Amino acid sequence of Acanthamoeba actin

By amino acid sequence studies, only one form of cytoplasmic actin was detected in Acanthamoeba castellanii. Its amino acid sequence is very similar to the sequences of Dictyostelium and Physarum actins, from which Acanthamoeba actin differs in only nine and seven residues, respectively, including the deletion of the first residue. Acanthamoeba actin is unique in containing a blocked NH2-terminal neutral amino acid (glycine), while all other actins sequenced thus far have a blocked acidic amino acid (aspartic or glutamic) at the NH2 terminus. Acanthamoeba actin is also unique in that it contains an N epsilon-trimethyllysine residue at position 326. Like other actins, Acanthamoeba actin contains an NT-methylhistidine residue at position 73. The protein sequence is in complete agreement with the sequence derived from the nucleotide sequence of an expressed actin gene.     

Characterization of cytoplasmic actin isolated from Acanthamoeba castellanii by a new method.

Cytoplasmic actin has been isolated from Acanthamoeba castellanii by a new method, employing chromatography on DEAE-cellulose, that improves the yield by more than 20-fold over the previously reported method. This procedure should be particularly useful for isolating actin from cells in which it is present in relatively low concentration because the method does not depend initially on the polymerization of actin or its interaction with myosin. Systematic comparison of the properties of purified Acanthamoeba actin and rabbit skeletal muscle actin shows them to be similar in many ways: viscosity of F-actin, stoichiometry of bound nucleotide, stoichiometry of binding of muscle heavy meromyosin and myosin subfragment 1 in the absence of ATP, and ability to inhibit the KATPase activity of heavy meromyosin. The amino acid compositions of Acanthamoeba and muscle actin are also quite similar, but significant differences, especially the presence of epsilon-N-methyllysines in Acanthamoeba actin, have been confirmed. In addition to this structural difference, we find that Acanthamoeba actin is only one-third as effective as muscle actin as an activator of the MgATPase of muscle heavy meromyosin and subfragment 1. For Acanthamoeba actin, as for muscle actin, this activation exhibits hyperbolic dependence on actin concentration; i.e. the double reciprocal plot of ATPase activation versus actin concentration is linear. From these plots we find that the two actins give the same extrapolated ATPase activity at infinite actin concentration (Vmax) but differ by a factor of three in the concentration of actin needed to produce half-maximal activation (Kapp).     

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.     

Preferential association of acidic actin with nuclei and Nuclear matrix from mouse leukemia L5178Y cells

Nuclear matrix prepared from mouse leukemia L5178Y cells contained not only the two common actin isomers, beta and gamma actins, but also two additional acidic species of actin (pI 5.1 and 5.3). An anti-actin antibody recognized these acidic species as well as beta and gamma actins on a nitrocellulose filter following western blotting of two-dimensional electrophoresis. These acidic species were co-purified with beta and gamma actins using DNase I-Sepharose affinity chromatography on the nuclear matrix. Limited digestion of the acidic actin with protease V8 or trypsin gave very similar peptide fragments as did digestion of beta and gamma actins. These acidic actins were found to be distributed in the nuclear fraction, but were scarcely detectable in the cytoplasmic fraction. One of the acidic actins (pI 5.3) was found in all subnuclear fractions (DNase extract, high-salt extract and nuclear matrix), while the other species, the most acidic actin (pI 5.1), was localized predominantly in the nuclear matrix.     

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 isoeletric 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.     

Polymerization of Acanthamoeba actin. Kinetics, thermodynamics, and co-polymerization with muscle actin.

The kinetics and thermodynamics for the polymerization of purified Acanthamoeba actin were studied and compared to muscle actin. Polymerization was qualitatively similar for the two actins with a rate-limiting nucleation step followed by rapid polymer extension. Polymerization occurred only above a threshold critical concentration which varied with polymerization conditions for each actin. In the presence of 2 mM MgCl2, nucleation of both actins was rapid and their critical concentrations were similarly low and not detectably dependent on temperature. In 0.1 M KCl, the rates of nucleation of both actins were much slower than when Mg2+ was present and were significantly different from each other. Also, under these conditions, the critical concentrations of Acanthamoeba and muscle actin were significantly different and both varied markedly with temperature. These quantitative differences between the two actins could be attributed to differences in both their enthalpies and entropies of polymerization, Acanthamoeba actin having the more positive deltaH and delta S. Co-polymerization of the two actins was also demonstrated. Overall, however, there were no qualitative differences between Acanthamoeba and muscle actin that would suggest a unique role for the monomer-polymer equilibrium of cytoplasmic actin in cell motility.     

