Isolation of newt lung ciliated cell models: Characterization of motility and coordination...

Cell Motility 5355-375 (1985)

Isolation of Newt Lung Ciliated Cell Models: Characterization of Motility and Coordination Thresholds

Alice Weaver and Robert Hard

Department of Zoology, Oregon State University, Corvallis

Dernembranated ciliated cell models are useful for studying mechanisms respon- sible for the regulation of ciliary coordination and waveform. This paper describes procedures for isolating ciliated cells from the newt, Taricha granulosa, by trypsin dissociation, their subsequent demembranation by Triton X- 100, and their reactivation with MgATP to produce highly motile, coordinated, ciliated cell models. Reactivation of cell models with a high degree of mechanochemical coupling depended on avoiding mechanical damage and maintaining optimal conditions during all stages of isolation and reactivation. Highly motile models were prepared from cells incubated in trypsin, treated briefly with EDTA, separated by gentle agitation, and concentrated by centrifugation at low gravitational forces. Optimal demembranation and reactivation conditions were similar to those described previously for isolated newt lung axonemes. Under these conditions, nearly 100% of the models were reactivated when provided with MgATP and 90-95 % beat with coordinated waves. The ciliary tufts beat at frequencies within the range measured in living cells and their reactivated motility was stable for at least 30 min at constant MgATP. These highly coupled models were used to show (1) that development of coordination in the ciliary tuft occurs at a higher substrate concentration range (10-25 pM) than that required to initiate motility per se (2-10 pM; (2) that outer dynein arms may not contribute to beat frequency at substrate concentrations below 35 pM; and (3) that vanadate has effects both on beat frequency and coordination of the tufts.

Key words: Newt, lung, cilia, cell models, ciliary coordination

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

Robert Hard’s current address is Robert Hard, Department of Anatomical Sciences, SUNY at Buffalo, Buffalo, N Y 14214. Address reprint requests there.

0 1985 Alan R. Liss, Inc.

356 Weaver and Hard

INTRODUCTION

Mucociliary transport continuously rids the respiratory system of foreign material, thus serving as a defense against damage or disease. The mucociliary epithelium may be envisioned as a vast field of densely packed, oscillating cilia, whose beat cycles are coordinated in regular spatial and temporal patterns for efficient mucus transport. These patterns assure that at any instant many cilia are providing motive force, so that continuity of forward flow is maintained. Metachrony is a common feature of coordinated groups of cilia [Machemer, 1972a,b]. Although various types of rnetachrony have been described and well documented in living cells [Knight- Jones, 1954; Machemer, 1974; Sleigh, 1974; Aiello and Sleigh, 1972; Sanderson and Sleigh, 19811, the mechanism by which coordination occurs among cilia is not fully understood. The most widely accepted hypothesis proposes that synchrony and metachrony result from hydrodynamic servoregulation which provides the least interference between individual cilia beating near a common frequency [Machemer, 1972al.

On approach to studying factors affecting coordination is through the use of demembranated ciliated cell models, i.e., isolated ciliary tufts. Such models have the advantage that upon removal of the cell membrane, axonemal components are in direct contact with the surrounding reactivating solution. Thus factors thought to affect motility or coordination can be tested directly be manipulating solution conditions or other variables. The ultimate usefulness of any ciliated cell model depends on the extent to which its motile capabilities correspond to those of the living cells from which it has been isolated. Ciliary models isolated previously from a number of cell types have made important contributions to our understanding of ciliary movement, although their motile characteristics have not always been thoroughly documented [Eckert and Murakami, 1972; Naitoh and Kaneko, 1973; Torres et al, 1977; Tsuchiya, 1977; Walter and Satir, 1978; Wais-Steider and Satir, 1979; Dirksen and Zeira 19811.

The purpose of the present study is to describe procedures for isolating and reactivating highly motile, coordinated ciliary tufts from newt lungs and to evaluate their motile capabilities. The newt system possesses several advantages for model studies of mucociliary transport, including large cells, relatively long cilia for tissue cells, and a temperature optimum near 20°C [Hard and Weaver, 19831. Ciliary axonemes with a very high degree of mechanochemical coupling already have been isolated from this system [Hard et al, 1985; Hard and Cypher, 19851.

These highly coupled cell models have been used in attempts to answer the following questions: (1) what is the specific threshold MgATP level required to initiate motility? (2) Does coordinated beating of cilia develop at the same threshold MgATP level required to initiate motility? In addition, the effect of high salt extraction and vanadate treatment on the motility and coordination of the models has been investigated. Other aspects of the motile behavior of the models, including the effect of MgATP on ciliary beat frequency and waveform, are presented elsewhere [Weaver and Hard, 19851.

