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Vazquez-Victorio, Genaro; Gonzalez-Espinosa, Claudia; Espinosa-Riquer, Zyanya P; Macias-Silva, Marina (2016)


Methods Cell Biol. 132():165-188
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A multitude of physiological processes regulated by G protein-coupled receptors (GPCRs) signaling are accomplished by the participation of active rearrangements of the cytoskeleton. In general, it is common that a cross talk occurs among networks of microfilaments, microtubules, and intermediate filaments in order to reach specific cell responses. In particular, actin-cytoskeleton dynamics regulate processes such as cell shape, cell division, cell motility, and cell polarization, among others. This chapter describes the current knowledge about the regulation of actin-cytoskeleton dynamic by diverse GPCR signaling pathways, and also includes some protocols combining immunofluorescence and confocal microscopy for the visualization of the different rearrangements of the actin-cytoskeleton. We report how both the S1P-GPCR/G12/13/Rho/ROCK and glucagon-GPCR/Gs/cAMP axes induce differential actin-cytoskeleton rearrangements in epithelial cells. We also show that specific actin-binding molecules, like phalloidin and LifeAct, are very useful to analyze F-actin reorganization by confocal microscopy, and also that both molecules show similar results in fixed cells, whereas the anti-actin antibody is useful to detect both the G- and F-actin, as well as their compartmentalization. Thus, it is highly recommended to utilize different approaches to investigate the regulation of actin dynamics by GPCR signaling, with the aim to get a better picture of the phenomenon under study.