THE EFFECT OF PARTIAL EXTRACTION OF DYNEIN ARMS ON THE MOVEMENT OF REACTIVATED SEA-URCHIN SPERM

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1 J. Cell Sd. 13, (i973) 337 Printed in Great Britain THE EFFECT OF PARTIAL EXTRACTION OF DYNEIN ARMS ON THE MOVEMENT OF REACTIVATED SEA-URCHIN SPERM BARBARA H. GIBBONS AND I. R. GIBBONS Pacific Biomedical Research Center, University of Hawaii, 41 Ahui Street, Honolulu, Hawaii 96813, U.S.A. SUMMARY Sea-urchin sperm were extracted with 0-5 M KC1 for 45 s at room temperature in the presence of Triton X-ioo, and then transferred to reactivating solution containing 1 mm ATP. The flagellar beat frequency of these KCl-extracted sperm (16 beats/s) was only about half that of control Triton-extracted sperm that had not been exposed to 0-5 M KC1 (31 beats/s), although the form of their bending waves was not significantly altered. Examination by electron microscopy showed that the extraction with 0-5 M KC1 removed the majority of the outer arms from the doublet tubules, leaving the inner arms apparently intact. By varying the duration of the KCl-extraction, it was shown that the rate of decrease in beat frequency paralleled the rate of disappearance of the arms. Prolonging the extraction time beyond 45 3 at room temperature, or 4 min at o C C, had little further effect on beat frequency. ATPase measurements suggested that % of th e dynein in the original axonemes had been solubilized when the extraction with KC1 was permitted to go to completion. These results indicate that the generation and propagation of flagellar bending waves of essentially typical form are not prevented by the removal of the outer row of dynein arms from the doublet tubules. In terms of the sliding filament model of flagellar bending, the results suggest that the rate of sliding between tubules under these conditions is proportional to the number of dynein arms present. The lack of significant change in wave form implies that the total amount of sliding that occurs during each bending cycle is not affected by the reduced number of dynein arms, but is regulated independently in some manner by the elastic forces generated by other structures in the bent axoneme. INTRODUCTION It has recently been demonstrated that ATP induces active sliding movements between adjacent doublet tubules in trypsin-treated axonemes of sea-urchin sperm flagella (Summers & Gibbons, 1971). Since the chemical specificity requirements for generating this sliding closely resemble those for reactivating normal bending waves in demembranated sperm that have not been exposed to trypsin (Gibbons, B. H. & Gibbons, 1972), it seems likely that the same ATP-induced shearing force is involved in both cases. These findings, together with the earlier report of Satir (1968) that the overall length of the tubules appears to remain constant during bending, strongly support a sliding-tubule model offlagellarmovement, in which the normal propagated bending waves are the result of localized sliding movements between tubules (Summers & Gibbons, 1971). Several theoretical models have been proposed to explain how such localized sliding might generate propagated bending waves (Brokaw, 1971, 22 C E L 13

2 H. Gibbons and I. R. Gibbons 1972; Rikmenspoel, 1971; Lubliner & Blum, 1972). However, although the experimental basis of the sliding-tubule model is now well established, there has been little direct evidence to indicate how ATP generates the sliding movements between tubules, and how these movements are coordinated to produce propagated bending waves. Several lines of indirect evidence have suggested that the arms on the doublet tubules may be involved in the transduction of chemical to mechanical energy. The ATPase protein, dynein, has been localized in the arms (Gibbons, I. R., 1965 a). The conditions necessary for dynein ATPase activity resemble those for the reactivation of motility in demembranated cilia andflagella (Brokaw, 1961; Gibbons, I. R., 1965A; Raff & Blum, 1969; Gibbons, B. H. & Gibbons, 1972; Gibbons, I. R. & Fronk, 1972; Ogawa & Mohri, 1972). Motility of demembranated sperm is accompanied by a rapid hydrolysis of the ATP in the medium, with about 70% of this ATPase activity being tightly coupled to motility (Brokaw & Benedict, 1968a, b; Gibbons, B. H. & Gibbons, 1972), and treatment of the sperm with agents that uncouple ATP hydrolysis from motility results in a substantial change in the enzymic properties of dynein (Brokaw & Benedict, 1971; Gibbons, I. R. & Fronk, 1972). The above evidence all tends to implicate the dynein arms in the mechanism of motility, but it is insufficient to define the nature of their role. In this paper we report the effects of partial removal of dynein arms on the beat frequency and waveform of reactivated sea-urchin sperm. Our results provide direct evidence for an active role of the dynein arms in generating the sliding movements between tubules. MATERIALS AND METHODS Sperm of the sea urchin Colobocentrotus atratus were collected by injecting 0-5 M KCI into the body of the animals. The undiluted semen with a covering layer of seawater was stored at 4 C. For use, semen was diluted with vol. of seawater to give a protein concentration of mg/ml, and stored at o C. All preparations of sperm were extracted with a solution containing Triton X-100 in order to remove their membranes, as described in detail below. The concentration of KCI in this extraction solution was made 0-5 M to obtain preparations of' KCl-extracted sperm' from which the dynein had been partially extracted, and was 0-15 M in control preparations where no extraction of the dynein was desired. The standard preparations of KCl-extracted sperm were made by adding a 50-fil sample of the diluted sperm suspension in seawater to o-6 ml of extracting solution containing 0-04 % (w/v) Triton X-100, 0-5 M KCI, 4 mm MgSO 4, 0-5 mm ethylenediaminetetra-acetate (EDTA) 1 mm dithiothreitol (DTT), and 2 mm tris-hydroxymethylaminomethane (Tris)-HCl buffer, ph 8-o, at room temperature, and gently mixing for The extraction was then terminated by diluting the sperm suspension into a relatively large volume of reactivating solution containing 0-15 M KCI, 2 mm MgSO 4, 0-5 mm EDTA, 1 mm DTT, 1 mm ATP, 2 % polyethylene glycol (mol. wt. =20000), 20 mm Tris-HCl buffer, ph 8-o, at room temperature. When required by particular experiments, the conditions of extraction were varied as described in the Results. On some occasions, the extraction was terminated by centrifuging the sperm suspension, and resuspending the pellet in reactivating solution. In control preparations, the composition of the extracting solution was the same as above except that the concentration of KCI was reduced to 0-15 M. Extraction was performed for about 30 s at room temperature. The conditions of extraction and reactivation are essentially the same as those described previously (Gibbons, B. H. & Gibbons, 1972).