MATERIALS AND METHODS Experimental Animals

dechlorinated water at 15°C. Taricha granulosa were captured locally and stored in a large tank of running,

Newt Lung Ciliated Cell Models 357

Trypsinization of Lungs

The lung mucociliary epithelium of T granulosa is confined to areas overlying the large, ventral pulmonary vein and the proximal portions of its paired, afferent tributary vessels [Hard and Weaver, 1983; Hard et al, 19851. To reduce the numbers of nonciliated cells present in dissociated tissue, strips of lung containing these areas were isolated in sterile culture medium consisting of 60% Leibovitz medium [Leibov- itz, 19631, to which 5% whole egg ultrafiltrate, 10% fetal calf serum, 1 % penicillin/ streptomycin (l0,OOO units per rnl/lO,OOO pg per ml), 5 mM BES, and 5 mM HEPES buffers were added. The pH was adjusted to 7.2 with NaOH and the medium was sterilized by filtration. The lung strips were minced into small pieces, cooled on ice, and then placed into cold, sterile, 0.5% trypsin (type IX, Sigma Chemical Co., St. Louis, MO) in culture medium and stored at 4°C for 48 h. This trypsinization protocol produced healthier cells than did a number of alternative procedures tested (see Results).

Following trypsinization, the lung fragments were treated for 5 min with 0.5% Soybean Trypsin Inhibitor (Sigma Chemical Co., St. Louis, MO) in culture medium. The trypsin inhibitor then was replaced with fresh, cold, sterile culture medium and the trypsinized lung fragments were stored on ice and used the same day.

Isolation and Reactivation of Cell Models For each preparation, several trypsinized lung fragments were placed in a 6 x

50 mm tube containing Ca2+/Mg2+-free saline and 2 mM EDTA. After 5 min at room temperature, the fragments gently were drawn up and down 10-20 times in a Pasteur pipet to free the epithelial cells from the underlying tissues. The tissue fragments then were discarded. The contents of the tube were transferred to a 0.4 ml polyethylene tube and spun with a hand centrifuge (Wm. Boekel and Co., Inc., Philadelphia, PA) at 550-600 rpm for 1 min. The wash, demembranating, and reactivating solutions used in subsequent steps were those described previously [Hard and Cypher, 19851, except that the demembranating solution contained 2 % Triton X- 100. The pellet of cells was rinsed twice with wash solution (pH 7.0; 120 mM KPIPES buffer, 1 mM DTT, 2.5 mM MgS04) to remove all traces of the Ca2+/ Mg2+-free saline. After washing, the cells were placed into two drops of demembranating solution (pH 7.0) consisting of 120 mM KPIPES buffer, 2.5 mM MgS04, 1 mM DTT, 2% Triton X-100.

Immediately following demembranation, the models were placed on a coverglass and a drop of reactivating solution was added. The reactivating solution was identical to the wash solution except that it contained 0.5 mM EDTA, and ATP was added with or without additional Mg2+ to produce desired MgATP concentrations that were calculated from a series of simultaneous equations describing the multiple equilibria involved [Hard and Cypher, 19851. After allowing 1-2 min for models to adhere, the coverglass was inverted onto spacers made of Silastic elastomer (DOW Corning Corp., Midland, MI), which were placed on the top coverglass of a temperature-controlled observation chamber [Hard and Cypher, 19851. A large excess of reactivating solution containing the desired MgATP concentration was perfused slowly through the preparation with a filter paper wick.

High Salt Extraction and Vanadate Treatment of Cell Models In several experiments we compared the behavior of normal models with those

bearing cilia whose outer dynein arms were removed or inhibited. Outer arm dynein

358 Weaver and Hard

was removed by perfusing preparations of demembranated models with an excess of a high salt solution consisting of 0.46 M KCI, 2.5 mM MgS04, 1.0 mM DTT, 0.5 mM EDTA, and 15 mM KPIPES buffer (pH 7.0). After 1 min the high salt solution was replaced with fresh reactivating solution.

Outer arm dynein was inhibited by perfusing freshly prepared models with reactivating solutions containing different concentrations of Na3 VO4 10 H20 (Mathe- son, Coleman and Bell, Cincinnati, OH). A stock solution (10 mM) was prepared in wash solution. All experiments were conducted at 21 "C.

Beat Frequency Measurements

Reactivated cell models were observed using a Wild-Heerbrugg microscope equipped with phase-contrast optics. Beat frequencies were measured stroboscopically with a Model 136 Point Source Strobex (Chadwick-Helmuth CO., Elmonte, CA). The frequency of the stroboscope was varied until it matched that of a reactivated model, at which point the model appeared to be completely stationary. Care was taken to be sure that measurements reflected the true beat frequency rather than some harmonic. Beat frequencies below 4 Hz were measured using a stopwatch. Only single models and pairs were selected for observation and measurement. Any models showing obvious structural damage, marked loss of cilia, or impediments to ciliary beating were not used for measurements.

