RESUMO
The kinetochore is the primary site of interaction between chromosomes and microtubules of the mitotic spindle during chromosome segregation. Kinetochores are composed of more than 100 proteins that transiently assemble during mitosis at a single epigenetically defined region on each chromosome, known as the centromere. Kinetochore assembly and activity must be tightly regulated to ensure proper microtubule interaction and faithful chromosome segregation. Kinetochore malfunction can result in chromosome segregation defects leading to aneuploidy and cell death. As such, cell free and reconstituted systems to analyze kinetochore formation and function are invaluable in probing the biochemical activities of kinetochores. In vitro approaches to studying kinetochores have enabled the manipulation of kinetochore protein structure, function, interactions, and regulation that are not possible in cells. Here we outline a cell-free approach for the assembly of centromeres and recruitment of functional kinetochores that enables their manipulation and analysis.
Assuntos
Cinetocoros/metabolismo , Animais , Pontos de Checagem do Ciclo Celular , Extratos Celulares , Centrômero/genética , Centrômero/metabolismo , Proteína Centromérica A/genética , Proteína Centromérica A/metabolismo , Cromatina/genética , Cromatina/metabolismo , Feminino , Expressão Gênica , Histonas/química , Histonas/metabolismo , Metáfase , Microtúbulos/química , Microtúbulos/metabolismo , Mitose , Nucleossomos/genética , Nucleossomos/metabolismo , Oócitos/metabolismo , Multimerização Proteica , Fuso Acromático/metabolismo , XenopusRESUMO
In this issue of Developmental Cell, Yu et al. (2015) demonstrate that CENP-A phosphorylation by CDK1 inhibits its association with the chaperone protein HJURP and that the removal of this modification at mitotic exit is a key regulatory event that controls the timing of new CENP-A nucleosome formation at centromeres.
Assuntos
Autoantígenos/metabolismo , Ciclo Celular/fisiologia , Centrômero/fisiologia , Proteínas Cromossômicas não Histona/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteína Fosfatase 1/metabolismo , Serina/metabolismo , Proteína Quinase CDC2 , Proteína Centromérica A , HumanosRESUMO
Nucleosomes with histone H3 replaced by CENP-A direct kinetochore assembly. CENP-A nucleosomes from human and Drosophila have been reported to have reduced heights as compared to canonical octameric H3 nucleosomes, thus suggesting a unique tetrameric hemisomal composition. We demonstrate that octameric CENP-A nucleosomes assembled in vitro exhibit reduced heights, indicating that they are physically distinct from H3 nucleosomes and negating the need to invoke the presence of hemisomes.
Assuntos
Autoantígenos/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Nucleossomos/metabolismo , Nucleossomos/ultraestrutura , Proteína Centromérica A , Humanos , Microscopia de Força AtômicaRESUMO
The E4 (also called E1--E4) and E2 proteins of human papillomavirus type 16 are thought to be expressed within the same cells of a lesion, and their open reading frames overlap, suggesting that they may have a functional relationship. We have examined the effect of co-expression of these two proteins and found that each enhances the level of the other. We also identified the N-terminus of E2 as the first example of a viral protein that directly binds the HPV16 E1--E4 protein. This appears to result in the E2 becoming less soluble and promotes its relocation from the nucleus to the cytoplasm. In addition, the turnover of the E2 protein is decreased in the presence of E1--E4. All this raises the possibility that E1--E4 acts to influence E2 activity by varying the amount of available E2 in the cell.
