Abnormalities of lysosomes in human diploid fibroblasts from patients with Farber's disease

An accumulation of ceramide associated with the deficiency of acid ceramidase has been demonstrated in cultured diploid skin fibroblasts from a patient with Farber's disease. We extend this observation to investigate the lysosomal localization of accumulated ceramide and the abnormalities of lysosomes caused by this ceramide accumulation in Farber's diseased fibroblasts. We have found that the lysosomal fraction isolated from Farber's diseased fibroblasts by a subcellular fractionation procedure is markedly low in density compared with that of normal fibroblasts and is separated from other subcellular organellers. Ultrastructural studies of the isolated lysosomal fraction from Farber's diseased fibroblasts showed in mixed population of intact and swollen vesicles with a lysosomal appearance. Examination under high magnification clearly demonstrated lysosomal inclusions which contain lamellar and curvilinear membranes and resembled those seen in the intact fibroblasts. Subcellular localization of Farber's fibroblasts showed that the accumulated [³H]ceramide from the culture medium was predominantly localized in the lysosomal fraction with a markedly low density and very little was found to be associated with other cellular membranes. Our finding that ceramide is accumulated in the lysosomal fraction of Farber's fibroblasts and that these cells also show membranous inclusions strongly suggests that the accumulation of ceramide is directly involved in the formation of lysosomal inclusions.     

Defective lysosomal enzyme secretion in kidneys of Chediak-Higashi (beige) mice

The beige mouse is an animal model for the human Chediak-Higashi syndrome, a disease characterized by giant lysosomes in most cell types. In mice, treatment with androgenic hormones causes a 20-50-fold elevation in at least one kidney lysosomal enzyme, beta-glucuronidase. Beige mice treated with androgen had significantly higher kidney beta-glucuronidase, beta-galactosidase, and N-acetyl-beta-D-glucosaminidase (hexosaminidase) levels than normal mice. Other androgen-inducible enzymes and enzyme markers for the cytosol, mitochondria, and peroxisomes were not increased in kidney of beige mice. No significant lysosomal enzyme elevation was observed in five other organs of beige mice with or without androgen treatment, nor in kidneys of beige females not treated with androgen. Histochemical staining for glucuronidase together with subcellular fractionation showed that the higher glucuronidase content of beige mouse kidney is caused by a striking accumulation of giant glucuronidase-containing lysosomes in tubule cells near the corticomedullary boundary. In normal mice lysosomal enzymes are coordinately released into the lumen of the kidney tubules and appreciable amounts of lysosomal enzymes are present in the urine. Levels of urinary lysosomal enzymes are much lower in beige mice than in normal mice. It appears that lysosomes may accumulate in beige mice because of defective exocytosis resulting either from decreased intracellular motility of lysosomes or from their improper fusion with the plasma membrane. A similar defect could account for characteristics of the Chediak-Higashi syndrome.     

Membrane Cholesterol Removal Changes Mechanical Properties of Cells and Induces Secretion of a Specific Pool of Lysosomes

In a previous study we had shown that membrane cholesterol removal induced unregulated lysosomal exocytosis events leading to the depletion of lysosomes located at cell periphery. However, the mechanism by which cholesterol triggered these exocytic events had not been uncovered. In this study we investigated the importance of cholesterol in controlling mechanical properties of cells and its connection with lysosomal exocytosis. Tether extraction with optical tweezers and defocusing microscopy were used to assess cell dynamics in mouse fibroblasts. These assays showed that bending modulus and surface tension increased when cholesterol was extracted from fibroblasts plasma membrane upon incubation with MβCD, and that the membrane-cytoskeleton relaxation time increased at the beginning of MβCD treatment and decreased at the end. We also showed for the first time that the amplitude of membrane-cytoskeleton fluctuation decreased during cholesterol sequestration, showing that these cells become stiffer. These changes in membrane dynamics involved not only rearrangement of the actin cytoskeleton, but also de novo actin polymerization and stress fiber formation through Rho activation. We found that these mechanical changes observed after cholesterol sequestration were involved in triggering lysosomal exocytosis. Exocytosis occurred even in the absence of the lysosomal calcium sensor synaptotagmin VII, and was associated with actin polymerization induced by MβCD. Notably, exocytosis triggered by cholesterol removal led to the secretion of a unique population of lysosomes, different from the pool mobilized by actin depolymerizing drugs such as Latrunculin-A. These data support the existence of at least two different pools of lysosomes with different exocytosis dynamics, one of which is directly mobilized for plasma membrane fusion after cholesterol removal.     

