The role of intraorganellar Ca(2+) in late endosome-lysosome heterotypic fusion and in the reformation of lysosomes from hybrid organelles

We have investigated the requirement for Ca(2+) in the fusion and content mixing of rat hepatocyte late endosomes and lysosomes in a cell-free system. Fusion to form hybrid organelles was inhibited by 1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA), but not by EGTA, and this inhibition was reversed by adding additional Ca(2+). Fusion was also inhibited by methyl ester of EGTA (EGTA-AM), a membrane permeable, hydrolyzable ester of EGTA, and pretreatment of organelles with EGTA-AM showed that the chelation of lumenal Ca(2+) reduced the amount of fusion. The requirement for Ca(2+) for fusion was a later event than the requirement for a rab protein since the system became resistant to inhibition by GDP dissociation inhibitor at earlier times than it became resistant to BAPTA. We have developed a cell-free assay to study the reformation of lysosomes from late endosome-lysosome hybrid organelles that were isolated from the rat liver. The recovery of electron dense lysosomes was shown to require ATP and was inhibited by bafilomycin and EGTA-AM. The data support a model in which endocytosed Ca(2+) plays a role in the fusion of late endosomes and lysosomes, the reformation of lysosomes, and the dynamic equilibrium of organelles in the late endocytic pathway.     

Combinatorial SNARE complexes with VAMP7 or VAMP8 define different late endocytic fusion events

Both heterotypic and homotypic fusion events are required to deliver endocytosed macromolecules to lysosomes and remodel late endocytic organelles. A trans-SNARE complex consisting of Q-SNAREs syntaxin 7, Vti1b and syntaxin 8 and the R-SNARE VAMP8 has been shown by others to be responsible for homotypic fusion of late endosomes. Using antibody inhibition experiments in rat liver cell-free systems, we confirmed this result, but found that the same Q-SNAREs can combine with an alternative R-SNARE, namely VAMP7, for heterotypic fusion between late endosomes and lysosomes. Co-immunoprecipitation demonstrated separate syntaxin 7 complexes with either VAMP7 or VAMP8 in solubilized rat liver membranes. Additionally, overexpression of the N-terminal domain of VAMP7, in cultured fibroblastic cells, inhibited the mixing of a preloaded lysosomal content marker with a marker delivered to late endosomes. These data show that combinatorial interactions of SNAREs determine whether late endosomes undergo homotypic or heterotypic fusion events.     

Membrane traffic to and from lysosomes

In the late endocytic pathway, it has been proposed that endocytosed macromolecules are delivered to a proteolytic environment by 'kiss-and-run' events or direct fusion between late endosomes and lysosomes. To test whether the fusion hypothesis accounts for delivery to lysosomes in living cells, we have used confocal microscopy to examine content mixing between lysosomes loaded with rhodamine-dextran and endosomes subsequently loaded with Oregon-Green-dextran. Both kissing and explosive fusion events were recorded. Data from cell-free content-mixing assays have suggested that fusion is initiated by tethering, which leads to formation of a trans-SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) protein complex and then release of lumenal Ca(2+), followed by membrane bilayer fusion. We have shown that the R-SNARE (arginine-containing SNARE) protein VAMP (vesicle-associated membrane protein) 7 is necessary for heterotypic fusion between late endosomes and lysosomes, whereas a different R-SNARE, VAMP 8 is required for homotypic fusion of late endosomes. After fusion of lysosomes with late endosomes, lysosomes are re-formed from the resultant hybrid organelles, a process requiring condensation of content and the removal/recycling of some membrane proteins.     

The Arf-like GTPase Arl8 Mediates Delivery of Endocytosed Macromolecules to Lysosomes in Caenorhabditis elegans

Late endocytic organelles including lysosomes are highly dynamic acidic organelles. Late endosomes and lysosomes directly fuse for content mixing to form hybrid organelles, from which lysosomes are reformed. It is not fully understood how these processes are regulated and maintained. Here we show that the Caenorhabditis elegans ARL-8 GTPase is localized primarily to lysosomes and involved in late endosome-lysosome fusion in the macrophage-like coelomocytes. Loss of arl-8 results in an increase in the number of late endosomal/lysosomal compartments, which are smaller than wild type. In arl-8 mutants, late endosomal compartments containing endocytosed macromolecules fail to fuse with lysosomal compartments enriched in the aspartic protease ASP-1. Furthermore, loss of arl-8 strongly suppresses formation of enlarged late endosome-lysosome hybrid organelles caused by mutations of cup-5, which is the orthologue of human mucolipin-1. These findings suggest that ARL-8 mediates delivery of endocytosed macromolecules to lysosomes by facilitating late endosome-lysosome fusion.     

