Name: analysis of the gap junction-dependent transfer of mirna with 3d-frap microscopy
Uploaded: 2017-06-19T14:00:00
Description: Watch this Scientific Journal Video about Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy at JoVE.com

This work was supported by the Federal Ministry of Education and Research Germany (FKZ 0312138A and FKZ 316159), the State Mecklenburg-Western Pomerania with EU Structural Funds (ESF/IVWM-B34-0030/10 and ESF/IVBM-B35-0010/12), the DFG (DA1296-1), and the German Heart Foundation (F/01/12). In addition, R.D. is supported by the FORUN Program of Rostock University Medical Centre (889001), the DAMP Foundation, and the BMBF (VIP+ 00240).

All steps in this protocol involving neonatal mice were performed per the ethical guidelines for animal care of the Rostock University Medical Centre.
1. Preparation of Cell Culture Dishes and the Medium for Cardiomyocyte Culture
<ol>
	<li>Coat a cell culture plate with 0.1% gelatin in PBS and incubate at 37 &#176;C for 4 h or at 4 &#176;C overnight. Remove the gelatin and allow it to dry under sterile laminar air flow.</li>
	<li>Prepare cell culture medium composed of 50 mL of DMEM suppleme...

<ol>
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miRNAs are key players in cellular physiology and were shown to act as signaling molecules by using&#8212;among others&#8212;GJs as a pathway for intercellular exchange11,12,22. The current protocol presents an in vitro live-cell imaging technique to characterize this GJ-dependent shuttling using fluorescent miRNAs within cell clusters.
The protocol was developed on cardiomyocytes as a cell m...

Small noncoding RNAs are important players in cellular gene regulation. These molecules are composed of 20-25 nucleotides that bind to a specific target mRNA, leading to the blockage of translation or to mRNA degradation1,2. The gene regulatory process undertaken by small RNAs, such as miRNA and siRNA, is a highly conserved mechanism that has been found in many different species3. In particular, miRNA molecules are of crucial importance to a variety of physiological processes, including proliferation, differentiation, and regeneration4,5. In addition, the dysregulation of miRNA expression is attributed to many pathological disorders. Correspondingly, miRNAs have been demonstrated to be suitable as biomarkers for diagnosis and as therapeutic agents for gene therapy6,7.
Gap junctions (GJs) are specialized protein structures in the plasma membrane of two adjacent cells that allow the diffusional exchange of molecules with a molecular weight of up to 1 kD. They have been shown to be important to tissue development, differentiation, cell death, and pathological disorders such as cancer or cardiovascular disease8,9,10. Several molecules have been described as being capable of crossing GJ channels, including ions, metabolites, and nucleotides. Interestingly, GJs were also found to provide a pathway for the intercellular movement of small RNAs11,12. Thus, miRNAs can act not only within the cell in which they are produced, but also within recipient cells. This highlights the role of miRNAs in the intercellular signal transduction system. At the same time, the data demonstrate that gap junctional intercellular communication is closely linked to miRNA function. Because of the significant impact of miRNA and GJs on tissue homeostasis, pathology, and diagnosis, a comprehensive understanding of the function of GJs and the related intercellular dynamics of miRNA will help to clarify the mechanisms of miRNA-based diseases and to develop new strategies for miRNA therapies.
Depending on the extent of gap junctional coupling, the transfer of miRNA molecules between cells can be a very rapid process. Therefore, a methodology that allows the visualization and quantification of the fast intercellular movement of these regulatory signaling molecules is required. Commonly, flow cytometry and molecular biological techniques have been applied to demonstrate the shuttling of small RNAs11,12,13,14. However, as opposed to FRAP microscopy, these approaches lack high temporal resolution, which is mandatory when analyzing the exchange of miRNA via GJs. Moreover, FRAP microscopy is less invasive and therefore represents a powerful and novel live-cell imaging technique to evaluate the GJ-dependent exchange of molecules in several cell types15,16,17.
Here, we present a detailed protocol describing the application of 3D-FRAP to assess miRNA shuttling between cardiomyocytes. For this purpose, cardiomyocytes were transfected with fluorescently labeled miRNA. A cell marked with this miRNA was photobleached, and the gap junctional miRNA re-influx from adjacent cells was recorded in a time-dependent manner. The high temporal resolution of FRAP experiments offers the possibility to perform kinetic studies for the precise evaluation of the intercellular transfer of miRNA and siRNA between living cells. Moreover, as small RNAs can be exchanged via different mechanisms with highly different kinetics, FRAP microscopy can help to clarify the extent to which GJs are involved in the respective shuttling processes18. In addition, 3D-FRAP can be used to investigate physiological and pathological alterations in GJ permeability and its impact on small RNA transfer15,19.

