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Biomedical subjects

J Bryan

Publications and source records attributed to J Bryan.

At least 37 records · Page 2Linked to original sources
PubMed1990

Caldesmon: fragments, sequence, and domain mapping.

A summary of the available binding site data is shown in Figure 6 and should be compared with the predicted structures in Figure 5. The blocks of amino acid sequence were those known at the time the cDNA sequencing was being done; they now include the entire head region. The phosphorylation site, indicated here, is a potential site, a consensus sequence, -AXYS(T)-, for the multifunctional calmodulin-dependent kinase. It has not been established that this site is utilized, and it is not clearly related to any of the established binding domains. Exactly how caldesmon regulates the actomyosin interaction and whether it contributes to the latch state is unclear. The organization of the molecule suggests, however, that it could function as a bridge between thick and thin filaments. In support of this idea, our recent efforts to map the smaller non-muscle form of caldesmon indicate that it retains the myosin and the calmodulin approximately actin binding domains, but is missing the central repeated region, arguing this region may serve to space the N and C terminal-binding domains in smooth muscle. Work from several laboratories has demonstrated that smooth muscle caldesmon is an elongated molecule with a calmodulin, tropomyosin, and actin-binding region at the C-terminus and a myosin-binding domain at the N-terminus. Sequence determination has shown that smooth muscle caldesmon is smaller than previously believed, has demonstrated similarities between caldesmon and troponin T, and has suggested possible calmodulin-binding peptides. The available sequence and domain mapping studies on smooth muscle caldesmon are reviewed.

Actins↗
PubMed1989

Cloning and expression of a smooth muscle caldesmon.

Caldesmon is a smooth muscle and nonmuscle regulatory protein that interacts with actin, myosin, tropomyosin, and calmodulin. Two overlapping clones, isolated from a chicken oviduct cDNA plasmid library and a chicken gizzard cDNA lambda NM1149 library, were used to generate a 4108-base pair sequence coding for one caldesmon. Expression of the coding sequence confirms this is one of the large smooth muscle caldesmons. The deduced protein molecular weight is 86.974, significantly less than the molecular weights estimated by sodium dodecyl sulfate gel electrophoresis. The protein has a high content of Gly, Lys, Arg, and Ala; there are two cysteine residues, one at either end of the molecule. Comparison with the Protein Identification Resource database demonstrates a similarity with a tropomyosin binding domain of troponin T, but none with any calmodulin or actin binding proteins. The center of the protein has an 8-fold repeat of a 13 amino acid sequence whose general motif is -Glu3-(Lys/Arg)2-Ala2-Glu2-(Lys/Arg)1-X-(Lys/Arg)1-Ala1-, where X is Glu, Gln, or Ala. Comparison with peptide sequences from a chymotryptic fragment that binds actin and calmodulin places this domain on the C terminus of caldesmon adjacent to the troponin T similarity. A tentative map of the major binding domains is proposed on the basis of available data.

Amino Acid Sequence↗
PubMed1989

Caldesmon, acidic amino acids and molecular weight determinations.

Recent estimates of molecular weight and cDNA sequencing indicate that smooth muscle caldesmons are considerably smaller than previously thought. The anomalous behaviour of these proteins during SDS-polyacrylamide gel electrophoresis can be correlated with their high acidic amino acid content. The results suggest a need to re-evaluate the stoichiometric relations of caldesmon to tropomyosin and actin in thin filaments and its presumed 1:1 interaction with calmodulin.

Amino Acids↗
PubMed1989

Validation of an imaging system: steps to evaluate and validate a microscope imaging system for quantitative studies.

The processing methods described in this article are general and can be used with any computer with adequate software without reference to a specific experimental apparatus. To achieve appropriately corrected images, the operations must be performed correctly and in a specific order to extract meaningful information from digital images. The operations are subtraction of the dark current, a shading correction after low pass filtering of the shading mask, a geometric correction with a reference grid obtained at the longest wavelength of interest, and finally registration. Since the digital data sets are too large for manual evaluation, statistical criteria must be used to define the analytical capabilities and error limits of the system before the more interesting cellular studies can be undertaken.

Algorithms↗
PubMed1988

Localization and mobility of gelsolin in cells.

