Protein Microarrays pp 277-287 | Cite as
Cell Arrays and High-Content Screening
Abstract
Endocytosis is one of the most essential cellular processes, which enables cells to internalise diverse material. It is crucial for regulation of receptor activity and signalling, cell polarisation, attachment and motility, and a great number of other cellular functions. A number of diverse endocytosis pathways are described by now; however, their specificity for different cellular cargoes is poorly resolved. Only few of endocytosis regulators are well-characterised and even less are attributed to the specific cargo. That is very true for the integrin endocytosis pathway, which is a key process in cell migration, adhesion, and signalling. The recent advent of quantitative fluorescent microscopy and cell arrays opened an exciting possibility to systematically characterise molecules playing a role in this crucially important process. Here, we describe a fluorescent screening microscopy-based assay to identify regulators of integrin α2 internalisation. The experimental procedure is the best suited for a highly parallel screening format, such as cell arrays, albeit can be used in single experiments. We provide protocols for sample preparation, fabrication of cell arrays and quantification of integrin α2 internalisation. The approach can be modified to quantify endocytosis of other cargo, and can be used under the conditions of knock-down and knock-in as well as for chemical screening.
Key words
Reverse transfection Cell arrays Fluorescent screening microscopy RNA interference Endocytosis IntegrinsNotes
Acknowledgments
The authors would like to acknowledge funding within the Forsys-ViroQuant consortium, Project no. 0313923, as well as by the Federal Ministry of Education and Research (BMBF). The ViroQuant-CellNetworks RNAi Screening core facility is supported by CellNetworks – Cluster of Excellence (EXC81). A.E. is supported by a Landesgraduiertenförderung fellowship from University of Heidelberg, and by the Hartmut Hoffmann-Berling International Graduate School of Molecular and Cellular Biology, University of Heidelberg.
References
- 1.Pellinen, T., and Ivaska, J. (2006) Integrin traffic. J. Cell Sci. 119, 3723–3731.PubMedCrossRefGoogle Scholar
- 2.Upla, P., Marjomaki, V., Kankaanpaa, P., Ivaska, J., Hyypia, T., van der Goot, F. G., and Heino, J. (2004) Clustering induces a lateral redistribution of α2β1 integrin from membrane rafts to caveolae and subsequent protein kinase C-dependent internalization. Mol. Biol. Cell 15, 625–636.PubMedCrossRefGoogle Scholar
- 3.Powelka, A. M., Sun, J., Li, J., Gao, M., Shaw, L. M., Sonnenberg, A., and Hsu, V. W. (2004) Stimulation-dependent recycling of integrin β1 regulated by ARF6 and Rab11. Traffic 5, 20–36.PubMedCrossRefGoogle Scholar
- 4.Winograd-Katz, S. E., Itzkovitz, S., Kam, Z. and Geiger, B. (2009) Multiparametric analysis of focal adhesion formation by RNAi-mediated gene knockdown. J. Cell Biol. 186, 423–436.PubMedCrossRefGoogle Scholar
- 5.Snijder, B., Sacher, R., Ramo, P., Damm, E.-M., Liberali, P., and Pelkmans, L. (2009) Population context determines cell-to-cell variability in endocytosis and virus infection. Nature 461, 520–523.PubMedCrossRefGoogle Scholar
- 6.Erfle, H., Neumann, B., Liebel, U., Rogers, P., Held, M., Walter, T., Ellengerb, J., and Pepperkok, R. (2007) Reverse transfection on cell arrays for high content screening microscopy. Nat. Protocols 2, 392–399.CrossRefGoogle Scholar
- 7.Ziauddin, J., and D.M. Sabatini. 2001. Microarrays of cells expressing defined cDNAs. Nature 411, 107–110.PubMedCrossRefGoogle Scholar
- 8.Baghdoyan, S., Y. Roupioz, A. Pitaval, D. Castel, E. Khomyakova, A. Papine, F. Soussaline, and X. Gidrol. 2004. Quantitative analysis of highly parallel transfection in cell microarrays. Nucleic Acids Res. 32, e77.PubMedCrossRefGoogle Scholar
- 9.Bartz, F., Kern, L., Erz, D., Zhu, M., Gilbert, D., Meinhof, T., Wirkner, U., Erfle, H., Muckenthaler, M., Pepperkok, R., and Runz, H. (2009) Idenitfication of cholesterol-regulating genes by targeted RNAi screening. Cell Metab. 10, 1–13.CrossRefGoogle Scholar
- 10.Neumann, B., Held, M., Liebel, U., Erfle, H., Rogers, P., Pepperkok, R., and Ellenberg, J. (2006). High-throughput RNAi screening by time-lapse imaging of live human cells. Nat. Methods 3, 385–390.PubMedCrossRefGoogle Scholar
- 11.Starkuviene, V., Seitz, A., Erfle, H. and Pepperkok, R. (2008) Measuring Secretory Membrane Traffic. Methods in Molecular Biology 457, 1–9.CrossRefGoogle Scholar
- 12.Chao, W.-T., and Kunz, J. (2009) Focal adhesion disassembly requires clathrin-dependent endocytosis of integrins. FEBS Letters 583, 1337–1343.PubMedCrossRefGoogle Scholar