Journal articles on the topic 'Endocytic maturation'
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1
Thilo, L., E. Stroud, and T. Haylett. "Maturation of early endosomes and vesicular traffic to lysosomes in relation to membrane recycling." Journal of Cell Science 108, no. 4 (April 1, 1995): 1791–803. http://dx.doi.org/10.1242/jcs.108.4.1791.
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The controversy whether endocytic processing occurs by organellar maturation or by vesicular traffic has not been resolved. It is also not clear whether maturation continues to the stage of lysosomes, to what extent it involves a decrease in organellar fusogenicity, and how it relates to membrane recycling. Maturation and vesicular traffic imply distinct kinetics for the intermingling of endocytic markers after sequential endocytic uptake. We have studied the kinetics of intermingling of fluid-phase markers (fluorescein-labelled dextran and horseradish peroxidase) and cell surface-derived membrane (labelled by galactosylation) in organelles at early and late stages of the endocytic pathway in macrophage-like P388D1 cells. Intermingling declined by sigmoid kinetics, indicating that endosomes matured within about 3 minutes to become non-fusogenic towards early endosomes. During maturation about 60% of internalized membrane was recycled with T1/2 approximately 2 minutes. Whereas matured endosomes were non-fusogenic towards early endosomes and towards each other, a second phase of intermingling was observed upon delivery to lysosomes. This intermingling occurred by a first-order process (T1/2 approximately 4 minutes), concurrent with recycling of the remaining 40% of internalized membrane marker. These kinetic observations suggest a model for endocytic processing which reconciles maturation of early endosomes with the known function of carrier vesicles: Endocytic carrier vesicles do not bud off from permanent early endosomes as proposed for vesicular traffic, but are derived, together with recycling vesicles, from the maturation of early endosomes which are consumed by this process; these carrier vesicles subsequently mediate delivery to lysosomes by vesicular traffic during which the remaining surface-derived membrane is recycled.
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Carroll, Susheela Y., Helen E. M. Stimpson, Jasper Weinberg, Christopher P. Toret, Yidi Sun, and David G. Drubin. "Analysis of yeast endocytic site formation and maturation through a regulatory transition point." Molecular Biology of the Cell 23, no. 4 (February 15, 2012): 657–68. http://dx.doi.org/10.1091/mbc.e11-02-0108.
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The earliest stages of endocytic site formation and the regulation of endocytic site maturation are not well understood. Here we analyzed the order in which the earliest proteins are detectable at endocytic sites in budding yeast and found that an uncharacterized protein, Pal1p/Ydr348cp, is also present at the initial stages of endocytosis. Because Ede1p (homologue of Eps15) and clathrin are the early-arriving proteins most important for cargo uptake, their roles during the early stages of endocytosis were examined more comprehensively. Ede1p is necessary for efficient recruitment of most early-arriving proteins, but not for the recruitment of the adaptor protein Yap1802p, to endocytic sites. The early-arriving proteins, as well as the later-arriving proteins Sla2p and Ent1/2p (homologues of Hip1R and epsins), were found to have longer lifetimes in CLC1-knockout yeast, which indicates that clathrin light chain facilitates the transition from the intermediate to late coat stages. Cargo also arrives during the early stages of endocytosis, and therefore its effect on endocytic machinery dynamics was investigated. Our results are consistent with a role for cargo in regulating the transition of endocytic sites from the early stages of formation to the late stages during which vesicle formation occurs.
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Lu, Rebecca, and David G. Drubin. "Selection and stabilization of endocytic sites by Ede1, a yeast functional homologue of human Eps15." Molecular Biology of the Cell 28, no. 5 (March 2017): 567–75. http://dx.doi.org/10.1091/mbc.e16-06-0391.
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During clathrin-mediated endocytosis (CME), endocytic-site maturation can be divided into two stages corresponding to the arrival of the early and late proteins at the plasma membrane. The early proteins are required to capture cargo and position the late machinery, which includes proteins involved in actin assembly and membrane scission. However, the mechanism by which early-arriving proteins select and stabilize endocytic sites is not known. Ede1, one of the earliest proteins recruited to endocytic sites, facilitates site initiation and stabilization. Deletion of EDE1 results in fewer CME initiations and defects in the timing of vesicle maturation. Here we made truncation mutants of Ede1 to better understand how different domains contribute to its recruitment to CME sites, site selection, and site maturation. We found that the minimal domains required for efficient Ede1 localization at CME sites are the third EH domain, the proline-rich region, and the coiled-coil region. We also found that many strains expressing ede1 truncations could support a normal rate of site initiation but still had defects in site-maturation timing, indicating separation of Ede1 functions. When expressed in yeast, human Eps15 localized to the plasma membrane, where it recruited late-phase CME proteins and supported productive endocytosis, identifying it as an Ede1 functional homologue.
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Liu, Guojun, Paulomi Sanghavi, Kathryn E. Bollinger, Libby Perry, Brendan Marshall, Penny Roon, Tsubasa Tanaka, Akira Nakamura, and Graydon B. Gonsalvez. "Efficient Endocytic Uptake and Maturation inDrosophilaOocytes Requires Dynamitin/p50." Genetics 201, no. 2 (August 10, 2015): 631–49. http://dx.doi.org/10.1534/genetics.115.180018.
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Stevens-Hernandez, Christian J., and Lesley J. Bruce. "Reticulocyte Maturation." Membranes 12, no. 3 (March 10, 2022): 311. http://dx.doi.org/10.3390/membranes12030311.
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Changes to the membrane proteins and rearrangement of the cytoskeleton must occur for a reticulocyte to mature into a red blood cell (RBC). Different mechanisms of reticulocyte maturation have been proposed to reduce the size and volume of the reticulocyte plasma membrane and to eliminate residual organelles. Lysosomal protein degradation, exosome release, autophagy and the extrusion of large autophagic–endocytic hybrid vesicles have been shown to contribute to reticulocyte maturation. These processes may occur simultaneously or perhaps sequentially. Reticulocyte maturation is incompletely understood and requires further investigation. RBCs with membrane defects or cation leak disorders caused by genetic variants offer an insight into reticulocyte maturation as they present characteristics of incomplete maturation. In this review, we compare the structure of the mature RBC membrane with that of the reticulocyte. We discuss the mechanisms of reticulocyte maturation with a focus on incomplete reticulocyte maturation in red cell variants.
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Dumas, Audrey, Gabrielle Lê-Bury, Florence Marie-Anaïs, Floriane Herit, Julie Mazzolini, Thomas Guilbert, Pierre Bourdoncle, et al. "The HIV-1 protein Vpr impairs phagosome maturation by controlling microtubule-dependent trafficking." Journal of Cell Biology 211, no. 2 (October 26, 2015): 359–72. http://dx.doi.org/10.1083/jcb.201503124.
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Human immunodeficiency virus type 1 (HIV-1) impairs major functions of macrophages but the molecular basis for this defect remains poorly characterized. Here, we show that macrophages infected with HIV-1 were unable to respond efficiently to phagocytic triggers and to clear bacteria. The maturation of phagosomes, defined by the presence of late endocytic markers, hydrolases, and reactive oxygen species, was perturbed in HIV-1–infected macrophages. We showed that maturation arrest occurred at the level of the EHD3/MICAL-L1 endosomal sorting machinery. Unexpectedly, we found that the regulatory viral protein (Vpr) was crucial to perturb phagosome maturation. Our data reveal that Vpr interacted with EB1, p150Glued, and dynein heavy chain and was sufficient to critically alter the microtubule plus end localization of EB1 and p150Glued, hence altering the centripetal movement of phagosomes and their maturation. Thus, we identify Vpr as a modulator of the microtubule-dependent endocytic trafficking in HIV-1–infected macrophages, leading to strong alterations in phagolysosome biogenesis.
