Direction of sliding and relative sliding velocities within trypsinized axonemes of Gallus domesticus

DAVID M. WOOL LEY and ANDREW BRAMMALL*

Department ofl'liysioloi>y. The Medical School, L'niversity of Bristol, Bristol BSS ITD, L'K •Present address: MRC Experimental Embryology and Teratology Unit, Medical Research Council Laboratories, Carshalton, Surrev SMS 4EF, UK

Summary

Trypsin digestion of demembranated fowl sper- orderly way by inter-doublet sliding. All the matozoa caused a longitudinal splitting of the doublets of the axoneme could be reactivated and distal part of the axoneme. The resulting strands, in all instances the direction of sliding was the consisting of groups of doublet , same as that reported for the cilia of Tetrahy- formed left-handed helices. On the evidence of tnena. Within the groups of doublets the electron micrographs, the digestion had caused measured inter-doublet displacements were gen- the loss of arms from the outer row; it is erally similar, suggesting that the rates of sliding assumed that the doublets remained linked had been equivalent. These findings are con- together by dynein arms of the inner row. When trasted with the differential pattern of activation such helices were mechanically detached from that is assumed to occur in vivo. the proximal and reactivated with ad- Key words: Gallus domestic/is, sperm axonemes, sliding enosine triphosphate, they lengthened in an velocity.

