TORC2-Gad8-dependent myosin phosphorylation modulates regulation by calcium
The actin cytoskeleton underpins cellular organization by maintaining cell shape through the transmission of mechanical signals between the cell periphery and the nucleus , thereby influencing protein expression , protein organization and cellular architecture in response to the needs of the cell . Myosins , which are actin-associated motor-proteins , work in collaboration with an array of actin-binding proteins to facilitate global cytoskeletal reorganization and a plethora of other processes including cell migration , intracellular transport , tension sensing and cell division ( O'Connell et al . , 2007 ) . Each of the many classes of myosin contain three distinct domains: an actin-binding ATPase motor domain that exerts force against actin , a lever arm or neck region that contains light-chain-binding IQ motifs , and a tail region that specifies cargo binding and other molecular interactions . Although the different classes of myosin perform very different cellular functions , they all operate through the same basic mechanism: the motor domain undergoes cyclical interactions with actin , which are coupled to the breakdown of ATP . Each molecule of ATP that is converted to ADP and inorganic phosphate can generate movement along actin of 5–25 nm and force of up to 5 pN . The regulation of acto-myosin motility is multi-faceted ( Heissler and Sellers , 2016a ) , combining regulatory pathways that operate through the actin track ( historically called thin-filament regulation ) and myosin-linked regulation ( historically called thick-filament regulation ) . This latter control is often mediated by phosphorylation of the heavy chain or light chain ( s ) , or by calcium-regulation of light-chain binding ( Heissler and Sellers , 2016b ) . Phosphorylation at the conserved ‘TEDS’ site motif , which is located within the myosin motor domain of class one myosin , affects acto-myosin interaction ( Bement and Mooseker , 1995 ) , whereas phosphorylation within the tail region of class five myosin controls cargo binding ( Rogers et al . , 1999 ) . By contrast , phosphorylation of class two myosin light chains and/or heavy chain can change the folded state of the heavy chain , thereby altering actin interaction and the ability to form filaments ( Redowicz , 2001; Kendrick-Jones et al . , 1987; Pasapera et al . , 2015 ) . Thus , phosphoregulation of myosin can occur in the head , neck and tail regions , as well as in the light chains , and its impact varies across myosin classes and between paralogues within the same class . The impact of phosphorylation upon the motile function of most myosins remains to be established . The genome of the fission yeast Schizosaccharomyces pombe encodes five myosin heavy chains from classes 1 , 2 , and 5 ( Win et al . , 2002 ) . The single class one myosin ( UniProt Accession: Q9Y7Z8 ) , here termed Myo1 , is a 135-kDa protein with a motor domain , a neck region ( containing two canonical IQ motifs ) , and a 49-kDa tail region containing a myosin tail-homology-2 domain ( MYTH-2 ) , a membrane-binding pleckstrin homology ( PH ) domain , an SH3 domain and a carboxyl-terminal acidic region . The acidic region associates with , and activates , the Arp2/3 complex to nucleate actin polymerization ( Lee et al . , 2000 ) . The myosin motor has a conserved TEDS site , which is phosphorylated to modulate the protein’s ability to associate with actin ( Attanapola et al . , 2009 ) . Myo1 associates with membranes , primarily at sites of cell growth , where it is required for endocytosis , actin organization and spore formation ( Sirotkin et al . , 2005; Lee et al . , 2000; Itadani et al . , 2006 ) . Calmodulin or calmodulin-like light chains associate with the IQ motifs within the myosin neck to regulate both the length and the stiffness of the lever arm ( Trybus et al . , 2007 ) and the behavior of the motor domain ( Adamek et al . , 2008 ) . Calmodulins are ubiquitous calcium-binding proteins that associate with and regulate the cellular function of diverse proteins . Calcium associates with up to four EF hand motifs within the calmodulin molecule to instigate a conformational change that modulates the molecule's affinity for IQ motifs ( Crivici and Ikura , 1995 ) . Schizosaccharomyces pombe has two calmodulin-like proteins , Cam1 and Cam2 ( Takeda and Yamamoto , 1987; Itadani et al . , 2006 ) . Cam1 is a typical calmodulin that associates with IQ-domain-containing proteins in a calcium-dependent manner to modulate functions as diverse as endocytosis , spore formation , cell division and spindle pole body integrity ( Takeda and Yamamoto , 1987; Moser et al . , 1995; Moser et al . , 1997; Itadani et al . , 2010 ) . Although Cam2 shares Cam1’s ability to regulate Myo1 , Cam2 differs from Cam1 in two important respects: Cam2 is not essential for viability and is predicted to be insensitive to calcium ( Sammons et al . , 2011; Itadani et al . , 2006 ) . Furthermore , although cells that lack Cam2 show defects in spore formation following sexual differentiation , they have no significant growth-associated phenotypes during the vegetative growth cycle . From yeast to man , TOR ( Target of Rapamycin ) signaling plays a key role in modulating cell growth in response to changes in cell-cycle status and environmental conditions ( Laplante and Sabatini , 2012; Hartmuth and Petersen , 2009 ) . The mTOR kinase forms two distinct protein complexes , TOR complex 1 ( TORC1 ) and TOR complex 2 ( TORC2 ) , which are each defined by unique components that are highly conserved across species . TORC1 contains the Regulatory Associated Protein of mTOR ( RAPTOR ) , whereas in TORC2 , RAPTOR is replaced with the Rapamycin-Insensitive Companion of mTOR ( RICTOR ) . Both TORC1 and TORC2 complexes control cell migration and F-actin organization ( Liu and Parent , 2011 ) . TORC2 plays a key role in regulating the actin cytoskeleton in yeasts , Dictyostelium discoideum and mammalian cells , modulating actin organization and growth in response to cell-cycle progression and the cellular environment ( Jacinto et al . , 2004; Baker et al . , 2016; Lee et al . , 2005 ) . In S . pombe , TORC2 recruits and phosphorylates the fission yeast AGC kinase Gad8 ( Matsuo et al . , 2003 ) , a homologue of human SGK1/2 kinase , to regulate cell proliferation , the switch to bipolar cell growth , cell fusion during mating , and the subsequent meiosis ( Du et al . , 2016 ) . The basic principles of the control of the calcium signaling and phosphorylation signaling pathways are understood , but little is known about the interplay between these parallel modes of regulation . We have used molecular cell biological , biochemical and single-molecule biophysical techniques to identify and characterize a novel TORC2−Gad8-dependent system that regulates the calcium-dependent switching of the binding of different calmodulin light chains to the neck region of Myo1 . We define the contribution that each calmodulin makes to the regulation of this conserved motor protein and describe how they affect the conformation of the Myo1 lever arm . We propose that a concerted mode of regulation involving calcium and phosphorylation controls the motility and function of Myo1 in response to cell-cycle progression .
