Optogenetics: optical control of a photoactivatable Rac in living cells.
Recent developments in optogenetics have extended optical control of signaling to intracellular proteins, including Rac, a small G protein in the Rho family. A blue light-sensing LOV (light, oxygen, or voltage) domain derived from Avena sativa (oat) phototropin was fused to the N-terminus of a constitutively active mutant of Rac, via an α-helix (Jα) that is conserved among plant phototropins. The fused LOV domain occluded binding of downstream effectors to Rac in the dark. Exposure to blue light caused a conformational change of the LOV domain and unwinding of the Jα helix, relieving steric inhibition. The LOV domain incorporates a flavin as the photon-absorbing cofactor and can be activated by light in a reversible and repeatable fashion. In cultured cells, global illumination with blue light rapidly activated Rac and led to cell spreading and membrane ruffling. Localized and pulsed illumination generated a gradient of Rac activity and induced directional migration. In this chapter, we will describe the techniques in detail and present some examples of applications of using photoactivatable Rac (PA-Rac) in living cells.
Robert D. Berlin Center for Cell Analysis and Modeling, University of Connecticut Health Center, 400 Farmington Avenue, Farmington, CT, 06030, USA.
Taofei
T
Yin
Yi I
YI
Wu
D000818:Animals; D018554:Avena / Q000737:chemistry; D003584:Cytological Techniques / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D008027:Light; D008029:Lighting; D051379:Mice; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods*; D062308:Optogenetics / Q000379:methods*; D056949:Phototropins / Q000032:analysis / Q000235:genetics; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D015398:Signal Transduction / Q000502:physiology*; D020744:rac GTP-Binding Proteins / Q000648:ultrastructure*
D056949:Phototropins; D020744:rac GTP-Binding Proteins