IC2 participates in the cooperative activation of outer arm dynein densely attached to microtubules.

Ciliary outer-arm dynein (OAD) consists of heavy chains (HCs), intermediate chains (ICs), and light chains (LCs), of which HCs are the motor proteins that produce force. Studies using the green alga Chlamydomonas have revealed that ICs and LCs form a complex (IC/LC tower) at the base of the OAD tail and play a crucial role in anchoring OAD to specific sites on the microtubule. In this study, we isolated a novel slow-swimming Chlamydomonas mutant deficient in the IC2 protein.

Bld10p/Cep135 determines the number of triplets in the centriole independently of the cartwheel.

The conserved nine-fold structural symmetry of the centriole is thought to be generated by cooperation between two mechanisms, one dependent on and the other independent of the cartwheel, a sub-centriolar structure consisting of a hub and nine spokes. However, the molecular entity of the cartwheel-independent mechanism has not been elucidated. Here, using Chlamydomonas reinhardtii mutants, we show that Bld10p/Cep135, a conserved centriolar protein that connects cartwheel spokes and triplet microtubules, plays a central role in this mechanism.

The roles of a flagellar HSP40 ensuring rhythmic beating.

HSP40s are regarded as cochaperones, perpetually shuttling client polypeptides to HSP70s for refolding. However, many HSP40s that are central for disparate processes diverge from this paradigm. To elucidate the noncanonical mechanisms, we investigated HSP40 in the radial spoke (RS) complex in flagella.

Holliday junction resolvases mediate chloroplast nucleoid segregation.

Holliday junctions, four-stranded DNA structures formed during homologous recombination, are disentangled by resolvases that have been found in prokaryotes and eukaryotes but not in plant organelles. Here, we identify monokaryotic chloroplast 1 (MOC1) as a Holliday junction resolvase in chloroplasts by analyzing a green alga mutant defective in chloroplast nucleoid (DNA-protein complex) segregation. is structurally similar to a bacterial Holliday junction resolvase, resistance to ultraviolet (Ruv) C, and genetically conserved among green plants.

Identification of the mutation responsible for negative phototaxis in a "wild-type" strain of .

The unicellular green alga is a model organism for various studies in biology. CC-124 is a laboratory strain widely used as a wild type. However, this strain is known to carry mutation, which causes cells to swim away from the light source (negative phototaxis), in contrast to the cells of other wild-type strains, which swim toward the light source (positive phototaxis).

Eyespot-dependent determination of the phototactic sign in Chlamydomonas reinhardtii.

The biflagellate green alga Chlamydomonas reinhardtii exhibits both positive and negative phototaxis to inhabit areas with proper light conditions. It has been shown that treatment of cells with reactive oxygen species (ROS) reagents biases the phototactic sign to positive, whereas that with ROS scavengers biases it to negative. Taking advantage of this property, we isolated a mutant, lts1-211, which displays a reduction-oxidation (redox) dependent phototactic sign opposite to that of the wild type.

SAS-6 engineering reveals interdependence between cartwheel and microtubules in determining centriole architecture.

Centrioles are critical for the formation of centrosomes, cilia and flagella in eukaryotes. They are thought to assemble around a nine-fold symmetric cartwheel structure established by SAS-6 proteins. Here, we have engineered Chlamydomonas reinhardtii SAS-6-based oligomers with symmetries ranging from five- to ten-fold.

Reduced tubulin polyglutamylation suppresses flagellar shortness in Chlamydomonas.

Ciliary length control is an incompletely understood process essential for normal ciliary function. The flagella of Chlamydomonas mutants lacking multiple axonemal dyneins are shorter than normal; previously it was shown that this shortness can be suppressed by the mutation suppressor of shortness 1 (ssh1) via an unknown mechanism. To elucidate this mechanism, we carried out genetic analysis of ssh1 and found that it is a new allele of TPG2 (hereafter tpg2-3), which encodes FAP234 functioning in tubulin polyglutamylation in the axoneme.

Algal dual-specificity tyrosine phosphorylation-regulated kinase, triacylglycerol accumulation regulator1, regulates accumulation of triacylglycerol in nitrogen or sulfur deficiency.

Although microalgae accumulate triacylglycerol (TAG) and starch in response to nutrient-deficient conditions, the regulatory mechanisms are poorly understood. We report here the identification and characterization of a kinase, triacylglycerol accumulation regulator1 (TAR1), that is a member of the yeast (Saccharomyces cerevisiae) Yet another kinase1 (Yak1) subfamily in the dual-specificity tyrosine phosphorylation-regulated kinase family in a green alga (Chlamydomonas reinhardtii). The kinase domain of TAR1 showed auto- and transphosphorylation activities.

Space-dependent formation of central pair microtubules and their interactions with radial spokes.

