Curated Optogenetic Publication Database

Abstract: Aberrant aggregation of the prion-like, RNA-binding protein TDP-43 underlies several debilitating neurodegenerative proteinopathies, including amyotrophic lateral sclerosis (ALS). Here, we define how short, specific RNAs antagonize TDP-43 aggregation. Short, specific RNAs engage and stabilize the TDP-43 RNA-recognition motifs, which allosterically destabilizes a conserved helical region in the prion-like domain, thereby promoting aggregationresistant conformers. By mining sequence space, we uncover short RNAs with enhanced activity against TDP-43 and diverse disease-linked variants. The solubilizing activity of enhanced short RNA chaperones corrects aberrant TDP-43 phenotypes in optogenetic models and ALS patientderived neurons. Remarkably, an enhanced short RNA chaperone mitigates TDP-43 proteinopathy and neurodegeneration in mice. Our studies reveal mechanisms of short RNA chaperones and pave the way for the development of short RNA therapeutics for fatal TDP-43 proteinopathies.

Abstract: The endoplasmic reticulum (ER) links to multiple organelles through membrane contact sites (MCS), which play critical roles in signal transduction, cell homeostasis and stress response. However, studying the behaviour and functions of MCS in plants is still challenging, partially due to the lack of site-specific markers. Here, we used an optogenetic reporter, LiMETER (Light-inducible Membrane-Tethered cortical ER), to study the structure and dynamics of ER-PM contact sites (EPCS) in plants. Upon blue light activation, LiMETER is recruited to the EPCS rapidly, while this process is reversible when blue light is turned off. Compared with other EPCS reporters, LiMETER specifically and reversibly labels the contact sites, causing little side-effects on the ER structure and plant development. With its help, we re-examined the formation of ER-PM connections induced by cell-intrinsic factors or extracellular stimuli. We found that EPCSs are preferably localised at ER tubules and the edge of ER cisternae, and their number increased significantly under abiotic stress conditions. The abundance of ER and PM interaction is also developmental dependent, suggesting a direct link between ER-PM interaction, ER function and cell homeostasis. Taken together, we showed that LiMETER is an improved marker for functional and microscopical studies of ER-PM interaction, demonstrating the effectiveness of optogenetic tools in future research.

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: Mutations of the cyclin-dependent kinase-like 5 (CDKL5) gene, which encodes a serine/threonine protein kinase, can cause the CDKL5 deficiency disorder (CDD), a severe neurodevelopmental disease characterized by epileptic encephalopathy and neurocognitive impairment. The CDKL5 kinase consists of a catalytic N-terminal domain (NTD) and a less characterized C-terminal domain (CTD). Numerous disease-related mutations truncate CDKL5, leaving the NTD intact while variably shortening the CTD, which highlights the importance of the CTD for CDKL5 function. By systematically analyzing CDKL5 compositional features and evolutionary dynamics, we found that the CTD is a low-complexity region (LCR) highly enriched in serine residues and with a high propensity to undergo liquid-liquid phase separation (LLPS), a biophysical process of condensation controlling protein localization and function. Using a combination of super-resolution imaging, electron microscopy, and molecular and cellular approaches, including optogenetic LLPS induction, we discovered that CDKL5 undergoes LLPS, predominantly driven by its CTD, forming membraneless condensates in neuronal and non-neuronal cells. A CTD internal fragment (CTIF) plays a pivotal LLPS-promoting role, along with the distal portion of the protein. Indeed, two disease-related truncating mutations (S726X and R781X), eliding variable portions of the CTIF, significantly impair LLPS. This impairment is paralleled at the functional level by a reduction in the CDKL5-dependent phosphorylation of EB2, a known CDKL5 target. These findings demonstrate that CDKL5 undergoes LLPS, driven by a CTD region elided by most disease-related truncating mutations. Its loss––through the impairment of CDKL5 LLPS and functional activity––may play a key role in the molecular pathogenesis of CDD.

