N-ethylmaleimide-modified heavy meromyosin. A probe for actomyosin interactions

Treatment of rabbit skeletal muscle heavy meromyosin (HMM) with the sulfhydryl reagent N-ethylmaleimide (NEM) produces a species of HMM which remains tightly bound to actin in the presence of MgATP. NEM-HMM forms characteristic "arrowhead" complexes with actin which persist despite rinses with MgATP. NEM-HMM inhibits the actin activation of native HMM-ATPase activity, the superprecipitation of actomyosin, the contraction of glycerinated muscle myofibrils, and the contraction of cytoplasmic strands of the soil amoeba Chaos carolinensis. However, NEM-HMM does not interfere with in vitro microtubule polymerization or beating of demembranated cilia.     

A STUDY OF PHAGOCYTOSIS IN THE AMEBA CHAOS CHAOS

The process of phagocytosis was investigated by observing the interactions between the ameba Chaos chaos and its prey (Paramecium aurelia), by studying food cup formation in the living cell, and by studying the fine structure of the newly formed cup using electron microscopy of serial sections. The cytoplasm surrounding the food cup was found to contain structures not seen elsewhere in the ameba. The results are discussed in relation to the mechanisms which operate during food cup formation.     

A comparison of the polycation receptors of Paramecium tetraurelia and Tetrahymena thermophila

Chemorepellents are compounds that cause ciliated protozoans to reorient their swimming direction. A number of chemorepellents have been studied in the ciliated protozoans, Paramecium and Tetrahymena. Chemorepellents, such as polycations, cause the organism to exhibit "avoidance behavior," a swimming behavior characterized by jerky movements and other deviations from normal forward swimming, which result from ciliary reversal. One well-characterized chemorepellent pathway in Tetrahymena is that of the proposed polycation receptor that is activated by lysozyme and pituitary adenylate cyclase activating polypeptide (PACAP). In this study, we compare the response of Paramecium to the chemorepellents lysozyme, vasoactive intestinal peptide (VIP), and PACAP to the previously studied polycation response in Tetrahymena. Our results indicate that lysozyme, VIP, and PACAP are all chemorepellents in Paramecium, just as they are in Tetrahymena. However, the signaling pathways involved appear to be different. While previous pharmacological characterization indicates that G-proteins are involved in polycation signaling in Tetrahymena, we present evidence that similar reception in Paramecium involves activation of a tyrosine kinase pathway in order for lysozyme avoidance to occur. Polycation responses of both organisms are inhibited by neomycin sulfate. While PACAP is the most effective of the three chemorepellents in Tetrahymena, lysozyme is the most effective chemorepellent in Paramecium.     

Mutations of tubulin glycylation sites reveal cross-talk between the C termini of alpha- and beta-tubulin and affect the ciliary matrix in Tetrahymena

Two types of polymeric post-translational modifications of alpha/beta-tubulin, glycylation and glutamylation, occur widely in cilia and flagella. Their respective cellular functions are poorly understood. Mass spectrometry and immunoblotting showed that two closely related species, the ciliates Tetrahymena and Paramecium, have dramatically different compositions of tubulin post-translational modifications in structurally identical axonemes. Whereas the axonemal tubulin of Paramecium is highly glycylated and has a very low glutamylation content, the axonemal tubulin of Tetrahymena is glycylated and extensively glutamylated. In addition, only the alpha-tubulin of Tetrahymena undergoes detyrosination. Mutations of the known glycylation sites in Tetrahymena tubulin affected the level of each polymeric modification type in both the mutated and nonmutated subunits, revealing cross-talk between alpha- and beta-tubulin. Ultrastructural analyses of glycylation site mutants uncovered defects in the doublet B-subfiber of axonemes and revealed an accumulation of dense material in the ciliary matrix, reminiscent of intraflagellar transport particles seen by others in Chlamydomonas. We propose that polyglycylation and/or polyglutamylation stabilize the B-subfiber of outer doublets and regulate the intraflagellar transport.     

