Curated Optogenetic Publication Database

Abstract: Background Cytoplasmic aggregation of TAR DNA binding protein 43 (TDP-43) in neurons is one of the hallmarks of TDP-43 proteinopathy. Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are closely associated with TDP-43 proteinopathy; however, it remains uncertain whether TDP-43 aggregation initiates the pathology or is a consequence of it. Methods To demonstrate the pathology of TDP-43 aggregation, we applied the optoDroplet technique in Caenorhabditis elegans (C. elegans), which allows spatiotemporal modulation of TDP-43 phase separation and assembly. Results We demonstrate that optogenetically induced TDP-43 aggregates exhibited insolubility similar to that observed in TDP-43 proteinopathy. These aggregates increased the severity of neurodegeneration, particularly in GABAergic motor neurons, and exacerbated sensorimotor dysfunction in C. elegans. Conclusions We present an optogenetic C. elegans model of TDP-43 proteinopathy that provides insight into the neuropathological mechanisms of TDP-43 aggregates. Our model serves as a promising tool for identifying therapeutic targets for TDP-43 proteinopathy.

Abstract: Transcription factors relay information from the external environment to gene regulatory networks that control cell physiology. To confer signalling specificity, robustness and coordination, these signalling networks use temporal communication codes, such as the amplitude, duration or frequency of signals. Although much is known about how temporal information is encoded, a mechanistic understanding of how gene regulatory networks decode signalling dynamics is lacking. Recent advances in our understanding of phase separation of transcriptional condensates provide new biophysical frameworks for both temporal encoding and decoding mechanisms. In this Perspective, we summarize the mechanisms by which transcriptional condensates could enable temporal decoding through signal adaptation, memory and persistence. We further outline methods to probe and manipulate dynamic communication codes of transcription factors and condensates to rationally control gene activation.

Abstract: Post-translational modifications (PTMs) are indispensable modulators of protein activity. Most cellular behaviors, from cell division to cytoskeletal organization, are controlled by PTMs, their misregulation being associated with a plethora of human diseases. Traditionally, the role of PTMs has been studied employing biochemical techniques. However, these approaches fall short when studying PTM dynamics in vivo. In recent years, functionalized protein binders have allowed the PTM of endogenous proteins by bringing an enzymatic domain in close proximity to the protein they recognize. To date, most of these methods lack the temporal control necessary to understand the complex effects triggered by PTMs. In this study, we have developed a method to phosphorylate endogenous Myosin in a light-inducible manner. The method relies both on nanobody-targeting and light-inducible activation in order to achieve both tight specificity and temporal control. We demonstrate that this technology is able to disrupt cytoskeletal dynamics during Drosophila embryonic development. Together, our results highlight the potential of combining optogenetics and protein binders for the study of the proteome in multicellular systems.

Abstract: p53 protein, a crucial transcription factor in cellular responses to a wide variety of stress, regulates multiple target genes involved in tumor suppression, senescence induction, and metabolic functions. However, it remains unclear how diverse cellular phenotypes are modulated by p53. In this study, we developed an optogenetic tool, Opto-p53, to control p53 signaling by light. Opto-p53 was designed to trigger p53 signaling by reconstituting p53 N-terminal and C-terminal fragments with a light-inducible dimerization (LID) system. Upon light exposure, cells expressing Opto-p53 demonstrated p53 transcriptional activation, resulting in cell death and cell cycle arrest. We further enhanced the efficacy of light-induced p53 activation by introducing specific mutations into Opto-p53 fragments. Our findings unveil the capability of Opto-p53 to serve as a powerful tool for dissecting the complex roles of p53 in cellular processes, thereby contributing to the field of synthetic biology and providing general design principles for optogenetic tools using endogenous transcription factors.

Abstract: G protein-coupled receptors (GPCRs) are the largest class of receptors in the genome and control many signaling cascades essential for survival. GPCR signaling is regulated by β-arrestins, multifunctional adapter proteins that direct receptor desensitization, internalization, and signaling. While at many GPCRs, β-arrestins interact with a wide array of signaling effectors, it is unclear how β-arrestins promote such varied functions. Here we show that β-arrestins undergo liquid-liquid phase separation (LLPS) to form condensates that regulate GPCR function. We demonstrate that β-arrestin oligomerization occurs in proximity to the GPCR and regulates GPCR functions such as internalization and signaling. This model is supported by a cryoEM structure of the adhesion receptor ADGRE1 in a 2:2 complex with β-arrestin 1, with a β-arrestin orientation that can promote oligomerization. Our work provides a paradigm for β-arrestin condensates as regulators of GPCR function, with LLPS serving as an important promoter of signaling compartmentalization at GPCRs.

