Two-input protein logic gate for computation in living cells

Design of ChOp-FAK

To develop a protein that functions as a two-input logic OR gate, we engineered a dual-regulated kinase ChOp-FAK with two rationally incorporated regulatory sensor modules (Fig. 1). The chemogenetic (Ch) module is uniRapR, which activates the protein in response to its binding partner rapamycin (Supplementary Fig. 1a). The second module is the optogenetic (Op) LOV2 domain, which inactivates the protein in response to blue light (Supplementary Fig. 1b). Rapamycin and light serve as the input signals, and kinase function is the output signal. The FAK structure includes an N-terminal FERM domain, central kinase domain, and C-terminal FAT domain. The FERM and kinase domains play direct roles in the catalytic regulation of FAK¹⁸. Therefore, to build ChOp-FAK and to enable dual regulation, we rationally introduced chemo and opto orthogonal switches into the kinase domain and FERM domain, respectively.

Chop-FAK is allosterically regulated by inserted sensor domains uniRapR and LOV2, which serve as input response elements. Rapamycin and light are the input signals for uniRapR and LOV2, respectively. From the sensor domains signals propagate through the amino acid core network (shown as contact network) of the protein. The output is FAK activation.

Design, preparation, and validation of Ch-FAK

We first built the chemogenetically controlled FAK (Ch-FAK) by integrating the rapamycin-binding uniRapR domain into the kinase domain (Fig. 2a). Kinases containing the uniRapR domain remain catalytically inactive until the domain binds rapamycin^7,19. Rapamycin stabilizes the uniRapR domain structure, which, in turn, results in activation of the kinase. Our previous study demonstrated that FAK activity could be allosterically regulated by the insertion of a regulatory domain into the kinase domain⁷. Hence, we introduced the uniRapR domain into the previously identified allosteric site on the kinase domain of FAK. Short GPG linkers were introduced on both the sides of the uniRapR domain to optimize regulation (Supplementary Table 2). We performed DMD simulations for Ch-FAK in presence and absence of rapamycin. Rapamycin-bound structure displayed an open conformation, corresponding to the active conformation of FAK, whereas simulations without rapamycin showed a closed confirmation of the protein, suggesting the inactive form (Supplementary Fig. 2). We also incorporated two reported mutations, Y180A and M183A, into Ch-FAK to rule out the possibility of FAK activation by endogenous upstream factors⁷.

a Top, schematic of Ch-FAK. 50 nM rapamycin is the input signal for Ch-FAK. Output is measured as Ch-FAK activation. Bottom, schematic of a cell plated on fibronectin-coated glass surface showing stress fibers, focal adhesions and the location of activated Ch-FAK. b Top, time-lapse fluorescent imaging data for the mCherry-tagged Ch-FAK demonstrating rapamycin-induced activation in HeLa cells. 50 nm rapamycin was added at 30th min to induce the activation. Enlarged, late focal adhesions are indicated by arrows. Bottom, time-lapse bright-field images of HeLa cells that express Ch-FAK before and after addition of rapamycin. Dorsal ruffles are indicated by arrows. Center, zoomed-in regions of indicated images. c Normalized quantification of average size and total number of focal adhesions before and after the rapamycin addition. Control is kinase-dead mutant of FAK. Data represent box plots and individual data points. Box plots show the median (center line), first and third quartiles (box edges), while the whiskers going from each quartile to the minimum or maximum. n = 11 cells for total focal adhesions (FAs) and n = 14 cells for average size of FAs from 3 independent experiments; ^****P = 2.6 × 10⁻⁸ for number of FAs, ^****P = 2.7 × 10⁻⁸ for size of FAs in Ch-FAK. P = 0.872 for number of FAs and P = 0.1464 for FAs size in control conditions calculated by unpaired two-tailed Student’s t-test. NS, not significant. d Depth analysis for the time-lapse imaging data for rapamycin-induced activation in Ch-FAK-expressing HeLa cells. Analysis was performed by measuring the distance between the glass surface and each optical planes of the cell. Distances are indicated by a color scale. Scale bar, 40 μm. FAs indicates focal adhesions. Source data are provided as a Source Data file.

