Curated Optogenetic Publication Database

Abstract: The mechanical properties of biological tissues are key to their physical integrity and function. Although external loading or biochemical treatments allow the estimation of these properties globally, it remains difficult to assess how such external stimuli compare with cell-generated contractions. Here we engineer microtissues composed of optogenetically-modified fibroblasts encapsulated within collagen. Using light to control the activity of RhoA, a major regulator of cellular contractility, we induce local contractions within microtissues, while monitoring microtissue stress and strain. We investigate the regulation of these local contractions and their spatio-temporal distribution. We demonstrate the potential of our technique for quantifying tissue elasticity and strain propagation, before examining the possibility of using light to create and map local anisotropies in mechanically heterogeneous microtissues. Altogether, our results open an avenue to guide the formation of tissues while non-destructively charting their rheology in real time, using their own constituting cells as internal actuators.

Abstract: Adherent cells use actomyosin contractility to generate mechanical force and to sense the physical properties of their environment, with dramatic consequences for migration, division, differentiation, and fate. However, the organization of the actomyosin system within cells is highly variable, with its assembly and function being controlled by small GTPases from the Rho family. To understand better how activation of these regulators translates into cell-scale force generation in the context of different physical environments, here we combine recent advances in non-neuronal optogenetics with micropatterning and traction force microscopy on soft elastic substrates. We find that, after whole-cell RhoA activation by the CRY2/CIBN optogenetic system with a short pulse of 100 ms, single cells contract on a minute timescale in proportion to their original traction force, before returning to their original tension setpoint with near perfect precision, on a longer timescale of several minutes. To decouple the biochemical and mechanical elements of this response, we introduce a mathematical model that is parametrized by fits to the dynamics of the substrate deformation energy. We find that the RhoA response builds up quickly on a timescale of 20 s, but decays slowly on a timescale of 50 s. The larger the cells and the more polarized their actin cytoskeleton, the more substrate deformation energy is generated. RhoA activation starts to saturate if optogenetic pulse length exceeds 50 ms, revealing the intrinsic limits of biochemical activation. Together our results suggest that adherent cells establish tensional homeostasis by the RhoA system, but that the setpoint and the dynamics around it are strongly determined by cell size and the architecture of the actin cytoskeleton, which both are controlled by the extracellular environment.

Abstract: To migrate efficiently, neutrophils must polarize their cytoskeletal regulators along a single axis of motion. This polarization process is thought to be mediated through local positive feedback that amplifies leading edge signals and global negative feedback that enables sites of positive feedback to compete for dominance. Though this two-component model efficiently establishes cell polarity, it has potential limitations, including a tendency to “lock” onto a particular direction, limiting the ability of cells to reorient. We use spatially-defined optogenetic control of a leading edge organizer (PI3K) to probe how cells balance “decisiveness” needed to polarize in a single direction with the flexibility needed to respond to new cues. Underlying this balancing act is a local Rac inhibitor that destabilizes the leading edge to promote exploration. We show that this local inhibitor enables cells to process input signal dynamics, linking front stability and orientation to local temporal increases in input signals.

Abstract: Integration of collective cell direction and coordination is believed to ensure collective guidance for efficient movement. Previous studies demonstrated that chemokine receptors PVR and EGFR govern a gradient of Rac1 activity essential for collective guidance of Drosophila border cells, whose mechanistic insight is unknown. By monitoring and manipulating subcellular Rac1 activity, here we reveal two switchable Rac1 pools at border cell protrusions and supracellular cables, two important structures responsible for direction and coordination. Rac1 and Rho1 form a positive feedback loop that guides mechanical coupling at cables to achieve migration coordination. Rac1 cooperates with Cdc42 to control protrusion growth for migration direction, as well as to regulate the protrusion-cable exchange, linking direction and coordination. PVR and EGFR guide correct Rac1 activity distribution at protrusions and cables. Therefore, our studies emphasize the existence of a balance between two Rac1 pools, rather than a Rac1 activity gradient, as an integrator for the direction and coordination of collective cell migration.

