scholarly journals Annexin A2–dependent actin bundling promotes secretory granule docking to the plasma membrane and exocytosis

2015 ◽  
Vol 210 (5) ◽  
pp. 785-800 ◽  
Author(s):  
Marion Gabel ◽  
Franck Delavoie ◽  
Valérie Demais ◽  
Cathy Royer ◽  
Yannick Bailly ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Spatial Organization ◽  
Electron Tomography ◽  
Secretory Granules ◽  
Membrane Lipid ◽  
Lipid Binding ◽  
Annexin A2 ◽  
Cortical Actin ◽  
Lipid Microdomains ◽  
Lipid Domain

Annexin A2, a calcium-, actin-, and lipid-binding protein involved in exocytosis, mediates the formation of lipid microdomains required for the structural and spatial organization of fusion sites at the plasma membrane. To understand how annexin A2 promotes this membrane remodeling, the involvement of cortical actin filaments in lipid domain organization was investigated. 3D electron tomography showed that cortical actin bundled by annexin A2 connected docked secretory granules to the plasma membrane and contributed to the formation of GM1-enriched lipid microdomains at the exocytotic sites in chromaffin cells. When an annexin A2 mutant with impaired actin filament–bundling activity was expressed, the formation of plasma membrane lipid microdomains and the number of exocytotic events were decreased and the fusion kinetics were slower, whereas the pharmacological activation of the intrinsic actin-bundling activity of endogenous annexin A2 had the opposite effects. Thus, annexin A2–induced actin bundling is apparently essential for generating active exocytotic sites.

scholarly journals Annexin A2 Egress during Calcium-Regulated Exocytosis in Neuroendocrine Cells

Cells ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 2059 ◽  
Author(s):  
Marion Gabel ◽  
Cathy Royer ◽  
Tamou Thahouly ◽  
Valérie Calco ◽  
Stéphane Gasman ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Chromaffin Cells ◽  
Spatial Organization ◽  
Lipid Binding ◽  
Annexin A2 ◽  
Neuroendocrine Cells ◽  
Outer Face ◽  
Tissue Plasminogen

Annexin A2 (AnxA2) is a calcium- and lipid-binding protein involved in neuroendocrine secretion where it participates in the formation and/or stabilization of lipid micro-domains required for structural and spatial organization of the exocytotic machinery. We have recently described that phosphorylation of AnxA2 on Tyr23 is critical for exocytosis. Considering that Tyr23 phosphorylation is known to promote AnxA2 externalization to the outer face of the plasma membrane in different cell types, we examined whether this phenomenon occurred in neurosecretory chromaffin cells. Using immunolabeling and biochemical approaches, we observed that nicotine stimulation triggered the egress of AnxA2 to the external leaflets of the plasma membrane in the vicinity of exocytotic sites. AnxA2 was found co-localized with tissue plasminogen activator, previously described on the surface of chromaffin cells following secretory granule release. We propose that AnxA2 might be a cell surface tissue plasminogen activator receptor for chromaffin cells, thus playing a role in autocrine or paracrine regulation of exocytosis.

Barrier role of actin filaments in regulated mucin secretion from airway goblet cells

2005 ◽  
Vol 288 (1) ◽  
pp. C46-C56 ◽  
Author(s):  
Camille Ehre ◽  
Andrea H. Rossi ◽  
Lubna H. Abdullah ◽  
Kathleen De Pestel ◽  
Sandra Hill ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Actin Filaments ◽  
Fluorescent Protein ◽  
Secretory Granules ◽  
Yellow Fluorescent Protein ◽  
Goblet Cells ◽  
Actin Binding ◽  
Secretory Cells ◽  
Cortical Actin ◽  
Mucin Secretion

