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 activity and (5) Broad range of applicability to various target proteins. Notably, LightR uniquely integrates all these benefits into a single tool, thereby providing a significant step forward.
The design of LightR as an allosteric switch offers several advantages. The ability of LightR to control protein activity when inserted into a small flexible loop provides considerable versatility for application across various proteins with diverse structures and functions. Being an allosteric switch, LightR regulates only the target domain of the protein of interest and does not affect the other domains of the protein. Hence, this method allows for specific regulation of activity without compromising essential functions of the protein, such as interactions with its binding partners or native localization within the cells. Two-component optogenetic systems have been successfully applied for the regulation of protein localization, interactions, or other essential functions by using light-mediated control of homo- or heterodimerization29,30,31,32,33,34,35. However, these systems often require optimized equimolar expression of two proteins, making their application more challenging. Allosteric regulation using the LightR system is achieved within a single protein and thus provides a much more versatile approach for the interrogation of cell signaling.
The wide range of applicability of the LightR switch to different protein classes is, in part, achieved through the optimization of different modules within the LightR domain. The linkers connecting LightR to the protein of interest and the linker connecting two VVD domains within LightR (inter-VVD linker) can influence the efficiency of regulation. The choice of the inter-VVD flexible linker (GGS)4G(GGS)3 provides sufficient flexibility and length to ensure the association and dissociation of the VVD monomers within the LightR domain. This linker has ensured the successful regulation of all targeted proteins so far. However, in cases when the LightR domain does not sufficiently inactivate the protein in the dark, a more rigid linker can be introduced that will keep VVD domains further apart to enhance the opening of the LightR clamp, thereby keeping the enzyme inactive. The linker should still retain sufficient flexibility to enable dimerization of the VVDs and activation of the targeted protein. To connect LightR to the protein of interest, we use short flexible linkers, GPGGSGG and GSGGPG, added to the N- and C-termini of the LightR domain. They provide sufficient flexibility in the connection, thus preventing irreversible disruption of the target protein. Yet, they are short enough to enable distortion of the targeted protein in the dark while facilitating the restoration of protein function in the light. These linkers support regulation by LightR for many protein applications. However, in certain cases when these linkers may not provide sufficient inactivation of the protein in the dark, shortening of the linkers could be beneficial. Such Linker shortening could reduce flexibility and enhance distortion caused by the open LightR clamp in the dark, thereby minimizing any leakiness. Nevertheless, this adjustment might compromise the enzyme&#39;s maximum activity due to potential structural distortions in the lit state. Alternatively, replacing multiple amino acids in the insertion loop could provide similar benefits of balanced flexibility in cases where conformational instability is not a significant concern.
Many critical signaling processes are activated transiently or regulated in an oscillatory manner36,37,38,39,40,41,42,43. The tunability of the LightR tool allows us to mimic complex signaling patterns and thus provides a powerful approach for the interrogation of temporally controlled biological processes. We achieve this by modulating the off kinetics of LightR through the introduction of the I85V mutation into both VVD domains. This mutation reduces the half-life of VVD dimer in the dark state from 18,000 s to 780 s19 thus facilitating faster inactivation in FastLightR-Enzyme.
Fast inactivation kinetics also enables the regulation of protein activity at subcellular resolution. The LightR enzymes, with slow-off kinetics, could make it difficult to observe the effects of subcellular activation due to intracellular diffusion of the activated construct. With fast-off kinetics, any FastLightR-enzyme that diffuses beyond the zone of local illumination will be quickly inactivated. By selectively activating specific subcellular regions, researchers can gain insights into the local functions of the proteins for a better understanding of their contribution to the complex cellular pathways and signaling networks.
While FastLightRs are invaluable for spatially and temporally confined optical control of protein activities within cells, there are still applications where slow-off kinetics might be desirable. LightR with slow-off kinetics could be implemented in studies where sustained and enhanced blue light-inducible activity is required. These applications will require only infrequent illumination pulses to maintain continuous optogenetic activity, thus also minimizing issues such as phototoxicity. For different scenarios, a wide range of kinetic modifications10,18could be implemented alone or in tandem with each other to offer a promising solution for tuning LightR performance to optimize its application for a specific experiment.
