Curated Optogenetic Publication Database

Abstract: Spatiotemporal localization of protein function is essential for physiological processes from subcellular to tissue scales. Genetic and pharmacological approaches have played instrumental roles in isolating molecular components necessary for subcellular machinery. However, these approaches have limited capabilities to reveal the nature of the spatiotemporal regulation of subcellular machineries like those of cytoskeletal organelles. With the recent advancement of optogenetic probes, the field now has a powerful tool to localize cytoskeletal stimuli in both space and time. Here, we detail the use of tunable light-controlled interacting protein tags (TULIPs) to manipulate RhoA signaling in vivo. This is an optogenetic dimerization system that rapidly, reversibly, and efficiently directs a cytoplasmic RhoGEF to the plasma membrane for activation of RhoA using light. We first compare this probe to other available optogenetic systems and outline the engineering logic for the chosen recruitable RhoGEFs. We also describe how to generate the cell line, spatially control illumination, confirm optogenetic control of RhoA, and mechanically induce cell-cell junction deformation in cultured tissues. Together, these protocols detail how to probe the mechanochemical circuitry downstream of RhoA signaling. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Generation of a stable cell line expressing TULIP constructs Basic Protocol 2: Preparation of collagen substrate for imaging Basic Protocol 3: Transient transfection for visualization of downstream effectors Basic Protocol 4: Calibration of spatial illumination Basic Protocol 5: Optogenetic activation of a region of interest.

Abstract: We describe a detailed procedure for the use of LOVTRAP, an approach to reversibly sequester and release proteins from cellular membranes using light. In the application described here, proteins that act at the plasma membrane are held at mitochondria in the dark, and reversibly released by irradiation. The technique relies on binding of an engineered Zdk domain to a LOV2 domain, with affinity <30 nM in the dark and >500 nM upon irradiation between 400 and 500 nm. LOVTRAP can be applied to diverse proteins, as it requires attaching only one member of the Zdk/LOV2 pair to the target protein, and the other to the membrane where the target protein is to be sequestered. Light-induced protein release occurs in less than a second, and the half-life of return can be adjusted using LOV point mutations (∼2 to 500 sec). © 2016 by John Wiley & Sons, Inc.

Abstract: Genetically encoded actuators that allow control of protein-protein interactions using light, termed 'optical dimerizers', are emerging as new tools for experimental biology. In recent years, numerous new and versatile dimerizer systems have been developed. Here we discuss the design of optical dimerizer experiments, including choice of a dimerizer system, photoexcitation sources, and the coordinate use of imaging reporters. We provide detailed protocols for experiments using two dimerization systems we previously developed, CRY2/CIB and UVR8/UVR8, for use in controlling transcription, protein localization, and protein secretion using light. Additionally, we provide instructions and software for constructing a pulse-controlled LED device for use in experiments requiring extended light treatments.