Tubulin post-translational modifications in protists - Tiny models for solving big questions.

Protists are an exceptionally diverse group of mostly single-celled eukaryotes. The organization of the microtubular cytoskeleton in protists from various evolutionary lineages has different levels of sophistication, from a network of microtubules (MTs) supporting intracellular trafficking as in Dictyostelium, to complex structures such as basal bodies and cilia/flagella enabling cell motility, and lineage-specific adaptations such as the ventral disc in Giardia. MTs building these diverse structures have specific properties partly due to the presence of tubulin post-translational modifications (PTMs). Among them there are highly evolutionarily conserved PTMs: acetylation, detyrosination, (poly)glutamylation and (poly)glycylation. In some protists also less common tubulin PTMs were identified, including phosphorylation, methylation, Δ2-, Δ5- of α-tubulin, polyubiquitination, sumoylation, or S-palmitoylation. Not surprisingly, several single-celled organisms become models to study tubulin PTMs, including their effect on MT properties and discovery of the modifying enzymes. Here, we briefly summarize the current knowledge on tubulin PTMs in unicellular eukaryotes and highlight key findings in protists as model organisms.

Evolutionary divergence of enzymatic mechanisms for posttranslational polyglycylation.

Polyglycylation is a posttranslational modification that generates glycine side chains on proteins. Here we identify a family of evolutionarily conserved glycine ligases that modify tubulin using different enzymatic mechanisms. In mammals, two distinct enzyme types catalyze the initiation and elongation steps of polyglycylation, whereas Drosophila glycylases are bifunctional. We further show that the human elongating glycylase has lost enzymatic activity due to two amino acid changes, suggesting that the functions of protein glycylation could be sufficiently fulfilled by monoglycylation. Depletion of a glycylase in Drosophila using RNA interference results in adult flies with strongly decreased total glycylation levels and male sterility associated with defects in sperm individualization and axonemal maintenance. A more severe RNAi depletion is lethal at early developmental stages, indicating that protein glycylation is essential. Together with the observation that multiple proteins are glycylated, our functional data point towards a general role of glycylation in protein functions.

Ccdc113/Ccdc96 complex, a novel regulator of ciliary beating that connects radial spoke 3 to dynein g and the nexin link.

Ciliary beating requires the coordinated activity of numerous axonemal complexes. The protein composition and role of radial spokes (RS), nexin links (N-DRC) and dyneins (ODAs and IDAs) is well established. However, how information is transmitted from the central apparatus to the RS and across other ciliary structures remains unclear. Here, we identify a complex comprising the evolutionarily conserved proteins Ccdc96 and Ccdc113, positioned parallel to N-DRC and forming a connection between RS3, dynein g, and N-DRC. Although Ccdc96 and Ccdc113 can be transported to cilia independently, their stable docking and function requires the presence of both proteins. Deletion of either CCDC113 or CCDC96 alters cilia beating frequency, amplitude and waveform. We propose that the Ccdc113/Ccdc96 complex transmits signals from RS3 and N-DRC to dynein g and thus regulates its activity and the ciliary beat pattern.

LF4/MOK and a CDK-related kinase regulate the number and length of cilia in Tetrahymena.

The length of cilia is controlled by a poorly understood mechanism that involves members of the conserved RCK kinase group, and among them, the LF4/MOK kinases. The multiciliated protist model, Tetrahymena, carries two types of cilia (oral and locomotory) and the length of the locomotory cilia is dependent on their position with the cell. In Tetrahymena, loss of an LF4/MOK ortholog, LF4A, lengthened the locomotory cilia, but also reduced their number. Without LF4A, cilia assembled faster and showed signs of increased intraflagellar transport (IFT). Consistently, overproduced LF4A shortened cilia and downregulated IFT. GFP-tagged LF4A, expressed in the native locus and imaged by total internal reflection microscopy, was enriched at the basal bodies and distributed along the shafts of cilia. Within cilia, most LF4A-GFP particles were immobile and a few either diffused or moved by IFT. We suggest that the distribution of LF4/MOK along the cilium delivers a uniform dose of inhibition to IFT trains that travel from the base to the tip. In a longer cilium, the IFT machinery may experience a higher cumulative dose of inhibition by LF4/MOK. Thus, LF4/MOK activity could be a readout of cilium length that helps to balance the rate of IFT-driven assembly with the rate of disassembly at steady state. We used a forward genetic screen to identify a CDK-related kinase, CDKR1, whose loss-of-function suppressed the shortening of cilia caused by overexpression of LF4A, by reducing its kinase activity. Loss of CDKR1 alone lengthened both the locomotory and oral cilia. CDKR1 resembles other known ciliary CDK-related kinases: LF2 of Chlamydomonas, mammalian CCRK and DYF-18 of C. elegans, in lacking the cyclin-binding motif and acting upstream of RCKs. The new genetic tools we developed here for Tetrahymena have potential for further dissection of the principles of cilia length regulation in multiciliated cells.

