Optogenetic control of cofilin and αTAT in living cells using Z-lock

Abstract

Here we introduce Z-lock, an optogenetic approach for reversible, light-controlled steric inhibition of protein active sites. The 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 upon irradiation.

Engineering Photosensory Modules of Non-Opsin-Based Optogenetic Actuators

“…Upon illumination, the binding is disrupted by the conformational change of the LOV2 domain, resulting in the uncaging of biofunctional domain by freeing the protein to move to its site of action. The LOVTRAP system, and its recently reported derivative Z-lock [ 52 ], have been used to mediate activation of different signaling protein that control cell-edge protrusion (Vav2, Rac1, RhoA, cofilin, and αTAT) and represent a versatile approach for reversibly photo-caging and uncaging.…”

“…The schematic representations in this figure are inspired and adapted from previously reported studies, which are labelled in parentheses below the corresponding schematic. The citations are as follow: ChR2 [ 2 , 34 , 35 ]; eNpHR [ 2 , 34 , 36 ]; eBR [ 34 , 37 ]; OptoXR [ 2 , 34 , 38 ]; LightOn [ 24 ]; CRY2oligo [ 44 , 45 ]; Dronpa145N [ 46 ]; Magnets [ 18 , 47 , 48 ]; TULIPs [ 49 ]; LOVTRAP [ 50 , 51 , 52 ]; CRY2-CIB1 [ 27 , 28 , 53 ]; PhyB-PIF [ 54 , 55 ]; BphP1-PpsR2 [ 25 , 56 ]; LINuS [ 20 , 21 ]; PA-Rac1 [ 57 ]; and PhoCl [ 58 ].…”

Tools of the trade: studying actin in zebrafish

“…The F actin network can also be remodelled by genetically modified cofilins that are fused to light activated domains. Such approaches would further enhance the genetic toolkit of actin regulators in zebrafish (Hughes and Lawrence 2014 ; Stone et al 2019 ).…”

Advances, Perspectives and Potential Engineering Strategies of Light-Gated Phosphodiesterases for Optogenetic Applications

“…Previous work has demonstrated that attaching Zdk and LOV2 to the C and N termini of cofilin respectively could effectively occlude the cofilin active site in dark. Upon irradiation, the dissociation of Zdk and LOV frees the active site [ 98 ].…”