To investigate the physiologic role of gelsolin in cells, we have studied the location and mobility of gelsolin in a mouse fibroblast cell line (C3H). Gelsolin was localized by immunofluorescence of fixed and permeabilized cells and by fluorescent analog cytochemistry of living cells and cells that were fixed and/or permeabilized. Overall, the images show that in living cells gelsolin has a diffuse cytoplasmic distribution, but in fixed cells a minor fraction is associated with regions of the cell that are rich in actin filaments. The latter fraction is more prominent after permeabilization of the fixed cells because some diffuse gelsolin is not fixed and is therefore lost during permeabilization, confirmed by immunoblots. To determine quantitatively whether gelsolin is bound to actin filaments in living cells, we measured the mobility of microinjected fluorescent gelsolin by fluorescence photobleaching recovery. Gelsolin is fully mobile with a diffusion coefficient similar to that of control proteins. As a positive control, fluorescent phalloidin, which binds actin filaments, is totally immobile. These results are supported by immunoblots on cells permeabilized with detergent. All the endogenous gelsolin is extracted, and the half-time for the extraction is approximately 5 s, which is about the rate predicted for diffusion. Therefore, gelsolin is not tightly bound to actin filaments in cells. The most likely interpretation of the difference between living and fixed cells is that fixation traps a fraction of gelsolin that is associated with actin filaments in short-lived complexes.

Animals↗
PubMed1988

Gelsolin has three actin-binding sites.

Gelsolin, a Ca2+-modulated actin filament-capping and -severing protein, complexes with two actin monomers. Studies designed to localize binding sites on proteolytic fragments identify three distinct actin-binding peptides. 14NT, a 14-kD fragment that contains the NH2 terminal, will depolymerize F-actin. This peptide forms a 1:1 complex with G-actin which blocks the exchange of etheno-ATP from bound actin. The estimated association and dissociation rates for this complex are 0.3 microM-1 s-1 and 1.35 x 10(-6) s-1 which gives a maximum calculated Kd = 4.5 x 10(-12) M. 26NT, the adjacent peptide on the NH2-terminal half of gelsolin, binds to both G- and F-actin. This fragment has little or no intrinsic severing activity and will bind to F-actin to nearly stoichiometric ratios. The interactions of 14NT and 26NT with actin are largely Ca2+ independent and one of these sites, probably 14NT, is the EGTA-stable site identified in the intact protein. 41CT, the COOH-terminal half of gelsolin, forms a rapidly reversible 1:1 complex with actin, Kd = 25 nM, that slows but does not block etheno-ATP exchange. This interaction is Ca2+ dependent and is the exchangeable site in the intact protein. One of these sites is hidden in the intact protein, but cleavage into half fragments exposes all three and removes the Ca2+ dependence of severing.

Actins↗
PubMed1988

Geometric correction of digital images using orthonormal decomposition.

We have developed an algorithm which can be used to correct the geometric distortion of digital images. The method uses an orthonormal decomposition and a two-dimensional Horner's scheme to construct and evaluate a polynomial equation of arbitrary degree in two independent variables. This numerical scheme for geometric correction combines several methods selected on the basis of their computation efficiency and numerical stability. The differences and advantages of this numerical scheme are compared with methods found in the image processing literature. The algorithm presented here has a reduced number of mathematical operations, is flexible and numerically stable. Based on the least-squares criteria, the algorithm provides corrected pixel positions with an accuracy equal to or better than the pixel size.

Algorithms↗
PubMed1987

Domain mapping of chicken gizzard caldesmon.

