SummaryCohesin is a highly conserved multisubunit complex that holds sister chromatids together in mitotic cells. At the metaphase to anaphase transition, proteolytic cleavage of the α kleisin subunit (Rad21) by separase causes cohesin's dissociation from chromosomes and triggers sister-chromatid disjunction. To investigate cohesin's function in postmitotic cells, where it is widely expressed, we have created fruit flies whose Rad21 can be cleaved by TEV protease. Cleavage causes precocious separation of sister chromatids and massive chromosome missegregation in proliferating cells, but not disaggregation of polytene chromosomes in salivary glands. Crucially, cleavage in postmitotic neurons is lethal. In mushroom-body neurons, it causes defects in axon pruning, whereas in cholinergic neurons it causes highly abnormal larval locomotion. These data demonstrate essential roles for cohesin in nondividing cells and also introduce a powerful tool by which to investigate protein function in metazoa.
Chromosome segregation during meiosis and mitosis depends on the assembly of functional kinetochores within centromeric regions. Centromeric DNA and kinetochore proteins show surprisingly little sequence conservation despite their fundamental biological role. However, our identification in Drosophila melanogaster of the most diverged orthologs identified so far, which encode components of a kinetochore protein network including the Ndc80 and Mis complexes, further emphasizes the notion of a shared eukaryotic kinetochore design. To determine its spatial organization, we have analyzed by quantitative light microscopy hundreds of native chromosomes from transgenic Drosophila strains coexpressing combinations of red and green fluorescent fusion proteins, fully capable of providing the essential wild-type functions. Thereby, Cenp-A/Cid, Cenp-C, Mis12 and the Ndc80 complex were mapped along the inter sister kinetochore axis with a resolution below 10 nm. The C terminus of Cenp-C was found to be near but well separated from the innermost component Cenp-A/Cid. The N terminus of Cenp-C is further out, clustered with Mis12 and the Spc25 end of the rod-like Ndc80 complex, which is known to bind to microtubules at its other more distal Ndc80/Nuf2 end.
SummaryPropagation of centromere identity during cell cycle progression in higher eukaryotes depends critically on the faithful incorporation of a centromere-specific histone H3 variant encoded by CENPA in humans and cid in Drosophila. Cenp-A/Cid is required for the recruitment of Cenp-C, another conserved centromere protein. With yeast three-hybrid experiments, we demonstrate that the essential Drosophila centromere protein Cal1 can link Cenp-A/Cid and Cenp-C. Cenp-A/Cid and Cenp-C interact with the N-and C-terminal domains of Cal1, respectively. These Cal1 domains are sufficient for centromere localization and function, but only when linked together. Using quantitative in vivo imaging to determine protein copy numbers at centromeres and kinetochores, we demonstrate that centromeric Cal1 levels are far lower than those of Cenp-A/Cid, Cenp-C and other conserved kinetochore components, which scale well with the number of kinetochore microtubules when comparing Drosophila with budding yeast. Rather than providing a stoichiometric link within the mitotic kinetochore, Cal1 limits centromeric deposition of Cenp-A/Cid and Cenp-C during exit from mitosis. We demonstrate that the low amount of endogenous Cal1 prevents centromere expansion and mitotic kinetochore failure when Cenp-A/Cid and Cenp-C are present in excess.
Cyclin J is a cyclin family member that appears to have evolved before the metazoan radiation. Its evolutionary conservation argues for an important role but functional characterizations of Cyclin J have remained very limited. In Drosophila, Cyclin J is expressed only in females. Using transgenic Drosophila lines expressing Cyclin J versions with N- or C-terminal GFP extensions, we demonstrate that it is expressed exclusively in the germline. After low level expression in all nuclei within the germarium, it gets highly enriched in the germinal vesicle within the oocyte until stage 12 of oogenesis, followed by disappearance after germinal vesicle breakdown before the first meiotic division. Surprisingly, Cyclin J is not required for female fertility. Chromosome segregation during female meiosis, as well as the rapid early embryonic cell cycles after fertilization, occurs normally in the complete absence of Cyclin J. Cyclin J with EGFP fused at either N- or C-terminus binds to Cdk1 and not to Cdk2. However, in contrast to the other known Cdk1 partners, the A- and B-type cyclins, Cyclin J is not degraded during mitosis.
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