Centromeres provide a region of chromatin upon which kinetochores are assembled in mitosis
1, 2. Centromeric protein C (CENP-C) is a core component of this centromeric chromatin
3, 4 that, when ...depleted, prevents the proper formation of both centromeres and kinetochores
5–10. CENP-C localizes to centromeres throughout the cell cycle via its C-terminal part
6, 8, whereas its N-terminal part appears necessary for recruitment of some but not all components of the Mis12 complex of the kinetochore
8. We now find that all kinetochore proteins belonging to the KMN (
KNL1/Spc105, the
Mis12 complex, and the
Ndc80 complex) network
1 bind to the N-terminal part of
Drosophila CENP-C. Moreover, we show that the Mis12 complex component Nnf1 interacts directly with CENP-C in vitro. To test whether CENP-C's N-terminal part was sufficient to recruit KMN proteins, we targeted it to the centrosome by fusing it to a domain of Plk4 kinase
11. The Mis12 and Ndc80 complexes and Spc105 protein were then all recruited to centrosomes at the expense of centromeres, leading to mitotic abnormalities typical of cells with defective kinetochores. Thus, the N-terminal part of
Drosophila CENP-C is sufficient to recruit core kinetochore components and acts as the principal linkage between centromere and kinetochore during mitosis.
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► The amino-terminal part of the centromeric protein CENP-C binds kinetochore proteins ► The CENP-C's N-terminus is sufficient to recruit KMN proteins even to ectopic sites ► The expression of this fragment of CENP-C in culture cells leads to mitotic defects ► In mitosis, CENP-C functions as a linker between centromeres and kinetochores
Centromeres are essential chromosomal structures that mediate accurate chromosome segregation during cell division. Centromeres are specified epigenetically by the heritable incorporation of the ...centromeric histone H3 variant CENP-A. While many of the primary factors that mediate centromeric deposition of CENP-A are known, the chromatin and DNA requirements of this process have remained elusive. Here, we uncover a role for transcription in Drosophila CENP-A deposition. Using an inducible ectopic centromere system that uncouples CENP-A deposition from endogenous centromere function and cell-cycle progression, we demonstrate that CENP-A assembly by its loading factor, CAL1, requires RNAPII-mediated transcription of the underlying DNA. This transcription depends on the CAL1 binding partner FACT, but not on CENP-A incorporation. Our work establishes RNAPII passage as a key step in chaperone-mediated CENP-A chromatin establishment and propagation.
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•CENP-A deposition is coupled with transcription•CAL1 recruits RNAPII onto DNA during CENP-A deposition•CAL1 interacts directly with the histone chaperone FACT•FACT depletion causes loss of transcription and defective CENP-A deposition
Centromeres are specified epigenetically by chromatin containing the histone H3 variant CENP-A. Chen et al. shed light on CENP-A deposition mechanisms, showing that the CENP-A chaperone CAL1 recruits FACT and RNAPII to CENP-A assembly sites, where they trigger transcription. In the absence of FACT, transcription ceases and CENP-A deposition is defective.
Accurate chromosome segregation is a prerequisite for the maintenance of the genomic stability. Consequently, elaborate molecular machineries and mechanisms emerged during the course of evolution in ...order to ensure proper division of the genetic material. The kinetochore, an essential multiprotein complex assembled on mitotic or meiotic centromeres, is an example of such machinery. Recently considerable progress has been made in understanding their composition, the recruitment hierarchy of their components, and the principles of their regulation. However, these advances are accompanied by a growing number of unanswered questions about the function of the individual subunits and of how the structure of the different subcomplexes relates to function. Here we review our rapidly growing knowledge on interacting networks of structural and regulatory proteins of the metazoan mitotic kinetochore: its centromeric foundations, its structural core, its components that interact with spindle microtubules and the spindle assembly checkpoint.
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.
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.
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.
Polarised epithelial cell divisions represent a fundamental mechanism for tissue maintenance and morphogenesis. Morphological and mechanical changes in the plasma membrane influence the organisation ...and crosstalk of microtubules and actin at the cell cortex, thereby regulating the mitotic spindle machinery and chromosome segregation. Yet, the precise mechanisms linking plasma membrane remodelling to cell polarity and cortical cytoskeleton dynamics to ensure accurate execution of mitosis in mammalian epithelial cells remain poorly understood. Here, we manipulated the density of mammary epithelial cells in culture, which led to several mitotic defects. Perturbation of cell-cell adhesion formation impairs the dynamics of the plasma membrane, affecting the shape and size of mitotic cells and resulting in defects in mitotic progression and the generation of daughter cells with aberrant architecture. In these conditions, F- actin-astral microtubule crosstalk is impaired, leading to mitotic spindle misassembly and misorientation, which in turn contributes to chromosome mis-segregation. Mechanistically, we identify S100 Ca2+-binding protein A11 (S100A11) as a key membrane-associated regulator that forms a complex with E-cadherin (CDH1) and the leucine-glycine-asparagine repeat protein LGN (also known as GPSM2) to coordinate plasma membrane remodelling with E-cadherin-mediated cell adhesion and LGN-dependent mitotic spindle machinery. Thus, plasma membrane-mediated maintenance of mammalian epithelial cell identity is crucial for correct execution of polarised cell divisions, genome maintenance and safeguarding tissue integrity.
