scholarly journals Inhibition of Very Long Chain Fatty Acids Synthesis Mediates PI3P Homeostasis at Endosomal Compartments

2021 ◽  
Vol 22 (16) ◽  
pp. 8450
Author(s):  
Yoko Ito ◽  
Nicolas Esnay ◽  
Louise Fougère ◽  
Matthieu Pierre Platre ◽  
Fabrice Cordelières ◽  
...  
Keyword(s):  
Small Gtpase ◽  
Animal Cells ◽  
Anionic Phospholipid ◽  
Intracellular Traffic ◽  
Cellular Processes ◽  
Late Endosomes ◽  
Early Endosomes ◽  
A Minor ◽  
Golgi Network ◽  
Long Chain

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.

scholarly journals Recycling pathways of glucosylceramide in BHK cells: distinct involvement of early and late endosomes

1992 ◽  
Vol 103 (4) ◽  
pp. 1139-1152
Author(s):  
J.W. Kok ◽  
K. Hoekstra ◽  
S. Eskelinen ◽  
D. Hoekstra
Keyword(s):  
Plasma Membrane ◽  
Intracellular Ph ◽  
Brefeldin A ◽  
Fluid Phase ◽  
Endocytic Pathway ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Recycling Pathway ◽  
Golgi Network ◽  
Extensive Involvement

Recycling pathways of the sphingolipid glucosylceramide were studied by employing a fluorescent analog of glucosylceramide, 6(-)[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]hexanoylglucosyl sphingosine (C6-NBD-glucosylceramide). Direct recycling of the glycolipid from early endosomes to the plasma membrane occurs, as could be shown after treating the cells with the microtubule-disrupting agent nocodazole, which causes inhibition of the glycolipid's trafficking from peripheral early endosomes to centrally located late endosomes. When the microtubuli are intact, at least part of the glucosylceramide is transported from early to late endosomes together with ricin. Interestingly, also N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine (N-Rh-PE), a membrane marker of the fluid-phase endocytic pathway, is transported to this endosomal compartment. However, in contrast to both ricin and N-Rh-PE, the glucosylceramide can escape from this organelle and recycle to the plasma membrane. Monensin and brefeldin A have little effect on this recycling pathway, which would exclude extensive involvement of early Golgi compartments in recycling. Hence, the small fraction of the glycolipid that colocalizes with transferrin (Tf) in the Golgi area might directly recycle via the trans-Golgi network. When the intracellular pH was lowered to 5.5, recycling was drastically reduced, in accordance with the impeding effect of low intracellular pH on vesicular transport during endocytosis and in the biosynthetic pathway. Our results thus demonstrate the existence of at least two recycling pathways for glucosylceramide and indicate the relevance of early endosomes in recycling of both proteins and lipids.

scholarly journals The R-SNARE Endobrevin/VAMP-8 Mediates Homotypic Fusion of Early Endosomes and Late Endosomes

2000 ◽  
Vol 11 (10) ◽  
pp. 3289-3298 ◽  
Author(s):  
Wolfram Antonin ◽  
Claudia Holroyd ◽  
Ritva Tikkanen ◽  
Stefan Höning ◽  
Reinhard Jahn
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Subcellular Fractionation ◽  
Immunogold Electron Microscopy ◽  
Fusion Reactions ◽  
Coated Pits ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Golgi Network

Endobrevin/VAMP-8 is an R-SNARE localized to endosomes, but it is unknown in which intracellular fusion step it operates. Using subcellular fractionation and quantitative immunogold electron microscopy, we found that endobrevin/VAMP-8 is present on all membranes known to communicate with early endosomes, including the plasma membrane, clathrin-coated pits, late endosomes, and membranes of thetrans-Golgi network. Affinity-purified antibodies that block the ability of endobrevin/VAMP-8 to form SNARE core complexes potently inhibit homotypic fusion of both early and late endosomes in vitro. Fab fragments were as active as intact immunoglobulin Gs. Recombinant endobrevin/VAMP-8 inhibited both fusion reactions with similar potency. We conclude that endobrevin/VAMP-8 operates as an R-SNARE in the homotypic fusion of early and late endosomes.

