We thank the entire Chernoff laboratory for contributing to the optimization and validation of this protocol, in particular Maria Radu and Galina Semenova. We also thank Andrey Efimov of the Cell Imaging Facility at Fox Chase Cancer Center. This work was supported by a grant from the NIH (R01 CA148805) to JC.

1. Preparation of Solutions
<ol>
	<li>Prepare fixative solution: 4% paraformaldehyde (PFA) in 1x PBS. For 10 mL, take 2.5 mL of 16% PFA and add 7.5 mL of 1x PBS. 
	Hazards: PFA is carcinogenic at low doses. Fumes and skin contact are hazardous. Store at -20 &#176;C.</li>
	<li>Prepare permeabilization solution: 0.1% Triton X-100 in 1x PBS. For 100 mL of solution, add 100 &#181;L of Triton X-100 into 100 mL of 1x PBS. Store at room temperature (RT).</li>
	<li>Prepare Wash Buffer: 1x TBST. For 1 L, take 100 mL of 10x TBS, 890 mL of dH2O, and 10 mL of Tween 20 (10%).</li>
	<li>Prepare blocking solution as supplied by kit. Alternatively, use 1x PBS solution containing 2% BSA.</li>
	<li>Prepare the antibody diluent as supplied by kit. Alternatively, use 1x PBS solution containing 1% BSA.</li>
</ol>
2. Plating of Cells
NOTE: In this study, we used immortalized Human Schwann Cells (iHSC) and Human Embryonic Kidney 293 cells (HEK 293); however, this method can be used for many types of adherent cells.
<ol>
	<li>Culture HEK 293 in DMEM supplemented with 10% FBS, 0.2% Glucose, 2 mM L-Glutamine, 100 U/mL penicillin G and 100 &#181;g/mL streptomycin on tissue culture-treated plates at 37 &#176;C in 5% CO2. Maintain iHSC in 10% FBS/DMEM supplemented with Pen/Strep and 2 &#181;M forskolin. Routinely test cells for mycoplasma. No cell line authentication was performed.</li>
	<li>Coat a 16-well chamber slide with 50 &#181;L of 10 &#181;g/mL natural mouse laminin or 0.01% poly-L-lysine solution and incubate for 30 min at 37 &#176;C in 5% CO2. Split cells using 0.04% trypsin-EDTA.</li>
	<li>Plate iHSC and HEK-293 in 100 &#181;L of medium at 80% confluence (15,000-25,000 cells/well).</li>
	<li>Incubate the cells for 12 -24 hours at 37 &#176;C in a humidified, 5% CO2 incubator.</li>
</ol>
3. Fixation and Permeabilization
<ol>
	<li>Remove the medium from the wells and wash with 100 &#181;L of 1x PBS. Aspirate with a micropipette to minimize the risk of removing the sample.</li>
	<li>Fix cells by adding 50 &#181;L of 4% PFA per well and incubate for 10 min at RT, without agitation. Cells are sensitive to detachment, so avoid pipetting the solutions directly onto the cells.</li>
	<li>Wash the cells with 0.05% TBST three times for 5 min each. Aspirate with a micropipette to minimize the risk of removing the sample.</li>
	<li>Treat the cells with permeabilization solution (0.1% Triton x-100 in 1x PBS) for 10 min without agitation at RT.</li>
	<li>Wash cells with TBST three times for 5 min each with agitation.</li>
</ol>
4. Blocking
<ol>
	<li>Tap off the TBST (very gently with a micropipette). Visualize the slide in a microscope to see if cells remain attached.</li>
	<li>Add one drop (50-60 &#181;L) of 1x Blocking Solution to each sample.</li>
	<li>Pre-heat a humidity chamber (empty tip box with water) at 37 &#176;C and incubate the slides in it for 1 h at 37 &#176;C.</li>
</ol>
5. Primary Antibodies
<ol>
	<li>Dilute the primary antibodies (1:100) in the Antibody Diluent (see Table of Materials). Prepare 40 &#181;L antibody solution per well.</li>
	<li>Remove blocking solution from the slides by tapping off the liquid. Do not allow cells to dry.</li>
	<li>Vortex and add 40 &#181;L of the antibody solutions to each well.</li>
	<li>Incubate 1 h at 37 &#176;C in a preheated humidity chamber.</li>
</ol>
6. Proximity Ligation Assay Probes
NOTE: PLA probes are provided as part of a kit (see Table of Materials). The choice of probes will depend on the species of primary antibodies used to detect the proteins of interest.
<ol>
	<li>Dilute the two PLA probes (anti-Mouse MINUS and anti-Rabbit PLUS) 1:5 in the Antibody Diluent. For each sample, prepare 40 &#181;L of PLA probe. Incubate the mixture for 20 min at RT.</li>
	<li>Tap off the primary antibody solution from the slides. Wash the slides twice for 5 min each with Wash Buffer at RT.</li>
	<li>Gently tap of the Wash Buffer from the slides and add the diluent PLA probe solution to wells (40 &#181;L/well).</li>
	<li>Incubate the slides in a pre-heated humidity chamber for 1 h at 37 &#176;C.</li>
</ol>
7. Ligation
NOTE: The in situ Detection Reagents Red, Ligase, and Ligation Solution are provided as part of a kit (see Table of Materials).
<ol>
	<li>Use the in situ Detection Reagents Red.</li>
	<li>Immediately before use, vortex and dilute the required volumes of the 5x ligation stock 1:5 in high purity water. 
	NOTE: Do not store diluted reagents.</li>
	<li>Tap off the PLA probe solution from the slides. Wash the slides in 1x Wash Buffer twice for 5 min each at RT.</li>
	<li>Immediately before addition to the samples, vortex and add Ligase to the Ligation solution at 1:40 dilution and vortex again.</li>
	<li>Tap off the Wash Buffer from the slides and add the Ligation/Ligase solution to each well (40 &#181;L/well).</li>
	<li>Incubate the slides in a pre-heated humidity chamber for 30 min at 37 &#176;C.</li>
</ol>
8. Amplification
NOTE: The amplification stock is provided as part of a kit (see Table of Materials). Reagents are light sensitive; thus avoid exposing the slides to light.
<ol>
	<li>Vortex and dilute the required volumes of the 5x Amplification stock 1:5 in high purity water and mix.</li>
	<li>Tap off the Ligation/Ligase solution from the slides. Wash the slides in 1x Wash Buffer twice for 2 min each at RT.</li>
	<li>Vortex and add the polymerase to the Amplification solution at 1:80 dilution and vortex again.</li>
	<li>Tap off Wash Buffer from the slides and add the Amplification/Polymerase solution to each well (40 &#181;L/well). Incubate the slides in a pre-heated humidity chamber for 100 min at 37 &#176;C.</li>
</ol>
9. Preparation for Imaging
NOTE: These are light sensitive reagents. Keep the slides protected from light.
<ol>
	<li>Tap off the Amplification-Polymerase solution from the slides and wash twice for 10 min each in 1x Wash Buffer at RT.</li>
	<li>Remove the chambers and silicone around the wells from the slide completely. Scrape off the remaining bits of silicone with a razor. Draw a grid on the slide separating each well.</li>
	<li>Add ~40 &#181;L of the mounting medium with DAPI. Be careful to avoid trapping air bubbles under the cover slip. The edges of the cover slip can be sealed with nail polish.</li>
	<li>Wait for at least 20 min before performing image analysis using a confocal microscope. 
	NOTE: The protocol can be paused here.</li>
	<li>After imaging, store the slides at -20 &#176;C in the dark.</li>
</ol>

