scholarly journals Exocyst subcomplex functions in autophagosome biogenesis by regulating Atg9 trafficking

2018 ◽  
Author(s):  
Sunaina Singh ◽  
Sarika Chinchwadkar ◽  
Amol Aher ◽  
Saravanan Matheshwaran ◽  
Ravi Manjithaya
Keyword(s):  
Plasma Membrane ◽  
Membrane Fusion ◽  
De Novo ◽  
Membrane Vesicles ◽  
Vesicle Transport ◽  
Secretory Vesicles ◽  
Double Membrane ◽  
Primary Function

AbstractDuring autophagy, double membrane vesicles called autophagosomes capture and degrade the intracellular cargo. The de novo formation of autophagosomes requires several vesicle transport and membrane fusion events which are not completely understood. We studied the involvement of Exocyst- an octameric tethering complex, which has a primary function in tethering post-Golgi secretory vesicles to plasma membrane, in autophagy. Our findings indicate not all subunits of exocyst are involved in selective and general autophagy. We show that in the absence of autophagy specific subunits, autophagy arrest is accompanied by accumulation of incomplete autophagosome-like structures. In these mutants, impaired Atg9 trafficking leads to decreased delivery of membrane to the site of autophagosome biogenesis thereby impeding the elongation and completion of the autophagosomes. The subunits of exocyst which are dispensable for autophagic function do not associate with the autophagy specific subcomplex of exocyst.

scholarly journals HIV-cell membrane fusion intermediates are restricted by Serincs as revealed by cryo-electron and TIRF microscopy

2020 ◽  
Vol 295 (45) ◽  
pp. 15183-15195 ◽  
Author(s):  
Amanda E. Ward ◽  
Volker Kiessling ◽  
Owen Pornillos ◽  
Judith M. White ◽  
Barbie K. Ganser-Pornillos ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Membrane ◽  
Membrane Fusion ◽  
Electron Tomography ◽  
Membrane Vesicles ◽  
Lipid Binding ◽  
Plasma Membrane Vesicles ◽  
Tirf Microscopy ◽  
Host Restriction ◽  
Whole Cells

To enter a cell and establish infection, HIV must first fuse its lipid envelope with the host cell plasma membrane. Whereas the process of HIV membrane fusion can be tracked by fluorescence microscopy, the 3D configuration of proteins and lipids at intermediate steps can only be resolved with cryo-electron tomography (cryoET). However, cryoET of whole cells is technically difficult. To overcome this problem, we have adapted giant plasma membrane vesicles (or blebs) from native cell membranes expressing appropriate receptors as targets for fusion with HIV envelope glycoprotein-expressing pseudovirus particles with and without Serinc host restriction factors. The fusion behavior of these particles was probed by TIRF microscopy on bleb-derived supported membranes. Timed snapshots of fusion of the same particles with blebs were examined by cryo-ET. The combination of these methods allowed us to characterize the structures of various intermediates on the fusion pathway and showed that when Serinc3 or Serinc5 (but not Serinc2) were present, later fusion products were more prevalent, suggesting that Serinc3/5 act at multiple steps to prevent progression to full fusion. In addition, the antifungal amphotericin B reversed Serinc restriction, presumably by intercalation into the fusing membranes. Our results provide a highly detailed view of Serinc restriction of HIV-cell membrane fusion and thus extend current structural and functional information on Serinc as a lipid-binding protein.

Epithelial paraphagy is a luminal lipophilic signal sensor and promotes transepithelial cholesterol clearance

2021 ◽  
Author(s):  
Bao Li Zhang (Co-first) ◽  
Bao Ying Liu (Co-First) ◽  
Da Yuan Gao (Co-First) ◽  
Xin Zhou ◽  
Xin Yu Xu ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Tight Junction ◽  
Membrane Vesicles ◽  
Male Mice ◽  
Reproductive Systems ◽  
Cholesterol Accumulation ◽  
Double Membrane ◽  
Physiological Homeostasis ◽  
Epithelial Tight Junction ◽  
Dependent Processes

Abstract Epithelial endocytosis is essential for physiological homeostasis. The current dogma is that all endocytotic mechanisms involve only single-membrane vesicles at the plasma membrane. Here, we report a previously undescribed LC3-associated phagocytosis mechanism which forms double-membrane phagosomes from the epithelial tight-junction paracellular membranes, which we term “paraphagy”. We observed that paraphagy is present in epithelia of the renal, respiratory, circulatory, and male reproductive systems. In the epididymis, paraphagy takes up lipophilic cargo, including apolipoproteins, by involving the surface low-affinity IgG-receptor Fcgr2b and occludin-bound intracellular HDL-receptor ATP5b. In this way, extracellular lipophilic signals are sensed and intracellular phagolysosomes are maintained. Occludin-null male mice show arrested paraphagy and impaired phagolysosome in proximal epididymal compartments, accompanying cholesterol accumulation, ApoJ deposition and dysregulated metabolic-dependent processes in distal compartments, including redox-promoted VK-dependent MGP-carboxylation. We propose that paraphagy senses and distinguishes the luminal lipophilic signals in epithelia and modulates their function via crosstalk across physiological compartments.

scholarly journals Unconventional Myosin Myo1c Promotes Membrane Fusion in a Regulated Exocytic Pathway

2004 ◽  
Vol 24 (12) ◽  
pp. 5447-5458 ◽  
Author(s):  
Avirup Bose ◽  
Stacey Robida ◽  
Paul S. Furcinitti ◽  
Anil Chawla ◽  
Kevin Fogarty ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Fusion ◽  
Glucose Transporter ◽  
Kinase Inhibitor ◽  
Microscopic Analysis ◽  
Membrane Vesicles ◽  
Cell Cortex ◽  
Unconventional Myosin ◽  
Membrane Ruffling ◽  
Cytoskeletal Network