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.     

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.     

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.

     

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 Chediak-Higashi protein interacts with SNARE complex and signal transduction proteins

BACKGROUND:Chediak-Higashi syndrome (CHS) is an inherited immunodeficiency disease characterized by giant lysosomes and impaired leukocyte degranulation. CHS results from mutations in the lysosomal trafficking regulator (LYST) gene, which encodes a 425-kD cytoplasmic protein of unknown function. The goal of this study was to identify proteins that interact with LYST as a first step in understanding how LYST modulates lysosomal exocytosis. MATERIALS AND METHODS: Fourteen cDNA fragments, covering the entire coding domain of LYST, were used as baits to screen five human cDNA libraries by a yeast two-hybrid method, modified to allow screening in the activation and the binding domain, three selectable markers, and more stringent confirmation procedures. Five of the interactions were confirmed by an in vitro binding assay. RESULTS: Twenty-one proteins that interact with LYST were identified in yeast two-hybrid screens. Four interactions, confirmed directly, were with proteins important in vesicular transport and signal transduction (the SNARE-complex protein HRS, 14-3-3, and casein kinase II). CONCLUSIONS:On the basis of protein interactions, LYST appears to function as an adapter protein that may juxtapose proteins that mediate intracellular membrane fusion reactions. The pathologic manifestations observed in CHS patients and in mice with the homologous mutation beige suggest that understanding the role of LYST may be relevant to the treatment of not only CHS but also of diseases such as asthma, urticaria, and lupus, as well as to the molecular dissection of the CHS-associated cancer predisposition.     

Cellular expression of the beige mouse mutation and its correction in hybrids with control human fibroblasts

Summary Fibroblasts from a beige mouse (C57BL/6J;bg ^J bg^J) have been established and maintained in culture for more than 3 yr. At early passages, the mutant cells were distinguishable from C57BL/6J control mouse fibroblasts at the ultrastructural level by the presence of enlarged cytoplasmic granules. After continuous passaging, this distinguishing feature was lost from the mutant cells, correlated with their increased growth rate. Clustered, perinuclear distribution of lysosomes was retained, however, and was quantitatively different at any passage number of the beige cell line from the dispersed distribution of these organelles in control mouse fibroblasts, as analyzed by computer-aided, video-enhanced light microscopy. In somatic cell hybrids between the established beige cell line and a control human diploid fibroblast cell strain, seven uncorrected hybrid lines retained a lysosomal dispersion pattern statistically indistinguishable from that of the beige mouse cell lines. Three corrected hybrid lines had lysosomal dispersion patterns that were significantly different from the beige parent line and indistinguishable from that of the control mouse fibroblast line. Thus, lysosomal dispersion can be used objectively and quantitatively to distinguish mutant beige and control mouse fibroblasts and corrected vs. uncorrected cell hybrids made from the beige/control human somatic cell crosses.     

Ultrastructural and immunocytochemical study of skin fibroblasts from normal and sialidosis patients

The objectives of this study were to analyze morphologically, morphometrically and immunocytochemically the lysosomal compartment of normal fibroblasts and of fibroblasts with neuraminidase deficiency. The immunocytochemical analyses consisted of quantifying the distribution of saposins and -galactosidase in the lysosomes of these cells to test the hypothesis that neuraminisdase deficiency is associated with an impairment in the transport of these proteins to the lysosomal compartment. To test this idea, cultured skin fibroblasts of patients with or without sialidosis were prepared for electron microscopy and probed with antibodies against lysosomal -galactosidase and lysosomal saposins. The lysosomes of the affected cells had an abnormal accumulation of incompletely digested membranes which was associated with a significant lowering in the density of antigenic sites per lysosome. However, due to a significant increase in the number of lysosomes per affected cell, the total number of antigenic sites in control and neuraminidase deficient cells was similar. This presumably compensatory effect indicates that although the rate of production of -galactosidase and saposins remains unchanged, the transport of these molecules to the lysosomes is somehow affected. Our data also indicate that in the fibroblasts, lysosomes require a normal concentration of the three enzymes to maintain neuraminidase activity and sphingolipid degradation.     

