Journal articles on the topic 'Endosomal maturation'
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1
Abenza, Juan F., Antonio Galindo, Mario Pinar, Areti Pantazopoulou, Vivian de los Ríos, and Miguel A. Peñalva. "Endosomal maturation by Rab conversion in Aspergillus nidulans is coupled to dynein-mediated basipetal movement." Molecular Biology of the Cell 23, no. 10 (May 15, 2012): 1889–901. http://dx.doi.org/10.1091/mbc.e11-11-0925.
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We exploit the ease with which highly motile early endosomes are distinguished from static late endosomes in order to study Aspergillus nidulans endosomal traffic. RabSRab7 mediates homotypic fusion of late endosomes/vacuoles in a homotypic fusion- and vacuole protein sorting/Vps41–dependent manner. Progression across the endocytic pathway involves endosomal maturation because the end products of the pathway in the absence of RabSRab7 are minivacuoles that are competent in multivesicular body sorting and cargo degradation but retain early endosomal features, such as the ability to undergo long-distance movement and propensity to accumulate in the tip region if dynein function is impaired. Without RabSRab7, early endosomal Rab5s—RabA and RabB—reach minivacuoles, in agreement with the view that Rab7 homologues facilitate the release of Rab5 homologues from endosomes. RabSRab7 is recruited to membranes already at the stage of late endosomes still lacking vacuolar morphology, but the transition between early and late endosomes is sharp, as only in a minor proportion of examples are RabA/RabB and RabSRab7 detectable in the same—frequently the less motile—structures. This early-to-late endosome/vacuole transition is coupled to dynein-dependent movement away from the tip, resembling the periphery-to-center traffic of endosomes accompanying mammalian cell endosomal maturation. Genetic studies establish that endosomal maturation is essential, whereas homotypic vacuolar fusion is not.
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Delevoye, Cédric, Ilse Hurbain, Danièle Tenza, Jean-Baptiste Sibarita, Stéphanie Uzan-Gafsou, Hiroshi Ohno, Willie J. C. Geerts, et al. "AP-1 and KIF13A coordinate endosomal sorting and positioning during melanosome biogenesis." Journal of Cell Biology 187, no. 2 (October 19, 2009): 247–64. http://dx.doi.org/10.1083/jcb.200907122.
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Specialized cell types exploit endosomal trafficking to deliver protein cargoes to cell type–specific lysosome-related organelles (LROs), but how endosomes are specified for this function is not known. In this study, we show that the clathrin adaptor AP-1 and the kinesin motor KIF13A together create peripheral recycling endosomal subdomains in melanocytes required for cargo delivery to maturing melanosomes. In cells depleted of AP-1 or KIF13A, a subpopulation of recycling endosomes redistributes to pericentriolar clusters, resulting in sequestration of melanosomal enzymes like Tyrp1 in vacuolar endosomes and consequent inhibition of melanin synthesis and melanosome maturation. Immunocytochemistry, live cell imaging, and electron tomography reveal AP-1– and KIF13A-dependent dynamic close appositions and continuities between peripheral endosomal tubules and melanosomes. Our results reveal that LRO protein sorting is coupled to cell type–specific positioning of endosomes that facilitate endosome–LRO contacts and are required for organelle maturation.
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Kim, Sungsu, Yogesh P. Wairkar, Richard W. Daniels, and Aaron DiAntonio. "The novel endosomal membrane protein Ema interacts with the class C Vps–HOPS complex to promote endosomal maturation." Journal of Cell Biology 188, no. 5 (March 1, 2010): 717–34. http://dx.doi.org/10.1083/jcb.200911126.
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Endosomal maturation is critical for accurate and efficient cargo transport through endosomal compartments. Here we identify a mutation of the novel Drosophila gene, ema (endosomal maturation defective) in a screen for abnormal synaptic overgrowth and defective protein trafficking. Ema is an endosomal membrane protein required for trafficking of fluid-phase and receptor-mediated endocytic cargos. In the ema mutant, enlarged endosomal compartments accumulate as endosomal maturation fails, with early and late endosomes unable to progress into mature degradative late endosomes and lysosomes. Defective endosomal down-regulation of BMP signaling is responsible for the abnormal synaptic overgrowth. Ema binds to and genetically interacts with Vps16A, a component of the class C Vps–HOPS complex that promotes endosomal maturation. The human orthologue of ema, Clec16A, is a candidate susceptibility locus for autoimmune disorders, and its expression rescues the Drosophila mutant demonstrating conserved function. Characterizing this novel gene family identifies a new component of the endosomal pathway and provides insights into class C Vps–HOPS complex function.
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Szatmári, Zsuzsanna, Viktor Kis, Mónika Lippai, Krisztina Hegedűs, Tamás Faragó, Péter Lőrincz, Tsubasa Tanaka, Gábor Juhász, and Miklós Sass. "Rab11 facilitates cross-talk between autophagy and endosomal pathway through regulation of Hook localization." Molecular Biology of the Cell 25, no. 4 (February 15, 2014): 522–31. http://dx.doi.org/10.1091/mbc.e13-10-0574.
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During autophagy, double-membrane autophagosomes deliver sequestered cytoplasmic content to late endosomes and lysosomes for degradation. The molecular mechanism of autophagosome maturation is still poorly characterized. The small GTPase Rab11 regulates endosomal traffic and is thought to function at the level of recycling endosomes. We show that loss of Rab11 leads to accumulation of autophagosomes and late endosomes in Drosophila melanogaster. Rab11 translocates from recycling endosomes to autophagosomes in response to autophagy induction and physically interacts with Hook, a negative regulator of endosome maturation. Hook anchors endosomes to microtubules, and we show that Rab11 facilitates the fusion of endosomes and autophagosomes by removing Hook from mature late endosomes and inhibiting its homodimerization. Thus induction of autophagy appears to promote autophagic flux by increased convergence with the endosomal pathway.
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Casanova, James E., and Bettina Winckler. "A new Rab7 effector controls phosphoinositide conversion in endosome maturation." Journal of Cell Biology 216, no. 10 (September 19, 2017): 2995–97. http://dx.doi.org/10.1083/jcb.201709034.
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Endosome maturation requires a coordinated change in the Rab GTPase and phosphoinositide composition of the endosomal membrane. In this issue, Liu et al. (2017. J. Cell Biol. https://doi.org/10.1083/jcb.201705151) identify WDR91 as a ubiquitous Rab7 effector that inhibits phosphatidylinositol 3-kinase activity on endosomes and is critical for endosome maturation, viability, and dendrite growth of neurons in vivo.
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Chotard, Laëtitia, Ashwini K. Mishra, Marc-André Sylvain, Simon Tuck, David G. Lambright, and Christian E. Rocheleau. "TBC-2 Regulates RAB-5/RAB-7-mediated Endosomal Trafficking inCaenorhabditis elegans." Molecular Biology of the Cell 21, no. 13 (July 2010): 2285–96. http://dx.doi.org/10.1091/mbc.e09-11-0947.
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During endosome maturation the early endosomal Rab5 GTPase is replaced with the late endosomal Rab7 GTPase. It has been proposed that active Rab5 can recruit and activate Rab7, which in turn could inactivate and remove Rab5. However, many of the Rab5 and Rab7 regulators that mediate endosome maturation are not known. Here, we identify Caenorhabditis elegans TBC-2, a conserved putative Rab GTPase-activating protein (GAP), as a regulator of endosome to lysosome trafficking in several tissues. We show that tbc-2 mutant animals accumulate enormous RAB-7–positive late endosomes in the intestine containing refractile material. RAB-5, RAB-7, and components of the homotypic fusion and vacuole protein sorting (HOPS) complex, a RAB-7 effector/putative guanine nucleotide exchange factor (GEF), are required for the tbc-2(−) intestinal phenotype. Expression of activated RAB-5 Q78L in the intestine phenocopies the tbc-2(−) large late endosome phenotype in a RAB-7 and HOPS complex-dependent manner. TBC-2 requires the catalytic arginine-finger for function in vivo and displays the strongest GAP activity on RAB-5 in vitro. However, TBC-2 colocalizes primarily with RAB-7 on late endosomes and requires RAB-7 for membrane localization. Our data suggest that TBC-2 functions on late endosomes to inactivate RAB-5 during endosome maturation.
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Sun, Ming, Gary Luong, Faiz Plastikwala, and Yue Sun. "Abstract 155: An endosomal type Igamma PIP 5-kinase controls endosome maturation, lysosome function, and autophagy by modulating Rab7a." Cancer Research 82, no. 12_Supplement (June 15, 2022): 155. http://dx.doi.org/10.1158/1538-7445.am2022-155.
