The major focus for the next period of this grant will be spindle, kinetochore and centromere protein mechanisms that act to achieve accurate chromosome segregation. Accuracy is critical because the mis- segregation of even one chromosome produces aneuploidy that can lead to cancer or developmental defects. Kinetochores have at least five key roles in assuring accurate segregation: 1) they produce a diffusible signal for the spindle checkpoint to delay anaphase until sister kinetochores are properly attached by MTs to opposite poles and aligned on the metaphase plate;2) they provide stable, but dynamic, attachment to MT plus ends to turn off spindle checkpoint activity and prevent errors in MT attachment;3) they act as a force- generating depolymerase for movement of chromosomes poleward coupled to plus-end depolymerization of kMTs at the kinetochore;4) they provide a tension-sensitive slip clutch, generating tension from the poleward flux of kMTs while maintaining attachment to polymerizing plus ends of MTs during kinetochore movements away from the pole, and 5) they correct errors in MT attachment so that the formation of kMTs to opposite poles (merotelic orientation) does not result in lagging chromosomes and mis-segregation in anaphase. Centromere passenger proteins, which are located on the inner centromere behind the kinetochore, also appear to regulate kMT attachment and MT-dependent signaling of the cortical site for cytokinesis. Most of our studies focus on protein function in mammalian tissue cells, but budding yeast mitotic kinetochores and shmoo tips are useful genetic models for understanding protein function at dynamic plus-end attachment sites. A major strength of our program has been, and will continue to be, the development and application of new microscopy techniques for measurements of protein function in living cells and reconstituted preparations.
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