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J V Small

Publications and source records attributed to J V Small.

At least 55 records · Page 3Linked to original sources
PubMed1992

Calponin and SM 22 isoforms in avian and mammalian smooth muscle. Absence of phosphorylation in vivo.

Calponin is a basic smooth-muscle-specific protein capable of binding to F-actin, tropomyosin and calmodulin in vitro. Using two-dimensional gel electrophoresis, we show that calponin exists as multiple isoelectric variants in avian and mammalian tissues. During chick embryogenesis, one isoform is expressed in gizzard that shows a pI identical to the most basic adult alpha variant; around 10 d after hatching multiple isoforms then appear. SM 22 [Pearlstone, J. R., Weber, M., Lees-Miller, J. P., Carpenter, M. R. & Smillie, L. B. (1987) J. Biol. Chem. 262, 5985-5991], which has sequence-motifs related to calponin, displays a similar isoform pattern during development; one isoform (alpha) is present in the embryo and three in the adult. In living smooth-muscle strips from chicken gizzard and guinea pig taenia coli, labelled with 32PO4, no phosphate incorporation could be detected in any of the calponin or SM 22 isoforms during either contraction or relaxation. From the additional observation that antibodies against phosphoserine also failed to label calponin and SM 22 in two-dimensional gel immunoblots, we conclude that the multiple isoforms do not arise via differential phosphorylation. These results support the claim [Barany, M., Rokolya, A. & Barany, K. (1991) FEBS Lett. 279, 65-68] that calponin phosphorylation is not involved in smooth muscle regulation in vivo, as has been suggested from in vitro studies [Winder, S. J. & Walsh, M. J. (1990) J. Biol. Chem. 265, 10148-10155]. In vitro translation of porcine and chicken smooth-muscle mRNA produced only a single (alpha) isoform of calponin, suggesting that the adult isoforms do not derive from multiple gene products; in the same assay two polypeptides appeared in the position of SM 22, one corresponding to the alpha isoform and a second more basic spot, not observed in tissue samples. Whereas calponin and SM 22 appear synchronously during smooth muscle differentiation in vivo, SM 22 is not fully down-regulated like calponin, metavinculin and heavy-caldesmon in smooth muscle cells in culture, pointing to a differential regulation of expression of the alpha SM 22 isoform during smooth-muscle phenotype modulation in vitro.

Animals↗
PubMed1991

Calponin. Developmental isoforms and a low molecular weight variant.

Two-dimensional gel analysis of basic proteins in developing human smooth muscle identifies calponin as a prominent marker of the differentiated phenotype. Adult tissue (human and mouse) typically expresses up to four calponin isoforms, three of which appear sequentially during fetal development: adult myometrial cells express the same three isoforms in primary culture in vitro and these are down-regulated, in reverse order, during the subsequent modulation of phenotype. Monospecific, polyclonal antibodies against calponin identify a lower molecular weight variant of calponin (L-calponin) that is strongly and specifically expressed in adult smooth muscles of the human urogenital tract. L-calponin is down-regulated in benign smooth muscle derived tumors (leiomyoma) and is not expressed in primary cultures of normal uterine tissue.

Animals↗
PubMed1991

Leading edge movement and ultrastructure in mouse macrophages.

The first event in the process of translocation of a cell over a substrate is the forward protrusion of a thin layer of cytoplasm, sometimes referred to as the leading edge. To gain more direct information on structural reorganizations associated with protrusion we have documented the ultrastructure of the actin cytoskeleton of mouse macrophages whose history of locomotion prior to fixation for electron microscopy had been recorded by video microscopy. It is shown that rapid protrusion is associated with the formation of a dense, diagonal network of actin filaments, lacking organized bundles. In cell edges that showed minor fluctuations back and forth over a period of 30 sec or more no dense meshworks were found: instead, a loose peripheral bundle of actin filaments was commonly observed. Cell edges that first protruded and then retracted showed a similar ultrastructure to those that exhibited only forward movement, but the width of the leading edge meshwork was, by comparison, reduced. Measurements showed that there was an approximate correlation between the leading edge mesh width and the net forward translocation observed during the terminal 30 sec, up to fixation. The results are discussed in relation to present concepts of the protrusion mechanism.