ABSTRACT Glucose homeostasis is controlled in part by regulation of glucose uptake into muscle and adipose tissue. Intracellular membrane vesicles containing the GLUT4 glucose transporter move towards the cell cortex in response to insulin and then fuse with the plasma membrane. Here we show that the fusion step is retarded by the inhibition of phosphatidylinositol (PI) 3-kinase. Treatment of insulin-stimulated 3T3-L1 adipocytes with the PI 3-kinase inhibitor LY294002 causes the accumulation of GLUT4-containing vesicles just beneath the cell surface. This accumulation of GLUT4-containing vesicles near the plasma membrane prior to fusion requires an intact cytoskeletal network and the unconventional myosin motor Myo1c. Remarkably, enhanced Myo1c expression under these conditions causes extensive membrane ruffling and overrides the block in membrane fusion caused by LY294002, restoring the display of GLUT4 on the cell exterior. Ultrafast microscopic analysis revealed that insulin treatment leads to the mobilization of GLUT4-containing vesicles to these regions of Myo1c-induced membrane ruffles. Thus, localized membrane remodeling driven by the Myo1c motor appears to facilitate the fusion of exocytic GLUT4-containing vesicles with the adipocyte plasma membrane.

New membrane assembly during exocytosis

1987 ◽  
Vol 45 ◽  
pp. 782-783
Author(s):  
Elsa A. Schmauder-Chock ◽  
Stephen P. Chock
Keyword(s):  
Plasma Membrane ◽  
De Novo ◽  
Membrane Vesicles ◽  
Aqueous Environment ◽  
Ige Receptor ◽  
Early Step ◽  
Ige Antibodies ◽  
Membrane Assembly ◽  
Physiological Conditions ◽  
Mast Cell Granule

A rapid membrane assembly process has been postulated as an early step in the mechanism of mast cell granule exocytosis. This theory explains how an activated granule can rapidly enlarge prior to contact and fusion with the plasma membrane. Some evidence supporting this theory includes experiments which showed the ability of the purified granule to generate new membrane in an aqueous environment after removal of its perigranular membrane (PGM). The formation and the fusion of these newly assembled membrane vesicles with the PGM has also been observed in mast cells stimulated with saponin. Under physiological conditions, histamine release is triggered by antigen-specific crosslinking of receptor-bound IgE antibodies on the plasma membrane. Thus it is important to see if the process of de novo membrane generation also occurs in this IgE-receptor mediated exocytosis.

scholarly journals In vitro fusion between Saccharomyces cerevisiae secretory vesicles and cytoplasmic-side-out plasma membrane vesicles

2003 ◽  
Vol 370 (2) ◽  
pp. 641-649 ◽  
Author(s):  
Lorena ARRASTUA ◽  
Eider SAN SEBASTIAN ◽  
Ana F. QUINCOCES ◽  
Claude ANTONY ◽  
Unai UGALDE
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Secretory Pathway ◽  
Membrane Vesicles ◽  
Fusion Process ◽  
Temperature Sensitive ◽  
Secretory Vesicles ◽  
Plasma Membrane Vesicles ◽  
Fusion Event ◽  
Cytoplasmic Side

The final step in the secretory pathway, which is the fusion event between secretory vesicles and the plasma membrane, was reconstructed using highly purified secretory vesicles and cytoplasmic-side-out plasma membrane vesicles from the yeast Saccharomyces cerevisiae. Both organelle preparations were obtained from a sec 6-4 temperature-sensitive mutant. Fusion was monitored by means of a fluorescence assay based on the dequenching of the lipophilic fluorescent probe octadecylrhodamine B-chloride (R18). The probe was incorporated into the membrane of secretory vesicles, and it diluted in unlabelled cytoplasmic-side-out plasma membrane vesicles as the fusion process took place. The obtained experimental dequenching curves were found by mathematical analysis to consist of two independent but simultaneous processes. Whereas one of them reflected the fusion process between both vesicle populations as confirmed by its dependence on the assay conditions, the other represented a non-specific transfer of the probe. The fusion process may now be examined in detail using the preparation, validation and analytical methods developed in this study.

scholarly journals Asnc1Endocytosis Mutant: Phenotypic Analysis and Suppression by Overproduction of Dihydrosphingosine Phosphate Lyase

2000 ◽  
Vol 11 (12) ◽  
pp. 4051-4065 ◽  
Author(s):  
Eric Grote ◽  
Greg Vlacich ◽  
Marc Pypaert ◽  
Peter J. Novick
Keyword(s):  
Plasma Membrane ◽  
Structural Gene ◽  
De Novo ◽  
Secretory Vesicle ◽  
Snare Proteins ◽  
Secretory Vesicles ◽  
Phenotypic Analysis ◽  
Alternative Protein ◽  
Secretion Rates ◽  
Enhanced Degradation

The v-SNARE proteins Snc1p and Snc2p are required for fusion of secretory vesicles with the plasma membrane in yeast. Mutation of a methionine-based sorting signal in the cytoplasmic domain of either Sncp inhibits Sncp endocytosis and prevents recycling of Sncp to the Golgi after exocytosis. snc1-M43A mutant yeast have reduced growth and secretion rates and accumulate post-Golgi secretory vesicles and fragmented vacuoles. However, cells continue to grow and secrete for several hours after de novo Snc2-M42A synthesis is repressed. DPL1, the structural gene for dihydrosphingosine phosphate lyase, was selected as a high copy numbersnc1-M43A suppressor. Because DPL1 also partially suppresses the growth and secretion phenotypes of asnc deletion, we propose that enhanced degradation of dihydrosphingosine-1-phosphate allows an alternative protein to replace Sncp as the secretory vesicle v-SNARE.

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