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 treatments, such as siRNA treatment and/or DNA plasmid transfection, before mitochondrial staining and PLA.</li>
	<li>Mitochondrial staining
	<ol>
		<li>Wash the cells once in serum-free medium pre-warmed at 37 &#176;C. Incubate the cells with pre-warmed serum-free medium for 10 min at 37 &#176;C with 5% CO2.</li>
		<li>Prepare 1 &#181;M red mitochondrial marker in pre-warmed serum-free medium.</li>
		<li>Incubate the cells with 500 &#181;L of mitochondrial marker solution for 30 min at 37 &#176;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.</li>
		<li>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.</li>
		<li>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 &#181;L of 1x PBS (using disposable pipets or a vacuum system connected to a pump).</li>
		<li>Remove the 1x PBS, and incubate the coverslips with 500 &#181;L of 50mM NH4Cl for 15 min at room temperature (in the dark). Wash 1x for 2 min in 500 &#181;L of 1x PSB using a vacuum system.</li>
		<li>Wash 3x for 2 min in 500 &#181;L 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.</li>
		<li>Transfer the coverslips from the 24-well plate into a humidity chamber. After transferring the coverslips into the chamber, add 100 &#181;L 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.</li>
	</ol>
	</li>
	<li>Proximity ligation assay (PLA) 
	NOTE: The PLA protocol follows the manufacturer&#39;s instructions with slight modifications.
	<ol>
		<li>Primary antibody incubation
		<ol>
			<li>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 &#181;L of primary antibodies solution per coverslip (e.g., 0.27 &#181;L of rabbit anti-ORP5, 0.2 &#181;L of mouse anti-ORP8, 39.53 &#181;L of BB1).</li>
			<li>Remove the BB from the previous wash (step 1.2.8) using a vacuum system, and add 40 &#181;L of primary antibody solution to the coverslips. Incubate for 1 h at room temperature in a humidity chamber protected from light.</li>
		</ol>
		</li>
		<li>Wash coverslips 3x for 5 min with 100 &#181;L of the corresponding BB using the vacuum system.</li>
		<li>PLA probe incubation
		<ol>
			<li>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 &#181;L of PLA probe solution (e.g., 8 &#181;L of rabbit PLUS PLA probe, 8 &#181;L of mouse MINUS PLA probe, 24 &#181;L of BB). Allow the PLA probe solution to sit for 20 min at room temperature before use.</li>
			<li>Remove the BB from the coverslips (from step 1.3.2) using a vacuum system, and add 40 &#181;L of PLA probe solution to the coverslips. Incubate for 1 h at 37&#176;C in a humidity chamber protected from the light.</li>
		</ol>
		</li>
		<li>Wash the coverslips 2x for 5 min in 100 &#181;L of 1x wash buffer A at room temperature.</li>
		<li>Ligation
		<ol>
			<li>Dilute 5x ligation buffer to 1:5 in ultra-pure water and mix. For each coverslip, prepare (at least) 40 &#181;L of ligation solution (8 &#181;L of 5x ligation buffer, 31 &#181;L of ultra-pure water).</li>
			<li>Add the ligase (1 U/&#181;L, supplied in the PLA kit) to 1x ligation buffer prepared in the previous step at a dilution of 1:40, and mix.</li>
			<li>Remove 1x wash buffer A from the coverslips (from step 1.3.4) using a vacuum system, add 40 &#181;L of ligase solution to the coverslips, and incubate for 30 min at 37 &#176;C in a humidity chamber protected from the light.</li>
		</ol>
		</li>
		<li>Wash the coverslips 2x for 2 min in 100 &#181;L of 1x wash buffer A at room temperature using the vacuum system.</li>
		<li>Polymerization
		<ol>
			<li>Dilute 5x amplification buffer 1:5 in ultra-pure water, and mix. For each coverslip, prepare (at least) 40 &#181;L of amplification solution (8 &#181;L of 5x amplification buffer, 31.5 &#181;L of ultra-pure water).</li>
			<li>Add the polymerase (10 U/&#181;L, supplied in the PLA kit) to the 1x polymerization buffer prepared in the previous step at a dilution of 1:80, and mix.</li>
			<li>Remove 1x wash buffer A from the coverslips (from step 1.3.6) using a vacuum system, add 40 &#181;L of polymerase solution to the coverslips, and incubate for 1 h 40 min at 37 &#176;C in a humidity chamber protected from the light.</li>
		</ol>
		</li>
		<li>Remove polymerase solution from coverslips, and wash 2x for 10 min in 100 &#181;L of 1x wash buffer B at room temperature in a humidity chamber protected from the light.</li>
		<li>Wash the coverslips 1x for 1 min in 100 &#181;L of 0.001x wash buffer B at room temperature in a humidity chamber protected from the light.</li>
		<li>Mount the coverslips on glass slides for microscopy using mounting medium with DAPI (concentration between 1.6-0.4 &#181;g/mL). Seal with nail polish.</li>
	</ol>
	</li>
</ol>
2. Image acquisition
<ol>
	<li>Observe the PLA results, and acquire images using fluorescence confocal microscopy with a 63x oil immersion objective using the accompanying software.</li>
	<li>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 (&#955;ex = 488 nm, &#955;em = 505-560 nm) and mitochondria (&#955;ex = 543 nm, &#955;em = 606-670 nm).</li>
</ol>
3. Image processing and assessment of PLA spots associated with mitochondria
<ol>
	<li>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).</li>
	<li>Installation of the 3D distance map extension
	<ol>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Import the images into the software
	<ol>
		<li>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.</li>
		<li>After the importation, check that images remain properly calibrated. Click on Edit &#62; Image Properties &#62; 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.</li>
		<li>Adjust the contrast of the different channels in the menu Display Adjustment by clicking on Edit &#62; 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.</li>
		<li>Limit the analysis to a single cell by cropping the image using the options Edit &#62; Crop 3D. To analyze another cell in the same field of view, open the same image once again, and crop it differently.</li>
	</ol>
	</li>
	<li>ORP5-ORP8 spot detection
	<ol>
		<li>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.</li>
		<li>Select the channel on which the spot detection should be performed.</li>
		<li>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.</li>
		<li>Click on Background Subtraction to remove the image background prior to spot detection to enhance the local contrast around the objects of interest.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Mitochondrial network detection
	<ol>
		<li>Detect the mitochondrial network to generate a surface rendering by&#160;clicking on Add new Surfaces to create a new object, and open the surface detection wizard.</li>
		<li>Select the channel on which the surface creation should be performed.</li>
		<li>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.</li>
		<li>Perform a background subtraction to enhance the local contrasts and to help with the threshold step.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Generation of the 3D distance map around the mitochondria
	<ol>
		<li>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 &#62; Surfaces Function &#62; 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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Extraction of the objects&#39; distances from the closest mitochondrion
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>Export the data as a .csv file by clicking on the Floppy disk icon at the bottom left of the window.</li>
		<li>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.</li>
		<li>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 &#181;m and the upper threshold to 0.380 &#181;m. 
		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).</li>
		<li>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.</li>
	</ol>
	</li>
</ol>

