Curated Optogenetic Publication Database

Search precisely and efficiently by using the advantage of the hand-assigned publication tags that allow you to search for papers involving a specific trait, e.g. a particular optogenetic switch or a host organism.

Showing 1 - 25 of 61 results

Coupling between protrusion dynamics and polarized trafficking steers persistent cell migration.

blue iLID hTERT RPE-1 Control of cytoskeleton / cell motility / cell shape
bioRxiv, 22 Mar 2021 DOI: 10.1101/2021.03.20.436273 Link to full text
Abstract: Migrating cells present a variety of paths, from non-persistent random walks to highly directional trajectories. While random movement can be easily explained by an intrinsic basal activity of the cell, persistent movement requires the cell to be stably polarized. It remains unclear how this is achieved from the regulation of underlying subcellular processes. In the context of mesenchymal migration, the ability of cells to migrate persistently over several hours require a mechanism stabilizing their protruding activity at their front. Here, we address this mechanism using human RPE1 cell line as our model. We measure, manipulate, and quantitatively perturb cell protrusive activity of the cortex as well as intracellular organization of the endomembrane trafficking system using dynamic micropatterning, pharmacological and trafficking assays, optogenetics and live-cell imaging with tracking. First, we demonstrate that the Nucleus-Golgi axis aligns with the direction of migration and its alignment with the protrusive activity leads to efficient cell movement. Then, using low doses of Nocodazole to disrupt internal cell organization, we show that long-lived polarity breaks down and migration becomes random. Next, we indicate that a flow of vesicles is directed towards the protrusive activity with a delay of 20 min. Eventually, by applying a sustained optogenetic activation, we prove that a localized Cdc42 gradient is able to orient the Nucleus-Golgi axis over a couple of hours. Taken together, our results suggest that the internal polarity axis, provided by the polarized trafficking of vesicles, is stabilizing the protrusive activity of the cell, while the protrusive activity biases this polarity axis. Using a novel minimal physical model, we show that this feedback is sufficient by itself to recapitulate the quantitative properties of cell migration in the timescale of hours. Our work highlights the importance of the coupling between high-level cellular functions in stabilizing the direction of migration over long timescales.

Optogenetic manipulation of YAP cellular localisation and function.

blue AsLOV2 HEK293T HFF-1 MKN28 zebrafish in vivo Signaling cascade control
bioRxiv, 19 Mar 2021 DOI: 10.1101/2021.03.19.436118 Link to full text
Abstract: YAP, an effector of the Hippo signalling pathway, promotes organ growth and regeneration. Prolonged YAP activation results in uncontrolled proliferation and cancer. Therefore, exogenous regulation of YAP activity has potential translational applications. We present a versatile optogenetic construct (optoYAP) for manipulating YAP localisation, and consequently its activity and function. We attached a LOV2 domain that photocages a nuclear localisation signal (NLS) to the N-terminus of YAP. In 488 nm light, the LOV2 domain unfolds, exposing the NLS, which shuttles optoYAP into the nucleus. Nuclear import of optoYAP is reversible and tuneable by light intensity. In cell culture, activated optoYAP promotes YAP target gene expression, cell proliferation, and anchorage-independent growth. Similarly, we can utilise optoYAP in zebrafish embryos to modulate target genes. OptoYAP is functional in both cell culture and in vivo, providing a powerful tool to address basic research questions and therapeutic applications in regeneration and disease.

Asymmetric Contraction of Adherens Junctions arises through RhoA and E-cadherin feedback.

