Assessing in situ phosphoinositide-protein interactions through fluorescence proximity ligation assay in cultured cells

Abstract

Proximity ligation assay (PLA) is a well-established method for detecting in situ interactions between two epitopes with high resolution and specificity. Notably, PLA is not only a robust method for studying protein-protein interaction, but also an efficient approach to characterize and validate protein post-translational modifications (PTM) using one antibody against the core protein and one against the PTM residue. Therefore, it could be applied as a powerful approach to detect specific interactions of endogenous phosphoinositides and their binding proteins within cells. Importantly, we have specifically detected the PLA signal between PtdIns(4,5)P₂ and its binding effector p53 in the nucleus. This cutting-edge method fully complements other conventional approaches for studying phosphoinositide-protein interactions and provides important localization signals and robust quantitation of the detected interactions. Here, we present the PLA fluorescence protocol for detecting in situ phosphoinositide-protein interactions in cultured cells and is semi-quantitative for interactions that are regulated by cellular signaling.

1. Introduction

Proximity ligation assay (PLA) is a robust method for detecting and quantifying interactions between two epitopes with high resolution (<40 nm, traditionally considered as direct interaction) and specificity because interactions between endogenous proteins are detected in their cellular context at physiological expression levels [1,2]. Since its development by Fredriksson et al. in 2002 [3], PLA has been increasingly used to detect the interaction between two proteins [4–8]. In addition to those studies, we have also applied PLA for validating protein-protein interactions suggested by traditional methods, including pull-down assay followed by mass-spectrometry, co-immunoprecipitation, in vitro protein binding assay, enzyme-linked immunosorbent assay (ELISA), and protein-protein colocalization post immunofluorescence staining [9–11].

Notably, PLA is not only a robust method for studying protein-protein interactions, but also an efficient approach to characterize and quantify protein post-translational modifications (PTM) using one antibody against the core protein and one against the PTM residue. For example, the covalent modification of proteins can be studied in situ owing to the dual recognition format provided by PLA [12]. Therefore, it could be applied as a powerful approach to detect specific interaction of endogenous phosphoinositides and their binding proteins within cells. Importantly, we have first introduced PLA into the field of phosphoinositide signaling by specifically detecting the PLA signal between PtdIns(4,5)P₂ and its binding effector-p53 in the nucleus, which was enhanced by the genotoxic agent cisplatin, and diminished by deletion of PIPKIα, the kinase responsible for PtdIns(4,5)P₂ generation [13]. This cutting-edge method fully complements other conventional approaches for studying phosphoinositide-protein interactions, such as lipid strip assay and liposome sedimentation assay, and provides semi-quantitative subcellular localization of the detected interactions.

Here, we present the PLA protocol, modified from the Duolink^® Proximity Ligation Assay procedure (Millipore Sigma), the only commercial resource currently available, for detecting the in situ phosphoinositide-protein interactions in the nucleus (Figure 1). Briefly, cultured cells are fixed, permeabilized, and blocked as per traditional immunofluorescence staining procedure. Next, two primary antibodies raised in different species are used to detect a specific phosphoinositide and its potential binding effector. A pair of PLA probes, oligonucleotide-labeled secondary antibodies raised in corresponding species, then bind to the primary antibodies. Only PLA probes located in close proximity (less than 40 nm) are able to be joined by the hybridizing connector oligos and ligase to form a closed circular DNA template, which is required for rolling-circle amplification (RCA). The PLA probe then functions as the primer for DNA polymerase to generate concatemeric sequences during RCA. This reaction results in up to 1000-fold amplification of the signal, thereby enabling in situ detection of phosphoinositide-protein interaction. Lastly, fluorophore-labeled oligos hybridize to the complementary repeating sequences in the amplicon. These PLA signals are visualized as discrete spots by fluorescence microscopy that can be quantified by NIH ImageJ analysis to provide precise intracellular localization of the phosphoinositide-protein interaction.

Figure 1: Schematic illustration of protein-phosphoinositide PLA reaction.

First, two primary antibodies recognize the specific epitopes of the protein-phosphoinositide (PI) complex in the cell. Then secondary antibodies coupled with oligonucleotides (PLA probes) bind to the primary antibodies. Next, the connector oligos join the PLA probes located in close proximity and become ligated. The resulting circular, closed DNA template becomes amplified by the DNA polymerase. Complementary detection oligos conjugated with fluorochromes hybridize to repeating sequences in the amplicons. Lastly, PLA signals are detected by fluorescent microscopy as discrete punctate foci and provide the intracellular localization of the protein-PI complex. The example image shows the PLA signals of p53-PtdIns(4,5)P₂ complex (Red) locate at the nucleus (DAPI, Blue) of MDA-MB-231 cells.