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Abstract Endosomes are major protein-sorting stations in cells. Defects in endosomal sorting are implicated in various human pathologies, such as cancer and neurodegenerative disorders. The small GTPase Ras-related protein Rab-7a (Rab7a) serves as a key organizer of the endosomal-lysosomal system. However, molecular mechanisms controlling Rab7a activation levels and subcellular translocation are still poorly defined. Here, we demonstrate that type Igamma phosphatidylinositol phosphate 5-kinase i5 (PIPKIgammai5), an endosome-localized enzyme that produces phosphatidylinositol 4,5-bisphosphate (PI4,5P2), directly interacts with Rab7a and plays critical roles in the control of endosomal-lysosomal system. The loss of PIPKIgammai5 blocks Rab7a recruitment to early endosomes, which prevents the maturation of early to late endosomes. In this way, PIPKIgammai5 is required for Epidermal Growth Factor Receptor (EGFR) sorting from endosomes to lysosomes, which controls the down-regulation of EGFR signaling. Furthermore, PIPKIgammai5 loss disturbs retromer complex connection with Rab7a, which blocks the retrograde sorting of Cation-independent Mannose 6-Phosphate Receptor (CI-MPR) from late endosomes. This leads to the decreased sorting of hydrolases to lysosomes. Thus, loss of PIPKIgammai5 causes lysosome dysfunction and reduces the autophagic degradation. By modulating retromer-Rab7a connection, PIPKIgammai5 is also required for the recruitment of the GTPase-activating protein (GAP) TBC1 domain family member 5 (TBC1D5) to late endosomes, which controls the conversion of Rab7a from the active state to the inactive state. Altogether, PIPKIgammai5 is critical for the modulation of Rab7a activity, localization, and function in the endosomal-lysosomal system. Citation Format: Ming Sun, Gary Luong, Faiz Plastikwala, Yue Sun. An endosomal type Igamma PIP 5-kinase controls endosome maturation, lysosome function, and autophagy by modulating Rab7a [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 155.
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He, Jing, Jennifer L. Johnson, Jlenia Monfregola, Mahalakshmi Ramadass, Kersi Pestonjamasp, Gennaro Napolitano, Jinzhong Zhang, and Sergio D. Catz. "Munc13-4 interacts with syntaxin 7 and regulates late endosomal maturation, endosomal signaling, and TLR9-initiated cellular responses." Molecular Biology of the Cell 27, no. 3 (February 2016): 572–87. http://dx.doi.org/10.1091/mbc.e15-05-0283.
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The molecular mechanisms that regulate late endosomal maturation and function are not completely elucidated, and direct evidence of a calcium sensor is lacking. Here we identify a novel mechanism of late endosomal maturation that involves a new molecular interaction between the tethering factor Munc13-4, syntaxin 7, and VAMP8. Munc13-4 binding to syntaxin 7 was significantly increased by calcium. Colocalization of Munc13-4 and syntaxin 7 at late endosomes was demonstrated by high-resolution and live-cell microscopy. Munc13-4–deficient cells show increased numbers of significantly enlarged late endosomes, a phenotype that was mimicked by the fusion inhibitor chloroquine in wild-type cells and rescued by expression of Munc13-4 but not by a syntaxin 7–binding–deficient mutant. Late endosomes from Munc13-4-KO neutrophils show decreased degradative capacity. Munc13-4–knockout neutrophils show impaired endosomal-initiated, TLR9-dependent signaling and deficient TLR9-specific CD11b up-regulation. Thus we present a novel mechanism of late endosomal maturation and propose that Munc13-4 regulates the late endocytic machinery and late endosomal–associated innate immune cellular functions.
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Arlt, Henning, Kathrin Auffarth, Rainer Kurre, Dominik Lisse, Jacob Piehler, and Christian Ungermann. "Spatiotemporal dynamics of membrane remodeling and fusion proteins during endocytic transport." Molecular Biology of the Cell 26, no. 7 (April 2015): 1357–70. http://dx.doi.org/10.1091/mbc.e14-08-1318.
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Organelles of the endolysosomal system undergo multiple fission and fusion events to combine sorting of selected proteins to the vacuole with endosomal recycling. This sorting requires a consecutive remodeling of the organelle surface in the course of endosomal maturation. Here we dissect the remodeling and fusion machinery on endosomes during the process of endocytosis. We traced selected GFP-tagged endosomal proteins relative to exogenously added fluorescently labeled α-factor on its way from the plasma membrane to the vacuole. Our data reveal that the machinery of endosomal fusion and ESCRT proteins has similar temporal localization on endosomes, whereas they precede the retromer cargo recognition complex. Neither deletion of retromer nor the fusion machinery with the vacuole affects this maturation process, although the kinetics seems to be delayed due to ESCRT deletion. Of importance, in strains lacking the active Rab7-like Ypt7 or the vacuolar SNARE fusion machinery, α-factor still proceeds to late endosomes with the same kinetics. This indicates that endosomal maturation is mainly controlled by the early endosomal fusion and remodeling machinery but not the downstream Rab Ypt7 or the SNARE machinery. Our data thus provide important further understanding of endosomal biogenesis in the context of cargo sorting.
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Friedman, Jonathan R., Jared R. DiBenedetto, Matthew West, Ashley A. Rowland, and Gia K. Voeltz. "Endoplasmic reticulum–endosome contact increases as endosomes traffic and mature." Molecular Biology of the Cell 24, no. 7 (April 2013): 1030–40. http://dx.doi.org/10.1091/mbc.e12-10-0733.
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The endosomal pathway is responsible for plasma membrane cargo uptake, sorting, and, in many cases, lysosome targeting. Endosome maturation is complex, requiring proper spatiotemporal recruitment of factors that regulate the size, maturity, and positioning of endosomal compartments. In animal cells, it also requires trafficking of endosomes on microtubules. Recent work has revealed the presence of contact sites between some endosomes and the endoplasmic reticulum (ER). Although these contact sites are believed to have multiple functions, the frequency, dynamics, and physical attributes of these contacts are poorly understood. Here we use high-resolution three-dimensional electron microscopy to reveal that ER tubules wrap around endosomes and find that both organelles contact microtubules at or near membrane contact sites. As endosomes traffic, they remain bound to the ER, which causes the tubular ER to rearrange its structure around dynamic endosomes at contact sites. Finally, as endosomes transition through steps of maturation, they become more tightly associated with the ER. The major implication of these results is that endosomes mature and traffic while coupled to the ER membrane rather than in isolation.
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Wang, ShiYu, Zechuan Zhao, and Avital A. Rodal. "Higher-order assembly of Sorting Nexin 16 controls tubulation and distribution of neuronal endosomes." Journal of Cell Biology 218, no. 8 (June 28, 2019): 2600–2618. http://dx.doi.org/10.1083/jcb.201811074.
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The activities of neuronal signaling receptors depend heavily on the maturation state of the endosomal compartments in which they reside. However, it remains unclear how the distribution of these compartments within the uniquely complex morphology of neurons is regulated and how this distribution itself affects signaling. Here, we identified mechanisms by which Sorting Nexin 16 (SNX16) controls neuronal endosomal maturation and distribution. We found that higher-order assembly of SNX16 via its coiled-coil (CC) domain drives membrane tubulation in vitro and endosome association in cells. In Drosophila melanogaster motor neurons, activation of Rab5 and CC-dependent self-association of SNX16 lead to its endosomal enrichment, accumulation in Rab5- and Rab7-positive tubulated compartments in the cell body, and concomitant depletion of SNX16-positive endosomes from the synapse. This results in accumulation of synaptic growth–promoting bone morphogenetic protein receptors in the cell body and correlates with increased synaptic growth. Our results indicate that Rab regulation of SNX16 assembly controls the endosomal distribution and signaling activities of receptors in neurons.
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Cabrera, Margarita, and Christian Ungermann. "Guiding Endosomal Maturation." Cell 141, no. 3 (April 2010): 404–6. http://dx.doi.org/10.1016/j.cell.2010.04.013.
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Abenza, Juan F., Antonio Galindo, Areti Pantazopoulou, Concha Gil, Vivian de los Ríos, and Miguel A. Peñalva. "Aspergillus RabBRab5 Integrates Acquisition of Degradative Identity with the Long Distance Movement of Early Endosomes." Molecular Biology of the Cell 21, no. 15 (August 2010): 2756–69. http://dx.doi.org/10.1091/mbc.e10-02-0119.
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Aspergillus nidulans early endosomes display characteristic long-distance bidirectional motility. Simultaneous dual-channel acquisition showed that the two Rab5 paralogues RabB and RabA colocalize in these early endosomes and also in larger, immotile mature endosomes. However, RabB-GTP is the sole recruiter to endosomes of Vps34 PI3K (phosphatidylinositol-3-kinase) and the phosphatidylinositol-3-phosphate [PI(3)P] effector AnVps19 and rabBΔ, leading to thermosensitivity prevents multivesicular body sorting of endocytic cargo. Thus, RabB is the sole mediator of degradative endosomal identity. Importantly, rabBΔ, unlike rabAΔ, prevents early endosome movement. As affinity experiments and pulldowns showed that RabB-GTP recruits AnVps45, RabB coordinates PI(3)P-dependent endosome-to-vacuole traffic with incoming traffic from the Golgi and with long-distance endosomal motility. However, the finding that Anvps45Δ, unlike rabBΔ, severely impairs growth indicates that AnVps45 plays RabB-independent functions. Affinity chromatography showed that the CORVET complex is a RabB and, to a lesser extent, a RabA effector, in agreement with GST pulldown assays of AnVps8. rabBΔ leads to smaller vacuoles, suggesting that it impairs homotypic vacuolar fusion, which would agree with the sequential maturation of endosomal CORVET into HOPS proposed for Saccharomyces cerevisiae. rabBΔ and rabAΔ mutations are synthetically lethal, demonstrating that Rab5-mediated establishment of endosomal identity is essential for A. nidulans.