blue TULIP Caco-2 Control of cell-cell / cell-material interactions
bioRxiv, 26 Feb 2021 DOI: 10.1101/2021.02.26.433093 Link to full text
Abstract: Tissue morphogenesis often arises from the culmination of discrete changes in cell-cell junction behaviors, namely ratcheted junction contractions that lead to collective cellular rearrangements. Mechanochemical signaling in the form of RhoA underlies these ratcheted contractions, which occur asymmetrically as one highly motile vertex contracts toward a relatively less motile tricellular vertex. The underlying mechanisms driving asymmetric vertex movement remains unknown. Here, we use optogenetically controlled RhoA in model epithelia together with biophysical modeling to uncover the mechanism lending to asymmetric vertex motion. We find that both local and global RhoA activation leads to increases in junctional tension, thereby facilitating vertex motion. RhoA activation occurs in discrete regions along the junction and is skewed towards the less-motile vertex. At these less-motile vertices, E-cadherin acts as an opposing factor to limit vertex motion through increased frictional drag. Surprisingly, we uncover a feedback loop between RhoA and E-cadherin, as regional optogenetic activation of specified junctional zones pools E-cadherin to the location of RhoA activation. Incorporating this circuit into a mathematical model, we find that a positive feedback between RhoA-mediated tension and E-cadherin-induced frictional drag on tricellular vertices recapitulates experimental data. As such, the location of RhoA determines which vertex is under high tension, pooling E-cadherin and increasing the frictional load at the tricellular vertex to limit its motion. This feedback drives a tension-dependent intercellular “clutch” at tricellular vertices which stabilizes vertex motion upon tensional load.

Optical regulation of endogenous RhoA reveals switching of cellular responses by signal amplitude.

blue cyan CRY2/CIB1 Dronpa145K/N pdDronpa1 TULIP HEK293A rat hippocampal neurons U-87 MG Endogenous gene expression
bioRxiv, 7 Feb 2021 DOI: 10.1101/2021.02.05.430013 Link to full text
Abstract: Precise control of the timing and amplitude of protein activity in living cells can explain how cells compute responses to complex biochemical stimuli. The small GTPase RhoA can promote either focal adhesion (FA) growth or cell edge retraction, but how a cell chooses between these opposite outcomes is poorly understood. Here, we developed a photoswitchable RhoA guanine exchange factor (psRhoGEF) to obtain precise optical control of endogenous RhoA activity. We find that low levels of RhoA activation by psRhoGEF induces edge retraction and FA disassembly, while high levels of RhoA activation induces both FA growth and disassembly. We observed that mDia-induced Src activation at FAs occurs preferentially at lower levels of RhoA activation. Strikingly, inhibition of Src causes a switch from FA disassembly to growth. Thus, rheostatic control of RhoA activation reveals how cells use signal amplitude and biochemical context to select between alternative responses to a single biochemical signal.

Single-component optogenetic tools for inducible RhoA GTPase signaling.

blue BcLOV4 HEK293T Control of cytoskeleton / cell motility / cell shape
bioRxiv, 2 Feb 2021 DOI: 10.1101/2021.02.01.429147 Link to full text
Abstract: We created optogenetic tools to control RhoA GTPase, a central regulator of actin organization and actomyosin contractility. RhoA GTPase, or its upstream activating GEF effectors, were fused to BcLOV4, a photoreceptor that can be dynamically recruited to the plasma membrane by a light-regulated protein-lipid electrostatic interaction with the inner leaflet. Direct membrane recruitment of these effectors induced potent contractile signaling sufficient to separate adherens junctions in response to as little as one pulse of blue light. Cytoskeletal morphology changes were dependent on the alignment of the spatially patterned stimulation with the underlying cell polarization. RhoA-mediated cytoskeletal activation induced YAP nuclear localization within minutes and subsequent mechanotransduction, verified by YAP-TEAD transcriptional activity. These single-component tools, which do not require protein binding partners, offer spatiotemporally precise control over RhoA signaling that will advance the study of its diverse regulatory roles in cell migration, morphogenesis, and cell cycle maintenance.

