Additional Projects

In addition to continued studies on Ipl and Cse4, we have initiated the following new studies aimed at gaining a better understanding of chromosome segregation. These studies should continue to elucidate new details about the mechanisms of chromosome segregation and thus aid in understanding the generation of aneuploidy and disease progression:

*We have developed a minichromosome purification assay to dissect the requirements for kinetochore assembly and function. We purify minichromosomes using a lactose repressor fusion protein that binds to lactose operator sequences in the minichromosome. The purified minichromosomes are analyzed by mass spectrometry to identify kinetochore proteins. Using this technique, we have identified greater than 95% of structural kinetochore proteins as well as novel kinetochore proteins. We are currently identifying post-translational and kinetochore composition changes that occur when kinetochores are mono-oriented vs. bi-oriented. In addition, we are able to purify the centromeric nucleosome and analyze its physical properties using this assay. In collaboration with Chip Asbury's lab, we are using single molecule optical trap assays to determine how tension alters microtubule binding properties. Our future goals will be to identify features of centromeres that contribute to the assembly and stability of the kinetochore, to purify the meiosis I kinetochore, and to use the purified minichromosomes to develop a fluorescent assay to monitor kinetochore binding to microtubules in vitro. We are also using the assay to study the requirements for centromeric chromatin and Ipl1 phosphorylation in kinetochore function.

*We have developed a second method to purify kinetochores through affinity purification of a kinetochore component and we are using this material for a number of assays.  First, we are analyzing the structure of the kinetochore by EM in collaboration with Tamir Gonen's lab to determine how kinetochores can carry out their various functions.  Second, we are using this material to elucidate details about the mechanism of spindle checkpoint activation.  Finally, the Asbury lab is also using this material to explore the biophysical properties of kinetochores using optical trap and TIRF microscopy.

*We are studying spindle repair to determine how cells respond to and correct spindle defects. We have performed a genetic screen to identify mutants that are defective in spindle repair and we are currently characterizing the mutants to learn how cells cope with spindle damage to prevent errors in cell division.

*We are identifying histone modifications that are important for chromosome segregation and the spindle checkpoint using genetic and biochemical approaches.