Determination of ciliary polarity precedes differentiation in the epithelial cells of quail oviduct.

In quail oviduct epithelium, as in all metazoan and protozoan ciliated cells, cilia beat in a coordinated cycle. They are arranged in a polarized pattern oriented according to the anteroposterior axis of the oviduct and are most likely responsible for transport of the ovum and egg white proteins from the infundibulum toward the uterus. Orientation of ciliary beating is related to that of the basal bodies, indicated by the location of the lateral basal foot, which points in the direction of the active stroke of ciliary beating. This arrangement of the ciliary cortex occurs as the ultimate step in ciliogenesis and following the oviduct development. Cilia first develop in a random orientation and reorient later, simultaneously with the development of the cortical cytoskeleton. In order to know when the final orientation of basal bodies and cilia is determined in the course of oviduct development, microsurgical reversal of a segment of the immature oviduct was performed. Then, after hormone-induced development and ciliogenesis, ciliary orientation was examined in the inverted segment and in normal parts of the ciliated epithelium. In the inverted segment, orientation was reversed, as shown by a video recording of the direction of effective flow produced by beating cilia, by the three-dimensional bending forms of cilia immobilized during the beating cycle and screened by scanning electron microscopy, and by the position of basal body appendages as seen in thin sections by transmission electron microscopy. These results demonstrate that basal body and ciliary orientation are irreversibly determined prior to development by an endogenous signal present early in the cells of the immature oviduct, transmitted to daughter cells during the proliferative phase and expressed at the end of ciliogenesis.     

Hyperactivation is the mode conversion from constant-curvature beating to constant-frequency beating under a constant rate of microtubule sliding

Flagellar beating of hyperactivated golden hamster spermatozoa was analyzed in detail using digital image analysis and was compared to that of nonhyperactivated (activated) spermatozoa in order to understand the change in flagellar beating during hyperactivation and the active microtubule sliding that brought about the change in flagellar beating. Hyperactivated flagellar beating, which was characterized by a sharp bend in the proximal midpiece and low beat frequency, was able to alter the waveform with little change in beat frequency (constant-frequency beating), whereas activated flagellar beating, which was characterized by a slight bend in the proximal midpiece and high beat frequency, was able to alter beat frequency with little change in the waveform (constant-curvature beating). These results demonstrate that flagellar beating of hyperactivated and activated spermatozoa were essentially different modes and that hyperactivation was the mode conversion from constant-curvature beating to constant-frequency beating. Detailed analysis of flagellar bends revealed that the increase in curvature in the proximal midpiece during hyperactivation was due to the increase in total length of microtubule sliding in a nearly straight region between bends, while the rate of microtubule sliding remained almost constant.     

An electro-optic monitor of the behavior of Chlamydomonas reinhardtii cilia

The unicellular green alga Chlamydomonas reinhardtii steers through water with a pair of cilia (eukaryotic flagella). Long-term observation of the beating of its cilia with controlled stimulation is improving our understanding of how a cell responds to sensory inputs. Here we describe how to record ciliary motion continuously for long periods. We also report experiments on the network of intracellular signaling that connects the environment inputs with response outputs. Local spatial changes in ciliary response on the time scale of the underlying biochemical dynamics are observed. Near-infrared light monitors the cells held by a micropipette. This condition is tolerated well for hours, not interfering with ciliary beating or sensory transduction. A computer integrates the light stimulation of the eye of Chlamydomonas with the ciliary motion making possible long-term correlations. Measures of ciliary responses include the beating frequency, stroke velocity, and stroke duration of each cilium, and the relative phase of the cis and trans cilia. The stationarity and dependence of the system on light intensity was investigated. About 150,000,000 total beat cycles and up to 8 h on one cell have been recorded. Each beat cycle is resolved so that each asynchronous beat is detected. Responses extend only a few hundred milliseconds, but there is a persistence of momentary changes that last much longer. Interestingly, we see a response that is linear with absolute light intensity as well as different kinds of response that are clearly nonlinear, implying two signaling pathways from the cell body to the cilia.     

