scholarly journals Stabilization of the SNARE Core by Complexin-1 Facilitates Fusion Pore Expansion

2021 ◽  
Vol 8 ◽  
Author(s):  
Josh Pierson ◽  
Yeon-Kyun Shin
Keyword(s):  
Neurotransmitter Release ◽  
Fluorescence Probe ◽  
Mechanistic Model ◽  
Reciprocal Relationship ◽  
Fusion Pore ◽  
Vesicle Recycling ◽  
Binding Core ◽  
Fusion Pores ◽  
Single Vesicle ◽  
Pore Expansion

In the neuron, neurotransmitter release is an essential function that must be both consistent and tightly regulated. The continuity of neurotransmitter release is dependent in large part on vesicle recycling. However, the protein factors that dictate the vesicle recycling pathway are elusive. Here, we use a single vesicle-to-supported bilayer fusion assay to investigate complexin-1 (cpx1)’s influence on SNARE-dependent fusion pore expansion. With total internal reflection (TIR) microscopy using a 10 kDa polymer fluorescence probe, we are able to detect the presence of large fusion pores. With cpx1, however, we observe a significant increase of the probability of the formation of large fusion pores. The domain deletion analysis reveals that the SNARE-binding core domain of cpx1 is mainly responsible for its ability to promote the fusion pore expansion. In addition, the results show that cpx1 helps the pore to expand larger, which results in faster release of the polymer probe. Thus, the results demonstrate a reciprocal relationship between event duration and the size of the fusion pore. Based on the data, a hypothetical mechanistic model can be deduced. In this mechanistic model, the cpx1 binding stabilizes the four-helix bundle structure of the SNARE core throughout the fusion pore expansion, whereby the highly curved bilayer within the fusion pore is stabilized by the SNARE pins.

scholarly journals Membrane Binding of α-Synuclein Stimulates Expansion of SNARE-Dependent Fusion Pore

2021 ◽  
Vol 9 ◽  
Author(s):  
Ryan Khounlo ◽  
Brenden J. D. Hawk ◽  
Tung-Mei Khu ◽  
Gyeongji Yoo ◽  
Nam Ki Lee ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Membrane Binding ◽  
Snare Complex ◽  
Fusion Pore ◽  
Fusion Inhibitors ◽  
Important Regulator ◽  
Fusion Pores ◽  
Single Vesicle ◽  
Pore Expansion

SNARE-dependent membrane fusion is essential for neurotransmitter release at the synapse. Recently, α-synuclein has emerged as an important regulator for membrane fusion. Misfolded α-synuclein oligomers are potent fusion inhibitors. However, the function of normal α-synuclein has been elusive. Here, we use the single vesicle-to-supported bilayer fusion assay to dissect the role of α-synuclein in membrane fusion. The assay employs 10 kD Rhodamine B-dextran as the content probe that can detect fusion pores larger than ∼6 nm. We find that the SNARE complex alone is inefficient at dilating fusion pores. However, α-synuclein dramatically increases the probability as well as the duration of large pores. When the SNARE-interacting C-terminal region of α-synuclein was truncated, the mutant behaves the same as the wild-type. However, the double proline mutants compromising membrane-binding show significantly reduced effects on fusion pore expansion. Thus, our results suggest that α-synuclein stimulates fusion pore expansion specifically through its membrane binding.

scholarly journals Fusion pore expansion is a slow, discontinuous, and Ca2+-dependent process regulating secretion from alveolar type II cells

2001 ◽  
Vol 155 (2) ◽  
pp. 279-290 ◽  
Author(s):  
Thomas Haller ◽  
Paul Dietl ◽  
Kristian Pfaller ◽  
Manfred Frick ◽  
Norbert Mair ◽  
...  
Keyword(s):  
Pore Diameter ◽  
Fusion Pore ◽  
Alveolar Type ◽  
Type Ii Cells ◽  
Type Ii ◽  
Alveolar Type Ii Cells ◽  
Intact Cells ◽  
Fusion Pores ◽  
Pyridinium Dibromide ◽  
Pore Expansion

