A500 buffer .", "The use of two distinct protease cleavage sites allowed the isolation of stable holoenzyme complex using a two-stage elution approach ( Figure 1—figure supplement 1 ) .", "First-stage TEV cleavage led to the release of free Mediator , with Mediator retention occurring through complex formation with uncleaved RNA pol II .", "Subsequent cleavage with 3C PreScission protease led to the release of both free RNA pol II and holoenzyme complex .", "The order of protease cleavage could be reversed with equivalent yields of holoenzyme complex ( ∼5 mg/3 Kg dry cell mass ) .", "The holoenzyme was further purified from free RNA pol II ( TEV-3C ) or Mediator ( 3C-TEV ) using ion exchange ( 75–600 mM ( NH4 ) 2SO4; HiTrap FFQ ) and size-exclusion ( 200 mM ( NH4 ) 2SO4 , 25 mM HEPES , pH 7 . 8 , 5% glycerol , 2 mM DTT; Superose 6 ) chromatography steps .", "In the case of the TEV-3C cleavage order , the holoenzyme eluted from the IE column as a leading shoulder in the large peak due to free RNA pol II .", "Using the 3C-TEV cleavage order , the holoenzyme was retained longer in IE than free Mediator and could be resolved as an independent peak .", "Prior to cross-linking , holoenzyme samples were exchanged into phosphate buffer containing: 150 mM NaPO4 , 150 mM KOAc , pH 7 . 5 , 5% glycerol and 2 mM DTT by dialyzing against three changes of buffer .", "Cross-linking was then performed by one of two procedures: MS Peaklists were generated such that all monoisotopic ions detected within the selection window of the linear ion trap ( 4 m/z units ) were annotated as possible precursors to a given product ion spectrum , as described previously ( Robinson et al . , 2012; Trnka et al . , 2014 ) .", "Peaklists were first searched against the full SwissProt database to check the composition of the sample .", "Cross-link searches were then carried out against a target database consisting of the 52 components of the S . cerevisiae Preinitiation Complex .", "For each polypeptide sequence , 10 randomized versions were generated and concatenated to the target database for a total of 572 sequences ( 52 target and 520 decoy ) .", "Decoy protein sequences were the same length as their corresponding target sequences and sampled from the natural distribution of S . cerevisiae amino acid frequency .", "Peaklists were then searched against this database using Protein Prospector ( version 5 . 13 . 1 ) to assign putative cross-linked peptides ( Trnka et al . , 2014 ) .", "Carbamidomethylation of cysteine was considered as a fixed modification .", "N-terminal methionine loss with and without acetylation , peptide N-terminal glutamine conversion to pyroglutamate , oxidation of methionine , and ‘dead-end’ modification of lysine and the protein N-terminus by semi-hydrolyzed BS3 were considered as variable modifications in addition to cross-linking by BS3 .", "Up to 3 variable modifications per peptide were considered .", "Mass tolerances of 13 ppm and 20 ppm were used for precursor and product ions respectively .", "Trypsin specificity with 3 missed cleavages was used to generate theoretical peptides .", "85 product ion signals from each MS2 spectrum were used for assessment .", "For experiments involving isotopic mixtures of cross-linking reagents , the searches were performed twice , once specifying heavy BS3 and once with light BS3 .", "The search results were later merged .", "Both light and heavy dead-end modifications were considered in each of these database searches .", "For all experiments , cross-link spectral matches ( CSM ) were discarded if the following Protein Prospector parameters fell outside the threshold values: peptide score below 20 , peptide/protein/worse_peptide expectation values above 50 , and score_difference below 0 .", "A linear support vector machine ( SVM ) model was constructed to classify CSMs between decoy and target classes ( Trnka et al . , 2014 ) .", "Briefly , the CSMs were split evenly into two sets .", "SVM models were trained on half of the data and evaluated for their ability to correctly classify true negatives ( hits to the decoy database ) on the test set .", "CSMs were considered decoy matches if either of the two constituent peptides came from the decoy database .", "The final SVM model included a metric reflecting the number of product ions matched to the less fragmented of the two peptides ( score_difference ) and a metric for the overall match of the spectrum ( percentage of the total ion current annotated ) .", "The inclusion of additional metrics did not increase the predictive power of the model .", "An SVM decision value of 0 was taken as the acceptance threshold for reporting cross-linked peptides .", "FDRs were calculated by dividing the number of decoy hits at this threshold by 10 ( to account for the larger decoy database size ) and dividing by the number of hits to the target database .", "CSMs where either peptide was less than 4-amino acids long were discarded , with a few manually validated exceptions .", "When spectra could be interpreted by multiple cross-linked peptide-pairs , CSMs were considered to be unambiguous if the second best match was further than 0 . 3 SVM decision value units from the top match .", "Intra-protein cross-links were selected over inter-protein cross-links if there were still conflicts .", "The remaining decoy matches were also removed from consideration .", "Following these selection criteria , most CSMs identified a single cross-linked peptide-pair .", "The remaining spectral redundancy came exclusively from ambiguous site localizations , where there was sufficient evidence to identify both peptides , but the position of the modified amino acid remained ambiguous .", "In these cases , all possible sites were reported using a spectral identifier and used in subsequent modelling steps .", "Cross-linking results and annotated HCD spectra may be viewed online using Protein Prospector's MS-Viewer program: http://prospector2 . ucsf . edu/prospector/cgi-bin/msform . cgi ?", "=msviewer .", "Search key: qzpxihtngx All raw mass spectrometry files and peaklists used in the study have been deposited to the MassIVE proteomics repository and are available through the ProteomeXchange consortium .", "MassIVE accession: MSV000079237 ProteomeXchange accession: PXD002723 Our integrative structure modeling of RNA Pol II and Mediator complexes proceeds through four stages ( Lasker et al . , 2010; Fernandez-Martinez et al . , 2012; Lasker et al . , 2012 ) ( Figure 2 ) : ( 1 ) gathering of data , ( 2 ) representation of subunits and translation of the data into spatial restraints , ( 3 ) configurational sampling to produce an ensemble of models that satisfies the restraints , and ( 4 ) analysis and assessment of the ensemble .", "The modelling protocol ( i . e . , stages 2 , 3 , and", "4 ) was scripted using the Python Modelling Interface ( https://github . com/salilab/pmi ) , version be72c15 , a library to model macromolecular complexes based on our open source IMP package ( http://salilab . org/imp/ ) , version 829c3f0 ( Russel et al . , 2012 ) .", "Files containing the input data , scripts , and output models are available at http://salilab . org/mediator .", "In the IMP modeling procedure we perform a large number of independent modeling runs , each starting from a random configuration and undergoing thousands of rounds of small random model perturbations , in order to achieve exhaustive sampling of conformational space .", "As we are interested only in models that best satisfy the restraints , we select a small fraction of the models representing the very best scoring solutions ( Figure 2—figure supplement 3A ) .", "These models then undergo pairwise-RMSD clustering to separate out divergent structural states ( Figure 2—figure supplement 3B ) .", "In our study , most of the best scoring models ( 92% , n = 500 ) gave a unique subunit arrangement for 18 of the 21 Mediator subunits .", "The divergent clusters differed only in the relative locations of Med5 , Med15 and Med16 within the Tail module ( Figure 2—figure supplement 3C ) .", "We compared these three clusters in terms of crosslink violation statistics ( Figure 2—figure supplement 3E ) and consistency with prior EM localization experiments ( Figure 2—figure supplement 3H ) .", "A single cluster showed both consistency with earlier localizations and significantly better violation statistics and was therefore deemed to be the Mediator architecture with the highest confidence .", "Modeling studies of RNA pol II employed cross-link data from two sources: 156 cross-links from previous experiments conducted with the free pol II fraction isolated during our holoenzyme SEC purification ( Trnka et al . , 2014 ) , and 108 cross-links from a previous study ( Chen et al . , 2010 ) .", "These sources had 63 cross-links in common .", "Hence , the current study used 201 unique cross-links .", "The atomic structures for the twelve-subunit yeast pol II have been previously determined by X-ray crystallography ( PDB accession ID: 1WCM , [Armache et al . , 2005] ) .", "The crystallographic structure covers 89% of the residues in the pol II complex .", "A 20 . 9 Å resolution density map from single particle EM reconstruction of the pol II complex was also considered ( EMDB accession id: 1883 , [Czeko et al . , 2011] ) .", "The 12 pol II subunits were represented as 15 domains , where Rpb1 was decomposed into 3 domains ( residues: 1–1140 , 1141–1274 , 1275–1733 ) , Rpb2 was decomposed into 2 domains ( residues 1–1102 , 1103–1224 ) , and the remaining subunits were represented as single domains .", "Regions without a crystal structure were modeled as beads containing up to 5 amino acid residues .", "We followed the same 4-stage procedure as described for the Mediator complex above ." ] ]

[ "The 21-subunit Mediator complex transduces regulatory information from enhancers to promoters , and performs an essential role in the initiation of transcription in all eukaryotes .", "Structural information on two-thirds of the complex has been limited to coarse subunit mapping onto 2-D images from electron micrographs .", "We have performed chemical cross-linking and mass spectrometry , and combined the results with information from X-ray crystallography , homology modeling , and cryo-electron microscopy by an integrative modeling approach to determine a 3-D model of the entire Mediator complex .", "The approach is validated by the use of X-ray crystal structures as internal controls and by consistency with previous results from electron microscopy and yeast two-hybrid screens .", "The model shows the locations and orientations of all Mediator subunits , as well as subunit interfaces and some secondary structural elements .", "Segments of 20–40 amino acid residues are placed with an average precision of 20 Å .", "The model reveals roles of individual subunits in the organization of the complex ." ]

[ "Inside a cell , proteins are made from instructions encoded by DNA .", "To produce a particular protein , a section of DNA within a gene is copied into a molecule of messenger ribonucleic acid ( or mRNA ) .", "This process is called transcription and is carried out by an enzyme known as RNA polymerase .", "Transcription begins in a region of DNA called a promoter , which is found at the start of the gene .", "RNA polymerase is brought to the DNA by many proteins , including the so-called Mediator complex .", "Mediator receives signals from within the cell and from the environment , processes the information , and instructs RNA polymerase whether to transcribe the gene or not .", "Mediator performs this important role in all organisms from yeast to humans , but it is not clear how it works .", "A crucial step towards the solution of this problem is to understand the three-dimensional structure of the complex .", "Previous research using a technique called ‘electron microscopy’ showed that Mediator is composed of three modules , referred to as Head , Middle and Tail .", "The images from electron microscopy were not sufficiently detailed to reveal the organization of the proteins within these modules .", "An open-source Integrative Modeling Platform ( IMP for short ) was recently developed to arrive at structural models of large protein complexes from a combination of experimental data and computer models .", "Now , Robinson , Trnka , Pellarin et al . have used this platform to study the Mediator complex .", "First , Robinson , Trnka , Pellarin et al . collected experimental data on the structure of the Mediator complex using two approaches called ‘chemical cross-linking’ and ‘mass spectrometry’ .", "This data was combined with biochemical and structural information from previous studies to generate a three-dimensional model of the structure of the entire Mediator using IMP .", "The model is detailed enough to show the location and orientation of all the proteins in the complex .", "For example , a protein called Med17 connects the Head and Middle modules , while another subunit—known as Med14—spans the entire complex and makes extensive contacts with other proteins in all three modules ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "neuroscience" ]

