Loss of RPGR disrupts motile cilia and causes primary ciliary dyskinesia by affecting F-actin dynamics
Yang Wu, Erika Tavares, Binrun Liang, Wallace B. Wee, Vito Mennella, Han-Chao Feng, Jiaying Cao, Pui Yee Wong, Jiayi Zheng, Mu He, Kirk AJ Stephenson, Liran Hanan Hochma, Janice Min Li, Nan-Peng Chen, Sharon D. Dell, Elise Heon, Zhen Liu
Yang Wu, Erika Tavares, Binrun Liang, Wallace B. Wee, Vito Mennella, Han-Chao Feng, Jiaying Cao, Pui Yee Wong, Jiayi Zheng, Mu He, Kirk AJ Stephenson, Liran Hanan Hochma, Janice Min Li, Nan-Peng Chen, Sharon D. Dell, Elise Heon, Zhen Liu
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Research Article Cell biology Pulmonology

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

Abstract

Cilia are cellular organelles that extrude 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 had cilia and airway anomalies. All RPGR variants affected motile cilia in some way, and the mechanisms involved the accumulation of apical F-actin.

Authors

Yang Wu, Erika Tavares, Binrun Liang, Wallace B. Wee, Vito Mennella, Han-Chao Feng, Jiaying Cao, Pui Yee Wong, Jiayi Zheng, Mu He, Kirk AJ Stephenson, Liran Hanan Hochma, Janice Min Li, Nan-Peng Chen, Sharon D. Dell, Elise Heon, Zhen Liu

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Figure 1

MCCs with pathological RPGR variants presented with short cilia and decreased ciliation.

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MCCs with pathological RPGR variants presented with short cilia and decr...
(A) Cartoon showing RPGR as the causative gene for RP and PCD, as well as the focus of this study. (B) Distributions of variants of the 32 patients participating in this study. Red indicates variants that affect both isoforms and correspond to LoF or splicing; pink corresponds to variants with missense effect; and purple corresponds to variants affecting only RPGRORF15. Variants in patients 9 and 15 are predicted to be missense and affect splicing. (C) The proposed workflow for this study. (D) Immunostaining of human nasal cells of control individuals and patients with RPGR variants. Green represents RPGR and red represents α-tubulin. MIP, maximum intensity projection. Scale bar: 10 μm. (E) Cilia length measurements for the nasal MCCs from control participants and patients with RP. Individuals with PCD are distinguished with a red asterisk. (F) Cilia length measurements for the MCCs from control participants and patients with RP cultured at the ALI for 8 weeks. (G) Immunostaining of basal body (POC1B) and cilia (acetylated tubulin) showed reduced ciliation in the MCCs of a patient. Scale bar: 5 μm. (H) Ciliation was reduced for the MCCs bearing pathological RPGRex1–19 or RPGRORF15 variants. Data represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by 2-tailed t test (E, F, and H). For (E), we performed 1 experiment in a subset of the cohort that included 6 healthy control participants and 28 patients. For (F–H), we performed 3 independent replicates for a subset of the cohort that included 3 healthy control participants and 18 patients.