In eukaryotic cilia and flagella, various types of axonemal dyneins orchestrate their distinct fu... more In eukaryotic cilia and flagella, various types of axonemal dyneins orchestrate their distinct functions to generate oscillatory bending of axonemes. The force-generating mechanism of dyneins has recently been well elucidated, mainly in cytoplasmic dyneins, thanks to progress in single-molecule measurements, X-ray crystallography, and advanced electron microscopy. These techniques have shed light on several important questions concerning what conformational changes accompany ATP hydrolysis and whether multiple motor domains are coordinated in the movements of dynein. However, due to the lack of a proper expression system for axonemal dyneins, no atomic coordinates of the entire motor domain of axonemal dynein have been reported. Therefore, a substantial amount of knowledge on the molecular architecture of axonemal dynein has been derived from electron microscopic observations on dynein arms in axonemes or on isolated axonemal dynein molecules. This review describes our current knowl...
Genomics and proteomics studies in Chlamydomonas have revealed that an axoneme is composed of 200... more Genomics and proteomics studies in Chlamydomonas have revealed that an axoneme is composed of 200-600 types of proteins, including uncharacterized proteins collectively named flagellar-associated proteins (FAPs). Nine FAPs contain the EF-hand motif; however, they have not yet been well characterized. To find components responsible for Chlamydomonas-specific waveform changes coupled with intracellular Ca 2+ concentrations, we focused on FAP85, an EF-hand motif-containing FAP specific to Chlamydomonas and its relatives. We cloned the cDNA encoding FAP85, expressed it in Escherichia coli cells, and generated a polyclonal antibody against the expressed protein. Immunoblotting showed that FAP85 was present in every axoneme of several flagellar mutants lacking major axonemal components. Immuno-electron microscopy revealed that anti-FAP85 antibodies were found only on the inner wall of A-tubules of the doublets exposed by N-lauroylsarcosine (Sarkosyl) treatment. The zero-length cross-linker 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) applied to 0.6 M KCl-extracted axonemes generated a 75-kDa complex containing β-tubulin and FAP85. Further characterization of FAP85 and its effects on microtubule dynamics showed that FAP85 binds to tubulin and stabilized microtubules. According to these results, we conclude that FAP85 is a novel member of microtubulebinding proteins, localizing on the inner wall of the A-tubule and stabilizing microtubules.
{ODA14). ODA-DC binds to specific loci on the outer-doublets or mutant axonemes that iack outer a... more {ODA14). ODA-DC binds to specific loci on the outer-doublets or mutant axonemes that iack outer arm dynein, suggesting that it is responsible for the binding ofouter arrn dyneins to specific sites. To understand the basis for this crucial functjon of the ODA-DC, we exarnined the structural and biechemical properties of ODA-DC using both TecDmbinant and native pToteins. Low-angle retary shadewing indicated that the recembinant ODA-DC has an oval shape,-25 nm in ]ength. The Tecombinant ODA-DC binds to cytoplasmic microtubules in an alt-or-none manner, suggesting a cooperative binding evcnt. Chemical ¢ rosslinking ofaxonemes frorn a mutant lacking outer-arms revea]ed that the native ODA-DCs associate with each other on the doublet rnicTotubutes. These data suggest that the periodic and cooperative binding of the ODA-DC rnolecules on the doubtet microtubules is based on an end-to-end association of the ODA-DCs. Furthermore, chemical cross]inking a]so reveated that the ODA-DC is associated with three uncharacterized novel proteins on the axoneme. These proteins possibly specify the binding site of the ODA-DC on the outer doublet microtubules. o7E Fitxtumw4,g.t opstpty.r=yopmesurtEMgzas yJtofimaregfimopneiji Analysis of protein-proteill interactions required for the assembty of outer arm dyneins on the axonemal microtubllles in Chlamydomonas ,
Japan General IncorporatedAssociation iate <O 5nm x O 5nmlpixel) Congequently thL imagL taLlmg at... more Japan General IncorporatedAssociation iate <O 5nm x O 5nmlpixel) Congequently thL imagL taLlmg at tsLeep iamp off aTea was reduced Tbpographs of dyllem c were aligned classified and e]ass dveraged
