Actin cytoskeleton and complex cell architecture in an Asgard archaeon.

Asgard archaea are considered to be the closest known relatives of eukaryotes. Their genomes contain hundreds of eukaryotic signature proteins (ESPs), which inspired hypotheses on the evolution of the eukaryotic cell1-3. A role of ESPs in the formation of an elaborate cytoskeleton and complex cellular structures has been postulated4-6, but never visualized. Here we describe a highly enriched culture of 'Candidatus Lokiarchaeum ossiferum', a member of the Asgard phylum, which thrives anaerobically at 20 °C on organic carbon sources. It divides every 7-14 days, reaches cell densities of up to 5 × 107 cells per ml and has a significantly larger genome compared with the single previously cultivated Asgard strain7. ESPs represent 5% of its protein-coding genes, including four actin homologues. We imaged the enrichment culture using cryo-electron tomography, identifying 'Ca. L. ossiferum' cells on the basis of characteristic expansion segments of their ribosomes. Cells exhibited coccoid cell bodies and a network of branched protrusions with frequent constrictions. The cell envelope consists of a single membrane and complex surface structures. A long-range cytoskeleton extends throughout the cell bodies, protrusions and constrictions. The twisted double-stranded architecture of the filaments is consistent with F-actin. Immunostaining indicates that the filaments comprise Lokiactin-one of the most highly conserved ESPs in Asgard archaea. We propose that a complex actin-based cytoskeleton predated the emergence of the first eukaryotes and was a crucial feature in the evolution of the Asgard phylum by scaffolding elaborate cellular structures.

The actin cytoskeleton orchestra in Entamoeba histolytica.

Years of evolution have kept actin conserved throughout various clades of life. It is an essential protein starring in many cellular processes. In a primitive eukaryote named Entamoeba histolytica, actin directs the process of phagocytosis. A finely tuned coordination between various actin-binding proteins (ABPs) choreographs this process and forms one of the virulence factors for this protist pathogen. The ever-expanding world of ABPs always has space to accommodate new and varied types of proteins to the earlier existing repertoire. In this article, we report the identification of 390 ABPs from Entamoeba histolytica. These proteins are part of diverse families that have been known to regulate actin dynamics. Most of the proteins are primarily uncharacterized in this organism; however, this study aims to annotate the ABPs based on their domain arrangements. A unique characteristic about some of the ABPs found is the combination of domains present in them unlike any other reported till date. Calponin domain-containing proteins formed the largest group among all types with 38 proteins, followed by 29 proteins with the infamous BAR domain in them, and 23 proteins belonging to actin-related proteins. The other protein families had a lesser number of members. Presence of exclusive domain arrangements in these proteins could guide us to yet unknown actin regulatory mechanisms prevalent in nature. This article is the first step to unraveling them.

Genome-wide identification, phylogenetic classification, and exon-intron structure characterization of the tubulin and actin genes in flax (Linum usitatissimum).

Flax (Linum usitatissimum L.) is a valuable food and fiber crop cultivated for its quality fiber and seed oil. α-, ß-, γ-tubulins and actins are the main structural proteins of the cytoskeleton. α- and γ-tubulin and actin genes have not been characterized yet in the flax genome. In this study, we have identified 6 α-tubulin genes, 13 ß-tubulin genes, 2 γ-tubulin genes, and 15 actin genes in the flax genome and analyzed the phylogenetic relationships between flax and Arabidopsis thaliana tubulin and actin genes. Six α-tubulin genes are represented by three paralogous pairs, among 13 ß-tubulin genes 7 different isotypes can be distinguished, 6 of which are encoded by two paralogous genes each. γ-tubulin is represented by a paralogous pair of genes one of which may be not functional. Fifteen actin genes represent seven paralogous pairs-seven actin isotypes and a sequentially duplicated copy of one of the genes of one of the isotypes. Exon-intron structure analysis has shown intron length polymorphism within the ß-tubulin genes and intron number variation among the α-tubulin gene: three or four introns are found in two or four genes, respectively. Intron positioning occurs at conservative sites, as observed in numerous other plant species. Flax actin genes show both intron length polymorphisms and variation in the number of intron that may be two or three. These data will be useful to support further studies on the specificity, functioning, regulation, and evolution of the flax cytoskeleton proteins.

Actin genes and their expression in pacific white shrimp, Litopenaeus vannamei.

