Stripping of Extenral Cell-Associated Fibronectin from Mitotic Fibroblasts

Abstract

ABSTRACT. Using a sensitive method to determine the amount of fibronectin (FN), we investigated the release of cellular FN in C3H mouse fibroblasts, which depends upon the progress of the cell cycle. Cells first labeled with [14C]leucine and arrested in confluent culture were trypsinized and replated in fresh medium containing [14C]leucine with or without 1 mM hydroxyurea (HU). The cell number in the untreated culture increased 1.3-fold 36 h after replating, whereas HU-treated cells neither entered the S phase nor divided. The synthesis, turnover and rebinding of FN to cells were almost the same for both cultures. The amounts of FN released into the medium also were the same for untreated and HU-treated cultures. These results indicate that FN was not released into the medium specifically before or during the rounding up of mitotic cells. Immunofluorescence observations using a specific antibody directed against FN showed the presence of FN networks on the surface of the substratum, which surrounded the point at which mitotic cells were attached. In addition, for about half the population of mitotic cells no FN was detected on the cell surfaces. Because of these results, we suggest that when mitotic cells become round, they leave FN in networks on the substratum.Fibronectin (FN) is a high molecular weight glycoprotein present on the surfaces of cells, in connective tissues, and in extracellular fluids (20,23). One function of this protein is to mediate cell adhesion (25). An addition of FN to transformed cells increases their adhesiveness to the substratum, an adhesiveness that had decreased because of transformation; it therefore restores normal cell morphology (2, 25). Treatment of cultured fibroblasts with trypsin induces the rounding of these cells, probably because FN and other adhesive proteins have been lost due to trypsin treatment (12). It also has been reported that cytochalasin B induces the release of FN into medium, which may be related to alterations in cell morphology after treat-* Present address: