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.     

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.     

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).     

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.     

Identification of a novel actin-related protein in Tetrahymena cilia

Actin is an ancient cytoskeletal protein that plays many essential roles in cell motility. In eukaryotes, its gene belongs to a highly conserved gene family, while the protein is also a member of an actin superfamily comprising various kinds of actin-related proteins (Arps). A ciliate, Tetrahymena, has a unique conventional actin. Data from the TIGR Tetrahymena genome project and our own research suggest the existence of 12 actin-like sequences: four conventional actins, two of Arp4, one each of Arp1, Arp2, Arp3, Arp5, and Arp6, and a novel actin-related protein, tArp. We cloned the entire cDNA sequences of Tetrahymena Arp2 (tArp2), Tetrahymena Arp3 (tArp3), and tArp for the work described herein. In phylogenetic analyses, tArp was not included in any Arp subfamily. Unlike other known Arps, tArp localizes in cilia, and its expression was upregulated after deciliation. To see the precise localization of tArp, cilia were fractionated and analyzed using specific antibodies. tArp was observed preferentially in the "outer-doublet" fraction, while actin was found in the "crude-dynein" fraction. In immunoelectron microscopy, most of the gold particles were found either on the outer-doublet or central-pair microtubules. These results suggest that tArp is a ciliary component and that it has a unique function in the formation and maintenance of cilia.     

Amino acid sequence of Acanthamoeba actin

By amino acid sequence studies, only one form of cytoplasmic actin was detected in Acanthamoeba castellanii. Its amino acid sequence is very similar to the sequences of Dictyostelium and Physarum actins, from which Acanthamoeba actin differs in only nine and seven residues, respectively, including the deletion of the first residue. Acanthamoeba actin is unique in containing a blocked NH2-terminal neutral amino acid (glycine), while all other actins sequenced thus far have a blocked acidic amino acid (aspartic or glutamic) at the NH2 terminus. Acanthamoeba actin is also unique in that it contains an N epsilon-trimethyllysine residue at position 326. Like other actins, Acanthamoeba actin contains an NT-methylhistidine residue at position 73. The protein sequence is in complete agreement with the sequence derived from the nucleotide sequence of an expressed actin gene.     

Evolutionarily divergent, unstable filamentous actin is essential for gliding motility in apicomplexan parasites

Apicomplexan parasites rely on a novel form of actin-based motility called gliding, which depends on parasite actin polymerization, to migrate through their hosts and invade cells. However, parasite actins are divergent both in sequence and function and only form short, unstable filaments in contrast to the stability of conventional actin filaments. The molecular basis for parasite actin filament instability and its relationship to gliding motility remain unresolved. We demonstrate that recombinant Toxoplasma (TgACTI) and Plasmodium (PfACTI and PfACTII) actins polymerized into very short filaments in vitro but were induced to form long, stable filaments by addition of equimolar levels of phalloidin. Parasite actins contain a conserved phalloidin-binding site as determined by molecular modeling and computational docking, yet vary in several residues that are predicted to impact filament stability. In particular, two residues were identified that form intermolecular contacts between different protomers in conventional actin filaments and these residues showed non-conservative differences in apicomplexan parasites. Substitution of divergent residues found in TgACTI with those from mammalian actin resulted in formation of longer, more stable filaments in vitro. Expression of these stabilized actins in T. gondii increased sensitivity to the actin-stabilizing compound jasplakinolide and disrupted normal gliding motility in the absence of treatment. These results identify the molecular basis for short, dynamic filaments in apicomplexan parasites and demonstrate that inherent instability of parasite actin filaments is a critical adaptation for gliding motility.     

Isolation and identification of an actin gene from Babesia gibsoni

Actin is a ubiquitous and highly conserved microfilament protein that is hypothesized to play a mechanical force-generating role in the unusual gliding motility of sporozoan zoites and their active penetration of host cells. We have identified and isolated an actin gene from a Babesia gibsoni cDNA library by random sequencing. The complete nucleotide sequence of the actin gene is 1,243 bp; a single open reading frame encodes a polypeptide of 377 amino acid residues. The deduced amino acid sequence showed a high homology with actins from other species, especially with reported apicomplexan protozoans. The antiserum against recombinant actin expressed in Escherichia coli recognizes a 42-kDa native protein, which is consistent with its expected size. Immunofluorescence and confocal microscopic observation revealed that the protein is diffusely distributed throughout the B. gibsoni parasites.     

