Mitotic abnormalities were first recognised in human tumours towards the end of the nineteenth century, but only recently have oncogenic lesions have been demonstrated in genes that regulate mitotic checkpoints controlling the onset of anaphase or entry into cytokinesis. Moreover levels of the human counterparts of the mitotic kinases polo and aurora, which our laboratory first identified in Drosophila1,2, have been shown to be elevated in tumour cells and over-expression of both enzymes leads to oncogenic transformation in cultured cells. Perhaps it should come as no surprise to learn that the morphology of centrosome, the organelle at the spindle poles, is abnormal in a wide variety of tumours and tumour cell lines, and that such cells usually have abnormal numbers of centrosomes.
Formation of the mitotic spindle first requires changes in the dynamic behaviour of microtubules brought about through the activity of the major mitotic kinase, cyclin dependent kinase 1. The centrosomes dramatically increase in size upon mitotic entry to facilitate nucleation of the spindle microtubules. The centrosomes and their 'maturation' at mitotic entry were described by Boveri in the 1890s. Now, over a century later we can account for some, but by no means all, of these events in molecular terms. We have shown that centrosome maturation requires the activity of Polo kinase that activates the Abnormal Spindle protein (Asp) and promotes recruitment of the γ-tubulin ring complex. Together these molecules nucleate the minus ends of microtubules in the centrosome. We can study this process in living cells and in vitro. The Aurora-A kinase also participates by phosphorylating the conserved D-TACC protein and promoting its association with the centrosome in complex with Minispindles, a homologue of the microtubule associated protein ch-TOG, upregulated in several tumour types3. Mutants of aurora A have monopolar spindles reflecting a function of the Aurora kinase in activating one of the major mitotic motors that maintains spindle bipolarity.
We also discovered that another protein kinase related to Polo, SAK (also called PLK4), is required for the replication of centrioles, a barrel-like structure of microtubules found in the core of the centrosome and at the base of cilia and flagellae. Curiously we found Drosophila cells can organise their mitotic spindle poles in the absence of centrioles and continue to divide whereas human cells arrest and undergo cell death4.
Bundles of kinetochore microtubules may be either captured or independently nucleated by the kinetochores of the condensed chromosomes. A specific microtubule associated protein, Orbit (also known as Mast or CLASP), promotes addition of tubulin sub-units at the plus ends of microtubules associated with the kinetochore. This activity is opposed by a microtubule destabilising kinesin that effectively removes tubulin subunits from the minus ends5.
The metaphase-anaphase transition marks a turning point in mitosis. The B-type Aurora kinase required for the phosphorylation of histone H3 and recruitment of condensins upon chromosome condensation is transferred from the chromosomes to the central spindle at the metaphase-anaphase transition where it is subsequently required for cytokinesis6. The spindle undergoes several changes in these late mitotic stages. First it elongates during late anaphase and then undergoes a more profound re-organisation with the formation of the central spindle, an overlapping array of microtubules that will orchestrate cytokinesis. We wish to understand how various motor proteins and microtubule associated proteins help mediate these transitions that are in part regulated by the Aurora and Polo kinases. The Orbit protein, for example, is re-used for later mitotic stages by becoming incorporated into central spindle microtubules but only those in the interior part of the cell. Consistently, in orbit mutants, initiation of the cleavage furrow occurs when peripheral central spindle micortubules contact the cell cortex, but it cannot be sustained as, in the absence of Orbit, the interior central spindle collapses7.
In the cytokinesis of a typical animal cell, a cleavage furrow forms at the cell's equator and ingresses inward to bisect the dividing cell. We are studying how central spindle microtubules deliver the kinesin-like protein, Pavarotti8, and its associated signalling molecule RacGAP to initiate furrow formation. We also aim to understand how the Rho and Rac GTPases antagonistically regulate functions of the STICKY/citron kinase9 to coordinate the correct organisation of F-actin and Anillin in the contractile ring. Ingression of the furrow also requires addition of new plasma membrane regulated through the activities of phosphatidyl-inositol lipids in the final stages of membrane addition10.
Sunkel CE and Glover DM (1988)
polo, a mitotic mutant of Drosophila displaying abnormal spindle poles.
Journal of Cell Science 89:25-38
Glover DM, Leibowitz MH, McLean DA and Parry H (1995)
Mutations in aurora prevent centrosome separation leading to the formation of monopolar spindles.
Giet R, McLean D, Descamps S, Lee MJ, Raff JW, Prigent C and Glover DM (2002)
Drosophila Aurora A kinase is required to localize D-TACC to centrosomes and to regulate astral microtubules.
Journal of Cell Biology 156:437-451
Bettencourt-Dias M, Rodrigues-Martins A, Carpenter L, Riparbelli M, Lehmann L, Gatt MK, Carmo N, Balloux F, Callaini G and Glover DM (2005)
SAK/PLK4 is required for centriole duplication and flagella development.
Current Biology 15:2199-2207
Laycock JE, Savoian MS and Glover DM (2006)
Antagonistic activities of Klp10A and Orbit regulate spindle length, bipolarity and function in vivo.
Journal of Cell Science 119:2354-2361
Giet R and Glover DM (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.
Journal of Cell Biology 152:669-682
Inoue YH, Savoian MS, Suzuki T, Mathe E, Yamamoto MT and Glover DM (2004)
Mutations in orbit/mast reveal that the central spindle is comprised of two microtubule populations, those that initiate cleavage and those that propagate furrow ingression.
Journal of Cell Biology 166:49-60
Adams RR, Tavares AA, Salzberg A, Bellen HJ and Glover DM (1998)
pavarotti encodes a kinesin-like protein required to organize the central spindle and contractile ring for cytokinesis.
Genes and Development 12:1483-1494
D'Avino PP, Savoian MS and Glover DM (2004)
Mutations in sticky lead to defective organization of the contractile ring during cytokinesis and are enhanced by Rho and suppressed by Rac.
Journal of Cell Biology 166:61-71
Gatt MK and Glover DM (2006)
The Drosophila phosphatidylinositol transfer protein encoded by vibrator is essential to maintain cleavage-furrow ingression in cytokinesis.
Journal of Cell Science 119:2225-2235