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Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2017

Dynamic Current Clamp Experiments Define The Functional Roles Of IK1 And Ito,f In Human Induced Pluripotent Stem Cell Derived Cardiomyocytes

The transient outward potassium current (Ito) plays a key, albeit incompletely defined, role in cardiomyocyte physiology and pathophysiology. In light of the technical challenges of studying adult human cardiomyocytes, this study examines the use of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) as a system which potentially preserves the native cellular milieu of human cardiomyocytes. ISPC-CMs express a robust Ito with slow recovery kinetics and fail to express the rapidly recovering Ito,f which is implicated in human disease. Overexpression of the accessory subunit KChIP2 (which is not expressed in iPSC-CMs) resulted in restoration of a rapid component of recovery. To define the functional role of Ito, dynamic current clamp was used to introduce computationally modeled currents into iPSC-CMs while recording action potentials. However, iPSC-CMs exhibit action potentials with multiple immature physiological properties, including slow upstroke velocity, heterogeneous action potential waveforms, and the absence of a phase 1 notch, thus potentially limiting the utility of these cells as a model of adult cardiomyocytes. Importantly, the introduction of modeled inwardly rectified current (IK1) ameliorated these immature properties by restoring a hyperpolarized resting membrane potential. In this context of normalized action potential morphologies, dynamic current clamp experiments introducing Ito,f demonstrated that there is significant cell-to-cell heterogeneity and that the functional effect of Ito,f is highly sensitive to the action potential plateau voltage in each cell.

cell biology↗

bioRxiv2017

CRISPR-Cas9 Genome Editing In Human Cells Works Via The Fanconi Anemia Pathway

CRISPR-Cas9 genome editing creates targeted double strand breaks (DSBs) in eukaryotic cells that are processed by cellular DNA repair pathways. Co-administration of single stranded oligonucleotide donor DNA (ssODN) during editing can result in high-efficiency (>20%) incorporation of ssODN sequences into the break site. This process is commonly referred to as homology directed repair (HDR) and here referred to as single stranded template repair (SSTR) to distinguish it from repair using a double stranded DNA donor (dsDonor). The high efficacy of SSTR makes it a promising avenue for the treatment of genetic diseases1,2, but the genetic basis of SSTR editing is still unclear, leaving its use a mostly empiric process. To determine the pathways underlying SSTR in human cells, we developed a coupled knockdown-editing screening system capable of interrogating multiple editing outcomes in the context of thousands of individual gene knockdowns. Unexpectedly, we found that SSTR requires multiple components of the Fanconi Anemia (FA) repair pathway, but does not require Rad51-mediated homologous recombination, distinguishing SSTR from repair using dsDonors. Knockdown of FA genes impacts SSTR without altering break repair by non-homologous end joining (NHEJ) in multiple human cell lines and in neonatal dermal fibroblasts. Our results establish an unanticipated and central role for the FA pathway in templated repair from single stranded DNA by human cells. Therapeutic genome editing has been proposed to treat genetic disorders caused by deficiencies in DNA repair, including Fanconi Anemia. Our data imply that patient genotype and/or transcriptome profoundly impact the effectiveness of gene editing treatments and that adjuvant treatments to bias cells towards FA repair pathways could have considerable therapeutic value.

cell biology↗

bioRxiv2017

A New Quantitative Cell Microscopy Approach Reveals Mechanistic Insights Into Clearance Of Membrane Substrates By The AAA Protein Msp1

Msp1 is a conserved AAA ATPase in budding yeast localized to mitochondria where it prevents accumulation of mistargeted tail-anchored (TA) proteins, including the peroxisomal TA protein Pex15. Msp1 also resides on peroxisomes but it remains unknown how native TA proteins on mitochondria and peroxisomes evade Msp1 surveillance. We used live-cell quantitative cell microscopy tools and drug-inducible gene expression to dissect Msp1 function. We found that a small fraction of peroxisomal Pex15, exaggerated by overexpression, is turned over by Msp1. Kinetic measurements guided by theoretical modeling revealed that Pex15 molecules at mitochondria display age-independent Msp1 sensitivity. By contrast, Pex15 molecules at peroxisomes are rapidly converted from an initial Msp1-sensitive to an Msp1-resistant state. Lastly, we show that Pex15 interacts with the peroxisomal membrane protein Pex3, which shields Pex15 from Msp1-dependent turnover. In sum, our work argues that Msp1 selects its substrates on the basis of their solitary membrane existence.