Phospho-proteomic studies of the fission yeast S . pombe ( Carpy et al . , 2014; Wilson-Grady et al . , 2008 ) have revealed a conserved phosphoserine residue that is located within the IQ-motif-containing neck region of class I and V myosins ( Figure 1A ) . The location of this AGC family kinase consensus phosphoserine site ( Pearce et al . , 2010 ) has the potential to impact myosin activity and function by affecting light-chain binding and the conformation of the lever arm . We generated polyclonal antibodies that recognized S . pombe myosin-1 when phosphorylated at this conserved serine at position 742 ( Myo1S742 ) . Myo1S742 phosphorylation was significantly reduced in cells lacking Ste20 ( the fission yeast homolog of the core TORC2 component , RICTOR ) , and abolished in cells lacking the downstream AGC kinase , Gad8 . Thus , Myo1S742 is phosphorylated in a TORC2–Gad8-kinase-dependent manner ( Figure 1B ) . Within cells , Gad8 kinase activity is reduced through the phosphorylation of a conserved threonine ( T6 ) residue ( Du et al . , 2016; Hálová et al . , 2013 ) ( Figure 1C ) . A significant reduction of Myo1S742 phosphorylation was observed in cells expressing phospho-mimetic Gad8 . T6D ( Figure 1D ) , which has reduced Gad8 kinase activity ( Du et al . , 2016 ) . Schizosaccharomyces pombe cells lacking either TORC2 or Gad8 display defects in actin organization , polarized growth regulation and the control of cell-cycle progression ( Petersen and Nurse , 2007; Du et al . , 2016 ) . Similarly , replacing Myo1 serine 742 with a phosphorylation-resistant alanine residue in myo1 . S742A cells blocked the division of cells that were cultured for an extended period in restricted-growth medium ( mean length ± SEM ( µm ) : 6 . 67 ± 0 . 3 for wildtype cells; 18 . 50 ± 1 . 3 for myo1 . S742A cells ( n > 300 ) ) ( Figure 1E ) . Therefore , although Gad8 may not directly phosphorylate Myo1S742 , phosphorylation of this residue is dependent upon the TORC2–Gad8 signaling pathway . We conclude that TORC2-directed Gad8-dependent phosphorylation at S742 regulates Myo1 activity . As serine 742 lies within the IQ motif of the Myo1 neck region , we asked whether Myo1S742 phosphorylation alters calmodulin binding and the conformation of the neck region . Isoforms of the Ca2+-sensitive fission yeast calmodulin ( wild type Cam1 and a Cam1 . T6C cysteine mutant , allowing conjugation to a fluorescent probe ) were purified from bacteria co-expressing the fission yeast NatA amino-α-acetyl-transferase complex in their native amino-terminally ( Nt ) acetylated forms ( Eastwood et al . , 2017 ) . Two methods were used to measure Ca2+-dependent changes in Cam1 conformation . First , a Förster resonance energy transfer ( FRET ) -based sensor was generated consisting of N-terminal CyPet donor and C-terminal YPet acceptor fluorophores fused in-frame with Cam1 ( Nguyen and Daugherty , 2005 ) ( Figure 2A ) . Second , Nt-acetylated Cam1 . T6C was conjugated to a cysteine-reactive synthetic fluorophore 2- ( 4'- ( iodoacetamido ) anilino naphthalene-6-sulfonic acid ( IAANS ) ) . IAANS fluorescence changes in response to changes in its local environment , so the fluorescence emission of this fusion will change in response to calcium-induced changes in Cam1 conformation . The Ca-binding affinity reported by the Cam1-FRET sensor ( Figure 2C , pCa50: 6 . 12 ) reflects the global change in Cam1 conformation , whereas the Ca2+-dependent change in IAANS’ fluorescence signal ( Figure 2C-inset , pCa50: 6 . 54 ) reflects changes in the local environment of the amino lobe of Cam1 . Together these probes demonstrated that Ca2+ binding induced a change in Cam1 conformation . The rate of Ca2+ ion release from Cam1 was independently measured by monitoring changes in the fluorescence of the Ca2+ indicator Quin-2 ( Tsien , 1980 ) . The time-course of Ca2+ ion release exhibited three phases , fast , medium and slow , of approximately equal amplitude ( rate constants 137 , 12 . 9 and 2 . 0 s−1 , respectively ) , indicating that the cation has different affinities for each Ca2+ binding lobe of Cam1 ( Figure 2D ) . To characterize Cam1 binding to the IQ neck region of Myo1 , recombinant FRET constructs were produced in which CyPet and YPet were separated by one of the two Myo1 IQ motifs or by both of these motifs ( Myo1IQ1-FRET , Myo1IQ2-FRET or Myo1IQ12-FRET ) ( Figure 2B and Figure 2—figure supplement 1 ) . Cam1 binding to the IQ motif ( s ) stabilizes the α-helix and results in a drop in FRET signal in the absence of calcium ( Figure 2E–G ) . This drop in signal correlates with a Cam1-bound IQ12 neck region length of 4 . 6 nm ( Wu and Brand , 1994 ) , close to the 4 . 7 nm length predicted from the modeled structure ( based upon PDB structure 4R8G ) . Analysis of interactions between Cam1 and Myo1IQ12–FRET revealed two distinct phases to the association of Cam1 molecules with the combined Myo1IQ12 motifs . Each phase contributed 50% of the overall change in signal ( Figure 2F ) . The first Cam1–Myo1IQ12-binding event corresponded to an affinity of less than 0 . 1 µM ( binding was too tight to calculate affinity with higher precision ) , whereas the second event correlated with an approximately 10-fold weaker binding affinity ( 0 . 68 µM ) . This association was sensitive to calcium ( pCa of 5 . 87 ) ( Figure 2C ) , indicating that Cam1 can only associate with both Myo1 IQ motifs at low cellular Ca2+ concentrations . Interestingly , while Cam1 bound tightly to a single , isolated , Myo1IQ1 alone ( Kd <0 . 1 µM ) , no detectable association was observed for the equivalent single Myo1IQ2 motif ( Figure 2E ) . Together these data are consistent with a sequential cooperative binding mechanism in which the stable residency of Cam1 in the first IQ position is required before calmodulin can bind to Myo1IQ2 . Replacing serine 742 within the IQ neck region with a phosphomimetic aspartic acid residue had no significant impact upon the affinity , calcium sensitivity or cooperative nature of the interaction between Myo1 and Cam1 ( Figure 2F ) . However , the S742D replacement resulted in a change in maximum FRET signal upon Cam1 binding ( Fmax 46 . 05 vs 31 . 