<ol>
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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MAM+(mitochondria-associated+membranes)+in+mammalian+cells:+Lipids+and+beyond.">MAM (mitochondria-associated membranes) in mammalian cells: Lipids and beyond.</a> Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids. 1841 (4), 595-609 (2014).</li><li>Giordano, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-vesicular+lipid+trafficking+at+the+endoplasmic+reticulum-mitochondria+interface.">Non-vesicular lipid trafficking at the endoplasmic reticulum-mitochondria interface.</a> Biochemical Society Transactions. 46 (2), 437-452 (2018).</li><li>S&#246;derberg, O., 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=Direct+observation+of+individual+endogenous+protein+complexes+in+situ+by+proximity+ligation.">Direct observation of individual endogenous protein complexes in situ by proximity ligation.</a> Nature Methods. 3 (12), 995-1000 (2006).</li><li>Gomez-Suaga, P., 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=The+ER-mitochondria+tethering+complex+VAPB-PTPIP51+regulates+autophagy.">The ER-mitochondria tethering complex VAPB-PTPIP51 regulates autophagy.</a> Current Biology. 27 (3), 371-385 (2017).</li><li>Han, 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=The+role+of+Mfn2+in+the+structure+and+function+of+endoplasmic+reticulum-mitochondrial+tethering+in+vivo.">The role of Mfn2 in the structure and function of endoplasmic reticulum-mitochondrial tethering in vivo.</a> Journal of Cell Science. 134 (13), jcs253443 (2021).</li><li>Stoica, 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=ALS/FTD-associated+FUS+activates+GSK-3&#946;+to+disrupt+the+VAPB-PTPIP51+interaction+and+ER-mitochondria+associations.">ALS/FTD-associated FUS activates GSK-3&#946; to disrupt the VAPB-PTPIP51 interaction and ER-mitochondria associations.</a> EMBO reports. 17 (9), 1326-1342 (2016).</li><li>Tubbs, E., Rieusset, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+of+endoplasmic+reticulum+and+mitochondria+interactions+by+in+situ+proximity+ligation+assay+in+fixed+cells.">Study of endoplasmic reticulum and mitochondria interactions by in situ proximity ligation assay in fixed cells.</a> Journal of Visualized Experiments. (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>