The role of mVps18p in clustering, fusion, and intracellular localization of late endocytic organelles

Delivery of endocytosed macromolecules to mammalian cell lysosomes occurs by direct fusion of late endosomes with lysosomes, resulting in the formation of hybrid organelles from which lysosomes are reformed. The molecular mechanisms of this fusion are analogous to those of homotypic vacuole fusion in Saccharomyces cerevisiae. We report herein the major roles of the mammalian homolog of yeast Vps18p (mVps18p), a member of the homotypic fusion and vacuole protein sorting complex. When overexpressed, mVps18p caused the clustering of late endosomes/lysosomes and the recruitment of other mammalian homologs of the homotypic fusion and vacuole protein sorting complex, plus Rab7-interacting lysosomal protein. The clusters were surrounded by components of the actin cytoskeleton, including actin, ezrin, and specific unconventional myosins. Overexpression of mVps18p also overcame the effect of wortmannin treatment, which inhibits membrane traffic out of late endocytic organelles and causes their swelling. Reduction of mVps18p by RNA interference caused lysosomes to disperse away from their juxtanuclear location. Thus, mVps18p plays a critical role in endosome/lysosome tethering, fusion, intracellular localization and in the reformation of lysosomes from hybrid organelles.     

Membrane dynamics and the biogenesis of lysosomes (Review)

Lysosomes are dynamic organelles receiving membrane traffic input from the biosynthetic, endocytic and autophagic pathways. They may be regarded as storage organelles for acid hydrolases and are capable of fusing with late endosomes to form hybrid organelles where digestion of endocytosed macromolecules occurs. Reformation of lysosomes from the hybrid organelles involves content condensation and probably removal of some membrane proteins by vesicular traffic. Lysosomes can also fuse with the plasma membrane in response to cell surface damage and a rise in cytosolic Ca 2+ concentration. This process is important in plasma membrane repair. The molecular basis of membrane traffic pathways involving lysosomes is increasingly understood, in large part because of the identification of many proteins required for protein traffic to vacuoles in the yeast Saccharomyces cerevisiae. Mammalian orthologues of these proteins have been identified and studied in the processes of vesicular delivery of newly synthesized lysosomal proteins from the trans-Golgi network, fusion of lysosomes with late endosomes and sorting of membrane proteins into lumenal vesicles. Several multi-protein oligomeric complexes required for these processes have been identified. The present review focuses on current understanding of the molecular mechanisms of fusion of lysosomes with both endosomes and the plasma membrane and on the sorting events required for delivery of newly synthesized membrane proteins, endocytosed membrane proteins and other endocytosed macromolecules to lysosomes.     

Membrane dynamics and the biogenesis of lysosomes

Lysosomes are dynamic organelles receiving membrane traffic input from the biosynthetic, endocytic and autophagic pathways. They may be regarded as storage organelles for acid hydrolases and are capable of fusing with late endosomes to form hybrid organelles where digestion of endocytosed macromolecules occurs. Reformation of lysosomes from the hybrid organelles involves content condensation and probably removal of some membrane proteins by vesicular traffic. Lysosomes can also fuse with the plasma membrane in response to cell surface damage and a rise in cytosolic Ca(2+) concentration. This process is important in plasma membrane repair. The molecular basis of membrane traffic pathways involving lysosomes is increasingly understood, in large part because of the identification of many proteins required for protein traffic to vacuoles in the yeast Saccharomyces cerevisiae. Mammalian orthologues of these proteins have been identified and studied in the processes of vesicular delivery of newly synthesized lysosomal proteins from the trans-Golgi network, fusion of lysosomes with late endosomes and sorting of membrane proteins into lumenal vesicles. Several multi-protein oligomeric complexes required for these processes have been identified. The present review focuses on current understanding of the molecular mechanisms of fusion of lysosomes with both endosomes and the plasma membrane and on the sorting events required for delivery of newly synthesized membrane proteins, endocytosed membrane proteins and other endocytosed macromolecules to lysosomes.     

