Abstract
Studies using the social amoeba Dictyostelium discoideum have greatly contributed to the current understanding of the signaling network that underlies chemotaxis. Since directed migration is essential for normal D. discoideum multicellular development, mutants with chemotactic impairments are likely to have abnormal developmental morphologies. We have used multicellular development as a readout in a screen of mutants to identify new potential regulators of chemotaxis. In this chapter, we describe how mutants generated by restriction enzyme-mediated integration (REMI) are analyzed, from assessment of development to detailed characterization of 3′,5′-cyclic adenosine monophosphate (cAMP)-induced responses. Two complementary approaches, plating cells either clonally on a bacterial lawn or as a population on non-nutrient agar, are used to evaluate multicellular development. Once mutants with aberrant developmental phenotypes are identified, their chemotaxis toward cAMP is assessed by both small population and micropipette assays. Furthermore, mutants are tested for defects in both general and specific signaling pathways by examining the recruitment of actin-binding LimEΔcoil or PIP3-binding PH domains to the plasma membrane in response to cAMP stimulation.
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Acknowledgments
The authors would like to thank Huaqing Cai for helpful comments and suggestions. KFS is supported by the American Heart Association. This work was supported by National Institutes of Health Grants GM34933 and GM28007 to PND.
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Artemenko, Y., Swaney, K.F., Devreotes, P.N. (2011). Assessment of Development and Chemotaxis in Dictyostelium discoideum Mutants. In: Wells, C., Parsons, M. (eds) Cell Migration. Methods in Molecular Biology, vol 769. Humana Press. https://doi.org/10.1007/978-1-61779-207-6_20
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DOI: https://doi.org/10.1007/978-1-61779-207-6_20
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