Molecular Analysis of Core Kinetochore Composition and Assembly in Drosophila melanogaster

Abstract

BackgroundKinetochores 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.Methodology/Principal FindingsWe 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/SignificanceThe 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.

KNL1 and the CENP-H/I/K Complex Coordinately Direct Kinetochore Assembly in Vertebrates

“…However, with the exception of CENP-C, no counterparts to the CCAN have been found to date in Drosophila, which has monocentric chromosomes. The reported dependency of Ndc80 complex localization on the KNL1 ortholog in Drosophila (Przewloka et al, 2007) suggests that, as in C. elegans, the CCAN protein group may have been lost in this lineage, further work is needed to test if this indeed the case.…”

“…A key player in kinetochore assembly in C. elegans is KNL-1, which functions downstream of the inner kinetochore components such as CENP-A, but upstream of all known outer kinetochore proteins (Desai et al, 2003;Cheeseman et al, 2004). KNL-1 belongs to a conserved protein family that includes budding yeast Spc105 (Nekrasov et al, 2003), fission yeast Spc7 (Kerres et al, 2004), Drosophila Spc105R (Przewloka et al, 2007), and a novel protein previously identified in human cells known as AF15q14, D40, CASC5, or Blinkin (Cheeseman et al, 2004;Obuse et al, 2004;Kiyomitsu et al, 2007). AF15q14 is present in all vertebrates including chicken (Gallus gallus).…”

“…Here we will refer to the human and chicken proteins as hKNL1 and ggKNL1, respectively. The C. elegans, human, Drosophila, and fission yeast KNL-1 proteins directly interact with the Ndc80 and Mis12 complexes (Cheeseman et al, 2004;Obuse et al, 2004;Liu et al, 2005;Przewloka et al, 2007). We have termed this interacting protein set that is conserved throughout eukaryotes the KNL-1/Mis12 complex/Ndc80 complex (KMN) network .…”

Drosophila Nnf1 paralogs are partially redundant for somatic and germ line kinetochore function

“…D. melanogaster provides a well-studied example illustrating the evolutionary plasticity of centromere and kinetochore organization. All 16 centromere proteins of the so-called CCAN (constitutive centromere associated network) with the exception of Cenp-C are absent in the fly (Heeger et al 2005;Przewloka et al 2007;Schittenhelm et al 2007;Westermann and Schleiffer 2013). Cenp-C, which binds directly to Cenp-A/Cid, recruits the kinetochore proteins of the KMN network composed of Knl-1/Spc105 and the heterotetrameric Mis12-and Ndc80-complexes.…”

“…Moreover, the Drosophila Mis12 complex lacks the Dsn1 subunit present in yeast and 5 vertebrates (Przewloka and Glover 2009;Przewloka et al 2007;Schittenhelm et al 2007). …”