Abstract: The spatial organization of cell-surface receptors is fundamental for the coordination of biological responses to physical and biochemical cues of the extracellular matrix. How serine/threonine kinase receptors, ALK3-BMPRII, cooperate with integrins upon BMP2 to drive cell migration is unknown. Whether the dynamics between integrins and BMP receptors intertwine in space and time to guide adhesive processes is yet to be elucidated. We found that BMP2 stimulation controls the spatial organization of BMPRs by segregating ALK3 from BMPRII into β3 integrin-containing focal adhesions. The selective recruitment of ALK3 to focal adhesions requires β3 integrin engagement and ALK3 activation. BMP2 controls the partitioning of immobilized ALK3 within and outside focal adhesions according to single-protein tracking and super-resolution imaging. The spatial control of ALK3 in focal adhesions by optogenetics indicates that ALK3 acts as an adhesive receptor by eliciting cell spreading required for cell migration. ALK3 segregation from BMPRII in integrin-based adhesions is a key aspect of the spatio-temporal control of BMPR signaling.

Abstract: During cell migration and polarization, numerous signal transduction and cytoskeletal components self-organize to generate localized protrusions. Although biochemical and genetic analyses have delineated many specific interactions, how the activation and localization of so many different molecules are spatiotemporally orchestrated at the subcellular level has remained unclear. Here we show that the regulation of negative surface charge on the inner leaflet of the plasma membrane plays an integrative role in the molecular interactions. Surface charge, or zeta potential, is transiently lowered at new protrusions and within cortical waves of Ras/PI3K/TORC2/F-actin network activation. Rapid alterations of inner leaflet anionic phospholipids-such as PI(4,5)P2, PI(3,4)P2, phosphatidylserine and phosphatidic acid-collectively contribute to the surface charge changes. Abruptly reducing the surface charge by recruiting positively charged optogenetic actuators was sufficient to trigger the entire biochemical network, initiate de novo protrusions and abrogate pre-existing polarity. These effects were blocked by genetic or pharmacological inhibition of key signalling components such as AKT and PI3K/TORC2. Conversely, increasing the negative surface charge deactivated the network and locally suppressed chemoattractant-induced protrusions or subverted EGF-induced ERK activation. Computational simulations involving excitable biochemical networks demonstrated that slight changes in feedback loops, induced by recruitment of the charged actuators, could lead to outsized effects on system activation. We propose that key signalling network components act on, and are in turn acted upon, by surface charge, closing feedback loops, which bring about the global-scale molecular self-organization required for spontaneous protrusion formation, cell migration and polarity establishment.

Abstract: Microtubules are cytoskeletal polymers that separate chromosomes during mitosis and serve as rails for intracellular transport and organelle positioning. Manipulation of microtubules is widely used in cell and developmental biology, but tools for precise subcellular spatiotemporal control of microtubules are currently lacking. Here, we describe a light-activated system for localized recruitment of the microtubule-severing enzyme katanin. This system, named opto-katanin, uses targeted illumination with blue light to induce rapid, localized, and reversible microtubule depolymerization. This tool allows precise clearing of a subcellular region of microtubules while preserving the rest of the microtubule network, demonstrating that regulation of katanin recruitment to microtubules is sufficient to control its severing activity. The tool is not toxic in the absence of blue light and can be used to disassemble both dynamic and stable microtubules in primary neurons as well as in dividing cells. We show that opto-katanin can be used to locally block vesicle transport and to clarify the dependence of organelle morphology and dynamics on microtubules. Specifically, our data indicate that microtubules are not required for the maintenance of the Golgi stacks or the tubules of the endoplasmic reticulum but are needed for the formation of new membrane tubules. Finally, we demonstrate that this tool can be applied to study the contribution of microtubules to cell mechanics by showing that microtubule bundles can exert forces constricting the nucleus.

Abstract: The emerging field of synthetic developmental biology proposes bottom-up approaches to examine the contribution of each cellular process to complex morphogenesis. However, the shortage of tools to manipulate three-dimensional (3D) shapes of mammalian tissues hinders the progress of the field. Here we report the development of OptoShroom3, an optogenetic tool that achieves fast spatiotemporal control of apical constriction in mammalian epithelia. Activation of OptoShroom3 through illumination in an epithelial Madin-Darby Canine Kidney (MDCK) cell sheet reduces the apical surface of the stimulated cells and causes displacements in the adjacent regions. Light-induced apical constriction provokes the folding of epithelial cell colonies on soft gels. Its application to murine and human neural organoids leads to thickening of neuroepithelia, apical lumen reduction in optic vesicles, and flattening in neuroectodermal tissues. These results show that spatiotemporal control of apical constriction can trigger several types of 3D deformation depending on the initial tissue context.

