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 28 results
1.

Optogenetic control of Neisseria meningitidis Cas9 genome editing using an engineered, light-switchable anti-CRISPR protein.

blue AsLOV2 HEK293T Nucleic acid editing
bioRxiv, 29 Nov 2019 DOI: 10.1101/858589 Link to full text
Abstract: Optogenetic control of CRISPR-Cas9 systems has significantly improved our ability to perform genome perturbations in living cells with high precision in time and space. As new Cas orthologues with advantageous properties are rapidly being discovered and engineered, the need for straightforward strategies to control their activity via exogenous stimuli persists. The Cas9 from Neisseria meningitidis (Nme) is a particularly small and target-specific Cas9 orthologue, and thus of high interest for in vivo genome editing applications. Here, we report the first optogenetic tool to control NmeCas9 activity in mammalian cells via an engineered, light-dependent anti-CRISPR (Acr) protein. Building on our previous Acr engineering work, we created hybrids between the NmeCas9 inhibitor AcrIIC3 and the LOV2 blue light sensory domain from Avena sativa. Two AcrIIC3-LOV2 hybrids from our collection potently blocked NmeCas9 activity in the dark, while permitting robust genome editing at various endogenous loci upon blue light irradiation. Structural analysis revealed that, within these hybrids, the LOV2 domain is located in striking proximity to the Cas9 binding surface. Together, our work demonstrates optogenetic regulation of a type II-C CRISPR effector and might suggest a new route for the design of optogenetic Acrs.
2.

Construction of light-activated neurotrophin receptors using the improved Light-Induced Dimerizer (iLID) .

blue iLID PC-12 Signaling cascade control
bioRxiv, 21 Nov 2019 DOI: 10.1101/850412 Link to full text
Abstract: Receptor tyrosine kinases (RTKs) play crucial roles in human health, and their misregulation is implicated in disorders ranging from neurodegenerative disorders to cancers. The highly conserved mechanism of activation of RTKs makes them especially appealing candidates for control via optogenetic dimerization methods. This work offers a strategy for using the improved Light-Induced Dimer (iLID) system with a constructed tandem-dimer of its binding partner nano (tdnano) to build light-activatable versions of RTKs. In the absence of light, the iLID-RTK is cytosolic, monomeric and inactive. Under blue light, the iLID + tdnano system recruits two copies of iLID-RTK to tdnano, dimerizing and activating the RTK. We demonstrate that iLID opto-iTrkA and opto-iTrkB are capable of reproducing downstream ERK and Akt signaling only in the presence of tdnano. We further show with our opto-iTrkA that the system is compatible with multi-day and population-level activation of TrkA in PC12 cells. By leveraging genetic targeting of tdnano, we achieve RTK activation at a specific subcellular location even with whole-cell illumination, allowing us to confidently probe the impact of context on signaling outcome.
3.

Optogenetic lac operon to control chemical and protein production in Escherichia coli with light.

blue YtvA E. coli Transgene expression
bioRxiv, 16 Nov 2019 DOI: 10.1101/845453 Link to full text
Abstract: Control of the lac operon with IPTG has been used for decades to regulate gene expression in E. coli for countless applications, including metabolic engineering and recombinant protein production. However, optogenetics offers unique capabilities such as easy tunability, reversibility, dynamic induction strength, and spatial control that are difficult to obtain with chemical inducers. We developed an optogenetic lac operon in a series of circuits we call OptoLAC. With these circuits, we control gene expression from various IPTG-inducible promoters using only blue light. Applying them to metabolic engineering improves mevalonate and isobutanol production by 24% and 27% respectively, compared to IPTG induction, in light-controlled fermentations scalable to at least 2L bioreactors. Furthermore, OptoLAC circuits enable light control of recombinant protein production, reaching yields comparable to IPTG induction, but with enhanced tunability of expression and spatial control. OptoLAC circuits are potentially useful to confer light controls over other cell functions originally engineered to be IPTG-inducible.
4.