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Chirivino, Dafne, Laurence Del Maestro, Etienne Formstecher, Philippe Hupé, Graça Raposo, Daniel Louvard, and Monique Arpin. "The ERM proteins interact with the HOPS complex to regulate the maturation of endosomes." Molecular Biology of the Cell 22, no. 3 (February 2011): 375–85. http://dx.doi.org/10.1091/mbc.e10-09-0796.
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In the degradative pathway, the progression of cargos through endosomal compartments involves a series of fusion and maturation events. The HOPS (homotypic fusion and protein sorting) complex is part of the machinery that promotes the progression from early to late endosomes and lysosomes by regulating the exchange of small GTPases. We report that an interaction between subunits of the HOPS complex and the ERM (ezrin, radixin, moesin) proteins is required for the delivery of EGF receptor (EGFR) to lysosomes. Inhibiting either ERM proteins or the HOPS complex leads to the accumulation of the EGFR into early endosomes, delaying its degradation. This impairment in EGFR trafficking observed in cells depleted of ERM proteins is due to a delay in the recruitment of Rab7 on endosomes. As a consequence, the maturation of endosomes is perturbed as reflected by an accumulation of hybrid compartments positive for both early and late endosomal markers. Thus, ERM proteins represent novel regulators of the HOPS complex in the early to late endosomal maturation.
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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.
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Margiotta, Azzurra, Dominik M. Frei, Ingrid Hegnes Sendstad, Lennert Janssen, Jacques Neefjes, and Oddmund Bakke. "Invariant chain regulates endosomal fusion and maturation through an interaction with the SNARE Vti1b." Journal of Cell Science 133, no. 19 (September 9, 2020): jcs244624. http://dx.doi.org/10.1242/jcs.244624.
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ABSTRACTThe invariant chain (Ii, also known as CD74) is a multifunctional regulator of adaptive immune responses and is responsible for sorting major histocompatibility complex class I and class II (MHCI and MHCII, respectively) molecules, as well as other Ii-associated molecules, to a specific endosomal pathway. When Ii is expressed, endosomal maturation and proteolytic degradation of proteins are delayed and, in non-antigen presenting cells, the endosomal size increases, but the molecular mechanisms underlying this are not known. We identified that a SNARE, Vti1b, is essential for regulating these Ii-induced effects. Vti1b binds to Ii and is localized at the contact sites of fusing Ii-positive endosomes. Furthermore, truncated Ii lacking the cytoplasmic tail, which is not internalized from the plasma membrane, relocates Vti1b to the plasma membrane. Knockout of Ii in an antigen-presenting cell line was found to speed up endosomal maturation, whereas silencing of Vti1b inhibits the Ii-induced maturation delay. Our results suggest that Ii, by interacting with the SNARE Vti1b in antigen-presenting cells, directs specific Ii-associated SNARE-mediated fusion in the early part of the endosomal pathway that leads to a slower endosomal maturation for efficient antigen processing and MHC antigen loading.
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Razi, Minoo, Edmond Y. W. Chan, and Sharon A. Tooze. "Early endosomes and endosomal coatomer are required for autophagy." Journal of Cell Biology 185, no. 2 (April 13, 2009): 305–21. http://dx.doi.org/10.1083/jcb.200810098.
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Autophagy, an intracellular degradative pathway, maintains cell homeostasis under normal and stress conditions. Nascent double-membrane autophagosomes sequester and enclose cytosolic components and organelles, and subsequently fuse with the endosomal pathway allowing content degradation. Autophagy requires fusion of autophagosomes with late endosomes, but it is not known if fusion with early endosomes is essential. We show that fusion of AVs with functional early endosomes is required for autophagy. Inhibition of early endosome function by loss of COPI subunits (β′, β, or α) results in accumulation of autophagosomes, but not an increased autophagic flux. COPI is required for ER-Golgi transport and early endosome maturation. Although loss of COPI results in the fragmentation of the Golgi, this does not induce the formation of autophagosomes. Loss of COPI causes defects in early endosome function, as both transferrin recycling and EGF internalization and degradation are impaired, and this loss of function causes an inhibition of autophagy, an accumulation of p62/SQSTM-1, and ubiquitinated proteins in autophagosomes.
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John Peter, Arun T., Jens Lachmann, Meenakshi Rana, Madeleine Bunge, Margarita Cabrera, and Christian Ungermann. "The BLOC-1 complex promotes endosomal maturation by recruiting the Rab5 GTPase-activating protein Msb3." Journal of Cell Biology 201, no. 1 (April 1, 2013): 97–111. http://dx.doi.org/10.1083/jcb.201210038.
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Membrane microcompartments of the early endosomes serve as a sorting and signaling platform, where receptors are either recycled back to the plasma membrane or forwarded to the lysosome for destruction. In metazoan cells, three complexes, termed BLOC-1 to -3, mediate protein sorting from the early endosome to lysosomes and lysosome-related organelles. We now demonstrate that BLOC-1 is an endosomal Rab-GAP (GTPase-activating protein) adapter complex in yeast. The yeast BLOC-1 consisted of six subunits, which localized interdependently to the endosomes in a Rab5/Vps21-dependent manner. In the absence of BLOC-1 subunits, the balance between recycling and degradation of selected cargoes was impaired. Additionally, our data show that BLOC-1 is both a Vps21 effector and an adapter for its GAP Msb3. BLOC-1 and Msb3 interacted in vivo, and both mutants resulted in a redistribution of active Vps21 to the vacuole surface. We thus conclude that BLOC-1 controls the lifetime of active Rab5/Vps21 and thus endosomal maturation along the endocytic pathway.
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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.
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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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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.
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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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Barrès, Céline, Lionel Blanc, Pascale Bette-Bobillo, Sabine André, Robert Mamoun, Hans-Joachim Gabius, and Michel Vidal. "Galectin-5 is bound onto the surface of rat reticulocyte exosomes and modulates vesicle uptake by macrophages." Blood 115, no. 3 (January 21, 2010): 696–705. http://dx.doi.org/10.1182/blood-2009-07-231449.
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Abstract Reticulocytes release small membrane vesicles termed exosomes during their maturation into erythrocytes. Exosomes are intraluminal vesicles of multivesicular endosomes released into the extracellular medium by fusion of these endosomal compartments with the plasma membrane. This secretion pathway contributes to reticulocyte plasma membrane remodeling by eliminating certain membrane glycoproteins. We show in this study that galectin-5, although mainly cytosolic, is also present on the cell surface of rat reticulocytes and erythrocytes. In addition, in reticulocytes, it resides in the endosomal compartment. We document galectin-5 translocation from the cytosol into the endosome lumen, leading to its secretion in association with exosomes. Galectin-5 bound onto the vesicle surface may function in sorting galactose-bearing glycoconjugates. Fittingly, we found that Lamp2, a major cellular glycoprotein presenting galectin-reactive poly-N-acetylactosamine chains, is lost during reticulocyte maturation. It is associated with released exosomes, suggestive of binding to galectin-5. Finally, we reveal that the uptake of rat reticulocyte exosomes by macrophages is dependent on temperature and the mechanoenzyme dynamin and that exosome uptake is decreased by adding galectin-5. These data imply galectin-5 functionality in the exosomal sorting pathway during rat reticulocyte maturation.
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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.
Full textAbstract:
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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Kelley, Victoria A., and Jeffrey S. Schorey. "Mycobacterium's Arrest of Phagosome Maturation in Macrophages Requires Rab5 Activity and Accessibility to Iron." Molecular Biology of the Cell 14, no. 8 (August 2003): 3366–77. http://dx.doi.org/10.1091/mbc.e02-12-0780.
Full textAbstract:
Many mycobacteria are intramacrophage pathogens that reside within nonacidified phagosomes that fuse with early endosomes but do not mature to phagolysosomes. The mechanism by which mycobacteria block this maturation process remains elusive. To gain insight into whether fusion with early endosomes is required for mycobacteria-mediated inhibition of phagosome maturation, we investigated how perturbing the GTPase cycles of Rab5 and Rab7, GTPases that regulate early and late endosome fusion, respectively, would affect phagosome maturation. Retroviral transduction of the constitutively activated forms of both GTPases into primary murine macrophages had no effect on Mycobacterium avium retention in an early endosomal compartment. Interestingly, expression of dominant negative Rab5, Rab5(S34N), but not dominant negative Rab7, resulted in a significant increase in colocalization of M. avium with markers of late endosomes/lysosomes and increased mycobacterial killing. This colocalization was specific to mycobacteria since Rab5(S34N) expressing cells showed diminished trafficking of endocytic tracers to lysosomes. We further demonstrated that maturation of M. avium phagosomes was halted in Rab5(S34N) expressing macrophages supplemented with exogenous iron. These findings suggest that fusion with early endosomes is required for mycobacterial retention in early phagosomal compartments and that an inadequate supply of iron is one factor in mycobacteria's inability to prevent the normal maturation process in Rab5(S34N)-expressing macrophages.