In alveolar type II cells, the release of surfactant is considerably delayed after the formation of exocytotic fusion pores, suggesting that content dispersal may be limited by fusion pore diameter and subject to regulation at a postfusion level. To address this issue, we used confocal FRAP and N-(3-triethylammoniumpropyl)-4-(4-[dibutylamino]styryl) pyridinium dibromide (FM 1-43), a dye yielding intense localized fluorescence of surfactant when entering the vesicle lumen through the fusion pore (Haller, T., J. Ortmayr, F. Friedrich, H. Volkl, and P. Dietl. 1998. Proc. Natl. Acad. Sci. USA. 95:1579–1584). Thus, we have been able to monitor the dynamics of individual fusion pores up to hours in intact cells, and to calculate pore diameters using a diffusion model derived from Fick's law. After formation, fusion pores were arrested in a state impeding the release of vesicle contents, and expanded at irregular times thereafter. The expansion rate of initial pores and the probability of late expansions were increased by elevation of the cytoplasmic Ca2+ concentration. Consistently, content release correlated with the occurrence of Ca2+ oscillations in ATP-treated cells, and expanded fusion pores were detectable by EM. This study supports a new concept in exocytosis, implicating fusion pores in the regulation of content release for extended periods after initial formation.

scholarly journals The role of the C terminus of the SNARE protein SNAP-25 in fusion pore opening and a model for fusion pore mechanics

2008 ◽  
Vol 105 (40) ◽  
pp. 15388-15392 ◽  
Author(s):  
Qinghua Fang ◽  
Khajak Berberian ◽  
Liang-Wei Gong ◽  
Ismail Hafez ◽  
Jakob B. Sørensen ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Mechanistic Model ◽  
Snare Complex ◽  
Fusion Pore ◽  
Snare Protein ◽  
C Terminus ◽  
Target Membrane ◽  
Pore Opening ◽  
Fusion Pores

Formation of a fusion pore between a vesicle and its target membrane is thought to involve the so-called SNARE protein complex. However, there is no mechanistic model explaining how the fusion pore is opened by conformational changes in the SNARE complex. It has been suggested that C-terminal zipping triggers fusion pore opening. A SNAP-25 mutant named SNAP-25Δ9 (lacking the last nine C-terminal residues) should lead to a less-tight C-terminal zipping. Single exocytotic events in chromaffin cells expressing this mutant were characterized by carbon fiber amperometry and cell-attached patch capacitance measurements. Cells expressing SNAP-25Δ9 displayed smaller amperometric “foot-current” currents, reduced fusion pore conductances, and lower fusion pore expansion rates. We propose that SNARE/lipid complexes form proteolipid fusion pores. Fusion pores involving the SNAP-25Δ9 mutant will be less tightly zipped and may lead to a longer fusion pore structure, consistent with the observed decrease of fusion pore conductance.

scholarly journals Intracellular curvature-generating proteins in cell-to-cell fusion

2011 ◽  
Vol 440 (2) ◽  
pp. 185-193 ◽  
Author(s):  
Jean-Philippe Richard ◽  
Evgenia Leikina ◽  
Ralf Langen ◽  
William Mike Henne ◽  
Margarita Popova ◽  
...  
Keyword(s):  
Cell Fusion ◽  
Cell Metabolism ◽  
Syncytium Formation ◽  
Dominant Negative ◽  
Fusion Pore ◽  
Intracellular Membrane ◽  
Expansion Stage ◽  
Membrane Bending ◽  
Fusion Pores ◽  
Pore Expansion

Cell-to-cell fusion plays an important role in normal physiology and in different pathological conditions. Early fusion stages mediated by specialized proteins and yielding fusion pores are followed by a pore expansion stage that is dependent on cell metabolism and yet unidentified machinery. Because of a similarity of membrane bending in the fusion pore rim and in highly curved intracellular membrane compartments, in the present study we explored whether changes in the activity of the proteins that generate these compartments affect cell fusion initiated by protein fusogens of influenza virus and baculovirus. We raised the intracellular concentration of curvature-generating proteins in cells by either expressing or microinjecting the ENTH (epsin N-terminal homology) domain of epsin or by expressing the GRAF1 (GTPase regulator associated with focal adhesion kinase 1) BAR (Bin/amphiphysin/Rvs) domain or the FCHo2 (FCH domain-only protein 2) F-BAR domain. Each of these treatments promoted syncytium formation. Cell fusion extents were also influenced by treatments targeting the function of another curvature-generating protein, dynamin. Cell-membrane-permeant inhibitors of dynamin GTPase blocked expansion of fusion pores and dominant-negative mutants of dynamin influenced the syncytium formation extents. We also report that syncytium formation is inhibited by reagents lowering the content and accessibility of PtdIns(4,5)P2, an important regulator of intracellular membrane remodelling. Our findings indicate that fusion pore expansion at late stages of cell-to-cell fusion is mediated, directly or indirectly, by intracellular membrane-shaping proteins.

