scholarly journals A Chlamydomonas outer arm dynein mutant with a truncated beta heavy chain

1993 ◽  
Vol 122 (3) ◽  
pp. 653-661 ◽  
Author(s):  
H Sakakibara ◽  
S Takada ◽  
SM King ◽  
GB Witman ◽  
R Kamiya
Keyword(s):  
Cross Sections ◽  
Heavy Chain ◽  
Swimming Velocity ◽  
Wild Type ◽  
Terminal Part ◽  
Electron Microscopic ◽  
Heavy Chains ◽  
Dynein Arms ◽  
Alpha Beta ◽  
Arm Function

A new allele of the Chlamydomonas oda4 flagellar mutant (oda4-s7) possessing abnormal outer dynein arms was isolated. Unlike the previously described oda4 axoneme lacking all three (alpha, beta, and gamma) outer-arm dynein heavy chains, the oda4-s7 axoneme contains the alpha and gamma heavy chains and a novel peptide with a molecular mass of approximately 160 kD. The peptide reacts with a mAb (18 beta B) that recognizes an epitope on the NH2-terminal part of the beta heavy chain. These observations indicate that this mutant has a truncated beta heavy chain, and that the NH2-terminal part of the beta heavy chain is important for the stable assembly of the outer arms. In averaged electron microscopic images of outer arms from cross sections of axonemes, the mutant outer arm lacks its mid-portion, producing a forked appearance. Together with our previous finding that the mutant oda11 lacks the alpha heavy chain and the outermost portion of the arm (Sakakibara, H., D. R. Mitchell, and R. Kamiya. 1991. J. Cell Biol. 113:615-622), this result defines the approximate locations of the three outer arm heavy chains in the axonemal cross section. The swimming velocity of oda4-s7 is 65 +/- 8 microns/s, close to that of oda4 which lacks the entire outer arm (62 +/- 8 microns/s) but significantly lower than the velocities of wild type (194 +/- 23 microns/s) and oda11 (119 +/- 17 microns/s). Thus, the lack of the beta heavy chain impairs outer-arm function more seriously than does the lack of the alpha heavy chain, suggesting that the alpha and beta chains play different roles in outer arm function.

scholarly journals Functional reconstitution of Chlamydomonas outer dynein arms from alpha-beta and gamma subunits: requirement of a third factor.

1994 ◽  
Vol 126 (3) ◽  
pp. 737-745 ◽  
Author(s):  
S Takada ◽  
R Kamiya
Keyword(s):  
Cross Section ◽  
Cross Sections ◽  
Attachment Site ◽  
The Other ◽  
Wild Type ◽  
Heavy Chains ◽  
Sds Page ◽  
Gamma Subunits ◽  
Dynein Arms ◽  
Alpha Beta

The outer dynein arm of Chlamydomonas flagella, when isolated under Mg(2+)-free conditions, tends to dissociate into an 11 to 12S particle (12S dynein) containing the gamma heavy chain and a 21S particle (called 18S dynein) containing the alpha and beta heavy chains. We show here that functional outer arms can be reconstituted by the addition of 12S and 18S dyneins to the axonemes of the outer armless mutants oda1-oda6. A third factor that sediments at integral 7S is required for efficient reconstitution of the outer arms on the axonemes of oda1 and oda3. However, this factor is not necessary for reconstitution on the axonemes of oda2, oda4, oda5, and oda6. SDS-PAGE analysis indicates that the axonemes of the former two mutants lack a integral of 70-kD polypeptide that is present in those of the other mutants as well as in the 7S fraction from the wild-type extract. Furthermore, electron micrographs of axonemal cross sections revealed that the latter four mutants, but not oda1 or oda3, have small pointed structures on the outer doublets, at a position in cross section where outer arms normally occur. We suggest that the 7S factor constitutes the pointed structure on the outer doublets and facilitates attachment of the outer arm. The discovery of this structure raises a new question as to how the attachment site for the outer arm dynein is determined within the axoneme.

scholarly journals Ifm(2)2 is a myosin heavy chain allele that disrupts myofibrillar assembly only in the indirect flight muscle of Drosophila melanogaster.

