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 - 22 of 22 results
1.

OptoREACT: Optogenetic Receptor Activation on Nonengineered Human T Cells.

red PhyB/PIF6 HEK293T human T cells Jurkat Signaling cascade control Extracellular optogenetics
ACS Synth Biol, 9 Feb 2024 DOI: 10.1021/acssynbio.3c00518 Link to full text
Abstract: Optogenetics is a versatile and powerful tool for the control and analysis of cellular signaling processes. The activation of cellular receptors by light using optogenetic switches usually requires genetic manipulation of cells. However, this considerably limits the application in primary, nonengineered cells, which is crucial for the study of physiological signaling processes and for controlling cell fate and function for therapeutic purposes. To overcome this limitation, we developed a system for the light-dependent extracellular activation of cell surface receptors of nonengineered cells termed OptoREACT (Optogenetic Receptor Activation) based on the light-dependent protein interaction of A. thaliana phytochrome B (PhyB) with PIF6. In the OptoREACT system, a PIF6-coupled antibody fragment binds the T cell receptor (TCR) of Jurkat or primary human T cells, which upon illumination is bound by clustered phytochrome B to induce receptor oligomerization and activation. For clustering of PhyB, we either used tetramerization by streptavidin or immobilized PhyB on the surface of cells to emulate the interaction of a T cell with an antigen-presenting cell. We anticipate that this extracellular optogenetic approach will be applicable for the light-controlled activation of further cell surface receptors in primary, nonengineered cells for versatile applications in fundamental and applied research.
2.

Controlling the Potency of T Cell Activation Using an Optically Tunable Chimeric Antigen Receptor.

blue LOVTRAP Jurkat
Methods Mol Biol, 2024 DOI: 10.1007/978-1-0716-3834-7_5 Link to full text
Abstract: The ability of biological systems to convert inputs from their environment into information to guide future decisions is central to life and a matter of great importance. While we know the components of many of the signaling networks that make these decisions, our understanding of the dynamic flow of information between these parts remains far more limited. T cells are an essential white blood cell type of an adaptive immune response and can discriminate between healthy and infected cells with remarkable sensitivity. This chapter describes the use of a synthetic T-cell receptor (OptoCAR) that is optically tunable within cell conjugates, providing control over the duration, and intensity of intracellular T-cell signaling dynamics. Optical control can also provide control over signaling with high spatial precision, and the OptoCAR is likely to find application more generally when modulating T-cell function with imaging approaches.
3.

Nano-optogenetic CAR-T Cell Immunotherapy.

blue iLID Jurkat mouse in vivo
Methods Mol Biol, 2024 DOI: 10.1007/978-1-0716-3593-3_17 Link to full text
Abstract: Chimeric antigen receptor (CAR)-T cell immunotherapy emerges as an effective cancer treatment. However, significant safety concerns remain, such as cytokine release syndrome (CRS) and "on-target, off-tumor" cytotoxicity, due to a lack of precise control over conventional CAR-T cell activity. To address this issue, a nano-optogenetic approach has been developed to enable spatiotemporal control of CAR-T cell activity. This system is comprised of synthetic light-sensitive CAR-T cells and upconversion nanoparticles acting as an in situ nanotransducer, allowing near-infrared light to wirelessly control CAR-T cell immunotherapy.
4.

Engineering of an Optogenetic T Cell Receptor Compatible with Fluorescence-Based Readouts.

red PhyB/PIF6 Jurkat Signaling cascade control
ACS Synth Biol, 2 Oct 2023 DOI: 10.1021/acssynbio.3c00429 Link to full text
Abstract: Optogenetics offers a set of tools for the precise manipulation of signaling pathways. Here we exploit optogenetics to experimentally change the kinetics of protein-protein interactions on demand. We had developed a system in which the interaction of a modified T cell receptor (TCR) with an engineered ligand can be controlled by light. The ligand was the plant photoreceptor phytochrome B (PhyB) and the TCR included a TCRβ chain fused to GFP and a mutated PhyB-interacting factor (PIFS), resulting in the GFP-PIFS-TCR. We failed to engineer a nonfluorescent PIFS-fused TCR, since PIFS did not bind to PhyB when omitting GFP. Here we tested nine different versions of PIFS-fused TCRs. We found that the SNAP-PIFS-TCR was expressed well on the surface, bound to PhyB, and subsequently elicited activation signals. This receptor could be combined with a GFP reporter system in which the expression of GFP is driven by the transcription factor NF-AT.
5.

