Anne Baroin-Tourancheau

Isolation and characterization of libraries of monoclonal antibodies directed against various forms of tubulin in Paramecium

Summary— Ciliates are very good models for studying post‐translationally generated tubulin heterogeneity because they exhibit highly differentiated microtubular networks in combination with reduced genetic diversity. We have approached the analysis of tubulin heterogeneity in Paramecium through extensive isolation and characterization of monoclonal antibodies using various antigens and several immunization protocols. Eight monoclonal antibodies and 10 hybridoma supernatants were characterized by: i) immunoblotting on ciliate and pig brain tubulins as well as on peptide maps of Paramecium axonemal tubulin; ii) immunoblotting on ciliate tubulin fusion peptides generated in E coli, a procedure which allows in principle to discriminate antibodies that are directed against tubulin sequence (reactive on fusion peptides) from those directed against a post‐translational epitope (non‐reactive); and iii) immunofluorescence on Paramecium, 3T3 and PtK2 cells. Twelve antibodies labeled all microtubules in Paramecium cells and were found to be directed against tubulin primary sequences (nine of them being located in the α N‐terminal domain, one in the β C‐terminal one, and two in α and β central stretches). The remaining ones decorated only a specific subset of microtubules within the cell and were presumably directed against post‐translational modifications. Among these, three antibodies are directed against an N‐terminal acetylated epitope of α‐tubulin whereas the epitopes of three other ones (TAP 952°, AXO 58 and AXO 49°) apparently correspond to still unidentified post‐translational modifications, located in the C‐terminal domain of both α‐ and β‐tubulins. The AXO 49° specificity is similar to that of a previously described polyclonal serum raised against Paramecium axonemal tubulin [2]. The results are discussed in terms of identification and accessibility of the epitopes and immunogenicity of ciliate tubulin with reference to mammalian and ciliate tubulin sequences.

Plateins: A Novel Family of Signal Peptide-Containing Articulins in Euplotid Ciliates1

Abstract In euplotid ciliates, the cortex is reinforced by alveolar plates—proteinaceous scales located within the membranous alveolar sacs, forming a monolayer just below the plasma membrane. This system appears to play a cytoskeletal role analogous to that provided by the fibrous epiplasm found beneath the cortical alveoli in other ciliates. In Euplotes aediculatus, the major alveolar plate proteins (termed α-, β-, and γ-plateins) have been identified. Using anti-platein antibodies, an expression library of Euplotes genes was screened, and a platein gene identified, cloned, and completely sequenced. Comparison of its derived amino acid sequence with microsequences obtained directly from purified plateins identified this gene as encoding one of the closely related β- or γ-plateins. The derived protein, of 644 amino acids (74.9 kDa), is very acidic (pI = 4.88). Microsequences from authentic α-platein were then used to design oligonucleotide primers, which yielded, via a PCR-based approach, the sequences of two α-platein genes from E. aediculatus. Even more acidic proteins, the derived α1- and α2-plateins contain 536 and 501 residues, respectively. Analyses of their amino acid sequences revealed the plateins to be members of the articulin superfamily of cytoskeletal proteins, first described in Euglena and now identified in the ciliate Pseudomicrothorax and in Plasmodium. The hallmark articulin repetitive motifs (based on degenerate valine- and proline-rich 12-mers) are present in all three plateins. In β/γ-platein this primary motif domain (27 repeats) is central in the molecule, whereas the primary repeats in the α-plateins lie near their C-termini. A cluster of proline-rich pentameric secondary repeats is found in the C-terminus of β/γ-platein, but near the N-terminus of α-plateins. All three plateins contain canonical N-terminal signal sequences, unique among known cytoskeletal proteins. The presence of start-transfer sequences correlates well with the final intra-alveolar location of these proteins. This feature, and significant differences from known articulins in amino acid usage and arrangement within the repeat domains, lead us to propose that the plateins comprise a new family of articulin-related proteins. Efforts to follow microscopically the assembly of plateins into new alveolar plates during pre-fission morphogenesis are underway.

Searching for Excystment-Regulated Genes in Sterkiella histriomuscorum (Ciliophora, Oxytrichidae): A mRNA Differential Display Analysis of Gene Expression in Excysting Cells

Abstract In the absence of food, the oxytrichid Sterkiella histriomuscorum transforms like many ciliates into resting cysts. When transferred back into feeding medium, the cyst re-transforms into a vegetative cell. The entry into and exit from the dormant cyst stage are complex developmental processes still poorly investigated at the molecular level. Assuming that these changes in state could involve changes in gene expression, we have used the technique of mRNA differential display to detect differentially expressed genes in cysts and two different stages of excysting cell. Variation in the temporal expression pattern of transcripts could be detected and, in using an inverse-PCR strategy on circularized macronuclear DNA, we have sequenced the macronuclear genes of three of the isolated cDNAs, which correspond to 1) a nucleotide-binding domain-encoding gene, 2) a DHHC-domain-carrying gene, and 3) a phosphatase type 2C-encoding gene. For the first two genes, Northern blot analyses supported an excystment-associated regulated gene expression. We discuss their possible role during excystment and we show that the combination of differential display and inverse PCR constitutes a powerful approach to isolate excystment-regulated genes in hypotrichs.

