High lumenal chloride in the lysosome is critical for lysosome function
Chloride is the most abundant , soluble anion in the body . Cytosolic chloride can be as low as ~45 mM , while extracellular chloride is ~110 mM ( Treharne et al . , 2006 ) , ( Sonawane et al . , 2002 ) . Chloride concentration values thus span a wide range and yet , in each compartment , it is quite tightly regulated ( Sonawane and Verkman , 2003 ) . For example , in early endosomes it is ~40 mM , late endosomes it is ~70 mM and lysosomes it is ~108 mM ( Hara-Chikuma et al . , 2005; Saha et al . , 2015; Sonawane et al . , 2002 ) . Chloride levels are stringently regulated by chloride channels such as cystic fibrosis transmembrane regulator ( CFTR ) , the CLC family of channels or calcium activated chloride channels , and their dysregulation is directly linked to several diseases including cystic fibrosis , myotonia , epilepsy , hyperekplexia or deafness ( Planells-Cases and Jentsch , 2009 ) . Chloride is largely considered to function as a counter ion only to balance changes in cation fluxes related to signaling ( Scott and Gruenberg , 2011 ) . In one form , this balancing function serves to reset the membrane potential of depolarized neurons through the operation of plasma membrane resident chloride channels/exchangers ( Chen , 2005 ) . In another form , it serves to continuously facilitate organelle acidification , through the operation of intracellular chloride channels ( Stauber and Jentsch , 2013 ) . Despite its importance in cell function , intracellular chloride has never been visualized or quantitated in vivo . DNA nanotechnology has offered creative , functional imaging solutions to quantitate second messengers as well as image organelles in real time in living cells and in genetic model organisms ( Bhatia et al . , 2016; Chakraborty et al . , 2016; Krishnan and Bathe , 2012; Surana et al . , 2015 ) . Here , using a previously developed , pH-independent , DNA-based fluorescent chloride reporter called Clensor , we have made the first measure of chloride in a live multicellular organism , creating in vivo chloride maps of lysosomes in C . elegans . Our investigations reveal that lysosomal chloride levels in vivo are even higher than extracellular chloride levels . Others and we have shown that lysosomes have the highest lumenal acidity and the highest lumenal chloride , among all endocytic organelles ( Saha et al . , 2015; Weinert et al . , 2010 ) . Although lumenal acidity has been shown to be critical to the degradative function of the lysosome ( Appelqvist et al . , 2013; Eskelinen et al . , 2003 ) , the necessity for such high lysosomal chloride is unknown . In fact , in many lysosomal storage disorders , lumenal hypoacidification compromises the degradative function of the lysosome leading to the toxic build-up of cellular cargo targeted to the lysosome for removal , resulting in lethality ( Guha et al . , 2014 ) . Lysosomal storage disorders ( LSDs ) are a diverse collection of ~70 different rare , genetic diseases that arise due to dysfunctional lysosomes ( Samie and Xu , 2014 ) . Dysfunction in turn arises from mutations that compromise protein transport into the lysosome , the function of lysosomal enzymes , or lysosomal membrane integrity ( Futerman and van Meer , 2004 ) . Importantly , for a sub-set of lysosomal disorders like osteopetrosis or neuronal ceroid lipofuscinoses ( NCL ) , lysosomal hypoacidification is not observed ( Kasper et al . , 2005 ) . Both these conditions result from a loss of function of the lysosomal H+-Cl- exchange transporter CLC-7 ( Kasper et al . , 2005 ) . In both mice and flies , lysosomal pH is normal , yet both mice and flies were badly affected ( Poët et al . , 2006; Weinert et al . , 2010 ) . The lysosome performs multiple functions due to its highly fusogenic nature . It fuses with the plasma membrane to bring about plasma membrane repair as well as lysosomal exocytosis , it fuses with the autophagosome to bring about autophagy , it is involved in nutrient sensing and it fuses with endocytic cargo to bring about cargo degradation ( Appelqvist et al . , 2013; Xu and Ren , 2015 ) . To understand which , if any , of these functions is affected by chloride dysregulation , we chose to study genes related to osteopetrosis in the versatile genetic model organism Caenorhabditis elegans . By leveraging the DNA scaffold of Clensor as a natural substrate along with its ability to quantitate chloride , we could simultaneously probe the degradative capacity of the lysosome in vivo and then in cultured mammalian cells . Our findings reveal that depleting lysosomal chloride showed a direct correlation with loss of the degradative function of the lysosome . We found that lowering lysosomal chloride also reduced the level of Ca2+ released from the lysosome . We also observed that reduction of lysosomal chloride inhibited the activity of specific lysosomal enzymes such as cathepsin C and arylsulfatase B . The role of chloride in defective lysosomal degradation has been hypothesized in the past ( Stauber and Jentsch , 2013; Wartosch and Stauber , 2010; Wartosch et al . , 2009 ) , and our studies provide the first mechanistic proof of a broader role for chloride in lysosome function .
In this study we use two DNA nanodevices , called the I-switch and Clensor , to fluorescently quantitate pH and chloride respectively ( Modi et al . , 2009; Saha et al . , 2015 ) . The I-switch is composed of two DNA oligonucleotides . One of these can form an i-motif , which is an unusual DNA structure formed by protonated cytosines ( Gehring et al . , 1993 ) . In the I-switch , intrastrand i-motif formation is used to bring about a pH-dependent conformational change , that leverages fluorescence resonance energy transfer ( FRET ) to create a ratiometric fluorescent pH reporter . ( Figure 1—figure supplement 2 ) The DNA-based chloride sensor , Clensor , is composed of three modules: a sensing module , a normalizing module and a targeting module ( Figure 1a ) ( Saha et al . , 2015; Prakash et al . , 2016 ) . The sensing module is a 12 base long peptide nucleic acid ( PNA ) oligomer conjugated to a fluorescent , chloride-sensitive molecule 10 , 10′-Bis[3-carboxypropyl]−9 , 9′-biacridinium dinitrate ( BAC ) , ( Figure 1a ) ( Sonawane et al . , 2002 ) . The normalizing module is a 38 nt DNA sequence bearing an Alexa 647 fluorophore that is insensitive to Clˉ . The targeting module is a 26 nt double stranded DNA domain that targets it to the lysosome via the endolysosomal pathway by engaging the scavenger receptor or ALBR pathway . In physiological environments , BAC specifically undergoes collisional quenching by Clˉ , thus lowering its fluorescence intensity ( G ) linearly with increasing Clˉ concentrations . In contrast , the fluorescence intensity of Alexa 647 ( R ) remains constant ( Figure 1b ) . This results in R/G ratios of Clensor emission intensities varying linearly with [Clˉ] over the entire physiological regime of [Clˉ] . Since the response of Clensor is insensitive to pH changes , it enables the quantitation of lumenal chloride in organelles of living cells regardless of their lumenal pH ( Saha et al . , 2015 ) . 10 . 7554/eLife . 28862 . 003Figure 1 . Clensor recapitulates its chloride sensing characteristics in vivo . ( a ) Schematic of the ratiometric , fluorescent chloride ( Clˉ ) reporter Clensor . It bears a Clˉ sensitive fluorophore , BAC ( green star ) and a Clˉ insensitive fluorophore , Alexa 647 ( red circle ) ( b ) Calibration profile of Clensor in vitro ( grey ) and in vivo ( red ) given by normalized Alexa 647 ( R ) and BAC ( G ) intensity ratios versus [Cl-] . ( c ) Receptor mediated endocytic uptake of Clensor in coelomocytes post injection in C . elegans . ( d ) Clensor is trafficked by the anionic ligand binding receptor ( ALBR ) from the early endosome ( EE ) to the late endosome ( LE ) and then lysosome ( LY ) . ( e ) Colocalization of ClensorA647 ( red channel ) microinjected in the pseudocoelom with GFP-labeled coelomocytes ( green channel ) . Scale bar: 5 μm . ( f ) Representative fluorescence images of endosomes in coelomocytes labeled with Clensor and clamped at the indicated Clˉ concentrations ( [Cl-] ) . Images are acquired in the Alexa 647 ( R ) and BAC ( G ) channels from which corresponding pseudocolored R/G images are generated . The in vivo calibration profile is shown in ( b ) . Scale bar: 5 µm . Error bars indicate s . e . m . ( n = 15 cells , ≥50 endosomes ) ( g ) In vitro ( grey ) and in vivo ( red ) fold change in R/G ratios of Clensor from 5 mM to 80 mM [Clˉ] . DOI: http://dx . doi . org/10 . 7554/eLife . 28862 . 00310 . 7554/eLife . 28862 . 004Figure 1—figure supplement 1 . ( a ) Quantification of co-localization between DNA nanodevices and GFP in arIs37 worms . Mean of n = 10 cells . ( b ) Coelomocyte labeling efficiency with I-switch ( I4A647 , grey ) and ClensorA647 ( red ) in the absence ( -mBSA ) or presence of 150 or 300 equivalents excess of maleylated bovine serum albumin ( mBSA ) . Error bars indicate s . e . m . ( n = 10 worms ) ( c ) Images showing colocalization of I-switch ( red ) 1 hr post injection in the pseudocoelom of arIs37 [pmyo-3::ssGFP] worms ( green ) . Scale bar: 5 μm . ( d ) RT-PCR analysis of total RNA isolated from C . elegans pre- and post- RNAi . Lanes correspond to PCR-amplified cDNA of the indicated gene product isolated from wild type without RNAi treatment ( denoted by gene name ) and the corresponding dsRNA-fed worms ( denoted as . ‘— ncr-1’ , ‘— chl-6’ etc ) . DOI: http://dx . doi . org/10 . 7554/eLife . 28862 . 00410 . 7554/eLife . 28862 . 005Figure 1—figure supplement 2 . ( a ) Schematic of a DNA nanodevice , I-switch , that functions as a fluorescent pH reporter based on a pH triggered conformational change that is transduced to photonic changes driven by differential fluorescent resonance energy transfer between donor ( D , green ) and acceptor ( A , red ) fluorophores ( b ) pH calibration curve of I4cLYA488/A647 in vivo ( red ) and in vitro ( grey ) showing normalized D/A ratios versus pH . Error bars indicate s . e . m . ( n = 15 cells , ≥60 endosomes ) ( c ) in vitro ( grey ) and in vivo ( red ) fold change in D/A ratios of I4cLYA488/A647 from pH 4 . 0 to pH 7 . 