Curated Optogenetic Publication Database

Abstract: Optogenetics holds great potential for diverse biological applications, including fundamental research, tissue engineering, and regenerative medicine, by enabling the precise spatial and temporal control of cellular signaling pathways. Transforming growth factor-beta (TGFβ), a multifunctional cytokine, is a critical regulator of cell proliferation, differentiation, and particularly chondrogenesis. Although TGFβ signaling is necessary for effective chondrogenic differentiation, previous studies have primarily relied on recombinant TGFβ ligand supplementation. In this study, we established an advanced optogenetic platform by knocking-in opto-TGFβ receptors in the AAVS1 locus of human embryonic stem cells (hESCs), enabling precise optogenetic activation of endogenous TGFβ signaling. Blue light illumination specifically activated TGFβ signaling, indicated by enhanced SMAD2 phosphorylation. Employing a three-dimensional pellet culture system, we demonstrated that direct optogenetic activation of TGFβ receptors, without exogenous ligand supplementation, is sufficient for robust chondrogenic differentiation of hESC-derived mesenchymal stem cells. The efficiency of optogenetic differentiation was comparable to conventional recombinant TGFβ protein treatment, evidenced by the expression of chondrogenic markers and deposition of cartilage-specific extracellular matrix components, including aggrecan and type II collagen. Our findings directly confirm the sufficiency and critical role of TGFβ receptor activation itself in chondrogenesis. Furthermore, this optogenetic approach provides a theoretical advantage by enabling noninvasive external modulation of TGFβ signaling post-transplantation, potentially facilitating further maturation and functional integration of transplanted chondrocytes. Thus, our results highlight a promising recombinant-protein-free strategy for use in cartilage tissue engineering and regenerative medicine.

Abstract: CRISPR-Cas systems enable powerful gene editing and regulation, yet single-modality control often fails to achieve orthogonal, spatiotemporally precise regulation of multiple endogenous genes. We engineered OREC, an orthogonal platform integrating chemogenetic and optogenetic modalities for precise, reversible, multiplex gene control. OREC comprises two components: ORECC regulated by doxycycline (Dox) and ORECo controlled by light. By assembling catalytically dead Cpf1 (dCpf1), gene regulatory elements, and crRNA arrays on single transcripts, OREC enables robust simultaneous manipulation of multiple genes. We demonstrated OREC's therapeutic potential in vitro for osteoblast function modulation and in vivo for osteoporotic fracture repair. OREC effectively activated Bmp2 while inhibiting Dkk1, significantly enhancing bone formation and fracture healing in mouse models. These results establish OREC as a versatile platform for precise multiplex gene regulation, offering significant advancement for CRISPR-based gene therapy applications in complex tissues where coordinated control of multiple therapeutic targets is essential.

Abstract: Autophagy, a conserved recycling process, manages intracellular quality control to mitigate stress. To determine the rapid effects of autophagy perturbation, we developed the first optogenetic tool to rapidly inhibit autophagy, termed ASAP. Our approach selectively inhibits autophagy within 5 minutes, providing a precise and dynamic approach to study autophagy regulation. Proteomic profiling with ASAP revealed the most tightly regulated autophagy substrates along with novel, previously unidentified substrates, including nuclear pore complex (NPC) proteins. Interestingly, autophagy regulates quality control of incomplete NPCs still in the cytoplasm via specific LC3-interacting regions (LIRs), sparing NPCs embedded in the nuclear envelope. Upon rapid autophagy inhibition, incomplete NPCs accumulate and instead of undergoing autophagic degradation, cytoplasmic NPCs aggregate in processing bodies. Using ASAP, we demonstrate rapid and specific inhibition of autophagy, revealing that the nuclear pore complex is a tightly regulated autophagy substrate.

