Cilia-Mediated Oriented Chemokinesis in Tetrahymena thermophila

The role of the cilia in the locomotion (“gliding”) of Tetrahymena thermophila in a semi-solid medium has been studied when cells were migrating in gradients of attractant. Video recordings and computer-aided motion analysis of migrating cells and their ciliary activity show that Tetrahymena thermophila migrate by swimming forward in semi-solid methyl cellulose, using their cilia. Ciliary reversals occur at certain intervals and cause a termination (“stop”) of cellular migration. Cells with reversed cilia resume forward migration when normal ciliary beating resumes. In gradients of attractants, cells migrating towards the attractant suppress ciliary reversals, which leads to longer runs between stops than in control cells. Cells migrating away from the attractant have a higher frequency of ciliary reversals than the control cells resulting in shorter runs. Stimulated cells adapt to a particular ambient concentration of attractant several times during migration in the gradient. Adaptation is followed by de-adaptation, which occurs during the “stop”. In the presence of cycloheximide, a strong inhibitor of chemoattraction, the attractant-induced suppression of ciliary reversal is abolished (cells become desensitized to the attractant). It is concluded that Tetrahymena has a short-term memory during adaptation. This is important for the efficiency of migration towards an attractant.     

Ciliary Membrane Proteins of Tetrahymena thermophila

SYNOPSIS SDS polyacrylamide gels of the ciliary membrane proteins of Tetrahymena thermophila revealed 5 major peaks and 11 minor protein peaks ranging in molecular weight from below 20,000 to above 250,000. The peaks resembled those found for ciliary membrane proteins of Paramecium aurelia. .     

The dynein heavy chain gene family in Tetrahymena thermophila

The dynein ATPases are a family of motor enzymes that drive microtubule sliding in cilia and flagella and contribute to microtubule-based transport inside cells. The multi-dynein hypothesis makes two predictions: 1) Axonemes contain multiple dynein heavy chain (DHC) isoforms, each encoded by a different gene; 2) Each isoform performs a specific role in ciliary beating. We used PCR-based techniques to clone thirteen different DHC sequences from Tetrahymena genomic DNA. All thirteen genes appeared to be expressed in growing cells. Comparisons of the deduced amino acid sequences of the thirteen DHCs with other known DHCs suggested that we have cloned three outer arm DHCs, two cytoplasmic DHCs, and eight inner arm DHCs.     

Cytoplasmic dynein-2: from molecules to human diseases

The dynein motor protein family is involved in a wide variety of functions in eukaryotic cells. The axonemal dynein class and cytoplasmic dynein-1 subclass have been well characterized. However, the cytoplasmic dynein-2 subclass of the family has only recently begun to be understood. We describe the entire dynein family but focus on cytoplasmic dynein-2. Dynein-2 consists of a heavy, an intermediate, a light intermediate, and a light chain. The complex appears to function primarily as the retrograde motor for intraflagellar transport. This process is important for the formation and maintenance of cilia and flagella. Additionally, dynein-2 has roles in the control of ciliary length and in non-ciliary functions. Mutations in the human dynein-2 heavy chain lead to cilia-related diseases.     

Bend propagation drives central pair rotation in Chlamydomonas reinhardtii flagella

Regulation of motile 9+2 cilia and flagella depends on interactions between radial spokes and a central pair apparatus. Although the central pair rotates during bend propagation in flagella of many organisms and rotation correlates with a twisted central pair structure, propulsive forces for central pair rotation and twist are unknown. Here we compared central pair conformation in straight, quiescent flagella to that in actively beating flagella using wild-type Chlamydomonas reinhardtii and mutants that lack radial spoke heads. Twists occur in quiescent flagella in both the presence and absence of spoke heads, indicating that spoke--central pair interactions are not needed to generate torque for twisting. Central pair orientation in propagating bends was also similar in wild type and spoke head mutant strains, thus orientation is a passive response to bend formation. These results indicate that bend propagation drives central pair rotation and suggest that dynein regulation by central pair--radial spoke interactions involves passive central pair reorientation to changes in bend plane.     

Effects of taxol treatment on the microtubular system and mitochondria of Tetrahymena

Complex investigation was done using immunocytochemical confocal microscopy, electron microscopy and flow cytometry on the effect of taxol to the microtubular arrangement and dynamics. The most interesting phenomenon was the rapid disappearance of transversal microtubule bands, while longitudinal microtubule bands remained and were submitted to the known effects of taxol. There was a broad variation in mitochondrial effect, some of them remained normal, while others swollen, desintegrated and their tubules disoriented. Treatment with 50 nM taxol significantly reduced the binding of anti alpha-tubulin antibody and a lesser degree anti-acetylated tubulin antibody. The difference between the transversal and longitudinal microtubules is emphasized by the results and the paper discusses the possibilities of indirect effects of taxol to the transversal microtubules (tubulin-GTP interaction, faster turnover, mitochondrial interaction). Polyglutamylation of tubulin has not a role in this difference.     

