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: 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: 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: 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: 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: Cortical positioning of the meiotic spindle within an oocyte is required to expel chromosomes into polar bodies to generate a zygote with the correct number of chromosomes. In C. elegans, yolk granules and mitochondria are packed inward, away from the cortex, while the spindle moves outward, both in a kinesin-dependent manner. The kinesin-dependent inward packing of yolk granules suggests the existence of microtubules with minus ends at the cortex and plus ends extending inward, making it unclear how kinesin moves the spindle outward. We hypothesize that the inward packing of organelles might indirectly force the spindle outward by volume exclusion. To test this hypothesis, we generate a strain in which the only kinesin consists of motor domains with no cargo-binding tail optogenetically attached to mitochondria. This mitochondria-only kinesin packs mitochondria into a tight ball and efficiently moves the meiotic spindle to the cortex, supporting the volume exclusion hypothesis.

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

Abstract: Keeping condensates in liquid-like states throughout the biosynthesis process in microbial cell factories remains an ongoing challenge. Here, we present a light-controlled phase regulator, which maintains the liquid-like features of synthetic condensates on demand throughout the biosynthesis process upon light induction, as demonstrated by various live cell-imaging techniques. Specifically, the tobacco etch virus (TEV) protease controlled by light cleaves intrinsically disordered proteins (IDPs) to alter their valency and concentration for controlled phase transition and programmable fluidity of cellular condensates. As a proof of concept, we harness this capability to significantly improve the production of squalene and ursolic acid (UA) in engineered Saccharomyces cerevisiae. Our work provides a powerful approach to program the solid-liquid phase transition of biomolecular condensates for improved biosynthesis.

Abstract: Millions of ribosomes are packed within mammalian cells, yet we lack tools to visualize them in toto and characterize their subcellular composition. In this study, we present ribosome expansion microscopy (RiboExM) to visualize individual ribosomes and an optogenetic proximity-labeling technique (ALIBi) to probe their composition. We generated a super-resolution ribosomal map, revealing subcellular translational hotspots and enrichment of 60S subunits near polysomes at the endoplasmic reticulum (ER). We found that Lsg1 tethers 60S to the ER and regulates translation of select proteins. Additionally, we discovered ribosome heterogeneity at mitochondria guiding translation of metabolism-related transcripts. Lastly, we visualized ribosomes in neurons, revealing a dynamic switch between monosomes and polysomes in neuronal translation. Together, these approaches enable exploration of ribosomal localization and composition at unprecedented resolution.

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

Abstract: The balance of mitochondrial fission and fusion plays an important role in maintaining the stability of cellular homeostasis. Abnormal mitochondrial fission and fragmentation have been shown to be associated with oxidative stress, which causes a variety of human diseases from neurodegeneration disease to cancer. Therefore, the induction of mitochondrial aggregation and fusion may provide an alternative approach to alleviate these conditions. Here, an optogenetic-based mitochondrial aggregation system (Opto-MitoA) developed, which is based on the CRY2clust/CIBN light-sensitive module. Upon blue light illumination, CRY2clust relocates from the cytosol to mitochondria where it induces mitochondrial aggregation by CRY2clust homo-oligomerization and CRY2clust-CIBN hetero-dimerization. Our functional experiments demonstrate that Opto-MitoA-induced mitochondrial aggregation potently alleviates niclosamide-caused cell dysfunction in ATP production. This study establishes a novel optogenetic-based strategy to regulate mitochondrial dynamics in cells, which may provide a potential therapy for treating mitochondrial-related diseases.

Abstract: Abstract Tauopathy is a spectrum of diseases characterized by fibrillary tau aggregate formation in neurons and glial cells in the brain. Tau aggregation originates in the brainstem and entorhinal cortex and then spreads throughout the brain in Alzheimer’s disease (AD), which is the most prevalent type of tauopathy. Understanding the mechanism by which locally developed tau pathology propagates throughout the brain is crucial for comprehending AD pathogenesis. Therefore, a novel model of tau pathology that artificially induces tau aggregation in targeted cells at specific times is essential. This study describes a novel optogenetic module, OptoTau, which is a human tau with the P301L mutation fused with a photosensitive protein CRY2olig, inducing various forms of tau according to the temporal pattern of blue light illumination pattern. Continuous blue light illumination for 12 h to Neuro2a cells that stably express OptoTau (OptoTauKI cells) formed clusters along microtubules, many of which eventually accumulated in aggresomes. Conversely, methanol-resistant tau aggregation was formed when alternating light exposure and darkness in 30-min cycles for 8 sets per day were repeated over 8 days. Methanol-resistant tau was induced more rapidly by repeating 5-min illumination followed by 25-min darkness over 24 h. These results indicate that OptoTau induced various tau aggregation stages based on the temporal pattern of blue light exposure. Thus, this technique exhibits potential as a novel approach to developing specific tau aggregation in targeted cells at desired time points.

