1998 Abstracts

Glover DM, Hagan IM and Tavares AA

Polo-like kinases: a team that plays throughout mitosis.

When the first mutant allele of the Drosophila gene polo was first characterized over 10 years ago, attention focused on the defects that centrosome behavior exhibited at various stages of development. The subsequent realization that the serine-threonine kinase it encodes is highly conserved from yeasts to humans has provoked a flurry of investigation into the function of the enzyme. A role for the polo-like kinases (plks) in regulating centrosome behavior has been borne out in several organisms, and the enzymes have attracted further attention recently with the realization that they regulate multiple stages of mitotic progression. In this article we review the current status of our understanding of the functions of plks from the time of commitment to M phase in the activation of Cdc25, through the activation of the anaphase promoting complex (APC), to the regulation of late mitotic events essential for cytokinesis. We discuss how to reconcile the sometimes apparently disparate observations made upon plk function in different organisms.

Genes and Development 12:3777-3787

Gonzalez C, Sunkel CE and Glover DM

Interactions between mgr, asp, and polo: asp function modulated by polo and needed to maintain the poles of monopolar and bipolar spindles.

We describe genetic interactions between mutations in mgr, asp, and polo genes required for the correct behaviour of the spindle poles in Drosophila. The phenotype of a polo1 mgr double mutant is more similar to mgr than polo1, but the frequency of circular monopolar figures (CMFs) seen with either mutant alone is additive, suggesting that the two gene products are required for independent functions in the formation of bipolar spindles. The aspE3 mgr double mutant arrests much earlier in development than either mutant alone, indicative of a strong block to cell proliferation. We discuss whether the lack of microtubular structures in these cells reflects an extended mitotic arrest, or if it is a more direct consequence of the double mutant combination. A polo1 aspE3 double mutant shows a dramatic synergistic increase in mitotic frequency. The loss of CMFs normally associated with the polo1 phenotype suggests that the Asp microtubule-associated protein is required to maintain the structure of spindle poles. We speculate that Asp protein might be a substrate for the serine-threonine protein kinase encoded by polo.

Chromosoma 107:452-460

Wianny F, Tavares A, Evans MJ, Glover DM and Zernicka-Goetz M

Mouse polo-like kinase 1 associates with the acentriolar spindle poles, meiotic chromosomes and spindle midzone during oocyte maturation.

We have examined the dynamics of the localisation of the polo-like kinase 1 (Plk1) during maturation of the mouse oocyte. Levels of Plk1 protein increase following germinal vesicle breakdown, at which time the enzyme begins to accumulate at discrete positions on the condensing chromosomes and, subsequently, at the poles of the meiotic spindle, which moves towards the cortex of the egg. Interestingly, at metaphase in both meiotic divisions, Plk1 shows a punctate localisation along the broad spindle poles. Moreover, the punctate distribution of Plk1 on the meiotic chromosomes appears at early anaphase to correspond to the centromeric regions. The protein relocates to the spindle midzone during late anaphase and then associates with the midbody at telophase. We have confirmed the specific pattern of immuno-localisation seen in fixed preparations by observing the distribution of Plk1 tagged with green fluorescent protein in living oocytes. We discuss the localisation of the enzyme in light of the structure of the spindle poles, which are known to lack centrioles, and the highly asymmetric nature of the meiotic divisions.

Chromosoma 107:430-439

Endow S and Glover DM (Eds)

Dynamics of Cell Division (Second edition)

This volume focuses on the structural aspects of cell division, ranging from nuclear envelope breakdown to cytokinesis and partitioning of the cytoplasm. It examines spindle assembly and chromosome behaviour in mitosis and meiosis, centromere and kinetochore structure and regulation, telomeres, the role of centrosomes, and mechanisms by which overall regulation is achieved. The up-to-date reviews of each topic provide perspectives on recent important findings. Each chapter presents models and new ideas that accommodate available information.

Frontiers in Molecular Biology series OUP, Oxford

Hagan I, Gull K and Glover DM

Poles apart: yeast spindle pole bodies and animal cell centrosomes share common function and regulation.

No abstract available

In S Endow and DM Glover (Eds), Dynamics of Cell Division, OUP, Oxford

Carmena M, Riparbelli MG, Minestrini G, Tavares AM, Adams R, Callaini G and Glover DM

Drosophila polo kinase is required for cytokinesis.

A number of lines of evidence point to a predominance of cytokinesis defects in spermatogenesis in hypomorphic alleles of the Drosophila polo gene. In the pre-meiotic mitoses, cytokinesis defects result in cysts of primary spermatocytes with reduced numbers of cells that can contain multiple centrosomes. These are connected by a correspondingly reduced number of ring canals, structures formed by the stabilization of the cleavage furrow. The earliest defects during the meiotic divisions are a failure to form the correct mid-zone and mid-body structures at telophase. This is accompanied by a failure to correctly localize the Pavarotti kinesin-like protein that functions in cytokinesis, and of the septin Peanut and of actin to be incorporated into a contractile ring. In spite of these defects, cyclin B is degraded and the cells exit M phase. The resulting spermatids are frequently binuclear or tetranuclear, in which case they develop either two or four axonemes, respectively. A significant proportion of spermatids in which cytokinesis has failed may also show the segregation defects previously ascribed to polo1 mutants. We discuss these findings in respect to conserved functions for the Polo-like kinases in regulating progression through M phase, including the earliest events of cytokinesis.

