nNOS regulates ciliated cell polarity, ciliary beat frequency, and directional flow in mouse trachea

Abstract

Clearance of the airway is dependent on directional mucus flow across the mucociliary epithelium, and deficient flow is implicated in a range of human disorders. Efficient flow relies on proper polarization of the multiciliated cells and sufficient ciliary beat frequency. We show that NO, produced by nNOS in the multiciliated cells of the mouse trachea, controls both the planar polarity and the ciliary beat frequency and is thereby necessary for the generation of the robust flow. The effect of nNOS on the polarity of ciliated cells relies on its interactions with the apical networks of actin and microtubules and involves RhoA activation. The action of nNOS on the beat frequency is mediated by guanylate cyclase; both NO donors and cGMP can augment fluid flow in the trachea and rescue the deficient flow in nNOS mutants. Our results link insufficient availability of NO in ciliated cells to defects in flow and ciliary activity and may thereby explain the low levels of exhaled NO in ciliopathies.

Quantifying cilia beat frequency using high‐speed video microscopy: Assessing frame rate requirements when imaging different ciliated tissues

“…Ns = not significantly different (p > 0.05). Mateos-Quiros et al, 2021;Mikhailik et al, 2021;Reula et al, 2021;Smith et al, 2012;Zahid et al, 2020). The lack of a standardized fps for imaging motile cilia is especially problematic because it hinders high-speed video microscopy from being clinically adapted as a diagnostic tool to help uncover motile cilia diseases, such as primary ciliary dyskinesia (Bricmont et al, 2021;Shapiro et al, 2018).…”

“…The easiest way to assess motile cilia function is by quantifying CBF via highspeed video microscopy (Francis & Lo, 2013 ; O'Callaghan et al, 2012 ; Sisson et al, 2003 ) and this forms a sizable chunk of the cilia literature. However, no standardized frame rate exists when imaging cilia motility for subsequent CBF calculation, this has resulted in a large range of frames rates used to image cilia motility within the published literature (Abdelhamed et al, 2018 ; Abdelhamed et al, 2020 ; Bustamante‐Marin et al, 2019 ; Chen et al, 2016 ; Hagiwara et al, 2020 ; Hennessy et al, 1986 ; Liu et al, 2021 ; Mateos‐Quiros et al, 2021 ; Mikhailik et al, 2021 ; Reula et al, 2021 ; Smith et al, 2012 ; Zahid et al, 2020 ). The lack of a standardized fps for imaging motile cilia is especially problematic because it hinders high‐speed video microscopy from being clinically adapted as a diagnostic tool to help uncover motile cilia diseases, such as primary ciliary dyskinesia (Bricmont et al, 2021 ; Shapiro et al, 2018 ).…”

“…Our data matches previously published findings by highlighting the significant difference in CBF between airway and ependymal cilia and by emphasizing how CBF is modulated by changes in temperature. Murine ependymal cilia have been previously shown to beat significantly faster than airway cilia (Francis et al, 2009 ; Hagiwara et al, 2020 ; Mahuzier et al, 2018 ; Mikhailik et al, 2021 ), while increased incubation temperature is well recognized to result in a linear increase in mean CBF (Christopher et al, 2014 ; Schipor et al, 2006 ; Sisson et al, 2003 ; Welchering et al, 2015 ).…”

“…The most used method to assess motile cilia function is to quantify cilia beat frequency (CBF) via high‐speed video microscopy (Reula et al, 2021 ; Rubbo et al, 2019 ; Shapiro et al, 2018 ). However, a large heterogeneity exists within the published literature as to what frame rate per second (fps) to use when imaging cilia motility for subsequent CBF calculation; with published frame rates varying between 28–500 fps (Abdelhamed et al, 2018 ; Abdelhamed et al, 2020 ; Bustamante‐Marin et al, 2019 ; Chen et al, 2016 ; Hagiwara et al, 2020 ; Liu et al, 2021 ; Mateos‐Quiros et al, 2021 ; Mikhailik et al, 2021 ; Reula et al, 2021 ; Smith et al, 2012 ; Zahid et al, 2020 ). The large variation in sampling rates suggests some studies may be under‐sampling or oversampling cilia motility, which could result in inaccurate quantification of CBF or generation of large volumes of oversampled data requiring analysis and storage.…”

Genomic and ecologic characteristics of the airway microbial-mucosal complex

“…Nitric oxide (NO) is a central host signalling molecule in the airways, where it mediates bronchodilation, vasodilation, and ciliary beating 29 . NO exhibits cytostatic or cytocidal activity against many pathogenic microorganisms 30 and NO elevation in exhaled breath is used as a clinical marker for lower airway inflammation.…”