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Immunofluorescence and confocal microscopy were used to monitor the positioning of microtubule-organizing centers (MTOCs) during directional migration of chicken embryo fibroblasts on planar substrata and within three-dimensional collagen gels. Homologous assay conditions based on the radial emigration of cells from cell aggregates were used in both cases. Whereas approximately 70% of the cells migrating directionally on glass and at least 60% on other planar substrata have their MTOCs anterior to the nucleus, MTOCs are randomly distributed around the nucleus in cells within collagen gels. The anterior location of the MTOC in cells on glass is attained gradually during the first 4 hr of directional migration. Cells on oriented planar substrata, manufactured by photolithographic etching of narrow parallel grooves into the glass surface, also have a random position of the MTOC, although the cells themselves assume a highly polarized cell shape parallel to the grooves. This environment mimics the partial orientation of the collagen fibers produced by the tractive forces of the cells within collagen networks. These findings demonstrate a difference in MTOC positioning between fibroblasts on planar substrata and within a quasinatural environment.

The position of the microtubule-organizing center in directionally migrating fibroblasts depends on the nature of the substratum.