Dimensional and mechanical dynamics of active and stable edges in motile fibroblasts investigated by using atomic force microscopy

The atomic force microscope (AFM) was employed to investigate the extension and retraction dynamics of protruding and stable edges of motile 3T3 fibro blasts in culture. Such dynamics closely paralleled the results of earlier studies employing video microscopy that indicated that the AFM force-mappin g technique does not appreciably perturb these dynamics, Force scans permit ted height determinations of active and stable edges, Whereas the profiles of active edges are flat with average heights of 0.4-0.8 mu m, stable edges smoothly ascend to 2-3 mu m within about 6 mu m of the edge. In the region of the leading edge, the height fluctuates up to 50% (SD) of the mean valu e, much more than the stable edge; this fluctuation presumably reflects dif ferences in underlying cytoskeletal activity. In addition, force mapping yi elds an estimate of the local Young's modulus or modulus of elasticity (E, the cortical stiffness). This stiffness will be related to "cortical tensio n," can be accurately calculated for the stable edges, and is approximate t o 12 kPa in this case, The thinness of the leading edge precludes accurate estimation of the E values, but within 4 mu m of the margin it is considera bly smaller than that for stable edges, which have an upper limit of 3-5 kP a. Although blebbing cannot absolutely be ruled out as a mechanism of exten sion, the data are consistent with an actin polymerization and/or myosin mo tor mechanism in which the average material properties of the extending mar gin would be nearly constant to the edge. Because the leading edge is softe r than the stable edge, these data also are consistent with the notion that extension preferentially occurs in regions of lower cortical tension.