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doi: 10.1242/10.1242/jcs.00463

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Drosophila skpA, a component of SCF ubiquitin ligases, regulates centrosome duplication independently of cyclin E accumulation

Department of Embryology, Carnegie Institution of Washington, Baltimore, MD 21210, USA

View larger version (26K): [in a new window]   Fig. 1. The Drosophila genome contains six Skp1-related genes. (A) Partial phylogenetic tree of eukaryotic Skp1 homologs. ORFs homologous to human Skp1 and the related ElonginC gene were identified from the genomes of Saccharomyces cerevisiae (Sc), Schizosaccharomyces pombe (Sp), Caenorhabditis elegans (Ce), Arabidopsis thaliana (At), Drosophila melanogaster (Dm), and humans (Hs), and the predicted protein sequences were aligned using ClustalW and displayed as a phenogram. The six Drosophila Skp1-related proteins share 43-76% identity with human Skp1. Only one human Skp1 gene is known to be complete and expressed. Clusters of Skp1-related genes restricted to a single species have been grouped for clarity. (B) Expression of Drosophila skp genes during development. Northern blots of mRNA from different developmental stages (0-2- and 2-20-hour-old embryos, 3rd instar larvae, adult females, and adult males, respectively) were probed for skpA, skpB or a mixture of skpC and skpD (combined because the two transcripts could not be reliably distinguished by hybridization). Signal from the skpB probe in embryos may result from cross-hybridization with the abundant skpA transcripts. Two images of each blot are shown, with the signal scaling increased 100-fold in the bottom images to compensate for the differences in signal levels. Relative amounts of mRNA in each lane were determined by quantifying the signal obtained using an rp49 probe. (C) Representation of Drosophila skp genes in EST databases. cDNAs corresponding to five of the six Drosophila skp genes were identified, indicating that they are bona fide genes, although skpA ESTs were 10- to 80-fold more common than the other skp genes.

View larger version (27K): [in a new window]   Fig. 2. Null mutations in skpA result in larval lethality. (A) Structure of the skpA gene, transcripts and mutations. The skpA transcript structures, site of the EP(X)1423 P-element insertion, and extent of the skpA deletion alleles are shown relative to the skpA genomic region. The skpA1 deletion breakpoints were precisely mapped by sequencing; the extents of the other deletions are not known. P-element transformation of a 4 kb genomic fragment completely rescued the lethality associated with all four skpA alleles. (B) Survival of skpA– larvae. Four-hour collections of embryos from the cross skpA1/FM7, GFP x FM7, GFP/Y were aged from 1 to 5 days AED, and scored for the percentage of GFP– larvae. Approximately 25% of larvae from a viable precise excision allele are GFP– (skpA+/Y). Similarly, 25% of larvae from skpA mutant crosses are GFP– immediately after hatching; however, this percentage steadily declines, indicating that skpA–/Y larvae die at various points during larval development. (C) Growth of skpA– larvae. The average cross-sectional area of GFP– larvae from the previous cross are graphed (error bars equal one standard deviation). Wildtype and GFP+ sibling larvae increased in size 17-fold over 4 days, whereas skpA– larvae only grew 6-fold.

View larger version (103K): [in a new window]   Fig. 3. skpA mutants exhibit cell cycle defects. (A-E) Quantification of cell cycle parameters. The CNSs from wildtype and skpA– larvae of the indicated ages were assayed for different cell cycle parameters. (A) Mitotic index is the number of phosphorylated-histone H3 (P-histone H3)-positive cells per squashed field observed with a 100x objective. (B) S phase index is the number of BrdU-incorporating cells relative to the volume of DNA staining determined from whole mount confocal analyses. (C) 2C:4C ratio is the ratio of cells with a 2C versus a 4C DNA content, determined from CCD images of DAPI-stained squashed CNSs. (D) Apoptosis index is the number of TUNEL-positive cells relative to the volume of DNA staining. (E) S phase (%) is the percentage of BrdU-incorporating salivary gland or fat body nuclei relative to the total number of nuclei from larvae 3.5 days AED. Error bars in B, D and E represent one standard deviation (n4 CNSs). (F-H) Examples of BrdU incorporation and TUNEL-labeling in wildtype and mutant tissues. Projections of confocal sections through entire CNSs (F,G) or salivary glands and fat bodies (H) are shown. Note the skpA– imaginal disc (I.D.) with many TUNEL+ cells (G'). No BrdU-incorporating nuclei were observed in skpA– fat bodies (F.B.), in contrast with the neighboring salivary glands (S.G.) (H'). Bar, 30 µm.

