Role of delta-tubulin and the C-tubule in assembly of Paramecium basal bodies.

BACKGROUND: A breakthrough in the understanding of centriole assembly was provided by the characterization of the UNI3 gene in Chlamydomonas. Deletion of this gene, found to encode a novel member of the tubulin superfamily, delta-tubulin, results in the loss of the C-tubule, in the nine microtubule triplets which are the hallmark of centrioles and basal bodies. Delta-tubulin homologs have been identified in the genomes of mammals and protozoa, but their phylogenetic relationships are unclear and their function is not yet known. RESULTS: Using the method of gene-specific silencing, we have inactivated the Paramecium delta-tubulin gene, which was recently identified. This inactivation leads to loss of the C-tubule in all basal bodies, without any effect on ciliogenesis. This deficiency does not directly affect basal body duplication, but perturbs the cortical cytoskeleton, progressively leading to mislocalization and loss of basal bodies and to altered cell size and shape. Furthermore, additional loss of B- and even A-tubules at one or more triplet sites are observed: around these incomplete cylinders, the remaining doublets are nevertheless positioned according to the native ninefold symmetry. CONCLUSIONS: The fact that in two distinct phyla, delta-tubulin plays a similar role provides a new basis for interpreting phylogenetic relationships among delta-tubulins. The role of delta-tubulin in C-tubule assembly reveals that tubulins contribute subtle specificities at microtubule nucleation sites. Our observations also demonstrate the existence of a prepattern for the ninefold symmetry of the organelle which is maintained even if less than 9 triplets develop.

Growth and form of secretory granules involves stepwise assembly but not differential sorting of a family of secretory proteins in Paramecium.

Paramecium trichocysts are voluminous secretory vesicles consisting of a spindle-shaped body surmounted by a tip that serves to anchor them at exocytotic sites in the plasma membrane. This constrained shape is conferred by the proteins stored in the vesicles, which form an insoluble three-dimensional crystalline array. The constituent polypeptides (Trichocyst Matrix Proteins, TMPs), which assemble during trichocyst biogenesis, are produced by proteolytic processing of soluble proproteins encoded by a large multigene family. In order to investigate the functional significance of the TMP multigene family, which assures the synthesis of a mixture of related polypeptides, we have designed synthetic genes for heterologous expression of three different mature polypeptides, which were used to obtain sequence-specific rabbit antisera. We used these antisera to carry out immunolocalization experiments with wild-type trichocysts at different stages of development and found that the trichocyst matrix consists of two concentric layers containing different TMPs, and that the assembly of each layer corresponds to a distinct phase of trichocyst growth. Examination of mutant trichocysts created by targeted gene silencing of different TMP genes showed that the layer containing the products of the silenced genes is specifically affected, as are all subsequently assembled parts of the structure, consistent with an ordered assembly pathway. This stepwise assembly is not controlled by differential sorting of the TMPs, as single and double label experiments provided evidence that the different TMPs are delivered together to post-Golgi vesicles and developing trichocysts. We present a model for trichocyst biogenesis in which TMP assembly is controlled by protein processing.

Basal body-associated nucleation center for the centrin-based cortical cytoskeletal network in Paramecium.

The infraciliary lattice, a contractile cortical cytoskeletal network of Paramecium, is composed of a small number of polypeptides including centrins. Its overall pattern reflects a hierarchy of structural complexity, from assembly and bundling of microfilaments to formation of polygonal meshes arranged in a continuous network subtending the whole cell surface, with local differentiations in the shape and size of the meshes. To analyse how the geometry of this complex network is generated and maintained, we have taken two approaches. Firstly, using monoclonal antibodies raised against the purified network, we have shown that all the component polypeptides colocalize, in agreement with previous biochemical data indicating that the infraciliary lattice is formed of large complexes comprising all the component polypeptides. Secondly, by taking advantage of different experimental conditions leading to disassembly of the network, we have followed its reassembly. Cytological analysis of the process revealed 1) that the network regrows exclusively from specific infraciliary lattice organizing centers (ICLOC), precisely localized near each basal body and, 2) that the global organization is not precisely controlled by genetic information but by the basal body pattern. Finally, slight ultrastuctural differences between reassembled and control lattices suggest that the organization of the filament bundles is partly templated by that of the preexisting ones.

Selective and reversible effects of vinca alkaloids on Trypanosoma cruzi epimastigote forms: blockage of cytokinesis without inhibition of the organelle duplication.

