The oral apparatus of Tetrahymena pyriformis, strain WH-6. IV. Observations on the organization of microtubules and filaments in the isolated oral apparatus and the differential effect of potassium chloride on the stability of oral apparatus microtubules.

This report is an ultrastructural analysis of the organization of the isolated oral apparatus of Tetrahymena pyriformis, strain WH-6, syngen 1. Attention has been focused on the organization of microtubules and filaments in oral apparatus membranelles. Oral apparatus membranellar basal bodies were characterized with respect to structural differentiations at the distal and proximal ends. The distal region of membranellar basal bodies contains the basal plate, accessory microtubules and filaments. The proximal end contains a dense material from which emanate accessory microtubules and filaments. There are at least two possibly three different arrangements of accessory structures at the proximal end of membranellar basal bodies. All membranellar basal bodies appear to have a dense material at the proximal end from which filaments emanate. Some of these basal bodies have accessory microtubules and filaments emanating from this dense material. A possible third arrangement is represented by basal bodies which have lateral projections, from the proximal end, of accessory microtubules and filaments which constitute cross or peripheral connectives. There are at least three examples of direct associations between oral apparatus microtubules and filaments: (1) filaments which form links between basal body triplet microtubules, (2) filaments which link the material of the basal plate to internal basal body microtubules, (3) filaments which link together microtubule bundles from membranellar connectives. KCl extraction of the isolated oral apparatus resulted in the selective solubilization of oral apparatus basal bodies, remnants of ciliary axonemes and fused basal plates. Based on their response to KCl extraction two distinct sets of morphologically similar micro tubules can be identified: (a) microtubules which constitute the internal structure of basal bodies and ciliary axonemes, (b) microtubules which constitute the fiber connectives between basal bodies.     

A novel rapid oral bacteria detection apparatus for effective oral care to prevent pneumonia

doi: 10.1111/j.1741‐2358.2011.00517.x A novel rapid oral bacteria detection apparatus for effective oral care to prevent pneumonia Objective: To clarify the oral environment, we evaluated the usefulness and clinical applicability of a new apparatus developed for the simple and rapid quantification of oral bacteria. Background: Professional oral health care can reduce the number of oral bacteria and days of fever and inhibit the development of pneumonia. A novel detection apparatus was developed by applying the dielectrophoretic impedance measurement method. Methods: First, to determine the accuracy of this apparatus, employing standard samples of Escherichia coli. Next, to evaluate the oral environment, samples were taken from the tongue in elderly (mean age: 86.6 years) in nursing home. Results: In the first study, a good correlation was observed between the two methods (R = 0.999). In the second study, there were significant correlations between measurement values obtained using this apparatus and those obtained by the culture method (R = 0.852), as well as those obtained by the FM method (R = 0.885). Conclusion: Our data showed that this rapid oral bacterial detection apparatus is effective in evaluating the oral hygiene to prevent pneumonia in the elderly.     

THE SYNTHESIS OF MICROTUBULE AND OTHER PROTEINS OF THE ORAL APPARATUS IN TETRAHYMENA PYRIFORMIS

Several proteins, including microtubule proteins, have been isolated from the oral apparatus of the ciliate Tetrahymena. The synthesis of these proteins has been studied in relation to formation of this organelle system by the cell. Electron microscopy has shown that the isolated oral apparatus consists primarily of basal bodies, pellicular membranes, and a system of subpellicular microtubules and filaments. Cilia were removed during the isolation; therefore none of the proteins studied was from these structures. Evidence was obtained from the study of total oral apparatus protein which indicates that at least some of the proteins involved in formation of this organelle system may be synthesized and stored in the cytoplasm for use over long periods. This pattern of regulation was found for three individual proteins isolated from the oral apparatus fraction after extraction with a phenol-acetic acid solvent. A different pattern of regulation was found for microtubule proteins isolated from the oral apparatus of Tetrahymena. The data suggest that microtubule proteins, at least in logarithmically growing cells, are not stored in a cytoplasmic pool but are synthesized in the same cell cycle in which they are assembled into oral structures.     

