Thapsigargin distinguishes membrane fusion in the late stages of endocytosis and autophagy

A close relationship exists between autophagy and endocytosis with both sharing lysosomes as their common end-point. Autophagy even requires a functional endocytic pathway. The point at which the two pathways merge, i.e., fusion of autophagosomes and endosomes with lysosomes is poorly understood. Early work in yeast and more recent studies in mammalian cells suggested that conventional membrane trafficking pathways control the fusion of autophagosomes with lysosomes; Rab GTPases are required to recruit tethering proteins which in turn coordinate the SNARE family of proteins that directly drive membrane fusion. Some components required for endosomes to fuse with lysosomes are also shared by autophagosomes; both are thought to require the GTPase Rab7 and the homotypic fusion and vacuole protein sorting (HOPS) complex. Essentially, the autophagosome becomes endosome-like, allowing it to recruit the common fusion machinery to deliver its contents to the lysosome. This raises an interesting question of how the cell determines when the autophagosome is ready to fuse with the endocytic system and bestows upon it the properties required to recruit the fusion machinery. Our recent work has highlighted this conundrum and shown that autophagosome fusion with lysosomes has specific distinctions from the parallel endosomal-lysosomal pathway.     

Beclin1-binding UVRAG targets the class C Vps complex to coordinate autophagosome maturation and endocytic trafficking

Autophagic and endocytic pathways are tightly regulated membrane rearrangement processes that are crucial for homeostasis, development and disease. Autophagic cargo is delivered from autophagosomes to lysosomes for degradation through a complex process that topologically resembles endosomal maturation. Here, we report that a Beclin1-binding autophagic tumour suppressor, UVRAG, interacts with the class C Vps complex, a key component of the endosomal fusion machinery. This interaction stimulates Rab7 GTPase activity and autophagosome fusion with late endosomes/lysosomes, thereby enhancing delivery and degradation of autophagic cargo. Furthermore, the UVRAG-class-C-Vps complex accelerates endosome-endosome fusion, resulting in rapid degradation of endocytic cargo. Remarkably, autophagosome/endosome maturation mediated by the UVRAG-class-C-Vps complex is genetically separable from UVRAG-Beclin1-mediated autophagosome formation. This result indicates that UVRAG functions as a multivalent trafficking effector that regulates not only two important steps of autophagy - autophagosome formation and maturation - but also endosomal fusion, which concomitantly promotes transport of autophagic and endocytic cargo to the degradative compartments.     

Distinct autophagosomal-lysosomal fusion mechanism revealed by thapsigargin-induced autophagy arrest

Autophagy, a catabolic pathway that delivers cellular components to lysosomes for degradation, can be activated by stressful conditions such as nutrient starvation and endoplasmic reticulum (ER) stress. We report that thapsigargin, an ER stressor widely used to induce autophagy, in fact blocks autophagy. Thapsigargin does not affect autophagosome formation but leads to accumulation of mature autophagosomes by blocking autophagosome fusion with the endocytic system. Strikingly, thapsigargin has no effect on endocytosis-mediated degradation of epidermal growth factor receptor. Molecularly, while both Rab7 and Vps16 are essential regulatory components for endocytic fusion with lysosomes, we found that Rab7 but not Vps16 is required for complete autophagy flux, and that thapsigargin blocks recruitment of Rab7 to autophagosomes. Therefore, autophagosomal-lysosomal fusion must be governed by a distinct molecular mechanism compared to general endocytic fusion.     

Drosophila Rab2 controls endosome-lysosome fusion and LAMP delivery to late endosomes

Rab2 is a conserved Rab GTPase with a well-established role in secretory pathway function and phagocytosis. Here we demonstrate that Drosophila Rab2 is recruited to late endosomal membranes, where it controls the fusion of LAMP-containing biosynthetic carriers and lysosomes to late endosomes. In contrast, the lysosomal GTPase Gie/Arl8 is only required for late endosome-lysosome fusion, but not for the delivery of LAMP to the endocytic pathway. We also find that Rab2 is required for the fusion of autophagosomes to the endolysosomal pathway, but not for the biogenesis of lysosome-related organelles. Surprisingly, Rab2 does not rely on HOPS-mediated vesicular fusion for recruitment to late endosomal membranes. Our work suggests that Drosophila Rab2 is a central regulator of the endolysosomal and macroautophagic/autophagic pathways by controlling the major heterotypic fusion processes at the late endosome.     

