Dr. David Glover
University of Cambridge

Developmental Regulation of Cell Division in Model Organisms and in Cancer Cells

Minority groups suffer disproportionately from cancer, and disparities exist in both mortality and incidence rates.

Model organisms provide rapid insight into the mechanisms and control of cell cycle since they facilitate both genetic and cell free studies. Drosophila offers an excellent model for studying fundamental aspects of cell division cycle control and how these might differ between tissues throughout development. The organisation of the mitotic spindle and its function are also much closer to that of human cells than the yeasts, the other major genetic model for studying cell cycle control. The value of Drosophila as a model for studying cancer comes not only from our discoveries that many conserved cell cycle regulators are mis-regulated in cancer cells, but also that the cellular processes of the centrosome duplication cycle and of chromosome segregation go awry in tumour cells. We are able to use Drosophila as a developmental system in which to model these processes. Our genetical and cell biological studies on regulatory mitotic proteins in Drosophila have highlighted their multiple roles in mitotic and meiotic progression and how these can vary in different tissues. The group continues to identify components of these regulatory networks. We study their interactions both genetically and biochemically, and follow their behaviour in cells using both conventional and confocal microscopy on fixed and living specimens. As the mitotic and meiotic apparati are dynamic molecular machines, such time lapse imaging of the behaviour of individual molecular components is particularly valuable. The development of RNAi techniques enables us to study virtually any protein from the fly or human genomes. We have the capability to carry our whole genome RNAi screens in cultured Drosophila cells to identify regulatory proteins, and then study the roles of their human counterparts in normal and tumour cells.

Projects

Dissection of the multiple roles of Polo and Aurora kinases in mitotic progression using chemical genetics.
The evolutionarily-conserved mitotic kinases Polo and Auroras A and B have been shown to play multiple roles in mitotic progression. However, dissection of these functions has not been easily amenable using traditional genetics, due to the prolonged period before mutations produce a discernable phenotype. For this reason a chemical genetics approach has been proposed. This entails using small molecules that specifically target and perturb the function of the above kinases, acting immediately. The effects of inhibition of these kinases on mitotic progression will be examined in both normal and cancerous mammalian cell lines stably expressing fluorescently labelled components of the mitotic apparatus. Comparative studies will be applied to living Drosophila embryos, and cultured cells from larval brains and testes. In this way the effects of these compounds can be studied in concert with those of various mutations affecting cell cycle progression.

In vitro studies of microtubule nucleation by centrosomes
We have previously used an in vitro system to study the nucleation of microtubules by preparations of partially purified centrosomes. This has enabled us to study some of the roles of Polo kinase in recruiting and/or activating molecules associated with the minus ends of microtubules to increase the nucleating capacity of the centrosome on mitotic entry. We propose to further our understanding of the roles of protein kinases and protein chaperones using this in vitro system.

Protein kinases with mitotic functions
Mitotic progression is regulated through the concerted action of cycles of protein phosphorylation and dephosphorylation coupled to controlled proteolysis of key regulatory molecules. We discovered three of the principal regulatory mitotic protein kinases, Polo and the Auroras, through our work in Drosophila. We recently completed a screen of the entire fly kinome by knocking down the function of all of the protein kinases in the genome (239 enzymes) and examining the effects on cell cycle progression. This screen identified a number of protein kinases with mitotic roles that we now wish to study in detail using a combination of biochemical, genetic, and cell biological approaches.

Selected References
· Avides, M.C., and Glover, D.M. (1999) Abnormal spindle protein, Asp, and the integrity of mitotic centrosomal organising centers. Science 283: 1733-1735
· Cullen, C.F., Deak, P., Glover, D.M., and Ohkura, H. (1999) mini spindles: a gene encoding a conserved microtubule associated protein required for the integrity of the mitotic spindle in Drosophila. J. Cell Biol.146: 1005-1018
· Inoue, Y., Shiraki, M., Deak, P., Avides, M.C., Yamaguchi, M., Nishimoto, Y., Matsukage, A., and Glover, D.M. (2000) Orbit, a novel microtubule associated protein essential for mitosis in Drosophila. J. Cell Biol. 149: 153-165
· Barbosa, V., Yamamoto, R., Carpenter, A.T.C., Henderson, D.S., and Glover, D.M. (2000) Mutation of a Drosophila Gamma Tubulin Ring Complex Sub-Unit Encoded by discs degenerate-4 Differentially Disrupts Centrosomal Proteins. Genes & Dev. 14: 3126-3139
· Giet, R., and Glover, D.M. (2001) Drosophila Aurora B kinase is required for histone H3 phosphorylation and condensin recruitment during chromosome condensation and to organise the central spindle during cytokinesis J. Cell Biol. 152: 669-682
· Avides, M.C., Tavares, A., and Glover, D.M. (2001) Polo kinase and Asp are required to promote mitotic centrosomal organising activity. Nature Cell Biology 3: 421 424
· Donaldson, M.M., Tavares, A.A.M., Ohkura, H., Deak, P., and Glover, D.M. (2001) Checkpoint arrest after anaphase onset in polo mutants of Drosophila J. Cell Biol. 153: 663-677
· de Cárcer, G., Avides, M do C., Lallena, M.J. Glover, D.M. and González, C. (2001) Requirement of Hsp90 for centrosomal function reflects its regulation of Polo kinase stability EMBOJ 20: 2878-2884
· Giet, R., McLean, D., Descamps, S., Lee, M.J., Raff, J.W., Prigent C., and Glover, D.M. (2002) Drosophila Aurora A kinase is required to localise D-TACC to centrosomes and to regulate astral microtubules J. Cell Biol. 156: 437-451
· Barbosa, V., Gatt, M., Rebollo, E., Gonzalez, C., and Glover, D.M. (2003) Spermatocyte spindle bipolarity is affected in Drosophila dd4 mutants of the gTuRC but asters still form. J. Cell Sci. 116:929-941
· Blagden, S.P., and Glover, D.M. (2003) Polar Expeditions-provisioning the centrosome for mitosis Nature Cell Biology 5: 505-511
· Minestrini, G., Harley, A.S., and Glover, D.M. (2003) Localisation of Pavarotti-KLP in living Drosophila embryos suggests roles in re-organising the cortical cytoskeleton during the mitotic cycle. Mol. Biol. Cell 14:4028-38
· Deák, P., Donaldson, M., and Glover, D.M. (2003) Mutations in mákos, a Drosophila gene encoding the Cdc27 subunit of the anaphase promoting complex enhance centrosomal defects in polo and are suppressed by mutations in twins/aar encoding a regulatory sub-unit of PP2A J. Cell. Sci. 116:4147-58.