2. Materials

1. Microscope cover glass (22×22 mm) (see Note 1)

2. 6-well plate

3. 70% ethanol

4. Phosphate Buffered Saline (PBS) (137 mM NaCl, 10 mM Phosphate, 2.7 mM KCl, pH 7.4)

5. Coating solution: dissolve 0.2% gelatin in ddH₂O, incubate in 60~70°C water bath for 1 h to make sure complete dissolution, filter by 0.22 μm filter before use, store at 4°C.

6. 4% paraformaldehyde (PFA) solution in PBS (4% PFA)

7. 0.3% Triton X-100 in PBS

8. Humidity chamber: 15 cm cell culture dish with damp tissue on the bottom, covered by PARAFILM to create a clean dry surface and pre-warmed to 37°C before use.

9. Blocking buffer: 1% Bovine Serum Albumin (BSA) in PBS, store at 4°C.

10. Buffer A: 0.01 M Tris, 0.15 M NaCl, 0.05% Tween 20, pH 7.4, filter by 0.22 μm filter before use, store at 4°C. Bring the solution to room temperature before use.

11. Buffer B: 0.2 M Tris, 0.1 M NaCl, pH 7.5, filter by 0.22 μm filter before use, store at 4°C. Bring the solution to room temperature before use.

12. Buffer C: 10 ml of Buffer B, 990 ml high purity water, store at 4°C. Bring the solution to room temperature before use.

13. Primary antibodies against the phosphoinositides or proteins of interest (see Note 2)

14. PLA PLUS probe: Duolink^® in situ PLA Probe anti-Rabbit PLUS (Millipore Sigma)

15. PLA MINUS probe: Duolink^® in situ PLA Probe anti-Mouse MINUS (Millipore Sigma)

16. Antibody diluent: provided in the above Duolink^® in situ PLA Probes (Millipore Sigma)

17. Duolink^® in situ detection reagents Red kit (Millipore Sigma)

    1. 5x Ligation buffer (see Note 3)
    2. Ligase
    3. 5x Amplification buffer (see Note 3)
    4. Polymerase

18. 4′,6-diamidino-2-phenylindole (DAPI)-containing mounting medium

19. Glass microscope slides

20. Nail polish

21. Incubator at 37°C

22. Freeze block for enzymes

23. Shaker

24. Water bath

25. Fume hood

26. Fluorescence microscope

27. Analysis software (such as NIH ImageJ)

3. Methods

3.1. Cell Culture and Cover Glass Preparation

1. Place a microscope cover glass into each well of a 6-well plate.

2. Add 2 ml of 70% ethanol to each well and incubate for 10 min.

3. Remove the 70% ethanol and wash the wells with 3 ml of PBS 3 times.

4. Add 2 ml of the coating solution to each well and incubate overnight at 4°C.

5. Remove the coating solution, seed and treat cells with desired experimental conditions to reach 50% confluency before fixation.

3.2. Prepare Cells for PLA

1. Remove the media from the cells and wash the cells once with PBS.

2. In a fume hood, add 1 ml of 4% PFA in PBS to each well and put on a rocker at low speed for 30 min at room temperature.

3. In a fume hood, remove the 4% PFA in PBS and wash the cells 3 times with 2 ml of PBS.

4. Add 500 μl of 0.3% Triton X-100 in PBS to each well to permeabilize cells for 10 min at room temperature with gentle rocking (see Note 4).

5. Remove the 0.3% Triton X-100 in PBS and wash 3 times with 2 ml of PBS.

6. Add 500 μl of blocking buffer to each well for 1 hour at room temperature with gentle rocking to block the cells.

7. Prepare primary antibody mixes by diluting the primary antibodies, one from mouse and one from rabbit, in 40 μl of blocking buffer per cover glass at a 1:50 to 1:300 dilution depending on the antibody for each reaction.

8. Add the 40 μl primary antibody mixes on top of the PARAFILM in the humidity chamber.

9. Take out the cover glasses from the wells and place them cell side down on the antibody mixes in the humidity chamber so the cells are touching the mixture (see Note 5 and Note 6).