CreLite: An Optogenetically Controlled Cre/loxP System Using Red Light.

red PhyB/PIF6 zebrafish in vivo
bioRxiv, 30 Oct 2019 DOI: 10.1101/823971 Link to full text
Abstract: Precise manipulation of gene expression with temporal and spatial control is essential for functional studies and the determination of cell lineage relationships in complex biological systems.The Cre-loxP system is commonly used for gene manipulation at desired times and places. However, specificity is dependent on the availability of tissue- or cell-specific regulatory elements used in combination with Cre or CreER (tamoxifen-inducible). Here we present CreLite, an optogenetically-controlled Cre system using red light in developing zebrafish embryos. Cre activity is disabled by splitting Cre and fusing the inactive halves with the Arabidopsis thaliana red light-inducible binding partners, PhyB and PIF6. In addition, PhyB-PIF6 binding requires phycocyanobilin (PCB), providing an additional layer of control. Upon exposure to red light (660 nm) illumination, the PhyB-CreC and PIF6-CreN fusion proteins come together in the presence of PCB to restore Cre activity. Red-light exposure of transgenic zebrafish embryos harboring a Cre-dependent multi-color fluorescent protein reporter (ubi:zebrabow) injected with CreLite mRNAs and PCB, resulted in Cre activity as measured by the generation of multi-spectral cell labeling in various tissues, including heart, skeletal muscle and epithelium. We show that CreLite can be used for gene manipulations in whole embryos or small groups of cells at different stages of development. CreLite provides a novel optogenetic tool for precise temporal and spatial control of gene expression in zebrafish embryos that may also be useful in cell culture, ex vivo organ culture, and other animal models for developmental biology studies.
5.

Composition dependent phase separation underlies directional flux through the nucleolus.

blue CRY2olig HeLa Organelle manipulation
bioRxiv, 22 Oct 2019 DOI: 10.1101/809210 Link to full text
Abstract: Intracellular bodies such as nucleoli, Cajal bodies, and various signaling assemblies, represent membraneless organelles, or condensates, that form via liquid-liquid phase separation (LLPS)1,2. Biomolecular interactions, particularly homotypic interactions mediated by self-associating intrinsically disordered protein regions (IDRs), are thought to underlie the thermodynamic driving forces for LLPS, forming condensates that can facilitate the assembly and processing of biochemically active complexes, such as ribosomal subunits within the nucleolus. Simplified model systems3–6 have led to the concept that a single fixed saturation concentration (Csat) is a defining feature of endogenous LLPS7–9, and has been suggested as a mechanism for intracellular concentration buffering2,7,8,10. However, the assumption of a fixed Csat remains largely untested within living cells, where the richly multicomponent nature of condensates could complicate this simple picture. Here we show that heterotypic multicomponent interactions dominate endogenous LLPS, and give rise to nucleoli and other condensates that do not exhibit a fixed Csat. As the concentration of individual components is varied, their partition coefficients change, in a manner that can be used to extract thermodynamic interaction energies, that we interpret within a framework we term polyphasic interaction thermodynamic analysis (PITA). We find that heterotypic interactions between protein and RNA components stabilize a variety of archetypal intracellular condensates, including the nucleolus, Cajal bodies, stress granules, and P bodies. These findings imply that the composition of condensates is finely tuned by the thermodynamics of the underlying biomolecular interaction network. In the context of RNA processing condensates such as the nucleolus, this stoichiometric self-tuning manifests in selective exclusion of fully-assembled RNP complexes, providing a thermodynamic basis for vectorial ribosomal RNA (rRNA) flux out of the nucleolus. The PITA methodology is conceptually straightforward and readily implemented, and it can be broadly utilized to extract thermodynamic parameters from microscopy images. These approaches pave the way for a deep understanding of the thermodynamics of multi-component intracellular phase behavior and its interplay with nonequilibrium activity characteristic of endogenous condensates.
6.