Photomicrography

Photomicrographs were taken with a Nikon camera affixed to a Zeiss UEM microscope equipped with Nomarski differential interference-contrast (DIC) and phase-contrast optics. Kodak Technical Pan and Kodak Panatomic X (35 mm) films were used and developed in HC110. The film was exposed by a single flash (duration 20 or 55 ps) from the stroboscope.

High-speed movies were taken with a Redlake Locam Model 51 high-speed 16- mm movie camera (Redlake Corporation, Campbell, CA) using Kodak Technical Pan 16 mm film developed in HC110. The stroboscope was synchronized to the framing rate of the camera.

Electron Microscopy

Electron microscopy was used to confirm that high salt extraction caused depletion of outer dynein arms. Ciliated cells were isolated at room temperature from lung fragments as described above and pelleted in BEEM capsules using a hand centrifuge operated at approximately 1,OOO rpm. The pellet of cells was rinsed with wash solution and agitated in demembranating solution. The models were pelleted at 1,OOO rpm and either immersed in reactivating solution or agitated in high salt solution. Those extracted with high salt were pelleted once more and then washed with reactivating solution. The pelleted tufts were fixed and embedded according to the procedures of Hard and Rieder [1983] except that Epon-Araldite resin was used in place of Spurr's resin.

Thin sections were cut using a diamond knife and a Sorvall MT-2 ultramicro- tome. The sections were stained with lead citrate and uranyl acetate and examined with a Philips 300 transmission electron microscope operated at 60 kV.


RESULTS

Isolation of Ciliated Cells

The newt lung mucociliary epithelium was relatively resistant to dissociation with trypsin. High trypsin concentrations or long incubation times were required to free the epithelial cells from the underlying connective tissue. Models with the highest motility were obtained from cells that had been incubated in 0.5% trypsin for 48 h at 4°C. Trypsin concentrations greater than 0.5%, incubation times longer than 48 h, or temperatures above 4°C reduced the agitation needed to free the cells, but models prepared from these cells showed reduced motility.

Gentle agitation of the trypsinized tissue fragments released cell aggregates of various sizes and a few single cells (Fig. 1A). The yield of single, dissociated cells was increased by incubating the trypsinized tissue for 5 min at 20°C in Ca2+/Mg2+- free saline containing 2 mM EDTA prior to agitation (Fig. IB). This treatment also minimized spontaneous detachment of the cilia from basal bodies during demembranation and had no deleterious effects on the motility of the reactivated models. Concentration of the cells by hand-centrifugation ensured a large number of models in the final preparation, while minimizing the irreversible losses of motility found at higher centrifugal forces. Rinsing cells with wash solution prior to demembranation removed traces of the Ca2+/Mg2+-free saline and reduced the pH to 7.0, the optimal pH for reactivation [Hard et al, 19851.

Motile Capabilities of Demembranated Models

Cell models prepared from the isolated cells consisted of the intact ciliary tuft and underlying cortical material (Fig. 2). The nucleus typically remained attached to the cortical material, although occasionally the nucleus was absent. No difference in function was noted in these anucleate models, so they were included in the measurements described below. The ultrastructural basis for the integrity of demembranated models is presented in detail elsewhere [Hard and Rieder, 19831.

Ciliary tufts were not motile following demembranation with Triton X- 100. However, nearly 100% of the models reactivated with MgATP. Approximately 90- 95% of the reactivated models exhibited ciliary beat cycles with bending waves of normal amplitude and shape. The remaining 5-10% of the motile models possessed obvious structural damage or cilia that exhibited abnormal, fibrillating movements. When reactivated with 1.25 mM MgATP, nearly all of the models with waveforms of normal shape and amplitude also were coordinated: ie, the spatial and temporal relationships among ciliary beat cycles within a model were constant so that synchrony or metachrony were observed, depending upon the direction of observation. To assure some degree of quality control, preparations were discarded if less than 90% of the reactivated models beat with waves of normal shape and amplitude or exhibited coordinated beating.

As shown in Table I, ciliary tufts reactivated with 1.25 mM MgATP beat at frequencies between 24 and 27 Hz. These beat frequencies were comparable to those measured previously for isolated axonemes from newt lungs [Hard et al, 19851 and were within the range measured for living, cultured, newt lung ciliated cells at 21°C (Table I). Therefore, 1.25 mM MgATP was adopted as a standard substrate concen-

360 Weaver and Hard

Fig. I. Nomarski differential interference-contrast (DIC) micrographs of ciliated cells isolated from newt lungs. A. Agitation of trypsinizcd lung fragments releases some single cells, but mostly cell aggregates (arrows) of various sizes. X330. B. The yield of single, dissociated cells is increased by incubating ciliated cell aggregates in Ca2+/Mg2+-free Ringers containing 2 rnM EDTA. X 330.

tration in reactivating solutions used to evaluate other motile characteristics of the cell models.

