The authors acknowledge Dr. Mark Shaaya for his contribution to the development of LightR enzymes and associated protocols. pCAG-iCre was a gift from Wilson Wong (Addgene plasmid #89573), pcDNA3.1 Floxed-STOP mCherry was a gift from Mositoshi Sato (Addgene plasmid #122963), bRaf-Venus construct bearing V600E mutation was a gift from Dr. John O&#39;Bryan (MUSC); ERK2 gene from pCEFL-ERK2 (a gift from Dr. Channing Der&#39;s lab, UNC) was cloned into mCherry-C1 backbone to obtain mCherry-ERK2 plasmid; and pmiRFP670-N1 was a gift from Vladislav Verkhusha (Addgene plasmid # 79987). The work was supported by NIH grants R33CA258012, R35GM145318, and P01HL151327 to AK. This work was further supported by the T32 VBST training fellowship T32HL144459 to NL.

Optogenetic Control of Enzyme Activity Using Light-Regulated Allosteric Switch

The authors have nothing to disclose.

Our study presents an optogenetic approach for the investigation of diverse signaling pathways and demonstrates its wide applicability in addressing different biological questions. The LightR tool system provides several essential advantages: (1) Allosteric regulation of protein activity, (2) Tight temporal control of activity that can be tuned to achieve different kinetics of activation and inactivation, (3) Spatial resolution of activity at the subcellular level, (4) Specificity of signaling modulation and biological a...

The ability of the cell to interpret external signals and convert them into specific responses in physiological or pathological contexts is directed by dedicated groups of proteins. The contribution of any protein to such complex responses is often defined by its subcellular location, level of expression, and the timing of transient, sustained, or oscillatory activation. Dissecting the role of these individual parameters in the regulation of signaling demands methods capable of replicating intricate spatiotemporal control of protein activity at the subcellular level. Traditional techniques such as genetic manipulation and small molecule inhibitors fall short in this regard. In contrast, optogenetic techniques promise the potential for the dissection of biological processes by manipulating or mimicking physiological and pathological processes. However, current tools often lack broad applicability or subcellular control. Several existing strategies achieve tight regulation of protein localization and interactions; but lack direct control over enzymatic activity1,2,3,4. Others enable regulation of enzymatic activity but may lack subcellular control or have limited applicability across different enzyme classes5,6,7,8,9. In this protocol paper, we describe a novel protein engineering method that combines the advantages of optogenetics into one tool: tight temporal regulation, tunable kinetics, subcellular control, and broad enzyme applicability10. We engineered a Light-regulated (LightR) domain that functions as an allosteric switch when inserted into the target protein of interest. This strategy enables tight spatiotemporal control of the activation and deactivation of a protein of interest in living cells.
Here, the design and application strategy for the LightR optogenetic tool across several enzyme classes is discussed. This study offers a step-by-step protocol for the development, characterization, and application of light-regulated tyrosine kinase Src (LightR-Src). The study further demonstrates the tunability of LightR switch inactivation kinetics. The slow-inactivating LightR switch can maintain enzyme activity with reduced frequency of illumination, whereas fast cycling version, FastLightR, requires more frequent illumination for activation but shows fast inactivation when illumination is turned off. Activation/inactivation of FastLightR-Src in living cells induces cycles of cell spreading and retraction. FastLightR-Src-induced cell spreading agrees with the physiological role of Src kinase11,12. Due to fast inactivation kinetics, FastLightR-Src can also be regulated at a subcellular level, resulting in stimulation of local protrusions and cell polarization. To demonstrate the applicability of the LightR tool to other types of enzymes, we briefly walk the readers through similar success with engineered light-regulated kinase bRaf (LightR-bRaf, FastLightR-bRaf) and a site-specific DNA recombinase Cre (LightR-Cre). Overall, this approach has the potential to advance our understanding of complex signaling pathways shaping the pathophysiology of diverse diseases.

