Curated Optogenetic Publication Database

Abstract: Extracellular-signal-regulated kinase (ERK) signaling controls development and homeostasis and is genetically deregulated in human diseases, including neurocognitive disorders and cancers. Although the list of ERK functions is vast and steadily growing, the full spectrum of processes controlled by any specific ERK activation event remains unknown. Here, we show how ERK functions can be systematically identified using targeted perturbations and global readouts of ERK activation. Our experimental model is the Drosophila embryo, where ERK signaling at the embryonic poles has thus far only been associated with the transcriptional patterning of the future larva. Through a combination of live imaging and phosphoproteomics, we demonstrated that ERK activation at the poles is also critical for maintaining the speed and synchrony of embryonic cleavages. The presented approach to interrogating phosphorylation networks identifies a hidden function of a well-studied signaling event and sets the stage for similar studies in other organisms.

Abstract: In recent decades, the field of synthetic biology has witnessed remarkable progress, driving advances in both research and practical applications. One pivotal area of development involves the design of transgene switches capable of precisely regulating specified outputs and controlling cell behaviors in response to physical cues, which encompass light, magnetic fields, temperature, mechanical forces, ultrasound, and electricity. In this review, we delve into the cutting-edge progress made in the field of physically controlled protein expression in engineered mammalian cells, exploring the diverse genetic tools and synthetic strategies available for engineering targeting cells to sense these physical cues and generate the desired outputs accordingly. We discuss the precision and efficiency limitations inherent in these tools, while also highlighting their immense potential for therapeutic applications.

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: TANGO1, TANGO1-Short, and cTAGE5 form stable complexes at the endoplasmic reticulum exit sites (ERES) to preferably export bulky cargoes. Their C-terminal proline-rich domain (PRD) binds Sec23A and affects COPII assembly. The PRD in TANGO1-Short was replaced with light-responsive domains to control its binding to Sec23A in U2OS cells (human osteosarcoma). TANGO1-ShortΔPRD was dispersed in the ER membrane but relocated rapidly, reversibly, to pre-existing ERES by binding to Sec23A upon light activation. Prolonged binding between the two, concentrated ERES in the juxtanuclear region, blocked cargo export and relocated ERGIC53 into the ER, minimally impacting the Golgi complex organization. Bulky collagen VII and endogenous collagen I were collected at less than 47% of the stalled ERES, whereas small cargo molecules were retained uniformly at almost all the ERES. We suggest that ERES are segregated to handle cargoes based on their size, permitting cells to traffic them simultaneously for optimal secretion.

Abstract: Memory processes rely on a molecular signaling system that balances the interplay between positive and negative modulators. Recent research has focused on identifying memory-regulating genes and their mechanisms. Phospholipase C beta 1 (PLCβ1), highly expressed in the hippocampus, reportedly serves as a convergence point for signal transduction through G protein-coupled receptors. However, the detailed role of PLCβ1 in memory function has not been elucidated. Here, we demonstrate that PLCβ1 in the dentate gyrus functions as a memory suppressor. We reveal that mice lacking PLCβ1 in the dentate gyrus exhibit a heightened fear response and impaired memory extinction, and this excessive fear response is repressed by upregulation of PLCβ1 through its overexpression or activation using a newly developed optogenetic system. Last, our results demonstrate that PLCβ1 overexpression partially inhibits exaggerated fear response caused by traumatic experience. Together, PLCβ1 is crucial in regulating contextual fear memory formation and potentially enhancing the resilience to trauma-related conditions.

