Faculty Opinions recommendation of Synaptotagmin-1 utilizes membrane bending and SNARE binding to drive fusion pore expansion.

Cell-to-cell fusion plays an important role in normal physiology and in different pathological conditions. Early fusion stages mediated by specialized proteins and yielding fusion pores are followed by a pore expansion stage that is dependent on cell metabolism and yet unidentified machinery. Because of a similarity of membrane bending in the fusion pore rim and in highly curved intracellular membrane compartments, in the present study we explored whether changes in the activity of the proteins that generate these compartments affect cell fusion initiated by protein fusogens of influenza virus and baculovirus. We raised the intracellular concentration of curvature-generating proteins in cells by either expressing or microinjecting the ENTH (epsin N-terminal homology) domain of epsin or by expressing the GRAF1 (GTPase regulator associated with focal adhesion kinase 1) BAR (Bin/amphiphysin/Rvs) domain or the FCHo2 (FCH domain-only protein 2) F-BAR domain. Each of these treatments promoted syncytium formation. Cell fusion extents were also influenced by treatments targeting the function of another curvature-generating protein, dynamin. Cell-membrane-permeant inhibitors of dynamin GTPase blocked expansion of fusion pores and dominant-negative mutants of dynamin influenced the syncytium formation extents. We also report that syncytium formation is inhibited by reagents lowering the content and accessibility of PtdIns(4,5)P2, an important regulator of intracellular membrane remodelling. Our findings indicate that fusion pore expansion at late stages of cell-to-cell fusion is mediated, directly or indirectly, by intracellular membrane-shaping proteins.

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Conserved arginine residues in synaptotagmin 1 regulate fusion pore expansion through membrane contact

Nature Communications ◽

10.1038/s41467-021-21090-x ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Sarah B. Nyenhuis ◽

Nakul Karandikar ◽

Volker Kiessling ◽

Alex J. B. Kreutzberger ◽

Anusa Thapa ◽

...

Keyword(s):

Binding Sites ◽

Membrane Surface ◽

Fusion Pore ◽

Fusion Event ◽

Fusion Probability ◽

Arginine Residues ◽

Synaptotagmin 1 ◽

Membrane Contact ◽

Curved Membrane ◽

Pore Expansion

AbstractSynaptotagmin 1 is a vesicle-anchored membrane protein that functions as the Ca2+ sensor for synchronous neurotransmitter release. In this work, an arginine containing region in the second C2 domain of synaptotagmin 1 (C2B) is shown to control the expansion of the fusion pore and thereby the concentration of neurotransmitter released. This arginine apex, which is opposite the Ca2+ binding sites, interacts with membranes or membrane reconstituted SNAREs; however, only the membrane interactions occur under the conditions in which fusion takes place. Other regions of C2B influence the fusion probability and kinetics but do not control the expansion of the fusion pore. These data indicate that the C2B domain has at least two distinct molecular roles in the fusion event, and the data are consistent with a model where the arginine apex of C2B positions the domain at the curved membrane surface of the expanding fusion pore.

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Conserved Arginine Residues in Synaptotagmin 1 Regulate Fusion Pore Expansion Through Membrane Contact

10.1101/2020.09.17.301820 ◽

2020 ◽

Author(s):

Sarah B. Nyenhuis ◽

Nakul Karandikar ◽

Volker Kiessling ◽

Alex Kreutzberger ◽

Anusa Thapa ◽

...

Keyword(s):

Membrane Surface ◽

Fusion Pore ◽

Fusion Event ◽

Arginine Residues ◽

Synaptotagmin 1 ◽

Membrane Contact ◽

Curved Membrane ◽

Kinetics Of ◽

Novel Model ◽

Pore Expansion

AbstractSynaptotagmin 1 is a vesicle-anchored membrane protein that functions as the Ca2+ sensor for synchronous neurotransmitter release. In this work, an arginine containing region in the second C2 domain of synaptotagmin 1 (C2B) is shown to control the expansion of the fusion pore and thereby the concentration of neurotransmitter released. This arginine apex, which is opposite the Ca2+ binding sites, interacts with membranes or membrane reconstituted SNAREs; however, only the membrane interactions occur under the conditions in which fusion takes place. Other regions of C2B influence the probably and kinetics of fusion but do not control the expansion of the fusion pore. These data indicate that the C2B domain has at least two distinct molecular roles in the fusion event, and the data are consistent with a novel model where the arginine apex of C2B positions the domain at the curved membrane surface of the expanding fusion pore.