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Lennon-Duménil, Ana-Maria, Arnold H. Bakker, René Maehr, Edda Fiebiger, Herman S. Overkleeft, Mario Rosemblatt, Hidde L. Ploegh, and Cécile Lagaudrière-Gesbert. "Analysis of Protease Activity in Live Antigen-presenting Cells Shows Regulation of the Phagosomal Proteolytic Contents During Dendritic Cell Activation." Journal of Experimental Medicine 196, no. 4 (August 19, 2002): 529–40. http://dx.doi.org/10.1084/jem.20020327.
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Here, we describe a new approach designed to monitor the proteolytic activity of maturing phagosomes in live antigen-presenting cells. We find that an ingested particle sequentially encounters distinct protease activities during phagosomal maturation. Incorporation of active proteases into the phagosome of the macrophage cell line J774 indicates that phagosome maturation involves progressive fusion with early and late endocytic compartments. In contrast, phagosome biogenesis in bone marrow–derived dendritic cells (DCs) and macrophages preferentially involves endocytic compartments enriched in cathepsin S. Kinetics of phagosomal maturation is faster in macrophages than in DCs. Furthermore, the delivery of active proteases to the phagosome is significantly reduced after the activation of DCs with lipopolysaccharide. This observation is in agreement with the notion that DCs prevent the premature destruction of antigenic determinants to optimize T cell activation. Phagosomal maturation is therefore a tightly regulated process that varies according to the type and differentiation stage of the phagocyte.
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Mettlen, Marcel, Ping-Hung Chen, Saipraveen Srinivasan, Gaudenz Danuser, and Sandra L. Schmid. "Regulation of Clathrin-Mediated Endocytosis." Annual Review of Biochemistry 87, no. 1 (June 20, 2018): 871–96. http://dx.doi.org/10.1146/annurev-biochem-062917-012644.
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Clathrin-mediated endocytosis (CME) is the major endocytic pathway in mammalian cells. It is responsible for the uptake of transmembrane receptors and transporters, for remodeling plasma membrane composition in response to environmental changes, and for regulating cell surface signaling. CME occurs via the assembly and maturation of clathrin-coated pits that concentrate cargo as they invaginate and pinch off to form clathrin-coated vesicles. In addition to the major coat proteins, clathrin triskelia and adaptor protein complexes, CME requires a myriad of endocytic accessory proteins and phosphatidylinositol lipids. CME is regulated at multiple steps—initiation, cargo selection, maturation, and fission—and is monitored by an endocytic checkpoint that induces disassembly of defective pits. Regulation occurs via posttranslational modifications, allosteric conformational changes, and isoform and splice-variant differences among components of the CME machinery, including the GTPase dynamin. This review summarizes recent findings on the regulation of CME and the evolution of this complex process.
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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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Merrill, Nathan M., Joshua L. Schipper, Jonathan B. Karnes, Audra L. Kauffman, Katie R. Martin, and Jeffrey P. MacKeigan. "PI3K-C2α knockdown decreases autophagy and maturation of endocytic vesicles." PLOS ONE 12, no. 9 (September 14, 2017): e0184909. http://dx.doi.org/10.1371/journal.pone.0184909.
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Mettlen, Marcel, Miriam Stoeber, Dinah Loerke, Costin N. Antonescu, Gaudenz Danuser, and Sandra L. Schmid. "Endocytic Accessory Proteins Are Functionally Distinguished by Their Differential Effects on the Maturation of Clathrin-coated Pits." Molecular Biology of the Cell 20, no. 14 (July 15, 2009): 3251–60. http://dx.doi.org/10.1091/mbc.e09-03-0256.
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Diverse cargo molecules (i.e., receptors and ligand/receptor complexes) are taken into the cell by clathrin-mediated endocytosis (CME) utilizing a core machinery consisting of cargo-specific adaptors, clathrin and the GTPase dynamin. Numerous endocytic accessory proteins are also required, but their differential roles and functional hierarchy during CME are not yet understood. Here, we used a combination of quantitative live-cell imaging by total internal reflection fluorescence microscopy (TIR-FM), and decomposition of the lifetime distributions of clathrin-coated pits (CCPs) to measure independent aspects of CCP dynamics, including the turnover of abortive and productive CCP species and their relative contributions. Capitalizing on the sensitivity of this assay, we have examined the effects of specific siRNA-mediated depletion of endocytic accessory proteins on CME progression. Of the 12 endocytic accessory proteins examined, we observed seven qualitatively different phenotypes upon protein depletion. From this data we derive a temporal hierarchy of protein function during early steps of CME. Our results support the idea that a subset of accessory proteins, which mediate coat assembly, membrane curvature, and cargo selection, can provide input into an endocytic restriction point/checkpoint mechanism that monitors CCP maturation.
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Desjardins, M., N. N. Nzala, R. Corsini, and C. Rondeau. "Maturation of phagosomes is accompanied by changes in their fusion properties and size-selective acquisition of solute materials from endosomes." Journal of Cell Science 110, no. 18 (September 15, 1997): 2303–14. http://dx.doi.org/10.1242/jcs.110.18.2303.
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Maturation of phagosomes is characterized by changes in their polypeptides, phosphorylated proteins and phospholipid composition. Kinetic analyses have shown that a variety of proteins associate and dissociate from latex-containing phagosomes at precise intervals during phagolysosome biogenesis. In an attempt to link these temporal biochemical modifications to functional changes, we have examined the in vivo fusion properties of aging endosomes and phagosomes. Using an in vivo fusion assay at the electron microscope, we measured the rate of exchange of bovine serum albumin-gold (5 and 16 nm particles) between endosomes and latex-bead-containing phagosomes. The results obtained indicate that the maturation of phagosomes is accompanied by changes of their fusion properties. Early phagosomes were shown to fuse preferentially with early endocytic organelles and to gradually acquire the ability to fuse with late endocytic organelles. Furthermore, the transfer of bovine serum albumin-gold from endosomes to phagosomes is size-dependent, a process also modulated by the maturation of these organelles, in agreement with the concept that transient fusion events occur between endosomes and phagosomes. Biochemical analysis showed variations in the levels of rab proteins associated with phagosomes during maturation while other ‘fusion’ proteins, including synaptobrevin1 and synaptobrevin2, remained constant.
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Santic, Marina, Maëlle Molmeret, and Yousef Abu Kwaik. "Maturation of the Legionella pneumophila-Containing Phagosome into a Phagolysosome within Gamma Interferon-Activated Macrophages." Infection and Immunity 73, no. 5 (May 2005): 3166–71. http://dx.doi.org/10.1128/iai.73.5.3166-3171.2005.
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ABSTRACT Legionella pneumophila is an intracellular pathogen that modulates the biogenesis of its phagosome to evade endocytic vesicle traffic. The Legionella-containing phagosome (LCP) does not acquire any endocytic markers and is remodeled by the endoplasmic reticulum during early stages. Here we show that intracellular replication of L. pneumophila is inhibited in gamma interferon (IFN-γ)-activated, bone marrow-derived mouse macrophages and IFN-γ-activated, human monocyte-derived macrophages in a dose-dependent manner. This inhibition of intracellular replication is associated with the maturation of the LCP into a phagolysosome, as documented by the acquisition of LAMP-2, cathepsin D, and lysosomal tracer Texas Red ovalbumin, and with the failure of the LCP to be remodeled by the rough endoplasmic reticulum. We conclude that IFN-γ-activated macrophages override the ability of L. pneumophila to evade endocytic fusion and that the LCP is processed through the “default” endosomal-lysosomal degradation pathway.