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.     

Actin amino-acid sequences. Comparison of actins from calf thymus, bovine brain, and SV40-transformed mouse 3T3 cells with rabbit skeletal muscle actin.

Actin was purified from calf thymus, bovine brain and SV40-transformed mouse 3T3 cells grown in tissue culture. Isoelectric focusing analysis showed the presence of the two actin polypeptides beta and gamma typical for non-muscle actins in all three actins. Tryptic and thermolytic peptides accounting for the complete amino-acid sequence of the cytoplasmic actins were separated and isolated by preparative fingerprint techniques. All peptides were characterized by amino-acid analysis and compared with the corresponding peptides from rabbit skeletal muscle actin. Peptides which differed in amino-acid composition from the corresponding skeletal muscle actin peptides were subjected to sequence analysis in order to localize the amino-acid replacement. The results obtained show that all three mammalian cytoplasmic actins studied contain the same amino-acid exchanges indicating that mammalian cytoplasmic actins are very similar if not identical in amino-acid sequence. The presence of two different isoelectric species beta and gamma in cytoplasmic actins from higher vertebrates is acccounted for by the isolation of two very similar but not identical amino-terminal peptides in all three actin preparations. The nature of the amino-acid replacements in these two peptides not only accounts for the different isoelectric forms but also shows that beta and gamma cytoplasmic actins are the products of two different structural genes expressed in the same cell. The total number of amino-acid replacements so far detected in the comparison of these cytoplasmic actins and skeletal muscle actin is 25 for the beta chain and 24 for the gamma chain. With the exception of the amino-terminal three or four residues, which are responsible for the isoelectric differences, the replacements do not involve charged amino acids. The exchanges are not randomly distributed. No replacements were detected in regions 18--75 and 299--356 while the regions between residues 2--17 and 259--298 show a high number of replacements. In addition documentation for a few minor revisions of the amino acid sequence of rabbit skeletal muscle actin is provided.     

The covalent structure of Acanthamoeba actobindin

Actobindin is a protein from Acanthamoeba castellanii with bivalent affinity for monomeric actin. Because it can bind two molecules of actin, actobindin is a substantially more potent inhibitor of the early phase of actin polymerization than of F-actin elongation. The complete amino acid sequence of 88 residues has been deduced from the determined sequences of overlapping peptides obtained by cleavage with trypsin, Staphylococcus V8 protease, endoproteinase Asp-N, and CNBr. Actobindin contains 2 trimethyllysine residues and an acetylated NH2 terminus. About 76% of the actobindin molecule consists of two nearly identical repeated segments of approximately 33 residues each. This could explain actobindin's bivalent affinity for actin. The circular dichroism spectrum of actobindin is consistent with 15% alpha-helix and 22% beta-sheet structure. A hexapeptide with sequence LKHAET, which occurs at the beginning of each of the repeated segments of actobindin, is very similar to sequences found in tropomyosin, muscle myosin heavy chain, paramyosin, and Dictyostelium alpha-actinin. A longer stretch in each repeated segment is similar to sequences in mammalian and amoeba profilins. Interestingly, the sequences around the trimethyllysine residues in each of the repeats are similar to the sequences flanking the trimethyllysine residue of rabbit reticulocyte elongation factor 1 alpha, but not to the sequences around the trimethyllysine residues in Acanthamoeba actin and Acanthamoeba profilins I and II.     