3 Partial extraction of dynein arms 3 39 Sperm were observed at room temperature (23-26 C) by dark-field light microscopy in buffered reactivating solution, in glass Petri dishes, or in trough slides 1 mm deep. The beat frequencies of the sperm tails were measured with a stroboscopic flash unit (Chadwick- Helmuth Co., Monrovia, California). Most measurements were performed on sperm which were attached to the bottom of the dish by the tip of their heads. Measured values of beat frequency were corrected to a standard temperature of 25 C as described earlier (Gibbons, B. H. & Gibbons, 1972). Each value given represents the average for at least 15 sperm in a preparation. Michaelis constants for frequency were calculated by the weighted least squares procedure of Wilkinson (1961). Samples of sperm to be examined by electron microscopy were collected by centrifuging the suspension of sperm in reactivating solution at g for 5 min. The resultant pellets were fixed in a solution containing 0-15 M KCl, 2 mil MgSO 4, 10 mm phosphate buffer and 2 % glutaraldehyde, ph 7-8. After 1 h, the pellets were washed 3 times in the same buffer without glutaraldehyde, and then postfixed with 1 % osmium tetroxide. The pellets were dehydrated with acetone, embedded in Araldite epoxy resin, and thin-sectioned. Sections were stained first with uranyl acetate, and then with lead citrate. Electron micrographs were taken with a Philips EM 300 at 80 kv. An enzymic method was used to estimate what fraction of the dynein was removed from the axonemes by the standard KCl extraction. Previous work has shown that the enzymic properties of dynein change when it is extracted from the axonemes into solution. However, if the ATPase assays are performed in 0-05 M KCl at ph 8-o in the absence of Triton, the specific activity of dynein remains unchanged upon extraction, so that the relative ATPase activity in different fractions provides an approximate measure of the quantity of dynein present (Gibbons, I. R. & Fronk, 1972). Since this assay requires the absence of Triton, the standard KCl-extraction procedure was modified by first treating the sperm with a solution containing 004% Triton, 0-15 M KCl, 4 mm MgSO 4, 1 mm DTT, and 05 mm EDTA to remove the membranes, and then centrifuging at 800 g for 5 min. The pellet of demembranated sperm was suspended in modified KCl-extracting solution containing 0-5 M KCl, 2 mm MgSO 4, 1 mm DTT, 0-5 mm EDTA, 01 mm ATP and 2 mm Tris-HCl buffer, ph 80. After 1 min, the extraction was terminated by centrifuging at 3000 g for 5 min, and the resulting supernatant and pellet assayed for ATPase activity using an assay solution containing 005 M KCl, 2 mm MgSO 4, 1 mm ATP, 0-5 mm EDTA, and 100 fig oligomycin in the ph-stat at ph 80 (Gibbons, B. H. & Gibbons, 1972; Gibbons, I. R. & Fronk, 1972). Appropriate controls were performed by diluting a small sample of the KCl-extracted sperm into regular reactivating solution and measuring their beat frequency, and by fixing a pellet of the extracted sperm for electron microscopy, in order to ensure that the extraction had proceeded normally. Sources of chemicals and the preparation of seawater were the same as given in our earlier paper (Gibbons, B. H. & Gibbons, 1972). RESULTS Structure of control and KCl-extracted sperm flagella As reported earlier, treatment of the sperm with 0-04% Triton X-100 removes the flagellar membrane, leaving the resultant naked axoneme accessible to exogenous ATP. In the control sperm (Fig. 3), the structure of the axoneme appears essentially intact, and the 2 dynein arms are present on almost all of the outer doublet tubules. However, brief extraction of the sperm with 0-5 M KCl causes a characteristic change in the structure of the axoneme, with the outer dynein arms being removed from most of the doublet tubules, while the inner dynein arms remain apparently intact. This change in structure is seen most clearly when the KCl-extraction is terminated by centrifugation, for in this case the solubilized dynein has no opportunity to become rebound when the extracted sperm are suspended in reactivating solution prior to fixation. In such centrifuged preparations almost all the outer dynein arms appear

