Nanostructured polymer-titanium composites and titanium oxide through polymer swelling in titania precursor.

Polymer (XAD7HP)/Ti4+ nanocomposites were prepared through the swelling of polymer in titanium (IV) ethoxide as a titanium dioxide precursor. The nanocomposite beads exhibit relatively high porosity different than the porosity of the initial polymer. Thermal treatment of composite particles up to 200 °C in vacuum causes the change of their internal structure. At higher temperature, the components of composite become more tightly packed. Calcination at 600 °C and total removal of polymer produce spherically shaped TiO2 condensed phase as determined by XRD. Thermally treated composites show the substantial change of pore dimensions within micro- and mesopores. The presence of micropores and their transformation during thermal processing was studied successfully by positron annihilation lifetime spectroscopy (PALS). The results derived from PALS experiment were compared with those obtaining from low-temperature nitrogen adsorption data.

Septins stabilize mitochondria in Tetrahymena thermophila.

We describe phylogenetic and functional studies of three septins in the free-living ciliate Tetrahymena thermophila. Both deletion and overproduction of septins led to vacuolization of mitochondria, destabilization of the nuclear envelope, and increased autophagy. All three green fluorescent protein-tagged septins localized to mitochondria. Specific septins localized to the outer mitochondrial membrane, to septa formed during mitochondrial scission, or to the mitochondrion-associated endoplasmic reticulum. The only other septins known to localize to mitochondria are human ARTS and murine M-septin, both alternatively spliced forms of Sep4 (S. Larisch, Cell Cycle 3:1021-1023, 2004; S. Takahashi, R. Inatome, H. Yamamura, and S. Yanagi, Genes Cells 8:81-93, 2003). It therefore appears that septins have been recruited to mitochondrial functions independently in at least two eukaryotic lineages and in both cases are involved in apoptotic events.

KIN241: a gene involved in cell morphogenesis in Paramecium tetraurelia reveals a novel protein family of cyclophilin-RNA interacting proteins (CRIPs) conserved from fission yeast to man.

In this study, we report cloning, by functional complementation of the KIN241 gene involved in Paramecium cell morphogenesis, cortical organization and nuclear reorganization. This gene is predicted to encode a protein of a novel type, comprising a cyclophilin-type, peptidyl-prolyl isomerase domain, an RNA recognition motif, followed by a region rich in glutamate and lysine (EK domain) and a C-terminal string of serines. As homologues of this protein are present in the genomes of Schizosaccharomyces pombe, Caenorhabditis elegans, Drosophila melanogaster, Arabidopsis thaliana and Homo sapiens, the Kin241p predicted sequence defines a new family of proteins that we propose to call 'CRIP', for cyclophilin-RNA interacting protein. We demonstrate that, in Paramecium, Kin241p is localized in the nucleus and that deletion of some nuclear localization signals (NLSs) decreases transport of the protein into the nucleus. No Kin241-1 protein is present in mutant cells, suggesting that the C-terminal serine-rich region is responsible for protein stability.

The dynamics of filamentous structures in the apical band, oral crescent, fission line and the postoral meridional filament in Tetrahymena thermophila revealed by monoclonal antibody 12G9.