<table><tbody><tr><td>#1.5 Glass Coverslips 25 mm Round</td><td>Warner Instruments&amp;nbsp;</td><td>64-0715&amp;nbsp;</td><td></td></tr><tr><td>1.5 mL Tubes&amp;nbsp;</td><td>USA Scientific&amp;nbsp;</td><td>cc7682-3394</td><td></td></tr><tr><td>2x Laemmli Buffer</td><td></td><td></td><td>For 500 mL: 5.18 g of&amp;nbsp; Tris-HCL, 131.5 mL of glycerol, 52.5 mL of 20% SDS, 0.5 g of bromophenol blue, final pH 6.8</td></tr><tr><td>4-20% Mini-PROTEAN TGX Precast Gel</td><td>Biorad</td><td>4561096</td><td></td></tr><tr><td>5x Phusion Plus Buffer&amp;nbsp;</td><td>Thermo Scientific&amp;nbsp;</td><td>F538L</td><td></td></tr><tr><td>60 LED Microscope Ring Light</td><td>Boli Optics</td><td>ML46241324</td><td>Blue LED, 60 mm diameter, 5 W</td></tr><tr><td>Agarose&amp;nbsp;</td><td>GoldBiotech</td><td>A-201</td><td></td></tr><tr><td>Anti-Erk 1/2 Antibody</td><td>Cell Signaling</td><td>9102</td><td></td></tr><tr><td>Anti-GAPDH Antibody</td><td>invitrogen</td><td>AM4300</td><td></td></tr><tr><td>Anti-GFP</td><td>Clontech</td><td>632380</td><td></td></tr><tr><td>Anti-mCherry Antibody</td><td>invitrogen</td><td>M11217</td><td></td></tr><tr><td>Anti-MEK</td><td>Cell Signaling</td><td>9122</td><td></td></tr><tr><td>Anti-p130Cas</td><td>BD Biosciences</td><td>610271</td><td></td></tr><tr><td>Anti-paxillin</td><td>Cell Signaling</td><td>2542</td><td></td></tr><tr><td>Anti-phospho-Erk 1/2 T202/Y204 Antibody</td><td>Cell Signaling</td><td>9101</td><td></td></tr><tr><td>Anti-phospho-pY249 p130Cas</td><td>Cell Signaling</td><td>4014</td><td></td></tr><tr><td>Anti-phospho-Y118 Paxillin</td><td>Cell Signaling</td><td>2541</td><td></td></tr><tr><td>Anto-phospho-S217/221 MEK</td><td>Cell Signaling</td><td>9121</td><td></td></tr><tr><td>Arduino Compatable Power Supply</td><td>Corporate Computer</td><td>LJH -186</td><td></td></tr><tr><td>Arduino Uno Rev3</td><td>Arduino</td><td>&amp;lrm;A000066</td><td></td></tr><tr><td>Attofluor Cell Chamber</td><td>invitrogen</td><td>A7816</td><td></td></tr><tr><td>Benchmark Fetal Bovine Serum (FBS)&amp;nbsp;</td><td>Gemini Bio-products</td><td>100-106</td><td>Heat Inactivated Triple 0.1 &amp;micro;m sterile-filtered&amp;nbsp;</td></tr><tr><td>bRaf-V600E-Venus</td><td></td><td></td><td>Gift from Dr. John O&#039;Bryan, MUSC</td></tr><tr><td>BSA&amp;nbsp;</td><td>GoldBiotech</td><td>A-420&amp;nbsp;</td><td></td></tr><tr><td>Carbenicillin (Disodium)</td><td>Gold Biotechnology</td><td>C-103-25</td><td></td></tr><tr><td>CellMask Deep Red plasma membrane dye</td><td>invitrogen</td><td>c10046</td><td></td></tr><tr><td>Colony Screen MasterMix&amp;nbsp;</td><td>Genesee</td><td>42-138</td><td></td></tr><tr><td>DH5a competent cells&amp;nbsp;</td><td>NEB&amp;nbsp;</td><td>C2987H</td><td></td></tr><tr><td>DMEM&amp;nbsp;</td><td>Corning&amp;nbsp;</td><td>15-013-CV</td><td></td></tr><tr><td>DNA Ladder&amp;nbsp;</td><td>GoldBio&amp;nbsp;</td><td>D010-500</td><td></td></tr><tr><td>dNTPs&amp;nbsp;</td><td>NEB</td><td>N04475</td><td></td></tr><tr><td>Dpn1 Enzyme&amp;nbsp;</td><td>NEB</td><td>R01765</td><td></td></tr><tr><td>DTT</td><td>GoldBio</td><td>DTT10</td><td>DL-Dithiothreitol, Cleland&#039;s Reagents&amp;nbsp;</td></tr><tr><td>EGTA</td><td>Acros&amp;nbsp;</td><td>409910250</td><td></td></tr><tr><td>FastLightR-bRaf-mVenus</td><td>Addgene</td><td>#162155</td><td></td></tr><tr><td>Fibronectin from bovine plasma&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>F1141</td><td></td></tr><tr><td>FuGENE(R) 6 Transfection Reagent&amp;nbsp;</td><td>Promega</td><td>E2692</td><td>Transfection reagent</td></tr><tr><td>Gel Green Nucleic Acid Stain&amp;nbsp;</td><td>GoldBio</td><td>G-740-500</td><td></td></tr><tr><td>Gel Loading Dye Purple 6x&amp;nbsp;</td><td>NEB</td><td>B7024A</td><td></td></tr><tr><td>GeneJET