A synthetic switch based on orange carotenoid protein to control blue light responses in chloroplasts.

blue OCP A. thaliana in vivo A. thaliana leaf protoplasts
bioRxiv, 30 Jan 2021 DOI: 10.1101/2021.01.27.428448 Link to full text
Abstract: Synthetic biology approaches to engineer light‐responsive system are widely used, but their applications in plants are still limited, due to the interference with endogenous photoreceptors. Cyanobacteria, such as Synechocystis spp., possess a soluble carotenoid associated protein named Orange Carotenoid binding Protein (OCP) that, when activated by blue‐green light, undergoes reversible conformational changes that enable photoprotection of the phycobilisomes. Exploiting this system, we developed a new chloroplast‐localized synthetic photoswitch based on a photoreceptor‐associated protein‐fragment complementation assay (PCA). Since Arabidopsis thaliana does not possess the prosthetic group needed for the assembly of the OCP2 protein, we implemented the carotenoid biosynthetic pathway with a bacterial β‐carotene ketolase enzyme (crtW), to generate keto‐carotenoids producing plants. The novel photoswitch was tested and characterized in Arabidopsis protoplasts with experiments aimed to uncover its regulation by light intensity, wavelength, and its conversion dynamics. We believe that this pioneer study establishes the basis for future implementation of plastid optogenetics to regulate organelle responses, such as gene transcription or enzymatic activity, upon exposure to specific light spectra.

Transcription activation is enhanced by multivalent interactions independent of liquid-liquid phase separation.

blue CRY2/CIB1 HeLa U-2 OS Transgene expression
bioRxiv, 28 Jan 2021 DOI: 10.1101/2021.01.27.428421 Link to full text
Abstract: Transcription factors (TFs) consist of a DNA binding and an activation domain (AD) that are considered to be independent and exchangeable modules. However, recent studies conclude that also the physico-chemical properties of the AD can control TF assembly at chromatin via driving a phase separation into “transcriptional condensates”. Here, we dissected the mechanism of transcription activation at a reporter gene array with real-time single-cell fluorescence microscopy readouts. Our comparison of different synthetic TFs reveals that the phase separation propensity of the AD correlates with high transcription activation capacity by increasing binding site occupancy, residence time and the recruitment of co-activators. However, we find that the actual formation of phase separated TF liquid-like droplets has a neutral or inhibitory effect on transcription induction. Thus, our study suggests that the ability of a TF to phase separate reflects the functionally important property of the AD to establish multivalent interactions but does not by itself enhance transcription.

Random sub-diffusion and capture of genes by the nuclear pore reduces dynamics and coordinates interchromosomal movement.

blue CRY2/CIB1 S. cerevisiae
bioRxiv, 17 Jan 2021 DOI: 10.1101/2021.01.16.426968 Link to full text
Abstract: Hundreds of genes interact with the yeast nuclear pore complex (NPC), localizing at the nuclear periphery and clustering with co-regulated genes. Dynamic tracking of peripheral genes shows that they cycle on and off the NPC and that interaction with the NPC slows their sub-diffusive movement. Furthermore, NPC-dependent inter-chromosomal clustering leads to coordinated movement of pairs of loci separated by hundreds of nanometers. We developed Fractional Brownian Motion simulations for chromosomal loci in the nucleoplasm and interacting with NPCs. These simulations predict the rate and nature of random sub-diffusion during repositioning from nucleoplasm to periphery and match measurements from two different experimental models, arguing that recruitment to the nuclear periphery is due to random subdiffusion, collision, and capture by NPCs. Finally, the simulations do not lead to inter-chromosomal clustering or coordinated movement, suggesting that interaction with the NPC is necessary, but not sufficient, to cause clustering.

High levels of Dorsal transcription factor downregulate, not promote, snail expression by regulating enhancer action.