Structure and expression of an actin gene of Physarum polycephalum

Physarum polycephalum (strain M3CVIII) contains four unlinked actin gene loci, each with two alleles (ardA1, ardA2, ardB1, ardB2, ardC1, ardC2, ardD1 and ardD2). The 4800 base HindIII fragment of the ardC2 allele was previously isolated as a recombinant phage lambda. We now report the structure of the actin gene sequences (C-actin gene). The gene, which contains four intervening sequences, codes for the principal actin isotype of plasmodia and it is expressed in both the haploid myxamoebal and diploid plasmodial phases of the life cycle. The C-actin isotype is closely related to actins of Dictyostelium, Acanthamoebae, Drosophila, sea urchin and mammalian cytoplasmic actin, and more distantly related to actins of yeast, Entamoebae and Tetrahymena. The ardC1 and ardC2 alleles differ by a 700(+/- 100) base-pair insertion/deletion in the vicinity of the 3' end of the transcribed region of the gene.     

Induction of anti-actin drug resistance in Tetrahymena

Both cytochalasin D and latrunculin B reversibly inhibited Tetrahymena phagocytosis at concentrations similar to those effective in mammalian systems, even though ciliate actins are known to be highly divergent from mammalian actins. Overnight exposure to relatively low (0.25 microM) concentrations of latrunculin B induced resistance in Tetrahymena to the inhibitory effects of that drug, as well as cross-resistance to cytochalasin D. However, much higher (> 30 microM) concentrations of cytochalasin D were required for induction of cross-resistance to latrunculin B. Anti-actin drug resistance in Tetrahymena may involve a general multidrug resistance mechanism and/or specific feedback regulation of F-actin assembly and stability.     

Tetrahymena fimbrin localized in the division furrow bundles actin filaments in a calcium-independent manner

In cytokinesis, the contractile ring constricts the cleavage furrow. However, the formation and properties of the contractile ring are poorly understood. Fimbrin has two actin-binding domains and two EF-hand Ca(2+)-binding motifs. Ca(2+) binding to the EF-hand motifs inhibits actin-binding activity. In Tetrahymena, fimbrin is localized in the cleavage furrow during cytokinesis. In a previous study, Tetrahymena fimbrin was purified with an F-actin affinity column. However, the purified Tetrahymena fimbrin was broken in to a 60 kDa fragment of a 70 kDa full length fimbrin. In this study, we investigated the properties of recombinant Tetrahymena fimbrin. In an F-actin cosedimentation assay, Tetrahymena fimbrin bound to F-actin and bundled it in a Ca(2+)-independent manner, with a K(d) of 0.3 micro M and a stoichiometry at saturation of 1:1.4 (Tetrahymena fimbrin: actin). In the presence of 1 molecule of Tetrahymena fimbrin to 7 molecules of actin, F-actin was bundled. Immunofluorecence microscopy showed that a dotted line of Tetrahymena fimbrin along the cleavage furrow formed a ring structure. The properties and localization of Tetrahymena fimbrin suggest that it bundles actin filaments in the cleavage furrow and plays an important role in contractile ring formation during cytokinesis.     

Evolution of the chordate muscle actin gene

Summary The ascidians Styela plicata, S. clava, and Mogula citrina are urochordates. The larvae of urochordates are considered to morphologically resemble the ancestral vertebrate. We asked whether larval and adult ascidian muscle actin sequences are nonmusclelike as in lower invertebrates, musclelike as in vertebrates, or possess characteristics of both. Nonmuscle and muscle actin cDNA clones from S. plicata were sequenced. Based on 27 diagnostic amino acids, which distinguish vertebrate muscle actin from other actins, we found that the deduced protein sequences of ascidian muscle actins exhibit similarities to both invertebrate and vertebrate muscle actins. A comparison to muscle actins from different vertebrate and invertebrate phylogenetic groups suggested that the urochordate muscle actins represent a transition from a nonmusclelike sequence to a vertebrate musclelike sequence. The ascidian adult muscle actin is more similar to skeletal actin and the larval muscle actin is more similar to cardiac actin, which indicates that the divergence of the skeletal and cardiac isoforms occurred before the emergence of urochordates. The muscle actin gene may be a powerful probe for investigating the chordate lineage. Offprint requests to: C.R. Tomlinson     