Abstract: The signaling events controlling proliferation, survival, and apoptosis during mammary epithelial acinar morphogenesis remain poorly characterized. By imaging single-cell ERK activity dynamics in MCF10A acini, we find that these fates depend on the average frequency of non-periodic ERK pulses. High pulse frequency is observed during initial acinus growth, correlating with rapid cell motility and proliferation. Subsequent decrease in motility correlates with lower ERK pulse frequency and quiescence. Later, during lumen formation, coordinated multicellular ERK waves emerge, correlating with high and low ERK pulse frequencies in outer surviving and inner dying cells, respectively. Optogenetic entrainment of ERK pulses causally connects high ERK pulse frequency with inner cell survival. Acini harboring the PIK3CA H1047R mutation display increased ERK pulse frequency and inner cell survival. Thus, fate decisions during acinar morphogenesis are coordinated by different spatiotemporal modalities of ERK pulse frequency.

Abstract: Lattice lightsheet microscopy (LLSM) featuring three-dimensional recording is improved to manipulate cellular behavior with subcellular resolution through optogenetic activation (optoLLSM). A position-controllable Bessel beam as a stimulation source is integrated into the LLSM to achieve spatiotemporal photoactivation by changing the spatial light modulator (SLM) patterns. Unlike the point-scanning in a confocal microscope, the lattice beams are capable of wide-field optical sectioning for optogenetic activation along the Bessel beam path.We show that the energy power required for optogenetic activations is lower than 1 nW (or 24 mWcm-2) for time-lapses of CRY2olig clustering proteins, and membrane ruffling can be induced at different locations within a cell with subcellular resolution through light-triggered recruitment of phosphoinositide 3-kinase. Moreover, with the epidermal growth factor receptor (EGFR) fused with CRY2olig, we are able to demonstrate guided cell migration using optogenetic stimulation for up to 6 h, where 463 imaging volumes are collected, without noticeable cellular damages.

Abstract: BAR (Bin, Amphiphysin and Rvs) protein domains are responsible for the generation of membrane curvature and represent a critical mechanical component of cellular functions. Thus, BAR domains have great potential as components of membrane-remodeling tools for cell biologists. In this work, we describe the design and implementation of a family of versatile light-gated I-BAR (inverse-BAR) domain containing tools derived from the fusion of the A. thaliana Cryptochrome 2 photoreceptor and I-BAR protein domains ('CRY-BARs') with applications in the remodeling of membrane architectures and the control of cellular dynamics. By taking advantage of the intrinsic membrane binding propensity of the I-BAR domain, CRY-BARs can be used for spatial and temporal control of cellular processes that require induction of membrane protrusions. Using cell lines and primary neuron cultures, we demonstrate here that the CRY-BAR optogenetic tool evokes membrane dynamics changes associated with cellular activity. Moreover, we provide evidence that ezrin, an actin and PIP2 binding protein, acts as a relay between the plasma membrane and the actin cytoskeleton and therefore is an important mediator of switch function. Overall, we propose that CRY-BARs hold promise as a useful addition to the optogenetic toolkit to study membrane remodeling in live cells.

Abstract: The expulsion of dying epithelial cells requires well-orchestrated remodelling steps to maintain tissue sealing. This process, named cell extrusion, has been mostly analysed through the study of actomyosin regulation. Yet, the mechanistic relationship between caspase activation and cell extrusion is still poorly understood. Using the Drosophila pupal notum, a single layer epithelium where extrusions are caspase-dependent, we showed that the initiation of cell extrusion and apical constriction are surprisingly not associated with the modulation of actomyosin concentration and dynamics. Instead, cell apical constriction is initiated by the disassembly of a medio-apical mesh of microtubules which is driven by effector caspases. Importantly, the depletion of microtubules is sufficient to bypass the requirement of caspases for cell extrusion, while microtubule stabilisation strongly impairs cell extrusion. This study shows that microtubules disassembly by caspases is a key rate-limiting step of extrusion, and outlines a more general function of microtubules in epithelial cell shape stabilisation.

