Chicken Gizzard Smooth Muscle Cells Research Articles

Acidic Calponin controls ERK1/2 translocation and l-Caldesmon phosphorylation in fibroblasts

Calponin is an actin binding, troponin-like molecule, first isolated from chicken gizzard smooth muscle cells where it is involved in regulation of contractility. So far there are three known isoforms, basic, acidic and neutral Calponin, each encoded by a separate gene. Expression of the acidic Calponin isoform is, unlike basic Calponin, not restricted to smooth muscle cells but is expressed in a variety of tissues and cell types, especially in the brain. For basic Calponin it is known that the molecule affects smooth muscle cell contractility by regulation of the PKC/ERK signaling pathway. Here we could show that acidic Calponin co-precipitates together with ERK1/2 and PKCalpha in the cultured rat fibroblast cell line REF52.2. Moreover it colocalises with ERK1/2 and PKCalpha in podosome-like structures and at the cell cortex after Phorbol-12,13-Dibutyrate (PDBu) treatment. Knockdown of acidic Calponin expression in these cells results in inhibition of PDBu-mediated ERK1/2 translocation to these structures, whereas PKCalpha targeting is not affected. Both endogenous ERK1/2 activity and phosphorylation of the ERK1/2 substrate l-Caldesmon are blocked by acidic Calponin knockdown, indicating an important role for the acidic Calponin isoform in regulation of ERK1/2 function in REF52.2 cells.Abstract sponsored by John Gergely, Boston Biomedical Research Institute, Watertown 02472, MA, USASupport: NIH grants HL80003, HL86655

Unzipping the Role of Myosin Light Chain Phosphatase in Smooth Muscle Cell Relaxation

Recently, it has been hypothesized that myosin light chain (MLC) phosphatase is activated by cGMP-dependent protein kinase (PKG) via a leucine zipper-leucine zipper (LZ-LZ) interaction through the C-terminal LZ in the myosin-binding subunit (MBS) of MLC phosphatase and the N-terminal LZ of PKG (Surks, H. K., Mochizuki, N., Kasai, Y., Georgescu, S. P., Tang, K. M., Ito, M., Lincoln, T. M., and Mendelsohn, M. E. (1999) Science 286, 1583-1587). Alternative splicing of a 3'-exon produces a LZ+ or LZ- MBS, and the sensitivity to cGMP-mediated smooth muscle relaxation correlates with the relative expression of LZ+/LZ- MBS isoforms (Khatri, J. J., Joyce, K. M., Brozovich, F. V., and Fisher, S. A. (2001) J. Biol. Chem. 276, 37250 -37257). In the present study, we determined the effect of LZ+/LZ- MBS isoforms on cGMP-induced MLC20 dephosphorylation. Four avian smooth muscle MBS-recombinant adenoviruses were prepared and transfected into cultured embryonic chicken gizzard smooth muscle cells. The expressed exogenous MBS isoforms were shown to replace the endogenous isoform in the MLC phosphatase holoenzyme. The interaction of type I PKG (PKGI) with the MBS did not depend on the presence of cGMP or the MBS LZ. However, direct activation of PKGI by 8-bromo-cGMP produced a dose-dependent decrease in MLC20 phosphorylation (p<0.05) only in smooth muscle cells expressing a LZ+ MBS. These results suggest that the activation of MLC phosphatase by PKGI requires a LZ+ MBS, but the binding of PKGI to the MBS is not mediated by a LZ-LZ interaction. Thus, the relative expression of LZ+/LZ- MBS isoforms could explain differences in tissue sensitivity to NO-mediated vasodilatation.