Formins filter modified actin subunits during processive elongation

Fission yeast cells reject actin subunits tagged with a fluorescent protein from the cytokinetic contractile ring, so cytokinesis fails and the cells die when the native actin gene is replaced by GFP-actin. The lack of a fluorescent actin probe has prevented a detailed study of actin filament dynamics in contractile rings, and left open questions regarding the mechanism of cytokinesis. To incorporate fluorescent actin into the contractile ring to study its dynamics, we introduced the coding sequence for a tetracysteine motif (FLNCCPGCCMEP) at 10 locations in the fission yeast actin gene and expressed the mutant proteins from the native actin locus in diploid cells with wild-type actin on the other chromosome. We labeled these tagged actins inside live cells with the FlAsH reagent. Cells incorporated some of these labeled actins into actin patches at sites of endocytosis, where Arp2/3 complex nucleates all of the actin filaments. However, the cells did not incorporate any of the FlAsH-actins into the contractile ring. Therefore, formin Cdc12p rejects actin subunits with a tag of ~2 kDa, illustrating the stringent structural requirements for this formin to promote the elongation of actin filament barbed ends as it moves processively along the end of a growing filament.     

The Molecular Evolution of Actin

We have investigated the molecular evolution of plant and nonplant actin genes comparing nucleotide and amino acid sequences of 20 actin genes. Nucleotide changes resulting in amino acid substitutions (replacement substitutions) ranged from 3-7% for all pairwise comparisons of animal actin genes with the following exceptions. Comparisons between higher animal muscle actin gene sequences and comparisons between higher animal cytoplasmic actin gene sequences indicated less than 3% divergence. Comparisons between plant and nonplant actin genes revealed, with two exceptions, 11-15% replacement substitution. In the analysis of plant actins, replacement substitution between soybean actin genes SAc1, SAc3, SAc4 and maize actin gene MAc1 ranged from 8-10%, whereas these members within the soybean actin gene family ranged from 6-9% replacement substitution. The rate of sequence divergence of plant actin sequences appears to be similar to that observed for animal actins. Furthermore, these and other data suggest that the plant actin gene family is ancient and that the families of soybean and maize actin genes have diverged from a single common ancestral plant actin gene that originated long before the divergence of monocots and dicots. The soybean actin multigene family encodes at least three classes of actin. These classes each contain a pair of actin genes that have been designated kappa (SAc1, SAc6), lambda (SAc2, SAc4) and mu (SAc3, SAc7). The three classes of soybean actin are more divergent in nucleotide sequence from one another than higher animal cytoplasmic actin is divergent from muscle actin. The location and distribution of amino acid changes were compared between actin proteins from all sources. A comparison of the hydropathy of all actin sequences, except from Oxytricha, indicated a strong similarity in hydropathic character between all plant and nonplant actins despite the greater number of replacement substitutions in plant actins. These protein sequence comparisons are discussed with respect to the demonstrated and implicated roles of actin in plants and animals, as well as the tissue-specific expression of actin.     

Subcellular sorting of isoactins: selective association of gamma actin with skeletal muscle mitochondria

We describe two subpopulations of actin antibodies isolated by affinity chromatography from a polyclonal antibody to chicken gizzard actin. One subpopulation recognizes gamma actins from smooth muscle and nonmuscle cells, but does not recognize alpha actin from skeletal muscle. The other subpopulation recognizes determinants that are common to alpha actin from skeletal muscle and the two gamma actin isotypes. Neither antibody recognizes cytoplasmic beta actin. Both antibodies recognize only actins or molecules with determinants that are also present in actins. By immunofluorescence we found that the anti-gamma actin colocalizes with mitochondria in fibers of mouse diaphragm, and that it does not bind detectably to the 1 bands of sarcomeres. The antibody that recognizes both alpha and gamma actins stains 1 bands intensely, as expected. We interpret these observations as preliminary evidence for selective association of gamma actin with skeletal muscle mitochondria and, more broadly, as evidence for subcellular sorting of isoactins.