Optogenetic modulation of guanine nucleotide exchange factors of Ras superfamily proteins directly controls cell shape and movement

In this article, we provide detailed protocols on using optogenetic dimerizers to acutely perturb activities of guanine nucleotide exchange factors (GEFs) specific Ras, Rac or Rho small GTPases the migratory networks in various mammalian and amoeba cell lines. These GEFs are crucial components signal transduction which link upstream G-protein coupled receptors downstream cytoskeletal help cells migrate through their dynamic microenvironment. Conventional approaches examine these signaling networks, such as gene knockout overexpression, protracted allows readjust expression changes. Moreover, tools lack spatial resolution probe effects local network activations. To overcome challenges, blue light-inducible cryptochrome- LOV domain-based dimerization systems have been recently developed control events a spatiotemporally precise manner. We illustrate that, within minutes global membrane recruitment full-length catalytic domains only, widespread increases decreases F-actin rich protrusions size occur, depending particular node targeted. Additionally, demonstrate localized GEF robust assay system study activation-driven changes polarity directed migration. Altogether, optical confirmed Ras superfamily regulators shape, actin dynamics, polarity. Furthermore, toolbox may be exploited perturbing complex interactions varied physiological contexts including embryogenesis.