Trypanosoma,Paramecium and Tetrahymena: From genomics to flagellar and ciliary structures and cytoskeleton dynamics

Cilia and flagella play an important role in motility, sensory perception, and the life cycles of eukaryotes, from protists to humans. However, much critical information concerning cilia structure and function remains elusive. The vast majority of ciliary and flagellar proteins analyzed so far are evolutionarily conserved and play a similar role in protozoa and vertebrates. This makes protozoa attractive biological models for studying cilia biology. Research conducted on ciliated or flagellated protists may improve our general understanding of cilia protein composition, of cilia beating, and can shed light on the molecular basis of the human disorders caused by motile cilia dysfunction. The Symposium “From genomics to flagellar and ciliary structures and cytoskeleton dynamics” at ECOP2019 in Rome presented the latest discoveries about cilia biogenesis and the molecular mechanisms of ciliary and flagellum motility based on studies in Paramecium, Tetrahymena, and Trypanosoma. Here, we review the most relevant aspects presented and discussed during the symposium and add our perspectives for future research.     

Induction of digoxin-like material production, and the digoxin binding in the unicellular organism Tetrahymena by digitoxin.

Thin layer chromatographic, and laser-confocal microscopic analyses with a monoclonal antibody to digoxin also displaying high affinity to digoxigenin, were used to determine the presence and localization of cardioactive glycosides. Tetrahymena pyriformis was found to possess digitoxigenin-like material, but digoxin, digitoxin, digoxigenin, gitoxin and lanatoside C were not detected. Digitoxin treatment elicited the appearance of a digoxin-like material in the progeny generations. Digoxin was taken up by untreated Tetrahymena, especially strongly 24 h after digitoxin treatment. While the cardenolide was localized in vesicles of the cell body in untreated Tetrahymena, the engulfed digoxin appeared in the epiplasmic layer and also in the cilia after digitoxin pretreatment. Digoxin pretreatment did not increase digoxin uptake. These data indicate that Tetrahymena has: (1) the capacity to discriminate between closely related molecules; (2) the ability to induce digoxin-like material production; and/or (3) enzymes that can effect a digitoxin-digoxin transformation.     

The nucleotide sequences of 5S rRNAs from three ciliated protozoa.

The nucleotide sequences of 5S rRNAs from three ciliated protozoa, Paramecium tetraurelia, Tetrahymena thermophila and Blepharisma japonicum have been determined. All of them are 120 nucleotides long and the sequence of probable tRNA binding site of position 41-44 is GAAC which is characteristic of the plant 5S rRNAs. The sequence similarity percents are 87% (Paramecium/Tetrahymena), 86% (Paramecium/Blepharisma) and 79% (Tetrahymena/Blepharisma), suggesting a close relationship of these three ciliates.     

Pellicle of Paramecium caudatum as Revealed by Freeze Etching

SYNOPSIS. Freeze etching study of Paramecium caudatum surface reveals a regular pattern formed by depressions equipped with a single or paired cilia. This picture is consistent with the generally accepted concept of Paramecium cortex based on conventional methods of study. The surface of the ciliate is covered with small globular particles ∼14 nm in diameter, which have been found also on the plasma membrane of other cells. In the areas where the tips of trichocysts are attached to the pellicle, the globular particles are arranged into circles 0.4 μm in diameter. The whole surface of Paramecium is covered by a smooth thin layer which, always chipped off during fracturing, is detectable only after etching.     

Kinesin-II Is Preferentially Targeted to Assembling Cilia and Is Required for Ciliogenesis and Normal Cytokinesis in Tetrahymena

We cloned two genes, KIN1 and KIN2, encoding kinesin-II homologues from the ciliate Tetrahymena thermophila and constructed strains lacking either KIN1 or KIN2 or both genes. Cells with a single disruption of either gene showed partly overlapping sets of defects in cell growth, motility, ciliary assembly, and thermoresistance. Deletion of both genes resulted in loss of cilia and arrests in cytokinesis. Mutant cells were unable to assemble new cilia or to maintain preexisting cilia. Double knockout cells were not viable on a standard medium but could be grown on a modified medium on which growth does not depend on phagocytosis. Double knockout cells could be rescued by transformation with a gene encoding an epitope-tagged Kin1p. In growing cells, epitope-tagged Kin1p preferentially accumulated in cilia undergoing active assembly. Kin1p was also detected in the cell body but did not show any association with the cleavage furrow. The cell division arrests observed in kinesin-II knockout cells appear to be induced by the loss of cilia and resulting cell paralysis.     