A role for Rab5 activity in the biogenesis of endosomal and lysosomal compartments

Rab5 is a small GTPase that plays roles in the homotypic fusion of early endosomes and regulation of intracellular vesicle transport. We show here that expression of GFP-tagged GTPase-deficient form of Rab5b (Rab5bQ79L) in NRK cells results in the sequential formation of three morphologically and functionally distinct types of endosomes. Expression of GFP-Rab5bQ79L initially caused a homotypic fusion of early endosomes accompanying a redistribution of the TGN-resident cargo molecules, and subsequent fusion with late endosomes/lysosomes, leading to the formation of giant hybrid organelles with features of early endosomes and late endosomes/lysosomes. Surprisingly, the giant endosomes gradually fragmented and shrunk, leading to the accumulation of early endosome clusters and concurrent reformation of late endosomes/lysosomes, a process accelerated by treatment with a phosphatidylinositol-3-kinase (PI(3)K) inhibitor, wortmannin. We postulate that such sequential processes reflect the biogenesis and maintenance of late endosomes/lysosomes, presumably via direct fusion with early endosomes and subsequent fission from hybrid organelles. Thus, our findings suggest a regulatory role for Rab5 in not only the early endocytic pathway, but also the late endocytic pathway, of membrane trafficking in coordination with PI(3)K activity.     

Endosome-lysosome fusion

The delivery of endocytosed cargo to lysosomes occurs through kissing and direct fusion of late endosomes/MVBs (multivesicular bodies) and lysosomes. Live-cell and electron microscopy experiments together with cell-free assays have allowed us to describe the characteristics of the delivery process and determine the core protein machinery required for fusion. The ESCRT (endosomal sorting complex required for transport) machinery is required for MVB biogenesis. The HOPS (homotypic fusion and vacuole protein sorting) complex is required for endosome-lysosome tethering and a trans-SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor) complex including the R-SNARE VAMP7 (vesicle-associated membrane protein 7) mediates endosome-lysosome membrane fusion. Protein-binding partners of VAMP7 including the clathrin adaptors AP-3 (adaptor protein 3) and Hrb (HIV Rev-binding protein) are required for its correct intracellular localization and function. Overall, co-ordination of the activities of ESCRT, HOPS and SNARE complexes are required for efficient delivery of endocytosed macromolecules to lysosomes. Endosome-lysosome fusion results in a hybrid organelle from which lysosomes are re-formed. Defects in fusion and/or lysosome reformation occur in a number of lysosome storage diseases.     

Endocytic delivery to lysosomes mediated by concurrent fusion and kissing events in living cells

In mammalian cells, macromolecules internalized by endocytosis are transported via endosomes for digestion by lysosomal acid hydrolases . The mechanism by which endosomes and lysosomes exchange content remains equivocal . However, lysosomes are reusable organelles because they remain accessible to endocytic enzyme replacement therapies and undergo content mixing with late endosomes . The maturation model, which proposes that endosomes mature into lysosomes , cannot explain these observations. Three mechanisms for content mixing have been proposed. The first is vesicular transport, best supported by a yeast cell-free assay . The second suggests that endosomes and lysosomes engage in repeated transient fusions termed "kiss-and-run" . The third is that endosomes and lysosomes fuse completely, yielding hybrid compartments from which lysosomes reform , termed "fusion-fission" . We utilized time-lapse confocal microscopy to test these hypotheses in living cells. Lysosomes were loaded with rhodamine dextran by pulse-chase, and subsequently late endosomes were loaded with Oregon green 488 dextran. Direct fusions were observed between endosomes and lysosomes, and one such event was captured by correlative electron microscopy. Fluorescence intensity analyses of endosomes that encountered lysosomes revealed a gradual accumulation of lysosomal content. Our data are compatible with a requirement for direct contact between organelles before content is exchanged.     

