Relationship Between Spatial Pattern of Basal Bodies and Membrane Skeleton (Epiplasm) During the Cell Cycle of Tetrahymena: cdaA Mutant and Anti-Membrane Skeleton Immunostaining

Microtubular basal bodies and epiplasm (membrane skeleton) are the main components of the cortical skeleton of Tetrahymena. The aim of this report was to study functional interactions of basal bodies and epiplasm during the cell cycle. The cortex of Tetrahymena cells was stained with anti-epiplasm antibody. This staining produced a bright epiplasmic layer with a dark pattern of unstained microtubular structures. The fluorescence of the anti-epiplasm antibody disappeared at sites of newly formed microtubular structures, so the new basal body domains and epiplasmic layer could be followed throughout the cell cycle. Different patterns of deployment of new basal bodies were observed in early and advanced dividers. In advanced dividers the fluorescence of the epiplasmic layer diminished locally within the forming fission line where the polymerization of new basal bodies largely extincted. In wild type Tetrahymena, the completion of the micronuclear metaphase/anaphase transition was associated with a transition from the pattern of new basal body deployment and epiplasm staining of the early divider to the pattern of the advanced dividers. The signal for the fission line formation in Tetrahymena (absent in cdaA1 Tetrahymena mutationally arrested in cytokinesis) brings about 1) transition of patterns of deployment of basal bodies and epiplasmic layer on both sides of the fission line; and 2) coordination of cortical divisional morphogenesis with the micronuclear mitotic cycle.     

Inter-strain Variability of Structural Proteins in Tetrahymena*

SYNOPSIS The high molecular weight proteins found in isolated pellicles of Tetrahymena have been compared in several individual strains within the genus using SDS polyacrylamide gel electrophoresis. Three variants of the B-protein of epiplasm (MW 174,000; 155,000; 145,000) and 2 of the C-protein (MW 140,000; 122,000) were found among the strains examined. No variation was observed in the major kinetodesmal fiber protein (MW 250,000). The variation found between strains in the proteins of a structure which is (as far as we know) the same in all strains indicates a disjunction between evolutionary change at the 2 levels of organization. The taxonomic implications of the observed variation in structural proteins in Tetrahymena are discussed.     

Mitochondrial Associations with Specific Components of the Cortex of Tetrahymena thermophila Nanney & McCoy, 1976: Microtubules and the Epiplasm1

Ultrastructural observations of the cortically-located mitochondria of Tetrahymena thermophila revealed associations not only between the mitochondria and certain of the cortical microtubule bands, but also between the mitochondria and the epiplasm of the cortex. Most of the distal mitochondrial surface is close and parallel to the epiplasm; favorable views show bridge-like structures spanning the 20–10 nm gap between the mitochondrion and the epiplasm. Previous studies have shown that the placement of mitochondria in the cortex appears to be determined by certain of the cortical microtubule bands. This study, however, shows that mitochondrion-microtubule interactions account for only a small proportion of the total mitochondrial area associated with the cortex; the rest is accounted for by the epiplasm. A possible analogue of the spectrin layer of erythrocyte membranes, the epiplasm may be important in helping to arrange the intricately organized components of the ciliate cortex. Its involvement in apparently helping to “moor” mitochondria to their cortical sites is the first suggestion of any role in cell patterning played by the epiplasm.     

Solution NMR structures of the C‐domain of Tetrahymena cytoskeletal protein Tcb2 reveal distinct calcium‐induced structural rearrangements

Tcb2 is a calcium‐binding protein that localizes to the membrane‐associated skeleton of the ciliated protozoan Tetrahymena thermophila with hypothesized roles in ciliary movement, cell cortex signaling, and pronuclear exchange. Tcb2 has also been implicated in a unique calcium‐triggered, ATP‐independent type of contractility exhibited by filamentous networks isolated from the Tetrahymena cytoskeleton. To gain insight into Tcb2's structure‐function relationship and contractile properties, we determined solution NMR structures of its C‐terminal domain in the calcium‐free and calcium‐bound states. The overall architecture is similar to other calcium‐binding proteins, with paired EF‐hand calcium‐binding motifs. Comparison of the two structures reveals that Tcb2‐C's calcium‐induced conformational transition differs from the prototypical calcium sensor calmodulin, suggesting that the two proteins play distinct functional roles in Tetrahymena and likely have different mechanisms of target recognition. Future studies of the full‐length protein and the identification of Tcb2 cellular targets will help establish the molecular basis of Tcb2 function and its unique contractile properties. Proteins 2016; 84:1748–1756. © 2016 Wiley Periodicals, Inc.     

