scholarly journals Redistribution of cholesterol from vesicle to plasmalemma controls fusion pore geometry

2020 ◽  
Author(s):  
Boštjan Rituper ◽  
Alenka Guček ◽  
Marjeta Lisjak ◽  
Urszula Gorska ◽  
Aleksandra Šakanović ◽  
...  
Keyword(s):  
Pore Diameter ◽  
Membrane Lipids ◽  
Radial Force ◽  
Fusion Pore ◽  
Pore Geometry ◽  
Cholesterol Depletion ◽  
List Type ◽  
Vesicle Membrane ◽  
Membrane Pore ◽  
Outer Leaflet

ABSTRACTEukaryotic vesicles fuse with the plasmalemma to form the fusion pore, previously considered to be unstable with widening of the pore diameter. Recent studies established that the pore diameter is stable, reflecting balanced forces of widening and closure. Proteins are considered key regulators of the fusion pore, whereas the role of membrane lipids remains unclear. Super-resolution microscopy revealed that lactotroph secretory vesicles discharge cholesterol after stimulation of exocytosis; subsequently, vesicle cholesterol redistributes to the outer leaflet of the plasmalemma. Cholesterol depletion in lactotrophs and astrocytes evokes release of vesicle hormone, indicating that cholesterol constricts the fusion pore. A new model of cholesterol-dependent fusion pore diameter regulation is proposed. High-resolution measurements of fusion pore conductance confirmed that the fusion pore widens with cholesterol depletion and constricts with cholesterol enrichment. In fibroblasts lacking the Npc1 protein, in which cholesterol accumulates in vesicles, the fusion pore is narrower than in controls, showing that cholesterol regulates fusion pore geometry.Graphical AbstractTop: stages through which a vesicle interacts with the plasmalemma. Stage A denotes hemifusion, which proceeds to stage B, with a narrow fusion pore, which can then reversibly open (stage C), before widening fully (stage D). Bottom: redistribution of cholesterol from the vesicle to the outer leaflet of the plasmalemma controls fusion pore constriction.In BriefA membrane pore is formed when the vesicle membrane fuses with the plasmalemma. Proteins were considered key regulators of the opening and closing of this fusion pore. Here, evidence is provided to show that cholesterol, a membrane constituent, determines a radial force constricting the fusion pore, revealing that the fusion pore functions as a proteolipidic structure.HighlightsIntravesicular cholesterol redistributes to the outer leaflet of the plasmalemma.Cholesterol depletion widens the fusion pore, whereas cholesterol enrichment constricts the fusion pore.A model of cholesterol-dependent force preventing fusion pore widening is developed.Disease-related increase in vesicle cholesterol constricts the fusion pore.

scholarly journals Fusion Pore Diameter Regulation by Cations Modulating Local Membrane Anisotropy

2012 ◽  
Vol 2012 ◽  
pp. 1-7 ◽  
Author(s):  
Doron Kabaso ◽  
Ana I. Calejo ◽  
Jernej Jorgačevski ◽  
Marko Kreft ◽  
Robert Zorec ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Continuum Model ◽  
Pore Diameter ◽  
Fusion Pore ◽  
Spontaneous Curvature ◽  
Local Concentration ◽  
Vesicle Membrane ◽  
Membrane Elasticity ◽  
Opening And Closing ◽  
Pore Dynamics

The fusion pore is an aqueous channel that is formed upon the fusion of the vesicle membrane with the plasma membrane. Once the pore is open, it may close again (transient fusion) or widen completely (full fusion) to permit vesicle cargo discharge. While repetitive transient fusion pore openings of the vesicle with the plasma membrane have been observed in the absence of stimulation, their frequency can be further increased using a cAMP-increasing agent that drives the opening of nonspecific cation channels. Our model hypothesis is that the openings and closings of the fusion pore are driven by changes in the local concentration of cations in the connected vesicle. The proposed mechanism of fusion pore dynamics is considered as follows: when the fusion pore is closed or is extremely narrow, the accumulation of cations in the vesicle (increased cation concentration) likely leads to lipid demixing at the fusion pore. This process may affect local membrane anisotropy, which reduces the spontaneous curvature and thus leads to the opening of the fusion pore. Based on the theory of membrane elasticity, we used a continuum model to explain the rhythmic opening and closing of the fusion pore.