Abstract: Studying the physical properties of sub-cellular components is increasingly important in understanding cell mechanics. This review focuses on the most advanced techniques available for investigating intracellular mechanics. We distinguish methods that act as force generators and those that act as force sensors. We highlight six state-of-the-art techniques, with increased spatial and temporal resolutions: optogenetics, Brillouin microscopy, bacterial cells and nanorobots, optical tweezers, membrane tension probes, and magnetic particles.

Abstract: Adrenoceptors (ARs) play a vital role in various physiological processes and are key therapeutic targets. The advent of optical control techniques, including optogenetics and photopharmacology, offers the potential to modulate AR signaling with precise temporal and spatial resolution. In this review, we summarize the latest advancements in the optical control of AR signaling, encompassing optogenetics, photocaged compounds, and photoswitchable compounds. We also discuss the limitations of current tools and provide an outlook on the next generation of optogenetic and photopharmacological tools. These emerging optical technologies not only enhance our understanding of AR signaling but also pave the way for potential therapeutic developments.

Abstract: Pyroptosis, an inflammatory form of cell death, is associated with large cell swelling and plasma membrane rupture. Recently, such swelling has been shown to occur in a two steps fashion, but the precise molecular and biophysic mechanisms driving the process remain elusive. We demonstrate through advanced quantitative microscopy that, between the two swelling phases, cell volume stabilizes, while plasma membrane permeability to ions and small molecules is markedly elevated due to the formation of pores. From a biophysical perspective, how such a volume plateau exists is puzzling as ion pumps should not regulate the cell volume in these conditions. To address this, we developed a physical model based on an ions pump-and-leak framework, incorporating the dynamics of non-selective pore formation. We experimentally identify two distinct pore permeability dynamics, associated to an increase in the water filtration coefficient and to an ion selectivity decrease due to pore opening. Altogether our results suggest the existence of two mechanistically different pore types, likely driven by separate molecular players. Our findings provide fundamental insights into the biophysics of cell death and may have broader implications for understanding membrane rupture in other pathological contexts. Significance Statement: Among various programmed lytic cell death, pyropytosis is marked by dramatic changes in cell shape and large fluctuations in volume, fundamentally altering the cell’s physical properties. These biophysical changes are not mere byproducts but integral components of the death process, closely interacting with molecular events. By combining optogenetics, quantitative microscopy, and modeling, we show that a progressive increase in plasma membrane permeability alone drives cell swelling and membrane lysis. We therefore demonstrate that a deeper understanding of these dynamic cell modifications and their consequences will shed light on the molecular and biophysical mechanisms driving different forms of cell death.

Abstract: Ferroptosis is a lytic, iron-dependent form of regulated cell death characterized by excessive lipid peroxidation and associated with necrosis spread in diseased tissues through unknown mechanisms. Using a novel optogenetic system for light-driven ferroptosis induction via degradation of the anti-ferroptotic protein GPX4, we show that lipid peroxidation and ferroptotic death can spread to neighboring cells through their closely adjacent plasma membranes. Ferroptosis propagation is dependent on cell distance and completely abolished by disruption of α-catenin-dependent intercellular contacts or by chelation of extracellular iron. Remarkably, bridging cells with a lipid bilayer or increasing contacts between neighboring cells enhances ferroptosis spread. Reconstitution of iron-dependent spread of lipid peroxidation between pure lipid, contacting liposomes provides evidence for the physicochemical mechanism involved. Our findings support a model in which iron-dependent lipid peroxidation propagates across proximal plasma membranes of neighboring cells, thereby promoting the transmission of ferroptotic cell death with consequences for pathological tissue necrosis spread.