Gel extraction Kit&amp;nbsp;</td><td>Thermo Scientific&amp;nbsp;</td><td>K0692</td><td>Gel Extraction Kit&amp;nbsp;</td></tr><tr><td>GeneJET Plasmid Miniprep Kit</td><td>Thermo Scientific&amp;nbsp;</td><td>K0502</td><td></td></tr><tr><td>Glutamax</td><td>Gibco</td><td>35050-061</td><td>GlutaMAX-l (100x) 100 mL&amp;nbsp;</td></tr><tr><td>HEK 293T Cells&amp;nbsp;</td><td>ATCC&amp;nbsp;</td><td>CRL-11268</td><td></td></tr><tr><td>HeLa Cells&amp;nbsp;</td><td>ATCC&amp;nbsp;</td><td>CRM-CCL-2</td><td></td></tr><tr><td>HEPES</td><td>Fischer&amp;nbsp;</td><td>BP310-500</td><td></td></tr><tr><td>Iot Relay</td><td>Digital Loggers</td><td>DLI 705020645490</td><td>AC/DC control relay for illumination</td></tr><tr><td>Kanamycin Monosulfate</td><td>Gold Biotechnology</td><td>K-120-25</td><td></td></tr><tr><td>KCl</td><td>Sigma&amp;nbsp;</td><td>P-4504</td><td></td></tr><tr><td>L-15 1x</td><td>Corning&amp;nbsp;</td><td>10-045-CV&amp;nbsp;</td><td></td></tr><tr><td>LB Agar&amp;nbsp;</td><td>Fisher</td><td>BP1425-2</td><td></td></tr><tr><td>LED Grow Light System</td><td>HQRP</td><td>884667106091218</td><td>LED panel lamp system&amp;nbsp;</td></tr><tr><td>LightR-bRaf-mVenus</td><td>Addgene</td><td>#162154</td><td></td></tr><tr><td>LightR-iCre-miRFP670</td><td>Addgene</td><td>#162158</td><td></td></tr><tr><td>MATLAB</td><td>Mathworks</td><td>R2024a</td><td>Software for running CellGEO Scripts</td></tr><tr><td>Metamorph</td><td>Molecular Devices</td><td></td><td>Imaging Analysis Software</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;&amp;nbsp;</td><td>Fisher Chemical</td><td>M33-500</td><td></td></tr><tr><td>Mineral Oil</td><td>Sigma&amp;nbsp;</td><td>M5310</td><td></td></tr><tr><td>MiniPrep Kit&amp;nbsp;</td><td>Gene Choice&amp;nbsp;</td><td>96-308</td><td></td></tr><tr><td>Mini-PROTEAN TGX Precast Gels 12 well</td><td>Bio-Rad</td><td>4561085</td><td></td></tr><tr><td>Molecular Biology Grade Water&amp;nbsp;</td><td>Corning&amp;nbsp;</td><td>46-000-CV&amp;nbsp;</td><td></td></tr><tr><td>Multiband Polychroic Mirror</td><td>89903BS</td><td>Chroma</td><td></td></tr><tr><td>NaCl&amp;nbsp;</td><td>Fisher Chemical</td><td>S271-3&amp;nbsp;</td><td></td></tr><tr><td>PBS w/o Ca and Mg&amp;nbsp;</td><td>Corning&amp;nbsp;</td><td>21-031-CV</td><td></td></tr><tr><td>pCAG-iCre</td><td>Addgene</td><td>#89573</td><td></td></tr><tr><td>pcDNA3.1_Floxed-STOP mCherry</td><td>Addgene</td><td>#122963</td><td></td></tr><tr><td>pCEFL-ERK2</td><td></td><td></td><td>Gift from Dr. Channing Der&#039;s Lab, UNC</td></tr><tr><td>PCR Tubes&amp;nbsp;</td><td>labForce</td><td>1149Z65</td><td>0.2 mL 8-Strip Tubes and Caps, Rigid Strip Individually Attached Dome Caps&amp;nbsp;</td></tr><tr><td>Phusion Plus DNA Polymerase</td><td>Thermo Scientific&amp;nbsp;</td><td>F630S</td><td></td></tr><tr><td>pmiRFP670-N1</td><td>Addgene</td><td>#79987</td><td></td></tr><tr><td>Polygon 400 Patterned Illuminator</td><td>Mightex</td><td>DSI-G-00C</td><td></td></tr><tr><td>Primers&amp;nbsp;</td><td>IDT</td><td></td><td></td></tr><tr><td>PVDF Membranes</td><td>BioRad</td><td>1620219</td><td>Immun-Blot PVDF/Filter Paper Sandwiches&amp;nbsp;</td></tr><tr><td>T0.25% Trypsin, 2.21 mM, eDTA, 1x [-] sodium</td><td>Corning&amp;nbsp;</td><td>25-053-CI</td><td></td></tr><tr><td>Tris-Acetate-EDTA (TAE) 50x&amp;nbsp;</td><td>Fischer&amp;nbsp;</td><td>BP1332-1&amp;nbsp;</td><td>For electrophoresis&amp;nbsp;</td></tr><tr><td>UPlanSApo 40x Microscope Objective</td><td>Olympus</td><td>1-U2B828</td><td></td></tr><tr><td>USB TTL Box</td><td>National Instruments</td><td>6501</td><td>For TTL interface</td></tr><tr><td>&amp;beta;-Mercaptoethanol</td><td>Fisher Chemical&amp;nbsp;</td><td>O3446I-100</td><td></td></tr></tbody></table>