4 340 B. H. Gibbons and I. R. Gibbons to have been removed (Fig. 4). The KCl-extraction appears to cause no significant disruption or extraction of the other structures of the axoneme, such as the inner arms, the radial spokes, the central sheath, and the central and outer tubules. This effect of KCl-extraction on the axonemal structure is essentially the same as that reported previously by Gibbons, I. R. & Fronk (1972), although a much longer extraction time of 30 min was used in this earlier work. In preparations where the KCl-extraction is terminated by dilution, about 75 % of the solubilized dynein becomes rebound to the sperm when the salt concentration is lowered (see below). However, under our conditions, most of this dynein appears to be bound with little specificity. Electron micrographs of these preparations show (Fig. 5) an average of only about one normally positioned outer arm per axonemal cross-section. Most axonemes appear to possess, in addition, some outer arms of low density that point in an abnormal direction away from the axonemal cylinder and there is also irregular flurry material associated with some of the tubules. This fluffy material and the abnormally directed arms are not present in the control or the centrifuged preparations of sperm (Figs. 3, 4), and they presumably represent dynein that has rebound non-specifically or in an incorrect location. The results presented in the next section indicate that this non-specifically bound dynein is functionally inactive. Motility of KCl-extracted sperm The sperm that have been extracted with 0-5 M KC1 can be reactivated with ATP in a manner similar to that of the control sperm. After being transferred to reactivating solution containing 1 mm ATP, the KCl-extracted sperm immediately begin swimming through the medium, and soon accumulate at the bottom of the dish and at the meniscus where they mostly move in repeated circles. Many of the sperm become attached to the glass by the tip of their head, while their tail continues to beat, parallel and close to the glass surface. This general pattern of movement is similar to that of live sperm (Gray, 1955) and of control reactivated sperm (Gibbons, B. H. & Gibbons, 1972). Both the control and the KCl-extracted preparations show a high degree of uniformity, with % of the sperm becoming motile. The most immediately apparent and striking difference between the movement of the KCl-extracted and the control sperm is in beat frequency. The flagella of the KCl-extracted sperm beat at an average frequency of about 16 beats/s in reactivating solution containing 1 mm ATP at 25 C, while those of the control sperm beat at 31 beats/s under the same conditions. The reduction in beat frequency resulting from KCl-extraction is highly uniform, and the frequencies of individual sperm within a preparation show a scatter of only about ±10%, which is about the same as that in preparations of control sperm. The reduction is also highly reproducible from one preparation to another, and in 13 preparations the average frequency has ranged from 14-8 to 16-8 beats/s, while that of the corresponding preparations of control sperm ranged from 28 to 32 beats/s. This uniformity and reproducibility result from the fact that the beat frequency of the KCl-extracted sperm is not sensitive to small variations in the extraction conditions. For example, prolonging the duration of

5 Partial extraction of dynein arms 341 extraction from the usual 45 s up to 5 min did not cause any significant further decrease in frequency. In spite of the considerable difference in beat frequency between KCl-extracted and control sperm, there is remarkably little difference in the form of their bending waves (Figs. 6, 7). The slight differences between them do not exceed the magnitude of the differences between individual sperm in a preparation of a given type. This similarity in waveform is consistently observed so long as the duration of the KCl-extraction is kept brief (i.e. less than 1 min). After longer extraction times, however, the waveform of KCl-extracted sperm tends to deteriorate, with the bending waves dying away before they propagate the full length of the axoneme. After 2 min extraction, the waves usually die away about half way along the axoneme, and the distal portion appears stiff. A similar abnormal waveform has been observed previously in some preparations of reactivated sperm which have not been extracted with 0-5 M KCl, and it is believed to be due partly to oxidation of axonemal sulphydryl groups, since it develops more quickly when DTT is omitted from the reactivating solution (Gibbons, B. H. & Gibbons, 1972). Whether the apparent distal stiffness that develops during the longer KCl extraction is also due to oxidation is not known, but it was not prevented by using more highly purified KCl or by increasing the DTT concentration to 50 mm. Whatever may be the cause of this change in waveform, its effect can be avoided by using a suitably short extraction time, 45 s in our standard procedure. Although prolonged KCl extraction causes a pronounced decrease in wave amplitude toward the distal region of the axoneme, this change in waveform does not appear to result in a significant change in beat frequency. The frequency of sperm which have been extracted for 45 s and have normal waveform is essentially the same as the frequency of sperm extracted for 5 min which have quite abnormal waveforms. We have compared the beating of KCl-extracted sperm in which the extraction was terminated by centrifugation with that of sperm in which the extraction was terminated by dilution. When the durations of the extraction were matched as nearly a? possible, the waveforms of the 2 groups of sperm appeared identical, while the beat frequency of the centrifuged sperm averaged 14-8 beats/s as compared to 16-0 beats/s for the diluted sperm. This small difference in frequency (8%) was only marginally significant, indicating that only a minor fraction of the dynein that becomes rebound on dilution is functionally active. In our standard procedure, we chose to terminate the extraction with KCl by dilution because this permitted us to use shorter and more accurately defined extraction times. Effect of variation in KCl-extraction time upon beat frequency and axonemal structure We wished to make detailed studies of the changes in beat frequency and axonemal structure as a function of the time of extraction with 0-5 M KCl. For these experiments, we slowed the process down by extracting at o C because at room temperature the extraction process was largely complete within s > an^ it was not feasible to make reliable observations at shorter times. In order to compensate for the change in ph that results from the temperature coefficient of Tris-buffer, the ph of the

6 342 B. H. Gibbons and I. R. Gibbons Extraction time, min Fig. i. Effect of time of KCl-extraction at o C on the fiagellar beat frequency (O O), and on the number of arms remaining on the outer doublet tubules (0 %). For both curves, the values obtained with control reactivated sperm were taken as the zero extraction time points, i.e. 31 beats/s for frequency, and 18 arms. The data for other times were then expressed as percentages of these values. All frequencies were measured at room temperature and corrected to 25 C. Each point on the curve for number of arms remaining represents the average count for axonemal cross-sections, except the 4-min point which represents the average of 12 sections. extracting solution, measured at room temperature, was lowered from 8-o to 7-2. Control experiments showed that demembranation of the sperm flagella by Triton was complete within 30 s at o C. Fig. 1 gives the results of an experiment in which samples of sperm were extracted with 0-5 M KC1 at o C for times ranging from 25 s to 8 min. At the end of the appropriate extraction time, the sperm were diluted into reactivating solution at room temperature. A portion of the diluted suspension was examined by light microscopy for measurement of beat frequency; the remainder of the suspension was centrifuged, and the resultant pellet fixed immediately for electron microscopy. The observations show that at o C about 4 min extraction are required for the beat frequency to reach its lower level of about 16 beats/s, and that the frequency then remains approximately constant with longer extraction times up to 8 min. As before, the beat frequency was not affected by the gradual change in waveform at the distal ends of the flagella after prolonged extraction. It is notable that although the speed of extraction is considerably slower at o C than at room temperature, the final frequency of 16 beats/s is the same in both cases, with more prolonged extraction causing little further decrease in frequency. Examination of electron micrographs of these same preparations showed that

