Tetrahymena Actin Research Articles

Expression of GFP-actin leads to failure of nuclear elongation and cytokinesis in Tetrahymena thermophila.

Green fluorescent protein (GFP)-tagged actin was used to investigate the distribution and function of actin in Tetrahymena. A strain that expresses both GFP-actin and endogenous actin was developed by transformation of Tetrahymena thermophila with a ribosomal DNA-based replicative vector. Confocal microscopy of living cells and immunogold electron microscopy confirmed localization of GFP-actin to basal bodies and the contractile ring. Incorporation of the fusion protein into these and other actin-related structures correlated with severe impairment of macronuclear elongation and cytokinesis. At 30 degrees C macronuclear elongation failed to occur in 25% of the transformants despite completion of micronuclear division. At 20 degrees C macronuclear elongation failed to occur in 2% of the population. Arrest of cytokinesis coincided with failure of macronuclear elongation. Arrested cells developed into homopolar doublets with two sets of oral structures. This study indicates a requirement for actin in nuclear elongation and cytokinesis. Although GFP-actin can interfere with the functioning of actin-containing structures, the GFP-actin transformant strain can be used to monitor actin distribution and dynamics and is therefore an important new tool for further studies of Tetrahymena actin.

Recovery of flagellar inner-arm dynein and the fertilization tubule in Chlamydomonas ida5 mutant by transformation with actin genes.

The ida5 mutant of Chlamydomonas, first isolated as a mutant lacking a subset of axonemal inner-arm dyneins, has recently been shown to lack conventional actin owing to a serious mutation in its gene. It lacks inner-arm dyneins probably because actin is an essential subunit for their assembly. In addition, male gametes of ida5 are unable to produce the fertilization tubule, a structure that contains a core of actin filament bundles. To establish that those observed deficiencies are solely attributable to the loss of actin, and to provide a basis for future studies on the actin function in this organism, we examined in this study whether transformation of this mutant with cloned actin genes can rescue the mutant phenotypes. Cotransformation of the double mutant ida5arg2 with the wild-type actin gene and arginino-succinate lyase gene that suppresses the arg2 mutation yielded several transformants that displayed increased motility. All of them were found to have acquired the introduced actin gene in the genome and the product actin in the flagella, and regained the missing inner-arm dyneins and wild-type motility. In addition, most transformants also became able to grow the fertilization tubule when mating reaction was induced. In addition to the wild-type actin gene, we also used a chimeric actin gene in which the N-terminal 12 amino-acid sequence of Chlamydomonas actin was replaced by that of the greatly divergent Tetrahymena actin. Transformants with this gene also resulted in recovery of inner-arm dynein and 70-80% of the wild-type level of motility. These results established that the lack of inner-arm dynein and the fertilization tubule in ida5 are consequences of its loss of conventional actin. Furthermore, they demonstrate that Chlamydomonas offers an excellent experimental system with which to study the structure-function relationship of actin by means of mutant analysis.

Tropomyosin-binding site(s) on the Dictyostelium actin surface as identified by site-directed mutagenesis.

To identify tropomyosin-binding site(s) on the surface of actin molecule, we examined the effect of mutagenesis introduced to subdomain 4 of actin. Because the sequence of Gln228-Ser232 of Dictyostelium actin differs from that of Tetrahymena actin that does not bind tropomyosin, the Dictyostelium/Tetrahymena chimeric actin was produced. Also, Lys238 and Glu241 were replaced with alanine (mutant 645) to study the role of charged residues which are located at both ends of a beta-sheet. As a control experiment, a negative charge was introduced near to the N-terminus (mutant 663). To facilitate the separation of mutant actins without affecting the normal function, Glu360 was replaced with histidine. As a control mutant to such mutants, the mutant 647 (E360H) was produced. Mutant actins were expressed in Dictyostelium cells. All mutant actins were functional: they (i) polymerize and (ii) activate ATPase activity of rabbit skeletal myosin subfragment-1 (S1). The mutant 663 (G2E) showed tropomyosin binding and activated myosin ATPase almost as well as rabbit skeletal actin. However, the tropomyosin binding of the mutant 645 (K238A/E241A/E360H) became magnesium dependent. The chimeric actin (mutant 646: QTAAS-to-KAYKE replacement and E360H) showed decreased tropomyosin binding even in the presence of magnesium ions. These results indicate that the tropomyosin-binding sites of "on"-state actin are on subdomain 4. Surprisingly, the chimeric actin showed more cooperative calcium regulation than rabbit skeletal actin in the presence of tropomyosin-troponin. The mutant actin 645 can hardly activate S1 ATPase irrespective of calcium concentration in the presence of tropomyosin-troponin, even though this actin by itself can activate S1 ATPase. The steric blocking or cooperative/allosteric mechanism of thin filament regulation is discussed.

