Acquisition of cell polarity during cell cycle and oral replacement in Tetrahymena

The aim of this study was to search for a mechanism responsible for the acquisition of cell polarity in a ciliate Tetrahymena. Homologs of the mammalian genes coding for CDC42-GSK3beta- MARK/PAR1-MAPs proteins were found in the Tetrahymena genome (Eisen et al., 2006, and this study). These proteins belong to a pathway which controls assembly and disassembly of microtubule bundles and cell polarity in neural cells. In Tetrahymena, there are two types of morphogenesis: divisional and oral replacement (OR). In divisional morphogenesis, an elongation of longitudinal microtubule bundles (LMs) takes place during cell division. In contrast, in OR type morphogenesis, which occurs in starved non-dividing cells, a polar retraction of LMs occurs. In T. pyriformis, the frequency of developmental switch to OR morphogenesis increases in the presence of wortmannin, an inhibitor of the CDC42-GSK3beta-MARK pathway. In contrast, wortmannin when applied to dividing cells does not affect divisional morphogenesis. Using immunostaining with the antibody against mammalian mitotic phosphoproteins (MPM-2) we show that these proteins co-localize with the LMs and are distributed along the anterior-posterior gradient. In addition, we show that during OR type morphogenesis, the fate of LMs correlates with the anterior-posterior gradient of instability of the cortical structures. We used the conditional mouth-less mutant of T. thermophila (Tiedtke et al., 1988) to test if the presence of the oral apparatus is required for the maintenance of cell polarity. We discuss our results in relation to the hypothesis of GSK3-beta-MARK pathway involvement in the acquisition of cell polarity in Tetrahymena.     

Tetrahymena 14-nm filament-forming protein has citrate synthase activity

The Tetrahymena 14-nm filament-forming protein (49K protein) is a structural protein involved in oral morphogenesis and in pronuclear behavior during conjugation. Cloning the 49K protein gene from a Tetrahymena thermophila cDNA library, we found that its primary structure exhibits a high sequence identity (51.5%) with porcine heart citrate synthase and retains functional domains. The 49K protein actually possesses citrate synthase activity, and is detected in mitochondria. These results suggest that the 49K protein has dual functions as both a respiratory enzyme and a structural protein in the cytoskeleton.     

Localization of a new fiber-forming protein within Tetrahymena pyriformis

As a step to study the biological function of a new protein, fiber-forming protein-38,000 (FFP-38), which was previously isolated from Tetrahymena pyriformis, its intracellular localization was investigated by using antiserum specific for FFP-38. Double immunodiffusion tests revealed that the anti-FFP-38 serum reacted with the protein(s) in isolated organelles such as oral apparatus, pellicles and mitochondria, to give rise to a precipitin line confluent with the line formed between the antiserum and the FFP-38. Furthermore, indirect fluorescent antibody staining showed that fluorescence was intensely localized in the oral apparatus and faintly distributed throughout the cytoplasm. In particular, distinct fluorescence was found along the division furrow of dividing Tetrahymena.     

Localization of Tubulin and a Centrin-Homologue in Vegetative Cells and Developing Gametangia of Ectocarpus siliculosus (Dillw.) Lyngb. (Phaeophyceae,Ectocarpales)

The distribution of tubulin and centrin in vegetative cells and during gametogenesis of Ectocarpus siliculosus was studied by immunofluorescence. In interphase cells bundles of microtubules are focused on the centriolar region near the nuclear surface. Some of the bundles ensheath the nucleus while others traverse the cytoplasm in various directions, sometimes reaching the cell cortex. Evaluation of serial optical sections by confocal laser scanning microscopy (CLSM) revealed that the perinuclear and “cytoplasmic” microtubule bundles presumably constitute a single complex. In interphase cells centrin is localized as a single bright spot in the centriolar region. In dividing cells duplication and separation of the microtubular complex and the centrin spot takes place. In post-mitotic cells with two nuclei, the centrioles are located at opposite cell poles, short microtubule bundles emanate from them and partially encompass the nucleus. During gametogenesis a gradual transformation of the vegetative cytoskeleton to the gametic flagellar apparatus occurs.     

The oral apparatus of Tetrahymena pyriformis, mating type 1, variety 1. I. Solubilization and electrophoretic separation of oral apparatus proteins

The protein composition of the oral apparatus of Tetrahymena pyriformis has been investigated with urea-acrylamide gel electrophoresis. The data show that the oral apparatus is composed of numerous proteins, two of which appear to be microtubule proteins (tubulins 1 and 2).     

