The ultrastructural composition of basement membranes in vivo
The ultrastructure of basement membranes has a homogeneous appearance. The enormous cell biological importance of basement membranes and their components f o r c e l l proliferation, migration and differentiation implies that their composition is more complex than their structure suggests. To elucidate the molecular composition of basement membranes in vivo, we optimised immunogold histochemistry to allow the determination of the molecular arrangement of matrix molecules.
Basically, we apply a mild fixation and embed the tissues in the hydrophilic LR-Gold. This reserves the basement membrane with a quality similar to freeze substitution. The application of two antibodies directed toward the C- and N-terminal ends of a molecule and coupled to gold particles of different sizes allows determination of the orientation of a molecule within the basement membrane. We were able to demonstrate that the molecular orientation of the laminin-l molecule changes in the basement membrane according to cell biological needs. We also showed that ultrastructurally identical basement membranes like the ones of the proximal and distal tubules of the kidney have a differing molecular arrangement. Integrin α7 influences the molecular composition of the basement membranes at the myotendinous junction. With the help of double labelling at the ultrastructural level we could show that nidogen-l is CO-localised with laminin-l and only found in fully developed, mature basement membranes. In general, laminin-l, nidogen-l and collagen type IV are localised over the entire width of basement membranes, with laminin-l and nidogen-l CO-localised, in accordance with the current basement membrane models.
Incidentally, our investigations warn us, that not every matrix protein found at the light microscopic level as a linear staining pattern underneath an epithelium (basement membrane zone) is a real basement membrane component when investigated at the ultrastructural level. Instead, one and the same molecule, e.g. endostatin, can be a basement membrane component in one organ and a matrix molecule in another.