Studies on intercellular LETS glycoprotein matrices

Chen, Lan Bo; Murray, Andrew S.; Segal, Rosalind A.; Bushnell, Anne; Walsh, Marcia L.

doi:10.1016/0092-8674(78)90123-x

Cited by 312 publications

(157 citation statements)

References 41 publications

Supporting

Mentioning

146

Contrasting

Unclassified

Order By: Relevance

“…1B,C), in agreement with previously published images of such artificial Fn fibers (Ejim et al, 1993;Wojciak-Stothard et al, 1997). Indeed, cryo-scanning electron microscopic images suggest that Fn fibers exist as 'cables' comprised of individual fibrous strands of ~5-15 nm in diameter and larger (Chen et al, 1978;Dzamba and Peters, 1991;Peters et al, 1998;Singer, 1979) which are proposed to be held together by hydrogen bonds, intermolecular beta-strand swapping (Briknarova et al, 2003;Litvinovich et al, 1998), disulfide bonds which are potentially formed by cryptic disulfide isomerase activity (Langenbach and Sottile, 1999), and other weak electrostatic interactions (Chen and Mosher, 1996;Morla et al, 1994). Although the exact location and properties of these bonds are unknown, it has been observed that they are strong enough to render cell-derived Fn fibers irreversibly insoluble in 1% de-oxycholate (McKeown-Longo and Mosher, 1983), which is a phenomenon we observed with manually deposited Fn fibers as well (data not shown).…”

Section: Manually Deposited Fn Fibers Bundle Into Fiber Cables Similasupporting

confidence: 90%

“…To briefly characterize the morphology of our deposited fibers, it should be noted that the average diameter (3.7± 1.0 µm) of fibers deposited from a 0.76 mg/mL Fn solution, as observed via fluorescence confocal microscopy, is similar to that of the thickest fibers and branching areas found in cell culture matrices (Chen et al, 1978). The average fiber diameter can be moderately adjusted via the pulling procedure.…”

Section: Manually Deposited Fn Fibers Bundle Into Fiber Cables Similamentioning

confidence: 83%

“…Fibers used in this study were generally 1 to 2 cm long. Fibronectin fibers in 24-h Fn matrix produced by NIH-3T3 fibroblasts in cell culture form a mesh-like network where smaller fibrils merge to form bundles and later branched again (Chen et al, 1978).…”

Section: Manually Deposited Fn Fibers Bundle Into Fiber Cables Similamentioning

confidence: 99%

See 2 more Smart Citations

Assay to mechanically tune and optically probe fibrillar fibronectin conformations from fully relaxed to breakage

et al. 2008

View full text Add to dashboard Cite

In response to growing needs for quantitative biochemical and cellular assays that address whether the extracellular matrix (ECM) acts as a mechanochemical signal converter to co-regulate cellular mechanotransduction processes, a new assay is presented where plasma fibronectin fibers are manually deposited onto elastic sheets, while force-induced changes in protein conformation are monitored by fluorescence resonance energy transfer (FRET). Fully relaxed assay fibers can be stretched at least 5-6 fold, which involves Fn domain unfolding, before the fibers break. In native fibroblast ECM, this full range of stretch-regulated conformations coexists in every field of view confirming that the assay fibers are physiologically relevant model systems. Since alterations of protein function will directly correlate with their extension in response to force, the FRET vs. strain curves presented herein enable the mapping of fibronectin strain distributions in 2D and 3D cell cultures with high spatial resolution. Finally, cryptic sites for fibronectin's N-terminal 70-kD fragment were found to be exposed at relatively low strain, demonstrating the assay's potential to analyze stretch-regulated protein-rotein interactions.

