Kinesin-Like Proteins in the Mitotic Mechanism

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Intellectual Merit: Microtubule kinesin-like proteins are crucial to the mitotic mechanism. To be effective, their actions must also integrate temporally in the spindle mechanism to ensure chromosome partitioning before mitotic exit. Kinesin-14 family proteins are ubiquitous in eukaryotes and able to effect mitotic progression through control of microtubule organization, dynamics and anchoring of spindle proteins. This project will provide insights into three important aspects of the mitotic mechanism: (1) the molecular nature of kinesin-tubulin interactions; (2) Kinesin-14 Pkl1 controlled spindle and microtubule parameters and required protein complexes; and (3) Kinesin-14 Pkl1p control of post-metaphase Mad2p transitions (bipolar to unipolar to equatorial) and their importance to mitotic exit. With regard to aspect (1), Dr. Paluh will test the hypothesis that kinesin-gamma-tubulin interactions at poles are reserved for Kinesin-14 family proteins, while microtubule tubulins contain additional information for binding of multiple kinesin families. No information is currently available at the molecular level that defines kinesin-tubulin interactions, which is a critical mechanistic component for the field. With regard to aspect (2), the underlying mechanism of bipolar spindle assembly remains fundamentally unknown. Dr. Paluh's prior research work identified a complex of Kinesin 14 Pkl1p and gamma-tubulin in this process. With regard to aspect (3), Mad2p is a key regulator of ubiquitin-mediated proteolysis and provides temporal control of mitotic progression by inhibiting the APC/C, anaphase promoting complex/cyclosome. The APC/C ubiquitin ligase regulates both sister chromatid cohesion and mitotic exit after anaphase. Mad2p at kinetochores monitors chromosome-microtubule attachment and tension preceding anaphase. Dr. Paluh's prior research work links Mad2p conserved movement to poles and SIN-like spindle transitions in mitotic exit pathways. Dr. Paluh has extensive cell cycle expertise in analysis of all aspects of the mitotic mechanism and reagents are in hand to monitor chromosome segregation, microtubule dynamics, spindle proteins, and checkpoint mechanisms. She will use a variety of different approaches, including comparative sequence analysis, structural analysis, mutagenesis, time-lapse video microscopy of mitosis, genetic analysis and molecular biochemical approaches that employ GST-pull downs, co-immunoprecipitation, and yeast two-hybrid assays. Broader Impact: The research will actively link new discoveries in the mitotic mechanism with undergraduate training that has a track record for promoting student participation at all levels of scientific investigation and strives for minority involvement. Dr. Paluh has an exemplary record of training undergraduates in teaching and research, and the students previously trained by Dr. Paluh are continuing in productive and recognized science careers. Eleven undergraduate researchers have been trained by Dr. Paluh to date. Of these, six students were able to attend national and international scientific meetings, including one who presented the work as a speaker at the meeting. Undergraduate researchers contributed extensively to preliminary findings that support this project. Seven students are coauthors on meeting abstracts or manuscripts related to this project. Two minority women undergraduates have been trained; one of these, Adrianna Rodriguez, was recently awarded the Boston College Young Alumni in Science Award. Under Dr. Paluh's direct tutelage, students acquire a comprehensive understanding of cell cycle control mechanisms while developing the rigors of scientific methodology. The range of experimental methodologies used in this project are accessible by undergraduate researchers, allowing them to work semi-independently.