The applicability of LightR-enzymes is determined by their ability to mimic the biological function of their endogenous counterparts. As such, it is critical to assess their substrate specificity, downstream signaling, and subcellular localization. The specificity of LightR-Src activity is confirmed through the phosphorylation of Src substrates, including paxillin and p130Cas22,23. FastLightR-Src shuttles between the perinuclear region and focal adhesions under cyclic illumination, resulting in cell spreading. Such a phenomenon successfully resembles earlier observations made with native Src kinase and further confirms the effectiveness of the present tool in exploring Src biology11,12,44,45,46,47. LightR-bRaf also demonstrates specificity in targeting signal complexes such as MEK and ERK kinases24,25. Additionally, FastLightR-bRaf exhibits cyclic oscillations of the ERK nuclear shuttle in response to blue light, which is a known consequence of bRaf activation28. The ability of LightR-enzymes to closely mimic the functions of endogenous proteins provides an invaluable tool for interrogation of their function.
The above discussion depicts the capacity of LightR kinases to replicate precise cellular signaling pathways within defined cellular compartments. In alignment with such success with kinases, LightR-Cre serves as an illustrative model demonstrating the functionality of the original Cre recombinase derived from the P1 bacteriophage. The Cre recombinase belongs to the integrase family of site-specific recombinases, and it works by facilitating recombination between two recognition sites called loxP on target DNA. Such recombination results in DNA rearrangement through a crossover event. Based on the directional orientation of the loxP site on the target DNA, the crossover event can lead to either deletion, duplication, or translocation of chromosomal elements48. Cre recombinase has been widely used for the generation of in vivo models of inducible gene regulation49,50. However, other spontaneous, chemically inducible, or optogenetically inducible cre recombinase systems provide limited control of when and where DNA recombination is induced in the animal model. LightR-Cre overcomes these limitations by enabling tight temporal and spatial control of DNA recombination. Furthermore, the tunability of LightR allows us to eliminate unwanted leaky activity and ensure the regulation of Cre with high precision. These capabilities open new opportunities for the modeling of disease pathology and the development of new therapeutics.
The precise regulation of LightR in kinases and DNA recombinase further provides proof that this tool can be applied to several proteins with the same strategy. In addition to activating various enzyme classes, LightR can potentially be attached to other protein types, where the allosteric modulation could be harnessed to localize proteins to specific subcellular regions, induce protein-protein interactions, engineer blue-light inducible biosensors, and more. Due to the allosteric advantages of specific domain targeting, LightRs can also be combined with other chemogenetic and optogenetic approaches in the same protein to achieve a higher degree of tunability to study biological processes.
We have successfully applied LightR to multiple other proteins (manuscripts under preparation). Despite this success, the development of a functional LightR protein may face potential challenges, particularly in selecting an appropriate insertion site and accurately mimicking the target protein&#39;s biological function. When the crystal structure is unavailable, researchers must rely on predicted structures or amino acid sequence information, necessitating more troubleshooting to engineer proteins with multiple insertion sites or amino acid substitutions. Additionally, the engineered protein activity must be finely tuned to replicate the biological function within the cell accurately. Success in these experiments depends on the precise functional mimicry of the engineered protein, with its endogenous homolog, which often requires rigorous troubleshooting and can vary based on cell type, target protein class, and type, as well as the strength and duration of blue light activation.
In conclusion, the LightR system represents a powerful tool for achieving precise spatiotemporal control over protein function. Its robustness, tunability, and versatility make it invaluable for studying complex cellular processes, unraveling signaling pathways, and elucidating gene regulatory mechanisms. By providing researchers with unprecedented control over protein activity, the LightR system promises to advance our understanding of fundamental biological processes and pave the way for innovative therapeutic interventions.