scholarly journals CD13 tethers the IQGAP1-ARF6-EFA6 complex to the plasma membrane to promote ARF6 activation, β1 integrin recycling, and cell migration

2019 ◽  
Vol 12 (579) ◽  
pp. eaav5938 ◽  
Author(s):  
Mallika Ghosh ◽  
Robin Lo ◽  
Ivan Ivic ◽  
Brian Aguilera ◽  
Veneta Qendro ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Migration ◽  
Cell Adhesion ◽  
Cell Attachment ◽  
Leading Edge ◽  
Small Gtpase ◽  
Β1 Integrin ◽  
Recycling Endosomes ◽  
Cellular Processes ◽  
Early Endosomes

Cell attachment to the extracellular matrix (ECM) requires a balance between integrin internalization and recycling to the surface that is mediated by numerous proteins, emphasizing the complexity of these processes. Upon ligand binding in various cells, the β1 integrin is internalized, traffics to early endosomes, and is returned to the plasma membrane through recycling endosomes. This trafficking process depends on the cyclical activation and inactivation of small guanosine triphosphatases (GTPases) by their specific guanine exchange factors (GEFs) and their GTPase-activating proteins (GAPs). In this study, we found that the cell surface antigen CD13, a multifunctional transmembrane molecule that regulates cell-cell adhesion and receptor-mediated endocytosis, also promoted cell migration and colocalized with β1 integrin at sites of cell adhesion and at the leading edge. A lack of CD13 resulted in aberrant trafficking of internalized β1 integrin to late endosomes and its ultimate degradation. Our data indicate that CD13 promoted ARF6 GTPase activity by positioning the ARF6-GEF EFA6 at the cell membrane. In migrating cells, a complex containing phosphorylated CD13, IQGAP1, GTP-bound (active) ARF6, and EFA6 at the leading edge promoted the ARF6 GTPase cycling and cell migration. Together, our findings uncover a role for CD13 in the fundamental cellular processes of receptor recycling, regulation of small GTPase activities, cell-ECM interactions, and cell migration.

scholarly journals Role of the RAB7 Protein in Tumor Progression and Cisplatin Chemoresistance

Cancers ◽  
2019 ◽  
Vol 11 (8) ◽  
pp. 1096 ◽  
Author(s):  
Guerra ◽  
Bucci
Keyword(s):  
Cancer Progression ◽  
Extracellular Vesicle ◽  
Endocytic Pathway ◽  
Cellular Processes ◽  
Molecular Events ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Cellular Context ◽  
Guanosine Triphosphatase

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.

scholarly journals Rab5-mediated endosome formation is regulated at the trans-Golgi network

2019 ◽  
Vol 2 (1) ◽  
Author(s):  
Makoto Nagano ◽  
Junko Y. Toshima ◽  
Daria Elisabeth Siekhaus ◽  
Jiro Toshima
Keyword(s):  
Plasma Membrane ◽  
Early Endosome ◽  
Vesicle Transport ◽  
Nucleotide Exchange ◽  
Trafficking Pathway ◽  
Transport Vesicles ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Critical Location ◽  
Golgi Network

AbstractEarly endosomes, also called sorting endosomes, are known to mature into late endosomes via the Rab5-mediated endolysosomal trafficking pathway. Thus, early endosome existence is thought to be maintained by the continual fusion of transport vesicles from the plasma membrane and the trans-Golgi network (TGN). Here we show instead that endocytosis is dispensable and post-Golgi vesicle transport is crucial for the formation of endosomes and the subsequent endolysosomal traffic regulated by yeast Rab5 Vps21p. Fittingly, all three proteins required for endosomal nucleotide exchange on Vps21p are first recruited to the TGN before transport to the endosome, namely the GEF Vps9p and the epsin-related adaptors Ent3/5p. The TGN recruitment of these components is distinctly controlled, with Vps9p appearing to require the Arf1p GTPase, and the Rab11s, Ypt31p/32p. These results provide a different view of endosome formation and identify the TGN as a critical location for regulating progress through the endolysosomal trafficking pathway.

