Identification and Characterization of Mitotic Defect‐related Proteins (MDr1 and MDr2) Necessary for Bipolar Spindle Assembly

During mitosis chromosome segregation relies on the proper formation and stabilization of the mitotic spindle. Although many proteins have been identified that contribute to spindle formation, presumably many others are involved. Therefore, we sought to identify novel proteins involved in mitotic spindle formation. To this end, mitotic HeLa cell extracts were used for in vitro microtubule polymerization reactions and proteins co‐pelleting with microtubules were identified by mass spectrometry. We generated a Dharmacon siRNA library directed against 600 microtubule binding proteins for use in various high throughput genetic screens, including mitotic spindle assembly, mitotic arrest, mitotic bypass, and apoptosis. Among the positive hits from these screens were a 55‐kDa protein MDr1 and a 42‐kDa protein MDr2. To characterize these proteins, I am taking both cellular and biochemical approaches. I have developed inducible GFP‐tagged MDr1 and MDr2 protein cell lines and show that MDr1 and MDr2 only co‐localize during mitosis. I also showed that recombinant MDr1 and MDr2 form a stable complex by in‐vitro binding experiments. The results suggested that MDr1 and MDr2 are regulated and only form a functional complex when entering mitosis.