blue AsLOV2 D. melanogaster in vivo Developmental processes
bioRxiv, 11 Jan 2021 DOI: 10.1101/2021.01.11.426256 Link to full text
Abstract: In Drosophila embryos, genes expressed along the dorsal-ventral axis are responsive to concentration of the Dorsal (Dl) transcription factor, which varies in space; however, levels of this morphogen also build over time. Since expression of high-threshold Dl target genes such as snail (sna) is supported before Dl levels peak, it is unclear what role increasing levels have if any. Here we investigated action of two enhancers that control sna expression in embryos, demonstrating using genome editing that Dl binding sites within one enhancer located promoter proximally, sna.prox, can limit the ability of the other distally-located enhancer, sna.dis, to increase sna levels. In addition, MS2-MCP live imaging was used to study sna transcription rate in wildtype, dl heterozygote, and a background in which a photo-sensitive degron is fused to Dl (dl-BLID). The results demonstrate that, when Dl levels are high, Dl acts through sna.prox to limit the activity of sna.dis and thereby influence sna transcription rate. In contrast, when Dl levels are kept low using dl-BLID, sna.prox positively influences sna transcription rate. Collectively, our data support the view that Dl’s effect on gene expression changes over time, switching from promoting sna expression at low concentration to dampening sna expression at high concentration by regulating enhancer interactions. We propose this differential action of the Dl morphogen is likely supported by occupancy of this factor first to high and then low affinity binding sites over time as Dl levels rise to coordinate action of these two co-acting enhancers.

Synthetic gene networks recapitulate dynamic signal decoding and differential gene expression.

blue CRY2/CIB1 EL222 S. cerevisiae Transgene expression
bioRxiv, 7 Jan 2021 DOI: 10.1101/2021.01.07.425755 Link to full text
Abstract: Cells live in constantly changing environments and employ dynamic signaling pathways to transduce information about the signals they encounter. However, the mechanisms by which dynamic signals are decoded into appropriate gene expression patterns remain poorly understood. Here, we devise networked optogenetic pathways that achieve novel dynamic signal processing functions that recapitulate cellular information processing. Exploiting light-responsive transcriptional regulators with differing response kinetics, we build a falling-edge pulse-detector and show that this circuit can be employed to demultiplex dynamically encoded signals. We combine this demultiplexer with dCas9-based gene networks to construct pulsatile-signal filters and decoders. Applying information theory, we show that dynamic multiplexing significantly increases the information transmission capacity from signal to gene expression state. Finally, we use dynamic multiplexing for precise multidimensional regulation of a heterologous metabolic pathway. Our results elucidate design principles of dynamic information processing and provide original synthetic systems capable of decoding complex signals for biotechnological applications.

Physically asymmetric division of the C. elegans zygote ensures invariably successful embryogenesis.

blue TULIP C. elegans in vivo Developmental processes
bioRxiv, 7 Jan 2021 DOI: 10.1101/2021.01.04.425207 Link to full text
Abstract: Asymmetric divisions that yield daughter cells of different sizes are frequent during early embryogenesis, but the importance of such a physical difference for successful development remains poorly understood. Here, we investigated this question using the first division of C. elegans embryos, which yields a large AB cell and a small P1 cell. We equalized AB and P1 sizes using acute genetic inactivation or optogenetic manipulation of the spindle positioning protein LIN-5. We uncovered that only some embryos tolerated equalization, and that there was a size asymmetry threshold for viability. Cell lineage analysis of equalized embryos revealed an array of defects, including faster cell cycle progression in P1 descendants, as well as defects in cell positioning, division orientation and cell fate. Moreover, equalized embryos were more susceptible to external compression. Overall, we conclude that unequal first cleavage is essential for invariably successful embryonic development of C. elegans.

Endosomal cAMP production broadly impacts the cellular phosphoproteome.

blue bPAC (BlaC) HEK293 Signaling cascade control Immediate control of second messengers
bioRxiv, 6 Jan 2021 DOI: 10.1101/2021.01.06.425636 Link to full text
Abstract: Endosomal signaling from G protein-coupled receptors (GPCRs) has emerged as a novel paradigm with important pharmacological and physiological implications. Yet, our knowledge of the functional consequences of activating intracellular GPCRs is incomplete. To address this gap, we combined an optogenetic approach for site-specific generation of the prototypical second messenger cyclic AMP (cAMP) with unbiased mass spectrometry-based analysis of phosphoproteomic effects. We identified 218 unique, high-confidence sites whose phosphorylation is either increased or decreased in response to cAMP production. We next determined that cAMP produced from endosomes led to more robust changes in phosphorylation than cAMP produced from the plasma membrane. Remarkably, this was true for the entire repertoire of identified targets, and irrespective of their annotated sub-cellular localization. Furthermore, we identified a particularly strong endosome bias for a subset of proteins that are dephosphorylated in response to cAMP. Through bioinformatics analysis, we established these targets as putative substrates for protein phosphatase 2A (PP2A), and we propose compartmentalized activation of PP2A-B56δ as the likely underlying mechanism. Altogether, our study extends the concept that endosomal signaling is a significant functional contributor to cellular responsiveness by establishing a unique role for localized cAMP production in defining categorically distinct phosphoresponses.