<ol>
	<li>Zhou, D., 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+Nore1B/Mst1+complex+restrains+antigen+receptor-induced+proliferation+of+na&#239;ve+T+cells.">The Nore1B/Mst1 complex restrains antigen receptor-induced proliferation of na&#239;ve T cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 105 (51), 20321-20326 (2008).</li><li>Praskova, M., Xia, F., Avruch, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MOBKL1A/MOBKL1B+phosphorylation+by+MST1+and+MST2+inhibits+cell+proliferation.">MOBKL1A/MOBKL1B phosphorylation by MST1 and MST2 inhibits cell proliferation.</a> Current Biology. 18 (5), 311-321 (2008).</li><li>Dan, I., Watanabe, N. M., Kusumi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Ste20+group+kinases+as+regulators+of+MAP+kinase+cascades.">The Ste20 group kinases as regulators of MAP kinase cascades.</a> Trends in Cell Biology. 11 (5), 220-230 (2001).</li><li>Rawat, S. 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=H-ras+Inhibits+the+Hippo+Pathway+by+Promoting+Mst1/Mst2Heterodimerization.">H-ras Inhibits the Hippo Pathway by Promoting Mst1/Mst2Heterodimerization.</a> Current Biology. 26 (12), 1556-1563 (2016).</li><li>Creasy, C. L., Ambrose, D. M., Chernoff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Ste20-like+protein+kinase,+Mst1,+dimerizes+and+contains+an+inhibitory+domain.">The Ste20-like protein kinase, Mst1, dimerizes and contains an inhibitory domain.</a> Journal of Biological Chemistry. 271 (35), 21049-21053 (1996).</li><li>Buntru, A., Trepte, P., Klockmeier, K., Schnoegl, S., Wanker, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+Approaches+Toward+Quantitative+Mapping+of+the+Interactome.">Current Approaches Toward Quantitative Mapping of the Interactome.</a> Frontiers in Genetics. 74 (7), (2016).</li><li>Greenwood, C., Ruff, D., Kirvell, S., Johnson, G., Dhillon, H. S., Bustin, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proximity+assays+for+sensitive+quantification+of+proteins.">Proximity assays for sensitive quantification of proteins.</a> Biomolecular Detection and Quantification. 4, 10-16 (2015).</li><li>Fredriksson, 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=Protein+detection+using+proximity-dependent+DNA+ligation+assays.">Protein detection using proximity-dependent DNA ligation assays.</a> Nature Biotechnology. 20 (5), 473-477 (2002).</li><li>Leuchowius, K. 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=Parallel+visualization+of+multiple+protein+complexes+in+individual+cells+in+tumor+tissue.">Parallel visualization of multiple protein complexes in individual cells in tumor tissue.</a> Molecular &amp; Cellular Proteomics. 12 (6), 1563-1571 (2013).</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></ol>