Abstract: There is currently a lack of tools capable of perturbing genes in both a precise and a spatiotemporal fashion. The flexibility of CRISPR (clustered regularly interspaced short palindromic repeats), coupled with light's unparalleled spatiotemporal resolution deliverable from a controllable source, makes optogenetic CRISPR a well-suited solution for precise spatiotemporal gene perturbations. Here, we present a new optogenetic CRISPR tool (Blue Light-inducible Universal VPR-Improved Production of RGRs, BLU-VIPR) that diverges from prevailing split-Cas design strategies and instead focuses on optogenetic regulation of guide RNA (gRNA) production. We engineered BLU-VIPR around a new potent blue-light activated transcription factor (VPR-EL222) and ribozyme-flanked gRNA. The BLU-VIPR design is genetically encoded and ensures precise excision of multiple gRNAs from a single messenger RNA transcript. This simplified spatiotemporal gene perturbation and allowed for several types of optogenetic CRISPR, including indels, CRISPRa, and base editing. BLU-VIPR also worked in vivo with cells previously intractable to optogenetic gene editing, achieving optogenetic gene editing in T lymphocytes in vivo.

Abstract: While optogenetic tools have recently opened new avenues for controlling and understanding cellular behavior, Suh et al.1 present an effective strategy to regulate tissue densification and outgrowth through optogenetic control of EGFR. Their work ultimately uncovers fundamental principles that pave the way for improved tissue engineering approaches.

Abstract: Precise regulation of protein abundance is critical for cellular homeostasis, whose dysfunction may directly lead to human diseases. Optogenetics allows rapid and reversible control of precisely defined cellular processes, which has the potential to be utilized for regulation of protein dynamics at various scales. Here, we developed a novel optogenetics-based protein degradation system, namely Peptide-mediated OptoTrim-Away (POT) which employs expressed small peptides to effectively target endogenous and unmodified proteins. By engineering the light-induced oligomerization of the E3 ligase TRIM21, POT can rapidly trigger protein degradation via the proteasomal pathway. Our results showed that the developed POT-PI3K and POT-GPX4 modules, which used the iSH2 and FUNDC1 domains to specifically target phosphoinositide 3-kinase (PI3K) and glutathione peroxidase 4 (GPX4) respectively, were able to potently induce the degradation of these endogenous proteins by light. Both live-cell imaging and biochemical experiments validated the potency of these tools in downregulating cancer cell migration, proliferation, and even promotion of cell apoptosis. Therefore, we believe the POT offers an alternative and practical solution for rapid manipulation of endogenous protein levels, and it could potentially be employed to dissect complex signaling pathways in cell and for targeted cellular therapies.

Abstract: Lipids are a major class of biological molecules, the primary components of cellular membranes, and critical signaling molecules that regulate cell biology and physiology. Due to their dynamic behavior within membranes, rapid transport between organelles, and complex and often redundant metabolic pathways, lipids have traditionally been considered among the most challenging biological molecules to study. In recent years, a plethora of tools bridging the chemistry-biology interface has emerged for studying different aspects of lipid biology. Here, we provide an overview of these approaches. We discuss methods for lipid detection, including genetically encoded biosensors, synthetic lipid analogs, and metabolic labeling probes. For targeted manipulation of lipids, we describe pharmacological agents and controllable enzymes, termed membrane editors, that harness optogenetics and chemogenetics. To conclude, we survey techniques for elucidating lipid-protein interactions, including photoaffinity labeling and proximity labeling. Collectively, these strategies are revealing new insights into the regulation, dynamics, and functions of lipids in cell biology.

Abstract: Calcium (Ca2+) release from intracellular stores, Ca2+ entry across the plasma membrane, and their coordination via store-operated Ca2+ entry (SOCE) are critical for receptor-activated Ca2+ oscillations. However, the precise mechanism of Ca2+ oscillations and whether their control loop resides at the plasma membrane or intracellularly remain unresolved. By examining the dynamics of stromal interaction molecule 1 (STIM1), an endoplasmic reticulum (ER)-localized Ca2+ sensor that activates the Orai1 channel on the plasma membrane for SOCE and in mast cells, we found that a significant proportion of cells exhibited STIM1 oscillations with the same periodicity as Ca2+ oscillations. These cortical oscillations, occurring in the cell's cortical region and shared with ER-plasma membrane (ER-PM) contact site proteins, were only detectable using total internal reflection fluorescence microscopy (TIRFM). Notably, STIM1 oscillations could occur independently of Ca2+ oscillations. Simultaneous imaging of cytoplasmic Ca2+ and ER Ca2+ with SEPIA-ER revealed that receptor activation does not deplete ER Ca2+, whereas receptor activation without extracellular Ca2+ influx induces cyclic ER Ca2+ depletion. However, under such nonphysiological conditions, cyclic ER Ca2+ oscillations lead to sustained STIM1 recruitment, indicating that oscillatory Ca2+ release is neither necessary nor sufficient for STIM1 oscillations. Using optogenetic tools to manipulate ER-PM contact site dynamics, we found that persistent ER-PM contact sites reduced the amplitude of Ca2+ oscillations without alteration of oscillation frequency. Together, these findings suggest an active cortical mechanism governs the rapid dissociation of ER-PM contact sites, thereby controlling the amplitude of oscillatory Ca2+ dynamics during receptor-induced Ca2+ oscillations.