Limited proteolysis, affinity chromatography, and immunoblotting have been used to define the domains of chicken gizzard caldesmon, caldesmon120, that interact with calmodulin, F-actin, and a monoclonal antibody prepared using human platelet caldesmon. Treatment of caldesmon120 with chymotrypsin produces groups of fragments near 100, 80, 60, 38, and 20 kDa. Further digestion produces peptides between 40 and 50 kDa. The 100- and 80-kDa peptides cross-react with the monoclonal antibody; the smaller polypeptides do not. The kinetics of cleavage and the antibody studies indicate that the 38- and 80-kDa fragments are the two major pieces of the 120-kDa protein. The 38-kDa fragment, purified by high performance liquid chromatography, and several of its subfragments at 21 and 25 kDa sediment with F-actin, bind to calmodulin-Sepharose in the presence of Ca2+, and are displaced from F-actin by Ca2+-calmodulin. The 80-kDa fragments did not interact with F-actin or calmodulin. We have tentatively placed the 38-kDa fragment at the C-terminal using polyclonal antibodies selected against a beta-galactosidase-caldesmon120 fusion protein produced by a lambda gt11 lysogen. The 38-, 25-, and 21-kDa fragments cross-react with these antibodies; the 80- and 60-kDa fragments do not. Caldesmon77 from human platelets also cross-reacts with these selected antibodies. The results suggest that interacting calmodulin and F-actin binding sites are localized on a 38-kDa C-terminal fragment of caldesmon. The smallest subfragment of this peptide that binds to both F-actin and calmodulin-Sepharose is about 21 kDa. The monoclonal antibody epitope is tentatively localized near the N-terminal of caldesmon77 and must be within 50 kDa of the N-terminal on caldesmon120.

Animals↗
PubMed1987

Microinjection of gelsolin into living cells.

Gelsolins are actin-binding proteins that cap, nucleate, and sever actin filaments. Microinjection of cytoplasmic or plasma gelsolin into living fibroblasts and macrophages did not affect the shape, actin distribution, deformability, or ruffling activity of the cells. Gelsolin requires calcium for activity, but the NH2-terminal half is active without calcium. Microinjection of this proteolytic fragment had marked effects: the cells rounded up, stopped ruffling, became soft, and stress fibers disappeared. These changes are similar to those seen with cytochalasin, which also caps barbed ends of actin filaments. Attempts to raise the cytoplasmic calcium concentration and thereby activate the injected gelsolin were unsuccessful, but the increases in calcium concentration were minimal or transient and may not have been sufficient. Our interpretation of these results is that at the low calcium concentrations normally found in cells, gelsolin does not express the activities observed in vitro at higher calcium concentrations. We presume that gelsolin may be active at certain times or places if the calcium concentration is elevated to a sufficient level, but we cannot exclude the existence of another molecule that inhibits gelsolin. Microinjection of a 1:1 gelsolin/actin complex had no effect on the cells. This complex is stable in the absence of calcium and has capping activity but no severing and less nucleation activity as compared with either gelsolin in calcium or the NH2-terminal fragment. The NH2-terminal fragment-actin complex also has capping and nucleating activity but no severing activity. On microinjection it had the same effects as the fragment alone. The basis for the difference between the two complexes is unknown. The native molecular weight of rabbit plasma gelsolin is 82,500, and the extinction coefficient at 280 nm is 1.68 cm2/mg. A new simple procedure for purification of plasma gelsolin is described.

Animals↗
PubMed1986

The effects of a 45 000 molecular weight protein from unfertilized sea urchin eggs and its 1:1 actin complex on actin filaments.

A 45 kDa actin-binding protein (SU45) has been isolated previously from egg extracts of the Hawaiian sea urchin Tripneustes gratilla by DEAE Sephacel, Sephadex G-75 and hydroxyapatite chromatography. Using pyrene-labelled rabbit skeletal muscle actin, we have found that when SU45 is added to actin in the presence of calcium and the salt concentration is increased, the initial rate of actin assembly is accelerated. Moreover, the final polymer concentration is reduced indicating that SU45 caps the preferred end of actin filaments shifting the critical concentration (Cc) to that of the nonpreferred end. Determination of the Cc as a function of the concentration of SU45 gave an apparent KD of 1 nM. Dilution of F-actin to below its Cc, into buffers containing SU45 and Ca2+ resulted in a sharp increase in the rate of depolymerization; reducing the Ca2+ concentration attenuated this effect. Incubation of SU45 with rabbit skeletal muscle G-actin yielded a 1:1 complex which held 45Ca2+ tightly with a dissociation half-time of 10.8 days. By kinetic analyses of assembly in the presence of the SU45-actin complex and dilution-induced disassembly of filaments precapped with complex, we have estimated both the association rate constant (4.0 X 10(4)M-1s-1) and the dissociation rate constant (0.05s-1) for the nonpreferred ends of actin filaments. Finally, dilution of F-actin to below its Cc, into complex in either Ca2+ or EGTA resulted in a much slower depolymerization consistent with a rapid capping of the preferred end by the SU45-actin complex.