show abstract

Section: Manually Deposited Fn Fibers Bundle Into Fiber Cables Similasupporting

confidence: 90%

Section: Manually Deposited Fn Fibers Bundle Into Fiber Cables Similamentioning

confidence: 83%

See 1 more Smart Citation

Assay to mechanically tune and optically probe fibrillar fibronectin conformations from fully relaxed to breakage

et al. 2008

View full text Add to dashboard Cite

show abstract

“…Three major types of investigation have been pursued in this area: (a) at the biochemical level, extracellular matrix molecules such as fibronectin (for review, see reference 42) and intracellular cytoskeletal molecules such as a-actinin (50,55) (12,26) have been isolated and implicated in the formation of such contacts; (b) at the light microscopic level, interference reflection microscopy has been used to define different sites of contact of cells to substrata (20,46,47), and immunofluorescence microscopy to provide information at the resolution of the light microscope about some of the molecular species associated with those sites (4,8,19,23,26,34,45,57,69) (c) at the electron microscopic level, the ultrastructural morphology of the contact sites has been investigated (2, 6, 1 I, 37-39, 54), and immunoelectron microscopic studies of the extracellular components of these sites have been initiated (13,15,24,25,40,56). While much has been learned from these investigations, there is still considerable uncertainty about the molecular ultrastructure of different types of contact sites and particularly about their transmembrane relationships.…”

mentioning

confidence: 99%

Immunoelectron microscopic studies of the sites of cell-substratum and cell-cell contacts in cultured fibroblasts.

Chen¹,

Singer²

1982

The Journal of Cell Biology

351

199

View full text Add to dashboard Cite

Our object was to obtain information about the molecular structures present at cell-substratum and cell-cell contact sites formed by cultured fibroblasts. We have carried out double irnmunoelectron-microscopic labeling experiments on ultrathin frozen sections cut through such contact sites to determine the absolute and relative dispositions of the three proteins fibronectin, vinculin, and a-actinin with respect to these sites.(a) Three types of cell-substratum and cell-cell contact sites familiar from plastic sections could also be discriminated in the frozen sections by morphological criteria alone, i.e., the gap distances between the two surfaces, and the presence of submembranous densities. These types were: (i) focal adhesions (FA); (ii) close contacts (CC); and (iii) extracellular matrix contacts (ECM). This morphological typing of the contact sites allowed us to recognize and assign distinctive immunolabeling patterns for the three proteins to each type of site on the frozen sections.(b) FA sites were immunolabeled intracellularly for vinculin and a-actinin, with vinculin labeling situated closer to the membrane than a-actinin. Fibronectin was not labeled in the narrow gap between the cell surface and the substratum, or between two cells, at FA sites. Control experiments showed that this could not be ascribed to inaccessibility of the FA narrow gap to the immunolabeling reagents but indicated an absence or severe depletion of fibronectin from these sites.(c) CC sites were labeled intracellularly for a-actinin but not vinculin and were labeled extracellularly for fibronectin.(d) ECM sites were characterized by large separations (often >100 nm) between the cell and substratum or between two cells, which were connected by long cables of extracellular matrix components, including fibronectin. In late (24-36 h) cultures, ECM contacts predominated over the other types. ECM sites appeared to be of two kinds, one labeled intracellularly for both aactinin and vinculin, the other for a-actinin alone.(e) From these and other results, a coherent but tentative scheme is proposed for the molecular ultrastructure of these contacts sites, and specific functional roles are suggested for fibronectin, vinculin, and a-actinin in cell adhesion and in the linkage of intracellular microfilaments to membranes at the different types of contact sites.The nature and molecular structure of the sites of contact of fibroblastic cells with one another and with the substrata on which they grow are subjects of much recent interest. Three major types of investigation have been pursued in this area: (a) at the biochemical level, extracellular matrix molecules such as fibronectin (for review, see reference 42) and intracellular cytoskeletal molecules such as a-actinin (50,55) (12,26) have been isolated and implicated in the formation of such contacts; (b) at the light microscopic level, interference reflection microscopy has been used to define different sites of contact of cells to substrata (20,46,47), and immunofluorescence micro...

