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High-content screening of yeast mutant librariesHigh-content screening assays have enabled the efficient analysis of cellular events in a large number of experimental samples and with high throughput. Screening approaches based on light microscopic techniques have been applied e.g. to characterize morphological phenotypes of cells caused by mutations or by treatment of cells with chemical inhibitors or drugs. In addition, screening assays using fluorescence microscopy particularly in combination with green-fluorescent-protein (GFP) technology have provided a more global view of the spatial and temporal behavior of subcellular structures in the context of the living cell.
We have recently developed a high-resolution cell-based assay in intact yeast cells to systematically screen yeast mutant strain libraries for abnormal morphology and distribution of fluorescently labeled subcellular structures. The method was validated in a pilot screen assaying abnormal morphology and distribution of fluorescently labeled peroxisomes of yeast. The modular assay enables reliable imaging-based analysis of fluorescently labeled subcellular structures of entire yeast strain collections (1,2).
Flow chart of the established cell-based assay in intact yeast cells
for high-content mutant and drug screens.
-Saito, T. L., Ohtani, M., Sawai, H., Sano, F., Saka, A., Watanabe, D., Yukawa, M., Ohya, Y. and Morishita, S. (2004). SCMD: Saccharomyces cerevisiae Morphological Database. Nucleic Acids Res 32, D319-22.
-Ohtani, M., Saka, A., Sano, F., Ohya, Y. and Morishita, S. (2004). Development of image processing program for yeast cell morphology. J Bioinform Comput Biol 1, 695-709.
-Ohya, Y., Sese, J., Yukawa, M., Sano, F., Nakatani, Y., Saito, T. L., Saka, A., Fukuda, T., Ishihara, S., Oka, S. et al. (2005). High-dimensional and large-scale phenotyping of yeast mutants. Proc Natl Acad Sci U S A 102, 19015-20.
-Saito, T. L., Sese, J., Nakatani, Y., Sano, F., Yukawa, M., Ohya, Y. and Morishita, S. (2005). Data mining tools for the Saccharomyces cerevisiae morphological database. Nucleic Acids Res 33, W753-7.
-Suzuki, G., Sawai, H., Ohtani, M., Nogami, S., Sano-Kumagai, F., Saka, A., Yukawa, M., Saito, T. L., Sese, J., Hirata, D. et al. (2006). Evaluation of image processing programs for accurate measurement of budding and fission yeast morphology. Curr Genet 49, 237-47.
-Ohnuki, S., Nogami, S., Kanai, H., Hirata, D., Nakatani, Y., Morishita, S. and Ohya, Y. (2007). Diversity of Ca2+-induced morphology revealed by morphological phenotyping of Ca2+-sensitive mutants of Saccharomyces cerevisiae. Eukaryot Cell 6, 817-30.
-Wolinski H, Petrovic U, Mattiazzi M, Petschnigg J, Heise B, Natter K, Kohlwein SD (2009). Imaging-based live cell yeast screen identifies novel factors involved in peroxisome assembly. J Proteome Res. 2009 Jan;8(1):20-7.
-Wolinski H, Natter K & Kohlwein SD. 2009. The fidgety yeast: focus on high-resolution live yeast cell microscopy. in Methods Mol Biol 548:75-99.
-Ohnuki, S., Oka, S., Nogami, S. and Ohya, Y. High-content, image-based screening for drug targets in yeast. PLoS One 5, 2010, e10177.
-Tavassoli S, Chao JT, Loewen C. A high-throughput method to globally study the organelle morphology in S. cerevisiae.J Vis Exp. 2009 Mar 2;(25). pii: 1224. doi: 10.3791/1224.
-Vizeacoumar FJ, van Dyk N, S Vizeacoumar F, Cheung V, Li J, Sydorskyy Y, Case N, Li Z, Datti A, Nislow C, Raught B, Zhang Z, Frey B, Bloom K, Boone C, Andrews BJ.Integrating high-throughput genetic interaction mapping and high-content screening to explore yeast spindle morphogenesis. Cell Biol. 2010 Jan 11;188(1):69-81.
-Natter K, Leitner P, Faschinger A, Wolinski H, McCraith S, Fields S, Kohlwein SD, The spatial organization of lipid synthesis in the yeast Saccharomyces cerevisiae derived from large scale green fluorescent protein tagging and high resolution microscopy 2005;4(5):662-72.
-Nicholson RL, Welch M, Ladlow M, Spring DR. Small-molecule screening: advances in microarraying and cell-imaging technologies. ACS chemical biology 2007;2(1):24-30.
-Lederman L. High-content screening. Biotechniques 2007;43(1):25, 7, 9.
-Krausz E. High-content siRNA screening. Molecular bioSystems 2007;3(4):232-40.
-Korn K, Krausz E. Cell-based high-content screening of small-molecule libraries. Curr Opin Chem Biol 2007.
-Wolff M, Wiedenmann J, Nienhaus GU, -Valler M, Heilker R. Novel fluorescent proteins for high-content screening. Drug discovery today 2006;11(23-24):1054-60.
-Rines DR, Tu B, Miraglia L, et al. High-content screening of functional genomic libraries. Methods Enzymol 2006;414:530-65.
-Neumann B, Held M, Liebel U, et al. High-throughput RNAi screening by time-lapse imaging of live human cells. Nature methods 2006;3(5):385-90.
-Lee S, Howell BJ. High-content screening: emerging hardware and software technologies. Methods Enzymol 2006;414:468-83.
-Lang P, Yeow K, Nichols A, Scheer A. Cellular imaging in drug discovery. Nat Rev Drug Discov 2006;5(4):343-56.
-A.H. Tong, M. Evangelista, A.B. Parsons, H. Xu, G.D. Bader, N. Page, M. Robinson, S.Raghibizadeh, C.W. Hogue, H. Bussey, B. Andrews, M. Tyers, C. Boone, Systematic
genetic analysis with ordered arrays of yeast deletion mutants, Science 294 (2001) 2364-
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