Showing 1 - 25 of 116 results
Optogenetic control of integrin-matrix interaction.
Optogenetic approaches have gathered momentum in precisely modulating and interrogating cellular signalling and gene expression. The use of optogenetics on the outer cell surface to interrogate how cells receive stimuli from their environment, however, has so far not reached its full potential. Here we demonstrate the development of an optogenetically regulated membrane receptor-ligand pair exemplified by the optically responsive interaction of an integrin receptor with the extracellular matrix. The system is based on an integrin engineered with a phytochrome-interacting factor domain (OptoIntegrin) and a red light-switchable phytochrome B-functionalized matrix (OptoMatrix). This optogenetic receptor-ligand pair enables light-inducible and -reversible cell-matrix interaction, as well as the controlled activation of downstream mechanosensory signalling pathways. Pioneering the application of optogenetic switches in the extracellular environment of cells, this OptoMatrix–OptoIntegrin system may serve as a blueprint for rendering matrix–receptor interactions amendable to precise control with light.
Luminescence-activated nucleotide cyclase regulates spatial and temporal cAMP synthesis.
cAMP is a ubiquitous second messenger that regulates cellular proliferation, differentiation, attachment, migration, and several other processes. It has become increasingly evident that tight regulation of cAMP accumulation and localization confers divergent yet specific signaling to downstream pathways. Currently, few tools are available that have sufficient spatial and temporal resolution to study location-biased cAMP signaling. Here, we introduce a new fusion protein consisting of a light-activated adenylyl cyclase (bPAC) and luciferase (nLuc). This construct allows dual activation of cAMP production through temporally precise photostimulation or chronic chemical stimulation that can be fined-tuned to mimic physiological levels and duration of cAMP synthesis to trigger downstream events. By targeting this construct to different compartments, we show that cAMP produced in the cytosol and nucleus stimulates proliferation in thyroid cells. The bPAC-nLuc fusion construct adds a new reagent to the available toolkit to study cAMP-regulated processes in living cells.
Liquid Nuclear Condensates Mechanically Sense and Restructure the Genome.
Phase transitions involving biomolecular liquids are a
fundamental mechanism underlying intracellular organization.
In the cell nucleus, liquid-liquid phase
separation of intrinsically disordered proteins (IDPs)
is implicated in assembly of the nucleolus, as well
as transcriptional clusters, and other nuclear bodies.
However, it remains unclear whether and how physical
forces associated with nucleation, growth, and
wetting of liquid condensates can directly restructure
chromatin. Here, we use CasDrop, a novel
CRISPR-Cas9-based optogenetic technology, to
show that various IDPs phase separate into liquid
condensates that mechanically exclude chromatin
as they grow and preferentially form in low-density,
largely euchromatic regions. A minimal physical
model explains how this stiffness sensitivity arises
from lower mechanical energy associated with deforming
softer genomic regions. Targeted genomic
loci can nonetheless be mechanically pulled together
through surface tension-driven coalescence. Nuclear
condensates may thus function as mechanoactive
chromatin filters, physically pulling in targeted
genomic loci while pushing out non-targeted regions
of the neighboring genome.
Mapping Local and Global Liquid Phase Behavior in Living Cells Using Photo-Oligomerizable Seeds.
Liquid-liquid phase separation plays a key role in the
assembly of diverse intracellular structures. However,
the biophysical principles by which phase separation
can be precisely localized within subregions
of the cell are still largely unclear, particularly for
low-abundance proteins. Here, we introduce an oligomerizing
biomimetic system, ‘‘Corelets,’’ and utilize
its rapid and quantitative light-controlled
tunability to map full intracellular phase diagrams,
which dictate the concentrations at which phase
separation occurs and the transition mechanism, in
a protein sequence dependent manner. Surprisingly,
both experiments and simulations show that while
intracellular concentrations may be insufficient for
global phase separation, sequestering protein ligands
to slowly diffusing nucleation centers can
move the cell into a different region of the phase diagram,
resulting in localized phase separation. This
diffusive capture mechanism liberates the cell from
the constraints of global protein abundance and is
likely exploited to pattern condensates associated
with diverse biological processes.
