Immunocytochemistry of the acellular slime mold Physarum polycephalum. III. Distribution of myosin and the actin-modulating protein (fragmin) in sandwiched plasmodia

The acellular slime mold Physarum forms very thin plasmodia when sandwiched between two agar sheets. After extraction with glycerol-containing buffers, suitable objects for immunofluorescence microscopy are obtained, and an analysis of the cytoskeletal and contractile system of Physarum becomes possible. Plasmodia were stained with antibodies against myosin and fragmin, a protein factor involved in actin filament length regulation. The microanatomy and topography of cellular structures containing these proteins were investigated at the light and electron microscopic levels. The patterns obtained with the two antibodies are closely related to those obtained with actin antibody [25]. In both cases the complex system of cytoplasmic fibrils is stained selectively. The fibrils form a more or less regular network in the advancing front zone with the fibrils being interconnected by focal nodes. In the posterior region of the plasmodium, where endoplasmic pathways and protoplasmic veins are differentiated, larger fibrils are detected, running obliquely or longitudinally to the veins. With both antibodies the fluorescent pattern of the fibrils is continuous without indications of periodic interruptions or striations, which would be expected in the case of sarcomere-like subunits. With anti-myosin unstained patches are frequently seen at or close to the nodes of the fibrillar network in the anterior region. The small lobopodia, which are rich in actin, are apparently not stained by the myosin antibody, a result similar to the situation in "ruffling edges? of cultured vertebrate cells. Electron microscopic investigations of antibody-labeled fibrils in embedded and sectioned plasmodia allow the identification of antibody molecules at specific sites along the fibrils with a different distribution pattern for each of the two antibodies.     

A novel 36,000-dalton actin-binding protein purified from microfilaments in Physarum plasmodia which aggregates actin filaments and blocks actin-myosin interaction

In the plasmodia of Physarum polycephalum, which show a cyclic contraction-relaxation rhythm of the gel layer, huge aggregates of entangled actin microfilaments are formed at about the onset of the relaxation (R. Nagai, Y. Yoshimoto, and N. Kamiya. 1978. J. Cell Sci. 33:205-225). By treating the plasmodia with Triton X-100, we prepared a demembranated cytoskeleton consisting of entangled actin filaments and found that the actin filaments hardly interact with rabbit skeletal myosin. From the cytoskeleton we purified a novel actin-binding protein which binds stoichiometrically to actin and makes actin filaments curled and aggregated. It also inhibits the ATPase activity as well as the superprecipitation of reconstituted rabbit skeletal muscle actomyosin. This protein has a polypeptide molecular weight of 36,000 and binds 7 mol of actin/mol 36,000 polypeptide.     

Physarum amoebae express a distinct fragmin-like actin-binding protein that controls in vitro phosphorylation of actin by the actin-fragmin kinase.

Amoebae and plasmodia constitute the two vegetative growth phases of the Myxomycete Physarum. In vitro and in vivo phosphorylation of actin in plasmodia is tightly controlled by fragmin P, a plasmodium-specific actin-binding protein that enables actin phosphorylation by the actin-fragmin kinase. We investigated whether amoebal actin is phosphorylated by this kinase, in spite of the lack of fragmin P. Strong actin phosphorylation was detected only following addition of recombinant actin-fragmin kinase to cell-free extracts of amoebae, suggesting that amoebae contain a protein with properties similar to plasmodial fragmin. We purified the complex between actin and this protein to homogeneity. Using an antibody that specifically recognizes phosphorylated actin, we demonstrate that Thr203 in actin can be phosphorylated in this complex. A full-length amoebal fragmin cDNA was cloned and the deduced amino acid sequence shows 65% identity with plasmodial fragmin. However, the fragmins are encoded by different genes. Northern blots using RNA from a developing Physarum strain demonstrate that this fragmin isoform (fragmin A) is not expressed in plasmodia. In situ localization showed that fragmin A is present mainly underneath the plasma membrane. Our results indicate that Physarum amoebae express a fragmin P-like isoform which shares the property of binding actin and converting the latter into a substrate for the actin-fragmin kinase.     

