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-lipid droplet (LD) contacts¹. Among them, those formed between the ER and the mitochondria (MERCSs) are among the most studied as they are involved in the regulation of several cellular functions, including lipid and calcium homeostasis². As mitochondria are largely excluded from the classical vesicular transport pathways, they rely on MERCS and on their molecular constituents to import key lipids or lipid precursors from the ER. The non-vesicular transport of these lipids across MERCSs ensures the maintenance of proper mitochondrial lipid composition, as well as their functional and structural integrity³.

Given the crucial involvement of MCSs in various cellular functions, the interest in providing a deeper understanding of their molecular components has greatly increased in the last years. Several types of imaging-based approaches have been used to advance the knowledge on MCSs. Among them, the fluorescence probe-based proximity ligation assay (PLA) has been widely used as an indicator of the abundance of MCSs by detecting inter-organelle protein-protein interactions (in a detection range of 40 nm) at endogenous levels⁴. For instance, MERCSs have been visualized and quantified by using PLA between several mitochondria-ER proteins pairs, including VDAC1-IP3R, GRP75-IP3R, CypD-IP3, and PTPIP51-VAPB^5,6,7,8. Although this technology has been used to detect and quantify inter-organelle protein-protein interactions that are present at the MCS^5,7,9,10,11, most of the studies did not combine PLA with organelle staining. Consequently, a quantitative method that allows the measurement of the proximity between PLA interactions and associated organelles has not been developed yet. Thus, so far, in the case of ER proteins, their interaction within membrane subdomains in contact with other organelles has not been distinguished from their interaction within the widely distributed ER network.

Here, we describe a protocol to detect PLA interactions between proteins that reside in the membrane of the same organelle and to analyze their proximity to the membrane of the partner organelle at the MCS. This protocol was developed based on two premises: 1) previous studies showing that, in overexpression conditions, the ER lipid transfer proteins ORP5 and ORP8 co-localize and interact at ER-mitochondria and ER-PM MCSs^12,13,14,15 and that ORP5 localizes at ER-LD contacts^16,17; 2) existing technologies, including PLA, confocal microscopy, organelle labeling, and 3D imaging analysis.