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Guiducci, Cristiana, Gary Ott, Jean H. Chan, Emily Damon, Carlo Calacsan, Tracy Matray, Kyung-Dall Lee, Robert L. Coffman, and Franck J. Barrat. "Properties regulating the nature of the plasmacytoid dendritic cell response to Toll-like receptor 9 activation." Journal of Experimental Medicine 203, no. 8 (July 24, 2006): 1999–2008. http://dx.doi.org/10.1084/jem.20060401.
Full textAbstract:
Human plasmacytoid dendritic cells (PDCs) can produce interferon (IFN)-α and/or mature and participate in the adaptive immune response. Three classes of CpG oligonucleotide ligands for Toll-like receptor (TLR)9 can be distinguished by different sequence motifs and different abilities to stimulate IFN-α production and maturation of PDCs. We show that the nature of the PDC response is determined by the higher order structure and endosomal location of the CpG oligonucleotide. Activation of TLR9 by the multimeric CpG-A occurs in transferrin receptor (TfR)-positive endosomes and leads exclusively to IFN-α production, whereas monomeric CpG-B oligonucleotides localize to lysosome-associated membrane protein (LAMP)-1–positive endosomes and promote maturation of PDCs. However, CpG-B, when complexed into microparticles, localizes in TfR-positive endosomes and induces IFN-α from PDCs, whereas monomeric forms of CpG-A localize to LAMP-1–positive endosomes accompanied by the loss of IFN-α production and a gain in PDC maturation activity. CpG-C sequences, which induce both IFN-α and maturation of PDCs, are distributed in both type of endosomes. Encapsulation of CpG-C in liposomes stable above pH 5.75 completely abrogated the IFN-α response while increasing PDC maturation. This establishes that the primary determinant of TLR9 signaling is not valency but endosomal location and demonstrates a strict compartmentalization of the biological response to TLR9 activation in PDCs.
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Snyder, Anthony J., Andrew T. Abad, and Pranav Danthi. "A CRISPR-Cas9 screen reveals a role for WD repeat-containing protein 81 (WDR81) in the entry of late penetrating viruses." PLOS Pathogens 18, no. 3 (March 23, 2022): e1010398. http://dx.doi.org/10.1371/journal.ppat.1010398.
Full textAbstract:
Successful initiation of infection by many different viruses requires their uptake into the endosomal compartment. While some viruses exit this compartment early, others must reach the degradative, acidic environment of the late endosome. Mammalian orthoreovirus (reovirus) is one such late penetrating virus. To identify host factors that are important for reovirus infection, we performed a CRISPR-Cas9 knockout (KO) screen that targets over 20,000 genes in fibroblasts derived from the embryos of C57/BL6 mice. We identified seven genes (WDR81, WDR91, RAB7, CCZ1, CTSL, GNPTAB, and SLC35A1) that were required for the induction of cell death by reovirus. Notably, CRISPR-mediated KO of WD repeat-containing protein 81 (WDR81) rendered cells resistant to reovirus infection. Susceptibility to reovirus infection was restored by complementing KO cells with human WDR81. Although the absence of WDR81 did not affect viral attachment efficiency or uptake into the endosomal compartments for initial disassembly, it reduced viral gene expression and diminished infectious virus production. Consistent with the role of WDR81 in impacting the maturation of endosomes, WDR81-deficiency led to the accumulation of reovirus particles in dead-end compartments. Though WDR81 was dispensable for infection by VSV (vesicular stomatitis virus), which exits the endosomal system at an early stage, it was required for VSV-EBO GP (VSV that expresses the Ebolavirus glycoprotein), which must reach the late endosome to initiate infection. These results reveal a previously unappreciated role for WDR81 in promoting the replication of viruses that transit through late endosomes.
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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.
Full textAbstract:
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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Cuesta-Geijo, Miguel Ángel, Isabel García-Dorival, Ana del Puerto, Jesús Urquiza, Inmaculada Galindo, Lucía Barrado-Gil, Fátima Lasala, et al. "New insights into the role of endosomal proteins for African swine fever virus infection." PLOS Pathogens 18, no. 1 (January 26, 2022): e1009784. http://dx.doi.org/10.1371/journal.ppat.1009784.
Full textAbstract:
African swine fever virus (ASFV) infectious cycle starts with the viral adsorption and entry into the host cell. Then, the virus is internalized via clathrin/dynamin mediated endocytosis and macropinocytosis. Similar to other viruses, ASF virion is then internalized and incorporated into the endocytic pathway. While the endosomal maturation entails luminal acidification, the decrease in pH acts on the multilayer structure of the virion dissolving the outer capsid. Upon decapsidation, the inner viral membrane is exposed to interact with the limiting membrane of the late endosome for fusion. Viral fusion is then necessary for the egress of incoming virions from endosomes into the cytoplasm, however this remains an intriguing and yet essential process for infection, specifically for the egress of viral nucleic acid into the cytoplasm for replication. ASFV proteins E248R and E199L, located at the exposed inner viral membrane, might be implicated in the fusion step. An interaction between these viral proteins and cellular endosomal proteins such as the Niemann-Pick C type 1 (NPC1) and lysosomal membrane proteins (Lamp-1 and -2) was shown. Furthermore, the silencing of these proteins impaired ASFV infection. It was also observed that NPC1 knock-out cells using CRISPR jeopardized ASFV infection and that the progression and endosomal exit of viral cores was arrested within endosomes at viral entry. These results suggest that the interactions of ASFV proteins with some endosomal proteins might be important for the membrane fusion step. In addition to this, reductions on ASFV infectivity and replication in NPC1 KO cells were accompanied by fewer and smaller viral factories. Our findings pave the way to understanding the role of proteins of the endosomal membrane in ASFV infection.
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Schluter, Cayetana, Karen K. Y. Lam, Jochen Brumm, Bella W. Wu, Matthew Saunders, Tom H. Stevens, Jennifer Bryan, and Elizabeth Conibear. "Global Analysis of Yeast Endosomal Transport Identifies the Vps55/68 Sorting Complex." Molecular Biology of the Cell 19, no. 4 (April 2008): 1282–94. http://dx.doi.org/10.1091/mbc.e07-07-0659.
Full textAbstract:
Endosomal transport is critical for cellular processes ranging from receptor down-regulation and retroviral budding to the immune response. A full understanding of endosome sorting requires a comprehensive picture of the multiprotein complexes that orchestrate vesicle formation and fusion. Here, we use unsupervised, large-scale phenotypic analysis and a novel computational approach for the global identification of endosomal transport factors. This technique effectively identifies components of known and novel protein assemblies. We report the characterization of a previously undescribed endosome sorting complex that contains two well-conserved proteins with four predicted membrane-spanning domains. Vps55p and Vps68p form a complex that acts with or downstream of ESCRT function to regulate endosomal trafficking. Loss of Vps68p disrupts recycling to the TGN as well as onward trafficking to the vacuole without preventing the formation of lumenal vesicles within the MVB. Our results suggest the Vps55/68 complex mediates a novel, conserved step in the endosomal maturation process.
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Jalagadugula, Gauthami S., Guangfen Mao, Lawrence E. Goldfinger, Jeremy Wurtzel, Michele P. Lambert, and Angara Koneti Rao. "RAB31-Mediated Endosomal Trafficking Is Defective in RUNX1 Haplodeficiency." Blood 132, Supplement 1 (November 29, 2018): 519. http://dx.doi.org/10.1182/blood-2018-99-118312.