Abstract: Chronic pain is a prevalent problem that plagues modern society, and better understanding its mechanisms is critical for developing effective therapeutics. Nerve growth factor (NGF) and its primary receptor, Tropomyosin receptor kinase A (TrkA), are known to be potent mediators of chronic pain, but there is a lack of established methods for precisely perturbing the NGF/TrkA signaling pathway in the study of pain and nociception. Optobiological tools that leverage light-induced protein-protein interactions allow for precise spatial and temporal control of receptor signaling. Previously, our lab reported a blue light-activated version of TrkA generated using light-induced dimerization of the intracellular TrkA domain, opto-iTrkA. In this work, we show that opto-iTrkA activation is able to activate endogenous ERK and Akt signaling pathways and causes the retrograde transduction of phospho-ERK signals in dorsal root ganglion (DRG) neurons. Opto-iTrkA activation also sensitizes the transient receptor potential vanilloid 1 (TRPV1) channel in cellular models, further corroborating the physiological relevance of the optobiological stimulus. Finally, we show that opto-iTrkA enables light-inducible potentiation of mechanical sensitization in mice. Light illumination enables nontraumatic and reversible (<2 days) sensitization of mechanical pain in mice transduced with opto-iTrkA, which provides a platform for dissecting TrkA pathways for nociception in vitro and in vivo.

Abstract: The question of how changes in chemoattractant concentration translate into the chemotactic response of immune cells serves as a paradigm for the quantitative understanding of how cells perceive and process temporal and spatial information. Here, using a microfluidic approach, we analyzed the migration of neutrophil-like HL-60 cells to a traveling wave of the chemoattractants fMLP and leukotriene B4 (LTB4). We found that under a pulsatile wave that travels at a speed of 95 and 170 µm/min, cells move forward in the front of the wave but slow down and randomly orient at the back due to temporal decrease in the attractant concentration. Under a slower wave, cells re-orient and migrate at the back of the wave; thus, cell displacement is canceled out or even becomes negative as cells chase the receding wave. FRET-based analysis indicated that these patterns of movement correlated well with spatiotemporal changes in Cdc42 activity. Furthermore, pharmacological perturbations suggested that migration in front of the wave depends on Cdc42, whereas that in the back of the wave depends more on PI3K/Rac and ROCK. These results suggest that pulsatile attractant waves may recruit or disperse neutrophils, depending on their speed and degree of cell polarization.

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: Hydrophilic and biocompatible hydrogels are widely applied as ideal scaffolds in tissue engineering. The "smart" gelation material can alter its structural, physiochemical, and functional features in answer to various endo/exogenous stimuli to better biomimic the endogenous extracellular matrix for the engineering of cells and tissues. Light irradiation owns a high spatial-temporal resolution, complete biorthogonal reactivity, and fine-tunability and can thus induce physiochemical reactions within the matrix of photoresponsive hydrogels with good precision, efficiency, and safety. Both gel structure (e.g., geometry, porosity, and dimension) and performance (like conductivity and thermogenic or mechanical properties) can hence be programmed on-demand to yield the biochemical and biophysical signals regulating the morphology, growth, motility, and phenotype of engineered cells and tissues. Here we summarize the strategies and mechanisms for encoding light-reactivity into a hydrogel and demonstrate how fantastically such responsive gels change their structure and properties with light irradiation as desired and thus improve their applications in tissue engineering including cargo delivery, dynamic three-dimensional cell culture, and tissue repair and regeneration, aiming to provide a basis for more and better translation of photoresponsive hydrogels in the clinic.

Abstract: Microtubules regulate cell polarity and migration via local activation of focal adhesion turnover, but the mechanism of this process is insufficiently understood. Molecular complexes containing KANK family proteins connect microtubules with talin, the major component of focal adhesions. Here, local optogenetic activation of KANK1-mediated microtubule/talin linkage promoted microtubule targeting to an individual focal adhesion and subsequent withdrawal, resulting in focal adhesion centripetal sliding and rapid disassembly. This sliding is preceded by a local increase of traction force due to accumulation of myosin-II and actin in the proximity of the focal adhesion. Knockdown of the Rho activator GEF-H1 prevented development of traction force and abolished sliding and disassembly of focal adhesions upon KANK1 activation. Other players participating in microtubule-driven, KANK-dependent focal adhesion disassembly include kinases ROCK, PAK, and FAK, as well as microtubules/focal adhesion-associated proteins kinesin-1, APC, and αTAT. Based on these data, we develop a mathematical model for a microtubule-driven focal adhesion disruption involving local GEF-H1/RhoA/ROCK-dependent activation of contractility, which is consistent with experimental data.