Tetrahymena actin: localization and possible biological roles of actin in Tetrahymena cells

Though actin is ubiquitous in eukaryotes, its existence has not been clearly proven in Tetrahymena. Recently, we have succeeded in cloning and sequencing the Tetrahymena actin gene using a Dictyostelium actin probe (Hirono, M. et al. (1987) J. Mol. Biol. 194, 181-192). The primary structure of the Tetrahymena actin deduced from the nucleotide sequence of its gene is greatly divergent from those of other known actins, making it necessary to ascertain whether the predicted Tetrahymena actin is indeed an actin. In this paper, we investigated the localization of the predicted Tetrahymena actin by an immunofluorescence technique using antibody against its synthetic N-terminal peptide, in order to elucidate its possible biological roles. The results showed that immunofluorescence was localized in the division furrow of the dividing cell, and in the intranuclear filament bundles formed in cells exposed to heat shock or DMSO. In addition, the oral apparatus and the proximity of the cytoproct, which are organelles involved in endocytosis and exocytosis, respectively, also fluoresced. Thus, we conclude that the Tetrahymena actin we identified is indeed an actin and plays the same biological roles as ubiquitous actins do, although it is considerably divergent in its amino acid sequence.     

Oral filament proteins and their regulation in Tetrahymena pyriformis

Two proteins from the Triton X-100-insoluble fraction of Tetrahymena pyriformis have been isolated and shown by immunological methods to be major components of a pervasive system of filaments localized within the oral apparatus. These proteins, OF-1 and OF-2, have apparent molecular weights (MWapp) in polyacrylamide gels of 87,000 and 80,000 D, respectively. Peptide maps obtained and the absence of immunological cross-reactivity suggest that these proteins are not closely related to each other. Indirect immunofluorescence studies on dividing cells have shown that the oral filament system forms late in the cell cycle. The filaments appeared first after the basal bodies in the oral primordium had organized into groups and the fission furrow had begun to form. Dedifferentiation of the oral filament system in the anterior (old) oral apparatus was also observed at this point in the cell cycle. Following this, the oral filament systems in both old and new oral apparatuses completed development synchronously. Proteins showing antigenic similarity to OF-1 were found in a number of other cell types. Tests with heterologous antisera failed to demonstrate a relationship between vertebrate cytoskeletal proteins and the oral filament proteins of Tetrahymena.     

Tetrahymena elongation factor-1 alpha is localized with calmodulin in the division furrow

Translation elongation factor 1 alpha (EF-1 alpha) catalyzes the GTP-dependent binding of amino-acyl-tRNA to ribosomes. We previously reported that Tetrahymena EF-1 alpha induced the formation of bundles of rabbit skeletal muscle filamentous actin (F-actin) as well as Tetrahymena F-actin [Kurasawa et al. (1996) Zool. Sci. (Tokyo) 13, 371-375], and that Ca(2+)/calmodulin (CaM) regulated the F-actin-bundling activity of EF-1 alpha [Kurasawa et al. (1996) J. Biochem. 119, 791-798]. In the present study, we investigated the binding between Tetrahymena EF-1 alpha and CaM using a Tetrahymena EF-1 alpha affinity column, and the localization of EF-1 alpha and CaM by indirect immunofluorescence. Only CaM in the Tetrahymena cell extract bound to Tetrahymena EF-1 alpha in a Ca(2+)-dependent manner. In interphase Tetrahymena cells, EF-1 alpha and CaM are colocalized in the crescent structure of the oral apparatus and the apical ring, while in dividing cells, they are colocalized in the division furrow. This is the first report describing the coexistence of EF-1 alpha and CaM in the division furrow, suggesting that EF-1 alpha and CaM are involved in the organization of contractile ring microfilaments during cytokinesis.     

