Figure 2. Syt1 promotes fusion and expands fusion pores in a calcium and PI(4,5)P₂ dependent manner, and soluble Syt1 C2AB largely recapitulates these effects.

(A) The rate at which current bursts appeared (pore nucleation rate) for the conditions indicated (error bars represent ± S.E.M.). SNARE-induced pores appeared more frequently in the presence of Syt1 or C2AB, when both calcium and PI(4,5)P₂ were also present. Student's t-test (one-tailed) was used to assess significant differences between the 'no Syt1' group and the rest. *, **, *** indicate p<0.05, 0.01, and 0.001, respectively. There is no difference between the Syt1 and C2AB groups in the presence of calcium and exogenous PI(4,5)P₂ (Student’s t-test: $p=0.18$). (B) Mean single fusion pore conductance, $⟨G_{po}⟩$, for different conditions as indicated (± S.E.M.). $⟨G_{po}⟩$ was three-fold larger in the presence of Syt1 or C2AB, when both calcium and PI(4,5)P₂ were also present. Two-sample Kolmogorov-Smirnov test was used to assess significant differences between the 'no Syt1' group and the rest. The same asterisk notation as in A was used. There is no difference between the Syt1 and C2AB groups in the presence of calcium and exogenous PI(4,5)P₂ (two-sample Kolmogorov-Smirnov test: $p=0.29$). (C) Probability density functions (PDFs) for point-by-point open-pore conductances (see Materials and methods) for pores induced in the presence of Syt1, PI(4,5)P₂ and with 0 or 100 μM calcium. Notice the higher density at larger conductance values in the presence of 100 μM calcium. (D) Probability density functions for pore radii, calculated from the conductance PDFs in C, assuming a 15-nm long cylindrical pore (Hille, 2001). (E) Apparent free energy profiles for Syt1 and soluble Syt1 C2AB domains in the absence or presence of calcium. These profiles were calculated from the pore radii PDFs as in D (see text and Materials and methods) (Wu et al., 2017b). The profiles were shifted vertically for clarity. (F) Cumulative density functions (CDFs) for mean single-pore conductances for the conditions indicated. Soluble C2AB recapitulated effects of full-length Syt1 co-reconstituted into NLPs.