Actin is a multi-functional gene family that can be divided into muscle-type actins and non-muscle-type actins. In this study, 37 unigenes encoding actins were identified from RNA-Seq data of Pacific white shrimp, Litopenaeus vannamei. According to phylogenetic analysis, four and three cDNAs belong to cytoplasmic- and heart-type actins and were named LvActinCT and LvActinHT, respectively. 10 cDNAs belong to the slow-type skeletal muscle actins, and 18 belong to the fast-type skeletal muscle actins; they were designated LvActinSSK and LvActinFSK, respectively. Some muscle actin genes formed gene clusters in the genome. Multiple alternative transcription starts sites (ATSSs) were found for LvActinCT1. Based on the early developmental expression profile, almost all LvActins were highly expressed between the early limb bud and post-larval stages. Using LvActinSSK5 as probes, slow-type muscle was localized in pleopod muscle and superficial ventral muscle. We also found three actin genes that were down-regulated in the hemocytes of white spot syndrome virus (WSSV)- and Vibrio parahaemolyticus-infected L. vannamei. This study provides valuable information on the actin gene structure of shrimp, furthers our understanding of the shrimp muscle system and helps us develop strategies for disease control and sustainable shrimp farming.

Molecular phylogeny of the Achatinoidea (Mollusca: Gastropoda).

This study presents a multi-gene phylogenetic analysis of the Achatinoidea and provides an initial basis for a taxonomic re-evaluation of family level groups within the superfamily. A total of 5028 nucleotides from the nuclear rRNA, actin and histone 3 genes and the 1st and 2nd codon positions of the mitochondrial cytochrome c oxidase subunit I gene were sequenced from 24 species, representing six currently recognised families. Results from maximum likelihood, neighbour joining, maximum parsimony and Bayesian inference trees revealed that, of currently recognised families, only the Achatinidae are monophyletic. For the Ferussaciidae, Ferussacia folliculus fell separately to Cecilioides gokweanus and formed a sister taxon to the rest of the Achatinoidea. For the Coeliaxidae, Coeliaxis blandii and Pyrgina umbilicata did not group together. The Subulinidae was not resolved, with some subulinids clustering with the Coeliaxidae and Thyrophorellidae. Three subfamilies currently included within the Subulinidae based on current taxonomy likewise did not form monophyletic groups.

Insights into the origin of metazoan filopodia and microvilli.

Filopodia are fine actin-based cellular projections used for both environmental sensing and cell motility, and they are essential organelles for metazoan cells. In this study, we reconstruct the origin of metazoan filopodia and microvilli. We first report on the evolutionary assembly of the filopodial molecular toolkit and show that homologs of many metazoan filopodial components, including fascin and myosin X, were already present in the unicellular or colonial progenitors of metazoans. Furthermore, we find that the actin crosslinking protein fascin localizes to filopodia-like structures and microvilli in the choanoflagellate Salpingoeca rosetta. In addition, homologs of filopodial genes in the holozoan Capsaspora owczarzaki are upregulated in filopodia-bearing cells relative to those that lack them. Therefore, our findings suggest that proteins essential for metazoan filopodia and microvilli are functionally conserved in unicellular and colonial holozoans and that the last common ancestor of metazoans bore a complex and specific filopodial machinery.

Molecular phylogeny, divergence times and biogeography of spiders of the subfamily Euophryinae (Araneae: Salticidae).

We investigate phylogenetic relationships of the jumping spider subfamily Euophryinae, diverse in species and genera in both the Old World and New World. DNA sequence data of four gene regions (nuclear: 28S, Actin 5C; mitochondrial: 16S-ND1, COI) were collected from 263 jumping spider species. The molecular phylogeny obtained by Bayesian, likelihood and parsimony methods strongly supports the monophyly of a Euophryinae re-delimited to include 85 genera. Diolenius and its relatives are shown to be euophryines. Euophryines from different continental regions generally form separate clades on the phylogeny, with few cases of mixture. Known fossils of jumping spiders were used to calibrate a divergence time analysis, which suggests most divergences of euophryines were after the Eocene. Given the divergence times, several intercontinental dispersal events are required to explain the distribution of euophryines. Early transitions of continental distribution between the Old and New World may have been facilitated by the Antarctic land bridge, which euophryines may have been uniquely able to exploit because of their apparent cold tolerance. Two hot-spots of diversity of euophryines are discovered: New Guinea and the Caribbean Islands. Implications of the molecular phylogeny on the taxonomy of euophryines, and on the evolution of unusual genitalic forms and myrmecophagy, are also briefly discussed.

[Telomere length and phylogenetic relationship of Baikal and Siberian planarians (Turbellaria, Tricladida)].

Dynamics of the telomeric DNA (tDNA) and the phylogeny of the Baikal and Siberian planarians have been studied based on the analysis of the 18S rDNA and beta-actin gene fragments. A relationship between tDNA and the planarians size has been demonstrated. Giant planarians with a minor exception have longer tDNA than little planarians. Phylogenetic affinity between the species that have the stretched tracks of tDNA, big size and similar habitats may indicate possible role of tDNA in the development of the indefinite regenerative capacity of planarians.