64 ) ( Figure 2F ) , indicating that Myo1S742 phosphorylation changes the conformation of the lever arm upon Cam1 binding , rather than modulating the affinity of the neck region for Cam1 . Immunofluorescence using Myo1S742 phospho-specific antibodies confirmed the presence of serine-742 phosphorylated Myo1 at cortical foci ( Figure 3A ) . To explore how this phosphorylation affected Myo1 and calmodulin dynamics in vivo , we generated prototroph S . pombe strains in which endogenous myo1 , cam1 , or cam2 genes were fused to cDNA encoding monomeric fluorescent proteins ( Figure 3—figure supplement 1 ) . Using high-speed ( 20 fps ) single-molecule total internal reflection fluorescence ( TIRF ) imaging , we explored how Myo1S742 phosphorylation impacts Myo1 and Cam1 dynamics and function in vivo . Myo1 and Cam1 associated with the cell membrane in two distinct ways: we observed both ( i ) rapid , transient , binding of single Myo1 molecules to the cell membrane , characterized by low-intensity , single , stepwise , changes in intensity ( Video 1 ) , alongside ( ii ) longer endocytic events that were much brighter and took much longer ( Video 2 ) . The rapid , single-molecule , interactions of Myo1 and Cam1 with the membrane had low mobility ( 0 . 03 µm2 . s−1 ) , ~10 times slower than the diffusion of integral membrane proteins ( Mashanov et al . , 2010 ) . The individual , diffraction-limited fluorescent spots appeared and disappeared at the cell membrane in a single step . The durations of these short single-molecule events ( defined as the period over which individual objects were observed and their paths tracked ) were exponentially distributed with mean lifetime of ~8 s−1 ( n = 152 ) ( Video 1 ) . The distribution of the durations of individual Myo1 events is shown in Figure 3—figure supplement 1 . By contrast , during endocytic events , the fluorescence signal increased gradually ( at a rate corresponding to ~13 molecules . s−1 ) to a peak amplitude corresponding to ~45 molecules of mNeongreen . Myo1 , which persisted for ~6 s ( plateau phase ) , before decaying back to baseline level ( at a rate of ~14 molecules . s−1 ) ( Figure 3B , Video 2 ) . The duration ( Tdur ) of endocytic events ( measured as described in the Materials and methods ) was 13 . 84 s ± 0 . 39 ( mean ± SEM , n = 50 ) ( Figure 3C ) . Although there was significant variation in the maximum mNeongreen . Myo1 intensity ( 2373 ± 155 AU ) , there was no correlation between maximum intensity ( measured during the plateau phase ) and event duration ( not shown ) . The fluorescence intensity dynamics of Cam1 . GFP during endocytic events were similar to those of mNeongreen . Myo1 , but Tdur was significantly shorter for Cam1 . GFP ( p<0 . 0001 ) , 10 . 99 s +/– 0 . 21 ( n = 52 ) while the peak ( plateau ) intensity for Cam1 . GFP was roughly double that measured for mNeongreen . Myo1 and equivalent to ~90 GFP molecules ( Figure 3C ) , consistent with the occupation of both IQ sites within the Myo1 neck region by Cam1 . The briefer event duration observed for Cam1 is best explained by the dissociation of Cam1 from Myo1 before Myo1 leaves the endocytic patch . This process was confirmed by two-color imaging of mNeongreen . myo1 cam1 . mCherry cells , which revealed how Myo1 and Cam1 arrived simultaneously at the endocytic patch , before Cam1 . mCherry disassociated ~3 s before mNeongreen . Myo1 ( Figure 3D , Figure 3—figure supplement 1 ) . Myo1 and Cam1 dynamics in myo1 . S742A cells during endocytosis revealed how Myo1S742A had average assembly and disassembly rates and peak intensity measurements that were identical to those of wild-type Myo1 , yet the duration of the signal ( Tdur ) was 1 . 5 s shorter for Myo1S742A ( 12 . 3 s + /- 0 . 31 n = 67 ) ( Figure 3E and Figure 3—figure supplement 1 ) . Consistent with the in vitro data , the myo1 . S742A mutation did not impact on the ability of Cam1 molecules to bind both IQ motifs , as the average assembly and disassembly rates and the plateau intensity for Cam1 were the same in both wild-type and myo1 . S742A cells . However , Myo1S742A and Cam1 proteins disassociated simultaneously and somewhat earlier during the endocytic event than in otherwise isogenic wild-type cells . Upon cell division , fission yeast cells grow exclusively from the old cell end that existed in the parental cell . At a point during interphase ( called New End Take Off ( NETO ) ) , there is a transition to bipolar growth ( Mitchison and Nurse , 1985 ) . This cell-cycle switch in growth pattern correlates precisely with a parallel redistribution of endocytic actin patches ( Marks and Hyams , 1985 ) . The TIRF imaging data were consistent with widefield , 3D , time-lapse imaging that showed that the lifetimes of Myo1 and Cam1 foci were shorter in myo1 . S742A cells than in myo1+ cells ( Supplementary file 1 Table 1 ) . By contrast , while the myo1 . S742A allele did not affect the accumulation of Cam2 or LifeACT at sites of endocytosis ( Supplementary file 1 Table 1 ) , the rate of endocytosis ( as measured by actin foci lifetimes ) differed significantly ( p<0 . 01 ) between the old and new ends of myo1-S742A cells but not of wild-type cells ( lifetimes at old end and new cell end: 11 . 96 ± 2 . 28 and 11 . 39 ± 1 . 07 for wild-type cells; 14 . 17 ± 3 . 3 and 11 . 09 ± 1 . 29 s for myo1 . S742A cells ( mean ± s . d . ) ) . Therefore , although Myo1S742 phosphorylation does not impact the assembly of Myo1–Cam1 endocytic foci , it regulates myosin-1 to modulate the activity and function of the ensemble of endocytic proteins during bipolar growth . As the myo1 . S742A allele only has affected actin dynamics at the old-cell end during bipolar growth , we examined whether this post-translational modification was subject to cell-cycle-dependent variance . Analysis of extracts from cell-division cycle mutants arrested in G1 ( cdc10 . v50 cells ) prior to NETO ( Marks et al . , 1986 ) or in late G2 ( cdc25 . 22 cells ) after NETO revealed that Myo1S742 is phosphorylated in a cell-cycle-dependent manner ( Figure 4A ) . This was confirmed by monitoring Myo1S742 phosphorylation in cells that were synchronized with respect to cell-cycle progression ( Figure 4B-D ) . These data established that , at its peak in early interphase ( prior to the transition to a bipolar growth pattern ) , approximately half of cellular Myo1 is phosphorylated on S742 , before dropping to undetectable levels by the end of late G2 ( the Cdc25 execution point ) , prior to entry into mitosis . myo1 . S742A cells have a longer average length than wild-type cells ( 9 . 77 ± 1 . 77 µm for myo1+; 13 . 2 ± 2 . 47 µm for myo1 . S742A; t-test >99% significance; n > 500 ) . In addition to the NETO phenotype , a significant proportion of myo1 . S742A cells exhibited significant issues with their ability to maintain , linear , polarized , growth , as 24 . 