Inhibitory effect of intracellular lipid load on macroautophagy

Degradation of intracellular components via macroautophagy is a complex multi-step process that starts with the sequestration of cytosolic cargo in a de novo formed double-membrane vesicle or autophagosome. This compartment acquires the hydrolases required for cargo digestion by fusion with lysosomes. In contrast to the detailed molecular dissection of the components that participate in the induction, regulation and execution of the early steps in macroautophagy, through the engulfment of cargo in autophagosomes, the mechanisms involved in the lysosomal clearance of autophagosomes have been poorly characterized in mammals. One of the major limitations in this respect has been the fact that autophagosome-lysosome fusion in intact cells involves several independent steps, namely binding of the molecular motors associated to the surface of the vesicles with the cytoskeletal network, directional vesicular trafficking and fusion between the two vesicular compartments. Furthermore, both lysosomes and autophagosomes are very dynamic organelles that can fuse with different vesicular structures involved in macroautophagy, but also along the endocytic and phagocytic pathways. To resolve these limitations and directly analyze the fusion step between autophagosomes and different compartments of the endocytic-lysosomal pathway, we have recently developed an in vitro fusion assay with autophagosomes, lysosomes and endosomes isolated from cells or tissues. Fluorescent labeling of these compartments allows for the tracking of fusion events by fluorescence microscopy or by fluorescence activated cell sorting (FACS). Labeling of either membrane proteins on the surface of the organelles or dye-loading of the vesicles permits the monitoring of hemi-membrane fusion and complete vesicular fusion (cargo mixing).     

Commentary: ATG16L1 meets ATG9 in recycling endosomes: Additional roles for the plasma membrane and endocytosis in autophagosome biogenesis

Autophagosomes are formed by double-membraned structures, which engulf portions of cytoplasm. Autophagosomes ultimately fuse with lysosomes, where their contents are degraded. The origin of the autophagosome membrane may involve different sources, such as mitochondria, Golgi, endoplasmic reticulum, plasma membrane, and recycling endosomes. We recently observed that ATG9 localizes on the plasma membrane in clathrin-coated structures and is internalized following a classical endocytic pathway through early and then recycling endosomes. By contrast, ATG16L1 is also internalized by clathrin-mediated endocytosis but via different clathrin-coated pits, and appears to follow a different route to the recycling endosomes. The R-SNARE VAMP3 mediates the coalescence of the 2 different pools of vesicles (containing ATG16L1 or ATG9) in recycling endosomes. The heterotypic fusion between ATG16L1- and ATG9-containing vesicles strongly correlates with subsequent autophagosome formation. Thus, ATG9 and ATG16L1 both traffic from the plasma membrane to autophagic precursor structures and provide 2 routes from the plasma membrane to autophagosomes.     

ATG16L1 meets ATG9 in recycling endosomes

Autophagosomes are formed by double-membraned structures, which engulf portions of cytoplasm. Autophagosomes ultimately fuse with lysosomes, where their contents are degraded. The origin of the autophagosome membrane may involve different sources, such as mitochondria, Golgi, endoplasmic reticulum, plasma membrane, and recycling endosomes. We recently observed that ATG9 localizes on the plasma membrane in clathrin-coated structures and is internalized following a classical endocytic pathway through early and then recycling endosomes. By contrast, ATG16L1 is also internalized by clathrin-mediated endocytosis but via different clathrin-coated pits, and appears to follow a different route to the recycling endosomes. The R-SNARE VAMP3 mediates the coalescence of the 2 different pools of vesicles (containing ATG16L1 or ATG9) in recycling endosomes. The heterotypic fusion between ATG16L1- and ATG9-containing vesicles strongly correlates with subsequent autophagosome formation. Thus, ATG9 and ATG16L1 both traffic from the plasma membrane to autophagic precursor structures and provide 2 routes from the plasma membrane to autophagosomes.     