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: Alpha-synuclein (α-syn) aggregation is a defining feature of Parkinson's disease (PD) and related synucleinopathies. Despite significant research efforts focused on understanding α-syn aggregation mechanisms, the early stages of this process remain elusive, largely due to limitations in experimental tools that lack the temporal resolution to capture these dynamic events. Here, we introduce UltraID-LIPA, an innovative platform that combines the Light-Inducible Protein Aggregation (LIPA) system with the UltraID proximity-dependent biotinylation assay to identify α-syn-interacting proteins and uncover key mechanisms driving its oligomerization. UltraID-LIPA successfully identified 38 α-syn-interacting proteins, including both established and novel candidates, highlighting the accuracy and robustness of the approach. Notably, a strong interaction with endolysosomal and membrane-associated proteins was observed, supporting the hypothesis that interactions with membrane-bound organelles are pivotal in the early stages of α-syn aggregation. This powerful platform provides new insights into dynamic protein aggregation events, enhancing our understanding of synucleinopathies and other proteinopathies.

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: Lamellipodia are sheet-like protrusions essential for migration and endocytosis, yet the ultrastructure of the actin cytoskeleton during lamellipodia formation remains underexplored. Here, we combined the optogenetic tool PA-Rac1 with cryo-ET to enable ultrastructural analysis of newly formed lamellipodia. We successfully visualized lamellipodia at various extension stages, representing phases of their formation. In minor extensions, several unbundled actin filaments formed “Minor protrusions” at the leading edge. For moderately extended lamellipodia, cross-linked actin filaments formed small filopodia-like structures, termed “mini filopodia.” In fully extended lamellipodia, filopodia matured at multiple points, and cross-linked actin filaments running nearly parallel to the leading edge increased throughout the lamellipodia. These observations suggest that actin polymerization begins in specific plasma membrane regions, forming mini filopodia that either mature into full filopodia or detach from the leading edge to form parallel filaments. This actin turnover likely drives lamellipodial protrusion, providing new insights into actin dynamics and cell migration.

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: When mammalian cells are exposed to extracellular stress, they coordinate the condensation of stress granules (SGs) through the action of key nucleating proteins G3BP1 and G3BP2 (G3BPs) and, simultaneously, undergo a massive reduction in translation.1-5 Although SGs and G3BPs have been linked to this translation response, their overall impact has been unclear. Here, we investigate the longstanding question of how, and indeed whether, G3BPs and SGs shape the stress translation response. We find that SGs are enriched for mRNAs that are resistant to the stress-induced translation shutdown. Although the accurate recruitment of these stress-resistant mRNAs does require the context of stress, a combination of optogenetic tools and spike-normalized ribosome profiling demonstrates that G3BPs and SGs are necessary and sufficient to both help prioritize the translation of their enriched mRNAs and help suppress cytosolic translation. Together these results support a model in which G3BPs and SGs reinforce the stress translation program by prioritizing the translation of their resident mRNAs.

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: Disintegration of organelle membranes induces various cellular responses and has pathological consequences, including autoinflammatory diseases and neurodegeneration. Establishing methods to induce membrane rupture of organelles of interest is essential to analyze the downstream effects of membrane rupture; however, the spatiotemporal induction of rupture of specific membranes remains challenging. Here, we develop a series of optogenetic tools to induce organelle membrane rupture by using engineered Bcl-2-associated X protein (BAX), whose primary function is 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 target 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: 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.

Abstract: Intracellular tau aggregation requires a local protein concentration increase, referred to as "droplets". However, the cellular mechanism for droplet formation is poorly understood. Here, we expressed OptoTau, a P301L mutant tau fused with CRY2olig, a light-sensitive protein that can form homo-oligomers. Under blue light exposure, OptoTau increased tau phosphorylation and was sequestered in aggresomes. Suppressing aggresome formation by nocodazole formed tau granular clusters in the cytoplasm. The granular clusters disappeared by discontinuing blue light exposure or 1,6-hexanediol treatment suggesting that intracellular tau droplet formation requires microtubule collapse. Expressing OptoTau-ΔN, a species of N-terminal cleaved tau observed in the Alzheimer's disease brain, formed 1,6-hexanediol and detergent-resistant tau clusters in the cytoplasm with blue light stimulation. These intracellular stable tau clusters acted as a seed for tau fibrils in vitro. These results suggest that tau droplet formation and N-terminal cleavage are necessary for neurofibrillary tangles formation in neurodegenerative diseases.

