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4- Microscopy

Analysis of pathway-dependent Ste2 endocytosis by fluorescence microscopy was performed as follows: cultures were grown to early log phase (OD600=0.1-0.3) in complete synthetic dropout media and then transferred to a Concavaline-A treated 96-well fluorescence plate. Individual wells were observed at 100x in a Nikon Eclipse T2000 microscope. Ste2 endocytosis was induced by addition of 1 μM α-factor (Zymo Research) to activate the pathway. Time-lapse experiments were done by taking pictures every 1.5 minutes, for a total of up to 2 hours.