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 A continuum model of docking of synaptic vesicle to plasma membrane

2015 ◽  
Vol 12 (102) ◽  
pp. 20141119 ◽  
Author(s):  
Tianshu Liu ◽  
Pankaj Singh ◽  
James T. Jenkins ◽  
Anand Jagota ◽  
Maria Bykhovskaia ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Synaptic Vesicle ◽  
Neurotransmitter Release ◽  
Pore Formation ◽  
Continuum Model ◽  
Synaptic Cleft ◽  
Fusion Pore ◽  
Neuronal Membrane ◽  
Electrostatic Repulsion ◽  
Synaptic Vesicle Fusion

Neurotransmitter release from neuronal terminals is governed by synaptic vesicle fusion. Vesicles filled with transmitters are docked at the neuronal membrane by means of the SNARE machinery. After a series of events leading up to the fusion pore formation, neurotransmitters are released into the synaptic cleft. In this paper, we study the mechanics of the docking process. A continuum model is used to determine the deformation of a spherical vesicle and a plasma membrane, under the influence of SNARE-machinery forces and electrostatic repulsion. Our analysis provides information on the variation of in-plane stress in the membranes, which is known to affect fusion. Also, a simple model is proposed to study hemifusion.

Fusion Pore: An Evolutionary Invention of Nucleated Cells

2010 ◽  
Vol 18 (3) ◽  
pp. 347-364 ◽  
Author(s):  
N. Vardjan ◽  
M. Stenovec ◽  
J. Jorgačevski ◽  
M. Kreft ◽  
R. Zorec
Keyword(s):  
Plasma Membrane ◽  
Blood Stream ◽  
Fusion Pore ◽  
The Novel ◽  
Vesicle Membrane ◽  
Signalling Molecules ◽  
Fundamental Biological Process ◽  
Chemical Synapses ◽  
Fusion Pores ◽  
Single Vesicle

This article outlines the lecture presented by Robert Zorec at the Academia Europea meeting in Liverpool on 19 September 2008, four decades after the Sherrington Lecture of Bernard Katz who, together with his colleagues, developed a number of paradigms addressing vesicles in chemical synapses. Vesicles are subcellular organelles that evolved in eukaryotic cells 1000 to 2000 million years ago. They store signalling molecules such as chemical messengers, which are essential for the function of neurons and endocrine cells in supporting the communication between tissues and organs in the human body. Upon a stimulus, the vesicle-stored signalling molecules (neurotransmitters or hormones) are released from cells. This event involves exocytosis, a fundamental biological process, consisting of the merger of the vesicle membrane with the plasma membrane. The two fusing membranes lead to the formation of an aqueous channel – the fusion pore – through which signalling molecules exit into the extracellular space or blood stream. The work of Bernard Katz and colleagues considered that vesicle cargo discharge initially requires the delivery of vesicles to the plasma membrane, where vesicles dock and get primed for fusion with the plasma membrane, and that stimulation initiates the formation of the transient fusion pore through which cargo molecules leave the vesicle lumen in an all-or-none-fashion. However, recent studies indicate that this may not be so simple. Here we highlight the novel findings which indicate that fusion pores are subject to regulations, which affect the release competence of a single vesicle. At least in pituitary lactotrophs, which are the subject of research in our laboratories, single vesicle release of peptide signalling molecules involves modulation of fusion pore diameter and fusion pore kinetics.

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 TRIM67 Regulates Exocytic Mode and Neuronal Morphogenesis via SNAP47

2020 ◽  
Author(s):  
Fabio L. Urbina ◽  
Shalini Menon ◽  
Dennis Goldfarb ◽  
Reginald Edwards ◽  
M. Ben Major ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Ubiquitin Ligase ◽  
Cortical Neurons ◽  
Fusion Pore ◽  
Membrane Material ◽  
New Classification ◽  
Vesicle Membrane ◽  
Neuronal Morphogenesis ◽  
Neuronal Synapses ◽  
Developing Neurons

AbstractNeuronal morphogenesis involves dramatic plasma membrane expansion, likely fueled by SNARE-mediated exocytosis. Distinct fusion modes described at neuronal synapses include full-vesicle-fusion (FVF) and kiss-and-run fusion (KNR). During FVF, lumenal cargo is secreted and vesicle membrane incorporates into the plasma membrane. During KNR a transient fusion pore secretes cargo, but closes without membrane addition. In contrast, fusion modes are not described in developing neurons where plasma membrane expansion is significant. Here, we resolve individual exocytic events in developing murine cortical neurons and use new classification tools to identify four distinguishable fusion modes: two FVF-like modes that insert membrane material and two KNR-like modes that do not. Discrete fluorescence profiles suggest distinct behavior of the fusion pore with each mode. Simulations and experiments agree that FVF-like exocytosis provides sufficient membrane material for morphogenesis. We find the E3 ubiquitin ligase TRIM67 promotes FVF-like exocytosis. Our data suggest this is accomplished in part by limiting incorporation of the Qb/Qc SNARE SNAP47 into SNARE complexes and thus, SNAP47 involvement in exocytosis.

