Multiple phosphorylation sites on γ-tubulin are essential and contribute to the biogenesis of basal bodies in Tetrahymena.

The mechanisms that regulate γ-tubulin, including its post-translational modifications, are poorly understood. γ-Tubulin is important for the duplication of centrioles and structurally similar basal bodies (BBs), organelles which contain a ring of nine triplet microtubules. The ciliate Tetrahymena thermophila carries hundreds of cilia in a single cell and provides an excellent model to specifically address the role of γ-tubulin in the BBs assembly and maintenance. The genome of Tetrahymena contains a single γ-tubulin gene. We show here that there are multiple isoforms of γ-tubulin that are likely generated by post-translational modifications. We identified evolutionarily conserved serine and threonine residues as potential phosphosites of γ-tubulin, including S80, S129, S131, T283, and S360. Several mutations that either prevent (S80A, S131A, T283A, S360A) or mimic (T283D) phosphorylation were conditionally lethal and at a higher temperature phenocopied a loss of γ-tubulin. Cells that overproduced S360D γ-tubulin displayed phenotypes consistent with defects in the microtubule-dependent functions, including an asymmetric division of the macronucleus and abnormalities in the pattern of BB rows, including gaps, fragmentation, and misalignment. In contrast, overexpression of S129D γ-tubulin affected the orientation, docking, and structure of the BBs, including a loss of either the B- or C-subfibers or the entire triplets. We conclude that conserved potentially phosphorylated amino acids of γ-tubulin are important for either the assembly or stability of BBs.

The conserved centrosomal protein FOR20 is required for assembly of the transition zone and basal body docking at the cell surface.

Within the FOP family of centrosomal proteins, the conserved FOR20 protein has been implicated in the control of primary cilium assembly in human cells. To ascertain its role in ciliogenesis, we have investigated the function of its ortholog, PtFOR20p, in the multiciliated unicellular organism Paramecium. Using combined functional and cytological analyses, we found that PtFOR20p specifically localises at basal bodies and is required to build the transition zone, a prerequisite to their maturation and docking at the cell surface and hence to ciliogenesis. We also found that PtCen2p (one of the two basal body specific centrins, an ortholog of HsCen2) is required to recruit PtFOR20p at the developing basal body and to control its length. By contrast, the other basal-body-specific centrin PtCen3p is not needed for assembly of the transition zone, but is required downstream, for basal body docking. Comparison of the structural defects induced by depletion of PtFOR20p, PtCen2p or PtCen3p, respectively, illustrates the dual role of the transition zone in the biogenesis of the basal body and in cilium assembly. The multiple potential roles of the transition zone during basal body biogenesis and the evolutionary conserved function of the FOP proteins in microtubule membrane interactions are discussed.

PHLP2 is essential and plays a role in ciliogenesis and microtubule assembly in Tetrahymena thermophila.

Recent studies have implicated the phosducin-like protein-2 (PHLP2) in regulation of CCT, a chaperonin whose activity is essential for folding of tubulin and actin. However, the exact molecular function of PHLP2 is unclear. Here we investigate the significance of PHLP2 in a ciliated unicellular model, Tetrahymena thermophila, by deleting its single homolog, Phlp2p. Cells lacking Phlp2p became larger and died within 96 h. Overexpressed Phlp2p-HA localized to cilia, basal bodies, and cytosol without an obvious change in the phenotype. Despite similar localization, overexpressed GFP-Phlp2p caused a dominant-negative effect. Cells overproducing GFP-Phlp2p had decreased rates of proliferation, motility and phagocytosis, as compared to wild type cells or cells overproducing a non-tagged Phlp2p. Growing GFP-Phlp2p-overexpressing cells had fewer cilia and, when deciliated, failed to regenerate cilia, indicating defects in cilia assembly. Paclitaxel-treated GFP-Phlp2p cells failed to elongate cilia, indicating a change in the microtubules dynamics. The pattern of ciliary and cytosolic tubulin isoforms on 2D gels differed between wild type and GFP-Phlp2p-overexpressing cells. Thus, in Tetrahymena, PhLP2 is essential and under specific experimental conditions its activity affects tubulin and microtubule-dependent functions including cilia assembly.

