Curated Optogenetic Publication Database

Abstract: In migrating cells, the GTPase Rac organizes a protrusive front, whereas Rho organizes a contractile back. How these GTPases are positioned at opposite poles remains unclear. We leverage optogenetics, mechanical perturbations, and mathematical modelling to reveal a surprising mechanochemical long-range mutual activation between front and back polarity programmes that complements their well-known local mutual inhibition. Rac-based protrusions elevate membrane tension, stimulating an mTORC2-dependent activation of Rho at the opposite side of the cell. Conversely, Rho-mediated contractility induces cortical-flow-based regulation of phosphoinositide signalling that triggers Rac activation distally. We develop a minimal mechanochemical model to explain how long-range facilitation, together with local inhibition, enables robust Rho and Rac partitioning. Our findings demonstrate how the actin cortex and plasma membrane interact as an integrated mechanochemical system for long-range Rac-Rho patterning. This circuit is required for efficient polarity and migration in primary human T cells and is conserved in epithelial cells, highlighting the generality of this mechanism.

Abstract: In canonical developmental patterning, the embryo is exposed to gradients of signaling activators that elicit different cellular responses depending on the activator's concentration. Recent optogenetic studies of terminal ERK signaling downstream of Torso receptor tyrosine kinase in the early Drosophila embryo reveal that even a brief, 5-minute ERK stimulus is sufficient to rescue the development of larval "tail" structures. Here, we reveal components of the molecular network that defines this sensitive developmental fate response. We find that low ERK doses produce sustained Abdominal-B ( Abd-B ) expression comparable to that of wild-type embryos. Abd-B expression is adjacent to, but non-overlapping with, two other transcriptional repressors: the ERK effector Tailless (Tll) and the gap gene Giant (Gt). Analysis of gene expression patterns in response to optogenetic perturbations suggests that the Tll-dependent repression of gt constitutes the sensitive ERK-responsive step: even low tll expression leads to potent repression of gt in nearby regions, with Abd-B expression arising in a stripe between the tll and gt domains. Our work suggests that the spectrum of phenotypes produced through optogenetic manipulation can be used to define how robust patterning can arise from low doses of inductive signals.

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: 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: 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.

Abstract: Endoplasmic reticulum-plasma membrane (ER-PM) contact sites are essential signaling hubs that regulate lipid transport, calcium homeostasis, and spatially organized signal transduction. Emerging evidence indicates that not only the presence but also the dynamics, stability, and geometry of ER-PM contacts critically shape cellular functions; however, tools that enable simultaneous high-fidelity visualization and reversible, quantitative control of these contacts in living cells remain limited. Here, we introduce a modular toolkit for inducible ER-PM contact-site reconstitution based on complementary chemical and optical dimerization strategies. We develop a nontoxic and reversible abscisic acid (ABA)-inducible system using the plant-derived ABIcs/PYLcs pair, and a rapidly reversible optogenetic system based on the iLID/SspB module, both of which allow robust visualization and dose-dependent control over contact-site formation kinetics, increasing contact-site density and total area fraction per cell without altering the size of individual contacts. In contrast, systematic variation of rigid α-helical linker length or inducible tether abundance selectively tunes the lateral growth, stability, and lifetime of individual contact sites, without changing their density. By combining these two orthogonal strategies, we achieve independent control of both individual contact-site size and overall contact-site density, providing complementary mechanisms to adjust total contact area per cell. This versatile platform enables quantitative dissection of ER-PM contact site structure-function relationships and offers broad utility in studies of lipid exchange, calcium signaling, membrane repair, metabolic regulation, and disease-relevant dysregulation.

