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 897 results
1.

Color Sensing and Signal Transmission Diversity of Cyanobacterial Phytochromes and Cyanobacteriochromes.

green red Cyanobacteriochromes Phytochromes Review
Mol Cells, 22 May 2020 DOI: 10.14348/molcells.2020.0077 Link to full text
Abstract: To perceive fluctuations in light quality, quantity, and timing, higher plants have evolved diverse photoreceptors including UVR8 (a UV-B photoreceptor), cryptochromes, phototropins, and phytochromes (Phys). In contrast to plants, prokaryotic oxygen-evolving photosynthetic organisms, cyanobacteria, rely mostly on bilin-based photoreceptors, namely, cyanobacterial phytochromes (Cphs) and cyanobacteriochromes (CBCRs), which exhibit structural and functional differences compared with plant Phys. CBCRs comprise varying numbers of light sensing domains with diverse color-tuning mechanisms and signal transmission pathways, allowing cyanobacteria to respond to UV-A, visible, and far-red lights. Recent genomic surveys of filamentous cyanobacteria revealed novel CBCRs with broader chromophore-binding specificity and photocycle protochromicity. Furthermore, a novel Cph lineage has been identified that absorbs blue-violet/yellow-orange light. In this minireview, we briefly discuss the diversity in color sensing and signal transmission mechanisms of Cphs and CBCRs, along with their potential utility in the field of optogenetics.
2.

CofActor: A light- and stress-gated optogenetic clustering tool to study disease-associated cytoskeletal dynamics in living cells.

blue CRY2/CIB1 HeLa Control of cytoskeleton / cell motility / cell shape
J Biol Chem, 18 May 2020 DOI: 10.1074/jbc.ra119.012427 Link to full text
Abstract: The hallmarks of neurodegenerative diseases, including neural fibrils, reactive oxygen species (ROS), and cofilin-actin rods, present numerous challenges in the development of in vivo diagnostic tools. Biomarkers such as amyloid β (Aβ) fibrils and Tau tangles in Alzheimer's disease (AD) are accessible only via invasive cerebrospinal fluid assays, and ROS can be fleeting and challenging to monitor in vivo. Although remaining a challenge for in vivo detection, the protein-protein interactions underlying these disease-specific biomarkers present opportunities for the engineering of in vitro pathology-sensitive biosensors. These tools can be useful for investigating early-stage events in neurodegenerative diseases in both cellular and animal models and may lead to clinically useful reagents. Here, we report a light- and cellular stress-gated protein switch based on cofilin-actin rod formation, occurring in stressed neurons in the AD brain and following ischemia. By coupling the stress-sensitive cofilin-actin interaction with the light-responsive Cry2-CIB blue-light switch, referred to hereafter as the "CofActor," we accomplished both light- and energetic/oxidative stress-gated control of this interaction. Site-directed mutagenesis of both cofilin and actin revealed residues critical for sustaining or abrogating the light- and stress-gated response. Of note, the switch response varied, depending on whether cellular stress was generated via glycolytic inhibition or by both glycolytic inhibition and azide-induced ATP depletion. We also demonstrate light- and cellular stress-gated switch function in cultured hippocampal neurons. CofActor holds promise for the tracking of early-stage events in neurodegeneration and for investigating actin's interactions with other proteins during cellular stress.
3.

Optical Activation of TrkB Signaling.

blue CRY2/CIB1 CRY2/CRY2 VfAU1-LOV NIH/3T3 PC-12 Signaling cascade control
J Mol Biol, 15 May 2020 DOI: 10.1016/j.jmb.2020.05.002 Link to full text
Abstract: Brain-derived neurotrophic factor (BDNF), via activation of tropomyosin receptor kinase B (TrkB), plays a critical role in neuronal proliferation, differentiation, survival, and death. Dysregulation of TrkB signaling is implicated in neurodegenerative disorders and cancers. Precise activation of TrkB signaling with spatial and temporal resolution is greatly desired to study the dynamic nature of TrkB signaling and its role in related diseases. Here we develop different optogenetic approaches that use light to activate TrkB signaling. Utilizing the photosensitive protein Arabidopsis thaliana cryptochrome 2 (CRY2), the light-inducible homo-interaction of the intracellular domain of TrkB (iTrkB) in the cytosol or on the plasma membrane is able to induce the activation of downstream MAPK/ERK and PI3K/Akt signaling as well as the neurite outgrowth of PC12 cells. Moreover, we prove that such strategies are generalizable to other optical homo-dimerizers by demonstrating the optical TrkB activation based on the light-oxygen-voltage domain of aureochrome 1 from Vaucheria frigida. The results open up new possibilities of many other optical platforms to activate TrkB signaling to fulfill customized needs. By comparing all the different strategies, we find that the CRY2-integrated approach to achieve light-induced cell membrane recruitment and homo-interaction of iTrkB is most efficient in activating TrkB signaling. The optogenetic strategies presented are promising tools to investigate BDNF/TrkB signaling with tight spatial and temporal control.
4.