Cortical morphogenesis in Paramecium: a transcellular wave of protein phosphorylation involved in ciliary rootlet disassembly

In Paramecium, the morphogenesis of the cortex at cell division, which assures reconstruction of shape and surface pattern, has been shown to involve transcellular signals which spread across the cortex like a wave, originating principally from the oral apparatus. One of the events these signals control is the reorganization of the ciliary rootlets through a cycle of regression and regrowth. The ciliary rootlets are nucleated on the ciliary basal bodies and form a scaffold extending over the entire cell surface that is important in aligning the basal bodies and the unit territories organized around them in longitudinal rows. We present evidence that the mechanism underlying their reorganization is cell-cycle-dependent phosphorylation of the structural proteins which compose the ciliary rootlets. We have isolated the rootlets and prepared a polyclonal antibody against them. In situ immunofluorescence of dividing cells with the anti rootlet antibody, and with the monoclonal antibody MPM-2 specific for phosphoproteins shows that a wave of phosphorylation of the ciliary rootlets spreads across the cell at division and just precedes their regression. Two-dimensional Western blot analysis of cytoskeleton and isolated rootlets along with alkaline phosphatase treatment demonstrates that the rootlets are composed of phosphoproteins, while experiments with interphase and dividing cells provide direct evidence that hyperphosphorylation of these proteins at division brings about disassembly of the structure.     

慈姑根尖细胞微管骨架的免疫荧光观察

分别用考马斯亮蓝染色和间接免疫荧光标记,并运用荧光倒置显微镜和激光共聚焦显微镜,对慈姑根尖固定后酶解获得的去壁细胞和细胞团块以及根尖细胞分裂周期中微管骨架列阵进行详细观察,以探索高等植物微管周期的普遍性。结果表明:慈姑根尖固定后酶解可获得大量结构完整的去壁细胞与细胞团块;考马斯亮蓝染色观察可见,慈姑根尖细胞中丰富的蛋白物质以及处于不同分裂期的细胞核染色体;免疫荧光观察可见,慈姑根尖细胞周期中微管骨架保存较好,主要有周质微管、早前期带微管、纺缍体微管和成膜体微管4种循序变化的排列方式,构成了高等水生植物分裂细胞中典型的微管周期。实验结果证明,高等水生植物与陆生植物微管周期具有相似性,为植物微管周期概念提供了新的实例。     

Confocal Microscopy of Microtubule Cytoskeleton in the Pollen Protoplast of Narcissus

Pollen protoplasts were isolated from the mature pollen grains of Narcissus cyclamineus using cellulase Onozuka'R-10 and pectinase in Bs medium. The microtubule cytoskeleton in the pollen protoplasts was studied using immunofluorescence and confocal microscopy. In the cortical region there was a very complex microtubule network. The network contained numerous whirl-like arrays. The microtubule bundles in the whirl-like arrays were connected with each other by smaller bundles indicating that the arrangement of the whirl-like bundles were quite well organized and not at random. From the cortex to the centre of the protoplast another microtubule network having a structure different from the one in the cortical region was present. This network was much loosely packed than the cortical network. The arrangement of the microtubule bundles near the vegetative nucleus was again different. Numerous granules appeared outside the nuclear membrane. From these granules microtubule bundles radiated towards the cytoplasm. The arrangement of the microtubule network around the generative cell showed no specialized features. But inside the cell three types of microtubule arrays were present. 1. parallel arrays, 2. network, and 3. a mixture of the two. In the bursted pollen protoplast (as a result of osmotic shock treatment )some microtubule bundles could still be found attached to the ghost. The microtubule bundles associated with the ghost were much fragmented. But some still retained their branches and junctions. In the dry cleaved samples,a number of organelles still remained attached to the membrane and they included : microtubules, microfilaments, coated vesicles, endoplasmic reticulum and numerous honey-comb-like apparatus. The honey-comb-like apparatus was named as coated pits by Traas (1984). But we feel that it is more appropriate to call this organelle the honey-comb apparatus and we also believe that this organelle may be involved in microtubule and/or microfilament organization.     

Localization of a kinesin-like protein in generative cells of tobacco

Summary We have obtained immunofluorescence and immunoblot evidence for the presence of kinesin-like protein (KLP) in pollen tubes of tobacco using an antibody generated against peptides encoded by theKATA gene ofArabidopsis. This antibody recognizes an M_r 140,000 polypeptide inArabidopsis seedlings, and stains the mitotic apparatus in this species as well as in tobacco suspension cells. In tobacco pollen tubes prepared for dual immunofluorescence localizations of KLP and -tubulin, the antibody binds transiently to microtubule (Mt) bundles and the nucleus in premitotic generative cells; it then stains the developing mitotic apparatus as the nuclear envelope breaks down. By metaphase, fluorescence is located over kinetochore fibers and associated Mts. Localization of KLP is concentrated in the midzone during anaphase, and by early cytokinesis, it closely brackets the cell plate. Phragmoplast fluorescence then spreads along the phragmoplast distal to the cell plate. Punctate staining is also detected along vegetative Mts. No KLP localization is seen in pollen tubes treated with antibody after it had been preadsorbed to the antigenic peptides. The antibody recognizes an M_r 110,000 polypeptide in extracts of tobacco pollen tubes, and a polypeptide of somewhat lower M_r inTradescantia pollen tubes. Our results show that KLP(s) related to KatAp are present in tobacco generative cells and may play roles in the organization and/or operation of the mitotic apparatus and phragmoplast.Abbreviations KLP kinesin-like protein - Mt microtubule - MA mitotic apparatus Dedicated to Professor Eldon H. Newcomb in recognition of his contributions to cell biology     

