Name: detection of heterodimerization of protein isoforms using an in situ proximity ligation assay
Uploaded: 2018-10-20T12:30:00
Description: Watch this Scientific Journal Video about Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay at JoVE.com

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, t...

<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>

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 ...

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 border="0" cellpadding="0" cellspacing="0" style="width:849px;" width="849">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Chamber slides</td>
			<td style="width:123px;">Thermo Fisher Scientific</td>
			<td style="width:344px;">178599</td>
			<td style="width:167px;">16 well, glass slide</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">PLA probe Anti-Mouse Minus</td>
			<td>Sigma-Aldrich</td>
			<td>DUO92004</td>
			<td>Contains 1x Blocking solution, 1x antibody Diluent</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">PLA Probe Anti-Rabbit PLUS</td>
			<td>Sigma-Aldrich</td>
			<td>DUO92002</td>
			<td>Contains 1x Blocking solution, 1x antibody Diluent</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Wash Buffers, Flurescence</td>
			<td>Sigma-Aldrich</td>
			<td>DUO82049</td>
			<td>Contains Wash Buffer A and Wash Buffer B</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Mounting Medium with DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>DUO82040</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">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 height="40">
			<td height="40" style="height:40px;">p44/42 MAPK (Erk1/2) Antibody</td>
			<td>Cell Signaling</td>
			<td>9102s</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Phospho-p44/42 MAPK (Erk1) (Tyr204)/(Erk2) (Tyr187)</td>
			<td>Cell Signaling</td>
			<td>5726s</td>
			<td>pERK antibody</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">MST2 antibody</td>
			<td>Cell Signaling</td>
			<td>3952s</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Krs-2 (RJ-5)</td>
			<td>Santa Cruz</td>
			<td>sc-100449</td>
			<td>MST1 antibody</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">16% Paraformaldehyde</td>
			<td>Electron microscopy sciences</td>
			<td>15710</td>
			<td>Dilute to 4% PFA in PBS for fixing solution</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">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 height="40">
			<td height="40" style="height:40px;">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...

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 ...

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