The significance of the laminin/nidogen-1 interaction for basement membrane formation and stability in embryoid bodies

Basement membranes are thin layers of extracellular matrix which separate epithelial and endothelial cells from underlying connective tissue and surround nerve, muscle and fat cells. Biochemical data indicate the important role of nidogen-1, a 150kD sulfated glycoprotein, in supporting basement membrane stability by connecting the laminin and type IV collagen network (Aumailley et al., 1989; Yurchenco and Schittny, 1990). The aim of this study was to analyse the significance of the laminin/nidogen-1 interaction for basement membrane formation and stability in a more complex system. For that purpose F9 embryoid bodies, a cell culture model for basement membrane formation, were grown in the cell spin system after comparison with the conventional hanging drop method. F9 cells are mouse teratocarcinoma cells which differentiate and develop a basement membrane when cultured with retinoic acid. Recombinant expression of the nidogen-binding site, located in LE module 4 of the γ1 chain of the trimeric laminin molecule, as the γ1III3-5 fragment and its binding to free nidogen-1 molecules should interfere with the laminin/nidogen-1 interaction. As controls, F9 cells were transfected with the empty expression vector, the γ1V1-3 and the γ1III3-5mut fragment. γ1V1-3 is similar to the γ1III3-5 fragment in secondary structure and size while γ1III3-5mut carries a point mutation drastically reducing its affinity to nidogen-1. For detection of recombinant expression and for purification purposes all constructs had been modified with the FLAG peptide at their N-terminal end. Analysis of basement membrane formation by fluorescence microscopy revealed a different pattern in clones expressing the nidogen-binding site than in the controls. While cells carrying the empty expression vector and such expressing the γ1V1-3 or the γ1III3-5mut fragment developed embryoid bodies with completely or partially continuous basement membranes, only a punctate distribution of basement membrane components could be detected in cell aggregates expressing the nidogen-binding site. This indicated effective blocking of the laminin/nidogen-1 interaction by recombinant expression of the γ1III3-5 fragment. Additional studies on differentiation showed patchy distribution of cells expressing genes specific for visceral endoderm all over the embryoid body. In the controls these cells were organised into an peripheral epithelium which suggests a role of basement membranes in regulating the differentiation of these cells. To evaluate further functional aspects of the basement membrane discontinuity a diffusion assay was developed in which permeability properties could be tested. This revealed higher permeabilities among clones with comparable embryoid body morphologies when the basement membrane was disrupted. To exclude the possibility that endogenous recombinant expression or the genetic manipulation during transfection was responsible for the observed phenotypes, experiments were also performed with the extraneous addition of the recombinant nidogen-binding site. Wild type F9 cells were grown in suspension culture and treated with recombinantly expressed, affinity purified nidogen-binding site fragment. Microscopy demonstrated a similar basement membrane breakdown upon exogenous addition of the nidogen-binding site as observed before in embryoid bodies derived from F9 cells recombinantly expressing the γ1III3-5 fragment.