A high molecular weight actin binding protein: Its localization in the cortex of the sea urchin egg

From the Triton-treated cortex fraction of sea urchin eggs, a high molecular weight actin binding protein (260K protein) was solubilized by a high salt solution and purified. A cosedimentation assay revealed that the 260K protein binds to actin filaments in a concentration-dependent manner. The low-shear viscosity of actin solutions largely increased in a concentration-dependent manner after addition of 260K protein. Electron microscopy showed that this protein induces the formation of large curled bundles of actin filaments. Different from fascin-induced actin bundles, no clear striations were observed within the actin bundles formed by the 260K protein. Antibodies against the 260K protein were raised in a rabbit and affinity purified. Immunoblotting analysis of Triton-solubilized cortex and various subcellular fractions showed that first only a single band reacted with the antibody and second that the 260K protein exclusively localized in the cortex fraction. Indirect immunofluorescence microscopy localized the protein in the cortex and the region of the cleavage furrow. After double staining, the fluorescence images for actin filaments and the 260K protein well correlate with each other.     

The cotton kinesin-like calmodulin-binding protein associates with cortical microtubules in cotton fibers

Microtubules in interphase plant cells form a cortical array, which is critical for plant cell morphogenesis. Genetic studies imply that the minus end-directed microtubule motor kinesin-like calmodulin-binding protein (KCBP) plays a role in trichome morphogenesis in Arabidopsis. However, it was not clear whether this motor interacted with interphase microtubules. In cotton (Gossypium hirsutum) fibers, cortical microtubules undergo dramatic reorganization during fiber development. In this study, cDNA clones of the cotton KCBP homolog GhKCBP were isolated from a cotton fiber-specific cDNA library. During cotton fiber development from 10 to 21 DPA, the GhKCBP protein level gradually decreases. By immunofluorescence, GhKCBP was detected as puncta along cortical microtubules in fiber cells of different developmental stages. Thus our results provide evidence that GhKCBP plays a role in interphase cell growth likely by interacting with cortical microtubules. In contrast to fibers, in dividing cells of cotton, GhKCBP localized to the nucleus, the microtubule preprophase band, mitotic spindle, and the phragmoplast. Therefore KCBP likely exerts multiple roles in cell division and cell growth in flowering plants.     

Cloning,localization, and axonemal function of Tetrahymena centrin

Centrin, an EF hand Ca(2+) binding protein, has been cloned in Tetrahymena thermophila. It is a 167 amino acid protein of 19.4 kDa with a unique N-terminal region, coded by a single gene containing an 85-base pair intron. It has > 80% homology to other centrins and high homology to Tetrahymena EF hand proteins calmodulin, TCBP23, and TCBP25. Specific cellular localizations of the closely related Tetrahymena EF hand proteins are different from centrin. Centrin is localized to basal bodies, cortical fibers in oral apparatus and ciliary rootlets, the apical filament ring and to inner arm (14S) dynein (IAD) along the ciliary axoneme. The function of centrin in Ca(2+) control of IAD activity was explored using in vitro microtubule (MT) motility assays. Ca(2+) or the Ca(2+)-mimicking peptide CALP1, which binds EF hand proteins in the absence of Ca(2+), increased MT sliding velocity. Antibodies to centrin abrogated this increase. This is the first demonstration of a specific centrin function associated with axonemal dynein. It suggests that centrin is a key regulatory protein for Tetrahymena axonemal Ca(2+) responses, including ciliary reversal or chemotaxis.     