Lysosomes can fuse with a late endosomal compartment in a cell-free system from rat liver

The passage of pulse doses of asialoglycoproteins through the endosomal compartments of rat liver hepatocytes was studied by subcellular fractionation and EM. The kinetics of disappearance of radiolabeled asialofetuin from light endosomes prepared on Ficoll gradients were the same as the kinetics of disappearance of asialoorosomucoid-horse radish peroxidase reaction products from intracellular membrane-bound structures in the blood sinusoidal regions of hepatocytes. The light endosomes were therefore identifiable as being derived from the peripheral early endosome compartment. In contrast, the labeling of dense endosomes from the middle of the Ficoll gradient correlated with EM showing large numbers of reaction product-containing structures in the nonsinusoidal parts of the hepatocyte. In cell-free, postmitochondrial supernatants, we have previously observed that dense endosomes, but not light endosomes, interact with lysosomes. Cell-free interaction between isolated dense endosomes and lysosomes has now been reconstituted and analyzed in three ways: by transfer of radiolabeled ligand from endosomal to lysosomal densities, by a fluorescence dequenching assay which can indicate membrane fusion, and by measurement of content mixing. Maximum transfer of radiolabel to lysosomal densities required ATP and GTP plus cytosolic components, including N-ethylmaleimide-sensitive factor(s). Dense endosomes incubated in the absence of added lysosomes did not mature into vesicles of lysosomal density. Content mixing, and hence fusion, between endosomes and lysosomes was maximal in the presence of cytosol and ATP and also showed inhibition by N-ethyl-maleimide. Thus, we have demonstrated that a fusion step is involved in the transfer of radiolabeled ligand from an isolated endosome fraction derived from the nonsinusoidal regions of the hepatocyte to preexisting lysosomes in a cell-free system.     

Reconstitution of an endosome-lysosome interaction in a cell-free system

A cell-free model for the transfer of endocytosed material to lysosomes is described. Rat liver late endosomes, loaded in vivo with radiolabeled ligand by intravenous injection shortly before killing the animal, showed a specific interaction with lysosomes when incubated at 37 degrees C in the presence of cytosol and an ATP regenerating system. The location of the ligand, generally asialofetuin, was analyzed by isopycnic centrifugation on Nycodenz gradients. Appearance of radiolabel in the lysosomal position on such gradients was maximal after approximately 30 min at 37 degrees C and required the provision of undamaged cytosol, lysosomes, and an ATP regenerating system. It could not be accounted for by nonspecific bulk aggregation of membranes. Transfer occurred only from late endosomes; radiolabel in early endosomes was unaffected. Digestion of the asialofetuin, as shown by the appearance of TCA-soluble radioactivity, occurred on incubation at 37 degrees C and was increased by the provision of an ATP regenerating system.     

Purification of autophagosomes from rat hepatocytes

To facilitate the purification of rat liver autophagosomes, isolated rat hepatocytes are first incubated for 2 h at 37°C with vinblastine, which induces autophagosome accumulation by blocking the fusion of these organelles with endosomes and lysosomes. The hepatocytes are then electrodisrupted and homogenized, and the various cellular organelles sequentially removed by subcellular fractionation. A brief incubation of the homogenate with the cathepsin C substrate, glycyl-phenylalanine-naphthylamide (GPN), causes rapid osmotic disruption of the lysosomes due to intralysosomal accumulation of GPN cleavage products. Nuclei are removed by differential centrifugation, and the postnuclear supernatant subsequently fractionated on a two-step Nycodenz density gradient. Autophagosomes are recovered in an intermediate density fraction, free from cytosol and mitochondria. The autophagosomes are finally separated from the membranes and vesicles of the endoplasmic reticulum, Golgi, endosomes, etc. by sieving through a density gradient of colloidal silica particles (Percoll). The final preparation contains about 95% autophagosomes and 5% amphisomes according to morphological and biochemical criteria.     

Endosomal density shift is related to a decrease in fusion capacity.

