Curated Optogenetic Publication Database

Abstract: Here we introduce Z-lock, an optogenetic approach for reversible, light-controlled steric inhibition of protein active sites. The light oxygen voltage (LOV) domain and Zdk, a small protein that binds LOV selectively in the dark, are appended to the protein of interest where they sterically block the active site. Irradiation causes LOV to change conformation and release Zdk, exposing the active site. Computer-assisted protein design was used to optimize linkers and Zdk-LOV affinity, for both effective binding in the dark, and effective light-induced release of the intramolecular interaction. Z-lock cofilin was shown to have actin severing ability in vitro, and in living cancer cells it produced protrusions and invadopodia. An active fragment of the tubulin acetylase αTAT was similarly modified and shown to acetylate tubulin on irradiation.

Abstract: Optogenetic tools provide a level of spatial and temporal resolution needed to shed new light on dynamic intercellular processes. In this chapter we outline specific protocols for applying these tools to cell motility (optogenetic cofilin), apoptosis [optogenetic Bcl-like protein 4 (Bax)], and protein kinase-mediated signaling pathways [optogenetic cAMP-dependent protein kinase (PKA)]. The activity of these optogenetic species is regulated by the light-mediated dimerization of a cryptochrome/Cib protein pair, which controls the intracellular positioning of the protein of interest. The light induced recruitment of cofilin to the cytoskeleton is utilized for directed migration studies and filopodial dynamics. Light-triggered migration of Bax to the outer mitochondrial membrane induces cellular collapse and eventual apoptosis. Finally, the light-mediated movement of PKA to specific intracellular compartments offers the means to assess the consequences of PKA activity in a site-specific fashion via phosphoproteomic analysis.

Abstract: Cellular optogenetics employs light-regulated, genetically encoded protein actuators to perturb cellular signaling with unprecedented spatial and temporal control. Here, we present a potentially generalized approach for transforming a given protein of interest (POI) into an optogenetic species. We describe the rational and methods by which we developed three different optogenetic POIs utilizing the Cry2-Cib photodimerizing pair. The process pipeline is highlighted by (1) developing a low level, constitutively active POI that is independent of endogenous regulation, (2) fusion of the mutant protein of interest to an optogenetic photodimerizing system, and (3) light-mediated recruitment of the light-responsive POI to specific subcellular regions.

Abstract: An optogenetic Bax has been designed that facilitates light-induced apoptosis. We demonstrate that mitochondrial recruitment of a genetically encoded light-responsive Bax results in the release of mitochondrial proteins, downstream caspase-3 cleavage, changes in cellular morphology, and ultimately cell death. Mutagenesis of a key phosphorylatable residue or modification of the C-terminus mitigates background (dark) levels of apoptosis that result from Bax overexpression. The mechanism of optogenetic Bax-mediated apoptosis was explored using a series of small molecules known to interfere with various steps in programmed cell death. Optogenetic Bax appears to form a mitochondrial apoptosis-induced channel analogous to that of endogenous Bax.

Abstract: Genetically encoded, light-activatable proteins provide the means to probe biochemical pathways at specific subcellular locations with exquisite temporal control. However, engineering these systems in order to provide a dramatic jump in localized activity, while retaining a low dark-state background remains a significant challenge. When placed within the framework of a genetically encodable, light-activatable heterodimerizer system, the actin-remodelling protein cofilin induces dramatic changes in the F-actin network and consequent cell motility upon illumination. We demonstrate that the use of a partially impaired mutant of cofilin is critical for maintaining low background activity in the dark. We also show that light-directed recruitment of the reduced activity cofilin mutants to the cytoskeleton is sufficient to induce F-actin remodeling, formation of filopodia, and directed cell motility.