Immune Localization of Calmodulin of Hamster Tracheal Epithelium in the Ciliated Cells

Metachronal beating of cilia of epithelial surfaces of most respiratory airways moves the overlying mucous layer in a caudal direction. The molecular mechanisms controlling ciliary beat remain largely unknown. Calcium, an element in its cationic form, is ubiquitous in biological functions and its concentration is critical for ciliary beating. Calmodulin, a calciumbinding protein which regulates the activity of many enzymes and cellular processes, may regulate ciliary beating by controlling enzymes responsible for mechanochemical movement between adjacent peripheral microtubule doublets composing the ciliary axoneme. As a first step in describing a calmodulin-related controlling mechanism for ciliary beating, calmodulin was localized in the ciliated cells lining the respiratory tracts of hamsters by electron microscopy, using an indirect immunoperoxidase technique with anticalmodulin antibodies as the molecular probe. Thin-sections revealed calmodulin located on microtubules and dynein arms of the ciliary shaft, basal body, apical cytoskeletal microtubules, and plasma membranes in specimens fixed with 1 mM Ca +2. Specimens fixed with less Ca ÷2 (1 #M), Mn +2, Mg +2, and EGTA showed a diffuse pattern of calmodulin with loci of greatest densities on basal body microtubule triplets. Demembranated specimens showed a less specific localization on axonemal microtubules but only on cells fixed with Ca +2. Calmodulin, by binding calcium, may function in ciliary beating in the respiratory tract of mammals either directly or indirectly through its effects on the energy-producing enzymes and by control of Ca ÷2 flux through plasma membranes. The epithelial lining of respiratory airways contains a large number of ciliatcd cells responsible for mucous clearance mediated by metachronai ciliary beating. The cilia on the surface of these cells are anchored into the apical cytoplasm by basal bodies, centriole counterparts (5, 26). Microtubules and microfilaments form an integrated system linking the ciliary axoneme and basal body to the cytoskeleton (8). The mechanism(s) controlling ciliary beat initiation, frequency, and synchronization is unknown, but changes in beat pattern of cilia and flagella are due to calcium ion concentrations, e.g., ciliary reversal in Paramecium (4) and Tetrahymena (29), ciliary arrest in molluscan gill (24), flagellar wave form change in Chlamydomonas (25), reversal of the flagellar wave propagation in Crithidia (13), asymmetrical (2) and intermittent (6, 7) flagellar beating in sea urchin sperm, mammalian ciliary beat frequency change (3) and activation (21) and in vitro (14). Ohnishi et al. (23) suggested that calcium ion effect may be a two-step phenomenon: (a) an influx of Ca +2 into the cilia or flagella and (b) a specific effect on the motile apparatus of the cilia or flagella. Calmodulin may be a specific intermediate target molecule for the calcium effects on cilia and flagella described above. Calmodulin--a small, acidic calcium-binding protein that modulates numerous enzymatic reactions and the assembly and disassembly of microtubules, specifically in the mitotic spindle (20)--may participate in the mechanism of ciliary beating. A protein isolated from Tetrahymena cilia had characteristics similar to those of calmodulin (15-17, 27). Ohnishi et al. (23) isolated calmodulin from Tetrahymena cilia and in isolated axonemal preparations showed its location on interdoublet cross-bridges. Previously, calmodulin has been demonstrated in cilia of protozoa by immunofluorescence (18, 28) and in centrioles by immunofluorescence and immunoperoxidase at the electron microscopic level (21). In this paper, we present evidence that a calmodulin or a calmodulin-like molecule observed by immunoelectron microscopy using an antiTHE JOURNAL OF CELL BIOLOGY • VOLUMe 95 OCTOBER 1982 57-63 © The Rockefeller University Press • 0021-9525/82/10/0057/07 $I.00 5 7 on Jne 1, 2017 D ow nladed fom Published October 1, 1982