Influence of extremely low frequency electromagnetic fields on the swimming behavior of ciliates

Different species of ciliates (Paramecium biaurelia, Loxodes striatus, Tetrahymena thermophila) have been taken as model systems to study the effects of extremely low-frequency electromagnetic fields (50 Hz, 0.5–2.0 mT) on the cellular level. A dose-dependent increase in the mean swimming velocity and a decrease in the linearity of cell tracks were observed in all wild-type cells. In contrast, field-exposure did not increase the number of directional turns of the Paramecium tetraurelia pawn mutant (d4–500r), which is characterized by defective Ca²⁺-channels. The described changes indicate a direct effect of low frequency electromagnetic fields on the transport mechanisms of the cell membrane for ions controlling the motile activity of cilia. Bioelectromagnetics 18:491–498, 1997. © 1997 Wiley-Liss, Inc.     

1001 model organisms to study cilia and flagella

Most mammalian cell types have the potential to assemble at least one cilium. Immotile cilia participate in numerous sensing processes, while motile cilia are involved in cell motility and movement of extracellular fluid. The functional importance of cilia and flagella is highlighted by the growing list of diseases due to cilia defects. These ciliopathies are marked by an amazing diversity of clinical manifestations and an often complex genetic aetiology. To understand these pathologies, a precise comprehension of the biology of cilia and flagella is required. These organelles are remarkably well conserved throughout eukaryotic evolution. In this review, we describe the strengths of various model organisms to decipher diverse aspects of cilia and flagella biology: molecular composition, mode of assembly, sensing and motility mechanisms and functions. Pioneering studies carried out in the green alga Chlamydomonas established the link between cilia and several genetic diseases. Moreover, multicellular organisms such as mouse, zebrafish, Xenopus, Caenorhabditis elegans or Drosophila, and protists such as Paramecium, Tetrahymena and Trypanosoma or Leishmania each bring specific advantages to the study of cilium biology. For example, the function of genes involved in primary ciliary dyskinesia (due to defects in ciliary motility) can be efficiently assessed in trypanosomes.     

Codon Usage in Tetrahymena and Other Ciliates

Codon usage in ciliates was examined by analyzing the coding regions of 22 ciliate genes corresponding to a total of 26, 142 nucleotides (8, 714 codons). It was found that Tetrahymena, Paramecium and the hypotrichs ( Oxytricha and Stylonychia ) differed in which synonymous codons were used most frequently by their genes. In fact, the codon choices in highly expressed Tetrahymena genes were more similar to those of yeast genes than those of Paramecium genes. The ciliates do not appear to have unusually strong biases in codon usage frequency when compared to other protists such as yeast. The analysis of the Tetrahymena genes indicated that genes which are highly expressed during normal cell growth have a stronger bias towards using the "preferred" codons than those expressed at lower levels during growth or for brief periods during processes such as conjugation. This conforms to what is found in other protists.     

Codon usage in Tetrahymena and other ciliates

Codon usage in ciliates was examined by analyzing the coding regions of 22 ciliate genes corresponding to a total of 26,142 nucleotides (8,714 codons). It was found that Tetrahymena, Paramecium and the hypotrichs (Oxytricha and Stylonychia) differed in which synonymous codons were used most frequently by their genes. In fact, the codon choices in highly expressed Tetrahymena genes were more similar to those of yeast genes than those of Paramecium genes. The ciliates do not appear to have unusually strong biases in codon usage frequency when compared to other protists such as yeast. The analysis of the Tetrahymena genes indicated that genes which are highly expressed during normal cell growth have a stronger bias towards using the "preferred" codons than those expressed at lower levels during growth or for brief periods during processes such as conjugation. This conforms to what is found in other protists.     