Actins↗
PubMed1986

Immuno-identification of Ca2+-induced conformational changes in human gelsolin and brevin.

Gelsolin is a 90,000-mol-wt protein with two actin and two high affinity calcium-binding sites that can form complexes with Ca2+ ions and monomeric actin. These complexes will nucleate filament growth and cap the barbed end of filaments, but will not fragment F-actin. Uncomplexed gelsolin severs F-actin. (Bryan, J., and L. M. Coluccio, 1985, J. Cell Biol., 101:1236-1244). These associations with actin are modulated by Ca2+. We have purified and characterized monoclonal antibodies that recognize Ca2+-induced conformational changes in human platelet gelsolin (G) and human plasma brevin (B), a closely related protein. Two hybridomas, 8G5 and 4F8, were adapted to growth in serum-free medium. 8G5 was found to secrete an IgG; 4F8 secretes an IgA. On immunoblots, both antibodies gave a strong reaction if Ca2+ was present, but gave barely detectable reactions if EGTA was used. 8G5 IgG-Sepharose columns retained gelsolin (as GCa2) or brevin (as BCa2) in 0.1 mM CaCl2 containing buffers, but released these molecules when eluted with 4 mM EGTA. 8G5 IgG-Sepharose columns also retained gelsolin-actin-Ca2+ complexes, as GA1Ca2 or higher oligomers from platelet extracts containing 0.1 mM CaCl2. Elution with 4 mM EGTA released material that gel filtration showed to be the EGTA-stable 130,000-mol-wt gelsolin-actin complex, GA1Ca1. The results demonstrate that the 8G5 IgG recognizes a conformation of gelsolin or brevin induced by binding of an easily exchangeable Ca2+ ion. Actin is not required for this conformational change, and the antibody discriminates, for example, GCa2 from G and GCa1. A 4F8 IgA-Sepharose column retained brevin or gelsolin in 0.1 mM CaCl2-containing buffers, but, like the 8G5 IgG, released these molecules when eluted with 4 mM EGTA. The 4F8 IgA column also retained gelsolin or brevin-actin-Ca2+ complexes, for example, as BA1Ca2, or higher oligomers, in 0.1 mM CaCl2. No protein was recovered, however, upon elution with 4 mM EGTA, but elution with 0.1 M glycine-HCl, pH 2.8, released bound brevin or gelsolin and actin. Similarly, preformed brevin-actin-Ca2+ complex, equilibrated with EGTA, was retained by 4F8 IgA-Sepharose. The results demonstrate that the 4F8 IgA recognizes a conformation of gelsolin or brevin that is maintained and presumably induced by binding of a nonexchangeable Ca2+ ion that is trapped in the complex.

Antibodies, Monoclonal↗
PubMed1986

A re-evaluation of cytoplasmic gelsolin localization.

Gelsolin is a 90,000-mol-wt Ca2+-binding, actin-associated protein that can nucleate actin filament growth, sever filaments, and cap barbed filament ends. Brevin is a closely related 92,000-mol-wt plasma protein with similar properties. Gelsolin has been reported to be localized on actin filaments in stress fibers, in cardiac and skeletal muscle I-bands, and in cellular regions where actin filaments are known to be concentrated. Previous localization studies have used sera or antibody preparations that contain brevin. Using purified brevin-free IgG and IgA monoclonal antibodies or affinity-purified polyclonal antibodies for gelsolin and brevin, we find no preferential stress fiber staining in cultured human fibroblasts or I-band staining in isolated rabbit skeletal muscle sarcomeres. Cardiac muscle frozen sections show no pronounced I-band staining, except in local areas where brevin may have penetrated from adjacent blood vessels. Spreading platelets show endogenous gelsolin localized at the cell periphery, in the central cytoplasmic mass and on thin fibers that radiate from the central cytoplasm. Addition of 3-30 micrograms/ml of brevin to the antibodies restores intense stress fiber and I-band staining. We see no evidence for large-scale severing and removal of filaments in stress fibers in formaldehyde-fixed, acetone-permeabilized cells even at brevin concentrations of 30 micrograms/ml. The added brevin or brevin antibody complex binds to actin filaments and is detected by the fluorescently tagged secondary antibody. Brevin binding occurs in either Ca2+ or EGTA, but is slightly more intense in EGTA suggesting some severing and filament removal may occur in Ca2+. The I-band staining is limited to the region where actin and myosin do not overlap. In addition, brevin does not appear to bind at the Z-line. A comparison of cells double-labeled with fluorescein-phallotoxin, exogenous brevin, and a monoclonal antibody, detected with a rhodamine-labeled secondary antibody, shows almost complete co-localization of F-actin with the brevin-gelsolin-binding sites. A major exception is in the area of the adhesion plaque. A quantitative comparison of the fluorescein-rhodamine fluorescence intensities along a stress fiber and into the adhesion plaque shows that the fluorescein signal, associated with F-actin, increases while the rhodamine signal decreases. We infer that exogenous brevin or endogenous gelsolin can bind to and potentially sever most actin filaments, but that actin-associated proteins in the adhesion plaque can prevent binding and severing.(ABSTRACT TRUNCATED AT 400 WORDS)