The beat frequency of the ciliated models was quite stable over time. In the two preparations shown in Figure 3, beat frequency remained constant for the first 30 min following reactivation and decreased by less than 10% in the ensuing 60 min, provided that the MgATP concentration was maintained by occasionally perfusing fresh reactivating solution. The percentage of models propagating coordinated waves within the

Fig. 2. A typical Triton-extracted cell after reactivation with 1.25 mM MgATP. This particular model was beating at 26.5 Hz at the time it was photographed. The intact ciliary tuft (C), underlying accessory structures (AS), and the demembranated nucleus (N) are shown. Note the coordinated beating o f cilia making up the tuft. X5,OOO.

TABLE I. A Comparison of Beat Frequencies of Living Newt Lung Cells and Reactivated Models

Source N Beat frequency (X k SE)d

Culture No. 1 15 25.51 f 3.08 2 15 28.63 f 3.24 3 15 24.44 f 3.52

Isolated axonernesa,h 25 26.62 k 2.41 Cell modelsb,' 25 25.52 f 2.72

"Axonemes were isolated and reactivated as described in Hard et al [ 19851. %he reactivating solution contained I .25 mM MgATP. 'Cell models were isolated and reactivated as described in Materials and Methods dT = 21°C.

362 Weaver and Hard

- 100

- 80

- 60

- 40

B

0 30 60 90

TIME (Min.)

Fig. 3 . The percentage of models propagating coordinated waves and their beat frequency as a function of time following reactivation. Two preparations of models (A,B) were isolated and reactivated (1.25 mM MgATP, 21°C) as described in the Materials and Methods. The preparations were prevented from drying during the experiments by perfusing excess reactivating solution at short intervals. In both preparations, the beat frequency of 25 coordinated models was measured over a course of 90 min. In each case, beat frequency remained constant for the first 30 rnin and declined by less than 10% during the next 60 min (0). The percentage of models propagating coordinated waves (%C,A) was constant for the first 30 min, but decreased rapidly within the next 60 min.

ciliary tuft also remained stable for the first 30 min following reactivation. However, in the next 60 min the percentage of coordinated models decreased by more than 50%. On the basis of these results, all experiments described below were completed within 30 min of reactivating any given preparation.

Motility and Coordination Thresholds of Reactivated Models

Within seconds of introducing the demembranating solution into a preparation of isolated cells, the tufts appeared to become uncoordinated just before motility finally ceased. This was investigated further by supplying increasing amounts of MgATP to freshly prepared, immotile models and observing their subsequent behavior in an effort to answer the question: does coordinated beating of cilia develop at the same MgATP level as that required to initiate motility?

As the MgATP concentration was increased, the motile behavior of the ciliary tufts changed continuously. We subdivided this continuous process into four distinct categories with respect to waveform and the degree of coordination. Each category constituted a stage through which a model passed as the MgATP supplied to it increased. The four categories are illustrated in Figure 4 and defined as follows:

Low amplitude (LA). Cilia were motile, but had an extremely reduced amplitude of beat. In many cases, only the distal ends of the cilia were motile. No coordination existed between cilia.


A

LOW AMPLITUDE

B

UNCOORDINATED

I PARTLY COORDINATED I I D 1

I COORDINATED I Fig. 4. Diagrammatic representation of the four stages through which models pass as MgATP concentration is increased from 2 pM to 35 pM. See text for details.

Uncoordinated (U). The amplitude of ciliary beat cycles was normal, but beat cycles within a model were not spatially or temporally related to one another. The tufts appeared “tangled.”

Partly coordinated (PC). Beat amplitude was normal, but the ciliary tuft exhibited only transient areas of synchrony. The coordination in such areas lasted for a few seconds, then was lost. Synchrony or metachrony never was seen over the entire tuft at any one time.

Coordinated (C). Beat amplitude was normal and the spatial and temporal relationships among the cilia were constant so that synchrony and metachrony were observed.

Freshly prepared models were treated with a series of MgATP concentrations (2-35 pM) and at each MgATP concentration the number of models in each descrip- tive category was recorded. The percentage of models in each category was calculated as follows:

Percent motility was defined as the number of models showing ciliary motion of any kind divided by the total number of undamaged models,

%M = LA + + pc + x 100, TOTAL

percent motility with full amplitude (normal) waveforms,

364 Weaver and Hard

+ pc + x 1 0 , TOTAL %MFA =

percent uncoordinated models,

x loo, U U + P C + C

%U =

percent partly coordinated models,

%PC = pc x loo, U + P C + C

and percent coordinated models,

%C = x loo U + P C + C

(3)

(4)

Figure 5 shows how the proportion of models exhibiting each behavior changed with increasing MgATP concentration. Motility first was observed at 2 pM MgATP (%M, Fig. 5A) and was characterized by very slow, extremely low-amplitude mo- tions, usually involving only the distal portions of the cilia. At 5 pM MgATP, almost 100% of the models showed ciliary motion, usually involving the whole length of the cilia. However, fewer than 50% had beat cycles of full amplitude at 5 pM. This percentage increased, until at 10 pM MgATP, the %MFA reached a plateau at a level of 90-95%. In the 5-10% of the models that never developed full-amplitude waveforms, ciliary movement consisted of rapid, low-amplitude fibrillations.

