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ABSTRACT: Axonemal dyneins must be precisely regulated and coordinated to produce ordered ciliary/flagellar motility, but how this is achieved is not understood. We analyzed two Chlamydomonas reinhardtii mutants, mia1 and mia2, which display slow swimming and low flagellar beat frequency. We found that the MIA1 and MIA2 genes encode conserved coiled-coil proteins, FAP100 and FAP73, respectively, which form the modifier of inner arms (MIA) complex in flagella. Cryo-electron tomography of mia mutant axonemes revealed that the MIA complex was located immediately distal to the intermediate/light chain complex of I1 dynein and structurally appeared to connect with the nexin-dynein regulatory complex. In axonemes from mutants that lack both the outer dynein arms and the MIA complex, I1 dynein failed to assemble, suggesting physical interactions between these three axonemal complexes and a role for the MIA complex in the stable assembly of I1 dynein. The MIA complex appears to regulate I1 dynein and possibly outer arm dyneins, which are both essential for normal motility.
The Journal of Cell Biology 04/2013; · 10.26 Impact Factor
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ABSTRACT: The tpg1 mutant of Chlamydomonas lacks the tubulin polyglutamylase TTLL9 and is deficient in flagellar tubulin polyglutamylation. It exhibits slow swimming, whereas the double mutant with oda2 (a slow-swimming mutant that lacks outer-arm dynein) is completely nonmotile. Thus, tubulin polyglutamylation must be important for the functioning of inner-arm dynein(s). In this study, we show that the tpg1 mutation only slightly affects the motility of mutants that lack dynein "e," one of the seven species of major inner-arm dyneins, whereas it greatly reduces the motility of mutants lacking other inner-arm dynein species. This suggests that dynein e is the main target of motility regulation by tubulin polyglutamylation. Furthermore, the motility of various mutants in the background of the tpg1 mutation raises the possibility that tubulin polyglutamylation also affects the dynein regulatory complex, a dynein e-associated key regulator of flagellar motility, which possibly constitutes the interdoublet (nexin) link. Tubulin polyglutamylation thus may play a central role in the regulation of ciliary and flagellar motility. © 2012 Wiley Periodicals, Inc.
Cytoskeleton 10/2012;
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ABSTRACT: Understanding the molecular architecture of the flagellum is crucial to elucidate the bending mechanism produced by this complex organelle. The current known structure of the flagellum has not yet been fully correlated with the complex composition and localization of flagellar components. Using cryoelectron tomography and subtomogram averaging while distinguishing each one of the nine outer doublet microtubules, we systematically collected and reconstructed the three-dimensional structures in different regions of the Chlamydomonas flagellum. We visualized the radial and longitudinal differences in the flagellum. One doublet showed a distinct structure, whereas the other eight were similar but not identical to each other. In the proximal region, some dyneins were missing or replaced by minor dyneins, and outer-inner arm dynein links were variable among different microtubule doublets. These findings shed light on the intricate organization of Chlamydomonas flagella, provide clues to the mechanism that produces asymmetric flagellar beating, and pose a new challenge for the functional study of the flagella.
The Journal of Cell Biology 09/2012; 198(5):913-25. · 10.26 Impact Factor
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Hannah M Mitchison,
Miriam Schmidts,
Niki T Loges,
Judy Freshour,
Athina Dritsoula,
Rob A Hirst,
Christopher O'Callaghan,
Hannah Blau,
Maha Al Dabbagh,
Heike Olbrich,
Philip L Beales, Toshiki Yagi,
Huda Mussaffi,
Eddie M K Chung,
Heymut Omran,
David R Mitchell
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ABSTRACT: Primary ciliary dyskinesia most often arises from loss of the dynein motors that power ciliary beating. Here we show that DNAAF3 (also known as PF22), a previously uncharacterized protein, is essential for the preassembly of dyneins into complexes before their transport into cilia. We identified loss-of-function mutations in the human DNAAF3 gene in individuals from families with situs inversus and defects in the assembly of inner and outer dynein arms. Knockdown of dnaaf3 in zebrafish likewise disrupts dynein arm assembly and ciliary motility, causing primary ciliary dyskinesia phenotypes that include hydrocephalus and laterality malformations. Chlamydomonas reinhardtii PF22 is exclusively cytoplasmic, and a PF22-null mutant cannot assemble any outer and some inner dynein arms. Altered abundance of dynein subunits in mutant cytoplasm suggests that DNAAF3 (PF22) acts at a similar stage as other preassembly proteins, for example, DNAAF2 (also known as PF13 or KTU) and DNAAF1 (also known as ODA7 or LRRC50), in the dynein preassembly pathway. These results support the existence of a conserved, multistep pathway for the cytoplasmic formation of assembly competent ciliary dynein complexes.
