Search PubMed⌕ Search

Biomedical subjects

C Chaponnier

Publications and source records attributed to C Chaponnier.

At least 55 records · Page 3Linked to original sources
PubMed1987

Reversibility of gelsolin/actin interaction in macrophages. Evidence of Ca2+-dependent and Ca2+-independent pathways.

We have developed an immunoadsorption technique for quantitating EGTA-resistant gelsolin/actin complexes in macrophages extracted with Triton X-100. We report here that the proportion of gelsolin complexed irreversibly to actin is low in freshly harvested macrophages. The amount of the EGTA-resistant complex increases spontaneously during incubation of the cells in suspension at 37 degrees C, or after exposure to the Ca2+ ionophore ionomycin. On the other hand, exposure of suspended cells to the chemotactic oligopeptide, FMLP, or plating of the cells onto tissue culture dishes causes the EGTA-resistant complex to dissociate rapidly. Plating even prevents Ca2+ ionomycin-treated cells with elevated intracellular Ca2+ from inducing this complex. Therefore, our results suggest that macrophages possess a mechanism, not directly involving Ca2+, for dissociating actin/gelsolin EGTA-resistant complexes. This mechanism may be a Ca2+-independent signal for leukocyte activation.

Actins↗
PubMed1987

Reversible binding of actin to gelsolin and profilin in human platelet extracts.

This paper documents the reversible appearance of high-affinity complexes of profilin and gelsolin with actin in extracts of platelets undergoing activation and actin assembly. Sepharose beads coupled to either monoclonal anti-gelsolin antibodies or to polyproline were used to extract gelsolin and profilin, respectively, from EGTA-containing platelet extracts and determine the proportion of these molecules bound to actin with sufficient affinity to withstand dilution (high-affinity complexes). Resting platelets (incubated for 30 min at 37 degrees C after gel filtration) contained nearly no high-affinity actin/gelsolin or actin/profilin complexes. Thrombin, within seconds, caused quantitative conversion of platelet profilin and gelsolin to high-affinity complexes with actin, but these complexes were not present 5 min after stimulation. The calcium-dependent actin filament-severing activity of platelet extracts, a function of free gelsolin, fell in concert with the formation of EGTA-stable actin/gelsolin complexes, and rose when the adsorption experiments indicated that free gelsolin was restored. The dissociation of high-affinity complexes was temporally correlated with the accumulation of actin in the Triton-insoluble cytoskeleton.

Actins↗
PubMed1987

Cytoskeletal organization of peripheral blood normal and leukemic lymphocytes and lymphoblasts.

Content of actin and vimentin and organization of actin were studied in peripheral blood lymphocytes from healthy donors, circulating lymphocytes from patients with chronic lymphocytic leukemia (CLL) and lymphoblasts from children with common acute lymphoblastic leukemia (c-ALL). Actin and vimentin were quantified as percentage of total proteins by densitometric scanning of SDS-polyacrylamide gels loaded with Triton-X-100 cell lysates. In addition, the amounts of F- and G-actin were evaluated by ultracentrifugation of total lysates prior to electrophoresis. Compared to peripheral blood lymphocytes from healthy donors, CLL lymphocytes, but not blasts expressing c-ALL antigen, show a decrease in total actin and vimentin contents. Analysis of SDS gels after ultracentrifugation shows that F-actin is diminished of about 50% in both CLL and ALL cells compared to peripheral lymphocytes from healthy donors. The changes in actin and vimentin content and of actin organization may account for some of the functional abnormalities observed in leukemic cell populations.

Actin Cytoskeleton↗
PubMed1986

The actin filament-severing domain of plasma gelsolin.

Gelsolin, a multifunctional actin-modulating protein, has two actin-binding sites which may interact cooperatively. Native gelsolin requires micromolar Ca2+ for optimal binding of actin to both sites, and for expression of its actin filament-severing function. Recent work has shown that an NH2-terminal chymotryptic 17-kD fragment of human plasma gelsolin contains one of the actin-binding sites, and that this fragment binds to and severs actin filaments weakly irrespective of whether Ca2+ is present. The other binding site is Ca2+ sensitive, and is found in a chymotryptic peptide derived from the COOH-terminal two-thirds of plasma gelsolin; this fragment does not sever F-actin or accelerate the polymerization of actin. This paper documents that larger thermolysin-derived fragments encompassing the NH2-terminal half of gelsolin sever actin filaments as effectively as native plasma gelsolin, although in a Ca2+-insensitive manner. This result indicates that the NH2-terminal half of gelsolin is the actin-severing domain. The stringent Ca2+ requirement for actin severing found in intact gelsolin is not due to a direct effect of Ca2+ on the severing domain, but indirectly through an effect on domains in the COOH-terminal half of the molecule to allow exposure of both actin-binding sites.