7 Partial extraction of dynein arms 343 1/ATP, mri Fig. 2. Double-reciprocal plots for the variation of beat frequency with ATP concentration for control reactivated sperm (#), and standard KCl-extracted sperm (O). Values for the effective 'Michaelis constant' and maximal frequency were calculated using the weighted least-squares procedure of Wilkinson (1961). there is a rapid decrease in the number of outer arms present on the doublet tubules as the KCl-extraction progresses (Fig. 8). In order to obtain unbiased counts of the number of outer arms remaining after different extraction times, the micrographs were all mixed together and identified by code numbers, before the arms were counted. An arm of normal position and density was counted as one; an arm of normal position but of lower density than normal was counted as half; arms that were misplaced or misdirected were not counted at all. Stubby arms of about half normal length were observed quite frequently in partially extracted axonemes, and these also were not counted. The results of these counts were tabulated, and plotted together with the frequency data derived from the same preparation of extracted sperm (Fig. 1). Within the limits of experimental error, the rate of disappearance of the arms parallels the rate of decrease in beat frequency. In both cases, the process is about half complete after 1 min, and essentially fully complete after 4 min, with no significant change occurring during the next 4 min. A repeat of this whole experiment with another batch of sperm gave essentially the same result. 20 Determination of the effective Michaelis constant for frequency, K lllf The beat frequency of reactivated sperm varies with the concentration of ATP in the medium, and double-reciprocal plots of beat frequency against ATP concentration are linear (Brokaw, 1967). The effective 'Michaelis constant' thus obtained for beat frequency has been denoted K mf, and is about 0-2 mm for reactivated sperm (Gibbons, B. H. & Gibbons, 1972). In the present work we have determined K m/ for standard KCl-extracted sperm (Fig. 2) and also repeated measurements of K inf for control reactivated sperm. For KCl-extracted sperm, the average values from 2

8 344 B- H. Gibbons and I. R. Gibbons separate preparations were o-i4mm + o-oi and 17-7 beats/s + o-3, for K m j and frequency max, respectively. For the control reactivated sperm, equivalent values were 0-21 mm + o-oi and 36-2 beats/s + o-6. Thus, at high ATP concentration the beat frequency of KCl-extracted sperm is half that of control sperm, while at low ATP concentration the KCl-extraction causes a relatively smaller decrease in frequency (Fig. 2). As the ATP concentration was decreased below o-i mm, the shape of the bending wave of the KCl-extracted sperm showed less resemblance to that of the control reactivated sperm than at the higher ATP concentrations. Under these conditions the speed of forward progression of the sperm head became less uniform than usual, with a marked forward surge at one particular stage of the beat cycle and little forward movement occurring during the rest of the cycle. Estimation of the fraction of dynein extracted by KCl The fraction of dynein solubilized by 0-5 M KCl was estimated by determining the relative ATPase activity of the supernatant and pellet obtained by centrifugation of the extracted sperm suspension. The supernatant of solubilized dynein was assayed directly in the ph-stat as described in Methods. The pellet of KCl-extracted sperm was resuspended in KCl-extraction solution, homogenized to prevent motility, and then assayed for ATPase activity by the same procedure. The results obtained from 2 of these experiments showed that % f the total ATPase activity was in the supernatant of the KCl-extraction, with the other % remaining associated with the pellet. A similar procedure has also been used to estimate how much of the ATPase activity that is solubilized by the KCl extraction becomes rebound to the sperm when the KCl concentration is lowered by dilution. In this case, the KCl extraction was terminated by diluting the suspension into a relatively large volume of ph-stat assay solution with a final KCl concentration of 0-05 M. After about 10 min, the suspension was centrifuged at g for 5 min, and the ATPase activity remaining in the supernatant was assayed. This ATPase activity was less than the supernatant activity when the KCl-extraction was terminated by centrifugation as described above, and the difference provided a measure of the amount of ATPase activity that was rebound to the axonemes upon dilution. The average results from 2 experiments indicated that about 75 % of the solubilized ATPase activity became rebound on dilution. To the extent that the amount of ATPase activity can be equated with the quantity of dynein present, these results indicate that % f the dynein is present in the outer arms and is solubilized by KCl extraction. The rebinding experiments indicate that about 75 % of this solubilized dynein becomes rebound to the sperm when the KCl extraction is terminated by dilution. However, the total number of enzyme units present did not remain constant upon extraction in these experiments as they did in the earlier experiments with isolated axonemes (Gibbons, I. R. & Fronk, 1972). For this reason, the above figures for dynein must be regarded only as approximate, and they require confirmation by other methods. (See note added in proof, p. 351.)