THE METACHRONAL WAVE OF LATERAL CILIA OF MYTILUS EDULIS

The form of beat of cilia and the structure of the metachronal wave on the lateral gill epithelium of Mytulus edulis have been studied on living material by interference-contrast microscopy and stroboscopic illumination, and compared with the same features in rapid-fixed preparations studied by light microscopy and with the scanning electron microscope. The most striking finding is that the beat of the cilia is not planar, as previously assumed, but involves a sideways movement in the recovery stroke Previous reports on nonplanar ciliary beating from protozoan examples describe a planar effective stroke and a counterclockwise rotation in the recovery stroke; in this molluscan example there is a clockwise rotation in the recovery stroke The lateral inclination of the cilia in the recovery stroke is in the same direction as the propagation of the waves, and the orientation of cilia in the recovery stroke is thought to determine whether the waves move to the left or right of the direction of the effective stroke     

Regulation of outer-arm-dynein activity by phosphorylation and control of ciliary beat frequency

Summary Ciliary beating is empowered by a mechanochemical enzyme, dynein, which appears as two rows of projections on doublet microtubules. While inner-arm dyneins modulate beat form, outer-arm dynein empowers ciliary beat and sets beat frequency. Beat frequency is controlled via phosphorylation of outer-arm dynein. UsingParamecium tetraurelia as model system, we have previously identified a regulatory light chain of outer-arm dynein (22S dynein), M_r29 (p29), whose phosphorylation is cAMP-dependent. The phosphorylation state of the p29 in 22 S dynein determines in vitro microtubule translocation velocity. Although in vitro phosphorylation of p29 takes place in a short time, the percent change ist significantly less than the percent change in dynein activation, or in ciliary beat frequency. A potential mechanism that explains how a few activated dyneins can change ciliary beating is discussed.     

Extracellular ATP induces hyperpolarization and motility stimulation of ciliary cells.

Cellular membrane potential and ciliary motility were examined in tissues cultures prepared from frog palate and esophagus epithelia. Addition of micromolar concentrations of extracellular ATP caused membrane hyperpolarization and enhanced the beat frequency. These two effects of ATP were 1) dose dependent, reaching a maximum at 10 microM ATP; 2) dependent on the presence of extracellular Ca2+ or Mg2+; 3) insensitive to inhibitors of voltage-gated calcium channels; 4) abolished after depleting the intracellular Ca2+ stores with thapsigargin; 5) attenuated by quinidine (1 mM), Cs+ (5-20 mM), and replacement of extracellular Na+ by K+; 6) insensitive to charybdotoxin (5-20 nM), TEA (1-20 microM), and apamin (0.1-1 microM); 7) independent of initial membrane potential; and 8) unaffected by amiloride. In addition, extracellular ATP induced an appreciable rise in intracellular Ca2+. Addition of thapsigargin caused an initial enhancement of the ciliary beat frequency and membrane hyperpolarization. These results strongly suggest the involvement of calcium-dependent potassium channels in the response to ATP. The results show that moderate hyperpolarization is closely associated with a sustained enhancement of ciliary beating by extracellular ATP.     

Membrane control of ciliary movement in ciliates

Ciliary movement is generated in the axoneme by the unidirectional sliding of the outer doublets of microtubules produced by the adenosine triphosphate (ATP)-energized dynein arms. It is composed of an effective stroke phase and a passive recovery stroke phase. Two parameters are modulated to determine swimming characteristics of the cell (speed and direction): beat frequency; direction of the effective stroke. They are linked to the internal Ca++ level and to the membrane potential. The membrane governs the internal Ca++ level by regulating Ca++ influx and efflux. It contains voltage-sensitive Ca++ channels through which a passive Ca++ influx, driven by the electrochemical gradient, occurs during step depolarization. The rise of the Ca++ level, up to 6.10-7M triggers ciliary reversal and enhances beat frequency. Ca+ is extruded from cilia by active transport. Ca++ also activates a multistep enzymatic process, the first component of which is a membrane calmodulin-dependent guanylate cyclase. cGMP interacts with Ca++ to modulate the parameters of the ciliary beat. The phosphorylation-dephosphorylation cycle of axoneme and membrane proteins seems to play a major role in controlling ciliary movement. Hyperpolarization of the membrane enhances beat frequency by an unknown mechanism. It could be a modification of the ratio of axonemal bound Ca++ and Mg++, or activation by cyclic adenosine monophosphate (cAMP) produced by a membrane adenylate cyclase. The ciliary membrane behaves as a receptor able to detect modifications of external parameters, and as a transductor transmitting the detected signal by a second or third messengers toward the interior of the cilia. These messengers. acting at different levels, modulate the parameters of the mechanism that generates ciliary movement.     