7% of these cells developed a bent morphology ( i . e . growth deviates by >5° from the longitudinal axis ) ( Figure 4E-F ) . The myo1 . S742A allele did not have an additive effect on the growth-polarity defects associated with cells lacking Tea4 , a polarity determinant protein that plays an important role in integrating actin cytoskeleton function with the regulation of polarised cell growth ( Martin et al . , 2005; Tatebe et al . , 2005 ) ( Figure 5A ) . Consistently , cell-wall staining revealed a significantly higher than normal proportion of myo1 . S742A cells that exhibited monopolar growth ( when compared to equivalent wild type ) , indicating a disruption in the switch from monopolar to bipolar growth ( Figure 4E-F ) . This was confirmed by tracking the cellular distribution of the actin-patch marker , Sla2/End4 , following cell division . Sla2 failed to redistribute to the newly divided end of myo1 . S742A cells during interphase ( Figure 5B ) . This failure of myo1 . S742A cells to switch to bipolar growth , a and restrict growth upon nutrient depletion ( Figure 1E ) is consistent with the reduced growth rate at the end of the log phase and with growth to an overall higher density upon reaching the stationary phase ( Figure 5C ) . We conclude that cell-cycle-dependent changes in Myo1S742 phosphorylation modulate the ability of the myosin lever arm region to regulate endocytosis and polarized growth . Myo1 has been reported to associate with a second calmodulin-like protein , Cam2 , via its second IQ motif ( Sammons et al . , 2011 ) . However , our data indicate that Cam1 occupies both Myo1 IQ motifs during endocytosis . Widefield microscopy revealed that Myo1 and Cam1 dynamics ( Figure 6A ) at endocytic foci differ significantly from Cam2 dynamics at these foci . Cam2 is recruited to sites of endocytosis later than Myo1 and Cam1 , but prior to vesicle scission/budding , whereupon , like CAPZAAcp1 , Sla2 and actin , Cam2 remains associated with laterally oscillating , internalized , endosomes ( Figure 6B–C ) . Similarly , simultaneous imaging of Cam1 and Cam2 in cam1 . mCherry cam2 . gfp cells revealed how each protein localizes to a significant proportion of foci lacking the other calmodulin , thereby highlighting the different timings of the engagement of each molecule with the endocytic machinery ( Figure 6D ) . Finally , although Cam1 recruitment to endocytic foci is abolished when Myo1 is absent ( Figure 6E ) , the intensity , volume and number of Cam2 foci actually increases in the absence of Myo1 ( Figure 6F Supplementary file 1 Table 1 ) , even though the internalization and lateral ‘oscillating’ dynamics of Cam2 and actin were dependent on Myo1 ( Figure 6F & G ) . We assume that this arises from the requirement for prior action of Cam1 for vesicle budding . TIRF imaging revealed that , on average , a total of ~30 Cam2 molecules were recruited to each endocytic focus ( compared to 45 and 90 molecules observed for Myo1 and Cam1 , respectively ) , and that the kinetics of Cam2 recruitment to foci differed significantly to those observed for both Myo1 and Cam1 . The Cam2 signal often increased steadily , before an abrupt decline ( Figure 7A ) , which contrasts with the more gradual ( sigmoidal ) rise and decay in intensity observed for Myo1 and Cam1 ( Figure 3C , E ) . TIRF microscopy ( TIRFM ) confirmed that Cam2 continued to be associated with the endocytic vesicles after they were internalized and their connection with the cell membrane was broken ( Video 3 ) . Background-corrected intensity traces of Cam2 dynamics at the membrane patch before , during , and after the end of endocytosis showed that the signal rapidly dropped to baseline ( <1 s ) ( Figure 7A ) , with the Cam2-labelled vesicles remaining visible close to the membrane but moved inwardly , away from the location of the endocytic event . A large number of these mobile , internalized Cam2-labelled vesicles were seen moving within the cytoplasm with relatively low cytosolic background signal ( Video 3 ) , indicating that much of the Cam2 was associated with endocytic vesicles and remained bound to mature endosomes . We conclude that endocytosis was inhibited , with Cam2 persisting on the endosome while Myo1 remained at the plasma membrane during and after endosome abscission , as previously reported ( Figure 6A , Video 2 ) ( Sirotkin et al . , 2010; Berro and Pollard , 2014; Picco et al . , 2015 ) . Thus , although Cam1 and Cam2 both localize to sites of endocytosis , they appear to do so at different times , and each have different Myo1 dependencies . To correlate Myo1-Cam1 dynamics at sites of endocytosis with the internalization of the mature endosome into the cytoplasm , we followed Cam1 and Cam2 dynamics simultaneously in cam1 . mCherry cam2 . gfp cells ( Video 4 ) . An average curve ( Figure 7B ) , generated from profiles of 65 two-color individual endocytic events , synchronized relative to the Tstart of Cam1 ( see Figure 3B , C ) , shows that Cam2 moves away from the cell surface shortly after Cam1 leaves but before Myo1 leaves , with the time of abscission ( Tscis ) occurring on average 11 . 4 s after the event starts ( Tstart ) . Therefore endosome scission takes place immediately prior to the Myo1 disassembly phase ( Figure 3B ) , and around the time when Cam1 dissociates from Myo1 ( Figure 3C ) . Intriguingly , although the overall distribution of Myo1 and Cam1 appeared to be unaffected in cam2∆ cells , the number , volume and intensity of foci were significantly reduced ( Figure 7C , D; Supplementary file 1 Table 1 ) . In vitro analysis revealed how two Cam2 molecules can associate with the unphosphorylated Myo1IQ12 region ( Figure 2F ) in a process that has two distinct phases . In contrast to Cam1 , in which the two Myo1IQ12 binding events contributed equally to the change in FRET signal , for Cam2 , 70% of the signal change was brought about by a single binding event , associated with an affinity of 1 . 10 µM . The smaller amplitude and tighter binding signal is not accurately measurable , but the combined change in signal is consistent with two binding events . As predicted from sequence analysis , Cam2 failed to associate with calcium ( Figure 2D ) , and its conformation and interactions with Myo1 were insensitive to the divalent cation ( Figure 2G ) . Like Cam1 , Cam2 had a higher affinity for the first IQ motif ( 0 . 