We expressed Ch-FAK in HeLa cells, and cultured these cells on a coverslip in a glass-bottomed dish. Addition of 50 nM rapamycin to the Ch-FAK-expressing cells increased the membrane dynamics of the cells as shown by imaging of live cells over time (Fig. 2b). After rapamycin addition, dorsal ruffles formed, and we observed localization of FAK within these membrane ruffles (Supplementary Fig. 3). These results are in agreement with our previous findings⁷. The dorsal ruffle phenotype was observed in only 10 of 30 cells, therefore we looked for alternate output signals.

We observed that rapamycin treatment promoted translocation of Ch-FAK from the cytoplasmic regions to focal adhesions resulting in their enlargement (Fig. 2b, Supplementary Fig. 4, and Supplementary Movie 1). The conversion from small, early focal adhesions to large, late focal adhesions was very rapid, occurring about 10 min post-rapamycin treatment. Quantitative analysis showed an approximately 1.6-fold increase (P < 0.0001 by unpaired Student’s t-test) in the total number of focal adhesions per cell post-rapamycin addition (Fig. 2c). Importantly, there was an approximately 1.9-fold increase (P < 0.0001 by unpaired Student’s t-test) in the average size of the focal adhesions upon rapamycin treatment (Fig. 2c). In cells that expressed the control construct, Ch-FAK-YM-KD, which is a mutant of FAK lacking catalytic activity, we did not observe any significant changes in the focal adhesions upon rapamycin addition (Fig. 2c). The majority of the large, late focal adhesions were located on the side of the cells next to the culture plate (Fig. 2d). FAK and Paxillin phosphorylation studies suggested that Ch-FAK’s catalytic activity was activated upon rapamycin treatment (Supplementary Fig. 5).

Previous reports have implicated FAK in cell motility and spreading²⁰. To test such effects, FAK^−/− fibroblasts were cultured on an architecturally standardized microenvironmental platform using 2D biomimetic collagen type-1 fibers as micropatterns²¹. Ch-FAK-transfected FAK^−/− fibroblasts exhibited a spindle morphology (Fig. 3a, left and Supplementary Fig. 6, left), very similar to wild-type fibroblast morphology is observed in 3D collagen ECM²². Upon treatment with rapamycin, there was a change from uniaxial spindle organization toward complex multiaxial cell architectures indicating a higher responsiveness to the cell microenvironments (Fig. 3a, right and Supplementary Fig. 6, right). These changes were evaluated by standardized cell morphometric analysis (Fig. 3b). Ch-FAK activation resulted in change in cell morphometric organization as indicated by increases in lengths, widths, and apicality along the rhomboid anisotropic biomimetic collagen “fibers” (Fig. 3c, d). We did not observe any morphological changes in Ch-FAK non-transfected cells upon rapamycin treatment (Supplementary Fig. 7). We also evaluated effect of Ch-FAK on cell motility and speed in MDA-MB-231 cells (Supplementary Movies 2 and 3). Treatment of these cells with rapamycin significantly reduced motility compared to untreated cells (Fig. 3e, f). Thus, we reasoned that Ch-FAK activation increases cellular adhesion and architectural complexity, which decreases cell motility.

