Simultaneous measurement of ciliary beating and intracellular calcium.

A novel system for measuring, simultaneously, ciliary beating and intracellular free calcium is presented. The advantages and dynamic nature of the system are demonstrated by measuring the effects of the calcium ionophore lonomycin and of extracellular ATP on ciliated rabbit trachea. The results are discussed with regard to the ciliary and calcium stimulation.     

Spectral characterization of ciliary beating. Temperature dependence on spectral parameters

Ciliary beating was optically examined in tissue cultures from frog palate epithelium. Consecutive segments of the analog signal were Fast-Fourier transformed. The three main parameters which define the spectrum, position of the peak maximum (f), width of the spectral line (S.D.), and area (A) under the spectrum, were all measured as a function of temperature. These measurements were also examined as a function of the number of cilia by varying the examined area from 1.2 to 122 microns2. It was found that: all the parameters were exponentially temperature dependent; and the average frequency was independent of the number of cilia examined, while S.D. was dependent on it. On a physiological level, we demonstrated that the ciliary fluctuation in frequency is temperature dependent, increasing with increase in temperature. Moreover, it was shown that where a relatively small number of cilia were measured (d = 1.24 micron), the area A under the observed spectrum was directly proportional to the amplitude of ciliary beating. Increasing the temperature decreases the amplitude and vice versa. According to our suggested model the dependence of A on f was predicted and verified experimentally. A mathematical model which simulates the S.D. as a function of examined area and temperature is suggested. The calculated results from the model are in a good agreement with our experimental findings.     

Calcium and the regulation of mammalian ciliary beating

Summary This report summarizes our recent work on the role of intracellular Ca²⁺ ([Ca²⁺]_i) in regulating mammalian ciliary beat frequency (CBF). CBF from a single ovine cilium and [Ca²⁺]_i from the same cell were measured by digital video phase contrast microscopy and fura-2 ratiometric imaging video microscopy, respectively. Cells were stimulated with two exposures to 10 M acetylcholine (ACh). CBF was recorded during the first and [Ca²⁺]_i during the second stimulation. ACh increased [Ca²⁺]_i and CBF transiently with indistinguishable kinetics and, early in culture, even induced [Ca²⁺]_i oscillations and ciliary frequency modulations with the same peak-to-peak time interval. Cells treated with 1 M thapsigargin, an inhibitor of the endoplasmic-reticulum Ca²⁺-ATPase, showed transient [Ca²⁺]_i and CBF increases, again with similar kinetics, which often remained at an elevated plateau. Application of ACh to cells pretreated with thapsigargin produced decreases in both [Ca²⁺]_i and CBF. Finally, changing extracellular Ca²⁺-concentrations induced corresponding changes in [Ca²⁺]_i that were associated with kinetically similar CBF changes. These data strongly suggested that [Ca²⁺]_i is a critical signal to regulate CBF in mammalian tracheal epithelial cells. In an initial effort to provide constraints on the number and type of reactions that link changes in [Ca²⁺]_i to changes in CBF, simultaneous recordings of both signals from a single cell were analyzed. Such recordings provided higher resolution of the kinetic responses of CBF and [Ca²⁺]_i to ACh as well as they allowed direct assessment of the coupling between [Ca²⁺]_i and CBF. Simultaneous measurements revealed that [Ca²⁺]_i and CBF were perfectly correlated within the CBF measurement time resolution, except for the period of the fastest changes in both signals during the initial ACh exposure. There, changes in CBF lagged the changes in [Ca²⁺]_i by 1–3 ciliary beat cycles (ca. 150–450 ms).     

On self synchronization--aspects of flagellar and ciliary beating

Mathematical models involving hydrodynamically coupled oscillators are proposed to explain some self-synchronization phenomena observed in flagella and cilia. It is shown using elementary mathematical analysis and computer simulations that the behaviors of the models proposed are similar to those observed in experiment.     

Effect of Pseudomonas aeruginosa rhamnolipids on mucociliary transport and ciliary beating.

