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1
Liu, Kai, Ruxiao Xing, Youli Jian, Zhiyang Gao, Xinli Ma, Xiaojuan Sun, Yang Li, et al. "WDR91 is a Rab7 effector required for neuronal development." Journal of Cell Biology 216, no. 10 (August 31, 2017): 3307–21. http://dx.doi.org/10.1083/jcb.201705151.
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Early-to-late endosome conversion, which is essential for delivery of endosomal cargoes to lysosomes, requires switching of early endosome–specific Rab5 and PtdIns3P to late endosome–specific Rab7 and PtdIns(3,5)P2. In this study, we identify the WD40-repeat protein WDR91 as a Rab7 effector that couples Rab switching with PtdIns3P down-regulation on endosomes. Loss of WDR91 greatly increases endosomal PtdIns3P levels, arresting endosomes at an intermediate stage and blocking endosomal–lysosomal trafficking. WDR91 is recruited to endosomes by interacting with active guanosine triphosophate–Rab7 and inhibits Rab7-associated phosphatidylinositol 3-kinase activity. In mice, global Wdr91 knockout causes neonatal death, whereas brain-specific Wdr91 inactivation impairs brain development and causes postnatal death. Mouse neurons lacking Wdr91 accumulate giant intermediate endosomes and exhibit reduced neurite length and complexity. These phenotypes are rescued by WDR91 but not WDR91 mutants that cannot interact with Rab7. Thus, WDR91 serves as a Rab7 effector that is essential for neuronal development by facilitating endosome conversion in the endosome–lysosome pathway.
2
Wozniak, Karen L., and Stuart M. Levitz. "Cryptococcus neoformans Enters the Endolysosomal Pathway of Dendritic Cells and Is Killed by Lysosomal Components." Infection and Immunity 76, no. 10 (August 4, 2008): 4764–71. http://dx.doi.org/10.1128/iai.00660-08.
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ABSTRACT Cryptococcus neoformans is an opportunistic fungal pathogen that primarily causes disease in immunocompromised individuals. Dendritic cells (DCs) can phagocytose C. neoformans, present cryptococcal antigen, and kill C. neoformans. However, early events following C. neoformans phagocytosis by DCs are not well defined. We hypothesized that C. neoformans traffics to the endosome and the lysosome following phagocytosis by DCs and is eventually killed in the lysosome. Murine bone marrow-derived DCs (BMDCs) or human monocyte-derived DCs (HDCs) were incubated with live, encapsulated C. neoformans yeast cells and opsonizing antibody. Following incubation, DCs were intracellularly stained with antibodies against EEA1 (endosome) and LAMP-1 (late endosome/lysosome). As assessed by confocal microscopy, C. neoformans trafficked to endosomal compartments of DCs within 10 min and to lysosomal compartments within 30 min postincubation. For HDCs, the studies were repeated using complement-sufficient autologous plasma for the opsonization of C. neoformans. These data showed results similar to those for antibody opsonization, with C. neoformans localized to endosomes within 20 min and to lysosomes within 60 min postincubation. Additionally, the results of live real-time imaging studies demonstrated that C. neoformans entered lysosomal compartments within 20 min following the initiation of phagocytosis. The results of scanning and transmission electron microscopy demonstrated conventional zipper phagocytosis of C. neoformans by DCs. Finally, lysosomal extracts were purified from BMDCs and incubated with C. neoformans to determine their potential to kill C. neoformans. The extracts killed C. neoformans in a dose-dependent manner. This study shows that C. neoformans enters into endosomal and lysosomal pathways following DC phagocytosis and can be killed by lysosomal components.
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Allison, Rachel, James R. Edgar, Guy Pearson, Tania Rizo, Timothy Newton, Sven Günther, Fiamma Berner, et al. "Defects in ER–endosome contacts impact lysosome function in hereditary spastic paraplegia." Journal of Cell Biology 216, no. 5 (April 7, 2017): 1337–55. http://dx.doi.org/10.1083/jcb.201609033.
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Contacts between endosomes and the endoplasmic reticulum (ER) promote endosomal tubule fission, but the mechanisms involved and consequences of tubule fission failure are incompletely understood. We found that interaction between the microtubule-severing enzyme spastin and the ESCRT protein IST1 at ER–endosome contacts drives endosomal tubule fission. Failure of fission caused defective sorting of mannose 6-phosphate receptor, with consequently disrupted lysosomal enzyme trafficking and abnormal lysosomal morphology, including in mouse primary neurons and human stem cell–derived neurons. Consistent with a role for ER-mediated endosomal tubule fission in lysosome function, similar lysosomal abnormalities were seen in cellular models lacking the WASH complex component strumpellin or the ER morphogen REEP1. Mutations in spastin, strumpellin, or REEP1 cause hereditary spastic paraplegia (HSP), a disease characterized by axonal degeneration. Our results implicate failure of the ER–endosome contact process in axonopathy and suggest that coupling of ER-mediated endosomal tubule fission to lysosome function links different classes of HSP proteins, previously considered functionally distinct, into a unifying pathway for axonal degeneration.
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Arighi, Cecilia N., Lisa M. Hartnell, Ruben C. Aguilar, Carol R. Haft, and Juan S. Bonifacino. "Role of the mammalian retromer in sorting of the cation-independent mannose 6-phosphate receptor." Journal of Cell Biology 165, no. 1 (April 12, 2004): 123–33. http://dx.doi.org/10.1083/jcb.200312055.
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The cation-independent mannose 6-phosphate receptor (CI-MPR) mediates sorting of lysosomal hydrolase precursors from the TGN to endosomes. After releasing the hydrolase precursors into the endosomal lumen, the unoccupied receptor returns to the TGN for further rounds of sorting. Here, we show that the mammalian retromer complex participates in this retrieval pathway. The hVps35 subunit of retromer interacts with the cytosolic domain of the CI-MPR. This interaction probably occurs in an endosomal compartment, where most of the retromer is localized. In particular, retromer is associated with tubular–vesicular profiles that emanate from early endosomes or from intermediates in the maturation from early to late endosomes. Depletion of retromer by RNA interference increases the lysosomal turnover of the CI-MPR, decreases cellular levels of lysosomal hydrolases, and causes swelling of lysosomes. These observations indicate that retromer prevents the delivery of the CI-MPR to lysosomes, probably by sequestration into endosome-derived tubules from where the receptor returns to the TGN.
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Luzio, J. P., N. A. Bright, and P. R. Pryor. "The role of calcium and other ions in sorting and delivery in the late endocytic pathway." Biochemical Society Transactions 35, no. 5 (October 25, 2007): 1088–91. http://dx.doi.org/10.1042/bst0351088.
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The passage of endocytosed receptor-bound ligands and membrane proteins through the endocytic pathway of mammalian cells to lysosomes occurs via early and late endosomes. The latter contain many luminal vesicles and are often referred to as MVBs (multivesicular bodies). The overall morphology of endosomal compartments is, in major part, a consequence of the many fusion events occurring in the endocytic pathway. Kissing events and direct fusion between late endosomes and lysosomes provide a means of delivery to lysosomes. The luminal ionic composition of organelles in the endocytic pathway is of considerable importance both in the trafficking of endocytosed ligands and in the membrane fusion events. In particular, H+ ions play a role in sorting processes and providing an appropriate environment for the action of lysosomal acid hydrolases. Na+/H+ exchangers in the endosomal membrane have been implicated in the formation of MVBs and sorting into luminal vesicles. Ca2+ ions are required for fusion events and luminal content condensation in the lysosome. Consistent with an important role for luminal Ca2+ in traffic through the late endocytic pathway, mutations in the gene encoding mucolipin-1, a lysosomal non-specific cation channel, result in abnormalities in lipid traffic and are associated with the autosomal recessive lysosomal storage disease MLIV (mucolipidosis type IV).
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Gallon, Matthew, and Peter J. Cullen. "Retromer and sorting nexins in endosomal sorting." Biochemical Society Transactions 43, no. 1 (January 26, 2015): 33–47. http://dx.doi.org/10.1042/bst20140290.
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The evolutionarily conserved endosomal retromer complex rescues transmembrane proteins from the lysosomal degradative pathway and facilitates their recycling to other cellular compartments. Retromer functions in conjunction with numerous associated proteins, including select members of the sorting nexin (SNX) family. In the present article, we review the molecular architecture and cellular roles of retromer and its various functional partners. The endosomal network is a crucial hub in the trafficking of proteins through the cellular endomembrane system. Transmembrane proteins, here termed cargos, enter endosomes by endocytosis from the plasma membrane or by trafficking from the trans-Golgi network (TGN). Endosomal cargo proteins face one of the two fates: retention in the endosome, leading ultimately to lysosomal degradation or export from the endosome for reuse (‘recycling’). The balance of protein degradation and recycling is crucial to cellular homoeostasis; inappropriate sorting of proteins to either fate leads to cellular dysfunction. Retromer is an endosome-membrane-associated protein complex central to the recycling of many cargo proteins from endosomes, both to the TGN and the plasma membrane (and other specialized compartments, e.g. lysosome-related organelles). Retromer function is reliant on a number of proteins from the SNX family. In the present article, we discuss this inter-relationship and how defects in retromer function are increasingly being linked with human disease.
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Alvarez-Dominguez, C., R. Roberts, and P. D. Stahl. "Internalized Listeria monocytogenes modulates intracellular trafficking and delays maturation of the phagosome." Journal of Cell Science 110, no. 6 (March 15, 1997): 731–43. http://dx.doi.org/10.1242/jcs.110.6.731.
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Previous studies have shown that early phagosome-endosome fusion events following phagocytosis of Listeria monocytogenes are modulated by the live organism. In the present study, we have characterized more fully the intracellular pathway of dead and live Listeria phagosomes. To examine access of endosomal and lysosomal markers to phagosomes containing live and dead Listeria, quantitative electron microscopy was carried out with intact cells using internalized BSA-gold as a marker to quantify transfer of solute from endosomal and lysosomal compartments to phagosomes. To monitor the protein composition of phagosomal membranes and to quantify transfer of HRP from endosomes and lysosomes to phagosomes, highly enriched phagosomes containing live and dead Listeria were isolated. Enriched phagosomal membranes were used for western blotting experiments with endosomal and lysosomal markers. In this study, we used a listeriolysin-deficient mutant, Listeria(hly-), that is retained within the phagosome following phagocytosis. Western blotting experiments indicate that early endosomal markers (mannose receptor, transferrin receptor) and key fusion factors necessary for early events (NSF, alpha/beta-SNAP) but not late endosomal markers (cation dependent mannose 6-phosphate receptor) or lysosomal proteins (cathepsin D or lamp-1) accumulate on the live-Listeria phagosomal membranes. On the contrary, phagosomes containing dead-Listeria are readily accessible by both endocytic and lysosomal markers. Studies with radiolabeled dead- and live-Listeria(hly-) indicate that, following phagocytosis, degradation of the live microorganism is substantially delayed. These findings indicate that dead-Listeria containing phagosomes rapidly mature to a phagolysosomal stage whereas live-Listeria(hly-) prevents maturation, in part, by avoiding fusion with lysosomes. The data suggest that by delaying phagosome maturation and subsequent degradation, Listeria prolongs survival inside the phagosome/endosome assuring bacterial viability as a prelude to escape into the cytoplasm.