spatiotemporal control of protein activity through optogenetic allosteric regulation

Optogenetics offers the potential for mimicking complex spatiotemporal control of enzyme activity down to a subcellular resolution. However, most optogenetic approaches often face significant challenges in integrating multiple capabilities in a single tool applicable to a wide range of target proteins. Achieving precise control over ON/OFF kinetics, ensuring minimum leakiness in the dark, and demonstrating efficient performance in mammalian cells with subcellular precision are some of the most common challenges faced in this field. A promising solution lies in the application of rationally designed light-sensitive domains to allosterically control protein activity. Using that strategy, we generated an optogenetic method combining all the desired features. The approach involves the incorporation of the Light-regulated allosteric switch module (LightR) in the target protein to regulate enzyme activity using blue (465 nm) light. The LightR domain is generated by linking two Vivid (VVD) photoreceptor domains, creating a light-sensitive clamp that can be incorporated into a small flexible loop within the catalytic domain of an enzyme. In its dark state, LightR clamp is open, thus distorting the enzyme&#39;s catalytic domain and inactivating it. Upon exposure to blue light, the LightR domain closes and restores the catalytic domain&#39;s structure and enzyme activity. In this manuscript, we discuss design strategies&#160;to generate a light-regulated protein kinase and demonstrate its control by blue light, reversibility, kinetics, and precise regulation at the subcellular level, enabling tight spatiotemporal precision. Utilizing Src tyrosine kinase as a model, we showcase a protocol for effectively regulating LightR-Src kinase activity. We also demonstrate LightR applicability across different enzyme classes, expanding the utility of the tool system in addressing mechanistic questions of signaling pathways in different diseases.

