Curated Optogenetic Publication Database

Abstract: The small GTPase Rac1 controls cell protrusion for a wide variety of critical cell functions. Its regulation by upstream guanine exchange factors (GEFs) has been the focus of multiple studies, but regulation by the GTPase RhoG remains poorly understood. RhoG is known to activate the ELMO/DOCK180 GEF complex, which in turn interacts with Rac1. It is unclear which aspects of protrusion are controlled by RhoG, and which of RhoG’s effects on protrusion are mediated by Rac1. To address these questions, we developed biosensors and optogenetic tools to activate one GTPase while observing another, and to simultaneously visualize the activity of two GTPases. New tools included a photoactivable RhoG, a RhoG biosensor, and red shifted biosensors of RhoG and Rac1. RhoG and Rac1 activation events in protrusions were spatio-temporally correlated with one another and with protrusion velocity. Causal inference indicated that RhoG indeed unidirectionally activated Rac1. Photoactivation of RhoG and Rac1 indicated that specific aspects of protrusion behavior were controlled by RhoG, and only some via Rac1. Further dissection of RhoG to Rac1 signaling through simultaneous GTPase activation and biosensor visualization showed that PA-RhoG activates Rac1 predominantly through DOCK180 and that PA-RhoG can activate Cdc42 independently of Rac1.

Abstract: Methylotrophic yeasts such as Pichia pastoris are widely used for heterologous protein production because they contain strong and tightly regulated promoters. However, the use of methanol as an inducer presents several practical challenges, including toxicity, flammability, high oxygen demand during fermentation, and increased production costs. To overcome these limitations, researchers have been working on redesigning the AOX1 regulatory system and developing alternative induction strategies that do not rely on methanol. One promising approach is optogenetics, which uses light to control gene expression in a non-invasive way. These systems rely on light-sensitive proteins such as phytochromes, cryptochromes, LOV-domain proteins, and UVR8, allowing gene activity to be regulated in a precise and reversible manner without adding chemical inducers to the culture medium. This review brings together key advances in yeast optogenetics, with a focus on the EL222 system, highlighting its implementation for light-controlled heterologous protein production in P. pastoris and its broad application in synthetic biology and metabolic engineering in Saccharomyces cerevisiae. The growing versatility and scalability of EL222-based circuits highlight their potential to reshape both fundamental research and industrial bioprocessing through safer, more controllable, and energy-efficient gene regulation strategies.

Abstract: Cre recombination is a widely used technique for mechanistic insights in physiology and disease. However, available constitutive and inducible Cre systems present challenges that can be prohibitive for some study designs. For example, Cre expression can result in cell types targeted across numerous tissues and organs, or when a gene is expressed across multiple cell types in a tissue, Cre-Lox restricted knockout will not enable ablation across an entire tissue or organ. Photoactivatable Cre (PA-Cre) systems enable temporally and spatially restricted gene expression control in delimited anatomical regions, typically requiring a micro-LED or fibre optic implantation. Here, we report as proof-of-concept the effective knockout of BDNF in subcutaneous adipose tissue after PA-Cre activation through external blue light illumination in awake, freely moving mice. We demonstrated that for mice with black fur, shaving can be used to anatomically limit PA-Cre activation. BDNF protein expression was decreased by 87% in the inguinal scWAT after blue light exposure, with no effect observed in the perigonadal (deep) or axillary subcutaneous (non-shaved) adipose tissues. We propose blue light induction of PA-Cre as safe and effective to study adipose tissue physiology and pathology across models. Considerations for applying this tool to future studies are also presented.

