Newt lung ciliated cell models: Effect of MgATP on beat frequency and waveforms

Cell Motility 5377-392 (1985)

Newt Lung Ciliated Cell Effect of MgATP on Beat and Waveforms

Models: Frequency

Alice Weaver and Robert Hard

Department of Zoology, Oregon State University, Corvallis

Highly coupled newt lung ciliated cell models were used to study the effects of MgATP concentration on ciliary beat frequency and waveform. Models were prepared from ciliated lung cells of the newt Taricha granulosa by trypsin dissociation of the epithelium, demembranation with Triton X- 100, and reactivation with MgATP, as described previously [Weaver and Hard, 19851. Beat frequencies were measured stroboscopically. Ciliary waveforms of reactivated models and intact mucociliary epithelial sheets were determined by single frame analysis of high-speed movies. Waveform parameters calculated included the durations of the effective and recovery strokes, the angular swings and angular velocities of the ciliary base and tip, the position of the bend along the ciliary shaft during the recovery stroke, the velocity of recovery stroke bend propagation, and the ratio of the duration of recovery stroke bend propagation to the duration of the recovery stroke itself. We found that beat frequency varied biphasically in response to MgATP at 2I0C, as shown previously for isolated, individual, newt lung axonemes. Apparent F,,, (maximum beat frequency) and K, values of 25 Hz and 0.14 mM, and 35 Hz and 0.47 mM, respectively, were obtained for each linear segment of the biphasic double reciprocal plot. Demembranation did not alter either ciliary waveform or the pattern of coordination. In this system, metachrony is antilaeoplectic and ciliary waveform appears to be regulated independent of beat frequency.

Key words: newt, lung, cilia, beat frequency, waveform, models

Received February 4, 1985; accepted July 8, 1985.

Robcrl Hard is now at Department of Anatomical Sciences, S U N Y at Bullalo, But'lalo. NY 14214. Address reprint requests there.

0 1985 Alan R. Liss, Inc.

378 Weaver and Hard

INTRODUCTION

Ciliary movements, unlike the undulations of flagella, clearly are biphasic: the effective stroke is characterized by a high angular velocity and an extended, rigid ciliary profile; whereas in the slower recovery stroke, propagation of a bend along the shaft results in a less extended, flexed profile. Ciliary motility also is characterized by spatial and temporal phase shifts in the beat cycles of adjacent cilia, which produce a travelling wavefront, or metachronal wave.

Careful descriptions of ciliary beat cycles and metachrony have appeared previously [Cheung, 1981; Machemer, 1974; Sanderson and Sleigh, 1981; Sleigh, 1968, 1974; Wilson, Jan, and Fonseca, 19751. However, most descriptions of beat parameters have been limited to water transporting and compound cilia [Sleigh, 19681, since the small size, dense packing, and high-beat frequencies of simple cilia make them relatively difficult to visualize. These problems are exacerbated in mucociliary systems, where the cilia generally are even shorter and more densely packed on an opaque mucosa.

However, we previously isolated highly motile ciliated models from the mucociliary system present in newt lungs. These models have several features that are well suited for studies of ciliary waveforms [Hard and Weaver, 19831. We have partially characterized their motility and demonstrated that they possess a high degree of mechanochemical coupling based on the following criteria: 1) a high percentage of reactivation-at MgATP concentrations above 5 pM, the ciliary tufts of nearly 100% of the models are motile; 2) a high percentage of coordination-above 25 pM MgATP, more than 90% of the motile models produce coordinated waves in the ciliary tuft; 3) beat frequencies correspond to those measured in living, cultured cells and populations of isolated, individual axonemes; 4) a high degree of stability following reactivation-the beat frequencies of reactivated models do not change over a time course of 90 minutes and the percentage of coordination is constant for 30 minutes [Weaver and Hard, 19851.

To our knowledge, complete waveform analyses never have been undertaken for any demembranated ciliary model system. In the present study, we use these ciliary models to address the following specific questions: I ) How does the beat frequency of models vary with MgATP concentration? 2) Does removal of the cell membrane alter the waveforms of models induced to beat with MgATP? 3) Do the waveforms of the models also vary with MgATP or are beat frequency and other waveform parameters regulated independently?

