Proximity Ligation Assay Maps Spatial Protein Interactions at Subciliary Compartments

A research team led by Thorsten Pfirrmann, Ulrich Rüther, and Christoph Gerhardt has presented an optimized protocol applying the in situ proximity ligation assay to map protein proximities within subciliary compartments, reporting their methodology in BMC Molecular and Cell Biology. Primary cilia are hair-like organelles projecting from the surface of nearly all vertebrate cells, coordinating essential biochemical signaling cascades. Structurally, the primary cilium is divided into three compartments: the basal body, the microtubule-based axoneme, and an intermediate transition zone measuring roughly 0.5 micrometers. The transition zone functions as a critical regulatory gatekeeper controlling protein trafficking into and out of the ciliary shaft. Genetic mutations affecting transition zone proteins cause a broad group of human developmental disorders termed ciliopathies. Although motile cilia execute familiar transport activities like mucus clearance and sperm propulsion, defective primary cilia cause a higher number of ciliopathies, highlighting the need to elucidate spatial protein organization within subciliary compartments.

Deciphering the functional landscape of the ciliary transition zone requires robust techniques capable of capturing physical protein-protein interactions. In recent years, proximity labeling technologies including BioID, TurboID, and APEX have mapped broad protein interaction networks across ciliary compartments. The BioID method fuses a ciliary protein to an engineered Escherichia coli biotin ligase known as BirA*, which biotinylates adjacent lysine residues over several hours before proteins are isolated for mass spectrometry. TurboID utilizes an engineered biotin ligase with substantially higher catalytic activity, shortening the necessary labeling timeframe to several minutes. The APEX technique utilizes an ascorbate peroxidase fusion partner that generates reactive radicals upon brief exposure to biotin-phenol and hydrogen peroxide, achieving protein biotinylation within seconds. Although BioID, TurboID, and APEX successfully identify candidate interactors across ciliary proteomes, these mass spectrometry-coupled approaches completely lack spatial resolution, preventing researchers from identifying the precise subcellular microenvironments where the physical associations occur.

To address the lack of spatial resolution in traditional interactomic screens, researchers turn to the in situ proximity ligation assay, originally introduced by Söderberg and colleagues in 2006. In situ proximity ligation assay permits the direct detection of protein proximities closer than forty nanometers within fixed cells and tissues with high spatial precision. Because the method detects native, endogenous proteins, it bypasses the requirement for fluorescent fusion constructs that often complicate Förster resonance energy transfer experiments in clinical samples. Furthermore, in situ proximity ligation assay serves as a spatial complement to conventional biochemical methods like yeast two-hybrid screens, co-immunoprecipitation, and tandem affinity purification. While classical biochemical techniques determine whether two candidate proteins physically interact in bulk cellular lysates, they strip away intracellular compartmentalization. By contrast, in situ proximity ligation assay preserves intact cellular morphology, allowing direct visualization of whether protein interactions take place specifically at the transition zone, the basal body, or elsewhere in the cytoplasm.

The molecular mechanism of the in situ proximity ligation assay relies on dual antibody recognition coupled to localized rolling circle DNA amplification. The procedure begins by incubating fixed biological specimens with two primary antibodies derived from different host species, each binding selectively to one target protein. Species-specific secondary antibodies conjugated to short, unique single-stranded oligonucleotides, termed proximity ligation probes, are subsequently introduced to bind the primary antibodies. If the targeted proteins reside within forty nanometers of each other, the attached oligonucleotides are positioned close enough to hybridize with two circle-forming connector oligonucleotides added to the solution. An enzymatic ligation reaction then covalently seals the hybridized oligonucleotides into a closed circular DNA template. A rolling circle amplification reaction initiated from this circular molecule generates an extended single-stranded DNA concatemeric product that remains physically tethered to the interaction site. Fluorescently labeled complementary oligonucleotides hybridize to this amplified strand, producing bright fluorescent punctae detectable under a fluorescence microscope.

In the documented protocol, the authors demonstrated the workflow using mouse embryonic fibroblasts derived from wild-type and mutant embryos at embryonic day 12.5. Animal housing and experimental procedures followed national guidelines under approval permit number O18/99 from Heinrich Heine University Düsseldorf, utilizing C3H background mice obtained from Charles River Laboratories. Primary fibroblasts were cultured in Dulbecco’s Modified Eagle Medium supplemented with ten percent fetal calf serum, sodium pyruvate, non-essential amino acids, L-glutamine, and penicillin-streptomycin at thirty-seven degrees Celsius with five percent carbon dioxide. To stimulate primary ciliogenesis, confluent fibroblast cultures underwent serum starvation in medium containing 0.5 percent fetal calf serum for at least twenty-four hours. Because standard indirect immunofluorescence interferes with the proximity ligation reaction, the researchers fluorescently labeled cilia by transiently transfecting cells with Lipofectamine 2000 using an IFT88-EYFP expression construct driven by the SV40 promoter within the pJAG368 plasmid vector.

The authors detailed a precise step-by-step staining workflow on glass slides to ensure assay consistency and experimental reproducibility across trials. Cells are washed three times in phosphate-buffered saline for one minute each, permeabilized with 0.5 percent Triton X-100 in phosphate-buffered saline for ten minutes at room temperature, and washed again. To prevent non-specific background binding, samples are incubated for at least ten minutes at room temperature with a blocking solution containing ten percent donkey serum and 0.1 percent Triton X-100 in phosphate-buffered saline. Primary antibodies diluted in a compatible custom diluent are immediately applied after removing the blocking solution and incubated overnight at four degrees Celsius within a sealed humid chamber. The following morning, proximity ligation probes are diluted one-to-five in antibody diluent, pre-incubated at room temperature for twenty minutes, and added to the cells following three ten-minute agitated washes in phosphate-buffered saline, followed by incubation for one hour at thirty-seven degrees Celsius.