Ciliary sieving and active ciliary response in capture of particles by suspension-feeding brachiopod larvae

Larvae of a brachiopod, Glottidia pyramidata, used at least two ciliary mechanisms to capture algal cells upstream from the lateral band of cilia that produces a feeding/swimming current. (1) Filtration: the larvae retained algal cells on the upstream (frontal) side of a sieve composed of a row of stationary laterofrontal cilia. Movement of the laterofrontal cilia could not be observed during capture or rejection of particles, but the laterofrontal cilia can bend toward the beating lateral cilia, a possible mechanism for releasing rejected particles from the ciliary sieve. (2) Localized changes of ciliary beat: the larvae may also concentrate particles by a local change in beat of lateral cilia in response to particles. The evidence is that the beat of lateral cilia changed coincident with captures of algal cells and that captured particles moved on paths consistent with a current redirected toward the frontal side of the tentacle by an induced local reversal of the lateral cilia. The change of beat of lateral cilia could have been an arrest rather than a reversal of ciliary beat, however. The similar ciliary bands in adult and larval lophophorates (brachiopods, phoronids, and bryozoans) suggest that these animals share a range of ciliary behaviours. The divergent accounts of ciliary feeding of lophophorates could be mostly the result of different authors observing different aspects of ciliary feeding.     

Flagellar and ciliary beating in trypanosome motility

The single flagellum of Leishmania and Trypanosoma parasites is becoming an increasingly attractive model for the analysis of flagellar function-driven largely by the abundance of genomic and proteomic information available for the organelle, the genetic manipulability of the organisms and the importance of motility for the parasite lifecycle. However, as yet, there is a paucity of published data on the beating of any genetically malleable trypanosomatid species. Here we undertook an in-depth analysis using high-speed videomicroscopy of the beating of free-swimming Leishmania major cells in comparison to Crithidia species (for which there is some existing literature). In so doing, we describe a simple and generally-applicable technique to facilitate the quantitative analysis of free-swimming cells. Our analysis thoroughly defines the parameters of the expected tip-to-base symmetrical flagellar beat in these species. It also describes beat initiation from points other than the flagellum tip and a completely different, base-to-tip highly-asymmetric beat that represents a ciliary beat of trypanosomatid flagella. Moreover, detailed analysis of parameter interrelationships revealed an unexpected dependency of wavelength on oscillator length that may be the result of reversible constraint of doublet sliding at the tip or resonance of the flagellar beat.     

A numerical study of muco-ciliary transport under the condition of diseased cilia

Structural and functional disorders of pulmonary cilia may result from genetic disorders and acquired insults. A two-dimensional numerical model based on the immersed boundary method coupled with the projection method is used to study the flow physics of muco-ciliary transport of the human respiratory tract under various abnormalities of cilia. The effects of the cilia beat pattern (CBP), ciliary length, immotile cilia, beating amplitude and uncoordinated beating of cilia are investigated. As expected, the mucus velocity decreases as the beating amplitude reduces. The windscreen wiper motion and rigid planar motion, which are two abnormal CBPs owing to genetic disorders, greatly reduce or almost stop the mucus transport. If the ciliary length varies from its standard length, the mucus velocity would decrease. The mucus velocity decreases rather linearly if the number of uniformly distributed immotile cilia increases. The numerical results show that the mucus velocity would be further reduced marginally when the uniformly distributed immotile cilia are rearranged as a cluster of immotile cilia. Furthermore, if half of the cilia are immotile and uniformly distributed and motile cilia beat at reduced amplitude, the incoordination between the active motile cilia would not significantly affect the mucus velocity.     