scholarly journals Roles for the SNAP25 linker domain in the fusion pore and a dynamic plasma membrane SNARE “acceptor” complex

2020 ◽  
Vol 152 (9) ◽  
Author(s):  
Ronald W. Holz ◽  
Mary A. Bittner
Keyword(s):  
Plasma Membrane ◽  
Protein Interactions ◽  
Fusion Reaction ◽  
Snare Complex ◽  
Fusion Pore ◽  
Protein Protein Interactions ◽  
Vesicle Membrane ◽  
Acceptor Complex ◽  
Linker Domain ◽  
Pore Expansion

Central to the exocytotic release of hormones and neurotransmitters is the interaction of four SNARE motifs in proteins on the secretory granule/synaptic vesicle membrane (synaptobrevin/VAMP, v-SNARE) and on the plasma membrane (syntaxin and SNAP25, t-SNAREs). The interaction is thought to bring the opposing membranes together to enable fusion. An underlying motivation for this Viewpoint is to synthesize from recent diverse studies possible new insights about these events. We focus on a recent paper that demonstrates the importance of the linker region joining the two SNARE motifs of the neuronal t-SNARE SNAP25 for maintaining rates of secretion with roles for distinct segments in speeding fusion pore expansion. Remarkably, lipid-perturbing agents rescue a palmitoylation-deficient mutant whose phenotype includes slow fusion pore expansion, suggesting that protein–protein interactions have a role not only in bringing together the granule or vesicle membrane with the plasma membrane but also in orchestrating protein–lipid interactions leading to the fusion reaction. Unexpectedly, biochemical investigations demonstrate the importance of the C-terminal domain of the linker in the formation of the plasma membrane t-SNARE “acceptor” complex for synaptobrevin2. This insight, together with biophysical and optical studies from other laboratories, suggests that the plasma membrane SNARE acceptor complex between SNAP25 and syntaxin and the subsequent trans-SNARE complex with the v-SNARE synaptobrevin form within 100 ms before fusion.

scholarly journals Roles for the SNAP25 Linker Domain in the Fusion Pore and a Dynamic Plasma Membrane SNARE Acceptor Complex

2020 ◽  
Author(s):  
Ronald Holz ◽  
Mary Bittner
Keyword(s):  
Plasma Membrane ◽  
Protein Interactions ◽  
Fusion Reaction ◽  
Snare Complex ◽  
Fusion Pore ◽  
Protein Protein Interactions ◽  
Vesicle Membrane ◽  
Acceptor Complex ◽  
Linker Domain ◽  
Pore Expansion

A recent paper demonstrates the importance of the linker region joining the two SNARE motifs of the neuronal t-SNARE SNAP25 for maintaining rates of secretion with roles for distinct segments in speeding fusion pore expansion (Shaaban et al., 2019, Elife. 8). Remarkably, lipid perturbing agents rescue a palmitoylation-deficient phenotype that includes slow fusion pore expansion, suggesting that protein-protein interactions have a role not only in bringing together the granule or vesicle membrane with the plasma membrane but also in orchestrating protein-lipid interactions leading to the fusion reaction. Furthermore, biochemical investigations demonstrate the importance of the C-terminal domain of the linker in the formation of the plasma membrane t-SNARE acceptor complex for synaptobrevin2 (Jiang, et al., 2019, FASEB J. 33:7985-7994;Shaaban et al., 2019, Elife. 8). This insight, together with biophysical and optical studies from other laboratories (Wang, et al., 2008, Molecular Biology of the Cell. 19:3944-3955; Zhao, et al., 2013, Proc Natl Acad Sci U S A. 110:14249-14254) suggests that the plasma membrane SNARE acceptor complex between SNAP25 and syntaxin and the resulting trans SNARE complex with the v-SNARE synaptobrevin form just milliseconds before fusion.

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 Partial purification of presynaptic plasma membrane by immunoadsorption.