The authors have nothing to disclose.

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 ...

visualization-quantification-endogenous-intra-organelle-protein

Research

JoVE Journal

Biology

1.4K Views.  Université Paris-Saclay. 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.

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

Imaging Protein-protein Interactions in vivo

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, preventing their identification and analysis through traditional biochemical methods such as co-immunoprecipitation. In this regard, the genetically encodable fluorescent proteins (GFP, RFP, etc.) and their associated overlapping fluorescence spectrum have revolutionized our ability to monitor weak interactions in vivo using F&ouml;rster resonance energy transfer (FRET)1-3. Here, we detail our use of a FRET-based proximity assay for monitoring receptor-receptor interactions on the endothelial cell surface.

Imaging Protein-protein Interactions in vivo

This protocol describes how to image protein-protein interactions using a FRET-based proximity assay.

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, ...

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular fluorescence complementation (BiFC) is an in vivo method to monitor such interactions in plant cells. In the presented protocol the investigated candidate proteins are fused to complementary halves of fluorescent proteins and the respective constructs are introduced into plant cells via agrobacterium-mediated transformation. Subsequently, the proteins are transiently expressed in tobacco leaves and the restored fluorescent signals can be detected with a confocal laser scanning microscope in the intact cells. This allows not only visualization of the interaction itself, but also the subcellular localization of the protein complexes can be determined. For this purpose, marker genes containing a fluorescent tag can be coexpressed along with the BiFC constructs, thus visualizing cellular structures such as the endoplasmic reticulum, mitochondria, the Golgi apparatus or the plasma membrane. The fluorescent signal can be monitored either directly in epidermal leaf cells or in single protoplasts, which can be easily isolated from the transformed tobacco leaves. BiFC is ideally suited to study protein-protein interactions in their natural surroundings within the living cell. However, it has to be considered that the expression has to be driven by strong promoters and that the interaction partners are modified due to fusion of the relatively large fluorescence tags, which might interfere with the interaction mechanism. Nevertheless, BiFC is an excellent complementary approach to other commonly applied methods investigating protein-protein interactions, such as coimmunoprecipitation, in vitro pull-down assays or yeast-two-hybrid experiments.

Formation of protein complexes in vivo can be visualized by bimolecular fluorescence complementation. Interaction partners are fused to complementary parts of fluorescent tags and transiently expressed in tobacco leaves, resulting in a reconstituted fluorescent signal upon close proximity of the two proteins.

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular ...