Abstract: Receptor tyrosine kinases (RTKs) play key roles in coordinating cell movement at both single-cell and tissue scales. The recent development of optogenetic tools for controlling RTKs and their downstream signaling pathways suggests that these responses may be amenable to engineering-based control for sculpting tissue shape and function. Here, we report that a light-controlled epidermal growth factor (EGF) receptor (OptoEGFR) can be deployed in epithelial cells for precise, programmable control of long-range tissue movements. We show that in OptoEGFR-expressing tissues, light can drive millimeter-scale cell rearrangements to densify interior regions or produce rapid outgrowth at tissue edges. Light-controlled tissue movements are driven primarily by phosphoinositide 3-kinase (PI3K) signaling, rather than diffusible ligands, tissue contractility, or ERK kinase signaling as seen in other RTK-driven migration contexts. Our study suggests that synthetic, light-controlled RTKs could serve as a powerful platform for controlling cell positions and densities for diverse applications, including wound healing and tissue morphogenesis.

Abstract: Light-based immunotherapy uses specific wavelengths of light to activate or modulate immune responses. It primarily employs two mechanisms: direct activation of immune cells and indirect modulation of the tumor microenvironment (TME). Several light-based technologies are under investigation or clinical use in immunotherapy, including photodynamic immunotherapy (PDIT) and photothermal therapy (PTT). Optogenetic tools have the potential to precisely control T-cell receptor activation, cytokine release, or the activity of other immune effector cells. Light-based technologies present innovative opportunities within the realm of immunotherapy. The ability to precisely regulate immune cell activation via optogenetics, alongside the improved targeting of cancer cells through photoimmunotherapy, signifies a transformative shift in our strategies for immune modulation. Although many of these technologies remain in the experimental stage for various applications, initial findings are encouraging, especially concerning cancer treatment and immune modulation. Continued research and clinical trials are essential to fully harness the capabilities of light technology in the context of immune cell therapy.

Abstract: Proteins phase-separate to form condensates that partition and concentrate biomolecules into membraneless compartments. These condensates can exhibit dichotomous behaviors in biology by supporting cellular physiology or instigating pathological protein aggregation1–3. Tau and α- synuclein (αSyn) are neuronal proteins that form heterotypic (Tau:αSyn) condensates associated with both physiological and pathological processes. Tau and αSyn functionally regulate microtubules8–12, but are also known to misfold and co-deposit in aggregates linked to various neurodegenerative diseases4,5,6,7, which highlights the paradoxically ambivalent effect of Tau:αSyn condensation in health and disease. Here, we show that tubulin modulates Tau:αSyn condensates by promoting microtubule interactions, competitively inhibiting the formation of homotypic and heterotypic pathological oligomers. In the absence of tubulin, Tau-driven protein condensation accelerates the formation of toxic Tau:αSyn heterodimers and amyloid fibrils. However, tubulin partitioning into Tau:αSyn condensates modulates protein interactions, promotes microtubule polymerization, and prevents Tau and αSyn oligomerization and aggregation. We distinguished distinct Tau and αSyn structural states adopted in tubulin-absent (pathological) and tubulin-rich (physiological) condensates, correlating compact conformations with aggregation and extended conformations with function. Furthermore, using various neuronal cell models, we showed that loss of stable microtubules, which occurs in Alzheimer’s disease and Parkinsons disease patients13,14, results in pathological oligomer formation and loss of neurites, and that functional condensation using an inducible optogenetic Tau construct resulted in microtubule stablization. Our results identify that tubulin is a critical modulator in switching Tau:αSyn pathological condensates to physiological, mechanistically relating the loss of stable microtubules with disease progression. Tubulin restoration strategies and Tau-mediated microtubule stabilization can be potential therapies targeting both Tau-specific and Tau/αSyn mixed pathologies.