Abstract: Mitochondrial quality control is tightly associated with aging-related neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), and frontotemporal dementia (FTD). Previous studies reported that ALS/FTD-associated protein p62 drives "mitochondrial clustering" (perinuclear clustering of fragmented and swollen mitochondria) during PINK1/Parkin-mediated mitophagy, but the underlying molecular mechanism, especially the precise role of p62 in mitochondrial clustering during mitophagy and the potential relationship between the mitochondrial quality control mediated by p62 and disease pathogenesis of ALS/FTD, remains unclear. Here, using cell biology in combination with an optogenetic tool, we show that the phase separation (condensation) of p62 mediates the clustering of damaged mitochondria to form "grape-like" clusters during PINK1/Parkin-mediated mitophagy, which is tightly associated with aging-related neurodegenerative diseases. In addition, our data suggest this mitochondrial clustering process is an arrest mechanism driven by p62 condensation (beyond the function of other autophagy receptors in mitophagy), which acts as a "brake" to reduce the surface area of dysfunctional mitochondria within cytoplasm for minimizing mitochondrial turnover in cells. Moreover, ALS/FTD-related pathological mutations perturb p62 condensation, thereby inhibiting mitochondrial clustering and destroying the "brake" machinery of mitochondrial quality control. Together, our data highlight how p62 condensation modulates organelle quality control in cell biology, and the important role of p62 condensation in both physiology and pathology.

Abstract: G protein-coupled receptors (GPCRs) perceive spatially and temporally diverse stimuli and activate G protein heterotrimers comprising α, β, and γ subunits, which broadcast signals through a broad range of effectors at various subcellular compartments. Therefore, understanding endogenous G protein activity dynamics at the subcellular level, thereby recapitulating in vivo signaling paradigms, will facilitate the identification of pathological signaling pathways. However, the lack of sensors for endogenous G proteins has been an obstacle. Here, we demonstrate the engineering of sensors to probe endogenous GαiGTP and GαqGTP. Compared to examining overexpressed and fluorescently tagged Gα, our sensors capture the magnitude and kinetics of endogenous GαGTP dynamics, including their generation, equilibrium signaling, and hydrolysis, with native fidelity. Using the translocation-based GαiGTP sensor, we show that heterotrimer dissociation upon Gi-GPCR activation is Gγ-subtype dependent. Confirming our previous findings, the GαqGTP sensor showed that Gαq expression is low and tightly regulated in most cells. Using optogenetic tools, we demonstrate that our sensors detect GαGTP generation and hydrolysis during asymmetric GPCR-G protein activation, a capability that will be particularly useful in morphologically diverse cells such as neurons. Therefore, our engineered novel GαGTP sensors can be highly beneficial in decoding subcellularly resolved endogenous G protein signaling dynamics.

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: Chimeric antigen receptor (CAR) cellular therapy, particularly CAR-T cells, has revolutionized the treatment of hematologic malignancies. However, these therapies show limited efficacy against solid tumors, in part due to the inefficient trafficking of effector cells to the tumor. This review explores the potential of engineering natural and synthetic G protein-coupled receptors (GPCRs) to overcome this migratory hurdle. Chemokine receptors have been the most used GPCR family in this setting. Engineering effector immune cells to express chemokine receptors that match tumor-derived chemokines has been shown to increase their chemotaxis and to improve antitumor efficacy in preclinical models. In addition to improved migration, chemokine receptor engineering can also have additional benefits, such as remodeling of the tumor microenvironment and metabolic rewiring of engineered cells. However, the effectiveness of this approach is limited by the tumor-specific and heterogeneous chemokine milieu. Emerging strategies make use of synthetic GPCRs and could overcome some of these limitations using chemogenetic and optogenetic approaches. Here, mutated GPCRs binding only to specific and orthogonal ligands or light-sensitive channels are used for cell modulation and trafficking. Equipping cells with these synthetic GPCRs allows for precise and stimulus-controlled immune cell migration. Together, natural and synthetic GPCR engineering form promising approaches to enhance immune cell trafficking, persistence, and efficacy.