Abstract: Cells in many naturally occurring organisms routinely cooperate to control their extracellular pH in a dynamic and reversible manner, but this capability has been underexplored in synthetic biology. Here, we sought to engineer a microbial system that switches between two states -high and low extracellular pH- with minimal human intervention. We accomplished this by combining: (1) a genetic circuit that produces recombinant urease under the control of a light-inducible promoter; (2) a degradation tag on urease to accelerate the high-to-low pH transition; and (3) optimization of several environmental factors, including media composition, replenishment rate, and light exposure patterns. The system raises the pH when urease is produced and hydrolyzes urea in the media to produce ammonia; it lowers the pH as a byproduct of the cell's native metabolism when urease production ceases. We demonstrate that the optimized system cycles continuously for up to 14 days with minimal performance loss. Overall, our system demonstrates synthetic pH control in an engineered living system and highlights challenges and potential solutions for using such systems outside of the context of typical laboratory manipulation.

Abstract: Here, we present extrabody, an activatable, modular antibody platform that enables optogenetic or chemical reassembly of split antibody fragments for inducible extracellular antigen recognition. We demonstrate compatibility across diverse targets, including GFP, mCherry, and the tumor-associated antigens EGFR and HER2, and show that both nanobody- and scFv-derived fragments support light-dependent reconstitution. Extrabody enables input-gated cell-cell interactions and antigen transfer, providing external control over intercellular communication. Integration with synNotch receptors and chimeric antigen receptors (CARs) further allows dual-input regulation of downstream responses, including gene expression, cytokine release, and cytotoxicity. Together, these results establish extrabody as a versatile and generalizable interface for externally controlled cellular communication and synthetic signaling.

Abstract: Optogenetic tools-light-responsive proteins that enable to regulate specific cellular activities, study biological processes, and develop new therapies-are attractive approaches for achieving endogenous gene regulation under minimally invasive conditions. Our first step in constructing an optogenetic system to regulate endogenous Drosophila gene expression was to identify inhibitory anti-CRISPR (Acr) proteins that block CRISPRa-mediated activation. Next, we inserted optogenetic protein LOV2 into these Acrs, tested for their ability to optogenetically modulate endogenous gene upregulation through the CRISPRa-based flySAM system in Drosophila, and found that the photoswitchability of these prototypes was weak. We therefore engineered an optimized Acr-LOV2 fusion module by refining length of intrinsically disordered and ordered regions (IDR and IOR) of Acrs. This optimization yielded a variant with significantly greater sensitivity to blue-light-induced endogenous gene upregulation than the prototypes, leading to new in vivo discoveries. In addition, this work provides insights for in vivo functional characterization of the IDR and the IOR of these small-sized proteins. Together, these findings establish a robust optogenetic toolbox for precise, light-controlled endogenous gene regulation in Drosophila.

Abstract: In developmental biology, cellular events must be orchestrated at precise times and locations in the embryo. Many classic discoveries were achieved by perturbing developmental organization using approaches ranging from tissue transplantation to local, acute heat shock. A growing suite of optogenetic tools is now available with fine spatiotemporal control, opening the door to perturbation experiments with unprecedented precision. Here we highlight these tools, review their application in developmental contexts, and discuss their current challenges and future promise.

Abstract: The nuclear pore complex regulates nucleocytoplasmic transport. It was recently shown that the global mechanical stability of proteins regulates their nuclear import rate. On the basis of these findings, we hypothesize that the main principles governing protein translocation through narrow biological pores-in which locally unstructured and unfolded regions determine cargo orientation and translocation kinetics-can help rationalize protein trafficking across the nuclear pore complex. Inspired by single-molecule studies showing that proteins exhibit different mechanical stability when pulled from different termini, here we show that the rate of both nuclear import and export is enhanced when the translocating protein is threaded through the nuclear pore from the specific region exhibiting lower local nanomechanical stability and increased structural disorder. We demonstrate this for a range of model proteins with different folds and stabilities by combining single-molecule magnetic tweezers with single-cell optogenetic experiments, complemented by steered molecular dynamics simulations and biochemical binding assays. Our bioinformatics survey then shows that in human transcription factors, the termini containing the nuclear localization signal sequence exhibit a higher degree of structural disorder. We propose that protein orientation might offer an additional layer of structural and mechanical control of the kinetics of nuclear transport.