Abstract: Proper orientation of the mitotic spindle plays a crucial role in embryos, during tissue development, and in adults, where it functions to dissipate mechanical stress to maintain tissue integrity and homeostasis. While mitotic spindles have been shown to reorient in response to external mechanical stresses, the subcellular cues that mediate spindle reorientation remain unclear. Here, we used a combination of optogenetics and computational modeling to investigate how mitotic spindles respond to inhomogeneous tension within the actomyosin cortex. Strikingly, we found that the optogenetic activation of RhoA only influences spindle orientation when it is induced at both poles of the cell. Under these conditions, the sudden local increase in cortical tension induced by RhoA activation reduces pulling forces exerted by cortical regulators on astral microtubules. This leads to a perturbation of the balance of torques exerted on the spindle, which causes it to rotate. Thus, spindle rotation in response to mechanical stress is an emergent phenomenon arising from the interaction between the spindle positioning machinery and the cell cortex.

Abstract: Microtubules tightly regulate various cellular activities. Our understanding of microtubules is largely based on experiments using microtubule-targeting agents, which, however, are insufficient to dissect the dynamic mechanisms of specific microtubule populations, due to their slow effects on the entire pool of microtubules. To overcome this technological limitation, we have used chemo and optogenetics to disassemble specific microtubule subtypes, including tyrosinated microtubules, primary cilia, mitotic spindles, and intercellular bridges, by rapidly recruiting engineered microtubule-cleaving enzymes onto target microtubules in a reversible manner. Using this approach, we show that acute microtubule disassembly swiftly halts vesicular trafficking and lysosomal dynamics. It also immediately triggers Golgi and ER reorganization and slows the fusion/fission of mitochondria without affecting mitochondrial membrane potential. In addition, cell rigidity is increased after microtubule disruption owing to increased contractile stress fibers. Microtubule disruption furthermore prevents cell division, but does not cause cell death during interphase. Overall, the reported tools facilitate detailed analysis of how microtubules precisely regulate cellular architecture and functions.

Abstract: Most bacteria lack membrane-enclosed organelles to compartmentalize cellular processes. In lieu of physical compartments, bacterial proteins are often recruited to macromolecular scaffolds at specific subcellular locations to carry out their functions. Consequently, the ability to modulate a protein’s subcellular location with high precision and speed bears the potential to manipulate its corresponding cellular functions. Here we demonstrate that the CRY2/CIB1 system from Arabidopsis thaliana can be used to rapidly direct proteins to different subcellular locations inside live E. coli cells including the nucleoid, the cell pole, membrane, and the midcell division plane. We further show that such light-induced re-localization can be used to rapidly inhibit cytokinesis in actively dividing E. coli cells. Finally, we demonstrate that the CRY2/CIBN binding kinetics can be modulated by green light, adding a new dimension of control to the system.

Abstract: Cells migrate through crowded microenvironments within tissues during normal development, immune response, and cancer metastasis. Although migration through pores and tracks in the extracellular matrix (ECM) has been well studied, little is known about cellular traversal into confining cell-dense tissues. We find that embryonic tissue invasion by Drosophila macrophages requires division of an epithelial ectodermal cell at the site of entry. Dividing ectodermal cells disassemble ECM attachment formed by integrin-mediated focal adhesions next to mesodermal cells, allowing macrophages to move their nuclei ahead and invade between two immediately adjacent tissues. Invasion efficiency depends on division frequency, but reduction of adhesion strength allows macrophage entry independently of division. This work demonstrates that tissue dynamics can regulate cellular infiltration.

Abstract: Cells probe their microenvironment using membrane protrusion-retraction cycles. Spatiotemporal coordination of Rac1 and RhoA GTP-binding activities initiates and reinforces protrusions and retractions, but the control of their finite lifetime remains unclear. We examined the relations of Rac1 and RhoA GTP-binding levels to key protrusion and retraction events, as well as to cell-ECM traction forces at physiologically relevant ECM stiffness. High RhoA-GTP preceded retractions and Rac1-GTP elevation before protrusions. Notable temporal Rac1-GTP nadirs and peaks occurred at the maximal edge velocity of local membrane protrusions and retractions, respectively, followed by declined edge velocity. Moreover, altered local Rac1-GTP consistently preceded similarly altered traction force. Local optogenetic Rac1-GTP perturbations defined a function of Rac1 in restricting protrusions and retractions and in promoting local traction force. Together, we show that Rac1 plays a fundamental role in restricting the size and durability of protrusions and retractions, plausibly in part through controlling traction forces.