Immunological detection of actin in the 14S ciliary dynein of Tetrahymena

The association of actin with Tetrahymena ciliary dyneins was examined using a polyclonal antibody against Tetrahymena actin. Western blotting shows that actin is present in the 14S dynein fraction, but not in the 22S dynein fraction, which comprises the outer arm. By anion-exchange chromatography, 14S dynein can be further separated into three major fractions that contain four distinct heavy chains in total. When each fraction was tested by anti-actin immunoblotting, all three fractions contained actin in nearly stoichiometric amounts with the heavy chain. Since Tetrahymena actin differs significantly from acting of other species, the association with inner-arm dynein may be a conserved property of actin.

Localization of actin in the Tetrahymena basal body-cage complex.

In the ciliate cytoskeleton, basal bodies are contained within separate, filamentous cages which are closely associated with basal body microtubules. We have used two polyclonal anti-actin antibodies to localize actin within the basal body-cage complex of Tetrahymena. An antiserum against a Tetrahymena oral apparatus fraction enriched for basal body proteins was produced in rabbits. Agarose-linked chicken muscle actin was used to affinity-purify anti-Tetrahymena actin antibodies from the anti-oral apparatus antiserum. Agarose-linked chicken muscle actin was used to affinity-purify anti-chicken muscle actin antibodies from a commercially available antiserum against chicken muscle actin. Both affinity-purified antibodies were monospecific for Tetrahymena actin on immunoblots containing total oral apparatus protein. The anti-actin antibodies were localized to both somatic and oral basal bodies in Tetrahymena by immunofluorescence microscopy. At the ultrastructural level with the immunogold technique, these antibodies labeled actin epitopes in four distinct regions of the basal body-cage complex: (a) basal body walls, (b) basal plate filaments, (c) proximal-end filaments and (d) cage wall filaments. In addition, the antibody labeled filament bundles that interconnect groups of basal bodies (membranelles) within the oral apparatus. Identical labeling patterns were observed with basal bodies in the isolated oral apparatus, basal bodies in the in situ oral apparatus and somatic basal bodies in situ. Quantitative analysis of gold particle distribution was used to demonstrate the specificity of the antibodies for the basal body-cage complex and to show that non-specific binding of the antibodies was negligible. Preadsorption of the antibody with muscle actin effectively eliminated the capacity of the antibody to bind to proteins on immunoblots and to basal body structures with the immunogold labeling technique. These results provide evidence for actin in the basal body-cage complex and raise the possibility of a contractile system associated with basal bodies.

A chimeric actin carrying N-terminal portion of tetrahymena actin does not bind to DNAse I

A chimeric actin gene was constructed from Tetrahymena actin sequence corresponding to residues 1-83 and Dictyostelium actin sequence corresponding to residues 84-375, and the gene was expressed in Dictyostelium cells. Using DNase I-affinity column, we revealed that the product of the chimeric actin gene was not retained in the column whereas intrinsic actin was retained. In conjunction with our previous data that Tetrahymena actin does not interact with DNase I [Hirono, M., Kumagai, Y., Numata, O., & Watanabe Y. (1989) Proc. Natl. Acad. Sci. U.S. 86, 75-79], we suggest that the binding site of DNase I in an ubiquitous actin is located in N-terminal region (residues 1-83).

Expression of tetrahymena actin in mammalian cells

We previously revealed that Tetrahymena actin can copolymerize with rabbit skeletal muscle actin whereas it has a very divergent primary structure and some unusual properties. To investigate the effects of coexistence of this unusual Tetrahymena actin in mammalian cells, we here transfected Tetrahymena actin gene on an expression vector into COS-1 cells. From the results of immunofluorescence microscopy, it was found that Tetrahymena actin expressed in COS-1 cells copolymerized with intrinsic actin, and it was conspicuously localized to the tips of microfilament core bundles in microspikes. On the other hand, increase in cell number tended to cease temporarily about 24 hr after transfection with Tetrahymena actin gene, implying the inhibition of cytokinesis by Tetrahymena actin coexistence.

