Narrow linear strips of adhesive substratum are powerful inducers of both growth and total focal contact area

ABSTRACT The stimulus to growth that occurs when cells attach to the substratum can be studied with small adhesive islands. Large numbers of these islands can be grouped together into arrays made up of various sizes, and the response of cells to incubation on these arrays allows the anchorage stimulus to be measured. Past work has shown that single isolated cells can be stimulated to proliferate under these circumstances quite as freely as they do in ordinary cultures, and that the maximum response is given by islands whose size is less than 5000μm2. This anchorage stimulus might be mediated by the cytoskeleton, which assembles rapidly around the points of attachment to the substratum. One possible approach to testing this hypothesis is to expose cells to islands of different shapes, and to search for common factors among the different arrangements of the cytoskeleton that these different islands cause. Circular islands induced a relatively disordered arrangement of actin fibres. The fibres were attached at one end to foci of vinculin, which sometimes became arranged in a ring around the margin of the island. Triangular islands showed a more orderly arrangement of actin, in three bands parallel to the sides. In this case, the vinculin accumulated at the apices. Long islands only 3μm wide could also provide effective attachment for the cells. In this shape the actin accumulated in two bands 2 μm or more apart and up to 5 μm high, and the vinculin similarly collected in parallel interrupted bands along the margins of the island. The number of vinculin foci differed on these three different island shapes, and the total area of vinculin was more than three times greater on long islands than on circles or triangles of the same size. Despite these differences, all three different shapes of island were capable of inducing up to 100μm2 of vinculin foci in each cell. Round and triangular islands induced this maximum amount of vinculin when their size was 5000 μm2. Linear islands induced the same amount when they were only 1000 μm2. The effect of different shapes on total vinculin focal area was paralleled by their effects on growth. All three shapes could support a similar amount of proliferation. Round and triangular islands induced the maximum amount of proliferation when they were 5000 μm2 in area, and linear islands when they were only 1000μm2. Similarly, linear islands induced as many cells to undergo DNA synthesis as did circles more than twice as large. These experiments support the idea that focal contacts might act as tranducers of the anchorage stimulus to growth. They show that total focal contact area is consistently and quantitatively related to growth, and suggest that the arrangement of the foci or their number may have less importance. Island substrata of different shapes offer a new approach towards the understanding of the involvement of the cytoskeleton in the mechanism of growth regulation.