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[Expression of an Arabidopsis actin-depolymerizing factor 4 gene (AtADF4) in tobacco causes morphological change of plants].

The actin-depolymerizing factor 4 gene (ADF4) of Arabidopsis thaliana was cloned and sequenced (Figs.1, 2). The plant expression vector with ADF4 was constructed and transformed into tobacco by Agrobacterium tumefaciens. Molecular identification showed that the ADF4 gene was integrated into the genome of tobacco and expressed in transgenic tobaccos assayed by PCR and RT-PCR (Fig.3). Expression of an Arabidopsis thaliana ADF4 gene in tobacco resulted in morphological change of plants. The effects of ADF4 on transgenic tobaccos growth were as follows: the hypocotyls of transgenic plants were wavy, especially in darkness, whereas those of the control were straight (Fig.4A); the root hairs of transgenic plants were less than the control, and they were also wavy (Fig.4B); the parenchyma cells of transgenic plants were larger than the control and the arrangement of vascular bundle was out of order (Fig.4C); the flowering time of T(2) line was at least 7 days later than the control; the pollen tubes of transgenic plants were shorter than those of the control (Fig.4D).

Agrobacterium tumefaciens↗

Purification and characterization of aginactin, a newly identified agonist-regulated actin-capping protein from Dictyostelium amoebae.

Amoeboid chemotaxis involves a regulated increase in actin nucleation activity that is correlated with an increase in actin polymerization occurring seconds after chemotactic stimulation (Carson, M., Weber, A., and Zigmond, S. H. (1986) J. Cell Biol. 103, 2707-2714; Hall, A. L., Warren, V., Dharmawardhane, S., and Condeelis, J. (1989) J. Cell Biol. 109, 2207-2213). We report the isolation and characterization of an agonist-regulated capping protein, aginactin, from Dictyostelium that may regulate these changes in actin nucleation activity. Aginactin is isolated from low speed supernatants of starved amoebae by sequential anion exchange, hydrophobic interaction, fast protein liquid chromatography anion exchange, and hydroxyapatite chromatography. Aginactin migrates with an apparent molecular weight of 70,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and gel filtration columns, suggesting that it is a globular monomer. Aginactin is a barbed-end capping protein by several criteria. It inhibits the rate and final extent of actin polymerization and increases the apparent critical concentration at substoichiometric ratios to actin. It also inhibits depolymerization of F-actin and inhibits polymerization at the barbed end of Limulus acrosomal bundles. Aginactin is unaffected by micromolar Ca2+, and it neither severs F-actin nor nucleates actin polymerization in either the presence or absence of Ca2+. Aginactin binds to and cosediments with F-actin and has an apparent Kd for capping F-actin of 2.7 nM.

Actin Capping Proteins↗

[Organization of stress fibrils in cultured fibroblasts studied by using an actin filament-fragmenting protein].

Earlier we isolated a 1:1 complex of 90 kD-protein and actin from bovine brain. This complex was able to fragment actin filaments. Effects of this complex on the cytoskeleton of mouse and quail embryo fibroblasts are described. The cells were extracted with Triton X-100, and the resulting cytoskeletons were treated with the complex. Tetramethylrhodaminylphalloin and actin antibody staining failed to detect actin in preparations treated with the 90 kD protein-actin complex. Electrophoretic data confirmed actin solubilization upon this treatment. Immunofluorescent microscopy showed that actin solubilization was accompanied by extraction of vinculin and alpha-actinin from focal contacts and stress-fibers. In contrast, myosin distribution in treated cytoskeletons did not differ significantly from that in control extracted cells: in both the cases myosin was found mainly in the stress-fibers. Thus, myosin localization in stress-fibers does not depend on actin and is probably controlled by some other cytoskeletal component(s).

Actin Cytoskeleton↗

Ca2+-independent F-actin capping proteins. Cap 32/34, a capping protein from Dictyostelium discoideum, does not share sequence homologies with known actin-binding proteins.

Polyclonal and monoclonal antibodies against purified 32/34-kDa F-actin-capping protein from Dictyostelium discoideum were generated and used to isolate clones coding for both subunits from a lambda gt11 expression library. In addition genomic clones were isolated for the 34-kDa subunit. The sequences of the corresponding inserts were determined and the amino acid sequences of the proteins deduced. The amino acid sequences have been confirmed by sequencing tryptic peptides of both subunits. As judged from immunological data and sequence comparison, the subunits are completely different. Each of them is encoded by a single copy gene. The mRNAs are present throughout all stages of development. Severing proteins, which exert also capping activity, show no sequence similarity with the cap 32/34-kDa protein. However, a pronounced homology between the 32-kDa subunit and a hypothetical yeast protein was found indicating the presence of a protein homologous to the 32-kDa subunit of the D. discoideum capping protein in yeast.

Actin Depolymerizing Factors↗

cDNAs encoding the beta subunit of cap Z, the actin-capping protein of the Z line of muscle.