Katanin regulates dynamics of microtubules and biogenesis of motile cilia.

The in vivo significance of microtubule severing and the mechanisms governing its spatial regulation are not well understood. In Tetrahymena, a cell type with elaborate microtubule arrays, we engineered null mutations in subunits of the microtubule-severing complex, katanin. We show that katanin activity is essential. The net effect of katanin on the polymer mass depends on the microtubule type and location. Although katanin reduces the polymer mass and destabilizes the internal network of microtubules, its activity increases the mass of ciliary microtubules. We also show that katanin reduces the levels of several types of post-translational modifications on tubulin of internal and cortical microtubules. Furthermore, katanin deficiencies phenocopy a mutation of beta-tubulin that prevents deposition of polymodifications (glutamylation and glycylation) on microtubules. We propose that katanin preferentially severs older, post-translationally modified segments of microtubules.

Functional study of genes essential for autogamy and nuclear reorganization in Paramecium.

Like all ciliates, Paramecium tetraurelia is a unicellular eukaryote that harbors two kinds of nuclei within its cytoplasm. At each sexual cycle, a new somatic macronucleus (MAC) develops from the germ line micronucleus (MIC) through a sequence of complex events, which includes meiosis, karyogamy, and assembly of the MAC genome from MIC sequences. The latter process involves developmentally programmed genome rearrangements controlled by noncoding RNAs and a specialized RNA interference machinery. We describe our first attempts to identify genes and biological processes that contribute to the progression of the sexual cycle. Given the high percentage of unknown genes annotated in the P. tetraurelia genome, we applied a global strategy to monitor gene expression profiles during autogamy, a self-fertilization process. We focused this pilot study on the genes carried by the largest somatic chromosome and designed dedicated DNA arrays covering 484 genes from this chromosome (1.2% of all genes annotated in the genome). Transcriptome analysis revealed four major patterns of gene expression, including two successive waves of gene induction. Functional analysis of 15 upregulated genes revealed four that are essential for vegetative growth, one of which is involved in the maintenance of MAC integrity and another in cell division or membrane trafficking. Two additional genes, encoding a MIC-specific protein and a putative RNA helicase localizing to the old and then to the new MAC, are specifically required during sexual processes. Our work provides a proof of principle that genes essential for meiosis and nuclear reorganization can be uncovered following genome-wide transcriptome analysis.

Hyperglutamylation of tubulin can either stabilize or destabilize microtubules in the same cell.

In most eukaryotic cells, tubulin is subjected to posttranslational glutamylation, a conserved modification of unclear function. The glutamyl side chains form as branches of the primary sequence glutamic acids in two biochemically distinct steps: initiation and elongation. The length of the glutamyl side chain is spatially controlled and microtubule type specific. Here, we probe the significance of the glutamyl side chain length regulation in vivo by overexpressing a potent side chain elongase enzyme, Ttll6Ap, in Tetrahymena. Overexpression of Ttll6Ap caused hyperelongation of glutamyl side chains on the tubulin of axonemal, cortical, and cytoplasmic microtubules. Strikingly, in the same cell, hyperelongation of glutamyl side chains stabilized cytoplasmic microtubules and destabilized axonemal microtubules. Our observations suggest that the cellular outcomes of glutamylation are mediated by spatially restricted tubulin interactors of diverse nature.

Glutamylation on alpha-tubulin is not essential but affects the assembly and functions of a subset of microtubules in Tetrahymena thermophila.

