Studies In Chlamydomonas Research Articles

Regulation of motor activity of ciliary outer-arm dynein by the light chain 1; Implications from the structure of the light chain bound to the microtubule-binding domain of the heavy chain.

Ciliary bending movements are powered by motor protein axonemal dyneins. They are largely classified into two groups, inner-arm dynein and outer-arm dynein. Outer-arm dynein, which is important for the elevation of ciliary beat frequency, has three heavy chains (α, β, and γ), two intermediate chains, and more than 10 light chains in green algae, Chlamydomonas. Most of intermediate chains and light chains bind to the tail regions of heavy chains. In contrast, the light chain LC1 was found to bind to the ATP-dependent microtubule-binding domain of outer-arm dynein γ-heavy chain. Interestingly, LC1 was also found to interact with microtubules directly, but it reduces the affinity of the microtubule-binding domain of γ-heavy chain for microtubules, suggesting the possibility that LC1 may control ciliary movement by regulating the affinity of outer-arm dyneins for microtubules. This hypothesis is supported by the LC1 mutant studies in Chlamydomonas and Planaria showing that ciliary movements in LC1 mutants were disordered with low coordination of beating and low beat frequency. To understand the molecular mechanism of the regulation of outer-arm dynein motor activity by LC1, X-ray crystallography and cryo-electron microscopy have been used to determine the structure of the light chain bound to the microtubule-binding domain of γ-heavy chain. In this review article, we show the recent progress of structural studies of LC1, and suggest the regulatory role of LC1 in the motor activity of outer-arm dyneins. This review article is an extended version of the Japanese article, The Complex of Outer-arm Dynein Light Chain-1 and the Microtubule-binding Domain of the Heavy Chain Shows How Axonemal Dynein Tunes Ciliary Beating, published in SEIBUTSU BUTSURI Vol. 61, p. 20-22 (2021).

Flagellar-associated Protein FAP85 Is a Microtubule Inner Protein That Stabilizes Microtubules.

Genomics and proteomics studies in Chlamydomonas have revealed that an axoneme is composed of 200-600 types of proteins, including uncharacterized proteins collectively named flagellar-associated proteins (FAPs). Nine FAPs contain the EF-hand motif; however, they have not yet been well characterized. To find components responsible for Chlamydomonas-specific waveform changes coupled with intracellular Ca2+ concentrations, we focused on FAP85, an EF-hand motif-containing FAP specific to Chlamydomonas and its relatives. We cloned the cDNA encoding FAP85, expressed it in Escherichia coli cells, and generated a polyclonal antibody against the expressed protein. Immunoblotting showed that FAP85 was present in every axoneme of several flagellar mutants lacking major axonemal components. Immuno-electron microscopy revealed that anti-FAP85 antibodies were found only on the inner wall of A-tubules of the doublets exposed by N-lauroylsarcosine (Sarkosyl) treatment. The zero-length cross-linker 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) applied to 0.6 M KCl-extracted axonemes generated a 75-kDa complex containing β-tubulin and FAP85. Further characterization of FAP85 and its effects on microtubule dynamics showed that FAP85 binds to tubulin and stabilized microtubules. According to these results, we conclude that FAP85 is a novel member of microtubule-binding proteins, localizing on the inner wall of the A-tubule and stabilizing microtubules.Key words: Chlamydomonas, flagella, doublet microtubule, microtubule inner proteins.

The Deep Thioredoxome in Chlamydomonas reinhardtii: New Insights into Redox Regulation

Thiol-based redox post-translational modifications have emerged as important mechanisms of signaling and regulation in all organisms, and thioredoxin plays a key role by controlling the thiol-disulfide status of target proteins. Recent redox proteomic studies revealed hundreds of proteins regulated by glutathionylation and nitrosylation in the unicellular green alga Chlamydomonas reinhardtii, while much less is known about the thioredoxin interactome in this organism. By combining qualitative and quantitative proteomic analyses, we have comprehensively investigated the Chlamydomonas thioredoxome and 1188 targets have been identified. They participate in a wide range of metabolic pathways and cellular processes. This study broadens not only the redox regulation to new enzymes involved in well-known thioredoxin-regulated metabolic pathways but also sheds light on cellular processes for which data supporting redox regulation are scarce (aromatic amino acid biosynthesis, nuclear transport, etc). Moreover, we characterized 1052 thioredoxin-dependent regulatory sites and showed that these data constitute a valuable resource for future functional studies in Chlamydomonas. By comparing this thioredoxome with proteomic data for glutathionylation and nitrosylation at the protein and cysteine levels, this work confirms the existence of a complex redox regulation network in Chlamydomonas and provides evidence of a tremendous selectivity of redox post-translational modifications for specific cysteine residues.

