Sea urchin egg preparations as systems for the study of calcium‐triggered exocytosis

Zimmerberg, Joshua; Coorssen, Jens R.; Vogel, Steven S.; Blank, Paul S.

doi:10.1111/j.1469-7793.1999.00015.x

Cited by 27 publications

(20 citation statements)

References 51 publications

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“…Settle CV-CV fusion assays revealed no differences between fresh and incubated eggs, indicating that CV were able to form critical inter-membrane attachments necessary for a robust fusion response (Fig. 2b) [8,26,27,31,38,39,72,84,97]. Overlapping activity curves and kinetic responses found for both CV-CV and CV-PM fusion ( Fig.…”

Section: Resultsmentioning

confidence: 93%

“…Unlike mammalian secretory vesicles, CV retain Ca 2+ sensitivity and fusion competence in vitro hours after isolationwashed free of soluble components, CV are 'locked' in the docked, fusion-ready state, requiring only an increase in [Ca 2+ ] free to trigger fusion [30,57,75,80,90]. Being amenable to biochemical manipulations, CSC and CV have proven invaluable in dissecting molecular mechanisms underlying docking, Ca 2+ -triggering, and membrane fusion; comparable quantitative manipulations are unfeasible in other secretory systems [30,76,97].…”

Section: Introductionmentioning

confidence: 99%

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A new approach to the molecular analysis of docking, priming, and regulated membrane fusion

Rogasevskaia

Coorssen

2011

J Chem Biol

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Studies using isolated sea urchin cortical vesicles have proven invaluable in dissecting mechanisms of Ca 2+ -triggered membrane fusion. However, only acute molecular manipulations are possible in vitro. Here, using selective pharmacological manipulations of sea urchin eggs ex vivo, we test the hypothesis that specific lipidic components of the membrane matrix selectively affect defined late stages of exocytosis, particularly the Ca 2+ -triggered steps of fast membrane fusion. Egg treatments with cholesterol-lowering drugs resulted in the inhibition of vesicle fusion. Exogenous cholesterol recovered fusion extent and efficiency in cholesterol-depleted membranes; α-tocopherol, a structurally dissimilar curvature analogue, selectively restored fusion extent. Inhibition of phospholipase C reduced vesicle phosphatidylethanolamine and suppressed both the extent and kinetics of fusion. Although phosphatidylinositol-3-kinase inhibition altered levels of polyphosphoinositide species and reduced all fusion parameters, sequestering polyphosphoinositides selectively inhibited fusion kinetics. Thus, cholesterol and phosphatidylethanolamine play direct roles in the fusion pathway, contributing negative curvature. Cholesterol also organizes the physiological fusion site, defining fusion efficiency. A selective influence of phosphatidylethanolamine on fusion kinetics sheds light on the local microdomain structure at the site of docking/fusion. Polyphosphoinositides have modulatory upstream roles in priming: alterations in specific polyphosphoinositides likely represent the terminal priming steps defining fully docked, release-ready vesicles. Thus, this pharmacological approach has the potential to be a robust high-throughput platform to identify molecular components of the physiological fusion machine critical to docking, priming, and triggered fusion.

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Section: Resultsmentioning

confidence: 93%

Section: Introductionmentioning

confidence: 99%

A new approach to the molecular analysis of docking, priming, and regulated membrane fusion

Rogasevskaia

Coorssen

2011

J Chem Biol

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“…Urchin cortical vesicles (CV) are a well-established, stage-specific model for the analysis of the molecular mechanisms underlying fast, Ca 2+ -triggered native membrane fusion [1][2][3]. In the intact egg, an increase in intracellular free Ca 2+ concentration ([Ca 2+ ] free ) triggers all the fully docked, primed, and fusion-ready CV to fuse with the plasma membrane (PM); the released content forms the fertilization envelope which prevents polyspermy.…”

Section: Introductionmentioning

confidence: 99%

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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“…(2) In contrast to starfish oocytes arrested in meiosis I, early development cannot be induced in G1-arrested sea urchin eggs by only inactivating MEK. (3) Elevation of a thin fertilization envelope in some eggs that had been treated with MEK inhibitor suggests changes in Ca 2+ i levels, because cortical exocytosis is Ca 2+ -dependent (Zimmerberg et al, 1999).…”

Section: Inactivation Of Erk-lp Triggers Mitotic Events But Not Embrymentioning

confidence: 99%

Inactivation of MAPK in mature oocytes triggers progression into mitosis via a Ca2+-dependent pathway but without completion of S phase

Zhang

Huitorel

Genevière

et al. 2006

Journal of Cell Science

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Unfertilized sea urchin eggs that are arrested at G1 phase after completion of meiosis contain a highly phosphorylated mitogen-activated protein (MAP) kinase (MAPK), the ERK-like protein (ERK-LP). Several data including our previous results show that ERK-LP is inactivated after fertilization, which agrees with results obtained in other species including Xenopus, starfish and mammals. The question is to elucidate the function of a high MAPK activity in sea urchin eggs. We report here that dephosphorylation of ERK-LP with very low concentrations of two MEK inhibitors, PD98059 or U0126, triggers entry into mitosis. Under these conditions, recurrent oscillations of the phosphorylation of ERK-LP and of a tyrosine residue in Cdc2 occur, and the intracellular Ca2+ level (Ca2+i) progressively and slowly increases. Nuclear envelope breakdown and all mitotic events initiated after dephosphorylation of ERK-LP are inhibited when changes in Ca2+i are prevented; however, they are independent of the intracellular pH. These results suggest that inactivation of a MEK-ERK pathway, normally induced after fertilization of sea urchin eggs, triggers entry into mitosis by altering Ca2+i but cannot trigger full DNA replication. We discuss the hypothesis that neither inactivation nor activation of a MEK-ERK pathway is required for S phase completion in sea urchin egg.

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Sea urchin egg preparations as systems for the study of calcium‐triggered exocytosis

Cited by 27 publications

References 51 publications

A new approach to the molecular analysis of docking, priming, and regulated membrane fusion

A new approach to the molecular analysis of docking, priming, and regulated membrane fusion

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

Inactivation of MAPK in mature oocytes triggers progression into mitosis via a Ca2+-dependent pathway but without completion of S phase

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