title: Molecular analysis of core kinetochore composition and assembly in Drosophila melanogaster.
abstract: BACKGROUND
  Kinetochores are large multiprotein complexes indispensable for proper chromosome segregation. Although Drosophila is a classical model organism for studies of chromosome segregation, little is known about the organization of its kinetochores.
  
  
  RESULTS
  We employed bioinformatics, proteomics and cell biology methods to identify and analyze the interaction network of Drosophila kinetochore proteins. We have shown that three Drosophila proteins highly diverged from human and yeast Ndc80, Nuf2 and Mis12 are indeed their orthologues. Affinity purification of these proteins from cultured Drosophila cells identified a further five interacting proteins with weak similarity to subunits of the SPC105/KNL-1, MIND/MIS12 and NDC80 kinetochore complexes together with known kinetochore associated proteins such as dynein/dynactin, spindle assembly checkpoint components and heterochromatin proteins. All eight kinetochore complex proteins were present at the kinetochore during mitosis and MIND/MIS12 complex proteins were also centromeric during interphase. Their down-regulation led to dramatic defects in chromosome congression/segregation frequently accompanied by mitotic spindle elongation. The systematic depletion of each individual protein allowed us to establish dependency relationships for their recruitment onto the kinetochore. This revealed the sequential recruitment of individual members of first, the MIND/MIS12 and then, NDC80 complex.
  
  
  CONCLUSIONS
  The Drosophila MIND/MIS12 and NDC80 complexes and the Spc105 protein, like their counterparts from other eukaryotic species, are essential for chromosome congression and segregation, but are highly diverged in sequence. Hierarchical dependence relationships of individual proteins regulate the assembly of Drosophila kinetochore complexes in a manner similar, but not identical, to other organisms.
authors.affiliation: Cancer Research UK, Cell Cycle Genetics Research Group, Department of Genetics, University of Cambridge, Cambridge, United Kingdom. mrp50@cam.ac.uk
authors.forename: Marcin R
authors.initials: MR
authors.lastname: Przewloka
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
authors.forename: Patricia
authors.initials: P
authors.lastname: Costa
authors.forename: Vincent
authors.initials: V
authors.lastname: Archambault
authors.forename: Pier Paolo
authors.initials: PP
authors.lastname: D'Avino
authors.forename: Kathryn S
authors.initials: KS
authors.lastname: Lilley
authors.forename: Ernest D
authors.initials: ED
authors.lastname: Laue
authors.forename: Andrew D
authors.initials: AD
authors.lastname: McAinsh
authors.forename: David M
authors.initials: DM
authors.lastname: Glover
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D029721:Drosophila Proteins / Q000737:chemistry / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D018386:Kinetochores; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D040901:Proteomics; D017386:Sequence Homology, Amino Acid
chemical_list: D029721:Drosophila Proteins