Mucolipidosis type IV: the importance of functional lysosomes for efficient autophagy

Mucolipidosis IV (MLIV) is a lysosomal storage disorder characterized by severe neurological and ophthalmologic abnormalities. In contrast with most lysosomal storage disorders, which are attributed to the absence of specific lysosomal hydrolases, accumulation of material in MLIV results from defects in membrane transport along the late endocytic pathway. Mutations in MCOLN1 are the cause of MLIV; however, how the lack of MCOLN1 function ultimately leads to neurodegeneration remains largely unknown. We found that MCOLN1 is required for efficient fusion of both late endosomes and autophagosomes with lysosomes. Impaired autophagosome degradation results in accumulation of autophagosomes in MLIV fibroblasts. In addition, we found increased levels and aggregation of p62, suggesting that abnormal accumulation of ubiquitinated protein inclusions may contribute to the neurodegenerative phenotype observed in MLIV patients. These findings corroborate recent evidence indicating that defects in autophagy may be a common feature of many neurodegenerative disorders.     

Rab11 facilitates cross-talk between autophagy and endosomal pathway through regulation of Hook localization

During autophagy, double-membrane autophagosomes deliver sequestered cytoplasmic content to late endosomes and lysosomes for degradation. The molecular mechanism of autophagosome maturation is still poorly characterized. The small GTPase Rab11 regulates endosomal traffic and is thought to function at the level of recycling endosomes. We show that loss of Rab11 leads to accumulation of autophagosomes and late endosomes in Drosophila melanogaster. Rab11 translocates from recycling endosomes to autophagosomes in response to autophagy induction and physically interacts with Hook, a negative regulator of endosome maturation. Hook anchors endosomes to microtubules, and we show that Rab11 facilitates the fusion of endosomes and autophagosomes by removing Hook from mature late endosomes and inhibiting its homodimerization. Thus induction of autophagy appears to promote autophagic flux by increased convergence with the endosomal pathway.     

In vitro reconstitution of fusion between immature autophagosomes and endosomes

Autophagy is a highly conserved degradative pathway whereby a double membrane engulfs cytoplasmic constituents to form an autophagic vacuole or autophagosome. An essential requirement for efficient autophagy is the acquisition of an adequate degradative capacity by the autophagosomes. To acquire this capacity the immature autophagic vacuoles (AVis) obtain lysosomal hydrolases by fusion with endosomes. The current models suggest that at least two types of endosomes, early and late, fuse with AVis to form mature, degradative AVds. This fusion and maturation requires proteins also involved in endosome maturation such as Rab7. However, it is not known if there are molecular requirements unique to AVi-endosome fusion. To identify and investigate the molecular requirements of this fusion we developed a cell-free fusion assay based on content mixing, which occurs after fusion of isolated AVis and different endosomal fractions. Our assay shows that isolated AVis can fuse to a similar extent in vitro with both early and late endosomes. Furthermore, fusion between autophagosomes and endosomes requires cytosolic and endosomal proteins, but does not show a nucleotide-dependence, and is partially N-ethylmaleimide sensitive. We also demonstrate that the lipidated form of the autophagosomal protein LC3 is dispensable for this fusion event.     

Dynein-mediated vesicle transport controls intracellular Salmonella replication

Salmonella typhimurium survives and replicates intracellular in a membrane-bound compartment, the Salmonella-containing vacuole (SCV). In HeLa cells, the SCV matures through interactions with the endocytic pathway, but Salmonella avoids fusion with mature lysosomes. The exact mechanism of the inhibition of phagolysosomal fusion is not understood. Rab GTPases control several proteins involved in membrane fusion and vesicular transport. The small GTPase Rab7 regulates the transport of and fusion between late endosomes and lysosomes and associates with the SCV. We show that the Rab7 GTPase cycle is not affected on the SCV. We then manipulated a pathway downstream of the small GTPase Rab7 in HeLa cells infected with Salmonella. Expression of the Rab7 effector RILP induces recruitment of the dynein/dynactin motor complex to the SCV. Subsequently, SCV fuse with lysosomes. As a result, the intracellular replication of Salmonella is inhibited. Activation of dynein-mediated vesicle transport can thus control intracellular survival of Salmonella.     

