A genetically-encoded photoactivatable Rac controls the motility of living cells

By Yi I. Wu, D. Frey, Oana I. Lungu, A. Jaehrig, I. Schlichting, Brian Kuhlman, K. Hahn. 2009

1,078 citations recorded by the source corpus.

The precise spatio-temporal dynamics of protein activity are often critical in determining cell behaviour, yet for most proteins they remain poorly understood; it remains difficult to manipulate protein activity at precise times and places within living cells. Protein activity has been controlled by light, through protein derivatization with photocleavable moieties or using photoreactive small-molecule ligands. However, this requires use of toxic ultraviolet wavelengths, activation is irreversible, and/or cell loading is accomplished via disruption of the cell membrane (for example, through microinjection). Here we have developed a new approach to produce genetically encoded photoactivatable derivatives of Rac1, a key GTPase regulating actin cytoskeletal dynamics in metazoan cells. Rac1 mutants were fused to the photoreactive LOV (light oxygen voltage) domain from phototropin, sterically blocking Rac1 interactions until irradiation unwound a helix linking LOV to Rac1. Photoactivatable Rac1 (PA-Rac1) could be reversibly and repeatedly activated using 458- or 473-nm light to generate precisely localized cell protrusions and ruffling. Localized Rac activation or inactivation was sufficient to produce cell motility and control the direction of cell movement. Myosin was involved in Rac control of directionality but not in Rac-induced protrusion, whereas PAK was required for Rac-induced protrusion. PA-Rac1 was used to elucidate Rac regulation of RhoA in cell motility. Rac and Rho coordinate cytoskeletal behaviours with seconds and submicrometre precision. Their mutual regulation remains controversial, with data indicating that Rac inhibits and/or activates Rho. Rac was shown to inhibit RhoA in mouse embryonic fibroblasts, with inhibition modulated at protrusions and ruffles. A PA-Rac crystal structure and modelling revealed LOV–Rac interactions that will facilitate extension of this photoactivation approach to other proteins.

Subfields: Cellular and Molecular Neuroscience, Molecular Biology, Plant Science

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Karl Deisseroth

Citations in the corpus

Control of protein signaling using a computationally designed GTPase/GEF orthogonal pair (2012, background): “Recent pioneering examples include engineered control of input/output relationships in protein circuits (4, 5), protein-based logic gates (6), and control of protein activity in biological processes by light (7 – 9).”
Cell adhesion: integrating cytoskeletal dynamics and cellular tension (2010, background): “Importantly, local activation of PA-RAC1 inhibited RHOA activation in protrusions of migrating fibroblast”
The Development and Application of Optogenetics (2011, background): “Optical control over small GTPases was next achieved in cultured cells by several different laboratories (Levskaya et al. 2009, Wu et al. 2009, Yazawa et al. 2009) using optically modulated protein-protein interactions; although these have not yet been shown to express or display single-component…”
Life at the leading edge. (2011, background): “Activation of Rac1 by itself, using a photoactivatable Rac1, is sufficient to induce lamellipodium extension (Wu et al., 2009), and it would be interesting to know whether this involves RhoA and Cdc42.”