The evolutionary conserved proteins CEP90, FOPNL, and OFD1 recruit centriolar distal appendage proteins to initiate their assembly.

In metazoa, cilia assembly is a cellular process that starts with centriole to basal body maturation, migration to the cell surface, and docking to the plasma membrane. Basal body docking involves the interaction of both the distal end of the basal body and the transition fibers/distal appendages, with the plasma membrane. Mutations in numerous genes involved in basal body docking and transition zone assembly are associated with the most severe ciliopathies, highlighting the importance of these events in cilium biogenesis. In this context, the ciliate Paramecium has been widely used as a model system to study basal body and cilia assembly. However, despite the evolutionary conservation of cilia assembly events across phyla, whether the same molecular players are functionally conserved, is not fully known. Here, we demonstrated that CEP90, FOPNL, and OFD1 are evolutionary conserved proteins crucial for ciliogenesis. Using ultrastructure expansion microscopy, we unveiled that these proteins localize at the distal end of both centrioles/basal bodies in Paramecium and mammalian cells. Moreover, we found that these proteins are recruited early during centriole duplication on the external surface of the procentriole. Functional analysis performed both in Paramecium and mammalian cells demonstrate the requirement of these proteins for distal appendage assembly and basal body docking. Finally, we show that mammalian centrioles require another component, Moonraker (MNR), to recruit OFD1, FOPNL, and CEP90, which will then recruit the distal appendage proteins CEP83, CEP89, and CEP164. Altogether, we propose that this OFD1, FOPNL, and CEP90 functional module is required to determine in mammalian cells the future position of distal appendage proteins.

OrbiSIMS Imaging Identifies Molecular Constituents of the Perialgal Vacuole Membrane of Paramecium bursaria with Symbiotic Chlorella variabilis.

The protist (mostly single-celled organisms), Paramecium bursaria, forms an intracellular symbiotic relationship with the single-celled algae, Chlorella variabilis, where P. bursaria provides nutrients (i.e., Ca2+, Mg2+, and K+), carbon dioxide for photosynthesis and protection from viruses, while C. variabilis provides oxygen, carbon fixation, and nutrients. Key to this successful relationship is the perialgal vacuole (PV) membrane, which surrounds C. variabilis and protects it from digestion by P. bursaria. The membrane is fragile and difficult to analyze using conventional methods therefore very little is known about the molecular composition. We used the OrbiSIMS, a new high-resolution mass spectrometer with subcellular resolution imaging, to study the compartmentalization of endosymbionts and elucidate biomolecular interactions between the host and endosymbiont. Ions from the region of interest, close to C. variabilis, and specific to the target samples containing PVs were found based on the chemical mapping and masses of the ions. We show chemical localizations of oligosaccharides in close proximity of C. variabilis endosymbionts in P. bursaria. These oligosaccharides are detected in host-endosymbiont samples containing PV membrane-bound algae and absent in free-living algae and digestive vacuole (DV) membrane-bound algae in P. bursaria.

An UPF3-based nonsense-mediated decay in Paramecium.

Nonsense-mediated decay recognises mRNAs containing premature termination codons. One of its components, UPF3, is a molecular link bridging through its binding to the exon junction complex nonsense-mediated decay and splicing. In protists UPF3 has not been identified yet. We report that Paramecium tetraurelia bears an UPF3 gene and that it has a role in nonsense-mediated decay. Interestingly, the identified UPF3 has not conserved the essential amino acids required to bind the exon junction complex. Though, our data indicates that this ciliate bears genes coding for core proteins of the exon junction complex.

Protection capabilities of nanostructured shells toward cell encapsulation: a Saccharomyces/Paramecium model.

In this work we report the formation of nanostructured hybrid objects, made up of living cells encapsulated in a protective multilayer shell assembly of nanostructured polyelectrolyte. Such constructs can be hosted on nanostructured surfaces or can be installed around living organisms, at the right time. Their construction is based on the layer-by-layer (LbL) self-assembly of two oppositely alternated charged polyelectrolytes (PEs) on cell membranes as earlier done for nanocapsules or fuzzy structured nanoshells. This communication reports the optimal conditions for cell encapsulation in terms of nanoshell design and construction.

Mass spectrometry analysis of C-terminal posttranslational modifications of tubulins.

