Curated Optogenetic Publication Database

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: Recent advances in experimental model systems have improved our ability to study cardiovascular development, function, and disease with high spatial and temporal resolution. The zebrafish (Danio rerio) has emerged as a powerful vertebrate model for cardiovascular research due to its transparency, genetic tractability, and conserved cardiac physiology, similar to humans. These features allow real-time in vivo imaging, the functional assessment of cardiac performance, and the tracking of signaling pathways that are fundamental in cardiovascular development and disease. Recent advances in nanotechnology and optogenetics have introduced complementary tools for probing and manipulating cardiovascular systems with high spatial and temporal precision. Nanoparticle-based platforms enable the tunable delivery of drugs, nucleic acids, and imaging agents, while optogenetic systems allow the light-mediated control of gene expression, signaling pathways, and cardiac electrophysiology. In this review, we summarize recent progress in the application of nanoparticle-based technologies and the emerging optogenetic tools in zebrafish cardiovascular research, including the optical control of cardiac signaling and electrophysiology. We briefly discuss emerging complementary efforts toward nanoparticle and optogenetic approaches, how to overcome key technical limitations, such as light penetration and gene delivery, and how to facilitate the development of fully optical platforms for cardiovascular disease modeling and drug screening.

Abstract: Evolving dynamic, multi-state, and computational protein functionalities is challenging because it requires selection pressure on all the states of a protein of interest (POI) and the transitions between them. To create a continuous directed evolution paradigm for such properties, we genetically engineered budding yeast for optogenetic input to switch a POI "on" and "off," which, in turn, controls a Cdk1 cyclin that is essential for one cell-cycle stage but detrimental for another. The method, "optovolution," generates dynamic selection pressure on POI cycling at the timescale of tens of minutes. We used it to evolve 19 new variants of the LOV transcription factor El222, including in vivo green-light-responsive variants allowing LOV color-multiplexing. Evolving the PhyB-Pif3 optogenetic system, we discovered that loss of YOR1 makes supplementing phycocyanobilin (PCB) unnecessary. Finally, we demonstrated the generality of the method by evolving a non-light-responsive AND gate (PEST-rtTA). Optovolution makes difficult-to-engineer protein functionalities continuously evolvable.

Abstract: As an interdisciplinary frontier integrating optical technologies and genetic principles, optogenetics enables precise spatiotemporal control of gene expression and neuronal activity via light-sensitive molecular assemblies, thereby driving transformative advancements in biomedical fields. Red/far-red light optogenetic tools, by virtue of the advantages of long wavelengths, have emerged as powerful platforms for deep-tissue manipulations for both basic researches and clinical applications. Although a number of in-depth studies on various red/far-red light optogenetic tools and their biomedical applications have been published, there has not yet been a comprehensive review that systematically summarizes the advancements of diverse researches on this type of optogenetics. This article systematically delineates the technology of red/far-red light optogenetics, focusing on the molecular mechanisms and biomedical applications of two core photoreceptor protein families: phytochromes and channelrhodopsins. Phytochromes distributed in plants, bacteria and fungi undergo reversible red/far-red light-driven conformational conversion, initiating downstream signaling cascades that support various optogenetic technologies. Channelrhodopsins, originally microalgal blue-light-gated cation channels, are engineered into red-shifted variants, enabling rapid and non-invasive red/far-red light-controlled neuronal excitability manipulation at precise spatiotemporal resolution. The representative case studies of applications of phytochromes-based optogenetic tools in gene editing, transcriptional regulation, light-gated drug delivery and deep tissue imaging and diagnosis; as well as applications of red-shifted channelrhodopsins-based optogenetic tools in spatiotemporally precise neuromodulation are discussed in detail. Moreover, the main technical challenges in the utilization of red/far-red light optogenetic tools are analyzed. With continuous advancements of wavelength-optimized actuators and closed-loop control architectures, red/far-red light optogenetic techniques are poised to drive multidisciplinary convergence, offering unprecedented tools for decoding cellular dynamics and accelerating therapeutic discoveries.