Neuraminidase 1 is a negative regulator of lysosomal exocytosis

Lysosomal exocytosis is a Ca2+-regulated mechanism that involves proteins responsible for cytoskeletal attachment and fusion of lysosomes with the plasma membrane. However, whether luminal lysosomal enzymes contribute to this process remains unknown. Here we show that neuraminidase NEU1 negatively regulates lysosomal exocytosis in hematopoietic cells by processing the sialic acids on the lysosomal membrane protein LAMP-1. In macrophages from NEU1-deficient mice, a model of the disease sialidosis, and in patients' fibroblasts, oversialylated LAMP-1 enhances lysosomal exocytosis. Silencing of LAMP-1 reverts this phenotype by interfering with the docking of lysosomes at the plasma membrane. In neu1-/- mice the excessive exocytosis of serine proteases in the bone niche leads to inactivation of extracellular serpins, premature degradation of VCAM-1, and loss of bone marrow retention. Our findings uncover an unexpected mechanism influencing lysosomal exocytosis and argue that exacerbations of this process form the basis for certain genetic diseases.     

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.     

Binding and degradation of α2-macroglobulin by cultured fibroblasts

We studied the interactions of α₂-macroglobulin, a major protease inhibitor of plasma and of serum-containing culture medium, with cultured fibroblasts. Iodinated human α₂-macroglobulin bound specifically to washed cell layers of cultured human fibroblasts. At 0–4°C, binding was saturated at a concentration of 10–20 μg/ml. At 37°C, radiolabel appered in the medium in a form soluble in 10% trichloroacetic acid. Sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that ingested iodinated α₂-macroglobulin transiently forms a complex with a trypsin-like protease. Indirect immunofluorescence demonstrated α₂-macroglobulin in vacuoles of fibroblasts grown in 10% human serum or incubated with purified α₂-macroglobulin. Fibroblasts transformed by SV-40 (VA-13 cells) bound and degraded less ¹²⁵I-labeled α₂-macroglobulin than non-transformed fibroblasts and had fewer vacuoles containing α₂-macroglobulin. These observations indicate that cultured fibroblasts bind, take up by endocytosis, and degrade α₂-macroglobulin. Binding and endocytosis of α₂-macroglobulin by a cell may be a means of modulating proteases in the micro-environment of the cell and during endocytosis.     

The effects of sucrose loading on lysosomal hydrolases

Summary The addition of 88 mM sucrose to the culture medium of human skin fibroblasts from normal subjects caused remarkable increase in the intracellular lysosomal hydrolase activities. The mechanism of this induction by sucrose loading was carefully studied with several fibroblast strains of different inherited lysosomal storage disorders. In single lysosomal hydrolase defect such as GM₁-gangliosidosis, mannosidosis and Sandhoff disease, no induction of the deficient hydrolase was found with 88 mM sucrose loading. In contrast, sucrose loading caused normalization of intracellular lysosomal hydrolase activities in I-cell disease fibroblasts and cytoplasmic inclusion materials disappeared. Subsequent investigations reveal that I-cell disease cells are classified into three subgroups by the degree of hydrolase induction by sucrose loading; a high responding, an intermediate responding and a no-response group. The heterogeneity may be based upon different induction by sucrose loading of the enzyme, probably the residual phosphotransferase which is involved in the processing steps of lysosomal enzyme molecules. With the addition of mannose-6-phosphate and 10 mM NH₄Cl to cultured skin fibroblasts, it was shown that sucrose loading caused increased synthesis of lysosomal enzyme proteins. The result of the test with 2,4-dinitrophenol suggests that sucrose is indeed pinocytosed by cultured human skin fibroblasts and localized in lysosomes and that this event is the essential factor to trigger the induction of lysosomal hydrolases. Simultaneous loading of both invertase and sucrose in cultured cells caused no induction of -mannosidase activity. This result indicates that invertase is also pinocytosed, reaches the lysosomes and hydrolyzes sucrose in the lysosomes. Lysosomal overloading with sucrose resulted in induction of lysosomal hydrolases and invertase blocked the induction of -mannosidase activity. However, some induction still exists in -galactosidase and -fucosidase activity. Thus it is very likely that the induction of lysosomal hydrolases demands a complicated process.In this article, we investigated the effects of sucrose on the lysosomal hydrolases in cultured human skin fibroblasts of several inherited lysosomal storage disorders and normal subjects and discuss the possible mechanism. of the induction of lysosomal hydrolase activities by sucrose loading.     