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Mordue, D. G., and L. D. Sibley. "Intracellular fate of vacuoles containing Toxoplasma gondii is determined at the time of formation and depends on the mechanism of entry." Journal of Immunology 159, no. 9 (November 1, 1997): 4452–59. http://dx.doi.org/10.4049/jimmunol.159.9.4452.
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Abstract We have characterized the intracellular fate of Toxoplasma in bone marrow-derived macrophages following two disparate modes of uptake: phagocytosis vs active invasion. The fate of parasite-containing vacuoles was followed by immunofluorescence localization of endogenous endocytic markers to track phagocytic processing in pulse-infected cells. During uptake of both opsonized and dead parasites, host cell plasma membrane MHC class II molecules and FcR were internalized into the phagosome and then gradually lost. Maturation of phagosomes containing Toxoplasma was a rapid, dynamic process of sequential fusion with early endosomes, late endosomes, and lysosomes that was complete within 15 min. Toxoplasma-containing phagosomes were transiently positive for transferrin receptor between 0 and 2.5 min, then contained the cation-independent mannose 6-phosphate receptor between 2.5 and 7.5 min, and finally matured to lysosome-like compartments containing lysosomal membrane glycoprotein 1 and the proton pump, but lacking cation-independent mannose 6-phosphate receptor. Toxoplasma-containing phagosomes also sequentially acquired host proteins that regulate endocytic fusion including rab5, N-ethylmaleimide-sensitive factor, and rab7. In marked contrast, MHC class II molecules and FcR were excluded from the parasitophorous vacuole formed by active parasite invasion. The parasitophorous vacuole also failed to acquire any host compartmental markers or fusion proteins analyzed. Our results indicate that Toxoplasma evades endocytic processing due to an absence of host regulatory proteins necessary to drive endocytic fusion, and that this divergence from normal maturation occurs during the formation of the primary vacuole.
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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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Vines, James H., and Jason S. King. "The endocytic pathways of Dictyostelium discoideum." International Journal of Developmental Biology 63, no. 8-9-10 (2019): 461–71. http://dx.doi.org/10.1387/ijdb.190236jk.
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The formation and processing of vesicles from the cell surface serves many important cellular functions ranging from nutrient acquisition to regulating the turnover of membrane components and signalling. In this article, we summarise the endocytic pathways of the social amoeba Dictyostelium from the clathrin-dependent and independent internalisation of surface components to the engulfment of bacteria or fluid by phagocytosis and macropinocytosis respectively. Due to similarities with the professional phagocytes of the mammalian immune system Dictyostelium has been extensively used to investigate the complex remodelling and trafficking events that occur as phagosomes and macropinosomes transit through the cell. Here we discuss what is known about this maturation process in order to kill any potential pathogens and obtain nutrients for growth. Finally, we aim to put these studies in evolutionary context and highlight some of the many questions that remain in our understanding of these complex and important pathways.
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Illinger, D., and J. G. Kuhry. "The kinetic aspects of intracellular fluorescence labeling with TMA-DPH support the maturation model for endocytosis in L929 cells." Journal of Cell Biology 125, no. 4 (May 15, 1994): 783–94. http://dx.doi.org/10.1083/jcb.125.4.783.
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TMA-DPH (1-(4-trimethylammonium)-6-phenyl-1,3,5-hexatriene), a hydrophobic fluorescent membrane probe, interacts with living cells by instantaneous incorporation into the plasma membrane, where it becomes fluorescent. It then follows the intracellular constitutive membrane traffic and acts as a bulk membrane marker of the endocytic pathway (Illinger, D., P. Poindron, P. Fonteneau, M. Modolell, and J. G. Kuhry. 1990. Biochim. Biophys. Acta. 1030:73-81; Illinger, D., P. Poindron, and J. G. Kuhry. 1991. Biol. Cell. 73:131-138). As such, TMA-DPH displays particular properties mainly due to partition between membranes and aqueous media. From these properties, original arguments can be inferred in favor of the maturation model for the endocytic pathway, against that of pre-existing compartments, in L929 cultured mouse fibroblasts. (a) TMA-DPH labeling is seen to progress from the cell periphery to perinuclear regions during endocytosis without any noticeable loss in fluorescence intensity; with a vesicle shuttle model this evolution would be accompanied by probe dilution with a decrease in the overall intracellular fluorescence intensity, and the labeling of the inner (late) compartments could in no way become more intense than that of the peripheral (early) ones. (b) From TMA-DPH fluorescence anisotropy assays, it is concluded that membrane fluidity is the same in the successive endocytic compartments as in the plasma membrane, which probably denotes a similar phospholipidic membrane composition, as might be expected in the maturation model. (c) TMA-DPH internalization and release kinetics are more easily described with the maturation model.
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Tabata, Keisuke, Kohichi Matsunaga, Ayuko Sakane, Takuya Sasaki, Takeshi Noda, and Tamotsu Yoshimori. "Rubicon and PLEKHM1 Negatively Regulate the Endocytic/Autophagic Pathway via a Novel Rab7-binding Domain." Molecular Biology of the Cell 21, no. 23 (December 2010): 4162–72. http://dx.doi.org/10.1091/mbc.e10-06-0495.
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The endocytic and autophagic pathways are involved in the membrane trafficking of exogenous and endogenous materials to lysosomes. However, the mechanisms that regulate these pathways are largely unknown. We previously reported that Rubicon, a Beclin 1–binding protein, negatively regulates both the autophagic and endocytic pathways by unidentified mechanisms. In this study, we performed database searches to identify potential Rubicon homologues that share the common C-terminal domain, termed the RH domain. One of them, PLEKHM1, the causative gene of osteopetrosis, also suppresses endocytic transport but not autophagosome maturation. Rubicon and PLEKHM1 specifically and directly interact with Rab7 via their RH domain, and this interaction is critical for their function. Furthermore, we show that Rubicon but not PLEKHM1 uniquely regulates membrane trafficking via simultaneously binding both Rab7 and PI3-kinase.
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Ganley, Ian G. "Autophagosome maturation and lysosomal fusion." Essays in Biochemistry 55 (September 27, 2013): 65–78. http://dx.doi.org/10.1042/bse0550065.
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Compartmentalization is essential in the eukaryotic cell and this is most often achieved by sequestering specific components that perform a related function in a membrane-bound organelle. To function normally these organelles must transiently fuse with other compartments in order to transfer protein and lipid that is needed for them to function. These events must be highly coordinated otherwise non-specific fusion could occur leading to loss of compartment identity and function. The autophagosome is a specialized membrane compartment that delivers cytosolic components to the lysosome for degradation. Likewise, this delivery is coordinated so that only when the autophagosome is fully formed is it imparted with the information to allow it to specifically fuse with the endocytic system and deliver its contents to the lysosome. In the present chapter, I discuss our current understanding of how this happens.
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Lőrincz, Péter, Sarolta Tóth, Péter Benkő, Zsolt Lakatos, Attila Boda, Gábor Glatz, Martina Zobel, et al. "Rab2 promotes autophagic and endocytic lysosomal degradation." Journal of Cell Biology 216, no. 7 (May 8, 2017): 1937–47. http://dx.doi.org/10.1083/jcb.201611027.