Introduction We have therefore made use of these preparations to study two aspects of the sliding behaviour relevant to It is well established that the motion of eukaryotic cilia the question of control and coordination. First, we and flagella depends upon active sliding between the have confirmed the important finding of Sale & Satir doublet microtubules of the 9+2 axoneme. The rel- (1977) that the active sliding has a single polarity under evant ATPase activity resides in the dynein these circumstances. Second, we have measured the that project as inner and outer rows of arms from the A within-group displacements to discover whether the individual doublets achieve similar sliding velocities; tubule of each doublet and interact cyclically with the B these data augment the short series published for sea- tubule of the adjacent doublet (reviewed by Gibbons, urchin sperm axonemes by Takahashi el al. (1982). 1981). The control, spatial and temporal, of this sliding Finally, from the absence of the outer row of dynein so that it gives rise to a pattern of bending, bend arms from our electron micrographs, we attribute the propagation and oscillation remains to be elucidated. sliding behaviour of these preparations to the activity of In this work we have used spermatozoa from the the inner row of dynein arms. domestic fowl (Gallus domesticus) as a convenient source of long lengths of simple 9+2 axoneme. Apply- Materials and methods ing the standard procedures to induce active sliding and axonemal disintegration (Summers & Gibbons, Collection of spennatozoa 1971), we noted that exposure to trypsin caused Cocks of a commercial broiler strain (Ross) were obtained at longitudinal splitting of the axoneme and that sub- 50 weeks of age as ex-breeding stock. They were fed a pelleted sequent treatment with ATP produced an orderly diet and maintained on a 16 h: 8 h (light/dark) cycle. After an sliding apart of these subsets of the original nine initial period of training, the most productive animals were doublets. kept and used as semen donors every 2 or 3 days. The Journal of Science 88, 361-371 (1987) Printed in Great Untam © The Company of Biologists Limited 1987 361 collection of semen by massage followed the standard To obtain thin sections for electron microscopy, the cells or 2-operator technique described by Lake & Stewart (1978). fragments were routinely fixed for 30 min in suspension by Immediately after collection into graduated 10-ml glass adding an equal volume of 5 % glutaraldehyde in 01M- tubes, the semen was held at 40°C, then allowed to cool to sodium cacodylate, then centrifuged (2000 £ for 20min), room temperature over 30min. Samples that were noticeably washed in buffer for 20 min, treated with 1 % OsO.) in the contaminated with faecal and urinary excretion were dis- same buffer for 1 h, dehydrated in ethanol and embedded in carded. In general the semen was not pooled before use. The Epon 812 by conventional methods. In addition, some 'ejaculates' thus obtained ranged in volume from 015 to preparations were fixed in a Hepes-buffered glutaraldehyde 1-0 ml and contained typically about 109 spermatozoa (sperm solution containing 1 % tannic acid, exactly according to the density was not measured routinely). method of Warner et al. (1985). Silver-gold sections of the pellets were stained with uranyl acetate and lead citrate and examined in a Philips EM300. Preparation of gmups ofaxonemal doublets To prepare negatively stained axonemes, the fixed suspen- A stock suspension of spermatozoa was initially made by sions were first dialysed against distilled water for 2-3 h to diluting the semen 1:20 (v/v) in Hanks' balanced salt remove the salts. The droplets were placed for 4 min on 300- solution at room temperature. (For observations on the mesh grids (Pioloform-coated, carbon-stabilized), which had motility of intact sperm the Hanks' solution contained been treated immediately before with an aqueous solution of 3mgml~' of bovine serum albumin.) Demembranation was poly-L-lysine (l-5mgml~') rinsed off with de-ionized water. achieved by adding O'OS ml of the sperm suspension to 1 ml of The suspension was then drained off the grid and immedi- a 'reactivation solution' containing 0-04% Triton X-100. The ately replaced by a drop of negative stain. This was then fully reactivation solution was based on that used for mammalian drained off and the gnd allowed to dry. Uranyl acetate (3 % spermatozoa (Yeung & Woolley, 1984) and contained aqueous), the initial choice of negative stain, was later given 230 mM-sucrose, 15 mM-potassium glutamate, 3mM-MgSO.t, up in favour of a 2% aqueous solution of methylamine OSmM-EDTA and 20mM-Tris • HC1 buffer, adjusted to tungstate (Agar Aids Ltd). The quality of the negatively pH 7-9. Naturalistic flagellar beating could be induced in this stained grids was variable and the yield of well-displayed medium by adding 1 mM-ATP, though it lasted only specimens was low. The extended axonemes were photo- 5-10 min. The longitudinal splitting of the distal axoneme graphed on several plates at between X4500 and X 10000, was achieved by adding 20j(gml~' trypsin to the suspension with higher magnification micrographs of the regions where of demembranated sperm for 4min at 23°C and then doublets terminated. Prints (X2-5 enlargement) were as- terminating the digestion with 40j(gml~' trypsin-inhibitor sembled into montages and estimates of the inter-doublet (from soybean; Sigma). displacements were then made by hand, using a map The split, distal portions of the axoneme were then measurer read to l/16 inch. mechanically detached from the proximal axoneme by several passages through a standard Pasteur pipette. Reactivation of these separated groups of doublets was achieved by adding Results ATP (250/JM-l mM, varying according to preparation). Ac- tive sliding was arrested after 5-15 s by mixing the suspen- General observations on flagellar structure and sion with an equal volume of 5 % glutaraldehyde in 0-1 M- sodium cacodylate (pH 7-4). The results of this were moni- motility tored by dark-field light microscopy before proceeding to The structure of the cock has been electron microscopy. described and reviewed by Lake (1981). As a basis for the present study, we illustrate again the main features Microscopy of the flagellum. Its length is 70jUm. There is a short mitochondrial section or midpiece (4^tm), containing Suspensions of living spermatozoa were placed in 100/mi deep chambers, made by supporting covershps on strips of an elongated distal proximally and beyond double-sided tape. For viewing trypsin-treated axonemes, a this a 9 + 2 axoneme surrounded by nine very short \2-fi\ droplet of suspension was covered by an unsupported accessory fibres (Fig. 1). Distal to the midpiece is the 22 mm X 22 mm coverslip giving a depth of approximately principal piece, a 9 + 2 axoneme surrounded, for the 25 /.im. These preparations were viewed using a Leitz Ortho- first approximately 30fxm only, by a tapering - lux II microscope fitted with a X40 oil-immersion planapo- aceous sheath; the sheath is symmetrical in cross- chromat (N.A. l'00), an oil-immersion concentric dark-field section and apparently unattached to the axonemal condenser (N.A. 1-2-1-4), and a heat filter. For photomicro- doublet microtubules (Figs 2, 3). We can add the graphy, short exposures were required to arrest movement information that at the tip of the flagellum the A-tubule and to obviate the drifting of immobile specimens: therefore a flash unit discharging 200 Ws of input energy in 500 f/s was loses its dynein arms and the electron density of its used, as described (Woolley & Osborn, 1984). Images were interior. Subsequently, the B-tubule terminates as a recorded on Kodak Tri-X 35 mm film; development was in loose strand, leaving the A-tubule to continue as a 'Acutol' (Paterson), for twice the normal time. Some images singlet to the tip (Figs 4, 5, 24). The reduced doub- of living spermatozoa were recorded on videotape using the lets are apparently attached to the two-colour dark-field technique for three-dimensional analy- (Figs 4, 5). These features of the tip are fairly typical sis of flagellar movement (Woolley, 1981). of flagella in general (see Woolley & Nickels, 1985). An

