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 topography was investigated using confocal scanning microscopy. This was carried out with normal cells and cells treated with the cytoskeleton modifiers cytochalasin D, colcemid, and taxol. Actin was localised with fluorescent phalloidin. Tubulin, vinculin, and intracellular adhesion molecule-1 were visualised by indirect immunofluorescence. The spreading, elongation, and orientation of the cells after 24 h of culture in these conditions were measured on grooves of 5, 10, and 25 microns width and 0.5, 1, 2, and 5 microns depth. We have also observed events over the first 30 min of cell attachment. Five minutes after cell attachment, F-actin condensations were seen close to the intersection of groove wall and ridge top, that is, at a topographic discontinuity. The condensations were often at right angles to the groove edge and showed a periodicity of 0.6 microns. Vinculin arrangement at the early stages of cell spreading was similar to that of actin. Organisation of the microtubule system followed later, becoming obvious at about 30 min after cell plating. The Curtis and Clark theory (that cells react to topography primarily 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 spreading and reduced cell orientation and elongation. Cytochalasin D reduced cell spreading, orientation, and elongation. Taxol reduced cell elongation but did not affect cell spreading and orientation. We conclude that the aggregation of actin along groove/ridge boundaries is a primary driving event in determining fibroblast orientation on microgrooved substrata.