Biochemical and Functional Studies of Cortical Vesicle Fusion: The SNARE Complex and Ca2+ Sensitivity

Abstract

Cortical vesicles (CV) possess components critical to the mechanism of exocytosis. The homotypic fusion of CV centrifuged or settled into contact has a sigmoidal Ca2+ activity curve comparable to exocytosis (CV–PM fusion). Here we show that Sr2+ and Ba2+ also trigger CV–CV fusion, and agents affecting different steps of exocytotic fusion block Ca2+, Sr2+, and Ba2+-triggered CV–CV fusion. The maximal number of active fusion complexes per vesicle, <n\>Max, was quantified by NEM inhibition of fusion, showing that CV–CV fusion satisfies many criteria of a mathematical analysis developed for exocytosis. Both <n\>Max and the Ca2+ sensitivity of fusion complex activation were comparable to that determined for CV–PM fusion. Using Ca2+-induced SNARE complex disruption, we have analyzed the relationship between membrane fusion (CV–CV and CV–PM) and the SNARE complex. Fusion and complex disruption have different sensitivities to Ca2+, Sr2+, and Ba2+, the complex remains Ca2+- sensitive on fusion-incompetent CV, and disruption does not correlate with the quantified activation of fusion complexes. Under conditions which disrupt the SNARE complex, CV on the PM remain docked and fusion competent, and isolated CV still dock and fuse, but with a markedly reduced Ca2+ sensitivity. Thus, in this system, neither the formation, presence, nor disruption of the SNARE complex is essential to the Ca2+-triggered fusion of exocytotic membranes. Therefore the SNARE complex alone cannot be the universal minimal fusion machine for intracellular fusion. We suggest that this complex modulates the Ca2+ sensitivity of fusion.

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

“…CSC and CV preparations, treatments, and fusion assays CSC/CV were isolated from fresh and 20-h incubated eggs using standard protocols [27,30,31]. Acute treatments and fusion assays were carried out in baseline intracellular media (BIM-210 mM potassium glutamate, 500 mM glycine, 10 mM NaCl, 10 mM 1,4-piperazinediethanesulfonic acid (PIPES), 1 mM MgCl 2 , 0.05 mM CaCl 2 , 1 mM ethyleneglycoltetraacetic acid (EGTA); pH 6.7) with the addition of protease inhibitors and 2.5 mM adenosine triphosphate (ATP) [30].…”

“…αT was delivered in hexadecane (<0.1% solvent, final) [26,27]. CV/CSC end-point and kinetic fusion assays and CV 'settle' fusion assays to assess inter-membrane attachment were as described [27,31,41,87,98]. Briefly, suspensions of free-floating CV were aliquoted into multiwell plates and low speed centrifugation was used to bring the CV gently into contact at the bottom of the wells.…”

“…For the 'settle' fusion assay, aliquoted CV suspensions were simply allowed to settle into contact for 60 min before Ca 2+ addition. OD measurements (initial suspension OD 405 =0.4 for CV and OD 405 =0.3 for CSC) were taken before and after the addition of various Ca 2+ stocks, and the final [Ca 2+ ] free were measured with a Ca 2+ -sensitive electrode (Nicossensors, Huntington Valley, PA, USA), as described [31]. All fusion measurements were carried out using a Victor 2 V microplate-reader (Perkin Elmer, Boston, MA, USA).…”

“…The resulting Ca 2+ -activity curves were fit using the sigmoidal cumulative log-normal model to establish fusion extent and Ca 2+ sensitivity; the applicability of a log-normal cumulative distribution function to exocytotic survival and thus Ca 2+ -activity curves has been clearly demonstrated [8,49,84]-the fraction of CV that fuse at a given [Ca 2+ ] free is solely a function of the average number of active fusion complexes (<n>) at a docking site. As CV-CV fusion proceeds through the same fusion complexes as exocytosis, it is fully described by the same analysis developed for CV-PM fusion, yielding comparable fitting parameters for the sigmoidal relationship between <n> and [Ca 2+ ] free [8,27,31,84]. Curves for controls were thus fit to a two-parameter model with 100% fusion extent, and curves for treated conditions were fit to a three-parameter model (TableCurve-2D, v.5.0, SYSTAT, Richmond, CA, USA).…”

“…This 'cell' type plays a central role in studies of membrane-associated events, including regulated exocytosis. Shearing yields cell surface complexes (CSC)-plasma membrane (PM) sheets with docked, fusion-ready cortical vesicles (CV), from which high purity CV are isolated [29,31,85,96]. 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].…”