Optogenetic modulation of TDP-43 oligomerization fast-forwards ALS-related pathologies in the spinal motor neurons.

blue CRY2olig zebrafish in vivo
bioRxiv, 1 Oct 2019 DOI: 10.1101/789057 Link to full text
Abstract: Cytoplasmic aggregation of TDP-43 characterizes degenerating neurons in most cases of amyotrophic lateral sclerosis (ALS), yet the mechanisms and cellular outcomes of TDP-43 pathology remain largely elusive. Here, we develop an optogenetic TDP-43 variant (opTDP-43), whose multimerization status can be modulated in vivo through external light illumination. Using the translucent zebrafish neuromuscular system, we demonstrate that short-term light stimulation reversibly induces cytoplasmic opTDP-43 mislocalization, but not aggregation, in the spinal motor neuron, leading to an axon outgrowth defect associated with myofiber denervation. In contrast, opTDP-43 forms pathological aggregates in the cytoplasm after longer-term illumination and seeds non-optogenetic TDP-43 aggregation. Furthermore, we find that an ALS-linked mutation in the intrinsically disordered region (IDR) exacerbates the light-dependent opTDP-43 toxicity on locomotor behavior. Together, our results propose that IDR-mediated TDP-43 oligomerization triggers both acute and long-term pathologies of motor neurons, which may be relevant to the pathogenesis and progression of ALS.
7.

Optogenetic rescue of a developmental patterning mutant.

blue iLID D. melanogaster in vivo Signaling cascade control Developmental processes
bioRxiv, 1 Oct 2019 DOI: 10.1101/776120 Link to full text
Abstract: Animal embryos are partitioned into spatial domains by complex patterns of signaling and gene expression, yet it is still largely unknown what features of a developmental signal are essential. Part of the challenge arises because it has been impossible to “paint” arbitrary signaling patterns on an embryo to test their sufficiency. Here we demonstrate exactly this capability by combining optogenetic control of Ras/Erk signaling with the genetic loss of terminal signaling in early Drosophila embryos. Simple all-or-none light inputs at the embryonic termini were able to completely rescue normal development, generating viable larvae and fertile adults from this otherwise-lethal genetic mutant. Systematically varying illumination parameters further revealed that at least three distinct developmental programs are triggered by different cumulative doses of Erk. These results open the door to the targeted design of complex morphogenetic outcomes as well as the ability to correct patterning errors that underlie developmental defects.
8.

Green, orange, red, and far-red optogenetic tools derived from cyanobacteriochromes.

green red Am1 c0023g2/BAm green Am1 c0023g2/BAm red in vitro S. cerevisiae
bioRxiv, 14 Sep 2019 DOI: 10.1101/769422 Link to full text
Abstract: Existing optogenetic tools for controlling protein-protein interactions are available in a limited number of wavelengths thereby limiting opportunities for multiplexing. The cyanobacteriochrome (CBCR) family of photoreceptors responds to an extraordinary range of colors, but light-dependent binding partners for CBCR domains are not currently known. We used a phage-display based approach to develop small (~50-residue) monomeric binders selective for the green absorbing state (Pg), or for the red absorbing state (Pr) of the CBCR Am1_c0023g2 with a phycocyanobilin chromophore and also for the far-red absorbing state (Pfr) of Am1_c0023g2 with a biliverdin chromophore. These bind in a 1:1 mole ratio with KDs for the target state from 0.2 to 2 μM and selectivities from 10 to 500-fold. We demonstrate green, orange, red, and far-red light-dependent control of protein-protein interactions in vitro and also in vivo where these multicolor optogenetic tools are used to control transcription in yeast.
9.

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, 28 Aug 2019 DOI: 10.1101/747030 Link to full text
Abstract: Protein Kinase A (PKA) is an important cellular signaling hub whose activity has long been assumed to monotonically depend on the level of cyclic adenosine monophosphate (cAMP). Using an optogenetic tool that can introduce precise amounts of cAMP in MDCKI cells, we demonstrate that PKA activity is instead characterized by a biphasic response, in which PKA activity increases and then decreases as a function of cAMP. 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 on PKA to inhibit it. These interactions and their ensuing biphasic PKA profile have important physiological repercussions, triggering two distinct transcriptional programs elicited by low and high cAMP doses. These transcriptional responses in turn influence the ability of MDCKI cells to proliferate and form acini. Our data, supported by computational analyses, 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.
10.