Timing of formation of Tetrahymena contractile ring microfilaments investigated by inhibition with skeletal muscle actin

AbstractUnderstanding the mechanism that determines the cell division plane is one of the most important problems in the fields of cell and developmental biology. Studying the timing and site of formation of contractile ring (CR) micro‐filaments provides key information for solving the problem. We tried to create a nonfunctional CR in Tetrahymena by microinjecting rabbit skeletal muscle actin, which can copolymerize with Tetrahymena actin but has properties different from those of Tetrahymena actin. When skeletal muscle actin was injected in a predivision stage, before the onset of furrow constriction, long‐term arrest of cell division was observed. Muscle actin did not cause any delay in cell division when the actin was injected at any stage other than the predivision stage. In all cases, muscle actin had little affect on other actin‐related functions. Injected skeletal muscle actin polymerized near the equatorial division plane in cases of cell division arrest; it polymerized at other nonspecific locations when cell division was observed. Arrest occurred when the microinjection took place in the 17‐min period just before the start of furrowing. This period coincides with the occurrence of equatorial deposits of p85, which is also suggested to be required for the determination of the division plane. The present experimental results are consistent with the idea that p85 is a crucial factor for determining the cell division plane and also functions as a polymerization nucleus for CR microfilaments. © 1992 Wiley‐Liss, Inc.

An unusual actin-encoding gene in Physarum polycephalum

Actin is one of the most conserved proteins in eukaryotic organisms. In the present work, we cloned and determined the nucleotide sequence of an unusual actin-encoding gene, ardD, from the slime mold, Physarum polycephalum. The ardD gene encodes an ArdD protein containing 367 amino acids (aa) instead of the 375–376 aa found in a typical actin. The nine missing aa are accounted for by deletions of three aa in the first exon, five in the fifth exon and one in the sixth exon. These deletions in the coding sequence were observed in a polymerase chain reaction (PCR)-generated cDNA fragment, which excludes the possibility of a cloning artifact. In addition, ArdD contains numerous aa substitutions distributed throughout the protein. The ArdD aa sequence was compared with published actin sequences. The most identity is seen with the P. polycephalum ArdA, ArdB and ArdC (84%) and Acanthamoeba (82%) actins, while the least identity is found with Tetrahymena actin (67%). The expression of the ardD gene is developmentally regulated. The highest levels of ardD mRNA were found in spherules, less was seen in plasmodia and no detectable transcripts were observed in amoebae. The PCR amplification of an ardD cDNA from spherules confirmed the presence of mRNA in this developmental stage. The aa deletions and substitutions in the predicted ArdD aa sequence make it one of the most distinctive actins known.

Purification and characterization of Tetrahymena profilin

We subjected Tetrahymena cell extract to a poly(L-proline) affinity column for isolating profilin and obtained a protein of 12.8 kDa. Purified 12.8 kDa protein dose-dependently inhibited the polymerization of Tetrahymena actin more strongly than that of rabbit skeletal muscle actin. Because the 12.8 kDa protein fulfills properties common to profilins, the protein is considered to be Tetrahymena profilin. The present paper is the first report of the isolation of an actin-binding protein from Tetrahymena.

Tetrahymena Actin: Copolymerization with Skeletal Muscle Actin and Interactions with Muscle Actin-Binding Proteins

We have previously shown that actin from Tetrahymena pyriformis has a very divergent primary structure (Hirono, M., Endoh, H., Okada, N., Numata, O., & Watanabe, Y. (1987) J. Mol. Biol. 194, 181-192) and that though it shares essential properties with skeletal muscle actin, it does not interact at all with phalloidin or DNase I (Hirono, M., Kumagai, Y., Numata, O., & Watanabe, Y. (1989) Proc. Natl. Acad. Sci. U.S. 86, 75-79). In this study, we investigated the copolymerization of this actin with skeletal muscle actin by direct observation of the heteropolymers formed from the two actins by means of electron microscopy. We also examined the binding of actin-binding proteins from skeletal muscle or smooth muscle to Tetrahymena actin by means of a cosedimentation assay. The results show that (i) Tetrahymena actin copolymerizes with skeletal muscle actin and that (ii) muscle myosin subfragment 1 binds to it in the absence of ATP, like skeletal muscle actin. However, it was also shown that (iii) muscle alpha-actinin hardly binds to Tetrahymena actin and that (iv) muscle tropomyosin does not bind to it at all. The results show that Tetrahymena actin has both properties similar and dissimilar to those of skeletal muscle actin.

Purification of Tetrahymena actin reveals some unusual properties.

