Active Negative Control of Collagen Fibrillogenesis in Vivo: INTRACELLULAR CLEAVAGE OF THE TYPE I PROCOLLAGEN PROPEPTIDES IN TENDON FIBROBLASTS WITHOUT INTRACELLULAR FIBRILS

Sally M Humphries,Yinhui Lu,Elizabeth G Canty,Karl E Kadler

doi:10.1074/jbc.m708198200

Abstract

It is established fact that type I collagen spontaneously self-assembles in vitro in the absence of cells or other macromolecules. Whether or not this is the situation in vivo was unknown. Recent evidence shows that intracellular cleavage of procollagen (the soluble precursor of collagen) to collagen can occur in embryonic tendon cells in vivo, and when this occurs, intracellular collagen fibrils are observed. A cause-and-effect relationship between intracellular collagen and intracellular fibrils was not established. Here we show that intracellular cleavage of procollagen to collagen occurs in postnatal murine tendon cells in situ. Pulse-chase analyses showed cleavage of procollagen to collagen via its two propeptide-retained intermediates. Furthermore, immunoelectron microscopy, using an antibody that recognizes the triple helical domain of collagen, shows collagen molecules in large-diameter transport compartments close to the plasma membrane. However, neither intracellular fibrils nor fibripositors (collagen fibril-containing plasma membrane protrusions) were observed. The results show that intracellular collagen occurs in murine tendon in the absence of intracellular fibrillogenesis and fibripositor formation. Furthermore, the results show that murine postnatal tendon cells have a high capacity to prevent intracellular collagen fibrillogenesis.

Highlights

The functional integrity of connective tissues is dependent upon the production of an extensive and highly organized network of extracellular collagen fibrils
Fibril diameter differences between C3H and C57BL/6 strains were not significant (p ϭ 0.579). These data show that differences in intracellular processing of type I procollagen in C57BL/6 tendons do not appear to affect collagen fibril diameter. It was in the 1970s that type I collagen was shown to be synthesized as a soluble precursor procollagen containing Nand C-terminal propeptides [8]
It has been shown that cleavage of the C-terminal propeptides by procollagen C-proteinase is essential for the assembly of collagen fibrils [9] and that, following cleavage, spontaneous fibrillogenesis occurs

Summary

Active Negative Control of Collagen Fibrillogenesis in Vivo

INTRACELLULAR CLEAVAGE OF THE TYPE I PROCOLLAGEN PROPEPTIDES IN TENDON FIBROBLASTS WITHOUT INTRACELLULAR FIBRILS*. It is established fact that type I collagen spontaneously selfassembles in vitro in the absence of cells or other macromolecules. We show that intracellular cleavage of procollagen to collagen occurs in postnatal murine tendon cells in situ. The results show that intracellular collagen occurs in murine tendon in the absence of intracellular fibrillogenesis and fibripositor formation. Actin-stabilized plasma membrane processes (fibripositors) protrude into the ECM to organize the newly assembled collagen fibrils in parallel alignment with the existing matrix [5, 6]. We show using pulsechase analysis of procollagen processing that the intracellular conversion of procollagen to collagen can occur within the cell during postnatal stages of tendon development in the absence of intracellular collagen fibrils and fibripositors. PNcollagen, procollagen lacking the C-propeptides but retaining the N-propeptides; *collagen, a collective term for all procollagen, pCcollagen, pNcollagen, and collagen molecules or polypeptide chains of these molecules

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION