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Address correspondence to Karl E. Kadler, Wellcome Trust Centre for Cell-Matrix Research, School of Biological Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT UK. Tel.: 44-161-275-5086. Fax: 44-161-275-1505. email: karl.kadler{at}man.ac.uk
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Abstract |
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Key Words: 3-D reconstruction; collagen; fibril; GPC; procollagen
Abbreviations used in this paper: 3-D, three-dimensional; ADAMTS, a disintegrin and metalloprotease (reprolysin type) with thrombospondin motifs; BMP, bone morphogenetic protein; dpc, days post coital; GPC, Golgi to plasma membrane carrier; GPC+cf, GPC containing one or more 67-nm periodic collagen fibrils; pCcollagen, a naturally occurring intermediate in the cleavage of procollagen to collagen that contains the C-propeptides and not the N-propeptides; PM, plasma membrane; pNcollagen, a naturally occurring intermediate in the cleavage of procollagen to collagen that contains the N-propeptides and not the C-propeptides.
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Introduction |
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A primary function of the secretory pathway is to transport macromolecules from their site of synthesis in the ER to the plasma membrane (PM). A critically important node in this pathway is the Golgi complex with its associated TGN consisting of a complex network of anastomosing tubules (Rambourg et al., 1979). The TGN mediates the final modification of N-linked oligosaccharides to the complex form (Roth et al., 1985) and is involved in both the transport and sorting of membrane and secretory proteins (Griffiths and Simons, 1986; Taatjes and Roth, 1986; Orci et al., 1987). Previous works have shown that GFP fusion proteins are transported from the Golgi to the cell surface in tubular-saccular compartments, which travel along microtubules (Sciaky et al., 1997; Hirschberg et al., 1998; Toomre et al., 1999; Polishchuk et al., 2000; Puertollano et al., 2003). (In this paper, the authors distinguish between vesicles and compartments or carriers. The terms "compartment" and "carrier" refer to any membrane-bound transport container within the cell, regardless of its size, position, or function in the secretory pathway. The term "vesicle" refers to any compartment that is spherical or near spherical in shape, regardless of its size, position, or function in the secretory pathway.) These pleiomorphic Golgi to PM carriers (GPCs) can be 0.51.7 µm in length (Polishchuk et al., 2000) and have also been called transport containers (Toomre et al., 1999) and post-Golgi carriers (Hirschberg et al., 1998). A recent in vitro study has shown that exit from the TGN occurs by the formation of a tubular-reticular TGN domain that is a precursor structure to the release of tubular-saccular GPCs (Polishchuk et al., 2003).
Tendon fibrils are predominately comprised of collagen I, which is the most abundant collagen in vertebrates (Boot-Handford et al., 2003). It is synthesized in the ER as procollagen I, which comprises two pro1(I) chains and one pro
2(I) chains folded into an uninterrupted 300-nm-long triple helix flanked by globular N- and C-propeptides. Procollagen molecules are too large to fit into conventional 6080-nm transport vesicles and traverse the Golgi complex of these cells by cisternal maturation (Bonfanti et al., 1998). We were particularly interested to know if procollagen occurs in GPCs en route to the ECM. Of particular relevance to this question is that proteolytic cleavage of the N- and C-propeptides results in spontaneous collagen fibril formation. N-propeptide removal is catalyzed by the procollagen N-proteinases, which include a disintegrin and metalloprotease (reprolysin type) with thrombospondin motifs (ADAMTS)-2, -3, and -14 (Colige et al., 1997, 2002; Fernandes et al., 2001), whereas C-proteinase activity is possessed by all members of the tolloid family of zinc metalloproteinases including bone morphogenetic protein-1 (BMP-1; Scott et al., 1999). Both BMP-1 and ADAMTS-2 are activated by a furin-like proprotein convertase, and in the case of proBMP-1, activation has been shown to occur in the TGN (Leighton and Kadler, 2003; Wang et al., 2003). Furin itself undergoes autocatalytic activation and is thought to cycle between the TGN, the cell surface, and the endosomal system (Molloy et al., 1999; Thomas, 2002).