Abstract: Precise control of gene expression is one of the fundamental goals of synthetic biology. Whether the objective is to modify endogenous cellular function or induce the expression of molecules for diagnostic and therapeutic purposes, gene regulation remains a key aspect of biological systems. Over time, advances in protein engineering and molecular biology have led to the creation of gene circuits capable of inducing the expression of specific proteins in response to external stimulus such as light. These optogenetic, or light-activated circuits hold significant potential for gene therapy as a tool for regulating the expression of therapeutic genes within cells. However, the applications of optogenetic systems can be limited by the lack of efficient ways to deliver light into cells or tissue. Our approach to address this challenge is to harness the power of bioluminescence to produce light directly inside cells using a luminescent enzyme. Combined with a photosensitive transcription factor, we report the development of a genetically encoded optogenetic circuit for the control of gene expression. Furthermore, we utilized a magneto-sensitive protein to engineer a split-protein version of this luminescent enzyme, where its reconstitution is driven by a 50 mT magnetic stimulus. Thus, resulting in a gene circuit activated by a combination of light and magnetic stimulus. We expect this work to advance the implementation of light-controlled systems without the need of external light sources, as well as serve as a basis for the development of future magneto-sensitive tools.

Abstract: The bacterial flagellar motor drives bacterial swimming and chemotaxis by rotating helical flagellar filaments. When Escherichia coli navigates chemical gradients, the motor switches from counterclockwise (CCW) during forward swimming to clockwise (CW) during direction-changing tumbles. The motor responds indirectly to extracellular chemosensory input to membrane-bound chemoreceptors using an intervening intracellular signaling pathway. How the motor responds to its direct input signal-the diffusible messenger phosphorylated CheY (CheY-P)-remains poorly understood. Steady-state motor measurements have been modeled as an allosteric switch between CCW/CW states that depends on mean CheY-P levels. Allosteric models have suggested that as many as 20 CheY-P molecules can be bound to the motor when it switches rotational direction. But steady-state models cannot predict the sensitivity of the motor to dynamic changes in CheY-P that essentially modulate chemotactic behavior. We present an optogenetic reagent that precisely controls the direct dynamical input signal to the motor. We designed a "caged" molecule, Opto-CheY, that is transiently activated by photon absorption. We find that activation and binding of one to three additional CheY-P molecules is sufficient to switch the motor from the CCW to CW state. The sensitivity of the motor to small changes in CheY-P occupancy helps resolve a long-standing paradox about the high sensitivity of the chemotactic response to external sensory input. Optogenetic biochemistry by light-activated uncaging of signal molecules is a new strategy to dissect information-processing in the living cell.

Abstract: Light is a pivotal environmental signal regulating diverse plant developmental and physiological processes, including seed germination, hypocotyl elongation, phototropism, metabolite biosynthesis, stress resistance, temperature response, and circadian rhythms. Multiple signal transduction pathways of ultraviolet, blue light, and red/far-red light as well as related protein interaction networks in plants have been identified. Deciphering the mechanisms of light perception and signal transduction is of great significance to crop breeding and optogenetic manipulation. Structural biology has profoundly advanced the studies of light signal transduction by elucidating high-resolution three-dimensional (3D) structures of photoreceptors and their downstream signaling components. These studies uncover the molecular basis underlying perception and transduction of different light signals by plants. This review summarizes key structural findings of plant light signal transduction, highlighting the architectures and molecular functions of photoreceptors and associated signaling factors. We also outline the mechanisms underlying photoreceptor activation, inhibition, and regulatory interactions within light signaling networks and discuss the challenges in this field.

Abstract: An optogenetic split-GAL4 system, ShineGAL4, allows genes to be manipulated with unprecedented spatiotemporal precision. Here, we convert a panel of 14 GAL4 drivers widely used in Drosophila research into their ShineGAL4 counterparts. Homology assisted CRISPR knock-in (HACK) is used to replace GAL4 with the GAL4 DNA binding domain fused to a Magnet photoswitch. We show that the resulting ShineGAL4 drivers enable gene expression to be rapidly induced by light specifically in fat body, muscles, enterocytes, oenocytes, Malpighian tubules, neurons, neuroblast lineages, glial subtypes or in all glia. We also develop an optogenetic cassette for photoactivation of GAL4 in 'silent' FLP-out clones. This panel of optogenetic tools will enable precise spatiotemporal control of gene expression in a wide range of different Drosophila tissues and cell-types.

