Analysis of Ni 2+ -induced arrest of Paramecium axonemes

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1 Analysis of Ni 2+ -induced arrest of Paramecium axonemes J0RGEN LARSEN 1 ' 2 and PETER SATIR 1 'Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA ^Institute of Cell Biology and Anatomy, University of Copenhagen, Copenhagen 0, Denmark Summary Thi9 study examines the molecular basis for paralysis of ciliary motility by Ni 2+. At concentrations above 0.1 mm, Ni 2+ slowed and subsequently stopped swimming of living, axenically grown Paramecium tetraurelia. However, some cilia still beat in the presence of 0.1 mm Ni 2+. When permeabilized and reactivated with 4mM ATP at pca>7, cells resumed ciliary beat and swam forward at approximately 170±28/ims~ 1 ; swimming speed increased in the presence of 10 ftm cyclic AMP. Addition of Ni 2+ (pni<5) caused rapid arrest of all ciliary beat in a single position. This was fully reversible when EGTA was added to raise the pni. Axonemes were then isolated and sliding was observed in the presence of trypsin and ATP. When pni was lowered to about 5, sliding was reduced dramatically. This too was reversible with EGTA. Dynein was then extracted from the axonemes and used for in vitro translocation assays. At concentrations of Ni 2+ where microtubule-sliding and axonemal beat were greatly inhibited or absent, microtubule translocation in vitro by 22 S dynein was only slightly affected. However, translocation by 14 S dynein was stopped completely. When pni was raised by repeated washing with solutions containing EGTA, microtubule translocation by 14 S dynein resumed. We conclude that Ni 2+ induces a reversible paralysis by a direct effect on 14 S dynein while 22 S dynein is not a primary target. Key words: microtubule-based motility, cilia, dynein Introduction Paramecium is an excellent system for studying the effects of various agents on the control of ciliary activity, since biochemical and mutational analysis can be combined to dissect the regulatory pathways (Hinrichsen et al. 1986; Saimi and Kung, 1987; Bonini and Nelson, 1988; Satir et al. 1988). Since the original work of Gelei (1935) and Tartar (1950), it has been well established that Ni 2+ gradually inhibits ciliary activity in different ciliated cells, including Paramecium, but the molecular basis of Ni 2+ action has not been understood. This may prove important, since Ni 2+ is effective in reversing the high Ca block in certain axonemes to a fixed beat position (Tiands down'), in which one axonemal switch that controls arm activity during beat is thought to be blocked (Satir et al. 1990). The present study has investigated whether Ni 2+ acts directly on the Paramecium ciliary axoneme and, if so, whether the site of inhibition is dynein itself. Detergent-permeabilized cells have proven valuable in addressing the first question (Naitoh and Kaneko, 1973; Bonini and Nelson, 1988; Lieberman et al. 1988). This treatment compromises the cell membrane so that, as we will demonstrate, low concentrations of Ni 2+ can directly and reversibly block axonemal motility. In order to clarify the mechanism of the immobilizing action of Ni 2+ on demembranated cilia, we first investigated the effect of Ni 2+ on the ATP-induced sliding of microtubules in trypsin-treated axonemes of Paramecium. We will demonstrate that Ni 2+ directly and reversibly inhibits sliding. However, the uncertain effects of protease Journal of Cell Science 99, (1991) Printed in Great Britain The Company of Biologists Limited 1991 digestion and the complexity of structural interaction in the axonemes limit the information obtainable about the direct effect of Ni 2+ on dynein itself in such studies. An in vitro motility assay has recently been introduced by which the properties of isolated dynein in translocating microtubules can be studied directly (Paschal et al. 1987; Vale and Toyoshima, 1988; Sale and Fox, 1988). This assay is an improvement over existing motility assays, because only purified dynein and microtubules are involved. We have used this assay to investigate the effect of Ni 2+ on microtubule translocation induced by the two dyneins (22 S and 14 S) that we have isolated from Paramecium axonemes (Larsen et al. 1991). The effect of Ni 2+ on dynein ATPase activity has also been determined. We will demonstrate that Ni 2+ inhibits beat and sliding at the same concentration, that it