Identification of dynein as the outer arms of sea urchin

Transcription

1 Proc. Natl. Acad. Sci. USA Vol 74, No. 11, pp , November 1977 Cell Biology Identification of dynein as the outer arms of sea urchin sperm axonemes (flagella/adenosinetriphosphatase/motility/immunoelectron microscopy) KAZUO OGAWA*, TOSHIKO MOHRIt, AND HIDEO MOHRItI * Department of Biology, Tokyo Metropolitan University, Setagaya-ku, Tokyo 158; and t Department of Biology, University of Tokyo, Meguro-ku, Tokyo 153, Japan Communicated by Don W. Fawcett, July 29,1977 ABSTRACT The location of dynein, the main flagellar ATPase, within the sea urchin sperm axoneme was investigated by the use of immunofluorescence and immunoelectron microscopy, employing an antiserum against a t tic fragment of dynein 1 (Fragment 1A) purified from sea urchin sperm flagella. The axonemes were found to be stained with the antiserum when examined by an indirect immunofluorescence technique. Immunoelectron microscopy with the antiserum and a ferritin-conjugated IgG fraction of goat antiserum to rabbit IgG revealed that, among the structures within the axoneme, only the outer arms were labeled with ferritin particles. With either the normal serum or antiserum absorbed with Fragment 1A, there were no ferritin particles within the axonemes. When the outer arms were extracted with 0.5 M NaCl, leaving the inner arms intact, again no ferritin dots were detected. Furthermore, it was found that the outer arm on the no. 5 doublet microtubule, which connects with the extra arm projection backward from the no. 6 doublet, had no attached ferritin particles. From these observations, it can be concluded that the outer arm consists of dynein (at least dynein 1) and that Fragment 1A, containing the active site for ATPase activity of dynein 1, is located at the distal end of the outer arms. The significance of the present findings is considered in connection with flagellar movement. Flagellar movement is a particular, rhythmic, propagating bending. The sliding-microtubule model, principally based on the sliding of the arms against the neighboring outer doublet microtubules, has been presented for interpreting the mechanisms that are responsible for producing this bending (1-4). Experimental evidence for such a sliding process was reported by Summers and Gibbons (5), who observed that when ATP was added to trypsin-treated axonemes, one to several of the outer doublets were extruded from these axonemes. We have succeeded in preparing an antiserum against a tryptic fragment of dynein 1 [Fragment 1A; the new terminology for dynein (6) will be used in this paper] believed to be the arms (7, 8). When this antiserum to Fragment 1A was added to Triton X-100-extracted spermatozoa reactivated with ATP, both beat frequency and bend angle were decreased and the spermatozoa finally ceased to move (8-10). If we can demonstrate that (i) antibody specifically binds to the arms, not to other axonemal structures such as the radial spokes, and (ii) the active site -for ATPase activity of dynein faces toward the neighboring outer microtubule, then the model which holds that interaction between the arms and the outer microtubules is enough to produce a typical flagellar movement would be confirmed. As described in the following sections, immunoelectron microscopy with the antiserum to Fragment 1A and a ferritinconjugated IgG fraction of goat antiserum to rabbit IgG re- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C solely to indicate this fact vealed that, among the structures within the axoneme, only the outer arms were labeled with ferritin particles and that the enzymatically active site is located in the distal part of the outer arm. MATERIALS AND METHODS Spermatozoa of the sea urchin Anthocidaris crassispina were used in this study. Some points were corroborated with other species, Pseudocentrotus depressus and Hemicentrotus pulcherrimus. Shedding was induced by introducing a few drops of 0.5 M KCI into the body cavity. The spermatozoan plasma membrane- was removed by adding 20 volumes of 