scholarly journals Cdc42 controls the dilation of the exocytotic fusion pore by regulating membrane tension

2014 ◽  
Vol 25 (20) ◽  
pp. 3195-3209 ◽  
Author(s):  
Marine Bretou ◽  
Ouardane Jouannot ◽  
Isabelle Fanget ◽  
Paolo Pierobon ◽  
Nathanaël Larochette ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Small Gtpase ◽  
Dominant Negative ◽  
Neuroendocrine Cells ◽  
Fusion Pore ◽  
Membrane Tension ◽  
Release Process ◽  
Vesicle Exocytosis ◽  
Pore Dilation ◽  
Pore Enlargement

Membrane fusion underlies multiple processes, including exocytosis of hormones and neurotransmitters. Membrane fusion starts with the formation of a narrow fusion pore. Radial expansion of this pore completes the process and allows fast release of secretory compounds, but this step remains poorly understood. Here we show that inhibiting the expression of the small GTPase Cdc42 or preventing its activation with a dominant negative Cdc42 construct in human neuroendocrine cells impaired the release process by compromising fusion pore enlargement. Consequently the mode of vesicle exocytosis was shifted from full-collapse fusion to kiss-and-run. Remarkably, Cdc42-knockdown cells showed reduced membrane tension, and the artificial increase of membrane tension restored fusion pore enlargement. Moreover, inhibiting the motor protein myosin II by blebbistatin decreased membrane tension, as well as fusion pore dilation. We conclude that membrane tension is the driving force for fusion pore dilation and that Cdc42 is a key regulator of this force.

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 SCAMP2 Interacts with Arf6 and Phospholipase D1 and Links Their Function to Exocytotic Fusion Pore Formation in PC12 Cells

2005 ◽  
Vol 16 (10) ◽  
pp. 4463-4472 ◽  
Author(s):  
Lixia Liu ◽  
Haini Liao ◽  
Anna Castle ◽  
Jie Zhang ◽  
James Casanova ◽  
...  
Keyword(s):  
Pc12 Cells ◽  
Pore Formation ◽  
Plasma Membranes ◽  
Secretory Vesicle ◽  
Small Gtpase ◽  
Fusion Pore ◽  
Dense Core Vesicle ◽  
Nucleotide Exchange ◽  
Phospholipase D1 ◽  
Vesicle Exocytosis

SNAP receptor (SNARE)-mediated fusion is regarded as a core event in exocytosis. Exocytosis is supported by other proteins that set up SNARE interactions between secretory vesicle and plasma membranes or facilitate fusion pore formation. Secretory carrier membrane proteins (SCAMPs) are candidate proteins for functioning in these events. In neuroendocrine PC12 cells, SCAMP2 colocalizes on the cell surface with three other proteins required for dense-core vesicle exocytosis: phospholipase D1 (PLD1), the small GTPase Arf6, and Arf6 guanine nucleotide exchange protein ARNO. Arf6 and PLD1 coimmunoprecipitate (coIP) with SCAMP2. These associations have been implicated in exocytosis by observing enhanced coIP of Arf6 with SCAMP2 after cell depolarization and in the presence of guanosine 5′-O-(3-thio)triphosphate and by inhibition of coIP by a SCAMP-derived peptide that inhibits exocytosis. The peptide also suppresses PLD activity associated with exocytosis. Using amperometry to analyze exocytosis, we show that expression of a point mutant of SCAMP2 that exhibits decreased association with Arf6 and of mutant Arf6 deficient in activating PLD1 have the same inhibitory effects on early events in membrane fusion. However, mutant SCAMP2 also uniquely inhibits fusion pore dilation. Thus, SCAMP2 couples Arf6-stimulated PLD activity to exocytosis and links this process to formation of fusion pores.

scholarly journals Modification of the Cytoplasmic Domain of Influenza Virus Hemagglutinin Affects Enlargement of the Fusion Pore

2000 ◽  
Vol 74 (16) ◽  
pp. 7529-7537 ◽  
Author(s):  
Christine Kozerski ◽  
Evgeni Ponimaskin ◽  
Britta Schroth-Diez ◽  
Michael F. G. Schmidt ◽  
Andreas Herrmann
Keyword(s):  
Influenza Virus ◽  
Membrane Fusion ◽  
Transmembrane Domain ◽  
Expression System ◽  
Fusion Pore ◽  
Target Cells ◽  
Fusion Activity ◽  
Wild Type ◽  
Influenza Virus Hemagglutinin ◽  
Pore Enlargement

