Spatiotemporal Control of Protein Activity through Optogenetic Allosteric Regulation

Summary

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.

Abstract

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's catalytic domain and inactivating it. Upon exposure to blue light, the LightR domain closes and restores the catalytic domain's structure and enzyme activity. In this manuscript, we discuss design strategies 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.

Introduction

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 activity^1,2,3,4. Others enable regulation of enzymatic activity but may lack subcellular control or have limited applicability across different enzyme classes^5,6,7,8,9. 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 kinase^11,12. 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.

Protocol

1. Design and development of LightR ( Figure 1)

1. Strategic planning
   NOTE: The LightR domain is comprised of two tandemly connected Vivid (VVD) photoreceptor domains from Neurospora crassa^13,14,15,16. VVDs homodimerize in the presence of light, and such dimerization involves a conformational change that brings the N-terminus of one VVD monomer next to the C-terminus of another VVD monomer (Figure 1A). Connecting two VVD domains with a flexible linker creates a clamp-like domain that will be open in the dark and closed upon illumination with blue light. Integrating this clamp into an enzyme's catalytic domain enables light-mediated regulation of activity. In darkness, the open clamp distorts the domain, deactivating the enzyme; illumination with the blue light causes the clamp to close, restoring enzyme's activity (Figure 1B). This concept can be applied to enzymes from various families, including protein kinases and DNA recombinases¹⁰ (Figure 1C).
   1. Design of LightR
      1. To connect two VVD molecules, use a flexible 22-amino acid linker (GGS)4G(GGS)3 between each monomer.
         NOTE: The linker provides sufficient flexibility and length to accommodate the association and dissociation of VVD monomers.
      2. Add GPGGSGG and GSGGPG linkers to the N- and C-termini of the LightR domain, respectively.
         NOTE: The length and the composition of the linkers may need to be adjusted for a specific protein. Shorter GSG and single Gly linkers are routinely used when tighter regulation is needed. The size of the insertion loop and its proximity to catalytic residues will also influence the dynamic range of regulation. Shorter loops are more likely to provide tighter regulation of catalytic activity but may result in lower activity after illumination. The sequence of the LightR domain is provided in Supplementary Table 1.
   2. Insertion of LightR
      NOTE: A suitable LightR insertion site will enable tight regulation of the targeted domain function without sterically blocking its interactions or causing irreversible structural changes that will affect its biological role. A crystal structure of the target protein is an ideal guide in the identification of such flexible loop regions for the insertion of LightR. If necessary, the crystal structure of a close homolog can suffice. Some general criteria to consider are provided below.
      1. Ensure that the insertion loop is structurally coupled to the critical catalytic elements of the enzyme. Ensure that the selected insertion loop is not an existing binding site for the protein and that the insertion of LightR does not potentially disrupt any functional interactions due to steric effects.
      2. As an initial strategy, replace one amino acid in the insertion loop when inserting LightR. Target the middle of the insertion loop containing a polar or small amino acid (e.g., Glu, Arg, Lys, Gly) exposed to the solvent and not involved in intramolecular interactions.
         NOTE: When one amino acid replacement does not result in a functional protein, optimization of the LightR construct will require the replacement of multiple amino acids or the entire insertion loop. Evaluate the functionality of multiple insertion sites for LightR within the defined loop.
      3. Ensure that the target enzyme operates independently of endogenous regulation. Achieve this by using a constitutively active mutant version of the enzyme for the insertion template.
         NOTE: Wild-type enzymes can also be used to generate LightR constructs, but this may result in an AND-gated system that will be regulated by endogenous mechanisms and light.
      4. Positive and negative controls are vital for LightR-enzyme activity analysis. Use constitutively active mutants of endogenous proteins as positive controls and catalytically inactive mutant versions of the targeted LightR enzyme as negative controls.
         NOTE: When selecting inactivating mutations, it is crucial to ensure that the mutation is sufficiently spaced from the LightR insertion site to avoid disrupting the irreversibility of LightR-enzyme folding. Based on the above criteria, the LightR insertion site in Src kinase is selected in a flexible loop region that is structurally coupled to the highly conserved GXGXXG motif in the ATP-binding G-loop/glycine-rich loop region via a beta-strand. Importantly, it is positioned away from the ATP binding pocket and substrate binding region^10,17 (Figure 1C (i)).
         NOTE: Due to structural homology between kinases, the LightR insertion site in bRaf is selected in the same structural loop as the insertion site in Src (Figure 1C (ii)).
         NOTE: Based on the principles stated in Section 1.1.2., the LightR insertion site in Cre recombinase, a non-kinase, is picked in one of the flexible loops distant from the catalytic core, i.e., the DNA binding site. However, the insertion loop is still structurally coupled to critical catalytic residues within the DNA binding domain through an α-helix to ensure the success of LightR-Cre functionality (Figure 1C (iii)). All insertion sites with positive and negative controls for different enzymes described in the current protocol are detailed in Supplementary Table 2.
