Curated Optogenetic Publication Database

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: Macrophage-based adoptive cell therapies hold promise for solid tumors, but spatiotemporally controlling macrophage polarization within the immunosuppressive tumor microenvironment remains challenging. Here, we aimed to validate an optogenetic strategy using the LOV2-STIM1 system to achieve light-induced, sustained M1 polarization of macrophages. Upon blue light stimulation, engineered macrophages robustly exhibited M1 phenotypes, suppressed melanoma cell proliferation, migration, and invasion in vitro, and recapitulated the antitumor functions of M1 macrophages. Notably, combining light-activated engineered macrophages with temozolomide in melanoma models resulted in synergistic inhibition of tumor growth. This synergy is accompanied by a profound remodeling of the tumor immune microenvironment, characterized by M1-driven reversal of chemoresistance and enhanced infiltration of cytotoxic CD8+ T cells. Our findings establish a proof-of-concept for optogenetic regulation of macrophage polarization and demonstrate its feasibility for enhancing antitumor effects and chemosensitivity in melanoma models, providing a promising and controllable platform for macrophage-based immunotherapy.

Abstract: Precise control of covalent protein binding and cleavage in mammalian cells is crucial for manipulating cellular processes but remains challenging due to dark background, poor stability, low efficiency, or requirement of unnatural amino acids in current optogenetic tools. We introduce a photoswitchable intein (PS Intein) engineered by allosterically modulating a small autocatalytic gp41-1 intein with tandem Vivid photoreceptor. PS Intein exhibits superior functionality and low background in cells compared to existing tools. PS Intein-based systems enable light-induced covalent binding, cleavage, and release of proteins for regulating gene expression and cell fate. The high responsiveness and ability to integrate multiple inputs allow for intersectional cell targeting using cancer- and tumor microenvironment-specific promoters. PS Intein tolerates various fusions and insertions, facilitating its application in diverse cellular contexts. This versatile technology offers efficient light-controlled protein manipulation, providing a powerful tool for adding functionalities to proteins and precisely controlling protein networks in living cells.

Abstract: Localized translation broadly enables spatiotemporal control of gene expression. Here, we present LOV-domain-controlled ligase for translation localization (LOCL-TL), an optogenetic approach for monitoring translation with codon resolution at any defined subcellular location under physiological conditions. Application of LOCL-TL to mitochondrially localized translation revealed that ∼20% of human nuclear-encoded mitochondrial genes are translated on the outer mitochondrial membrane (OMM). Mitochondrially translated messages form two classes distinguished by encoded protein length, recruitment mechanism, and cellular function. An evolutionarily ancient mechanism allows nascent chains to drive cotranslational recruitment of long proteins via an unanticipated bipartite targeting signal. Conversely, mRNAs of short proteins, especially eukaryotic-origin electron transport chain (ETC) components, are specifically recruited by the OMM protein A-kinase anchoring protein 1 (AKAP1) in a translation-independent manner that depends on mRNA splicing. AKAP1 loss lowers ETC levels. LOCL-TL thus reveals a hierarchical strategy that enables preferential translation of a subset of proteins on the OMM.

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: Nucleocytoplasmic transport (NCT) is essential for maintaining cellular homeostasis, and its disruption is involved in various diseases, including neurodegenerative disorders and amyotrophic lateral sclerosis. This underscores the need to develop tools to monitor and quantify NCT. Amongst these tools, the fast and reversible optogenetics probes, LEXY (light-inducible nuclear export system) and LINuS (light-inducible nuclear localization signal), allow the measurement of NCT dynamics in live cells. The original publications describe manual segmentation and quantification of the fluorescent probe signal in the nucleus and cytosol upon transfection of LEXY and LINuS constructs in live-cell imaging. However, both transfection and manual segmentation limit the number of cells that can be analyzed and are subject to imprecision due to potential user-dependent errors. While the high speed and reversibility provided by optogenetics should, in principle, allow for high sensitivity in detecting changes in NCT dynamics, it depends on the acquisition parameters and analysis of a sufficient number of cells. We have therefore established lentiviral vectors expressing LEXY and LINuS to create stable cell lines, tested live imaging markers and control conditions, and implemented a semi-automated image analysis pipeline that allows for the analysis of hundreds of cells. This analysis method uses the open-access software FIJI, is accessible to beginners in bioinformatics, and does not require advanced computer setups. Here we provide a step-by-step protocol to set up LEXY as an example of these optogenetic tools to monitor nuclear export, from preparation of the samples to live-cell imaging acquisition and automated analysis, while demonstrating how to adapt the protocol for other conditions, controls, or models in any lab. All plasmids and cell lines used in this protocol will be made available to the scientific community, therefore further increasing the accessibility of the method.

