Name: visualization and quantification of endogenous intra-organelle protein interactions at er-mitochondria contact sites by proximity ligation assays
Uploaded: 2023-10-20T13:20:00
Description: Watch this Scientific Journal Video about Visualization and Quantification of Endogenous Intra-Organelle Protein Interactions at ER-Mitochondria Contact Sites by Proximity Ligation Assays at JoVE.com

This work was supported by the ANR Jeune Chercheur (ANR0015TD), the ATIP-Avenir Program, the Fondation pour la Recherche Medicale (n&#176;206548), and the Fondation Vaincre Alzheimer (eOTP:669122 LS 212527), I&#39;Agence Nationale de la Recherche (ANR-11-EQPX-0029/Morphoscope, ANR-10-INBS-04/FranceBioImaging; ANR-11-IDEX-0003-02/Saclay Plant Sciences ANR-22-CE11-0024-01/MADE to FG) and AFM Telethon (Project AFM 23778).

1. Mitochondrial staining and proximity ligation assay (PLA)
<ol>
	<li>Plate 0.5 x 105-2 x 105 HeLa cells, maintained in Dulbecco&#39;s modified Eagle medium (DMEM) supplemented with 10% FBS, 1% penicillin/streptomycin, and 1% non-essential amino acids at 37 &#176;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 ...

<ol>
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(118), e54899 (2016).</li><li>Cuello, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impairment+of+the+ER/mitochondria+compartment+in+human+cardiomyocytes+with+PLN+p.Arg14del+mutation.">Impairment of the ER/mitochondria compartment in human cardiomyocytes with PLN p.Arg14del mutation.</a> EMBO Molecular Medicine. 13 (6), e13074 (2021).</li><li>Paillusson, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#945;-Synuclein+binds+to+the+ER-mitochondria+tethering+protein+VAPB+to+disrupt+Ca2++homeostasis+and+mitochondrial+ATP+production.">&#945;-Synuclein binds to the ER-mitochondria tethering protein VAPB to disrupt Ca2+ homeostasis and mitochondrial ATP production.</a> Acta Neuropathologica. 134 (1), 129-149 (2017).</li><li>Tubbs, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria-associated+endoplasmic+reticulum+membrane+(MAM)+integrity+is+required+for+insulin+signaling+and+is+implicated+in+hepatic+insulin+resistance.">Mitochondria-associated endoplasmic reticulum membrane (MAM) integrity is required for insulin signaling and is implicated in hepatic insulin resistance.</a> Diabetes. 63 (10), 3279-3294 (2014).</li><li>Chung, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PI4P/phosphatidylserine+countertransport+at+ORP5-+and+ORP8-mediated+ER-plasma+membrane+contacts.">PI4P/phosphatidylserine countertransport at ORP5- and ORP8-mediated ER-plasma membrane contacts.</a> Science. 349 (6246), 428-432 (2015).</li><li>Galmes, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ORP5/ORP8+localize+to+endoplasmic+reticulum-mitochondria+contacts+and+are+involved+in+mitochondrial+function.">ORP5/ORP8 localize to endoplasmic reticulum-mitochondria contacts and are involved in mitochondrial function.</a> EMBO Reports. 17 (6), 800-810 (2016).</li><li>Ghai, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ORP5+and+ORP8+bind+phosphatidylinositol-4,+5-biphosphate+(PtdIns(4,5)P+2)+and+regulate+its+level+at+the+plasma+membrane.">ORP5 and ORP8 bind phosphatidylinositol-4, 5-biphosphate (PtdIns(4,5)P 2) and regulate its level at the plasma membrane.</a> Nature Communications. 8, 757 (2017).</li><li>Monteiro-Cardoso, V. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ORP5/8+and+MIB/MICOS+link+ER-mitochondria+and+intra-mitochondrial+contacts+for+non-vesicular+transport+of+phosphatidylserine.">ORP5/8 and MIB/MICOS link ER-mitochondria and intra-mitochondrial contacts for non-vesicular transport of phosphatidylserine.</a> Cell Reports. 40 (12), 111364 (2022).</li><li>Du, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ORP5+localizes+to+ER-lipid+droplet+contacts+and+regulates+the+level+of+PI(4)P+on+lipid+droplets.">ORP5 localizes to ER-lipid droplet contacts and regulates the level of PI(4)P on lipid droplets.</a> Journal of Cell Biology. 219 (1), e201905162 (2020).</li><li>Guyard, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ORP5+and+ORP8+orchestrate+lipid+droplet+biogenesis+and+maintenance+at+ER-mitochondria+contact+sites.">ORP5 and ORP8 orchestrate lipid droplet biogenesis and maintenance at ER-mitochondria contact sites.</a> The Journal of Cell Biology. 221 (9), e202112107 (2022).</li><li>Ran, F. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+engineering+using+the+CRISPR-Cas9+system.">Genome engineering using the CRISPR-Cas9 system.</a> Nature Protocols. 8 (11), 2281-2308 (2013).</li></ol>

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...