8
Press, Barry, Yan Feng, Bernard Hoflack, and Angela Wandinger-Ness. "Mutant Rab7 Causes the Accumulation of Cathepsin D and Cation-independent Mannose 6–Phosphate Receptor in an Early Endocytic Compartment." Journal of Cell Biology 140, no. 5 (March 9, 1998): 1075–89. http://dx.doi.org/10.1083/jcb.140.5.1075.
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Stable BHK cell lines inducibly expressing wild-type or dominant negative mutant forms of the rab7 GTPase were isolated and used to analyze the role of a rab7-regulated pathway in lysosome biogenesis. Expression of mutant rab7N125I protein induced a dramatic redistribution of cation-independent mannose 6–phosphate receptor (CI-MPR) from its normal perinuclear localization to large peripheral endosomes. Under these circumstances ∼50% of the total receptor and several lysosomal hydrolases cofractionated with light membranes containing early endosome and Golgi markers. Late endosomes and lysosomes were contained exclusively in well-separated, denser gradient fractions. Newly synthesized CI-MPR and cathepsin D were shown to traverse through an early endocytic compartment, and functional rab7 was crucial for delivery to later compartments. This observation was evidenced by the fact that 2 h after synthesis, both markers were more prevalent in fractions containing light membranes. In addition, both were sensitive to HRP-DAB– mediated cross-linking of early endosomal proteins, and the late endosomal processing of cathepsin D was impaired. Using similar criteria, the lysosomal membrane glycoprotein 120 was not found accumulated in an early endocytic compartment. The data are indicative of a post-Golgi divergence in the routes followed by different lysosome-directed molecules.
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Dores, Michael R., May M. Paing, Huilan Lin, William A. Montagne, Adriano Marchese, and JoAnn Trejo. "AP-3 regulates PAR1 ubiquitin-independent MVB/lysosomal sorting via an ALIX-mediated pathway." Molecular Biology of the Cell 23, no. 18 (September 15, 2012): 3612–23. http://dx.doi.org/10.1091/mbc.e12-03-0251.
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The sorting of signaling receptors within the endocytic system is important for appropriate cellular responses. After activation, receptors are trafficked to early endosomes and either recycled or sorted to lysosomes and degraded. Most receptors trafficked to lysosomes are modified with ubiquitin and recruited into an endosomal subdomain enriched in hepatocyte growth factor–regulated tyrosine kinase substrate (HRS), a ubiquitin-binding component of the endosomal-sorting complex required for transport (ESCRT) machinery, and then sorted into intraluminal vesicles (ILVs) of multivesicular bodies (MVBs)/lysosomes. However, not all receptors use ubiquitin or the canonical ESCRT machinery to sort to MVBs/lysosomes. This is exemplified by protease-activated receptor-1 (PAR1), a G protein–coupled receptor for thrombin, which sorts to lysosomes independent of ubiquitination and HRS. We recently showed that the adaptor protein ALIX binds to PAR1, recruits ESCRT-III, and mediates receptor sorting to ILVs of MVBs. However, the mechanism that initiates PAR1 sorting at the early endosome is not known. We now report that the adaptor protein complex-3 (AP-3) regulates PAR1 ubiquitin-independent sorting to MVBs through an ALIX-dependent pathway. AP-3 binds to a PAR1 cytoplasmic tail–localized tyrosine-based motif and mediates PAR1 lysosomal degradation independent of ubiquitination. Moreover, AP-3 facilitates PAR1 interaction with ALIX, suggesting that AP-3 functions before PAR1 engagement of ALIX and MVB/lysosomal sorting.
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Clemens, D. L., and M. A. Horwitz. "Characterization of the Mycobacterium tuberculosis phagosome and evidence that phagosomal maturation is inhibited." Journal of Experimental Medicine 181, no. 1 (January 1, 1995): 257–70. http://dx.doi.org/10.1084/jem.181.1.257.
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We have used the cryosection immunogold technique to study the composition of the Mycobacterium tuberculosis phagosome. We have used quantitative immunogold staining to determine the distribution of several known markers of the endosomal-lysosomal pathway in human monocytes after ingestion of either M. tuberculosis, Legionella pneumophila, or polystyrene beads. Compared with the other phagocytic particles studied, the M. tuberculosis phagosome exhibits delayed clearance of major histocompatibility complex (MHC) class I molecules, relatively intense staining for MHC class II molecules and the endosomal marker transferrin receptor, and relatively weak staining for the lysosomal membrane glycoproteins, CD63, LAMP-1, and LAMP-2 and the lysosomal acid protease, cathepsin D. In contrast to M. tuberculosis, the L. pneumophila phagosome rapidly clears MHC class I molecules and excludes all endosomal-lysosomal markers studied. In contrast to both live M. tuberculosis and L. pneumophila phagosomes, phagosomes containing either polystyrene beads or heat-killed M. tuberculosis stain intensely for lysosomal membrane glycoproteins and cathepsin D. These findings suggest that (a) M. tuberculosis retards the maturation of its phagosome along the endosomal-lysosomal pathway and resides in a compartment with endosomal, as opposed to lysosomal, characteristics; and (b) the intraphagosomal pathway, i.e., the pathway followed by several intracellular parasites that inhibit phagosome-lysosome fusion, is heterogeneous.
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Guerra and Bucci. "Role of the RAB7 Protein in Tumor Progression and Cisplatin Chemoresistance." Cancers 11, no. 8 (August 1, 2019): 1096. http://dx.doi.org/10.3390/cancers11081096.
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RAB7 is a small guanosine triphosphatase (GTPase) extensively studied as regulator of vesicular trafficking. Indeed, its role is fundamental in several steps of the late endocytic pathway, including endosome maturation, transport from early endosomes to late endosomes and lysosomes, clustering and fusion of late endosomes and lysosomes in the perinuclear region and lysosomal biogenesis. Besides endocytosis, RAB7 is important for a number of other cellular processes among which, autophagy, apoptosis, signaling, and cell migration. Given the importance of RAB7 in these cellular processes, the interest to study the role of RAB7 in cancer progression is widely grown. Here, we describe the current understanding of oncogenic and oncosuppressor functions of RAB7 analyzing cellular context and other environmental factors in which it elicits pro and/or antitumorigenic effects. We also discuss the role of RAB7 in cisplatin resistance associated with its ability to regulate the late endosomal pathway, lysosomal biogenesis and extracellular vesicle secretion. Finally, we examined the potential cancer therapeutic strategies targeting the different molecular events in which RAB7 is involved.
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Holleman, Justine, and Adriano Marchese. "The ubiquitin ligase deltex-3l regulates endosomal sorting of the G protein–coupled receptor CXCR4." Molecular Biology of the Cell 25, no. 12 (June 15, 2014): 1892–904. http://dx.doi.org/10.1091/mbc.e13-10-0612.
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G protein–coupled receptor (GPCR) sorting into the degradative pathway is important for limiting the duration and magnitude of signaling. Agonist activation of the GPCR CXCR4 induces its rapid ubiquitination and sorting to lysosomes via the endosomal sorting complex required for transport (ESCRT) pathway. We recently reported that ESCRT-0 ubiquitination is linked to the efficiency with which CXCR4 is sorted for lysosomal degradation; however mechanistic insight is lacking. Here we define a novel role for the really interesting new gene–domain E3 ubiquitin ligase deltex-3-like (DTX3L) in regulating CXCR4 sorting from endosomes to lysosomes. We show that DTX3L localizes to early endosomes upon CXCR4 activation and interacts directly with and inhibits the activity of the E3 ubiquitin ligase atrophin-1 interacting protein 4. This serves to limit the extent to which ESCRT-0 is ubiquitinated and is able to sort CXCR4 for lysosomal degradation. Therefore we define a novel role for DTX3L in GPCR endosomal sorting and reveal an unprecedented link between two distinct E3 ubiquitin ligases to control the activity of the ESCRT machinery.
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Robinson, L. J., F. Aniento, and J. Gruenberg. "NSF is required for transport from early to late endosomes." Journal of Cell Science 110, no. 17 (September 1, 1997): 2079–87. http://dx.doi.org/10.1242/jcs.110.17.2079.
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Protein transport between early and late endosomes is a major membrane trafficking pathway in the cell followed by many proteins, including all down-regulated receptors. Yet, little is known at the molecular level about the mechanisms regulating membrane interactions in the endocytic pathway beyond early endosomes. In this study, we have used an in vitro transport assay to study the biochemical properties of endosome docking/fusion events. Our data demonstrate that N-ethylmaleimide (NEM) sensitive factor (NSF) and its soluble associated proteins (SNAPs) are required for transport from early to late endosomes, as well as at all other steps of endosomal membrane transport. We also find that these proteins are enriched on endosomal membranes. In addition, our studies suggest that besides NSF/SNAPs, another NEM-sensitive component may also be involved in docking/fusion at this late stage of the pathway. Finally, we find that, in contrast to Golgi membranes, NSF association to both early and late endosomal membranes occurs via an ATP-independent mechanism, indicating that the binding properties of endosomal and biosynthetic NSF are different. Our data thus show that NSF/SNAPs, perhaps together with another NEM-sensitive factor, are part of the basic molecular machinery which controls docking/fusion events during transport from early to late endosomes, along the lysosomal degradation pathway.
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Metcalf, Daniel, and Adrian M. Isaacs. "The role of ESCRT proteins in fusion events involving lysosomes, endosomes and autophagosomes." Biochemical Society Transactions 38, no. 6 (November 24, 2010): 1469–73. http://dx.doi.org/10.1042/bst0381469.
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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.
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Rosales-Reyes, Roberto, Celia Alpuche-Aranda, María de la Luz Ramírez-Aguilar, Angel Denisse Castro-Eguiluz, and Vianney Ortiz-Navarrete. "Survival of Salmonella enterica Serovar Typhimurium within Late Endosomal-Lysosomal Compartments of B Lymphocytes Is Associated with the Inability To Use the Vacuolar Alternative Major Histocompatibility Complex Class I Antigen-Processing Pathway." Infection and Immunity 73, no. 7 (July 2005): 3937–44. http://dx.doi.org/10.1128/iai.73.7.3937-3944.2005.
