The underlying theme behind the work of our laboratory is to study the basic biology of mitotic cell division, in particular the aspects regulating the formation of a bipolar mitotic spindle and the connection between centrosomes and cytokinesis. The ultimate goal is to understand how modifications of these processes contribute to the transformation of normal into cancer cells.
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Knockdown of one of our kinetochore genes. Chromosome fails to align during metaphase and cell goes into apoptosis.
Cytokinesis failure and regression
We have recently found that SGT1, a highly conserved protein, is required for Polo protein stability and kinetochore assembly. Sgt1 has been found to associate with the molecular chaperone HSP90, and the Skp1 subunit of SCF (E3 ubiquitin ligase). We found Sgt1 to be required for proper centrosome maturation and mitotic spindle assembly in mitosis. Mutant analysis show that Sgt1 is an essencial gene, and lack of Sgt1 results in block in a prometaphase/metaphase stage due to activation of the spindle assembly checkpoint. However, we found that most mutant neuroblasts dont ever reach mitosis due to the activation of the DNA damage checkpoint. Moreover, lack of Sgt1 severely compromises spindle assembly, centrosome maturation and centrosome number. Importantly, we show that protein levels of several centrosomal components, including Polo and g-tubulin, are severely reduced in the Sgt1 mutant.
IDENTIFICATION AND CHARACTERIZATION OF MITOTIC KINETOCHORE PROTEINS. We have developed a new method to obtain enriched mitotic kinetochore preparations from Drosophila embryos and cultured cells. This way we have identified 92 proteins of which 26 correspond to previously uncharacterized genes. Expression of these proteins as GFP-fusions in cultured cells, showed that 10 do accumulate on the mitotic kinetochores. In addition, their downregulation by RNAi results in severe chromosome congression and segregation defects. In agreement with these results, preliminary mutant analysis for four of new genes show that they are essential in early embryonic or larval development. Finally, we have identified the human homologues for some of the new genes and downregulation of these in HeLa cells also resulted in severely defective chromosome congression and segregation during mitosis.
Dynamic kinetochore EMBO Workshop
June, 28- July 1, 2022