Airway goblet cells secrete mucin onto mucosal surfaces under the regulation of an apical, phospholipase C/Gq-coupled P2Y2receptor. We tested whether cortical actin filaments negatively regulate exocytosis in goblet cells by forming a barrier between secretory granules and plasma membrane docking sites as postulated for other secretory cells. Immunostaining of human lung tissues and SPOC1 cells (an epithelial, mucin-secreting cell line) revealed an apical distribution of β- and γ-actin in ciliated and goblet cells. In goblet cells, actin appeared as a prominent subplasmalemmal sheet lying between granules and the apical membrane, and it disappeared from SPOC1 cells activated by purinergic agonist. Disruption of actin filaments with latrunculin A stimulated SPOC1 cell mucin secretion under basal and agonist-activated conditions, whereas stabilization with jasplakinolide or overexpression of β- or γ-actin conjugated to yellow fluorescent protein (YFP) inhibited secretion. Myristoylated alanine-rich C kinase substrate, a PKC-activated actin-plasma membrane tethering protein, was phosphorylated after agonist stimulation, suggesting a translocation to the cytosol. Scinderin (or adseverin), a Ca2+-activated actin filament severing and capping protein was cloned from human airway and SPOC1 cells, and synthetic peptides corresponding to its actin-binding domains inhibited mucin secretion. We conclude that actin filaments negatively regulate mucin secretion basally in airway goblet cells and are dynamically remodeled in agonist-stimulated cells to promote exocytosis.

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.

scholarly journals Eisosome proteins assemble into a membrane scaffold

2011 ◽  
Vol 195 (5) ◽  
pp. 889-902 ◽  
Author(s):  
Lena Karotki ◽  
Juha T. Huiskonen ◽  
Christopher J. Stefan ◽  
Natasza E. Ziółkowska ◽  
Robyn Roth ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Self Assembly ◽  
Spatial Organization ◽  
Protein Complexes ◽  
Lipid Binding ◽  
Yeast Cells ◽  
Core Components ◽  
The Many ◽  
Plasma Membrane Domain ◽  
Membrane Scaffold

Spatial organization of membranes into domains of distinct protein and lipid composition is a fundamental feature of biological systems. The plasma membrane is organized in such domains to efficiently orchestrate the many reactions occurring there simultaneously. Despite the almost universal presence of membrane domains, mechanisms of their formation are often unclear. Yeast cells feature prominent plasma membrane domain organization, which is at least partially mediated by eisosomes. Eisosomes are large protein complexes that are primarily composed of many subunits of two Bin–Amphiphysin–Rvs domain–containing proteins, Pil1 and Lsp1. In this paper, we show that these proteins self-assemble into higher-order structures and bind preferentially to phosphoinositide-containing membranes. Using a combination of electron microscopy approaches, we generate structural models of Pil1 and Lsp1 assemblies, which resemble eisosomes in cells. Our data suggest that the mechanism of membrane organization by eisosomes is mediated by self-assembly of its core components into a membrane-bound protein scaffold with lipid-binding specificity.

scholarly journals Exophilin8 transiently clusters insulin granules at the actin-rich cell cortex prior to exocytosis

2011 ◽  
Vol 22 (10) ◽  
pp. 1716-1726 ◽  
Author(s):  
Kouichi Mizuno ◽  
José S. Ramalho ◽  
Tetsuro Izumi
Keyword(s):  
Plasma Membrane ◽  
Secretory Granules ◽  
Small Gtpase ◽  
Cell Cortex ◽  
Cortical Actin ◽  
Insulin Granules ◽  
Myosin Va ◽  
Exogenous Expression ◽  
Mutation Analyses

Exophilin8/MyRIP/Slac2-c is an effector protein of the small GTPase Rab27a and is specifically localized on retinal melanosomes and secretory granules. We investigated the role of exophilin8 in insulin granule trafficking. Exogenous expression of exophilin8 in pancreatic β cells or their cell line, MIN6, polarized (exophilin8-positive) insulin granules at the cell corners, where both cortical actin and the microtubule plus-end–binding protein, EB1, were present. Mutation analyses indicated that the ability of exophilin8 to act as a linker between Rab27a and myosin Va is essential for its granule-clustering activity. Moreover, exophilin8 and exophilin8-associated insulin granules were markedly stable and immobile. Total internal reflection fluorescence microscopy indicated that exophilin8 restricts the motion of insulin granules at a region deeper than that where another Rab27a effector, granuphilin, accumulates docked granules directly attached to the plasma membrane. However, the exophilin8-induced immobility of insulin granules was eliminated upon secretagogue stimulation and did not inhibit evoked exocytosis. Furthermore, exophilin8 depletion prevents insulin granules from being transported close to the plasma membrane and inhibits their fusion. These findings indicate that exophilin8 transiently traps insulin granules into the cortical actin network close to the microtubule plus-ends and supplies them for release during the stimulation.