Abstract: Single-cell resolution studies have transformed our understanding of microbial systems, revealing substantial cell-to-cell heterogeneity and complex dynamic behaviors. This review describes recent advances in using optogenetics, where light-sensitive proteins control cellular processes, to investigate microbial behavior at the individual cell level. We discuss studies where optogenetic approaches have enabled high-resolution analysis of properties such as relative cell positioning, subcellular localization, morphology, and gene expression dynamics. In addition, we highlight emerging feedback and event-driven control methods that dynamically modulate cellular states using light signals. By leveraging light's unique capabilities for spatial and temporal manipulation, researchers can now probe cellular characteristics with unprecedented precision. We anticipate significant advances as researchers introduce more sophisticated dynamically patterned light signals for single-cell microbial research.

Abstract: Mitochondria besides being the powerhouse of the cell are also involved in performing a multitude of critical cellular functions. Any failure in maintenance of these organelles is implicated in multiple human pathologies, including neurodegenerative disorders. Over the past two decades, significant efforts have been made to investigate the pharmacodynamic propensity of various potential compounds, which could be engaged as efficient therapeutic approach in modulating mitochondrial dynamics during neuronal dysfunctions.

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: Phosphoinositides, comprising less than 10% of membrane lipids, function as 'lipid codes' within cellular compartments through seven species formed by myo-inositol headgroup phosphorylation. This review examines their diverse roles in endocytic transport, encompassing endocytosis, endosomal sorting, degradation, and recycling, as well as specialized mechanisms, such as caveolin-mediated endocytosis. The review also investigates the involvement of specific kinases and phosphatases in these processes. Additionally, it discusses the impact of technological advancements, such as fluorescent biosensors, super-resolution microscopy, optogenetics, and synthetic biology, on elucidating phosphoinositide dynamics during endocytic trafficking. Perturbations in phosphoinositide metabolism have been associated with human diseases, including cancer and neurodegenerative disorders. Exploring these pathways may unveil potential therapeutic targets, with subsequent research focusing on their spatiotemporal regulation, tissue-specific metabolism, the synergistic effects of phosphoinositides with other lipids, and the incorporation of systems biology to bridge basic cell biology with translational medicine.

Abstract: Cellular lipid metabolism is subject to strong homeostatic regulation, but the players involved in and mechanisms underlying these pathways remain largely uncharacterized. Here we develop a 'feeding-fishing' approach coupling membrane editing using optogenetic lipid-modifying enzymes (feeding) with organelle membrane proteomics through proximity labeling (fishing) to elucidate molecular players and pathways involved in the homeostasis of phosphatidic acid (PA), a multifunctional lipid central to glycerolipid metabolism. This approach identified several PA-metabolizing enzymes and lipid transfer proteins enriched in and depleted from PA-fed membranes. Mechanistic analysis revealed that PA homeostasis in the cytosolic leaflets of the plasma membrane and lysosomes is mediated by both local PA metabolism and the action of lipid transfer proteins that carry out interorganelle lipid transport before subsequent metabolism. More broadly, the interfacing of membrane editing to controllably modify membrane lipid composition with organelle membrane proteomics using proximity labeling represents a strategy for revealing mechanisms governing lipid homeostasis.

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: During vertebrate development, the segmentation clock drives oscillatory gene expression in the presomitic mesoderm (PSM), leading to the periodic formation of somites. Oscillatory gene expression is synchronized at the cell population level; inhibition of Delta-Notch signaling results in the loss of synchrony and the fusion of somites. However, it remains unclear how cell-cell signaling couples oscillatory gene expression and controls synchronization. Here, we report that synthetic cell-cell signaling using designed ligand-receptor pairs can induce synchronized oscillations in PSM organoids. Optogenetic assays uncovered that the intracellular domains of synthetic ligands play key roles in dynamic cell-cell communication. Oscillatory coupling using synthetic cell-cell signaling recovered the synchronized oscillation in PSM cells deficient for Delta-Notch signaling; nonoscillatory coupling did not induce recovery. This study reveals the mechanism by which ligand-receptor molecules coordinate the synchronization of the segmentation clock and provides a way to program temporal gene expression in organoids and artificial tissues.

