scholarly journals Nascent fusion pore opening monitored at single-SNAREpin resolution

2021 ◽  
Vol 118 (5) ◽  
pp. e2024922118
Author(s):  
Paul Heo ◽  
Jeff Coleman ◽  
Jean-Baptiste Fleury ◽  
James E. Rothman ◽  
Frederic Pincet
Keyword(s):  
Energy Landscape ◽  
Three Dimensional ◽  
Fusion Pore ◽  
Fusion Event ◽  
Direct Estimate ◽  
Target Membrane ◽  
Pore Opening ◽  
Two Peaks ◽  
Fusion Pores ◽  
Second Peak

Vesicle fusion with a target membrane is a key event in cellular trafficking and ensures cargo transport within the cell and between cells. The formation of a protein complex, called SNAREpin, provides the energy necessary for the fusion process. In a three-dimensional microfluidic chip, we monitored the fusion of small vesicles with a suspended asymmetric lipid bilayer. Adding ion channels into the vesicles, our setup allows the observation of a single fusion event by electrophysiology with 10-μs precision. Intriguingly, we identified that small transient fusion pores of discrete sizes reversibly opened with a characteristic lifetime of ∼350 ms. The distribution of their apparent diameters displayed two peaks, at 0.4 ± 0.1 nm and 0.8 ± 0.2 nm. Varying the number of SNAREpins, we demonstrated that the first peak corresponds to fusion pores induced by a single SNAREpin and the second peak is associated with pores involving two SNAREpins acting simultaneously. The pore size fluctuations provide a direct estimate of the energy landscape of the pore. By extrapolation, the energy landscape for three SNAREpins does not exhibit any thermally significant energy barrier, showing that pores larger than 1.5 nm are spontaneously produced by three or more SNAREpins acting simultaneously, and expand indefinitely. Our results quantitatively explain why one SNAREpin is sufficient to open a fusion pore and more than three SNAREpins are required for cargo release. Finally, they also explain why a machinery that synchronizes three SNAREpins, or more, is mandatory to ensure fast neurotransmitter release during synaptic transmission.

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 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 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.

scholarly journals The initial fusion pore induced by baculovirus GP64 is large and forms quickly.

1996 ◽  
Vol 135 (6) ◽  
pp. 1831-1839 ◽  
Author(s):  
I Plonsky ◽  
J Zimmerberg
Keyword(s):  
Wide Distribution ◽  
Fusion Pore ◽  
High Time Resolution ◽  
Viral Fusion ◽  
Time Resolved ◽  
Cell Recording ◽  
Resolution Model ◽  
Pore Opening ◽  
Lag Times ◽  
Fusion Pores

The formation of the fusion pore is the first detectable event in membrane fusion (Zimmerberg, J., R. Blumenthal, D.P. Sarkar, M. Curran, and S.J. Morris. 1994. J. Cell Biol. 127:1885-1894). To date, fusion pores measured in exocytosis and viral fusion have shared features that include reversible closure (flickering), highly fluctuating semistable stages, and a lag time of at least several seconds between the triggering and the pore opening. We investigated baculovirus GP64-induced Sf9 cell-cell fusion, triggered by external acid solution, using two different electrophysiological techniques: double whole-cell recording (for high time resolution, model-independent measurements), and the more conventional time-resolved admittance recordings. Both methods gave essentially the same results, thus validating the use of the admittance measurements for fusion pore conductance calculations. Fusion was first detected by abrupt pore formation with a wide distribution of initial conductance, centered around 1 nS. Often the initial fusion pore conductance was stable for many seconds. Fluctuations in semistable conductances were much less than those of other fusion pores. The waiting time distribution, measured between pH onset and initial pore appearance, fits best to a model with many (approximately 19) independent elements. Thus, unlike previously measured fusion pores, GP64-mediated pores do not flicker, can have large, stable initial pore conductances lasting up to a minute, and have typical lag times of &lt; 1 s. These findings are consistent with a barrel-shaped model of an initial fusion pore consisting of five to eight GP64 trimers that is lined with lipid.