In mammalian brain and ciliary axonemes from ciliates, alpha- and beta-tubulins exhibit an extraordinary heterogeneity due to a combination of multigene family expression and numerous posttranslational modifications (PTMs). The combination of several PTMs located in the C-terminal tail of tubulins plays a major role in this important polymorphism of tubulin: polyglutamylation, polyglycylation, detyrosination, tyrosination, removal of the penultimate glutamate residue, and phosphorylation. In order to document the relationship and functions of these PTMs, we have developed a tubulin C-terminal Peptide Mass Fingerprinting (PMF) method. Using simplified microtubule proteins and tubulin C-terminal peptides purifications, direct matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) analysis can generate a complete picture of all tubulin isotype-specific C-terminal peptides together with their respective PTMs. This chapter will illustrate the capability of this approach to compare tubulin isoform compositions and document the changes in PTMs between samples with different tubulin assembly properties or consecutively to inactivation of modification sites or modification enzymes. Complementary MS-based approaches useful to document the structure of the highly heterogeneous posttranslational polymodifications will also be presented.

Secondary structural analyses of ITS1 in Paramecium.

The nuclear ribosomal RNA gene operon is interrupted by internal transcribed spacer (ITS) 1 and ITS2. Although the secondary structure of ITS2 has been widely investigated, less is known about ITS1 and its structure. In this study, the secondary structure of ITS1 sequences for Paramecium and other ciliates was predicted. Each Paramecium ITS1 forms an open loop with three helices, A through C. Helix B was highly conserved among Paramecium, and similar helices were found in other ciliates. A phylogenetic analysis using the ITS1 sequences showed high-resolution, implying that ITS1 is a good tool for species-level analyses.

Identification and cloning of first cadmium metallothionein like gene from locally isolated ciliate, Paramecium sp.

First cadmium metallothionein like gene PMCd1 of a ciliate, Paramecium sp., isolated from industrial wastewater has been cloned and sequenced. PMCd1 is an intronless gene, encoding 612 nucleotides, with TAA coding for glutamine. The coding region of PMCd1 comprises 203 amino acids, including 37 cysteine residues with a conserved structural pattern in the form of recurring structural motifs, arranged in 17 x-cys-x-y-cys-x, 1 x-cys-cys-x and x-cys-x contexts. Both, the deduced amino acids and nucleotide sequence differ, not only from other animal metallothioneins (MTs), but also from the previously characterized Tetrahymena Cu and Cd-MTs. The translated protein of PMCd1 contains conserved cysteine residues, peculiar characteristic of stress inducible metallothionein genes of ciliates and other groups of organisms.

Identification and characterization of a 38 kDa glycoprotein functionally associated with mating activity of Paramecium primaurelia.

In Paramecium primaurelia mating interactions take place immediately after mixing mating-competent cells of opposite mating types. The cells clump in clusters (mating reaction) and then separate in pairs. Previous results have shown that sialic acid-containing glycoconjugates are present on the cell surface and are involved in mating-cell pairing. In order to identify the sialic acid-containing glycoprotein(s), we first metabolically radiolabelled non-mating-competent cells with D-[6-(3)H]galactose, and then analyzed the radiolabelled proteins by anion exchange chromatography. We characterized a 38 kDa (gp38) sialic acid-containing glycoprotein and raised the corresponding polyclonal antibody by means of which we localized the antigen at the level of the oral region of non-mating-competent cells and on the ciliary surface of mating-competent cells. Immunoblot analysis of the ciliary protein fraction showed that the anti-gp38 serum interacted with a 38 kDa protein in both mating types I and II cells. We also demonstrated the functional activity of gp38 in the mating reaction by means of anti-gp38 antibody competition assays.

Altered toxicities of fatty acid salts in green paramecia cultured in different waters.

Detergents including fatty acid salts act as surface-active agents and thus possibly damage the plasma membrane structures of aquatic organisms. Therefore, when excess, the house-used and industrial outflows of such detergents into aquatic environments may have considerable impacts on the ecosystem. In this study, we propose the use of green paramecia (Paramecium bursaria) for assessing the acute toxicity of eight fatty acid salts (Na and K salts of oleate, palmitate, laurate and myristate) under various water conditions. The Paramecium in the stationary phase were used for a toxicity assay carried out on 12-well microplates and the median lethal concentration (LC50) was determined for each fatty acid salt. In the low mineral culture medium prepared with ultra-pure water, the LC50 for each fatty acid ranged from 5.8 to 144 ppm (w/v). The toxic levels of fatty acid salts differed in the following order: laurate, myristate > or = oleate, palmitate. The toxic levels of oleate and palmitate salts were ca. 10-fold lower than those of laurate and myristate salts. When river water and local tap water instead of ultra-pure water were used for culturing, the toxic levels of all fatty acid salts were drastically lowered compared to the low mineral condition by 30- to 100-fold (198-660 ppm, w/v). Similar detoxification effect was observed when Ca or Mg was added to the low mineral culture media, indicating that the toxicity of fatty acid salts can be notably lowered as the mineral content increases. As we demonstrated that toxicities of fatty acid salts can be lowered in river water and tap water compared to the low mineral condition, some chemical substances behave differently in the different water conditions. Therefore, the use of natural waters reflecting the real environmental conditions in further collection of data on the ecotoxicity impacts of variety of chemicals is highly encouraged.

