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

Cilia are small, hair-like structures on the surface of most eukaryotic cells. They are composed of distinct substructures: the basal body, the transition zone, and the axoneme. Proper ciliary function is crucial for human health, and defects can result in a group of disorders known as ciliopathies. Many ciliopathy-associated mutations affect genes encoding proteins of the ciliary transition zone, a key structural and regulatory region at the base of the cilium. Understanding the molecular composition and interactions within subciliary compartments, such as the transition zone, is essential to elucidate their role in ciliary function and disease.

Protein interaction studies have played a central role in uncovering the functional landscape of subciliary compartments. In this context, the in situ proximity ligation assay (in situ PLA) has emerged as a valuable tool to investigate whether two proteins are located in close proximity (less than 40 nm) within the cellular environment, implying potential interaction. In situ PLA uses primary antibodies to recognise target proteins, followed by secondary antibodies conjugated with oligonucleotides (PLA probes). When two probes are sufficiently close, added circle-forming oligonucleotides can hybridise and ligate to form a circular DNA strand. This DNA circle serves as a template for rolling circle amplification, which is then detected through hybridisation with fluorescently labelled oligonucleotides. The resulting signals can be visualised using fluorescence microscopy, enabling precise spatial mapping of protein proximities in cells.

The in situ PLA technique offers a powerful means of detecting protein proximities in subciliary compartments with high spatial resolution. This method supports the identification of novel protein interactions and contributes to a deeper understanding of subciliary architecture and its disruption in ciliopathies.