Ultrastructural localization of the major components of the extracellular matrix in normal rat nerve.

In this study we determined the ultrastructural distribution of the various components of the extracellular matrix (laminin, fibronectin, Type I, III, and IV collagens) of the normal peripheral nerve in adult rat. The localization of these macromolecules was investigated in basement membranes as well as in different areas of epi-, peri-, and endoneurium, by use of a pre-embedding immunoperoxidase method.     

Motility of L 5222 rat leukemia cells in the flattened state. Evidence against emperipolesis.

Emperipolesis is the term for the assumed penetration of living cells into other living cells. As reported earlier, L 5222 rat leukemia cells, migrating in vitro, change from a spherical to a spread configuration when they meet flat cells, and continue to move in this shape within the contours of the target cells. Whether or not this close cellular association corresponded to emperipolesis could not be determined with phase and interference contrast cinemicrography alone. In combination with transmission electron microscopy, it could be demonstrated that the compartment, in which the spread leukemia cells move, is not the cytoplasm of the target cells, but the narrow space created by the target cells and the underlying glass surface. Thus, emperipolesis could be ruled out for L 5222 leukemia cells. On this basis the reported observations on emperipolesis are reviewed, and a critical attitude regarding the occurrence of emperiopolesis in general is advocated.     

Production of components of extracellular matrix by cultured rat Sertoli cells

Sertoli cells from rats aged 15, 20, and 25 days were cultured in plastic dishes and extracted with Triton X-100 (0.1 percent w/v) or sodium deoxycholate (2 percent w/v). Residues left after extraction were found to contain three proteins characteristic of extracellular matrix (fibronectin, collagen IV, and laminin). These proteins were identified by four methods: indirect immunofluorescence, co-migration with standard proteins on electrophoresis in polyacrylamide gels, immunoblotting (Western blots), and immunoprecipitation after incubating the Sertoli cells with [35S]methionine. In addition, fibronectin was identified by immunoelectron microscopy with a second antibody conjugated to colloidal gold. In the same cell residues, heparan sulfate was tentatively identified by the first of these methods. The cells used in these studies were shown, by electron microscopy, to be essentially pure cultures of Sertoli cells (greater than 95% pure). Since 100 percent of the cells examined showed positive and specific immunofluorescent staining with well-characterized antibodies to the four components of the extracellular matrix, and since studies with colloidal gold revealed the presence of fibronectin closely associated with and inside cells identified by electron microscopy as Sertoli cells, it must be concluded that Sertoli cells synthesize these four proteins and presumably heparan sulfate. Evidently, cultured Sertoli cells can synthesize and secrete some of the components of an extracellular matrix.     

Differential expression of extracellular matrix components in rat Sertoli cells.

We studied expression of laminin, fibronectin, and Type IV collagen in the testis by means of immunofluorescence and immunoblot analysis and also examined gene expression of fibronectin using the ribonuclease protection assay. By immunofluorescence on sections from 20-day-old rats, laminin, fibronectin, and Type IV collagen were found in the basement membrane of the seminiferous tubules and in the interstitial regions of the testis. No localization of any extracellular matrix components was found inside the sectioned cells. However, when Sertoli cells were cultured on glass coverslips, laminin and Type IV collagen were both found inside the cells, suggesting new synthesis. In cultured peritubular cells, Type IV collagen, laminin, and fibronectin were found within the cells. When examined by immunoblot analysis, freshly isolated Sertoli and peritubular cells from 20-day-old rats did not demonstrate production of laminin or fibronectin. After 5 days in culture, peritubular cells produced both laminin and fibronectin, whereas cultured Sertoli cells produced only laminin. In contrast, freshly isolated and cultured Sertoli and peritubular cells all produced Type IV collagen. Moreover, the ribonuclease protection assay indicated that the bulk of fibronectin gene expression occurs within the first 10 days of postnatal development, with lower maintenance levels occurring thereafter. These results indicate that in the testis the highest levels of expression of laminin and fibronectin occur during development and in primary cell culture, whereas expression of Type IV collagen is higher at later stages.     