ABSTRACT The fusion activity of chimeras of influenza virus hemagglutinin (HA) (from A/fpv/Rostock/34; subtype H7) with the transmembrane domain (TM) and/or cytoplasmic tail (CT) either from the nonviral, nonfusogenic T-cell surface protein CD4 or from the fusogenic Sendai virus F-protein was studied. Wild-type or chimeric HA was expressed in CV-1 cells by the transient T7-RNA-polymerase vaccinia virus expression system. Subsequently, the fusion activity of the expression products was monitored with red blood cells or ghosts as target cells. To assess the different steps of fusion, target cells were labeled with the fluorescent membrane label octadecyl rhodamine B-chloride (R18) (membrane fusion) and with the cytoplasmic fluorophores calcein (molecular weight [MW], 623; formation of small aqueous fusion pore) and tetramethylrhodamine-dextran (MW, 10,000; enlargement of fusion pore). All chimeric HA/F-proteins, as well as the chimera with the TM of CD4 and the CT of HA, were able to mediate the different steps of fusion very similarly to wild-type HA. Quite differently, chimeric proteins with the CT of CD4 were strongly impaired in mediating pore enlargement. However, membrane fusion and formation of small pores were similar to those of wild-type HA, indicating that the conformational change of the ectodomain and earlier fusion steps were not inhibited. Various properties of the CT which may affect pore enlargement are considered. We surmise that the hydrophobicity of the sequence adjacent to the transmembrane domain is important for pore dilation.

scholarly journals Age-dependent Preferential Dense-Core Vesicle Exocytosis in Neuroendocrine Cells Revealed by Newly Developed Monomeric Fluorescent Timer Protein

2010 ◽  
Vol 21 (1) ◽  
pp. 87-94 ◽  
Author(s):  
Takashi Tsuboi ◽  
Tetsuya Kitaguchi ◽  
Satoshi Karasawa ◽  
Mitsunori Fukuda ◽  
Atsushi Miyawaki
Keyword(s):  
Time Course ◽  
Secretory Pathway ◽  
Molecular Mechanisms ◽  
Small Gtpase ◽  
Neuroendocrine Cells ◽  
Tissue Type ◽  
Dense Core Vesicle ◽  
Secretory Vesicles ◽  
Age Dependent ◽  
Vesicle Exocytosis

Although it is evident that only a few secretory vesicles accumulating in neuroendocrine cells are qualified to fuse with the plasma membrane and release their contents to the extracellular space, the molecular mechanisms that regulate their exocytosis are poorly understood. For example, it has been controversial whether secretory vesicles are exocytosed randomly or preferentially according to their age. Using a newly developed protein-based fluorescent timer, monomeric Kusabira Green Orange (mK-GO), which changes color with a predictable time course, here we show that small GTPase Rab27A effectors regulate age-dependent exocytosis of secretory vesicles in PC12 cells. When the vesicles were labeled with mK-GO–tagged neuropeptide Y or tissue-type plasminogen activator, punctate structures with green or red fluorescence were observed. Application of high [K+] stimulation induced exocytosis of new (green) fluorescent secretory vesicles but not of old (red) vesicles. Overexpression or depletion of rabphilin and synaptotagmin-like protein4-a (Slp4-a), which regulate exocytosis positively and negatively, respectively, disturbed the age-dependent exocytosis of the secretory vesicles in different manners. Our results suggest that coordinate functions of the two effectors of Rab27A, rabphilin and Slp4-a, are required for regulated secretory pathway.

scholarly journals SERINC Inhibits HIV-1 Env Induced Membrane Fusion and Slows Fusion Pore Enlargement

2017 ◽  
Vol 112 (3) ◽  
pp. 79a
Author(s):  
Ruben M. Markosyan ◽  
Shan-Lu Liu ◽  
Fred S. Cohen
Keyword(s):  
Membrane Fusion ◽  
Fusion Pore ◽  
Induced Membrane ◽  
Hiv 1 ◽  
Pore Enlargement

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 ROCK and mDia1 antagonize in Rho-dependent Rac activation in Swiss 3T3 fibroblasts