Association of Calponin with Desmin Intermediate Filaments

Our previous immunoelectron microscopy studies of chicken gizzard smooth muscle cells showed that in certain areas the distribution of anti-calponin exhibits a high degree of overlap with beta-actin, filamin, and in particular, desmin, suggesting that in situ a fraction of calponin may be associated with intermediate filaments of the cytoskeleton. In this work we further explore this idea by studying the interaction between calponin and desmin. We found that at physiological salt concentrations, calponin bound only weakly to synthetic desmin intermediate filaments. On the other hand, calponin bound strongly to nonfilamentous desmin tetramers and was incorporated into intermediate filaments when the two proteins were mixed in a buffer containing 6 M urea and dialyzed into a buffer containing 0.15 M NaCl. Anti-calponin was found to label a portion of intermediate filaments and dense bodies isolated from gizzard tissues. Our findings suggest that in chicken gizzard smooth muscle cells, calponin may be an integral component of desmin intermediate filaments in the vicinity of dense bodies. Since calponin is also known to bind actin, we hypothesize that one of the functions of calponin might be to bridge intermediate filaments with actin in dense bodies.

A 260-kDa filamin/ABP-related protein in chicken gizzard smooth muscle cells is a new component of the dense plaques and dense bodies of smooth muscle.

A 260-kDa protein, termed cgABP260, which localized in the dense plaques and dense bodies of smooth muscle cells, was found in a low-salt alkaline extract of chicken gizzard smooth muscle. An antibody against cgABP260 was used to screen a chicken gizzard cDNA library, and the nucleotide sequence of the partial cDNA encoding this protein was determined. Comparison of predicted amino acid sequences revealed that the protein had significant homology with human ABP-280 and chicken retina filamin [Barry, C.P. et al. (1993) J. Biol. Chem. 268, 25577-25586], but despite the high homology, cgABP260 was immunologically distinguishable from filamin. Immunoblot analysis showed that an anti-cgABP260 antibody reacted exclusively with the cgABP260 band of smooth, skeletal, and cardiac muscle tissues. By indirect immunofluorescence, the membrane-affinity-purified antibody against cgABP260 intensely stained the dense plaques of the isolated smooth muscle cells. Immunoelectron microscopy showed that immunogold particles representing cgABP260 were found abundantly on the dense plaques and less abundantly on the dense bodies. Its amino acid sequence, molecular size, immunological reactivity, and localization in smooth muscle thus indicated that cgABP260 is a new component of the dense plaques and dense bodies of smooth muscle cells.

A 36-kDa Protein of the Dense Bodies of Smooth Muscle Cells

We purified a 36-kDa protein from a low-salt, alkaline extract of chicken gizzard smooth muscle by sequential column chromatography using DEAE-Cellurofine A-800 m, hydroxylapatite, and CM-Cellurofine C-500. This protein decreased the low-shear viscosity of actin filaments and coprecipitated with them by centrifugation at 18,500 x g. Electron microscopy showed that the 36-kda peptide bundled actin filaments. Immunoblot analysis revealed that an affinity-purified antibody against the 36-kDa protein reacted exclusively with the 36-kDa protein band of smooth muscle. In indirect immunofluorescence microscopy, the affinity-purified anti-36-kDa protein antibody stained the dotty structures of isolated smooth muscle cells, while in post-embedding immunoelectron microscopy, most of the colloidal gold particles representing the 36-kDa protein were found on the dense bodies of ultrathin sections of chicken gizzard smooth muscle cells. The antibody did not stain the dense plaques of isolated smooth muscle cells. Judging from its molecular weight, the 36-kDa protein is concluded to be a new component of the dense bodies of smooth muscle.

Actin isoform compartments in chicken gizzard smooth muscle cells.

Differentiated smooth muscle cells typically contain a mixture of muscle (alpha and gamma) and cytoplasmic (beta and gamma) actin isoforms. Of the cytoplasmic actins the beta-isoform is the more dominant, making up from 10% to 30% of the total actin complement. Employing an antibody raised against the N-terminal peptide specific to beta-actin, which labels only the beta-isoform on two-dimensional gel immunoblots, we have shown that this isoform has a restricted localisation in smooth muscle. Using double-label immunofluorescence and immunoelectron microscopy of ultrathin sections of chicken gizzard, beta-actin was localised in the dense bodies and in longitudinal channels linking consecutive dense bodies that were also occupied by desmin. It was additionally found in the membrane-associated dense plaques, but was excluded from the actomyosin-containing regions of the contractile apparatus. Taken together with earlier results these findings identify a cytoskeletal compartment containing intermediate filaments, cytoplasmic actin and the actin cross-linking protein filamin. Using an antibody specific only for muscle actin, labelling was found generally around the myosin filaments of the contractile apparatus, but was absent from the core of the dense bodies that contained beta-actin. Thus, if dense bodies act as dual-purpose anchorage sites, for the cytoskeletal actin and the contractile actin, the thin filaments of the contractile apparatus must be anchored at the periphery of the dense bodies. A model of the structural organisation of the cell is presented and the possible roles of the cytoskeleton are discussed.