Opto-APC: Engineering of cells that display phytochrome B on their surface for optogenetic studies of cell-cell interactions.

red PhyB/PIF6 HEK293T Jurkat Raji Control of cell-cell / cell-material interactions Extracellular optogenetics
Front Mol Biosci, 20 Feb 2023 DOI: 10.3389/fmolb.2023.1143274 Link to full text
Abstract: The kinetics of a ligand-receptor interaction determine the responses of the receptor-expressing cell. One approach to experimentally and reversibly change this kinetics on demand is optogenetics. We have previously developed a system in which the interaction of a modified receptor with an engineered ligand can be controlled by light. In this system the ligand is a soluble Phytochrome B (PhyB) tetramer and the receptor is fused to a mutated PhyB-interacting factor (PIFS). However, often the natural ligand is not soluble, but expressed as a membrane protein on another cell. This allows ligand-receptor interactions in two dimensions. Here, we developed a strategy to generate cells that display PhyB as a membrane-bound protein by expressing the SpyCatcher fused to a transmembrane domain in HEK-293T cells and covalently coupling purified PhyB-SpyTag to these cells. As proof-of-principle, we use Jurkat T cells that express a GFP-PIFS-T cell receptor and show that these cells can be stimulated by the PhyB-coupled HEK-293T cells in a light dependent manner. Thus, we call the PhyB-coupled cells opto-antigen presenting cells (opto-APCs). Our work expands the toolbox of optogenetic technologies, allowing two-dimensional ligand-receptor interactions to be controlled by light.
6.

Progressive enhancement of kinetic proofreading in T cell antigen discrimination from receptor activation to DAG generation.

blue LOVTRAP Jurkat Signaling cascade control Extracellular optogenetics
Elife, 20 Sep 2022 DOI: 10.7554/elife.75263 Link to full text
Abstract: T cells use kinetic proofreading to discriminate antigens by converting small changes in antigen binding lifetime into large differences in cell activation, but where in the signaling cascade this computation is performed is unknown. Previously, we developed a light-gated immune receptor to probe the role of ligand kinetics in T cell antigen signaling. We found significant kinetic proofreading at the level of the signaling lipid diacylglycerol (DAG) but lacked the ability to determine where the multiple signaling steps required for kinetic discrimination originate in the upstream signaling cascade (Tischer and Weiner, 2019). Here we uncover where kinetic proofreading is executed by adapting our optogenetic system for robust activation of early signaling events. We find the strength of kinetic proofreading progressively increases from Zap70 recruitment to LAT clustering to downstream DAG generation. Leveraging the ability of our system to rapidly disengage ligand binding, we also measure slower reset rates for downstream signaling events. These data suggest a distributed kinetic proofreading mechanism, with proofreading steps both at the receptor and at slower resetting downstream signaling complexes that could help balance antigen sensitivity and discrimination.
7.

Nano-optogenetic engineering of CAR T cells for precision immunotherapy with enhanced safety.

blue CRY2/CIB1 iLID human T cells Jurkat mouse T cells Signaling cascade control
Nat Nanotechnol, 25 Oct 2021 DOI: 10.1038/s41565-021-00982-5 Link to full text
Abstract: Chimeric antigen receptor (CAR) T cell-based immunotherapy, approved by the US Food and Drug Administration, has shown curative potential in patients with haematological malignancies. However, owing to the lack of control over the location and duration of the anti-tumour immune response, CAR T cell therapy still faces safety challenges arising from cytokine release syndrome and on-target, off-tumour toxicity. Herein, we present the design of light-switchable CAR (designated LiCAR) T cells that allow real-time phototunable activation of therapeutic T cells to precisely induce tumour cell killing. When coupled with imaging-guided, surgically removable upconversion nanoplates that have enhanced near-infrared-to-blue upconversion luminescence as miniature deep-tissue photon transducers, LiCAR T cells enable both spatial and temporal control over T cell-mediated anti-tumour therapeutic activity in vivo with greatly mitigated side effects. Our nano-optogenetic immunomodulation platform not only provides a unique approach to interrogate CAR-mediated anti-tumour immunity, but also sets the stage for developing precision medicine to deliver personalized anticancer therapy.
8.

