Curated Optogenetic Publication Database

Abstract: The microtubule-associated protein tau aggregates into oligomeric complexes that highly correlate with Alzheimer’s disease (AD) progression. Increasing evidence suggests that nuclear membrane disruption occurs in AD and related tauopathies, but whether this is a cause or consequence of neurodegeneration remains unclear. Using the optogenetically inducible 4R1N Tau::mCherry::Cry2Olig (optoTau) system in iPSC-derived neurons, we demonstrate that tau oligomerization triggers nuclear rupture and nuclear membrane invagination. Pathological tau accumulates at sites of invagination, inducing structural abnormalities in the nuclear envelope and piercing into the nuclear space. These findings were confirmed in the humanized P301S tau (PS19) transgenic mouse model, where nuclear envelope disruption appeared as an early-onset event preceding neurodegeneration. Further validation in post-mortem AD brain tissues revealed nuclear lamina disruption correlating with pathological tau emergence in early-stage patients. Notably, electron microscopy shows that tau-induced nuclear invagination triggers global chromatin reorganization, potentially driving aberrant gene expression and protein translation associated with AD. These findings suggest that nuclear membrane disruption is an early and possibly causative event in tau-mediated neurodegeneration, establishing a mechanistic link between tau oligomerization and nuclear stress. Further investigation into nuclear destabilization could inform clinical strategies for mitigating AD pathogenesis.

Abstract: The Arabidopsis blue light photoreceptor cryptochrome 2 (CRY2) responds to blue light to initiate a variety of plant light-based behaviors and has been widely used for optogenetic engineering. Despite these important biological functions, the precise photoactivation mechanism of CRY2 remains incompletely understood. In light, CRY2 undergoes tetramerization and binds to partner proteins, including the transcription factor CIB1. Here we used yeast-two hybrid screening and deep mutational scanning to identify CRY2 amino acid changes that result in constitutive interaction with CIB1 in dark. The majority of CRY2 variants show constitutive CIB1 interaction mapped to two regions, one near the FAD chromophore and a second region located near the ATP binding site. Further testing of CRY2 variants from each region revealed three mapping near to the FAD binding pocket (D393S, D393A, and M378R) that also form constitutive CRY2-CRY2 homomers in dark, suggesting they adopt global conformational changes that mimic the photoactive state. Characterization of D393S in the homolog pCRY from Chlamydomonas reinhardtii using time-resolved UV-vis spectroscopy revealed that the FAD chromophore fails to form the neutral radical as signaling state upon illumination. Size exclusion chromatography of D393S shows the presence of homomers instead of a monomer in the dark, providing support for a hyperactive variant decoupled from the FAD. Our work provides new insight into photoactivation mechanisms of plant cryptochromes relevant for physiology and optogenetic application by revealing and localizing distinct activation pathways for light-driven CRY2-CIB1 and CRY2-CRY2 interactions.

Abstract: Optogenetic systems offer precise control over gene expression, but leaky activity in the dark limits their dynamic range and, consequently, their applicability. Here, we enhanced an optogenetic system based on a split T7 RNA polymerase fused to blue-light-inducible Magnets by incorporating a tet-controlled riboregulatory module. This module exploits the photosensitivity of anhydrotetracycline and the designability of synthetic small RNAs to digitize light-controlled gene expression, implementing a repressive action over the translation of a polymerase fragment gene that is relieved with blue light. Our engineered system exhibited 13-fold improvement in dynamic range upon blue light exposure, which even raised to 23-fold improvement when using cells preadapted to chemical induction. As a functional demonstration, we implemented light-controlled antibiotic resistance in bacteria. Such integration of regulatory layers represents a suitable strategy for engineering better circuits for light-based biotechnological applications.

Abstract: Light-inducible regulatory proteins are powerful tools to interrogate fundamental mechanisms driving cellular behavior. In particular, genetically encoded photosensory domains fused to split proteins can tightly modulate protein activity and gene expression. While light-inducible split protein systems have performed well individually, few multichromatic and orthogonal gene regulation systems exist in mammalian cells. The design space for multichromatic circuits is limited by the small number of orthogonally addressable optogenetic switches and the types of effectors that can be actuated by them. We developed a library of red light-inducible recombinases and directed patterned myogenesis in a mesenchymal fibroblast-like cell line. To address the limited number of light-inducible domains (LIDs) responding to unique excitation spectra, we multiplexed light-inducible recombinases with our "Boolean logic and arithmetic through DNA excision" (BLADE) platform. Multiplexed optogenetic tools will be transformative for understanding the role of multiple interacting genes and their spatial context in endogenous signaling networks.

