Curated Optogenetic Publication Database

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

Abstract: Cofilin is a key regulator of actin dynamics that, along with a myriad of other actin-binding proteins, controls the balance of F- and G-actin in numerous cell types. While prior structural studies of the cofilin-actin binding interface have delineated many critical interactions between cofilin and actin, the roles of some residues within the cofilin-actin binding interface remain poorly defined. In this study, we investigate the role of cofilin S119 in the cofilin-actin interaction. Despite its unique position within the cofilin-actin interface and its putative role as a phosphorylation site, relatively little direct evidence exists to define whether it plays an important role in cofilin-actin dynamics. Using site-directed mutagenesis, we demonstrate that mutation of S119 to aromatic amino acids (W, F, Y) results in cofilins with strong actin bundling activity in living cells. This activity can be countered by the incorporation of mutants that disfavor actin rod forming activity (R21Q). Mutation of S119 to phospho-mimic (E) and non-phosphorylated (A) residues either strongly inhibits (E) or modestly increases (A) actin bundling activity. Expression of the S119W mutant in neurons reveals its impacts on spine length and size, while FRAP studies show that its mobile fraction is intermediate between that of LifeAct and WT cofilin. Finally, it is shown that the strong actin bundling phenotype associated with S119W inhibits the progression of optogenetically induced apoptosis.

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

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

Abstract: Neuronal activity robustly engages the extracellular signal-regulated kinase (ERK) signaling pathway through Ca2+-dependent mechanisms; however, whether ERK can acutely and causally modulates ongoing neuronal activity remains unsolved due to complex upstream regulation and diverse subcellular functions. Here, we directly address this question using an optogenetic ERK activator, opto-miniRaf, that enables selective, rapid, graded, and reversible control of ERK signaling. Combining this AAV-compatible system with calcium imaging and electrophysiology, we interrogate ERK functions across biological scales, from cultured neurons, acute brain slices, and the intact brain. Acute optogenetic activation of ERK enhances synchronized network burst activity in cultured rat cortical neurons and increases calcium activity of cortical pyramidal neurons in awake and moving mice following non-invasive light stimulation. Together, these results establish ERK signaling as an acute modulator of neuronal and network activity, positioning opto-miniRaf as a generalizable platform for precise spatiotemporal control of intracellular kinase signaling in complex biological systems.

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

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

Abstract: Embryonic cells must interpret morphogen signals that vary in both time and space, but the rules by which they decode these dynamics remain unclear. Here we combine optogenetics with human 2D gastruloids to define minimal WNT signaling rules for germ-layer patterning. We block endogenous WNT secretion to create a “blank canvas” and reconstitute signaling using light-gated LRP6. Systematic temporal scans reveal a narrow competence window when the onset and duration of WNT signaling specify mesoderm; this window is shifted by cell density and amplified by BMP priming, whereas identical WNT inputs outside it invert germ-layer order or generate alternative mesodermal subtypes. Using micromirror-based illumination, we restricted WNT activation to a mid-ring during this temporal window; combined with BMP4, this fully restored germ layer domains with boundaries sharper than those generated by ligand stimulation. Thus, precise spatiotemporal control of a single pathway is sufficient to optically rebuild germ-layer architecture and reveals WNT as a temporal morphogen.

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 for light delivery inside 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 fully genetically encoded optogenetic circuit for 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 50mT magnetic stimulus. Thus, resulting in a first-of-its-kind 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: Regulation of cancer cells by their environment contributes to tumorigenesis and drug response, though the extent to which the oncogenic state can alter a cell's perception of its environment is not clear. Prior studies found that EML4-ALK, a receptor tyrosine kinase (RTK) fusion oncoprotein, suppresses transmembrane receptor signaling through EGFR. Moreover, suppression was reversed with targeted ALK inhibition, thereby promoting survival and drug tolerance. Here we tested whether such modulation of EGFR was common among other RTK fusions, which collectively are found in ∼5% of all cancers. Using live- and fixed-cell microscopy in isogenic and patient-derived cell lines, we found that a wide variety of RTK fusions suppress transmembrane EGFR and sequester essential adaptor proteins in the cytoplasm, as evidenced by the localization of endogenous Grb2. Targeted therapies rapidly released Grb2 from sequestration and potentiated EGFR. Synthetic optogenetic analogs of RTK fusions confirmed that cytoplasmic sequestration of Grb2 was sufficient to suppress perception of extracellular EGF and could do so without driving signaling from the synthetic fusion itself, demonstrating that fusion signaling and suppression of EGFR could be functionally decoupled. Our study uncovers that a large number of RTK fusions simultaneously act as both activators and suppressors of signaling, the mechanisms of which could be exploited for new biomimetic therapies that enhance cell killing and suppress drug tolerance.