a Representative cell fluorescent micrographs for Ch-FAK-expressing FAK^−/− cells with (right) and without (left) rapamycin treatment on 2D collagen type-1 micropatterns. Red fluorescence indicates Ch-FAK. Scale bar, 20 μm. b Schematic of the cell morphometric analysis. Arrows indicate apices (apicality) of the cell. c Width of Ch-FAK-transfected FAK^−/− fibroblasts with and without rapamycin treatment versus Ch-FAK expression levels. Data represent scatter plot, n = 50 cells for controls and n = 67 cells for rapamycin-treated conditions from 3 independent experiments. d Length, width, and apicality of Ch-FAK-transfected FAK^−/− fibroblasts with and without rapamycin treatment. Data represent box plots, violin plots and individual data points. Data represent box plots and individual data points. Box plots show the median (center line), first and third quartiles (box edges), while the whiskers going from each quartile to the minimum or maximum. n = 50 cells for controls and n = 67 cells for rapamycin-treated conditions from 3 independent experiments. ^****P = 1.5 × 10⁻⁵ for length, ^****P = 3.6 × 10⁻⁹ for width, and ^****P = 4.7 × 10⁻¹¹ for apicality by unpaired two-tailed Student’s t-test. e Migration tracks of rapamycin-treated and untreated Ch-FAK-transfected MDA-MB-231 cells along the collagen fibers. Data represent migration tracks, n = 4 cells for control and rapamycin-treated conditions. f Migration speeds of the rapamycin-treated and untreated Ch-FAK-transfected MDA-MB-231 cells along the collagen fibers on 2D collagen type-1 micropatterns. n = 4 cells for control and rapamycin-treated conditions; ^***P = 0.001 by unpaired two-tailed Student’s t-test. Source data are provided as a Source Data file.

To confirm the role of Ch-FAK activation in the formation of enlarged focal adhesions, we treated the Ch-FAK-transfected, rapamycin-treated FAK^−/− fibroblasts with FAK inhibitor 14 and performed live-cell imaging. FAK inhibitor 14 treatment resulted in rapid degradation of enlarged, late focal adhesions only in the Ch-FAK-transfected cells (Fig. 4a and Supplementary Figs. 8 and 9). Focal adhesion growth depends on stress fibers²³, which are crucial for mechanotransduction²⁴. To verify the role of stress fibers in the effects of rapamycin on Ch-FAK-transfected FAK^−/− fibroblasts, we stained the cells for phalloidin to visualize the stress fibers during the Ch-FAK activation process. In these cells, focal adhesions were visible as large clusters at the stress fiber termini, and activation of Ch-FAK altered the distribution of intracellular stress fibers (Fig. 4b). When we treated rapamycin-activated cells with 50 µM of blebbistatin, a myosin-II inhibitor, the central and peripheral stress fibers disintegrated and focal adhesions were disrupted within 20 min, and the cells underwent drastic changes in morphology (Fig. 4c and Supplementary Fig. 10). These data demonstrate that Ch-FAK activation is responsible for the stress fiber-mediated alterations in focal adhesions in the Ch-FAK-transfected cells.

a Images of rapamycin-treated Ch-FAK-expressing FAK^−/− fibroblasts treated with control (DMSO) and FAK inhibitor 14. Top panel shows the Ch-FAK-activated FAK^−/− cells treated with control. Arrows indicate focal adhesions. Bottom panel shows same set of cells treated with FAK inhibitor 14. Scale bar, 20 μm. b Images of Ch-FAK-expressing FAK^−/− fibroblasts without (top) and with (bottom) rapamycin stained with phalloidin to reveal stress fibers (magenta). Large focal adhesions formed by the activation of Ch-FAK are visible as red clusters (mCherry fluorescence) at the ends of stress fibers in bottom panel. Nuclei were stained with Hoechst (blue). Individual channels are shown to the right of the merged images. Scale bar, 20 μm. c Images of rapamycin-treated Ch-FAK-expressing FAK^−/− fibroblasts treated with control (DMSO) (top) or blebbistatin (bottom). Red indicates phalloidin stained stress fibers and green indicates Hoechst-stained nucleus. Scale bar, 40 μm.

Design, preparation, and validation of Op-FAK

We next introduced the light-inducible LOV2 domain from Avena sativa into a loop of the FERM domain of FAK. The 10 Å spacing between the N and C termini of LOV2 is ideal as it results in minimal perturbation of the structure of the loop of the protein into which it is inserted². Blue light exposure leads to the unfolding of the C-terminal Jα helix of LOV2. This light-induced conformational change in LOV2 leads to the distortion of the FAK and resulting in its inactivation.