Abstract: CRISPR-Cas technologies have revolutionized life sciences by enabling programmable genome editing across diverse organisms. Achieving dynamic and precise control over CRISPR-Cas activity with exogenous triggers, such as light or chemical ligands, remains an important need. Existing tools for CRISPR-Cas control are often limited to specific Cas orthologs or selected applications, restricting their versatility. Anti-CRISPR (Acr) proteins are natural inhibitors of CRISPR-Cas systems and provide a flexible regulatory layer but are constitutively active in their native forms. In this study, we built on our previously reported concept for optogenetic CRISPR-Cas control with engineered, light-switchable anti-CRISPR proteins and expanded it from ortholog-specific Acrs towards AcrIIA5 and AcrVA1, broad-spectrum inhibitors of CRISPR-Cas9 and CRISPR-Cas12a, respectively. We then conceived and implemented a novel, chemogenetic anti-CRISPR platform based on engineered, circularly permuted ligand receptor domains, that together respond to six clinically relevant drugs. The resulting toolbox achieves both optogenetic and chemogenetic control of genome editing in human cells with a wide range of CRISPR-Cas effectors, including type II-A and II-C CRISPR-Cas9s, and CRISPR-Cas12a. In sum, this work establishes a versatile platform for the multidimensional control of CRISPR-Cas systems, with immediate applications in basic research and biotechnology, and with the potential for therapeutic use in the future.

Abstract: The cofactor LIM-domain-binding protein 1 (Ldb1) is linked to many processes in gene regulation, including enhancer-promoter communication, interchromosomal interactions, and enhanceosome-cofactor-like activity. However, its functional requirement and molecular role during embryogenesis remain unclear. Here, we used optogenetics (iLEXY) to rapidly deplete Drosophila Ldb1 (Chip) from the nucleus at precise time windows. Remarkably, this pinpointed the essential window of Chip's function to just 1 h of embryogenesis, overlapping zygotic genome activation (ZGA). We show that Zelda, a pioneer factor essential for ZGA, recruits Chip to chromatin, and both factors regulate concordant changes in gene expression, suggesting that Chip is a cofactor of Zelda. Chip does not significantly impact chromatin architecture at these stages, but instead recruits CBP, and is essential for H3K27ac deposition at enhancers and promoters, and for the proper expression of co-regulated genes. These data identify Chip as a functional bridge between Zelda and the coactivator CBP to regulate gene expression in early embryogenesis.

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: 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: 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.

Abstract: To address the need for methods for tagging and manipulating neuronal ensembles underlying specific behaviors, we present an improved version of FLARE, termed cytoFLARE (cytosol-expressed FLARE). cytoFLARE incorporates cytosolic tethering of a transcription factor and expression of a more sensitive pair of calcium-sensing domains. We show that cytoFLARE captures more calcium- and light-dependent signals in HEK293T cells and higher signal-to-background ratios in neuronal cultures. We further establish cytoFLARE transgenic Drosophila models and apply cytoFLARE to label activated neurons upon sensory or optogenetic stimulation within a defined time window. Notably, through the cytoFLARE-driven expression of optogenetic actuators, we successfully reactivated and inhibited neurons involved in the larval nociceptive system. Our findings demonstrate the characterization and application of time-gated calcium integrators for both recording and manipulating neuronal activity in Drosophila larvae.