The authors declare that they have no competing financial interests.

We found it useful to use glass chamber slides for this experiment, as it is very convenient to perform experiment with several (14-16) cell lines and there is no need to change the sample every time during microscopy analysis. A few complications may arise, such as an increased risk for cross-contamination with antibodies. Therefore, we suggest washing every well individually instead of using a Coplin jar, despite the increased duration of the experiment. In addition, removal of silicone insert is a delicate affair and must be done with care and patience. Even with meticulous technique, it is sometimes possible to see small amounts of silicone debris after removal the insert. For this reason, one must remove the silicone insert scrupulously, using a razorblade if necessary, as too much unremoved silicone can alter the confocal distance during microscopy. It can also be useful to draw lines as borders of wells after removing the silicon insert to help find cells under microscope.
It is imperative to use non-cross reactive primary antibodies that each recognize only one member of the heterodimer (e.g., the MST1 antibodies should not also recognize MST2 and vice-versa). Also, it is important to remember to use different tips to avoid cross-contamination of primary antibodies and PLA probes, to avoid touching the bottom of the well and to avoid pipetting directly over samples. We found that for iHSC, it is better to coat chamber slides with 0.01% poly-L-lysine solution, and for HEK-293 it is better to coat with mouse 10 &#181;g/mL laminin.
As with any procedure, there are some limitations of this method. The PLA assay, while a powerful method for all the reasons listed above, is limited by the specificity and sensitivity of the antibodies. Furthermore, antibody concentrations and cell culture conditions should be finely tuned before laboratory experiments are set up to reduce the costs of the assays. In that regard, an alternative to reduce the cost of the assays is to use uncoated 35-mm glass-bottom dish instead of chamber slides.
An advantage of the assay is that intensity and number of fluorescent dots can be quantified using various computer software programs such as the Blob-finder software, the Duolink Image Tool, and th Olink Bioscience.

