scholarly journals Molecular mechanism of fusion pore formation driven by the neuronal SNARE complex

2018 ◽  
Vol 115 (50) ◽  
pp. 12751-12756 ◽  
Author(s):  
Satyan Sharma ◽  
Manfred Lindau
Keyword(s):  
Pore Formation ◽  
Structural Model ◽  
Snare Complex ◽  
Coarse Grained ◽  
Fusion Pore ◽  
Protein Mutants ◽  
Experimental Values ◽  
Dynamics Simulations ◽  
External Forces ◽  
Fusion Pores

Release of neurotransmitters from synaptic vesicles begins with a narrow fusion pore, the structure of which remains unresolved. To obtain a structural model of the fusion pore, we performed coarse-grained molecular dynamics simulations of fusion between a nanodisc and a planar bilayer bridged by four partially unzipped SNARE complexes. The simulations revealed that zipping of SNARE complexes pulls the polar C-terminal residues of the synaptobrevin 2 and syntaxin 1A transmembrane domains to form a hydrophilic core between the two distal leaflets, inducing fusion pore formation. The estimated conductances of these fusion pores are in good agreement with experimental values. Two SNARE protein mutants inhibiting fusion experimentally produced no fusion pore formation. In simulations in which the nanodisc was replaced by a 40-nm vesicle, an extended hemifusion diaphragm formed but a fusion pore did not, indicating that restricted SNARE mobility is required for rapid fusion pore formation. Accordingly, rapid fusion pore formation also occurred in the 40-nm vesicle system when SNARE mobility was restricted by external forces. Removal of the restriction is required for fusion pore expansion.

scholarly journals The fusion kinetics of influenza hemagglutinin expressing cells to planar bilayer membranes is affected by HA density and host cell surface.

1995 ◽  
Vol 106 (5) ◽  
pp. 783-802 ◽  
Author(s):  
G B Melikyan ◽  
W D Niles ◽  
F S Cohen
Keyword(s):  
Cell Surface ◽  
Fusion Protein ◽  
Pore Formation ◽  
Surface Proteins ◽  
Fusion Pore ◽  
Planar Bilayer ◽  
Bilayer Membranes ◽  
Pore Evolution ◽  
Fusion Pores ◽  
Kinetics Of

Time-resolved admittance measurements were used to follow formation of individual fusion pores connecting influenza virus hemagglutinin (HA)-expressing cells to planar bilayer membranes. By measuring in-phase, out-of-phase, and dc components of currents, pore conductances were resolved with millisecond time resolution. Fusion pores developed in stages, from small pores flickering open and closed, to small successful pores that remained open until enlarging their lumens to sizes greater than those of viral nucleocapsids. The kinetics of fusion and the properties of fusion pores were studied as functions of density of the fusion protein HA. The consequences of treating cell surfaces with proteases that do not affect HA were also investigated. Fusion kinetics were described by waiting time distributions from triggering fusion, by lowering pH, to the moment of pore formation. The kinetics of pore formation became faster as the density of active HA was made greater or when cell surface proteins were extensively cleaved with proteases. In accord with this faster kinetics, the intervals between transient pore openings within the flickering stage were shorter for higher HA density and more extensive cell surface treatment. Whereas the kinetics of fusion depended on HA density, the lifetimes of open fusion pores were independent of HA density. However, the lifetimes of open pores were affected by the proteolytic treatment of the cells. Faster fusion kinetics correlated with shorter pore openings. We conclude that the density of fusion protein strongly affects the kinetics of fusion pore formation, but that once formed, pore evolution is not under control of fusion proteins but rather under the influence of mechanical forces, such as membrane bending and tension.

scholarly journals HIV-1 Envelope Proteins Complete Their Folding into Six-helix Bundles Immediately after Fusion Pore Formation

2003 ◽  
Vol 14 (3) ◽  
pp. 926-938 ◽  
Author(s):  
Ruben M. Markosyan ◽  
Fredric S. Cohen ◽  
Grigory B. Melikyan
Keyword(s):  
Fusion Proteins ◽  
Pore Formation ◽  
Temperature Jump ◽  
Fusion Pore ◽  
Target Cells ◽  
Pore Growth ◽  
Essential Function ◽  
Intermediate Value ◽  
Fusion Pores ◽  
Hiv 1

