Biochemical and Functional Studies of Cortical Vesicle Fusion: The SNARE Complex and Ca 2 (cid:49) Sensitivity

Cortical vesicles (CV) possess components critical to the mechanism of exocytosis. The homotypic fusion of CV centrifuged or settled into contact has a sigmoidal Ca 2 (cid:49) activity curve comparable to exocytosis (CV—PM fusion). Here we show that Sr 2 (cid:49) and Ba 2 (cid:49) also trigger CV—CV fusion, and agents affecting different steps of exocytotic fusion block Ca 2 (cid:49) , Sr 2 , and Ba 2 (cid:49) 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 Ca 2 (cid:49) sensitivity of fusion complex activation were comparable to that determined for CV—PM fusion. Using Ca 2 (cid:49) 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 Ca 2 (cid:49) , Sr 2 (cid:49) , and Ba 2 (cid:49) , the complex remains Ca 2 (cid:49) - 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 Ca 2 (cid:49) sensitivity. Thus, in this system, neither the formation, presence, nor disruption of the SNARE complex is essential to the Ca 2 (cid:49) -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 Ca 2 (cid:49) sensitivity of fusion. sigmoidal curves (translational in- variance) is best demonstrated by comparing the fitting parameters obtained when data sets have the same numbers of cases and data points. Since these varied with our experimental conditions, an alternative method was adopted. First, the ED 50 was determined using linear interpo- lation between data points above and below 50% fusion. (offset) was calculated subtracting ordinate values 50 Translational sets. determinations of significance P Two variations of the CV—CV fusion assay were also examined. One variation was used to test whether CV membrane constituents themselves could initiate membrane contact that would support fusion without centrifugation. This assay is similar to that described above except that CV were permitted to settle into contact for 60 min before Ca 2 (cid:49) addition, and then further processed using a single centrifugation step to facilitate the dispersal of vesicle contents from the collapsed, fused CV. To test the effects of exposing CV to Ca 2 (cid:49) during the initiation of CV—CV contact rather than after contact had already been established, 100- (cid:109) l aliquots of CV suspension were added directly to 100- (cid:109) l aliquots of the cation-IM buffer stocks and initial A 405 was immediately measured. The microtiter plates containing the suspended CV were then centrifuged twice for com- parison to results obtained in the standard assay format and the turbidity was measured.