To identify regions of the FERM domain that could be connected to LOV2 to result in allosteric control, we mapped the allosteric connectivity between the loops and target region using Ohm (Fig. 5a). We also considered three other parameters: sequence conservation, surface exposure, and loop tightness (Fig. 5b). Based on these criteria, loop 2, between S264 and S265, was selected as a potential insertion site. We used DMD simulations to analyze conformations of Op-dark-FAK and Op-lit-FAK, with mutants of LOV2 locked in the dark and light states, respectively, inserted at loop 2 (Fig. 5c). We observed a closed conformation in Op-lit-FAK, corresponding to an inactive conformation of FAK, whereas Op-dark-FAK had an open, active conformation. In design of Op-FAK, we also considered linkers of different lengths and sequences for connection of the LOV2 domain to FERM. A short GP linker was optimal (Fig. 6a and Supplementary Table 2).

a Ribbon diagrams of FAK from the published crystal structure (PDB ID: 2JOJ). Loop 1 is the insertion site for uniRapR (left), and Loop 2 is the insertion site for LOV2 (bottom). Green paths represent signal propagation pathways from the insertion sites to the target region (circled). b Contact map computed from the published crystal structure (PDB ID: 2JOJ) for sequence conservation and surface exposure. Loop 2 (L2) has low sequence conservation and high surface exposure. Bottom figure demonstrates the β-hairpin loop (loop 2) within the FERM domain. c Top, snapshots from the DMD simulations showing conformations of Op-lit-FAK and Op-dark-FAK. The distance between the two domains in Op-dark-FAK is indicative of activation. Bottom, frequencies of structures with given distances between FERM and kinase domains in Op-lit-FAK and Op-dark-FAK quantified during DMD simulations. Source data are provided as a Source Data file.

a Schematic representation of the Op-FAK. LOV2 domain is inserted into the FERM domain of FAK. Light is used as input signal. Dark condition activates FAK and light conditions inactivates FAK. b Images of cells transfected with Op-dark-FAK (left) and Op-lit-FAK (right) on a fibronectin-coated glass surface. Inset shows the cell edges with arrows highlighting focal adhesions. Scale bar, 40 μm. c Normalized quantification of average size and total number of focal adhesions in Op-dark-FAK- and Op-lit-FAK-expressing FAK^−/− cells on a fibronectin-coated glass surface. Data represent bar plots with mean±s.d. and individual data points, n = 10 cells for dark and lit mutants from 3 independent experiments; ^****P = 6.2 × 10⁻¹¹ for number of focal adhesion (FAs), ^**P = 0.0014 for size of FAs in mutants calculated by unpaired two-tailed Student’s t-test. NS, not significant. d Images of Op-lit-FAK- (left) and Op-dark-FAK-expressing (right) FAK^−/− cells on PAA gels printed with fluorescently labeled fibronectin grids. Zoomed-in images show the well-formed focal adhesions along the grid lines for Op-dark-FAK. Scale bar, 40 μm; zoomed-in images, 14 μm. e Top, time-lapse images of FAK^−/− fibroblasts that express Op-FAK exposed to light for the indicated periods of time. Bottom, magnification of region boxed in the upper left panel as a function of time in response to blue light (488 nm). Arrows indicate focal adhesions. Scale bar, 40 μm. f Normalized quantification of average size and total number of focal adhesions during light-induced inactivation of FAK^−/− cells that express Op-FAK or Op-dark-FAK (control). FAs indicates focal adhesions. Data represent box plots and individual data points. Data represent box plots and individual data points. Box plots show the median (center line), first and third quartiles (box edges), while the whiskers going from each quartile to the minimum or maximum. n = 10 cells for total FAs and average size of FAs from 3 independent experiments; ^***P = 0.0003 for number of FAs, ^***P = 0.0001 for size of FAs in Op-FAK. P = 0.7737 for number of FAs and P = 0.4617 for FAs size in control conditions calculated by unpaired two-tailed Student’s t-test. NS, not significant. Source data are provided as a Source Data file.

