Manipulation and Analysis of Cell Cycle-Dependent Processes in Budding Yeast.

Eukaryotic cells follow a conserved cell cycle that regulates diverse processes, including DNA maintenance and organelle homeostasis. Studying cellular processes in a cell cycle-dependent manner is often necessary to properly interpret experimental results. There are chemical and genetic methods available to produce cell cycle synchronization in cultured cells across a wide swath of organisms, including vertebrate models, enabling the study of cell cycle-dependent processes. However, among model organisms, budding yeast remains a powerhouse for cell cycle analysis due to its particularly robust synchronization methods, short generation time, and genetic tractability. Yeast shares core cell cycle machinery with other eukaryotes, which has enabled landmark discoveries in cell cycle regulation. This protocol details methods for cell cycle analysis in yeast, focusing on G1 arrest-release and mitotic arrest-release experiments, including strain construction, culture preparation, and microscopy. PCR tagging methods for producing suitable strains for cell cycle arrests and fluorescence microscopy are presented. A G1 arrest is achieved using the peptide pheromone α-factor, and brief washes result in synchronous release and cell cycle progression. Samples are taken at different time points following release into the cell cycle and fixed for microscopy. A second method arrests yeast cells in mitosis by depleting the cell cycle regulator Cdc20 to achieve a metaphase-arrested population, as well as optional release into anaphase. Samples are fixed and prepared for imaging pre- and post-release, and are imaged and analyzed. Image analysis focuses on cataloging dynamic localization and population abundance changes of proteins in the cell cycle. These synchronization methods are suitable for diverse cell cycle manipulations, and while their use in imaging fixed cells is highlighted here, they can be adapted for many other analyses, including live cell imaging as well as biochemical and molecular assays.