Potassium channel-based optogenetic silencing.
Optogenetics enables manipulation of biological processes with light at high spatio-temporal resolution to control the behavior of cells, networks, or even whole animals. In contrast to the performance of excitatory rhodopsins, the effectiveness of inhibitory optogenetic tools is still insufficient. Here we report a two-component optical silencer system comprising photoactivated adenylyl cyclases (PACs) and the small cyclic nucleotide-gated potassium channel SthK. Activation of this 'PAC-K' silencer by brief pulses of low-intensity blue light causes robust and reversible silencing of cardiomyocyte excitation and neuronal firing. In vivo expression of PAC-K in mouse and zebrafish neurons is well tolerated, where blue light inhibits neuronal activity and blocks motor responses. In combination with red-light absorbing channelrhodopsins, the distinct action spectra of PACs allow independent bimodal control of neuronal activity. PAC-K represents a reliable optogenetic silencer with intrinsic amplification for sustained potassium-mediated hyperpolarization, conferring high operational light sensitivity to the cells of interest.
Membrane dynamics induced by a PIP3 optogenetic tool.
Membrane dynamic structures such as filopodia, lamellipodia, and ruffles have important cellular functions in phagocytosis and cell motility, and in pathological states such as cancer metastasis. Phosphatidylinositol 3,4,5-trisphosphate (PIP3) is a crucial lipid that regulates PIP3 dynamics. Investigations of how PIP3 is involved in these functions have mainly relied on pharmacological interventions, and therefore have not generated detailed spatiotemporal information of membrane dynamics upon PIP3 production. In the present study, we applied an optogenetic approach using the CRY2–CIBN system. Using this system, we revealed that local PIP3 generation induced directional cell motility and membrane ruffles in COS7 cells. Furthermore, combined with structured illumination microscopy (SIM), membrane dynamics were investigated with high spatial resolution. We observed PIP3-induced apical ruffles and unique actin fiber behavior in that a single actin fiber protruded from the plasma membrane was taken up into the plasma membrane without depolymerization. This system has the potential to investigate other high-level cell motility and dynamic behaviors such as cancer cell invasion and wound healing with high spatiotemporal resolution, and could provide new insights of biological sciences for membrane dynamics.
Engineered anti-CRISPR proteins for optogenetic control of CRISPR-Cas9.
Anti-CRISPR proteins are powerful tools for CRISPR-Cas9 regulation; the ability to precisely modulate their activity could facilitate spatiotemporally confined genome perturbations and uncover fundamental aspects of CRISPR biology. We engineered optogenetic anti-CRISPR variants comprising hybrids of AcrIIA4, a potent Streptococcus pyogenes Cas9 inhibitor, and the LOV2 photosensor from Avena sativa. Coexpression of these proteins with CRISPR-Cas9 effectors enabled light-mediated genome and epigenome editing, and revealed rapid Cas9 genome targeting in human cells.
RalB directly triggers invasion downstream Ras by mobilizing the Wave complex.
The two Ral GTPases, RalA and RalB, have crucial roles downstream Ras oncoproteins in human cancers; in particular, RalB is involved in invasion and metastasis. However, therapies targeting Ral signalling are not available yet. By a novel optogenetic approach, we found that light-controlled activation of Ral at plasma-membrane promotes the recruitment of the Wave Regulatory Complex (WRC) via its effector exocyst, with consequent induction of protrusions and invasion. We show that active Ras signals to RalB via two RalGEFs (Guanine nucleotide Exchange Factors), RGL1 and RGL2, to foster invasiveness; RalB contribution appears to be more important than that of MAPK and PI3K pathways. Moreover, on the clinical side, we uncovered a potential role of RalB in human breast cancers by determining that RalB expression at protein level increases in a manner consistent with progression toward metastasis. This work highlights the Ras-RGL1/2-RalB-exocyst-WRC axis as appealing target for novel anti-cancer strategies.
