National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda
Zheng, C., Petralia, R. S., Wang, Y., Kachar, B. Fluorescence Recovery After Photobleaching (FRAP) of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons. J. Vis. Exp. (50), e2568, doi:10.3791/2568 (2011).
FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached in the region of interest. The fluorescence of the region recovers when the unbleached GFP-tagged protein from outside of the region diffuses into the region of interest. The mobility of the protein is then analyzed by measuring the fluorescence recovery rate. This technique could be used to characterize protein mobility and turnover rate.
In this study, we express the (enhanced green fluorescent protein) EGFP vector in cultured hippocampal neurons. Using the Zeiss 710 confocal microscope, we photobleach the fluorescence signal of the GFP protein in a single spine, and then take time lapse images to record the fluorescence recovery after photobleaching. Finally, we estimate the percentage of mobile and immobile fractions of the GFP in spines, by analyzing the imaging data using ImageJ and Graphpad softwares.
This FRAP protocol shows how to perform a basic FRAP experiment as well as how to analyze the data.
1. Neuron transfection
2. FRAP experiment on a spine
3. Data analysis
4. Representative Results:
In this study, we perform a FRAP experiment on mature hippocampal neurons. At 18-22 DIV, mushroom spines are already formed. Using our method, the dynamic changes of the fluorescence intensity in a small region, such as a spine, can be recorded.
To analyze the fluorescence recovery process of EGFP, we take 5 images as controls before bleaching and then 1 image every 1 second immediately after bleaching for 15 seconds. The resolution of the image is sufficent for quantitative analysis. The fluorescence recovery profiles of tagged fluorescence proteins are highly reproducible.
We also briefly show how to define the mobile and immobile fractions of a fluorescence protein, using ImageJ and Graphpad Prism software. The FRAP method and analysis we show here can be broadly used in neuroscience, cell biology and other studies.
Figure 1. FRAP measurements of EGFP fluorescence in a spine from a cultured hippocampal neuron. The red arrowheads indicate the time of photobleaching. Photographs represent the same area before (Pre) and at 0, 1, 5, 10, 15 seconds after photobleaching. The region of the spine, control and background are marked with letters S, C and B, respectively. Neurons were maintained at 37°C during the experiment. Scale bar, 1 μm.
Figure 2. FRAP curves of EGFP fluorescence over a 15-second period. The green line shows the original curve; the red line shows the normalized curve. The dots on curves show the FRAP every 1 second. The curves were fitted by one-phase exponential equations. The average fluorescence before photobleaching was counted as 100%. In this experiment, the mobile fraction (MF) is 94% and the immobile fraction (IF) is 6%.
FRAP analysis has been broadly used in vivo and in vitro1-2studies. This technique commonly utilizes GFP fusion proteins, although it could also use red alga fusion proteins3. This analysis is sensitive and can be used to characterize the mobility of GFP-tagged proteins.
To produce meaningful FRAP analysis, it is important to avoid unnecessary photobleaching before and during the FRAP experiment. There are two ways to achieve this. First, the process to search and observe the experimental neuron should be fast. Especially, observation of neurons with a 100X objective for a long time significantly bleaches the fluorescence. Second, high laser power and frequent scanning often increase the possibility of photobleaching. Thus, it will be necessary to calculate the photobleaching rate in a control region and then normalize the FRAP curve in the experimental region. The normalizing method has been described in the protocol (see step 3.3-3.5 for details).
The photobleaching step is also critical for ensuring good FRAP results. In this experiment, we bleach the spine of interest 10 times at 100% laser transmission. This condition is sufficient to bleach the fluorescence of a spine to background level in a fixed preparation. Thus, we set the fluorescence intensity to the same number as background at 0 seconds after photobleaching. Depending on the speed of the first scan, a significant amount of fluorescence recovery might already be detected when the protein of interest is highly mobile.
Many fluorescent protein probes have been developed to study protein dynamics with FRAP. A complementary approach is, for example, to use photoactivatable GFP (PA-GFP), or photoconvertible variants. Together with FRAP technique, these tools are becoming indispensible for live cell imaging studies4-6.
No conflicts of interest declared.
This work was supported by the National Institute on Deafness and Other Communication Disorders (NIDCD) Intramural Program.
|35 mm glass-bottom dishes||MatTek Corp.||P35GC-1.0-14-C|
|CalPhos Mammalian Transfection Kit||Clontech Laboratories||631312|
|pEGFP-N1 plasmid||Clontech Laboratories||6085-1|
|Zeiss confocal microscope||Carl Zeiss, Inc.||LSM 710|
|Zeiss TempModule system||Carl Zeiss, Inc.||N.A.|
|ImageJ software||National Institutes of Health||N.A.|
|Graphpad Prism software||GraphPad Software Inc.||N.A.|