2006 Abstracts

D'Avino PP, Savoian MS, Capalbo L, Glover DM.

RacGAP50C is sufficient to signal cleavage furrow formation during cytokinesis.

Several studies indicate that spindle microtubules determine the position of the cleavage plane at the end of cell division, but their exact role in triggering the formation and ingression of the cleavage furrow is still unclear. Here we show that in Drosophila depletion of either the GAP (GTPase-activating protein) or the kinesin-like subunit of the evolutionary conserved centralspindlin complex prevents furrowing without affecting the association of astral microtubules with the cell cortex. Moreover, timelapse imaging indicates that astral microtubules serve to deliver the centralspindlin complex to the equatorial cortex just before furrow formation. However, when the GAPsignaling component was mislocalized around the entire cortex using a membrane-tethering motif, this caused ectopic furrowing even in the absence of its motor partner. Thus, the GAP component of centralspindlin is both necessary and sufficient for furrow formation and ingression and astral microtubules provide a route for its delivery to the cleavage site

Journal of Cell Science 119:4402-8

Carpenter ATC

Monte Moses and Adelaide Carpenter: Duke, 1974-1976.

This memoir recounts the scientific threads in Monte Moses’s laboratory during 1974–1976.

Chromosoma 115:155-157

McInnes C, Mazumdar A, Mezna M, Meades C, Midgley C, Scaerou F, Carpenter L, Mackenzie M, Taylor P, Walkinshaw M, Fischer PM, Glover D.

Inhibitors of Polo-like kinase reveal roles in spindle-pole maintenance.

Polo-like kinases (Plks) have several functions in mitotic progression and are upregulated in many tumor types. Small-molecule Plk inhibitors would be valuable as tools for studying Plk biology and for developing antitumor agents. Guided by homology modeling of the Plk1 kinase domain, we have discovered a chemical series that shows potent and selective Plk1 inhibition. The effects of one such optimized benzthiazole N-oxide, cyclapolin 1 (1), on purified centrosomes indicate that Plks are required to generate MPM2 epitopes, recruit g-tubulin and enable nucleation of microtubules. The compound can also promote loss of centrosome integrity and microtubule nucleating ability apparently through increased accessibility of protein phosphatases. We show that treatment of living S2 cells with cyclapolin 1 leads to collapsed spindles, in contrast to the metaphase-arrested bipolar spindles observed after RNAi. This different response to protein depletion and protein inhibition may have significance in the development of antitumor agents.

Nat Chem Biol 2:608-17

Pearson J, Godinho SA, Tavares A and Glover DM

Heterologous expression of mammalian Plk1 in Drosophila reveals divergence from Polo during late mitosis.

Drosophila Polo kinase is the founder member of a conserved kinase family required for multiple stages of mitosis. We assessed the ability of mouse Polo-like kinase 1 (Plk1) to perform the multiple mitotic functions of Polo kinase, by expressing a Plk1-GFP fusion in Drosophila. Consistent with the previously reported localization of Polo kinase, Plk1-GFP was strongly localized to centrosomes and recruited to the centromeric regions of condensing chromosomes during early mitosis. However, in contrast to a functional Polo-GFP fusion, Plk1-GFP failed to localize to the central spindle midzone in both syncytial embryo mitosis and the conventional mitoses of cellularized embryos and S2 cells. Moreover, unlike endogenous Polo kinase and Polo-GFP, Plk1-GFP failed to associate with the contractile ring. Expression of Plk1-GFP enhanced the lethality of hypomorphic polo mutants and disrupted the organization of the actinomyosin cytoskeleton in a dominant-negative manner. Taken together, our results suggest that endogenous Polo kinase has specific roles in regulating actinomyosin rearrangements during Drosophila mitoses that its mammalian counterpart, Plk1, cannot fulfil. Consistent with this hypothesis, we observed defects in the cortical recruitment of myosin and myosin regulatory light chain in Polo deficient cells

Experimental Cell Research 312:770-781

Petretti C, Savoian M, Montembault E, Glover DM, Prigent C and Giet R

The PITSLRE/CDK11p58 protein kinase promotes centrosome maturation and bipolar spindle formation.

