W1 confocal scanhead

Mid log-phase S. cerevisiae cells were mounted on an agarose pad made from SC media. For Figures 6 and 7, live cells endogenously modified to express fluorescently labeled DYN1-3XGFP, CFP-TUB1, and SPC110-tdTomato were imaged using a Yokogawa W1 confocal scanhead mounted to a Nikon Ti2 microscope with an Apo TIRF 100 × 1.49 NA objective. The microscope was run with NIS Elements using the 488 nm and 561 nm lines of a six-line (405 nm, 445 nm, 488 nm, 515 nm, 561 nm, and 640 nm) LUN-F-XL laser engine and Prime95B cameras (Photometrics). The localization of DYN1-3XGFP foci was assessed and the number of foci on each location (SPB, microtubule plus end, and cell cortex) was counted.

Single colonies were picked and grown at 30 °C. Log-phase live S. cerevisiae cells were mounted on a thin agarose pad made from SC medium pressed between two glass slides. Live cells genetically modified to express fluorescently labeled DYN1-3XGFP, CFP-TUB1, and SPC110-tdTomato were imaged using a Yokogawa W1 confocal scanhead mounted to a Nikon Ti2 microscope with an Apo TIRF 100 ×1.49 NA objective (Nikon, Plano Apo). The microscope was run with NIS Elements using the 488 nm 515 nm and 561 nm lines of a six-line (405 nm, 445 nm, 488 nm, 515 nm, 561 nm, and 640 nm) LUN-F-XL laser engine and Prime95B cameras (Photometrics). The DYN1-3×GFP foci localizing to the spindle pole body (SPB), microtubule plus end, and cell cortex were outlined as regions of interest in Fiji^{66 (link)}, recorded, and analyzed for three replicates of at least 120 cells for each sample.