Skp1 is localized to centrosomes throughout the cell cycle in vertebrate cells (Freed, 1999; Gstaiger, 1999), suggesting that it may act directly at the centrosome to regulate duplication. To determine if SKPa shows a similar localization pattern in Drosophila cells, two polyclonal antibodies were raised against recombinant SKPa. Both antisera predominantly recognize a single 24 kDa band in embryo, larval and adult extracts which is absent from skpA- larval extracts, indicating that the antisera specifically recognize SKPa (Murphy, 2003).
Immunofluorescence analyses reveal that SKPa is localized throughout the cytoplasm and nucleus in diploid tissues such as the CNS and during embryogenesis. Triple labeling cells for SKPa, DNA and centrosomes suggests that SKPa does not preferentially associate with centrosomes or chromosomes at any point during the cell cycle. SKPa is slightly more concentrated in the nucleus of some diploid cells, and is predominantly nuclear in salivary gland and fat body cells. No signal was observed in skpA- cells from mosaic or homozygous mutant larvae, demonstrating that the staining pattern specifically represents the localization of SKPa in CNS cells (Murphy, 2003).
The relatively uniform distribution of Drosophila SKPa in diploid tissues is dramatically different from the centrosomal localization observed in vertebrate cells. In Drosophila, SKPa may regulate centrosome duplication by transiently associating with the centrosome; alternatively, it may function indirectly by acting on cytoplasmic or nuclear proteins. In contrast, the pronounced localization of SKPa to the nucleus of polyploid cells suggests that it may function directly on nuclear proteins involved in endoreduplication (Murphy, 2003).
Extensive biochemical analyses in yeast and vertebrates have shown that Skp1 homologs primarily function as part of SCF complexes that regulate the ubiquitination and subsequent degradation of various proteins in the cell. One known target is cyclin E, which is degraded via an SCF complex in vitro and in vivo and is necessary for centrosome duplication in some vertebrate cell assays. Together, these data suggest a model in which skpA- cells accumulate high levels of cyclin E that drive extra rounds of centrosome duplication (Murphy, 2003).
To test this model, cyclin E levels were quantified at different points of the cell cycle using immunofluorescence correlated with nuclear DNA content and morphology. Wildtype cells in G1 phase (2C DNA content) show low levels of cyclin E staining that are, on average, greater than the background staining observed in cycE- cells, which probably reflects cyclin E beginning to accumulate in late-G1 cells. Cyclin E staining intensity is increased in S- and G2-phase cells, and is highest in mitotic cells (2.3-fold higher than in G1). Because cyclin E levels are low in G1 phase, cyclin E must normally be degraded at the end of mitosis (after anaphase) in the CNS (Murphy, 2003).
As predicted by the model, some skpA- cells accumulate higher levels of cyclin E than wildtype. SkpA- cells in G1 and early S phase have cyclin E levels similar to wildtype; however, cells in late S, G2 and M phase stain 1.5 to 2-fold more intensely than similarly-staged wildtype cells. As in wildtype, cyclin E levels are highest in mitotic cells (4-fold higher than in G1). Cyclin E levels were also measured in extracts of newly-eclosed wildtype and skpA- larvae by Western blotting. Fivefold more cyclin E was detected in skpA- larval extracts than wildtype, confirming that loss of skpA function results in the accumulation of cyclin E (Murphy, 2003).
These results indicate that skpA function is required to properly regulate cyclin E levels in the CNS. However, the overall pattern of cyclin E accumulation during S, G2 and M phases and subsequent degradation at the end of mitosis is not perturbed. One possibility is that many skpA- cells are arrested in G1 with low cyclin E levels while a subpopulation of cells manage to proceed through the cell cycle and produce two G1 cells with high cyclin E levels; however, this is unlikely to be the case because no small population of G1 cells with high cyclin E levels was observed. Therefore, CNS cells probably have a skpA-independent mechanism to degrade cyclin E at the end of mitosis, and only require skpA to prevent the accumulation of abnormally high levels of cyclin E during the cell cycle (Murphy, 2003).
If the elevated levels of cyclin E in skpA- cells are necessary to induce centrosome overduplication, then mutations in cyclin E should suppress the skpA- phenotype. To test this prediction, cyclin E levels were reduced with a P element allele [l(2)k05007, hereafter referred to as cycEk05007] that results in larval lethality and a growth defect similar to, but more severe than, loss of skpA function. Immunofluorescence staining of cyclin E verified that the cycEk05007 mutation dramatically reduces levels of cyclin E in all cells of the CNS. Most CNS cells in cycEk05007 larvae have a 2C DNA content suggesting that they are arrested in G1 phase. However, a few cells are able to proceed through the cell cycle and enter mitosis; these cells have slightly higher levels of cyclin E than seen in G1 phase cells, suggesting that some cycEk05007 cells still produce a small amount of cyclin E that is sufficient to proceed through the cell cycle. Therefore, cycEk05007 is a strong hypomorphic mutation that dramatically reduces but does not eliminate cyclin E from CNS cells (Murphy, 2003).
Centrosome staining and quantification in cycEk05007 and skpA1; cycEk05007 cells reveals that low levels of cyclin E are sufficient for centrosome overduplication. CycEk05007 cells that have proceeded into mitosis invariably have two centrosomes and replicated chromosomes, indicating that the low levels of cyclin E in cycling cycEk05007 cells are sufficient for centrosome duplication to occur. Mitotic cells in skpA1; cycEk05007 larvae were even rarer than in cycEk05007 larvae. Nonetheless, supernumerary centrosomes are observed in 56% of skpA1; cycEk05007 mitotic cells 3.5 days AED, similar to the 60% frequency seen in skpA1 cells. The difficulty of generating skpA1; cycEk05007 larvae precluded direct measurements of cyclin E levels; however, the fact that loss of skpA function does not increase the frequency of cycling cells compared to cycEk05007 larvae suggests that cyclin E levels are still limiting for entry into S phase and must be lower than in wildtype cells. Therefore, the elevated levels of cyclin E found in skpA- cells are not necessary for centrosome overduplication to occur (Murphy, 2003).
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date revised: 12 February 2004
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