Tension
Chromosome segregation requires sister kinetochores to attach to microtubules from opposite poles. When kinetochores achieve biorientation, they come under tension due to microtubule pulling forces and the attachments are stabilized. However, kinetochores often form inappropriate attachments that lack tension and must be corrected prior to anaphase to prevent aneuploidy. The lack of tension triggers microtubule detachment, giving the kinetochores another chance to form proper connections. This selective destabilization is due in part to an error correction system in which phosphorylation of kinetochore components by the Aurora B kinase weakens the interaction between the kinetochore and MT. Kinetochores also engage a highly conserved signal transduction system called the spindle checkpoint to halt the cell cycle when kinetochore-MT interactions are aberrant. Kinetochores are required to trigger and to amplify the signal that ultimately inhibits the cell cycle until every pair of chromosomes is bioriented.
Projects
What are the kinetochore substrates of Aurora B, Mps1 and other kinetochore kinases that are required for biorientation?
We are mapping phosphorylation sites on kinetochore proteins by MS and then studying the corresponding mutants in vivo. We also analyze the effect of phosphorylation on kinetochore-microtubule attachments in vitro in collaboration with the Asbury lab.
What additional mechanisms regulate biorientation?
In collaboration with Chip Asbury's lab at UW, we found that tension directly stabilizes reconstituted kinetochore-microtubule attachments. We recently discovered this intrinsic tension-sensing activity requires the Stu2 kinetochore protein. We are currently exploring the mechanism of Stu2 action as well as identifying additional mechanisms whereby tension stabilizes attachments through genetic and cell biological approaches. Our collaboration with the Asbury lab also led to the recent discovery that kinetochores recognize the polarity of the microtubule lattice.
How does tension regulate Aurora B and Mps1 kinase activity?
We have used an optical trapping assay to determine that tension suppresses the ability of Aurora B to detach kinetochores from microtubules. We are exploring the underlying mechanism and testing how tension regulates Mps1 using similar assays.
How is the spindle checkpoint activated and silenced?
We are analyzing the effects of tension and attachment on kinetochore-microtubule attachments using a reconstituted system. Our long-term goal is to reconstitute APC inhibition with purified kinetochore particles in vitro to fully dissect the specific requirements for checkpoint signaling.
Model illustrating how conformational changes within a kinetochore could inhibit Aurora B-triggered detachment
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