[ [ "Working memory ( WM ) can hold information for a short period of time to allow further processing in the absence of sensory input ( Cowan , 2017; Oberauer et al . , 2018 ) .", "By bridging this gap between the immediate sensory environment and behavior , WM is a cornerstone for complex cognition .", "It is a very flexible memory system , yet severely limited in its capacity .", "While this capacity is often seen as a general cognitive bottleneck , for simple stimuli , like colors , the capacity is very similar between humans , monkeys , and crows ( Balakhonov and Rose , 2017; Buschman et al . , 2011; Cowan , 2001; Luck and Vogel , 1997 ) .", "Different models have been proposed to conceptualize how this capacity limit arises .", "This work motivated many psychophysical and electrophysiological experiments that in turn led to a spectrum of more refined models of WM ( Ma et al . , 2014 ) .", "‘Discrete models’ of WM argue that a fixed number of items can be stored .", "Once this capacity is reached , an additional item can only be maintained if it replaces a previous item ( Awh et al . , 2007; Fukuda et al . , 2010; Luck and Vogel , 1997; Vogel and Machizawa , 2004; Zhang and Luck , 2008 ) .", "‘Continuous models’ describe WM as a flexible resource that is allocated to individual items .", "A minimum amount of this resource has to be allocated to each item for successful retention , thereby resulting in a capacity limit ( Bays and Husain , 2008; van den Berg et al . , 2012; Wilken and Ma , 2004 ) .", "On the neurophysiological level , models of WM capacity suggest that interference between memory representations ( ‘items’ ) within the neuronal network is a source of information loss and capacity limitation ( Bouchacourt and Buschman , 2019; Lundqvist et al . , 2016; Lundqvist et al . , 2011; Schneegans et al . , 2020 ) .", "Interference may arise due to divisive normalization that appears as competition between items , related to oscillatory dynamics ( Lundqvist et al . , 2016; Lundqvist et al . , 2011 ) , WM flexibility ( Bouchacourt and Buschman , 2019 ) , and neuronal information sampling ( Schneegans et al . , 2020 ) .", "Divisive normalization is a computational principle that acts upon neurons when presenting multiple stimuli simultaneously , it normalizes neuronal responses by creating ‘a ratio between the response of an individual neuron and the summed activity of a pool of neurons’ ( Carandini and Heeger , 2011 , p . 51 ) .", "An effect related to divisive normalization can be observed when two stimuli are presented either individually or simultaneously within the receptive field of a visual sensory neuron .", "The neuron’s firing rate when the stimuli are presented simultaneously becomes normalized by the populations’ responses to each individual stimulus ( Carandini et al . , 1997; Heeger , 1992 ) .", "This effect also occurs in relation to attentive processes and has been formalized into the ‘normalization model of attention’ ( Reynolds et al . , 1999; Reynolds and Heeger , 2009 ) .", "Normalization of neuronal responses is commonly observed in many species throughout the animal kingdom , not just in sensory , but also in cognitive domains ( Carandini and Heeger , 2011 ) .", "Investigations into WM capacity and model predictions focus mostly on humans and monkeys .", "By extending this work to include birds , one can gain a unique comparative perspective .", "Crows have a similar limit in WM capacity and neuronal correlates of WM are comparable to monkeys’ ( Balakhonov and Rose , 2017; Nieder , 2017 ) .", "But while the neuronal architecture of sensory areas is similar between birds and mammals , higher associative areas , critical for WM , do not share a common architecture between the species ( Stacho et al . , 2020 ) .", "Therefore , an outstanding question is whether modern models of WM such as the ‘flexible model’ capture WM capacity in general , or if their predictions ( e . g . , divisive normalization ) are confined to the mammalian neocortex .", "To resolve this , it is crucial to investigate the avian brain to understand how its different organization can produce such similar behavioral and neurophysiological results .", "While the neuronal correlates of WM maintenance in birds have been investigated in some detail ( Diekamp et al . , 2002a; Hartmann et al . , 2018; Rinnert et al . , 2019; Rose and Colombo , 2005; Veit et al . , 2014 ) , a neurophysiological investigation of WM capacity limitation is still lacking .", "The avian forebrain structure , nidopallium caudolaterale ( NCL ) is a critical component of avian WM .", "The NCL is considered functionally equivalent to the mammalian prefrontal cortex ( PFC ) ( Güntürkün and Bugnyar , 2016; Nieder , 2017 ) , as it receives projections from all sensory modalities ( Kröner and Güntürkün , 1999 ) , projects to premotor areas ( Kröner and Güntürkün , 1999 ) , and is a target of dopaminergic innervation ( Waldmann and Güntürkün , 1993 ) .", "To investigate the neurophysiology of WM capacity in birds , we adopted a task design developed for monkeys ( Buschman et al . , 2011 ) to use it with carrion crows ( Corvus corone ) .", "Our animals were trained to memorize an array of colors and to indicate which color had changed after a short memory delay , while we performed extracellular recordings of individual neurons in the NCL using multi-channel probes .", "We expected to find a clear correlate of WM representations in NCL neurons and a load-dependent response modulation based on divisive normalization of neuronal responses .", "This would allow us to evaluate if the behavioral WM capacity observations of crows fit a ‘discrete’ or ‘continuous’ WM resource model .", "If the neuronal responses also fit the contemporary neurophysiological models of WM capacity limitations ( Bouchacourt and Buschman , 2019; Lundqvist et al . , 2016; Lundqvist et al . , 2011; Schneegans et al . , 2020 ) , it would further suggest that crows and monkeys have convergently evolved a similar neurophysiological basis for WM capacity despite a different architecture of the critical forebrain structures ." ], [ "The behavioral performance was influenced by the number of colored squares on the screen .", "It significantly decreased with an increasing number of ipsilateral squares ( median performances , load 1: 95 . 88% , load 2: 78 . 31% , load 3: 58 . 21%; Friedman test: Χ² = 92 . 00 , p < 0 . 001 , Figure 1B ) .", "We ran a generalized linear model with ipsilateral load ( i . e . , load of hemifield where a color changed ) , contralateral load ( i . e . , load of hemifield without a color change ) and their interaction as predictors for performance ( R2adj 0 . 78 , F ( 460 , 456 ) = 555 . 00 , p < 0 . 001 ) .", "We found that the number of ipsilateral colors significantly reduced performance ( βipsi –0 . 177 , t ( 459 ) = –18 . 00 , p < 0 . 001 ) , whereas the number of contralateral colors did not ( βcontra –0 . 021 , t ( 459 ) = –1 . 77 , p = 0 . 0772; Figure 1C ) .", "There was also a significant interaction between ipsilateral and contralateral load ( β = –0 . 024 , t ( 458 ) = –4 . 28 , p < 0 . 001 ) .", "We compared this model to a reduced model , where we omitted the non-significant βcontra and found that this reduced model ( R2adj 0 . 78 , F ( 460 , 457 ) = 828 . 00 , p < 0 . 001 ) explained the performance equally as well ( |ΔLLR| = 0 . 0102 ) .", "Therefore , we conclude that contralateral load by itself did not significantly affect performance .", "We calculated the capacity K ( see Materials and methods ) for all full WM loads ( i . e . , two to five items ) .", "The capacity K peaks at four items ( mean ± SEM: 3 . 05± . 038 , Figure 1—figure supplement 1 ) .", "These observations are very similar to observations made in primates ( Buschman et al . , 2011 ) and fully reproduce our earlier behavioral findings ( Balakhonov and Rose , 2017 ) .", "We recorded 362 neurons from the NCL of two crows performing the WM task ( delayed change localization ) .", "All reported effects were also present in each individual bird ( Figure 3—figure supplements 1–2 ) , we , therefore , pooled the data for population analysis .", "A large subset of neurons responded to the presence of a color ( i . e . , at load 1 ) by substantially increasing or decreasing their firing rate relative to baseline .", "This change in firing rate occurred selectively , depending on the presented color either in the sample ( Figure 2A ) or the delay period ( Figure 2—figure supplement 1 ) .", "For most neurons , this difference in firing rate between the two possible colors became attenuated when the load increased from one to two colors , and it was further attenuated from two to three colors .", "To quantify this effect , we calculated the amount of information about the color identity at a neuron's favorite location as the percent explained variance ( PEV ) during a memory load of one , two , or three items in bins of 200 ms ( see Materials and methods for details ) .", "Most neurons did not sustain information about color ( measured as a significant PEV , henceforth ‘information’ ) throughout the entire sample or memory delay but rather had shorter periods in which the information was significant ( Figure 2A bottom ) .", "To better capture the time points when the individual neurons carried information , we performed a hierarchical clustering analysis of the PEV values of the individual neurons at load 1 ( see Materials and methods for details ) .", "We found a total of seven clusters that were organized into two overarching groups ( Figure 3A ) .", "Group 1 contained neurons ( n = 227 ) that showed peak information during the sample and early delay phase , while group 2 contained neurons ( n = 135 ) that showed peak information during the delay phase .", "For each neuron , we then calculated if it carried a significant amount of color information by applying a permutation test ( for all bins at load 1 , see Materials and methods ) .", "The individual neurons were then further classified into three groups depending on the phase in which they had a significant amount of information ( Figure 3B ) .", "Overall , 37 . 57% ( n = 136 ) of neurons were significant during the sample phase , 9 . 39% ( n = 34 ) of neurons were significant during the memory delay , and 14 . 64% ( n = 53 ) of neurons were significant during both the sample phase and the memory delay ( all proportions of neurons were significantly higher than expected by chance [binomial test , see Materials and methods , all p < 0 . 001] ) .", "Refer to Figure 2A for an example neuron , significant at load 1 with a large differentiation in firing rate between color identities ( a large PEV ) and a loss of differentiation with increasing ipsilateral load .", "Further inspection of individual neuronal activity revealed , however , that a substantial number of neurons responded differently .", "Instead of losing information at higher loads , many neurons gained information ( e . g . , Figure 2B , Figure 2—figure supplement 2 ) .", "Thus , we additionally performed the permutation testing for loads 2 and 3 to determine which neurons had significant information ( see Materials and methods ) .", "We found that many of the neurons that did not have significant information at load 1 did have significant information at load 2 and load 3 ( Figure 3C ) .", "For the memory delay , more than half of the significant neurons we detected were only significant for either load 2 or load 3 , compared to only 36% of neurons that were significant at load 1 ( Figure 3C middle ) .", "By including the higher loads in our analysis , we found a total of 249 ( 68 . 78% ) sample neurons and 94 ( 25 . 97% ) delay neurons .", "For the population analyses , we subsequently pooled all significant neurons into three groups ( one per load ) .", "These pooled groups were then each subdivided into sample and delay neurons ( i . e . , ‘sample-load1’ , ‘delay-load1’ , ‘sample-load2’ , etc . , see Table 1 in the Materials and methods for an overview ) .", "The clustering analysis indicated that the population of neurons as a whole did sustain the color information throughout the entire trial ( Figure 3A ) .", "Plotting the information averages of each of the three ‘sample populations’ and the ‘delay populations’ over time confirmed this result ( Figure 4A , Figure 4—figure supplement 1 ) .", "After the onset of the stimulus array , the average information exhibited a sharp increase that peaked roughly 400 ms after stimulus onset and remained at an elevated level throughout the memory delay , until the choice array appeared .", "Results obtained from neurons of the lateral PFC of monkeys indicated distinct hemispheric independence of WM capacity ( Buschman et al . , 2011 ) .", "This means that increasing ipsilateral load ( i . e . , load in the hemifield containing the target for which information is assessed ) should affect neuronal processing while increasing contralateral load should not .", "This effect might be further emphasized in birds due to the full decussation of their optic nerve ( Husband and Shimizu , 2001 ) .", "Parallel to the behavioral results and in line with the results from monkeys , we found a strong effect of ipsilateral load on the information maintenance , as there was a sharp drop in information when the load increased from one item to two items ( Figure 4A , blue and yellow curves ) .", "The addition of a third item only slightly decreased the maintained information further ( Figure 4A , red curve ) .", "The load dependence was much more pronounced during the sample period than during the memory delay where the information remained at a lower elevated level .", "Notably , the load effect was only present for ipsilateral manipulations .", "If the number of items on the contralateral side was increased , the information encoded about the colors at the favorite location did not change ( Figure 4A , right ) .", "To compare our results to the results obtained in monkeys we also applied the method of Buschman et al . , 2011 for testing the ipsilateral load effect during the sample and delay phase , by splitting each phase into an early and a late portion ( first and second 400 ms of the sample , and first and second 500 ms for the delay ) .", "We did find a significant drop in information with an increase in load from one through three in the early ( F ( 2 , 537 ) = 18 . 73 , p < 0 . 001 , ω² = 0 . 0616 ) and late ( F ( 2 , 536 ) = 20 . 07 , p < 0 . 001 , ω² = 0 . 0661 ) sample period and the early ( F ( 2 , 267 ) = 6 . 88 , p = 0 . 0012 , ω² = 0 . 0417 ) and late ( F ( 2 , 267 ) = 3 . 85 , p = 0 . 0225 , ω² = 0 . 0207 ) delay period ( Figure 4B ) .", "There was a large and significant drop between one and two items ( post hoc Bonferroni corrected multiple comparisons: early and late sample p < 0 . 001 , early delay p < 0 . 001 , late delay p = 0 . 0198 ) and one and three items ( post hoc Bonferroni corrected multiple comparisons: early and late sample p < 0 . 001 , early delay p = 0 . 019 , late delay p > 0 . 05 ) but no difference between loads 2 and 3 ( post hoc Bonferroni corrected multiple comparisons: all p > 0 . 05 ) .", "The maintenance of a significant amount of information at higher loads ( even for three items , early sample t ( 156 ) = 7 . 55 , p < 0 . 001; late sample t ( 156 ) = 8 . 73 , p < 0 . 001; early delay t ( 87 ) = 3 . 84 , p < 0 . 001; late delay t ( 87 ) = 4 . 73 , p < 0 . 001 ) and its gradual reduction when items were added to the corresponding hemifield are indicative of a flexible resource allocation and not an all-or-nothing slot-like WM .", "Furthermore , if there is a flexible resource , in error trials a small but insufficient amount of resource might still be allocated to an item .", "Indeed , error trial analysis ( applying correct trial sub-sampling , see Materials and methods ) for the load 2 and 3 conditions further supported this interpretation .", "The amount of information in the early and late sample phase remained above zero ( load 2: early , t ( 186 ) = 3 . 25 , p = 0 . 0014; late , t ( 186 ) = 5 . 33 , p < 0 . 001; load 3: t ( 156 ) = 4 . 21 , p < 0 . 001; Figure 4B asterisks ) , and was significantly smaller than in correct trials ( load 2: late , t ( 186 ) = 2 . 81 , p = 0 . 0055 , d = 0 . 26; load 3: late t ( 156 ) = 2 . 55 , p = 0 . 0117 , d = 0 . 23 ) .", "Additionally , there was no further maintenance throughout the memory delay at any load ( Figure 4B , PEV at loads 2 and 3 in error trials delay , all non-significant ) .", "This indicates that a failure to report which color had changed at higher loads ( two and three ipsilateral items ) resulted from a smaller amount of information encoding during the sample phase that was not maintained throughout the delay .", "A possible alternative ‘slot-model’ explanation would be that , on error trials , the color information was completely lost after the sample phase , because it was not successfully transferred into a slot ( or that a slot was not available to take on information ) .", "The graded amount of information on correct trials is not compatible with the simple ( all or none ) slot model , but could fit the ‘slots and averaging model’ ( Zhang and Luck , 2008 ) .", "We next wanted to understand the neuronal mechanisms behind the information loss at higher WM loads .", "For that , we analyzed how the responses of individual sample and delay neurons changed when the load increased from one color to two colors .", "For the ‘sample populations’ and the ‘delay populations’ , an increasing number of items reduced the amount of encoded information about the color identity ( Figure 4 ) .", "This effect was due to neurons that had a large difference of firing rates between the color A and color B at load 1 ( high PEV , i . e . , information about color ) , and reduced differentiation at load 2 ( small PEV , no or little information about color , e . g . , Figure 2A ) .", "‘Divisive-normalization-like regularization’ ( DNR; Carandini and Heeger , 2011 ) can explain this effect .", "DNR describes the computation that takes place when two stimuli are presented simultaneously .", "In a simplified case a neuronal response becomes normalized , analogous to vector normalization , with a normalization factor consisting of the simultaneous stimuli ( Carandini and Heeger , 2011 ) .", "Applied to our context , a consequence of DNR would be a reduced differentiation between two color identities at load 2 because differences in firing rate ( for each stimulus by itself ) at load 1 would be normalized at load 2 ( resulting in information loss , e . g . , Figure 2A ) .", "We , therefore , hypothesized that DNR was observable for neurons with significant information at load", "1 . We tested for DNR in the NCL by calculating a selectivity index ( SE ) and a sensory interaction index ( SI ) for each neuron for the sample phase and the memory delay phase ( Reynolds et al . , 1999 , see Materials and methods for details ) .", "SE indicates how strongly the neuronal response is driven by a color at the favorite location of the neuron ( reference ) in relation to a selected probe color ( when either is presented alone ) .", "SI indicates how the probe color interacts with the reference color when both are presented simultaneously .", "Values of both indices , SE and SI , lie between –1 and +1 .", "The addition of a probe color influences the response to the reference color by either suppressing the firing rate of the reference color ( if the reference elicits a higher firing rate than the probe , i . e . , SE <0 ) , or increasing the firing rate for the reference color ( if the probe elicits a higher firing rate than the reference , i . e . , SE >0 ) .", "If DNR was present , this influence to suppress or enhance neuronal responses should be an even mixture at the population level , resulting in a significant regression between SE and SI with a slope of around 0 . 5 ( Bouchacourt and Buschman , 2019 ) .", "We compared regressions for the sample and delay phase ( each as one bin , see Materials and methods for details ) for two groups of neurons: information-carrying neurons ( significant information at load 1 ) , and non-informative neurons ( no information at load 1 or at load 2; Figure 5—figure supplement 1 ) .", "We found that DNR was present in both sample and delay phases ( Figure 5A ) .", "Information-carrying sample neurons had a fitted slope of 0 . 47 ( R2adj 0 . 39 , F ( 1 , 838 ) = 547 . 69 , p < 0 . 001 , CI = [0 . 43 0 . 51] ) and delay neurons had a slope of 0 . 50 ( R2adj 0 . 34 , F ( 1 , 342 ) = 175 . 60 , p < 0 . 001 , CI = [0 . 43 0 . 58] ) .", "As the slopes were not significantly different from 0 . 5 , this indicates that reference and probe color had an equal influence on neuronal responses .", "We thus show that DNR was observable in the neuronal population , and as a consequence of this computation , neurons had generally less information about the color identity at load", "2 . Some neurons showed encoding of color identity at higher loads , instead of loss of information .", "These neurons were abundant in both the sample phase and the delay phase ( Figure 3C ) .", "For example , the neuron shown in Figure 2B did not differentiate between color identities at load 1 but did so for load 2 , thus , representing a case of information gain ( instead of loss ) at a higher load .", "We wanted to understand if DNR , the mechanism that we found reduced color information at load 2 , could also produce color differentiation .", "The ‘normalization model of attention’ ( Reynolds and Heeger , 2009 ) incorporates divisive normalization , and can explain how attention can modulate neuronal responses .", "By attending a preferred ( or non-preferred ) second colored square in the load 2 condition the neuronal response of a neuron to the target location ( i . e . , to color A and to color B at the favorite location ) might be altered .", "As a result a difference between color A and B may arise even though each color by itself elicited a similar response .", "In other words , the interaction between the additional color and the target color is unequal .", "Neurons without a color differentiation at load 1 that gained differentiation at load 2 through this process ( e . g . , if the interaction of probe color A with reference color A is larger than the interaction of probe color A with reference color B , see Figure 5—figure supplement 2 for an example ) should have a population regression slope smaller than 0 . 5 .", "We thus hypothesized that the population of neurons showing information at load 2 , but not at load 1 ( e . g . , Figure 2B ) , would have a smaller slope than the neurons that lost information ( Figure 5A ) .", "Sample neurons had a slope of 0 . 19 ( R2adj 0 . 05 , F ( 1 , 446 ) = 23 . 0 , p < 0 . 001 , CI = [0 . 11 0 . 27] , Figure 5B ) , and delay neurons had a slope of –0 . 04 ( R2adj –0 . 015 , F ( 1 , 62 ) = 0 . 08 , p = 0 . 78 , CI = [–0 . 29 0 . 22] , Figure 5B ) .", "Both slopes were significantly smaller than 0 . 5 and smaller than the slopes of the non-informative neurons ( Figure 5—figure supplement 1 ) .", "This indicates that these neurons were influenced more strongly by the reference color , and that the addition of the probe color at load 2 resulted in an unequal interaction .", "Therefore , DNR was also computationally responsible for a gain of information at load 2 , in a specific subset of neurons ." ], [ "Our results confirm behavioral findings that have been discussed in detail in an earlier study ( Balakhonov and Rose , 2017 ) .", "In brief , we found that the WM capacity of crows is limited to about four items , and that the two visual hemifields are largely independent ( i . e . , the number of items on one side does not affect change detection performance on the other side ) .", "Within each hemifield , performance dropped gradually with the addition of a second and third item but remained above chance .", "Fittingly , on the neuronal level , we found a markedly reduced amount of color information when the number of colored squares was increased from one to two ( roughly 50% reduction in correct trials ) .", "This suggests that WM could be conceptualized as a continuous resource that has to be divided between the two items ( Bays and Husain , 2008; van den Berg et al . , 2012; Wilken and Ma , 2004 ) , rather than two ‘simple’ slots that would each have the same amount of information irrespective of the memory load .", "This is also consistent with results of human neuroimaging that report decreased signal amplitude and precision with increasing memory load ( Emrich et al . , 2013; Sprague et al . , 2014 ) .", "In contrast , the hemispheric independence we observed would fit a slot-like model , in which the hemispheres as a whole act like discrete slots .", "A more nuanced version of the slot model ( ‘slots and averaging’; Zhang and Luck , 2008 ) could also account for graded amounts of information within a limited number of slots ( Fukuda et al . , 2010; Zhang and Luck , 2008 ) , as we found here .", "The mix of discrete and independent hemispheres with a graded allocation of information between items that we found is comparable to results by Buschman et al . , 2011 , observed in monkey PFC .", "On the neuronal level , recurrent connections between neurons within a hemisphere may reduce item differentiation when multiple items are present simultaneously , creating capacity limitations within the hemisphere ( Matsushima and Tanaka , 2014 ) .", "A lack of interhemispheric recurrent connections would make processing in the other hemisphere independent .", "Like in monkeys , WM capacity in crows may therefore result from neuronal activity patterns governed by multiple individual items .", "We probed the WM capacity of crows using colored squares , based on the task design of Buschman et al . , 2011 .", "Using the identical task allowed us to directly compare our neuronal results of WM capacity from NCL to results from PFC of monkeys .", "Task similarity is very important for such cross species comparisons as even small changes in task parameters may introduce substantial differences in neuronal responses , leading to potentially different conclusions .", "In a task similar to the one used here , Lara and Wallis , 2014 , have found that neurons in the PFC of monkeys encoded nearly no information about color , but instead about location .", "In their task monkeys had to memorize the color of squares at two locations on a screen , and were again confronted with a colored square at one of the two locations after a delay .", "The monkeys then had to indicate if the color at that location had changed .", "Lara and Wallis , 2014 , discuss the absence of color information in the neurons they recorded in relation to the task of Buschman et al . , 2011 , who like us , did find color information .", "In brief , the exact task design may determine the neuronal encoding of task relevant information ( Lara and Wallis , 2014 ) .", "Similar to the complex contribution of PFC neurons to WM , neurons of NCL can also encode a wide range of very different task relevant aspects , like color ( this study ) , spatial locations ( Rinnert et al . , 2019; Veit et al . , 2017 ) , and more abstract items like rules ( Veit and Nieder , 2013 ) and numerosities ( Ditz and Nieder , 2015 ) .", "One way to circumvent WM failure when item load increases is to allocate attention .", "Our results suggest that attention may play an important role in crow WM .", "Capacity limitation became apparent during encoding , as the amount of information at the end of the sample period was affected by the stimulus load .", "Adding a second and third item to the ipsilateral stimulus array reduced the amount of color information encoded by NCL neurons that carried over into the memory delay .", "Furthermore , neuronal activity in trials in which the birds made an incorrect response showed only weak encoding during the sample phase without information maintenance during the memory delay .", "This fits studies of human WM that have shown attentive filtering during encoding of stimuli influencing WM capacity ( Bays and Husain , 2008; Vogel et al . , 2005; Vogel and Machizawa , 2004 ) , and neuronal correlates of this have been reported for monkeys as well ( Buschman et al . , 2011 ) .", "Beyond the domain of sensory signals , attention and WM may be directly linked .", "Neuronal correlates of WM and attention overlap in PFC neurons , for example , Lebedev et al . , 2004 , found that a substantial amount of PFC neurons encode either an attentional signal , or a memory signal , and some ( hybrid ) neurons do both .", "A purely mnemonic function of PFC thereby seems unlikely .", "Indeed , very recently , Panichello and Buschman , 2021 , have reported that at the population level neurons of PFC encode ‘both the selection of items from working memory and attention to sensory inputs’ ( p . 2 ) , rather than just memory content .", "The independence of hemifields that we observed on the behavioral level ( this study and Balakhonov and Rose , 2017 ) and found in the neuronal responses could also be related to attention .", "Adding stimuli in the contralateral hemifield affected neither performance nor information maintained by NCL neurons , whereas additional ipsilateral stimuli strongly reduced both .", "This fits the influence of attention on WM and hemifield independence , which is consistently accentuated in studies in which attention had to be divided between the two hemifields ( Alvarez and Cavanagh , 2005; Buschman et al . , 2011; Cavanagh and Alvarez , 2005; Delvenne , 2005; Delvenne et al . , 2011 ) .", "Finally , the DNR computation may explain the responses of the neurons that gained information at load 2 through attentional processes predicted by the ‘normalization model of attention’ ( Reynolds and Heeger , 2009 ) .", "This may appear counter-intuitive and contradictory , considering that the same process is also responsible for the loss of information .", "However , when attention is overtly directed to a specific ( preferred or non-preferred ) item within the receptive field of a neuron , the DNR computation shifts its weighting of the normalized response toward the response of the attended item ( Reynolds et al . , 1999; Reynolds and Heeger , 2009 ) .", "This weighted normalization can produce a difference in the neuronal response to both color identities at load 2 , even if the neuronal response was non-informative at load 1 .", "At the population level we were able to observe such an effect as the reduced slope of the selectivity/interaction fit .", "Thus , an attentive process might have enhanced information in WM at higher loads .", "It is important to clarify that , as we did not use any form of attentional cueing in our study , we cannot explicitly test for such an attention effect .", "However , we do know that the animals participating in this study can use attentional cues to enhance their WM ( Fongaro and Rose , 2020 ) .", "The attention cues used by Fongaro and Rose , 2020 , positively affected not only encoding but also the maintenance and retrieval of the information held in WM , comparable to results from monkeys and humans ( Brady and Hampton , 2018; Souza and Oberauer , 2016 ) .", "We , therefore , want to emphasize that our data is in line with the interpretation that the birds possibly attended a load 2 stimulus array differently than a load 1 stimulus array in order to enhance their performance in trials with higher loads .", "Our neuronal recordings offer a mechanistic explanation for the behavioral effects , as we found clear evidence of DNR governing the neuronal responses tied to WM capacity that is in accordance with mammalian models of WM capacity ( Bouchacourt and Buschman , 2019; Lundqvist et al . , 2016; Lundqvist et al . , 2011 ) .", "The loss of information about color identity ( i . e . , neuronal response differentiation between colors ) can be accounted for by DNR when an item is added to a neuron’s receptive field .", "The normalization of neuronal firing rate diminishes the differentiation between color identities .", "As such it is analogous to neurophysiological responses from visual areas ( Carandini et al . , 1997; Reynolds et al . , 1999 ) and to the PFC during spatial WM ( Matsushima and Tanaka , 2014 ) .", "The WM model of Bouchacourt and Buschman , 2019 , is based solely on data from monkey electrophysiology , and thus implicitly tied to the layered columns of the neocortex .", "The results we report here show that the model also fits the neurophysiology of WM in crows .", "However , the picture is incomplete since important aspects of monkeys’ WM are still not investigated in crows .", "Oscillations of local field potentials are relevant for how information enters WM and how it is maintained ( Miller et al . , 2018 ) , and have been tied to normalization and competition between items in WM ( Lundqvist et al . , 2018b ) .", "Thus , the oscillatory interplay of the layers and different regions of the mammalian neocortex are important fields of research to further our understanding of WM .", "Such aspects are so far completely unknown in crows and their non-layered associative areas .", "This encourages further investigation into the neuronal circuits of WM in birds .", "There is also ongoing debate about the role of sustained activity during delay periods and how it relates to WM ( Constantinidis et al . , 2018; Lundqvist et al . , 2018a; Miller et al . , 2018 ) .", "We cannot report of any neuron that showed persistent activity comparable to those reported by classical WM studies in PFC ( e . g . , Funahashi et al . , 1989; Fuster and Alexander , 1971 ) , or in NCL ( Diekamp et al . , 2002b; Veit et al . , 2014; Veit and Nieder , 2013 ) .", "This may be reconcilable with some other contemporary models of WM .", "One major type of those models implements ‘synfire chains’ , where individual neurons fire sequentially ( and transiently ) to bridge temporal gaps and maintain task relevant contents ( Rajan et al . , 2016 ) .", "This has , for example , been reported to be the case in posterior parietal cortex of mice performing a T-maze task that required WM for cued spatial locations to be maintained ( Harvey et al . , 2012 ) .", "The transient activity of neurons that we report ( Figures 2 and 3A ) might fit into such models .", "However , our results can only be compared very cautiously to this ( since even small changes in task design significantly alter neuronal responses , e . g . , Lara and Wallis , 2014 ) .", "Therefore , while we cannot , yet , fully equate crow and monkey WM , our results raise two important questions about how WM is implemented on the level of neuronal networks that have implications for our comparative view of crow WM .", "The first regards the neuronal computations underlying WM .", "Is there a common canonical computation governing WM , or are there different solutions based on different neuronal architectures ?", "Recent work has shown that the sensory areas of mammals and birds show remarkably similar circuit organization ( Stacho et al . , 2020 ) .", "However , higher-order associative areas involved in WM , like the LPFC in mammals and the NCL in birds , have distinctly different architectures ( Stacho et al . , 2020 ) .", "The fact that differently organized areas like LPFC and NCL produce strikingly similar physiological responses points to shared computational principles .", "Modeling work already suggests that the competing WM capacity models can be accommodated into a unifying framework based on theoretical neuronal information sampling , where stochastic information sampling ( assumed for continuous resource models ) can account for item limitations better than fixed information sampling ( assumed by the slots and averaging models ) ( Schneegans et al . , 2020 ) .", "Similarly , DNR is already considered to be a general , canonical computation of the nervous system , present in evolutionarily distant phyla , for example , fruit flies and monkeys ( Carandini and Heeger , 2011 ) .", "The second question regards the tradeoff between WM flexibility and capacity ( Bouchacourt and Buschman , 2019 ) .", "Is the WM of a crow as flexible as that of a monkey ?", "Our results show that the computations by individual neurons that result in WM capacity limitations are virtually the same in crows and monkeys , highlighting a further aspect of WM that is similar between these animal groups ( Nieder , 2017 ) .", "Ultimately , our results were in line with different modern models of WM that implement DNR to explain capacity ( Bouchacourt and Buschman , 2019; Lundqvist et al . , 2016; Lundqvist et al . , 2011; Schneegans et al . , 2020 ) .", "However , the data we presented cannot carry a definitive conclusion about which of the different models fits best .", "For example , a tradeoff between flexibility and capacity ( Bouchacourt and Buschman , 2019 ) might be present , but further investigation into the models’ predictions is required .", "We do , however , show that mammalian models of WM are in line with WM in birds , which implies that fundamental aspects of WM are shared between these animal groups .", "Together , all these facets of crow WM capacity suggest that the different intricate neuronal architectures that carry out the computations in monkeys and crows have likely been shaped by convergent evolution – into systems that yield similar cognitive performances .", "The systems may share the same basic mechanisms and thus limitations .", "Further investigation into the oscillatory dynamics of WM in the avian brain may elucidate if birds also share the prominent limitation of a tradeoff between flexibility and capacity ." ], [ "Two hand-raised carrion crows ( C . corone ) of 2 years of age served as subjects in this study .", "The birds were housed in spacious aviaries in social groups .", "During the experimental procedures , the animals were held on a controlled food protocol with ad libitum access to water and grit .", "All experimental procedures and housing conditions were carried out in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals and were authorized by the national authority ( LANUV ) .", "We used operant training chambers ( 50 × 50 . 5 × 77 . 5 cm3 , width × depth × height ) equipped with an acoustic pulse touchscreen ( 22’’ , ELO 2200L APR , Elo Touch Solutions Inc , Milpitas , CA ) and an infrared camera ( Sygonix , Nürnberg , Germany ) for remote monitoring .", "The birds sat on a wooden perch so that the distance between the bird’s eye and the touchscreen was 8 cm .", "Food pellets were delivered as a reward via a custom-made automatic feeder ( plans available at http://www . jonasrose . net/ ) .", "The position of the animal’s head was tracked online during the experiment by two open-source computer vision cameras ( ‘Pixy’ , CMUcam5 , Charmed Labs , Austin , TX ) that reported the location and angle between two LEDs .", "For tracking , we surgically implanted a lightweight head-post and used a lightweight 3D-printed mount with LEDs that was removed after each experimental session .", "The system reported the head location at a frame rate of 50 Hz and data was smoothed by integrating over two frames in MATLAB using custom programs on a control PC .", "All experiments were controlled by custom programs in MATLAB using the Biopsychology ( Rose et al . , 2008 ) and Psychophysics toolboxes ( Brainard , 1997 ) .", "Digital input and output of the control PC were handled by a microcontroller ( ODROID C1 , Hardkernel co . Ltd , Anyang , South Korea ) connected through a gigabit network running custom software ( available at http://www . jonasrose . net/ ) .", "The behavioral protocol was identical to the one described in Balakhonov and Rose , 2017 .", "We trained the birds to perform a delayed change localization task that had previously been used to test the performance under different WM loads in primates ( Buschman et al . , 2011 ) .", "Each trial started after a 2 s inter-trial-interval , with the presentation of a red dot centered on the touchscreen ( for a maximum of 40 s ) .", "The animals initiated the trial by centering their head in front of the red dot for 160 ms . This caused the red dot to disappear and a stimulus array of two to five colored squares to appear ( Figure 1A , ‘sample’ ) .", "The colored squares were presented for a period of 800 ms , during which the animals had to hold their head still and center their gaze on the screen ( ‘hold gaze’ , no more than 2 cm horizontal or vertical displacement , and no more than 20 degrees horizontal or vertical rotation ) .", "Failure to hold the head in this position resulted in an aborted trial .", "This sample phase was followed by a memory delay of 1000 ms after which the stimulus array reappeared with one color exchanged .", "The animal had to indicate the location of the color change by pecking the respective square .", "Correct responses were rewarded probabilistically ( BEO special pellets , in 55% of correct trials , additional 2 s illumination of the food receptacle in 100% of correct trials ) .", "Incorrect responses to colors that had not changed or a failure to respond within 4 s resulted in a brief screen flash and a 10 s timeout .", "The stimuli were presented at six fixed locations on the screen ( 1–6 , Figure 1A ) .", "In each session , one pair of colors was assigned to each of the six locations .", "Each location had its own distinct pair .", "These pairs were randomly chosen from a pool of 14 colors ( two color combinations were excluded since the animals did not discriminate them equally well during a pre-training ) .", "Let us consider Figure 1A as an example .", "The color change occurs in the middle-left where turquois ( T ) is presented during the sample and orange ( O ) during the choice .", "In this particular session the middle-left could thus show either of the following colors during the sample and choice: T-O ( shown in Figure 1A ) ; O-T; O-O; T-T; None-None .", "On the next session a new random pair of colors was displayed at this location .", "For identification and analysis an arbitrary label was assigned to each of the randomly drawn colors at the start of each session ( i . e . , left middle location: ‘color A’ , or ‘color B’; left top location: ‘color A’ , or ‘color B’; etc . ) .", "These indices do not refer to the order of presentation of the colors at any time , but were held constant for neuronal analysis .", "The order of presentation of colors within a pair , the target location ( where the color change occurred ) , and the number of stimuli in the array ( two to five ) were randomized and balanced across trials so that each condition had an equal likelihood to appear .", "The order of presentation of colors within a pair , the target location ( where the color change occurred ) , and the number of stimuli in the array ( two to five ) were randomized and balanced across trials so that each condition had an equal likelihood to appear .", "The color squares had a width of 10 degrees of visual angle ( DVA ) and were placed on the horizontal meridian of the screen and at 45 . 8 DVA above or below the meridian at a distance of 54 and 55 . 4 DVA from the center .", "This arrangement in combination with the head tracking ensured that all stimuli appeared outside of the binocular visual field of crows ( 37 . 6 DVA; Troscianko et al . , 2012 ) .", "Both animals were chronically implanted with a lightweight head-post to attach a small LED holder during the experiments .", "Before surgery , animals were deeply anesthetized with ketamine ( 50 mg/kg ) and xylazine ( 5 mg/kg ) .", "Once deeply anesthetized , animals were placed in a stereotaxic frame .", "After attaching the small head-post with dental acrylic , a microdrive with a multi-channel microelectrode was stereotactically implanted at the craniotomy ( Neuronexus Technologies Inc , Ann Arbor MI , DDrive ) .", "The electrode was positioned in NCL ( AP 5 . 0 , ML 13 . 0 ) of the left hemisphere ( coordinates for the region based on histological studies on the localization of NCL in crows; Veit and Nieder , 2013 ) .", "After the surgery , the crows received analgesics .", "Extracellular single neuron recordings were performed using chronically implanted multi-channel microelectrodes .", "The distance between recording sites was 50 µm .", "The signal was amplified , filtered , and digitized using Intan RHD2000 headstages and a USB-Interface board ( Intan Technologies LLC , Los Angeles , CA ) .", "The system also recorded digital event codes that were sent from the behavioral control PC using a custom IO device ( details available at http://www . jonasrose . net/ ) .", "Before each recording session , the electrodes were advanced manually using the microdrive .", "Recordings were started 20 min after the advancement , and each recording site was manually checked for neuronal signals .", "The signals were recorded at a sampling rate of 30 kHz and filtered with a band-pass filter at recording ( 0 . 5–7 . 5 kHz ) .", "The recorded neuronal signals were not pre-selected for task involvement .", "We performed spike sorting using the semi-automatic Klusta-suite software ( Rossant et al . , 2016 ) , which uses the high electrode count and their close spacing to isolate signals of single neurons .", "For spike sorting , we filtered with a high pass of 500 Hz and a low pass of 7125 Hz .", "The software utilizes the spatial distribution of the recorded signal along the different recording sites to untangle overlapping signals and separate signals with similar waveforms but different recording depths .", "All statistical analyses were performed in MATLAB ( 2018b , Mathworks Inc ) using commercially available toolboxes ( Curve Fitting Toolbox Version 3 . 5 . 3 , Statistics and Machine Learning Toolbox Version 10 . 2 ) and custom code .", "For all statistical tests , we assumed a significance level of α = 0 . 05 , unless stated otherwise .", "Trials were classified as error trials if the bird chose a location where no change of colors had appeared .", "Trials in which the bird did not choose any location or failed to maintain head fixation were not analyzed .", "All correct trials were included in the analysis of neural data .", "Depending on the analysis we refer to different ‘load conditions’ relative to referential sides of the screen which are either ipsilateral ( same side ) or contralateral ( opposite side ) , each with a possible load between one and three items .", "For the behavioral analyses the terms ipsilateral and contralateral refer to the location of the change that had to be detected .", "For the neurophysiological analyses the terms ipsilateral and contralateral refer to the respective neuron’s favorite location ( described in the following section ) .", "Because there were only very few error trials in the load one condition , we performed error trial analysis only for the load 2 and load 3 conditions .", "The behavioral data were analyzed as described in our previous study ( Balakhonov and Rose , 2017 ) , estimating the WM capacity K for each load by Equation 1 . ( 1 ) K=n*p where p is the percentage correct and n is the number of items in WM .", "This estimate has been applied to similar primate data and in studies with humans ( Johnson et al . , 2013; Kornblith et al . , 2016 ) .", "Based on a one-way ANOVA of color identity at a given location , we calculated a PEV statistic to measure the effect size of neuronal modulation .", "Its main parameter ω² is a measurement for the percentage to which the tested factor can explain the variance of the data , and it is calculated from the sum of squares of the effect ( SSeffect ) and the mean squares of the within-group ( error ) variance ( MSerror ) ( Equation 2 ) .", "( 2 ) ω2=SSeffect-df*MSerrorSStotal+MSerror For each neuron , we determined a ‘favorite location’ , which was defined as the location with the highest cumulative PEV , of the three possible locations on the right half of the screen , that is , opposite to the implanted hemisphere , across four non-overlapping bins during the sample phase ( bin size 200 ms , advanced in steps of 200 ms , from start till the end of the sample phase ) .", "The significance of calculated effect size values was determined by a permutation test .", "We ran the permutation to calculate the likelihood of getting an explained variance value bigger than the one calculated from the actual distribution of the data by randomly permuting the color identity labels and calculating the PEV 1000 times .", "The test thereby does not assume any distribution of the data and returns an unbiased estimate of the likelihood of generating an effect size within the data randomly .", "The measured value of explained variance from the actual dataset was assumed to be significant if the likelihood of randomly generating a bigger value was below 5% .", "We chose to not correct for the multiple comparisons at this level , as we reasoned that if we were to only include those neurons that had the most information at the individual loads ( i . e . , those with the highest PEV values that are significant even under very stringent statistical criteria ) we would have artificially inflated the amount of information present at each load .", "By including neurons that encoded less information too ( i . e . , at the uncorrected p-value ) our analysis population was more resistant to such outlier effects .", "We additionally performed our analyses using a more stringent statistical criterion ( significance of two consecutive non-overlapping bins ) and found the same results ( Figure 4—figure supplement 2 ) .", "We tested the proportions of significant neurons we found for the different trial phases by performing a binomial test , assuming a significance level α = 0 . 05 ( Equation 3 ) .", "( 3 ) PX=i=Bp0 , n=nkp0i1-p0n-i Calculating the probability p , of finding X significant neurons , given a total amount of i ( 362 ) neurons , and a probability p0 of 5% finding a significance by chance .", "We considered neuronal significance ( i . e . , significant PEV as determined above ) for each load independently .", "This means , we tested if the PEV of a neuron was significant three times with the permutation method described above: once for each of the three load conditions .", "Therefore , we can report seven groups of significance ( Table 1 , Figure 3C ) .", "Subsequently , we created three pooled groups ( Table", "1 ) from all neurons with a significant PEV at each individual load .", "We used these pooled groups for the population analyses ( Figures 4 and 5 ) .", "Neurons of these pooled groups , with a significant PEV during the sample phase were assigned to the ‘sample population’ , and neurons with a significant amount of information during the memory-delay phase were assigned to the ‘delay population’ ( significance criterion: one significant 200 ms bin , at α = 0 . 05 , see above for our reasoning not to correct for multiple comparison at this point ) .", "Thus , neurons with significant PEV during both the sample and delay phase were included in both subpopulations .", "We corrected for the unequal amount of correct and error trials when comparing information about color ( PEV ) between the trial conditions , by sub-sampling correct trials with the number of error trials 1000 times for each neuron .", "The resulting PEV values of correct trials were then averaged for each neuron , this population of averaged PEV values was then statistically tested against the PEV values of error trials ( of the same neurons ) using a dependent t-test .", "We tested for the presence of divisive normalization using the method of Reynolds et al . , 1999 .", "Three conditions were considered: ( 1 ) neuronal response to stimulus A , ( 2 ) neuronal response to stimulus B , and ( 3 ) neuronal response to the simultaneity of stimuli A and B . As we wanted to relate this to the information about color identity , we selected subsets of the favorite location and the additional two ipsilateral locations .", "To test how the neurons altered their response when multiple stimuli were presented simultaneously , we calculated the color selectivity index ( SE ) and the sensory interaction index ( SI ) of each neuron .", "SEi was calculated by subtracting the normalized firing rate for the chosen reference color i ( REFi ) at the neuron’s favorite location , from a second color j ( PROBEj ) at a different location ( ipsilateral to the favorite location , Equation 4 ) .", "( 4 ) SEi=PROBEj-REFi The resulting selectivity index lies between –1 ( completely selective for the reference color ) and 1 ( completely selective for the probe color ) .", "SI was calculated ( Equation 5 ) by subtracting the normalized firing rate for REFi from the normalized firing rate of the combination of REFi and PROBEj ( PAIRi , j ) .", "( 5 ) SIi , j=PAIRi , j-REFi This interaction index also lies between –1 ( full suppression of reference stimulus by the probe stimulus ) and 1 ( full enhancement of the reference stimulus by the probe stimulus ) .", "As each of the three locations had two possible colors , we calculated eight SE and SI indices per neuron and performed a linear regression for all indices .", "This is required as each stimulus combination is informative about the normalization .", "The effects of divisive normalization were compared between the sample and the delay phase .", "Therefore , SE and SI indices were calculated across the entire sample ( 800 ms ) and memory delay ( 1000 ms ) phase .", "Neurons with significant information were accordingly identified over the entire sample and delay as one bin , using the permutation test described in the section ‘information about color identity’ .", "We considered the entire sample and delay phase because we wanted to analyze the population response as a whole , irrespective of highly diverse response profiles of individual neurons .", "To visualize the different groups of neurons that encoded and maintained information about the color identity during different phases of the trial , we performed a hierarchical clustering analysis in MATLAB on the normalized PEV values of individual neurons throughout the trial .", "We used a ( 1 − correlation ) distance metric and an average distance linkage function for a maximum of seven clusters .", "The maximum number of clusters was first determined by calculating the clustering for different amounts of clusters ( 1–10 ) and subsequently calculating the within-cluster sum-of-squares .", "This resulted in a graph that allowed us to visually inspect the tradeoff between cluster number and fit improvement , from which we estimated the inflection point ( elbow method ) .", "A cluster number of seven presented the best tradeoff that allowed visualization of the different groups at an acceptable clustering success .", "We then ordered the neuron clusters to minimize the average distance between the clusters in the dendrogram ." ] ]

[ "Complex cognition relies on flexible working memory , which is severely limited in its capacity .", "The neuronal computations underlying these capacity limits have been extensively studied in humans and in monkeys , resulting in competing theoretical models .", "We probed the working memory capacity of crows ( Corvus corone ) in a change detection task , developed for monkeys ( Macaca mulatta ) , while we performed extracellular recordings of the prefrontal-like area nidopallium caudolaterale .", "We found that neuronal encoding and maintenance of information were affected by item load , in a way that is virtually identical to results obtained from monkey prefrontal cortex .", "Contemporary neurophysiological models of working memory employ divisive normalization as an important mechanism that may result in the capacity limitation .", "As these models are usually conceptualized and tested in an exclusively mammalian context , it remains unclear if they fully capture a general concept of working memory or if they are restricted to the mammalian neocortex .", "Here , we report that carrion crows and macaque monkeys share divisive normalization as a neuronal computation that is in line with mammalian models .", "This indicates that computational models of working memory developed in the mammalian cortex can also apply to non-cortical associative brain regions of birds ." ]

[ "Working memory is the brain’s ability to temporarily hold and manipulate information .", "It is essential for carrying out complex cognitive tasks , such as reasoning , planning , following instructions or solving problems .", "Unlike long-term memory , information is not stored and recalled , but held in an accessible state for brief periods .", "However , the capacity of working memory is very limited .", "Humans , for example , can only hold around four items of information simultaneously .", "There are various competing theories about how this limitation arises from the network of neurons in the brain .", "These models are based on studies of humans and other primates .", "But memory limitations are not exclusive to mammals .", "Indeed , the working memory of some birds , such as crows , has a similar capacity to humans despite the architecture of their brains being very different to mammals .", "So , how do brains with such distinct structural differences produce working memories with similar capacities ?", "To investigate , Hahn et al . probed the working memory of carrion crows in a change detection task developed for macaque monkeys .", "Crows were trained to memorize varying numbers of colored squares and indicate which square had changed after a one second delay when the screen went blank .", "While the crows performed the task , Hahn et al . measured the activity of neurons in an area of the brain equivalent to the prefrontal cortex , the central hub of cognition in mammals .", "The experiments showed that neurons in the crow brain responded to the changing colors virtually the same way as neurons in monkeys .", "Hahn et al . also noticed that increasing the number of items the crows had to remember affected individual neurons in a similar fashion as had previously been observed in monkeys .", "This suggests that birds and monkeys share the same central mechanisms of , and limits to , working memory despite differences in brain architecture .", "The similarities across distantly related species also validates core ideas about the limits of working memory developed from studies of mammals ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "epidemiology and global health", "immunology and inflammation" ]