Biochemical Analysis of a Mutant Tetrahymena Lacking Outer Dynein Arms

ABSTRACT. Tetrahymena thermophila mutants homozygous for the oad mutation become nonmotile when grown at the restrictive temperature, and axonemes isolated from nonmotile mutants lack approximately 90% of their outer dynein arms. Electrophoretic analyses of axonemes isolated from nonmotile mutants ( oad axonemes) indicate they contain significantly fewer of the 22 S dynein heavy chains that axonemes isolated from wild-type cells (wild-type axonemes) contain. The 22 S dynein heavy chains that remain in axonemes isolated from nonmotile, oad mutants are assembled into 22 S dynein particles that exhibit wild-type levels of ATPase activity. Two-dimensional gel electrophoresis of oad axonemes show that they are deficient in no proteins other than those proteins thought to be components of 22 S dynein. This report is the first formal proof that outer dynein arms in Tetrahymena cilia are composed of 22 S dynein.     

Long-Term Maintenance of Tetrahymena Spp.

SYNOPSIS. Simple media for Tetrahymena, using rat gut or soybean as the nutrient source are described. Cultures can be maintained in these media up to one year at room temperature.     

梨形四膜虫在环境毒理学研究中的应用

总结了以四膜虫为生物模型的环境毒理学研究,对各种环境化学毒物胁迫下四膜虫群体生长动力学,形态结构,行为特征以及生理生化特性变化等进行了综述。     

Ciliary polypeptides and glycoconjugates of wild-type and mutant Tetrahymena thermophila: Starved versus nonstarved

To investigate the role of cilia in mating interactions of Tetrahymena thermophila, ciliary membrane-rich fractions were isolated from two wild-type strains, a non-discharge mucocyst mutant which possesses mating behavior similar to wild-type, and a mating mutant which is able to costimulate cells of complementary mating type but cannot enter into pair formation. In each case, proteins from the ciliary membrane-rich fractions of starved, mating-competent (“initiated”) cells were compared with those from non-starved, mating-incompetent (“non-initiated”) cells, by gel electro-phoresis and lectin blotting. In stained gels, a 43 kDa polypeptide was reduced or absent in initiated cells but present in non-initiated cells, in all strains. In silver-stained gels, a 25 kDa polypeptide was present in all strains, both initiated and non-initiated. In blots probed with Con A-peroxidase, a 25 kDa glycoprotein was present in ciliary membrane fractions from non-initiated cells and absent in membranes of initiated cells of the two wild-type strains and the mucocyst mutant, but is present in initiated and non-initiated cells of the mating mutant (several hypotheses are presented to explain these findings). In addition, ciliary proteins of the mating mutant included at least two unique Con A-binding polypeptides. Our results support the idea that development of mating competence during starvation involves an extensive remodeling of ciliary membranes, and identify a 25 kDa glyco-conjugate as having a potential role in control of pair formation during mating. © 1992 Wiley-Liss, Inc.     

Remodeling and activation mechanisms of outer arm dyneins revealed by cryo‐EM

Cilia are thin microtubule‐based protrusions of eukaryotic cells. The swimming of ciliated protists and sperm cells is propelled by the beating of cilia. Cilia propagate the flow of mucus in the trachea and protect the human body from viral infections. The main force generators of ciliary beating are the outer dynein arms (ODAs) which attach to the doublet microtubules. The bending of cilia is driven by the ODAs'' conformational changes caused by ATP hydrolysis. Here, we report the native ODA complex structure attaching to the doublet microtubule by cryo‐electron microscopy. The structure reveals how the ODA complex is attached to the doublet microtubule via the docking complex in its native state. Combined with coarse‐grained molecular dynamic simulations, we present a model of how the attachment of the ODA to the doublet microtubule induces remodeling and activation of the ODA complex.     