5 . ( d ) Representative images of coelomocytes labelled with I4cLYA488/A647 , clamped at the indicated pH . Images are acquired in donor channel ( D ) , acceptor channel ( A ) from which the respective pseudocolored D/A image is obtained . Scale bar , 5 μm . ( e ) Histograms showing typical spread of D/A ratios of endosomes clamped at pH 4 ( lavender ) and pH 7 . 5 ( salmon ) ; ( n = 10 cells , ≥100 endosomes ) . We use the I switch for pH measurements in the lysosomes of coelomocytes ( Figure 1—figure supplement 2 ) . We first validate the sensor in vivo . For this we first generate an in vivo calibration profile for the sensor using previous methods standardized by our lab ( Surana et al . , 2011 ) . Briefly worms are injected with 500 nM I-switch and were placed in clamping buffers of varying pH 1 hr post injection . Post 75 mins incubation , worms were imaged in the Donor Channel ( D ) and Acceptor Channel ( A ) ( Figure 1—figure supplement 2d ) . On plotting the mean D/A against varying pH we generate a calibration curve which shows that the sensor’s in vivo performance is similar to the in vitro profile ( Figure 1—figure supplement 2b and c ) . DOI: http://dx . doi . org/10 . 7554/eLife . 28862 . 00510 . 7554/eLife . 28862 . 006Figure 1—figure supplement 3 . Selectivity of Clensor ( 200 nM ) in terms of its fold change in R/G from ~0 to 100 mM of each indicated anion unless otherwise indicated . ( ** ~ 0 to 10 mM ) . Since Cl− channels are known to exhibit poor anion selectivity , we investigate the selectivity of Clensor to demonstrate the signal change in cell is attributed by the change of chloride ions level . The selectivity of Clensor to various anions in the form of their sodium salts . This reveals that the sensitivity of Clensor to diverse biologically abundant anions including NO3- and PO43- is negligible . DOI: http://dx . doi . org/10 . 7554/eLife . 28862 . 006 Coelomocytes of C . elegans are known to endocytose foreign substances injected in the body cavity ( Fares and Greenwald , 2001 ) . The polyanionic phosphate backbone of DNA can be co-opted to target it to scavenger receptors and thereby label organelles on the endolysosomal pathway in tissue macrophages and coelomocytes in C . elegans ( Figure 1c and d ) ( Bhatia et al . , 2011; Modi et al . , 2009; Saha et al . , 2015; Surana et al . , 2011 ) . Alexa 647 labelled I-switch ( I4cLY ) and Clensor were each injected in the pseudocoelom of 1-day-old adult worms expressing pmyo-3::ssGFP . In these worms , soluble GFP synthesized in muscles and secreted into the pseudocoelom is actively internalized by the coelomocytes resulting in GFP labeling of the coelomocytes ( Fares and Greenwald , 2001 ) . After 1 hr , both devices quantitatively colocalize with GFP indicating that they specifically mark endosomes in coelomocytes ( Figure 1e and Figure 1—figure supplement 1c ) . Endocytic uptake of DNA nanodevices was performed in the presence of 30 equivalents of maleylated bovine serum albumin ( mBSA ) , a well-known competitor for the anionic ligand binding receptor ( ALBR ) pathway ( Gough and Gordon , 2000 ) . Coelomocyte labeling by I4cLYor Clensor were both efficiently competed out by mBSA indicating that both reporters were internalized by ALBRs and trafficked along the endolysosomal pathway ( Figure 1—figure supplement 1b ) ( Surana et al . , 2011 ) . Next , the functionality of I4cLY and Clensor were assessed in vivo . To generate an in vivo calibration curve for the I-switch I4cLY , coelomocytes labeled with I4cLY were clamped at various pH values between pH 4 and 7 . 5 as described previously and in the supporting information ( Surana et al . , 2011 ) . This indicated that , as expected , the I-switch showed in vitro and in vivo performance characteristics that were extremely well matched ( Figure 1—figure supplement 2b–e ) . To assess the in vivo functionality of Clensor , a standard Clˉ calibration profile was generated by clamping the lumenal [Clˉ] to that of an externally added buffer containing known [Clˉ] as described previously for cultured cells ( Saha et al . , 2015 ) . Endosomes of coelomocytes were labeled with Clensor and fluorescence images were acquired in the BAC channel ( G ) as well as Alexa 647 channel ( R ) as described ( see Materials and methods ) , from which were obtained R/G ratios of every endosome clamped at a specific [Clˉ] ( Figure 1b ) . Endosomal R/G ratios showed a linear dependence on [Clˉ] with ~2 fold change in R/G values from 5 mM to 80 mM [Clˉ] ( Figure 1f and g ) . This is very well matched with its in vitro fold change in R/G over the same regime of [Clˉ] . Before performing quantitative chloride imaging in various mutant nematodes , we checked whether lysosomal targeting of Clensor and the I-switch were preserved in a variety of genetic backgrounds of our interest . Clensor was injected into LMP-1::GFP worms treated with RNAi against specific lysosomal storage disorder ( LSD ) -related genes or genes linked to osteopetrosis . We observed significant colocalization ( >74% ) of Clensor with LMP-1-GFP labeled lysosomes in these coelomocytes ( Figure 3—figure supplement 2b and c ) . Given that both Clensor and the I-switch robustly labeled lysosomes of coelomocytes in wild type worms ( N2 ) , mutants and RNAi knockdowns of a range of LSD-related genes , we explored whether these devices could report on alterations , if any , in the lumenal ionicity in these lysosomes , and thereby possibly report on lysosome dysfunction . As an initial study , we focused on C . elegans nematodes in which genes related to osteopetrosis are mutated . Osteopetrosis results from non-functional osteoclasts that lead to increased bone mass and density due to a failure in bone resorption ( Sobacchi et al . , 2013 ) . In humans , osteopetrosis results from mutations in a lysosomal chloride-proton antiporter CLCN7 , and its auxiliary factor OSTM1 ( Figure 2a ) ( Kornak et al . , 2001; Lange et al . , 2006 ) . It also results from mutations in TCIRG1 , which is the a3 subunit of a lysosomal V-ATPase ( Kornak et al . , 2000 ) and SNX-10 , a sorting nexin implicated in lysosome transport to form the ruffled border of osteoclasts , which is critical for osteoclast function ( Aker et al . , 2012 ) ( Figure 2a ) . Lysosomes of CLC7 knockout mice show normal lumenal pH , yet the mice manifest osteopetrosis as well as neurodegeneration , indicating that despite the apparently normal lumenal milieu , the organelle is still dysfunctional ( Kasper et al . , 2005 ) . The C . elegans homologs for these genes are clh-6 ( CLCN7 ) , F42A8 . 3 ( ostm-1; OSTM1 ) , unc-32 ( TCIRG1 ) and snx-3 ( SNX10 ) ( Figure 2a ) . 10 . 7554/eLife . 28862 . 007Figure 2 . Dysregulation in lysosomal [Cl-] correlates with reduced lysosomal degradation . ( a ) Schematic depicting protein players involved in autosomal recessive osteopetrosis . ( b ) Representative images of Clensor in lysosomes of coelomocytes , in the indicated genetic backgrounds acquired in the Alexa 647 ( R ) and BAC ( G ) channels and their corresponding pseudocolored R/G images . Scale bar , 5 μm . ( c ) Lysosomal Cl- concentrations ( [Cl-] ) measured using Clensor in indicated genetic background ( n = 10 worms , ≥100 lysosomes ) . ( d ) Degradative capacity of lysosomes of coelomocytes in nematodes with the indicated genetic backgrounds as given by the observed half-life of Clensor . Error bars indicate s . e . m . DOI: http://dx . doi . org/10 . 7554/eLife . 28862 . 00710 . 7554/eLife . 28862 . 008Figure 2—figure supplement 1 . ( a ) Representative images of coelomocyte lysosomes labeled with Clensor one hour post injection , in the indicated genetic backgrounds acquired in the Alexa 647 ( R ) and BAC ( G ) channels and the corresponding pseudocolored R/G images . Scale bar , 5 μm . ( b ) Histograms comparing the spread of R/G in coelomocytes in different RNAi background . ( n = 10 cells; >100 lysosomes ) . ( c ) Lysosomal pH measured using I4cLYA488/A647 in the indicated genetic backgrounds ( n = 10 worms , ≥100 lysosomes ) . Figure 2—figure supplement 1a shows representative images of chloride measurements in lysosomes of coelomocytes of worms where various genes related to osteopetrosis are either knocked out or knocked downs . Figure 2—figure supplement 1b shows a histogram comparing the spread of R/G values reporting lysosomal chloride in the indicated genetic backgrounds . We observe that the R/G values are lower in the case of clh-6 mutant and ostm-1 RNAi whereas that of snx-3 is similar to wild type . DOI: http://dx . doi . org/10 . 7554/eLife . 28862 . 00810 . 7554/eLife . 28862 . 009Figure 2—figure supplement 2 . ( a ) Plots showing mean whole cell intensity of I4A647 per coelomocyte , as a function of time , post-injection in indicated genetic backgrounds . Error bars indicate s . e . m . ( n ≥ 10 cells ) . ( b ) Schematic depicting protein players involved in autosomal recessive osteopetrosis . Also shown is clh-4 , a plasma membrane resident chloride channel , that is used as a control . The above figure shows the stability of I switch in the lysosomes of the coelomocytes . Worms were injected with I4A647 and the number of labeled coelomocytes were plotted as a function of time . This gives an exponential decay profile , with progressively more coelomocytes losing their labeling as the DNA device is degraded in the coelomocyte . An exponential is fitted that gives the half-life of the device . In N2 worms , the half-life of the DNA reporters is ~6 . 5 hr , with maximal labelling occurring at 2 hr post injection . DOI: http://dx . doi . org/10 . 7554/eLife . 28862 . 