[ [ "Renewed emphasis on malaria control has resulted in substantial reductions in overall malaria prevalence and incidence in many endemic countries ( World Health Organization , 2015 ) .", "However , where transmission persists , it is highly heterogeneous even on small spatial scales ( Bousema et al . , 2012 ) .", "Individual exposure is further influenced by factors such as use of bednets , attractiveness to mosquitoes , or behavioural differences .", "In Papua New Guinea ( PNG ) , malaria prevalence has sharply declined in the last decade , largely as a result of two nationwide distributions of long-lasting insecticide treated bednets ( LLIN ) ( Hetzel et al . , 2015; Hetzel et al . , 2014; Hetzel et al . , 2012 ) .", "P . vivax and P . falciparum PCR-prevalence in the general population was reduced from 32% and 39% in 2006 to 13% and 18% in 2010 ( Koepfli et al . , 2015 ) .", "Already before this decline in malaria prevalence , studies in PNG had reported significant heterogeneity in malaria transmission attributed to local population structure and geographical diversity ( Hetzel et al . , 2015; Cattani et al . , 1986; Genton et al . , 1995; Müller et al . , 2003; Mueller et al . , 2009a ) .", "Prior to the up-scaling of malaria control , P . vivax endemicity in PNG was among the highest worldwide ( Hetzel et al . , 2015 ) .", "Clinical immunity to P . vivax was acquired very rapidly in PNG children , and the incidence of P . vivax clinical episodes peaked in children younger than two years with only very few P . vivax clinical episodes reported in children older than 5 years or adults ( Genton et al . , 2008; Michon et al . , 2007; Lin et al . , 2010; Betuela et al . , 2012 ) .", "In contrast , the risk for uncomplicated P . falciparum clinical episodes increased during early childhood ( Lin et al . , 2010 ) and significant reductions in incidence of clinical episodes or high-density infections were only observed in children aged 5 years and older ( Michon et al . , 2007 ) .", "Compared to the incidence of clinical malaria , prevalence of P . falciparum and P . vivax peaked in older age groups , with asymptomatic infections remaining common until adulthood in PNG ( Koepfli et al . , 2015; Mueller et al . , 2009a ) .", "Concordant with the species-specific pattern in the burden of clinical episodes , P . vivax prevalence peaked in younger age groups than P . falciparum prevalence ( Koepfli et al . , 2015; Mueller et al . , 2009a ) .", "As malaria transmission declines , it is important to understand the resulting changes in malaria prevalence and clinical incidence patterns , as well as the extent of heterogeneity in transmission within malaria endemic regions so that high-risk areas can be identified and targeted ( Mosha et al . , 2014 ) .", "Most attempts to delineate high and low transmission areas made to-date , by both researchers and control programs , have used passive case surveillance or cross-sectional malaria indicator surveys .", "These surveillance strategies result in clinical incidence and prevalence estimates , both of which are surrogate markers for transmission .", "A more accurate understanding of the relationship between exposure to new infections and malaria prevalence or clinical incidence is needed to determine how accurately these surrogate markers represent heterogeneity in transmission at local scales .", "In addition , quantifying clinical incidence in relation to exposure to blood-stage infections can increase our insight into the development and maintenance of immunity to malaria in a setting of sustained malaria control ( Battle et al . , 2015; Cameron et al . , 2015 ) .", "The molecular force of blood-stage infection ( molFOB ) describes the number of new genotypes observed in consecutive blood samples from cohort participants over time ( Mueller et al . , 2012; Koepfli et al . , 2013 ) .", "Genotyping of highly polymorphic markers detects superinfecting parasite clones in asymptomatic ( but parasitaemic ) or symptomatic individuals .", "molFOB thus provides a longitudinal , individual and quantitative measure for exposure to new blood-stage malaria infections ( Mueller et al . , 2012; Koepfli et al . , 2013 ) .", "For P . falciparum , molFOB is closely linked to the number of infective mosquito bites and therefore is a direct proxy for the actual force of infection ( FOI ) and thus for transmission in endemic settings ( Smith et al . , 2010 ) .", "For P . vivax , clones appearing in the blood-stream can either originate directly from an infective mosquito bite or from a relapsing liver hypnozoite ( Koepfli et al . , 2013 ) .", "For P . vivax , molFOB is thus a compound measure of exposure to newly acquired infections from mosquito bites and relapsing blood-stage infections .", "The usefulness of molFOB as a surrogate marker of individual exposure was validated originally in a cohort of young PNG children 1–4 years of age , in which species-specific molFOB was the most important predictor of clinical incidence for both species ( Mueller et al . , 2012; Koepfli et al . , 2013 ) .", "Although some spatial heterogeneity of transmission was observed in that study for both species , due to the high level of transmission the species-specific difference in rate of immune acquisition was the predominant feature in that study .", "While P . vivax molFOB ( Pv-molFOB ) did not change with age , the incidence of P . vivax clinical episodes decreased significantly with age , with a faster rate of decrease in children with high Pv-molFOB [12] .", "P . falciparum molFOB ( Pf-molFOB ) in that cohort was lower compared to Pv-molFOB and the incidence of P . falciparum clinical episodes increased in parallel with an increasing Pf-molFOB in children 1–3 years , reaching a plateau thereafter ( Lin et al . , 2010; Mueller et al . , 2012 ) .", "These earlier results indicated that", "( i ) immunity to P . vivax is acquired more rapidly in children with higher cumulative exposure , that", "( ii ) this developing immunity led to proportionally fewer clinical P . vivax episodes in older children despite similar exposure to new P . vivax blood-stage infections , and that", "( iii ) higher exposure to P . vivax blood-stage infections , compared to P . falciparum , resulted in a more advanced immunity to P . vivax in this age group compared to P . falciparum ( Doolan et al . , 2009; Longley et al . , 2016 ) .", "The major challenge to these cross-species comparisons lies within the intrinsic differences of Pf- and Pv-molFOB: whereas Pf-molFOB is a direct marker of mosquito-borne transmission , Pv-molFOB is a composite measure reflecting both newly acquired infections and those caused by relapses of previously acquired infections .", "In this study , we extend the analysis of molFOB’s relationship with incidence of clinical malaria episodes to older PNG children and a lower transmission scenario .", "In addition , given the unique study design that randomized blood-stage only or blood- plus liver-stage treatment at enrolment ( Robinson et al . , 2015 ) , we are now , for the first time , able to compare the incidence of newly acquired P . falciparum infections with both the incidence of newly acquired P . vivax infections and relapsing P . vivax infections .", "Advancing on previous studies that investigated each species individually ( Mueller et al . , 2012; Koepfli et al . , 2013 ) , we now provide a comparative analysis of P . falciparum and P . vivax , directly exploring the role of exposure to multiple Plasmodium species in the development of clinical immunity to P . falciparum and P . vivax malaria .", "We quantify in detail the extent of heterogeneity in molFOB on a small geographical scale and relate this to heterogeneity in clinical episode incidence to investigate effects of small-scale variation in malaria transmission on local malaria epidemiology .", "By combining in-depth molecular parasitological data with demographic and clinical data , this study thus provides detailed insights into the changing epidemiology of malaria in PNG in response to intense malaria control efforts ." ], [ "This study was conducted in six villages in Maprik district , East Sepik Province , PNG between August 2009 and May 2010 ( Robinson et al . , 2015 ) .", "524 children aged 5–10 years were enrolled and randomized to receive either chloroquine ( CQ ) , artemeter-lumefantrine ( AL ) and primaquine ( PQ ) ; or CQ , AL and placebo .", "Demographic parameters of the 466 children that completed the full course of randomized treatment with PQ/CQ/AL ( n = 233 ) or placebo/CQ/AL ( n = 233 ) , and were thereafter closely followed for 8 months , were comparable between the six villages ( Table 1 ) .", "P . vivax was the most common infection at enrolment with 48% of children positive by quantitative PCR ( qPCR ) , followed by P . falciparum ( 24% ) , P . malariae ( 15% ) and P . ovale ( 3%; Table 2 ) .", "39% of children were not infected with any Plasmodium species at enrolment .", "The vast majority of P . malariae ( 75% ) and almost all P . ovale infections ( 93% ) occurred in children co-infected with either P . vivax and/or P . falciparum ( Table 2 ) .", "Prevalence of each Plasmodium species varied between villages ( P .", "falciparum , 9–71%; P . vivax , 38–67%; P . malariae , 8–40%; P . ovale , 0–11%; Table", "2 ) and was highest in Bolumita for all species .", "Accordingly , mixed-species infections were also most prevalent in Bolumita ( Table 2 ) .", "The multiplicity of infection ( MOI ) , that is , the number of parasite genotypes per infection , also varied between villages for both species ( mean P . falciparum MOI , 1 . 1–2 . 2 clones/infection; mean P . vivax MOI , 1 . 6–2 . 9 clones/infection ) and children from Bolumita carried more multi-clone infections with P . vivax and P . falciparum than children in other villages ( Table 2 ) .", "Mean P . falciparum parasite density was almost two- to six-fold higher in Bolumita ( 331 18S rRNA gene copies/µl ) than in other villages ( 56–192 18S rRNA gene copies/µl , Table 2 ) .", "Children who had received PQ for clearance of P . vivax hypnozoites experienced similar numbers of new blood-stage infections with P . falciparum and P . vivax during follow-up ( mean Pf-molFOB = 1 . 5 CI95 [1 . 3–1 . 7] new blood-stage clones/year , Pv-molFOB = 1 . 6 [1 . 4–1 . 9] new blood-stage clones/year , Figure 1A , Figure 1—figure supplement 1 ) .", "Pf-molFOB in the placebo arm was comparable to the PQ arm ( mean Pf-molFOB = 1 . 4 [1 . 2–1 . 6] new blood-stage clones/year ) , whereas due to the hypnozoite reservoir Pv-molFOB was more than three times higher in the placebo arm compared to the PQ arm ( mean Pv-molFOB = 5 . 4 [4 . 9–5 . 8] new blood-stage clones/year ) .", "Pv-molFOB in the placebo arm showed a pronounced peak at months 2–3 of follow-up , which likely represents a wave of fast-relapsing hypnozoites in children who did not receive PQ ( Figure 1A ) .", "P . vivax prevalence in the PQ arm was comparable to P . falciparum prevalence throughout the study and increased steadily , irrespective of the diagnostic method used ( Figure 1B and C ) .", "P . vivax prevalence increased more rapidly in the placebo arm until month 3 of follow-up and dropped thereafter , similar to patterns in Pv-molFOB in the same arm .", "Prevalence as measured by qPCR did not reach pre-treatment levels until the end of the study for any of the four Plasmodium species ( Figure 1B , Figure 1—figure supplement 2 ) .", "At the end of follow-up , P . vivax prevalence by qPCR in the placebo arm was 25% [19–31%] , and therefore more than two-fold higher than in the PQ arm ( 9% [6–14%]; Figure 1B ) .", "Also , throughout follow-up , P . vivax prevalence in the placebo arm was 2–3 fold higher compared to the PQ arm , suggesting that at least 50% of the overall P . vivax prevalence in this cohort can be attributed to the contribution of relapses .", "Similarly , throughout and at the end of follow-up P . vivax prevalence in the placebo arm was 2–3 fold higher compared to P . falciparum ( irrespective of treatment arm; P . falciparum prevalence at end of follow-up , 10% [8–14%] ) , which is in agreement with the prevalence pattern at enrolment .", "Assuming equal transmission from mosquitoes for both species , which was corroborated by a comparable Pf-molFOB and Pv-molFOB in the PQ arm , P . vivax relapses have contributed to a P . vivax prevalence twice as high as that of P . falciparum .", "In the present study design , recurrent blood-stage infection can either originate from a new transmission event ( both arms and all species ) or for P . vivax and P . ovale also from a relapse of any previous infection ( placebo arm only ) .", "After adjusting for the effect of PQ treatment ( Robinson et al . , 2015 ) , village of residence and infection status by qPCR at enrolment were the main predictors for the risk of recurrent Plasmodium spp .", "during follow-up ( Table 3 ) .", "Interestingly , in addition to a protective effect against recurrent P . vivax and P . ovale , the risk of recurrent P . falciparum was also reduced by 27% [0–48%] after PQ treatment ( p=0 . 064 ) .", "The risk of a recurrent infection ( measured by qPCR ) with P . falciparum , P . vivax and P . ovale varied more than 7-fold between villages , with a higher risk observed in Bolumita ( 78% , 77% , and 15% with recurrent P . vivax , P . falciparum and P . ovale , respectively ) compared to the other villages ( recurrent P . vivax , range 25–73%; recurrent P . falciparum , range 12–44%; recurrent P . ovale , range 0–7% ) .", "For P . falciparum and P . vivax , a mixed infection at enrolment as measured by qPCR was further associated with up to a two-fold increased risk of recurrent infection ( P . falciparum: AHR = 2 . 08 [1 . 25–3 . 48] , p=0 . 005; P . vivax: AHR = 1 . 74 [1 . 14–2 . 65] , p=0 . 010; Table 3 ) , supporting the idea that focal transmission within villages leads to the presence of high-risk and low-risk individuals .", "For P . malariae , the infection status at enrolment was a stronger predictor of risk of recurrent infection than village of residence .", "An infection with P . falciparum , P . malariae or a mixed infection at enrolment as measured by qPCR was associated with up to a 6-fold increase in risk of recurrent P . malariae ( AHRPf-enrol = 3 . 54 [0 . 85–14 . 72] , p=0 . 083; AHRPm-enrol = 6 . 35 [1 . 31–30 . 81] , p=0 . 022; AHRmixed = 3 . 37 [0 . 88–12 . 90] , p=0 . 076; Table 3 ) .", "Reported use of a LLIN during the night previous to enrolment was associated with a reduced risk of recurrent P . vivax and P . falciparum in univariate analyses ( Supplementary file 1 - Table 1 ) but to a lesser extent in multivariable analyses ( P . vivax: AHR = 0 . 62 [0 . 39–0 . 98] , p=0 . 043 , P . falciparum: AHR = 0 . 84 [0 . 49–144] , p=0 . 531 ) .", "Haemoglobin ( Hb ) level at enrolment was negatively associated with the risk of recurrent infection with P . vivax ( AHR = 0 . 88 [0 . 80–0 . 98] , p=0 . 019 ) and P . falciparum ( AHR = 0 . 90 [0 . 80–1 . 02] , p=0 . 099 ) .", "Patterns in the risk of recurrent infections with P . falciparum and P . vivax as measured by light microscopy ( LM , Supplementary file", "2 ) were similar to those observed for re-infection as measured by qPCR .", "When based on LM observation ( but not as measured by qPCR ) , increasing age was associated with a reduced risk of recurrent P . vivax ( AHR = 0 . 85 [0 . 77–0 . 95] , p=0 . 004; Supplementary file", "2 ) but an increased risk of recurrent P . falciparum ( AHR = 1 . 16 [1 . 01–1 . 33] , p=0 . 037; Supplementary file 2 ) .", "The incidence of new P . falciparum and P . vivax blood-stage clones detected during follow-up , that is molFOB , was highly variable between individual children and ranged from 0 to 18 new clones/year for P . falciparum ( Figure 2A ) and 0 to 36 or 23 new blood-stage clones/year for P . vivax in the placebo or PQ arm , respectively ( Figure 2B ) .", "Mean Pf- and Pv-molFOB varied significantly between villages and were higher in Bolumita ( Pf-molFOB = 4 . 9 new blood-stage clones/year , Pv-molFOBPQ arm=4 . 4 new blood-stage clones/year , Pv-molFOBplacebo arm=12 . 1 new blood-stage clones/year; Table 4; Figure", "3 ) than in the other villages ( Pf-molFOB , range 0 . 7–1 . 8 new blood-stage clones/year; Pv-molFOBPQ arm , range 0 . 03–2 . 2 new blood-stage clones/year; Pv-molFOBplacebo arm , range 2 . 3–7 . 4 new blood-stage clones/year ) .", "In univariate analyses , new P . vivax infections were strongly associated with new P . falciparum infections per sampling interval and vice versa , suggesting concurrent exposure to the two species ( Supplementary file 1 – Table 2 ) .", "However , these effects were reduced when other variables of varying exposure such as village of residence or infection at enrolment were included in the multivariable model ( P . vivax: IRRPQ arm=1 . 32 [0 . 92–1 . 89] , p=0 . 134; IRRPlacebo arm=1 . 10 [0 . 85–1 . 42] , p=0 . 466; P . falciparum: IRR = 1 . 15 [0 . 97–1 . 36] , p=0 . 100 , Table 4 ) .", "LLIN use , although strongly associated with lower Pf- and Pv-molFOB in univariate analyses ( Supplementary file 1 – Table 2 ) , remained significantly associated in multivariable models only for P . vivax in the placebo arm , where sleeping under a LLIN in the night previous to enrolment was associated with a 38% [9–57%] reduction in Pv-molFOB ( p=0 . 013 , Table 4 ) .", "Each additional year of age was associated with a 14% [0–26%] reduction Pv-molFOB per sampling interval in the PQ arm ( p=0 . 059 ) , while no age effect was observed in the placebo arm or for P . falciparum ( Table 4 ) .", "Hb level at enrolment was negatively associated with Pf- and Pv-molFOB ( P . vivax: IRRPQ arm=0 . 85 [0 . 72–1 . 01] , p=0 . 063; IRRPlacebo arm=0 . 91 [0 . 85–0 . 99] , p=0 . 025; P . falciparum: IRR = 0 . 85 [0 . 75–0 . 97] , p=0 . 013 ) , suggesting anaemia in individuals continuously exposed to blood-stage infections .", "A total of 98 clinical malaria episodes , here defined as fever plus presence of LM-detectable parasites , were observed during the study period .", "Of these , 64 ( 65% ) exceeded the previously established pyrogenic thresholds of 2500 and 500 parasites/µl per LM for P . falciparum and P . vivax , respectively ( Mueller et al . , 2009b ) .", "P . falciparum was the most common cause of clinical malaria episodes ( P . falciparum , 64 clinical episodes; P . vivax , 31 clinical episodes; mixed P . falciparum/P . vivax by LM , 3 clinical episodes ) , despite lower incidence of new P . falciparum blood-stage clones compared with P . vivax ( P . falciparum , 342 new P . falciparum blood-stage clones; P . vivax , 849 new blood-stage clones ) .", "Including clinical episodes with mixed infection as determined by LM in the estimates for both species , clinical incidence rate ( IR ) was 0 . 28 [0 . 21–0 . 35] P . falciparum episodes/year and 0 . 12 [0 . 08–0 . 17] P . vivax episodes/year .", "At least one new blood-stage clone was detected in 70% ( 47/67 ) of samples from P . falciparum and 71% ( 24/34 ) of samples from P . vivax clinical episodes .", "Of these clinical episodes with new blood-stage clones , 96% ( 45/47 ) and 83% ( 20/24 ) carried only the new but no persistent P . falciparum and P . vivax clones , respectively .", "P . vivax clinical episodes occurred mainly in the placebo arm shortly after directly observed treatment ( DOT ) ( Robinson et al . , 2015 ) , the time of peak Pv-molFOB due to relapsing hypnozoites ( Figure 1A ) .", "On an individual level , Pv-molFOB was positively associated with the risk of clinical episodes and each additional blood-stage P . vivax clone increased the risk of experiencing a P . vivax clinical episode slightly ( AHR = 1 . 07 [1 . 04–1 . 09] , p<0 . 001; Table 5 ) .", "No significant differences in P . vivax clinical episode risk were observed between villages after adjusting for individual molFOB .", "The risk for a P . vivax clinical episode decreased significantly with age ( AHR = 0 . 62 [0 . 46–0 . 84] , p=0 . 002; Table 5 ) .", "This was paralleled by a decrease in P . vivax densities with age ( by qPCR , exp ( β ) =0 . 90 [0 . 83–0 . 98] , p=0 . 016; Supplementary file", "3 ) indicative of more advanced immunity against P . vivax and thus better control of P . vivax densities in older children .", "Patterns in the occurrence of P . falciparum clinical episodes during follow-up were more complex .", "Between-village variation in P . falciparum clinical episode risk remained significant even after adjusting for individual exposure .", "This effect was mainly apparent in Numangu , where children were at three- to six-fold higher risk for clinical episodes than children in other villages ( Numangu AHR = 4 . 29 [2 . 06–8 . 97] , other villages range AHR = 0 . 65 [0 . 20–2 . 08] to 1 . 39 [0 . 59–3 . 30]; Table 5 ) .", "Overall , Pv-molFOB was positively associated with the risk of clinical episodes and each additional P . falciparum blood-stage clone slightly increased the risk for P . falciparum clinical episodes ( AHR = 1 . 15 [1 . 11–1 . 21] , p<0 . 001; Table 5 ) ; however , relative to the number of new blood-stage clones , P . falciparum clinical episodes were less frequent in highly exposed children compared to low-exposed children ( Figure 4 ) .", "One clinical episode per three new blood-stage clones was detected in the least exposed children ( Pf-molFOB <4 new blood-stage clones/year ) , but only one clinical episode per 15 blood-stage clones in the highest exposed children ( Pf-molFOB >9 new blood-stage clones/year , Fisher’s exact test p<0 . 001 ) .", "Age was not associated with the risk of P . falciparum clinical episodes .", "LLIN use at enrolment was associated with a 56% [13–78%] reduced risk of P . falciparum clinical episodes ( p=0 . 018; Table 5 ) .", "A higher risk for P . falciparum clinical episodes in children that had received PQ treatment for clearance of P . vivax liver stages was observed ( AHR = 1 . 79 [1 . 05–3 . 03] , p=0 . 031; Table 5 ) , suggesting a potential protective effect of P . vivax infections against P . falciparum clinical episodes .", "Analysis to further explore this revealed that a concurrent or recent infection ( i . e . , at the same or preceding follow-up visit ) with P . vivax reduced the odds of a P . falciparum clinical episode by 65% [22-85%] ( p=0 . 011; Table 6 ) .", "Further indications for a potential interaction between the two species was also observed when analyzing P . falciparum parasite densities , which were reduced by 55% [19–75%] ( p=0 . 008; Supplementary file", "3 ) in mixed P . falciparum/P .", "vivax infections compared to P . falciparum single infections , indicative of suppression of one of the species in mixed infections ." ], [ "In the present study , we describe striking heterogeneity in malaria transmission not only between closely neighboring communities in Maprik district , PNG , but also substantial differences in exposure between individual children from the same village .", "On village level this heterogeneity is apparent both when using traditional markers such as prevalence of infection , as well as when using the novel `reference standard` marker of individual exposure molFOB .", "The increased resolution provided by molFOB further allows quantifying heterogeneity in exposure to new blood-stage infections between individual children .", "Extending an earlier study in a neighboring area in which younger children had been enrolled , and that had identified molFOB as the most important predictor of malaria clinical episodes ( Mueller et al . , 2012; Koepfli et al . , 2013 ) , we confirmed that molFOB remains significantly associated with the risk for clinical episodes , but other factors such as age ( P . vivax ) , or a mixed Pf/Pv infection and village factors not captured by any of the other parameters assessed ( P . falciparum ) have a stronger effect on the risk for clinical malaria ( Table 5 and 6 ) .", "Malaria transmission is often estimated by investigating the more accessible human host rather than the mosquito vector ( Tusting et al . , 2014 ) .", "Because P . falciparum blood-stage infections are a direct outcome of mosquito-to-human transmission , infection parameters assessed in the human blood closely reflect P . falciparum transmission .", "In contrast , relapses arising from dormant hypnozoites contribute substantially to P . vivax blood-stage infections ( Robinson et al . , 2015 ) , thus complicating the assessment of mosquito-to-human P . vivax transmission via infection parameters measured in the human blood .", "The unique design of this study , that combined clearance of hypnozoites in half of the study participants with subsequent measurement of Pv-molFOB , allowed us to identify the burden of P . vivax infections due to mosquito-to-human transmission ( in hypnozoite-cleared individuals ) and compare it to the total burden of P . vivax infections .", "We found a highly similar incidence and comparable temporal and spatial heterogeneity of P . falciparum and P . vivax infection acquired through renewed exposure to infected mosquito bites .", "In children that experienced the full burden of relapses we found two-fold higher P . vivax infection prevalence and 4-times higher incidence ( molFOB ) compared to P . falciparum .", "This first quantitative comparative assessment of P . falciparum and P . vivax transmission using non-entomological molecular parameters thus indicates that the observed differences in epidemiology between the two species are largely due to the high burden of relapsing P . vivax blood-stage infections ( Robinson et al . , 2015 ) .", "Our molecular results thus support recent entomological data from Dreikikir district , 50 km from Maprik in East Sepik Province ( Reimer et al . , 2016 ) as well as earlier studies in East Sepik ( Hii et al . , 2001 ) that found similar sporozoite rates for P . falciparum and P . vivax .", "Our previous analysis of this cohort had investigated the contribution of the hypnozoite reservoir to P . vivax infection and disease in order to inform strategies for achieving a sustained reduction of the P . vivax burden in PNG ( Robinson et al . , 2015 ) .", "Here , we now describe the post-treatment re-infection dynamics in higher temporal detail .", "Through a detailed comparison of these patterns for P . vivax and P . falciparum in PQ and placebo-treated children we further elucidate the contribution of relapses to P . vivax prevalence and clinical incidence , which are the most commonly used parameters for planning and monitoring of malaria control strategies .", "P . vivax relapses accounted for more than half of the observed P . vivax prevalence in this cohort , which is lower than what was previously estimated as the contribution of relapses towards Pv-molFOB by comparison of treatment arms ( 77% , Robinson et al . , 2015 ) .", "This difference can be accounted for by the higher number of P . vivax multiple clone infections that will accumulate more rapidly in the placebo-arm , where additional parasite clones from relapses and/or new infections may overlap with without a corresponding change in overall prevalence .", "While we previously described a sustained effect of PQ treatment with significant reductions in Pv-molFOB observed up to eight months post treatment ( Robinson et al . , 2015 ) , here , we describe temporal variation in relapse rate with a rapid and wave-like recurrence of P . vivax in children from the placebo arm , who had retained their hypnozoites ( Figure 1A ) .", "The concurrent , modest peaks in Pf-molFOB and Pv-molFOB in the PQ arm represent seasonal variation in transmission , which is highest in December and January in the study area ( Mueller et al . , 2012 ) ; corresponding to weeks 8–14 of follow-up ) .", "A much higher peak and subsequent drop in appearance of new clones within three months after blood-stage only treatment , which was mirrored by a corresponding peak and drop in P . vivax prevalence ( Figure 1B andC ) , suggests that the incidence of relapse infections in the blood was not constant during follow-up .", "P . vivax infections are often observed following treatment of P . falciparum malaria ( Douglas et al . , 2011 ) , and it can thus been hypothesized that the frequency of relapses may be temporarily increased after blood-stage antimalarial treatment ( White and Imwong , 2012 ) .", "It is thus conceivable that the blood-stage antimalarial at baseline either triggered P . vivax relapses directly , or indirectly by allowing more hypnozoites to establish blood-stage infections in parasite-free hosts .", "Alternatively , blood-stage P . vivax infections from hypnozoites relapsing shortly after baseline treatment ( during a period when antimalarial drugs were present at sub-curative levels ) may be suppressed to sub-detectable densities until complete waning of drug levels , resulting in simultaneous proliferation and detection of many new blood-stage clones within the first weeks after treatment ( Douglas et al . , 2011; Tarning et al . , 2014 ) .", "More detailed modeling of the dynamics of individual P . vivax blood-stage infections and their association with potential triggers such as treatment or febrile illness will be required to determine the existence and importance of proposed relapse-triggers .", "Malaria transmission showed high micro-spatial heterogeneity with more than 10-fold differences in Pf- and Pv-molFOB ( in the PQ arm ) between villages despite an overall high LLIN use by the study participants ( during follow-up; village average use , >90%; individual use , >50% ) .", "Individual LLIN use at enrolment was nevertheless associated with a reduced risk of recurrent P . falciparum and P . vivax in univariate analyses .", "However , after adjustment for other related variables ( i . e . , village of residence or infection status ) this association became non-significant .", "Children living in Bolumita , where both P . falciparum and P . vivax molFOB and prevalence were highest , had a modestly lower LLIN use ( mean during follow-up , 92%; at enrolment , 77% ) compared to children from other villages ( mean during follow-up , 97–100%; at enrolment , 91–100% ) .", "It is conceivable that LLIN use in the Bolumita community may be less effective in reducing malaria transmission ( Killeen et al . , 2007; Smith et al . , 2009 ) .", "Potential differences between villages in mosquito density , behavior , sporozoite rate , proximity of house or play areas to mosquito breeding sites , or human behavioral factors ( related to LLIN use or other risk factors ) are however likely to be more important determinants for exposure to infective bites .", "Small-scale variations in vector species and distribution between and within villages in PNG have been described previously ( Cattani et al . , 1986; Reimer et al . , 2016; Charlwood et al . , 1986; Hii et al . , 1997; Burkot et al . , 1988 ) and likely account in a large part for the micro-geographic heterogeneity in malariological parameters observed in this and other studies .", "Assessing the incidence of new infections from consecutive blood samples using molecular methods ( as is necessary to determine molFOB ) , is complicated by fluctuating densities of clonal parasitemia that may temporarily fall below the limit of detection of the genotyping PCR , leading to imperfect detectability of clones ( Bretscher et al . , 2010; Felger et al . , 2012; Koepfli et al . , 2011 ) .", "For P . falciparum , periodical sequestration of clones and absence from the peripheral blood at time of sampling may further contribute to imperfect detectability .", "For P . vivax , generally low parasite densities aggravate the problem of imperfect detectability , and dis- and re-appearance of clones may be a result of imperfect detectability or relapsing hypnozoites .", "The overall estimates of molFOB presented here may thus be biased .", "Accurately assessing the effects of this imperfect detectability on parameters estimated from longitudinal genotyping data , such as molFOB , requires complex mathematical modeling ( Bretscher et al . , 2010; Felger et al . , 2012; Sama et al . , 2005; Sama et al . , 2006 ) .", "However , although clonal detectability has been shown to decrease with age ( Felger et al . , 2012; Sama et al . , 2006 ) and MOI ( Koepfli et al . , 2011 ) , it is unlikely to vary substantially within our cohort’s age range and transmission setting .", "Hence the observed differences in molFOB are likely to accurately reflect the relative differences in individual exposure as well as in population transmission levels within the study area .", "Evaluating the impact of malaria control efforts requires monitoring changes in malariological metrics over extended periods of time .", "Drawing comparisons between studies performed at different times in different age groups is particularly challenging because of the interplay of past and current exposure to infective bites and the resulting anti-malarial immunity in the study population of a certain age .", "In our cohort , fewer P . vivax clinical episodes than P . falciparum clinical episodes were detected despite a higher incidence of P . vivax blood-stage infections , which is consistent with earlier studies in children of similar age ( Michon et al . , 2007 ) .", "The very low incidence of clinical P . vivax episodes in our cohort , at 0 . 16 clinical episodes/year ( placebo arm [Robinson et al . , 2015] ) , contrasts drastically with that of 2 . 46 P . vivax clinical episodes/year observed in an earlier observational cohort of younger children aged 1–4 years from the same area ( Lin et al . , 2010 ) .", "The 3-fold difference in Pv-molFOB between the two cohorts seems modest when compared to the 15-fold difference in the incidence of clinical Pv episodes ( Koepfli et al . , 2013 ) .", "This suggests that the much lower incidence of P . vivax clinical illness in 5–10 years old children of this study is more likely explained by an advanced state of immunity to P . vivax compared to the younger children of the earlier cohort than the drop in P . vivax transmission .", "Consistently , age emerged as the strongest factor associated with protection against P . vivax clinical episodes , Pv-molFOB and P . vivax parasite density .", "Like in the previous cohort of younger children from neighboring villages ( Lin et al . , 2010 ) the incidence clinical P . vivax clinical episodes dropped significantly with age .", "Unlike in the previous cohort of younger children ( Koepfli et al . , 2013 ) , in this cohort we additionally observed a drop in Pv-molFOB as well as P . vivax densities with age ( Table 4 and Supplementary file 3 ) .", "This is a further indication of the substantial clinical immunity to P . vivax acquired during years of past exposure in the children of this study , which is still ongoing after the age of five .", "In sharp contrast to the age-dependent decline of P . vivax clinical episode incidence , no age-dependent decrease in the incidence of clinical episodes was observed for P . falciparum .", "In a previous cohort study conducted in 2004 in 5–14 year old children in an area from PNG with substantially higher transmission levels ( mean incidence risk 5 . 0 versus 0 . 8 infections/year , Michon et al . , 2007; Robinson et al . , 2015 ) , the risk of moderate- to high-density P . falciparum infections decreased significantly with age ( Michon et al . , 2007 ) .", "Clinical immunity to P . falciparum in children of this earlier cohort was not only significantly further advanced compared to children of this cohort , but in addition , transmission was more homogeneous in the area of that study .", "As a consequence age was a much better marker of life-time exposure and thus immune status compared to the present cohort .", "In this cohort , exposure to P . falciparum infections was highly heterogeneous between study participants .", "Mathematical modeling suggests that at heterogeneous transmission , changes of parasite prevalence and clinical episode incidence with age are less pronounced compared to settings with homogeneous transmission ( Ross and Smith , 2010 ) .", "Although no age trends were observed for P . falciparum in this cohort , when children were stratified into groups ranging from low to high exposure we found that the proportion of P . falciparum clinical episodes relative to new infections decreased with increasing exposure .", "This could either reflect the development of clinical immunity in highly exposed children , or premunition , a proposed mechanism by which established infections help to control superinfections by immunological cross-protection ( Sergent and Parrot , 1935; Smith et al . , 1999 ) .", "In settings of decreasing and heterogeneous transmission , age alone may therefore not be a suitable marker of immunity to P . falciparum .", "Instead , combining age and molFOB to estimate cumulative life-time exposure may provide a more accurate surrogate measure of the extent of acquired clinical immunity .", "With P . falciparum transmission declining in PNG due to successful malaria control strategies ( Koepfli et al . , 2015 ) , it is conceivable that immunity against P . falciparum will develop more slowly , shifting the burden of disease towards older age groups or towards more complex , non-linear age patterns .", "This delay in immune acquisition is however more than compensated by the overall much lower incidence of clinical malaria clinical episodes: in cohort studies in children younger than 4 years from Maprik district , clinical P . falciparum incidence had dropped from 2 . 56 clinical episodes/year before ( observational cohort , [Lin et al . , 2010; Mueller et al . , 2012] ) to 0 . 67 clinical episodes/year immediately after the free LLIN distribution campaign ( placebo arm , [Betuela et al . , 2012] ) .", "Finally , given that four Plasmodium species co-exist in PNG , there has long been considerable interest in potential mechanisms of cross-species immunity and mixed species interactions ( Mueller et al . , 2009a; Bruce et al . , 2000; Smith et al . , 2001; Mehlotra et al . , 2000 ) .", "However , there is so far no consistent evidence for the presence or absence of cross-protection among Plasmodium species .", "P . vivax and P . falciparum infections in our study were concentrated in the same children and villages ( Figure 3 ) , likely due to overlapping focal transmission for P . falciparum and P . vivax and thus potentially high co-infection rates in mosquitoes .", "Contrary to an earlier cohort in younger PNG children that found a decreased risk of P . falciparum clinical episodes after PQ radical cure ( Betuela et al . , 2012 ) , we found indications for an increased risk of P . falciparum illness after clearance of P . vivax hypnozoites using PQ .", "Our data suggests that in individuals with substantial clinical immunity against P . vivax , a concurrent P . vivax infection may provide protection against P . falciparum clinical episodes by limiting P . falciparum densities ( Table 6 , Supplementary file 3 ) .", "However , the comparably small number of clinical episodes in this and the earlier contrasting study does not allow an in-depth analysis of causal relationships and therefore does not allow firm conclusions on the potential effects and mechanisms of cross-species interactions in mixed infections .", "In conclusion , this study provides detailed insight into the changing epidemiology of malaria in PNG children under sustained malaria control , by using molFOB as a powerful measure to quantitatively investigate patterns of new mosquito-derived P . falciparum and P . vivax infections versus those for P . vivax relapsing infections , as well as spatial and age trends in exposure to these infections .", "Striking heterogeneity in malaria transmission between villages as well as in individual exposure to new P . falciparum and P . vivax infections persisted in our study area despite very high use of LLINs .", "This presents a significant challenge for on-going malaria control efforts .", "The comparable patterns of new mosquito-derived P . falciparum and P . vivax infections indicate that sustained use of LLINs does result in a comparable reduction in transmission of both species .", "The higher incidence and prevalence of P . vivax infections observed in our data is thus directly linked to its ability to cause relapsing infections , highlighting the crucial role of hypnozoites for P . vivax epidemiology and the need to effectively intervene against these hidden stages .", "Together , these insights provide a crucial link to evaluate the level of P . vivax mosquito-based transmission against that of P . falciparum and serve to calibrate other standard malaria indicators such as parasite prevalence or incidence of clinical episodes and to ultimately inform new approaches to surveillance and response systems ." ], [ "This study was conducted in six villages in the Albinama and Balif areas , Maprik district , East Sepik Province , PNG between August 2009 and May 2010 .", "The area is serviced by the Albinama health sub-center , Balif aid post and a network of health workers in all study villages .", "The study design has been described in detail elsewhere ( Robinson et al . , 2015 ) .", "Briefly , 524 children aged 5–10 years whose parents provided written informed consent for their participation were enrolled and randomized to receive either chloroquine ( CQ , days 1–3 , total dose 25 mg/kg ) , artemeter-lumefantrine ( Coartem , AL , days 11–13 , 2 mg/kg A , 12 mg/kg L ) and primaquine ( PQ , days 1–20 , 0 . 5 mg/kg/day ) ; or CQ ( days 1–3 ) , AL ( days 11–13 ) , and placebo ( days 1–20 ) over 20 days of directly observed treatment ( DOT1-20 ) in a double-blinded manner .", "Children were actively visited and examined for signs and symptoms of malaria fortnightly at their schools for 8 months .", "In addition , passive surveillance was provided by the local health centre , aid post and village health workers throughout the study period .", "Finger-prick blood samples ( 250 µl ) were collected at fortnightly active-follow-up visits in the first 12 weeks and monthly thereafter , as well as from symptomatic children detected during active or passive morbidity surveillance .", "Symptomatic children were tested for malaria infection with rapid diagnostic test ( RDT , CareStartMalaria pLDH/HRP2 Combo , AccessBio , USA ) , and only RDT and or LM-confirmed Plasmodium infections of any density were treated with a 3 day course of AL .", "Household , village and health facility location data was collected using a handheld GPS receiver ( Garmin GPSmap62sc ) and maps were prepared using ArcGIS 10 . 2 ( Esri , USA ) .", "The study received ethical clearance from the PNG IMR Institutional Review Board ( 0908 ) , the PNG Medical Advisory Committee ( 09 . 11 ) , the Ethics Committee of Basel 237/11 and was conducted in full concordance with the Declaration of Helsinki .", "The study was registered on ClinicalTrials . gov ( NCT02143934 ) .", "All blood samples were examined by LM and qPCR for detection and speciation of Plasmodium infections as described earlier ( Robinson et al . , 2015 ) .", "Each blood slide was read independently by two skilled microscopists and re-read by an expert microscopist in case of discrepancies in positivity , speciation or density ( ≥2 x log10 difference ) .", "Thick blood films were examined by LM for 200 fields ( 1000x magnification ) before being declared parasite-negative .", "Parasite density was converted from the number of parasites per 200–500 white blood cells ( WBC ) to parasites/µl assuming 8000 WBC/µl ( WHO malaria microscopy training guide ) and calculated as the geometric mean of all positive reads .", "DNA was extracted from the red blood cell pellet using the FavorPrep 96-well genomic DNA extraction kit ( Favorgen ) .", "Samples carrying any Plasmodium spp .", "infection were identified using a generic qPCR ( Wampfler et al . , 2013 ) and positives were subsequently tested in species-specific qPCRs ( Rosanas-Urgell et al . , 2010; Wampfler et al . , 2013 ) .", "All qPCRs targeted the small subunit ( 18S ) ribosomal RNA gene and were performed as simplex ( P . vivax and P . falciparum ) or duplex qPCR ( P . malariae , P . ovale ) .", "The concentration of target copies per µl of DNA was determined relative to a dilution row of standard plasmid as previously described ( Rosanas-Urgell et al . , 2010 ) .", "The qPCR limit of detection ( LOD ) was determined using a standard plasmid dilution row and defined as the last point with more than 50% of replicates positive .", "The LOD was 2 target copies/µl DNA , equaling 4 target copies/reaction , for all qPCRs .", "All samples that crossed the fluorescence threshold were scored as positive for species-specific qPCRs .", "In all samples positive in P . falciparum and/or P . vivax qPCRs , individual parasite clones were distinguished by genotyping the length-polymorphic Pf-msp2 or Pv-msp1F3 marker genes using capillary electrophoresis for highly precise fragment sizing ( Koepfli et al . , 2013; Koepfli et al . , 2011; Falk et al . , 2006; Schoepflin et al . , 2009 ) .", "MOI was determined by counting the number of detected Pf-msp2 or Pv-msp1F3 alleles per sample .", "molFOB was calculated from the number of new parasite clones detected per child or per sampling interval in the peripheral blood , divided by the individual time at risk or length of the interval .", "A new infection was defined as a Pf-msp2 or Pv-msp1F3 allele not present in the two preceding genotyping-positive samples collected during active or passive surveillance ( Figure 1—figure supplement 1 ) .", "Imperfect diagnostic detectability was not further adjusted for .", "Children were considered at risk for clinical malaria clinical episodes until the end of the study or until they were censored ( on the last visit before two consecutively missed scheduled follow-up visits [Robinson et al . , 2015] ) .", "For clinical endpoints , time-at-risk ( TAR ) was not further adjusted for interim missed follow-up visits because the intense active and passive case detection presumably led to detection of all malaria clinical episodes .", "In contrast , TAR for analysis of molecular data ( e . g . , molFOB ) was reduced by the duration of the missed interval if a child was not seen by the study team for six weeks or more ( ≥42 days ) .", "Children with a TAR of less than 3 months ( <84 days ) were excluded .", "This resulted in an analyzed population of 466 children ( characterized in Table 1 ) of which 430 ( 92 . 3% ) completed the whole follow-up period , with a median of 15 ( IQR: 13–17 ) study contacts and mean TAR of 186 days ( IQR 168–223 days ) .", "Time to first Plasmodium infection by qPCR and LM and its association with covariates were modeled using Cox regression , and the proportional hazards assumption was checked using the test based on the Schoenfeld residuals .", "Multiple failure Cox regression was used to model the time to P . vivax and P . falciparum clinical episodes .", "For statistical analysis , a malaria clinical episode was defined as fever ( >37 . 5°C axillary ) plus the presence of LM-detectable parasites , irrespective of RDT result or antimalarial treatment during the field visit .", "Negative binomial generalized estimating equations ( GEE ) with log link function using an exchangeable correlation structure were used to model incidence of new infections with P . falciparum and P . vivax per sampling interval .", "For these analyses , the time at risk was restricted to the intervals where molFOB could be estimated ( i . e . , starting in the third follow-up interval ) .", "A binomial GEE with logit link function using an exchangeable correlation structure was used to model the odds of a P . falciparum clinical episode per interval .", "Gaussian GEEs with log link function using an exchangeable correlation matrix were used to model log-transformed qPCR parasite densities in qPCR-positive samples , measured as 18S rRNA copy numbers/µl blood .", "In the GEE and Cox models where molFOB was a covariate , it was included as a time-varying covariate .", "When modelling molFOB ( Table 4 ) and the odds of clinical episodes ( Table 6 ) using GEEs , molFOB was calculated for each follow-up interval and used as predictor .", "In Cox models investigating the risk of clinical episodes ( Table 5 ) , molFOB was calculated based on the new infections up to the time of failure and used as predictor .", "In exploratory preliminary analyses we tested for a wide variety of interactions between covariates including interactions between all combinations of molFOB , enrolment infection status , age , village and bednet-usage .", "All analyses were done using STATA v14 and R . Maps were drawn using Arcgis 10 . 1 ( Esri Inc . ) .", "Ordinary kriging was used to generate the contour maps .", "Semivariograms were used as the mathematical forms used to express autocorrelation .", "Input variables for the spatial models were", "( i ) molFOB ( prediction of independent variable , molFOB ) using the model presented in Table 4 ( negative binomial GEE ) for Pf and Supplementary file 4 for Pv ( same as that shown in Table 4 but with primaquine and placebo arms combined ) , resulting in Figure 3 Panels A and B;", "( ii ) relative risk of clinical episodes as predicted by the model shown in Table 5 , resulting in Panels C and D of Figure 3 .", "Relative standard error maps were generated by dividing the absolute standard error map by the model prediction map ." ] ]