Positive feedback between the T cell kinase Zap70 and its substrate LAT acts as a clustering-dependent signaling switch.

blue CRY2/CRY2 iLID HEK293T Jurkat NIH/3T3 SYF Signaling cascade control Organelle manipulation
Cell Rep, 22 Jun 2021 DOI: 10.1016/j.celrep.2021.109280 Link to full text
Abstract: Protein clustering is pervasive in cell signaling, yet how signaling from higher-order assemblies differs from simpler forms of molecular organization is still poorly understood. We present an optogenetic approach to switch between oligomers and heterodimers with a single point mutation. We apply this system to study signaling from the kinase Zap70 and its substrate linker for activation of T cells (LAT), proteins that normally form membrane-localized condensates during T cell activation. We find that fibroblasts expressing synthetic Zap70:LAT clusters activate downstream signaling, whereas one-to-one heterodimers do not. We provide evidence that clusters harbor a positive feedback loop among Zap70, LAT, and Src-family kinases that binds phosphorylated LAT and further activates Zap70. Finally, we extend our optogenetic approach to the native T cell signaling context, where light-induced LAT clustering is sufficient to drive a calcium response. Our study reveals a specific signaling function for protein clusters and identifies a biochemical circuit that robustly senses protein oligomerization state.
9.

Cross-TCR Antagonism Revealed by Optogenetically Tuning the Half-Life of the TCR Ligand Binding.

red PhyB/PIF6 Jurkat Signaling cascade control
Int J Mol Sci, 6 May 2021 DOI: 10.3390/ijms22094920 Link to full text
Abstract: Activation of T cells by agonistic peptide-MHC can be inhibited by antagonistic ones. However, the exact mechanism remains elusive. We used Jurkat cells expressing two different TCRs and tested whether stimulation of the endogenous TCR by agonistic anti-Vβ8 antibodies can be modulated by ligand-binding to the second, optogenetic TCR. The latter TCR uses phytochrome B tetramers (PhyBt) as ligand, the binding half-life of which can be altered by light. We show that this half-life determined whether the PhyBt acted as a second agonist (long half-life), an antagonist (short half-life) or did not have any influence (very short half-life) on calcium influx. A mathematical model of this cross-antagonism shows that a mechanism based on an inhibitory signal generated by early recruitment of a phosphatase and an activating signal by later recruitment of a kinase explains the data.
10.

Circularly permuted LOV2 as a modular photoswitch for optogenetic engineering.

blue AsLOV2 cpLID cpLOV2 cpLOVTRAP iLID LOVTRAP HEK293T HeLa human T cells in vitro Jurkat mouse in vivo NIH/3T3
Nat Chem Biol, 6 May 2021 DOI: 10.1038/s41589-021-00792-9 Link to full text
Abstract: Plant-based photosensors, such as the light-oxygen-voltage sensing domain 2 (LOV2) from oat phototropin 1, can be modularly wired into cell signaling networks to remotely control protein activity and physiological processes. However, the applicability of LOV2 is hampered by the limited choice of available caging surfaces and its preference to accommodate the effector domains downstream of the C-terminal Jα helix. Here, we engineered a set of LOV2 circular permutants (cpLOV2) with additional caging capabilities, thereby expanding the repertoire of genetically encoded photoswitches to accelerate the design of optogenetic devices. We demonstrate the use of cpLOV2-based optogenetic tools to reversibly gate ion channels, antagonize CRISPR-Cas9-mediated genome engineering, control protein subcellular localization, reprogram transcriptional outputs, elicit cell suicide and generate photoactivatable chimeric antigen receptor T cells for inducible tumor cell killing. Our approach is widely applicable for engineering other photoreceptors to meet the growing need of optogenetic tools tailored for biomedical and biotechnological applications.
11.

Optogenetic Control of Non-Apoptotic Cell Death.

blue cpLOV2 cpLOVTRAP CRY2/CRY2 LOVTRAP 786-O B16-F0 E. coli HEK293T HeLa Jurkat Signaling cascade control Cell death
Adv Biology, 6 May 2021 DOI: 10.1002/advs.202100424 Link to full text
Abstract: Herein, a set of optogenetic tools (designated LiPOP) that enable photoswitchable necroptosis and pyroptosis in live cells with varying kinetics, is introduced. The LiPOP tools allow reconstruction of the key molecular steps involved in these two non-apoptotic cell death pathways by harnessing the power of light. Further, the use of LiPOPs coupled with upconversion nanoparticles or bioluminescence is demonstrated to achieve wireless optogenetic or chemo-optogenetic killing of cancer cells in multiple mouse tumor models. LiPOPs can trigger necroptotic and pyroptotic cell death in cultured prokaryotic or eukaryotic cells and in living animals, and set the stage for studying the role of non-apoptotic cell death pathways during microbial infection and anti-tumor immunity.
12.