scholarly journals The evidence for open and closed exocytosis as the primary release mechanism

2016 ◽  
Vol 49 ◽  
Author(s):  
Lin Ren ◽  
Lisa J. Mellander ◽  
Jacqueline Keighron ◽  
Ann-Sofie Cans ◽  
Michael E. Kurczy ◽  
...  
Keyword(s):  
Fusion Pore ◽  
Release Mechanism ◽  
Vesicle Membrane ◽  
Membrane Pore ◽  
Initial Formation ◽  
Quantitative Evidence ◽  
Fundamental Process ◽  
The Subject ◽  
Chemical Messenger ◽  
Partial Release

AbstractExocytosis is the fundamental process by which cells communicate with each other. The events that lead up to the fusion of a vesicle loaded with chemical messenger with the cell membrane were the subject of a Nobel Prize in 2013. However, the processes occurring after the initial formation of a fusion pore are very much still in debate. The release of chemical messenger has traditionally been thought to occur through full distention of the vesicle membrane, hence assuming exocytosis to be all or none. In contrast to the all or none hypothesis, here we discuss the evidence that during exocytosis the vesicle-membrane pore opens to release only a portion of the transmitter content during exocytosis and then close again. This open and closed exocytosis is distinct from kiss-and-run exocytosis, in that it appears to be the main content released during regular exocytosis. The evidence for this partial release via open and closed exocytosis is presented considering primarily the quantitative evidence obtained with amperometry.

scholarly journals The C2b Domain of Synaptotagmin Is a Ca2+–Sensing Module Essential for Exocytosis

2000 ◽  
Vol 150 (5) ◽  
pp. 1125-1136 ◽  
Author(s):  
Radhika C. Desai ◽  
Bimal Vyas ◽  
Cynthia A. Earles ◽  
J. Troy Littleton ◽  
Judith A. Kowalchyck ◽  
...  
Keyword(s):  
Synaptic Vesicle ◽  
Pc12 Cells ◽  
Conformational Changes ◽  
Cytoplasmic Domain ◽  
Dominant Negative ◽  
Fusion Pore ◽  
Vesicle Membrane ◽  
C2 Domains ◽  
Essential Step ◽  
Synaptotagmin I

The synaptic vesicle protein synaptotagmin I has been proposed to serve as a Ca2+ sensor for rapid exocytosis. Synaptotagmin spans the vesicle membrane once and possesses a large cytoplasmic domain that contains two C2 domains, C2A and C2B. Multiple Ca2+ ions bind to the membrane proximal C2A domain. However, it is not known whether the C2B domain also functions as a Ca2+-sensing module. Here, we report that Ca2+ drives conformational changes in the C2B domain of synaptotagmin and triggers the homo- and hetero-oligomerization of multiple isoforms of the protein. These effects of Ca2+ are mediated by a set of conserved acidic Ca2+ ligands within C2B; neutralization of these residues results in constitutive clustering activity. We addressed the function of oligomerization using a dominant negative approach. Two distinct reagents that block synaptotagmin clustering potently inhibited secretion from semi-intact PC12 cells. Together, these data indicate that the Ca2+-driven clustering of the C2B domain of synaptotagmin is an essential step in excitation-secretion coupling. We propose that clustering may regulate the opening or dilation of the exocytotic fusion pore.