Actin from Tetrahymena pyriformis has been purified by monitoring the presence of the actin gene product with an antiserum against a synthetic N-terminal peptide deduced from the nucleotide sequence of the Tetrahymena actin gene that we cloned previously. This highly purified Tetrahymena actin shares many essential properties with ubiquitous actin, including ion-dependent polymerization to microfilaments, binding with muscle heavy meromyosin to form arrowheads, and activation of the Mg2+-ATPase of muscle myosin subfragment 1. On the other hand, some properties of this purified Tetrahymena actin clearly differ from those of muscle actin: (i) Tetrahymena actin has 8 times less ability to activate the Mg2+-ATPase of muscle myosin subfragment 1 than muscle actin; (ii) Tetrahymena actin did not bind to phalloidin at all; (iii) Tetrahymena actin did not inhibit DNase I activity at all. In general, Tetrahymena actin has very unusual properties when compared to other actins described so far. This actin is expected to provide important clues for elucidating problems concerning the relationships between the structural and functional domains in an actin molecule.

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.

Tetrahymena actin : Cloning and sequencing of the Tetrahymena actin gene and identification of its gene product

Actin is ubiquitous in eukaryotes, nevertheless its existence has not yet been clearly proven in Tetrahymena. Here we report the cloning and sequencing of an actin gene from the genomic library of Tetrahymena pyriformis using a Dictyostelium actin gene as a probe. The Tetrahymena actin gene has no intron. The predicted actin is composed of 375 amino acids like other actins and its molecular weight is estimated as 41,906. Both T. pyriformis and T. thermophila possess a single species of actin genes which differ in their restriction patterns. Northern hybridization analysis revealed that the actin gene was actively transcribed in vivo. To detect the gene product, we synthesized an N-terminal peptide of the deduced sequence and prepared its antibody. Using an immunoblotting technique, we identified Tetrahymena actin on a two-dimensional gel electrophoretic plate. The actin spot migrated near an added spot of rabbit skeletal muscle actin, but clearly differed from the latter in its isoelectric point and apparent molecular weight. The primary structure of Tetrahymena actin shares about 75% homology equally with those of other representative actins. This value is extremely low as a homology rate between known actins. Tetrahymena actin diverges not only in relatively variable regions of other actins, but also in relatively constant regions. The hydrophilicity levels of two regions (residues 190 to 200 and residues 225 to 235) are also quite different between the Tetrahymena actin and skeletal muscle actin. Thus, we conclude that actin is present in Tetrahymena, but it is one of the most unique actins among the actins known hereto.

In vivo identification of Tetrahymena actin probed by DMSO induction of nuclear bundles

We report the first successful identification of actin, an ubiquitous contractile protein, in Tetrahymena pyriformis (strain W). We employed dimethyl sulfoxide (DMSO) as a probe to induce the formation of actin bundles in the cell nucleus [1, 2] through disruption of cytoplasmic microfilament organization [3, 4]. The cells were incubated for 30 min at 22 °C in the inorganic medium of Prescott & James [5] containing 10% DMSO, and observed under a transmission electron microscope (TEM). Microfilarment bundles were formed in interphase macronuclei, and these microfilaments, approx. 6 nm in diameter, could be decorated by rabbit skeletal muscle heavy meromyosin (HMM) in the glycerinated model. In many cases, the bundles formed closely parallel to natively existing bundles of microtubules. Interestingly, these microtubules had prominent striation with 15–16 nm periodicity. SDS-polyacrylamide gel electrophoresis was designed to show the low actin content of Tetrahymena cells in comparison with that of Dictyostelium. Actin was suggested to comprise less than 1.7% of the total protein in Tetrahymena, whereas as much as 6% was actin in Dictyostelium cells. In assessing the physiological significance of the bundle formation, we further performed HMM and myosin subfragment-1 (S1)-binding studies to clarify the organization process and the polarity of the DMSO-induced nuclear actin filaments by using the tannic acid staining technique [6]. Randomly oriented short filaments appeared in the nucleus treated with 10% DMSO for 10 min. These filaments became elongated and associated with each other to form loose bundles in the following 10 min. With 30-min treatment, the filaments were organized and large bundles with single axes developed. With these well-developed bundles, the Student's t-test was performed on 172 pairs of neighboring filaments and the probability ( p) of the deviation from random polarity was 0.08, suggesting that the filaments were organized in an anti-parallel manner. The results show that the DMSO induction of nuclear actin is a powerful tool to demonstrate the existence of cellular actin in vivo and to study the mechanism of microfilament organization in relation to cell physiological activities.