Abstract: Light is a critical environmental factor that governs the growth and development of plants. Plants have specialised photoreceptor proteins, which allow them to sense both quality and quantity of light and drive a wide range of responses critical for optimising growth, resource use and adaptation to changes in environment. Understanding the role of these photoreceptors in plant biology has opened up potential avenues for engineering crops with enhanced productivity by engineering photoreceptor activity and/or action. The ability to manipulate plant genomes through genetic engineering and synthetic biology approaches offers the potential to unlock new agricultural innovations by fine-tuning photoreceptors or photoreceptor pathways that control plant traits of agronomic significance. Additionally, optogenetic tools which allow for precise, light-triggered control of plant responses are emerging as powerful technologies for real-time manipulation of plant cellular responses. As these technologies continue to develop, the integration of photoreceptor engineering and optogenetics into crop breeding programs could potentially revolutionise how plant researchers tackle challenges of plant productivity. Here we provide an overview on the roles of key photoreceptors in regulating agronomically important traits, the current state of plant photoreceptor engineering, the emerging use of optogenetics and synthetic biology, and the practical considerations of applying these approaches to crop improvement. This review seeks to highlight both opportunities and challenges in harnessing photoreceptor engineering approaches for enhancing plant productivity. In this review, we provide an overview on the roles of key photoreceptors in regulating agronomically important traits, the current state of plant photoreceptor engineering, the emerging use of optogenetics and synthetic biology, and the practical considerations of applying these approaches to crop improvement.

Abstract: Red light, characterized by superior tissue penetration and minimal phototoxicity, represents an ideal wavelength for optogenetic applications. However, the existing tools for reversible protein inhibition by red light remain limited. Here, we introduce R-LARIAT (red light-activated reversible inhibition by assembled trap), a novel optogenetic system enabling precise spatiotemporal control of protein function via 660 nm red-light-induced protein clustering. Our system harnesses the rapid and reversible binding of engineered light-dependent binders (LDBs) to the bacterial phytochrome DrBphP, which utilizes the endogenous mammalian biliverdin chromophore for red light absorption. By fusing LDBs with single-domain antibodies targeting epitope-tagged proteins (e.g., GFP), R-LARIAT enables the rapid sequestration of diverse proteins into light-responsive clusters. This approach demonstrates high light sensitivity, clustering efficiency, and sustained stability. As a proof of concept, R-LARIAT-mediated sequestration of tubulin inhibits cell cycle progression in HeLa cells. This system expands the optogenetic toolbox for studying dynamic biological processes with high spatial and temporal resolution and holds the potential for applications in living tissues.

Abstract: Bacterial therapy has emerged as a promising approach for disease treatment due to its environmental sensitivity, immunogenicity, and modifiability. However, the clinical application of engineered bacteria is limited by differences of expression levels in patients and possible off-targeting. Optogenetics, which combines optics and genetics, offers key advantages such as remote controllability, non-invasiveness, and precise spatiotemporal control. By utilizing optogenetic tools, the behavior of engineered bacteria can be finely regulated, enabling on-demand control of the dosage and location of their therapeutic products. In this review, we highlight the latest advancements in the optogenetic engineering of bacteria for light-controlled disease theranostics and therapeutic regulation. By constructing a three-dimensional analytical framework of “sense-produce-apply”, we begin by discussing the key components of bacterial optogenetic systems, categorizing them based on their photosensitive protein response to blue, green, and red light. Next, we introduce innovative light-producing tools that extend beyond traditional light sources. Then, special emphasis is placed on the biomedical applications of optogenetically engineered bacteria in treating diseases such as cancer, intestinal inflammation and systemic disease regulation. Finally, we address the challenges and future prospects of bacterial optogenetics, outlining potential directions for enhancing the safety and efficacy of light-controlled bacterial therapies. This review aims to provide insights and strategies for researchers working to advance the application of optogenetically engineered bacteria in drug delivery, precision medicine and therapeutic regulation.

