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 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.

Actin filaments are required for fibripositor-mediated collagen fibril alignment in tendon.

Cells in tendon deposit parallel arrays of collagen fibrils to form a functional tissue, but how this is achieved is unknown. The cellular mechanism is thought to involve the formation of intracellular collagen fibrils within Golgi to plasma membrane carriers. This is facilitated by the intracellular processing of procollagen to collagen by members of the tolloid and ADAMTS families of enzymes. The carriers subsequently connect to the extracellular matrix via finger-like projections of the plasma membrane, known as fibripositors. In this study we have shown, using three-dimensional electron microscopy, the alignment of fibripositors with intracellular fibrils as well as an orientated cable of actin filaments lining the cytosolic face of a fibripositor. To demonstrate a specific role for the cytoskeleton in coordinating extracellular matrix assembly, cytochalasin was used to disassemble actin filaments and nocodazole or colchicine were used to disrupt microtubules. Microtubule disruption delayed procollagen transport through the secretory pathway, but fibripositor numbers were unaffected. Actin filament disassembly resulted in rapid loss of fibripositors and a subsequent disappearance of intracellular fibrils. Procollagen secretion or processing was not affected by cytochalasin treatment, but the parallelism of extracellular collagen fibrils was altered. In this case a significant proportion of collagen fibrils were found to no longer be orientated with the long axis of the tendon. The results suggest an important role for the actin cytoskeleton in the alignment and organization of the collagenous extracellular matrix in embryonic tendon.

A complete domain structure of Drosophila tolloid is required for cleavage of short gastrulation.

Drosophila tolloid (TLD) is a member of a family of proteinases that play important roles in development and includes mammalian tolloid (mTLD) and bone morphogenetic protein (BMP)-1. TLD accentuates the activity of decapentaplegic (DPP), a transforming growth factor beta superfamily growth factor, by cleaving its antagonist Short gastrulation (Sog). Similarly, the activity of BMP-2/4 (vertebrate homologues of DPP) is augmented by cleavage of chordin. However, whereas TLD is an effective Sogase, mTLD is a poor chordinase and is functionally replaced by its smaller splice variant BMP-1, which lacks the most C-terminal epidermal growth factor (EGF)-like and CUB domains of mTLD. Moreover, the minimal chordinase activity resides in the N-terminal half of BMP-1. This study showed that the proteolytic activity of TLD is considerably enhanced by Ca2+ and tested the hypothesis that the Sogase activity of TLD resides in the N-terminal half of the proteinase. Unexpectedly, it was found that TLD lacking the CUB4 and CUB5 domains and/or the EGF-like domains was unable to cleave Sog. Loss of function mutations have been reported in the tld gene that result in amino acid substitutions at E835K (in CUB4), S915L (in CUB5), and N760I (in EGF2) in TLD. The CUB mutants were found to be ineffective Sogases, but the activity of the EGF2 mutant was unchanged. The results show that substrate recognition and cleavage by Drosophila tolloid and mTLD are different despite their identical domain structure and homologous functions in patterning. The result that the N760I mutant has full Sogase activity suggests that novel substrates for TLD exist.

Procollagen trafficking, processing and fibrillogenesis.

Collagen fibrils in the extracellular matrix allow connective tissues such as tendon, skin and bone to withstand tensile forces. The fibrils are indeterminate in length, insoluble and form elaborate three-dimensional arrays that extend over numerous cell lengths. Studies of the molecular basis of collagen fibrillogenesis have provided insight into the trafficking of procollagen (the precursor of collagen) through the cellular secretory pathway, the conversion of procollagen to collagen by the procollagen metalloproteinases, and the directional deposition of fibrils involving the plasma membrane and late secretory pathway. Fibril-associated molecules are targeted to the surface of collagen fibrils, and these molecules play an important role in regulating the diameter and interactions between the fibrils.

Coalignment of plasma membrane channels and protrusions (fibripositors) specifies the parallelism of tendon.

The functional properties of tendon require an extracellular matrix (ECM) rich in elongated collagen fibrils in parallel register. We sought to understand how embryonic fibroblasts elaborate this exquisite arrangement of fibrils. We show that procollagen processing and collagen fibrillogenesis are initiated in Golgi to plasma membrane carriers (GPCs). These carriers and their cargo of 28-nm-diam fibrils are targeted to previously unidentified plasma membrane (PM) protrusions (here designated "fibripositors") that are parallel to the tendon axis and project into parallel channels between cells. The base of the fibripositor lumen (buried several microns within the cell) is a nucleation site of collagen fibrillogenesis. The tip of the fibripositor is the site of fibril deposition to the ECM. Fibripositors are absent at postnatal stages when fibrils increase in diameter by accretion of extracellular collagen, thereby maintaining parallelism of the tendon. Thus, we show that the parallelism of tendon is determined by the late secretory pathway and interaction of adjacent PMs to form extracellular channels.