Tubulin undergoes glutamylation, a conserved posttranslational modification of poorly understood function. We show here that in the ciliate Tetrahymena, most of the microtubule arrays contain glutamylated tubulin. However, the length of the polyglutamyl side chain is spatially regulated, with the longest side chains present on ciliary and basal body microtubules. We focused our efforts on the function of glutamylation on the alpha-tubulin subunit. By site-directed mutagenesis, we show that all six glutamates of the C-terminal tail domain of alpha-tubulin that provide potential sites for glutamylation are not essential but are needed for normal rates of cell multiplication and cilium-based functions (phagocytosis and cell motility). By comparative phylogeny and biochemical assays, we identify two conserved tubulin tyrosine ligase (TTL) domain proteins, Ttll1p and Ttll9p, as alpha-tubulin-preferring glutamyl ligase enzymes. In an in vitro microtubule glutamylation assay, Ttll1p showed a chain-initiating activity while Ttll9p had primarily a chain-elongating activity. GFP-Ttll1p localized mainly to basal bodies, while GFP-Ttll9p localized to cilia. Disruption of the TTLL1 and TTLL9 genes decreased the rates of cell multiplication and phagocytosis. Cells lacking both genes had fewer cortical microtubules and showed defects in the maturation of basal bodies. We conclude that glutamylation on alpha-tubulin is not essential but is required for efficiency of assembly and function of a subset of microtubule-based organelles. Furthermore, the spatial restriction of modifying enzymes appears to be a major mechanism that drives differential glutamylation at the subcellular level.

Members of the NIMA-related kinase family promote disassembly of cilia by multiple mechanisms.

The genome of Tetrahymena thermophila contains 39 loci encoding NIMA-related kinases (NRKs), an extraordinarily large number for a unicellular organism. Evolutionary analyses grouped these sequences into several subfamilies, some of which have orthologues in animals, whereas others are protist specific. When overproduced, NRKs of three subfamilies caused rapid shortening of cilia. Ultrastructural studies revealed that each NRK triggered ciliary resorption by a distinct mechanism that involved preferential depolymerization of a subset of axonemal microtubules, at either the distal or proximal end. Overexpression of a kinase-inactive variant caused lengthening of cilia, indicating that constitutive NRK-mediated resorption regulates the length of cilia. Each NRK preferentially resorbed a distinct subset of cilia, depending on the location along the anteroposterior axis. We also show that normal Tetrahymena cells maintain unequal length cilia. We propose that ciliates used a large number of NRK paralogues to differentially regulate the length of specific subsets of cilia in the same cell.

Cell context-specific effects of the beta-tubulin glycylation domain on assembly and size of microtubular organelles.

Tubulin glycylation is a posttranslational modification found in cells with cilia or flagella. The ciliate Tetrahymena has glycylation on ciliary and cortical microtubules. We showed previously that mutating three glycylation sites on beta-tubulin produces immotile 9 + 0 axonemes and inhibits cytokinesis. Here, we use an inducible glycylation domain mutation and epitope tagging to evaluate the potential of glycylation-deficient tubulin for assembly and maintenance of microtubular systems. In axonemes, the major defects, including lack of the central pair, occurred during assembly, and newly made cilia were abnormally short. The glycylation domain also was required for maintenance of the length of already assembled cilia. In contrast to the aberrant assembly of cilia, several types of cortical organelles showed an abnormally high number of microtubules in the same mutant cells. Thus, the consequences of deficiency in tubulin glycylation are organelle type specific and lead to either insufficient assembly (cilia) or excessive assembly (basal bodies and cortical microtubules). We suggest that the diverse functions of the beta-tubulin glycylation domain are executed by spatially restricted microtubule-associated proteins.

The CSC proteins FAP61 and FAP251 build the basal substructures of radial spoke 3 in cilia.