009 Clensor was injected into N2 , clh-6 and unc-32 mutants and RNAi knockdowns of ostm-1 and snx-3 . Chloride concentrations in the lysosomes of each genetic background at 60 min post injection were obtained ( Figure 2b , c and Figure 2—figure supplement 1 ) . N2 worms showed a chloride concentration of ~75 mM . Knocking down clh-6 and ostm-1 resulted in a dramatic decrease of lysosomal chloride to ~45 mM due to the loss of function in chloride transport . Lumenal pH in the lysosomes of these mutants was normal , consistent with findings in both flies and mice ( Saha et al . , 2015; Weinert et al . , 2010 ) . As a control , knocking down a plasma membrane resident CLC channel such as clh-4 showed no effect on either lysosomal chloride or pH ( Schriever et al . , 1999 ) . unc-32c is a non-functional mutant of the V-ATPase a sub-unit , while unc-32f is a hypomorph ( Pujol et al . , 2001 ) . Interestingly , a clear inverse correlation with unc-32 functionality was obtained when comparing their lysosomal chloride levels i . e . , ~55 mM and ~65 mM for unc-32c and unc-32f respectively . Importantly , snx-3 knockdowns showed lysosomal chloride levels that mirrored those of wild type lysosomes . In all genetic backgrounds , we observed that lysosomal chloride concentrations showed no correlation with lysosome morphology ( Figure 3—figure supplement 1d ) . Dead and necrotic bone cells release their endogenous chromatin extracellularly - thus duplex DNA constitutes cellular debris and is physiologically relevant cargo for degradation in the lysosome of phagocytic cells ( Elmore , 2007; Luo and Loison , 2008 ) . Coelomocytes are phagocytic cells of C . elegans , and thus , the half-life of Clensor or I4cLY in these cells constitutes a direct measure of the degradative capacity of the lysosome ( Tahseen , 2009 ) . We used a previously established assay to measure the half-life of I-switches in lysosomes ( Surana et al . , 2013 ) . Worms were injected with 500 nM I4cLY and the fluorescence intensity obtained in 10 cells at each indicated time point was quantitated as a function of time . The I-switch I4cLY had a half-life of ~6 hr in normal lysosomes , which nearly doubled when either clh-6 or ostm-1 were knocked down ( Figure 2d and Figure 2—figure supplement 2 ) . Both unc-32c and unc-32f mutants showed near-normal lysosome degradation capacity , inversely correlated with their lysosomal chloride values ( Figure 2d and Figure 2—figure supplement 2 ) . In this context , data from snx-3 and unc-32f mutants support that high lysosomal chloride is critical to the degradation function of the lysosome . In humans , SNX10 is thought to be responsible for the vesicular sorting of V-ATPase from the Golgi or for its targeting to the ruffled border ( Aker et al . , 2012 ) . Non-functional SNX10 can thus be considered a 'secondary V-ATPase deficiency' , phenocopying a V-ATPase deficiency and showing osteoclasts without ruffled borders due to defective lysosomal transport ( Aker et al . , 2012 ) . Importantly , lysosomal pH in snx-3 knockdowns was compromised by 0 . 3 pH units , while that in unc-32 knockdowns was compromised by 0 . 2 pH units ( Figure 2—figure supplement 1 ) ( Chen et al . , 2012 ) . Yet both these genetic backgrounds showed completely normal lysosomal degradation capacity , that is consistent with their normal lumenal chloride levels , rather than their defective pH levels . This further supports that high lysosomal chloride is a sensitive correlate of the degradative function of the lysosome . Since lysosomal chloride dysregulation correlated with a loss of degradative ability of the lysosome , we wondered whether the converse was true , i . e . , whether lysosomes known to be defective in degradation as seen in lysosomal storage disorders , showed depleted chloride levels . Given that in higher organisms such as mice and humans , high acidity has also been shown to be essential for proper lysosome function ( Mindell , 2012 ) , we measured both lysosomal pH and lysosomal chloride in C . elegans mutants and RNAi knockdowns for a range of genes that are known to cause lysosomal storage disorders . These included a selection of diseases due to dysfunctional enzymes that metabolize sugar derivatives , such as mannose and glycosaminoglycans , as well as lipids such as sphingomyelin and glucosylceramide . Lysosomal pH and chloride measurements were made with I4cLY and Clensor respectively , in each genetic background at 60 min post injection ( Figure 3a and b ) . We found that in C . elegans mutants for Gaucher’s disease , Batten disease , different forms of NCL , MPS VI and Niemann Pick A/B disease , lysosomal chloride levels were severely compromised ( Figure 3a and b ) . Dysfunctional lysosomes showed three types of ion profiles , those where either lysosomal acidity or chloride levels were reduced , and those where both lysosomal acidity and chloride were reduced . The magnitude of proton dysregulation in these defective lysosomes ranged between 1 . 9–2 . 8 µM . However , the magnitude of lysosomal chloride showed a stark drop , decreasing by 19–34 mM in most mutants . Importantly , in mammalian cell culture models for many of these diseases example for Gaucher's disease , NCL , MPS VI , etc . , only pH dysregulation has been reported ( Bach et al . , 1999; Holopainen et al . , 2001; Sillence , 2013 ) . Yet we find that in C . elegans models of these diseases that chloride levels are highly compromised . Chloride decreases by nearly three orders of magnitude more than proton decrease , and the percentage changes of both ions are similar . 10 . 7554/eLife . 28862 . 010Figure 3 . Lysosomal chloride dysregulation is observed in nematode models in several pH-related lysosomal storage disorders . ( a ) Representative pH maps of lysosomes in coelomocytes labelled with a DNA-based pH reporter , I4cLYA488/A647 , in the indicated genetic backgrounds . Images were acquired in the donor ( D , magenta ) and acceptor ( A , cyan ) channels and the corresponding pseudocolored D/A images . Scale bar , 5 μm ( b ) Representative [Cl-] maps of lysosomes acquired in these genetic backgrounds using Clensor . Images are acquired in the Alexa 647 ( R ) and BAC ( G ) channels and the corresponding pseudocolored R/G images are shown . Scale bar , 5 μm . ( c ) Quantification of lysosomal pH and lysosomal Cl- in C . elegans mutants or RNAi knockdowns of genes responsible for the indicated lysosomal storage diseases in humans . Mutants are grouped according to dysregulation only in lysosomal pH ( purple box ) ; only in lysosomal chloride ( green box ) and both lysosomal pH and chloride ( pink box ) for n = 10 worms ( ≥100 lysosomes ) Error bars indicate s . e . m . DOI: http://dx . doi . org/10 . 7554/eLife . 28862 . 01010 . 7554/eLife . 28862 . 011Figure 3—figure supplement 1 . ( a ) Representative images of LMP-1::GFP marked coelomocytes in the background of indicated RNAi . Scale bar: 5 μm . ( b ) Histograms comparing the spread in size of LMP-1::GFP positive vesicles in coelomocytes in the indicated RNAi background ( n = 20 cells ) . ( c ) D/A values obtained for individual pH measurements in lysosomes as a function of area of each vesicle in the indicated genetic background . The linear regression is shown in red . The coefficient of regression ( p = ‒0 . 14 ) shows no correlation ( n > 100 lysosomes ) . ( d ) R/G values obtained for individual Cl- measurement in lysosomes as a function of area of each vesicle in the indicated genetic background . The linear regression line is shown in red . The coefficient of regression ( p= ‒0 . 18 ) shows no correlation ( n > 100 lysosomes ) . A key phenotype observed in cells derived from patients suffering from lysosomal storage disorders ( LSD ) is the presence of enlarged lysosomes ( Filocamo and Morrone , 2011; Platt et al . , 2012 ) . LSD-related gene knockdowns in worms show subtle to no observable whole organismal phenotypes ( de Voer et al . , 2008 ) . However , on knocking down various LSD-related genes in the background of LMP-1::GFP worms we observed that the morphology of LMP-1 positive vesicles were altered and that the coelomocytes contained enlarged lysosomes , some as large as eight microns in diameter ( Figure 3—figure supplement 1 ) . Figure 3—figure supplement 1b represents a plot of the diameter distribution for LMP-1 positive vesicles under each indicated genetic background . Worms with LSD-related gene knockdowns show a broader distribution of vesicle sizes compared to a more tightly regulated size distribution in wild type nematodes or non-LSD related mutants . example for when clh-4 , a plasma membrane resident chloride channel was knocked down , lysosomal morphology is not affected . We carried out pH and chloride measurements for worms from various genetic backgrounds and checked for correlations between lysosomal D/A ( or R/G ) and lysosome size . On plotting D/A values obtained from pH measurements in ncr-1 RNAi worms , against the area of each vesicle; we observe no correlation between the two parameters ( Figure 3—figure supplement 1c ) . A similar observation is seen in the case of chloride R/G measurements ( Figure 3—figure supplement 1d ) . Thus assaying only for lysosome size shows no correlation with lysosome functionality . However lumenal chloride concentration is the best correlate of lysosome dysfunction , irrespective of size . DOI: http://dx . doi . org/10 . 7554/eLife . 28862 . 01110 . 7554/eLife . 28862 . 012Figure 3—figure supplement 2 . ( a ) Worms expressing LMP-1::GFP in coelomocytes were injected with I4cLYA647 or ClensorA647 ( red ) and show maximal colocalization with LMP-1::GFP vesicles ( green ) at 60 min . Scale bar: 5 μm ( b ) Representative images of worms expressing LMP-1::GFP ( green ) in the background of various indicated RNAi's , which were injected with ClensorA647 ( red ) and imaged 60 mins post-injection . Scale bar: 5 μm . ( c ) Quantification of colocalization between the ClensorA647 and GFP in arIs37 worms . Mean of n = 10 cells . To check whether our DNA nanodevices can mark the lysosomes of coelomocytes in wild type worms , we injected 500 nM of I4cLYA647 or ClensorA647 into worms containing LMP1::GFP marker ( Figure 3—figure supplement 2a ) . The worms were imaged 1 hr post injection in the GFP channel ( green ) and in Alexa 647 channel ( red ) to visualize the lysosomal marker and DNA reporter respectively . Merged images show colocalization of DNA devices with the lysosomal marker , similar to previous studies ( Surana et al . , 2011 ) . We then proceeded to validate the devices in the lysosomes in coelomocytes of various LSD-related genes knocked down worms . LMP1::GFP positive worms that were RNAi-ed for indicated genes were injected with ClensorA647 and imaged 1 hr post injection ( Figure 3—figure supplement 2b ) . We observe that , in worms , Clensor reliably marks the lysosomes in all LSD- related gene knockdowns with over 74% colocalization ( Figure 3—figure supplement 2c ) . DOI: http://dx . doi . org/10 . 7554/eLife . 28862 . 01210 . 7554/eLife . 