Journal of Cell Biology 143:659-671

Warbrick E, Heatherington W, Lane DP and Glover DM

PCNA binding proteins in Drosophila melanogaster: the analysis of a conserved PCNA binding domain.

The eukaryotic polymerase processivity factor, PCNA, interacts with cell cycle regulatory proteins such as p21WAF1/Cip1 and Gadd45, as well as with proteins involved in the mechanics of DNA repair and replication. A conserved PCNA-binding motif is found in a subset of PCNA-interacting proteins, including p21, suggesting that the regulation of these interactions is important for the co-ordination of DNA replication and repair. We have identified several classes of protein which bind to Drosophila PCNA. Two of these proteins contain the consensus PCNA-binding domain: one is the Dacapo protein, a Drosophila homologue of p21WAF1/Cip1, and the second is the transposase encoded by the Pogo DNA transposon. A conserved PCNA-binding domain is also present in a human relative of Pogo, named Tigger, suggesting that this domain has a functional role in this class of transposable element. This raises interesting possibilities for a novel method of transposition in which the transposase might be targeted to replicating DNA. Finally, we have investigated the use of this conserved PCNA-binding domain as a predictor of PCNA-binding capacity

Nucleic Acids Research 26:3925-3932

Adams RR, Tavares AA, Salzberg A, Bellen HJ and Glover DM

pavarotti encodes a kinesin-like protein required to organize the central spindle and contractile ring for cytokinesis.

Mutations in the Drosophila gene pavarotti result in the formation of abnormally large cells in the embryonic nervous system. In mitotic cycle 16, cells of pav mutant embryos undergo normal anaphase but then develop an abnormal telophase spindle and fail to undertake cytokinesis. We show that the septin Peanut, actin, and the actin-associated protein Anillin, do not become correctly localized in pav mutants. pav encodes a kinesin-like protein, PAV-KLP, related to the mammalian MKLP-1. In cellularized embryos, the protein is localized to centrosomes early in mitosis, and to the midbody region of the spindle in late anaphase and telophase. We show that Polo kinase associates with PAV-KLP with which it shows an overlapping pattern of subcellular localization during the mitotic cycle and this distribution is disrupted in pav mutants. We suggest that PAV-KLP is required both to establish the structure of the telophase spindle to provide a framework for the assembly of the contractile ring, and to mobilize mitotic regulator proteins.

Genes and Development 12:1483-1494

Inoue YH and Glover DM

Involvement of the rolled/MAP kinase gene in Drosophila mitosis: interaction between genes for the MAP kinase cascade and abnormal spindle.

We have found that mutations that lead to loss of rolled/MAP kinase function result in a reduced mitotic index in the larval central nervous system, consistent with an interphase block to cell cycle progression, associated with a low frequency of cells showing chromosome over-condensation in mitosis and abnormal anaphase figures. In contrast to wild-type tissue, such rolled mutants do not show a significant increase in accumulation of mitotic cells when treated with colchicine. We have studied double mutant combinations between mutations affecting the activity of rolled/MAP kinase and several genes that are essential to the establishment of a bipolar spindle during progression through mitosis, and find no interactions with mutations in polo, mgr,or aurora. However, partial loss-of-function mutations in rolled enhance the abnormal spindle (asp) phenotype, whereas gain-of function mutations in rolled or in the gene encoding its activating kinase Dsor1, act as suppressors. We discuss these findings in relation to the proposed role of MAP kinase in mediating the spindle integrity checkpoint.

Molecular and General Genetics 258:334-341

Alphey L, Jimenez J and Glover D

A Drosophila homologue of oxysterol binding protein (OSBP) - implications for the role of OSBP.

The identification of a Drosophila homologue (OSBP-Dm) of mammalian oxysterol binding protein (OSBP) is reported. OSBP-Dm was identified by its ability to overcome the cell cycle arrest induced by over-expression of Wee1p in fission yeast. OSBP-Dm has an overall sequence identity of 52% with mammalian OSBP, and shows a number of highly conserved regions of functional significance. Insects are unable to biosynthesize the steroid core, relying instead on dietary sterols to satisfy their requirements. It is therefore unlikely that OSBP-Dm is involved in feedback inhibition of the mevalonate pathway, as has previously been suggested for its mammalian homologues.

Biochimica et Biophysica Acta 1395:159-164

Henderson DS and Glover DM

Chromosome fragmentation resulting from an inability to repair transposase-induced DNA double-strand breaks in PCNA mutants of Drosophila.

Proliferating cell nuclear antigen (PCNA) has several roles in progression through S phase: it is required for the function of DNA polymerases d and e and physically associates with the structure-specific nuclease FEN-1 that is essential for Okazaki fragment processing. The cyclin dependent kinase inhibitor p21 appears to displace FEN-1 from PCNA to inhibit DNA replication and possibly permit participation of PCNA in nucleotide excision repair. Here we show that PCNA is also indispensable for repair of DNA double-strand breaks (DSBs), lesions which are not corrected by excision repair processes. When PCNA-deficient Drosophila mutants are incorporated into a genetic system that induces chromosomal site-specific DSBs upon mobilization of transposable P elements they fail to undertake DSB repair. This has dominant lethal effects: DSBs are converted into chromosome breaks that can be seen at mitosis

Mutagenesis 13:57-60


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