scholarly journals Role of Plasma Membrane Lipid Microdomains in Respiratory Syncytial Virus Filament Formation

2003 ◽  
Vol 77 (3) ◽  
pp. 1747-1756 ◽  
Author(s):  
Lewis H. McCurdy ◽  
Barney S. Graham
Keyword(s):  
Plasma Membrane ◽  
Respiratory Syncytial Virus ◽  
Fusion Protein ◽  
Syncytium Formation ◽  
Membrane Lipid ◽  
Small Gtpase ◽  
Cellular Proteins ◽  
Lipid Microdomains ◽  
Protein F ◽  
Syncytial Virus

ABSTRACT The fusion protein (F) of respiratory syncytial virus (RSV) is the envelope glycoprotein responsible for the characteristic cytopathology of syncytium formation. RSV has been shown to bud from selective areas of the plasma membrane as pleomorphic virions, including both filamentous and round particles. With immunofluorescent microscopy, we demonstrated evidence of RSV filaments incorporating the fusion protein F and colocalizing with a lipid microdomain-specific fluorescent dye, 1,1-dihexadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate. Western blot analysis of Triton X-100 cold-extracted membrane fractions confirmed the presence of RSV proteins within the lipid microdomains. RSV proteins also colocalized with cellular proteins associated with lipid microdomains, caveolin-1, and CD44, as well as with RhoA, a small GTPase. ADP-ribosylation of RhoA by Clostridium botulinum exotoxin inactivated RhoA signaling and resulted in the absence of RSV-induced syncytia despite no significant change in viral titer. We demonstrated an overall decrease in both the number and length of the viral filaments and a shift in the localization of F to nonlipid microdomain regions of the membrane in the presence of C3 toxin. This suggests that the selective incorporation of RSV proteins into lipid microdomains during virus assembly may lead to critical interactions of F with cellular proteins, resulting in microvillus projections necessary for the formation of filamentous virus particles and syncytium formation. Thus, manipulation of membrane lipid microdomains may lead to alterations in the production of viral filaments and RSV pathogenesis and provide a new pharmacologic target for RSV therapy.

scholarly journals Signal Integration by Lipid-Mediated Spatial Cross Talk between Ras Nanoclusters

2013 ◽  
Vol 34 (5) ◽  
pp. 862-876 ◽  
Author(s):  
Yong Zhou ◽  
Hong Liang ◽  
Travis Rodkey ◽  
Nicholas Ariotti ◽  
Robert G. Parton ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Spatial Organization ◽  
Signal Transmission ◽  
Signal Integration ◽  
Ras Proteins ◽  
Cortical Actin ◽  
Mediated Communication ◽  
Ras Gtpases ◽  
Ras Isoforms ◽  
Lipid Assemblies

Lipid-anchored Ras GTPases form transient, spatially segregated nanoclusters on the plasma membrane that are essential for high-fidelity signal transmission. The lipid composition of Ras nanoclusters, however, has not previously been investigated. High-resolution spatial mapping shows that different Ras nanoclusters have distinct lipid compositions, indicating that Ras proteins engage in isoform-selective lipid sorting and accounting for different signal outputs from different Ras isoforms. Phosphatidylserine is a common constituent of all Ras nanoclusters but is only an obligate structural component of K-Ras nanoclusters. Segregation of K-Ras and H-Ras into spatially and compositionally distinct lipid assemblies is exquisitely sensitive to plasma membrane phosphatidylserine levels. Phosphatidylserine spatial organization is also modified by Ras nanocluster formation. In consequence, Ras nanoclusters engage in remote lipid-mediated communication, whereby activated H-Ras disrupts the assembly and operation of spatially segregated K-Ras nanoclusters. Computational modeling and experimentation reveal that complex effects of caveolin and cortical actin on Ras nanoclustering are similarly mediated through regulation of phosphatidylserine spatiotemporal dynamics. We conclude that phosphatidylserine maintains the lateral segregation of diverse lipid-based assemblies on the plasma membrane and that lateral connectivity between spatially remote lipid assemblies offers important previously unexplored opportunities for signal integration and signal processing.

scholarly journals Vesicular trafficking through cortical actin during exocytosis is regulated by the Rab27a effector JFC1/Slp1 and the RhoA-GTPase–activating protein Gem-interacting protein

