Saccharomyces cerevisiae microtubule and kinetochore dynamics - Project Summary/Abstract For replicated chromosomes to be segregated to two daughter cells accurately, the microtubule (MT) cytoskeleton must be completely remodeled to form a bipolar spindle. A large, dynamic protein complex called the kinetochore attaches replicated chromosomes to microtubules emanating from opposite spindle poles. Proper kinetochore-microtubule attachment is vital for preservation of genomic integrity and prevention of cancer and birth defects. Therefore, mitotic spindle assembly and kinetochore attachment must be well coordinated. Challenging understanding of these processes and their coordination is the fact that mitotic spindles and kinetochores are extremely complex molecular machines (kinetochores contain >60 proteins), that they are targets of phosphoregulation by multiple protein kinases, and that they possess a striking range of biochemical activities. Kinetochore activities include: (1) lateral MT binding, (2) translocation along the MT lattice to the plus end, (3) conversion from lateral to end-binding, (4) association with dynamic MT ends while tubulin subunits are exchanged, and (5) serving as force-coupling devices between chromosomes and MT plus ends during anaphase A. Understanding how the kinetochore performs its various functions, the structural underpinnings of these activities, and how these activities are regulated post-translationally, is far from complete. A biochemical cell-lysate assay recently developed in the Barnes laboratory combines, for the first time, two of the most powerful approaches for studies of microtubule dynamics: biochemical extract studies and genetics. Dynamics of single microtubules and single kinetochores associated with these microtubules are revealed and quantitatively analyzed by highly sensitive Total Internal Reflection Fluorescence microscopy. Cell lysates synchronized to specific cell-cycle stages are made from budding yeast mutants of specific mitotic proteins. Quantitative analysis of MT dynamics and kinetochore activities will establish how these parameters are regulated in the cell cycle, and these studies will identify the specific proteins that carry out the specific behaviors. Since mitosis is a highly conserved process, lessons learned from these studies are expected to apply broadly. Unlike many other assays, this assay exclusively uses homologous sources of tubulin and interacting proteins, avoiding artefacts that arise from species mismatch incompatibilities. Proposed studies build upon recent unique observations of microtubule dynamics regulation and kinetochore dynamic activity in this lysate system. The objectives are: (1) To investigate biochemical activities of intact kinetochores and their regulation, and to relate kinetochore structure to function; and (2) To investigate how microtubule dynamics are regulated through the cell cycle in both the nucleus and the cytoplasm, focusing on Kar3 and Kip3 kinesins.
