The effect of antidynein 1 serum on the movement of reactivated sea urchin sperm.

Gibbons, Barbara; Ogawa, Kazuo; Gibbons, I. R.

doi:10.1083/jcb.71.3.823

Cited by 92 publications

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“…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)(9)(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model for flagellar movement, was also inhibited by this antiserum (H. Masuda, K. Ogawa and T. Miki-Noumura, unpublished data).…”

Section: Resultsmentioning

confidence: 99%

“…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)(9)(10) 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.…”

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Identification of dynein as the outer arms of sea urchin sperm axonemes.

Ogawa¹,

Mohri²,

Mohri³

1977

Proc. Natl. Acad. Sci. U.S.A.

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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) 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 METHODSSpermatozoa 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 fla...

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Identification of dynein as the outer arms of sea urchin sperm axonemes.

Ogawa¹,

Mohri²,

Mohri³

1977

Proc. Natl. Acad. Sci. U.S.A.

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“…dynein. For example, an antiserum that inhibits the ATPase activity of sea urchin sperm dynein caused a corresponding decrease in the beat frequency of reactivated sea urchin sperm, supporting the hypothesis that the dynein ATPase drives axonemal motility (Ogawa and Mohri, 1975;Okuno et al, 1976;Gibbons et al, 1976). In addition, polyclonal and monoclonal myosin antibodies have been used to inhibit the actin activated ATPase activity of myosin, (Mabuchi and Okuno, 1977;Kiehart and Pollard, 1984b;Peltz et al, 1985) to block myosin-driven motility in vitro (Kiehart and Pollard, 1984b;Flicker et al, 1985) to examine the relationship between myosin polymerization and ATPase activity as well as to probe the conformation and polymerization state of myosin (Citi and KendrickJones, 1987).…”

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confidence: 79%

Inhibition of kinesin-driven microtubule motility by monoclonal antibodies to kinesin heavy chains.

Ingold

Cohn

Scholey

1988

The Journal of Cell Biology

174

153

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Abstract, We have prepared and characterized seven mouse monoclonal antibodies (SUK 1-7) to the 130-kD heavy chain of sea urchin egg kinesin. On immunoblots, SUK 3 and SUK 4 cross-reacted with Drosophila embryo ll6-kD heavy chains, and SUK 4, SUK 5, SUK 6, and SUK 7 bound to the 120-kD heavy chains of bovine brain kinesin. Three out of seven monoclonal antikinesins (SUK 4, SUK 6, and SUK 7) caused a dose-dependent inhibition of sea urchin egg kinesininduced microtubule translocation, whereas the other four monoclonal antibodies had no detectable effect on this motility. The inhibitory monoclonal antibodies (SUK 4, SUK 6, and SUK 7) appear to bind to spatially related sites on an ATP-sensitive microtubule binding 45-kD chymotryptic fragment of the 130-kD heavy chain, whereas SUK 2 binds to a spatially distinct site. None of the monoclonal antikinesins inhibited the microtubule activated MgATPase activity of kinesin, suggesting that SUK 4, SUK 6, and SUK 7 uncouple this MgATPase activity from motility.V ARIOUS forms of intracellular transport are thought to depend upon microtubule-associated mechanochemical ATPases (Vale, 1987). One such enzyme, kinesin (Vale et al., 1985a), binds microtubules in a nucleotide sensitive fashion, and displays a microtubule-activated ATPase activity that drives movement of kinesin-coated objects towards the plus end of microtubules (MTs) ~ in vitro (Vale et al., 1985a, b;Scholey et al., 1985;Brady, 1985; Kuznetsoy and Gelfand, 1986;Porter et al., 1987;Cohn et al., 1987;Saxton et al., 1988;Gelles et al., 1988). Kinesin from a variety of animals contains a major polypeptide with an apparent molecular mass of 110-140 kD, encoded by a single gene in Drosophila (Yang et al., 1988), plus copurifying light chains of Mr = 40-80 kD (Vale et al., 1985a,b;Amos, 1987;Kuznetsov and Gelfand, 1986;Saxton et al., 1988). Bovine brain kinesin is an tt2132 heterotetramer consisting of two 120-kD heavy chains plus two 60-kD light chains, assembled into an elongated molecule (Amos, 1987;Kuznetsov et al., 1988;Bloom et al., 1988). Kinesin is proposed to participate in organelle/vesicle transport, in organizing the endomembrane system, and in mitosis (Vale et al., 1986;Vale, 1987;Leslie et al., 1987;Schroer and Sheetz, 1988), but direct evidence concerning the biological functions of kinesin is currently lacking.Antibodies that inhibit mechanochemical activity have been extremely useful in probing the functions of myosin and 1. Abbreviations used in this paper: MT(s), microtubule(s); PMEG, 0.9 M glycerol, 0.1 M Pipes, pH 6.9, 5 mM EGTA, 2.5 mM MgSO4, 0.5 mM EDTA, 1 mM DTT, 100 Ixg/ml soybean trypsin inhibitor, 1 mg/mlp-tosyl-Larginine methyl ester hydrochloride, 10 Ixg/ml leupeptin, pepstatin, and aprotinin; RT, room temperature. dynein. For example, an antiserum that inhibits the ATPase activity of sea urchin sperm dynein caused a corresponding decrease in the beat frequency of reactivated sea urchin sperm, supporting the hypothesis that the dynein ATPase drives axonemal motility (Ogawa and Mohri, 1975;O...