Focal contacts: transmembrane links between the extracellular matrix and the cytoskeleton

The sites of tightest adhesion that form between cells and substrate surfaces in tissue culture are termed focal contacts. The external faces of focal contacts include specific receptors, belonging to the integrin family of proteins, for fibronectin and vitronectin, two common components of extracellular matrices. On the internal (cytoplasmic) side of focal contacts, several proteins, including talin and vinculin, mediate interactions with the actin filament bundles of the cytoskeleton. The changes that occur in focal contacts as a result of viral transformation are discussed.     

Organization of rat mesenteric artery after removal of cells or extracellular matrix components

Summary Rat mesenteric arteries, perfusion fixed in relaxed or contracted conditions, were digested with acid and elastase, bleach (sodium hypochlorite), or alkali to selectively remove collagen, elastin, or cells. Scanning electron microscopy was used to study the three-dimensional organization of the remaining cells or extracellular components. Smooth muscle cells of the tunica media were elongated and circumferentially oriented. Superior mesenteric artery cells had an irregular surface with numerous projections and some ends were forked. Small mesenteric artery cells were spindle shaped with longitudinal surface ridges, and showed extensive corrugations upon contraction. Elastin was present both as laminae and as an interconnected fibrous meshwork. Collagen was arranged in an irregular network of individual fibrils and small bundles of fibrils that formed nests around the cells in both arteries. This irregular arrangement persisted, with no apparent reordering or loss of order, upon contraction. The lack of an ordered arrangement or specialized organization at the cell ends suggests mechanical coupling of the cells to elastin or collagen throughout the length of the cell, allowing for force transmission in a number of directions. The tunica media is thus a composite material consisting of cells, elastin, and collagen. The isotropic network of fibers is well suited for transmitting the shearing forces placed on it by contraction of smooth muscle cells and by pressure-induced loading.     

Cooperativity between Sertoli cells and testicular peritubular cells in the production and deposition of extracellular matrix components

We examined the synthesis and deposition of extracellular matrix (ECM) components in cultures of Sertoli cells and testicular peritubular cells maintained alone or in contact with each other. Levels of soluble ECM components produced by populations of isolated Sertoli cells and testicular peritubular cells were determined quantitatively by competitive enzyme-linked immunoabsorbent assays, using antibodies shown to react specifically with Type I collagen, Type IV collagen, laminin, or fibronectin. Peritubular cells in monoculture released into the medium fibronectin (432 to 560 ng/microgram cell DNA per 48 h), Type I collagen (223 to 276 ng/microgram cell DNA per 48 h), and Type IV collagen (350 to 436 ng/microgram cell DNA per 48 h) during the initial six days of culture in serum-free medium. In contrast, Sertoli cells in monoculture released into the medium Type IV collagen (322 to 419 ng/microgram cell DNA per 48 h) but did not form detectable amounts of Type I collagen or fibronectin during the initial six days of culture. Neither cell type produced detectable quantities of soluble laminin. Immunocytochemical localization investigations demonstrated that peritubular cells in monoculture were positive for fibronectin, Type I collagen, and Type IV collagen but negative for laminin. In all monocultures most of the ECM components were intracellular, with scant deposition as extracellular fibrils. Sertoli cells were positive immunocytochemically for Type IV collagen and laminin but negative for fibronectin and Type I collagen. Co-cultures of peritubular cells and Sertoli cells resulted in interactions that quantitatively altered levels of soluble ECM components present in the medium. This was correlated with an increased deposition of ECM components in extracellular fibrils. The data correlated with an increased deposition of ECM components in extracellular fibrils. The data presented here we interpret to indicate that the two cell types in co-culture act cooperatively in the formation and deposition of ECM components. Results are discussed with respect to the nature of interactions between mesenchymal peritubular cell precursors and adjacent epithelial Sertoli cell precursors in the formation of the basal lamina of the seminiferous tubule.     

Suggestive evidence for a functional unit between mast cells and substance P fibers in the rat diaphragm and mesentery

Summary A close spatial relationship between serotonin-containing mast cells and substance P-containing nerves was shown by immunohistochemistry using a combination of antisera specific for serotonin and substance P. This supports earlier morphological results suggesting an innervation of mast cells and pharmacological studies which postulate an influence of substance P on the release of histamine from mast cells.International Research Fellow, awarded by Fogarty International Center, Fellowship number 1 FO5 TWO 3293-01 BI     

Suggestive evidence for a functional unit between mast cells and substance P fibers in the rat diaphragm and mesentery

A close spatial relationship between serotonin-containing mast cells and substance P-containing nerves was shown by immunohistochemistry using a combination of antisera specific for serotonin and substance P. This supports earlier morphological results suggesting an innervation of mast cells and pharmacological studies which postulate an influence of substance P on the release of histamine from mast cells.     

