2002 Abstracts

Anderson M, Ng SS, Marchesi V, MacIver FH, Stevens FE, Riddell T, Glover DM, Hagan IM and McInerny CJ

Plo1+ regulates gene transcription at the M-G1 interval during the fission yeast mitotic cell cycle.

The regulation of gene expression plays an important part in cell cycle controls. We describe the molecular machinery that co-ordinates gene transcription at the M-G1 interval during the fission yeast mitotic cell cycle. A sequence is identified in the cdc15+ promoter that we call a PCB (pombe cell cycle box), which confers M-G1-specific transcription. Sequences similar to the PCB are present in the promoters of seven other genes, spo12+, cdc19+, fin1+, sid2+, ppb1+, mid1+/dmf1+ and plo1+, which we find to be transcribed at M-G1. A transcription factor complex is identified that binds to the PCB sequence, which we name PBF, for PCB-binding factor. Finally, we show that PBF binding activity and consequent gene transcription are regulated by the Plo1p protein kinase, thus invoking a potential auto-feedback loop mechanism that regulates mitotic gene transcription and passage through septation and cytokinesis

EMBO Journal 21:5745-5755

Plusa B, Grabarek JB, Piotrowska K, Glover DM and Zernicka-Goetz M

Site of the previous meiotic division defines cleavage orientation in the mouse embryo.

The conservation of early cleavage patterns in organisms as diverse as echinoderms and mammals suggests that even in highly regulative embryos such as the mouse, division patterns might be important for development. Indeed, the first cleavage divides the fertilized mouse egg into two cells: one cell that contributes predominantly to the embryonic part of the blastocyst, and one that contributes to the abembryonic part. Here we show, by removing, transplanting or duplicating the animal or vegetal poles of the mouse egg, that a spatial cue at the animal pole orients the plane of this initial division. Embryos with duplicated animal, but not vegetal, poles show abnormalities in chromosome segregation that compromise their development. Our results show that localized factors in the mammalian egg orient the spindle and so define the initial cleavage plane. In increased dosage, however, these factors are detrimental to the correct execution of division

Nature Cell Biology 4:811-815

Grabarek JB, Plusa B, Glover DM and Zernicka-Goetz M

Efficient delivery of dsRNA into zona-enclosed mouse oocytes and preimplantation embryos by electroporation.

Conditions for the electroporation of mouse oocytes and preimplantation embryos have been optimised by following the incorporation of rhodamine labeled dextran. This procedure includes a step to weaken but not remove the zona pellucida that helps achieve good survival. This approach has been applied to introduce double-stranded RNA for c-mos into oocytes and green fluorescent protein (GFP) into transgenic GFP-expressing embryos at the 1- and 4-cell stages. In both cases we were able to observe sequence-specific interference with the expression of the target gene - a failure of oocytes to arrest at metaphase II and a loss in the green fluorescence of embryos by the morula or blastocyst stages. These effects could be observed in multiple oocytes or embryos allowed to develop together following electroporation.

Genesis 32:269-276

Riparbelli MG, Callaini G, Glover DM and Avides MdC

A requirement for the Abnormal Spindle protein to organise microtubules of the central spindle for cytokinesis in Drosophila.

Drosophila abnormal spindle (asp) mutants exhibit a mitotic metaphase checkpoint arrest with abnormal spindle poles, which reflects a requirement for Asp for the integrity of microtubule organising centres (MTOCs). In male meiosis, the absence of a strong spindle integrity checkpoint enables asp mutant cells to proceed through anaphase and telophase. However, the central spindle region is not correctly organised and cells frequently fail to complete cytokinesis. This contrasts with meiosis in wild-type males where at late anaphase a dense array of microtubules forms in the central spindle region that has Asp localised at its border. We speculate that Asp is associated with the minus ends of microtubules that have been released from the spindle poles to form the central spindle. A parallel situation arises in female meiosis where Asp not only associates with the minus ends of microtubules at the acentriolar poles but also with the central spindle pole body that forms between the two tandem spindles of meiosis II. Upon fertilisation, Asp is also recruited to the MTOC that nucleates the sperm aster. Asp is required for growth of the microtubules of the sperm aster, which in asp mutants remains diminutive and so prevents migration of the pronuclei.