Translational control and the cytoskeleton in Physarum polycephalum

Translationally active plasmodia of the syncytial slime mold Physarum polycephalum develop into translationally dormant sclerotia during starvation. Although functional mRNA and ribosomes exist in sclerotia, protein synthesis is suppressed at the level of initiation. To test the possibility that alterations in the cytoskeleton may limit protein synthesis, we have examined the distribution of polysomes and actin mRNA in the cytoskeletal (CSK) and soluble (SOL) fractions of Triton X-100-extracted plasmodia and sclerotia. Most of the polysomes and actin mRNA were located in the CSK of plasmodia, while most of the ribosomes and actin mRNA were located in the SOL of sclerotia. The results suggest that ribosomes and mRNA shift from the CSK to the SOL as protein synthesis is suppressed during starvation. Plasmodia and sclerotia can be induced to accumulate excess polysomes by treatment with low levels of the elongation inhibitor cycloheximide. Treatment of plasmodia with cycloheximide caused excess polysomes to accumulate in the SOL, suggesting that the CSK contains a limited capacity for binding translational components and that the association of polysomes with the cytoskeleton is not required for protein synthesis. Treatment of sclerotia with cycloheximide, however, caused polysomes and actin mRNA to accumulate in the CSK, suggesting that the sclerotial cytoskeleton, although depleted in ribosomes and mRNA, is capable of binding translational components. It is concluded that alterations in the sclerotial cytoskeleton are not involved in translational control.     

Actomyosin content of Physarum plasmodia and detection of immunological cross-reactions with myosins from related species

The content of myosin in plasmodia of the myxomycete Physarum polycephalum was measured by an immunological technique, quantitative microcomplement (C') fixation. Migrating plasmodia (starved after growth on rolled oats) contained 0.60 +/- 0.08 (SD) mg myosin per g fresh plasmodia. Myosin comprised 0.77% +/- 0.05 (SD) of the total plasmodial protein. When total plasmodial proteins were separated by electrophoresis on SDS-polyacrylamide gels, a large amount of protein appeared in a band comigrating with muscle actin. Densitometry performed after Coomassie blue staining indicated that as much as 15- 25% of the total protein in the plasmodium could be actin. This gives an actin/myosin ratio by weight in the myxomycete plasmodium as high as 19-33, a very "actin-rich" actomyosin compared with rabbit skeletal muscle actomyosin with an actin/myosin ratio of 0.6. Starvation stimulates rapid migration and is correlated with a higher percent of both myosin and actin in the total protein of the plasmodium compared with normally growing cultures. Immunological cross-reaction of myosins from a variety of species was measured by C' fixation using an antiserum produced against purified native myosin from P. polycephalum. Although myxomycete and vertebrate striated muscle myosins have very similar morphological and biochemical properties, and apparently possess similar binding properties to F-actin, only myosins from myxomycetes in the order Physarales, rather closely related to P. polycephalum, gave detectable cross-reactions. This finding suggests that many amino acid sequences in myosin have been variable during evolution.     

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.     

Naegleria actin elicits species-specific antibodies

Actin, the major protein of amebae of Naegleria gruberi, proved to be strongly immunogenic in rabbits. The resulting precipitating antibodies are specific to actin of Naegleria. In a competitive solid-phase radioimmunoassay, these antibodies bound similarly to Naegleria G- and F-actin. Actins from amebae of Acanthamoeba and Dictyostelium, plasmodia of Physarum, sea urchin eggs, and vertebrate muscles gave no competition in the radioimmunoassay. Estimates of the amount of actin in Naegleria amebae ranged from a minimum of 5% of the total cell protein by radioimmunoassay to a maximum of 16% by electrophoresis. The unusual species specificity of these antibodies indicates that Naegleria actin, although conserved in many properties, is different enough to have unique antigenic determinants.     