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ABSTRACT Gamma interferon (IFN-γ)-activated macrophages use an alternative processing mechanism to present Salmonella antigens to CD8+ T lymphocytes. This pathway involves processing of antigen in a vacuolar compartment followed by secretion and loading of antigenic peptides to major histocompatibility complex class I (MHC-I) molecules on macrophage cell surface and bystander cells. In this study, we have shown that B lymphocytes are not able to process Salmonella antigens using this alternative pathway. This is due to differences in Salmonella enterica serovar Typhimurium-containing vacuoles (SCV) when comparing late endosomal-lysosomal processing compartments in B lymphocytes to those in macrophages. The IFN-γ-activated IC21 macrophage cell line and A-20 B-cell line were infected with live or dead Salmonella enterica serovar Typhimurium. The SCV in B cells were in a late endosomal-lysosomal compartment, whereas SCV in macrophages were remodeled to a noncharacteristic late endosomal-lysosomal compartment over time. Despite the difference in SCV within macrophages and B lymphocytes, S. enterica serovar Typhimurium survives more efficiently within the IFN-γ-activated B cells than in activated macrophage cell lines. Similar results were found during in vivo acute infection. We determined that a lack of remodeling of late endosomal-lysosomal compartments by live Salmonella infection in B lymphocytes is associated with the inability to use the alternative MHC-I antigen-processing pathway, providing a survival advantage to the bacterium. Our data also suggest that the B lymphocyte late endosome-lysosome environment allows the expression of Salmonella virulence mechanisms favoring B lymphocytes in addition to macrophages and dendritic cells as a reservoir during in vivo infection.
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Polanco, Juan Carlos, Gabriel Rhys Hand, Adam Briner, Chuanzhou Li, and Jürgen Götz. "Exosomes induce endolysosomal permeabilization as a gateway by which exosomal tau seeds escape into the cytosol." Acta Neuropathologica 141, no. 2 (January 8, 2021): 235–56. http://dx.doi.org/10.1007/s00401-020-02254-3.
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AbstractThe microtubule-associated protein tau has a critical role in Alzheimer’s disease and other tauopathies. A proposed pathomechanism in the progression of tauopathies is the trans-synaptic spreading of tau seeds, with a role for exosomes which are secretory nanovesicles generated by late endosomes. Our previous work demonstrated that brain-derived exosomes isolated from tau transgenic rTg4510 mice encapsulate tau seeds with the ability to induce tau aggregation in recipient cells. We had also shown that exosomes can hijack the endosomal pathway to spread through interconnected neurons. Here, we reveal how tau seeds contained within internalized exosomes exploit mechanisms of lysosomal degradation to escape the endosome and induce tau aggregation in the cytosol of HEK293T-derived ‘tau biosensor cells’. We found that the majority of the exosome-containing endosomes fused with lysosomes to form endolysosomes. Exosomes induced their permeabilization, irrespective of the presence of tau seeds, or whether the exosomal preparations originated from mouse brains or HEK293T cells. We also found that permeabilization is a conserved mechanism, operating in both non-neuronal tau biosensor cells and primary neurons. However, permeabilization of endolysosomes only occurred in a small fraction of cells, which supports the notion that permeabilization occurs by a thresholded mechanism. Interestingly, tau aggregation was only induced in cells that exhibited permeabilization, presenting this as an escape route of exosomal tau seeds into the cytosol. Overexpression of RAB7, which is required for the formation of endolysosomes, strongly increased tau aggregation. Conversely, inhibition of lysosomal function with alkalinizing agents, or by knocking-down RAB7, decreased tau aggregation. Together, we conclude that the enzymatic activities of lysosomes permeabilize exosomal and endosomal membranes, thereby facilitating access of exosomal tau seeds to cytosolic tau to induce its aggregation. Our data underscore the importance of endosomal membrane integrity in mechanisms of cellular invasion by misfolded proteins that are resistant to lysosomal degradation.
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Ludwig, T., G. Griffiths, and B. Hoflack. "Distribution of newly synthesized lysosomal enzymes in the endocytic pathway of normal rat kidney cells." Journal of Cell Biology 115, no. 6 (December 15, 1991): 1561–72. http://dx.doi.org/10.1083/jcb.115.6.1561.
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We have investigated the distribution of newly synthesized lysosomal enzymes in endocytic compartments of normal rat kidney (NRK) cells. The mannose-6-phosphate (Man6-P) containing lysosomal enzymes could be iodinated in situ after internalization of lactoperoxidase (LPO) by fluid phase endocytosis and isolated on CI-MPR affinity columns. For EM studies, the ectodomain of the CI-MPR conjugated to colloidal gold was used as a probe specific for the phosphomannosyl marker of the newly synthesized hydrolases. In NRK cells, approximately 20-40% of the phosphorylated hydrolases present in the entire pathway were found in early endocytic structures proximal to the 18 degrees C temperature block including early endosomes. These structures were characterized by a low content of endogenous CI-MPR and were accessible to fluid phase markers internalized for 5-15 min at 37 degrees C. The bulk of the phosphorylated lysosomal enzymes was found in late endocytic structures distal to the 18 degrees C block, rich in endogenous CI-MPR and accessible to endocytic markers internalized for 30-60 min at 37 degrees C. The CI-MPR negative lysosomes were devoid of phosphorylated hydrolases. This distribution was unchanged in cells treated with Man6-P to block recapture of secreted lysosomal enzymes. However, lysosomal enzymes were no longer detected in the early endosomal elements of cells treated with cycloheximide. Immunoprecipitation of cathepsin D from early endosomes of pulse-labeled cells showed that this hydrolase is a transient component of this compartment. These data indicate that in NRK cells, the earliest point of convergence of the lysosomal biosynthetic and the endocytic pathways is the early endosome.
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Marshall, Karen E., Devkee M. Vadukul, Kevin Staras, and Louise C. Serpell. "Misfolded amyloid-β-42 impairs the endosomal–lysosomal pathway." Cellular and Molecular Life Sciences 77, no. 23 (February 5, 2020): 5031–43. http://dx.doi.org/10.1007/s00018-020-03464-4.
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Abstract Misfolding and aggregation of proteins is strongly linked to several neurodegenerative diseases, but how such species bring about their cytotoxic actions remains poorly understood. Here we used specifically-designed optical reporter probes and live fluorescence imaging in primary hippocampal neurons to characterise the mechanism by which prefibrillar, oligomeric forms of the Alzheimer’s-associated peptide, Aβ42, exert their detrimental effects. We used a pH-sensitive reporter, Aβ42-CypHer, to track Aβ internalisation in real-time, demonstrating that oligomers are rapidly taken up into cells in a dynamin-dependent manner, and trafficked via the endo-lysosomal pathway resulting in accumulation in lysosomes. In contrast, a non-assembling variant of Aβ42 (vAβ42) assayed in the same way is not internalised. Tracking ovalbumin uptake into cells using CypHer or Alexa Fluor tags shows that preincubation with Aβ42 disrupts protein uptake. Our results identify a potential mechanism by which amyloidogenic aggregates impair cellular function through disruption of the endosomal–lysosomal pathway.
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Palmulli, Roberta, and Guillaume van Niel. "To be or not to be... secreted as exosomes, a balance finely tuned by the mechanisms of biogenesis." Essays in Biochemistry 62, no. 2 (May 1, 2018): 177–91. http://dx.doi.org/10.1042/ebc20170076.
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The release of extracellular vesicles such as exosomes provides an attractive intercellular communication pathway. Exosomes are 30- to 150-nm membrane vesicles that are generated in endosomal compartment and act as intercellular mediators in both physiological and pathological context. Despite the growing interest in exosome functions, the mechanisms responsible for their biogenesis and secretion are still not completely understood. Knowledge about these mechanisms is important because they control the composition, and hence the function and secretion, of exosomes. Exosomes are produced as intraluminal vesicles in extremely dynamic endosomal organelles, which undergo various maturation processes in order to form multivesicular endosomes. Notably, the function of multivesicular endosomes is balanced between exosome secretion and lysosomal degradation. In the present review, we present and discuss each intracellular trafficking pathway that has been reported or proposed as regulating exosome biogenesis, with a particular focus on the importance of endosomal dynamics in sorting out cargo proteins to exosomes and to the secretion of multivesicular endosomes. An overall picture reveals several key mechanisms, which mainly act at the crossroads of endosomal pathways as regulatory checkpoints of exosome biogenesis.
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Beaumelle, B. D., A. Gibson, and C. R. Hopkins. "Isolation and preliminary characterization of the major membrane boundaries of the endocytic pathway in lymphocytes." Journal of Cell Biology 111, no. 5 (November 1, 1990): 1811–23. http://dx.doi.org/10.1083/jcb.111.5.1811.
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Plasma membrane, coated pits, endosomes, and lysosomes were isolated from a mouse T lymphoma cell line using a density shift protocol in which these compartments were selectively loaded with gold conjugates. The plasma membrane was prepared after selective labeling for 1 h at 2 degrees C with gold-ricin and gave a yield of 40% according to enzymatic and antigenic markers. Endosomes were obtained by loading the cells for 2 h at 22 degrees C with gold complexed to an antimouse transferrin receptor mAb. Coated pits were isolated using a similar procedure, but after an incubation at 10 degrees C, which allowed deep invagination of the pits but prevented internalization. The yield (calculated using the recovery of [125I]transferrin) was 32% for endosomes and 10% for coated pits. Finally lysosomes were prepared by loading the cells for 18 h at 37 degrees C with gold low density lipoproteins (LDLs) followed by a 3-h chase at 37 degrees C with LDL alone. The final lysosome yield (based on the recovery of lysosomal enzymes) was 16%. Studies of the protein composition of these cellular compartments on two-dimensional gels showed that while some major proteins are present throughout the pathway, specific proteins can be identified in each of the isolated fractions. The greatest change in the pattern of protein constituents seen along the pathway was between endosomal and lysosomal preparations.
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Zhang, Ming, Li Chen, Shicong Wang, and Tuanlao Wang. "Rab7: roles in membrane trafficking and disease." Bioscience Reports 29, no. 3 (April 27, 2009): 193–209. http://dx.doi.org/10.1042/bsr20090032.