Cilia and flagella contain nine outer doublet microtubules and a pair of central microtubules. The central pair of microtubules (CP) is important for cilia/flagella beating, as clearly shown by primary ciliary dyskinesia resulting from the loss of the CP. The CP is thought to regulate axonemal dyneins through interaction with radial spokes (RSs).

Cartwheel assembly.

The cartwheel is a subcentriolar structure consisting of a central hub and nine radially arranged spokes, located at the proximal end of the centriole. It appears at the initial stage of the centriole assembly process as the first ninefold symmetrical structure. The cartwheel was first described more than 50 years ago, but it is only recently that its pivotal role in establishing the ninefold symmetry of the centriole was demonstrated.

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.

FAP20 is an inner junction protein of doublet microtubules essential for both the planar asymmetrical waveform and stability of flagella in Chlamydomonas.

The axoneme-the conserved core of eukaryotic cilia and flagella-contains highly specialized doublet microtubules (DMTs). A long-standing question is what protein(s) compose the junctions between two tubules in DMT. Here we identify a highly conserved flagellar-associated protein (FAP), FAP20, as an inner junction (IJ) component.

Isolation and characterization of novel high-CO2-requiring mutants of Chlamydomonas reinhardtii.

Aquatic microalgae induce a carbon-concentrating mechanism (CCM) to maintain photosynthetic activity in low-CO2 (LC) conditions. Although the molecular mechanism of the CCM has been investigated using the single-cell green alga Chlamydomonas reinhardtii, and several CCM-related genes have been identified by analyzing high-CO2 (HC)-requiring mutants, many aspects of the CO2-signal transduction pathways remain to be elucidated. In this study, we report the isolation of novel HC-requiring mutants defective in the induction of CCM by DNA tagging.

The simplest integrated multicellular organism unveiled.

Volvocine green algae represent the "evolutionary time machine" model lineage for studying multicellularity, because they encompass the whole range of evolutionary transition of multicellularity from unicellular Chlamydomonas to >500-celled Volvox. Multicellular volvocalean species including Gonium pectorale and Volvox carteri generally have several common morphological features to survive as integrated multicellular organisms such as "rotational asymmetry of cells" so that the cells become components of the individual and "cytoplasmic bridges between protoplasts in developing embryos" to maintain the species-specific form of the multicellular individual before secretion of new extracellular matrix (ECM). However, these morphological features have not been studied in the four-celled colonial volvocine species Tetrabaena socialis that is positioned in the most basal lineage within the colonial or multicellular volvocine greens.

A conserved flagella-associated protein in Chlamydomonas, FAP234, is essential for axonemal localization of tubulin polyglutamylase TTLL9.

Tubulin undergoes various posttranslational modifications, including polyglutamylation, which is catalyzed by enzymes belonging to the tubulin tyrosine ligase-like protein (TTLL) family. A previously isolated Chlamydomonas reinhardtii mutant, tpg1, carries a mutation in a gene encoding a homologue of mammalian TTLL9 and displays lowered motility because of decreased polyglutamylation of axonemal tubulin. Here we identify a novel tpg1-like mutant, tpg2, which carries a mutation in the gene encoding FAP234, a flagella-associated protein of unknown function.

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

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.

The role of retrograde intraflagellar transport in flagellar assembly, maintenance, and function.

The maintenance of flagellar length is believed to require both anterograde and retrograde intraflagellar transport (IFT). However, it is difficult to uncouple the functions of retrograde transport from anterograde, as null mutants in dynein heavy chain 1b (DHC1b) have stumpy flagella, demonstrating solely that retrograde IFT is required for flagellar assembly. We isolated a Chlamydomonas reinhardtii mutant (dhc1b-3) with a temperature-sensitive defect in DHC1b, enabling inducible inhibition of retrograde IFT in full-length flagella.

Scaffolding function of the Chlamydomonas procentriole protein CRC70, a member of the conserved Cep70 family.

Centriole duplication occurs once per cell cycle through the assembly of daughter centrioles on the side wall of pre-existing centrioles. Little is known about the molecules involved in the assembly of new centrioles. Here, we identify CRC70 as a Chlamydomonas protein with an important role in the accumulation of centriole proteins at the site of assembly.

Structures of SAS-6 suggest its organization in centrioles.

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.

Discrete PIH proteins function in the cytoplasmic preassembly of different subsets of axonemal dyneins.

Axonemal dyneins are preassembled in the cytoplasm before being transported into cilia and flagella. Recently, PF13/KTU, a conserved protein containing a PIH (protein interacting with HSP90) domain, was identified as a protein responsible for dynein preassembly in humans and Chlamydomonas reinhardtii. This protein is involved in the preassembly of outer arm dynein and some inner arm dyneins, possibly as a cofactor of molecular chaperones.

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.