The CDK11 (cyclin-dependent kinase 11) gene has an internal ribosome entry site (IRES), allowing the expression of two protein kinases. The longer 110-kDa isoform is expressed at constant levels during the cell cycle and the shorter 58-kDa isoform is expressed only during G2 and M phases. By means of RNA interference (RNAi), we show that the CDK11 gene is required for mitotic spindle formation. CDK11 RNAi leads to mitotic checkpoint activation. Mitotic cells are arrested with short or monopolar spindles. ?-Tubulin as well as Plk1 and Aurora A protein kinase levels are greatly reduced at centrosomes, resulting in microtubule nucleation defects. We show that the mitotic CDK11p58 isoform, but not the CDK11p110 isoform, associates with mitotic centrosomes and rescues the phenotypes resulting from CDK11 RNAi. This work demonstrates for the first time the role of CDK11p58 in centrosome maturation and bipolar spindle morphogenesis.

EMBO Reports 7:418-424

Gatt MK and Glover DM

The Drosophila phosphatidylinositol transfer protein encoded by vibrator is essential to maintain cleavage-furrow ingression in cytokinesis.

Cytokinesis requires the coordination of cytoskeletal and plasma membrane dynamics. A role for phosphatidylinositol lipids has been proposed for the successful completion of cytokinesis but this is still poorly characterised. Here, we show mutants of the gene vibrator, previously found to encode the Drosophila phosphatidylinositol transfer protein, produce multinucleate cells indicative of cytokinesis failure in male meiosis. Examination of fixed preparations of mutant spermatocytes showed contractile rings of anillin and actin that were of normal appearance at early stages but were larger and less well organised at later stages of cytokinesis than in wild-type cells. Time-lapse imaging revealed sequential defects in cytokinesis of vibrator spermatocytes. In cells that fail cytokinesis, central spindle formation occurred correctly, but furrow ingression was delayed and the central spindle did not become compressed to the extent seen in wild-type cells. Cells then stalled at this point before the apparent connection between the constricted cytoskeleton and the plasma membrane was lost; the furrow then underwent elastic regression. We discuss these defects in relation to multiple functions of phosphoinositol lipids in regulating actin dynamics and membrane synthesis.

Journal of Cell Science 119:2225-2235

Laycock JE, Savoian MS and Glover DM

Antagonistic activities of Klp10A and Orbit regulate spindle length, bipolarity and function in vivo.

The metaphase-spindle steady-state length occurs as spindle microtubules 'flux', incorporating new subunits at their plus ends, while simultaneously losing subunits from their minus ends. Orbit/Mast/CLASP is required for tubulin subunit addition at kinetochores, and several kinesins regulate spindle morphology and/or flux by serving as microtubule depolymerases. Here, we use RNA interference in S2 cells to examine the relationship between Orbit and the four predicted kinesin-type depolymerases encoded by the Drosophila genome (Klp10A, Klp59C, Klp59D and Klp67A). Single depletion of Orbit results in monopolar spindles, mitotic arrest and a subsequent increase in apoptotic cells. These phenotypes are rescued by co-depleting Klp10A but none of the other three depolymerases. Spindle bipolarity is restored by preventing the spindle collapse seen in cells that lack Orbit, leading to functional spindles that are similar to controls in shape and length. We conclude that Klp10A exclusively antagonises Orbit in the regulation of bipolar spindle formation and maintenance.

Journal of Cell Science 119:2354-2361

Plusa B, Hadjantonakis AK, Gray D, Piotrowska-Nitsche K, Jedrusik A, Papaioannou VE, Glover DM and Zernicka-Goetz M

Embryology: Does prepatterning occur in the mouse egg? (Reply).

Hiiragi et al. compare our model of the developing mouse egg with theirs. They seem to present patterning as equivalent to determination, but this is confusing as patterning does not have to mean determination. We have never stated that mouse embryo development is determined. Mouse development is regulative rather than determinative, and this can be explained in two ways: first, development could be entirely unbiased, generating identical cells; second, there could be some developmental bias or, in other words, pattern from the beginning that is not constraining. There is evidence for early bias from several laboratories but this does not mean that cells have localized determinants fixing their fates. Regulative development does not exclude bias, which indicates inclination, not determination

Nature 442:E4


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