Abstract: Microbial optogenetic tools can regulate gene expression with spatial and temporal precision, offering excellent potential for single-cell resolution studies. However, bacterial optogenetic systems have primarily been deployed for population-level experiments. It is not always clear how these tools perform in single cells, where stochastic effects can be substantial. In this study, we focus on optogenetic Cre recombinase and compare the performance of three variants (OptoCre-REDMAP, OptoCre-Vvd, and PA-Cre) for their population-level and single-cell activity. We quantify recombination efficiency, expression variability, and activation dynamics using reporters which produce changes in fluorescence or antibiotic resistance following light-induced Cre activity. We find that optogenetic recombinase performance can be reporter-dependent. Further, single-cell analysis reveals highly heterogeneous activity, with substantial variation in the efficiency and timing of recombinase activity from cell to cell. These findings suggest important criteria for selecting optogenetic recombinases and indicate areas for optimization to improve single-cell capabilities of bacterial optogenetic tools.

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: The genome folds inside the cell nucleus into hierarchical architectural features, such as chromatin loops and domains. If and how this genome organization influences the regulation of gene expression remains only partially understood. The structure-function relationship of genomes has traditionally been probed by population-wide measurements after mutation of crucial DNA elements or by perturbation of chromatin-associated proteins. To circumvent possible pleiotropic effects of such approaches, we have developed OptoLoop, an optogenetic system that allows direct manipulation of chromatin contacts by light in a controlled fashion. OptoLoop is based on the fusion between a nuclease-dead SpCas9 protein and the light-inducible oligomerizing protein CRY2. We demonstrate that OptoLoop can bring together genomically distant, repetitive DNA loci. As a proof-of-principle application of OptoLoop, we probed the functional role of DNA looping in the regulation of the human telomerase gene TERT. By analyzing the extent of chromatin looping and nascent RNA production at individual alleles, we find evidence for looping-mediated repression of TERT. In sum, OptoLoop represents a novel means for the interrogation of structure-function relationships in the genome.

Abstract: Estrogen receptor (ER)-mediated genomic actions are crucial for maintaining various physiological functions, and their dysfunction is associated with numerous human diseases. Traditional estrogen replacement therapy (ERT) is commonly used to manage estrogen deficiency-related conditions, such as vulvovaginal atrophy during menopause, but its systemic effects pose notable risks. This study introduces OptoER, an optogenetic tool engineered to precisely modulate ER-mediated genomic pathways through light-induced transcription regulation, offering spatial-temporal control over ER-dependent gene expression. Our in vitro studies demonstrate that OptoER significantly enhances ER-specific gene transcription and protein synthesis, leading to improved cell proliferation and migration. In a proof-of-principle study using ovariectomized (OVX) mice, OptoER demonstrated considerable therapeutic potential for vaginal atrophy, with observed improvement in epithelial thickness and keratinization. These findings suggest that OptoER provides a targeted therapeutic strategy for estrogen deficiency conditions, with significant implications for treating vaginal atrophy and promoting regenerative healing in estrogen-deprived tissues.

Abstract: Synthetic microbial consortia (SMCs) represent a paradigm shift from monocultures to multi-strain systems that leverage ecological interactions for enhanced environmental adaptation and bioproduction. This review systematically sorts out engineering strategies for constructing stable SMCs, focusing on three core principles regarding host selection based on obligate mutualism (e.g., auxotrophs), pathway modularization to resolve metabolic conflicts, and dynamic regulation using tools like quorum sensing and optogenetics. We demonstrate the efficacy of SMCs in diverse applications including high-value compound synthesis and lignocellulosic biomass conversion through consolidated bioprocessing and inhibitor mitigation. SMCs enabling advanced functions in engineered living materials, environmental remediation, and biomedical innovation via division of labor are also described. Despite such progress, challenges in scalability and real-time control of SMCs under industrial conditions remain. We conclude that SMCs serve to bridge evolutionary ecology and biotechnology, offering robust solutions for sustainable biomanufacturing and beyond.

