Articulins and epiplasmins: two distinct classes of cytoskeletal proteins of the membrane skeleton in protists.

The cortex of ciliates, dinoflagellates and euglenoids comprises a unique structure called the epiplasm, implicated in pattern-forming processes of the cell cortex and in maintaining cell shape. Despite significant variation in the structural organization of their epiplasm and cortex, a novel type of cytoskeletal protein named articulin is the principal constituent of the epiplasm in the euglenoid Euglena and the ciliate Pseudomicrothorax. For another ciliate, Paramecium, epiplasmins, a group of polypeptides with common biochemical properties, are the major constituents of the epiplasm. Using molecular tools and affinity purification we have selected polyclonal antibodies and identified epitopes of monoclonal antibodies that identify epitopes characteristic of articulins and epiplasmins. With these antibodies we have analysed the occurrence of the two types of cytoskeletal proteins in a dinoflagellate, a euglenoid and several ciliates. Our results indicate that both articulins and epiplasmins are present in these organisms, suggesting that both contribute to the organization of the membrane skeleton in protists. Articulins and epiplasmins represent two distinct classes of cytoskeletal proteins, since different polypeptides were labeled by articulin core domain-specific or epiplasmin epitope-specific antibodies in each organism studied. In one case, a polypeptide in Pseudomicrothorax was identified that reacts with both articulin core domain-specific and with anti-epiplasmin monoclonal antibodies; however, the epiplasmin monoclonal antibody epitope was mapped to the C terminus of the polypeptide, well outside the central VPV-repeat core domain that contains the articulin monoclonal antibody epitope and that is the hallmark of the articulins.

Characterisation of two articulins, the major epiplasmic proteins comprising the membrane skeleton of the ciliate Pseudomicrothorax.

Most protists possess a unique membrane skeleton, the epiplasm, which is involved in pattern forming processes of the cell cortex and functions in maintaining cell shape. Articulins, a novel class of cytoskeletal proteins, are major constituents of the epiplasm. We have isolated cDNAs encoding the two major articulins of the ciliate Pseudomicrothorax dubius. Peptide sequence data confirm the identity of the cloned cDNAs encoding articulins 1 and 4. With the data presented here sequence information for all major articulins of ciliates as well as the distantly related euglenoids is available. Sequence comparison of the two newly characterised ciliate articulins with the previously determined sequences of p60, a minor articulin of the same species, and the two euglenoid articulins reveals general sequence principles and uncovers new features of this protein family. The hallmark of articulins is a central core domain of repetitive motifs of alternating valine and proline residues, the VPV-motif. These VPV-motif repeats are either 12-residues, or in some places, six residues long. Positively and negatively charged residues segregate in register with valine and proline positions. The VPV-motif is unique to articulins. The terminal domains flanking the core are generally hydrophobic and contain a series of hexa- or heptapeptide repeats rich in glycine and hydrophobic residues. The sequences of these short repeats are very similar in articulins of the same species but are not conserved between euglenoids and ciliates.

Major epiplasmic proteins of ciliates are articulins: cloning, recombinant expression, and structural characterization.

The cytoskeleton of certain protists comprises an extensive membrane skeleton, the epiplasm, which contributes to the cell shape and patterning of the species-specific cortical architecture. The isolated epiplasm of the ciliated protist Pseudomicrothorax dubius consists of two major groups of proteins with molecular masses of 78-80 kD and 11-13 kD, respectively. To characterize the structure of these proteins, peptide sequences of two major polypeptides (78-80 kD) as well as a cDNA representing the entire coding sequence of a minor and hitherto unidentified component (60 kD; p60) of the epiplasm have been determined. All three polypeptides share sequence similarities. They contain repeated valine- and proline-rich motifs of 12 residues with the consensus VPVP--V-V-V-. In p60 the central core domain consists of 24 tandemly repeated VPV motifs. Within the repeat motifs positively and negatively charged residues, when present, show an alternating pattern in register with the V and P positions. Recombinant p60 was purified in 8 M urea and dialyzed against buffer. Infrared spectroscopic measurements indicate 30% beta-sheet. Electron microscopy reveals short filamentous polymers with a rather homogenous diameter (approximately 15-20 nm), but variable lengths. The small polymers form thicker filaments, ribbons, and larger sheets or tubes. A core domain similar to that of P. dubius p60 is also found in the recently described epiplasmic proteins of the flagellate Euglena, the so-called articulins. Our results show that the members of this protein family are not restricted to flagellates, but are also present in the distantly related ciliates where they are major constituents of the epiplasm. Comparison of flagellate and ciliate articulins highlights common features of this novel family of cytoskeletal proteins.

Immunological characterization of trichocyst proteins in the ciliate Pseudomicrothorax dubius.

