The bound nucleotides of the isolated microtubules of sea-urchin sperm flagella and their possible role in flagellar movement

Yanagisawa, Tomio; Hasegawa, Sanaé; Mohri, Hideo

doi:10.1016/0014-4827(68)90549-1

Cited by 50 publications

(13 citation statements)

References 21 publications

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“…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.…”

Section: Methodsmentioning

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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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: Methodsmentioning

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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“…Studies on both sea urchin sperm and Chlamydomonas revealed that demembranated flagella exhibit NDKase activity, suggesting that nucleotides other than ATP may be of functional importance (Yanagisawa et al, 1968;Kobayashi et al, 1976;Watanabe and Flavin, 1976). However, apart from the identification of NDK modules in the IC1 polypeptide from sea urchin sperm outer arm dynein (Ogawa et al, 1996), the intraflagellar location of these enzymes and the role they might play in motility has remained almost completely unexplored.…”

Section: Discussionmentioning

confidence: 99%

“…Furthermore, isolated Chlamydomonas flagella have been reported to exhibit two distinct nucleoside diphosphate kinase 1 (NDKase) activities, which catalyze the general reaction ATP ϩ NDP 3 ADP ϩ NTP; at least one of these enzymes has a marked preference for generating GTP (Watanabe and Flavin, 1976). Similarly, NDKase activity has been detected in sea urchin sperm flagella (Yanagisawa et al, 1968;Kobayashi et al, 1976), and there are also several mammalian NDKs (nm23-H5, -H7, -H8, and Article published online ahead of print. Mol.…”

Section: Introductionmentioning

confidence: 99%

Flagellar Radial Spokes Contain a Ca²⁺-stimulated Nucleoside Diphosphate Kinase

Patel‐King

Gorbatyuk

Takebe

et al. 2004

MBoC

104

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The radial spokes are required for Ca 2؉ -initiated intraflagellar signaling, resulting in modulation of inner and outer arm dynein activity. However, the mechanochemical properties of this signaling pathway remain unknown. Here, we describe a novel nucleoside diphosphate kinase (NDK) from the Chlamydomonas flagellum. This protein (termed p61 or RSP23) consists of an N-terminal catalytic NDK domain followed by a repetitive region that includes three IQ motifs and a highly acidic C-terminal segment. We find that p61 is missing in axonemes derived from the mutants pf14 (lacks radial spokes) and pf24 (lacks the spoke head and several stalk components) but not in those from pf17 (lacking only the spoke head). The p61 protein can be extracted from oda1 (lacks outer dynein arms) and pf17 axonemes with 0.5 M KI, and copurifies with radial spokes in sucrose density gradients. Furthermore, p61 contains two classes of calmodulin binding site: IQ1 interacts with calmodulin-Sepharose beads in a Ca 2؉ -independent manner, whereas IQ2 and IQ3 show Ca 2؉ -sensitive associations. Wild-type axonemes exhibit two distinct NDKase activities, at least one of which is stimulated by Ca 2؉ . This Ca 2؉ -responsive enzyme, which accounts for ϳ45% of total axonemal NDKase, is missing from pf14 axonemes. We found that purified radial spokes also exhibit NDKase activity. Thus, we conclude that p61 is an integral component of the radial spoke stalk that binds calmodulin and exhibits Ca 2؉ -controlled NDKase activity. These observations suggest that nucleotides other than ATP may play an important role in the signal transduction pathway that underlies the regulatory mechanism defined by the radial spokes. INTRODUCTIONThe eukaryotic cilium/flagellum is a highly conserved motile organelle built around nine outer doublet microtubules and a central pair of singlet microtubules that form the 9 ϩ 2 axoneme. Individual flagella provide for the motility of single cells such as mammalian sperm and Chlamydomonas, and ciliated epithelia are essential for fluid/mucus transport in many organs. Moreover, nodal cilia are required to define the left-right axis during mammalian development (Supp et al., 1997;Nonaka et al., 1998). Many cells also contain primary cilia that seem to play critical sensory roles during normal tissue function and development . The power for ciliary/flagellar movement is generated by the inner and outer rows of dynein arms that associate with the A tubule of each microtubule doublet and transiently interact with the B tubule of the adjacent doublet to produce a linear force vector that results in the microtubules sliding with respect to each other. This sliding motion is then converted to a flagellar bend by other axonemal components, including the radial spoke and central pair microtubule complexes (see Mitchell (2000) for a review of Chlamydomonas flagellar structure and function).In Chlamydomonas, control of flagellar waveform is mediated through Ca 2ϩ signaling pathways (Bessen et al., 1980;Kamiya and Witman, 1984). Reactivatio...

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“…Sea urchin sperm flagella show NDK activity to produce GTP from GDP plus ATP. 69 Also, NDK has a marked preference for generating GTP in Chlamydomonas flagella. 70 The presence of these modules suggests a requirement for GTP either directly by dynein and/or by other axonemal components.…”

Section: Domains Related To Ca 2+ and Ndpmentioning

confidence: 99%

Molecular Basis of Sperm Flagellar Axonemes

Inaba

2007

Annals of the New York Academy of Sciences

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The axonemes serve as motile machineries in sperm flagella. Although atypical axonemal structures are observed in some cases, 9 + 2 microtubule structure of the axoneme is predominant in many organisms. Several structures are bound to these microtubules and comprise a highly organized protein network. Extensive proteomic analysis of the axonemes has led to find several repeats, domains, and motifs in axonemal proteins. Molecular comparison of subunit composition of axonemal substructures between the ascidian Ciona intestinalis and the green algae Chlamydomonas reinhardtti leads to an intriguing molecular aspect concerning the evolution of intracellular functional complex: The architecture of the axonemes has been well conserved through evolution, but the molecular structure of each axonemal component is not always conserved. In light of domain structure in the axonemal proteins, substructures like outer arm dynein and radial spoke contain a set of domain structures, although some domain-containing subunits are different between these two organisms. Thus, conservation of protein domains within a substructure seems to take precedence over that of each protein ("module-dominant conservation"), which may ultimately result in morphological and functional conservation of the axonemes through evolution.

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The bound nucleotides of the isolated microtubules of sea-urchin sperm flagella and their possible role in flagellar movement

Cited by 50 publications

References 21 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.

Flagellar Radial Spokes Contain a Ca²⁺-stimulated Nucleoside Diphosphate Kinase

Molecular Basis of Sperm Flagellar Axonemes

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The bound nucleotides of the isolated microtubules of sea-urchin sperm flagella and their possible role in flagellar movement

Cited by 50 publications

References 21 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.

Flagellar Radial Spokes Contain a Ca2+-stimulated Nucleoside Diphosphate Kinase

Molecular Basis of Sperm Flagellar Axonemes

Contact Info

Product

Resources

About

Flagellar Radial Spokes Contain a Ca²⁺-stimulated Nucleoside Diphosphate Kinase