Pseudomonas aeruginosa rhamnolipid causes ciliostasis and cell membrane damage to rabbit tissue, is a secretagogue in cats, and inhibits epithelial ion transport in sheep tissue. It could therefore perturb mucociliary clearance. We have investigated the effect of rhamnolipid on mucociliary transport in the anesthetized guinea pig and guinea pig and human respiratory epithelium in vitro. Application of rhamnolipid to the guinea pig tracheal mucosa reduced tracheal mucus velocity (TMV) in vivo in a dose-dependent manner: a 10-microgram bolus caused cessation of TMV without recovery; a 5-micrograms bolus reduced TMV over a period of 2 h by 22.6% (P = 0.037); a 2.5-microgram bolus caused no overall changes in TMV. The ultrastructure of guinea pig tracheal epithelium exposed to 10 micrograms of rhamnolipid in vivo was normal. Application of 1,000 micrograms/ml rhamnolipid had no effect on the ciliary beat frequency (CBF) of guinea pig tracheal rings in vitro after 30 min, but 250 micrograms/ml stopped ciliary beating after 3 h. Treatment with 100 micrograms/ml rhamnolipid caused immediate slowing of the CBF (P less than 0.01) of human nasal brushings (n = 7), which was maintained for 4 h. Mono- and dirhamnolipid had equivalent effects. The CBF of human nasal turbinate organ culture was also slowed by 100 micrograms/ml rhamnolipid, but only after 4 h (CBF test, 9.87 +/- 0.41 Hz; control, 11.48 +/- 0.27 Hz; P less than 0.05, n = 6), and there was subsequent recovery by 14 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

Towards an atomic model of a beating ciliary axoneme

The axoneme of motile cilia and eukaryotic flagella is an ordered assembly of hundreds of proteins that powers the locomotion of single cells and generates flow of liquid and particles across certain mammalian tissues. The symmetric and organized structure of the axoneme has invited structural biologists to unravel its intricate architecture at different scales. In the last few years, single-particle cryo-electron microscopy provided high-resolution structures of axonemal complexes that comprise dozens of proteins and are key to cilia function. This review summarizes unique structural features of the axoneme and the framework they provide to understand cilia assembly, the mechanism of ciliary beating, and clinical conditions associated with impaired cilia motility.     

Simulations of three-dimensional ciliary beats and cilia interactions.

A new set of equations describing the time evolution of torsion and curvature for an inextensible curve is developed. Combined with our recently developed Slender Body Theory approach to such problems, these equations were applied to simulate three-dimensional ciliary beats, while allowing for cilia interactions. The computer animation technique, which was originally designed to display two-dimensional beats, has been enhanced to accommodate the new three-dimensional results.     

Luminal fluid tonicity regulates airway ciliary beating by altering membrane stretch and intracellular calcium

The coordinated, directional beating of airway cilia drives airway mucociliary clearance. Here we explore the hypothesis that airway surface liquid osmolarity is a key regulator of ciliary beating. Cilia in freshly isolated human and murine airways visualized with streaming video-microscopy exhibited a reciprocal dependence on a physiological range of luminal fluid osmolarities, across the entire range of ciliary activity (0-20 beats per sec). Increasing osmolarity slowed or completely abrogated, while lower osmolarity dramatically stimulated ciliary beating. In parallel, epithelial cell height and importantly, intracellular calcium levels (as judged by fluorescence imaging) also changed. Moreover, ciliary beating was stimulated by isosmotic solutions containing membrane permeant osmolytes, suggesting that cell size and membrane stretch (governed by apical fluid tonicity), rather than osmolarity itself, contribute to the activation. These findings shed light on the pathophysiology of diseases of mucociliary clearance such as cystic fibrosis and other chronic inflammatory lung diseases.     

Simulation of ciliary beating by an excitable dynein model: oscillations, quiescence and mechano-sensitivity

Murase et al. (1989, J. theor. Biol. 139, 413) showed that an excitable dynein model can generate flagellar-like bending waves of low amplitude along an axoneme suspended in a viscous fluid. Either regular base-to-tip and irregular tip-to-base propagating waves can be produced. The present study shows that if the force-vs.-distance functions (or the potential energy functions as their integral form) that represent the functional properties of dyneins differ in the basal region, as compared with the rest of the active length of a short axoneme, and also differ between the opposing doublets, ciliary-like repetitive beats can be simulated. Depending on the parameter values, a cilium beats once and then becomes resting or quiescent, at the end of either its recovery or effective stroke. Interestingly, a quiescent cilium exhibits repetitive beats when a steady flow of water is applied to a part of the cilium in a suitable direction and at an appropriate speed. This kind of responsiveness to external stimuli, called directional mechano-sensitivity, may account for metachronal waves over a layer of cilia. As in the previous model for flagellar movement, the present model requires a passive region at the tip, but does not need a curvature feedback control, to generate ciliary-like beating patterns.     

