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 6

Mature RPGR LoF MCCs presented with condensed apical F-actin meshwork that did not dissolve.

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Mature RPGR LoF MCCs presented with condensed apical F-actin meshwork th...
(A) Distribution of F-actin in 4-week control and RPGR KO MCCs. Scale bar: 5 μm. (B) Distribution of F-actin in 8-week control cells and RPGR KO MCCs. Scale bar: 5 μm. (C) STORM imaging of the apical F-actin in both RPGR KO cells and healthy control cells. Scale bars: 5 μm; insert, 1 μm. (A–C) Findings from 4 biological replicates are represented. (D and E) Immunoblotting showed increased F-actin in 4-week RPGR KO MCCs. Results were summarized from 3 biological replicates. Mr, relative molecular mass. (F and G) Immunoblotting showed increased F-actin in 8-week RPGR KO MCCs. Results were summarized from 3 biological replicates. (H) STED imaging of the apical F-actin in MCCs from patients with RP and healthy control cells. Scale bars: 5 μm; insert, 1 μm. (I and J) Apical gelsolin was diminished in 4-week RPGR LoF MCCs. (I) RPGR KO HBEC MCCs. (J) MCCs from 1 patient with RP. Scale bar: 2 μm. Data represent mean ± SEM. **P < 0.01, ***P < 0.001 by 2-tailed t test (E and G). (H) represents results from 8 patients with RP (see Supplemental Figure 11, C and D, for details). (I) represents results from 5 biological replicates. (J) represents similar results from 17 patients (see Supplemental Figure 13, B and C, for details). (K) The proposed model of this study: without RPGR, F-actin meshwork persists at the apical surface, preventing ciliation and cilia elongation; cilia beat is severely impaired.