4 µM ) than for both IQ1 and IQ2 together , and failed to bind IQ2 alone ( Figure 2E ) . Cam1 calcium binding , as measured by IAANS labeling or by change in Quin-2 fluorescence were unaffected by Cam2 , whereas gel filtration and fluorescence binding assays provided no evidence of a direct physical interaction between the two proteins ( Figure 2—figure supplement 2 ) . Interestingly , a difference in fluorescence amplitudes between Cam1 and Cam2 binding to the IQ12 motif indicated an impact upon the conformation of the lever arm ( Figure 2G ) , providing a potential mechanism to control Myo1 motor activity directly . However , Myo1S742 phosphorylation had no measurable impact upon the dynamics and distribution of Cam2 within S . pombe cells undergoing normal vegetative growth ( Figure 8A; Supplementary file 1 Table 1 ) . Calcium levels within log phase yeast cells are relatively low ( 100–200 nM ) ( Ma et al . , 2011; Miseta et al . , 1999 ) , and so provide conditions that favor the association of Cam1 with Myo1 ( pCa , 5 . 87 ) . Analysis of cell fluorescence indicated the relative abundance of Myo1: Cam1: Cam2 within the S . pombe cell to be 0 . 25: 1 . 56: 1 ( Supplementary file 1 Table 1 ) , which is similar to the ratio defined by quantitative proteomic analysis of 0 . 45: 1 . 56: 1 ( Marguerat et al . , 2012 ) . Similarly , image analysis of Cam1–GFP fluorescence showed how 1 . 7% of Cam1 associated with discrete foci within cells ( Supplementary file 1 Table 1 ) , 40% of which was dependent upon Myo1 , with the majority associating with the SPB ( Figure 6E ) . This indicates that ~0 . 68% of cellular Cam1 associates with Myo1 at dynamic endocytic foci . These relative protein levels , binding affinities and low Ca2+ concentrations favor Cam1 binding to Myo1 , over Cam2 at both IQ sites ( Figure 8B ) , consistent with in vivo observations . Although Ca2+ levels are low during vegetative growth , sporadic prolonged calcium bursts occur upon pheromone release during mating ( Carbó et al . , 2017; Iida et al . , 1990 ) , and levels elevate significantly ( ~10 fold ) during the subsequent meiosis and sporulation ( Suizu et al . , 1995 ) . Cam1 would be less likely to bind to Myo1 in these conditions ( pCa , 5 . 87 ) . We observed that Myo1S742 is phosphorylated in mating and meiotic cells ( Figure 8C ) . Cam2 abundance simultaneously increases significantly in relation to Cam1 upon starvation , mating and entry into meiosis ( Mata and Bähler , 2006; Mata et al . , 2002 ) . These conditions favor interactions between Myo1 and Cam2 over an association of Cam1 with Myo1 ( Figure 8B ) , which is consistent with important roles for both Myo1 and Cam2 at the leading edge of forespore membrane formation during meiosis ( Toya et al . , 2001; Itadani et al . , 2006 ) . Consistent with this prediction , the lifetimes and dynamics of Myo1 , Cam1 and Cam2 foci differ significantly from those observed in vegetative cells ( p<0 . 0001 ) , with foci lasting significantly longer ( >1 min ) in meiotic and sporulating cells ( Supplementary file 1 Table 1 ) . In contrast to vegetative cells , in cells undergoing meiosis and subsequent spore formation , cortical foci containing accumulations of Cam2 and actin ( like those containing like Myo1 and Cam1 ) were less dynamic , lacking any oscillation and remaining in a fixed position , and had a significantly longer lifetime than foci within actively growing cells ( Figure 8D , Supplementary file 1 Table 1 , Videos 5–8 ) . Consistent with this , endocytosis is significantly diminished in fusing and meiotic cells ( Figure 8E ) containing the stable Myo1 , Cam1 , Cam2 or actin foci , indicating that the reduction in foci dynamics minimizes endocytosis in meiotic cells . Finally , we used the myo1 . S742A allele to monitor the impact of Myo1S742 phosphorylation on Myo1 , Cam1 and Cam2 dynamics during meiosis . In contrast to wild-type cells , the lifetime of Myo1 and Cam1 foci were not significantly different to each other in myo1 . S742A cells . In addition , the lifetime of the Myo1 and Cam1 foci in myo1 . S742A cells were significantly reduced when compared to those in the wild type . Cam2 dynamics did not correlate with Myo1 in myo1 . S742A cells , which is in contrast to those in the wild type ( Supplementary file 1 Table 1 ) . Myo1 and Cam1 foci were also seen to be shorter in cam2∆ cells during meiosis , when compared to those in wild type cells ( Supplementary file 1 Table 1 ) . These data indicate that Myo1S742 phosphorylation is required for Cam2 to interact with the Myo1 IQ motif and thereby reduce Myo1 foci dynamics . The majority of Cam2 foci in meiotic cells lacking Myo1S742 phosphorylation remained present in the cell for longer than two mins . Such timing differs significantly from the dynamics of non-phosphorylatable Myo1S742A , indicating that normal Cam1 and Cam2 interactions with Myo1 were abolished in Myo1S742A cells . Consistent with observations of myo1 . S742A cells grown to stationary phase ( Figure 1E ) , heterothallic ( h90 ) nitrogen-starved G1-arrested myo1 . S742A cells failed to inhibit polar growth ( Figure 8F ) . 27 . 9% of mating myo1 . S742A cells continued to grow at their mating ( schmooing ) tips ( vs of myo1+ 1 . 8% cells; n > 100 ) , and meioses frequently produced asci with an abnormal number of unequally sized spores ( Figure 8F , arrowheads ) ( 0 . 9% of myo1+ asci , 13 . 1% of myo1 . S742A asci; n > 100 ) . This spore defect phenotype is reminiscent of the meiotic phenotype of cam2∆ cells ( Itadani et al . , 2006 ) , which supports the view that increases in cellular Ca2+ and Myo1S742 phosphorylation are both key for Cam2 association with and regulation of Myo1 . These data support a model in which changes in calcium levels and TORC2-dependent phosphorylation status provides a simple two-stage mechanism for modulating motor activity . In this mechanism , modification of lever arm conformation and a switch in calmodulin light-chain preference co-ordinate myosin function with the changing environmental and cell-cycle-dependent needs of the cell ( Figure 8B , F ) .
Myosins are subject to diverse modes of regulation , including modulation of the composition of the actin track , changes to cargo and light-chain interactions , and phosphorylation that changes the core physical properties of the motor . Here , we describe a newly discovered mechanism through which phosphorylation of the myosin heavy chain ( Figure 1 ) regulates light chain specificity , and lever arm conformation and flexibility , to impact upon cellular function . During the vegetative life cycle , at basal levels of cellular calcium , S . pombe Myo1 preferentially associates with two molecules of the calcium-regulated calmodulin light-chain Cam1 ( Figures 2 and 3 ) . During early stages of the cell cycle , phosphorylation of the Myo1 neck region ( Figure 8G ) changes the conformation of the Cam1-associated lever arm to moderate motor activity , thereby regulating the rate of endocytosis , and a switch from monopolar to bipolar growth ( Figure 5 ) . There is a significant increase in TORC2 and Gad8 activity upon starvation , which promotes the onset of the meiotic lifecycle ( Hálová et al . , 2013; Laboucarié et al . , 2017; Martín et al . , 2017 ) . Upon starvation , there is an increase in Myo1 serine 472 phosphorylation ( Figure 8 ) , and myo1 . S742A cells fail to arrest growth in response to starvation ( Figures 1E and Figure 8F ) . Phosphorylation of the IQ region , combined with an increase in cytosolic Ca2+ levels observed during G1 , starvation and meiosis , switches light chain preference to favor the recruitment of a single molecule of the calcium-insensitive calmodulin-like Cam2 . However , it is worth noting that there are currently differences of opinion on the relationship between levels of Gad8 activity and cytosolic calcium ( Cohen et al . , 2014 ) . The structures of the IQ region of Myosin-1 and calmodulin ( Lu et al . , 2015 ) suggest that phosphorylation of Myo1S742 is likely to impact Cam2 binding at the 1st IQ position . Furthermore , our data reveal that CaM is unable to associate with IQ2 alone , as occupancy of IQ1 is required before a second CaM can bind to IQ2 ( i . e . regulatory cooperative binding occurs ) . This switch in light-chain occupancy may provide a mechanism to change the stiffness of the Myo1 neck region ( i . e . the ‘lever arm’ ) and might thereby modulate the movement and force that is produced by this region during the acto-myosin ATPase cycle and/or the load-sensitivity of its actin-bound form . Observations within budding yeast indicate that motor activity from a ring of myosins at the lip of the endosome ( Mund et al . , 2018 ) is necessary for endocytic internalization , but the mechanism by which the myosin interacts with actin to facilitate this localized activation is unknown ( Sun et al . , 2006 ) . Here we find that the size of the early endocytic patch determines the number of Myo1 molecules necessary to generate a critical local concentration of Arp2/3-nucleated actin filaments ( Barker et al . , 2007 ) . At the critical concentration , myosin heads are able to interact with actin filaments nucleated from either adjacent Myo1 tails or WASP-activated Arp2/3 complexes , which are tethered to the membrane via molecules such as the Talin-like Sla2 ( Sirotkin et al . , 2005; Sirotkin et al . , 2010 ) . The Myo1 is then primed to act as a tension sensor against the actin filament as it pushes against the membrane of the internalized endosome and grows against the significant 0 . 85 MPa ( 8 . 3 atm ) turgor pressure within the cell ( Minc et al . , 2009 ) . The number of Myo1 molecules at the plasma membrane focus remains constant as the membrane is internalized until 2 s after Cam1 disassociates from Myo1 . The trigger for Cam1 release is unknown , but the speed at which the event takes place indicates that it is likely to be initiated by a rapid localized spike in calcium . This could perhaps be driven by a critical level of membrane deformation coupled to calcium influx , similar to processes proposed for mechano-transduction and the role of mammalian myosin-1 within the stereocilia of the inner ear ( Adamek et al . , 2008; Batters et al . , 2004 ) . Once Cam1 detaches from the Myo1 molecule , the neck loses rigidity , reducing tension between the myosin motor and the actin filament , to promote detachment from F-actin ( Lewis et al . , 2012; Mentes et al . , 2018 ) . Single molecules of Myo1 do not reside for long at the plasma membrane ( off rate is ~8 sec−1 , Video 1 ) , so without an interaction with actin , Myo1 would leave the endocytic patch a second or so after losing its Cam1 light chain . Therefore , after Cam1 release , there is a 2 s delay in the disappearance of Myo1 signal as it disassociates from the endocytic machinery ( Figure 3C , D ) . The conformation and rigidity of the Myo1 lever arm therefore play a key role in modulating the tension-sensing properties of the motor domain . This is consistent with our data showing that wild-type phosphorylation-competent Myo1 resides at the membrane ~1 . 8 s longer than does Myo1 mutant protein that cannot be phosphorylated at serine 742 ( Myo1S742A ) ( Figure 3—figure supplement 1 ) . Phosphorylation-dependent changes in the conformation of the myosin neck provide a simple mechanism to modulate the rate of endocytosis according to the size and needs of the cell . Similarly , in the presence of Ca2+ and Myo1S742 phosphorylation , a single Cam2 resides at the IQ1 motif of the neck ( Figure 8B ) . While bringing about a change in the conformation of the first half of the myosin lever arm ( adjacent to the motor domain ) , the vacant IQ2 motif allows flexibility within the carboxyl half of the neck region . This would provide a relatively tension-insensitive motor that stalls against the actin polymer , which would therefore persist significantly longer at the endocytic foci , as observed to occur here in meiotic cells ( Figure 8D , Supplementary file 1 Table 1 ) . These changes in lever-arm properties change the overall rate of endocytosis , as observed in differences in the time taken for endosomes to internalize within the cytoplasm ( Supplementary file 1 Tables 1 and 2 ) . Thus , phosphorylation-dependent changes in the calcium-regulated conformation and rigidity of the myosin lever arm could provide a universal mechanism for regulating the diverse cytoplasmic activities and functions of myosin motors within all cells .
Cell culture and maintenance were carried out according to Moreno et al . ( 1991 ) using Edinburgh minimal medium with glutamic acid nitrogen source ( EMMG ) unless specified otherwise . Cells were cultured at 25°C unless stated otherwise and cells were maintained as early to mid-log phase cultures for 48 hr before being used for analyses . Genetic crosses were undertaken on MSA plates ( Egel et al . , 1994 ) . All strains used in this study were prototrophs . They are listed in Supplementary file 1 . cam1+ ( SPAC3A12 . 14 ) , cam1 . T6C and cam2+ ( SPAC29A4 . 