Nature Genetics 03/2012; 44(4):381-9, S1-2. · 35.53 Impact Factor
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Erik F Y Hom,
George B Witman,
Elizabeth H Harris,
Susan K Dutcher,
Ritsu Kamiya,
David R Mitchell,
Gregory J Pazour,
Mary E Porter,
Winfield S Sale,
Maureen Wirschell, Toshiki Yagi,
Stephen M King
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ABSTRACT: The formation and function of eukaryotic cilia/flagella require the action of a large array of dynein microtubule motor complexes. Due to genetic, biochemical, and microscopic tractability, Chlamydomonas reinhardtii has become the premier model system in which to dissect the role of dyneins in flagellar assembly, motility, and signaling. Currently, 54 proteins have been described as components of various Chlamydomonas flagellar dyneins or as factors required for their assembly in the cytoplasm and/or transport into the flagellum; orthologs of nearly all these components are present in other ciliated organisms including humans. For historical reasons, the nomenclature of these diverse dynein components and their corresponding genes, mutant alleles, and orthologs has become extraordinarily confusing. Here, we unify Chlamydomonas dynein gene nomenclature and establish a systematic classification scheme based on structural properties of the encoded proteins. Furthermore, we provide detailed tabulations of the various mutant alleles and protein aliases that have been used and explicitly define the correspondence with orthologous components in other model organisms and humans.
Cytoskeleton 09/2011; 68(10):555-65.
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ABSTRACT: Tubulin polyglutamylation is a modification that adds multiple glutamates to the gamma-carboxyl group of a glutamate residue in the C-terminal tails of alpha- and beta-tubulin [1, 2]. This modification has been implicated in the regulation of axonal transport and ciliary motility. However, its molecular function in cilia remains unknown. Here, using a novel Chlamydomonas reinhardtii mutant (tpg1) that lacks a homolog of human TTLL9, a glutamic acid ligase enzyme [3], we found that the lack of a long polyglutamate side chain in alpha-tubulin moderately weakens flagellar motility without noticeably impairing the axonemal structure. Furthermore, the double mutant of tpg1 with oda2, a mutation that leads to loss of outer-arm dynein, completely lacks motility. More surprisingly, when treated with protease and ATP, the axoneme of this paralyzed double mutant displayed faster microtubule sliding than the motile oda2 axoneme. These and other results suggest that polyglutamylation directly regulates microtubule-dynein interaction mainly by modulating the function of inner-arm dyneins.
Current biology: CB 02/2010; 20(5):441-5. · 10.99 Impact Factor
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ABSTRACT: Regulation of cell cycle progression in changing environments is vital for cell survival and maintenance, and different regulation mechanisms based on cell size and cell cycle time have been proposed. To determine the mechanism of cell cycle regulation in the unicellular green algae Chlamydomonas reinhardtii, we developed an on-chip single-cell cultivation system that allows for the strict control of the extracellular environment. We divided the Chlamydomonas cell cycle into interdivision and division phases on the basis of changes in cell size and found that, regardless of the amount of photosynthetically active radiation (PAR) and the extent of illumination, the length of the interdivision phase was inversely proportional to the rate of increase of cell volume. Their product remains constant indicating the existence of an 'interdivision timer'. The length of the division phase, in contrast, remained nearly constant. Cells cultivated under light-dark-light conditions did not divide unless they had grown to twice their initial volume during the first light period. This indicates the existence of a 'commitment sizer'. The ratio of the cell volume at the beginning of the division phase to the initial cell volume determined the number of daughter cells, indicating the existence of a 'mitotic sizer'.
Journal of Nanobiotechnology 01/2010; 8:23. · 5.09 Impact Factor
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ABSTRACT: The movements of cilia and flagella are driven by multiple species of dynein heavy chains (DHCs), which constitute inner- and outer-dynein arms. In Chlamydomonas, 11 DHC proteins have been identified in the axoneme, but 14 genes encoding axonemal DHCs are present in the genome. Here, we assigned each previously unassigned DHC gene to a particular DHC protein and found that DHC3, DHC4 and DHC11 encode novel, relatively low abundance DHCs. Immunofluorescence microcopy revealed that DHC11 is localized exclusively to the proximal approximately 2 microm region of the approximately 12 microm long flagellum. Analyses of growing flagella suggested that DHC3 and DHC4 are also localized to the proximal region. By contrast, the DHC of a previously identified inner-arm dynein, dynein b, displayed an inverse distribution pattern. Thus, the proximal portion of the flagellar axoneme apparently differs in dynein composition from the remaining portion; this difference might be relevant to the special function performed by the flagellar base.
