Characterization of chicken gizzard calcyclin and examination of its interaction with caldesmon

Using a procedure developed to purify calcyclin from mouse Ehrlich ascites tumor cells calcyclin was purified from smooth muscle of chicken gizzard. Chicken gizzard calcyclin bound to phenyl-Sepharose in a calcium dependent manner as did mouse EAT cells and rabbit lung calcyclin but appeared to be more acidic than its mammalian counterparts as revealed by ion exchange chromatography on Mono Q. Chicken gizzard calcyclin bound ⁴⁵Ca²⁺ on nitrocellulose filters and exhibited a shift in electrophoretic mobility on urea-PAGE depending on Ca²⁺ concentration. Crosslinking experiments with BS³ showed that chicken gizzard calcyclin was able to form noncovalent dimers. As indicated by a decrease in maximum tryptophan fluorescence emission of caldesmon (about 14% at 1:1 molar ratio) and displacement of calmodulin from its complex with caldesmon, chicken gizzard calcyclin binds caldesmon. This binding was, however, much weaker than that of calmodulin and could not influence the interaction of caldesmon with actin. In consequence, calcyclin was unable to reverse the inhibitory effect of caldesmon on actin-activated Mg²⁺-ATPase activity of myosin in the presence of Ca²⁺.     

Direct binding studies of 125I-α-bungarotoxin and 3H-quinuclidinyl benzilate interaction with axon plasma membrane fragments. Evidence for nicotinic and muscarinic binding sites

The attachment of ¹²⁵I-α-bungarotoxin (BgTx) which is reportedly bound exclusively to “nicotinic” acetylcholine receptors, as well as ³H-atropine and ³H-3-quinuclidinyl benzilate (QNB), which reportedly bind exclusively to “muscarinic” receptors, was measured in isolated lobster axon plasma membrane fragments and in the soluble axonal protein fraction. ¹²⁵I-α-BgTx binding was also measured in lysolecithin-solubilized fragments. Binding assays were adapted for these studies and are described in detail. High affinity, saturable binding of all three ligands to membrane fragments was observed, as well as binding of BgTx to a macromolecule present in both the soluble fraction and the membrane fragments. These experiments provide the first evidence for the very tight binding of both “nicotinic” and “muscarinic” ligands in peripheral nerve.     

Contact-dependent regulation of vinculin expression in cultured fibroblasts: a study with vinculin-specific cDNA probes.

Vinculin specific cDNA clones were isolated from chicken embryo fibroblast (CEF) cDNA library in lambda gt11. The clones, ranging in size from 2.8 to 5.0 kb, were initially selected by rabbit antibodies to vinculin. Their identity was further confirmed by their specific reactivities with a battery of different vinculin-specific monoclonal antibodies. Southern blot analysis of restriction enzyme digested chicken spleen DNA suggested that all the isolated cDNA clones correspond to the same gene(s). Northern blot hybridization revealed that the vinculin-specific cDNA clones react with a single 6.5 kb mRNA in total cellular RNA preparations of CEF, whole chicken embryos and chicken gizzard smooth muscle. Moreover, fractionation of CEF poly(A)+ RNA by sucrose gradient centrifugation followed by translation in cell free system indicated that the mRNA coding for vinculin has a size of about 6.0-7.0 kb. The identity of these clones was finally confirmed by selection hybridization assay. The isolated vinculin-specific cDNA probes were subsequently used in order to study the effect of substrate adhesiveness on the expression of vinculin. We show here that cells cultured on highly adhesive substrate, such as endothelial extracellular matrix (ECM), form large vinculin-rich focal contacts, while cells grown on poorly adhesive substrate poly(2-hydroxyethyl methacrylate) [poly(HEMA)] contain only small distorted vinculin spots. These morphological differences were accompanied by over 5-fold reduction in vinculin synthesis in cells growing on poly(HEMA), compared to those cultured on the ECM and over 7.5-fold decrease in the levels of vinculin-specific mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Paxillin: a new vinculin-binding protein present in focal adhesions

