416 Biochemical Society Transactions (2004) Volume 32, part 3 Hierarchical assembly of cell–matrix adhesion complexes R. Zaidel-Bar*, M. Cohen*, L. Addadi† and B. Geiger* 1 *Department of Molecular Cell Biology, the Weizmann Institute of Science, Rehovot, Israel, and †Department of Structural Biology, the Weizmann Institute of Science, Rehovot, Israel Abstract The adhesion of cells to the extracellular matrix is a dynamic process, mediated by a series of cell-surface and matrix-associated molecules that interact with each other in a spatially and temporally regulated manner. These interactions play a major role in tissue formation, cellular migration and the induction of adhesion- mediated transmembrane signals. In this paper, we show that the formation of matrix adhesions is a hierarchical process, consisting of several sequential molecular events. One of the earliest steps in surface recognition is mediated, in some cells, by a 1 μm-thick cell-surface hyaluronan coat, which precedes the establishment of stable, cytoskeleton-associated adhesions. The earliest forms of these integrin-mediated contacts are dot-shaped FXs (focal complexes), which are formed under the protrusive lamellipodium of migrating cells. These adhesions recruit, sequentially, different anchor proteins that are involved in binding the actin cytoskeleton to the membrane. Conspicuous in its absence from FXs is zyxin, which is recruited to these sites only on retraction of the leading edge and the transformation of the FXs into a focal adhesion. Continuing application of force to focal adhesions results in the formation of fibrillar adhesions and reorganization of the extracellular matrix. The formation of these adhesions depends on actomyosin contractility and matrix pliability. Introduction Cell adhesion to the ECM (extracellular matrix) plays key roles in the assembly of cells into functional multicellular organisms. At the same time, such adhesions are involved in transmembranal signalling processes that regulate cell behaviour and fate. Adhesive interactions occur via a variety of molecular systems. These include different integrin receptors that bind to ECM molecules via their extracellular domains and interact via their cytoplasmic moieties with the actin cytoskeleton [1,2]. Adhesive interactions are mediated by a network of ‘anchor proteins’, some of which directly mediate the linkage between the actin and the membrane, whereas others play a regulatory role (reviewed in [3–5]). Adhesion is also mediated via a variety of membrane- or matrix-bound glycosaminoglycan molecules [6,7]. Integrin-mediated adhesions are molecularly hetero- geneous, appearing in different forms such as ‘classical’ FAs (focal adhesions), FBs (fibrillar adhesions) and FXs (focal complexes). Each one of these has a typical morphology and molecular composition (see [8] and Figure 1). In motile cells, the spatially and temporally regulated formation and dissociation of matrix adhesions play a central role in the motile process. In this paper, we will consider the molecular steps involved in cell–matrix adhesion, including hyaluronan-mediated Key words: cell adhesion, fibrillar adhesion, focal adhesion, focal complex. Abbreviations used: ECM, extracellular matrix; ESEM, environmental scanning electron microscopy; FA, focal adhesion; FB, fibrillar adhesion; FX, focal complex; GFP, green fluorescent protein. 1 To whom correspondence should be addressed (email benny.geiger@weizmann.ac.il). attachment and the sequential formation of FX, FA and FB. We will discuss the mechanisms underlying the formation of each of these adhesion sites and the interdependence between them. Hyaluronan-mediated adhesions: the first encounters ECM consists of a rich variety of macromolecules that are recognized by a comparably large variety of cell-surface re- ceptors. Whereas integrins and their ligands appear to play a major role in the formation of cell–matrix adhesion, other adhesive systems have also been described, including matrix- and membrane-bound glycosaminoglycans. Recently, we have shown that cell-associated hyaluronan plays a central role in mediating early stages in the attachment of cells to external surfaces [9–13]. In a series of studies on cell adhesion to molecularly defined crystal surfaces and to conventional cell adhesion substrates (glass and tissue culture dishes), it was found that rapid (time scale of seconds) attachment of A6 epithelial cells was mediated by cell-associated hy- aluronan [13]. This glycosaminoglycan is a large linear polymer of [D-N-acetylglucosamine-β -D-glucuronate] that can be associated either with the ECM or with the plasma membrane. Its role in surface adhesion was demonstrated by the drastic inhibition of the rapid attachment to compatible crystal surfaces, observed after treating the cells with hyaluronidase. Subsequent addition of pure hyaluronan to the hyaluronidase-treated cells or to the matrix could restore adhesion. Interestingly, the presence of hyaluronan on both the surfaces inhibited cell adhesion [13]. C  2004 Biochemical Society