362 D. M. Woollev and A. Brammall unusual feature is that the central pair of microtubules by the displacements of the head, the axis of which is at terminates before the doublets, becoming merged into an angle of approximately 35° to that of the midpiecc. a central electron-dense fusiform mass (Figs 4, 23). The rotation occurs even when spermatozoa are swim- The most striking aspect of the movement of the ming close to surfaces and it persists in moribund spermatozoon is the rapid rotation of the cell about the spermatozoa that are executing bends of scarcely ap- progression axis. This rotation or rolling is evidenced preciable amplitude and progressing at very low vel- ocity. Using two-colour videomicroscopy, we have followed, frame-by-frame, the head displacements in such moribund cells and established that in every one of a sample of 25 cells the direction of rotation was anti- clockwise (as would be seen from an imaginary position in advance of the sperm head). In view of this rotation, it is not surprising that the flagellar wave appears to be three-dimensional in short-exposure micrographs of progressively swimming sperm (Figs 6, 7). As yet, we have not attempted a systematic analysis of the wave- form and so only a brief indication of the bend parameters can be given. Considering only those 'in- stantaneous' micrographs in which the tail appears to m be helicoidal, 2-2i cycles of bending are generally seen on the flagellum. The proximal part of the principal 1 piece tends to remain straight, with bends reaching their greatest amplitude in mid-flagellum and reducing in amplitude towards the tip. The sperm photographed for these preliminary observations were swimming progressively at linear velocities within the range 20-30 fims"' at 22°C. A maximum velocity of 165 (Urn s"1 was observed at 40°C (the range of body temperature is 40-43 °C). Data for beat and roll frequency were not obtained.

Effects of treatment with Triton X-100 and tn'psin Thin sections showed that the treatment with Triton X-100 completely removed the cell membrane (Fig. 14) and caused swelling and eventual loss of the

Figs 1-5. The ultrastructure of the intact fowl spermatozoon seen in transverse sections. X91 700. Fig. 1. The midpiece, distal to the distal centriole. The axoneme is surrounded by nine accessory (dense) fibres and, outside these, a cylindrical grouping of mitochondria

("')• Fig. 2. The axoneme in the proximal principal piece, surrounded by a fibrous sheath. The sheath tapers in thickness until it is eventually lost. Its greatest thickness, at the junction with the midpiece, is about twice that seen in this section. Fig. 3. The distal principal piece, beyond the limit of the fibrous sheath. The axoneme has the typical 9 + 2 structure. Fig. 4. This section (centre) passes close to the tip of the axoneme. The doublet microtubules show reduced structure and the central pair of microtubules are replaced by an amorphous mass (see also Figs 23 and 24). Fig. 5. At a level nearer the tip than that in Fig. 4 most of the doublet microtubules are further reduced to singlets. As in Fig. 4, they tend to adhere to the overlying plasmalemma. Bar, 0-2/ini.

Sliding in Gallus spenn axonewes 363 Figs 6-11. Dark-field micrographs, exposure time 500[is. X885. Bar, 25/im. Figs 6, 7. Examples of the 'helicoidal' waveforms commonly seen in progressively swimming spermatozoa. Fig. 8. Motionless spermatozoon after treatment with Triton X-100. The thinness of the midpiece, due to the loss of mitochondria, is apparent at the left of the figure. Figs 9—11. Three micrographs of one spermatozoon showing the effects of trypsin over approximately 2min. In Fig. 9 the nucleus is already dispersed and axonemal splitting is beginning at the tip. It progresses until it is halted by the presence of the fibrous sheath around the axoneme, as in Fig. 11. mitochondria. By light microscopy the immobile fla- The pitch of the helices became gradually reduced gella were seen to have a variety of shapes, usually distally, as did the diameter (Figs 16, 18). A mean irregular and not resembling the images obtained of pitch (p) was measured for individual helices, and then swimming cells (Fig. 8). averaged for a population. In eight experiments, the Exposure of the demembranated sperm to trypsin mean value for p ranged between 3-93 ±S.E. 0-08 fim caused swelling and then complete disruption of the (7? =18 helices) and 5-19±0-12jum (« = 30). The nucleus. The effect on the flagellum was to induce a helices were approximately 3-5 ^im in diameter, mid- longitudinal splitting of the distal part of the axoneme, way along their length. beginning at the tip and progressing during 2 min as far Electron microscopy revealed that these helical as the distal limit of the proteinaceous sheath strands were indeed subsets of the nine doublets. Most (Figs 9-11). In most cases the axoneme split into two often there were four or five doublets in each helix, parts, though sometimes there were three and some- indicating a single plane of splitting. With more times the axonemal cylinder 'opened out' rather than prolonged exposure to trypsin a larger proportion of splitting into separate strands. Occasionally a further, the helices consisted of fewer than four doublets. The very thin strand was just visible, which was sub- doublets were held together by structures in the sequently confirmed by electron microscopy to be the position of the inner dynein arms; no outer dynein central pair of microtubules. The major strands pro- arms were present (Figs 12-15). The radial spokes and duced by splitting all had the form of sinistral helices. spoke-heads remained attached to the A-tubules. They

