During the growth of somatic cells, mitotic divisions partition one copy of each chromosome to each daughter cell. The production of gametes requires a special form of chromosome partitioning, meiosis, in which two sequential rounds chromosome segregation yield gametes with half the number of chromosomes of the somatic cells. The meiotic process is accomplished in part by layering specialized levels of meiosis-specific control upon the cellular machinery that is used for mitosis. Identifying these meiosis-specific controls is central to deciphering the mechanisms that are used to achieve high fidelity chromosome segregation in meiosis. MPS1 encodes a conserved, essential, kinase that has been shown be involved in several key steps in chromosome segregation in both mitotic and meiotic cells in many species. Mps1 is involved in: 1) duplication of the spindle pole body (in budding yeast), the structure that organizes the assembly of microtubules into the spindle that will pull the segregating chromosomes apart, 2) participation in the spindle assembly checkpoint mechanism that signals to the cell that a chromosome is attached inappropriately to microtubules, 3) mediating the release of these inappropriate attachments so that new ones can form, and 4) (in budding yeast) promoting the proper assembly of spore walls. Because of the multiple roles of Mps1 protein, MPS1 mutants often have complex phenotypes that are not informative for genetic studies. The recent discovery of a specific mutation, mps1-R170S has opened the door to exploring meiosis-specific roles for Mps1. mps1-R170S mutants have very mild defects in mitotic growth but catastrophic defects in chromosome segregation in meiosis. msp1-R170S mutants exhibit very high levels of chromosome segregation errors in both the first and second meiotic division. Thus the mps1-R170S destroys only some functions of Mps1 (meiosis-specific functions) and provides the opportunity to study those defects in ways that should reveal the role of the Mps1 protein in meiosis. Preliminary data suggest two hypotheses for the role of Mps1 in meiosis. This project will test these hypotheses. Either hypothesis, or a combination of the two, would explain the observed meiotic behavior mps1-R170S mutants. The first hypothesis is that Mps1 plays an essential meiotic role in dissolving kinetochore-microtubule attachments. By this hypothesis, kinetochores are attached early in both meiotic divisions to microtubules from only one spindle pole body and Mps1 is required to release these attachments so that attachments to both spindle pole bodies can be formed. The second hypothesis is that Mps1 is required to release an association between sister chromatids in meiosis. According to this hypothesis a persistent association of sister chromatids locks homologous chromosomes together in meiosis I, forcing them to move together to one spindle pole at meiosis I, and forcing sister chromatids to move to one pole at meiosis II. A final objective of this proposal is to use genetic approaches to identify the partners with which Mps1 interacts in meiosis.

Broader Impacts: Because Mps1 is highly conserved, these studies of Mps1 in yeast meiosis may reveal general principles of Mps1 function, and mitotic and meiotic chromosome behavior, that are widely applicable to most eukaryotic organisms. The project will provide a training opportunity for one post-doctoral researcher. In addition, this project will be used to provide summer students, from a local high school and undergraduate training program, with independent research projects. In this program the students will learn fundamentals of genetics while contributing to the analysis of genes that interact with Mps1 in meiosis. The post-doctoral researcher will not only perform most of the experiments on the project but will serve as comentor for the undergraduate/high school trainees.

Project Report

In both mitosis and meiosis, correct chromosome segregation depends on creating connections between chromosomes and the microtubules that pull chromosomes during the segregation process. In mitosis, equal chromosome sets are partitioned to opposite sides of the cell before it divides to create two daughters with identical chromosome sets. In meiosis, two sequential rounds of chromosome segregation occur to produce gametes with half to normal chromosome content such that fusion of two gametes creates a new zygote with the normal chromosome number. The microtubules radiate from the opposite ends, or poles of a structure called the spindle. In mitosis each chromosome is replicated to form a pair of joined sister chromatids that are pulled away from each other to opposite poles of the spindle. In meiosis, each chromosome pairs with its homologous partner and the homologs attach to microtubules and are then segregated. In each case, if the partners fail to segregate away from each other and instead migrate to the same pole then cells are created with the wrong number of chromosomes. In humans such errors are associated with cancer and are the cause of infertility and most birth defects. The goal of this study was to explore the role of a regulatory protein, Mps1, which is conserved across eukaryotic species and critical for high fidelity chromosome segregation. Previous work had shown that yeast cells with mutations in the Mps1 gene had severe defects in meiotic chromosome segregation, suggesting that by studying this process in yeast we might be able to elucidate the role of Mps1 in controlling chromosome segregation. Most of the studies were done by affixing a fluorescent protein to one yeast chromosome pair, then using microscopy to study the behavior of that pair. The behavior of chromosomes and the microtubules were examined throughout meiosis to determine the steps used to position chromosomes in the cell. Cells with Mps1 inactivated at different times in meiosis were studied as were mutants in the Ipl1 protein which is also a conserved protein required for proper chromosomes segregation. Defects in either gene resulted in catastrophic errors in meiosis producing gametes with incorrect chromosome numbers. The experiments revealed a number of new features of the meiotic process and the roles of Ipl1 and Mps1 in that process. Cells began meiosis with all chromosomes attached together to an organelle (the spindle pole body) that organizes the growth of microtubules. Ipl1 could release the chromosomes so that homologous partners were free to be re-positioned so they could pair with one another. The experiments revealed that in most cases, the paired yeast chromosomes initially attached to microtubules in such a way that if they proceeded, they would migrate to the same pole. In wildtype yeast cells, chromosomes moved back and forth across the spindle multiple times pulled by microtubules connected to one partner or the other. After several attempts they ended up correctly positioned in the middle of the spindle with microtubules attached to both partners so the could ultimately be pulled apart from each other. In this process, Ipl1 was found to release, improper attachments that pulled both partners to one pole, and Mps1 was necessary to induce new attachments that would position the chromosomes for correct segregation. Together the results showed that Ipl1 and Mps1 have opposite jobs (disconnecting and reconnecting microtubules from chromosomes) and that eliminating either one resulted in missegregation of the chromosomes.

National Science Foundation (NSF)
Division of Molecular and Cellular Biosciences (MCB)
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Gregory W. Warr
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Oklahoma Medical Research Foundation
Oklahoma City
United States
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