Fusion proteins of many viruses, including HIV-1 envelope protein (Env), fold into six-helix bundle structures. Fusion between individual Env-expressing cells and target cells was studied by fluorescence microscopy, and a temperature jump technique, to determine whether folding of Env into a bundle is complete by the time fusion pores have formed. Lowering temperature to 4°C immediately after a pore opened halted pore growth, which quickly resumed when temperature was raised again. HIV gp41-derived peptides that inhibit bundle formation (C34 or N36) caused the cold-arrested pore to quickly and irreversibly close, demonstrating that bundle formation is not complete by the time a pore has formed. In contrast, lowering the temperature to an intermediate value also halted pore growth, but the pore was not closed by the bundle-inhibiting peptides, and it enlarged when temperature was again elevated. This latter result shows that bundle formation is definitely required for the fusion process, but surprisingly, some (if not all) bundle formation occurs after a pore has formed. It is concluded that an essential function of the bundle is to stabilize the pore against collapse and ensure its growth.

scholarly journals Fusion Pore Formation Observed During SNARE-Mediated Vesicle Fusion with Pore-Spanning Membranes

2020 ◽  
Author(s):  
P. Mühlenbrock ◽  
K. Herwig ◽  
L. Vuong ◽  
I. Mey ◽  
C. Steinem
Keyword(s):  
Fluorescence Microscopy ◽  
Pore Formation ◽  
Three Dimensional ◽  
Rare Event ◽  
Vesicle Fusion ◽  
Fusion Process ◽  
Fusion Pore ◽  
Dual Color ◽  
Fusion Pores

ABSTRACTPlanar pore-spanning membranes (PSMs) have been shown to be a versatile tool to resolve docking and elementary steps of the fusion process with single large unilamellar vesicles (LUVs). However, in previous studies, we monitored only lipid mixing and did not gather information about the formation of fusion pores. To address this important step of the fusion process, we entrapped sulforhodamine B at self-quenching concentrations into LUVs containing the v-SNARE synaptobrevin 2, which were docked and fused with lipid-labeled PSMs containing the t-SNARE acceptor complex ΔN49 prepared on porous silicon substrates. By dual color spinning disc fluorescence microcopy with a time resolution of 20 ms, we could unambiguously distinguish between bursting vesicles and fusion pore formation. Owing to the aqueous compartment underneath the PSMs, vesicle bursting turned out to be an extremely rare event (< 0.01 %). From the time-resolved dual color fluorescence time traces, we were able to identify different fusion pathways including remaining three-dimensional postfusion structures with released content and flickering fusion pores. Our results on fusion pore formation and lipid diffusion from the PSM into the fusing vesicle let us conclude that the content release, i.e., fusion pore formation follows the merger of the two lipid membranes by only about 40 ms.STATEMENT OF SIGNIFICANCEDespite great efforts to develop in vitro fusion assays to understand the process of neuronal fusion, there is still a huge demand to provide single vesicle fusion assays that simultaneously report on all intermediate states including three-dimensional postfusion structures and fusion pore formation including flickering pores without the underlying artifact of vesicle bursting. Here, we show that pore-spanning membranes (PSMs) are ideal candidates to fulfill these demands. Owing to their planarity and the second aqueous compartments, they are readily accessible by fluorescence microscopy and provide sufficient space so that vesicle bursting becomes negligible. Dual color fluorescence microscopy allows distinguishing between different fusion intermediates and fusion pathways such as “kiss and run” fusion as well as flickering fusion pores.

scholarly journals The neuronal calcium sensor Synaptotagmin-1 and SNARE proteins cooperate to dilate fusion pores

2019 ◽  
Author(s):  
Zhenyong Wu ◽  
Nadiv Dharan ◽  
Sathish Thiyagarajan ◽  
Ben O’Shaughnessy ◽  
Erdem Karatekin
Keyword(s):  
Membrane Fusion ◽  
Snare Complex ◽  
Snare Proteins ◽  
Fusion Pore ◽  
Calcium Sensor ◽  
Fusion Reactions ◽  
Synaptotagmin 1 ◽  
Pore Opening ◽  
Pore Dilation ◽  
Fusion Pores

