Functional recombination of outer dynein arms with outer arm-missing flagellar axonemes of a Chlamydomonas mutant

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1 Functional recombination of outer dynein arms with outer arm-missing flagellar axonemes of a Chlamydomonas mutant HITOSHI SAKAKIBARA and RITSU KAMIYA Department of Molecular Biology, Faculty of Science, Nagoya University, Nagoya , Japan Summary A flagellar mutant of Chlamydomonas,, lacks the entire outer dynein arm but can swim at a speed of one third to half of that of the wild type. We found that the addition of a high-salt extract of wild-type axonemes to demembranated cell models restored up to 83% of the outer arms normally present on the outer-doublet microtubules of wild-type axonemes. Furthermore, when reactivated in the presence of ATP after being mixed with the extract, the cell models gained a higher level of motility, close to that of the wild type. The increase in flagellar beat frequency parallelled the increase in the number of restored outer dynein arms. These observations indicate that the axoneme of the mutant retains the binding sites for the outer dynein arms, and that the outer arms solubilized with high salt are functionally active. This in vitro recombination system with the mutant should be useful as an assay system for various preparations of outer-arm dynein. Evidence is presented that the two axonemes on an cell model beat at the same frequency, whereas those on a wild-type model beat at different frequencies. The two axonemes beat at the same frequency even after the higher level of motility has been restored by addition of crude dynein extract. We propose that a heterogeneity in the outer dynein arms is responsible for the frequency imbalance between the two flagella of wild-type Chlamydomonas. Key words: cilia, flagella, dynein, Chlamydomonas, cell model. Introduction The inner and outer dynein arms in cilia and flagella are the key moleculear assemblies responsible for force production. Both types of arm are capable of causing sliding movement between adjacent outer-doublet microtubules through a mechanochemical process accompanying ATP hydrolysis (Gibbons, 1981; Summers & Gibbons, 1971; Yano & Miki-Noumura, 1981; Okagaki & Kamiya, 1986; Paschal et al. 1987; Vale & Yano- Toyoshima, 1988). However, the mechanism that converts microtubule sliding into the cyclical bending movement of axonemes has not been established. Nor is it known whether the inner and outer dynein arms contribute to this mechanism. Gibbons & Gibbons (1973) showed that outer dynein arms were selectively solubilized from demembranated sea-urchin sperm flagella by extraction with a high-salt solution, and that, upon addition of MgATP, the extracted axonemes beat at a frequency of about half of that of the unextracted control axonemes. Furthermore, they showed that the solubilized outer dynein arms can rebind to extracted axonemes and restore the normal beat frequency (Gibbons & Gibbons, 1976, 1979). These results not only provided evidence that outer dynein arms Journal of Cell Science 92, (1989) Printed in Great Britain The Company of Biologists Limited 1989 are directly involved in the flagellar beating mechanism, but also demonstrated that flagella can beat with the inner dynein arms only. These results have been taken to imply that the inner and outer arms are functionally similar. However, recent studies have shown that the two types of arms differ greatly in protein composition (Huang et al. 1979; Sale et al. 1988), morphology (Goodenough & Heuser, 1984, 1985), and possibly in function: waveform analyses of Chlamydomonas axonemes have indicated that mutations in outer arms result in decrease in flagellar beat frequency, whereas those in inner arms result in a reduction in the shear amplitude (Brokaw & Kamiya, 1987). Therefore, the function specific to each type of dynein arm must be reassessed. Chlamydomonas is very important in the study of dynein structure and function, because it is the only known organism that offers several types of mutants with different degrees of difficiency in the inner and outer dynein arms (Huang et al. 1979; Kamiya & Okamoto, 1985; Mitchell & Rosenbaum, 1985; Brokaw & Kamiya, 1987; Kamiya et al. 1988). While pursuing attempts to isolate new types of dynein mutants, we thought that our study on dynein arm function would be greatly facilitated by the development of an in vitro system to recombine isolated dynein arms with mutant axonemes lacking