1988 ◽  
Vol 107 (6) ◽  
pp. 2613-2621 ◽  
Author(s):  
M Chun ◽  
S Falkenthal
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Flight Muscle ◽  
Microscopic Analysis ◽  
Thick Filament ◽  
Indirect Flight Muscle ◽  
Chain Gene ◽  
Wild Type ◽  
Electron Microscopic ◽  
Sarcomere Assembly

Using a combination of molecular and genetic techniques we demonstrate that Ifm(2)2 is an allele of the single-copy sarcomeric myosin heavy chain gene. Flies homozygous for this allele accumulate wild-type levels of mRNA and protein in tubular muscle of adults, but fail to accumulate detectable amounts of myosin heavy chain mRNA or protein in the indirect flight muscle. We propose that the mutation interferes with either transcription of the gene or splicing of the primary transcript in the indirect flight muscle and not in other muscle tissues. Biochemical and electron microscopic analysis of flies homozygous for this mutation has revealed that thick filament assembly is abolished in the indirect flight muscle resulting in the instability of wild-type thick filament proteins. In contrast, thin filament and Z disc assembly are marginally affected. We discuss a working hypothesis for sarcomere assembly and define and experimental approach to test the predictions of this proposed pathway for sarcomere assembly.

scholarly journals Three distinct inner dynein arms in Chlamydomonas flagella: molecular composition and location in the axoneme.

1990 ◽  
Vol 110 (2) ◽  
pp. 379-389 ◽  
Author(s):  
G Piperno ◽  
Z Ramanis ◽  
E F Smith ◽  
W S Sale
Keyword(s):  
Ionic Strength ◽  
Sedimentation Coefficient ◽  
High Ionic Strength ◽  
Molecular Composition ◽  
Electron Microscopic ◽  
Heavy Chains ◽  
Dynein Arms ◽  
And Function ◽  
Atp Binding And Hydrolysis ◽  
A Site

The molecular composition and organization of the row of axonemal inner dynein arms were investigated by biochemical and electron microscopic analyses of Chlamydomonas wild-type and mutant axonemes. Three inner arm structures could be distinguished on the basis of their molecular composition and position in the axoneme as determined by analysis of pf30 and pf23 mutants. The three inner arm structures repeat every 96 nm and are referred to here as inner arms I1, I2, and I3. I1 is proximal to the radial spoke S1, whereas I2 and I3 are distal to spokes S1 and S2, respectively. The mutant pf30 lacks I1 whereas the mutant pf23 lacks both I1 and I2 but has a normal inner arm I3. Each of the six heavy chains that was identified as an inner dynein arm subunit has a site for ATP binding and hydrolysis. Two of the heavy chains together with a polypeptide of 140,000 molecular weight form the inner arm I1 and were extracted from the axoneme as a complex that had a sedimentation coefficient close to 21S at high ionic strength. Different subsets of two of the remaining four heavy chains form the inner arms I2 and I3. These arms at high ionic strength are dissociated as 11S particles that include one heavy chain, one intermediate chain, two light chains, and actin. These and other lines of evidence indicate that the inner arm I1 is different in structure and function from the inner arms I2 and I3.

scholarly journals Asymmetry of inner dynein arms and inter-doublet links in Chlamydomonas flagella

2009 ◽  
Vol 186 (3) ◽  
pp. 437-446 ◽  
Author(s):  
Khanh Huy Bui ◽  
Hitoshi Sakakibara ◽  
Tandis Movassagh ◽  
Kazuhiro Oiwa ◽  
Takashi Ishikawa
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Heavy Chain ◽  
Single Particle ◽  
Heavy Chains ◽  
Molecular Arrangement ◽  
Dynein Arms ◽  
Electron Cryotomography ◽  
Microtubule Doublet ◽  
Asymmetrical Bending ◽  
Dynein Heavy Chains