Abstract: During development, epithelia function as malleable substrates that undergo extensive remodeling to shape developing embryos. Optogenetic control of Rho signaling provides an avenue to investigate the mechanisms of epithelial morphogenesis, but transgenic optogenetic tools can be limited by variability in tool expression levels and deleterious effects of transgenic overexpression on development. Here, we use CRISPR/Cas9 to tag Drosophila RhoGEF2 and Cysts/Dp114RhoGEF with components of the iLID/SspB optogenetic heterodimer, permitting light-dependent control over endogenous protein activities. Using quantitative optogenetic perturbations, we uncover a dose-dependence of tissue furrow depth and bending behavior on RhoGEF recruitment, revealing mechanisms by which developing embryos can shape tissues into particular morphologies. We show that at the onset of gastrulation, furrows formed by cell lateral contraction are oriented and size-constrained by a stiff basal actomyosin layer. Our findings demonstrate the use of quantitative, 3D-patterned perturbations of cell contractility to precisely shape tissue structures and interrogate developmental mechanics.

Abstract: Cells generate a highly diverse microtubule network to carry out different activities. This network is comprised of distinct tubulin isotypes, tubulins with different post-translational modifications, and many microtubule-based structures. Defects in this complex system cause numerous human disorders. However, how different microtubule subtypes in this network regulate cellular architectures and activities remains largely unexplored. Emerging tools such as photosensitive pharmaceuticals, chemogenetics, and optogenetics enable the spatiotemporal manipulation of structures, dynamics, post-translational modifications, and cross-linking with actin filaments in target microtubule subtypes. This review summarizes the design rationale and applications of these new approaches and aims to provide a roadmap for researchers navigating the intricacies of microtubule dynamics and their post-translational modifications in cellular contexts, thereby opening new avenues for therapeutic interventions.

Abstract: Phospholipase C (PLC) is a key enzyme that regulates physiological processes via lipid and calcium signaling. Despite advances in protein engineering, no tools are available for direct PLC control. Here, we developed a novel optogenetic tool, light-controlled PLCβ (opto-PLCβ). Opto-PLCβ uses a light-induced dimer module, which directs an engineered PLC to the plasma membrane in a light-dependent manner. Our design includes an autoinhibitory capacity, ensuring stringent control over PLC activity. Opto-PLCβ triggers reversible calcium responses and lipid dynamics in a restricted region, allowing precise spatiotemporal control of PLC signaling. Using our system, we discovered that phospholipase D-mediated phosphatidic acid contributes to diacylglycerol clearance on the plasma membrane. Moreover, we extended its applicability in vivo, demonstrating that opto-PLCβ can enhance amygdala synaptic plasticity and associative fear learning in mice. Thus, opto-PLCβ offers precise spatiotemporal control, enabling comprehensive investigation of PLC-mediated signaling pathways, lipid dynamics, and their physiological consequences in vivo.

Abstract: Biomolecular condensates are dynamic subcellular compartments that lack surrounding membranes and can spatiotemporally organize the cellular biochemistry of eukaryotic cells. However, such dynamic organization has not been realized in prokaryotes that naturally lack organelles, and strategies are urgently needed for dynamic biomolecular compartmentalization. Here we develop a light-switchable condensate system for on-demand dynamic organization of functional cargoes in the model prokaryotic Escherichia coli cells. The condensate system consists of two modularly designed and genetically encoded fusions that contain a condensation-enabling scaffold and a functional cargo fused to the blue light-responsive heterodimerization pair, iLID and SspB, respectively. By appropriately controlling the biogenesis of the protein fusions, the condensate system allows rapid recruitment and release of cargo proteins within seconds in response to light, and this process is also reversible and repeatable. Finally, the system is demonstrated to dynamically control the subcellular localization of a cell division inhibitor, SulA, which enables the reversible regulation of cell morphologies. Therefore, this study provides a new strategy to dynamically control cellular processes by harnessing light-controlled condensates in prokaryotic cells.

Abstract: Molecular optogenetics utilizes genetically encoded, light-responsive protein switches to control the function of molecular processes. Over the last two years, there have been notable advances in the development of novel optogenetic switches, their utilization in elucidating intricate signaling pathways, and their progress toward practical applications in biotechnological processes, material sciences, and therapeutic applications. In this review, we discuss these areas, offer insights into recent developments, and contemplate future directions.

