Proposed function of the accumulation of plasma membrane-type Ca2+-ATPase mRNA in resting cysts of the ciliate Sterkiella histriomuscorum.

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.

Localization of centrins in the hypotrich ciliate Paraurostyla weissei.

Centrins are ubiquitous cytoskeletal proteins that are generally associated with the centrosome and form large cytoskeletal networks in protists. To obtain more data on the respective role of different centrin proteins, we studied their distribution and behavior in one ciliate species, Paraurostyla weissei, using specific antibodies. In this species, only two major proteins of 21 and 24 kDa corresponding to centrins, were identified by 1D and 2D electrophoresis. Immunofluorescence analysis showed that these two proteins displayed non-overlapping localization in the interphase cell and during morphogenesis. Both centrin proteins localize on the fibrous network linking the oral basal bodies in the interphase cell and in the form of marginal dots, which correspond to the proximal ends of the striated rootlets; the 21 kDa centrin was also detected within the basal bodies, whereas the 24 kDa centrin allowed identifying new structures, the frontal dashes. During morphogenesis, the 21 kDa centrin locates at the basal bodies, while the 24 kDa centrin is detected along the striated rootlets and in close association with the basal bodies pairs. These data are discussed in terms of the potential roles of the two centrins in different cellular functions.

Cysteine proteases and cell differentiation: excystment of the ciliated protist Sterkiella histriomuscorum.

The process of excystment of Sterkiella histriomuscorum (Ciliophora, Oxytrichidae) leads in a few hours, through a massive influx of water and the resorption of the cyst wall, from an undifferentiated resting cyst to a highly differentiated and dividing vegetative cell. While studying the nature of the genes involved in this process, we isolated three different cysteine proteases genes, namely, a cathepsin B gene, a cathepsin L-like gene, and a calpain-like gene. Excystation was selectively inhibited at a precise differentiating stage by cysteine proteases inhibitors, suggesting that these proteins are specifically required during the excystment process. Reverse transcription-PCR experiments showed that both genes display differential expression between the cyst and the vegetative cells. A phylogenetic analysis showed for the first time that the cathepsin B tree is paraphyletic and that the diverging S. histriomuscorum cathepsin B is closely related to its Giardia homologues, which take part in the cyst wall breakdown process. The deduced cathepsin L-like protein sequence displays the structural signatures and phylogenetic relationships of cathepsin H, a protein that is known only in plants and animals and that is involved in the degradation of extracellular matrix components in cancer diseases. The deduced calpain-like protein sequence does not display the calcium-binding domain of conventional calpains; it belongs to a diverging phylogenetic cluster that includes Aspergillus palB, a protein which is involved in a signal transduction pathway that is sensitive to ambient pH.

Structural inheritance in Paramecium: ultrastructural evidence for basal body and associated rootlets polarity transmission through binary fission.

One main difference between basal bodies and centrioles resides in the expression of their polarity: centrioles display a structural nine-fold radial symmetry, whereas basal bodies express a circumferential polarity, thanks to their asymmetric set of rootlets. The origin of this polarity during organelle duplication still remains under debate: is it intrinsic to the nine-fold structure itself (i.e. the nine microtubular triplets are not equivalent) or imposed by its immediate environment at time of assembly? We have reinvestigated this problem using the Ciliate Paramecium, in which the pattern of basal body duplication is well known. In this cell, all basal bodies produced within ciliary rows appear immediately anterior to parental ones. Observations on cells fixed with the tannic acid protocol suggest that, to be competent for basal body assembly, parental basal bodies have to be individually associated with a complete set of rootlets (monokinetid structure). During pro-basal body assembly, full microtubular triplets were detected according to a random circumferential sequence; during the whole process, the new basal body and its associated rootlets maintained structural relations with the parental monokinetid structure by way of specific links. These results strongly suggest that basal body and associated rootlets (kinetid) polarity is driven by its immediate environment and provide a basis for the structural heredity property observed by Sonneborn some decades ago.