PCD Genes-From Patients to Model Organisms and Back to Humans.

Primary ciliary dyskinesia (PCD) is a hereditary genetic disorder caused by the lack of motile cilia or the assembxly of dysfunctional ones. This rare human disease affects 1 out of 10,000-20,000 individuals and is caused by mutations in at least 50 genes. The past twenty years brought significant progress in the identification of PCD-causative genes and in our understanding of the connections between causative mutations and ciliary defects observed in affected individuals. These scientific advances have been achieved, among others, due to the extensive motile cilia-related research conducted using several model organisms, ranging from protists to mammals. These are unicellular organisms such as the green alga Chlamydomonas, the parasitic protist Trypanosoma, and free-living ciliates, Tetrahymena and Paramecium, the invertebrate Schmidtea, and vertebrates such as zebrafish, Xenopus, and mouse. Establishing such evolutionarily distant experimental models with different levels of cell or body complexity was possible because both basic motile cilia ultrastructure and protein composition are highly conserved throughout evolution. Here, we characterize model organisms commonly used to study PCD-related genes, highlight their pros and cons, and summarize experimental data collected using these models.

Central Apparatus, the Molecular Kickstarter of Ciliary and Flagellar Nanomachines.

Motile cilia and homologous organelles, the flagella, are an early evolutionarily invention, enabling primitive eukaryotic cells to survive and reproduce. In animals, cilia have undergone functional and structural speciation giving raise to typical motile cilia, motile nodal cilia, and sensory immotile cilia. In contrast to other cilia types, typical motile cilia are able to beat in complex, two-phase movements. Moreover, they contain many additional structures, including central apparatus, composed of two single microtubules connected by a bridge-like structure and assembling numerous complexes called projections. A growing body of evidence supports the important role of the central apparatus in the generation and regulation of the motile cilia movement. Here we review data concerning the central apparatus structure, protein composition, and the significance of its components in ciliary beating regulation.

Ciliary proteins Fap43 and Fap44 interact with each other and are essential for proper cilia and flagella beating.

Cilia beating is powered by the inner and outer dynein arms (IDAs and ODAs). These multi-subunit macrocomplexes are arranged in two rows on each outer doublet along the entire cilium length, except its distal end. To generate cilia beating, the activity of ODAs and IDAs must be strictly regulated locally by interactions with the dynein arm-associated structures within each ciliary unit and coordinated globally in time and space between doublets and along the axoneme. Here, we provide evidence of a novel ciliary complex composed of two conserved WD-repeat proteins, Fap43p and Fap44p. This complex is adjacent to another WD-repeat protein, Fap57p, and most likely the two-headed inner dynein arm, IDA I1. Loss of either protein results in altered waveform, beat stroke and reduced swimming speed. The ciliary localization of Fap43p and Fap44p is interdependent in the ciliate Tetrahymena thermophila.

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.

Regulation of katanin activity in the ciliate Tetrahymena thermophila.

Katanin is a microtubule severing protein that functions as a heterodimer composed of an AAA domain catalytic subunit, p60, and a regulatory subunit, a WD40 repeat protein, p80. Katanin-dependent severing of microtubules is important for proper execution of key cellular activities including cell division, migration, and differentiation. Published data obtained in Caenorhabditis elegans, Xenopus and mammals indicate that katanin is regulated at multiple levels including transcription, posttranslational modifications (of both katanin and microtubules) and degradation. Little is known about how katanin is regulated in unicellular organisms. Here we show that in the ciliated protist Tetrahymena thermophila, as in Metazoa, the localization and activity of katanin requires specific domains of both p60 and p80, and that the localization of p60, but not p80, is sensitive to the levels of microtubule glutamylation. A prolonged overexpression of either a full length, or a fragment of p80 containing WD40 repeats, partly phenocopies a knockout of p60, indicating that in addition to its activating role, p80 could also contribute to the inhibition of p60. We also show that the level of p80 depends on the 26S proteasome activity.

[Ciliopaties - diseases caused by abnormal cilia functioning]. / Ciliopatie ­ choroby spowodowane nieprawidlowym funkcjonowaniem rzesek.