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Rab7 promotes fusion of autophagosomes and late endosomes with lysosomes in yeast and metazoan cells, acting together with its effector, the tethering complex HOPS. Here we show that another small GTPase, Rab2, is also required for autophagosome and endosome maturation and proper lysosome function in Drosophila melanogaster. We demonstrate that Rab2 binds to HOPS, and that its active, GTP-locked form associates with autolysosomes. Importantly, expression of active Rab2 promotes autolysosomal fusions unlike that of GTP-locked Rab7, suggesting that its amount is normally rate limiting. We also demonstrate that RAB2A is required for autophagosome clearance in human breast cancer cells. In conclusion, we identify Rab2 as a key factor for autophagic and endocytic cargo delivery to and degradation in lysosomes.
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Gabel, C. A., and S. A. Foster. "Postendocytic maturation of acid hydrolases: evidence of prelysosomal processing." Journal of Cell Biology 105, no. 4 (October 1, 1987): 1561–70. http://dx.doi.org/10.1083/jcb.105.4.1561.
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The mannose 6-phosphate (Man 6-P) receptor operates to transport both endogenous newly synthesized acid hydrolases and extracellular enzymes to the lysosomal compartment. In a previous study (Gabel, C. A., and S. A. Foster, 1986, J. Cell Biol., 103:1817-1827), we noted that beta-glucuronidase molecules internalized by mouse L-cells via the Man 6-P receptor undergo a proteolytic cleavage and a limited dephosphorylation. In this report, we present evidence that indicates that the postendocytic alterations of the acid hydrolase molecules occur at a site through which the enzymes pass en route to the lysosomal compartment. Mouse L-cells incubated at 20 degrees C with beta-glucuronidase (isolated from mouse macrophage secretions) internalize the enzyme in a process that is inhibited by Man 6-P but unaffected by cycloheximide. As such, the linear accumulation of the ligand observed at 20 degrees C appears to occur through the continued recycling of the cell surface Man 6-P receptor. The subcellular distribution of the internalized ligands was assessed after homogenization of the cells and fractionation of the extracts by density gradient centrifugation. In contrast to the accumulation of the ligand within lysosomes at 37 degrees C, the beta-glucuronidase molecules internalized by the L cells at 20 degrees C accumulate within a population of vesicles that sediment at the same density as endocytic vesicles. Biochemical analysis of the internalized ligands indicates that: (a) the subunit molecular mass of both beta-glucuronidase and beta-galactosidase decrease upon cell association relative to the input form of the enzymes, and (b) the beta-glucuronidase molecules experience a limited dephosphorylation such that high-mannose-type oligosaccharides containing two phosphomonoesters are converted to single phosphomonoester forms. The same two post-endocytic alterations occur after the internalization of beta-glucuronidase by human I-cell disease fibroblasts, despite the low acid hydrolase content of these cells. The results indicate, therefore, that acid hydrolases internalized via the Man 6-P receptor are processed within the endocytic compartment. In that endogenous newly synthesized acid hydrolases display similar alterations during their maturation, the results further suggest that the endosomal compartment is involved in the sorting of ligands transported via both the cell surface and intracellular Man 6-P receptor.
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Driessen, Christoph, Rebecca A. R. Bryant, Ana-Maria Lennon-Duménil, José A. Villadangos, Paula Wolf Bryant, Guo-Ping Shi, Harold A. Chapman, and Hidde L. Ploegh. "Cathepsin S Controls the Trafficking and Maturation of Mhc Class II Molecules in Dendritic Cells." Journal of Cell Biology 147, no. 4 (November 15, 1999): 775–90. http://dx.doi.org/10.1083/jcb.147.4.775.
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Before a class II molecule can be loaded with antigenic material and reach the surface to engage CD4+ T cells, its chaperone, the class II-associated invariant chain (Ii), is degraded in a stepwise fashion by proteases in endocytic compartments. We have dissected the role of cathepsin S (CatS) in the trafficking and maturation of class II molecules by combining the use of dendritic cells (DC) from CatS−/− mice with a new active site–directed probe for direct visualization of active CatS. Our data demonstrate that CatS is active along the entire endocytic route, and that cleavage of the lysosomal sorting signal of Ii by CatS can occur there in mature DC. Genetic disruption of CatS dramatically reduces the flow of class II molecules to the cell surface. In CatS−/− DC, the bulk of major histocompatibility complex (MHC) class II molecules is retained in late endocytic compartments, although paradoxically, surface expression of class II is largely unaffected. The greatly diminished but continuous flow of class II molecules to the cell surface, in conjunction with their long half-life, can account for the latter observation. We conclude that in DC, CatS is a major determinant in the regulation of intracellular trafficking of MHC class II molecules.
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Shah, Ankur H., Nicholas L. Cianciola, Jeffrey L. Mills, Frank D. Sönnichsen, and Cathleen Carlin. "Adenovirus RIDα regulates endosome maturation by mimicking GTP-Rab7." Journal of Cell Biology 179, no. 5 (November 26, 2007): 965–80. http://dx.doi.org/10.1083/jcb.200702187.
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The small guanosine triphosphatase Rab7 regulates late endocytic trafficking. Rab7-interacting lysosomal protein (RILP) and oxysterol-binding protein–related protein 1L (ORP1L) are guanosine triphosphate (GTP)–Rab7 effectors that instigate minus end–directed microtubule transport. We demonstrate that RILP and ORP1L both interact with the group C adenovirus protein known as receptor internalization and degradation α (RIDα), which was previously shown to clear the cell surface of several membrane proteins, including the epidermal growth factor receptor and Fas (Carlin, C.R., A.E. Tollefson, H.A. Brady, B.L. Hoffman, and W.S. Wold. 1989. Cell. 57:135–144; Shisler, J., C. Yang, B. Walter, C.F. Ware, and L.R. Gooding. 1997. J. Virol. 71:8299–8306). RIDα localizes to endocytic vesicles but is not homologous to Rab7 and is not catalytically active. We show that RIDα compensates for reduced Rab7 or dominant-negative (DN) Rab7(T22N) expression. In vitro, Cu2+ binding to RIDα residues His75 and His76 facilitates the RILP interaction. Site-directed mutagenesis of these His residues results in the loss of RIDα–RILP interaction and RIDα activity in cells. Additionally, expression of the RILP DN C-terminal region hinders RIDα activity during an acute adenovirus infection. We conclude that RIDα coordinates recruitment of these GTP-Rab7 effectors to compartments that would ordinarily be perceived as early endosomes, thereby promoting the degradation of selected cargo.
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Bhave, Madhura, Rosa E. Mino, Xinxin Wang, Jeon Lee, Heather M. Grossman, Ashley M. Lakoduk, Gaudenz Danuser, Sandra L. Schmid, and Marcel Mettlen. "Functional characterization of 67 endocytic accessory proteins using multiparametric quantitative analysis of CCP dynamics." Proceedings of the National Academy of Sciences 117, no. 50 (November 30, 2020): 31591–602. http://dx.doi.org/10.1073/pnas.2020346117.
