A monoclonal antibody (anti-alpha sm-1) recognizing exclusively alpha-smooth muscle actin was selected and characterized after immunization of BALB/c mice with the NH2-terminal synthetic decapeptide of alpha-smooth muscle actin coupled to keyhole limpet hemocyanin. Anti-alpha sm-1 helped in distinguishing smooth muscle cells from fibroblasts in mixed cultures such as rat dermal fibroblasts and chicken embryo fibroblasts. In the aortic media, it recognized a hitherto unknown population of cells negative for alpha-smooth muscle actin and for desmin. In 5-d-old rats, this population is about half of the medial cells and becomes only 8 +/- 5% in 6-wk-old animals. In cultures of rat aortic media SMCs, there is a progressive increase of this cell population together with a progressive decrease in the number of alpha-smooth muscle actin-containing stress fibers per cell. Double immunofluorescent studies carried out with anti-alpha sm-1 and anti-desmin antibodies in several organs revealed a heterogeneity of stromal cells. Desmin-negative, alpha-smooth muscle actin-positive cells were found in the rat intestinal muscularis mucosae and in the dermis around hair follicles. Moreover, desmin-positive, alpha-smooth muscle actin-negative cells were identified in the intestinal submucosa, rat testis interstitium, and uterine stroma. alpha-Smooth muscle actin was also found in myoepithelial cells of mammary and salivary glands, which are known to express cytokeratins. Finally, alpha-smooth muscle actin is present in stromal cells of mammary carcinomas, previously considered fibroblastic in nature. Thus, anti-alpha sm-1 antibody appears to be a powerful probe in the study of smooth muscle differentiation in normal and pathological conditions.

/pdf/a-monoclonal-antibody-against-alpha-smooth-muscle-actin-a-3piknl7au6.pdf

A monoclonal antibody against alpha-smooth muscle actin: a new probe for smooth muscle differentiation.

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.

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

A monoclonal antibody, 6-11B-1, specific for acetylated alpha-tubulin (Piperno, G., and M. T. Fuller, 1985, J. Cell Biol., 101:2085-2094) was used to study the distribution of this molecule in interphase cells of Chlamydomonas reinhardtii. Double-label immunofluorescence was performed using 6-11B-1, and 3A5, an antibody specific for all alpha-tubulin isoforms. It was found that acetylated alpha-tubulin is not restricted to the axonemes, but is also present in basal bodies and in a subset of cytoplasmic microtubules that radiate from the basal bodies just beneath the plasma membrane. Immunoblotting experiments of basal body polypeptide components using 6-11B-1 as a probe confirmed that basal bodies contain acetylated alpha-tubulin. In the cell body, 6-11B-1 stained an average of 2.2 microtubules/cell, while 3A5 stained an average of 6.5 microtubules. Although exposure to 0 degrees C depolymerized both types of cytoplasmic microtubules, exposure to various concentrations of colchicine or nocodazole showed that the acetylated microtubules are much more resistant to drug-induced depolymerization than nonacetylated microtubules. Axonemes and basal bodies are already known to be colchicine-resistant. All acetylated microtubules appear, therefore, to be more drug-resistant than nonacetylated microtubules. The acetylation of alpha-tubulin may be part of a mechanism that stabilizes microtubules.

/pdf/cytoplasmic-microtubules-containing-acetylated-alpha-tubulin-3wsad5sb0s.pdf

Cytoplasmic microtubules containing acetylated alpha-tubulin in Chlamydomonas reinhardtii: spatial arrangement and properties.

Cyclase-associated protein (CAP) is an evolutionarily conserved regulator of the G-actin/F-actin ratio and, in yeast, is involved in regulating the adenylyl cyclase activity. We show that cell polarization, F-actin organization, and phototaxis are altered in a Dictyostelium CAP knockout mutant. Furthermore, in complementation assays we determined the roles of the individual domains in signaling and regulation of the actin cytoskeleton. We studied in detail the adenylyl cyclase activity and found that the mutant cells have normal levels of the aggregation phase-specific adenylyl cyclase and that receptor-mediated activation is intact. However, cAMP relay that is responsible for the generation of propagating cAMP waves that control the chemotactic aggregation of starving Dictyostelium cells was altered, and the cAMP-induced cGMP production was significantly reduced. The data suggest an interaction of CAP with adenylyl cyclase in Dictyostelium and an influence on signaling pathways directly as well as through its function as a regulatory component of the cytoskeleton.

The cyclase-associated protein CAP as regulator of cell polarity and cAMP signaling in Dictyostelium.

When Dictyostelium cells that have initiated their developmental program upon starvation are returned to growth medium, there is a rapid and transient de novo tyrosine phosphorylation of a 43-kilodalton protein. This protein was found to be actin. Most of the phosphorylation occurred in a single, minor acidic isoform of actin. Developing cells that had been returned to growth medium lost their pseudopod extensions, became round, and had reduced adhesion to the substratum. These effects occurred with kinetics that matched the increase in tyrosine phosphorylation of actin. In mutant cell lines in which the gene for the phosphotyrosine phosphatase PTP1 had been disrupted, tyrosine phosphorylation of actin was rapid and more prolonged. These cells responded with proportionally accelerated kinetics of cell rounding. Cell lines overexpressing PTP1 had diminished amplitude and duration of actin tyrosine phosphorylation and exhibited diminished cell-shape change and an accelerated return to the extended cell-shape morphology seen in starved cells.

https://science.sciencemag.org/content/sci/259/5092/241.full.pdf

Tyrosine phosphorylation of actin in Dictyostelium associated with cell-shape changes

Three mouse monoclonal antibodies, Act I, Act II, and Act IV, against actin from the cellular slime mold Dictyostelium discoideum, have been made and characterized. All three antibodies are IgG1 and share the following properties: They form stable complexes with monomeric Dictyostelium actin, which prevents polymerization of the actin into filaments. On addition to preformed actin filaments, they cause a reduction in filament size and in the viscosity of the actin solution. They cross-react strongly with actins from the lower eucaryotes Physarum and Acanthamoeba, but not with alpha-actins from rabbit and human muscle or beta- and gamma-actins from human erythrocytes and a human B lymphoid cell line. Act II and Act IV recognize a similar antigenic determinant that is topographically distinct from that identified by Act I. In protein immunoblotting, only Act I bound strongly to Dictyostelium actin. Analysis of actin fragments with this technique showed that amino acids 13 to about 50 are required for Act I binding to actin. A comparison of the amino acid sequences of actins from lower eucaryotes and higher vertebrates implicates threonine 41 as a critical residue in the Act I antigenic site. The properties of Act II and Act IV suggest that they recognize antigenic sites involving the NH2-terminal six residues.

/pdf/monoclonal-antibodies-prepared-against-dictyostelium-actin-1qreyt4tcr.pdf

Monoclonal antibodies prepared against Dictyostelium actin: characterization and interactions with actin.

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Monoclonal antibodies prepared against Dictyostelium actin: characterization and interactions with actin.