MATERIALS AND METHODS Isolation of Single Newt Lung Ciliated Cells

The procedures used for isolating populations of dissociated newt lung ciliated cells have been described previously [Weaver and Hard, 19851. Briefly, minced lungs were incubated for 48 hours at 4°C in culture medium (pH 7.2) containing 0.5% trypsin (type IX; Sigma Chemical Co., St. Louis, MO). The lung fragments then were placed for 5 minutes in 0.5% soybean trypsin inhibitor (Sigma Chemical Co.) followed by fresh culture medium. Cell aggregates were removed from the underlying tissues by agitation with a Pasteur pipette and dissociated further by incubation in Ca2+/Mg2+-free saline with 2 mM EDTA.

Ciliary Beat Frequency and Waveform 379

Isolation of Sheets of Lung Mucociliary Epithelium

The procedures used to isolate large sheets of newt lung mucociliary epithelium were presented in detail elsewhere [Hard and Rieder, 19831. Minced lungs were incubated for 24 hours at 4°C in 5% trypsin (1:250; Difco Laboratories, Detroit, Mi) in culture medium at pH 7.2. After trypsin inhibition (3.0% soybean trypsin inhibitor in culture medium), the lung fragments were placed in fresh culture medium and gently agitated to release sheets of mucociliary epithelium from the underlying tissues.

Reactivation of Cell Models

isolated, ciliated cells were demembranated and reactivated according to procedures detailed elsewhere [Weaver and Hard, 19851. Wash, demembranating, and reactivating solutions also were the same as those used previously. isolated cells were rinsed twice in wash solution, mixed with demembranating solution, and incorporated into a temperature-controlled observation chamber [Hard and Cypher, 19851. Excess reactivating solution containing the desired MgATP concentration was added to preparations by perfusion under a coverslip separated from the chamber by spacers made of silastic elastomer (Dow Corning Corp., Midland, Mi). All beat frequency measurements and high-speed movies were taken within 30 minutes following reactivation, since the motile characteristics of the models remain unchanged over that time span [Weaver and Hard, 19851.

Beat Frequency Measurements and Photomicrography

Reactivated models and isolated mucociliary epithelial sheets were observed using either a Wild-Heerbrugg microscope equipped with phase contrast optics or a Zeiss UEM microscope (Carl Zeiss, inc., New York, NY) equipped with phase contrast and Nomarslu differential interference contrast (DiC) optics. Beat frequencies were measured stroboscopically with a Model 136 Point Source Strobex modified to extend its frequency range to 512 Hz (Chadwick-Helmuth Co., Elmonte, CA). Care was taken to be sure that measurements reflected the true frequency rather than some harmonic.

Photomicrographs were taken with a Nikon camera system (Nikon Inc., Garden City, NY) using Kodak Technical Pan or Kodak Panatomic X 35-mm films. The film was exposed by a single flash (20-55 psec) from the stroboscope. High-speed movies were taken using a Redlake Locam Model 51 high-speed 16-mm movie camera (Redlake Corp., Campbell, CA) and Kodak Technical Pan film. For this purpose, the stroboscope was synchronized to the framing rate of the camera. All film was processed in Kodak HCllO developer. Framing rates were 200 framedsec for intact cells and models reactivated with 50 pM MgATP and 400 framedsec for models at higher MgATP levels.

Analysis of Ciliary Waveforms

Ciliary waveforms were determined by single-frame analysis of high-speed movies using an LW Photoanalyzer (LW Photo, Inc., Van Nuys, CA). Preliminary analyses of high-speed movies, strobe-illuminated models, and cultured cells clearly showed that the ciliary waveform was planar throughout the beat cycle regardless of the orientation of observations. Cilia selected for detailed analysis were beating in the plane of focus so that the entire beat cycle could be followed at high magnification,

380 Weaver and Hard

but care was used to be sure that their motion was not constrained by coverglass surfaces. Cilia were followed through at least four beat cycles and their profiles traced every one or two frames. The angles of the ciliary base and tip with respect to the cell surface during the beat cycle and the progression of the ciliary bend during the recovery stroke were measured using a Zeiss MOP-3 digitizing pad (Carl Zeiss Inc .) .