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

blue LOVTRAP HEK293T human T cells Jurkat Signaling cascade control
Mol Syst Biol, May 2021 DOI: 10.15252/msb.202010091 Link to full text
Abstract: T cells discriminate between healthy and infected cells with remarkable sensitivity when mounting an immune response, which is hypothesized to depend on T cells combining stimuli from multiple antigen-presenting cell interactions into a more potent response. To quantify the capacity for T cells to accomplish this, we have developed an antigen receptor that is optically tunable within cell conjugates, providing control over the duration, and intensity of intracellular T-cell signaling. We observe limited persistence within the T-cell intracellular network on disruption of receptor input, with signals dissipating entirely in ~15 min, and directly show sustained proximal receptor signaling is required to maintain gene transcription. T cells thus primarily accumulate the outputs of gene expression rather than integrate discrete intracellular signals. Engineering optical control in a clinically relevant chimeric antigen receptor (CAR), we show that this limited signal persistence can be exploited to increase CAR-T cell activation threefold using pulsatile stimulation. Our results are likely to apply more generally to the signaling dynamics of other cellular networks.
13.

Light-regulated voltage-gated potassium channels for acute interrogation of channel function in neurons and behavior.

blue VfAU1-LOV CHO-K1 Cos-7 HEK293 Jurkat mouse CGN Xenopus oocytes Neuronal activity control
PLoS ONE, 23 Mar 2021 DOI: 10.1371/journal.pone.0248688 Link to full text
Abstract: Voltage-gated potassium (Kv) channels regulate the membrane potential and conductance of excitable cells to control the firing rate and waveform of action potentials. Even though Kv channels have been intensely studied for over 70 year, surprisingly little is known about how specific channels expressed in various neurons and their functional properties impact neuronal network activity and behavior in vivo. Although many in vivo genetic manipulations of ion channels have been tried, interpretation of these results is complicated by powerful homeostatic plasticity mechanisms that act to maintain function following perturbations in excitability. To better understand how Kv channels shape network function and behavior, we have developed a novel optogenetic technology to acutely regulate Kv channel expression with light by fusing the light-sensitive LOV domain of Vaucheria frigida Aureochrome 1 to the N-terminus of the Kv1 subunit protein to make an Opto-Kv1 channel. Recording of Opto-Kv1 channels expressed in Xenopus oocytes, mammalian cells, and neurons show that blue light strongly induces the current expression of Opto-Kv1 channels in all systems tested. We also find that an Opto-Kv1 construct containing a dominant-negative pore mutation (Opto-Kv1(V400D)) can be used to down-regulate Kv1 currents in a blue light-dependent manner. Finally, to determine whether Opto-Kv1 channels can elicit light-dependent behavioral effect in vivo, we targeted Opto-Kv1 (V400D) expression to Kv1.3-expressing mitral cells of the olfactory bulb in mice. Exposure of the bulb to blue light for 2-3 hours produced a significant increase in sensitivity to novel odors after initial habituation to a similar odor, comparable to behavioral changes seen in Kv1.3 knockout animals. In summary, we have developed novel photoactivatable Kv channels that provide new ways to interrogate neural circuits in vivo and to examine the roles of normal and disease-causing mutant Kv channels in brain function and behavior.
14.

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.
15.

Optogenetic Tuning of Ligand Binding to The Human T cell Receptor Using The opto-ligand-TCR System.

red PhyB/PIF6 Jurkat
Bio Protoc, 5 Mar 2020 DOI: 10.21769/bioprotoc.3540 Link to full text
Abstract: T cells are one major cell type of the immune system that use their T cell antigen receptor (TCR) to bind and respond to foreign molecules derived from pathogens. The ligand-TCR interaction half-lives determine stimulation outcome. Until recently, scientists relied on mutating either the TCR or its ligands to investigate how varying TCR-ligand interaction durations impacted on T cell activation. Our newly created opto-ligand-TCR system allowed us to precisely and reversibly control ligand binding to the TCR by light illumination. This system uses phytochrome B (PhyB) tetramers as a light-regulated TCR ligand. PhyB can be photoconverted between a binding (ON) and non-binding (OFF) conformation by 660 nm and 740 nm light illumination, respectively. PhyB ON is able to bind to a synthetic TCR, generated by fusing the PhyB interacting factor (PIF) to the TCRβ chain. Switching PhyB to the OFF conformation disrupts this interaction. Sufficiently long binding of PhyB tetramers to the PIF-TCR led to T cell activation as measured by calcium influx. Here, we describe protocols for how to generate the tetrameric ligand for our opto-ligand-TCR system, how to measure ligand-TCR binding by flow cytometry and how to quantify T cell activation via calcium influx.
16.

