Fig. 5. The effects of synaptotagmin mutations in C2B on secretory granule fusion and SNARE orientation.

a Single granule supported membrane TIRF fusion assay (left) and sdFLIC microscopy of the ternary SNARE complex (right). b Fluorescence intensity traces of single granules interacting with supported membranes in a TIRF microscope. After a sudden increase in fluorescence, indicating binding, the intensity stays constant if the granule never fuses (top) or the trace shows a characteristic dip and peak after different delay times (bottom) if the granule membrane fuses with the reconstituted supported membrane. c Averaged traces (20 events per trace) showing a fast mode of fusion in the presence of C2AB (red) or slow mode of fusion in the absence of C2AB (blue). Both conditions were in the presence of 100 µM Ca²⁺. d Granules depleted of synaptotagmins (Syt1 and Syt9) do not fuse in response to calcium. 0.4 μM soluble C2AB stimulates fusion of synaptotagmin knockdown granules to a similar level as granules containing endogenous synaptotagmin in the presence of 100 µM Ca²⁺ (membrane composition was 32:32:15:20:1 bPC:bPE:bPS: Chol:PIP₂). e Structural changes in the orientation of the ternary SNARE complex in the presence of calcium and 0.4 μM C2AB. f The effects of RQ, RQRQ, or KAKA mutations in C2AB on granule binding, fusion, release characteristics, SNARE orientation, and fusion kinetics in the presence of 100 µM Ca²⁺ (membrane composition was 32:32:15:20:1 bPC:bPE:bPS:Chol:PIP₂). The bar plots show mean ± standard error or the mean. The boxplots show mean, 2nd and 3rd quartile, and outliers of the data.