Controlling lifetimes of exocytic events using the CherryTemp system
Yeast cells strains expressing a GFP labeled exocyst complex component were grown overnight in standard yeast peptone dextrose medium (YPD) and were cultured at 30°C with continuous shaking at 280 rpm. After overnight culture, cells were washed 3x with H2O and diluted 10-fold in SC-All media for 2h at 30°C. Afterwards cells were mounted on glass cover slips coated with Concanavalin A.
An iMIC-based microscopy (Thermo/Till Photonics) with an Olympus x100 1.45 NA objective was used for image acquisition.
Images were taken with Andor iXON DU-897 EMCCD using the LiveAcquisition software (Thermo).
Shifts between temperatures were performed using the CherryTemp device with a 1 min delay to allow for cell adaption.
Recorded timelapses of exocytosis were analyzed via kymographs to determine the lifetimes of exocytic events at different temperatures.