Ciliopathies are a group of genetic diseases caused by defects in the function of cilia, that are cellular processes composed of a microtubule-based core. Ciliopathies present with pathological changes in one or many organs at the same time. Symptoms of ciliopathies depend on the type of damaged tissues and organs. The most common are polycystic kidney and liver, blindness, dysfunction of neural tube, brain anomalies, mental retardation, abnormalities in skeletal system from polydactyly to abnormal short ribs and limbs, abnormalities in ectoderms, obesity, situs inversus, infertility and infections of the upper airways. Both basic and clinical studies provide data regarding novel ciliary proteins the lack or mutation of which are associated with cilia dysfunction and which, in consequence, may give rise to ciliopathies. The number of ciliopathies (35 known at present) is still increasing due to identification of additional genes (187 identified up to now) directly connected with these diseases. In this work, the most important mechanisms responsible for abnormal cilia formation and functioning, that constitute the primary cause of ciliopathies, are presented.

Tubulin Post-Translational Modifications and Microtubule Dynamics.

Microtubules are hollow tube-like polymeric structures composed of α,ß-tubulin heterodimers. They play an important role in numerous cellular processes, including intracellular transport, cell motility and segregation of the chromosomes during cell division. Moreover, microtubule doublets or triplets form a scaffold of a cilium, centriole and basal body, respectively. To perform such diverse functions microtubules have to differ in their properties. Post-translational modifications are one of the factors that affect the properties of the tubulin polymer. Here we focus on the direct and indirect effects of post-translational modifications of tubulin on microtubule dynamics.

MEC-17 is an alpha-tubulin acetyltransferase.

In most eukaryotic cells, subsets of microtubules are adapted for specific functions by post-translational modifications (PTMs) of tubulin subunits. Acetylation of the epsilon-amino group of K40 on alpha-tubulin is a conserved PTM on the luminal side of microtubules that was discovered in the flagella of Chlamydomonas reinhardtii. Studies on the significance of microtubule acetylation have been limited by the undefined status of the alpha-tubulin acetyltransferase. Here we show that MEC-17, a protein related to the Gcn5 histone acetyltransferases and required for the function of touch receptor neurons in Caenorhabditis elegans, acts as a K40-specific acetyltransferase for alpha-tubulin. In vitro, MEC-17 exclusively acetylates K40 of alpha-tubulin. Disruption of the Tetrahymena MEC-17 gene phenocopies the K40R alpha-tubulin mutation and makes microtubules more labile. Depletion of MEC-17 in zebrafish produces phenotypes consistent with neuromuscular defects. In C. elegans, MEC-17 and its paralogue W06B11.1 are redundantly required for acetylation of MEC-12 alpha-tubulin, and contribute to the function of touch receptor neurons partly via MEC-12 acetylation and partly via another function, possibly by acetylating another protein. In summary, we identify MEC-17 as an enzyme that acetylates the K40 residue of alpha-tubulin, the only PTM known to occur on the luminal surface of microtubules.

[Microtubule severing proteins - structure and activity regulation]. / Bialka tnace mikrotubule - struktura i regulacja aktywnosci.

Microtubule severing proteins, katanin, spastin and fidgetin cause local destabilization of the microtubules structure. This ATP-dependent activity leads to the shortening or disassembly of the existing microtubules. The generated short microtubule fragments may serve as templates to polymerize new microtubules and in consequence, the activity of the microtubule severing proteins leads to the reorganization of the microtubular cytoskeleton. This review summarizes current knowledge concerning structural organization of the microtubule severing proteins, the molecular mechanism of their action, factors that regulate the level of the katanin and spastin within the cells and their microtubule severing activity.

[Role of microtubule severing proteins in cytoskeleton reorganization]. / Rola bialek tnacych mikrotubule w reorganizacji cytoszkieletu.

ATP-dependent severing activity of microtubule severing proteins leads to the local destabilization of the microtubule structure and causes shortening or disassembly of the existing microtubules or formation of the numerous short microtubule fragments that serve as templates during new microtubule polymerization. Microtubule severing protein-dependent rearrangement of the microtubular cytoskeleton plays an important role in the numerous cellular processes including chromosome segregation during meiosis and mitosis, cells migration, dendrites and axon formation, cilia assembly and arrangement of the cortical microtubules in plant cells.

ADF/cofilin is not essential but is critically important for actin activities during phagocytosis in Tetrahymena thermophila.