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 darkness for 16-18 h post-transfection. Furthermore, the catalytically inactive mutant of LightR-Src with D388R mutation does not induce phosphorylation of Src substrate, demonstrating that phosphorylation is carried out by active LightR-Src and not caused by nonspecific effect of blue light illumination (Figure 2B). Acknowledging reports of oscillatory activity of Src27 kinase in living cells, we investigated whether LightR-Src could be tuned to exhibit cyclic activation. The original LightR-Src displayed slow deactivation kinetics (Figure 2C), prompting the generation of a new variant, Fast-LightR-Src, harboring the I85V mutation in both VVDs. Figure 2D illustrates that Fast-LightR-Src shows markedly faster deactivation kinetics and completely inactivates within 10 min of turning off the light, compared to over an hour for LightR-Src. Using FastLightR-Src, we were also able to achieve repeated cycles of activation/inactivation (Figure 2E). These results collectively demonstrate the ability of the LightR tool to achieve specific and tightly controlled regulation of Src activity in living cells.
The application of FastLightR-Src also allows for assessing the effects of transient Src activation on cell morphodynamics. Activation of FastLightR-Src in HeLa cells induces cell spreading, and this process stops as soon as FastLightR-Src is inactivated. Upon activation, we observed the translocation of FastLightR-Src from the perinuclear compartment to focal adhesions and plasma membrane, a behavior exhibited by wild-type Src kinase11,12. Illuminating cells expressing the catalytically inactive mutant D388RLightR-Src fail to show such spreading effects (Figure 3B,C). This further confirms that FastLightR-Src functions similarly to its wild-type counterpart. Local illumination of a cell region results in localized activation of FastLightR-Src indicated by local accumulation LightR Src in focal adhesion and accompanied by the formation of local membrane protrusions (Figure 4B (i), (ii)) and cell centroid shift (Figure 4C (i), (ii)) resulting in cell movement towards light. Having established the application of the LightR tool for the regulation of Src kinase, we showcase LightR&#39;s wide-ranging utility extended to the regulation of bRaf kinase and DNA recombinase Cre. Analysis of LightR-bRaf shows light-induced phosphorylation of the endogenous substrate of bRaf, MEK1, and downstream phosphorylation of ERK1/2 kinases24,25 at levels comparable to those of the constitutively active bRaf mutant (V600E) (Figure 5A). Conversely, the catalytically inactive D575RLightR-bRaf demonstrates no MEK1 and ERK1/2 phosphorylation, comparable to non-illuminated LightR-bRaf samples, thus confirming the specificity of LightR-bRaf regulation. Live cell imaging demonstrated that repeated cycles of FastLightR-bRaf activation/inactivation induce repeated translocation of ERK2 in and out of the nucleus, corroborating with the known outcomes of bRaf activation28 (Figure 5B). The activation of LightR-Cre results in the successful recombination of a reporter mCherry gene, demonstrating the applicability of LightR to different classes of enzymes (Figure 5C).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67261/67261fig01.jpg" /> 
Figure 1:&#160;Development strategy of LightR approach. (A) Crystal structures of two Vivid monomers in the dark state (PDB: 2PD7) and the dimer in the lit state (PDB: 3RH8). (B) Cartoon representation of LightR design. Two tandemly connected VVD photoreceptors inserted in the catalytic domain disrupt the catalytic activity of the protein in the dark. Dimerization of VVD in response to blue light restores the protein activity. (C) Crystal structures of (i) Src (PDB 1Y57), and (ii) bRaf (PDB 4MNF) catalytic domains. (ii) Structure of Cre recombinase (PDB 1MA7). Yellow arrows indicate the LightR insertion site and indicate substituted amino acid. (D) Schematic diagram of cloning strategy for LightR domain insertion into a target enzyme sequence19. In Step 1, specifically designed primers are used for PCR synthesis of a LightR fragment flanked by sites annealing in the target gene before and after the insertion site. In Step 2, The LightR fragment thus produced is shown to be used as a &#34;megaprimer&#34; for insertion of LightR into the target enzyme gene by Quick change site-directed mutagenesis. Panels A-C have been adapted and modified with permission from Shaaya et al.10. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67261/67261fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/67261/67261fig02.jpg" /> 