Abstract: Biopolymers that separate into condensed and dilute phases in solution also prewet membranes when one or more components couple to membrane lipids. Here we demonstrate that this prewetting transition becomes exquisitely sensitive to lipid composition when membranes have compositions near the boundary of liquid-ordered/liquid-disordered phase coexistence in both simulation and in reconstitution when polyelectrolytes are coupled to model membranes. In cells, we use an optogenetic tool to characterize prewetting at both the plasma membrane (PM) and the endoplasmic reticulum (ER) and find that prewetting is potentiated or inhibited by perturbations of membrane composition. Prewetting can also mediate membrane adhesion, with avidity dependent on membrane composition, as demonstrated in cells through the potentiation or inhibition of ER-PM contact sites. The strong correspondence of results in simulation, reconstitution and cells reveals a new role for membrane lipids in regulating the recruitment and assembly of soluble proteins.

Abstract: Molecular biology has benefited enormously from repurposed tools-many enzymes and antibodies evolved for other functions but are now essential for interrogating biological function by manipulating proteins or nucleic acids. In contrast, lipids have remained technically difficult to visualize or manipulate in cells. This review introduces tools that bring lipid biology into reach for molecular cell biologists, using familiar experimental approaches. We first describe adaptations of immunofluorescence and live-cell imaging of fluorescent molecules to track lipids. Then, we discuss tools for manipulating lipid levels, including pharmacologic inhibitors, synthetic biology platforms for inducible lipid generation or degradation, and optogenetic systems for precise temporal control. While some methods remain technically demanding, most tools are now broadly accessible. Our goal is to offer a practical framework for integrating lipid biology into mainstream cell biology experiments.

Abstract: The spatial organization of the cell relies on biomolecular condensates formed via liquid-liquid phase separation (LLPS). The dysregulation of this physicochemical order drives a growing class of human pathologies. Here, we champion the unifying term "Condensatopathies" and establish a rigorous framework for their classification based on three core criteria: genetic/environmental triggers, demonstrable biophysical defects, and causal toxicity. We synthesize the pathogenic landscape into two distinct yet interconnected mechanisms: Loss-of-Function (LOF), where essential condensates fail to form or harden; and Toxic Gain-of-Function (TGOF), characterized by the formation of aberrant, often solid-like aggregates or oncogenic hubs that hijack cellular machinery. By analyzing representative cases-from the biophysical maturation of TDP-43 in neurodegeneration to the chromatin hijacking by NUP98 fusions in leukemia-we reveal how the loss of "tunable metastability" underpins these disorders. Furthermore, we review how emerging technologies like optogenetics and cryo-ET are decoding these mechanisms. Finally, we propose an integrated "See-and-Treat" theranostic paradigm, utilizing the unique material properties of condensates to design specific diagnostic probes and "molecular scalpels" for precision intervention.

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: The dynamics of the early steps of protein aggregation remain poorly understood, particularly in the case of α-synuclein (α-syn) aggregation, the hallmark of synucleinopathies. Here, we present a protocol that combines light-inducible protein aggregation (LIPA) with proximity biotinylation using an UltraID construct. We describe the workflow from protein expression to biochemical validation, including the purification of biotinylated proteins prior to liquid chromatography-mass spectrometry (LC-MS) analysis and subsequent validation. This platform provides a powerful strategy to identify proteins interacting with nascent α-syn aggregates. For complete details on the use and execution of this protocol, please refer to Teixeira et al.1.

Abstract: Although considerable research has focused on enhancing the apoptotic function of BAX for several decades, inhibition of its functionality remains relatively underexplored, despite intensive BAX activation occurring in various neurodegenerative diseases. Here we present a protein engineering approach to modulate BAX integration into the mitochondrial outer membrane, establishing a tunable strategy for antiapoptosis. Utilizing optogenetic methods that employ cryptochrome 2 and its binding partner cryptochrome-interacting basic helix loop helix 1, we achieved precise spatial control over BAX localization, a critical determinant of its function. Our results demonstrate that the engineered BAX variant is effectively incapacitated in its apoptotic function while also modulating endogenous BAX activity to enhance cellular resistance to apoptosis. These findings not only advance our understanding of BAX regulation but also offer promising prospects for the development of therapeutic strategies against apoptosis-related diseases.

