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

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Zhenyong Wu ◽  
Nadiv Dharan ◽  
Zachary A McDargh ◽  
Sathish Thiyagarajan ◽  
Ben O'Shaughnessy ◽  
...  
Keyword(s):  
Snare Complex ◽  
Snare Proteins ◽  
Fusion Pore ◽  
C2 Domain ◽  
Calcium Sensor ◽  
Fusion Reactions ◽  
Small Pore ◽  
Synaptotagmin 1 ◽  
Pore Dilation ◽  
Fusion Pores

All membrane fusion reactions proceed through an initial fusion pore, including calcium-triggered release of neurotransmitters and hormones. Expansion of this small pore to release cargo 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. Pore dilation relied on calcium-induced insertion of the tandem C2 domain hydrophobic loops of Syt1 into the membrane, previously shown to reorient the C2 domain. Mathematical modelling suggests that C2B reorientation rotates a bound SNARE complex so that it exerts force on the membranes in a mechanical lever action that increases the height of the fusion pore, provoking pore dilation to offset the bending energy penalty. We conclude that Syt1 exerts novel non-local calcium-dependent mechanical forces on fusion pores that dilate pores and assist neurotransmitter and hormone release.

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 Peptide Hormone Release Monitored From Single Vesicles in “Membrane Lawns” of Differentiated Male Pituitary Cells: SNAREs and Fusion Pore Widening

Endocrinology ◽  
2013 ◽  
Vol 154 (3) ◽  
pp. 1235-1246 ◽  
Author(s):  
Matjaž Stenovec ◽  
Paula P. Gonçalves ◽  
Robert Zorec
Keyword(s):  
Fluorescent Protein ◽  
Peptide Hormone ◽  
Dominant Negative ◽  
Snare Complex ◽  
Snare Proteins ◽  
Fusion Pore ◽  
Pituitary Cells ◽  
Intact Cells ◽  
Protein Receptor ◽  
Pore Widening

Abstract In this study we used live-cell immunocytochemistry and confocal microscopy to study the release from a single vesicle in a simplified system called membrane lawns. The lawns were prepared by exposing differentiated pituitary prolactin (PRL)-secreting cells to a hypoosmotic shear stress. The density of the immunolabeled ternary soluble N-ethylmaleimide-sensitive factor-attachment protein receptor (SNARE) complexes that bind complexin was approximately 10 times lower than the PRL-positive, lawn-resident vesicles; this indicates that some but not all vesicles are associated with ternary SNARE complexes. However, lawn-resident PRL vesicles colocalized relatively well with particular SNARE proteins: synaptobrevin 2 (35%), syntaxin 1 (22%), and 25-kDa synaptosome associated protein (6%). To study vesicle discharge, we prepared lawn-resident vesicles, derived from atrial natriuretic peptide tagged with emerald fluorescent protein (ANP.emd)-transfected cells, which label vesicles. These maintained the structural passage to the exterior because approximately 40% of ANP.emd-loaded vesicles were labeled by extracellular PRL antibodies. Cargo release from the lawn-resident vesicles, monitored by the decline in the ANP.emd fluorescence intensity, was similar to that in intact cells. It is likely that SNARE proteins are required for calcium-dependent release from these vesicles. This is because the expression of the dominant-negative SNARE peptide, which interferes with SNARE complex formation, reduced the number of PRL-positive spots per cell (PRL antibodies placed extracellularly) significantly, from 58 ± 9 to 4 ± 2. In dominant-negative SNARE-treated cells, the PRL-positive area was reduced from 0.259 ± 0.013 to 0.123 ± 0.014 μm2, which is consistent with a hindered vesicle luminal access for extracellular PRL antibodies. These results indicate that vesicle discharge is regulated by SNARE-mediated fusion pore widening.

Pep3p/Pep5p Complex: A Putative Docking Factor at Multiple Steps of Vesicular Transport to the Vacuole of Saccharomyces cerevisiae

Genetics ◽  
2000 ◽  
Vol 156 (1) ◽  
pp. 105-122 ◽  
Author(s):  
Amit Srivastava ◽  
Carol A Woolford ◽  
Elizabeth W Jones
Keyword(s):  
Vacuolar Membrane ◽  
Snare Complex ◽  
Snare Proteins ◽  
Fusion Reactions ◽  
Hybrid Analysis ◽  
Transport Pathways ◽  
Transport Vesicles ◽  
Oligomeric Complex ◽  
Two Hybrid

Abstract Pep3p and Pep5p are known to be necessary for trafficking of hydrolase precursors to the vacuole and for vacuolar biogenesis. These proteins are present in a hetero-oligomeric complex that mediates transport at the vacuolar membrane. PEP5 interacts genetically with VPS8, implicating Pep5p in the earlier Golgi to endosome step and/or in recycling from the endosome to the Golgi. To understand further the cellular roles of Pep3p and Pep5p, we isolated and characterized a set of pep3 conditional mutants. Characterization of mutants revealed that pep3ts mutants are defective in the endosomal and nonendosomal Golgi to vacuole transport pathways, in the cytoplasm to vacuole targeting pathway, in recycling from the endosome back to the late Golgi, and in endocytosis. PEP3 interacts genetically with two members of the endosomal SNARE complex, PEP12 (t-SNARE) and PEP7 (homologue of mammalian EEA1); Pep3p and Pep5p associate physically with Pep7p as revealed by two-hybrid analysis. Our results suggest that a core Pep3p/Pep5p complex promotes vesicular docking/fusion reactions in conjunction with SNARE proteins at multiple steps in transport routes to the vacuole. We propose that this complex may be responsible for tethering transport vesicles on target membranes.

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 Fusion Pore Dilation by Synaptotagmin-1

2019 ◽  
Vol 116 (3) ◽  
pp. 526a
Author(s):  
Zhenyong Wu ◽  
Nadiv Dharan ◽  
Sathish Thiyagarajan ◽  
Ben O'Shaughnessy ◽  
Erdem Karatekin
Keyword(s):  
Fusion Pore ◽  
Synaptotagmin 1 ◽  
Pore Dilation

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 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 Fusion Pores Are Cooperatively Dilated by the Neuronal Calcium Sensor Syt1 and SNARE Proteins in a Mechanical Lever Action

2020 ◽  
Vol 118 (3) ◽  
pp. 488a-489a
Author(s):  
Nadiv Dharan ◽  
Sathish Thiyagarajan ◽  
Zhenyong Wu ◽  
Erdem Karatekin ◽  
Ben O'Shaughnessy
Keyword(s):  
Snare Proteins ◽  
Calcium Sensor ◽  
Neuronal Calcium Sensor ◽  
Lever Action ◽  
Fusion Pores

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.

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