Polo-like kinases control multiple events during cell division, including mitotic entry, centrosome organization, spindle formation, chromosome segregation and cytokinesis. Their roles during ...cytokinesis, however, are not well understood because the requirement of these kinases during early stages of mitosis complicates the study of their functions after anaphase onset.
We used time-lapse microscopy to analyze the dynamics of Polo::GFP in Drosophila tissue culture cells during mitosis. After anaphase onset, Polo::GFP concentrated at the spindle midzone, but also diffused along the entire length of the central spindle. Using RNA interference we demonstrate that the microtubule-associated proteins Feo and Klp3A are required for Polo recruitment to the spindle midzone, but not the kinesin Pavarotti as previously thought. Moreover, we show that Feo and Klp3A form a complex and that Polo co-localizes with both proteins during cytokinesis.
Our results reveal that the Feo/Klp3A complex is necessary for Polo recruitment to the spindle midzone. A similar finding has also been recently reported in mammalian cells 1, suggesting that this basic mechanism has been conserved during evolution, albeit with some differences. Finally, since cleavage furrow formation and ingression are unaffected following feo RNAi, our data imply that Polo recruitment to the central spindle is not required for furrowing, but some other aspect of cytokinesis.
The kinetochore provides a physical connection between microtubules and the centromeric regions of chromosomes that is critical for their equitable segregation. The trimeric Mis12 sub-complex of the ...Drosophila kinetochore binds to the mitotic centromere using CENP-C as a platform. However, knowledge of the precise connections between Mis12 complex components and CENP-C has remained elusive despite the fundamental importance of this part of the cell division machinery. Here, we employ hydrogen–deuterium exchange coupled with mass spectrometry to reveal that Mis12 and Nnf1 form a dimer maintained by interacting coiled-coil (CC) domains within the carboxy-terminal parts of both proteins. Adjacent to these interacting CCs is a carboxy-terminal domain that also interacts with Nsl1. The amino-terminal parts of Mis12 and Nnf1 form a CENP-C-binding surface, which docks the complex and thus the entire kinetochore to mitotic centromeres. Mutational analysis confirms these precise interactions are critical for both structure and function of the complex. Thus, we conclude the organization of the Mis12–Nnf1 dimer confers upon the Mis12 complex a bipolar, elongated structure that is critical for kinetochore function.
Cytokinesis, the final step of cell division, usually ends with the abscission of the two daughter cells. In some tissues, however, daughter cells never completely separate and remain interconnected ...by intercellular bridges or ring canals. In this paper, we report the identification and analysis of a novel ring canal component, Nessun Dorma (Nesd), isolated as an evolutionarily conserved partner of the centralspindlin complex, a key regulator of cytokinesis. Nesd contains a pectin lyase-like domain found in proteins that bind to polysaccharides, and we present evidence that it has high affinity for β-galactosides in vitro. Moreover, nesd is an essential gene in Drosophila melanogaster, in which it is required for completion of cytokinesis during male meiosis and possibly in female germline cells. Our findings indicate that Nesd is a novel carbohydrate-binding protein that functions together with centralspindlin in late cytokinesis, thus highlighting the importance of glycosylation in this process.
KNL1 is a large intrinsically disordered kinetochore (KT) protein that recruits spindle assembly checkpoint (SAC) components to mediate SAC signaling. The N-terminal region (NTR) of KNL1 possesses ...two activities that have been implicated in SAC silencing: microtubule (MT) binding and protein phosphatase 1 (PP1) recruitment. The NTR of
KNL1 (Spc105) has never been shown to bind MTs or to recruit PP1. Furthermore, the phosphoregulatory mechanisms known to control SAC protein binding to KNL1 orthologues is absent in
. Here, these apparent discrepancies are resolved using in vitro and cell-based assays. A phosphoregulatory circuit that utilizes Aurora B kinase promotes SAC protein binding to the central disordered region of Spc105 while the NTR binds directly to MTs in vitro and recruits PP1-87B to KTs in vivo. Live-cell assays employing an optogenetic oligomerization tag and deletion/chimera mutants are used to define the interplay of MT and PP1 binding by Spc105 and the relative contributions of both activities to the kinetics of SAC satisfaction.
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