From these measurements, the following waveform parameters were calculated for each beat cycle: 1) the duration (in msec) of the effective stroke (tES); 2) the duration (in msec) of the recovery stroke (tRs); 3) the angular swing (in degrees) of the ciliary base during the effective stroke (BES) and recovery stroke (BRs); 4) the angular velocity (in degreedmsec) of the base during the effective stroke (mEsB) and recovery stroke (aRSB); 5 ) the angular velocity (in degreedmsec) of the tip during the effective stroke (aEST) and recovery stroke (sJRST); 6) the position of the bend (in pm) from the base during the recovery stroke (DBp); 7) the average velocity (in pml sec) of bend propagation during the recovery stroke (VBp); and 8) the proportion of the beat cycle duration occupied by the effective stroke (aEs), the recovery stroke (aRs), and by bend propagation (aBp).

RESULTS Metachrony of Intact Cells and Cell Models

Profile views of the demembranated ciliary tufts are shown in Figure lA, and those of living cells in large sheets of mucociliary epithelium are shown in Figure IB. High-speed movies confirmed that both models and intact cells exhibited antilaeoplectic metachrony [Knight-Jones, 19541. The antiplectic component was observed when the beat cycle was oriented from side to side in the field of view, and the metachronal wave appeared to move opposite the direction of the effective stroke. The laeoplectic component was seen when the beat cycle was directed in and out of the plane of the field of view, toward the viewer, and the metachronal wave appeared to move to the left.

Beat Frequency of the Cell Models in Response to Varying MgATP A biphasic beat frequency response to varying MgATP has been reported for

isolated, individual newt lung axonemes reactivated at 20°C [Hard and Cypher, 19851. The question can be posed: Is this biphasic response inherent to the axoneme itself, or is it only acquired following the separation of axonemes from the ciliary tuft? This question can be answered by comparing the response of reactivated, intact, ciliary tufts to that of individual axonemes.

Figure 2 shows how the beat frequency of the demembranated tufts varied with MgATP over the concentration range 0.125-5.0 mM. Beat frequency increased with increasing MgATP concentration, reaching a plateau at approximately 2.5 mM MgATP (Fig. 2A). A double reciprocal plot of these data showed distinct non- Michaelis-Menten kinetics (Fig. 2B). Two separate, linear regions could be identified, one at low MgATP levels (below 0.5 mM) and one at higher MgATP levels (above 0.8 mM). From the regression line calculated for the low MgATP region (0.125-0.5 mM) in the double reciprocal plot, the apparent F,,,,, was 25.4 Hz and the apparent K,,, was 0.14 mM. For the high substrate region (0.8-5.0 mM), the apparent F,,,, was 3.5 Hz and the apparent K,, was 0.47 mM. The biphasic nature of the response


Fig. I. Nomarski DIC micrographs of a demembranated cell model and a normal mucociliary sheet isolated from newt lungs. A) A typical Triton-extracted cell model after reactivation with MgATP. The intact ciliary tuft (C), underlying accessory structures (AS), and the demembranated nucleus (N) are shown ( ~ 2 , 5 0 0 ) . B) A sheet of living mucociliary epithelial cells isolated from the lung. Mucous cells (M) are interspersed among the ciliated cells (CC) ( x 1,400).

was further emphasized when the data were arranged as an Eadie-Scatchard plot (Fig. 2C).

Ciliary Beat Cycle Analysis It has been estimated that there are 300-400 cilia on each ciliated newt lung cell

[Hard, Cypher, and Schabtach, 19851. Although some cilia are lost upon dernernbra-

382 Weaver and Hard

v z W

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a

L L m

I 2 3 4 5 0 20 30

M g A T P (mM) BEAT FREQUENCY (HZ)

2 4 6 8 -8 -6 -4 -2 0 I /MgATP (mh4-l)