0.025% (vol/vol) Triton X-100 in 0.15 M KCI/4 mm MgSO4/1 mm CaCl2/2 mm EDTA/5 mm 2- mercaptoethanol/2 mm Tris-HCl, ph 8.2. After 1 min at room temperature, the sperm suspension was diluted 6-fold with the above medium without Triton X-100, and then centrifuged at 3000 rpm for 5 min. (Centrifugations were in a Hitachi instrument.) The sedimented spermatozoa were washed three times with the same medium and finally suspended in a small volume of 0.1 M sodium phosphate buffer, ph 7.5. In order to remove the outer arms from the flagellar axoneme, the spermatozoa were first suspended in 0.04% Triton X-100/0.15 M NaCl/4 mm MgSO4/0.5 mm EDTA/1 mm dithiothreitol/2 mm Tris-HCI, ph 8.0, and homogenized to detach the flagella from the heads. The homogenate was centrifuged at 4000 rpm for 10 min to remove the heads, the presence of which was found to be undesirable in the following procedure, and the supernatant was centrifuged at 12,000 rpm for 10 min at 4. The sedimented axonemes were treated with 0.5 M NaCl/4 mm MgSO4/1 mm dithiothreitol/2 mm Tris- HCl, ph 8.0, for 10 min at room temperature, and then immediately centrifuged at 12,000 rpm for 10 min at 4. The final precipitate was suspended in 0.1 M sodium phosphate buffer, ph 7.5. Antiserum against a tryptic fragment of dynein 1 (Fragment 1A) purified from sperm flagella of Anthocidaris crassispina was prepared according to the method of Ogawa and Mohri (7, 11), using a rabbit. The antiserum specifically reacted with dynein 1 (A-polypeptide), judging from the results of an immunodiffusion test, inhibition of ATPase activity, and sodium dodecyl sulfate/electrophoresis of the antigen-antibody complex (6, 7). For indirect immunofluorescence microscopy, spermatozoa were either glycerinated as described previously (12), or demembranated with Triton X-100 as described above. In some cases, the demembranated flagella were isolated from the heads and collected by centrifugation. A drop of sperm suspension or flagellar suspension was placed on a glass slide and Abbreviation: PBS, phosphate-buffered saline. t To whom reprint requests should be addressed.

2 Cell Biology: Ogawa et al. FIG. 1. Indirect immunofluorescent staining of sea urchin spermatozoa with anti-fragment 1A serum. (a) Triton-treated spermatozoa (Anthocidaris crassispina). (X800.) (b) Isolated axonemes (Pseudocentrotus depressus). (X600.) air dried. After fixing with 5% formalin in phosphate-buffered saline (PBS), the specimen was washed with distilled deionized water and dried again. Then, a 1:20 or 1:40 dilution of the antiserum with PBS was applied to the specimen overnight at 40 in a moist chamber. After washing with PBS, the specimen was incubated with a 1:40 dilution of fluorescein-conjugated goat antiserum against rabbit 7S IgG (Hyland) in PBS, washed with PBS, and enclosed in 50% (vol/vol) glycerol/pbs with a cover slip. Fluorescence was observed with a Nikon FK fluorescence microscope. Pictures were taken on Kodak Tri-X films and developed with Microdol X (Kodak) for an ASA of 400. Immunoelectron microscopy with ferritin was conducted as follows. A commercial ferritin (Nutritional Biochemicals, "Cd trace") was further purified by repeated fractionation with ammonium sulfate, followed by dialysis against distilled deionized water and 0.1 M sodium phosphate buffer, ph 7.5, at 4. The ferritin was then precipitated by centrifugation at 100,000 X g for 2 hr and dissolved in a small volume of 0.1 M sodium phosphate buffer, ph 7.5. The purified ferritin was conjugated with IgG fraction of goat antiserum to rabbit IgG (Miles-Yeda), using p,p'-difluoro-m,m'-dinitrodiphenyl sulfonate (FNPS) as the intermediate coupling molecule after the method of SriRam et al. (13) with slight modification. Samples such as demembranated spermatozoa were pre-fixed with 2.5% glutaraldehyde in the phosphate buffer for 1 hr at 00 and Proc. Natl. Acad. Sc. USA 74 (1977) 5007 washed well three times with the buffer. To the samples suspended in PBS, l/lo to '/3o volume of the anti-fragment 1A serum was added, and the mixture was