At least six different actins are expressed in a higher mammal: An analysis based on the amino acid sequence of the amino-terminal tryptic peptide

The protein chemical characterization of the amino-terminal tryptic peptide of actin from different bovine tissues shows that at least six different actin structural genes are expressed in this mammal.Unique amirio acid sequences are found for actin from skeletal muscle, for actin from heart muscle, for two different actin species from smooth muscle, and for two different actin species typical of non-muscle tissues such as brain and thymus. The presence of more than one actin species in the same tissue (e.g. nonmuscle tissues and smooth muscles) is demonstrated by different amino-terminal peptides which, however, are closely related. The actins from the sarcomeric muscles (e.g. skeletal muscle and heart muscle) show unique but extremely similar amino-terminal peptides. A limited comparison of bovine and avian actins involving smooth and skeletal muscles emphasizes that among higher vertebrates actin divergence involves tissue rather than species specificity.For the lower eukaryotic organism Physarum polycephalum a single actin amino-terminal peptide is found, indicating that only one actin species is present during the plasmodial stage. The amino acid sequence of this peptide although unique reveals a high degree of homology with the corresponding mammalian cytoplasmic actin peptides.Different actin extraction and purification procedures have been compared by the relative yields of the different amino-terminal peptides. The results indicate that the various actin species obtained by the current purification procedures are a true reflection of the actual actins present in the tissue. In addition we compare the resolution provided by either isoelectric focusing analysis of different actins or by the protein chemical characterization of the amino-terminal peptides of different actins. We show that the latter procedure is more suitable for recording changes in actin expression during evolution and differentiation.     

Purification of microvilli and an analysis of the protein components of the microfilament core bundle

A fast and convenient method for the purification of microvilli from chicken intestinal brush borders is described. The microvilli appear morphologically very similar to those found on intact brush borders. Removal of the microvillus membrane from the microvilli by Triton X-100 treatment reveals compact bundles of microfilaments with small regularly spaced projections along their length. SDS-polyacrylamide gel analysis of the protein components of the brush border, the microvilli and the microvillus core bundles shows that little or no tropomyosin, myosin or filamin is found in the microvillus, whereas polypeptide chains with mobilities characteristic for these proteins are present in the whole brush border. The majority of the microvillus core protein is actin, and the other major protein present has a polypeptide molecular weight of 95 000. Total actin from both brush borders and microvilli, characterized by isoelectric focussing analysis, contained about 40% β actin and 60% γ actin. The presence of both the β and γ cytoplasmic actins in the highly ordered parallel arrays of microfilaments of the microvilli is discussed in light of hypotheses for different functional roles of these two actin species.     

MICROHETEROGENEITY OF BRAIN CYTOPLASMIC AND SYNAPTOPLASMIC ACTINS

Abstract— Actin present in whole rat brain cytoplasm and in synaptosomes was purified by DNase I affinity chromatography. By use of two-dimensional gels and one-dimensional isoelectric focusing gels, brain actin was shown to be composed of two isomeric forms. By comparison with muscle actins, brain actins were identified as the β and γ isomers. Muscle type α actin is not present in brain. Synaptosomal protein with high affinity for DNase I is primarily composed of β and γ actin, however, two minor synaptosomal proteins, S₁ and S₂, with similar DNase I affinity were also isolated. S₁1 and S₂ have the same apparent molecular weight as whole brain actin, are more acidic than the major actin forms and are distinct from a actin. Relative to β and γ actin, the content of S₁ and S₂ is 3-fOld greater in synaptosomes when compared to similar non-synaptosomal species. The results demonstrate heterogeneity of brain actins and compartmentalization of brain proteins with high affinity for DNase I at the synapse. It was also shown that tubulin has selective affinity for the DNase I-actin complex.     

Conserved tissue-restricted expression of HUC 1-1 actin phenotype among eumetazoan organisms.

Actin has evolved from a single protein into a family of more than six distinct isoforms in mammals. Based on amino acid sequence data, actins segregate into two major classes, the "cytoplasmic" or nonmuscle actins, present in all animals, and the "a-" or muscle actins, a group restricted to vertebrate muscle. We have recently identified two unique actin isoforms in rat intestinal brush border which combine features of these two classes. The amino terminal regions of these actins indicate that they are of a cytoplasmic type and yet the carboxy terminal regions contain an epitope (defined by Mab HUC 1-1) which, among mammalian actins, is restricted to the muscle isoforms. We report here that in addition to the rat, all species thus far examined which have an intestinal "brush border" express actins containing the HUC 1-1 epitope in this region. Furthermore, we show that the actins present in the muscle tissue of nonvertebrate eumetazoans, which are all of the cytoplasmic type, also contain this epitope. Thus these findings suggest that the HUC 1-1 epitope appeared early on a subset of cytoplasmic-type actins and was retained among actins expressed in muscle tissue throughout the evolutionary divergence of these cytoplasmic-type actins to the "a-" muscle actins.     