Fig. 2. The beat frequency of cell models reactivated at 21°C with 0.125-5.0 mM MgATP. A) The mean beat frequency f SE of three replicates of the experiment are plotted vs MgATP. Each replicate used a separate pool of trypsinized lungs from three newts and freshly made solutions. The beat frequency of 25 models was measured for each replicate at each MgATP concentration. Beat frequency increased with increasing MgATP, reaching a plateau at about 2.5 mM MgATP. B) A double reciprocal plot of the data shown in A is nonlinear over the range studied. The data can be fit by two separate linear regions of different slope. The high MgATP region (0.833-2.5 rnM) has an apparent F,,,, of 34.9 Hz and an apparent K, of 0 .47 mM. The low MgATP region (0.125-0.5 mM) has an apparent F,,,, of 25.4 Hz and an apparent K, of 0.14 mM. The points at 3.33 and 5.0 mM were omitted from regressions because of substrate inhibition at these concentrations. C) An Eadie-Scatchard plot further emphasizes the deviation from linearity seen in B. The apparent F,,,,, and K,, values for the two linear segments calculated from this plot were essentially the same as those obtained from the double reciprocal plot.

nation, the cilia in the tufts remain densely packed. Newt lung cilia, 13-14 pm long, are substantially longer than anuran (6 pm) and mammalian (5 pm) cilia, other mucus-transporting cilia commonly studied [Hard and Weaver, 19831, so that many individual cilia are distinguishable and can be followed through successive beat cycles. Profile views of cilia showed that the recovery and effective strokes are coplanar in both demembranated models and in intact cells of isolated, mucociliary


epithelial sheets. We included different orientations of observation on many different cells and models. Never did we see the cilium in the recovery stroke follow a path outside the plane of the effective stroke. This planar waveform further facilitated analysis.

We sought to answer two questions with the waveform analysis: 1) Does the removal of the plasma membrane alter ciliary waveform? 2) Do the waveforms of the ciliary models vary with beat frequency? Therefore, we compared the waveforms of intact cells with those of demembranated cell models reactivated with various MgATP concentrations. MgATP levels were chosen to span the low, transition, and high substrate regions of the biphasic beat frequency vs MgATP plot (Fig. 2B). Beat cycle parameters were calculated as described in Materials and Methods. These parameters are shown in Table I and Figures 3 through 7.

Temporal changes of the basal inclination angle and the progression of the bend along the ciliary shaft during the recovery stroke provide a means for describing the ciliary beat cycle [for details see Sleigh, 19681. Figure 3 shows the movements of representative cilia in intact cells and in models reactivated at four different MgATP concentrations. Each graph begins at the left with an effective stroke and shows the angle of basal inclination increasing to a maximum at the end of the effective stroke. The basal inclination angle then decreases during the recovery stroke, reaching a minimum that is maintained until the next effective stroke begins. The propagation of the bend along the ciliary shaft, which starts at the beginning of the recovery stroke, continues through the rest of the recovery stroke and is not completed until the cilium already has begun its forward swing during the ensuing effective stroke.

The respective durations of the effective (tES) and recovery strokes (tRS) are shown in Figure 4 (see also Table I). As beat frequency increased, the duration of both portions of the cycle decreased. Models reactivated with 50 pM MgATP had the lowest beat frequency and the longest tES and tRS. Models reactivated with 0.5 mM and 2.5 mM MgATP had much higher beat frequencies and correspondingly shorter tES and tRS. The beat frequencies of cilia of h e cells in the mucociliary epithelial sheets tend to be only one half to two thirds of those recorded in primary explant cultures in which trypsin is not used in the isolation process [Weaver and Hard, 19851. With the lower beat frequencies, the tES and tRS of cilia on the sheets were between those of models reactivated at 50 pM and 0.5 mM MgATP.

The angular swing of the base during the recovery stroke was the same as that during the effective stroke, as might be expected. However, for all groups, the angle traversed by the axonemal tip during the effective stroke (&ST) was greater than that traversed by the base (OESB, &SB) (Fig. 5A; Table I). This was due to a reverse flexure in the axoneme that appeared proximal to the propagating bend during the recovery stroke. It also was due to the fact that the distal portion of the axoneme continued moving forward after the basal portion had stopped moving near the end of the effective stroke. Figure 5 also shows that &SB, ORSB, and 6EsT did not vary significantly among the groups, indicating that there was no detectable effect of demembranation or of MgATP concentration on these parameters.

The angular velocities of the basal portion of the axoneme in the recovery stroke (mRSB) and the effective stroke (~JESB) and of the tip in the effective stroke (WEST) were found to vary with beat frequency (Fig. 5B; Table I). Models reactivated with 50 pM MgATP had the lowest vRSB, wESB, and oEST. Angular velocities were greater in intact cilia and in models reactivated at the higher MgATP concentrations.

