Curated Optogenetic Publication Database

Abstract: p53 protein, a crucial transcription factor in cellular responses to a wide variety of stress, regulates multiple target genes involved in tumor suppression, senescence induction, and metabolic functions. However, it remains unclear how diverse cellular phenotypes are modulated by p53. In this study, we developed an optogenetic tool, Opto-p53, to control p53 signaling by light. Opto-p53 was designed to trigger p53 signaling by reconstituting p53 N-terminal and C-terminal fragments with a light-inducible dimerization (LID) system. Upon light exposure, cells expressing Opto-p53 demonstrated p53 transcriptional activation, resulting in cell death and cell cycle arrest. We further enhanced the efficacy of light-induced p53 activation by introducing specific mutations into Opto-p53 fragments. Our findings unveil the capability of Opto-p53 to serve as a powerful tool for dissecting the complex roles of p53 in cellular processes, thereby contributing to the field of synthetic biology and providing general design principles for optogenetic tools using endogenous transcription factors.

Abstract: There is currently a lack of tools capable of perturbing genes in both a precise and a spatiotemporal fashion. The flexibility of CRISPR (clustered regularly interspaced short palindromic repeats), coupled with light's unparalleled spatiotemporal resolution deliverable from a controllable source, makes optogenetic CRISPR a well-suited solution for precise spatiotemporal gene perturbations. Here, we present a new optogenetic CRISPR tool (Blue Light-inducible Universal VPR-Improved Production of RGRs, BLU-VIPR) that diverges from prevailing split-Cas design strategies and instead focuses on optogenetic regulation of guide RNA (gRNA) production. We engineered BLU-VIPR around a new potent blue-light activated transcription factor (VPR-EL222) and ribozyme-flanked gRNA. The BLU-VIPR design is genetically encoded and ensures precise excision of multiple gRNAs from a single messenger RNA transcript. This simplified spatiotemporal gene perturbation and allowed for several types of optogenetic CRISPR, including indels, CRISPRa, and base editing. BLU-VIPR also worked in vivo with cells previously intractable to optogenetic gene editing, achieving optogenetic gene editing in T lymphocytes in vivo.

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: Talin regulates the adhesion and migration of cells in part by promoting the affinity of integrins for extracellular matrix proteins, a process that in cells such as endothelial cells and platelets requires the direct interaction of talin with both the small GTPase Rap1 bound to GTP (Rap1-GTP) and the integrin β3 cytoplasmic tail. To study this process in more detail, we employed an optogenetic approach in living, immortalized endothelial cells to be able to regulate the interaction of talin with the plasma membrane. Previous studies identified talin as the Rap1-GTP effector for β3 integrin activation. Surprisingly, optogenetic recruitment of talin-1 (TLN1; herein referred to as talin) to the plasma membrane also led to the localized activation of Rap1 itself, apparently by talin competing for Rap1-GTP with SHANK3, a protein known to sequester Rap1-GTP and to block integrin activation. Rap1 activation by talin was localized to the cell periphery in suspension cells and within lamellipodia and pseudopodia in cells adherent to fibronectin. Thus, membrane-associated talin can play a dual role in regulating integrin function in endothelial cells: first, by releasing Rap1-GTP from its sequestration by SHANK3, and second, by serving as the relevant Rap1 effector for integrin activation.

Abstract: G-protein-coupled receptors (GPCRs) are efficient guanine nucleotide exchange factors (GEFs) and exchange GDP to GTP on the Gα subunit of G-protein heterotrimers in response to various extracellular stimuli, including neurotransmitters and light. GPCRs primarily broadcast signals through activated G proteins, GαGTP and free Gβγ and are major disease drivers. Evidence shows that the ambient low threshold signalling required for cells is likely supplemented by signalling regulators such as non-GPCR GEFs and guanine nucleotide dissociation inhibitors (GDIs). Activators of G-protein signalling 3 (AGS3) are recognized as a GDI involved in multiple health and disease-related processes. Nevertheless, understanding of AGS3 is limited, and no significant information is available on its structure-function relationship or signalling regulation in living cells. Here, we employed in silico structure-guided engineering of a novel optogenetic GDI, based on the AGS3's G-protein regulatory motif, to understand its GDI activity and induce standalone Gβγ signalling in living cells on optical command. Our results demonstrate that plasma membrane recruitment of OptoGDI efficiently releases Gβγ, and its subcellular targeting generated localized PIP3 and triggered macrophage migration. Therefore, we propose OptoGDI as a powerful tool for optically dissecting GDI-mediated signalling pathways and triggering GPCR-independent Gβγ signalling in cells and in vivo.