Clustering of the ζ-Chain Can Initiate T Cell Receptor Signaling.

blue CRY2/CRY2 Cos-7 Jurkat Signaling cascade control
Int J Mol Sci, 15 May 2020 DOI: 10.3390/ijms21103498 Link to full text
Abstract: T cell activation is initiated when ligand binding to the T cell receptor (TCR) triggers intracellular phosphorylation of the TCR-CD3 complex. However, it remains unknown how biophysical properties of TCR engagement result in biochemical phosphorylation events. Here, we constructed an optogenetic tool that induces spatial clustering of ζ-chain in a light controlled manner. We showed that spatial clustering of the ζ-chain intracellular tail alone was sufficient to initialize T cell triggering including phosphorylation of ζ-chain, Zap70, PLCγ, ERK and initiated Ca2+ flux. In reconstituted COS-7 cells, only Lck expression was required to initiate ζ-chain phosphorylation upon ζ-chain clustering, which leads to the recruitment of tandem SH2 domain of Zap70 from cell cytosol to the newly formed ζ-chain clusters at the plasma membrane. Taken together, our data demonstrated the biophysical relevance of receptor clustering in TCR signaling.
5.

CLIC4 is a cytokinetic cleavage furrow protein that regulates cortical cytoskeleton stability during cell division.

blue CRY2/CIB1 CRY2/CRY2 HeLa Control of cytoskeleton / cell motility / cell shape
J Cell Sci, 14 May 2020 DOI: 10.1242/jcs.241117 Link to full text
Abstract: During mitotic cell division, the actomyosin cytoskeleton undergoes several dynamic changes that play key roles in progression through mitosis. Although the regulators of cytokinetic ring formation and contraction are well established, proteins that regulate cortical stability during anaphase and telophase have been understudied. Here, we describe a role for CLIC4 in regulating actin and actin regulators at the cortex and cytokinetic cleavage furrow during cytokinesis. We first describe CLIC4 as a new component of the cytokinetic cleavage furrow that is required for successful completion of mitotic cell division. We also demonstrate that CLIC4 regulates the remodeling of the sub-plasma-membrane actomyosin network within the furrow by recruiting MST4 kinase (also known as STK26) and regulating ezrin phosphorylation. This work identifies and characterizes new molecular players involved in regulating cortex stiffness and blebbing during the late stages of cytokinetic furrowing.
6.

LITESEC-T3SS - Light-controlled protein delivery into eukaryotic cells with high spatial and temporal resolution.

blue iLID LOVTRAP HEp-2 Y. enterocolitica Cell death
Nat Commun, 13 May 2020 DOI: 10.1038/s41467-020-16169-w Link to full text
Abstract: Many bacteria employ a type III secretion system (T3SS) injectisome to translocate proteins into eukaryotic host cells. Although the T3SS can efficiently export heterologous cargo proteins, a lack of target cell specificity currently limits its application in biotechnology and healthcare. In this study, we exploit the dynamic nature of the T3SS to govern its activity. Using optogenetic interaction switches to control the availability of the dynamic cytosolic T3SS component SctQ, T3SS-dependent effector secretion can be regulated by light. The resulting system, LITESEC-T3SS (Light-induced translocation of effectors through sequestration of endogenous components of the T3SS), allows rapid, specific, and reversible activation or deactivation of the T3SS upon illumination. We demonstrate the light-regulated translocation of heterologous reporter proteins, and induction of apoptosis in cultured eukaryotic cells. LITESEC-T3SS constitutes a new method to control protein secretion and translocation into eukaryotic host cells with unparalleled spatial and temporal resolution.
7.