   3. Development of FastLightR.
      NOTE: The introduction of I85V mutation to both VVDs in LightR enables a faster activation-inactivation dynamic, attributed to the rapid conversion of the mutant into the dark state^18,19. The generation of FastLightR-Src and FastLightR-bRaf with fast inactivation kinetics enables tight temporal control of activation and inactivation. It allows for achieving subcellular regulation of LightR-Src in living cells.
      NOTE: Cre recombinase activity results in irreversible DNA recombination; hence, the reversibility properties of LightR are not applicable.
      NOTE: To simplify the detection of the LightR construct, a fluorescent protein or any other suitable tag can be attached to the N- or C- terminus of the target protein. This study used mCherry-myc for LightR-Src, Venus for LightR-bRaf, and miRFP670 for LightR-Cre recombinase.
2. Cloning Strategy
   NOTE: The cloning approach, unlike traditional approaches, is based on site-directed mutagenesis that does not rely on specific restriction sites^10,20.
   1. Generation of a "Megaprimer"
      1. Codon-optimize the LightR genes to ensure stable expression of the two tandem VVD DNA sequences of fungal origin in mammalian cells and to make the sequences as different as possible for errorless cloning using PCR. To perform codon optimization follow steps 1.2.1.2-1.2.1.3.
      2. Align the amino acid sequence of VVDs and the selected linkers side by side to create the theoretical sequence map of LightR.
      3. Paste the sequence in any online codon optimization platform that utilizes an advanced algorithm for codon optimization to determine the best sequence option with minimal complexity. Ensure the target organism is selected, the reading frame is maintained, and the VVD sequences are selected for optimization.
      4. Obtain LightR DNA as a custom synthesized fragment (See Supplementary Table 1) or from a previously published LightR construct¹⁰.
      5. Amplify the LightR gene to generate "Megaprimer" using the previously described²⁰ strategy outlined in Figure 1D. Synthesize the LightR "Megaprimer" via PCR following the manufacturer's recommendations for the specific DNA polymerase and using the following PCR reaction mixture:
              5x DNA Polymerase compatible buffer: 10 µL
              100 µM Forward megaprimer: 0.5 µL
              100 µM Reverse megaprimer: 0.5 µL
              10 nM dNTP mix: 2 µL
              DNA polymerase (2000 units/mL): 1 µl (0.02 U/µL)
              50 ng Template DNA: 1 µL
              PCR grade water: 35 µL
         ​NOTE: Here, high fidelity, hot start DNA polymerase with universal primer annealing temperatures are used.
      6. Separate the resulting megaprimer by agarose gel electrophoresis. The product will be approximately 1000 nucleotides in length. Excise the megaprimer from the agarose gel and purify using a gel extraction kit, following the manufacturer's recommendations or using an analogous technique.
   2. Insertion of LightR gene using site-directed mutagenesis: Insert the LightR, replacing a desired amino acid or a length of amino acids. The template plasmid will be the one where the LightR domain is inserted. Perform PCR reaction as previously described²⁰ using the following mixture.
           5x DNA Polymerase compatible buffer: 5 µL
           10 nM dNTP mix: 1 µL
           DNA polymerase (2000 units/mL): 1 µL (0.02 U/µL)
           50 ng Target Template DNA: 1 µL
           Extracted Megaprimer (calculated mass): 10 µL
           DMSO: 2.5 µL
           PCR grade water: 2.5 µL
      ​NOTE: Megaprimer mass (ng) = desired insert/vector molar ratio x mass of vector x ratio of insert to vector lengths. The desired insert/vector molar ratio used is 3/1.
   3. After completion of the PCR reaction, add 1 µL of DpnI (2000 units/mL) enzyme to selectively digest only the excess and methylated DNA and incubate the mixture at 37 °C for 1-1.5 h. This will significantly increase the yield of the modified construct.
   4. Transform 1-2 µL of the PCR reaction into DH5α competent cells following manufacturer protocols.
   5. Plate transformed bacteria on Luria Broth (LB)-agar plate with appropriate antibiotic for selection. Incubate plate at 37 °C overnight or at room temperature (RT; 25-30 °C ) for 72 h.
      NOTE: LB plates with colonies grown on them can be stored at 4 °C for up to 1 month, and protocol can be paused.
   6. Colony screen PCR
      1. For PCR-based colony screening, design primers that anneal in the LightR insert and target DNA to generate approximately 400-700 bp fragments.
      2. Add a small amount of a colony from the LB plate to the PCR mix provided below and proceed to thermocycling following manufacturer's recommendations for the specific DNA polymerase.
              2x Taq RED Master Mix: 5 µL
              PCR grade water : 5 µL
              100 µM Forward primer : 0.5 µL
              100 ​µM Reverse primer: 0.5 µL
      3. Check for positive colonies by agarose gel electrophoresis.
      4. Pick the colonies that generated the desired PCR product size and inoculate them individually into 3 mL of LB broth with the antibiotic, matching the antibiotic resistance in the plasmid backbone. Then, incubate and shake at 37 °C overnight.
      5. Extract plasmid DNA from liquid culture following the manufacturer's instructions of the plasmid purification kit for plasmid DNA extraction and verify the presence of the insert by sequencing.
         NOTE: Cloning primers are summarized in Supplementary Table 3.