scholarly journals Solidified Fillings of Nanopores

2005 ◽  
Vol 876 ◽  
Author(s):  
Patrick Huber ◽  
Klaus Knorr
Keyword(s):  
Pore Diameter ◽  
Stacking Faults ◽  
Pore Geometry ◽  
X Ray Diffraction ◽  
X Ray ◽  
Medium Length ◽  
Close Packed Structure ◽  
Packed Structure ◽  
Diffraction Patterns ◽  
Selection Of

AbstractWe present a selection of x-ray diffraction patterns of spherical (He, Ar), dumbbell- (N2, CO), and chain-like molecules (n-C9H20, n-C19H40) solidified in nanopores of silica glass (mean pore diameter 7nm). These patterns allow us to demonstrate how key principles governing crystallization have to be adapted in order to accomplish solidification in restricted geometries. 4He, Ar, and the spherical close packed phases of CO and N2 adjust to the pore geometry by introducing a sizeable amount of stacking faults. For the pore solidified, medium-length chainlike n-C19H40 we observe a close packed structure without lamellar ordering, whereas for the short-chain C9H20 the layering principle survives, albeit in a modified fashion compared to the bulk phase.

scholarly journals Improved red blood cell preservation correlates with decreased loss of bands 3, 4.1, acetylcholinestrase, and lipids in microvesicles

Blood ◽  
1996 ◽  
Vol 87 (4) ◽  
pp. 1612-1616 ◽  
Author(s):  
UJ Dumaswala ◽  
RU Dumaswala ◽  
DS Levin ◽  
TJ Greenwalt
Keyword(s):  
Fluorescence Anisotropy ◽  
Ache Activity ◽  
Membrane Lipids ◽  
Final Concentration ◽  
Vesicle Membrane ◽  
Rbc Membrane ◽  
Additive Solution ◽  
Cell Preservation

In earlier studies we have shown that a final concentration of 0.69% glycerol in blood mixed with an experimental additive solution, EAS 25, improves the in vitro quality and in vivo survival of red blood cells (RBCs). The objective of this study was to determine if the better preservation of RBCs in EAS 25 is correlated with the improved maintenance of membrane lipids and proteins and decreased vesiculation. Split units of RBCs were stored in Adsol or EAS 25 (mmol/L: adenine 2/2, dextrose 122/110, mannitol 42/55, glycerol 0/150, NaCl 154/50). After 12 weeks storage, RBC and microvesicle membranes were analyzed for cholesterol, phospholipid, diphenyl hexatriene fluorescence anisotropy, and acetylcholinesterase (AchE) activity. Bands 3 and 4.1 were identified in the microvesicle membranes by immunoblotting. The RBC membrane cholesterol, phospholipids, and AchE remained higher in EAS 25 than in Adsol (P < .001). Vesicle membrane lipids and AchE in EAS 25 were significantly less than in Adsol (P < .001). The fluidity of stored cells in both the solutions was greater than the prestorage samples. Immunoblotting analyses showed that bands 3 and 4.1 were greatly reduced in the microvesicle membranes shed by the RBCs stored in EAS 25 compared with those formed in Adsol.

scholarly journals Cellular Lipid Composition Affects Sensitivity of Plant Pathogens to Fengycin, an Antifungal Compound Produced by Bacillus subtilis Strain CU12

2014 ◽  
Vol 104 (10) ◽  
pp. 1036-1041 ◽  
Author(s):  
Cody Wise ◽  
Justin Falardeau ◽  
Ingrid Hagberg ◽  
Tyler J. Avis
Keyword(s):  
Bacillus Subtilis ◽  
Lipid Composition ◽  
Plant Pathogens ◽  
Membrane Lipids ◽  
Acyl Chain ◽  
Fatty Acyl ◽  
Subtilis Strain ◽  
Antifungal Compound ◽  
Membrane Pore ◽  
Ergosterol Content