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Clathrin-mediated endocytosis (CME) begins with the nucleation of clathrin assembly on the plasma membrane, followed by stabilization and growth/maturation of clathrin-coated pits (CCPs) that eventually pinch off and internalize as clathrin-coated vesicles. This highly regulated process involves a myriad of endocytic accessory proteins (EAPs), many of which are multidomain proteins that encode a wide range of biochemical activities. Although domain-specific activities of EAPs have been extensively studied, their precise stage-specific functions have been identified in only a few cases. Using single-guide RNA (sgRNA)/dCas9 and small interfering RNA (siRNA)-mediated protein knockdown, combined with an image-based analysis pipeline, we have determined the phenotypic signature of 67 EAPs throughout the maturation process of CCPs. Based on these data, we show that EAPs can be partitioned into phenotypic clusters, which differentially affect CCP maturation and dynamics. Importantly, these clusters do not correlate with functional modules based on biochemical activities. Furthermore, we discover a critical role for SNARE proteins and their adaptors during early stages of CCP nucleation and stabilization and highlight the importance of GAK throughout CCP maturation that is consistent with GAK’s multifunctional domain architecture. Together, these findings provide systematic, mechanistic insights into the plasticity and robustness of CME.
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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.
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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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HENKEL, M. Kerstin, Gregory POTT, Andreas W. HENKEL, Luiz JULIANO, Chih-Min KAM, James C. POWERS, and Alex FRANZUSOFF. "Endocytic delivery of intramolecularly quenched substrates and inhibitors to the intracellular yeast Kex2 protease1." Biochemical Journal 341, no. 2 (July 8, 1999): 445–52. http://dx.doi.org/10.1042/bj3410445.
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Kex2 in the yeast Saccharomyces cerevisiae is a transmembrane, Ca2+-dependent serine protease of the subtilisin-like pro-protein convertase (SPC) family with specificity for cleavage after paired basic amino acids. At steady state, Kex2 is predominantly localized in late Golgi compartments and initiates the proteolytic maturation of pro-protein precursors that transit the distal secretory pathway. However, Kex2 localization is not static, and its itinerary apparently involves transiting out of the late Golgi and cycling back from post-Golgi endosomal compartments during its lifetime. We tested whether the endocytic pathway could deliver small molecules to Kex2 from the extracellular medium. Here we report that intramolecularly quenched fluorogenic substrates taken up into intact yeast revealed fluorescence due to specific cleavage by Kex2 protease in endosomal compartments. Furthermore, the endocytic delivery of protease inhibitors interfered with Kex2 activity for precursor protein processing. These observations reveal that the endocytic pathway does intersect with the cycling itinerary of active Kex2 protease. This strategy of endocytic drug delivery has implications for modulating SPC protease activity needed for hormone, toxin and viral glycoprotein precursor processing in human cells.
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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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Long, Kimberly R., Katherine E. Shipman, Youssef Rbaibi, Elizabeth V. Menshikova, Vladimir B. Ritov, Megan L. Eshbach, Yu Jiang, Edwin K. Jackson, Catherine J. Baty, and Ora A. Weisz. "Proximal tubule apical endocytosis is modulated by fluid shear stress via an mTOR-dependent pathway." Molecular Biology of the Cell 28, no. 19 (September 15, 2017): 2508–17. http://dx.doi.org/10.1091/mbc.e17-04-0211.
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Cells lining the proximal tubule (PT) have unique membrane specializations that are required to maintain the high-capacity ion transport and endocytic functions of this nephron segment. PT cells in vivo acutely regulate ion transport in response to changes in glomerular filtration rate (GFR) to maintain glomerulotubular balance. PT cells in culture up-regulate endocytic capacity in response to acute changes in fluid shear stress (FSS); however, it is not known whether GFR modulates PT endocytosis to enable maximally efficient uptake of filtered proteins in vivo. Here, we show that cells cultured under continuous FSS develop an expanded apical endocytic pathway and increased endocytic capacity and lysosomal biogenesis. Furthermore, endocytic capacity in fully differentiated cells is rapidly modulated by changes in FSS. PT cells exposed to continuous FSS also acquired an extensive brush border and basolateral membrane invaginations resembling those observed in vivo. Culture under suboptimal levels of FSS led to intermediate phenotypes, suggesting a threshold effect. Cells exposed to FSS expressed higher levels of key proteins necessary for PT function, including ion transporters, receptors, and membrane-trafficking machinery, and increased adenine nucleotide levels. Inhibition of the mechanistic target of rapamycin (mTOR) using rapamycin prevented the increase in cellular energy levels, lysosomal biogenesis, and endocytic uptake, suggesting that these represent a coordinated differentiation program. In contrast, rapamycin did not prevent the FSS-induced increase in Na+/K+-ATPase levels. Our data suggest that rapid tuning of the endocytic response by changes in FSS may contribute to glomerulotubular balance in vivo. Moreover, FSS provides an essential stimulus in the differentiation of PT cells via separate pathways that up-regulate endocytosis and ion transport capacity. Variations in FSS may also contribute to the maturation of PT cells during kidney development and during repair after kidney injury.
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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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Baltierra-Uribe, Shantal Lizbeth, Manuel de Jesús García-Vásquez, Nayeli Shantal Castrejón-Jiménez, Mayra Patricia Estrella-Piñón, Julieta Luna-Herrera, and Blanca Estela García-Pérez. "Mycobacteria entry and trafficking into endothelial cells." Canadian Journal of Microbiology 60, no. 9 (September 2014): 569–77. http://dx.doi.org/10.1139/cjm-2014-0087.
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Endothelial cells are susceptible to infection by mycobacteria, but the endocytic mechanisms that mycobacteria exploit to enter host cells and their mechanisms of intracellular transport are completely unknown. Using pharmacological inhibitors, we determined that the internalization of Mycobacterium tuberculosis (MTB), Mycobacterium smegmatis (MSM), and Mycobacterium abscessus (MAB) is dependent on the cytoskeleton and is differentially inhibited by cytochalasin D, nocodazole, cycloheximide, wortmannin, and amiloride. Using confocal microscopy, we investigated their endosomal trafficking by analyzing Rab5, Rab7, LAMP-1, and cathepsin D. Our results suggest that MSM exploits macropinocytosis to enter endothelial cells and that the vacuoles containing these bacteria fuse with lysosomes. Conversely, the entry of MTB seems to depend on more than one endocytic route, and the observation that only a subset of the intracellular bacilli was associated with phagolysosomes suggests that these bacteria are able to inhibit endosomal maturation to persist intracellularly. The route of entry for MAB depends mainly on microtubules, which suggests that MAB uses a different trafficking pathway. However, MAB is also able to inhibit endosomal maturation and can replicate intracellularly. Together, these findings provide the first evidence that mycobacteria modulate proteins of host endothelial cells to enter and persist within these cells.
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Jeschke, Andreas, Nicole Zehethofer, Buko Lindner, Jessica Krupp, Dominik Schwudke, Ina Haneburger, Marko Jovic, et al. "Phosphatidylinositol 4-phosphate and phosphatidylinositol 3-phosphate regulate phagolysosome biogenesis." Proceedings of the National Academy of Sciences 112, no. 15 (March 30, 2015): 4636–41. http://dx.doi.org/10.1073/pnas.1423456112.
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Professional phagocytic cells ingest microbial intruders by engulfing them into phagosomes, which subsequently mature into microbicidal phagolysosomes. Phagosome maturation requires sequential fusion of the phagosome with early endosomes, late endosomes, and lysosomes. Although various phosphoinositides (PIPs) have been detected on phagosomes, it remained unclear which PIPs actually govern phagosome maturation. Here, we analyzed the involvement of PIPs in fusion of phagosomes with various endocytic compartments and identified phosphatidylinositol 4-phosphate [PI(4)P], phosphatidylinositol 3-phosphate [PI(3)P], and the lipid kinases that generate these PIPs, as mediators of phagosome–lysosome fusion. Phagosome–early endosome fusion required PI(3)P, yet did not depend on PI(4)P. Thus, PI(3)P regulates phagosome maturation at early and late stages, whereas PI(4)P is selectively required late in the pathway.