Actin Cytoskeleton↗
PubMed1985

Interactions of gelsolin and gelsolin-actin complexes with actin. Effects of calcium on actin nucleation, filament severing, and end blocking.

Gelsolin is a calcium binding protein that shortens actin filaments. This effect occurs in the presence but not in the absence of micromolar calcium ion concentrations and is partially reversed following removal of calcium ions. Once two actin molecules have bound to gelsolin in solutions containing Ca2+, one of the actins remains bound following chelation of calcium, so that the reversal of gelsolin's effect cannot be accounted for simply by its dissociation from the ends of the shortened filaments to allow for elongation. In this paper, the interactions with actin of the ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) stable 1:1 gelsolin-actin complexes are compared with those of free gelsolin. The abilities of free or complexed gelsolin to sever actin filaments, nucleate filament assembly, bind to the fast growing (+) filament ends, and lower the filament size distribution in the presence of either Ca2+ or EGTA were examined. The results show that both free gelsolin and gelsolin-actin complexes are highly dependent on Ca2+ concentration when present in a molar ratio to actin less than 1:50. The gelsolin-actin complexes, however, differ from free gelsolin in that they have a higher affinity for (+) filament ends in EGTA and they cannot sever filaments in calcium. The limited reversal of actin-gelsolin binding following removal of calcium and the calcium sensitivity of nucleation by complexes suggest an alternative to reannealing of shortened filaments that involves redistribution of actin monomers and may account for the calcium-sensitive functional reversibility of the solation of actin by gelsolin.

Actin Cytoskeleton↗
PubMed1985

Human plasma actin-depolymerizing factor. Purification, biological activity and localization in leukocytes and platelets.

The plasma and serum of humans and various animal species exert an actin-depolymerizing activity. Human actin-depolymerizing factor (ADF) has been purified by ammonium sulfate fractionation, DEAE-cellulose and blue-Sepharose chromatography. It is a single polypeptide of approximately 90 kDa, with a pI between 6.0 and 6.5. ADF is heat and trypsin-sensitive, inactivated by EGTA, not stained by HIO4/Schiff on sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE), and not retained on a concanavalin-A-Sepharose column. Incubation of ethanol-fixed cultured cells or unfixed cryostat tissue sections with ADF abolishes immunofluorescent actin staining, by a mechanism which involves extraction of actin from the preparations. ADF promotes fragmentation and depolymerization of actin filaments as shown by electron microscopy, differential ultracentrifugation and DNAse I inhibition assay. This depolymerized actin retains its mobility on SDS/PAGE and is able to repolymerize in the presence of EGTA. Human white blood cells and platelets (but neither human fibroblasts nor white blood cells and platelets from pig, rat and rabbit) contain a 90-kDa protein reacting with an antibody raised in rabbit against human ADF as judged by immunofluorescence and immunoblotting techniques. Immunoblots of human granulocyte subcellular fractions suggest that the protein reacting with ADF antibody is present in the soluble cytoplasmic fraction. ADF may play a role in solubilization of plasma actin and in the intracellular organization of actin, and should be useful for the evaluation of the relative stability of cytoplasmic actin filaments in various physiological and pathological processes.

Actin Depolymerizing Factors↗
PubMed1984

Abnormal properties of skin fibroblasts from patients with breast cancer.

The growth properties of fibroblasts from the thoracic skin of patients with mammary cancers were compared to those of fibroblastic cultures from patients with benign lesions or having undergone surgery for non-neoplastic diseases. As expected, an inverse correlation was found between the doubling potential of fibroblasts in vitro and the donor's age for cells from patients with benign lesions; however no correlation, was found with cultures from cancer patients. Moreover, the latter group responded in an abnormal way to three biological parameters: anchorage dependence, colony formation on monolayers of normal human epithelial cells and saturation densities in overcrowded culture conditions. Skin fibroblasts from one patient with a benign lesion, whose mother had developed a breast cancer, displayed all the abnormal growth properties. Periodic controls of this patient resulted in the early detection of a carcinoma 3 years after the first operation for a benign microcystic lesion. Finally, we found that multiple subcultivations in overcrowded culture conditions cause the selection of a fibroblastic cell subset with greater growth potential which, in the cell strain tested, could invade foreign tissue in vitro.

Adult↗
PubMed1983

Abnormal behavior of cultured fibroblasts from nodule and nonaffected aponeurosis of Dupuytren's disease.