Insect muscle actins differ distinctly from invertebrate and vertebrate cytoplasmic actins

Summary Invertebrate actins resemble vertebrate cytoplasmic actins, and the distinction between muscle and cytoplasmic actins in invertebrates is not well established as for vertebrate actins. However, Bombyx and Drosophila have actin genes specifically expressed in muscles. To investigate if the distinction between muscle and cytoplasmic actins evidenced by gene expression analysis is related to the sequence of corresponding genes, we compare the sequences of actin genes of these two insect species and of other Metazoa. We find that insect muscle actins form a family of related proteins characterized by about 10 muscle-specific amino acids. Insect muscle actins have clearly diverged from cytoplasmic actins and form a monophyletic group emerging from a cluster of closely related proteins including insect and vertebrate cytoplasmic actins and actins of mollusc, cestode, and nematode. We propose that muscle-specific actin genes have appeared independently at least twice during the evolution of animals: insect muscle actin genes have emerged from an ancestral cytoplasmic actin gene within the arthropod phylum, whereas vertebrate muscle actin genes evolved within the chordate lineage as previously described.Offprint requests to.: N. Mounier     

Two monoclonal antibodies to actin: one muscle selective and one generally reactive

Two IgG1, kappa monoclonal antibodies (Mab) against actin have been obtained from a fusion in which chicken gizzard actin was used as the immunogen. One Mab, designated B4, shows a preferential reactivity toward enteric smooth muscle actin but also cross-reacts with skeletal, cardiac, and aorta actins on the basis of immunoblots, ELISA assays, and indirect immunofluorescence. However, this antibody does not react with either cytoplasmic actin in any of these assay systems. A second Mab, designated C4, reacts with all six known vertebrate isoactins as well as Dictyostelium discoideum and Physarum polycephalum actins. Thus B4 Mab appears to react with an epitope that is at least partially shared among the muscle actins but not found in cytoplasmic actins, while C4 Mab binds to an antigenic determinant that has been highly conserved among the actins. The binding sites of both Mabs on skeletal actin overlap that of pancreatic DNase I. Both antibodies bind a SV8 proteolytic product comprising the amino-terminal two-thirds of the actin molecule, and their epitopes appear to overlap since C4 can compete for the binding of B4 to skeletal actin. Neither antibody is able to prevent actin polymerization.     

Isolation and partial characterization of a 110-kD dimer actin-binding protein

Two Triton-insoluble fractions were isolated from Acanthamoeba castellanii. The major non-membrane proteins in both fractions were actin (30-40%), myosin II (4-9%), myosin I (1-5%), and a 55-kD polypeptide (10%). The 55-kD polypeptide did not react with antibodies against tubulins from turkey brain, paramecium, or yeast. All of these proteins were much more concentrated in the Triton-insoluble fractions than in the whole homogenate or soluble supernatant. The 55-kD polypeptide was extracted with 0.3 M NaCl, fractionated by ammonium sulfate, and purified to near homogeneity by DEAE-cellulose and hydroxyapatite chromatography. The purified protein had a molecular mass of 110 kD and appeared to be a homodimer by isoelectric focusing. The 110-kD dimer bound to F-actin with a maximal binding stoichiometry of 0.5 mol/mol of actin (1 mol of 55-kD subunit/mol of actin). Although the 110-kD protein enhanced the sedimentation of F-actin, it did not affect the low shear viscosity of F-actin solutions nor was bundling of F-actin observed by electron microscopy. The 110-kD dimer protein inhibited the actin-activated Mg2+-ATPase activities of Acanthamoeba myosin I and myosin II in a concentration-dependent manner. By indirect immunofluorescence, the 110-kD protein was found to be localized in the peripheral cytoplasm near the plasma membrane which is also enriched in F-actin filaments and myosin I.     