28862 . 013Figure 3—figure supplement 3 . ( a ) Histograms comparing the spread of D/A in coelomocytes in different RNAi background . ( n = 10 cells; >100 lysosomes ) . ( b ) Histograms comparing the spread of R/G in coelomocytes in different RNAi background . ( n = 10 cells; >100 lysosomes ) . DOI: http://dx . doi . org/10 . 7554/eLife . 28862 . 013 To check whether such chloride decrease is observed also in higher organisms , we made pH and chloride measurements in mammalian cell culture models of two relatively common lysosomal storage disorders . Macrophages are a convenient cell culture system to study lysosomal storage disorders as they can be isolated from blood samples and have a lifetime of 3 weeks in culture ( Vincent et al . , 1992 ) . We re-created two widely used murine and human cell culture models of Gaucher’s disease by inhibiting β-glucosidase with its well-known inhibitor conduritol β epoxide ( CBE ) in murine and human macrophages namely , J774A . 1 and THP-1 cells respectively ( Hein et al . , 2013 , 2007; Schueler et al . , 2004 ) . We also recreated common mammalian cell culture models of Niemann-Pick A/B disease by inhibiting acid sphinogomyelinase ( SMPD1 ) in J774A . 1 and THP-1 cells with a widely used inhibitor amitriptyline hydrochloride ( AH ) ( Aldo et al . , 2013; Jones et al . , 2008 ) . First we confirmed that Clensor and our DNA-based pH reporter localized exclusively in lysosomes . In both cell lines , DNA nanodevices ( 500 nM ) were uptaken from the extracellular milieu by the scavenger receptors , followed the endolysosomal pathway and showed quantitative colocalization with lysosomes that were pre-labelled with TMR-Dextran ( Figure 4—figure supplement 3a and b ) . In-cell calibration curves of both pH ( Figure 4—figure supplement 1 ) and chloride reporters ( Figure 4a ) were well matched with their in vitro calibration profiles , indicating that both sensor integrity and performance were quantitatively preserved at the time of making lysosomal pH and chloride measurements in these cells . Both human and murine lysosomes in normal macrophages showed chloride concentrations close to ~118 mM , revealing that lysosomes have the highest chloride levels compared to any other endocytic organelle ( Saha et al . , 2015; Sonawane et al . , 2002 ) . This is nearly 10–15% higher than even extracellular chloride concentrations , which reaches only up to 105–110 mM ( Arosio and Ratto , 2014 ) . 10 . 7554/eLife . 28862 . 014Figure 4 . Lysosomal chloride is substantially depleted in mammalian cell culture models of lysosomal storage diseases . ( a ) Calibration profile of Clensor in cells ( red ) and in vitro ( grey ) showing normalized Alexa 647 ( R ) and BAC ( G ) intensity ( R/G ) ratios versus [Cl-] . Error bars indicate s . e . m . ( n = 20 cells , ≥100 endosomes ) ( b ) Fold change in R/G ratios of Clensor in vitro ( grey ) and in cells ( red ) from 5 mM to 120 mM [Clˉ] ( c ) Representative [Cl-] maps of Clensor in lysosomes of J774A . 1 cells treated with the indicated lysosomal enzyme inhibitor . Images of the Alexa 647 ( R ) channel and pseudocolored R/G images are shown . Scalebar: 10 μm . ( d ) Bar graphs of lysosomal Cl- values obtained in THP-1 and J774A . 1 cells treated with the indicated inhibitors . NPPB ( 50 μM ) , Amitryptiline , AH ( 10 μM ) , Conduritol β-epoxide , CBE ( 400 μM ) were used to model Niemann Pick A/B and Gaucher's diseases in both cell types . Error bars indicate s . e . m . ( n = 10 cells , ≥60 endosomes ) . ( e ) Bar graphs of lysosomal pH values obtained in THP-1 and J774A . 1 cells treated with the indicated inhibitors . NPPB ( 50 μM ) , Amitryptiline , AH ( 10 μM ) , Conduritol β-epoxide , CBE ( 400 μM ) were used to model Niemann Pick A/B and Gaucher's diseases respectively in both cell types . Error bars indicate s . e . m . ( n = 10 cells , ≥50 endosomes ) . DOI: http://dx . doi . org/10 . 7554/eLife . 28862 . 01410 . 7554/eLife . 28862 . 015Figure 4—figure supplement 1 . ( a ) Structure of Oregon Green ( OG ) and schematic of ImLy ( b ) Fluorescence emission spectra of ImLy at the indicated pH obtained using λExOG = 494 nm ( green ) and λEx Atto 647N = 650 nm ( red ) . ( c ) Representative 12% denaturing PAGE stained with Ethidium bromide ( EtBr ) showing conjugation of OG ( green ) to thiol labelled DNA ( purple arrow ) imaged in the ETBr and OG channels respectively ( d ) Representative 20% PAGE showing formation of ImLy ( e ) pH calibration curve of ImLy in cellulo ( red ) and in vitro ( black ) showing normalized G/R ratios versus pH . Error bars indicate s . e . m . ( n = 15 cells , ≥60 endosomes ) ( f ) in vitro and in cellulo fold change in G/R ratios of ImLy from 3 . 5 to pH 6 . 4 . To create a pH sensor for mammalian lysosomes we conjugated a pH sensitive dye ‒ Oregon green ( OG ) which pKa is 4 . 9 ( green chemical structure ) to thiol labelled DNA using a maleimide linker ( blue chemical structure ) . The complementary strand contains a normalizing dye , Atto 647N ( red circle ) . The conjugation of OG was confirmed by 12% denaturing PAGE ( Figure 4—figure supplement 1 ) . Furthermore , the formation of ImLy was confirmed by a gel mobility shift assay using native polyacrylamide gel electrophoresis . We annealed 10 μM of both components in equimolar ratios in 10 mM sodium phosphate buffer , pH 7 . 4 and investigated the sample by PAGE . The formation of ImLy was revealed by its lower electrophoretic mobility with respect to its component single strands ( Figure 4—figure supplement 1d ) . The fluorescence of OG increases with the pH while the fluorescence of normalizing dye Atto 647N remains unchanged ( Figure 4—figure supplement 1b ) . Furthermore , the in cellulo calibration were well matched with their in vitro calibration profiles ( Figure 4—figure supplement 1e‒f ) , indicating that both sensor integrity and performance were quantitatively preserved at the time of making lysosomal pH a measurements in these cells . DOI: http://dx . doi . org/10 . 7554/eLife . 28862 . 01510 . 7554/eLife . 28862 . 016Figure 4—figure supplement 2 . Plots showing mean whole cell intensity ( wci , black line ) of ClensorA647 in cells imaged as a function of indicated times in ( a ) J774A . 1 cells and ( b ) THP-1 cells . Blue lines show wci traces of individual cells as a function of time . Error bars indicate s . e . m . ( n ≥ 25 cells ) . Representative images of ( c ) J774A . 1 cells and ( d ) THP-1 cells at indicated time points . Scale bar: 10 µm . To examine the stability of Clensor in cellulo , we pulse 500 nM Clensor for 30 min and chase for other 60 min to J774A . 1 cells and THP-1 cells . The whole cell intensity of ClensorA647 was unchanged upon 60 min chasing and decrease significantly from 60 min to 90 min for J774A . 1 cell while no significant change was observed for THP-1 cells . The result indicates that the DNA device is stable for lysosomal pH measurement using our protocol ( 30 min pulse follow by 60 min chasing ) . DOI: http://dx . doi . org/10 . 7554/eLife . 28862 . 01610 . 7554/eLife . 28862 . 017Figure 4—figure supplement 3 . ( a ) Representative images showing colocalization of ImLyAT647 with TMR-Dex in J774A . 1 and THP-1 macrophages ( b ) Macrophage labeling efficiency with ClensorA647 ( A647 ) in the absence or presence of 50 equivalents excess of maleylated bovine serum albumin ( mBSA ) in comparison to TMR Dextran . Error bars indicate s . e . m . ( n = 20 cells ) . To verify whether ImLy was endocytosized to lysosome by anionic ligand binding receptor ( ALBR ) pathyway , cells were first pre-labelled with TMR-Dextran a marker of lysosome through fluid phase ( 0 . 5 mg/mL; 1 hr pulse followed by 16 hr chase ) . Cells were then pulse ImLy for 30 min and chased for 60 min . Co-localization of ImLy with TMR-Dextran was observed . Next , competition experiments with 30 equivalents of maleylated BSA ( mBSA ) ‒ a well-known competitor for the anionic ligand , were performed . In the presence of mBSA , only the intensity of ImLy was decreased significantly thus confirming that ImLy was endocytosized via ALBR pathway . DOI: http://dx . doi . org/10 . 7554/eLife . 28862 . 01710 . 7554/eLife . 28862 . 018Figure 4—figure supplement 4 . Co-localization of Clensor ( red ) with lysosomes labelled with TMR–dextran ( green ) in J774A . 1 cells treated with the indicated lysosomal enzyme inhibitors . ( a ) Representative images of lysosomes of J774A . 1 cells labelled with TMR dextran ( TMR; G ) and Clensor ( Alexa 647; R ) in the absence of any inhibitor , 50 μM NPPB , 10 μM Amitryptiline hydrochloride ( AH ) 400 μM of Conduritol β-epoxide ( CBE ) or 10 μM of U18666A . Scale bar , 5 μm ( b ) Pearson's correlation coefficient for the colocalization between Clensor and TMR Dextran . To eliminate the possibility that trafficking defect in LSD may contributes to the observed reduction in lysosomal [Cl-] . We pre-labelled lysosomes with TMR-Dextran ( 0 . 5 mg/mL ) using literature protocols , treated cells with U18666A and Conduritol β-epoxide ( CBE ) to induce Niemann Pick C and Gaucher's cellular phenotype respectively , only then pulse Clensor for 30 min , chased for 60 min , and scored for co-localization with TMR-Dextran . Amitriptyline ( AH ) and NPPB were added 30 min after the pulsing and chasing of Clensor to the lysosome . We observed that Clensor colocalized with lysosomes under each of these conditions , indicating that they do not suffer trafficking defects in these cell culture models of lysosomal storage diseases . DOI: http://dx . doi . org/10 . 7554/eLife . 28862 . 01810 . 7554/eLife . 28862 . 019Figure 4—figure supplement 5 . Representative pH and [Cl-] maps of ImLy and Clensor in lysosomes of J774A . 1 cells in the absence and presence of 10 μM U18666A that gives a cell culture model of NP-C . ( A ) Representative images of lysosomes labeled with Clensor in the Alexa 647 ( R ) channel and pseudocolored R/G images are shown . Scale bar: 10 μm ( B ) Representative images of lysosomes labeled with pH sensor ImLy in the Oregon Green ( OG ) channel ( G ) , Atto647 channel ( R ) and pseudocolored G/R images are shown . Scale bar: 10 μm ( C ) pH and Cl- levels in lysosomes in the presence and absence of 10 μM U18666A . Error bars indicate s . e . m . ( n = 10 cells , ≥50 endosomes ) . To directly compare the results from ncr-1 knockdowns that yielded a worm model of Niemann Pick C in Figure 3 , we also employed U18666A a selective inhibitor for NPC1 protein to induce NP-C cell model . We first confirmed that Clensor could traffic to the lysosome in this cell culture model ( Figure 4—figure supplement 5 ) . We found a dramatic lysosomal hypoacidification and no significant change of lysosomal [Cl-] . This matches our observation in a C . elegans model for NP-C . DOI: http://dx . doi . org/10 . 