Full textAbstract:
The endocytosis pathway controls multiple cellular and physiological events. The lysosome is the destination of newly synthesized lysosomal hydrolytic enzymes. Internalized molecules or particles are delivered to the lysosome for degradation through sequential transport along the endocytic pathway. The endocytic pathway is also emerging as a signalling platform, in addition to the well-known role of the plasma membrane for signalling. Rab7 is a late endosome-/lysosome-associated small GTPase, perhaps the only lysosomal Rab protein identified to date. Rab7 plays critical roles in the endocytic processes. Through interaction with its partners (including upstream regulators and downstream effectors), Rab7 participates in multiple regulation mechanisms in endosomal sorting, biogenesis of lysosome [or LRO (lysosome-related organelle)] and phagocytosis. These processes are closely related to substrates degradation, antigen presentation, cell signalling, cell survival and microbial pathogen infection. Consistently, mutations or dysfunctions of Rab7 result in traffic disorders, which cause various diseases, such as neuropathy, cancer and lipid metabolism disease. Rab7 also plays important roles in microbial pathogen infection and survival, as well as in participating in the life cycle of viruses. Here, we give a brief review on the central role of Rab7 in endosomal traffic and summarize the studies focusing on the participation of Rab7 in disease pathogenesis. The underlying mechanism governed by Rab7 and its partners will also be discussed.
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Rabinowitz, S., H. Horstmann, S. Gordon, and G. Griffiths. "Immunocytochemical characterization of the endocytic and phagolysosomal compartments in peritoneal macrophages." Journal of Cell Biology 116, no. 1 (January 1, 1992): 95–112. http://dx.doi.org/10.1083/jcb.116.1.95.
Full textAbstract:
We have used endocytic and phagocytic tracers in an EM immunocytochemical study to define the compartments of the phagocytic and endocytic pathways in mouse peritoneal macrophages. Endocytosed BSA-gold appeared successively in early endosomes, spherical endosomal vesicles, a late endosomal tubuloreticular compartment (TC), and terminal lysosomes. The TC appeared as an elaborate structure enriched for the lysosomal membrane glycoproteins Lamp 1 and Lamp 2, and expressing significant levels of rab7, a late endosome-specific GTP-binding protein. The cation-independent mannose-6-phosphate receptor was restricted to specialized regions of the TC that were predominantly adjacent to the Golgi complex. Both the early endosome and the TC had coated bud structures whose composition and function are presently unknown. Phagolysosomes containing latex beads expressed the same membrane antigens and received endocytic tracers simultaneously with the TC. Since the membrane surrounding both organelles was also in direct continuity, we assume that both structures form one functional compartment. Macrosialin, an antigen confined to macrophages and dendritic cells, was heavily expressed in TC and phagolysosomal membranes with low levels being detected in other endosomal compartments and on the cell surface. Treatment of cells with wheat germ agglutinin drastically altered the morphology of the TC, giving rise to sheets of tightly adherent membrane and greatly expanded vesicles, in which cell-associated wheat germ agglutinin was concentrated. The spherical endosomal carrier vesicles loaded with internalized gold tracers clustered nearby, often making contact without fusing. Since the delivery of endocytic tracer to the TC was significantly delayed these experiments suggest that the lectin is somehow preventing the endosome vesicles from fusing with the TC. Collectively, our data argue first that the PLC is equivalent to the "tubular lysosomes" commonly described in macrophages, and second that the meeting of the phagocytic and endocytic pathway occurs in this compartment.
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Gullapalli, Anuradha, Breann L. Wolfe, Courtney T. Griffin, Terry Magnuson, and JoAnn Trejo. "An Essential Role for SNX1 in Lysosomal Sorting of Protease-activated Receptor-1: Evidence for Retromer-, Hrs-, and Tsg101-independent Functions of Sorting Nexins." Molecular Biology of the Cell 17, no. 3 (March 2006): 1228–38. http://dx.doi.org/10.1091/mbc.e05-09-0899.
Full textAbstract:
Sorting nexin 1 (SNX1) and SNX2 are the mammalian homologues of the yeast Vps5p retromer component that functions in endosome-to-Golgi trafficking. SNX1 is also implicated in endosome-to-lysosome sorting of cell surface receptors, although its requirement in this process remains to be determined. To assess SNX1 function in endocytic sorting of protease-activated receptor-1 (PAR1), we used siRNA to deplete HeLa cells of endogenous SNX1 protein. PAR1, a G-protein-coupled receptor, is proteolytically activated by thrombin, internalized, sorted predominantly to lysosomes, and efficiently degraded. Strikingly, depletion of endogenous SNX1 by siRNA markedly inhibited agonist-induced PAR1 degradation, whereas expression of a SNX1 siRNA-resistant mutant protein restored agonist-promoted PAR1 degradation in cells lacking endogenous SNX1, indicating that SNX1 is necessary for lysosomal degradation of PAR1. SNX1 is known to interact with components of the mammalian retromer complex and Hrs, an early endosomal membrane-associated protein. However, activated PAR1 degradation was not affected in cells depleted of retromer Vps26/Vps35 subunits, Hrs or Tsg101, an Hrs-interacting protein. We further show that SNX2, which dimerizes with SNX1, is not essential for lysosomal sorting of PAR1, but rather can regulate PAR1 degradation by disrupting endosomal localization of endogenous SNX1 when ectopically expressed. Together, our findings establish an essential role for endogenous SNX1 in sorting activated PAR1 to a distinct lysosomal degradative pathway that is independent of retromer, Hrs, and Tsg101.
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van Kerkhof, Peter, Cristina M. Alves dos Santos, Martin Sachse, Judith Klumperman, Guojun Bu, and Ger J. Strous. "Proteasome Inhibitors Block a Late Step in Lysosomal Transport of Selected Membrane but not Soluble Proteins." Molecular Biology of the Cell 12, no. 8 (August 2001): 2556–66. http://dx.doi.org/10.1091/mbc.12.8.2556.
Full textAbstract:
The ubiquitin-proteasome pathway acts as a regulator of the endocytosis of selected membrane proteins. Recent evidence suggests that it may also function in the intracellular trafficking of membrane proteins. In this study, several models were used to address the role of the ubiquitin-proteasome pathway in sorting of internalized proteins to the lysosome. We found that lysosomal degradation of ligands, which remain bound to their receptors within the endocytic pathway, is blocked in the presence of specific proteasome inhibitors. In contrast, a ligand that dissociates from its receptor upon endosome acidification is degraded under the same conditions. Quantitative electron microscopy showed that neither the uptake nor the overall distribution of the endocytic marker bovine serum albumin-gold is substantially altered in the presence of a proteasome inhibitor. The data suggest that the ubiquitin-proteasome pathway is involved in an endosomal sorting step of selected membrane proteins to lysosomes, thereby providing a mechanism for regulated degradation.
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Tjelle, T. E., A. Brech, L. K. Juvet, G. Griffiths, and T. Berg. "Isolation and characterization of early endosomes, late endosomes and terminal lysosomes: their role in protein degradation." Journal of Cell Science 109, no. 12 (December 1, 1996): 2905–14. http://dx.doi.org/10.1242/jcs.109.12.2905.
Full textAbstract:
Although endosomal proteolysis has been reported (e.g. for peptide hormones and lysosomal enzymes), lysosomes are believed to be the main site of degradation in the endocytic pathway. We have studied the separate roles of lysosomes and prelysosomal endocytic organelles in the degradation of ovalbumin in J774 cells. The ovalbumin was labelled with 125I-tyramine cellobiose (125I-TC-ova). The labelled degradation products formed from this probe are trapped at the site of formation. To separate lysosomes efficiently from prelysosomal endocytic organelles we allowed the cells to endocytose a pulse of colloidal gold particles complexed with ovalbumin. By combining this density shift technique with subcellular fractionation of a postnuclear supernatant in Percoll gradients we could isolate three fractions that were sequentially involved in the endocytic pathway: a light Percoll fraction, a dense Percoll fraction and a gold fraction. The light Percoll fraction contained early endosomes since it was transferrin positive and received endocytic markers such as ovalbumin and horseradish peroxidase (HRP) early (< 5 minutes) after internalization. The dense Percoll fraction was transferrin negative, rab7 positive and received endocytic markers after 10–15 minutes of internalization. The gold-filled fraction was negative for both transferrin and rab7 but highly enriched in the lysosomal enzyme beta-hexosaminidase and was therefore defined as a lysosome. To study the role of endosomes and lysosomes in the degradation of endocytosed material we allowed the cells to take up (via the mannose receptor) 125I-TC-ova. It was found that the main degradation of 125I-TC-ova (measured as acid soluble radioactivity trapped in the organelle) took place in the late endosomes (and not in the lysosomes containing the bulk of the lysosomal enzymes). Our data therefore suggest that the late endosomes operate as an early lysosomal compartment. The terminal lysosomes may serve as storage bodies for acid hydrolases that may be called upon when needed (for instance during phagocytosis).
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Goebeler, Verena, Michaela Poeter, Dagmar Zeuschner, Volker Gerke, and Ursula Rescher. "Annexin A8 Regulates Late Endosome Organization and Function." Molecular Biology of the Cell 19, no. 12 (December 2008): 5267–78. http://dx.doi.org/10.1091/mbc.e08-04-0383.
Full textAbstract:
Different classes of endosomes exhibit a characteristic intracellular steady-state distribution governed by interactions with the cytoskeleton. Late endosomes, organelles of the degradative lysosomal route, seem to require associated actin filaments for proper localization and function. We show here that the F-actin and phospholipid binding protein annexin A8 is associated specifically with late endosomes. Altering intracellular annexin A8 levels drastically affected the morphology and intracellular distribution of late endosomes. Trafficking through the degradative pathway was delayed in the absence of annexin A8, resulting in attenuated ligand-induced degradation of the epidermal growth factor receptor and prolonged epidermal growth factor-induced activation of mitogen-activated protein kinase. Depletion of annexin A8 reduced the association of late endosomal membranes with actin filaments. These results indicate that the defective cargo transport through the late endocytic pathway and the imbalanced signaling of activated receptors observed in the absence of annexin A8 results from the disturbed association of late endosomal membranes with the actin network, resulting in impaired actin-based late endosome motility.
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Block, Marc R., Molly Brunner, Théo Ziegelmeyer, Dominique Lallemand, Mylène Pezet, Genevieve Chevalier, Philippe Rondé, Cécile Gauthier-Rouviere, Bernhard Wehrle-Haller, and Daniel Bouvard. "The mechano-sensitive response of β1 integrin promotes SRC-positive late endosome recycling and activation of Yes-associated protein." Journal of Biological Chemistry 295, no. 39 (July 19, 2020): 13474–87. http://dx.doi.org/10.1074/jbc.ra120.013503.