Abstract: Bis(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) plays a crucial role in bacterial signaling pathways, allowing bacterial cells to respond to various environmental stimuli. The prevalence of c-di-GMP and its potential applications underscore the necessity for developing tools and methods to regulate intracellular c-di-GMP levels. Optogenetic control of c-di-GMP dynamics is particularly attractive because it enables tunable and spatiotemporal regulation of c-di-GMP metabolism. The development of sensitive optogenetic control systems requires highly active, light-responsive c-di-GMP synthases. Here, we report an engineered, highly active photosensitive c-di-GMP synthase, BphS-13. This engineered c-di-GMP synthase was developed from a near-infrared (NIR) light-activable bacteriophytochrome c-di-GMP synthase, BphS, using a three-step directed evolution process that included error-prone PCR, in vitro homologous recombination, and site-directed mutagenesis. After two rounds of this directed evolution strategy, we generated a BphS variant with 13 mutations, referred to as BphS-13. The diguanylate cyclase (DGC) activity of BphS-13 was approximately 13 times higher than that of the original BphS, and it exhibited tightly regulated DGC activity in response to NIR light with minimal leakage in the dark. We then demonstrated the effectiveness of BphS-13 in controlling biofilm dynamics. Overall, this study highlights BphS-13 as a highly active and photosensitive tool for optogenetic applications in biotechnology and suggests its future potential application in mammalian systems for precise control of gene expression, particularly given the lack of native c-di-GMP signaling pathways in mammalian cells.

Abstract: Light-induced membrane fusion has become a pivotal technique for constructing and functionalizing synthetic cells by enabling precise control over membrane merging events. Traditional fusion approaches that rely on chemical, physical, and mechanical stimuli frequently lack both specificity and reversibility, limiting their utility in mimicking dynamic cellular processes. Here, we review advances employing photosensitive molecules and optogenetic tools that facilitate spatiotemporally controlled fusion of lipid and polymer vesicles, enabling dynamic content exchange and membrane remodeling. These approaches have enhanced synthetic cell assembly, molecular transport, and signal transduction, with applications extending to drug delivery and biosensing. Despite challenges in efficiency and biocompatibility, ongoing innovations in photosensitizer design and light activation strategies promise to expand the capabilities of synthetic biology platforms. This work underscores the potential of light-induced fusion to advance the development of intelligent nanomaterials and functional synthetic cellular systems.

Abstract: System identification approaches are commonly used in engineering to infer simple yet predictive models of complex systems from their responses to time-dependent perturbations. Here, we apply this strategy at the whole organism scale, establishing a predictive model of commitment to metamorphosis in Drosophila. At this critical point in animal development, the larva stops feeding and proceeds to take on the adult form. The neuroendocrine circuits governing commitment to metamorphosis assess the growth and patterning programs, eventually triggering the production of systemic hormones that terminate growth and initiate metamorphosis. Previous studies of these circuits relied on relatively static genetic perturbations and starvation experiments. Here, we take advantage of optogenetic approaches in Drosophila to flexibly perturb a key signaling node within the endocrine gland in otherwise undisturbed larvae. We used this approach to infer parameters in a compact mathematical model and demonstrate that it makes accurate predictions of larval commitment to metamorphosis. Our work paves the way for quantitative studies of other juvenile-to-adult transitions, including mammalian puberty, which relies on strikingly similar mechanisms.

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: Numerous organisms have evolved the ability to utilize light through photoreceptor proteins that mediate diverse biological processes. Currently, several optogenetic sensor systems are widely used in yeast. However, when these systems are applied for gene repression to regulate endogenous yeast gene expression, they typically require the insertion of corresponding target sites near the native promoter of the gene of interest to achieve precise modulation. To address these constraints, a novel blue light-inducible optogenetic tool designated iLight9 was developed, a single-component optogenetic biosensor integrated with the CRISPR-dCas9 platform. The stability of the iLight9 system was further enhanced by employing a strategy involving the addition of a protein degradation tag. The resulting system was designated as iLight9O, which facilitated programmable regulation of distinct genes through the introduction of specific sgRNAs. Subsequently, systematic metabolic engineering strategies were employed to construct an efficient patchoulol-producing cell factory in Saccharomyces cerevisiae. Moreover, a two-step isoprenol utilization (IU) pathway was introduced into the recombinant strain to enhance its capacity for patchoulol biosynthesis. Crucially, the iLight9O system was adopted to dynamically downregulate squalene synthase, a key enzyme in the competing squalene biosynthetic pathway. This optogenetic flux control strategy increased patchoulol titers by 66 % in the IU-optimized strain and 24 % in the MVAIU2 strain, demonstrating significant improvements over static engineering approaches.