2. Cell plating and biochemical analysis of LightR enzyme activity (Figure 2A)

1. For Biochemical analysis of LightR-kinases (LightR Src, FastLightR Src, LightR bRaf), plate 1 x 10⁶ LinXE cells per 3.5 cm cell culture dish for each experimental group. See Supplementary Table 4.
2. Incubate cells at 37 °C and 5 % CO₂ for 16-18 h.
3. The following day, transfect the cells with the selected DNA construct, using a suitable transfection reagent following the manufacturer's recommendations (Supplementary Table 4).
   NOTE: The optimal transfection reagent will depend on the construct, vector, and type of cells used. This study used 2 µg of DNA in a 3.5 cm dish using a transfection reagent. Empirical testing should be done to determine the optimal transfection reagent. Since transfected constructs will express proteins regulated by blue light, all subsequent steps handling transfected cells must be performed under red light. Wrap the plates with transfected cells with aluminum foil before placing them in the incubator to prevent accidental illumination.
4. After 16-18 h of transfection expose the cells to blue light¹⁰. To perform this, place a 465 nm light-emitting diode (LED) panel lamp system in the tissue culture incubator. Place a perforated plexiglass panel 10 cm above the lamp to obtain the desired illumination of 3 mW/cm² (Supplementary File 1). A perforated panel is needed to maintain uninterrupted air circulation in the incubator. Illuminate cells for the desired period.
5. For activation of LightR-Src and LightR-bRaf, use continuous illumination. For continuous illumination, turn the LED panel on and off manually.
6. For maintaining the activation/inactivation cycle of FastLightR Src, use ON/OFF cycles either controlled manually or by a microcontroller.
   NOTE: Several software toolkits that provide an Integrated Development Environment (IDE) for writing and uploading the required code can be utilized. These toolkits support C/C++ programming and offer general-purpose input/output (GPIO) access, which is essential for controlling the pulsed illumination. A detailed description of the protocols and code used for these purposes is described in Supplementary File 1.
7. At the end of the respective time points of the experiment (Supplementary Table 4), harvest the cells under safe red lights. Aspirate the media and wash the cells with cold PBS.
8. To isolate protein, lyse cells with 500 µL of 2x Laemmli sample buffer supplemented with 5% v/v 2-Mercaptoethanol. Incubate the lysate at 100 °C for 5 min. Analyze cell lysates by protein gel electrophoresis and Western blotting²¹.
   NOTE: The composition of 2x Laemmli buffer is as follows: For 500 mL: 5.18 g of Tris-HCL, 131.5 mL of glycerol, 52.5 mL of 20% SDS, 0.5 g of bromophenol blue, final pH 6.8.
9. Assess LightR-Src activity by evaluating the phosphorylation level of endogenous p130cas on Tyr249 and paxillin on TyrY118^22,23(Figure 2). Assess LightR-bRaf activity by evaluating the phosphorylation level of MEK1 on Ser217/221 and ERK1/2 on TyrY202/204^24,25.