Dominant negative mutant actins identified in flightless Drosophila can be classified into three classes.

Strongly dominant negative mutant actins, identified by An and Mogami (An, H. S., and Mogami, K. (1996) J. Mol. Biol. 260, 492-505), in the indirect flight muscle of Drosophila impaired its flight, even when three copies of the wild-type gene were present. Understanding how these strongly dominant negative mutant actins disrupt the function of wild-type actin would provide useful information about the molecular mechanism by which actin functions in vivo. Here, we expressed and purified six of these strongly dominant negative mutant actins in Dictyostelium and classified them into three groups based on their biochemical phenotypes. The first group, G156D, G156S, and G268D actins, showed impaired polymerization and a tendency to aggregate under conditions favoring polymerization. G63D actin of the second group was also unable to polymerize but, unlike those in the first group, remained soluble under polymerizing conditions. Kinetic analyses using G63D actin or G63D actin.gelsolin complexes suggested that the pointed end surface is defective, which would alter the polymerization kinetics of wild-type actin when mixed and could affect formation of thin filament structures in indirect flight muscle. The third group, R95C and E226K actins, was normal in terms of polymerization, but their motility on heavy meromyosin surfaces in the presence of tropomyosin-troponin indicated altered sensitivity to Ca(2+). Cofilaments in which R95C or E226K actins were copolymerized with a 3-fold excess of wild-type actin also showed altered Ca(2+) sensitivity in the presence of tropomyosin-troponin.

Actin isoforms.

The actin supergene family encodes a number of structurally related, but perhaps functionally distinct, protein isoforms that regulate contractile potential in muscle tissues and help to control the shape as well as the motility of non-muscle cells. In spite of the documented conservation amongst isoactin genes and their encoded proteins, recent results of biochemical, antibody localization, molecular mutagenesis and isoactin gene replacement studies lend credence to the notion that functional differences amongst muscle and non-muscle actin isoforms exist. Furthermore, the discovery of a new class of actin isoforms, the actin-related proteins, reveals that the actin gene and protein isoform family is more complex than was previously believed.

The complexity and diversity of the actin cytoskeleton of trypanosomatids.

Trypanosomatids are a monophyletic group of parasitic flagellated protists belonging to the order Kinetoplastida. Their cytoskeleton is primarily made up of microtubules in which no actin microfilaments have been detected. Although all these parasites contain actin, it is widely thought that their actin cytoskeleton is reduced when compared to most eukaryotic organisms. However, there is increasing evidence that it is more complex than previously thought. As in other eukaryotic organisms, trypanosomatids encode for a conventional actin that is expected to form microfilament-like structures, and for members of three conserved actin-related proteins probably involved in microfilament nucleation (ARP2, ARP3) and in gene expression regulation (ARP6). In addition to these canonical proteins, also encode for an expanded set of actins and actin-like proteins that seem to be restricted to kinetoplastids. Analysis of their amino acid sequences demonstrated that, although very diverse in primary sequence when compared to actins of model organisms, modelling of their tertiary structure predicted the presence of the actin fold in all of them. Experimental characterization has been done for only a few of the trypanosomatid actins and actin-binding proteins. The most studied is the conventional actin of Leishmania donovani (LdAct), which unusually requires both ATP and Mg2+ for polymerization, unlike other conventional actins that do not require ATP. Additionally, polymerized LdAct tends to assemble in bundles rather than in single filaments. Regulation of actin polymerization depends on their interaction with actin-binding proteins. In trypanosomatids, there is a reduced but sufficient core of actin-binding proteins to promote microfilament nucleation, turnover and stabilization. There are also genes encoding for members of two families of myosin motor proteins, including one lineage-specific. Homologues to all identified actin-family proteins and actin-binding proteins of trypanosomatids are also present in Paratrypanosoma confusum (an early branching trypanosomatid) and in Bodo saltans (a closely related free-living organism belonging to the trypanosomatid sister order of Bodonida) suggesting they were all present in their common ancestor. Secondary losses of these genes may have occurred during speciation within the trypanosomatids, with salivarian trypanosomes having lost many of them and stercorarian trypanosomes retaining most.

Permanently proliferating rat vascular smooth muscle cell with maintained expression of smooth muscle characteristics, including actin of the vascular smooth muscle type.