Actins↗
PubMed1990

Smooth muscle specific expression of calponin.

Calponin is an actin-, calmodulin-, and tropomyosin-binding protein that has been isolated from smooth muscle tissue. Using a monoclonal antibody specific for avian calponin, we demonstrate a differentiation-linked increase in calponin expression in embryonic chick gizzard. Cultivation of gizzard smooth muscle cells in vitro resulted in a down-regulation of calponin expression after the first 48 h that was paralleled by a loss of synthesis of metavinculin and the high molecular weight isoform of caldesmon. In early cultures of smooth muscle cells calponin was localised in the actin-containing stress fibres but labelling was restricted to the central parts of the actin cytoskeleton. Calponin expression is suggested as a potentially useful index of smooth muscle differentiation.

Animals↗
PubMed1990

Supercontracted state of vertebrate smooth muscle cell fragments reveals myofilament lengths.

Isolated cell preparations from chicken gizzard smooth muscle typically contain a mixture of cell fragments and whole cells. Both species are spontaneously permeable and may be preloaded with externally applied phalloidin and antibodies and then induced to contract with Mg ATP. Labeling with antibodies revealed that the cell fragments specifically lacked certain cytoskeletal proteins (vinculin, filamin) and were depleted to various degrees in others (desmin, alpha-actinin). The cell fragments showed a unique mode of supercontraction that involved the protrusion of actin filaments through the cell surface during the terminal phase of shortening. In the presence of dextran, to minimize protein loss, the supercontracted products were star-like in form, comprising long actin bundles radiating in all directions from a central core containing myosin, desmin, and alpha-actinin. It is concluded that supercontraction is facilitated by an effective uncoupling of the contractile apparatus from the cytoskeleton, due to partial degradation of the latter, which allows unhindered sliding of actin over myosin. Homogenization of the cell fragments before or after supercontraction produced linear bipolar dimer structures composed of two oppositely polarized bundles of actin flanking a central bundle of myosin filaments. Actin filaments were shown to extend the whole length of the bundles and their length averaged integral to 4.5 microns. Myosin filaments in the supercontracted dimers averaged 1.6 microns in length. The results, showing for the first time the high actin to myosin filament length ratio in smooth muscle are readily consistent with the slow speed of shortening of this tissue. Other implications of the results are also discussed.

Actin Cytoskeleton↗
PubMed1990

The cytoskeletal and contractile apparatus of smooth muscle: contraction bands and segmentation of the contractile elements.

Confocal laser scanning microscopy of isolated and antibody-labeled avian gizzard smooth muscle cells has revealed the global organization of the contractile and cytoskeletal elements. The cytoskeleton, marked by antibodies to desmin and filamin is composed of a mainly longitudinal, meandering and branched system of fibrils that contrasts with the plait-like, interdigitating arrangement of linear fibrils of the contractile apparatus, labeled with antibodies to myosin and tropomyosin. Although desmin and filamin were colocalized in the body of the cell, filamin antibodies labeled additionally the vinculin-containing surface plaques. In confocal optical sections the contractile fibrils showed a continuous label for myosin for at least 5 microns along their length: there was no obvious or regular interruption of label as might be expected for registered myosin filaments. The cytoplasmic dense bodies, labeled with antibodies to alpha-actinin exhibited a regular, diagonal arrangement in both extended cells and in cells shortened in solution to one-fifth of their extended length: after the same shortening, the fibrils of the cytoskeleton that showed colocalization with the dense bodies in extended cells became crumpled and disordered. It is concluded that the dense bodies serve as coupling elements between the cytoskeletal and contractile systems. After extraction with Triton X-100, isolated cells bound so firmly to a glass substrate that they were unable to shorten as a whole when exposed to exogenous Mg ATP. Instead, they contracted internally, producing integral of 10 regularly spaced contraction nodes along their length. On the basis of differences of actin distribution two types of nodes could be distinguished: actin-positive nodes, in which actin straddled the node, and actin-negative nodes, characterized by an actin-free center flanked by actin fringes of 4.5 microns minimum length on either side. Myosin was concentrated in the center of the node in both cases. The differences in node morphology could be correlated with different degrees of coupling of the contractile with the cytoskeletal elements, effected by a preparation-dependent variability of proteolysis of the cells. The nodes were shown to be closely related to the supercontracted cell fragments shown in the accompanying paper (Small et al., 1990) and furnished further evidence for long actin filaments in smooth muscle. Further, the segmentation of the contractile elements pointed to a hierarchial organization of the myofilaments governed by as yet undetected elements.

