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.
Searching for Drosophila Dsn1 kinetochore protein Przewloka, Marcin R.; Venkei, Zsolt; Glover, David M.
Cell cycle (Georgetown, Tex.),
4/15/2009, 2009/04/15, 2009-Apr-15, 2009-04-15, 20090415, Letnik:
8, Številka:
8
Journal Article
Recenzirano
Odprti dostop
The Mis12/MIND kinetochore complex is composed of 4 subunits of which the Dsn1 protein is a crucial component in all organisms where it has been identified. In Caenorhabditis elegans, depletion of ...Dsn1 results in a so-called "kinetochore null" phenotype, hence Dsn1's alternative name KNL3. In human, Dsn1 is required to shape an interface between the Mis12 complex and Blinkin, the counterpart of Spc105. In Drosophila however, despite many efforts using sequence comparisons and proteomics-based studies, a Dsn1 ortholog has not been found. Here we speculate that Drosophila Spc105R, a protein very much diverged from its counterparts in other species, might not only be playing the role of Spc105 itself but also of Dsn1.
The identification of all the individual components that constitute the plethora of complexes in each cell type represents perhaps the most exciting challenge of postgenomic biology. This is ...particularly important in the study of events such as mitosis and cytokinesis, in which rapid and precise protein–protein interactions regulate both the direction and accuracy of these intricate processes. Here we describe an experimental strategy to isolate protein complexes involved in mitosis and cytokinesis in cultured Drosophila cells. This method involves the tagging of the bait protein with two IgG binding domains of Protein A and the isolation of the tagged bait along with its interacting partners by a single affinity purification step. These isolated complexes can then be analysed by several methods including mass spectrometry and Western blotting. Although this method has proven very successful in isolating mitotic and cytokinetic complexes, it can also be used to characterise protein complexes involved in many other cellular processes.
Triple-negative breast cancer (TNBC) is the most aggressive type of breast cancer that lacks the oestrogen receptor, progesterone receptor and human epidermal growth factor receptor 2, making it ...difficult to target therapeutically. Targeting synthetic lethality is an alternative approach for cancer treatment. TNBC shows frequent loss of phosphatase and tensin homologue (PTEN) expression, which is associated with poor prognosis and treatment response. To identify PTEN synthetic lethal interactions, TCGA analysis coupled with a whole-genome siRNA screen in isogenic PTEN-negative and -positive cells were performed. Among the candidate genes essential for the survival of PTEN-inactive TNBC cells, WDHD1 (WD repeat and high-mobility group box DNA-binding protein 1) expression was increased in the low vs. high PTEN TNBC samples. It was also the top hit in the siRNA screen and its knockdown significantly inhibited cell viability in PTEN-negative cells, which was further validated in 2D and 3D cultures. Mechanistically, WDHD1 is important to mediate a high demand of protein translation in PTEN-inactive TNBC. Finally, the importance of WDHD1 in TNBC was confirmed in patient samples obtained from the TCGA and tissue microarrays with clinic-pathological information. Taken together, as an essential gene for the survival of PTEN-inactive TNBC cells, WDHD1 could be a potential biomarker or a therapeutic target for TNBC.
The nucleosome remodeling and deacetylase (NuRD) complex is a widely conserved transcriptional co-regulator that harbors both nucleosome remodeling and histone deacetylase activities. It plays a ...critical role in the early stages of ES cell differentiation and the reprogramming of somatic to induced pluripotent stem cells. Abnormalities in several NuRD proteins are associated with cancer and aging. We have investigated the architecture of NuRD by determining the structure of a subcomplex comprising RbAp48 and MTA1. Surprisingly, RbAp48 recognizes MTA1 using the same site that it uses to bind histone H4, showing that assembly into NuRD modulates RbAp46/48 interactions with histones. Taken together with other results, our data show that the MTA proteins act as scaffolds for NuRD complex assembly. We further show that the RbAp48-MTA1 interaction is essential for the in vivo integration of RbAp46/48 into the NuRD complex.
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.
Nucleoplasmin is a histone chaperone that consists of a pentameric N-terminal domain and an unstructured C-terminal tail. The pentameric core domain, a doughnut-like structure with a central pore, is ...only found in the nucleoplasmin family. Here, we report the first structure of a nucleoplasmin-like domain (NPL) from the unrelated Drosophila protein, FKBP39, and we present evidence that this protein associates with chromatin. Furthermore, we show that two other chromatin proteins, Arabidopsis thaliana histone deacetylase type 2 (HD2) and Saccharomyces cerevisiae Fpr4, share the NPL fold and form pentamers, or a dimer of pentamers in the case of HD2. Thus, we propose a new family of proteins that share the pentameric nucleoplasmin-like NPL domain and are found in protists, fungi, plants and animals.
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•The FKBP39 N-terminal domain has the nucleoplasmin fold and forms a homo-pentamer.•FKBP39 binds to chromatin, interacts with histones and binds divalent metal ions.•Other proteins such as the yeast Fpr4 and plant HD-tuins are expected to have the same N-terminal domain with similar properties.
The kinetochore is a dynamic multiprotein complex assembled at the centromere in mitosis. Exactly how the structure of the kinetochore changes during mitosis and how its individual components ...contribute to chromosome segregation is largely unknown. Here we have focused on the contribution of the Mis12 complex to kinetochore assembly and function throughout mitosis in Drosophila. We show that despite the sequential kinetochore recruitment of Mis12 complex subunits Mis12 and Nsl1, the complex acts as a single functional unit. mis12 and nsl1 mutants show strikingly similar developmental and mitotic defects in which chromosomes are able to congress at metaphase, but their anaphase movement is strongly affected. While kinetochore association of Ndc80 absolutely depends on both Mis12 and Nsl1, BubR1 localization shows only partial dependency. In the presence of residual centromeric BubR1 the checkpoint still responds to microtubule depolymerization but is significantly weaker. These observations point to a complexity of the checkpoint response that may reflect subpopulations of BubR1 associated with residual kinetochore components, the core centromere, or elsewhere in the cell. Importantly our results indicate that core structural elements of the inner plate of the kinetochore have a greater contribution to faithful chromosome segregation in anaphase than in earlier stages of mitosis.
Barrier-to-Autointegration Factor (BAF) is a conserved nuclear envelope (NE) component that binds chromatin and helps its anchoring to the NE. Cycles of phosphorylation and dephosphorylation control ...BAF function. Entering mitosis, phosphorylation releases BAF from chromatin and facilitates NE-disassembly. At mitotic exit, PP2A-mediated dephosphorylation restores chromatin binding and nucleates NE-reassembly. Here, we show that in Drosophila a small fraction of BAF (cenBAF) associates with centromeres. We also find that PP4 phosphatase, which is recruited to centromeres by CENP-C, prevents phosphorylation and release of cenBAF during mitosis. cenBAF is necessary for proper centromere assembly and accurate chromosome segregation, being critical for mitosis progression. Disrupting cenBAF localization prevents PP2A inactivation in mitosis compromising global BAF phosphorylation, which in turn leads to its persistent association with chromatin, delays anaphase onset and causes NE defects. These results suggest that, together with PP4 and CENP-C, cenBAF forms a centromere-based mechanism that controls chromosome segregation and mitosis progression.
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.
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.
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.
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