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

Abstract

Immunofluorescence and confocal microscopy were used to monitor the positioning of microtubuleorganizing 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 =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 Jibroblasts on planar substrata and within a quasinatural environment.Microtubules play important roles in the life of a eukaryotic cell. Originating from the centrosome, the cell's microtubuleorganizing center (MTOC), they are involved in cell division, intracellular transport, the development and maintenance of cell asymmetry, and cell migration (1). An involvement in the expression of a locomotory phenotype is revealed in the experimental observation that microtubule depolymerization impairs cell locomotion (e.g., see refs. 2 and 3), and the morphological finding that, in certain migrating cells, the MTOC is located ahead of the nucleus and behind the advancing lamellipodium (4-6). This intriguing correlation was interpreted to mean that MTOC reorientation to the front of the cell not only accompanies the onset of cell migration but actually "may play a role in determining the direction of cell movement" (ref. 4; for reviews, see refs. 7-9).Fibroblasts cultured on glass or plastic substrata have served as important models for some of these studies (3,5,10). However, the advantage of two-dimensional surfaces for microscopic observation is counterbalanced by the disadvantage that these conditions are clearly unrepresentative of a fibroblast's natural environment, a three-dimensional collagen network (11, 12). Observations on fibroblasts in hydrated collagen gels in vitro and in situ demonstrate a number of differences in morphology and behavior from their counterparts on planar substrata (e.g., see refs. 11, and 13-17). It would seem important to ascertain that findings on cells cultured on planar substrata apply to cells in a more natural environment as well, particularly with respect to the question of MTOC positioning during cell migration.We have used a convenient assay for determining directional migration of large numbers of cells that does no...

In migrating cells, the Golgi complex and the position of the centrosome depend on geometrical constraints of the substratum

“…However, mixed front-back positions have been observed in CHO cells during wound healing (Yvon et al, 2002), in endothelial cells under shear stress (Coan et al, 1993) and in fibroblasts grown on grooved substrates or grown in collagen gels (Schütze et al, 1991). These results challenge the view that the Golgi complex -similar to the centrosome -is always at the leading edge of a migratory cell.…”

Roles of microtubules, cell polarity and adhesion in electric-field-mediated motility of 3T3 fibroblasts

“…Our experiments tested the hypothesis that directional migration requires that fibroblasts reorient in the direction of movement, as they do during spontaneous or wound-healing migration (Palazzo et al, 2001b;Ueda et al, 1997). MTOC reorientation does not occur in fibroblasts migrating within three-dimensionally oriented collagen gels (Schutze et al, 1991) or in wound-healing Journal of Cell Science 117 (8) (b) show that most PAK4 transfectants were flatter and more bipolar than controls, in which 20-25% of cells appear spherical before EF application. Primary fibroblasts from mouse embryos whose PAK4 expression was knocked out by homologous recombination (PAK4 -/-; c) and PAK4-expressing heterozygotes (PAK4 +/-; d) were morphologically indistinguishable.…”

“…For example, MTs are unnecessary for fibroblast locomotion in an oriented, three-dimensional extracellular matrix (ECM) (Schutze et al, 1991) or for rapid migration of tiny fish keratocytes (Euteneuer and Schliwa, 1984), or keratocyte fragments (Verkhovsky et al, 1999). Fibroblasts, however, require MTs (Gail and Boone, 1971;Vasiliev et al, 1970), specifically dynamic MTs (Liao et al, 1995), for spontaneous and wound-healing migration.…”

In vitro large-wound re-endothelialization. Inhibition of centrosome redistribution by transient inhibition of transcription after wounding prevents rapid repair.

“…29 These studies on Dictyostelium discoideum 28 and fibroblasts 29 suggest that polarization of the centrosome is not required for directional migration in three-dimensional gels. However, large-vessel ECs differ from fibroblasts because the cells migrate on a substratum that defines a luminal and an abluminal surface of the cell, and the cells migrate only in a two-dimensional plane.…”

“…28 Thus, in Dictyostelium discoideum the position of the centrosome is dependent on the conditions of migration and the nature of cell-cell adhesion. 28 In a study that used chicken embryo fibroblasts, 29 it was found that the centrosomes of cells grown on glass coverslips reoriented in the direction of cell migration, whereas In vitro large-wound re-endothelialization. Inhibition of centrosome redistribution by…”