Dynein motors and regulatory complexes repeat every 96 nm along the length of motile cilia. Each repeat contains three radial spokes, RS1, RS2, and RS3, which transduct signals between the central microtubules and dynein arms. Each radial spoke has a distinct structure, but little is known about the mechanisms of assembly and function of the individual radial spokes. In Chlamydomonas, calmodulin and spoke-associated complex (CSC) is composed of FAP61, FAP91, and FAP251 and has been linked to the base of RS2 and RS3. We show that in Tetrahymena, loss of either FAP61 or FAP251 reduces cell swimming and affects the ciliary waveform and that RS3 is either missing or incomplete, whereas RS1 and RS2 are unaffected. Specifically, FAP251-null cilia lack an arch-like density at the RS3 base, whereas FAP61-null cilia lack an adjacent portion of the RS3 stem region. This suggests that the CSC proteins are crucial for stable and functional assembly of RS3 and that RS3 and the CSC are important for ciliary motility.

Effect of alteration in the global body plan on the deployment of morphogenesis-related protein epitopes labeled by the monoclonal antibody 12G9 in Tetrahymena thermophila.

We have employed monoclonal antibodies to reinvestigate the janus mutants of the ciliate Tetrahymena thermophila, which cause reversal of circumferential polarity on the dorsal surface of the cell. This reversal brings about frequent ectopic expression of ventral cortical landmarks, such as a "secondary" oral apparatus, on the dorsal surface. The principal antibody employed, FXXXIX-12G9, immunolabels both transient cortical structures not directly associated with basal bodies (the fission line and the postoral meridional filament) and more permanent structures (apical band and oral crescent) that are associated with basal bodies. 12G9-immunolabeling of janus cells has revealed additional phenotypes, including disorder of ciliary rows. Further, this labeling has shown that the postoral meridional filament is often expressed and the apical band is frequently interrupted on the mid-dorsal surface of janus cells irrespective of whether or not these cells express a "secondary" oral apparatus. Of the permanent structures revealed by 12G9 immunofluorescence, modifications of the oral crescent (OC) are associated with prior modifications in the development of basal body-containing structures in the secondary oral apparatus. The formation of the apical band (AB) is also commonly abnormal in janus cells; analysis of specific abnormalities shows that the AB depends both on its initiation at a specific site near the anterior basal body of apical basal body couplets and on the normal location of these couplets just posterior to the fission line. We also have uncovered an intriguing difference in the reactivity of apical-band filaments to the 12G9 antibody in the two non-allelic janus mutants (janA1 and janC2) that we have investigated. Taken together, our observations indicate that the formation of new cellular structures at division depends both upon pre-existing cytoskeletal structures and upon the positional information provided by large-scale cellular polarities.

DisAp-dependent striated fiber elongation is required to organize ciliary arrays.

Cilia-organizing basal bodies (BBs) are microtubule scaffolds that are visibly asymmetrical because they have attached auxiliary structures, such as striated fibers. In multiciliated cells, BB orientation aligns to ensure coherent ciliary beating, but the mechanisms that maintain BB orientation are unclear. For the first time in Tetrahymena thermophila, we use comparative whole-genome sequencing to identify the mutation in the BB disorientation mutant disA-1. disA-1 abolishes the localization of the novel protein DisAp to T. thermophila striated fibers (kinetodesmal fibers; KFs), which is consistent with DisAp's similarity to the striated fiber protein SF-assemblin. We demonstrate that DisAp is required for KFs to elongate and to resist BB disorientation in response to ciliary forces. Newly formed BBs move along KFs as they approach their cortical attachment sites. However, because they contain short KFs that are rotated, BBs in disA-1 cells display aberrant spacing and disorientation. Therefore, DisAp is a novel KF component that is essential for force-dependent KF elongation and BB orientation in multiciliary arrays.

A Centrin3-dependent, transient, appendage of the mother basal body guides the positioning of the daughter basal body in Paramecium.