inhibits microtubule translocation by one of the dyneins (14 S), while it has a much more limited effect on translocation by isolated 22 S dynein, suggesting that Ni 2+ inhibits axonemal switching by an effect on 14 S dynein. Materials and methods Paramecium tetraurelia were grown axenically at 27 C in a medium adapted from Soldo et al. (1966). Cells were harvested at late log or early stationary phase by gentle centrifugation. Paramecia were then washed three times in different buffers and allowed to equilibrate for at least 30min prior to experimental use. Ni 2+ was added as nickel acetate from 10 mm or 100 mm stock solutions. When addition of Ni 2+ or other components to the 33

2 media caused a change in ph this was readjusted by addition of NaOH or HC1. To calculate pni for different solutions, we have used the Calcon computer program developed by Goldstein (1979), and modified by J. S. Tash. Stability constants are from Martell and Smith (1974). In preliminary experiments we investigated the effect of Ni 2+ on swimming behavior of living paramecia in different buffers: (1) in 2 mm KC1, 1 mm CaCl 2, 5 ra Tris-HCl, ph 7.2; (2) in a TECK buffer (4mM KC1, lmm CaCl 2, 0.1 mm EDTA, 10 mm Tris-HCl, ph 7.2); or (3) in a buffer without calcium (5 mm Pipes, 1 mm KC1, 20 mm MgCl2, ph7.2). For determination of the rate of motility, control and cation-exposed cells were placed in a small chamber and observed by dark-field or phase-contrast microscopy. Visual observations or motion analysis were used to estimate changes in swimming behavior caused by Ni 2+ Preparation and reactivation of permeabilized cells Paramecia were permeabilized with 0.01% Triton X-100 on ice according to the method of Naitoh and Kaneko (1972). When swimming and ciliary beating had ceased (25 min), the cells were washed free of detergent. Thin sections of such permeabilized cells indicate that the ciliary, cell and outer alveolar membranes are greatly disrupted or entirely missing (Lieberman et al. 1988). Permeabilized cells were reactivated at room temperature in a MgATP solution (4mM MgSO 4, 4mM ATP, 1.5mM EGTA, 10 mm Tris-maleate, 50 mm KC1, ph 7.0). In some cases, cyclic AMP was added to the reactivation medium to increase the swimming speed (Bonini and Nelson, 1988). Samples of 0.4ml were withdrawn and exposed to Ni 2+ and other divalent cations (Ca 2+, Mg 2 " 1 ", Co 2+ ) to determine their effect on swimming behavior and ciliary activity in reactivated cell models. After 2 mm exposure the effect was observed by phase-contrast and dark-field microscopy. In some experiments EGTA was added following cation exposure, in which case the effect on swimming behavior and ciliary activity was determined again 2 min after EGTA addition. Observation and recording of swimming behavior Swimming behavior was examined by dark-field microscopy and recorded on videotape for quantitative motion analysis. The videotapes were analyzed using a system introduced by Motion Analysis Systems (MAS), Inc. (Santa Rosa, CA), following the procedure of Lieberman et al. (1988), modified after Sundberg et al. (1986). Scanning electron microscopy Preparation for SEM was performed as described by Lieberman et al. (1988) with minor modifications. Living and permeabilized cells were quick-fixed for 30 s in 1 % OsO< in 10 mm cacodylate buffer (ph 7.0), followed by rapid addition of 2 % glutaraldehyde in 10 mm cacodylate buffer. After 10 min the OsO 4 -glutaraldehyde mixture was replaced by fresh glutaraldehyde (2 %) for 1 h, and washed twice with 10 mm cacodylate buffer. Fixed cells were then transferred to polylysine-coated coverslips and dehydrated through a graded series of ethanol. After dehydration the samples were critical-point dried and gold coated before examination in a JEOL S35 scanning electron microscope (SEM). Micrographs are representative of the sample of undistorted, well-ciliated cells. Isolation of axonemes To eliminate the possible effect of high Ca 2+ in the calcium shock treatment, in some cases axonemes were prepared by mechanical deciliation of permeabilized cells as described by Hamasaki et al. (1989). Results using this preparation are essentially identical to those obtained by a calcium shock deciliation (Adoutte et al. 1980), which was used routinely. After deciliation, cilia were isolated from cell bodies by centrifugation and purified according to methods of Hamasaki et al. (1989). Cilia