Fengycin is an antimicrobial cyclic lipopeptide produced by various Bacillus subtilis strains, including strain CU12. Direct effects of fengycin include membrane pore formation and efflux of cellular contents leading to cell death in sensitive microorganisms. In this study, four plant pathogens were studied in order to elucidate the role of membrane lipids in their relative sensitivity to fengycin. Inhibition of mycelial growth in these pathogens varied considerably. Analysis of membrane lipids in these microorganisms indicated that sensitivity correlated with low ergosterol content and shorter phospholipid fatty acyl chains. Sensitivity to fengycin also correlated with a lower anionic/zwitterionic phospholipid ratio. Our data suggest that decreased fluidity buffering capacity, as a result of low ergosterol content, and higher intrinsic fluidity afforded by short fatty acyl chain length may increase the sensitivity of microbial membranes to fengycin. Our results also suggest that lower content in anionic phospholipids may increase fengycin insertion into the membrane through reduced electrostatic repulsion with the negatively charged fengycin. The intrinsic membrane lipid composition may contribute, in part, to the observed level of antimicrobial activity of fengycin in various plant pathogens.

scholarly journals Kiss-and-Coat and Compartment Mixing: Coupling Exocytosis to Signal Generation and Local Actin Assembly

2006 ◽  
Vol 17 (4) ◽  
pp. 1495-1502 ◽  
Author(s):  
Anna M. Sokac ◽  
William M. Bement
Keyword(s):  
Cell Types ◽  
Secretory Vesicle ◽  
Signal Generation ◽  
General Mechanism ◽  
Fusion Pore ◽  
Secretory Vesicles ◽  
Actin Assembly ◽  
Vesicle Membrane ◽  
Regulated Exocytosis ◽  
Broad Variety

Regulated exocytosis is thought to occur either by “full fusion,” where the secretory vesicle fuses with the plasma membrane (PM) via a fusion pore that then dilates until the secretory vesicle collapses into the PM; or by “kiss-and-run,” where the fusion pore does not dilate and instead rapidly reseals such that the secretory vesicle is retrieved almost fully intact. Here, we describe growing evidence for a third form of exocytosis, dubbed “kiss-and-coat,” which is characteristic of a broad variety of cell types that undergo regulated exocytosis. Kiss-and-coat exocytosis entails prolonged maintenance of a dilated fusion pore and assembly of actin filament (F-actin) coats around the exocytosing secretory vesicles followed by direct retrieval of some fraction of the emptied vesicle membrane. We propose that assembly of the actin coats results from the union of the secretory vesicle membrane and PM and that this compartment mixing represents a general mechanism for generating local signals via directed membrane fusion.

scholarly journals Transient anchorage of cross-linked glycosyl-phosphatidylinositol–anchored proteins depends on cholesterol, Src family kinases, caveolin, and phosphoinositides

2006 ◽  
Vol 175 (1) ◽  
pp. 169-178 ◽  
Author(s):  
Yun Chen ◽  
William R. Thelin ◽  
Bing Yang ◽  
Sharon L. Milgram ◽  
Ken Jacobson
Keyword(s):  
Transmembrane Protein ◽  
Cross Linking ◽  
Cholesterol Depletion ◽  
Transmembrane Conductance Regulator ◽  
Membrane Biology ◽  
Glycosyl Phosphatidylinositol ◽  
Caveolin 1 ◽  
Outer Leaflet ◽  
Membrane Components ◽  
Open Question