Abstract: Liquid-liquid phase separation (LLPS) is responsible for the emergence of intracellular membrane-less organelles and the development of coacervate protocells. Benefitting from the advantages of simplicity, precision, programmability, and noninvasiveness, light has become an effective tool to regulate the assembly dynamics of LLPS, and mediate various biochemical processes associated with LLPS. In this review, recent advances in optically controlling membrane-less organelles within living organisms are summarized, thereby modulating a series of biological processes including irreversible protein aggregation pathologies, transcription activation, metabolic flux, genomic rearrangements, and enzymatic reactions. Among these, the intracellular systems (i.e., optoDroplet, Corelet, PixELL, CasDrop, and other optogenetic systems) that enable the photo-mediated control over biomolecular condensation are highlighted. The design of photoactive complex coacervate protocells in laboratory settings by utilizing photochromic molecules such as azobenzene and diarylethene is further discussed. This review is expected to provide in-depth insights into phase separation-associated biochemical processes, bio-metabolism, and diseases.

Abstract: Only a few short decades have passed since the sequencing of GFP, yet the modern repertoire of transgenically encoded optical tools implies an exponential proliferation of ever improving constructions to interrogate the subcellular environment. A myriad of tags for labeling proteins, RNA, or DNA have arisen in the last few decades, facilitating unprecedented visualization of subcellular components and processes. Development of a broad array of modern genetically encoded sensors allows real-time, in vivo detection of molecule levels, pH, forces, enzyme activity, and other subcellular and extracellular phenomena in ever expanding contexts. Optogenetic, genetically encoded optically controlled manipulation systems have gained traction in the biological research community and facilitate single-cell, real-time modulation of protein function in vivo in ever broadening, novel applications. While this field continues to explosively expand, references are needed to assist scientists seeking to use and improve these transgenic devices in new and exciting ways to interrogate development and disease. In this review, we endeavor to highlight the state and trajectory of the field of in vivo transgenic optical tools.

Abstract: Many essential biological processes are triggered by the proximity of molecules. Meanwhile, diverse approaches in synthetic biology, such as new biological parts or engineered cells, have opened up avenues to precisely control the proximity of molecules and eventually downstream signaling processes. This also applies to a main Ca2+ entry pathway into the cell, the so-called Ca2+ release-activated Ca2+ (CRAC) channel. CRAC channels are among other channels are essential in the immune response and are activated by receptor-ligand binding at the cell membrane. The latter initiates a signaling cascade within the cell, which finally triggers the coupling of the two key molecular components of the CRAC channel, namely the stromal interaction molecule, STIM, in the ER membrane and the plasma membrane Ca2+ ion channel, Orai. Ca2+ entry, established via STIM/Orai coupling, is essential for various immune cell functions, including cytokine release, proliferation, and cytotoxicity. In this review, we summarize the tools of synthetic biology that have been used so far to achieve precise control over the CRAC channel pathway and thus over downstream signaling events related to the immune response.

Abstract: The precise localization and activation of proteins at the cell membrane at a certain time gives rise to many cellular processes, including cell polarization, migration, and division. Thus, methods to recruit proteins to model membranes with subcellular resolution and high temporal control are essential when reproducing and controlling such processes in synthetic cells. Here, a method is described for fabricating light-regulated reversible protein patterns at lipid membranes with high spatiotemporal precision. For this purpose, we immobilize the photoswitchable protein iLID (improved light-inducible dimer) on supported lipid bilayers (SLBs) and on the outer membrane of giant unilamellar vesicles (GUVs). Upon local blue light illumination, iLID binds to its partner Nano (wild-type SspB) and allows the recruitment of any protein of interest (POI) fused to Nano from the solution to the illuminated area on the membrane. This binding is reversible in the dark, which provides dynamic binding and release of the POI. Overall, this is a flexible and versatile method for regulating the localization of proteins with high precision in space and time using blue light.

