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 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 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 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 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 Synaptotagmin-Ca2+triggers two sequential steps in regulated exocytosis in rat PC12 cells: fusion pore opening and fusion pore dilation

2006 ◽  
Vol 570 (2) ◽  
pp. 295-307 ◽  
Author(s):  
Chih-Tien Wang ◽  
Jihong Bai ◽  
Payne Y. Chang ◽  
Edwin R. Chapman ◽  
Meyer B. Jackson
Keyword(s):  
Pc12 Cells ◽  
Fusion Pore ◽  
Regulated Exocytosis ◽  
Pore Opening ◽  
Pore Dilation

scholarly journals SLY1 and Syntaxin 18 specify a distinct pathway for procollagen VII export from the endoplasmic reticulum

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Cristina Nogueira ◽  
Patrik Erlmann ◽  
Julien Villeneuve ◽  
António JM Santos ◽  
Emma Martínez-Alonso ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Fusion ◽  
Protein Secretion ◽  
Family Members ◽  
Retrograde Transport ◽  
Cytoplasmic Domain ◽  
Snare Complex ◽  
Snare Proteins ◽  
General Protein

TANGO1 binds and exports Procollagen VII from the endoplasmic reticulum (ER). In this study, we report a connection between the cytoplasmic domain of TANGO1 and SLY1, a protein that is required for membrane fusion. Knockdown of SLY1 by siRNA arrested Procollagen VII in the ER without affecting the recruitment of COPII components, general protein secretion, and retrograde transport of the KDEL-containing protein BIP, and ERGIC53. SLY1 is known to interact with the ER-specific SNARE proteins Syntaxin 17 and 18, however only Syntaxin 18 was required for Procollagen VII export. Neither SLY1 nor Syntaxin 18 was required for the export of the equally bulky Procollagen I from the ER. Altogether, these findings reveal the sorting of bulky collagen family members by TANGO1 at the ER and highlight the existence of different export pathways for secretory cargoes one of which is mediated by the specific SNARE complex containing SLY1 and Syntaxin 18.

scholarly journals Otoferlin is a calcium sensor that directly regulates SNARE-mediated membrane fusion

2010 ◽  
Vol 191 (1) ◽  
pp. 187-197 ◽  
Author(s):  
Colin P. Johnson ◽  
Edwin R. Chapman
Keyword(s):  
Membrane Fusion ◽  
Calcium Binding ◽  
Dissociation Constants ◽  
Dependent Manner ◽  
Calcium Sensor ◽  
Fusion Reactions ◽  
Cochlear Hair Cells ◽  
C2 Domains ◽  
Synaptic Vesicle Exocytosis ◽  
Vesicle Exocytosis

Otoferlin is a large multi–C2 domain protein proposed to act as a calcium sensor that regulates synaptic vesicle exocytosis in cochlear hair cells. Although mutations in otoferlin have been associated with deafness, its contribution to neurotransmitter release is unresolved. Using recombinant proteins, we demonstrate that five of the six C2 domains of otoferlin sense calcium with apparent dissociation constants that ranged from 13–25 µM; in the presence of membranes, these apparent affinities increase by up to sevenfold. Using a reconstituted membrane fusion assay, we found that five of the six C2 domains of otoferlin stimulate membrane fusion in a calcium-dependent manner. We also demonstrate that a calcium binding–deficient form of the C2C domain is incapable of stimulating membrane fusion, further underscoring the importance of calcium for the protein’s function. These results demonstrate for the first time that otoferlin is a calcium sensor that can directly regulate soluble N-ethyl-maleimide sensitive fusion protein attachment protein receptor–mediated membrane fusion reactions.

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.

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.

Sign in / Sign up

Export Citation Format

Share Document