Abstract: Hsp70 prevents protein aggregation and is cytoprotective, but sustained Hsp70 overexpression is problematic. Therefore, we characterized small molecule agonists that augment Hsp70 activity. Because cumbersome assays were required to assay agonists, we developed cell-based and in vivo assays in which disease-associated consequences of Hsp70 activation can be quantified. One assay uses an optogenetic system in which the formation of TDP-43 inclusions can be controlled, and the second assay employs a zebrafish model for acute kidney injury (AKI). These complementary assays will facilitate future work to identify new Hsp70 agonists as well as optimized agonist derivatives.

Abstract: Dysfunction of the RNA binding protein heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) contributes to neurodegeneration, the primary cause of permanent disability in multiple sclerosis (MS). To better understand the role of hnRNP A1 dysfunction in the pathogenesis of neurodegeneration, we utilized optogenetics-driven hnRNP A1 clustering to model its dysfunction in neuron-like differentiated Neuro-2A cells. hnRNP A1 clustering activates the integrated stress response (ISR) and results in a neurodegenerative phenotype marked by decreased neuronal protein translation and neurite loss. Small molecule inhibition of the ISR with either PERKi (GSK2606414) or ISRIB (integrated stress response inhibitor) attenuated both the decrease in neuronal translation and neurite loss, without affecting hnRNP A1 clustering. We then confirmed a strong association between hnRNP A1 clustering and ISR activation in neurons from MS brains. These data illustrate that hnRNP A1 dysfunction promotes neurodegeneration by activation of the ISR in vitro and in vivo, thus revealing a novel therapeutic target to reduce neurodegeneration and subsequent disability in MS.

Abstract: Alternative splicing is a fundamental process that contributes to the functional diversity and complexity of proteins. The regulation of each alternative splicing event involves the coordinated action of multiple RNA-binding proteins, creating a diverse array of alternatively spliced products. Dysregulation of alternative splicing is associated with various diseases, including neurodegeneration. Here we demonstrate that CELF2, a splicing regulator and a GWAS-identified risk factor for Alzheimer’s disease, binds to mRNAs associated with neurodegenerative diseases, with a specific interaction observed in the intron adjacent to exon 10 on Tau mRNA. Loss of CELF2 in the mouse brain results in a decreased inclusion of Tau exon 10, leading to a reduced 4R:3R ratio. Further exploration shows that the hinge domain of CELF2 possesses an intrinsically disordered region (IDR), which mediates CELF2 condensation and function. The functionality of IDR in regulating CELF2 function is underscored by its substitutability with IDRs from FUS and TAF15. Using TurboID we identified proteins that interact with CELF2 through its IDR. We revealed that CELF2 co-condensate with NOVA2 and SFPQ, which coordinate with CELF2 to regulate the alternative splicing of Tau exon 10. A negatively charged residue within the IDR (D388), which is conserved among CELF proteins, is critical for CELF2 condensate formation, interactions with NOVA2 and SFPQ, and function in regulating tau exon 10 splicing. Our data allow us to propose that CELF2 regulates Tau alternative splicing by forming condensates through its IDR with other splicing factors, and that the composition of the proteins within the condensates determines the outcomes of alternative splicing events.

Abstract: Endogenous condensates with transient constituents are notoriously difficult to study with common biological assays like mass spectrometry and other proteomics profiling. Here, we report a method for light-induced targeting of endogenous condensates (LiTEC) in living cells. LiTEC combines the identification of molecular zip codes that target the endogenous condensates with optogenetics to enable controlled and reversible partitioning of an arbitrary cargo, such as enzymes commonly used in proteomics, into the condensate in a blue light-dependent manner. We demonstrate a proof of concept by combining LiTEC with proximity-based biotinylation (BioID) and uncover putative components of transcriptional condensates in mouse embryonic stem cells. Our approach opens the road to genome-wide functional studies of endogenous condensates.

Abstract: Synucleinopathies, including Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy, are triggered by α-synuclein aggregation, triggering progressive neurodegeneration. However, the intracellular α-synuclein aggregation mechanism remains unclear. Herein, we demonstrate that RNA G-quadruplex assembly forms scaffolds for α-synuclein aggregation, contributing to neurodegeneration. Purified α-synuclein binds RNA G-quadruplexes directly through the N terminus. RNA G-quadruplexes undergo Ca2+-induced phase separation and assembly, accelerating α-synuclein sol-gel phase transition. In α-synuclein preformed fibril-treated neurons, RNA G-quadruplex assembly comprising synaptic mRNAs co-aggregates with α-synuclein upon excess cytoplasmic Ca2+ influx, eliciting synaptic dysfunction. Forced RNA G-quadruplex assembly using an optogenetic approach evokes α-synuclein aggregation, causing neuronal dysfunction and neurodegeneration. The administration of 5-aminolevulinic acid, a protoporphyrin IX prodrug, prevents RNA G-quadruplex phase separation, thereby attenuating α-synuclein aggregation, neurodegeneration, and progressive motor deficits in α-synuclein preformed fibril-injected synucleinopathic mice. Therefore, Ca2+ influx-induced RNA G-quadruplex assembly accelerates α-synuclein phase transition and aggregation, potentially contributing to synucleinopathies.