Vigorous beating of Chlamydomonas axonemes lacking central pair/radial spoke structures in the presence of salts and organic compounds

Flagella of Chlamydomonas mutants lacking the central pair of microtubules or radial spokes do not beat; however, axonemes isolated from these mutants were found to display vigorous bending movements in the presence of ATP and various salts, sugars, alcohols, and other organic compounds. For example, about 15% of the total axonemes isolated from pf18, a mutant lacking the central pair, displayed beating in the presence of 10 mM MgSO(4) and 0.2 mM ATP at about 22 Hz, while none beat with the same concentration of ATP and < or = 5 mM or > or = 25 mM MgSO(4). The beat frequency and waveform of beating pf18 axonemes were similar to those of wild type axonemes beating under the same conditions. Similarly, 10-50% of the axonemes beat in the presence of 0.5 M sucrose, 2.0 M glycerol, or 1.7 M[10% (v/v)] ethanol. The appearance of motility did not correlate with the change in axonemal ATPase; however, these substances at those concentrations commonly increased the amplitude of nanometer-scale oscillation (hyper-oscillation) in pf18 axonemes, as well as the extent of ATP-induced sliding disintegration of protease-treated axonemes. Axonemes of double mutants lacking both the central pair and various subspecies of inner-arm dynein also beat at increased MgSO(4) concentrations, but axonemes lacking outer-arm dynein in addition to the central pair did not beat. These and other observations suggest that small molecules perturb the regulation of microtubule sliding through some change in water activity or osmotic stress. Axonemes must have an intrinsic ability to beat without the central pair/radial spokes under a variety of non-physiological solution conditions, as long as the outer dynein arms are present. Apparently, the major function of the central pair/radial spoke structures is to restore this activity under physiological conditions.     

Extraction of cilium beat parameters by the combined application of photoelectric measurements and computer simulation.

Photoelectric signals were created and used to investigate the features of the signals as a function of the ciliary beat parameters. Moreover, correlation between the simulated and the measured signals permitted measurement of the cilium beat parameters. The simulations of the signals were based on generation of a series of time-frozen top-view frames of an active ciliary area and determination of the amount of light passing through an observation area in each of these frames. All the factors that might contribute to the shape of the signals, namely, partial ciliary transmittance of light, three-dimensional ciliary beat (composed of recovery, effective, and pause parts), phase distribution on the ciliary surface, and the large number of cilia that contribute to the photoelectric signal, were taken into account in generation of the signals. Changes in the ciliary parameters influenced the shape of the photoelectric signals, and the different phases of the beat could not be directly and unequivocally identified in the signals. The degree of temporal asymmetry of the beat and the portion of the cycle occupied by the pause significantly influenced the shapes of both the lower and the upper parts of the signal and the slopes of the signal. Increases in the angle of the arc swept by the cilium during the effective stroke smoothed the signals and increased the duration of the upper part of the signal. The angle of the arc projected by the cilium onto the cell surface during the recovery stroke had minor effects on the signal's shape. Characteristics of the metachronal wave also influenced the signal's shape markedly. Decreases in ciliary spacing smoothed the signals, whereas ciliary length had a minor influence on the simulated photoelectric signals. Comparison of the simulated and the measured signals showed that the beat parameters of the best-fitting simulated signals converged to values that agree well with the accepted range of beat parameters in mucociliary systems.     

Microtubule displacements at the tips of living flagella

We have observed that the flagellar axoneme of the Chinese hamster spermatozoon undergoes periodic changes in length at the same frequency as the flagellar beat. The amplitude of the length oscillation recorded at the tip is maximally about 0.5 microm or 0.2% of the total length. In some favourable cells, it was possible to see the opposing "halves" of the axoneme moving at the tip in a reciprocating manner and 180 degrees out-of-phase. This behaviour, when analysed quantitatively, is broadly consistent with predictions made from the sliding-doublet theory of ciliary and flagellar motility and thus it constitutes an additional verification of the theory, for the first time in a living cell. However, on close examination, there is a partial mismatch between the timing of the length oscillation and the phase of the beat cycle. We deduce from this that there is some sliding at the base of the flagellum, sliding that is accommodated by elastic compression of the connecting piece. Micrographic evidence for such compression is presented.     