2 them; if this were possible, combinations of dynein arms and axonemes from different mutants, or those of artificially modified dynein and extracted axonemes, should give rise to a wide variety of axonemes with different dynein subunit compositions. As the first step, we attempted to recombine functionally the outer dynein arms to the axoneme of a mutant lacking them (odd), as Gibbons & Gibbons (1976) did with sea-urchin sperm axonemes. In this report, we show that addition of crude extract of wild-type axonemes to detergent-extracted 'cell models' can restore the outer arms and that those arms can restore the normal motility. Materials and methods Strains Chlamydomonas reinhardtii 137c mt + (wild type) and an outer dynein arm-deficient mutant l (, previously called 3S; Kamiya & Okamoto, 1985) were used. Crude dynein extract To prepare crude dynein extract, wild-type cells were grown on 1*5 % agar plates containing Tris-acetic acid-phosphate (TAP) medium (Gorman & Levine, 1965) under continuous illumination. Four plastic dishes of 800cm 2 were used. After one week of culture, cells on the plates were scraped off and suspended in 200 ml of liquid TAP medium. The cell suspension was kept aerated for 1 h to induce formation of flagellated cells. Flagellar axonemes and the high-salt extract of axonemes containing dynein were obtained by the methods of Witman et al. (1978) and Haimo (1982), respectively, with minor modifications. Briefly, flagella were detached from the cell body by addition of dibucaine-hc1, purified through a differential centrifugation series, and demembranated with HMDEK solution (lomm-hepes, 5 mm-mgso^, 1 mm-dithiothreitol, 1 mm-egta, 50mM-potassium acetate, ph74) containing 0-1% Nonidet P40 (NP40). For dynein extraction, the axonemes were precipitated by centrifugation, suspended in 0-4 ml of HMDEK containing 0-6M-KC1, left at 0 C for 15 mm, and then precipitated again. The supernatant fraction was desalted by centrifugation through Sephadex-G25 (Neal & Florini, 1973) swollen with HMDEK. This sample was used as the crude dynein extract in the following experiments. About 0*5 mg of sample was obtained from an ordinary culture of four plastic plates. Cell models Detergent-extracted cell models of wild-type and mutant strains were obtained by a method modified from that of Kamiya & Witman (1984). Cells were cultured in liquid TAP medium with aeration on a 16 h light/8 h dark cycle. When cell density reached 2X10 6 to 4xl0 6 mr 1, about 10ml of the culture was centrifuged at 1500 # for 4min at room temperature. The cells were washed in a solution containing 1 mm-egta and 4 % sucrose, centrifuged at 1500# for 4min, and suspended in 0-1 ml of ice-cold HMDEKP (HMDEK plus 05 % polyethylene glycol; M T ) containing 0-5 % NP40 to effect demembranation. This suspension of cell models can be stored for up to 5 h without noticeable change in their activity to undergo reactivation. Dynein recombination and reactivation Demembranated cells (cell models) were left to stand in various concentrations of crude dynein extract in HMDEK at a cell density of about 2xl0 8 ml~ at 0 C for 1 h. The sample was then mixed with a large volume (>10 times the volume of the cell model suspension) of reactivation medium (HMDEKP containing ATP) to induce flagellar movement. The final concentration of ATP was 1 mm. When the effect of Ca 2+ was examined, one of the Ca z+ -buffer solutions reported by Bessen et al. (1980) was used. The reactivated cell models were observed at 25 C with a dark-field microscope. Beat frequency measurement Flagellar beat frequency in reactivated cell models was measured by the method of Kamiya & Hasegawa (1987) using a fast Fourier transform (FFT) analyser. Briefly, dark-field microscope images of reactivated cell models were projected on a linearly graded filter placed on the film-plane of a microscope camera (Smyth & Berg, 1982). Because swimming Chlamydomonas cells move back and forth at the flagellar beat frequency, the intensity fluctuation of the light transmitted through this filter contains a high-frequency component (20-80 Hz) corresponding to the beat frequency. The frequency of this component was measured with a photomultiplier (R928, Hamamatsu Photonics Co., Hamamatsu, Japan) and an FFT analyser (model AD3522; A and D Co., Tokyo, Japan). Other methods The swimming velocity of the cell models was measured by using a dark-field microscope with a total magnification of X 100, a video recording system, and a personal computer. Fifty samples were measured to obtain the average velocity of a given sample. Axonemes in cell models were observed by electron microscopy, using fixation with 2 % glutaraldehyde in the presence of 1 % tannic acid. The samples were post-fixed with 1 % OsO 4, dehydrated through a series of ethanol solutions, embedded in Epon 812 and sectioned according to standard procedures. Sections were double-stained with aqueous solutions of uranyl acetate and lead citrate. A JEM100C microscope was used. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out by the method of Laemmli (1970) as modified by Jarvik & Rosenbaum (1980). The gel was composed of a 3 % to 5% acrylamide gradient and 3 M to 8M-urea gradient. A silver staining method was used (Merril et al. 1981). Protein concentrations of the axoneme and crude extract were determined by the method of Bradford (1976). Results Binding of outer dynein arms to axonemes We first examined whether outer dynein arms in the crude extract could bind to isolated axonemes. We mixed crude dynein extract with isolated axonemes at 0 C in the presence and absence of ATP, left the mixture at 0 C for 1 h, and then centrifuged it to precipitate axonemes. Binding of the outer-dynein arm was assessed by subjecting the precipitates to SDS-PAGE. Fig. 1 shows a high molecular weight portion of a silver-stained SDS-PAGE pattern obtained in such an experiment. The gel pattern of the crude extract (lane 1) indicates that it contained many proteins in addition to the outer-arm proteins, which are known to comprise three high molecular weight chains (HMWs) (Piperno & Luck, 1979; Pfister et al. 1982). Lanes 4 and 5 show the SDS-PAGE patterns of the precipitates obtained by 78 H. Sakakibara and R. Kamiya

3 a -0-7 Fig. 1. SDS-PAGE patterns demonstrating binding of outer dynein arms to axonemes. Stained with silver. Mixtures of axonemes and crude dynein extract were centrifuged at g for 15 min and the resulting pellet was subjected to SDS-PAGE analysis. Lane 1, crude dynein extract; lane 2, precipitates from crude dynein extract alone; lane 3, axonemes alone; lane 4, axonemes and crude dynein extract mixed in the absence of ATP; lane 5, axonemes and crude dynein extract mixed in the presence of 1 mmatp; lane 6, wild-type axoneme. Arrows indicate the a, fi and y HMW chains of the outer dynein arm. The heavily stained bands above the a HMW are those of contaminating membrane proteins. Concentrations of axonemes and crude dynein extract in the mixture were 0-3 mgml~ (axoneme) and 0-5 m g m r ' (extract). centrifugation of mixtures of crude dynein extract and axonemes in the presence (lane 4) and absence of ATP (lane 5). These patterns clearly indicate that outerarm HMWs were precipitated with the axonemes in both cases, although a significantly larger amount of HMWs was precipitated when ATP was absent than when it was present. No significant amount of outer-arm HMW was precipitated when the dynein sample alone was centrifuged (lane 2). These results suggest that the outer dynein arms contained in the crude extract can indeed bind to the axonemes, and that they have ATPsensitive and ATP-insensitive binding sites as reported (Takahashi & Tonomura, 1978; Haimo et al. 1979). Functional recombination of outer arms The above observations prompted us to investigate whether the outer dynein arms bound to axonemes can function as in the wild-type axonemes. For this functional assay, we used demembranated cell models instead of isolated axonemes, because axonemes on cell models underwent reactivation much more easily and reproducibly than isolated axonemes. Fig. 2 shows the averaged fast Fourier transform (FFT) patterns obtained from large populations of reactivated cell models. These F F T spectra (power spectra) had a peak corresponding to the flagellar beat frequency, in addition to a large lowfrequency component derived from the net translative movement of cell bodies (Kamiya & Hasegawa, 1987). The spectrum from cell models had a single peak at 30 Hz (Fig. 2A). However, after having been left with the crude dynein extract (concentration, loo/ugml"1) for min in the absence of ATP, cell models displayed a peak at a higher frequency of about 50 Hz (Fig. 2B). This frequency is similar to that of the wild type, although the wild type displayed two peaks at about 47 Hz and Hz, because the two axonemes on a single cell model beat at different frequencies (Kamiya & Hasegawa, 1987); the 40 Frequency (Hz) Fig. 2. Power spectra of light intensity fluctuation in microscope images of swimming cell models. These spectra show approximate distributions of flagellar beat frequency. A. cell models; B, cell models that had been mixed with crude dynein extract at 0 c C for min; C, wild-type