The degree of coordination also varied with MgATP concentration. At 4 pM MgATP, all of the MFA models were uncoordinated (Fig. 5B). As MgATP concentration was raised, %U dropped rapidly (Fig. 5B), whereas %PC increased steeply (Fig. 5C) and then subsequently declined. Fully coordinated beating was not observed until the MgATP concentration reached about 10 pM (Fig. 5D), and then %C rose steadily with increasing MgATP concentration to levels over 95 % . At ATP concentrations greater than 25 pM, little further change was observed in any of the categories.

It should be emphasized that at any single MgATP concentration between 5 pM and 25 pM, not all the models in the preparation showed the same behavior. Rather, at each MgATP concentration there was a mixture of models with different behavior types. As MgATP concentration increased, each model passed through the progres- sion of stages described, but there was variation among models in the MgATP concentrations at which specific transitions occurred.


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Fig. 5 . Motile behavior of reactivated models in the range 2-35 pM MgATP at 21°C. A. % M (8) and %MFA (0) as a function of MgATP concentration. Motility first was detected at 2 pM MgATP, and %M reached nearly 100% at 5 g M MgATP (%M = 2.5 pM). %MFA increased rapidly from 3 pM MgATP to a maximum (90-95%) at I0 pM (%MFA = 5-6 gM). B. % U (U) as a function of MgATP concentration. All of the full amplitude motile models were uncoordinated at 5 p M MgATP; %U dropped rapidly to less than 5% at 20 pM MgATP, and at 35 pM MgATP, % U was zero. C. %PC (x) rose to a plateau of 50-60% between 10 and 16.7 pM MgATP, then declined back to lzss than 5% at 25 gM. D. Fully coordinated models first appeared at 10 p M MgATP. %C (A) rose steadily to better than 95% at 35 p M MgATP

366 Weaver and Hard

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Fig. 6. Beat frequency of reactivated, coordinated models in the substrate range 12.5-35 pM MgATP. A. The beat frequencies of normal models increased as MgATP concentration was increased. The beat frequencies of high salt-extracted models (0) were essentially the same as those of unextracted models ( A ) . B. A double reciprocal plot of the data shown in (A) is linear over the substrate range examined. T = 21°C.

We defined the threshold for a particular behavior (eg, attainment of motility or coordination) as the MgATP concentration at which 50% of the models exhibited the behavior. Thus, the threshold for motility could be defined in two ways. If one considered only motility per se and a distinction was not made between ciliary motion of reduced and normal amplitude, the threshold of the models was 2-3 pM MgATP. When only models with full-amplitude waveforms (MFA models) were considered, the threshold was between 5 and 6 pM MgATP.

Similarly, a coordination threshold could be defined as the MgATP concentration at which 50% of the models were fully coordinated. For the newt lung models, this threshold was between 17 and 19 pM MgATP. Spatial and temporal coordination of the ciliary beat cycles developed more gradually in the population and at higher MgATP concentrations than those necessary to initiate motility. The development of coordination was characterized by a change in the proportions of U, PC and C models as MgATP concentration increased.

Beat frequency measurements were possible only with coordinated models. In uncoordinated models it was not possible to visually follow cilia through a series of beat cycles, so beat frequency could not be measured. In PC models the transient areas of synchrony did not persist long enough for beat frequency measurement. Figure 6 shows that the beat frequency of coordinated models increased as MgATP concentration was raised from 10 pM to 35 pM.

High Salt Extraction and Vanadate Treatment of Models

We next determined the effects of high salt extraction and vanadate treatment on the beat frequency and coordination of the newt lung ciliated cell models. Extrac- tion of the cell models with 0.46 M KCl selectively removed the outer dynein arms (Fig. 7). Of 50 randomly selected axonemes examined in two separate experiments (25 axonemes each), all were found to be missing their outer dynein arms, although 4-5 % of the axonemes still possessed some short fragments of electron-dense material that could possibly represent remnants of outer arm material. When reactivated with 1.25 mM MgATP, the beat frequency of high salt-extracted models was reduced to


Fig. 7. Typical cross sections of normal (A-C) and high salt-extracted (D-F) axonemes of demembranated ciliary tufts. Note the absence of outer dynein arms in the extracted axonemes in comparison to the untreated axonemes. x 125,000.

between 40% and 50% of unextracted controls (Fig. 8A). However, in the range of the coordination threshold (12.5 pM to 35 pM ATP), the beat frequency of high salt- extracted models was not reduced (Figs. 6, 8B). High salt extraction did not affect %M, %MFA, or %C either at 1.25 mM MgATP (Fig. 8A) or in the coordination threshold range (Figs. 8B, 9).

