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

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 cell^7,8,9,10.

1. Preparation of Solutions

1. 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 °C.
2. Prepare permeabilization solution: 0.1% Triton X-100 in 1x PBS. For 100 mL of solution, add 100 µL of Triton X-100 into 100 mL of 1x PBS. Store at room temperature (RT).
3. 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%).
4. Prepare blocking solution as supplied by kit. Alternatively, use 1x PBS solution containing 2% BSA.
5. Prepare the antibody diluent as supplied by kit. Alternatively, use 1x PBS solution containing 1% BSA.

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.

1. Culture HEK 293 in DMEM supplemented with 10% FBS, 0.2% Glucose, 2 mM L-Glutamine, 100 U/mL penicillin G and 100 µg/mL streptomycin on tissue culture-treated plates at 37 °C in 5% CO₂. Maintain iHSC in 10% FBS/DMEM supplemented with Pen/Strep and 2 µM forskolin. Routinely test cells for mycoplasma. No cell line authentication was performed.
2. Coat a 16-well chamber slide with 50 µL of 10 µg/mL natural mouse laminin or 0.01% poly-L-lysine solution and incubate for 30 min at 37 °C in 5% CO₂. Split cells using 0.04% trypsin-EDTA.
3. Plate iHSC and HEK-293 in 100 µL of medium at 80% confluence (15,000-25,000 cells/well).
4. Incubate the cells for 12 -24 hours at 37 °C in a humidified, 5% CO₂ incubator.

3. Fixation and Permeabilization

1. Remove the medium from the wells and wash with 100 µL of 1x PBS. Aspirate with a micropipette to minimize the risk of removing the sample.
2. Fix cells by adding 50 µL 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.
3. 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.
4. Treat the cells with permeabilization solution (0.1% Triton x-100 in 1x PBS) for 10 min without agitation at RT.
5. Wash cells with TBST three times for 5 min each with agitation.

4. Blocking

1. Tap off the TBST (very gently with a micropipette). Visualize the slide in a microscope to see if cells remain attached.
2. Add one drop (50-60 µL) of 1x Blocking Solution to each sample.
3. Pre-heat a humidity chamber (empty tip box with water) at 37 °C and incubate the slides in it for 1 h at 37 °C.

5. Primary Antibodies

1. Dilute the primary antibodies (1:100) in the Antibody Diluent (see Table of Materials). Prepare 40 µL antibody solution per well.
2. Remove blocking solution from the slides by tapping off the liquid. Do not allow cells to dry.
3. Vortex and add 40 µL of the antibody solutions to each well.
4. Incubate 1 h at 37 °C in a preheated humidity chamber.

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.

1. Dilute the two PLA probes (anti-Mouse MINUS and anti-Rabbit PLUS) 1:5 in the Antibody Diluent. For each sample, prepare 40 µL of PLA probe. Incubate the mixture for 20 min at RT.
2. Tap off the primary antibody solution from the slides. Wash the slides twice for 5 min each with Wash Buffer at RT.
3. Gently tap of the Wash Buffer from the slides and add the diluent PLA probe solution to wells (40 µL/well).
4. Incubate the slides in a pre-heated humidity chamber for 1 h at 37 °C.

7. Ligation

NOTE: The in situ Detection Reagents Red, Ligase, and Ligation Solution are provided as part of a kit (see Table of Materials).

1. Use the in situ Detection Reagents Red.
2. 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.
3. Tap off the PLA probe solution from the slides. Wash the slides in 1x Wash Buffer twice for 5 min each at RT.
4. Immediately before addition to the samples, vortex and add Ligase to the Ligation solution at 1:40 dilution and vortex again.
5. Tap off the Wash Buffer from the slides and add the Ligation/Ligase solution to each well (40 µL/well).
6. Incubate the slides in a pre-heated humidity chamber for 30 min at 37 °C.

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.

1. Vortex and dilute the required volumes of the 5x Amplification stock 1:5 in high purity water and mix.
2. Tap off the Ligation/Ligase solution from the slides. Wash the slides in 1x Wash Buffer twice for 2 min each at RT.
3. Vortex and add the polymerase to the Amplification solution at 1:80 dilution and vortex again.
4. Tap off Wash Buffer from the slides and add the Amplification/Polymerase solution to each well (40 µL/well). Incubate the slides in a pre-heated humidity chamber for 100 min at 37 °C.

9. Preparation for Imaging

NOTE: These are light sensitive reagents. Keep the slides protected from light.

1. Tap off the Amplification-Polymerase solution from the slides and wash twice for 10 min each in 1x Wash Buffer at RT.
2. 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.
3. Add ~40 µL 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.
4. Wait for at least 20 min before performing image analysis using a confocal microscope.
   NOTE: The protocol can be paused here.
5. After imaging, store the slides at -20 °C in the dark.

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).

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 µm. Please click here to view a larger version of this figure.

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 µg/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.