Dinitrophenol (DNP)-beta-glucuronidase and mannosylated anti-DNP IgG, which are endocytosed by the mannose receptor and delivered to lysosomes, were previously developed as probes for examination of fusion between early endosomes in a cell-free system. In this study, these probes were found to be transported by intact cells to endocytic vesicles with heavy buoyant density at different rates, as determined by Percoll gradient fractionation of cell homogenates. There was a concomitant loss of in vitro fusion activity as the ligands moved to dense compartments. In monensin-treated cells, DNP-beta-glucuronidase was retained in a light compartment corresponding to intracellular vesicles capable of fusion in vitro. Pulse-chase studies using a DNP-derivatized transferrin-alkaline phosphatase conjugate showed that a recycling ligand was always found in light intracellular vesicles that were capable of fusion to early endosomes in vitro. In contrast to cell-free systems, intact cells sequentially labeled with DNP-beta-glucuronidase and then mannosylated anti-DNP IgG showed ligand mixing in both early and late endocytic compartments. Treatment with nocodazole or colchicine did not affect the rate of DNP-beta-glucuronidase transport to heavy vesicles in intact cells, however, the extent of ligand mixing in late endosomes was decreased by microtubule disruption. Using sequentially labeled cells split into two groups, we directly compared ligand mixing in vitro to mixing by intact cells. Fusion alone does not mediate increases in vesicle density, since DNP-beta-glucuronidase/anti-DNP IgG complexes formed in vitro were found in light vesicles, while intact cells showed immune complexes predominantly in heavy vesicles. These results suggest that the density shift is an initial step in targeting to lysosomes.     

Atg5 regulates late endosome and lysosome biogenesis

Autophagy is an evolutionarily conserved lysosome-based degradation process.Atg5 plays a very important role in autophagosome formation.Here we show that Atg5 is required for biogenesis of late endosomes and lysosomes in an autophagy-independent manner.In Atg5 cells,but not in other essential autophagy genes defecting cells,recycling and retrieval of late endosomal components from hybrid organelles are impaired,causing persistent hybrid organelles and defective formation of late endosomes and lysosomes.Defective retrieval of late endosomal components from hybrid organelles resulting from impaired recruitment of a component of V1-ATPase to acidic organelles blocks the pH-dependent retrieval of late endosomal components from hybrid organelles.Lowering the intracellular pH restores late endosome/lysosome biogenesis in Atg5 cells.Our data demonstrate an unexpected role of Atg5 and shed new light on late endosome and lysosome biogenesis.     

The HOPS Proteins hVps41 and hVps39 Are Required for Homotypic and Heterotypic Late Endosome Fusion

The homotypic fusion and protein sorting (HOPS) complex is a multisubunit tethering complex that in yeast regulates membrane fusion events with the vacuole, the yeast lysosome. Mammalian homologs of all HOPS components have been found, but little is known about their function. Here, we studied the role of hVps41 and hVps39, two components of the putative human HOPS complex, in the endo‐lysosomal pathway of human cells. By expressing hemagglutinin (HA)‐tagged constructs, we show by immunoelectron microscopy (immunoEM) that both hVps41 and hVps39 associate with the limiting membrane of late endosomes as well as lysosomes. Small interference RNA (siRNA)‐mediated knockdown of hVps41 or hVps39 resulted in an accumulation of late endosomes, a depletion in the number of lysosomes and a block in the degradation of endocytosed cargo. Lysosomal pH and cathepsin B activity remained unaltered in these conditions. By immunoEM we found that hVps41 or hVps39 knockdown impairs homotypic fusion between late endosomes as well as heterotypic fusion between late endosomes and lysosomes. Thus, our data show that both hVps41 and hVps39 are required for late endosomal–lysosomal fusion events and the delivery of endocytic cargo to lysosomes in human cells.     

In vivo endocytosis by bristle coated pits of protein tracers and their intracellular transport in the endothelial cells lining the sinuses of the liver. II. The endosomal-lysosomal transformation

The events leading to lysosomal activity in the sinus endothelium of the rat liver have been studied by means of intravascularly injected ferritin at time intervals ranging from 0.5 min to 1 hr after administration. From 6 min on, the dense body-type lysosomes contain ferritin. There are direct luminal communications of transfer tubules with these lysosomes. In time, there is a marked progressive increase in the number of ferritin-containing dense body-type lysosomes. No formation of lysosomes de novo nor a direct fusion of endosomes with lysosomes has been observed. Endosomes, however, continue to be formed as endocytosis continues. These observations are interpreted as indicating a transport of hydrolytic enzymes by the transfer tubules to the newly formed ferritin containing endosomes, which in this way are transformed into ferritin containing lysosomes. The ferritin-containing lysosomes increase considerably in size by fusing with each other. Continued endocytosis of ferritin leads to an increase of ferritin density in the dense bodies. This increase in particle density cannot be explained solely on the basis of transport by luminal fusion of the endocytic organelles, but requires an active transport mechanism. Administration of low doses of ferritin shows that the bristle coated pits of the sinus endothelium have a high degree of in vivo affinity for protein and that this endothelium must be considered to be an avid catabolic endocytic system.     