Seminal studies in the early 1980s by Birk, Trelstad, and coworkers (Trelstad and Hayashi, 1979; Birk and Trelstad, 1984, 1986) suggested that collagen fibrils occur in deep PM recesses and that the recesses increase in diameter at distances from the cell to accommodate fibril bundles. Evidence from EM autoradiography indicated that newly synthesized collagen molecules pass through these recesses en route to the ECM. However, recent findings that proBMP-1 is converted to BMP-1 in the TGN, as well as studies of GPCs in cultured cells, prompted us to relate these new observations to the description of fibril formation in embryonic tendon described by Trelstad and colleagues (see references above). Here, we show that GPCs are indeed present in embryonic tendon fibroblasts and that some GPCs contain 28-nm-diam collagen fibrils (GPCs+cf ). Moreover, GCPs+cf are targeted to novel PM protrusions, which we have termed "fibripositors" (fibril depositors). In addition, we show that procollagen can be converted to collagen within the confines of the cell membrane, which is consistent with the observation of collagen fibrils in some GPCs and the known intracellular activation of BMP-1. A novel observation was that fibripositors are always oriented along the tendon axis, which establishes a link between intracellular transport and the organization of the ECM. Interestingly, fibripositor formation is not a constitutive process in procollagen-secreting cells but occurs only during a narrow window of embryonic development when tissue architecture is being established.
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Results |
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To confirm that cleavage of procollagen did not occur during the extraction procedure, tendons were subjected to the same extraction protocols as above but in the presence of partially purified 14C-labeled procollagen. No processing of pro1(I) and pro
2(I) chains occurred (Fig. 2 C), indicating that the labeled procollagen in the pulse-chase experiment must have been processed before extraction. To further confirm that intracellular collagen molecules could only be removed by extraction with buffer containing NP-40, pulse-chase experiments were performed in the presence of
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-dipyridyl and brefeldin A. These treatments are both known to result in the accumulation of procollagen within the ER. Two successive NaCl extractions (S1 and S2) were performed before the NP-40 extraction (N), which was found to contain only unprocessed procollagen (unpublished data). These results are consistent with cleavage of procollagen to collagen occurring in post-Golgi compartments and the results of EM showing collagen fibrils in GPCs+cf.
Appearance of PM protrusions coincides with onset of post-Golgi collagen fibril polymerization in 14.5-d mouse tail tendon
Transverse sections through the presumptive tail tendon of 13.5 dpc (days post coital) mouse embryos showed closely packed and apparently undifferentiated cells, which lacked evidence of GPCs+cf or a fibrillar ECM (Fig. 3 A). However, cells in the proximal region of tails from 14.5 dpc embryos were loosely packed and had large extracellular spaces that contained numerous collagen fibrils. Cellular projections (Fig. 3 B, open arrows) were an obvious feature at this stage of development. Some of the projections contained tubular carriers in which collagen fibrils were clearly visible (Fig. 3 B, closed arrows). Analysis of the distal region of the same tails showed spaces between cells and a conspicuous absence of collagen fibrils and cellular projections, indicating that the development of mouse tail tendon proceeds from the proximal to the distal end. Therefore, the occurrence of parallel collagen fibrils in the ECM coincided with the appearance of cellular projections having GPCs+cf.
