Molecular cloning of actin genes in Trichomonas vaginalis and phylogeny inferred from actin sequences

The parasitic protozoan Trichomonas vaginalis is known to contain the ubiquitous and highly conserved protein actin. A genomic library and a cDNA library have been screened to identify and clone the actin gene(s) of T. vaginalis. The nucleotide sequence of one gene and its flanking regions have been determined. The open reading frame encodes a protein of 376 amino acids. The sequence is not interrupted by any introns and the promoter could be represented by a 10 bp motif close to a consensus motif also found upstream of most sequenced T. vaginalis genes. The five different clones isolated from the cDNA library have similar sequences and encode three actin proteins differing only by one or two amino acids. A phylogenetic analysis of 31 actin sequences by distance matrix and parsimony methods, using centractin as outgroup, gives congruent trees with Parabasala branching above Diplomonadida.     

Identification of Atg8 Isoform in Encysting Acanthamoeba

Autophagy-related protein 8 (Atg8) is an essential component of autophagy formation and encystment of cyst-forming parasites, and some protozoa, such as, Acanthamoeba, Entamoeba, and Dictyostelium, have been reported to possess a type of Atg8. In this study, an isoform of Atg8 was identified and characterized in Acanthamoeba castellanii (AcAtg8b). AcAtg8b protein was found to encode 132 amino acids and to be longer than AcAtg8 protein, which encoded 117 amino acids. Real-time PCR analysis showed high expression levels of AcAtg8b and AcAtg8 during encystation. Fluorescence microscopy demonstrated that AcAtg8b is involved in the formation of the autophagosomal membrane. Chemically synthesized siRNA against AcAtg8b reduced the encystation efficiency of Acanthamoeba, confirming that AcAtg8b, like AcAtg8, is an essential component of cyst formation in Acanthamoeba. Our findings suggest that Acanthamoeba has doubled the number of Atg8 gene copies to ensure the successful encystation for survival when 1 copy is lost. These 2 types of Atg8 identified in Acanthamoeba provide important information regarding autophagy formation, encystation mechanism, and survival of primitive, cyst-forming protozoan parasites.     

Crystallization of a non-muscle actin

Eukaryotic cells contain a protein which specifically inhibits DNAsae I. This inhibitor protein is closely related to muscle actin. As shown here the inhibitor purified from calf spleen consists of two polypeptides: one which closely resembles muscle actin and a second (15,000 to 20,000 in molecular weight) whose combination with actin has not been recognized before. Crystals of the complex were obtained in ammonium sulphate. They belong to the orthorhombic space group P2₁2₁2₁, have unit cell dimensions of $\text{a = 187.4}\text{A}\text{?}\text{, b = 72.33}\text{A}\text{?}\text{and}\text{c = 38.19}\text{A}\text{?}$, and have one molecule of the spleen actin associated with one molecule of the low molecular weight protein per asymmetric unit.     

Molecular characterization of mutant actin genes which induce heat-shock proteins in Drosophila flight muscles

Heat-shock proteins (hsps) are constitutively induced by the mutant actins in the Drosophila indirect flight muscles (IFM). We compared primary structures of the mutant actin genes (KM75 and HH5) which induce hsps and of the non-inducing alleles (KM129 and KM88). The KM75 actin has lost 20 amino acids at the C-terminus. The HH5 actin has only one amino acid substitution, from Gly-336 to Ser. In KM129, the C-terminal part of actin is replaced by novel amino acids. KM88 is a null allele, with an amber mutation early in the coding region of the mutated actin gene. Although all of the KM75, HH5 and KM129 actins have defects near the C-terminus, only hsp-inducing mutant actins cause enlargement of the IFM nuclei as well as a disruption of myofibrils even in the presence of two copies of the normal genes. We further consider the underlying mechanisms linking these features of the hsp-inducing alleles.     

Genotyping, physiological features and proteolytic activities of a potentially pathogenic Acanthamoeba sp. isolated from tap water in Brazil

Acanthamoeba spp., known to cause keratitis and granulomatous encephalitis in humans, are frequently isolated from a variety of water sources. Here we report for the first time the characterization of an Acanthamoeba sp. (ACC01) isolated from tap water in Brazil. This organism is currently being maintained in an axenic growth medium. Phylogenetic analysis based on SSU rRNA gene sequences positioned the new isolate in genotype T4, closest to the keratitis-causing isolate, A. polyphaga ATCC 30461 (∼99% similarity). Acanthamoeba ACC01 and A. polyphaga 30461 both grew at 37 °C and were osmotically resistant, multiplying in hyperosmolar medium. Both isolates secreted comparable amounts of proteolytic enzymes, including serine peptidases that were optimally active at a near neutral/alkaline pH and resolved identically in gelatin gels. Incubation of gels at pH 4.0 with 2 mM DTT also indicated the secretion of similar cysteine peptidases. Altogether, the results point to the pathogenic potential of Acanthamoeba ACC01.     