Dual-controlled optogenetic system for the rapid down-regulation of protein levels in mammalian cells.
Optogenetic switches are emerging molecular tools for studying cellular processes as they offer higher spatiotemporal and quantitative precision than classical, chemical-based switches. Light-controllable gene expression systems designed to upregulate protein expression levels meanwhile show performances superior to their chemical-based counterparts. However, systems to reduce protein levels with similar efficiency are lagging behind. Here, we present a novel two-component, blue light-responsive optogenetic OFF switch (‘Blue-OFF’), which enables a rapid and quantitative down-regulation of a protein upon illumination. Blue-OFF combines the first light responsive repressor KRAB-EL222 with the protein degradation module B-LID (blue light-inducible degradation domain) to simultaneously control gene expression and protein stability with a single wavelength. Blue-OFF thus outperforms current optogenetic systems for controlling protein levels. The system is described by a mathematical model which aids in the choice of experimental conditions such as light intensity and illumination regime to obtain the desired outcome. This approach represents an advancement of dual-controlled optogenetic systems in which multiple photosensory modules operate synergistically. As exemplified here for the control of apoptosis in mammalian cell culture, the approach opens up novel perspectives in fundamental research and applications such as tissue engineering.
Light Control of the Tet Gene Expression System in Mammalian Cells.
Gene expression and its network structure are dynamically altered in multicellular systems during morphological, functional, and pathological changes. To precisely analyze the functional roles of dynamic gene expression changes, tools that manipulate gene expression at fine spatiotemporal resolution are needed. The tetracycline (Tet)-controlled gene expression system is a reliable drug-inducible method, and it is used widely in many mammalian cultured cells and model organisms. Here, we develop a photoactivatable (PA)-Tet-OFF/ON system for precise temporal control of gene expression at single-cell resolution. By integrating the cryptochrome 2-cryptochrome-interacting basic helix-loop-helix 1 (Cry2-CIB1) light-inducible binding switch, expression of the gene of interest is tightly regulated under the control of light illumination and drug application in our PA-Tet-OFF/ON system. This system has a large dynamic range of downstream gene expression and rapid activation/deactivation kinetics. We also demonstrate the optogenetic regulation of exogenous gene expression in vivo, such as in developing and adult mouse brains.
Increasing spatial resolution of photoregulated GTPases through immobilized peripheral membrane proteins.
Light-induced dimerizing systems, e.g. iLID, are an increasingly utilized optogenetics tool to perturb cellular signaling. The major benefit of this technique is that it allows external spatiotemporal control over protein localization with sub-cellular specificity. However, when it comes to local recruitment of signaling components to the plasmamembrane, this precision in localization is easily lost due to rapid diffusion of the membrane anchor. In this study, we explore different approaches of countering the diffusion of peripheral membrane anchors, to the point where we detect immobilized fractions with iFRAP on a timescale of several minutes. One method involves simultaneous binding of the membrane anchor to a secondary structure, the microtubules. The other strategy utilizes clustering of the anchor into large immobile structures, which can also be interlinked by employing tandem recruitable domains. For both approaches, the anchors are peripheral membrane constructs, which also makes them suitable for in vitro use. Upon combining these slower diffusing anchors with recruitable guanine exchange factors (GEFs), we show that we can elicit much more localized morphological responses from Rac1 and Cdc42 as compared to a regular CAAX-box based membrane anchor in living cells. Thanks to these new slow diffusing anchors, more precisely defined membrane recruitment experiments are now possible.
Spatiotemporal control of zebrafish (Danio rerio) gene expression using a light-activated CRISPR activation system.