The ciliate Tetrahymena thermophila possesses a multitude of cytoskeletal structures whose differentiation is related to the basal bodies - the main mediators of the cortical pattern. This investigation deals with immunolocalization using light and electron microscopy of filaments labeled by the monoclonal antibody 12G9, which in other ciliates identifies filaments involved in transmission of cellular polarities and marks cell meridians with the highest morphogenetic potential. In Tetrahymena interphase cells, mAb 12G9 localizes to the sites of basal bodies and to the striated ciliary rootlets, to the apical band of filaments and to the fine fibrillar oral crescent. We followed the sequence of development of these structures during divisional morphogenesis. The labeling of the maternal oral crescent disappears in pre-metaphase cells and reappears during anaphase, concomitantly with differentiation of the new structure in the posterior daughter cell. In the posterior daughter cell, the new apical band originates as small clusters of filaments located at the base of the anterior basal bodies of the apical basal body couplets during early anaphase. The differentiation of the band is completed in the final stages of cytokinesis and in the young post-dividing cell. The maternal band is reorganized earlier, simultaneously with the oral structure. The mAb 12G9 identifies two transient structures present only in dividing cells. One is a medial structure demarcating the two daughter cells during metaphase and anaphase, and defining the new anterior border of the posterior daughter cell. The other is a post-oral meridional filament marking the stomatogenic meridian in postmetaphase cells. Comparative analysis of immunolocalization of transient filaments labeled with mAb12G9 in Tetrahymena and other ciliates indicates that this antibody identifies a protein bound to filamentous structures, which might play a role in relying polarities of cortical domains and could be a part of a mechanism which governs the positioning of cortical organelles in ciliates.

A novel healing filament in ciliate regeneration.

The interphase cells of the hypotrich ciliate Paraurostyla weissei possess a complex fibrillar system surrounding basal bodies in the compound ciliary assemblages, cirri and membranelles. During replacement of the ciliature at cell division, transient filaments precede and accompany the development of ciliary primordia and participate in the formation of the fission furrow. Both fibrillar systems are recognized by monoclonal antibody FXXXIX 12G9. We studied regeneration of cellular fragments after transection employing the mAb 12G9 and found a new cytoskeletal structure involved in healing of the excisional wound. The healing filament is formed at the wound edge, distally and in connection with the bases of cirri closest to the wound. It is visible 5 min after transection. Concomitant with development of new ciliary primordia, the healing filament shrinks and finally disappears together with other transient fibers formed in this process. Ultrastructural analysis of immunolabeled regenerating cells revealed that structures recognized by mAb 12G9 contain fine filaments whose packing and arrangement depends on accompanying cytoplasmic elements and the developmental status of a fragment. Assembly of the healing fiber does not depend on microtubules and microfilaments since it develops in cellular fragments exposed to cold, nocodazole, and Cytochalasin D. On Western blots of whole cell and cytoskeletal extracts of P. weissei the 12G9 antibody identified one protein band whose molecular weight corresponds to 60 kDa.

Polarities of the centriolar structure: morphogenetic consequences.

Centrioles and basal bodies are two versions of the same conserved eukaryotic organelle and share two remarkable properties: nine-fold symmetry of their microtubular shaft and capacity to generate a new organelle in a fixed geometrical relationship to the mother organelle. It can thus be postulated that what is true for basal bodies is likely to be true also for centrioles. While the functions of centrioles are difficult to dissect, the functions of basal bodies are easier to approach. Over more than two decades, studies on protists have led to the notion that ciliary and flagellar basal bodies display polarities, not only a proximo-distal polarity, like in centrioles, but also a circumferential polarity accorded to the polarities of the cell and of its cytoskeleton. The major cytological and genetical data, mainly of Chlamydomonas, Paramecium and Tetrahymena, which support the notion that the microtubule triplets of basal bodies are non-equivalent, are reviewed. The morphogenetic implications of this circumferential anisotropy, perpetuated through the process of basal body duplication itself, are discussed. The question is raised of the possibility that centrioles also display a circumferential polarity, like basal bodies, and whether at least certain of their functions depend on such asymmetries.

Uncoupling of basal body duplication and cell division in crochu, a mutant of Paramecium hypersensitive to nocodazole.