364 D. M. Woollev and A. Brammall 13 Figs 12, 13. Thin sections of a conventionally fixed pellet of demembranated, trypsinized flagella. X95 000. Bar, 0-2^/m. Fig. 12. At the left of the figure is an intact axoneme, enclosed in its fibrous sheath. Elsewhere, there are ribbons of linked doublets and central pair microtubules. The outer dynein arms are generally absent but the radial spokes remain. Fig. 13. Longitudinal sections from the same preparation show inter-doublet links of irregular form. Figs 14, 15. Tannic acid fixation. X 195 000. Bar, 0-1 pirn. Fig. 14. A Triton-treated flagellum, not trypsinized, showing the doublet structure in detail and the presence of inner and outer dynein arms. Fig. 15. A ribbon of doublets results from trypsin treatment. The removal of the outer dynein arms is confirmed. The remaining inter-doublet links, presumably the inner anus, again appear irregular (see also Figs 25, 26). could be seen distinct from the inter-doublet links in most clearly seen in preparations under coverslips to edge-on views of negatively stained doublets at the which reactivation solution containing ATP was added torsion points resulting from the drying down of the from the side. Extensions to the helices, of diminishing helices (see Fig. 27). Their position on the convex side brightness, were observed emerging from both ends. of such twisted curves means that, in becoming helical, Under these conditions, some of the helices held the doublets bend in such a way that their inner together after the sliding process had reached its limit, surfaces (those bearing the spokes) project away from leaving short regions of greater brightness, represent- the helical axis. Although the spokes and the inner arm ing overlapping doublets, occasionally visible (Figs 19, structures could generally be recognized in negatively 20). When suspensions of detached helices were reacti- stained preparations, the images (e.g. see Fig. 26) have vated in a test tube and fixed during the sliding process not yet been sufficiently regular or distinct to permit a the fixative preserved the helical form of the doublets detailed assessment of their spacings. though it consistently caused an increase in mean pitch (62% greater than unfixed extended helices in each of Reactivation of the helical groups of doublets two experiments). The necessary mixing of solutions, When the split axonemes, still attached to the proximal however, meant that there were fewer intact speci- flagellum, were reactivated by adding 200 fiM- to 1 mM- mens. Nevertheless, a sample of 16 negatively stained ATP to the suspension, active sliding occurred within extended helices was found on which complete analysis both helices and led to extensive looping and entangle- of the displacements was possible (e.g. see Fig. 22). ment of the doublets (Fig. 17). But, after the helices For assessing the polarity of sliding, all extended had been mechanically detached, reactivation pro- helices were included provided that the tip ends of at duced an orderly lengthening of the helices. This was least one of the interacting doublets could be certainly

Sliding in Gallus spenn axonemes 365 established by the characteristic morphology at the tip within a group of doublets, all helices with more than (Fig. 25). (Other criteria of base-tip polarity, such as three doublets were included. spacings or dynein arm angulation were In the 16 specimens photographed and reconstructed not reliable enough in this material.) Also, any doublet in detail, all the sliding interactions had resulted from that did not show regions of regular close apposition to the inner dynein arms having pushed the adjacent B- the relevant neighbouring doublet was excluded from tubule towards the tip (Figs 25-27 and Table 1). The the study. For assessing the relative displacement same conclusion was drawn from a further sample of 21

Figs 16-20. Dark-field micrographs. X1122. Bar, 20;*m. Fig. 16. A trypsinized specimen showing typical features: a thin midpiece, and the distal axoneme split into two helical halves. Fig. 17. A similar specimen following reactivation with ATP. The helices are disrupted and the component doublets extensively entangled. Fig. 18. Helical groups of doublets detached by mechanical agitation. Figs 19, 20. Examples of detached helices after reactivation with ATP. The helices are extended in length and of reduced brightness.