show abstract

“…The cell-free, extracellular matrix was prepared from nearly confluent cultures of embryonic tibroblasts (BALB/c mouse, Chinese hamster or human (GM38)), using the detergent NP40, according to Chen [22].…”

Section: Preparation Of the Extracellular Matrixmentioning

confidence: 99%

Isolation and characterization of Chinese hamster cells defective in cell-cell coupling via gap junctions

Willecke¹,

Müller²,

Drüge³

et al. 1983

Experimental Cell Research

View full text Add to dashboard Cite

SUMMARYChinese hamster Wg3-h-o cells which were descended from DON cells have been mutagenized and selected for derivatives defective in metabolic cooperation via gap junctions (i.e., met-). The selection protocol included four consecutive cycles of cocultivating mutagenized cells, deficient in hypoxanthine phosphoribosyltransferase (HPRT) and wild-type cells in the presence of thioguanine (cf Slack, C, Morgan, R H M & Hooper, M L, Exp cell res 117 (1978) 195-205) [8]. We carried out the last two selection cycles in the presence of 1 mM dibutyryl cyclic adenosine monophosphate (db-CAMP). The isolated Chinese hamster CI-4 cells which expressed the met-phenotype most stringently showed the following characteristics:1. In standard culture medium no cell-cell coupling was detected among CI-4 cells when assayed by injections of the fluorescent dye Lucifer yellow or by electrical measurements. Between 73 and 100% of the met+ parental cells were coupled under these conditions. Up to 14% positive contacts were found between CI-4 cells and Chinese hamster Don cells (met'). Confluent CI-4 cells grown in the presence of 1 mM db-CAMP showed 9% coupled cells.2. No gap junction plaques were found on electron micrographs of freeze-fractured, confluent CI-4 cells. The met+ parental cells showed small gap junction plaques (0.013 % of the total cell surface analyzed).3. CI-4 cells exhibited 16% positive contacts and the parental Wg3-h-o cells showed 92 % positive contacts in autoradiographic measurements of metabolic cooperation with DON cells. On an extracellular matrix, prepared from normal embryonic libroblasts, metabolic cooperation between CI-4 and DON cells was autoradiographically measured to be 68 %. Other cells of spontaneous met-phenotype (for example mouse L cells or human fibrosarcoma HT1080 cells) also appeared to exhibit increased metabolic cooperation when grown on an extracellular matrix and assayed by autoradiographic measurements. When tested by Lucifer yellow injections, however, only very few positive contacts were found for CI-4/DON cell pairs and no positive contacts were found among mouse L cells grown on an extracellular matrix.4. The met-defect in the genome of CI-4 cells was cured in somatic cell hybrids with mouse embryonic fibroblasts or with mouse embryonal carcinoma cells. The results of isozyme and karyotype studies of met-, as well as met+ somatic cell hybrids suggest that mouse chromosome 16 may be involved in complementation of the met-defect.Most cells in organs and in tissue culture show cell-cell coupling via gap junctions which-can be demonstrated by the spreading of electrical signals, injected fluorescent dyes or radioactively labelled metabolites between contiguous cells [l, 21. In many cases gap junction plaques, i.e., arrays of particles of polygonal symmetry, have been demonstrated by different techniques of electron microscopy on apposed membranes of contiguous cells. Since the appearance of gap junction plaques and the demonstration of cell-cell coupling (by one or more of

show abstract

Studies on intercellular LETS glycoprotein matrices

Cited by 312 publications

References 41 publications

Assay to mechanically tune and optically probe fibrillar fibronectin conformations from fully relaxed to breakage

Assay to mechanically tune and optically probe fibrillar fibronectin conformations from fully relaxed to breakage

Immunoelectron microscopic studies of the sites of cell-substratum and cell-cell contacts in cultured fibroblasts.

Isolation and characterization of Chinese hamster cells defective in cell-cell coupling via gap junctions

Contact Info

Product

Resources

About