derived from Ca 2+ shock treatment were then treated with 0.8% Triton X-100 for 20 min on ice to remove membranes. Axonemal sliding experiments The axonemes were washed twice with 'activation buffer' (4mM MgS0 4, 1.5 mm EGTA, 50 mm KCl, 10 mm Tris-maleate, ph7.0 or 7.5) to remove Triton, resedimented, and resuspended in activation buffer. Following measurement of absorbance (O.D. units) of the suspension at 350 nm, a sample corresponding to 0.4 unit A350 was obtained and placed on ice for immediate use. In some experiments, this axonemal suspension was exposed to Ca 2+, Mg 2+, Co 2+ or Ni 2+ for 2 min and introduced into a 15/J perfusion chamber (Larsen et al. 1991). The axonemes attached to the glass were then activated by perfusion with activation buffer containing trypsin (l/;gml -1 ), ATP (O.lmM 4mM) and appropriate cations. In other sliding experiments, the axonemal suspension was first digested with trypsin. The digestion process was monitored turbidimetrically and halted with excess soybean trypsin inhibitor, when the A350 decreased to 80 % of the initial value. The axonemes were then applied to the perfusion chamber and unadsorbed axonemes were removed by perfusing activation buffer through the chamber. The adsorbed axonemes were exposed to the appropriate cations for 2 min and then perfused with activation buffer containing ATP. In some cases, experiments were performed in which EGTA was added following cation exposure. The proportion of disintegrated axonemes was estimated by direct observation of sliding in dark-field and recorded on videotape. Electron microscopy For determination of the effect of Ni 2+ on negatively stained preparations of digested axonemes two samples were withdrawn from the above mentioned cuvette. Ni 2+ was added to one sample, whereas the other served as control. Digested axonemes were applied to a sheet of Formvar-coated carbon-stabilized, copper grids. Sliding was initiated by diluting the sample with an equal volume of activation buffer containing ATP. The axonemes were incubated for 5 min in these solutions at room temperature, after which excess fluid was removed and 1 % aqueous uranyl acetate pipetted onto the grids for negative staining. The grids were drained, allowed to dry in air under cover, and observed in a JEOL 100CX electron microscope. Sliding images were identified by criteria discussed by Sale and Satir (1977) and Larsen et al. (1991). Preparation of dynein from paramecium This procedure essentially follows Larsen et al. (1991). Axonemes from 4-liter stationary-phase cultures were isolated as above, and resuspended in axoneme buffer (30 mm Hepes, 5mM MgS0 4> 0.5 mm EDTA, 20 mm KCl, lmm dithiothreitol (DTT), SOKTUmT 1 aprotinin, lo/zgrnp 1 leupeptin, ph7.6) with 0.6 M KCl to extract the dyneins. Samples of 0.2 ml containing high-salt extracted protein were layered on top of linear 12 ml 5 % to 30 % sucrose gradients prepared in axoneme buffer without leupeptin and aprotinin, and with DTT at O.lmM. The gradient was centrifuged for 15 h at revs min" 1 (4 C) in a SW41 Ti rotor. Fractions were collected from top to bottom of the tube. Each fraction was assayed for protein and ATPase activity, and those containing the 14 S and 22 S dynein were pooled separately. The purified dynein solutions were frozen for later use in in vitro motility assays or for measurement of ATPase activity. Dynein in vitro motility assay Motility assays were carried out essentially as described by Vale and Toyoshima (1988) and Larsen et al. (1991). 14 S or 22 S dynein samples were adjusted to approximately O.lmgml" 1 protein in axoneme buffer and a 20 ;d sample was applied to the assay chamber in two successive portions for 2-min incubation periods each. The unadsorbed dynein was removed by perfusing 20/d translocation buffer (50 mm K + -acetate, 10 mm Tris-acetate, ph7.5, lmm EGTA, 3rnM MgS0 4 ) through the chamber. Subsequently, translocation buffer containing taxol-stabilized bovine brain microtubules and 1 mm ATP was perfused through the chamber. This assay system has the advantage that the chamber that has dynein bound to the glass can be perfused several times to examine movement under different conditions. To test the effects of Ni 2+ on microtubule motility, Ni 2+ was added to the translocation medium in which the motility assay 34 J. Larsen and P. Satir

