Visualizing Microtubule Dynamics in Yeast

Budding yeast was grown in standard media and then manipulated and transformed by standard methods [40 ]. GFP-Tub1 fusions were integrated into the genome and expressed ectopically, in addition to the native $\alpha$ -tubulin genes TUB1 and TUB3 [41 (link)]. We estimate that GFP-Tub1 comprises approximately $25\%$ of the total $\alpha$ -tubulin expressed in these cells [42 (link)]. Cells were grown asynchronously to the early log phase in a nonfluorescent medium and adhered to slide chambers coated with concanavalin A [43 ]. Images were collected on a Nikon Ti-E microscope equipped with a 1.45 NA 100× CFI Plan Apo objective, piezoelectric stage (Physik Instrumente; Auburn, MA, USA), spinning disk confocal scanner unit (CSU10; Yokogawa, Musashino, Tokyo), 488 nm laser (Agilent Technologies; Santa Clara, CA, USA), and an EMCCD camera (iXon Ultra 897; Andor Technology; Belfast, UK) using NIS Elements software (Nikon, Minato City, Tokyo). During imaging, sample temperature was maintained at $37{}^{°}$ C as indicated using the CherryTemp system (CherryBiotech; Rennes, France). Z-stacks consisting of 12 images separated by 0.45 µm were collected at 5 second intervals for 10 minutes. All analyses were conducted in pre-anaphase cells, which typically exhibit one or two individual astral microtubules extending from each SPB [44 (link)].