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

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

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1129864.586951 ◽

2008 ◽

Author(s):

Sandra Bajjalieh

Keyword(s):

Fusion Pore ◽

Synaptotagmin 1 ◽

Membrane Bending ◽

Pore Expansion

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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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Phosphoinositides Regulate Membrane-dependent Actin Assembly by Latex Bead Phagosomes

Molecular Biology of the Cell ◽

10.1091/mbc.01-06-0314 ◽

2002 ◽

Vol 13 (4) ◽

pp. 1190-1202 ◽

Cited By ~ 51

Author(s):

Hélène Defacque ◽

Evelyne Bos ◽

Boyan Garvalov ◽

Cécile Barret ◽

Christian Roy ◽

...

Keyword(s):

Binding Sites ◽

Kinase Activity ◽

Kinase Inhibitors ◽

Membrane Surface ◽

Latex Bead ◽

Actin Assembly ◽

Wild Type ◽

Membrane Surfaces ◽

Organelle Membrane

Actin assembly on membrane surfaces is an elusive process in which several phosphoinositides (PIPs) have been implicated. We have reconstituted actin assembly using a defined membrane surface, the latex bead phagosome (LBP), and shown that the PI(4,5)P2-binding proteins ezrin and/or moesin were essential for this process ( Defacque et al., 2000b ). Here, we provide several lines of evidence that both preexisting and newly synthesized PI(4,5)P2, and probably PI(4)P, are essential for phagosomal actin assembly; only these PIPs were routinely synthesized from ATP during in vitro actin assembly. Treatment of LBP with phospholipase C or with adenosine, an inhibitor of type II PI 4-kinase, as well as preincubation with anti-PI(4)P or anti-PI(4,5)P2 antibodies all inhibited this process. Incorporation of extra PI(4)P or PI(4,5)P2 into the LBP membrane led to a fivefold increase in the number of phagosomes that assemble actin. An ezrin mutant mutated in the PI(4,5)P2-binding sites was less efficient in binding to LBPs and in reconstituting actin assembly than wild-type ezrin. Our data show that PI 4- and PI 5-kinase, and under some conditions also PI 3-kinase, activities are present on LBPs and can be activated by ATP, even in the absence of GTP or cytosolic components. However, PI 3-kinase activity is not required for actin assembly, because the process was not affected by PI 3-kinase inhibitors. We suggest that the ezrin-dependent actin assembly on the LBP membrane may require active turnover of D4 and D5 PIPs on the organelle membrane.

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The Synaptotagmin-1 C2B Domain Is a Key Regulator in the Stabilization of the Fusion Pore

Journal of Chemical Theory and Computation ◽

10.1021/acs.jctc.0c00734 ◽

2020 ◽

Vol 16 (12) ◽

pp. 7840-7851

Author(s):

Marcelo Caparotta ◽

Claudia N. Tomes ◽

Luis S. Mayorga ◽

Diego Masone

Keyword(s):

Fusion Pore ◽

Synaptotagmin 1

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Identification of β-synuclein on secretory granules in chromaffin cells and the effects of α- and β-synuclein on post-fusion BDNF discharge and fusion pore expansion

Neuroscience Letters ◽

10.1016/j.neulet.2019.01.056 ◽

2019 ◽

Vol 699 ◽

pp. 134-139 ◽

Cited By ~ 2

Author(s):

Prabhodh S. Abbineni ◽

Kevin P. Bohannon ◽

Mary A. Bittner ◽

Daniel Axelrod ◽

Ronald W. Holz

Keyword(s):

Chromaffin Cells ◽

Secretory Granules ◽

Fusion Pore ◽

Pore Expansion

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Topography of Lectin Binding Sites in Celiac Sprue

Canadian Journal of Gastroenterology ◽

10.1155/1992/567285 ◽

1992 ◽

Vol 6 (5) ◽

pp. 271-276 ◽

Cited By ~ 3

Author(s):

Hugh J Freeman

Keyword(s):

Small Intestine ◽

Binding Sites ◽

Lectin Binding ◽

Membrane Surface ◽

Celiac Sprue ◽

Intestinal Biopsy ◽

Small Intestinal Biopsy ◽

Sequential Addition ◽

Enterocytic Differentiation ◽

Lectin Binding Sites

Lectins are ubiquitious, naturally occurring proteins largely derived from planes that are present in the human diet. They are released from food during mastication and digestion to remain biologically intact during passage through the small intestine. In this study, the topographic distribution of binding sites in the small intestine for six different lectins (derived from wheat germ, castor bean, gorse, horsegram, jackbean and peanut) were explored in 16 patients with celiac sprue a long with controls. For each lectin, the pattern of binding in the small intestinal biopsy specimens was distinct and specifically inhibited by a different carbohydrate residue. Lectin labelling patterns were different for both controls and celiac patients. in addition, the epithelial cell membrane surface and goblet cells exhibited a gradient of fluorescence intensity along the crypt-villus column. This gradient appeared to parallel the degree of enterocytic differentiation consistent with the concept that sequential addition of sugar residues to form completed oligosaccharide side chains occurs with enterocytic differentiation and maturation. These results also suggest that the process of epithelial cellular differentiation, although present in celiac enterocytes, is altered substantially.

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