Abstract: Notch signalling is a critical regulator of multiple developmental processes through its ability to control gene expression and thereby influence cell fate specification and cell proliferation through direct cell-cell communication. Although Notch signalling has been implicated in myogenesis during late embryogenesis, its role in early mesoderm development has been largely unexplored. Endocytosis of the Notch ligand Delta and the Notch receptor extracellular domain, a critical step in Notch pathway activation, has been extensively observed in the ventral mesoderm of the early Drosophila embryo, indicating a potential for Notch signalling activity in this early germ layer. Here, we present evidence that genes critical to mesoderm development require and are responsive to Notch signalling activity. Using a novel light-inducible Optogenetic variant of the Notch intracellular domain (OptoNotch), which affords precise spatial and temporal control over ectopic activation of Notch signalling, in combination with high-resolution fluorescent RNA in situ hybridization and qPCR, we identified a set of mesodermal genes whose expression is directly regulated by Notch signalling. We also provide evidence that Notch signalling indirectly regulates the dorsal-ventral patterning program mediated by the Toll signalling pathway through the Dorsal/Twist/Snail gene network. Our findings demonstrate that Notch signalling regulates ventral mesoderm patterning and is critical for establishing the mesoderm-mesectoderm-ectoderm boundary by regulating gene expression patterns and providing negative feedback on the upstream patterning network.

Abstract: Cancer is known to be a disease of altered cellular signaling; however, the relationship between mutation-specific changes to signal transduction and the phenotypic consequences produced remains poorly understood. Here, we investigate two common breast cancer driver mutations, the PIK3CAH1047R mutation and the ErbB2 amplification, both of which activate the PI3K-Akt pathway but paradoxically drive distinct cellular outcomes. Indeed, in nontransformed mammary epithelial cells, PI3KH1047R expression induced features of epithelial-mesenchymal transition (EMT), while ErbB2amp cells exhibited a hyperproliferative phenotype. Characterization of PI3K axis signaling revealed that ErbB2amp cells display prolonged, stimulus-dependent PI3K activation, whereas PI3KH1047R cells show constitutive, ligand-independent signaling. To test whether these distinct dynamics contribute to the phenotypic responses, we employed an iLID-based optogenetic system that enables precise, tunable control of endogenous PI3K activity. Using this tool to mimic the mutation-specific dynamics in MCF10A mammary epithelial cells, we found that PI3K signaling patterns alone were sufficient to reproduce key features of the PIK3CA H1047R-associated EMT phenotype but not the ErbB2-associated proliferative phenotype. These findings suggest that the temporal encoding of pathway activity, not merely its magnitude, can drive some phenotypic changes in oncogenic progression, explain how distinct mutations within a common signaling pathway can produce divergent cellular phenotypes, and provide a workflow for interrogating the functional consequences of changes in signaling dynamics.

Abstract: RNA-binding proteins (RBPs) with prion-like domains (PrLDs), such as FUS and TDP-43, condense into functional liquids, which can transform into pathological fibrils that underpin fatal neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD). Here, we define short RNAs that prevent FUS fibrillization by promoting liquid phases and distinct short RNAs that prevent and reverse FUS condensation and fibrillization. These activities require interactions with multiple RNA-binding domains of FUS and are encoded by RNA sequence, length, and structure. We define a short RNA that dissolves cytoplasmic FUS aggregates, restores nuclear FUS, and mitigates FUS toxicity in optogenetic models and ALS patient-derived motor neurons. Another short RNA dissolves cytoplasmic TDP-43 aggregates, restores nuclear TDP-43, and mitigates TDP-43 toxicity. Since short RNAs can be effectively delivered to the human brain, these oligonucleotides could have utility for ALS/FTD and related disorders.

Abstract: CRISPR/Cas9 is a powerful tool for targeted genome editing experiments. Using CRISPR/Cas9, genes can be deleted or modified by inserting specific DNA sequences, encoding for fluorescent proteins, small peptide tags, or other modifications. Such experiments are essential for detailed gene and protein characterization. However, designing and cloning the corresponding constructs can be repetitive, time-consuming, and laborious. To assist users in CRISPR/Cas9-based genome engineering, we developed CrisprBuildr, an open-source, web-based application for designing modifications to their target genes. CrisprBuildr guides users through creating guide RNAs and repair template vectors to generate cloning maps. The application is designed for the Drosophila melanogaster genome but can serve as a template for other available genomes. We also created new tagging vectors using EGFP and mCherry combined with the small peptide SspB-Q73R for use in iLID-based optogenetic experiments.