Abstract: Protein-protein interactions (PPIs) and spatially restricted molecular contacts govern cellular function, yet many are poorly captured by classical biochemical approaches that rely on cell lysis or stable complex isolation. Proximity labeling (PL) technologies have transformed interactome analysis by enabling covalent tagging of biomolecular neighborhoods directly within intact cells, tissues, and living organisms. By generating short-lived reactive species, PL provides spatially and temporally resolved snapshots of molecular organization under native conditions. Recent advances across enzymatic, chemical, and photocatalytic PL platforms have expanded control over labeling radius, kinetics, and activation, while reducing background and enabling microenvironment-specific targeting. Hybrid genetic-chemical and optogenetic strategies further extend PL beyond mapping toward proximity-based signal amplification and functional interrogation. This review focuses on the most significant methodological and conceptual advances in proximity labeling reported over the past two years, highlighting how these developments have enabled discovery of previously inaccessible interaction networks, including membrane assemblies, chromatin complexes, and in vivo protein microenvironments. We conclude by outlining key challenges and future opportunities for proximity labeling in interactome mapping and amplification.

Abstract: Tumor recurrence, metastasis, and therapeutic resistance remain major challenges in oncology, driving the need for advanced therapeutic strategies with improved precision and controllability. Optogenetics, which enables light-mediated regulation of cellular functions, has emerged as a promising modality for cancer therapy by offering unparalleled spatiotemporal precision. This capability allows dynamic control of intracellular signaling and transgene expression, enabling selective targeting of malignant cells while minimizing damage to surrounding tissues. However, clinical translation is hindered by key challenges, including inefficient in vivo delivery of optogenetic components, limited tissue penetration of activating light, and suboptimal performance of existing tools. Addressing these barriers requires a convergence of molecular engineering and materials science, wherein advanced biomaterials play a critical role in enabling gene delivery and overcoming tissue-penetration limitations in complex tumor environments. In this review, we provide a comprehensive oriented overview of optogenetics in oncology. We first analyze the molecular mechanisms and engineering principles of representative optogenetic tools, with a focus on LOV- and CRY2-based systems. We then highlight recent advances in biomaterial-assisted optogene delivery and light delivery strategies, emphasizing their material-dependent mechanisms that enable precise spatiotemporal control in vivo. Furthermore, we summarize emerging preclinical applications in cancer immunotherapy, gene regulation, and intracellular signaling control. Finally, we discuss key challenges in biosafety, kinetic optimization, and clinical scalability, and outline future directions that integrate optogenetics with functional materials and intelligent design to realize clinically viable platforms. This review aims to provide a framework for the development of clinically viable optogenetic platforms for next-generation cancer therapy.

Abstract: The spatiotemporal dynamics and density of actin networks are key determinants of actin cytoskeleton-mediated cellular functions. In vitro reconstitution systems have been widely used to study actin cytoskeletal dynamics; however, many existing approaches offer limited flexibility in controlling the geometry, thickness, and density of the assembled actin networks. Here, we present an in vitro optogenetic protocol that enables precise control of actin network assembly on supported lipid bilayers using an improved light-induced dimer (iLID)-SspB-based light-inducible dimerization system. In this system, His-mEGFP-iLID is anchored to a Ni-NTA-containing lipid bilayer, while SspB-mScarlet-I-VCA, a nucleation-promoting factor fused with SspB, together with other actin cytoskeletal proteins, is supplied in bulk solution. Upon blue light illumination, SspB-mScarlet-I-VCA is recruited to the membrane in a spatially and temporally defined manner, inducing localized actin polymerization. By tuning illumination patterns and duration, actin networks with defined density, thickness, and geometry can be generated, and polymerization can be rapidly halted by stopping illumination. This protocol provides a versatile platform for reconstructing actin networks with controlled spatial organization and density, enabling quantitative analysis of density-dependent interactions between actin networks and actin-binding proteins. Key features • Actin networks with varying densities and arbitrary shapes can be formed on the same supported lipid bilayer by controlling blue light illumination through the objective lens. • Actin polymerization can be stopped simply by turning off blue light illumination, enabling the formation of actin networks with defined thicknesses. • This protocol requires purified actin and actin-binding proteins.