Following probe incubation, enzymatic ligation and amplification steps are executed under low-light conditions using commercial Duolink In Situ Detection Reagents. After two five-minute washes in Wash Buffer A, samples are treated with a ligation mixture containing ligation stock diluted one-to-five and ligase diluted one-to-forty, incubating for thirty minutes at thirty-seven degrees Celsius. After two further washes in Wash Buffer A, an amplification mixture containing one-to-five amplification stock, one-to-eighty DNA polymerase, and fluorescently labeled oligonucleotides is applied for one hundred minutes at thirty-seven degrees Celsius in the dark. The authors utilized Detection Reagents Orange, exhibiting excitation and emission peaks at 554 and 579 nanometers, while highlighting that Green, Red, and Far Red kits are also available. Samples are washed twice in Wash Buffer B for ten minutes, once in 0.01-times Wash Buffer B for one minute, air-dried in the dark, and mounted in Mowiol containing one microgram per milliliter DAPI before examination using a Zeiss Imager.A2 microscope with a 100-times oil immersion objective.

Applying this optimized experimental protocol allowed the researchers to detect key molecular interactions occurring specifically at the primary ciliary base. The authors previously uncovered that the transition zone protein Rpgrip1l regulates ciliary proteasome activity and identified the proteasomal subunit Psmd2 as an Rpgrip1l interactor through biochemical screening. Implementing in situ proximity ligation assay in IFT88-EYFP-marked fibroblasts revealed distinct red fluorescent punctae at the ciliary base, confirming that Rpgrip1l and Psmd2 reside within forty nanometers of each other in the ciliary compartment. The assay also verified close spatial proximity between Rpgrip1l and the basal body protein Rpgrip1. In contrast, when the assay was performed in mutant fibroblasts from Rpgrip1nmf247/nmf247 mice producing a truncated Rpgrip1 protein, proximity ligation signals were absent at primary cilia. Furthermore, technical specificity was confirmed by omitting the anti-Rpgrip1l primary antibody while applying only the anti-Rpgrip1 antibody, which completely abolished fluorescent signal generation at the cilium.

Despite its sensitivity and spatial precision, in situ proximity ligation assay possesses inherent technical constraints that necessitate rigorous protocol adherence. Because the technique relies on antibody binding within fixed cells and tissues, it is fundamentally restricted to endpoint analyses and cannot monitor dynamic interactions in living systems. When troubleshooting weak or absent fluorescent signals, investigators must evaluate primary antibody quality, affinity, and dilution, which should mirror conditions established for standard immunofluorescence. Fixation protocols must also match antibody preferences; the authors obtained optimal results using either four percent paraformaldehyde for one hour at four degrees Celsius or one hundred percent methanol for five minutes at minus twenty degrees Celsius. Furthermore, enzymatic reagents require careful handling: ligase and polymerase stocks must be stored at minus twenty degrees Celsius and freshly diluted before use, avoiding repeated freeze-thaw cycles. Residual wash solutions must be thoroughly removed using a suction apparatus to prevent reagent dilution during critical enzymatic incubations.

High background fluorescence represents another frequent technical challenge that can be mitigated through methodical troubleshooting. Non-specific signals often stem from inadequate blocking, requiring reagents compatible with both primary antibodies and probes, such as the ten percent donkey serum solution utilized by the authors. Incomplete paraffin clearing, agitated washing omissions, or accidental sample drying during protocol transitions can introduce intense fluorescent artifacts. Investigators should delineate reaction zones using a grease pencil rather than ink-based markers, which produce diffuse fluorescent mists, while clean working conditions prevent particulate contamination from dust or crystals. Regarding multi-protein ciliary visualization, the authors noted that directly conjugated primary antibodies, such as anti-gamma-tubulin-FITC, or co-expressed fluorescent fusion proteins, including IFT88-EYFP, gamma-tubulin-BFP, or OFD1-EYFP, offer viable pathways to delineate subciliary landmarks. Ultimately, by pinpointing whether truncated ciliopathy proteins, such as mutant forms of Rpgrip1l and Nphp4, retain their localized transition zone interactions or mislocalize elsewhere in the cell, in situ proximity ligation assay provides vital mechanistic insights into ciliopathy pathogenesis.

Subject of Research: In situ proximity ligation assay protocol for mapping spatial protein interactions at primary cilia

Article Title: In situ proximity ligation assay for analysing spatial interactions between ciliary proteins

Article References: Pfirrmann, T., Rüther, U., & Gerhardt, C. (2025). In situ proximity ligation assay for analysing spatial interactions between ciliary proteins. BMC Molecular and Cell Biology, 26(1), Article 30. https://doi.org/10.1186/s12860-025-00555-7

Image Credits: AI Generated

DOI: 10.1186/s12860-025-00555-7

Keywords: in situ proximity ligation assay, primary cilia, ciliary transition zone, ciliopathies, Rpgrip1l, Psmd2, Rpgrip1, rolling circle amplification, fluorescence microscopy, mouse embryonic fibroblasts, subciliary compartments, IFT88-EYFP, BioID, TurboID, APEX, protein-protein interactions, Duolink detection reagents