10. Cover the humidity chamber and incubate at 4°C overnight (see Note 7).

11. Place the cover glasses back into the 6-well plate with the cell side facing up and wash 3 times with 2 ml PBS.

3.3. Prepare PLA Probes

1. Dilute the two PLA probes in 1:5 dilutions with the antibody diluent provided by the detection reagents kit to a total volume of 40 μl per cover glass (8 μl PLUS probe and 8 μl MINUS probe in 24 μl antibody diluent per cover glass).

2. Add the 40 μl mixes on top of the PARAFILM in the humidity chamber.

3. Take out the cover glasses from the wells and place them cell side down on the antibody mixes in the humidity chamber so the cells are touching the mixture (see Note 5 and Note 6).

4. Cover the humidity chamber and transfer it into the 37°C incubator and incubate for 1 h.

3.4. Ligation

1. Dilute the ligation buffer in high purity water in a 1:5 dilution to a total volume of 40 μl per cover glass (5 μl ligation buffer in 32 μl of high purity water per cover glass).

2. Place the cover glasses back into the 6-well plate with the cell sides facing up.

3. Wash the cover glasses for 5 min twice in buffer A at room temperature with gentle rocking.

4. Clean the PARAFILM surface in the humidity chamber with 70% ethanol and high purity water and let dry.

5. Add ligase to the diluted ligation buffer in a 1:40 dilution to a total volume of 40 μl per cover glass (1 μl ligase in 39 μl diluted ligation buffer per cover glass).

6. Add the 40 μl ligation solutions to the surface of the PARAFILM in the humidity chamber and place the cover glasses on top with the cell side facing down after tapping off excess wash buffer (see Note 5 and Note 6).

7. Incubate in a 37°C incubator for 30 min.

3.5. Amplification

1. Dilute the amplification buffer in high purity water in a 1:5 dilution to a total volume of 40 μl per cover glass (8 μl amplification buffer in 32 μl high purity water per cover glass).

2. Place the cover glasses back into the 6-well plate with the cells facing up and wash the glasses for 5 min twice in Buffer A with gentle rocking at room temperature.

3. Clean the PARAFILM surface in the humidity chamber with 70% ethanol and high purity water and let dry.

4. Add the polymerase to the diluted amplification buffer in a 1:80 dilution to a total volume of 40 μl per cover glass (0.5 μl polymerase in 39.5 μl of diluted amplification buffer per cover glass) (see Note 9).

5. Add the 40 μl amplification solutions to the surface of the PARAFILM in the humidity chamber and add the cover glasses to them cell side down after tapping off excess buffer A (see Note 5 and Note 6).

6. Incubate in a 37°C incubator for 100 min.

3.6. Final Washing

1. Place the cover glasses back into the 6-well plate with the cells facing up and wash with buffer B for 10 min twice with gentle rocking at room temperature (see Note 10).

2. Wash the cover glasses with buffer C for 1 minute with gentle rocking at room temperature (see Note 11).

3.7. Mounting

1. Take out the cover glasses and place them on a dry tissue with cells side facing up.

2. Loosely cover them with aluminum foil and let dry for 20 min (see Note 12).

3. Add 30 μl of DAPI-containing mounting medium on to a glass microscope slide.

4. Place the cover glasses on top of the mounting medium cells down (see Note 13).

5. Seal the edges of the cover glasses by applying nail polish to them and dry the applied nail polish at room temperature with the slides protected from light (see Note 14).

6. Store the microscope slides at 4°C prior to imaging.

3.8. Imaging Acquisition and Quantification

1. Acquire images of the slides using a fluorescence microscope.

2. Make sure you use appropriate filters for the PLA channel. The fluorophore in the Amplification Red has an excitation/emission wavelength of 594 nm/624 nm and can be detected using the same filter as for Texas Red. Alternative detection channels are also available, such as Amplification Green, Orange, and Far Red reagents from Millipore Sigma, the only commercial resource for PLA reagent currently available.

3. Export PLA and DAPI channels separately.

4. The PLA signal can be counted either manually (if the number is not high) or by an analysis software such as NIH ImageJ.

5. When using ImageJ for PLA signal quantification, load the corresponding PLA and DAPI channels to ImageJ by using File>Open> function.