The orientation of ciliary basal bodies in quail oviduct is related to the ciliary beating cycle commencement

In mature ciliated cells, the basal feet associated to the basal bodies point out in the direction of the effective stroke of the ciliary beating. In contrast, during ciliogenesis, the basal feet of the newly anchored basal bodies are randomly oriented. The reorientation of basal bodies occurs during the beginning of the coordinated beating cycle of the cilia.     

Hyperpolarization-activated inward current associated with the frequency increase in ciliary beating of Paramecium

Summary In order to study the relationship between the inward Ca current activated by hyperpolarization and the frequency increase in ciliary beating, Paramecium cells were voltage clamped under conditions where K current was suppressed by use of CsCl electrodes and by extracellular tetraethyl ammonium. A 2-s pulse of hyperpolarization from the resting potential activated an inward current consisting of two components, an initial transient current peaking at 0.1–0.2 s (which had been identified as a Ca current) and a subsequent sustained current. The initial component was not associated with the frequency increase because the frequency increase was normally induced even when the peak current was almost completely inhibited by external addition of Ba²⁺. The second sustained current was closely correlated with the frequency increase. The frequency rose steeply with the sustained current and saturated at –0.6 nA. External addition of La³⁺ or replacement of Ca²⁺ by Mg²⁺ suppressed this current, and at the same time the frequency increase was inhibited. As the amplitude of the sustained current was not changed by deciliation, this current must pass through the somatic membrane. These results suggest that the frequency increase upon hyperpolarization is triggered by the voltage-activated inward current passing through the somatic membrane of the interciliary compartment.Abbreviations cAMP cyclic adenosine monophosphate - HEPES hydroxyethylpiperazine ethanesulfonate - TEA⁺ tetraethyl ammonium     

Coordinated ciliary beating requires Odf2-mediated polarization of basal bodies via basal feet

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Highlights? Mice expressing truncated Odf2 cough and sneeze due to primary ciliary dyskinesia ? Full-length Odf2 is needed for the formation of basal body-associated basal feet ? In the absence of basal feet, basal bodies fail to align with planar polarity cues ? Polarization of basal bodies by Odf2 is required for coordinated ciliary beating     

Halteria grandinella: a rapid swimming ciliate with a high frequency of ciliary beating

A ciliated protozoan, Halteria grandinella, swam backward rapidly with a migration distance per second attaining 100 times the cell size. This high swimming velocity was accompanied by a high frequency of ciliary beating. Recordings with a high-speed digital video (10(3) frames/s) revealed that the frequency during forward and backward swimming was, respectively, 105 +/- 10 Hz and 260 +/- 30 Hz. These frequencies are the highest among cilia and flagella reported to date. Electron microscopic observation of the ciliary structure confirmed normal 9 + 2 arrangements of the axoneme except that cilia for migration are bundled into membranelles. Ciliary beating of saponin-treated cells was reactivated by the addition of Mg2+ -ATP, although the beating amplitude was smaller than that of intact cells. Kinetic analysis of the ATP-dependent increase of beating frequency revealed that the maximal frequency in the presence of free Ca2+ and 0.9 microM Ca2+ was approximately 60 and 110 Hz, respectively. A possible mechanism to increase beating frequency with Ca2+ is discussed.     

Proliferation,differentiation and ciliary beating of human respiratory ciliated cells in primary culture

Summary The growth, differentiation, ciliary beating pattern and frequency of human respiratory ciliated cells in primary culture were studied by scanning and transmission electron microscopy and by videomicroscopy. The epithelial cells were obtained as outgrowth from explants of adult nasal polyps. When the explants were grown on type-I and type-IV collagen substrates in a standard serum-free, hormone-supplemented medium, a high percentage of ciliated cells (range 29±5% to 37±6%) was present within 2 days of culture. After 5 days of culture, the percentage of ciliated cells near the explant was 51±5%. Most of the cultured ciliated cells (85%) were characterized by individual cilia showing a coordinated movement during the beat cycle and a beating frequency (13.3±1.3 Hz) similar to that reported in vivo. In the other 15% of the ciliated cells, the dyskinetic cilia were aggregated into clumps and characterized by a rigid and planar bending movement and a lower (P<0.01) beating frequency (10.7±1.4 Hz). It is suggested that the latter type of cell, already described during fetal development, might be an intermediate type of ciliated cell which appears temporarily during the surface respiratory epithelial differentiation.     