A synthetic gene circuit for imaging-free detection of dynamic cell signaling.

blue iLID NIH/3T3
bioRxiv, 6 Jan 2021 DOI: 10.1101/2021.01.06.425615 Link to full text
Abstract: Cells employ intracellular signaling pathways to sense and respond to changes in their external environment. In recent years, live-cell biosensors have revealed complex pulsatile dynamics in many pathways, but studies of these signaling dynamics are limited by the necessity of live-cell imaging at high spatiotemporal resolution1. Here, we describe an approach to infer pulsatile signaling dynamics from just a single measurement in fixed cells using a pulse-detecting gene circuit. We computationally screened for circuit with pulse detecting capability, revealing an incoherent feedforward topology that robustly performs this computation. We then implemented the motif experimentally for the Erk signaling pathway using a single engineered transcription factor and fluorescent protein reporter. Our ‘recorder of Erk activity dynamics’ (READer) responds sensitively to both spontaneous and stimulus-driven Erk pulses. READer circuits thus open the door to permanently labeling transient, dynamic cell populations to elucidate the mechanistic underpinnings and biological consequences of signaling dynamics.

Mechanical frustration of phase separation in the cell nucleus by chromatin.

blue iLID U-2 OS Organelle manipulation
bioRxiv, 24 Dec 2020 DOI: 10.1101/2020.12.24.424222 Link to full text
Abstract: Liquid-liquid phase separation is a fundamental mechanism underlying subcellular organization. Motivated by the striking observation that optogenetically-generated droplets in the nucleus display suppressed coarsening dynamics, we study the impact of chromatin mechanics on droplet phase separation. We combine theory and simulation to show that crosslinked chromatin can mechanically suppress droplets’ coalescence and ripening, as well as quantitatively control their number, size, and placement. Our results highlight the role of the subcellular mechanical environment on condensate regulation.

Optogenetic inhibition and activation of Rac and Rap1 using a modified iLID system.

blue iLID HEK293T HeLa
bioRxiv, 11 Dec 2020 DOI: 10.1101/2020.12.11.421990 Link to full text
Abstract: The small GTPases Rac1 and Rap1 can fulfill multiple cellular functions because their activation kinetics and localization are precisely controlled. To probe the role of their spatio-temporal dynamics, we generated optogenetic tools that activate or inhibit endogenous Rac and Rap1 in living cells. An improved version of the light-induced dimerization (iLID) system [1] was used to control plasma membrane localization of protein domains that specifically activate or inactivate Rap1 and Rac (Tiam1 and Chimerin for Rac, RasGRP2 and Rap1GAP for Rap1 [2–5]). Irradiation yielded a 50-230% increase in the concentration of these domains at the membrane, leading to effects on cell morphodynamics consistent with the known roles of Rac1 and Rap1.

Improved Photocleavable Proteins with Faster and More Efficient Dissociation.

violet PhoCl HeLa Transgene expression Cell death
bioRxiv, 10 Dec 2020 DOI: 10.1101/2020.12.10.419556 Link to full text
Abstract: The photocleavable protein (PhoCl) is a green-to-red photoconvertible fluorescent protein that, when illuminated with violet light, undergoes main chain cleavage followed by spontaneous dissociation of the resulting fragments. The first generation PhoCl (PhoCl1) exhibited a relative slow rate of dissociation, potentially limiting its utilities for optogenetic control of cell physiology. In this work, we report the X-ray crystal structures of the PhoCl1 green state, red state, and cleaved empty barrel. Using structure-guided engineering and directed evolution, we have developed PhoCl2c with higher contrast ratio and PhoCl2f with faster dissociation. We characterized the performance of these new variants as purified proteins and expressed in cultured cells. Our results demonstrate that PhoCl2 variants exhibit faster and more efficient dissociation, which should enable improved optogenetic manipulations of protein localization and protein-protein interactions in living cells.

