LTE1 promotes exit from mitosis by multiple mechanisms

In budding yeast, alignment of the anaphase spindle along the mother–bud axis is crucial for maintaining genome integrity. If the anaphase spindle becomes misaligned in the mother cell compartment, cells arrest in anaphase because the mitotic exit network (MEN), an essential Ras-like GTPase signaling cascade, is inhibited by the spindle position checkpoint (SPoC). Distinct localization patterns of MEN and SPoC components mediate MEN inhibition. Most components of the MEN localize to spindle pole bodies. If the spindle becomes mispositioned in the mother cell compartment, cells arrest in anaphase due to inhibition of the MEN by the mother cell–restricted SPoC kinase Kin4. Here we show that a bud-localized activating signal is necessary for full MEN activation. We identify Lte1 as this signal and show that Lte1 activates the MEN in at least two ways. It inhibits small amounts of Kin4 that are present in the bud via its central domain. An additional MEN-activating function of Lte1 is mediated by its N- and C-terminal GEF domains, which, we propose, directly activate the MEN GTPase Tem1. We conclude that control of the MEN by spindle position is exerted by both negative and positive regulatory elements that control the pathway’s GTPase activity.National Institutes of Health (U.S.) (Grant HD085866)National Cancer Institute (U.S.)David H. Koch Institute for Integrative Cancer Research at MIT. Support (Core) (Grant P30-CA14051

The Mitotic Exit Network detects spindle position and anaphase entry

This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2019Cataloged from student-submitted PDF version of thesis.Includes bibliographical references.The Mitotic Exit Network (MEN), an essential GTPase signal-transduction cascade, controls mitotic exit in budding yeast. The MEN protects genomic integrity by ensuring chromosome segregation is complete prior to cytokinesis. Two signals are required for MEN activation: (1) movement of the nucleus into the daughter cell and (2) anaphase onset. These two events only coincide after anaphase chromosome segregation, ensuring that mitosis is complete prior to cytokinesis. The MEN is regulated by spindle position. The MEN GTPase, Tem1, is inhibited as long as the entire spindle resides in the mother cell. Tem1 becomes active when spindle elongation along the mother-daughter axis drives half of the nucleus into the bud. If spindle elongation fails to move part of the nucleus into the daughter cell, MEN activation is prevented, providing time to reposition the spindle. In addition to this spatial regulation, activation of the MEN is restricted to anaphase by inhibitory cyclin-dependent kinase (Cdk) phosphorylation of the MEN kinase cascade. During anaphase onset, Cdk activity decreases; creating a temporal signal that releases the MEN from inhibition. This temporal signal prevents MEN activation should the nucleus move into the daughter cell prior to anaphase. By integrating multiple inputs the MEN creates a regulated cell-cycle transition that is responsive to cell-cycle stage and spindle position.by Ian Winsten Campbell.Ph. D.Ph.D. Massachusetts Institute of Technology, Department of Biolog

The Mitotic Exit Network integrates temporal and spatial signals by distributing regulation across multiple components

GTPase signal transduction pathways control cellular decision making by integrating multiple cellular events into a single signal. The Mitotic Exit Network (MEN), a Ras-like GTPase signaling pathway, integrates spatial and temporal cues to ensure that cytokinesis only occurs after the genome has partitioned between mother and daughter cells during anaphase. Here we show that signal integration does not occur at a single step of the pathway. Rather, sequential components of the pathway are controlled in series by different signals. The spatial signal, nuclear position, regulates the MEN GTPase Tem1. The temporal signal, commencement of anaphase, is mediated by mitotic cyclin-dependent kinase (CDK) phosphorylation of the GTPase's downstream kinases. We propose that integrating multiple signals through sequential steps in the GTPase pathway represents a generalizable principle in GTPase signaling and explains why intracellular signal transmission is a multi-step process. Serial signal integration rather than signal amplification makes multi-step signal transduction necessary.Eunice Kennedy Shriver National Institute of Child Health and Human Development (U.S.) (Grant HD085866