Supplementary data figure of electrophysiological experiments depicted in Figures 5 and 6. (a) Loading protocol. Cells were incubated with cg-PI(4,5)P2 for 30 min leading to reliable uptake of cg-PI(4,5)P2 as indicated by the coumarin fluorescence (bright field and fluorescence images of a wildtype cell inserted, histogram shows mean intensity ± SEM, differences between means were tested by a Student’s two tailed t-test: p=0.533). (b) Physiological paradigm to investigate the effect of PI(4,5)P2 uncaging on exocytosis. (c) Average capacitance traces of exocytosis elicited during the pre-uncaging depolarization train (pre-pulse, left) and during the identical test pulse (right). The average exocytosis of wild type (light blue), Munc13-2 KO (magenta), CAPS1/−2 DKO (yellow) and syt-1 KO (red) which were subjected to UV uncaging in-between the pre- and the test pulse are shown together with their respective controls (grey, no uncaging). As expected, exocytosis in response to the pre-pulse was identical between cells in the uncaging group compared to their controls (due to identical treatment at this stage, the distinction in terms of light treatment occurs later). (d) Quantification (mean ±SEM) of data in (c), performed as outlined in Figure 5a for all genotypes. To investigate whether the magnitude of exocytosis influenced the PI(4,5)P2-sensitivity, we analyzed subsets of wild type cells with intrinsically low exocytosis (wild type low exo, with suitable maximal thresholds (0 < IRP < 20 fF, 0<(RRP-IRP)<80 fF, 0<(total-RRP)<65 fF) during pre-pulse) and of syt-1 KO cells with intrinsically higher exocytosis (syt-1 KO high exo, with suitable minimal thresholds (3 fF <IRP, 12 fF<(RRP-IRP), 8 fF<(total-RRP)) during pre-pulse). While release could still be augmented in wild type cells with low release, PI(4,5)P2 uncaging was without effect in syt-1 KO cells with high release. These data argue that the absence of potentiation by PI(4,5)P2 uncaging in syt-1 KO cells is not caused by the lower total exocytosis in these cells, which is in line with the augmenting effect observed in CAPS1/−2 DKO cells, which also have reduced exocytosis. Statistically significant differences between the means were tested using Student’s two-tailed t-test: pre-pulse wild type: (IRP) p=0.0439, (RRP-IRP) p=0.686, (total-RRP) p=0.857, (slope) p=0.868; test-pulse wild type (IRP) p=0.896, (RRP-IRP) p=0.000999, (total-RRP) p=0.256, (slope) p=0.00601; pre-pulse Munc13-2KO: (IRP) p=0.210, (RRP-IRP) p=0.183, (total-RRP) p=0.691, (slope) p=0.311; test-pulse Munc13-2KO (IRP) p=0.0391, (RRP-IRP) p=0.431, (total-RRP) p=0.999, (slope) p=0.650; pre-pulse CAPS DKO: (IRP) p=0.466, (RRP-IRP) p=0.840, (total-RRP) p=0.487, (slope) p=0.989; test-pulse CAPS DKO (IRP) p=0.0762, (RRP-IRP) p=0.0778, (total-RRP) p=0.0449, (slope) p=0.0200; pre-pulse Syt-1KO: (IRP) p=0.813, (RRP-IRP) p=0.149, (total-RRP) p=0.823, (slope) p=0.212; test-pulse Syt-1 KO (IRP) p=0.645, (RRP-IRP) p=0.537, (total-RRP) p=0.607, (slope) p=0.258; pre-pulse wild type low exo: (IRP) p=0.286, (RRP-IRP) p=0.930, (total-RRP) p=0.708; test-pulse wild type low exo (IRP) p=0.448, (RRP-IRP) p=0.00725, (total-RRP) p=0.356; pre-pulse Syt-1 KO high exo: (IRP) p=0.954, (RRP-IRP) p=0.593, (total-RRP) p=0.447; test-pulse Syt-1KO high exo (IRP) p=0.590, (RRP-IRP) p=0.417, (total-RRP) p=0.147. #p<0.08; *p<0.05, ***p<0.001. Scale bars 20 fF/1 s. n.d.: not determined. Number of cells (n) in electrophysiological recordings: n = 50 (wild type control), n = 49 (wild type PI(4,5)P2 uncaging), n = 21 (CAPS1/−2 DKO control), n = 20 (CAPS1/−2 DKO PI(4,5)P2 uncaging), n = 32 (Munc13-2 KO control), n = 37 (Munc13-2 KO PI(4,5)P2 uncaging), n = 33 (syt-1 KO control), n = 36 (syt-1 KO PI(4,5)P2 uncaging).