Study of Endoplasmic Reticulum and Mitochondria Interactions by In Situ Proximity Ligation Assay in Fixed Cells

Summary

Here, we describe a procedure to visualize and quantify with high sensitivity the endogenous interactions between the endoplasmic reticulum and mitochondria in fixed cells. The protocol features an optimized in situ proximity ligation assay targeting the inositol 1,4,5-triphosphate receptor/glucose-regulated protein 75/voltage-dependent anion channel/cyclophilin D complex at the mitochondria-associated membrane interface.

Abstract

Structural interactions between the endoplasmic reticular (ER) and mitochondrial membranes, in domains known as mitochondria-associated membranes (MAM), are crucial hubs for cellular signaling and cell fate. Particularly, these inter-organelle contact sites allow the transfer of calcium from the ER to mitochondria through the voltage-dependent anion channel (VDAC)/glucose-regulated protein 75 (GRP75)/inositol 1,4,5-triphosphate receptor (IP3R) calcium channeling complex. While this subcellular compartment is under intense investigation in both physiological and pathological conditions, no simple and sensitive method exists to quantify the endogenous amount of ER-mitochondria contact in cells. Similarly, MAMs are highly dynamic structures, and there is no suitable approach to follow modifications of ER-mitochondria interactions without protein overexpression. Here, we report an optimized protocol based on the use of an in situ proximity ligation assay to visualize and quantify endogenous ER-mitochondria interactions in fixed cells by using the close proximity between proteins of the outer mitochondrial membrane (VDAC1) and of the ER membrane (IP3R1) at the MAM interface. Similar in situ proximity ligation experiments can also be performed with the GRP75/IP3R1 and cyclophilin D/IP3R1 pairs of antibodies. This assay provides several advantages over other imaging procedures, as it is highly specific, sensitive, and suitable to multiple-condition testing. Therefore, the use of this in situ proximity ligation assay should be helpful to better understand the physiological regulations of ER-mitochondria interactions, as well as their role in pathological contexts.

Introduction

Mitochondria and endoplasmic reticulum (ER) are not independent organelles in the cell, but they interact structurally and functionally at contact sites defined as mitochondria-associated endoplasmic reticulum membranes (MAM). In fact, MAMs correspond to regions where the membranes of the ER and mitochondria are closely apposed, allowing interactions between proteins from both sides. Nonetheless, the membranes of these organelles do not fuse within these regions, so they maintain their separate entities. The MAMs play a crucial role in calcium (Ca²⁺) and phospholipid transfer from ER to mitochondria, impacting energy metabolism and cell survival^1-3

Protocol

1. Preparation of Solutions

1. Prepare 10% formaldehyde in PBS (low salt) by diluting 5.5 ml of 37% formaldehyde in 14.5 ml of PBS. Prepare 1 M glycine, pH 8.0, by dissolving 3.8 g of glycine in 50 ml of PBS; dilute this solution to obtain 100 mM glycine in 1x PBS.
2. Prepare 0.1% Triton-X100 in 1x PBS. Prepare 20x Saline Sodium Citrate (SSC) by using 3.0 M sodium chloride and 0.30 M trisodium citrate prepared in a deionized water buffer with pH 7.0 at 25 °C. Dilute this buffer to 1x and 0.01x using deionized water.

2. Fixation of Cells

NOTE: We used the HuH7 hepatocarcinoma cell line in thi....

Results

Based on our experience using this protocol, we can safely recommend this method for the visualization and quantification of ER-mitochondria interactions in fixed cells. Representative images of in situ proximity ligation assay-visualized ER-mitochondria interactions in the HuH7 hepatocarcinoma cell line, using several pairs of antibodies, are shown. As shown in Figure 2 by fluorescent microscopy, each red dot represents an interaction between the two targeted pr.......

Discussion

Collectively, our studies indicate that the in situ proximity ligation assay is truly a relevant strategy to follow and quantify endogenous ER-mitochondria interactions in fixed cells, without the need for using organelle-specific fluorophores or fluorescent proteins. The specific use of VDAC1/IP3R1 antibodies has been adapted to study ER-mitochondria interactions in HuH7 cells. However, alternative isoforms of VDAC and IP3R may be used, depending on the cell type. In this case, antibodies need to be validated b.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Formaldehyde	Sigma	F-8775
Glycine	Sigma	G-8898
Triton	Sigma	T8532
35 mm Glass bottom culture dishes	MatTeK corporation	P35G-0-14-C
Blocking solution	Sigma	DUO-92004 or DUO-92002	provided in the Duolink PLA probes, Sigma
VDAC1 antibody	Abcam	ab14734
IP3R1-H80 antibody	Santa Cruz	sc28614
CypD antibody	Abcam	ab110324
Grp75 antibody	Santa Cruz	sc13967
TBS 10x	euromedex	ET220	Dilute to obtain 1x
Tween 100x	euromedex	2001-B	dilute in TBS to obtain 0,01%
PLA Probes Mouse MINUS	Sigma	DUO-92004	Duolink, Sigma
PLA Probes Rabbit PLUS	Sigma	DUO-92002	Duolink, Sigma
Duolink detection reagents red	Sigma	DUO-92008	Duolink, Sigma
Ligation solution	Sigma	DUO-92008	Part of the Duolink detection reagents red, Sigma
Ligase	Sigma	DUO-92008	Part of the Duolink detection reagents red, Sigma
Amplification solution	Sigma	DUO-92008	Part of the Duolink detection reagents red, Sigma
Polymerase	Sigma	DUO-92008	Part of the Duolink detection reagents red, Sigma
Duolink Mounting Medium	Sigma	DUO80102	Duolink, Sigma
Softwares
Blob-finder software			BlobFinder is a freely distributed software that can perform calculations on cells from fluorescence microscopy images. This software can be downloaded for free from The Centre for Image Analysis at Uppsala University who have developed the software and the work was supported by the EU FP6 Project ENLIGHT and Olink Bioscience. http://www.cb.uu.se/~amin/BlobFinder/index_files/Page430.htm
ImageJ software			Can be downloaded for free from: http://rsb.info.nih.gov/ij/download.html