Three-dimensional reconstruction of Paramecium primaurelia oral apparatus through confocal laser scanning optical microscopy

The oral apparatus of the ciliate protozoan Paramecium primaurelia, a single-celled eukaryotic organism, is a highly organized structure whose arrangement is of important taxonomic, phylogenetic and developmental significance. This paper analyses oral structures by means of a confocal laser scanning optical microscope (CLSM), which allows their three-dimensional visualization and measurement. The extraction of the intrinsic three-dimensional information related to the biological objects under investigation can in turn be related to their functional state, according to the classical paradigms of structure to function relationship identification. In our experiments, we acquired different data sets. These are optical slices of the biological sample under investigation, acquired in a confocal situation, through epi-illumination, in reflection. For comparison with conventional microscopy, two-dimensional images were acquired via a standard TV camera coupled to the microscope itself. The volumes obtained by piling up the slices were rendered through different techniques, some of them directly implemented on the workstation controlling the CLSM system, some of them on a SUN SPARCstation 1, where the original data were transferred via an Ethernet link. In this last instance, original software has been developed for the visualization and animation of the three-dimensional structures, under UNIX and X-Window, according to a ray-tracing algorithm.     

A putative novel protein,DEPDC1B,is overexpressed in oral cancer patients,and enhanced anchorage-independent growth in oral cancer cells that is mediated by Rac1 and ERK

Background

The DEP domain is a globular domain containing approximately 90 amino acids, which was first discovered in 3 proteins: Drosophila disheveled, Caenorhabditis elegans EGL-10, and mammalian Pleckstrin; hence the term, DEP. DEPDC1B is categorized as a potential Rho GTPase-activating protein. The function of the DEP domain in signal transduction pathways is not fully understood. The DEPDC1B protein exhibits the characteristic features of a signaling protein, and contains 2 conserved domains (DEP and RhoGAP) that are involved in Rho GTPase signaling. Small GTPases, such as Rac, CDC42, and Rho, regulate a multitude of cell events, including cell motility, growth, differentiation, cytoskeletal reorganization and cell cycle progression.

Results

In this study, we found that it was a guanine nucleotide exchange factor and induced both cell migration in a cultured embryonic fibroblast cell line and cell invasion in cancer cell lines; moreover, it was observed to promote anchorage-independent growth in oral cancer cells. We also demonstrated that DEPDC1B plays a role in regulating Rac1 translocated onto cell membranes, suggesting that DEPDC1B exerts a biological function by regulating Rac1. We examined oral cancer tissue; 6 out of 7 oral cancer tissue test samples overexpressed DEPDC1B proteins, compared with normal adjacent tissue.

Conclusions

DEPDC1B was a guanine nucleotide exchange factor and induced both cell migration in a cultured embryonic fibroblast cell line and cell invasion in cancer cell lines; moreover, it was observed to promote anchorage-independent growth in oral cancer cells. We also demonstrated that DEPDC1B exerts a biological function by regulating Rac1. We found that oral cancer samples overexpressed DEPDC1B proteins, compared with normal adjacent tissue. Suggest that DEPDC1B plays a role in the development of oral cancer. We revealed that proliferation was linked to a novel DEPDC1B-Rac1-ERK1/2 signaling axis in oral cancer cell lines.     

Different polypeptides in two homologous cellular structures: the microstomal and macrostomal oral apparatus of Tetrahymena vorax

By two-dimensional electrophoresis it is demonstrated that the macrostomal oral apparatus of Tetrahymena vorax contains about 55 unique polypeptides in addition to a set of about 145 polypeptides also found in the microstomal oral apparatus.     

Fine oral filaments in Paramecium: a biochemical and immunological analysis

In Paramecium, several kinds of the oral networks of fine filaments are defined at the ultrastructural level. Using the sodium chloride-treated oral apparatus of Paramecium as an antigen to produce monoclonal antibodies, we have begun to identify the proteins constituting these networks. Immunoblotting showed that all positive antibodies were directed against three bands (70-, 75-and 83-kD), which corresponded to quantitatively minor components of the antigen; there was no antibody specific for the quantitatively major components (58- and 62-kD). Immunolocalization with four of these antibodies directed against one or several of these three bands showed that these proteins are components of the fine filaments supporting the oral area; a decoration of the basal bodies and the outer lattice was also observed on the cortex. Immunofluorescence on interphase cells suggested that the three proteins colocalized on the left side of the oral apparatus, whereas only the 70-kD band was detected on the right side. During division, the antigens of the antibodies were detected at different stages after oral basal body assembly. The antibodies cross-reacted with the tetrins, which are oral filament-forming proteins in Tetrahymena, demonstrating that tetrin-related proteins are quantitatively minor components of the oral and the somatic cytoskeleton of Paramecium.     

