RESUMO
The kinetochore proteins assemble onto centromeric chromatin and regulate DNA segregation during cell division. The inner kinetochore proteins bind centromeres while most outer kinetochore proteins assemble at centromeres during mitosis, connecting the complex to microtubules. The centromere-kinetochore complex contains specific nucleosomes and nucleosomal particles. CENP-A replaces canonical H3 in centromeric nucleosomes, defining centromeric chromatin. Next to CENP-A, the CCAN multi-protein complex settles which contains CENP-T/W/S/X. These four proteins are described to form a nucleosomal particle at centromeres. We had found the CENP-T C-terminus and the CENP-S termini next to histone H3.1 but not to CENP-A, suggesting that the Constitutive Centromere-Associated Network (CCAN) bridges a CENP-A- and a H3-containing nucleosome. Here, we show by in vivo FRET that this proximity between CENP-T and H3 is specific for H3.1 but neither for the H3.1 mutants H3.1(C96A) and H3.1(C110A) nor for H3.2 or H3.3. We also found CENP-M next to H3.1 but not to these H3.1 mutants. Consistently, we detected CENP-M next to CENP-S. These data elucidate the local molecular neighborhood of CCAN proteins next to a H3.1-containing centromeric nucleosome. They also indicate an exclusive position of H3.1 clearly distinct from H3.2, thus documenting a local, and potentially also functional, difference between H3.1 and H3.2.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Histonas/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Cromatina/química , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/química , Transferência Ressonante de Energia de Fluorescência , Células HeLa , Histonas/química , Histonas/genética , Humanos , Cinetocoros/química , Cinetocoros/metabolismo , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Alinhamento de Sequência , Proteína Vermelha FluorescenteRESUMO
DNA segregation in mammalian cells during mitosis is an essential cellular process that is mediated by a specific subchromosomal protein complex, the kinetochore. Malfunction of this complex results in aneuploidy and can cause cancer. A subkinetochore complex, the "inner kinetochore", is present at the centromere during the entire cell cycle. Its location seems to be defined by the settlement of CENP-A (CENH3), which replaces histone H3 in centromeric nucleosomes. This suggests that CENP-A can recruit further inner kinetochore proteins by direct binding. Surprisingly, intense in vitro studies could not identify an interaction of CENP-A with any other inner kinetochore protein. Instead, centromere identity seems to be maintained by a unique nucleosome, which might have a modified structure or epigenetic state that serves to distinguish the centromere from the rest of the chromosome. We investigated the association of CENP-A and CENP-B by fluorescence intensity and lifetime-based FRET measurements in living human HEp-2 cells. We observed Förster resonance energy transfer (FRET) between CENP-A and CENP-B at centromere locations; this indicates that these proteins are in the molecular vicinity (<10 nm) of each other. In addition, we analysed protein-protein interactions within the centromeric nucleosome. We could detect energy transfer between CENP-A and histone H4 as well as between CENP-A molecules themselves. On the other hand, no FRET was detected between CENP-A and H2A.1 or H3.1. Our data support the view that two CENP-A molecules are packed with H4, but not with H3, in a single centromeric nucleosome.
Assuntos
Autoantígenos/metabolismo , Proteína B de Centrômero/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Hepatócitos/citologia , Hepatócitos/metabolismo , Cinetocoros/química , Cinetocoros/metabolismo , Sobrevivência Celular , Proteína Centromérica A , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes , Histonas/metabolismo , HumanosRESUMO
At the centromere, a network of proteins, the kinetochore, assembles in order to grant correct chromatin segregation. In this study the dynamics and molecular interactions of the inner kinetochore protein CENP-T were analyzed employing a variety of fluorescence microscopy techniques in living human cells. Acceptor-bleaching FRET indicates that CENP-T directly associates with CENP-A and CENP-B. CENP-T exchange into centromeres is restricted to the S-phase of the cell cycle as revealed by FRAP, suggesting a coreplicational loading mechanism, as we have recently also demonstrated for CENP-I. These properties make CENP-T one of the basic inner kinetochore proteins with most further proteins binding downstream, suggesting a fundamental role of CENP-T in kinetochore function.
Assuntos
Autoantígenos/metabolismo , Proteína B de Centrômero/metabolismo , Centrômero/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Sequência de Bases , Ciclo Celular , Centrômero/ultraestrutura , Proteína Centromérica A , Proteínas Cromossômicas não Histona/genética , Primers do DNA/genética , Recuperação de Fluorescência Após Fotodegradação , Transferência Ressonante de Energia de Fluorescência , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Cinetocoros/metabolismo , Cinetocoros/ultraestrutura , Microscopia de Fluorescência , Fenômenos Ópticos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Espectrometria de FluorescênciaRESUMO
The inner kinetochore protein complex binds to centromeres during the whole cell cycle. It serves as the basis for the binding of further kinetochore proteins during mitosis. CENP-H is one of the inner kinetochore proteins which is conserved amongst many eukaryotes. By specific RNAi knockdown, we reduced the CENP-H protein level in human HEp-2 cells down to less than 5% of its normal value. In these CENP-H knocked-down cells, we observed severe mitotic phenotypes like misaligned chromosomes and multipolar spindles, however, no mitotic arrest. Strong reduction of CENP-H resulted in a slightly reduced CENP-C level at the kinetochores and normal localisation of hBubR1, indicating a functional mitotic checkpoint at the hBubR1 protein level. In CENP-H knocked-down human cells, the misaligned chromosomes contained only reduced levels of CENP-E. Our data clearly indicate that CENP-H has an important impact on the architecture and function of the human kinetochore complex.
Assuntos
Proteínas Cromossômicas não Histona/antagonistas & inibidores , Cinetocoros/fisiologia , Mitose/fisiologia , Interferência de RNA , Linhagem Celular , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Humanos , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases , Fuso Acromático/metabolismoRESUMO
We have employed a novel in vivo approach to study the structure and function of the eukaryotic kinetochore multiprotein complex. RNA interference (RNAi) was used to block the synthesis of centromere protein A (CENP-A) and Clip-170 in human cells. By coexpression, homologous kinetochore proteins from Saccharomyces cerevisiae were then tested for the ability to complement the RNAi-induced phenotypes. Cse4p, the budding yeast CENP-A homolog, was specifically incorporated into kinetochore nucleosomes and was able to complement RNAi-induced cell cycle arrest in CENP-A-depleted human cells. Thus, Cse4p can structurally and functionally substitute for CENP-A, strongly suggesting that the basic features of centromeric chromatin are conserved between yeast and mammals. Bik1p, the budding yeast homolog of human CLIP-170, also specifically localized to kinetochores during mitosis, but Bik1p did not rescue CLIP-170 depletion-induced cell cycle arrest. Generally, the newly developed in vivo complementation assay provides a powerful new tool for studying the function and evolutionary conservation of multiprotein complexes from yeast to humans.