384 Weaver and Hard

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Fig. 3. The change in the basal inclination angle and propagation of the recovery stroke bend during the ciliary beat cycle. These parameters were measured for cilia on mucociliary epithelial sheets (A) and models reactivated with 2.5 mM (B), 1.25 mM (C), 0.50 mM (D), and 50 pM MgATP (E). Ciliary profiles were traced at one or two frame intervals from high-speed movies, and the basal inclination angle in degrees (continuous plot) and the distance (in pm) of the bend along the shaft during the recovery stroke (discontinuous plot) were plotted against time (in msec). The movements of a typical cilium in each treatment group are shown. The basal inclination angle increases in the effective stroke. then decreases during the recovery stroke. Bend propagation during the recovery stroke continued into the following effective stroke. The beat frequencies of A-E were 15.2, 30.3, 25.6, 23.2, and 7.1 Hz, respectively.

Not surprisingly, the velocity of bend propagation (VBp) during the recovery stroke varied with beat frequency (Fig. 6A; Table I). The bend traveled most slowly along cilia reactivated with 50 pM MgATP. VBp was higher in intact cilia of the mucociliary sheets and increased in models treated with 0.5 mM and 2.5 mM MgATP. However, the total distance of bend propagation (DBp) did not appear to vary among groups in any consistent way (Fig. 6B; Table I). This parameter appeared to be greater for axonemes reactivated at 50 pM MgATP. The meaning of this difference is uncertain.

The ratio of the proportion of the beat cycle taken up by bend propagation (aBp) to the proportion of the beat cycle taken up by the recovery stroke (aRs) is slightly

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Fig. 4. The total duration of the effective stroke and the recovery stroke during the beat cycle. As the MgATP concentration was increased, the total duration (in msec) of both the effective stroke (tES) and the recovery stroke (tRS) decreased. The values shown represent the mean and standard error of 12 beat cycles measured for each group.

A

B

BASE-ES BASE-RS T IP - E S

Fig. 5 . The angular swing (8) and angular velocities (w ) of the ciliary base and tip during the beat cycle of the Same cilia measured in Figure 4. A) The total angle (degrees) traversed by the axonemal tip is much greater than the basal angular swing. This parameter did not vary among treatment groups. B) The angular velocity (degreedmsec) of the base is greater during the effective stroke than during the recovery stroke. The angular velocity of the tip during the effective stroke is greater than that of the base, except at 50 pM MgATP. As MgATP concentration was increased the angular velocity of the base and tip increased.


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1 I Fig. 6 . Parameters associated with bend propagation tipward. A) As the MgATP concentration (and therefore beat frequency) was increased, the velocity of bend propagation (VBP) increased. B) The distance of bend propagation along the shaft (DBP) did not vary in any consistent manner among groups. C) The ratio of the duration of bend propagation as a proportion of the beat cycle to the duration of the recovery stroke as a proportion of the beat cycle ( 0 l ~ ~ / 0 1 ~ ~ ) also did not vary among groups. This parameter is greater than 1, indicating that recovery stroke bend propagation continues into the following effective stroke.

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Fig. 7. Ratios of the angular velocities of the base (wRsB/ZEsB) and the total durations of effective and recovery stroke (tRS/tES). Neither G R ~ B / ~ E ~ B nor tRS/tES varied among the groups measured. tRS/tES iS

greater than 1, indicating that the recovery stroke occupies a greater proportion of the beat cycle than the effective stroke.

388 Weaver and Hard

DEGREES 90 I

10 5 0 5 10

I-cm Fig. 8. Diagrammatic representation of the beat cycle of a newt lung cilium. Since waveform parameters (8EsB, ORSB, BEST, DBP; see Table I) do not differ among treatment groups, a single diagram can illustrate the beat cycle of newt lung cilia over the beat frequency range examined. Ciliary profiles are drawn at intervals of one tenth of a beat cycle. They were constructed from the average 8 and DBp values in Table I, and the intermediate angles and bend distances as determined from single-frame cine analyses. The shapes and positions of the axoneme profiles are approximate owing to the variability of measured parameters. Positions 1-6 represent the recovery stroke and positions 7-10 represent the effective stroke.

greater than 1.0 (Fig. 6C; Table I). This was indicative of the completion of bend propagation during the ensuing effective stroke and also demonstrated that there was no rest period between beat cycles.