7554/eLife . 28862 . 019 Treating J774A . 1 cells and THP-1 cells with a global chloride ion channel blocker , such as NPPB ( 5-Nitro-2- ( 3-phenylpropylamino ) benzoic acid ) , lowered lysosomal chloride concentrations to 104 and 106 mM respectively , indicating that Clensor was capable of measuring pharmacologically induced lysosomal chloride changes , if any , in these cells . In Gaucher’s cell culture models , murine and human cells showed a substantial decrease in lysosomal chloride to ~101 mM and ~92 mM respectively . This is a drop of 15–25 mM ( 13‒21% change ) chloride , as compared to a drop of ~10 µM in lysosomal proton concentrations . In Niemann-Pick A/B cell culture models , murine and human macrophages showed an even more dramatic decrease in lysosomal chloride to ~77 mM and ~86 mM respectively . This is also a substantial decrease of 30–40 mM ( 25‒34% change ) chloride , as compared to a drop of ~9 µM in lysosomal proton concentrations . On average in these four cell culture models , we find that the magnitude of chloride concentration decrease is at least 3 orders of magnitude greater than proton decrease , indicating that lysosome dysfunction is easily and sensitively reflected in its lumenal chloride concentrations . A Niemann Pick C cell culture model using the inhibitor U18666A recapitulated our findings in nematode models , where only lysosomal pH , but not Cl- , was altered ( Figure 4—figure supplement 5 ) The ClC family protein CLC-7 is expressed mainly in the late endosomes and lysosomes ( Graves et al . , 2008; Jentsch , 2007 ) . The loss of either ClC-7 or its β-subunit Ostm1 does not affect lysosomal pH in any way , yet leads to osteopetrosis , resulting in increased bone mass , and severe degeneration of the brain and retina ( Lange et al . , 2006 ) . Along with our studies in nematodes , this reveals a role for high chloride in lysosome function that is beyond that of a mere counter-ion in the lysosome . We therefore probed whether it could indirectly affect lysosomal function by affecting lysosomal Ca2+ ( Luzio et al . , 2007; Rodr ? guez et al . , 1997; Shen et al . , 2012 ) . We also considered the possibility that lysosomal chloride could exert a direct effect , where its reduction could impede the function of lysosomal enzymes thus affecting its degradative capacity ( Baccino et al . , 1975; Cigic and Pain , 1999; Maurus et al . , 2005; Wartosch and Stauber , 2010 ) ( Figure 5a ) . 10 . 7554/eLife . 28862 . 020Figure 5 . ( a ) Schematic of potential roles for lysosomal chloride . Cl- ions can regulate lysosomal Ca2+ and/or affect lysosomal enzyme function . ( b ) Representative traces of Glycyl-L-phenylalanine 2-naphthylamide ( GPN ) ( 400 μM ) triggered lysosomal Ca2+ release in J774A . 1 cells ratiometrically imaged using Fura-2 ( F340/F380 ) in the presence and absence of 50 μM NPPB . ( c ) Representative traces of ML-SA1 ( 20 μM ) triggered lysosomal Ca2+ release in J774A . 1 cells ratiometrically imaged using Fura-2 ( F340/F380 ) in the presence and absence of 50 μM NPPB . ( d ) Quantification of lysosomal Ca2+ release from b ) and c ) given by ( Ft-F0/F0 ) ( ΔFura-2 ) for n = 15 cells . ( e ) Representative images of lysosomes of J774A . 1 cells labelled with TMR dextran ( TMR; G ) and LysoTracker Red ( LyT; R ) in the presence or absence of 50 μM NPPB , 200 μM GPN or 1 mM NH4Cl . Scale bar , 5 μm ( f ) Quantification of LysoTracker Red release from ( e ) ( n = 50 cells ) . ( g ) Quantification of activity of the enzymes arylsulfatase B ( ARSB ) and Cathepsin L ( Cath L ) in J774A . 1 cells in the absence and presence of 50 μM NPPB ( n = 70 cells ) . Error bars indicate s . e . m . P values are as follows; ** = p<0 . 001 , *** = p<0 . 0001 , n . s = non significant . DOI: http://dx . doi . org/10 . 7554/eLife . 28862 . 02010 . 7554/eLife . 28862 . 021Figure 5—figure supplement 1 . Representative fluorescence images of cleaved substrates of ARSB and cathepsin L ( E ) , DAPI ( D ) merge of E and D channels and respective pseudocolour E/D maps of J774A . 1 cells with and without 50 µM NPPB . Scale bar: 5 μm . To demonstrate chloride ion levels directly influence lysosome function , 50 µM of chloride channel inhibitor – NPPB was employed to decrease lysosomal chloride without affecting lysosomal pH ( Figure 5—figure supplement 1 ) . Upon incubation of 50 µM of NPPB for 30 min , the activity of arylsulfatase B ( ARSB ) was reduced ca . 50% while the activity of its upstream enzyme cathepsin L which activates cathepsin C , ( Dahl et al . , 2001 ) is still unchanged . It reveals that decrease of chloride ions level directly attenuate cathepsin C activity . DOI: http://dx . doi . org/10 . 7554/eLife . 28862 . 02110 . 7554/eLife . 28862 . 022Figure 5—figure supplement 2 . ( a ) Lysosomal pH and ( b ) chloride levels measured by ImLy and Clensor in J774A . 1 cells with increasing concentrations of NPPB . Here we show that by varying doses of NPPB that at < 100 µM of NPPB , we can selectively reduce lysosomal Cl- without changing in lysosomal pH . Consequently all further studies that use NPPB do so at 50 µM . DOI: http://dx . doi . org/10 . 7554/eLife . 28862 . 022 Lysosomes are also intracellular Ca2+ stores with free Ca2+ ranging between ~400–600 µM ( Christensen et al . , 2002; Lloyd-Evans et al . , 2008 ) . The principal Ca2+ channel responsible for lysosomal Ca2+ release is Mucolipin TRP channel 1 ( TRPML1 ) . We therefore sought to estimate lysosomal Ca2+ by measuring Ca2+ that is released from the lysosome using two different triggers under conditions of normal and reduced lysosomal Cl- . Glycyl-L-phenylalanine 2-naphthylamide ( GPN ) is a substrate for Cathepsin C , which when added to cells , gets cleaved in the lysosome to release naphthylamine that is known to compromise the integrity of the lysosomal membrane , leading to a leakage of ions such as Ca2+ into the cytosol ( Berg et al . , 1994; Jadot et al . , 1984; Morgan et al . , 2011 ) . This has been used to induce lysosomal Ca2+ release . The cytosol of J774A . 1 cells are labeled with ~3 µM Fura2-AM to ratiometrically image cytosolic Ca2+ elevation upon its release , if at all , from the lysosome . After addition of 400 µM GPN , cells were continuously imaged ratiometrically over 15–20 mins . Shortly after GPN addition , a burst of Ca2+ was observed in the cytosol , corresponding to released lysosomal Ca2+ ( Figure 5b ) . When the same procedure was performed on cells that had been incubated with 50 µM NPPB that reduces lysosomal Cl- , the amount of lysosomal Ca2+ released was significantly reduced ( Figure 5b–d ) We then performed a second , more targeted way to release lysosomal Ca2+ into the cytosol , by using 20 µM ML-SA1 which specifically binds to and opens the TRPML1 channel on lysosomes ( Shen et al . , 2012 ) . We found that when lysosomal Cl- was reduced with NPPB , lysosomal Ca2+ release into the cytosol was near negligible ( Figure 5c–d ) . Taken together this indicates that high lysosomal Cl- is necessary for effective lysosomal Ca2+ release , possibly by affect lysosomal Ca2+ accumulation . We next investigated whether reducing lysosomal chloride directly impacted the activity of any lysosomal enzymes . In vitro enzymology of Cathepsin C , a lysosome-resident serine protease has revealed that increasing Cl- increased its enzymatic activity ( Cigic and Pain , 1999; McDonald et al . , 1966 ) . Further , the crystal structure of Cathepsin C shows bound chloride ions close to the active site ( Cigic and Pain , 1999; Turk et al . , 2012 ) . We therefore used GPN cleavage to probe Cathepsin C activity in the lysosome upon reducing Cl- with NPPB . GPN cleavage by Cathepsin C releases naphthylamine which compromises lysosomal membrane integrity leading to proton leakage from the lysosome into the cytosol . This hypoacidifies the lysosomes resulting in reduced LysoTracker labeling as the labeling efficiency of the latter is directly proportional to compartment acidity . Lysosomes are pre-labeled with TMR-Dextran , and LysoTracker intensities are normalized to the fluorescence intensity of TMR-Dextran , given as G/R . Hypoacidifying lysosomes by addition of 1 mM NH4Cl indeed reduced LysoTracker labeling , as expected ( Figure 5e–f ) . A similar effect was also obtained upon GPN addition . The presence or absence of NPPB showed no change in LysoTracker labeling in cells ( Figure 5e–f ) , indicating that NPPB by itself caused no alteration in lysosomal pH . However , when GPN was added to NPPB treated cells LysoTracker staining was remarkably well preserved ( Figure 5e and f ) indicating preservation of lysosomal membrane integrity because GPN was no longer effectively cleaved by Cathepsin C when lysosomal Cl- was reduced . Unlike other cathepsins , Cathepsin C does not undergo autoactivation but requires processing by Cathepsin L and Cathepsin S to convert it into active Cathepsin C ( Dahl et al . , 2001 ) . We measured the activity of the upstream cathepsins such as Cathepsin L using fluorogenic substrates in the presence and absence of NPPB ( Figure 5g , Figure 5—figure supplement 1 ) . We observed no effect of chloride levels on Cathepsin L activity . This indicates that low Cathepsin C activity is not due to decreased amounts of mature Cathepsin C in the lysosome , but rather , reduced activity of mature Cathepsin C ( Figure 5g , Figure 5—figure supplement 1 ) . Based on reports suggesting that arylsulfatase B activity was also affected by low chloride ( Wojczyk , 1986 ) , we similarly investigated a fluorogenic substrate for arylsulfatase and found that NPPB treatment impeded arylsulfatase cleavage in the lysosome . Taken together , these results suggest that high lysosomal chloride is integral to the activity of key lysosomal enzymes and that reducing lysosomal chloride affects their function . The