scholarly journals Syntaxin 7 Is Localized to Late Endosome Compartments, Associates with Vamp 8, and Is Required for Late Endosome–Lysosome Fusion

2000 ◽  
Vol 11 (9) ◽  
pp. 3137-3153 ◽  
Author(s):  
Barbara M. Mullock ◽  
Chez W. Smith ◽  
Gudrun Ihrke ◽  
Nicholas A. Bright ◽  
Margaret Lindsay ◽  
...  
Keyword(s):  
Late Endosome ◽  
Endocytic Pathway ◽  
Snare Complex ◽  
Rab Gtpase ◽  
Animal Cells ◽  
Protein Traffic ◽  
Late Endosomes ◽  
Molecular Machinery ◽  
Golgi Network

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

Yeast Ypt51p and mammalian Rab5: counterparts with similar function in the early endocytic pathway

1995 ◽  
Vol 108 (11) ◽  
pp. 3509-3521 ◽  
Author(s):  
B. Singer-Kruger ◽  
H. Stenmark ◽  
M. Zerial
Keyword(s):  
Small Gtpase ◽  
Endocytic Pathway ◽  
Yeast Cells ◽  
Carboxypeptidase Y ◽  
Cell Fractionation ◽  
Animal Cells ◽  
Early Endosomes ◽  
Fractionation Analysis ◽  
Alpha Factor ◽  
Vacuolar Protein

Ypt51p, a small GTPase of Saccharomyces cerevisiae, has been previously identified as a structural homolog of mammalian Rab5. Although disruption analysis revealed that the protein is required for endocytic transport and for vacuolar protein sorting, the precise step controlled by Ypt51p was not determined. In this work we show that by heterologous expression in animal cells Ypt51p was targeted to Rab5-positive early endosomes and stimulated endocytosis. Furthermore, two Ypt51p mutants induced similar morphological alterations as the corresponding Rab5 mutants. Also in yeast cells Ypt51p was found to be required at an early step in endocytic membrane traffic, since alpha-factor accumulated in an early endocytic intermediate in the absence of Ypt51p. Cell fractionation analysis revealed cofractionation of Ypt51p with endocytic intermediates, while no association with the late Golgi compartment could be detected. Indirect immunofluorescence microscopy allowed us to morphologically identify the Ypt51p-containing compartment. Similar to the mammalian system larger Ypt51p-positive structures were revealed upon expression of Ypt51p Q66L. These structures were also positive for alpha-factor receptor and for carboxypeptidase Y, thus providing direct evidence for their endocytic nature and for the convergence of the vacuolar biosynthetic and endocytic pathways.

scholarly journals The phosphatidylinositol 3-phosphate-binding protein SNX4 controls ATG9A recycling and autophagy

2021 ◽  
Vol 134 (3) ◽  
pp. jcs250670 ◽  
Author(s):  
Anthony Ravussin ◽  
Andreas Brech ◽  
Sharon A. Tooze ◽  
Harald Stenmark
Keyword(s):  
Membrane Protein ◽  
Autophagic Flux ◽  
Dependent Manner ◽  
Phosphate Binding ◽  
Protein Traffic ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Phosphate Binding Protein ◽  
Golgi Network ◽  
Phosphatidylinositol 3

ABSTRACTLate endosomes and lysosomes (endolysosomes) receive proteins and cargo from the secretory, endocytic and autophagic pathways. Although these pathways and the degradative processes of endolysosomes are well characterized, less is understood about protein traffic from these organelles. In this study, we demonstrate the direct involvement of the phosphatidylinositol 3-phosphate (PI3P)-binding SNX4 protein in membrane protein recycling from endolysosomes, and show that SNX4 is required for proper autophagic flux. We show that SNX4 mediates recycling of the lipid scramblase ATG9A, which drives expansion of nascent autophagosome membranes, from endolysosomes to early endosomes, from where ATG9A is recycled to the trans-Golgi network in a retromer-dependent manner. Upon siRNA-mediated depletion of SNX4 or the retromer component VPS35, we observed accumulation of ATG9A on endolysosomes and early endosomes, respectively. Moreover, starvation-induced autophagosome biogenesis and autophagic flux were inhibited when SNX4 was downregulated. We propose that proper ATG9A recycling by SNX4 sustains autophagy by preventing exhaustion of the available ATG9A pool.This article has an associated First Person interview with the first author of the paper.