9 Partial extraction of dynein arms 345 Effects of changes in extraction conditions Our standard extraction conditions, using 0-5 M KC1 for 45 s at room temperature, are such as to mask 2 factors which appear to be necessary for a successful extraction. In our early experiments, we obtained occasional preparations of KCl-extracted sperm in which the flagellar bending waves were highly asymmetric. These sperm swam in circles of such narrow diameter as to make the measurement of beat frequency and observation of waveform impossible. This abnormal movement was traced to a requirement for a minimum level of 1 mm Ca 2+ during the KC1 extraction. For reasons that remain to be determined, the absence of a sufficient concentration of Ca 2+ during the extraction leads to formation of highly asymmetric bending waves when the sperm are subsequently reactivated. When the relative volumes of sperm in seawater and of KCl-extraction solution are as given above, an adequate concentration of Ca 2+ is provided by the seawater. Under other extraction conditions, it may be necessary to add additional CaCl a to the extraction solution to bring the Ca 2+ concentration up to 1 mm. The presence or absence of Ca 2+ in the extracting solution containing 0-15 M KC1 had no apparent effect on the waveform of the control reactivated sperm. A second factor which appears necessary for a good extraction is the presence of ATP in the extracting solution. This requirement was revealed in the experiments in which it was necessary to remove the Triton before the dynein was extracted. In these experiments, the sperm were first demembranated with Triton solution containing KC1, then centrifuged, and resuspended in extracting solution containing 0-5 M KC1 but no Triton. After extraction for 1 min at room temperature under such conditions we found that the reactivated sperm had frequencies of about 25 beats/s, about 80% that of the controls. Examination of electron micrographs showed that more than half of the outer arms were still present on the doublet tubules, indicating incomplete extraction of the dynein. We found that this problem could be cured by adding o-i mm ATP to the extraction solution. With this addition, the dynein extracted as readily as in the standard one-step procedure where the initial extracting solution contains both Triton and 0-5 M KC1. The requirement is not apparent in this standard procedure because the ATP contained within the live sperm contributes a concentration of about 0-05 M ATP to the extracting solution (Gibbons, B. H. & Gibbons, 1972), and this is apparently sufficient to facilitate the extraction. In addition to investigating the effects of Ca 2+ and of ATP, we have performed a limited number of experiments to determine the effects of other changes in extraction conditions. There is a fairly sharply defined minimum effective concentration for the KC1 in the extraction solution. If this concentration is lowered from the usual 0-5 M to 0-4 M, then little extraction of dynein occurs within a i-min extraction period, and the frequency of the reactivated sperm is only slightly lower than that of the controls. On the other hand, increasing the KC1 within the range M does not result in any significant increase in the amount of dynein extracted, and the frequency remains

10 346 B. H. Gibbons and I. R. Gibbons constant at about half the value of control sperm. The fact that increasing the KC1 concentration above 0-5 M, and prolonging the duration of extraction in 0-5 M KC1 beyond 45 s at room temperature, have no further effect on beat frequency or on the number of dynein arms removed indicates that the inner and outer dynein arms differ considerably in their extractability, with the inner arms being almost completely resistant to solubilization by KC1 under these conditions. The use of higher concentrations of KC1 than o-6 M proved impractical, because they caused extraction of DNA from the sperm heads. Tests of the extraction efficiency of other salts indicated that NaCl behaved essentially identically to KC1. The minimum effective concentration was about 0-5 M, and extraction for 45 s at room temperature reduced the frequency by half without changing the form of the bending waves. The minimum effective concentration of LiCl was also about 0-5 M, but slight modification of the extraction procedure was required to get optimum results in this case. The proportion of seawater to extracting solution had to be increased, so that o-i ml of sperm suspension in seawater was added to o-6 ml of extracting solution, and the time of extraction had to be limited to 30 s at room temperature. Under these conditions, the frequency was halved and the waveform remained normal. If the extraction was prolonged to 2 min, the sperm were only 50% motile and had an average frequency of about 10 beats/s when initially diluted into reactivating solution, but the percentage of motility increased up to nearly 100% within, a few minutes. The third salt tested was Na 2 SO 4, but it appeared to give inferior results; a concentration of only about 0-35 M was necessary to reduce the beat frequency of the sperm, but even this concentration caused some extraction of DNA from the sperm heads. We have made some preliminary experiments using more vigorous extracting conditions in an attempt to solubilize the inner dynein arms and obtain frequencies less than half those of the control sperm. As mentioned above, we obtained frequencies of about 10 beats/s, 30% that of the controls, after extracting with 0-5 M LiCl for 2 min at room temperature (24 C). Approximately the same results were obtained by extracting with 0-5 M LiCl for 30 s at 33 C, and by extracting with 0-5 M NaCl, ph 9-5 for 1 min at 24 C. However, in all cases, the frequencies were less uniform than normal, and many sperm showed abnormal waveforms. More detailed experiments will be needed to determine whether this further decrease in frequency results from partial extraction of the inner dynein arms. Dynein arms as cross-bridges between doublet tubules In electron micrographs of flagella and cilia, the dynein arms appear as a double row of projections extending from the A-tubule of each outer doublet toward the B-tubule of the adjacent doublet. In most previously published micrographs, the arms do not usually appear to bridge completely the space between tubules, and there is a gap 5-7 nm wide between the end of an arm and the B-tubule of the next doublet (Gibbons, I. R. & Grimstone, i960; Allen, 1968; Warner, 1970). Only in a few cases have the arms been observed to form a complete cross-bridge between tubules, and this bridge was present at only one particular position, between tubules numbers