scholarly journals Phosphatidylserine Regulation of Ca2+-triggered Exocytosis and Fusion Pores in PC12 Cells

2009 ◽  
Vol 20 (24) ◽  
pp. 5086-5095 ◽  
Author(s):  
Zhen Zhang ◽  
Enfu Hui ◽  
Edwin R. Chapman ◽  
Meyer B. Jackson
Keyword(s):  
Pc12 Cells ◽  
Gradual Increase ◽  
Fusion Pore ◽  
Dependent Manner ◽  
Synaptotagmin I ◽  
Pore Opening ◽  
Kinetic Effects ◽  
Two Phases ◽  
Fusion Pores ◽  
Kinetics Of

The synaptic vesicle protein synaptotagmin I (Syt I) binds phosphatidylserine (PS) in a Ca2+-dependent manner. This interaction is thought to play a role in exocytosis, but its precise functions remain unclear. To determine potential roles for Syt I-PS binding, we varied the PS content in PC12 cells and liposomes and studied the effects on the kinetics of exocytosis and Syt I binding in parallel. Raising PS produced a steeply nonlinear, saturating increase in Ca2+-triggered fusion, and a graded slowing of the rate of fusion pore dilation. Ca2+-Syt I bound liposomes more tightly as PS content was raised, with a steep increase in binding at low PS, and a further gradual increase at higher PS. These two phases in the PS dependence of Ca2+-dependent Syt I binding to lipid may correspond to the two distinct and opposing kinetic effects of PS on exocytosis. PS influences exocytosis in two ways, enhancing an early step leading to fusion pore opening, and slowing a later step when fusion pores dilate. The possible relevance of these results to Ca2+-triggered Syt I binding is discussed along with other possible roles of PS.

scholarly journals During entry of alphaviruses, the E1 glycoprotein molecules probably form two separate populations that generate either a fusion pore or ion-permeable pores

2004 ◽  
Vol 85 (6) ◽  
pp. 1695-1701 ◽  
Author(s):  
Gerd Wengler ◽  
Andreas Koschinski ◽  
Gisela Wengler ◽  
Holger Repp
Keyword(s):  
Fusion Protein ◽  
Semliki Forest Virus ◽  
Fusion Pore ◽  
Viral Membrane ◽  
Endosomal Membrane ◽  
Target Membrane ◽  
Membrane Reaction ◽  
Fusion Pores

Studies using the alphavirus Semliki Forest virus have indicated that the viral E1 fusion protein forms two types of pore: fusion pores and ion-permeable pores. The formation of ion-permeable pores has not been generally accepted, partly because it was not evident how the protein might form these different pores. Here it is proposed that the choice of the target membrane determines whether a fusion pore or ion-permeable pores are formed. The fusion protein is activated in the endosome and for steric reasons only a fraction of the activated molecules can interact with the endosomal membrane. This target membrane reaction forms the fusion pore. It is proposed that the rest of the activated molecules interact with the membrane in which the protein is anchored and that this self-membrane reaction leads to formation of ion-permeable pores, which can be detected in the target membrane after fusion of the viral membrane into the target membrane.

scholarly journals Reversible Merger of Membranes at the Early Stage of Influenza Hemagglutinin-mediated Fusion

2000 ◽  
Vol 11 (7) ◽  
pp. 2359-2371 ◽  
Author(s):  
Eugenia Leikina ◽  
Leonid V. Chernomordik
Keyword(s):  
Fusion Protein ◽  
Surface Density ◽  
Early Stage ◽  
Low Ph ◽  
Fusion Pore ◽  
Complete Fusion ◽  
Physiological Temperature ◽  
Influenza Hemagglutinin ◽  
Pore Opening ◽  
Fusion Pores