CRISPR activation (CRISPRa) system is the convenient tool for targeted-gene activation, it has been developed and combined with a lighting-based system that can control transcription initiation spatially and temporally by utilizing photoreceptor derived from plant Arabidopsis thaliana. A blue light photoreceptor the Cryptochrome 2 (CRY2), and its binding partner CIB1 will dimerize by exposure to the blue light and it has been applied to human cells. However, the application of a combination of these two systems to zebrafish cell is still not explored. We performed zebrafish gene activation using p65 and VP64 activators in the zebrafish cells (ZF4). Our study demonstrated that we have successfully controlled the transcription level of ASCL1a, BCL6a, and HSP70 genes using blue light-activated CRISPR activation system. The result showed that using this system, mRNA level expression of ASCL1a, BCL6a, and HSP70 genes increased after irradiated under blue light for several hours and significantly different to those which treated in the dark.
Directly light-regulated binding of RGS-LOV photoreceptors to anionic membrane phospholipids.
We report natural light-oxygen-voltage (LOV) photoreceptors with a blue light-switched, high-affinity (KD ∼ 10-7 M), and direct electrostatic interaction with anionic phospholipids. Membrane localization of one such photoreceptor, BcLOV4 from Botrytis cinerea, is directly coupled to its flavin photocycle, and is mediated by a polybasic amphipathic helix in the linker region between the LOV sensor and its C-terminal domain of unknown function (DUF), as revealed through a combination of bioinformatics, computational protein modeling, structure-function studies, and optogenetic assays in yeast and mammalian cell line expression systems. In model systems, BcLOV4 rapidly translocates from the cytosol to plasma membrane (∼1 second). The reversible electrostatic interaction is nonselective among anionic phospholipids, exhibiting binding strengths dependent on the total anionic content of the membrane without preference for a specific headgroup. The in vitro and cellular responses were also observed with a BcLOV4 homolog and thus are likely to be general across the dikarya LOV class, whose members are associated with regulator of G-protein signaling (RGS) domains. Natural photoreceptors are not previously known to directly associate with membrane phospholipids in a light-dependent manner, and thus this work establishes both a photosensory signal transmission mode and a single-component optogenetic tool with rapid membrane localization kinetics that approaches the diffusion limit.
Synthetic far-red light-mediated CRISPR-dCas9 device for inducing functional neuronal differentiation.
The ability to control the activity of CRISPR-dCas9 with precise spatiotemporal resolution will enable tight genome regulation of user-defined endogenous genes for studying the dynamics of transcriptional regulation. Optogenetic devices with minimal phototoxicity and the capacity for deep tissue penetration are extremely useful for precise spatiotemporal control of cellular behavior and for future clinic translational research. Therefore, capitalizing on synthetic biology and optogenetic design principles, we engineered a far-red light (FRL)-activated CRISPR-dCas9 effector (FACE) device that induces transcription of exogenous or endogenous genes in the presence of FRL stimulation. This versatile system provides a robust and convenient method for precise spatiotemporal control of endogenous gene expression and also has been demonstrated to mediate targeted epigenetic modulation, which can be utilized to efficiently promote differentiation of induced pluripotent stem cells into functional neurons by up-regulating a single neural transcription factor, NEUROG2 This FACE system might facilitate genetic/epigenetic reprogramming in basic biological research and regenerative medicine for future biomedical applications.
Light-controllable Transcription System by Nucleocytoplasmic Shuttling of a Truncated Phytochrome B.
Transcriptional regulation is a useful strategy for gene therapy and for biomedical research. Unlike chemically regulated transcriptional approaches, spatiotemporal control of transcription using optogenetic tools is a powerful technology for the analysis of single cells. For light to penetrate into tissues, it is desired to use photoreceptors absorbing red/far-red light with a low-molecular mass applicable for the use of virus vectors, and a photoswitch using the photoreceptor need to be constructed as a single expression vector. Herein, we describe an optogenetic tool based on Arabidopsis thaliana phytochrome (Phy) B and its binding partner, phytochrome-interacting factor (PIF) 6. We generated a truncated PhyB, which allowed for reversible association with PIF6 by red/far-red light illumination. The red light illumination only for 5 min induced PhyB translocation from cytoplasm into the nucleus by the association with PIF6, resulting in transcriptional activation based on Gal4 DNA-binding domain and the upstream activating sequence of Gal system. The nucleocytoplasmic shuttling vector using PhyB and PIF6 might be applicable for transcriptional regulation in tissue experiments. This article is protected by copyright. All rights reserved.
