Spatial organization of a ubiquitous eukaryotic kinetochore protein network in Drosophila chromosomes.
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.
Schittenhelm
Ralf B
RB
Department of Genetics, University of Bayreuth, Universitaetsstr. 30, 95447 Bayreuth, Germany.
Heeger
Sebastian
S
Althoff
Friederike
F
Walter
Anne
A
Heidmann
Stefan
S
Mechtler
Karl
K
Lehner
Christian F
CF
D000818:Animals; D030801:Animals, Genetically Modified; D002503:Centromere / Q000737:chemistry* / Q000648:ultrastructure; D000076247:Centromere Protein A; D002868:Chromosomal Proteins, Non-Histone / Q000032:analysis; D002875:Chromosomes / Q000737:chemistry / Q000648:ultrastructure; D004268:DNA-Binding Proteins / Q000032:analysis; D004330:Drosophila; D029721:Drosophila Proteins / Q000032:analysis* / Q000502:physiology; D006657:Histones / Q000032:analysis; D018386:Kinetochores / Q000737:chemistry* / Q000648:ultrastructure; D008869:Microtubule-Associated Proteins / Q000032:analysis; D008938:Mitosis; D009687:Nuclear Proteins / Q000032:analysis* / Q000502:physiology; D008941:Spindle Apparatus / Q000737:chemistry
D000076247:Centromere Protein A; D002868:Chromosomal Proteins, Non-Histone; C427237:Cid protein, Drosophila; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D006657:Histones; D008869:Microtubule-Associated Proteins; D009687:Nuclear Proteins; C060905:centromere protein C