The LightR-Src is designed and generated following the strategy described in Figure 1A,B. Biochemical analysis of LightR-Src accesses the phosphorylation of known endogenous Src substrates, p130Cas (Y249)22 and paxillin (Y118)23 in response to blue light at 60 min of continuous illumination (Figure 2B). Notably, no background activation of Src kinase is observed when LightR-Src is expressed only in...

Spatiotemporal Control of Protein Activity through Optogenetic Allosteric Regulation

This protocol describes the application of an engineered blue-light-activated allosteric switch (LightR) domain for reversible spatiotemporal control of protein activity. Utilizing Src tyrosine kinase as a model, this study offers an elaborate protocol for developing and characterizing light-regulated Src (LightR-Src). It demonstrates the versatility of this approach across enzyme classes.

Optogenetics offers the potential for mimicking complex spatiotemporal control of enzyme activity down to a subcellular resolution. However, most ...

spatiotemporal-control-protein-activity-through-optogenetic

Research

JoVE Journal

Biology

484 Views.  University of Illinois at Chicago. Ons algemene onderzoek richt zich op het ontwikkelen van optogenetische methoden die een nauwkeurige spatiotemporele controle van eiwitactiviteit door licht bereiken. We ontwikkelen lichtreguleerbare domeinen, lightR genaamd, voor allosterische regulatie, waardoor we kunnen bestuderen hoe spatiotemporeel gecontroleerde eiwitactiviteit de cellulaire signalering en functies beïnvloedt, wat helpt bij preklinische ziektemodellering en therapieontwikkeling. Optog...

Video: Spatiotemporele controle van eiwitactiviteit door optogenetische allosterische regulatie

Spatio-Temporal Manipulation of Small GTPase Activity at Subcellular Level and on Timescale of Seconds in Living Cells

Dynamic regulation of the Rho family of small guanosine triphosphatases (GTPases) with great spatiotemporal precision is essential for various cellular functions and events1, 2. Their spatiotemporally dynamic nature has been revealed by visualization of their activity and localization in real time3. In order to gain deeper understanding of their roles in diverse cellular functions at the molecular level, the next step should be perturbation of protein activities at a precise subcellular location and timing. 
To achieve this goal, we have developed a method for light-induced, spatio-temporally controlled activation of small GTPases by combining two techniques: (1) rapamycin-induced FKBP-FRB heterodimerization and (2) a photo-caging method of rapamycin. With the use of rapamycin-mediated FKBP-FRB heterodimerization, we have developed a method for rapidly inducible activation or inactivation of small GTPases including Rac4, Cdc424, RhoA4 and Ras5, in which rapamycin induces translocation of FKBP-fused GTPases, or their activators, to the plasma membrane where FRB is anchored. For coupling with this heterodimerization system, we have also developed a photo-caging system of rapamycin analogs. A photo-caged compound is a small molecule whose activity is suppressed with a photocleavable protecting group known as a caging group. To suppress heterodimerization activity completely, we designed a caged rapamycin that is tethered to a macromolecule such that the resulting large complex cannot cross the plasma membrane, leading to virtually no background activity as a chemical dimerizer inside cells6. Figure 1 illustrates a scheme of our system. With the combination of these two systems, we locally recruited a Rac activator to the plasma membrane on a timescale of seconds and achieved light-induced Rac activation at the subcellular level6.