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: Unambiguous targeting of cellular structures for in situ cryo-electron microscopy in the heterogeneous, dense, and compacted environment of the cytoplasm remains challenging. Here we have developed a cryogenic correlative light and electron microscopy (cryo-CLEM) workflow which combines thin cells grown on a mechanically defined substratum to rapidly analyse organelles and macromolecular complexes by cryo-electron tomography (cryo-ET). We coupled these advancements with optogenetics to redistribute perinuclear-localised organelles to the cell periphery, allowing visualisation of organelles otherwise positioned in cellular regions too thick for cryo-ET. This reliable and robust workflow allows for fast in situ analyses without the requirement for cryo-focused ion beam milling. Using this protocol, cells can be frozen, imaged by cryo-fluorescence microscopy and be ready for batch cryo-ET within a day.

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.

Abstract: Proteinaceous inclusions formed by C9orf72-derived dipeptide-repeat (DPR) proteins are a histopathological hallmark in ∼50% of familial amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD) cases. However, DPR aggregation/inclusion formation could not be efficiently recapitulated in cell models for four out of five DPRs. In this study, using optogenetics, we achieved chemical-free poly-PR condensation/aggregation in cultured cells including human motor neurons, with spatial and temporal control. Strikingly, nuclear poly-PR condensates had anisotropic, hollow-center appearance, resembling TDP-43 anisosomes, and their growth was limited by RNA. These condensates induced abnormal TDP-43 granulation in the nucleus without stress response activation. Cytoplasmic poly-PR aggregates forming under prolonged opto-stimulation were more persistent than its nuclear condensates, selectively sequestered TDP-43 in a demixed state and surrounded spontaneous stress granules. Thus, poly-PR condensation accompanied by nuclear TDP-43 dysfunction may constitute an early pathological event in C9-ALS/FTD. Anisosome-type condensates of disease-linked proteins may represent a common molecular species in neurodegenerative disease.

Abstract: Biomolecular condensates are broadly implicated in both normal cellular regulation and disease. Consequently, several chemical biology and optogenetic approaches have been developed to induce phase separation of a protein of interest. However, few tools are available to perform the converse function - dissolving a condensate of interest on demand. Such a tool would aid in testing whether the condensate plays specific functional roles. Here we show that light-gated recruitment of a solubilizing domain, maltose-binding protein (MBP), results in rapid and controlled dissolution of condensates formed from proteins of interest. Our optogenetic MBP-based dissolution strategy (OptoMBP) is rapid, reversible, and can be spatially controlled with subcellular precision. We also provide a proof-of-principle application of OptoMBP by disrupting condensation of the oncogenic fusion protein FUS-CHOP and reverting FUS-CHOP driven transcriptional changes. We envision that the OptoMBP system could be broadly useful for disrupting constitutive protein condensates to probe their biological functions.

Abstract: Signal processing by intracellular kinases controls near all biological processes but how signal pathway functions evolve with changed cellular context is poorly understood. Functional specificity of c-Jun N-terminal Kinases (JNK) are partly encoded by signal strength. Here we reveal that intracellular pH (pHi) is a significant component of the JNK network and defines signal response to specific stimuli. We show pHi regulates JNK activity in response to cell stress, with the relationship between pHi and JNK activity dependent on specific stimuli and upstream kinases activated. Using the optogenetic clustering tag CRY2, we show that an increase in pHi promotes the light-induced phase transition of ASK1 to augment JNK activation. While increased pHi similarly promoted CRY2-tagged JNK2 to form light-induced condensates, this attenuated JNK activity. Mathematical modelling of feedback signalling incorporating pHi and differential contributions by ASK1 and JNK2 condensates was sufficient to delineate signal responses to specific stimuli. Taking pHi and ASK1/JNK2 signal contributions into consideration may delineate oncogenic versus tumour suppressive JNK functions and cancer cell drug responses.

Abstract: High-performance biosensors play a crucial role in elucidating the intricate spatiotemporal regulatory roles and dynamics of membrane phospholipids. However, enhancing the sensitivity and imaging performance remains a significant challenge. Here, optogenetic-based strategies are presented to optimize phospholipid biosensors. These strategies involves presequestering unbound biosensors in the cell nucleus and regulating their cytosolic levels with blue light to minimize background signal interference in phospholipid detection, particularly under conditions of high expression levels of biosensor. Furthermore, optically controlled phase separation and the SunTag system are employed to generate punctate probes for substrate detection, thereby amplifying biosensor signals and enhancing visualization of the detection process. These improved phospholipid biosensors hold great potential for enhancing the understanding of the spatiotemporal dynamics and regulatory roles of membrane lipids in live cells and the methodological insights in this study might be valuable for developing other high-performance biosensors.