The ultrastructural organization and properties of the mouse tectorial membrane matrix

The polyphenolic compound tannic acid and the cationic stains ruthenium red, Alcian blue and lanthanum chloride have been used to reinvestigate the ultrastructural organization of the tectorial membrane matrix. Tannic acid treatment reveals that the matrix both in between and within the Type A protofibril bundle system has a high degree of structural organization. The basic unit of this matrix is best described as a 'striated sheet'. These striated sheets are formed by alternating 'dark' and 'light' fibrils which run parallel to one another and lie within the plane of each sheet. In sodium based buffers both light and dark fibrils have diameters of approximately 7 nm and the distance between each dark fibril in a sheet varies from 30 to 46 nm. Dark and light fibrils are coupled by periodic, staggered cross-bridges which occur at approximately 12 nm intervals along the fibrils. Fibril diameters in tectorial membranes prepared and fixed in potassium based buffers are from 10-20% greater than they are in tectorial membranes prepared and fixed in sodium based buffers. Fine fibrils can also be resolved in the matrix with the cationic stains lanthanum chloride and ruthenium red, but the organization of these fibrils into a regular matrix structure is most clearly resolved with tannic acid treatment. The striated sheets are largely destroyed by treating the tectorial membranes with neutral trypsin and are insensitive to treatment with bacterial collagenase. In contrast, the Type A protofibril system is trypsin resistant and collagenase sensitive. Treatment of tectorial membranes with salt solutions containing either 5 nM EDTA or 5 mM EGTA and 2 mM MgCl2 results in a complete loss of organized striated sheets and the appearance of randomly dispersed fibrillar material and small particles. Re-addition of Ca2+ ions causes the striated sheets to reform, indicating that the structure can undergo at least one cycle of depolymerization and polymerization in vitro. Reduction of disulphide bonds with beta-mercaptoethanol causes a loss of structural organization similar to that observed after EDTA or EGTA treatment. The results demonstrate that the non-collagenous components of the tectorial form a matrix with a degree of organization that has been previously unrecognised.