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 ...

Research

JoVE Journal

Biology

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion ...

Detection of miRNA Targets in High-throughput Using the 3'LIFE Assay

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an ...

Real-time Imaging of Plant Cell Surface Dynamics with Variable-angle Epifluorescence Microscopy

A plant&#8217;s cell surface is its interface for perceiving environmental cues; it responds with cell biological changes such as membrane trafficking and ...

Open-source Single-particle Analysis for Super-resolution Microscopy with VirusMapper

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm ...

Biological Sample Preparation by High-pressure Freezing, Microwave-assisted Contrast Enhancement, and Minimal Resin Embedding for Volume Imaging

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the ...

Tethered Bilayer Lipid Membranes to Monitor Heat Transfer between Gold Nanoparticles and Lipid Membranes

Here we report a protocol to investigate the heat transfer between irradiated gold nanoparticles (GNPs) and bilayer lipid membranes by electrochemistry ...

Application of Passive Head Motion to Generate Defined Accelerations at the Heads of Rodents

Exercise is widely recognized as effective for various diseases and physical disorders, including those related to brain dysfunction. However, molecular ...

Measurement of Liver Stiffness Using Atomic Force Microscopy Coupled with Polarization Microscopy

Matrix stiffening has been recognized as one of the key drivers of the progression of liver fibrosis. It has profound effects on various aspects of cell ...

Fluorescence Lifetime Macro Imager for Biomedical Applications

This paper presents a new photoluminescence lifetime imager designed to map the molecular oxygen (O2) concentration in different phosphorescent samples ...

Imaging of Mitochondrial Peroxide in Living Yeast Cells Using mtHyper7

Imaging of mtHyPer7, a Ratiometric Biosensor for Mitochondrial Peroxide, in Living Yeast Cells

Advancing Mitochondrial Research Â– mtHyper7 Biosensor for Subcellular Analysis (Video) | JoVE

Mitochondrial dysfunction, or functional alteration, is found in many diseases and conditions, including neurodegenerative and musculoskeletal disorders, ...