cAMP regulates Ca2+-dependent exocytosis of lysosomes and lysosome-mediated cell invasion by trypanosomes.

Ca2+-regulated exocytosis, previously believed to be restricted to specialized cells, was recently recognized as a ubiquitous process. In mammalian fibroblasts and epithelial cells, exocytic vesicles mobilized by Ca2+ were identified as lysosomes. Here we show that elevation in intracellular cAMP potentiates Ca2+-dependent exocytosis of lysosomes in normal rat kidney fibroblasts. The process can be modulated by the heterotrimeric G proteins Gs and Gi, consistent with activation or inhibition of adenylyl cyclase. Normal rat kidney cell stimulation with isoproterenol, a beta-adrenergic agonist that activates adenylyl cyclase, enhances Ca2+-dependent lysosome exocytosis and cell invasion by Trypanosoma cruzi, a process that involves parasite-induced [Ca2+]i transients and fusion of host cell lysosomes with the plasma membrane. Similarly to what is observed for T. cruzi invasion, the actin cytoskeleton acts as a barrier for Ca2+-induced lysosomal exocytosis. In addition, infective stages of T. cruzi trigger elevation in host cell cAMP levels, whereas no effect is observed with noninfective forms of the parasite. These findings demonstrate that cAMP regulates lysosomal exocytosis triggered by Ca2+ and a parasite/host cell interaction known to involve Ca2+-dependent lysosomal fusion.     

δ-Tocopherol Reduces Lipid Accumulation in Niemann-Pick Type C1 and Wolman Cholesterol Storage Disorders

Niemann-Pick disease type C (NPC) and Wolman disease are two members of a family of storage disorders caused by mutations of genes encoding lysosomal proteins. Deficiency in function of either the NPC1 or NPC2 protein in NPC disease or lysosomal acid lipase in Wolman disease results in defective cellular cholesterol trafficking. Lysosomal accumulation of cholesterol and enlarged lysosomes are shared phenotypic characteristics of both NPC and Wolman cells. Utilizing a phenotypic screen of an approved drug collection, we found that δ-tocopherol effectively reduced lysosomal cholesterol accumulation, decreased lysosomal volume, increased cholesterol efflux, and alleviated pathological phenotypes in both NPC1 and Wolman fibroblasts. Reduction of these abnormalities may be mediated by a δ-tocopherol-induced intracellular Ca²⁺ response and subsequent enhancement of lysosomal exocytosis. Consistent with a general mechanism for reduction of lysosomal lipid accumulation, we also found that δ-tocopherol reduces pathological phenotypes in patient fibroblasts from other lysosomal storage diseases, including NPC2, Batten (ceroid lipofuscinosis, neuronal 2, CLN2), Fabry, Farber, Niemann-Pick disease type A, Sanfilippo type B (mucopolysaccharidosis type IIIB, MPSIIIB), and Tay-Sachs. Our data suggest that regulated exocytosis may represent a potential therapeutic target for reduction of lysosomal storage in this class of diseases.     

Lysosomes Behave as Ca2+-regulated Exocytic Vesicles in Fibroblasts and Epithelial Cells

Lysosomes are considered to be a terminal degradative compartment of the endocytic pathway, into which transport is mostly unidirectional. However, specialized secretory vesicles regulated by Ca²⁺, such as neutrophil azurophil granules, mast cell–specific granules, and cytotoxic lymphocyte lytic granules, share characteristics with lysosomes that may reflect a common biogenesis. In addition, the involvement of Ca²⁺ transients in the invasion mechanism of the parasite Trypanosoma cruzi, which occurs by fusion of lysosomes with the plasma membrane, suggested that lysosome exocytosis might be a generalized process present in most cell types.

Here we demonstrate that elevation in the intracellular free Ca²⁺ concentration of normal rat kidney (NRK) fibroblasts induces fusion of lysosomes with the plasma membrane. This was verified by measuring the release of the lysosomal enzyme β-hexosaminidase, the appearance on the plasma membrane of the lysosomal glycoprotein lgp120, the release of fluid-phase tracers previously loaded into lysosomes, and the release of the lysosomally processed form of cathepsin D. Exposure to the Ca²⁺ ionophore ionomycin or addition of Ca²⁺containing buffers to streptolysin O–permeabilized cells induced exocytosis of ~10% of the total lysosomes of NRK cells. The process was also detected in other cell types such as epithelial cells and myoblasts. Lysosomal exocytosis was found to require micromolar levels of Ca²⁺ and to be temperature and ATP dependent, similar to Ca²⁺-regulated secretory mechanisms in specialized cells.