Table 1. Data obtained from electron micrographs of re-activated groups of axonemai doublets

Number Length of doublet (fim) Displacement at tip (/im) of doublets in sequence n, n + 1, n + 2.. in corresponding sequence

6 (?) 38-8, 38-2, 38-4, 38-4, 38-2 12-7, 11-7, 12-8, 13-0, 12-5 5 36 1, 36-3, 36-5, 36-3, 38-1 11-8, 110, 10-7, 9-8 5 (?) (?) 29-0, 29-4, 29-5 10-0, 10-4, 9-4, 10-2 4 44-1, 44-8, 44-7, >43-8 14-4, 15-0, 14-3 4 47-3, 47-0, 47-3, 47-0. 19-5, 20-3, 19-9 4 43-2, 43-5, 45-0, 43-3 8-6, 7-7, 7'2 4 42-3, 42-3, (?) (?) 14-4, 14-3, 13-1 4 38-4, 38-3, 38-4, 38-5 11-0, 11-6, 6-8 4 44-6, 44-7, 44-4, 44-4 7-6, 0-2, 2-6 4 28-4, 28-4, 28-0, 28-1 4-7, 4-2, 5-5 3 43-4, 43-4, 41-3 6-7, 6-5 3 (?) (?) (?) 7-9, 7-3 3 40-5, 38-5, 38-8 13-3, 14-1 3 34-6, 34-3, 33-7 11-5, 11-2 3 49-6, 49-0, 49-2 8-4, 7-9 3 28-5, 28-5, >27-9 8-4, 5-3

The displacements at the tip resulted from active sliding, with in all cases doublet;/ displacing doublet n + 1 towards the tip. In some instances the grid bars prevented an exact estimate of doublet length.

366 D. M. Woolly and A. Brammall Fig. 21. Electron micrograph of part of a negatively stained, unreactivated helix consisting of three doublets. The structure has the appearance expected of a flattened helix, with localized 180° torsions at the bends. X 12700. Bar, 2/.lm. Fig. 22. A similar preparation showing the changes in part of a detached helix resulting from reactivation. The entire extended helix of four doublets was available for analysis. In this sample micrograph the tip (/) of doublet ;; and base (b) of doublet n+3 are clearly distinguished. X24 300. Bar, 1/im.

Sliding in Gallus spertn axonemes 367 sliding interactions seen in less-complete specimens with standing 'displacements' resulting from irregular (from five experiments). Table 1 presents the data for breakage of the flagellum. From the data, it can be said the sliding displacements within the helices and also that the split distal flagellum is maximally about 49jum shows the number of doublets and their lengths. All the in length. The actual sliding velocities cannot be displacements were estimated as displacements be- obtained accurately from this type of experiment but tween the tip ends rather than between the base ends, must have been within the range 0-5-1-5 jums"1 for the where the active displacements might be confounded ATP concentrations applied (data not presented in

