Coorssen JR et al. (1998), Biochemical and functional studies of cortical ...

ECB-ART-37066

J Cell Biol 1998 Dec 28;1437:1845-57. doi: 10.1083/jcb.143.7.1845.

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Biochemical and functional studies of cortical vesicle fusion: the SNARE complex and Ca2+ sensitivity.

Coorssen JR , Blank PS , Tahara M , Zimmerberg J .

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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.

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Species referenced: Echinodermata
Genes referenced: LOC100887844 LOC100893907 LOC105438433 LOC594261 VAMP
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	Figure 4. SNARE complex disruption has different sensitivities to Ca2+, Sr2+, and Ba2+. (a) The sensitivity of SNARE complex disruption to concentrations of Ca2+, Sr2+, or Ba2+ that trigger maximal fusion was compared. Contacting CV were exposed to either IM buffer alone (Control), 150 μM [Ca2+]free (Ca2 +), 20 mM [Sr2+]free (Sr2 +), or 20 mM [Ba2+]free (Ba2 +). (b) The sensitivity of SNARE complex disruption to increasing concentrations of Ca2+ was examined. Contacting CV were exposed to either IM buffer alone (zero calcium), or to the indicated [Ca2+]free. In all cases, proteins were extracted with Triton X-114, dissolved in SDS sample buffer without boiling and then analyzed by SDS-PAGE on 12% gels followed by immunoblotting. Immunoblots were probed with anti-VAMP antibody to visualize both VAMP monomers (∼19 kD; data not shown) and the SNARE complex (∼70 kD; Tahara et al., 1998).
	Figure 5. The SNARE complex remains Ca2+ sensitive on fusion-incompetent CV. The Ca2+ sensitivity of SNARE complex disruption was tested with CV rendered nonfusogenic by a 1-h exposure to chaotropic buffer at 37°C. (A) After incubation in PKME buffer at 37°C or on ice (Control), CV suspensions were tested for fusion in response to Ca2+ using the standard assay format (Materials and Methods); each point is the mean ± SEM from three to seven separate determinations from five CV preparations. (B) Parallel samples were exposed to either buffer alone (−), or to 100 μM [Ca2+]free (+). CV in PKME buffer had previously been incubated either on ice for 1 h (Control), or at 37°C (37°C). Proteins were extracted with Triton X-114, dissolved in SDS sample buffer without boiling, and then analyzed by SDS-PAGE on 12% gels followed by immunoblotting. Immunoblots were probed with anti-VAMP antibody.
	Figure 6. There is no direct correlation between exocytosis and SNARE complex disruption. (a) The Ca2+ sensitivity of SNARE complex disruption on CSC was examined. CSC in suspension were exposed to either IM buffer alone (zero calcium) or to the indicated [Ca2+]free in assays analogous to those used to measure exocytosis from CSC (Fig. 1 a). Proteins were extracted and analyzed as described in Fig. 4. (b) Ca2+-triggered exocytosis was measured in the well-characterized egg cortex preparation using a double Ca2+ challenge paradigm. Egg cortices were isolated in a KCl-based buffer and exocytosis assayed in a rapid perfusion microscope chamber, as described (Blank et al., 1998). Cortices were first challenged with 14 μM [Ca2+]free (short arrow) and subsequently with 24 μM [Ca2+]free (tall arrow). The resulting light-scattering data were normalized such that ∼100 nM [Ca2+]free gave 0% fusion and 300 μM [Ca2+]free gave 100% fusion (Blank et al., 1998).

	Figure 8. CV remain firmly attached to the plasma membrane despite SNARE complex disruption. Isolated planar cortices from sea urchin eggs were monitored by quantitative light scattering in a rapid-flow perfusion chamber. After treatment of the isolated cortices for 10 min with 100 μM LPC in the presence of low [Ca2+]free, cortices were perfused successively with (i) LPC plus 100 μM [Ca2+]free, sufficient to fully disrupt SNARE complexes (low flow, 0–60 s); (ii) LPC plus 100 μM [Ca2+]free under high shear conditions (8 ml/s solution delivery, 65–70 s); and (iii) 1 mM [Ca2+]free at low flow (beginning at arrow). Representative of nine experiments.
	Figure 9. CV membrane components mediate docking and the SNARE complex may enhance the Ca2+ sensitivity of fusion. (a) CV allowed to settle into contact for 60 min before Ca2+ application (open circles; dotted line fit) undergo extensive fusion; centrifugation of these samples (solid circles; solid line fit) results in a curve slightly right-shifted relative to samples assayed using the standard format (curve to left, from Fig. 1 a). Each point is the mean ± SEM of 3–14 separate determinations, done in parallel with standard assays. (b) Exposure to Ca2+ while bringing CV into contact results in a significant loss of Ca2+ sensitivity for fusion. CV suspensions were added directly to cation buffer stocks before centrifugation (Materials and Methods). There is a significant rightward shift in the Ca2+ activity curve (open circles) relative to the standard assay (curve to left, from Fig. 1 a). Comparable assays involving Sr2+ (open squares) yield a less pronounced shift relative to the standard assay (Sr2+ curve from Fig. 1 a). Each point is the mean ± SEM of 3 –15 separate determinations for Ca2+ and eight or nine for Sr2+, done in parallel with standard assays. In all experiments, results have been normalized to the peak Ca2+-induced fusion in parallel standard assays. (c) The Ca2+ activity curves for the CV–CV fusion assays described in a (solid circles) and b (open symbols) show translational invariance and superimpose on a comparable curve for Ca2+-triggered exocytosis from the planar cortex (Blank et al., 1998). Dashed lines delimit the 99% confidence interval for the cortex data.

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