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Barone, Maria Vittoria, Merlin Nanayakkara, Giovanni Paolella, Mariantonia Maglio, Virginia Vitale, Raffaele Troiano, Maria Teresa Silvia Ribecco, et al. "Gliadin Peptide P31-43 Localises to Endocytic Vesicles and Interferes with Their Maturation." PLoS ONE 5, no. 8 (August 18, 2010): e12246. http://dx.doi.org/10.1371/journal.pone.0012246.
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MOUSAVI, Seyed Ali, Andreas BRECH, Trond BERG, and Rune KJEKEN. "Phosphoinositide 3-kinase regulates maturation of lysosomes in rat hepatocytes." Biochemical Journal 372, no. 3 (June 15, 2003): 861–69. http://dx.doi.org/10.1042/bj20021136.
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To obtain information about the role of phosphoinositide 3-kinase (PI3K) in the endocytic pathway in hepatocytes, the uptake and intracellular transport of asialo-orosomucoid (ASOR) was followed in cells treated with wortmannin or LY294002. The two inhibitors, at concentrations known to inhibit the enzyme, did not affect internalization or the number of surface asialoglycoprotein receptors, but they caused a paradoxical increase (approx. 50% above control values) in the degradation of ASOR labelled with [125I]tyramine cellobiose ([125I]TC). Wortmannin or LY204002 inhibited the autophagic sequestration of lactate dehydrogenase very effectively, and the enhanced degradation of [125I]TC-ASOR could be an indirect effect of reduced autophagy, as an amino acid mixture known to inhibit autophagy also caused increased degradation of [125I]TC-ASOR, and its effect was not additive to that of wortmannin or LY294002. Wortmannin or LY294002 had pronounced effects on the late parts of the endocytic pathway in the hepatocytes: first, dense lysosomes disappeared and were replaced by swollen vesicles; secondly, degradation of [125I]TC-ASOR took place in an organelle of lower buoyant density (in a sucrose gradient) than the bulk of lysosomes (identified in the gradient by lysosomal marker enzymes). With increasing length of incubation with wortmannin or LY294002, the density distributions of the lysosomal markers also shifted to lower density and gradually approached that of the labelled degradation products. The labelled degradation products formed from [125I]TC-labelled proteins were trapped at the site of formation, because they did not penetrate the vesicle membranes. The results obtained indicate that internalization and intracellular transport of ASOR to lysomes may take place in the absence of PI3K activity in rat hepatocytes. On the other hand, fusion of late endosomes with lysosomes seems to produce ‘hybrid organelles’ (active lysosomes) that are unable to mature into dense lysosomes.
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ROW, Paula E., Barbara J. REAVES, Jan DOMIN, J. Paul LUZIO, and Howard W. DAVIDSON. "Overexpression of a rat kinase-deficient phosphoinositide 3-kinase, Vps34p, inhibits cathepsin D maturation." Biochemical Journal 353, no. 3 (January 25, 2001): 655–61. http://dx.doi.org/10.1042/bj3530655.
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Lipid kinases and their phosphorylated products are important regulators of many cellular processes, including intracellular membrane traffic. The best example of this is provided by the class III phosphoinositide 3-kinase (PI-3K), Vps34p, which is required for correct targeting of newly synthesized carboxypeptidase Y to the yeast vacuole. A probable mammalian Vps34p orthologue has been previously identified, but its function in the trafficking of lysosomal enzymes has not been resolved. To investigate the possible role(s) of mammalian Vps34p in protein targeting to lysosomes, we have cloned the rat orthologue and overexpressed a kinase-deficient mutant in HeLa cells. Expression of the mutant protein inhibited both maturation of procathepsin D and basal secretion of the precursor. In contrast wortmannin, which also inhibited maturation, caused hypersecretion of the precursor. We propose that mammalian Vps34p plays a direct role in targeting lysosomal enzyme precursors to the endocytic pathway in an analogous fashion to its role in the fusion of early endocytic vesicles with endosomes. We further suggest that inhibition of a wortmannin-sensitive enzyme, other than mammalian Vps34p, is responsible for the failure to recycle unoccupied mannose 6-phosphate receptors to the trans-Golgi network, and consequent hypersecretion of lysosomal enzyme precursors observed in the presence of this drug.
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Singer-Krüger, B., H. Stenmark, A. Düsterhöft, P. Philippsen, J. S. Yoo, D. Gallwitz, and M. Zerial. "Role of three rab5-like GTPases, Ypt51p, Ypt52p, and Ypt53p, in the endocytic and vacuolar protein sorting pathways of yeast." Journal of Cell Biology 125, no. 2 (April 15, 1994): 283–98. http://dx.doi.org/10.1083/jcb.125.2.283.
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The small GTPase rab5 has been shown to represent a key regulator in the endocytic pathway of mammalian cells. Using a PCR approach to identify rab5 homologs in Saccharomyces cerevisiae, two genes encoding proteins with 54 and 52% identity to rab5, YPT51 and YPT53 have been identified. Sequencing of the yeast chromosome XI has revealed a third rab5-like gene, YPT52, whose protein product exhibits a similar identity to rab5 and the other two YPT gene products. In addition to the high degree of identity/homology shared between rab5 and Ypt51p, Ypt52p, and Ypt53p, evidence for functional homology between the mammalian and yeast proteins is provided by phenotypic characterization of single, double, and triple deletion mutants. Endocytic delivery to the vacuole of two markers, lucifer yellow CH (LY) and alpha-factor, was inhibited in delta ypt51 mutants and aggravated in the double ypt51ypt52 and triple ypt51ypt52ypt53 mutants, suggesting a requirement for these small GTPases in endocytic membrane traffic. In addition to these defects, the here described ypt mutants displayed a number of other phenotypes reminiscent of some vacuolar protein sorting (vps) mutants, including a differential delay in growth and vacuolar protein maturation, partial missorting of a soluble vacuolar hydrolase, and alterations in vacuole acidification and morphology. In fact, vps21 represents a mutant allele of YPT51 (Emr, S., personal communication). Altogether, these data suggest that Ypt51p, Ypt52p, and Ypt53p are required for transport in the endocytic pathway and for correct sorting of vacuolar hydrolases suggesting a possible intersection of the endocytic with the vacuolar sorting pathway.
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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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Reaves, B. J., P. E. Row, N. A. Bright, J. P. Luzio, and H. W. Davidson. "Loss of cation-independent mannose 6-phosphate receptor expression promotes the accumulation of lysobisphosphatidic acid in multilamellar bodies." Journal of Cell Science 113, no. 22 (November 15, 2000): 4099–108. http://dx.doi.org/10.1242/jcs.113.22.4099.