cell models. Concentrations of cell models and crude extract when mixed were 2 x l 0 8 m l ~ ' and loo^gml"'. Reactivation was carried out at 25 CC in the presence of 1 mmatp. Power spectra shown were those averaged about times for 2 min. observation that cell models display a single peak suggests that the two flagella beat at the same frequency in this case (see Discussion). The shape and position of the peaks in the wild-type F F T patterns did not change even if the wild-type cell models had been incubated with the crude dynein extract (Table 1). As a control experiment, we mixed cell models with a high-salt extract from axonemes under essentially identical conditions. However, the frequency of the peak did not increase in those experiments (Table 1). These results Recombination of outer dynein arms 79

4 Table 1. Flagellar beat frequency in cell models mixed ivith axoneme extracts Exp. no S Cell model Extract - Incubation time (min) Peak freq. (Hz) * 28 47, 47, Cell models of or wild type () were left with or without extract from wild type or axonemes at 0 C for the times indicated. The density of the cell models was 2x10*ml" 1 when left with the extract and lxlo'ml" 1 when reactivated. The protein concentration of the axoneme extracts when mixed with the cell models was 100 fig ml". Peak frequencies are the frequencies of the peaks in the power spectra from cell models reactivated with 1 mm- ATP. Cell models and extract were mixed in the presence of 1 mm- ATP. strongly suggest that the binding of exogenous outer dynein arms can restore the outer-arm function in the axoneme. In the above experiment, we mixed the cell models and crude extract in the absence of ATP. When 1 mm-atp was present at the time of mixing, the beat frequency of cell models reactivated after 1 h of incubation was lower by 3-6 Hz than the frequency attained by those that hadbeen mixed in the absence of ATP (Table 1). Thus, functional recombination of outer arms appears to proceed less efficiently in the presence of ATP than in its absence. A similar phenomenon has been observed in the experiment with sea-urchin sperm axonemes (Gibbons & Gibbons, 1976). In addition to measuring the beat frequency, we measured the swimming velocity of the reactivated cell models. The swimming velocity of cell models was 39-3 ± 5-8 Jims" 1 in the presence of 1 mm-atp at 25 C. This average velocity was 45 % of that of wild-type cell models (83-2 ± 7-6^ms~'). After having been left with the crude dynein extract in the absence of ATP, their average swimming velocity increased to 71*4 i 11-2^ms" 1, i.e. 85% of the wild-type velocity. Restored outer arms and beat frequency The beat frequency of reactivated cell models depended on the concentration (0-1 ng ml" 1 ) of the crude dynein with which they had been mixed. To correlate the number of restored outer arms and flagellar beat frequency, we fixed a portion of the reactivated cell model samples for electron microscopy, simultaneously with the frequency measurement. Fig. 3 shows representative cross-section images of axonemes in such reactivated samples. These photographs demonstrate that outer dynein arms appeared at the correct sites after cell models had been mixed with the crude extract and subsequently reactivated, and that the number of arms per axoneme increased with increasing concentrations of the extract. Fig. 4 shows the average number of outer arms and beat frequency recorded at each concentration of the extract. Here, we counted the number of outer arms in more than 20 cross-sections at each concentration, adopting a rule that an arm that looked fainter than normal was counted as 0-5 equivalent of a complete arm. The number of arms parallelled the increase in beat frequency. With excess dynein samples, an average of 6-6 arms occurred in a cross-section. This corresponds to 83% of that of the wild-type axonemes, since it is known that only eight outer doublet microtubules bear outer dynein arms in the wild-type Chlamydomonas axoneme (Huang et al. 1979; Hoops & Witman, 1983). It is interesting to note that, even with excess amounts of crude dynein, we did not observe axonemes with nine outer arms, nor did we observe outer arms attached at abnormal positions. Recovery of backward-swimming type beating The beating pattern of wild-type axonemes of Chlamydomonas changes from an asymmetric ciliary type (forward-swimming type) to a symmetrical flagellar type (backward-swimming type) when Ca 2+ is raised above 10~ 6 M (Hyams & Borisy, 1978; Bessen et al. 1980). Axonemes of, however, have difficulty in beating