Automated measurement of ciliary beat frequency

Measurements of ciliary beat frequency using video images are dependent on observer interpretation. To obtain objective estimates of ciliary beat frequency from video-image sequences, a computer-based method was developed. Regions of interest of video-image sequences were selected and digitized. Variations in numerical values representing light intensity resulting from cilia beating were extracted and analyzed using autocorrelation techniques. The ciliary beat frequencies obtained for 14 in vitro experiments on ciliated cells or epithelium from the frog palate (Rana catesbeiana) over the range of frequencies 2-25 Hz correlated well with independent observer measurements (r = 0.979). The addition of such computer-based methods to video observer-based systems allows more objective and efficient determinations of ciliary beat frequency.     

Analysis of three-dimensional ciliary beating by means of high-speed stereomicroscopy.

Results are presented on the analysis of three-dimensional motion of compound cilia or cirri in voltage-clamped specimens of the protozoan Stylonychia mytilus. Time series of three-dimensional data were obtained by using the anaxial illumination method for simultaneous recording of stereoscopic video images. Data processing involved the following steps: determination of a reference coordinate system based solely on features present in each stereo-pair; tracing of cirral axes in digitized images, conversion to parameter curves by means of least-squares polynomial approximation, conversion of pairs of two-dimensional data to a series of three-dimensional data; correction for distortion due to projective shortening and conversion to a series of polynomial triplets, and analysis of the periodical components of the motion pattern in the frequency domain. Reconstructed beating cycles show typical differences between hyperpolarization-induced ciliary activity and depolarization-induced ciliary activity. Reconstructions of the motion of the basal segment of a cirrus are in agreement with existing data. Analysis of the curvature and torsion of a cirral axis during beating does not reveal any simple pattern of propagated activity within the axoneme. The return stroke may be subdivided into two phases. First, a curvature peak develops proximally. Secondly, a region with increased torsion arises more distally and spreads out in proximal direction. Both curvature and torsion return to minimal values by the beginning of the power stroke.     

Electrophysiological control of ciliary motor responses in the ctenophorePleurobrachia

Prey capture by a tentacle of the ctenophore Pleurobrachia elicits a reversal of beat direction and increase in beat frequency of comb plates in rows adjacent to the catching tentacle (Tamm and Moss 1985). These ciliary motor responses were elicited in intact animals by repetitive electrical stimulation of a tentacle or the midsubtentacular body surface with a suction electrode. An isolated split-comb row preparation allowed stable intracellular recording from comb plate cells during electrically stimulated motor responses of the comb plates, which were imaged by high-speed video microscopy. During normal beating in the absence of electrical stimulation, comb plate cells showed no changes in the resting membrane potential, which was typically about -60 mV. Trains of electrical impulses (5/s, 5 ms duration, at 5-15 V) delivered by an extracellular suction electrode elicited summing facilitating synaptic potentials which gave rise to graded regenerative responses. High K+ artificial seawater caused progressive depolarization of the polster cells which led to volleys of action potentials. Current injection (depolarizing or release from hyperpolarizing current) also elicited regenerative responses; the rate of rise and the peak amplitude were graded with intensity of stimulus current beyond a threshold value of about -40 mV. Increasing levels of subthreshold depolarization were correlated with increasing rates of beating in the normal direction. Action potentials were accompanied by laydown (upward curvature of nonbeating plates), reversed beating at high frequency, and intermediate beat patterns. TEA increased the summed depolarization elicited by pulse train stimulation, as well as the size and duration of the action potentials. TEA-enhanced single action potentials evoked a sudden arrest, laydown and brief bout of reversed beating. Dual electrode impalements showed that cells in the same comb plate ridge experienced similar but not identical electrical activity, even though all of their cilia beat synchronously. The large number of cells making up a comb plate, their highly asymmetric shape, and their complex innervation and electrical characteristics present interesting features of bioelectric control not found in other cilia.     