Disruption of MST1/Hippo signaling has been connected to developmental disorders and carcinogenesis1. In mammals, the kinases MST1 and MST2 activate (phosphorylate) MOB1 and LATS1/2, the latter of which then phosphorylates and inactivates the transcriptional co-activator Yes-associated protein (YAP)2. In its active (unphosphorylated) form, YAP has oncogenic activity, enhancing transcription of cell proliferation genes; conversely, when YAP is inactivated by the Hippo pathway, cell proliferation is suppressed and apoptosis promoted3. In tissues, MST1 and MST2 exist mainly as active homodimers, but oncogenic stimuli can increase levels of MST1/MST2 heterodimers, and such heterodimers are inactive4. However, how MST1/MST2 heterodimerization is regulated remains poorly understood. Both homo- and heterodimers are mediated via interactions between C-terminal coiled-coil regions of MST1 and MST2 known as SARAH domains5. Using an in situ PLA demonstrated in this article, we show the presence of MST1/MST2 heterodimers in Human Schwann Cells (HSC) and Human Embryonic Kidney cells (HEK-293). PLA has an advantage over other protein/protein interaction detection methods because it allows the detection of endogenous protein-protein interactions, which can be identified and quantified without the need of transgene expression or the use of epitope tags6.
Signaling transduction pathways are largely controlled by the conditional association of component proteins. For example, stimulation of most receptor tyrosine kinases leads to their homo- or hetero-dimerization and subsequent association with additional intracellular signaling proteins, which themselves form further complexes. The purpose of the PLA method is to visualize proximity between the proteins in cells, provided that the proteins are less than 30-40 nm apart. Protein proximity is usually detected by first incubating the cells with appropriate primary antibodies raised in different species (e.g., rabbit and mouse) against each interacting protein, then adding species-specific secondary antibodies, pre-coupled to short DNA probes. If the DNA probes are in close proximity, a specific linking DNA oligonucleotide can simultaneously bind both of these probes, forming a platform for amplification by in situ PCR or by rolling circle mechanism. Fluorescent tags added to the amplification reaction allow visualization of the interacting proteins, which appear as fluorescent dots that can be readily quantified and localized to particular regions in the cell7,8,9,10.

<table><tbody><tr><td>Chamber slides</td><td>Thermo Fisher Scientific</td><td>178599</td><td>16 well, glass slide</td></tr><tr><td>PLA probe Anti-Mouse Minus</td><td>Sigma-Aldrich</td><td>DUO92004</td><td>Contains 1x Blocking solution, 1x antibody Diluent</td></tr><tr><td>PLA Probe Anti-Rabbit PLUS</td><td>Sigma-Aldrich</td><td>DUO92002</td><td>Contains 1x Blocking solution, 1x antibody Diluent</td></tr><tr><td>Wash Buffers, Flurescence</td><td>Sigma-Aldrich</td><td>DUO82049</td><td>Contains Wash Buffer A and Wash Buffer B</td></tr><tr><td>Mounting Medium with DAPI</td><td>Sigma-Aldrich</td><td>DUO82040</td><td></td></tr><tr><td>Detection Reagents Red</td><td>Sigma-Aldrich</td><td>DUO92008</td><td>Contains 5x Ligation, 1x Ligase, 5x Amlification Red, 1x Polymerase</td></tr><tr><td>p44/42 MAPK (Erk1/2) Antibody</td><td>Cell Signaling</td><td>9102s</td><td></td></tr><tr><td>Phospho-p44/42 MAPK (Erk1) (Tyr204)/(Erk2) (Tyr187)</td><td>Cell Signaling</td><td>5726s</td><td>pERK antibody</td></tr><tr><td>MST2 antibody</td><td>Cell Signaling</td><td>3952s</td><td></td></tr><tr><td>Krs-2 (RJ-5)</td><td>Santa Cruz</td><td>sc-100449</td><td>MST1 antibody</td></tr><tr><td>16% Paraformaldehyde</td><td>Electron microscopy sciences</td><td>15710</td><td>Dilute to 4% PFA in PBS for fixing solution</td></tr><tr><td>Triton x100</td><td>Fisher BioReagents</td><td>BP 151-500</td><td>To prepare 0.1% Triton x-100 in 1xPBS for Permeabilization solution</td></tr><tr><td>Confocal microscopy</td><td>Leica TCS SP8, 63x</td><td></td><td>Image analysis with ImageJ software</td></tr></tbody></table>

detection of heterodimerization of protein isoforms using an in situ proximity ligation assay