Identification of elongation factor-1alpha as a Ca2+/calmodulin-binding protein in Tetrahymena cilia

Calmodulin (CaM) is known to be a ciliary component. However, the function of CaM in cilia or flagella has not been well understood. Immunoelectron microscopy using anti-CaM antibody showed that CaM was localized on the axonemal microtubules (MTs) and matrix of Tetrahymena cilia. To investigate the signal transduction of Ca(2+)/CaM in cilia, we performed Ca(2+)/CaM-affinity column chromatography in the membrane and matrix fraction. Elongation factor-1alpha (EF-1alpha) was identified as a Ca(2+)/CaM-binding protein in cilia. EF-1alpha is a highly conserved protein and functions in protein translation. In addition, EF-1alpha has been reported to interact with MTs and F-actin in several organisms. Immunoelectron microscopy showed that EF-1alpha was localized on the axonemal MTs. However, in immunoblot analysis, EF-1alpha was mainly extracted in the membrane and matrix fraction from the axonemal MTs by 1% Triton X-100 extraction. These results suggest that interaction between EF-1alpha and axonemal MTs is weak and sensitive to treatment with 1% Triton X-100 and that EF-1alpha mediates between axonemal MTs and CaM in the presence of Ca(2+). Moreover, EF-1alpha was also localized in cilia of Paramecium, suggesting that EF-1alpha functions as a target protein of Ca(2+)/CaM in ciliate cilia.     

External GTP binding and induction of cell division in starved Tetrahymena thermophila

The extracellular nucleotide, guanosine 5'-triphosphate (GTP) is known to be a chemorepellent for ciliated protozoa such as Paramecium and Tetrahymena. Here, we studied the surface localization of GTP binding sites and also its effects on the cell division of Tetrahymena thermophila. When a ribose-modified and fluorescent analog of GTP, 2'-(or -3')-O-trinitrophenyl (TNP)-GTP was added to the cells starved in non-nutrient buffer, a remarkable fluorescence was observed at the compound cilia of the oral area, while it was weak at other cilia and the somatic membrane. Following transfer of the cells to the starvation medium, the intensity of TNP-GTP fluorescence at the oral area gradually increased and was saturated at 3-4 hours. Addition of GTP to the starved cell induced not only an avoiding reaction in swimming, but also induced a synchronous cell division that occurred 2 hours later. An attempt to search for other stimuli, which induced cell division, revealed that mechanical stimulation by a short period of centrifugation was almost as effective as the addition of GTP. The supernatant after centrifugation had the ability to induce cell division, and such activity was abolished after the supernatant was treated with the phosphatase, apyrase, suggesting the release of GTP by the mechanical stimulation. These results indicate that the released GTP binds mainly to the oral area and this then induces the cell division of starved T. thermophila.     

Epigenetic mechanisms affecting macronuclear development in Paramecium and Tetrahymena

Epigenetic inheritance includes all non-Mendelian inheritance, in fact any inheritance that does not arise from base changes. Ciliates, particularly Paramecium and Tetrahymena, undergo epigenetic changes to their macronuclei when they are formed at nuclear reorganization. Once set, however, they are reproduced in a constant fashion, except for allelic segregations, during vegetative fissions in Tetrahymena and certain life cycle changes in both Paramecium and Tetrahymena. This review is meant to be inclusive, discussing all the known cases of epigenetic changes in macronuclei. They involve virtually all traits. We find that these macronuclear changes are subject to a variety of modifications in the way that they are implemented. They constitute a major feature of ciliate genetics, probably because the separation of generative and vegetative functions to micronuclei and macronuclei makes such changes possible.     

Guanylyl cyclases with the topology of mammalian adenylyl cyclases and an N-terminal P-type ATPase-like domain in Paramecium, Tetrahymena and Plasmodium.

