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First published online 25 September 2007
doi: 10.1242/jcs.014290

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Microcephalin coordinates mitosis in the syncytial Drosophila embryo

Kathrin Brunk^1,2,3,*, Bertrand Vernay^3,*, Elen Griffith³, Natalie L. Reynolds³, David Strutt², Philip W. Ingham² and Andrew P. Jackson^3,

¹ Institute of Integrative and Comparative Biology, University of Leeds, LS2 9JT, UK
² MRC Centre for Developmental and Biomedical Genetics, University of Sheffield, S10 2TN, UK
³ MRC Human Genetics Unit, Western General Hospital, Edinburgh, EH4 2XU, UK

View larger version (38K): [in this window] [in a new window]   Fig. 1. Drosophila microcephalin. (A) Genomic organisation of mcph1. Schematic drawing illustrating exons (boxed), coding sequence (grey shading) and P{GawB}NP6229 insertion site (triangle). Translational start sites (arrows) are present in exon 1, and intron 1 (for transcripts with unspliced intron 1). Variable splicing in intron 7 results in a second premature stop codon (asterisk). Deleted regions in the mcph1d1 and mcph1d2 mutants, generated by imprecise excision of the P{GawB}NP6229 element are marked by black bars. The mcph1d1 deletion is 1210 bp from the P insertion site to coding base pair 626 in exon 3. mcph1d2 deletion extends 1649 bp from the P insertion site to intron 4. (B) Schematic drawings showing the organisation of the mcph1 transcripts and (C) proteins. The long (L) isoform contains three BRCT domains (black boxes), and the short (S) isoform contains a single complete BRCT domain near its N terminus. The human MCPH1 protein domain structure is shown for comparison. Transcripts with unspliced intron 1 (indicated by dashed lines) are predicted to utilise a second translational start site, resulting in proteins with the same domain structure as MCPH1-L and/or MCPH1-S, but 47 amino acids shorter at the N terminus. (N-terminally truncated proteins not shown.) (D) In situ hybridisation of mcph1 transcript in wild-type embryos using clone LD43341 as probe. (a-c) mcph1 sense probe. (d-f) mcph1 antisense probe, showing that Drosophila microcephalin transcript is present at its highest embryonic level during syncytial stages (d). (E) Immunoblot analysis of wild-type embryos (wt) and embryos from mcph1d2/d2 females (d2) using an affinity-purified rabbit polyclonal anti-MCPH1 antibody raised against a polypeptide at the N-terminal end of MCPH1 (AA 1-130), indicated by the antibody symbol in C. The antibody detects Microcephalin at 115 kDa in wild-type but not in mcph1d2/d2 embryos. Loading control, cyclin B. (F) Developmental western blot of MCPH1. The 115 kDa isoform of MCPH1 is highly expressed in embryos (0-2 hour AED) and ovaries, but is present at lower levels in third instar larval brain. (G) The 115 kDa endogenous protein migrates most similarly to the short isoform. Transgenically expressed epitope tagged (Myc x6) MCPH1(L) and MCPH1(S) isoforms compared with the lower 115 kDa endogenous MCPH1 protein present in all lanes except mcph1d2/d2 embryos. Loading control -tubulin (-Tub). (H) RT-PCR amplification of mcph1 exons 6-8 in wild-type 0- to 2-hour embryos, late embryos and testis. Primer locations indicated by arrowheads in A. The upper band corresponds to transcripts containing the unspliced intron 7, and the lower band, fully spliced exon 6-8. Control, Ribosomal protein RP49 transcript.

View larger version (46K): [in this window] [in a new window]   Fig. 2. MCPH1 cellular localisation in the syncytial embryo varies through the cell cycle. (A) Localisation of endogenous MCPH1 during phases of the cell cycle in syncytial blastoderm embryos. DNA (blue, DAPI); affinity purified anti-MCPH1 antibody (green). MCPH1 is detectable in prophase (Pro) and interphase (Inter) nuclei and is not detectable in prometaphase (Prometa), metaphase (Meta), anaphase (Ana) or telophase (Telo). Bar, 5 µm. (B) Microcephalin is expressed at constant levels throughout mitosis. Methanol fixed, DAPI stained embryos from cycles 8-10 were hand sorted, and 15 embryos per mitotic stage were analysed by SDS-PAGE and immunoblotting with anti-MCPH1 antibody.