incubated for 1 hr at room temperature with gentle shaking. After washing with the phosphate buffer three times, the ferritin-conjugated anti-rabbit IgG serum was added to the samples at the final ferritin concentration of about 10 mg/ml of PBS. The mixture was again incubated for 1 hr at room temperature and washed with phosphate buffer three times. The antiserum-treated samples were post-fixed with 1% osmium tetroxide, embedded in Epon 812, and thin-sectioned by the standard procedure. Sections were stained with uranyl acetate, or uranyl acetate and lead citrate. Observations were made with a Hitachi HS9 electron microscope. As control experiments, anti-fragment IA serum absorbed with a small excess of the antigen, the amount of which had been checked by an immunodiffusion test, or nonimmune serum obtained before immunizing the rabbit were applied instead of the antiserum. RESULTS Immunofluorescent Staining of Sea Urchin Spermatozoa. As shown in Fig. la, by the indirect immunofluorescence technique the flagella of Triton-treated sea urchin spermatozoa were brightly stained with the antiserum against Fragment 1A. The same was true with either the flagella of glycerinated spermatozoa or isolated flagellar axonemes (Fig. lb). Bright fluorescence was also detected in the sperm heads, especially at the acrosomal region and the base of the flagellum in the Triton-treated spermatozoa. The mitochondria, however, were usually lacking in the Triton-treated specimens. Although the spermatozoa stained with nonimmune serum as well as the untreated control emitted some fluorescence, it was grey and clearly distinguishable from the green of fluorescein. From these results, it can be concluded, at least, that the axonemes were stained with the anti-fragment IA serum, indicating the presence of the antigen, dynein 1, in the sperm axonemes; the reason why the heads were stained remains to be investigated. Immunoelectron Microscopy Concerning Localization of Dynein within the Axoneme. As the next step, axonemes la. II If t' a b. t FIG. 2. Immunoelectron microscopy of sea urchin (Anthocidaris crassispina) sperm axonemes treated with anti-fragment 1A serum and ferritin-conjugated IgG fraction of goat anti-rabbit IgG. (X100,000.) (a) Control with no serum. Numbering of the outer doublets is after Afzelius (14). (b) Experimental, with antiserum and ferritin-conjugated IgG. (c) With antiserum absorbed with Fragment 1A. (d) With nonimmune serum. d

3 5008 Cell Biology: Ogawa et al. Proc. Natl. Acad. Sci. USA 74 (1977) r ,at. -1 I I -.0 I.- 1: -14, FIG. 3. Immunoelectron microscopy of sea urchin (Anthocidaris crassispina) sperm axonemes after extraction of the outer arms and treatment with antiserum and ferritin-conjugated IgG. (X100,000.) (a) Experimental, with antiserum. (b) With nonimmune serum. treated with anti-fragment 1A serum and the ferritin-conjugated IgG fraction of goat anti-rabbit IgG were examined under the electron microscope, in order to determine where dynein 1 (Fragment 1A) is localized. On the cross sections of axonemes of Triton-treated sea urchin spermatozoa, the ferritin particles were detected between the outer doublets, in the vicinity of the distal end of the outer arms (Fig. 2b; compare with Fig. 2a). With appropriate concentrations of the ferritin-conjugated IgG, almost all outer arms were decorated with ferritin particles. On the other hand, no ferritin dots were observed near the inner arms or any other structures within the axoneme such as the microtubules, the spokes, the nexin links, or the central sheath. In longitudinal sections of the axonemes, only one side of the outer doublet was labeled with ferritin. It appeared that there was a minimum periodicity of approximately 20 nm in the labeling. When the antiserum was replaced with either the antiserum absorbed with Fragment 1A (Fig. 2c) or the nonimmune serum (Fig. 2d), no ferritin particles were in the axonemes. The same was true when free ferritin was incubated with the Triton-treated spermatozoa. It might be possible that the antiserum and/or the