These findings highlight a novel role for lysosomes in cellular membrane traffic and suggest that fusion of lysosomes with the plasma membrane may be an ubiquitous form of Ca²⁺-regulated exocytosis.

     

Site-1 protease-activated formation of lysosomal targeting motifs is independent of the lipogenic transcription control

Site-1 protease (S1P) cleaves membrane-bound lipogenic sterol regulatory element-binding proteins (SREBPs) and the α/β-subunit precursor protein of the N-acetylglucosamine-1-phosphotransferase forming mannose 6-phosphate (M6P) targeting markers on lysosomal enzymes. The translocation of SREBPs from the endoplasmic reticulum (ER) to the Golgi-resident S1P depends on the intracellular sterol content, but it is unknown whether the ER exit of the α/β-subunit precursor is regulated. Here, we investigated the effect of cholesterol depletion (atorvastatin treatment) and elevation (LDL overload) on ER-Golgi transport, S1P-mediated cleavage of the α/β-subunit precursor, and the subsequent targeting of lysosomal enzymes along the biosynthetic and endocytic pathway to lysosomes. The data showed that the proteolytic cleavage of the α/β-subunit precursor into mature and enzymatically active subunits does not depend on the cholesterol content. In either treatment, lysosomal enzymes are normally decorated with M6P residues, allowing the proper sorting to lysosomes. In addition, we found that, in fibroblasts of mucolipidosis type II mice and Niemann-Pick type C patients characterized by aberrant cholesterol accumulation, the proteolytic cleavage of the α/β-subunit precursor was not impaired. We conclude that S1P substrate-dependent regulatory mechanisms for lipid synthesis and biogenesis of lysosomes are different.     

Sensitivity to Lysosome-Dependent Cell Death Is Directly Regulated by Lysosomal Cholesterol Content

Alterations in lipid homeostasis are implicated in several neurodegenerative diseases, although the mechanisms responsible are poorly understood. We evaluated the impact of cholesterol accumulation, induced by U18666A, quinacrine or mutations in the cholesterol transporting Niemann-Pick disease type C1 (NPC1) protein, on lysosomal stability and sensitivity to lysosome-mediated cell death. We found that neurons with lysosomal cholesterol accumulation were protected from oxidative stress-induced apoptosis. In addition, human fibroblasts with cholesterol-loaded lysosomes showed higher lysosomal membrane stability than controls. Previous studies have shown that cholesterol accumulation is accompanied by the storage of lipids such as sphingomyelin, glycosphingolipids and sphingosine and an up regulation of lysosomal associated membrane protein-2 (LAMP-2), which may also influence lysosomal stability. However, in this study the use of myriocin and LAMP deficient fibroblasts excluded these factors as responsible for the rescuing effect and instead suggested that primarily lysosomal cholesterol content determineD the cellular sensitivity to toxic insults. Further strengthening this concept, depletion of cholesterol using methyl-β-cyclodextrin or 25-hydroxycholesterol decreased the stability of lysosomes and cells became more prone to undergo apoptosis. In conclusion, cholesterol content regulated lysosomal membrane permeabilization and thereby influenced cell death sensitivity. Our data suggests that lysosomal cholesterol modulation might be used as a therapeutic strategy for conditions associated with accelerated or repressed apoptosis.     

Membrane cholesterol regulates lysosome-plasma membrane fusion events and modulates Trypanosoma cruzi invasion of host cells

Background

Trypomastigotes of Trypanosoma cruzi are able to invade several types of non-phagocytic cells through a lysosomal dependent mechanism. It has been shown that, during invasion, parasites trigger host cell lysosome exocytosis, which initially occurs at the parasite-host contact site. Acid sphingomyelinase released from lysosomes then induces endocytosis and parasite internalization. Lysosomes continue to fuse with the newly formed parasitophorous vacuole until the parasite is completely enclosed by lysosomal membrane, a process indispensable for a stable infection. Previous work has shown that host membrane cholesterol is also important for the T. cruzi invasion process in both professional (macrophages) and non-professional (epithelial) phagocytic cells. However, the mechanism by which cholesterol-enriched microdomains participate in this process has remained unclear.