How outer leaflet plasma membrane components, including glycosyl-phosphatidylinositol–anchored proteins (GPIAPs), transmit signals to the cell interior is an open question in membrane biology. By deliberately cross-linking several GPIAPs under antibody-conjugated 40-nm gold particles, transient anchorage of the gold particle–induced clusters of both Thy-1 and CD73, a 5′ exonucleotidase, occurred for periods ranging from 300 ms to 10 s in fibroblasts. Transient anchorage was abolished by cholesterol depletion, addition of the Src family kinase (SFK) inhibitor PP2, or in Src-Yes-Fyn knockout cells. Caveolin-1 knockout cells exhibited a reduced transient anchorage time, suggesting the partial participation of caveolin-1. In contrast, a transmembrane protein, the cystic fibrosis transmembrane conductance regulator, exhibited transient anchorage that occurred without deliberately enhanced cross-linking; moreover, it was only slightly inhibited by cholesterol depletion or SFK inhibition and depended completely on the interaction of its PDZ-binding domain with the cytoskeletal adaptor EBP50. We propose that cross-linked GPIAPs become transiently anchored via a cholesterol-dependent SFK-regulatable linkage between a transmembrane cluster sensor and the cytoskeleton.

scholarly journals v-SNARE transmembrane domains function as catalysts for vesicle fusion

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Madhurima Dhara ◽  
Antonio Yarzagaray ◽  
Mazen Makke ◽  
Barbara Schindeldecker ◽  
Yvonne Schwarz ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Conformational Flexibility ◽  
Vesicle Fusion ◽  
Transmembrane Domains ◽  
Fusion Process ◽  
Snare Proteins ◽  
Fusion Pore ◽  
Structural Flexibility ◽  
Outer Leaflet ◽  
Pore Expansion

Vesicle fusion is mediated by an assembly of SNARE proteins between opposing membranes, but it is unknown whether transmembrane domains (TMDs) of SNARE proteins serve mechanistic functions that go beyond passive anchoring of the force-generating SNAREpin to the fusing membranes. Here, we show that conformational flexibility of synaptobrevin-2 TMD is essential for efficient Ca2+-triggered exocytosis and actively promotes membrane fusion as well as fusion pore expansion. Specifically, the introduction of helix-stabilizing leucine residues within the TMD region spanning the vesicle’s outer leaflet strongly impairs exocytosis and decelerates fusion pore dilation. In contrast, increasing the number of helix-destabilizing, ß-branched valine or isoleucine residues within the TMD restores normal secretion but accelerates fusion pore expansion beyond the rate found for the wildtype protein. These observations provide evidence that the synaptobrevin-2 TMD catalyzes the fusion process by its structural flexibility, actively setting the pace of fusion pore expansion.

scholarly journals Impact of Pore Geometry and Water Saturation on Gas Effective Diffusion Coefficient in Soil

2018 ◽  
Vol 8 (11) ◽  
pp. 2097 ◽  
Author(s):  
Wulong Hu ◽  
Yao Jiang ◽  
Daoyi Chen ◽  
Yongshui Lin ◽  
Qiang Han ◽  
...  
Keyword(s):  
Diffusion Coefficient ◽  
Power Law ◽  
Diffusion Coefficients ◽  
Effective Diffusion Coefficient ◽  
Pore Diameter ◽  
Water Saturation ◽  
Knudsen Diffusion ◽  
Effective Diffusion ◽  
Pore Geometry ◽  
Effective Diffusion Coefficients

Gas flow in soil plays a crucial role in terrestrial ecosystems, and numerical simulation of their movement needs to know their effective diffusion coefficients. How pore structure influences the effective diffusion coefficient has been studied intensively for dry porous media, but much remains unknown for unsaturated soils. Here, we employed the X-ray tomography technique at the pore scale to directly obtain the soil structures, the geometry of their pores and the water distribution under different water saturation levels were calculated using a morphological model. The results show that pore structures including porosity, interface area of gas–solid–water and pore diameter are closely related to water saturation. The increase of mean pore diameter with gas saturation can be fitted into a power law. We also investigated the impact of pore geometry and water saturation on the effective diffusion coefficients, which is independent of the molecular mass of gas after normalization. As the normalized effective Knudsen diffusion coefficient increases with average pore diameter following a power law, with the scaling factor related to pore geometry and the exponent is a constant, we explained and proved that the Knudsen diffusion coefficient increases with gas saturation, also following a power law.

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