Ejectable trichocysts were isolated from the ciliate Pseudomicrothorax dubius. Polyclonal antibodies were raised against three groups of trichocyst proteins: G1 (30-31 kDa), G2 (26-27 kDa) and G3 (15-20 kDa). By indirect immunofluorescence, the three antisera strongly label the shafts of ejected trichocysts and the proximal ends of condensed trichocysts within the cells. By immunogold labeling for electron microscopy, the three sera specifically recognize the shafts of both extended and condensed trichocysts and shaft precursors in pretrichocysts as well. On one-dimensional immunoblots of isolated trichocysts, anti-G1 serum recognizes the G1 proteins, anti-G2 serum detects G2 proteins and some G1 proteins, and anti-G3 serum reacts with 15 bands, mainly the G3 and (30-41)-kDa proteins. In cells with and without trichocysts, the sera recognize non-ejectable trichocyst proteins at 41-42 kDa and 47 kDa. On two-dimensional immunoblots of isolated trichocysts, anti-G1 serum labels proteins with a pI of 4.75-5.7, anti-G2 serum labels proteins with a pI of 4.75-6.25 and anti-G3 serum labels proteins with a pI of 4.7-6.6. Analyses of cells with and without trichocysts allow identification of possible precursors between 41 and 47 kDa. Some are in the same pI range as their putative products, but others, labeled by anti-G3 serum, are less acidic than most of their mature products.

Secretory proteins in a ciliate cell: identification of epitopes common to numerous polypeptides.

Secretory vesicles of the ciliate Pseudomicrothorax dubius, called trichocysts, are separated into > 40 proteins by two-dimensional gel electrophoresis. The trichocyst, composed of a shaft and four arms, is in a condensed state when docked in the cell cortex, and it elongates into an extended state during exocytosis. Monoclonal antibodies (mAbs) were raised against trichocyst proteins. Their reactivities were analysed: 1) on Western blots of extended, isolated trichocysts by immunolabeling; 2) on entire cells and extended trichocysts by indirect immunofluorescent binding assay (IFA); 3) on semi-thin sectioned cells by IFA; and 4) on ultra-thin sections of cells by immunogold labeling. mAb IV 4E5 labels major trichocyst proteins at 15-19, 22 and 24 kDa, pI 4.6-6.6. The epitope recognized by mAb IV 4E5 is common to as many as 30 proteins and suggests a family of proteins with possible sequence homology. By IFA, the shafts of extended trichocysts are labeled. The shafts of condensed trichocysts are labeled on both semi-thin sections in Lowicryl and ultrathin sections. On semi-thin Epon sections, the part of the trichocyst which is labeled is arm-like. mAb VI 2D12 labels three major trichocyst proteins at 31 kDa, pI 5.0-5.4. The arms of extended trichocysts are labeled by IFA, but are only weakly labeled on ultrathin sections. The shaft of extended trichocysts is labeled by IFA, and the shaft of condensed trichocysts is labeled on ultrathin sections.

Immunological similarities among trichocyst secretory proteins of Paramecium caudatum, Paramecium tetraurelia and Pseudomicrothorax dubius.

Secretory vesicles called trichocysts structurally show a body and a tip in Paramecium and a shaft and four arms in Pseudomicrothorax. Biochemical and immunological relationships between both trichocyst types were examined. Three polyclonal antibodies were produced against 3 groups of protein bands of isolated Pseudomicrothorax dubius trichocysts separated by one-dimensional gel electrophoresis: G 1 (30-31 kDa), G 2 (26-27 kDa) and G 3 (15-20 kDa). By indirect immunofluorescent binding assay, the three antisera label extended (i.e. discharged) and condensed (i.e. non-discharged) trichocysts of both Ps. dubius and Paramecium caudatum. By immunogold labeling on ultrathin sections, the three antisera label the trichocyst shaft of Ps. dubius and the trichocyst body of Pa. caudatum on both condensed and extended secretory vesicles. On two-dimensional immunoblots, remarkable antigenic similarities are shown by trichocysts of Ps. dubius, Paramecium tetraurelia nd6 and Pa. caudatum. Anti-G 1 serum detects proteins at 28-32 kDa, pI 5.0-5.7, in the three cells. Anti-G 3 serum labels one protein group at ~ 15-22 kDa and a second protein group at ~ 35-50 kDa in Ps. dubius, Pa. tetraurelia nd6 and Pa. caudatum. Anti-G 2 serum labels proteins at ~ 15-22 kDa, pI 4.7-5.2, in all three species. Anti-G 2 serum also detects bands at 24-28 kDa and 30-31 kDa, pI 4.7-6.1, in Ps. dubius. No equivalent M(r) proteins are observed in either Paramecium spp. Comparisons of immunoblots of proteins of entire cells with and without ejectable trichocysts allowed identification of non-ejectable trichocyst proteins, some of which may be precursors localized within pretrichocysts. Such proteins are at 41-47 kDa in Ps. dubius, and at ~ 45 kDa in Paramecium.

Evidence for common epitopes among proteins of the membrane skeleton of a ciliate, an euglenoid and a dinoflagellate.

