Two types of Chlamydomonas reinhardtii tlagellar mutants (idaA and idaB) lacking partial componen... more Two types of Chlamydomonas reinhardtii tlagellar mutants (idaA and idaB) lacking partial components of the inner-arm dynein were isolated by screening mutations that produce paralyzed phenotypes when present in a mutant missing outer-arm dynein. Of the currently identified three inner-arm subspecies I1, 12, and 13, each containing two heterologous heavy chains (Piperno, G., Z. Ramanis, E. E Smith, and W. S. Sale. 1990. J. Cell Biol. 110:379-389), idaA and idaB lacked I1 and 12, respectively. The 13 idaA isolates comprised three genetically different groups (idal, ida2, ida3) and the two idaB isolates comprised a single group (ida4). In averaged crosssection electron micrographs, inner dynein arms in wild-type axonemes appeared to have two projections pointing to discrete directions. In idal-3 and ida4 axonemes, on the other hand, either one of them was missing or greatly diminished. Both projections were weak in the double mutant idal-3 x ida4. These observations suggest that the inner dynein arms in Chlamydomonas axonemes are aligned not in a single straight row, but in a staggered row or two discrete rows. Both idal-3 and ida4 swam at reduced speed. Thus, the inner-arm subspecies missing in these mutants are not necessary for flagellar motility. However, the double mutants idal-3 x ida4 were nonmotile, suggesting that axonemes with significant defects in inner arms cannot function. The inner-arm dynein should be important for the generation of axonemal beating.
Beating of eukaTyotic fiagetla is based om the ccmrdinated force generation by outer and inner dy... more Beating of eukaTyotic fiagetla is based om the ccmrdinated force generation by outer and inner dynein arms, The outer dynein arin (ODA) is a eoiriplex composed of 2-3 heavy chains atid more than 10 smaller preteins (intenllediate and light chains). To facilitate experiments
the bound nucleot]de which Tg consistcnt wth LhLprcwous expelimentg Oii the ethu hand the po-er s... more the bound nucleot]de which Tg consistcnt wth LhLprcwous expelimentg Oii the ethu hand the po-er stroke of the lever ann wab fbund te be energettcatty unfavorablc We then chssect the potentiaT energy and idenhfy the key residues that largely contribuLL to the tree energy landscapes and "ill also addrcss the interdependLncy bctwccn the actm bindmg clett and the lever arm 3D1534 MmgvueJm i MayanagiKLushtt inst iDivision Struc Oi-rtMIwaFEV77-I[di6ditfiemzafiSre(i.xJbLfu LC4 oprc-Detection of genetic heametal labeled hght Lham 4 b} clectroncrNotomographN i Hani akiYanagisa-a2 Kotaro Koyasako] RitsuKamTya2 Kota
m a suTface irnmobilizedFi MPase could be medtsured A bead attached to the rotary shafi was pulle... more m a suTface irnmobilizedFi MPase could be medtsured A bead attached to the rotary shafi was pulled up-ard and tlte abm!pt displaLement Lorresponding to N20 pN wats obgcTved
Cilia and flagella have multiple dyneins in their inner and outer arms. Chlamydomonas inner-arm d... more Cilia and flagella have multiple dyneins in their inner and outer arms. Chlamydomonas inner-arm dynein contains at least seven major subspecies (dynein a to dynein g), of which all but dynein f (also called dynein I1) are the single-headed type that are composed of a single heavy chain, actin, and either centrin or a 28-kDa protein (p28). Dynein d was found to associate with two additional proteins of 38 kDa (p38) and 44 kDa (p44). Following the characterization of the p38 protein (R. Yamamoto, H. A. Yanagisawa, T. Yagi, and R. Kamiya, FEBS Lett. 580:6357-6360, 2006), we have identified p44 as a novel component of dynein d by using an immunoprecipitation approach. p44 is present along the length of the axonemes and is diminished, but not absent, in the ida4 and ida5 mutants, both lacking this dynein. In the ida5 axoneme, p44 and p38 appear to form a complex, suggesting that they constitute the docking site of dynein d on the outer doublet. p44 has potential homologues in other ciliated organisms. For example, the mouse homologue of p44, NYD-SP14, was found to be strongly expressed in tissues with motile cilia and flagella. These results suggest that inner-arm dynein d and its subunit organization are widely conserved.