Design of smart antibody mimetics with photosensitive switches.

blue AsLOV2 HEK293T HeLa Transgene expression Nucleic acid editing
bioRxiv, 4 Dec 2020 DOI: 10.1101/2020.12.03.410936 Link to full text
Abstract: As two prominent examples of intracellular single-domain antibodies or antibody mimetics derived from synthetic protein scaffolds, monobodies and nanobodies are gaining wide applications in cell biology, structural biology, synthetic immunology, and theranostics. We introduce herein a generally-applicable method to engineer light-controllable monobodies and nanobodies, designated as moonbody and sunbody, respectively. These engineered antibody-like modular domains enable rapid and reversible antibody-antigen recognition by utilizing light. By paralleled insertion of two LOV2 modules into a single sunbody and the use of bivalent sunbodies, we substantially enhance the range of dynamic changes of photo-switchable sunbodies. Furthermore, we demonstrate the use of moonbodies or sunbodies to precisely control protein degradation, gene transcription, and base editing by harnessing the power of light.

Regulating enzymatic reactions in Escherichia coli utilizing light-responsive cellular compartments based on liquid-liquid phase separation.

blue CRY2/CIB1 E. coli
bioRxiv, 29 Nov 2020 DOI: 10.1101/2020.11.26.395616 Link to full text
Abstract: Enzymatic reactions in cells are well organized into different compartments, among which protein-based membraneless compartments formed through liquid-liquid phase separation (LLPS) are believed to play important roles1,2. Hijacking them for our own purpose has promising applications in metabolic engineering3. Yet, it is still hard to precisely and dynamically control target enzymatic reactions in those compartments4. To address those problems, we developed Photo-Activated Switch in E. coli (PhASE), based on phase separating scaffold proteins and optogenetic tools. In this system, a protein of interest (POI) can be enriched up to 15-fold by LLPS-based compartments from cytosol within only a few seconds once activated by light, and become fully dispersed again within 15 minutes. Furthermore, we explored the potentiality of the LLPS-based compartment in enriching small organic molecules directly via chemical-scaffold interaction. With enzymes and substrates co-localized under light induction, the overall reaction efficiency could be enhanced. Using luciferin and catechol oxidation as model enzymatic reactions, we found that they could accelerate 2.3-fold and 1.6-fold, respectively, when regulated by PhASE. We anticipate our system to be an extension of the synthetic biology toolkit, facilitating rapid recruitment and release of POIs, and reversible regulation of enzymatic reactions.

Spatio-temporal Control of ERK Pulse Frequency Coordinates Fate Decisions during Mammary Acinar Morphogenesis.

blue CRY2/CIB1 CRY2/CRY2 MCF10A Signaling cascade control Cell differentiation Cell death
bioRxiv, 21 Nov 2020 DOI: 10.1101/2020.11.20.387167 Link to full text
Abstract: The signaling events controlling proliferation, survival, and apoptosis during mammary epithelial acinar morphogenesis remain poorly characterized. By imaging single-cell ERK activity dynamics in MCF10A acini, we find that these fates depend on the frequency of ERK pulses. High pulse frequency is observed during initial acinus growth, correlating with rapid cell motility. Subsequent decrease in motility correlates with lower ERK pulse frequency and quiescence. Later, during lumen formation, coordinated ERK waves emerge across multiple cells of an acinus, correlating with high and low ERK pulse frequency in outer surviving and inner dying cells respectively. A PIK3CA H1047R mutation, commonly observed in breast cancer, increases ERK pulse frequency and inner cell survival, causing loss of lumen formation. Optogenetic entrainment of ERK pulses causally connects high ERK pulse frequency with inner cell survival. Thus, fate decisions during acinar morphogenesis are fine-tuned by different spatio-temporal coordination modalities of ERK pulse frequency.