Abstract: Impaired angiogenesis is a central barrier in the treatment of chronic and deep tissue wounds, preventing progression through the normal healing cascade. While the combination of near-infrared (NIR) photobiomodulation and pro-angiogenic growth factors has shown synergistic therapeutic benefit, the clinical translation of growth factor therapy is hindered by high cost, instability and the need for localized dosing to avoid aberrant vasculature. Peptidomimetics such as the VEGF-derived QK peptide offer a more stable and predictable alternative, but still require a means for localized, tunable presentation. Here, we establish an engineered living material based delivery system that responds to clinically relevant NIR light to produce and releases a QK-Fusion protein directly at the target site. The probiotic Escherichia coli Nissle 1917 was engineered with an 800 nm-responsive optogenetic circuit and encapsulated within an optimized alginate core–shell hydrogel that ensures biocontainment while allowing controlled outward diffusion of the secreted peptide. The released peptide remains non-cytotoxic and capable of binding extracellular matrix analogs and promoting the formation of organized, branched capillary-like networks in endothelial cultures. We thus establish a strategy for developing engineered living materials towards remote-controlled angiogenic stimulation.

Abstract: Microtubules (MTs) form dynamic cytoskeletal scaffolds essential for intracellular transport, organelle positioning, and spatial organization of signaling. Their architecture and function are continuously remodeled through the concerted actions of microtubule-associated proteins (MAPs), post-translational modifications (PTMs), and molecular motors. To precisely interrogate these processes in living systems, we developed a genetically encoded optogenetic toolkit for spatiotemporal control of MT organization and dynamics. By replacing native multimerization motifs with a blue light-responsive oligoermization domain, we have engineered single-component probes, OptoMT and OptoTIP, that reversibly label MT polymers or track plus-ends with tunable kinetics from seconds to minutes. When coupled to enzymatic effectors, these modules enable localized tubulin acetylation or detyrosination, directly linking PTMs to MT stability. We further engineered OptoMotor, a light-activatable kinesin platform that reconstitutes tail-dependent cargo transport along MTs, and OptoSAW, a light-triggered severing actuator for controlled MT disassembly. Using these tools, we reveal how local MT integrity governs lysosomal trafficking and ER-associated signaling dynamics. Collectively, this versatile single-component toolkit bridges molecular design with cytoskeletal function, offering new avenues to illuminate how dynamic cytoskeletal architectures coordinate intracellular organization, transport, and signaling.

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

Abstract: Mitochondrial membrane potential (MMP) is essential for mitochondrial functions, yet current methods for modulating MMP lack precise spatial and temporal control. Here, we present an optogenetic system that enables reversible formation of inter-mitochondrial contacts (mito-contacts) with high spatiotemporal precision. Blue light stimulation induces rapid formation of mito-contacts, which fully dissipate upon cessation of illumination. These light-induced mito-contacts can enhance MMP, leading to increased ATP production under stress conditions. Moreover, in human retinal cells and C. elegans, high MMP induced by mito-contacts alleviates the deleterious effects of prolonged blue light exposure, restoring energy metabolism and extending organismal lifespan. This optogenetic approach provides a powerful tool for modulating MMP and offers potential therapeutic applications for diseases linked to mitochondrial dysfunction.

Abstract: The proper establishment of cell form, fate, and function during morphogenesis requires precise coordination between cell polarity and developmental cues. To achieve this, cells must establish polarity domains that are stable yet sensitive to guiding cues. Here we show that C. elegans germline blastomeres resolve this trade-off by creating a time-varying polarization landscape. Specifically, coupling the PAR polarity network to the cell-cycle kinase CDK-1 ensures that newborn cells operate in a low-feedback regime that lowers barriers to polarity state switching, allowing spatial cues to induce and orient PAR protein asymmetries. As CDK-1 activity rises at mitotic entry, increasing molecular feedback reinforces cue-induced asymmetries to yield robust and stable patterning of PAR domains. Consistent with this model, optogenetic and chemical perturbations show that low-CDK/low-feedback regimes destabilize PAR domains but are required for both de novo polarization and the reorientation of polarity in response to inductive cues. We propose that mitotic oscillations in cell polarity circuits dynamically optimize the polarization landscape to enable coordination of polarity with morphogenesis. Such temporal control of developmental networks is likely a general mechanism to balance robustness of cellular states with sensitivity to signal-induced state switching.