Robust Transgene Expression from Bicistronic mRNA in the Green Alga Chlamydomonas reinhardtii.

The unicellular green alga Chlamydomonas reinhardtii is a model organism that provides an opportunity to understand the evolution and functional biology of the lineage that includes the land plants, as well as aspects of the fundamental core biology conserved throughout the eukaryotic phylogeny. Although many tools are available to facilitate genetic, molecular biological, biochemical, and cell biological studies in Chlamydomonas, expression of unselected transgenes of interest (GOIs) has been challenging. In most methods used previously, the GOI and a selectable marker are expressed from two separate mRNAs, so that their concomitant expression is not guaranteed. In this study, we developed constructs that allow expression of an upstream GOI and downstream selectable marker from a single bicistronic mRNA. Although this approach in other systems has typically required a translation-enhancing element such as an internal ribosome entry site for the downstream marker, we found that a short stretch of unstructured junction sequence was sufficient to obtain adequate expression of the downstream gene, presumably through post-termination reinitiation. With this system, we obtained robust expression of both endogenous and heterologous GOIs, including fluorescent proteins and tagged fusion proteins, in the vast majority of transformants, thus eliminating the need for tedious secondary screening for GOI-expressing transformants. This improved efficiency should greatly facilitate a variety of genetic and cell-biological studies in Chlamydomonas and also enable new applications such as expression-based screens and large-scale production of foreign proteins.

Cellular organelles facilitate dimerization of a newly identified Arf from Chlamydomonas reinhardtii.

GTPases of the Ras superfamily regulate a wide variety of cellular processes including vesicular transport and various secretory pathways of the cell. ADP - ribosylation factor (ARF) belongs to one of the five major families of the Ras superfamily and serves as an important component of vesicle formation and transport machinery of the cells. The binding of GTP to these Arfs and its subsequent hydrolysis, induces conformational changes in these proteins leading to their enzymatic activities. The dimeric form of Arf is associated with membrane pinch-off during vesicle formation. In this report, we have identified an arf gene from the unicellular green alga Chlamydomonas reinhardtii, CrArf, and showed that the oligomeric state of the protein in C.renhardtii is modulated by the cellular membrane environment of the organism. Protein cross-linking experiments showed that the purified recombinant CrArf has the ability to form a dimer. Both the 20-kDa monomeric and 40-kDa dimeric forms of CrArf were recognized from Chlamydomonas total cell lysate (CrTLC) and purified recombinant CrArf by the CrArf specific antibody. The membranous environment of the cell appeared to facilitate dimerization of the CrArf, as dimeric form was found exclusively associated with the membrane bound organelles. The subcellular localization studies in Chlamydomonas suggested that CrArf mainly localized in the cytosol and was mislocalized in vesicle transport machinery inhibitor treated cells. This research sheds light on the importance of the cellular membrane environment for regulating the oligomeric state of CrArf protein in this organism and associated functional role.

Getting to the heart of intraflagellar transport using Trypanosoma and Chlamydomonas models: the strength is in their differences

Cilia and flagella perform diverse roles in motility and sensory perception, and defects in their construction or their function are responsible for human genetic diseases termed ciliopathies. Cilia and flagella construction relies on intraflagellar transport (IFT), the bi-directional movement of ‘trains’ composed of protein complexes found between axoneme microtubules and the flagellum membrane. Although extensive information about IFT components and their mode of action were discovered in the green algae Chlamydomonas reinhardtii, other model organisms have revealed further insights about IFT. This is the case of Trypanosoma brucei, a flagellated protist responsible for sleeping sickness that is turning out to be an emerging model for studying IFT. In this article, we review different aspects of IFT, based on studies of Chlamydomonas and Trypanosoma. Data available from both models are examined to ask challenging questions about IFT such as the initiation of flagellum construction, the setting-up of IFT and the mode of formation of IFT trains, and their remodeling at the tip as well as their recycling at the base. Another outstanding question is the individual role played by the multiple IFT proteins. The use of different models, bringing their specific biological and experimental advantages, will be invaluable in order to obtain a global understanding of IFT.