These results suggest that the flagellaT motor ofBactltus subttbs ]]iebumabl} gcneTateg torque by... more These results suggest that the flagellaT motor ofBactltus subttbs ]]iebumabl} gcneTateg torque by the electrostatic interactaon between the rotoi and the stator 3P 1 41 tn wtro motihty assay ic tst6 ua4sfiOfiti finM The tuiming tendenLy o{ thL microtubule dnven by the dynein subspeeies c Yngi Shitaka (1) Kazuhire Oiwa (1) Hiroaki KoJim" (1) ((l) National JnformattonandComniunicanons7lechnotosy) axoiiemdllnstittste tij We exammed the turnmg tendenLy of the miLrotabule dnven bv the axonemal inner arm dy"ein subspLuLs L m in-tro motility assdiy We found thdt the mTLrotubule tLnd to tuin to the nght with the angular uelocity ef O O16 r idts in the hTgh d}nem densTty Londition (2000 moleculei"m2) This tendency ib deLredsed by lowcnng the d)nem suTface density These observationg suggLst
elements that mfluenLe nucleotlde exchange iaLe of actin In ths work we characterized nuLleotide ... more elements that mfluenLe nucleotlde exchange iaLe of actin In ths work we characterized nuLleotide bmd]ng er carp (C}pnnus tarpio) muscle actin which Lontdms five ammo aeid subghtutions conipared to chicken rnuscle actm In carp actm beth the assoaatdon lnd the dTssociatTon rdte of E ATP wcre sigmfiLdlltly ]aTgc At high CaZ' concentrataon the dissouat]on iate Longtant ui E ATP was g 3 x 10 5 s i which was about
I.iposome-Cellular Communieation through Gap Junction OKazunari Akiy[}shi'', Wakike Asayamai", Sh... more I.iposome-Cellular Communieation through Gap Junction OKazunari Akiy[}shi'', Wakike Asayamai", Shin-;chiro M. NomLu'ai'], NUki Mori[aniS,TatsuyaSLtdai,L ilnst. of Biomaterials and Bjocnginccring,
In eukaryotic cilia and flagella, various types of axonemal dyneins orchestrate their distinct fu... more In eukaryotic cilia and flagella, various types of axonemal dyneins orchestrate their distinct functions to generate oscillatory bending of axonemes. The force-generating mechanism of dyneins has recently been well elucidated, mainly in cytoplasmic dyneins, thanks to progress in single-molecule measurements, X-ray crystallography, and advanced electron microscopy. These techniques have shed light on several important questions concerning what conformational changes accompany ATP hydrolysis and whether multiple motor domains are coordinated in the movements of dynein. However, due to the lack of a proper expression system for axonemal dyneins, no atomic coordinates of the entire motor domain of axonemal dynein have been reported. Therefore, a substantial amount of knowledge on the molecular architecture of axonemal dynein has been derived from electron microscopic observations on dynein arms in axonemes or on isolated axonemal dynein molecules. This review describes our current knowl...
Genomics and proteomics studies in Chlamydomonas have revealed that an axoneme is composed of 200... more Genomics and proteomics studies in Chlamydomonas have revealed that an axoneme is composed of 200-600 types of proteins, including uncharacterized proteins collectively named flagellar-associated proteins (FAPs). Nine FAPs contain the EF-hand motif; however, they have not yet been well characterized. To find components responsible for Chlamydomonas-specific waveform changes coupled with intracellular Ca 2+ concentrations, we focused on FAP85, an EF-hand motif-containing FAP specific to Chlamydomonas and its relatives. We cloned the cDNA encoding FAP85, expressed it in Escherichia coli cells, and generated a polyclonal antibody against the expressed protein. Immunoblotting showed that FAP85 was present in every axoneme of several flagellar mutants lacking major axonemal components. Immuno-electron microscopy revealed that anti-FAP85 antibodies were found only on the inner wall of A-tubules of the doublets exposed by N-lauroylsarcosine (Sarkosyl) treatment. The zero-length cross-linker 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) applied to 0.6 M KCl-extracted axonemes generated a 75-kDa complex containing β-tubulin and FAP85. Further characterization of FAP85 and its effects on microtubule dynamics showed that FAP85 binds to tubulin and stabilized microtubules. According to these results, we conclude that FAP85 is a novel member of microtubulebinding proteins, localizing on the inner wall of the A-tubule and stabilizing microtubules.