3 was performed. Translocation of microtubules was examined at room temperature using video-enhanced dark-field microscopy. Reversibility of inhibition was tested by perfusing additional translocation buffer without nickel through the chamber. ATPase and protein assays ATPase activities of the 22 S and 14 S dyneins were tested in the presence of Ca 2+, Mg 2+, Co 2+, Ni 2+ and the dynein inhibitor vanadate. In these experiments the dyneins were always washed free from sucrose using a 30000Mr cutoff centricon and resuspended in activation or translocation buffer before addition of the metal ions. Dynein ATPase activity (in the absence of microtubules) was analyzed by the orthophosphate determination method following Hayashi and Takahashi (1979), modified after Murphy and Riley (1962). Protein concentration was determined using the BioRad Bradford reagent (BioRad, Rockville Centre, NY) using bovine serum albumin as a standard. Results The effect ofni2+ on ciliary activity in living paramecia In TECK buffer Paramecium tetraurelia swam forward in a gently curving spiral path with a swimming velocity of about 360 ± 26/an s" 1 (mean of 3 experiments; swimming velocity determined by motion analysis of at least 30 randomly chosen cells in each experiment). These forward swimming cells showed characteristic metachronal wave patterns with a wavelength of approximately 11 fan and an effective stroke toward the posterior end of the cell (Fig. 1). Ni 2+ immobilized cell motility in a dose-dependent manner. In TECK buffer, concentrations above 0.1 mm Ni 2+ slowed and finally stopped swimming within 5 min as a result of uncoordinated ciliary activity. Such immobilized cells showed some sporadic ciliary motion, but had lost the coordinated beating characteristic of metachronal waves. After prolonged exposure to Ni 2+ concentrations above 0.1 mm an increasing deciliation was observed, but some cilia still beat, and the cells exhibited rocking movements. This residual ciliary activity was, however, limited and the cilia appeared stiff, in that their beat was restricted to a limited arc relative to the body surface. Ca 2+ has been reported to abolish the immobilizing effect of Ni 2+ in Paramecium (Kuznicki, 1963; Andrivon, 1972) and it could be that the residual beating we observed was due to the presence of 1 mm CaCl2 in TECK buffer. Therefore the effect of Ni 2+ on cell motility was tested in a buffer without added Ca 2+. This buffer did not affect the results. Effect of Ni2+ and other divalent cations on permeabilized cells In an appropriate reactivation medium, permeabilized paramecia were immobile in the absence of externally supplied ATP. After reactivation with Mg 2+ and ATP, ciliary activity resumed and most cell models swam. The few models that did not swim were reactivatable, since their cilia were beating rapidly, but they appeared to be attached firmly to the glass of the chamber. The average swimming velocity of the free-swimming cell models was approximately 170±28Jums~1 (mean of 3 experiments) and the models swam continuously forward with the metachronal wave pattern characteristic of living, forward-swimming" cells (Fig. 2A). Addition of cyclic AMP to the reactivation medium caused an increase in the swimming speed of the free-moving permeabilized cells, and an increase in the number of free-moving cells in the population. When the cells were reactivated in the presence of 10,UM cyclic AMP the swimming speed increased to about 200 jim s" 1 and virtually all of the cells in the population swam in straight or curved paths (Fig. 3). Ni 2+ at low free-ion concentration (30,UM) caused rapid arrest of all ciliary activity, resulting in a complete immobilization of the permeabilized cells (Fig. 3). Other divalent cations such as Co 2+ did not cause a comparable inhibition. When permeabilized cells were reactivated and exposed to approximately 30 /JM free Ni 2 + (pni 4.6), there was no deciliation. The metachronal wave was no longer observed in SEM and cilia were captured primarily in a single-stroke position (Fig. 2B). For most of their length, the cilia curved uniformly to point toward the posterior end of the cell in a position corresponding to the end of the effective stroke ('hands down', in the terminology of WaisSteider and Satir, 1979). The permeabilized cells resumed ciliary activity when EGTA was added, raising the pni to 6.4 or higher (Fig. 3; Table 1). Although beat was reactivated in the whole Fig. 1. SEM of quick-fixed living Paramecium. Metachronal waves are seen (A=ll/an). Anterior end of the cell to the left; effective stroke direction to the right. Bar, 10;<m. Effect of Ni2+ on axonemes 35