Full textAbstract:
Yes-associated protein (YAP) signaling has emerged as a crucial pathway in several normal and pathological processes. Although the main upstream effectors that regulate its activity have been extensively studied, the role of the endosomal system has been far less characterized. Here, we identified the late endosomal/lysosomal adaptor MAPK and mTOR activator (LAMTOR) complex as an important regulator of YAP signaling in a preosteoblast cell line. We found that p18/LAMTOR1-mediated peripheral positioning of late endosomes allows delivery of SRC proto-oncogene, nonreceptor tyrosine kinase (SRC) to the plasma membrane and promotes activation of an SRC-dependent signaling cascade that controls YAP nuclear shuttling. Moreover, β1 integrin engagement and mechano-sensitive cues, such as external stiffness and related cell contractility, controlled LAMTOR targeting to the cell periphery and thereby late endosome recycling and had a major impact on YAP signaling. Our findings identify the late endosome recycling pathway as a key mechanism that controls YAP activity and explains YAP mechano-sensitivity.
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Berg, T. O., P. E. Strømhaug, T. Løvdal, P. O. Seglen, and T. Berg. "Use of glycyl-l-phenylalanine 2-naphthylamide, a lysosome-disrupting cathepsin C substrate, to distinguish between lysosomes and prelysosomal endocytic vacuoles." Biochemical Journal 300, no. 1 (May 15, 1994): 229–36. http://dx.doi.org/10.1042/bj3000229.
Full textAbstract:
Lysosome-disrupting enzyme substrates have been used to distinguish between lysosomal and prelysosomal compartments along the endocytic pathway in isolated rat hepatocytes. The cells were incubated for various periods of time with 125I-labelled tyramine cellobiose (125I-TC) covalently coupled to asialoorosomucoid (AOM) (125I-TC-AOM); this molecule is internalized by receptor-mediated endocytosis and degraded in lysosomes, where the degradation products (acid-soluble, radio-labelled short peptides) accumulate, Glycyl-L-phenylalanine 2-naphthylamide (GPN) and methionine O-methyl ester (MOM), which are hydrolysed by lysosomal cathepsin C and a lysosomal esterase respectively, both diffused into hepatocytic lysosomes after electrodisruption of the cells. Intralysosomal accumulation of the hydrolysis products (amino acids) of these substrates caused osmotic lysis of more than 90% of the lysosomes, as measured by the release of acid-soluble radioactivity derived from 125I-TC-AOM degradation. The acid-soluble radioactivity coincided in sucrose-density gradients with a major peak of the lysosomal marker enzyme acid phosphatase at 1.18 g/ml; in addition a minor, presumably endosomal, acid phosphatase peak was observed around 1.14 g/ml. The major peak of acid phosphatase was almost completely released by GPN (and by MOM), while the minor peak seemed unaffected by GPN. Acid-insoluble radioactivity, presumably in endosomes, banded (after 1 h of 125I-TC-AOM uptake) as a major peak at 1.14 and a minor peak at 1.18 g/ml in sucrose gradients, and was not significantly released by GPN. GPN thus appears to be an excellent tool by which to distinguish between endosomes and lysosomes. MOM, on the other hand, released some radioactivity and acid phosphatase from endosomes as well as from lysosomes.
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Temesvari, L. A., J. M. Bush, M. D. Peterson, K. D. Novak, M. A. Titus, and J. A. Cardelli. "Examination of the endosomal and lysosomal pathways in Dictyostelium discoideum myosin I mutants." Journal of Cell Science 109, no. 3 (March 1, 1996): 663–73. http://dx.doi.org/10.1242/jcs.109.3.663.
Full textAbstract:
The role of myosin Is in endosomal trafficking and the lysosomal system was investigated in a Dictyostelium discoideum myosin I double mutant myoB-/C-, that has been previously shown to exhibit defects in fluid-phase endocytosis during growth in suspension culture (Novak et al., 1995). Various properties of the endosomal pathway in the myoB-/C- double mutant as well as in the myoB- and myoC- single mutants, including intravesicular pH, and intracellular retention time and exocytosis of a fluid phase marker, were found to be indistinguishable from wild-type parental cells. The intimate connection between the contractile vacuole complex and the endocytic pathway in Dictyostelium, and the localization of a myosin I to the contractile vacuole in Acanthamoeba, led us to also examine the structure and function of this organelle in the three myosin I mutants. No alteration in contractile vacuole structure or function was observed in the myoB-, myoC- or myoB-/C- cell lines. The transport, processing, and localization of a lysosomal enzyme, alpha-mannosidase, were also unaltered in all three mutants. However, the myoB- and myoB-/C- cell lines, but not the myoC- cell line, were found to oversecrete the lysosomal enzymes alpha-mannosidase and acid phosphatase, during growth and starvation. None of the mutants oversecreted proteins following the constitutive secretory pathway. Two additional myosin I mutants, myoA- and myoA-/B-, were also found to oversecrete the lysosomally localized enzymes alpha-mannosidase and acid phosphatase. Taken together, these results suggest that these myosins do not play a role in the intracellular movement of vesicles, but that they may participate in controlling events that occur at the actin-rich cortical region of the cell. While no direct evidence has been found for the association of myosin Is with lysosomes, we predict that the integrity of the lysosomal system is tied to the fidelity of the actin cortex, and changes in cortical organization could influence lysosomal-related membrane events such as internalization or transit of vesicles to the cell surface.
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Petersen, Wiebke, Werner Stenzel, Olivier Silvie, Judith Blanz, Paul Saftig, Kai Matuschewski, and Alyssa Ingmundson. "Sequestration of cholesterol within the host late endocytic pathway restricts liver-stage Plasmodium development." Molecular Biology of the Cell 28, no. 6 (March 15, 2017): 726–35. http://dx.doi.org/10.1091/mbc.e16-07-0531.
Full textAbstract:
While lysosomes are degradative compartments and one of the defenses against invading pathogens, they are also hubs of metabolic activity. Late endocytic compartments accumulate around Plasmodium berghei liver-stage parasites during development, and whether this is a host defense strategy or active recruitment by the parasites is unknown. In support of the latter hypothesis, we observed that the recruitment of host late endosomes (LEs) and lysosomes is reduced in uis4− parasites, which lack a parasitophorous vacuole membrane protein and arrest during liver-stage development. Analysis of parasite development in host cells deficient for late endosomal or lysosomal proteins revealed that the Niemann–Pick type C (NPC) proteins, which are involved in cholesterol export from LEs, and the lysosome-associated membrane proteins (LAMP) 1 and 2 are important for robust liver-stage P. berghei growth. Using the compound U18666A, which leads to cholesterol sequestration in LEs similar to that seen in NPC- and LAMP-deficient cells, we show that the restriction of parasite growth depends on cholesterol sequestration and that targeting this process can reduce parasite burden in vivo. Taken together, these data reveal that proper LE and lysosome function positively contributes to liver-stage Plasmodium development.
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Lu, Albert, Francesc Tebar, Blanca Alvarez-Moya, Cristina López-Alcalá, Maria Calvo, Carlos Enrich, Neus Agell, Takeshi Nakamura, Michiyuki Matsuda, and Oriol Bachs. "A clathrin-dependent pathway leads to KRas signaling on late endosomes en route to lysosomes." Journal of Cell Biology 184, no. 6 (March 16, 2009): 863–79. http://dx.doi.org/10.1083/jcb.200807186.
Full textAbstract:
Ras proteins are small guanosine triphosphatases involved in the regulation of important cellular functions such as proliferation, differentiation, and apoptosis. Understanding the intracellular trafficking of Ras proteins is crucial to identify novel Ras signaling platforms. In this study, we report that epidermal growth factor triggers Kirsten Ras (KRas) translocation onto endosomal membranes (independently of calmodulin and protein kinase C phosphorylation) through a clathrin-dependent pathway. From early endosomes, KRas but not Harvey Ras or neuroblastoma Ras is sorted and transported to late endosomes (LEs) and lysosomes. Using yellow fluorescent protein–Raf1 and the Raichu-KRas probe, we identified for the first time in vivo–active KRas on Rab7 LEs, eliciting a signal output through Raf1. On these LEs, we also identified the p14–MP1 scaffolding complex and activated extracellular signal-regulated kinase 1/2. Abrogation of lysosomal function leads to a sustained late endosomal mitogen-activated protein kinase signal output. Altogether, this study reveals novel aspects about KRas intracellular trafficking and signaling, shedding new light on the mechanisms controlling Ras regulation in the cell.
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Peden, Andrew A., Viola Oorschot, Boris A. Hesser, Cary D. Austin, Richard H. Scheller, and Judith Klumperman. "Localization of the AP-3 adaptor complex defines a novel endosomal exit site for lysosomal membrane proteins." Journal of Cell Biology 164, no. 7 (March 29, 2004): 1065–76. http://dx.doi.org/10.1083/jcb.200311064.
Full textAbstract:
The adaptor protein (AP) 3 adaptor complex has been implicated in the transport of lysosomal membrane proteins, but its precise site of action has remained controversial. Here, we show by immuno-electron microscopy that AP-3 is associated with budding profiles evolving from a tubular endosomal compartment that also exhibits budding profiles positive for AP-1. AP-3 colocalizes with clathrin, but to a lesser extent than does AP-1. The AP-3– and AP-1–bearing tubular compartments contain endocytosed transferrin, transferrin receptor, asialoglycoprotein receptor, and low amounts of the cation-independent mannose 6-phosphate receptor and the lysosome-associated membrane proteins (LAMPs) 1 and 2. Quantitative analysis revealed that of these distinct cargo proteins, only LAMP-1 and LAMP-2 are concentrated in the AP-3–positive membrane domains. Moreover, recycling of endocytosed LAMP-1 and CD63 back to the cell surface is greatly increased in AP-3–deficient cells. Based on these data, we propose that AP-3 defines a novel pathway by which lysosomal membrane proteins are transported from tubular sorting endosomes to lysosomes.
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Pieters, J., O. Bakke, and B. Dobberstein. "The MHC class II-associated invariant chain contains two endosomal targeting signals within its cytoplasmic tail." Journal of Cell Science 106, no. 3 (November 1, 1993): 831–46. http://dx.doi.org/10.1242/jcs.106.3.831.
Full textAbstract:
The oligomeric complex formed by major histocompatibility complex (MHC) class II alpha and beta chains and invariant chain (Ii) assembles in the endoplasmic reticulum and is then transported via the Golgi complex to compartments of the endocytic pathway. When Ii alone is expressed in CV1 cells it is sorted to endosomes. The Ii cytoplasmic tail has been found to be essential for targeting to these compartments. In order to characterize further the signals responsible for endosomal targeting, we have deleted various segments of the cytoplasmic tail. The Ii mutants were transiently expressed and the cellular location of the proteins was analyzed biochemically and morphologically. The cytoplasmic tail of Ii was found to contain two endosomal targeting sequences within its cytoplasmic tail; one targeting sequence was present within amino acid residues 12–29 and deletion of this segment revealed the presence of a second endosomal targeting sequence, located within the first 11 amino acid residues. The presence of a leucine-isoleucine pair at positions 7 and 8 within this sequence was found to be essential for endosomal targeting. In addition, the presence of this L-I motif lead to accumulation of Ii molecules in large endosomal vacuoles containing lysosomal marker proteins. Both wild type Ii and Ii mutant molecules containing only one endosomal targeting sequence were rapidly internalized from the plasma membrane. When the Ii cytoplasmic tail was fused to the membrane-spanning region of neuraminidase, a resident plasma membrane protein, the resulting chimera (INA) was found in endocytic compartments containing lysosomal marker proteins. Thus the cytoplasmic tail of Ii is sufficient for targeting to the endocytic/lysosomal pathway.