Methodology/Principal Finding

In the present work we show that cardiomyocytes treated with MβCD, a drug able to sequester cholesterol from cell membranes, leads to a 50% reduction in invasion by T. cruzi trypomastigotes, as well as a decrease in the number of recently internalized parasites co-localizing with lysosomal markers. Cholesterol depletion from host membranes was accompanied by a decrease in the labeling of host membrane lipid rafts, as well as excessive lysosome exocytic events during the earlier stages of treatment. Precocious lysosomal exocytosis in MβCD treated cells led to a change in lysosomal distribution, with a reduction in the number of these organelles at the cell periphery, and probably compromises the intracellular pool of lysosomes necessary for T. cruzi invasion.

Conclusion/Significance

Based on these results, we propose that cholesterol depletion leads to unregulated exocytic events, reducing lysosome availability at the cell cortex and consequently compromise T. cruzi entry into host cells. The results also suggest that two different pools of lysosomes are available in the cell and that cholesterol depletion may modulate the fusion of pre-docked lysosomes at the cell cortex.     

The microtubule-binding protein CLIP-170 coordinates mDia1 and actin reorganization during CR3-mediated phagocytosis

Microtubule dynamics are modulated by regulatory proteins that bind to their plus ends (+TIPs [plus end tracking proteins]), such as cytoplasmic linker protein 170 (CLIP-170) or end-binding protein 1 (EB1). We investigated the role of +TIPs during phagocytosis in macrophages. Using RNA interference and dominant-negative approaches, we show that CLIP-170 is specifically required for efficient phagocytosis triggered by αMβ2 integrin/complement receptor activation. This property is not observed for EB1 and EB3. Accordingly, whereas CLIP-170 is dynamically enriched at the site of phagocytosis, EB1 is not. Furthermore, we observe that CLIP-170 controls the recruitment of the formin mDia1, an actin-nucleating protein, at the onset of phagocytosis and thereby controls actin polymerization events that are essential for phagocytosis. CLIP-170 directly interacts with the formin homology 2 domain of mDia1. The interaction between CLIP-170 and mDia1 is negatively regulated during αMβ2-mediated phagocytosis. Our results unravel a new microtubule/actin cooperation that involves CLIP-170 and mDia1 and that functions downstream of αMβ2 integrins.     

OxLDL induces membrane structure rearrangement leading to biomechanics alteration and migration deficiency in macrophage

Cholesterol sequestration from plasma membrane has been shown to induce lipid packing disruption, causing actin cytoskeleton reorganization and polymerization, increasing cell stiffness and inducing lysosomal exocytosis in non-professional phagocytes. Similarly, oxidized form of low-density lipoprotein (oxLDL) has also been shown to disrupt lipid organization and packing in endothelial cells, leading to biomechanics alterations that interfere with membrane injury and repair. For macrophages, much is known about oxLDL effects in cell activation, cytokine production and foam cell formation. However, little is known about its impact in the organization of macrophage membrane structured domains and cellular mechanics, the focus of the present study. Treatment of bone marrow-derived macrophages (BMDM) with oxLDL not only altered membrane structure, and potentially the distribution of raft domains, but also induced actin rearrangement, diffuse integrin distribution and cell shrinkage, similarly to observed upon treatment of these cells with MβCD. Those alterations led to decreased migration efficiency. For both treatments, higher co-localization of actin cytoskeleton and GM1 was observed, indicating a similar mechanism of action involving raft-like domain dynamics. Lastly, like MβCD treatment, oxLDL also induced lysosomal spreading in BMDM. We propose that OxLDL induced re-organization of membrane/cytoskeleton complex in macrophages can be attributed to the insertion of oxysterols into the membrane, which lead to changes in lipid organization and disruption of membrane structure, similar to the effect of cholesterol depletion by MβCD treatment. These results indicate that oxLDL can induce physical alterations in the complex membrane/cytoskeleton of macrophages, leading to significant biomechanical changes that compromise cell behavior.     

