Amoeba Actin Research Articles

Organization and Ligand Binding Properties of the Tail ofAcanthamoeba Myosin-IA: IDENTIFICATION OF AN ACTIN-BINDING SITE IN THE BASIC (TAIL HOMOLOGY-1) DOMAIN

The Acanthamoeba myosin-IA heavy chain gene encodes a 134-kDa protein with a catalytic domain, three potential light chain binding sites, and a tail with separately folded tail homology (TH) -1, -2, and -3 domains. TH-1 is highly resistant to trypsin digestion despite consisting of 15% lysine and arginine. TH-2/3 is resistant to alpha-chymotrypsin digestion. The peptide link between TH-1 and TH-2/3 is cleaved by trypsin, alpha-chymotrypsin, and endo-AspN but not V8 protease. The CD spectra of TH-2/3 indicate predominantly random structure, turns, and beta-strands but no alpha-helix. The hydrodynamic properties of TH-2/3 (Stokes' radius of 3.0 nm, sedimentation coefficient of 1.8 S, and molecular mass of 21.6 kDa) indicate that these domains are as long as the whole myosin-I tail in reconstructions of electron micrographs. Furthermore, separately expressed and purified TH-1 binds with high affinity to TH-2/3. Thus we propose that TH-1 and TH-2/3 are arranged side by side in the myosin-IA tail. Separate TH-1, TH-2, and TH-2/3 each binds muscle actin filaments with high affinity. Salt inhibits TH-2/3 binding to muscle actin but not amoeba actin filaments. TH-1 enhances binding of TH-2/3 to muscle actin filaments at physiological salt concentration, indicating that TH-1 and TH-2/3 cooperate in actin binding. An intrinsic fluorescence assay shows that TH-2/3 also binds with high affinity to the protein Acan125 similar to the SH3 domain of myosin-IC. Phylogenetic analysis of SH3 sequences suggests that myosin-I acquired SH3 domain after the divergence of the genes for myosin-I isoforms.

Mechanism of Interaction of Acanthamoeba Actophorin (ADF/Cofilin) with Actin Filaments

We characterized the interaction of Acanthamoeba actophorin, a member of ADF/cofilin family, with filaments of amoeba and rabbit skeletal muscle actin. The affinity is about 10 times higher for muscle actin filaments (Kd = 0.5 microM) than amoeba actin filaments (Kd = 5 microM) even though the affinity for muscle and amoeba Mg-ADP-actin monomers (Kd = 0.1 microM) is the same (Blanchoin, L., and Pollard, T. D. (1998) J. Biol. Chem. 273, 25106-25111). Actophorin binds slowly (k+ = 0.03 microM-1 s-1) to and dissociates from amoeba actin filaments in a simple bimolecular reaction, but binding to muscle actin filaments is cooperative. Actophorin severs filaments in a concentration-dependent fashion. Phosphate or BeF3 bound to ADP-actin filaments inhibit actophorin binding. Actophorin increases the rate of phosphate release from actin filaments more than 10-fold. The time course of the interaction of actophorin with filaments measured by quenching of the fluorescence of pyrenyl-actin or fluorescence anisotropy of rhodamine-actophorin is complicated, because severing, depolymerization, and repolymerization follows binding. The 50-fold higher affinity of actophorin for Mg-ADP-actin monomers (Kd = 0.1 microM) than ADP-actin filaments provides the thermodynamic basis for driving disassembly of filaments that have hydrolyzed ATP and dissociated gamma-phosphate.

Interactions of Acanthamoeba profilin with actin and nucleotides bound to actin.

Three methods, fluorescence anisotropy of rhodamine-labeled profilin, intrinsic fluorescence and nucleotide exchange, give the same affinity, Kd = 0.1 microM, for Acanthamoeba profilins binding amoeba actin monomers with bound Mg-ATP. Replacement of serine 38 with cysteine created a unique site where labeling with rhodamine did not alter the affinity of profilin for actin. The affinity for rabbit skeletal muscle actin is about 4-fold lower. The affinity for both actins is 5-8-fold lower with ADP bound to actin rather than ATP. Pyrenyliodoacetamide labeling of cysteine 374 of muscle actin reduces the affinity for profilin 10-fold. The affinity of profilin for nucleotide-free actin is approximately 3-fold higher than for Mg-ATP-actin and approximately 24-fold higher than for Mg-ADP-actin. As a result, profilin binding reduces the affinity of actin 3-fold for Mg-ATP and 24-fold for Mg-ADP. Mg-ATP dissociates 8 times faster from actin-profilin than from actin and binds actin-profilin 3 times faster than actin. Mg-ADP dissociates 14 times faster from actin-profilin than from actin and binds actin-profilin half as fast as actin. Thus, profilin promotes the exchange of ADP for ATP. These properties allow profilin to bind a high proportion of unpolymerized ATP-actin in the cell, suppressing spontaneous nucleation but allowing free barbed ends to elongate at more than 500 subunits/second.