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Lally, Edward T., Kathleen Boesze-Battaglia, Anuradha Dhingra, Nestor M. Gomez, Jinery Lora, Claire H. Mitchell, Alexander Giannakakis, Syed A. Fahim, Roland Benz, and Nataliya Balashova. "Aggregatibacter actinomycetemcomitans LtxA Hijacks Endocytic Trafficking Pathways in Human Lymphocytes." Pathogens 9, no. 2 (January 21, 2020): 74. http://dx.doi.org/10.3390/pathogens9020074.
Full textAbstract:
Leukotoxin (LtxA), from oral pathogen Aggregatibacter actinomycetemcomitans, is a secreted membrane-damaging protein. LtxA is internalized by β2 integrin LFA-1 (CD11a/CD18)-expressing leukocytes and ultimately causes cell death; however, toxin localization in the host cell is poorly understood and these studies fill this void. We investigated LtxA trafficking using multi-fluor confocal imaging, flow cytometry and Rab5a knockdown in human T lymphocyte Jurkat cells. Planar lipid bilayers were used to characterize LtxA pore-forming activity at different pHs. Our results demonstrate that the LtxA/LFA-1 complex gains access to the cytosol of Jurkat cells without evidence of plasma membrane damage, utilizing dynamin-dependent and presumably clathrin-independent mechanisms. Upon internalization, LtxA follows the LFA-1 endocytic trafficking pathways, as identified by co-localization experiments with endosomal and lysosomal markers (Rab5, Rab11A, Rab7, and Lamp1) and CD11a. Knockdown of Rab5a resulted in the loss of susceptibility of Jurkat cells to LtxA cytotoxicity, suggesting that late events of LtxA endocytic trafficking are required for toxicity. Toxin trafficking via the degradative endocytic pathway may culminate in the delivery of the protein to lysosomes or its accumulation in Rab11A-dependent recycling endosomes. The ability of LtxA to form pores at acidic pH may result in permeabilization of the endosomal and lysosomal membranes.
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Urbé, S., J. McCullough, P. Row, I. A. Prior, R. Welchman, and M. J. Clague. "Control of growth factor receptor dynamics by reversible ubiquitination." Biochemical Society Transactions 34, no. 5 (October 1, 2006): 754–56. http://dx.doi.org/10.1042/bst0340754.
Full textAbstract:
Activated tyrosine kinase receptors acquire ubiquitin tags. Ubiquitination governs receptor down-regulation through interaction with components of the endosomal ESCRT (endosomal sorting complexes required for transport) machinery that shepherds receptors into luminal vesicles of multivesicular bodies en route to the lysosome. We have characterized two de-ubiquitinating enzymes that interact with components of this machinery. AMSH [associated molecule with the SH3 domain (Src homology 3 domain) of STAM (signal transducing adapter molecule)] shows specificity for Lys63- over Lys48-linked ubiquitin and may act to rescue receptors from taking the lysosomal pathway. In contrast, UBPY (ubiquitin-specific processing protease Y) does not discriminate between Lys48 and Lys63-linked chains and is required for lysosomal sorting.
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Rana, Meenakshi, Jens Lachmann, and Christian Ungermann. "Identification of a Rab GTPase-activating protein cascade that controls recycling of the Rab5 GTPase Vps21 from the vacuole." Molecular Biology of the Cell 26, no. 13 (July 2015): 2535–49. http://dx.doi.org/10.1091/mbc.e15-02-0062.
Full textAbstract:
Transport within the endocytic pathway depends on a consecutive function of the endosomal Rab5 and the late endosomal/lysosomal Rab7 GTPases to promote membrane recycling and fusion in the context of endosomal maturation. We previously identified the hexameric BLOC-1 complex as an effector of the yeast Rab5 Vps21, which also recruits the GTPase-activating protein (GAP) Msb3. This raises the question of when Vps21 is inactivated on endosomes. We provide evidence for a Rab cascade in which activation of the Rab7 homologue Ypt7 triggers inactivation of Vps21. We find that the guanine nucleotide exchange factor (GEF) of Ypt7 (the Mon1-Ccz1 complex) and BLOC-1 both localize to the same endosomes. Overexpression of Mon1-Ccz1, which generates additional Ypt7-GTP, or overexpression of activated Ypt7 promotes relocalization of Vps21 from endosomes to the endoplasmic reticulum (ER), which is indicative of Vps21 inactivation. This ER relocalization is prevented by loss of either BLOC-1 or Msb3, but it also occurs in mutants lacking endosome–vacuole fusion machinery such as the HOPS tethering complex, an effector of Ypt7. Importantly, BLOC-1 interacts with the HOPS on vacuoles, suggesting a direct Ypt7-dependent cross-talk. These data indicate that efficient Vps21 recycling requires both Ypt7 and endosome–vacuole fusion, thus suggesting extended control of a GAP cascade beyond Rab interactions.
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Vergne, Isabelle, Rutilio A. Fratti, Preston J. Hill, Jennifer Chua, John Belisle, and Vojo Deretic. "Mycobacterium tuberculosisPhagosome Maturation Arrest: Mycobacterial Phosphatidylinositol Analog Phosphatidylinositol Mannoside Stimulates Early Endosomal Fusion." Molecular Biology of the Cell 15, no. 2 (February 2004): 751–60. http://dx.doi.org/10.1091/mbc.e03-05-0307.
Full textAbstract:
Mycobacterium tuberculosis is a facultative intracellular pathogen that parasitizes macrophages by modulating properties of the Mycobacterium-containing phagosome. Mycobacterial phagosomes do not fuse with late endosomal/lysosomal organelles but retain access to early endosomal contents by an unknown mechanism. We have previously reported that mycobacterial phosphatidylinositol analog lipoarabinomannan (LAM) blocks a trans-Golgi network-to-phagosome phosphatidylinositol 3-kinase-dependent pathway. In this work, we extend our investigations of the effects of mycobacterial phosphoinositides on host membrane trafficking. We present data demonstrating that phosphatidylinositol mannoside (PIM) specifically stimulated homotypic fusion of early endosomes in an ATP-, cytosol-, and N-ethylmaleimide sensitive factor-dependent manner. The fusion showed absolute requirement for small Rab GTPases, and the stimulatory effect of PIM increased upon partial depletion of membrane Rabs with RabGDI. We found that stimulation of early endosomal fusion by PIM was higher when phosphatidylinositol 3-kinase was inhibited by wortmannin. PIM also stimulated in vitro fusion between model phagosomes and early endosomes. Finally, PIM displayed in vivo effects in macrophages by increasing accumulation of plasma membrane-endosomal syntaxin 4 and transferrin receptor on PIM-coated latex bead phagosomes. In addition, inhibition of phagosomal acidification was detected with PIM-coated beads. The effects of PIM, along with the previously reported action of LAM, suggest that M. tuberculosis has evolved a two-prong strategy to modify its intracellular niche: its products block acquisition of late endosomal/lysosomal constituents, while facilitating fusion with early endosomal compartments.
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Zhang, Jialin, Jianfei Chen, Da Shi, Hongyan Shi, Xin Zhang, Jianbo Liu, Liyan Cao, et al. "Porcine deltacoronavirus enters cells via two pathways: A protease-mediated one at the cell surface and another facilitated by cathepsins in the endosome." Journal of Biological Chemistry 294, no. 25 (May 8, 2019): 9830–43. http://dx.doi.org/10.1074/jbc.ra119.007779.
Full textAbstract:
Porcine deltacoronavirus (PDCoV) is a pathogen belonging to the genus Deltacoronavirus that in 2014 caused outbreaks of piglet diarrhea in the United States. To identify suitable therapeutic targets, a more comprehensive understanding of the viral entry pathway is required, particularly of the role of proteases. Here, we identified the proteases that activate the viral spike (S) glycoprotein to initiate cell entry and also pinpointed the host-cellular pathways that PDCoV uses for entry. Our results revealed that cathepsin L (CTSL) and cathepsin B (CTSB) in lysosomes and extracellular trypsin in cell cultures independently activate the S protein for membrane fusion. Pretreating the cells with the lysosomal acidification inhibitor bafilomycin-A1 (Baf-A1) completely inhibited PDCoV entry, and siRNA-mediated ablation of CTSL or CTSB expression significantly reduced viral infection, indicating that PDCoV uses an endosomal pathway for entry. Of note, trypsin treatment of cell cultures also activated PDCoV entry, even when the endosomal pathway was inhibited. This observation indicated that trypsin-induced S protein cleavage and activation in cell cultures enables viral entry directly from the cell surface. Our results provide critical insights into the PDCoV infection mechanism, uncovering two distinct viral entry pathways: one through cathepsin L and cathepsin B in the endosome and another via a protease at the cell surface. Because PDCoV infection sites represent a proteases-rich environment, these findings suggest that endosome inhibitor treatment alone is insufficient to block PDCoV entry into intestinal epithelial cells in vivo. Therefore, approaches that inhibit viral entry from the cell membrane should also be considered.
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Elgner, Fabian, Huimei Ren, Regina Medvedev, Daniela Ploen, Kiyoshi Himmelsbach, Klaus Boller, and Eberhard Hildt. "The Intracellular Cholesterol Transport Inhibitor U18666A Inhibits the Exosome-Dependent Release of Mature Hepatitis C Virus." Journal of Virology 90, no. 24 (October 5, 2016): 11181–96. http://dx.doi.org/10.1128/jvi.01053-16.