A method for spatio-temporal control of small GTPase activity by light is described. This method is based on rapamycin-induced FKBP-FRB heterodimerization and photo-caging systems. Optimization of light-irradiation enables the spatio-temporally controlled activation of small GTPases at the subcellular level.

Dynamic regulation of the Rho family of small guanosine triphosphatases (GTPases) with great spatiotemporal precision is essential for various cellular ...

Bioengineering

An Optogenetic Method to Control and Analyze Gene Expression Patterns in Cell-to-cell Interactions

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling pathway is one of such essential molecular machinery for cell-to-cell communications, which plays key roles in normal development of embryos. This pathway involves a cell-to-cell transfer of oscillatory information with ultradian rhythms, but despite the progress in molecular biology techniques, it has been challenging to elucidate the impact of multicellular interactions on oscillatory gene dynamics. Here, we present a protocol that permits optogenetic control and live monitoring of gene expression patterns in a precise temporal manner. This method successfully revealed that intracellular and intercellular periodic inputs of Notch signaling entrain intrinsic oscillations by frequency tuning and phase shifting at the single-cell resolution. This approach is applicable to the analysis of the dynamic features of various signaling pathways, providing a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

Here, we present a protocol to analyze cell-to-cell transfer of oscillatory information by optogenetic control and live monitoring of gene expression. This approach provides a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling ...

Genetics

Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development

Kinase activity is crucial for a plethora of cellular functions, including cell proliferation, differentiation, migration, and apoptosis. During early embryonic development, kinase activity is highly dynamic and widespread across the embryo. Pharmacological and genetic approaches are commonly used to probe kinase activities. Unfortunately, it is challenging to achieve superior spatial and temporal resolution using these strategies. Furthermore, it is not feasible to control the kinase activity in a reversible fashion in live cells and multicellular organisms. Such a limitation remains a bottleneck for achieving a quantitative understanding of kinase activity during development and differentiation. This work presents an optogenetic strategy that takes advantage of a bicistronic system containing photoactivatable proteins Arabidopsis thaliana cryptochrome 2 (CRY2) and the N-terminal domain of cryptochrome-interacting basic-helix-loop-helix (CIBN). Reversible activation of the mitogen-activated protein kinase (MAPK) signaling pathway is achieved through light-mediated protein translocation in live cells. This approach can be applied to mammalian cell cultures and live vertebrate embryos. This bicistronic system can be generalized to control the activity of other kinases with similar activation mechanisms and can be applied to other model systems.

Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development

This protocol describes an optogenetic strategy to modulate mitogen-activated protein kinase (MAPK) activity during cell differentiation and Xenopus embryonic development. This method allows for the reversible activation of the MAPK signaling pathway in mammalian cell culture and in multicellular live organisms, like Xenopus embryos, with high spatial and temporal resolution.

Kinase activity is crucial for a plethora of cellular functions, including cell proliferation, differentiation, migration, and apoptosis. During early ...

Developmental Biology

Design and Implementation of an Automated Illuminating, Culturing, and Sampling System for Microbial Optogenetic Applications

Optogenetic systems utilize genetically-encoded proteins that change conformation in response to specific wavelengths of light to alter cellular processes. There is a need for culturing and measuring systems that incorporate programmed illumination and stimulation of optogenetic systems. We present a protocol for building and using a continuous culturing apparatus to illuminate microbial cells with programmed doses of light, and automatically acquire and analyze images of cells in the effluent. The operation of this apparatus as a chemostat allows the growth rate and the cellular environment to be tightly controlled. The effluent of the continuous cell culture is regularly sampled and the cells are imaged by multi-channel microscopy. The culturing, sampling, imaging, and image analysis are fully automated so that dynamic responses in the fluorescence intensity and cellular morphology of cells sampled from the culture effluent are measured over multiple days without user input. We demonstrate the utility of this culturing apparatus by dynamically inducing protein production in a strain of Saccharomyces cerevisiae engineered with an optogenetic system that activates transcription.