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“…Chemical and functional equivalences have been shown between the outer and inner arms of sea urchin sperm axonemes on the basis of chemical dissection (6,14) . The present work, together with the previous report (22), has tried to demonstrate the immunological dissimilarity in protein component (dynein 1) between the two members of a pair of arms, since a major area of controversy in the eukaryotic flagellar motility at present is the question of whether they are identical in protein component and function .…”

Section: Discussionmentioning

confidence: 99%

Immunological dissimilarity in protein component (dynein 1) between outer and inner arms within sea urchin sperm axonemes

Ogawa¹,

Negishi²,

Obika³

1982

The Journal of Cell Biology

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The 0.5 M KCI-treatment solubilizes the outer arms from sea urchin sperm axonemes. -30% of A-polypeptide, corresponding to dynein 1 in SDS-polyacrylamide gel, was solubilized by this treatment (as SEA-dynein 1) . Electron microscopic observation indicated that the extracted axonemes lacked the outer arms in various degrees. The SEA-dynein 1 was purified and an antiserum against it was prepared in rabbits. The specificity of antiserum to dynein 1 was determined by immunoelectrophoresis and Ouchterlony's double-diffusion test . The anti-dynein 1 serum inhibited ATPase activity of purified SEA-dynein 1 by 95%. By the indirect peroxidase-conjugated antibody method, the loci of SEA-dynein 1 within the intact, salt-extracted and mechanically disrupted axonemes were determined to be the outer arms : deposition of electron-dense materials which represents their localization was detected at the distal ends of the outer arms, in the case of intact axonemes . The 5-6 cross-bridge was hardly decorated . No decoration was seen in the salt-extracted axonemes lacking all the outer arms . In disrupted axonemes, which consist of single to several peripheral doublets, electron-dense materials were deposited only at the outer arms .-73% of axonemal ATPase activity sensitive to antiserum was solubilized by repeated saltextractions . One-half of A-polypeptide (SEA-dynein 1 located at the outer arms) was contained in the pooled extracts . The extracted axonemes contained another half of A-polypeptide (SUAdynein 1 supposed to locate at the inner arms) and retained 31% of axonemal ATPase activity that was almost resistant to antiserum . Solubilized SUA-dynein 1 was immunologically the same as SEA-dynein 1 . This result indicates that in situ SUA-dynein 1 did not receive antidynein 1 antibodies, coinciding with the result obtained for salt-extracted axonemes lacking all the outer arms by the enzyme-antibody method mentioned above. These observations suggest the immunological dissimilarity in dynein 1 between outer and inner arms but do not tell us that the inner arms do not contain dynein 1 .

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The effect of antidynein 1 serum on the movement of reactivated sea urchin sperm.

Cited by 92 publications

References 14 publications

Identification of dynein as the outer arms of sea urchin sperm axonemes.

Identification of dynein as the outer arms of sea urchin sperm axonemes.

Inhibition of kinesin-driven microtubule motility by monoclonal antibodies to kinesin heavy chains.

Immunological dissimilarity in protein component (dynein 1) between outer and inner arms within sea urchin sperm axonemes

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