Electron-Microscopic Studies on a Double-Stranded Beaded Filament of Embryonic Collagen
Beaded filaments consisting of paired 40-Å strands in parallel coupled by 150-Å beads at periodic spacings of 640 Å have been isolated from embryonic pig dermis. These filaments are found in association with embryonic collagenous fibrils and exclusively at sites of active fibrillogenesis of collagen. They have been identified within sectioned native fibrils as well as in interfibrillar regions. Furthermore, they have been recovered from fibrils denatured by exposure to alkalinity or to heat. In those instances, the filaments are packed in parallel array and the component beads are registered to produce a banding pattern with a major 640 Å repeat. The strands of the complex are composed of non-rigid fibrous protein as deduced from their fixation with aldehyde fixatives and from their degradation by urea. The beads are also proteinaceous, as indicated by their susceptibility to tryptic digestion. Moreover, these beads contain lipid, since they stain with lipid stains and dissolve in lipid solvents. Additional evidence suggests the inclusion of polysaccharide in the beads: periodic acid destroys these structures, and in native fibrils silver staining for carbohydrate occurs at loci of registered beads. The structural integrity of the conjugated protein bead does not depend upon the lipid or polysaccharide components, since lipases or β-amylase do not digest the structures. The filament proper is collagenase-resistant, but trypsin-sensitive. The latter enzyme bisects the beaded filament, forming single-stranded filaments with alternating 640-Å and 200-Å segments between beads. The double-stranded filaments are soluble in acid solutions below pH 4 but are stable at higher pH's through 9-10. The beaded filament fits into the scheme of extracellular collagen fibrillogenesis as the primary stable fibril or protofibril of collagen. It is presented as a doublet of procollagen fibrils which serves as a template for the polymerization of tropocollagen into additional beaded filaments. Aligned in parallel array and in register as they are assembled, the aggregate of beaded filaments is built into an immature fibril. By the establishment of interfilamentous cross-links, the individuality of the beaded filament is lost within the progressively maturing fibril.