The oral apparatus of tadpoles of Rana dalmatina, Bombina variegata, Bufo bufo, and Bufo viridis (Anura)

The morphology and development of the larval oral apparatus of Rana dalmatina, Bombina variegata, Bufo bufo, and Bufo viridis are described and compared using scanning electron microscopy. The species show different arrangements of the mouthparts. The small oral apparatus of R. dalmatina larvae has three labial tooth rows on the upper labium, while there are four tooth rows on the lower labium with a medial gap in row proximal to the mouth. The margins of the oral apparatus are defined by papillae that encircle the lower labium. B. variegata tadpoles have two upper labial tooth rows and three lower labial tooth rows that are uninterrupted, unlike the ones of R. dalmatina. The mouth is encircled by papillae that are larger than those of R. dalmatina. The oral discs of tadpoles of both B. bufo and B. viridis are similar. They are defined by two upper labial tooth rows (the second of which is interrupted by a medial gap) and by three lower tooth rows that differ in lengths in the two Bufo species. Both species develop papillae on the mouth angles and in two rows on the upper labium. Some morphological differences among the oral discs of R. dalmatina, B. variegata, B. bufo, and B. viridis tadpoles can be attributed to phylogenetic differences, but most can be related to their varying feeding habits and/or to their dietary specializations.     

Reduction in Golgi apparatus dimension in the absence of a residential protein,N-acetylglucosaminyltransferase V

Various proteins are involved in the generation and maintenance of the membrane complex known as the Golgi apparatus. We have used mutant Chinese hamster ovary (CHO) cell lines Lec4 and Lec4A lacking N-acetylglucosaminyltransferase V (GlcNAcT-V, MGAT5) activity and protein in the Golgi apparatus to study the effects of the absence of a single glycosyltransferase on the Golgi apparatus dimension. Quantification of immunofluorescence in serial confocal sections for Golgi α-mannosidase II and electron microscopic morphometry revealed a reduction in Golgi volume density up to 49 % in CHO Lec4 and CHO Lec4A cells compared to parental CHO cells. This reduction in Golgi volume density could be reversed by stable transfection of Lec4 cells with a cDNA encoding Mgat5. Inhibition of the synthesis of β1,6-branched N-glycans by swainsonine had no effect on Golgi volume density. In addition, no effect on Golgi volume density was observed in CHO Lec1 cells that contain enzymatically active GlcNAcT-V, but cannot synthesize β1,6-branched glycans due to an inactive GlcNAcT-I in their Golgi apparatus. These results indicate that it may be the absence of the GlcNAcT-V protein that is the determining factor in reducing Golgi volume density. No dimensional differences existed in cross-sectioned cisternal stacks between Lec4 and control CHO cells, but significantly reduced Golgi stack hits were observed in cross-sectioned Lec4 cells. Therefore, the Golgi apparatus dimensional change in Lec4 and Lec4A cells may be due to a compaction of the organelle.     

A simple preparative polyacrylamide disc gel electrophoresis apparatus: purification of three branched-chain amino acid binding proteins from Escherichia coli

The equipment used for preparative polyacrylamide gel electrophoresis has been either difficult to construct or costly if purchased commercially. An inexpensive preparative acrylamide gel apparatus and peristaltic pump are described in this paper which are easy to use and may be constructed from readily available materials. The construction of the preparative gel apparatus requires no special machining or glass blowing.This report describes the use of the disc gel apparatus in the final purification step of three binding proteins which appear to be involved in the transport of the branched-chain amino acids in Escherichia coli. Two of these proteins have been described previously (1–4). The apparatus has also been successfully used in a number of other laboratories for the purification of a variety of other proteins (5–9).     

A golgi apparatus associated with mating in Tetrahymena pyriformis

The classical Golgi apparatus has not been observed in the several strains of Tetrahymena pyriformis examined in this laboratory at the ultrastructural level when the ciliates are grown vegetatively. However, sexually active strains, when starved for the purpose of inducing conjugation, contain stacked saccules in the oral region. When such opposite mating types are mixed for mating, the stacked saccules become swollen at their ends and vesicles appear to pinch off from them. These bodies possess the configuration of the classical Golgi apparatus of other eucells. Vesicles seem to be formed from the saccules just prior to, and toward the end of conjugation, suggesting a relationship with the mating process.     