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A number of recent studies have highlighted the importance of lipid domains within endocytic organelles in the sorting and movement of integral membrane proteins. In particular, considerable attention has become focussed upon the role of the unusual phospholipid lysobisphosphatidic acid (LBPA). This lipid appears to be directly involved in the trafficking of cholesterol and glycosphingolipids, and accumulates in a number of lysosomal storage disorders. Antibody-mediated disruption of LBPA function also leads to mis-sorting of cation-independent mannose 6-phosphate receptors. We now report that the converse is also true, and that spontaneous loss of cation-independent mannose 6-phosphate receptors from a rat fibroblast cell line led to the formation of aberrant late endocytic structures enriched in LBPA. Accumulation of LBPA was directly dependent upon the loss of the receptors, and could be reversed by expression of bovine cation-independent mannose 6-phosphate receptors in the mutant cell line. Ultrastructural analysis indicated that the abnormal organelles were electron-dense, had a multi-lamellar structure, accumulated endocytosed probes, and were distinct from dense-core lysosomes present within the same cells. The late endocytic structures present at steady state within any particular cell likely reflect the balance of membrane traffic through the endocytic pathway of that cell, and the rate of maturation of individual endocytic organelles. Moreover, there is considerable evidence which suggests that cargo receptors also play a direct mechanistic role in membrane trafficking events. Therefore, loss of such a protein may disturb the overall equilibrium of the pathway, and hence cause the accumulation of aberrant organelles. We propose that this mechanism underlies the phenotype of the mutant cell line, and that the formation of inclusion bodies in many lysosomal storage diseases is also due to an imbalance in membrane trafficking within the endocytic pathway.
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Ward, D. M., C. M. Perou, M. Lloyd, and J. Kaplan. ""Synchronized" endocytosis and intracellular sorting in alveolar macrophages: the early sorting endosome is a transient organelle." Journal of Cell Biology 129, no. 5 (June 1, 1995): 1229–40. http://dx.doi.org/10.1083/jcb.129.5.1229.
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Incubation of alveolar macrophages in hypoosmotic K(+)-containing buffers results in persistent cell swelling and an inability to undergo regulatory volume decrease. We demonstrate that cells incubated in hypo-K+ show an inhibition of endocytosis without any observed alteration in recycling. The inhibition of endocytosis affected all forms of membrane internalization, receptor and fluid phase. Both increased cell volume and the inhibition of endocytosis could be released upon return of cells to iso-Na+ buffers. The ability to synchronize the endocytic apparatus allowed us to examine hypotheses regarding the origin and maturation of endocytic vesicles. Incubation in hypo-K+ buffers had no effect on the delivery of ligands to degradative compartments or on the return of previously internalized receptors to the cell surface. Thus, membrane recycling and movement of internalized components to lysosomes occurred in the absence of continued membrane influx. We also demonstrate that fluorescent lipids, that had been incorporated into early endosomes, returned to the cell surface upon exposure of cells to hypo-K+ buffers. These results indicate that the early sorting endosome is a transient structure, whose existence depends upon continued membrane internalization. Our data supports the hypothesis that the transfer of material to lysosomes can best be explained by the continuous maturation of endosomes.
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Kong, Ying Ying, Kirsty Wilson, Vasso Apostolopoulos, and Magdalena Plebanski. "Dendritic Cells and Myeloid Derived Suppressor Cells Fully Responsive to Stimulation via Toll-Like Receptor 4 Are Rapidly Induced from Bone-Marrow Cells by Granulocyte-Macrophage Colony-Stimulating Factor." Vaccines 8, no. 3 (September 12, 2020): 522. http://dx.doi.org/10.3390/vaccines8030522.
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Dendritic cells (DCs) are commonly generated from bone marrow (BM) progenitor cells with granulocyte-macrophage colony-stimulating factor (GM-CSF) alone or in combination with interleukin 4 (IL-4). These cells are often harvested post day 5, when they acquire maturation markers and can stimulate T cells. Apart from DCs, myeloid derived suppressor cells (MDSCs) are also found within these cultures. However, little is known about the functional characteristics of DCs and MDSCs before day 5. Herein, using a murine model, it is shown that early DCs and MDSCs, even in cultures with GM-CSF alone, upregulate fully maturation and activation surface molecules in response to the toll-like receptor 4 (TLR4) ligand lipopolysaccharide (LPS) stimulation. Despite initially displaying lower marker expression levels, these cells efficiently induced T cell stimulation and cytokine production. Interestingly, Gr-1int MDSCs increased their T cell co-stimulatory activity upon TLR4 stimulation. Additionally, early DCs and MDSCs exhibited differential endocytic capacity for viral sized nanoparticles and bacterial sized microparticles. DCs internalized both particle sizes, whilst MDSCs only internalized the larger microparticles, with reduced endocytic activity over time in the culture. These findings have unveiled an important role for the rapid initiation of productive immunity by GM-CSF, with promising implications for future vaccine and DC immunotherapy developments.
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Kawauchi, Takeshi, Katsutoshi Sekine, Mima Shikanai, Kaori Chihama, Kenji Tomita, Ken-ichiro Kubo, Kazunori Nakajima, Yo-ichi Nabeshima, and Mikio Hoshino. "Rab GTPases-Dependent Endocytic Pathways Regulate Neuronal Migration and Maturation through N-Cadherin Trafficking." Neuron 67, no. 4 (August 2010): 588–602. http://dx.doi.org/10.1016/j.neuron.2010.07.007.
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Ramachandra, Lakshmi, Erika Noss, W. Henry Boom, and Clifford V. Harding. "Processing of Mycobacterium tuberculosis Antigen 85B Involves Intraphagosomal Formation of Peptide–Major Histocompatibility Complex II Complexes and Is Inhibited by Live Bacilli that Decrease Phagosome Maturation." Journal of Experimental Medicine 194, no. 10 (November 12, 2001): 1421–32. http://dx.doi.org/10.1084/jem.194.10.1421.
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Mycobacterium tuberculosis (MTB) inhibits phagosomal maturation to promote its survival inside macrophages. Control of MTB infection requires CD4 T cell responses and major histocompatibility complex (MHC) class II (MHC-II) processing of MTB antigens (Ags). To investigate phagosomal processing of MTB Ags, phagosomes containing heat-killed (HK) or live MTB were purified from interferon-γ (IFN-γ)–activated macrophages by differential centrifugation and Percoll density gradient subcellular fractionation. Flow organellometry and Western blot analysis showed that MTB phagosomes acquired lysosome-associated membrane protein-1 (LAMP-1), MHC-II, and H2-DM. T hybridoma cells were used to detect MTB Ag 85B(241–256)–I-Ab complexes in isolated phagosomes and other subcellular fractions. These complexes appeared initially (within 20 min) in phagosomes and subsequently (>20 min) on the plasma membrane, but never within late endocytic compartments. Macrophages processed HK MTB more rapidly and efficiently than live MTB; phagosomes containing live MTB expressed fewer Ag 85B(241–256)–I-Ab complexes than phagosomes containing HK MTB. This is the first study of bacterial Ag processing to directly show that peptide–MHC-II complexes are formed within phagosomes and not after export of bacterial Ags from phagosomes to endocytic Ag processing compartments. Live MTB can alter phagosome maturation and decrease MHC-II Ag processing, providing a mechanism for MTB to evade immune surveillance and enhance its survival within the host.
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Kessler, Tobias, Michael Reich, Gerhard Jahn, Eva Tolosa, Alexander Beck, Hubert Kalbacher, Herman Overkleeft, Susanne Schempp, and Christoph Driessen. "Human cytomegalovirus infection interferes with major histocompatibility complex type II maturation and endocytic proteases in dendritic cells at multiple levels." Journal of General Virology 89, no. 10 (October 1, 2008): 2427–36. http://dx.doi.org/10.1099/vir.0.2008/001610-0.