Spatiotemporal sensitivity of embryonic heart specification to FGFR signaling in Drosophila.

blue CRY2/CRY2 D. melanogaster in vivo Signaling cascade control Developmental processes
bioRxiv, 19 Nov 2020 DOI: 10.1101/2020.11.16.384123 Link to full text
Abstract: Development of the Drosophila embryonic mesoderm is controlled through both internal and external inputs to the mesoderm. One such factor is Heartless (Htl), a Fibroblast Growth Factor Receptor (FGFR) expressed in the mesoderm. Htl is involved in shaping the mesoderm at both early and later stages during embryogenesis. How Htl expression levels and timing of signaling affect mesoderm morphogenesis after spreading remains elusive. We have developed an optogenetic tool (Opto-htl) to control the activation of Htl signaling with precise spatiotemporal resolution in vivo. We find that the embryo is most sensitive to Htl over-activation within a developmental window of ~4 hours ranging from late stage 10 until early stage 13, which corresponds to early stages of heart morphogenesis. Opto-htl restores heart cells in htl mutants upon light activation, independent of its role in early mesoderm shaping events. We also successfully generated spatially distinct regions of Htl activity in the mesoderm using light-sheet microscopy. The developing tissue was unable to correct for the ensuing asymmetries in cell fate. Overall, Opto-htl is a powerful tool for studying spatiotemporal regulation of Htl signaling during embryogenesis.

Synthetic protein condensates that recruit and release protein activity in living cells.

bioRxiv, 10 Nov 2020 DOI: 10.1101/2020.11.09.375766 Link to full text
Abstract: Compartmentation of proteins into biomolecular condensates or membraneless organelles formed by phase separation is an emerging principle for the regulation of cellular processes. Creating synthetic condensates that accommodate specific intracellular proteins on demand would have various applications in chemical biology, cell engineering and synthetic biology. Here, we report the construction of synthetic protein condensates capable of recruiting and/or releasing proteins of interest in living mammalian cells in response to a small molecule or light. We first present chemogenetic protein-recruiting and -releasing condensates, which rapidly inhibited and activated signaling proteins, respectively. An optogenetic condensate system was successfully constructed that enables reversible release and sequestration of protein activity using light. This proof-of-principle work provides a new platform for chemogenetic and optogenetic control of protein activity in mammalian cells and represents a step towards tailor-made engineering of synthetic protein condensates with various functionalities.

Biphasic Response of Protein Kinase A to Cyclic Adenosine Monophosphate Triggers Distinct Epithelial Phenotypes.

blue bPAC (BlaC) MDCK Signaling cascade control Immediate control of second messengers
bioRxiv, 3 Nov 2020 DOI: 10.1101/747030 Link to full text
Abstract: Despite the large diversity of the proteins involved in cellular signaling, many intracellular signaling pathways converge onto one of only dozens of small molecule second messengers. Cyclic adenosine monophosphate (cAMP), one of these second messengers, is known to regulate activity of both Protein Kinase A (PKA) and the Extracellular Regulated Kinase (ERK), among other signaling pathways. In its role as an important cellular signaling hub, intracellular cAMP concentration has long been assumed to monotonically regulate its known effectors. Using an optogenetic tool that can introduce precise amounts of cAMP in MDCKI cells, we identify genes whose expression changes biphasically with monotonically increasing cAMP levels. By examining the behavior of PKA and ERK1/2 in the same dose regime, we find that these kinases also respond biphasically to increasing cAMP levels, with opposite phases. We reveal that this behavior results from an elaborate integration by PKA of many cellular signals triggered by cAMP. In addition to the direct activation of PKA, cAMP also modulates the activity of p38 and ERK, which then converge to inhibit PKA. These interactions and their ensuing biphasic PKA profile have important physiological repercussions, influencing the ability of MDCKI cells to proliferate and form acini. Our data, supported by computational modeling, synthesize a set of network interconnections involving PKA and other important signaling pathways into a model that demonstrates how cells can capitalize on signal integration to create a diverse set of responses to cAMP concentration and produce complex input-output relationships.

