2001 Abstracts

Carpenter ATC

Attached-X and other compound chromosomes.

No abstract available

In S Brenner and JH Miller (Eds), Encylopedia of Genetics, Academic Press, San Diego

Glover DM

...still cycling.

No abstract available

Journal of Cell Science 114:3953-2954

Donaldson MM, Tavares AAM, Hagan IM, Nigg EA and Glover DM

The mitotic roles of Polo-like kinase.

The Polo-like protein kinases (Plks) are a conserved family of enzymes that play a variety of roles in the passage of cells through M phase. Named after the Drosophila polo gene originally identified through a recessive maternal effect lethal mutation, conserved Plk homologues have been identified in yeast, Xenopus, C. elegans and mammals. The interactions presented here represent information drawn from all these systems and integrated to form an overall picture. As with any undertaking of this type, there will be slight inconsistencies between specific roles for Plks in different species.

Journal of Cell Science 114:2357-2358

de Carcer G, Avides MdC, Lallena MJ, Glover DM and Gonzalez C

Requirement of Hsp90 for centrosomal function reflects its regulation of Polo kinase stability.

We have previously shown that the molecular chaperone heat shock protein 90 (Hsp90) is required to ensure proper centrosome function in Drosophila and vertebrate cells. This observation led to the hypothesis that this chaperone could be required for the stability of one or more centrosomal proteins. We have found that one of these is Polo, a protein kinase known to regulate several aspects of cell division including centrosome maturation and function. Inhibition of Hsp90 results in the inactivation of Polo kinase activity. It also leads to a loss in the ability of cytoplasmic extracts to complement the failure of salt-stripped preparations of centrosomes to nucleate microtubules. This effect can be rescued upon addition of active recombinant Polo. We also show that Polo and Hsp90 are part of a complex and conclude that stabilization of Polo is one of the mechanisms by which Hsp90 contributes to the maintenance of functional centrosomes.

EMBO Journal 20:2878-2884

Donaldson MM, Tavares AAM, Ohkura H, Deak P and Glover DM

Metaphase arrest with centromere separation in polo mutants of Drosophila.

The Drosophila gene polo encodes a conserved protein kinase known to be required to organize spindle poles and for cytokinesis. Here we report two strongly hypomorphic mutations of polo that arrest cells of the larval brain at a point in metaphase when the majority of sister kinetochores have separated by between 20-50% of the total spindle length in intact cells. In contrast, analysis of sister chromatid separation in squashed preparations of cells indicates that some 83% of sisters remain attached. This suggests the separation seen in intact cells requires the tension produced by a functional spindle. The point of arrest corresponds to the spindle integrity checkpoint; Bub1 protein and the 3F3/2 epitope are present on the separated kinetochores and the arrest is suppressed by a bub1 mutation. The mutant mitotic spindles are anastral and have assembled upon centrosomes that are associated with Centrosomin and the abnormal spindle protein (Asp), but neither with ?-tubulin nor CP190. We discuss roles for Polo kinase in recruiting centrosomal proteins and in regulating progression through the metaphase-anaphase checkpoint.

Journal of Cell Biology 153:663-677

Benos PV, Gatt MK, Murphy L, Harris D, Barrell B, Ferraz C, Vidal S, Brun C, Demailles J, Cadieu E, Dreano S, Gloux S, Lelaure V, Mottier S, Galibert F, Borkova D, Minana B, Kafatos FC, Louis C, Siden-Kiamos I, Bolshakov S, Papagiannakis G, Spanos L, Madueno E, de Pablos B, Modolell J, Peter A, Schottler P, Werner M, Mourkioti F, Beinert N, Dowe G, Schafer U, Jackle H, Bucheron A, Callister D, Campbell L, Henderson NS, McMillan PJ, Salles C, Tait E, Valenti P, Pachter L, Saunders RDC, Glover DM and Ashburner M

From first base: The sequence of the tip of the X chromosome of D. melanogaster, a comparison of two sequencing strategies.

We present the sequence of a contiguous 2.63 Mb of DNA extending from the tip of the X chromosome of Drosophila melanogaster. Within this sequence, we predict 277 protein coding genes, of which 94 had been sequenced already in the course of studying the biology of their gene products, and examples of 12 different transposable elements. We show that an interval between bands 3A2 and 3C2, believed in the 1970s to show a correlation between the number of bands on the polytene chromosomes and the 20 genes identified by conventional genetics, is predicted to contain 45 genes from its DNA sequence. We have determined the insertion sites of P-elements from 111 mutant lines, about half of which are in a position likely to affect the expression of novel predicted genes, thus representing a resource for subsequent functional genomic analysis. We compare the European Drosophila Genome Project sequence with the corresponding part of the independently assembled and annotated Joint Sequence determined through "shotgun" sequencing. Discounting differences in the distribution of known transposable elements between the strains sequenced in the two projects, we detected three major sequence differences, two of which are probably explained by errors in assembly; the origin of the third major difference is unclear. In addition there are eight sequence gaps within the Joint Sequence. At least six of these eight gaps are likely to be sites of transposable elements; the other two are complex. Of the 275 genes in common to both projects, 60% are identical within 1% of their predicted amino-acid sequence and 31% show minor differences such as in choice of translation initiation or termination codons; the remaining 9% show major differences in interpretation.