Vinculin is a permanent component of the membrane skeleton and is incorporated into the (re)organising cytoskeleton upon platelet activation

Vinculin, a 130-kDa protein discovered in chicken gizzard smooth-muscle cells and subsequently also described in platelets, is believed to be involved in membrane-cytoskeleton interactions. In this study we investigated vinculin distribution in human blood platelets. Two skeletal fractions and a remaining cytosolic fraction were prepared with a recently described Triton X-100 lysis buffer causing minimal post-lysis breakdown by proteolysis. The presence of vinculin was demonstrated in the membrane skeleton and cytosol of resting and thrombin-activated human platelets. Upon thrombin stimulation vinculin also appeared in the cytoskeleton. this cytoskeletal incorporation was completed during the early stages of platelet aggregation and secretion, when the uptake of myosin, actin-binding protein and talin was still not maximal. We conclude therefore, that vinculin may play an important role in the structural (re)organisation of the human platelet cytoskeleton upon platelet activation.

Immunoelectron microscope studies of membrane-microfilament interactions: distributions of alpha-actinin, tropomyosin, and vinculin in intestinal epithelial brush border and chicken gizzard smooth muscle cells.

The ultrastructural localization of three cytoskeletal proteins, alpha-actinin, tropomyosin, and vinculin, in the brush border of epithelial cells of chicken small intestine and the smooth muscle cells of chicken gizzard was studied by immunofluorescence and immunonelectron microscope labeling of frozen sections of lightly fixed, intact tissues. In the immunoelectron microscope studies, a recently described new type of electron-dense antibody conjugate, imposil-antibody, has been successfully used, along with ferritin-antibody conjugates, in single and double immunolabeling experiments. In the intestinal brush border shows that vinvulin is sharply confined to the junctional complex close to the membrane region of the zonula adherens, in distinct contrast to the more diffuse distributions of the other two proteins. In the smooth muscle cells, the labeling patterns show that vinculin is sharply confined to the membrane-associated dense plaques, closer to the membrane than the alpha-Actinin is also present in the cytoplastic dense bodies, from which vinculin is absent. Tropomyosin is present diffusely distributed in the cytoplasm, but absent from both dense plaques and dense bodies. These findings with the muscle cells demonstrate, therefore, that the dense plaques and dense bodies are chemically and structurally distinct entities. The results with both tissues, along with those in previous papers (Geiger, 1979, Cell. 18:193-205.; Geiger et al., 1980, Proc. Natl. Acad. Sci. U. S. A. 77:4127-4131), suggest that vinculin may play an important and widespread role in the linkage of actin-containing microfilament bundles to membranes.

Striated myofibrils in anti-myosin stained, isolated chicken gizzard smooth muscle cells.

Highly purified chicken gizzard myosin was used to induce antibody production in rabbits. The IgG fraction was separated from the antisera and coupled to fluorescein isothiocyanate (FITC). Specific antibody (AGM) was isolated from the IgG fraction by affinity purification. Comparisons of the specificity of IgG and AGM for chicken smooth muscle myosin revealed a much greater specificity by AGM. Staining with IgG led to an apparent cross-reactivity with guinea pig smooth muscles which was not seen with AGM staining. Therefore, staining of cells for localization of myosin was performed with AGM. Isolated cells were obtained from chicken gizzards either by collagenase digestion or by agitation of glycerinated pieces. Stained cells and cell fragments revealed the presence of myofibrils as structural units with diameters of about 1.0 micrometer. Stained myofibrils occasionally displayed regular banding patterns with a repeating period of about 1.5 +/- 0.2 micrometer. The presence of banded myofibrils in non-cultured cells shows that the organization of the contractile material is similar to that previously reported for cultured cells by Gröschel-Stewart.