[ "The molecular force of blood-stage infection ( molFOB ) is a quantitative surrogate metric for malaria transmission at population level and for exposure at individual level .", "Relationships between molFOB , parasite prevalence and clinical incidence were assessed in a treatment-to-reinfection cohort , where P . vivax ( Pv ) hypnozoites were eliminated in half the children by primaquine ( PQ ) .", "Discounting relapses , children acquired equal numbers of new P . falciparum ( Pf ) and Pv blood-stage infections/year ( Pf-molFOB = 0–18 , Pv-molFOB = 0–23 ) resulting in comparable spatial and temporal patterns in incidence and prevalence of infections .", "Including relapses , Pv-molFOB increased >3 fold ( relative to PQ-treated children ) showing greater heterogeneity at individual ( Pv-molFOB = 0–36 ) and village levels .", "Pf- and Pv-molFOB were strongly associated with clinical episode risk .", "Yearly Pf clinical incidence rate ( IR = 0 . 28 ) was higher than for Pv ( IR = 0 . 12 ) despite lower Pf-molFOB .", "These relationships between molFOB , clinical incidence and parasite prevalence reveal a comparable decline in Pf and Pv transmission that is normally hidden by the high burden of Pv relapses .", "Clinical trial registration: ClinicalTrials . gov NCT02143934" ]

[ "Malaria is caused by five different species of parasites that are transmitted to humans by bites from parasite-carrying mosquitos .", "Once in human blood , the parasites rapidly multiply .", "People who live in countries where malaria is common may become infected and never show any symptoms because their immune systems are able to keep parasite numbers low .", "Repeated infections , or infection with more than one species of malaria parasite also are common .", "Some species of malaria , including Plasmodium vivax , can hibernate in the liver for weeks or months after the infection and only become active later .", "Asymptomatic infections , multi-parasite infections , and reactivating parasites make it hard to measure how often new malaria infections occur .", "One way scientists can determine if a new infection has occurred is by genotyping the parasites in a person’s blood .", "Genotyping involves looking for small differences in the parasite DNA .", "For example , a study in Papua New Guinea , where P . vivax is very common , showed that reactivations of hibernating parasites were more common than new infections .", "Now , Hofmann et al . use the same study in Papua New Guinea to compare the frequency and consequences of new infections with P . vivax and another malaria parasite , Plasmodium falciparum .", "In the study , 466 children from 6 villages were followed for 8 months with tests every 2 to 4 weeks to genotype the parasites in their blood .", "Some of the children were treated with antimalarial drugs to help wipe out any existing parasites including hibernating ones .", "While P . vivax was about twice as common in blood samples—likely due to reactivation—genotyping showed that new infections with the two parasites occur at equal rates and often at the same times and locations .", "Hofmann et al . also showed that some villages and some children had much higher rates of infection than others .", "This difference could not fully be explained by use of bednets or other preventive measures .", "Children were more likely to become ill from P . falciparum than P . vivax even though P . vivax was more common .", "But children with more frequent infections with P . falciparum seemed better able to manage the parasites and were less likely to develop symptoms that those with infrequent infections .", "The experiments show that genotyping may help scientists better track new malaria infections and develop better strategies to prevent or treat malaria ." ]

[ "Introduction", "Results and discussion", "Materials and methods" ]

[ "cell biology", "physics of living systems" ]