Regulated protein-protein interactions are a guiding principle for many signaling events, and the detection of such events is an important element in understanding how such pathways are organized and how they function. There are many methods to detect protein-protein interactions in cells, but relatively few can be used to detect interactions between endogenous proteins. One such method, the proximity ligation assay (PLA), has several advantages to recommend its use. Compared to other common methods of protein-protein interaction analysis, PLA has relatively high sensitivity and specificity, can be performed with minimal cell manipulation, and, in the protocol described herein, requires only two target-specific antibodies derived from different species (e.g., from mouse and rabbit) and one specialized reagent: a set of secondary antibodies that are covalently linked to specific oligonucleotides that, when brought in close proximity of one another, create an amplifiable platform for in situ PCR or rolling circle amplification. In this presentation, we show how to apply the PLA technique to visualize changes in MST1 and MST2 proximity in fixed cells. The technique described in this manuscript is particularly applicable for the analysis of cell signaling studies.

We used the PLA assay to test the interaction between MST1 and MST2 in HEK-293 and iHSC. The cells were fixed, permeabilized, and stained with various antibodies, followed by in situ amplification according to the PLA protocol (Figure 1). To document the level of MST1/MST2 heterodimerization, cells were stained with MST1 and MST2 antibodies (Figure 1A, 1C, and 1G). As a positive control, we also used ERK and pERK antibodies that are expected to be in close proximity in cells with activated ERK (Figure 1B and 1F). Cells lacking MST1 and MST2 show no PLA signal (Figure 1C). Cells stained with only one of the two antibodies likewise show no PLA signal (Figure 1G). These results suggest that HEK-293 cells and iHSC cells contain MST1/MST2 heterodimers, consistent with previous findings from our laboratory4. As an additional negative control we incubated cells with ERK and MST2 primary antibodies to show specificity of signals (Figure 1D and 1H).
To confirm levels of MST1 and MST2 expression, extracts from WT and MST1/MST2 knockout HEK-293 cells, respectively, were analysed by immunoblot with the indicated antibodies (Figure 1I).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57755/57755fig1.jpg" /> 
Figure 1: Representative PLA data. (A-H) Cells were fixed and stained with the indicated antibodies followed by the PLA procedure. Blue: DAPI, RED: PLA signal. Photomicrographs were obtained with a Leica SP5 confocal microscope. (I) Immunoblot for MST1 and MST2. Scale bar is 10 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57755/57755fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay at JoVE.com

Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

Here, we show how to use a Proximity Ligation Assay (PLA) to visualize MST1/MST2 heterodimerization in fixed cells with high sensitivity.

Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

Regulated protein-protein interactions are a guiding principle for many signaling events, and the detection of such events is an important element in ...

detection-heterodimerization-protein-isoforms-using-an-situ-proximity

Nanyang Technological University

Research

JoVE Journal

Biochemistry

7.4K Views.  Fox Chase Cancer Center. De in situ Proximity Ligation Assay of PLA, is een technologie die in staat is om interacties tussen eiwitten, aangebracht weefsel en cellen te detecteren. De associaties tussen eiwitten kunnen worden geïdentificeerd en gekwantificeerd zonder de noodzaak van transgene expressie of extra techs. De enige vereiste is de beschikbaarheid van specifieke hoog-efficiënte antilichamen die met DNA oligonucleotiden kunnen worden gewijzigd.In weefsels MST1 en MST2 bestaan voornamelijk als actiev...

Video: Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay

Protein-protein interactions are involved in thousands of cellular processes and occur in distinct spatial context. Traditionally, co-immunoprecipitation is a popular technique to detect protein-protein interactions. Subsequent Western blot analysis is the most common method to visualize co-immunoprecipitated proteins. Recently, the proximity ligation assay has become a powerful tool to visualize protein-protein interactions in situ and provides the possibility to quantify protein-protein interactions by this method. Similar to conventional immunocytochemistry, the proximity ligation assay technique is also based on the accessibility of primary antibodies to the antigens, but in contrast, proximity ligation assay detects protein-protein interactions with a unique technique involving rolling-circle PCR, while conventional immunocytochemistry only shows co-localization of proteins.
Nuclear factor 90 (NF90) and RNA-binding motif protein 3 (RBM3) have been previously demonstrated as interacting partners. They are predominantly localized in the nucleus, but also migrate into the cytoplasm and regulate signaling pathways in the cytoplasmic compartment. Here, we compared NF90-RBM3 interaction in both the nucleus and the cytoplasm by co-immunoprecipitation and proximity ligation assay. In addition, we discussed the advantages and limitations of these two techniques in visualizing protein-protein interactions in respect to spatial distribution and the properties of protein-protein interactions.

Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay

Protein-protein interactions can occur in both the nucleus and the cytoplasm of a cell. To investigate these interactions, traditional co-immunoprecipitation and modern proximity ligation assay are applied. In this study, we compare these two methods to visualize the distribution of NF90-RBM3 interactions in the nucleus and the cytoplasm.

Protein-protein interactions are involved in thousands of cellular processes and occur in distinct spatial context. Traditionally, co-immunoprecipitation ...

Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of programmed DNA breaks in lymphocytes. The Ataxia-Telangiectasia Mutated kinase (ATM), ATM- and Rad3-Related kinase (ATR) and the catalytic subunit of DNA-dependent Protein Kinase (DNA-PKcs) are among the first that are activated upon induction of DNA damage, and are central regulators of a network that controls DNA repair, apoptosis and cell survival. As part of a tumor-suppressive pathway, ATM and ATR activate p53 through phosphorylation, thereby regulating the transcriptional activity of p53. DNA damage also results in the formation of so-called ionizing radiation-induced foci (IRIF) that represent complexes of DNA damage sensor and repair proteins that accumulate at the sites of DNA damage, which are visualized by fluorescence microscopy. Co-localization of proteins in IRIFs, however, does not necessarily imply direct protein-protein interactions, as the resolution of fluorescence microscopy is limited. 
In situ Proximity Ligation Assay (PLA) is a novel technique that allows the direct visualization of protein-protein interactions in cells and tissues with unprecedented specificity and sensitivity. This technique is based on the spatial proximity of specific antibodies binding to the proteins of interest. When the interrogated proteins are within ~40 nm an amplification reaction is triggered by oligonucleotides that are conjugated to the antibodies, and the amplification product is visualized by fluorescent labeling, yielding a signal that corresponds to the subcellular location of the interacting proteins. Using the established functional interaction between ATM and p53 as an example, it is demonstrated here how PLA can be used in suspension cell cultures to study the direct interactions between proteins that are integral parts of the DNA damage response.

Here, it is demonstrated how the in situ Proximity Ligation Assay (PLA) can be used to detect and visualize the direct protein-protein interactions between ATM and p53 in suspension cell cultures exposed to genotoxic stress.

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of ...

In Situ Detection of Ribonucleoprotein Complex Assembly in the C. elegans Germline using Proximity Ligation Assay

Understanding when and where protein-protein interactions (PPIs) occur is critical to understanding protein function in the cell and how broader processes such as development are affected. The Caenorhabditis elegans germline is a great model system for studying PPIs that are related to the regulation of stem cells, meiosis, and development. There are a variety of well-developed techniques that allow proteins of interest to be tagged for recognition by standard antibodies, making this system advantageous for proximity ligation assay (PLA) reactions. As a result, the PLA is able to show where PPIs occur in a spatial and temporal manner in germlines more effectively than alternative approaches. Described here is a protocol for the application and quantification of this technology to probe PPIs in the C. elegans germline.

In Situ Detection of Ribonucleoprotein Complex Assembly in the C. elegans Germline using Proximity Ligation Assay

This protocol demonstrates use of the proximity ligation assay to probe for protein-protein interactions in situ in the C. elegans germline.

Understanding when and where protein-protein interactions (PPIs) occur is critical to understanding protein function in the cell and how broader processes ...