The cytoskeleton of many protists comprises an extensive submembranous epiplasm which contributes to cell shape and integration of cell membranes with underlying structures according to the species-specific cortical architecture. Using various extraction procedures, epiplasm-enriched fractions have been isolated from the ciliate Pseudomicrothorax dubius, the euglenoid Euglena acus and the dinoflagellate Noctiluca scintillans. Comparative gel electrophoretic analysis of such preparations reveals heterogeneity of protein composition, the major polypeptides differing in size. Antibodies raised against epiplasmic proteins from these three organisms have permitted the confirmation of submembranous localization of the antigens by immunoelectron microscopy. Heterologous reactions performed by means of combined immunocytochemical and immunoblotting procedures indicate the existence of common epitopes among major proteins making up the bulk of the epiplasm of the three species examined. These findings suggest that proteins of the epiplasm have significantly diverged during evolution while conserving structural domains essential for their cytoskeletal function. It is postulated that these common domains may underly the ability of epiplasmic proteins to assemble into an ordered spatial organization, typical of the highly differentiated cortex of unicellular micro-organisms.

Microtubules and microfilaments as major components of a phagocytic apparatus: the cytopharyngeal basket of the ciliate Pseudomicrothorax dubius.

The cytopharyngeal basket of Pseudomicrothorax dubius, through which filamentous blue-green algae are ingested, consists of 22 (+/- 3) nemadesmata and nemadesmal lamellae, in the form of a tube. A cytostome, delimited by the cell membrane and surrounded by 22 (+/- 3) major and minor cortical corrugations, covers the end of the basket where the latter is attached to the cell cortex. Each nemadesm, at its greatest diameter, consists of about 200 microtubules which are joined together by sheet-like cross-bridges. The cross-bridges appear to be responsible for the high structural resilience of the nemadesmata. Each nemadesmal lamella is a ribbon of 20--30 microtubules, with two arm-like structures associated with one side of each microtubule. The arms are partially embedded in a fine filamentous layer. Except for a perforated zone, the wall of the basket is completely closed due to the presence of a filamentous sheath which extends between adjacent nemadesmata. Absence of the sheath allows movement of vesicles between the cytoplasm and the lumen of the basket in the perforated zone. The sheath is capable of elastic stretching during food uptake.

The ultrastructure of the somatic cortex of Pseudomicrothorax dubius: structure and function of the epiplasm in ciliated protozoa.

The ultrastructure of the somatic cortex of the ciliate Pseudomicrothorax dubius is studied with emphasis on the epiplasm layer which lies immediately under the inner alveolar membrane and is continuous with the terminal plates of cortical basal bodies. In addition to a clearly demonstrable cytoskeletal role, the epiplasm appears to function as a comenting substance which integrates numerous cortical fibres and membranes. The kinetodesmal, postciliary and transverse fibre systems which originate at the proximal ends of basal bodies extend toward the cell surface and end at or in the epiplasm. Inner alveolar membranes and trichocyst membranes are attached to the epiplasm. Basal bodies are anchored into the epiplasm via their terminal plates. The epiplasm appears to be morphogenetically important as a matrix into which newly formed basal bodies insert. Electron-opaque arms occur at the terminal plate level of new basal bodies, and these arms fuse with the epiplasm when basal body insertion occurs. The position of trichocysts in the cortex is specified by the epiplasm. Evidence from numerous other ciliates tends to confirm both structural and morphogenetic roles of the epiplasm.

Cortical ultrastructure of the Scuticociliates Dexiotricha media and Dexiotricha colpidiopsis (Hymenostomata).

Cortical ultrastructure of the scuticociliates Dexiotricha media and Dexiotricha colpidiopsis was investigated. The following elements of the somatic cortex were studied: the cell membrane, alveolar membranes and the epiplasm, kinetodesmal fibers, postciliary and transverse microtubular ribbons, and transverse fibers associated with single and paired kinetosomes; mitochondria and single microtubules located in interkinetal ridges; mature and early extrusion stages of mucocysts; the expulsion vacuole pore and tube, the nephridioplasm and the cytoproct. In the buccal cortex, the paroral kinety-ribbed wall complex, the 3 polykineties, and the cytostome-cytopharynx were investigated. Comparative survey of ciliate ultrastructure indicates 2 principal orientation patterns for kinetodesmal and postciliary fibers, recognition of which leads to reevaluation of the theory of paroral kinety formation and the ideas of homology based on this theory. Ultrastructurally, the scuticociliates are not distinct from tetrahymenines and peniculines; the 3 groups appear to be 1 assemblage.

[The importance of electron microscopy in the systematics of unicellular organisms: for example, the ciliates of the genus Cyclogramma]. / Importance de la microscopie électronique dans les études de systématique chez les Unicellulaires; un exemple: les Ciliés du genre Cyclogramma

With a parorale of "stichodyade-type", and three adoral organelles like peniculus, the genus Cyclogramma may be included in the Oligohymenophora class, subclass Hymenostomata. The presence of a basket as cytopharyngeal apparatus, and the semi-autonomous stomatogenesis justify, for this genus, the establishment of the new order Parahymenostomatida.