[ [ "Cytokinesis begins in late anaphase , triggered by regulatory pathways with feedback from the mitotic spindle that ensure appropriate positioning of the molecular machinery ( reviewed in Fededa and Gerlich , 2012; Green et al . , 2012 ) .", "As a consequence , the small regulatory GTPase RhoA ( Piekny et al . , 2005; Tse et al . , 2012 ) and the molecular motor myosin ( Shelton et al . , 1999; Werner and Glotzer , 2008 ) are enriched in an equatorial band ( Piekny et al . , 2005; Werner and Glotzer , 2008 ) .", "Myosin can directly organize the actin network ( Miller et al . , 2012; Soares e Silva et al . , 2011; Vavylonis et al . , 2008 ) , and if myosin-based active alignment ( e . g . via search-and-capture [Vavylonis et al . , 2008] ) or flow-based compression as proposed by White and Borisy ( White and Borisy , 1983; Rappaport , 1996 ) drive furrow formation and cytokinesis in eukaryotes remains unclear ( Green et al . , 2012; Bray and White , 1988; Levayer and Lecuit , 2012; Mendes Pinto et al . , 2013; Cao and Wang , 1990; Murthy and Wadsworth , 2005; Zhou and Wang , 2008 ) ." ], [ "We set out to address this question in the one cell embryo of the nematode C . elegans which forms two constricting ingressions , first a pseudocleavage furrow during polarity establishment , and then a cytokinetic furrow during cytokinesis ( Figure 1—figure supplement 1a , Video 1 ) ( Munro and Bowerman , 2009; Rose et al . , 1995 ) .", "Notably , the pseudocleavage furrow is linked to cortical flow ( Mayer et al . , 2010; Munro et al . , 2004 ) and lacks regulatory control from the mitotic spindle ( Werner and Glotzer , 2008 ) .", "We first characterized the actomyosin cortical network during pseudocleavage and cytokinesis .", "For this , we generated a Lifeact::mKate2 fluorescent line of C . elegans by coupling the Lifeact peptide to the far-red fluorophore mKate2 that was codon optimized for C . elegans ( Redemann et al . , 2011 ) ( see Materials and methods and Video 2 ) .", "This allows us to quantify cortical actin filament orientation in space and time , as well as cortical flow velocity fields by Particle Image Velocimetry ( PIV , Figures 1 and 2 , Figure 2—figure supplement 1 ) ( Mayer et al . , 2010 ) .", "We find that actin filaments change from a randomly oriented and isotropic gel before cortical flows initiate , into a more circumferentially aligned organization at the future site of furrow ingression during both pseudocleavage and cytokinesis ( Figure 1b , Figure 1—figure supplement 1 and Videos 1 and 3 ) .", "We studied how pseudocleavage and cytokinesis differ in the spatial distribution of molecular regulation and contractility within the equatorial region ( Figure 1 ) .", "Non-muscle myosin II ( NMY-2 ) is the essential molecular motor that drives both cortical flow and cytokinesis ( Shelton et al . , 1999; Guo and Kemphues , 1996 ) , whereas the small GTPase RhoA ( Piekny et al . , 2005; Tse et al . , 2012 ) , when active , is responsible for its local activation .", "At the onset of cytokinesis , active RhoA and NMY-2 locally increase at the position of the contractile ring , while for pseudocleavage we observed no such increase ( Figure 1 ) .", "Instead , the equatorial region in pseudocleavage corresponds to a transition zone with a gradual decrease of RhoA and myosin between the anterior region of high and the posterior region of low contractility ( Figure 1 ) ( Mayer et al . , 2010 ) .", "The actin nucleator formin CYK-1 is downstream of RhoA ( Piekny et al . , 2005 ) and follows a similar pattern of localization , and actin bundling via anillin ( Piekny and Glotzer , 2008 ) or plastin also does not appear to be increased in the equatorial region during pseudocleavage ( Figure 2—figure supplement 5e–f ) .", "To conclude , the pseudocleavage furrow ingresses with a circumferential alignment of filaments in the gel , but without a local zone of RhoA activation and the corresponding local increase in myosin and formin density .", "Since search-and-capture like mechanisms require a localized band of myosin and actin nucleators at the equator to drive myosin-based active alignment ( Vavylonis et al . , 2008 ) , this suggests that actin filaments in pseudocleavage align via flow-based compression and not via active alignment . 10 . 7554/eLife . 17807 . 003Video 1 . Actomyosin gel dynamics in the C . elegans zygote . Cortical and medial planes of an embryo expressing both Lifeact::mKate2 and endogenous NMY-2::GFP .", "Left panel , medial plane Lifeact:mKate2 , center , cortical NMY-2::GFP , right panel , cortical Lifeact:mKate2 ( min:s ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 17807 . 00310 . 7554/eLife . 17807 . 004Video 2 . Three-dimensional reconstruction of an early one-cell embryo of C . elegans expressing Lifeact::mKate2 . The back of the embryo is dimmer due to imaging . DOI: http://dx . doi . org/10 . 7554/eLife . 17807 . 00410 . 7554/eLife . 17807 . 005Figure 1 . Flow and ingression during pseudocleavage and cytokinesis .", "( a ) Average active RhoA biosensor intensity profiles along the AP axis ( 0 represents the embryo center; N = 14 embryos for pseudocleavage and N = 7 for cytokinesis; errors represent the standard error of the mean ) .", "( b ) Actomyosin cortical organization at the onset of pseudocleavage and cytokinesis in embryos expressing both Lifeact::mKate2 and NMY-2::GFP .", "( c–d )", "Average actin , myosin and ingression profiles as a function of position along the AP axis ( N = 10 embryos for pseudocleavage and N = 22 for cytokinesis; errors represent the standard error of the mean ) .", "Scale bars , 10 μm .", "The following figure supplement is available for Figure 1:DOI: http://dx . doi . org/10 . 7554/eLife . 17807 . 00510 . 7554/eLife . 17807 . 006Figure 1—figure supplement 1 . Details of actin organization during the polarization flow phase .", "( a ) Actomyosin gel dynamics in the C . elegans zygote .", "Single cortical and medial planes from a timelapse sequence of a representative embryo expressing both Lifeact::mKate2 and NMY-2::GFP .", "Anterior is to the left , posterior to the right .", "White arrows indicate the position of pseudocleavage and cytokinesis ingressions .", "Red arrows indicate flow directions .", "( b ) Close up look at the filaments dynamics near the site of flow initiation .", "Cortical plane from a timelapse sequence of an embryo expressing both Lifeact::mKate2 and NMY-2::GFP .", "( c ) Top: Actin network organization from an embryos expressing Lifeact::mKate2 .", "Times rescaled to 0 at flow initiation .", "Green arcs indicate the smooth cleared zone during the polarization process .", "Red lines schematize filaments maximum vertical alignment positions .", "White arrows underlie pseudocleavage ingression .", "Bottom: color-coded representation of the orientation of filaments .", "( d ) Example from an embryo for which the polarization process starts off centered from the physical pole of the embryo due to a mispositioning of the sperm pronucleus ( dashed circle on medial plane ) .", "Alignment direction is observed to follow perpendicularly to the direction of the flow as it reorients along the long axis of the embryo ( also see Appendix ) .", "Red arrows represent flow direction , red lines filaments main orientation .", "Scale bars , 10 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 17807 . 00610 . 7554/eLife . 17807 . 007Figure 2 . Cortical flows compress the gel to generate alignment .", "( a ) Filament orientation is quantified by a nematic order parameter; flow fields are obtained from Particle Image Velocimetry ( PIV ) analysis .", "( b ) Gel flow , compression rate , filament orientation and cell ingression fields .", "Top row , schematic representations .", "Middle row , kymographs of the time evolution of the profiles along the AP axis of flow velocity , compression rate , nematic order parameter and outer ingression distance for pseudocleavage ( N = 10 ) .", "Bottom row , cytokinesis ( N = 12 ) .", "Orange dashed lines indicate the times where all four fields are stationary .", "Flow , compression and nematic order were determined from bottom-section and ingression from mid-section views of Lifeact::mKate2 labelled embryos ( see Materials and methods and Appendix ) .", "( c ) Spatial correlation between compression , alignment and ingression ( see Materials and methods ) .", "Peak value positions ( estimated by a Gaussian fitting of the 1D correlation curve ) are indicated .", "See Figure 2—figure supplement 2 for spatiotemporal correlation functions .", "( d ) Table of the temporal delays obtained from the correlation peak value positions using the mean values of the velocity in the 10 μm central region ( mean values for pseudocleavage: v = 6 . 16 ±1 . 23 μm/min; for cytokinesis: v = 4 . 01±1 . 92 μm/min; mean ± SD ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 17807 . 00710 . 7554/eLife . 17807 . 008Figure 2—figure supplement 1 . Quantification methods developed .", "( a ) Example of the filaments alignment quantification ( red lines ) and the flow velocity field ( yellow arrows ) , as well as nematic order parameters conventions .", "( b ) Time evolution of flow ( light green ) and compression rate ( dark green ) as well as ingression ( grey ) measured at a fixed position along the AP axis ( −20 μm for velocity , 0 μm or central position of the embryo for compression and 3 μm for ingression , measured from the center ) .", "Error bars represent the standard error of the mean .", "Bottom , fit using the error function ( red line ) of the time evolution of the velocity from a single embryo ( blue dots ) used to determine the reference time point ( Time = 0 ) .", "( c ) Time evolution of the mean velocity and compression rate along the AP axis and nematic order parameter , all taken at fixed positions along the AP axis .", "Error bars represent the standard error of the mean .", "In orange is represented the period chosen as stationary flow phase .", "( d ) Mean profiles along the AP axis of the velocities ( vx light green and vy yellow ) , compression rate and nematic order parameters ( Q = −Qxx in dark blue and Qxy in cyan ) during the stationary flow phase .", "( See Appendix for further explanations . ) DOI: http://dx . doi . org/10 . 7554/eLife . 17807 . 00810 . 7554/eLife . 17807 . 009Figure 2—figure supplement 2 . Spatiotemporal correlations . Plots of the spatiotemporal correlations between the compression , alignment and ingression data sets taken during the pseudocleavage stationary phase", "( a ) and cytokinesis onset stationary phase", "( b ) .", "Data is restricted to the 30 μm central region of the embryo . DOI: http://dx . doi . org/10 . 7554/eLife . 17807 . 00910 . 7554/eLife . 17807 . 010Figure 2—figure supplement 3 . Nematic order parameter quantification of artificial images with and without directional bias .", "( a ) Example of a simulated isotropic actin meshwork ( see Appendix for details ) .", "( b ) Example of a simulated actin meshwork for which the angle of the starting direction is biased toward vertical ( see Appendix ) .", "( c ) Cumulative distribution function ( CDF ) used and example of an image and its nematic order map ( red lines indicate the preferential orientation inside a template or orientation vector ) obtained for different values of the starting parameter B that controls the bias of the filaments orientation .", "B = 0 leads to an isotropic orientation .", "( d ) Variation of the average nematic order parameter obtained for several synthetic meshworks while increasing the vertical bias of the filaments orientation .", "The saturation is caused by the fact that we here bias only the initial direction of growth . DOI: http://dx . doi . org/10 . 7554/eLife . 17807 . 01010 . 7554/eLife . 17807 . 011Figure 2—figure supplement 4 . Impact of image quality on the nematic order parameter quantification .", "( a ) An artificial filamentous network is obtained by a random iteration process with controllable parameters in Matlab ( see Appendix for more details ) .", "( b-d )", "We chose to maintain a vertical bias in the orientation of the filaments in all the following cases ( B = 1 ) .", "Red lines indicate the preferential orientation inside a template or orientation vector .", "A profile of the mean nematic order parameter along the x-axis as well as the angular distribution are shown in all cases .", "Note that as we do not measure the directionality of filaments thus we obtain values modulo π .", "( b ) Impact of the filament density ( ranging from 60 to 120 filaments per image ) on the nematic order parameter quantification .", "The profile of nematic order is to first order independent of network density .", "( c ) Impact of the signal to noise ratio .", "High signal to noise ratio leads to higher values of the nematic order parameter , low signal to noise ratio leads to a poor detection of the direction of the filaments and artificially small values of the nematic order parameter . DOI: http://dx . doi . org/10 . 7554/eLife . 17807 . 01110 . 7554/eLife . 17807 . 012Figure 2—figure supplement 5 . Comparison of different actin labeling strains and actin-binding protein localization .", "( a–c )", "Average AP profiles of flow velocity ( light green ) and nematic order parameter ( blue ) at the time of stationary flow during pseudocleavage of respectively 22 embryos expressing Lifeact:GFP , NMY:RFP , 9 embryos expressing Lifeact:RFP , NMY-2:GFP and 16 embryos expressing Lifeact:mKate2 , NMY-2:GFP .", "Overlaid in red is the velocity profile of worms expressing only labeled NMY-2 ( N = 5 ) .", "Error bars represent the standard error of the mean .", "( d ) Images of NMY-2:GFP in the early zygote .", "Left strain without Lifeact , right strain expressing Lifeact:mKate2 .", "The density , size and dynamics are similar in both strains .", "Right panel , the average radial autocorrelation coefficient of the myosin intensity over a minute in early embryos gives an average myosin foci size .", "( e ) Localization of the actin bundling protein plastin during pseudocleavage and cytokinesis .", "Plastin localizes in dense bundles of actin filaments and is particularly abundant in the cytokenetic ring and could act to stabilize actin bundles in these regions .", "( f ) Localization of the actin nucleating agent formin during pseudocleavage and cytokinesis .", "CYK-1 is present throughout the cell cortex during pseudocleavage , with higher densities in foci regions .", "It is recruited to the cell equator during cytokinetic ring formation and ingression .", "Scale bars , 10 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 17807 . 01210 . 7554/eLife . 17807 . 013Video 3 . Cortical dynamics at flow initiation . Left panel , cortical NMY-2::GFP , center cortical Lifeact:mKate2 and a zoom is shown in the right panel ( min:s ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 17807 . 013 We next sought to test if flow-based compression drives actin filament alignment .", "To this end , we developed tools for the spatiotemporal quantification of compression by flow , of filament orientation , and of cortex ingression distance within the gel both at the onset of pseudocleavage and cytokinesis ( see Appendix as well as Figure 2a and Figure 2—figure supplement 1 ) .", "We find that the flow compression rate along the antero-posterior direction ( AP axis or x axis ) , given by the spatial gradient of the velocity field −∂xv ( Figure 2b ) , increases with time during the very early stages of pseudocleavage .", "The compression rate peaks in the central region of the embryo , with a maximum of ~0 . 4 min−1 .", "Notably , this peak is stable for several minutes until pseudocleavage flows cease entirely ( Figure 2b ) .", "For cytokinesis , we find that the early flow also proceeds in a unidirectional manner from the posterior toward the anterior ( Figure 2b ) .", "Similar to pseudocleavage , the compression rate increases with time over the very early stages of cytokinesis , with the compression profile peaking at the center of the embryo with a maximum rate of ~0 . 7 min−1 ( Figure 2b ) .", "We characterized the average actin filaments orientation by a nematic order tensor Q ( Figure 2—figure supplement 1a ) .", "This analysis relies on the fact that the intensity of the Fourier transformed image encodes geometric characteristics such as its main orientation pattern ( see Appendix ) .", "We find that vertical ( orthogonal to the direction of flow ) alignment appears coincidental in space and time with the local increase of the compression rate ( Figure 2b , compare column 2 and 3 ) .", "Notably , changing the direction of flow also changes the direction of alignment: off-site sperm entry leads to flows along the short axis of the egg ( Goldstein et al . , 1993 ) and actin filaments still align in the direction determined by compression ( Figure 1—figure supplement 1d , Video 4 and Appendix ) .", "Finally , the ingressing furrow forms in the region where filaments are aligned ( Figure 2b , column 4 ) .", "Our quantifications reveal remarkable similarities between the compression rate fields , the pattern of alignment , and the ingression distance between pseudocleavage and cytokinesis ( Figure 2b ) , suggesting common mechanisms .", "To conclude , actin filament alignment arises at locations of significant compression by flow . 10 . 7554/eLife . 17807 . 014Video 4 . Cortical dynamics of an embryo in which flow was initiated at the side ( bottom ) and far from the posterior pole of the embryo . Actin filament alignment follows flow and compression , thus transitions from a horizontal to a vertical direction ( min:s ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 17807 . 014 If flow-based compression aligns actin filaments for forming an ingression , we would expect compression to precede or to be concomitant with alignment and ingression .", "The 1D crosscorrelations between compression , filament alignment and ingression distance , calculated for the time that flows are essentially stationary ( Figure 2b and Figure 2—figure supplement 1 ) , show that for pseudocleavage both compression rate and alignment peak at about the same location , while the ingression field peaks ~ 3 µm further to the anterior ( Figure 2c and Figure 2—figure supplement 2 for the spatiotemporal crosscorrelations ) .", "The situation is similar at cytokinesis , except that the compression field peaks furthest to the posterior .", "We next translated the characteristic distances between peaks in stationary flow to temporal delays between events ( see Materials and methods for further explanation ) .", "We find that for pseudocleavage , filament alignment is essentially concomitant with compression , while for cytokinesis compression precedes alignment by 0 . 40 ± 0 . 16 min ( Figure 2d ) .", "Furthermore , ingression arises approximately 0 . 5 min after compression for pseudocleavage and 0 . 9 min after compression for cytokinesis .", "To conclude , filament alignment appears to rapidly follow compression while ingression arises with a delay .", "We next sought to provide a physical explanation for flow-based alignment ( Salbreux et al . , 2009 ) .", "The cell cortex in this and other systems can be thought of as a gel that forms a thin layer underneath the plasma membrane .", "Recent advances in physical theory show that this gel is active and generates the forces that drive cell shape changes ( Salbreux et al . , 2009; Gowrishankar , 2012; Kruse et al . , 2005; Turlier et al . , 2014 ) .", "We describe the actin cortical layer as a thin film of a nematic gel under shear and compression by flow ( Salbreux et al . , 2009; Kruse et al . , 2005; Prost et al . , 2015 ) .", "In the x-direction , the time evolution of the local nematic order Q depends on advection ( first term on the right hand side in Equation 1 ) , compression in gel flow ( second term ) , a process of relaxation to an isotropic configuration possibly via turnover ( third term ) , and a tendency of filaments to align with each other over space ( last term , see also Figure 3a ) ( 1 ) ∂tQ=−v∂xQ−β2∂xv−1τQ+ℓ2τ∂x2Q . 10 . 7554/eLife . 17807 . 015Figure 3 . Theory predicts an alignment peak .", "( a ) Schematic representation of flow-based alignment .", "For full theory refer to Appendix .", "( b ) Average AP profiles of gel flow ( light green , smoothened ) , compression rates ( dark green ) and nematic order parameter ( blue ) at the time of stationary flow ( Figure 2C ) during pseudocleavage ( left , N = 16 ) and cytokinesis onset ( right , N = 32 ) .", "Error bars represent the standard error of the mean .", "Dashed red line indicates the respective best-fit theory predictions for the nematic order parameter ( blue ) .", "( c ) Table of the material parameters .", "See also Figure 3—figure supplement 1 and Table 1 , Table 2 .", "( d ) Illustration of the cortical tension related to the ingression distance .", "( e ) Outer ingression profiles at the onset of pseudocleavage ( brown ) and cytokinesis ( yellow ) .", "The theoretical profiles ( thick dashed red lines: active tension depends on alignment and can be anisotropic; thin dashed lines: active tension is isotropic ) are determined by simultaneous fitting of both the pseudocleavage and cytokinesis datasets with a single fit parameter ( see Appendix ) using the myosin density , flow velocity and nematic order parameter fields . DOI: http://dx . doi . org/10 . 7554/eLife . 17807 . 01510 . 7554/eLife . 17807 . 016Figure 3—figure supplement 1 . Influence of the gel material parameters for the nematic order parameter .", "( a ) Nematic order parameter profiles obtained when varying independently each of the fitting parameters τ , βτ , ℓ away from their the best fit values obtained for cytokinesis .", "Dashed red line , best fit result ( τ = 2 . 34 min , βτ = 0 . 654 min , ℓ =1 . 69 μm ) .", "For each set of parameters τ , βτ , ℓ , a fit is performed for the fitting parameters C1 and C2 only ( see Appendix ) .", "( b ) Nematic order parameter profiles obtained when varying the fitting parameter τ while keeping βτ , ℓ fixed and equal to their best-fit values .", "Appropriate fits to experimental curve are obtained for all values of τ < 0 . 5 min .", "For each value of τ , a fit is performed for the fitting parameters C1 and C2 only . DOI: http://dx . doi . org/10 . 7554/eLife . 17807 . 01610 . 7554/eLife . 17807 . 017Figure 3—figure supplement 2 . Contribution of active alignment to the nematic order parameter profile .", "( a ) Average AP profiles of gel flow ( light green ) , compression rates ( dashed dark green ) , nematic order parameter ( blue ) and myosin fluorescence ( magenta ) at the time of stationary flow during pseudocleavage ( left , N = 16 ) and cytokinesis onset ( right , N = 32 ) .", "Error bars represent the standard error of the mean .", "Dashed red lines indicate the best-fit theory predictions for the nematic order parameter in absence of active alignment , dashed black lines are the fits obtained while including the myosin-based active alignment , with λ′τ=4 . 1 10−8±0 . 54 for pseudocleavage and λ′τ=0 . 34± 0 . 45 for cytokinesis .", "( b ) Contribution of the individual terms from Equation 3 in the main text ( red: −β2τ∂xv , green: −τv∂xQ , blue: ℓ2 ∂x2Q , magenta: λ′τ c ( x ) /c0Q ) .", "Note that myosin-based active alignment ( magenta ) is insignificant for pseudocleavage and is low for cytokinesis compared to compression-based alignment ( red ) .", "The theoretical alignment profile ( sum of all the terms shown in black ) overlays the experimental data points ( dots ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 17807 . 01710 . 7554/eLife . 17807 . 018Figure 3—figure supplement 3 . Ingression through anisotropic stress generation in the aligned gel .", "( a ) Top , a representative embryo is used to visualize the time course of ingression .", "Bottom , Kymograph obtained from the region specified by the dashed white box in the upper pannel .", "The pseudocleavage and cytokinesis ingressions are visible .", "Yellow arrow indicates the inner ingression distance during polarizing flow phase , magenta dashed line indicates the ingressing membrane and blue arrow indicates the outer ingression .", "Red doted lines represent the position of the eggshell .", "Scale bar 10 μm .", "( b ) Top , illustration of the cortical tension during ingression .", "Bottom , outer ingression profiles ( grey ) averaged over the time periods indicated by the thin horizontal white lines in ( a , bottom ) during pseudocleavage ( left ) and cytokinesis ( right ) .", "The theoretical profiles ( dashed red lines ) are determined by simultaneous fitting of both the pseudocleavage and cytokinesis datasets with a single fit parameter ( see Appendix ) using the myosin density ( magenta ) , flow velocity ( green ) and nematic order parameter ( blue ) fields .", "( c ) and", "( d ) Illustration of the coordinate and reference systems used for this theoretical description .", "( e ) Comparison of the theoretically obtained profile of the outer ingression without the anisotropic stress induced by nematic order ( dashed red , obtained by imposing p3 = 0 ) during pseudocleavage and cytokinesis phases with the experimental data ( black ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 17807 . 01810 . 7554/eLife . 17807 . 019Figure 3—figure supplement 4 . Shape and ingression distance quantification . Left column , plots of the cell shape ( mean radius for all embryos during the stationary flow phase and eggshell reference ) for pseudocleavage", "( a ) and cytokinesis", "( b ) .", "Error bars , the standard error of the mean .", "Right column , ingression distance for several embryos and mean value ( cyan ) during the stationary phase of pseudocleavage and cytokinesis . DOI: http://dx . doi . org/10 . 7554/eLife . 17807 . 019 The characteristic time for relaxation to an isotropic state via turnover is determined by τ , while β is a dimensionless coefficient that describes how local compression −∂xv impacts nematic ordering .", "Finally , ℓ is a length scale that determines the distance over which actin filaments tend to point in the same direction ( Salbreux et al . , 2009 ) .", "In our experiments , we observe a stationary phase ( Figure 2b ) , and the profile of nematic order Q at steady-state is given by ( 2 ) Q=−τv∂xQ−β2τ∂xv+ℓ2 ∂x2Q .", "We next determined the theoretical alignment field from the experimental flow field v and compression field ∂xv .", "We used nonlinear least-square fitting to evaluate parameter values for which the theoretical alignment profile best matched the experimental one .", "There are five unknowns ( three parameters that characterize emergent material properties , τ , ℓ , and β τ , as well as two boundary values of nematic order ) but three spatial fields , hence the fitting procedure is significantly constrained and the best fit parameters are uniquely defined in most cases ( see Appendix and Figure 3—figure supplement 1 ) .", "For cytokinesis the calculated alignment profile ( dashed red lines in Figure 3b ) best matches the experimentally measured one for β τ =0 . 64 ( 0 . 548 , 0 . 713 ) min ( unless otherwise noted we indicate the median value together with the standard 68% confidence interval of the distribution of the bootstrap data , see Appendix ) , τ = 2 . 3 ( 1 . 89 , 2 . 71 ) min and ℓ = 1 . 7 ( 1 . 37 , 2 . 44 ) μm ( Table 1 and Table 2 ) .", "For pseudocleavage , β τ was similar ( 0 . 6 ( 0 . 577 , 0 . 7 ) min ) , τ was determined to be smaller than 0 . 5 min , and ℓ = 4 . 7 ( 3 . 12 , 6 . 09 ) μm larger ( Table 1 and Table 2 ) .", "We note that in both cases , we obtain good agreement between the theoretical and experimental alignment field , allowing us to conclude that compressive gel flow can account for alignment .", "Further , we observe a small τ for pseudocleavage and a larger τ for cytokinesis , which is consistent with the observation that alignment appears concomitant with compression in pseudocleavage but arises with a short delay during cytokinesis ( Figure 2d ) .", "We speculate that the changes in physical parameters of the actomyosin cortical layer between pseudocleavage and cytokinesis ( higher τ and smaller ℓ ) reflect the fact that the cell forms a strong and more pronounced ring of aligned filaments during cytokinesis .", "To conclude , our results are consistent with a scenario in which actin filament alignment arises in a disordered network through compression by flow ( White and Borisy , 1983; Salbreux et al . , 2009; Turlier et al . , 2014 ) . 10 . 7554/eLife . 17807 . 020Table 1 . Best fit gel material parameters , bootstrapping .", "The median values together with the standard 68% confidence bounds of the distribution of the bootstrap data are given . DOI: http://dx . doi . org/10 . 7554/eLife . 17807 . 020Pseudocleavage Cytokinesis non RNAi non RNAi τ , min< 0 . 5 ( n . d . ) †2 . 26 ( 1 . 89 , 2 . 71 ) β τ , min0 . 626 ( 0 . 577 , 0 . 7 ) 0 . 646 ( 0 . 548 , 0 . 713 ) ℓ , μm4 . 72 ( 3 . 12 , 6 . 09 ) 1 . 74 ( 1 . 37 , 2 . 44 ) nop-1 RNAi nop-1 RNAi τ , minn . d .", "*< 0 . 5 ( n . d . ) †β τ , minn . d .", "*0 . 771 ( 0 . 694 , 2 . 75 ) ℓ , μmn . d .", "*2 . 67 ( 1 . 7 , 11 . 5 ) ani-1 RNAi ani-1 RNAi τ , min< 0 . 5 ( n . d . ) †0 . 989 ( 0 . 555 , 1 . 79 ) β τ , min1 . 45 ( 0 . 969 , 18 ) ‡0 . 724 ( 0 . 636 , 0 . 929 ) ℓ , μm12 . 3 ( 6 . 39 , 58 . 4 ) ‡3 . 38 ( 2 . 97 , 6 . 16 ) mlc-4 ( 5–7 hr ) RNAi mlc-4 ( 5–7 hr ) RNAi τ , min< 0 . 5 ( n . d . ) †4 . 76 ( 3 . 67 , 6 . 6 ) β τ , min0 . 467 ( 0 . 451 , 0 . 503 ) 0 . 637 ( 0 . 567 , 0 . 768 ) ℓ , μm2 . 75 ( 2 . 38 , 4 . 1 ) 6 . 58 ( 5 . 31 , 6 . 85 ) mlc-4 ( 7–9 hr ) RNAi mlc-4 ( 7–9 hr ) RNAi τ , minn . d .", "*< 0 . 5 ( n . d . ) †β τ , minn . d .", "*0 . 916 ( 0 . 82 , 1 . 04 ) ℓ , μmn . d .", "*3 . 12 ( 2 . 49 , 4 . 18 ) *n . d . denotes parameters which could not be determined , †< 0 . 5 ( n . d . ) denotes parameters which could not be determined , but could be determined to be below 0 . 5 ( see Figure 3—figure supplement 1b ) , ‡denotes values that could be determined but that were not well constrained . 10 . 7554/eLife . 17807 . 021Table 2 . Best fit gel material parameters , no bootstrapping .", "The mean values together with its 95% confidence bounds are given . DOI: http://dx . doi . org/10 . 7554/eLife . 17807 . 021PseudocleavageCytokinesisnon RNAi non RNAi τ , min< 0 . 5 ( n . d . ) †2 . 34 ( 1 . 74 , 3 . 14 ) β τ , min0 . 59 ( 0 . 513 , 0 . 806 ) 0 . 652 ( 0 . 541 , 0 . 764 ) ℓ , μm5 . 38 ( 3 . 27 , 8 . 85 ) 1 . 69 ( 1 . 04 , 2 . 75 ) nop-1 RNAi nop-1 RNAi τ , minn . d .", "*< 0 . 5 ( n . d . ) †β τ , minn . d .", "*0 . 738 ( 0 . 0 . 614 , 0 . 862 ) ℓ , μmn . d .", "*1 . 79 ( 0 . 64 , 5 . 03 ) ani-1 RNAi ani-1 RNAi τ , min< 0 . 5 ( n . d . ) †1 . 01 ( 0 . 989 , 1 . 03 ) β τ , min1 . 31 ( 0 . 428 , 2 . 19 ) 0 . 74 ( 0 . 588 , 0 . 892 ) ℓ , μm10 . 4 ( 4 . 91 , 21 . 9 ) ‡4 . 08 ( 2 . 68 , 6 . 2 ) mlc-4 ( 5–7 hr ) RNAi mlc-4 ( 5–7 hr ) RNAi τ , min< 0 . 5 ( n . d . ) †**4 . 37 ( 3 . 08 , 6 . 21 ) β τ , min0 . 454 ( 0 . 437 , 0 . 47 ) 0 . 614 ( 0 . 501 , 0 . 727 ) ℓ , μm2 . 24 ( 1 . 87 , 2 . 67 ) 5 . 81 ( 5 . 02 , 6 . 72 ) mlc-4 ( 7–9 hr ) RNAi mlc-4 ( 7–9 hr ) RNAi τ , minn . d .", "*< 0 . 5 ( n . d . ) †β τ , minn . d .", "*0 . 97 ( 0 . 931 , 1 . 01 ) ℓ , μmn . d .", "*2 . 98 ( 2 . 88 , 3 . 08 ) *n . d . denotes parameters which could not be determined , †< 0 . 5 ( n . d . ) denotes parameters which could not be determined , but could be determined to be below 0 . 5 ( see Figure 3—figure supplement 1b ) , ‡denotes values that could be determined but that were not well constrained .", "Caption for Videos 1Videos 1 to 7 Until now , we have not considered processes of myosin-based active alignment in our theory ( Vavylonis et al . , 2008 ) .", "This simplification is probably appropriate for pseudocleavage , but this is less clear for cytokinesis given that there is a clear band of myosin enrichment at the equator when the cell divides ( Figure 1c , d ) .", "We next consider active alignment by myosin , and describe the cortical layer as a thin film of an active nematic gel .", "For stationary flows the profile of nematic order Q is now given by ( 3 ) Q=−τv∂xQ−β2τ∂xv+ℓ2 ∂x2Q+λQ , where the right-most term describes active alignment by myosin molecular motors , with λ an alignment parameter dependent on the local myosin concentration .", "In our modified fitting procedure , we now evaluate the respective contributions of myosin-based active and flow-based passive alignment to the stationary Q profile , under the assumption that λ is proportional to local myosin concentration .", "We find that considering active alignment does not increase the overall agreement between calculated and measured alignment profiles for both pseudocleavage and cytokinesis ( Figure 3—figure supplement 2 ) .", "Notably , the contribution of active alignment to pseudocleavage is insignificant , while active alignment contributes to enhancing compression-induced alignment during cytokinesis ( Figure 3—figure supplement 2b; see Appendix ) , albeit to a small degree .", "We conclude that compression by flow and not myosin-based active alignment is the driving force of ring formation in both pseudocleavage and cytokinesis .", "A key prediction from our model is that reducing flow speeds and compression rates should give rise to less filament alignment and a possible inhibition of pseudocleavage furrow formation .", "To test this prediction , we performed weak-perturbation RNAi experiments ( Naganathan et al . , 2014 ) of the regulatory myosin light chain of non-muscle myosin ( mlc-4 ( RNAi ) ( Craig et al . , 1983 ) to mildly reduce actomyosin flow speeds without significant changes in overall actomyosin organization ( Figure 4—figure supplement 1 ) .", "Short feeding times ( 5–7 hr ) lead to a small reduction in gel flow and compression rates ( Figure 4a ) , but actin filaments still aligned in the central region and the pseudocleavage furrow still formed and ingressed .", "Consistent with our hypothesis , longer feeding times ( 7–9 hr ) lead to a complete abolishment of cortical flow and a loss of both actin alignment and pseudocleavage furrow ( Figure 4b ) .", "Actomyosin foci are not required for compression-based alignment since anillin- depleted embryos ( ani-1 ( RNAi ) , 24 hr ) still show significant alignment under compression in the absence of these dense foci ( Figure 4c ) ( Maddox et al . , 2005; Piekny and Maddox , 2010; Tse et al . , 2011 ) .", "Reduced flow speeds and compression rates can also account for the absence of alignment and pseudocleavage in nop-1 ( RNAi ) embryos , in which RhoA-dependent processes are affected and pseudocleavage formation abrogated ( Figure 4d , Figure 4—figure supplement 1 and 2 ) ( Tse et al . , 2012; Rose et al . , 1995; Zhang and Glotzer , 2015 ) .", "Overall , these experiments lead us to conclude that reducing gel flow and compression rates affects alignment in a manner that is consistent with Equation 2 .", "Finally , a complete loss of flow and compression abolishes pseudocleavage entirely ( Figure 4b;d and Figure 4—figure supplement 1 ) .", "Taken together , our results suggest that the pseudocleavage furrow arises as a by-product of compressive flows , mechanically aligning actin filaments into a ring even in the absence of localized RhoA activation . 10 . 7554/eLife . 17807 . 022Figure 4 . Flows and compression are required to generate alignment .", "( a-d )", "Average AP profiles of gel flow ( light green , smoothened ) , compression rates ( dark green ) and nematic order parameter ( blue ) at the time of stationary flow during pseudocleavage onset for mlc-4 5–7 hr and 7–9 hr , nop-1 and ani-1 RNAi .", "Error bars represent the standard error of the mean ( N = 17 , 14 , 10 , 22 for a-d , respectively ) .", "Dashed red line indicate the respective best-fit theory predictions for the nematic order parameter ( blue ) .", "For ani-1 and mlc-4 ( 5–7 hr ) , τ is small and is determined to be smaller than 0 . 5 min .", "For nop-1 and mlc-4 ( 7–9 hr ) , theory profiles were generated using the parameters obtained for the unperturbed non-RNAi pseudocleavage , since insufficient compression rates did not constrain the physical parameters .", "Scale bar , 10 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 17807 . 02210 . 7554/eLife . 17807 . 023Figure 4—figure supplement 1 . Cytoskeletal organization under RNAi perturbation .", "( a-d )", "Actin organization and myosin distribution under nop-1 ( RNAi ) , ani-1 ( RNAi ) and mlc-4 ( RNAi ) .", "Inserts with fluorescence intensity profiles during pseudocleavage and cytokinesis onset are overlaid next to the corresponding images ( actin in blue , myosin in magenta ) .", "Note that ANI-1 depleted embryos had more elongated and thin anterior halves , with no localized dense actin ring like structure and no clear and stable central furrow with membrane overlapping .", "Also note that the asymmetry in the distribution of actin ( usually denser in the anterior half of the embryo ) during cytokinesis was reduced for both nop-1 and long mlc-4 ( RNAi ) .", "Right , combined images and zoom ( green Lifeact , red NMY-2 ) .", "Scale bar , 10 μm ( full images ) ; 5 μm ( zoom ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 17807 . 02310 . 7554/eLife . 17807 . 024Figure 4—figure supplement 2 . Flows , compression and alignment at cytokinesis onset .", "( a-d )", "Results of nop-1 ( RNAi ) , ani-1 ( RNAi ) and mlc-4 ( RNAi ) .", "Average AP profiles of gel flow ( light green , smoothened ) , compression rates ( dark green ) and nematic order parameter ( blue ) at the time of stationary flow during cytokinesis onset .", "Error bars represent the standard error of the mean ( for nop-1 , ani-1 , mlc-4 5–7 hr and 7–9 hr , N = 9 , 18 , 9 , 13 , respectively ) .", "Dashed red line indicates the respective best-fit theory predictions for the nematic order parameter ( blue ) .", "For cytokinesis mlc-4 ( 7–9 hr ) tau is small and is determined to be smaller than 0 . 5 min . ( e-f ) Overlay of the different profiles at pseudocleavage and cytokinesis onset , non-RNAi is shown in blue and compression rates in dashed lines .", "Note that the level of outer ingression was similar to the non-RNAi case for ani-1 ( RNAi ) pseudocleavage , but with improper location and no membrane overlapping , thus no full pseudocleavage furrowing was obtained but a remaining initial anterior ingression was achieved in the presence of some filaments alignment . DOI: http://dx . doi . org/10 . 7554/eLife . 17807 . 024 In this context we wondered if the cortex is specifically modified to favor ring formation in cytokinesis .", "We used our fitting procedure to determine the parameters that characterize emergent material properties of the cortex under mlc-4 ( RNAi ) .", "Notably , we observe that 7–9 hr of mlc-4 ( RNAi ) leads to a reduction of the alignment relaxation time scale τ during cytokinesis .", "The value obtained is similar to the alignment relaxation time scale during pseudocleavage under non-RNAi conditions ( Table 1 and Table 2 ) , leading us to speculate that an increase in myosin activity via Rho signaling is responsible for the observed increase of the characteristic relaxation time τ in cytokinesis .", "Importantly , increasing τ allows the cell to form a pronounced ring of aligned filaments by compressive cortical flow during cytokinesis , as predicted by theory ( Figure 3—figure supplement 1 ) .", "Finally , we sought to find support from theory that aligned filaments in the equatorial region drive anisotropic stress generation ( Mayer et al . , 2010 ) to form an ingression ( White and Borisy , 1983; Salbreux et al . , 2009; Turlier et al . , 2014 ) .", "By use of a theory that connects anisotropic active stress generation in the active nematic gel to changes in cell shape ( see Appendix and Figure 3d ) we show that we cannot appropriately account for the observed ingression profiles unless we consider anisotropic active stress generation in an aligned gel ( Figure 3e , Figure 3—figure supplement 3 ) .", "This suggests that anisotropic active stress generation in a network of aligned actin filaments is important for forming an ingression .", "To conclude , we find that compression by flow drives actomyosin ring formation in both pseudocleavage and cytokinesis , as originally proposed by White and Borisy ( White and Borisy , 1983 ) ( Figure 5 ) .", "For this , we provide a general framework for determining emergent material parameters of the actomyosin gel .", "This characterizes flow-alignment coupling and allows for capturing essential aspects of the mechanics of furrow generation .", "Our analysis of the pseudocleavage ingression in C . elegans demonstrates that constricting rings can form in the absence of equatorial RhoA activation .", "This reveals that cortical flow functions as a central organizer of network architecture , mechanically aligning filaments to form a constricting furrow in the equatorial region without a localized increase of actin nucleation ( Figure 2—figure supplement 5 , f ) and myosin contractility ( Figure 1b-d ) .", "Finally , it will be interesting to investigate if a cortex that is aligned by compressive flow favors the recruitment of specific actin binding proteins such as bundling or motor proteins ( Robinson et al . , 2002 ) , comprising an interesting mechanism of positive feedback for stabilizing and enhancing the ring during cytokinesis .", "Our work highlights that determining emergent material properties of actomyosin is important for understanding cytokinesis , and a challenge for the future is to link emergent material properties at the larger scale with molecular mechanisms ( Mendes Pinto et al . , 2013; Eggert et al . , 2006 ) ." ], [ "Existing reagents did not allow for detailed and long-term imaging of actin filaments in embryos , which is critical for reliably quantifying their orientation .", "Hence , we generated a new Lifeact transgenetic line with enhanced actin filament labeling ( SWG001 ) .", "A codon optimized for C . elegans far-red fluorophore , mKate2 kindly shared by Henrik Bringman ( Redemann et al . , 2011 ) ( 26 kDa , 588/633 nm ) was added to the actin probe with a linker ( 66 bp ) and cloned into MOSCI vector containing unc119 rescue gene ( Hyman Lab ) under the control of the mex-5 promoter .", "Stable integration was obtained by bombardment of this plasmid in DP38 strain .", "This strain was then backcrossed with N2 .", "In this line , we observed within the cortical plane bright actin filament labeling with a high signal-to-noise ratio and little photobleaching .", "Importantly , overall cortical organization and flow dynamics appeared to not be affected by this reagent , foci lifetime , spacing , cortical flow velocities were similar to previous measurement with other fluorescent lines .", "A dual colored transgenic line was obtained in order to image simultaneously actin and myosin dynamics by crossing Lifeact::mKate2 strain with LP133 ( NMY-2::GFP obtained by CRISPR ( Dickinson et al . , 2013 ) , SWG007 ) .", "The RhoA biosensor used for Figure 1 was developed by the Glotzer lab ( GFP::AHPH , C . elegans strain MG617 , Tse et al . , 2012 ) .", "This sensor consists of GFP fused to the C-terminal portion of C . elegans anillin , which contain its conserved region ( AH ) and pleckstrin homology ( PH ) domain .", "It lacks the N-terminal myosin and actin-binding domains but retains its RhoA-binding domain .", "The strains expressing CYK-1:GFP ( SWG004 ) and PLST-1:GFP ( SWG005 ) were obtained by the insertion of a GFP tag at the endogenous locus in their C-terminal region using CRISPR according to Dickinson et al ( Dickinson et al . , 2013 ) .", "C . elegans worms were cultured on OP50-seeeded NGM agar plates as described ( Brenner , 1974 ) .", "L4 mothers were maintained overnight at 20°C before dissection in M9 buffer .", "For imaging , one-cell embryos were mounted on 2% agar pads , thereby slightly compressed to increase the cortical surface visible in a confocal plane .", "Images were acquired with spinning disk confocal microscope ( Zeiss C-Apochromat , 63X/1 . 2 NA or 100X/1 . 42 NA , Yokogawa CSU-X1 scan head and Hamamatsu Orca-Flash4 . 0 camera ) every 2 s on two or three different planes ( one or two at the bottom for a cortical section , and one 12 µm above for a medial section of the embryo ) .", "For 3D acquisitions , Z-stacks were acquired every 0 . 2 μm .", "We assume the shape to be rotationally symmetrical to the longer axis , thus the outline of one medial section of the embryo reflects faithfully cell shape changes and ingression distance .", "RNAi experiments were performed by feeding ( Timmons et al . , 2001 ) .", "L4 worms were grown at 20°C on feeding plate ( NGM agar containing 1 mM isopropyl-β-D-thiogalactoside and 50 μg ml−1 ampicillin ) for the required number of hours before imaging .", "Spatiotemporal correlation functions were calculated for all time points during the stationary period , in the 30 μm central region of the embryo .", "The plot of the spatial correlation only ( 1D correlation function ) is shown in Figure 2c between the compression , alignment and ingression data sets .", "Peak value positions are obtained by a Gaussian fitting procedure and allow the calculation of positional differences between the different data sets .", "Because the flow field appears stationary , we expect this distance between peaks of compression , alignment and ingression to arise due to temporal delays between these events in the reference frame co-moving with the cortical flows .", "We thus translated the characteristic distances between stationary peaks to temporal delays using the average flow velocity in the −10 μm to 0 central region ( 6 . 16 ± 1 . 23 µm/min for pseudocleavage and 4 . 01 ± 1 . 92 µm/min for cytokinesis ) ( Figure 2d ) . 10 . 7554/eLife . 17807 . 025Figure 5 . Compression drives alignment for furrowing . Schematic illustration of flows , cytoskeletal organization and cell shape changes during pseudocleavage and at the onset of cytokinesis . DOI: http://dx . doi . org/10 . 7554/eLife . 17807 . 02510 . 7554/eLife . 17807 . 026Video 5 . Actomyosin gel dynamics in the C . elegans zygote after 24 hr nop-1 ( RNAi ) .", "Cortical and medial planes of an embryo expressing both Lifeact::mKate2 and endogenous NMY-2::GFP .", "Left panel , medial plane Lifeact:mKate2 , center , cortical NMY-2::GFP , right panel , cortical Lifeact:mKate2 ( min:s ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 17807 . 02610 . 7554/eLife . 17807 . 027Video 6 . Actomyosin gel dynamics in the C . elegans zygote after 26 hr ani-1 ( RNAi ) .", "Cortical and medial planes of an embryo expressing both Lifeact::mKate2 and endogenous NMY-2::GFP .", "Left panel , medial plane Lifeact:mKate2 , center , cortical NMY-2::GFP , right panel , cortical Lifeact:mKate2 ( min:s ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 17807 . 02710 . 7554/eLife . 17807 . 028Video 7 . Actomyosin gel dynamics in the C . elegans zygote after 14 hr mlc-4 ( RNAi ) .", "Cortical and medial planes of an embryo expressing both Lifeact::mKate2 and endogenous NMY-2::GFP .", "Left panel , medial plane Lifeact:mKate2 , center , cortical NMY-2::GFP , right panel , cortical Lifeact:mKate2 ( min:s ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 17807 . 028" ] ]

[ "Cytokinesis in eukaryotic cells is often accompanied by actomyosin cortical flow .", "Over 30 years ago , Borisy and White proposed that cortical flow converging upon the cell equator compresses the actomyosin network to mechanically align actin filaments .", "However , actin filaments also align via search-and-capture , and to what extent compression by flow or active alignment drive furrow formation remains unclear .", "Here , we quantify the dynamical organization of actin filaments at the onset of ring assembly in the C . elegans zygote , and provide a framework for determining emergent actomyosin material parameters by the use of active nematic gel theory .", "We characterize flow-alignment coupling , and verify at a quantitative level that compression by flow drives ring formation .", "Finally , we find that active alignment enhances but is not required for ring formation .", "Our work characterizes the physical mechanisms of actomyosin ring formation and highlights the role of flow as a central organizer of actomyosin network architecture ." ]

[ "Just under the surface of every animal cell , a thin and dynamic network of filaments called the cell cortex acts as a scaffold and determines the cell’s shape .", "When the cell divides , this material re-organizes to make a ring of filaments – known as the cytokinetic ring – across the middle of the cell .", "This ring then constricts to split the cell into two separate daughter cells .", "The filaments are guided to form the ring by specific proteins around the middle of the cell .", "A process called cortical flow – the mechanical compression of filaments towards the middle – also influences the shape of the ring .", "However , it is not clear to what degree cortical flow actually helps the ring to form .", "A tiny worm called Caenorhabditis elegans is often used to study how animal cells divide and grow .", "When the C . elegans embryo is made of just a single cell , two rings of filaments form consecutively as this cell prepares to divide .", "Reymann et al . used microscopy to investigate how filaments are arranged in C . elegans embryos as the rings assemble .", "The experiments showed that filaments are arranged into rings in locations where the filaments are being mechanically compressed by cortical flow .", "The first ring forms and partially constricts , and then relaxes once the cell is polarized; that is , once the cell has developed two distinct ends .", "A second ring then forms during cytokinesis and constricts to divide the cell into two .", "To understand the physical changes occurring , Reymann et al . compared the experimental data with a mathematical model of the cortical network .", "This model assumed that the cortical network acts as a thin film in which the orientation of the filaments is coupled to the flow of the fluid .", "Reymann et al . used this model to demonstrate that the observed arrangements of the filaments in both rings can be explained by cortical flow .", "Together , the findings of Reymann et al . highlight the central role that cortical flow plays in organizing rings of filaments in C . elegans .", "Future studies will explore whether cortical flow is linked to other mechanisms that affect the formation of the cytokinetic ring ." ]

[ "Introduction", "Results", "Discussion", "Materials and methods" ]

[ "chromosomes and gene expression", "computational and systems biology" ]