ABSTRACT eLife digest Sperm cells have tails called flagella that propel them towards an egg. Oth... more ABSTRACT eLife digest Sperm cells have tails called flagella that propel them towards an egg. Other cells have similar, but shorter, structures called cilia that sway back and forth on their surface. In addition to sweeping dust and debris out of our lungs and airways, cilia have a number of other crucial roles during development. This means that faulty cilia can lead to serious birth defects, as well as diseases of the kidneys and respiratory system. Cilia and flagella are made from proteins that are assembled in a process called intraflagellar transport or IFT for short. Around 20 proteins are thought to be involved in this process, but the precise role of many of these proteins remains unclear. Now Ishikawa et al. have compared the versions of one of these proteins, called TTC26, that are found in zebrafish, mouse cells, and a single-celled alga called Chlamydomonas reinhardtii that uses a pair of flagella to move around. This protein localizes to the cilia of mice cells and can be seen to move along these cilia in a manner typical of other IFT proteins. Ishikawa et al. then blocked production of TTC26 in zebrafish embryos, which caused these embryos to fail to develop the correct left–right asymmetry, and these fish also had problems with their eyes, ears, and kidneys. Furthermore and although cilia were present in the affected zebrafish, these cilia were shortened and moved abnormally. Ishikawa et al. also found that algae that had a mutation in the gene that codes for TTC26 had short cilia that moved in an abnormal way. The findings of Ishikawa et al. suggest that TTC26 may help to transport a specific subset of proteins into the cilia. If other IFT proteins are also shown to carry distinct subsets of cargo, this might explain why as many as 20 different proteins are involved in the IFT process. DOI: http://dx.doi.org/10.7554/eLife.01566.002
Chlamydomonas, an organism that offers a variety of flagella-deficient mutants, has been very imp... more Chlamydomonas, an organism that offers a variety of flagella-deficient mutants, has been very important for studies of cilia and flagella. Motility assessment of mutant flagella at various levels helps us understand the function of specific axonemal proteins and structures for flagellar function. Measurements of gross cell movements are useful to assess the overall flagellar activity, analyses of demembranated and reactivated cells ("cell models") enable us to study the regulatory mechanism, and measurements of microtubule sliding velocity in vitro provide important information about dynein-microtubule interactions. This chapter describes fundamental techniques for these measurements.
Biochimica Et Biophysica Acta - Bioenergetics, 1972
When rabbit actin was polymerized by 2-3 m3/[ MgC12 in the presence of/3actinin, 5-1o °,0 of acti... more When rabbit actin was polymerized by 2-3 m3/[ MgC12 in the presence of/3actinin, 5-1o °,0 of actin by weight, aggregates very similar to the Mg-polymer of plasmodium actin were formed: reduced viscosity was low, I-2 dl/g, and the sedimentation coefficient was 31-33 S. The ATPase activity, although lower than that of the plasmodium Mg-polymer, was clearly demonstrated. Electron microscopic observations revealed that the Mg-polymer consisted of globular aggregates and random aggregates of short F-actin particles in negatively stained samples. However, when preparations were fixed with glutaraldehyde and then negatively stained with uranyl acetate, short F-actin particles alone were observed. It is considered that the Mg-polymer consists of short fragile F-actin particles. The Mg-polymer was transformed into short F-actin filaments when incubated for 5 rain at 45 ° in the presence of ATP. When incubated for 25-30 min at 55 °, elongation of F-actin particles occurred. This was explained by partial inactivation of/¢-actinin, where some recombination of F-actin particles must have taken place.
