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.

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			<td>#1.5 Glass Coverslips 25 mm Round</td>
			<td>Warner Instruments&#160;</td>
			<td>64-0715&#160;</td>
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			<td>1.5 mL Tubes&#160;</td>
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			<td>cc7682-3394</td>
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			<td>2x Laemmli Buffer</td>
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			<td>4-20% Mini-PROTEAN TGX Precast Gel</td>
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			<td>5x Phusion Plus Buffer&#160;</td>
			<td>Thermo Scientific&#160;</td>
			<td>F538L</td>
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			<td>60 LED Microscope Ring Light</td>
			<td>Boli Optics</td>
			<td>ML46241324</td>
			<td>Blue LED, 60 mm diameter, 5 W</td>
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			<td>Agarose&#160;</td>
			<td>GoldBiotech</td>
			<td>A-201</td>
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			<td>Anti-Erk 1/2 Antibody</td>
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			<td>9102</td>
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			<td>Anti-GAPDH Antibody</td>
			<td>invitrogen</td>
			<td>AM4300</td>
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			<td>Anti-GFP</td>
			<td>Clontech</td>
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			<td>Anti-mCherry Antibody</td>
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			<td>M11217</td>
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			<td>Anti-MEK</td>
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			<td>9122</td>
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			<td>Anti-p130Cas</td>
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			<td>Anti-paxillin</td>
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			<td>Anti-phospho-Erk 1/2 T202/Y204 Antibody</td>
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			<td>Anti-phospho-pY249 p130Cas</td>
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			<td>4014</td>
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			<td>Anti-phospho-Y118 Paxillin</td>
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			<td>2541</td>
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			<td>Anto-phospho-S217/221 MEK</td>
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			<td>9121</td>
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			<td>Arduino Compatable Power Supply</td>
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			<td>Arduino Uno Rev3</td>
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			<td>&#8206;A000066</td>
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			<td>Attofluor Cell Chamber</td>
			<td>invitrogen</td>
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			<td>Benchmark Fetal Bovine Serum (FBS)&#160;</td>
			<td>Gemini Bio-products</td>
			<td>100-106</td>
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			<td>bRaf-V600E-Venus</td>
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			<td>Gift from Dr. John O&#39;Bryan, MUSC</td>
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			<td>BSA&#160;</td>
			<td>GoldBiotech</td>
			<td>A-420&#160;</td>
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			<td>Carbenicillin (Disodium)</td>
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			<td>C-103-25</td>
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			<td>CellMask Deep Red plasma membrane dye</td>
			<td>invitrogen</td>
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			<td>Colony Screen MasterMix&#160;</td>
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			<td>42-138</td>
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			<td>DH5a competent cells&#160;</td>
			<td>NEB&#160;</td>
			<td>C2987H</td>
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			<td>DMEM&#160;</td>
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			<td>15-013-CV</td>
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			<td>DNA Ladder&#160;</td>
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			<td>D010-500</td>
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			<td>dNTPs&#160;</td>
			<td>NEB</td>
			<td>N04475</td>
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			<td>Dpn1 Enzyme&#160;</td>
			<td>NEB</td>
			<td>R01765</td>
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			<td>DTT</td>
			<td>GoldBio</td>
			<td>DTT10</td>
			<td>DL-Dithiothreitol, Cleland&#39;s Reagents&#160;</td>
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		<tr>
			<td>EGTA</td>
			<td>Acros&#160;</td>
			<td>409910250</td>
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			<td>FastLightR-bRaf-mVenus</td>
			<td>Addgene</td>
			<td>#162155</td>
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		<tr>
			<td>Fibronectin from bovine plasma&#160;</td>
			<td>Sigma&#160;</td>
			<td>F1141</td>
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			<td>FuGENE(R) 6 Transfection Reagent&#160;</td>
			<td>Promega</td>
			<td>E2692</td>
			<td>Transfection reagent</td>
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		<tr>