Gene structure of the ciliate Sterkiella histriomuscorum based on a combined analysis of DNA and cDNA sequences from 21 macronuclear chromosomes

Macronuclear deoxyribonucleic acid (DNA) in hypotrichous ciliates consists of a set of linear molecules ranging in size from 0.5 to several tens of kilobases and typically carrying a single gene. Each minichromosome is present at a ploidy of ≥1,000 per macronucleus. These molecules are known as gene-sized molecules. Multigene molecules are also present, but are still poorly described. In analyzing the encystment–excystment cycle of Sterkiella histriomuscorum, we have characterized a set of 21 macronuclear molecules both at the DNA and complementary DNA (cDNA) levels. On a total of 23 validated coding sequences, we mapped the 5′ and 3′ untranslated regions for a subset of 10 and 18 transcripts, respectively. A combination of DNA and cDNA data allows us to precisely determine several structural features of macronuclear chromosomes, such as the organization of multigene molecules, an intron content higher than expected, and a conserved sequence surrounding the initiation transcription site. It also reveals one coding sequence containing a transcribed 10-bp element that displays the characteristic features of internal eliminated sequences (IES). Its presence in a fraction of the minichromosomes carrying this gene raises the possibility of an incomplete IES excision process during the development of the S. histriomuscorum macronucleus.

Multiple Uses of Lys63-Polyubiquitination in the Ciliate Sterkiella histriomuscorum

Poly-ubiquitination through Lys63, a post-translational modification system, is emerging as a mechanism implicated in a variety of non-proteolytic functions in eukaryotic cells. This modification is mainly carried out via a unique complex formed between a canonical ubiquitin-conjugating enzyme (E2), Ubc13, and an E2-variant, Uev. To date, a wealth of functional information has been obtained mostly from animals and yeast. Here, we show that the ciliate Sterkiella histriomuscorum harbors a Ubc13 gene that encodes a putative protein exhibiting 60-64% identity with Ubc13 from organisms of diverse eukaryotic phyla. A yeast double-hybrid assay and 3D-structure modelling gave evidence that ShUbc13 is able to physically interact with ShUev; the existence of such a complex in a ciliate attests for its ancient origin. Western blotting reveals that Ubc13 is present in different stages of the vegetative cycle of S. histriomuscorum. Immunolocalization of Ubc13 and K63 polyubiquitinated proteins shows three main locations, namely the nucleus, cilia and plasma membrane. Possible functions of ShUbc13 and K63 polyubiquitination in this single-celled organism are considered with reference to current knowledge. In particular their likely involvement in DNA replication and DNA damage response is especially discussed.

Proposed Function of the Accumulation of Plasma Membrane-Type Ca2+-ATPase mRNA in Resting Cysts of the Ciliate Sterkiella histriomuscorum

ABSTRACT From an mRNA differential-display analysis of the encystment-excystment cycle of the ciliate Sterkiella histriomuscorum, we have isolated an expressed sequence tag encoding a plasma membrane-type Ca2+-ATPase (PMCA). PMCAs are located either in the plasma membranes or in the membranes of intracellular organelles, and their function is to pump calcium either out of the cell or into the intracellular calcium stores, respectively. The S. histriomuscorum macronuclear PMCA gene (ShPMCA) and its corresponding cDNA were cloned; it is the first member of the Ca2+-ATPase family identified in Sterkiella. The predicted protein of 1,065 amino acids exhibits 37% identity with PMCAs of diverse organisms. A phylogenetic analysis showed its relatedness to homologs of two alveolates: the ciliate Paramecium tetraurelia and the apicomplexan Toxoplasma gondii. Overexpression of the protein ShPMCA failed to rescue the wild-type phenotype of three Ca2+-ATPase-defective mutant strains of Saccharomyces cerevisiae; this failure contrasts with the reported ability of the PMCAs of parasites to complement defects in yeast. ShPMCA mRNA is markedly accumulated during encystment and in resting cysts, suggesting a function during excystment. To address the possibility of a signaling role for calcium at excystment, the capacity of calcium to induce excystment was examined.