Cells of an established clonal line (RVF-SMC) derived from rat vena cava are described by light and electron microscope methods and biochemical analysis of the major proteins. The cells are flat, and they moderately elongate and form monolayers. They are characterized by prominent cables of microfilaments bundles decoratable with antibodies to actin and alpha-actinin. These bundles contain numerous densely stained bodies and are often flanked by typical rows of surface caveolae and vesicles. The cells are rich in intermediate-sized filaments of the vimentin type but do not show detectable amounts of desmin and cytokeratin filaments. Isoelectric focusing and protein chemical studies have revealed actin heterogeneity. In addition to the two cytoplasmic actins, beta and gamma, common to proliferating cells, two smooth muscle-type actins (an acidic alpha-like and a gamma-like) are found. The major (alpha-type) vascular smooth muscle actin accounts for 28% of the total cellular actin. No skeletal muscle or cardiac muscle actin has been detected. The synthesis of large amounts of actin and vimentin and the presence of at least three actins, including alpha-like actin, have also been demonstrated by in vitro translation of isolated poly(A)+ mRNAs. This is, to our knowledge, the first case of expression of smooth muscle-type actin in a permanently growing cell. We conclude that permanent cell growth and proliferation is compatible with the maintained expression of several characteristic cell features of the differentiated vascular smooth muscle cell including the formation of smooth muscle-type actin.

Heterogeneity of microvascular pericytes for smooth muscle type alpha-actin.

Microvascular pericytes are believed to be involved in various functions such as regulation of capillary blood flow and endothelial proliferation. Since pericytes represent a morphologically heterogeneous cell population ranging from circular smooth musclelike to elongated fibroblast-like morphology it is possible that regulation of blood flow (via contractility) and control of endothelial proliferation (as well as other metabolic functions) may be accomplished by different subsets of pericytes. In the present study we provide evidence for heterogeneity of pericytes at the molecular level by using two novel technical approaches. These are (a) immunostaining of whole mounts of the microvascular beds of the rat mesentery and bovine retina and (b) immunoblotting studies of microdissected retinal microvessels. We show that pericytes of true capillaries (midcapillaries) apparently lack the smooth muscle isoform of alpha-actin whereas transitional pericytes of pre- and postcapillary microvascular segments do express this isoform. Thus, regulation of capillary blood flow may be accomplished by the smooth muscle-related pre- and postcapillary pericytes whereas the nonmuscle pericytes of true capillaries may play a role in other functions.

The evolution of new structures: clues from plant cytoskeletal genes.

How large numbers of genes were recruited simultaneously to build new organ structures is one of the greatest puzzles in evolutionary biology. Here, we present data suggesting that the vegetative and reproductive classes of actins and other cytoskeletal proteins arose concurrently with the macroevolutionary divergence of leaves and reproductive structures in the earliest land plants. That the cytoskeleton is essential for physically programming the development of organs and tissues is well established. Thus, we propose that this regulatory dichotomy represents an ancient landmark event in the global regulation of hundreds of higher-plant genes, an event that is linked to the macroevolution of plant vegetative and reproductive organs. The recent availability of sequence and expression data for large numbers of plant genes should make it possible to dissect this and other major macroevolutionary events.

Mouse testes contain two size classes of actin mRNA that are differentially expressed during spermatogenesis.

Using several actin isotype-specific cDNA probes, we found actin mRNA of two size classes, 2.1 and 1.5 kilobases (kb), in extracts of polyadenylated and nonpolyadenylated RNA from sexually mature CD-1 mouse testes. Although the 2.1-kb sequence was present in both meiotic and postmeiotic testicular cell types, it decreased manyfold in late haploid cells. The 1.5-kb actin sequence was not detectable in meiotic pachytene spermatocytes (or in liver or kidney cells), but was present in round and elongating spermatids and residual bodies. To differentiate between the beta- and gamma-actin mRNAs, we isolated a cDNA, pMGA, containing the 3' untranslated region of a mouse cytoplasmic actin that has homology to the 3' untranslated region of a human gamma-actin cDNA but not to the 3' untranslated regions of human alpha-, beta-, or cardiac actins. Dot blot hybridizations with pMGA detected high levels of presumptive gamma-actin mRNA in pachytene spermatocytes and round spermatids, with lower amounts found in elongating spermatids. Hybridization with the 3' untranslated region of a rat beta-actin probe revealed that round spermatids contained higher levels of beta-actin mRNA than did pachytene spermatocytes or residual bodies. Both probes hybridized to the 2.1-kb actin mRNA but failed to hybridize to the 1.5-kb mRNA.

Human cytoplasmic actin proteins are encoded by a multigene family.