Journal of Cell Science 115:913-922

Minestrini G, Mathe E and Glover DM

Domains of the Pavarotti kinesin-like protein that direct its subcellular distribution: effects of mislocalisation on the tubulin and actin cytoskeleton during Drosophila oogenesis.

The kinesin-like protein encoded by pavarotti (Pav-KLP) is essential for cytokinesis and associates with the central part of the late mitotic spindle and interphase nuclei in somatic cells. Here we define regions of the molecule that regulate its subcellular localisation and study the consequences of overexpressing mutant forms of the protein during oogenesis in Drosophila. Pav-KLP normally associates with the oocyte nucleus, but when over-expressed at moderate levels, its GFP tagged form also accumulates in nurse cell nuclei. At high expression levels this leads to loss of the microfilaments that tether these nuclei, so that they block the ring canals and prevent the 'dumping' of nurse cell cytoplasm into the oocyte, which results in sterility. Localisation to these nuclei is prevented by mutations in either the conserved ATP-binding site of the motor domain or the nuclear localisation sequences in the C-terminal domain. Both such mutations lead to the formation of stable arrays of cytoplasmic microtubules and the progressive disruption of the actin cytoskeleton. The latter is evident by a breakdown of the cortical actin causing disruption of cell membranes; this breakdown ultimately results in the accumulation of cytoplasmic aggregates containing tubulin, actin and at least some of their binding proteins. Pav-KLP is also found associated with the ring canals, actin-rich structures built from remnants of the cytokinesis ring. The stalk domain alone is sufficient for the exclusive association of Pav-KLP to these structures, and this has no consequences for fertility. We discuss whether disruption of actin structures by full-length cytoplasmic forms of Pav-KLP is a consequence of the resulting stabilised cytoplasmic microtubules per se or accumulation of the motor protein at ectopic cortical sites to sequester molecules that regulate actin behaviour.

Journal of Cell Science 115:725-736

Giet R, McLean D, Descamps S, Lee MJ, Raff JW, Prigent C and Glover DM

Drosophila Aurora A kinase is required to localize D-TACC to centrosomes and to regulate astral microtubules.

Disruption of the function of the A-type Aurora kinase of Drosophila by mutation or RNAi leads to a reduction in the length of astral microtubules in syncytial embryos, larval neuroblasts, and cultured S2 cells. In neuroblasts, it can also lead to loss of an organized centrosome and its associated aster from one of the spindle poles, whereas the centrosome at the other pole has multiple centrioles. When centrosomes are present at the poles of aurA mutants or aurA RNAi spindles, they retain many antigens but are missing the Drosophila counterpart of mammalian transforming acidic coiled coil (TACC) proteins, D-TACC. We show that a subpopulation of the total Aurora A is present in a complex with D-TACC, which is a substrate for the kinase. We propose that one of the functions of Aurora A kinase is to direct centrosomal organization such that D-TACC complexed to the MSPS/XMAP215 microtubule-associated protein may be recruited, and thus modulate the behavior of astral microtubules.

Journal of Cell Biology 156:437-451

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

Mapping and identification of essential gene functions on the X chromosome of Drosophila.

The Drosophila melanogaster genome consists of four chromosomes that contain 165 Mb of DNA, 120 Mb of which are euchromatic. The two Drosophila Genome Projects, in collaboration with Celera Genomics Systems, have sequenced the genome, complementing the previously established physical and genetic maps. In addition, the Berkeley Drosophila Genome Project has undertaken large-scale functional analysis based on mutagenesis by transposable P element insertions into autosomes. Here, we present a large-scale P element insertion screen for vital gene functions and a BAC tiling map for the X chromosome. A collection of 501 X-chromosomal P element insertion lines was used to map essential genes cytogenetically and to establish short sequence tags (STSs) linking the insertion sites to the genome. The distribution of the P element integration sites, the identified genes and transcription units as well as the expression patterns of the P-element-tagged enhancers is described and discussed.

EMBO Reports 3:34-38

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