The phosphoinositide-associated protein Rush hour regulates endosomal trafficking in Drosophila

Endocytosis regulates multiple cellular processes, including the protein composition of the plasma membrane, intercellular signaling, and cell polarity. We have identified the highly conserved protein Rush hour (Rush) and show that it participates in the regulation of endocytosis. Rush localizes to endosomes via direct binding of its FYVE (Fab1p, YOTB, Vac1p, EEA1) domain to phosphatidylinositol 3-phosphate. Rush also directly binds to Rab GDP dissociation inhibitor (Gdi), which is involved in the activation of Rab proteins. Homozygous rush mutant flies are viable but show genetic interactions with mutations in Gdi, Rab5, hrs, and carnation, the fly homologue of Vps33. Overexpression of Rush disrupts progression of endocytosed cargo and increases late endosome size. Lysosomal marker staining is decreased in Rush-overexpressing cells, pointing to a defect in the transition between late endosomes and lysosomes. Rush also causes formation of endosome clusters, possibly by affecting fusion of endosomes via an interaction with the class C Vps/homotypic fusion and vacuole protein-sorting (HOPS) complex. These results indicate that Rush controls trafficking from early to late endosomes and from late endosomes to lysosomes by modulating the activity of Rab proteins.     

A fluorescence resonance energy transfer-based approach for investigating late endosome-lysosome retrograde fusion events

Traditionally, lysosomes have been considered to be a terminal endocytic compartment. Recent studies suggest that lysosomes are quite dynamic, being able to fuse with other late endocytic compartments as well as with the plasma membrane. Here we describe a quantitative fluorescence energy transfer (FRET)-based method for assessing rates of retrograde fusion between terminal lysosomes and late endosomes in living cells. Late endosomes were specifically labeled with 800-nm latex beads that were conjugated with streptavidin and Alexa Fluor 555 (FRET donor). Terminal lysosomes were specifically labeled with 10,000-MW dextran polymers conjugated with biotin and Alexa Fluor 647 (FRET acceptor). Following late endosome-lysosome fusion, the strong binding affinity between streptavidin and biotin brought the donor and acceptor fluorophore molecules into close proximity, thereby facilitating the appearance of a FRET emission signal. Because apparent size restrictions in the endocytic pathway do not permit endocytosed latex beads from reaching terminal lysosomes in an anterograde fashion, the appearance of the FRET signal is consistent with retrograde transport of lysosomal cargo back to late endosomes. We assessed the efficiency of this transport step in fibroblasts affected by different lysosome storage disorders—Niemann-Pick type C, mucolipidosis type IV, and Sandhoff’s disease, all of which have a similar lysosomal lipid accumulation phenotype. We report here, for the first time, that these disorders can be distinguished by their rate of transfer of lysosome cargos to late endosomes, and we discuss the implications of these findings for developing new therapeutic strategies.     

Fate of asialofetuin endocytosed by rat liver

We have investigated the endocytosis by rat liver of asialofetuin coupled to [125I] tyramine cellobiose: [125I] TCASF. Subcellular distribution of radioactive compounds was established after differential and isopycnic centrifugation and by analysing the fractions by SDS electrophoresis. Labelling secondary lysosomes was performed by injecting rats with Triton WR 1339 four days before injecting the protein. Results show that after being associated with endosomes [125I] TCASF is recovered in organelles where they are subjected to a first degradation, the density of these organelles is practically not affected by Triton WR 1339 injection. Later the degradation products are associated with lysosomes whose density is markedly lowered by Triton WR 1339 treatment. These observations suggest that the first intracellular organelles where [125I] TCASF is subjected to digestion are distinct from the secondary lysosome population. This could be in agreement with the hypothesis that supposes that endosomes acquire enzymes from primary lysosomes before fusion with secondary lysosomes.