Actinin↗
PubMed1989

Unique geometry of actin-membrane anchorage sites in avian gizzard smooth muscle cells.

Adherens junctions in isolated avian gizzard smooth muscle cells appear as short longitudinal streaks or chevrons that are arranged in periodic, mainly transverse bands along the cell surface. This barrel-like geometry, revealed by antibodies to either vinculin or talin, was seen also in teased gizzard strips by confocal laser-scanning microscopy and contrasted with the rib-like surface pattern observed here and previously in other avian and mammalian smooth muscles. There were on average 67 transverse bands per gizzard cell and an estimated total of around 800 vinculin/talin sites. The longitudinal spacing between the transverse bands of vinculin streaks in the gizzard cells changed from 4-5 microns in extended cells to around 1 micron in shortened cells and the bands remained essentially transverse at all cell lengths, inconsistent with a screw-like mode of cell shortening as has been invoked for smooth muscle cells by others. The absence of rotation on shortening was confirmed by observations on isolated and bead-decorated skinned cells that were induced to contract with ATP. Counterlabelling of cells with alpha-actinin antibodies produced more or less exclusive staining of the cytoplasmic dense bodies, and little surface label: the total number of dense bodies per cell, estimated from confocal microscope through focal series was in the range of 3000. The data are consistent with a periodic anchorage of actin filaments to the cell surface and, in turn, with the existence of regularly spaced contractile assemblies.

Animals↗
PubMed1988

Porcine vinculin and metavinculin differ by a 68-residue insert located close to the carboxy-terminal part of the molecule.

Metavinculin is a higher mol. wt variant of vinculin expressed only in muscle tissue. Using amino acid sequencing methods on the intact molecules and their proteolytic subfragments, together with a polyclonal antibody specific only for metavinculin from porcine stomach, we have been able to identify and sequence the difference peptide in the porcine metavinculin molecule. By alignment with the complete sequence of chick fibroblast vinculin (communicated by G.J. Price, P. Jones, M.D. Davison, R. Bendori, S. Griffiths, B. Patel, B. Geiger and D.R. Critchley, prior to publication) the exact location of the insert could be determined. In porcine metavinculin, this insert lies between the 90-kd protease-resistant amino-terminal core and the carboxy terminus of the molecule. It contains 68 amino acids and is flanked by KWSSK sequences, one of which is present in vinculin. The identity of the mapped vinculin and metavinculin sequences outside this difference peptide is consistent with the two proteins arising via alternative splicing at the mRNA level. The lack of reactivity of the porcine metavinculin antibody with metavinculin from chicken as well as the finding of different proteolytic cleavage sites in avian metavinculin indicate a species-specific amino acid sequence in the difference piece of the metavinculin molecule.

Amino Acid Sequence↗
PubMed1988

Cytoskeleton-associated plectin: in situ localization, in vitro reconstitution, and binding to immobilized intermediate filament proteins.

The association and interaction of plectin (Mr 300,000) with intermediate filaments and filament subunit proteins were studied. Immunoelectron microscopy of whole mount cytoskeletons from various cultured cell lines (rat glioma C6, mouse BALB/c 3T3, and Chinese hamster ovary) and quick-frozen, deep-etched replicas of Triton X-100-extracted rat embryo fibroblast cells revealed that plectin was primarily located at junction sites and branching points of intermediate filaments. These results were corroborated by in vitro recombination studies using vimentin and plectin purified from C6 cells. Filaments assembled from mixtures of both proteins were extensively crosslinked by oligomeric plectin structures, as demonstrated by electron microscopy of negatively stained and rotary-shadowed specimens as well as by immunoelectron microscopy; the binding of plectin structures on the surface of filaments and cross-link formation occurred without apparent periodicity. Plectin's cross-linking of reconstituted filaments was also shown by ultracentrifugation experiments. As revealed by the rotary-shadowing technique, filament-bound plectin structures were oligomeric and predominantly consisted of a central globular core region of 30-50 nm with extending filaments or filamentous loops. Solid-phase binding to proteolytically degraded vimentin fragments suggested that plectin interacts with the helical rod domain of vimentin, a highly conserved structural element of all intermediate filament proteins. Accordingly, plectin was found to bind to the glial fibrillar acidic protein, the three neurofilament polypeptides, and skin keratins. These results suggest that plectin is a cross-linker of vimentin filaments and possibly also of other intermediate filament types.