cell biology↗

bioRxiv2017

Scc2/Nipbl Hops Between Chromosomal Cohesin Rings After Loading

The cohesin complex mediates DNA-DNA interactions both between (sister chromatid cohesion) and within chromosomes (DNA looping) via a process thought to involve entrapment of DNAs within its tripartite ring. It has been suggested that intra- chromosome loops are generated through processive extrusion of DNAs through the lumen of cohesins ring. Scc2 (Nipbl) is essential for loading cohesin onto chromosomes but not for maintaining sister chromatid cohesion following DNA replication. It has therefore been assumed that Scc2 is involved exclusively in the cohesin loading process. However, it is possible that the stimulation of cohesins ABC-like ATPase by Scc2 also has a post-loading function, for example driving loop extrusion. Using fluorescence recovery after photobleaching (FRAP) and single-molecule tracking, we show that Scc2 binds dynamically to chromatin, principally through an association with cohesin. Scc2s movement within chromatin is consistent with a \"stop-and-go\" or \"hopping\" motion. We suggest that a low diffusion coefficient, a low stoichiometry relative to cohesin, and a high affinity for chromosomal cohesin enables Scc2 to move rapidly from one chromosomal cohesin complex to another, performing a function distinct from loading.

cell biology↗

bioRxiv2017

Arginase 1 Is Negatively Regulated By β-Catenin Signaling In The Lung

We previously demonstrated that mice lacking the Wnt co-receptor, Lrp5, had attenuated pulmonary fibrosis in the bleomycin model. We found that Arginase 1 (Arg1), an enzyme that converts L-arginine to urea and ornithine, was markedly elevated in Lrp5-/- lungs compared with wild-type mice after bleomycin injury. We show that this induction is not apparently due to the expression of Th2 cytokines, IL-4 and IL-13, but instead is due to Wnt/{beta}-catenin signaling, which negatively regulates Arg1 expression in lung macrophages. Although Arg1 expression in macrophages has been used to define an alternatively activated phenotype, flow cytometry analysis of alveolar and interstitial macrophage sub-populations in Lrp5-/- lungs 14 days after bleomycin injury revealed no clear evidence of skewing from a classical to an alternatively activated phenotype. Upregulation of Arg1 expression and arginase activity might diminish lung arginine levels with consequent alterations in collagen or cytokine production. However, dietary supplementation of bleomycin-treated Lrp5-/- mice with the Arg1 substrate, L-arginine, failed to alter lung collagen content or cytokine levels 21 days after bleomycin injury. These findings demonstrate that Arg1 is negatively regulated by {beta}-catenin signaling in macrophages, raising the possibility that Wnt signaling directs alterations in immune cell metabolism that may be relevant to lung repair after injury.

cell biology↗

bioRxiv2017

Tacrolimus Rescues Endothelial ALK1 Loss-Of-Function Signaling And Improves HHT Vascular Pathology

Hereditary hemorrhagic telangiectasia (HHT) is a genetic vascular disorder arising from endothelial cell (EC) proliferation and hypervascularization, for which no cure exists. Because HHT is caused by loss-of-function mutations in BMP9-ALK1-Smad1/5/8 signaling, interventions aimed at activating this pathway are of therapeutic value. By screening FDA-approved drug libraries, we identified tacrolimus (FK-506) as a potent activator of Smad1/5/8 in BMP9-challenged reporter cells. In primary ECs, tacrolimus activated Smad1/5/8 to oppose the pro-angiogenic gene expression signature associated with ALK1 loss-of-function, by notably reducing Dll4 expression. In these cells, tacrolimus also inhibited Akt and p38 stimulation by VEGF. In the BMP9/10-immunodepleted postnatal retina--a mouse model of HHT vascular pathology--tacrolimus activated endothelial Smad1/5/8 and prevented the Dll4 overexpression and hypervascularization associated with this model. Finally, tacrolimus stimulated Smad1/5/8 in cells transfected with BMP9-unresponsive ALK1 HHT mutants and in HHT patient blood outgrowth ECs. We propose that tacrolimus repurposing has therapeutic potential in HHT.