Abstract: Disintegration of organelle membranes induces various cellular responses and has pathological consequences, including autoinflammatory diseases and neurodegeneration. Establishing methods to induce membrane rupture of specific organelles is essential to analyze the downstream effects of membrane rupture; however, the spatiotemporal induction of organelle membrane rupture remains challenging. Here, we develop a series of optogenetic tools to induce organelle membrane rupture by using engineered Bcl-2-associated X protein (BAX), which primarily functions to form membrane pores in the outer mitochondrial membrane (OMM) during apoptosis. When BAX is forced to target mitochondria, lysosomes, or the endoplasmic reticulum (ER) by replacing its C-terminal transmembrane domain (TMD) with organelle-targeting sequences, the BAX mutants rupture their targeted membranes. To regulate the activity of organelle-targeted BAX, the photosensitive light-oxygen-voltage-sensing 2 (LOV2) domain is fused to the N-terminus of BAX. The resulting LOV2-BAX fusion protein exhibits blue light-dependent membrane-rupture activity on various organelles, including mitochondria, the ER, and lysosomes. Thus, LOV2-BAX enables spatiotemporal induction of membrane rupture across a broad range of organelles, expanding research opportunities on the consequences of organelle membrane disruption.

Abstract: Motile cilia perform crucial functions during embryonic development and in adult tissues. They are anchored by an apical actin network that forms microridge-like structures on the surface of multiciliated cells. Using Xenopus as a model system to investigate the mechanisms underlying the formation of these specialized actin structures, we observed stochastic bursts of intracellular calcium concentration in developing multiciliated cells. Through optogenetic manipulation of calcium signaling, we found that individual calcium bursts triggered the fusion and extension of actin structures by activating non-muscle myosin. Repeated cycles of calcium activation promoted assembly and coherence of the maturing apical actin network. Inhibition of the endogenous inositol triphosphate-calcium pathway disrupted the formation of apical actin/microridge-like structures by reducing local centriolar RhoA signaling. This disruption was rescued by transient expression of constitutively active RhoA in multiciliated cells. Our findings identify repetitive calcium bursts as a driving force that promotes the self-organization of the highly specialized actin cytoskeleton of multiciliated cells.

Abstract: The actin cytoskeleton and its nanoscale organization are central to all eukaryotic cells-powering diverse cellular functions including morphology, motility, and cell division-and is dysregulated in multiple diseases. Historically studied largely with purified proteins or in isolated cells, tools to study cell type-specific roles of actin in multicellular contexts are greatly needed. DeActs are recently created, first-in-class genetic tools for perturbing actin nanostructures and dynamics in specific cell types across diverse eukaryotic model organisms. Here, ChiActs are introduced, the next generation of actin-perturbing genetic tools that can be rapidly activated in cells and optogenetically targeted to distinct subcellular locations using light. ChiActs are composed of split halves of DeAct-SpvB, whose potent actin disassembly-promoting activity is restored by chemical-induced dimerization or allosteric switching. It is shown that ChiActs function to rapidly induce actin disassembly in several model cell types and are able to perturb actin-dependent nano-assembly and cellular functions, including inhibiting lamellipodial protrusions and membrane ruffling, remodeling mitochondrial morphology, and reorganizing chromatin by locally constraining actin disassembly to specific subcellular compartments. ChiActs thus expand the toolbox of genetically-encoded tools for perturbing actin in living cells, unlocking studies of the many roles of actin nano-assembly and dynamics in complex multicellular systems.

Abstract: Light-controlled transcriptional activation is a commonly used optogenetic strategy that allows researchers to regulate gene expression with high spatiotemporal precision. The vast majority of existing tools are, however, limited to light-triggered induction of gene expression. Here, we inverted this mode of action and created optogenetic systems capable of efficiently terminating transcriptional activation in response to blue light. First, we designed highly compact regulators by photo-controlling the VP16 (pcVP16) transactivation peptide. Then, applying a two-hybrid strategy, we engineered LOOMINA (light off-operated modular inductor of transcriptional activation), a versatile transcriptional control platform for mammalian cells that is compatible with various effector proteins. Leveraging the flexibility of CRISPR systems, we combined LOOMINA with dCas9 to control transcription with blue light from endogenous promoters with exceptionally high dynamic ranges in multiple cell lines. Functionally and mechanistically, the versatile LOOMINA platform and the exceptionally compact pcVP16 transactivator represent valuable additions to the optogenetic repertoire for transcriptional regulation.

