Background:

Motile cilia dysfunction leads to Primary Ciliary Dyskinesia (PCD), a disease characterised by abnormal mucociliary flow. Drug screening for PCD is challenging due to the unavailability of high-throughput assays that can evaluate ciliary flow generation. Here, we describe the development of a unique assay that enables the direct evaluation of mucociliary flow as well as high-throughput screening of potential therapeutic agents.

Methods:

The assay relies on the ability of Xenopus tadpoles to promote mixing of a two-phase differential density aqueous mixture, through the robust flow generated by the mucociliary epithelium on their epidermis, assessed by real-time monitoring of fluorescence intensity. The assay was assessed for its ability to detect changes in flow generation in focal adhesion kinase (FAK) morphants that display defects in multiciliated cells (MCC) function, in the presence of different ciliary beat frequency (CBF) modulating compounds as well as in the presence of cytochalasin D or nocodazole that bind to the sub-apical actin cytoskeleton and microtubule network respectively, and disrupt cilia polarity.

Results:

The rate of phase mixing was proportional to the rate of cilia-driven flow and therefore it directly represents the effectiveness of flow generation. In addition, the assay was able to detect changes in ciliary flow elicited by defects in MCC differentiation, CBF modulation and rotational polarity. Importantly, the assay demonstrated that CBF modulating drugs improved flow generation and could thus be used as a potential therapeutic approach in PCD patients.

Conclusion:

This novel assay represents a new tool for research and drug development in motile ciliopathies.