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: 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.     

Tetrahymena elongation factor-1 alpha is localized with calmodulin in the division furrow

Translation elongation factor 1 alpha (EF-1 alpha) catalyzes the GTP-dependent binding of amino-acyl-tRNA to ribosomes. We previously reported that Tetrahymena EF-1 alpha induced the formation of bundles of rabbit skeletal muscle filamentous actin (F-actin) as well as Tetrahymena F-actin [Kurasawa et al. (1996) Zool. Sci. (Tokyo) 13, 371-375], and that Ca(2+)/calmodulin (CaM) regulated the F-actin-bundling activity of EF-1 alpha [Kurasawa et al. (1996) J. Biochem. 119, 791-798]. In the present study, we investigated the binding between Tetrahymena EF-1 alpha and CaM using a Tetrahymena EF-1 alpha affinity column, and the localization of EF-1 alpha and CaM by indirect immunofluorescence. Only CaM in the Tetrahymena cell extract bound to Tetrahymena EF-1 alpha in a Ca(2+)-dependent manner. In interphase Tetrahymena cells, EF-1 alpha and CaM are colocalized in the crescent structure of the oral apparatus and the apical ring, while in dividing cells, they are colocalized in the division furrow. This is the first report describing the coexistence of EF-1 alpha and CaM in the division furrow, suggesting that EF-1 alpha and CaM are involved in the organization of contractile ring microfilaments during cytokinesis.     

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.     

Investigation of actin in Tetrahymena cells. A comparison with skeletal muscle actin by a devised two-dimensional gel electrophoresis method

Total protein constituents of Tetrahymena thermophila strain B1868 III were studied by two-dimensional agarose-polyacrylamide gel electrophoresis to detect actin among the constituents. In the attempts to prepare a whole-cell extract of Tetrahymena, it was found that protease activity in the extract was so high that high molecular components were quickly digested with the endogenous protease into small peptides unless the homogenization and heat-treatment in a sodium dodecylsulfate solution were performed within 5 s. It was eventually found that employment of 8 M guanidine hydrochloride (HCl) in the homogenization of cells perfectly prevented the degradation of protein components, even through a long preparation procedure. A devised two-dimensional agarose-polyacrylamide gel electrophoresis of the guanidine HCl extract gave a 'protein map' on which most proteins were located in their respective positions, including proteins with more than 200,000 mol. wt. Addition of rabbit skeletal muscle actin on the protein map revealed that no protein with isoelectric point and molecular weight identical with those of the actin was contained in the whole Tetrahymena extract, suggesting that Tetrahymena actin may have characteristics far different from those of skeletal muscle actin.     

Tetrahymena profilin is localized in the division furrow.

Localization of Tetrahymena profilin was examined by an immunofluorescence method. In interphase Tetrahymena cells, immunofluorescence for profilin was diffusely distributed in the cytoplasm, while in dividing cells, additional intense fluorescence was observed in the division furrow. From the result of immunofluorescence localization using cytoskeletal cell models, a significant fraction of profilin appeared to become insoluble in association with a cytoskeletal structure just beneath the division furrow during cytokinesis, although remaining profilin existed as a soluble form in the cytoplasm. Double immunofluorescence staining with anti-profilin and anti-actin antibodies revealed that the localization of profilin in the division furrow coincided with that of contractile ring microfilaments in terms of both position and timing. This is the first report describing the coexistence of profilin with actin filaments in the division furrow, implying the possible involvement of profilin in assembly and disassembly of contractile ring microfilaments in the process of cytokinesis.     

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.     

C-terminal 15 kDa fragment of cytoskeletal actin is posttranslationally N-myristoylated upon caspase-mediated cleavage and targeted to mitochondria

To detect the posttranslational N-myristoylation of caspase substrates, the susceptibility of the newly exposed N-terminus of known caspase substrates to protein N-myristoylation was evaluated by in vivo metabolic labeling with [(3)H]myristic acid in transfected cells using a fusion protein in which the query sequence was fused to a model protein. As a result, it was found that the N-terminal nine residues of the newly exposed N-terminus of the caspase-cleavage product of cytoskeletal actin efficiently direct the protein N-myristoylation. Metabolic labeling of COS-1 cells transiently transfected with cDNA coding for full-length truncated actin (tActin) revealed the efficient incorporation of [(3)H]myristic acid into this molecule. When COS-1 cells transiently transfected with cDNA coding for full-length actin were treated with staurosporine, an apoptosis-inducing agent, an N-myristoylated tActin was generated. Immunofluorescence staining coupled with MitoTracker or fluorescence tagged-phalloidin staining revealed that exogenously expressed tActin colocalized with mitochondria without affecting cellular and actin morphology. Taken together, these results demonstrate that the C-terminal 15 kDa fragment of cytoskeletal actin is posttranslationally N-myristoylated upon caspase-mediated cleavage during apoptosis and targeted to mitochondria.     