lysosome is the most acidic organelle within the cell . This likely confers on it a unique ionic microenvironment , reinforced by its high lumenal chloride , that is critical to its function ( Xu and Ren , 2015 ) . Using a DNA-based , fluorescent reporter called Clensor we have been able to create quantitative , spatial maps of chloride in vivo and measured lysosomal chloride . We show that , in C . elegans , lysosomes are highly enriched in chloride and that when lysosomal chloride is depleted , the degradative function of the lysosome is compromised . Intrigued by this finding , we explored the converse: whether lysosomes that had lost their degradative function – as seen in lysosomal storage disorders - showed lower lumenal chloride concentrations . In a host of C . elegans models for various lysosomal storage disorders , we found that this was indeed the case . In fact , the magnitude of change in chloride concentrations far outstrips the change in proton concentrations by at least three orders of magnitude . To see whether chloride dysregulation correlated with lysosome dysfunction more broadly , we studied murine and human cell culture models of Gaucher’s disease , Niemann-Pick A/B disease and Niemann Pick C . We found that in mammalian cells too , lysosomes are particularly rich in chloride , surpassing even extracellular chloride levels . Importantly , chloride values in all the mammalian cell culture models revealed magnitudes of chloride dysregulation that were similar to that observed in C . elegans . Our findings suggest more widespread and as yet unknown roles for the single most abundant , soluble physiological anion in regulating lysosome function . Decrease in lysosomal chloride impedes the release of calcium from the lysosome implicating an interplay between these two ions in the lysosome . It is also possible that chloride accumulation could facilitate lysosomal calcium enrichment through the coupled action of multiple ion channels . The ability to quantitate lysosomal chloride enables investigations into the broader mechanistic roles of chloride ions in regulating multiple functions performed by the lysosome . As such , given that chloride dysregulation shows a much higher dynamic range than hypoacidification , quantitative chloride imaging can provide a much more sensitive measure of lysosome dysfunction in model organisms as well as in cultured cells derived from blood samples that can be used in disease diagnoses and screening applications .
All fluorescently labeled oligonucleotides were HPLC-purified and obtained from IBA-GmBh ( Germany ) and IDT ( Coralville , IA , USA ) . Unlabeled oligonucleotides were purchased from IDT ( Coralville , IA , USA ) . The peptide nucleic acids ( PNA ) oligomer , P was synthesized using standard solid phase Fmoc chemistry on Nova Syn TGA resin ( Novabiochem , Germany ) using analytical grade reagents ( Applied Biosystems , USA ) , purified by reverse phase HPLC ( Shimadzu , Japan ) as previously reported and stored at −20°C until further use ( Prakash et al . , 2016 ) . Bovine serum albumin ( 66 kDalton ) , nigericin , valinomycin , monensin , chloride ionophore I , Isopropyl β-D-1-thiogalactopyranoside ( IPTG ) , amitriptyline hydrochloride , 5-nitro-2- ( 3-phenylpropylamino ) benzoic acid ( NPPB ) and conduritol β epoxide ( CBE ) were obtained from Sigma ( USA ) . LysoTracker Deep Red , TMR-Dextran ( 10 kDa ) and Oregon Green 488 maleimide was obtained from Molecular Probes , Invitrogen ( USA ) . Lysosomal enzyme kits namely lysosomal sulfatase assay kit was purchased from Marker Gene ( USA ) ; Magic Red Cathepsin L assay kit from Immunochemistry Technologies . Gly-Phe β-naphthylamide was purchased from Santa Cruz Biotechnology ( USA ) . All other reagents were purchased from Sigma-Aldrich ( USA ) unless otherwise specified . BSA was maleylated according to a previously published protocol ( Haberland and Fogelman , 1985 ) . Trizol was purchased from Invitrogen ( U . S . A . ) . All oligonucleotides were ethanol precipitated and quantified by their UV absorbance . For I-switch ( I4cLYA488/A647 ) sample preparation , 5 μM of I4 and I4′ were mixed in equimolar ratios in 20 mM potassium phosphate buffer , pH 5 . 5 containing 100 mM KCl . The resulting solution was heated to 90°C for 5 min , cooled to the room temperature at 5°C/15 mins and equilibrated at 4°C overnight . Samples were diluted and used within 7 days of annealing . A sample of Clensor was similarly prepared using HPLC purified oligonucleotides and PNA oligomer at a final concentration of 10 μM by mixing D1 , D2 and P ( see Table S1 for sequence information ) in equimolar ratios in 10 mM sodium phosphate buffer , pH 7 . 2 and annealed as described above . For ImLy , Oregon Green maleimide was first conjugated to the thiol labeled oligonucleotide ( Hermanson , 2008 ) . Briefly , to 10 µM thiol labelled oligonucleotide in HEPES pH 7 . 4 , 500 µM of TCEP ( tris-carboxyethylphosphine ) was added to reduce the disulfide bonds . After 1 hr at room temperature , 50 µM Oregon Green Maleimide was added and the reaction was kept overnight at room temperature . The reaction mixture was purified using an Amicon cutoff membrane filter ( 3 kDa , Millipore ) to remove unreacted dye ( Figure 4—figure supplement 1 ) . A sample of ImLy was similarly prepared using HPLC purified oligonucleotides at a final concentration of 5 μM by mixing ImLY OG and ImLYAT647 ( see Table S1 for sequence information ) in equimolar ratios in 10 mM sodium phosphate buffer , pH 7 . 2 and annealed as described above . Prior to use , all buffer stock solutions were filtered using 0 . 22 μm disk filters ( Millipore , Germany ) . Standard methods were followed for the maintenance of C . elegans . Wild type strain used was the C . elegans isolate from Bristol , strain N2 ( Brenner , 1974 ) . Strains used in the study , provided by the Caenorhabditis Genetics Center ( CGC ) , are RRID:WB-STRAIN:RB920 clh-6 ( ok791 ) , RRID:WB-STRAIN:FF451 unc-32 ( f131 ) , RRID:WB-STRAIN:CB189 unc-32 ( e189 ) , RRID:WB-STRAIN:MT7531 ppk-3 ( n2835 ) , RRID:WB-STRAIN:VC3135 gba-3 ( gk3287 ) , RRID:WB-STRAIN:VC183 ppt-1 ( gk139 ) , and RRID:WB-STRAIN:XT7 cln-3 . 2 ( gk41 ) I; cln-3 . 3 ( gk118 ) cln-3 . 1 ( pk479 ) . Transgenics used in this study , also provided by the CGC , are RRID:WB-STRAIN:GS1912 arIs37 [pmyo-3::ssGFP] , a transgenic strain that expresses ssGFP in the body wall muscles , which is secreted in the pseudocoelom and endocytosed by coelomocytes and RRID:WB-STRAIN:RT258 pwIs50 [lmp-1::GFP + Cb-unc-119 ( + ) ] , a transgenic strain expressing GFP-tagged lysosomal marker LMP-1 . Genes , for which mutants were unavailable , were knocked down using Ahringer library based RNAi methods ( Kamath and Ahringer , 2003 ) . The RNAi clones used were: L4440 empty vector control , ncr-1 ( clone F02E8 . 6 , Ahringer Library ) , ostm1 ( clone F42A8 . 3 , Ahringer Library ) , snx-3 ( clone W06D4 . 5 , Ahringer Library ) , manba ( clone C33G3 . 4 , Ahringer Library ) , aman-1 ( clone F55D10 . 1 , Ahringer Library ) , sul-3 ( clone C54D2 . 4 , Ahringer Library ) , gba-3 ( clone F11E6 . 1 , Ahringer Library ) and asm1 ( clone B0252 . 2 , Ahringer Library ) . Coelomocyte labeling and competition experiments were carried out with I4cLYA647 , and ClensorA647 as described previously by our lab ( Surana et al . , 2011 ) . For microinjections , the samples were diluted to 100 nM using 1X Medium 1 ( 150 mM NaCl , 5 mM KCl , 1 mM CaCl2 , 1 mM MgCl2 , 20 mM HEPES , pH 7 . 2 ) . Injections were performed , in the dorsal side in the pseudocoelom , just opposite to the vulva , of one-day old wild type hermaphrodites using an Olympus IX53 Simple Inverted Microscope ( Olympus Corporation of the Americas , Center Valley , PA ) equipped with 40X , 0 . 6 NA objective , and microinjection setup ( Narishige , Japan ) . Injected worms were mounted on 2 . 0% agarose pad and anesthetized using 40 mM sodium azide in M9 buffer . In all cases labeling was checked after 1 hr incubation at 22°C . I4cLYA647 or ClensorA647 sample was diluted to 100 nM using 1X Medium 1 and injected in 10 arIs37 [pmyo-3::ssGFP] hermaphrodites as described previously by our lab ( Surana et al . , 2011 ) . Imaging and quantification of the number of coelomocytes labeled , after 1 hr of incubation , was carried out on the Leica TCS SP5 II STED laser scanning confocal microscope ( Leica Microsystems , Inc . , Buffalo Grove , IL ) using an Argon ion laser for 488 nm excitation and He-Ne laser for 633 excitation with a set of dichroics , excitation , and emission filters suitable for each fluorophore . Cross talk and bleed-through were measured and found to be negligible between the GFP/Alexa 488/BAC channel and Alexa 647 channel . Bacteria from the Ahringer RNAi library expressing dsRNA against the relevant gene was fed to worms , and measurements were carried out in one-day old adults of the F1 progeny ( Kamath and Ahringer , 2003 ) . RNA knockdown was confirmed by probing mRNA levels of the candidate gene , assayed by RT-PCR . Briefly , total RNA was isolated using the Trizol-chloroform method; 2 . 5 μg of total RNA was converted to cDNA using oligo-dT primers . 5 μL of the RT reaction was used to set up a PCR using gene-specific primers . Actin mRNA was used as a control . PCR products were separated on a 1 . 5% agarose-TAE gel . Genes in this study that were knocked down by RNAi correspond to clh-6 , ncr-1 and ostm-1 that showed expected 1 . 1 kb ( clh-6 ) ; 1 . 1 kb ( ncr-1 ) ; 0 . 9 kb ( ostm-1 ) etc ( Figure 1—figure supplement 1 ) . Fluorescence spectra were measured on a FluoroMax-4 Scanning Spectrofluorometer ( Horiba Scientific , Edison , NJ , USA ) using previously established protocols ( Modi et al . , 2009; Saha et al . , 2015 ) . Briefly , I4cLYA488/A647 was diluted to 50 nM in 1X pH clamping buffer of desired pH for all in vitro fluorescence experiments . All samples were vortexed and equilibrated for 30 min at room temperature . The samples were excited at 488 nm and emission collected between 505–750 nm . A calibration curve was obtained by plotting the ratio of donor emission intensity ( D ) at 520 nm and acceptor intensity ( A ) at 669 nm ( for A488/A647 ) as a function of pH . Mean of D/A from three independent experiments and their s . e . m were plotted for each pH value . For in vitro calibration of ImLy , 50 nM of the sensor is diluted into 1X pH clamping buffer of desired pH . Oregon Green and Atto 647N are excited at 490 nm and 645 nm respectively . Emission spectra for Oregon Green and Atto 647N were collected between 500–550 nm and 650–700 nm respectively . A calibration curve was obtained by plotting the ratio of Oregon Green ( G ) at 520 nm and Atto 647N ( R ) at 665 nm ( for G/R ) as a function of pH . Mean of G/R from three independent experiments and their s . e . m were plotted for each pH value . For chloride measurements , 10 μM stock of Clensor was diluted to a final concentration of 200 nM using 10 mM sodium phosphate buffer , pH 7 . 