cell biology↗

bioRxiv2017

Loss Of PTEN Promotes Formation Of Signaling-Specific Clathrin-Coated Pits

Defective endocytosis and vesicular trafficking of signaling receptors has recently emerged as a multifaceted hallmark of malignant cells. Clathrin-coated pits (CCPs), the fundamental unit of clathrin-mediated endocytosis, display highly heterogeneous dynamics on the plasma membrane where they can take from 20 seconds to over a minute to form cytosolic coated-vesicles. Despite the large number of cargo molecules that traffic through CCPs, it is not well understood whether signaling receptors activated in cancer, such as epidermal growth factor receptor (EGFR), are regulated through a specific subset of CCPs. The signaling lipid phosphatidylinositol (3,4,5)-triphosphate (PI(3,4,5)P3), which is dephosphorylated by phosphatase tensin homolog (PTEN), is a potent tumorigenic signaling lipid that is present in excess in many types of cancers. Using total internal reflection fluorescence microscopy and automated tracking and detection of CCPs, we find PTEN and EGF bound EGFR are enriched in a distinct subset of short-lived CCPs that corresponded with clathrin-dependent EGF-induced signaling. By deleting PTEN using CRISPR-Cas9 and reconstituting PTEN, we demonstrate that PTEN plays a role in the regulation of CCP dynamics; this appears to recapitulate CCP dynamics in highly metastatic PTEN-deleted cancer cells where we find a larger proportion of short-lived CCPs and higher initiation density compared to the normal cells. Furthermore, increased PI(3,4,5)P3 results in higher proportion of short-lived CCPs, an effect that recapitulates PTEN deletion. Our findings provide strong evidence for the existence of short-lived signaling-capable CCPs. Altogether, these findings demonstrate the importance of PTEN and PI(3,4,5)P3 in regulating CCP dynamics and assign a new function to PTEN as a modulator of signaling-capable CCPs.

cell biology↗

bioRxiv2017

Analysis Of Interphase Node Proteins In Fission Yeast By Quantitative And Super Resolution Fluorescence Microscopy

We used quantitative confocal microscopy and FPALM super resolution microscopy of live fission yeast to investigate the structures and assembly of two types of interphase nodes, multiprotein complexes associated with the plasma membrane that merge together and mature into the precursors of the cytokinetic contractile ring. During the long G2 phase of the cell cycle seven different interphase node proteins maintain constant concentrations as they accumulate in proportion to cell volume. During mitosis the total numbers of type 1 node proteins (cell cycle kinases Cdr1p, Cdr2p, Wee1p, and anillin Mid1p) are constant even when the nodes disassemble. Quantitative measurements provide strong evidence that both types of nodes have defined sizes and numbers of constituent proteins, as observed for cytokinesis nodes. Type 1 nodes assemble in two phases, a burst at the end of mitosis, followed by steady increase during interphase to double the initial number. Type 2 nodes containing Blt1p, Rho-GEF Gef2p, and kinesin Klp8p remain intact throughout the cell cycle and are constituents of the contractile ring. They are released from the contractile ring as it disassembles and then associate with type 1 nodes around the equator of the cell during interphase.\n\nHighlight summaryFPALM super resolution microscopy and quantitative confocal microscopy reveal that interphase nodes, the precursors to the fission yeast cytokinetic contractile ring, are discrete unitary structures with defined sizes and ratios of component proteins. Type 1 nodes disassemble during mitosis, but type 2 nodes remain intact throughout the cell cycle.