3. Functional analysis of LightR-Cre activity

1. For the functional analysis of LightR-Cre activity, plate 1 x 10⁶ LinXE cells per 3.5 cm cell culture dish. Incubate cells at 37 °C and 5% CO₂ for 16-18 h.
2. Transfect the cells following the protocol outlined in section 2, Figure 2A, and Supplementary Table 5. Co-transfect a total of 2 µg DNA, with a 1:9 ratio of LightR-Cre-iRFP670 and a Reporter plasmid Floxed-STOP-mCherry².
3. Following 16-18 h after transfection, illuminate cells. An efficient LightR-Cre activation needs prolonged illumination. Therefore, to avoid phototoxicity, use pulsed illumination for 8 h with 2 s ON and 20 s OFF cycles utilizing a 465 nm LED panel lamp system controlled by a microcontroller as detailed in section 2 and Supplementary File 1.
4. Perform imaging using epifluorescence microscopy with a 20x air objective. For visualization of LightR-Cre-iRFP670, use Cy5 filter set, and for visualization of mCherry from Floxed-mCherry expression, use RFP filter set.
   NOTE: For Imaging, an epifluorescence microscope equipped with Cy5/RFP light cubes is used.

4. Preparation of samples for live cell imaging

1. Plate 2 x 10⁵ HeLa cells per 35 mm tissue culture dish in cell culture media and incubate for 2 h at 37 °C and 5% CO₂.
2. Once the cells have attached and the confluency is approximately 60%-70%, co-transfect the HeLa cells with one of the following mixes: (i) 0.85 µg of FastLightR-Src-mCherry-myc/0.15 µg of Stargazin-iRFP, or (ii) 0.75 µg of FastLightR-bRaf-Venus/0.25 µg of mCherry-ERK2
   NOTE: In FastLightR-Src experiments, the Stargazin-iRFP is used to label plasma membrane for cell spreading analysis. If higher FastLightR-Src expression is desired, stargazin-iRFP can be omitted, and 1 µg of FastLightR-Src can be used instead. In these cases, a membrane dye should be used to visualize the cells. Plasma membrane stains are routinely used for labeling cell membranes.
3. Cover the dish with aluminum foil to avoid accidental illumination of the cells and incubate for 16-18 h at 37 °C and 5% CO₂.
4. Complete all the following steps under red light illumination to avoid inadvertent activation of the constructs. Exposure to white light will induce the activation of LightR-kinases and could affect subsequent results.
5. On the same day, coat 3 round glass coverslips (25 mm diameter, 0.17 mm thickness) overnight with 5 mg/L fibronectin in PBS at 37 °C.
6. Rinse the coverslips with PBS 16-18 h after transfection and plate ~1 x 10⁵ transfected HeLa cells onto each coverslip. Incubate in cell culture media for 2 h at 37 °C and 5% CO₂.
   NOTE: The low seeding density is necessary to ensure that cells in the field of view are not touching each other.
7. Prepare the imaging media by adding FBS to Levobitz's L-15 imaging media to a final concentration of 5%. Filter the solution through a 0.22 µm filter and warm it to 37 °C.
8. Prewarm mineral oil to 37 °C.
9. Wash the coverslips containing the cells with PBS two times. If using a membrane dye, stain the cells following the manufacturer's instructions before washing the coverslips.
10. Carefully place the coverslip into a live cell imaging chamber. Add 1 mL of L-15 imaging media to the chamber.
11. Add 1 mL of mineral oil on top of the media to prevent evaporation during the imaging process.
12. Keep the chamber protected from light at 37 °C until prepared to image.

