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Phenotypic profiling of the human genome by time-lapse microscopy reveals cell division genes.

ABSTRACT: 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.

SUBMITTER: Neumann B

PROVIDER: S-EPMC3108885 | biostudies-literature | 2010 Apr

REPOSITORIES: biostudies-literature

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Phenotypic profiling of the human genome by time-lapse microscopy reveals cell division genes.

Neumann Beate B Walter Thomas T Hériché Jean-Karim JK Bulkescher Jutta J Erfle Holger H Conrad Christian C Rogers Phill P Poser Ina I Held Michael M Liebel Urban U Cetin Cihan C Sieckmann Frank F Pau Gregoire G Kabbe Rolf R Wünsche Annelie A Satagopam Venkata V Schmitz Michael H A MH Chapuis Catherine C Gerlich Daniel W DW Schneider Reinhard R Eils Roland R Huber Wolfgang W Peters Jan-Michael JM Hyman Anthony A AA Durbin Richard R Pepperkok Rainer R Ellenberg Jan J

Nature 20100401 7289

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 ...[more]

PMID: 20360735

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Project description:UnlabelledBackgroundCholesterol is an important membrane component, but our knowledge about its transport in cells is sparse. Previous imaging studies using dehydroergosterol (DHE), an intrinsically fluorescent sterol from yeast, have established that vesicular and non-vesicular transport modes contribute to sterol trafficking from the plasma membrane. Significant photobleaching, however, limits the possibilities for in-depth analysis of sterol dynamics using DHE. Co-trafficking studies with DHE and the recently introduced fluorescent cholesterol analog BODIPY-cholesterol (BChol) suggested that the latter probe has utility for prolonged live-cell imaging of sterol transport.ResultsWe found that BChol is very photostable under two-photon (2P)-excitation allowing the acquisition of several hundred frames without significant photobleaching. Therefore, long-term tracking and diffusion measurements are possible. Two-photon temporal image correlation spectroscopy (2P-TICS) provided evidence for spatially heterogeneous diffusion constants of BChol varying over two orders of magnitude from the cell interior towards the plasma membrane, where D?~?1.3 ?m2/s. Number and brightness (N&B) analysis together with stochastic simulations suggest that transient partitioning of BChol into convoluted membranes slows local sterol diffusion. We observed sterol endocytosis as well as fusion and fission of sterol-containing endocytic vesicles. The mobility of endocytic vesicles, as studied by particle tracking, is well described by a model for anomalous subdiffusion on short time scales with an anomalous exponent ??~?0.63 and an anomalous diffusion constant of D??=?1.95 x 10-3 ?m2/s?. On a longer time scale (t?>?~5 s), a transition to superdiffusion consistent with slow directed transport with an average velocity of v?~?6 x 10-3 ?m/s was observed. We present an analytical model that bridges the two regimes and fit this model to vesicle trajectories from control cells and cells with disrupted microtubule or actin filaments. Both treatments reduced the anomalous diffusion constant and the velocity by ~40-50%.ConclusionsThe mobility of sterol-containing vesicles on the short time scale could reflect dynamic rearrangements of the cytoskeleton, while directed transport of sterol vesicles occurs likely along both, microtubules and actin filaments. Spatially varying anomalous diffusion could contribute to fine-tuning and local regulation of intracellular sterol transport.

Dataset Information

Phenotypic profiling of the human genome by time-lapse microscopy reveals cell division genes.

Publications

Phenotypic profiling of the human genome by time-lapse microscopy reveals cell division genes.

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