Actins↗
PubMed1986

Definition of an N-terminal actin-binding domain and a C-terminal Ca2+ regulatory domain in human brevin.

Brevin is a Ca2+-modulated actin-associated protein that will sever F-actin and cap barbed filament ends. Limited proteolysis with chymotrypsin or subtilisin cleaves the molecule approximately in half. Cleavage is approximately 10-fold more rapid in Ca2+ than in EGTA. The two fragments are readily separated from each other and from undigested brevin by high pressure liquid chromatography on a DEAE resin. A 40,000-mol-wt fragment from the N-terminal is not retained by DEAE, while a 45,000-mol-wt C-terminal fragment binds more tightly than brevin. The N-terminal fragment retains approximately 10% of the nucleation activity, caps barbed ends, and retains 50% of the total severing activity defined by dilution induced depolymerization of pyrenyl actin, but, in contrast to brevin, none of these functions are affected by Ca2+. Fluorescent actin binding studies and gel-filtration demonstrate that the 40,000-mol-wt fragment binds two actin monomers. The 45,000-mol-wt C-terminal fragment has no severing, nucleating, or capping activity. Cross-reaction with two monoclonal antibodies against two specific Ca2+-induced conformations of human platelet gelsolin suggest that both Ca2+ binding sites are located on the carboxyl half of the brevin molecule. One epitope, defined as the rapidly exchanging Ca2+ binding site in the gelsolin-actin complex, is lost when a 20,000-mol-wt fragment is cleaved from the carboxyl terminal. The second epitope, related to the poorly exchanging Ca2+ binding site in the complex, is nearer the middle of the brevin molecule.

Actins↗
PubMed1986

Identification by monoclonal antibodies and characterization of human platelet caldesmon.

Actin-based gels were prepared from clarified high-salt extracts of human platelets by dialysis against physiological salt buffers. The gel was partially solubilized with 0.3 M KCl. Mice were immunized with the 0.3 M KCl extract of the actin gel, and hybridomas were produced by fusion of spleen cells with myeloma cells. Three hybridomas were generated that secrete antibodies against an 80-kD protein. These monoclonal antibodies stained stress fibers in cultured cells and cross-reacted with proteins in several tissue types, including smooth muscle. The cross-reacting protein in chicken gizzard smooth muscle had an apparent molecular weight of 140,000 and was demonstrated to be caldesmon, a calmodulin and actin-binding protein (Sobue, K., Y. Muramoto, M. Fujita, and S. Kakiuchi, Proc. Natl. Acad. Sci. USA, 78:5652-5655). No proteins of molecular weight greater than 80 kD were detectable in platelets by immunoblotting using the monoclonal antibodies. The 80-kD protein is heat stable and was purified using modifications of the procedure reported by Bretscher for the rapid purification of smooth muscle caldesmon (Bretscher, A., 1985, J. Biol. Chem., 259:12873-12880). The 80-kD protein bound to calmodulin-Sepharose in a Ca++-dependent manner and sedimented with actin filaments, but did not greatly increase the viscosity of F-actin solutions. The actin-binding activity was inhibited by calmodulin in the presence of calcium. Except for the molecular weight difference, the 80-kD platelet protein appears functionally similar to 140-kD smooth muscle caldesmon. We propose that the 80-kD protein is platelet caldesmon.

Animals↗
PubMed1985

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.

Actins↗