Receptors for gonadotropins and prostaglandins in lysosomes of bovine corpora lutea.

The total mitochondrial fraction of bovine corpus luteum specifically bound [³H]prostaglandin (PG) E₁, [³H] PGF_2α, and ¹²⁵I-labeled human lutropin (hLH) despite very little 5′-nucleotidase activity, a marker for plasma membranes. Since the total mitochondrial fraction isolated by conventional centrifugation techniques contains both mitochondria and lysosomes, it was subfractionated into mitochondria and lysosomes to ascertain the relative contribution of these fractions to the binding. Subfractionation resulted in an enrichment of cytochrome c oxidase (a marker for mitochondria) in mitochondria and of acid phosphatase (a marker for lysosomes) in lysosomes. The lysosomes exhibited little or no contamination with Golgi vesicles, rough endoplasmic reticulum, or peroxisomes as assessed by their appropriate marker enzymes. Subfractionation also re ulted in [³H] PGE₁, [³H] PGF_2α, and ¹²⁵I-labeled hLH binding enrichment with respect to homogenate in lysosomes but not in mitochondria. The lysosomal binding enrichment and recovery were, however, lower than in plasma membranes. The ratios of marker enzyme to binding, an index of organelle contamination, revealed that plasma membrane and lysosomal receptors were intrinsic to these organelles. Freezing and thawing had markedly increased lysosomal binding but had no effect on plasma membrane binding. Exposure to 0.05% Triton X-100 resulted in a greater loss of plasma membrane compared to lysosomal binding. In summary, the above results suggest that lysosomes, but not mitochondria, in addition to plasma membranes, intrinsically contain receptors for PGs and gonadotropins. Furthermore, lysosomes overall contain a greater number of PGs and gonadotropin receptors compared to plasma membranes and these receptors are associated with the membrane but not the contents of lysosomes.     

Molecular pathology of Fabry's disease physical and kinetic properties of α-galactosidase a in cultured human endothelial cells

Fabry's disease (α-galactosidase A deficiency) is characterized by the progressive lysosomal accumulation of trihexosyl ceramide primarily in the vascular endothelium. The pathophysiologic mechanisms responsible for this preferential site of deposition were investigated in primary endothelial cell cultures established from veins of Fabry hemizygotes and umbilical veins from normal newborns. Morphologically, cultured endothelial cells from Fabry hemizygotes were strikingly different from normal cultured cells; they contained numerous, large, birefringent, cytoplasmic inclusions, presumably substrate-engorged lysosomes, observable by phase-contrast microscopy. The physical and kinetic properties of α-galactosidase A activity in normal cultured endothelial cells were determined, including (1) thermal inactivation at 50°C, $\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{≈ 15}\text{min}$; (2) elution profile on DEAE-cellulose; (3) pH optimum, 4.7; (4) specific activity, 349 nmol/h per mg protein; and (5) kinetics, apparent K_m = 2.6 mM with the 4-methylumbelliferyl substrate. These results were similar to the α-galactosidase A properties reported for other enzyme sources. In addition, preliminary glycosphingolipid analysis demonstrated relatively low levels of trihexosyl ceramide in normal cultured endothelial cells. Based on these results, a mechanism for the molecular pathology of Fabry's disease was postulated consistent with previously reported findings. It is suggested that in Fabry hemizygotes the accumulated, circulating trihexosyl ceramide may gain access to the lysosomal apparatus of the vascular endothelium by receptor-mediated lipoprotein uptake, where it is accumulated due to the defective α-galactosidase A activity.     

