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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.

We systematically evaluated the performance and reliability of several widely used, commercially available actin-filament probes in a highly motile breast adenocarcinoma cell line to optimize the visualization of F-actin-rich dynamic lamellipodia. We evaluated four Phalloidin-fluorophores, two anti-actin antibodies, and three live-cell actin probes in five fixation conditions across three imaging platforms as a basis for the design of optimized protocols. Of the fluorescent phalloidin-dye conjugates tested, Alexa Fluor-488 Phalloidin ranked best in overall labeling of the actin cytoskeleton and maintenance of the fluorescence signal over time. Use of actin monoclonal antibodies revealed significant limitations under a variety of fixation–permeabilization conditions. Evaluation of commonly used live-cell probes provides evidence for actin filament bias, with TagRFP-Lifeact excluded from lamellipodia, but not mEGFP-Lifeact or F-tractin-EGFP.

Summary We describe an approach to study the conformation of individual proteins during single particle tracking (SPT) in living cells. "Binder/tag" is based on incorporation of a 7-mer peptide (the tag) into a protein where its solvent exposure is controlled by protein conformation. Only upon exposure can the peptide specifically interact with a reporter protein (the binder). Thus, simple fluorescence localization reflects protein conformation. Through direct excitation of bright dyes, the trajectory and conformation of individual proteins can be followed. Simple protein engineering provides highly specific biosensors suitable for SPT and FRET. We describe tagSrc, tagFyn, tagSyk, tagFAK, and an orthogonal binder/tag pair. SPT showed slowly diffusing islands of activated Src within Src clusters and dynamics of activation in adhesions. Quantitative analysis and stochastic modeling revealed in vivo Src kinetics. The simplicity of binder/tag can provide access to diverse proteins.

Actomyosin supracellular networks emerge during development and tissue repair. These cytoskeletal structures are able to generate large scale forces that can extensively remodel epithelia driving tissue buckling, closure and extension. How supracellular networks emerge, are controlled and mechanically work still remain elusive. During Drosophila oogenesis, the egg chamber elongates along the anterior-posterior axis. Here we show that a dorsal-ventral polarized supracellular F-actin network, running around the egg chamber on the basal side of follicle cells, emerges from polarized intercellular filopodia that radiate from basal stress fibers and extend penetrating neighboring cell cortexes. Filopodia can be mechanosensitive and function as cell-cell anchoring sites. The small GTPase Cdc42 governs the formation and distribution of intercellular filopodia and stress fibers in follicle cells. Finally, our study shows that a Cdc42-dependent supracellular cytoskeletal network provides a scaffold integrating local oscillatory actomyosin contractions at the tissue scale to drive global polarized forces and tissue elongation., During development, organs undergo large scale forces driven by the cytoskeleton but the precise molecular regulation of cytoskeletal networks remains unclear. Here, the authors report a Cdc42-dependent supracellular cytoskeletal network integrates local actomyosin contraction at tissue scale and drives global tissue elongation.

The spatial and temporal organization of protein activity is an essential but poorly understood aspect of cellular signaling. Optogenetics is emerging as a powerful tool for precise modulation of specific protein's activity within living cells and animals. For non-channel proteins, optical control of protein activity has been accomplished through light-regulated protein dimerization, oligomerization, and by light-controlled steric block of a protein or peptide active site using the LOV (light-oxygen-voltage) domain. Illumination triggers a conformational change in LOV that reversibly releases the steric block. A challenge in this approach has been the difficulty of controlling the orientation of LOV in the dark to efficiently block the protein site. Recently our lab has generated a novel LOV-binding protein based on z-affibodies, a class of engineered protein scaffold. Our z-affibody (termed Zdk) binds selectively to the dark state of LOV ( 4 μM affinity in the light). We show that Zdk can be used to control the orientation of LOV in the dark state and efficiently block the actin-binding site of cofilin in a light-dependent, reversible manner. To make photoactivatable cofilin (PA-cofilin), Zdk was fused to the N-terminus of cofilin and LOV was fused to the C-terminus. We used Rosetta-based molecular modeling to rationally design linkers that oriented the Zdk-LOV complex over the actin-binding interface and also to rationally design Zdk variants with a range of affinities for LOV. Using a Zdk of the optimum affinity for LOV was found to be critical in the design. PA-cofilin undergoes a five-fold increase in actin-binding in response to light, demonstrating the tight control of protein activity that can be achieved using Zdk. Ongoing studies using PA-cofilin to probe the role of cofilin in polarized migration will be described.

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.