The 68-kD protein (paxillin) is a cytoskeletal component that localizes to the focal adhesions at the ends of actin stress fibers in chicken embryo fibroblasts. It is also present in the focal adhesions of Madin-Darby bovine kidney (MDBK) epithelial cells but is absent, like talin, from the cell-cell adherens junctions of these cells. Paxillin purified from chicken gizzard smooth muscle migrates as a diffuse band on SDS-PAGE gels with a molecular mass of 65-70 kD. It is a protein of multiple isoforms with pIs ranging from 6.31 to 6.85. Using purified paxillin, we have demonstrated a specific interaction in vitro with another focal adhesion protein, vinculin. Cleavage of vinculin with Staphylococcus aureus V8 protease results in the generation of two fragments of approximately 85 and 27 kD. Unlike talin, which binds to the large vinculin fragment, paxillin was found to bind to the small vinculin fragment, which represents the rod domain of the molecule. Together with the previous observation that paxillin is a major substrate of pp60src in Rous sarcoma virus-transformed cells (Glenney, J. R., and L. Zokas. 1989. J. Cell Biol. 108:2401-2408), this interaction with vinculin suggests paxillin may be a key component in the control of focal adhesion organization.     

An ordered precipitate of polyadenylic acid formed by freezing at acidic pH: comparison of X-ray diffraction and other properties of the precipitate with those of fibers or direct acid-precipitates

Poly A was found to precipitate upon freezing acidic solutions at p^H values where it is normally soluble; this precipitate tends to have the form of small thin plates of irregular outline (“plates”). The X-ray diffraction pattern and solubility properties of the “plates” were compared with those of poly A precipitated solely by exposure to lower p^H values, and with fibers drawn from acidic solution. There is considerable molecular order in each of these three types of preparation. In all cases, the diffraction patterns are consistent with the presence of the double-stranded helical structure proposed by Rich, Davies, Crick, and Watson (J. Mol. Biol., 3 , 71 (1961)) based on fiber diffraction data. The diffraction pattern from the “plates” is compared in detail with that of the fibers, and is shown to be in accord with a packing scheme having the chain axis of the molecular structure confined to the plane of the “plate,” but oriented randomly in that plane.     

Interaction of the 47-kDa talin fragment and the 32-kDa vinculin fragment with acidic phospholipids: a computer analysis.

In recent in vitro experiments, it has been demonstrated that the 47-kDa fragment of the talin molecule and the 32-kDa fragment of the vinculin molecule interact with acidic phospholipids. By using a computer analysis method, we determined the hydrophobic and amphipathic stretches of these fragments and, by applying a purpose-written matrix method, we ascertained the molecular amphipathic structure of alpha-helices. Calculations for the 47-kDa mouse talin fragment (residues 1-433; NH2-terminal region) suggest specific interactions of residues 21-39, 287-342, and 385-406 with acidic phospholipids and a general lipid-binding domain for mouse talin (primary amino acid sequence 385-401) and for Dictyostelium talin (primary amino acid sequence 348-364). Calculations for the 32-kDa chicken embryo vinculin fragment (residues 858-1066; COOH-terminal region) and from nematode vinculin alignment indicate for chicken embryo vinculin residues 935-978 and 1020-1040 interactions with acidic phospholipids. Experimental confirmation has been given for vinculin (residues 916-970), and future detailed experimental analyses are now needed to support the remaining computational data.     

