Abstract. We have correlated the motility of the leading edge of fibroblasts, monitored by phase-contrast cinematography, with the relative distributions of several cytoskeletal elements (vinculin, tubulin, and actin) as well as with the contact patterns determined by interference reflection microscopy. This analysis has revealed the involvement of both ruffles and microspikes, as well as microtubules in the initiation of focal contact formation. Nascent vinculin sites within the leading edge or at its base, taken as primordial cell-substrate contacts, were invariably colocalized with sites that showed a history of transient, prolonged, or cyclic ruffling activity. Extended microspike structures, often preceded the formation of ruffles. Immunofluorescent labeling indicated that some of these primordial contacts were in close apposition to the ends of microtubules that penetrated into the leading edge. By fluorescence and electron microscopy short bundles of actin filaments found at the base of the leading edge were identified as presumptive, primordial contacts. It is concluded that ruffles and microspikes, either independently or in combination, initiate and mark the sites for future contact. Plaque proteins then accumulate (within 10-30 s) at the contact site and, beneath ruffles, induce localized bundling of actin filaments. We propose that all primordial contacts support traction for leading edge protrusion but that only some persist long enough to nucleate stress fiber assembly. Microtubules are postulated as the elements that select, stabilize, and potentiate the formation of these latter, long-lived contacts.

In this article we discuss three aspects of cell contact formation : (a) the molecular architecture of the cytomatrix in cell-to-substrate focal contacts, (b) the dynamic properties of membrane-and microfilament-associated proteins in the contact areas, and (c) the involvement of microtubules in the coordinated and directed formation of new substrate contacts during cell locomotion . We show that different microfilament-associated proteins exhibit distinct patterns of association with focal contacts : some proteins are specifically associated with focal contacts (vinculin and talin) ; a-actinin is enriched in the contact areas but also is present along the stress fibers and in the lamellipodium; actin and filamin are detected throughout the contact areas but in apparently reduced amounts compared with the associated stress fibers ; and tropomyosin, myosin, and spectrin are either absent from the endofacial surfaces of contact areas or are present in only very small amounts. Fluorescence photobleaching recovery analyses performed with living cells microinjected with fluorescently labeled actin, vinculin, and a-actinin indicate that each of these proteins maintains a dynamic equilibrium between a soluble cytoplasmic pool and a membrane-bound fraction . Correlation of the distribution of vinculin and tubulin in motile fibroblasts to local movements of the leading edge of the same cells indicates that free-end microtubules extend into actively ruffling areas along the lamellipodium and that new vinculin-containing contacts are preferentially formed in these protruding regions .Cells move by cycles ofanterior membrane protrusion, establishment ofcontact with the underlying substrate, and retraction of the posterior trailing edge (for reviews, see references 1 and 2). The spatial and temporal coordination ofthese three major phases of the locomotory cycle is an essential element of directional cell motility.Apparently, cells sense and identify external stimuli that affect their motility. These include chemotactic stimuli, contacts with neighboring cells, and changes in the texture or adhesivity of the substrate . In addition, a central mechanism probably exists that integrates these stimuli and coordinates the dynamic molecular events that occur in different domains of the cell. The most active region is the leading lamellipodium. This area is very rich in actin filaments in the form of dense webs and small bundles (3-7). Besides actin, the leading lamella contains a-actinin (8-10) and, often, filamin . The THE JOURNAL OF CELL BIOLOGY " VOLUME 99 No . 1 Pt . 2 JULY 1984 83s-91s 0 The Rockefeller University Press -0021-9525/84/07/083s/09 $1 .00 focal contacts with the substrate that are formed under the leading lamella are particularly rich in the cytoskeletal protein vinculin and eventually, as they mature, become associated with the termini of stress fibers (9,(11)(12)(13)(14)(15)(16) . In fact, we suggest that focal contacts and the associated vinculin serve as organizing centers for the assembly of actin-c...

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