05 ) genes were amplified as Nde1 - BamH1 fragments from genomic S . pombe DNA using o226/o227 and o393/o394 primers and cloned into pGEM-T-Easy ( Promega , Madison , WI , USA ) . After sequencing , the subsequent genes were cloned into pJC20 ( Clos and Brandau , 1994 ) to generate bacterial calmodulin-expression constructs . DNA encoding for the FRET optimized fluorophores CyPet and YPet ( Nguyen and Daugherty , 2005 ) were each amplified using primers o405/o406 and o403/o404 , respectively . o406 also incorporated DNA at the 3′ end of the CyPet ORF that encodes the first IQ motif of the Myo1 neck region , whereas o404 included DNA encoding a Gly3His6 tag at the 3′ of the YPet ORF . The two DNA fragments were cloned into pGEM-T-Easy in a three-way ligation reaction to generate pGEM-CyPet–Myo1IQ1–YPet . The CyPet–Myo1IQ1–YPet DNA was subsequently sequenced and cloned as a Nde1 - BamH1 fragment into pJC20 ( Clos and Brandau , 1994 ) to generate pJC20CyPet–Myo1IQ1–YPet . Complementary oligonucleotides o425 and o426 were annealed together and ligated into BglII – Xho1 cut pJC20CyPet–Myo1IQ1–YPet to generate pJC20CyPet–Myo1IQ12–YPet . Similarly , the complementary oligonucleotides o429 and o430 were annealed together and subsequently ligated into Sal1-BglII cut pJC20CyPet–Myo1IQ1–YPet and the subsequent Xho1 fragment was excised to generate pJC20CyPet–Myo1IQ2–YPet . Site-directed mutagenesis was carried out using the pJC20CyPet–Myo1IQ12–YPet template and o427 and o428 primers to generate pJC20CyPet–Myo1IQ12S742D–YPet . Complementary oligonucleotides o449 and o450 were annealed together and ligated into Nru1 – Xho1 digested pJC20CyPet–Myo1IQ12S742D–YPet to generate pJC20CyPet–Myo1IQ12S742A–YPet . All plasmids were sequenced upon construction . Strains with fluorophore-tagged alleles of cam1+ and cam2+ were generated as described previously using appropriate templates and primers ( Bähler et al . , 1998 ) . Strains in which the myo1 . S742A , myo1 . S742D , mNeongreen-myo1 , mNeongreen-myo1 . S742A , or mNeongreen-myo1 . S742D alleles replaced the endogenous myo1+ gene ( SPBC146 . 13c ) were generated using a marker switching method ( MacIver et al . , 2003 ) . Oligonucleotides are described in Supplementary file 2 . All recombinant proteins were expressed and purified from BL21 DE3 Escherichia coli cells , except Cam1 proteins for which BL21 DE3 pNatA cells ( Eastwood et al . , 2017 ) were used to allow amino-terminal acetylation ( Figure 2—figure supplement 1 ) . For calmodulin purification , cell lysates were resuspended in Buffer A ( 50 mM Tris , 2 mM EDTA , 1 mM DTT , 0 . 1 mM PMSF ( pH 7 . 5 ) ) and precleared by high-speed centrifugation ( 48 , 500 RCF; 30 min; 4°C ) , before ammonium sulphate was added to the supernatant at 35% saturation , and the mixture incubated for 30 min at 4°C . Precipitated proteins were removed by centrifugation ( 48 , 500 RCF; 30 min; 4°C ) . For Cam1 purifications , the precipitation-cleared supernatant was added to a pre-equilibrated 10 ml phenyl sepharose ( CL-4B ) column ( Buffer B: 50 mM Tris , 1 mM DTT , 1 mM NaN3 , 5 mM CaCl2 ( pH 8 . 0 ) ) , then washed in four volumes of Buffer B before being eluted as fractions in Buffer C ( 50 mM Tris , 1 mM DTT , 1 mM NaN3 , 5 mM EGTA ( pH 8 . 0 ) ) . For Cam2 purification , the precipitation-cleared supernatant underwent a second round of ammonium sulphate precipitation and clearing , and the subsequent supernatant was subjected to isoelectric precipitation ( pH 4 . 3 ) and centrifugation ( 48 , 500 RCF: 30 min; 4°C ) . The resultant pellet was resuspended in Buffer A and heated to 80°C for 5 min , before denatured proteins were removed by centrifugation ( 16 , 000 RCF; 5 min ) . His-tagged proteins were purified in native conditions using prepacked , pre-equilibrated 5 ml Ni2+ columns . Standard immunological methods were used as described ( Harlow and Lane , 1988 ) . Serine 742 phosphorylation-state specific anti-Myo1 antibodies were raised against phosphate-conjugated peptide encompassing Myo1 serine 742 in SPF rabbits ( Eurogentec , Seraing , Belgium ) . These antibodies were subsequently affinity-purified . Protein extracts were prepared and analyzed as described elsewhere ( Baker et al . , 2016 ) . For western blot analysis , anti-Myo1 sera was diluted 1:1000 , whereas Myo1 serine 742 phosphorylation state specific antibodies were used at a dilution of 1:50 . Gel densitometry was undertaken using ImageJ software . All transient kinetics were carried out using a HiTech Scientific DF-61 DX2 Stopped Flow apparatus ( TgK Scientific , Bradford-upon-Avon , UK ) at 20°C . All data were acquired as the average of 3–5 consecutive shots and analyzed using the KineticStudio software supplied with the equipment . Quin-2 fluorescence was excited at 333 nm and a Schott GG445 cut off filter was used to monitor fluorescence above 445 nm . IAANS ( 2- ( 4'- ( iodoacetamido ) anilino ) -naphthalene-6-sulfonic acid ) was excited at 335 nm and fluorescence was monitored through a GG455 filter . For the FRET measurements , CyPet was excited at 435 nm and YPet emission was monitored through a combination of a Wrattan Gelatin No12 ( Kodak ) filter with a Schott GG495 nm filter to monitor fluorescence at 525–530 nm . Emission spectra were obtained using a Varian Cary Eclipse Fluorescence Spectrophotometer ( Agilent Technologies , Santa Clara , CA ) using a 100 µl quartz cuvette . For FRET measurements , samples were excited at 435 nm ( CyPet excitation ) and emission was monitored from 450 to 600 nm with both slits set to 1 nm . Affinity experiments were carried out using 1 µM IQ-FRET protein with varying concentrations of Cam1 or Cam2 in a final volume of 100 µl in analysis buffer of 140 mM KCl , 20 mM MOPS ( pH 7 . 0 ) , with or without 2 mM MgCl2 and with 2 mM of EGTA , CaCl2 or Ca2+-EGTA as required . Distances between FRET fluorophores were calculated as described previously ( Wu and Brand , 1994 ) using an CyPet–YPet R0 value of 53 . 01 . Live cell widefield fluorescence imaging was undertaken as described previously ( Baker et al . , 2016 ) . For total internal reflection fluorescence microscopy ( TIRFM ) , S . pombe cells were immobilized on №1 , Ø 25 mm lectin-coated coverslips and placed into imaging chambers filled with EMMG medium . A previously described custom TIRF microscope ( Mashanov et al . , 2003 ) was used to image individual cells at a rate of 20 fps in either single of dual color mode . Lasers were 488 nm/100 mW and 561 nm/150 mW ( Omicron , Germany ) ; emission filters 525/50 nm and 585/29 nm; dichroic mirror 552 nm ( Semrock , NY ) ; all other lenses and mirrors were from Thorlabs ( NJ ) , except two Ø3 mm mirrors ( Comar Optics , UK ) which directed light in and out of the 100 × 1 . 45 NA objective lens ( Olympus , Japan ) . Sequences of images were captured using one or two iXon897BV cameras ( Andor Technology , UK ) with custom-made acquisition software . 