The methylome of motile cilia.

Cilia are highly complex motile, sensory, and secretory organelles that contain perhaps 1000 or more distinct protein components, many of which are subject to various posttranslational modifications such as phosphorylation, N-terminal acetylation, and proteolytic processing. Another common modification is the addition of one or more methyl groups to the side chains of arginine and lysine residues. These tunable additions delocalize the side-chain charge, decrease hydrogen bond capacity, and increase both bulk and hydrophobicity. Methylation is usually mediated by S-adenosylmethionine (SAM)-dependent methyltransferases and reversed by demethylases. Previous studies have identified several ciliary proteins that are subject to methylation including axonemal dynein heavy chains that are modified by a cytosolic methyltransferase. Here, we have performed an extensive proteomic analysis of multiple independently derived cilia samples to assess the potential for SAM metabolism and the extent of methylation in these organelles. We find that cilia contain all the enzymes needed for generation of the SAM methyl donor and recycling of the S-adenosylhomocysteine and tetrahydrofolate byproducts. In addition, we find that at least 155 distinct ciliary proteins are methylated, in some cases at multiple sites. These data provide a comprehensive resource for studying the consequences of methyl marks on ciliary biology.

Methylation of ciliary dynein motors involves the essential cytosolic assembly factor DNAAF3/PF22.

Axonemal dynein motors drive ciliary motility and can consist of up to twenty distinct components with a combined mass of ~2 MDa. In mammals, failure of dyneins to assemble within the axonemal superstructure leads to primary ciliary dyskinesia. Syndromic phenotypes include infertility, rhinitis, severe bronchial conditions, and situs inversus. Nineteen specific cytosolic factors (Dynein Axonemal Assembly Factors; DNAAFs) are necessary for axonemal dynein assembly, although the detailed mechanisms involved remain very unclear. Here, we identify the essential assembly factor DNAAF3 as a structural ortholog of S-adenosylmethionine-dependent methyltransferases. We demonstrate that dynein heavy chains, especially those forming the ciliary outer arms, are methylated on key residues within various nucleotide-binding sites and on microtubule-binding domain helices directly involved in the transition to low binding affinity. These variable modifications, which are generally missing in a Chlamydomonas null mutant for the DNAAF3 ortholog PF22 (DAB1), likely impact on motor mechanochemistry fine-tuning the activities of individual dynein complexes.

N-Terminal Processing and Modification of Ciliary Dyneins.

Axonemal dyneins are highly complex microtubule motors that power ciliary motility. These multi-subunit enzymes are assembled at dedicated sites within the cytoplasm. At least nineteen cytosolic factors are specifically needed to generate dynein holoenzymes and/or for their trafficking to the growing cilium. Many proteins are subject to N-terminal processing and acetylation, which can generate degrons subject to the AcN-end rule, alter N-terminal electrostatics, generate new binding interfaces, and affect subunit stoichiometry through targeted degradation. Here, we have used mass spectrometry of cilia samples and electrophoretically purified dynein heavy chains from Chlamydomonas to define their N-terminal processing; we also detail the N-terminal acetylase complexes present in this organism. We identify four classes of dynein heavy chain based on their processing pathways by two distinct acetylases, one of which is dependent on methionine aminopeptidase activity. In addition, we find that one component of both the outer dynein arm intermediate/light chain subcomplex and the docking complex is processed to yield an unmodified Pro residue, which may provide a setpoint to direct the cytosolic stoichiometry of other dynein complex subunits that contain N-terminal degrons. Thus, we identify and describe an additional level of processing and complexity in the pathways leading to axonemal dynein formation in cytoplasm.

Outer-arm dynein light chain LC1 is required for normal motor assembly kinetics, ciliary stability, and motility.

Light chain 1 (LC1) is a highly conserved leucine-rich repeat protein associated with the microtubule-binding domain of the Chlamydomonas outer-dynein arm γ heavy chain. LC1 mutations in humans and trypanosomes lead to motility defects, while its loss in oomycetes results in aciliate zoospores. Here we describe a Chlamydomonas LC1 null mutant (dlu1-1). This strain has reduced swimming velocity and beat frequency, can undergo waveform conversion, but often exhibits loss of hydrodynamic coupling between the cilia. Following deciliation, Chlamydomonas cells rapidly rebuild cytoplasmic stocks of axonemal dyneins. Loss of LC1 disrupts the kinetics of this cytoplasmic preassembly so that most outer-arm dynein heavy chains remain monomeric even after several hours. This suggests that association of LC1 with its heavy chain-binding site is a key step or checkpoint in the outer-arm dynein assembly process. Similarly to strains lacking the entire outer arm and inner arm I1/f, we found that loss of LC1 and I1/f in dlu1-1 ida1 double mutants resulted in cells unable to build cilia under normal conditions. Furthermore, dlu1-1 cells do not exhibit the usual ciliary extension in response to lithium treatment. Together, these observations suggest that LC1 plays an important role in the maintenance of axonemal stability.

Developmental changes in ciliary composition during gametogenesis in Chlamydomonas.

Chlamydomonas reinhardtii transitions from mitotically dividing vegetative cells to sexually competent gametes of two distinct mating types following nutrient deprivation. Gametes of opposite mating type interact via their cilia, initiating an intraciliary signaling cascade and ultimately fuse forming diploid zygotes. The process of gametogenesis is genetically encode, and a previous study revealed numerous significant changes in mRNA abundance during this life-cycle transition. Here we describe a proteomic analysis of cilia derived from vegetative and gametic cells of both mating types in an effort to assess the global changes that occur within the organelle during this process. We identify numerous membrane- and/or matrix-associated proteins in gametic cilia that were not detected in cilia from vegetative cells. This includes the pro-protein from which the GATI-amide gametic chemotactic modulator derives, as well as receptors, a dynamin-related protein, ammonium transporters, two proteins potentially involved in the intraciliary signaling cascade-driven increase in cAMP, and multiple proteins with a variety of interaction domains. These changes in ciliary composition likely directly affect the functional properties of this organelle as the cell transitions between life-cycle stages.

Heme-binding protein CYB5D1 is a radial spoke component required for coordinated ciliary beating.

Coordinated beating is crucial for the function of multiple cilia. However, the molecular mechanism is poorly understood. Here, we characterize a conserved ciliary protein CYB5D1 with a heme-binding domain and a cordon-bleu ubiquitin-like domain. Mutation or knockdown of Cyb5d1 in zebrafish impaired coordinated ciliary beating in the otic vesicle and olfactory epithelium. Similarly, the two flagella of an insertional mutant of the CYB5D1 ortholog in Chlamydomonas (Crcyb5d1) showed an uncoordinated pattern due to a defect in the cis-flagellum. Biochemical analyses revealed that CrCYB5D1 is a radial spoke stalk protein that binds heme only under oxidizing conditions. Lack of CrCYB5D1 resulted in a reductive shift in flagellar redox state and slowing down of the phototactic response. Treatment of Crcyb5d1 with oxidants restored coordinated flagellar beating. Taken together, these data suggest that CrCYB5D1 may integrate environmental and intraciliary signals and regulate the redox state of cilia, which is crucial for the coordinated beating of multiple cilia.

WDR92 is required for axonemal dynein heavy chain stability in cytoplasm.

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.