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 Temperature Dependence of Fusion Kinetics and Fusion Pores in Ca2+-triggered Exocytosis from PC12 Cells

2008 ◽  
Vol 131 (2) ◽  
pp. 117-124 ◽  
Author(s):  
Zhen Zhang ◽  
Meyer B. Jackson
Keyword(s):  
Temperature Dependence ◽  
Pc12 Cells ◽  
Bulk Diffusion ◽  
Fusion Pore ◽  
Spike Shape ◽  
Fusion Probability ◽  
Temperature Dependences ◽  
Membrane Components ◽  
Fusion Pores ◽  
Single Vesicle

The temperature dependence of Ca2+-triggered exocytosis was studied using carbon fiber amperometry to record the release of norepinephrine from PC12 cells. Single-vesicle fusion events were examined at temperatures varying from 12 to 28°C, and with release elicited by depolarization. Measurements were made of the initial and maximum frequencies of exocytotic events, of fusion pore lifetime, flux through the open fusion pore, kiss-and-run versus full-fusion probability, and parameters associated with the shapes of amperometric spikes. The fusion pore open-state flux, and all parameters associated with spike shape, including area, rise time, and decay time, had weak temperature dependences and activation energies in the range expected for bulk diffusion in an aqueous solution. Kiss-and-run events also varied with temperature, with lower temperatures increasing the relative probability of kiss-and-run events by ∼50%. By contrast, kinetic parameters relating to the frequency of exocytotic events and fusion pore transitions depended much more strongly on temperature, suggesting that these processes entail structural rearrangements of proteins or lipids or both. The weak temperature dependence of spike shape suggests that after the fusion pore has started to expand, structural transitions of membrane components are no longer kinetically limiting. This indicates that the content of a vesicle is expelled completely after fusion pore expansion.

scholarly journals ‘Full fusion’ is not ineluctable during vesicular exocytosis of neurotransmitters by endocrine cells

2017 ◽  
Vol 473 (2197) ◽  
pp. 20160684 ◽  
Author(s):  
Alexander Oleinick ◽  
Irina Svir ◽  
Christian Amatore
Keyword(s):  
Cell Membrane ◽  
Endocrine Cells ◽  
Fusion Pore ◽  
Spike Time ◽  
Vesicle Membrane ◽  
Analytical Formulation ◽  
Vesicular Exocytosis ◽  
Time Courses ◽  
Fusion Pores ◽  
Pore Expansion

Vesicular exocytosis is an essential and ubiquitous process in neurons and endocrine cells by which neurotransmitters are released in synaptic clefts or extracellular fluids. It involves the fusion of a vesicle loaded with chemical messengers with the cell membrane through a nanometric fusion pore. In endocrine cells, unless it closes after some flickering (‘Kiss-and-Run’ events), this initial pore is supposed to expand exponentially, leading to a full integration of the vesicle membrane into the cell membrane—a stage called ‘full fusion’. We report here a compact analytical formulation that allows precise measurements of the fusion pore expansion extent and rate to be extracted from individual amperometric spike time courses. These data definitively establish that, during release of catecholamines, fusion pores enlarge at most to approximately one-fifth of the radius of their parent vesicle, hence ruling out the ineluctability of ‘full fusion’.

scholarly journals Deletion of the Cytoplasmic Tail of the Fusion Protein of the Paramyxovirus Simian Virus 5 Affects Fusion Pore Enlargement

2001 ◽  
Vol 75 (11) ◽  
pp. 5363-5369 ◽  
Author(s):  
Rebecca Ellis Dutch ◽  
Robert A. Lamb
Keyword(s):  
Cytoplasmic Tail ◽  
Simian Virus ◽  
Fusion Pore ◽  
Wild Type ◽  
Direct Role ◽  
F Protein ◽  
Simian Virus 5 ◽  
Fusion Pores ◽  
Pore Enlargement ◽  
Pore Expansion