<table border="0" cellpadding="0" cellspacing="0" width="698">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">neonatal NMRI mice</td>
			<td style="width:159px;">Charles River</td>
			<td style="width:119px;"></td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Gelatin</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:119px;">G7041</td>
			<td style="width:179px;">0.1% solution in PBS, sterilized</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">PBS</td>
			<td style="width:159px;">Pan Biotech</td>
			<td style="width:119px;">P04-53500</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Dulbecco&#180;s modified medium</td>
			<td style="width:159px;">Pan Biotech</td>
			<td style="width:119px;">P04-03550</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Penicillin/Streptomycin</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">15140122</td>
			<td style="width:179px;">100 U/ml, 100&#181;g/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Fetal bovine serum</td>
			<td style="width:159px;">Pan Biotech</td>
			<td style="width:119px;">P30-3306</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Cell culture plastic</td>
			<td style="width:159px;">TPP</td>
			<td style="width:119px;"></td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Primary Cardiomyocyte Isolation Kit</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">88281</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:243px;">HBSS</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">88281</td>
			<td style="width:179px;">included in primary cardiomyocyte isolation kit</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Trypan blue</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">15250061</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:243px;">miRIDIAN Dy547 labeled microRNA Mimic&#160;</td>
			<td style="width:159px;">Dharmacon</td>
			<td style="width:119px;">CP-004500-01-05</td>
			<td style="width:179px;">dissolve in RNAse free water, stock solution 20&#181;M</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:243px;">Sodium phosphate monobasic</td>
			<td style="width:159px;">Sigma</td>
			<td style="width:119px;">S3139</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Sodium phosphate dibasic</td>
			<td style="width:159px;">Carl Roth</td>
			<td style="width:119px;">T877.2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Potassium chloride</td>
			<td style="width:159px;">Sigma</td>
			<td style="width:119px;">P9333</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Magnesium chloride</td>
			<td style="width:159px;">Serva</td>
			<td style="width:119px;">28305.01</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Sodium succinate</td>
			<td style="width:159px;">Carl Roth</td>
			<td style="width:119px;">3195.1</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">0.05% Trypsin/EDTA solution</td>
			<td style="width:159px;">Merck</td>
			<td style="width:119px;">L2153</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">4-well- Glass bottom chamber slides</td>
			<td style="width:159px;">IBIDI</td>
			<td style="width:119px;">80827</td>
			<td style="width:179px;">coat with 0.1% gelatin solution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Amaxa Nucleofector II</td>
			<td style="width:159px;">Lonza</td>
			<td style="width:119px;"></td>
			<td style="width:179px;">program G-009 was used for Electroporation&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">LSM 780 ELYRA PS.1 system</td>
			<td style="width:159px;">Zeiss</td>
			<td style="width:119px;"></td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Excel software</td>
			<td style="width:159px;">Microsoft</td>
			<td style="width:119px;"></td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">&#945;-actinin antibody</td>
			<td style="width:159px;">Abcam</td>
			<td style="width:119px;">ab9465</td>
			<td style="width:179px;">dilution 1:200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Connexin43 antibody</td>
			<td style="width:159px;">Santa Cruz</td>
			<td style="width:119px;">sc-9059</td>
			<td style="width:179px;">dilution 1:200</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">goat anti-mouse Alexa 594 antibody</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td>A-11005</td>
			<td>dilution 1:300</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">goat anti-rabbit Alexa 488 antibody&#160;</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td>A-11034</td>
			<td>dilution 1:300</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Connexin43 siRNA</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">AM16708 ID158724</td>
			<td>&#160;final concentration 250nM</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Near-IR Live/Dead Cell Stain Kit</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">L10119</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">CellTrace Calcein Red-Orange</td>
			<td style="width:159px;">Thermo Scientific</td>
			<td style="width:119px;">C34851</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">DAPI nuclear stain</td>
			<td style="width:159px;">Thermo Scientific</td>
			<td style="width:119px;">D1306</td>
			<td style="width:179px;"></td>
		</tr>
	</tbody>
</table>

analysis of the gap junction-dependent transfer of mirna with 3d-frap microscopy

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents in the treatment of several diseases. The development of new, innovative strategies for miRNA/siRNA therapy is based on an extensive knowledge of the underlying mechanisms. Recent data suggest that small RNAs are exchanged between cells in a gap junction-dependent manner, thereby inducing gene regulatory effects in the recipient cell. Molecular biological techniques and flow cytometric analysis are commonly used to study the intercellular exchange of miRNA. However, these methods do not provide high temporal resolution, which is necessary when studying the gap junctional flux of molecules. Therefore, to investigate the impact of miRNA/siRNA as intercellular signaling molecules, novel tools are needed that will allow for the analysis of these small RNAs at the cellular level. The present protocol describes the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) microscopy to elucidating the gap junction-dependent exchange of miRNA molecules between cardiac cells. Importantly, this straightforward and non-invasive live-cell imaging approach allows for the visualization and quantification of the gap junctional shuttling of fluorescently labeled small RNAs in real time, with high spatio-temporal resolution. The data obtained by 3D-FRAP confirm a novel pathway of intercellular gene regulation, where small RNAs act as signaling molecules within the intercellular network.

Here, we present 3D-FRAP microscopy as a non-invasive technique to study the gap junctional shuttling of fluorescent miRNA within neonatal cardiomyocytes. The isolated cardiomyocytes revealed the typical striated &#945;-actinin pattern and contained large plaques of Cx43 along the cell-cell borders (Figure 1A, white arrowheads), which allowed for the high intercellular flux of molecules. The purity of isolated cardiomyocytes was assessed by the microscopic qu...

Watch this Scientific Journal Video about Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy at JoVE.com

Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy

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.

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents ...

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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