27

368 D. M. Wooliev and A. Brammall detail). The relative sliding velocities are assumed to be Tetrahymena cilia without trypsinization (Takasaki & proportional to the displacements within a given helix. Miki-Noumura, 1982) and the present authors have The similarity of these displacements may be judged mimicked the trypsin effect on fowl sperm axonemes from the right-hand column of Table 1. by damaging them osmotically (unpublished data). Recently, the helical coiling of split axonemes from Discussion Anguilla sperm has been observed with or without trypsin digestion. The pitch was 7-12jum and the chirality reported to be dextral (Gibbons et al. 1985). The first finding made in this study of fowl spermato- Even though endogenous proteases may be active in zoa was that as groups of axonemal doublets separate non-trypsinized preparations, there is now consider- from the axoneme they adopt a left-handed helical able evidence, including an extensive series of invert- form. After complete sliding distintegration, the indi- ebrate examples presented by Costello (1973), that a vidual doublets keep their helical shape, so it can be genuine inherent coiling tendency exists in individual concluded that the helical form of the groups is due to doublet microtubules. Doublets held together in cylin- the torsion and bending of the doublets per se and not drical 9+2 arrays have no torsion, nor apparently do the result of interactions between them. One possible groups of fewer than nine doublets, provided they arc interpretation is that the formation of helices reflects arranged as incomplete cylinders (Sale, 1986). torsional tensions present in the intact axoneme. Another explanation is that trypsinization has pro- We have studied these mechanical interactions by duced an asymmetrical denaturation of the doublets, constructing a simple model of the axoneme using giving an artefactual coiling. Most previous reports of open-end zip fasteners stitched together side-by-sidc the phenomenon have been based on studies of trypsin- and then assembled as a cylinder, the interlocking teeth digested material. Thus, Summers & Gibbons (1971) representing inter-doublet bridges. To add the tor- observed that doublets from ATP-disintegrated sea- sional component, strips of helically formed thermo- urchin sperm tended to coil into helices; Zobel (1973) plastic (Armourguard cable protection) were stapled noted the pitch of such helices in various solutions into the overlapping fabric between each fastener. It (1-8 ^m) and claimed that both sinistral and dextral was found that the cylindrical assembly had some types were present; Miki-Noumura & Kamiya (1976) interesting properties. It had no overall torsion, even concluded that doublets from sea-urchin sperm were though each element when separate and the whole uniformly helical (pitch 0-8 fim) and always sinistral. group when opened out as a ribbon had left-handed Later, these authors reported that the helical pitch helical shapes. However, when torsions were imposed increased when the calcium concentration was lowered on the assembled structure, there was a strong resist- (Miki-Noumura & Kamiya, 1979). However, in some ance to a dextral torsion so that the system, when of these later experiments they obtained helices from released, sprang back to its untwisted condition; but dynein-depleted axonemes that had not been exposed there was virtually no resistance to an imposed sinistral to trypsin. Similarly, helices have been prepared from torsion. A further property of the model was that when the thermoplastic spring strips were applied so as to Fig. 23. Negatively stained, unfixed fowl spermatozoon cause the 'doublets' to curve away from the cylindrical showing structures at the tip. Generalized membrane axis, then the model tended to collapse, i.e. not to damage is seen but the persistence of a membrane cap over maintain a circular cross-section. As our results show, the tip is typical. An amorphous fusiform structure is this is the direction of curvature seen in the split present in the midst of the structurally reduced doublets: axonemes. Thus, one role for the radial spokes could be this novel structure also appears in Fig. 4. X58 300. that of supporting the doublet cylinder against a Fig. 24. Negatively stained, glutaraldehyde-fixed, unreactivated helix (5 doublets) showing the structural tendency to collapse inwards. changes in the doublets at the tip of the flagellum. Concerning the direction in which dynein arms exert X58 300. Bar, 0-5/un. force, our findings agree unequivocally with those of Figs 25-27. Negatively stained helices after brief Sale & Satir (1977), who studied reactivated cilia from reactivation and fixation with glutaraldehyde. The Tetrahymena. Since then the only published work distinction between the A tubule (wider, electron-lucent, relating to the matter has been that of Mogami & bearing projections) and the B tubule of each doublet is Takahashi (1983), who demonstrated that the polarity seen in each figure. By recognizing the doublet tip (in of sliding is uniform and uninfluenced by [Ca +] in Fig. 25) and the doublet bases (Figs 26, 27) it follows that disintegrating cilia from Paramecium, but were unable the direction of active displacement, by the inner arms of the A tubules, has been towards the tip of the flagellum. to confirm the actual direction of sliding. A minor X72 600. Fig. 27 includes a localized torsion, where it can question, arising from the attachment of the axonemes be seen that the projections from the doublets (including to a substrate in Sale & Satir's study was whether all presumably the radial spokes) occur only on the outer edge nine doublets were in a re-activatable state. In the of the bend, as arrowed (see text). Bar, 0-5/.im. present study they clearly were, since both halves of the