11 Partial extraction of dynein arms and 6 (Afzelius, 1959; Gibbons, I. R., 1961; the system of numbering the tubules is given in these references). During the present studies, we have incidentally observed that in many of our preparations the axonemes show a substantial number of dynein arms forming complete cross-bridges between tubules. In a typical preparation (Fig. 9), counts of 12 randomly selected sections showed an average of 4-5 cross-bridges per axonemal cross-section. The occurrence of the bridges was not equally frequent at all positions, but most commonly there would be 2 groups of tubules bridged together, one group consisting of about 4 tubules around positions 4, 5, 6, 7, and the other group of about 3 tubules around positions 1, 2, 3. However, this pattern was not invariant, and 1 or 3 groups of bridged tubules were observed in some sections. The most common position for a bridge was between tubules 5 and 6, where it was present in almost all sections. The least common positions were between tubules 3 and 4 and between tubules 7, 8, and 9. The average spacing between tubules appears to be rather less when a bridge is present than when it is not, suggesting that formation of a crossbridge is associated with a moving together of the affected doublet tubules. However, the measurements show appreciable scatter, possibly as a result of the distortions introduced during sectioning of the specimen for microscopy, and the possibility of some change in the length of the dynein arms cannot be excluded. It must be noted that the average number of cross-bridges varied significantly from one preparation of sperm to another. In occasional preparations, such as the one illustrated in Fig. 10, there was only an average of about o-6 cross-bridge per axonemal cross-section. We have not yet been able to determine whether this variation results from differences in physiological state of the sperm at the time of fixation, or from small inadvertent changes in the technique of fixation. Until the factors causing this variability are resolved, any interpretation of the significance of the cross-bridges must be made with considerable caution. Nevertheless, we believe that these preliminary results constitute evidence that the dynein arms do form crossbridges between tubules at some stage of their cyclic activity, and that, in favourable conditions, these cross-bridges can be preserved by fixation for examination by electron microscopy. DISCUSSION Our results confirm the previous report (Gibbons, I. R. & Fronk, 1972) that the dynein from axonemes of sea-urchin sperm consists of 2 distinct fractions which differ substantially in extractability, the more readily extracted fraction being located in the outer arm3 on the doublet tubules, and the less readily extracted fraction presumably being located in the inner arms. When the extraction of the axonemes with 0-5 M KC1 is permitted to go to completion, essentially all the outer arms are removed from the doublet tubules, while the inner arms and all the other axonemal structures remain apparently intact. Preliminary enzymic measurements suggest that about 60 % of the dynein is extracted from the axonemes under these conditions, although this value needs to be confirmed by other more direct methods. The

12 348 B. H. Gibbons and I. R. Gibbons relationship of these two solubility fractions of dynein to those obtained by dialysis of scallop gill cilia (Linck, 1970), and to the 14s and 30s dynein fractions obtained from Tetrahymena cilia (Gibbons, I. R., 1966), remains to be determined. The most striking and important of our new observations is that the KCl-extracted sperm, with their outer dynein arms removed, can be reactivated with 1 mm ATP to produce bending waves whose shape is essentially the same as that of control demembranated sperm, but whose frequency is only half that of the controls. When the KC1 extraction is terminated before completion, the decrease in beat frequency is approximately proportional to the fraction of total number of arms that has been removed. After complete extraction, both the beat frequency and the number of arms are reduced to about half of their original values. Prolonging the extraction time beyond 45 s at room temperature, or 4 min at o C, has little further effect on the beat frequency or on the number of arms remaining. This parallel relationship of beat frequency and number of dynein arms, together with the fact that no other structural component of the axoneme appears to be affected by the KC1 extraction, and that dynein appears to be the only protein present in significant quantity in electrophoresis gels of the fraction solubilized by KC1 under these conditions (H. L. Kincaid, B. H. Gibbons & I. R. Gibbons, unpublished results), strongly suggests that the decrease in beat frequency is a direct result of the reduced number of dynein arms on the tubules. When interpreted in terms of the slidingtubule model of motility, it implies that the rate of sliding between tubules, under these conditions, is proportional to the number of functional dynein arms present. The inner and outer dynein arms have been shown to differ in fine structure (Allen, 1968), and as we have seen they differ substantially in their extractability, but in spite of these differences, our results suggest that the inner and outer arms are functionally equivalent in their role of inducing sliding between tubules. Since bending waves of normal form can be produced when only the inner arms are present, it is probable that any polarity which the arms may possess with respect to the proximal-distal axis of the tubules lies in the same direction for both inner and outer arms. The formation and propagation of planar bending waves implies that some mechanism exists to control and coordinate the amount of sliding that occurs between particular doublet tubules at different stages of the bending cycle. The geometrical relationships are such that, if the tubules are inextensible, the amount of sliding between tubules in a bent region of the axoneme is proportional to the angle of the bend, and is independent of the curvature. The bend angles usually observed are about radians, and bends of tiiis magnitude require a sliding of about nm between tubules on opposite sides of the axoneme, with at least nm between adjacent doublets. The production of bending waves of normal form when the outer arms have been removed indicates that the total amount of sliding between tubules that occurs during each cycle of bending is not affected by the removal of these arms, or by the reduced speed of sliding. This finding suggests that the mechanism regulating the amount of sliding involves elastic forces resulting from the strain of some axonemal component other than the dynein arms, and also suggests