Abstract: How do cells maintain robust, yet flexible polarization for directed motion? Recent optogenetic experiments by Town and Weiner on neutrophil-like HL-60 cells strongly point to the essential role of a Rac-inhibitor (downstream of the small GTPase Rac) in shaping requisite negative feedback that allows cells to respond to rapidly changing directional cues. Here we adapt a previous mathematical model for cell polarity to model interactions of Rac, its putative inhibitor, and upstream PIP3 (a product of the optogenetically stimulated PI3K). We fit parameters in our partial differential equation (PDE) model to temporal and spatial experimental data. Cell shapes, motility, and stimulus responses are modeled in 2D simulations, with PDEs solved along the cell edge. We show that the Rac-inhibitor-PIP3 circuit accounts for the optogenetic data (including exotic cell trajectories), that it is the minimal circuit to do so, and that it improves gradient sensing under noisy or dynamic conditions.

Abstract: Primordial germ cells (PGCs) are the first cells specified in the Drosophila embryo and serve as precursors to the germline. Their formation requires suppression of somatic fates, a process achieved by excluding the receptor tyrosine kinase Torso from the posterior pole through degradation mediated by the ubiquitin ligase adaptor Germ Cell-Less (GCL). Although Torso is known to antagonize PGC formation, the underlying mechanism has remained unclear. Here, we combine optogenetic Ras activation and Ras effector loop mutants to show that Ras signaling suppresses PGC formation independently of the canonical Raf/MEK/ERK pathway. We identify an unexpected early role for Torso in activating phosphoinositide 3-kinase (PI3K), generating posterior membrane domains enriched in phosphatidylinositol (3,4,5)-trisphosphate (PIP3). Elevated PI3K activity disrupts PGC formation, while reduced PI3K activity leads to ectopic PGCs. We further demonstrate that GCL remodels the posterior pole membrane by suppressing Torso-dependent PI3K activation. Clearing PIP3 enables Myosin II enrichment, thereby constricting the pole bud for PGC formation. Together, our findings reveal how antagonistic Torso and GCL activities establish the soma-germline boundary by regulating cortical lipid organization.

Abstract: Smart microscopy is transforming biological imaging by integrating real-time analysis with adaptive acquisition to enhance imaging efficiency. Whereas many emerging implementations are event-driven and focus on on-demand data acquisition to reduce phototoxicity, we here present 'outcome-driven' microscopy, a framework combining smart microscopy with optogenetics to control cell biological processes and achieve predefined outcomes. We validate this approach using light-based control of cell migration and nucleocytoplasmic transport, demonstrating robust spatiotemporal control of cellular behaviour in single cells and in cell populations.

Abstract: Multimerization and phase separation represent two paradigms for organizing receptor tyrosine kinases (RTKs). However, their functional distinctions from the perspective of biomolecular organization remain unclear. Here, we present CORdensate, a light-controllable condensation system combining two synergistic photoactuators: oligomeric Cry2 and heterodimeric LOVpep/ePDZ. Engineering single-chain photoswitches, we achieve four biomolecular organization patterns ranging from monomerization to phase separation. CORdensate exhibits constant assembly and disassembly kinetics. Applying CORdensate to mimic pathogenic RTK granules establishes the role of phase separation in activating ALK and RET. Moreover, assembling ALK and RET through varying organization patterns, we highlight the superior organizational ability of phase separation over multimerization. Additionally, CORdensate-based RTK granules suggest that phase separation broadly and robustly activates RTKs. This study introduces a optogenetic tool for investigating biomolecular condensation.

Abstract: Small GTPases are critical regulators of cellular processes, such as cell migration, and comprise a family of over 167 proteins in the human genome. Importantly, the location-dependent regulation of small GTPase activity is integral to coordinating cellular signaling. Currently, there are no generalizable methods for directly controlling the activity of these signaling enzymes with subcellular precision. To address this issue, we introduce a modular, optogenetic platform for the spatial control of small GTPase activity within living cells, termed spLIT-small GTPases. This platform enabled spatially precise control of cytoskeletal dynamics such as filopodia formation (spLIT-Cdc42) and directed cell migration (spLIT-Rac1). Furthermore, a spLIT-RhoA system uncovered previously unreported long-range RhoA signaling in HeLa cells, resulting in bipolar membrane retraction. These results establish spLIT-small GTPases as a versatile platform for the direct, spatial control of small GTPase signaling and demonstrate the ability to uncover spatially defined aspects of small GTPase signaling.