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-VAPB5,6,7,8. Although this technology has been used to detect and quantify inter-organelle protein-protein interactions that are present at the MCS5,7,9,10,11, 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 MCSs12,13,14,15 and that ORP5 localizes at ER-LD contacts16,17; 2) existing technologies, including PLA, confocal microscopy, organelle labeling, and 3D imaging analysis.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1X Dulbecco&#39;s Phosphate Buffered Saline (1X DPBS)</td>
			<td>Gibco</td>
			<td>14190-094</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium chloride (NH4Cl)</td>
			<td>VWR</td>
			<td>21236.291</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>Circular glass coverslips 13mm no. 1.5</td>
			<td>Agar Scientific</td>
			<td>L46R13-15</td>
			<td></td>
		</tr>
		<tr>
			<td>CMXRos red MitoTracker</td>
			<td>Invitrogen</td>
			<td>M7512</td>
			<td>red mitochondrial marker</td>
		</tr>
		<tr>
			<td>Confocal inverted microscope SP8-X</td>
			<td>Leica</td>
			<td>DMI 6000</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Costar TC-Treated 24-Well Plates</td>
			<td>Merck</td>
			<td>CLS3526</td>
			<td></td>
		</tr>
		<tr>
			<td>Duolink In Situ Detection Reagents Green&#160;</td>
			<td>Sigma</td>
			<td>DUO92002</td>
			<td></td>
		</tr>
		<tr>
			<td>Duolink In Situ Mounting Medium with DAPI&#160;</td>
			<td>Sigma</td>
			<td>DUO82040</td>
			<td></td>
		</tr>
		<tr>
			<td>Duolink In Situ PLA Probe Anti-Mouse MINUS&#160;</td>
			<td>Sigma</td>
			<td>DUO92004</td>
			<td></td>
		</tr>
		<tr>
			<td>Duolink In Situ PLA Probe Anti-Rabbit PLUS</td>
			<td>Sigma</td>
			<td>DUO92014</td>
			<td></td>
		</tr>
		<tr>
			<td>Duolink In Situ Wash Buffers, Fluorescence&#160;</td>
			<td>Sigma</td>
			<td>DUO82049</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco Opti-MEM I Reduced Serum Medium, GlutaMAX Supplement&#160;</td>
			<td>Gibco</td>
			<td>51985026</td>
			<td>serum free medium</td>
		</tr>
		<tr>
			<td>Imaris software v 9.3</td>
			<td>Bitplane</td>
			<td>N/A</td>
			<td>cell imaging software</td>
		</tr>
		<tr>
			<td>Incubator UINCU-line IL10</td>
			<td>VWR</td>
			<td>390-0384</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slide StarFrost (3&#8220; x 1&#8220;)&#160;</td>
			<td>Knittel Glass</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>mouse anti-ORP8&#160;</td>
			<td>Santa Cruz</td>
			<td>134409</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>Sigma</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>rabbit anti-ORP5&#160;</td>
			<td>Sigma</td>
			<td>HAP038712</td>
			<td></td>
		</tr>
		<tr>
			<td>Saponin</td>
			<td>Sigma</td>
			<td>84510</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra Pure Distilled Water, DNase/RNase free</td>
			<td>Invitrogen</td>
			<td>10977-035</td>
			<td></td>
		</tr>
	</tbody>
</table>

visualization and quantification of endogenous intra-organelle protein interactions at er-mitochondria contact sites by proximity ligation assays

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 (&gt;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.

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...

Watch this Scientific Journal Video about Visualization and Quantification of Endogenous Intra-Organelle Protein Interactions at ER-Mitochondria Contact Sites by Proximity Ligation Assays at JoVE.com

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

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.

Membrane contact sites (MCSs) are areas of close membrane proximity that allow and regulate the dynamic exchange of diverse biomolecules (i.e., calcium ...