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3-D reconstruction showed that tendon fibroblasts are roughly cylindrical in shape with their long axis parallel to the axis of the tendon. Moreover, the PM adopts a novel conformation to generate cylindrical channels that are parallel to the tendon-long axis. The fibripositors were also parallel to the long axis of the tendon and to the collagen fibril bundles (Figs. 6 and 7). Fibripositors projected into channels that were formed by close contacts between the PMs of adjacent cells (Fig. 7 B, open circles). The longitudinal axes of channels, which had smooth concave surfaces, were always parallel to the tendon-long axis (Fig. 7, A and B). Channels presumably provide a confined environment for the supramolecular organization of collagen fibrils into parallel bundles. Fibrils were observed exiting fibripositors in either direction along the tendon axis and branched fibripositors and branched lumen were also observed (Fig. 7 D).
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Discussion |
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This work has shown that processing of procollagen to collagen begins in Golgi to PM compartments that are refractory to extraction with high salt buffer but which can be solubilized using NP-40 detergent, suggesting that they are within the cell. In addition, we have shown that GPCs+cf are tubular in shape and are completely enclosed within the fibroblast PM. A question of interest is, once GPCs+cf have fused to the PM to form a fibripositor, is the lumen of the fibripositor accessible to salt extraction buffer or does it require NP-40 for solubilization? Incubation of tendons with HRP indicates that diffusion of exogenous molecules (such as trypsin, HRP, and salt ions) into the lumen does occur (Fig. 8 B). Some compartments did not contain DAB-reactive HRP, which is consistent with the presence of GPCs+cf that are enclosed within the cell. It is unlikely that fibripositor lumen close during the extraction process and then become refractory to salt extraction because the compartments that are accessible to salt extraction are also accessible to trypsin (Fig. 2 B). Trypsin digestion was performed directly after labeling and before the salt extractions when tissue shrinkage might be expected to occur. Therefore, it is likely that the material in the NP-40 extract is derived from ER, Golgi, GPCs+cf, and potentially from material at the very base of the fibripositor lumen where short early fibrils are found (Fig. 5 B). pNcollagen was absent from the salt extract indicating that the N-propeptides are removed before secretion into the lumen of the fibripositor or ECM and that pCcollagen is the major intermediate used for the extension (embryonic) or broadening (6 wk) of pre-existing fibrils.
Two lines of evidence indicate that collagen fibril polymerization during embryogenesis begins in the TGN or in TGN exit sites, although future studies are needed to identify the origin of collagen-fibril-containing vesicles and precursors of the GPCs+cf. First, GPCs+cf near to the Golgi stacks contained cross-banded collagen fibrils. Second, processing of procollagen to collagen was completely prevented in the presence of brefeldin A. The idea that intracellular procollagen processing could be mediated by N- and C-proteinases, which are concomitantly synthesized and trafficked with procollagen is supported by recent work in our laboratory showing intracellular activation of BMP1 in the TGN (Leighton and Kadler, 2003). Cleavage of procollagen would require neutral pH and a concentration of free calcium ions between 2 and 5 mM (Hojima et al., 1985). Alternatively, the procollagen proteinases could be targeted to the ECM and/or the base of the fibripositors and a cycling mechanism, similar to that used by furin (Molloy et al., 1999), could be used to localize the enzymes to the transface of the TGN. Fusion of procollagen containing GPCs with vesicles containing the processing enzymes would then trigger fibrillogenesis and the formation of new GPCs+cf.
At 6 wk of development very little cleavage of procollagen to collagen occurs within the cell: the NP-40soluble compartments contain procollagen and pCcollagen, whereas the NaCl-soluble compartments contain pCcollagen and collagen. No GPCs+cf are observed despite immunolocalization of triple helical collagen to GPCs. Unfortunately, it is not possible to determine which intermediates are present within the GPCs because antibodies that are directed to the collagen triple helix, terminal propeptides, or cleaved neoepitopes will inevitably recognize at least two procollagen intermediates. Further studies are needed to explain the observed difference of N- and C-proteinase activity in embryonic and older tendon fibroblasts. The low abundance and high sequence homology between the various gene products has so far complicated the use of specific antibodies for immunolocalization studies.