4 Fig. 2. SEM of quick-fixed permeabilized Paramecium in reactivation medium. (A) Triton-permeabilized reactivated control cell with normal metachronal waves. (B) After 26 /.IM free Ni 2+ is added, virtually all cilia point toward the posterior of the cell. Metachronal waves are absent. Bar, 10 fun. population, both the number of swimming cells and their average swimming velocity fell substantially (Fig. 3). Even after all treatment, however, some cells resumed swimming at rates greater than 200(ms" 1, suggesting that for these cells, Ni 2 + inhibition was completely reversible (Fig. 4). The effect of Ni2+ on axonemal sliding The axonemes obtained from isolated cilia of Para36 J. Larsen and P. Satir mecium were not uniform in length and short fragments were commonly found. Without trypsin treatment, in ATP Paramecium axonemes did not normally resume beating and only slid to a limited extent. Trypsin treatment caused an increase in the proportion of axonemes that disintegrated by sliding upon ATP addition. Ni 2 + had a marked effect on the ATP-induced disintegration of trypsintreated axonemes. When pni was lowered to about 5, the number of axonemes sliding was greatly reduced or sliding

5 B pni % Swimming cells Swimming speed (/ans~') 0 0 <5 0 0 > Fig. 3. Swimming tracks of permeabilized Paramecium generated by motion analysis. Swimming paths represent distances travelled in 1 s (11 frames). For clarity, only frames 1, 4, 7 and 10 are displayed. (A) Cells in reactivation medium containing 10 (JM cyclic AMP; (B) after addition of 2mM Ni 2+ to A; (C) After addition of 2mM EGTA to B. Table 1. Reversal ofni 2+ inhibition of beat of permeabilized cells by EGTA Conditions before EGTA addition Final conditions pni Beat pni 7.7* 6.4* 9.6t Beat} *+2mM EGTA. t+4ram EGTA. } + + +, Comparable to untreated permeabilized cells; + +, slight inhibition; -, no beat " oj- P" Swimming speed (/ims" 1 ) 30- Fig. 4. Distribution of swimming speeds for free-moving permeabilized paramecia reactivated in medium containing 10 tat cyclic AMP. (A) Control. (B) Cells stopped by addition of 2 mm Ni 2+ and restarted by addition of 2 mm EGTA (protocol as in Fig. 3). 10- Table 2. Effects of different cations on ATP-induced sliding of axonemes Cation pca' Shdingt Control Ni Co Mg Ca > Axonemes in buffer (ph7 or 7.6) treated with trypsin before or simultaneously with addition of ATP. * Free ion concentration with 2 mm of cation added. t Direct observation of sliding in darkfleld on videotape: + + +, marked increase in number sliding versus control; + +, comparable to control; +, decrease versus control; -, little or no sliding. was abolished. In contrast, marked sliding occurred at pca 4. Sliding also occurred when Co 2+ was substituted for Ni 2+ (Table 2). The Ni 2+ inhibition of sliding could be reversed by subsequent addition of EGTA. Electron microscopy of negatively stained preparations of disintegrated axonemes showed that both the dynein arms and the spoke groups were easily identified after trypsin treatment. Sliding configurations of ciliary axonemes were readily identified (Larsen et al. 1991) (Fig. 5A). When pni was lowered to about 5, sliding configurations could no longer be found, but axonemes sometimes opened to show periodic interdoublet linkages (Fig. 5B). The effect of on dynein-mediated microtubule translocation When dyneins were extracted from Paramecium axonemea with 0.6 M KC1 and further purified by sucrose density gradient centrifugation, two peaks of ATPase activity, corresponding to 22 S dynein and 14 S dynein, were obtained. In in vitro motility assays, the translocation velocity depends on the buffer used. Under standard conditions with the translocation buffer (see Materials and methods) the isolated 22 S dynein induced attachment and translocated MAP-free, taxol-stabilized, calf brain microtubules with an average velocity of 2.71(±0.92)/zms~ 1 at 22 C in the presence of 1 mm ATP (Table 3) (Larsen et al. 1991). Addition of Ni 2+, to pni 4.6, sufficient to inhibit beat and sliding, affected translocation velocity significantly, but did not stop translocation (Table 3). In some cases, translocation by 22 S dynein could still be observed at free Ni 2+ concentrations at least 10 times higher than those necessary to inhibit beat completely in permeabilized cells. In contrast, under the same standard conditions at pni>7, 14 S dynein translocated microtubules at velocities of 0.79(±0.33)/ans" 1 (Table 3) (Larsen et al. 1991). However, translocation was inhibited at pni 5.6 and Effect ofni 2+ on axonemes 37