Vinca alkaloids, vincristine and vinblastine, produce differential effects on the cell division of Trypanosoma cruzi epimastigote forms depending on drug concentrations. These effects are related to different microtubule-based mechanisms. For 15 microM vinblastine and 50 microM vincristine, the drugs inhibit both nuclear division and cytokinesis, and affect cell shape. At 3 microM vinblastine and 10 microM vincristine, however, cytokinesis is inhibited without major effect on the progression of the cell cycle; this yields giant cells having multiple nuclei, kinetoplasts and flagella. Cultures maintained over 1 week with daily drug replacement produced cells with more than 16 nuclei and 24 kinetoplasts, indicating that an equivalent of a fifth cell cycle was initiated. The ultrastructure of the multinucleate cells showed a basic organization closely similar to that of trypanosomes. Cytokinesis inhibition by vinca alkaloids seems to result from modulations of interactions between microtubules and associated proteins, rather than from an inhibition of microtubule dynamics as is usually proposed for vinca alkaloids. Cytokinesis inhibition is reversible: after removing the drug, epimastigotes emerge from the multinucleate cells. The emerging process follows a precise axis and polarity which are determined by the position of the flagellum/kinetoplast complex. This region could play an essential role in cell morphogenesis since zoids (cells without a nucleus) are frequently observed.

Genetic evidence for a role of centrin-associated proteins in the organization and dynamics of the infraciliary lattice in Paramecium.

Within the superfamily of "EF-hand Ca2+-modulated proteins," centrins constitute a family of cytoskeletal proteins that are highly conserved from lower eukaryotes to man. Their cytoskeletal specialization is manifest in their capacity to form filamentous contractile arrays of various shapes and functions and by their association with microtubule organizing centres (MTOCs). While the latter property has been conserved throughout the evolution of eukaryotes, centrin-based contractile structures are only found in protists where they form arrays of widely diverse organization and function. In the ciliate Paramecium tetraurelia, three centrin genes have been characterized, which may be part of a larger centrin gene family [Madeddu et al., 1996: Eur J. Biochem. 238:121-128]. The products of these genes were originally identified as components of the infraciliary lattice, a contractile cytoskeletal network [Garreau de Loubresse et al., 1991: Biol. Cell 71:217-225]. We show here that centrins are localized not only in this lattice but also in basal bodies and in the cord, a filamentous structure associated with the oral apparatus. We demonstrate that in the infraciliary lattice, but not in basal bodies, centrins are associated with high-molecular-weight proteins (ca. 350 kD). Their role in the biogenesis of the infraciliary lattice is documented by cytological and biochemical properties of the mutant "démaillé" (dem1) characterized by altered centrin-associated proteins and abnormal organization and dynamics of the infraciliary lattice.

Evidence for defects in membrane traffic in Paramecium secretory mutants unable to produce functional storage granules.

The ciliated protozoan Paramecium has a regulated secretory system amenable to genetic analysis. The secretory storage granules, known as trichocysts, enclose a crystalline matrix with a genetically determined shape whose biogenesis involves proteolytic maturation of a family of precursor molecules into a heterogeneous set of small acidic polypeptides that crystallize within the maturing vesicles. We have developed an original pulse-chase protocol for monoxenic Paramecium cultures using radiolabeled bacteria to study the processing of trichocyst matrix proteins in wild-type and mutant cells. In wild-type cells, proteolytic processing is blocked in the presence of monensin and otherwise rapidly completed after approximately 20 min of chase, suggesting that the conversion occurs in the trans-Golgi and/or in small vesicles soon after sorting to the regulated pathway, probably before crystallization begins. In trichless mutant cells, which contain no visible trichocysts, secretory proteins are synthesized but not processed and we report constitutive secretion of the uncleaved precursor molecules. The mutation thus appears to affect sorting to the regulated pathway and should prove useful for analysis of the sorting machinery and of the relationship between sorting and proteolytic processing of secretory proteins. In mutants bearing misshapen trichocysts with poorly crystallized contents (tam33, tam38, stubbyA), the proteolytic processing of the trichocyst matrix proteins appears to be normal, while both pulse-chase and morphological data indicate that intracellular transport is perturbed, probably between ER and Golgi. Precursor molecules are present in the mutant trichocysts but not in wild-type trichocysts and may account for the defective crystallization. Our analysis of these mutants suggests that the temporal coordination of intracellular traffic plays a regulatory role in granule maturation.

Protein processing and morphogenesis of secretory granules in Paramecium.

The ciliated protozoan Paramecium provides a model system for the study of regulated secretion, featuring architecturally complex secretory storage granules-trichocysts-docked at the plasma membrane, ready to respond to an exocytotic stimulus. The trichocysts are characterized by crystalline contents that confer upon the organelle a defined shape which can be altered by single gene mutation. The crystalline trichocyst contents are built up from a heterogeneous set of small acidic polypeptides generated by proteolytic maturation of a family of precursor molecules, suggesting an important role for protein processing in this system. We have recently shown that the primary defect in several secretory mutants lacking functional trichocysts is in intracellular trafficking rather than protein processing. However, analysis of how these defects lead to altered trichocyst shape supports the notion that the protein processing is essential for morphogenesis. Preliminary results of a cloning project reveal that an extensive multigene family (approximately 100 genes) codes for the trichocyst matrix proteins. Deduced amino acid sequences of putative processing sites indicate that (at least) two distinct processing reactions are probably involved in the maturation of these proteins, and allow us to speculate that each reaction may control a key event of trichocyst biogenesis.