Abstract: Phospholipase C-γ1 (PLC-γ1) signaling is required for mesenchymal chemotaxis, but is it sufficient to bias motility? PLC-γ1 enzyme activity is basally autoinhibited, and light-controlled membrane recruitment of wild-type (WT) PLC-γ1 (OptoPLC-γ1) in Plcg1-null fibroblasts does not trigger lipid hydrolysis, complicating efforts to isolate its contribution. Utilizing cancer-associated mutations to investigate the regulatory logic of PLC-γ1, we demonstrate that the canonical hallmark of enzyme activity, phosphorylated Tyr783 (pTyr783), is not a proxy for activity level, but is rather a marker of dysregulated autoinhibition. Accordingly, OptoPLC-γ1 with a deregulating mutation (P867R, S345F, or D1165H) exhibits elevated phosphorylation, and membrane localization of such is sufficient to activate substrate hydrolysis and concomitant motility responses. In particular, local recruitment of OptoPLC-γ1 S345F polarizes cell motility on demand. This response is spatially dose-sensitive and only partially reduced by blocking canonical PLC-γ1 signaling yet is lipase-dependent. Our findings reframe the interpretation of PLC-γ1 regulation and demonstrate that local activation of PLC-γ1 is sufficient to direct cell motility.

Abstract: The actin cytoskeleton forms a meshwork that drives cellular deformation. Network properties, determined by density and actin-binding proteins, are crucial, yet how density governs protein penetration and dynamics remains unclear. Here, we report an in vitro optogenetic system, named OptoVCA, enabling Arp2/3 complex-mediated actin assembly on lipid membranes. By tuning illumination power, duration, and pattern, OptoVCA flexibly manipulates the density, thickness, and shape of the actin network. Taking these advantages, we examine how network density affects two actin-binding proteins: myosin and ADF/cofilin. We find that even modest increases in density strictly inhibit myosin filament penetration by steric hindrance. Penetrated myosin filaments generate directional actin flow in networks with density gradients. In contrast, ADF/cofilin accesses networks regardless of density, yet network disassembly is markedly reduced by increased density. Thus, OptoVCA reveals that network density differentially regulates actin-binding protein penetration and activity. These findings advance understanding of cell mechanics through precise, light-based manipulation of cytoskeletal structure.

Abstract: During development, epithelia function as malleable sheets that undergo extensive remodeling to shape developing embryos. Optogenetic control of Rho signaling provides an avenue to investigate mechanisms of epithelial morphogenesis, but transgenic optogenetic tools can be limited by variability in expression levels and deleterious effects of transgenic overexpression on development. Here, we use CRISPR/Cas9 to tag Drosophila RhoGEF2 and Cysts/Dp114RhoGEF with components of the iLID/SspB optogenetic heterodimer, permitting light-dependent control over endogenous protein activities. Using quantitative optogenetic perturbations, we uncover a dose-dependence of tissue furrow depth and bending behavior on RhoGEF recruitment, revealing mechanisms by which developing embryos can shape tissues into particular morphologies. We show that at the onset of gastrulation, furrows formed by cell lateral contraction are oriented and size-constrained by basal actomyosin. Our findings demonstrate the use of quantitative, 3D-patterned perturbations of cell contractility to precisely shape tissue structures and interrogate developmental mechanics.