cell biology↗

bioRxiv2017

Intercellular mRNA Trafficking Via Membrane Nanotubes In Mammalian Cells

RNAs have been shown to undergo transfer between mammalian cells, though the mechanism behind this phenomenon and its overall importance to cell physiology is not well understood. Numerous publications have suggested that RNAs (microRNAs and incomplete mRNAs) undergo transfer via extracellular vesicles (e.g. exosomes). However, in contrast to a diffusion-based transfer mechanism, we find that full-length mRNAs undergo direct cell-cell transfer via cytoplasmic extensions, called membrane nanotubes (mNTs), which connect donor and acceptor cells. By employing a simple co-culture experimental model and using single-molecule imaging, we provide quantitative data showing that mRNAs are transferred between cells in contact. Examples of mRNAs that undergo transfer include those encoding GFP, mouse {beta}-actin, and human Cyclin D1, BRCA1, MT2A, and HER2. We show that intercellular mRNA transfer occurs in all co-culture models tested (e.g. between primary cells, immortalized cells, and in co-cultures of immortalized human and murine cells). Rapid mRNA transfer is dependent upon actin, but independent of de novo protein synthesis, and is modulated by stress conditions and gene expression levels. Hence, this work supports the hypothesis that full-length mRNAs undergo transfer between cells through a refined structural connection. Importantly, unlike the transfer of miRNA or RNA fragments, this process of communication transfers genetic information that could potentially alter the acceptor cell proteome. This phenomenon may prove important for the proper development and functioning of tissues, as well as host-parasite or symbiotic interactions.\n\nSignificanceMessenger RNA (mRNA) molecules convey genetic information within cells, beginning from genes in the nucleus to ribosomes in the cell body, where they are translated into proteins. Here, we show a novel mode of transferring genetic information from one cell to another. Contrary to previous publications suggesting that mRNAs transfer via extracellular vesicles, we provide visual and quantitative data showing that mRNAs transfer via membrane nanotubes and direct cell-to-cell contact. We predict that this process has a major role in regulating local cellular environments with respect to tissue development and maintenance, cellular responses to stress, interactions with parasites, tissue transplants, and the tumor microenvironment.\n\nAuthor contributionsG.H., A.R. and R.H.S. conceived the research and designed the experiments; C.M.E. performed and analyzed the experiments with WM983b+/-GFP, including transwell and exosomes; M.C.D. and E.E. performed and analyzed the WM983b/NIH393 co-culture experiments; G.H. performed and analyzed all other experiments; and G.H., J.E.G, A.R. and R.H.S. wrote the paper.

cell biology↗

bioRxiv2017

Aminoglycoside Antibiotics Perturb Physiologically Important MicroRNA Contributing To Drug Toxicity

miRNAs are key non-protein coding regulators of gene expression in various pathophysiological conditions. Targeting miRNA with small molecules offer an unconventional approach, where clinically active compounds with RNA binding activity can be tested for their ability to modulate miRNA levels and thus for drug repositioning. Aminoglycoside antibiotics are highly effective microbicidal RNA binding molecules that bind to prokaryotic rRNA secondary structures. Here, we report that specific subsets of miRNA can be modulated by aminoglycosides. However, ototoxicity (cochlear and vestibular) and nephrotoxicity of multiple origins resulting from prolonged use are a well-known disadvantage of aminoglycosides. Mature non-coding RNAs and their precursors can present off-target sites, by forming secondary structures that resemble ribosomal RNA, thus providing an additional molecular basis for the toxicity of aminoglycosides. Using in vitro, in cellulae and physiological responses, we provide evidence for the direct functional perturbation of the miR- 96 cluster leading to selective cell death in neuromasts- the zebrafish equivalent of cochlear hair cells, by Streptomycin, a prototype aminoglycoside antibiotic, thus contributing to the observed ototoxicity. Our observations, collectively underscore the importance of re- evaluating RNA binding drugs for their off-targeting effects in the context of miRNA and other functional non-coding RNA.

cell biology↗

bioRxiv2017

A Theory That Predicts Behaviors Of Disordered Cytoskeletal Networks

Morphogenesis in animal tissues is largely driven by tensions of actomyosin networks, generated by an active contractile process that can be reconstituted in vitro. Although the network components and their properties are known, the requirements for contractility are still poorly understood. Here, we describe a theory that predicts whether an isotropic network will contract, expand, or conserve its dimensions. This analytical theory correctly predicts the behavior of simulated networks consisting of filaments with varying combinations of connectors, and reveals conditions under which networks of rigid filaments are either contractile or expansile. Our results suggest that pulsatility is an intrinsic behavior of contractile networks if the filaments are not stable but turn over. The theory offers a unifying framework to think about mechanisms of contractions or expansion. It provides a foundation for the study of a broad range of processes involving cytoskeletal networks, and a basis for designing synthetic networks.