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

Abstract: In cell biology, optical techniques are increasingly used to measure cells' internal states (biosensors) and to stimulate cellular responses (optogenetics). Yet the design of all-optical experiments is often manual: a pre-determined stimulus pattern is applied to cells, biosensors are measured over time, and the resulting data is processed off-line. With the advent of machine learning for segmentation and tracking, it becomes possible to envision closed-loop experiments where real-time information about cells' positions and states are used to dynamically determine optogenetic stimuli to alter or control their behavior. Here, we develop PyCLM, a Python-based suite of tools to enable real-time measurement, image segmentation, and optogenetic control of thousands of cells per experiment. PyCLM is designed to be as simple for the end user as possible, and multipoint experiments can be set up that combine a wide variety of imaging, image processing, and stimulation modalities without any programming. We showcase PyCLM on diverse applications: studying the effect of epidermal growth factor receptor activity waves on epithelial tissue movement, simultaneously stimulating ~1,000 single cells to guide tissue flows, and performing real-time feedback control of cell-to-cell fluorescence heterogeneity. This tool will enable the next generation of dynamic experiments to probe cell and tissue properties, and provides a first step toward precise control of cell states at the tissue scale.

Abstract: Methods for monitoring physiological changes in cellular Ca2+ levels have been in high demand for their utility in monitoring neuronal signaling. Recently, we introduced SCANR (Split-Tobacco Etch Virus (TEV) protease Calcium-regulated Neuron Recorder), which reports on Ca2+ changes in cells through the binding of calmodulin and M13 to reconstitute an active TEV protease. First-generation SCANR marked all of the Ca2+ spikes that occur throughout the lifetime of the cell, but it did not have a mechanism for controlling the time window in which recording of physiological changes in Ca2+ occurred. Here, we explore both chemical and light-based strategies for controlling the time and place in which Ca2+ recording occurs. We describe the adaptation of six popular chemo- and opto-genetics methods for controlling protein activity and subcellular localization to the SCANR system. We report two successful strategies, one that leverages the LOV-Jα optogenetics system for sterically controlling protein interactions and another that employs chemogenetic manipulation of subcellular protein distribution using the FKBP/FRB rapamycin binding pair.

Abstract: Inositol-requiring enzyme 1 (IRE1) is one of three known sensor proteins that respond to homeostatic perturbations in the metazoan endoplasmic reticulum. The three sensors collectively initiate an intertwined signaling network called the Unfolded Protein Response (UPR). Although IRE1 plays pivotal roles in human health and development, understanding its specific contributions to the UPR remains a challenge due to signaling crosstalk from the other two stress sensors. To overcome this problem, we engineered a light-activatable version of IRE1 and probed the transcriptomic effects of IRE1 activity in isolation from the other branches of the UPR. We demonstrate that 1) oligomerization alone is sufficient to activate IRE1 in human cells, 2) IRE1's transcriptional response evolves substantially under prolonged activation, and 3) the UPR induces major changes in mRNA splice isoform abundance in an IRE1-independent manner. Our data reveal previously unknown targets of IRE1 transcriptional regulation and direct degradation. Additionally, the tools developed here will be broadly applicable for precise dissection of signaling networks in diverse cell types, tissues, and organisms.

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

Abstract: Mitochondria contain double membranes that enclose their contents. Within their interior, the mitochondrial genome and its RNA products are condensed into ∼100 nm sized (ribo)nucleoprotein complexes. How these endogenous condensates maintain their roughly uniform size and spatial distributions within membranous mitochondria remains unclear. Here, we engineered an optogenetic tool (mt-optoIDR) that allowed for controlled formation of synthetic condensates upon light activation in live mitochondria. Using live cell super-resolution microscopy, we visualized the nucleation of small, yet elongated condensates (mt-opto-condensates), which recapitulated the morphologies of endogenous mitochondrial condensates. We decoupled the contribution of the double membranes from the environment within the matrix by overexpressing the dominant negative mutant of a membrane fusion protein (Drp1K38A). The resulting bulbous mitochondria had significantly more dynamic condensates that coarsened into a single, prominent droplet. These observations inform how mitochondrial membranes can limit the growth and dynamics of the condensates they enclose, without the need of additional regulatory mechanisms.

Abstract: Microbial optogenetic tools can regulate gene expression with high 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 systematically 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. Our results indicate that optogenetic recombinase performance can be reporter-dependent, suggesting that this is an important consideration in system design. Further, our single-cell analysis reveals highly heterogeneous activity across cells. Although general trends match expectations for mean levels of light-dependent recombination, we found substantial variation in this behavior across individual cells. In addition, our results show that the timing of recombinase activity is highly variable from cell to cell. These findings suggest critical criteria for selecting appropriate optogenetic recombinase systems and indicate areas for optimization to improve the single-cell capabilities of bacterial optogenetic tools.

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.