Although tES, tRS (Fig. 4), SJESB, and SJRSB (Fig. 5B) varied with beat frequency, the ratios ~JRSB/SJESB, and tRS/tES remained constant among groups (Fig. 7; Table I). For each group G+SB was slightly greater than GJRSB, although the differences were not significant owing to the large variability within each group. In all groups tRS/tES was significantly greater than 1, indicating that the recovery stroke occupied a greater proportion of the beat cycle than did the effective stroke. This did not change upon demembranation or with MgATP concentration.

It should be emphasized that those parameters that contribute to ciliary waveform (ie, t&B, &SB, OEST, DBP, tRS/tES, and (YB~/(YRS) did not vary significantly among groups. Using these parameters and the ciliary profiles traced from high-speed movies, a generalized diagram of the newt lung ciliary waveform has been constructed (Fig. 8).

DISCUSSION Beat Frequency Vs MgATP

The beat frequencies of both flagellar and ciliary axonemes have been shown to obey Michaelis-Menten kinetics in response to varying MgATP [Gibbons and Gib- bons, 1972, 1973; Okuno and Brokaw, 1979; Torres, Reynaud, and Portocarrero, 19771. The beat frequency of the newt lung models studied here also varied with MgATP, but they clearly did not obey simple saturation kinetics, as shown by biphasic double reciprocal and Eadie-Scatchard plots (Fig. 2).


A biphasic beat frequency response to MgATP also has been found for populations of individual, reactivated, newt lung axonemes [Hard and Cypher, 19851. It was proposed that an intraaxonemal mechanism controls an equilibrium between two distinct conformational states. Each conformational state is characterized by a different apparent F,,, and the equilibrium between the two states can be shifted by temperature and MgATP. It was suggested that this switching mechanism may be mediated by the in situ activation of outer dynein arms, analogous to the activation of isolated, latent outer arm dynein (LAD-1) ATPase activity that has been described both for flagella [Gibbons and Fronk, 19791 and cilia [Blum and Hayes, 19771, although other possible mechanisms exist as well, given that the dynein arm has been shown to be a heteropolymer of distinct ATPases [Tang et al., 1982; Piperno and Luck, 1979; Huang, Piperno, and Luck, 1979; Pfister and Witman, 19841.

The procedures used here for isolating ciliary tufts were only slightly altered from those used to obtain populations of individual axonemes from the same source [Hard, Cypher, and Schabtach, 19851. Therefore, the biphasic response of the ciliary tufts likely reflects the same mechanism that occurs in isolated axonemes. The transition substrate concentration of the tufts (in the range 0.5-0.8 mM MgATP at 21°C) corresponds to that of isolated axonemes (approximately 0.5 mM MgATP at 21°C). In the low substrate region the extrapolated apparent F,,, of the cell models was slightly higher than that found previously for isolated axonemes (25.3 Hz as opposed to 21.99 Hz), and in the high substrate region the apparent F,,, of the cell models was lower than that of isolated axonemes (34.6 Hz as opposed to 44.8 Hz). The latter F,,,, which describes the upper beat frequency limit of the models, may indicate slightly reduced mechanochemical coupling as compared to that of isolated axonemes .

Maintenance of Constant Ciliary Waveforms

Sleigh 119681 has noted a problem inherent in ciliary waveform analyses. Ciliary beat cycles appear to be quite variable. This variability renders description of the movement difficult and precise interpretation troublesome. It is an especially impor- tant issue when dealing with cell models, where it must be determined whether the variability encountered is inherent to the cilia or whether it is the result of the procedures used to isolate functional models.

In the present study, we have compared the beat cycles of intact cells in isolated mucociliary epithelial sheets with the beat cycles of demembranated models reactivated at several MgATP concentrations. Both living cells and reactivated models showed within group variations for most beat cycle parameters that were greater than 10% of the mean. Because the variability of these parameters was comparable both for intact cilia and models reactivated at several MgATP concentrations, we conclude that demenibranation and reactivation was not the cause of the variability.

