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Abstract:
To maintain genome integrity, the genetic material has to be equally distributed during cell division. The spindle assembly checkpoint is a conserved surveillance mechanism that delays anaphase as long as any of the chromosomes is not properly attached to the mitotic spindle. Malfunction of this checkpoint causes erroneous chromosome segregation and has been implicated in tumorigenesis. Despite a wealth of information on the localization and interaction of checkpoint proteins, the in vivo signaling mechanism is only partially understood. A complex between the proteins Mad1 and Mad2 is crucial for the spindle assembly checkpoint. Kinetochore-bound Mad1:Mad2 promotes binding of Mad2 to the anaphase activator Cdc20, which ultimately prevents anaphase. By performing live cell imaging in fission yeast, we show that loss of the Mad1 C-terminus preserves the interaction between Mad1 and Mad2, but destroys checkpoint function. Previous experiments in budding yeast hint to an interaction between the Mad1 C-terminus and the checkpoint protein Bub1 and it was shown that Bub1 is required for kinetochore localization of Mad1. Our experiments revealed that deletion or specific mutations of the Mad1 C-terminus impaired kinetochore recruitment of Mad1. However, artificial tethering of a localization deficient Mad1 mutant to the kinetochore did not restore checkpoint function. We currently explore whether the checkpoint defect results from a loss of the Mad1-Bub1 interaction or whether the Mad1 C-terminus and Bub1 have other roles in checkpoint signaling.
