Condensins and mitotic chromosome structure: functional and dynamic analysis in Drosophila Melanogaster

Tese de doutoramento em Bioquímica (Biologia Molecular) apresentada à Fac. de Ciências e Tecnologia de Coimbra ; The condensed state of mitotic chromosomes is crucial for the faithful segregation of the genome during cell division. Chromosome condensation not only allows the physical compaction of chromatin but also promotes the resolution of topological problems such as intertwines between sister chromatids and different chromosomes. Key factors implicated in the formation of mitotic chromosomes are the condensin I and II complexes. However, the exact contribution of these complexes and the molecular mechanisms involved are far from being understood. The work reported in this thesis aims to further our understanding on the role of condensins in the structure of mitotic chromosome in Drosophila melanogaster. The first part of the thesis describes the phenotypic analysis of S2 cells in which the condensin I subunit Barren/CAP-H was depleted. The results showed that mitotic chromosomes are able to condense but fail to resolve sister chromatids. Additionally, Barren/CAP-H-depleted cells show chromosome congression defects that are not associated to abnormal kinetochoremicrotubule interaction. Instead, the centromeric and pericentromeric heterochromatin of Barren/CAP-H-depleted chromosomes shows severe structural abnormalities. The data suggests that centromeric heterochromatin organized in the absence of Barren/CAP-H cannot withstand the forces exerted by the mitotic spindle and undergoes irreversible distortion. The second part of the thesis reports the in vivo analysis of the dynamic behavior of condensin I during early embryonic divisions. We find that Barren-EGFP associates with chromatin early in prophase concomitantly with the initiation of chromosome condensation. Barren-EGFP loading starts at the centromeric region from where it spreads distally reaching maximum accumulation at metaphase/early anaphase. Furthermore, FRAP analysis indicates that most of the bound protein exchanges rapidly with the ...