YIPF5 and YIF1A recycle between the ER and the Golgi apparatus and are involved in the maintenance of the Golgi structure

Yip1p/Yif1p family proteins are five-span transmembrane proteins localized in the Golgi apparatus and the ER. There are nine family members in humans, and YIPF5 and YIF1A are the human orthologs of budding yeast Yip1p and Yif1p, respectively. We raised antisera against YIPF5 and YIF1A and examined the localization of endogenous proteins in HeLa cells. Immunofluorescence, immunoelectron microscopy and subcellular fractionation analysis suggested that YIPF5 and YIF1A are not restricted to ER exit sites but also localized in the ER-Golgi intermediate compartment (ERGIC) and some in the cis-Golgi at steady state. Along with ERGIC53, YIPF5 and YIF1A remained in the cytoplasmic punctate structures after brefeldin A treatment, accumulated in the ERGIC and the cis-Golgi after treatment with AlF₄^- and accumulated in the ER when ER to Golgi transport was inhibited by Sar1(H79G). These results supported the localization of YIPF5 and YIF1A in the ERGIC and the cis-Golgi, and strongly suggested that they are recycling between the ER and the Golgi apparatus. Analysis by blue native PAGE and co-immunoprecipitation showed that YIPF5 and YIF1A form stable complexes of three different sizes. Interestingly, the knockdown of YIPF5 or YIF1A caused partial disassembly of the Golgi apparatus suggesting that YIPF5 and YIF1A are involved in the maintenance of the Golgi structure.     

The tip growth apparatus of Aspergillus nidulans

Hyphal tip growth in fungi is important because of the economic and medical importance of fungi, and because it may be a useful model for polarized growth in other organisms. We have investigated the central questions of the roles of cytoskeletal elements and of the precise sites of exocytosis and endocytosis at the growing hyphal tip by using the model fungus Aspergillus nidulans. Time-lapse imaging of fluorescent fusion proteins reveals a remarkably dynamic, but highly structured, tip growth apparatus. Live imaging of SYNA, a synaptobrevin homologue, and SECC, an exocyst component, reveals that vesicles accumulate in the Spitzenkörper (apical body) and fuse with the plasma membrane at the extreme apex of the hypha. SYNA is recycled from the plasma membrane by endocytosis at a collar of endocytic patches, 1–2 μm behind the apex of the hypha, that moves forward as the tip grows. Exocytosis and endocytosis are thus spatially coupled. Inhibitor studies, in combination with observations of fluorescent fusion proteins, reveal that actin functions in exocytosis and endocytosis at the tip and in holding the tip growth apparatus together. Microtubules are important for delivering vesicles to the tip area and for holding the tip growth apparatus in position.     

Organization and composition of the striated roots supporting the Golgi apparatus,the so-called parabasal apparatus,in parabasalid flagellates

Summary— In parabasalid flagellates, trichomonads and hypermastigids, the stack of cisternae of the Golgi apparatus are supported by striated roots attached to the basal bodies of flagella forming the so-called parabasal apparatus. Monoclonal antibodies raised for several trichomonad species, Monocercomonas, Trichomonas and Tetratrichomonas, label the parabasal fibre in immunofluorescence or immunogold staining and protein bands in immunoblotting. Several antibodies cross-react between trichomonad species, and one of them labels the homologous parabasal fibre in the hypermastigids: Trichonympha, Joenia, Pseudotrichonympha and Holomastigotoides. Considering the molecular mass range of the labelled proteins (100–135 kDa) and the lack of antibody cross-reactivity with the striated root proteins (centrin, assemblin, kinetodesmal protein, ciliary root proteins of epithelial ciliated cells) of other organisms, these proteins recognized by these antibodies seem to represent a new class of protein forming striated roots. The occurrence and significance of parabasal organization in eukaryogenesis is discussed.     

