Advanced Light Microscopy FacilityPublications
Genome-wide RNAi screening identifies human proteins with a regulatory function in the early secretory pathway.
Simpson, J.C., Joggerst, B., Laketa, V., Verissimo, F., Cetin, C., Erfle, H., Bexiga, M.G., Singan, V.R., Heriche, J.K., Neumann, B., Mateos, A., Blake, J., Bechtel, S., Benes, V., Wiemann, S., Ellenberg, J. & Pepperkok, R.
Nat Cell Biol. 2012 Jun 3;14(7):764-74. doi: 10.1038/ncb2510.
The secretory pathway in mammalian cells has evolved to facilitate the transfer of cargo molecules to internal and cell surface membranes. Use of automated microscopy-based genome-wide RNA interference screens in cultured human cells allowed us to identify 554 proteins influencing secretion. Cloning, fluorescent-tagging and subcellular localization analysis of 179 of these proteins revealed that more than two-thirds localize to either the cytoplasm or membranes of the secretory and endocytic pathways. The depletion of 143 of them resulted in perturbations in the organization of the COPII and/or COPI vesicular coat complexes of the early secretory pathway, or the morphology of the Golgi complex. Network analyses revealed a so far unappreciated link between early secretory pathway function, small GTP-binding protein regulation, actin cytoskeleton organization and EGF-receptor-mediated signalling. This work provides an important resource for an integrative understanding of global cellular organization and regulation of the secretory pathway in mammalian cells.
At the cutting edge: applications and perspectives of laser nanosurgery in cell biology.
Ronchi, P., Terjung, S. & Pepperkok, R.
Biol Chem. 2012 Apr;393(4):235-48. doi: 10.1515/hsz-2011-0237.
Laser-mediated nanosurgery has become popular in the last decade because of the previously unexplored possibility of ablating biological material inside living cells with sub-micrometer precision. A number of publications have shown the potential applications of this technique, ranging from the dissection of sub-cellular structures to surgical ablations of whole cells or tissues in model systems such as Drosophila melanogaster or Danio rerio . In parallel, the recent development of micropatterning techniques has given cell biologists the possibility to shape cells and reproducibly organize the intracellular space. The integration of these two techniques has only recently started yet their combination has proven to be very interesting. The aim of this review is to present recent applications of laser nanosurgery in cell biology and to discuss the possible developments of this approach, particularly in combination with micropattern-mediated endomembrane organization.
Micropilot: automation of fluorescence microscopy-based imaging for systems biology.
Conrad, C., Wunsche, A., Tan, T.H., Bulkescher, J., Sieckmann, F., Verissimo, F., Edelstein, A., Walter, T., Liebel, U., Pepperkok, R. & Ellenberg, J.
Nat Methods. 2011 Mar;8(3):246-9. doi: 10.1038/nmeth.1558. Epub 2011 Jan 23.
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.
Phenotypic profiling of the human genome by time-lapse microscopy reveals cell division genes.
Neumann, B., Walter, T., Heriche, J.K., Bulkescher, J., Erfle, H., Conrad, C., Rogers, P., Poser, I., Held, M., Liebel, U., Cetin, C., Sieckmann, F., Pau, G., Kabbe, R., Wünsche, A., Satagopam, V., Schmitz, M.H., Chapuis, C., Gerlich, D.W., Schneider, R., Eils, R., Huber, W., Peters, J.M., Hyman, A.A., Durbin, R., Pepperkok, R. & Ellenberg, J.
Nature. 2010 Apr 1;464(7289):721-7.
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.
Work flow for multiplexing siRNA assays by solid-phase reverse transfection in multiwell plates.
Erfle, H., Neumann, B., Rogers, P., Bulkescher, J., Ellenberg, J. & Pepperkok, R.
J Biomol Screen. 2008 Aug;13(7):575-80. Epub 2008 Jul 3.
Solid-phase reverse transfection on cell microarrays is a high-throughput method for the parallel transfection of mammalian cells. However, the cells transfected in this way have been restricted so far to microscopy-based analyses. Analysis methods such as reverse transcriptase-polymerase chain reaction (RT-PCR) and access to higher cell numbers for statistical reasons in microscopy-based assays are not possible with solid-phase reverse transfection on cell microarrays. We have developed a quick and reliable protocol for automated solid-phase reverse transfection of human cells with siRNAs in multiwell plates complementing solid-phase reverse transfection on cell microarrays. The method retains all advantages of solid-phase reverse transfection such as long-term storage capacity after fabrication, reduced cytotoxicity, and reduced cost per screen compared with liquid-phase transfection in multiwell plates. The protocol has been tested for the RNAi-mediated knockdown of several genes in different cell lines including U20S, RPE1, A549, and HeLa cells. We show that even 3 months after production of the "ready to transfect" multiwell plates, there is no reduction in their transfection efficiency as assessed by RT-PCR and nuclear phenotyping by fluorescence microscopy. We conclude that solid-phase reverse transfection in multiwell plates is a cost-efficient and flexible tool for multiplexing cellular assays.