Full textAbstract:
Abstract RAB GTPases are key players in vesicle trafficking, granule targeting of proteins, granule biogenesis and secretion. RAB31 (Ras-related protein 31) has been implicated in the regulation of vesicular trafficking between the Golgi/TGN and endosomes, and from early endosome (EE) to late endosome (LE). Studies in neuronal cells have implicated RAB31 in the transport of epidermal growth factor receptor (EGFR) from EE to LE. RUNX1, a major hematopoietic transcription factor, plays a critical role in megakaryocyte (MK) maturation and platelet production. Patients with RUNX1 haplodeficiency have thrombocytopenia, abnormal platelet function, and impaired granule contents and secretion. Our studies in a patient with a heterozygous RUNX1 mutation (c.969-323G>T ) (Sun et al, Blood 103: 948-54, 2004) associated with thrombocytopenia, platelet dysfunction, granule deficiency and impaired platelet responses revealed decreased platelet expression of RAB31. The role of RAB31 in MK and platelets is unknown. We addressed the hypothesis that RAB31 is a transcriptional target of RUNX1. We studied the role of RAB31 on vesicle transport in MK cells in human erythroleukemia (HEL) cells transformed into megakaryocytic cells by PMA (phorbol 12-myristate 13-acetate). RAB31 mRNA was decreased on platelet expression profiling of the patient (fold change: 0.28, p<0.0076, Sun et al J Thromb Haemost 5: 146-154). With real-time PCR platelet platelet RAB31 mRNA was decreased compared to 5 healthy controls by 60-80% in our patient (P1) and in two additional unrelated patients (siblings, P2 and P3) with RUNX1 mutation (c.508+1G>A). Platelet RAB31 protein was decreased compared to that in 5 healthy controls in patients P2 and P3. RAB31 promoter region (-2023/-1bp from the ATG) revealed 4 RUNX1 consensus sites: site I (-813/-808), site II (-972/-967), site III (-1500/-1495) and site IV (-2007/-2002). Chromatin immunoprecipitation (ChIP) revealed RUNX1 binding to RAB31 sites II and IV but not to other sites. Electrophoretic mobility shift assays using HEL cell proteins showed RUNX1 binding to sites II and IV. In luciferase reporter assays, mutation of individual sites II and IV decreased promoter activity indicating that they are functional sites. RAB31 promoter activity and protein expression were inhibited by RUNX1 siRNA and enhanced by RUNX1 overexpression. These indicate that RAB31 is a direct RUNX1 target, providing a mechanism for decreased RAB31 in patient platelets. We investigated putative roles of RUNX1 and its target RAB31 in endosomal dynamics in PMA treated HEL cells. We used immunofluorescent staining for markers of early endosomes (EE, EEA1) and late endosomes/multivesicular bodies (LE/MVB, CD63) in RUNX1- or RAB31-depleted cells by siRNA transfection. Either RUNX1 or RAB31 siRNA yielded a striking enlargement of early endosomes, as indicated by the EE marker EEA1. This finding suggests a role for RUNX1/RAB31 in EE maturation, either by mediating vesicle fission, or maturation to late endosomes by fusion with other endosomal vesicles. This effect of RUNX1 knockdown on EE enlargement was partially reversed by reconstitution of RAB31 by plasmid co-transfection, indicating that RAB31 is a significant but non-exclusive contributor to this RUNX1 function. Conclusions: These studies provide the first evidence that RAB31 is a direct transcriptional target of RUNX1 and a mechanism for RAB31 downregulation in RUNX1 haplodeficient patients. Downregulation of RAB31 or RUNX1 results in impaired endosomal maturation/trafficking, and this may contribute to the defective handling of α-granule proteins recognized in patients with RUNX1 mutations. Disclosures Lambert: Sysmex: Consultancy; Rigel: Consultancy; Bayer: Membership on an entity's Board of Directors or advisory committees; Educational Concepts in Medicine: Consultancy; CSL: Consultancy; Novartis: Membership on an entity's Board of Directors or advisory committees; Summus: Consultancy; Amgen: Membership on an entity's Board of Directors or advisory committees; Shionogi: Consultancy.
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Ito, Yoko, Nicolas Esnay, Louise Fougère, Matthieu Pierre Platre, Fabrice Cordelières, Yvon Jaillais, and Yohann Boutté. "Inhibition of Very Long Chain Fatty Acids Synthesis Mediates PI3P Homeostasis at Endosomal Compartments." International Journal of Molecular Sciences 22, no. 16 (August 6, 2021): 8450. http://dx.doi.org/10.3390/ijms22168450.
Full textAbstract:
A main characteristic of sphingolipids is the presence of a very long chain fatty acid (VLCFA) whose function in cellular processes is not yet fully understood. VLCFAs of sphingolipids are involved in the intracellular traffic to the vacuole and the maturation of early endosomes into late endosomes is one of the major pathways for vacuolar traffic. Additionally, the anionic phospholipid phosphatidylinositol-3-phosphate (PtdIns (3)P or PI3P) is involved in protein sorting and recruitment of small GTPase effectors at late endosomes/multivesicular bodies (MVBs) during vacuolar trafficking. In contrast to animal cells, PI3P mainly localizes to late endosomes in plant cells and to a minor extent to a discrete sub-domain of the plant’s early endosome (EE)/trans-Golgi network (TGN) where the endosomal maturation occurs. However, the mechanisms that control the relative levels of PI3P between TGN and MVBs are unknown. Using metazachlor, an inhibitor of VLCFA synthesis, we found that VLCFAs are involved in the TGN/MVB distribution of PI3P. This effect is independent from either synthesis of PI3P by PI3-kinase or degradation of PI(3,5)P2 into PI3P by the SUPPRESSOR OF ACTIN1 (SAC1) phosphatase. Using high-resolution live cell imaging microscopy, we detected transient associations between TGNs and MVBs but VLCFAs are not involved in those interactions. Nonetheless, our results suggest that PI3P might be transferable from TGN to MVBs and that VLCFAs act in this process.
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Brett, Christopher L., Deepali N. Tukaye, Sanchita Mukherjee, and Rajini Rao. "The Yeast Endosomal Na+(K+)/H+ Exchanger Nhx1 Regulates Cellular pH to Control Vesicle Trafficking." Molecular Biology of the Cell 16, no. 3 (March 2005): 1396–405. http://dx.doi.org/10.1091/mbc.e04-11-0999.
Full textAbstract:
The relationship between endosomal pH and function is well documented in viral entry, endosomal maturation, receptor recycling, and vesicle targeting within the endocytic pathway. However, specific molecular mechanisms that either sense or regulate luminal pH to mediate these processes have not been identified. Herein we describe the use of novel, compartment-specific pH indicators to demonstrate that yeast Nhx1, an endosomal member of the ubiquitous NHE family of Na+/H+ exchangers, regulates luminal and cytoplasmic pH to control vesicle trafficking out of the endosome. Loss of Nhx1 confers growth sensitivity to low pH stress, and concomitant acidification and trafficking defects, which can be alleviated by weak bases. Conversely, weak acids cause wild-type yeast to present nhx1Δ trafficking phenotypes. Finally, we report that Nhx1 transports K+ in addition to Na+, suggesting that a single mechanism may responsible for both pH and K+-dependent endosomal processes. This presents the newly defined family of eukaryotic endosomal NHE as novel targets for pharmacological inhibition to alleviate pathological states associated with organellar alkalinization.
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Sriram, V., K. S. Krishnan, and Satyajit Mayor. "deep-orange and carnation define distinct stages in late endosomal biogenesis in Drosophila melanogaster." Journal of Cell Biology 161, no. 3 (May 12, 2003): 593–607. http://dx.doi.org/10.1083/jcb.200210166.
Full textAbstract:
Endosomal degradation is severely impaired in primary hemocytes from larvae of eye color mutants of Drosophila. Using high resolution imaging and immunofluorescence microscopy in these cells, products of eye color genes, deep-orange (dor) and carnation (car), are localized to large multivesicular Rab7-positive late endosomes containing Golgi-derived enzymes. These structures mature into small sized Dor-negative, Car-positive structures, which subsequently fuse to form tubular lysosomes. Defective endosomal degradation in mutant alleles of dor results from a failure of Golgi-derived vesicles to fuse with morphologically arrested Rab7-positive large sized endosomes, which are, however, normally acidified and mature with wild-type kinetics. This locates the site of Dor function to fusion of Golgi-derived vesicles with the large Rab7-positive endocytic compartments. In contrast, endosomal degradation is not considerably affected in car1 mutant; fusion of Golgi-derived vesicles and maturation of large sized endosomes is normal. However, removal of Dor from small sized Car-positive endosomes is slowed, and subsequent fusion with tubular lysosomes is abolished. Overexpression of Dor in car1 mutant aggravates this defect, implicating Car in the removal of Dor from endosomes. This suggests that, in addition to an independent role in fusion with tubular lysosomes, the Sec1p homologue, Car, regulates Dor function.
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Dunn, KW, and FR Maxfield. "Delivery of ligands from sorting endosomes to late endosomes occurs by maturation of sorting endosomes." Journal of Cell Biology 117, no. 2 (April 15, 1992): 301–10. http://dx.doi.org/10.1083/jcb.117.2.301.
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After endocytosis, lysosomally targeted ligands pass through a series of endosomal compartments. The endocytic apparatus that accomplishes this passage may be considered to take one of two forms: (a) a system in which lysosomally targeted ligands pass through preexisting, long-lived early sorting endosomes and are then selectively transported to long-lived late endosomes in carrier vesicles, or (b) a system in which lysosomally targeted ligands are delivered to early sorting endosomes which themselves mature into late endosomes. We have previously shown that sorting endosomes in CHO cells fuse with newly formed endocytic vesicles (Dunn, K. W., T. E. McGraw, and F. R. Maxfield. 1989. J. Cell Biol. 109:3303-3314) and that previously endocytosed ligands lose their accessibility to fusion with a half-time of approximately 8 min (Salzman, N. H., and F. R. Maxfield. 1989. J. Cell Biol. 109:2097-2104). Here we have studied the properties of individual endosomes by digital image analysis to distinguish between the two mechanisms for entry of ligands into late endosomes. We incubated TRVb-1 cells (derived from CHO cells) with diO-LDL followed, after a variable chase, by diI-LDL, and measured the diO content of diI-containing endosomes. As the chase period was lengthened, an increasing percentage of the endosomes containing diO-LDL from the initial incubation had no detectable diI-LDL from the second incubation, but those endosomes that contained both probes showed no decrease in the amount of diO-LDL per endosomes. These results indicate that (a) a pulse of fluorescent LDL is retained by individual sorting endosomes, and (b) with time sorting endosomes lose the ability to fuse with primary endocytic vesicles. These data are inconsistent with a preexisting compartment model which predicts that the concentration of ligand in sorting endosomes will decline during a chase interval, but that the ability of the stable sorting endosome to receive newly endocytosed ligands will remain high. These data are consistent with a maturation mechanism in which the sorting endosome retains and accumulates lysosomally directed ligands until it loses its ability to fuse with newly formed endocytic vesicles and matures into a late endosome. We also find that, as expected according to the maturation model, new sorting endosomes are increasingly labeled during the chase period indicating that new sorting endosomes are continuously formed to replace those that have matured into late endosomes.(ABSTRACT TRUNCATED AT 400 WORDS)
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Muriel, Olivia, Alejandra Tomas, Cameron C. Scott, and Jean Gruenberg. "Moesin and cortactin control actin-dependent multivesicular endosome biogenesis." Molecular Biology of the Cell 27, no. 21 (November 2016): 3305–16. http://dx.doi.org/10.1091/mbc.e15-12-0853.