Perhaps the high variability was due to trypsinization. The beat frequencies measured for cilia on the trypsinized mucociliary epithelial sheets were significantly lower than those of cultured cells [Weaver and Hard, 19851, suggesting that trypsin had effects in addition to dissociation of the epithelium. It is possible that cilia in vivo are less variable. Perhaps studies using nontrypsinized primary cell cultures can resolve this question.

Beat cycle parameters that describe ciliary waveform ( B E S B , 0 ~ ~ 6 , OEsr, and DBP), the proportion of time spent in each stage of the beat cycle (shown by tRs/tes)?

390 Weaver and Hard

the overlap between one recovery stroke and the next effective stroke (shown by aRs), and the ratio of basal angular velocities (zJRSB/GJESB) did not vary with beat frequency. These data suggest that ciliary waveform and beat frequency are regulated independently as shown previously for flagella [Gibbons, 19741. In addition, since there were no significant differences in these parameters between living cells and reactivated models, it also can be concluded that removal of the plasma membrane and subsequent reactivation did not cause major changes in ciliary waveform. There has been speculation about the role of the cell membrane and microtubule-membrane bridges in the regulation of ciliary motility [Dentler, 1981; Dentler, Pratt, and Stephens, 19801. Our data leave this question unanswered.

To our knowledge, these conclusions have not been documented previously for cilia. Sperm flagella have been shown to maintain a relativzly constant wavelength and wave amplitude at varying beat frequencies [Brokaw and Josslin, 1973; Gibbons and Gibbons, 19731. However, at very low frequencies, below one fourth of the frequency of intact sperm, the wavelength of reactivated flagellar axonemes increases [Brokaw and Josslin, 19731. The ciliary beat cycle parameters analogous to flagellar wavelength and amplitude are D B p and 8. Like wavelength and amplitude in sperm flagella, these remained relatively constant at different beat frequencies. We noted a slight increase in D B p in models reactivated with 50 ~ L M MgATP, although the large standard error associated with this parameter casts some uncertainty on its interpretation.

Metachrony and Other Aspects of the Beat Cycle The type of metachronism in a field of cilia and the path followed by each

cilium in the recovery stroke seem to be related [Machemer, 1972a,b, 1974; Sleigh, 19741. Orthoplectic metachronism, where the direction of the ciliary effective stroke and the direction of metachronal wave transmission occur in the same plane, consis- tently is observed in systems in which the effective stroke and the recovery stroke are coplanar. Laeoplectic metachronism has been reported in systems in which the recovery stroke gyrates clockwise with respect to the effective stroke plane. Newt lung ciliated cells and models apparently do not fit this pattern. Because the recovery and effective strokes are coplanar, some type of orthoplectic metachronism would be expected, but instead we observed antilaeoplectic metachronism in both the mucociliary epithelial sheets and the models. According to the generally accepted hypothesis, the direction of metachrony tends to be determined by the smallest vector of force in the ciliary field [Machemer, 1972a,b]. The patterns observed in other systems agree with the predictions of this hypothesis, but the relationship between the beat cycle and metachronal pattern in newt lung cells does not.

Although relatively few mucociliary systems have been described in detail, some comparisons can be made. The presence of a rest period at the end of each effective stroke has been described in frog palate and rabbit tracheal epithelial cilia and is thought to be characteristic of mucus-propelling cilia [Sanderson and Sleigh, 19811. However, newt lung cilia do not rest between beat cycles. The newt lung mucociliary epithelium is similar to other mucociliary systems in the antilaeoplectic metachrony present [Sleigh, 1974; Marino and Aiello, 19821. However, the presence of coplanar beat cycles displaying no rest period is different from other mucociliary systems in which these parameters were studied.


ACKNOWLEDGMENTS

We thank Drs. Ralph Quatrano and John Morris for their comments regarding this manuscript. We are indebted to Suzi Sargent for her secretarial assistance and to Kathy Blaustein for her technical assistance. This work was supported by grants to R.H. from the MedicaI Research Foundation of Oregon, NIH Biomedical Research Support grant RR07079 and NIH grant HL29233. Support to A.W. from Sigma Xi and the Department of Zoology, Oregon State University, is gratefully acknowledged.

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Newt lung ciliated cell models: Effect of MgATP on beat frequency and waveforms

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