Abstract: Cells under high confinement form highly polarized hydrostatic pressure-driven, stable leader blebs that enable efficient migration in low adhesion, environments. Here we investigated the basis of the polarized bleb morphology of metastatic melanoma cells migrating in non-adhesive confinement. Using high-resolution time-lapse imaging and specific molecular perturbations, we found that EGF signaling via PI3K stabilizes and maintains a polarized leader bleb. Protein activity biosensors revealed a unique EGFR/PI3K activity gradient decreasing from rear-to-front, promoting PIP3 and Rac1-GTP accumulation at the bleb rear, with its antagonists PIP2 and RhoA-GTP concentrated at the bleb tip, opposite to the front-to-rear organization of these signaling modules in integrin-mediated mesenchymal migration. Optogenetic experiments showed that disrupting this gradient caused bleb retraction, underscoring the role of this signaling gradient in bleb stability. Mathematical modeling and experiments identified a mechanism where, as the bleb initiates, CD44 and ERM proteins restrict EGFR mobility in a membrane-apposed cortical actin meshwork in the bleb rear, establishing a rear-to-front EGFR-PI3K-Rac activity gradient. Thus, our study reveals the biophysical and molecular underpinnings of cell polarity in bleb-based migration of metastatic cells in non-adhesive confinement, and underscores how alternative spatial arrangements of migration signaling modules can mediate different migration modes according to the local microenvironment.

Abstract: Microtubule acetylation is implicated in regulating cell motility, yet its physiological role in directional migration and the underlying molecular mechanisms have remained unclear. This knowledge gap has persisted primarily due to a lack of tools capable of rapidly manipulating microtubule acetylation in actively migrating cells. To overcome this limitation and elucidate the causal relationship between microtubule acetylation and cell migration, we developed a novel optogenetic actuator, optoTAT, which enables precise and rapid induction of microtubule acetylation within minutes in live cells. Using optoTAT, we observed striking and rapid responses at both molecular and cellular level. First, microtubule acetylation triggers release of the RhoA activator GEF-H1 from sequestration on microtubules. This release subsequently enhances actomyosin contractility and drives focal adhesion maturation. These subcellular processes collectively promote sustained directional cell migration. Our findings position GEF-H1 as a critical molecular responder to microtubule acetylation in the regulation of directed cell migration, revealing a dynamic crosstalk between the actin and microtubule cytoskeletal networks.

Abstract: Morphogen gradients convey essential spatial information during tissue patterning. While both concentration and timing of morphogen exposure are 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), homologue of NF-κB, which orchestrates dorso-ventral 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 spatio-temporal 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 is 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: Transcriptional control is fundamental to cellular function. However, despite knowing that transcription factors can repress or activate specific genes, how these functions are implemented at the molecular level has remained elusive, particularly in the endogenous context of developing animals. Here, we combine optogenetics, single-cell live-imaging, and mathematical modeling to study how a zinc-finger repressor, Knirps, induces switch-like transitions into long-lived quiescent states. Using optogenetics, we demonstrate that repression is rapidly reversible (~1 min) and memoryless. Furthermore, we show that the repressor acts by decreasing the frequency of transcriptional bursts in a manner consistent with an equilibrium binding model. Our results provide a quantitative framework for dissecting the in vivo biochemistry of eukaryotic transcriptional regulation.

Abstract: Chimeric antigen receptor T cell therapies have achieved great success in eradicating some liquid tumors, whereas the preclinical results in treating solid tumors have proven less decisive. One of the principal challenges in solid tumor treatment is the physical barrier composed of a dense extracellular matrix, which prevents immune cells from penetrating the tissue to attack intratumoral cancer cells. Here, we improve immune cell infiltration into solid tumors by manipulating septin-7 functions in cells. Using protein allosteric design, we reprogram the three-dimensional structure of septin-7 and insert a blue light-responsive light-oxygen-voltage-sensing domain 2 (LOV2), creating a light-controllable septin-7-LOV2 hybrid protein. Blue light inhibits septin-7 function in live cells, inducing extended cell protrusions and cell polarization, enhancing cell transmigration efficiency through confining spaces. We genetically edited human natural killer cell line (NK92) and mouse primary CD8+ T-cells expressing the engineered protein, and we demonstrated improved penetration and cytotoxicity against various tumor spheroid models. Our proposed strategy to enhance immune cell infiltration is compatible with other methodologies and therefore, could be used in combination to further improve cell-based immunotherapies against solid tumors.