Genome Research 11:710-730

Avides MdC, Tavares A and Glover DM

Polo kinase and Asp are required to promote mitotic centrosomal organising activity.

Interfering with the activity of polo-like kinases can lead to the formation of monopolar spindles. Polo-like kinases also regulate mitotic entry, activation of the anaphase-promoting complex and the necessary preconditions for cytokinesis. Similarities between the phenotypes of the Drosophila mutants asp and polo point towards a common role in spindle pole function. The abnormal spindles of asp mutants are bipolar but have disorganized broad poles at which ?-tubulin has an abnormal distribution. Moreover, the synergism of polo1 aspE3 double mutants indicates a possible involvement of these genes in a common process. Asp is a microtubule-associated protein of relative molecular mass 220,000 (Mr 220K) found at the face of the centrosome that contacts spindle microtubules. In partially purified centrosomes, it is required with ?-tubulin to organize microtubule asters. Here, we show that Asp is the previously identified Mr 220K substrate of Polo kinase. Polo phosphorylates Asp in vitro, converting it into an MPM2 epitope. Polo and Asp proteins immunoprecipitate together and exist as part of a 25-38S complex. Extracts of polo-derived embryos are unable to restore the ability of salt-stripped centrosomes to nucleate microtubule asters. This can be rescued by addition of phosphorylated Asp or active Polo kinase.

Nature Cell Biology 3:421-424

Tanaka K, Petersen J, MacIver F, Mulvihill DP, Glover DM and Hagan IM

The role of Plo1 kinase in mitotic commitment and septation in Schizosaccharomyces pombe.

Plo1-associated casein kinase activity peaked during mitosis before septation. Phosphatase treatment abolished this activity. Mitotic Plo1 activation had a requirement for prior activation of M-phase promoting factor (MPF), suggesting that Plo1 does not act as a mitotic trigger kinase to initiate MPF activation during mitotic commitment. A link between Plo1 and the septum initiating network (SIN) has been suggested by the inability of plo1 cells to septate and the prolific septation following plo1+ overexpression. Interphase activation of Spg1, the G protein that modulates SIN activity, induced septation but did not stimulate Plo1-associated kinase activity. Conversely, SIN inactivation did not affect the mitotic stimulation of Plo1-associated kinase activity. plo1.ts4 cells formed a misshapen actin ring, but rarely septated at 36°C. Forced activation of Spg1 enabled plo1.ts4 mutant cells, but not cells with defects in the SIN component Sid2, to convert the actin ring to a septum. The ability of plo1+ overexpression to induce septation was severely compromised by SIN inactivation. We propose that Plo1 acts before the SIN to control septation.

EMBO Journal 20:1259-1270

Giet R and Glover DM

Drosophila Aurora B kinase is required for histone H3 phosphorylation and condensin recruitment during chromosome condensation and to organise the central spindle during cytokinesis.

Aurora/Ipl1-related kinases are a conserved family of enzymes that have multiple functions during mitotic progression. Although it has been possible to use conventional genetic analysis to dissect the function of aurora, the founding family member in Drosophila, the lack of mutations in a second aurora-like kinase gene, aurora B, precluded this approach. We now show that depleting Aurora B kinase using double-stranded RNA interference in cultured Drosophila cells results in polyploidy. aurora B encodes a passenger protein that associates first with condensing chromatin, concentrates at centromeres, and then relocates onto the central spindle at anaphase. Cells depleted of the Aurora B kinase show only partial chromosome condensation at mitosis. This is associated with a reduction in levels of the serine 10 phosphorylated form of histone H3 and a failure to recruit the Barren condensin protein onto chromosomes. These defects are associated with abnormal segregation resulting from lagging chromatids and extensive chromatin bridging at anaphase, similar to the phenotype of barren mutants. The majority of treated cells also fail to undertake cytokinesis and show a reduced density of microtubules in the central region of the spindle. This is accompanied by a failure to correctly localize the Pavarotti kinesin-like protein, essential for this process. We discuss these conserved functions of Aurora B kinase in chromosome transmission and cytokinesis.

Journal of Cell Biology 152:669-682


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