Tetrahymena pyriformis as a Model System for Membrane Studies*†

SYNOPSIS. Tetrahymena pyriformis is an exceptionally useful subject for studying metabolic interrelationships among intracellular membranes. Its advantages include the striking differences in lipid composition among the cell's various functionally distinct membrane systems, indicating a pronounced lipid specificity at the membrane sites. The magnitude of these differences permits analysis of the mechanisms underlying the specificity. Even more valuable is the unique physical isolation of ciliate surface membranes from the cytoplasm of the cell. In contrast to the almost immediate equilibration of newly made lipids with preexisting lipids found in most cells, Tetrahymena surface membranes have a lipid turnover slow enough to be conveniently analyzed. Finally, the well-studied responses of Tetrahymena to such physiological stresses as heat and starvation may be used to evaluate the effects of environmental factors on membrane formation.     

Localization of hormone receptors inTetrahymena

Summary It can be shown by light and electron microscopic autoradiography that¹²⁵I triiodothyronine and¹²⁵I thyroxine bind to the cell membrane ofTetrahymena. For thyroxine the finer localization is at the cilia as well as in pinocytotic and food vacuoles. Some grains also appear above the nucleus. On the other hand, insulin only binds to the membrane ofTetrahymena, and binding can be inhibited by unlabelled insulin.     

THE TETRAHYMENA HOMOLOG OF BACTERIAL AND MAMMALIAN 4-HYDROXYPHENYLPYRUVATE DIOXYGENASES LOCALIZES TO MEMBRANES OF THE ENDOPLASMIC RETICULUM

The expression and intracellular localization of the Tetrahymena homolog of 4-hydroxyphenylpyruvate dioxygenase (HPPD) were investigated in wild-type Tetrahymena thermophila strain B1868 VII and the mutant strains IIG8, defective in food vacuole formation, MS-1, blocked in secretion of lysosomal enzymes, and SB 281, defective in mucocyst maturation. Immunoelectron microscopy and confocal laser scanning microscopy demonstrated that Tetrahymena HPPD primarily localized to membranes of the endoplasmic reticulum. In addition, Tetrahymena HPPD was detected in association with membranes of the Golgi apparatus, and transport vesicles in exponentially growing wild-type and mutant strains. In starved cells, Tetrahymena HPPD localized exclusively to membranes of small vesicles. Since no de novo synthesis ofTetrahymena HPPD takes place in cells starved for more than 30min, these results suggest that there is a flow ofTetrahymena HPPD from the endoplasmic reticulum to small vesicles, possibly via the Golgi apparatus, and thatTetrahymena HPPD contains a signal for vesicle membrane retrieval or retention.     

Probing the adaptive response of Escherichia coli to extracellular Zn(II)

The adaptive response of Escherichia coli cells to differing intracellular and extracellular Zn(II) concentrations was evaluated by two-dimensional gel electrophoresis and peptide identifications. Twenty-one Zn(II)-responsive proteins, which were previously not known to be associated with Zn(II), were identified. Most of the proteins were related to cellular metabolism and include membrane transporters and glycolytic and TCA-associated enzymes. The expression levels of no known Zn(II) transporters were identified with these studies. The results of these studies suggest a role of Zn(II) in the expression levels of several E. coli proteins, and the results are discussed in light of recent genomic profiling studies on the adaptive response of E. coli cells to stress by Zn(II) excess.     

Cytoskeletal proteins of the cell surface in Tetrahymena : I. Identification and localization of major proteins

Isolated pellicles (cell ‘ghosts’) have been prepared from Tetrahymena thermophila strain B by two different methods. Using differential solubilization in combination with polyacrylamide gel electrophoresis and electron microscopy, we have tentatively identified the major proteins found in the surface-associated cytoskeleton. The ‘epiplasm’, a continuous layer of fibrous material found just beneath the surface membranes, appears to contain two major proteins. The smaller of the two (mol. wt 122 000 D) is believed to be present throughout the layer, whereas the larger protein (mol. wt 145 000 D) appears to be localized in the regions where ciliary basal bodies connect to the epiplasmic layer and to surface membranes. Evidence is presented which suggests that actin may also be present in this structure. Tubulin has been isolated from the cytosol of Tetrahymena and compared with cytoskeletal tubulin and porcine brain tubulin. A major protein of mol. wt 250 000 D which is found in Tetrahymena pellicles appears to be the major component of kinetodesmal fibers (striated elements which attach to the ciliary basal bodies).     

Cholesterol domains in biological membranes

Membrane cholesterol is distributed asymmetrically both within the cell or within cellular membranes. Elaboration of intracellular cholesterol trafficking, targeting and intramembrane distribution has been spurred by both molecular and structural approaches. The expression of recombinant sterol carrier proteins in L-cell fibroblasts has been especially useful in demonstrating for the first time that such proteins actually elicit intracellular and intra-plasma membrane redistribution of sterol. Additional advances in the use of native fluorescent sterols allowed resolution of transbilayer and lateral cholesterol domains in plasma membranes from cultured fibroblasts, brain synaptosomes and erythrocytes. In all three cell surface membranes, cholesterol is enriched in the inner, cytofacial leaflet. Up to three different cholesterol domains have been identified in the lateral plane of the plasma membrane: a fast exchanging domain comprising less than 10% of cholesterol, a slowly exchanging domain comprising about 30% of cholesterol, and a very slowly or non-exchangeable sterol domain comprising 50–60.

Of plasma membrane cholesterol. Factors modulating plasma membrane cholesterol domains include polyunsaturated fatty acids, expression of intracellular sterol carrier proteins, drugs such as ethanol, and several membrane pathologies (systemic lupus erythematosus, sickle cell anaemia and aging). Disturbances in plasma membrane cholesterol domains after transbilayer fluidity gradients in plasma membranes. Such changes are associated with decreased Ca²⁺ -ATPase and Na ⁺, K⁺ -ATPase activity. Thus, the size, dynamics and distribution of cholesterol domains within membranes not only regulate cholesterol efflux/influx but also modulate plasma membrane protein functions and receptor-effector coupled systems.     

TpMRK regulates cell division of Tetrahymena in response to oxidative stress

TpMRK was identified as a stress‐responsive mitogen activated protein kinase (MAPK)‐related kinase and has been shown to play a critical role in the stress signalling in Tetrahymena cells. Here, we found that the mRNA expression of TpMRK was correlated with cell division of Tetrahymena with decreased expression occurring in cells prior to entering synchronous cell division induced by heat treatment. Notably, cell division was delayed with a lower division index of 40% after exposure to hydrogen peroxide while 85% of cells underwent cell division synchronously at 75 min after heat treatment without the oxidative exposure. Furthermore, inactivation of TpMRK signalling by p38 MAPK inhibitor SB203580 or MEK inhibitor PD 98059 partially derepressed cell division induced by hydrogen peroxide. Our data suggest that oxidative stimuli might cause aberration of synchronous cell division of Tetrahymena through activating the TpMRK cascade. Copyright © 2009 John Wiley & Sons, Ltd.     

Purification and Characterization of a Novel Chemorepellent Receptor from Tetrahymena thermophila

Chemosensory transduction and adaptation are important aspects of signal transduction mechanisms in many cell types, ranging from prokaryotes to differentiated tissues such as neurons. The eukaryotic ciliated protozoan, Tetrahymena thermophila, is capable of responding to both chemoattractants (O'Neill et al., 1985; Leick, 1992; Kohidai, Karsa & Csaba, 1994, 1995) and chemorepellents (Francis & Hennessey, 1995; Kuruvilla, Kim & Hennessey, 1997). An example of a nontoxic, depolarizing chemorepellent in Tetrahymena is extracellular lysozyme (Francis & Hennessey, 1995; Hennessey, Kim & Satir, 1995). Lysozyme is an effective chemorepellent at micromolar concentrations, binds to a single class of externally facing membrane receptors and prolonged exposure (10 min) produces specific chemosensory adaptation (Kuruvilla et al., 1997). We now show that this lysozyme response is initiated by a depolarizing chemoreceptor potential in Tetrahymena and we have purified the membrane lysozyme receptor by affinity chromatography of solubilized Tetrahymena membrane proteins. The solubilized, purified protein is 42 kD and it exhibits saturable, high affinity lysozyme binding. Polyclonal antibodies raised against this 42 kD receptor block the in vivo lysozyme chemoresponse. This is not only the first time that a chemoreceptor potential has been recorded from Tetrahymena but also the first time that a chemorepellent receptor has been purified from any unicellular eukaryote. Received: 28 July 1997/Revised: 14 November 1997     