A beta-actinin-like protein was isolated from plasmodia of the slime mold. The chain weight was t... more A beta-actinin-like protein was isolated from plasmodia of the slime mold. The chain weight was the same as that of actin (43,000), but the amino acid composition was significantly different. The action of plasmodium beta-actinin on actin was the same as that of beta-actinin from rabbit skeletal muscle: inhibition of the recombination of F-actin fragments; formation of Mg polymer; inhibition of interfilamental interaction of F-actin and retardation of depolymerization of F-actin. The only difference observed was its sensitivity to trypsin: plasmodium actinin was less quickly digested by trypsin than rabbit beta-actinin.
A nonmotile mutant of Salmonella typhimurium, SJW1254, has very short flagella (<0.1 pm long) due... more A nonmotile mutant of Salmonella typhimurium, SJW1254, has very short flagella (<0.1 pm long) due to a mutation in the structural gene of flagellin (H2). When ammonium sulfate was added to the culture medium of SJW1254 grown to the late-log phase, a large amount of protein precipitated. Gel electrophoresis and immunodiffusion showed that more than 90% (wt/wt) of the precipitated protein was flagellin. The mutant flagellin appeared to be excreted in the monomeric form, in an amount comparable to the amount in the flagellar filaments of wildtype bacteria. No such precipitate was obtained from the medium of wild-type bacteria. The mutant flagellin had the same apparent molecular weight (55,000) and isoelectric point (5.3) as the wild-type flagellin, but differed in mobility in polyacrylamide gel electrophoresis under nondenaturing conditions. Moreover, the mutant flagellin did not polymerize in vitro under various conditions in which wild-type flagellin polymerized. These results suggested that the mutant bacteria excreted flagellin because the flagellin polymerized poorly and therefore could not be trapped at the tip of the flagellar filament. This short-flagella mutant should be useful for studying the mechanism of flagellin transport.
Two types of Chlamydomonas reinhardtii tlagellar mutants (idaA and idaB) lacking partial componen... more Two types of Chlamydomonas reinhardtii tlagellar mutants (idaA and idaB) lacking partial components of the inner-arm dynein were isolated by screening mutations that produce paralyzed phenotypes when present in a mutant missing outer-arm dynein. Of the currently identified three inner-arm subspecies I1, 12, and 13, each containing two heterologous heavy chains (Piperno, G., Z. Ramanis, E. E Smith, and W. S. Sale. 1990. J. Cell Biol. 110:379-389), idaA and idaB lacked I1 and 12, respectively. The 13 idaA isolates comprised three genetically different groups (idal, ida2, ida3) and the two idaB isolates comprised a single group (ida4). In averaged crosssection electron micrographs, inner dynein arms in wild-type axonemes appeared to have two projections pointing to discrete directions. In idal-3 and ida4 axonemes, on the other hand, either one of them was missing or greatly diminished. Both projections were weak in the double mutant idal-3 x ida4. These observations suggest that the inner dynein arms in Chlamydomonas axonemes are aligned not in a single straight row, but in a staggered row or two discrete rows. Both idal-3 and ida4 swam at reduced speed. Thus, the inner-arm subspecies missing in these mutants are not necessary for flagellar motility. However, the double mutants idal-3 x ida4 were nonmotile, suggesting that axonemes with significant defects in inner arms cannot function. The inner-arm dynein should be important for the generation of axonemal beating.