13 Partial extraction of dynein arms 349 that the mechanism is relatively independent of forces resulting from the internal and external viscous resistances. The latter conclusion is further supported by the fact that the waveform undergoes little change when the beat frequency is changed 10-fold by varying the ATP concentration (Gibbons, B. H. & Gibbons, 1972). The details of the mechanism regulating the sliding between tubules are not yet known, but since the plane of the bending waves is correlated with and perpendicular to the plane of the 2 central tubules (Gibbons, I. R., 1961; Tamm & Horridge, 1970), it seems possible that the central tubules and sheath, together with the spokes that connect them to the doublet tubules, are involved in the coordination process. The proportionality between beat frequency and number of arms is observed only at high ATP concentrations, such as the 1 mm used in our standard reactivating solution. At low concentrations of ATP, the decrease in the number of dynein arms has relatively less effect on the frequency (Fig. 2). It is not known whether the diminished effect of the decreased number of arms results from the fact that the amount of viscous mechanical work performed by the flagellum per cycle of bending decreases as the beat frequency falls at low ATP concentrations, or whether it results from the changed waveform under these conditions. The former hypothesis would be analogous to the situation in a muscle sarcomere, where the number of ATP molecules hydrolysed for a given amount of shortening decreases as the load on the muscle is decreased (Kushmerick & Davies, 1969). When the KC1 extractions were terminated by dilution, about 75 % of the extracted dynein became rebound to the axonemes. However, very little of this rebound dynein regained its functional activity, for the beat frequency of sperm in which the extraction was terminated by dilution was only about 8 % greater than when the extraction was terminated by centrifugation to remove the solubilized dynein. This slight difference in beat frequency is consistent with the fact that the diluted sperm contained an average of about one normally positioned outer arm per axonemal crosssection, while the centrifuged sperm contained almost none. The greater part of the dynein which rebinds upon dilution presumably does so in a relatively nonspecific manner, and it probably accounts for the misplaced arms and fluffy material visible around the axonemes in these preparations. It is also possible that some dynein becomes bound to the sperm heads. The overall percentage of dynein that becomes rebound is similar to that reported previously for sea-urchin sperm by Ogawa & Mohri (1972) and by Hayashi & Higashi-Fujime (1972), but these workers did not examine the functional activity of the rebound dynein. The rebinding of dynein to flagella of sea-urchin sperm appears to occur with less specificity than it does to cilia of Tetrahymena, where about 60% of the arms are restored to their original positions, and functional activity is substantially regained (Gibbons, I. R., 1965 a, c). The lower specificity of rebinding to sea-urchin flagella may result from the fact that this dynein is solubilized wholly in the 14s monomeric form (Gibbons, I. R., 1965a; Gibbons, I. R. & Fronk, 1972), while it is the 30s polymeric form that binds best in Tetrahymena cilia (Gibbons, I. R., 1965 a). However, the possibility that greater specificity of rebinding to sea-urchin flagella could be achieved by varying the experimental conditions merits further study.

14 350 B. H. Gibbons and I. R. Gibbons Quite apart from these detailed considerations, our general finding of a relationship between the removal of dynein arms and the decrease in beat frequency provides direct evidence for a functional role of the arms in the mechanism of motility. Taken together with the previous evidence for the similarity of the conditions necessary for enzymic activity of dynein to the conditions necessary for reactivating motility in demembranated cilia and flagella (Brokaw, 1961; Gibbons, I. R., 19656, 1966; Raff & Blum, 1969; Gibbons, B. H. & Gibbons, 1972; Gibbons, I. R. & Fronk, 1972; Ogawa & Mohri, 1972) and for inducing sliding between tubules in trypsintreated flagella (Summers & Gibbons, 1971), it strongly suggests that the dynein arms are responsible for generating the shearing force that leads to sliding between tubules. Such a role for the dynein arms implies that the arms on one doublet are capable of binding chemically to specific sites on the B-tubule of the adjacent doublet, and thus forming cross-bridges between the 2 doublets. Our electronmicroscopic evidence indicates that it is possible, under favourable circumstances, to preserve these cross-bridges for examination by electron microscopy. Further study of the factors controlling and coordinating the formation of these crossbridges may provide information concerning the mechanism by which the sliding between tubules is coordinated to produce and propagate the bending waves characteristic of normal motility. We are grateful to Mrs Lina Guillory for her skilful help with the electron microscopy and to Dr Charles Lindemann for his comments on the manuscript. This work has been supported in part by NIH grants GM and HD REFERENCES AFZELIUS, B. (1959). Electron microscopy of the sperm tail. Results obtained with a new fixative. J. biophys. biochem. Cytol. 5, ALLEN, R. D. (1968). A reinvestigation of cross-sections of cilia. J. Cell Biol. 37, BROKAW, C. J. (1961). Movement and nucleoside polyphosphatase activity of isolated flagella from Polytoma uvella. Expl Cell Res. 22, BROKAW, C. J. (1967). Adenosine triphosphate usage by flagella. Science, N.Y. 156, BROKAW, C. J. (1971). Bend propagation by a sliding filament model for flagella. J. exp. Biol. 55, BROKAW, C. J. (1972). Flagellar movement: a sliding filament model. Science, N.Y. 178, BROKAW, C. J. & BENEDICT, B. (1968a). Mechanochemical coupling in flagella, I. Movementdependent dephosphorylation of ATP by glycerinated spermatozoa. Archs Biochem. Biophys. 1*5, 77O-778. BROKAW, C. J. & BENEDICT, B. (19686). Mechanochemical coupling in flagella, II. Effects of viscosity and thiourea on metabolism and motility of Ciona spermatozoa. J. gen. Physiol. 52, BROKAW, C. J. & BENEDICT, B. (1971). Mechanochemical coupling in flagella, III. Effects of some uncoupling agents on properties of the flagellar ATPase. Archs Biochem. Biophys. \\i, GIBBONS, B. H. & GIBBONS, I. R. (1972). Flagellar movement and adenosine triphosphatase activity in sea urchin sperm extracted with Triton X-100. J. Cell Biol. 54, GIBBONS, I. R. (1961). The relationship between the fine structure and direction of beat in gill cilia of a lamellibranch mollusc. J. biophys. biochem. Cytol. 11, GIBBONS, I. R. (1965 a). Chemical dissection of cilia. Archs Biol., Liege 76, GIBBONS, I. R. (19656). Reactivation of glycerinated cilia from Tetrahymena pyriformis. J. Cell Biol. 25,