ferritinconjugated IgG could not reach the inside of the axonemes because of the presence of the outer arms. To examine this possibility, axonemes from which the outer arms had been extracted were also processed as described above. As shown in Fig. 3, no ferritin particles were present near the inner arms or inside the axoneme in this case. Whenever ferritin dots were found, there were always outer arms that had not been removed by extraction. Conversely, in a few cases of Triton-treated spermatozoa where all the outer arms were lacking accidentally, no ferritin labeling was observed. Furthermore, only the outer arms, not the inner arms, were labeled in the cases where the structures of Triton-treated sperm axonemes appeared as opened or fragmented arrays owing to breakdown of the inter-doublet links. On inspecting the electron micrographs, it was noted that the ferritin labeling was quite rare on the outer arm projecting from the no. 5 outer doublet. The results of the counts are summarized in Table 1 (see also Fig. 2b). Whereas there was almost complete labeling of the outer arms on other doublets (an average of 94.9%), fewer than one-fourth of the outer arms on no. 5 were labeled. The labeling percentage of the no. 4 and no. 6 outer arms was also somewhat lower than the percentages of the other doublets, which averaged 95.9%, probably reflecting an error in identifying the no. 1 doublet. If this is taken into account, the real occurrence of labeling of the no. 5 outer arms would be further reduced. It has been reported that both the outer and the inner arm on the no. 5 doublet are bridged with the extra projections from the no. 6 doublet (14). This suggests that binding of the antibody against Fragment 1A to the outer arm was prevented by the presence of the projection. Apart from the axoneme, ferritin particles were found on the membrane remaining around the acrosomal region of Tritontreated spermatozoa; this is consistent with the bright staining of this region in the immunofluorescence experiment. The same was true at the basal region of the head. Little labeling with ferritin was obtained in other parts of the sperm head, which lacked the plasma membrane and nuclear membrane. Very few dots of ferritin were found in detached mitochondria, not surrounded by the plasma membrane, if they remained intact. DISCUSSION In both immunofluorescence microscopy and immunoelectron microscopy of Triton-treated sea urchin spermatozoa, the apical and basal regions of the sperm heads were labeled with the anti-fragment LA serum. Under the electron microscope, the plasma membrane was found to be preserved in these regions. Several possibilities are conceivable concerning the labeling of the plasma membrane: (i) dynein 1 or related ATPase protein is actually present on the sperm plasma membrane in addition to the outer arms in the axonemes; (ii) the anti-fragment 1A serum, although it specifically reacts with dynein 1 in an immunodiffusion test or inhibits the ATPase activity of dynein 1, still contains antibodies against some sort of protein(s) in the plasma membrane; (i) the antiserum is merely absorbed to the plasma membrane., In a preliminary experiment, we examined the effect of anti-fragment 1A serum on ATPase activity of the supernatant obtained after sedimenting glycerinated spermatozoa, which seemed to contain fragments of the plasma membrane. The activity was almost completely inhibited by the antiserum. Furtbermore, we have found that the fluorescent anti-fragment 1A serum stained the peripheral area of cleaving sea urchin eggs together with their mitotic apparatus (15), and that Mg-ATPase activity of the isolated egg cortex was considerably reduced by the antiserum, suggesting the presence of dynein 1 in the stained area (Y. Kobayashi, K. Ogawa, and H. Mohri, unpublished data). Regardless of the reason for the Table 1. Occurrence of ferritin labeling on outer arms of sea urchin sperm flagella Outer doublet no Count of ferritin labeling Count X 100/total count The number of axonemes examined (total count) was 387.