Although the widely shared “9 + 2” structure of axonemes is thought to be highly symmetrical, axonemes show asymmetrical bending during planar and conical motion. In this study, using electron cryotomography and single particle averaging, we demonstrate an asymmetrical molecular arrangement of proteins binding to the nine microtubule doublets in Chlamydomonas reinhardtii flagella. The eight inner arm dynein heavy chains regulate and determine flagellar waveform. Among these, one heavy chain (dynein c) is missing on one microtubule doublet (this doublet also lacks the outer dynein arm), and another dynein heavy chain (dynein b or g) is missing on the adjacent doublet. Some dynein heavy chains either show an abnormal conformation or were replaced by other proteins, possibly minor dyneins. In addition to nexin, there are two additional linkages between specific pairs of doublets. Interestingly, all these exceptional arrangements take place on doublets on opposite sides of the axoneme, suggesting that the transverse functional asymmetry of the axoneme causes an in-plane bending motion.

scholarly journals The sup-pf-2 mutations of Chlamydomonas alter the activity of the outer dynein arms by modification of the gamma-dynein heavy chain.

1996 ◽  
Vol 135 (6) ◽  
pp. 1853-1865 ◽  
Author(s):  
G Rupp ◽  
E O'Toole ◽  
L C Gardner ◽  
B F Mitchell ◽  
M E Porter
Keyword(s):  
Linkage Analysis ◽  
Heavy Chain ◽  
Biochemical Analysis ◽  
Beat Frequency ◽  
Wild Type ◽  
Dynein Heavy Chain ◽  
Flagellar Beat ◽  
Regulatory Mutations ◽  
Components Analysis ◽  
Dynein Arms

The sup-pf-2 mutation is a member of a group of dynein regulatory mutations that are capable of restoring motility to paralyzed central pair or radial spoke defective strains. Previous work has shown that the flagellar beat frequency is reduced in sup-pf-2, but little else was known about the sup-pf-2 phenotype (Huang, B., Z. Ramanis, and D.J.L. Luck. 1982. Cell. 28:115-125; Brokaw, C.J., and D.J.L. Luck. 1985. Cell Motil. 5:195-208). We have reexamined sup-pf-2 using improved biochemical and structural techniques and by the analysis of additional sup-pf-2 alleles. We have found that the sup-pf-2 mutations are associated with defects in the outer dynein arms. Biochemical analysis of sup-pf-2-1 axonemes indicates that both axonemal ATPase activity and outer arm polypeptides are reduced by 40-50% when compared with wild type. By thin-section EM, these defects correlate with an approximately 45% loss of outer dynein arm structures. Interestingly, this loss is biased toward a subset of outer doublets, resulting in a radial asymmetry that may reflect some aspect of outer arm assembly. The defects in outer arm assembly do not appear to result from defects in either the outer doublet microtubules or the outer arm docking structures, but rather appear to result from defects in outer dynein arm components. Analysis of new sup-pf-2 mutations indicates that the severity of the outer arm assembly defects varies with different alleles. Complementation tests and linkage analysis reveal that the sup-pf-2 mutations are alleles of the PF28/ODA2 locus, which is thought to encode the gamma-dynein heavy chain subunit of the outer arm. The sup-pf-2 mutations therefore appear to alter the activity of the outer dynein arms by modification of the gamma-dynein heavy chain.

scholarly journals WDR92 is required for axonemal dynein heavy chain stability in cytoplasm

2019 ◽  
Vol 30 (15) ◽  
pp. 1834-1845 ◽  
Author(s):  
Ramila S. Patel-King ◽  
Miho Sakato-Antoku ◽  
Maya Yankova ◽  
Stephen M. King
Keyword(s):  
Heavy Chain ◽  
Beat Frequency ◽  
Gel Filtration ◽  
Dynein Heavy Chain ◽  
Heavy Chains ◽  
Assembly Factor ◽  
Axonemal Dynein ◽  
Dynein Arms ◽  
Phylogenetic Signature ◽  
Dynein Heavy Chains

