Intracellular Cl− Regulation of Ciliary Beating in Ciliated Human Nasal Epithelial Cells: Frequency and Distance of Ciliary Beating Observed by High-Speed Video Microscopy

Abstract

Small inhaled particles, which are entrapped by the mucous layer that is maintained by mucous secretion via mucin exocytosis and fluid secretion, are removed from the nasal cavity by beating cilia. The functional activities of beating cilia are assessed by their frequency and the amplitude. Nasal ciliary beating is controlled by intracellular ions (Ca2+, H+ and Cl−), and is enhanced by a decreased concentration of intracellular Cl− ([Cl−]i) in ciliated human nasal epithelial cells (cHNECs) in primary culture, which increases the ciliary beat amplitude. A novel method to measure both ciliary beat frequency (CBF) and ciliary beat distance (CBD, an index of ciliary beat amplitude) in cHNECs has been developed using high-speed video microscopy, which revealed that a decrease in [Cl−]i increased CBD, but not CBF, and an increase in [Cl−]i decreased both CBD and CBF. Thus, [Cl−]i inhibits ciliary beating in cHNECs, suggesting that axonemal structures controlling CBD and CBF may have Cl− sensors and be regulated by [Cl−]i. These observations indicate that the activation of Cl− secretion stimulates ciliary beating (increased CBD) mediated via a decrease in [Cl−]i in cHNECs. Thus, [Cl−]i is critical for controlling ciliary beating in cHNECs. This review introduces the concept of Cl− regulation of ciliary beating in cHNECs.

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

“…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 large disparity in frame rates used to image cilia motility is partly technology dependant, with early studies generally being restricted to lower fps imaging technology (2–25 Hz) (Clary‐Meinesz et al, 1992 ; Hennessy et al, 1986 ), while more recent studies with access to much faster and cheaper imaging technology generally having higher fps (≥500 fps) (Smith et al, 2012 ; Yasuda et al, 2020 ). Thus, the need exists to determine the optimum frame rate needed for imaging cilia using video microscopy for subsequent CBF calculation.…”

“…Finally, it should be stressed that this study was designed to determine the influence of movie fps on CBF measurement. For cilia beat pattern analysis (e.g., normal cilia motion vs. dyskinetic cilia motion) higher fps movies (i.e., ≥500 Hz) may be required to fully observe the fine movements of cilia (Yasuda et al, 2020 ).…”

Essential requirement of complex number for oscillatory phenomenon in intracellular trafficking process

“…Since the time resolution of these techniques is not so high, it is generally difficult to detect time-dependent oscillatory changes in the amounts of proteins including receptors in intracellular states with a high time resolution. The electrophysiological and imaging techniques have advantages with a high time resolution, thus the technique provides us with phenomena showing oscillatory changes in: the membrane potentials of cardiac cells [19] and pancreatic beta cells [18] ; intracellular Ca 2+ concentration in pancreatic alpha cells [21] , beta cells [22] , and adipocytes [20] ; movements of granules including insulin in pancreatic beta cells [23] ; regular rhythms of the brain [24] ; delayed electroretinogram potential in diabetic rats [24] ; ciliary beat frequently [27] , [28] . However, we have little information on the quantitative change in protein amounts in the intracellular state.…”

Ciliary Videomicroscopy: A Long Beat from the European Respiratory Society Guidelines to the Recognition as a Confirmatory Test for Primary Ciliary Dyskinesia

“…Indeed, the respiratory epithelium can be sampled using a brush, a curette or forceps, usually from the nose [ 34 ]. Moreover, ciliary function varies under differing environmental and physiological conditions, such as pH [ 35 , 36 , 37 ], osmolarity [ 36 , 37 ], oxygen [ 38 ], the presence of drugs [ 39 ], vitamins [ 40 ] or ions [ 41 ] within the medium, the temperature [ 35 , 42 ] (with some centers measuring at 37 °C [ 26 , 43 , 44 , 45 ] and others at lower temperatures [ 7 , 9 , 46 ]) and the time between sampling and video recording [ 47 ]. These conditions need to be strictly regulated, since minor variations may affect ciliary function.…”