Physarum actinin previously isolated [Hatano, S., & Owaribe, K. (1976) in Cell Motility (Goldman, R., Pollard, T., & Rosenbaum, J., Eds.) Vol. 3, Book B, p 499, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY] was found to be a 1:1 complex of actin and fragmin which is a regulatory factor in the formation of actin filaments. Since fragmin did not contain a cysteine residue, it was purified from the complex by the selective cleavage of actin with 2-nitro-5-thiocyanobenzoic acid, followed by column chromatography. Fragmin had nearly the same molecular weight as actin, but had a quite different amino acid composition. When added to G-actin before polymerization, fragmin accelerated the initial viscosity increase of actin solutions induced by salts, but kept the final viscosity much lower than normal F-actin. When added to F-actin after polymerization, fragmin drastically reduced the viscosity of actin solutions. In both cases, the final products of reaction of fragmin with actin were short F-actin filaments. The number average length of the filaments decreased with the increasing molar ratio of fragmin to actin, and the length distribution was always exponential. Fragmin required for its activity a concentration of free Ca2+ higher than 10(-6) M. When the concentration of free Ca2+ was lower than 10(-7) M, fragmin affected neither actin polymerization nor F-actin. The regulation by Ca2+ was reversible.

Accurately estimating the spectral reflectance of art paintings from low-dimensional multichannel images requires that both image-acquisition hardware with appropriate spectral characteristics and appropriate estimation software be applied to the captured multichannel image. In this study, a system that incorporates both factors is designed and developed on the basis of the minimum-mean-square error criterion. The accuracy of spectral estimation by use of this system is evaluated, and the system's high performance is demonstrated.

The possible role of a 140K membrane-associated protein complex (140K) in fibronectin-cytoskeleton associations has been examined. The 140K was identifed by the monoclonal antibody JG22E. Monoclonal and polyclonal antibodies to the 140K showed identical patterns of binding to the cell membranes of fixed and permeabilized chicken embryonic fibroblasts; localization was diffuse, but with marked concentration in cell-toextracellular matrix contact sites. Correlative localization with interference reflection microscopy and double-label or triple-label immunofluorescence showed that 140K co-distributed with extracellular fibronectin fibrils and intracellular a-actinin in microfilament bundles at extracellular matrix contact sites but tended not to co-localize with tropomyosin present in bundles at sites farther from adhesion sites. In addition, binding of antibodies to 140K, aactinin, and fibronectin was excluded from vinculin-rich focal adhesion sites at the cellular periphery. A progressive development of cell surface a-actinin-140K-fibronectin associations was observed in early spreading cells. The anti-140K monoclonal antibody JG22E inhibited the attachment and spreading of both normal and Rous sarcoma virus-transformed chicken embryonic fibroblasts to a fibronectin substratum. However, the anti-140K monoclonal antibody became a positive mediator of cell attachment and spreading if it was adsorbed or cross-linked to the substratum. Our results provide the first description of a membraneassociated protein complex that co-localizes with fibronectin and microfilament bundles, and they suggest that the 140K complex may be part of a cell surface linkage between fibronectin and the cytoskeleton.Interactions of cells with extracellular materials are critically important events during embryonic development and for the maintenance of normal tissue functions. Fibronectin has been shown to promote the adhesion and spreading of cells ~ on a variety of materials including plastic, collagen, gelatin, and fibrin (for reviews, see references 15, 22, 26, 31, 37, and 47). Concomitant with the spreading induced by added fibronectin, cells often acquire highly ordered intracellular microfilament bundles (MFBs) I (1, 45). In highly spread cells, extracellular matrix (ECM) fibers that contain fibronectin are often Abbreviations used in this paper: CEF, chicken embryo fibroblasts; CEL, chicken embryonic lung; ECM, extracellular matrix; IRM, interference reflection microscopy; 140K, a three component, membrane-associated protein complex; MFB, microfilament bundle; RSV, Rous sarcoma virus.observed to correspond in their arrangement with intracellular MFBs (20,23,39). At sites where ECM fibrils appear to attach to the plasma membrane, there is a co-distribution of actin and a-actinin (and sometimes vinculin) inside cells and fibronectin outside cells (4,9,24,40, 41).Immunoelectron microscopy has shown a spatial relationship between fibronectin and a-actinin at membrane attachment sites in spread fibroblasts, which were termed ECM...

Abstract. The dynamic process of embryonic cell motility was investigated by analyzing the lateral mobility of the fibronectin receptor in various locomotory or stationary avian embryonic cells, using the technique of fluorescence recovery after photobleaching. The lateral mobility of fibronectin receptors, labeled by a monoclonal antibody, was defined by the diffusion coeIficient and mobile fraction of these receptors. Even though the lateral diffusion coefficient did not vary appreciably (2 × 10 -~° cm2/s ~< D ~< 4 × 10 -I° cm2/s) with the locomotory state and the cell type, the mobile fraction was highly dependent on the degree of cell motility. In locomoting cells, the population of fibronectin receptors, which was uniformly distributed on the cell surface, displayed a high mobile fraction of 66 + 19% at 25°C (82 + 14% at 37°C). In contrast, in nonmotile cells, the population of receptors was concentrated in focal contacts and fibrillar streaks associated with microfilament bundles and, in these sites, the mobile fraction was small (16 __+ 8%). When cells were in a stage intermediate between highly motile and stationary, the population of fibronectin receptors was distributed both in focal contacts with a small mobile fraction and in a diffuse pattern with a reduced mobile fraction (33 + 9%) relative to the diffuse population in highly locomotory cells. The mobile fraction of the fibronectin receptor was found to be temperature dependent in locomoting but not in stationary cells. The mobile fraction could be modulated by affecting the interaction between the receptor and the substratum. The strength of this interaction could be increased by growing cells on a substratum coated with polyclonal antibodies to the receptor. This caused the mobile fraction to decrease. The interaction could be decreased by using a probe, monoclonal antibodies to the receptor known to perturb the adhesion of certain cell types which caused the mobile fraction to increase. From these results, we conclude that in locomoting embryonic cells, most fibronectin receptors can readily diffuse in the plane of the membrane. This degree of lateral mobility may be correlated to the labile adhesions to the substratum presumably required for high motility. In contrast, fibronectin receptors in stationary cells are immobilized in focal contacts and fibrillar streaks which are in close association with both extracellular and cytoskeletal structures; these stable complexes appear to provide firm anchorage to the substratum.URING early embryonic development, certain groups of cells can transiently express locomotory properties that allow them to migrate long distances from their sites of origin and populate other areas of the embryo (for reviews, see references 16,41,56,57). There is a large body of evidence that suggests that cell motility results from the conjunction of environmental influences and intrinsic properties of cells. For example, the extracellular matrix glycoprotein fibronectin is known to promote cell locomotion in vitro (3,46,47,61...