Effects of hormones and nucleotides on ciliary beating in frog esophagus and guinea pig trachea

The $\text{in vivo}$ effects of various hormones, nucleotides and related substances on the rate of ciliary beating in frog esophagus and guinea pig trachea were studied using both particle transport and photoelectric methods. Frog esophageal ciliary beating $\text{in vitro}$ was greatly accelerated by acetycholine, eserine, prostaglandin A₁ and E₂, N⁶- 2′-0-dibutyryl-adenosine--3′,5′-cyclic monophosphate, all tri- and di-phosphonucleotides (ATP, ADP, UTP, UDP, etc.), EDTA, EGTA, and calcium-free medium. Adenosine monophosphate, epinephrine, serotonin at low concentrations, 3,3′5-L-triiodothyronine, creative phosphate, and phosphoenolpyruvate were inactive or only minimally stimulatory in the frog. All substances tested, including those trachea. Furthermore, guinea pig ciliary activity was unaffected by hyperthyroidism or hypothyroidism induced in the intact animal before testing.     

Study of development of ciliary folds and Zinn's zonules in Rana temporaria L. by means of scanning electron microscopy]

The development of ciliary folds and zonul of Zinn has been studied in the eyes of the common frog Rana temporaria L. by means of scanning electron microscopy. The development of ciliary folds begins at the stage 45 by the flexure of the external layer in the ciliary zone. At the stage 46 this process involves the internal layer and the folds become two-layered. The zonules of Zinn form before the folds of internal layer of the ciliary epithelium begin to form as separate bundles of fibers. At the stage 45 they are already distinct. Later the ciliary filaments fold in 2 felt-like layers -- zonula which pass from the equatorial lens zone and attach near orbiculum ciliaris. In the place of attachment the margin of zonul repeats the relief of folds, thus attaching to their whole surface, and individual filaments go farther, to orbiculum ciliaris. All these processes take place in they eye prior to the beginning of metamorphosis.     

An endogenous SCP-related peptide modulates ciliary beating in the gills of a venerid clam, Mercenaria mercenaria

The activities of both the lateral and frontal cilia of Mercenaria mercenaria were unaffected, either by the two endogenous SCP-related peptides AMSFYFPRMamide and YFAFPRQamide, or by FMRFamide (all at 10(-6) M). Dopamine (DA) inhibited the lateral cilia; the mean EC50 was 2 x 10(-6) M. The peptide YFAFPRQamide--but neither AMSFYFPRMamide nor FMRFamide--antagonized the inhibition induced by DA; this effect was dependent on both time and dose. At a DA concentration of 5 x 10(-7) M, the effect of YFAFPRQamide appeared within 20 min and became maximal within 40-60 min; the mean EC50 at these times was 4.7 x 10(-11) M. If the concentration of DA was increased to 10(-6) M, the maximal effect of the peptide was delayed to 50 min, and the mean EC50 increased to 1.1 x 10(-7) M. Particle transport by the frontal cilia was inhibited by 5-hydroxytryptamine (5HT); the mean EC50 was 5.7 x 10(-7) M. Again, only YFAFPRQamide had an antagonistic effect on the 5HT-induced inhibition. At a 5HT concentration of 10(-6) M, the effects of YFAFPRQamide did not appear until 45 min; the mean EC50 was 10(-6) M. When radioimmunoassayed with an SCP antiserum, the elution profile of a gill extract overlapped those of the SCP-related peptides that had previously been identified in extracts of whole animals. These data suggest that all three SCP analogs occur in the gill. Immunohistochemistry of the gill, carried out with a monoclonal antibody raised to SCPB, stained many varicose neuronal fibers. Most of these were associated with the gill musculature, but a sparse innervation of the filaments underlying the cilia was also observed. Some fluorescent nerve cell bodies were also seen in the gill tissue. Our results are consistent with the hypothesis that YFAFPRQamide modulates branchial activities--muscular as well as ciliary--that are associated with feeding.