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Chromosome dynamics during cell divisions in Drosophila melanogaster: The role of Rad21 in meiotic cohesion and dynamic analysis of the condensin subunit CapG in early embryonic mitotic divisions
(2010)
- Faithful segregation of genetic material is an essential hallmark of cell division. In eukaryotic cells, the DNA is replicated during S phase into two identical copies, which reside intimately paired (cohesed) in the nucleus as dispersed and entangled interphase chromatin fibers. At the onset of mitosis, the chromatin fibers start to resolve and by the end of metaphase they are compacted and individualized into a pair of cylindrical structures called sister chromatids, which remain connected until anaphase onset by residual sister chromatid cohesion in their centromeric regions. The compaction process is known as chromosome condensation, which is a prerequisite for accurate segregation of sister chromatids in anaphase. Chromosome condensation and sister chromatid cohesion require multisubunit protein complexes, the condensin and the cohesin complexes, respectively. Both complexes are composed of two core SMC subunits and a set of non-SMC subunits, which are conserved among most eukaryotes. In the first part of my thesis, I have analyzed the localization and dynamic behavior of a functional, EGFP-fused variant of CapG, one of the non-SMC subunits of the condensin I complex in Drosophila melanogaster. In vivo fluorescence microscopy of early embryonic mitotic divisions revealed that CapG-EGFP is mainly nuclear during interphase and that it starts to enrich at centromeric proximal regions in late interphase. Thereafter, CapG-EGFP spreads onto the chromosome arms concomitantly with the initiation of chromosome condensation (ICC) and loading is complete already in prophase at the time of nuclear envelope breakdown. Furthermore, FRAP analyses revealed that a major proportion of CapG-EGFP is stably bound to chromatin during metaphase and only a minor fraction shows a dynamic association with chromatin. These results are similar, but not identical, to findings previously obtained for another non-SMC subunit, CapH/Barren, suggesting interactions of the individual non-SMC subunits with chromatin outside a bona fide condensin complex. Since a non-SMC cohesin subunit homologous to the typical meiotic Rec8 protein found in other eukaryotes appears to be missing in Drosophila, I have assessed in the second part of my thesis a possible cohesive role for the mitotic subunit Rad21 during female meiosis. Furthermore, a potential redundancy during oogenesis between Rad21 and another candidate cohesin subunit, C(2)M, was analyzed. Forced proteolysis of Rad21 during oogenesis resulted in delocalization of the canonical cohesin core subunit Smc1 from oocyte chromatin. Furthermore, immunofluorescence and fluorescence in situ hybridization analyses revealed a high proportion of premature homolog disjunction and premature sister chromatid separation in the developing mutant oocytes and also during the meiotic divisions. Moreover, it was established that Rad21 has a role in the maintenance of the synaptonemal complex (SC), as shown by delocalization of the transversal SC component C(3)G. Taken together, these results suggest that Rad21 is indeed involved in sister chromatid cohesion during female meiosis in D. melanogaster. Since in the absence of Rad21 and the concomitant presence of C(2)M meiotic sister chromatid cohesion is compromised, Rad21 appears to play the major role in meiotic sister chromatid cohesion in D. melanogaster and a functional redundancy between C(2)M and Rad21 is unlikely.