"Novel cytoskeletal proteins in protists": introductory remarks.

Protists provide the opportunity to integrate analyses from a low (molecular) to a high (organism) level of complexity within a broad evolutionary framework. The perpectives they offer in the cytoskeletal field are discussed with respect to emerging concepts of cellular biology.

Plateins: a novel family of signal peptide-containing articulins in euplotid ciliates.

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 alpha-, beta-, and gamma-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 beta- or gamma-plateins. The derived protein, of 644 amino acids (74.9 kDa), is very acidic (pI = 4.88). Microsequences from authentic alpha-platein were then used to design oligonucleotide primers, which yielded, via a PCR-based approach, the sequences of two alpha-platein genes from E. aediculatus. Even more acidic proteins, the derived alpha1- and alpha2-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 beta/gamma-platein this primary motif domain (27 repeats) is central in the molecule, whereas the primary repeats in the alpha-plateins lie near their C-termini. A cluster of proline-rich pentameric secondary repeats is found in the C-terminus of beta/gamma-platein, but near the N-terminus of alpha-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.

Cytoskeletal proteins with N-terminal signal peptides: plateins in the ciliate Euplotes define a new family of articulins.

Protistan cells employ a wide variety of strategies to reinforce and give pattern to their outermost cortical layers. Whereas some use common cytoskeletal elements such as microtubules, others are based on novel cytoskeletal proteins that are as-yet-unknown in higher eukaryotes. The hypotrich ciliate Euplotes possesses a continuous monolayer of scales or plates, located within flattened membranous sacs ('alveoli') just below the plasma membrane, and this provides rigidity and form to the cell. Using immunological techniques, the major proteins comprising these 'alveolar plates' have been identified and termed alpha-, beta-, and gamma-plateins. The present report describes work leading to the molecular characterization of three plateins, alpha 1 and alpha 2 (predicted M(r)s of 61 and 56 kDa) and a beta/gamma form (M(r)=73 kDa). All three proteins have features that are hallmarks of articulins, a class of cytoskeletal proteins that has been identified in the cortex of a wide variety of protistan cells, including certain flagellates, ciliates, dinoflagellates and PLASMODIUM: Chief among these common features are a prominent primary domain of tandem 12-amino acid repeats, rich in valine and proline, and a secondary domain of fewer, shorter repeating units. However, variations in amino acid use within both primary and secondary repetitive domains, and a much more acidic character (predicted pIs of 4.7-4.9), indicate that the plateins represent the first proteins in a new subclass or family of articulins. This conclusion is supported by another novel feature of the plateins, the presence of a canonical hydrophobic signal peptide at the N-terminus of each derived platein sequence. This correlates well with the final cellular location of the plateins, which are assembled into plates within the membrane-limited alveolar sacs. To our knowledge, this is the first report in any eukaryote of cytoskeletal proteins with such start-transfer sequences. Confocal immunofluorescence microscopy, using antibodies to the plateins as probes, reveals that new alveolar plates (enlarging in cortical zones undergoing morphogenesis) label more faintly than mature parental plates. During plate assembly (or polymerization), the plateins thus appear to exist in a more soluble form.

Functional role of epsilon-tubulin in the assembly of the centriolar microtubule scaffold.

Centrioles and basal bodies fascinate by their spectacular architecture, featuring an arrangement of nine microtubule triplets into an axial symmetry, whose biogenesis relies on yet elusive mechanisms. However, the recent discovery of new tubulins, such as delta-, epsilon-, or eta-tubulin, could constitute a breakthrough for deciphering the assembly steps of this unconventional microtubule scaffold. Here, we report the functional analysis in vivo of epsilon-tubulin, based on gene silencing in Paramecium, which demonstrates that this protein, which localizes at the basal bodies, is essential for the assembly and anchorage of the centriolar microtubules.