In Paramecium the development of cell shape and surface pattern during division depends on a precise spatial and temporal pattern of duplication of the ciliary basal bodies which are the organizers of the cortical cytoskeleton. According to their localization, basal bodies will duplicate once, more than once or not all and this duplication is coupled with cell division, as is centrosomal duplication in metazoan cells. We describe here a monogenic nuclear recessive mutation, crochu1 (cro1), resulting in abnormal cell shape and cortical pattern and hypersensitivity to nocodazole. The cytological analysis, by immunofluorescence and electron microscopy, demonstrates that the mutation causes hyper duplication of basal bodies and releases both spatial and temporal control of duplication as basal bodies continue to proliferate in interphase and do so at ectopic locations, beneath the surface and in cortical territories where no duplication occurs in the wild type. However, the abnormal surface organization of cro1 cells does not affect the program of basal body duplication during division. By genetic analysis, no interaction was detected with the sm19 mutation which impairs basal body duplication. In contrast, the cro1 mutation suppresses the nocodazole resistance conferred by nocr1, a mutation in a beta-tubulin gene. This interaction suggests that the primary effect of the mutation bears on microtubule dynamics, whose instability, normally increased during division, would persist throughout the interphase and provide a signal for constitutive basal body duplication.

The effects of lithium chloride on pattern formation in Tetrahymena thermophila.

Lithium ions have long been known to exert dramatic effects on the specification of cell fates in multicellular systems. We have analyzed the effects of Li+ on intracellular patterning in a complex unicellular organism, the ciliate Tetrahymena thermophila. LiCl does not affect the locations of major structural landmarks in the cortical region of wild-type cells and does not modify the phenotype of pattern-mutant cells. However, in all strains studied LiCl differentially affects early stages of oral development. It initially triggers a slow regression of oral primordia, which is followed by an excessive proliferation of basal bodies that leads to a hypertrophy of the ciliature of the cell's feeding organelle. This hypertrophy mimics the effects of the membranellar-pattern-D mutation, the phenotype of which is enhanced in the presence of LiCl. These effects were partially reversed by myo-inositol; however, neomycin failed to mimic the effects of LiCl. Thus, although lithium ions have major cellular effects on Tetrahymena, they do not influence the specification of the body plan in a manner analogous to that observed in multicellular organisms and may work in part through mechanisms other than the now-classical inositol-phosphate cycle.

Cellular polarity in ciliates: persistence of global polarity in a disorganized mutant of Tetrahymena thermophila that disrupts cytoskeletal organization.

Much of the cell surface on the ciliate Tetrahymena thermophila is covered by a polarized lattice of cytoskeletal structures that are associated with basal bodies of the ciliary rows. Unique structural landmarks, including an oral apparatus and contractile vacuole pores, develop before cell division in localized domains located, respectively, posterior and anterior to the transverse fission zone. All of these structures can be visualized by specific monoclonal antibodies. A single-locus recessive mutation, disorganized-A (disA), primarily affects the striated rootlets of the ciliary-row basal bodies and brings about a severe disorganization in the positioning and orientation of these basal bodies and associated cytoskeletal elements. Nonetheless, the new oral apparatus, contractile vacuole pores, and other unique structures appeared at or near their normal sites along the anteroposterior axis of disA cells, indicating that the positioning of these localized structures is not dependent on the integrity of the ciliary rows. Abnormalities were present in the details of construction of some of the localized structures and in aspects of cell shape that may be influenced by these details. In the main, however, analysis of disA mutant cells indicates that intracellular domains near the cell poles develop independently of the vectorial polarity of the ciliary rows.

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.

Ultrastructural studies on the development of cortical structures in the ciliary pattern mutants of the hypotrich ciliate Paraurostyla weissei.