Control of development of the oral apparatus of Paramecium during sexual reproduction: an embryological perspective

This study shows that development of the new soma during sexual reproduction in ciliates can be conceptualized on the same basis as embryogenesis in multicellular organisms. In conjugating Paramecium, development of a new oral apparatus takes place during fertilization and the first three divisions of the zygotic nucleus and completes well before the postsexual cell undergoes the first cell fission. The control of oral development is analyzed by microsurgical removal of the zygotic nucleus or the postzygotic nuclei from conjugants. The enucleated exconjugants can pass through an early hurdle in oral development (the initiation of oral membranelle assembly) and subsequently develop an oral apparatus. Such oral apparatuses nevertheless exhibit structural and functional abnormalities including fragmentation and misalignment of oral membranelles, absence of the postoral microtubular bundle, reduction in the length of buccal cavity, and impaired phagocytosis. Other stomatogenic aspects, such as the arrangement of basal bodies in the oral membranelles, remain unaffected. The two groups of exconjugants, one derived from cells enucleated at the zygotic stage, and the other at the postzygotic stage, exhibit the same types of oral abnormality. We conclude that (i) the zygotic nucleus is not essential for the initiation of oral membranelle assembly. The existence of zygotic signals for subsequent oral development is not ruled out, but these are insufficient. (ii) Postzygotic nuclei, as well as maternal nuclei (the old somatic nucleus and meiotic derivatives of the germ nucleus), control oral development. This reveals a parallelism between postsexual development in ciliates and the early embryology of multicellular organisms, in their reliance on information provided by maternal, as well as early postzygotic nuclei. (iii) The activity of the old somatic nucleus alone is not sufficient for the later stages of oral development. Probably, some stomatogenic functions of the old somatic nucleus normally utilized for the later stages of oral development in binary fission are inactivated during sexual reproduction. Alternatively, the old somatic nucleus may rely on some critical conditions prescribed by the postzygotic nuclei in order to act.     

Abnormal micronuclear telomeres lead to an unusual cell cycle checkpoint and defects in Tetrahymena oral morphogenesis

Telomere mutants have been well studied with respect to telomerase and the role of telomere binding proteins, but they have not been used to explore how a downstream morphogenic event is related to the mutated telomeric DNA. We report that alterations at the telomeres can have profound consequences on organellar morphogenesis. Specifically, a telomerase RNA mutation termed ter1-43AA results in the loss of germ line micronuclear telomeres in the binucleate protozoan Tetrahymena thermophila. These cells also display a micronuclear mitotic arrest, characterized by an extreme delay in anaphase with an elongated, condensed chromatin and a mitotic spindle apparatus. This anaphase defect suggests telomere fusions and consequently a spindle rather than a DNA damage checkpoint. Most surprisingly, these mutants exhibit unique, dramatic defects in the formation of the cell's oral apparatus. We suggest that micronuclear telomere loss leads to a “dynamic pause” in the program of cortical development, which may reveal an unusual cell cycle checkpoint.     

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.     

A convenient apparatus and tracking dye for anaerobic analytical polyacrylamide-gel electrophoresis

An anaerobic modification of conventional polyscrylamide-gel electrophoretic equipment is described. The modified apparatus has been applied to the separation of Azotobacter vinelandii nitrogenase components and should prove useful in the analysis of other O₂-sensitive proteins. Electrophoresis in reducing gels can be followed with a dithionite-resistant tracking dye, potassium gualazulene-1-sulfonate.     

Involvement of Tetrahymena intermediate filament protein, a 49K protein, in the oral morphogenesis

To study the biological function of Tetrahymena intermediate-type filament protein (a 49K protein), we examined the immunofluorescence localization of 49K protein within Tetrahymena cells. The results showed that the immunofluorescence was localized in the oral apparatus, mitochondria and mucocysts. Among them, the fluorescence in the oral apparatus was of high interest in its unique region and vicissitude in the cell cycle: a tau-shaped region of the oral apparatus intensely fluoresced during interphase, but the fluorescence completely disappeared during dividing phase. The tau-shaped region corresponded to 'posterior connectives' and the root part of 'deep fiber', to the conjunction parts of microtubule bundles. In the those parts, there was electron-dense material in the microtubule bundles. Hence, it is conceivable that 49K protein corresponds to the dense material and has a function of microtubule bundle conjunction. On the other hand, disappearance of immunofluorescence from the old oral apparatus of most dividing cells reflected the oral apparatus regression and remodelling which have been known as necessary sequential events in the cell cycle. We observed that oral fluorescence disappeared concurrently with the onset of oral regression and of constriction of division furrow, whereas at a late dividing stage immunofluorescence began to appear simultaneously in both new and old oral apparatus. Thus, the 49K protein may play a crucial role(s) not only in the morphogenesis of oral primordia but also in the transient morphogenesis in the old oral system.