Identifying Protein-protein Interaction Sites Using Peptide Arrays

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may result in disease, making protein interactions important drug targets. It is thus highly important to understand these interactions at the molecular level. Protein interactions are studied using a variety of techniques ranging from cellular and biochemical assays to quantitative biophysical assays, and these may be performed either with full-length proteins, with protein domains or with peptides. Peptides serve as excellent tools to study protein interactions since peptides can be easily synthesized and allow the focusing on specific interaction sites. Peptide arrays enable the identification of the interaction sites between two proteins as well as screening for peptides that bind the target protein for therapeutic purposes. They also allow high throughput SAR studies. For identification of binding sites, a typical peptide array usually contains partly overlapping 10-20 residues peptides derived from the full sequences of one or more partner proteins of the desired target protein. Screening the array for binding the target protein reveals the binding peptides, corresponding to the binding sites in the partner proteins, in an easy and fast method using only small amount of protein.
In this article we describe a protocol for screening peptide arrays for mapping the interaction sites between a target protein and its partners. The peptide array is designed based on the sequences of the partner proteins taking into account their secondary structures. The arrays used in this protocol were Celluspots arrays prepared by INTAVIS Bioanalytical Instruments. The array is blocked to prevent unspecific binding and then incubated with the studied protein. Detection using an antibody reveals the binding peptides corresponding to the specific interaction sites between the proteins.

Peptide array screening is a high throughput assay for identifying protein-protein interaction sites. This allows mapping multiple interactions of a target protein and can serve as a method for identifying sites for inhibitors that target a protein. Here we describe a protocol for screening and analyzing peptide arrays.

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may ...

Preparation, Imaging, and Quantification of Bacterial Surface Motility Assays

Bacterial surface motility, such as swarming, is commonly examined in the laboratory using plate assays that necessitate specific concentrations of agar and sometimes inclusion of specific nutrients in the growth medium. The preparation of such explicit media and surface growth conditions serves to provide the favorable conditions that allow not just bacterial growth but coordinated motility of bacteria over these surfaces within thin liquid films. Reproducibility of swarm plate and other surface motility plate assays can be a major challenge. Especially for more &#8220;temperate swarmers&#8221; that exhibit motility only within agar ranges of 0.4%-0.8% (wt/vol), minor changes in protocol or laboratory environment can greatly influence swarm assay results. &#8220;Wettability&#8221;, or water content at the liquid-solid-air interface of these plate assays, is often a key variable to be controlled. An additional challenge in assessing swarming is how to quantify observed differences between any two (or more) experiments. Here we detail a versatile two-phase protocol to prepare and image swarm assays. We include guidelines to circumvent the challenges commonly associated with swarm assay media preparation and quantification of data from these assays. We specifically demonstrate our method using bacteria that express fluorescent or bioluminescent genetic reporters like green fluorescent protein (GFP), luciferase (lux operon), or cellular stains to enable time-lapse optical imaging. We further demonstrate the ability of our method to track competing swarming species in the same experiment.

Swarming motility is influenced by physical and environmental factors. We describe a two-phase protocol and guidelines to circumvent the challenges commonly associated with swarm assay preparation and data collection. A macroscopic imaging technique is employed to obtain detailed information on swarm behavior that is not provided by current analysis techniques.

Bacterial surface motility, such as swarming, is commonly examined in the laboratory using plate assays that necessitate specific concentrations of agar ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

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

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.

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

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.

Structural interactions between the endoplasmic reticular (ER) and mitochondrial membranes, in domains known as mitochondria-associated membranes (MAM), ...

Advanced Confocal Microscopy Techniques to Study Protein-protein Interactions and Kinetics at DNA Lesions

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological approach to analyzing protein kinetics at DNA lesions over time or protein-protein interactions on locally microirradiated chromatin. We also show how to recognize individual phases of the cell cycle using the Fucci cellular system to study cell-cycle-dependent protein kinetics at DNA lesions. A methodological description of the use of two UV lasers (355 nm and 405 nm) to induce different types of DNA damage is also presented. Only the cells microirradiated by the 405-nm diode laser proceeded through mitosis normally and were devoid of cyclobutane pyrimidine dimers (CPDs). We also show how microirradiated cells can be fixed at a given time point to perform immunodetection of the endogenous proteins of interest. For the DNA repair studies, we additionally describe the use of biophysical methods including FRAP (Fluorescence Recovery After Photobleaching) and FLIM (Fluorescence Lifetime Imaging Microscopy) in cells with spontaneously occurring DNA damage foci. We also show an application of FLIM-FRET (Fluorescence Resonance Energy Transfer) in experimental studies of protein-protein interactions.