Full textAbstract:
We used in vivo and in vitro strategies to study the mechanisms of multivesicular endosome biogenesis. We found that, whereas annexinA2 and ARP2/3 mediate F-actin nucleation and branching, respectively, the ERM protein moesin supports the formation of F-actin networks on early endosomes. We also found that moesin plays no role during endocytosis and recycling to the plasma membrane but is absolutely required, much like actin, for early-to-late-endosome transport and multivesicular endosome formation. Both actin network formation in vitro and early-to-late endosome transport in vivo also depend on the F-actin–binding protein cortactin. Our data thus show that moesin and cortactin are necessary for formation of F-actin networks that mediate endosome biogenesis or maturation and transport through the degradative pathway. We propose that the primary function of endosomal F-actin is to control the membrane remodeling that accompanies endosome biogenesis. We also speculate that this mechanism helps segregate tubular and multivesicular membranes along the recycling and degradation pathways, respectively.
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Laman, Jon, Marvin van Luijn, Karim Kreft, Marlieke Jongsma, Steven Mes, Annet Wierenga-Wolf, Marjan van Meurs, et al. "The autoimmunity-associated gene CLEC16A controls HLA-II expression by participating in the molecular machinery of late endosomal maturation (HUM2P.337)." Journal of Immunology 192, no. 1_Supplement (May 1, 2014): 53.10. http://dx.doi.org/10.4049/jimmunol.192.supp.53.10.
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Abstract In GWA studies, CLEC16A has been identified as a risk gene for several major autoimmune diseases, including multiple sclerosis (MS). We show that CLEC16A is increased in MS and functionally links to HLA-II, and uncovered the molecular mechanism of CLEC16A as key regulator of HLA-II antigen presentation. We used the human melanoma cell line MelJuSo and primary moDC as APC models to silence CLEC16A with different siRNA and lentiviral shRNA. CLEC16A knockdown in MelJuSo cells and moDC resulted in cytoplasmic dispersion and strong accumulation of HLA-II+ late endosomes, suggesting impaired formation of multivesicular late endosomal compartments, i.e. MIIC. Indeed, in CLEC16A shRNA transductants, MIIC contained no or a few abnormally enlarged internal vesicles and showed an increased number of HLA-II molecules as compared to scrambled shRNA transductants of MelJuSo cells. CLEC16A associated with Rab7-interacting lysosomal protein (RILP) and the homotypic fusion and protein sorting (HOPS) complex, two members of the dynein motor complex regulating late endosomal trafficking and maturation. In addition, CLEC16A silencing disrupted RILP-mediated recruitment of HLA-II+ late endosomes to perinuclear regions. We reveal direct involvement of CLEC16A in the machinery regulating late endosomal processing of HLA-II. This novel C-type lectin function supports a pathogenic role of CLEC16A in autoimmune disease by promoting HLA-II antigen presentation via the formation of MIIC.
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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.
Full textAbstract:
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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Fratti, Rutilio A., Jonathan M. Backer, Jean Gruenberg, Silvia Corvera, and Vojo Deretic. "Role of phosphatidylinositol 3-kinase and Rab5 effectors in phagosomal biogenesis and mycobacterial phagosome maturation arrest." Journal of Cell Biology 154, no. 3 (August 6, 2001): 631–44. http://dx.doi.org/10.1083/jcb.200106049.
Full textAbstract:
Phagosomal biogenesis is a fundamental biological process of particular significance for the function of phagocytic and antigen-presenting cells. The precise mechanisms governing maturation of phagosomes into phagolysosomes are not completely understood. Here, we applied the property of pathogenic mycobacteria to cause phagosome maturation arrest in infected macrophages as a tool to dissect critical steps in phagosomal biogenesis. We report the requirement for 3-phosphoinositides and acquisition of Rab5 effector early endosome autoantigen (EEA1) as essential molecular events necessary for phagosomal maturation. Unlike the model phagosomes containing latex beads, which transiently recruited EEA1, mycobacterial phagosomes excluded this regulator of vesicular trafficking that controls membrane tethering and fusion processes within the endosomal pathway and is recruited to endosomal membranes via binding to phosphatidylinositol 3-phosphate (PtdIns[3]P). Inhibitors of phosphatidylinositol 3′(OH)-kinase (PI-3K) activity diminished EEA1 recruitment to newly formed latex bead phagosomes and blocked phagosomal acquisition of late endocytic properties, indicating that generation of PtdIns(3)P plays a role in phagosomal maturation. Microinjection into macrophages of antibodies against EEA1 and the PI-3K hVPS34 reduced acquisition of late endocytic markers by latex bead phagosomes, demonstrating an essential role of these Rab5 effectors in phagosomal biogenesis. The mechanism of EEA1 exclusion from mycobacterial phagosomes was investigated using mycobacterial products. Coating of latex beads with the major mycobacterial cell envelope glycosylated phosphatidylinositol lipoarabinomannan isolated from the virulent Mycobacterium tuberculosis H37Rv, inhibited recruitment of EEA1 to latex bead phagosomes, and diminished their maturation. These findings define the generation of phosphatidylinositol 3-phosphate and EEA1 recruitment as: (a) important regulatory events in phagosomal maturation and (b) critical molecular targets affected by M. tuberculosis. This study also identifies mycobacterial phosphoinositides as products with specialized toxic properties, interfering with discrete trafficking stages in phagosomal maturation.
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Via, L. E., R. A. Fratti, M. McFalone, E. Pagan-Ramos, D. Deretic, and V. Deretic. "Effects of cytokines on mycobacterial phagosome maturation." Journal of Cell Science 111, no. 7 (April 1, 1998): 897–905. http://dx.doi.org/10.1242/jcs.111.7.897.
Full textAbstract:
One of the major mechanisms permitting intracellular pathogens to parasitize macrophages is their ability to alter maturation of the phagosome or affect its physical integrity. These processes are opposed by the host innate and adaptive immune defenses, and in many instances mononuclear phagocytes can be stimulated with appropriate cytokines to restrict the growth of the microorganisms within the phagosomal compartment. Very little is known about the effects that cytokines have on phagosome maturation. Here we have used green fluorescent protein (GFP)-labeled mycobacteria and a fixable acidotropic probe, LysoTracker Red DND-99, to monitor maturation of the mycobacterial phagosome. The macrophage compartments that stained with the LysoTracker probe were examined first. This dye was found to colocalize preferentially with the late endosomal and lysosomal markers rab7 and Lamp1, and with a fluid phase marker chased into the late endosomal compartments. In contrast, LysoTracker showed only a minor overlap with the early endosomal marker rab5. Pathogenic mycobacteria are believed to reside in nonacidified vacuoles sequestered away from late endosomal compartments as a part of their intracellular survival strategy. We examined the status of mycobacterial phagosomes in macrophages from IL-10 knockout mice, in quiescent cells, and in mononuclear phagocytes stimulated with the macrophage-activating cytokine IFN-(gamma). When macrophages were derived from the bone marrow of transgenic IL-10 mice lacking this major deactivating cytokine, colocalization of GFP-fluorescing mycobacteria with the LysoTracker staining appeared enhanced, suggestive of increased acidification of the mycobacterial phagosome relative to macrophages from normal mice. When bone marrow-derived macrophages from normal mice or a J774 murine macrophage cell line were stimulated with IFN-(gamma) and LPS, this resulted in increased colocalization of mycobacteria and LysoTracker, but no statistically significant enhancement was observed in IL-10 transgenic animals. These studies are consistent with the interpretation that proinflammatory and anti-inflammatory cytokines affect maturation of mycobacterial phagosomes. Although multiple mechanisms are likely to be at work, we propose the existence of a direct link between cytokine effects on the host cell and phagosome maturation in the macrophage.
