The overall goal of this procedure is to visualize plasma membrane insertion of neuronal proteins. This is accomplished by first infecting cultured hippocampal neurons with sinus virus to express the protein of interest, which is tagged with super ecliptic, Florin or sep. Next, a region of interest or ROI is photo bleached.
Then flanking segments are repetitively photo bleached while the original region of interest is allowed to fluorescently recover. Finally, the fluorescence recovery in the region of interest is analyzed. Ultimately, this adaptation to conventional frap experiments is used to show the pattern of exocytosis events of the target protein in the plasma membrane.
The main advantage of this technique over existing methods like surface bio tonation or antibody feeding, is that it enables the dynamics of membrane insertion events to be visualized. These methods can help addressing key questions in protein trafficking sets us where and how exocytosis events occurs. I first had the idea of this meted when I was looking for an alternative to turf aging and to be able to study exocytosis sevens in spines in live cell experiments I To begin culture high density hippocampal neurons from embryonic day 18 wrapped pups on polyol lysine coated glass cover slips for 14 to 25 days in vitro six to 24 hours prior to the live experiment.
Transduce the cells with symbis virus encoding the membrane protein of interest, typed with the SUP ecliptic fluorine osep. Add the pseudo virion, contain medium directly to the cover slip containing one milliliter of conditioned medium and return the cells to the culture incubator. To image the cells switch on a zes axia vert LSM five 10 meta confocal microscope with 100%laser output for at least 20 minutes prior to imaging.
To minimize power fluctuations during imaging and to prewarm the stage. When the microscope is ready, select the 63 times objective, then transfer the cover slip to the imaging chamber, and immediately replace the culture medium. With Prewarm extracellular recording solution.
Place the chamber on the preheated microscope stage. The most difficult aspect of this procedure is ensuring that the health of the cells is not compromised during the recording process. As this protocol requires extensive photo bleaching to ensure success, make sure all imaging parameters are optimized before beginning large scale experiments and favor rapid acquisition over high quality images.
To define the imaging capture parameters, identify a neuron expressing a recombinant protein of interest, and bring it into focus. Scan the entire cell using a 4 88 nanometer laser at low power. To minimize photobleaching.
Use a fast nominal speed and low pixel resolution. Keeping the total scan speed at less than one second. Select a portion of the dendrite to image and using an optical zoom of 1.5 to 2.5.
Capture a frame containing the region of interest to determine if non-specific photo bleaching is occurring. Try to ensure the field of view contains several processes so that measurements from reference dendrites can be obtained. Using a range indicator palette, adjust the imaging parameters to enable maximal fluorescence from minimal laser excitation, but with limited saturation.
A large pinhole diameter is recommended to maximize photon collection. The detector gain should be strong enough to detect small fluorescence increments such that the very first images before the photobleaching do not overcome 10%of saturated pixels. Adjust the focus to ensure the midline focal plane is achieved.
After setting the configuration, save it to be used for the pre and postle recovery phases of the experiment. Next, define the photo bleach regions. By selecting a region of interest or ROI for the initial photo bleach and flanking regions for the subsequent repetitive photobleaching phase.
For the initial photo bleach, both the central and flanking ROIs will be photo bleached. Whereas for the repetitive photobleach, only the flanking ROIs are selected. Ensure that the flanking regions are wide enough to prevent recovery by diffusion between scans.
Adjust the bleach parameters for both photobleach ROIs. The initial photo bleach should be rapid requiring between one to five iterations, depending on the optical zoom and the volume of the ROI For the flanking regions, the laser excitation should be adjusted to ensure continuous photo bleaching, but without phototoxic damage. Once the parameters have been set, perform a FRA flip experiment as a variable time-lapse image sequence by first acquiring three to 10 preble baseline images at low laser power with no time delay.
Next, photobleach the central ROI at full laser power for one to five iterations. Then acquire three to 10 post bleach recovery images. Perform a repetitive photo bleach of the flanking regions of medium laser power with image capture at a typical time interval of one to five seconds depending on the recovery rate of the protein under investigation.
Finally, to quench the fluorescence, replace the extracellular recording solution with recording solution buffered at pH six, or to reveal the protein in low pH intracellular stores, use a buffer complimented with 50 millimolar ammonium chloride. Collect at least 10 to 20 data sets for each recombinant protein to enable statistical analysis to avoid bias results, ensure imaging conditions are maintained across replicates. To analyze the images, open them.
Using Image J software, align the stacks to account for small fluctuations in the XY plane that may have occurred throughout the time series. Using the stack reg plugin for images taken on a zes confocal, use the LSM toolbox plugin to report the time values as a text file and import these values into an analysis spreadsheet to measure the fluorescence fluctuations. During the FRA flip experiment, divide the photo bleach segment into individual pixel regions using the image ROI manager analysis tool to select multiple ROIs.
Next, use the plot Z axis profile command to measure the mean fluorescence per pixel At each time point. Repeat the process to measure the mean fluorescence intensity of a background non-flu fluorescent region. Normalize the fluorescence intensity at each time point by subtracting the background values to remove experimental noise and divide all values by the mean pre bleached baseline measure or the immediate post bleach value.
Examine the recovery trace for each ROI plotted against time to calculate if significant recovery has taken place. In an ROI calculate delta F by subtracting the mean of the first five to 10 measures of the photobleach recovery sequence from the mean of the final five to 10 measures. Then determine the statistical significance of the delta F and characterize the recovering and non recovering R ois to assess the pattern of exocytosis.
That is exocytosis hotspots or spine versus shaft recovery. This video shows the representative outcome of a typical frap flip experiment using SERP glue. A two.
A region of dendrite is photo bleached once followed by a one minute recovery period. The repetitive photo bleaching of the flanking regions is then initiated, indicated by the blue arrows. The red arrow highlights a small region of interest where fluorescence recovery is observed, shown enlarged in the right hand panel of the screen.
The recovery is tracked in the graph below with an overall increase in fluorescence marked as delta F.The increase in fluorescence observed over this sequence can be attributed to insertion of glue A two in the dendritic shaft. The large changes in fluorescence intensity at the end of the experiment show the effect of the low pH and the pH 7.4 plus ammonium chloride washes on the sep fluorescence signal. In this example, the neurons have been infected with SEP glue K two.
Here, a large ROI with multiple dendrites in the field of view has been photo bleached, followed by selective repetitive photobleaching of only one dendrite. These neurons were treated with cyclo heide for two hours prior to imaging to block protein synthesis. As such, fluorescence recovery in the respective measured ROIs reveals the proportion of recovery due to lateral diffusion and recycling in the standard frap dendrite versus recycling alone in the dendrites subjected to the frap flip protocol.
Comparing the delta F values from the frap versus frap flip recovery curves, the relative contributions of recycling versus lateral diffusion can be inferred due to the inhibition of protein synthesis. The recovery shows a subsequent drop-off in signal as the available receptor pool is depleted. After watching this video, you should have a good understanding of how to set up, perform, and analyze successful imaging experiments in neurons.
Using this combination of photobleaching techniques, you can study membrane insertion evidence for your protein of interest.