Keeping good friends close – The surface and secreted proteomes of a probiotic bacterium provide candidate proteins for intestinal attachment and communication with the host

Bacteria use cell surface proteins and secreted proteins to interact with host tissues. Several dozen previously published proteomics studies have identified cell surface proteins for pathogens. In this issue, Celebioglu and Svensson (Proteomics 2017, 17, 1700019) use 2D gel electrophoresis and mass spectrometry to identify secreted and cell surface proteins of a commensual gut bacterium, Lactobacillus acidophilus NCFM. Some of the proteins are known to have functions in the cytoplasm, and their presence on the cell surface suggests they might be moonlighting proteins. In addition, comparisons of proteins used by pathogenic and probiotic species to interact with their hosts could lead to improved treatments of infections and chronic diseases that are associated with an imbalance of pathogenic and probiotic gut bacteria.     

Global proteomic pattern of Tropheryma whipplei: A Whipple's disease bacterium

The proteome of Tropheryma whipplei, the intracellular bacterium responsible for Whipple's disease (WD), was analyzed using two complementary approaches: 2‐DE coupled with MALDI‐TOF and SDS‐PAGE with nanoLC‐MS/MS. This strategy led to the identification of 206 proteins of 808 predicted ORFs, resolving some questions raised by the genomic sequence of this bacterium. We successfully identified antibiotic targets and proteins with predicted N‐terminal signal sequences. Additionally, we identified a family of surface proteins (known as T. whipplei surface proteins (WiSPs)), which are encoded by a unique group of species‐specific genes and serve as both coding regions and DNA repeats that promote genomic recombination. Comparison of the protein expression profiles of the intracellular facultative host‐associated WD bacterium with other host‐associated, intracellular obligate, and environmental bacteria revealed that T. whipplei shares a proteomic expression profile with other host‐associated facultative intracellular bacteria. In summary, this study describes the global protein expression pattern of T. whipplei and reveals some specific features of the T. whipplei proteome.     

Ultrastructure of the Fusion Area during Conjugation in the Suctorian Heliphrya erhardi (Rieder) Matthes*

SYNOPSIS. The suctorian Heliophrya erhardi (Rieder) Matthes is attached to the substrate by the flattened ventral side of the cell body. The dorsal is covered by a pellicle composed of 3 unit membranes. Below the pellicle is a 0.4–0.8-μm thick epiplasm composed of 6–8-nm thick fibrils. Microtubules form a network beneath the epiplasm. The epipalsm is penetrated by tube-like pellicular pits, which are lined by the cell membrane and end beneath the epiplasm in a saccule-like enlargement. During conjugation, 2 neighboring organisms form cytoplasmic processes which come into contact and fuse, thus forming a cytoplasmic bridge between the 2 cells. Around the bridge the pellicles of both organisms fuse, and the partners become united by a continuous common membrane system. Across the entire conjugation bridge the 2 fused epiplasms form a septum. Tube-like structures can be seen lying partly in the epiplasmic septum and partly in the adjacent cytoplasm. These structures are open at both ends and represent remnants of the pellicular pits. No trace of the original pellicular membranes can be found at the fusion area within the epiplasmic septum. The cytoplasm of the conjugation partners is separated only by the fused epiplasms forming the epiplasmic septum.     

Variation In Surface Proteins In Tetrahymena*

SYNOPSIS. Surface proteins of Tetrahymena were identified by lactoperoxidase iodination, and comparisons were made between a number of strains and species within the genus. an adequate procedure for strain comparisons was found to be solubilization of whole cells following iodination, separation of total cell protein using polyacrylamide gel electrophoresis, and identification of surface proteins by autoradiography of dried gels. the results obtained in the present study show the existence of both interspecific and intraspecific variation in surface proteins of Tetrahymena, but the differences tend to be small within species and large between species. the relation of these cell surface fingerprints to the present taxonomic designations within the genus is discussed. Questions are raised about the functional significance of these surface proteins.     