Demonstration of the cell cycle positions of taxol-induced "asters" and "bundles" by sequential measurements of tubulin immunofluorescence, DNA content, and autoradiographic labeling of taxol-sensitive and -resistant cells

We used reliable and relatively inexpensive equipment to make sequential sets of measurements of antitubulin immunofluorescence, Feulgen staining, and autoradiography on the same cells. This was done to evaluate tubulin conformations, DNA content, and [3H]-thymidine incorporation in cell lines sensitive (HL60) and resistant (K562) to the novel anti-tubulin chemotherapeutic agent taxol. Numbers of cells with microtubule bundles have been found to correlate with sensitivity to taxol by clonogenic assay for several leukemic cell lines. We have found that cells with "asters" produced by taxol exposure are in mitosis and that cells with taxol-induced "bundles" are in G0/G1, S, and G2 phases. We further found that S-phase cells with microtubule bundles in both sensitive (HL60) and resistant (K562) cell lines were able to incorporate [3H]-thymidine after 4-hr exposure to taxol. As microtubule bundles and asters occur in cells of the same cell cycle phases in both lines, we conclude that the greater frequency of cells with microtubule bundles reported for sensitive cells after taxol treatment cannot result from drug exclusion nor from different effects of the drug on cell microtubules in these two leukemic lines.     

Properties of microtubule proteins in different organelles in Tetrahymena pyriformis

Attempts were made to elucidate whether or not microtubules within cilia, oral apparatus and macronuclei in Tetrahymena pyriformis include common proteins, by making use of antiserum to microtubule proteins of cilia. The microtubule fraction containing two protein components was used as antigen and the antiserum to the microtubule proteins was proved to be specific by analysing electrophoretic patterns in the antigen absorption experiments. The antiserum reacted with the dissolved proteins of isolated oral apparatus or macronuclei, forming precipitin lines common to those of cilia. Furthermore, the two organelles were positively stained with the fluorescein-labelled antiserum. These results offered important clues to understand multifariousness in function and behavior of morphologically identical microtubules; that is, various microtubules in the cell appear to include a common protein(s) one another.     

Golgi 58K-like protein in pollens and pollen tubes ofLilium davidii

In animal cells, Golgi apparatus is located near the microtubule organizing center (MTOC) and its position is determined partly by 58K protein. By sodium dodecyl-sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immuno-blotting methods, a 58K-like protein has been found in pollen grains and pollen tubes of Lilium davidii. Its molecular weight is very similar to that of the 58K protein of animal cells. By immunofluorescence labeling, under a confocal laser scanning microscope (CLSM), the animal 58K antibody revealed a punctate staining in pollen grains and pollen tubes, which is consistent with the distribution of Golgi apparatus in plant cells. In addition, immuno-gold labeling and transmission electron microscopy showed that the 58K-like protein bound mainly to the membrane of vesicles-like structure near Golgi apparatus. This is the first demonstration of the 58K-like protein in plant cells.     

Golgi 58K-like protein in pollens and pollen tubes of Lilium davidii

In animal cells, Golgi apparatus is located near the microtubule organizing center (MTOC) and its position is determined partly by 58K protein. By sodium dodecyl-sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immuno-blotting methods, a 58K-like protein has been found in pollen grains and pollen tubes of Lilium davidii. Its molecular weight is very similar to that of the 58K protein of animal cells. By immunofluorescence labeling, under a confocal laser scanning microscope (CLSM), the animal 58K antibody revealed a punctate staining in pollen grains and pollen tubes, which is consistent with the distribution of Golgi apparatus in plant cells. In addition, immuno-gold labeling and transmission electron microscopy showed that the 58K-like protein bound mainly to the membrane of vesicles-like structure near Golgi apparatus. This is the first demonstration of the 58K-like protein in plant cells.     

Involvement of 14-nm Filament-Forming Protein and Tubulin in Gametic Pronuclear Behavior during Conjugation in Tetrahymena

We have studied in detail the immunofluorescence localizations of Tetrahymena 14-nm filament-forming protein (49-kDa protein) in relation to tubulin in conjugating wild-type Tetrahymena thermophila (B strain) pairs and in pairs between B strain and star strains with defective micronuclei. The results suggest that germ nuclear behavior during conjugation may involve the following cytoskeletal structures: (1) during meiosis, microtubule structures are involved in micronuclear elongation and meiotic division; (2) at the postmeiotic stage, 49-kDa protein network structures that are formed independently of the existence of pronuclei are involved in the selection and the survival of one of four meiotic products; (3) during the third prezygotic division, gametic pronuclear transfer, and zygote formation, a cytoskeletal structure in which the 49-kDa protein colocalizes with microtubules and which is dependent on the existence of a normal gametic pronucleus is involved in gametic pronuclear behavior, and (4) during the postzygotic divisions, the microtubules are involved in nuclear behavior.