Our PLA protocol allows to identify and to quantify endogenous interaction between proteins at membrane contact sites. We have used it to study the interaction between two endoplasmic reticulum proteins at endoplasmic reticulum mitochondrial contact sites. This technique combines PLA with organelle stainings and analysis of organelles'distances.It allows to localize and to quantify in interaction between proteins at inter-organelles'membrane contact sites. After performing proximity ligation assay, or PLA, and image acquisition, set up the cell analysis software to start MATLAB and launch an extension by clicking the option Imaris in the Mac operating system or edit menu in Windows. Select the preferences, change to the custom tools panel, and set the path.To import the images into the software, convert the confocal stack images into a IMS file, either directly through the arena section or using the standalone file converter that allows batch conversion. After the importation, click on edit, go to image properties, and select image geometry to check the image calibration. Check that the voxel size corresponds to the pixel size expected for the actual image in X and Y, and the step applied by the microscope to generate the Z stack in Z.To adjust the contrast of the different channels click edit in the menu, go to the display adjustment, and select show display adjustment.Adjust each channel independently to optimize the display of each color. This step is essential to set precise thresholds or detect weak objects. Then, click edit and select crop 3D to limit the analysis to a single cell by cropping the image.To analyze another cell in the same field of view, open the same image again and crop it differently. 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. Select the channel on which the spot detection should be performed.Adjust the estimated XY diameter to help the spot detection algorithm to find the objects of interest. If the chosen value is too high, the nearby objects will fuse, and if the value is too small, aberrant signals may be detected. Now, click on background subtraction to remove the image background before spot detection to enhance the local contrast around the objects of interest.Adjust the spot detection threshold by keeping the quality as the threshold parameter in 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. Then, 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.Select the channel on which the surface creation should be performed. Apply Gaussian filter to obtain a smoother surface by clicking the smooth checkbox, and setting a threshold indicating the minor details observable on the surface. Afterward, perform a background subtraction to enhance the local contrasts and adjust the threshold to detect the mitochondrial network based on the intensity of the signal.Apply a filter on the surface to remove small residuals from the threshold by selecting 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. To generate a distance map outside the created mitochondrial surface, select the mitochondria surface in the scene tree box.Click image processing and select surfaces function, followed by distance transformation. A MATLAB extension asking the user to choose whether the map should be computed outside or inside the object surface will show up. Select outside surface object to measure the distance between the PLA spots and the surface of the mitochondria.Once the map is generated, it 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. Select the spots previously generated in the scene tree box to measure the distance from each point to the closest mitochondrion, and identify and visualize the closest ones.Now, select specific values and center intensity of the channel corresponding to the distance map in the statistics and detailed log to measure the value of each spot center, which corresponds to its distance to the closest to mitochondrion in the distance map. Export the data as a CSV file by clicking on the floppy disk icon at the bottom left of the window. 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.Add a new filter based on the center intensity of the channel corresponding to the distance map in this window, and extract the spots less than 380 nanometers away from the mitochondria by setting the lower threshold to zero micrometers and the upper threshold to 0.380 micrometers. Perform a duplication step by pressing the duplicate selection to new spots button to focus on the selected spots. Confocal images of endogenous ORP5-ORP8 PLA showed interactions in the HeLa cells in the reticular ER, cortical ER, and ER subdomains in close contact with mitochondria, commonly referred to as mitochondria-associated ER membranes.3D imaging analysis revealed that about 50%of endogenous ORP5-ORP8 PLA interactions were detected at mitochondria-associated ER membranes. The percentage of ORP5-ORP8 PLA interactions occurring at mitochondria-associated ER membranes in cells co-overexpressing ORP5 and ORP8 was similar to that observed in cells where these proteins were expressed at endogenous levels. ORP5 and ORP8 downregulation induced a massive decrease in the total number of PLA signals, while their co-overexpression increased PLA.PLA signals were detected in ORP5-PTPIP51 and ORP8-PTPIP51 couples, and their average numbers were similar to the ORP5-ORP8 PLA couple, confirming ORP5 and ORP8 localization at ER-mitochondria membrane contact sites. Further, the interaction of ORP5-ORP8 PLA at ER plasma membrane contacts in a three-way contact site between mitochondria, the ER, and lipid droplets are identified using HeLa cells transfected with PHPLCD-RFP or mCherry-PLN1. As in any image analysis method, the image quality is a key point, so the optimization of the signal is decisive for the automatic detection of the objects.This technique can be also used to study the associations between two or multiple cellular organelles, as we did in our recent study on ORP5 and ORP8 function at the three-party ER, mitochondria, lipid droplets contact sites. This technique can be helpful in other studies in the intracellular communication field to identify novel multipartite inter-organelle associations.

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