ABSTRACT
The CENP-A nucleosome is a key structure for kinetochore assembly. Once the CENP-A nucleosome is established in the centromere, additional proteins recognize the CENP-A nucleosome to form a kinetochore. CENP-C and CENP-N are CENP-A binding proteins. We previously demonstrated that vertebrate CENP-C binding to the CENP-A nucleosome is regulated by CDK1-mediated CENP-C phosphorylation. However, it is still unknown how the phosphorylation of CENP-C regulates its binding to CENP-A. It is also not completely understood how and whether CENP-C and CENP-N act together on the CENP-A nucleosome. Here, using cryo-electron microscopy (cryo-EM) in combination with biochemical approaches, we reveal a stable CENP-A nucleosome-binding mode of CENP-C through unique regions. The chicken CENP-C structure bound to the CENP-A nucleosome is stabilized by an intramolecular link through the phosphorylated CENP-C residue. The stable CENP-A-CENP-C complex excludes CENP-N from the CENP-A nucleosome. These findings provide mechanistic insights into the dynamic kinetochore assembly regulated by CDK1-mediated CENP-C phosphorylation.
Subject(s)
Centromere Protein A/metabolism , Centromere/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Cryoelectron Microscopy/methods , Kinetochores/metabolism , Nucleosomes/metabolism , Animals , Centromere/ultrastructure , Centromere Protein A/ultrastructure , Chickens , Chromosomal Proteins, Non-Histone/ultrastructure , Kinetochores/ultrastructure , Models, Molecular , Nucleosomes/ultrastructure , Phosphorylation , Protein ConformationABSTRACT
Here we describe methods for (a) collecting starfish during their breeding period; (b) maintaining adults with fully grown gonads in laboratory aquaria; (c) rearing fertilized eggs to brachiolaria larvae, and (d) inducing larvae to metamorphose into juveniles under laboratory conditions. Such protocols should facilitate various analyses of starfish development throughout the entire life cycle of these model organisms.
Subject(s)
Asterina/growth & development , Animals , Aquaculture/instrumentation , Aquaculture/methods , Asterina/embryology , Equipment Design , Female , Larva/growth & development , Male , Metamorphosis, Biological , Oocytes/cytology , OogenesisABSTRACT
The kinetochore is essential for faithful chromosome segregation during mitosis. To form a functional kinetochore, constitutive centromere-associated network (CCAN) proteins are assembled on the centromere chromatin that contains the centromere-specific histone CENP-A. CENP-C, a CCAN protein, directly interacts with the CENP-A nucleosome to nucleate the kinetochore structure. As CENP-C is a hub protein for kinetochore assembly, it is critical to address how the CENP-A-CENP-C interaction is regulated during cell cycle progression. To address this question, we investigated the CENP-C C-terminal region, including a conserved CENP-A-binding motif, in both chicken and human cells and found that CDK1-mediated phosphorylation of CENP-C facilitates its binding to CENP-A in vitro and in vivo. We observed that CENP-A binding is involved in CENP-C kinetochore localization during mitosis. We also demonstrate that the CENP-A-CENP-C interaction is critical for long-term viability in human RPE-1 cells. These results provide deeper insights into protein-interaction network plasticity in centromere proteins during cell cycle progression.
Subject(s)
CDC2 Protein Kinase/metabolism , Centromere Protein A/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Kinetochores/metabolism , Animals , Centromere/metabolism , Chickens , Cyclin B/metabolism , Cytoskeletal Proteins/metabolism , Female , Humans , Mitosis , Mutation , Oocytes/metabolism , Phosphorylation , Protein Binding , Recombinant Proteins/metabolism , StarfishABSTRACT
In the version of this Article originally published, the 'ON' and 'OFF' labels in panel c of Fig. 6 were incorrect. For the Tet treated cells (+Tet) in both image panels, CENP-T should have been 'OFF' and CENP-T Δ90 should have been 'ON'. For the cells untreated with Tet (-Tet) in both graph panels, CENP-T Δ90 should have been 'ON'. This has now been amended.
ABSTRACT
To establish a functional kinetochore, the constitutive centromere-associated network (CCAN) forms a foundation on the centromere and recruits the KMN network, which directly binds to spindle microtubules. The CENP-C and CENP-T pathways in the CCAN recruit the KMN network to kinetochores, independently. The CENP-C pathway has been considered the major scaffold for the KMN network in vertebrate CCAN. However, we demonstrate that it is mainly the CENP-T pathway that recruits the KMN network onto the kinetochores and that CENP-T-KMN interactions are essential in chicken DT40 cells. By contrast, less Ndc80 binds to the CENP-C pathway in mitosis and the Mis12-CENP-C association is decreased during mitotic progression, which is consistent with the finding that the Mis12 complex-CENP-C binding is dispensable for cell viability. Furthermore, we find that multiple phosphoregulations of CENP-T and the Mis12 complex make the CENP-T pathway dominant. These results provide key insights into kinetochore dynamics during mitotic progression.
Subject(s)
Centromere/metabolism , Kinetochores/metabolism , Microtubules/metabolism , Signal Transduction , Spindle Apparatus/metabolism , Animals , Cell Line, Tumor , Chickens , Chromosomal Proteins, Non-Histone/metabolism , Microtubule-Associated Proteins/metabolism , Mitosis , Multiprotein Complexes/metabolism , Phosphorylation , Protein BindingABSTRACT
The Cdc2-cyclin B kinase has a central role in regulating the onset of M phase. In starfish oocytes, Cdc2-cyclin B begins to be activated approximately 10 min after application of maturation hormone, followed by accumulation in the nucleus then nuclear envelope breakdown. By immunofluorescence and by expressing a green fluorescent (GFP) chimera of cyclin B, we find that cyclin B is present in aggregates in the cytoplasm of immature oocytes. The aggregates disperse at approximately 10 min, suggesting that the dispersal is closely related to the activation of the kinase. Using cyclin B-GFP, the dispersion begins from the region containing the centrosomes. Extractability of Cdc2-cyclin B changes with similar kinetics during maturation. Active Cdc25 phosphatase released Cdc2-cyclin B from the detergent-insoluble fraction independently of its phosphatase activity. Live cell imaging also showed that Cdc2-cyclin B begins to accumulate in the nucleus before changes in nuclear pore permeability, consistent with Cdc2-cyclin B-induced disassembly of the pores.