with the symmetrical waveform and do not beat (Kamiya & Okamoto, 1985) or, at best, beat with a very small amplitude at higher Ca 2+ concentrations (Mitchell & Rosenbaum, 1985). When cell models were reactivated at 10~ 4 M-Ca 2+ after they had been mixed with the crude dynein extract, some of the axonemes were observed to beat with a symmetrical waveform similar to the one displayed by the wild-type axoneme. Since axonemes beating with a symmetrical waveform were never observed in control cell models that had not been mixed with the crude extract, we concluded that the ability to swim backwards was restored by the restoration of the outer dynein arm in the axonemes. Recombination with other dynein-ann mutants Ten genetically different groups of mutants (\-\0) have been isolated in our laboratory (Kamiya, 1988). We examined whether any strains differ in the ability to recover normal motility upon incubation with the crude dynein extract. We found that all of the strains of the 10 different groups underwent functional recovery equally. Thus, the defects in all the mutants seem to be similar, if not identical. Interestingly, with />/13a and pf22, which are non-motile mutants missing the outer arm (Huang et al. 1979), we could not observe motile cell models even if the mutant models had been mixed with crude dynein extract. Electron microscopy confirmed that the addition of the crude extract restored the outer arms in axonemes of these paralysed mutants (data not shown). Hence it is likely that pf\3a and pf22 have second defects in addition to the absence of the outer arms. We also looked for functional recombination of inner dynein arms with axonemes of two types of mutants, ida and idb, which lack different subsets of the inner dynein arms and swim more slowly than the wild type (Kamiya et al. 1988). However, we were unable to obtain clear 80 H. Sakakibara and R. Kamiva

5 3A Fig. 3. Appearance of outer dynein arms in axonemes after incubation with crude dynein extract. Concentrations of the crude dynein extract were 0 (A), 16 (B), 32 (C) and 1/igml" 1 (D). E. An example of the wild-type axoneme. Arrows in B and C indicate outer arms attached. X Protein concentration (/igml ) Fig. 4. Average number of outer arms per axoneme crosssection and flagellar beat frequency at different concentrations of the crude extract. ( ) Average numbers of outer arms per cross-section obtained from more than 20 counts at each concentration. Vertical bars represent standard deviations. ( ) Average number of outer arms per crosssection in wild-type cell models. (O O) Median frequencies of the high-frequency peak in power spectra as shown in Fig. 2. evidence that the motility in the cell models of these mutants was improved by addition of crude extract from wild-type or axonemes. Discussion We have shown that outer dynein arms in the high-salt extract from wild-type axonemes can bind to axonemes and increase the beat frequency. Although the dynein sample used in this study was crude, the increase in beat frequency of axonemes was most probably due to the binding of the outer dynein arms to their normal positions, because the number of recombined outer arms parallelled the increase in beat frequency and addition of crude extract from axonemes did not increase the beat frequency of reactivated axonemes. The present results indicate that the binding sites for outer arms are retained in the axoneme of, which lacks the outer arm due to mutation, and that the outer dynein arm solubilized from wild-type axonemes by highsalt solutions is functionally active. When observed after addition of ATP, the exogenous outer arms were bound to axonemes only at sites where they would normally be located in wild-type axonemes. In our preliminary observations on samples fixed before the addition of ATP, a few examples showed outer arms bound to abnormal sites such as the outer side of the doublet microtubules, although most of about 30 cross-sections photographed showed outer arms bound to normal sites. Since we have not observed binding at abnormal positions after addition of ATP, it is likely that dynein arms attached to abnormal sites become detached in the presence of ATP. A similar ATP-dependent recombination of outer dynein arms to outer arm-missing axonemes has been studied by Hyams (1985) using demembranated spermatozoids of the water fern Marsilea and Tetrahymena dynein. In agreement with the present study, he reported that the addition of dynein to Marsilea axonemes restored the outer arms, while subsequent addition of ATP reduced the number of outer arms