Haru-aki Yanagisawa

Structures of sas-6 suggest its organization in centrioles

Self-assembly of a centriolar protein may contribute to organizing the cartwheel-like hub and establishing centriole symmetry. Centrioles are cylindrical, ninefold symmetrical structures with peripheral triplet microtubules strictly required to template cilia and flagella. The highly conserved protein SAS-6 constitutes the center of the cartwheel assembly that scaffolds centrioles early in their biogenesis. We determined the x-ray structure of the amino-terminal domain of SAS-6 from zebrafish, and we show that recombinant SAS-6 self-associates in vitro into assemblies that resemble cartwheel centers. Point mutations are consistent with the notion that centriole formation in vivo depends on the interactions that define the self-assemblies observed here. Thus, these interactions are probably essential to the structural organization of cartwheel centers.

Tubulin Polyglutamylation Regulates Axonemal Motility by Modulating Activities of Inner-Arm Dyneins

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.

A molecular ruler determines the repeat length in eukaryotic cilia and flagella

Existence of cellular structures with specific size raises a fundamental question in biology: How do cells measure length? One conceptual answer to this question is by a molecular ruler, but examples of such rulers in eukaryotes are lacking. In this work, we identified a molecular ruler in eukaryotic cilia and flagella. Using cryo-electron tomography, we found that FAP59 and FAP172 form a 96–nanometer (nm)–long complex in Chlamydomonas flagella and that the absence of the complex disrupted 96-nm repeats of axonemes. Furthermore, lengthening of the FAP59/172 complex by domain duplication resulted in extension of the repeats up to 128 nm, as well as duplication of specific axonemal components. Thus, the FAP59/172 complex is the molecular ruler that determines the 96-nm repeat length and arrangements of components in cilia and flagella. A protein complex controls the length and assembly of repeat structures in eukaryotic cilia and flagella. Molecular ruler rules cilia and flagella length Cilia and flagella contain within their ultrastructure repeating structures at regularly spaced intervals. How does the cell measure length with nanometer precision? Oda et al. identify a flagella protein complex in Chlamydomonas that appears to act as a sort of molecula ruler to define repeat length. Genetic changes that would change the length of this protein led to corresponding changes in the length of repeats within the resulting flagella. Science, this issue p. 857

Systematic comparison of in vitro motile properties between chlamydomonas wild-type and mutant outer arm dyneins each lacking one of the three heavy chains

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 α, β, and γ 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 μm/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 β heavy chain lowered both the gliding velocity and ATPase activity, whereas the absence of the γ heavy chain increased both activities. Strikingly, the absence of the α 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.

The MIA complex is a conserved and novel dynein regulator essential for normal ciliary motility

The MIA complex, composed of FAP100 and FAP73, interacts with I1 dynein components and is required for normal ciliary beat frequency.

TTC26/DYF13 is an intraflagellar transport protein required for transport of motility-related proteins into flagella

Cilia/flagella are assembled and maintained by the process of intraflagellar transport (IFT), a highly conserved mechanism involving more than 20 IFT proteins. However, the functions of individual IFT proteins are mostly unclear. To help address this issue, we focused on a putative IFT protein TTC26/DYF13. Using live imaging and biochemical approaches we show that TTC26/DYF13 is an IFT complex B protein in mammalian cells and Chlamydomonas reinhardtii. Knockdown of TTC26/DYF13 in zebrafish embryos or mutation of TTC26/DYF13 in C. reinhardtii, produced short cilia with abnormal motility. Surprisingly, IFT particle assembly and speed were normal in dyf13 mutant flagella, unlike in other IFT complex B mutants. Proteomic and biochemical analyses indicated a particular set of proteins involved in motility was specifically depleted in the dyf13 mutant. These results support the concept that different IFT proteins are responsible for different cargo subsets, providing a possible explanation for the complexity of the IFT machinery. DOI: http://dx.doi.org/10.7554/eLife.01566.001

Identification of the Outer-Inner Dynein Linker as a Hub Controller for Axonemal Dynein Activities

BACKGROUND In flagella, the outer dynein arm (ODA) and inner dynein arm (IDA) play distinct roles in generating beating motion. However, functional communications between the two dyneins have not been investigated. RESULTS Here, we demonstrated by cryo-electron microscopy and chemical crosslinking that intermediate chain 2 (IC2) of ODAs interacts with the dynein regulatory complex in the axoneme and constitutes part of the outer-inner dynein (OID) linker. Furthermore, we identified IC2 as a functional hub between ODAs and IDAs based on the phenotypes of Chlamydomonas mutants expressing biotinylation-tagged IC2. The flagella of the IC2 mutant showed activated microtubule sliding and enhanced ATPase activities of ODAs, as well as an altered waveform, indicating attenuated IDA activity. CONCLUSIONS We concluded that the OID linker controls both ODAs and IDAs and regulates flagellar beating.

Novel 44-Kilodalton Subunit of Axonemal Dynein Conserved from Chlamydomonas to Mammals

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.

A novel subunit of axonemal dynein conserved among lower and higher eukaryotes

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.

Detailed Structural and biochemical characterization of the nexin-dynein regulatory complex

The nexin-dynein regulatory complex (N-DRC) is a microtubule-cross-bridging structure in cilia/flagella. The precise 3D positions of N-DRC subunits are identified using cryo–electron tomography and structural labeling. The N-DRC is purified and its composition and microtubule-binding properties were characterized.