Abstract
The cell cycle is an ordered series of events by which cells grow and divide to give rise to two daughter cells. In eukaryotes, cyclin-cyclin-dependent kinase (cyclin-Cdk) complexes act as master regulators of the cell division cycle by phosphorylating numerous substrates. Their activity and expression profiles are regulated in time. The budding yeast S. cerevisiae was one of the pioneering model organisms to study the cell cycle. Its genetic amenability continues to make it a favorite model to decipher the principles of how changes in cyclin-Cdk activity translate into the intricate sequence of substrate phosphorylation events that govern the cell cycle. In this chapter, we introduce robust and straightforward methods to analyze cell cycle progression in S. cerevisiae. These techniques can be utilized to describe cell cycle events and to address the effects of perturbations on accurate and timely cell cycle progression.
Highlights
The eukaryotic cell cycle is a highly regulated process that coordinates the doubling of all cellular content, especially genome duplication during DNA replication, with chromosome segregation and cell division
The timing of events and their unidirectional progression are controlled by the master cell cycle regulator, the cyclin– Cdk complexes
How are the phosphorylation and dephosphorylation timings of each cyclinCdk substrate controlled to coordinate the multitude of molecular events that are required for successful cell growth and division, and for maintaining the integrity of the genome? Here we report a series of techniques which allow the analysis of cell cycle progression in the unicellular model eukaryote S. cerevisiae
Summary
The eukaryotic cell cycle is a highly regulated process that coordinates the doubling of all cellular content, especially genome duplication during DNA replication, with chromosome segregation and cell division. The cyclical expression of cell cycle stage-specific cyclins results in oscillations of cyclin-Cdk activity This allows the step by step phosphorylation of many cyclinCdk substrates throughout the cell cycle [1]. A ubiquitin ligase complex, the APC/C (anaphase promoting complex/ cyclosome) is activated that ubiquitinates cyclins and thereby targets them for degradation This causes cyclin-Cdk activity to fall, facilitating the dephosphorylation of cyclin-Cdk substrates by counteracting phosphatases and leading to completion of cell division and return of the cell cycle to G1. Together with the budding index, this is a useful marker to follow DNA replication and cell division This is followed by a protocol for reliable protein extraction for Western blotting, used to visualize both protein abundance changes and electrophoretic mobility shifts that often go hand in hand with cyclin-Cdk phosphorylation. We turn to the cytological observation of nuclei and the microtubule cytoskeleton, which yields additional information on the progression through mitosis
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