Engineering light-controllable CAR T cells for cancer immunotherapy.

blue AsLOV2 CRY2/CIB1 HEK293T human T cells Jurkat mouse in vivo Endogenous gene expression
Sci Adv, 19 Feb 2020 DOI: 10.1126/sciadv.aay9209 Link to full text
Abstract: T cells engineered to express chimeric antigen receptors (CARs) can recognize and engage with target cancer cells with redirected specificity for cancer immunotherapy. However, there is a lack of ideal CARs for solid tumor antigens, which may lead to severe adverse effects. Here, we developed a light-inducible nuclear translocation and dimerization (LINTAD) system for gene regulation to control CAR T activation. We first demonstrated light-controllable gene expression and functional modulation in human embryonic kidney 293T and Jurkat T cell lines. We then improved the LINTAD system to achieve optimal efficiency in primary human T cells. The results showed that pulsed light stimulations can activate LINTAD CAR T cells with strong cytotoxicity against target cancer cells, both in vitro and in vivo. Therefore, our LINTAD system can serve as an efficient tool to noninvasively control gene activation and activate inducible CAR T cells for precision cancer immunotherapy.
17.

An AND-Gated Drug and Photoactivatable Cre-loxP System for Spatiotemporal Control in Cell-Based Therapeutics.

blue Magnets HEK293T Jurkat
ACS Synth Biol, 8 Oct 2019 DOI: 10.1021/acssynbio.9b00175 Link to full text
Abstract: While engineered chimeric antigen receptor (CAR) T cells have shown promise in detecting and eradicating cancer cells within patients, it remains difficult to identify a set of truly cancer-specific CAR-targeting cell surface antigens to prevent potentially fatal on-target off-tumor toxicity against other healthy tissues within the body. To help address this issue, we present a novel tamoxifen-gated photoactivatable split-Cre recombinase optogenetic system, called TamPA-Cre, that features high spatiotemporal control to limit CAR T cell activity to the tumor site. We created and optimized a novel genetic AND gate switch by integrating the features of tamoxifen-dependent nuclear localization and blue-light-inducible heterodimerization of Magnet protein domains (nMag, pMag) into split Cre recombinase. By fusing the cytosol-localizing mutant estrogen receptor ligand binding domain (ERT2) to the N-terminal half of split Cre(2-59aa)-nMag, the TamPA-Cre protein ERT2-CreN-nMag is physically separated from its nuclear-localized binding partner, NLS-pMag-CreC(60-343aa). Without tamoxifen to drive nuclear localization of ERT2-CreN-nMag, the typically high background of the photoactivation system was significantly suppressed. Upon blue light stimulation following tamoxifen treatment, the TamPA-Cre system exhibits sensitivity to low intensity, short durations of blue light exposure to induce robust Cre-loxP recombination efficiency. We finally demonstrate that this TamPA-Cre system can be applied to specifically control localized CAR expression and subsequently T cell activation. As such, we posit that CAR T cell activity can be confined to a solid tumor site by applying an external stimulus, with high precision of control in both space and time, such as light.
18.

Flotillins promote T cell receptor sorting through a fast Rab5-Rab11 endocytic recycling axis.

blue CRY2/CIB1 CRY2clust Jurkat Control of vesicular transport Organelle manipulation
Nat Commun, 26 Sep 2019 DOI: 10.1038/s41467-019-12352-w Link to full text
Abstract: The targeted endocytic recycling of the T cell receptor (TCR) to the immunological synapse is essential for T cell activation. Despite this, the mechanisms that underlie the sorting of internalised receptors into recycling endosomes remain poorly understood. To build a comprehensive picture of TCR recycling during T cell activation, we developed a suite of new imaging and quantification tools centred on photoactivation of fluorescent proteins. We show that the membrane-organising proteins, flotillin-1 and -2, are required for TCR to reach Rab5-positive endosomes immediately after endocytosis and for transfer from Rab5- to Rab11a-positive compartments. We further observe that after sorting into in Rab11a-positive vesicles, TCR recycles to the plasma membrane independent of flotillin expression. Our data suggest a mechanism whereby flotillins delineate a fast Rab5-Rab11a endocytic recycling axis and functionally contribute to regulate the spatial organisation of these endosomes.
19.

