Centromeric binding and activity of Protein Phosphatase 4

The cell division cycle requires tight coupling between protein phosphorylation and dephosphorylation. However, understanding the cell cycle roles of multimeric protein phosphatases has been limited by the lack of knowledge of how their diverse regulatory subunits target highly conserved catalytic subunits to their sites of action. Phosphoprotein phosphatase 4 (PP4) has been recently shown to participate in the regulation of cell cycle progression. We now find that the EVH1 domain of the regulatory subunit 3 of Drosophila PP4, Falafel (Flfl), directly interacts with the centromeric protein C (CENP-C). Unlike other EVH1 domains that interact with proline-rich ligands, the crystal structure of the Flfl amino-terminal EVH1 domain bound to a CENP-C peptide reveals a new target-recognition mode for the phosphatase subunit. We also show that binding of Flfl to CENP-C is required to bring PP4 activity to centromeres to maintain CENP-C and attached core kinetochore proteins at chromosomes during mitosis

Centromeric binding and activity of Protein Phosphatase 4

The cell division cycle requires tight coupling between protein phosphorylation and dephosphorylation. However, understanding the cell cycle roles of multimeric protein phosphatases has been limited by the lack of knowledge of how their diverse regulatory subunits target highly conserved catalytic subunits to their sites of action. Phosphoprotein phosphatase 4 (PP4) has been recently shown to participate in the regulation of cell cycle progression. We now find that the EVH1 domain of the regulatory subunit 3 of Drosophila PP4, Falafel (Flfl), directly interacts with the centromeric protein C (CENP-C). Unlike other EVH1 domains that interact with proline-rich ligands, the crystal structure of the Flfl amino-terminal EVH1 domain bound to a CENP-C peptide reveals a new target-recognition mode for the phosphatase subunit. We also show that binding of Flfl to CENP-C is required to bring PP4 activity to centromeres to maintain CENP-C and attached core kinetochore proteins at chromosomes during mitosis

Spatiotemporal dynamics of Spc105 regulates the assembly of the Drosophila kinetochore

The formation of kinetochores shortly before each cell division is a prerequisite for proper chromosome segregation. The synchronous mitoses of Drosophila syncytial embryos have provided an ideal in vivo system to follow kinetochore assembly kinetics and so address the question of how kinetochore formation is regulated. We found that the nuclear exclusion of the Spc105/KNL1 protein during interphase prevents precocious assembly of the Mis12 complex. The nuclear import of Spc105 in early prophase and its immediate association with the Mis12 complex on centromeres are thus the first steps in kinetochore assembly. The cumulative kinetochore levels of Spc105 and Mis12 complex then determine the rate of Ndc80 complex recruitment commencing only after nuclear envelope breakdown. The carboxy-terminal part of Spc105 directs its nuclear import and is sufficient for the assembly of all core kinetochore components and CENP-C, when localized ectopically to centrosomes. Super-resolution microscopy shows that carboxy-terminus of Spc105 lies at the junction of the Mis12 and Ndc80 complexes on stretched kinetochores. Our study thus indicates that physical accessibility of kinetochore components plays a crucial role in the regulation of Drosophila kinetochore assembly and leads us to a model in which Spc105 is a licensing factor for its onset

DAPPER: a data-mining resource for protein-protein interactions.

BACKGROUND: The identification of interaction networks between proteins and complexes holds the promise of offering novel insights into the molecular mechanisms that regulate many biological processes. With increasing volumes of such datasets, especially in model organisms such as Drosophila melanogaster, there exists a pressing need for specialised tools, which can seamlessly collect, integrate and analyse these data. Here we describe a database coupled with a mining tool for protein-protein interactions (DAPPER), developed as a rich resource for studying multi-protein complexes in Drosophila melanogaster. RESULTS: This proteomics database is compiled through mass spectrometric analyses of many protein complexes affinity purified from Drosophila tissues and cultured cells. The web access to DAPPER is provided via an accelerated version of BioMart software enabling data-mining through customised querying and output formats. The protein-protein interaction dataset is annotated with FlyBase identifiers, and further linked to the Ensembl database using BioMart's data-federation model, thereby enabling complex multi-dataset queries. DAPPER is open source, with all its contents and source code are freely available. CONCLUSIONS: DAPPER offers an easy-to-navigate and extensible platform for real-time integration of diverse resources containing new and existing protein-protein interaction datasets of Drosophila melanogaster.This work was supported financially by grants from the Cancer Research UK (CRUK), the Biotechnology and Biological Sciences Research Council and the Medical Research Council to DMG (C3/A11431, BB/I013938/1, G1001696), by a Cancer Research UK Career Development Fellowship to YK (C40697/A12874), and by Cancer Research UK grants to PPD (C12296/A8039 and C12296/A12541). ZL is on leave from the Biological Research Centre of the Hungarian Academy of Sciences (Institute of Biochemistry, Szeged, Hungary) and was supported by a Long-Term Fellowship of the Federation of European Biochemical Societies (FEBS)

Network of protein interactions within the Drosophila inner kinetochore.

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.The work was funded by the Foundation for Polish Science via an International PhD Projects Programme grant to MR and MD; Polish National Science Center via collaborative Harmonia 5 grant to MD and DMG (2013/10/M/NZ2/00298), and by the Medical Research Council and Cancer Research UK via programme grants to DMG. ZL was on leave from the Institute of Biochemistry, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary, and supported by the FEBS Long Term fellowship. We are grateful to Magdalena Kaus-Drobek and Kinga Fituch for help with the structural characterisation of peptides. We thank Andrea Musacchio and his group for sharing their data before publication.This is the final version of the article. It first appeared from Royal Society Publishing via http://dx.doi.org/10.1098/rsob.15023

Molecular analysis of core kinetochore composition and assembly in Drosophila melanogaster.

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. METHODOLOGY/PRINCIPAL FINDINGS: 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/SIGNIFICANCE: 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

Establishment of Centromeric Chromatin by the CENP-A Assembly Factor CAL1 Requires FACT-Mediated Transcription

SummaryCentromeres 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