Lysosomal membranes from beige mice contain higher than normal levels of endoplasmic reticulum proteins

Chediak-Higashi syndrome is characterized by dysfunctional giant organelles of common origin, that is, lysosomes, melanosomes, and platelet dense bodies. Its defective gene LYST encodes a large molecular weight protein whose function is unknown. The Beige mouse also defective in Lyst is a good model of the human disease. Purified lysosomes from Beige and normal black mouse livers were used to carry out a proteomics study. Two-dimensional gel electrophoretic separation of soluble lysosomal proteins of Beige and normal mice revealed no major differences. The cleavable isotope-coded affinity tag (cICAT) technique was used to compare the composition of Beige and normal lysosomal membrane proteins. While the levels of common proteins, that is, Lamp1, Lamp2, and Niemann-Pick type C1, were decreased in Beige mice, there was an increase in the levels of endoplasmic reticulum (ER) resident proteins, for example, cytochrome P450, NADPH-cytochrome P450 oxidoreductase, and flavin-containing monooxygenase. Confocal microscopy confirmed that another ER protein, calnexin, colocalizes with Lamp1 on membranes of giant lysosomes from fibroblasts of Chediak-Higashi syndrome patient. Our results suggest that LYST may play a role in either preventing inappropriate incorporation of proteins into the lysosomal membrane or in membrane recycling/maturation.     

A Voltage-Gated Calcium Channel Regulates Lysosomal Fusion with Endosomes and Autophagosomes and Is Required for Neuronal Homeostasis

Autophagy helps deliver sequestered intracellular cargo to lysosomes for proteolytic degradation and thereby maintains cellular homeostasis by preventing accumulation of toxic substances in cells. In a forward mosaic screen in Drosophila designed to identify genes required for neuronal function and maintenance, we identified multiple cacophony (cac) mutant alleles. They exhibit an age-dependent accumulation of autophagic vacuoles (AVs) in photoreceptor terminals and eventually a degeneration of the terminals and surrounding glia. cac encodes an α1 subunit of a Drosophila voltage-gated calcium channel (VGCC) that is required for synaptic vesicle fusion with the plasma membrane and neurotransmitter release. Here, we show that cac mutant photoreceptor terminals accumulate AV-lysosomal fusion intermediates, suggesting that Cac is necessary for the fusion of AVs with lysosomes, a poorly defined process. Loss of another subunit of the VGCC, α2δ or straightjacket (stj), causes phenotypes very similar to those caused by the loss of cac, indicating that the VGCC is required for AV-lysosomal fusion. The role of VGCC in AV-lysosomal fusion is evolutionarily conserved, as the loss of the mouse homologues, Cacna1a and Cacna2d2, also leads to autophagic defects in mice. Moreover, we find that CACNA1A is localized to the lysosomes and that loss of lysosomal Cacna1a in cerebellar cultured neurons leads to a failure of lysosomes to fuse with endosomes and autophagosomes. Finally, we show that the lysosomal CACNA1A but not the plasma-membrane resident CACNA1A is required for lysosomal fusion. In summary, we present a model in which the VGCC plays a role in autophagy by regulating the fusion of AVs with lysosomes through its calcium channel activity and hence functions in maintaining neuronal homeostasis.     

The small chemical vacuolin-1 inhibits Ca(2+)-dependent lysosomal exocytosis but not cell resealing

Resealing after wounding, the process of repair following plasma membrane damage, requires exocytosis. Vacuolins are molecules that induce rapid formation of large, swollen structures derived from endosomes and lysosomes by homotypic fusion combined with uncontrolled fusion of the inner and limiting membranes of these organelles. Vacuolin-1, the most potent compound, blocks the Ca(2+)-dependent exocytosis of lysosomes induced by ionomycin or plasma membrane wounding, without affecting the process of resealing. In contrast, other cell structures and membrane trafficking functions including exocytosis of enlargeosomes are unaffected. Because cells heal normally in the presence of vacuolin-1, we suggest that lysosomes are dispensable for resealing.