6. Click the opened images, remove the background by using Image>Adjust>Brightness/Contrast function. Drag the adjusting bars for Maximum/Minimum/Brightness/Contrast until the PLA signals are recognized as fluorescent puncta. An individual signal is of sub-micrometer size.

7. To create the merged image, overlay the PLA and DAPI channels by using Image>Color>Merge Channels function. Remember to check the box for keeping source images.

8. Use the DAPI channel as the reference to quantify PLA signals per cell.

9. To quantify the PLA signal, select the opened PLA channel, adjust the threshold using Image>Adjust>Color Threshold function, choose the default threshold method and click select. Then use the Analyze>Analyze Particles> function to get the PLA signal count.

10. To count the PLA signal in each cell, crop the image before analyzing particles.

4. Notes

1. Commercially available cover glasses come in a variety of thicknesses, but the thickness closest to 0.17 mm is a No. 1.5 coverslip. For the newer, high- to super-resolution optical microscopes, a No. 1.5H (high performance) is the required thickness. Using the incorrect thickness for cover glasses can greatly reduce the ability to maximize information from prepared samples using an optical microscope.

2. Use verified phosphoinositide/protein antibodies suitable for immunofluorescence staining in the fluorescence PLA procedure. For example, the anti-PtdIns(4,5)P₂ antibody (Echelon Biosciences) used in our recent study to show the PtdIns(4,5)P₂-p53 interaction through PLA was verified for immunofluorescence staining, which detected nuclear PtdIns(4,5)P₂ in nuclear speckles, nucleoplasmic foci, and nucleoli, and was neutralized by preincubation with PtdIns(4,5)P₂ [13,14]. Also, the two selected primary antibodies must be raised from different species, such as one from mouse and one from rabbit, and correspond to the subsequently used PLA probes.

3. Aliquot the 5x Ligation buffer and 5x Amplification buffer into 50 μl per tube and keep those tubes in −20°C before use to avoid multiple freeze and thaw cycles. Wrap all the 5x Amplification buffer tubes with aluminum foil to protect them from light.

4. Permeabilization with a detergent such as Triton X-100 or NP-40 which could penetrate the nuclear membrane is required for detecting the PLA signal of interacting phosphoinositide-protein in the nucleus.

5. When taking out the cover glasses from the 6-well plate for incubation in the humidity chamber, save the 6-well plate for subsequent washings.

6. Make sure the reaction area corresponds to the reaction volume. The droplet must cover the reaction area.

7. If no or few signals in positive samples occur due to insufficient binding of primary antibodies, try to enhance the incubation condition of primary antibodies by incubating the cover glass in a humidity chamber at room temperature overnight or in a 37°C incubator for 1 h. This all depends on the specific antibodies used.

8. Keep the ligase in a freezer block (−20°C), and only add it immediately before the addition to the sample.

9. Keep the polymerase in a freezer block (−20°C), and only add it immediately before the reaction. The amplification buffer is light-sensitive, so protect all amplification buffer solutions from light.

10. Keep cover glasses protected from light while washing.

11. Additional staining steps for extra proteins/lipids of interest or subcellular/subnuclear structures using fluorophore direct-conjugated antibodies or dyes, such as fluorophore-conjugated antibodies against SC-35 for nuclear speckles and phosphatidylserine (PS) for nuclear envelope [13] can be added from this step by following conventional immunofluorescence staining procedure. Make sure they are conjugated with different fluorophores from the one used for PLA detection.

12. Make sure the cover glasses are dry before mounting.

13. Use approximately 30 μl mounting media for each cover glass. The mounting media is viscous, so cut the pipette tip before pipetting the mounting media. Apply the mounting media to the slide, hold the cover glass with the side of the cells facing the mounting media, tilt the cover glass, touch one side to the mounting media, slowly place the cover glass down and allow the mounting media to spread throughout the entire cover glass. Try to avoid bubbles. If there are bubbles, gently push or rotate the cover glass to slide off the microscope slide to release those bubbles. After bubbles are removed, push or rotate the cover glass back. Then gently remove excessive mounting media at the edge of the cover glass using tissue paper.

14. Apply half of the nail polish on the cover glass and half of the nail polish on the slides. Leave no gap to join every side of the cover glass and slide, and make sure the cover glass is completely sealed by the nail polish onto the slides. This can be repeated once when the first-time applied nail polish is dry. Also, protect the slides from light while drying them at room temperature.