Roles of extracellular matrix components in differentiating teratocarcinoma cells

F9 embryonal carcinoma cells treated with 5 X 10(-8) M retinoic acid and cultured in suspension for 8 days form aggregates consisting of an outer epithelial layer of alpha-fetoprotein-producing visceral endoderm cells. We have previously shown (Grover, A., Oshima, R. G., and Adamson, E. D. (1983) J. Cell Biol. 96, 1690-1696) that the differentiation of F9 cells to visceral endoderm is accompanied by the activation of several genes, and increased laminin synthesis is one of the earliest events. Here we analyze in detail the syntheses and secretion of fibronectin, type IV collagen, and laminin during the 8-day process. Employing immunoprecipitation and enzyme-linked immunosorbent assay, we show that the levels of all three components change with different patterns. Unstimulated F9 cells synthesize and secrete relatively high levels of fibronectin and low levels of type IV collagen. Fibronectin synthesis and secretion decreases to 10% of its original level whereas type IV collagen synthesis rises approximately 3-fold during the differentiation process. Laminin synthesis also rises at least 2-fold, and the proportions of its subunits change as the syntheses of B1 and A accelerate starting on day 2. However, unlike fibronectin and type IV collagen, laminin is largely accumulated in the aggregates. The data suggest that fibronectin has a role in aggregation whereas laminin is important in the differentiation process.     

Deposition of extracellular matrix along the pathways of migrating fibroblasts

Summary Fibroblasts from rat, mouse and chick embryos cultured on poly-lysine/fibronectin- or poly-lysine/laminin-coated dishes were stained with antibodies directed to extracellular matrix molecules. The staining showed that cells had migrated during culture and deposited extracellular matrix components along their migration trails. Depending on the antigen, the staining of the matrix revealed fibrils, spots or a diffuse smear along the migration pathways. The major matrix components were fibronectin and heparan sulfate proteoglycan; however, laminin nidogen, tenascin, glia-derived nexin (GDN) and chondroitin-4-sulfate proteoglycan were also found. The migration trails were also detectable by scanning electron microscopy. Here, the fibrils were the prominent structures. The deposition of matrix was independent from the substratum: fibronectin was deposited on laminin, plain poly-lysine, basal lamina and even on fibronectin. Functional assays using anti-fibronectin or an antiserum to embryonic pigment epithelium basement membrane disturbed the formation of matrix fibrils, but did not inhibit cell attachment and translocation. Likewise, heparin in the culture medium only partially inhibited cell migration, despite the fact that it disturbed the formation of proper matrix fibrils. Our results suggest that the deposition of extracellular matrix by cells may not be mandatory for attachment and translocation. However, the deposition of matrix along defined trails might be important for the pathfinding of cells or nerve fibers that appear later in development.     

Regulation of rat mammary gene expression by extracellular matrix components

In the mammary gland the induction and maintenance of differentiation are dependent on both lactogenic hormones and the extracellular matrix (ECM). Since mammary epithelial cells differentiate on a basement membrane in vivo we have examined the effects of basement membrane components on the expression of milk protein genes in primary rat mammary cultures. We examined the effects of a basement membrane gel derived from the Englebreth-Holm-Swarm tumor as well as its major component, laminin, on the expression of a group of milk protein genes. We demonstrate that the basement membrane gel induces alpha-casein and alpha-lactalbumin (alpha-LA) accumulation up to 160- and 70-fold, respectively, of that on tissue culture plastic. Laminin, a major component of the basement membrane, also caused significant induction of these same proteins. In order to determine whether these ECM effects occurred at a translational or post-translational level, pulse-chase experiments were performed. These experiments demonstrated that a laminin substratum selectively effects milk protein turnover and secretion. In order to demonstrate whether ECM effects occurred at the level of steady state accumulation of mRNA we performed dot blot and Northern analyses using cloned cDNA probes for alpha-, beta-, and gamma-caseins and alpha-LA. These studies demonstrated that ECM components induced alpha- and beta-caseins up to 10-fold, and alpha-LA up to 3-fold, with no significant effect on gamma-casein. These results demonstrate that milk protein genes are not coordinately regulated by ECM components. Furthermore, since the amount of induction of milk proteins exceeds the amount of induction of mRNAs for these proteins, we conclude that in our system a major effect of ECM components is at the translational and/or post-translational levels. Based on these findings we propose a model in which basement membrane components effect mammary gene expression at multiple levels.     