Abstract: Optogenetics is an interdisciplinary field wherein optical and genetic engineering methods are employed together to impart photounresponsive cells (usually of higher animals) the ability to respond to light through expression of light-sensitive proteins sourced generally from algae or bacteria. It enables precise spatiotemporal control of various cellular activities through light stimulation. Recently, emerging as a synthetic biology-based approach for diabetes management, optogenetics can provide user-control of hormonal secretion by photoactivation of a suitably modified cell. For around a decade, studies have been performed on the applicability of various light-sensitive proteins and their incorporation into pancreatic and nonpancreatic cells for photoinduced insulin secretion. Further, in vivo studies demonstrated amelioration of diabetes in mouse models through photoactivation of the implanted engineered cells. Here, we attempt to highlight the various optogenetic approaches explored in terms of influencing the insulin secretion pathway at different points in light of the natural insulin secretion pathway in pancreatic β cells. We also discuss how transgenic cells of both pancreatic as well as nonpancreatic origin are exploited for photoinduced secretion of insulin. Recent advances on integration of “smart” technologies for remote control of light irradiation and thereby insulin secretion from implanted engineered cells in preclinical models are also described. Additionally, the need for further comprehensive studies on irradiation parameters, red-shifted opsins, and host–cell interaction is stressed to realize the full potential of optogenetics as a clinically applicable modality providing user-controlled “on demand” hormonal secretion for better management of diabetes.

Abstract: Lamellipodia are sheet-like protrusions essential for cell migration and endocytosis, but their ultrastructural dynamics remain poorly understood because conventional electron microscopy lacks temporal resolution. Here, we combined optogenetics with cryo-electron tomography (cryo-ET) to visualize the actin cytoskeleton and membrane structures during lamellipodia formation with temporal precision. Using photoactivatable-Rac1 (PA-Rac1) in COS-7 cells, we induced lamellipodia formation with a 2-min blue light irradiation, rapidly vitrified samples, and analyzed their ultrastructure with cryo-ET. We obtained 16 tomograms of lamellipodia at different degrees of extension from three cells. These revealed small protrusions with unbundled actin filaments, "mini filopodia" composed of short, bundled actin filaments at the leading edge, and actin bundles running nearly parallel to the leading edge within inner regions of lamellipodia, suggesting dynamic reorganizations of the actin cytoskeleton. This approach provides a powerful framework for elucidating the ultrastructural dynamics of cellular processes with precise temporal control.

Abstract: Cell signaling regulates a wide range of biological processes including development, homeostasis, and disease. Accessible technologies to precisely manipulate signaling have important applications in basic and translational research. Here, we introduce an optogenetic toolkit comprised of 1) a zebrafish-optimized FGF signaling activator, 2) a single-transcript Nodal signaling activator, and 3) a previously established BMP signaling activator. We thoroughly characterize this suite of tools in zebrafish embryos and show that they provide tunable, light-dependent spatiotemporal control of signaling in vivo. In response to blue light (∼455 nm), receptor kinase domains fused to blue light-dimerizing LOV domains enable robust signaling activation with minimal ectopic activity in the dark or at wavelengths over 495 nm. Optogenetic activation by each tool is pathway-specific and results in increased expression of known target genes. Signaling is activated with rapid on/off kinetics, and activation strength depends on light irradiance. Finally, we demonstrate spatially localized signaling activation with our optimized FGF activator. Together, our results establish this optogenetic toolkit as a potent experimental platform to rapidly, directly, and adjustably activate FGF, BMP, and Nodal signaling in zebrafish embryos.

Abstract: Near-infrared (NIR) fluorescent proteins and optogenetic tools derived from bacterial phytochromes' photosensory core modules (PCMs) operate within the first (NIR-I) tissue transparency window under single-photon activation. Leveraging two-photon (2P) light in the second transparency window (NIR-II) for photoswitching bacterial phytochromes between Pr and Pfr absorption states offers significant advantages, including enhanced tissue penetration, spatial resolution, and signal-to-noise ratio. However, 2P photoconversion of bacterial phytochromes remains understudied. Here, we study the non-linear Pr to Pfr photoconversion's dependence on irradiation wavelength (1180–1360 nm) and energy fluence (41–339 mJ/cm2) for the PCM of DrBphP bacterial phytochrome. Our findings reveal substantially higher photoconversion efficiency for the engineered monomeric DrBphP-PCM (73%) compared to the natural dimeric DrBphP-PCM (57%). Molecular mechanical calculations, based on experimentally determined 2P absorption cross-section coefficients for the monomer (167 GM) and dimer (170 GM), further verify these results. We demonstrate both short- (SWE) and long-wavelength excitation (LWE) fluorescence of the Soret band using 405 and 810–890 nm laser sources, respectively. Under LWE, fluorescence emission (724 nm) exhibits saturation at a peak power density of 1.5 GW/cm2. For SWE, we observe linear degradation of fluorescence for both DrBphP-PCMs, decreasing by 32% as the temperature rises from 19 to 38°C. Conversely, under LWE, the monomeric DrBphP-PCM's brightness increases up to 182% (at 37°C), surpassing the dimeric form's fluorescence rise by 39%. These findings establish the monomeric DrBphP-PCM as a promising template for developing NIR imaging and optogenetic probes operating under the determined optimal parameters for its 2P photoconversion and LWE fluorescence.