Abstract: Migrating cells present a variety of paths, from random to highly directional ones. While random movement can be explained by basal intrinsic activity, persistent movement requires stable polarization. Here, we quantitatively address emergence of persistent migration in (hTERT)-immortalizedRPE1 (retinal pigment epithelial) cells over long timescales. By live cell imaging and dynamic micropatterning, we demonstrate that the Nucleus-Golgi axis aligns with direction of migration leading to efficient cell movement. We show that polarized trafficking is directed toward protrusions with a 20-min delay, and that migration becomes random after disrupting internal cell organization. Eventually, we prove that localized optogenetic Cdc42 activation orients the Nucleus-Golgi axis. Our work suggests that polarized trafficking stabilizes the protrusive activity of the cell, while protrusive activity orients this polarity axis, leading to persistent cell migration. Using a minimal physical model, we show that this feedback is sufficient to recapitulate the quantitative properties of cell migration in the timescale of hours.

Abstract: Epithelial folding mediated by apical constriction serves as a fundamental mechanism to convert flat epithelial sheets into multilayered structures. It remains unknown whether additional mechanical inputs are required for apical constriction-mediated folding. Using Drosophila mesoderm invagination as a model, we identified an important role for the non-constricting, lateral mesodermal cells adjacent to the constriction domain ('flanking cells') in facilitating epithelial folding. We found that depletion of the basolateral determinant Dlg1 disrupts the transition between apical constriction and invagination without affecting the rate of apical constriction. Strikingly, the observed delay in invagination is associated with ineffective apical myosin contractions in the flanking cells that lead to overstretching of their apical domain. The defects in the flanking cells impede ventral-directed movement of the lateral ectoderm, suggesting reduced mechanical coupling between tissues. Specifically disrupting the flanking cells in wild-type embryos by laser ablation or optogenetic depletion of cortical actin is sufficient to delay the apical constriction-to-invagination transition. Our findings indicate that effective mesoderm invagination requires intact flanking cells and suggest a role for tissue-scale mechanical coupling during epithelial folding.

Abstract: Apical constriction driven by actin and non-muscle myosin II (actomyosin) provides a well-conserved mechanism to mediate epithelial folding. It remains unclear how contractile forces near the apical surface of a cell sheet drive out-of-the-plane bending of the sheet and whether myosin contractility is required throughout folding. By optogenetic-mediated acute inhibition of actomyosin, we find that during Drosophila mesoderm invagination, actomyosin contractility is critical to prevent tissue relaxation during the early, 'priming' stage of folding but is dispensable for the actual folding step after the tissue passes through a stereotyped transitional configuration. This binary response suggests that Drosophila mesoderm is mechanically bistable during gastrulation. Computer modeling analysis demonstrates that the binary tissue response to actomyosin inhibition can be recapitulated in the simulated epithelium that undergoes buckling-like deformation jointly mediated by apical constriction in the mesoderm and in-plane compression generated by apicobasal shrinkage of the surrounding ectoderm. Interestingly, comparison between wild-type and snail mutants that fail to specify the mesoderm demonstrates that the lateral ectoderm undergoes apicobasal shrinkage during gastrulation independently of mesoderm invagination. We propose that Drosophila mesoderm invagination is achieved through an interplay between local apical constriction and mechanical bistability of the epithelium that facilitates epithelial buckling.

Abstract: Cytoplasmic dynein, a major minus-end directed microtubule motor, plays essential roles in eukaryotic cells. Drosophila oocyte growth is mainly dependent on the contribution of cytoplasmic contents from the interconnected sister cells, nurse cells. We have previously shown that cytoplasmic dynein is required for Drosophila oocyte growth and assumed that it simply transports cargoes along microtubule tracks from nurse cells to the oocyte. Here, we report that instead of transporting individual cargoes along stationary microtubules into the oocyte, cortical dynein actively moves microtubules within nurse cells and from nurse cells to the oocyte via the cytoplasmic bridges, the ring canals. This robust microtubule movement is sufficient to drag even inert cytoplasmic particles through the ring canals to the oocyte. Furthermore, replacing dynein with a minus-end directed plant kinesin linked to the actin cortex is sufficient for transporting organelles and cytoplasm to the oocyte and driving its growth. These experiments show that cortical dynein performs bulk cytoplasmic transport by gliding microtubules along the cell cortex and through the ring canals to the oocyte. We propose that the dynein-driven microtubule flow could serve as a novel mode of fast cytoplasmic transport.

