Studying the Role of the Mitotic Exit Network in Cytokinesis

In budding yeast cells, cytokinesis is achieved by the successful division of the cytoplasm into two daughter cells, but the precise mechanisms of cell division and its regulation are still rather poorly understood. The Mitotic Exit Network (MEN) is the signaling cascade that is responsible for the release of Cdc14 phosphatase leading to the inactivation of the kinase activity associated to cyclin-dependent kinases (CDK), which drives exit from mitosis and a rapid and efficient cytokinesis. Mitotic CDK impairs the activation of MEN before anaphase, and activation of MEN in anaphase leads to the inactivation of CDK, which presents a challenge to determine the contribution that each pathway makes to the successful onset of cytokinesis. To determine CDK and MEN contribution to cytokinesis irrespectively of each other, here we present methods to induce cytokinesis after the inactivation of CDK activity in temperature sensitive mutants of the MEN pathway. An array of methods to monitor the cellular events associated with the successful cytokinesis is included.