Cholesterol Down-Regulates BK Channels Stably Expressed in HEK 293 Cells

Cholesterol is one of the major lipid components of the plasma membrane in mammalian cells and is involved in the regulation of a number of ion channels. The present study investigates how large conductance Ca²⁺-activated K⁺ (BK) channels are regulated by membrane cholesterol in BK-HEK 293 cells expressing both the α-subunit hKCa1.1 and the auxiliary β1-subunit or in hKCa1.1-HEK 293 cells expressing only the α-subunit hKCa1.1 using approaches of electrophysiology, molecular biology, and immunocytochemistry. Membrane cholesterol was depleted in these cells with methyl-β-cyclodextrin (MβCD), and enriched with cholesterol-saturated MβCD (MβCD-cholesterol) or low-density lipoprotein (LDL). We found that BK current density was decreased by cholesterol enrichment in BK-HEK 293 cells, with a reduced expression of KCa1.1 protein, but not the β1-subunit protein. This effect was fully countered by the proteasome inhibitor lactacystin or the lysosome function inhibitor bafilomycin A1. Interestingly, in hKCa1.1-HEK 293 cells, the current density was not affected by cholesterol enrichment, but directly decreased by MβCD, suggesting that the down-regulation of BK channels by cholesterol depends on the auxiliary β1-subunit. The reduced KCa1.1 channel protein expression was also observed in cultured human coronary artery smooth muscle cells with cholesterol enrichment using MβCD-cholesterol or LDL. These results demonstrate the novel information that cholesterol down-regulates BK channels by reducing KCa1.1 protein expression via increasing the channel protein degradation, and the effect is dependent on the auxiliary β1-subunit.     

Activation of p61Hck triggers WASp- and Arp2/3-dependent actin-comet tail biogenesis and accelerates lysosomes

Secretory lysosomes exist in few cell types, but various mechanisms are involved to ensure their mobilization within the cytoplasm. In phagocytes, lysosome exocytosis is a regulated phenomenon at least in part under the control of the phagocyte-specific and lysosome-associated Src-kinase p61Hck (hematopoietic cell kinase). We show here that p61Hck activation triggered polymerization of actin at the membrane of lysosomes, which resulted in F-actin structures similar to comet tails observed on endocytic vesicles. We correlated this actin-comet biogenesis to a 35% acceleration of p61Hck-lysosomes in cells, which was dependent on actin polymerization and required an intact microtubular network. It was possible to initiate the formation of actin tails on p61Hck-positive lysosomes and on p61Hck-associated latex beads incubated in human phagocyte cytosolic extracts. The in vitro reconstitution on beads indicated that other lysosomal proteins were dispensable in this mechanism. The de novo actin polymerization process was functionally dependent on the kinase activity of Hck, WASp, the Arp2/3 complex, and Cdc42 but not Rac or Rho. Thus, we identified p61Hck as the first lysosomal protein able to recruit the molecular machinery responsible for actin tail formation. Altogether, our results suggest a new mechanism for lysosome motility involving p61Hck, actin-comet tail biogenesis, and the microtubule network.     

Methyl-β-cyclodextrin Suppresses Hyaluronan Synthesis by Down-regulation of Hyaluronan Synthase 2 through Inhibition of Akt

Hyaluronan synthases (HAS1–3) are integral plasma membrane proteins that synthesize hyaluronan, a cell surface and extracellular matrix polysaccharide necessary for many biological processes. It has been shown that HAS is partly localized in cholesterol-rich lipid rafts of MCF-7 cells, and cholesterol depletion with methyl-β-cyclodextrin (MβCD) suppresses hyaluronan secretion in smooth muscle cells. However, the mechanism by which cholesterol depletion inhibits hyaluronan production has remained unknown. We found that cholesterol depletion from MCF-7 cells by MβCD inhibits synthesis but does not decrease the molecular mass of hyaluronan, suggesting no major influence on HAS stability in the membrane. The inhibition of hyaluronan synthesis was not due to the availability of HAS substrates UDP-GlcUA and UDP-GlcNAc. Instead, MβCD specifically down-regulated the expression of HAS2 but not HAS1 or HAS3. Screening of signaling proteins after MβCD treatment revealed that phosphorylation of Akt and its downstream target p70S6 kinase, both members of phosphoinositide 3-kinase-Akt pathway, were inhibited. Inhibitors of this pathway suppressed hyaluronan synthesis and HAS2 expression in MCF-7 cells, suggesting that the reduced hyaluronan synthesis by MβCD is due to down-regulation of HAS2, mediated by the phosphoinositide 3-kinase-Akt-mTOR-p70S6K pathway.     

Synaptotagmin VII restricts fusion pore expansion during lysosomal exocytosis

Synaptotagmin is considered a calcium-dependent trigger for regulated exocytosis. We examined the role of synaptotagmin VII (Syt VII) in the calcium-dependent exocytosis of individual lysosomes in wild-type (WT) and Syt VII knockout (KO) mouse embryonic fibroblasts (MEFs) using total internal reflection fluorescence microscopy. In WT MEFs, most lysosomes only partially released their contents, their membrane proteins did not diffuse into the plasma membrane, and inner diameters of their fusion pores were smaller than 30 nm. In Syt VII KO MEFs, not only was lysosomal exocytosis triggered by calcium, but all of these restrictions on fusion were also removed. These observations indicate that Syt VII does not function as the calcium-dependent trigger for lysosomal exocytosis. Instead, it restricts the kinetics and extent of calcium-dependent lysosomal fusion.     