Abstract: Talin regulates the adhesion and migration of cells in part by promoting the affinity of integrins for extracellular matrix proteins, a process that in cells such as endothelial cells and platelets requires the direct interaction of talin with both the small GTPase Rap1 bound to GTP (Rap1-GTP) and the integrin β3 cytoplasmic tail. To study this process in more detail, we employed an optogenetic approach in living, immortalized endothelial cells to be able to regulate the interaction of talin with the plasma membrane. Previous studies identified talin as the Rap1-GTP effector for β3 integrin activation. Surprisingly, optogenetic recruitment of talin-1 (TLN1; herein referred to as talin) to the plasma membrane also led to the localized activation of Rap1 itself, apparently by talin competing for Rap1-GTP with SHANK3, a protein known to sequester Rap1-GTP and to block integrin activation. Rap1 activation by talin was localized to the cell periphery in suspension cells and within lamellipodia and pseudopodia in cells adherent to fibronectin. Thus, membrane-associated talin can play a dual role in regulating integrin function in endothelial cells: first, by releasing Rap1-GTP from its sequestration by SHANK3, and second, by serving as the relevant Rap1 effector for integrin activation.

Abstract: Optogenetics has substantially enhanced our understanding of biological processes by enabling high-precision tracking and manipulation of individual cells. It relies on photosensitive proteins to monitor and control cellular activities, thereby paving the way for significant advancements in complex system research. Photosensitive proteins play a vital role in the development of optogenetics, facilitating the establishment of cutting-edge methods. Recent breakthroughs in protein design have opened up opportunities to develop protein-based tools that can precisely manipulate and monitor cellular activities. These advancements will significantly accelerate the development and application of optogenetic tools. This article emphasizes the pivotal role of protein design in the development of optogenetic tools, offering insights into potential future directions. We begin by providing an introduction to the historical development and fundamental principles of optogenetics, followed by an exploration of the operational mechanisms of key photosensitive domains, which includes clarifying the conformational changes they undergo in response to light, such as allosteric modulation and dimerization processes. Building on this foundation, we reveal the development of protein design tools that will enable the creation of even more sophisticated optogenetic techniques.

Abstract: In cancer cells, ATR signaling is crucial to tolerate the intrinsically high damage levels that normally block replication fork progression. Assembly of TopBP1, a multifunctional scaffolding protein, into condensates is required to amplify ATR kinase activity to the levels needed to coordinate the DNA damage response and manage DNA replication stress. Many ATR inhibitors are tested for cancer treatment in clinical trials, but their overall effectiveness is oven compromised by the emergence of resistance and toxicities. In this proof-of-concept study, we propose to disrupt the ATR pathway by targeting TopBP1 condensation. First, we screened a molecule-based library using a previously developed optogenetic approach and identified several TopBP1 condensation inhibitors. Amongst them, AZD2858 disrupted TopBP1 assembly induced by the clinically relevant topoisomerase I inhibitor SN-38, thereby inhibiting the ATR/Chk1 signaling pathway. We found that AZD2858 exerted its effects by disrupting TopBP1 self-interaction and binding to ATR in mammalian cells, and by increasing its chromatin recruitment n cell-free Xenopus laevis egg extracts. Moreover, AZD2858 prevented S-phase checkpoint induction by SN-38, leading to increased DNA damage and apoptosis in a colorectal cancer cell line. Lastly, AZD2858 showed synergistic effect in combination with the FOLFIRI chemotherapy regimen in a spheroid model of colorectal cancer.

Abstract: Cells process dynamic signaling inputs to regulate fate decisions during development. While oscillations or waves in key developmental pathways, such as Wnt, have been widely observed the principles governing how cells decode these signals remain unclear. By leveraging optogenetic control of the Wnt signaling pathway in both HEK293T cells and H9 human embryonic stem cells, we systematically map the relationship between signal frequency and downstream pathway activation. We find that cells exhibit a minimal response to Wnt at certain frequencies, a behavior we term anti-resonance. We developed both detailed biochemical and simplified hidden variable models that explain how anti-resonance emerges from the interplay between fast and slow pathway dynamics. Remarkably, we find that frequency directly influences cell fate decisions involved in human gastrulation; signals delivered at anti-resonant frequencies result in dramatically reduced mesoderm differentiation. Our work reveals a previously unknown mechanism of how cells decode dynamic signals and how anti-resonance may filter against spurious activation. These findings establish new insights into how cells decode dynamic signals with implications for tissue engineering, regenerative medicine, and cancer biology.