Discovery of myosin I and Pollard-san

In this short review, I describe a brief history of the discovery of myosin I isolated from Acanthamoeba in 1973 by Tom Pollard and Ed Korn. Today, myosins form a large “family tree” that includes more than 30 types of myosins. I discuss the importance of the relationship among actin, myosin, and other actin-binding proteins, many of which were pioneered by Pollard-san (“-san” is a Japanese honorific suffix showing respect, politeness and friendship). At the first conference devoted to actin, Pollard-san, Korn-san, and I discussed the importance of the nucleotide bound at the two ends of the actin filament. I conclude that life is a dynamic accumulation of molecule-molecule bindings, and although we do not yet know how they coordinate with each other to operate a living cell, many enthusiastic and excellent researchers like Pollard-san will unveil mechanisms that will show us what life really looks like.     

Oxidation and reduction of actin: Origin,impact in vitro and functional consequences in vivo

Actin is among the most abundant proteins in eukaryotic cells and assembles into dynamic filamentous networks regulated by many actin binding proteins. The actin cytoskeleton must be finely tuned, both in space and time, to fulfill key cellular functions such as cell division, cell shape changes, phagocytosis and cell migration. While actin oxidation by reactive oxygen species (ROS) at non-physiological levels are known for long to impact on actin polymerization and on the cellular actin cytoskeleton, growing evidence shows that direct and reversible oxidation/reduction of specific actin amino acids plays an important and physiological role in regulating the actin cytoskeleton. In this review, we describe which actin amino acid residues can be selectively oxidized and reduced in many different ways (e.g. disulfide bond formation, glutathionylation, carbonylation, nitration, nitrosylation and other oxidations), the cellular enzymes at the origin of these post-translational modifications, and the impact of actin redox modifications both in vitro and in vivo. We show that the regulated balance of oxidation and reduction of key actin amino acid residues contributes to the control of actin filament polymerization and disassembly at the subcellular scale and highlight how improper redox modifications of actin can lead to pathological conditions.     

Molecular evolution of two actin genes from carrot

We have isolated and sequenced two full-length cDNA clones encoding actin from carrot. The two carrot clones are almost identical at the nucleotide level, and are quite homologous to each other and to other plant actins at the amino acid level. In those regions where amino acid variation exists between the two genes from carrot, the differences have arisen from very simple changes at the nucleotide level. The most common changes are nucleotide insertion(s) coupled to the deletion of a different nucleotide(s) nearby in the DNA sequence, resulting in the restoration of the proper reading frame for the protein; thus, these changes can be viewed as multiple or coupled frameshift mutations. There are almost no base substitutions between the two carrot genes. In contrast to this, when the carrot actin nucleotide sequences are compared to those of a soybean actin gene or a maize actin gene, many base substitutions are observed (ca. 21.8% and 23.5%), more than half of which are third base changes which do not alter the protein sequence. At the amino acid level, both carrot genes show greater similarity to maize actin than they do to soybean actin, thus reinforcing the idea that plant actin genes diverged from a single common ancestral actin gene prior to the divergence of monocots and dicots.     

Treadmilling of actin at physiological salt concentrations: An analysis of the critical concentrations of actin filaments

Treadmilling of actin was investigated at physiological salt concentrations (100 mm-KCl, 0.5 to 2.0 mm-MgCl₂, 200 μm-ethyleneglycol-bis(β-aminoethyl ether)N,N′-tetraacetic acid or 50 μm-CaCl₂ at 37 °C. The concentration at which monomers bind to the lengthening end of filaments with the same rate as subunits are released (low critical concentration c₁ was determined by mixing unmodified actin filaments with various concentrations of monomeric actin labeled with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. Above a monomeric actin concentration of about 0.12 μm, incorporation of actin molecules into filaments was detected, whereas below this concentration no incorporation was found (c₁ = 0.12 μm). Combination of various concentrations of labeled monomers with labeled filaments permitted determination of the net critical concentration ($\text{c}{}_1$) at which filaments lengthen at one end with the same rate as they shorten at the other end ($\text{c}{}_1\text{= 0.16}\text{μm}$). A lower limit of the high critical concentration at the shortening end (c_h) was estimated by measuring the release of subunits from labeled filaments in the presence of various concentrations of unlabeled monomers (c_h >0.5 μm). The differences in the three critical concentrations demonstrate that under physiological conditions actin filaments lengthen at one end by, on the average, one subunit during the time that four association reactions take place at the two ends (efficiency parameters $\text{s =}\text{1}\text{4}$). The small difference between the low and the net critical concentration suggests that the rates of both association and dissociation are considerably greater at the lengthening end than at the shortening end of actin filaments.     

A Gly65Val substitution in an actin,GhACT_LI1, disrupts cell polarity and F‐actin organization resulting in dwarf,lintless cotton plants

Actin polymerizes to form part of the cytoskeleton and organize polar growth in all eukaryotic cells. Species with numerous actin genes are especially useful for the dissection of actin molecular function due to redundancy and neofunctionalization. Here, we investigated the role of a cotton (Gossypium hirsutum) actin gene in the organization of actin filaments in lobed cotyledon pavement cells and the highly elongated single‐celled trichomes that comprise cotton lint fibers. Using mapping‐by‐sequencing, virus‐induced gene silencing, and molecular modeling, we identified the causative mutation of the dominant dwarf Ligon lintless Li₁ short fiber mutant as a single Gly65Val amino acid substitution in a polymerization domain of an actin gene, GhACT_LI1 (Gh_D04G0865). We observed altered cell morphology and disrupted organization of F‐actin in Li₁ plant cells by confocal microscopy. Mutant leaf cells lacked interdigitation of lobes and F‐actin did not uniformly decorate the nuclear envelope. While wild‐type lint fiber trichome cells contained long longitudinal actin cables, the short Li₁ fiber cells accumulated disoriented transverse cables. The polymerization‐defective Gly65Val allele in Li₁ plants likely disrupts processive elongation of F‐actin, resulting in a disorganized cytoskeleton and reduced cell polarity, which likely accounts for the dominant gene action and diverse pleiotropic effects associated with the Li₁ mutation. Lastly, we propose a model to account for these effects, and underscore the roles of actin organization in determining plant cell polarity, shape and plant growth.     

Construction of EST Database for Comparative Gene Studies of Acanthamoeba

The genus Acanthamoeba can cause severe infections such as granulomatous amebic encephalitis and amebic keratitis in humans. However, little genomic information of Acanthamoeba has been reported. Here, we constructed Acanthamoeba expressed sequence tags (EST) database (Acanthamoeba EST DB) derived from our 4 kinds of Acanthamoeba cDNA library. The Acanthamoeba EST DB contains 3,897 EST generated from amebae under various conditions of long term in vitro culture, mouse brain passage, or encystation, and downloaded data of Acanthamoeba from National Center for Biotechnology Information (NCBI) and Taxonomically Broad EST Database (TBestDB). The almost reported cDNA/genomic sequences of Acanthamoeba provide stand alone BLAST system with nucleotide (BLAST NT) and amino acid (BLAST AA) sequence database. In BLAST results, each gene links for the significant information including sequence data, gene orthology annotations, relevant references, and a BlastX result. This is the first attempt for construction of Acanthamoeba database with genes expressed in diverse conditions. These data were integrated into a database (http://www.amoeba.or.kr).     

Genomic organization and evolution of actin genes in the amphioxus Branchiostoma belcheri and Branchiostoma floridae

We previously described the cDNA cloning and expression patterns of actin genes from amphioxus Branchiostoma floridae (Kusakabe, R., Kusakabe, T., Satoh, N., Holland, N.D., Holland, L.Z., 1997. Differential gene expression and intracellular mRNA localization of amphioxus actin isoforms throughout development: implications for conserved mechanisms of chordate development. Dev. Genes Evol. 207, 203–215). In the present paper, we report the characterization of cDNA clones for actin genes from a closely related species, Branchiostoma belcheri, and the exon–intron organization of B. floridae actin genes. Each of these two amphioxus species has two types of actin genes, muscle and cytoplasmic. The coding and non-coding regions of each type are well-conserved between the two species. A comparison of nucleotide sequences of muscle actin genes between the two species suggests that a gene conversion may have occurred between two B. floridae muscle actin genes BfMA1 and BfMA2. From the conserved positions of introns between actin genes of amphioxus and those of other deuterostomes, the evolution of deuterostome actin genes can be inferred. Thus, the presence of an intron at codon 328/329 in vertebrate muscle and cytoplasmic actin genes but not in any known actin gene in other deuterostomes suggests that a gene conversion may have occurred between muscle and cytoplasmic actin genes during the early evolution of the vertebrates after separation from other deuterostomes. A Southern blot analysis of genomic DNA revealed that the amphioxus genome contains multiple muscle and cytoplasmic actin genes. Some of these actin genes seem to have arisen from recent duplication and gene conversion. Our findings suggest that the multiple genes encoding muscle and cytoplasmic actin isoforms arose independently in each of the three chordate lineages and that gene duplications and gene conversions established the extant actin multigene family during the evolution of chordates.     