Vimentin-positive, desmin-negative cells were established in culture from the nodule and from apparently normal palmar aponeurosis of a patient with Dupuytren's disease and compared with normal human embryonic and adult fibroblasts or sarcomatous cells. Cells from the nodule display in vitro biological properties that are intermediate between those expressed by normal fibroblasts and sarcoma cells or cells from the nodule transformed with SV40 virus. Thus, they represent an interesting in vitro model of partially transformed human cells. This behavior is not evolutive and justifies the classification of Dupuytren's disease among the benign mesenchymal tumors. The production of high level of plasminogen activator probably explains the local reactive pathology, and could act as a mitogenic stimulus for the proliferation of the nodule itself. Cultures derived from the apparently normal palmar aponeurosis show some but not all the abnormal growth properties of cells from nodules; this may help to explain the onset of local recurrences. Our results suggest that Dupuytren's disease is not strictly local and limited to the nodules, but affects, at least partially, the whole aponeurosis. Dupuytren's nodules could be considered as a model of tumor progression in a benign situation.

Blood↗
PubMed1982

Effect of phalloidin on liver actin distribution, content, and turnover.

Phalloidin increases F-actin microfilament content and actin-directed immunofluorescence in hepatocytes in vivo and also increases actin polymerization and the stability of F-actin in vitro. We studied the sensitivity of immunofluorescent staining of actin to an actin depolymerizing factor (ADF) as well as actin content, degree of polymerization, and turnover in livers of in vivo phalloidin-treated rats. Pretreatment with ADF abolished anti-actin antibody (AAA) staining of normal liver but did not modify staining of livers from phalloidin-treated animals. Planimetric analyses of SDS-polyacrylamide gels showed the percent actin of total protein was increased by approximately 40% and the absolute amount of actin by approximately 43%, ten days after daily phalloidin treatment (50 micrograms/100 gm body weight). Similar but smaller changes could be seen after one day of treatment. Ultracentrifugational analyses of liver extracts indicated no change in the amount or proportion of G-actin but a 194% increase in the proportion of F-actin in ten-day treated animals, changes also apparent in one day animals. Neither the relative fractional rate of actin synthesis nor its synthesis as a percent of total protein synthesis was altered either at one-day or ten-day post-phalloidin treatment. Dualisotope experiments indicated that the rate of actin degradation was decreased selectively in the one- to three-day period following drug treatment. Thus, phalloidin appears to stabilize actin against the depolymerizing actions of ADF, increases the proportion of F-actin without altering the size of the G-actin pool, and causes accumulation of actin by decreasing its relative rate of degradation.

Actin Depolymerizing Factors↗
PubMed1982

Actin organization and fibrin-clot retractile activity of cultured mouse fibroblasts.

It is known that human and animal fibroblasts are able to induce the retraction of a fibrin clot. In the present study the correlation between (i) fibrin-fibroblasts during growth, (ii) the number of actin stress-lines in mouse fibroblasts during growth in culture, and (iii) the sensitivity of actin stress-lines to a powerful actin-depolymerizing factor (ADF), present in plasma and serum of humans and laboratory animals was investigated. Fibroblasts at early passages (2-4) were tested for these parameters at various intervals after seeding (24, 96, and 168 h). The number of actin stress-lines was progressively higher, while the sensitivity to ADF action was progressively lower in cells cultured from 24 to 168 h; the FCR capacity was significantly decreased at 168 h. These data suggest that cells containing weakly polymerized and/or stabilized actin are more active than those containing highly polymerized and/or stabilized actin in triggering fibroblast contraction.

Actin Depolymerizing Factors↗
PubMed1982

The area of attachment of cytotoxic T lymphocytes to their target cells shows high motility and polarization of actin, but not myosin.

Conjugates of cytotoxic T lymphocytes attached to their target cells were studied by double immunofluorescence on fixed smears to detect simultaneously the localization of actin and myosin within the cells. Actin was found to be polarized in the area of attachment of the lymphocytes (but not of the target cells), whereas myosin remained evenly distributed in the cytoplasm. When living conjugates were brought to 37 degrees C to induce cytotoxicity, this pattern remained unchanged, but observation by interference reflexion microcinematography revealed a high motility of the lymphocytes in the contact area. This localized motility in the area of attachment associated with a peculiar actin polarization, which has no equivalent in any type of cell contact presently known, could represent a necessary step in the sequence of events leading to target cell killing by cytotoxic T lymphocytes.

Actins↗
PubMed1981

Vascular smooth muscle cells differ from other smooth muscle cells: predominance of vimentin filaments and a specific alpha-type actin.