Nucleated transcriptional condensates amplify gene expression.

blue CRY2olig NIH/3T3 Endogenous gene expression Organelle manipulation
bioRxiv, 21 Aug 2019 DOI: 10.1101/737387 Link to full text
Abstract: Liquid-liquid phase separation is thought to underly gene transcription, through the condensation of the large-scale nucleolus, or in smaller assemblies known as transcriptional hubs or condensates. However, phase separation has not yet been directly linked with transcriptional output, and our biophysical understanding of transcription dynamics is poor. Here, we utilize an optogenetic approach to control condensation of key FET-family transcriptional regulators, particularly TAF15. We show that amino acid sequence-dependent phase separation of TAF15 is enhanced significantly due to strong nuclear interactions with the C-terminal domain (CTD) of RNA Pol II. Nascent CTD clusters at primed genomic loci lower the energetic barrier for nucleation of TAF15 condensates, which in turn further recruit RNA Pol II to drive transcriptional output. These results suggest a model in which positive feedback between key transcriptional components drives intermittent dynamics of localized phase separation, to amplify gene expression.
11.

Optogenetic control of protein binding using light-switchable nanobodies.

blue AsLOV2 HEK293T in vitro NIH/3T3 Signaling cascade control
bioRxiv, 18 Aug 2019 DOI: 10.1101/739201 Link to full text
Abstract: A growing number of optogenetic tools have been developed to control binding between two engineered protein domains. In contrast, relatively few tools confer light-switchable binding to a generic target protein of interest. Such a capability would offer substantial advantages, enabling photoswitchable binding to endogenous target proteins in vivo or light-based protein purification in vitro. Here, we report the development of opto-nanobodies (OptoNBs), a versatile class of chimeric photoswitchable proteins whose binding to proteins of interest can be enhanced or inhibited upon blue light illumination. We find that OptoNBs are suitable for a range of applications: modulating intracellular protein localization and signaling pathway activity and controlling target protein binding to surfaces and in protein separation columns. This work represents a first step towards programmable photoswitchable regulation of untagged, endogenous target proteins.
12.

CLIC4, a new component of the cytokinetic ring, regulates actin cytoskeleton dynamics during the anaphase-to-telophase transition.

blue CRY2/CIB1 CRY2/CRY2 HeLa Control of cytoskeleton / cell motility / cell shape
bioRxiv, 14 Aug 2019 DOI: 10.1101/723940 Link to full text
Abstract: During mitotic cell division, the actin cytoskeleton undergoes several dynamic changes that lead to cell rounding during metaphase, the formation and ingression of the cytokinetic contractile ring during anaphase, and finally the formation of the intercellular bridge during telophase. These dramatic changes in the organization of the actomyosin cytoskeleton play a key role in progression through mitosis and are regulated by a number of proteins. While the regulators of cytokinetic ring formation and contraction are well-established, very little is known about the proteins that are responsible for the changing actin dynamics during the transition from an actively ingressing cytokinetic furrow to the stable intercellular bridge connecting two daughter cells during telophase. Here, we describe a role for CLIC4 in regulating this anaphase-to-telophase transition. We first describe CLIC4 as a new component of the cytokinetic ring that is required for successful completion of mitotic cell division. We also show that RhoA recruits CLIC4 to the cytokinetic ring and that CLIC4regulates the formation of a stable intercellular bridge by preventing regression of the cytokinetic furrow. Finally, we demonstrate that CLIC4 regulates the remodeling of sub-plasma membrane actomyosin network within the furrow by recruiting MST4 kinase and regulating ezrin phosphorylation. This work identifies and characterizes new molecular players involved in the transition from the contracting cytokinetic ring to the intercellular bridge during cytokinesis.
13.

Targeted cell ablation in zebrafish using optogenetic transcriptional control.

blue VVD zebrafish in vivo Transgene expression Cell death
bioRxiv, 10 Aug 2019 DOI: 10.1101/730507 Link to full text
Abstract: Cell ablation is a powerful method forelucidatingthe contributions of individual cell populations toembryonicdevelopment and tissue regeneration. Targeted cell lossin whole organisms has been typically achieved through expression of a cytotoxic or prodrug-activating gene productin the cell type of interest. This approach depends on the availability of tissue-specific promoters, and it does not allow further spatial selectivity within the promoter-defined region(s). To address this limitation, we have developedablative methodsthat combine genetically encoded toxins, thetissue specificity afforded by cis-regulatory elements,and the conditionalityof optogenetics.Using this integrative approach, we have ablated cellsin zebrafish embryoswith spatial and temporal precision.
14.