2002 ◽  
Vol 157 (5) ◽  
pp. 819-830 ◽  
Author(s):  
Takahiro Tsuji ◽  
Toshimasa Ishizaki ◽  
Muneo Okamoto ◽  
Chiharu Higashida ◽  
Kazuhiro Kimura ◽  
...  
Keyword(s):  
Focal Adhesions ◽  
Small Gtpase ◽  
Dominant Negative ◽  
Stress Fibers ◽  
Induced Membrane ◽  
3T3 Fibroblasts ◽  
C3 Exoenzyme ◽  
Swiss 3T3 ◽  
Swiss 3T3 Fibroblasts ◽  
Membrane Ruffle

The small GTPase Rho acts on two effectors, ROCK and mDia1, and induces stress fibers and focal adhesions. However, how ROCK and mDia1 individually regulate signals and dynamics of these structures remains unknown. We stimulated serum-starved Swiss 3T3 fibroblasts with LPA and compared the effects of C3 exoenzyme, a Rho inhibitor, with those of Y-27632, a ROCK inhibitor. Y-27632 treatment suppressed LPA-induced formation of stress fibers and focal adhesions as did C3 exoenzyme but induced membrane ruffles and focal complexes, which were absent in the C3 exoenzyme-treated cells. This phenotype was suppressed by expression of N17Rac. Consistently, the amount of GTP-Rac increased significantly by Y-27632 in LPA-stimulated cells. Biochemically, Y-27632 suppressed tyrosine phosphorylation of paxillin and focal adhesion kinase and not that of Cas. Inhibition of Cas phosphorylation with PP1 or expression of a dominant negative Cas mutant inhibited Y-27632–induced membrane ruffle formation. Moreover, Crk-II mutants lacking in binding to either phosphorylated Cas or DOCK180 suppressed the Y-27632–induced membrane ruffle formation. Finally, expression of a dominant negative mDia1 mutant also inhibited the membrane ruffle formation by Y-27632. Thus, these results have revealed the Rho-dependent Rac activation signaling that is mediated by mDia1 through Cas phosphorylation and antagonized by the action of ROCK.

scholarly journals Activity-Dependent Fusion Pore Dilation Mediated by a Dynamin I-Syndapin Pathway

2011 ◽  
Vol 100 (3) ◽  
pp. 408a
Author(s):  
Prattana Samasilp ◽  
Bryan Doreian ◽  
Shyue-An Chan ◽  
Corey Smith
Keyword(s):  
Fusion Pore ◽  
Pore Dilation ◽  
Dynamin I ◽  
Activity Dependent

scholarly journals The C2b Domain of Synaptotagmin Is a Ca2+–Sensing Module Essential for Exocytosis

2000 ◽  
Vol 150 (5) ◽  
pp. 1125-1136 ◽  
Author(s):  
Radhika C. Desai ◽  
Bimal Vyas ◽  
Cynthia A. Earles ◽  
J. Troy Littleton ◽  
Judith A. Kowalchyck ◽  
...  
Keyword(s):  
Synaptic Vesicle ◽  
Pc12 Cells ◽  
Conformational Changes ◽  
Cytoplasmic Domain ◽  
Dominant Negative ◽  
Fusion Pore ◽  
Vesicle Membrane ◽  
C2 Domains ◽  
Essential Step ◽  
Synaptotagmin I

The synaptic vesicle protein synaptotagmin I has been proposed to serve as a Ca2+ sensor for rapid exocytosis. Synaptotagmin spans the vesicle membrane once and possesses a large cytoplasmic domain that contains two C2 domains, C2A and C2B. Multiple Ca2+ ions bind to the membrane proximal C2A domain. However, it is not known whether the C2B domain also functions as a Ca2+-sensing module. Here, we report that Ca2+ drives conformational changes in the C2B domain of synaptotagmin and triggers the homo- and hetero-oligomerization of multiple isoforms of the protein. These effects of Ca2+ are mediated by a set of conserved acidic Ca2+ ligands within C2B; neutralization of these residues results in constitutive clustering activity. We addressed the function of oligomerization using a dominant negative approach. Two distinct reagents that block synaptotagmin clustering potently inhibited secretion from semi-intact PC12 cells. Together, these data indicate that the Ca2+-driven clustering of the C2B domain of synaptotagmin is an essential step in excitation-secretion coupling. We propose that clustering may regulate the opening or dilation of the exocytotic fusion pore.

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