Journal of Cell Science 05/2009; 122(Pt 9):1306-14. · 6.11 Impact Factor
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ABSTRACT: Outer arm dynein (OAD) of cilia and flagella contains two or three distinct heavy chains, each having a motor function. To elucidate their functional difference, we compared the in vitro motile properties of Chlamydomonas wild-type OAD containing the alpha, beta, and gamma heavy chains and three kinds of mutant OADs, each lacking one of the three heavy chains. For systematic comparison, a method was developed to introduce a biotin tag into a subunit, LC2, which served as the specific anchoring site on an avidin-coated glass surface. Wild-type OAD displayed microtubule gliding in the presence of ATP and ADP, with a maximal velocity of 5.0 mum/s, which is approximately 1/4 of the microtubule sliding velocity in the axoneme. The duty ratio was estimated to be as low as 0.08. The absence of the beta heavy chain lowered both the gliding velocity and ATPase activity, whereas the absence of the gamma heavy chain increased both activities. Strikingly, the absence of the alpha heavy chain lowered the gliding velocity but increased the ATPase activity. Thus, the three heavy chains are likely to play distinct roles and regulate each other to achieve coordinated force production.
Journal of Biological Chemistry 02/2009; 284(9):5927-35. · 4.77 Impact Factor
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Toshiki Yagi
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ABSTRACT: Multiple dynein heavy chain (DHC) genes are found in the genomes of organisms with motile cilia and flagella. Phylogenetic analyses classify these into several groups, each of which may be associated with a specific function. The Chlamydomonas genome contains 16 DHC genes, of which 15 genes have been correlated with particular DHC proteins. The functional properties of Chlamydomonas DHCs have been extensively studied by biochemical and genetic methods. Therefore, the phylogenetic classification of Chlamydomonas DHC genes can serve as the standard for DHC gene classification in other organisms. Here, I classify Chlamydomonas DHC genes by phylogenetic analysis and then show how to use this information to classify dyneins from other species that lack biochemical and genetic characterization. As an example, I classify the 16 human DHC genes into functional groups using the Chlamydomonas genes as references. Many of the human DHC genes have a closely related counterpart in Chlamydomonas, suggesting that the human genes will have functional properties similar to what has been described in Chlamydomonas.
Methods in cell biology 01/2009; 92:1-9. · 2.05 Impact Factor
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Heymut Omran,
Daisuke Kobayashi,
Heike Olbrich,
Tatsuya Tsukahara,
Niki T Loges,
Haruo Hagiwara,
Qi Zhang,
Gerard Leblond,
Eileen O'Toole,
Chikako Hara, [......], Toshiki Yagi,
Sumito Koshida,
Atsushi Miyawaki,
Hanswalter Zentgraf,
Horst Seithe,
Richard Reinhardt,
Yoshinori Watanabe,
Ritsu Kamiya,
David R Mitchell,
Hiroyuki Takeda
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ABSTRACT: Cilia and flagella are highly conserved organelles that have diverse roles in cell motility and sensing extracellular signals. Motility defects in cilia and flagella often result in primary ciliary dyskinesia. However, the mechanisms underlying cilia formation and function, and in particular the cytoplasmic assembly of dyneins that power ciliary motility, are only poorly understood. Here we report a new gene, kintoun (ktu), involved in this cytoplasmic process. This gene was first identified in a medaka mutant, and found to be mutated in primary ciliary dyskinesia patients from two affected families as well as in the pf13 mutant of Chlamydomonas. In the absence of Ktu/PF13, both outer and inner dynein arms are missing or defective in the axoneme, leading to a loss of motility. Biochemical and immunohistochemical studies show that Ktu/PF13 is one of the long-sought proteins involved in pre-assembly of dynein arm complexes in the cytoplasm before intraflagellar transport loads them for the ciliary compartment.