An actin-modulating protein from Physarum polycephalum. I. Isolation and purification

High-speed centrifugation supernatants from slime mould plasmodia show considerable activities to inhibit the polymerization of actin as revealed by viscosity measurements. By following increasing inhibitory activities an actin modulating protein (AM-protein) has been isolated and purified which affects the polymer state of actin. AM-protein has a peptide chain weight of 42 000 and is thus indistinguishable from actin by SDS-electrophoresis, but can be clearly distinguished by isoelectric focussing. Peptide maps from partial proteolytic digests of AM-protein and Physarum actin reveal no similarities thereby excluding that AM-protein is a denatured or modified form of actin. The protein is isolated from crude extracts as a heterodimer with actin to which it strongly binds. This heterodimer affects the polymerization of large amounts of actin by inducing oligomeric or low-polymer actin complexes and thus inhibiting the formation of long actin filaments. The AM-protein/actin heterodimer has only a slight effect of F-actin. It partially depolymerized F-actin within several hours. By ion exchange chromatography in 8 M urea the AM-protein is separated from the actin. The purified AM-protein monomer is renatured and inhibits the polymerization of actin like the heterodimer but additionally, depolymerizes actin filaments very rapidly and effectively by breaking them into oligomer or low-polymer complexes. The addition of less than 1% AM-protein causes a decrease of the specific viscosity of an F-actin solution by 50%. The degree of polymerization inhibition and depolymerization of actin is strictly dependent on the amount of AM-protein added; therefore a catalytic type of reaction between both proteins can be excluded.     

Actin-fragmin interactions as revealed by chemical cross-linking

A one to one complex of actin and fragmin (a capping protein from Physarum polycephalum plasmodia) was cross-linked with 1-ethyl-3-[3-(dimethylamino)propyl] carbodiimide. The cross-linking reaction generated two cross-linked products with slightly different molecular weights (88 000 and 90 000) as major species. They were cross-linked products of one actin and one fragmin. The cross-linking site of fragmin in the actin sequence was determined by peptide mappings [Sutoh, K. (1982) Biochemistry 21, 3654-3661] after partial chemical cleavages of cross-linked products with hydroxylamine. The results indicated that the N-terminal segment of actin spanning residues 1-12 participated in cross-linking with fragmin. The cross-linker used in this study covalently bridges lysine side chains and side chains of acidic residues when they are in direct contact. Therefore, it seems that acidic residues in the N-terminal segment of actin (Asp-1, Glu-2, Asp-3, Glu-4, and Asp-11), at least some of them, are in the binding site of fragmin. It has already been shown that the same acidic segment of actin is in the binding site of myosin or depactin (an actin-depolymerizing protein isolated from starfish oocytes). We suggest that the unusual amino acid sequence of the N-terminal segment of actin makes its N-terminal region a favorable anchoring site for various types of actin-binding proteins.     

Naegleria Actin Elicits Species-Specific Antibodies1

ABSTRACT. Actin, the major protein of amebae of Naegleria gruberi, proved to be strongly immunogenic in rabbits. The resulting precipitating antibodies are specific to actin of Naegleria. In a competitive solid-phase radioimmunoassay, these antibodies bound similarly to Naegleria G- and F-actin. Actins from amebae of Acanthamoeba and Dictyostelium, plasmodia of Physarum, sea urchin eggs, and vertebrate muscles gave no competition in the radioimmunoassay. Estimates of the amount of actin in Naegleria amebae ranged from a minimum of 5% of the total cell protein by radioimmunoassay to a maximum of 16% by electrophoresis. The unusual species specificity of these antibodies indicates that Naegleria actin, although conserved in many properties, is different enough to have unique antigenic determinants.     

Physarum action. Observations on its presence, stability, and assembly in plasmodial extracts and development of an improved purification procedure.