Optogenetic inhibition of Gαq protein signaling reduces calcium oscillation stochasticity.
As fast terminators of G-protein coupled receptor (GPCR) signaling, regulators of G-protein signaling (RGS) serve critical roles in fine-tuning second messenger levels and, consequently, cellular responses to external stimuli. Here, we report the creation of an optogenetic RGS2 (opto-RGS2) that suppresses agonist-evoked calcium oscillations by the inactivation of Gαq protein. In this system, cryptochrome-mediated hetero-dimerization of the catalytic RGS2-box with its N-terminal amphipathic helix reconstitutes a functional membrane-localized complex that can dynamically suppress store-operated release of calcium. Engineered opto-RGS2 cell lines were used to establish the role of RGS2 as a key inhibitory feedback regulator of the stochasticity of the Gαq-mediated calcium spike timing. RGS2 reduced the stochasticity of carbachol-stimulated calcium oscillations, and the feedback inhibition was coupled to the global calcium elevation by calmodulin/RGS2 interactions. The identification of a critical negative feedback circuit exemplifies the utility of optogenetic approaches for interrogating RGS/GPCR biology and calcium encoding principles through temporally precise molecular gain-of-function.
Activation of EphB2 Forward Signaling Enhances Memory Consolidation.
EphB2 is involved in enhancing synaptic transmission and gene expression. To explore the roles of EphB2 in memory formation and enhancement, we used a photoactivatable EphB2 (optoEphB2) to activate EphB2 forward signaling in pyramidal neurons in lateral amygdala (LA). Photoactivation of optoEphB2 during fear conditioning, but not minutes afterward, enhanced long-term, but not short-term, auditory fear conditioning. Photoactivation of optoEphB2 during fear conditioning led to activation of the cAMP/Ca2+ responsive element binding (CREB) protein. Application of light to a kinase-dead optoEphB2 in LA did not lead to enhancement of long-term fear conditioning memory or to activation of CREB. Long-term, but not short-term, auditory fear conditioning memory was impaired in mice lacking EphB2 forward signaling (EphB2lacZ/lacZ). Activation of optoEphB2 in LA of EphB2lacZ/lacZ mice enhanced long-term fear conditioning memory. The present findings show that the level of EphB2 forward signaling activity during learning determines the strength of long-term memory consolidation.
Filopodia Conduct Target Selection in Cortical Neurons Using Differences in Signal Kinetics of a Single Kinase.
Dendritic filopodia select synaptic partner axons by interviewing the cell surface of potential targets, but how filopodia decipher the complex pattern of adhesive and repulsive molecular cues to find appropriate contacts is unknown. Here, we demonstrate in cortical neurons that a single cue is sufficient for dendritic filopodia to reject or select specific axonal contacts for elaboration as synaptic sites. Super-resolution and live-cell imaging reveals that EphB2 is located in the tips of filopodia and at nascent synaptic sites. Surprisingly, a genetically encoded indicator of EphB kinase activity, unbiased classification, and a photoactivatable EphB2 reveal that simple differences in the kinetics of EphB kinase signaling at the tips of filopodia mediate the choice between retraction and synaptogenesis. This may enable individual filopodia to choose targets based on differences in the activation rate of a single tyrosine kinase, greatly simplifying the process of partner selection and suggesting a general principle.
A green light-responsive system for the control of transgene expression in mammalian and plant cells.
