Abstract
The model organism D. discoideum is well suited to investigate basic questions of molecular and cell biology, particularly those related to the structure, regulation, and dynamics of the cytoskeleton, signal transduction, cell-cell adhesion, and development. D. discoideum cells make use of Rho-regulated signaling pathways to reorganize the actin cytoskeleton during chemotaxis, endocytosis, and cytokinesis. In this organism the Rho family encompasses 20 members, several belonging to the Rac subfamily, but there are no representatives of the Cdc42 and Rho subfamilies. Here we present protocols suitable for monitoring the actin polymerization response and the activation of Rac upon stimulation of aggregation-competent cells with the chemoattractant cAMP, and for monitoring the localization and dynamics of Rac activity in live cells.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Kessin R (2001) Dictyostelium—evolution, cell biology, and the development of multicellularity. Cambridge Univ. Press, Cambridge
Müller-Taubenberger A, Kortholt A, Eichinger L (2013) Simple system - substantial share: the use of Dictyostelium in cell biology and molecular medicine. Eur J Cell Biol 92:45–53
Eichinger L, Rivero F (eds) (2006) Dictyostelium discoideum protocols. Humana Press, Springer Verlag, Heidelberg
Eichinger L, Rivero F (eds) (2013) Dictyostelium discoideum protocols, 2nd edn. Humana Press, Springer Verlag, Heidelberg
Vlahou G, Rivero F (2006) Rho GTPase signaling in Dictyostelium discoideum: insights from the genome. Eur J Cell Biol 85:947–959
Rivero F, Xiong H (2016) Rho signaling in Dictyostelium discoideum. Int Rev Cell Mol Biol 322:61–122
Hall A, Schlein A, Condeelis J (1988) Relationship of pseudopod extension to chemotactic hormone-induced actin polymerization in amoeboid cells. J Cell Biochem 37:285–299
Howard T, Oresajo C (1985) The kinetics of chemotactic peptide-induced change in F-actin content, F-actin distribution, and the shape of neutrophils. J Cell Biol 101:1078–1085
Park KC, Rivero F, Meili R, Lee S, Apone F, Firtel RA (2004) Rac regulation of chemotaxis and morphogenesis in Dictyostelium. EMBO J 23:4177–4189
Han JW, Leeper L, Rivero F, Chung CY (2006) Role of RacC for the regulation of WASP and phosphatidylinositol 3-kinase during chemotaxis of Dictyostelium. J Biol Chem 281:35224–35234
Somesh BP, Vlahou G, Iijima M, Insall RH, Devreotes P, Rivero F (2006) RacG regulates morphology, phagocytosis, and chemotaxis. Eukaryot Cell 5:1648–1663
Vlahou G, Schmidt O, Wagner B, Uenlue H, Dersch P, Rivero F, Weissenmayer BA (2009) Yersinia outer protein YopE affects the actin cytoskeleton in Dictyostelium discoideum through targeting of multiple Rho family GTPases. BMC Microbiol 9:138
Bernard V, Bohl B, Bokoch G (1999) Characterization of rac and cdc42 activation in chemoattractant-stimulated human neutrophils using a novel assay for active GTPases. J Biol Chem 274:13198–13204
Chung CY, Feoktistov A, Hollingsworth RJ, Rivero F, Mandel NS (2013) An attenuating role of a WASP-related protein, WASP-B, in the regulation of F-actin polymerization and pseudopod formation via the regulation of RacC during Dictyostelium chemotaxis. Biochem Biophys Res Commun 436:719–724
Veltman DM, King JS, Machesky LM, Insall RH (2012) SCAR knockouts in Dictyostelium: WASP assumes SCAR's position and upstream regulators in pseudopods. J Cell Biol 198:501–508
de la Roche M, Mahasneh A, Lee SF, Rivero F, Côté GP (2005) Cellular distribution and functions of wild-type and constitutively activated Dictyostelium PakB. Mol Biol Cell 16:238–247
Filić V, Marinović M, Faix J, Weber I (2012) A dual role for Rac1 GTPases in the regulation of cell motility. J Cell Sci 125:387–398
Marinović M, Šoštar M, Filić V, Antolović V, Weber I (2016) Quantitative imaging of Rac1 activity in Dictyostelium cells with a fluorescently labelled GTPase-binding domain from DPAKa kinase. Histochem Cell Biol 146:267–279
Filić V, Marinović M, Faix J, Weber I (2014) The IQGAP-related protein DGAP1 mediates signaling to the actin cytoskeleton as an effector and a sequestrator of Rac1 GTPases. Cell Mol Life Sci 71:2775–2785
Dumontier M, Hocht P, Mintert U, Faix J (2000) Rac1 GTPases control filopodia formation, cell motility, endocytosis, cytokinesis and development in Dictyostelium. J. Cell Sci 113:2253–2265
Veltman DM, Akar G, Bosgraaf L, van Haastert PJM (2009) A new set of small, extrachromosomal expression vectors for Dictyostelium discoideum. Plasmid 61:110–118
Faix J, Linkner J, Nordholz B, Platt JL, Liao XH, Kimmel AR (2013) The application of the Cre-loxP system for generating multiple knock-out and knock-in targeted loci. Methods Mol Biol 983:249–267
Dormann D, Libotte T, Weijer CJ, Bretschneider T (2002) Simultaneous quantification of cell motility and protein-membrane-association using active contours. Cell Motil Cytoskeleton 52:221–230
Condeelis J, Hall A (1991) Measurement of actin polymerization and cross-linking in agonist-stimulated cells. Methods Enzymol 196:486–496
Acknowledgments
M.M. and I.W. were supported by the Croatian Science Foundation under the project IP-2014-09-4753. We thank Marko Šoštar for help in preparing Fig. 3 .
Author information
Authors and Affiliations
Corresponding authors
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Marinović, M., Xiong, H., Rivero, F., Weber, I. (2018). Assaying Rho GTPase-Dependent Processes in Dictyostelium discoideum . In: Rivero, F. (eds) Rho GTPases. Methods in Molecular Biology, vol 1821. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8612-5_25
Download citation
DOI: https://doi.org/10.1007/978-1-4939-8612-5_25
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-8611-8
Online ISBN: 978-1-4939-8612-5
eBook Packages: Springer Protocols