We designed a continuous culturing apparatus for use with optogenetic systems to illuminate cultures of microbes and regularly image cells in the effluent with an inverted microscope. The culturing, sampling, imaging, and image analysis are fully automated so that dynamic responses to illumination can be measured over several days.

Optogenetic systems utilize genetically-encoded proteins that change conformation in response to specific wavelengths of light to alter cellular ...

Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to photo-regulate endogenous proteins expressed in their native environment. Here, we present an approach to optically control the function of a neuronal protein directly in neurons using a genetically encoded unnatural amino acid (Uaa). By using an orthogonal tRNA/aminoacyl-tRNA synthetase pair to suppress the amber codon, a photo-reactive Uaa 4,5-dimethoxy-2-nitrobenzyl-cysteine (Cmn) is site-specifically incorporated in the pore of a neuronal protein Kir2.1, an inwardly rectifying potassium channel. The bulky Cmn physically blocks the channel pore, rendering Kir2.1 non-conducting. Light illumination instantaneously converts Cmn into a smaller natural amino acid Cys, activating Kir2.1 channel function. We express these photo-inducible inwardly rectifying potassium (PIRK) channels in rat hippocampal primary neurons, and demonstrate that light-activation of PIRK ceases the neuronal firing due to the outflux of K+ current through the activated Kir2.1 channels. Using in utero electroporation, we also express PIRK in the embryonic mouse neocortex in vivo, showing the light-activation of PIRK in neocortical neurons. Genetically encoding Uaa imposes no restrictions on target protein type or cellular location, and a family of photoreactive Uaas is available for modulating different natural amino acid residues. This technique thus has the potential to be generally applied to many neuronal proteins to achieve optical regulation of different processes in brains. The current protocol presents an accessible procedure for intricate Uaa incorporation in neurons in vitro and in vivo to achieve photo control of neuronal protein activity on the molecular level.

Here, a procedure to selectively activate a neuronal protein with a short pulse of light by genetically encoding a photo-reactive unnatural amino acid into a target neuronal protein expressed in neurons in culture or in vivo is presented.

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to ...

Neuroscience

Spatial and Temporal Control of T Cell Activation Using a Photoactivatable Agonist

T lymphocytes engage in rapid, polarized signaling, occurring within minutes following TCR activation. This induces formation of the immunological synapse, a stereotyped cell-cell junction that regulates T cell activation and directionally targets effector responses. To study these processes effectively, an imaging approach that is tailored to capturing fast, polarized responses is necessary. This protocol describes such a system, which is based on a photoactivatable peptide-major histocompatibility complex (pMHC) that is non-stimulatory until it is exposed to ultraviolet light. Targeted decaging of this reagent during videomicroscopy experiments enables precise spatiotemporal control of TCR activation and high-resolution monitoring of subsequent cellular responses by total internal reflection (TIRF) imaging. This approach is also compatible with genetic and pharmacological perturbation strategies. This allows for the assembly of well-defined molecular pathways that link TCR signaling to the formation of the polarized cytoskeletal structures that underlie the immunological synapse.

This protocol describes an imaging-based method to activate T lymphocytes using photoactivatable peptide-MHC, enabling precise spatiotemporal control of T cell activation.

T lymphocytes engage in rapid, polarized signaling, occurring within minutes following TCR activation. This induces formation of the immunological ...

