Figure 1. Doc2B requires synaptotagmin-1 and Munc13-2 to increase the Readily Releasable Pool (RRP) size.

(A) Expression of Doc2B in Doc2B knockout (KO) mouse adrenal chromaffin cells. Top panels: Intracellular [Ca²⁺] (mean ±SEM) after and (insert) shortly before Ca²⁺ uncaging (uncaging light for 1–2 ms at red arrow). Middle panel: average capacitance traces from Ca²⁺ uncaging experiments in Doc2B KO cells (magenta traces, n = 40 cells) and after overexpression of Doc2B WT in Doc2B KO cells (grey traces, n = 36 cells). Bottom panel: amperometric measurements (mean traces) from the same cells. Traces showing a release transient after the uncaging stimulus are the amperometric currents (left ordinate axes). Monotonically increasing traces display the time-integral of the amperometric current (i.e. the charge), scaled to the right ordinate axis. Note that the time-integrals display the same general shape as the capacitance traces, as expected. (Ai) The amplitude (mean ±SEM) of the fast burst was increased by Doc2B (***p<0.001, Mann-Whitney test), whereas (Aii) the amplitude of the slow burst component remained constant. Fast and slow burst correspond to the sizes of the RRP and the SRP, respectively. (Aiii) The rate of the near-linear sustained component (D) was strongly reduced by Doc2B (***p<0.001, Mann-Whitney test). (Aiv) The fusion time constant for the fast burst, and (Av) fusion time constant for the slow burst were unaffected by Doc2B. (B) Ca²⁺ uncaging experiment in syt-1 KO cells (n = 18) and syt-1 KO cells overexpressing Doc2B WT (n = 20). Panels (B–Bv) are arranged as in A. Doc2B expression led to a highly significant reduction in sustained release, but no significant changes in the fast or slow burst sizes. ***p<0.001, Mann-Whitney test. (C) Ca²⁺ uncaging experiment in syt-7 KO cells (n = 17) and syt-7 KO cells overexpressing Doc2B WT (n = 17). Panels (C–Cv) are arranged as in A. Doc2B expression led to a significant increase in fast burst size (**p<0.01, Mann Whitney test), and a reduction in sustained release (**p<0.01, unpaired, two-tailed Student’s t test), as in control experiments (panel A). (D) Ca²⁺ uncaging experiment in Munc13-2 KO cells (n = 16) and Munc13-2 KO cells overexpressing Doc2B WT (n = 18). Panels (D–Dv) are arranged as in A. All components of release were reduced by overexpression of Doc2B WT in the absence of Munc13-2 (**p<0.01, ***p<0.001, Mann Whitney test; # p=0.0575, unpaired two-tailed Student’s t-test). The time constant of the slow component was increased (***p<0.001, unpaired two-tailed Student's t-test).

Figure 1—figure supplement 1. Overexpression of Doc2B in wild-type cells increases the burst size and decreases the sustained release.

(A) Calcium uncaging experiments in CD1 mouse adrenal chromaffin cells (n = 20 cells) and CD1 cells overexpressing Doc2B (n = 20 cells). Top panel: intracellular [Ca²⁺] (mean ±SEM) obtained by microfluorimetry just after and (insert) before calcium uncaging (uncaging light for 1–2 ms at red arrow). Middle panel: Average capacitance traces of CD1 adrenal chromaffin cells (black trace) and CD1 cells overexpressing Doc2B WT (red trace). Bottom panel: amperometric measurements (average traces) from the same cells. The left ordinate axis represent the amperometric current, the right axis represent amperometric charged. (Ai) The amplitude of the fast burst (mean ±SEM) and the slow burst (Aii) were increased by overexpression Doc2B WT in CD1 cells (Mann-Whitney test, **p<0.01; ***p<0.001). The rate of the sustained component (Aiii) was strongly reduced by overexpressing Doc2B WT in CD1 cells (Mann-Whitney test, ***p<0.001). The fusion time constant of the fast (Aiv) and slow (Av) burst of release were unaffected. (B) Calcium uncaging experiments in Black6 mouse adrenal chromaffin cells (n = 25 cells) and Black6 cells overexpressing Doc2B (n = 26 cells). (Bi) The amplitude of the fast burst (mean ±SEM) was increased by overexpression of Doc2B WT in Black6 cells (Mann-Whitney test, *p<0.05) while the slow burst (Bii) was unchanged. In Black6 mouse chromaffin cells, the sustained rate was also dramatically reduced by overexpressing Doc2B WT (Mann-Whitney test, **p<0.01) and the fast (Biv) and slow (Bv) burst time constants were unaffected.

Figure 1—figure supplement 2. No difference of expression of Doc2B in Syt-1 and Syt-7 WT vs KO mouse adrenal glands.

(A) Western blot analysis of Doc2B expression in Syt-1 and Syt-7 (WT and KO) adrenal glands. Syt-1 and Syt-7 antibodies were used as a control for the genotype. VCP antibody was used as a loading control. (B) Quantification of Doc2B signal in Syt-1 and Syt-7 mouse adrenal glands showing no difference of Doc2b expression. (n = 1 animal/condition, no statistical test).