Beating of eukaTyotic fiagetla is based om the ccmrdinated force generation by outer and inner dy... more Beating of eukaTyotic fiagetla is based om the ccmrdinated force generation by outer and inner dynein arms, The outer dynein arin (ODA) is a eoiriplex composed of 2-3 heavy chains atid more than 10 smaller preteins (intenllediate and light chains). To facilitate experiments
the bound nucleot]de which Tg consistcnt wth LhLprcwous expelimentg Oii the ethu hand the po-er s... more the bound nucleot]de which Tg consistcnt wth LhLprcwous expelimentg Oii the ethu hand the po-er stroke of the lever ann wab fbund te be energettcatty unfavorablc We then chssect the potentiaT energy and idenhfy the key residues that largely contribuLL to the tree energy landscapes and "ill also addrcss the interdependLncy bctwccn the actm bindmg clett and the lever arm 3D1534 MmgvueJm i MayanagiKLushtt inst iDivision Struc Oi-rtMIwaFEV77-I[di6ditfiemzafiSre(i.xJbLfu LC4 oprc-Detection of genetic heametal labeled hght Lham 4 b} clectroncrNotomographN i Hani akiYanagisa-a2 Kotaro Koyasako] RitsuKamTya2 Kota
m a suTface irnmobilizedFi MPase could be medtsured A bead attached to the rotary shafi was pulle... more m a suTface irnmobilizedFi MPase could be medtsured A bead attached to the rotary shafi was pulled up-ard and tlte abm!pt displaLement Lorresponding to N20 pN wats obgcTved
Cilia and flagella have multiple dyneins in their inner and outer arms. Chlamydomonas inner-arm d... more Cilia and flagella have multiple dyneins in their inner and outer arms. Chlamydomonas inner-arm dynein contains at least seven major subspecies (dynein a to dynein g), of which all but dynein f (also called dynein I1) are the single-headed type that are composed of a single heavy chain, actin, and either centrin or a 28-kDa protein (p28). Dynein d was found to associate with two additional proteins of 38 kDa (p38) and 44 kDa (p44). Following the characterization of the p38 protein (R. Yamamoto, H. A. Yanagisawa, T. Yagi, and R. Kamiya, FEBS Lett. 580:6357-6360, 2006), we have identified p44 as a novel component of dynein d by using an immunoprecipitation approach. p44 is present along the length of the axonemes and is diminished, but not absent, in the ida4 and ida5 mutants, both lacking this dynein. In the ida5 axoneme, p44 and p38 appear to form a complex, suggesting that they constitute the docking site of dynein d on the outer doublet. p44 has potential homologues in other ciliated organisms. For example, the mouse homologue of p44, NYD-SP14, was found to be strongly expressed in tissues with motile cilia and flagella. These results suggest that inner-arm dynein d and its subunit organization are widely conserved.
ABSTRACT eLife digest Sperm cells have tails called flagella that propel them towards an egg. Oth... more ABSTRACT eLife digest Sperm cells have tails called flagella that propel them towards an egg. Other cells have similar, but shorter, structures called cilia that sway back and forth on their surface. In addition to sweeping dust and debris out of our lungs and airways, cilia have a number of other crucial roles during development. This means that faulty cilia can lead to serious birth defects, as well as diseases of the kidneys and respiratory system. Cilia and flagella are made from proteins that are assembled in a process called intraflagellar transport or IFT for short. Around 20 proteins are thought to be involved in this process, but the precise role of many of these proteins remains unclear. Now Ishikawa et al. have compared the versions of one of these proteins, called TTC26, that are found in zebrafish, mouse cells, and a single-celled alga called Chlamydomonas reinhardtii that uses a pair of flagella to move around. This protein localizes to the cilia of mice cells and can be seen to move along these cilia in a manner typical of other IFT proteins. Ishikawa et al. then blocked production of TTC26 in zebrafish embryos, which caused these embryos to fail to develop the correct left–right asymmetry, and these fish also had problems with their eyes, ears, and kidneys. Furthermore and although cilia were present in the affected zebrafish, these cilia were shortened and moved abnormally. Ishikawa et al. also found that algae that had a mutation in the gene that codes for TTC26 had short cilia that moved in an abnormal way. The findings of Ishikawa et al. suggest that TTC26 may help to transport a specific subset of proteins into the cilia. If other IFT proteins are also shown to carry distinct subsets of cargo, this might explain why as many as 20 different proteins are involved in the IFT process. DOI: http://dx.doi.org/10.7554/eLife.01566.002
Chlamydomonas, an organism that offers a variety of flagella-deficient mutants, has been very imp... more Chlamydomonas, an organism that offers a variety of flagella-deficient mutants, has been very important for studies of cilia and flagella. Motility assessment of mutant flagella at various levels helps us understand the function of specific axonemal proteins and structures for flagellar function. Measurements of gross cell movements are useful to assess the overall flagellar activity, analyses of demembranated and reactivated cells (&amp;amp;amp;amp;quot;cell models&amp;amp;amp;amp;quot;) enable us to study the regulatory mechanism, and measurements of microtubule sliding velocity in vitro provide important information about dynein-microtubule interactions. This chapter describes fundamental techniques for these measurements.