Abstract: Reversibly photoswitchable fluorescent proteins (rsFPs) represent a unique class of genetically encoded probes that undergo light-driven transitions between non-fluorescent OFF and emissive ON states. Their distinctive switching properties enable repeated, non-destructive control of fluorescence and have become central to advanced bioimaging approaches. In this review, we provide a critical overview of the molecular mechanisms underlying rsFP function, focusing on GFP-like proteins and fluorogen-activating systems that employ external chromophores. We describe switching kinetics, ON/OFF contrast, and fatigue as fundamental performance parameters, and highlight mechanistic insights from spectroscopy, crystallography, and computational studies. The three subclasses of GFP-like rsFPs-negative, positive, and decoupled types-are discussed in detail, alongside external-chromophore systems such as FAST, UnaG, FbFPs, and biliverdin-binding near-infrared proteins. We further survey a wide range of applications, including super-resolution microscopy, functional biosensing, multiplex discrimination, anisotropy-based analyses, diffusion and transport studies, optical data storage, and optogenetic control. Finally, we outline emerging strategies for improving brightness, photostability, spectral diversity, and switching robustness, emphasizing opportunities for rational protein engineering guided by structural and computational approaches. Together, these developments establish rsFPs as versatile, chemically tunable tools that expand the frontiers of fluorescence imaging and quantitative biology.

Abstract: Photoreceptor proteins regulate fundamental biological processes such as vision, photosynthesis and circadian rhythms1. A large photoreceptor subfamily uses vitamin B12 derivatives for light sensing2, contrasting with the well-established mode of action of these organometallic derivatives in thermally activated enzymatic reactions3. The exact molecular mechanism of B12 photoreception and how this differs from the thermal pathways remains unknown. Here we provide a detailed description of photoactivation in the prototypical B12 photoreceptor CarH4,5 from nanoseconds to seconds, combining time-resolved and temperature-resolved structural and spectroscopic methods with quantum chemical calculations. Building on the crystal structures of the initial tetrameric dark and final monomeric light-activated states5, our structural snapshots of key intermediates in the truncated B12-binding domain illustrate how photocleavage of a cobalt-carbon (Co-C) bond within the B12 chromophore adenosylcobalamin triggers a series of structural changes that propagate throughout CarH. Breakage of the photolabile Co-C5' bond leads to the formation of a previously unknown adduct that links the C4' position of the adenosyl moiety to the Co ion and can subsequently be cleaved thermally over longer timescales to allow release of the adenosyl group, ultimately causing tetramer dissociation4,5. This adduct, which differentiates CarH from thermally activated B12 enzymes, steers the photoactivation pathway and acts as the molecular bridge between photochemical and photobiological timescales. The biological relevance of our study is corroborated by kinetic data on full-length CarH in the presence of DNA. Our results offer a spatiotemporal understanding of CarH photoactivation and pave the way for designing B12-dependent photoreceptors for optogenetic applications.

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: Despite its central role in signaling, the influence of protein architecture on phytochrome structure and reactivity remains poorly understood. Here, we test how removal of the PHY domain reshapes the far-red-absorbing Pfr energy landscape and photochemical branching in the knotless phytochrome All2699g1g2.

Abstract: The light-mediated, specific, and precise control of cell functions enabled by optogenetics has become a versatile method for investigating and combatting cancer. An increasing set of optogenetic tools enables tightly controlled regulation of ion flux across biological membranes, gene expression, gene editing, and protein-protein interactions and is being used to interrogate hallmark traits of cancer at the cellular, subcellular, and organismic level. This enables, on the one hand, the identification of critical signaling circuits required for cancer development and progression in vitro and in animal models and can flag potential intervention points for pharmacologic interference. On the other hand, optogenetics can improve the level of control in cell-based therapeutics. The current article provides a review of optogenetic tools and approaches used in the cancer research field and their multiple applications for improving our understanding of signal transduction pathways, modulating immune functions in the tumor microenvironment, facilitating drug screening, or directly attacking cancer cells. Key advantages and achievements of optogenetics in the cancer research field and remaining barriers for clinical applications are discussed.

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.