RESUMEN
Cyclin-dependent kinases comprise the conserved machinery that drives progress through the cell cycle, but how they do this in mammalian cells is still unclear. To identify the mechanisms by which cyclin-cdks control the cell cycle, we performed a time-resolved analysis of the in vivo interactors of cyclins E1, A2, and B1 by quantitative mass spectrometry. This global analysis of context-dependent protein interactions reveals the temporal dynamics of cyclin function in which networks of cyclin-cdk interactions vary according to the type of cyclin and cell-cycle stage. Our results explain the temporal specificity of the cell-cycle machinery, thereby providing a biochemical mechanism for the genetic requirement for multiple cyclins in vivo and reveal how the actions of specific cyclins are coordinated to control the cell cycle. Furthermore, we identify key substrates (Wee1 and c15orf42/Sld3) that reveal how cyclin A is able to promote both DNA replication and mitosis.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Ciclo Celular , Ciclina A2/metabolismo , Ciclina B1/metabolismo , Quinasas Ciclina-Dependientes/fisiología , Proteínas Nucleares/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Secuencia de Aminoácidos , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/fisiología , Línea Celular , Ciclina A2/química , Ciclina A2/fisiología , Ciclina B1/química , Ciclina B1/fisiología , Ciclina E/química , Ciclina E/metabolismo , Ciclina E/fisiología , Quinasas Ciclina-Dependientes/química , Quinasas Ciclina-Dependientes/metabolismo , Replicación del ADN , Células HeLa , Humanos , Inmunoprecipitación , Espectrometría de Masas , Datos de Secuencia Molecular , Proteínas Oncogénicas/química , Proteínas Oncogénicas/metabolismo , Proteínas Oncogénicas/fisiología , Fosforilación , Proteómica/métodos , Alineación de Secuencia , Especificidad por SustratoRESUMEN
Human pluripotent stem cells (hPSCs) have the potential to generate any human cell type, and one widely recognized goal is to make pancreatic ß cells. To this end, comparisons between differentiated cell types produced in vitro and their in vivo counterparts are essential to validate hPSC-derived cells. Genome-wide transcriptional analysis of sorted insulin-expressing (INS(+)) cells derived from three independent hPSC lines, human fetal pancreata, and adult human islets points to two major conclusions: (i) Different hPSC lines produce highly similar INS(+) cells and (ii) hPSC-derived INS(+) (hPSC-INS(+)) cells more closely resemble human fetal ß cells than adult ß cells. This study provides a direct comparison of transcriptional programs between pure hPSC-INS(+) cells and true ß cells and provides a catalog of genes whose manipulation may convert hPSC-INS(+) cells into functional ß cells.
Asunto(s)
Diferenciación Celular/fisiología , Células Secretoras de Insulina/citología , Páncreas/citología , Células Madre Pluripotentes/citología , Adulto , Diferenciación Celular/genética , Feto/citología , Feto/metabolismo , Citometría de Flujo , Perfilación de la Expresión Génica , Humanos , Células Secretoras de Insulina/metabolismo , Análisis por Micromatrices , Células Madre Pluripotentes/metabolismoRESUMEN
We describe a method to help overcome restrictions on the differentiation propensities of human pluripotent stem cells. Culturing pluripotent stem cells in dimethylsulfoxide (DMSO) activates the retinoblastoma protein, increases the proportion of cells in the early G1 phase of the cell cycle and, in more than 25 embryonic and induced pluripotent stem cell lines, improves directed differentiation into multiple lineages. DMSO treatment also improves differentiation into terminal cell types in several cell lines.