Flotillin-upregulation acts as an epithelial-mesenchymal transition driver by promoting sphingosine kinase 2-dependent AXL stabilization.

blue CRY2/CIB1 MCF10A
bioRxiv, 13 May 2020 DOI: 10.1101/2020.05.12.090571 Link to full text
Abstract: Epithelial-to-mesenchymal transition (EMT) is critical for cancer cell dissemination and metastasis formation. Here, we found that flotillin 1 and 2 upregulation, which is observed in many aggressive cancers, is sufficient to induce EMT in non-tumoral mammary cells. We identified that the Upregulated Flotillin-Induced Trafficking (UFIT) pathway promotes the endocytosis of several transmembrane receptors in flotillin-positive late endosomes, thus modifying their destiny and leading to the the activation of oncogenic pathways that promotes EMT. The receptor tyrosine kinase AXL, a key actor during EMT and metastasis formation, is stabilized by the UFIT pathway and is required for EMT induced by flotillin upregulation. Moreover, sphingosine kinase 2, a lipid kinase recruited in flotillin-positive plasma membrane domains and vesicles, is required to stabilize AXL. These findings show that flotillin upregulation is a key EMT inducer. The UFIT pathway, through sphingosine kinase 2 activity, targets membrane receptors, such as AXL, to flotillin-positive vesicles, allowing their stabilization and the activation of EMT-linked signaling pathways.
8.

The oligomeric structures of plant cryptochromes.

blue Cryptochromes Background
Nat Struct Mol Biol, 11 May 2020 DOI: 10.1038/s41594-020-0420-x Link to full text
Abstract: Cryptochromes (CRYs) are a group of evolutionarily conserved flavoproteins found in many organisms. In plants, the well-studied CRY photoreceptor, activated by blue light, plays essential roles in plant growth and development. However, the mechanism of activation remains largely unknown. Here, we determined the oligomeric structures of the blue-light-perceiving PHR domain of Zea mays CRY1 and an Arabidopsis CRY2 constitutively active mutant. The structures form dimers and tetramers whose functional importance is examined in vitro and in vivo with Arabidopsis CRY2. Structure-based analysis suggests that blue light may be perceived by CRY to cause conformational changes, whose precise nature remains to be determined, leading to oligomerization that is essential for downstream signaling. This photoactivation mechanism may be widely used by plant CRYs. Our study reveals a molecular mechanism of plant CRY activation and also paves the way for design of CRY as a more efficient optical switch.
9.

Using optogenetics to tackle systems-level questions of multicellular morphogenesis.

blue red Cryptochromes LOV domains Phytochromes Review
Curr Opin Cell Biol, 11 May 2020 DOI: 10.1016/j.ceb.2020.04.004 Link to full text
Abstract: Morphogenesis of multicellular systems is governed by precise spatiotemporal regulation of biochemical reactions and mechanical forces which together with environmental conditions determine the development of complex organisms. Current efforts in the field aim at decoding the system-level principles underlying the regulation of developmental processes. Toward this goal, optogenetics, the science of regulation of protein function with light, is emerging as a powerful new tool to quantitatively perturb protein function in vivo with unprecedented precision in space and time. In this review, we provide an overview of how optogenetics is helping to address system-level questions of multicellular morphogenesis and discuss future directions.
10.

Light-powered Escherichia coli cell division for chemical production.

blue red BphS EL222 E. coli Cell cycle control Endogenous gene expression Immediate control of second messengers Multichromatic
Nat Commun, 8 May 2020 DOI: 10.1038/s41467-020-16154-3 Link to full text
Abstract: Cell division can perturb the metabolic performance of industrial microbes. The C period of cell division starts from the initiation to the termination of DNA replication, whereas the D period is the bacterial division process. Here, we first shorten the C and D periods of E. coli by controlling the expression of the ribonucleotide reductase NrdAB and division proteins FtsZA through blue light and near-infrared light activation, respectively. It increases the specific surface area to 3.7 μm-1 and acetoin titer to 67.2 g·L-1. Next, we prolong the C and D periods of E. coli by regulating the expression of the ribonucleotide reductase NrdA and division protein inhibitor SulA through blue light activation-repression and near-infrared (NIR) light activation, respectively. It improves the cell volume to 52.6 μm3 and poly(lactate-co-3-hydroxybutyrate) titer to 14.31 g·L-1. Thus, the optogenetic-based cell division regulation strategy can improve the efficiency of microbial cell factories.
11.

