RESUMEN
Quantitative microscopy relies on imaging of large cell numbers but is often hampered by time-consuming manual selection of specific cells. The 'Micropilot' software automatically detects cells of interest and launches complex imaging experiments including three-dimensional multicolor time-lapse or fluorescence recovery after photobleaching in live cells. In three independent experimental setups this allowed us to statistically analyze biological processes in detail and is thus a powerful tool for systems biology.
Asunto(s)
Recuperación de Fluorescencia tras Fotoblanqueo/métodos , Microscopía Fluorescente/métodos , Programas Informáticos , Biología de Sistemas/métodos , Homólogo de la Proteína Chromobox 5 , Proteínas Cromosómicas no Histona/análisis , Proteínas Fluorescentes Verdes/análisis , Células HeLa , HumanosRESUMEN
Despite our rapidly growing knowledge about the human genome, we do not know all of the genes required for some of the most basic functions of life. To start to fill this gap we developed a high-throughput phenotypic screening platform combining potent gene silencing by RNA interference, time-lapse microscopy and computational image processing. We carried out a genome-wide phenotypic profiling of each of the approximately 21,000 human protein-coding genes by two-day live imaging of fluorescently labelled chromosomes. Phenotypes were scored quantitatively by computational image processing, which allowed us to identify hundreds of human genes involved in diverse biological functions including cell division, migration and survival. As part of the Mitocheck consortium, this study provides an in-depth analysis of cell division phenotypes and makes the entire high-content data set available as a resource to the community.
Asunto(s)
División Celular/genética , Genoma Humano/genética , Microscopía Fluorescente/métodos , Fenotipo , Animales , Movimiento Celular/genética , Supervivencia Celular/genética , Color , Técnicas de Silenciamiento del Gen , Genes/genética , Células HeLa , Humanos , Cinética , Ratones , Mitosis/genética , Interferencia de ARN , Reproducibilidad de los Resultados , Huso Acromático/genética , Huso Acromático/metabolismo , Factores de TiempoRESUMEN
Live-cell imaging allows detailed dynamic cellular phenotyping for cell biology and, in combination with small molecule or drug libraries, for high-content screening. Fully automated analysis of live cell movies has been hampered by the lack of computational approaches that allow tracking and recognition of individual cell fates over time in a precise manner. Here, we present a fully automated approach to analyze time-lapse movies of dividing cells. Our method dynamically categorizes cells into seven phases of the cell cycle and five aberrant morphological phenotypes over time. It reliably tracks cells and their progeny and can thus measure the length of mitotic phases and detect cause and effect if mitosis goes awry. We applied our computational scheme to annotate mitotic phenotypes induced by RNAi gene knockdown of CKAP5 (also known as ch-TOG) or by treatment with the drug nocodazole. Our approach can be readily applied to comparable assays aiming at uncovering the dynamic cause of cell division phenotypes.