Ultrastructural evidence for sinusoid spaces and coupling between pituicytes in the rat

Summary The pericapillary palisade of the rat neurohypophysis was examined by means of thin-section and freeze-etch electron-microscopy. Special attention was given to pituicyte processes intermingled with neurosecretory terminals. These processes are identified by the presence of lipid droplets and ribosomes.Extracellular spaces are conspicuously enlarged in circumscribed regions between fingerlike protrusions of pituicyte processes. Neurosecretory axons seem to have free access to these enlarged spaces. Zonulae occludentes often combined with small gap junctions are found at the border of these sinusoid spaces. Gap junctions and occasionally intermediate junctions are seen between pituicyte processes. The topographic relationship and the functional significance of these structural features remain to be further elucidated.Supported by Grants of the Dr. Eric Slack-Gyr Foundation, Zürich, the Swiss National Foundation for Scientific Research Nrs. 3.823.72, 3.774.72, 3.712.72 and 3.045.73, the EMDO-Foundation and the Hartmann-Müller-Foundation for Medical Research at the University of Zürich. A short account has been presented at the meeting of the Union of Swiss Societies for Experimental Biology, April 1975 (Experientia 1975, in press).     

TNF-alpha associated with extracellular matrix fibronectin provides a stop signal for chemotactically migrating T cells

The migration of T cells into extravascular sites of inflammation is regulated by information derived from the molecular structure of the invaded tissue and from chemokine and cytokine gradients in the context of the extracellular matrix (ECM). Although recent studies have highlighted the role of particular chemoattractants in leukocyte migration, to date little is known about how specific combinations of contextual signals control the migration of leukocytes and their localization at sites of inflammation. Here we studied the interplay between a pleiotropic cytokine, TNF-alpha, and two prototypic chemoattractants, RANTES and stromal cell-derived factor-1alpha (SDF-1alpha), on human CD45RO+ T cells migrating within an ECM-like context. For this purpose, we used a newly constructed three-dimensional gel system designed to follow, in real time, the migration of individual leukocytes along chemotactic gradients in vitro. We found that TNF-alpha, which binds the ECM protein fibronectin and lacks adhesion- and migration-promoting effects of its own, can act as a proadhesive cytokine on T cells exposed to RANTES and SDF-1alpha. Furthermore, fibronectin-complexed TNF-alpha provided anchorage signals to the T cells as they moved directionally along chemoattractive gradients. This effect of TNF-alpha required an intact TNF-alpha receptor II subtype on the migrating T cells. The anchoring effect of TNF-alpha appears to be specific; IL-2, an integrin-activating proadhesive cytokine, does not transmit stoppage signals to T cell migration induced by RANTES. Thus, TNF-alpha present in the ECM at sites of inflammation may function to anchor T cells recruited to these sites by chemotactic signals.     

Ultrastructural aspects of the production of extracellular matrix components by the chick embryonic notochord in vitro

The morphology of extracellular matrix (ECM) components and of the cell organelles, particularly the Golgi complex and its derived structures, implicated in the production of ECM in the chick embryonic notochord have been studied by transmission electron microscopy. Isolated notochordal fragments were cultured in suspension in liquid medium. Native striated collagen fibrils with a period of 540 A were observed in the perinotochordal sheath. Fine granular and filamentous materials suggestive of proteoglycans have been observed in intercellular spaces and under the basal lamina of the notochordal sheath. Golgi mature vesicles with structures resembling the previously described segment-long-spacing (SLS)-like aggregates and secretory vesicles probably containing proteoglycans or condensed collagen precursors have also been observed.