Developmental Biology

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval neuromuscular junction (NMJ) in Drosophila. Previous studies have shown that the postsynaptic distribution of Dlg at the larval NMJ overlaps with that of Hu-li tai shao (Hts), a homologue to the mammalian adducins. In addition, Dlg and Hts are observed to form a complex with each other based on co-immunoprecipitation experiments involving whole adult fly lysates. Due to the nature of these experiments, however, it was unknown whether this complex exists specifically at the NMJ during larval development.
Proximity Ligation Assay (PLA) is a recently developed technique used mostly in cell and tissue culture that can detect protein-protein interactions in situ. In this assay, samples are incubated with primary antibodies against the two proteins of interest using standard immunohistochemical procedures. The primary antibodies are then detected with a specially designed pair of oligonucleotide-conjugated secondary antibodies, termed PLA probes, which can be used to generate a signal only when the two probes have bound in close proximity to each other. Thus, proteins that are in a complex can be visualized. Here, it is demonstrated how PLA can be used to detect in situ protein-protein interactions at the Drosophila larval NMJ. The technique is performed on larval body wall muscle preparations to show that a complex between Dlg and Hts does indeed exist at the postsynaptic region of NMJs.

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

This protocol demonstrates how Proximity Ligation Assay can be used to detect in situ protein-protein interactions at the Drosophila larval neuromuscular junction. With this technique, Discs large and Hu-li tai shao are shown to form a complex at the postsynaptic region, an association previously identified through co-immunoprecipitation.

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval ...

Neuroscience

Detection of Signaling Effector-Complexes Downstream of BMP4 Using in situ PLA, a Proximity Ligation Assay

BMPs are responsible for a wide range of developmental and biological effects. BMP receptors activate (phosphorylate) the Smad1/5/8 effectors, which then, form a complex with Smad4 and translocate to the nucleus where they function as transcription factors to initiate BMP specific downstream effects 1. Traditional immuno-fluorescence techniques with antibodies against phospho-Smad peptides exhibit low sensitivity, high background and offer gross quantification as they rely on intensity of the antibody signal particularly if this is photosensitive fluorescent. In addition, phospho-Smads may not all be in complex with Smad4 and engaged in active transcription.
In situ PLA is a technology capable of detecting protein interactions with high specificity and sensitivity 2-4. This new technology couples antibody recognition with the amplification of DNA surrogate of the protein. It generates a localized, discrete signal in a form of spots revealing the exact position of the recognition event. The number of signals can be counted and compared providing a measurement. We applied in situ PLA, using the Duolink kit, with a combination of antibodies that allows the detection of the BMP signaling effectors phospho-Smad1/5/8 and Smad4 only when these are in proximity i.e. in a complex, which occurs only with signaling activation. This allowed for the first time, the visualization and measurement of endogenous BMP signaling with high specificity and sensitivity in a time course experiment under BMP4 stimulation.

Detection of Signaling Effector-Complexes Downstream of BMP4 Using in situ PLA, a Proximity Ligation Assay

Here we show how to use Proximity Ligation Assay (PLA), with a combination of antibodies to visualize Bone Morphogenetic Protein (BMP) signaling in fixed cells. This technique allowed us to follow the nuclear accumulation of endogenous BMP activated effector-complexes and quantify their levels over time under BMP4 stimulation.

BMPs are responsible for a wide range of developmental and biological effects. BMP receptors activate (phosphorylate) the Smad1/5/8 effectors, which then, ...

Biology

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ryanodine receptor (RyR) is necessary for the formation of a functional Ca2+ release channel pore. Here, we describe detailed protocols for the assessment of protein self-association, including yeast two-hybrid (Y2H), co-immunoprecipitation (co-IP) and chemical cross-linking assays. In the Y2H system, protein self-interaction is detected by &#946;-galactosidase assay in yeast co-expressing GAL4 bait and target fusions of the test protein. Protein self-interaction is further assessed by co-IP using HA- and cMyc-tagged fusions of the test protein co-expressed in mammalian HEK293 cells. The precise stoichiometry of the protein homo-oligomer is examined by cross-linking and SDS-PAGE analysis following expression in HEK293 cells. Using these different but complementary techniques, we have consistently observed the self-association of the RyR N-terminal domain and demonstrated its intrinsic ability to form tetramers. These methods can be applied to protein-protein interaction and homo-oligomerization studies of other mammalian integral membrane proteins.

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization of the ryanodine receptor, a homo-tetrameric ion channel mediating Ca2+ release from intracellular stores, is critical for skeletal and cardiac muscle contraction. Here, we present complementary in vivo and in vitro methods to detect protein self-association and determine homo-oligomer stoichiometry.

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ...

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), ...