Biochimica Et Biophysica Acta - Bioenergetics, 1972
When rabbit actin was polymerized by 2-3 m3/[ MgC12 in the presence of/3actinin, 5-1o °,0 of acti... more When rabbit actin was polymerized by 2-3 m3/[ MgC12 in the presence of/3actinin, 5-1o °,0 of actin by weight, aggregates very similar to the Mg-polymer of plasmodium actin were formed: reduced viscosity was low, I-2 dl/g, and the sedimentation coefficient was 31-33 S. The ATPase activity, although lower than that of the plasmodium Mg-polymer, was clearly demonstrated. Electron microscopic observations revealed that the Mg-polymer consisted of globular aggregates and random aggregates of short F-actin particles in negatively stained samples. However, when preparations were fixed with glutaraldehyde and then negatively stained with uranyl acetate, short F-actin particles alone were observed. It is considered that the Mg-polymer consists of short fragile F-actin particles. The Mg-polymer was transformed into short F-actin filaments when incubated for 5 rain at 45 ° in the presence of ATP. When incubated for 25-30 min at 55 °, elongation of F-actin particles occurred. This was explained by partial inactivation of/¢-actinin, where some recombination of F-actin particles must have taken place.
A beta-actinin-like protein was isolated from plasmodia of the slime mold. The chain weight was t... more A beta-actinin-like protein was isolated from plasmodia of the slime mold. The chain weight was the same as that of actin (43,000), but the amino acid composition was significantly different. The action of plasmodium beta-actinin on actin was the same as that of beta-actinin from rabbit skeletal muscle: inhibition of the recombination of F-actin fragments; formation of Mg polymer; inhibition of interfilamental interaction of F-actin and retardation of depolymerization of F-actin. The only difference observed was its sensitivity to trypsin: plasmodium actinin was less quickly digested by trypsin than rabbit beta-actinin.
A nonmotile mutant of Salmonella typhimurium, SJW1254, has very short flagella (<0.1 pm long) due... more A nonmotile mutant of Salmonella typhimurium, SJW1254, has very short flagella (<0.1 pm long) due to a mutation in the structural gene of flagellin (H2). When ammonium sulfate was added to the culture medium of SJW1254 grown to the late-log phase, a large amount of protein precipitated. Gel electrophoresis and immunodiffusion showed that more than 90% (wt/wt) of the precipitated protein was flagellin. The mutant flagellin appeared to be excreted in the monomeric form, in an amount comparable to the amount in the flagellar filaments of wildtype bacteria. No such precipitate was obtained from the medium of wild-type bacteria. The mutant flagellin had the same apparent molecular weight (55,000) and isoelectric point (5.3) as the wild-type flagellin, but differed in mobility in polyacrylamide gel electrophoresis under nondenaturing conditions. Moreover, the mutant flagellin did not polymerize in vitro under various conditions in which wild-type flagellin polymerized. These results suggested that the mutant bacteria excreted flagellin because the flagellin polymerized poorly and therefore could not be trapped at the tip of the flagellar filament. This short-flagella mutant should be useful for studying the mechanism of flagellin transport.
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