5. Global activation and imaging of FastLightR-Src (Figure 3)

1. Attach a blue LED microscopy ring light to the microscope condenser, positioning it approximately 1.5 cm above the sample holder. Connect the AC/DC control relay for the ring light to the microscope computer.
   NOTE: For the experiments in this study, the illumination was controlled through a Transistor-Transistor Logic (TTL) interface via the imaging software. Epifluorescent illumination may also be used to globally activate LightR constructs if an external illumination source is not available. GFP excitation wavelengths, such as the 490/20 nm excitation filter, are effective in activating LightR.
2. Place the chamber onto a microscope stage pre-heated to 37 °C and select cells expressing FastLightR-Src-mCherry and Stargazin-iRFP (Figure 3B) Or FastLightR-bRaf-Venus and mCherry ERK-2 (Figure 5B).
3. For the study of global illumination and imaging experiments, use microscope-compatible, high-performance objectives (40x) with apochromatic correction, flat field correction, and high numerical aperture.
   1. For global illumination-based studies of Src kinase, use 561/10 nm excitation and 595/40 nm emission filters to visualize FastLightR-Src-mCherry and use 640/20 nm excitation and 655LP emission filter to visualize stargazin-iRFP.
   2. For global illumination-based studies of bRaf kinase, use 514/10 nm excitation and 540/21 nm emission filters to visualize FastLightR-bRaf-Venus and use 561/10 nm excitation and 595/40 nm emission filters to visualize mCherry-ERK-2.
   3. Use a multiband polychroic mirror in all fluorescent imaging channels.
4. Depending on experimental conditions, consider the following recommendations.
   1. Image the cells for at least 10 min prior to illumination to establish a baseline activity of the cells.
      NOTE: This baseline is required to determine whether activation of FastLightR kinase by illumination induces any changes in the cell behavior or protein localization. One can also perform longer periods of basal imaging to provide greater statistical significance.
   2. Due to the rapid inactivation kinetics of FastLightRs, including many cells for imaging may cause the deactivation of Src/bRaf between stage positions and can attenuate its response. To avoid such error, image 4 cells/min while analyzing FastLightR-Kinase activity in the described protocol and image each selected cell every minute for a total of 110 min (Supplementary File 2).
   3. Use pulsed illumination over continuous illumination to reduce the potential phototoxic effects of blue light. Illumination for 12 s for each of the 4 cells/min is effective (Supplementary File 2, Figure 3A).
      NOTE: The illumination cycles should be empirically determined for different constructs and illumination source types.
   4. Turn off the Illumination during cell imaging to avoid bleed-through in the fluorescent channels.
      NOTE: Imaging too many cells at once will decrease the total illumination time available for the sample, leading to an attenuated response from insufficient activation.
5. After imaging, save the movies as a .TIF stack file format for analysis.
   NOTE: Due to the less uniform illumination with GFP epifluorescence compared to ring light, experiments using GFP epifluorescence will require more frequent illumination. This results in fewer cells being imaged per experiment.

