High Resolution Immunoelectron Microscopic Localization of Functional Domains of Laminin , Nidogen , and Heparan Sulfate Proteoglycan in Epithelial Basement Membrane of Mouse Cornea Reveals Different Topological Orientations

Thin and ultrathin cryosections of mouse cornea were labeled with affinity-purified antibodies directed against either laminin, its central segments (domain 1), the end of its long arm (domain 3), the end of one of its short arms (domain 4), nidogen, or low density heparan sulfate proteoglycan. All basement membrane proteins are detected by indirect immunofluorescence exclusively in the epithelial basement membrane, in Descemet's membrane, and in small amorphous plaques located in the stroma. Immunoelectron microscopy using the protein A-gold technique demonstrated laminin domain 1 and nidogen in a narrow segment of the lamina densa at the junction to the lamina lucida within the epithelial basement membrane. Domain 3 shows three preferred locations at both the cellular and stromal boundaries of the epithelial basement membrane and in its center. Domain 4 is located predominantly in the lamina lucida and the adjacent half of the lamina densa. The low density heparan sulfate proteoglycan is found all across the basement membrane showing a similar uniform distribution as with antibodies against the whole laminin molecule. In Descemet's membrane an even distribution was found with all these antibodies. It is concluded that within the epithelial basement membrane the center of the laminin molecule is located near the lamina densa/lamina lucida junction and that its long arm favors three major orientations. One is close to the cell surface indicating binding to a cell receptor, while the other two are directed to internal matrix structures. The apparent codistribution of laminin domain 1 and nidogen agrees with biochemical evidence that nidogen binds to this domain. B ASEMENT membranes are specialized pericellular matrix structures which underlay endothelial and epithelial cells and surround fat cells, muscle fibers, and peripheral nerves (for a review see reference 64). Basement membranes are responsible for the maintenance and compartmentalization of tissue architecture and their status determines repair after injury. They also provide anchorage for adjacent cells and maintain their polarized and differentiated state. Other functions include the control of cell migration and invasion. Some specialized basement membranes (17) serve as a selective barrier in the filtration of macromolecules. Basement membranes are composed of glycoproteins, proteoglycans, and collagen. The best characterized components are laminin, nidogen/entactin, the low and high density forms of heparan sulfate proteoglycans, BM-40/SPARC/osteonectin, and collagen IV (for reviews see references 15, 33, 44, 64). Some biologically important functions of basement membranes have been attributed to individual components. Collagen IV forms network structures both in vitro (67, 70) and in vivo (69). These are believed to provide the scaffold onto which the other components assemble. Laminin is a large multidomain protein of crosslike shape with three short arms and one long arm composed of rodlike and globular structural elements (16). This elongated structure may allow the connection of various matrix components including a tight binding to nidogen (12, 45). Laminin is in addition a well-characterized cell-binding protein (33, 64). Cell adhesion was correlated with inner segments of the short arms (domain 1) and the peripheral portion of the long arm (domain 8) (7, 23, 65). Other important functions of laminin include promotion of neurite outgrowth (13) due to a site localized in the long arm (domain 8), glowth promotion (14, 28) and nidogen binding (45) localized in domain 1, and heparan sulfate binding localized in a globular structure at the tip of the long arm (domain 3) which is a substructure of domain 8 (43, 45). Cross-sectioned basement membranes are visualized at the EM level as continuous thin sheets with a rather amorphous appearance. The epithelial basement membrane of © The Rockefeller University Press, 0021-9525/88/10/1599/12 $2.00 The Journal of Cell Biology, Volume 107, October 1988 1599-161