cell biology↗

bioRxiv2017

Filopodyan: An Open-Source Pipeline For The Analysis Of Filopodia

Filopodia have important sensory and mechanical roles in motile cells. The recruitment of actin regulators, such as ENA/VASP proteins, to sites of protrusion underlies diverse molecular mechanisms of filopodia formation and extension. We developed Filopodyan (filopodia dynamics analysis) in Fiji and R to measure fluorescence in filopodia and at their tips and bases concurrently with their morphological and dynamic properties. Filopodyan supports high-throughput phenotype characterization as well as detailed interactive editing of filopodia reconstructions through an intuitive graphical user interface. Our highly customizable pipeline is widely applicable, capable of detecting filopodia in four different cell types in vitro and in vivo. We use Filopodyan to quantify the recruitment of ENA and VASP preceding filopodia formation in neuronal growth cones, and uncover a molecular heterogeneity whereby different filopodia display markedly different responses to changes in the accumulation of ENA and VASP fluorescence in their tips over time.\n\neTOC summary: Urban[c]i[c] et al. developed an open-source platform called Filopodyan (filopodia dynamics analysis) in Fiji and R to measure fluorescence in filopodia, at their tips, and bases, concurrently with their morphological and dynamic properties. This customizable tool therefore enables researchers to determine the relationship between protein localization and filopodium behavior.

cell biology↗

bioRxiv2017

Natural Chromatin Is Heterogeneous And Self Associates In Vitro

The 30-nm fiber is commonly found in oligonucleosome arrays in vitro but rarely found in chromatin within nuclei. To determine how chromatin high-order structure is controlled, we used cryo-ET to study the undigested natural chromatin released from cells that do not have evidence of 30-nm fibers in vivo: picoplankton and yeast. In the presence of divalent cations, most of the chromatin from both organisms is compacted into a large mass. Rare irregular 30-nm fibers do form at the periphery of this mass, some of which include face-to-face interactions. In the absence of divalent cations, picoplankton chromatin decondenses into open zigzags. By contrast, yeast chromatin mostly remains compact with looser nucleosome packing, even after treatment with histone-deacetylase inhibitor. The 3-D configuration of natural chromatin is therefore sensitive to the local environment, but generally nonpermissive of regular motifs, even at the level of oligonucleosomes.

cell biology↗

bioRxiv2017

NQO1 Binds And Supports SIRT1 Function

Silent information regulator 2-related enzyme 1 (SIRT1) is an NAD+-dependent class III deacetylase and a key component of the cellular metabolic sensing pathway. The requirement of NAD+ for SIRT1 activity led us to assume that NQO1, an NADH oxidoreductase producing NAD+, regulates SIRT1 activity. We show here that SIRT1 is capable of increasing NQO1 (NAD(P)H Dehydrogenase Quinone 1) transcription and protein levels. NQO1 physically interacts with SIRT1 but not with an enzymatically dead SIRT1 H363Y mutant. The interaction of NQO1 with SIRT1 is markedly increased under mitochondrial inhibition. Interestingly, under this condition the nuclear pool of NQO1 is elevated. Depletion of NQO1 compromises the role of SIRT1 in inducing transcription of several target genes and eliminates the protective role of SIRT1 following mitochondrial inhibition. Our results suggest that SIRT1 and NQO1 form a regulatory loop where SIRT1 regulates NQO1 expression and NQO1 binds and mediates the protective role of SIRT1 during mitochondrial stress. The interplay between an NADH oxidoreductase enzyme and an NAD+ dependent deacetylase may act as a rheostat in sensing mitochondrial stress.

cell biology↗

bioRxiv2017

Identification Of A Native Novel Oncolytic Immunoglobulin On Exfoliated Colon Epithelial Cells: A Bispecific Heterodimeric Chimera Of IgA/IgG