6 Table 4. Effects of different divalent cations on 14 S and 22 S dynein Mg2*-ATPase Activity Relative activity (.9, >) Addition 22 S 14 S Control 10/(M vanadate 2mM Ca 2+ 3 HIM Ca 2+ 2mM Mg243m M Nig2* 2mM Ni 2+ 3mMNi $4 m S Mean of 3 experiments; s.d. less than 10 %. was almost completely abolished at pni 4.6, i.e. the same concentration of Ni 2 + that blocked ciliary beat in MgATPreactivated permeabilized cells and sliding of isolated trypsin-treated axonemes. When pni was raised by repeated perfusion with translocation buffer (containing EGTA), translocation by 14 S dynein resumed at control velocities. Effect of divalent cations on dynein ATPase activity The Mg2+-ATPase activity of purified 22 S or 14 S dynein was measured before and after addition of Ni 2 +. In translocation buffer, at pni values below 5, dynein activity decreased to about 80 % of the control value for both 22 S and 14 S dynein (Table 4). Comparable addition of Ca 2+ had no effect on ATPase activity. Addition of 10,UM vanadate nearly abolished activity, as anticipated. Thus at pni values where motility, sliding and microtubule translocation by 14 S dynein were greatly inhibited, ATPase activity of dynein was much less affected. Discussion B Fig. 5. Negative-stain images of axonemes treated with trypsin and ATP. (A) Control. Sliding configurations are readily found. Note normal (+) direction displacement of doublet N+l. Dynein arms are indicated by arrow. Bar, 0.1/on. (B) With Ni 2+ at pni< 5, sliding configurations are absent. Axonemes are largely intact. Inset shows that axonemes sometimes unroll. Spoke groups repeat is indicated. Doublet displacement is not seen. Bar, 0.5 fira. Table 3. Measurements of dynein-mediated microtubule translocation Velocity of translocation pni 22 S 14 S >7 (control) 6.8 5,6 2.71± ± ± ±0.09 4* The effect on Ni on 22 S and 14 S dynein-induced microtubule translocation. Translocation of microtubules was examined at room temperature using video-enhanced dark-field microscopy. Measurements were made on at least 30 microtubules. Values are given in (ons"1±s.d. * Translocation almost completely abolished. 38 J. Larsen and P. Satir Ni 2+ has long been known to inhibit ciliary activity of ciliates (Gelei, 1935; Kuznicki, 1963) and other cells, although the precise mechanism involved was unknown. In living paramecia, Ni 2 + inhibits ciliary activity in a gradual and diffuse way, probably acting first at the cell membrane, by competing with other divalent cations at membrane channel sites. The use of detergent-permeabilized, MgATP-reactivated cells has permitted experimental access to the axonemal effects of Ni 2 +. In contrast to its effects on living cells, Ni 2 + rapidly arrests ciliary activity in reactivated Triton-extracted cells (Naitoh and Kaneko, 1973; Andrivon, 1974). Hence Ni 2 + appears to act directly on the axoneme. In SEM, almost all nonbeating cilia of Ni2+-immobilized permeabilized paramecia point posteriorly ('hands down'). Naitoh and Kaneko (1973) showed that Ni2+-immobilized cilia could be moved into a 'hands up' position by addition of Ca 2+ (above 1 0 ~ 4 M ). This behavior would be consistent with the switching of mammalian sperm cells by Ca 2+ and Ni 2 + (Lindemann and Goltz, 1988) and of 'hands down' mussel gill cilia (Wais-Steider and Satir, 1979; Reed and Satir, 1986). In cilia, Ni 2 + affects a switch point of axonemal activity so that cilia are blocked in a position corresponding to the final stage of an effective stroke, while Ca acts to block motility at the end of a recovery stroke (Satir et al. 1990). ATP-induced sliding of trypsin-treated axonemes is inhibited by the same concentration of Ni 2+ that inhibits the motility of demembranated cells under the same

