Visualization and Quantification of Endogenous Intra-Organelle Protein Interactions at ER-Mitochondria Contact Sites by Proximity Ligation Assays

Summary

The need for new approaches to study membrane contact sites (MCSs) has grown due to increasing interest in studying these cellular structures and their components. Here, we present a protocol that integrates previously available microscopy technologies to identify and quantify intra-organelle and inter-organelle protein complexes that reside at MCSs.

Abstract

Membrane contact sites (MCSs) are areas of close membrane proximity that allow and regulate the dynamic exchange of diverse biomolecules (i.e., calcium and lipids) between the juxtaposed organelles without involving membrane fusion. MCSs are essential for cellular homeostasis, and their functions are ensured by the resident components, which often exist as multimeric protein complexes. MCSs often involve the endoplasmic reticulum (ER), a major site of lipid synthesis and cellular calcium storage, and are particularly important for organelles, such as the mitochondria, which are excluded from the classical vesicular transport pathways. In the last years, MCSs between the ER and mitochondria have been extensively studied, as their functions strongly impact cellular metabolism/bioenergetics. Several proteins have started to be identified at these contact sites, including membrane tethers, calcium channels, and lipid transfer proteins, thus raising the need for new methodologies and technical approaches to study these MCS components. Here, we describe a protocol consisting of combined technical approaches, that include proximity ligation assay (PLA), mitochondria staining, and 3D imaging segmentation, that allows the detection of proteins that are physically close (>40 nm) to each other and that reside on the same membrane at ER-mitochondria MCSs. For instance, we used two ER-anchored lipid transfer proteins, ORP5 and ORP8, which have previously been shown to interact and localize at ER-mitochondria and ER-plasma membrane MCSs. By associating the ORP5-ORP8 PLA with cell imaging software analysis, it was possible to estimate the distance of the ORP5-ORP8 complex from the mitochondrial surface and determine that about 50% of ORP5-ORP8 PLA interaction occurs at ER subdomains in close proximity to mitochondria.

Introduction

Inter-organelle communication is a defining characteristic of eukaryotic cells. One way in which organelles communicate is by forming membrane contact sites (MCSs), which are close membrane oppositions between two organelles that are maintained by structural and functional proteins, such as tethers, lipid transfer proteins, and calcium channels¹. MCSs can be established between similar or different organelles, and they mediate the exchange of cellular components, which is important for maintaining cellular homeostasis. To date, several MCSs have been identified, including endoplasmic reticulum (ER)-mitochondria, ER-plasma membrane (PM), and ER-....

Protocol

1. Mitochondrial staining and proximity ligation assay (PLA)

1. Plate 0.5 x 10⁵-2 x 10⁵ HeLa cells, maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% FBS, 1% penicillin/streptomycin, and 1% non-essential amino acids at 37 °C with 5% CO₂, in 13 mm glass coverslips in 1.5 in 24-well plates at a dilution that allows 75%-90% cell confluence on the day of the procedure.
   NOTE: Of note, HeLa cells can be submitted to additional treatments, such as siRNA treatment and/or DNA plasmid transfection, before mitochondrial staining and PLA.
2. Mitochondrial staining
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Results

Using the protocol described above, we detected the sites of interaction of two ER-anchored lipid transfer proteins, ORP5 and ORP8, and assessed their occurrence at ER membrane subdomains in contact with other organelles, in particular, with the mitochondria. For that, the mitochondrial network in HeLa cells was stained with a red mitochondrial marker, and ORP5-ORP8 PLA green spots were detected after fixation using the primary antibodies anti-ORP5 and anti-ORP8, whose specificity was previously tested by immunofluoresce.......

Discussion

This protocol was designed to identify and quantify inter-organelle protein PLA interactions at MCSs, in particular at MERCSs. The novelty of the protocol is that it combines PLA with the labeling of multiple organelles, confocal microscopy, and 3D image analysis to localize and quantify PLA interactions between two proteins residing in the same membrane, in this case within the ER membrane in close proximity with the membrane of mitochondria (MAM) or with the MAM and LDs simultaneously. This protocol can be used as a to.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1X Dulbecco's Phosphate Buffered Saline (1X DPBS)	Gibco	14190-094
Ammonium chloride (NH4Cl)	VWR	21236.291
Bovine serum albumin (BSA)	Sigma	A7906
Circular glass coverslips 13mm no. 1.5	Agar Scientific	L46R13-15
CMXRos red MitoTracker	Invitrogen	M7512	red mitochondrial marker
Confocal inverted microscope SP8-X	Leica	DMI 6000
Corning Costar TC-Treated 24-Well Plates	Merck	CLS3526
Duolink In Situ Detection Reagents Green	Sigma	DUO92002
Duolink In Situ Mounting Medium with DAPI	Sigma	DUO82040
Duolink In Situ PLA Probe Anti-Mouse MINUS	Sigma	DUO92004
Duolink In Situ PLA Probe Anti-Rabbit PLUS	Sigma	DUO92014
Duolink In Situ Wash Buffers, Fluorescence	Sigma	DUO82049
Gibco Opti-MEM I Reduced Serum Medium, GlutaMAX Supplement	Gibco	51985026	serum free medium
Imaris software v 9.3	Bitplane	N/A	cell imaging software
Incubator UINCU-line IL10	VWR	390-0384
Microscope slide StarFrost (3“ x 1“)	Knittel Glass
mouse anti-ORP8	Santa Cruz	134409
Paraformaldehyde (PFA)	Sigma	P6148
rabbit anti-ORP5	Sigma	HAP038712
Saponin	Sigma	84510
Ultra Pure Distilled Water, DNase/RNase free	Invitrogen	10977-035