Apo tirf 100 1.49 na objective

Single colonies were picked and grown at 30 °C. Log-phase S. cerevisiae cells growing at 30 °C were transferred to 16 °C for 16 h. Cells were fixed with 75% ethanol for 1 h, sonicated for 5 s at 40% amplitude, and mounted in medium containing DAPI. Imaging was performed using an Apo TIRF 100 ×1.49 NA objective (Nikon, Plano Apo) on a Nikon Ti2 microscope with a Yokogawa-X1 spinning disk confocal system, MLC400B laser engine (Agilent), Prime 95B back-thinned sCMOS camera (Teledyne Photometrics), and a piezo Z-stage (Mad City Labs). The samples were blinded during imaging. The percentage of aberrant binucleate cells was calculated as the number of binucleate cells divided by the sum of wild-type and binucleate cells. Eight to 12 biological replicates from independent colonies were done for Figure 4c, and three were done for Figure 4d,i. At least 200 cells were counted for each biological replicate per condition.

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.