Curated Optogenetic Publication Database

Abstract: The small GTPase Rac1 controls cell protrusion for a wide variety of critical cell functions. Its regulation by upstream guanine exchange factors (GEFs) has been the focus of multiple studies, but regulation by the GTPase RhoG remains poorly understood. RhoG is known to activate the ELMO/DOCK180 GEF complex, which in turn interacts with Rac1. It is unclear which aspects of protrusion are controlled by RhoG, and which of RhoG’s effects on protrusion are mediated by Rac1. To address these questions, we developed biosensors and optogenetic tools to activate one GTPase while observing another, and to simultaneously visualize the activity of two GTPases. New tools included a photoactivable RhoG, a RhoG biosensor, and red shifted biosensors of RhoG and Rac1. RhoG and Rac1 activation events in protrusions were spatio-temporally correlated with one another and with protrusion velocity. Causal inference indicated that RhoG indeed unidirectionally activated Rac1. Photoactivation of RhoG and Rac1 indicated that specific aspects of protrusion behavior were controlled by RhoG, and only some via Rac1. Further dissection of RhoG to Rac1 signaling through simultaneous GTPase activation and biosensor visualization showed that PA-RhoG activates Rac1 predominantly through DOCK180 and that PA-RhoG can activate Cdc42 independently of Rac1.

Abstract: This chapter provides an overview of the technologies we have developed to control proteins with light. First, we focus on the LOV domain, a versatile building block with reversible photo-response, kinetics tunable through mutagenesis, and ready expression in a broad range of cells and animals. Incorporation of LOV into proteins produced a variety of approaches: simple steric block of the active site released when irradiation lengthened a linker (PA-GTPases), reversible release from sequestration at mitochondria (LOVTRAP), and Z-lock, a method in which a light-cleavable bridge is placed where it occludes the active site. The latter two methods make use of Zdk, small engineered proteins that bind selectively to the dark state of LOV. In order to control endogenous proteins, inhibitory peptides are embedded in the LOV domain where they are exposed only upon irradiation (PKA and MLCK inhibition). Similarly, controlled exposure of a nuclear localization sequence and nuclear export sequence is used to reversibly send proteins into the nucleus. Another avenue of engineering makes use of the heterodimerization of FKBP and FRB proteins, induced by the small molecule rapamycin. We control rapamycin with light or simply add it to target cells. Incorporation of fused FKBP-FRB into kinases, guanine exchange factors, or GTPases leads to rapamycin-induced protein activation. Kinases are engineered so that they can interact with only a specific substrate upon activation. Recombination of split proteins using rapamycin-induced conformational changes minimizes spontaneous reassembly. Finally, we explore the insertion of LOV or rapamycin-responsive domains into proteins such that light-induced conformational changes exert allosteric control of the active site. We hope these design ideas will inspire new applications and broaden our reach towards dynamic biological processes that unfold when studied in vivo.