The Proline-rich Domain Promotes Tau Liquid Liquid Phase Separation in Cells.

blue CRY2olig SH-SY5Y Control of cytoskeleton / cell motility / cell shape Organelle manipulation
bioRxiv, 5 May 2020 DOI: 10.1101/2020.05.04.076968 Link to full text
Abstract: Tau protein in vitro can undergo liquid liquid phase separation (LLPS); however, observations of this phase transition in living cells are limited. To investigate protein state transitions in living cells we found that Cry2 can optogentically increase the association of full lengh tau with microtubules. To probe this mechanism, we identified tau domains that drive tau clustering on microtubules in living cells. The polyproline rich domain (PRD) drives LLPS and does so under the control of phosphorylation. These readily observable cytoplasmic condensates underwent fusion and fluorescence recovery after photobleaching consistent with the ability of the PRD to undergo LLPS in vitro. In absence of the MTBD, the tau PRD co-condensed with EB1, a regulator of plus-end microtubule dynamic instability. The specific domain properties of the MTBD and PRD serve distinct but mutually complementary roles that utilize LLPS in a cellular context to implement emergent functionalities that scale their relationship from binding alpha-beta tubulin heterodimers to the larger proportions of microtubules.
12.

A STIMulating journey into optogenetic engineering.

blue Cryptochromes LOV domains Review
Cell Calcium, 4 May 2020 DOI: 10.1016/j.ceca.2020.102197 Link to full text
Abstract: Genetically-encoded calcium actuators (GECAs) stemmed from STIM1 have enabled optical activation of endogenous ORAI1 channels in both excitable and non-excitable tissues. These GECAs offer new non-invasive means to probe the structure-function relations of calcium channels and wirelessly control the behavior of awake mice.
13.

The mitotic protein NuMA plays a spindle-independent role in nuclear formation and mechanics.

blue LOVTRAP hTERT RPE-1
bioRxiv, 3 May 2020 DOI: 10.1101/2020.05.02.070680 Link to full text
Abstract: Eukaryotic cells typically form a single, round nucleus after mitosis, and failures to do so can compromise genomic integrity. How mammalian cells form such a nucleus remains incompletely understood. NuMA is a spindle protein whose disruption results in nuclear fragmentation. What role NuMA plays in nuclear integrity, or whether its perceived role stems from its spindle function, is unclear. Here, we use live imaging to demonstrate that NuMA plays a spindle-independent role in forming a single, round nucleus. NuMA keeps the decondensing chromosome mass compact at mitotic exit, and promotes a mechanically robust nucleus. NuMA’s C-terminus binds DNA in vitro and chromosomes in interphase, while its coiled-coil acts as a regulatory and structural hub: it prevents NuMA from binding chromosomes at mitosis, regulates its nuclear mobility and is essential for nuclear formation. Thus, NuMA plays a long-range structural role in building and maintaining an intact nucleus, as it does for the spindle, playing a protective role over the cell cycle.
14.

A Light-Inducible Strain for Genome-Wide Histone Turnover Profiling in Neurospora crassa.

blue VVD N. crassa Epigenetic modification
Genetics, 1 May 2020 DOI: 10.1534/genetics.120.303217 Link to full text
Abstract: In chromatin, nucleosomes are composed of about 146 base pairs of DNA wrapped around a histone octamer, and are highly dynamic structures subject to remodeling and exchange. Histone turnover has previously been implicated in various processes including regulation of chromatin accessibility, segregation of chromatin domains, and dilution of histone marks. Histones in different chromatin environments may turnover at different rates, possibly with functional consequences.Neurospora crassasports a chromatin environment that is more similar to that of higher eukaryotes than yeasts, which have been utilized in the past to explore histone exchange. We constructed a simple light-inducible system to profile histone exchange in N. crassaon a 3xFLAG-tagged histone H3 under the control of the rapidly inducible vvdpromoter. After induction with blue light, incorporation of tagged H3 into chromatin occurred within 20 minutes. Previous studies of histone turnover involved considerably longer incubation periods and relied on a potentially disruptive change of medium for induction. We used this reporter to explore replication-independent histone turnover at genes and examine changes in histone turnover at heterochromatin domains in different heterochromatin mutant strains. In euchromatin, H3-3xFLAG patterns were almost indistinguishable from that observed in wild type in all mutant backgrounds tested, suggesting that loss of heterochromatin machinery has little effect on histone turnover in euchromatin. However, turnover at heterochromatin domains increased with loss of H3K9me3 or HP1, but did not depend on DNA methylation. Our reporter strain provides a simple yet powerful tool to assess histone exchange across multiple chromatin contexts.
15.