Actin is readily extracted from plasmodia of Physarum polycephalum by low ionic strength solutions which do not solubilize the plasmodial myosin. The actin in such extracts exists predominantly as a monomer which slowly denatures, apparently via removal of bound nucleotide, and is subsequently proteolyzed. However, the native monomeric actin can be induced to assemble into polymeric arrays under appropriate solvent conditions. Actin assembly is dependent on the addition of ATP and is a function of KCl and CaCl2 concentrations. These observations have allowed the development of an improved actin purification scheme which is simple, rapid, and efficient, yielding approximately 60 mg of protein from 100 g of plasmodium. The actin thus obtained is pure, stable, and comparable to that obtained by previously described procedlres. Furthermore, the observations suggest that actin polymers may be metastably assembled in vivo and raise the possibility that actin assembly, and plasmodial movements, could be regulated via alterations in intracellular concentrations of nucleotide and/or divalent cation.     

Cephalic region damage in the nemertean Lineus ruber by the orthonectid Intoshia linei

Previous investigators believed, that the plasmodia of Intoshia linei can affect all part of nemerteans body except the cephalic lobe. Our results indicate that in strongly infected worms mature plasmodia can settle inside of the cephalic region and form the large conglomerates. Common places of plasmodia localization in hosts are not only the tegument, connective tissue, gonads, muscles of body wall and proboscis, but also the nervous tissue. We found a lot of the mature plasmodia in all four lobes of the nemerteans brain and in the lateral nerve trunks. Furthermore the plasmodia can settle in the cerebral organs, in the walls of blood lacunae and rhynchcoel. In spite of the strong invasion of the cephalic lobe we have never observed a plasmodial outgrowths in the tegument of this region. This phenomena can be explained by the assumption, that plasmodia usually intrude into the cephalic region relatively late and have no time to form outgrowths up to the moment when starts the emission of adult males and females in the rest parts of nemertean body. Moreover, single parasite cells were repeatedly found in the host tissue near the mature plasmodium. These cells had few cytoplasm and large vesicular nuclei, which were very similar in size to the nuclei of the host cells. General morphology of single cells was very similar to the generative cells of the plasmodium. At present, the function of these cells is not evident, but we believe, they appearance is concerned with the proliferation of plasmodiuma and agglomeration of the parasite in the body of Lineus ruber.     

Fragmin60 encodes an actin-binding protein with a C2 domain and controls actin Thr-203 phosphorylation in Physarum plasmodia and sclerotia

We report the isolation of a cDNA clone encoding a 60-kDa protein termed fragmin60 that cross-reacts with fragmin antibodies. Unlike other gelsolin-related proteins, fragmin60 contains a unique N-terminal domain that shows similarity with C2 domains of aczonin, protein kinase C, and synaptotagmins. The fragmin60 C2 domain binds three calcium ions, one with nanomolar affinity and two with micromolar affinity. Actin binding by fragmin60 requires higher calcium concentrations than does binding of actin by a fragmin60 mutant lacking the C2 domain, suggesting that the C2 domain secures the actin binding moiety in a conformation preventing actin binding at low calcium concentrations. The fragmin60 C2 domain does not bind phospholipids but interacts with the endogenous homologue of Saccharomyces cerevisiae S-phase kinase-associated protein (Skp1), as shown by pull-down assays and co-expression in mammalian cells. Recombinant fragmin60 promotes in vitro phosphorylation of actin Thr-203 by the actin-fragmin kinase. We further show that in vivo phosphorylation of actin in the fragmin60-actin complex occurs in sclerotia, a dormant stage of Physarum development, as well as in plasmodia. Our findings indicate that we have cloned a novel type of gelsolin-related actin-binding protein that is involved in controlling regulation of actin phosphorylation in vivo.     

