B. Wojciakstothard et al., ROLE OF THE CYTOSKELETON IN THE REACTION OF FIBROBLASTS TO MULTIPLE GROOVED SUBSTRATA, Cell motility and the cytoskeleton, 31(2), 1995, pp. 147-158
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.