[ [ "Transcription factors ( TFs ) perform the last step in signal transduction pathways .", "They thus serve key roles in central processes such as growth , stress response , and development , and their mutation or misregulation underlies many human diseases ( Spitz and Furlong , 2012 ) .", "A TF includes a sequence-specific DNA-binding domain ( DBD ) and an effector domain that regulates nearby gene transcription .", "Activation domains ( ADs ) – effector domains that increase transcription – have long been of particular interest due to their roles as oncogenic drivers and use as scientific tools ( Bradner et al . , 2017; Brückner et al . , 2009; Dominguez et al . , 2016 ) .", "ADs were discovered as regions that could independently stimulate transcription when ectopically recruited to a gene promoter ( Brent and Ptashne , 1985 ) .", "Early experiments showed that ADs were unlike structured domains because progressive truncations showed graded reductions in activity ( Hope and Struhl , 1986; Hope et al . , 1988 ) .", "Subsequent studies showed that ADs were disordered and had few similarities in their primary sequence ( Mitchell and Tjian , 1989 ) .", "Instead , ADs were classified based on their enrichment of certain residues , whether acidic , glutamine-rich , or proline-rich .", "Acidic ADs are the most common and best characterized .", "Acidic ADs retain activity when transferred between yeast and animals , pointing to a conserved eukaryotic mechanism ( Fischer et al . , 1988; Struhl , 1988 ) .", "While some have found that acidic residues are necessary for activation , others have found that they are dispensable ( Brzovic et al . , 2011; Pacheco et al . , 2018; Staller et al . , 2018; Warfield et al . , 2014 ) .", "Besides their negative charge , acidic ADs are rich in bulky hydrophobic residues .", "Mutating these hydrophobic residues reduces activation , often in proportion to the number mutated .", "Because AD sequences are highly diverse and poorly conserved , only a small fraction of all ADs in eukaryotic TFs have likely been annotated .", "Sequence motifs have been proposed based on analysis of select ADs but have not been used for large-scale prediction ( Piskacek et al . , 2007 ) .", "Screens of random sequences in yeast identified many activating sequences that represented as many as 1–4% of elements tested ( Hackett et al . , 2020; Ravarani et al . , 2018 ) .", "Actual protein sequences are , however , highly non-random .", "Direct screening of protein sequences has identified relatively few ADs at low resolution ( Arnold et al . , 2018; Tycko et al . , 2020 ) .", "There is a need for methods to experimentally detect or computationally predict all ADs .", "ADs stimulate transcription of genes by recruiting coactivator complexes , especially the Mediator complex , which interacts with RNA Polymerase II and regulates its transcription initiation ( Kornberg , 2005 ) .", "Mediator is necessary for TF-dependent activation in vitro and in vivo and is required for regulation by enhancers in all eukaryotes ( Allen and Taatjes , 2015 ) .", "Mediator and TFs are concentrated at strong enhancers , perhaps in a phase-separated state , which may play a role in gene activation ( Boija et al . , 2018; Chong et al . , 2018; Sabari et al . , 2018; Shrinivas et al . , 2019; Whyte et al . , 2013 ) .", "Beyond Mediator , TFs have been suggested to recruit other conserved multiprotein complexes , including TFIID , SAGA , and SWI/SNF , that play roles in activation of various genes ( Hahn and Young , 2011; Mitchell and Tjian , 1989 ) .", "While many such TF interactions have been described , their occurrence and roles remain to be determined .", "Biochemical studies of TF-coactivator interaction have given insight into the mechanism of activation .", "Nuclear magnetic resonance ( NMR ) studies revealed short alpha helices formed by acidic ADs of yeast Gcn4 protein , with hydrophobic faces contacting hydrophobic surfaces of the Med15 subunit of Mediator ( Brzovic et al . , 2011; Tuttle et al . , 2018 ) .", "NMR constraints were consistent with multiple possible AD binding poses , suggesting a dynamic ‘fuzzy complex’ ( Tompa and Fuxreiter , 2008 ) .", "Further consistent with these ideas , solvent exposure of aromatic residues was associated with activation in a screen of Gcn4 AD variants ( Staller et al . , 2018 ) .", "Here , we combine quantitative , high-throughput measurements of in vivo activation and in vitro interaction with computational modeling to characterize ADs .", "We identify 150 ADs in budding yeast and describe their shared sequence attributes .", "We extend the analysis by training and validating a neural network that predicts new ADs across eukaryotes .", "Guided by predictions , we design and measure activation of thousands of AD mutants to derive a deeper understanding of the principles underlying activation .", "Correlating activation with measurements of binding of ADs to Mediator in vitro identifies the key protein interactions driving activation , for which we predict atomic structures using a peptide-docking algorithm .", "We derive structural features common to AD-Mediator interactions and use them to explain measurements of Mediator recruitment kinetics in vitro .", "In total , we develop an integrated model for function , sequence determinants , molecular interactions , and kinetic features of TF ADs ." ], [ "We identified ADs across all TFs in budding yeast with the use of a quantitative , uniform , and high-throughput activation assay .", "Variability due to protein expression , activation duration , and secondary genetic effects was minimized by fusing domains of interest to a three-part artificial TF ( aTF ) that ( 1 ) is tracked by an mCherry tag , ( 2 ) localizes to the nucleus only upon induction with estrogen , and ( 3 ) binds uniquely through its mouse DBD in the promoter of a chromosomally-integrated GFP reporter gene ( Figure 1A; McIsaac et al . , 2013; Staller et al . , 2018 ) .", "In this assay , three known ADs stimulated GFP expression greater than 100-fold upon estrogen treatment ( Figure 1—figure supplement 1A ) .", "We used a pooled screen to measure activation by 7460 protein segments , each 53 amino acids ( aa ) in length , that tiled all 164 TFs ( identified by Gene Ontology terms ) with a step size of 12–13 aa ( 3 . 8-fold average coverage; Figure 1B ) .", "Outgrowth of transformed yeast for 5 days ensured that each cell contained a unique aTF expression plasmid due to mechanisms maintaining it at single-copy levels ( Materials and methods ) ( Scanlon et al . , 2009 ) .", "After induction with estrogen , cells with a defined level of aTF expression were selected by mCherry signal and sorted into eight bins based on GFP expression ( Figure 1—figure supplement 1B ) .", "By next-generation sequencing , the distribution of each protein tile across the bins was determined and the mean value was used to calculate activation—namely , the fold increase in GFP relative to background ( Figure 1—source data 1 , Materials and methods ) .", "Activation was highly concordant between distinct DNA sequences encoding the same protein fragment ( Figure 1—figure supplement 1C ) .", "GFP distributions of three known ADs in the pooled screen exactly reproduced measurements from individual pure populations ( Figure 1—figure supplement 1D ) .", "Controlling by a defined aTF expression level was critical for precisely measuring activation ( Figure 1—figure supplement 1E–F ) .", "Activation measurements of fragments shared between two independently cloned and assayed libraries were reproducible ( Pearson's r = 0 . 953 , Figure 1—figure supplement 1G ) .", "Over 2 , 850 , 000 cells were assayed in total , giving excellent coverage of library elements ( approximately 99% of TF tiles were observed in at least 10 cells each ) .", "Our assay exhibited high signal-to-noise: tiles in previously known and newly identified ADs activated nearly 200-fold while 88% of fragments activated less than twofold ( Figure 1C ) .", "Across the library , 451 tiles showed significant activation ( p<0 . 0001 by Z-test ) .", "When plotted by protein position , activating tiles clustered into discrete , well-defined ADs ( Figure 1D ) .", "Using a positional activation score , we identified 150 ADs in 96 TFs ( Figure 1E and Figure 1—figure supplement 1H , Figure 1—source data 2 , Materials and methods ) .", "These ADs overlapped 75% of all previously-reported ADs in TFs ( Figure 1—source data 2 ) .", "Furthermore , the 53-aa tile length was not limiting , since our screen successfully identified ADs in over 85% of TFs that activated in a previous one-hybrid screen testing full-length proteins ( Figure 1—source data 2; Titz et al . , 2006 ) .", "These results show that our screen is both sensitive and comprehensive .", "Three-quarters ( 112 ) of ADs identified here were previously unknown ( Figure 1—source data 2 ) .", "While 63 TFs contained just a single AD , 33 TFs had multiple , including up to seven distinct ADs in Adr1 ( Figure 1D and F ) .", "C-terminal ADs were common—found in nearly half of all AD-containing TFs—and were stronger and shorter on average than other ADs ( Figure 1—figure supplement 1I ) .", "Consistent with AD function in their native context , TFs that contained ADs upregulated a higher proportion of downstream genes than TFs without ADs ( Figure 1—figure supplement 1J; Hackett et al . , 2020 ) .", "Consistent with the diverse functions of TFs beyond transcriptional activation ( e . g . repression ) , 68 TFs did not contain any ADs ( Figure 1F ) .", "ADs were enriched in negative charge and hydrophobic content , and activation was strongest for fragments that were high in both ( Figure 2A and Figure 2—figure supplement 1A–B; Wimley-White hydrophobicity scale ) ( Wimley and White , 1996 ) .", "Past experiments have clearly shown the importance of bulky hydrophobic residues but presented conflicting evidence for acidic residues in activation ( Brzovic et al . , 2011; Cress and Triezenberg , 1991; Drysdale et al . , 1995; Jackson et al . , 1996; Pacheco et al . , 2018; Staller et al . , 2018; Sullivan et al . , 1998; Warfield et al . , 2014 ) .", "To determine the role of negative charge , we took the strongest-activating fragment from each AD and measured its activation with all acidic residues mutated .", "Except for a single example that activated just as strongly at +7 net charge without its acidic residues ( the Gln3 C-terminal AD ) , activation was abolished in all ADs , showing definitively that acidic residues are necessary in budding yeast ADs ( Figure 2B and Figure 2—figure supplement 1C ) .", "We also noticed that activation of wild-type tiles was more strongly correlated with the number of Asp residues than the number of Glu residues , and hypothesized that Asp promotes activation more strongly than Glu ( Figure 2—figure supplement 1D ) .", "Indeed , ADs with all Glu residues mutated to Asp consistently activated as well as or better than ADs with all Asp residues mutated to Glu ( Figure 2—figure supplement 1E ) .", "Having identified the important residue types , we looked for sequence motifs that could predict activation .", "However , AD sequences appeared to share no common motifs upon inspection .", "The 9aaTAD motif , originally proposed based on yeast ADs ( Piskacek et al . , 2007 ) , predicted ADs poorly: only 10% of 9aaTAD-containing tiles activated in our assay , which was not significantly more predictive than scrambled versions of the motif ( Figure 2C ) .", "A de novo search for motifs using DREME also found none that were shared by more than two ADs ( Bailey , 2011 ) .", "Evidently , ADs are diverse in sequence and not predictable from simple motifs .", "The lack of motifs suggests that activation is determined not by positions of individual residues but by distributed or redundant features .", "We pursued this point by measuring activation of a panel of designed mutants of eight ADs .", "Indeed , 94% of scanning single-amino acid mutants affected activation by less than twofold , and all still activated more than 10-fold ( Figure 2—figure supplement 1F–G ) .", "To produce larger effects , we varied the acidic and hydrophobic content of the ADs by mutating successively larger subsets of acidic or aromatic residues ( Figure 2D , Materials and methods ) .", "Activation was gradually , and finally completely , eliminated as either characteristic was reduced .", "It is noteworthy that the Gcn4 AD , a focus of many past studies , uniquely tolerated mutation of up to six acidic residues .", "Most notably , activation by acidic and hydrophobic mutants were related to the number , rather than position , of mutated residues ( Figure 2D and Figure 2—figure supplement 1H ) .", "Thus , activation strength is determined in large part by acidic and hydrophobic content , not by motifs .", "Since motifs predicted ADs poorly , we turned to more sophisticated approaches using machine learning .", "We first trained a simple neural network to predict activation based solely on tiles’ amino acid ( aa ) composition ( Materials and methods ) .", "When tested on TF tiles withheld from training , this neural network performed remarkably well , explaining 66% of observed variation ( Figure 3—figure supplement 1A ) .", "However , this algorithm was limited by its lack of information about residue positions .", "To experimentally disentangle the contributions of aa positioning and aa composition , we measured activation of eight scrambled sequences from each of the eight ADs tested before .", "Despite their shared abundance of acidic and hydrophobic residues , these mutants spanned a wide range of activation strengths , and some mutants activated stronger than wild-type while others failed to activate entirely ( Figure 3A ) .", "Clearly , full sequence information beyond aa composition is needed to predict activation .", "We therefore trained a deep convolutional neural network ( CNN ) to predict activation based on protein sequence , predicted secondary structure , and predicted disorder ( Materials and methods ) .", "CNNs evaluate sequences by hierarchically integrating matches to a suite of learned patterns and have recently found great success in many genomic prediction tasks ( Ching et al . , 2018; Kelley et al . , 2016; Wang et al . , 2016 ) .", "Our Predictor of Activation Domains using Deep Learning in Eukaryotes , or ‘PADDLE’ , explained 81% of observed variation in TF tiles withheld from training ( alternatively , area under the precision-recall curve of 0 . 805; Figure 3—figure supplement 1B–C , Figure 3—source data 1 ) , markedly better than the aa composition-based predictor .", "De novo , PADDLE accurately predicted the activation strength of ( 1 ) new ADs within TFs omitted from training ( R2 score = 0 . 81 across tiles from 22 TFs; example predictions for Arg81 in Figure 3B ) ; ( 2 ) scrambled AD sequences , despite their identical amino acid composition ( R2 score = 0 . 61 ) ; and ( 3 ) 232 mutants and 178 orthologs of the Pdr1 AD ( R2 score = 0 . 90 ) ( Figure 3—figure supplement 1B ) .", "Altogether , the performance of PADDLE was validated across a wide range of both wild-type and mutant sequences in yeast .", "Classic experiments showed that many acidic ADs retained activity even when transferred between yeast and animals ( Fischer et al . , 1988; Struhl , 1988 ) , so we investigated whether PADDLE could identify ADs in human TFs that would activate in human cells .", "Using PADDLE , we predicted 236 high-strength and 366 moderate-strength ADs , together spanning 462 ( 27% ) human TFs ( Figure 3C , Figure 3—source data 1 ) .", "These predicted ADs overlapped many known ADs of TFs from diverse families , including p53 , NFkB , Myc , Klf4 , Fos , PPARA , SREBF1 , E2F proteins , and the glucocorticoid receptor ( Figure 3—figure supplement 1D ) .", "PADDLE also predicted 41 high-strength and 45 moderate-strength ADs from among 419 transcription-regulating viral proteins ( Figure 3—source data 1; Liu et al . , 2020 ) .", "We randomly selected 25 high-strength predicted ADs from human TFs and measured their activation individually using a luciferase reporter in HEK293T cells .", "Remarkably , 23 domains ( 92% ) activated luciferase expression ( Figure 3D ) .", "While other classes of human ADs , such as glutamine-rich or proline-rich ADs , are not predictable by PADDLE since they are not present in S . cerevisiae , acidic activation mechanisms are evidently conserved and the patterns learned by PADDLE are generalizable across eukaryotes .", "To uncover the principles of acidic activation learned by PADDLE , we analyzed predictions on designed mutant sequences and developed hypotheses , which we then experimentally tested in a second library .", "We noticed from predictions on sequences less than 30 aa that yeast ADs contained short , independently activating regions , which we term core ADs ( cADs ) ( Figure 3—figure supplement 1E ) .", "As an experimental test , we measured in vivo activation of 13-aa-long fragments tiling ten ADs in 1-aa steps .", "Predictions proved highly accurate ( R2 score = 0 . 84 , Figure 3—figure supplement 1F ) , and both predictions and experiments identified at least one significantly activating 13-aa cAD within each AD ( Figure 3E; p<0 . 001 ) .", "Across all yeast TFs , PADDLE predicted that the minimal region for activation could be localized to within a 20-aa cAD in 85% of ADs ( Figure 3F , Figure 3—source data 1 ) .", "These cADs still shared no motifs ( Figure 3—figure supplement 1G ) , but instead had an especially high density of acidic and hydrophobic residues , even more so than the full ADs ( Figure 3G ) .", "To quantify the contribution of each residue to activation , we predicted the impact of mutating each position in each cAD to alanine ( Figure 3—figure supplement 1H ) .", "Single-aa mutants in these short cADs had a large predicted impact on activation , unlike in the longer 53-aa ADs ( Figure 2—figure supplement 1F–G ) , and showed clear trends when grouped by residue identity .", "Mutating the bulkiest hydrophobic residues led to the greatest decreases in predicted activation , and the three most important were Trp , Phe , and Leu ( Figure 3—figure supplement 1I ) .", "Mutating acidic or basic residues had opposite but comparable effects on predicted activation .", "Together , these analyses identify the regions and residues across ADs that contribute most to activation .", "Having identified the core AD regions , we sought to understand the sequence properties that drive their activation .", "We focused on the 28 strongest 13-aa cADs ( Figure 3—source data 1 ) , and first experimentally quantified the importance of their aa composition by comparing each cAD with 33 random scrambles of its sequence .", "Remarkably , only nine cADs ( 32% ) showed activation by the wild-type sequence greater than threefold greater than the average activation by scrambled sequences ( Figure 4A ) .", "In these nine cADs , maximal activation evidently requires a specific positioning of residues .", "On the other hand , 18 cADs ( 64% ) showed activation by the wild-type sequence within twofold of the scrambled sequence average , indicating that their activation is primarily determined by aa composition and not by a unique positioning of residues .", "The wild-type-to-scramble ratio was not correlated with wild-type activation strength ( Figure 4—figure supplement 1A , Pearson’s r = 0 . 32 , p=0 . 09 ) .", "Instead , cADs with the greatest hydrophobic content had the lowest wild-type-to-scramble ratios ( Figure 4B , Pearson’s r = −0 . 51 , p=0 . 006 ) .", "Thus , the majority of cADs studied here activate by a composition-driven mechanism that exploits an excess of bulky hydrophobic residues .", "Alpha-helical folding is thought to be important for activation , which is at odds with the prevalence of composition-driven cADs .", "We directly determined whether the 28 strongest 13-aa cADs require helical folding by measuring the impact of inserting a helix-breaking proline .", "Within the seven central positions of each 13-aa cAD , we individually mutated each non-hydrophobic residue to alanine or proline and asked whether proline inhibited activation relative to alanine ( Figure 4C ) .", "Proline mutations inhibited activation more than threefold over alanine in nine ADs ( 32% ) , including up to an 11-fold drop in activation in the Tda9 cAD .", "However , proline mutations inhibited activation by less than twofold in 16 cADs ( 58% ) .", "These effects were consistent between different positions within the same cAD ( Figure 4—figure supplement 1B ) .", "Interestingly , all three cADs that contained a basic residue ( Pul4 , Tda9 , and Rsf2:586 ) were strongly inhibited by proline , suggesting that a helix is necessary to position their inhibitory positive charge away from the coactivator binding interface .", "Most notably , the magnitude of proline disruption was tightly correlated with the wild-type-to-scramble ratio ( Figure 4D , Pearson’s r = 0 . 842 ) , showing that constraints on structure and sequence , or a lack thereof , were closely linked .", "Thus , composition-driven cADs typically do not need to form a helix and likely sample disordered conformations , while cADs that require their wild-type sequence typically also require helical folding , employing a structure-driven mechanism .", "To understand these two contrasting mechanisms , we studied them in a simplified system of artificial cADs that used only the four most activation-promoting residues .", "We examined 9-aa sequences ( denoted ‘9mers’ ) consisting entirely of only two types of amino acids each—Asp and either Leu , Phe , or Trp—so that all possible such sequences ( 3 × 29 = 1536 ) could be systematically assayed ( Figure 4E ) .", "As expected , activation required a balance of hydrophobic and acidic residues ( Figure 4—figure supplement 1C ) , so we focused analysis on aa compositions with the strongest median activation: 9mers with five or six hydrophobic residues .", "Within these , nearly all Phe 9mers and Trp 9mers activated regardless of their sequence ( Figure 4F ) , characteristic of a composition-driven mechanism and consistent with the highly hydrophobic nature of Phe and Trp residues .", "However , Leu 9mers spanned a wide range of activity with only a minority that were strongly activating , characteristic of structure-driven activation requiring a specific ordering of residues .", "To see what allows only certain Leu 9mers to activate , we examined all possible short motifs containing two or three Leu residues .", "The only motif strongly associated with activation was LxxLL ( Figure 4G and Figure 4—figure supplement 1D; p<10−8 by Kolmogorov-Smirnov test ) .", "This motif was strictly required for activation in these 9mers , present in two copies in the three strongest 9mers , found in two wild-type structure-driven cADs ( Put3 and Crz1 ) , and previously described in ADs of many nuclear receptors and other TFs ( Plevin et al . , 2005 ) .", "This motif also suggests an explanation for why helical folding was necessary for cADs with less hydrophobicity: by placing Leu residues along one face of an alpha helix , the motif efficiently forms a hydrophobic binding surface .", "The analogous FxxFF and WxxWW motifs were also significantly but more weakly associated with activation of Phe 9mers and Trp 9mers ( Figure 4—figure supplement 1E ) .", "However , their strongest predictor of activation was the average position of acidic residues , with N-terminal skew highly favored ( Figure 4H ) .", "Most dramatically , DDDFFFFFF activated 20-fold while its reverse sequence FFFFFFDDD activated just 1 . 3-fold .", "One role of N-terminal negative charge could be to neutralize the macroscopic dipole that arises from alpha helix backbone hydrogen bonds , stabilizing helical conformations ( Rocklin et al . , 2017 ) .", "Even though a helix is not necessary for composition-driven activation , this effect could reduce the entropic cost of binding to coactivators by promoting transient folding .", "Despite the importance of hydrophobic residues for activation , PADDLE also predicted that some TFs contained cADs whose activation was inhibited by nearby clusters of hydrophobic residues .", "We confirmed this experimentally for Rds1 , in which residues 239–263 alone activated 6 . 8-fold but extending the sequence by eight aa , five of them hydrophobic , abolished activation ( Figure 4I ) .", "Inhibition by hydrophobic residues also explains the weak activation of some scrambled 53-aa ADs ( Figure 3A , Figure 4—figure supplement 1F ) .", "To systematically quantify inhibition , we measured the effect on activation when the Pdr1 cAD was placed next to a library of 2177 random 20-aa sequences chosen to span a wide range of net charge and hydrophobic content ( Figure 4J ) .", "As expected , negative charge with moderate hydrophobicity bolstered activation .", "Positive charge without hydrophobicity inhibited activation , but even variable regions with +8 charge ( +4 net charge ) still activated an average of 4 . 4-fold .", "Therefore , the local clustering of negative charges near hydrophobic residues renders the Pdr1 cAD resistant to global changes in net charge , suggesting that interactions between opposite charges within the peptide do not fully prevent binding to coactivators .", "In contrast , hydrophobic residues at high density inhibited activation completely .", "This suggests that inhibitory hydrophobic clusters interact with hydrophobic residues in the cAD , completely preventing their interaction with coactivators .", "Consistent with two independent mechanisms of inhibition , basic and hydrophobic residues together reduced activation additively .", "In total , these experiments mapped the biochemical and structural properties that drive both activation and inhibition .", "The surprising ability of simple biochemical features to explain a large and diverse set of ADs suggests that these features may also drive interactions with coactivator complexes .", "To gain a mechanistic understanding of the AD-coactivator contacts that underlie activation , we mapped the interactions of wild-type and mutant ADs with Mediator .", "We focused on Med15 , the primary subunit of Mediator targeted by many ADs in budding yeast , which contains four tandem activator-binding domains ( ABDs ) ( Herbig et al . , 2010; Jedidi et al . , 2010; Thakur et al . , 2008 ) .", "We isolated the N-terminal portion of Med15 consisting of its four ABDs ( hereafter just called 'Med15' ) , confirmed its in vitro interaction with Gcn4 , and used it for binding experiments ( Figure 5—figure supplement 1A ) .", "To measure binding in high-throughput , we used mRNA display ( Takahashi and Roberts , 2009 ) , expressing our library of TF tiles as a pool of protein fragments covalently tagged with their mRNA sequences , and using this pool in Med15 pull-down experiments ( Figure 5A ) .", "Direct counts of Med15-bound and input protein molecules were obtained by amplifying and sequencing their mRNA tags and using unique molecular identifiers ( UMIs ) to remove PCR duplicates .", "Finally , the fractional pull-down of each protein fragment was computed by its abundance in the Med15-bound sample relative to input , normalized to total library concentrations measured by qPCR ( Materials and methods ) .", "Pull-down measurements were reproducible between replicates ( Pearson’s r > 0 . 90 ) and consistent between identical protein fragments encoded by distinct but synonymous mRNA sequences ( Figure 5—figure supplement 1B–C ) .", "Fragments known to bind Med15 enriched up to 17-fold above random sequence controls and 1000-fold higher than in mock pull-downs with beads alone ( Figure 5—figure supplement 1D , Figure 5—source data 1 ) .", "Binding to Med15 was widespread among ADs and coincident with activation activity .", "In total , 324 tiles bound Med15 significantly more than negative controls ( p<0 . 001 by Z-test , Figure 5—figure supplement 1E , Materials and methods ) .", "When plotted by protein position , Med15-binding tiles clustered into 153 discrete domains , the vast majority of which were not previously known to bind Mediator ( Figure 5B , Figure 5—source data 1 ) .", "ADs and Med15-binding domains overlapped substantially: 73% of ADs bound Med15 , and 71% of Med15-binding domains functioned as ADs ( Figure 5C ) .", "Moreover , ADs that did not bind Med15 tended to activate weakly ( Figure 5—figure supplement 1F ) , so it is possible that many bind Med15 at levels below the detection limit of our pull-down assay , which was less sensitive than our activation assay ( compare Figure 1C and Figure 5—figure supplement 1E ) .", "Just as with activation , high acidic and hydrophobic content were associated with Med15 binding ( Figure 5—figure supplement 1G ) .", "The Gln3 AD that did not require acidic residues also did not bind Med15 substantially ( 2 . 1-fold enrichment ) , suggesting that it activates through other mechanisms .", "Most notably , activation strength and Med15 binding of tiles were directly proportional ( Figure 5D ) .", "To test this relationship further , we measured Med15 binding of the set of AD mutants in which aromatic residues were systematically removed ( Figure 2D ) .", "In every case , Med15 binding and activation were strongly correlated ( Figure 5E ) , showing that the hydrophobic features that drive activation similarly underlie Med15 binding .", "Moreover , the ability for binding of a single protein in vitro to quantitatively explain activation in vivo suggests that Mediator recruitment is a key driver for gene activation and determined largely by AD binding to its Med15 subunit .", "ADs can bind different coactivators , though the extent of such interaction is unknown .", "To explore the possibility , we also examined binding of TF tiles to TFIID , a key factor in the transcription of nearly all genes ( Warfield et al . , 2017 ) .", "Yeast TFIID contains three known AD-binding subunits , Taf4 , Taf5 , and Taf12 , which form a subcomplex with Taf6 and Taf9 ( Layer et al . , 2010; Wright et al . , 2006 ) .", "We isolated this TFIID subcomplex and confirmed its interaction with the VP16 AD ( Figure 5—figure supplement 1H ) .", "Pull-downs of the TF tiling library with the TFIID subcomplex enriched fragments up to 6 . 7-fold above random sequence controls ( Figure 5—figure supplement 1I–J , Figure 5—source data 1 ) .", "In total , 27 ADs ( 18% ) across 26 TFs bound the TFIID subcomplex ( p<0 . 001 ) , greatly expanding its repertoire of known interactions ( Figure 5B–C ) .", "However , all ADs that bound TFIID also bound Med15 , and despite the identical pull-down conditions , ADs bound more weakly to TFIID than to Med15 ( Figure 5—figure supplement 1K ) .", "While more TFIID interactions may be discoverable if assay sensitivity can be further optimized , these results suggest that TFIID has lower affinity for most ADs and provide no evidence for its specific targeting as a coactivator .", "Since TFIID binding by ADs is redundant and less frequent , its role in most TF-directed activation is secondary to that of Mediator .", "What structural features of Med15 enable its promiscuous yet functional interaction with diverse AD sequences ?", "In the best studied example , the Gcn4 AD interacts with hydrophobic patches and basic residues on multiple Med15 activator-binding domains ( ABDs ) in a large number of binding poses to form a ‘fuzzy’ complex ( Brzovic et al . , 2011; Tuttle et al . , 2018 ) .", "We addressed the question by using FlexPepDock , a peptide docking algorithm from the Rosetta suite ( Leaver-Fay et al . , 2011; Raveh et al . , 2011 ) , to systematically build structural models of ABD-AD interactions , made computationally tractable by our identification of short cADs .", "Our modeling focused on two ABDs with known structures: the KIX domain , which has homology to human Med15 and the p300/CBP coactivator family , and ABD1 ( Figure 6A; Brzovic et al . , 2011; Thakur et al . , 2008 ) .", "To sample diverse sequences , aa composition , and secondary structure , we modeled interactions of the 28 13-aa cADs described above ( Figure 4A–D ) with the KIX domain and ABD1 .", "For each interaction , 50 , 000 candidate structural models were sampled and ranked by the Rosetta energy score , and the 10 best-scoring models from each interaction were used in subsequent analyses ( Figure 6—source data 1 and Figure 6—source data 2 ) .", "We validated our structural modeling by comparison to experimental data in two ways .", "First , KIX domain residues previously shown by NMR to be important for interacting with the Pdr1 AD were recapitulated by the best-scoring model of this interaction ( Figure 6—figure supplement 1A; Thakur et al . , 2008 ) .", "Second , the degree of alpha helix formation across the 28 cADs was consistent with the effect of proline insertion on in vivo activation ( Figure 6—figure supplement 1B ) .", "As expected , cADs that were inhibited by proline predominantly bound both domains as an alpha helix , and conversely cADs that bound both domains in disordered conformations were minimally affected by proline insertion .", "Some cADs that were minimally affected by proline insertion were modeled as alpha helical , possibly because disordered regions are underrepresented in protein structure databases on which Rosetta was trained .", "A unifying feature of the interactions was the prominent role of hydrophobic contacts at the protein interface .", "Hydrophobicity-driven interactions between proteins often employ high shape complementarity to maximize interface area and minimize solvent-exposed area .", "However , the flat surface of the KIX domain was ineffective in burying hydrophobic cAD residues , which on average had as much surface area exposed to solvent as area contacting the KIX domain ( Figure 6B , top ) .", "For example , the best-scoring Pdr1 AD model used Ile and Leu residues along one helical face to contact the KIX domain , but this left Trp and Tyr residues on the opposite face exposed to solvent ( Figure 6—figure supplement 1C ) .", "The poor hydrophobic shape complementarity of the KIX domain was frequently mitigated by multiple salt bridges and cation-pi interactions ( Figure 6—figure supplement 1D ) .", "ABD1 , which formed fewer ionic interactions , engaged cADs through a larger surface formed by its contoured hydrophobic ridge ( Figure 6B , bottom ) .", "Nevertheless , hydrophobic residues of most cADs were incompletely buried , with those residues showing greater than 20% of area exposed to solvent in over half of all structures .", "Thus , despite the central role of hydrophobic AD residues in activation and Med15 binding , hydrophobic shape complementarity is not an important feature of ABD-cAD interactions .", "Consistent with weak constraints on the shape of the binding interface , all cADs bound in diverse poses .", "For example , the 10 best-scoring models of the War1 cAD bound the KIX domain with its helix axis at different orientations and with different helical faces presented toward the interaction surface ( Figure 6C ) .", "To quantify this diversity , we defined the unique binding poses of each ABD-cAD interaction by clustering similar structures based on cAD backbone root mean square distance ( 2 Å distance cutoff ) , and then counted the number of poses adopted by the 10 best-scoring models .", "The 10 best-scoring models of nearly every cAD , when interacting with either domain , adopted six or more distinct poses ( Figure 6D ) .", "To ensure that this did not result from insufficient sampling , we generated tenfold more candidate models ( 500 , 000 total ) for six cADs binding the KIX domain .", "For all six cADs , the 10 best-scoring models still adopted seven or more distinct poses , despite spanning a narrower range of Rosetta scores ( Figure 6—figure supplement 1E–F ) .", "Disordered cADs , lacking helical content , adopted especially many distinct poses; also , poses were frequently sampled only once , suggesting that disordered cADs inhabit an extremely large conformational space that may reduce the entropic cost of binding ( Figure 6—figure supplement 1G–H ) .", "Altogether , these structures point to a shape-agnostic nature of Med15 ABD interaction surfaces and support the idea of a multi-pose , fuzzy mode of interaction .", "To explore the consequence of fuzzy binding for amino acid sequence constraints , we took Leu , Phe , and Asp residues from models of all cADs and plotted their binding positions on the KIX and ABD1 interfaces ( Figure 6E ) .", "As expected , the hydrophobic Leu and Phe residues occupied regions distinct from the negatively charged Asp residues .", "However , the Leu and Phe distributions were similar to each other: there was no binding pocket that selectively preferred one residue over the other , despite large differences in their size and shape .", "The diversity of AD sequences may thus be understood in terms of the fuzzy nature of the AD-ABD interaction , which only loosely constrains the characteristics of hydrophobic AD residues .", "Pull-down experiments with individual ABDs showed no appreciable binding to any of nine ADs ( Figure 7—figure supplement 1A ) , consistent with the previous suggestion that multiple ABDs are required for strong binding to Med15 ( Brzovic et al . , 2011; Tuttle et al . , 2018 ) .", "Multiple interaction sites were also common within ADs: PADDLE identified 42 ADs ( 28% ) that contained two or more non-overlapping cADs ( Figure 7—figure supplement 1B , Figure 3—source data 1 , Materials and methods ) .", "We took 47 pairs of adjacent cADs and measured activation by the two cADs individually or in tandem .", "For 40 of the pairs , the tandem cADs activated more strongly than expected from the combined activation of the individual cADs ( 4 . 0-fold median increase , Figure 7A ) .", "Thus , an increased valence of interaction sites in both Med15 and ADs drives stronger binding and activation .", "At larger scales , the number of interaction sites is further multiplied because some TFs contain multiple ADs , many TFs bind DNA as dimers , and many genes have several TF binding sites ( Hahn and Young , 2011; Spitz and Furlong , 2012 ) .", "To understand the advantages conferred by the high valence of TF-Mediator interactions , we studied the kinetics in vitro of the initial step of activation: TF-driven recruitment of Mediator to DNA .", "We performed these experiments with Gcn4 , which forms homodimers and has an extended AD consisting of three cADs .", "We isolated budding yeast Mediator complex , coupled DNA containing a Gcn4 motif to a NeutrAvidin-coated surface , added Gcn4 , and measured real-time binding of Mediator by surface plasmon resonance ( Figure 7B , Figure 7—figure supplement 1C ) .", "Mediator bound to the Gcn4-DNA complex with an apparent dissociation constant of KD = 14 nM ± 2 nM and an interaction half-life of 2 . 7 ± 0 . 2 min .", "We repeated the experiment with DNA containing 2 , 4 , or 6 copies of the Gcn4 motif .", "Mediator bound the resulting Gcn4-DNA complexes with identical on-rates .", "Mediator was also recruited more efficiently , with more Mediator bound per mole of Gcn4 than on templates with only one motif ( Figure 7—figure supplement 1D ) .", "The interaction half-life , however , increased with additional copies of the Gcn4 motif—up to 16 min when six motifs were present ( Figure 7B–C ) .", "If all copies bound Mediator independently , then the half-life should be the same , regardless of the number of copies .", "The presence of additional , neighboring ADs retards dissociation by enabling recapture of Mediator released from one AD through binding to another .", "A slower dissociation rate corresponds to an increase in apparent affinity constant .", "Conversely , we found that the multiplicity of ABDs in Mediator facilitated binding of Gcn4 ADs .", "When the surface plasmon resonance experiment was repeated with large excess Gcn4 in solution , there was almost no change in the measured on-rate .", "Even 160-fold excess of Gcn4 had only minimal effect on the rate of Mediator binding and final level achieved ( Figure 7—figure supplement 1E ) .", "To maintain stable TF-DNA complexes without TFs in solution , we repeated these experiments using a fusion of the Gcn4 AD to nuclease deficient EcoRI ( E111Q ) , which resides on DNA for many hours and also forms homodimers ( Wright et al . , 1989 ) .", "Still , Mediator with no Gcn4 competitor or with 250-fold excess of Gcn4 competitor bound similarly ( Figure 7D ) .", "Excess competitor Gcn4-DNA complexes also failed to slow Mediator binding to the surface .", "Evidently , binding of Gcn4 to Mediator does not appreciably block binding of other Gcn4 molecules .", "This behavior may be explained by the occurrence of multiple Gcn4-binding sites on Mediator and weak interaction of a cAD with any one site .", "Rapid association-dissociation equilibrium of a cAD allows a second Gcn4 molecule to interact with Gcn4-bound Mediator , increasing the frequency of exchange of one Gcn4 molecule by another ( Figure 7E ) .", "Such facilitated exchange provides a rationale for both the fuzzy nature of the AD-ABD interaction and the multiplicity of ABDs .", "PADDLE predictions and previous experiments ( Liu and Myers , 2015 ) identified two strong ADs in the Med2 subunit of Mediator , which is adjacent to Med15 in the Mediator complex , suggesting a broader role for ADs beyond TF proteins ( Figure 7—figure supplement 1F ) .", "We used PADDLE to search for ADs across all nuclear-localized non-TF proteins in yeast and selected 1485 fragments that showed potential for activation ( Materials and methods ) .", "Upon pooled in vivo testing , we found 290 fragments across 229 proteins that activated significantly ( p<0 . 001 by Z-test , Figure 1—source data 1 ) .", "To see how often PADDLE fails to identify acidic ADs , we also included 291 control fragments that had extremely high acidic and hydrophobic content but were predicted not to activate .", "Zero control fragments activated ( Figure 7—figure supplement 1G ) , showing that PADDLE likely identified all acidic ADs that exist and again demonstrating that AD function cannot be predicted from amino acid composition alone .", "ADs were remarkably widespread and were found in proteins associated with diverse functions including transcriptional regulation , RNA splicing , DNA repair , ribosome biogenesis , and protein degradation .", "We focused on the ADs in transcription-associated proteins .", "Fourteen ADs were found in proteins that bind DNA and regulate pathway-specific genes; these proteins were not included in the original tiling library because they bind DNA only through a TF partner .", "The two predicted Med2 ADs were also confirmed to activate , 36-fold and 25-fold in our assay .", "Notably , ADs were identified in all major coactivator and chromatin modifying complexes , including Mediator , TFIID , cohesin , condensin , SAGA , RSC , SWI/SNF , ISWI , NuA4 , and FACT ( Figure 7F ) .", "These complexes contained 2 . 9-fold more activating fragments than expected by chance ( p<10−12 compared to randomly shuffled controls ) , suggesting a functional role .", "Domains that activate on their own could be inactive in their natural context if buried within a protein or protein complex .", "To investigate this possibility , we cross-referenced coactivator ADs with all available structures in the Protein Data Bank ( PDB ) and with the Database of Disordered Protein Predictions ( D2P2 ) ( Berman et al . , 2000; Oates et al . , 2013 ) .", "In fact , 23 ADs were predicted in D2P2 to be highly disordered ( more than 30% ) or were predominantly disordered or unresolved in PDB structures ( Figure 7F ) , and so may be exposed and active in vivo .", "For example , cohesin , which binds Mediator and forms enhancer-promoter contacts through loop extrusion ( Davidson et al . , 2019; Kagey et al . , 2010; Kim et al . , 2019; Sanborn et al . , 2015 ) , contained strong and likely-disordered ADs in its subunits Scc1 ( RAD21 homolog ) , Scc3 ( STAG1/2 homolog ) , and Smc3 , as well as multiple ADs on the meiotic-specific subunit Rec8 .", "These cohesin ADs , as well as ADs within other coactivators , may play a role in gene regulation by driving interactions with Mediator ." ], [ "We obtained high-throughput measurements of activation strength that were both quantitative and precise .", "This enabled us to detect—and predict with PADDLE—how activation strength depends on amino acid sequence , amino acid composition , and valence .", "Quantitation was achieved by inducing artificial TF ( aTF ) binding for a brief , defined period so that GFP levels were representative of transcriptional activation ( McIsaac et al . , 2013 ) , and by partitioning the range of GFP signal into eight levels for sorting .", "Others have measured activation only as an on-off binary system or screened for non-quantitative measures of activation , such as cell survival or pull-down of a cell surface marker ( Arnold et al . , 2018; Ravarani et al . , 2018; Tycko et al . , 2020 ) .", "We achieved high precision by restricting measurements to cells with a defined level of aTF expression .", "Controlling for this frequently overlooked confounding variable was critical because activation was strongly dependent on aTF abundance , especially at low levels of expression ( Figure 1—figure supplement 1E ) .", "Another approach is to account for TF expression by sorting based on the GFP reporter signal divided by aTF expression ( Staller et al . , 2018; Staller et al . , 2021 ) ; this introduces substantial variability , because activation has a non-linear relationship with aTF expression ( Figure 1—figure supplement 1E ) , and because this ratio can be systematically biased by features that affect aTF expression ( Figure 1—figure supplement 1F ) .", "This may be why it has been concluded that acidic residues are not necessary for Gcn4 activation , whereas using a similar reporter system we found definitive evidence to the contrary .", "By this approach , we found that all ADs in S . cerevisiae employ a shared acidic and hydrophobic basis and trained a neural network termed PADDLE to predict sequences that activate on this basis .", "PADDLE predicted ADs in human TFs with 92% accuracy , indicative of broad conservation of the acidic activation mechanism , and identified hundreds of new ADs in human TFs and virus proteins .", "PADDLE could further be used to interpret the functional impact of cancer-associated and naturally occurring mutations in human ADs .", "Predicted ADs from multiple species and instructions for running PADDLE are available online at paddle . stanford . edu .", "During our development of PADDLE , a predictor of yeast activation named 'ADpred'’ was published ( Erijman et al . , 2020b ) .", "ADpred , a shallow neural network trained on random protein sequences , achieved a prediction accuracy of 93% on random sequences .", "However , its accuracy on wild-type sequences was much lower: only 33% of ADs predicted by ADpred activated in our experiments , and 29% of the ADs we identified were not predicted by ADpred , only 3 . 1-fold and 2 . 3-fold better than random predictions , respectively ( alternatively , tile-wise AUPRC of 0 . 