{ODA14). ODA-DC binds to specific loci on the outer-doublets or mutant axonemes that iack outer a... more {ODA14). ODA-DC binds to specific loci on the outer-doublets or mutant axonemes that iack outer arm dynein, suggesting that it is responsible for the binding ofouter arrn dyneins to specific sites. To understand the basis for this crucial functjon of the ODA-DC, we exarnined the structural and biechemical properties of ODA-DC using both TecDmbinant and native pToteins. Low-angle retary shadewing indicated that the recembinant ODA-DC has an oval shape,-25 nm in ]ength. The Tecombinant ODA-DC binds to cytoplasmic microtubules in an alt-or-none manner, suggesting a cooperative binding evcnt. Chemical ¢ rosslinking ofaxonemes frorn a mutant lacking outer-arms revea]ed that the native ODA-DCs associate with each other on the doublet rnicTotubutes. These data suggest that the periodic and cooperative binding of the ODA-DC rnolecules on the doubtet microtubules is based on an end-to-end association of the ODA-DCs. Furthermore, chemical cross]inking a]so reveated that the ODA-DC is associated with three uncharacterized novel proteins on the axoneme. These proteins possibly specify the binding site of the ODA-DC on the outer doublet microtubules. o7E Fitxtumw4,g.t opstpty.r=yopmesurtEMgzas yJtofimaregfimopneiji Analysis of protein-proteill interactions required for the assembty of outer arm dyneins on the axonemal microtubllles in Chlamydomonas ,
Japan General IncorporatedAssociation iate <O 5nm x O 5nmlpixel) Congequently thL imagL taLlmg at... more Japan General IncorporatedAssociation iate <O 5nm x O 5nmlpixel) Congequently thL imagL taLlmg at tsLeep iamp off aTea was reduced Tbpographs of dyllem c were aligned classified and e]ass dveraged
These results suggest that the flagellaT motor ofBactltus subttbs ]]iebumabl} gcneTateg torque by... more These results suggest that the flagellaT motor ofBactltus subttbs ]]iebumabl} gcneTateg torque by the electrostatic interactaon between the rotoi and the stator 3P 1 41 tn wtro motihty assay ic tst6 ua4sfiOfiti finM The tuiming tendenLy o{ thL microtubule dnven by the dynein subspeeies c Yngi Shitaka (1) Kazuhire Oiwa (1) Hiroaki KoJim" (1) ((l) National JnformattonandComniunicanons7lechnotosy) axoiiemdllnstittste tij We exammed the turnmg tendenLy of the miLrotabule dnven bv the axonemal inner arm dy"ein subspLuLs L m in-tro motility assdiy We found thdt the mTLrotubule tLnd to tuin to the nght with the angular uelocity ef O O16 r idts in the hTgh d}nem densTty Londition (2000 moleculei"m2) This tendency ib deLredsed by lowcnng the d)nem suTface density These observationg suggLst
elements that mfluenLe nucleotlde exchange iaLe of actin In ths work we characterized nuLleotide ... more elements that mfluenLe nucleotlde exchange iaLe of actin In ths work we characterized nuLleotide bmd]ng er carp (C}pnnus tarpio) muscle actin which Lontdms five ammo aeid subghtutions conipared to chicken rnuscle actm In carp actm beth the assoaatdon lnd the dTssociatTon rdte of E ATP wcre sigmfiLdlltly ]aTgc At high CaZ' concentrataon the dissouat]on iate Longtant ui E ATP was g 3 x 10 5 s i which was about
I.iposome-Cellular Communieation through Gap Junction OKazunari Akiy[}shi'', Wakike Asayamai", Sh... more I.iposome-Cellular Communieation through Gap Junction OKazunari Akiy[}shi'', Wakike Asayamai", Shin-;chiro M. NomLu'ai'], NUki Mori[aniS,TatsuyaSLtdai,L ilnst. of Biomaterials and Bjocnginccring,
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Papers by Kazuhiro Oiwa