Brucella intracellular replication requires trafficking through the late endosomal/lysosomal compartment

Upon entry into mammalian cells, the intracellular pathogen Brucella abortus resides within a membrane-bound compartment, the Brucella -containing vacuole (BCV), the maturation of which is controlled by the bacterium to generate a replicative organelle derived from the endoplasmic reticulum (ER). Prior to reaching the ER, Brucella is believed to ensure its intracellular survival by inhibiting fusion of the intermediate BCV with late endosomes and lysosomes, although such BCVs are acidic and accumulate the lysosomal-associated membrane protein (LAMP-1). Here, we have further examined the nature of intermediate BCVs using confocal microscopy and live cell imaging. We show that BCVs rapidly acquire several late endocytic markers, including the guanosine triphosphatase Rab7 and its effector Rab-interacting lysosomal protein (RILP), and are accessible to fluid-phase markers either delivered to the whole endocytic pathway or preloaded to lysosomes, indicating that BCVs interact with late endosomes and lysosomes. Consistently, intermediate BCVs are acidic and display proteolytic activity up to 12 h post-infection. Expression of dominant-negative Rab7 or overexpression of RILP significantly impaired the ability of bacteria to convert their vacuole into an ER-derived organelle and replicate, indicating that BCV maturation requires interactions with functional late endosomal/lysosomal compartments. In cells expressing dominant-negative Rab7[T22N], BCVs remained acidic, yet displayed decreased fusion with lysosomes. Taken together, these results demonstrate that BCVs traffic along the endocytic pathway and fuse with lysosomes, and such fusion events are required for further maturation of BCVs into an ER-derived replicative organelle.     

Roles of ESCRT in autophagy-associated neurodegeneration

Autophagy is a regulated pathway for bulk degradation of cytoplasmic contents and organelles, an important process involved in many physiological and pathological conditions in multiple organs, including the nervous system. It has been proposed that developing autophagosomes fuse with late endosomal compartments before their fusion with lysosomes; however, little is known about the functional relationship between the autophagy and endocytic pathways. In the endosomal-lysosomal pathway, a key step in sorting transmembrane cargo proteins is regulated by multimeric complexes called ESCRT (endosomal sorting complex required for transport). We recently reported that dysfunction of ESCRT-III, either by depletion of its essential subunit mSnf7-2 or by expression of a mutant CHMP2B protein associated with frontotemporal dementia linked to chromosome 3 (FTD3), caused autophagosome accumulation and dendritic retraction before neurodegeneration in cultured mature cortical neurons. This defect is likely a result of an abnormal fusion process between autophagosomes and endosomal compartments or lysosomes. This study suggests that defects in the late steps of the autophagy pathway may contribute to the pathogenesis of FTD and potentially other neurodegenerative diseases.     

The role of ESCRT proteins in fusion events involving lysosomes, endosomes and autophagosomes

ESCRT (endosomal sorting complex required for transport) proteins were originally identified for their role in delivering endocytosed proteins to the intraluminal vesicles of late-endosomal structures termed multivesicular bodies. Multivesicular bodies then fuse with lysosomes, leading to degradation of the internalized proteins. Four ESCRT complexes interact to concentrate cargo on the endosomal membrane, induce membrane curvature to form an intraluminal bud and finally pinch off the bud through a membrane-scission event to produce the intraluminal vesicle. Recent work suggests that ESCRT proteins are also required downstream of these events to enable fusion of multivesicular bodies with lysosomes. Autophagy is a related pathway required for the degradation of organelles, long-lived proteins and protein aggregates which also converges on lysosomes. The proteins or organelle to be degraded are encapsulated by an autophagosome that fuses either directly with a lysosome or with an endosome to form an amphisome, which then fuses with a lysosome. A common machinery is beginning to emerge that regulates fusion events in the multivesicular body and autophagy pathways, and we focus in the present paper on the role of ESCRT proteins. These fusion events have been implicated in diseases including frontotemporal dementia, Alzheimer's disease, lysosomal storage disorders, myopathies and bacterial pathogen invasion, and therefore further examination of the mechanisms involved may lead to new insight into disease pathogenesis and treatments.     

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.     

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.     

Syntaxin 7 is localized to late endosome compartments, associates with Vamp 8, and Is required for late endosome-lysosome fusion

Protein traffic from the cell surface or the trans-Golgi network reaches the lysosome via a series of endosomal compartments. One of the last steps in the endocytic pathway is the fusion of late endosomes with lysosomes. This process has been reconstituted in vitro and has been shown to require NSF, alpha and gamma SNAP, and a Rab GTPase based on inhibition by Rab GDI. In Saccharomyces cerevisiae, fusion events to the lysosome-like vacuole are mediated by the syntaxin protein Vam3p, which is localized to the vacuolar membrane. In an effort to identify the molecular machinery that controls fusion events to the lysosome, we searched for mammalian homologues of Vam3p. One such candidate is syntaxin 7. Here we show that syntaxin 7 is concentrated in late endosomes and lysosomes. Coimmunoprecipitation experiments show that syntaxin 7 is associated with the endosomal v-SNARE Vamp 8, which partially colocalizes with syntaxin 7. Importantly, we show that syntaxin 7 is specifically required for the fusion of late endosomes with lysosomes in vitro, resulting in a hybrid organelle. Together, these data identify a SNARE complex that functions in the late endocytic system of animal cells.     