Smooth muscle cells of the digestive, respiratory, and urogenital tracts contain desmin as their major, if not exclusive, intermediate-size filament constituent and also show a predominance of gamma-type smooth muscle actin. We have now examined smooth muscle tissue of different blood vessels (e.g., aorta, small arteries, arterioles, venules, and vena cava) from various mammals (man, cow, pig, rabbit, rat) by one- and two-dimensional gel electrophoresis of cell proteins and by immunofluorescence microscopy using antibodies to different intermediate-sized filament proteins. Intermediate-sized filaments of vascular smooth muscle cells contain abundant amounts of vimentin and little, if any, desmin. On gel electrophoresis, vascular smooth muscle vimentin appears as two isoelectric variants of apparent pI values of 5.30 and 5.29, shows the characteristic series of proteolytic fragments, and is one of the major cell proteins. Thus vimentin has been demonstrated in a smooth muscle cell present in the body. Vascular smooth muscle cells are also distinguished by the predominance of a smooth muscle-specific alpha-type actin, whereas gamma-type smooth muscle actin is present only as a minor component. It is proposed that the intermediate filament and actin composition of vascular smooth muscle cells reflects a differentiation pathway separate from that of other smooth muscle cells and may be related to special functions and pathological disorders of blood vessels.

Actins↗
PubMed1981

Organization of actin in epithelial cells during regenerative and neoplastic conditions. Correlation of morphologic, immunofluorescent, and biochemical findings.

To study the distribution and the degree of polymerization of actin in regenerating and neoplastic cells we have examined: (1) the immunofluorescent staining of these cells with human antiactin antibodies (AAA), (2) the sensitivity of cellular actin to AAA staining after treatment with the actin-depolymerizing factor present in plasma or serum of several animal species, and (3) the total and relative amounts of F- and G-actin in tissue preparations, determined by means of differential centrifugation and densitometric analysis of stained sodium dodecyl sulfate-polyacrylamide gels. There was little or no difference in staining intensity between normal and regenerating or tumoral tissues when treated with AAA, but AAA staining of normal tissues was abolished, whereas staining of regenerating and tumoral tissues remained intense after incubation with actin-depolymerizing factor. Gel analysis showed that normal, regenerating, and neoplastic epidermal cells contained similar amounts of actin. Compared to controls, regenerating liver tissue contained slightly more total actin, nearly the same amount of G-actin, but a substantially increased amount of F-actin. These results suggest that: (1) compared to controls, a greater proportion of regenerating and neoplastic tissue actin is stabilized against the action of actin-depolymerizing factor; (2) changes involving actin in regenerating and neoplastic epithelial cells reflect changes in its degree of polymerization rather than its total amount.

Actins↗
PubMed1979

Intranuclear injection of anti-actin antibodies into Xenopus oocytes blocks chromosome condensation.

The role of contractile proteins in the structural organisation of the interphase nucleus and of metaphase chromosomes is largely unknown. Actin has been found in interphase nuclei of different species, especially in association with condensed chromatin. In the germinal vesicle (nucleus) of Xenopus oocytes, actin has been localised in the nuclear gel supporting the chromosomes and the extrachromosomal nucleoli. It has been reported that the premeiotic lampbrush chromosomes in these germinal vesicles are positively stained for actin and tubulin by the immunoperoxidase technique. Moreover, the longitudinal contraction of these chromosomes is ATP dependent. Therefore it has been suggested that actin participates in the structural organisation of the highly specialised lampbrush chromosomes. However, actin is not a major component of the metaphase chromosome scaffold. The results reported here suggest that actin is involved in the condensation of Xenopus chromosomes.

Actins↗
PubMed1979

An actin-destabilizing factor is present in human plasma.

Plasma and serum of humans or experimental animals contain a factor which destabilizes F-actin. The factor has no DNAse or thrombin activity and after incubation with F-actin does not modify the position of the actin band on a SDS polyacrylamide gel. Hence it probably depolymerizes F-actin.

Actins↗
PubMed1979

Actin microfilaments, cell shape, and secretory processes in isolated rat hepatocytes. Effect of phalloidin and cytochalasin D.

The effects of phalloidin and cytochalasin D, drugs which, respectively, stabilize and destabilize actin microfilaments, have been tested on isolated rat hepatocytes. Both drugs produced a modification of cell shape, characterized by protrusions bulging from the cytoplasm. In phalloidin-treated hepatocytes, an accumulation of actin microfilamentous network was detectable at the base of each protrusion by electron microscopy, immunofluorescence, and HMM decoration. This accumulation of microfilaments was absent in cytochalasin D-treated cells. The release of triglycerides, an index of very low density lipoprotein secretion, was inhibited by phalloidin or cytochalasin D, and accompanied by an increase in cellular triglycerides. At the electron microscope examination, triglyceride accumulation was represented by fat droplets and vesicle-enclosed, very low density lipoprotein-like particles. Total protein and albumin secretion was only very slightly modified by either one of these drugs. With the use of various phalloidin analogs, a correlation was observed between their respective ability to stabilize F-actin in vitro, and their effects on cell shape and triglyceride secretion. In conclusion, phalloidin, and cytochalasin D: (a) modify the shape of isolated hepatocytes; (b) inhibit lipoprotein secretion. These effects possibly result from a modification of actin microfilament function.

Actins↗