Properiy segregating genetic material in mitosis and meiosis is crucial for cell survival and normal development. During cell cycle progression in mitosis, distinct molecular mechanisms ensure condensation, orientation and segregation of replicated chromosomes. The mitotic microtubule spindle array. Its interaction with chromosomes via the kinetochore and the multiple components of the spindle-assembly checkpoint (SAC) guarantees that the structural and chronological orchestration of events happen normally and repetitively in actively dividing cells. The SAC proteins, including the kinesin motor protein centromere associated protein E (CENP-E), are localized at the kinetochore of unattached mitotic chromosomes. While it is clear that depletion of CENP-E in cells leads to chromosome instability and subsequently tumor formation, how CENP-E's role in the congression of mono-oriented chromosomes to the metaphase plate is integrated into overall SAC function remains pooriy understood. Moreover very little is known about the SAC in plants. The proposed pilot project will test three key hypotheses in plant cell cycle control. The first evaluates whether plant (Arabidopis thaliana) CENP-E is Involved in growth control via the SAC, more specifically in regulating the timing of chromosome metaphase plate alignment. Second, the loss of CENP-E function will lead to aberrant chromosome alignment, and support one ofthe following models: 1) CENP-E will attach chromosomes to bipolar spindle microtubule fibers and align chromosomes at the metaphase plate;2) CENP-E will move chromosomes attached to monooriented spindle microtubule fibers towards the metaphase plate, 3) plant CENP-Es can do both 1 and 2, and this will be revealed with the analysis of both AtCENP-E1 and AtCENPE2 from Arabidopsis, or 4) CENP-E in plants does neither 1 nor 2, but rather has a distinct function. Thirdly, cytokinesis is not affected in loss-of function mutants, indicating that CENP-E functions primarily in prometaphase and metaphase of the cell cycle. Testing the above hypotheses will not only advance the plant cell cycle field but also contribute to the general understanding of motor protein function, check point control, ploidy and cancer development. The collaboration between Adan Colon-Carmona, PhD, and David Pellman, MD, provides a unique opportunity for Dr. Colon-Carmona's career development, and the training of students will be central to the project.
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