The ever-increasing complexity of synthetic gene networks and applications of synthetic biology requires precise and orthogonal gene expression systems. Of particular interest are systems responsive to light as they enable the control of gene expression dynamics with unprecedented resolution in space and time. While broadly used in mammalian backgrounds, however, optogenetic approaches in plant cells are still limited due to interference of the activating light with endogenous photoreceptors. Here, we describe the development of the first synthetic light-responsive system for the targeted control of gene expression in mammalian and plant cells that responds to the green range of the light spectrum in which plant photoreceptors have minimal activity. We first engineered a system based on the light-sensitive bacterial transcription factor CarH6 and its cognate DNA operator sequence CarO from Thermus thermophilus to control gene expression in mammalian cells. The system was functional in various mammalian cell lines, showing high induction (up to 350-fold) along with low leakiness, as well as high reversibility. We quantitatively described the systems characteristics by the development and experimental validation of a mathematical model. Finally, we transferred the system into A. thaliana protoplasts and demonstrated gene expression in response to green light. We expect that this system will provide new opportunities in applications based on synthetic gene networks and will open up perspectives for optogenetic studies in mammalian and plant cells.
Analysis of the CaMKIIα and β splice-variant distribution among brain regions reveals isoform-specific differences in holoenzyme formation.
Four CaMKII isoforms are encoded by distinct genes, and alternative splicing within the variable linker-region generates additional diversity. The α and β isoforms are largely brain-specific, where they mediate synaptic functions underlying learning, memory and cognition. Here, we determined the α and β splice-variant distribution among different mouse brain regions. Surprisingly, the nuclear variant αB was detected in all regions, and even dominated in hypothalamus and brain stem. For CaMKIIβ, the full-length variant dominated in most regions (with higher amounts of minor variants again seen in hypothalamus and brain stem). The mammalian but not fish CaMKIIβ gene lacks exon v3Nthat encodes the nuclear localization signal in αB, but contains three exons not found in the CaMKIIα gene (exons v1, v4, v5). While skipping of exons v1 and/or v5 generated the minor splice-variants β', βe and βe', essentially all transcripts contained exon v4. However, we instead detected another minor splice-variant (now termed βH), which lacks part of the hub domain that mediates formation of CaMKII holoenzymes. Surprisingly, in an optogenetic cellular assay of protein interactions, CaMKIIβH was impaired for binding to the β hub domain, but still bound CaMKIIα. This provides the first indication for isoform-specific differences in holoenzyme formation.
Rewiring Calcium Signaling for Precise Transcriptional Reprogramming.
Tools capable of modulating gene expression in living organisms are very useful for interrogating the gene regulatory network and controlling biological processes. The catalytically inactive CRISPR/Cas9 (dCas9), when fused with repressive or activating effectors, functions as a versatile platform to reprogram gene transcription at targeted genomic loci. However, without temporal control, the application of these reprogramming tools will likely cause off-target effects and lack strict reversibility. To overcome this limitation, we report herein the development of a chemical or light-inducible transcriptional reprogramming device that combines photoswitchable genetically encoded calcium actuators with dCas9 to control gene expression. By fusing an engineered Ca2+-responsive NFAT fragment with dCas9 and transcriptional coactivators, we harness the power of light to achieve photoinducible transcriptional reprogramming in mammalian cells. This synthetic system (designated CaRROT) can also be used to document calcium-dependent activity in mammals after exposure to ligands or chemicals that would elicit calcium response inside cells.
Near-infrared light-controlled gene expression and protein targeting in neurons and non-neuronal cells.