Important relationships between Rab and MICAL proteins in endocytic trafficking

The internalization of essential nutrients, lipids and receptors is a crucial process for all eukaryotic cells. Accordingly, endocytosis is highly conserved across cell types and species. Once internalized, small cargo-containing vesicles fuse with early endosomes (also known as sorting endosomes), where they undergo segregation to distinct membrane regions and are sorted and transported on through the endocytic pathway. Although the mechanisms that regulate this sorting are still poorly understood, some receptors are directed to late endosomes and lysosomes for degradation, whereas other receptors are recycled back to the plasma membrane; either directly or through recycling endosomes. The Rab family of small GTP-binding proteins plays crucial roles in regulating these trafficking pathways. Rabs cycle from inactive GDP-bound cytoplasmic proteins to active GTP-bound membrane-associated proteins, as a consequence of the activity of multiple specific GTPase-activating proteins (GAPs) and GTP exchange factors (GEFs). Once bound to GTP, Rabs interact with a multitude of effector proteins that carry out Rab-specific functions. Recent studies have shown that some of these effectors are also interaction partners for the C-terminal Eps15 homology (EHD) proteins, which are also intimately involved in endocytic regulation. A particularly interesting example of common Rab-EHD interaction partners is the MICAL-like protein, MICAL-L1. MICAL-L1 and its homolog, MICAL-L2, belong to the larger MICAL family of proteins, and both have been directly implicated in regulating endocytic recycling of cell surface receptors and junctional proteins, as well as controlling cytoskeletal rearrangement and neurite outgrowth. In this review, we summarize the functional roles of MICAL and Rab proteins, and focus on the significance of their interactions and the implications for endocytic transport.     

Small but mighty: Atg8s and Rabs in membrane dynamics during autophagy

Autophagy is a degradative pathway during which autophagosomes are formed that enwrap cytosolic material destined for turnover within the lytic compartment. Autophagosome biogenesis requires controlled lipid and membrane rearrangements to allow the formation of an autophagosomal seed and its subsequent elongation into a fully closed and fusion-competent double membrane vesicle. Different membrane remodeling events are required, which are orchestrated by the distinct autophagy machinery. An important player among these autophagy proteins is the small lipid-modifier Atg8. Atg8 proteins facilitate various aspects of autophagosome formation and serve as a binding platform for autophagy factors. Also Rab GTPases have been implicated in autophagosome biogenesis. As Atg8 proteins interact with several Rab GTPase regulators, they provide a possible link between autophagy progression and Rab GTPase activity. Here, we review central aspects in membrane dynamics during autophagosome biogenesis with a focus on Atg8 proteins and selected Rab GTPases.     

Mucolipidosis type IV: The importance of functional lysosomes for efficient autophagy

Mucolipidosis IV (MLIV) is a lysosomal storage disorder characterized by severe neurological and ophthalmologic abnormalities. In contrast with most lysosomal storage disorders, which are attributed to the absence of specific lysosomal hydrolases, accumulation of material in MLIV results from defects in membrane transport along the late endocytic pathway. Mutations in MCOLN1 are the cause of MLIV; however, how lack of MCOLN1 function ultimately leads to neurodegeneration remains largely unknown. We found that MCOLN1 is required for efficient fusion of both late endosomes and autophagosomes with lysosomes. Impaired autophagosome degradation results in accumulation of autophagosomes in MLIV fibroblasts. In addition, we found increased levels and aggregation of p62, suggesting that abnormal accumulation of ubiquitinated protein inclusions may contribute to the neurodegenerative phenotype observed in MLIV patients. These findings corroborate recent evidence indicating that defects in autophagy may be a common feature of many neurodegenerative disorders.

Addendum to: Vergarajauregui S, Connelly PS, Daniels MP, and Puertollano R. Autophagic dysfunction in mucolipidosis type IV patients. Hum Mol Genet 2008; DOI: 10.1093/hmg/ddn174.