			<td>Gel Green Nucleic Acid Stain&#160;</td>
			<td>GoldBio</td>
			<td>G-740-500</td>
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		<tr>
			<td>Gel Loading Dye Purple 6x&#160;</td>
			<td>NEB</td>
			<td>B7024A</td>
			<td></td>
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			<td>GeneJET Gel extraction Kit&#160;</td>
			<td>Thermo Scientific&#160;</td>
			<td>K0692</td>
			<td>Gel Extraction Kit&#160;</td>
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		<tr>
			<td>GeneJET Plasmid Miniprep Kit</td>
			<td>Thermo Scientific&#160;</td>
			<td>K0502</td>
			<td></td>
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			<td>Glutamax</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td>GlutaMAX-l (100x) 100 mL&#160;</td>
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		<tr>
			<td>HEK 293T Cells&#160;</td>
			<td>ATCC&#160;</td>
			<td>CRL-11268</td>
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		<tr>
			<td>HeLa Cells&#160;</td>
			<td>ATCC&#160;</td>
			<td>CRM-CCL-2</td>
			<td></td>
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		<tr>
			<td>HEPES</td>
			<td>Fischer&#160;</td>
			<td>BP310-500</td>
			<td></td>
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			<td>Iot Relay</td>
			<td>Digital Loggers</td>
			<td>DLI 705020645490</td>
			<td>AC/DC control relay for illumination</td>
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			<td>Kanamycin Monosulfate</td>
			<td>Gold Biotechnology</td>
			<td>K-120-25</td>
			<td></td>
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		<tr>
			<td>KCl</td>
			<td>Sigma&#160;</td>
			<td>P-4504</td>
			<td></td>
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		<tr>
			<td>L-15 1x</td>
			<td>Corning&#160;</td>
			<td>10-045-CV&#160;</td>
			<td></td>
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		<tr>
			<td>LB Agar&#160;</td>
			<td>Fisher</td>
			<td>BP1425-2</td>
			<td></td>
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			<td>LED Grow Light System</td>
			<td>HQRP</td>
			<td>884667106091218</td>
			<td>LED panel lamp system&#160;</td>
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		<tr>
			<td>LightR-bRaf-mVenus</td>
			<td>Addgene</td>
			<td>#162154</td>
			<td></td>
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		<tr>
			<td>LightR-iCre-miRFP670</td>
			<td>Addgene</td>
			<td>#162158</td>
			<td></td>
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		<tr>
			<td>MATLAB</td>
			<td>Mathworks</td>
			<td>R2024a</td>
			<td>Software for running CellGEO Scripts</td>
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		<tr>
			<td>Metamorph</td>
			<td>Molecular Devices</td>
			<td></td>
			<td>Imaging Analysis Software</td>
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		<tr>
			<td>MgCl2&#160;</td>
			<td>Fisher Chemical</td>
			<td>M33-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Mineral Oil</td>
			<td>Sigma&#160;</td>
			<td>M5310</td>
			<td></td>
		</tr>
		<tr>
			<td>MiniPrep Kit&#160;</td>
			<td>Gene Choice&#160;</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&#160;</td>
			<td>Corning&#160;</td>
			<td>46-000-CV&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Multiband Polychroic Mirror</td>
			<td>89903BS</td>
			<td>Chroma</td>
			<td></td>
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		<tr>
			<td>NaCl&#160;</td>
			<td>Fisher Chemical</td>
			<td>S271-3&#160;</td>
			<td></td>
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		<tr>
			<td>PBS w/o Ca and Mg&#160;</td>
			<td>Corning&#160;</td>
			<td>21-031-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>pCAG-iCre</td>
			<td>Addgene</td>
			<td>#89573</td>
			<td></td>
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		<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&#39;s Lab, UNC</td>
		</tr>
		<tr>
			<td>PCR Tubes&#160;</td>
			<td>labForce</td>
			<td>1149Z65</td>
			<td>0.2 mL 8-Strip Tubes and Caps, Rigid Strip Individually Attached Dome Caps&#160;</td>
		</tr>
		<tr>
			<td>Phusion Plus DNA Polymerase</td>
			<td>Thermo Scientific&#160;</td>
			<td>F630S</td>
			<td></td>
		</tr>
		<tr>
			<td>pmiRFP670-N1</td>
			<td>Addgene</td>
			<td>#79987</td>
			<td></td>
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			<td>Polygon 400 Patterned Illuminator</td>
			<td>Mightex</td>
			<td>DSI-G-00C</td>
			<td></td>
		</tr>
		<tr>
			<td>Primers&#160;</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&#160;</td>
		</tr>
		<tr>
			<td>T0.25% Trypsin, 2.21 mM, eDTA, 1x [-] sodium</td>
			<td>Corning&#160;</td>
			<td>25-053-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-Acetate-EDTA (TAE) 50x&#160;</td>
			<td>Fischer&#160;</td>
			<td>BP1332-1&#160;</td>
			<td>For electrophoresis&#160;</td>
		</tr>
		<tr>
			<td>UPlanSApo 40x Microscope Objective</td>
			<td>Olympus</td>
			<td>1-U2B828</td>
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			<td>USB TTL Box</td>
			<td>National Instruments</td>
			<td>6501</td>
			<td>For TTL interface</td>
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		<tr>
			<td>&#946;-Mercaptoethanol</td>
			<td>Fisher Chemical&#160;</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.