Nature 01/2009; 456(7222):611-6. · 36.28 Impact Factor
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ABSTRACT: How ciliary and flagellar motility is regulated is a challenging problem. The flagellar movement in Chlamydomonas reinhardtii is in part regulated by phosphorylation of a 138 kD intermediate chain (IC138) of inner arm dynein f (also called I1). In the present study, we found that the axoneme of mutants lacking dynein f lacks a novel protein having ankyrin repeat motifs, registered as FAP120 in the flagellar proteome database. FAP120 is also missing or decreased in the axonemes of bop5, a mutant that has a mutation in the structural gene of IC138 but assembles the dynein f complex. Intriguingly, the amounts of FAP120 in the axonemes of different alleles of bop5 and several dynein f-lacking mutants roughly parallel their contents of IC138. These results suggest a weak but stoichiometric interaction between FAP120 and IC138. We propose that FAP120 functions in the regulatoryprocess as part of a protein complex involving IC138. Cell Motil. Cytoskeleton 2008. (c) 2008 Wiley-Liss, Inc.
Cell Motility and the Cytoskeleton 12/2008; 66(8):448-56. · 4.19 Impact Factor
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ABSTRACT: The outer dynein arm of Chlamydomonas flagella contains three heavy chains (alpha, beta, and gamma), each of which exhibits motor activity. How they assemble and cooperate is of considerable interest. Here we report the isolation of a novel mutant, oda2-t, whose gamma heavy chain is truncated at about 30% of the sequence. While the previously isolated gamma chain mutant oda2 lacks the entire outer arm, oda2-t retains outer arms that contain alpha and beta heavy chains, suggesting that the N-terminal sequence (corresponding to the tail region) is necessary and sufficient for stable outer-arm assembly. Thin-section electron microscopy and image analysis localize the gamma heavy chain to a basal region of the outer-arm image in the axonemal cross section. The motility of oda2-t is lower than that of the wild type and oda11 (lacking the alpha heavy chain) but higher than that of oda2 and oda4-s7 (lacking the motor domain of the beta heavy chain). Thus, the outer-arm dynein lacking the gamma heavy-chain motor domain is partially functional. The availability of mutants lacking individual heavy chains should greatly facilitate studies on the structure and function of the outer-arm dynein.
Eukaryotic Cell 08/2008; 7(7):1136-45. · 3.60 Impact Factor
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ABSTRACT: 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.
Eukaryotic Cell 02/2008; 7(1):154-61. · 3.60 Impact Factor
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ABSTRACT: To elucidate the subunit composition of axonemal inner-arm dynein, we examined a 38 kDa protein (p38) co-purified with a Chlamydomonas inner arm subspecies, dynein d. We found it is a novel protein conserved among a variety of organisms with motile cilia and flagella. Immunoprecipitation using specific antibody verified its association with a heavy chain, actin and a previously identified light chain (p28). Unexpectedly, mutant axonemes lacking dynein d and other dyneins retained reduced amounts of p38. This finding suggests that p38 is involved in the docking of dynein d to specific loci.
FEBS Letters 12/2006; 580(27):6357-60. · 3.54 Impact Factor
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ABSTRACT: Experiments were carried out to see if isolated inner arm dyneins could functionally combine with axonemes lacking them. High-salt extract from the axoneme of Chlamydomonas oda1 mutant lacking outer-arm dynein was added to the demembranated cell models of ida1oda1 lacking inner arm dynein f (dynein I1) and outer arm dynein. After incubation, the originally paralyzed ida1oda1 axonemes recovered the ability to beat in the presence of ATP. A similar good motility recovery after incubation with crude oda1 extract was observed in ida9oda2 lacking outer arm and inner arm dynein c, and partial recovery in ida4oda1 lacking outer arm and inner arm species a, c, and d. These observations indicate that dynein f and dynein c can functionally bind with mutant axonemes lacking them. A method for combining isolated inner arm dyneins with axonemes in a functionally active manner should provide a powerful experimental tool with which to study the mechanism of beating.
Cell Motility and the Cytoskeleton 06/2006; 63(5):258-65. · 4.19 Impact Factor
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ABSTRACT: Ciliary and flagellar axonemes contain multiple inner arm dyneins of which the functional difference is largely unknown. In this study, a Chlamydomonas mutant, ida9, lacking inner arm dynein c was isolated and shown to carry a mutation in the DHC9 dynein heavy chain gene. The cDNA sequence of DHC9 was determined, and its information was used to show that >80% of it is lost in the mutant. Electron microscopy and image analysis showed that the ida9 axoneme lacked electron density near the base of the S2 radial spoke, indicating that dynein c localizes to this site. The mutant ida9 swam only slightly slower than the wild type in normal media. However, swimming velocity was greatly reduced when medium viscosity was modestly increased. Thus, dynein c in wild type axonemes must produce a significant force when flagella are beating in viscous media. Because motility analyses in vitro have shown that dynein c is the fastest among all the inner arm dyneins, we can regard this dynein as a fast yet powerful motor.