Abstract: Mitochondria provide cells with energy and regulate the cellular metabolism. Almost all mitochondrial proteins are nuclear-encoded, translated on ribosomes in the cytoplasm, and subsequently transferred to the different subcellular compartments of mitochondria. Here, we developed OptoMitoImport, an optogenetic tool to control the import of proteins into the mitochondrial matrix via the presequence pathway on demand. OptoMitoImport is based on a two-step process: first, light-induced cleavage by a TEV protease cuts off a plasma membrane-anchored fusion construct in close proximity to a mitochondrial targeting sequence; second, the mitochondrial targeting sequence preceding the protein of interest recruits to the outer mitochondrial membrane and imports the protein fused to it into mitochondria. Upon reaching the mitochondrial matrix, the matrix processing peptidase cuts off the mitochondrial targeting sequence and releases the protein of interest. OptoMitoImport is available as a two-plasmid system as well as a P2A peptide or IRES sequence-based bicistronic system. Fluorescence studies demonstrate the release of the plasma membrane-anchored protein of interest through light-induced TEV protease cleavage and its localization to mitochondria. Cell fractionation experiments confirm the presence of the peptidase-cleaved protein of interest in the mitochondrial fraction. The processed product is protected from proteinase K treatment. Depletion of the membrane potential across the inner mitochondria membrane prevents the mitochondrial protein import, indicating an import of the protein of interest by the presequence pathway. These data demonstrate the functionality of OptoMitoImport as a generic system with which to control the post-translational mitochondrial import of proteins via the presequence pathway.

Abstract: The human nucleoporin RanBP2/Nup358 interacts with SUMO1-modified RanGAP1 and the SUMO E2 Ubc9 at the nuclear pore complex (NPC) to promote export and disassembly of exportin Crm1/Ran(GTP)/cargo complexes. In mitosis, RanBP2/SUMO1-RanGAP1/Ubc9 remains intact after NPC disassembly and is recruited to kinetochores and mitotic spindles by Crm1 where it contributes to mitotic progression. Interestingly, RanBP2 binds SUMO1-RanGAP1/Ubc9 with motifs that also catalyze SUMO E3 ligase activity. Here, we resolve cryo-EM structures of a RanBP2 C-terminal fragment bound to Crm1, SUMO1-RanGAP1/Ubc9, and two molecules of Ran(GTP), one bound to Crm1 and the other bound to RanGAP1 and RanBP2. These structures reveal several unanticipated interactions with Crm1 including a nuclear export signal (NES) for RanGAP1, the deletion of which mislocalizes RanGAP1 and the Ran GTPase in cells. Our structural and biochemical results support models in which RanBP2 E3 ligase activity is dependent on Crm1, the RanGAP1 NES and Ran GTPase cycling.

Abstract: The Notch receptor is a pleiotropic signaling protein that translates intercellular ligand interactions into changes in gene expression via the nuclear localization of the Notch intracellular Domain (NICD). Using a combination of immunohistochemistry, RNA in situ, Optogenetics and super-resolution live imaging of transcription in human cells, we show that the N1ICD can form condensates that positively facilitate Notch target gene expression. We determined that N1ICD undergoes Phase Separation Coupled Percolation (PSCP) into transcriptional condensates, which recruit, enrich, and encapsulate a broad set of core transcriptional proteins. We show that the capacity for condensation is due to the intrinsically disordered transcriptional activation domain of the N1ICD. In addition, the formation of such transcriptional condensates acts to promote Notch-mediated super enhancer-looping and concomitant activation of the MYC protooncogene expression. Overall, we introduce a novel mechanism of Notch1 activity in which discrete changes in nuclear N1ICD abundance are translated into the assembly of transcriptional condensates that facilitate gene expression by enriching essential transcriptional machineries at target genomic loci.