Rabbit Skeletal Muscle Actin Behaves Differently Than Acanthamoeba Actin When Added to Solube Extracts of Acanthamoeba castellanii

Cold extracts of Acantharnoeba castellanii in polymerizing buffer contain 32 μM unpolymerized actin of which about 20% polymerizes (as measured by ultracentrifugation) when the extract is warmed to 22°C. As quantified by the increase in fluorescence of pyrene-labeled actin, 16% of muscle G-actin and 46% of Acanthamoeba G-actin polynterized when 0.8 μM of each was added to warm extracts of Acanthamoeba. Added muscle F-actin (1.2 μM) rapidly and totally depolymerized and then partially repolymerized whereas 1.2 μM added Acanthamoeba F-actin was stable indefinitely. Furthermore, muscle actin subunits were completely removed from copolymers of muscle and Acanthamoeba F-actin while all the amoeba actin remained polymerized when the copolymers contained at least 50% amoeba actin. These results suggest that exogenous tracer actin may not be an accurate indicator of the dynamics of endogenous actin in extracts and cells.

Roles of target cell membrane carbohydrate and lipid in Entamoeba histolytica interaction with mammalian cells

Latex beads and liposomes carrying glycoproteins with carbohydrate sequences recognized by an Entamoeba histolytica galactose-specific binding protein were assessed for their ability to adhere to trophozoites and to stimulate amoeba actin polymerization. Glycoprotein-conjugated beads bound significantly to amoebae but did not stimulate actin polymerization. Glycoprotein-bearing liposomes bound to amoebae and did enhance actin polymerization, as do recognized glycosphingolipid-bearing liposomes (G. B. Bailey, E. D. Nudelman, D. B. Day, C. F. Harper, and J. R. Gilmour, Infect. Immun. 58:43-47, 1990). Liposome-stimulated actin polymerization occurred only if the vesicle contained negatively charged phospholipid. It was concluded that both glycoprotein and glycosphingolipid glycans on the target cell surface are involved in attachment to E. histolytica but do not themselves induce the transmembrane signals that lead to cytoskeleton activation and target destruction. This requires interaction with lipids of the target membrane bilayer.

Specificity of glycosphingolipid recognition by Entamoeba histolytica trophozoites

The ability of purified glycosphingolipids to enhance liposome-stimulated Entamoeba histolytica actin polymerization was assessed as a means of defining the specificity of mammalian cell membrane lipid glycan recognition by this parasite. Synthetic liposomes containing a variety of individual glycosphingolipids bearing neutral, straight-chain oligomeric glycans with galactose or N-acetylgalactosamine termini stimulated rapid (90-s) polymerization of amoeba actin. Glycans with terminal N-acetylglucosamine residues were not stimulatory at all or were only weakly stimulatory. Glycans with glucose, N-acetylglucosamine, galactose, and N-acetylgalactosamine as the penultimate residue were recognized. Attachment of N-acetylneuraminate to the terminal residue of a stimulatory glycosphingolipid eliminated activity; attachment of fucose to the penultimate sugar reduced activity. Glycans with a terminal beta 1-4 or 1-3 glycosidic bond were most effective; glycans with terminal alpha 1-4 or 1-3 glycosides were less effective. The activity of glycans with both beta- and alpha-linked terminal glycosides was inhibited by lactose, suggesting recognition of both configurations by a single amoeba protein. The ability of liposomes to stimulate actin polymerization reflected the extent of liposome phagocytosis.

Antigenic reactivity of a monoclonal antibody against Amoeba proteus actin.

The reactivity of a monoclonal antibody against actin of Amoeba proteus with actins from other sources was examined. The monoclonal antibody cross-reacted with actins from vertebrate muscles, human erythrocytes, and Acanthamoeba castellanii, but it did not react with Naegleria gruberi actin. The amoeba actin was resolved into 3 bands with isoelectric points of 5.96, 6.03 and 6.10 in electrofocusing gels and they corresponded to 3 peptide spots reacting with the antibody on 2-dimensional immunoblots.

Phosphorylation of Amoeba G-actin and its effect on actin polymerization.