Seed and feed mechanism of ECM assembly
Evidence from in vitro studies indicates that collagen fibrillogenesis is a nucleation-propagation process in which the formation of a thermodynamically unstable nucleus occurs slowly but once formed, the nucleus propagates rapidly in size by accretion of collagen molecules (Wood and Keech, 1960; Holmes and Chapman, 1979; Kadler et al., 1987, 1990; Silver et al., 1992). This assembly mechanism predicts the formation of a nucleus having a high fidelity structure because it contains the structural blueprint for the final fibril. The data from embryonic tendon showing the formation of early fibrils at the base of fibripositors, and, the fact that the fibrils exhibit the same diameter as the fibrils in the ECM, are strongly suggestive that a nucleation-propagation assembly mechanism occurs in vivo. An important observation was that fibrils exceeding 10 µm in length could be traced from the center of a fiber bundle within the ECM to the lumen of a fibripositor deep within the cell. There is no evidence that collagen fibrils of this length can be synthesized de novo and deposited whole. Thus, we propose that the nucleation step occurs in GPCs and at the base of the fibripositors, at least in embryonic fibroblasts in situ. Further studies are needed to identify the site of fibril propagation, although the pulse-chase observations of pCcollagen being cleaved to collagen in a NaCl-extractable compartment are consistent with propagation occurring in the ECM or in open fibripositors. The increase in fibril diameter between embryonic and 6-wk stages of development and the subsequent absence of GPCs+cf is consistent with switching off of the nucleation step of fibrillogenesis at postnatal stages (Fig. 10, schematic). The results of immunoEM at 6 wk unequivocally showed the presence of procollagen and/or pCcollagen in 350-nm-diam compartments, which presumably are responsible for delivering procollagen and pCcollagen to the ECM.
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Perspectives
The 3-D serial section reconstruction studies by Trelstad and colleagues (Trelstad and Hayashi, 1979; Birk and Trelstad, 1984, 1986) in the 1970s and 80s used high voltage transmission electron microscopes to image relatively thick sections. These approaches were so far beyond the technical expertise of most collagen biologists at the time, and the data were so compelling, that few people contemplated extending these studies. However, recent development of state-of-the-art software such as IMOD has made it possible to revisit this approach using larger numbers of thinner sections imaged on a conventional transmission electron microscope. The results of our work and those obtained by Trelstad, Birk, and colleagues differ in several respects (see references above). First, it was speculated that only uncleaved procollagen was transported to the PM recesses. Our results show that procollagen can be converted to collagen within the cell and that fibril formation can occur in closed intracellular carriers. Furthermore, the recesses were envisioned to broaden at increasing distances from the body of the cell, and it was in these wider zones that fibrils formed into bundles. Our results show that the recesses are long channels in the PM that do not protrude into the cell but run down the surface of the cell. We also show that fibroblasts exhibit fibripositors that are finger-like protrusions from the PM.
Although the study of the cellular aspects of tissue assembly has been simplified by improvements in image analysis software such as IMOD, there remains a major technical hurdle concerning the cells. Fibroblasts flatten when plated out in culture and consequently the PM channels and fibripositors disappear. It is possible to culture whole tendons for up to 3 h and still observe normal procollagen processing but beyond this time the cells lose their embryonic phenotype. Therefore, dissection of the molecular mechanisms involved in PM channel formation and fibripositor biogenesis will rely on the development of novel cell or organ culture methods that preserve the 3-D shape of the cell and provide additional signals needed to specify continued cell differentiation.
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Materials and methods |
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Protein production, purification, and Western blotting
14C-Labeled procollagen I was purified from human skin fibroblasts as described previously (Kadler et al., 1987). Recombinant human BMP-1 was prepared as described previously (Hartigan et al., 2003). Recombinant ADAMTS-2 was obtained from 293-EBNA cells transfected with a cDNA encoding ADAMTS-2 (a gift from A. Colige, Universite de Liege, Liege, Belgium). Protein extracts were examined by standard Western blot procedures and optimal antibody dilutions determined empirically. Anticalnexin CT, antimembrin and anti-hsp47 antibodies were purchased from StressGen Biotechnologies. Anti-BiP and anticaveolin antibodies were purchased from Santa Cruz. The anti-ß1 integrin antibody was from Sigma-Aldrich.
