We have determined the structure of plasma fibronectin by electron microscopy of Shadowed specimens . The 440,000 molecular weight, dimeric molecule appears to be a long, thin, highly flexible strand . The contour length of the most extended molecules is 160 nm, but a distribution of lengths down to 120 nm was observed, indicating flexibility in extension as well as in bending. The average diameter of the strand is 2 nm and there are no large globular domains. The large fragments produced by limited digestion with plasmin are not globular domains but are segments of the strand, whose length corresponds to the molecular weight of the polypeptide chain. We conclude that each polypeptide chain of the dimeric molecule spans half the length of the strand, with their carboxyl termini joined at the center of the strand and their amino termini at the ends . This model is supported by images of fibronectinfibrinogen complexes, in which the fibrinogen is always attached to an end of the fibronectin strand .Fibronectin is a high molecular weight glycoprotein that is found in a soluble form in blood and other extracellular tissue fluids, and in an insoluble form in connective tissues and attached to cell surfaces . Fibronectin is thought to mediate the attachment of cells to the other components of the extracellular matrix, in particular to collagen and, where it occurs, fibrinogen or fibrin (for reviews see 14,16,22,26). Both plasma fibronectin and the cell surface or extracellular matrix forms are dimers, comprising two subunits of 220,000 mol wt, covalently linked by a single disulfide bond near their carboxyl termini . The two polypeptide chains are identical by most criteria but are frequently separated as a closely spaced doublet in gel electrophoresis ; the basis for this separation is not known (11) . Cell surface fibronectin differs from plasma fibronectin in carbohydrate content and solubility and exists as oligomers of the basic dimeric molecule, but the two forms are very similar in amino acid composition and are immunologically indistinguishable (14) .Plasma fibronectin has a sedimentation coefficient of 8 to 13S, depending on the pH and ionic strength (1) . This is too small for a globular protein of 440,000 mol wt, and suggests that the molecule has an elongated shape (1) . Studies with proteases have shown that fibronectin can be cleaved into a number of large fragments or domains, and it has been found that the different binding functions are retained by the separated domains . By analogy with the well established trinodular THE JOURNAL OF CELL BIOLOGY " VOLUME 91 DECEMBER 1981 673-678 © The Rockefeller University Press -0021-9525/81/12/0673/06 $1 .00 structure of fibrinogen, it has been suggested (1, 16) that fibronectin may have a nodular structure, consisting of large globular domains connected by flexible linking segments that can be readily attacked by proteases .Techniques of electron microscopy are now well established for determining the structure of large proteins . Molecular structures de...

We identified the two-stranded fibrin protofibril and studied its structure in electron micrographs of negatively stained specimens. Based on these images and on considerations of symmetry, we constructed a model of the protofibril in which the two strands oftrinodular fibrin molecules are related by a twofold screw axis between the strands and two-fold axes perpendicular to them. The two strands are held together by staggered lateral contacts between the central nodules of one strand and outer nodules of the other. The molecules within a strand are joined by longitudinal contacts between outer nodules. This interpretation of the structure of protofibrils is supported by images of trimer complexes whose preparation and structure are described here, in which the central nodule of a fibrin monomer is attached to the crosslinked outer nodules of two other molecules. We conclude that the association of protofibrils to form thicker fibers must involve a second type of lateral contact, probably between outer nodules ofadjacent, in-register strands. In total, we identify three intermolecular contacts involved in the polymerization of fibrin.The plasma protein fibrinogen, the major structural component of the blood clot, is an elongated molecule whose shape is a trinodular rod, 45 nm long (1, 2). Evidence has been presented recently for a finer subdivision into seven nodules (3), but the description of two 7-nm-diameter outer nodules connected by thin linking rods to a 5-nm-diameter central nodule is sufficient for the present work. Fibrinogen is composed of two identical half molecules, which are probably related by an exact or approximate two-fold axis through the central nodule (4, 5). The two-fold molecular symmetry is consistent with the overall shape of the molecule, with the identification of the nodules with specific parts of the sequence (6, 7), and with the measurements of periodicities in fibrin and crystalline aggregates (3,4,8). The central nodule is the site of activation by the protease thrombin, which cleaves off the small fibrinopeptides to form fibrin monomer. The fibrin monomer is essentially identical in structure to fibrinogen, but instead of being soluble, it spontaneously polymerizes to form fibrin fibers.The most informative structural feature seen in electron micrographs offibrin fibers is a pattern oftransverse bands, which repeat every 22.5 nm or exactly one-half the length of the molecule. The generally accepted interpretation of this banding pattern is that the rod-like molecules are parallel to the fiber axis; the molecules are arranged end-to-end to form strands, which are one molecule thick; and alternate strands are staggered by one-half the length of the molecule (8).Ferry identified a two-stranded polymer of fibrin, which he called a protofibril, and proposed a two-step mechanism for the polymerization of fibrin (9). In the first step, fibrin monomers polymerize to form protofibrils; in the second step, these protofibrils associate laterally to form the thicker fibrin fibers. ...

In light of a previous report suggesting that the brains of tenascin- deficient animals are grossly normal, we have studied the somatosensory cortical barrel field and injured cerebral cortex in postnatal homozygous tenascin knockout, heterozygote, and normal wild-type mice. Nissl staining, cytochrome oxidase, and Dil axonal tracing of thalamocortical axonal projections to the somatosensory cortex, all reveal the formation of normal barrels in the first postnatal week in homozygous knockout mice that cannot be distinguished from heterozygote or normal wild-type barrels. In addition to confirming the absence of tenascin in knockout animals, and reporting apparently reduced levels of the glycoprotein in barrel boundaries of heterozygote animals using well-characterized antibodies and immunocytochemistry, we also studied the DSD-1-PG proteoglycan, another developmentally regulated molecule known to be associated with transient glial/glycoconjugate boundaries that surround developing barrels; DSD-1-PG was also found to be expressed in barrel boundaries in apparently normal time frames in tenascin knockout mice. Peanut agglutinin (PNA) binding of galactosyl- containing glycoconjugates also revealed barrel boundaries in all three genotypes. We also examined the expression of tenascin-R, a paralog of tenascin-C (referred to here simply as tenascin). As previously reported, tenascin-R is prominently expressed in subcortical white matter, and we found it was not expressed in the barrel boundaries in any of the genotypes. Thus, the absence of tenascin does not result in a compensatory expression of tenascin-R in the barrel boundaries. Finally, we studied wounds of the cerebral cortex in the late postnatal mouse. The astroglial scar formed, for the most part, in the same time course and spatial distribution in the wild-type and tenascin knockout mice. However, there may be some differences in the extent of gliosis between the knockout and the wild type that warrant further study. Roles for boundary molecules like tenascin during brain pattern formation and injury are reconsidered in light of these findings on barrel development and cortical lesions in tenascin-deficient mice.

A stable population of fibrinogen dimers cross-linked by Factor XIIIa has been prepared and examined in the electron microscope. The trinodular fibrinogen molecules are cross-linked through their outer nodules in an end-to-end, non-overlapping fashion. These dimers form normal banded fibers after treatment with the clotting enzyme, thrombin.