Multiple tubulin forms in ciliated protozoan Tetrahymena and Paramecium species.

Tetrahymena and Paramecium species are widely used representatives of the phylum Ciliata. Ciliates are particularly suitable model organisms for studying the functional heterogeneity of tubulins, since they provide a wide range of different microtubular structures in a single cell. Sequencing projects of the genomes of members of these two genera are in progress. Nearly all members of the tubulin superfamily (alpha-, beta-, gamma-, delta-, epsilon-, eta-, theta-, iota-, and kappa-tubulins) have been identified in Paramecium tetraurelia. In Tetrahymena spp., the functional consequences of different posttranslational tubulin modifications (acetylation, tyrosination and detyrosination, phosphorylation, glutamylation, and glycylation) have been studied by different approaches. These model organisms provide the opportunity to determine the function of tubulins found in ciliates, as well as in humans, but absent in some other model organisms. They also give us an opportunity to explore the mechanisms underlying microtubule diversity. Here we review current knowledge concerning the diversity of microtubular structures, tubulin genes, and posttranslational modifications in Tetrahymena and Paramecium species.

Effect of Japanese Paramecium bursaria extract on photosynthetic carbon fixation of symbiotic algae.

To investigate the relationship between the Japanese Paramecium bursaria host and its symbiont, we studied the effect of a host cell-free extract on carbon fixation and photosynthate release of the symbiont. The host extract enhanced symbiotic algal carbon fixation about 3-fold at an increased concentration; however, release of photosynthate hardly changed. Since the enhancing effect was not affected by elimination of carbon dioxide from the host extract, the existence of a host factor that stimulates algal carbon fixation was made clear. The host factor is a heat-stable, low molecular weight substance. In relation to the pH dependence, the extract improved carbon fixation at acidic and neutral pH and showed almost no effect at pH 9.0. Therefore, the stimulation of carbon fixation by the host factor is unlikely to be caused by intracellular pH change. The extract also improved carbon fixation of several Chlorella species, symbiotic and free-living, and apparently exhibited no species specificity. Therefore, the host seems to regulate the photosynthesis of the symbiont via a specific compound.

GABAA receptor subunits identified in Paramecium by immunofluorescence confocal microscopy.

The presence of opioid, beta-adrenergic and cholinergic receptors has been demonstrated in ciliated protozoa, but little is known about gamma-aminobutyric acid (GABA) receptors. In this study we have analyzed the distribution of GABA(A)-type receptor subunits in Paramecium. Confocal laser microscopy using antibodies specific for alpha(1)-, alpha(2)-, alpha(3)-, alpha(6)-, beta(2/3)-, gamma(2)-, epsilon-, lambda-, and theta-subunits showed that most receptors are aggregated in clusters and are distributed both on cell surface and in the cytoplasm. The intensity of labelling of the alpha(6)-, beta(2/3)- and gamma(2)-subunits was more intense than the alpha(1)-, epsilon-, and theta-subunits, suggesting that the former are present in higher concentrations than the latter.

Calcium-induced conformational switching of Paramecium calmodulin provides evidence for domain coupling.

Calmodulin (CaM) is an intracellular calcium-binding protein essential for many pathways in eukaryotic signal transduction. Although a structure of Ca(2+)-saturated Paramecium CaM at 1.0 A resolution (1EXR.pdb) provides the highest level of detail about side-chain orientations in CaM, information about an end state alone cannot explain driving forces for the transitions that occur during Ca(2+)-induced conformational switching and why the two domains of CaM are saturated sequentially rather than simultaneously. Recent studies focus attention on the contributions of interdomain linker residues. Electron paramagnetic resonance showed that Ca(2+)-induced structural stabilization of residues 76-81 modulates domain coupling [Qin and Squier (2001) Biophys. J. 81, 2908-2918]. Studies of N-domain fragments of Paramecium CaM showed that residues 76-80 increased thermostability of the N-domain but lowered the Ca(2+) affinity of sites I and II [Sorensen et al. (2002) Biochemistry 41, 15-20]. To probe domain coupling during Ca(2+) binding, we have used (1)H-(15)N HSQC NMR to monitor more than 40 residues in Paramecium CaM. The titrations demonstrated that residues Glu78 to Glu84 (in the linker and cap of helix E) underwent sequential phases of conformational change. Initially, they changed in volume (slow exchange) as sites III and IV titrated, and subsequently, they changed in frequency (fast exchange) as sites I and II titrated. These studies provide evidence for Ca(2+)-dependent communication between the domains, demonstrating that spatially distant residues respond to Ca(2+) binding at sites I and II in the N-domain of CaM.