Optogenetics reveals Cdc42 local activation by scaffold-mediated positive feedback and Ras GTPase.

blue CRY2/CIB1 S. cerevisiae S. pombe Control of cytoskeleton / cell motility / cell shape
bioRxiv, 22 Jul 2019 DOI: 10.1101/710855 Link to full text
Abstract: The small GTPase Cdc42 is critical for cell polarization. Scaffold-mediated positive feedback regulation was proposed to mediate symmetry-breaking to a single active zone in budding yeast cells. In rod-shaped fission yeast S. pombe cells, active Cdc42-GTP localizes to both cell poles, where it promotes bipolar growth. Here, we implement the CRY2-CIBN optogenetic system for acute light-dependent protein recruitment to the plasma membrane, which allowed to directly demonstrate positive feedback. Indeed, optogenetic recruitment of constitutively active Cdc42 leads to co-recruitment of the GEF Scd1, in a manner dependent on the scaffold protein Scd2. We show that Scd2 function is completely bypassed and positive feedback restored by an engineered interaction between the GEF and a Cdc42 effector, the Pak1 kinase. Remarkably, such re-wired cells are viable and grow in a bipolar manner even when lacking otherwise essential Cdc42 activators. Interestingly, these cells reveal that Ras1 GTPase plays a dual role in localizing and activating the GEF, thus potentiating the feedback. We conclude that scaffold-mediated positive feedback, gated by Ras activity, is minimally required for rod-shape formation.
15.

m6A-binding YTHDF proteins promote stress granule formation by modulating phase separation of stress granule proteins.

blue CRY2olig U-2 OS
bioRxiv, 7 Jul 2019 DOI: 10.1101/694455 Link to full text
Abstract: Diverse RNAs and RNA-binding proteins form phase-separated, membraneless granules in cells under stress conditions. However, the role of the prevalent mRNA methylation, m6A, and its binding proteins in stress granule (SG) assembly remain unclear. Here, we show that m6A-modified mRNAs are enriched in SGs, and that m6A-binding YTHDF proteins are critical for SG formation. Depletion of YTHDF1/3 inhibits SG formation and recruitment of m6A-modified mRNAs to SGs. Both the N-terminal intrinsically disordered region and the C-terminal m6A-binding YTH domain of YTHDF proteins are crucial for SG formation. Super-resolution imaging further reveals that YTHDF proteins are in a super-saturated state, forming clusters that reside in the periphery of and at the junctions between SG core clusters, and promote SG phase separation by reducing the activation energy barrier and critical size for condensate formation. Our results reveal a new function and mechanistic insights of the m6A-binding YTHDF proteins in regulating phase separation.
16.

Ran GTPase regulates non-centrosomal microtubule nucleation and is transported by actin waves towards the neurite tip.

blue LOVTRAP HeLa primary mouse cortical neurons primary mouse hippocampal neurons Control of cytoskeleton / cell motility / cell shape
bioRxiv, 28 Jun 2019 DOI: 10.1101/684720 Link to full text
Abstract: Microtubule (MT) is the most abundant cytoskeleton in neurons and controls multiple facets of their development. While the organizing center of MTs in mitotic cells is typically located at the centrosome, MT nucleation in post-mitotic neurons switches to non-centrosomal sites. A handful of proteins and organelle have been shown to promote non-centrosomal MT formation in neurons, yet the regulation mechanism remains unknown. Here we demonstrate that the small GTPase Ran is a key regulator of non-centrosomal MT nucleation in neurons. The GTP-bound Ran (RanGTP) localizes to the neurite tips and around the soma. Using the RanGTP- and RanGDP-mimic mutants, we show that RanGTP promotes MT nucleation at the tip of the neurite. To demonstrate that RanGTP can promote MT nucleation in regions other than the neurite tip, an optogenetic tool called RanTRAP was constructed to enable light-induced local production of RanGTP in the neuronal cytoplasm. An increase of non-centrosomal MT nucleation can be observed by elevating the RanGTP level along the neurite using RanTRAP, establishing a new role for Ran in regulating neuronal MTs. Additionally, the mechanism of RanGTP enrichment at the neurite tip was examined. We discovered that actin waves drive the anterograde transport of RanGTP towards the neurite tip. Pharmacological disruption of actin waves abolishes the enrichment of RanGTP and reduces the non-centrosomal MT nucleation at the neurite tip. These observations provide a novel regulation mechanism of MTs and an indirect connection between the actin and MT cytoskeletons in neurons.
17.

