Tie inverted stand

Cells were plated in fibronectin-coated, glass-bottomed plates (Mattek) 24 hours before imaging. Widefield timelapse imaging was carried out using a Nikon Ti inverted microscope or a Zeiss Axiovert 200M microscope at 5 minute timepoints using a 20x or 40x objective. Live confocal imaging was carried out using a Nikon TiE inverted stand attached to a Yokogawa CSU-X1 spinning disc scan head using a 100x objective. Cell shape descriptors were measured using FIJI (Schindelin et al., 2012 (link), https://fiji.sc/) following manual segmentation of cell area.

Fluorescence microscopy experiments were performed on mid-log yeast cultures in synthetic media at the indicated temperatures. Live yeast cell imaging data in all Figures were acquired with a 100× CFI Plan Apochromat VC oil-immersion objective lens (1.4 NA), using a PerkinElmer Ultraview Vox spinning disk confocal microscope that consists of a Nikon TiE inverted stand attached to a Yokogawa CSU-X1 spinning disk scan head, a Hamamatsu C9100-13 EMCCD camera, Prior NanoscanZ piezo focus, and a Nikon Perfect Focus System (PFS). All images were collected as square images with 512 × 512 pixels. The number of cells observed in experiments is reported in the figures and figure legends. The brightness and contrast of images were linearly adjusted and cropped in Photoshop (Adobe) for presentation.
For vacuole staining in Fig S3, 5 μM FM4-64 (Invitrogen) was added to mid-log cell cultures in YPD media for 15 min at 26°C. Cultures were then resuspended in fresh YND media and incubated for 1 h in at 26°C. 0.1 mM CellTracker Blue CMAC (Invitrogen) was then added 15 min before heat stress and imaging. For the myriocin treatment in Fig S6, 2 μM myriocin was added to mid-log cell cultures in YND media for 1 h at 26°C before heat stress and imaging.