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 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 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 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 Synaptotagmin Expands Membrane Fusion Pore by Facilitating SNARE-Complex Formation

2011 ◽  
Vol 100 (3) ◽  
pp. 185a
Author(s):  
Jiajie Diao ◽  
Janghyun Yoo ◽  
Han-Ki Lee ◽  
Yoosoo Yang ◽  
Dae-Hyuk Kweon ◽  
...  
Keyword(s):  
Complex Formation ◽  
Membrane Fusion ◽  
Snare Complex ◽  
Fusion Pore ◽  
Snare Complex Formation

scholarly journals Sec1p directly stimulates SNARE-mediated membrane fusion in vitro

2004 ◽  
Vol 167 (1) ◽  
pp. 75-85 ◽  
Author(s):  
Brenton L. Scott ◽  
Jeffrey S. Van Komen ◽  
Hassan Irshad ◽  
Song Liu ◽  
Kirilee A. Wilson ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Membrane Fusion ◽  
Membrane Trafficking ◽  
Snare Complex ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Bud Neck ◽  
Precise Role

Sec1 proteins are critical players in membrane trafficking, yet their precise role remains unknown. We have examined the role of Sec1p in the regulation of post-Golgi secretion in Saccharomyces cerevisiae. Indirect immunofluorescence shows that endogenous Sec1p is found primarily at the bud neck in newly budded cells and in patches broadly distributed within the plasma membrane in unbudded cells. Recombinant Sec1p binds strongly to the t-SNARE complex (Sso1p/Sec9c) as well as to the fully assembled ternary SNARE complex (Sso1p/Sec9c;Snc2p), but also binds weakly to free Sso1p. We used recombinant Sec1p to test Sec1p function using a well-characterized SNARE-mediated membrane fusion assay. The addition of Sec1p to a traditional in vitro fusion assay moderately stimulates fusion; however, when Sec1p is allowed to bind to SNAREs before reconstitution, significantly more Sec1p binding is detected and fusion is stimulated in a concentration-dependent manner. These data strongly argue that Sec1p directly stimulates SNARE-mediated membrane fusion.

scholarly journals Membrane lysis during biological membrane fusion: collateral damage by misregulated fusion machines

2008 ◽  
Vol 183 (2) ◽  
pp. 181-186 ◽  
Author(s):  
Alex Engel ◽  
Peter Walter
Keyword(s):  
Membrane Fusion ◽  
Biological Membrane ◽  
Membrane Integrity ◽  
Fusion Pore ◽  
Mechanistic Models ◽  
Collateral Damage ◽  
Vacuole Fusion ◽  
Membrane Lysis

In the canonical model of membrane fusion, the integrity of the fusing membranes is never compromised, preserving the identity of fusing compartments. However, recent molecular simulations provided evidence for a pathway to fusion in which holes in the membrane evolve into a fusion pore. Additionally, two biological membrane fusion models—yeast cell mating and in vitro vacuole fusion—have shown that modifying the composition or altering the relative expression levels of membrane fusion complexes can result in membrane lysis. The convergence of these findings showing membrane integrity loss during biological membrane fusion suggests new mechanistic models for membrane fusion and the role of membrane fusion complexes.

scholarly journals FUS1Regulates the Opening and Expansion of Fusion Pores between Mating Yeast

2006 ◽  
Vol 17 (5) ◽  
pp. 2439-2450 ◽  
Author(s):  
Scott Nolan ◽  
Ann E. Cowan ◽  
Dennis E. Koppel ◽  
Hui Jin ◽  
Eric Grote
Keyword(s):  
Cell Wall ◽  
Plasma Membrane ◽  
Membrane Fusion ◽  
Cell Fusion ◽  
Yeast Cells ◽  
Fusion Pore ◽  
Sudden Increase ◽  
Fluorescent Markers ◽  
Fold Reduction ◽  
Fusion Pores