Abstract: Precise temporal control of gene expression is a prerequisite for disentangling timing-specific effects of gene function within the life cycle of Drosophila melanogaster. Here, we implement light-inducible FLPase reconstitution (LIFR) as a conditional gene expression system in flies, which combines blue light-responsive Magnet photoswitches and split-FLPase to remove an FRT-flanked stop cassette and irreversibly switch on transgene expression in response to light. This system is highly efficient, has virtually no transgene leakage, and a single light pulse is sufficient to induce long-term transgene expression. We demonstrate that LIFR in adulthood overcomes the developmental lethality elicited by constitutive pan-neuronal overexpression of neurodegeneration-causing mutants TDP43G298S and HTTQ97. We also illustrate that LIFR can help trace specific cell-type fates across developmental stages. Thus, we demonstrate proof of principle that LIFR is a versatile platform to conditionally activate long-lasting gene expression without the side effects of existing systems, thereby extending the Drosophila melanogaster genetic toolbox.

Abstract: Optogenetic tools have revolutionized the control of gene expression with high spatial and temporal resolution. Here we present a Single-chain Light-Activatable Transcriptional Reporter (SLATR), a system capable of fluorescently tagging target cells with minutes of white light stimulation. In its inactive, or dark state, a transcriptional factor is cytosolically bound, preventing nuclear translocation. White light irradiation triggers its release through the protease cleavage of a site that is sterically caged by the circularly permuted Avena sativa LOV2 (cpAsLOV2) domain. We discovered that cpAsLOV2 cages the cleavage site more efficiently than AsLOV2, achieving low background in the SLATR design. We demonstrate that SLATR exhibits a signal-to-background ratio between 3.4 and 36 and achieves reporter activation within 60 min of light stimulation. Furthermore, SLATR outperforms the only other single-chain light-activatable transcriptional reporter, LAUNCHER, with faster kinetics, greater light sensitivity, and markedly lower background under identical stimulation conditions. Our single-chain light-activatable transcriptional system expands the optogenetic toolkit though providing a simpler system for regulating gene expression with precise spatiotemporal control.

Abstract: Allostery, the transmission of locally induced conformational changes to distant functional sites, is a key mechanism for protein regulation. Artificial allosteric effectors enable remote manipulation of cell function; their engineering, however, is hampered by our limited understanding of allosteric residue networks. Here, we introduce a phage-assisted evolution platform for in vivo optimization of allosteric proteins. It applies opposing selection pressures to enhance activity and switchability of phage-encoded effectors and leverages retron-based recombineering to broadly explore fitness landscapes, introducing point mutations, insertions, and deletions. Applying this framework to the transcription factor AraC yielded near-binary optogenetic switches, with light-controlled activity spanning ~1000-fold dynamic range. Long-read sequencing across selection cycles enabled high-resolution tracking of evolving variant pools, revealing adaptive trajectories and context-dependent residue interactions. Mechanistically, we find that linker mutations promoting α-helix extension at the sensor-effector junction enhance conformational coupling between LOV2 and AraC. These variants emerge consistently across independently evolved pools, underscoring their functional relevance. Together, we develop a framework for the directed evolution of programmable allosteric switches in vivo. By coupling dynamic selection with deep mutational scanning and temporal sequencing, it enables both functional optimization and mechanistic insight into allosteric networks.