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Marcelić, Marina, Hana Mahmutefendić Lučin, Antonija Jurak Begonja, Gordana Blagojević Zagorac, and Pero Lučin. "Early Endosomal Vps34-Derived Phosphatidylinositol-3-Phosphate Is Indispensable for the Biogenesis of the Endosomal Recycling Compartment." Cells 11, no. 6 (March 11, 2022): 962. http://dx.doi.org/10.3390/cells11060962.
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Phosphatidylinositol-3-phosphate (PI3P), a major identity tag of early endosomes (EEs), provides a platform for the recruitment of numerous cellular proteins containing an FYVE or PX domain that is required for PI3P-dependent maturation of EEs. Most of the PI3P in EEs is generated by the activity of Vps34, a catalytic component of class III phosphatidylinositol-3-phosphate kinase (PI3Ks) complex. In this study, we analyzed the role of Vps34-derived PI3P in the EE recycling circuit of unperturbed cells using VPS34-IN1 (IN1), a highly specific inhibitor of Vps34. IN1-mediated PI3P depletion resulted in the rapid dissociation of recombinant FYVE- and PX-containing PI3P-binding modules and endogenous PI3P-binding proteins, including EEA1 and EE sorting nexins. IN1 treatment triggered the rapid restructuring of EEs into a PI3P-independent functional configuration, and after IN1 washout, EEs were rapidly restored to a PI3P-dependent functional configuration. Analysis of the PI3P-independent configuration showed that the Vps34-derived PI3P is not essential for the pre-EE-associated functions and the fast recycling loop of the EE recycling circuit but contributes to EE maturation toward the degradation circuit, as previously shown in Vps34 knockout and knockdown studies. However, our study shows that Vps34-derived PI3P is also essential for the establishment of the Rab11a-dependent pathway, including recycling cargo sorting in this pathway and membrane flux from EEs to the pericentriolar endosomal recycling compartment (ERC). Rab11a endosomes of PI3P-depleted cells expanded and vacuolized outside the pericentriolar area without the acquisition of internalized transferrin (Tf). These endosomes had high levels of FIP5 and low levels of FIP3, suggesting that their maturation was arrested before the acquisition of FIP3. Consequently, Tf-loaded-, Rab11a/FIP5-, and Rab8a-positive endosomes disappeared from the pericentriolar area, implying that PI3P-associated functions are essential for ERC biogenesis. ERC loss was rapidly reversed after IN1 washout, which coincided with the restoration of FIP3 recruitment to Rab11a-positive endosomes and their dynein-dependent migration to the cell center. Thus, our study shows that Vps34-derived PI3P is indispensable in the recycling circuit to maintain the slow recycling pathway and biogenesis of the ERC.
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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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Khaled, Annette, Shannon Ruppert, and Adina Carlson. "Bim is a component of the endosomal-lysosomal interface in Interleukin-7 dependent T cells (57.10)." Journal of Immunology 186, no. 1_Supplement (April 1, 2011): 57.10. http://dx.doi.org/10.4049/jimmunol.186.supp.57.10.
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Abstract Bim is a key protein in T cell homeostasis. Bim deficiency prevents activated T cell death, and protects splenic T cells in IL-7R/Bim double knockout mice through an undetermined mechanism. We propose a novel function for Bim in T cells involving lysosomal development in both apoptotic and non-apoptotic cells. Using IL-7 dependent T cell lines, Bim-containing and Bim-deficient (Bim-/-), we determined that the transcription of bim increased in the absence of IL-7 and the loss of Bim protected cells from apoptosis induced by IL-7 withdrawal. Treatment of Bim-containing cells with Bim siRNA confirmed these results. Bim-/- cells had increased endosomal and decreased lysosomal proteins, suggesting that Bim plays a role in endosomal to lysosomal trafficking and maturation. Subcellular organelle fractionation revealed that the BimL isoform associated with both endosomal and lysosomal fractions in non-apoptotic cells. However, during IL-7 withdrawal, the BimEL isoform associated with lysosomal fractions and less so with endosomal fractions. In fact, reduced co-localization of endosomes and lysosomes in the absence of Bim was observed by confocal microscopy, suggesting a defect in an endocytic compartment. Overexpression of the individual Bim isoforms restored acidic organelle distribution with BimL contributing the most in non-apoptotic cells. These results demonstrate that Bim has both apoptotic and non-apoptotic activities in T cells that affects lysosomal equilibrium.
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Grissom, James H., Verónica A. Segarra, and Richard J. Chi. "New Perspectives on SNARE Function in the Yeast Minimal Endomembrane System." Genes 11, no. 8 (August 6, 2020): 899. http://dx.doi.org/10.3390/genes11080899.
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Saccharomyces cerevisiae is one of the best model organisms for the study of endocytic membrane trafficking. While studies in mammalian cells have characterized the temporal and morphological features of the endocytic pathway, studies in budding yeast have led the way in the analysis of the endosomal trafficking machinery components and their functions. Eukaryotic endomembrane systems were thought to be highly conserved from yeast to mammals, with the fusion of plasma membrane-derived vesicles to the early or recycling endosome being a common feature. Upon endosome maturation, cargos are then sorted for reuse or degraded via the endo-lysosomal (endo-vacuolar in yeast) pathway. However, recent studies have shown that budding yeast has a minimal endomembrane system that is fundamentally different from that of mammalian cells, with plasma membrane-derived vesicles fusing directly to a trans-Golgi compartment which acts as an early endosome. Thus, the Golgi, rather than the endosome, acts as the primary acceptor of endocytic vesicles, sorting cargo to pre-vacuolar endosomes for degradation. The field must now integrate these new findings into a broader understanding of the endomembrane system across eukaryotes. This article synthesizes what we know about the machinery mediating endocytic membrane fusion with this new model for yeast endomembrane function.
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Tomaš, Maja Ilić, Natalia Kučić, Hana Mahmutefendić, Gordana Blagojević, and Pero Lučin. "Murine Cytomegalovirus Perturbs Endosomal Trafficking of Major Histocompatibility Complex Class I Molecules in the Early Phase of Infection." Journal of Virology 84, no. 21 (August 18, 2010): 11101–12. http://dx.doi.org/10.1128/jvi.00988-10.
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ABSTRACT Murine cytomegalovirus (MCMV) functions interfere with protein trafficking in the secretory pathway. In this report we used Δm138-MCMV, a recombinant virus with a deleted viral Fc receptor, to demonstrate that MCMV also perturbs endosomal trafficking in the early phase of infection. This perturbation had a striking impact on cell surface-resident major histocompatibility complex class I (MHC-I) molecules due to the complementary effect of MCMV immunoevasins, which block their egress from the secretory pathway. In infected cells, constitutively endocytosed cell surface-resident MHC-I molecules were arrested and retained in early endosomal antigen 1 (EEA1)-positive and lysobisphosphatidic acid (LBPA)-negative perinuclear endosomes together with clathrin-dependent cargo (transferrin receptor, Lamp1, and epidermal growth factor receptor). Their progression from these endosomes into recycling and degradative routes was inhibited. This arrest was associated with a reduction of the intracellular content of Rab7 and Rab11, small GTPases that are essential for the maturation of recycling and endolysosomal domains of early endosomes. The reduced recycling of MHC-I in Δm138-MCMV-infected cells was accompanied by their accelerated loss from the cell surface. The MCMV function that affects cell surface-resident MHC-I was activated in later stages of the early phase of viral replication, after the expression of known immunoevasins. MCMV without the three immunoevasins (the m04, m06, and m152 proteins) encoded a function that affects endosomal trafficking. This function, however, was not sufficient to reduce the cell surface expression of MHC-I in the absence of the transport block in the secretory pathway.
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Sturgill-Koszycki, Sheila, and Michele S. Swanson. "Legionella pneumophila Replication Vacuoles Mature into Acidic, Endocytic Organelles." Journal of Experimental Medicine 192, no. 9 (October 30, 2000): 1261–72. http://dx.doi.org/10.1084/jem.192.9.1261.
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After ingestion by macrophages, Legionella pneumophila inhibits acidification and maturation of its phagosome. After a 6–10-h lag period, the bacteria replicate for 10–14 h until macrophage lysis releases dozens of progeny. To examine whether the growth phase of intracellular L. pneumophila determines the fate of its phagosome, interactions between the endosomal network and pathogen vacuoles were analyzed throughout the primary infection period. Surprisingly, as L. pneumophila replicated exponentially, a significant proportion of the vacuoles acquired lysosomal characteristics. By 18 h, 70% contained lysosomal-associated membrane protein 1 (LAMP-1) and 40% contained cathepsin D; 50% of the vacuoles could be labeled by endocytosis, and the pH of this population of vacuoles averaged 5.6. Moreover, L. pneumophila appeared to survive and replicate within lysosomal compartments: vacuoles harboring more than five bacteria also contained LAMP-1, inhibition of vacuole acidification and maturation by bafilomycin A1 inhibited bacterial replication, bacteria within endosomal vacuoles responded to a metabolic inducer by expressing a gfp reporter gene, and replicating bacteria obtained from macrophages, but not broth, were acid resistant. Understanding how L. pneumophila first evades and then exploits the endosomal pathway to replicate within macrophages may reveal the mechanisms governing phagosome maturation, a process also manipulated by Mycobacteria, Leishmania, and Coxiella.