Lights, cytoskeleton, action: Optogenetic control of cell dynamics.

blue cyan red Cryptochromes Fluorescent proteins LOV domains Phytochromes Review
Curr Opin Cell Biol, 1 May 2020 DOI: 10.1016/j.ceb.2020.03.003 Link to full text
Abstract: Cell biology is moving from observing molecules to controlling them in real time, a critical step towards a mechanistic understanding of how cells work. Initially developed from light-gated ion channels to control neuron activity, optogenetics now describes any genetically encoded protein system designed to accomplish specific light-mediated tasks. Recent photosensitive switches use many ingenious designs that bring spatial and temporal control within reach for almost any protein or pathway of interest. This next generation optogenetics includes light-controlled protein-protein interactions and shape-shifting photosensors, which in combination with live microscopy enable acute modulation and analysis of dynamic protein functions in living cells. We provide a brief overview of various types of optogenetic switches. We then discuss how diverse approaches have been used to control cytoskeleton dynamics with light through Rho GTPase signaling, microtubule and actin assembly, mitotic spindle positioning and intracellular transport and highlight advantages and limitations of different experimental strategies.
16.

Photoactivatable Cre recombinase 3.0 for in vivo mouse applications.

blue CRY2/CIB1 FKF1/GI iLID Magnets HEK293T isolated MEFs mouse in vivo mouse neural progenitor cells
Nat Commun, 1 May 2020 DOI: 10.1038/s41467-020-16030-0 Link to full text
Abstract: Optogenetic genome engineering tools enable spatiotemporal control of gene expression and provide new insight into biological function. Here, we report the new version of genetically encoded photoactivatable (PA) Cre recombinase, PA-Cre 3.0. To improve PA-Cre technology, we compare light-dimerization tools and optimize for mammalian expression using a CAG promoter, Magnets, and 2A self-cleaving peptide. To prevent background recombination caused by the high sequence similarity in the dimerization domains, we modify the codons for mouse gene targeting and viral production. Overall, these modifications significantly reduce dark leak activity and improve blue-light induction developing our new version, PA-Cre 3.0. As a resource, we have generated and validated AAV-PA-Cre 3.0 as well as two mouse lines that can conditionally express PA-Cre 3.0. Together these new tools will facilitate further biological and biomedical research.
17.

Nano-positioning and tubuline conformation determine transport of mitochondria along microtubules.

blue TULIP primary mouse hippocampal neurons Control of cytoskeleton / cell motility / cell shape Organelle manipulation
bioRxiv, 28 Apr 2020 DOI: 10.1101/2020.04.27.064766 Link to full text
Abstract: Correct spatiotemporal distribution of organelles and vesicles is crucial for healthy cell functioning and is regulated by intracellular transport mechanisms. Controlled transport of bulky mitochondria is especially important in polarized cells such as neurons that rely on these organelles to locally produce energy and buffer calcium. Mitochondrial transport requires and depends on microtubules which fill much of the available axonal space. How mitochondrial transport is affected by their position within the microtubule bundles is not known. Here, we found that anterograde transport, driven by kinesin motors, is susceptible to the molecular conformation of tubulin both in vitro and in vivo. Anterograde velocities negatively correlate with the density of elongated tubulin dimers, similar to GTP-tubulin, that are more straight and rigid. The impact of the tubulin conformation depends primarily on where a mitochondrion is positioned, either within or at the rim of microtubule bundle. Increasing elongated tubulin levels lowers the number of motile anterograde mitochondria within the microtubule bundle and increases anterograde transport speed at the microtubule bundle rim. We demonstrate that the increased kinesin step processivity on microtubules consisting of elongated dimers underlies increased mitochondrial dynamics. Our work indicates that the molecular conformation of tubulin controls mitochondrial motility and as such locally regulates the distribution of mitochondria along axons.
18.