Mass culture of Amoeba proteus enabled us to do biochemical studies on this organism. Actin and profilin were purified from Amoeba to examine actin phosphorylation and polymerization. The apparent molecular weight of Amoeba actin was 44,000, and its isoelectric point was 5.8. The apparent molecular weight of Amoeba profilin was 12,000, and its isoelectric point was 4.9. It reduced the rate of actin polymerization as reported in the cases of profilins from other organisms. A protein of Mr = 44,000 (44 K protein) was phosphorylated in a Ca2+-dependent manner in cell homogenate of Amoeba without being inhibited by calmodulin antagonists. Using the homogenate as a kinase, purified Amoeba G-actin could be phosphorylated in proportion to the amount of actin. However, neither Amoeba F-actin nor rabbit skeletal muscle G-actin was phosphorylated. The phosphorylation of Amoeba actin with a kinase partially purified from A. proteus increased with dilution of the actin concentration. When Amoeba profilin was added, more than 80% of the actin was phosphorylated. By viscometry, electron microscopy, and ultracentrifugation analysis it was demonstrated that Amoeba G-actin phosphorylated in the presence of profilin and kinase did not polymerize in this solution. High-performance liquid chromatography analysis showed that phosphorylated Amoeba actin remained in a monomeric state even under conditions favorable for actin polymerization.

Purification and characterization of actophorin, a new 15,000-dalton actin-binding protein from Acanthamoeba castellanii.

Actophorin is a new actin-binding protein from Acanthamoeba castellanii that consists of a single polypeptide with a molecular weight of 15,000. The isoelectric point is 6.1, and amino acid analysis shows an excess of acidic residues over basic residues. The phosphate content is less than 0.2 mol/mol. There is 0.4 +/- 0.1 mg of actophorin/g of cells, so that the molar ratio of actin to actophorin is about 10:1 in the cell. Unique two-dimensional maps of tryptic and chymotryptic peptides and complete absence of antibody cross-reactivity show that Acanthamoeba actophorin, profilin, capping protein, and actin are separate gene products with minimal homology. Actophorin has features of both an actin monomer-binding protein and an actin filament-severing protein. Actophorin reduces the extent of actin polymerization at steady state in a concentration-dependent fashion and forms a complex with pyrene-labeled actin that has spectral properties of unpolymerized actin. During ultracentrifugation a complex of actophorin and actin sediments more rapidly than either actin monomers or actophorin. Although actophorin inhibits elongation at both ends of actin filaments, it accelerates the late stage of spontaneous polymerization like mechanical shearing and theoretical predictions of polymer fragmentation. Low concentrations of actophorin decrease the length and the low shear viscosity of actin filaments. High concentrations cause preformed filaments to shorten rapidly. Ca2+ is not required for any of these effects. Muscle and amoeba actin are equally sensitive to actophorin.

Rapid polymerization of Entamoeba histolytica actin induced by interaction with target cells.

Within 5 s of challenge of Entamoeba histolytica trophozoites with red blood cells (RBC), attachment and deformation of target cells occurred at multiple sites on the amoeba surface. Many trophozoite-target interfaces were outlined with a ring of polymerized amoeba actin, revealed by rhodamine-phalloidin staining of glutaraldehyde-fixed and Triton-X 100-extracted cells. The beginnings of phagocytic pseudopods rimmed many targets. The phagocytic membrane and underlying actin network grew uniformly about a target cell, which became dramatically elongated and constricted, sometimes severed, as it entered the amoeba. Total engulfment of RBC targets occurred within 10 s. By methanol extraction and spectrofluorimetric measurement of bound rhodamine-phalloidin we were able to quantitate polymerized actin in amoebae. Interaction with target cells was accompanied by a net increase of up to twofold in the average polymerized actin content of trophozoites. This reached a maximum during the period of most active phagocytosis (4 min after challenge at 25 degrees C), and declined as phagocytic activity diminished (8-16 min). Challenge with latex beads of similar size and number, which E. histolytica phagocytized more slowly than RBC, induced neither a detectable increase in polymerized actin content nor appearance of polymerized actin at the contact interface. RBC inhibited phagocytosis of latex beads, but the reverse did not occur. The results demonstrate a rapid, recognition-specific stimulation of reorganization of the actin cytoskeleton of E. histolytica induced by binding to target cells. Vigorous phagocytic activity is frequently an immediate consequence of cell-cell contact, which emphasizes the importance of this process in the contact-mediated attack mechanism of this pathogen. The quantitative assay of polymerized actin may be useful in further studies of this mechanism.

