Showing papers by "Elizabeth D. Hay published in 1999"

Biogenesis and organization of extracellular matrix.

Abstract: It may come as a surprise to you that Keith Porter had a deep interest in the structure and biogenesis of extracellular matrix. As early as 1949, Porter and Vanamee began to use the electron microscope (EM) to follow the formation of extracellular matrix by fibroblasts (1), and, in 1959, Porter and George Pappas called attention to the close association with the cell surface of extracellular fibrils that they believed to be composed of collagen in the process of polymerizing on the fibroblast plasmalemma (2). Observations such as these (2–5) led Porter to propose and defend for some time the theory that collagen fibrils form by a process of ecdysis or direct shedding of cytoplasmic filaments through the plasmalemma onto the mesenchymal cell surface. This hypothesis required newly synthesized collagen to be released from ribosomes directly into the cytoplasm, rather then to take the route from the RER through the Golgi apparatus that was being demonstrated at the same time and the same institute for other secretory proteins by Porter’s colleagues, Philip Seikevitz and George Palade (6). Inspired by the latter study (6), Jean Paul Revel and I did an autoradiographic study at the EM level in 1963 using H proline as a precursor for collagen, a protein that consists of 25% proline and hydroxyproline (7). Our data showing tritium localization to the RER, followed by transport to the Golgi apparatus, were consistent with the idea that chondrocytes use the same secretory pathways as do pancreatic acinar cells, and, indeed, it was subsequently shown by EM immunohistochemistry that procollagen is present only in the RER, Golgi apparatus, and secretory vacuoles/vesicles of mesenchymal cells (8). While quantitation of autoradiographic studies of intracellular collagen transport proved difficult (9), the conclusion that the newly synthesized proline-rich products do not significantly accumulate on the cell surface was unquestionable. Rather, the tritium-labeled extracellular matrix proteins seem to diffuse through the cartilage matrix to polymerize some distance from the cells (7), and a similar sequence of events was shown by autoradiography for deposition of matrix by fibroblasts (9, 10) and odontoblasts (11, 12). Thus, it seems clear that newly forming collagen is not polymerizing on the plasmalemma of mesenchymal cells to any significant degree. What are we to conclude from the convincing demonstration by Porter and others of the close association of extracellular matrix fibrils with the fibroblast cell surface? Surely, what was being observed for the first time at the EM level was the phenomenon that we now call ‘cell-matrix interaction’ (13, 14). In oblique sections across the plasmalemma, the extracellular fibrils appear to be continuous with filamentous cortical cytoplasmic material (2–5), and this observation was extended by Hynes and Destree (15) using immunohistochemistry to demonstrate codistribution with intracellular actin of fibronectin fibrils on the fibroblast cell surface. The functional consequences of actin-matrix interaction were subsequently demonstrated in mesenchymal cells by Tomasek and Hay (16) and Harkin and Hay (17), and in epithelium by Sugrue and Hay (18) and others (13, 19). The major family of transmembrane extracellular matrix receptors that link actin filaments to the matrix are termed integrins, and they form cytoplasmic complexes with focal adhesion and Src kinases that transduce signals to the nucleus via MAP kinases and other proteins (19, 20). Thus, cell and extracellular matrix form a mutually dependent continuum in the body of the multicellular organism. The manner in which epithelial cells might control the polymerization of collagen fibrils at some distance from their cell surface was a subject that also intrigued Porter. In the 1950s, Paul Weiss called attention to the elegant orthogonal lattice of collagen fibrils in the acellular dermis created by the epidermis of aquatic vertebrates. Later, Porter extended this observation to include the subepidermal matrix of the lamprey eel and collagenous cuticle of the annelid worm (5, 13). We showed in 1963 by EM autoradiography that the newly synthesized collagen of the amphibian subepidermal orthogonal gridwork is deposited near the basal lamina (basement membrane) and is subsequently displaced inward by unlabeled matrix after the tritiated pulse has passed (21). Weiss et al. (13) proposed that a cell-switching mechanism allows each layer to be deposited in a plywood fashion at right angles to the one previously formed, but Porter pointed out the many shortcomings of this hypothesis. With his colleagues, Joe Nadol and John Gibbons, he proposed in 1969 (22) the ‘shingle’ or ‘scindulene’ hypothesis,