Abstract: The regulation of PKC epsilon (PKCε) and its downstream effects is still not fully understood, making it challenging to develop targeted therapies or interventions. A more precise tool that enables spatiotemporal control of PKCε activity is thus required. Here, we describe a photo-activatable optogenetic PKCε probe (Opto-PKCε) consisting of an engineered PKCε catalytic domain and a blue-light inducible dimerization domain. Molecular dynamics and AlphaFold simulations enable rationalization of the dark-light activity of the optogenetic probe. We first characterize the binding partners of Opto-PKCε, which are similar to those of PKCε. Subsequent validation of the Opto-PKCε tool is performed with phosphoproteome analysis, which reveals that only PKCε substrates are phosphorylated upon light activation. Opto-PKCε could be engineered for recruitment to specific subcellular locations. Activation of Opto-PKCε in isolated hepatocytes reveals its sustained activation at the plasma membrane is required for its phosphorylation of the insulin receptor at Thr1160. In addition, Opto-PKCε recruitment to the mitochondria results in its lowering of the spare respiratory capacity through phosphorylation of complex I NDUFS4. These results demonstrate that Opto-PKCε may have broad applications for the studies of PKCε signaling with high specificity and spatiotemporal resolution.

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: Most bacteria lack membrane-enclosed organelles and rely on macromolecular scaffolds at different subcellular locations to recruit proteins for specific functions. Here, we demonstrate that the optogenetic CRY2-CIB1 system from Arabidopsis thaliana can be used to rapidly direct proteins to different subcellular locations with varying efficiencies in live Escherichia coli cells, including the nucleoid, the cell pole, the membrane, and the midcell division plane. Such light-induced re-localization can be used to rapidly inhibit cytokinesis in actively dividing E. coli cells. We further show that CRY2-CIBN binding kinetics can be modulated by green light, adding a new dimension of control to the system. Finally, we test this optogenetic system in three additional bacterial species, Bacillus subtilis, Caulobacter crescentus, and Streptococcus pneumoniae, providing important considerations for this system's applicability in bacterial cell biology.

Abstract: This volume explores the latest advancements in the field of optogenetics and how it uses cellular light-sensing components and genetic engineering to control proteins and biological processes. The book chapters are organized into four parts. Part One focuses on intracellular optogenetic components for control of specific cell functions; Part Two looks at externally supplied light regulators that do not require genetic manipulation of target cells; Part Three highlights optogenetic control of organelles, and Part Four introduces technical tools required for light induction in optogenetic experiments, as well as a method for performing and analyzing optogenetic cell-cell adhesion. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Cutting-edge and practical, Optogenetics: Methods and Protocols is a valuable resource to help researchers understand and apply the concepts of optogenetics and the underlying bioengineering principles, and establish the required technical light-illumination setups for administering light inputs and analysis of experimental outcomes.

Abstract: Development of neuronal connections is spatially and temporally controlled by extracellular cues which often activate their cognate cell surface receptors and elicit localized cellular responses. Here, we demonstrate the use of an optogenetic tool to activate receptor signaling locally to induce actin-mediated growth cone remodeling in neurons. Based on the light-induced interaction between Cryptochrome 2 (CRY2) and CIB1, we generated a bicistronic vector to co-expresses CRY2 fused to the intracellular domain of a guidance receptor and a membrane-anchored CIB1. When expressed in primary neurons, activation of the growth inhibitory PlexA4 receptor induced growth cone collapse, while activation of the growth stimulating TrkA receptor increased growth cone size. Moreover, local activation of either receptor not only elicited the predicted response in light-activated growth cones but also an opposite response in neighboring no-light-exposed growth cones of the same neuron. Finally, this tool was used to reorient growth cones toward or away from the site of light activation and to stimulate local actin polymerization for branch initiation along axonal shafts. These studies demonstrate the use of an optogenetic tool for precise spatial and temporal control of receptor signaling in neurons and support its future application in investigating cellular mechanisms of neuronal development and plasticity.