Detection of molecules related to the GABAergic system in a single-cell eukaryote, Paramecium primaurelia.

Gamma-aminobutyric acid (GABA)-related molecules were identified in Paramecium primaurelia by immunocytochemical methods, and GABA(A) receptors by their histochemical BODIPY-binding sites. Confocal microscope analysis showed different localizations according to the stages of the developmental cycle. A comparison was made with the cholinergic molecules, such as the acetylcholine biosynthetic enzyme (choline acetyltransferase), in double-labelled cells by confocal microscopy. In vivo experiments suggested the involvement of GABA-related molecules in cell-cell interaction.

Crystal structure analysis of the exocytosis-sensitive phosphoprotein, pp63/parafusin (phosphoglucomutase), from Paramecium reveals significant conformational variability.

During exocytosis of dense-core secretory vesicles (trichocysts) in Paramecium, the protein pp63/parafusin (pp63/pf) is transiently dephosphorylated. We report here the structures of two crystal forms of one isoform of this protein which has a high degree of homology with rabbit phosphoglucomutase, whose structure has been reported. As expected, both proteins possess highly similar structures, showing the same four domains forming two lobes with an active-site crevice in between. The two X-ray structures that we report here were determined after crystallization in the presence of sulfate and tartrate, and show the lobes arranged as a closed and an open conformation, respectively. While both conformations possess a bound divalent cation, only the closed (sulfate-bound) conformation shows bound sulfate ions in the "phosphate-transfer site" near the catalytic serine residue and in the "phosphate-binding site". Comparison with the open form shows that the latter dianion is placed in the centre of three arginine residues, one contributed by subunit II and two by subunit IV, suggesting that it causes a contraction of the arginine triangle, which establishes the observed conformational closure of the lobes. It is therefore likely that the closed conformation forms only when a phosphoryl group is bound to the phosphate-binding site. The previously published structure of rabbit phosphoglucomutase is intermediate between these two conformers. Several of the known reversible phosphorylation sites of pp63/pf-1 are at positions critical for transition between the conformations and for binding of the ligands and thus give hints as to possible roles of pp63/pf-1 in the course of exocytosis.

Glycophosphatidylinositol-anchored proteins in Paramecium tetraurelia: possible role in chemoresponse.

We have begun to characterize the glycophosphatidylinositol (GPI)-anchored proteins of the Paramecium tetraurelia cell body surface where receptors and binding sites for attractant stimuli are found. We demonstrate here (i) that inositol-specific exogenous phospholipase C (PLC) treatment of the cell body membranes (pellicles) removes proteins with GPI anchors, (ii) that, as in P. primaurelia, there is an endogenous lipase that responds differently to PLC inhibitors compared with its response to an exogenous PLC, (iii) that salt and ethanol treatment of cells removes GPI-anchored proteins from whole, intact cells, (iv) that Triton X-114 phase partitioning shows that many GPI-anchored proteins are cleaved from pellicles by the endogenous lipase and enter the aqueous phase, and (v) that integral membrane proteins are not among those cleaved with PLC or in the salt/ethanol wash. Antisera against the proteins removed by the salt/ethanol washing procedure include antibodies against large surface antigens, which we confirm in this species to be GPI-anchored, and against an array of proteins of smaller molecular mass. These antisera specifically block the chemoresponse to some stimuli, such as folate, which we suggest are signaled through GPI-anchored receptors. Responses to cyclic AMP, which we believe involve an integral membrane protein receptor, and to NH4Cl, which requires no receptor, are not affected by the antisera. Antiserum against a mammalian GPI-anchored folate-binding protein recognizes a single band among the GPI-anchored salt and ethanol wash proteins. The same antiserum specifically blocks the chemoresponse to folate.

Alterations in the protein pattern of subcellular fractions isolated from Paramecium cells suppressed in phagocytosis.