2 and incubated for 30 min at room temperature prior to experiments . BAC and Alexa 647 were excited at 435 nm for BAC and 650 nm for Alexa 647 respectively . Emission spectra of BAC and Alexa 647 were collected between 495–550 nm and 650–700 nm respectively . In order to study the chloride sensitivity of Clensor , final chloride concentrations ranging between 5 mM to 80 mM were achieved by addition of microliter aliquots of 1 M stock of NaCl to 400 μL of sample . Emission intensity of BAC at 505 nm ( G ) was normalized to emission intensity of Alexa 647 at 670 nm ( R ) . Fold change in R/G ratio was calculated from the ratio of R/G values at two specific values of [Clˉ] , either 5 mM and 80 mM or 5 mM and 120 mM as mentioned in the text . Clamping and real time measurement experiments were carried out with I4cLYA488/A647 as described by our lab previously ( Modi et al . , 2009; Surana et al . , 2011 ) . For microinjections , the I-switch sample was diluted to 500 nM using 1X Medium 1 . Briefly , worms were incubated at 22˚C for 1 hr post microinjection and then immersed in clamping buffers ( 120 mM KCl , 5 mM NaCl , 1 mM MgCl2 , 1 mM CaCl2 , 20 mM HEPES ) of varying pH , containing 100 µM nigericin and 100 µM monensin . In order to facilitate entry of the buffer into the body , the cuticle was perforated at three regions of the body using a microinjection needle . After 75 mins incubation in the clamping buffer , coelomocytes were imaged using wide field microscopy . Three independent measurements , each with 10 worms , were made for each pH value . Chloride clamping and real time measurements were carried out using Clensor . Worms were injected with 2 μM of Clensor and incubated at 22˚C for 2 hr . To obtain the chloride calibration profile , the worms were then immersed in the appropriate chloride clamping buffer containing a specific concentration of chloride , 100 μM nigericin , 100 μM valinomycin , 100 μM monensin and 10 μM chloride ionophore I for 45 mins at room temperature . Chloride calibration buffers containing different chloride concentrations were prepared by mixing the 1X chloride positive buffer ( 120 mM KCl , 20 mM NaCl , 1 mM CaCl2 , 1 mM MgCl2 , 20 mM HEPES , pH , 7 . 2 ) and 1X chloride negative buffer ( 120 mM KNO3 , 20 mM NaNO3 , 1 mM Ca ( NO3 ) 2 , 1 mM Mg ( NO3 ) 2 , 20 mM HEPES , pH 7 . 2 ) in different ratios . For real-time lysosomal pH or chloride measurements , 10 hermaphrodites were injected with I4cLYA488/A647 or Clensor respectively and incubated at 22°C for 1 hr . Worms were then anaesthetized and imaged on a wide field inverted microscope for pH measurements and confocal microscope for chloride measurements . Mouse alveolar macrophage J774A . 1 cells were a kind gift from Prof Deborah Nelson , Department of Pharmacological and Physiological Sciences , the University of Chicago , cultured in Dulbecco’s Modified Eagle’s Medium/F-12 ( 1:1 ) ( DMEM-F12 ) ( Invitrogen Corporation , USA ) containing 10% heat inactivated Fetal Bovine Serum ( FBS ) ( Invitrogen Corporation , USA ) . THP-1 monocyte cell line was obtained from late Professor Janet Rowley’s Lab at the University of Chicago . Cells were cultured in RPMI 1640 containing 10% heat-inactivated FBS , 10 mM HEPES , 2 mM glutamine , 100 U/ml penicillin , and 100 μg/ml streptomycin , and maintained at 37°C under 5% CO2 . All reagents and medium were purchased from ( Invitrogen Corporation , USA ) . THP-1 monocytic cells were differentiated into macrophages in 60 mm dishes containing 3 ml of the RPMI 1640 medium containing 10 nM PMA over 48 hr . These cells are not on the list of commonly misidentified cell lines maintained by the International Cell Line Authentication Committee . The sources of each cell line used in this study are as mentioned above and were used directly by us without additional authentication beyond that provided by the sources . All cells were regularly checked for mycoplasma contamination and were found to be negative for contamination as assayed by DAPI staining . Chloride clamping and measurements were carried out using Clensor using a previously published protocol from our lab ( Saha et al . , 2015 ) . J774A . 1 and THP-1 cells were pulsed and chased with 2 μM of Clensor . Cells are then fixed with 200 μL 2 . 5% PFA for 2 min at room temperature , washed three times and retained in 1X PBS . To obtain the intracellular chloride calibration profile , perfusate and endosomal chloride concentrations were equalized by incubating the previously fixed cells in the appropriate chloride clamping buffer containing a specific concentration of chloride , 10 μM nigericin , 10 μM valinomycin , and 10 μM tributyltin chloride ( TBT-Cl ) for 1 hr at room temperature . Chloride calibration buffers containing different chloride concentrations were prepared by mixing the 1X chloride positive buffer ( 120 mM KCl , 20 mM NaCl , 1 mM CaCl2 , 1 mM MgCl2 , 20 mM HEPES , pH , 7 . 2 ) and 1X - chloride negative buffer ( 120 mM KNO3 , 20 mM NaNO3 , 1 mM Ca ( NO3 ) 2 , 1 mM Mg ( NO3 ) 2 , 20 mM HEPES , pH 7 . 2 ) in different ratios . For real-time chloride measurements , cells are pulsed with 2 μM of Clensor followed by a 60 min chase . Cells are then washed with 1X PBS and imaged . To see whether Clensor can detect changes in Clˉ accumulation under perturbed conditions , we treated cells with 50 µM NPPB , which is a well-known non-specific Clˉ channel blocker . Cells were labeled with 2 μM Clensor for 30 mins and chased for 30 mins at 37°C . The cells were then chased for 30 mins in media containing 50 µM NPPB and then imaged . To estimate the chloride accumulation in the lysosomes of Gaucher’s Disease in two cell models that is murine J774A . 1 and human THP-1 cells , glucosylceramide storage was induced catalytically inactivating the enzyme acid β-glucosidase , using its well-known inhibitor conduritol β epoxide ( CBE ) ( Grabowski et al . , 1986; Schueler et al . , 2004 ) . These are both well-documented murine and human cell culture models of Gaucher's disease . Macrophage cells were cultured with 400 µM CBE for 48 hr . Cells were then pulsed and chased with 2 μM Clensor as previously described . To estimate chloride accumulation in the lysosomes of Niemann Pick A/B disease , the same murine and human cell lines were used , and the activity of acid sphingomyelinase ( ASM ) in these macrophage cell lines was inhibited using the well-known inhibitor , amitriptyline hydrochloride ( Beckmann et al . , 2014; Kornhuber et al . , 2010 ) . Cells were labeled with 2 μM Clensor for 30 mins and chased for 30 mins at 37°C . The cells were then chased for 30 mins in media containing 10 µM amitriptyline hydrochloride and then imaged . In cellulo pH clamping and measurement experiments were carried out with ImLy with modifications to protocols described by our lab previously ( Modi et al . , 2013 , 2009 ) . J774A . 1 and THP-1 cells were pulsed and chased with 500 nM of ImLy . Cells are then fixed with 200 μL 2 . 5% PFA for 20 mins at room temperature , washed three times and retained in 1X PBS . To obtain the intracellular pH calibration profile , perfusate and endosomal pH were equalized by incubating the previously fixed cells in the appropriate pH clamping buffer clamping buffers ( 120 mM KNO3 , 5 mM NaNO3 , 1 mM Mg ( NO3 ) 2 , 1 mM Ca ( NO3 ) 2 , 20 mM HEPES , MES and NaOAc ) of varying pH , containing 25 µM nigericin and 25 µM monensin for 30 mins at room temperature . For real-time pH measurements , cells are pulsed with 500 nM of ImLy followed by a 60 mins chase . Cells are then washed with 1X PBS and imaged . pH measurements in the lysosomes of Gaucher’s Disease and of Niemann Pick A/B disease , in the two cell models that is murine J774A . 1 and human THP-1 cells , were carried out similar to the protocol above using 500 nM of ImLy . Wide field microscopy was carried out on IX83 research inverted microscope ( Olympus Corporation of the Americas , Center Valley , PA , USA ) using a 60X , 1 . 42 NA , phase contrast oil immersion objective ( PLAPON , Olympus Corporation of the Americas , Center Valley , PA , USA ) and Evolve Delta 512 EMCCD camera ( Photometrics , USA ) . Filter wheel , shutter and CCD camera were controlled using Metamorph Premier Ver 7 . 8 . 12 . 0 ( Molecular Devices , LLC , USA ) , suitable for the fluorophores used . Images on the same day were acquired under the same acquisition settings . All the images were background subtracted taking mean intensity over an adjacent cell free area . Mean intensity in each endosome was measured in donor ( D ) and acceptor ( A ) channels . Alexa 488 channel images ( D ) were obtained using 480/20 band pass excitation filter , 520/40 band pass emission filter and a 89016- ET - FITC/Cy3/Cy5 dichroic filter . Alexa 647 channel images ( A ) were obtained using 640/30 band pass excitation filter , 705/72 band pass emission filter and 89016- ET - FITC/Cy3/Cy5 dichroic filter . For FRET channel images were obtained using the 480/20 band pass excitation filter , 705/72 band pass emission filter and 89016- ET - FITC/Cy3/Cy5 dichroic filter . Mean intensity in each endosome was measured in donor and acceptor channels . A ratio of donor to acceptor intensities ( D/A ) was obtained from these readings . Pseudocolor images were generated by calculating the D/A ratio per pixel . Confocal images were captured with a Leica TCS SP5 II STED laser scanning confocal microscope ( Leica Microsystems , Inc . , Buffalo Grove , IL , USA ) equipped with 63X , 1 . 