Cell type-dependent expression of tubulins in Physarum

Three alpha-tubulins and two beta-tubulins have been resolved by two-dimensional gel electrophoresis of whole cell lysates of Physarum myxamoebae or plasmodia. Criteria used to identify the tubulins included migration on two-dimensional gels with myxamoebal tubulins purified by self-assembly into microtubules in vitro, peptide mapping with Staphylococcus V8 protease and with chymotrypsin, immunoprecipitation with a monoclonal antibody specific for beta-tubulin, and, finally, hybrid selection of specific mRNA by cloned tubulin DNA sequences, followed by translation in vitro. Differential expression of the Physarum tubulins was observed. The alpha 1- and beta 1-tubulins were detected in both myxamoebae and plasmodia; alpha 2 and beta 2 were detected only in plasmodia, alpha 3 was detected only in the myxamoebal phase, and may be specific to the flagellate. Observation of more tubulin species in plasmodia than in myxamoebae was remarkable; the only microtubules detected in plasmodia are those of the mitotoic spindle, whereas myxamoebae display cytoplasmic, centriolar, flagellar, and mitotic-spindle microtubules. In vitro translation of myxamoebal and plasmodial RNAs indicated that there are distinct mRNAs, and therefore probably separate genes, for the alpha 1-, alpha 2-, beta 1-, and beta 2-tubulins. Thus, the different patterns of tubulin expression in myxamoebae and plasmodia reflect differential expression of tubulin genes.     

Multiple forms of actin in Physarum polycephalum

Actin in the acellular slime mold Physarum polycephalum consists of three major forms closely spaced at isoelectric point (IP) 4.7 and a minor form at IP 5.1. Amino acid analysis has shown the IP 5.1 actin to be nearly identical to the 4.7 actins. In actin purified from acetone powder, both actin forms were present. Both forms bound to DNase I and have the same molecular weight of about 43 000 on sodium dodecyl sulfate (SDS) polyacrylamide gels. On 2-D gels of nuclear proteins, both forms of actin were present. The IP 4.7 actins account for 8.6% of total plasmodial protein, and the IP 5.1 form for about 0.7%. In the nucleus the IP 4.7 actins comprise 2.7% of total nuclear protein, and the 5.1 actin about 0.4%. No cell cycle-associated change in the concentration of actins was observed in either total plasmodial extracts or in isolated nuclei. Pulse-labelling experiments have shown that in total plasmodia actin synthesis occurs throughout the cell cycle, with no relative changes in the rate of synthesis. In isolated nuclei labelled during mitosis and early S-phase, there is about twice as much labelled actin as in nuclei labelled prior to mitosis. This result may indicate an increase in the transport of actin into the nucleus.     

The crystal structure of the Physarum polycephalum actin-fragmin kinase: an atypical protein kinase with a specialized substrate-binding domain.

Coordinated temporal and spatial regulation of the actin cytoskeleton is essential for diverse cellular processes such as cell division, cell motility and the formation and maintenance of specialized structures in differentiated cells. In plasmodia of Physarum polycephalum, the F-actin capping activity of the actin-fragmin complex is regulated by the phosphorylation of actin. This is mediated by a novel type of protein kinase with no sequence homology to eukaryotic-type protein kinases. Here we present the crystal structure of the catalytic domain of the first cloned actin kinase in complex with AMP at 2.9 A resolution. The three-dimensional fold reveals a catalytic module of approximately 160 residues, in common with the eukaryotic protein kinase superfamily, which harbours the nucleotide binding site and the catalytic apparatus in an inter-lobe cleft. Several kinases that share this catalytic module differ in the overall architecture of their substrate recognition domain. The actin-fragmin kinase has acquired a unique flat substrate recognition domain which is supposed to confer stringent substrate specificity.     

Immunocytochemical identification of actin in rabbit spermiogenesis and spermatozoa

Actin was localized in testicular spermatids and in spermatozoa of rabbit by using a monoclonal anti-actin antibody and a specific antiserum against actin, labeled with colloidal gold. The antibody reactivity with sperm homogenates was determined by immunoblotting of one-dimensional gels. With on-grid postembedding immunostaining of Lowicryl K4M sections, actin was identified in the subacrosomal region of differentiating spermatids, and in four bulges situated between the inner acrosomal membrane and the nuclear envelope and in the anterior part of the postacrosomal region of ejaculated spermatozoa. Sperm actin was identified on two-dimensional gels as two spots in the isoelectric point and molecular weight corresponding to gamma and beta-isoforms of actin. Immunoblots stained with specific antibodies demonstrated that rabbit spermatozoa express gamma and beta-actin isoforms.     