View larger version (72K): [in a new window]   Fig. 4. SkpA– larval CNS cells accumulate supernumerary centrosomes. (A-D) DNA and centrosome staining in wildtype and skpA– cells. (A-B) SkpA– interphase cells 3.5 days AED have abnormally condensed chromatin (arrowhead) compared to wildtype. DNA that labeled with P-histone H3 antibodies is marked (M). (C) skpA– metaphase cell with four centrosomes labeled with antibodies against -tubulin and centrosomin (cnn). (D) Polyploid (16C) skpA– nucleus from larva 5.5 days AED that partially labeled with P-histone H3 antibodies and contained 24 centrosomes. Bars, 5 µm. (E) Quantification of centrosomes in mitotic wildtype and skpA– cells. The distribution of P-histone H3-positive cells with different numbers of centrosomes is shown. (F) Quantification of polyploidy in mitotic wildtype and skpA– cells. Polyploid P-histone H3-positive cells were identified from measurements of their total DNA content. Error bar represents one standard error. (G) Electron micrographs of skpA– cell from 5.5 days AED larval CNS with four pairs of centrioles in nine 105 nm sections. A low magnification section with two pairs of centrioles is shown, with high magnification views of centrioles from this or adjacent sections shown in the insets. Bars, 500 nm.

View larger version (53K): [in a new window]   Fig. 5. Supernumerary centrosomes in skpA– cells nucleate microtubules. (A-F) Projections of confocal sections of mitotic skpA– neuroblasts (A-E) or ganglion mother cell (F) labeled for DNA (P-histone H3 or TOTO-3), centrosomes (cnn) and microtubules (-tubulin). Cells have the following number of centrosomes: (A) 4, prometaphase; (B) 10, metaphase; (C) 12, metaphase; (D) 8, multipolar prometaphase; (E), 4 and 6, anaphase; and (F) 6, metaphase. Note the two chromosomes pulled off the metaphase plate by supernumerary centrosomes (arrowheads, B), and the spindle pole not associated with any centrosomes (arrow, E). Bar, 2 µm. (G) Quantification of centrosome clustering during mitotic stages. Supernumerary centrosomes in P-histone H3-positive cells from 3.5-day AED larvae were scored as clustered if they were within 2 µm of another centrosome. Mitotic stages were determined from chromosome and spindle morphology. Error bar represents one standard error. The average number and range (solid line) of centrosomes per cell is also shown for each stage. (H) Ratios of metaphase to anaphase cells from larvae of different ages.

View larger version (72K): [in a new window]   Fig. 6. SKPa protein is found in the cytoplasm and nucleus. (A) Western analysis of extracts from 4-8-hours-old embryos or newly hatched skpA+ or skpA– larvae with SKPa antisera. Approximately 30 larvae or 100 µl of embryos were used to make each extract. Relative loading was determined from a coomassie-stained gel prepared with the same samples. (B-D) Localization of SKPa in CNS and salivary gland cells. (B) Clones of skpA– cells generated using FLP-mediated mitotic recombination were positively marked with GFP using the MARCM system, and stained with SKPa antisera. Only background staining is detected in skpA– cells. (C) SKPa is found in the cytoplasm and nucleus of CNS cells, and fails to co-localize with centrosomes (cnn), DNA (TOTO-3) or mitotic chromosomes (arrowhead). SKPa is partially concentrated in the nucleus of some cells (arrow). (D) SKPa is found exclusively in the nucleus of salivary gland cells. B and D are single confocal sections, C is a projection of 5 µm of confocal sections. Bar, 30 µm (B,D) or 5 µm (C).

View larger version (50K): [in a new window]   Fig. 7. skpA– cells accumulate cyclin E. (A-B) Cyclin E staining in wildtype and skpA– cells from larvae 3.5 days AED. Stronger cyclin E staining was detected in mitotic cells (M), and in some skpA– cells (arrowheads). Most cytoplasmic proteins were extracted with these fixation conditions; samples prepared without extraction also had elevated levels of cyclin E in the cytoplasm. Cyclin E did not co-localize with centrosomes in either wildtype or skpA– cells. Equivalent exposures of cyclin E staining are shown. Bar, 5 µm. (C) Quantification of cyclin E intensity relative to cell cycle stage. DNA content was determined from the total DAPI intensity of each nucleus and correlated with the average cyclin E intensity for the corresponding region. Mitotic cells with a 4C DNA content are shown separately. Significantly higher levels of cyclin E were detected in skpA– cells with a 3C or more DNA content. Almost no signal was detected in cells from cycEk05007 larvae. (D) Western analysis of cyclin E levels in skpA+ and skpA– larvae. Cyclin E was detected on a blot identical to the one used for Fig. 6A. Both maternal (77 kDa) and zygotic (67 kDa) cyclin E are detected in extracts from 4-8-hours-old embryos.

View larger version (53K): [in a new window]   Fig. 8. A cyclin E mutation fails to suppress skpA-induced centrosome overduplication in the CNS from larvae 3.5 days AED. (A-B) Supernumerary centrosomes in skpA1; cycEk05007 mitotic cells. Shown are cycEk05007 and skpA1; cycEk05007 mutant cells with two and six centrosomes, respectively. (C) Quantification of mitotic index and centrosome numbers in different genotypes. Mitotic index and centrosome numbers were calculated as in Fig. 3A and 5G, respectively. Bars, 5 µm.