Abstract: Store-operated Ca2+ release-activated Ca2+ (CRAC) channels, composed of STIM and ORAI, are essential for immune and developmental processes, and their dysregulation underlies channelopathies such as Stormorken syndrome. Here, we report the engineering of genetically encoded CRAC channel inhibitory binders (CRABs) derived from the ORAI C-terminal tail. Guided by deep mutational scanning, we optimize a membrane-anchored CRAB variant that potently inhibits Ca2+ influx and NFAT signaling, and rescues thrombocytopenia-like phenotypes in a zebrafish model of Stormorken syndrome. To enable tunable inhibition, we further design oligomeric, optogenetic (Opto-CRAB), and chemogenetic (Chemo-CRAB) variants, providing graded and real-time control of CRAC activity. Chemo-CRAB further suppresses Ca2+ signaling downstream of RTKs, GPCRs, and CAR-T cell activation, establishing broad applicability across physiological and synthetic contexts. Together, these programmable peptide-based inhibitors provide a versatile platform to dissect SOCE dynamics and hold promise as a therapeutic strategy against autoimmune, inflammatory, and neoplastic disorders driven by CRAC channel hyperactivity.

Abstract: Emerging evidence suggests that Guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) bind to the cytoskeleton or focal adhesions (FAs), controlling spatio-temporal Rho GTPase activity through feedback mechanisms. We explore such feedback in the Rho-specific GAP Deleted in Liver Cancer 1 (DLC1), which binds to FAs through mechanosensitive interactions. Using a FRET biosensor, we show that DLC1 loss of function leads to globally increased Rho activity and contractility in fibroblasts. Although Rho activity appears macroscopically steady, individual molecules undergo 'signaling flux'-a dynamic cycle of activation and deactivation. To measure this flux, we built a genetic circuit that enables both optogenetic activation of Rho and simultaneous readout of Rho activity. In cells at mechanical steady state, this reveals that DLC1 globally controls the rate of Rho deactivation, both at FAs and at the plasma membrane. Transient induction of local contractility, however, shows DLC1 associating with and dissociating from FAs during their reinforcement and relaxation, which might provide local positive feedback on Rho activity for robust FA disassembly. Together, our results indicate that DLC1 regulates Rho activity both globally at steady state and locally at FAs under tension, highlighting the complexity of spatio-temporal Rho GTPase signaling.

Abstract: In this study, we describe a rationally designed light-inducible RNA-releasing protein (LIRP) capable of inhibiting mRNA translation in the dark while permitting gene expression upon exposure to blue or ambient light. This LIRP-dependent gene switch is compatible with various delivery routes of gene- and cell-based therapy, such as subcutaneous implantation of microencapsulated light-sensitive cells or expression in various light-accessible body sites using single adeno-associated virus (AAV) vectors. To exemplify a gene therapy approach that directly harnesses ambient light as a natural illumination source to induce therapeutic action, we show how intradermal delivery of AAV2 vectors carrying a LIRP-regulated gene switch controlling murine thymic stromal lymphopoietin expression was effective in enabling light-dependent prevention and treatment of diet-induced obesity. To describe another therapeutic scenario, we engineered AAV2 vectors for LIRP-dependent expression of Vascular endothelial growth factor (VEGF) inhibitors for the treatment of retinal neovascular diseases. Upon intravitreal delivery into mice suffering from wet macular degeneration, VEGF inhibitors were constantly produced when animals were exposed to daylight, but therapeutic actions could be flexibly interrupted either by exposure to dark environments or by administration of a selective blue light filter at any point in time. When compared to conventional treatment strategies based on constitutive VEGF inhibition over the course of 3 months, we show that a regulated gene therapy approach through LIRP-dependent optogenetics was advantageous in maintaining a normal retina thickness. This work not only provides a valuable addition to the optogenetic toolbox but also offers a perspective to translate light-dependent gene switches toward therapeutic usage.

Abstract: Transcription factor nucleocytoplasmic dynamics play a key role in developmental gene regulation. In Dictyostelium , the transcription factor GtaC exhibits nucleocytoplasmic shuttling, but its shuttling trajectory across multicellular aggregation has not been systematically quantified. Using a knock-in strain, we tracked GtaC dynamics from starvation through aggregation and quantified developmental changes in shuttling period, amplitude, and synchrony. Notably, brief-pulse optogenetic activation of cAMP at higher input frequencies reproduced the reported attenuation of GtaC shuttling amplitude with high temporal precision and minimal phototoxicity. Together, long-term quantification and brief-pulse optogenetic cAMP perturbation show that GtaC nucleocytoplasmic shuttling is developmentally tuned in a frequency-dependent manner.