Varying concentrations of vanadate were added to models reactivated with 20 pM MgATP (near the coordination threshold). In the range of 0.1-1.25 pM vanadate, the beat frequency of coordinated models remained the same as that of untreated controls (Fig. 10). However, %M, %MFA, and %C were reduced by increasing vanadate, so that at 1.25 pM, %M was 20% lower than untreated models, %MFA was 68% lower, and %C was reduced by 77% (Fig. 10). When models reactivated with 1.25 mM MgATP were treated with 1.25 pM vanadate, the beat frequency was 40% lower than that of untreated models, %M remained the same, %MFA was 42% lower, and %C was 53% lower (Fig. 11A). The effects of vanadate treatment at this concentration were rapidly reversed by perfusion with vanadate-free reactivating solution (Fig. 11A). Cilia treated with vanadate concentrations > 2 pM first beat at reduced frequencies with low-amplitude waveforms and then rapidly were completely inhibited.

The effects of vanadate also were tested on high salt-extracted models. High salt-extracted models were reactivated first with vanadate-free reactivating solution. After beat frequency, %M, %MFA, and %C were recorded, 1.25 pM vanadate in reactivating solution was perfused through the preparation. The effects of vanadate were recorded and the models again were perfused with normal reactivating solution to remove the vanadate. As noted before (Fig. 8A), at 1.25 mM MgATP, the beat frequency of the KCI-extracted models was half that of unextracted models, while

368 Weaver and Hard

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I- z W 0 n w a

100

60

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25

15

- I"

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C HS C HS C HS C HS

X M X MFA %C BF

Fig. 8. Effect of high salt extraction on %M, %MFA, %C, and beat frequency, BF, of models following reactivation with 1.25 mM or 20 pM MgATP at 21°C. Demembranated models were extracted with 0.46 M KCI, then reactivated. Control models were reactivated immediately following demembranation. A. At 1.25 mM MgATP, %M, %MFA, and %C of high salt-extracted models, HS, did not differ significantly from those of controls (C). However, the beat frequency of high salt-extracted models was reduced to half that of unextracted models. B. At 20 pM MgATP, %M, %MFA, %C, and beat frequency did not differ significantly from those of unextrdcted controls.

100 -

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Fig. 9. The effect of high salt extraction of models on % C following reactivation with MgATP (10-35 pM) at 21°C; % C did not differ significantly between high salt-extracted models (0 ) and unextracted controls (A).

%M, %MFA, and %C were unaffected. Vanadate treatment reduced the beat frequency another 12%, and reduced both %MFA and %C by 46% (Fig. 11B). At 20 p M MgATP, the beat frequency of the KC1-extracted models was not reduced by treatment with 1.25 pM vanadate. However, %MFA was reduced by 45%, and %C by 48% (Fig. 1lC). All of these effects were reversible upon removal of the vanadate by perfusion with vanadate-free reactivating solution (Fig. llB,C).


I I II

I 1 I

0 0.25 0.75 1.25

VANADATE (OM)

Fig. 10. The effect of vanadate on %M, %MFA, %C, and beat frequency of models reactivated with 20 pM MgATP at 21 " C . Reactivating solutions containing 0.1-1.25 pM vanadate were perfused through freshly demembranated and reactivated preparations. At 20 pM MgATP beat frequency ( W ) was not affected by vanadate up to a concentration of 1.25 pM; %M (O) , %MFA (0). and %C ( A ) decreased with increasing vanadate concentration.

DISCUSSION

The newt lung ciliated models isolated by the methods described here had high motile capabilities. Their percent motility and percent coordination were well over 90%. At 1.25 mM MgATP, the beat frequencies measured for the reactivated models corresponded to those measured for newt lung ciliated cells in culture and populations of isolated, individual axonemes [Hard et al, 19851. Finally, the reactivated motility was quite stable with respect to beat frequency and percent coordination.

The three-dimensional relationships of the cilia in the models were not altered significantly upon demembranation, since both synchrony and metachrony were observed in reactivated models. Analyses of both ciliary waveforms and metachrony indicate no significant differences from those observed in situ [Weaver and Hard, 19851. These findings reconfirm previous findings that the cell membrane is not necessary for axonemal motility per se [Gibbons and Gibbons, 19721, nor for the generation and maintenance of coordination among cilia [Naitoh and Kaneko, 1973; Torres et a!, 1977; Dirksen and Zeira, 1981; Satir, 19781. The structural integrity of the newt lung demembranated ciliary tufts is maintained by interactions between accessory structures and cytoskeletal elements [Hard and Rieder, 19831.