Optogenetic control of the Bone Morphogenetic Protein signalling pathway through engineered blue light-sensitive receptors.

blue VfAU1-LOV HEK293T hESCs SW 1353 T/C28a2 Signaling cascade control
bioRxiv, 28 Apr 2020 DOI: 10.1101/2020.04.27.063073 Link to full text
Abstract: Bone Morphogenetic Proteins (BMPs) are members of the Transforming Growth Factor β (TGFβ) superfamily and have crucial roles during development; including mesodermal patterning and specification of renal, hepatic and skeletal tissues. In vitro developmental models currently rely upon costly and unreliable recombinant BMP proteins that do not enable dynamic or precise perturbation of the BMP signalling pathway. Here, we develop a novel optogenetic BMP signalling system (optoBMP) that enables rapid induction of the canonical BMP signalling pathway through illumination with blue light. We demonstrate the utility of the optoBMP system in multiple human cell lines to initiate signal transduction through phosphorylation and nuclear translocation of SMAD1/5, leading to upregulation of BMP target genes including Inhibitors of DNA binding ID2 and ID4. Furthermore, we demonstrate how the optoBMP system can be used to fine-tune activation of the BMP signalling pathway through variable light stimulation. Optogenetic control of BMP signalling will enable dynamic and high-throughput intervention across a variety of applications in cellular and developmental systems.
19.

Phosphatidylinositol 4,5-bisphosphate directly interacts with the β and γ subunits of the sodium channel ENaC.

blue CRY2/CIB1 HEK293 Organelle manipulation
J Biol Chem, 27 Apr 2020 DOI: 10.1074/jbc.ra120.012606 Link to full text
Abstract: The plasma membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) regulates the activity of diverse ion channels to include the epithelial Na+ channel ENaC. Whether PIP2 regulation of ENaC is due to a direct phospholipid-protein interaction, though, remains obscure. To date, possible interaction of PIP2 with ENaC primarily has been tested indirectly through assays of channel function. A fragment-based biochemical analysis approach is used here to directly quantify possible PIP2-ENaC interactions. We find using the CIBN-CRY2 optogenetic dimerization system that the phosphoryl group positioned at carbon 5 of PIP2 is necessary for interaction with ENaC. Previous studies have implicated conserved basic residues in the cytosolic portions of β- and γ-ENaC subunits as being important for PIP2-ENaC interactions. To test this, we used synthetic peptides of these regions of β- and γ-ENaC. Steady state intrinsic fluorescence spectroscopy demonstrated that phosphoinositides change the local conformation of the N terminus of β-ENaC, and two sites of γ-ENaC adjacent to the plasma membrane, suggesting direct interactions of PIP2 with these three regions. Microscale thermophoresis elaborated PIP2 interactions with the amino termini of β- (Kd ~5.2 µM) and γ-ENaC (Kd ~13 µM). A weaker interaction site within the carboxy terminus of γ-ENaC (Kd ~800 µM) was also observed. These results support that PIP2 regulates ENaC activity by directly interacting with at least three distinct regions within the cytoplasmic domains of the channel that contain conserved basic residues. These interactions are probably electrostatic in nature, and are likely to bear a key structural role in support of channel activity.
20.

Construction of Light-Activated Neurotrophin Receptors Using the Improved Light-Induced Dimerizer (iLID).

blue iLID PC-12 Signaling cascade control
J Mol Biol, 23 Apr 2020 DOI: 10.1016/j.jmb.2020.04.018 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 diseases 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.
21.

Nanobody-directed targeting of optogenetic tools to study signaling in the primary cilium.

blue red bPAC (BlaC) LAPD HEK293 mIMCD-3 Control of cytoskeleton / cell motility / cell shape Immediate control of second messengers
bioRxiv, 15 Apr 2020 DOI: 10.1101/2020.02.04.933440 Link to full text
Abstract: Compartmentalization of cellular signaling forms the molecular basis of cellular behavior. The primary ciliumconstitutesa subcellular compartment thatorchestrates signal transduction independentfrom the cell body.Ciliary dysfunction causes severe diseases, termed ciliopathies.Analyzing ciliary signaling and function has been challenging due to the lack of tools to temporarily manipulate and analyze ciliary signaling. Here, we describe ananobody-based targeting approach foroptogenetic tools that is applicable in vitroand in vivoandallows to specifically analyze ciliary signaling and function.Thereby,we overcomethe loss of protein function observed after direct fusion to a ciliary targeting sequence.Wefunctionally localizedmodifiers of cAMP signaling, i.e. the photo-activated adenylate cyclase bPACandthe light-activated phosphodiesterase LAPD, as well asthe cAMP biosensor mlCNBD-FRET tothe cilium. Using this approach, we studiedthe contributionof spatial cAMP signaling in controllingcilia length.Combining optogenetics with nanobody-based targeting will pave the way to the molecular understanding of ciliary function in health and disease.
22.