Basal bodies are tightly controlled not only for their time of duplication but also for their movements, which ensure proper division and morphogenesis. However, the mechanisms underlying these movements only begin to be explored. We describe here a novel basal body appendage in Paramecium, the anterior left filament (ALF), which develops transiently from the mother basal body before duplication and disassembles once the new basal body is docked at the surface. By comparing the ultrastructure of dividing wild type cells to that of cells defective in basal body duplication, either by depletion of conserved proteins required for basal body assembly, or by mutation, we showed 1) that assembly of the ALF requires PtCen3p, one of the two basal body specific centrins and 2) that absence of the ALF correlates with a failure of the newly assembled basal bodies to tilt up to their docking site at the surface. This correlation suggests that the function of the ALF consists in anchoring centrin-containing contractile fibers which pull up the new basal body toward its site of docking. The presence in T. thermophila of an ALF-like appendage suggests the conservation of an ancestral mechanism ensuring the coupling of basal body duplication and cell morphogenesis.

Basal body duplication in Paramecium: the key role of Bld10 in assembly and stability of the cartwheel.

Basal bodies which nucleate cilia and flagella, and centrioles which organize centrosomes share the same architecture characterized by the ninefold symmetry of their microtubular shaft. Among the conserved proteins involved in the biogenesis of the canonical 9-triplet centriolar structures, Sas-6 and Bld10 proteins have been shown to play central roles in the early steps of assembly and in establishment/stabilization of the ninefold symmetry. Using fluorescent tagged proteins and RNAi to study the localization and function of these two proteins in Paramecium, we focused on the early effects of their depletion, the consequences of their overexpression and their functional interdependence. We find that both genes are essential and their depletion affects cartwheel assembly and hence basal body duplication. We also show that, contrary to Sas6p, Bld10p is not directly responsible for the establishment of the ninefold symmetry, but is required not only for new basal body assembly and stability but also for Sas6p maintenance at mature basal bodies. Finally, ultrastructural analysis of cells overexpressing either protein revealed two types of early assembly intermediates, hub-like structures and generative discs, suggesting a conserved scaffolding process.

TTLL3 Is a tubulin glycine ligase that regulates the assembly of cilia.

In most ciliated cell types, tubulin is modified by glycylation, a posttranslational modification of unknown function. We show that the TTLL3 proteins act as tubulin glycine ligases with chain-initiating activity. In Tetrahymena, deletion of TTLL3 shortened axonemes and increased their resistance to paclitaxel-mediated microtubule stabilization. In zebrafish, depletion of TTLL3 led to either shortening or loss of cilia in several organs, including the Kupffer's vesicle and olfactory placode. We also show that, in vivo, glutamic acid and glycine ligases oppose each other, likely by competing for shared modification sites on tubulin. We propose that tubulin glycylation regulates the assembly and dynamics of axonemal microtubules and acts either directly or indirectly by inhibiting tubulin glutamylation.

The origin of mirror-image symmetry doublet cells in the hypotrich ciliateParaurostyla weissei.

Mirror-image doublets of the hypotrich ciliateParaurostyla weissei were induced by modifying culture conditions. Successive steps of doublet formation involve inhibiting the separation of daughter cells during cell division and the shifting of these to attain a parallel configuration. The posterior part of the adoral band of membranelles in the right component then turns to the left and fuses with the distal terminal of the membranellar band in the left component. In effect, part of the adoral band and some of the paroral membranelles become apposed upside down, and the paroral membranelles of the right component are located on the left side of the adoral membranelles. A new site of oral primordium formation is initiated at the junction of the two oral apparatuses, the ciliature of which is arranged in a mirror-image pattern. During further cortical reorganization, the whole body ciliature of the right component becomes organized as a mirror-image of the normal left component.Both components of the doublet show the same ultrastructure of body ciliature and lack the right marginal cirri; the symmetry-reversal half, however, possesses multiple rows of left marginal cirri. The individual adoral membranelles and paroral membranelles in the symmetry-reversal component are rotated anteroposteriorly. Some aspects of the patterning of cortical structures are discussed.