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.     

Connexin 43 is localized with actin in tenocytes

Varieties of cell-matrix or cell-cell adhesions are associated with the actin cytoskeleton. However, for gap junctions, which are both channels and adhesions, there has been little evidence for such an association. The purpose of this study was to determine if connexin 43 (Cx43) associates with actin and to determine if this association is altered under mechanical load in tenocytes, a mechanically sensitive cell. Avian tenocytes were subjected to multiple cyclic strain regimens and then fixed and examined immunohistochemically at various times poststrain to determine where Cx43 protein was localized within the cells in relation to actin filaments. Four percent of tenocytes had colocalization of actin filaments and Cx43, which was significantly increased with 5% cyclic strain. To confirm this phenomenon, human tenocytes and COS-7 cells were also subjected to cyclic strain and then fixed at the same times after strain. As with avian tenocytes, Cx43 was colocalized with actin in human tenocytes and COS-7 cells. The colocalization increased significantly after cyclic strain in human tenocytes but not in COS-7 cells. The lack of detectable changes in COS-7 cells may indicate that they are less mechanosensitive than tenocytes perhaps due to the less robust actin cytoskeleton seen in the COS-7 cells when compared to the tenocytes. Furthermore, inhibiting myosin II activity greatly reduced the immunohistochemically-detectable Cx43 on actin filaments. Connexins may associate with actin to stabilize gap junctions at the plasma membrane, ensuring that tenocytes remain coupled during periods of prolonged or intense mechanical loading.     

An unusual fibrillarin gene and protein: structure and functional implications.

The diploid germinal nucleus of the ciliated protozoan Tetrahymena thermophila is unusual among eukaryotes in that it encodes a single copy of the gene for rRNA allowing identification of cis-acting mutations in rDNA affecting rRNA structure, function, and processing. The generally conserved nucleolar protein fibrillarin has been characterized from a number of systems and is involved in pre-rRNA processing. We have demonstrated that Tetrahymena has fibrillarin and have analyzed the cDNA and the genomic DNA encoding this protein. The derived amino acid sequence of the N-terminal region of Tetrahymena fibrillarin shows little similarity with the generally highly conserved glycine/arginine-rich N-terminal domain of other eukaryotic fibrillarins. The remainder of the amino acid sequence of the molecule is more conserved. Polyclonal antibodies generated against the full-length Tetrahymena fibrillarin expressed in bacteria recognize a protein of M(r) approximately 32,000 in whole-cell or nucleolar preparations. Immunocytochemistry localizes fibrillarin to nucleoli in the somatic macronuclei of vegetative cells. Transformation experiments demonstrate that fibrillarin is an essential protein in Tetrahymena. The Tetrahymena fibrillarin is expressed but does not complement a NOP1 null mutation when transformed into the yeast Saccharomyces cerevisiae, indicating less functional conservation among fibrillarins than previously suggested.     

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.     

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.     

The actin gene ACT1 is required for phagocytosis, motility, and cell separation of Tetrahymena thermophila

A previously identified Tetrahymena thermophila actin gene (C. G. Cupples and R. E. Pearlman, Proc. Natl. Acad. Sci. USA 83:5160-5164, 1986), here called ACT1, was disrupted by insertion of a neo3 cassette. Cells in which all expressed copies of this gene were disrupted exhibited intermittent and extremely slow motility and severely curtailed phagocytic uptake. Transformation of these cells with inducible genetic constructs that contained a normal ACT1 gene restored motility. Use of an epitope-tagged construct permitted visualization of Act1p in the isolated axonemes of these rescued cells. In ACT1Delta mutant cells, ultrastructural abnormalities of outer doublet microtubules were present in some of the axonemes. Nonetheless, these cells were still able to assemble cilia after deciliation. The nearly paralyzed ACT1Delta cells completed cleavage furrowing normally, but the presumptive daughter cells often failed to separate from one another and later became reintegrated. Clonal analysis revealed that the cell cycle length of the ACT1Delta cells was approximately double that of wild-type controls. Clones could nonetheless be maintained for up to 15 successive fissions, suggesting that the ACT1 gene is not essential for cell viability or growth. Examination of the cell cortex with monoclonal antibodies revealed that whereas elongation of ciliary rows and formation of oral structures were normal, the ciliary rows of reintegrated daughter cells became laterally displaced and sometimes rejoined indiscriminately across the former division furrow. We conclude that Act1p is required in Tetrahymena thermophila primarily for normal ciliary motility and for phagocytosis and secondarily for the final separation of daughter cells.     