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: Biomolecular networks can dynamically encode information, generating time-varying patterns of activity in response to an input. Here we find that dynamic encoding can also be performed by individual proteins. BcLOV4 is an optogenetic protein that uniquely displays pulsatory activation in response to a step input of light, and response dynamics can be shaped by both light and temperature. However, how the BcLOV4 protein generates this step-to-pulse response is not understood. Here we combined live cell imaging and simulations to find that the activity pulse results from an intramolecular incoherent feedforward loop (IFFL) implemented by competitive interactions between protein domains that separately respond to light or temperature. We identified these light- and temperature-sensitive regions and found that they implement the IFFL by competitively caging an activation region. Structural and sequence analysis revealed temperature-responsive regions of BcLOV4 which allowed experimental tuning of activation dynamics and suggested that tuning has also occurred throughout evolution. These findings enabled the generation of more thermostable optogenetic tools and identified a modular thermosensitive domain that endowed thermogenetic control over unrelated proteins. Our findings uncover principles of dynamic and combinatorial signal processing in individual proteins that will fuel development of more sophisticated and compact synthetic systems.

Abstract: S-Palmitoylation is a reversible post-translational modification that tunes the localization, stability, and function of an impressive array of proteins including ion channels, G-proteins, and synaptic proteins. Indeed, altered protein palmitoylation is linked to various human diseases including cancers, neurodevelopmental and neurodegenerative diseases. As such, strategies to selectively manipulate protein palmitoylation with enhanced temporal and subcellular precision are sought after to both delineate physiological functions and as potential therapeutics. Here, we develop chemogenetically and optogenetically inducible engineered depalmitoylases to manipulate the palmitoylation status of target proteins. We demonstrate that this strategy is programmable allowing selective depalmitoylation in specific organelles, triggered by cell-signaling events, and of individual protein complexes. Application of this methodology revealed bidirectional tuning of neuronal excitability by distinct depalmitoylases. Overall, this strategy represents a versatile and powerful method for manipulating protein palmitoylation in live cells, providing insights into their regulation in distinct physiological contexts.

Abstract: During mating in budding yeast, cells use pheromones to locate each other and fuse. This model system has shaped our current understanding of signal transduction and cell polarization in response to extracellular signals. The cell populations producing extracellular signal landscapes themselves are, however, less well understood, yet crucial for functionally testing quantitative models of cell polarization and for controlling cell behavior through bioengineering approaches. Here we engineered optogenetic control of pheromone landscapes in mating populations of budding yeast, hijacking the mating-pheromone pathway to achieve spatial control of growth, cell morphology, cell-cell fusion, and distance-dependent gene expression in response to light. Using our tool, we were able to spatially control and shape pheromone gradients, allowing the use of a biophysical model to infer the properties of large-scale gradients generated by mating populations in a single, quantitative experimental setup, predicting that the shape of such gradients depends quantitatively on population parameters. Spatial optogenetic control of diffusible signals and their degradation provides a controllable signaling environment for engineering artificial communication and cell-fate systems in gel-embedded cell populations without the need for physical manipulation.

Abstract: Cytokinesis challenges epithelial tissue homeostasis by generating forces that pull on neighboring cells. Junction reinforcement at the furrow in Xenopus epithelia regulates the speed of furrowing, suggesting that cytokinesis is subject to resistive forces from epithelial neighbors. We show that contractility factors accumulate near the furrow in neighboring cells, and increasing neighbor cell stiffness slows furrowing. Optogenetically increasing contractility in one or both neighbor cells slows furrowing or induces cytokinetic failure. Uncoupling mechanotransduction between dividing cells and their neighbors increases the furrow ingression rate, alters topological cell packing following cytokinesis, and impairs barrier function at the furrow. Computational modeling validates our findings and provides additional insights about epithelial mechanics during cytokinesis. We conclude that forces from the cytokinetic array must be carefully balanced with restraining forces generated by neighbor cells to regulate the speed and success of cytokinesis and maintain epithelial homeostasis.