Isolation and partial characterization of human platelet vinculin

A 130,000 Mr protein was isolated from human platelets by sequential DEAE-Sephacel and Sepharose Cl-4B chromatography. Low shear viscometric measurements showed that the enriched protein after DEAE-Sephacel chromatography inhibited actin polymerization. This effect was somewhat greater in the presence of EGTA than in the presence of calcium. Further purification by Sepharose Cl-4B chromatography resulted in a complete loss of this inhibitory effect. Studies with fluorescent actin detected no nucleation or "+" end capping activity in either the DEAE-Sephacel- or Sepharose Cl-4B-purified vinculin. Antibodies raised in mice against the 130,000-mol-wt protein were shown to cross-react with chicken gizzard vinculin and a similar molecular weight protein was detected in WI38 cells and, Madin-Darby canine kidney cells. Lysis experiments with the Madin-Darby canine kidney cells indicated that most of the vinculin was soluble in Triton X-100, although some was found associated with the insoluble cytoskeletal residue. By immunofluorescence, vinculin in WI38 cells was localized to adhesion plaques as described by others. Discrete localization in platelets was also detected and appeared to depend on their state of adhesion and spreading. The results of these experiments suggest that human platelets contain a protein similar to vinculin. It is not clear if platelet vinculin is associated with structures analogous to adhesion plaques found in other cell types. The data indicate that the previously reported effects of nonmuscle vinculins on actin polymerization may be due to a contaminant or contaminants.     

Identification and isolation of vinculin from platelets

A vinculin-like protein was identified in chicken as well as in bovine platelets by ELISA competitive binding assay using antibodies against vinculin from chicken gizzard. By a modified procedure (J. Biol. Chem. (1980) 255, 1194–1199) we succeeded in isolating bovine platelet vinculin to apparent homogeneity. The structural identity of platelet and chicken gizzard vinculin was demonstrated by circular dichroism analysis. It was also shown that platelet vinculin induces a significant decrease in the low shear viscosity of F-actin. Vinculin, in all probability, plays an important role in the organization of actin filaments in platelets, especially in the linkages of microfilaments to the membrane.     

A 130K protein from chicken gizzard: Its localization at the termini of microfilament bundles in cultured chicken cells

A protein with a molecular weight of 130,000 (130K protein) was extracted from chicken gizzard and purified to homogeneity by ammonium sulfate fractionation and ion-exchange chromatography. Antibodies prepared against the pure protein were used for its immunochemical characterization and immunofluorescent visualization in cultured chicken cells. Both peptide mapping and immunochemical analysis indicated that the 130K protein is not related either structurally or antigenically to other mechanochemical proteins, including α-actinin, actin, myosin, tropomyosin, filamin and tubulin. Immunofluorescent labeling of different cultured embryonic chicken cells (from skin, heart and gizzard) indicated that the label was predominantly organized in intracellular plaques at the bottom of the cells and in some areas of cell-cell contact. Immunoprecipitation of the 130K protein from biosynthetically ³⁵S-methionine-labeled cultured cells, using the pure antibodies and Staphylococcus aureus, resulted in the specific isolation of a single labeled electrophoretic band indistinguishable from the chicken gizzard 130K protein. The 130K protein-rich plaques were found, by interference-reflection microscopy, to coincide with cell substrate adhesion plaques. Double immunofluorescent labeling for the 130K protein and other cytoskeletal proteins (actin, α-actinin and tropomyosin) indicated that the 130K protein-rich areas are localized at the termini of stress fibers. α-Actinin was found in close association with the 130K protein, while tropomyosin was usually excluded from those areas.     

Spatial and temporal relationships between vinculin and talin in the developing chicken gizzard smooth muscle

The spatiotemporal relationships between vinculin and talin in developing chicken gizzard smooth muscle were investigated. Immunofluorescence and immunoelectron-microscopic labeling revealed that both proteins are associated with membrane-bound dense plaques in muscle cells; however, the most intense labeling for vinculin was located rather closer to the membrane than that for talin. The localization of vinculin and talin in embryonic chicken gizzards indicated that both are primarily cytoplasmic during the first 2 embryonic weeks. Only around days 16-18 does talin apparently become associated with the plasma membrane, this being concomitant with the appearance of distinct myofilament-bound dense plaques. Vinculin, on the other hand, remains primarily cytoplasmic and appears in the plaques only 1-3 days after hatching. It is thus proposed that the interactions of the dense plaque with myofilaments or with the membrane do not depend on the presence of vinculin in the plaque. Electrophoretic analyses indicated that, during development, there is no major change in the differential expression of specific vinculin isoforms. Quantitative immunoblotting analysis indicated that the vinculin content (relative to total extracted protein) is virtually constant during the last week of embryonic life. However, within 3 days of hatching, the vinculin concentration increases remarkably to over twice the embryonic level, and then slowly increases until it reaches the adult levels, which are three to four times higher than the embryonic level. The concentration of metavinculin (a 160-Kd vinculin-related protein) showed only a limited increase after hatching. We discuss the possible roles of vinculin and talin in the assembly of membrane-bound dense plaques during the different phases of smooth-muscle development.     