Monoclonal Antibodies Reveal Complex Structure in the Membrane Skeleton of Tetrahymena

ABSTRACT. Twelve monoclonal antibodies were raised that are specific for the membrane skeleton of Tetrahymena . Five were directed against T. pyrifomis and seven were directed against T. thermophila . Some cross-reactivity between species was found. Each monoclonal antibody recognized one of the three major components of epiplasm, i.e. the bands A, B, and C identified in electrophoretic separations of epiplasmic proteins. It was found, using these antibodies, that the epiplasmic proteins A, B, and C have overlapping but independent distributions within the cell.     

Elucidation of clathrin-mediated endocytosis in tetrahymena reveals an evolutionarily convergent recruitment of dynamin

Ciliates, although single-celled organisms, contain numerous subcellular structures and pathways usually associated with metazoans. How this cell biological complexity relates to the evolution of molecular elements is unclear, because features in these cells have been defined mainly at the morphological level. Among these ciliate features are structures resembling clathrin-coated, endocytic pits associated with plasma membrane invaginations called parasomal sacs. The combination of genome-wide sequencing in Tetrahymena thermophila with tools for gene expression and replacement has allowed us to examine this pathway in detail. Here we demonstrate that parasomal sacs are sites of clathrin-dependent endocytosis and that AP-2 localizes to these sites. Unexpectedly, endocytosis in Tetrahymena also involves a protein in the dynamin family, Drp1p (Dynamin-related protein 1). While phylogenetic analysis of AP subunits indicates a primitive origin for clathrin-mediated endocytosis, similar analysis of dynamin-related proteins suggests, strikingly, that the recruitment of dynamin-family proteins to the endocytic pathway occurred independently during the course of the ciliate and metazoan radiations. Consistent with this, our functional analysis suggests that the precise roles of dynamins in endocytosis, as well as the mechanisms of targeting, differ in metazoans and ciliates.     

Cytoskeletal proteins of the cell surface in Tetrahymena : II. Turnover of major proteins

The turnover of surface membrane-associated cytoskeletal proteins has been studied in Tetrahymena. These proteins undergo rapid turnover and appear to be in equilibrium with precursor pools within the cytosol. Carefully controlled pulse-chase experiments, alone and in combination with cycloheximide, have shown that labeled proteins accumulated in the cytoskeleton after they were no longer being synthesized within the cell. The movement of subunits from the cytoskeleton to the cytosol was also demonstrated using tubulin isolated from these two compartments within the cell.     

The Fine Structure of the Colonial Kinetoplastid Flagellate Cephalothamnium cyclopum Stein

Cell structure, cell adhesion, and stalk formation have been examined by electron microscopy in the colonial flagellate, Cephalothamnium cyclopum. Each cell is obconical or spindle-shaped, pointed posteriorly and truncated anteriorly. The cell membrane is underlain by epiplasm 0.1 μm thick in the posterior region, but bands of microtubules support the anterior region which is differentiated into a flagellar pocket, oral apparatus and contractile vacuole. Each of 2 flagella, joined a short way above their bases by an interflagellar connective, has a paraxial rod and mastigonemes. One flagellum is free and is important in food gathering while the other is recurrent and lies in a shallow groove on the ventral cell surface but projects posteriorly into the stalk. The basal bodies of these flagella are bipartite structures connected by a pair of striated rootlets with accessory microtubular fibers. The oral apparatus consists of a funnel-shaped buccal cavity and cytostome. It is supported by helical and longitudinal microtubules and also has nearby striated and microtubular fibers. Possible roles of associated oral vesicles in relation to ingestion are discussed. A reticulate mitochondrion houses a massive kinetoplast which has a fibrillar substructure resembling that of dinoflagellate chromosomes. Adjacent flagellates adhere by laminate extensions of their posterior regions and attach by their recurrent flagella to a communally secreted stalk composed of finely fibrillar material. This study indicates that Cephalothamnium belongs in the order Kinetoplastida, and has many features in common with members of the family Bodonidae.