100% laser power ( 488 nm ) was used to image individual mNeongreen–Myo1 and Cam1–GFP molecules . The laser intensity was reduced to 20% during endocytosis imaging experiments to minimize photobleaching . All imaging was undertaken at 23 °С . Immunofluorescence microscopy was performed as described previously ( Hagan and Hyams , 1988 ) , except gluteraldehyde was omitted . Images were captured using the above widefield imaging system . Anti-Myo1 sera ( Attanapola et al . , 2009 ) were used at a dilution of 1:100 , whereas affinity-purified Myo1 serine 742 phosphorylation state-specific antibodies were used at a dilution of 1:10 . Widefield data were analyzed using Autoquant software ( MediaCybernetics , Rockville , MD ) . All 3D image stacks were subjected to blind 3D deconvolution before analysis . Average size , number and cellular distribution of foci were calculated from all foci present within ≥30 cells for each sample examined . Timing of foci events were calculated from kymographs generated in Metamorph software ( Molecular Devices , Sunnyvale , CA ) . The proportion of cells displaying a bent cell phenotype was determined from more than 350 calcofluor ( 1 mg . ml−1 ) stained cells for each strain . Bent cells were defined by a deviation in the direction of growth of >5° from the longitudinal axis . TIRF data analyses , including single-molecule detection and tracking , were undertaken using GMimPro software ( Mashanov and Molloy , 2007 ) . Endocytic events were identified by creating an image representing the standard deviation of each pixel over the whole video sequence ( known as a ‘z-projection’ ) . Bright spots in this image correspond to regions of the yeast cell that showed large intensity fluctuations associated with endocytic activity . Intensity trajectories in these regions of interest ( ROIs ) ( 0 . 5 µm diameter , 5 × 5 pixels ) were saved for future analysis . To correct for local variation in background signal , the average intensity in a region of 1 . 5 µm diameter around the endocytosis site ( but not including the central region of interest ) was subtracted . Data from ROIs that were contaminated by other endocytosis events , occurring in close proximity and close in time , were manually excluded from the analysis . It was critical to identify the start and end of each endocytosis event accurately so that individual traces could be averaged . To facilitate this , the rising and falling phases of the intensity trace were fitted with a straight line ( 60 data points , 3 s duration ) , see Figure 3C for example . The intercept of this line with the baseline intensity gave the tstart and tend values and event duration ( Tdur = Tend Tstart ) ( see Figure 7A ) . The amplitude ( intensity ) of the event Aav was measured at the middle of the event by averaging 60 data points from 5th to 8th second from the Tstart ( gray bar on Figure 3B ) . Intensity traces for each given condition were synchronized to the starting point ( Tstart ) and averaged . ( Except Cam2-GFP traces , which were synchronized using tstart measured from simultaneously acquired Cam1-mCherry signal . ) Similarly , traces were synchronized to their end point ( Tend ) and averaged . The mean duration of the events ( Tdur ) for each condition was then used to reconstruct the mean intensity changes with calculated errors for event amplitude and timing ( Supplementary file 1 Table 2 ) . We used the results of single-molecule imaging experiments to calculate the number of single fluorescent molecules contributing to the spot intensity at a given time . As the falling and rising phases of most events fitted well to a simple linear equation , the slope of the fitted lines was used to estimate the rate of accumulation and dissociation of the fluorescent molecules ( Figure 3C ) . As Cam2-GFP remained bound to the endocytic vesicle , when vesicle scission occurred , intensity fell rapidly to zero as the vesicle diffused from the TIRF evanescent field; the time of scission was defined as Tscis ( Figure 7A ) . Single-particle tracking was performed using GMimPro ( Mashanov and Molloy , 2007 ) ( ASPT module ) so that the paths ( or trajectories ) of individual Myo1 molecules bound to cell membrane could be traced . Trajectories were analyzed to yield mean intensities for individual mNeonGreen- and eGFP-labeled proteins , which could be used to estimate the number of fluorescently tagged molecules associated with each endocytotic event . Intensity-versus-time plots were generated from averages of >30 foci for each protein in each genetic background examined .
Cells respond to changes in their environment through signaling networks that modulate cytoskeleton and membrane organization to coordinate cell-cycle progression , polarized cell growth and multicellular development . Here , we define a novel regulatory mechanism by which the motor activity and function of the fission yeast type one myosin , Myo1 , is modulated by TORC2-signalling-dependent phosphorylation . Phosphorylation of the conserved serine at position 742 ( S742 ) within the neck region changes both the conformation of the neck region and the interactions between Myo1 and its associating calmodulin light chains . S742 phosphorylation thereby couples the calcium and TOR signaling networks that are involved in the modulation of myosin-1 dynamics to co-ordinate actin polymerization and membrane reorganization at sites of endocytosis and polarised cell growth in response to environmental and cell-cycle cues .
The cells of animals , yeast and other eukaryotes all contain a network of filaments known as the actin cytoskeleton that helps to maintain cell shape . When a cell grows , it needs to carefully regulate its actin cytoskeleton to allow the cell to increase in size . A family of proteins known as the myosins bind to actin filaments and act as motors to undertake many cellular activities , which include reorganising the cytoskeleton and moving cargo around the cell . Myosin proteins contain three distinct sections: a motor domain that exerts force against actin filaments , a long region known as the lever arm and a tail region that binds to cargo and other molecules . Cells control the activities of myosin proteins in several ways . For example , a protein known as calmodulin binds to the lever arm of myosin to regulate how long and stiff it is , and it can also alter how the motor domain behaves . Furthermore , proteins known as kinases add molecules called phosphate groups to specific parts of the myosin proteins in response to changes in the environment surrounding the cell . Previous studies reported that one of these sites is within a section of the lever arm region that binds to calmodulin . However , it remained unclear whether the action of the protein kinase affects how calmodulin regulates myosin . Baker et al . used biochemical approaches to study a myosin protein from fission yeast called myosin I . The experiments showed that a protein kinase system called TORC2 added phosphate groups to myosin I affecting how the protein interacted with calmodulin and also alter the shape of the lever arm . This in turn directly affected the ability of the yeast cells to modulate their actin cytoskeleton and grow in response to changes in the nutrients available in the surrounding environment . Myosin proteins and TORC2 play critical roles in many processes happening in healthy cells and these proteins are often badly regulated in many types of cancer . Therefore , these findings may benefit research into human health and disease in the future .