Roles of Ezrin in regulation of ciliary beating in lung multiciliated cell

Abstract

The mucociliary clearance is the first line of defense in the lungs. The function is given by the beating cilia on the surface of ciliated cells cooperating with the protective mucous layer. The beating cilia plays removing inhaled particles and pathogens (viruses, bacteria and fungi). Thus, the beating cilia are the key apparatus to conduct the mucociliary clearance. For ciliary beating to function properly, localization of ciliary proteins that regulate ciliary beating and formation of epithelial cell polarity are important. Ezrin/Radixin/Moesin (ERM) proteins function as general cross-linkers between apical membrane proteins and the actin cytoskeleton and are involved in the functional expression of apical membrane proteins. Especially, ezrin may play the important role of apical localization of β2-adrenergic receptor (β2AR) and cAMP-dependent protein kinase (PKA) in multiciliated cells. Here we examined roles of ezrin in the regulation of ciliary beating in lung multiciliated cells by using ezrin-knockdown (Vil2kd/kd) mice. To evaluate ciliary function, we analyzed ciliary beat frequency (CBF) and ciliary bend distance (CBD, and index of amplitude) in lung multiciliated cells of mice. The multiciliated cells were isolated by an elastase treatment. Ciliary beatings of lung multiciliated cells were observed by the videomicroscopy equipped with high speed camera (500 fps) and CBF and CBD were measured by an image analysis program using recorded images. We performed immunofluorescence microscopy analysis to evaluate the localization of β2AR, Na+/H+ exchanger regulatory factor (NHERF) 1, which is a PSD-95/Discs-large/ZO-1 (PDZ) scaffold protein, and PKA. In addition, we also performed cell surface protein biotinylation assay. In the results, stimulation with 1 nM procaterol, which is selective β2AR agonist, increased the ratios of CBF and CBD by about 80% in WT multiciliated cells. However, in Vil2kd/kd, stimulation with 1 nM procaterol increased the ratios of CBF and CBD by only 40%. While it is no significant difference that the increase of CBF and CBD by stimulation with 100 μM IBMX, which is a broad-spectrum phosphodiesterase inhibitor, in WT and Vil2kd/kd. In addition, the cell surface localization of NHERF1 and β2AR is disturbed in Vil2kd/kd multiciliated cells. There were no apparent changes in the subcellular localizations of PKA RIα and RIIα, which are PKA regulatory subunits, in Vil2kd/kd multiciliated cells. These results suggest that ezrin regulates the ciliary beating in lung multiciliated cells by promoting the apical localization of β2AR.