Spatiotemporal Control of Protein Activity through Optogenetic Allosteric Regulation

Our overall research focuses on developing optogenetic methods that achieve precise spatiotemporal control of protein activity by light. We engineer light regulatable domains called lightR for allosteric regulation, allowing us to study how spatiotemporally controlled protein activity impacts cellular signaling and functions aiding in preclinical disease modeling and therapy development. Optogenetics faces several challenges including achieving precise and reversible control of target protein activity and accurately mimicking endogenous signaling kinetics.Key issues are preventing unwanted activation, enabling targeted subcellular activation, and managing phototoxicity to preserve cell viability. Additionally, developing universally compatible and robust tools is crucial for improving versatility and reproducibility in applications. Our protocol for engineering lightR tools uniquely combines multiple advanced features into a single system, including allosteric regulation, high sensitivity, spatial resolution, tight temporal control, and precise signaling specificity.This integration allows for tunability across diverse target proteins, addressing the gaps in other methods that may lack one or more of these critical capabilities. We are interested in defining key signaling and structural processes regulating cell migration. We aim to use our optogenetic technology to dissect regulations of endothelial cell migration and interactions.Understand how remodeling of the extracellular matrix is mediated by endothelial cells, and determine how dysregulation of these processes contribute to disease development. For biochemical analysis of lightR kinases plate one times 10 to the power of six lin XE cells for 3.5 centimeter cell culture dish for each experimental group, Incubate the cells at 37 degrees Celsius and 5%carbon dioxide for 16 to 18 hours. The following day, transfect the cells with the selected DNA construct using an appropriate transfection reagent.Cover the dish with aluminum foil and return it to the incubator. After 16 to 18 hours of transfection place a 465 nanometer LED panel lamp system inside the tissue culture incubator. Position a perforated plexiglass panel 10 centimeters above the lamp to achieve illumination of three milliwatts per square centimeter.Use continuous illumination in cells expressing LightR-Src and D388 R-LightR-Src. Turn the LED panel on and off manually to control illumination. At the end of the experimental time points, harvest the cells under safe red light, aspirate the media and wash the cells with cold PBS.Global illumination of lin XE cells with engineered LightR-Src for 60 minutes shows phosphorylation of Src substrates, endogenous paxillin, and p130Cas cells expressing the catalytic inactive D388 RLightR-Src mutant show no phosphorylation of Src substrates at global illumination. To begin, plate two times 10 to the power of five hela cells per 35 millimeter tissue culture dish in cell culture media. Incubate the cells for two hours at 37 degrees Celsius and 5%carbon dioxide.Once the cells have attached and reached 60 to 70%confluency, cotransfect the hella cells with the given mix. Cover the dish with aluminum foil to prevent accidental illumination of the cells. Then incubate the cells for 16 to 18 hours at 37 degrees Celsius and 5%carbon dioxide.Next, place covered glasses with a diameter of 25 millimeters and a thickness of 0.17 millimeters into a six well plate chamber. Coat three round glass cover slips with five milligrams per liter of fibronectin in PBS and incubate at 37 degrees celsius overnight. Then rinse the cover slips with PBS 16 to 18 hours after transfection.Collect the transfected hela cells in safe, red light, then played approximately one times 10 to the power of five transfected hela cells onto each cover slip under safe, red light. Cover the plate with aluminum foil and incubate in cell culture media for two hours at 37 degrees Celsius and 5%carbon dioxide. Next, warm the prepared imaging media and mineral oil to 37 degrees Celsius.Then wash the cover slips containing the cells with PBS two times. After staining and washing the cells, carefully place the cover slip into a live cell imaging chamber. Add one milliliter of L15 imaging media to the chamber.Add one milliliter of pre-warmed mineral oil to the media to prevent evaporation during imaging. Keep the chamber protected from light at 37 degrees Celsius until ready to image. Place the chamber onto a microscope stage preheated to 37 degrees Celsius.Select a single cell expressing both fast LightR-Src cherry, and Src an IRFP. Select specific regions of interest within the cell to illuminate. For this study, choose a small area at the periphery of the selected cell.Image the selected cell every minute for 20 minutes in the basal state before illumination. Continue imaging for 50 minutes while illuminating locally followed by 20 minutes after activation for a total of 90 minutes. After imaging, save the movies in tif stack file format for analysis.Global illumination of hela cells led to the localization of fast LightR-Src to focal adhesions, which were reversed once the blue light was turned off. This illumination also caused a significant increase in cell spreading, which stopped once the blue light was turned off. The catalytic inactive D388 R-LightR-Src mutant showed no cell spreading.Localized illumination of hela cells expressing fast LightR-Src resulted in the accumulation of the construct in focal adhesions, leading to localized membrane protrusions with 50 minute illumination. No further area gain was observed in 20 minute dark after illumination. Fast LightR-Src gradually disappeared from focal adhesions in the dark phase.The cell centroid shifted toward the illuminated region. Following fast LightR-Src activation, indicating directed cell movement.

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...

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

349 Views.  University of Illinois at Chicago.  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.

Optogenetic Control of Enzyme Activity Using Light-Regulated Allosteric Switch

Summary