The Armadillo Repeat Protein PF16 Is Essential for Flagellar Structure and Function in Plasmodium Male Gametes

Malaria, caused by the apicomplexan parasite Plasmodium, threatens 40% of the world's population. Transmission between vertebrate and insect hosts depends on the sexual stages of the life-cycle. The male gamete of Plasmodium parasite is the only developmental stage that possesses a flagellum. Very little is known about the identity or function of proteins in the parasite's flagellar biology. Here, we characterise a Plasmodium PF16 homologue using reverse genetics in the mouse malaria parasite Plasmodium berghei. PF16 is a conserved Armadillo-repeat protein that regulates flagellar structure and motility in organisms as diverse as green algae and mice. We show that P. berghei PF16 is expressed in the male gamete flagellum, where it plays a crucial role maintaining the correct microtubule structure in the central apparatus of the axoneme as studied by electron microscopy. Disruption of the PF16 gene results in abnormal flagellar movement and reduced fertility, but does not lead to complete sterility, unlike pf16 mutations in other organisms. Using homology modelling, bioinformatics analysis and complementation studies in Chlamydomonas, we show that some regions of the PF16 protein are highly conserved across all eukaryotes, whereas other regions may have species-specific functions. PF16 is the first ARM-repeat protein characterised in the malaria parasite genus Plasmodium and this study opens up a novel model for analysis of Plasmodium flagellar biology that may provide unique insights into an ancient organelle and suggest novel intervention strategies to control the malaria parasite.

Redistribution of the kinesin-II subunit KAP from cilia to nuclei during the mitotic and ciliogenic cycles in sea urchin embryos

KAP is the non-motor subunit of the heteromeric plus-end directed microtubule (MT) motor protein kinesin-II essential for normal cilia formation. Studies in Chlamydomonas have demonstrated that kinesin-II drives the anterograde intraflagellar transport (IFT) of protein complexes along ciliary axonemes. We used a green fluorescent protein (GFP) chimera of KAP, KAP-GFP, to monitor movements of this kinesin-II subunit in cells of sea urchin blastulae where cilia are retracted and rebuilt with each mitosis. As expected if involved in IFT, KAP-GFP localized to apical cytoplasm, basal bodies, and cilia and became concentrated on basal bodies of newly forming cilia. Surprisingly, after ciliary retraction early in mitosis, KAP-GFP moved into nuclei before nuclear envelope breakdown, was again present in nuclei after nuclear envelope reformation, and only decreased in nuclei as ciliogenesis reinitiated. Nuclear transport of KAP-GFP could be due to a putative nuclear localization signal and nuclear export signals identified in the sea urchin KAP primary sequence. Our observation of a protein involved in IFT being imported into the nucleus after ciliary retraction and again after nuclear envelope reformation suggests KAP115 may serve as a signal to the nucleus to reinitiate cilia formation during sea urchin development.

ISOLATION AND CHARACTERIZATION OF DOMINANT TUNICAMYCIN RESISTANCE MUTATIONS IN CHLAMYDOMONAS REINHARDTII (CHLOROPHYCEAE)

Seven independent mutations that confer resistance to the nucleoside antibiotic tunicamycin in the unicellular green alga Chlamydomonas reinhardtii were isolated in wild-type diploid cells. Upon resolution to haploidy, all the mutations segregated as single mutations and were semi-dominant when retested in heterozygous diploids. In addition, the TUN1-3 allele affects the ability of cells to agglutinate during conjugation in the absence of tunicamycin. The seven mutations map to a single locus on linkage group VIII. These mutations may be useful as a selectable marker in transformation studies of Chlamydomonas and in studies of processes that require asparagine-linked glycosylation.