ABSTRACTAll membrane fusion reactions proceed through an initial fusion pore, including calcium-triggered vesicular release of neurotransmitters and hormones. Expansion of this small pore to release cargo molecules is energetically costly and regulated by cells, but the mechanisms are poorly understood. Here we show that the neuronal/exocytic calcium sensor Synaptotagmin-1 (Syt1) promotes expansion of fusion pores induced by SNARE proteins, beyond its established role in coupling calcium influx to fusion pore opening. Our results suggest that fusion pore dilation by Syt1 requires interactions with SNAREs, PI(4,5)P2, and calcium. Pore opening was abolished by a mutation of the tandem C2 domain (C2AB) hydrophobic loops of Syt1, suggesting that their calcium-induced insertion into the membrane is required for pore opening. We propose that loop insertion is also required for pore expansion, but through a distinct mechanism. Mathematical modelling suggests that membrane insertion re-orients the C2 domains bound to the SNARE complex, rotating the SNARE complex so as to exert force on the membranes in a mechanical lever action that increases the intermembrane distance. The increased membrane separation provokes pore dilation to offset a bending energy penalty. We conclude that Syt1 assumes a critical role in calcium-dependent fusion pore dilation during neurotransmitter and hormone release.SIGNIFICANCE STATEMENTMembrane fusion is a fundamental biological process, required for development, infection by enveloped viruses, fertilization, intracellular trafficking, and calcium-triggered release of neurotransmitters and hormones when cargo-laden vesicles fuse with the plasma membrane. All membrane fusion reactions proceed through an initial, nanometer-sized fusion pore which can flicker open-closed multiple times before expanding or resealing. Pore expansion is required for efficient cargo release, but underlying mechanisms are poorly understood. Using a combination of single-pore measurements and quantitative modeling, we suggest that a complex between the neuronal calcium sensor Synaptotagmin-1 and the SNARE proteins together act as a calcium-sensitive mechanical lever to force the membranes apart and enlarge the pore.

scholarly journals How Could SNARE Proteins Open a Fusion Pore?

Physiology ◽  
2014 ◽  
Vol 29 (4) ◽  
pp. 278-285 ◽  
Author(s):  
Qinghua Fang ◽  
Manfred Lindau
Keyword(s):  
Conformational Change ◽  
Pore Formation ◽  
Vesicle Fusion ◽  
Neurosecretory Cells ◽  
Snare Complex ◽  
Snare Proteins ◽  
Fusion Pore ◽  
Receptor Complex ◽  
Attachment Protein ◽  
Protein Receptor

The SNARE (Soluble NSF Attachment protein REceptor) complex, which in mammalian neurosecretory cells is composed of the proteins synaptobrevin 2 (also called VAMP2), syntaxin, and SNAP-25, plays a key role in vesicle fusion. In this review, we discuss the hypothesis that, in neurosecretory cells, fusion pore formation is directly accomplished by a conformational change in the SNARE complex via movement of the transmembrane domains.

scholarly journals Inner but Not Outer Membrane Leaflets Control the Transition from Glycosylphosphatidylinositol-anchored Influenza Hemagglutinin-induced Hemifusion to Full Fusion

1997 ◽  
Vol 136 (5) ◽  
pp. 995-1005 ◽  
Author(s):  
Grigory B. Melikyan ◽  
Sofya A. Brener ◽  
Dong C. Ok ◽  
Fredric S. Cohen
Keyword(s):  
Outer Membrane ◽  
Negative Curvature ◽  
Pore Formation ◽  
Transmembrane Domain ◽  
Positive Curvature ◽  
Fusion Pore ◽  
Spontaneous Curvature ◽  
Wild Type ◽  
Influenza Hemagglutinin ◽  
Fusion Pores