WDR92 associates with a prefoldin-like cochaperone complex and known dynein assembly factors. WDR92 has been very highly conserved and has a phylogenetic signature consistent with it playing a role in motile ciliary assembly or activity. Knockdown of WDR92 expression in planaria resulted in ciliary loss, reduced beat frequency and dyskinetic motion of the remaining ventral cilia. We have now identified a Chlamydomonas wdr92 mutant that encodes a protein missing the last four WD repeats. The wdr92-1 mutant builds only ∼0.7-μm cilia lacking both inner and outer dynein arms, but with intact doublet microtubules and central pair. When cytoplasmic extracts prepared by freeze/thaw from a control strain were fractionated by gel filtration, outer arm dynein components were present in several distinct high molecular weight complexes. In contrast, wdr92-1 extracts almost completely lacked all three outer arm heavy chains, while the IFT dynein heavy chain was present in normal amounts. A wdr92-1 tpg1-2 double mutant builds ∼7-μm immotile flaccid cilia that completely lack dynein arms. These data indicate that WDR92 is a key assembly factor specifically required for the stability of axonemal dynein heavy chains in cytoplasm and suggest that cytoplasmic/IFT dynein heavy chains use a distinct folding pathway.

scholarly journals The substructure of isolated and in situ outer dynein arms of sea urchin sperm flagella.

1985 ◽  
Vol 101 (4) ◽  
pp. 1400-1412 ◽  
Author(s):  
W S Sale ◽  
U W Goodenough ◽  
J E Heuser
Keyword(s):  
Sea Urchin ◽  
Heavy Chain ◽  
Heavy Chains ◽  
Low Salt ◽  
Spherical Head ◽  
Dynein Arms ◽  
Sea Urchin Sperm ◽  
Globular Head

Outer-arm dynein from the sperm of the sea urchin S. purpuratus was adsorbed to mica flakes and visualized by the quick-freeze, deep-etch technique. Replicas reveal particles comprised of two globular heads joined by two irregularly shaped stems which make contact along their length. One head is pear-shaped (18.5 X 12.5 nm) and the other is spherical (14.5-nm diam). The stems are decorated by a complex of bead-like subunits. The same two-headed protein is found in the 21S dynein-1 fraction of sucrose gradients. The beta-heavy chain/intermediate chain 1 (beta/IC-1) dynein subfraction, produced by low-salt dialysis and zonal centrifugation of the high-salt-extracted dynein-1, contains only single-headed molecules with single stems. These heads are predominantly pear-shaped (18.5 X 12.5 nm). Since 21S dynein-1 contains two heavy chains (alpha and beta), and the beta/IC-1 subfraction is comprised of only the beta-heavy chain (Tang et al., 1982, J. Biol. Chem. 257: 508-515), we conclude that each head is formed by a heavy chain, that the pear-shaped head contains the beta-heavy chain, and that the spherical head contains the alpha-heavy chain. The in situ outer dynein arms of demembranated sperm were also studied by the quick-freeze, deep-etch method. When frozen in reactivation buffer devoid of ATP, each arm consists of a large globular head that attaches to the A-microtubule by distally skewed subunits and attaches to the B-microtubule by a slender stalk. In ATP, this head shifts its orientation such that it can be seen to be constructed from two globular domains. We offer possible correlates between the in situ and the in vitro images, and we compare the structure of sea-urchin dynein with dynein previously described from Chlamydomonas and Tetrahymena.

scholarly journals A Chlamydomonas outer arm dynein mutant missing the alpha heavy chain.

1991 ◽  
Vol 113 (3) ◽  
pp. 615-622 ◽  
Author(s):  
H Sakakibara ◽  
D R Mitchell ◽  
R Kamiya
Keyword(s):  
Cross Section ◽  
Heavy Chain ◽  
Fragment Length Polymorphism ◽  
Beat Frequency ◽  
Polymorphism Analysis ◽  
Wild Type ◽  
Heavy Chains ◽  
Flagellar Beat ◽  
Section Electron ◽  
In The Wild