Sliding in Gallus sperm axonemes 369 split axonemcs, when still attached to the proximal the doublets 1-9 are always arranged clockwise when flagellum, become fully entangled upon re-activation. viewed from the base of the axoneme (Gibbons, 1961). Furthermore, because the detached helices were free in Whichever doublets are activated in a segment of suspension it has been possible to demonstrate the axoneme, it is evident that for a planar, torsion-free same direction of active sliding produced by as many as bend to develop the individual intcr-doublet displace- five doublets from a single axoneme. The recorded ments will not be equal but will depend on their polarity of active sliding therefore applies to all the topographical relationship to the neutral surface (the doublets in the preparation. We suggest also that the surface at 90° to the plane of bending, see Holwill el al. polarity seen is a property of the inner dynein arms. 1979). In measuring inter-doublet displacements This assumes that the absence of outer arms from the within axonemes we have examined the possibility that electron micrographs is not a fixation artefact but there may be intrinsic differences in the rate of sliding, indicates a differential action of trypsin on the inner which could determine the bend plane. We suggest that and outer . Quantitative biochemical data will the data in Table 1 do not support such an idea. Given be required to substantiate this. Mutants of Chlamy- that processes of mixing and diffusion were involved in clomonas are known that contain the inner but not the both starting and arresting the sliding, we do not outer rows of dynein arms. Three such genotypes attribute any significance to the differences in displace- undergo active disintegration following proteolysis ment found. This part of the work is thus in agreement (Okagaki & Kamiya, 1986) and one of them is capable with the short series of estimates for trypsinized of coordinated motility in the intact state, suggesting axonemes made cinematographically by Takahashi el that the inner arm dyneins can be sufficient for al. (1982). axonemal beating (Kamiya & Okamoto, 1985). In summary, the splitting of the fowl sperm axoneme into two regularly arranged groups of doublets has The finding that activated dynein arms displace the given a convenient preparation for studying the active adjacent doublet only towards the tip has implications sliding behaviour of the inner row (apparently) of for the intact axoneme. It can be shown from the dynein arms. It has not been possible, however, to geometrical displacements within a planar, torsion-free discover any functional differentiation between the bend that only four of the nine doublets displace their doublets, either in terms of their capacity for reacti- neighbours towards the tip; the other five must be vation, their polarity of sliding or their rate of sliding. either inactivated or in some way ineffective. In detail, The differential behaviour believed to occur in the the hypothesis is that where the plane of bending passes intact axoneme may well be due to trypsin-sensitive through doublet 1 and between doublets 5 and 6, cycles components involved in controlling either the cross- of bending result from a switching of activation be- bridge cycle or the compliances within the system. tween the dynein arms on the doublet subgroups 1-4 Therefore, a search for alternatives to trypsin digestion and 6—9, with doublet 5 considered inactive through- for preparing subgroups of interacting doublets may be out (Wais-Steider & Satir, 1979). One way to test this worthwhile. Two observations of split axonemes, pre- hypothesis is to see which doublets are extruded from pared without trypsin, have indicated that oscillating the axoneme (at tip or base) using appropriate actively movement is possible (Nakamura & Kamiya, 1978; disintegrating preparations: for example, either of the Kamiya & Okagaki, 1986). groups 2-5 or 7-1 should emerge at the tip if the basal On the other hand it may be worthwhile considering anchorages are destroyed. In the macrocilia of a that all nine doublets are active in a given length of ctenophore, the extrusion pattern seen by Tanim & axoneme. As stated above, equal tipward displace- Tamm (1984) would imply activation of groups 9-2 or ments can occur between the doublets by the develop- 5-7. In the flagellum of the rat sperm, the extrusions ment of a dextral torsion. This torsion would be noted by Olson & Linck (1977) and Mohri & Yano resisted by the supposed inherent coiling tendency of (1982) imply a synchronous activation of the group the doublets, as shown in our mechanical model of the 4-7. And the pattern of axonemal splitting seen in sea- system, and inactivation of the dynein-tubulin sliding urchin sperm by Sale (1986) seems to have involved the would lead to a passive return to the untwisted state. It activation of 7 alone. The question of which doublets is not clear how such a system could create the are active in a given segment is therefore still unsettled. untwisted bends that must be generated if net displace- We reiterate the point made by Sale & Satir (1977) that ments at the axoneme tip are to occur in accordance the trypsin treatment in their work and ours may have with the current sliding-filament hypothesis (Satir, reduced the functional capabilities of the sliding sys- 1968; reviewed by Gibbons, 1981). However, there is tem. However, we also point out that synchronous evidence that bend propagation in mammalian sperma- activation of all nine doublets at a locus, with tipward tozoa is three-dimensional and involves inter-bend displacement of all nine, is geometrically possible but regions that contain dextral torsions (Woollcy & that it would create a right-handed torsion, given that Osborn, 1984; Yeung & Woolley, 1984).