Figure 2:&#160;Biochemical characterization of engineered LightR-Proteins. (A) Schematic representation of experimental protocol for Biochemical characterization of LightR Src. (B) LinXE cells transiently expressing the LightR Src construct bearing an mCherry and a myc tag at the C-terminus were continuously illuminated with blue light for 60 min, using a 465 nm LED setup. Cell lysates were probed for phosphorylation of Src substrates, paxillin, and p130Cas. Western Blot results show phosphorylation of endogenous paxillin and p130Cas in response to activation of LightR-Src in living cells. (C,D) LinXE cells transiently expressing the indicated LightR-Src constructs bearing tandem mCherry-myc tag at the C-terminus were continuously exposed to blue light for specified times (0 min and 30 min) and then placed in the dark for indicated periods of time. Cell lysates were probed for phosphorylation of Src substrates, showing the comparison of the activation and deactivation kinetics between (C) LightR-Src and (D) FastLightR-Src. (E) LinXE cells transiently expressing the indicated LightR-Src constructs bearing tandem mCherry-myc tag at the C-terminus were continuously exposed to blue light for 20 min followed by repeated incubation in the dark for 10 min and in the light for 20 min. Cell lysates were collected and probed for phosphorylation of Src substrates, showing the efficiency of Src kinase cycling between activation and inactivation states. All experiments were repeated at least three times with similar results. The numbers displayed beside each western blot panels B-E represent the molecular weights of the proteins, measured in kilodaltons (kDa). Panels B-E have been adapted and modified with permission from Shaaya et al.10. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67261/67261fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/67261/67261fig03v3.jpg" /> 
Figure 3:&#160;Global and temporal illumination control and effect on FastLightR-Src activity. (A) Cartoon representation of the experimental scheme of global and temporal cyclic illumination and the subsequent data analyses. (B,C) Regulation of cell morphology and FastLightR-Src localization by blue light is depicted in representative images of HeLa cells obtained from a time-lapse series using widefield fluorescence microscopy. These cells were transiently co-expressing FastLightR-Src-mCherry-myc (N = 10 cells) or catalytically inactive D388RLightR-Src-mCherry-myc (N = 9 cells) along with Stargazin-iRFP670 (plasma membrane marker). Live-cell imaging was conducted every minute while cells were globally illuminated with blue light (blue outlines). (B) Yellow arrows highlight FastLightR-Src localization at structures resembling focal adhesions. The images were obtained from a time-lapse series of HeLa cells using widefield fluorescence microscopy. (C) The quantification of changes in cell area induced by activation of FastLightR-Src vs. D388RLightR-Src is shown on each panel. The illumination window is indicated as blue rectangles. Graphs represent mean &#177; 90% confidence intervals. Panels B and C have been adapted and modified with permission from Shaaya et al.10. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67261/67261fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/67261/67261fig04.jpg" /> 
Figure 4:&#160;Local subcellular illumination control and effect on FastLightR-Src activity. (A) Cartoon representation of experimental scheme of local Subcellular illumination control. (B) (i) Time-lapse sequence illustrating a HeLa cell expressing FastLightR-Src-mCherry, locally illuminated with blue light (depicted by a blue circle). Images were captured every minute using total internal reflection fluorescence microscopy (60x objective). The images are presented as inverted contrast images obtained at specified time intervals. (ii) Cell projection images displaying protrusions formed during specified time intervals. Thresholding and masking the cell morphology over the experimental timeline represents the platform to analyze cell spreading and cell edge dynamics. The specified illuminated area of the cell is outlined by a blue circle. (C) (i) Polar plot for cells representing the centroid&#39;s migration distance (d) and angle of deviation (&#952;) relative to local blue light illumination. Black dots represent individual HeLa cells expressing catalytically inactive D388RLightR-Src (N = 6 cells) or FastLightR-Src (N = 10 cells). (ii) Schematic representation demonstrating the centroid shift distance (d) traveled by the cell&#39;s centroid from the initiation (1) to the conclusion (2) of local blue light stimulation. The angle of deviation (&#952;) indicates the extent of the cell&#39;s centroid movement divergence from the location of blue light. The light grey shading indicates the region protruded by the cell in response to local FastLightR-Src stimulation. The specified illuminated area of the cell is indicated by a solid blue circle. Panels B and C have been adapted and modified from Shaaya et al.10. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67261/67261fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/67261/67261fig05.jpg" /> 
