Abstract
Rho GTPases, a family of molecular switches, are essential for the assembly and rearrangement of the cellular actin network. Actin remodeling is a central component of many important biological phenomena including chemotaxis, immunological synapse formation, and phagocytosis. Proper execution of these processes requires careful modulation of Rho GTPase activity in space and time. This is accomplished by delicate coordination of Rho GTPase activation and inactivation by Rho guanine nucleotide exchange factors (RhoGEFs) and Rho GTPase-activating proteins (RhoGAPs), respectively. Elucidating the function of these Rho GTPase modulators is complicated by their diversity, varied expression across different tissues, and multiplicity of substrates. To overcome some of these hurdles, we describe here a systematic and unbiased screening approach consisting of three sequential steps: (1) monitoring the subcellular localization of a library of Rho GTPase modulators; (2) assessing endogenous levels of expression of the suitably localized candidates in the cell type of interest; and (3) validating the functional relevance of the identified candidates by siRNA, followed by determining the effects of gene silencing on Rho GTPase activity and actin polymerization. To this end, we describe the expression and visualization of fluorescent Rho GTPase modulators, and the use of genetically encoded biosensors for active Rac/Cdc42 and of fluorescent probes of polymerized actin. Phagocytosis by macrophages is used in this chapter as an experimental paradigm, but the methods described herein can be easily extended to other cells and actin-dependent processes.
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
Schaefer AW, Schoonderwoert VTG, Ji L, Mederios N, Danuser G, Forscher P (2008) Coordination of actin filament and microtubule dynamics during neurite outgrowth. Dev Cell 15:146–162
Pertz OC, Wang Y, Yang F, Wang W, Gay LJ, Gristenko MA, Clauss TR, Anderson DJ, Liu T, Auberry KJ, Camp DG 2nd, Smith RD, Klemke RL (2008) Spatial mapping of the neurite and soma proteomes reveals a functional Cdc42/Rac regulatory network. Proc Natl Acad Sci U S A 105:1931–1936
Flynn KC, Hellal F, Neukirchen D, Jacob S, Tahirovic S, Dupraz S, Stern S, Garvalov BK, Gurniak C, Shaw AE, Meyn L, Wedlich-Söldner R, Bamburg JR, Small JV, Witke W, Bradke F (2012) ADF/Cofilin-mediated actin retrograde flow directs neurite formation in the developing brain. Neuron 76:1091–1107
Pestonjamasp KN, Forster C, Sun C, Gardiner EM, Bohl B, Weiner O, Bokoch GM, Glogauer M (2006) Rac1 links leading edge and uropod events through rho and myosin activation during chemotaxis. Blood 108:2814–2820
Glogauer M, Marchal CC, Zhu F, Worku A, Clausen BE, Foerster I, Marks P, Downey GP, Dinauer M, Kwiatkowski DJ (2003) Rac1 deletion in mouse neutrophils has selective effects on neutrophil functions. J Immunol 170:5652–5657
Treanor B, Depoil D, Bruckbauer A, Batista FD (2011) Dynamic cortical actin remodeling by ERM proteins controls BCR microcluster organization and integrity. J Exp Med 208:1055–1068
Scott CC, Dobson W, Botelho RJ, Coady-Osberg N, Chavrier P, Knecht DA, Heath C, Stahl P, Grinstein S (2005) Phosphatidylinositol-4,5-bisphosphate hydrolysis directs actin remodeling during phagocytosis. J Cell Biol 169:139–149
Freeman SA, Grinstein S (2014) Phagocytosis: receptors, signal integration, and the cytoskeleton. Immunol Rev 262:193–215
Laux T, Fukami K, Thelen M, Golub T, Frey D, Caroni P (2000) GAP43, MARCKS, and CAP23 modulate PI(4,5)P(2) at plasmalemmal rafts, and regulate cell cortex actin dynamics through a common mechanism. J Cell Biol 149:1455–1472
Sechi AS, Wehland J (2000) The actin cytoskeleton and plasma membrane connection: PtdIns(4,5)P(2) influences cytoskeletal protein activity at the plasma membrane. J Cell Sci 113:3685–3695
Etienne-Manneville S, Hall A (2002) Rho GTPases in cell biology. Nature 420:629–635
Moon SY, Zheng Y (2003) Rho GTPase-activating proteins in cell regulation. Trends Cell Biol 13:13–22
Cook DR, Rossman KL, Der CJ (2014) Rho guanine nucleotide exchange factors: regulators of rho GTPase activity in development and disease. Oncogene 33:4021–4035
Tcherkezian J, Lamarche-Vane N (2007) Current knowledge of the large RhoGAP family of proteins. Biol Cell 99:67–86
Schlam D, Bagshaw RD, Freeman SA, Collins RF, Pawson T, Fairn GD, Grinstein S (2015) Phosphoinositide 3-kinase enables phagocytosis of large particles by terminating actin assembly through Rac/Cdc42 GTPase-activating proteins. Nat Commun 6:8623
Srinivasan S, Wang F, Glavas S, Hofmann F, Aktories K, Kalman D, Bourne HR (2003) Rac and Cdc42 play distinct roles in regulating PI(3,4,5)P3 and polarity during neutrophil chemotaxis. J Cell Biol 160:375–385
Hoppe AD, Swanson JA (2004) Cdc42, Rac1, and Rac2 display distinct patterns of activation during phagocytosis. Mol Biol Cell 15:3509–3519
Riedl J, Crevenna AH, Kessenbrock K, Yu JH, Neukirchen D, Bista M, Bradke F, Jenne D, Holak TA, Werb Z, Sixt M, Wedlich-Soldner R (2008) Lifeact: a versatile marker to visualize F-actin. Nat Methods 5:605–607
Yi J, Wu XS, Crites T, Hammer JA (2012) Actin retrograde flow and actomyosin II arc contraction drive receptor cluster dynamics at the immunological synapse in Jurkat T cells. Mol Biol Cell 23:834–852
Wagner W, Brenowitz SD, Hammer JA (2011) Myosin-Va transports the endoplasmic reticulum into the dendritic spines of Purkinje neurons. Nat Cell Biol 13:40–48
Author information
Authors and Affiliations
Corresponding author
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
Schlam, D., Grinstein, S., Freeman, S.A. (2018). Screening for Rho GTPase Modulators in Actin-Dependent Processes Exemplified by Phagocytosis. 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_8
Download citation
DOI: https://doi.org/10.1007/978-1-4939-8612-5_8
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