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

This protocol details two methods of yeast cell cycle arrest and optional release, and elaborates on the use of fluorescence microscopy to study cell cycle-dependent processes in S. cerevisiae.

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.

Eukaryotic cell division is highly regulated through a program called the cell cycle. The highly conserved and dynamic processes occurring in the cell cycle make it interesting to study in-and-of-itself, but also have wide-spread implications that inform investigations of other cell biological processes-for example, many organelles undergo dramatic remodeling during cell division, and the abundance and localization of many proteins is highly regulated throughout^1,2,3. Although there are some additional layers of complexity present in metazoan systems compared to yeast, includ....

1. Construction of strains for cell cycle analysis and imaging

1. Design primers to C-terminally tag the gene of interest using Pringle Tagging Plasmids (pFA6a plasmids)²⁴. In short, design F2 and R1 primers that, when used with a pFA6a-series plasmid, produce a PCR product that can be directly transformed into yeast to generate a 'tagged' version of the gene of interest. Use the primer pairs and plasmids contained in Table 1 to tag the genes of interest in this protocol. Plasmid and primer design strategies for C-terminal tagging of yeast genes are described in detail by Longtine et al.²⁴

Analyzing changes in cell cycle-dependent protein localization by fluorescence microscopy can be readily accomplished using the methods we describe here. Our group has long been interested in the dynamic regulation and function of the mitotic spindle. In yeast, spindle pole bodies (marked by component Spc110) function as microtubule organizing centers from which microtubule filaments emanate to create the structure of the mitotic spindle³⁰. The microtubule binding .......

Utilizing cell cycle synchronization in budding yeast enables studying important mechanisms for a variety of cellular processes. The use of G1 arrest-releases with α-factor treatment allows synchronous progression of a population of cells through the stages of the cell cycle, and as we showed, can reveal dynamic localization patterns of cellular regulators like Stu2³⁶. Cell cycle arrests can also be accomplished using genetic means via depletion of cell cycle regulators like Cdc20, w.......

The authors declare no competing financial interests.

We thank the University of Utah Cell Imaging Core for maintaining the Delta Vision microscope facility. This work was supported in part by NIH grants F31CA2717405 (to M.G.S) and T32GM141848 (to M.G.S. and T.C.S.), 5 For the Fight (to M.P.M.), Pew Biomedical Scholars (to M.P.M.), and NIH grant R35GM142749 (to M.P.M.).