Abstract: Reprogramming tumor-associated macrophages (TAMs) from the pro-tumoral M2-like state to the immunostimulatory M1-like phenotype has emerged as a promising strategy for tumor therapy. However, most M2-like TAMs are preferentially located in hypoxic regions of the tumor, which are poorly accessible to many advanced drug delivery systems, posing a significant challenge to effective TAM reprogramming. Here, leveraging the tropism of facultative anaerobic bacteria to localize and propagate in the hypoxic tumor, an optogenetically engineered Escherichia coli Nissle 1917 strain conjugated with murine STING agonist (EcNflaB@UPD) was developed for cancer-specific immunotherapy. Upon near-infrared light illumination, the blue and UV emissions from upconversion nanoparticles (UCNPs) simultaneously activate the expression of Toll-like receptor (TLR) agonist, flaB, from EcNflaB, and the release of photocaged murine STING agonist, DMXAA, respectively. This spatiotemporally synchronized dual release ensures co-localized STING and TLR5 agonists inside the hypoxic niche, repolarizing TAMs from the M2 to the M1 phenotype via synergistic TLR5-MAPK1-NF-κB and STING-NF-κB signaling. The polarization of TAMs enhances their antigen-presenting capacity and, more importantly, activates the cytotoxic, stem-like and memory CD8+ T cells responses. This subsequently inhibits tumor growth, relapse, and metastasis in the murine 4T1 tumor model. Collectively, our work introduces the bacteria-based system that uses near-infrared light to dual-release immunotherapeutics for systemic anti-tumor immunity, opening new avenues for precise and effective cancer immunotherapy.

Abstract: Directed migration and rapid process extension-retraction allow microglia to continuously survey the brain and efficiently identify and phagocytose apoptotic neurons. Defining how cytoskeletal regulators coordinate these behaviors could guide strategies for targeted modulation of microglial activity. To this aim, using in vivo imaging in zebrafish, we identified a mechanistic framework in which myosin II-dependent contractility governs transitions between surveillance, migratory, and phagocytic states. Building on this, we engineered an optogenetic RhoA actuator (opto-ArhGEF25) that enables reversible, spatiotemporally precise manipulation of microglial behavior in vivo. We show that patterned RhoA activation can modulate process dynamics and induce front-rear polarity that drives rapid repulsive migration away from the applied light source, overriding injury-evoked cues and preventing migration toward lesions. Together, these results establish optogenetic control of the cytoskeleton as a powerful approach to probe and ultimately modulate microglial function in the living brain.

Abstract: The cytoskeleton is a dynamic intracellular network that governs cell shape, migration, division, and mechanotransduction. Precise spatiotemporal control of cytoskeletal regulation is essential for understanding how these processes are coordinated in physiology and disease, yet conventional pharmacological and genetic approaches often lack sufficient resolution or reversibility. Optogenetic technologies provide a powerful alternative by enabling light-controlled, noninvasive manipulation of cytoskeletal regulators with high temporal precision and subcellular specificity. This review summarizes recent advances in genetically encoded optogenetic tools for interrogating cytoskeletal dynamics. We discuss core design strategies, including allosteric regulation, light-induced oligomerization, heterodimerization, and dissociation, and highlight representative applications targeting actin filaments, microtubules, and upstream signaling pathways such as Rho family GTPases. We conclude by outlining current limitations and emerging directions, including improved tissue penetration, reduced phototoxicity, and multiplexed optical control, which are expected to further expand the utility of optogenetics in cytoskeleton research.

