Abstract
We have described a protocol for performing high-throughput immunofluorescence microscopy on microarrays of yeast cells. This approach employs immunostaining of spheroplasted yeast cells printed as high-density cell microarrays, followed by imaging using automated microscopy. A yeast spheroplast microarray can contain more than 5,000 printed spots, each containing cells from a given yeast strain, and is thus suitable for genome-wide screens focusing on single cell phenotypes, such as systematic localization or co-localization studies or genetic assays for genes affecting probed targets. We demonstrate the use of yeast spheroplast microarrays to probe microtubule and spindle defects across a collection of yeast strains harboring tetracycline-down-regulatable alleles of essential genes.
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References
Lockhart, D. J., Winzeler, E. A. (2000) Genomics, gene expression and DNA arrays. Nature 405, 827–836.
Aebersold, R., Mann, M. (2003) Mass spectrometry-based proteomics. Nature 422, 198–207.
Levsky, J. M., Shenoy, S. M., Pezo, R. C., Singer, R. H. (2002) Single-cell gene expression profiling. Science 297, 836–840.
Narayanaswamy, R., Niu, W., Scouras, A. D., Hart, G. T., Davies, J., Ellington, A. D., Iyer, V. R., Marcotte, E. M. (2006) Systematic profiling of cellular phenotypes with spotted cell microarrays reveals mating-pheromone response genes. Genome Biol 7, R6.
Davierwala, A. P., Haynes, J., Li, Z., Brost, R. L., Robinson, M. D., Yu, L., Mnaimneh, S., Ding, H., Zhu, H., Chen, Y., et al. (2005) The synthetic genetic interaction spectrum of essential genes. Nat Genet 37, 1147–1152.
Winsor, B., Schiebel, E. (1997) Review: an overview of the Saccharomyces cerevisiae microtubule and microfilament cytoskeleton. Yeast 13, 399–434.
Kilmartin, J. V., Adams, A. E. (1984) Structural rearrangements of tubulin and actin during the cell cycle of the yeast saccharomyces. J Cell Biol 98, 922–933.
Knop, M., Pereira, G., Schiebel, E. (1999) Microtubule organization by the budding yeast spindle pole body. Biol Cell 91, 291–304.
Zheng, L., Chen, Y., Lee, W. H. (1999) Hec1p, an evolutionarily conserved coiled-coil protein, modulates chromosome segregation through interaction with SMC proteins. Mol Cell Biol 19, 5417–5428.
Wigge, P. A., Kilmartin, J. V. (2001) The ndc80p complex from Saccharomyces cerevisiae contains conserved centromere components and has a function in chromosome segregation. J Cell Biol 152, 349–360.
Burke, D., Dawson, D., Stearns, T. (2000) Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Acknowledgments
This work was supported by grants from the N.S.F., N.I.H., and Welch (F-1515) and Packard Foundations to E.M.M.
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Niu, W., Hart, G.T., Marcotte, E.M. (2011). High-Throughput Immunofluorescence Microscopy Using Yeast Spheroplast Cell-Based Microarrays. In: Palmer, E. (eds) Cell-Based Microarrays. Methods in Molecular Biology, vol 706. Humana Press. https://doi.org/10.1007/978-1-61737-970-3_7
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DOI: https://doi.org/10.1007/978-1-61737-970-3_7
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Online ISBN: 978-1-61737-970-3
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