Immunology and Infection

Reliably Engineering and Controlling Stable Optogenetic Gene Circuits in Mammalian Cells

Reliable gene expression control in mammalian cells requires tools with high fold change, low noise, and determined input-to-output transfer functions, regardless of the method used. Toward this goal, optogenetic gene expression systems have gained much attention over the past decade for spatiotemporal control of protein levels in mammalian cells. However, most existing circuits controlling light-induced gene expression vary in architecture, are expressed from plasmids, and utilize variable optogenetic equipment, creating a need to explore characterization and standardization of optogenetic components in stable cell lines. Here, the study provides an experimental pipeline of reliable gene circuit construction, integration, and characterization for controlling light-inducible gene expression in mammalian cells, using a negative feedback optogenetic circuit as a case example. The protocols also illustrate how standardizing optogenetic equipment and light regimes can reliably reveal gene circuit features such as gene expression noise and protein expression magnitude. Lastly, this paper may be of use for laboratories unfamiliar with optogenetics who wish to adopt such technology. The pipeline described here should apply for other optogenetic circuits in mammalian cells, allowing for more reliable, detailed characterization and control of gene expression at the transcriptional, proteomic, and ultimately phenotypic level in mammalian cells.

Reliably controlling light-responsive mammalian cells requires the standardization of optogenetic methods. Toward this goal, this study outlines a pipeline of gene circuit construction, cell engineering, optogenetic equipment operation, and verification assays to standardize the study of light-induced gene expression using a negative-feedback optogenetic gene circuit as a case study.

Reliable gene expression control in mammalian cells requires tools with high fold change, low noise, and determined input-to-output transfer functions, ...

Light-Controlled Fermentations for Microbial Chemical and Protein Production

Microbial cell factories offer a sustainable alternative for producing chemicals and recombinant proteins from renewable feedstocks. However, overburdening a microorganism with genetic modifications can reduce host fitness and productivity. This problem can be overcome by using dynamic control: inducible expression of enzymes and pathways, typically using chemical- or nutrient-based additives, to balance cellular growth and production. Optogenetics offers a non-invasive, highly tunable, and reversible method of dynamically regulating gene expression. Here, we describe how to set up light-controlled fermentations of engineered Escherichia coli and Saccharomyces cerevisiae for the production of chemicals or recombinant proteins. We discuss how to apply light at selected times and dosages to decouple microbial growth and production for improved fermentation control and productivity, as well as the key optimization considerations for best results. Additionally, we describe how to implement light controls for lab-scale bioreactor experiments. These protocols facilitate the adoption of optogenetic controls in engineered microorganisms for improved fermentation performance.

Optogenetic control of microbial metabolism offers flexible dynamic control over fermentation processes. The protocol here shows how to set up blue light-regulated fermentations for chemical and protein production at different volumetric scales.

Microbial cell factories offer a sustainable alternative for producing chemicals and recombinant proteins from renewable feedstocks. However, ...

Yeast Optogenetic Response Analysis Using the Lustro Automation Technique

High-Throughput Optogenetics Experiments in Yeast Using the Automated Platform Lustro

Optogenetics offers precise control over cellular behavior by utilizing genetically encoded light-sensitive proteins. However, optimizing these systems to achieve the desired functionality often requires multiple design-build-test cycles, which can be time-consuming and labor-intensive. To address this challenge, we have developed Lustro, a platform that combines light stimulation with laboratory automation, enabling efficient high-throughput screening and characterization of optogenetic systems.
Lustro utilizes an automation workstation equipped with an illumination device, a shaking device, and a plate reader. By employing a robotic arm, Lustro automates the movement of a microwell plate between these devices, allowing for the stimulation of optogenetic strains and the measurement of&#160;their response. This protocol provides a step-by-step guide on using Lustro to characterize optogenetic systems for gene expression control in the budding yeast Saccharomyces cerevisiae. The protocol covers the setup of Lustro&#39;s components, including the integration of the illumination device with the automation workstation. It also provides detailed instructions for programming the illumination device, plate reader, and robot, ensuring smooth operation and data acquisition throughout the experimental process.

Author Spotlight: Advancing Optogenetics Research Using Lustro 

This protocol outlines the steps for utilizing the automated platform Lustro to perform high-throughput characterization of optogenetic systems in yeast.

Optogenetics offers precise control over cellular behavior by utilizing genetically encoded light-sensitive proteins. However, optimizing these systems to ...

Spatiotemporal Control of Protein Activity through Optogenetic Allosteric Regulation

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