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

Podsumowanie

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

Streszczenie

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.

Wprowadzenie

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.

Protokół

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.

Wyniki

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...

Dyskusje

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 ...

Materiały

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