Biology
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Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy
Chapters
Summary June 19th, 2017
Here, we describe the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) for the analysis of the gap junction-dependent shuttling of miRNA. In contrast to commonly applied methods, 3D-FRAP allows for the quantification of the intercellular transfer of small RNAs in real time, with high spatio-temporal resolution.
Transcript
The overall goal of this procedure is to investigate the gap junction-dependent transfer of micro RNA in living cardiomyocytes. This method can help to address the role of micro RNA for the intercellular signal transaction system. The main advantage of this technique is that the gap junction exchange of micro RNA can be visualized and quantified in life cells with high spatial temporal resolution.
After isolating cardiomyocytes according to the text protocol, plate the cells on six well plates at a density of three times 10 to the fifth cells per square centimeter. Incubate the cells overnight at 37 degrees celsius and 5%carbon dioxide. The following day, add 0.5 trypzine to the cells and incubate the cultures for five minutes to detach the cells.
Inactivate the trypzine by adding cell culture medium. After counting the cells, centrifuge them at 300 times g for 10 minutes. Then use electroporation buffer to re-suspend the cells at four times 10 to the fifth cells per 100 microliters.
Next, mix 100 microliters of cell suspension with fluorescent micro RNA in a tube and load the mixture into an electroporation cuvette. Then carry out electroporation using the G009 program on the electroporator. Add 500 microliters of pre-warmed cell culture medium and transfer the entire volume of cell suspension into a well of a four well glass bottom chamber slide.
Culture the cells for one day at 37 degrees celsius, in a 5%carbon dioxide atmosphere. After pre-warming the microscope incubator and turning on the confucal system, insert the chamber slide into the stage sample holder. Use a 1.4 na oil objective and 561 nanometer laser excitation light, at low laser power, with the detection range of 570 to 680 nanometers to find the cluster of transvected cardiomyocytes.
Next, in the setup manager, activate the z-stack, time series, bleaching and regions buttons. Then, to define the frap parameters, in the acquisition mode menu, set the frame size to 512 by 512, the line step to one and the scan time to less than one second. In the channels menu, adjust the laser power, offset and gain settings to obtain maximal fluorescence from minimal laser excitation, to avoid intensity saturation.
Then, set the pinhole size to two micrometers. Next, in the regions menu, select the roy drawing tool and use the cursor to mark the target cell, the reference cell and the background area. If required, select several target cells for photo bleaching.
In the bleaching menu, adjust the photo bleaching settings. Then, in the z-stack menu, define the limits for z-stack acquisition, depending on the thickness of the cells. Adjust the number of z layers to 12 tp 15, then, start the frap experiment and record the fluorescence recovery.
To analyze the data, create maximum projection of the acquired z-stacks using microscope specific software, click processing, maximum intensity projection, select file, apply. Finally, copy the fluorescence intensity data at all time points from the target, the background and the reference cell, into a spreadsheet and perform data analysis according to the text protocol. Shown here, are isolate cardiomyocytes with the typical striated alpha actinin pattern and large plaques of connexin43, along the cell to cell borders.
Some non-cardiomyocytes were present in the culture. As seen here, by flow cytometry, a 45%transvection efficiency was reached. In addition, a live-dead essay, revealed that the majority of cells demonstrated high viability, which is important when analyzing gap junctional communication.
For frap analysis, target cells are selected within a cell cluster and are photo bleached with 100%laser power, leading to reduced micro RNA fluorescence in the selected cells. Quantitative analysis and normalization of the acquired data, showed an average recovery of 20%In this experiment, siRNA mediated knockdown of connexin43, led to reduced protein expression and the inhibition of gap junctional communication. Fluorescence recovery was reduced by more than 50%Indicating that the efficiency of miro RNA transfer is strongly dependent of gap junctional coupling between cells.
This figure shows a 3D surface rendering of a frap experiment. Compared to maximum projections, the acquisition of z-stacks can be used to create 3D reconstructions to investigate the localisation and mobility of micro RNA within cells. Once mastered, frap measurements can be done in two to three hours if it is performed properly.
While attempting this procedure, it is important to choose target cells which has the similar number of neighboring cells. Moreover, my bleaching parameters and image acquisition conditions should be selected to avoid phototoxic effects. After watching this video, you should have a good understanding of how to use frap microscopy, in order to quantify and visualize the movement of micro RNA within cellular clusters.
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