Optogenetic control shows that kinetic proofreading regulates the activity of the T cell receptor.

red PhyB/PIF6 Jurkat Signaling cascade control Immediate control of second messengers
Elife, 5 Apr 2019 DOI: 10.7554/elife.42475 Link to full text
Abstract: The immune system distinguishes between self and foreign antigens. The kinetic proofreading (KPR) model proposes that T cells discriminate self from foreign ligands by the different ligand binding half-lives to the T cell receptor (TCR). It is challenging to test KPR as the available experimental systems fall short of only altering the binding half-lives and keeping other parameters of the interaction unchanged. We engineered an optogenetic system using the plant photoreceptor phytochrome B (PhyB) as a ligand to selectively control the dynamics of ligand binding to the TCR by light. This opto-ligand-TCR system was combined with the unique property of PhyB to continuously cycle between the binding and non-binding states under red light, with the light intensity determining the cycling rate and thus the binding duration. Mathematical modeling of our experimental datasets showed that indeed the ligand-TCR interaction half-life is the decisive factor for activating downstream TCR signaling, substantiating KPR.
20.

Light-based tuning of ligand half-life supports kinetic proofreading model of T cell signaling.

blue LOVTRAP Jurkat Signaling cascade control
Elife, 5 Apr 2019 DOI: 10.7554/elife.42498 Link to full text
Abstract: T cells are thought to discriminate self from foreign peptides by converting small differences in ligand binding half-life into large changes in cell signaling. Such a kinetic proofreading model has been difficult to test directly, as existing methods of altering ligand binding half-life also change other potentially important biophysical parameters, most notably the mechanical stability of the receptor-ligand interaction. Here we develop an optogenetic approach to specifically tune the binding half-life of a chimeric antigen receptor without changing other binding parameters and provide direct evidence of kinetic proofreading in T cell signaling. This half-life discrimination is executed in the proximal signaling pathway, downstream of ZAP70 recruitment and upstream of diacylglycerol accumulation. Our methods represent a general tool for temporal and spatial control of T cell signaling and extend the reach of optogenetics to probe pathways where the individual molecular kinetics, rather than the ensemble average, gates downstream signaling.
21.

A mobile endocytic network connects clathrin-independent receptor endocytosis to recycling and promotes T cell activation.

blue CRY2/CIB1 CRY2clust Jurkat Signaling cascade control
Nat Commun, 23 Apr 2018 DOI: 10.1038/s41467-018-04088-w Link to full text
Abstract: Endocytosis of surface receptors and their polarized recycling back to the plasma membrane are central to many cellular processes, such as cell migration, cytokinesis, basolateral polarity of epithelial cells and T cell activation. Little is known about the mechanisms that control the organization of recycling endosomes and how they connect to receptor endocytosis. Here, we follow the endocytic journey of the T cell receptor (TCR), from internalization at the plasma membrane to recycling back to the immunological synapse. We show that TCR triggering leads to its rapid uptake through a clathrin-independent pathway. Immediately after internalization, TCR is incorporated into a mobile and long-lived endocytic network demarked by the membrane-organizing proteins flotillins. Although flotillins are not required for TCR internalization, they are necessary for its recycling to the immunological synapse. We further show that flotillins are essential for T cell activation, supporting TCR nanoscale organization and signaling.
22.

An optogenetic gene expression system with rapid activation and deactivation kinetics.

blue EL222 HEK293T Jurkat zebrafish in vivo Transgene expression
Nat Chem Biol, 12 Jan 2014 DOI: 10.1038/nchembio.1430 Link to full text
Abstract: Optogenetic gene expression systems can control transcription with spatial and temporal detail unequaled with traditional inducible promoter systems. However, current eukaryotic light-gated transcription systems are limited by toxicity, dynamic range or slow activation and deactivation. Here we present an optogenetic gene expression system that addresses these shortcomings and demonstrate its broad utility. Our approach uses an engineered version of EL222, a bacterial light-oxygen-voltage protein that binds DNA when illuminated with blue light. The system has a large (>100-fold) dynamic range of protein expression, rapid activation (<10 s) and deactivation kinetics (<50 s) and a highly linear response to light. With this system, we achieve light-gated transcription in several mammalian cell lines and intact zebrafish embryos with minimal basal gene activation and toxicity. Our approach provides a powerful new tool for optogenetic control of gene expression in space and time.
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