6. Subcellular activation and imaging of FastLightR-Src (Figure 4)

1. Place the chamber onto a microscope stage pre-heated to 37 °C and select a single cell expressing both FastLightR-Src-mCherry and Stargazin-iRFP.
2. Select the specific regions (ROI) within the cell to illuminate. This study chooses a small area at the periphery of the selected cell.
   NOTE: For local illumination, a 445 nm laser focused by a TIRF module in FRAP mode or micro-mirror device controlled through TTL interface via the imaging software is used. Any patterned illumination system will work for these experiments. If a scanning-based system is used, determine the laser intensity and homogeneity for sufficient activation without damage to the cell.
3. Image the selected cell every minute for 20 min in the basal state before illumination, 50 min while illuminating locally, then 20 min after activation for a total of 90 min (Supplementary File 2).
4. For activation and deactivation, provide an appropriate amount of time suitable for the target protein to allow complete deactivation of the LightR protein. For FastLightR-Src, allow at least 10 min of deactivation for residual activity to ablate and allow 20 min deactivation in the demonstrated experimental setup for local illumination.
5. After imaging, save the movies as .TIF stack file format for analysis.

7. Cell spreading analysis

1. Prepare images for data processing and analysis. Ensure that the images are in a .TIF stack format. Save these directly from the imaging software or convert them using an open-source program.
2. Download the CellGeo script package from the supplementary data zipped files²⁶.
   NOTE: The CellGeo package contains several different scripts. Choose the desired package from the list based on the type of analysis as covered in protocol sections 7-9.
3. Open and run the script titled MovThresh to create a mask of the cell.
   1. Select File > Import (.tif) and select the Stargazin-iRFP images of the cells.
   2. Scan through the frames using the scroll bar in the bottom left corner. If the suggested threshold is appropriate, go to step 7.4.
   3. If the suggested threshold does not align with the cell's behavior, choose smoothed or custom curves underneath Curve Selection. Create the custom curve by scrolling through the frames and then adjusting the threshold on the right-hand side of the window.
   4. Click Re-Threshold.
   5. Click File > Save as masks, Change the name of the file, and save it using the Save as option.
4. Open the newly created stack file in an open-source image analysis program.
5. Select Image > Adjust > Threshold > Apply.
6. Select Analyze > Analyze particles > Check display results -> OK.
7. Calculate the change in area for each cell by dividing the area at any given time by the average area of the same cell prior to blue light irradiation.
8. Plot the area change as a line or bar graph.
9. Calculate the average value and 90% confidence intervals for each time point of all cells treated under the same conditions.

8. Cell edge dynamics analysis

1. Prepare the stack files using the steps described in protocol section 7.
2. Open and run the script titled ProActive.
   1. Select File > Import > Masks (.tif) and select the mask created through the MovThresh script.
   2. Select the activity of interest in analyzing in the bottom right corner (Protrusive, Retractive, or Total).
   3. Select the type of normalization. Typically, area normalization is used for these analyses.
   4. Select the beginning frame of reference using the slider on the left. The lag slider on the right will determine the time lag between twotime points used to measure the area gained and lost by the cell.
   5. If the selected frame and lag number are additively greater than the number of frames in the movie, the preview image will not display. Reset these values to equal less than the total number of frames.
3. Modify the thresholding for protrusion or retraction by checking the box for Smooth Curve under the Results section. This averages the thresholds over a specific window, which can resolve issues where minor fluctuations are detected as activity.
4. Select Run the Range to calculate the activity over the time specified by the frame and lag sliders.
5. Save the numerical data by selecting File > Save As > Results(.mat) and use it to determine mean protrusive or retractive activity.
6. Determine the 90% confidence intervals for each time point.
   NOTE: This will save an array of several different values. The "How to use ProActive" text file included with the script provides a detailed description of what each value represents and how they have been determined²⁶.
7. Save the visual representation of the activity by selecting File > Save As > Images (.tif).