Digital image analysis of the flagellar beat of activated and hyperactivated suncus spermatozoa

The flagellar beat of hyperactivated Suncus spermatozoa was analyzed by digital imaging and was compared to that of the nonhyperactivated (activated) spermatozoa in order to examine the function of the accessory fibers during the flagellar beat and the sliding filament mechanism inducing the motility of the hyperactivated spermatozoa. Unusual large and long characteristics of the accessory fibers were involved in generating the gently curved bends and a low beat frequency. Examination of the motility parameters of the flagellar beat of the activated and hyperactivated spermatozoa attached to a slide glass by their heads revealed that there were two beating modes: a frequency-curvature dependent mode in the activated flagellar beat and a nearly constant frequency mode in the hyperactivated flagellar beat. The hyperactivated flagellar beat was characterized by sharp bends in the proximal midpiece and a low beat frequency. The sharp bends in the proximal midpiece were induced by the increase in the total length of the microtubule sliding at the flagellar base. The rate of microtubule sliding (sliding velocity) in the axoneme remained almost constant in the flagellar beat of both the activated and hyperactivated spermatozoa. Comparison of the sliding velocity in Suncus, golden hamster, monkey, and sea urchin sperm flagella with their stiffness suggests that the sliding velocity is determined by the stiffness at the flagellar base and that the same sliding microtubule system functions in both mammalian and echinoderm spermatozoa.     

The forces applied by cilia depend linearly on their frequency due to constant geometry of the effective stroke

Mucus propelling cilia are excitable by many stimulants, and have been shown to increase their beating frequency up to threefold, by physiological extracellular stimulants, such as adenosine-triphosphate, acetylcholine, and others. This is thought to represent the evolutionary adaptation of mucociliary systems to the need of rapid and efficient cleansing the airways of foreign particles. However, the mucus transport velocity depends not only on the beat frequency of the cilia, but on their beat pattern as well, especially in the case of mucus bearing cilia that beat in a complex, three-dimensional fashion. In this study, we directly measured the force applied by live ciliary tissues with an atomic force microscope, and found that it increases linearly with the beating frequency. This implies that the arc swept by the cilia during their effective stroke remains unchanged during frequency increase, thus leading to a linear dependence of transport velocity on the beat frequency. Combining the atomic force microscope measurements with optical measurements, we have indications that the recovery stroke is performed on a less inclined plane, leading to an effective shortening of the overall path traveled by the cilia tip during this nontransporting phase of their beat pattern. This effect is observed to be independent of the type of stimulant (temperature or chemical), chemical (adenosine-triphosphate or acetylcholine), or concentration (1 μM-100 μM), indicating that this behavior may result from internal details of the cilium mechanical structure.     

On metachronism in ciliary systems: a model describing the dependence of the metachronal wave properties on the intrinsic ciliary parameters

A mathematical model is proposed to explain the dependence of the direction and the length of the metachronal wave on parameters that characterize the ciliary beat, the dimensions of the cilia, and the geometry of their arrangement on the ciliated surface. The metachronal wave is decomposed into two mutually perpendicular components, which are chosen in such a way that the direction of one of them is in the direction of the effective stroke. The magnitudes of the two components are determined by using the concept of the time of delay between adjacent cilia. The properties of the metachronal wave are then calculated as a function of the ciliary parameters. The results obtained with the present model predict that the direction of the wave propagation is strongly dependent on the type of metachronism in the direction of the effective stoke and the polarization in time and in space of the ciliary beat. The metachronal wavelength is found to depend on four parameters: the ciliary length, the angle of the arc projected on the cell surface by the ciliary tip during the recovery stroke, the degree of asymmetry of ciliary beat, and the portion of the cycle occupied by the pause. The metachronal wavelength is also found to be only weakly dependent on the ciliary frequency. At this stage there exists relatively little experimental information with which to characterize fully the metachronal properties of ciliary systems. Even when only partial information exists, the model allows prediction, to within a certain range, of the direction of the wave propagation. It also suggests a possible mechanism for the influence of changes in environmental conditions on wave direction and wavelength. In several cases in which full information does exist, good agreement between the experimental findings and the predictions of the model is found. According to this model it will be worthwhile to invest more effort in measuring the time and space polarization of ciliary beating and times of delay between cilia.