Unusual kinetic and structural properties control rapid assembly and turnover of actin in the parasite Toxoplasma gondii

Toxoplasma is a protozoan parasite in the phylum Apicomplexa, which contains a number of medically important parasites that rely on a highly unusual form of motility termed gliding to actively penetrate their host cells. Parasite actin filaments regulate gliding motility, yet paradoxically filamentous actin is rarely detected in these parasites. To investigate the kinetics of this unusual parasite actin, we expressed TgACT1 in baculovirus and purified it to homogeneity. Biochemical analysis showed that Toxoplasma actin (TgACT1) rapidly polymerized into filaments at a critical concentration that was 3-4-fold lower than conventional actins, yet it failed to copolymerize with mammalian actin. Electron microscopic analysis revealed that TgACT1 filaments were 10 times shorter and less stable than rabbit actin. Phylogenetic comparison of actins revealed a limited number of apicomplexan-specific residues that likely govern the unusual behavior of parasite actin. Molecular modeling identified several key alterations that affect interactions between monomers and that are predicted to destabilize filaments. Our findings suggest that conserved molecular differences in parasite actin favor rapid cycles of assembly and disassembly that govern the unusual form of gliding motility utilized by apicomplexans.     

Gene transfer to mammalian cells using genetically targeted filamentous bacteriophage.

We have genetically modified filamentous bacteriophage to deliver genes to mammalian cells. In previous studies we showed that noncovalently attached fibroblast growth factor (FGF2) can target bacteriophage to COS-1 cells, resulting in receptor-mediated transduction with a reporter gene. Thus, bacteriophage, which normally lack tropism for mammalian cells, can be adapted for mammalian cell gene transfer. To determine the potential of using phage-mediated gene transfer as a novel display phage screening strategy, we transfected COS-1 cells with phage that were engineered to display FGF2 on their surface coat as a fusion to the minor coat protein, pIII. Immunoblot and ELISA analysis confirmed the presence of FGF2 on the phage coat. Significant transduction was obtained in COS-1 cells with the targeted FGF2-phage compared with the nontargeted parent phage. Specificity was demonstrated by successful inhibition of transduction in the presence of excess free FGF2. Having demonstrated mammalian cell transduction by phage displaying a known gene targeting ligand, it is now feasible to apply phage-mediated transduction as a screen for discovering novel ligands.     

A conditional secretory mutant in an Ig L chain is caused by replacement of tyrosine/phenylalanine 87 with histidine.

The kappa-chain of the myeloma MOPC 21 is an unusual L chain, in that it is not secreted unless complexed with a H chain. This nonsecreted kappa-chain seems to be retained in the endoplasmic reticulum in association with the protein BiP/GRP78, both in myeloma cells and when expressed in COS-1 fibroblasts. By assaying the fate of the MOPC 21 kappa-chain and its mutated derivatives in COS-1 cells, we show that the cause of the nonsecreted phenotype is the presence of histidine in position 87 of the variable domain. When this amino acid is changed back to the tyrosine that usually occupies position 87, secretion of the unassembled kappa-chain is restored. As in B lymphoid cells, co-expression of gamma H chains in COS-1 cells complements the mutation in the L chain, and rescues secretion of the arrested kappa. Thus, the presence of histidine at position 87 creates a conditional L chain secretory mutant: it is not compatible with normal transport of free L chain, but can be rescued in the presence of H chain.     

Deafness mutation in the MYO3A motor domain impairs actin protrusion elongation mechanism

Class III myosins are actin-based motors proposed to transport cargo to the distal tips of stereocilia in the inner ear hair cells and/or to participate in stereocilia length regulation, which is especially important during development. Mutations in the MYO3A gene are associated with delayed onset deafness. A previous study demonstrated that L697W, a dominant deafness mutation, disrupts MYO3A ATPase and motor properties but does not impair its ability to localize to the tips of actin protrusions. In the current study, we characterized the transient kinetic mechanism of the L697W motor ATPase cycle. Our kinetic analysis demonstrates that the mutation slows the ADP release and ATP hydrolysis steps, which results in a slight reduction in the duty ratio and slows detachment kinetics. Fluorescence recovery after photobleaching (FRAP) of filopodia tip localized L697W and WT MYO3A in COS-7 cells revealed that the mutant does not alter turnover or average intensity at the actin protrusion tips. We demonstrate that the mutation slows filopodia extension velocity in COS-7 cells which correlates with its twofold slower in vitro actin gliding velocity. Overall, this work allowed us to propose a model for how the motor properties of MYO3A are crucial for facilitating actin protrusion length regulation.