SDS-PAGE and quantitative densitometric analysis revealed alterations in the protein pattern of subcellular fractions (100,000 x g) isolated from Paramecium aurelia (299s axenic) cells suppressed in phagocytosis as compared with the control. Two different agents were used to block phagocytosis: the beta-adrenergic antagonist-1-propranolol (200 microM) and inhibitor of calmodulin-dependent processes--trifluoperazine (20 microM). More than 40 polypeptides were identified in the cytosolic (soluble) fractions S1 and S2. A considerable decrease in band intensity was found for three polypeptides: by 60% for 87 kDa band, 52% for 75 kDa and 37% for 42 kDa in comparison to the control, when S2 fractions from propranolol-treated cells of equal load were quantified. TFP treatment evoked only a small decrease in the intensity of the same bands: 9%, 10% and 6%, respectively. The 42 kDa band was identified by Western blot analysis and chemiluminiscent detection to be actin. This result suggests that actin may be a primary target of pharmacological agents used in this study to inhibit Paramecium phagocytic activity.

Fine oral filaments in Paramecium: a biochemical and immunological analysis.

In Paramecium, several kinds of the oral networks of fine filaments are defined at the ultrastructural level. Using the sodium chloride-treated oral apparatus of Paramecium as an antigen to produce monoclonal antibodies, we have begun to identify the proteins constituting these networks. Immunoblotting showed that all positive antibodies were directed against three bands (70-, 75-and 83-kD), which corresponded to quantitatively minor components of the antigen; there was no antibody specific for the quantitatively major components (58- and 62-kD). Immunolocalization with four of these antibodies directed against one or several of these three bands showed that these proteins are components of the fine filaments supporting the oral area; a decoration of the basal bodies and the outer lattice was also observed on the cortex. Immunofluorescence on interphase cells suggested that the three proteins colocalized on the left side of the oral apparatus, whereas only the 70-kD band was detected on the right side. During division, the antigens of the antibodies were detected at different stages after oral basal body assembly. The antibodies cross-reacted with the tetrins, which are oral filament-forming proteins in Tetrahymena, demonstrating that tetrin-related proteins are quantitatively minor components of the oral and the somatic cytoskeleton of Paramecium.

Ligand binding in the ferric and ferrous states of Paramecium hemoglobin.

The unicellular protozoan Paramecium caudatum contains a monomeric hemoglobin (Hb) that has only 116 amino acid residues. This Hb shares the simultaneous presence of a distal E7 glutamine and a B10 tyrosine with several invertebrate Hbs. In the study presented here, we have used ligand binding kinetics and resonance Raman spectroscopy to characterize the effect of the distal pocket residues of Paramecium Hb in stabilizing the heme-bound ligands. In the ferric state, the high-spin to low-spin (aquo-hydroxy) transition takes place with a pK(a) of approximately 9.0. The oxygen affinity (P(50) = 0.45 Torr) is similar to that of myoglobin. The oxygen on- and off-rates are also similar to those of sperm whale myoglobin. Resonance Raman data suggest hydrogen bonding stabilization of bound oxygen, evidenced by a relatively low frequency of Fe-OO stretching (563 cm(-1)). We propose that the oxy complex is an equilibrium mixture of a hydrogen-bonded closed structure and an open structure. Oxygen will dissociate preferentially from the open structure, and therefore, the fraction of open structure population controls the rate of oxygen dissociation. In the CO complex, the Fe-CO stretching frequency at 493 cm(-1) suggests an open heme pocket, which is consistent with the higher on- and off-rates for CO relative to those in myoglobin. A high rate of ligand binding is also consistent with the observation of an Fe-histidine stretching frequency at 220 cm(-1), indicating the absence of significant proximal strain. We postulate that the function of Paramecium Hb is to supply oxygen for cellular oxidative processes.

The alpha1/2 helical backbone of the prodomains defines the intrinsic inhibitory specificity in the cathepsin L-like cysteine protease subfamily.

Proregions of papain-like cysteine proteases are potent and often highly selective inhibitors of their parental enzymes. The molecular basis of their selectivity is poorly understood. For two closely related members of the cathepsin L-like subfamily we established strong selectivity differences. The propeptide of cathepsin S was observed to inhibit cathepsin L with a K(i) of 0.08 nM, yet cathepsin L propeptide inhibited cathepsin S only poorly. To identify the respective structural correlates we engineered chimeric propeptides and compared their inhibitory specificity with the wild-types. Specificity resided in the N-terminal part, strongly suggesting that the backbone of the prodomain was the underlying structure.