Figure 5:&#160;Broad applicability of LightR approach. (A) Regulation of LightR-bRaf. LinXE cells transiently expressing LightR-bRaf-Venus, catalytically inactive D575R LightR-bRaf Venus, or constitutively active bRaf (V600E)-Venus was exposed to continuous blue light for the specified period of time. Cell lysates were probed for the phosphorylation of indicated proteins. (B) Representative images obtained from a time-lapse series of live cell imaging using widefield fluorescence microscopy illustrate the regulation of global blue light response in LinXE cells transiently co-expressing FastLightR-bRaf-Venus and mCherry-ERK2. These cells were globally illuminated with blue light (50 ms pulses every second, 50 pulses per minute) for specified durations outlined in blue. Live-cell imaging of mCherry-ERK2 was conducted every minute. Yellow arrows highlight stages of nuclear ERK2 localization induced by blue light activation of engineered bRaf. (C) LinXE cells were transiently co-transfected with a Floxed-stop-mCherry reporter and LightR-Cre-iRFP670. Cells were subjected to either dark conditions or pulsing blue light (2 s on, 10 s off) for up to 8 h. Images for all time points of each channel were acquired using a widefield fluorescence microscope (10x objective) at identical settings and were adjusted to consistent brightness and contrast levels to facilitate comparison of protein expression levels across samples. The experiment was repeated at least three times, yielding similar results. The figure has been adapted and modified from Shaaya et al.10. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67261/67261fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Table 1: LightR sequence. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67261/Supplementary Table 1.xlsx" target="_blank">Please click here to download this File.</a>
Supplementary Table 2: Description of LightR insertion sites and checklist for LightR-Protein design. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67261/Supplementary Table 2.xlsx" target="_blank">Please click here to download this File.</a>
Supplementary Table 3: Primers for LightR cloning. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67261/Supplementary Table 3.xlsx" target="_blank">Please click here to download this File.</a>
Supplementary Table 4: Transfection conditions for biochemical analysis of LightR-Protein activity. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67261/Supplementary Table 4.xlsx" target="_blank">Please click here to download this File.</a>
Supplementary Table 5: Transfection conditions for imaging-based analysis of LightR-Protein activity. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67261/Supplementary Table 5.xlsx" target="_blank">Please click here to download this File.</a>
Supplementary File 1: Pulsed illumination using Integrated Development Environment (IDE). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67261/Supplementary File 1.zip" target="_blank">Please click here to download this File.</a>
Supplementary File 2: Microscope illumination settings. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67261/Supplementary File 2_Updated.zip" target="_blank">Please click here to download this File.</a>

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

Nanyang Technological University

Research

JoVE Journal

Biology

445 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.

Video: Spatiotemporal Control of Protein Activity through Optogenetic Allosteric Regulation

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

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

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

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

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

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

Reliably Engineering and Controlling Stable Optogenetic Gene Circuits in Mammalian Cells

Light-Controlled Fermentations for Microbial Chemical and Protein Production

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

Regulacja allosteryczna