RESUMO
Eukaryotic chromosome segregation requires the kinetochore, a megadalton-sized machine that forms on specialized centromeric chromatin containing CENP-A, a histone H3 variant. CENP-A deposition requires a chaperone protein HJURP that targets it to the centromere, but it has remained unclear whether HJURP has additional functions beyond CENP-A targeting and why high AT DNA content, which disfavors nucleosome assembly, is widely conserved at centromeres. To overcome the difficulties of studying nucleosome formation in vivo, we developed a microscopy assay that enables direct observation of de novo centromeric nucleosome recruitment and maintenance with single molecule resolution. Using this assay, we discover that CENP-A can arrive at centromeres without its dedicated centromere-specific chaperone HJURP, but stable incorporation depends on HJURP and additional DNA-binding proteins of the inner kinetochore. We also show that homopolymer AT runs in the yeast centromeres are essential for efficient CENP-A deposition. Together, our findings reveal requirements for stable nucleosome formation and provide a foundation for further studies of the assembly and dynamics of native kinetochore complexes.
Assuntos
Proteínas Cromossômicas não Histona , Nucleossomos , Proteína Centromérica A/genética , Proteína Centromérica A/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Autoantígenos/genética , Autoantígenos/metabolismo , Centrômero/genética , Centrômero/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Many of the cell's macromolecular machines contain multiple components that transiently associate with one another. This compositional and dynamic complexity presents a challenge for understanding how these machines are constructed and function. Colocalization single molecule spectroscopy enables simultaneous observation of individual components of these machines in real-time and grants a unique window into processes that are typically obscured in ensemble assays. Colocalization experiments can yield valuable information about assembly pathways, compositional heterogeneity, and kinetics that together contribute to the development of richly detailed reaction mechanisms. This review focuses on recent advances in colocalization single molecule spectroscopy and how this technique has been applied to enhance our understanding of transcription, RNA splicing, and translation.
Assuntos
Corantes Fluorescentes/análise , Microscopia de Fluorescência/métodos , Espectrometria de Fluorescência/métodos , Animais , Desenho de Equipamento , Humanos , Microscopia de Fluorescência/instrumentação , Biossíntese de Proteínas , Splicing de RNA , RNA Mensageiro/análise , RNA Mensageiro/genética , Espectrometria de Fluorescência/instrumentação , Transcrição GênicaRESUMO
Eukaryotic chromosome segregation requires the kinetochore, a megadalton-sized machine that forms on specialized centromeric chromatin containing CENP-A, a histone H3 variant. CENP-A deposition requires a chaperone protein HJURP that targets it to the centromere, but it has remained unclear whether HJURP has additional functions beyond CENP-A targeting and why high AT DNA content, which disfavors nucleosome assembly, is widely conserved at centromeres. To overcome the difficulties of studying nucleosome formation in vivo, we developed a microscopy assay that enables direct observation of de novo centromeric nucleosome recruitment and maintenance with single molecule resolution. Using this assay, we discover that CENP-A can arrive at centromeres without its dedicated centromere-specific chaperone HJURP, but stable incorporation depends on HJURP and additional DNA-binding proteins of the inner kinetochore. We also show that homopolymer AT runs in the yeast centromeres are essential for efficient CENP-A deposition. Together, our findings reveal requirements for stable nucleosome formation and provide a foundation for further studies of the assembly and dynamics of native kinetochore complexes.
RESUMO
By estimating the absolute levels of tension at kinetochores in dividing yeast cells and relating these measurements to kinetochore detachment probability, Mukherjee et al. (2019) quantify in this issue of Developmental Cell the force sensitivity of the mitotic error correction system.