Abstract: Ras has traditionally been regarded as a positive regulator and therapeutic target due to its role in cell proliferation, but recent findings indicate a more nuanced role in cell migration, where suppressed Ras activity can unexpectedly promote migration. To clarify this complexity, we systematically modulate Ras activity using various RasGEF and RasGAP proteins and assess their effects on migration dynamics. Leveraging optogenetics, we assess the immediate, nontranscriptional effects of Ras signaling on migration. Local RasGEF recruitment to the plasma membrane induces protrusions and new fronts to effectively guide migration, even in the absence of GPCR/G-protein signaling, whereas global recruitment causes immediate cell spreading halting cell migration. Local RasGAP recruitment suppresses protrusions, generates new backs, and repels cells, whereas global relocation either eliminates all protrusions to inhibit migration or preserves a single protrusion to maintain polarity. Consistent local and global increases or decreases in signal transduction and cytoskeletal activities accompany these morphological changes. Additionally, we performed cortical tension measurements and found that Ras activity is regulated by guanine nucleotide exchange factors generally increase cortical tension while Ras activity is regulated by GTPase-activating proteins decrease it. Our results reveal a biphasic relationship between Ras activity and cellular dynamics, reinforcing our previous findings that optimal Ras activity and cortical tension are critical for efficient migration.

Abstract: Intermediate filaments (IFs) are a key component of the cytoskeleton, essential for regulating cell mechanics, maintaining nuclear integrity, organelle positioning, and modulating cell signaling. Current insights into IF function primarily come from studies using long-term perturbations, such as protein depletion or mutation. Here, we present tools that allow rapid manipulation of vimentin IFs in the whole cytoplasm or within specific subcellular regions by inducibly coupling them to microtubule motors, either pharmacologically or using light. Rapid perinuclear clustering of vimentin had no major immediate effects on the actin or microtubule organization, cell spreading, or focal adhesion number, but it reduced cell stiffness. Mitochondria and endoplasmic reticulum (ER) sheets were reorganized due to vimentin clustering, whereas lysosomes were only briefly displaced and rapidly regained their normal distribution. Keratin moved along with vimentin in some cell lines but remained intact in others. Our tools help to study the immediate and local effects of vimentin perturbation and identify direct links of vimentin to other cellular structures.

Abstract: Mitochondrial fission is controlled by dynamin-like proteins, the dysregulation of which is correlated with diverse diseases. Fission dynamin-like proteins are GTP hydrolysis-driven mechanoenzymes that self-oligomerize into helical structures that constrict membranes to achieve fission while also remodeling membranes by inducing negative Gaussian curvature, which is essential for the completion of fission. Despite advances in optical and electron imaging technologies, the underlying mechanics of mitochondrial fission remain unclear due to the multiple times involved in the dynamics of mechanoenzyme activity, oligomer disassembly, and membrane remodeling. Here, we examine how multiscale phenomena in dynamin Drp1 synergistically influence membrane fission using a mechanical model calibrated with small-angle X-ray scattering structural data and informed by a machine learning analysis of the Drp1 sequence, and tested the concept using optogenetic mechanostimulation of mitochondria in live cells. We find that free dynamin-like proteins can trigger a "snap-through instability" that enforces a shape transition from an oligomer-confined cylindrical membrane to a drastically narrower catenoid-shaped neck within the spontaneous hemi-fission regime, in a manner that depends critically on the length of the confined tube. These results indicate how the combination of assembly and paradoxically disassembly of dynamin-like proteins can lead to diverse pathways to scission.

Abstract: This study explores how the small GTPase RhoA modulates plasma membrane lipid nanodomains, particularly cholesterol-rich ordered membrane domains (OMDs). These nanodomains play a critical role in regulating ion channel activity and neuronal excitability. However, due to their nanoscale dimensions, OMDs remain challenging to visualize using conventional light microscopy. Here, we used fluorescently labeled cholera toxin B (CTxB) and the palmitoylated peptide Lck-10 (L10) as probes to visualize OMDs and quantified their size via confocal fluorescence lifetime imaging microscopy (FLIM)-based Förster resonance energy transfer (FRET). Pharmacological inhibition of RhoA significantly reduced OMD sizes in both human cell lines and dorsal root ganglion (DRG) neurons. To achieve better spatiotemporal control of specific RhoA activation, we employed an improved light-inducible dimerization (iLID) system. Optogenetic activation of RhoA rapidly increased FRET efficiency between CTxB probes, indicating OMD coalescence. Functionally, RhoA inhibition potentiated hyperpolarization-activated cyclic nucleotide-gated (HCN) channel activity in nociceptive DRG neurons, increasing spontaneous action potential firing. Conversely, in a spared nerve injury rat model, RhoA activation expanded OMDs in nociceptive DRG neurons. Constitutive RhoA activation suppressed HCN channel activity and decreased membrane excitability. These findings support a neuroprotective role for RhoA activation, where it restores OMD size and suppresses pathological hyperexcitability in neuropathic pain.