Mechanism of action of Acanthamoeba profilin: demonstration of actin species specificity and regulation by micromolar concentrations of MgCl2.

Acanthamoeba profilin strongly inhibits in a concentration-dependent fashion the rate and extent of Acanthamoeba actin polymerization in 50 mM KCl. The lag phase is prolonged indicating reduction in the rate of nucleus formation. The elongation rates at both the barbed and pointed ends of growing filaments are inhibited. At steady state, profilin increases the critical concentration for polymerization but has no effect on the reduced viscosity above the critical concentration. Addition of profilin to polymerized actin causes it to depolymerize until a new steady-state, dependent on profilin concentration, is achieved. These effects of profilin can be explained by the formation of a 1:1 complex with actin with a dissociation constant of 1 to 4 microM. MgCl2 strongly inhibits these effects of profilin, most likely by binding to the high-affinity divalent cation site on the actin. Acanthamoeba profilin has similar but weaker effects on muscle actin, requiring 5 to 10 times more profilin than with amoeba actin.

Biochemical and Structural Studies of Actomyosin-like Proteins from Non-Muscle Cells: II. PURIFICATION, PROPERTIES, AND MEMBRANE ASSOCIATION OF ACTIN FROM AMOEBAE OF DICTYOSTELIUM DISCOIDEUM

Abstract Actin, an apparently universal component of eukaryotic cells, has been purified from Dictyostelium amoebae to electrophoretic homogeneity. Purification was facilitated by newly discovered solubility properties of actomyosin in 30% sucrose. A quantitative assay for the Dictyostelium actin is described which is based on its ability to activate muscle heavy meromyosin ATPase activity. The specific activity of purified amoeba actin for activation of heavy meromyosin is nearly the same as that of purified muscle actin. The molecular weight of the Dictyostelium actin is identical with that of muscle actin, as judged by co-electrophoresis on sodium dodecyl sulfate acrylamide gels. The amoeba actin, like muscle actin, forms Mg2+-paracrystals with a helical repeat of about 360 A. Some of the Dictyostelium actin is associated with membrane in a MgATP-stable linkage. Myosin is found in membrane preparations in a MgATP-labile linkage, presumably due to association with the membranelinked actin.

Effect of skeletal muscle native tropomyosin on the interaction of amoeba actin with heavy meromyosin.

ACTIN and myosin, recently identified in protozoa and myxomycetes, are presumably responsible for the motility of these organisms1–5. Control of this motility is, however, not at all understood. In skeletal muscle, contraction is partly under the control of the protein complex, native tropomyosin which is believed to bind to the actin filaments6,7. In the presence of Ca2+, native tropomyosin allows the interaction of actin and myosin filaments to occur wiiereas in the absence of Ca2+ native tropomyosin inhibits this interaction6, probably by inhibiting the binding of actin to myosin8. No protein like native tropomyosin has yet been identified in a primitive contractile system, but we know that Acanthamoeba actin activates the ATPase of skeletal muscle heavy meromyosin (HMM) and we have used this fact to investigate the effect of skeletal muscle native tropomyosin on the interaction of Acanthamoeba actin with HMM. We have found that native tropomyosin in the presence of Ca2+ increases the activation of HMM ATPase by Acanthamoeba actin, while in the absence of Ca2+ the native tropomyosin inhibits this activation.

Epsilon-N-dimethyllysine in amoeba actin.

AMOEBA actin, a protein that shares many of the chemical, physical, and ultrastructural properties of muscle actin, has recently been isolated from Acanthamoeba castellanii1,2. The final steps in the purification procedure are the sedimentation of polymerized amoeba F-actin, its depolymerization by prolonged dialysis against a solution of ATP, and separation by molecular filtration on ‘Sephadex G-200’ of a minor excluded fraction and a major included fraction (amoeba G-actin). The product is monodisperse as judged by sedimentation equilibrium centrifugation in 5 M guanidine1 and has a molecular weight of about 39,500. Polyacrylamide gel electrophoresis at pH. 9.5 in 8.5 M urea of the reduced, carboxymethylated protein shows one major band that accounts for more than 90 per cent of the stainable protein. The amino-acid composition of amoeba actin was found to be very similar to that of other act ins including approximately 1 mole of the rare amino-acid 3-methylhistidine (3-MH) per mole of protein1. In addition, an unidentified component was detected which had the same elution time as e-N-methyllysine in the chromatographic system used1 which does not separate monomethyllysine from dimethyllysine.