Properties, intracellular localization, and stage-specific expression of membrane-bound beta-glucosidase, BglM1, from Physarum polycephalum

Physarum polycephalum expresses a membrane-bound beta-glucosidase (BglM1) with a molecular mass of 130 kDa. The primary structure of BglM1 consists of a glycosyl hydrolase family 3 domain at an amino-terminal domain and a carboxyl-terminal region without homology to the sequence of known glycosidases. The latter region contains two calx-beta motifs known as Ca(2+)-binding sites; an RGD sequence, which is known to be a cell attachment sequence; and a transmembrane region. The molecular mass calculated from the amino acid sequence is 130 kDa, but that in the crude extract was estimated by SDS-PAGE to be 230 kDa, and decreased to 130 kDa during purification. However, when BglM1 was purified in the presence of calcium ion, the molecular mass remained 230 kDa. The biochemical characteristics of the 130- and 230-kDa BglM1 forms were analyzed: differences were found in the kinetic data for some substrates specific for both these enzymes; however, no difference was found in their intrinsic characteristics such as optimum pH and temperature. In addition, the molecular mass of native BglM1 with a calcium ion was estimated to be 1,000 kDa or larger by gel filtration. These results suggest that the calcium ion influences the conformation of BglM1. The evidence that BglM1 localizes on the plasma membrane of plasmodia was confirmed using immunofluorescence microscopy. Although Physarum BglM1 was expressed in microplasmodia and plasmodia, little expression was detected in other stages. BglM1 may have some function only in multinuclear cells.     

Myxozoan pathogens in cultured Malaysian fishes. II. Myxozoan infections of redtail catfish Hemibagrus nemurus in freshwater cage cultures

Cage-cultured Asian redtail catfish Hemibagrus nemurus (Valenciennes, 1840), a popular food fish in Southeast Asia, proved to be infected by 3 myxozoan species. All the 3 species belonged to the genus Henneguya: 2 were identified as H. mystusia Sarkar, 1985 and H. hemibagri Tchang et Ma, 1993, while the other was described as H. basifilamentalis sp. n. All plasmodia were found in the gills and were characterised by a specific site selection. H. mystusia formed plasmodia in the multi-layered epithelium between the gill lamellae and in the non-lamellar edge of the gill filaments, while H. hemibagri developed in the capillary network of the lamellae. H. basifilamentalis sp. n. had large oval plasmodia located deep among the filaments just above the gill arch.     

Ca2+-sensitivity of actomyosin ATPase purified from Physarum polycephalum.

A contractile protein closely resembling natural actomyosin (myosin B) of rabbit skeletal muscle was extracted from plasmodia of the slime mold, Physarum polycephalum, by protecting the SH-groups with beta-mercaptoethanol or dithiothreitol. Superprecipitation of the protein induced by Mg2+-ATP at low ionic strength was observed only in the presence of very low concentrations of free Ca2+ ions, and the Mg2+-ATPase [EC 3.6.1.3] reaction was activated 2- to 6-fold by 1 muM of free Ca2+ ions. Crude myosin and actin fractions were separated by centrifuging plasmodium myosin B in the presence of Mg2+-PPi at high ionic strength. The crude myosin showed both EDTA- and Ca2+-activated ATPase activities. The Mg2+-ATPase activity of crude myosin from plasmodia was markedly activated by the addition of pure F-actin from rabbit skeletal muscle. Addition of the F-action-regulatory protein complex prepared from rabbit skeletal muscle as well as the actin fraction of plasmodium caused the same degree of activation as the addition of pure F-actin only in the presence of very low concentrations of Ca2+ ion