ROLE OF THE CYTOSKELETON IN THE REACTION OF FIBROBLASTS TO MULTIPLE GROOVED SUBSTRATA

The role of the cytoskeleton and cell attachments in the alignment of baby hamster kidney fibroblasts to ridge and groove substratum topogra phy was investigated using confocal scanning microscopy. This was carr ied out with normal cells and cells treated with the cytoskeleton modi fiers cytochalasin D, colcemid, and taxol. Actin was localised with fl uorescent phalloidin. Tubulin, vinculin, and intracellular adhesion mo lecule-1 were visualised by indirect immunofluorescence. The spreading , elongation, and orientation of the cells after 24 h of culture in th ese conditions were measured on grooves of 5, 10, and 25 mu m width an d 0.5, 1, 2, and 5 mu m depth. We have also observed events over the f irst 30 min of cell attachment. Five minutes after cell attachment, F- actin condensations were seen close to the intesection of groove wall and ridge top, that is, at a topographic discontinuity. The condensati ons were often at right angles to the groove edge and showed a periodi city of 0.6 mu m. Vinculin arrangement at the early stages of cell spr eading was similar to that of actin. Organisation of the microtubule s ystem followed later, becoming obvious at about 30 min after cell plat ing. The Curtis and Clark theory (that cells react to topography prima rily at lines of discontinuity in the substratum by actin nucleation) is supported by these results. The use of cytoskeletal poisons did not entirely abolish cell reaction to grooves. Colcemid increased cell sp reading and reduced cell orientation and elongation. Cytochalasin D re duced cell spreading, orientation, and elongation. Taxol reduced cell elongation but did not affect cell spreading and orientation. We concl ude that the aggregation of actin along groove/ridge boundaries is a p rimary driving event in determining fibroblast orientation on microgro oved substrata. (C) 1995 Wiley-Liss, Inc.