Abstract: Endogenous biomolecular condensates, composed of a multitude of proteins and RNAs, can organize into multiphasic structures with compositionally distinct phases. This multiphasic organization is generally understood to be critical for facilitating their proper biological function. However, the biophysical principles driving multiphase formation are not completely understood. Here we use in vivo condensate reconstitution experiments and coarse-grained molecular simulations to investigate how oligomerization and sequence interactions modulate multiphase organization in biomolecular condensates. We demonstrate that increasing the oligomerization state of an intrinsically disordered protein results in enhanced immiscibility and multiphase formation. Interestingly, we find that oligomerization tunes the miscibility of intrinsically disordered proteins in an asymmetric manner, with the effect being more pronounced when the intrinsically disordered protein, exhibiting stronger homotypic interactions, is oligomerized. Our findings suggest that oligomerization is a flexible biophysical mechanism that cells can exploit to tune the internal organization of biomolecular condensates and their associated biological functions.

Abstract: Protein clustering plays numerous roles in cell physiology and disease. However, protein oligomers can be difficult to detect because they are often too small to appear as puncta in conventional fluorescence microscopy. Here, we describe a fluorescent reporter strategy that detects protein clusters with high sensitivity called CluMPS (clusters magnified by phase separation). A CluMPS reporter detects and visually amplifies even small clusters of a binding partner, generating large, quantifiable fluorescence condensates. We use computational modeling and optogenetic clustering to demonstrate that CluMPS can detect small oligomers and behaves rationally according to key system parameters. CluMPS detected small aggregates of pathological proteins where the corresponding GFP fusions appeared diffuse. CluMPS also detected and tracked clusters of unmodified and tagged endogenous proteins, and orthogonal CluMPS probes could be multiplexed in cells. CluMPS provides a powerful yet straightforward approach to observe higher-order protein assembly in its native cellular context. A record of this paper's transparent peer review process is included in the supplemental information.

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 under-explored. Here, utilizing live-cell experiments in parallel with coarse-grained simulations, we investigate how chromatin interaction of the transcription factor 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.

Abstract: Phospholipase D (PLD) and phosphatidic acid (PA) play a spatio-temporal role in regulating diverse cellular activities. Although current methodologies enable optical control of the subcellular localization of PLD and by which influence local PLD enzyme activity, the overexpression of PLD elevates the basal PLD enzyme activity and further leads to increased PA levels in cells. In this study, we employed a split protein reassembly strategy and optogenetic techniques to modify superPLD (a PLDPMF variant with a high basal activity). We splited this variants into two HKD domains and fused these domains with optogenetic elements and by which we achieved light-mediated dimerization of the two HKD proteins and then restored the PLD enzymatic activity.

Abstract: Phosphatidic acid (PA) is a multifunctional lipid with important metabolic and signaling functions, and efforts to dissect its pleiotropy demand strategies for perturbing its levels with spatiotemporal precision. Previous membrane editing approaches for generating local PA pools used light-mediated induced proximity to recruit a PA-synthesizing enzyme, phospholipase D (PLD), from the cytosol to the target organelle membrane. Whereas these optogenetic PLDs exhibited high activity, their residual activity in the dark led to undesired chronic lipid production. Here, we report ultralow background membrane editors for PA wherein light directly controls PLD catalytic activity, as opposed to localization and access to substrates, exploiting a light–oxygen–voltage (LOV) domain-based conformational photoswitch inserted into the PLD sequence and enabling their stable and nonperturbative targeting to multiple organelle membranes. By coupling organelle-targeted LOVPLD activation to lipidomics analysis, we discovered different rates of metabolism for PA and its downstream products depending on the subcellular location of PA production. We also elucidated signaling roles for PA pools on different membranes in conferring local activation of AMP-activated protein kinase signaling. This work illustrates how membrane editors featuring acute, optogenetic conformational switches can provide new insights into organelle-selective lipid metabolic and signaling pathways.

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, a major question in cell biology and drug development. Here we report an optogenetic approach to selectively dissolve a condensate of interest in a reversible and spatially controlled manner. We show that light-gated recruitment of maltose-binding protein (MBP), a commonly used solubilizing domain in protein purification, 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, showing that disrupting condensation of the oncogenic fusion protein FUS-CHOP results in reversion of 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.