In Situ Monitoring of Transiently Formed Molecular Chaperone Assemblies in Bacteria, Yeast, and Human Cells

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the JDP family could form transient heterocomplexes in eukaryotes to fine-tune substrate selection for the 70 kDa heat shock protein (Hsp70) chaperone-based protein disaggregases. The JDP complexes target acute/chronic stress induced aggregated proteins and presumably help assemble the disaggregases by recruiting multiple Hsp70s to the surface of protein aggregates. The extent of the protein quality control (PQC) network formed by these physically interacting JDPs remains largely uncharacterized in vivo. Here, we describe a microscopy-based in situ protein interaction assay named the proximity ligation assay (PLA), which is able to robustly capture&#160;these transiently formed chaperone complexes in distinct cellular compartments of eukaryotic cells. Our work expands the employment of PLA from human cells to yeast (Saccharomyces cerevisiae) and bacteria (Escherichia coli), thus rendering an important tool to monitor the dynamics of transiently formed protein assemblies in both prokaryotic and eukaryotic cells.

Cognate J-domain proteins cooperate with the Hsp70 chaperone to assist in a myriad of biological processes ranging from protein folding to degradation. Here, we describe an in situ proximity ligation assay, which allows the monitoring of these transiently formed chaperone machineries in bacterial, yeast and human cells.

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the ...

Detection of Protein-Protein Interactions in Drosophila Larvae Using a Proximity Ligation Assay

Source: Wang, S., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/52139">Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay.</a> J. Vis. Exp. (2015).
This video demonstrates the visualization of protein-protein interactions at the Drosophila neuromuscular junction (NMJ) using Proximity Ligation Assay (PLA). After isolating the body walls, interacting proteins are probed with primary antibodies and PLA-conjugated secondary antibodies. The proximity of the interacting proteins facilitates the ligation of probes with connector oligonucleotides, forming circular DNA, which is then amplified by rolling circle amplification. Fluorescent probes bind to the amplified DNA, allowing the visualization of protein-protein interactions at the NMJ.

Source: Wang, S., et al. Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay. J. ...

JoVE Encyclopedia of Experiments

Neuropathology

Neuromuscular Disorders

Proximity Ligation Assay to Study In Situ Protein-Protein Interactions

Use the antibody diluent to dilute the primary antibodies. Apply 40 microliters of the diluted primary antibody solution per 14 by 14-millimeter space on the slides. Place the slides in a humid chamber, and incubate overnight at 4 degrees Celsius.
In the morning, wash the slides twice for 5 minutes each with 50 milliliters of 1x wash buffer A at room temperature in a Coplin jar. Set the Coplin jar on an orbital shaker set to 60 RPM. Place the slides on a paper towel to let wash buffer run off the slide, and gently wipe the edges.
Prepare a 40-microliter solution containing plus and minus probes. Add the solution to each 14 by 14-millimeter space. Incubate the slides in a humid chamber for 1 hour at 37 degrees Celsius. Wash the slides twice for 5 minutes each with 50 milliliters of 1x wash buffer A at room temperature in a Coplin jar.
Set the Coplin jar on an orbital shaker set to 60 RPM. Dilute the ligation buffer 1 to 5 with ultrapure water. Use this buffer to dilute the ligase 1 to 40 to prepare a working stock of ligation solution. Place the slide on a paper towel to let the wash buffer run off the sides, and gently wipe the edges.
Add 40 microliters of the ligation solution to each 14 by 14-millimeter space. Incubate the slides in a humid chamber for 30 minutes at 37 degrees Celsius. After washing and drying the slides as previously described, add 40 microliters of freshly-prepared amplification solution protected from light to each 14 by 14-millimeter space.
Incubate the slides in the humid dark chamber for 1 hour and 40 minutes at 37 degrees Celsius. Wash the slides twice for 10 minutes each with 50 milliliters of 1x wash buffer B, and wash the slides one time for 1 minute with 50 milliliters of 0.01x wash buffer B.
After drawing the epoxy-coated perimeter of the slide, add 10 microliters of mounting medium to each sample, and gently lay a coverslip on top, allowing for the mounting medium to spread out. Paint around the edge of the coverslip with nail polish to seal the coverslip and slide. Avoid moving the coverslip. Let the nail polish harden for at least 20 minutes at room temperature protected from light.

Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

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