Laser microirradiation is a useful tool for studies of DNA repair in living cells. A methodological approach for the use of UVA lasers to induce various DNA lesions is shown. We have optimized a method for local microirradiation that maintains the normal cell cycle; thus, irradiated cells proceed through mitosis.

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological ...

Visualization of DNA Repair Proteins Interaction by Immunofluorescence

Mammalian cells are constantly exposed to chemicals, radiations, and naturally occurring metabolic by-products, which create specific types of DNA insults. Genotoxic agents can damage the DNA backbone, break it, or modify the chemical nature of individual bases. Following DNA insult, DNA damage response (DDR) pathways are activated and proteins involved in the repair are recruited. A plethora of factors are involved in sensing the type of damage and activating the appropriate repair response. Failure to correctly activate and recruit DDR factors can lead to genomic instability, which underlies many human pathologies including cancer. Studies of DDR proteins can provide insights into genotoxic drug response and cellular mechanisms of drug resistance.
There are two major ways of visualizing&#160;proteins in vivo: direct observation, by tagging the protein of interest with a fluorescent protein and following it by live imaging, or indirect immunofluorescence on fixed samples. While visualization of fluorescently tagged proteins allows precise monitoring over time, direct tagging in N- or C-terminus can interfere with the protein localization or function. Observation of proteins in their unmodified, endogenous version is preferred. When DNA repair proteins are recruited to the DNA insult, their concentration increases locally and they form groups, or &#8220;foci&#8221;, that can be visualized by indirect immunofluorescence using specific antibodies.
Although detection of protein foci does not provide a definitive proof of direct interaction, co-localization of proteins in cells indicates that they regroup to the site of damage and can inform of the sequence of events required for complex formation. Careful analysis of foci spatial overlap in cells expressing wild type or mutant versions of a protein can provide precious clues on functional domains important for DNA repair function. Last, co-localization of proteins indicates possible direct interactions that can be verified by co-immunoprecipitation in cells, or direct pulldown using purified proteins.

Following DNA damage, human cells activate essential repair pathways to restore the integrity of their genome. Here, we describe the method of indirect immunofluorescence as a means to detect DNA repair proteins, analyze their spatial and temporal recruitment, and help interrogate protein-protein interaction at the sites of DNA damage.

Mammalian cells are constantly exposed to chemicals, radiations, and naturally occurring metabolic by-products, which create specific types of DNA ...

Three-dimensional Characterization of Interorganelle Contact Sites in Hepatocytes using Serial Section Electron Microscopy

Transmission electron microscopy has been long considered to be the gold standard for the visualization of cellular ultrastructure. However, analysis is often limited to two dimensions, hampering the ability to fully describe the three-dimensional (3D) ultrastructure and functional relationship between organelles. Volume electron microscopy (vEM) describes a collection of techniques that enable the interrogation of cellular ultrastructure in 3D at mesoscale, microscale, and nanoscale resolutions. 
This protocol provides an accessible and robust method to acquire vEM data using serial section transmission EM (TEM) and covers the technical aspects of sample processing through to digital 3D reconstruction in a single, straightforward workflow. To demonstrate the usefulness of this technique, the 3D ultrastructural relationship between the endoplasmic reticulum and mitochondria and their contact sites in liver hepatocytes is presented. Interorganelle contacts serve vital roles in the transfer of ions, lipids, nutrients, and other small molecules between organelles. However, despite their initial discovery in hepatocytes, there is still much to learn about their physical features, dynamics, and functions. 
Interorganelle contacts can display a range of morphologies, varying in the proximity of the two organelles to one another (typically ~10-30 nm) and the extent of the contact site (from punctate contacts to larger 3D cisternal-like contacts). The examination of close contacts requires high-resolution imaging, and serial section TEM is well suited to visualize the 3D ultrastructural of interorganelle contacts during hepatocyte differentiation, as well as alterations in hepatocyte architecture associated with metabolic diseases.

A simple and comprehensive protocol to acquire three-dimensional details of membrane contact sites between organelles in hepatocytes from the liver or cells in other tissues.

Transmission electron microscopy has been long considered to be the gold standard for the visualization of cellular ultrastructure. However, analysis is ...

Methods Article

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