7 conditions. When pni is lowered to about 5,* sliding is reduced dramatically or abolished. In contrast, sliding occurs at pca 4 or in comparable Co 2+ concentrations. Therefore, Ni 2+ specifically affects either dynein mechanochemistry and/or some trypsin-insensitive structural components of the axoneme. Lindemann et al. (1980) showed that low concentrations of Ni 2+ did not completely inhibit microtubule sliding in reactivated bull sperm flagella, but did inhibit beat. This suggests that the concentration thresholds for Ni 2+ inhibition of sliding and beat may not coincide exactly, although in our experiments they are in the same range. To investigate whether Ni 2+ affects dynein directly, we isolated 22 S and 14 S dynein from Paramecium axonemes. 22 S dynein is a three-headed bouquet with three heavy chains that are u.v. photocleavable in the presence of vanadate, while 14 S dynein is a single-headed species that may be heterogeneous (Larsen et al. 1991). Two u.v. photocleavable heavy chains are found in the 14 S region in our preparations. Travis and Nelson (1988) reported that a shoulder of the 22 S peak could be identified as 19 S dynein, but this is not apparent in our preparations where the fraction size is larger. Their 12 S peak probably corresponds to our 14 S dynein (see Larsen et al. 1991, for further details). Both 22 S dynein and 14 S dynein support translocation of purified bovine brain microtubules in an in vitro motility assay using a standard translocation buffer, but 22 S dynein translocates microtubules at a rate about three times as fast as 14 S dynein. Addition of Ni 2+ at concentrations sufficient to inhibit beat and sliding abolishes translocation by 14 S dynein in in vitro assays but only partially affects translocation by 22 S dynein. Translocation by 22 S dynein is observed at Ni 2+ concentrations at least ten times higher than those required for complete beat inhibition. In contrast, Vale and Toyoshima (1988) demonstrated that addition of vanadate completely inhibited 22 S dynein-induced microtubule translocation in Tetrahymena, indicating that Ni 2+ does not inhibit ciliary motility in the same way as vanadate, by abolishing general dynein ATPase activity. In fact ATPase activities of purified 22 S and 14 S dyneins are only weakly inhibited by Ni 2+, although the extent of inhibition seems dependent on the exact assay conditions. Using somewhat different conditions, Travis and Nelson (1988) showed that 14 S dynein ATPase activity was inhibited by Ni 2+ to a much greater extent than 22 S dynein. Therefore, inhibition of beat, sliding and probably ATPase activity by Ni 2+ correspond to the specific inhibition of 14 S dynein, as demonstrated by inhibition of microtubule translocation in in vitro assays. This is supported by the observation that the effect of Ni 2+ on translocation, as well as on beat and sliding, is reversible when EGTA is added to raise the pni. Unfortunately, the axonemal localization of 14 S dynein, which now seems critical to an understanding of switching activity in the axoneme, and of the Ni effects, is uncertain. Many investigators believe that, on the basis of rebinding studies, 14 S dynein is a component of the inner dynein arm (Warner et al. 1985). However, dyneins often rebind promiscuously to several structures in the axoneme (Satir et al. 1981). Another possibility is that 14 S dynein is a component of the spoke head. There is some suggestion that antibodies to 14 S Tetrahymena dynein are localized at either the spoke head or the outer midwall of the doublets. It is unlikely that dynein at the outer midwall could be responsible for switching arm activity, but the inner arm-spoke relationships are probably fundamental to this process. It would be useful to have the appropriate axonemal mutants in Paramecium to be able to pursue this question further. It is also interesting that 22 S dynein, which is found in the outer arm, is evidently not critically inhibited by Ni 2+ at low concentrations. This work was supported in part by grantsfromthe USPHS to P.S. and from the Danish Natural Science Research Council to J.L. We are grateful to J. Avolio, K. Barkalow and T. Hamasaki for their help with various aspects of the study and to M. Ann Holland for secretarial assistance. Assistance and support for the motion analysis work reported was kindly provided by Dr J. L. Spudich and his laboratory. References ADOUTTB, A., RAMANATHAN, R, LEWIS, R. M., DUTE, R. R., LING, K Y, KUNG, C. AND NELBON, D. L. (1980). Biochemical studies of the excitable membrane of Paramecium tetraurelia, HI. Proteins of cilia and ciliary membranes. J. Cell Bwl. 84, ANDRIVON, C. (1972). 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