scholarly journals The phosphatidylinositol 3-phosphate binding protein SNX4 controls ATG9A recycling and autophagy

2020 ◽  
Author(s):  
Anthony Ravussin ◽  
Sharon A. Tooze ◽  
Harald Stenmark
Keyword(s):  
Membrane Protein ◽  
Autophagic Flux ◽  
Dependent Manner ◽  
Phosphate Binding ◽  
Protein Traffic ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Phosphate Binding Protein ◽  
Golgi Network ◽  
Phosphatidylinositol 3

AbstractLate endosomes and lysosomes (endolysosomes) receive proteins and cargo from the secretory, endocytic and autophagic pathways. Whereas these pathways and the degradative processes of endolysosomes are well characterized, less is understood about protein traffic from these organelles. In this study, we demonstrate the direct involvement of the phosphatidylinositol 3-phosphate (PI3P) binding SNX4 protein in membrane protein recycling from endolysosomes, and show that SNX4 is required for proper autophagic flux. We show that SNX4 mediates recycling of the transmembrane autophagy machinery protein ATG9A from endolysosomes to early endosomes, from where ATG9A is recycled to the trans-Golgi network in a retromer-dependent manner. Upon siRNA-mediated depletion of SNX4 or the retromer component VPS35, we observed accumulation of ATG9A on endolysosomes and early endosomes, respectively. Moreover, starvation-induced autophagosome biogenesis and autophagic flux were inhibited when SNX4 was downregulated. Altogether, we propose that proper ATG9A recycling by SNX4 sustains autophagy by preventing exhaustion of the available ATG9A pool.

scholarly journals The Arabidopsis SAC9 Enzyme defines a cortical population of early endosomes and restricts PI(4,5)P2 to the Plasma Membrane

2021 ◽  
Author(s):  
Mehdi Doumane ◽  
Alexis Lebecq ◽  
Aurelie Fangain ◽  
Vincent Bayle ◽  
Frederique Rozier ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Cortex ◽  
Cellular Processes ◽  
Phosphoinositide Phosphatase ◽  
Early Endosomes ◽  
Wide Range ◽  
Intracellular Compartments ◽  
Functional Consequences ◽  
Src Homology ◽  
Golgi Network

Membranes lipids, and especially phosphoinositides, are differentially enriched within the eukaryotic endomembrane system. This generates a landmark code by modulating the properties of each membrane. Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] specifically accumulates at the plasma membrane in yeast, animal and plant cells, where it regulates a wide range of cellular processes including endocytosis. However, the functional consequences of mispatterning PI(4,5)P2 in plants are unknown. Here, we functionally characterized the phosphoinositide phosphatase SUPPRESSOR OF ACTIN9 (SAC9) in Arabidopsis thaliana (Arabidopsis). We found that SAC9 depletion led to the ectopic localization of PI(4,5)P2 on cortical intracellular compartments, which depends on PI4P and PI(4,5)P2 production at the plasma membrane. SAC9 localizes to a subpopulation of trans-Golgi Network/early endosomes that are spatially restricted to a region close to the cell cortex and that are coated with clathrin. Furthermore, it interacts and colocalizes with the endocytic component Src Homology 3 Domain Protein 2 (SH3P2). In the absence of SAC9, SH3P2 localization is altered and the clathrin mediated endocytosis rate is significantly reduced. Thus, SAC9 is required to maintain efficient endocytic uptake, highlighting the importance of restricting the PI(4,5)P2 pool at the plasma membrane for the proper regulation of endocytosis in plants.

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