Abstract: Optogenetics is an emerging technology that uses the light-responsive effects of photosensitive proteins to regulate the function of specific cells. This technique combines genetics with optics, allowing for the precise inhibition or activation of cell functions through the introduction of photosensitive proteins into target cells and subsequent light stimulation to activate these proteins. In recent years, numerous basic and clinical studies have demonstrated the unique advantages of this approach in the research and treatment of neurological disorders. This review aims to introduce the fundamental principles and techniques of optogenetics, as well as its applications in the research and treatment of neurological diseases.

Abstract: Necroptosis plays a crucial role in the progression of various diseases and has gained substantial attention for its potential to activate antitumor immunity. However, the complex signaling networks that regulate necroptosis have made it challenging to fully understand its mechanisms and translate this knowledge into therapeutic applications. To address these challenges, an optogenetically activatable necroptosis system is developed that allows for precise spatiotemporal control of key necroptosis regulators, bypassing complex upstream signaling processes. The system, specifically featuring optoMLKL, demonstrates that it can rapidly assemble into functional higher-order "hotspots" within cellular membrane compartments, independent of RIPK3-mediated phosphorylation. Moreover, the functional module of optoMLKL significantly enhances innate immune responses by promoting the release of iDAMPs and cDAMPs, which are critical for initiating antitumor immunity. Furthermore, optoMLKL exhibits antitumor effects when activated in patient-derived pancreatic cancer organoids, highlighting its potential for clinical application. These findings will pave the way for innovative cancer therapies by leveraging optogenetic approaches to precisely control and enhance necroptosis.

Abstract: No organism is an island: organisms of varying taxonomic complexity, including genetic variants of a single species, can coexist in particular niches, cooperating for survival while simultaneously competing for environmental resources. In recent years, synthetic biology strategies have witnessed a surge of efforts focused on creating artificial microbial communities to tackle pressing questions about the complexity of natural systems and the interactions that underpin them. These engineered ecosystems depend on the number and nature of their members, allowing complex cell communication designs to recreate and create diverse interactions of interest. Due to its experimental simplicity, the budding yeast Saccharomyces cerevisiae has been harnessed to establish a mixture of varied cell populations with the potential to explore synthetic ecology, metabolic bioprocessing, biosensing, and pattern formation. Indeed, engineered yeast communities enable advanced molecule detection dynamics and logic operations. Here, we present a concise overview of the state-of-the-art, highlighting examples that exploit optogenetics to manipulate, through light stimulation, key yeast phenotypes at the community level, with unprecedented spatial and temporal regulation. Hence, we envision a bright future where the application of optogenetic approaches in synthetic communities (optoecology) illuminates the intricate dynamics of complex ecosystems and drives innovations in metabolic engineering strategies.

Abstract: G-protein-coupled receptors (GPCRs) are efficient guanine nucleotide exchange factors (GEFs) and exchange GDP to GTP on the Gα subunit of G-protein heterotrimers in response to various extracellular stimuli, including neurotransmitters and light. GPCRs primarily broadcast signals through activated G proteins, GαGTP and free Gβγ and are major disease drivers. Evidence shows that the ambient low threshold signalling required for cells is likely supplemented by signalling regulators such as non-GPCR GEFs and guanine nucleotide dissociation inhibitors (GDIs). Activators of G-protein signalling 3 (AGS3) are recognized as a GDI involved in multiple health and disease-related processes. Nevertheless, understanding of AGS3 is limited, and no significant information is available on its structure-function relationship or signalling regulation in living cells. Here, we employed in silico structure-guided engineering of a novel optogenetic GDI, based on the AGS3's G-protein regulatory motif, to understand its GDI activity and induce standalone Gβγ signalling in living cells on optical command. Our results demonstrate that plasma membrane recruitment of OptoGDI efficiently releases Gβγ, and its subcellular targeting generated localized PIP3 and triggered macrophage migration. Therefore, we propose OptoGDI as a powerful tool for optically dissecting GDI-mediated signalling pathways and triggering GPCR-independent Gβγ signalling in cells and in vivo.