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J Vasiliev

Publications and source records attributed to J Vasiliev.

6 recordsLinked to original sources
PubMed2001

Focal adhesion features during myofibroblastic differentiation are controlled by intracellular and extracellular factors.

Transforming growth factor beta (TGFbeta), the most established promoter of myofibroblast differentiation, induces ED-A cellular fibronectin and alpha-smooth muscle actin expression in fibroblastic cells in vivo and in vitro. ED-A fibronectin exerts a permissive action for alpha-smooth muscle actin expression. A morphological continuity (called fibronexus), a specialized form of focal adhesion, has been described between actin stress fibers that contain alpha-smooth muscle actin, and extracellular fibronectin, which contains the ED-A portion, in both cultured fibroblasts and granulation tissue myofibroblasts. We have studied the development of these focal adhesions in TGFbeta-treated fibroblasts using confocal laser scanning microscopy, three-dimensional image reconstruction and western blots using antibodies against focal adhesion proteins. The increase in ED-A fibronectin expression induced by TGFbeta was accompanied by bundling of ED-A fibronectin fibers and their association with the terminal portion of alpha-smooth muscle actin-positive stress fibers. In parallel, the focal adhesion size was importantly increased, and tensin and FAK were neoexpressed in focal adhesions; moreover, vinculin and paxillin were recruited from the cytoplasmic pool into focal adhesions. We have evaluated morphometrically the length and area of focal adhesions. In addition, we have evaluated biochemically their content of associated proteins and of alpha-smooth muscle actin after TGFbeta stimulation and on this basis suggest a new focal adhesion classification, that is, immature, mature and supermature. When TGFbeta-induced alpha-smooth muscle actin expression was blocked by soluble recombinant ED-A fibronectin, we observed that the fragment was localised into the fibronectin network at the level of focal adhesions and that focal adhesion supermaturation was inhibited. The same effect was also exerted by the ED-A fibronectin antibody IST-9. In addition, the antagonists of actin-myosin contractility BDM and ML-7 provoked the dispersion of focal adhesions and the decrease of alpha-smooth muscle actin content in stress fibers of pulmonary fibroblasts, which constitutively show large focal adhesions and numerous stress fibers that contain alpha-smooth muscle actin. These inhibitors also decreased the incorporation of recombinant ED-A into fibronectin network. Our data indicate that a three-dimensional transcellular structure containing both ED-A fibronectin and alpha-smooth muscle actin plays an important role in the establishment and modulation of the myofibroblastic phenotype. The organisation of this structure is regulated by intracellularly and extracellularly originated forces.

Actins↗
PubMed2000

Changes in p53 expression in mouse fibroblasts can modify motility and extracellular matrix organization.

Effects of p53 expression on cell morphology and motility were studied using the derivatives of p53-null 10(1) mouse fibroblasts with tetracycline-regulated expression of exogenous human p53. Induction of p53 expression was accompanied by significant decrease in extracellular matrix (fibronectin) and reduction of matrix fibrils, diminution of the number and size of focal contacts, decrease of cell areas, establishment of more elongated cell shape and alterations of actin cytoskeleton (actin bundles became thinner, their number and size decreased). Expression of His175 and Gln22/ Ser23 p53 mutants caused no such effects. To study the influence of p53 expression on cell motility we used wound technique and videomicroscopy observation of single living cells. It was found that induction of p53 expression led to increase of lamellar activity of cell edge. However, in spite of enhanced lamellar activity p53-expressing cells migrated to shorter distance and filled the narrow wound in longer time as compared with their p53-null counterparts. Possible mechanisms of the influence of p53 expression on cell morphology and motility are discussed.

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PubMed1998

Rat fibroblasts cultured from various organs exhibit differences in alpha-smooth muscle actin expression, cytoskeletal pattern, and adhesive structure organization.

In vivo, alpha-smooth muscle actin (SMA) is expressed de novo and temporarily by fibroblastic cells during wound healing and correlates particularly with wound contraction. In culture, the presence of varying proportions of cells expressing and not expressing this actin isoform (alpha-SMA-positive and alpha-SMA-negative cells) is characteristic of fibroblastic populations from different tissues. It is possible that mechanisms controlling the expression of actin isoforms, and thus modulating cytoskeleton-related functions, play a major role in the organization of cell shape and motility. We have compared the cell shape as well as the cytoskeleton and focal contact organization in alpha-SMA-positive and alpha-SMA-negative rat fibroblasts from various organs (i.e., skeletal muscle, dermis, subcutaneous tissue, and lung). Within each category, i.e., alpha-SMA-positive or alpha-SMA-negative fibroblasts, no significant morphological differences were seen among populations derived from different tissues. In contrast, alpha-SMA-positive and alpha-SMA-negative fibroblasts were significantly different, independently of their origin: alpha-SMA-positive cells had larger average areas, higher numbers of narrow extensions at the edges, larger focal adhesions with the substratum, and a more important network of cellular fibronectin than alpha-SMA-negative cells. Thus, alpha-SMA-positive and alpha-SMA-negative variants naturally present in fibroblastic populations exhibit important phenotypic differences probably associated with distinct functional activities.

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PubMed1997

Changes in p53 expression can modify cell shape of ras-transformed fibroblasts and epitheliocytes.

p53 plays an important role in restriction of abnormal cell proliferation. Loss of this safeguard function induced by p53 mutations seems to be a key mechanism in oncogenesis. It cannot be excluded however, that in addition to elimination of p53-dependent checkpoints and/or apoptosis p53 mutations may cause additional effects that contribute to oncogenic transformation. In order to analyse the effects of wild-type (wt) and mutant p53 on expression of ras-induced morphological transformation we used the method of computer-assisted morphometry. The following parameters were determined: a) the area covered by the spread cells; b) dispersion and c) elongation of cell contours. The last two indices characterise cell shape. Elongation indicates the degree of bipolarity of cell contour and dispersion-the degree of its multipolarity. Transformation of Rat1 and mouse 10(3) fibroblasts by N-rasasp12 oncogene was accompanied by dramatic decrease of cell area and increase of dispersion and elongation. IAR-2 discoid epitheliocytes expressing exogenous ras oncogene transformed into polarised cells with decreased cell area. Fluorescent microscopic examination of actin cytoskeleton stained with rhodamine-phalloidin had shown that ras-induced transformation of IAR-2 cells is characterised by disappearance of circumferential actin bundle and straight fibers. Neither did we reveal actin stress-fibers in the ras-transformed Rat1 cells. Transduction of p53 cDNAs caused no significant changes in morphometric parameters of non-transformed parental Rat1, IAR-2 and 10(3) cells, but some of the p53 mutants modified cell shape of ras-transformed cells. p53-His273, unlike other tested p53 mutants (Tyr141, His194, Trp248), induced partial reversion of morphological transformation in both Rat1 fibroblasts and IAR-2 epitheliocytes. Its expression led to increase of average cell area, decrease of dispersion and elongation indices, and re-appearance of actin bundles. Exogenous wild-type p53 also caused some reversion of transformed phenotype of Rat/ras cells, but its effect was weaker than that of the p53-His273. In contrast, another p53 mutant p53-His175 was able to enhance ras-induced morphological transformation in p53-deficient murine 10(3) fibroblasts that is consistent with possible involvement of some gain of function activity of mutant p53 in modulation of cell shape. Possible pathways that might be responsible for p53-induced changes of cell morphology are discussed.

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