Mating yeast cells provide a genetically accessible system for the study of cell fusion. The dynamics of fusion pores between yeast cells were analyzed by following the exchange of fluorescent markers between fusion partners. Upon plasma membrane fusion, cytoplasmic GFP and DsRed diffuse between cells at rates proportional to the size of the fusion pore. GFP permeance measurements reveal that a typical fusion pore opens with a burst and then gradually expands. In some mating pairs, a sudden increase in GFP permeance was found, consistent with the opening of a second pore. In contrast, other fusion pores closed after permitting a limited amount of cytoplasmic exchange. Deletion of FUS1 from both mating partners caused a >10-fold reduction in the initial permeance and expansion rate of the fusion pore. Although fus1 mating pairs also have a defect in degrading the cell wall that separates mating partners before plasma membrane fusion, other cell fusion mutants with cell wall remodeling defects had more modest effects on fusion pore permeance. Karyogamy is delayed by >1 h in fus1 mating pairs, possibly as a consequence of retarded fusion pore expansion.

scholarly journals Cholesterol Promotes Hemifusion and Pore Widening in Membrane Fusion Induced by Influenza Hemagglutinin

2008 ◽  
Vol 131 (5) ◽  
pp. 503-513 ◽  
Author(s):  
Subrata Biswas ◽  
Shu-Rong Yin ◽  
Paul S. Blank ◽  
Joshua Zimmerberg
Keyword(s):  
Membrane Fusion ◽  
Membrane Lipid ◽  
Fusion Pore ◽  
Dye Transfer ◽  
Influenza Hemagglutinin ◽  
Human Red Blood Cells ◽  
Cholesterol Levels ◽  
Pore Opening ◽  
Two Stages ◽  
Pore Expansion

Cholesterol-specific interactions that affect membrane fusion were tested for using insect cells; cells that have naturally low cholesterol levels (<4 mol %). Sf9 cells were engineered (HAS cells) to express the hemagglutinin (HA) of the influenza virus X-31 strain. Enrichment of HAS cells with cholesterol reduced the delay between triggering and lipid dye transfer between HAS cells and human red blood cells (RBC), indicating that cholesterol facilitates membrane lipid mixing prior to fusion pore opening. Increased cholesterol also increased aqueous content transfer between HAS cells and RBC over a broad range of HA expression levels, suggesting that cholesterol also favors fusion pore expansion. This interpretation was tested using both trans-cell dye diffusion and fusion pore conductivity measurements in cholesterol-enriched cells. The results of this study support the hypothesis that host cell cholesterol acts at two stages in membrane fusion: (1) early, prior to fusion pore opening, and (2) late, during fusion pore expansion.

scholarly journals Reconciling the regulatory role of Munc18 proteins in SNARE-complex assembly

IUCrJ ◽  
2014 ◽  
Vol 1 (6) ◽  
pp. 505-513 ◽  
Author(s):  
Asma Rehman ◽  
Julia K. Archbold ◽  
Shu-Hong Hu ◽  
Suzanne J. Norwood ◽  
Brett M. Collins ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Transmembrane Domain ◽  
Blood Glucose Control ◽  
Vital Role ◽  
Snare Complex ◽  
Snare Proteins ◽  
Regulatory Role ◽  
Sm Proteins ◽  
Protein Receptor

Membrane fusion is essential for human health, playing a vital role in processes as diverse as neurotransmission and blood glucose control. Two protein families are key: (1) the Sec1p/Munc18 (SM) and (2) the solubleN-ethylmaleimide-sensitive attachment protein receptor (SNARE) proteins. Whilst the essential nature of these proteins is irrefutable, their exact regulatory roles in membrane fusion remain controversial. In particular, whether SM proteins promote and/or inhibit the SNARE-complex formation required for membrane fusion is not resolved. Crystal structures of SM proteins alone and in complex with their cognate SNARE proteins have provided some insight, however, these structures lack the transmembrane spanning regions of the SNARE proteins and may not accurately reflect the native state. Here, we review the literature surrounding the regulatory role of mammalian Munc18 SM proteins required for exocytosis in eukaryotes. Our analysis suggests that the conflicting roles reported for these SM proteins may reflect differences in experimental design. SNARE proteins appear to require C-terminal immobilization or anchoring, for example through a transmembrane domain, to form a functional fusion complex in the presence of Munc18 proteins.

Sign in / Sign up

Export Citation Format

Share Document