We have characterized a 140-kDa glycoprotein complex purified by a monoclonal antibody and implicated in cell adhesion to the extracellular molecule fibronectin. Three major polypeptide components were purified by monoclonal antibody JG22E, which had apparent molecular weights of 155,000 (band 1), 135,000 (band 2), and 120,000 (band 3). In two-dimensional gel electrophoresis, each subunit migrated as either a broad band or a series of spots at acidic isoelectric points. After treatment with neuraminidase, the spots became focused around pH 6.2 (band 1), pH 5.6 (band 2), and pH 5.3 (band 3). These three major bands were compared by two-dimensional peptide mapping in a series of pairwise combinations and were found to be distinct proteins. In sucrose gradients, these proteins co-migrated as a complex sedimenting at approximately 8.4 S either before or after affinity purification, whereas separated subunits migrated at 4.7 to 5.8 S. Amino acid analysis revealed no detectable hydroxyproline and a composition characterized by a substantial number of cysteine residues compared to the average protein. Our results suggest that a noncovalent complex of structurally distinct glycoproteins is involved in adhesive interactions of fibronectin with cells.

Abstract. Recent studies have shown that fibronectin and its 140K membrane receptor complex are spatially associated with microfilaments to form cell surface linkage complexes which are thought to mediate adhesive interactions between fibroblasts and their substrata. We examined the regulation of the organization of these cell surface structures in transformed and fibronectin-reconstituted cells as well as in cells treated with a competitive synthetic peptide inhibitor of fibronectin binding to its receptor. Correlative localization experiments with interference reflection microscopy and double-label or triple-label immunofluorescence revealed a concomitant loss of fibronectin, 140K receptor, and alpha-actinin colocalization at cell substratum extracellular matrix contact sites after transformation of chick fibroblasts by wild-type or temperature-sensitive Rous sarcoma viruses (RSV). Western and dot immunoblot analyses established that although similar total quantities of intact 140K molecules were present in the transformed cell cultures, significantly more was released into the culture medium of transformed cells. The 140K molecules on transformed cells were available for interaction with exogenously added fibronectin, which could reconstitute fibronectin-140K linkage complexes. In such fibronectin reconstitution experiments, many cells expressed both fibronectin-140K-actin linkage complexes and RSV pp60 s~, indicating that the morphological reversion could occur even in the continued presence of RSV transformation. The synthetic peptide Gly-ArgGly-Asp-Ser derived from the sequence of the cellbinding region of fibronectin could also prevent the organization of fibronectin-140K linkage complexes. Our results suggest that fibronectin interaction with cells regulates the organization of fibronectin receptor complexes and cytoskeletal components at the cell surface.

Fibronectin and other cell attachment proteins provide molecular models for beginning to unravel the complex interactions of the cell surface with the extracellular matrix. This area has been reviewed in considerable detail previously [I-lo]. Our brief review will therefore be selective rather than comprehensive, and it will focus on some recent generalizations about this class of proteins, as well as on recent advances in the molecular analysis of the functions of these proteins and their receptors. We shall also present various popular or provocative hypotheses and speculations about future work in the field. CLASSES AND SPECIFICITY OF ATTACHMENT PROTEINS of 1)An emerging generalization about this class of proteins is that they are composed separable functional regions, each specialized for specific binding activities (Fig. . Each appears to have one or more regions essential for binding to the cell surface. Fibronectin (Fig. 2) interacts with the fibroblast cell surface primarily through a region termed the cell-binding or cell-recognition site; recent molecular analysis of the function of this site will be discussed below. Although this site appears to be required for cell interactions with fibronectin [ 11,121, interactions at a heparin-binding domain may provide substantial strengthening of this interaction [ 131. In addition, neuronal cells may be capable of interacting with a heparin-binding domain elsewhere in the molecule in the process of axonal elongation; fibronectin-independent interactions with extracellular materials such as heparan-sulfate-containing molecules appear to be important in some aspects of this process [ 14,151.

A three-dimensional vertically-stacked flexible integrated circuit is demonstrated based on hybrid complementary inverters made of n-type In–Ga–Zn–O (a-IGZO) amorphous oxide thin-film transistors (TFTs) and p-type poly-(9,9-dioctylfluorene-co-bithiophene) (F8T2) polymer TFTs, where all the fabrication processes were performed at temperatures ≤120 °C. Saturation mobilities of the a-IGZO TFT and the F8T2 TFT are ∼3.2 and ∼1.7×10−3 cm2 V−1 s−1, respectively, from which we chose the appropriate dimensions of the TFTs so as to obtain a good balance for the inverter operation. The maximum voltage gain is ∼67, which is better than those reported for organic/oxide hybrid complementary inverters.