Light-inducible Deformation of Mitochondria in Live Cells.

blue CRY2/CIB1 iLID 3T3-L1 Cos-7 HeLa U-2 OS Organelle manipulation
bioRxiv, 2 Nov 2020 DOI: 10.1101/2020.11.01.363663 Link to full text
Abstract: Mitochondria, the powerhouse of the cell, are dynamic organelles that undergo constant morphological changes. Increasing evidence indicates that mitochondria morphologies and functions can be modulated by mechanical cues. However, the mechano-sensing and -responding properties of mitochondria and the correlation between mitochondrial morphologies and functions are unclear due to the lack of methods to precisely exert mechano-stimulation on and deform mitochondria inside live cells. Here we present an optogenetic approach that uses light to induce deformation of mitochondria by recruiting molecular motors to the outer mitochondrial membrane via light-activated protein-protein hetero-dimerization. Mechanical forces generated by motor proteins distort the outer membrane, during which the inner mitochondrial membrane can also be deformed. Moreover, this optical method can achieve subcellular spatial precision and be combined with other optical dimerizers and molecular motors. This method presents a novel mitochondria-specific mechano-stimulator for studying mitochondria mechanobiology and the interplay between mitochondria shapes and functions.

Quantifying signal persistence in the T cell signaling network using an optically controllable antigen receptor.

blue LOVTRAP HEK293T Jurkat Signaling cascade control
bioRxiv, 30 Oct 2020 DOI: 10.1101/2020.10.30.362194 Link to full text
Abstract: T cells discriminate between healthy and infected cells with remarkable sensitivity when mounting an immune response. It has been hypothesized that this efficient detection requires combining signals from discrete antigen-presenting cell interactions into a more potent response, requiring T cells to maintain a ‘memory’ of previous encounters. To quantify the magnitude of this phenomenon, we have developed an antigen receptor that is both optically and chemically tunable, providing control over the initiation, duration, and intensity of intracellular T-cell signaling within physiological cell conjugates. We observe very limited persistence within the T cell intracellular network on disruption of receptor input, with signals dissipating entirely in ~15 minutes, and directly confirm that sustained proximal receptor signaling is required to maintain active gene transcription. Our data suggests that T cells are largely incapable of integrating discrete antigen receptor signals but instead simply accumulate the output of gene expression. By engineering optical control in a clinically relevant chimeric antigen receptor, we show that this limited signal persistence can be exploited to increase the activation of primary T cells by ~3-fold by using pulsatile stimulation. Our results are likely to apply more generally to the signaling dynamics of other cellular networks.

Optogenetical control of infection signaling cascade of bacteria by an engineered light-responsive protein.

blue YtvA P. aeruginosa P. aeruginosa
bioRxiv, 29 Oct 2020 DOI: 10.1101/2020.10.28.358515 Link to full text
Abstract: Bacterial pathogens operate by tightly controlling the virulence to facilitate invasion and survival in host. Although pathways regulating virulence have been defined in detail and signals modulating these processes are gradually understood, a lack of controlling infection signaling cascades of pathogens when and whereabouts specificity limits deeper investigating of host-pathogen interactions. Here, we employed optogenetics to reengineer the GacS of Pseudomonas aeruginosa, sensor kinase of GacS/GacA TCS regulates the expression of virulence factors by directly mediating several sRNAs. The resultant protein YGS24 displayed significant light-dependent activity of GacS kinases in Pseudomonas aeruginosa. When introduced in Caenorhabditis elegans host systems, YGS24 stimulated the pathogenicity of PAO1 in BHI and of PA14 in SK medium progressively upon blue-light exposure. This optogenetic system provides an accessible way to spatiotemporally control bacterial pathogenicity in defined host even specific tissues to develop new pathogenesis systems, which may in turn expedite development of innovative therapeutics.
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