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First published online March 2, 2004
doi: 10.1242/10.1242/jcs.00979

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The Drosophila centrosome-associated protein CP190 is essential for viability but not for cell division

R. D. J. Butcher^1,4,*, S. Chodagam^2,*, R. Basto², J. G. Wakefield^2,, D. S. Henderson³, J. W. Raff² and W. G. F. Whitfield^4,

¹ NERC Center for Population Biology, Imperial College London, Silwood Park Campus, Ascot SL5 7PY, UK
² Wellcome Trust-Cancer Research UK Institute, Tennis Court Road, Cambridge CB2 1QR, UK
³ Department of Pharmacological Sciences, SUNY, Stony Brook, NY 11794-8651, USA
⁴ Biological Sciences Institute, Faculty of Life Sciences, University of Dundee, Dundee DD1 4HN, UK

View larger version (12K): [in a new window]   Fig. 1. RNAi depletion of CP190 or CP60 does not lead to growth or mitotic defects in tissue culture cells. (A) A Western blot showing the depletion of CP190 and CP60 in mock (lane 1), CP60 (lane 2) or CP190 (lane 3) RNAi-treated cells 96 hours after treatment. (B) Graph showing the total number of cells per ml during the 5-day time course of a typical RNAi-depletion experiment. (C) Graph showing the mitotic index (as judged by number of phospho-histone H3-positive cells) at the 96-hour time point of a typical RNAi-depletion experiment. The total numbers of cells counted were 811, 969, and 1052 for the control, CP60, and CP190 RNAi experiments, respectively. Similar results were obtained in two separate RNAi experiments (data not shown).

View larger version (76K): [in a new window]   Fig. 2. Microtubule organisation is not disrupted during mitosis in CP190 or CP60 RNAi-depleted cells. (A) The localisation of CP190 (black and white panels, blue in merged panels), microtubules (green in merged image), and DNA (red in merged image) in mock (left set of panels) and CP190 (right set of panels) RNAi-treated cells at various stages of the cell cycle. Interphase, top row; metaphase, second row; anaphase, third row; telophase, bottom row. (B) The localisation of CP60, microtubules and DNA in mock and CP60-depleted cells at various stages of the cell cycle [all labelling as in (A)]. (C) The localisation of CP190 in CP60-depleted cells (left panels) and CP60 in CP190-depleted cells (right panels) [all labelling as in (A)]. Scale bar: 5 µm.

View larger version (9K): [in a new window]   Fig. 3. The generation of mutations in the Cp190 gene. (A) A schematic representation of the Cp190 locus. Genes are highlighted in blue, and the position of the l(3)neo43 P-element insertion is shown in green. The extent of the Df(3R)P280NR27 is indicated by the black line. (B) A western blot showing the levels of CP190 and CP60 in wild type, and Cp1901 and Cp1902 homozygous mutant brains, and in Cp1902 homozygous mutant brains that also express either a Pubq-CP190 or Pubq-190M transgene (as indicated above each lane). Note that both CP190 and CP60 appeared to migrate slightly abnormally in the pUbq-CP190 over-expressing lane; this was because of a defect in this gel and it was not seen when this sample was re-run on a different gel.

View larger version (74K): [in a new window]   Fig. 4. Mitosis is not disrupted in Cp190 mutant larval brain cells. The distribution of microtubules (black and white panels, red in merged images), CP190 (green), and DNA (blue) in typical wild type (WT) and Cp1902 mutant larval brain cells in metaphase (left panels) and anaphase (right panels). Mitotic spindle organisation appears normal in the mutant cells even though CP190 is no longer detectable at centrosomes.

View larger version (74K): [in a new window]   Fig. 5. The centrosomal localisation of CP60 is specifically disrupted in Cp190 mutant cells. (A) The distribution of CP60 (black and white panels, red in merged images), microtubules (green), and DNA (blue) in typical wild type (WT) (left panels) and Cp190 mutant (right panels) brain cells in anaphase. CP60 is not detectable at the centrosomes of Cp190 mutant cells. (B) The centrosomal localisation of CNN (red in merged image) and -tubulin (green in merged image) is not disrupted in Cp190 mutant cells. DNA is shown in blue in the merged image. Scale bar: 10 µm.

View larger version (50K): [in a new window]   Fig. 6. CP190M does not interact with centrosomes or microtubules. (A) Schematic representation of the CP190 protein. The previously identified centrosomal and microtubule targeting domain is shown in red, whereas the region deleted in CP190M is indicated by the black bars above the protein (aa309-aa541). (B) A western blot of a microtubule spin-down experiment probed with anti-CP190 and anti-CP60 antibodies. In the absence of taxol in the embryo extract, microtubules do not form. Under these conditions, when the extract is centrifuged on a sucrose cushion, CP190, CP190M and CP60 all remain in the supernatant (S) and are not detectable in the pelleted material (P). In the presence of taxol, microtubules are polymerised in the extract. Under these conditions, both CP190 and CP60 co-sediment with microtubules through the sucrose cushion and are found in the pellet. CP190M, however, does not co-sediment with the microtubules and is not detectable in the pellet. (C) The localisation of CP190 (black and white panel, red in merged image), microtubules (green in merged image) and DNA (blue in merged image) in Cp190 mutant brains that also contain a transgene expressing either full-length CP190 (pUbq-CP190 – top panels) or CP190M (pUbq-CP190M – bottom panels). Only the full-length CP190 is concentrated at centrosomes. Scale bar: 10 µm.

View larger version (15K): [in a new window]   Fig. 7. CP190 and CP190M can both rescue the lethality associated with mutations in the Cp190 gene. A graph showing the number of hatching homozygous adults from crosses of Cp190 mutant flies on their own (Mut), or of mutant flies carrying a single copy of the pUbq-CP190 or pUbq-CP190M transgene (as indicated under the graph). The expected number of eclosing adults from each cross is normalised to 1. Thus, 85% and 80% of mutant flies expressing the CP190 or CP190M transgene, respectively, survive to adulthood. The graph shows the average figure from 3 independent experiments in which more than 100 surviving flies were counted for each genotype.

View larger version (48K): [in a new window]   Fig. 8. The overexpression of CP190 or CP190M is lethal, but this lethality can be rescued by the co-overexpression of CP60. (A) A western blot showing the levels of CP190 or CP190M in wild-type adults (lane 1), in three independent homozygous transgenic Pubq-CP190 lines (lanes 2-4), and in three independent heterozygous Pubq-CP190M transgenic lines. All three Pubq-CP190 transgenic lines are homozygous viable, but exhibit high levels of pupal mortality, whereas all three Pubq-CP190M lines are homozygous lethal. (B) A western blot showing the levels of CP190, CP190M or CP60 in wild-type adults (lane 1), or in adults that are homozygous for independent Pubq-CP190 insertions and a Pubq-CP60 insertion (lanes 2,3), or in adults homozygous for independent Pubq-CP190M insertions and a Pubq CP60 insertion (lanes 4,5). Note that both the Pubq-CP190 insertions and both the Pubq-CP190M insertions used in this experiment are normally homozygous lethal. The homozygous adults appear to be viable because they also overexpress CP60. The asterisk highlights a background band that is recognised by the anti-CP60 antibodies and is shown here as a loading control.

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