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

blue CRY2olig U-2 OS Organelle manipulation
Sci Adv, 15 Apr 2020 DOI: 10.1126/sciadv.aay6515 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.
23.

A Single-Objective Light-Sheet Microscope with 200 nm-Scale Resolution.

blue CRY2/CIB1 MV3 Control of cytoskeleton / cell motility / cell shape
bioRxiv, 9 Apr 2020 DOI: 10.1101/2020.04.07.030569 Link to full text
Abstract: We present a single-objective light-sheet microscope, also known as an oblique-plane microscope,that uses a bespoke glass-tipped tertiary objective andimproves the resolution, field of view, usability, and stability over previous variants. Owing to its highnumerical aperture optics, thismicroscope achieves the highest lateral resolution in light-sheet fluorescence microscopy, and its axial resolution is similar to that of Lattice Light-Sheet Microscopy.Given this performance, wedemonstrate high-resolution imaging of clathrin-mediated endocytosis, vimentin, the endoplasmic reticulum, membrane dynamics, and natural killer cell-mediated cell death. Furthermore, we image biological phenomena that would be otherwise challenging or impossibleto performin a traditional light-sheet microscope geometry, includingcell migration through a confined space within a microfluidic device, photoactivation of PI3K, and diffusionofcytoplasmic rheologicaltracersat a volumetric rate of 14 Hz.
24.

Exosome-based delivery of super-repressor IκBα relieves sepsis-associated organ damage and mortality.

blue CRY2/CIB1 HEK293T
Sci Adv, 8 Apr 2020 DOI: 10.1126/sciadv.aaz6980 Link to full text
Abstract: As extracellular vesicles that play an active role in intercellular communication by transferring cellular materials to recipient cells, exosomes offer great potential as a natural therapeutic drug delivery vehicle. The inflammatory responses in various disease models can be attenuated through introduction of super-repressor IκB (srIκB), which is the dominant active form of IκBα and can inhibit translocation of nuclear factor κB into the nucleus. An optogenetically engineered exosome system (EXPLOR) that we previously developed was implemented for loading a large amount of srIκB into exosomes. We showed that intraperitoneal injection of purified srIκB-loaded exosomes (Exo-srIκBs) attenuates mortality and systemic inflammation in septic mouse models. In a biodistribution study, Exo-srIκBs were observed mainly in the neutrophils, and in monocytes to a lesser extent, in the spleens and livers of mice. Moreover, we found that Exo-srIκB alleviates inflammatory responses in monocytic THP-1 cells and human umbilical vein endothelial cells.
25.

A Generalizable Optogenetic Strategy to Regulate Receptor Tyrosine Kinases during Vertebrate Embryonic Development.

blue CRY2/CIB1 VfAU1-LOV HEK293T PC-12 Xenopus in vivo Signaling cascade control Cell differentiation Developmental processes
J Mol Biol, 8 Apr 2020 DOI: 10.1016/j.jmb.2020.03.032 Link to full text
Abstract: Ligand-independent activation of receptor tyrosine kinases (RTKs) allows for dissecting out the receptor-specific signaling outcomes from the pleiotropic effects of the ligands. In this regard, RTK intracellular domains (ICD) are of interest due to their ability to recapitulate signaling activity in a ligand-independent manner when fused to chemical and optical dimerizing domains. A common strategy for synthetic activation of RTKs involves membrane tethering of dimerizer-RTK ICD fusions. Depending on the intrinsic signaling capacity, however, this approach could entail undesirable baseline signaling activity in the absence of stimulus, thereby diminishing the system's sensitivity. Here, we observed toxicity in early Xenopus laevis embryos when using such a conventional optogenetic design for the fibroblast growth factor receptor (FGFR). To surpass this challenge, we developed a cytoplasm-to-membrane translocation approach, where FGFR ICD is recruited from the cytoplasm to the plasma membrane by light, followed by its subsequent activation via homo-association. This strategy results in the optical activation of FGFR with low background activity and high sensitivity, which allows for the light-mediated formation of ectopic tail-like structure in developing Xenopus laevis embryos. We further generalized this strategy by developing optogenetic platforms to control three neurotrophic tropomyosin receptor kinases, TrkA, TrkB, and TrkC. We envision that these ligand-independent optogenetic RTKs will provide useful toolsets for the delineation of signaling sub-circuits in developing vertebrate embryos.
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