Detection of high molecular weight vinculin binding proteins in muscle and nonmuscle tissues with an electroblot-overlay technique

In this study, we examined binding of radiolabelled vinculin to proteins separated on sodium dodecyl sulfate-polyacrylamide gels, and then electrophoretically transferred onto nitrocellulose sheets. We detected saturable binding of vinculin to polypeptides with apparent Mr's of 215,000, 205,000 and 185,000 in a low ionic strength extract from chicken gizzard membranes. Binding of vinculin to proteins with apparent Mr's of 205,000, 185,000, and 165,000 in human platelets was also detected. In addition, we found that [125I]vinculin binds to unlabelled vinculin and to alpha-actinin, although these interactions appear to be of lower affinity than those with the higher molecular weight proteins.     

Isolation and characterization of a vinculin cDNA from chick-embryo fibroblasts.

A chick-embryo fibroblast lambda gt11 cDNA library was screened with affinity-purified antibodies to chick gizzard vinculin. One recombinant was purified to homogeneity and the fusion protein expressed in Escherichia coli strain C600. The fusion protein was unstable, but polypeptides that reacted with vinculin antibodies, but not non-immune immunoglobulin, were detected by Western blotting. The recombinant contained a single EcoRI fragment of 2891 bp with a single open reading frame. The deduced protein sequence could be aligned with that of six CNBr-cleavage peptides and two tryptic peptides derived from chicken gizzard vinculin. AUG-247 has tentatively been identified as the initiation codon, as it is contained within the consensus sequence for initiation sites of higher eukaryotes. The cDNA lacks 3' sequence and encodes 74% of the vinculin sequence, presuming the molecular mass of vinculin to be 130,000 Da. Analysis of the deduced sequence showed no homologies with other protein sequences, but it does display a triple internal repeat of 112 amino acid residues covering residues 259-589. The sequences surrounding the seven tyrosine residues in the available sequence were aligned with the tyrosine autophosphorylation consensus sequence found in protein tyrosine kinases. Tyr-822 showed a good match to this consensus, and may represent one of the two major sites of tyrosine phosphorylation by pp60v-sre. Northern blots showed that the 2.89 kb vinculin cDNA hybridized to one size of mRNA (approx. 7 kb) in chick-embryo fibroblasts, chick smooth muscle and chick skeletal muscle. Southern blots revealed multiple hybridizing bands in genomic DNA.     

Phosphorylation of filamin and other proteins in cultured fibroblasts.

Incubation of subcellular fractions of fibroblasts with [³²P]ATP demonstrated 10 phosphoproteins whose phosphorylation can be increased by cyclic AMP or cyclic AMP-dependent protein kinase. One of these phosphoproteins, MW 240,000, resembles the actin binding protein, filamin, and can be selectively precipitated by antibodies to chicken gizzard filamin. Furthermore chicken gizzard filamin can be phosphorylated by skeletal muscle protein kinase and cyclic AMP stimulates this reaction.     

Antibody mapping of functional domains in vinculin.

