Calcium-triggered Membrane Fusion Proceeds Independently of Specific Presynaptic Proteins

Szule, Joseph A.; Jarvis, Scott E.; Hibbert, Julie; Spafford, J. David; Braun, Janice E. A.; Zamponi, Gerald W.; Wessel, Gary M.; Coorssen, Jens R.

doi:10.1074/jbc.c300197200

Cited by 47 publications

(53 citation statements)

References 43 publications

(65 reference statements)

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“…CSC and CV preparations CV-PM (cell surface complex; CSC) and CV preparations were isolated from unfertilized sea urchin (Srongylocentrotus purpuratus, Westwind, BC, Canada) eggs as previously described [6][7][8][9][14][15][16]. All treatments and fusion assays were carried out in baseline intracellular medium (BIM; 210 mM potassium glutamate, 500 mM glycine, 10 mM NaCl, 10 mM Pipes, 0.05 mM CaCl 2 , 1 mM MgCl 2 , 1 mM EGTA; pH 6.7; [23]) supplemented with 2.5 mM ATP and protease inhibitors, unless otherwise stated.…”

Section: Methodsmentioning

confidence: 99%

“…Fusion assays Standard end-point and kinetic fusion assays were carried out as previously described [6,7,9,14]. For each experiment (n), all conditions were tested in replicates of three to four.…”

Section: Methodsmentioning

confidence: 99%

“…Similarly, the cell surface complex (CSC) isolated from the unfertilized egg, consisting of only the fully docked and primed CV attached to fragments of PM, requires no more than an increase in the [Ca 2+ ] free to trigger CV-PM fusion (e.g., exocytosis in vitro) in the absence of other cytosolic factors [4,5]. The system can be further refined as isolated CV undergo fast, Ca 2+ -triggered fusion with one another [6][7][8][9][10][11][12][13][14][15][16] and even with pure lipid membranes [12,17], indicating that CV contain the minimal essential molecular components for intermembrane attachment, Ca 2+ sensing, and membrane fusion. CV are thus a high purity, robust, stage-specific preparation of release-ready secretory vesicles with which to analyze the mechanisms essential to Ca 2+ -triggered membrane fusion [6,7,[14][15][16].…”

Section: Introductionmentioning

confidence: 99%

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Enhancement of the Ca2+-triggering steps of native membrane fusion via thiol-reactivity

2008

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Ca 2+ -triggered membrane fusion is the defining step of exocytosis. Isolated urchin cortical vesicles (CV) provide a stage-specific preparation to study the mechanisms by which Ca 2+ triggers the merger of two apposed native membranes. Thiol-reactive reagents that alkylate free sulfhydryl groups on proteins have been consistently shown to inhibit triggered fusion. Here, we characterize a novel effect of the alkylating reagent iodoacetamide (IA). IA was found to enhance the kinetics and Ca 2+ sensitivity of both CV-plasma membrane and CV-CV fusion. If Sr 2+ , a weak Ca 2+ mimetic, was used to trigger fusion, the potentiation was even greater than that observed for Ca 2+ , suggesting that IA acts at the Ca 2+ -sensing step of triggered fusion. Comparison of IA to other reagents indicates that there are at least two distinct thiol sites involved in the underlying fusion mechanism: one that regulates the efficiency of fusion and one that interferes with fusion competency.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Enhancement of the Ca2+-triggering steps of native membrane fusion via thiol-reactivity

2008

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“…This inhibition of exocytosis was not reversible with the addition of cytosol. We do not know the identity of the protein(s) that are targets for the thiol-reactive inhibitors that we used but it is unlikely that they are SNARE proteins alone, as previous studies of CV fusion have shown that SNAREs are not essential for fusion, although they might play regulatory functions Coorssen et al, 2003;Szule et al, 2003).…”

Section: Discussionmentioning

confidence: 99%

Membrane fusion of secretory vesicles of the sea urchin egg in the absence of NSF

Whalley

Timmers

Coorssen

et al. 2004

Journal of Cell Science

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The role of cytosolic ATPases such as N-ethylmaleimide (NEM)-sensitive fusion protein (NSF) in membrane fusion is controversial. We examined the physiology and biochemistry of ATP and NSF in the cortical system of the echinoderm egg to determine if NSF is an essential factor in membrane fusion during Ca2+-triggered exocytosis. Neither exocytosis in vitro, nor homotypic cortical vesicle (CV) fusion required soluble proteins or nucleotides, and both occurred in the presence of non-hydrolyzable analogs of ATP. While sensitive to thiol-specific reagents, CV exocytosis is not restored by the addition of cytosolic NSF, and fusion and NSF function are differentially sensitive to thiol-specific agents. To test participation of tightly bound, non-exchangeable NSF in CV-CV fusion, we cloned the sea urchin homolog and developed a species-specific antibody for western blots and physiological analysis. This antibody was without effect on CV exocytosis or homotypic fusion, despite being functionally inhibitory. NSF is detectable in intact cortices, cortices from which CVs had been removed and isolated CVs treated with ATP-γ-S and egg cytosol to reveal NSF binding sites. In contrast, isolated CVs, though all capable of Ca2+-triggered homotypic fusion, contain less than one hexamer of NSF per CV. Thus NSF is not a required component of the CV fusion machinery.

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“…Interestingly, although granuphilin (15,32) and Synt1A (14) knockout cells show a reduced number of docked granules, they do secrete insulin, suggesting that docking is not a prerequisite for fusion, as also suggested in other cell types (37,38). Synt1A-and granuphilin-deficient animals show virtually no first phase of insulin secretion, and almost all fusion events are due to new-comers (14,15).…”

mentioning

confidence: 98%

Newcomer insulin secretory granules as a highly calcium-sensitive pool

Pedersen

Sherman

2009

Proc. Natl. Acad. Sci. U.S.A.

118

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Insulin secretion is biphasic in response to a step in glucose stimulation. Recent experiments suggest that 2 different mechanisms operate during the 2 phases, with transient first-phase secretion due to exocytosis of docked granules but the second sustained phase due largely to newcomer granules. Another line of research has shown that there exist 2 pools of releasable granules with different Ca 2؉ sensitivities. An immediately releasable pool (IRP) is located in the vicinity of Ca 2؉ channels, whereas a highly Ca 2؉ -sensitive pool (HCSP) resides mainly away from Ca 2؉ channels. We extend a previous model of exocytosis and insulin release by adding an HCSP and show that the inclusion of this pool naturally leads to insulin secretion mainly from newcomer granules during the second phase of secretion. We show that the model is compatible with data from single cells on the HCSP and from stimulation of islets by glucose, including L-and R-type Ca 2؉ channel knockouts, as well as from Syntaxin-1A-deficient cells. We also use the model to investigate the relative contribution of calcium signaling and pool depletion in controlling biphasic secretion.␤-cells ͉ biphasic secretion ͉ exocytosis ͉ pancreatic islets ͉ vesicles

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Calcium-triggered Membrane Fusion Proceeds Independently of Specific Presynaptic Proteins

Cited by 47 publications

References 43 publications

Enhancement of the Ca2+-triggering steps of native membrane fusion via thiol-reactivity

Enhancement of the Ca2+-triggering steps of native membrane fusion via thiol-reactivity

Membrane fusion of secretory vesicles of the sea urchin egg in the absence of NSF

Newcomer insulin secretory granules as a highly calcium-sensitive pool

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