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ABSTRACT Hepatitis C virus (HCV) particles are described as lipoviroparticles which are released similarly to very-low-density lipoproteins (VLDLs). However, the release mechanism is still poorly understood; the canonical endoplasmic reticulum-Golgi intermediate compartment (ERGIC) pathway as well as endosome-dependent release has been proposed. Recently, the role of exosomes in the transmission of HCV has been reported. Only a minor fraction of the de novo -synthesized lipoviroparticles is released by the infected cell. To investigate the relevance of multivesicular bodies (MVBs) for viral morphogenesis and release, the MVB inhibitor U18666A was used. Intracellular trafficking was analyzed by confocal microscopy and electron microscopy. Moreover, an mCherry-tagged HCV variant was used. Conditions were established that enable U18666A-dependent inhibition of MVBs without affecting viral replication. Under these conditions, significant inhibition of the HCV release was observed. The assembly of viral particles is not affected. In U18666A-treated cells, intact infectious viral particles accumulate in CD63-positive exosomal structures and large dysfunctional lysosomal structures (multilamellar bodies). These retained particles possess a lower density, reflecting a misloading with lipids. Our data indicate that at least a fraction of HCV particles leaves the cell via the endosomal pathway. Endosomes facilitate the sorting of HCV particles for release or degradation. IMPORTANCE There are still a variety of open questions regarding morphogenesis and release of hepatitis C virus. The HCV-infected cell produces significant more viral particles that are released, raising the question about the fate of the nonreleased particles. Moreover, the relevance of the endosomal pathway for the release of HCV is under debate. Use of the MVB (multivesicular body) inhibitor U18666A enabled a detailed analysis of the impact of MVBs for viral morphogenesis and release. It was revealed that infectious, fully assembled HCV particles are either MVB-dependently released or intracellularly degraded by the lysosome. Our data indicate that at least a fraction of HCV particles leaves the cell via the endosomal pathway independent from the constitutive secretory pathway. Our study describes a so-far-unprecedented cross talk between two pathways regulating on the one hand the release of infectious viral particles and on the other hand the intracellular degradation of nonreleased particles.
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Molmeret, Maëlle, Marina Santic’, Rexford Asare, Reynold A. Carabeo, and Yousef Abu Kwaik. "Rapid Escape of the dot/icm Mutants of Legionella pneumophila into the Cytosol of Mammalian and Protozoan Cells." Infection and Immunity 75, no. 7 (April 16, 2007): 3290–304. http://dx.doi.org/10.1128/iai.00292-07.
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ABSTRACT The Legionella pneumophila-containing phagosome evades endocytic fusion and intercepts endoplasmic reticulum (ER)-to-Golgi vesicle traffic, which is believed to be mediated by the Dot/Icm type IV secretion system. Although phagosomes harboring dot/icm mutants are thought to mature through the endosomal-lysosomal pathway, colocalization studies with lysosomal markers have reported contradictory results. In addition, phagosomes harboring the dot/icm mutants do not interact with endocytosed materials, which is inconsistent with maturation of the phagosomes in the endosomal-lysosomal pathway. Using multiple strategies, we show that the dot/icm mutants defective in the Dot/Icm structural apparatus are unable to maintain the integrity of their phagosomes and escape into the cytoplasm within minutes of entry into various mammalian and protozoan cells in a process independent of the type II secretion system. In contrast, mutants defective in cytoplasmic chaperones of Dot/Icm effectors and rpoS, letA/S, and letE regulatory mutants are all localized within intact phagosomes. Importantly, non-dot/icm L. pneumophila mutants whose phagosomes acquire late endosomal-lysosomal markers are all located within intact phagosomes. Using high-resolution electron microscopy, we show that phagosomes harboring the dot/icm transporter mutants do not fuse to lysosomes but are free in the cytoplasm. Inhibition of ER-to-Golgi vesicle traffic by brefeldin A does not affect the integrity of the phagosomes harboring the parental strain of L. pneumophila. We conclude that the Dot/Icm transporter is involved in maintaining the integrity of the L. pneumophila phagosome, independent of interception of ER-to-Golgi vesicle traffic, which is a novel function of type IV secretion systems.
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Bush, J., L. Temesvari, J. Rodriguez-Paris, G. Buczynski, and J. Cardelli. "A role for a Rab4-like GTPase in endocytosis and in regulation of contractile vacuole structure and function in Dictyostelium discoideum." Molecular Biology of the Cell 7, no. 10 (October 1996): 1623–38. http://dx.doi.org/10.1091/mbc.7.10.1623.
Full textAbstract:
The small Mr Rab4-like GTPase, RabD, localizes to the endosomal pathway and the contractile vacuole membrane system in Dictyostelium discoideum. Stably transformed cell lines overexpressing a dominant negative functioning RabD internalized fluid phase marker at 50% of the rate of wild-type cells. Mutant cells were also slower at recycling internalized fluid. Microscopic and biochemical approaches indicated that the transport of fluid to large postlysosome vacuoles was delayed in mutant cells, resulting in an accumulation in acidic smaller vesicles, probably lysosomes. Also, RabD N121I-expressing cell lines missorted a small but significant percentage of newly synthesized lysosomal alpha-mannosidase precursor polypeptides. However, the majority of the newly synthesized alpha-mannosidase was transported with normal kinetics and correctly delivered to lysosomes. Subcellular fractionation and immunofluorescent microscopy indicated that in mutant cells contractile vacuole membrane proteins were associated with compartments morphologically distinct from the normal reticular network. Osmotic tests revealed that the contractile vacuole functioned inefficiently in mutant cells. Our results suggest that RabD regulates membrane traffic along the endosomal pathway, and that this GTPase may play a role in regulating the structure and function of the contractile vacuole system by facilitating communication with the endosomal pathway.
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Malik, Rohit, and Adriano Marchese. "Arrestin-2 Interacts with the Endosomal Sorting Complex Required for Transport Machinery to Modulate Endosomal Sorting of CXCR4." Molecular Biology of the Cell 21, no. 14 (July 15, 2010): 2529–41. http://dx.doi.org/10.1091/mbc.e10-02-0169.
Full textAbstract:
The chemokine receptor CXCR4, a G protein-coupled receptor, is targeted for lysosomal degradation via a ubiquitin-dependent mechanism that involves the endosomal sorting complex required for transport (ESCRT) machinery. We have reported recently that arrestin-2 also targets CXCR4 for lysosomal degradation; however, the molecular mechanisms by which this occurs remain poorly understood. Here, we show that arrestin-2 interacts with ESCRT-0, a protein complex that recognizes and sorts ubiquitinated cargo into the degradative pathway. Signal-transducing adaptor molecule (STAM)-1, but not related STAM-2, interacts directly with arrestin-2 and colocalizes with CXCR4 on early endosomal antigen 1-positive early endosomes. Depletion of STAM-1 by RNA interference and disruption of the arrestin-2/STAM-1 interaction accelerates agonist promoted degradation of CXCR4, suggesting that STAM-1 via its interaction with arrestin-2 negatively regulates CXCR4 endosomal sorting. Interestingly, disruption of this interaction blocks agonist promoted ubiquitination of hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) but not CXCR4 and STAM-1 ubiquitination. Our data suggest a mechanism whereby arrestin-2 via its interaction with STAM-1 modulates CXCR4 sorting by regulating the ubiquitination status of HRS.
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Malide, D., and S. W. Cushman. "Morphological effects of wortmannin on the endosomal system and GLUT4-containing compartments in rat adipose cells." Journal of Cell Science 110, no. 22 (November 15, 1997): 2795–806. http://dx.doi.org/10.1242/jcs.110.22.2795.
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Studies using functional and pharmacological approaches have implicated PI 3-kinase as a key intermediate in the glucose transport and GLUT4 translocation responses to insulin. Confocal microscopy was used to investigate the effects of the PI 3-kinase inhibitor wortmannin in isolated rat adipose cells. Independent of insulin, wortmannin induces the appearance of phase-lucent vacuoles containing the endosomal markers TfR, Rab4, M6PR, and cellubrevin. When added before or with insulin, wortmannin blocks insulin-stimulated GLUT4 translocation, but does not influence the basal VAMP2-containing GLUT4 compartment. These results substantiate the concept of a specialized basal GLUT4 compartment mostly distinct from that of the recycling receptors. However, when added after insulin, wortmannin induces a rapid redistribution of GLUT4 from the cell surface into those endosomal-derived vacuoles where the GLUT4 co-localize with TfR, Rab4, cellubrevin, and VAMP2, but not with clathrin, M6PR, Golgi complex markers TGN38-mannosidase II and gamma-adaptin, and lysosomal marker lgp-120. Therefore, wortmannin also disrupts insulin-stimulated GLUT4 traffic in the recycling endosomal pathway, at a step distal to the sorting of recycling proteins from late endosomal and TGN markers; wortmannin does not appear to affect internalization from the plasma membrane, and delivery from early to late endosomes or from late endosomes to the TGN. In combination with previous kinetic biochemical studies, these results suggest that: (i) insulin stimulates the exocytosis of GLUT4 through a direct pathway from a specialized basal compartment to the plasma membrane, (ii) during endocytosis in the presence of insulin, GLUT4 is sorted out of the TfR compartment into a separate recycling pathway back to the plasma membrane, and (iii) both of these pathways involve wortmannin sensitive enzymes.
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COLE, GREG M., LLORAINE BELL, QUANG B. TRUONG, and TSUNAO SAITOH. "An Endosomal-Lysosomal Pathway for Degradation of Amyloid Precursor Protein." Annals of the New York Academy of Sciences 674, no. 1 Proteases and (December 1992): 103–17. http://dx.doi.org/10.1111/j.1749-6632.1992.tb27480.x.
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Brunetti, Craig R., Kevin S. Dingwell, Cathy Wale, Frank L. Graham, and David C. Johnson. "Herpes Simplex Virus gD and Virions Accumulate in Endosomes by Mannose 6-Phosphate-Dependent and -Independent Mechanisms." Journal of Virology 72, no. 4 (April 1, 1998): 3330–39. http://dx.doi.org/10.1128/jvi.72.4.3330-3339.1998.
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ABSTRACT Herpes simplex virus (HSV) glycoprotein D (gD) is modified with mannose 6-phosphate (M6P) and binds to M6P receptors (MPRs). MPRs are involved in the well-characterized pathway by which lysosomal enzymes are directed to lysosomes via a network of endosomal membranes. Based on the impaired ability of HSV to form plaques under conditions in which glycoproteins could not interact with MPRs, we proposed that MPRs may function during HSV egress or cell-to-cell spread (C. R. Brunetti, R. L. Burke, B. Hoflack, T. Ludwig, K. S. Dingwell, and D. C. Johnson, J. Virol. 69:3517–3528, 1995). To further analyze M6P modification and intracellular trafficking of gD in the absence of other HSV proteins, adenovirus (Ad) vectors were used to express soluble and membrane-anchored forms of gD. Both membrane-bound and soluble gD were modified with M6P residues and were localized to endosomes that contained the 275-kDa MPR or the transferrin receptor. Similar results were observed in HSV-infected cells. Cell fractionation experiments showed that gD was not present in lysosomes. However, a mutant form of gD and another HSV glycoprotein, gI, that were not modified with M6P were also found in endosomes in HSV-infected cells. Moreover, a substantial fraction of the HSV nucleocapsid protein VP6 was found in endosomes, consistent with accumulation of virions in an endosomal compartment. Therefore, it appears that HSV glycoproteins and virions are directed to endosomes, by M6P-dependent as well as by M6P-independent mechanisms, either as part of the virus egress pathway or by endocytosis from the cell surface.