9. Centroid shift analysis

1. For centroid shift analysis, use any software that can determine centroid coordinates.
2. In the analysis software, open the masked image stack of the cell of interest as described in protocol section 7.
3. Select Measure > Integrated Morphometry Analysis and then click Preferences > Draw Centroid Mark > Check, then select Measure.
   1. Record centroid coordinates before movement (A).
   2. Record centroid coordinates at the end of the experiment (B).
   3. Record the coordinates for the center of the illumination pattern (C).
4. Now that the three points have been collected, determine the angle BAC⁸. θ₁ corresponds to the angle ABC and θ₂ corresponds to the angle CBA (Figure 4Cii). Do this using the following equation:
   cosθ = cos(θ₁ - θ₂) = cosθ₁cosθ₂ + sinθ₁sinθ₂
   NOTE: A calculator can be used to determine the angles and distances directly from the coordinates for ease of use.
5. Determine the centroid displacement by multiplying the distance that the cell centroid traveled in pixels by the conversion factor where 1 pixel is equal to 0.4 µm. The specific conversion for pixels to distance will depend on the magnification and camera setup on the system.
6. Once the centroid shift angle and distance have been determined, open a graphing software capable of producing images of polar coordinates.
7. Insert the centroid shift angle as the θ measurement and the distance traveled as the radius. Utilize ANOVA to test for 95% confidence intervals.
8. Plot the results and save the image.

Results

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 kinase^11,12. 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'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 kinases^24,25 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).

Figure 1: 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 "megaprimer" 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. Please click here to view a larger version of this figure.

Figure 2: 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. Please click here to view a larger version of this figure.

Figure 3: 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 ± 90% confidence intervals. Panels B and C have been adapted and modified with permission from Shaaya et al.¹⁰. Please click here to view a larger version of this figure.

Figure 4: 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's migration distance (d) and angle of deviation (θ) 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's centroid from the initiation (1) to the conclusion (2) of local blue light stimulation. The angle of deviation (θ) indicates the extent of the cell'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. Please click here to view a larger version of this figure.

Figure 5: 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. Please click here to view a larger version of this figure.

Supplementary Table 1: LightR sequence. Please click here to download this File.

Supplementary Table 2: Description of LightR insertion sites and checklist for LightR-Protein design. Please click here to download this File.

Supplementary Table 3: Primers for LightR cloning. Please click here to download this File.

Supplementary Table 4: Transfection conditions for biochemical analysis of LightR-Protein activity. Please click here to download this File.

Supplementary Table 5: Transfection conditions for imaging-based analysis of LightR-Protein activity. Please click here to download this File.

Supplementary File 1: Pulsed illumination using Integrated Development Environment (IDE). Please click here to download this File.

Supplementary File 2: Microscope illumination settings. Please click here to download this File.