We first expressed Op-dark-FAK and Op-lit-FAK in FAK^−/− fibroblasts and evaluated the responses. We observed large focal adhesions, similar to those observed upon expression of activated Ch-FAK, in Op-dark-FAK-transfected cells but not in Op-lit-FAK-transfected cells (Fig. 6b). Upon quantitation, we discovered that focal adhesions were approximately 2-fold larger in size (P < 0.01 by unpaired Student’s t-test) and that there were approximately 4.5-fold more focal adhesions (P < 0.0001 by unpaired Student’s t-test) in cells that expressed Op-dark-FAK than in those that expressed Op-lit-FAK (Fig. 6c). Moreover, on rigid PAA gels (G′ of 8.6 kPa) microprinted with fluorescent fibronectin grids to mimic the fibrous ECM, most Op-dark-FAK-transfected cells showed clear and well-formed focal adhesions along the fibronectin, whereas the Op-lit-FAK-transfected cells did not form focal adhesions (Fig. 6d).

We next tested cells that express the kinase with the wild-type LOV2 (Op-FAK). We performed live-cell imaging of the Op-FAK-transfected FAK^−/− fibroblasts for 30 min in the dark and then during 60 min of exposure to blue light. In the dark, cells had numerous, enlarged focal adhesions, similar to those formed by cells that express Op-dark-FAK. When we exposed the cells to blue light, the number and average size of the focal adhesions decreased by about 0.7 fold (P < 0.001 by unpaired Student’s t-test) within 35 min (Fig. 6e, f). As a control, we evaluated the effect of blue light on Op-dark-FAK-transfected cells but did not observe any significant changes in focal adhesions (Fig. 6f). Analysis of FAK and paxillin phosphorylation levels indicated that the Op-FAK’s catalytic activity was inhibited upon light irradiation (Supplementary Fig. 5).

Unlike the effects of Ch-FAK activation, which occurred very rapidly, inactivation of Op-FAK was a slow process. To improve the response, we tested the transfected cells under different conditions. Substrate stiffness influences the cellular responses²⁵. Therefore, we evaluated the light response of the Op-FAK-transfected cells on a soft elastic hydrogel surface (G′ of 2.3 kPa). There was not a significant improvement in the inactivation kinetics (Supplementary Fig. 11). We also substituted rested collagen type-1 coating of the glass surface but the response rate did not differ from that on a fibronectin-coated surface (Supplementary Fig. 12). Even though activation/inactivation kinetics are important when designing optogenetic or chemogenetic tools, the kinetics of Op-FAK were sufficient for us to build and test the gating functions.