4 Cell Biology: Ogawa et al. labeling of sperm heads or plasma membranes, the present conclusion is unaffected: dynein 1 is located at the outer arms of sea urchin sperm axonemes. Previously, both the outer and inner arms of Tetrahymena cilia and sea urchin sperm flagella were considered to consist of the same dynein molecule (16, 17). Furthermore, Gibbons and Gibbons (18) reported that reactivated spermatozoa of the sea urchin Colobocentrotus atratus, following brief extraction with 0.5 M KC1, will swim with normal wave form but with half-normal beat frequency. The KC1 treatment appeared to specifically extract the outer of each pair of arms, suggesting that both arms are functionally equivalent, although the result has not been confirmed in other sea urchins. The 0.5 M KCI extraction decreased both the amplitude and beat frequency and the long extraction (5-10 min) immobilized the Tritontreated spermatozoa (10). In the present work, on the other hand, no ferritin particles were observed near the inner arms, suggesting that the inner arm does not consist of dynein 1. Recently three bands (C-, D-, and B-polypeptides) around the dynein 1 band (A-polypeptide) were revealed by sodium dodecyl sulfate gel electrophoresis of the sea urchin axonemes, and one of them (D-polypeptide) corresponded to dynein 2, which is another axonemal ATPase immunologically distinct from dynein 1 (6, 10). Furthermore, recent studies have shown that 30S and 14S dyneins obtained from Tetrahymena axonemes, which had been thought to correspond to polymer and monomer of single dynein species, are distinct ATPases (19). Dynein 1 from sea urchin axonemes is functionally similar to 30S dynein from Tetrahymena axonemes, but the relation between dynein 2 and 14S dynein is still obscure. The Tris/ EDTA extract from Tetrahymena axonemes also contains a few other proteins with molecular sizes similar to those of dyneins. In the recombination experiments used to localize dynein previously, the possibility that these proteins were re-bound to the microtubules as the inner arms therefore cannot be ruled out. The inner arm may consist of dynein 2, although the amount of dynein 2 so far estimated is much smaller (one fifth) than that of dynein 1 and there is another possibility that dynein 2 may be located in the heads of the radial spokes (see ref. 6), or the inner arm may be composed of B-polypeptide with little ATPase activity, the amount of which is more comparable to that of A-polypeptide. It is also possible that the A-band appearing on the sodium dodecyl sulfate gel contains both dynein 1 and another similar polypeptide [as A1 and A2, postulated by Kincaid et al. (20)], the latter corresponding to the inner arm. Further studies are needed to clarify this point. In previous experiments, we found that only 45% of the ATPase activity of glycerinated spermatozoa (A. crassispina) was inhibited by an excess of anti-fragment IA serum (8), although the antiserum almost completely inhibited the ATPase activity of purified dynein 1 or Fragment la (7). Thus, it is conceivable that the residual activity is mostly due to the inner arms, which may consist of an ATPase protein equivalent to but immunologically distinct from dynein 1 (a different form of dynein 1?). However, K. Ogawa (unpublished) has found that the inhibition of ATPase activity of glycerinated spermatozoa by the antiserum is highly dependent on the KCl and ATP concentrations. Under the assay conditions of 2 mm MgCl2/10 mm Tris.HCl, ph 8.3, and 1 mm and 0.33 mm ATP, 55 and 68% inhibition at 0.1 M KC1, 60 and 74% inhibition at 50 mm KC1, and 64 and 80% inhibition at zero KCI were obtained, respectively, in H. pulcherrimus. Furthermore, we found that only the outer arms were labeled with the antiserum even when the reaction was made in 10mM Tris*HCl instead of PBS containing salts. These results appear to be inconsistent with the Proc. Natl. Acad. Sci. USA 74 (1977) 5009 idea that most of the ATPase activity remaining after the antiserum treatment is due to the dynein 1(?) constituting the inner arms. As already mentioned, the anti-fragment 1A serum