Abstract: Actin homeostasis is fundamental for cell structure and consumes a large portion of cellular ATP. It has been documented in the literature that certain glycolytic enzymes can interact with actin, indicating an intricate interplay between the cytoskeleton and cellular metabolism. Here we report that hyperosmotic stress triggers actin severing and subsequent phase separation of the actin-binding protein TPM4. TPM4 condensates glycolytic enzymes such as HK2, PFKM, and PKM2, and adhere to and wrap around actin filaments. Notably, the condensates of TPM4 and glycolytic enzymes are enriched of NADH and ATP, suggestive of their functional importance in cell metabolism. At cellular level, actin filaments assembly is enhanced upon hyperosmotic stress and TPM4 condensation, while depletion of TPM4 impaired osmolarity-induced actin reorganization. At tissue level, co-localized condensates of TPM4 and glycolytic enzymes are observed in renal tissues subjected to hyperosmotic stress. Together, our findings suggest that stress-induced actin perturbation may act on TPM4 to organize glycolytic hubs that tether energy production to cytoskeletal reorganization.

Abstract: A large percentage of proteins form higher-order structures in order to fulfill their function. These structures are crucial for the precise spatial and temporal regulation of the cellular signaling network. Investigation of this network requires sophisticated research tools, such as optogenetic tools, that allow dynamic control over the signaling molecules. Cryptochrome 2 and its variations are the best-characterized oligomerizing photoreceptors the optogenetics toolbox has to offer. Therefore, we utilized this switch and combined it with an eGFP-binding nanobody, to build a toolbox of optogenetic constructs that enables the oligomerization of any eGFP-tagged protein of interest. We further introduced the higher clustering variant Cry2olig and an intrinsically disordered region to create higher-order oligomers or phase-separated assemblies to investigate the impact of different oligomerization states on eGFP-tagged signaling molecules. We apply these constructs to cluster IKKα and IKKβ, which resemble the central signaling integrator of the NF-κB pathway, thereby engineer a potent, blue-light-inducible activator of NF-κB signaling.

Abstract: Receptor tyrosine kinases (RTKs) are thought to play key roles in coordinating cell movement at single-cell and tissue scales. The recent development of optogenetic tools for controlling RTKs and their downstream signaling pathways suggested these responses may be amenable to engineering-based control for sculpting tissue shape and function. Here, we report that a light-controlled EGF receptor (OptoEGFR) can be deployed in epithelial cell lines for precise, programmable control of long-range tissue movements. We show that in OptoEGFR-expressing tissues, light can drive millimeter-scale cell rearrangements to densify interior regions or produce rapid outgrowth at tissue edges. Light-controlled tissue movements are driven primarily by PI 3-kinase signaling, rather than diffusible signals, tissue contractility, or ERK kinase signaling as seen in other RTK-driven migration contexts. Our study suggests that synthetic, light-controlled RTKs could serve as a powerful platform for controlling cell positions and densities for diverse applications including wound healing and tissue morphogenesis.

Abstract: Nuclear compartments form via biomolecular phase separation, mediated through multivalent properties of biomolecules concentrated within condensates. Certain compartments are associated with specific chromatin regions, including transcriptional initiation condensates, which are composed of transcription factors and transcriptional machinery, and form at acetylated regions including enhancer and promoter loci. While protein self-interactions, especially within low-complexity and intrinsically disordered regions, are known to mediate condensation, the role of substrate-binding interactions in regulating the formation and function of biomolecular condensates is underexplored. Here, utilizing live-cell experiments in parallel with coarse-grained simulations, we investigate how chromatin interaction of the transcriptional activator BRD4 modulates its condensate formation. We find that both kinetic and thermodynamic properties of BRD4 condensation are affected by chromatin binding: nucleation rate is sensitive to BRD4–chromatin interactions, providing an explanation for the selective formation of BRD4 condensates at acetylated chromatin regions, and thermodynamically, multivalent acetylated chromatin sites provide a platform for BRD4 clustering below the concentration required for off-chromatin condensation. This provides a molecular and physical explanation of the relationship between nuclear condensates and epigenetically modified chromatin that results in their mutual spatiotemporal regulation, suggesting that epigenetic modulation is an important mechanism by which the cell targets transcriptional condensates to specific chromatin loci.