Near-infrared (NIR) light-inducible binding of bacterial phytochrome BphP1 to its engineered partner QPAS1 is used for optical protein regulation in mammalian cells. However, there are no data on the application of the BphP1-QPAS1 pair in cells derived from various mammalian tissues. Here, we tested functionality of two BphP1-QPAS1-based optogenetic tools, such as an NIR and blue light-sensing system for control of protein localization (iRIS) and an NIR light-sensing system for transcription activation (TA), in several cell types including cortical neurons. We found that the performance of these optogenetic tools often rely on physiological properties of a specific cell type, such as nuclear transport, which may limit applicability of blue light-sensitive component of iRIS. In contrast, the NIR-light-sensing part of iRIS performed well in all tested cell types. The TA system showed the best performance in HeLa, U-2 OS and HEK-293 cells. Small size of the QPAS1 component allows designing AAV viral particles, which were applied to deliver the TA system to neurons.
Unique Roles of β-Arrestin in GPCR Trafficking Revealed by Photoinducible Dimerizers.
Intracellular trafficking of G protein-coupled receptors (GPCRs) controls their localization and degradation, which affects a cell's ability to adapt to extracellular stimuli. Although the perturbation of trafficking induces important diseases, these trafficking mechanisms are poorly understood. Herein, we demonstrate an optogenetic method using an optical dimerizer, cryptochrome (CRY) and its partner protein (CIB), to analyze the trafficking mechanisms of GPCRs and their regulatory proteins. Temporally controlling the interaction between β-arrestin and β2-adrenergic receptor (ADRB2) reveals that the duration of the β-arrestin-ADRB2 interaction determines the trafficking pathway of ADRB2. Remarkably, the phosphorylation of ADRB2 by G protein-coupled receptor kinases is unnecessary to trigger clathrin-mediated endocytosis, and β-arrestin interacting with unphosphorylated ADRB2 fails to activate mitogen-activated protein kinase (MAPK) signaling, in contrast to the ADRB2 agonist isoproterenol. Temporal control of β-arrestin-GPCR interactions will enable the investigation of the unique roles of β-arrestin and the mechanism by which it regulates β-arrestin-specific trafficking pathways of different GPCRs.
Biosynthesis of Orthogonal Molecules Using Ferredoxin and Ferredoxin-NADP+ Reductase Systems Enables Genetically Encoded PhyB Optogenetics.
Transplanting metabolic reactions from one species into another has many uses as a research tool with applications ranging from optogenetics to crop production. Ferredoxin (Fd), the enzyme that most often supplies electrons to these reactions, is often overlooked when transplanting enzymes from one species to another because most cells already contain endogenous Fd. However, we have shown that the production of chromophores used in Phytochrome B (PhyB) optogenetics, is greatly enhanced in mammalian cells by expressing bacterial and plant Fds with ferredoxin-NADP+ reductases (FNR). We delineated the rate limiting factors and found that the main metabolic precursor, heme, was not the primary limiting factor for producing either the cyanobacterial or plant chromophores, phycocyanobilin or phytochromobilin, respectively. In fact, Fd is limiting, followed by Fd+FNR and finally heme. Using these findings, we optimized the PCB production system and for the first time, combined it with a tissue penetrating red/far-red sensing PhyB optogenetic gene switch in animal cells. We further characterized this system in several mammalian cell lines using red and far-red light. Importantly, we found that the light-switchable gene system remains active for several hours upon illumination, even with a short light pulse and requires very small amounts of light for maximal activation. Boosting chromophore production by matching metabolic pathways with specific ferredoxin systems will enable the unparalleled use of the many PhyB optogenetic tools and has broader implications for optimizing synthetic metabolic pathways.
Optogenetic Control of Endoplasmic Reticulum-Mitochondria Tethering.
The organelle interface emerges as a dynamic platform for a variety of biological responses. However, their study has been limited by the lack of tools to manipulate their occurrence in live cells spatiotemporally. Here, we report the development of a genetically encoded light-inducible tethering (LIT) system allowing the induction of contacts between endoplasmic reticulum (ER) and mitochondria, taking advantage of a pair of light-dependent heterodimerization called an iLID system. We demonstrate that the iLID-based LIT approach enables control of ER-mitochondria tethering with high spatiotemporal precision in various cell types including primary neurons, which will facilitate the functional study of ER-mitochondrial contacts.