Cell-in-the-loop pattern formation with optogenetically emulated cell-to-cell signaling.

blue EL222 S. cerevisiae
bioRxiv, 22 Jun 2019 DOI: 10.1101/679597 Link to full text
Abstract: Designing and implementing synthetic biological pattern formation remains a challenge due to underlying theoretical complexity as well as the difficulty of engineering multicellular networks bio-chemically. Here, we introduce a “cell-in-the-loop” approach where living cells interact through in silico signaling, establishing a new testbed to interrogate theoretical principles when internal cell dynamics are incorporated rather than modeled. We present a theory that offers an easy-to-use test to predict the emergence of contrasting patterns in gene expression among laterally inhibiting cells. Guided by the theory, we experimentally demonstrated spontaneous checkerboard patterning in an optogenetic setup where cell-to-cell signaling was emulated with light inputs calculated in silico from real-time gene expression measurements. The scheme successfully produced spontaneous, persistent checkerboard patterns for systems of sixteen patches, in quantitative agreement with theoretical predictions. Our research highlights how tools from dynamical systems theory may inform our understanding of patterning, and illustrates the potential of cell-in-the-loop for engineering synthetic multicellular systems.
18.

A live-cell screen for altered Erk dynamics reveals principles of proliferative control.

blue iLID mouse epidermal keratinocytes Signaling cascade control Cell cycle control
bioRxiv, 19 Jun 2019 DOI: 10.1101/675736 Link to full text
Abstract: Complex, time-varying responses have been observed widely in cell signaling, but how specific dynamics are generated or regulated is largely unknown. One major obstacle has been that high-throughput screens for identifying pathway components are typically incompatible with the live-cell assays used to monitor dynamics. Here, we address this challenge by performing a drug screen for altered Erk signaling dynamics in primary mouse keratinocytes. We screened a library of 429 kinase inhibitors, monitoring Erk activity over 5 h in more than 80,000 single live cells. The screen revealed both known and uncharacterized modulators of Erk dynamics, including inhibitors of non-EGFR receptor tyrosine kinases (RTKs) that increased Erk pulse frequency and overall activity. Using drug treatment and direct optogenetic control, we demonstrate that drug-induced changes to Erk dynamics alter the conditions under which cells proliferate. Our work opens the door to high-throughput screens using live-cell biosensors and reveals that cell proliferation integrates information from Erk dynamics as well as additional permissive cues.
19.

Engineered illumination devices for optogenetic control of cellular signaling dynamics.

blue CRY2/CRY2 hESCs Signaling cascade control Developmental processes
bioRxiv, 19 Jun 2019 DOI: 10.1101/675892 Link to full text
Abstract: Spatially and temporally varying patterns of morphogen signals during development drive cell fate specification at the proper location and time. However, current in vitro methods typically do not allow for precise, dynamic, spatiotemporal control of morphogen signaling and are thus insufficient to readily study how morphogen dynamics impact cell behavior. Here we show that optogenetic Wnt/β-catenin pathway activation can be controlled at user-defined intensities, temporal sequences, and spatial patterns using novel engineered illumination devices for optogenetic photostimulation and light activation at variable amplitudes (LAVA). The optical design of LAVA devices was optimized for uniform illumination of multi-well cell culture plates to enable high-throughput, spatiotemporal optogenetic activation of signaling pathways and protein-protein interactions. Using the LAVA devices, variation in light intensity induced a dose-dependent response in optoWnt activation and downstream Brachyury expression in human embryonic stem cells (hESCs). Furthermore, time-varying and spatially localized patterns of light revealed tissue patterning that models embryonic presentation of Wnt signals in vitro. The engineered LAVA devices thus provide a low-cost, user-friendly method for high-throughput and spatiotemporal optogenetic control of cell signaling for applications in developmental and cell biology.
20.