Cells that express wild-type influenza hemagglutinin (HA) fully fuse to RBCs, while cells that express the HA-ectodomain anchored to membranes by glycosylphosphatidylinositol, rather than by a transmembrane domain, only hemifuse to RBCs. Amphipaths were inserted into inner and outer membrane leaflets to determine the contribution of each leaflet in the transition from hemifusion to fusion. When inserted into outer leaflets, amphipaths did not promote the transition, independent of whether the agent induces monolayers to bend outward (conferring positive spontaneous monolayer curvature) or inward (negative curvature). In contrast, when incorporated into inner leaflets, positive curvature agents led to full fusion. This suggests that fusion is completed when a lipidic fusion pore with net positive curvature is formed by the inner leaflets that compose a hemifusion diaphragm. Suboptimal fusion conditions were established for RBCs bound to cells expressing wild-type HA so that lipid but not aqueous dye spread was observed. While this is the same pattern of dye spread as in stable hemifusion, for this “stunted” fusion, lower concentrations of amphipaths in inner leaflets were required to promote transfer of aqueous dyes. Also, these amphipaths induced larger pores for stunted fusion than they generated within a stable hemifusion diaphragm. Therefore, spontaneous curvature of inner leaflets can affect formation and enlargement of fusion pores induced by HA. We propose that after the HA-ectodomain induces hemifusion, the transmembrane domain causes pore formation by conferring positive spontaneous curvature to leaflets of the hemifusion diaphragm.

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 Mechanistic basis for motor-driven membrane constriction by dynamin

2020 ◽  
Author(s):  
Oleg Ganichkin ◽  
Renee Vancraenenbroeck ◽  
Gabriel Rosenblum ◽  
Hagen Hofmann ◽  
Alexander S. Mikhailov ◽  
...  
Keyword(s):  
Single Molecule ◽  
Structural Model ◽  
Molecular Motor ◽  
Coarse Grained ◽  
Gtp Hydrolysis ◽  
Single Molecule Fret ◽  
Membrane Tube ◽  
Mechanistic Basis ◽  
Dynamics Simulations ◽  
Gtpase Cycle

AbstractThe mechano-chemical GTPase dynamin assembles on membrane necks of clathrin-coated vesicles into helical oligomers that constrict and eventually cleave the necks in a GTP-dependent way. It remains not clear whether dynamin achieves this via molecular motor activity and, if so, by what mechanism. Here, we used ensemble kinetics, single-molecule FRET and molecular dynamics simulations to characterize dynamin’s GTPase cycle and determine the powerstroke strength. The results were incorporated into a coarse-grained structural model of dynamin filaments on realistic membrane templates. Working asynchronously, dynamin’s motor modules were found to collectively constrict a membrane tube. Force is generated by motor dimers linking adjacent helical turns and constriction is accelerated by their strain-dependent dissociation. Consistent with experiments, less than a second is needed to constrict a membrane tube to the hemi-fission radius. Thus, a membrane remodeling mechanism relying on cooperation of molecular ratchet motors driven by GTP hydrolysis has been revealed.

scholarly journals Structural transitions in the synaptic SNARE complex during Ca2+-triggered exocytosis

2006 ◽  
Vol 172 (2) ◽  
pp. 281-293 ◽  
Author(s):  
Xue Han ◽  
Meyer B. Jackson
Keyword(s):  
Plasma Membranes ◽  
Structural Transition ◽  
Snare Complex ◽  
Fusion Pore ◽  
Hydrophobic Core ◽  
Structural Transitions ◽  
Triggered Release ◽  
Rate Processes ◽  
Pore Opening ◽  
Fusion Pores

The synaptic SNARE complex is a highly stable four-helix bundle that links the vesicle and plasma membranes and plays an essential role in the Ca2+-triggered release of neurotransmitters and hormones. An understanding has yet to be achieved of how this complex assembles and undergoes structural transitions during exocytosis. To investigate this question, we have mutated residues within the hydrophobic core of the SNARE complex along the entire length of all four chains and examined the consequences using amperometry to measure fusion pore opening and dilation. Mutations throughout the SNARE complex reduced two distinct rate processes before fusion pore opening to different degrees. These results suggest that two distinct, fully assembled conformations of the SNARE complex drive transitions leading to open fusion pores. In contrast, a smaller number of mutations that were scattered through the SNARE complex but were somewhat concentrated in the membrane-distal half stabilized open fusion pores. These results suggest that a structural transition within a partially disassembled complex drives the dilation of open fusion pores. The dependence of these three rate processes on position within the SNARE complex does not support vectorial SNARE complex zipping during exocytosis.

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