Regulation of the Metaphase-Anaphase Transition – RMAT

The metaphase (or spindle) checkpoint inhibits anaphase onset until all the chromosomes are correctly attached to the spindle. The inhibition is mediated in part by Mad2 and BubR1 binding to and inhibiting Cdc20, a key cofactor of the Anaphase Promoting Complex (APC). When the checkpoint is turned off (following correct attachment of the last kinetochore), Cdc20 is freed to activate the APC, which then targets Cyclin B and Securin for destruction. This in turn inactivates cdc2 kinase and promotes sister chromatid separation, the two hallmarks of mitotic exit. The overall goal of this proposal is to better understand the mechanisms by which the metaphase checkpoint determines the timing of anaphase onset. Our approach is to combine live cell imaging with the genetic tools of Drosophila to examine in vivo the dynamics and functions of key checkpoint components (such as Mad1, Mad2, BubR1, RZZ complex, CenpE, Cdc20, Cyclin B) during normal and perturbed mitosis. We will focus in particular on the process of "shedding" (dynein-mediated removal of checkpoint proteins from kinetochores) which is presumed to be a major contributor to the mechanism by which the checkpoint is extinguished. By simultaneously monitoring in vivo the shedding of Mad2 and the degradation of fluorescent Cyclin B (an in vivo readout for checkpoint inactivation), we will put this model to a more rigorous test. We will ask how the relative kinetochores levels of Mad1 and Mad2 vary as a function of mitotic progression, to see if Mad1 shedding might also contribute to checkpoint shutoff. We will assay in vivo the contributions of Mad2 phosphorylation and of BubR1 checkpoint function in controlling mitotic timing. We will examine the role of Cenp-Meta, the putative CenpE homolog in Drosophila, in regulating BubR1 activity and investigate its role on the dynamics of RZZ shedding. Finally we will begin a mutational analysis of Cdc20.