α-Synuclein and Its A30P Mutant Affect Actin Cytoskeletal Structure and Dynamics

The function of α-synuclein, a soluble protein abundant in the brain and concentrated at presynaptic terminals, is still undefined. Yet, α-synuclein overexpression and the expression of its A30P mutant are associated with familial Parkinson''s disease. Working in cell-free conditions, in two cell lines as well as in primary neurons we demonstrate that α-synuclein and its A30P mutant have different effects on actin polymerization. Wild-type α-synuclein binds actin, slows down its polymerization and accelerates its depolymerization, probably by monomer sequestration; A30P mutant α-synuclein increases the rate of actin polymerization and disrupts the cytoskeleton during reassembly of actin filaments. Consequently, in cells expressing mutant α-synuclein, cytoskeleton-dependent processes, such as cell migration, are inhibited, while exo- and endocytic traffic is altered. In hippocampal neurons from mice carrying a deletion of the α-synuclein gene, electroporation of wild-type α-synuclein increases actin instability during remodeling, with growth of lamellipodia-like structures and apparent cell enlargement, whereas A30P α-synuclein induces discrete actin-rich foci during cytoskeleton reassembly. In conclusion, α-synuclein appears to play a major role in actin cytoskeletal dynamics and various aspects of microfilament function. Actin cytoskeletal disruption induced by the A30P mutant might alter various cellular processes and thereby play a role in the pathogenesis of neurodegeneration.     

β-Cyclodextrin-threaded Biocleavable Polyrotaxanes Ameliorate Impaired Autophagic Flux in Niemann-Pick Type C Disease

Niemann-Pick type C (NPC) disease is characterized by the lysosomal accumulation of cholesterols and impaired autophagic flux due to the inhibited fusion of autophagosomes to lysosomes. We have recently developed β-cyclodextrin (β-CD)-threaded biocleavable polyrotaxanes (PRXs), which can release threaded β-CDs in response to intracellular environments as a therapeutic for NPC disease. The biocleavable PRXs exhibited effective cholesterol reduction ability and negligible toxic effect compared with hydroxypropyl-β-CD (HP-β-CD). In this study, we investigated the effect of biocleavable PRX and HP-β-CD on the impaired autophagy in NPC disease. The NPC patient-derived fibroblasts (NPC1 fibroblasts) showed an increase in the number of LC3-positive puncta compared with normal fibroblasts, even in the basal conditions; the HP-β-CD treatment markedly increased the number of LC3-positive puncta and the levels of p62 in NPC1 fibroblasts, indicating that autophagic flux was further perturbed. In sharp contrast, the biocleavable PRX reduced the number of LC3-positive puncta and the levels of p62 in NPC1 fibroblasts through an mTOR-independent mechanism. The mRFP-GFP-LC3 reporter gene expression experiments revealed that the biocleavable PRX facilitated the formation of autolysosomes to allow for autophagic protein degradation. Therefore, the β-CD-threaded biocleavable PRXs may be promising therapeutics for ameliorating not only cholesterol accumulation but also autophagy impairment in NPC disease.     

Protein kinase Cdelta and calmodulin regulate epidermal growth factor receptor recycling from early endosomes through Arp2/3 complex and cortactin

The intracellular trafficking of the epidermal growth factor receptor (EGFR) is regulated by a cross-talk between calmodulin (CaM) and protein kinase Cδ (PKCδ). On inhibition of CaM, PKCδ promotes the formation of enlarged early endosomes and blocks EGFR recycling and degradation. Here, we show that PKCδ impairs EGFR trafficking due to the formation of an F-actin coat surrounding early endosomes. The PKCδ-induced polymerization of actin is orchestrated by the Arp2/3 complex and requires the interaction of cortactin with PKCδ. Accordingly, inhibition of actin polymerization by using cytochalasin D or by overexpression of active cofilin, restored the normal morphology of the organelle and the recycling of EGFR. Similar results were obtained after down-regulation of cortactin and the sequestration of the Arp2/3 complex. Furthermore we demonstrate an interaction of cortactin with CaM and PKCδ, the latter being dependent on CaM inhibition. In summary, this study provides the first evidence that CaM and PKCδ organize actin dynamics in the early endosomal compartment, thereby regulating the intracellular trafficking of EGFR.     