Basal Body Assembly in Ciliates: The Power of Numbers

Centrioles perform the dual functions of organizing both centrosomes and cilia. The biogenesis of nascent centrioles is an essential cellular event that is tightly coupled to the cell cycle so that each cell contains only two or four centrioles at any given point in the cell cycle. The assembly of centrioles and their analogs, basal bodies, is well characterized at the ultrastructural level whereby structural modules are built into a functional organelle. Genetic studies in model organisms combined with proteomic, bioinformatic and identifying ciliary disease gene orthologs have revealed a wealth of molecules requiring further analysis to determine their roles in centriole duplication, assembly and function. Nonetheless, at this stage, our understanding of how molecular components interact to build new centrioles and basal bodies is limited. The ciliates, Tetrahymena and Paramecium , historically have been the subject of cytological and genetic study of basal bodies. Recent advances in the ciliate genetic and molecular toolkit have placed these model organisms in a favorable position to study the molecular mechanisms of centriole and basal body assembly.     

Porphyrin Rings and Phospholipids: Stimulators of Cloning Efficiency in Certain Species of Tetrahymena

ABSTRACT. Species of Tetrahymena , including T. vorax, T. thermophila, T. pyriformis , and T. pigmentosa , were tested for cloning efficiency in proteose peptone and in synthetic nutrient media to which were added hemin, protoporphyrin IX, chlorophyllin, or asolectin, an impure mixture of phospholipids. All species could be cloned with high efficiency in the crude media. In unsupplemented synthetic medium the cloning efficiencies were 0–10%, around 50%, around 50%, and 90–100% for T. thermophila, T. vorax, T. pyriformis , and T. pigmentosa , respectively. The first three were all stimulated to 90–100% by addition of the porphyrin or phospholipid compounds mentioned above. Uroporphyrin III and coproporphyrin I and III had no effect. We suggest that cells unable to form clones suffer from a lack of cellular energy. This situation may be alleviated by our additions: certain porphyrin rings may be built into cytochromes and phospholipids may be used as fuel. Thus, the synthetic media used so far for these ciliates have not been optimal.     

Chlamydomonas flagella

Chlamydomonas reinhardtii Dangard has been widely adopted as a model system for studies of eukaryotic cilia and flagella. Here I review recent progress in understanding flagellar ultrastructure, the mechanisms that generate and regulate flagellar beating and gliding motility, the flagellar assembly process, basal body structure and function, and adhesion-based signaling, all advanced by work with this single-celled organism.     

Centriole-less pericentriolar material serves as a microtubule organizing center at the base of C. elegans sensory cilia

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嗜热四膜虫减数分裂研究进展

减数分裂是真核生物有性生殖过程的关键步骤,染色体的行为变化贯穿整个减数分裂的过程。近些年来,借助先进的分子生物学技术和细胞学实验手段,通过对突变细胞株的筛选和评价,单细胞真核模式生物原生动物嗜热四膜虫(Tetrahymena thermophila)减数分裂方面的研究取得了长足的进展。本文主要介绍嗜热四膜虫减数分裂的过程,以及在此过程中伴随染色体行为变化的相关基因的功能,从而为进一步探讨嗜热四膜虫减数分裂的分子机制提供有效信息。     

A cytoplasmic dynein heavy chain in sea urchin embryos.

By making the hypothesis that the pattern of conserved sequence residues in the vicinity of the hydrolytic ATP-binding site of dynein would resemble that in myosins from a broad variety of sources, we designed degenerate oligonucleotide primers capable of amplifying this region of multiple dynein isoforms from sea urchin embryo poly(A)+ RNA. Quantification of the expression of two of these dynein isoforms has shown that the level of mRNA encoding for the beta-heavy chain, like that of tubulin, increases 2-3-fold after deciliation of the embryos, whereas the expression of the second dynein isoform, like that of actin, is essentially unaffected. This second isoform is believed to be the cytoplasmic dynein of sea urchin embryos.     

Efficiency of Filter Feeding in Two Species of Tetrahymena*

SYNOPSIS. Rates of removal of suspended India ink particles from the surrounding medium by 2 ciliates, Tetrahymena pyriformis strain GL and Tetrahymena vorax strain V₂S, have been measured. It is evident from the results that the food vacuoles concentrate the suspended particles, clearing a volume of the surrounding suspension fluid 500 × greater than the total volume of food vacuoles made during the same period of time.     

Particle Ejection from the Cytoproct of Tetrahymena

SYNOPSIS. Egestion of carmine particle-containing food vacuoles from the cytoproct of Tetrahymena pyriformis has been analyzed by high-speed cinemicrography. The vacuole may enter into position in the cytoproct ∼ 7 sec before ejection, and forms a distinct bulge beyond the outline of the cell surface for over 2 sec prior to ejection. The ejection process itself requires 20–80 msec.