The high motile performance of the newt lung cell models appears to be due largely to two factors. First, the procedures for isolating ciliated cells and their subsequent demembranation and reactivation were gentle enough to prevent physical damage to the models. Vigorous agitation, either by pipet or vortex mixer, and excessive centrifugation at any step in the procedure reduced the motile capabilities of the models. Therefore such treatments, which proved useful in some previous isolations from tissue cells [Torres et al, 1977; Dirksen and Ziera, 19811, specifically were avoided.

The second factor that contributed to the high motile capabilities and structural integrity of the newt lung models was the composition of the demembranating and

370 Weaver and Hard

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4

2

Fig. 11. A comparison of the effect and reversibility of 1.25 pM vanadate treatment on %M, %MFA, %C, and beat frequency of normal and high salt-extracted, reactivated models. Models were reactivated with vanadate-free reactivating solution and %M, %MFA, %C, and beat frequency, BF, were recorded (0). Following perfusion of the same models with the same reactivating solution containing 1.25 pM vanadate, %M, %MFA, %C, and beat frequency again were recorded (+). The vanadate then was removed by perfusion with fresh, vanadate-free reactivating solution, and %M, %MFA, %C, and beat frequency were recorded a third time with the same models (a). A. At 1.25 mM MgATP, 1.25 pM vanadate greatly reduced %MFA, %C, and beat frequency. These effects were completely reversible. B. At 1.25 mM MgATP, high salt-extracted models also showed marked reduction of %MFA, and %C upon treatment with 1.25 pM vanadate. Vanadate caused only a slight reduction of beat frequency from its initial level of half that of unextracted models. Removal of vanadate again reversed these effects. C. At 20 pM MgATP, vanadate treatment of high salt-extracted models greatly reduced %MFA and %C, while beat frequency was unaffected. These effects were reversed by removal of vanadate.

reactivating solutions. The conditions used here were those previously defined as optimal for motility in isolated, individual axonemes of Taricha [Hard et al, 19851. Deviations from these conditions resulted in decreased motile capabilities. In addition to using those conditions optimal for reactivation of isolated axonemes, it was necessary to control Ca2' concentrations. Failure to do so resulted in the spontaneous


detachment of cilia upon demembranation, a phenomenon observed previously in other studies [Anderson, 1974; Blum, 19711.

Motility and Coordination Thresholds

The patterns of ciliary coordination in intact cells have been well documented [reviewed in Machemer, 1974; Sleigh, 1974; Aiello and Sleigh, 1972; Sanderson and Sleigh, 19811 and coordinated waves in reactivated cell models have been observed previously [Naitoh and Kaneko, 1973; Torres et al, 1977; Satir, 1978; Dirksen and Zeira, 19811. To date, reactivated ciliated cell models have not been used directly to study coordination. The response of the newt lung ciliated cell models to low MgATP concentrations showed that the threshold for coordinated beating was different from and higher than that for motility per se.

Thresholds for motility in other ciliary systems range from 60 to 100 pM ATP for Necturus oviduct ciliated cell models [Eckert and Murakami, 19721 to 16 pM ATP for Mytilus gill ciliated cell models [Tsuchiya, 19771. Lower motility thresholds have been reported for reactivated flagellar models: either 4-12 pM for sea urchin sperm flagella [Gibbons and Gibbons, 19721 or 2 pM when potassium acetate constituted the major salt in the reactivating solution [Gibbons et al, 19821, and 10 pM for Chlamydomonas axonemes [Hyams and Borisy, 19781. This probably reflects the higher mechanochemical coupling of flagellar model systems in relation to previously isolated ciliary models. The motility threshold value of 2-5 pM for our ciliated cell models is indicative of their high mechanochemical coupling and motile capabilities.

To our knowledge, the concept of a coordination threshold has not been described previously. The substrate range over which our models became coordinated was broad, with a lower limit of 10 pM MgATP and an upper limit of 25 pM MgATP. We have not yet determined whether intact, living cells behave similarly at low beat frequencies. Future work using low temperature to reduce the beat of living cells should answer this question.

High Salt and Vanadate Experiments

High-resolution gel electrophoresis analyses of wild-type Chlamydomonas and mutants lacking various axonemal components have shown that the inner and outer dynein arms contain different polypeptides and have different ATPase activities [Huang et al, 1979; Pfister and Witman, 1984; Piperno and Luck, 1979, 1981; Mitchell and Rosenbaum, 19851. Conclusions from these studies on the functional roles of the inner and outer dynein arms on axonemal motility have been limited, as yet, because many of these mutants are nonmotile.

Demembranated, reactivated axonemes provide another potentially useful system for determining the function of these structures, since removal or inhibition of particular axonemal structures can be correlated with alterations in motility upon reactivation.