restored. However, his study did not examine functional activities of those rebound outer arms. The two flagella on a wild-type cell (cis- and transflagella, distinguished by their position with respect to the eyespot) tend to beat at different frequencies (Ruffer & Nultsch, 1987) and, because of this, F F T spectra from wild-type cell models show two peaks (Kamiya & Hasegawa, 1987). However, the beat frequency of cell models had a single peak in F F T spectra, suggesting that the two flagella in models beat at the same frequency. Beat frequency measurements in uni-flagellated cells have further indicated that the two flagella of actually beat at the same frequency, whereas those of the wild type tend to beat at different frequencies (Kamiya & Hasegawa, 1987; Kamiya, unpublished observation). Interestingly, cell models displayed a single peak even when the higher beat frequency was restored by addition of crude dynein extract. These observations suggest that outer dynein arms are involved in the mechanism that causes the frequency difference between the two flagella. Although there may be several different explanations for these observations, we propose that the beat frequency difference between the two axonemes in the wild-type cell model is due to the existence of two types of outer dynein arms on different axonemes: cistype and trans-typt outer arms. The difference may arise from different levels of modification such as phosphorylation: an outer-arm HMW is known to be a phosphoprotein (Piperno & Luck, 1981). According to this hypothrecombination of outer dynein arms 81

6 esis, the two flagella on the models should beat with an identical frequency even after the models have been incubated with the crude dynein extract, since the extract contains both as-type and trans-type outer dynein arms, and they will bind unselectively to the two axonemes of. The present study demonstrates that cell models of the mutant can be used for functional assay of various outer arm preparations. With sea-urchin sperm flagella, Gibbons & Gibbons (1976) developed a method for recombining outer arm dynein isolated from Tripneustes gratilla to axonemes of another species (Colobocentrotus astratus) from which outer arms had been removed by treatment with a high-salt solution. The Chlamydomonas system reported here is simpler than the sea-urchin system in that we do not need to treat cell models with high-salt solutions, and that we can use the same species as donor and acceptor of arms. Although recent success in inducing dynein-based motihty in vitro (Paschal et al. 1987; Vale & Yano-Toyoshima, 1988) has opened a way to assay dynein subunits for their ability to cause microtubule sliding, experimental systems using whole axonemes are needed to understand the role of dynein subunits in axonemal motility, the mechanism of which should involve much more than microtubule sliding. In vitiv recombination of dynein arms, together with isolation of appropriate dynein mutants, would be a most powerful approach to revealing the function of inner and outer dynein arms and their subunits. We thank Professor Sho Asakura for his encouragement. This research has been supported by Grants-in-aid from the Ministry of Education, Science and Culture of Japan. References BESSEN, M., FAY, R. B. & WrrMAN, G. B. (1980). Calcium control of waveform in isolated flagellar axonemes of Chlamydomonas. y. Cell Biol. 80, BROKAW, C. J. & KAMIYA, R. (1987). Bending patterns of flagella: IV. 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7 SUMMERS, K. E. & GIBBONS, I. R. (1971). Adenosine triphosphate- WITMAN, G. B., PLUMMER, J. & SANDER, G. (1978). Chlamydomonas induced sliding of tubules in trypsin-treated flagella of sea urchin flagellar mutants lacking radial spokes and central tubules, sperm. Proc. natn. Acad. Sci. U.SA. 68, Structure, composition, and function of specific axonemal TAKAHASHI, M. & TONOMURA, Y. (1978). Binding of 30S dynein components, J. Cell Biol. 76, with the B-tubule of the outer doublet of axonemes from YANO, Y. & MIKI-NOUMURA, T. (1981). Recovery of sliding ability Tetrahymena pyriformis and adenosine tnphosphate-induced in arm-depleted flagellar axonemes after recombination with dissociation of the complex. J. Biochem., Tokyo 84, extracted dynein I. J. Cell Sci. 48, VALE, R. D. & YANO-TOYOSHIMA, Y. (1988). Rotation and translocation of microtubules in vitro induced by dyneins from Tetrahymena cilia. Cell 52, (Received 25July 1988-Accepted 13 October 198S) Recombination of outer dynein amis 83

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