Three cDNAs encoding the beta chain of Cap Z have been isolated from lambda gt11 chicken libraries screened with antibodies. The coding region, which is identical among the cDNAs, contains 831 basepairs encoding a protein with 277 amino acid residues and Mr = 31,352. The predicted protein sequence contains eight regions that match perfectly the NH2-terminal sequence of eight peptides isolated from muscle Cap Z beta. The amino acid compositions of the protein predicted from the cDNA sequence and Cap Z beta are similar. Comparison of the cDNA sequence and the predicted protein sequence with those of other actin-binding proteins and all sequences in the GenBank and NBRF Protein databases shows no remarkable similarities. Two of the three cDNAs contain the complete coding region. Both coding regions begin with a consensus translation start site; however, their 5'-untranslated regions are different. Northern analysis of whole chicken embryos and adult chicken tissues shows two mRNA species. The embryo and non-muscle tissues have transcripts of 1.35 and 2.00 kilobases, and the muscle tissues have transcripts of 2.15 and 1.45 kilobases. Southern analysis of chicken genomic DNA shows that there are probably two related genes, one more similar to the cDNA than the other.

Actin Capping Proteins↗

Amino acid sequence of starfish oocyte depactin.

The amino acid sequence of starfish oocyte depactin was determined by aligning lysyl endopeptidase, Staphylococcus aureus V8 protease, and cyanongen bromide peptides. Starfish oocyte depactin is composed of 150 amino acid residues and the N terminus is free proline. The molecular weight is calculated to be 17,590, in good agreement with the value estimated by sodium dodecyl-polyacrylamide gel electrophoresis. Prediction of the secondary structure shows that depactin contains 64% alpha-helix. Comparison of depactin sequence with those of other proteins shows no significant similarity.

Actin Depolymerizing Factors↗

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.

Actin Depolymerizing Factors↗

Isolation and characterization of a 30,000-dalton calcium-sensitive actin cross-linking protein from Dictyostelium discoideum.

A calcium-sensitive actin-binding protein having a subunit molecular mass of 30,000 daltons (30K protein) has been isolated from Dictyostelium discoideum. Structural, immunological, and functional analyses demonstrated that the 30K protein was distinct from other actin-binding proteins of D. discoideum. A native molecular mass of 31,700 daltons was determined by equilibrium sedimentation, indicating that the protein is monomeric. The Stokes radius was 30 A. The frictional coefficient calculated from these measurements was 1.44, indicating an asymmetric shape. The 30K protein induced an increase in the viscosity of a solution of F-actin. Bundles of actin filaments were observed in negatively stained mixtures of actin and the 30K protein. Both the formation of filament bundles and the increases in viscosity of actin induced by the 30K protein were observed in the presence of 1 X 10(-8) M but not 2 X 10(-6) M calcium. Variation of the pH from 6.6 to 7.8 had no effect on the activity of the 30K protein. Calcium induced neither a large change in quaternary structure of the 30K protein nor a restriction of the lengths of actin filaments by the 30K protein. The apparent affinity of the 30K protein for actin was decreased in the presence of calcium. Reversible cross-linking of actin filaments by the 30K protein may contribute to regulation of the consistency and contractility of cytoplasm in D. discoideum.

Actin Depolymerizing Factors↗

Differentiation of myeloid leukemia cells and changes in their contractile proteins.

In this report we tried to draw a sketch of our recent work on the changes in actomyosin system during the differentiation of a myeloid leukemia cell line (M1). Before the differentiation, the M1 cells do not have a locomotive and phagocytic activity but do after the differentiation. The rather peculiar properties of actin shown by the leukemic M1 cells, low polymerizability and low affinity to myosin ATPase as well as the presence of polymerization inhibitor may explain their inability of motility and phagocytosis. Enhanced binding of actin and myosin on the plasma membrane looks necessary to carry out those differentiated functions which must need vigorous movement of the membrane. The cell movement also anticipates cell deformability which accompanies gelation in a part of cytoplasm and solation in another part. We have already isolated several gelation-factors as well as inhibitors. Not only chemical modifications of actin and myosin molecules but also quantitative variations in those regulatory proteins must be taken into consideration. Thus, studies on the cellular contractile proteins are casting a new light on the process of differentiation of the M1 cell line.

Actin Depolymerizing Factors↗

Isolation of an inhibitor of actin polymerization from human polymorphonuclear leukocytes.

A large proportion of actin in extracts of human polymorphonuclear leukocytes is unpolymerized in the presence of 0.1 M KCl, a condition expected to promote maximal assembly of purified actin. Raising of the KCl concentration increases the extent of actin polymerization in leukocyte extracts, and, at KCl concentrations greater than or equal to 0.3 M, the polymer concentration is maximal and approaches that of purified muscle actin in 0.1 M KCl solution. After removal of the actin polymers from leukocyte extracts made 0.6 M in KCl, the supernatant fluid contains an activity which inhibits the extent of assembly of purified skeletal muscle actin in 0.1 M KCl solution. We have purified this activity by ion exchange and gel filtration chromatography. The purified inhibitor contains polypeptides of 65,000 and 62,000 daltons in a molar ratio of 1:2 as determined by polyacrylamide gel electrophoresis in dodecyl sulfate. The Stokes radius of 32 A, and S20,w of 4.8 of the native inhibitor suggest that the purified protein is a mixture of monomers of slightly different molecular weight. Substoichiometric concentrations of the inhibitor rapidly prevent the assembly of purified actin in 0.1 m KCl solution in a concentration-dependent manner, and the reduction in final viscosity remains constant over time. Raising the KCl concentration reverses the inhibition, as observed for actin polymerization in unfractionated extracts. The findings are consistent with the conclusion that this inhibitor protein is responsible for the large proportion of unpolymerized actin in extracts of polymorphonuclear leukocytes.

Actin Depolymerizing Factors↗