Zlock, a Broadly Applicable Optogenetic Method, Controls Cofilin in Living Cells

Abstract

The spatial and temporal organization of protein activity controls the flow of information through signaling networks. Optogenetics, the regulation of protein activity with light, has proven very useful to dissect the role of spatio-temporal control by inducing specific protein activities with seconds and microns resolution in living cells. However, current methods are not applicable to many important proteins. Here we describe Zlock, a broadly applicable optogenetic method to control protein or small-peptide activity in living cells using modifications of Zdark (Zdk), a small protein that binds only to the dark state of the LOV2 domain from Phototropin. The LOV2 domain undergoes a reversible conformational change induced by 400-500 nm light. Zdk, derived by screening a library of Z domain variants of protein A, binds selectively to the dark state of LOV2 with dark Kd 10 micromolar. In the “Zlock” system, Zdk and LOV2 are fused to the N- and C- termini of a target protein or small-peptide. In the dark, they bind to one another and cover or distort the active site. Upon irradiation they release one another in less than a second, freeing the active site for target interaction. We have engineered Zdk for the reversible intramolecular binding needed in the Zlock design. We have successfully applied Zlock to generate photoactivatable analogs of important cytoskeletal regulators including cofilin and Alpha Tubulin Acetyltransferase 1 (ATAT). Photoactivation of cofilin and ATAT in live cells controls cell protrusion and microtubule acetylation, and has demonstrated a clear role for cofilin in regulating directionality during cancer cell migration. Current work is focused on ‘caging’ photoactivatable small-peptide inhibitors of Cdc42, PP1, JNK and Gqα, to control endogenous proteins with light.