Design, construction, and validation of optogenetic proteins

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.

Keywords: Apoptosis; Biochemical signaling; Motility; Optogenetics; Protein design.

Fig. 1

Schematic for light-induced recruitment of a POI fused to Cry2-mCh to the OMM. Reproduced with permission from O’Banion, C. P., Priestman, M. A., Hughes, R. M., Herring, L. E., Capuzzi, S. J., & Lawrence, D. S. (2018). Design and profiling of a subcellular targeted optogenetic cAMP-dependent protein kinase. Cell Chemical Biology, 25(1), 100–109 e108. doi:10.1016/j.chembiol.2017.09.011.

Fig. 2

Light-Triggered optoPKA association with, and subsequent dissociation from, the OMM, cytoskeleton, and PM. All optoPKA constructs contain the mCh fluorescent protein: Cry2-mCh-C^W196R/E203A (A–D and F), Cry2-mCh (E), control, Cry2-mCh-C^W196R (E), Cry2-mCh-C^W196R/Y204A (F–H), and Cry2-mCh-C^W196R/F327A (F–H). The following Cib constructs were employed to recruit optoPKA to specific intracellular sites: Tom20MLS-Cib-GFP (OMM-Cib in A–F) at the OMM; LifeAct-GFP-Cib (LifeAct-Cib) in (G) at the actin cytoskeleton; Cib-GFP-CAAX (PM-Cib) in (H) at the PM. Visualization of the mCh label in PKA^196R/E203A (A) before and (B) 1min after stimulation with a 100ms, 488nm light pulse. (C) Visualization of the GFP label in OMM-Cib (Tom20MLS-Cib-GFP), where (D) is an overlay of (B and C). (E–H) Association and subsequent dissociation of optoPKA with and from the PM, OMM, and the cytoskeleton were monitored via mCh fluorescence. A single 100ms pulse (FITC cube) was employed to initiate recruitment of the optoPKA constructs to designated sites. Experiments were performed on either a wide-field (OMM-Cib, LifeAct-Cib) or confocal (PM-Cib) microscope. N=3 cells per group. Scale bar, 50 μm. Data expressed as meanSEM. Reproduced with permission from O’Banion, C. P., Priestman, M. A., Hughes, R. M., Herring, L. E., Capuzzi, S. J., & Lawrence, D. S. (2018). Design and profiling of a subcellular targeted optogenetic cAMP-dependent protein kinase. Cell Chemical Biology, 25(1), 100–109 e108. doi:10.1016/j.chembiol.2017.09.011.