EM
Freshly dissected chick metatarsal tendons were cut into 3-mm lengths and frozen to 196°C using an EM PACT high pressure freezer (Leica). Freeze substitution for ultrastructure was performed using an AFS system (Leica), starting at 90°C in 2% wt/vol osmium tetroxide in actone, going through pure acetone at 50°C and ending in several changes of Spurr's resin (Spurr, 1969). at 20°C. Polymerization in fresh resin was then performed at 60°C for 24 h. Freeze substitution for immunolabeling was performed using an AFS system (Leica) using pure acetone at 90°C, pure ethanol at 50°C in ethanol, and ending in several changes of HM20 Lowicryl resin at 50°C. UV polymerization in fresh resin was then performed at 50°C for 48 h and continued at 20°C for 48 h.
Embryonic mouse tails were fixed in 2% glutaraldehyde in 100 mM phosphate buffer, pH 7.0, for 30 min at RT. The tails were then diced and fixed for 2 h at 4°C in fresh fixative. After washing in 200 mM phosphate buffer they were fixed after in 1% glutaraldehyde and 1% OsO4 in 50 mM phosphate buffer, pH 6.2, for 40 min at 4°C. After a rinse in distilled water they were en bloc stained with 1% aqueous uranyl acetate for 16 h at 4°C, dehydrated and embedded in Spurrs' resin.
Ultra-thin sections for normal transmission electron microscopy were collected on uncoated copper 200 grids, serial sections for 3-D reconstruction on formvar-coated copper 1,000 µm slot grids (stabilized with carbon film) and ultra-thin sections (60 nm) for immunolabeling on formvar-coated nickel 400 grids. A postembedding labeling technique was used to detect type I collagen using a rabbit antichicken collagen-I antibody (Biodesign International) at a dilution of 1:500 followed by a gold-conjugated goat antirabbit antibody (British Biocell International) at a dilution of 1:200. All sections were subsequently stained with uranyl acetate and lead citrate, and examined using either a JEOL 1200EX, Philips EM 400, or Philips BioTwin transmission electron microscope. Images were recorded on 4489 film (Kodak) and scanned using an Imacon Flextight 848 scanner (Precision Camera & Video). Images from EM serial sections were aligned and reconstructed in IMOD for Linux (Kremer et al., 1996) and visualized using OpenSynu for Linux (Hessler et al., 1992).
Online supplemental material
Experimental procedures are available online concerning the differential extraction of secretory pathway proteins and ECM proteins. Fig. S1 depicts the differential extraction of ECM proteins and intracellular proteins. Methods were developed that facilitated the differential extraction of ECM proteins and proteins enclosed within membrane-bound compartments. Proteins in the ECM were solubilized in neutral buffers at 4°C, whereas those in membrane compartments were subsequently extracted in buffers containing NP-40. EM was used to examine the ultrastructure of the cells after each extraction. Western blot analysis was used to examine the protein composition of each extract. Video 1 depicts a 3-D reconstruction of part of a tendon fiber that runs down the mouse tail showing cells and associated fibripositors. Cells are color rendered. Some fibripositors are shown in yellow. The video shows that the cells are cylindrical in shape. Cylindrical channels occur between cells. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200312071/DC1.
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Acknowledgments |
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The work was supported by grants from The Wellcome Trust and the BBSRC (JREI fund), as well as a research collaborative grant from the EU (Framework 5). The EM was carried out in the EM Unit, School of Biological Sciences, University of Manchester, Manchester, UK.
Submitted: 10 December 2003
Accepted: 19 April 2004
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References |
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