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: 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: Chickens serve as an excellent model organism for developmental biology, offering unique opportunities for precise spatiotemporal access to embryos within eggs. Optogenes are light-activated proteins that regulate gene expression, offering a non-invasive method to activate genes at specific locations and developmental stages, advancing developmental biology research. This study employed the Magnet-Cre optogenetic system to control gene expression in developing chicken embryos. Magnet-Cre consists of two light-sensitive protein domains that dimerize upon light activation, each attached to an inactive half of the Cre recombinase enzyme, which becomes active upon dimerization. We developed an all-in-one plasmid containing a green fluorescent protein marker, the Magnet-Cre system, and a light-activated red fluorescent protein gene. This plasmid was electroporated into the neural tube of Hamburger and Hamilton (H&H) stage 14 chicken embryos. Embryo samples were cleared using the CUBIC protocol and imaged with a light sheet microscope to analyze optogenetic activity via red-fluorescent cells. We established a pipeline for Magnet-Cre activation in chicken embryos, demonstrating that a single 3-min exposure to blue light following incubation at 28 °C was sufficient to trigger gene activity within the neural tube, with increased activity upon additional light exposure. Finally, we showed a spatiotemporal control of gene activity using a localized laser light induction. This research lays the groundwork for further advancements in avian developmental biology and poultry research, enabling spatiotemporal control of genes in both embryos and transgenic chickens.

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: Morphogen gradients convey essential spatial information during tissue patterning. Although the concentration and timing of morphogen exposure are both crucial, how cells interpret these graded inputs remains challenging to address. We employed an optogenetic system to acutely and reversibly modulate the nuclear concentration of the morphogen Dorsal (DL), homolog of NF-κB, which orchestrates dorsoventral patterning in the Drosophila embryo. By controlling DL nuclear concentration while simultaneously recording target gene outputs in real time, we identified a critical window for DL action that is required to instruct patterning and characterized the resulting effect on spatiotemporal transcription of target genes in terms of timing, coordination and bursting. We found that a transient decrease in nuclear DL levels at nuclear cycle 13 leads to reduced expression of the mesoderm-associated gene snail (sna) and partial derepression of the neurogenic ectoderm-associated target short gastrulation (sog) in ventral regions. Surprisingly, the mispatterning elicited by this transient change in DL was detectable at the level of single-cell transcriptional bursting kinetics, specifically affecting long inter-burst durations. Our approach of using temporally resolved and reversible modulation of a morphogen in vivo, combined with mathematical modeling, establishes a framework for understanding the stimulus-response relationships that govern embryonic patterning.

Abstract: A near-infrared optogenetic system was developed for the controlled expression of therapeutics in engineered oncolytic bacteria, demonstrating significant anti-tumor efficacy in multiple tumor mouse models. This approach offers a non-invasive, customizable method for targeted solid tumor therapy and has broader applications in engineered living therapeutics.

Abstract: Cell migration is crucial for development and tissue homeostasis, while its dysregulation leads to severe pathologies. Cell migration is driven by the extension of actin-based lamellipodia protrusions, powered by actin polymerization, which is tightly regulated by signaling pathways, including Rho GTPases and Ca2+ signaling. While the importance of Ca2+ signaling in lamellipodia protrusions has been established, the molecular mechanisms linking Ca2+ to lamellipodia assembly are unknown. Here, we identify a novel Ca2+ signaling axis involving the mechano-gated channel TRPV4, which regulates lamellipodia protrusions in various cell types. Using Ca2+ and FRET imaging, we demonstrate that TRPV4-mediated Ca2+ influx upregulates RhoA activity within lamellipodia, which then facilitates formin-mediated actin assembly. Mechanistically, we identify CaMKII and TEM4 as key mediators relaying the TRPV4-mediated Ca2+ signal to RhoA. These data define a molecular pathway by which Ca2+ influx regulates small GTPase activity within a specific cellular domain – lamellipodia - and demonstrate the critical role in organizing the actin machinery and promoting cell migration in diverse biological contexts.

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.