The overall goal of the following experiment is to evaluate the presence of protein protein interactions on the cell surface. This is achieved by cloning two receptors of interest, a myo terminal to two fluorophores that have overlapping emission and spectra as a second step. The two vectors are simultaneously transfected into endothelial cells for receptor expression.
At this point, the fluorophores aid in evaluating the overall extent of expression as well as receptor localization. Next, the imaging parameters are established using single transfect as a control on the confocal microscope. Individually, transfected cells are selected based on their relative levels of receptor expression and localization.
Regions within these cells are then imaged in order to evaluate the extent of fret results are obtained. That show tie one and tie two interactions on the endothelial cell surface based on fre and confocal Microscopy. Hi, I'm Tom Seger from the laboratory of Dr.William Barton in the Department of Biochemistry and Molecular Biology at Virginia Commonwealth University.
Today we'll show you a procedure on evaluating a protein protein interactions in endothelial cells by a fret based confocal microscopy. In our lab, we use this to study receptor interactions during angiogenesis. So let's get started.
To image protein protein interactions using a fret based proximity assay begin by cloning your receptors of interest into the NHE one eco R one sites of the pcd, a 3.1 hydro or neo vector containing monomeric CFP or YFP. The determination of fret is empirical and is critically dependent on the length of the linker between the transmembrane spanning region and the start of the Fluor. Therefore, several different links of linker must be explored for each new receptor pair Under investigation, transform and amplify the vectors according to the accompanying protocol.
Then prepare transfection grade DNA, transform and amplify the vectors according to the accompanying protocol. Then prepare transfection grade DNA and dilute it to a working concentration of one microgram per microliter to prepare cells for transfection under a tissue culture hood split EAHY 92 6 cells grown in complete DMEM into cover slip line 35 millimeter dishes with two milliliters of medium. Prepare one dish to evaluate expression of each individual receptor as well as one dish for each receptor pair.
Allow the cells to incubate over 95%carbon dioxide atmosphere at 37 degrees Celsius the following day. The culture should be around 70 to 90%confluence on the day of transfection. Prepare nucleo lipid complexes by mixing two micrograms of the chimeric receptor vector DNA with eight microliters of Fuji N HD in 200 microliters of prewarm optimum for receptor pairs transfect with one microgram of each receptor for a total of two micrograms mixed gently by inversion and incubate it room temperature for 30 minutes.
During the incubation period, prepare cells for transfection aspirate off fresh media and wash each plate gently and briefly with warm PBS before the addition of two milliliters of pre-war complete DMEM without antibiotics. It is important to avoid the presence of penicillin and streptomycin as they're considerably more toxic to cells during treatment With FU gene HD at the complex dropwise to cells And incubate for an additional 20 to 24 hours. After 24 hours, Carefully aspirate the medium from the cells and pipette fresh complete DMEM with antibiotics following transfection.
Cell viability should remain relatively high. Few to no floating Cells should be observed. To perform live cell imaging, We use a like A-T-C-S-P two A OBS confocal laser scanning microscope equipped with blue diode argon and green, orange and red helium neon lasers, an H-C-X-P-I-A-O 63 x 1.3 NA glycerin immersion, a motorized XY stage and a temperature, humidity and carbon dioxide controlled stage incubator.
Experimental controls are crucial to eliminate Fluor crosstalk between the emission channels as well as to evaluate expression issues and non-specific Fluor four ization. As such, single fluoro four transfect are utilized for setting up every image session to eliminate crosstalk between the emission channels for CFP and YFP. Place the CFP expression control into the stage incubator using white light.
Adjust the Z plate to focus on the cells. Set SP window settings for emission detection of CFP to 465 to 505 and YFP to 525 to 600 nanometers while monitoring both emission channels excite the CFP at 458 nanometers. Using the A OTF set the laser power to eliminate appreciable bleed over of CFP into the YFP emission channel.
Save this setting. Perform the same control for YFP crosstalk in the CFP channel by adjusting the excitation power at 514 nanometers. The cells expressing only YFP save this setting.
Next, next place, cells coex expressing CFP and YFP into the stage incubator. Using the sequence setting for the LICO confocal software. Locate cells expressing similar levels of CFP and YFP with proper membrane localization using the LICO software except a software accepter photo bleaching program.
Set the donor and accepter settings to your saved CFP And YFP settings respectively. Zooming onto the cell, highlight a Region of interest or ROI in which the photo destruction of YFP is to occur on the cellular membrane And begin the program For photo destruction of the acceptor. Adjust the A OTF to 100%of 514 nanometers and allow the laser bleaching to continue until 70%of the YFP emission has been depleted.
To accelerate bleaching ROIs are typically chosen in the raster axis of the laser during photobleaching. Monitor the cellular movement and reject measurements obtained with appreciable movement in the XY plane. As these measurements can report false fret efficiencies, pre and post bleach images are recorded for both donor and acceptor and fret efficiency is calculated as fret efficiency equals open parent post minus DLO parentheses divided by depost for all depost greater than D pre where D, pre and D post are the donor intensities before and after photobleaching respectively.
JMP is used to determine the extent of Experimental variability. Individual receptors Should be fused to both CFP and YFP with varying lengths of juxta membrane sequence. To identify the best possible pair of receptor fret partners transfection efficiencies are typically between 30 to 40%despite previous findings by others.
We have not seen that transfection and expression of one vector favors that of the other. Indeed, we frequently observe exclusive expression of one flora, four KY mirror or the other. These representative images show an acceptor photobleaching analysis of fret occurring between CFP and YFP in an EAHY 9 26 cell.
The CFP and YFP emission channels were monitored separately before and after photobleaching photo bleaching and calculations were restricted to the region. Within the red box fret efficiency is displayed as an absolute range from high colored red at 1.0 to low colored purple at zero on a magnified overlay of A-C-F-P-Y-F-P merged image for orientation purposes only for the T receptor system. Typical fret efficiency values are 20 to 28%for epithelial cells and 19 to 23%for endothelial cells As shown here.
So once mastered, this technique will typically take three to four days if performed properly. While attempting this procedure, it's important to remember that the linker length between your target protein and fusion fluorescent protein, as well as their expression levels and cellular movement directly affect the fret measurement following their procedure. Further, biochemical assays regarding signal activation can be performed in order to gain insight into signal transduction pathways after its development.
This technique provides an assay in which protein protein interactions that are either weak or transient can be studied. After watching this video, you should have a good understanding as to how to perform your own fret based proximity assays and live cells. And don't forget that while working with cells in tissue culture, appropriate safety precautions should always be followed.
So thank you for watching and good luck with your future fret experiments.