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
   1. Wash the cells once in serum-free medium pre-warmed at 37 °C. Incubate the cells with pre-warmed serum-free medium for 10 min at 37 °C with 5% CO₂.
   2. Prepare 1 µM red mitochondrial marker in pre-warmed serum-free medium.
   3. Incubate the cells with 500 µL of mitochondrial marker solution for 30 min at 37 °C with 5% CO₂. During the mitochondrial marker treatment and the following steps of the protocol, keep the cells on coverslips protected from light as much as possible.
   4. Following the incubation, wash the cells 1x in pre-warmed serum free medium and 2x in 1x phosphate buffered saline (PBS). Perform this step as quickly as possible since long washing steps before fixation may affect the mitochondrial morphology.
   5. Remove the 1x PBS from the coverslips, and fix the cells by incubating them in freshly prepared 4% PFA (in 1x PBS) for 30 min at room temperature (in the dark) under the chemical hood. Wash 3x for 2 min in 500 µL of 1x PBS (using disposable pipets or a vacuum system connected to a pump).
   6. Remove the 1x PBS, and incubate the coverslips with 500 µL of 50mM NH₄Cl for 15 min at room temperature (in the dark). Wash 1x for 2 min in 500 µL of 1x PSB using a vacuum system.
   7. Wash 3x for 2 min in 500 µL of blocking buffer 1 (BB1, 1%BSA, 0.1% saponin in 1x PBS) for the ORP5-ORP8 PLA or blocking buffer 2 (BB2, 2% BSA, 0.1% normal goat serum, 0.1 saponin in 1x PBS) for the ORP8-PTPIP51 PLA.
   8. Transfer the coverslips from the 24-well plate into a humidity chamber. After transferring the coverslips into the chamber, add 100 µL of BB (BB1 or BB2, according to the pairs of antibodies used) to avoid dryness.
      NOTE: A humidity chamber protected from light can be easily and rapidly obtained by wrapping a Petri dish (plastic or glass) in aluminum foil and adding some pieces of wet paper towel in the periphery of the Petri dish.
3. Proximity ligation assay (PLA)
   NOTE: The PLA protocol follows the manufacturer's instructions with slight modifications.
   1. Primary antibody incubation
      1. Prepare the primary antibody working solution. Dilute rabbit anti-ORP5 (1:150) plus mouse anti-ORP8 (1:200) in BB1, mouse anti-ORP8 (1:200) plus rabbit anti-PTPIP51 (1:200) in BB2, and rabbit anti-ORP5 (1:150) plus mouse anti-PTPIP51 (1:200) in BB1. Prepare (at least) 40 µL of primary antibodies solution per coverslip (e.g., 0.27 µL of rabbit anti-ORP5, 0.2 µL of mouse anti-ORP8, 39.53 µL of BB1).
      2. Remove the BB from the previous wash (step 1.2.8) using a vacuum system, and add 40 µL of primary antibody solution to the coverslips. Incubate for 1 h at room temperature in a humidity chamber protected from light.
   2. Wash coverslips 3x for 5 min with 100 µL of the corresponding BB using the vacuum system.
   3. PLA probe incubation
      1. Prepare the PLA probes working solution. Dilute rabbit PLUS (1:5) and mouse MINUS (1:5) PLA probes in BB, and mix. For each coverslip, prepare (at least) 40 µL of PLA probe solution (e.g., 8 µL of rabbit PLUS PLA probe, 8 µL of mouse MINUS PLA probe, 24 µL of BB). Allow the PLA probe solution to sit for 20 min at room temperature before use.
      2. Remove the BB from the coverslips (from step 1.3.2) using a vacuum system, and add 40 µL of PLA probe solution to the coverslips. Incubate for 1 h at 37°C in a humidity chamber protected from the light.
   4. Wash the coverslips 2x for 5 min in 100 µL of 1x wash buffer A at room temperature.
   5. Ligation
      1. Dilute 5x ligation buffer to 1:5 in ultra-pure water and mix. For each coverslip, prepare (at least) 40 µL of ligation solution (8 µL of 5x ligation buffer, 31 µL of ultra-pure water).
      2. Add the ligase (1 U/µL, supplied in the PLA kit) to 1x ligation buffer prepared in the previous step at a dilution of 1:40, and mix.
      3. Remove 1x wash buffer A from the coverslips (from step 1.3.4) using a vacuum system, add 40 µL of ligase solution to the coverslips, and incubate for 30 min at 37 °C in a humidity chamber protected from the light.
   6. Wash the coverslips 2x for 2 min in 100 µL of 1x wash buffer A at room temperature using the vacuum system.
   7. Polymerization
      1. Dilute 5x amplification buffer 1:5 in ultra-pure water, and mix. For each coverslip, prepare (at least) 40 µL of amplification solution (8 µL of 5x amplification buffer, 31.5 µL of ultra-pure water).
      2. Add the polymerase (10 U/µL, supplied in the PLA kit) to the 1x polymerization buffer prepared in the previous step at a dilution of 1:80, and mix.
      3. Remove 1x wash buffer A from the coverslips (from step 1.3.6) using a vacuum system, add 40 µL of polymerase solution to the coverslips, and incubate for 1 h 40 min at 37 °C in a humidity chamber protected from the light.
   8. Remove polymerase solution from coverslips, and wash 2x for 10 min in 100 µL of 1x wash buffer B at room temperature in a humidity chamber protected from the light.
   9. Wash the coverslips 1x for 1 min in 100 µL of 0.001x wash buffer B at room temperature in a humidity chamber protected from the light.
   10. Mount the coverslips on glass slides for microscopy using mounting medium with DAPI (concentration between 1.6-0.4 µg/mL). Seal with nail polish.

2. Image acquisition

1. Observe the PLA results, and acquire images using fluorescence confocal microscopy with a 63x oil immersion objective using the accompanying software.
2. Set the fluorescence excitation using a 405 nm laser diode or a white light laser, and collect the spectral windows with GaAsP PMTs or hybrid detectors. At each focal plane (spanning 300 nm), acquire the fluorescence signals for PLA (λex = 488 nm, λem = 505-560 nm) and mitochondria (λex = 543 nm, λem = 606-670 nm).