294; Figure 3—figure supplement 1J–K , Materials and methods ) .", "This discrepancy may be because the random sequences on which ADpred was trained do not sufficiently represent the vastly larger space of actual protein sequences; for example , random sequences rarely form significant secondary structure .", "This failure to generalize highlights the importance of matching neural network training data to the prediction task and underscores the advantage of experiments on wild-type and related mutant sequences .", "PADDLE predictions were also crucial for generating , refining , and testing hypotheses for activation mechanisms .", "For example , we unexpectedly discovered that hydrophobic residues could inhibit activation by noticing that sub-domains of certain non-activating regions were predicted to activate on their own .", "More generally , predictions on AD subsequences identified the core domains responsible for activation at single-amino acid resolution , focusing subsequent experiments on these domains .", "By combining a high-throughput assay to measure and a machine learning algorithm to predict activity of protein sequences , we have demonstrated a design-build-test-learn cycle that could be applied to accelerate discovery of protein function in other areas of research .", "This approach yielded the most detailed view to date of the principles governing acidic AD sequence , which we summarize as follows .", "Activation arises from an abundance of acidic and bulky hydrophobic residues , especially Asp , Trp , Phe , and Leu .", "The most potent activators cluster these residues densely , forming short core ADs .", "In most ADs , and especially with high hydrophobic content or abundant Phe and Trp residues , this clustering is sufficient to activate in a composition-driven manner , regardless of sequence or secondary structure .", "ADs with lower hydrophobic content , particularly those with many Leu residues , instead must position their hydrophobic residues along one face of an alpha helix to activate .", "Overall , the loose constraints on sequence explain the remarkable diversity and lack of evolutionary conservation of ADs .", "The unusual plasticity in AD sequence has several advantages .", "It facilitates spontaneous evolution of new ADs , since an appreciable proportion of even random sequences can activate .", "This flexibility would allow organisms to develop new transcriptional circuits responsive to their unique needs .", "Furthermore , in contrast to classical interactions in which structure and function are dependent on a few key residues , activation strength can be adjusted gradually by mutation .", "Indeed , the observation that nearly all core ADs could increase their strength with simple mutations demonstrates that proper regulation requires precisely adjusted rather than maximized activation .", "Similarly , AD sequences can preserve their activity while evolving other features , such as in the Gal4 C-terminal AD , whose sequence is specifically bound and inhibited by Gal80 in the absence of galactose ( Johnston et al . , 1987; Ma and Ptashne , 1987 ) .", "With the use of mRNA display , we found that 73% of ADs bound the Med15 subunit of Mediator and that binding strength was strongly predictive of activation for thousands of wild-type and mutant protein sequences .", "In contrast , a five-protein TFIID subcomplex bound only 18% of ADs , all of which also bound Mediator .", "We conclude that Mediator recruitment by TFs is a key driver for gene activation , and Mediator recruitment is largely determined by affinity of ADs for the Med15 subunit .", "These findings are in keeping with reports that Mediator recruitment occurs independently of other coactivators and can stimulate the recruitment of other coactivators ( Ansari and Morse , 2013; Ansari et al . , 2014; Govind et al . , 2005 ) .", "How is Med15 , through its four activator-binding domains ( ABDs ) , able to interact with such a large diversity of AD sequences ?", "Despite the importance of hydrophobic interactions , our structural modeling showed that neither the Med15 KIX domain nor ABD1 provided enough shape complementarity to bury the hydrophobic residues of cADs .", "Consistent with this , most cADs did not need to form an alpha helix to activate .", "The lack of shape constraint has two important consequences .", "First , all modeled cADs bound each ABD in many distinct , equally favored poses .", "We therefore suggest that fuzzy binding to Med15 , previously demonstrated for the Gcn4 AD , is employed by all ADs .", "Second , neither ABD showed favored binding positions of Leu versus Phe residues of the cAD , suggesting that the shapes of those residues were unimportant .", "Thus , the loose constraints on binding shape explain the loose constraints on AD sequence .", "Instead , each ABD can be approximated as a hydrophobic surface , which engages hydrophobic AD residues through simple hydrophobic forces , flanked by basic residues , which form salt bridges and cation-pi interactions with acidic and aromatic AD residues .", "Clusters of hydrophobic residues adjacent to ADs inhibit activation , presumably because they compete for interaction with hydrophobic AD residues .", "Many experiments have shown that transcriptional initiation involves a cycle in which ( 1 ) TFs recruit Mediator to enhancers or upstream activating sites , ( 2 ) Mediator contacts the promoter and orchestrates a series of events starting with chromatin rearrangement , followed by pre-initiation complex formation and finally RNA polymerase II recruitment , and ( 3 ) Mediator facilitates phosphorylation of the polymerase C-terminal domain , which releases polymerase into transcriptional elongation and triggers dissociation of Mediator ( Jeronimo and Robert , 2014; Knoll et al . , 2018; Robinson et al . , 2016; Whyte et al . , 2013; Wong et al . , 2014 ) .", "Mediator release is apparently necessary because stably recruiting Mediator by fusing individual subunits to a DNA-binding domain failed to activate in nearly all subunits tested ( Wang et al . , 2010 ) .", "The cycling of Mediator complexes increases the responsiveness of transcriptional regulation in at least two ways .", "First , it requires certain steps to be repeated in order to continue initiating transcription , preventing the system from locking into an activated state .", "Second , it frees Mediator complexes to participate in regulation of other genes , which all depend upon and compete for a relatively limited supply of Mediator ( Flanagan et al . , 1991; Gill and Ptashne , 1988 ) .", "Thus , TFs must recruit Mediator in a manner that is specific and high-affinity but still dynamic .", "Our kinetic experiments showed that Mediator binds Gcn4-DNA complexes with high affinity but that excess Gcn4 does not impede this interaction , showing that interacting Mediator and Gcn4 molecules can exchange rapidly .", "If a bimolecular interaction occurs through a single site , a competing molecule cannot bind until the first molecule dissociates which , in a case of high affinity interaction , is a slow process .", "Our observations are indicative of facilitated exchange , arising from the multiple sites and fuzzy nature of the Gcn4-Mediator interaction ( Figure 7E ) .", "Fuzzy interactions allow for rapid association and dissociation , because a high proportion of random encounters lead to weak but productive binding ( Ferreira et al . , 2005; Sugase et al . , 2007; Tompa and Fuxreiter , 2008 ) .", "One among multiple binding sites will frequently be available for a second molecule , which can invade and facilitate release of the first .", "Similarly , accelerated dissociation or exchange of the E . coli sequence-flexible DNA-binding protein Fis in the presence of competitor proteins was proposed to occur by transitioning through a destabilizing Fis-DNA-Fis ternary complex ( Graham et al . , 2011; Kamar et al . , 2017 ) .", "These advantageous kinetics provide a rationale for the prevalence of multivalent fuzzy interactions among transcription proteins .", "ADs have primarily been characterized in TFs where they serve to recruit coactivators such as Mediator , TFIID , SAGA , SWI/SNF , and NuA4 ( Hahn and Young , 2011; Mitchell and Tjian , 1989 ) .", "Our finding that functional ADs are also present in all coactivator complexes suggests that ADs have broader roles .", "First , coactivator ADs could interact with ABDs within the same complex , limiting their weak or non-specific binding to TFs .", "These auto-inhibited coactivators could still bind to ADs clustered on DNA through facilitated exchange .", "For example , we found that two cADs activated more efficiently and two DNA-bound Gcn4 dimers recruited Mediator more efficiently in tandem than individually .", "Second , coactivator ADs could amplify activation by mediating interactions between coactivators ( Ansari and Morse , 2013; Liu and Myers , 2015 ) .", "For example , upon Mediator binding to TFs , the two ADs on Med2 would be liberated from auto-inhibition and could recruit other coactivators .", "Alternatively , Mediator and other coactivators could cluster in the nucleoplasm through AD-ABD cross interactions and bind together to promoters .", "Either mode of interaction would explain why recruitment of the Med2/Med3/Med15 subcomplex suffices to recruit SWI/SNF to the CHA1 promoter and suffices for activation of the ARG1 gene , while deletion of both Med2 ADs impairs transcription of galactose-inducible genes ( Ansari et al . , 2014; Liu and Myers , 2015; Zhang et al . , 2004 ) .", "Phase separation of Mediator and TFs has recently been demonstrated in vitro and is proposed to underlie enhancer-promoter clustering and transcriptional activation in vivo ( Boija et al . , 2018; Chong et al . , 2018; Sabari et al . , 2018; Shrinivas et al . , 2019 ) .", "Phase separation may occur when a protein makes dynamic self-interactions through two or more binding domains , ( Banani et al . , 2016 ) .", "Interactions between the four Med15 ABDs and the two Med2 ADs make possible phase separation of the Mediator tail subcomplex; other coactivators , with both ADs and ABDs , might phase separate as well .", "The functional role of such phase separation , if it occurs , is unclear .", "A parsimonious interpretation suggests that phase separation of coactivators and TFs is simply a macroscopic consequence of the high valence and dynamism of coactivator-TF interactions , features which may serve an altogether different purpose .", "ADs have long been enigmatic , due to the apparent mismatch of their structure and function: AD sequences are abundant among random polypeptides , and yet ADs bind their targets with high specificity; AD peptides are apparently disordered , and yet they bind with high affinity .", "We now understand that these structural features of ADs are ideally suited to their functions—they render AD-target interaction dynamic , through rapid yet weak binding to single sites and strong binding yet rapid displacement from multiple sites .", "Dynamic AD-target interaction may serve various purposes , such as a rapid response to changing conditions , and the recruitment of multiple AD-bearing proteins to a single target .", "For example , Mediator bound to RNA polymerase II at a promoter might interact transiently with TFIID , SAGA , SWI/SNF complex and other proteins during transcription .", "This mechanism may be employed with other unstructured sequences and high valence targets in other cellular processes ." ], [ "Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact , Roger Kornberg ( kornberg@stanford . edu ) .", "Raw sequencing data for in vivo activation and in vitro binding assays have been deposited in the Gene Expression Omnibus ( GEO ) database , accession number GSE173156 .", "The S . cerevisiae strain BY4711 ( ATCC 200873 ) with genotype MATalpha trp1delta63 was used for activation experiments .", "Standard cell growth conditions were 30°C incubation in YPD medium ( 1% w/v yeast extract , 2% w/v peptone , 2% w/v dextrose ) or synthetic complete ( SC ) medium ( Dunham , 2015 ) .", "Growth in SC medium lacking tryptophan ( SC-Trp ) was used to select for transformants with the pRS414 plasmid backbone .", "Selection for stable transformants harboring the natMX6 marker gene was performed by supplementing media with 100 µg/mL nourseothricin ( NAT ) .", "Selection for stable transformants harboring the KanMX marker gene was performed by supplementing media with 200 µg/mL geneticin ( G418 ) .", "In activation assays , overnight cultures were diluted to OD600 = 0 . 4 in SC-Trp-NAT-G418 with 10 nM beta-estradiol and grown for 4 hr at 30°C before fluorescence-activated cell sorting ( FACS ) .", "HEK293T cells ( ATCC CRL-3216 ) were cultured in DMEM with high glucose and GlutaMAX ( Thermo 10566016 ) supplemented with 10% fetal bovine serum ( Millipore TMS-013-B ) at 37°C in 5% CO2 .", "Yeast and human cells were used directly from ATCC , which authenticates all cell lines using STR profiling and tests for mycoplasma contamination .", "The artificial TF ( aTF ) expression cassette , which contains a KanMX gene and an ACT1 promoter driving expression of an aTF ( comprising an mCherry tag , mouse Zif269 DBD , and estrogen binding domain ) with a multiple cloning site for insertion of protein fragments of interest , was derived from pMVS142_pACT1_mCherry_Zif268_EBD_MCS_KAN , which was a gift from Barak Cohen ( Addgene plasmid # 99049 ) ( Staller et al . , 2018 ) .", "This cassette was cloned into the pRS414 backbone , which contains a TRP1 marker ( Sikorski and Hieter , 1989 ) .", "pRS414 plasmid was digested with EcoRI-HF and SacI , the aTF cassette was PCR-amplified using Phusion polymerase ( NEB ) , and DNA fragments were assembled through Gibson assembly and transformed into XL10-Gold Ultracompetent Cells ( Agilent ) .", "The final construct , called pRS414-TFchassis , was confirmed by Sanger sequencing .", "Oligo pools encoding all the protein fragments to be tested in the screen were synthesized at 12K scale ( Twist Bioscience ) .", "Each oligo was 200 nt in length and contained a 20-nt 5’ constant region and a 21-nt 3’ constant region which were used for PCR amplification .", "Different sub-libraries had different 3’ constant regions , which allowed specific amplification of the sub-library from the oligo pool .", "To enable cloning of the oligo pool into pRS414-TFchassis , 20 bp Gibson homology arms matching pRS414-TFchassis were added using PCR .", "To amplify each sub-library , 2 . 5 ng of oligo template was PCR-amplified for nine cycles using Phusion polymerase , followed by column cleanup .", "pRS414-TFchassis was digested with NheI-HF and AscI , the amplified oligo pool was cloned in by Gibson assembly , and the product was transformed in two replicates , each into 100 µL of Agilent XL10-Gold cells with 5 µL of Gibson mix .", "Transformed cells were plated on lysogeny broth ( LB ) plates with Carbenicillin ( Carb ) to estimate efficiency and also grown in 50 mL LB Carb overnight at 37°C for next-day plasmid extraction using Qiagen Midiprep kit .", "To generate positive control plasmids for the activation screen , IDT gBlocks containing genes expressing 53-aa regions from VP16 , Gcn4 , and Pho4 ADs were also cloned in the same manner as above .", "The uncloned pRS414-TFchassis , which lacks an introduced insert , was used as a negative control .", "The GFP reporter construct pMVS102_P3-GFP-NATMX6 was a gift from Barak Cohen ( Addgene plasmid # 99048 ) ( Staller et al . , 2018 ) .", "To construct a yeast GFP reporter cell line , a cassette from pMVS102_P3-GFP-NATMX6 comprising a natMX6 marker and a P3 promoter ( a modified GAL1 promoter with six copies of the Zif268 binding site ) that drives expression of a fast maturing EGFP variant ( McIsaac et al . , 2013 ) , was PCR-amplified using Phusion polymerase .", "PCR primers were designed to add 40 bp overhangs with homology to the dubious ORF YBR032W .", "The PCR product was transformed into yeast using a high-efficiency method ( Gietz et al . , 1992 ) .", "After overnight outgrowth at 30°C , transformed cells were plated onto YPD-NAT , colonies were picked , and integration into YBR032W was confirmed by PCR amplification of the integrant and Sanger sequencing .", "The in vivo activation screen requires a yeast library in which each cell contains one pRS414-TFchassis plasmid with a unique insert .", "The plasmid is maintained at single-copy or low levels due to its CEN/ARS origin of replication , so if a cell receives multiple different vectors upon transformation then repeated cell divisions will eventually separate the distinct vectors ( Scanlon et al . , 2009 ) .", "To ensure generation of a cellular library in which a unique pRS414-TFchassis plasmid was present in each cell , an assay was developed to quantify the fraction of cells initially receiving multiple plasmids and the time required for cells to reach single-copy plasmid levels by repeated cell divisions .", "First , using a high efficiency transformation protocol ( Gietz et al . , 1992 ) , approximately 50 million yeast cells in a volume of 360 µL were transformed with 2 µg total plasmid DNA , consisting of an equal mixture of aTF plasmids expressing one AD from VP16 , Gcn4 , or Pho4 .", "Transformed cells were sparsely plated on SC-Trp-NAT plates either immediately or after a 24 hr or 48 hr outgrowth in SC-Trp-NAT liquid media .", "For each plating condition , colonies were picked for DNA extraction , followed by qPCR to measure the abundance of VP16 , Gcn4 , and Pho4 plasmids .", "After 0 hr , 24 hr , and 48 hr of growth , 2 ( of 15 ) , 2 ( of 15 ) , and 1 ( of 24 ) colonies had multiple vectors present , respectively .", "Because this method cannot detect multiple transformation events of the same plasmid sequence in the same cell ( e . g . double VP16 transformation ) , a correction was applied , yielding multiple vector rates of 20% , 20% , and 6% for each outgrowth condition , respectively .", "The cloned pRS414-TFchassis plasmid library was transformed into the GFP reporter yeast strain using the same method as in the multiple transformant outgrowth assay above but scaled up 2 . 5-fold .", "To generate clonal control strains , cells were also separately transformed with a positive control plasmid expressing a VP16 , Gcn4 , or Pho4 AD or with a negative control plasmid , the pRS414-TFchassis plasmid without a protein fragment insert .", "Transformed cells were grown in 200 mL SC-Trp-NAT-G418 for 6 days ( first library ) or 5 days ( second library ) ; cells were diluted down to OD600 = 0 . 05 every day upon reaching saturation .", "After this period , cells were diluted to OD600 = 0 . 4 in SC-Trp-NAT-G418 containing 10 nM beta-estradiol and grown at 30°C for 4 hr .", "Cells were then pelleted and washed once in FACS buffer ( PBS , 2 mM EDTA , 0 . 1% BSA; sterile filtered ) , and finally suspended in FACS buffer at roughly 40 million cells/mL .", "Cells were analyzed and sorted on a BD Influx Cell Sorter at the Stanford Shared FACS Facility .", "Cells from different sub-libraries were pooled just prior to sorting .", "The 10–15% largest cells were excluded from sorting , and then an additional filter was applied to limit mCherry signal to within a twofold range .", "For the remaining cells , the full range of GFP signal was divided into eight evenly spaced bins in log scale; cells were first sorted into the four low bins and then sorted into the four high bins .", "In total , 2 . 85 million cells and 2 . 65 million cells were sorted for the first and second libraries , respectively .", "To define the background GFP signal , cells containing the negative control plasmid were also induced with 10 nM beta-estradiol and their distribution across the eight GFP bins defined above was measured .", "We found that these cells had slightly increased levels of GFP signal relative to untransformed cells , potentially because binding of the aTF alone slightly opens up chromatin around the GFP promoter .", "Cells expressing just one plasmid containing the VP16 , Gcn4 , or Pho4 AD were also individually analyzed ( by similarly measuring their distribution across same eight GFP bins ) and compared to results obtained for identical sequences in the pooled activation screen .", "Cells sorted into each of the 8 GFP bins and a sample of total input cells ( nine samples total ) were grown overnight and then pelleted .", "Pellets were treated with lyticase and then plasmid DNA was extracted using a Qiagen Miniprep kit .", "The region of the plasmid encoding the variable protein fragment was PCR-amplified for 11–20 cycles ( based on the OD600 of the culture ) using Phusion polymerase with PCR primers that added flanking sequences corresponding to the standard Illumina sequencing primers .", "After a column cleanup , a second PCR was performed with primers that added sample-specific barcodes and the P5 and P7 sequences for Illumina sequencing .", "This second PCR product was SPRI size-selected using AMPure XP beads ( Beckman Coulter ) at 1x dilution , and the different samples were pooled for paired-end sequencing on the Illumina NextSeq 550 .", "Roughly 2 million reads of 2 × 76 bp length were sequenced per sample .", "The pFN26A BIND plasmid ( Promega ) , which contains a Renilla luciferase gene used for normalization , was used to express domains of interest fused to a GAL4 DBD .", "Genes expressing 50 randomly chosen putative human ADs predicted by PADDLE were synthesized in a single oPool ( IDT ) with Gibson homology arms , amplified using Phusion polymerase , and cloned into pFN26A BIND ( digested with AsiSI and PmeI ) using Gibson assembly .", "The product was transformed into Agilent XL10 Gold cells , and then plated onto LB Carb plates .", "Forty-three colonies were picked , which yielded clones of 25 correct unique ADs .", "In addition , plasmids expressing a VP16 AD-positive control or three random protein sequences for negative controls were individually cloned in the same manner .", "HEK293T cells were plated at 24 , 000 cells per well of a 96-well plate .", "The next day , cells were transfected using Lipofectamine 3000 with 50 ng of cloned pFN26A BIND plasmid and 50 ng of pGL4 . 35 ( Promega ) , which contains a Firefly luciferase reporter gene under the control of a minimal promoter driven by nine upstream GAL4 DNA-binding sites .", "Each plasmid was transfected into triplicate wells .", "Seventy-two hours after transfection , luminescence from renilla and firefly luciferases were measured using the Dual-Glo Luciferase assay system ( Promega ) on a BioTek Synergy two plate reader .", "Measured activity of each predicted AD or control sequence was computed by dividing the Firefly luciferase signal by the Renilla signal and then averaging the triplicate values .", "Fold-activation was computed by dividing the activity of each predicted AD by the mean activity of the three negative controls , Z-scores were computed based on log-scale fold-activation by standardizing the negative controls to mean of 0 and variance of 1 , and p-values were computed with one tailed Z-tests .", "An mRNA display library was made for each of sub-library A , sub-library B , and six pooled controls ( three libraries total ) , using the following method .", "Sub-libraries of wild-type and mutant TF fragments were expressed as mRNA-tagged peptides using mRNA display with in vitro translation as described in Takahashi and Roberts , 2009 .", "Specifically , sub-library pools were PCR-amplified using primers that added an upstream T7 promoter sequence and a downstream linker and HA-tag but no stop codon .", "Thus , all encoded peptides were of the form MSGT[variable protein sequence]TSVGGSGSYPYDVPDYA and fused to mRNA at the C-terminus .", "In addition to the two sub-libraries , similar DNA sequences encoding positive control ADs from VP16 , Gcn4 , and Pho4 and three negative control random protein sequences were synthesized as gBlocks ( IDT ) ( containing an upstream T7 promoter sequence and a downstream linker and HA-tag but no stop codon ) .", "All subsequent work was performed in RNase-free conditions .", "mRNA was in vitro transcribed for 4 hr at 37°C using the MEGAscript T7 transcription kit ( Thermo ) and purified with RNeasy mini columns ( Qiagen ) .", "The PF30P oligo containing a poly-A sequence , spacers , and puromycin was synthesized by IDT and ligated to the 3’ end of the mRNA using Moore-sharp splinted ligation by T4 DNA ligase .", "The ligation product was desalted by three rounds of concentrating in 0 . 5 mL Amicon Ultracel 30K MWCO columns and diluting in water .", "The desalted product was run on a denaturing 4% polyacrylamide gel with 7 M urea .", "The band corresponding to full-length ligated mRNA was excised from the gel and recovered using the Model 422 Electro-Eluter with 3500 MWCO caps ( Bio Rad ) .", "After concentrating and desalting in the same manner , the ligated and purified mRNA was used in an in vitro translation reaction using the Retic Lysate IVT kit ( Thermo ) for 60 min at 30°C .", "MgCl2 and KCl were added to final concentrations of 60 mM and 500 mM , respectively , and the sample was incubated for 15 min at room temperature to encourage puromycin fusion .", "The sample was then diluted in binding buffer ( 20 mM Tris pH 8 . 0 , 100 mM KCl , 1 mM MgCl2 , 0 . 01% Tween-20 , 0 . 1 mg/mL BSA , 0 . 1 mM DTT ) and bound to Anti-HA magnetic beads ( Pierce ) for 30 min at room temperature with mixing .", "After washing three times for 5 min with binding buffer without BSA , peptides were eluted by incubating in 1x SuperScript II buffer ( 50 mM Tris-HCl pH 8 . 3 , 75 mM KCl , 3 mM MgCl2; Thermo ) with 0 . 01% Tween-20 and 0 . 5 mg/mL synthetic HA peptide ( Thermo ) for 1 hr at room temperature with mixing .", "The mRNA tag on the fused peptides was reverse transcribed using SuperScript II ( Thermo ) and a primer that contained ( 1 ) a sequence that hybridizes just downstream of variable protein sequence coding region , ( 2 ) a 12-nt random sequence that serves as a unique molecular identifier ( UMI ) , and ( 3 ) the Illumina read two sequencing primer sequence .", "Enzyme was heat inactivated by adding EDTA to 10 mM and heating to 65°C for 10 min .", "To remove aggregates , the library was fractionated in 20 mM Tris pH 8 . 0 , 100 mM KCl , 1 mM MgCl2 , 1 mM DTT over a 6 mL 10–30% sucrose gradient by spinning at 60 , 000 rpm in a SW60 rotor for 10 hr .", "Fractions of 100–200 µL were collected and the abundance of peptide-mRNA-cDNA fusion in each fraction was assayed using qPCR .", "The 3–4 peak fractions encompassing 80–90% of total input was pooled and used in pull-down experiments or flash frozen in aliquots and stored at −80°C .", "Biotinylated bait proteins ( 12 µg of Med15 or 8 µg of TFIID subcomplex ) were bound to 50 µL Dynabeads MyOne Streptavidin T1 ( Thermo ) for 15 min at room temperature in binding buffer ( 20 mM Tris pH 8 . 0 , 100 mM KCl , 10 mM MgCl2 , 0 . 01% Tween-20 , 1 mM DTT ) and then washed three times for 5 min in binding buffer .", "In the third wash , beads were blocked by also including 100 ng/µL salmon sperm DNA ( Thermo ) and 10 ng/µL BSA ( NEB ) in the buffer .", "mRNA display library input was prepared by mixing sub-library A , sub-library B , and the three positive and three negative controls .", "This input was then incubated with the bait-coated beads in binding buffer with salmon sperm DNA and BSA at 100 µL total volume for 8 min with mixing .", "Afterward , the beads were separated on a magnet , briefly washed once with cold binding buffer , and then immediately separated on a magnet again .", "cDNA from bound fragments was eluted by suspending the beads in 10 mM Tris pH 8 . 0 with proteinase K ( NEB ) and incubated for 30 min at room temperature .", "Proteinase K was heat inactivated at 95°C for 10 min , beads were separated on a magnet , and the eluate was collected .", "Total abundance of the library cDNA in the input , bound , and eluted samples was measured by qPCR .", "To measure the total abundance of the whole library ( without regard to the identity of the library elements ) , qPCR primers were designed to hybridize to the constant sequences flanking the variable region of the library .", "Additionally , this design enabled the use of different primers to separately track sub-library A and sub-library B , as well as each of the six control sequences individually .", "The controls were used to provide an estimate of binding efficiency .", "Binding percentage was calculated by dividing the abundance of each library in the bound sample by the abundance in the input sample .", "cDNA of the input and bound samples were prepared for sequencing in the same manner as the purified plasmids in the activation assay .", "Biotinylated 104 bp double-stranded DNA templates were assembled by annealing a 104-nt oligo to a 20-nt oligo with a 5’ biotin modification ( IDT ) and elongating using Klenow Fragment ( 3’-->5’ exo- ) ( NEB ) .", "The oligos were designed such that the TF DNA binding sites were placed away from the biotinylated end .", "SPR experiments were performed on the ProteOn XPR36 Surface Plasmon Resonance System using NeutrAvidin-coated NLC Sensor Chips ( Bio Rad ) .", "Mass of biomolecules bound to the chip surface was measured in arbitrary units ( AU ) approximately every 1 s .", "All plotted data were normalized to the first ligand channel and the first analyte channel which were used as negative controls in experiments as described below .", "In SPR experiments with GCN4 , 104 bp DNA templates with 0 , 1 , 2 , 4 , or 6 Gcn4 motifs were bound to the chip along the ligand channels , except for the first ligand channel , which was left without DNA for normalization .", "Binding buffer for all conditions was 20 mM Tris-HCl ( pH 7 . 5 ) , 100 mM KCl , 10 mM MgCl2 , 2 mM EDTA , 10 mM AmSO4 , 0 . 05% Tween-20 , with 1 mg/mL BSA ( NEB ) .", "Bound DNA was washed twice with 1 M NaCl .", "Gcn4 at 7 . 5 nM was then pre-bound to DNA for 480 s along the analyte channels , except for the first analyte channel , which was left without Gcn4 for normalization .", "After Gcn4 binding , Gcn4 at 7 . 5 nM with Mediator at 0 , 1 . 25 , 2 . 5 , 5 , or 10 nM were flowed across analyte channels 2–6 respectively for 300 s , then dissociation was observed for 20 min by flowing in binding buffer .", "Association , dissociation , and affinity constants of Mediator binding to Gcn4 on DNA were estimated by fitting to a Langmuir kinetic model using the ProteOn Manager Software ( Bio Rad ) .", "Reported values are the mean and standard deviation across the four different Mediator concentrations .", "Note that Mediator dissociation rates in conditions with multiple Gcn4 binding sites may be overestimated because it also includes the dissociation of Gcn4 from DNA .", "For SPR experiments with EcoRI , biotinylated 60 bp DNA templates containing 0 or 1 EcoRI motif were annealed and elongated as described above , and then bound along the ligand channels and washed with 1 M NaCl .", "Gcn4AD-EcoRI ( E111Q ) was bound along analyte channels at 27 nM monomer in 20 mM Tris-HCl ( pH 7 . 5 ) , 10 mM MgCl2 , 300 mM NaCl , 10 mM bME , 0 . 05% Tween-20 , 1 mg/mL BSA ( higher salt was used to reduce non-specific binding to DNA ) .", "Minimal dissociation was observed after binding .", "Then , Mediator at 5 nM with competitor Gcn4 protein ( in molar excess as specified in Figure 7D ) was flowed across the chip for 180 s in 20 mM Tris-HCl ( pH 7 . 5 ) , 10 mM MgCl2 , 100 mM NaCl , 10 mM AmSO4 , 10 mM bME , 0 . 05% Tween-20 , 1 mg/mL BSA .", "All protein positions reported in the text are 1-indexed and inclusive .", "The first library of protein sequences was composed of sub-library A , which contained wild-type tiles spanning all S . cerevisiae transcription factors , and sub-library B , which contains mutants of 8 known S . cerevisiae ADs .", "The second library was composed of sub-library D and sub-library E , which contained multiple collections of wild-type , mutant , and designed sequences .", "Sequences in sub-libraries A , B , and D were 53 amino acids in length and sequences in sub-library E were 30 amino acids in length .", "Protein and DNA sequences , as well as activation and pull-down data where available , are listed in Tables S1 and S4 .", "Libraries contained additional fragments which were included in experiments but are not reported here .", "In the reverse translation design process , we aimed to optimize our library DNA fragments for compatibility and consistency with our in vitro and in vivo assays , as well as with standard RNA-seq protocols .", "We also sought to build in redundancy for error-correcting reads .", "In particular , we used the Python package dnachisel 1 . 4 . 1 ( Zulkower and Rosser , 2020 ) to optimize the following objectives: To allow paired-end sequencing to uniquely identify each element , we additionally enforced an edit distance of 6 among the first 48 bases and last 48 bases of any two sequences in the same sub-library .", "This was performed in a randomized , brute-force , iterative approach , with each iteration consisting of the following steps: Finally , we verified that all sequences sharing the same sub-library primer ( e . g . all sequences in sub-library A , or all sequences in sub-library B ) had a paired-end edit distance ( sum of edit distance of 5’-most 50 bases and edit distance of 3’-most 50 bases ) of at least 6 .", "The use of sequencing primers unique to each sub-library enabled us to submit samples for sequencing in multiplexed format and accurately assign reads to the correct sub-library computationally .", "We further leveraged the edit distance margin built into the library to enable mapping of sequencing reads with a small number of errors .", "Sequencing read alignment was performed using custom bash script built on top of existing tools and additional custom scripts .", "It takes as input arguments the UMI length , the sub-library sequencing primer , the edit distance threshold for that sub-library , and raw FASTQ files .", "For pull-down experiments , unique molecular identifiers ( UMI ) were extracted from reads and appended to the read names using umi_tools 1 . 0 . 0 ( Smith et al . , 2017 ) .", "cutadapt 1 . 18 was used to discard reads without matching paired-end sub-library sequencing primers and trim the primers in reads with matching primers; the default error tolerance was used ( Martin , 2011 ) .", "bwa-mem 0 . 7 . 17-r1188 was used to perform a first-pass alignment of reads to the DNA fragment library ( Li , 2013 ) .", "Imperfectly mapped read pairs ( i . e . those without paired read SAM flags of 99 and 147 ) were re-mapped to the library sequence with minimal edit distance .", "This was necessary because bwa-mem did not always correctly map paired reads as a pair , a problem most evident in the mutant library with many similar sequences .", "Pairwise Levenshtein edit distance was computed using the Python package editdistance 0 . 5 . 3 ( https://github . com/roy-ht/editdistance , Tanaka , 2019 ) .", "Paired reads exceeding the edit distance threshold were discarded using reformat . sh from BBTools 38 . 61 ( https://sourceforge . net/projects/bbmap/ ) .", "Duplicate reads were identified and deduplicated using utmi_tools 1 . 0 . 0 .", "Finally , reads mapped to each DNA library fragment sequence were counted .", "Calculations of GFP signal and fold-activation required the following inputs: First , to make cell counts comparable between sorting bins 1–4 versus bins 5–8 , the number of cells sorted into each GFP bin were normalized based on the number of events assayed in each sort .", "Second , to estimate the number of cells sorted into each of the 8 GFP bins that expressed a given protein fragment F , the total number of cells sorted into each bin was multiplied by the fraction of sequencing reads for that bin that mapped to F . Any fragment represented by fewer than 10 cells were excluded from further analysis .", "Third , the average GFP signal of cells expressing F was calculated as a weighted average from the average GFP signal observed in each bin during sorting weighted by the distribution of the number of cells expressing F across the eight bins .", "Because GFP distributions cells expressing single AD sequences were normally distributed in FACS data when GFP signal ( arbitrary units ) was plotted in log-scale , all averages of GFP distributions were calculated in log-scale ( equivalently , a geometric mean of GFP values ) .", "The standard deviation of GFP signal of cells expressing F was similarly calculated in log-scale and used as a metric for measurement precision .", "All averages involving activation reported in the paper are calculated in log-scale ( equivalently , a geometric mean ) .", "Fourth , the fold-activation of F was calculated as the mean GFP signal of F divided by the mean GFP signal of negative control cells ( see below ) .", "Z-scores were calculated by standardizing the GFP signal of negative control cells to mean 0 and variance one in log scale and p-values were calculated with one tailed Z-tests .", "The mean and standard deviation GFP signal of negative control cells was defined in one of two ways .", "In the experiment with sub-libraries A and B , a sample of estrogen-induced cells containing only the empty pRS414-TFchassis plasmid was assayed under identical conditions and the mean and standard deviation GFP signal was computed from FACS data .", "In the experiment with sub-libraries D and E , these values were instead determined internally from the 50 random sequence negative controls in the pooled library .", "Since a small fraction of random sequences activate , outlier sequences were iteratively excluded if they had GFP signal greater than three standard deviations away from the mean , resulting in three sequences excluded from each sub-library .", "Then mean and standard deviation were computed from the sum of the negative control GFP distributions .", "In activation assays with sub-libraries D and E , we noticed that many sequencing reads that were mapped with a small number of errors corresponded to fragments with a very large GFP standard deviation .", "Upon closer examination of sequencing reads of example fragments , the same sequencing errors were consistently found , which likely corresponded to mutations that occurred in vivo that affected function of the protein fragment ( e . g . inactivation of a strong AD by a frameshift mutation ) .", "We therefore used only reads that aligned perfectly for this experiment .", "Furthermore , fragments with GFP standard deviation greater than 4 . 5 were excluded from further analysis .", "The following three metrics were tracked: the number of total unique fragments , number of fragments observed in at least one of the eight GFP bins , and number of fragments that passed filtering .", "For the activation assay with sub-libraries A and B , this was ( 7637 , 7604 , 7549 ) for sub-library A and ( 2900 , 2833 , 2813 ) for sub-library B . The same metrics for the activation assay with sub-libraries D and E was ( 7733 , 7716 , 7345 ) for sub-library D and ( 4261 , 4261 , 4252 ) for sub-library E . These numbers include additional fragments in the peptide libraries that are not reported in this paper .", "To define ADs across all TFs , at each protein position a mean Z-score was calculated from the Z-scores of all fragments overlapping that position .", "ADs were defined for all intervals of the protein that had a mean positional Z-score ( MPZ ) greater than 3 . 5 .", "Starting with intervals that had the highest maximal MPZ score , the start and stop positions of the AD were defined by the full width half maximum; that is , the positions in the protein at which the MPZ fell below half of the maximal MPZ value in that interval .", "If the AD defined in this manner includes a new region with an MPZ score higher than the original maximal value , this indicated that the MPZ peak was only small relative to nearby peaks , so the AD was then excluded .", "We compared our ADs with the Induction Dynamics gene Expression Atlas ( IDEA ) ( Hackett et al . , 2020 ) , which induced expression of every TF individually and measured ‘v_inter’ parameters that describe the resulting fold-change in the expression of all target genes .", "We said that a TF activated a gene if the v_inter > 1 , and for each TF that regulated at least five genes , we calculated the proportion of regulated genes that were activated .", "We then compared these proportions for TFs that had a strong AD ( overlapping a fragment with Z-score at least 6 ) versus TFs without ADs .", "Significance was calculated using a Kolmogorov–Smirnov test .", "Hydrophobicity measurements were calculated using the Wimley-White interfacial scale , which measures whole-residue ∆G for transfer from water to a water-octanol interface ( Wimley and White , 1996 ) .", "Because the goal was to quantify the total content of hydrophobic residues rather than the net hydrophobicity/hydrophilicity , hydrophobic content of a peptide is computed as the sum of the scores of only the hydrophobic residues ( i . e . those with negative ∆G ) .", "Net charge was computed by adding the number of Arg and Lys residues and subtracting the number of Asp and Glu residues .", "Motif finding using DREME was done through the web portal ( http://meme-suite . org/tools/dreme ) using default settings and shuffled input sequences as control ( Bailey , 2011 ) .", "Searching for 9aaTAD sequences was done by matching to the ‘most stringent pattern’ [MDENQSTYG]{KRHCGP}[ILVFWM]{KRHCGP}{CGP}{KRHCGP}[ILVFWM][ILVFWMAY]{KRHC} listed at the prediction portal https://www . med . muni . cz/9aaTAD/index . php ( Piskacek et al . , 2007 ) .", "ADpred was installed locally from https://github . com/FredHutch/adpred , Erijman , 2020a and predictions were computed for all 30-aa tiles on all yeast TFs ( Erijman et al . , 2020b ) .", "ADs predicted by ADpred were defined using the authors’ criteria , namely sites with five or more contiguous residues with a score >= 0 . 8 .", "An ADpred-predicted region was considered correct if it overlapped by at least five aa with a significantly activating tile ( p<0 . 0001 ) .", "With this approach , ADpred achieved a precision and recall of 0 . 366 and 0 . 692 in predicting experimental ADs , respectively .", "When ADpred-predicted regions were randomly shuffled 100 times to different proteins and positions , the average precision and recall of these shuffled predictions were 0 . 120 and 0 . 295 respectively .", "Alternatively , to compare ADpred predictions directly to our experimental measurements of activation , a representative ADpred score for each 53-aa TF tile in our library was calculated by taking the 80th percentile of ADpred scores of 30-aa tiles contained within the 53-aa tile .", "These scores were used for calculating tile-wise precision-recall curves for ADpred predictions ( Figure 3—figure supplement 1K ) .", "The fractional pull-down of each fragment F was calculated as ( the fraction of the total input library that bound to beads , measured from qPCR ) x ( the fraction of UMI-deduplicated sequencing reads in the bound sample matching F ) / ( the fraction of UMI-deduplicated sequencing reads in the input sample matching F ) .", "Fragments with fewer than 30 reads in the input sample or fewer than five reads in the bound sample were excluded from downstream analysis .", "Baseline binding was defined using the 50 random protein control sequences in each library , except the sequences that were outliers in the activation assay were excluded .", "Specifically , random control outliers were iteratively removed if their activation was more than three standard deviations away from the mean ( in log scale ) , resulting in the exclusion of random control #2 , 12 , 16 , 38 from sub-library A and #17 , 18 , 21 , 22 , 31 , 43 from sub-library B . Binding enrichment of each fragment was then computed as the fractional pull-down of the fragment divided by the mean fractional pull-down ( in log scale ) of the random controls .", "Because fractional pull-down and enrichment were normally distributed in log-scale , all averages involving pull-down or enrichment reported in the paper are calculated in log-scale ( equivalently , a geometric mean ) .", "Z-scores were computed by standardizing the binding enrichment of random controls to mean 0 and variance one in log-scale and P-values were calculated with one tailed Z-tests .", "Triplicate measurements for Med15 pull-downs and duplicate measurements for TFIID subcomplex pull-downs were combined by averaging the binding enrichment values .", "A small number of highly positively-charged fragments bound in Med15 pull-downs when the mRNA display library was purified on a sucrose gradient but did not bind when the sucrose gradient was omitted .", "Suspecting that this binding was artifactual , potentially through non-specific binding to mRNA tags , we excluded from analysis 92 fragments that bound with Z-score between −2 . 5 and 1 without the sucrose gradient and had a Z-score at least 3 . 5 higher with the sucrose gradient than without .", "Med15-binding domains were annotated in the same manner as ADs using a mean positional Z-score , but with a cutoff of 2 . 23 .", "An AD was considered to bind Med15 or TFIID subcomplex if it overlapped a fragment that bound significantly ( p<0 . 001 ) by at least 26 aa .", "A Med15-binding domain was considered to activate if it overlapped an AD .", "Neural network models were trained to predict activation Z-scores .", "Approximately 15% of sequences from sub-library A and B were randomly held-out as a test set , and the remaining library elements were split into 10 parts for training and cross-validation .", "Because adjacent TF tiles have significant overlap in sequence , all tiles from each protein were placed into the same train or test split .", "From sub-library B , all mutants and homologs of the Pdr1 AD and all scramble mutants were held-out in the test set and the remaining sequences were distributed evenly across the training splits .", "Models were developed in TensorFlow 2 . 2 ( Abadi et al . , 2016 ) and trained using CPUs on Stanford’s Sherlock computing cluster .", "All models used mean squared error as the loss function .", "Sequence encodings and activation Z-scores were standardized to mean 0 variance one across the training dataset for more efficient learning .", "For each architecture , 10 models were trained on each subset of nine training splits and validated on the 10th split .", "The best model architecture was chosen by the mean validation score across the 10 splits on sub-library A sequences only .", "For final evaluation on the test dataset , the mean prediction across all 10 models was used .", "Protein fragment sequences were each encoded as a single 20-element vector giving the proportions of each of the 20 amino acids .", "The best architecture , as determined by a grid search over hyperparameters , was a neural with three fully connected layers of width 40 with Swish activation ( Ramachandran et al . , 2017 ) , batch normalization , L2 weight penalty of 0 . 01 , and dropout rate of 0 . 4 .", "The model was trained with an initial learning rate of 0 . 0001 using the Adam optimizer for up to 300 epochs with two scheduling callbacks: reduction of the learning rate by fivefold if training loss did not improve for 20 epochs , and early stopping if no improvement on the validation loss ( averaged in a 10-epoch window ) was observed for 75 epochs , upon which the best model from previous epochs was saved .", "One-hot encodings were used to encode each 53-aa protein fragment sequence as a 53-by-20 matrix .", "Secondary structure predictions were generated using PSIPRED v . 4 . 01 without PSI-BLAST ( Jones , 1999 ) , and a 53-by-2 matrix of the alpha helix and coil predictions were also used as input .", "Disorder predictions were generated using IUPred2A ( Mészáros et al . , 2018 ) , and a 53-by-2 matrix of the disorder predictions in ‘short’ and ‘long’ mode were also used as input .", "In total , each 53-aa protein fragment was encoded as a 53-by-24 matrix .", "The best model architecture , as determined by a grid search over hyperparameters , was a convolutional neural network with nine convolutional layers with kernel size 10 and channel width 30 ( i . e . number of filters ) followed by max-pooling along the sequence-length dimension and two fully-connected layers of width 20 , with Swish activation .", "For regularization , the L2 weight penalty was 0 . 001 , dropout rate was 0 . 1 , and batch normalization was used .", "The model was trained with an initial learning rate of 0 . 001 in the same manner as the amino acid composition-based network , except for up to 500 epochs .", "After finding this optimal architecture , the test dataset was distributed across the 10 training datasets and 10 new models were trained , together comprising PADDLE .", "The mean value of these 10 predictors was used as the final prediction .", "This model was used for all predictions on yeast TFs and nuclear proteins , human TFs , and virus transcriptional regulators .", "Because secondary structure prediction was by far the slowest aspect of running PADDLE predictions , we trained a version of PADDLE called PADDLE-noSS which only used the one-hot sequence encoding for input , no secondary structure or disorder predictions .", "PADDLE-noSS was nearly as accurate as PADDLE ( R2 scores of 0 . 76 , 0 . 52 , and 0 . 89 for TF tiles , scramble mutants , and Pdr1 mutants and homologs respectively; compare to Figure 3—figure supplement 1B ) and was used for predictions of core ADs and mutant sequences in Figure 3E–G , Figure 3—figure supplement 1E–I , and Figure 4—figure supplement 1F .", "Human TFs were defined by Gene Ontology term GO:003700 ( DNA-binding transcription factor activity ) .", "Virus transcriptional regulators ( vTRs ) were taken from Figure 1—source data 1 from Liu et al . , 2020 .", "Within each human TF or vTR , predictions on 53-aa tiles at 1-aa resolution were generated using PADDLE and smoothed with a 9-aa moving average .", "High-strength ADs were defined as the union of at least 5 consecutive 53-aa tiles with a predicted Z-score greater than 6 .", "ADs defined in this way that overlapped by more than 26 aa were combined .", "Medium-strength ADs were defined similarly with a Z-score cutoff of 4 and were required to not overlap with high-strength ADs by more than 26 aa .", "All protein positions reported in the text are 1-indexed and inclusive; all annotations reported in supplemental tables are 0-indexed and inclusive of the start position but exclusive of the stop position .", "For testing in a luciferase assay , 50 predicted high-strength ADs from human TFs were chosen at random and the tile with the strongest ( smoothed ) predicted Z-score from each AD was used .", "Yeast nuclear proteins were defined by Gene Ontology term GO:0005634 ( Nucleus ) .", "Within each protein , predictions on 53-aa tiles at 1-aa resolution were generated using PADDLE and smoothed with a 9-aa moving average .", "All predicted Z-score local maxima higher than 3 . 09 ( p<0 . 001 ) were considered for experimental testing .", "The 53-aa tiles corresponding to the local maxima were included , starting from those with the largest predicted Z-score , unless they overlapped a previously-included tile by at least 15 aa .", "Analysis of experimentally-confirmed ADs focused on proteins that had experimental or high-throughput evidence for nuclear localization .", "Proteins involved in transcriptional regulation were defined by GO:0006355 ( regulation of transcription , DNA-templated ) .", "To calculate enrichment of activating tiles in coactivator proteins , we generated a null model in which ‘activating’ tiles , equal in number to actual activating tiles discovered in our screen , were randomly selected from among non-TF nuclear proteins .", "We then counted how many of these randomly-selected tiles were in coactivator proteins , and then repeated this sampling 10 , 000 times to generate a null distribution .", "The actual number of tiles , 42 , is 2 . 9-fold more than the mean of the null; p<10−12 by one tailed Z-test .", "Predicted disorder for each tile was computed from D2P2 annotations as the fraction of the tile’s residues that were annotated as disordered among all 8 of the predictors listed in D2P2 .", "To define core ADs , predictions of sequences shorter than 53 aa were done using PADDLE-noSS by embedding the shorter region in 100 different neutral sequences composed of AGSTNQV residues and averaging the predicted values .", "Predictions of all tiles 5–30 aa in length within every AD were computed in this manner , and used to define core ADs for Figure 3F–G , Figure 7A , and Figure 7—figure supplement 1B .", "Specifically , core ADs were defined as the shortest tiles with predicted Z-score greater than 3 . 09 ( p<0 . 001 ) that do not overlap another shorter core AD .", "In Figure 3—figure supplement 1I , the 20-aa region within each AD with the highest predicted Z-score was included if it had Z-score greater than 3 . 09 , and the effects of single AA mutants in these 20-aa core ADs were predicted in the same manner using PADDLE-noSS .", "Code for running PADDLE and PADDLE-noSS is available at https://github . com/asanborn/PADDLE ( Sanborn , 2021; copy archived at swh:1:rev:17aa36985e474d8593b99d593f7cc5e7c3e205a0 ) .", "Pre-generated PADDLE predictions from multiple species are available for download at http://paddle . stanford . edu .", "To generate structural models of binding , we took 28 core ADs peptides 13 residues in length and docked them to the ABDs using Rosetta3 ( Leaver-Fay et al . , 2011 ) , specifically its FlexPepDock ab initio pipeline ( Raveh et al . , 2011 ) .", "Core ADs were used as the peptide , and activator-binding domain as the receptor .", "Peptides were initialized in an extended conformation using Rosetta’s BuildPeptide application and manually placed near the binding pocket .", "For the KIX domain , NMR chemical shift data was used to aid in placement ( Thakur et al . , 2008 ) .", "For ABD1 , the AD of Gcn4 was used to aid in placement .", "For all models of ABD1 , the first conformer from PDB structure 2LPB was used ( Brzovic et al . , 2011 ) .", "For each AD-ABD pair , a set of 50 , 000 structural models were generated and ranked by FlexPepDock’s reweighted_sc score .", "Binding poses were generated by clustering the top 500 ranked models using Rosetta’s cluster application with the default clustering distance threshold of 2 Å .", "The best-scoring model from each cluster was designated as the cluster representative , and the cluster representatives that were also among the 10 best-scoring models were used for further analysis .", "Additionally , larger runs with 500 , 000 structural models and clustering on the top 5000 models were run for six individual ADs for the KIX ABD .", "The full list of ABDs and ADs used is available in Figure 6—source data", "1 . PDB files of 10 best-scoring models from different binding poses and the score , cluster number , and RMSD to best model for the 500 best-scoring models are available in Figure 6—source data", "2 . Due to the large number of peptides that needed to be docked , the FlexPepDock ab-initio pipeline was modified to run in a cluster setting at a large scale and used approximately 100 , 000 CPU hours on the Stanford Sherlock academic cluster .", "Secondary structure was calculated using DSSP ( Kabsch and Sander , 1983; Touw et al . , 2015 ) and annotations of G , H , or I were considered alpha-helical .", "Interaction types were computed with GetContacts ( Venkatakrishnan et al . , 2019 ) and per-AA accessible surface areas were computed with FreeSASA ( Mitternacht , 2016 ) .", "For each final docked structural model , buried peptide surface area was taken by subtracting the accessible surface area of the peptide in complex with the receptor from the accessible surface area of the peptide on its own .", "For Figure 6E , marked positions correspond to atom CG of Asp , atom CG of Leu , and the midpoint of the CG and CZ atoms in Phe ( the center of the aromatic ring ) .", "Residues are shown from all the 10 best models from different clusters for all core AD structures if the marked position is within 3 Angstroms of the KIX domain or ABD1 structure .", "Adapted FlexPepDock code , and wrapper code for GetContacts and FreeSASA are available at https://github . com/drorlab/med15 ." ] ]

