Recently initiated translational research on kinetochores is also discussed as kinetochores are being mined as a very rich target for the next generations of anti-cancer drugs. However, depending on which chromosome is affected, aneuploid organisms can be viable but will suffer from physical and mental disabilities, as exemplified by the Down syndrome. Activities During mitosis, kinetochores orchestrate chromosome transmission from the mother into the daughter cells. Structural protein studies are performed by hydrodynamics gel filtration plus glycerol gradient density velocity ultracentrifugation and crystallographic analysis of recombinant proteins following crystallization at our in-house crystallography facility. At the metaphase-anaphase transition, Cnn1 levels at kinetochores increase equaling those of the Mtw1 and Ndc80 complexes.
At the molecular level, we study yeast protein activity biochemically in yeast cell extracts or in in vitro assays using recombinant proteins produced in bacteria, yeast or insect cells. Hence, the segregation of the genome is coordinated in time and space. A screen for kinetochore-microtubule interaction inhibitors identifies novel antitubulin compounds. To identify new proteins involved in chromosome segregation we perform protein affinity purifications and yeast two-hybrid screens. Recently initiated translational research on kinetochores is also discussed as kinetochores are being mined as a very rich target for the next generations of anti-cancer drugs. In addition, the degradation of Mps1, decrease in Cdc28 activity due to cyclin degradation, and an increased dephosphorylation of Cnn1 allow the latter to bind to the Ndc80 complex, resulting in a looser kinetochore structure. As most solid tumors are aneuploid it has been hypothesized that chromosome missegregation drives or supports the cancer transformation process.
The latter may promote the binding of the Ndc80 complex to microtubules and to the microtubule-surrounding Dam1 ring complex, thereby supporting the transduction of forces generated by microtubule depolymerization that are required to mediate chromosome segregation. As chromosome segregation and the proteins involved are evolutionary conserved we study them in the yeast Saccharomyces cerevisiae. Most often, aneuploidy results in death e. To study chromosome segregation we implement a multi-dimensional approach. By integrating a variety of methodologies we wish to obtain a most detailed understanding of the proteins and regulators that drive chromosome segregation in yeast, and ultimately, in human cells. The binding of Cnn1 to Spc24-Spc25 is regulated by phosphorylation Cdc28 and Mps1 kinases and stabilizes the Ndc80 complex at the kinetochore. Our study of how Rio1 orchestrates chromosome segregation in time and space we will generate new insights into a most basic mitotic process and suggest how an anomalous expression of Rio1, as observed in various tumors, may underlie aneuploidy in dividing cells.
Kinetochores orchestrate the faithful transmission of chromosomes from one generation to the next. In addition, progression through the yeast cell cycle can easily be manipulated with small compounds or via mutations in regulatory proteins. However, to bind to the Ndc80 complex, the tail of Cnn1 must compete with the tail of Dsn1, a subunit of the tetrameric Mtw1 complex Mis12 complex in humans. Cold Spring Harbor Symposium in Quantitative Biology, 75:395-401. Importantly, insights obtained with yeast can be extrapolated to human cells. Its N-terminal tail extends inside the kinetochore and binds to the globular heads of the Spc24-Spc25 dimer of the Ndc80 complex further comprises Ndc80 and Nuf2.
We found that its nucleolar localization is dynamic and cell cycle stage dependent. Using yeast allows for a quick genetic identification of proteins involved in chromosome segregation and for dissecting functional relationships between the involved proteins. Errors made during chromosome segregation e. . We study at the molecular level how Cnn1 activity and levels at kinetochores are controlled during the cell cycle. From G1 to metaphase, the high copy number of the Mtw1 complex and phospho-inhibition of the Cnn1 tail favors Mtw1-Ndc80 complex binding, creating a rigid kinetochore structure.
Recently initiated translational research on kinetochores is also discussed as kinetochores are being mined as a very rich target for the next generations of anti-cancer drugs. By identifying, functionally dissecting, and structurally analyzing kinetochore proteins and their regulators we can better understand the basis of aneuploidy disease, including cancer, and convert major players into cancer biomarkers and anticancer drug targets. Evading Pgp activity in drug-resistant cancer cells: a structural and functional study of antitubulin furan metotica compounds. Recently initiated translational research on kinetochores is also discussed as kinetochores are being mined as a very rich target for the next generations of anti-cancer drugs. Molecular structures and interactions in the yeast kinetochore.
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