reduced both the beat frequency and amplitude of the reactivated spermatozoa and finally stopped their movement (8-10). Furthermore, the ATP-driven extrusion of outer doublets from trypsin-treated axonemes, which gives experimental evidence of the sliding microtubule model for flagellar movement, was also inhibited by this antiserum (H. Masuda, K. Ogawa and T. Miki-Noumura, unpublished data). The present result clearly demonstrated that the binding of antibody to the outer arms is enough to cause the inhibition. In other words, the interaction between the distal part of the outer arms (Fragment A portion containing an active site for ATPase activity) and the B-tubule of the neighboring outer doublets appears to produce a particular, rhythmic, propagating bending. Because the activation of ATP-dephosphorylation of dynein 1 by the microtubules (10, 21) occurs during this movement, the biochemical features of this interaction seem to be almost the same as those of myosin-actin interaction during muscular contraction. Flagellar bending occurs in the plane perpendicular to the central pairs of microtubules. In this connection, it should be noted that the outer arm projecting from the no. 5 outer doublet was only sparsely labeled with ferritin particles. As already mentioned, the paired arms of the no. 5 doublet are connected with the extra projections from the no. 6 doublet (14). It is interesting that, in an earlier cytochemical observation of ATPase localization in sperm flagella of Drosophila melanogaster, lead precipitates were found at positions very similar to the ferritin labelings in the present study (22), and that the no. 5 doublet appears to lack the lead precipitate (see figures 2 and 4 in ref. 22). From these facts together with the present findings, it is likely that the no. 5 and no. 6 doublets are firmly bound so that they do not move with respect to each other, and the arm(s), probably the outer arm, of the no. 5 doublet do not function as an active ATPase. In a sense, this would present further support for the sliding hypothesis, because if local contraction of the doublet microtubules is responsible for flagellar bending, the arm(s) on no. 5 (or no. 6) should produce more motive force than other doublets for bending in one direction, and the arm(s) on no. 1 in another direction. We thank Dr. Jean C. Dan and Prof. Y. Hiramoto for their critical reading of the manuscript. This work was supported in part by grants from the Ministry of Education of Japan and a grant from the Ford Foundation to H.M. 1. Satir, P. (1965) J. Cell Biol. 26, Brokaw, C. J. (1972) Science 178, Brokaw, C. J. (1975) in Molecules and Cell Movement, eds. Inoue, S. & Stephens, R. E. (Raven Press, New York), pp Gibbons, I. R. (1975) in Molecules and Cell Movement, eds. Inoue, S. & Stephens, R. E., (Raven Press, New York), pp Summers, K. E. & Gibbons, I. R. (1971) Proc. Natl. Acad. Sci. USA 68, Ogawa, K. & Gibbons, I. R. (1976) 1. Biol. Chem. 251, Ogawa, K. & Mohri, H. (1975) J. Biol. Chem. 250, Okuno, M., Ogawa, K. & Mohri, H. (1976) Biochem. Biophys. Res. Commun. 68, Gibbons, B. H., Ogawa, K. & Gibbons, I. R. (1976) J. Cell Biol. 71, Ogawa, K., Asai, D..J& Brokaw, C. J. (1977) J. Cell Bol. 73,

5 5010 Cell Biology: Ogawa et al. 11. Ogawa, K. & Mohri, H. (1972) Biochim. Biophys. Acta 293, Yanagisawa, T., Hasegawa, S. & Mohri, H. (1968) Exp. Cell Res. 52, SriRam, J., Tawde, S. S., Pierge, G. B. & Midgley, A. R. (1963) J. Cell Biol. 17, Afzelius, B. A. (1959) J. Biophys. Biochem. Cytol. 5, Mohri, H., Mohri, T., Mabuchi, I., Sakai, H., Yazaki, I. & Ogawa, K. (1976) Dev. Growth Differ. 18, Gibbons, I. R. (1963) Proc. Nati. Acad. Sci. USA 50, Proc. Nati. Acad. Sci. USA 74 (1977) 17. Mohri, H., Hasegawa, S., Yamamoto, M. & Murakami, S. (1969) Sci. Pap. Coll. Gen. Educ. Univ. Tokyo 19, Gibbons, B. H. & Gibbons, I. R. (1973) J. Cell Sci. 13, Mabuchi, I. & Shimizu, T. (1974) J. Biochem. (Tokyo) 76, Kincaid, H. L., Jr., Gibbons, B. H. & Gibbons, I. R. (1973) J. Supramol. Struct. 1, Ogawa, K. (1973) Biochim. Biophys. Acta 293, Daems, W. Th., Persijn, J.-P. & Tates, A. D. (1963) Exp. Cell Res. 32,