We characterized nine human actin genes that we isolated (Engel et al., Proc. Natl. Acad. Sci. U.S.A. 78:4674-4678, 1981) from a library of cloned human DNA. Measurements of the thermal stability of hybrids formed between each cloned actin gene and alpha-, beta-, and gamma-actin mRNA demonstrated that only one of the clones is most homologous to sarcomeric actin mRNA, whereas the remaining eight clones are most homologous to cytoplasmic actin mRNA. By the following criteria we show that these nine clones represent nine different actin gene loci rather than different alleles or different parts of a single gene: (i) the restriction enzyme maps of the coding regions are dissimilar; (ii) each clone contains sufficient coding region to encode all or most of an entire actin gene; and (iii) each clone contains sequences homologous to both the 5' and 3' ends of the coding region of a cloned chicken beta-actin cDNA. We conclude, therefore, that the human cytoplasmic actin proteins are encoded by a multigene family.

Actin's prokaryotic homologs.

Actin is one of the most abundant and conserved eukaryotic proteins. Remarkably, two prokaryotic homologs of actin, MreB and ParM, have only recently been identified. MreB and ParM polymerize into filaments and play important roles in the control of bacterial cell shape and plasmid segregation, respectively. Whereas the eukaryotic actins display a remarkable degree of conservation (e.g. no amino acid changes in muscle actin from chickens to humans), the two bacterial proteins have as much sequence similarity to each other ( approximately 11% sequence identity) as they do to actin. It is possible that the interesting properties of eukaryotic F-actin may account for the unusual degree of conservation among the actins, whereas the bacterial proteins have had fewer constraints over the course of evolution.

Tails of two Tirs: actin pedestal formation by enteropathogenic E. coli and enterohemorrhagic E. coli O157:H7.

Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli O157:H7 (EHEC) form characteristic lesions on infected mammalian cells called actin pedestals. Each of these two pathogens injects its own translocated intimin receptor (Tir) molecule into the plasma membranes of host cells. Interaction of translocated Tir with the bacterial outer membrane protein intimin is required to trigger the assembly of actin into focused pedestals beneath bound bacteria. Despite similarities between the Tir molecules and the host components that associate with pedestals, recent work indicates that EPEC and EHEC Tir are not functionally interchangeable. For EPEC, Tir-mediated binding of Nck, a host adaptor protein implicated in actin signaling, is both necessary and sufficient to initiate actin assembly. In contrast, for EHEC, pedestals are formed independently of Nck, and require translocation of bacterial factors in addition to Tir to trigger actin signaling.

Cloning of black carp beta-actin gene and primarily detecting the function of its promoter region.

A 3 338 bp DNA fragment including the open reading frame and 5'-flanking region of beta-actin gene for black carp genome was obtained through PCR amplification. Analysis of the sequencing results indicated the ORF of black carp beta-actin gene encoding a 375 amino acid protein that shares a high degree of conservation to other known actins. The black carp beta-actin sequence showed 100% identity to common carp, grass carp, and zebrafish, 99.2% identity to human and Norway rat beta-actin gene, 98.9% and 98.1% identity to chicken and Kenyan clawed frog beta-actin gene, respectively. The promoter region of black carp beta-actin gene was inserted into the promoterless pEGFP1 vector. The recombinant plasmid was microinjected into the fertilized eggs of mud loach before two-cell stage as well as transfected into HeLa cell line. GFP expression was found in 50% of mud loach embryos and 2/3 HeLa cells. The GFP expression could be observed in every part of the mud loach embryos, and in some embryos, the GFP was expressed in the whole body. Thus, the usefulness of black carp beta-actin promoter as a ubiquitous expression promoter was confirmed using the EGFP as a reporter gene.

Two-Photon Correlation Spectroscopy in Single Dendritic Spines Reveals Fast Actin Filament Reorganization during Activity-Dependent Growth.

Two-photon fluorescence correlation spectroscopy (2P-FCS) within single dendritic spines of living hippocampal pyramidal neurons was used to resolve various subpopulations of mobile F-actin during activity-dependent structural changes such as potentiation induced spine head growth. Two major classes of mobile F-actin were discovered: very dynamic and about a hundred times less dynamic F-actin. Spine head enlargement upon application of Tetraethylammonium (TEA), a protocol previously used for the chemical induction of long-term potentiation (cLTP) strictly correlated to changes in the dynamics and filament numbers in the different actin filament fractions. Our observations suggest that spine enlargement is governed by a mechanism in which longer filaments are first cut into smaller filaments that cooperate with the second, increasingly dynamic shorter actin filament population to quickly reorganize and expand the actin cytoskeleton within the spine head. This process would allow a fast and efficient spine head enlargement using a major fraction of the actin filament population that was already present before spine head growth.