Abstract: Cortical positioning of the meiotic spindle within an oocyte is required to expel chromosomes into polar bodies to generate a zygote with the correct number of chromosomes. In C. elegans, yolk granules and mitochondria are packed inward, away from the cortex, while the spindle moves outward, both in a kinesin-dependent manner. The kinesin-dependent inward packing of yolk granules suggests the existence of microtubules with minus ends at the cortex and plus ends extending inward, making it unclear how kinesin moves the spindle outward. We hypothesize that the inward packing of organelles might indirectly force the spindle outward by volume exclusion. To test this hypothesis, we generate a strain in which the only kinesin consists of motor domains with no cargo-binding tail optogenetically attached to mitochondria. This mitochondria-only kinesin packs mitochondria into a tight ball and efficiently moves the meiotic spindle to the cortex, supporting the volume exclusion hypothesis.

Abstract: Ras has traditionally been regarded as a positive regulator and therapeutic target due to its role in cell proliferation, but recent findings indicate a more nuanced role in cell migration, where suppressed Ras activity can unexpectedly promote migration. To clarify this complexity, we systematically modulate Ras activity using various RasGEF and RasGAP proteins and assess their effects on migration dynamics. Leveraging optogenetics, we assess the immediate, non-transcriptional effects of Ras signaling on migration. Local RasGEF recruitment to the plasma membrane induces protrusions and new fronts to effectively guide migration, even in the absence of GPCR/G-protein signaling whereas global recruitment causes immediate cell spreading halting cell migration. Local RasGAP recruitment suppresses protrusions, generates new backs, and repels cells whereas global relocation either eliminates all protrusions to inhibit migration or preserves a single protrusion to maintain polarity. Consistent local and global increases or decreases in signal transduction and cytoskeletal activities accompany these morphological changes. Additionally, we performed cortical tension measurements and found that RasGEFs generally increase cortical tension while RasGAPs decrease it. Our results reveal a biphasic relationship between Ras activity and cellular dynamics, reinforcing our previous findings that optimal Ras activity and cortical tension are critical for efficient migration.

Abstract: Corynebacterium glutamicum is a key industrial workhorse for producing amino acids and high-value chemicals. Balancing metabolic flow between cell growth and product synthesis is crucial for enhancing production efficiency. Developing dynamic, broadly applicable, and minimally toxic gene regulation tools for C. glutamicum remains challenging, as optogenetic tools ideal for dynamic regulatory strategies have not yet been developed. This study introduces an advanced light-controlled gene expression system using light-controlled RNA-binding proteins (RBP), a first for Corynebacterium glutamicum. We established a gene expression regulation system, 'LightOnC.glu', utilizing the light-controlled RBP to construct light-controlled transcription factors in C. glutamicum. Simultaneously, we developed a high-performance light-controlled gene interference system using CRISPR/Cpf1 tools. The metabolic flow in the synthesis network was designed to enable the production of chitin oligosaccharides (CHOSs) and chondroitin sulphate oligosaccharides A (CSA) for the first time in C. glutamicum. Additionally, a light-controlled bioreactor was constructed, achieving a CHOSs production concentration of 6.2 g/L, the highest titer recorded for CHOSs biosynthesis to date. Herein, we have established a programmable light-responsive genetic circuit in C. glutamicum, advancing the theory of dynamic regulation based on light signaling. This breakthrough has potential applications in optimizing metabolic modules in other chassis cells and synthesizing other compounds.