A novel Chlamydomonas flagellar mutant (oda-11) missing the alpha heavy chain of outer arm dynein but retaining the beta and gamma heavy chains was isolated. Restriction fragment length polymorphism analysis with an alpha heavy chain locus genomic probe indicated that the oda-11 mutation was genetically linked with the structural gene of the alpha heavy chain. In cross-section electron micrographs, the oda-11 axoneme lacked the outermost appendage of the outer arm, indicating that the alpha heavy chain should be located in this region in the wild-type outer arm. This mutant swam at 119 microns/s at 25 degrees C, i.e., at an intermediate speed between those of wild type (194 microns/s) and of oda-1 (62 microns/s), a mutant missing the entire outer dynein arm. The flagellar beat frequency (approximately 50 Hz) was also between those of wild type (approximately 60 Hz) and oda-1 (approximately 26 Hz). These results indicate that the outer dynein arm of Chlamydomonas can be assembled without the alpha heavy chain, and that the outer arm missing the alpha heavy chain retains partial function.

scholarly journals Two types of Chlamydomonas flagellar mutants missing different components of inner-arm dynein.

1991 ◽  
Vol 112 (3) ◽  
pp. 441-447 ◽  
Author(s):  
R Kamiya ◽  
E Kurimoto ◽  
E Muto
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Cross Section ◽  
Double Mutant ◽  
The Other ◽  
Flagellar Motility ◽  
Wild Type ◽  
Single Group ◽  
Heavy Chains ◽  
Section Electron ◽  
Dynein Arms

Two types of Chlamydomonas reinhardtii flagellar 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, I2, and I3, each containing two heterologous heavy chains (Piperno, G., Z. Ramanis, E. F. Smith, and W. S. Sale. 1990. J. Cell Biol. 110:379-389), idaA and idaB lacked I1 and I2, respectively. The 13 idA isolates comprised three genetically different groups (ida1, ida2, ida3) and the two idaB isolates comprised a single group (ida4). In averaged cross-section electron micrographs, inner dynein arms in wild-type axonemes appeared to have two projections pointing to discrete directions. In ida1-3 and ida4 axonemes, on the other hand, either one of them was missing or greatly diminished. Both projections were weak in the double mutant ida1-3 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 ida1-3 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 ida1-3 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.

scholarly journals Visualization of cardiac ventricular myosin heavy chain homodimers and heterodimers by monoclonal antibody epitope mapping.

1987 ◽  
Vol 105 (6) ◽  
pp. 3031-3037 ◽  
Author(s):  
C A Dechesne ◽  
P Bouvagnet ◽  
D Walzthöny ◽  
J J Léger
Keyword(s):  
Heavy Chain ◽  
Binding Sites ◽  
Epitope Mapping ◽  
Myosin Molecule ◽  
Myosin Heavy Chains ◽  
Cardiac Myosin ◽  
Heavy Chains ◽  
Ventricular Myosin ◽  
Alpha Beta ◽  
Myosin Rod

Two mAbs, one specific for cardiac alpha-myosin heavy chains (MHC) and the other specific for cardiac beta-MHC, were used to investigate the heavy-chain dimeric organization of rat cardiac ventricular myosin. Epitopes of the two mAbs were mapped on the myosin molecule by electron microscopy of rotary shadowed mAb-myosin complexes. mAbs were clearly identifiable by the different locations of their binding sites on the myosin rod. Thus, myosin molecules could be directly discriminated according to their alpha-or beta-MHC content. alpha alpha-MHC and beta beta-MHC homodimers were visualized in complexes consisting of two molecules of the same mAb bound to one myosin molecule. By simultaneously using the alpha-MHC-specific mAb and the beta-MHC-specific mAb, alpha beta-MHC heterodimers were visualized in complexes formed by one molecule of each of the two mAbs bound to one myosin molecule. Proportions of alpha alpha-and beta beta-MHC homodimers and alpha beta-MHC heterodimers were estimated from quantifications of mAb-myosin complexes and compared with the proportions given by electrophoreses under nondenaturing conditions. This visualization of cardiac myosin molecules clearly demonstrates the arrangement of alpha- and beta-MHC in alpha alpha-MHC homodimers, beta beta-MHC homodimers, and alpha beta-MHC heterodimers, as initially proposed by Hoh, J. F. Y., G. P. S. Yeoh, M. A. W. Thomas, and L. Higginbottom (1979).

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