A yeast optogenetic toolkit (yOTK) for gene expression control in Saccharomyces cerevisiae.

blue CRY2/CIB1 S. cerevisiae
bioRxiv, 16 Jun 2019 DOI: 10.1101/663393 Link to full text
Abstract: Optogenetic tools for controlling gene expression are ideal for tuning synthetic biological networks due to the exquisite spatiotemporal control available with light. Here we develop an optogenetic system for gene expression control and integrate it with an existing yeast toolkit allowing for rapid, modular assembly of light-controlled circuits in the important chassis organism Saccharomyces cerevisiae. We reconstitute activity of a split synthetic zinc-finger transcription factor (TF) using light-induced dimerization. We optimize function of this split TF and demonstrate the utility of the toolkit workflow by assembling cassettes expressing the TF activation domain and DNA-binding domain at different levels. Utilizing this TF and a synthetic promoter we demonstrate that light-intensity and duty-cycle can be used to modulate gene expression over the range currently available from natural yeast promoters. This work allows for rapid generation and prototyping of optogenetic circuits to control gene expression in Saccharomyces cerevisiae.
21.

Optogenetic control of Wnt signaling for modeling early embryogenic patterning with human pluripotent stem cells.

blue CRY2/CRY2 hESCs human IPSCs Signaling cascade control Control of cytoskeleton / cell motility / cell shape Cell differentiation
bioRxiv, 10 Jun 2019 DOI: 10.1101/665695 Link to full text
Abstract: The processes of cell proliferation, differentiation, migration, and self-organization during early embryonic development are governed by dynamic, spatially and temporally varying morphogen signals. Analogous tissue patterns emerge spontaneously in embryonic stem cell (ESC) models for gastrulation, but mechanistic insight into this self-organization is limited by a lack of molecular methods to precisely control morphogen signal dynamics. Here we combine optogenetic stimulation and single-cell imaging approaches to study self-organization of human pluripotent stem cells. Precise control of morphogen signal dynamics, achieved through activation of canonical Wnt/β-catenin signaling over a broad high dynamic range (>500-fold) using an optoWnt optogenetic system, drove broad transcriptional changes and mesendoderm differentiation of human ESCs at high efficiency (>95% cells). Furthermore, activating Wnt signaling in subpopulations of ESCs in 2D and 3D cultures induced cell self-organization and morphogenesis reminiscent of human gastrulation, including changes in cell migration and epithelial to mesenchymal transition. Our findings thus reveal an instructive role for Wnt in directing cell patterning in this ESC model for gastrulation.
22.

Mps1 releases Mad1 from nuclear pores to ensure a robust mitotic checkpoint and accurate chromosome segregation.

blue CRY2/CIB1 Schneider 2 Cell cycle control
bioRxiv, 4 Jun 2019 DOI: 10.1101/659938 Link to full text
Abstract: The strength of the Spindle Assembly Checkpoint (SAC) depends on the amount of the Mad1-C-Mad2 heterotetramer at kinetochores but also on its binding to Megator/Tpr at nuclear pore complexes (NPCs) during interphase. However, the molecular underpinnings controlling the spatiotemporal redistribution of Mad1-C-Mad2 as cells progress into mitosis remain elusive. Here, we show that Mps1-mediated phosphorylation of Megator/Tpr abolishes its interaction with Mad1 in vitro and in Drosophila cells. Timely activation of Mps1 during prophase triggers Mad1 release from NPCs, which we find to be required for competent kinetochore recruitment of Mad1-C-Mad2 and robust checkpoint response. Importantly, preventing Mad1 binding to Megator/Tpr rescues the fidelity of chromosome segregation and aneuploidy in larval neuroblasts of Drosophila mps1-null mutants. Our findings demonstrate that the subcellular localization of Mad1 is stringently coordinated with cell cycle progression by kinetochore-extrinsic activity of Mps1. This ensures that both NPCs in interphase and kinetochores in mitosis can generate anaphase inhibitors to efficiently preserve genomic stability.
23.