Abstract: Outer mitochondrial membranes (OMM) function as dynamic hubs for inter-organelle communication, integrating bidirectional signals, and coordinating organelle behavior in a context-dependent manner. However, tools for mapping mitochondrial surface proteomes with high spatial and temporal resolution remain limited. Here, we introduce an optogenetic proximity labeling strategy using LOV-Turbo, a light-activated biotin ligase, to profile mitochondrial surface proteomes with improved precision, temporal control, and reduced background. By fusing LOV-Turbo to a panel of variants of an OMM-anchored protein, Miro1, we generate spatially distinct baits that resolve modular architectures and regulatory states of the OMM proteomes across diverse conditions, a database we name MitoSurf. Building on this proteomic map, we present RiboLOOM, a platform that defines LOV-Turbo labeled ribosomes and their bound mRNAs at the mitochondrial surface. MitoSurf and RiboLOOM uncover a spatially distinct ribosome pool at the OMM that is maintained by Miro1, enabling local mRNA engagement and translation of mitochondria-related proteins. These findings establish Miro1 as a key organizer of mitochondrial protein biogenesis through spatial confinement of surface-associated ribosomes. Our platform reveals an uncharted layer of mitochondrial surface biology and provides a generalizable strategy to dissect dynamic RNA-protein-organelle interfaces in living cells.

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: When mammalian cells are exposed to stress, they co-ordinate the condensation of stress granules (SGs) through the action of proteins G3BP1 and G3BP2 (G3BPs) and, simultaneously, undergo a massive reduction in translation. Although SGs and G3BPs have been linked to this translation response, their overall impact has been unclear. Here we investigate the question of how, and indeed whether, G3BPs and SGs shape the stress translation response. We find that SGs are enriched for mRNAs that are resistant to the stress-induced translation shutdown. Although the accurate recruitment of these stress-resistant mRNAs does require the context of stress, a combination of optogenetic tools and spike-normalized ribosome profiling demonstrates that G3BPs and SGs are necessary and sufficient to both help prioritize the translation of their enriched mRNAs and help suppress cytosolic translation. Together, these results support a model in which G3BPs and SGs reinforce the stress translation programme by prioritizing the translation of their resident mRNAs.

Abstract: Light-sensitive proteins allow organisms to perceive and respond to their environment, and have diversified over billions of years. Among these, Light-Oxygen-Voltage (LOV) domains are widespread photosensors that control diverse physiological processes and are increasingly used in optogenetics. Yet, the evolutionary constraints that shaped their protein dynamics and thereby their functional diversity remain poorly resolved. Here we systematically characterize the dynamics of 21 natural LOV core domains, significantly extending the spectroscopically resolved catalog through the addition of 18 previously unstudied variants. Using time-resolved spectroscopy, we uncover an exceptional kinetic diversity spanning from picoseconds to days and identify distinct functional clusters within the LOV family. These clusters reflect evolutionary branching, including a divergence of ≈1.0 billion years between investigatedLOV variants from plants and ≈0.4 billion years of separation within one of these functional clusters. Individual variants with extreme photocycles emerge as promising anchor points for optogenetic applications, ranging from highly efficient adduct formation to ultrafast recovery. Beyond natural diversity, we introduce a LOV domain generated by artificial intelligence-guided protein design. Despite being sequentially remote from its maternal template, this variant retains core photocycle function while exhibiting unique biophysical properties, thereby occupying a new region on the biophysical landscape. Our work emphasizes how billions of years of evolution defined LOV protein dynamics, and how protein design can expand this repertoire, engineering next-generation optogenetic tools.