Animals↗
PubMed1988

Contact formation during fibroblast locomotion: involvement of membrane ruffles and microtubules.

We have correlated the motility of the leading edge of fibroblasts, monitored by phase-contrast cinematography, with the relative distributions of several cytoskeletal elements (vinculin, tubulin, and actin) as well as with the contact patterns determined by interference reflection microscopy. This analysis has revealed the involvement of both ruffles and microspikes, as well as microtubules in the initiation of focal contact formation. Nascent vinculin sites within the leading edge or at its base, taken as primordial cell-substrate contacts, were invariably colocalized with sites that showed a history of transient, prolonged, or cyclic ruffling activity. Extended microspike structures, often preceded the formation of ruffles. Immunofluorescent labeling indicated that some of these primordial contacts were in close apposition to the ends of microtubules that penetrated into the leading edge. By fluorescence and electron microscopy short bundles of actin filaments found at the base of the leading edge were identified as presumptive, primordial contacts. It is concluded that ruffles and microspikes, either independently or in combination, initiate and mark the sites for future contact. Plaque proteins then accumulate (within 10-30 s) at the contract site and, beneath ruffles, induce localized bundling of actin filaments. We propose that all primordial contacts support traction for leading edge protrusion but that only some persist long enough to nucleate stress fiber assembly. Microtubules are postulated as the elements that select, stabilize, and potentiate the formation of these latter, long-lived contacts.

Actins↗
PubMed1988

Coumarin-phalloidin: a new actin probe permitting triple immunofluorescence microscopy of the cytoskeleton.

7-Diethylamino-3-(4-isothiocyanotophenyl)-4-methylcoumarin (CPITC) was coupled to amino-methyldithiolanophalloidin to produce a new phalloidin derivative, coumarin-phalloidin, fluorescent in the blue region of the spectrum. Coumarin-phalloidin binds to actin with around 100-fold less affinity than unconjugated phalloidin, but with enough avidity to make it a useful stain for actin filaments. Appropriate filter combinations permit triple immunofluorescence microscopy of the cytoskeleton with fluorescein and rhodamine conjugates together with coumarin-phalloidin.

Actins↗
PubMed1987

Salt dependent dimerisation of caldesmon.

Using analytical gel filtration (FPLC) we show here that avian gizzard caldesmon (chain molecular mass 150 kDa) self-associates to form end-to-end dimers. Increasing salt concentration promotes dimerisation: at 150 mM KCl, about 40% of the caldesmon was dimeric. Freshly gel filtered caldesmon had an actin gelating activity which decreased with increasing ionic strength. At 150 mM KCl, caldesmon at a 1:90 molar ratio to actin doubled the low shear viscosity of F-actin. Sixfold less filamin was required to produce the same effect.

Animals↗
PubMed1987

Metavinculin and vinculin from mammalian smooth muscle: bulk isolation and characterization.

Metavinculin, a vinculin related protein found only in muscle, has been prepared in bulk amounts from porcine stomach by a new procedure: the same procedure is applicable to the purification of vinculin from porcine stomach and avian gizzard. A comparison of the mammalian and avian proteins by peptide mapping showed them all to contain a common protease resistant 90 kDa core; however both avian and mammalian vinculins were notably more resistant to proteolysis down to this core than their respective metavinculins. Despite the close similarities in the peptide maps, in molecular weight and amino acid composition neither of the mammalian proteins exhibited the head and tail morphology formerly described for gizzard vinculin and metavinculin; both porcine proteins appeared globular under the electron microscope. From the gross variability in the ratios of metavinculin to vinculin among smooth muscles of different origin as well as from the common detergent-independent solubility properties of both proteins during isolation, it is concluded that vinculin and metavinculin perform duplicatory roles as peripheral membrane components. No definitive evidence for the interaction of either protein with actin filaments was obtained.

Animals↗