Discussion

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 heterodimerization^{29,30,31,32,33,34,35}. 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'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 manner^{36,37,38,39,40,41,42,43}. 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 modifications^10,18could 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 p130Cas^22,23. 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 biology^{11,12,44,45,46,47}. LightR-bRaf also demonstrates specificity in targeting signal complexes such as MEK and ERK kinases^24,25. 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 regulation^49,50. 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'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.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
#1.5 Glass Coverslips 25 mm Round	Warner Instruments	64-0715
1.5 mL Tubes	USA Scientific	cc7682-3394
2x Laemmli Buffer			For 500 mL: 5.18 g of  Tris-HCL, 131.5 mL of glycerol, 52.5 mL of 20% SDS, 0.5 g of bromophenol blue, final pH 6.8
4-20% Mini-PROTEAN TGX Precast Gel	Biorad	4561096
5x Phusion Plus Buffer	Thermo Scientific	F538L
60 LED Microscope Ring Light	Boli Optics	ML46241324	Blue LED, 60 mm diameter, 5 W
Agarose	GoldBiotech	A-201
Anti-Erk 1/2 Antibody	Cell Signaling	9102
Anti-GAPDH Antibody	invitrogen	AM4300
Anti-GFP	Clontech	632380
Anti-mCherry Antibody	invitrogen	M11217
Anti-MEK	Cell Signaling	9122
Anti-p130Cas	BD Biosciences	610271
Anti-paxillin	Cell Signaling	2542
Anti-phospho-Erk 1/2 T202/Y204 Antibody	Cell Signaling	9101
Anti-phospho-pY249 p130Cas	Cell Signaling	4014
Anti-phospho-Y118 Paxillin	Cell Signaling	2541
Anto-phospho-S217/221 MEK	Cell Signaling	9121
Arduino Compatable Power Supply	Corporate Computer	LJH -186
Arduino Uno Rev3	Arduino	‎A000066
Attofluor Cell Chamber	invitrogen	A7816
Benchmark Fetal Bovine Serum (FBS)	Gemini Bio-products	100-106	Heat Inactivated Triple 0.1 µm sterile-filtered
bRaf-V600E-Venus			Gift from Dr. John O'Bryan, MUSC
BSA	GoldBiotech	A-420
Carbenicillin (Disodium)	Gold Biotechnology	C-103-25
CellMask Deep Red plasma membrane dye	invitrogen	c10046
Colony Screen MasterMix	Genesee	42-138
DH5a competent cells	NEB	C2987H
DMEM	Corning	15-013-CV
DNA Ladder	GoldBio	D010-500
dNTPs	NEB	N04475
Dpn1 Enzyme	NEB	R01765
DTT	GoldBio	DTT10	DL-Dithiothreitol, Cleland's Reagents
EGTA	Acros	409910250
FastLightR-bRaf-mVenus	Addgene	#162155
Fibronectin from bovine plasma	Sigma	F1141
FuGENE(R) 6 Transfection Reagent	Promega	E2692	Transfection reagent
Gel Green Nucleic Acid Stain	GoldBio	G-740-500
Gel Loading Dye Purple 6x	NEB	B7024A
GeneJET Gel extraction Kit	Thermo Scientific	K0692	Gel Extraction Kit
GeneJET Plasmid Miniprep Kit	Thermo Scientific	K0502
Glutamax	Gibco	35050-061	GlutaMAX-l (100x) 100 mL
HEK 293T Cells	ATCC	CRL-11268
HeLa Cells	ATCC	CRM-CCL-2
HEPES	Fischer	BP310-500
Iot Relay	Digital Loggers	DLI 705020645490	AC/DC control relay for illumination
Kanamycin Monosulfate	Gold Biotechnology	K-120-25
KCl	Sigma	P-4504
L-15 1x	Corning	10-045-CV
LB Agar	Fisher	BP1425-2
LED Grow Light System	HQRP	884667106091218	LED panel lamp system
LightR-bRaf-mVenus	Addgene	#162154
LightR-iCre-miRFP670	Addgene	#162158
MATLAB	Mathworks	R2024a	Software for running CellGEO Scripts
Metamorph	Molecular Devices		Imaging Analysis Software
MgCl2	Fisher Chemical	M33-500
Mineral Oil	Sigma	M5310
MiniPrep Kit	Gene Choice	96-308
Mini-PROTEAN TGX Precast Gels 12 well	Bio-Rad	4561085
Molecular Biology Grade Water	Corning	46-000-CV
Multiband Polychroic Mirror	89903BS	Chroma
NaCl	Fisher Chemical	S271-3
PBS w/o Ca and Mg	Corning	21-031-CV
pCAG-iCre	Addgene	#89573
pcDNA3.1_Floxed-STOP mCherry	Addgene	#122963
pCEFL-ERK2			Gift from Dr. Channing Der's Lab, UNC
PCR Tubes	labForce	1149Z65	0.2 mL 8-Strip Tubes and Caps, Rigid Strip Individually Attached Dome Caps
Phusion Plus DNA Polymerase	Thermo Scientific	F630S
pmiRFP670-N1	Addgene	#79987
Polygon 400 Patterned Illuminator	Mightex	DSI-G-00C
Primers	IDT
PVDF Membranes	BioRad	1620219	Immun-Blot PVDF/Filter Paper Sandwiches
T0.25% Trypsin, 2.21 mM, eDTA, 1x [-] sodium	Corning	25-053-CI
Tris-Acetate-EDTA (TAE) 50x	Fischer	BP1332-1	For electrophoresis
UPlanSApo 40x Microscope Objective	Olympus	1-U2B828
USB TTL Box	National Instruments	6501	For TTL interface
β-Mercaptoethanol	Fisher Chemical	O3446I-100