Understanding the nature of cell surface markers on exfoliated colonic cells is a crucial step in establishing criteria for a normally functioning mucosa. We have found that colonic cells isolated from stool samples (SCSR-010 Fecal Cell Isolation Kit, NonInvasive Technologies, Elkridge, MD), preserved at room temperature for up to one week, with viability of >85% and low levels of apoptosis (8% - 10%) exhibit two distinct cell size subpopulations, in the 2.5M- 5.0 M and 5.0M-8.0M range. In addition to IgA, about 60% of the cells expressed a novel heterodimeric IgA/IgG immunoglobulin that conferred a broad-spectrum cell mediated cytotoxicity against tumor cells. In a cohort of 58 subjects the exclusive absence of this immunoglobulin in two African-Americans was suggestive of a germline deletion. Serial cultures in stem cell medium retained the expression of this heterodimer. Since a majority of the cystic cells expressed the stem cell markers Lgr5 and Musashi-1 we termed these cells as gastrointestinal progenitor stem cells (GIP-C**). CXCR-4, the cytokine co-receptor for HIV was markedly expressed. These cells also expressed CD20, IgA, IgG, CD45, and COX-2. We assume that they originated from mature columnar epithelium by dedifferentiation. Our observations indicate that we have a robust noninvasive method to study mucosal pathophysiology and a direct method to create a database for applications in regenerative medicine.

cell biology↗

bioRxiv2017

Postmitotic Nuclear Pore Assembly Proceeds By Radial Dilation Of Small ER Membrane Openings

The nuclear envelope has to be reformed after mitosis to create viable daughter cells with closed nuclei. How membrane sealing of DNA and assembly of nuclear pore complexes (NPCs) are achieved and coordinated is poorly understood. Here, we reconstructed nuclear membrane topology and structure of assembling NPCs in a correlative three dimensional electron microscopy time-course of dividing human cells. Our quantitative ultrastructural analysis shows that nuclear membranes form from highly fenestrated ER sheets, whose shrinking holes are stabilized and then dilated into NPCs during inner ring complex assembly, forming thousands of transport channels within minutes. This mechanism is fundamentally different from interphase NPC assembly and explains how mitotic cells can rapidly establish a closed nuclear compartment while making it transport-competent at the same time.

cell biology↗

bioRxiv2017

Characterization Of Beta Tubulin Isotypes During Foam Cell Formation

Foam cells contribute to the development of a cardiovascular condition called atherosclerosis. They arise when monocytes become engorged and lipid-laden after exposure to native low-density lipoproteins (Falk, 2006). It is assumed that the cytoskeleton is responsible for the morphological changes observed during foam cell formation. Beta tubulin and alpha tubulin are proteins that dimerize and polymerize to form microtubules, which are an important component of the cytoskeleton (Joshi, 1998). Little is known regarding the changes in cytoskeletal composition, particularly that of beta tubulins, throughout foam cell induction. The purpose of this study was to elucidate the expression patterns of beta tubulin isotypes 1-4 in human THP-1 monocytes throughout foam cell formation and to determine what relationship exists between beta tubulin expression and foam cell lipid aggregation. Levels of beta tubulin 1-4 were measured by western blot and immunofluorescence throughout the stages of foam cell differentiation, and beta tubulin isotypes were manipulated by siRNA to determine the effects of diminished beta tubulin expression on foam cell formation. Regardless of isotype, beta tubulin was always present in the highest amounts in monocytes. Levels of beta tubulin-1 and -4 were significantly decreased in macrophage and foam cells relative to monocytes (p < 0.0079, p < 0.0208, respectively). Beta 3 levels also exhibited a decrease. Beta 2 levels remained low regardless of differentiation stage. The distribution of beta tubulin 1 was shown to be more spindle-like (stretching across cells), compared to beta tubulins 2, 3, and 4, which exhibited a more \"clumped,\" less interconnected arrangement. When expression of beta tubulin isotypes 1, 3, and 4 were reduced in monocytes, resulting foam cells appeared to have more lipid aggregates and were significantly larger (p<0.0001) when compared to the size of foam cells without siRNA following treatment with PMA + LDL. In conclusion, the distribution of beta tubulins 1, 3, and 4 changes throughout the stages of foam cell induction, and manipulation of beta tubulins altered foam cell formation. Unexpectedly, the silencing or decreasing of beta tubulin enhanced lipid aggregation. Information concerning how beta tubulin expression can effect foam cell formation may offer insight into how to reduce plaque formation in patients with atherosclerosis.

cell biology↗

bioRxiv2017

High lumenal chloride in the lysosome is critical for lysosome function.

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 ~104-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. -149 words.

cell biology↗