membranes are a subset of extracellular matrices made unique by their distinct composition and close association with selected cell surfaces. Laminin and type IV collagen polymers are thought to be key structural elements of the basement membrane and are usually accompanied by entactin/nidogen, perlecan, and other glycoproteins (65
). The laminins are a family of large multifunctional molecules thought to be essential for the development and stability of these specialized matrices. All laminins are heterotrimers composed of individual α, β, and γ subunits. Laminins assemble into trimers by forming a long coiled-coil interaction in the COOH-terminal half of the protein, designated as the long arm, whereas the NH2
-terminal regions remain independent and make up the three short arms. There are currently five laminin alpha chains, three beta chains, and three gamma chains that were found in at least 11 different heterotrimer combinations in vivo (37
). Depending on the tissue or organ, different laminin subunits appear to be required at crucial stages of embryogenesis, as well as for normal function in mature tissues such as muscle and skin (36
It is becoming clear that different laminins have both overlapping and unique functions. To understand the mechanisms underlying the requirement for so many laminins, it will be important to determine how these different laminins locally alter the function of basement membranes and exert effects on adjacent cell layers. For instance, it was shown that various laminin isoforms differ in their ability to form polymers. Laminin isoforms 1–4 are capable of forming three-dimensional polymers through interactions among their three NH2
-terminal short arms, whereas laminins 5–7, with one or more truncated short arms, are not (7
). An independent laminin polymer in conjunction with the collagen IV network provides structure and support for adjacent cells (7
). Furthermore, laminins provide these cells with signals and cues through multiple cell surface receptor interactions, influencing processes such as cell survival, proliferation, and migration. However, the relationship and possible interplay between the structural role of laminin (i.e., its architectural state) and its many cell interactive functions has remained unexplored.
In vitro laminin polymerization was shown to be a reversible process in which each of the three short arms interacts to form the basic polymer bond (7
). This nucleation/propagation assembly process occurs in solution and has a critical concentration of 70–140 nm (66
). Although laminins and collagens both form polymers in solution, laminin polymers are generated through weaker interactions, forming a more plastic polymer better suited to dynamic processes such as tissue remodeling and mechanochemical signaling. Studies using laminin in solution have been useful for elucidating the domain requirements and kinetics of laminin polymerization, but alone cannot describe how the process occurs in a living organism. For instance, it was observed that basement membranes do not form in just any empty space between cells, but are found closely adjacent to particular cell types or even particular regions of a cell type, such as the basolateral surface of epithelial cells. In fact, basement membrane constituents may be synthesized by one cell type but end up in the basement membrane of another, more distal cell type (26
). An earlier study found that as laminin attached to synthetic phospholipid membranes, it polymerized at concentrations well below the solution critical concentration (32
). These results hinted at a mechanism by which laminin– receptor interactions would generate very high local surface concentrations of laminins, thereby driving it above its critical concentration on the plasma membrane surface. Supporting a role for receptor interactions in basement membrane assembly, several mice with mutations in cell surface receptors were found to have various basement membrane defects (17
). Although these basement membrane defects were not characterized at the level of laminin polymer formation, and none of these receptors (α3 and β1 integrin subunits, and dystroglycan) exclusively binds laminin, together these studies strongly suggested that cells might use receptors to facilitate and target laminin polymerization.
Recently, attention has focused on the role of laminin and its interactions in skeletal muscle. Many severe congenital muscular dystrophies are caused by mutations in the LAMA2 gene and are marked by basement membrane defects in both muscle and the nervous system (46
). In the muscle sarcolemmal basement membrane, laminin has been proposed to provide a linkage between the extracellular matrix and the dystroglycan–glycoprotein complex. In fact, mutations in virtually any component of the dystroglycan–glycoprotein complex can result in some form of muscular dystrophy, from Duchenne's (dystrophin) to muscular dystrophies of the Limb-Girdle type (sarcoglycans; ref. 14). Another binding partner for laminin in skeletal muscle is the α7β1 integrin, particularly at the myotendinous junction. Mutations in the α7 integrin subunit can also cause skeletal muscle pathologies, although these appear to be less severe and are classified as myopathies, not dystrophies (28
). Interestingly, several LAMA2 mutations result in the expression of partially functional laminin molecules that maintain their basement membrane localization (1
). One such mutation occurs in the dy2J
mouse, where the laminin-2 expressed by these mice has been found to be defective in its ability to polymerize, even though it remains localized to the basement membrane (Colognato, H., and P.D. Yurchenco, manuscript in preparation).
While these disorders have established an important role for basement membranes, the underlying mechanisms behind the requirement for laminins have remained unclear. In the case of the dy2J
mouse, laminin remains linked to the cell through receptor interactions and to the basement membrane through its interaction with entactin/ nidogen. Therefore, it seems likely that laminin is required not only to provide an anchor to neighboring tissues, but to somehow alter these tissues, and we should consider that polymerization may play a role in this process. To address these issues it will be important to understand how the many and diverse functions of laminin might cooperate to promote matrix assembly and to provide mechanical and chemical signals to adjacent cells. An emerging paradigm is that the architecture itself transmits information to cells, through mechanisms such as matrix rigidity, spatial arrangement of cell receptors, and tension exerted between the matrix and receptor (8
). The structural properties and multiple functions of the laminin molecule would make it well suited to modulate cell interactions through these mechanisms.
In this study, we have evaluated the effects that laminin, as a monomeric or polymeric ligand, has on surface receptors and cortical cytoskeletal components using a muscle cell culture system (60
). We present evidence that laminin polymerization occurs preferentially on cell surfaces through specific receptor interactions, providing a targeting mechanism for the assembly of basement membranes. Furthermore, we show that laminin polymerization, above and beyond receptor occupancy, is required to induce reorganization of laminin, its receptors, and cytoskeletal components. This polymer-mediated rearrangement may represent a general mechanism used wherever basement membranes are assembling or remodeling. In fact, a specific disruption in this architecture may be the molecular basis for many congenital muscular dystrophies.