Abstract: Chromosome segregation is accomplished by the mitotic spindle, a bipolar micromachine built primarily from microtubules. Different microtubule populations contribute to spindle function: kinetochore microtubules attach and transmit forces to chromosomes, antiparallel interpolar microtubules support spindle structure, and astral microtubules connect spindle poles to the cell cortex.1,2 In mammalian cells, end-binding (EB) proteins associate with all growing microtubule plus ends throughout the cell cycle and serve as adaptors for diverse +TIPs that control microtubule dynamics and interactions with other intracellular structures.3 Because binding of many +TIPs to EB1 and thus microtubule-end association is switched off by mitotic phosphorylation,4-6 the mitotic function of EBs remains poorly understood. To analyze how EB1 and associated +TIPs on different spindle microtubule populations contribute to mitotic spindle dynamics, we use a light-sensitive EB1 variant, π-EB1, that allows local, acute, and reversible inactivation of +TIP association with growing microtubule ends in live cells.7 We find that acute π-EB1 photoinactivation results in rapid and reversible metaphase spindle shortening and transient relaxation of tension across the central spindle. However, in contrast to interphase, π-EB1 photoinactivation does not inhibit microtubule growth in metaphase but instead increases astral microtubule length and number. Yet in the absence of EB1 activity, astral microtubules fail to engage the cortical dynein/dynactin machinery, and spindle poles move away from regions of π-EB1 photoinactivation. In conclusion, our optogenetic approach reveals mitotic EB1 functions that remain hidden in genetic experiments, likely due to compensatory molecular systems regulating vertebrate spindle dynamics.

Abstract: Secretory trafficking from the endoplasmic reticulum (ER) is subject to regulation by extrinsic and intrinsic factors. While much of the focus has been on biochemical triggers, little is known whether and how the ER is subject to regulation by mechanical signals. Here, we show that COPII-dependent ER-export is regulated by mechanical strain. Mechanotransduction to the ER was mediated via a previously unappreciated ER-localized pool of the small GTPase Rac1. Mechanistically, we show that Rac1 interacts with the small GTPase Sar1 to drive budding of COPII carriers and stimulate ER-to-Golgi transport. Altogether, we establish an unprecedented link between mechanical strain and export from the ER.

Abstract: Actomyosin contractility generated cooperatively by nonmuscle myosin II and actin filaments plays essential roles in a wide range of biological processes, such as cell motility, cytokinesis, and tissue morphogenesis. However, subcellular dynamics of actomyosin contractility underlying such processes remains elusive. Here, we demonstrate an optogenetic method to induce relaxation of actomyosin contractility at the subcellular level. The system, named OptoMYPT, combines a protein phosphatase 1c (PP1c)-binding domain of MYPT1 with an optogenetic dimerizer, so that it allows light-dependent recruitment of endogenous PP1c to the plasma membrane. Blue-light illumination is sufficient to induce dephosphorylation of myosin regulatory light chains and a decrease in actomyosin contractile force in mammalian cells and Xenopus embryos. The OptoMYPT system is further employed to understand the mechanics of actomyosin-based cortical tension and contractile ring tension during cytokinesis. We find that the relaxation of cortical tension at both poles by OptoMYPT accelerated the furrow ingression rate, revealing that the cortical tension substantially antagonizes constriction of the cleavage furrow. Based on these results, the OptoMYPT system provides opportunities to understand cellular and tissue mechanics.

Abstract: Advances in protein design have brought us within reach of developing a nanoscale programming language, in which molecules serve as operands and their conformational states function as logic gates with precise input and output behaviors. Combining these nanoscale computing agents into larger molecules and molecular complexes will allow us to write and execute "code". Here, in an important step toward this goal, we report an engineered, single protein design that is allosterically regulated to function as a 'two-input logic OR gate'. Our system is based on chemo- and optogenetic regulation of focal adhesion kinase. In the engineered FAK, all of FAK domain architecture is retained and key intramolecular interactions between the kinase and the FERM domains are externally controlled through a rapamycin-inducible uniRapR module in the kinase domain and a light-inducible LOV2 module in the FERM domain. Orthogonal regulation of protein function was possible using the chemo- and optogenetic switches. We demonstrate that dynamic FAK activation profoundly increased cell multiaxial complexity in the fibrous extracellular matrix microenvironment and decreased cell motility. This work provides proof-of-principle for fine multimodal control of protein function and paves the way for construction of complex nanoscale computing agents.