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Lumenal Protein within Secretory Granules Affects Fusion Pore Expansion

Biophysical Journal ◽

10.1016/j.bpj.2014.04.064 ◽

2014 ◽

Vol 107 (1) ◽

pp. 26-33 ◽

Cited By ~ 19

Author(s):

Annita Ngatchou Weiss ◽

Arun Anantharam ◽

Mary A. Bittner ◽

Daniel Axelrod ◽

Ronald W. Holz

Keyword(s):

Secretory Granules ◽

Fusion Pore ◽

Pore Expansion

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Chromogranin A, the major lumenal protein in chromaffin granules, controls fusion pore expansion

The Journal of General Physiology ◽

10.1085/jgp.201812182 ◽

2018 ◽

Vol 151 (2) ◽

pp. 118-130 ◽

Cited By ~ 3

Author(s):

Prabhodh S. Abbineni ◽

Mary A. Bittner ◽

Daniel Axelrod ◽

Ronald W. Holz

Keyword(s):

Plasma Membrane ◽

Small Molecules ◽

Chromogranin A ◽

Chromaffin Cells ◽

Fusion Pore ◽

Chromaffin Granules ◽

Chromaffin Granule ◽

Chromogranin B ◽

Tissue Plasminogen ◽

Pore Expansion

Upon fusion of the secretory granule with the plasma membrane, small molecules are discharged through the immediately formed narrow fusion pore, but protein discharge awaits pore expansion. Recently, fusion pore expansion was found to be regulated by tissue plasminogen activator (tPA), a protein present within the lumen of chromaffin granules in a subpopulation of chromaffin cells. Here, we further examined the influence of other lumenal proteins on fusion pore expansion, especially chromogranin A (CgA), the major and ubiquitous lumenal protein in chromaffin granules. Polarized TIRF microscopy demonstrated that the fusion pore curvature of granules containing CgA-EGFP was long lived, with curvature lifetimes comparable to those of tPA-EGFP–containing granules. This was surprising because fusion pore curvature durations of granules containing exogenous neuropeptide Y-EGFP (NPY-EGFP) are significantly shorter (80% lasting <1 s) than those containing CgA-EGFP, despite the anticipated expression of endogenous CgA. However, quantitative immunocytochemistry revealed that transiently expressed lumenal proteins, including NPY-EGFP, caused a down-regulation of endogenously expressed proteins, including CgA. Fusion pore curvature durations in nontransfected cells were significantly longer than those of granules containing overexpressed NPY but shorter than those associated with granules containing overexpressed tPA, CgA, or chromogranin B. Introduction of CgA to NPY-EGFP granules by coexpression converted the fusion pore from being transient to being longer lived, comparable to that found in nontransfected cells. These findings demonstrate that several endogenous chromaffin granule lumenal proteins are regulators of fusion pore expansion and that alteration of chromaffin granule contents affects fusion pore lifetimes. Importantly, the results indicate a new role for CgA. In addition to functioning as a prohormone, CgA plays an important role in controlling fusion pore expansion.

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Doc2b promotes GLUT4 exocytosis by activating the SNARE-mediated fusion reaction in a calcium- and membrane bending–dependent manner

Molecular Biology of the Cell ◽

10.1091/mbc.e12-11-0810 ◽

2013 ◽

Vol 24 (8) ◽

pp. 1176-1184 ◽

Cited By ~ 35

Author(s):

Haijia Yu ◽

Shailendra S. Rathore ◽

Eric M. Davis ◽

Yan Ouyang ◽

Jingshi Shen

Keyword(s):

Glucose Transporter ◽

Fusion Reaction ◽

Vesicle Fusion ◽

Dependent Manner ◽

Membrane Bilayer ◽

Protein Receptors ◽

Synaptotagmin 1 ◽

Membrane Bending ◽

Glucose Transporter Glut4 ◽

Dependent Mechanism

The glucose transporter GLUT4 plays a central role in maintaining body glucose homeostasis. On insulin stimulation, GLUT4-containing vesicles fuse with the plasma membrane, relocating GLUT4 from intracellular reservoirs to the cell surface to uptake excess blood glucose. The GLUT4 vesicle fusion reaction requires soluble N-ethylmaleimide–sensitive factor attachment protein receptors (SNAREs) as the core fusion engine and a group of regulatory proteins. In particular, the soluble C2-domain factor Doc2b plays a key role in GLUT4 vesicle fusion, but its molecular mechanism has been unclear. Here we reconstituted the SNARE-dependent GLUT4 vesicle fusion in a defined proteoliposome fusion system. We observed that Doc2b binds to GLUT4 exocytic SNAREs and potently accelerates the fusion kinetics in the presence of Ca2+. The stimulatory activity of Doc2b requires intact Ca2+-binding sites on both the C2A and C2B domains. Using electron microscopy, we observed that Doc2b strongly bends the membrane bilayer, and this membrane-bending activity is essential to the stimulatory function of Doc2b in fusion. These results demonstrate that Doc2b promotes GLUT4 exocytosis by accelerating the SNARE-dependent fusion reaction by a Ca2+- and membrane bending–dependent mechanism. Of importance, certain features of Doc2b function appear to be distinct from how synaptotagmin-1 promotes synaptic neurotransmitter release, suggesting that exocytic Ca2+ sensors may possess divergent mechanisms in regulating vesicle fusion.

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