Fusion mediated by influenza hemagglutinin (HA), a prototype fusion protein, is commonly detected as lipid and content mixing between fusing cells. Decreasing the surface density of fusion-competent HA inhibited these advanced fusion phenotypes and allowed us to identify an early stage of fusion at physiological temperature. Although lipid flow between membranes was restricted, the contacting membrane monolayers were apparently transiently connected, as detected by the transformation of this fusion intermediate into complete fusion after treatments known to destabilize hemifusion diaphragms. These reversible connections disappeared within 10–20 min after application of low pH, indicating that after the energy released by HA refolding dissipated, the final low pH conformation of HA did not support membrane merger. Although the dynamic character and the lack of lipid mixing at 37°C distinguish the newly identified fusion intermediate from the intermediate arrested at 4°C described previously, both intermediates apparently belong to the same family of restricted hemifusion (RH) structures. Because the formation of transient RH structures at physiological temperatures was as fast as fusion pore opening and required less HA, we hypothesize that fusion starts with the formation of multiple RH sites, only a few of which then evolve to become expanding fusion pores.

scholarly journals Regulation of Exocytosis and Fusion Pores by Synaptotagmin-Effector Interactions

2010 ◽  
Vol 21 (16) ◽  
pp. 2821-2831 ◽  
Author(s):  
Zhen Zhang ◽  
Enfu Hui ◽  
Edwin R. Chapman ◽  
Meyer B. Jackson
Keyword(s):  
Pc12 Cells ◽  
Lipid Bilayers ◽  
Fusion Pore ◽  
Fusion Event ◽  
Event Frequency ◽  
Synaptic Release ◽  
Sensitive Factor ◽  
Fusion Pores ◽  
Kinetics Of ◽  
Stable Fusion

Synaptotagmin (syt) serves as a Ca2+ sensor in the release of neurotransmitters and hormones. This function depends on the ability of syt to interact with other molecules. Syt binds to phosphatidylserine (PS)-containing lipid bilayers as well as to soluble N-ethylmaleimide sensitive factor receptors (SNAREs) and promotes SNARE assembly. All these interactions are regulated by Ca2+, but their specific roles in distinct kinetic steps of exocytosis are not well understood. To explore these questions we used amperometry recording from PC12 cells to investigate the kinetics of exocytosis. Syt isoforms and syt I mutants were overexpressed to perturb syt-PS and syt-SNARE interactions to varying degrees and evaluate the effects on fusion event frequency and the rates of fusion pore transitions. Syt I produced more rapid dilation of fusion pores than syt VII or syt IX, consistent with its role in synchronous synaptic release. Stronger syt-PS interactions were accompanied by a higher frequency of fusion events and more stable fusion pores. By contrast, syt-SNARE interactions and syt-induced SNARE assembly were uncorrelated with rates of exocytosis. This associates the syt-PS interaction with two distinct kinetic steps in Ca2+ triggered exocytosis and supports a role for the syt-PS interaction in stabilizing open fusion pores.

scholarly journals Activity-evoked and spontaneous opening of synaptic fusion pores

2019 ◽  
Vol 116 (34) ◽  
pp. 17039-17044 ◽  
Author(s):  
Dinara Bulgari ◽  
David L. Deitcher ◽  
Brigitte F. Schmidt ◽  
M. Alexandra Carpenter ◽  
Christopher Szent-Gyorgyi ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Active Zone ◽  
Transmitter Release ◽  
Synaptic Vesicles ◽  
Pain Perception ◽  
Fusion Pore ◽  
Synaptic Release ◽  
Pore Opening ◽  
Active Zones ◽  
Fusion Pores

Synaptic release of neuropeptides packaged in dense-core vesicles (DCVs) regulates synapses, circuits, and behaviors including feeding, sleeping, and pain perception. Here, synaptic DCV fusion pore openings are imaged without interference from cotransmitting small synaptic vesicles (SSVs) with the use of a fluorogen-activating protein (FAP). Activity-evoked kiss and run exocytosis opens synaptic DCV fusion pores away from active zones that readily conduct molecules larger than most native neuropeptides (i.e., molecular weight [MW] up to, at least, 4.5 kDa). Remarkably, these synaptic fusion pores also open spontaneously in the absence of stimulation and extracellular Ca2+. SNARE perturbations demonstrate different mechanisms for activity-evoked and spontaneous fusion pore openings with the latter sharing features of spontaneous small molecule transmitter release by active zone-associated SSVs. Fusion pore opening at resting synapses provides a mechanism for activity-independent peptidergic transmission.

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