Actins from plant and animal sources tend not to form heteropolymers in vitro and function differently in plant cells

Summary. Pollen and skeletal muscle actins were purified and labeled with fluorescent dyes that have different emission wavelengths. Observation by electron microscopy shows that the fluorescent actins are capable to polymerize into filamentous actin in vitro, bind to myosin S-1 fragments, and have a critical concentration similar to unlabeled actin, indicating that they are functionally active. The globular actins from two sources were mixed and polymerized by the addition of ATP and salts. The copolymerization experiment shows that when excited by light of the appropriate wavelength, both red actin filaments (pollen actin) and green actin filaments (muscle actin) can be visualized under the microscope, but no filaments exhibiting both green and red colors are detected. Furthermore, coprecipitations of labeled pollen actin with unlabeled pollen and skeletal muscle actin were performed. Measurements of fluorescent intensity show that the amount of labeled pollen actin precipitating with pollen actin was much higher than that with skeletal muscle actin, indicating that pollen and muscle actin tend not to form heteropolymers. Injection of labeled pollen actin into living stamen hair cells results in the formation of normal actin filaments in transvacuolar strands and the cortical cytoplasm. In contrast, labeled skeletal muscle actin has detrimental effects on the cellular architecture. The results from coinjection of the actin-disrupting reagent cytochalasin D with pollen actin show that overexpression of pollen actin prolongs the displacement of the nucleus and facilitates the recovery of the nuclear position, actin filament architecture, and transvacuolar strands. However, muscle actin perturbs actin filaments when injected into stamen hair cells. Moreover, nuclear displacement occurs more rapidly when cytochalasin D and muscle actin are coinjected into the cell. It is concluded that actins from plant and animal sources behave differently in vitro and in vivo and that they are functionally not interchangeable.     

Immunological characterization of an anti-actin antibody specific for cytoplasmic actins and its use for the immunocytological localization of actin in Aplysia nervous tissue

Previous immunochemical and immunocytochemical studies have shown that an antibody to actin prepared from body wall muscle of the marine mollusc Aplysia californica is specific for vertebrate cytoplasmic actins. The ability of this anti-actin to distinguish between different forms of actin most likely reflects the recognition of amino acid sequences unique to cytoplasmic actins. We have confirmed the specificity of this antibody for cytoplasmic actins using nervous tissue as a source of cytoplasmic actin in further immunochemical studies. In addition to binding cytoplasmic actin in purified preparations, the antibody removed actin selectively from crude extracts of nervous tissue of some but not all of the species tested. Our results also suggest that tissue-specific differences in the distribution of cytoplasmic actins may exist. Immunofluorescence studies of Aplysia nervous tissue stained with anti-actin revealed that actin is present in the cell body and axonal processes of Aplysia neurons. Although the function of actin in nerve cells is not understood, the observed pattern of immunofluorescence staining is consistent with the idea that actin may be involved in movement within the axoplasm.     

The role of actin in the temperature-dependent gelation and contraction of extracts of Acanthamoeba

The temperature-dependent assembly and the interaction of Acanthamoeba contractile proteins have been studied in a crude extract. A cold extract of soluble proteins from Acanthamoeba castellanii is prepared by homogenizing the cells in a sucrose-ATP-ethyleneglycol-bis-(beta-aminoethyl ether) N,N'-tetraacetic acid buffer and centrifuging at 136,000 g for 1 h. When this supernate of soluble proteins is warmed to room temperature, it forms a solid gel. Upon standing at room temperature, the gel slowly contracts and squeezes out soluble components. The rates of gelation and contraction are both highly temperature dependent, with activation energies of about 20 kcal per mol. Gel formation is dependent upon the presence of ATP and Mg++. Low concentrations of Ca++ accelerate the contractile phase of this phenomenon. The major protein component of the gel is actin. It is associated with myosin, cofactor, a high molecular weight protein tentatively identfied as actin-binding protein, and several other unidentified proteins. Actin has been purified from these gels and was found to be capable of forming a solid gel when polymerized in the presence of ATP, MgCl3, and KCL. The rate of purified actin polymerication is very temperature dependent and is accelerated by the addition of fragments of muscle actin filaments. These data suggest that Acanthamoeba contractile proteins have a dual role in the cell; they may generate the forces for cellular movements and also act as cytoskeletal elements by controlling the consistency of the cytoplasm.     

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.