2012 ◽  
Vol 23 (10) ◽  
pp. 1902-1916 ◽  
Author(s):  
Jennifer L. Johnson ◽  
Jlenia Monfregola ◽  
Gennaro Napolitano ◽  
William B. Kiosses ◽  
Sergio D. Catz
Keyword(s):  
Plasma Membrane ◽  
Secretory Granules ◽  
Vesicular Transport ◽  
Gtpase Activating Protein ◽  
Cortical Actin ◽  
Interacting Protein ◽  
Actin Remodeling ◽  
Actin Depolymerization ◽  
Rhoa Activity ◽  
Rhoa Gtpase

Cytoskeleton remodeling is important for the regulation of vesicular transport associated with exocytosis, but a direct association between granular secretory proteins and actin-remodeling molecules has not been shown, and this mechanism remains obscure. Using a proteomic approach, we identified the RhoA-GTPase–activating protein Gem-interacting protein (GMIP) as a factor that associates with the Rab27a effector JFC1 and modulates vesicular transport and exocytosis. GMIP down-regulation induced RhoA activation and actin polymerization. Importantly, GMIP-down-regulated cells showed impaired vesicular transport and exocytosis, while inhibition of the RhoA-signaling pathway induced actin depolymerization and facilitated exocytosis. We show that RhoA activity polarizes around JFC1-containing secretory granules, suggesting that it may control directionality of granule movement. Using quantitative live-cell microscopy, we show that JFC1-containing secretory organelles move in areas near the plasma membrane deprived of polymerized actin and that dynamic vesicles maintain an actin-free environment in their surroundings. Supporting a role for JFC1 in RhoA inactivation and actin remodeling during exocytosis, JFC1 knockout neutrophils showed increased RhoA activity, and azurophilic granules were unable to traverse cortical actin in cells lacking JFC1. We propose that during exocytosis, actin depolymerization commences near the secretory organelle, not the plasma membrane, and that secretory granules use a JFC1- and GMIP-dependent molecular mechanism to traverse cortical actin.

scholarly journals Insulin-Induced Endothelial Cell Cortical Actin Filament Remodeling: A Requirement for Trans-Endothelial Insulin Transport

2012 ◽  
Vol 26 (8) ◽  
pp. 1327-1338 ◽  
Author(s):  
Hong Wang ◽  
Aileen X. Wang ◽  
Eugene J. Barrett
Keyword(s):  
Plasma Membrane ◽  
Lipid Rafts ◽  
Actin Filament ◽  
Pi3k Pathway ◽  
Membrane Lipid ◽  
Cortical Actin ◽  
Caveolin 1 ◽  
Actin Microfilament ◽  
Igf I ◽  
Insulin Uptake

Insulin's trans-endothelial transport (TET) is critical for its metabolic action on muscle and involves trafficking of insulin bound to its receptor (or at high insulin concentrations, the IGF-I receptor) via caveolae. However, whether caveolae-mediated insulin TET involves actin cytoskeleton organization is unknown. Here we address whether insulin regulates actin filament organization in bovine aortic endothelial cells (bAEC) and whether this affects insulin uptake and TET. We found that insulin induced extensive cortical actin filament remodeling within 5 min. This remodeling was inhibited not only by disruption of actin microfilament organization but also by inhibition of phosphatidylinositol 3-kinase (PI3K) or by disruption of lipid rafts using respective specific inhibitors. Knockdown of either caveolin-1 or Akt using specific small interfering RNA also eliminated the insulin-induced cortical actin filament remodeling. Blocking either actin microfilament organization or PI3K pathway signaling inhibited both insulin uptake and TET. Disruption of actin microfilament organization also reduced the caveolin-1, insulin receptor, and IGF-I receptor located at the plasma membrane. Exposing bAEC for 6 h to either TNFα or IL-6 blocked insulin-induced cortical actin remodeling. Extended exposure (24 h) also inhibited actin expression at both mRNA and protein levels. We conclude that insulin-induced cortical actin filament remodeling in bAEC is required for insulin's TET in a PI3K/Akt and plasma membrane lipid rafts/caveolae-dependent fashion, and proinflammatory cytokines TNFα and IL-6 block this process.

Sign in / Sign up

Export Citation Format

Share Document