It has been shown that treatment of demembranated axonemes of various types with high salt (0.4-0.6M KC1 or NaC1) solubilizes either the outer arms selectively, as in sea urchin sperm [Gibbons and Gibbons, 1973, 1979; Hata et al, 1980; Ogawa et al, 1982; Yano and Miki-Nomura, 19811, or solubilizes both inner and outer dynein arms, as in Chlamydomonas [Pfister and Witman, 1984; Piperno and Luck, 1979, 19811. The beat frequency of reactivated, high salt-extracted sea urchin axonemes is reduced in proportion to the number of arms lost, reaching approximately 50% of

372 Weaver and Hard

controls when all outer arms are absent [Gibbons and Gibbons, 1973, 19791. Vana- date, a potent inhibitor of dynein Mg-ATPases, reduces the beat frequency of reactivated sea urchin sperm flagella to between 50% and 80% of control values, presumably by inhibiting outer arm dynein activity [Gibbons et al, 1978; Sale and Gibbons, 19791. The beat frequency reduction is a function of the vanadate concentration. Above a critical vanadate concentration, itself a function of MgATP concentration, motility is completely inhibited.

In the present study, high salt extraction selectivity removed outer dynein arms, at least as verified by electron microscopy. In this respect, the newt lung axonemes responded like Colobocentrotus axonemes and differed from those of Chlamydomo- nas. Both high salt extraction and vanadate treatment reduced the beat frequency of models reactivated with 1.25 mM MgATP. The beat frequency of high salt-extracted models was reduced by 50-60% and that of vanadate treated models by 40%. However, in the range of 10-35 pM MgATP (the coordination threshold range), beat frequency was not reduced by either treatment. These results suggest either that outer dynein arms are nonfunctional below 35 pM MgATP and do not contribute to measured beat frequencies or that beat frequency at these low MgATP concentrations is limited by factors other than MgATP binding and hydrolysis. Our data do not distinguish between these alternatives.

The high salt and vanadate treatments had different effects on ciliary coordination. Ciliary coordination was unaffected by high salt extraction. Both at 1.25 mM MgATP and near the coordination threshold (10-35 pM MgATP), %C was the same for unextracted and high salt-extracted models. It can be concluded that removal of outer dynein arms does not affect coordination among cilia. Furthermore, if other specific axonemal components or accessory structures are involved in coordination, they are resistant to high salt extraction.

On the other hand, ciliary coordination was markedly decreased by vanadate treatment, both at 1.25 mM and 20 pM MgATP. This was not due to outer arm inhibition, since vanadate treatment (1.25 pM) of high salt-extracted models caused the same %C decrease as that recorded for unextracted models at both MgATP concentrations. It is possible that vanadate affects the localization and/or timing of regions of doublet sliding. This could result in altered waveforms during the beat cycle [Brokaw et al, 19821. If these changes were random, so that there was no longer any consistent shape of ciliary bending waves during the beat cycle, spatial and temporal ordering of all the beat cycles in a tuft (ie, coordination) would no longer be possible.

Vanadate could accomplish this by several possible mechanisms, such as (1) altering the patterns of activity of the inner dynein arms, thus altering the localization and timing of the doublet sliding; ( 2 ) affecting radial spoke-central pair interactions which could modify the bends formed by dynein arm activity [Brokaw et al, 19821; and (3) affecting basal bodies or other accessory structures. Basal bodies may [Gordon et al, 19801 or may not [Anderson and Floyd, 19801 contain contractile proteins. Although ATPase activity different from dynein ATPase activity has been observed in basal bodies from some sources [Anderson, 19771, there is no definitive evidence yet for an active role of these organelles or other accessory structures in coordination. In fact, observations in Chlorogonium, if they can be generalized, argue strongly against such a role [Hoops and Witman, 19851. It may be that the function of basal bodies in coordination is limited to a passive, anchoring role or to developmentally orienting the direction of the effective strokes of fields of cilia with different types of


metachrony [Anderson, 1974; Dirksen and Satir, 1972; Hard and Rieder, 1983; Holley, 19841.

In the present study, we did not explore whether the vanadate-sensitive switching mechanism that bas been described for mussel gill cilia [Wais-Steider and Satir, 19791 exists in the newt lung system, nor was the site of vanadate-induced disruption of coordination determined, although several possible targets exist, given the multiple ATPases present in dynein arms [Gibbons et al, 1976; Gibbons and Fronk, 1979; Piperno and Luck, 1981; Tang et al, 1982; Pfister and Witman, 19841.

ACKNOWLEDGMENTS

We wish to 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 Robert Hard from the Medical Research Foundation of Oregon, NIH Biomedical Research Support Grant RR07079, and NIH grant HL29233. Support to A. Weaver from Sigma Xi and the Department of Zoology, Oregon State University, is gratefully acknowledged.

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Isolation of newt lung ciliated cell models: Characterization of motility and coordination...

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