View larger version (67K): [in this window] [in a new window]   Fig. 3. GFP-MCPH1(S) and GFP-MCPH1(L) have different subcellular localisations during mitosis. (A,B), GFP-MCPH1(S) and GFP-MCPH(L) isoforms expressed in blastoderm stage embryos followed from interphase to subsequent interphase, show different localisations during mitosis. Time in minutes is shown at the top right of each panel. Bar, 10 µm. (A) After nuclear envelope breakdown GFP-MCPH1(S) is localised at the centrosomes, and faintly on the spindle (inset t=2:16, time in minutes:seconds). At later stages of mitosis, increased fluorescence is seen at the spindle poles, either representing increased protein at the spindle poles, or accumulation on decondensing chromosomes (t=2:42, arrow). (B) GFP-MCPH(L) is localised to the interphase nucleus, and this localisation is lost at the onset of mitosis. Several foci of GFP-MCPH1(L) then appear (t=2:38, representative nucleus, circled). These foci then appear to separate (t=3:03), and the fluorescent signal then expands to become decondensing. (C) In fixed blastoderm embryos nuclear GFP-MCPH1(S) staining is lost during prophase as DNA becomes histone H3 phosphorylated. (D) Anaphase and (E) telophase: GFP-MCPH1(S) reappears in decondensing telophase nuclei with loss of phosphoH3 staining. DNA (DAPI, blue), anti-GFP antibody (green), anti-phospho-histone H3 (red) in C,D and E. Bar, 10 µm.

View larger version (64K): [in this window] [in a new window]   Fig. 4. mcph1 has an early maternal effect lethal phenotype with cell cycle arrest in a metaphase-like state. (A) Time course of nuclear division in wild-type and mcph1d1/d1 embryos. Mitotic nuclei are stained with phosphoH3 antibody. mcph1d1/d1 embryos have a reduced number of nuclei, which become non-uniform in distribution and never migrate to the surface. By 2 hours, mcph1d1/d1 embryos begin to degenerate, exhibiting shape irregularities and disintegration of the yolk. (B) Number of nuclei per embryo. Mean number of nuclei is reduced in mcph1 mutants at all time points (AED at 25°C), indicating that nuclear division is markedly slowed. Number of nuclei in mutants is not 2n in the majority of embryos, suggesting loss of mitotic synchrony. Error bars, standard deviation. The numbers of wild-type nuclei plotted from 70 to 135 minutes are extrapolated from published data (Sullivan et al., 2000). (C-G) Terminal phenotype of mcph1d2/d2 embryos. DNA (DAPI, blue), microtubules (-tubulin, green), centrosomes [Centrosomin (Cnn), red]. (C) mcph1d2/d2 embryo: nuclei in a metaphase-like state, with multiple free centrosomes. (D) Detail from embryo shown in C (boxed). (E) Monopolar, monoastral, and (F) multipolar spindles are also seen. (G) Fragments of DNA also become distributed onto the acentrosomal anastral spindle. Asp localises on spindle poles (H) and gamma-tubulin is present at the centrosome (I) in mutant embryos. DNA (DAPI, blue), microtubules (-tubulin, green), Asp (H, red) or -tubulin (I, red). Bars, (C) 50 µm, (D) 10 µm, (E,F,G) 10 µm, (H,I) 8 µm.

View larger version (69K): [in this window] [in a new window]   Fig. 5. Centrosomal abnormalities are present from the first mitotic cycle. (A) Mitosis in cycle 1 in wild-type Oregon embryos. DNA (DAPI, blue), microtubules (-tubulin, green), centrosomes (Cnn, red). Centrosome separation occurs at telophase in wild-type embryos. (B) Cycle 1 mitosis in mcph1d2/d2 embryos. Nuclear compaction is less advanced at pronuclear fusion and prophase in mutant embryos (larger nuclear diameter than in wild type). Duplication of Centrosomin signal is also apparent from prophase, much earlier than in wild-type embryos. By metaphase, centrosomes are tandemly duplicated along the axis of the spindle, rather than side by side. Centrosome detachment also occurs, and increased -tubulin is seen at the centrosome, compared to that in wild-type embryos. The mitotic spindle is irregular and less substantial. Bar, 10 µm. (C) Centrosomal separation occurs earlier in mcph1d2/d2 embryos than wild-type Oregon cycle 1 embryos. n=66 mcph1, n=22 Oregon. (D) Proportion of cycle 1 embryos by mitotic stage, from 0-30 minute AED embryos collections of wild-type and mcph1d2/d2 females. There is an excess of mcph1d2/d2 embryos at prophase and metaphase in cycle 1 (P<0.001). Error bars, standard error. mcph1, total embryos scored n=601, unfertilised n=125, meiosis n=66, cycle 1 n=179, cycle 2 and later n=231 (38.44%). Oregon, total embryos scored n=656, unfertilised n=81, meiosis n=55, cycle 1 n=179, cycle 2 and later n=418 (63.72%).

View larger version (50K): [in this window] [in a new window]   Fig. 6. mcph1 mutant embryos have reduced inhibitory phosphorylation of Cdk1, and normal Cyclin protein levels. Immunoblot analysis of wild-type embryos and mcph1d2/d2 embryos 0-1 hour AED. Immunoblotted embryo extracts for Cyclin A, CyclinB, Cdk1 phospho-tyrosine 15 (p-Cdk1tyr15), Cdk1, Chk1, and Actin as a loading control. mcph1d2/d2 embryos have a reduced level of inhibitory Cdk1 phosphorylation compared to wild-type embryos, whereas other cell cycle proteins are apparently present at normal levels.