We have analyzed the functional domain structure of vinculin, a protein involved in linking microfilaments to the cytoplasmic face of cell membranes in animal cells. For this purpose, we used several monoclonal antibodies raised against chicken gizzard vinculin whose epitopes could be assigned to discrete regions in the vinculin sequence by immunoblotting of proteolytic fragments combined with N-terminal amino acid sequencing. Two of these antibodies induced the disruption of stress fibers and changed the number of morphology of focal contacts after microinjection in chicken embryo fibroblasts. Based on the location of its epitope in comparison with vinculin domains previously identified by other groups, we propose that one of these antibodies (15B7) interferes with the binding of vinculin to talin, the most peripheral of the microfilament proteins. The second antibody (14C10) binds within a region comprising three internal repeats and might therefore distort the inner architecture of vinculin. A third antibody (As3) inhibited the binding of F-actin to vinculin in an in vitro assay but had no effect on the microfilament system in cells. These data emphasize the role of vinculin as a key protein in microfilament-membrane linkage and support previous work on a direct interaction between vinculin and actin.     

Chicken gizzard filamin, retina filamin and cgABP260 are respectively, smooth muscle-, non-muscle- and pan-muscle-type isoforms: distribution and localization in muscles

We determined the full cDNA sequences of chicken gizzard filamin and cgABP260 (chicken gizzard actin-binding protein 260). The primary and secondary structures predicted by these sequences were similar to those of chicken retina filamin and human filamins. Like mammals, chickens have 3 filamin isoforms. Comparison of their amino acid sequences indicated that gizzard filamin, retina filamin, and cgABP260 were the counterparts of human FLNa (filamin a), b, and c, respectively. Antibodies against the actin-binding domain (ABD) of these 3 filamin isoforms were raised in rabbits. Using immunoabsorption and affinity chromatography, we prepared the monospecific antibody against the ABD of each filamin. In immunoblotting, the antibody against the gizzard filamin ABD detected a single band in gizzard, but not in striated muscles or brain. In brain, only the antibody against the retina filamin ABD produced a strong single band. The antibody against the cgABP260 ABD detected a single peptide band in smooth, skeletal, and cardiac muscle. In immunofluorescence microscopy of muscular tissues using these antibodies, the antibody against the gizzard filamin ABD only stained smooth muscle cells, and the antibody against the retina filamin ABD strongly stained endothelial cells of blood vessels and weakly stained cells in connective tissue. The antibody against the cgABP260 ABD stained the Z-lines and myotendinous junctions of breast muscle, the Z-lines and intercalated disks of cardiac muscle, and dense plaques of smooth muscle. These findings indicate that chicken gizzard filamin, retina filamin, and cgABP260 are, respectively, smooth muscle-type, non-muscle-type, and pan-muscle-type filamin isoforms.     

Differentiation of smooth muscle cells in chicken gizzard

During development of the chicken gizzard, a thick layer of undifferentiated cells (mesenchymal cells) is constructed, and the cells differentiate into smooth muscle cells or connective tissues. We found that the differentiation of smooth muscle cells occurred first near the outer surface of the gizzard and the differentiated area spread to the inside of the gizzard. Therefore, we assumed that the differentiation of most of the smooth muscle cells in the gizzard is induced by differentiated smooth muscle itself. When undifferentiated cells from gizzard of 7-day-old embryo (Hamburger and Hamilton's stages 26-27) were cultured on a coverglass coated with extract of gizzard that contained differentiated smooth muscle cells, the cells attached to the coverglass and differentiated into smooth muscle cells. On the other hand, extract of gizzard from 7-day-old embryo did not induce the differentiation of smooth muscle cells, though it induced the attachment of cells. We found that activity for the differentiation of smooth muscle cells appeared when differentiated smooth muscle cells appeared in developing gizzard. Gizzard contained higher activity for the differentiation of smooth muscle cells than the other tissues. Transforming growth factor-beta (TGF-beta), which induces the differentiation of vascular smooth muscle cells, did not induce the differentiation of smooth muscle cells in gizzard, though extract of aorta induced the differentiation of smooth muscle cells in gizzard. The results obtained here support evidence that the differentiation of most of the smooth muscle cells in gizzard is induced by a self-catalytic mechanism in which differentiated smooth muscle itself induces the differentiation of smooth muscle cells.     

