Dissociation of Actin Microfilament Organization from Acquisition and Maintenance of Elongated Shape of Human Dermal Fibroblasts in Three-Dimensional Collagen Gel

Abstract

Actin micro filaments of the fibroblasts cultured in a collagen gel were distributed along the inner surface of the entire cell membrane, in either spherical shape at an initial stage of culture or elongated shape at a later stage. The distribution was quite different from that of the fibroblast cultured on a two-dimensional surface, where actin microfilaments were found to be aligned essentially along the inner membrane which is in contact with a flat surface. Timing of morphological change from spherical shape to spread shape or elongated shape was also greatly affected by contact with substrates whether in two-dimension or in three-dimension: distinct morphological change was observed within 6 h on glass or on the collagen gel, and at 30 h or later within the collagen gel. The retardation of cell elongation in the gel was antagonized by a low dose (0.2 µM) of cytochalasin D, although the drug kept the cells in round shape at a concentration of 2 µM. Since a low concentration of cytochalasin was reported to induce actin polymerization in vitro, the organization of actin micro filaments was examined by rhodamine-phalloidin staining. It was found that actin filaments in elongated cells by low cytochalasin D were disrupted. These results suggest that accelerated acquisition of elongated shape by the treatment of a low dose of cytochalasin D might be initiated by destabilization of the actin microfilaments that may scaffold the spherical shape of the cell in the collagen gel. The elongated shape thus formed returned to spherical upon washing of the added free cytochalasin D. This is not the case with the elongated cells which had been established by a prolonged culture such as 40 h after starting the culture in the gel. That is, addition of cytochalasin D at a highest dose of 2.0 µM did not change the elongated shape in spite of the complete disruption of actin micro filaments of the fibroblasts. These findings suggest that actin microfilament organization is not actively involved in, or dissociated from, acquisition and maintenance of elongated shape of fibroblasts in the collagen gel.