Abstract: Starvation, which is associated with inactivation of the growth-promoting TOR complex 1 (TORC1), is a strong environmental signal for cell differentiation. In the fission yeast Schizosaccharomyces pombe, nitrogen starvation has distinct physiological consequences depending on the presence of mating partners. In their absence, cells enter quiescence, and TORC1 inactivation prolongs their life. In presence of compatible mates, TORC1 inactivation is essential for sexual differentiation. Gametes engage in paracrine pheromone signaling, grow towards each other, fuse to form the diploid zygote, and form resistant, haploid spore progenies. To understand the signaling changes in the proteome and phospho-proteome during sexual reproduction, we developed cell synchronization strategies and present (phospho-)proteomic data sets that dissect pheromone from starvation signals over the sexual differentiation and cell–cell fusion processes. Unexpectedly, these data sets reveal phosphorylation of ribosomal protein S6 during sexual development, which we establish requires TORC1 activity. We demonstrate that TORC1 is re-activated by pheromone signaling, in a manner that does not require autophagy. Mutants with low TORC1 re-activation exhibit compromised mating and poorly viable spores. Thus, while inactivated to initiate the mating process, TORC1 is reactivated by pheromone signaling in starved cells to support sexual reproduction.

Abstract: Controlling CRISPR/Cas9 gene editing at the spatiotemporal resolution level, especially for in vivo applications, remains a great challenge. Here, we developed a near-infrared (NIR) light-activated nanophotonic system (UCPP) for controlled CRISPR-Cas9 gene editing and synergistic photodynamic therapy (PDT). Lanthanide-doped upconversion nanoparticles are not only employed as carriers for intracellular plasmid delivery but also serve as the nanotransducers to convert NIR light (980 nm) into visible light with emission at 460 and 650 nm, which could result in simultaneous activation of gene editing and PDT processes, respectively. Such unique design not only achieves light-controlled precise gene editing of hypoxia-inducible factor 1α with minimal off-target effect, which effectively ameliorates the hypoxic state at tumor sites, but also facilitates the deep-seated PDT process with synergistic antitumor effect. This optogenetically activatable CRISPR-Cas9 nanosystem holds great potential for spatially controlled in vivo gene editing and targeted cancer therapy.

Abstract: Background Optogenetic systems use light-responsive proteins to control gene expression with the “flip of a switch”. One such tool is the light activated CRISPR effector (LACE) system. Its ability to regulate gene expression in a tunable, reversible, and spatially resolved manner makes it attractive for many applications. However, LACE relies on delivery of four separate components on individual plasmids, which can limit its use. Here, we optimize LACE to reduce the number of plasmids needed to deliver all four components. Results The two-plasmid LACE (2pLACE) system combines the four components of the original LACE system into two plasmids. Following construction, the behavior of 2pLACE was rigorously tested using optogenetic control of enhanced green fluorescent protein (eGFP) expression as a reporter. We optimized the ratio of the two plasmids, measured activation as a function of light intensity, and determined the frequency of the light to activate the maximum fluorescence. Overall, the 2pLACE system showed a similar dynamic range, tunability, and activation kinetics as the original four plasmid LACE (4pLACE) system. Interestingly, 2pLACE also had less variability in activation signal compared to 4pLACE. Conclusions This simplified system for optogenetics will be more amenable to biotechnology applications where variability needs to be minimized. By optimizing the LACE system to use fewer plasmids, 2pLACE becomes a flexible tool in multiple research applications.

Abstract: The use of optogenetic tools offers an excellent method for spatially and temporally regulated gene and protein expression in cell therapeutic approaches. This could be useful as a concomitant therapeutic measure, especially in small body compartments such as the inner ear, for example, during cochlea implantation, to enhance neuronal cell survival and function. Here, we used the blue light activatable CRY2/CIB system to induce transcription of brain-derived neurotrophic factor (BDNF) in human cells. Transfection with three plasmids, encoding for the optogenetic system and the target, as well as illumination protocols were optimized with luciferase as a reporter to achieve the highest protein expression in human embryonic kidney cells 293. Illumination was performed either with a light-emitting diode or with a scanning laser setup. The optimized protocols were applied for the production of BDNF. We could demonstrate a 64.7-fold increase of BNDF expression upon light induction compared to the basal level. Light-induced BDNF was biologically active and enhanced survival and neurite growth of spiral ganglion neurons. The optogenetic approach can be transferred to autologous cell systems, such as bone marrow-derived mesenchymal stem cells, and thus represents the first optogenetic neurotrophic therapy for the inner ear.

Abstract: Post-translational modifications (PTMs) are indispensable modulators of protein activity. Most cellular behaviours, from cell division to cytoskeletal organization, are controlled by PTMs, their miss-regulation being associated with a plethora of human diseases. Traditionally, the role of PTMs has been studied employing biochemical techniques. However, these approaches fall short when studying PTM dynamics in vivo. In recent years, functionalized protein binders have allowed the post-translational modification of endogenous proteins by bringing an enzymatic domain in close proximity to the protein they recognize. To date, most of these methods lack the temporal control necessary to understand the complex effects triggered by PTMs. In this study, we have developed a method to phosphorylate endogenous Myosin in a light-inducible manner. The method relies both on nanobody-targeting and light-inducible activation in order to achieve both tight specificity and temporal control. We demonstrate that this technology is able to disrupt cytoskeletal dynamics during Drosophila embryonic development. Together, our results highlight the potential of combining optogenetics and protein binders for the study of the proteome in multicellular systems.

Abstract: Gene delivery is fundamentally crucial for the genetic manipulation of stem cells. Here, we present a straightforward approach to create a library of charge-neutralized polyethylenimine (PEI)-lipid nanoparticles designed for stem cell transfection. These lipid nanoparticles were formulated using small, branched PEI and lipidic anhydrides. Remarkably, over 15% of the lipid nanoparticles demonstrated high transfection efficiency across various cell types, surpassing the efficiency of both Lipofectamine 2000 and FuGENE HD. A structure-activity analysis indicated that the length and ratio of hydrophobic alkyl substitutions were critical parameters for efficient gene delivery. Notably, the transfection efficiency was higher than that of the original cation PEI. Our optimized PEI-lipid system enabled highly effective plasmid DNA delivery and successfully co-transferred two plasmid DNAs into difficult-to-transfect human embryonic stem cells (hESCs), facilitating optogenetic manipulation within these cells.

Abstract: Collective cell migration is key during development, wound healing, and metastasis and relies on coordinated cell behaviors at the group level. Src kinase is a key signalling protein for the physiological functions of epithelia, as it regulates many cellular processes, including adhesion, motility, and mechanotransduction. Its overactivation is associated with cancer aggressiveness. Here, we take advantage of optogenetics to precisely control Src activation in time and show that its pathological-like activation slows the collective rotation of epithelial cells confined into circular adhesive patches. We interpret velocity, force, and stress data during period of non-activation and period of activation of Src thanks to a hydrodynamic description of the cell assembly as a polar active fluid. Src activation leads to a 2-fold decrease in the ratio of polar angle to friction, which could result from increased adhesiveness at the cell-substrate interface. Measuring internal stress allows us to show that active stresses are subdominant compared to traction forces. Our work reveals the importance of fine-tuning the level of Src activity for coordinated collective behaviors.

Abstract: The post-translational modification of intracellular proteins through O-linked β-N-acetylglucosamine (O-GlcNAc) is a conserved regulatory mechanism in multicellular organisms. Catalyzed by O-GlcNAc transferase (OGT), this dynamic modification has an essential role in signal transduction, gene expression, organelle function and systemic physiology. Here, we present Opto-OGT, an optogenetic probe that allows for precise spatiotemporal control of OGT activity through light stimulation. By fusing a photosensitive cryptochrome protein to OGT, Opto-OGT can be robustly and reversibly activated with high temporal resolution by blue light and exhibits minimal background activity without illumination. Transient activation of Opto-OGT results in mTORC activation and AMPK suppression, which recapitulate nutrient-sensing signaling. Furthermore, Opto-OGT can be customized to localize to specific subcellular sites. By targeting OGT to the plasma membrane, we demonstrate the downregulation of site-specific AKT phosphorylation and signaling outputs in response to insulin stimulation. Thus, Opto-OGT is a powerful tool for defining the role of O-GlcNAcylation in cell signaling and physiology.

Abstract: Epithelia are polarized layers of cells that line the outer and inner surfaces of organs. At the basal side, the epithelial cell layer is supported by a basement membrane, which is a thin polymeric layer of self-assembled extracellular matrix (ECM) that tightly adheres to the basal cell surface. Proper shaping of epithelial layers is an important prerequisite for the development of healthy organs during the morphogenesis of an organism. Experimental evidence suggests that local degradation of the basement membrane is one of the mechanisms that can drive epithelial folding. However, how folding emerges in the absence of tissue growth remains elusive. Here, we present a coarse-grained plate theory model of the basement membrane that assumes force balance between i) cell-transduced active forces and ii) deformation-induced elastic forces. We verify key assumptions of this model through experiments in the Drosophila wing disc epithelium and demonstrate that the model can explain the emergence of outward epithelial folds upon local plate degradation. The model accounts for local degradation of the basement membrane as a mechanism for the generation of epithelial folds in the absence of epithelial growth.

Abstract: Intracellular trafficking, an extremely complex network, dynamically orchestrates nearly all cellular activities. A versatile method that enables the manipulation of target transport pathways with high spatiotemporal accuracy in vitro and in vivo is required to study how this network coordinates its functions. Here, a new method called RIVET (Rapid Immobilization of target Vesicles on Engaged Tracks) is presented. Utilizing inducible dimerization between target vesicles and selective cytoskeletons, RIVET can spatiotemporally halt numerous intracellular trafficking pathways within seconds in a reversible manner. Its highly specific perturbations allow for the real-time dissection of the dynamic relationships among different trafficking pathways. Moreover, RIVET is capable of inhibiting receptor-mediated endocytosis. This versatile system can be applied from the cellular level to whole organisms. RIVET opens up new avenues for studying intracellular trafficking under various physiological and pathological conditions and offers potential strategies for treating trafficking-related disorders.

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: Cell contacts in epithelia are remodeled to regulate paracellular permeability and to control passage of migrating cells, but how barrier function is modulated while preserving epithelial integrity is not clear. In the follicular epithelium of Drosophila ovaries, tricellular junctions (TCJs) open transiently in a process termed patency to allow passage of externally produced yolk proteins for uptake by the oocyte. Here we show that modulation of actomyosin contractility at cell vertices controls TCJ permeability. Before patency, circumferential actomyosin bundles are anchored at apical follicle cell vertices, where tension-sensing junctional proteins, Rho-associated kinase (Rok), and active Myosin II accumulate and maintain vertices closed. TCJ opening is initiated by redistribution of Myosin II from circumferential bundles to a medial pool, accompanied by decreasing tension on vertices. This transition requires activation of Cofilin-dependent F-actin disassembly by the phosphatase Slingshot and Myosin II inactivation by Myosin light chain phosphatase, and is counteracted by Rok. Accordingly, constitutive activation of Myosin or of Rho signaling prevent vertex opening, whereas reduced Myosin II or Rok activity cause excessive and premature vertex opening. Thus, opening of intercellular gaps in the follicular epithelium does not require actomyosin-based forces, but relies on a reduction of actomyosin contractility. Conversely, F-actin assembly is required for closing intercellular gaps after patency. Our findings are consistent with a force transduction model in which TCJ integrity is maintained by vertex-anchored contractile actomyosin. We propose that the cell-type-specific organization of actomyosin at cell vertices determines the mode of contractility-dependent regulation of epithelial permeability.

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: Small-conductance Ca2+-activated K+ channels (SK, KCa2) are gated solely by intracellular microdomain Ca2+. The channel has emerged as a therapeutic target for cardiac arrhythmias. Calmodulin (CaM) interacts with the CaM binding domain (CaMBD) of the SK channels, serving as the obligatory Ca2+ sensor to gate the channels. In heterologous expression systems, phosphatidylinositol 4,5-bisphosphate (PIP2) coordinates with CaM in regulating SK channels. However, the roles and mechanisms of PIP2 in regulating SK channels in cardiomyocytes remain unknown. Here, optogenetics, magnetic nanoparticles, combined with Rosetta structural modeling, and molecular dynamics (MD) simulations revealed the atomistic mechanisms of how PIP2 works in concert with Ca2+-CaM in the SK channel activation. Our computational study affords evidence for the critical role of the amino acid residue R395 in the S6 transmembrane segment, which is localized in propinquity to the intracellular hydrophobic gate. This residue forms a salt bridge with residue E398 in the S6 transmembrane segment from the adjacent subunit. Both R395 and E398 are conserved in all known isoforms of SK channels. Our findings suggest that the binding of PIP2 to R395 residue disrupts the R395:E398 salt bridge, increasing the flexibility of the transmembrane segment S6 and the activation of the channel. Importantly, our findings serve as a platform for testing of structural-based drug designs for therapeutic inhibitors and activators of the SK channel family. The study is timely since inhibitors of SK channels are currently in clinical trials to treat atrial arrhythmias.