We cloned a guanylyl cyclase of 280 kDa from the ciliate Paramecium which has an N-terminus similar to that of a P-type ATPase and a C-terminus with a topology identical to mammalian adenylyl cyclases. Respective signature sequence motifs are conserved in both domains. The cytosolic catalytic C1a and C2a segments of the cyclase are inverted. Genes coding for topologically identical proteins with substantial sequence similarities have been cloned from Tetrahymena and were detected in sequences from Plasmodium deposited by the Malaria Genome Project. After 99 point mutations to convert the Paramecium TAA/TAG-Gln triplets to CAA/CAG, together with partial gene synthesis, the gene from Paramecium was heterologously expressed. In Sf9 cells, the holoenzyme is proteolytically processed into the two domains. Immunocytochemistry demonstrates expression of the protein in Paramecium and localizes it to cell surface membranes. The data provide a novel structural link between class III adenylyl and guanylyl cyclases and imply that the protozoan guanylyl cyclases evolved from an ancestral adenylyl cyclase independently of the mammalian guanylyl cyclase isoforms. Further, signal transmission in Ciliophora (Paramecium, Tetrahymena) and in the most important endoparasitic phylum Apicomplexa (Plasmodium) is, quite unexpectedly, closely related.     

Chemical stimulation of phagocytosis in Amoeba proteus and the influence of external calcium

Summary The process of phagocytosis in Amoeba proteus was examined by following the uptake of Tetrahymena pyriformis and agarose beads. The ciliates are taken up in a time dependent and saturable manner. T. pyriformis apparently emits a water-soluble substance that acts as a chemoattractant to the amoebae. Plain agarose beads are not engulfed by A. proteus, but those beads having reducedglutathione with the -SH group exposed are taken up almost to the same extent as T. pyriformis. Phagocytosis of the glutathione beads is calcium-dependent with maximum bead uptake at 10^-4M Ca⁺⁺. Glutathione applied to A. proteus brings about pseudopod formation, increased phagocytosis and displacement of surface-associated calcium.     

Computation of the internal forces in cilia: application to ciliary motion, the effects of viscosity, and cilia interactions.

This paper presents a simple and reasonable method for generating a phenomenological model of the internal mechanism of cilia. The model uses a relatively small number of parameters whose values can be obtained by fitting to ciliary beat shapes. Here, we use beat patterns observed in Paramecium. The forces that generate these beats are computed and fit to a simple functional form called the "engine." This engine is incorporated into a recently developed hydrodynamic model that accounts for interactions between neighboring cilia and between the cilia and the surface from which they emerge. The model results are compared to data on ciliary beat patterns of Paramecium obtained under conditions where the beats are two-dimensional. Many essential features of the motion, including several properties that are not built in explicitly, are shown to be captured. In particular, the model displays a realistic change in beat pattern and frequency in response to increased viscosity and to the presence of neighboring cilia in configurations such as rows of cilia and two-dimensional arrays of cilia. We found that when two adjacent model cilia start beating at different phases they become synchronized within several beat periods, as observed in experiments where two flagella are brought into close proximity. Furthermore, examination of various multiciliary configurations shows that an approximately antiplectic wave pattern evolves autonomously. This modeling evidence supports earlier conjectures that metachronism may occur, at least partially, as a self-organized phenomenon due to hydrodynamic interactions between neighboring cilia.     

Diaminobenzidine reactivity of mitochondria and peroxisomes in Tetrahymena and in wild-type and cytochrome oxidase-deficient Paramecium.

Reactivity of mitochondria and peroxisomes to diaminobenzidine was investigated in Tetrahymena pyriformis and in wild-type and cytochrome oxidase-deficient Paramecium aurelia. Wild-type and cytochrome oxidase-deficient Paramecium gave positive mitochondrial reactions in the absence of added H2O2, and the deposits were enhanced by the addition of H2O2, whereas Tetrahymena gave positive mitochondrial reactions only upon addition of H2O2. These results are discussed in the light of the current ideas concerning the mechanism of staining by diaminobenzidine. Peroxisome-like organelles which react positively to diaminobenzidine, the reaction being partially inhibited by aminotriazole, were identified in both protozoa.