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Mahnke, Karsten, Ming Guo, Sena Lee, Homero Sepulveda, Suzy L. Swain, Michel Nussenzweig, and Ralph M. Steinman. "The Dendritic Cell Receptor for Endocytosis, Dec-205, Can Recycle and Enhance Antigen Presentation via Major Histocompatibility Complex Class II–Positive Lysosomal Compartments." Journal of Cell Biology 151, no. 3 (October 30, 2000): 673–84. http://dx.doi.org/10.1083/jcb.151.3.673.
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Many receptors for endocytosis recycle into and out of cells through early endosomes. We now find in dendritic cells that the DEC-205 multilectin receptor targets late endosomes or lysosomes rich in major histocompatibility complex class II (MHC II) products, whereas the homologous macrophage mannose receptor (MMR), as expected, is found in more peripheral endosomes. To analyze this finding, the cytosolic tails of DEC-205 and MMR were fused to the external domain of the CD16 Fcγ receptor and studied in stable L cell transfectants. The two cytosolic domains each mediated rapid uptake of human immunoglobulin (Ig)G followed by recycling of intact CD16 to the cell surface. However, the DEC-205 tail recycled the CD16 through MHC II–positive late endosomal/lysosomal vacuoles and also mediated a 100-fold increase in antigen presentation. The mechanism of late endosomal targeting, which occurred in the absence of human IgG, involved two functional regions: a membrane-proximal region with a coated pit sequence for uptake, and a distal region with an EDE triad for the unusual deeper targeting. Therefore, the DEC-205 cytosolic domain mediates a new pathway of receptor-mediated endocytosis that entails efficient recycling through late endosomes and a greatly enhanced efficiency of antigen presentation to CD4+ T cells.
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Min, Sang H., Aae Suzuki, Timothy J. Stalker, Jessica Guzman, Liang Zhao, Lurong Lian, Ronghua Meng, Michael S. Marks, John K. Choi, and Charles S. Abrams. "Pikfyve Deletion In Platelets Causes Aberrant Platelet Lysosomal Storage Associated With Inappropriate Inflammatory Response." Blood 122, no. 21 (November 15, 2013): 24. http://dx.doi.org/10.1182/blood.v122.21.24.24.
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Abstract Platelets release different types of secretory granules into their local environment, and this allows them to contribute to a variety of physiologic processes. These platelet granules include alpha granules, dense granules, and lysosomes, which all derive from the endosomal-lysosomal system. However, the mechanism of the biogenesis of each type of granule is not completely understood. Phosphatidylinositol-3,5-bisphosphate [PtdIns(3,5)P2] is a membrane phosphoinositide that is essential for the regulation of membrane homeostasis, as well as for vesicle trafficking and cargo transport along the endolysosomal system in mammals. PtdIns(3,5)P2 is synthesized on endosomes by the lipid kinase PIKfyve. Given the role of PIKfyve-mediated PtdIns(3,5)P2 production in the endolysosomal pathway in other mammalian cells, we hypothesized that PtdIns(3,5)P2 was an essential regulator for the biogenesis of granules within platelets. To analyze the contribution of PtdIns(3,5)P2 to platelet granule biogenesis, we generated mice lacking PIKfyve kinase activity specifically in their platelets and in their megakaryocytes (PIKfyveflox/flox Pf4-Cre). We found that when compared with the control mice, PIKfyveflox/flox Pf4-Cre mice contained higher levels of the lysosomal enzyme, β-hexosaminidase, within their platelets and within their plasma. The PIKfyve-ablated platelets also released excessive β-hexosaminidase ex vivo upon stimulation with ADP, collagen or thrombin. However, the percentage of the total cellular β-hexosaminidase that was released from the PIKfyve-ablated platelets was comparable with that of control platelets. This suggests that the increased release of β-hexosaminidase from the PIKfyve-ablated platelets is due to the excessive storage of this enzyme within these platelets and not because of increased efficiency of lysosome secretion. In addition, we observed that PIKfyve-ablated platelets expressed increased amounts of Lysosomal Associated Membrane Protein 1 (LAMP-1), a marker of late endosomes and lysosomes. However, PIKfyve-ablated platelets expressed normal amounts of Early Endosome Antigen 1 (EEA-1), a marker of early endosomes. These results suggest that PIKfyve is critical for a component of platelet lysosome biology that occurs after the maturation of early endosomes. Together, these data demonstrate that PIKfyve is essential for the homeostasis of the endolysosomal system in platelets. Notably, the generation and secretion of the alpha granule components were intact in the PIKFyve-ablated platelets. This was shown by the normal expression of von Willebrand factor, platelet basic protein, and platelet factor 4. Likewise, secretion of ATP stored in the dense granules was similar between the PIKfyveflox/flox Pf4-Cre mice and their control littermates. Together, these results suggest that PIKfyve plays an essential regulatory role along the endolysosomal pathway in platelets, and the loss of PIKfyve in platelets can lead to an abnormality of lysosomal storage. Unexpectedly, we also found that the loss of PIKfyve exclusively within platelets triggers an inappropriate inflammatory response. This is shown by the massive tissue infiltration of aberrant vacuolated macrophages. In turn, this leads to multiple organ defects that impair development, body mass, and survival in mice. Moreover, mice lacking PIKfyve within their platelets developed accelerated arterial thrombosis in vivo, despite having normal platelet aggregation ex vivo. It is also remarkable that mice lacking PIKfyve in their platelets attenuated their organ defects when the secretion of their platelet lysosomes were inhibited in vivo. Collectively, our study demonstrates that PIKfyve is an essential regulator of platelet lysosome biogenesis. This study also highlights the previously unrecognized and important contributions of platelet lysosomal storage to inflammation, arterial thrombosis, and macrophage biology. Disclosures: No relevant conflicts of interest to declare.
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Katzmann, David J., Christopher J. Stefan, Markus Babst, and Scott D. Emr. "Vps27 recruits ESCRT machinery to endosomes during MVB sorting." Journal of Cell Biology 162, no. 3 (August 4, 2003): 413–23. http://dx.doi.org/10.1083/jcb.200302136.
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Down-regulation (degradation) of cell surface proteins within the lysosomal lumen depends on the function of the multivesicular body (MVB) sorting pathway. The function of this pathway requires the class E vacuolar protein sorting (Vps) proteins. Of the class E Vps proteins, both the ESCRT-I complex (composed of the class E proteins Vps23, 28, and 37) and Vps27 (mammalian hepatocyte receptor tyrosine kinase substrate, Hrs) have been shown to interact with ubiquitin, a signal for entry into the MVB pathway. We demonstrate that activation of the MVB sorting reaction is dictated largely through interactions between Vps27 and the endosomally enriched lipid species phosphatidylinositol 3-phosphate via the FYVE domain (Fab1, YGL023, Vps27, and EEA1) of Vps27. ESCRT-I then physically binds to Vps27 on endosomal membranes via a domain within the COOH terminus of Vps27. A peptide sequence in this domain, PTVP, is involved in the function of Vps27 in the MVB pathway, the efficient endosomal recruitment of ESCRT-I, and is related to a motif in HIV-1 Gag protein that is capable of interacting with Tsg101, the mammalian homologue of Vps23. We propose that compartmental specificity for the MVB sorting reaction is the result of interactions of Vps27 with phosphatidylinositol 3-phosphate and ubiquitin. Vps27 subsequently recruits/activates ESCRT-I on endosomes, thereby facilitating sorting of ubiquitinated MVB cargoes.
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Conzemius, Rick, Haleh Ganjian, Dieter Blaas, and Renate Fuchs. "ICAM-1 Binding Rhinoviruses A89 and B14 Uncoat in Different Endosomal Compartments." Journal of Virology 90, no. 17 (June 22, 2016): 7934–42. http://dx.doi.org/10.1128/jvi.00712-16.
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ABSTRACTHuman rhinovirus A89 (HRV-A89) and HRV-B14 bind to and are internalized by intercellular adhesion molecule 1 (ICAM-1); as demonstrated earlier, the RNA genome of HRV-B14 penetrates into the cytoplasm from endosomal compartments of the lysosomal pathway. Here, we show by immunofluorescence microscopy that HRV-A89 but not HRV-B14 colocalizes with transferrin in the endocytic recycling compartment (ERC). Applying drugs differentially interfering with endosomal recycling and with the pathway to lysosomes, we demonstrate that these two major-group HRVs productively uncoat in distinct endosomal compartments. Overexpression of constitutively active (Rab11-GTP) and dominant negative (Rab11-GDP) mutants revealed that uncoating of HRV-A89 depends on functional Rab11. Thus, two ICAM-1 binding HRVs are routed into distinct endosomal compartments for productive uncoating.IMPORTANCEBased on similarity of their RNA genomic sequences, the more than 150 currently known common cold virus serotypes were classified as species A, B, and C. The majority of HRV-A viruses and all HRV-B viruses use ICAM-1 for cell attachment and entry. Our results highlight important differences of two ICAM-1 binding HRVs with respect to their intracellular trafficking and productive uncoating; they demonstrate that serotypes belonging to species A and B, but entering the cell via the same receptors, direct the endocytosis machinery to ferry them along distinct pathways toward different endocytic compartments for uncoating.
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Barois, N., F. Forquet, and J. Davoust. "Actin microfilaments control the MHC class II antigen presentation pathway in B cells." Journal of Cell Science 111, no. 13 (July 1, 1998): 1791–800. http://dx.doi.org/10.1242/jcs.111.13.1791.
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Newly synthesised major histocompatibility complex class II molecules associate with invariant chains (Ii) to form nonameric complexes. These complexes are transported to endosomes, where proteolytic enzymes generate alphabeta class II dimers associated with nested Ii-derived peptides. These peptides are then exchanged with antigen peptide, and mature class II molecules reach the cell surface. The role of the actin cytoskeleton in the transport and maturation of class II molecules has not been studied. We show here that upon treatment with cytochalasin D (cyto D), the rate of Ii degradation is drastically reduced in B cells. Cyto D treatment also leads to a delayed appearance of stable forms of class II molecules, and a reduced presentation efficiency of antigen determinants requiring newly synthesised class II molecules. Under such conditions, we found that invariant chain fragments and class II molecules are accumulated in early and late endosomal compartments, whereas the leupeptin protease inhibitor induces their accumulation in lysosomal compartments. The addition of cyto D to leupeptin blocks the delivery of class II/invariant chain complexes to lysosomes, and further inhibits degradation of Ii. The dynamics of the actin cytoskeleton can therefore control the meeting point between newly synthesised class II molecules and lysosomal proteases, involved in Ii degradation and antigen peptide loading.