Figure 6.

Increasing [PIP₂] drives Ca²⁺-independent penetration by both C2 domains of Doc2β and syt1 and potentiates Ca²⁺-independent and -dependent vesicle fusion. A, NBD-labeled Doc2β C2AB was combined with liposomes harboring PS and increasing concentrations of PIP₂ in the absence of Ca²⁺, and NBD emission intensity was quantified. Increasing [PIP₂] drove substantial intensity increases from NBD labels on all four loops of Doc2β. This effect appeared to reach near-saturation at 5 mol % PIP₂. B, as in A but for syt1 C2AB. In addition to robust penetration by C2B loop 3, increasing [PIP₂] drove partial penetration by C2A loop 3. C–F, v-SNARE liposomes containing full-length syb2 and full-length syt1 were combined with t-SNARE liposomes containing full-length syntaxin-1A:SNAP-25B (syx:SN25) heterodimer and increasing mol % PIP₂. C, scheme of lipid-mixing assay. Fusion of vesicles was monitored by dequenching of NBD. D, results of lipid-mixing assays conducted with increasing mol % PIP₂ in the t-SNARE vesicles. Above, full traces; below, Ca²⁺-free portion of the trace shown on an expanded timescale. PIP₂ drove Ca²⁺-independent and -dependent lipid mixing in a dose-dependent manner. E, scheme of content-mixing assay. Fusion of vesicles was monitored by dequenching of sulforhodamine B. F, results of content-mixing assays conducted with increasing mol % PIP₂ in the t-SNARE vesicles. Above, full traces; below, Ca²⁺-free portion of the trace shown on an expanded timescale. As with lipid-mixing experiments, a dose-dependent effect of PIP₂ on Ca²⁺-independent and -dependent fusion was observed. Error bars, S.E. of four independent trials.