Formation of cell-to-substrate contacts during fibroblast motility: an interference-reflexion study

Abstract

The formatin of close contacts and focal contacts (ca. 30 nm and 10–15 nm separation distance respectively) has been studied during the movement of chick heart fibroblasts on planar substrates using interference-reflexion microscopy, and evaluated in the context of spreading and net movement. During spreading the overall advance of the margin of the close contact is steady, punctuated by periods in which it remains stationary, and only 5% of the time is spent withdrawing in contrast to the extreme leading edge. The close contact advances only where a lamellipodium has first extended free of the substrate (greater than or equal to 100 nm separation distance) ahead of the existing close contact. The new close contact is formed by the lamellipodium lowering to the substrate either progressively from its base forward or distally in patches which later join with the main close contact. New focal contacts are formed successively ahead of existing ones, either by microspikes or lamellipodia contacting the substrate locally ahead of the close contact, or within the close contact usually immediately, but not more than 1–2 microns, behind its margin. Examining the cell margin alternately with interference-reflexion and differential-interference contrast showed that the formation of the focal contact was preceded in 90% of the cases by the development of a linear structure in the form of a microspike (as expected), a short projection (< 2 microns long) of the lamellipodium, or a fibre within the lamellipodium, each of which could be traced to the cytoplasmic fibre typically associated with the focal contact. Stress fibres subsequently developed centripetally from these initial fibres. The different forms of the linear structure which preceded the focal contact were interchangeable, giving rise to one another, and we have evaluated that the structure common to each is probably a short bundle of microfilaments. The following features indicate that the close contact plays a primary role in marginal spreading: it is lost when spreading ceases; it is reformed when spreading resumes but only under the newly spread area; the advance of the margin of the thicker leading lamella closely follows that of the close contact; the advance of both can occur ahead of and is thus independent of existing focal contacts and associated stress fibres. We propose that the close contact provides the adhesion required to transmit to the substrate the forces involved in the forward movements of the marginal cytoplasm. The continual formation of focal contacts and stress fibres at the margin is consistent with their role, suggested by others, in drawing the bulk of the cell forward. These evaluations are discussed in the context of the form and distribution of contractile proteins in the cell margin. A primary role of the lamellipodia and microspikes in extending the cell margin and forming new adhesions, preparatory to further cytoplasmic movement, is established by this work.