Loss of RPGR disrupts motile cilia and causes primary ciliary dyskinesia by affecting F-actin dynamics

Cilia are cellular organelles extruding from the surface of various cell types, serving either sensory or motile functions. Retinitis pigmentosa GTPase regulator (RPGR) variants affect both photoreceptor sensory cilia and airway motile cilia, leading to retinitis pigmentosa (RP) and primary ciliary dyskinesia (PCD), respectively. Not all patients develop PCD, and it remains unclear which RPGR variants predispose patients to PCD. Here, we leverage 2D organoids, super-resolution microscopy, and live-cell imaging to characterize the multiciliated cells (MCCs) from patients with different RPGR variants and CRISPR-modified RPGR KO MCCs. We demonstrate that MCCs with RPGR variants have reduced ciliation, shorter cilia, impaired cilia beat, or cilia beat incoordination, potentially resulting in compromised mucociliary clearance and lung diseases. Moreover, we show that RPGR regulates motile cilia through interfering with F-actin dynamics, evidenced by the undissolved F-actin meshwork in RPGR-deficient MCCs, and the defects can be ameliorated with either Latrunculin A or Y27632 treatment. Though PCD was observed only in patients with variants that affect both isoforms, patients with RPGRORF15 variants also showed cilia and airway anomalies. All RPGR variants affect motile cilia one way or another, and the mechanisms involve the accumulation of apical F-actin.