Development of surface pattern during division in Paramecium. II. Defective spatial control in the mutant kin241.

kin241 is a monogenic nuclear recessive mutation producing highly pleiotropic effects on cell size and shape, generation time, thermosensitivity, nuclear reorganization and cortical organization. We have analyzed the nature of the cortical disorders and their development during division, using various specific antibodies labelling either one of the cortical cytoskeleton components, as was previously done for analysis of cortical pattern formation in the wild type. Several abnormalities in basal body properties were consistently observed, although with a variable frequency: extra microtubules in either the triplets or in the lumen; nucleation of a second kinetodesmal fiber; abnormal orientation of the newly formed basal body with respect to the mother one. The latter effect seems to account for the major observed cortical disorders (reversal, intercalation of supplementary ciliary rows). The second major effect of the mutation concerns the spatiotemporal map of cortical reorganization during division. Excess basal body proliferation occurs and is correlated with modified boundaries of some of the cortical domains identified in the wild type on the basis of their basal body duplication pattern. This is the first mutant described in a ciliate in which both the structure and duplication of basal bodies and the body plan are affected. The data support the conclusion that the mutation does not alter the nature of the morphogenetic signal(s) which pervade the dividing cell, nor the competence of cytoskeletal structures to respond to signalling, but affects the local interpretation of the signals.

Purification of the surface membrane-cytoskeleton complex (Cortex) of Paramecium and identification of several of its protein constituents.

In ciliates, the major morphogenetic events take place in the cortex, a complex of membranes and closely associated filamentous networks. To analyze the problems of assembly and morphogenesis at the molecular level in Paramecium, we have developed a method of purification of cortex fragments which retain their in situ organization and display a highly reproducible electrophoretic profile. The method used either a four-step sucrose gradient yielding a cortex + oral apparatus fraction or a six-step gradient which allowed the cortex fragments to be freed from the oral apparatuses (which were recovered separately). By comparative electrophoresis and immunological probing of these and other cell fractions or purified organelles, we could identify several of the major polypeptides resolved by SDS PAGE as components of specific cortical or oral structures. The purification method was successfully applied to morphological mutants, and the first case of a mutational modification of a cortical polypeptide was observed.

A mutation affecting basal body duplication and cell shape in Paramecium.

The thermosensitive mutant sm19 of Paramecium tetraurelia undergoes a progressive reduction in cell length and basal body number over successive divisions at the nonpermissive temperature of 35 degrees C. In spite of these defects, sm19 cells retain the same generation time as wild-type cells at 35 degrees C. Cytological observations at both electron and light microscopy levels reveal no other perturbation than the rarefaction of basal bodies and the rare (3%) absence of one or two microtubules in basal bodies or ciliary axonemes. The temperature-sensitive period, during the last 30 min of the cell cycle, corresponds to the phase of basal body duplication. Upon transfer back to the permissive temperature, all basal bodies are normally duplicated. The mutational defect is transiently restored by microinjection of wild-type cytoplasm or of a soluble proteic fraction from wild-type cell homogenates. Altogether, the cytological and physiological data support the conclusion that the sm19+ gene codes for a diffusible product required for the initiation of basal body duplication and would thus be the first identified gene involved in this process. Our data also indicate that in Paramecium basal body number is not coupled with control of the cell cycle, but helps determine the shape of the cell via the organization of the cytoskeleton.

Actin microfilaments in paramecium: localization and role in intracellular movements.

Using heavy meromyosin (HMM) or the fragment S1 of myosin as probes for actin microfilaments, we studied their organization in Paramecium both by fluorescence and electron microscopy. In interphasic cells, HMM decorates (a) most prominently the periphery of nascent and young food vacuoles and their route during the early phase of their intracellular transit; (b) a thin meshwork radiating from the gullet throughout the cytoplasm; (c) a small area beneath the pore of contractile vacuoles and beneath the cytoproct when open to release food residues. Most of these HMM-decorated structures are in close contact with microtubular arrays. All HMM decoration disappears in dividing cells and in cytochalasin-treated cells. In vivo, the drug immediately blocks food vacuole formation but does not affect cytokinesis, cyclosis, contractile vacuole pulsation, defecation, or nuclear movements. The data show that, as in the cells of other organisms, actin microfilaments form defined arrays that undergo physiologically controlled cycles of assembly/disassembly. These arrays contribute (at least in the phagocytotic process) to diverse types of movement: constriction, membrane fusion, and migration of food vacuoles. However, aside from their massive concentration along the phagocytotic tractus, actin microfilaments are neither major structural components of Paramecium cytoplasm nor the only cytoskeletal components ensuring motility or contractility processes.