Abstract: The regulation of the actin cytoskeleton is key to controlling cell shape and structure. While the Rho GTPase RhoA is well known to regulate the actomyosin cytoskeleton, its function in controlling the septin cytoskeleton remains unclear. As RhoA interactions can vary in both time and space, they can be challenging to discern from traditional bulk biochemical assays. Here, we use multiple optogenetic tools to spatially and temporally increase myosin localization, stimulate contractile force, and activate RhoA to investigate how RhoA and its downstream effector myosin impact the septin cytoskeleton. We find that neither local accumulation of myosin nor increased activity of myosin is sufficient to alter septin architecture. Local activation of RhoA, however, results in a local increase in septin accumulation. Importantly, this septin increase is independent of the scaffolding protein anillin, which can directly bind both septin and RhoA. Together, these data expand the potential role of septins in mediating RhoA signaling by stimulating the remodeling of the septin cytoskeleton.

Abstract: Microtubule acetylation is implicated in regulating cell motility, yet its physiological role in directional migration and the underlying molecular mechanisms have remained unclear. This knowledge gap has persisted primarily due to a lack of tools capable of rapidly manipulating microtubule acetylation in actively migrating cells. To overcome this limitation and elucidate the causal relationship between microtubule acetylation and cell migration, we developed a novel optogenetic actuator, optoTAT, which enables precise induction of microtubule acetylation within minutes in live cells. Implementing optoTAT in migration assays, we observed striking and rapid responses at both molecular and cellular levels. First, microtubule acetylation triggers release of the RhoA activator GEF-H1 from sequestration on microtubules. This release subsequently enhances actomyosin contractility and drives focal adhesion maturation. These subcellular processes collectively promote sustained directional migration. Our findings position GEF-H1 as a critical molecular responder to microtubule acetylation, enabling a dynamic crosstalk between the actin and microtubule cytoskeletal networks in the coordination of cellular motility.

Abstract: Photosensory protein domains, derived from nature, are foundational for optogenetic protein engineering. Tailoring their properties enables their full exploitation for optogenetic regulation in basic research and applied bioengineering applications. Here, we present a simple, yet powerful strategy based on random mutagenesis coupled to high-throughput screening that allowed altering the most fundamental properties of the widely used nMag/pMag photodimerization system: its light sensitivity and activation. Variants were characterized in vivo in bacteria by flow cytometry and during the entire growth curve by spectrofluorometry. We identify mutations that either increase or decrease the light sensitivity at sub-saturating light intensities, while also improving the light activation and dark-to-light fold change. Notably, light sensitivity and activation levels could be changed independently. In addition, we demonstrated that the shapes of the dose-response curves can be finely tuned. This broadens the applicability of the Magnets photosensors for optogenetic regulation strategies.

Abstract: Motile cells can sense and exert forces on the extracellular environment through dynamic actin networks. Increased stress against the polymerizing barbed ends of branched actin networks has been shown to lead to an increase in the density of these networks through a force feedback mechanism, though this phenomenon has not been explored through the examination of real-time responses of endogenous actin networks in cells. Here, we utilize mouse embryonic fibroblast CRISPR knock-in lines with labeled ARP2/3 complex to identify cellular and extracellular conditions that regulate branched actin density and enrichment at the leading edge of lamellipodial protrusions. A common theme shared among all branched actin density-increasing conditions is higher levels of interface stress between the plasma membrane and the barbed ends of the lamellipodial actin network. Among these conditions, we find that ARP2/3 is specifically required for robust spreading and protrusion in response to increased extracellular viscosity. Interestingly, time-lapse traction force microscopy of ARP2/3-dependent viscosity responses show significantly reduced changes in strain energy applied to the substrate when compared to spreading and motility through cell-matrix adhesion. In addition, we find that increased extracellular viscosity can bypass the need for extracellular matrix proteins to support lamellipodial protrusion driven by optogenetic Rac activation. Our studies provide strong support for in vitro models of branched actin force feedback responses and further characterize an essential role for branched actin in mediating dramatic cell shape changes in response to increased extracellular viscosity.