3. Image processing and assessment of PLA spots associated with mitochondria

1. Process the confocal images using a software for image analysis that generates distance maps between the cellular components. Follow the steps below to generate 3D reconstitutions of PLA spots and the mitochondrial network and to access the distance between them using cell imaging software (Figure 1).
2. Installation of the 3D distance map extension
   1. Install both the cell analysis software package with the XT option and the MATLAB software or only a MATLAB compiler runtime (MRC), which can be freely downloaded from the website.
   2. Set up the cell analysis software to start MATLAB when an XTension is launched as follows: from the option Imaris (Mac OS X) or Edit menu (Windows), select Preferences, change to the Custom Tools panel, and then set the path:
      C:\Program Files\MATLAB\R201Xa_x64\bin\win64\MATLAB.exe for Windows or/Applications/MATLAB_R201Xa.app/bin/matlab for Mac OS X.
3. Import the images into the software
   1. Convert the confocal stack images into an .IMS file, either directly through the Arena section or using the standalone File Converter that allows batch conversion.
   2. After the importation, check that images remain properly calibrated. Click on Edit > Image Properties > Image Geometry, and check that the voxel size corresponds in X and Y to the pixel size expected for the actual image (see image calibration in the acquisition software) and that the voxel size in Z corresponds to the step applied by the microscope to generate the Z-stack. If the values are not correct, modify them to ensure a correct estimation of the distances in the following steps.
   3. Adjust the contrast of the different channels in the menu Display Adjustment by clicking on Edit > Show Display Adjustment. Adjust each channel independently to optimize the display of each color. This step does not directly affect the image values but is essential to set precise thresholds or detect weak objects.
   4. Limit the analysis to a single cell by cropping the image using the options Edit > Crop 3D. To analyze another cell in the same field of view, open the same image once again, and crop it differently.
4. ORP5-ORP8 spot detection
   1. Detect the PLA signals generated at the location of ORP5 and ORP8 interaction by clicking on the Add New Spots option, which creates a new set of objects and opens the spot detection wizard.
   2. Select the channel on which the spot detection should be performed.
   3. Adjust the Estimated XY Diameter (the range has to be adapted if the object size differs) to help the spot detection algorithm to find the objects of interest. Note that if the chosen value is too high, the nearby objects will fuse. If the value is too small, one signal may be considered as multiple objects, or aberrant signals may be detected.
   4. Click on Background Subtraction to remove the image background prior to spot detection to enhance the local contrast around the objects of interest.
   5. Adjust the spot detection threshold by keeping the quality (intensity at the object center) as the threshold parameter and the software auto-threshold, or slightly modify this value to detect all the objects. Once the spot detection is finished, save the detection parameters, and reuse them to process other images.
5. Mitochondrial network detection
   1. Detect the mitochondrial network to generate a surface rendering by clicking on Add new Surfaces to create a new object, and open the surface detection wizard.
   2. Select the channel on which the surface creation should be performed.
   3. Apply a Gaussian filter to obtain a smoother surface by clicking the Smooth checkbox and by setting a threshold indicating the smallest details observable on the surface.
   4. Perform a background subtraction to enhance the local contrasts and to help with the threshold step.
   5. Adjust the threshold to detect the mitochondrial network based on the intensity of the signal. If it is difficult to properly set the threshold, it is recommended to go back to the previous step to adjust the degree of smoothness and background subtraction.
   6. If necessary, apply a filter on the surface to remove small residuals resulting from the threshold. To do this, select the Number of Voxels filter on the classify surface window, and play with the upper and lower thresholds to keep only the objects of interest. Once the surface creation is over, save the creation parameters, and reuse them to process other images.
6. Generation of the 3D distance map around the mitochondria
   1. Generate a distance map outside of the mitochondrial surface previously created as follows. Select the mitochondria surface in the scene tree box. Click on Image Processing > Surfaces Function > Distance Transformation. This will call a Matlab XTension that asks the user to choose whether the map should be computed outside or inside the object surface.
   2. Select Outside Surface Object to measure the distance between the PLA spots and the surface of the mitochondria. Once generated, the distance map appears as a new channel in the display adjustment panel. In this channel, every pixel has a value corresponding to the distance to the closest mitochondria.
7. Extraction of the objects' distances from the closest mitochondrion
   1. To measure the distance from each point to the closest mitochondrion and to identify and visualize the closest ones, select the spots previously generated in the scene tree box.
   2. In the statistics and detailed log, select Specific Values (to obtain one measurement for each spot). Select Center Intensity Ch=X (with X corresponding to the number of the distance map channel). This will measure the value of each spot center, which corresponds to its distance to the closest mitochondrion, in the distance map.
   3. Export the data as a .csv file by clicking on the Floppy disk icon at the bottom left of the window.
   4. To extract a subpopulation of spots based on their distances to mitochondria, select the spots in the scene tree, and click on the Filters tab.
   5. In this window, add a new filter based once again on the Center Intensity Ch=X, and extract the spots less than 380 nm away from mitochondria by setting the lower threshold to 0 µm and the upper threshold to 0.380 µm.
      NOTE: The threshold of 380 nm was estimated based on a PLA reaction including the association between the primary and secondary antibodies (30 nm) plus half of the full width at half maximum(FWHM) of the PLA amplification signals (350 nm).
   6. To focus on the selected spots, and for example, give them a distinct color, perform a duplication step by pressing the Duplicate Selection to New Spots button.

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