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Jia, Da, Timothy S. Gomez, Daniel D. Billadeau, and Michael K. Rosen. "Multiple repeat elements within the FAM21 tail link the WASH actin regulatory complex to the retromer." Molecular Biology of the Cell 23, no. 12 (June 15, 2012): 2352–61. http://dx.doi.org/10.1091/mbc.e11-12-1059.
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Wiskott–Aldrich syndrome protein (WASPs) control actin dynamics in cellular processes, including cell motility, receptor-mediated endocytosis, bacterial invasion, and vesicular trafficking. We demonstrated that WASH, a recently identified WASP family protein, colocalizes on endosomal subdomains with the cargo-selective complex (CSC) of the retromer, where it regulates retrograde sorting from endosomes in an actin-dependent manner. However, the mechanism of WASH recruitment to these retromer-enriched endosomal subdomains is unclear. Here we show that a component of the WASH regulatory complex (SHRC), FAM21, which contains 21 copies of a novel L-F-[D/E]3-10-L-F motif, directly interacts with the retromer CSC protein VPS35. Endosomal localization of FAM21 is VPS35 dependent and relies on multivalency of FAM21 repeat elements. Using a combination of pull-down assays and isothermal calorimetry, we demonstrate that individual repeats can bind CSC, and binding affinity varies among different FAM21 repeats. A high-affinity repeat can be converted into a low-affinity one by mutation of a hydrophobic residue within the motif. These in vitro data mirror the localization of FAM21 to retromer-coated vesicles in cells. We propose that multivalency enables FAM21 to sense the density of retromer on membranes, allowing coordination of SHRC recruitment, and consequent actin polymerization, with retromer sorting domain organization/maturation.
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Kuliawat, Regina, Elena Kalinina, Jason Bock, Lloyd Fricker, Timothy E. McGraw, Se Ryoung Kim, Jiayu Zhong, Richard Scheller, and Peter Arvan. "Syntaxin-6 SNARE Involvement in Secretory and Endocytic Pathways of Cultured Pancreatic β-Cells." Molecular Biology of the Cell 15, no. 4 (April 2004): 1690–701. http://dx.doi.org/10.1091/mbc.e03-08-0554.
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In pancreatic β-cells, the syntaxin 6 (Syn6) soluble N-ethylmaleimide-sensitive factor attachment protein receptor is distributed in the trans-Golgi network (TGN) (with spillover into immature secretory granules) and endosomes. A possible Syn6 requirement has been suggested in secretory granule biogenesis, but the role of Syn6 in live regulated secretory cells remains unexplored. We have created an ecdysone-inducible gene expression system in the INS-1 β-cell line and find that induced expression of a membrane-anchorless, cytosolic Syn6 (called Syn6t), but not full-length Syn6, causes a prominent defect in endosomal delivery to lysosomes, and the TGN, in these cells. The defect occurs downstream of the endosomal branchpoint involved in transferrin recycling, and upstream of the steady-state distribution of mannose 6-phosphate receptors. By contrast, neither acquisition of stimulus competence nor the ultimate size of β-granules is affected. Biosynthetic effects of dominant-interfering Syn6 seem limited to slowed intragranular processing to insulin (achieving normal levels within 2 h) and minor perturbation of sorting of newly synthesized lysosomal proenzymes. We conclude that expression of the Syn6t mutant slows a rate-limiting step in endosomal maturation but provides only modest and potentially indirect interference with regulated and constitutive secretory pathways, and in TGN sorting of lysosomal enzymes.
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Dardalhon, V. "FRACTIONATION ANALYSIS OF THE ENDOSOMAL COMPARTMENT DURING RAT RETICULOCYTE MATURATION." Cell Biology International 26, no. 8 (August 2002): 669–78. http://dx.doi.org/10.1006/cbir.2002.0917.
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McDaniel, Margaret, Charles Tracy, Helmut Kramer, and Chandrashekhar Pasare. "Role of Vps33B in regulation of inflammatory responses." Journal of Immunology 200, no. 1_Supplement (May 1, 2018): 46.10. http://dx.doi.org/10.4049/jimmunol.200.supp.46.10.
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Abstract Macrophages and dendritic cells use pattern recognition receptors (PRRs) to sense their surroundings and respond to pathogenic stimuli. Engagement of PRRs, such as Toll-like receptors (TLRs), results in accelerated phagocytosis of cargo, activation of anti-microbial responses, and induction of phagosomal and endosomal maturation. Endo/phagosomal maturation and endo/phagolysosome fusion likely plays a critical role in regulation of TLR signaling and outcome of inflammatory responses following microbial recognition. Previous work in the lab has shown that Vps (Vacuolar protein sorting) 33B specifically regulates endosomal maturation following TLR activation, thus influencing the outcome of downstream signaling. In the absence of Vps33B, drosophila hemocytes and mouse macrophages elicit an enormous inflammatory response to live or dead bacterial challenge, yet are unable to degrade the bacterial cargo. This exaggerated immune response is a direct result of accumulated TLRs and ligands that continue to signal while stuck in late endosomal compartments. We have now discovered that this inability to degrade pathogenic cargo has critical implications for adaptive immunity. Absence of Vps33B leads to enhanced maturation of DCs, yet these DCs are unable to effectively prime CD4 T cells, presumably due to lack of processing endocytosed cargo. Mice that specifically lack Vps33B in DCs were unable to activate antigen-specific CD4 T cells in vivo, leading to defective Th1 and Th17 priming. Collectively, our results suggest an important role for Vps33B in both innate and adaptive immune responses. Further work will elucidate the molecular mechanisms of regulation of Vps33B downstream of TLRs and its impact on host immunity.
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Mainou, Bernardo A., and Terence S. Dermody. "Transport to Late Endosomes Is Required for Efficient Reovirus Infection." Journal of Virology 86, no. 16 (June 6, 2012): 8346–58. http://dx.doi.org/10.1128/jvi.00100-12.
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Rab GTPases play an essential role in vesicular transport by coordinating the movement of various types of cargo from one cellular compartment to another. Individual Rab GTPases are distributed to specific organelles and thus serve as markers for discrete types of endocytic vesicles. Mammalian reovirus binds to cell surface glycans and junctional adhesion molecule-A (JAM-A) and enters cells by receptor-mediated endocytosis in a process dependent on β1 integrin. Within organelles of the endocytic compartment, reovirus undergoes stepwise disassembly catalyzed by cathepsin proteases, which allows the disassembly intermediate to penetrate endosomal membranes and release the transcriptionally active viral core into the cytoplasm. The pathway used by reovirus to traverse the endocytic compartment is largely unknown. In this study, we found that reovirus particles traffic through early, late, and recycling endosomes during cell entry. After attachment to the cell surface, reovirus particles and JAM-A codistribute into each of these compartments. Transfection of cells with constitutively active and dominant-negative Rab GTPases that affect early and late endosome biogenesis and maturation influenced reovirus infectivity. In contrast, reovirus infectivity was not altered in cells expressing mutant Rab GTPases that affect recycling endosomes. Thus, reovirus virions localize to early, late, and recycling endosomes during entry into host cells, but only those that traverse early and late endosomes yield a productive infection.
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Vieira, Otilia V., Rene E. Harrison, Cameron C. Scott, Harald Stenmark, David Alexander, Jun Liu, Jean Gruenberg, Alan D. Schreiber, and Sergio Grinstein. "Acquisition of Hrs, an Essential Component of Phagosomal Maturation, Is Impaired by Mycobacteria." Molecular and Cellular Biology 24, no. 10 (May 15, 2004): 4593–604. http://dx.doi.org/10.1128/mcb.24.10.4593-4604.2004.
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ABSTRACT Pathogenic mycobacteria survive within macrophages by precluding the fusion of phagosomes with late endosomes or lysosomes. Because the molecular determinants of normal phagolysosome formation are poorly understood, the sites targeted by mycobacteria remain unidentified. We found that Hrs, an adaptor molecule involved in protein sorting, associates with phagosomes prior to their fusion with late endosomes or lysosomes. Recruitment of Hrs required the interaction of its FYVE domain with phagosomal phosphatidylinositol 3-phosphate, but two other attachment sites were additionally involved. Depletion of Hrs by use of small interfering RNA impaired phagosomal maturation, preventing the acquisition of lysobisphosphatidic acid and reducing luminal acidification. As a result, the maturation of phagosomes formed in Hrs-depleted cells was arrested at an early stage, characterized by the acquisition and retention of sorting endosomal markers. This phenotype is strikingly similar to that reported to occur in phagosomes of cells infected by mycobacteria. We therefore tested whether Hrs is recruited to phagosomes containing mycobacteria. Hrs associated readily with phagosomes containing inert particles but poorly with mycobacterial phagosomes. Moreover, Hrs was found more frequently in phagosomes containing avirulent Mycobacterium smegmatis than in phagosomes with the more virulent Mycobacterium marinum. These findings suggest that the inability to recruit Hrs contributes to the arrest of phagosomal maturation induced by pathogenic mycobacteria.