1982 ◽  
Vol 94 (1) ◽  
pp. 88-96 ◽  
Author(s):  
G P Miljanich ◽  
A R Brasier ◽  
R B Kelly
Keyword(s):  
Plasma Membrane ◽  
Synaptic Vesicle ◽  
Nerve Terminal ◽  
Specific Activity ◽  
Electric Organ ◽  
Partial Purification ◽  
Vesicle Membrane ◽  
Specific Activities ◽  
Active Zones ◽  
Presynaptic Plasma Membrane

During transmitter release, synaptic vesicle membrane is specifically inserted into the nerve terminal plasma membrane only at specialized sites or "active zones." In an attempt to obtain a membrane fraction enriched in active zones, we have utilized the electric organ of the marine ray. From this organ, a fraction enriched in nerve terminals (synaptosomes) was prepared by conventional means. These synaptosomes were bound to microscopic beads by an antiserum to purified electric organ synaptic vesicles (anti-SV). The success of this immunoadsorption procedure was demonstrated by increased specific activities of bead-bound nerve terminal cytoplasmic markers and decreased specific activities of markers for contaminating membranes. To obtain a presynaptic plasma membrane (PSPM) fraction, we lysed the bead-bound synaptosomes by hypoosmotic shock and sonication, resulting in complete release of cytoplasmic markers. When the synaptosomal fraction was surface-labeled with iodine before immunoadsorption, 10% of this label remained bead-bound after lysis, compared with 2% of the total protein, indicating an approximately fivefold enrichment of bead-bound plasma membrane. Concomitantly, the specific activity of bead-bound anti-SV increased approximately 30-fold, indicating an enrichment of plasma membrane which contained inserted synaptic vesicle components. This PSPM preparation is not simply synaptic vesicle membrane since two-dimensional electrophoresis revealed that the polypeptides of the surface-iodinated PSPM preparation include both vesicle and numerous nonvesicle components. Secondly, antiserum to the PSPM fraction is markedly different from anti-SV and binds to external, nonvesicle, nerve terminal components.

scholarly journals Chromogranin A, the major lumenal protein in chromaffin granules, controls fusion pore expansion

2018 ◽  
Vol 151 (2) ◽  
pp. 118-130 ◽  
Author(s):  
Prabhodh S. Abbineni ◽  
Mary A. Bittner ◽  
Daniel Axelrod ◽  
Ronald W. Holz
Keyword(s):  
Plasma Membrane ◽  
Small Molecules ◽  
Chromogranin A ◽  
Chromaffin Cells ◽  
Fusion Pore ◽  
Chromaffin Granules ◽  
Chromaffin Granule ◽  
Chromogranin B ◽  
Tissue Plasminogen ◽  
Pore Expansion

Upon fusion of the secretory granule with the plasma membrane, small molecules are discharged through the immediately formed narrow fusion pore, but protein discharge awaits pore expansion. Recently, fusion pore expansion was found to be regulated by tissue plasminogen activator (tPA), a protein present within the lumen of chromaffin granules in a subpopulation of chromaffin cells. Here, we further examined the influence of other lumenal proteins on fusion pore expansion, especially chromogranin A (CgA), the major and ubiquitous lumenal protein in chromaffin granules. Polarized TIRF microscopy demonstrated that the fusion pore curvature of granules containing CgA-EGFP was long lived, with curvature lifetimes comparable to those of tPA-EGFP–containing granules. This was surprising because fusion pore curvature durations of granules containing exogenous neuropeptide Y-EGFP (NPY-EGFP) are significantly shorter (80% lasting <1 s) than those containing CgA-EGFP, despite the anticipated expression of endogenous CgA. However, quantitative immunocytochemistry revealed that transiently expressed lumenal proteins, including NPY-EGFP, caused a down-regulation of endogenously expressed proteins, including CgA. Fusion pore curvature durations in nontransfected cells were significantly longer than those of granules containing overexpressed NPY but shorter than those associated with granules containing overexpressed tPA, CgA, or chromogranin B. Introduction of CgA to NPY-EGFP granules by coexpression converted the fusion pore from being transient to being longer lived, comparable to that found in nontransfected cells. These findings demonstrate that several endogenous chromaffin granule lumenal proteins are regulators of fusion pore expansion and that alteration of chromaffin granule contents affects fusion pore lifetimes. Importantly, the results indicate a new role for CgA. In addition to functioning as a prohormone, CgA plays an important role in controlling fusion pore expansion.

scholarly journals Fusion Pore Dynamics Are Regulated by Synaptotagmin•t-SNARE Interactions

Neuron ◽  
2004 ◽  
Vol 41 (6) ◽  
pp. 929-942 ◽  
Author(s):  
Jihong Bai ◽  
Chih-Tien Wang ◽  
David A Richards ◽  
Meyer B Jackson ◽  
Edwin R Chapman
Keyword(s):  
Fusion Pore ◽  
Pore Dynamics
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