A new protein of adhesion plaques and ruffling membranes

A protein with a molecular weight on SDS polyacrylamide gels of 215,000 (referred to here as 215K) was purified from chicken gizzard smooth muscle. Antibodies against this protein localized it in fibroblasts to adhesion plaques (focal contacts), to regions underlying cell surface fibronectin, and to ruffling membranes. In the first two distributions it was similar to vinculin in cellular location, and this was confirmed by double-label immunofluorescence microscopy, but the concentration of 215K in membrane ruffles distinguished it from vinculin. There was no cross-reaction of the antibody against 215K with vinculin, and immunoprecipitation and antibody staining of SDS gels of whole cells revealed a single cross-reactive component with a molecular weight of 215,000. Immunoprecipitation from cultures labeled with [32P]phosphate revealed 215K to be a phosphoprotein. Transformation of rat or chicken fibroblasts by Rous sarcoma virus resulted in a reorganization of 215K, in some cases into complex intracellular structures. The localization of 215K where microfilament bundles terminate as well as in close relation to cell surface fibronectin and in membrane ruffles suggests that the protein has some function in the organization of actin filaments at or close to regions of actin-membrane attachment.     

Isoforms of the small non-catalytic subunit of smooth muscle myosin light chain phosphatase.

Chicken gizzard smooth muscle myosin light chain phosphatase is composed of a approximately 37 kDa catalytic subunit, a approximately 110 kDa myosin binding or targeting subunit and a approximately 20 kDa subunit (MPs) whose function is as yet undefined. It was reported previously that a cloned chicken gizzard MPs cDNA encodes a protein of 186 amino acids (aa) [Y.H. Chen, M.X. Chen, D.R. Alessi, D.G. Gampbell, C. Shanahan, P. Cohen, P.T.W. Cohen, FEBS Lett. 356 (1994) 51-55]. More recently, we obtained by PCR amplification another MPs cDNA that encodes a protein of only 161 aa [Y. Zhang, K. Mabuchi, T. Tao, Biochim. Biophys. Acta 1343 (1997) 51-58]. In this work we obtained cDNAs corresponding to both sequences using a different set of PCR primers, indicating that the two sequences correspond to isoforms that most likely arose from alternative splicing of the same gene. Using two polyclonal antibodies, one raised against the recombinant 161 aa isoform of chicken gizzard MPs and the other against a C-terminal polypeptide that is present only in the 186 aa isoform, we found that while the 161 aa isoform is the predominant one in chicken gizzard, in chicken aorta it is the 186 aa one; in chicken stomach both isoforms are present, and in mammalian tissues such as ferret and rat only the 186 aa isoform is detected. Furthermore, we purified the MPs associated with the chicken gizzard myosin light chain phosphatase holoenzyme and determined its molecular weight, amino acid composition and six residues of its C-terminal sequence. The results from these analyses showed conclusively that the predominant isoform in chicken gizzard is the 161 aa one.     

Biotransformation of testosterone and other androgens by suspension cultures of Nicotiana tabacum “Bright yellow”

It has been shown that the cultured cells of Nicotiana tabacum “Bright Yellow” are capable of transforming testosterone to Δ⁴-androstene-3, 17-dione, 5α-androstan-17β-ol-3-one, 5α-androstane-3β, 17β-diol, its dipalmitate and 3- and 17-monoglucosides, epiandrosterone, its palmitate and glucoside, testosterone glucoside. 5α-Androstane-3β, 17β-diol dipalmitate and 3- and 17-monoglucosides, epiandrosterone palmitate and glucoside, and testosterone glucoside have been found for the first time as metabolites of testosterone in plant systems. Δ⁴-Androstene-3,17-dione was converted to testosterone. 5α-Androstan-17β-ol-3-one, which has been recognized as an active form of testosterone in mammals, was also detected. It has also been demonstrated that [4-¹⁴C]testosterone is actively incorporated in these transformations.