4 NA , oil immersion objective . Alexa 488 was excited using an Argon ion laser for 488 nm excitation , Alexa 647 using He-Ne laser for 633 excitation and BAC using Argon ion laser for 458 nm excitation with a set of dichroics , excitation , and emission filters suitable for each fluorophore . Ratiometric calcium imaging of Fura-2 was carried out on an Olympus IX81 microscope equipped with a 40x objective , NA = 1 . 2 . Excitation of Fura-2 is performed using 340/26 and 380/10 nm excitation filters , equipped with a 455 nm dichroic mirror and a 535/40 nm emission filter . Exposure time was kept at 100 ms for all the imaging experiments to minimize phototoxicity . Images were analyzed with ImageJ ver 1 . 49 ( NIH , USA ) . For pH measurements Alexa 488 and Alexa 647 images were overlapped using ImageJ and endosomes showing colocalization were selected for further analysis . Intensity in each endosome was measured in donor ( D ) and FRET ( A ) channels and recorded in an OriginPro Sr2 b9 . 2 . 272 ( OriginLab Corporation , Northampton , MA , USA ) file from which D/A ratio of each endosome was obtained . The mean D/A of each distribution were converted to pH according to the intracellular calibration curve . Data was represented as mean pH value ± standard error of the mean . Data for pH clamping experiments was analysed similarly . For chloride measurements , regions of cells containing lysosomes in each Alexa 647 ( R ) image were identified and marked in the ROI plugin in ImageJ . The same regions were identified in the BAC ( G ) image recalling the ROIs and appropriate correction factor for chromatic aberration if necessary . After background subtraction , intensity for each endosome was measured and recorded in an Origin file . A ratio of R to G intensities ( R/G ) was obtained from these values by dividing the intensity of a given endosome in the R image with the corresponding intensity in the G image . For a given experiment , mean [Clˉ] of an organelle population was determined by converting the mean R/G value of the distribution to [Clˉ] values according to the intracellular calibration profile . Data was presented as mean of this mean [Clˉ] value ± standard error of the mean . Data for chloride clamping experiments was analyzed similarly . Colocalization of GFP and Alexa 647 was determined by counting the numbers of Alexa 647 positive puncta that colocalize with GFP and representing it as a Pearson's correlation coefficient . Temporal mapping of I-switch and Clensor was done in 10 worms of pwIs50 [lmp-1::GFP + Cb-unc-119 ( + ) ] as previously described by our lab ( Surana et al . , 2011 ) . Briefly , worms were injected with 500 nM of I4cLYA647 or ClensorA647 , incubated at 22°C for 1 hr , and then imaged using Leica TCS SP5 II STED laser scanning confocal microscope ( Leica Microsystems , Inc . , Buffalo Grove , IL , USA ) . Colocalization of GFP and I4cLYA647 or ClensorA647 was determined by counting the numbers of Alexa647 positive puncta that colocalize with GFP positive puncta and expressing them as a percentage of the total number of Alexa 647 positive puncta . In order to confirm lysosomal labeling in a given genetic background , the same procedure was performed on the relevant mutant or RNAi knockdown in pwIs50 [lmp-1::GFP + Cb-unc-119 ( + ) ] . All pH and chloride clamping experiments ( Figure 1b , Figure 1—figure supplement 2 , Figure 4—figure supplement 2 ) were performed in triplicates and the standard error of mean ( s . e . m ) values are plotted with the number of cells considered being mentioned in each legend . Experiment with murine macrophage , J774A . 1 and THP-1 cells ( Figure 4 ) has been performed in triplicates . Ratio of standard error of the mean is calculated for n = 20 cells and n = 10 cells and is plotted in Figure 4d and e respectively . All pH and chloride measurements in C . elegans of indicated genetic backgrounds ( Figures 2c and 3c and Figure 2—figure supplement 1c ) were carried out in n = 10 worms and the standard error of mean ( s . e . m ) values are plotted with the number of cells considered being mentioned in each legend . Coelomocyte labeling for stability assay were carried out with I4cLYA647 , and ClensorA647 . For microinjections , the samples were diluted to 500 nM using 1X Medium 1 ( 150 mM NaCl , 5 mM KCl , 1 mM CaCl2 , 1 mM MgCl2 , 20 mM HEPES , pH 7 . 2 ) . Post injection the worms are incubated at 22°C . After requisite time the injected worms are anesthetized in 40 mM sodium azide in M9 buffer and mounted on a glass slide containing 2% agarose pad . Worms were imaged using Olympus IX83 research inverted microscope ( Olympus Corporation of the Americas , Center Valley , PA , USA ) . The average whole cell intensity in the Alexa 647 channel was plotted as a function of time ( Figure 2—figure supplement 2 ) J774A . 1 and THP-1 macrophage cells labeling was carried out using 500 nM ClensorA647 using 1X Medium 1 ( 150 mM NaCl , 5 mM KCl , 1 mM CaCl2 , 1 mM MgCl2 , 20 mM HEPES , pH 7 . 2 ) . Cells were pulsed for 30 mins and then chased at 37°C for the indicated time points . The average whole cell intensity in the Alexa 647 channel was plotted as a function of time ( Figure 4—figure supplement 3 ) . Cells were loaded with 3 μM Fura-2 AM in HBSS ( 137 mM NaCl , 5 mM KCl , 1 . 4 mM CaCl2 , 1 mM MgCl2 , 0 . 25 mM Na2HPO4 , 0 . 44 mM KH2PO4 , 4 . 2 mM NaHCO3 and 10 mM glucose ) at 37°C for 60 min . Post incubation cells were washed with 1X PBS and then imaged in Low Ca2+ or Zero Ca2+ buffer ( 145 mM NaCl , 5 mM KCl , 3 mM MgCl2 , 10 mM glucose , 1 mM EGTA , and 20 mM HEPES ( pH 7 . 4 ) ) . Ca2+ concentration in the nominally free Ca2+ solution is estimated to be 1–10 μM . With 1 mM EGTA , the free Ca2+ concentration is estimated to be <10 nM based on the Maxchelator software ( http://maxchelator . stanford . edu/ ) . Florescence was recorded using two different wavelengths ( 340 and 380 nm ) and the ratio ( F340/F380 ) was used to calculate changes in intracellular [Ca2+] . Enzyme activity assays were performed in J774A . 1 cells . For Cathepsin C enzyme activity; we used Gly-Phe β-naphthylamide as a substrate . Lysosomes of J774A . 1 cells were pre-labeled with TMR-dextran ( 0 . 5 mg/mL; G ) for 1 hr and chased in complete medium for 16 hr at 37°C . The cells were then labeled with 50 nM LysoTracker in complete medium for 30 mins at 37°C . 50 μM NPPB or 200 μM GPN were then added to the cells and incubated for 30 mins at 37°C . The cells then washed and imaged in HBSS buffer containing either NPPB or GPN . The whole cell intensity ratio ( G/R ) was plotted to quantify the level of LysoTracker labelling of the endosomes . For Cathepsin L and Aryl Sulfatase enzyme activity Magic Red Cathepsin L assay kit ( Immunochemistry Technologies ) and Lysosomal sulfatase assay kit ( Marker Gene ) were used . The experiment was performed using the manufacture’s protocol . Briefly , cells were incubated with 1X Magic Red Cathepsin L assay probe or 200 μM Lysosomal sulfatase assay probe for 4 hr in complete medium . The cells were then labelled with 10 μM Hoechst stain for 10 mins at 37°C after which the cells were washed and imaged . For low chloride containing dishes; cells were preincubated with 50 μM NPPB before the addition of the enzyme probes . The ratio of enzyme substrate whole cell intensity to that of DAPI was used to quantify enzyme activity .
Lysosomes are organelles responsible for the breakdown and recycling of cellular machinery . Dysfunctional lysosomes give rise to lysosomal storage disorders as well as common neurodegenerative diseases . Here , we use a DNA-based , fluorescent chloride reporter to measure lysosomal chloride in Caenorhabditis elegans as well as murine and human cell culture models of lysosomal diseases . We find that the lysosome is highly enriched in chloride , and that chloride reduction correlates directly with a loss in the degradative function of the lysosome . In nematodes and mammalian cell culture models of diverse lysosomal disorders , where previously only lysosomal pH dysregulation has been described , massive reduction of lumenal chloride is observed that is ~103 fold greater than the accompanying pH change . Reducing chloride within the lysosome impacts Ca2+ release from the lysosome and impedes the activity of specific lysosomal enzymes indicating a broader role for chloride in lysosomal function .
In cells , worn out proteins and other unnecessary materials are sent to small compartments called lysosomes to be broken down and recycled . Lysosomes contain many different proteins including some that break down waste material into recyclable fragments and others that transport the fragments out of the lysosome . If any of these proteins do not work , waste products build up and cause disease . There are around 70 such lysosomal storage diseases , each arising from a different lysosomal protein not working correctly . A recently developed “nanodevice” called Clensor can measure the levels of chloride ions inside cells . Clensor is constructed from DNA , and its fluorescence changes when it detects chloride ions . Although chloride ions have many biological roles , chloride ion levels had not been measured inside a living organism . Now , Chakraborty et al . – including some of the researchers who developed Clensor – have used this nanodevice to examine chloride ion levels in the lysosomes of the roundworm Caenorhabditis elegans . This revealed that the lysosomes contain high levels of chloride ions . Furthermore , reducing the amount of chloride in the lysosomes made them worse at breaking down waste . Do lysosomes affected by lysosome storage diseases also contain low levels of chloride ions ? To find out , Chakraborty et al . used Clensor to study C . elegans worms and mouse and human cells whose lysosomes accumulate waste products . In all these cases , the levels of chloride in the diseased lysosomes were much lower than normal . This had a number of effects on how the lysosomes worked , such as reducing the activity of key lysosomal proteins . Chakraborty et al . also found that Clensor can be used to distinguish between different lysosomal storage diseases . This means that in the future , Clensor ( or similar methods that directly measure chloride ion levels in lysosomes ) may be useful not just for research purposes . They may also be valuable for diagnosing lysosomal storage diseases early in infancy that , if left undiagnosed , are fatal .