We have studied the ultrastructure of interphase and developing cells of mutants (mlm and mlmlpl) of Paraurostyla weissei that overproduce basal bodies in certain cortical domains and bring about coordinate modifications of the overall pattern [8, 25, 26]. In interphase cells, basal bodies and accessory microtubular organelles appear normal. In developing cells, overgrowth of adorai primordia, multiplication of ciliary primordia, differentiation of dorsal bristles on the ventral surface, and reversal of the orientation of oral and cirral promordia occur in spite of apparently normal assembly events of all studied ciliary structures. The three kinds of basal body pairs (paroral, adorai and somatic) have the same ultrastructure as in wild-type cells. Abnormal orientation of pairs occurs during the first wave of basal body proliferation. In the second wave, the new basal bodies complete new cirri and membranelles regardless of their orientation. As in wt cells, there is no second wave of basal body proliferation in the paroral membranes. Early steps of elimination of one category of cirri (FC) involve small defects in basal body assembly during the first wave of basal body proliferation. Thus modifications in the ciliary pattern in the mlm/pl mutants result from overduplication, abnormal location and orientation of basal bodies taking place during the first half of the process of morphogenesis in this ciliate.

Genetic approaches to ciliate pattern formation: from self-assembly to morphogenesis.

In the cortex of ciliates, thousands of basal bodies, with their associated cytoskeletal appendages and networks, are arranged in an elaborate pattern whose reproduction at division involves complex morphogenetic movements. Genetic analysis demonstrates that pattern formation relies both on local constraints imposed by the pre-existing organization, and on the differential interpretation of inductive signals by different cell territories whose individual developmental properties are reset at each division.

Defective spatial control in patterning of microtubular structures in mutants of the ciliate Paraurostyla: I. Morphogenesis in multi-left-marginal mutant.

The development of the primordia of ciliary structures was studied in a single-gene recessive mlm (multi-left-marginal) mutant of the hypotrich ciliate P. weissei. The modifications in morphogenesis consist of excessive production of basal bodies in the left half of the ciliate. In extreme cases, the formation of double sets of oral primordia (OP), production of multiple streaks of the left marginal cirri sometimes intermingled with the OP, and development of more than one streak in the left dorsal bristle rows is observed. All abnormalities in development of ciliary primordia are expressed most profoundly at the middle stages of morphogenesis. In late stages of morphogenesis, the excess basal bodies in the oral primordium are resorbed, and some of the ciliary primordia regress. Thus, the developmental phenotype of the mlm mutants involves a combination of broadening and overlapping of organellar domains in the left half of the ciliate surface layer, with occasional reversal of ciliary structures formed at the border between the two domains. The mlm mutation probably causes perturbations in the cell's ability to maintain discrete boundary conditions around the circumferential dimension.

Defective spatial control in patterning of microtubular structures in mutants of the ciliate Paraurostyla: II. Spatial coordinates in a double recessive mutant.

The abnormal ciliary pattern and its propagation was studied in the double recessive mutant 95 (mlm/mlm, pl/pl) in Paraurostyla weissei. In line 95, widening and concomitant overlapping of three organelle domains occurs at the right ventro-lateral part of the cell, in addition to the multi-left-marginal syndrome involving broadening of three organellar domains in the left ventro-lateral part of the cell (Dubielecka and Jerka-Dziadosz 1989). The modifications in positioning of ciliary primordia pervade both left and right parts of the cell, suggesting that the cell's ability to maintain discrete boundary conditions is affected. The way in which particular primordia of ciliature develop is consistent with an intercalation-overlapping model that postulates a cyclic instability of the system of positional information caused by a change in distribution of the ventral positional values and intercalation of intermediate values at the lateral parts of the cell. The developmental phenotype is a result of the ability of the morphogenetic machinery of the cell to record different phases of the postulated positional cycle.

Cytoskeleton-related structures in tetrahymena thermophila: microfilaments at the apical and division-furrow rings.