ADF/cofilin is a highly conserved actin-modulating protein. Reorganization of the actin cytoskeleton in vivo through severing and depolymerizing of F-actin by this protein is essential for various cellular events, such as endocytosis, phagocytosis, cytokinesis, and cell migration. We show that in the ciliate Tetrahymena thermophila, the ADF/cofilin homologue Adf73p associates with actin on nascent food vacuoles. Overexpression of Adf73p disrupted the proper localization of actin and inhibited the formation of food vacuoles. In vitro, recombinant Adf73p promoted the depolymerization of filaments made of T. thermophila actin (Act1p). Knockout cells lacking the ADF73 gene are viable but grow extremely slowly and have a severely decreased rate of food vacuole formation. Knockout cells have abnormal aggregates of actin in the cytoplasm. Surprisingly, unlike the case in animals and yeasts, in Tetrahymena, ADF/cofilin is not required for cytokinesis. Thus, the Tetrahymena model shows promise for future studies of the role of ADF/cofilin in vivo.

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.

A family of carboxypeptidases catalyzing α- and ß-tubulin tail processing and deglutamylation.

Tubulin posttranslational modifications represent an important mechanism involved in the regulation of microtubule functions. The most widespread among them are detyrosination, α∆2-tubulin, and polyglutamylation. Here, we describe a family of tubulin-modifying enzymes composed of two closely related proteins, KIAA0895L and KIAA0895, which have tubulin metallocarboxypeptidase activity and thus were termed TMCP1 and TMCP2, respectively. We show that TMCP1 (also known as MATCAP) acts as α-tubulin detyrosinase that also catalyzes α∆2-tubulin. In contrast, TMCP2 preferentially modifies ßI-tubulin by removing three amino acids from its C terminus, generating previously unknown ßI∆3 modification. We show that ßI∆3-tubulin is mostly found on centrioles and mitotic spindles and in cilia. Moreover, we demonstrate that TMCPs also remove posttranslational polyglutamylation and thus act as tubulin deglutamylases. Together, our study describes the identification and comprehensive biochemical analysis of a previously unknown type of tubulin-modifying enzymes involved in the processing of α- and ß-tubulin C-terminal tails and deglutamylation.

Integrated modeling of the Nexin-dynein regulatory complex reveals its regulatory mechanism.

Cilia are hairlike protrusions that project from the surface of eukaryotic cells and play key roles in cell signaling and motility. Ciliary motility is regulated by the conserved nexin-dynein regulatory complex (N-DRC), which links adjacent doublet microtubules and regulates and coordinates the activity of outer doublet complexes. Despite its critical role in cilia motility, the assembly and molecular basis of the regulatory mechanism are poorly understood. Here, utilizing cryo-electron microscopy in conjunction with biochemical cross-linking and integrative modeling, we localized 12 DRC subunits in the N-DRC structure of Tetrahymena thermophila . We also found that the CCDC96/113 complex is in close contact with the N-DRC. In addition, we revealed that the N-DRC is associated with a network of coiled-coil proteins that most likely mediates N-DRC regulatory activity.

Integrated modeling of the Nexin-dynein regulatory complex reveals its regulatory mechanism.

Cilia are hairlike protrusions that project from the surface of eukaryotic cells and play key roles in cell signaling and motility. Ciliary motility is regulated by the conserved nexin-dynein regulatory complex (N-DRC), which links adjacent doublet microtubules and regulates and coordinates the activity of outer doublet complexes. Despite its critical role in cilia motility, the assembly and molecular basis of the regulatory mechanism are poorly understood. Here, using cryo-electron microscopy in conjunction with biochemical cross-linking and integrative modeling, we localize 12 DRC subunits in the N-DRC structure of Tetrahymena thermophila. We also find that the CCDC96/113 complex is in close contact with the DRC9/10 in the linker region. In addition, we reveal that the N-DRC is associated with a network of coiled-coil proteins that most likely mediates N-DRC regulatory activity.

Native doublet microtubules from Tetrahymena thermophila reveal the importance of outer junction proteins.

Cilia are ubiquitous eukaryotic organelles responsible for cellular motility and sensory functions. The ciliary axoneme is a microtubule-based cytoskeleton consisting of two central singlets and nine outer doublet microtubules. Cryo-electron microscopy-based studies have revealed a complex network inside the lumen of both tubules composed of microtubule-inner proteins (MIPs). However, the functions of most MIPs remain unknown. Here, we present single-particle cryo-EM-based analyses of the Tetrahymena thermophila native doublet microtubule and identify 42 MIPs. These data shed light on the evolutionarily conserved and diversified roles of MIPs. In addition, we identified MIPs potentially responsible for the assembly and stability of the doublet outer junction. Knockout of the evolutionarily conserved outer junction component CFAP77 moderately diminishes Tetrahymena swimming speed and beat frequency, indicating the important role of CFAP77 and outer junction stability in cilia beating generation and/or regulation.