[ "Gene activator proteins comprise distinct DNA-binding and transcriptional activation domains ( ADs ) .", "Because few ADs have been described , we tested domains tiling all yeast transcription factors for activation in vivo and identified 150 ADs .", "By mRNA display , we showed that 73% of ADs bound the Med15 subunit of Mediator , and that binding strength was correlated with activation .", "AD-Mediator interaction in vitro was unaffected by a large excess of free activator protein , pointing to a dynamic mechanism of interaction .", "Structural modeling showed that ADs interact with Med15 without shape complementarity ( ‘fuzzy’ binding ) .", "ADs shared no sequence motifs , but mutagenesis revealed biochemical and structural constraints .", "Finally , a neural network trained on AD sequences accurately predicted ADs in human proteins and in other yeast proteins , including chromosomal proteins and chromatin remodeling complexes .", "These findings solve the longstanding enigma of AD structure and function and provide a rationale for their role in biology ." ]

[ "Cells adapt and respond to changes by regulating the activity of their genes .", "To turn genes on or off , they use a family of proteins called transcription factors .", "Transcription factors influence specific but overlapping groups of genes , so that each gene is controlled by several transcription factors that act together like a dimmer switch to regulate gene activity .", "The presence of transcription factors attracts proteins such as the Mediator complex , which activates genes by gathering the protein machines that read the genes .", "The more transcription factors are found near a specific gene , the more strongly they attract Mediator and the more active the gene is .", "A specific region on the transcription factor called the activation domain is necessary for this process .", "The biochemical sequences of these domains vary greatly between species , yet activation domains from , for example , yeast and human proteins are often interchangeable .", "To understand why this is the case , Sanborn et al . analyzed the genome of baker’s yeast and identified 150 activation domains , each very different in sequence .", "Three-quarters of them bound to a subunit of the Mediator complex called Med15 .", "Sanborn et al . then developed a machine learning algorithm to predict activation domains in both yeast and humans .", "This algorithm also showed that negatively charged and greasy regions on the activation domains were essential to be activated by the Mediator complex .", "Further analyses revealed that activation domains used different poses to bind multiple sites on Med15 , a behavior known as ‘fuzzy’ binding .", "This creates a high overall affinity even though the binding strength at each individual site is low , enabling the protein complexes to remain dynamic .", "These weak interactions together permit fine control over the activity of several genes , allowing cells to respond quickly and precisely to many changes .", "The computer algorithm used here provides a new way to identify activation domains across species and could improve our understanding of how living things grow , adapt and evolve .", "It could also give new insights into mechanisms of disease , particularly cancer , where transcription factors are often faulty ." ]