Figure 1.

In vitro single-vesicle content-mixing assay with Cpx. (a) Schematics of the In vitro single-vesicle content-mixing assay with Cpx. The flow chamber maintains constant Cpx concentration throughout the experiment by pretreating t-vesicles with Cpx prior to immobilization to the imaging surface. After the t-vesicles are immobilized on the imaging surface, unbound t-vesicles are washed out with buffer containing the designated Cpx concentration. Subsequent docking and washing of unbound v-vesicles are also performed in the presence of Cpx. Once the v-vesicles and t-vesicles are docked in the presence of Cpx, we inject Ca²⁺ into the flow chamber to evoke content-mixing which is detected by a sudden stepwise increase of fluorescent intensity. (b) A representative fluorescent intensity time trace is shown. The blue arrow indicates the time of Ca²⁺ injection and the red arrow depicts content-mixing.