A ring consisting of microfilaments was found in the apical region of Tetrahymena thermophila wild-type strain B and janus mutant. This ring, about 0.4 micrometer wide and 0.2 micrometer thick, is located at the bases of the anterior, non-ciliated basal bodies of the apical ciliary couplets. The apical ring is made of fine filaments showing a banded pattern, the distance between bands depending on the fixation procedure and ranging from 30-200 nm. The bands are made of small beads fastened to the filaments. The microfilaments of the apical ring are attached to the bases of the basel bodies. No connection with the cell membrane was found. In dividing cells in the incipient furrow region of filamentous band originates from the epiplasmic fibrogranular meshwork. This contractile ring is about 0.4 micrometer wide and 0.8 micrometer thick. It is formed by circumferentially aligned microfibrils. During constriction the contractile ring remains associated with the epiplasmic layer, which in turn adheres to the inner alveolar membrane. The microfilaments of both the apical and the division-furrow rings have diameters ranging from about 3.8-7.I nm.

Ultrastructural aspect of size dependent regulation of surface pattern of complex ciliary organelle in a protozoan ciliate.

A regulation is shown for size and number of the elements of complex ciliary structures forming the oral apparatus (OA) of a ciliate Paraurostyla weissei. Morphometric investigations were performed on oral ciliature of normal and size-reduced cells. Those constituents of the OA which exist as single structures, such as the inner and outer preoral membranelles, are not eliminated. Both shorten and the outer membranelle becomes narrower. Within the adornal zone of membranelles in size-reduced cells some frontal and ventral membranelles become eliminated, whereas the respective ratio of these types remain size invariant. In each individual adoral zone of membranelles there are membranelles of different length specifically located along the ventral part. Membranelles from small cells are significantly smaller than those of normal cells. The number of kinetosomes is reduced in all four rows constructing an adoral membranelle. The analysis showed that regardless of cell size, the number of kinetosomes in the two inner rows of a membranelle is linearly and proportionately related. Regulation of the size of all components of the oral ciliature in P. weissei occurs at the time when the primordia of oral ciliature are formed. The results are discussed in relation to recent ideas about pattern formation and size dependent regulation of the number and size of pattern elements.

A mutant of Tetrahymena thermophila with a partial mirror-image duplication of cell surface pattern. I. Analysis of the phenotype.

Cells of a mutant clone, CU-127, of Tetrahymena thermophila (formerly T. pyriformis, syngen 1) manifest three anatomical abnormalities. First, the stable number of ciliary meridians is 21-25, above the usual number (17-21) in this species. Second, up to 30% of the cells have two oral apparatuses (OAs), one normal and the other abnormal. Third, more than one-half of the cells possess two distinct sets of contractile vacuole pores (CVPs). In some living cells two contractile vacuoles are seen. These abnormalities have persisted unchanged during more than 500 generations of vegetative propagation, and are similarly expressed in subclones. The normal and abnormal OAs are topographically segregated, with normal OAs developing along the "primary oral axis" and abnormal OAs developing along a "secondary oral axis" that is situated 170 degrees of the cell circumference to the cell's right of the primary oral axis. CVPs always appear within this 170 degree arc and never within the complementary 190 degrees arc to the left of the primary oral axis. A unique feature of the CU-127 clone is the commonly expressed mirror image reversal of the structural pattern of OAs that develop along the secondary oral axis. The primordia of such OAs initially appear (as usual) to the cell's left of a ciliary meridian, but as membranelles develop the frequently come to be oriented in a mirror image of the normal pattern, and an undulating membrane sometimes develops on the wrong (left) side of the oral primordium. When two sets of CVPs are formed, their average positions are roughly equidistant with respect to the two oral axes, with the two sets located 50-60 degrees to the right and left respectively of the primary and secondary oral axis. Such cells are thus bilaterally symmetrical about a plane defined by the central longitudinal axis and the halfway point between the two CVP sets (see Fig. 25). This plane bisects the cell into a normal and a "reversed" half-cell. However, only oral asymmetry and large-scale CVP positioning are subject to such reversal; all ciliary meridians remain of normal asymmetry and all CVPs are situated on the left side of CVP meridians. The fact that major aspects of large-scale cellular organization can be reversed while the "fine-positioning" associated with the ciliary meridians remains normal indicates that the two aspects of cell organization are distinct.