Assembly and testing of ChOp-FAK

To build our final design, we engineered FAK with both chemo- and optogenetically regulated domains with uniRapR and LOV2 designs inserted into the kinase domain and the FERM domain, respectively (Fig. 7a). For these experiments, we used the ChOp-lit-FAK and ChOp-dark-FAK constructs for two reasons. When we attempted to start in the light conditions, phototoxicity was observed because of the prolonged exposure. In the dark conditions, FAK was activated and no difference was observed upon rapamycin addition. We therefore subjected cells that expressed ChOp-lit-FAK and ChOp-dark-FAK to input signals and measured the corresponding output signals (Fig. 7b). Rapamycin was the activating input condition for the Ch module, and the dark- and lit-state mutations served as activated and inactivated states for the Op module. In FAK^−/− cells transfected with ChOp-lit-FAK not treated with rapamycin, we anticipated that the two “OFF” signals would maintain the kinase and FERM domains in close proximity ensuring a strong inactivation. Indeed, only the small, early focal adhesions were observed in these cells, and these foci were comparable to those in cells in which Op-lit-FAK was expressed, suggesting inactivation of FAK. We quantified the number and the average size of the focal adhesions, and we used these parameters to identify activating conditions (Fig. 7c, Condition A). Next, we treated cells that expressed ChOp-lit-FAK with rapamycin, the input signal for the Ch module. Upon addition of 50 nM rapamycin, there was not a significant increase in the average size or total number of the focal adhesions compared to ChOp-lit-FAK not treated with rapamycin (Supplementary Fig. 13). At 100 nM rapamycin concentration, the average size did not change significantly, but there was a significant increase in the total number of focal adhesions (P < 0.05 by unpaired Student’s t-test) (Supplementary Fig. 13b). At 150 nM rapamycin, we observed significant increases in the average size (P < 0.001 by unpaired Student’s t-test) and the total number of focal adhesions (P < 0.05 by unpaired Student’s t-test) (Supplementary Fig. 13b), indicative of activation of the Ch module (Supplementary Fig. 13b). However, the activation was not very robust. To optimize the ChOp-FAK design, we modulated the strength of input via redesign of linkers. We introduced several linkers (differing in length and sequence) into ChOp-FAK and screened them using western blot (Supplementary Tables 1 and 2 and Supplementary Fig. 14). From the screening, we picked the optimized design (with linker “G”) and continued the evaluation. The results indicated that performance of the optimized ChOp-FAK design improved drastically in “Condition B” with ~2.9-fold increase (P < 0.0001 by unpaired Student’s t-test) in the average size of focal adhesions and ~4-fold increase (P < 0.0001 by unpaired Student’s t-test) in total number of focal adhesions compared to cells that expressed ChOp-lit-FAK not treated with rapamycin (Fig. 7c, Condition B). We next evaluated ChOp-dark-FAK expressed in FAK^−/− cells in the absence and presence of rapamycin. In the absence of rapamycin, the average size of focal adhesions was 3.3-fold higher (P < 0.0001 by unpaired Student’s t-test) and the number of focal adhesions was about 4.5-fold higher (P < 0.0001 by unpaired Student’s t-test) than in cells that expressed ChOp-lit-FAK not treated with rapamycin (Fig. 7c, Condition C). This suggested a near-complete activation of ChOp-FAK. When rapamycin was added to the ChOp-dark-FAK-expressing cells, sizes and numbers of focal adhesions were similar to those in the absence of rapamycin (Fig. 7c, Condition D).

a Left, schematic representation of ChOp-FAK. Validated Ch and Op modules were assembled to construct ChOp-FAK. Right, domain organization of ChOp-FAK. b Combinations of tested input conditions and the respective outputs. For Ch input, ChOp-FAK-expressing FAK^−/− cells were treated with 50 nM rapamycin for 30 min. For Op input, dark and lit mutants of LOV2 were used to mimic the dark and light conditions, respectively. c Quantification of average size and total number of focal adhesions in FAK^−/− cells expressing linker optimized ChOp-FAK. Pink box indicates inactivation and green box indicates activation of FAK. Data were normalized to Condition A. Focal adhesion (FAs) indicates focal adhesions. Data represent box plots and individual data points. Data represent box plots and individual data points. Box plots show the median (center line), first and third quartiles (box edges), while the whiskers going from each quartile to the minimum or maximum. n = 18 cells for Condition A, n = 16 cells for Condition B, n = 15 cells for Conditions C and D from 3 independent experiments. For total number of FAs, ^****P = 4.3 × 10⁻¹² for A and B, ^****P = 1.07 × 10⁻¹² for A and C, ^****P = 1.18 × 10⁻¹³ for A and D, P = 0.08 for C and D. For Average size of FAs, ^****P = 1.2 × 10⁻¹⁶ for A and B, ^****P = 7.25 × 10⁻²³ for A and C, ^****P = 5 × 10⁻²¹ for A and D, P = 0.768 for C and D conditions calculated by unpaired two-tailed Student’s t-test. NS, not significant. Source data are provided as a Source Data file.