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

Summary

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.

Abstract

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.

Introduction

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 degradation^1,2. 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 regeneration^4,

Protocol

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

1. Coat a cell culture plate with 0.1% gelatin in PBS and incubate at 37 °C for 4 h or at 4 °C overnight. Remove the gelatin and allow it to dry under sterile laminar air flow.
2. Prepare cell culture medium composed of 50 mL of DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S). Pre-warm it to 37 °C.

2. Isolation ....

Results

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 α-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 quantif.......

Discussion

miRNAs are key players in cellular physiology and were shown to act as signaling molecules by using—among others—GJs as a pathway for intercellular exchange^11,12,22. 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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
neonatal NMRI mice	Charles River
Gelatin	Sigma Aldrich	G7041	0.1% solution in PBS, sterilized
PBS	Pan Biotech	P04-53500
Dulbecco´s modified medium	Pan Biotech	P04-03550
Penicillin/Streptomycin	Thermo Fisher Scientific	15140122	100 U/mL, 100µg/mL
Fetal bovine serum	Pan Biotech	P30-3306
Cell culture plastic	TPP
Primary Cardiomyocyte Isolation Kit	Thermo Fisher Scientific	88281
HBSS	Thermo Fisher Scientific	88281	included in primary cardiomyocyte isolation kit
Trypan blue	Thermo Fisher Scientific	15250061
miRIDIAN Dy547 labeled microRNA Mimic	Dharmacon	CP-004500-01-05	dissolve in RNAse free water, stock solution 20µM
Sodium phosphate monobasic	Sigma	S3139
Sodium phosphate dibasic	Carl Roth	T877.2
Potassium chloride	Sigma	P9333
Magnesium chloride	Serva	28305.01
Sodium succinate	Carl Roth	3195.1
0.05% Trypsin/EDTA solution	Merck	L2153
4-well- Glass bottom chamber slides	IBIDI	80827	coat with 0.1% gelatin solution
Amaxa Nucleofector II	Lonza		program G-009 was used for Electroporation
LSM 780 ELYRA PS.1 system	Zeiss
Excel software	Microsoft
α-actinin antibody	Abcam	ab9465	dilution 1:200
Connexin43 antibody	Santa Cruz	sc-9059	dilution 1:200
goat anti-mouse Alexa 594 antibody	Thermo Fisher Scientific	A-11005	dilution 1:300
goat anti-rabbit Alexa 488 antibody	Thermo Fisher Scientific	A-11034	dilution 1:300
Connexin43 siRNA	Thermo Fisher Scientific	AM16708 ID158724	final concentration 250 nM
Near-IR Live/Dead Cell Stain Kit	Thermo Fisher Scientific	L10119
CellTrace Calcein Red-Orange	Thermo Scientific	C34851
DAPI nuclear stain	Thermo Scientific	D1306