title: Insights from the reconstitution of the divergent outer kinetochore of Drosophila melanogaster.
abstract: Accurate chromosome segregation during mitosis and meiosis is crucial for cellular and organismal viability. Kinetochores connect chromosomes with spindle microtubules and are essential for chromosome segregation. These large protein scaffolds emerge from the centromere, a specialized region of the chromosome enriched with the histone H3 variant CENP-A. In most eukaryotes, the kinetochore core consists of the centromere-proximal constitutive centromere-associated network (CCAN), which binds CENP-A and contains 16 subunits, and of the centromere-distal Knl1 complex, Mis12 complex, Ndc80 complex (KMN) network, which binds microtubules and contains 10 subunits. In the fruitfly, Drosophila melanogaster, the kinetochore underwent remarkable simplifications. All CCAN subunits, with the exception of centromeric protein C (CENP-C), and two KMN subunits, Dsn1 and Zwint, cannot be identified in this organism. In addition, two paralogues of the KMN subunit Nnf1 (Nnf1a and Nnf1b) are present. Finally, the Spc105R subunit, homologous to human Knl1/CASC5, underwent considerable sequence changes in comparison with other organisms. We combined biochemical reconstitution with biophysical and structural methods to investigate how these changes reflect on the organization of the Drosophila KMN network. We demonstrate that the Nnf1a and Nnf1b paralogues are subunits of distinct complexes, both of which interact directly with Spc105R and with CENP-C, for the latter of which we identify a binding site on the Mis12 subunit. Our studies shed light on the structural and functional organization of a highly divergent kinetochore particle.
authors.affiliation: Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Straße 11, 44227 Dortmund, Germany.
authors.forename: Yahui
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Straße 11, 44227 Dortmund, Germany.
authors.forename: Arsen
authors.initials: A
authors.lastname: Petrovic
authors.affiliation: Gene Center Munich, Ludwig-Maximilians-Universität München, Feodor-Lynen-Strasse 25, 81377 Munich, Germany.
authors.forename: Pascaline
authors.initials: P
authors.lastname: Rombaut
authors.affiliation: Department of Structural Biochemistry, Max Planck Institute of Molecular Physiology, Otto-Hahn-Straße 11, 44227 Dortmund, Germany.
authors.forename: Shyamal
authors.initials: S
authors.lastname: Mosalaganti
authors.affiliation: Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Straße 11, 44227 Dortmund, Germany.
authors.forename: Jenny
authors.initials: J
authors.lastname: Keller
authors.affiliation: Department of Structural Biochemistry, Max Planck Institute of Molecular Physiology, Otto-Hahn-Straße 11, 44227 Dortmund, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Raunser
authors.affiliation: Gene Center Munich, Ludwig-Maximilians-Universität München, Feodor-Lynen-Strasse 25, 81377 Munich, Germany.
authors.forename: Franz
authors.initials: F
authors.lastname: Herzog
authors.affiliation: Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Straße 11, 44227 Dortmund, Germany Centre for Medical Biotechnology, Faculty of Biology, University Duisburg-Essen, Universitätsstraße, 45141 Essen, Germany andrea.musacchio@mpi-dortmund.mpg.de.
authors.forename: Andrea
authors.initials: A
authors.lastname: Musacchio
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002868:Chromosomal Proteins, Non-Histone / Q000737:chemistry / Q000378:metabolism; D004331:Drosophila melanogaster / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D018386:Kinetochores / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D011994:Recombinant Proteins; D016415:Sequence Alignment
chemical_list: D002868:Chromosomal Proteins, Non-Histone; D011994:Recombinant Proteins; C060905:centromere protein C
keywords: KMN network; Mis12; Ndc80; Spc105; centromere; kinetochore