Optogenetic regulation of endogenous proteins

Abstract

Techniques of protein regulation, such as conditional gene expression, RNA interference, knock-in and knockout , lack sufficient spatiotemporal accuracy, while optogenetic tools suffer from non-physiological response due to overexpression artifacts. Here we present a near-infrared light-activatable optogenetic system, which combines the specificity and orthogonality of intrabodies with the spatiotemporal precision of optogenetics. We engineer optically-controlled intrabodies to regulate genomically expressed protein targets and validate the possibility to further multiplex protein regulation via dual-wavelength optogenetic control. We apply this system to regulate cytoskeletal and enzymatic functions of two nontagged endogenous proteins, actin and RAS GTPase, involved in complex functional networks sensitive to perturbations. The optogenetically-enhanced intrabodies allow fast and reversible regulation of both proteins, as well as simultaneous monitoring of RAS signaling with visiblelight biosensors, enabling all-optical approach. Growing number of intrabodies should make their incorporation into optogenetic tools the versatile technology to regulate endogenous targets.

Engineering Photosensory Modules of Non-Opsin-Based Optogenetic Actuators

“…Using this optimized variant, light-dependent interactions were demonstrated via protein translocation assay in cells [ 25 , 56 ] and in vivo light-induced gene expression in mice upon illumination of far-red light [ 25 ]. Recently, the utility and versatility of the BphP1-Q-PAS1 pair has been further demonstrated in combination with intrabodies [ 154 ]. The resulting light-controllable intrabodies enable multiplex protein regulation of endogenous protein (actin and RAS GTPase) with spatiotemporal precision.…”

Lights up on organelles: Optogenetic tools to control subcellular structure and organization

“…Therefore, optogenetic membrane recruitment of SOS cat leads to light‐sensitive control of local Ras activity. Another approach combines dual wavelength control with intrabodies targeted to actin, iB(actin), in order to recruit actin to the plasma membrane and the nucleus (Redchuk et al, 2020). This study targeted BphP1 to the plasma membrane and fused iB(actin) to both Q‐PAS1 and an As LOV2 caged NLS.…”

Light control of RTK activity: from technology development to translational research

“… 79 Both nanobodies could be fused to available optogenetic near-infrared heterodimerizers, 32 enabling light-activatable degradation of endogenous proteins, like RTKs or their downstream counterparts. 80 …”