370 D. M. Woollex and A. Brammall A.B. acknowledges receipt of an SERC Research Student- OLSON, G. E. & LINCK, R. W. (1977). Observations on the- ship. The authors are grateful to Miss D. Carter for expert structural components of flagellar axonemes and central technical assistance. pair microtubules from rat sperm. J. Vltrastruct. Res. 61, 21-43. SALE, W. S. (1986). The axonemal axis Ca2+-induced asymmetry of active sliding in sea urchin References sperm tails. J. Cell Biol. 102, 2042-2052. SALE, W. S. & SATIR, P. (1977). Direction of active sliding COSTELLO, D. P. (1973). A new theory on the mechanics of of microtubules in Tetrahvmena cilia. Proc. naln. Acad. ciliary and flagellar motility. I. Supporting observations. Sci. U.SA. 74, 2045-2049. Rial. Bull. mar. biol. Lab., Woods Hole 145, 279-291. SATIR, P. (1968). Studies on cilia. III. Further studies of GIBBONS, B. H., BACCETTI, B. & GIBBONS, I. R. (1985). the tip and a "sliding filament" model of ciliary Live and reactivated motility in the 9 + 0 flagellum of motility. J. Cell Biol. 39, 77-94. Anguilla sperm. Cell Molil. 5, 333-350. SUMMERS, K. E. & GIBBONS, I. R. (1971). Adenosine GIBBONS, 1. R. (1961). Structural asymmetry in cilia and tnphosphate-induced sliding of tubules in trypsin-trcated flagella. Nature, Land. 190, 1128-1129. flagella of sea-urchin sperm. Proc. natn. Acad. Sci. GIBBONS, I. R. (1981). Cilia and flagella of eukaryotes. J. U.SA. 68, 3092-3096. Cell Biol. 91, 107s-124s. TAKAHASHI, K., SHINGYOJI, C. & KAMIMURA, S. (1982). HOLWILL, M. E. J., COHEN, H. J. & SATIR, P. (1979). A Microtubule sliding in reactivated flagella. Svmp. Soc. sliding microtubule model incorporating axonemal twist exp. Biol. 35, 159-177. and compatible with three-dimensional ciliary beating. TAKASAKI, Y. & MIKI-NOUMURA, T. (1982). Shape of the J. ex p. Biol. 78, 265-280. ciliary doublet microtubule in solution..?, molec. Biol. KAMIYA, R. & OKAMOTO, M. (1985). A mutant of 158, 317-324. Chlamydomonas reinhardtii that lacks the flagellar outer TAMM, S. L. & TAMM, S. (1984). Alternate patterns of dyncin arm but can swim. J. Cell Sci. 74, 181-191. doublet microtubule sliding in ATP-disintegrated KAMIYA, R. A. & OKAGAKI, T. (1986). Cyclical bending of macrociha of the Ctenophore Beroe.jf. Cell Biol. 99, two outer-doublet microtubules in frayed axonemes of 1364-1371. Chlamydomonas. Cell Motil. 6, 580-585. WAIS-STEIDEL, J. & SATIR, P. (1979). Effect of vanadate on LAKE, P. E. (1981). Male genital organs. In Form and gill cilia: switching mechanism in ciliary beat. J. Function in Birds, vol. 2 (ed. A. S. King & J. supramolec. Struct. 2, 339-347. McLelland), pp. 1—61. London, New York: Academic WARNER, F. D., PEREAULT, J. G. & MCILVAIN, J. H. Press. (1985). Re-binding of Tetrahymena 13 S and 21 S dynein LAKE, P. E. & STEWART, J. M. (1978). Artificial ATPases to extracted doublet microtubules..7- Cell Sci. Insemination in Poultry. Ministry of Agriculture, 77, 263-287. Fisheries and Food (U.K.), Bulletin 213. London: WOOLLEY, D. M. (1981). A method for determining the H.M.S.O. three-dimensional form of active flagella, using two- MIKI-NOUMURA, T. & KAMIYA, R. (1976). Shape of colour darkground illumination. J. Microsc. 121, microtubules in solutions. Expl Cell Res. 97, 451-453. 241-244. MIKI-NOUMURA, T. & KAMIYA, R. (1979). Conformational WOOLLEY, D. M. & NICKELS, S. N. (1985). Microtubule changes in the outer doublet microtubules from sea termination patterns in mammalian sperm flagella. urchin sperm flagella. J. Cell Biol. 81, 355-360. J. Ultrastruct. Res. 90, 221-234. MOGAMI, Y. & TAKAHASHI, K. (1983). Calcium and WOOLLEY, D. M. & OSBORN, I. W. (1984). Three- microtubule sliding in ciliary axonemes isolated from dimensional geometry of motile hamster spermatozoa. Paramecium caudatum.J. Cell Sci. 61, 107-121. J. Cell Sci. 67, 159-170. MOHRI, H. & YANO, Y. (1982). Reactivation and YEUNG, C.-H. & WOOLLEY, D. M. (1984). Three- microtubule sliding in rodent spermatozoa. Cell Motil. dimensional bend propagation in hamster sperm models Supplement 1, 143-147. and the direction of roll in free-swimming cells. Cell NAKAMURA, S. & KAMIYA, R. (1978). Bending motion in Motil. 4, 215-226. split flagella of Chlamvdomonas. Cell Struct. Fund. 3, ZOBEL, C. R. (1973). Effect of solution composition and 141-144. proteolysis on the conformation of axonemal OKAGAKI, T. & KAMIYA, R. (1986). Microtubule sliding in components. J. Cell Biol. 59, 573-594. mutant Chlamydomonas axonemes devoid of outer or inner dynein arms..J. Cell Biol. 103, 1895-1902. (Received 22 April 1987 - Accepted 26 June 1987)

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