Journal of Biological Chemistry 12/2005; 280(50):41412-20. · 4.77 Impact Factor
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ABSTRACT: Recent indirect observations have suggested that various axonemal proteins in cilia and flagella of live cells undergo turnover independently of shortening or elongation of the axoneme. To gain direct evidence, here we examined using a FRAP (fluorescence recovery after photobleaching) technique whether actin, a subunit of inner arm dynein, is being turned over in Chlamydomonas flagella. Fluorescently labeled rabbit actin was introduced by electroporation into the cells of ida5oda1, a double mutant between oda1 lacking outer arm dynein and ida5 lacking several species of inner arm dyneins due to the absence of a conventional-type actin. In actin-loaded cells, flagella became motile and fluorescent due to incorporation of inner-arm dyneins containing the labeled actin. Cells were sandwiched between an agar layer and a coverslip so as to restrict flagellar movement. After a small portion of a flagellum was photobleached, the fluorescence intensity in the bleached area was monitored with a sensitive video camera. The fluorescence intensity in the photobleached region was found to recover 10-40% of the original level over several tens of minutes without changing its position. The time course and extent of the recovery varied greatly from one cell to another, suggesting that the turnover depends on cellular conditions. Western blot analysis indicated that 70-80% of flagellar actin was associated with the axoneme. Hence this experiment provides direct evidence that an axonemal component undergoes dynamic exchange in stationary flagella.
Cell Structure and Function 07/2004; 29(3):67-72. · 2.29 Impact Factor
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ABSTRACT: Dynein has four nucleotide binding sites, of which the functional significance is unknown except for the single catalytic site. To obtain clues to the function of non-catalytic nucleotide binding, we examined the effect of ADP on the in vitro motility of Chlamydomonas inner-arm dynein species 'a'. Upon continuous perfusion with ATP and ADP, microtubules glided on a dynein-coated glass surface with a velocity that gradually increased over a few minutes. The velocity increased faster at higher ADP concentrations. These results suggest that this dynein is activated by nucleotide binding to regulatory site(s) through an extremely slow process.
FEBS Letters 05/2004; 563(1-3):119-22. · 3.54 Impact Factor
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ABSTRACT: The outer dynein arm-docking complex (ODA-DC) targets the outer dynein arm to its correct binding site on the flagellar axoneme. The Chlamydomonas ODA-DC contains three proteins; loss of any one prevents normal assembly of the outer arm, leading to a slow, jerky swimming phenotype. We showed previously that the smallest ODA-DC subunit, DC3, has four EF-hands (Casey, D. M., Inaba, K., Pazour, G. J., Takada, S., Wakabayashi, K., Wilkerson, C. G., Kamiya, R., and Witman, G. B. (2003) Mol. Biol. Cell 14, 3650-3663). Two of the EF-hands fit the consensus pattern for calcium binding, and one of these contains two cysteine residues within its binding loop. To determine whether the predicted EF-hands are functional, we purified bacterially expressed wild-type DC3 and analyzed its calcium-binding potential in the presence and absence of dithiothreitol and Mg2+. The protein bound one calcium ion with an affinity (Kd) of approximately 1 x 10-5 m. Calcium binding was observed only in the presence of dithiothreitol and thus is redox-sensitive. DC3 also bound Mg2+ at physiological concentrations but with a much lower affinity. Changing the essential glutamate to glutamine in both EF-hands eliminated the calcium binding activity of the bacterially expressed protein. To investigate the role of the EF-hands in vivo, we transformed the modified DC3 gene into a Chlamydomonas insertional mutant lacking DC3. The transformed strain swam normally, assembled a normal number of outer arms, and had a normal photoshock response, indicating that the Glu to Gln mutations did not affect ODA-DC assembly, outer arm assembly, or Ca2+-mediated outer arm activity. Thus, DC3 is a true calcium-binding protein, but the function of this activity remains unknown.
Journal of Biological Chemistry 11/2003; 278(43):42652-9. · 4.77 Impact Factor