Steady-state kinetic studies on the actin activation of skeletal muscle heavy meromyosin subfragments: Effects of skeletal,smooth and non-muscle tropomyosins

The addition of either smooth muscle or brain tropomyosin to skeletal muscle actoheavy meromyosin (HMM) or acto-myosin subfragment-1 (SF1) produces an activation of the actin-activated ATPase activity up to 100%. This contrasts with the opposite, inhibitory effect produced by skeletal muscle tropomyosin. The degree of activation or inhibition depends on the ionic conditions, which influence the affinities of tropomyosin and HMM or SF1 for actin as well as on the molar ratio of actin to myosin.Enzyme kinetic analysis indicates that the inhibitory effect of skeletal muscle tropomyosin results from an approximately six- to tenfold increase in the apparent affinity (K_app) of the myosin head for the F-actin-tropomyosin complex with a concomitant six- to tenfold reduction in the maximal turnover rate (V_max). Thus, there is no direct competition of skeletal muscle tropomyosin and myosin for the same site on actin. Brain tropomyosin has an opposite effect, decreasing the apparent affinity with concomitant increase in the V_max.The effect of smooth muscle tropomyosin is more complex. At high ratios of myosin to actin this tropomyosin produces the same change in the K_app as skeletal muscle tropomyosin but yields a value of V_max that is about twofold higher. At lower molar ratios (below about 1 to 5 myosin subfragments to actin) the activating effect of this tropomyosin remains unchanged while the apparent affinity decreases to that observed for pure F-actin.On the basis of these data as well as from experiments carried out at fixed actin and varying SF1 concentrations, it is concluded that tropomyosins act in general as allosteric un-competitive inhibitors or activators of actomyosin by increasing or reducing the co-operative activation of myosin by actin at the level of product release.     

Stable transformation and actin visualization in callus cultures of dodder (Cuscuta europaea)

Agrobacterium tumefaciens-mediated transformation of callus culture, combined with a visual selection of GFP-tagged fimbrin actin binding domain (FABD₂) expression is described for parasitic species (Cuscuta europaea). The conditions for callus induction from 1 cm-long explants from the basal part of 7-day-old dodder seedlings were defined. We obtained light-green calli, which were transformed with A. tumefaciens bacterial strain GV3101 carrying plasmid pCB302 (35S::ABD₂:gfp) with neomycin phosphotransferase (nptII) gene. The limitations of selection procedures based on antibiotics were avoided using green fluorescent protein (GFP) detection, as a visual selection marker subcellularly targeted to the actin cytoskeleton. Fluorescence microscopy analyses demonstrated a network of nucleus-associated actin arrays and dense cortical actin arrangements in stably transformed Cuscuta callus cells. RT-PCR analyses confirmed gfp expression in transformed calli 7, 14 and 21 days after transformation. Although the GFP fluorescence associated with the actin cytoskeleton has retained for at least six months without silencing, no shoot regeneration was observed. It can be concluded that, C. europaea callus cells are competent for transformation, but under given conditions, these cells failed to realize their morphogenic and regeneration potentials.     

The effects of actin on the electron spin resonance of spin-labeled myosin

Myosin and heavy meromyosin have been spin labeled at either the S₁ or S₂ thiol groups, and their interaction with F-actin has been studied by electron spin resonance, both in the absence of substrate and during the hydrolysis of ATP. The spectrum of myosin labeled at either group indicates strong immobilization of the label. In the absence of substrate, actin added to S₁-labeled myosin slightly increases the separation of the outer spectral peaks, indicating a decrease in the mobility of the spin label. Actin also reduces the microwave power required to saturate the esr signal of S₁-labeled myosin or heavy meromyosin. The latter phenomenon is a more sensitive measure of the actin-myosin interaction than the spectral change seen in the absence of saturation. This suggests that saturation measurements may provide a more sensitive method of detecting changes in the environment of slowly tumbling nitroxide radicals than spectral measurements carried out in the absence of saturation. The decrease in the amplitude of the spectrum on adding actin at saturating microwave power was used to determine the stoichiometry of the interaction between actin and heavy meromyosin. This decrease is maximal when 2 moles of actin monomer are added per mole of heavy meromyosin and is reversed when actin and myosin are dissociated by ATP. During the steady state hydrolysis of ATP, actin had no detectable effect on the spectrum of S₁-labeled myosin. It can be concluded that spin labels bound to the S₁ groups are in a region of the myosin molecule that is affected by the interaction with actin. Actin does not affect the rate at which the bound spin label is reduced by dithiothreitol nor does the spin labeling of S₁ groups affect the activation by actin of the ATPase activity of myosin. These findings suggest that the most likely mechanism by which actin alters the mobility of labels on S₁ groups involves a change in the conformation of myosin. If a spin label is bound to the S₂ thiol groups rather than the S₁ groups, then actin has no detectable effect on the spectrum either in the presence or absence of ATP.