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