15 Partial extraction of dynein arms 351 GIBBONS, I. R. (1965 c). An effect of ATP on light scattered by suspensions of cilia. J. Cell Biol. 26, GIBBONS, I. R. (1966). Studies on the adenosine triphosphatase activity of 14S and 30S dynein from cilia of Tetrahymena. J. biol. Chem. 241, GIBBONS, I. R. & FRONK, E. (1972). Some properties of bound and soluble dynein from sea urchin sperm flagella. J. Cell Biol. 54, GIBBONS, I. R. & GRIMSTONE, A. V. (i960). On flagellar structure in certain flagellates. J. biophys. biochem. Cytol. 7, GRAY, J. (1955). The movement of sea-urchin spermatozoa. J. exp. Biol. 32, HAYASHI, M. & HIGASHI-FUJIME, S. (1972). Binding and adenosine triphosphatase of flagellar proteins from sea urchin sperm. Biochemistry, N.Y. 11, KUSHMERICK, M. J. & DAVIES, R. E. (1969). The chemical energetics of muscle contraction. II. The chemistry, efficiency, and power of maximally working sartorius muscle. Proc. R. Soc. B 174, LINCK, R. W. (1970). A biochemical comparison of ciliary and flagellar axonemes from the bay scallop, Aequipecten irradians. Biol. Bull. mar. biol. Lab., Woods Hole 139, 429 (abstr.). LUBLINER, J. & BLUM, J. J. (1972). Analysis of form and speed of flagellar waves according to a sliding filament model. J. Mechanochem. Cell Motility 1, OGAWA, K. & MOHRI, H. (1972). Studies on flagellar ATPase from sea urchin spermatozoa. I. Purification and some properties of the enzyme. Biochim. biophys. Ada 256, RAFF, E. C. & BLUM, J. J. (1969). The fractionation of glycerinated cilia by adenosine triphosphate. J. biol. Chem. 244, RIKMENSPOEL, R. (1971). Contractile mechanisms in flagella. Biophys. J. 11, SATIR, R. (1968). Studies on cilia. III. Further studies on the cilium tip and a 'sliding filament' model of ciliary motility. J. Cell Biol. 39, SUMMERS, K. E. & GIBBONS, I. R. (1971). Adenosine triphosphate-induced sliding of tubules in trypsin-treated flagella of sea-urchin sperm. Proc. natn. Acad. Sci. U.S.A. 68, TAMM, S. L. & HORRIDGE, G. A. (1970). The relation between the orientation of the central fibrils and the direction of beat in cilia of Opalina. Proc. R. Soc. B 175, WARNER, F. (1970). New observations on flagellar fine structure. The relationship between matrix structure and the microtubule component of the axoneme. J. Cell Biol. 47, WILKINSON, G. N. (1961). Statistical estimations in enzyme kinetics. Biochem. J. 80, NOTE ADDED IN PROOF {Received 3 January 1973) Recent quantitative analysis by gel electrophoresis has shown that extraction of isolated flagellar axonemes with 0-5 M salt solubilizes about hah 0 of the A-electrophoretic component of the dynein, while the other half of the A-component and all of the B-component remain bound to the extracted axonemes. Thus, the fraction of dynein solubilized does not appear simply to parallel the solubilization of ATPase activity (KINCAID, H. L., GIBBONS, B. H. & GIBBONS, I. R. (1973). The salt-extractable fraction of dynein from sea-urchin sperm flagella: an analysis by gel electrophoresis and by ATPase activity. J. Supramol. Struct, (in Press).)

16 B, H. Gibbons and I. R. Gibbons Fig. 3. Electron micrograph of control reactivated sperm. This field was selected to show cross-sections of flagellar axonemes, and it does not happen to contain any of the sperm heads in the preparation, x

17 Partial extraction of dynein arms 353 Fig. 4. Electron micrograph of KCl-extracted sperm from a preparation in which the extraction was terminated by centrifugation. x Fig. 5. Electron micrograph of KCl-extracted sperm from a preparation in which the extraction was terminated by dilution. The distorted axoneme near the centre of the field probably represents a developmental abnormality, x c EL 13

18 354 B. H. Gibbons and I. R. Gibbons Fig. 6. Dark-field light micrographs of a control reactivated sperm in buffered reactivating solution. The micrographs, which are all of the same sperm, have been arranged into a series to illustrate the form of the bending waves as they propagate. The sperm was circling at the underside of the coverglass with its tail beating parallel and close to the glass surface. Flash exposure; magnification x iooo. Beat frequency was 31/s. Fig. 7. Same as Fig. 6, but showing a KCl-extracted sperm. Beat frequency was 16/s.

19 Partial extraction of iyfiein arms

20 356 B. H. Gibbons and I. R. Gibbons Fig. 8. Typical examples of the cross-sections of sperm axonemes which were used for counting the number of arms remaining after extraction with 0-5 M KC1 at o C. The top row show control reactivated sperm, which represent zero extraction time. The sections shown in the middle row are from sperm which have been extracted with KC1 for 1 min, and those in the bottom row have been extracted for 8 min. x Fig. 9. Electron micrographs showing cross-sections of 2 flagella in a preparation of control reactivated sperm The demembranated sperm were suspended in reactivating solution containing 1 mm ATP, and centrifuged to form a pellet. Fixation was accomplished by layering 2 % glutaraldehyde solution over the pellet (see Methods), x Fig. 10. Same as Fig. 9, but a different preparation of sperm. The demembranated sperm were centrifuged into a pellet from Triton-extraction solution, x

21 Partial extraction of dynein arms \ 8 10