ABSTRACT The fusion (F) protein of the paramxyovirus simian parainfluenza virus 5 (SV5) promotes virus-cell and cell-cell membrane fusion. Previous work had indicated that removal of the SV5 F protein cytoplasmic tail (F Tail− or FΔ19) caused a block in fusion promotion at the hemifusion stage. Further examination has shown that although the F Tail− mutant is severely debilitated in promotion of fusion as measured by using two reporter gene assays and is debilitated in the formation of syncytia relative to the wild-type F protein, the F Tail− mutant is capable of promoting the transfer of small aqueous dyes. These data indicate that F Tail− is fully capable of promoting formation of small fusion pores. However, enlargement of fusion pores is debilitated, suggesting that either the cytoplasmic tail of the F protein plays a direct role in pore expansion or that it interacts with other components which control pore growth.

scholarly journals Dual-mode of insulin action controls GLUT4 vesicle exocytosis

2011 ◽  
Vol 193 (4) ◽  
pp. 643-653 ◽  
Author(s):  
Yingke Xu ◽  
Bradley R. Rubin ◽  
Charisse M. Orme ◽  
Alexander Karpikov ◽  
Chenfei Yu ◽  
...  
Keyword(s):  
Phospholipase D ◽  
Insulin Signaling ◽  
Insulin Action ◽  
Fusion Pore ◽  
Dual Mode ◽  
Vesicle Traffic ◽  
Storage Vesicles ◽  
Vesicle Exocytosis ◽  
Single Vesicle ◽  
Pore Expansion

Insulin stimulates translocation of GLUT4 storage vesicles (GSVs) to the surface of adipocytes, but precisely where insulin acts is controversial. Here we quantify the size, dynamics, and frequency of single vesicle exocytosis in 3T3-L1 adipocytes. We use a new GSV reporter, VAMP2-pHluorin, and bypass insulin signaling by disrupting the GLUT4-retention protein TUG. Remarkably, in unstimulated TUG-depleted cells, the exocytic rate is similar to that in insulin-stimulated control cells. In TUG-depleted cells, insulin triggers a transient, twofold burst of exocytosis. Surprisingly, insulin promotes fusion pore expansion, blocked by acute perturbation of phospholipase D, which reflects both properties intrinsic to the mobilized vesicles and a novel regulatory site at the fusion pore itself. Prolonged stimulation causes cargo to switch from ∼60 nm GSVs to larger exocytic vesicles characteristic of endosomes. Our results support a model whereby insulin promotes exocytic flux primarily by releasing an intracellular brake, but also by accelerating plasma membrane fusion and switching vesicle traffic between two distinct circuits.

scholarly journals The synaptotagmin C2B domain calcium-binding loops modulate the rate of fusion pore expansion

2018 ◽  
Vol 29 (7) ◽  
pp. 834-845 ◽  
Author(s):  
Mounir Bendahmane ◽  
Kevin P. Bohannon ◽  
Mazdak M. Bradberry ◽  
Tejeshwar C. Rao ◽  
Michael W. Schmidtke ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
In Silico ◽  
Calcium Binding ◽  
Chromaffin Cells ◽  
Fusion Pore ◽  
Structural Specificity ◽  
Fusion Pores ◽  
Kinetics Of ◽  
Pore Expansion

In chromaffin cells, the kinetics of fusion pore expansion vary depending on which synaptotagmin isoform (Syt-1 or Syt-7) drives release. Our recent studies have shown that fusion pores of granules harboring Syt-1 expand more rapidly than those harboring Syt-7. Here we sought to define the structural specificity of synaptotagmin action at the fusion pore by manipulating the Ca2+-binding C2B module. We generated a chimeric Syt-1 in which its C2B Ca2+-binding loops had been exchanged for those of Syt-7. Fusion pores of granules harboring a Syt-1 C2B chimera with all three Ca2+-binding loops of Syt-7 (Syt-1:7C2B123) exhibited slower rates of fusion pore expansion and neuropeptide cargo release relative to WT Syt-1. After fusion, this chimera also dispersed more slowly from fusion sites than WT protein. We speculate that the Syt-1:7 C2B123 and WT Syt-1 are likely to differ in their interactions with Ca2+ and membranes. Subsequent in vitro and in silico data demonstrated that the chimera exhibits a higher affinity for phospholipids than WT Syt-1. We conclude that the affinity of synaptotagmin for the plasma membrane, and the rate at which it releases the membrane, contribute in important ways to the rate of fusion pore expansion.

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