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Human cytomegalovirus (HCMV) infection suppresses cellular immunity and results in viral persistence. Dendritic cells (DCs) are susceptible to HCMV, and the development and immune function of HCMV-infected DCs are impaired in vitro. HCMV-derived proteins interfere with different aspects of major histocompatibility complex type II (MHC II) maturation and function in genetically engineered cellular models. This study directly analysed the effect of HCMV on the MHC II-associated antigen processing and presentation machinery in HCMV-infected human DCs in vitro. HCMV-infected DCs failed to mature newly synthesized MHC II to the final stage of SDS-stable MHC II αβ dimer/peptide complexes, in contrast to mock-infected controls. MHC II biosynthesis was delayed and reduced, whilst MHC II stability remained unchanged. MHC II surface expression was decreased in the late phase of HCMV infection. In addition, infected DCs decreased the transcription rate of the MHC II-associated proteases cathepsins S, Z, B, H and L and asparagine-specific endopeptidase (AEP). This translated into reduced protein expression of cathepsins H and S, as well as AEP, and less-efficient proteolytic degradation of a peptide substrate by endocytic proteases from HCMV-infected DCs in vitro. Thus, HCMV infection interferes with MHC II biosynthesis and maturation, as well as with the expression and function of endocytic proteases in infected DCs.
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Ghosh, Mallika, Beata McAuliffe, Jaganathan Subramani, Sreyashi Basu, and Linda H. Shapiro. "CD13 regulates dendritic cell cross-presentation and t cell responses by inhibiting receptor-mediated antigen uptake (106.16)." Journal of Immunology 188, no. 1_Supplement (May 1, 2012): 106.16. http://dx.doi.org/10.4049/jimmunol.188.supp.106.16.
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Abstract Dendritic cell (DC) antigen cross-presentation is generally associated with immune responses to tumors and viral antigens and enhancing this process is a focus of tumor vaccine design. In this study, we found that the myeloid cell surface peptidase CD13 is highly and specifically expressed on the subset of DCs responsible for cross-presentation, the CD8+ murine splenic DCs. In vivo studies indicated that lack of CD13 significantly enhanced T cell responses to soluble OVA antigen, although the development, maturation, antigen processing and presentation of DCs is normal in CD13KO mice. In vitro studies showed that CD13 regulates receptor-mediated, dynamin-dependent endocytosis of antigens such as OVA and transferrin but not fluid-phase or phagocytic antigen uptake. CD13 and antigen are co-internalized in DCs but CD13 did not co- immunoprecipitate with antigen receptors, suggesting that CD13 does not control internalization of specific receptors but regulates endocytosis at a more universal level. Mechanistically, we found that phosphorylation of the endocytic regulators p38MAPK and Akt was dysregulated in CD13KO DCs and blocking these kinases perturbed CD13-dependent endocytic uptake. Therefore, CD13 is a novel endocytic regulator that may be exploited to enhance antigen uptake and T cell activation to improve the efficacy of tumor-targeted vaccines.
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Kalaidzidis, Inna, Marta Miaczynska, Marta Brewińska-Olchowik, Anna Hupalowska, Charles Ferguson, Robert G. Parton, Yannis Kalaidzidis, and Marino Zerial. "APPL endosomes are not obligatory endocytic intermediates but act as stable cargo-sorting compartments." Journal of Cell Biology 211, no. 1 (October 12, 2015): 123–44. http://dx.doi.org/10.1083/jcb.201311117.
Full textAbstract:
Endocytosis allows cargo to enter a series of specialized endosomal compartments, beginning with early endosomes harboring Rab5 and its effector EEA1. There are, however, additional structures labeled by the Rab5 effector APPL1 whose role in endocytic transport remains unclear. It has been proposed that APPL1 vesicles are transport intermediates that convert into EEA1 endosomes. Here, we tested this model by analyzing the ultrastructural morphology, kinetics of cargo transport, and stability of the APPL1 compartment over time. We found that APPL1 resides on a tubulo-vesicular compartment that is capable of sorting cargo for recycling or degradation and that displays long lifetimes, all features typical of early endosomes. Fitting mathematical models to experimental data rules out maturation of APPL1 vesicles into EEA1 endosomes as a primary mechanism for cargo transport. Our data suggest instead that APPL1 endosomes represent a distinct population of Rab5-positive sorting endosomes, thus providing important insights into the compartmental organization of the early endocytic pathway.
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Yu, Fang, Lu Sun, and Khaled Machaca. "Constitutive recycling of the store-operated Ca2+ channel Orai1 and its internalization during meiosis." Journal of Cell Biology 191, no. 3 (November 1, 2010): 523–35. http://dx.doi.org/10.1083/jcb.201006022.
Full textAbstract:
The egg’s competency to activate at fertilization and transition to embryogenesis is dependent on its ability to generate a fertilization-specific Ca2+ transient. To endow the egg with this capacity, Ca2+ signals remodel during oocyte maturation, including inactivation of the primary Ca2+ influx pathway store-operated Ca2+ entry (SOCE). SOCE inactivation is coupled to internalization of the SOCE channel, Orai1. In this study, we show that Orai1 internalizes during meiosis through a caveolin (Cav)- and dynamin-dependent endocytic pathway. Cav binds to Orai1, and we map a Cav consensus–binding site in the Orai1 N terminus, which is required for Orai1 internalization. Furthermore, at rest, Orai1 actively recycles between an endosomal compartment and the cell membrane through a Rho-dependent endocytic pathway. A significant percentage of total Orai1 is intracellular at steady state. Store depletion completely shifts endosomal Orai1 to the cell membrane. These results define vesicular trafficking mechanisms in the oocyte that control Orai1 subcellular localization at steady state, during meiosis, and after store depletion.
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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.
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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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Leithe, Edward, Andreas Brech, and Edgar Rivedal. "Endocytic processing of connexin43 gap junctions: a morphological study." Biochemical Journal 393, no. 1 (December 12, 2005): 59–67. http://dx.doi.org/10.1042/bj20050674.
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Gap junctions are plasma membrane areas enriched in channels that provide direct intercellular communication. Gap junctions have a high turnover rate; however, the mechanisms by which gap junctions are degraded are incompletely understood. In the present study, we show that in response to phorbol ester treatment, the gap junction channel protein Cx43 (connexin43) is redistributed from the plasma membrane to intracellular vesicles positive for markers for early and late endosomes and for the endolysosomal protease cathepsin D. Immunoelectron microscopy studies indicate that the double membranes of internalized gap junctions undergo separation and cutting, resulting in multivesicular endosomes enriched in Cx43 protein. Using preloading of BSA–gold conjugates to mark lysosomes, we provide evidence suggesting that the degradation process of the double-membrane structure of annular gap junctions occurs prior to transport of Cx43 to the lysosome. The results further suggest that bafilomycin A1, an inhibitor of vacuolar H+-ATPases, causes accumulation of Cx43 in early endosomes. Taken together, these findings indicate that internalized gap junctions undergo a maturation process from tightly sealed double-membrane vacuoles to connexin-enriched multivesicular endosomes with a single limiting membrane. The results further suggest that along with the processing of the double-membrane structure of annular gap junctions, connexins are trafficked via early and late endosomes, finally resulting in their endolysosomal degradation.
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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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Aerbajinai, Wulin, Kevin Lee, Kyung Chin, and Griffin P. Rodgers. "Glia Maturation Factor-γ Negatively Modulates TLR4 Signaling by Facilitating TLR4 Endocytic Trafficking in Macrophages." Journal of Immunology 190, no. 12 (May 15, 2013): 6093–103. http://dx.doi.org/10.4049/jimmunol.1203048.
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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.
Full textAbstract:
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.