NK cells eliminate Epstein-Barr virus bound to B cells through a specific antibody-mediated uptake

Epstein Barr virus (EBV) causes a highly prevalent and lifelong infection contributing to the development of some malignancies. In addition to the key role played by T cells in controlling this pathogen, NK cells mediate cytotoxicity and IFNγ production in response to EBV-infected B cells in lytic cycle, both directly and through antibody (Ab)-dependent activation. We recently described that EBV-specific Ab-dependent NK cell interaction with viral particles (VP) bound to B cells triggered degranulation and TNFα secretion but not B cell lysis nor IFNγ production. In this report we show that NK cell activation under these conditions reduced B cell transformation by EBV. NK cells eliminated VP from the surface of B cells through a specific and active process which required tyrosine kinase activation, actin polymerization and Ca2+, being independent of proteolysis and perforin. VP were displayed at the NK cell surface before being internalized and partially shuttled to early endosomes and lysosomes. VP transfer was encompassed by a trogocytosis process including the EBV receptor CD21, together with CD19 and CD20. Our study reveals a novel facet of the antibody-dependent NK cell mediated response to this viral infection.     

Membrane Localization of β-Amyloid 1–42 in Lysosomes: A POSSIBLE MECHANISM FOR LYSOSOME LABILIZATION*

β-Amyloid peptide (Aβ42) is the core protein of amyloid plaque in Alzheimer disease. The intracellular accumulation of Aβ42 in the endosomal/lysosomal system has been under investigation for many years, but the direct link between Aβ42 accumulation and dysfunction of the endosomal/lysosomal system is still largely unknown. Here, we found that both in vitro and in vivo, a major portion of Aβ42 was tightly inserted into and a small portion peripherally associated with the lysosomal membrane, whereas its soluble portion was minimal. We also found that the Aβ42 molecules inserted into the membrane tended to form multiple oligomeric aggregates, whereas Aβ40 peptides formed only dimers. Neutralizing lysosomal pH in differentiated PC12 cells decreased the lysosomal membrane insertion of Aβ42 and moderated Aβ42-induced lysosomal labilization and cytotoxicity. Our findings, thus, suggest that the membrane-inserted portion of Aβ42 accumulated in lysosomes may destabilize the lysosomal membrane and induce neurotoxicity.     

Adenovirus RID-α activates an autonomous cholesterol regulatory mechanism that rescues defects linked to Niemann-Pick disease type C

Host–pathogen interactions are important model systems for understanding fundamental cell biological processes. In this study, we describe a cholesterol-trafficking pathway induced by the adenovirus membrane protein RID-α that also subverts the cellular autophagy pathway during early stages of an acute infection. A palmitoylation-defective RID-α mutant deregulates cholesterol homeostasis and elicits lysosomal storage abnormalities similar to mutations associated with Niemann-Pick type C (NPC) disease. Wild-type RID-α rescues lipid-sorting defects in cells from patients with this disease by a mechanism involving a class III phosphatidylinositol-3-kinase. In contrast to NPC disease gene products that are localized to late endosomes/lysosomes, RID-α induces the accumulation of autophagy-like vesicles with a unique molecular composition. Ectopic RID-α regulates intracellular cholesterol trafficking at two distinct levels: the egress from endosomes and transport to the endoplasmic reticulum necessary for homeostatic gene regulation. However, RID-α also induces a novel cellular phenotype, suggesting that it activates an autonomous cholesterol regulatory mechanism distinct from NPC disease gene products.     

Plasma membrane repair is mediated by Ca(2+)-regulated exocytosis of lysosomes

Plasma membrane wounds are repaired by a mechanism involving Ca(2+)-regulated exocytosis. Elevation in intracellular [Ca(2+)] triggers fusion of lysosomes with the plasma membrane, a process regulated by the lysosomal synaptotagmin isoform Syt VII. Here, we show that Ca(2+)-regulated exocytosis of lysosomes is required for the repair of plasma membrane disruptions. Lysosomal exocytosis and membrane resealing are inhibited by the recombinant Syt VII C(2)A domain or anti-Syt VII C(2)A antibodies, or by antibodies against the cytosolic domain of Lamp-1, which specifically aggregate lysosomes. We further demonstrate that lysosomal exocytosis mediates the resealing of primary skin fibroblasts wounded during the contraction of collagen matrices. These findings reveal a fundamental, novel role for lysosomes: as Ca(2+)-regulated exocytic compartments responsible for plasma membrane repair.