Nuclear actin regulates inducible transcription by enhancing RNA polymerase II clustering.

blue CRY2olig U-2 OS Organelle manipulation
bioRxiv, 30 May 2019 DOI: 10.1101/655043 Link to full text
Abstract: Gene expression in response to external stimuli underlies a variety of fundamental cellular processes. However, how the transcription machinery is regulated under these scenarios is largely unknown. Here, we discover a novel role of nuclear actin in inducible transcriptional regulation using next-generation transcriptome sequencing and super-resolution microscopy. The RNA-seq data reveal that nuclear actin is required for the establishment of the serum-induced transcriptional program. Using super-resolution imaging, we found a remarkable enhancement of RNA polymerase II (Pol II) clustering upon serum stimulation and this enhancement requires the presence of nuclear actin. To study the molecular mechanisms, we firstly observed that Pol II clusters co-localized with the serum-response genes and nuclear actin polymerized in adjacent to Pol II clusters upon serum stimulation. Furthermore, N-WASP and Arp2/3 are reported to interact with Pol II, and we demonstrated N-WASP is required for serum-enhanced Pol II clustering. Importantly, using an optogenetic tool, we revealed that N-WASP phase-separated with the carboxy-terminal domain of Pol II and nuclear actin. In addition to serum stimulation, we found nuclear actin also essential in enhancing Pol II clustering upon interferon-γ treatment. Taken together, our work unveils nuclear actin promotes the formation of transcription factory on inducible genes, acting as a general mechanism underlying the rapid response to environmental cues.
24.

The Glycolytic Protein Phosphofructokinase Dynamically Relocalizes into Subcellular Compartments with Liquid-like Properties in vivo.

blue CRY2olig C. elegans in vivo Organelle manipulation
bioRxiv, 13 May 2019 DOI: 10.1101/636449 Link to full text
Abstract: While much is known about the biochemical regulation of glycolytic enzymes, less is understood about how they are organized inside cells. Here we built a hybrid microfluidic-hydrogel device for use in Caenorhabditis elegans to systematically examine and quantify the dynamic subcellular localization of the rate-limiting enzyme of glycolysis, phosphofructokinase-1/PFK-1.1. We determine that endogenous PFK-1.1 localizes to distinct, tissue-specific subcellular compartments in vivo. In neurons, PFK-1.1 is diffusely localized in the cytosol, but capable of dynamically forming phase-separated condensates near synapses in response to energy stress from transient hypoxia. Restoring animals to normoxic conditions results in the dispersion of PFK-1.1 in the cytosol, indicating that PFK-1.1 reversibly organizes into biomolecular condensates in response to cues within the cellular environment. PFK-1.1 condensates exhibit liquid-like properties, including spheroid shapes due to surface tension, fluidity due to deformations, and fast internal molecular rearrangements. Prolonged conditions of energy stress during sustained hypoxia alter the biophysical properties of PFK-1.1 in vivo, affecting its viscosity and mobility within phase-separated condensates. PFK-1.1’s ability to form tetramers is critical for its capacity to form condensates in vivo, and heterologous self-association domain such as cryptochrome 2 (CRY2) is sufficient to constitutively induce the formation of PFK-1.1 condensates. PFK-1.1 condensates do not correspond to stress granules and might represent novel metabolic subcompartments. Our studies indicate that glycolytic protein PFK-1.1 can dynamically compartmentalize in vivo to specific subcellular compartments in response to acute energy stress via multivalency as phase-separated condensates.
25.

Optical induction of autophagy via Transcription factor EB (TFEB) reduces pathological tau in neurons.

blue EL222 HEK293T human IPSCs Neuro-2a Transgene expression
bioRxiv, 5 Apr 2019 DOI: 10.1101/600908 Link to full text
Abstract: Aggregation and accumulation of microtubule associated protein tau in neurons is major neuropathological hallmark of Alzheimer’s disease (AD) and related tauopathies. Attempts have been made to promote clearance of pathological tau (p-Tau) from neurons via autophagy. Over expression of transcription factor EB (TFEB), has shown to clear pTau from neurons via autophagy. However, sustained TFEB activation and/or autophagy can create burden on cellular bioenergetics and can be deleterious. Thus, we engineered a minimally invasive optical system that could transiently alter autophagic flux. We optimized and testedan optogenetic gene expression system derived from apreviouslyengineered bacterial transcription factor, EL222. For the first time, our group utilized this system not only to spatial-temporally control nuclear TFEB expression, we also show light-induced TFEB has the capacity to reduce p-Tau burden in AD patient-derived human iPSC-neurons. Together, these results suggest that optically-regulatable gene expression of TFEB unlocks opto-therapeutics to treat AD and other dementias.
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